In Vitro and in Vivo Pharmacology of Novel Phenylaminotetralin (PAT) Analogs at Serotonin 5-HT2 Receptors

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In Vitro and in Vivo Pharmacology of Novel Phenylaminotetralin (PAT) Analogs at Serotonin 5-HT2 Receptors Development of Drugs for Neuropsychiatric Disorders
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1 online resource (135 p.)
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english
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Kondabolu, Krishnakanth
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University of Florida
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Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Pharmaceutical Sciences, Medicinal Chemistry
Committee Chair:
Booth, Raymond G
Committee Members:
James, Margaret O
Sloan, Kenneth B
Morgan, Drake

Subjects

Subjects / Keywords:
5-ht2a -- 5-ht2b -- 5-ht2c -- drug-discovery -- gpcr -- pharmacology -- psychosis -- schizophrenia -- serotonin
Medicinal Chemistry -- Dissertations, Academic -- UF
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Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Novel 4-phenyl-N,N-dimethyl-1,2,3,4-tetrahydroanphthalene-2-amine(phenylaminotetrahydronaphthalin; PAT) analogssynthesized in our laboratories have been shown to specifically activate thehuman serotonin 5-HT2C receptors while acting as antagonists/inverseagonists at 5-HT2A and 5-HT2B receptors. This unique 5-HT2pharmacology was hypothesized to translate to efficacy in animal models ofpsychosis. The specific aims here were to characterize the molecular determinantsfor 5-HT2 affinity of new PAT derivatives synthesized before andduring the course of this thesis research (Aim 1), characterize the 5-HT2functional pharmacology of PATs meeting minimum affinity criteria (Aim 2), and,determine if PATs that demonstrate 5-HT2C specific agonism togetherwith 5-HT2A/2B antagonism/inverse agonism translate in preclinicalstudies to efficacious compounds to treat psychoses (Aim 3). Some noteworthy results are that meta- and para-substitutionof the 4-phenyl substituent impactedstereoselective high affinity of especially the trans-PAT analogs across 5-HT2 receptors. For example,(2S,4R)-(-)-enantiomer usually demonstrated highest affinity across 5-HT2receptors regarding meta-substituted trans-PATs, while, the (2R,4S)-(+)-enantiomerusually demonstrated highest affinity across 5-HT2 receptors regardingpara-substituted trans-PATs. Results from ligand docking studies conducted by acollaborator suggest that a conserved (across 5-HT2 receptors)serine S5.43 residue in the fifth transmembrane domain impacts binding of the4-para-substituted-PATs differentlythan the corresponding 4-(meta-substituted)-PATs.The (2S,4R)-(-)-trans-(meta-chloroand meta-bromo)-PAT analogs had thehighest affinity across 5-HT2 receptors and also demonstratedhighest potency and efficacy regarding 5-HT2C agonism together with5-HT2A/2B antagonism/inverse agonism in functional studies using humanrecombinant 5-HT2 receptors expressed in HEK clonal cells.Substitutions at the 6- and/or 7-position of tetrahydronaphthyl moiety, with orwithout concomitant halogen substitution at the 4-meta-phenyl-position, did not provide analogs with enhanced potencyor efficacy regarding functional activity. Accordingly, the trans-4-(meta-chloro and meta-bromo)-PATswere selected for study in translational studies that also included the trans-4-(para-chloro)-PATs for comparison. The PATs were assessed in three different rodent psycholocomotormodels of psychosis, which encompass the dopaminergic, glutamatergic andserotonergic neurotransmitter dysfunction thought to underlie thepathophysiology of psychoses. The (2S,4R)-(-)-trans-4-(meta-Cl and meta-Br)-PAT analogs demonstratedsuperior potency compared to (2S,4R)-(-)-trans- and (2R,4S)-(+)-trans-4-(para-Cl)-PATanalogs, suggesting that the in vitro medicinal chemistrystructure-activity relationship accurately translated to predict thepreclinical activity of PATs as antipsychotic drugs.
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In the series University of Florida Digital Collections.
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Includes vita.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Krishnakanth Kondabolu.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: Booth, Raymond G.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-05-31

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UFE0045235:00001


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1 IN VITRO AND IN VIVO PHARMACOLOGY OF NOVEL P HENYL A MINO T ETRALIN (PAT) ANALOGS AT SEROTONIN 5 HT 2 RECEPTORS: DEVELOPMENT OF DRUGS FOR NEUROPSYCHIATRIC DISORDERS By KRISHNAKANTH KONDABOLU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Krishnakanth Kondabolu

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3 To my mom, dad, brother wife and all those who have inspired and supported me through this journey

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4 ACKNOWLEDGMENTS I would like to express my most sincere gratitude to my committee chair, Dr. Raymond Booth, for his support and guidance through these five years in graduate school. His constant advice and encouragement were a tremendous source of inspiration for me in this project. I would also like to express my gratitude to our collaborator Dr. Drake Morgan for his time and patience in teaching me the ins and outs of in vivo experiments and disease modeling. I would like to thank Dr. Ken Sloan for his inputs about physical chemistry and also for making our TA sessions as fun as they were I would like to thank Dr. Margaret James for teaching me all I know about drug metabolism. I would like to thank all the post docs in my lab for helping me out during the rough patches of graduate school: Dr. Clinton Canal for his constant support and help whenever I had trouble with my experiments, Dr. Tania Cordova Sintjago for helping me make sense of my experiments, Dr. Rajeev Sakhuja and Dr. Myong Sang Kim for their expertise in organic synthesis. I would also like to thank all my colleagues in the department for their suppor t and encouragement Last but not least, I would like to thank my family for their constant support and patience. Special thanks to my wife Bindu for putting up with me during these years in graduate school

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREV IATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 G PROTEIN COUPLED RECEPTORS: MOLECULAR DETERMINANTS AND THERAPEUTIC RELEVANCE OF SEROTONIN 5 HT 2A AND 5 HT 2C RECEPTORS IN SCHIZOPHRENIA ................................ ................................ ....... 16 Introduction to G Protein Coupled Receptors ................................ ......................... 16 GPCR Structure ................................ ................................ ............................... 16 GPCR Signal Transduction ................................ ................................ .............. 17 Models of Ligand GPCR Interactions ................................ .............................. 18 Ballesteros and Weinstein GPCR Nomenclature ................................ ............. 19 Ligand Stabilized GPCR Conformations ................................ ........................... 20 Serotonin and 5 HT 2 GPCRs ................................ ................................ .................. 24 Serotonin Biosynthesis and Metabolism ................................ ........................... 24 Serotonin 5 HT 2 GPCRs ................................ ................................ ................... 24 5 HT 2 GPCR Signaling Pathways ................................ ................................ ..... 25 5 HT 2C GPCR Post Translational Modifications ................................ ............... 25 5 HT 2 GPCR Localization in Brain ................................ ................................ .... 27 Schizophrenia and Serotonin 5 HT 2 GPCRs ................................ .......................... 28 Relevance of 5 HT 2 GPCRs in Schizophrenia ................................ .................. 29 Serotonin 5 HT 2A Receptors and Schizophrenia ................................ .............. 29 Serotonin 5 HT 2B and 5 HT 2C Receptors and Schizophrenia ........................... 30 Antipsychotics Drugs and Obesity ................................ ................................ .... 31 Molecular Determinants for Ligand Binding at 5 HT 2 GPCRs .......................... 32 In Vivo Models to Screen for Antipsychotic Drug Efficacy ................................ 34 Central Hypothesis and Goals of this Dissertation ................................ .................. 37 AIM 1: Delineate Molecular Determinants for Binding of 4 phenyl N N dimethyl 1,2,3,4 tetrahydroanphthalene 2 amine (phenylaminotetralin, PAT) Derivatives at Human Recombinant 5 HT 2A and 5 HT 2C GPCRs ......... 38 AIM 2: Characterize the 5 HT 2 Functional Pharmacology of PATs .................. 38 AIM 3: Translational Studies: Assess ment of PATs Efficacy in rodent models of psychosis ................................ ................................ ...................... 39

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6 2 DELINEATE MOLECULAR DETERMINANTS FOR BINDING OF 4 PHENYL N N DIMETHYL 1,2,3,4 TETRAHYDROANPHTHALENE 2 AMINE (PHENYLAMINOTETRALIN; PAT) DERIVATIVES AT HUMAN RECOMBINANT 5 HT 2A AND 5 HT 2C GPCRs ................................ ................................ ................... 43 Specific Aim 1 ................................ ................................ ................................ ......... 43 Methodology ................................ ................................ ................................ ........... 44 Clonal Cell Culture and Transfection ................................ ................................ 44 Radioreceptor Competition Binding Assays ................................ ..................... 44 Results and Discussion for Competition Binding Assays ................................ ........ 45 Affinity of Unsubstituted PATs at the 5 HT 2A Receptor ................................ ..... 45 Affinity of Un substituted PATs at the 5 HT 2C Receptor ................................ ..... 45 Affinity of 4 ( ortho Substituted) PATs at the 5 HT 2A Receptor .......................... 46 Affinity of 4 ( ortho Substituted) PATs at the 5 HT 2C Receptor ......................... 47 Affinity of 4 ( meta Substituted) PATs at the 5 HT 2A Receptor .......................... 48 Affinity of 4 ( meta Substituted) PATs at the 5 HT 2C Receptor .......................... 52 Affinity of 4 ( para Substituted) PATs at the 5 HT 2A Receptor ........................... 55 Affinity of 4 ( para Substituted) PATs at the 5 HT 2C Receptor .......................... 5 8 Affinity of 4 ( meta Substituted Phenyl) and/or (6, 7 substituted Tetrahydronaphthalene) PATs at the 5 HT 2A Receptor ................................ 61 Affinity of 4 ( meta Substituted Phenyl) and/or (6, 7 substituted Tetrahydronaphthalene) PATs at the 5 HT 2C Receptor ................................ 64 Computational Chemistry and Molecular Modeling Studies: In silico Docking of PATs at 5 HT 2 receptors ................................ ................................ ........... 67 3 CHARACTERIZATION OF THE 5 HT 2 FUNCTIONAL PHARMACOLOGY OF PAT ANALOGS ................................ ................................ ................................ ...... 84 Specific Aim 2 ................................ ................................ ................................ ......... 84 Methodology ................................ ................................ ................................ ........... 84 Results and Discussion for Functional Assays ................................ ....................... 86 trans trans 4 ( meta halogenated) PATs at 5 HT 2 Receptors ................................ ................................ .............. 86 Functional Activity of (+) and ( ) t rans 4 ( p Cl) PATs at 5 HT 2 Receptors ..... 88 Functional Activity of ( ) t rans 4 ( m Cl) (6 OMe Tetrahydronaphthalene) PAT Analog at 5 HT 2A and 5 HT 2C Receptors ................................ ............... 88 4 TRANSLATIONAL STUDIES: ASSESSMENT OF PAT ANALOGS EFFICACY IN RODENT MODELS OF PSYCHOSIS ................................ ................................ 97 Specific Aim 3 ................................ ................................ ................................ ......... 97 Methodology ................................ ................................ ................................ ........... 97 In vivo Behavior al Pharmacology ................................ ................................ ..... 97 Psycholocomotor Activity (Head Twitch Response; HTR) Elicited by the Serotonin 5 HT 2 Agonist 2,5 dimethoxy 4 iodoamphetamine (DOI) .............. 98 Psycholocomotor Activity Elicited by the Glutamate Antagonist MK 801 ......... 98

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7 Psycholocomotor Activity Elicited by the Dopamine/Serotonin Agonist Amphetamine ................................ ................................ ................................ 99 Results for PAT Efficacy in Rodent Models of Psychosis ................................ ....... 99 5 CONCLUSION ................................ ................................ ................................ ...... 116 APPENDIX A AFFINITY OF 4 ( META OR PARA SUBSTITUTED) PATs at HISTAMINE H1 GPCRs ................................ ................................ ................................ .................. 118 B LIST OF PUBLICATIONS RESULTING FROM THE WORK IN THIS DISSERTATION ................................ ................................ ................................ ... 120 LIST OF REFERENCES ................................ ................................ ............................. 123 BIOGRAPH ICAL SKETCH ................................ ................................ .......................... 135

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8 LIST OF TABLES Table page 2 1 Shows changes in the bond length relative to changes in substituent. ............... 81 2 2 Binding affinities of parent PAT enantiomers and meta substituted PAT enantiomers at 5 HT 2A an d 5 HT 2C receptors. ................................ .................... 81 2 3 Binding affinities of para substituted PAT enantiomers at 5 HT 2A and 5 HT 2C receptors. ................................ ................................ ................................ ........... 82 2 4 Binding affinities of tetrahydronaphthalene ring with (or without) pendant phenyl ring substituted PAT analogs. ................................ ................................ 83 3 1 Functional activity of PAT analogs at 5 HT 2 receptors. ................................ ...... 96 4 1 Efficacy of PAT analogs ED 50 (95% CL) mg/kg at in vivo models of schizophrenia. ................................ ................................ ................................ .. 115

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9 LIST OF FIGURES Figure page 1 1 Generic 2 dimens ional structure of GPCR with amino acids numbered from amino terminal to carboxy terminal. ................................ ................................ .... 40 1 2 3 dimensional representation of GPCR.. ................................ ............................ 40 1 3 Extended ternary complex model. ................................ ................................ ...... 41 1 4 Ligands activati n g serotonin 5 HT 2 receptors. ................................ .................... 41 1 5 Structure of hallucinogens (phenylethylamines and tryptamines) acting on 5 HT 2 receptors. ................................ ................................ ................................ .... 41 1 6 Structure of M100,907. A selective antagonist of 5 HT 2A receptors. ................... 42 1 7 Ligands used in in vivo models of psychosis ................................ ...................... 42 2 1 Stereochemical relationship between PAT diastereomers and enantiomers. ..... 72 2 2 Structure of meta substituted PAT analogs. ................................ ....................... 73 2 3 Change in Ki value at 5 HT 2A receptors with increase in the size of substituent at meta position. ................................ ................................ ............... 73 2 4 Structure of para substituted PAT analogs ................................ ........................ 74 2 5 Effect of sterics on the affinity of para substituted PAT enantiomers at 5 HT 2A and 5 HT 2C receptors. ................................ ................................ ......................... 74 2 6 Single X ray crystallographic structure of (2 S ,4 R ) trans p Cl PAT. HCl ...... 75 2 7 Tetrahydronaphthalene and/or pendant phenyl ring substituted PAT analogs. .. 75 2 8 t rans PAT enantiomer docked at 5 HT 2A receptors with the corresponding amino acid interactions in binding pocket. ................................ .. 76 2 9 t rans PAT docked at 5 HT 2C receptors with the corresponding amino acid interactions in binding pocket. ................................ ................................ ............ 76 2 10 t rans m Br PAT enantiomer docked at 5 HT 2C receptors.. ........................... 77 2 11 (2 R ,4 S ) (+) trans and (2 S ,4 R ) ( ) trans p Cl PAT enantiomer docked at 5 HT 2A receptors ................................ ................................ ................................ ... 77 2 12 (2 R ,4 S ) (+) trans and (2 S ,4 R ) ( ) trans p Cl PAT enantiomer docked at 5 HT 2C receptors ................................ ................................ ................................ ... 78

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10 2 13 trans 6 OH 7 Cl PAT at 5 HT 2A 5 HT 2B and 5 HT 2C receptors ................................ ................................ ................................ 79 2 14 Trans 6 OH 7 Cl PAT and (+) trans 6 OH 7 Cl PAT docked at 5 HT 2A receptors. ................................ ................................ ................................ ........... 80 3 1 trans m Cl PAT enantiomer at 5 HT 2A receptor. ................................ ................................ ........... 90 3 2 trans m Cl PAT enantiomer at 5 HT 2B receptor. ................................ ................................ ........... 90 3 3 Functional trans m Cl PAT enantiomer at 5 HT 2C receptor. ................................ ................................ .......... 91 3 4 trans m Cl PAT at 5 HT 2A receptors. ................................ ...... 91 3 5 trans m Br PAT at 5 HT 2A 5 HT 2B and 5 HT 2C receptors. ................................ ................................ ................................ ........... 92 3 6 PLC trans p Cl PAT at 5 HT 2A receptors. ........ 92 3 7 trans p Cl PA T at 5 HT 2B receptors. ........ 93 3 8 trans p Cl PAT demonstrating partial agonism at 5 HT 2C receptors. ................................ ................................ ............. 93 3 9 Schild plot of (+) trans p Cl PAT at 5 HT 2A receptors. ................................ ........ 94 3 10 trans 6 OMe m Cl PAT enantiomer at 5 HT 2A and 5 HT 2C receptors. ................................ ................................ ................................ ... 94 3 11 trans m CF 3 PAT enantiomer at 5 HT 2C receptors. ................................ ................................ ................................ ........... 95 4 1 Clozapine decreases DOI induced HTR in a dose dependent manner. ........... 105 4 2 Clozapine 1 mg/kg dose attenuates both MK 801 and amphetamine (Amp) induced locomotion. ................................ ................................ .......................... 106 4 3 Attenuation of MK 801 induced loco trans PAT analogs ................................ ................................ ................................ ............ 107 4 4 Attenuation of amphetamine stimulated locomotor activity by PAT enantiom ers. ................................ ................................ ................................ ..... 108 4 5 Effect of highest dose of various ligands on locomotor activity ......................... 109

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11 4 6 Attenuation of MK trans m Br PAT. ................................ ................................ ................................ ................. 110 4 7 trans m Cl PAT. ................................ ................................ ................................ ................. 110 4 8 Attenuation of MK trans m Cl PAT ................................ ................................ ................................ ................. 111 4 9 Attenuation of HTR by (+) and trans p Cl PAT analogs. ............................ 111 4 10 trans p Cl PAT analogs 20 minu tes prior to DOI. ................................ ................................ ........................ 112 4 11 trans p Cl PAT on MK 801 (0.3 mg/kg) induced locomotor activity. ................................ ................................ ............................. 113 4 12 trans p Cl PAT on amphetamine (3 mg/kg) induced locomotor activity. ................................ ................................ ............................. 114

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12 LIST OF ABBREVIATIONS MSH Melanocyte stimulating hormone 5 HT serotonin ADAR adenosine deam inase actin g on ribonucleic acid CAM s constitutively active mutants CNS central nervous system DAG diacylglycerol DMA 2 5 dimethoxy phenylisopropylamine DMEM DOI 2 5 dimethoxy 4 iodoamphetamine EPS Extrapyramidal side effects GAP GTPase accelerating protein GDP guanine diphosphate GPCR G protein coupled receptor GTP guanine triphosphate HDL high dens ity lipoproteins HEK human embryonic kidney HPLC high pressure liquid chromatography HTR head twitch response HVD heart valve disease IP 3 inositol triphosphate MAO A monoamino oxidase A m CPP meta chlorophenylpiperazine NMDA N methyl D aspartate

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13 PAT phenylaminotetrahydronaphthalene PIP2 phosphatidylinositol bisphosphate POMC Pro opiomelanocortin RGS regulators of G protein RNA ribonucleic acid SAR structure activity relationship SN Substa ntia Nigra VTA Ventral tegmental area

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IN VITRO AND IN VIVO PHARMACOLOGY OF NOVEL P HENYL A MINO T ETRALIN (PAT) ANALOGS AT SE ROTONIN 5 HT 2 RECEPTORS: DEVELOPMENT OF DRUGS FOR NEUROPSYCHIATRIC DISORDERS By Krishnakanth Kondabolu M ay 2013 Chair: Raymond G. Booth Major: Pha r maceutical Sciences Medicinal Chemistry N ovel 4 phenyl N N dimethyl 1,2,3 ,4 tetrahydroanphthalene 2 amine ( p henyl a mino t etrahydronaphthalin ; PAT) analogs synthesized in our laboratories have been shown to specifical ly activate the human serotonin 5 HT 2C receptors while acting as antagonists/inverse agonists at 5 HT 2A and 5 HT 2B receptors. Th is unique 5 HT 2 pharmacology was hypothesized to translate to efficacy in animal models of psychosis. The specific aims here were to characterize the molecular determinants for 5 HT 2 affinity of new PAT derivatives synthesized before and durin g the course of this thesis research (Aim 1), characterize the 5 HT 2 functional pharmacology of PATs meeting minimum affinity criteria (Aim 2), and, determine if PATs that demonstrate 5 HT 2C specific agonism together with 5 HT 2A/2B antagonism/inverse agonism translate in preclinical studies to efficacious compounds to treat psychoses (Aim 3). Some noteworthy results are that meta and para substitution of the 4 phenyl substituent impacted stereoselective high affinity of especially the trans PAT analog s across 5 HT 2 receptors. For example, the (2 S ,4 R ) enantiomer usually demonstrated highest affinity across 5 HT 2 receptors regarding meta substituted trans PATs, while,

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15 the (2 R ,4 S ) (+) enantiomer usually demonstrated highest affinity across 5 HT 2 recep tors regarding para substituted trans PATs. Results from ligand docking and molecular modeling studies conducted by a collaborator suggest that a conserved (across 5 HT 2 receptors) serine S5.43 residue in the fifth transmembrane domain impacts binding of t he 4 para substituted PATs differently than for the corresponding 4 ( meta substituted) PATs. The (2 S ,4 R ) trans ( meta chloro and meta bromo ) PAT analogs had the highest affinity across 5 HT 2 receptors and these analogs also demonstrated highest potency and efficacy regarding 5 HT 2C agonism together with 5 HT 2A/2B antagonism/inverse agonism in functional studies using human recombinant 5 HT 2 receptors expressed in HEK clonal cells Substitutions at the 6 and/or 7 position of tetrahydronaphthyl moiety, wi th or without concomitant halogen substitution at the 4 meta phenyl position, did not provide analogs with enhanced potency or efficacy regarding functional activity. Accordingly, the trans 4 ( meta chloro and meta bromo) PATs were selected for study in tra nslational studies that also included the trans 4 ( para chloro) PATs for comparison. The PATs were assessed in three different rodent psycholocomotor models of psychosis, which encompass the dopaminergic, glutamatergic and serotonergic neurotransmitter dys function currently thought to underlie the pathophysiology of human psychoses. The (2 S ,4 R ) trans 4 ( meta Cl and meta Br) PAT analogs demonstrated superior potency compared to (2 S ,4 R ) trans and (2 R ,4 S ) (+) trans 4 ( para Cl) PAT analogs, suggesting that the in vitro medicinal chemistry structure activity relationship accurately translated to predict the preclinical activity of PATs as antipsychotic drugs.

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16 CHAPTER 1 G PROTEIN COUPLED RECEPTORS: M OLECULAR DETERMINANTS AND THERAPEUTIC RELEVANCE OF SEROTONIN 5 HT 2A AND 5 HT 2C RECEPTORS IN SCHIZOPHRENIA Introduction to G Protein Coupled Receptors G protein coupled receptors (GPCRs) constitut e the largest class of cell surface receptors in the human body representing, about one percent of the genome, and involved in regulating nearly all known physiological functions 1 GPCRs transduce signals from stimuli including neurotransmitters, peptides, small proteins and even photons of light intracellularly ac r o ss the membrane of cell 2 Genetic alt erations in GPCRs may lead to loss or gain in function at the receptor and account for many diseases like diabetes insipidus, dwarfism and hypothyroidism 3 5 GPCR Structure All GPCRs share a seven transmembrane structure despite the functional and chemical diversity of their signaling molecules. GPCRs a re made up of an extracellular N terminus, seven transmembrane domains and an intracellular C terminus. The seven transmembrane domains are connected to each other by extracellular and intracellular loops (Figure 1 1 and 1 2 ). The N terminus, extracellular loops and transmembrane domain play a role in ligand selection and prevent non specific ligands from binding to the receptor 6 ligand to dock and transmit the stimulus from the ligand to the G protein via the intracellular loops. This external stimulus is then converted into a chemical me ssenger and propagated intracellularly 7 Based on the homology of amino acid sequence, GPCRs can be cl assified into three classes: rhodopsin like receptors (Class 1), secretin like receptors (Class 2) and

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17 metabotropic glutamate like receptors (Class 3) 8 GPCRs within the same class share more than 25% sequence similarity. GPCRs can also be differentiated based on t he site of ligand binding: Class 1 receptors bind ligands in their transmembrane region, Class 2 receptors bind ligands in the extracellular loops and Class 3 receptors bind ligands in the N terminus (Venus fly trap) 9 GPCR Signal Transduction The G proteins couple to GPCR and mediate the transduction of the external stimuli into intracellular signaling molecules. G protein is a heterotrimeric protein unit is a Ras like protein and possesses GTPase activity. In its inacti unit binds to guanosine diphosphate units. Fatty acylation (palmitoyl group) anchoring G protein at th e interface of the plasma membrane 10 This anchoring of the G protein is done such that it is in close proximity with both the GPCR and effector systems 11 A GPCR system consists of a GPCR, a G protein and an effector 12 Activation of G protein by the GPCR re sults in the exchange of GTP for a GDP leading to the dissociation of the G units can now bind to different effector systems 13 G pr otein mediated activation of effectors produce secondary messengers that propagate the signal by activation or inactivation of a cascade of proteins. Activity of G GTP back to GDP. Following this the G pro tein collapses back into its inactive trimeric conformation 1 unit include phospholipase C, phospholipas e A 2 and adenyl cyclase. Mammals have about 20

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18 units that may complex units. These sub units can be arranged in different permutations and can give rise to a vast signaling diversity for the GPCR. unit is also capable of activating different unit regulates the responsiveness of sodium, potassium and calcium channels 11 14 Different G protein sub units are capable q leads to activation of phospholipase C that catalyzes the conversion of phosphatidylinositol bisphosphate (PIP 2 ) into inositol triphosphate (IP 3 ) and diac yl glycerol (DAG). In unit leads to activation of adenyl cyc lase enzyme that mediates the cyclization of ATP into cAMP. Models of Ligand GPCR I nteractions Ligands that bind to GPCRs to elicit a pharmacological response can be endogenous or exogenous in origin Agonist ligands stabilize an active conformation of the receptor that couples to G protein, whereas, antagonist ligands stabilize a conformation that does not favor the coupling of GPC R to G protein s 15 However, it was later discovered that GPCRs are capable of activating G proteins even in the absence of ligands 16 This property of the GPCRs is referred to as constitutive activity or basal activity 17 Ligands capable of decreasing this basal activity of the receptor are referred to as inverse agonists. The level of constitutive activity depends on the intrinsic property of the receptor and also on th e cellular milieu in which the receptor is present 18 For example, when both serotonin 5 HT 2A and 5 HT 2C receptors were expressed to the same density in a heterologous system, 5 HT 2A receptors showed a lower level of basal activity as compared to 5 HT 2C receptors 19

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19 The extended ternary complex model is the widely accepted model of GPCR activatio n 20 This theory postulates that a re ceptor exists in equilibrium between its inactive and active state conformations. GPCRs exist in equilibrium between active conformation, inactive conformation and active G protein coupled conformation A l igand may bind with different affinities to any of the se receptor conformation s (F igure 1 3) 21 Agonists have higher affinity for a GPCR in an active conformation as compared to inactive conformation The coupling of the G p rotein to an agonist s tabilized receptor is positively cooperative. This means that binding of a ligand to a GPCR enhances the interaction between GPCR and G protein. The activation of the G protein by substituting the GDP for a GTP is negatively cooperati ve. This is because the binding of GTP induces a conformational change in the G protein that is transmitted to the GPCR and negatively influences the binding of the agonist. This also initiates the uncoupling of the G protein from the GPCR. The constitut ive activity of the receptor and the affinity of the agonist depend on the amount of GPCR expressed on the membrane relative to the amount of G protein expressed in the cell system 18 Over e xpression of the GPCR in a heterolo gous system results in majority of the GPCR s to be in a G protein uncoupled state i.e., a low aff inity conformation for binding of agonist ligands. Ballesteros and Weinstein GPCR N omenclature Numbering the amino acids starting from the amino terminal and ending at the carboxy terminal does not allow for an efficient comparison of amino acid sequence b etween GPCRs. Significant diversity between GPCRs present in same class or family hinders such a system of nomenclature. This system of nomenclature also does no t give information about the position of the amino acid within the GPCR ternary structure, that is, whether it is present in the extracellular loop, transmembrane domain or

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20 intracellular loop. Ballesteros and Weinstein devised a numbering system for amino acids based on the characteristic conserved amino acid present in GPCR s belonging to the same class. In this system of nomenclature, each amino acid is identified first based on its location in the transmembrane domain. The most conserved residue in the transmem brane domain within the same family is then identified and assigned the number 50. The position of other amino acids is defined relative to this conserved amino acid 22 For example, asparagine amino acid in transmembrane 1 is the most conserved amino acid a cross class 1 GPCRs and hence is designated as N1.50, where 1 represents the transmembrane domain. G1.49 represents the glycine amino acid preceding the conserved asparagine when looking down from the extracellular region into the intracellular region 23 N umbering in this system increases as we move from the extracellular to the intracellular end of transmembrane 1, then, the numbering increases going from the intracellular to extracellular end of tr ansmembrane 2 and so on. Mutagenesis studies involve mutations of amino acids in the wild type receptor to a different amino acid. Ballesteros nomencl ature can be used to identify both the amino acid in the wild type receptor and the amino acid to which it has been mutated. For example, A5.46 represents an alanine amino acid present in the fifth transmembrane of the wild type serotonin 5 HT 2A receptor s When this amino acid is mutated to a serine it is represented as A5.46S. Ligand Stabilized GPCR C onformatio ns Binding of an agonist to a GPCR stabilizes a conformation of the GPCR that favors the coupling of a G protein to the receptor. In contrast, an i nverse agonist stabilizes a conformation of a GPCR that does not favor the binding of G proteins resulting in a signaling outcome that is less than basal constitutive signaling 24 X ray

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21 crys tal structures give a remarkable insight into the conformational changes involved in GPCR activation. O btaining a crystal structure o f a GPCR is difficult, however, due to GPCR inherent thermodynamic flexibility within the phospholipid membrane environment and a heterogeneity of receptor conformations 9 Purified GPCRs are not stable in detergents which are compatible with crystallography GPCRs possess structural heterogeneity because of the different post translational modifications such as phosphorylation, palmito ylation and glycosylation. This structural heterogeneity along with the conformational heterogeneity makes it difficult to standardize a common procedure for GPCR crystallization. Despite these difficulties a few chimeric GPCRs have been crystallized. Som e generalizations can be made about the organization and position of the amino acids in class 1 GPCRs based on the information provided by these crystal structures. For example, for all class 1 GPCRs the amino acids in the alpha helices at the interface be tween the membrane and the external milieu are comprised mainly of positively charged amino acids that interact with the negatively charged phospholipid heads of the lipid bilayer 23 The cytoplasmic end of the GPCR has critical residues that interact with G protein. A conserved DRY (aspartate, arginine, tyrosine) motif at the end of transmembrane domain 3 is surrounded by several hydrophobic residues from helix II (P2.38, L2.39), cytoplasmic loop II (F4.37), helix V (L5.61, V5.65) and helix VI (V6.33, M6.36). These residues together with amino acids from the intracellular loops are hypothesized to constitute the binding site of the G protein 25 The X 2 adrenergic receptor, adenosine 2A 1 adrenergic rece ptor (turkey), dopamine D 3 c hemokine CXCR 4 and

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22 histamine H 1 class 1 GPCRs have been determined 26 X ray crystal structures give an or. Molecular switches are non covalent interactions between amino acids present in the inactive receptor that are disrupted upon activation of the receptor. Some of the highly conserved motifs that act as molecular switches include the D/ERY motif (ionic lock) at the end of transmembrane 3 interacting with N6.30 at the end of transmembrane 6 and other switch es includes C6.47, W6.48 and F6.52 in transmembrane domain 6 (rotamer toggle switch) 27 a majority of class 1 GPCRs. As a consequence of this kink, the cytoplasmic end of transme mbrane 6 is angled towards the cytoplasmic end of transmembrane 3. A cluster of conserved aromatic amino acids F6.44, W6.48, F6.51 and F6.52 surround P6.50 and regulate the angle of the kink (rotamer toggle switch) This cluster of aromatic amino acids fac es the binding pocket and agonists interacting with these amino acids induce a conformation change such that the angle of the kink changes resulting in the activation of the receptor 28 An agonist when bound to a receptor can activate the receptors in two possible ways. It could disrupt existing amino acid interactions that hold the receptor in the inactive conformation, or it could stabilize a conformation of the receptor that is more active. Partial agonist and agonist differ in the conformation of the r eceptor they stabilize. For example, catechol, salbutamol and dopamine are partial agonists capable in the way they activate the receptor. Catechol stabilizes a conf ormation of the receptor where the rotamer toggle switch is activated, bu t the ionic lock is not broken.

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23 Salbutamol stabilizes a conformation where the ionic lock is broken, but the rotamer toggle switch is not activated. In contrast to this, dopamine acti vates both the rotamer toggle switch and breaks the ionic lock. Taken together these results indicate that different ligands activate different molecular switches thereby stabilizing different conformations of the receptor. They also show that there are m ore molecular switches yet to be discovered since dopamine, despite activating both the molecular switches, still behaves as a partial agonist 27 Crystal structures provide an understanding of GPCR ligand molecular interactions and give inferences about GPCR conformations that lead to agonist or inverse agonist or neutral antagonism functional outcomes. The GPCR molecular determinants involved in ligand recognition and GPCR conformational changes, in turn, provide inferences on how ligands interact with GPCRs to stabilize a particular conformation that leads to agonist, inverse agoni st or neutral antagonist function. The few known GPCR crystal structures provide templates for homology modeling of GPCRs that have not yet been crystallized. For example, ligand docking results presented in this thesis were based on the 5 HT 2 receptor hom ology models developed based on human 2 adrenergic GPCR crystal structure 29 The 5 HT 2 homology models h elped delineate the 3 dimensional arrangement of amino acids involved in binding of novel 4 phenyl N N dimethyl 1,2,3,4 tetrahydroanphthalene 2 amine (PAT) ligands 30 Results were used to design ligands that might better exploit hypothesized PAT 5 HT 2 molecular interactions toward higher affinity ligands. Results were corroborated by mutagenes is studies that helped to validate hypothesized PAT 5 HT 2 molecular interactions, thus, validating the 5 HT 2 homology models

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24 Serotonin and 5 HT 2 GPCRs Serotonin Biosynthesis and M etabolism Serotonin (5 hydroxytryptamine, 5 HT) is synthesized both in the ce ntral nervous system and the periphery from the aromatic amino acid tryptophan (Figure 1 4) Synthesis of serotonin involves a two step process; the first step involves hydroxylation of tryptophan by tryptophan hydroxylase. This hydroxylation step is the rate limiting step in the synthesis of serotonin. There are two isoforms of tryptophan hydro xylase: tryptophan hydroxylase 1 and 2. Tryptophan hydroxylase 1 is primarily localized in the periphery and tryptophan hydroxylase 2 regulates the synthesis of serotonin in the central nervous system 31 5 hydroxytryptophan, the product of trypt ophan hydroxylase, is decarboxylated by 5 hydroxytryptophan decarboxylase to produce serotonin. Serotonin is primarily metabolized by monoamine oxidase A (MAO A) into 5 hydroxyindoleacetic acid, an inert metabolite t hat is eliminated from the body Seroton in can also be metabolized by indole N methyl transferases to generate N methyl serotonin and N,N dimethyl seroto nin 32 Serotonin 5 HT 2 GPCRs Serotonin acts at 7 different families of GPCRs (5 HT 1 ,2,4 7 ), an ion channel family ( 5 HT 3 ) 33 and the serotonin neurotransporter (SERT), which terminates the action of serotonin released into the synapse and assists in recycling of serotonin 34 The work in this thesis focuses on the s erotonin 5 HT 2 class 1 GPCRs that are involved in the regulation of diverse physiological processes (e.g., vascular smooth muscle contraction, sleep cycle ) and psychological processes (e.g., psychoses, anxiety mood ) 35

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25 5 HT 2 GPCR Signaling P athway s The serotonin 5 HT 2 family consists of three GPCRs ( 5 HT 2A 5 HT 2B and 5 HT 2C ) that share significant structural homology and the same main signaling pathway For example 5 HT 2A and 5 HT 2C receptors share 80% sequence similarity within their transmembrane region and a 50% overall sequence similarity 36 Serotonin 5 HT 2 receptors also share similarity in signaling pathway due to the coupling with the same G proteins. These receptors couple to Gq G protein that in turn activates the phospholipase C. Phospholipase C catalyzes the conversion of phosphatidylinositol bisp hosphate (PIP 2 ) into water soluble inositol triphosphate (IP 3 ) and lipophilic diacyl glycerol (DAG). Inositol triphosphate binds to IP 3 receptors on the endoplasmic reticulum causing the release of calcium. DAG mediates the activation of protein kinases th at play a key role in the phosphorylation of numerous enzymes 37 Different ligands dose dependently modulate the activity of effector system to produce secondary messenger. These secondary messengers can be measured to determine the extent of 5 HT 2 receptor activation or inactivation by a test ligand. The serotonin 5 HT 2 receptors have been shown to also couple to other phospholipases like phospholipase D and phospholipase A 2 Activation of phospholipase A 2 catalyzes the cleavage of PIP 2 to produce a rachidonic acid and lysophospholipid 38 Phospholipase D catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid and choline 39 5 HT 2C GPCR Post Translational M odifications 5 HT 2C receptors are unique in their ability to undergo RNA editing. RNA editing is a type of post transcriptional modification, like splicing, which alters the primary nucleotide in the pre mRNA 40 RNA editing is characterized by the deamination of

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26 adenosine to form inosine and is catalyzed by adenosine deaminase that acts on RNA (ADAR). The ADAR enzymes can convert adenosine into inosine at 5 different locations on the pre mRNA of 5 HT 2C receptors. These 5 nucleotides correspond to 3 amino acids located on the second intracellular loop of 5 HT 2 C receptors. The unedited human 5 HT 2C receptor has an isoleucine, asparagine and isoleucine (INI) at position 156, 158 and 160 in the amino acid sequence 41 Editing these three amino acids result in 24 isoforms of the 5 HT 2C receptor with different amino acids at these positions 42 For example, the fully edited isoform replaces the INI amino acids with valine, glycine and valine (VGV) amino acids, and a partially edited isoform has valine, serine and valine (VSV) amino aci ds at these positions. The mRNA of partially edited VSV isoform is the most abundant isoform in the human brain (33%) where as the VNV isoform mRNA is the most abundant in the rat brain (33%). The second intracellular loop of GPCR plays an important role in coupling with G proteins to the GPCR. Therefore alterations of the amino acids at this position due to RNA editing result in reduction in the constitutive activity of the edited isoforms. The unedited INI isoform possesses the highest basal activity whi le the fully edited isoform VGV had a very low basal activity. The partially edited VSV isoform showed intermediate basal activity 43 These isoforms also differ in their functional profile, that is, the potency of an agonist to activate the different isoforms of 5 HT 2C receptors. Agonists like 5 HT, 2,5 dimethoxy 4 iodoamphetamine ( DOI ) and N N dimethyltryptamine ( DMT ) had a higher affinity and potency to activate the INI isoform relative to the VSV isofo rm and VGV isoforms. Post transcriptional RNA editing of the 5 HT 2C receptors contributes to its molecular diversity of signaling. The mRNA level of the edited isoforms were

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27 different in different regions of the brain indicating varying levels of basal act ivity of 5 HT 2C receptors in different brain regions 41 However, it is difficult to determine the level of 5 HT 2C receptor isoform protein expressed on the membrane as it is not proportiona l to the amount of mRNA detected 42 The INI (unedited wild type) isoform of 5 HT 2C receptors was used to assess the 5 HT 2C affinity and function of the PAT analogs in this thesis. Only the c DNA for the wild type receptor wa s available commercially. It is noted that the in vivo variation o f 5 HT 2C edited isoform expression in different mammalian brain regions is not accurately reflected by the in vitro molecular pharmacology studies here; in fact, as noted, the unedited wild type 5 HT 2C receptor is not the major isoform in rodents or humans. Nevertheless, the importance of not accounting for the possibility of 5 HT 2C post translational editing may not be so great in the transient transfection clonal cell system used in the cur rent studie s. This is because are greatly overexpressed relative to G protein, thus, G protein availability and not receptor G protein interaction may be the limiting factor in functional outcomes. In any event, the molecular pharmacology methods used here wer e successful to identify and translate PATs with potential antipsychotic activity (see Aim 3 Results). 5 HT 2 GPCR Localization in B rain The location of the 5 HT 2 receptors in the CNS has been characterized using radioligand binding and immunohistochemical studies 33 These studies revealed a high localization of the 5 HT 2A receptors in forebrain regions (particularly cortical areas like neocortex and pyriform cortex), caudate nucleus, nucleus accumbens and hippocampus. Serotonergic neurons a re clustered in the dorsal raphe nucleus and these neurons project from here to other parts of the brain. 5 HT 2A receptors are located

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28 post synaptically along the distribution of these axons from dorsal raphe nucleu s 44 The 5 HT 2A receptors are also widely distributed in the periphery. In contrast, 5 HT 2C receptors are exclusively present in the CNS. Serotonin 5 HT 2C receptors are distributed in limbic system (nucleus accumbens) ventral tegmental area (VTA) and basal ganglia (substantia nigra and caudate nucleus). Schizophrenia and S erotonin 5 HT 2 GPC R s Schizophrenia is a chronic mental disorder that is prevalent across 1 2% of world population perceptual and behavioral symptoms, and, cognitive symptoms. Positive perceptual and behav ioral symptoms manifest as hallucinations (mostly auditory) and delusions, whereas, negative symptoms manifest as apathy, s ocial withdrawal and anhedonia Cognitive symptoms include deficits in attention and memory 45 46 The p athophysiol ogy of schizophrenia is hypothes i z ed to include excessive stimulation of striatal dopamine D 2 receptors, a deficient activation of prefrontal dopamine D 1 receptors (dopamine theory) and alterations in glutamate rgic N methyl D aspartate (NMDA) receptor connectivity in prefrontal cortex (glutamate theory). Increased activation of mesolimbic dopaminergic pathway is hypothesized to mediate pos itive symptoms of schizophrenia 47 49 Mesocortical projections of dopamine neurons are hypothesized to be hypoactive in schizophrenia. This decreased activity is thou ght to contribute to negative and cognitive deficits in schizophrenia 50 The negative and cognitive deficits can also be attr ibuted to NMDA hypofunction in prefrontal cortex. Multiple lines of evidence implicat e the role of glutaminergic neurotransmission in schizophrenia : risk genes for schizophrenia affect functioning of NMDA receptor and a dministration of NMDA receptor antagonist to normal subjects produce symptoms similar to negative

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29 and cognitive deficits o bserved in schizophrenia 51 In addition to glutamate and dopamine neurotransmission, there is a significant role played by serotonergic neurotransmission in schizophrenia, including, specifically, serotonergic neurotransmission involving 5 HT 2A and 5 HT 2C GPCRs. Relevance of 5 HT 2 GPCRs in Schizophrenia Serotonin 5 HT 2A and 5 HT 2C receptors are localized in bra in regions (prefrontal cortex, VTA and accumbens) that regulate both glutamatergic and dopaminergic systems N umerous clinically approved newer atypical antipsychotics (olanzapine, c lozapine, aripiprazole) exert an antagonist action at 5 HT 2A receptors 52 Lack of motor side effects in the newer antipsychotics has been attributed to their action at serotonin receptors 53 Further evidence supporting involvement of 5 HT 2A receptors in schizophrenia is provided by similarities between hallucinogen induced psychosis and schizophrenia 54 55 Majority of the post mortem studies on brain tissue of schizophrenic patients reveal decreased levels of 5 HT 2A receptors However, it should be acknowledged that not all post mortem st udies found decreased 5 HT 2A receptor levels in schizophrenic patients. Serotonin 5 HT 2A receptor T102C and A1438G polymorphism has been reported to increase the risk of schizophrenia 56 57 Serotonin 5 H T 2A Receptors and S chizophrenia Serotonin 5 HT 2A receptors are primarily localized on pyramidal neurons in prefrontal cortex and can regulate the activity of corticotegmental neurons by enhancing glutamate release in VTA. This increased glutamate in turn stimulates glutamate receptors on VTA dopaminergic projections resulting in an increased activity of mesocortical projections 5 8 Administration of serotonin to a midbrain slice increases the firing of a large population of cells in the VTA and this firing was blocked by a selective

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30 an t agonist of 5 HT 2A receptors 59 60 This indicates the presence of 5 HT 2A receptors in VTA and demonstrates their ability to modulate dopaminergic output from VTA. 5 HT 2A receptors are localized o n t he dopaminergic neurons and are directly involved in their activation 61 Similar results were obtained in nucleus accumbens where administration of 5 HT 2A receptor antagonists decreased 5 HT induced dopamine release in accumbens 62 Administration of non competitive NMDA receptor antagonis t to norma l subjects results in a behavio ral response si milar to negative and cognitive deficits of schizophrenia 63 Both 5 HT 2A receptor antagonist s and atypical antipsychotic clozapine reversed this blockade of NMDA receptor in pyramidal neurons of medial prefrontal cortex. These findings suggest that 5 HT 2A antagonists may have a beneficial effect on cognitive deficits in schizophrenia 64 Serotonin 5 HT 2B and 5 HT 2C R eceptors and S chizophrenia There is no evidence implicating the 5 HT 2B receptors in schizophrenia. However, a ctivation of 5 HT 2B receptor s has been shown to produce cardiac valvulopathy or heart valve disease (HVD) in the periphery. HVD was found to be a major side effect of the drug combination fenfluramine phenteramine (Pondimin) (Figure: 1 4) This drug combination was therapeutically u seful for weight loss. The mechanism of action of fenfluramine is by indirect activation of serotonergic system by releasing serotonin from their presy naptic vesicles and also rever sing the function of serotonin transporter thereby increasing 5 HT concentr ation in the synapse 65 Neither of the fenfluramine enantiomers had a good affinity at 5 HT 2B receptors. However norfenfluramine, a metabolite of fenfluramine was f ound to be an agonist at 5 HT 2B receptors. Norfenfluramine mediated activation of 5 HT 2B receptor s leads to increased mitogenic

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31 activity resulting in matrix remodeling on the heart valves ultimately leading to HVD 66 Hence it is important to screen ligands for activity at 5 HT 2B receptors. Serotonin 5 HT 2C receptors are localized in both the VTA and substantia nigra (SN). Th is indicates their ability to modulate both mesolimbic and nigrostriatal dopaminergic pathways 67 Administration of a 5 HT 2B and 5 HT 2C receptor antagonist increases the firing rate of dopaminergic neurons in both VTA and SN 68 However, a 5 HT 2C receptor ag onist selectively decreased the firing of mesolimbic pathway and did not affect basal firing of nigrostriatal pathway 69 These data indicate that 5 HT 2C receptors could tonically inhibit dopaminergic output from nigrostriatal pathway either by constitutive activity or endogenous serotonin mediated activation. These 5 HT 2C receptors are hypothesized to be located on the GABAergic inter neurons of the VTA 70 There is relatively less evidence for the presence of 5 HT 2C receptor mRNA in prefrontal cortex. Studies demonstrate the presence of 5 HT 2C receptors in cingulate cortex 71 Hence, more studies focus on the effects of 5 HT 2C receptor ligands on me socortical dopaminergic pathway (also originating from VTA) Similar to mesolimbic pathway, 5 HT 2C receptors tonically inhibit the release of dopamine in mesocortical pathway and 5 HT 2C agonist further decrease the dopamine released in prefrontal cortex 72 Antipsychotics Drugs and O besity The major side effect of atypical antipsychotics is weight gain and the hypothesized neurotransmitter receptors implicated in this are 5 HT 2A 5 HT 2C histamine H 1 receptor, adrenergic 1 2 receptors and muscarinic M 3 receptor 73 This study by Kroeze et al showed the correlation between binding affinity of antipsychotics

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32 at these receptors and weight gain The correlation was found to be highest for the H 1 receptors followed by 1A followed by 5 HT 2C receptors. Transgenic mice with 5 HT 2C receptors kno cked out show ed an increase in weight 74 Further evidence for the role of 5 HT 2C receptors in weight gain was provided by FDA approval of 5 HT 2C receptor agonist, lorcaserin, to treat obesity 75 Several experimental ligands that act as agonist s at 5 HT 2C receptor also decrease food consumption in animals 76 77 The a norexic action s of serotonin 5 HT 2C receptors are hypothesized to be mediated by pro opiomelanocortin (POMC) neurons located in arcuate nucleus of hypothalamus. Activation of 5 HT 2C receptors triggers the conversion of POMC, a peptide precursor, into melanocyte stimulating hormone ( MSH). MSH is then released by the se activated neurons; this was detected by Fos like immunoreactivity detected in arcuate nucleus following administ ration of D fenfluramine 78 MSH interacts post synaptically with melanocortin 3 and 4 receptors to alter e nergy homeostasis and decrease food intake 79 80 Around 80% of MSH neurons were found to have 5 HT 2C mRNA indicating the modulation of melonocortin pathways by serotonin 5 HT 2C receptors. This mechanism of action explains the weight gain side effects observed following administration of atypical antipsychotic s which act as antagonist at 5 HT 2C receptors. Molecular Determinants for Ligand B inding at 5 HT 2 GPCRs The ligand binding pocket for serotonin 5 HT 2 receptors is well characterized for some ligands. Structural features of ligands binding at 5 HT 2 receptors give insight about the binding pocket based on the ligand GPCR interactions. Hallucinogens that bind to 5 HT 2 receptors can be classified into tryp tamines and phenylethylamines. The

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33 tryptamines include the structurally rigid ergolines like lysergic acid diethylamide (LSD) and the more flexible tryptamines like N,N dimethyl tryptamine (DMT). The phenylalkylamines also have structurally flexible ligand s like 2,5 dimethoxy 4 iodoamphetamine (DOI) that bind to 5 HT 2 receptors (F igure 1 5 ) 81 interacts with positively charged amines moiety of ligands bi nding at 5 HT 2 receptors 82 83 Mutat ions of this amino acid resulted in a loss of binding at 5 HT 2C receptors indicating that this interaction is essential for ligand binding 84 Aspartate D3.32 anchors the terminal amines of ligands and assists in binding at 5 HT 2 receptors. Serine amino acid, S3.36, is present one helical turn below the aspartate and also participates in ligand binding by forming hydrogen bonds with terminal amine of ligand. Mutations of this amino acid to alanine re sulted in a decrease in binding affinity for primary amines (5 HT). However, there was no change in affinity for tertiary amines like LSD or DMT at 5 HT 2A receptors 85 Similar results were obtained for 5 HT 2C receptors where a more structurally rigid tertiary amine (2 S 4 R ) trans 4 phenyl 2 N,N dimethyl 1,2,3,4 tetrahydronaphthalene 2 amine [ trans PAT] showed only a slight decrease in affinity 84 Similar to 2 adrenergic receptor, m olecular switches like the intracellular end of transmembrane 3 and intracellular end of transmembran e 6 hold 5 HT 2A receptors in inactive conformation 86 Mutation of the s erine residue S5.43A lead s to a decrease in affinity for serotonin a t 5 HT 2A receptors. The amino acid at 5.46 is a serine at 5 HT 2A receptors but is an alanine at 5 HT 2C receptors. Interchanging these amino acids switched the binding profiles of ergoline derivatives like mesulergine and

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34 LSD Their affinities at the corres ponding receptors were reversed, for example the affinity at 5 HT 2A receptor mutant S5.46A was similar to affinity at 5 HT 2C receptors 87 This position has also been shown to sterically modulate the efficacy of ligands by determining whether the ligand is an agonist or a partial agonist at 5 HT 2A receptors. The S5.46A mutat ion resulted in partial agonists, 1 N methyl 5 hydroxy tryptamine and 1 N methyl tryptamine acting as full agonists at 5 HT 2A receptor mutants 88 Two phenylalanines in transmembrane 5, F5.47 and F5.48 are also important for binding of ligands at 5 HT 2A re ceptors. Mutation of F5.47 to alanine decreased the affinity of ketanserin and ritanserin at 5 HT 2A receptors 89 A number of residues in transmembrane 6 (L6.37, N6.29, K6.32, C6.34 and V6.36) have been shown to be important for 5 HT 2A mediated phosphotidyl inositol hydrolysis 90 Some of the aromatic amino acids (W6.48, F6.51 and F6.52) present in transmembrane 6 have also been shown to be important for activation of 5 HT 2A receptors 91 93 These aromatic amino acids form a interaction with aromatic systems in ligand 89 Tyrosin e Y7.43 also had a significant effect in binding of 5 HT and ketanserin at 5 HT 2A receptors 91 I n V ivo Models to Screen for Antipsychotic Drug Efficacy The head twitch response (HTR) is an easily observable and quantifiable dose dependent response in rodents that is elicited by hallucinogens and is proposed as a model to screen for antipsychotic drug efficacy 55 94 Serotonin 5 HT 2 agonists that cause hallucinations in humans produce the HTR, however, 5 HT 2 agonists that do not produce ha llucinations in humans do not exhibit the HTR in rodents. It is noted, however, that the hallucinations produced by 5 HT 2 agonists in humans are primarily visual in nature as compared to positive symptoms of schizophrenia that are primarily

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35 auditory halluc inations 54 Nevertheless, 5 HT 2A receptors are thought to play a role in schizophrenia in part, because h allucinogens mediate their action by activating 5 HT 2A receptors in cortical regions of the brain 95 Also, as noted, most antipsychotic drugs act as antagonists at 5 H T 2 A receptors, and, correspondingly, these antipsychotic drugs possess the ability to decrease the hallucinogen induced HTR in rodents 96 97 Furthermore, there is a sensitization to the psychotic effects of amphetamine in schizophrenic patients, and, administration of amphetamine also results in sensitization of HTR in rodents 98 These observatio ns argue in favor of HTR as a model to screen compounds for antipsychotic activity. Interestingly, M100907 a selective antagonist of 5 HT 2A receptors, despite being effective in HTR model demonstrate s only limited efficacy as an antipsychotic (Figure 1 6) An important limitation of the HTR is that it does not model the negative and cognitive deficits observed in schizophrenia. The HTR also does not model the hypothesized genetically linked forms of schizophrenia 99 100 At best, the DOI induced HTR possesses some validity as a model to screen for drugs that may modulate the positive symptoms of schizophrenia. However, this model does show models of schizophrenia. The HTR model in the studies of this thesis inv olves 2,5 dimethoxy 4 iodoamphe t am ine (DOI) as the hallucinogen to induce the HTR in mice (Figure 1 7) DOI binds with a high affinity at 5 HT 2A 5 HT 2B and 5 HT 2C receptors and acts as an agonist or partial agonist at these receptors 101 Herein, the HTR is applied as a model to screen for drugs capable of modulating the serotonergic neurotransmission dysfunction thought to be part of the pathophysiology of psychoses i n humans.

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36 As mentioned, dysfunction of glutaminergic neurotransmission is also thought to contribute to the pathophysiology of schizophrenia Psychotomimetic drugs like MK 801 and ketamine are non competitive antagonist at NMDA receptors, and, in fact, adm inistration of ketamine to schizophrenic patients results in a dose related increase in both positive and negative symptoms (Figure 1 7) 102 105 Hence, the psycholocomotor behavioral responses in rodents that result from acute administration of NMDA antagonists (such as MK 801) have been used to screen for drugs with antipsychotic activity. These behavioral responses to acute NMDA antagonist administration include: hyperlocomotion, disrupted sensorimotor gating, stereotypic behavior and cognitive deficits 106 108 Cognitive and sensorimotor gating deficits are some of the well characterized symptoms of schizophrenia. Several studies have dem onstrated the efficacy of clinically approved antipsychotics to attenuated acute MK 801 induced hyperlocomotor activity 109 112 The d isrupted glutamatergic transmission model appears to have more validity than the HTR as a behavioral model for schizophrenia For example, g enetically linked forms of schizophrenia are thought to involve altered glutamatergic neurotransmission 100 Also, l igands acting on NMDA receptors have been shown to be beneficial in treating negative symptoms of schizophrenia 113 Accordingly, in addition to the HTR model, the studies in this thesis utilized MK 801 induced hyperlocomotor activity as model to test the efficacy PAT analogs as potential antipsyc hotics. Dysfunction of dopaminergic neurotransmission (mainly hyper dopaminergic neurotransmission) has long been thought to contribute to the pathophysiology of psychoses in humans. Amphetamine causes release of dopamine from intra neuronal

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37 storage vesicl es and also competitively inhibits dopamine reuptake by dopamine transporter (Figure 1 7) 114 This results in increased brain levels o f dopamine, primarily at the synapses in nucleus accumbens which is the terminal input of the mesolimbic dopaminergic pathway 115 Amphetamine induced hyperlocomotor activity is hypothesize d to model positive symptoms of schizophrenia 116 M odulation of the hyperlocomotor activity in rodents induced by acute administration of amphetamine has long been used as a model to screen drugs for antipsychot ic activity, and, is used in this thesis, along with the serotonergic HTR model and the MK 801 glutamate antagonist model, discussed above 117 119 Thus, multiple models, representing the serotonergic, glutaminergic, and dopaminergic neurotransmission dysfunction thought to be involved in the pathophysiology and endophenotypes of psychoses are used in the stud ies of this thesis to screen PATs for antipsychotic activity. Central H ypothesis and G oals of this D issertation The central hypothesis tested here is that compounds with serotonin 5 HT 2C agonist activity together with 5 HT 2A/2B antagonist/inverse agonist activity translate to efficacy in rodent models of psychosis and may suitable for development as novel antipsychotic drugs. The goal s of the dissertation are to characterize the molecular determinants for binding and function of novel 4 phenyl N N dimethyl 1,2,3,4 tetrahydroanphthalene 2 amine ( p henyl a mino t etralin; PAT) derivatives synthesized in our laboratories at human serotonin 5 HT 2A 5 HT 2B and 5 HT 2C In silico ligand 5 HT 2 receptor docking studies by Dr. Tania Cordova Sintjago complement the experimental binding studies to help identify molecular determinants for PAT binding and provide inferences of functional interaction at 5 HT 2A and 5 HT 2C receptors. In vivo antip sychotic efficacy of PAT analogs i s evaluated using three different rodent models of psychosis,

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38 the serotonin 5 HT 2 agonist HTR model, the MK 801 glutamate antagonist hyperactivity model, and amphetamine induced hyperactivity model The goals are pursued according to the following 3 Specific Aims: AIM 1: Delineate Molecular Determinants for Binding of 4 phenyl N N dimethyl 1,2,3,4 tetrahydroanphthalene 2 amine (phenylaminotetralin, PAT) Derivatives at Human R ecombinant 5 HT 2A and 5 HT 2C GPCRs PATs possesse d a unique attribute of being 5 HT 2C specific agonists while at the same time function ing as antagonist s/inverse agonists at 5 HT 2A and 5 HT 2B receptor s It is well established that there is no therapeutic relevance for compounds that activate 5 HT 2A and/o r 5HT 2B receptors, as a ctivation of these receptors is associated with deleterious CNS or cardiovascular events. In order to move forward drug development targeting 5 HT 2 receptors by characterizing the molecular determinants for 5 HT 2 receptor PAT binding, this aim will determine affinity ( Ki ) of PAT analogs with substituents at ortho, meta or para position of the 4 phenyl moiety of PATs. Another group of analogs assessed include substituents at the 6,7 position of tetrahydronaphthale ne ring, with, and without, concomitant substitution at the meta position of the 4 phenyl moiety. In silico ligand 5 HT 2 receptor docking studies by Dr. Tania Cordova Sintjago are used to complement the experimental binding studies to help identify molecul ar determinants for PAT binding at 5 HT 2A and 5 HT 2C receptors. PATs with Ki < 500 nM across 5HT 2 receptors are considered for additional in vitro molecular studies of Aim 2 to characterize their 5HT 2 functional pharmacology. AIM 2: Characterize the 5 HT 2 F unction al Pharmacology of PATs Selected PATs from Aim 1 that meet 5HT 2 affinity criteria, are assessed for their functional potency regarding 5 HT 2 receptor mediated activation of phospholipase C (PLC) signaling at human recombinant 5HT 2A 5HT 2B and 5HT 2C expressed in clonal

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39 HEK cells. PATs that demonstrate agonism at 5 HT 2C receptors together with antagonism or inverse agonism at 5 HT 2A and 5 HT 2B receptors and have EC 50 /IC 50 potency values <500 nM are considered for translational studies of Aim 3 to a ssess their preclinical efficacy in psychosis. AIM 3: Translational Studies: A sses sment of PATs Efficacy in rodent models of psychosis The h ead twitch response (HTR) is a characteristic response elicited when the hallucinogenic 5 HT 2A agonist () 2 5 dim ethoxy 4 iodoamphetamine (DOI) is administered to mice. MK 801 is a non competitive antagonis t at NMDA receptor and induced hyperlo co motion when administered to mice. Amphetamine targets dopaminergic neurocircuitry and increases dopamine level in synapse r esulting in hyperlocomotion when administered to mice. These responses, HTR and hyperlocomotion can be attenuated by ligands acting on 5 HT 2 receptors and other compounds that are known to possess antipsychotic activity. Thus, these assays provide a simpl e yet powerful in vivo model to identify compounds with antipsychotic or 5 HT 2 receptors mediated psychotherapeutic activity. Selected PAT analogs identified in Aim 2 as 5HT2C agonists with 5HT2A/2B inverse agonist/antagonist activity are administered oral ly or by intraperitoneal injection to mice and assessed for efficacy in the three different psychosis models Results of these studies also provide preliminary information on PAT absorption, distribution, metabolism, elimination, and toxicology (ADMET) in vivo

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40 Figure 1 1. Generic 2 dimens ional structure of GPCR with amino acids numbered from amino terminal to carboxy terminal Figure 1 2 3 dimensional representation of GPCR A) T he extracellular loops are present at the top and the intracellular loops at the bottom of the cartoon B) 3 dimensional representation of GPCR from a top down view T he extracellular loops are seen on the top and the binding pocket formed by the transmembrane domain is seen as the space betw een the helices.

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41 Figure 1 3. Extended ternary complex model. R, R and R G: represent the inactive state, active state and active G protein coupled state of GPCR respectively. K a and K G are the equilibrium constants for binding of ligand A and G protein G to GPCR. and are the cooperativity constants that modulate the equilibrium constants. L represents the isomerization constant of GPCR. A B C Figure 1 4. Ligands activati n g serotonin 5 HT 2 receptors. A ) 5 Hydroxytryptamine or serotonin the endogenous ligand for serotonin receptors. B ) and C ) Fenfluramine and phenteramine ligands respectively, used therapeutically for weight loss. Figure 1 5 Structure of hallucinogens ( phenylethylamines and tryptamines) acting on 5 HT 2 receptors AR AR* R* R R*G AR*G K a K a K a L L K G K G

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42 Figure 1 6 Structure of M100,907. A selective antagonist of 5 HT 2A receptors. A B C Figure 1 7 Ligands used in in vivo models of psychosis. A) Structure of 2,5 dimethoxy 4 iodoamphetamine (DOI), used in head twitch response model. B) and C) Structure of MK 801 and amphetamine used in hyperactivity model of psychosis.

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43 Chapter 2 DELINEATE MOLECULAR DETERMINANTS FOR BINDING OF 4 PHENYL N N DIMETHYL 1,2,3,4 TETRAHYDROANPHTHALENE 2 AMINE (PHENYLAMINOTETRALIN; PAT) DERIVATIVES AT HUMAN R ECOMBINANT 5 HT 2A AND 5 HT 2C GPCRs Specifi c A im 1 The lead compound trans PAT possesses a high affinity for the serotonin 5 HT 2 family of receptors. Since 5 HT 2A and 5HT 2C receptors are therapeutically relevant in a number of physiological conditions, it is important to determine the molecular determinants that influence PAT analog s b inding at these receptors 120 Serotonin 5 HT 2A and 5 HT 2C receptors share a high de gree of sequence homol ogy and similarity in their effector systems 121 122 The structure activity relationship (SAR) of meta and para substituted PAT analogs assisted in delineating the optimal functional group that achieved the desired selecti vity and affinity (PAT meta and para substituents were synthesized by Dr Rajeev Sakhuja and Dr Myong Sang Kim) The nature of functional group attached to PAT analogs was determined primarily by the ir ease of synthesis and also based on the potential interactions of the substituent with the surrounding amino acids in the binding pocket. Substituents were added to the PAT analogs at the 6 and/or 7 position of the tetrahydronaphthalene ring (synthesized by Dr. Zhuming Sun) These 6 and/or 7 substituted PAT analogs were further modified with the addition of a substituent at the meta position of pendant phenyl ring In silico docking of these substitutions assisted in providing more infor mation about the binding pocket surrounding these substituents. Since these analogs perturb a different region of the binding pocket as compared to the pendant phenyl ring substituents, we investigated the role of these substituents in determining the affi nity and selectivity of these substituents for the 5 HT 2A or 5 HT 2C

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44 receptors. Evaluating the structure activity relationship helped us glean more information about these substituents and their interactions at the 5 HT 2A and 5 HT 2C receptors Methodology Clonal Cell Culture and T ransfection Human embryonic kidney 293 cells (HEK, ATCC CRL 1573) were maintained in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal bovine serum and 1% penicillin streptomycin. Cells were grown in a humidified incubator at 37 C with 5% carbon dioxide. The cDNAs encoding the human 5 HT 2A 5 HT 2B and 5 HT 2C INI (unedited wild type isoform) wild type receptors were obtained from UMR cDNA Resource Center (Rolla, MO). HEK 293 cells were grown to 90% confluency in DMEM (10 013 CV, Mediatech, Manassas, VA), supplemented with 5% dialyzed fetal bovine serum in 10 cm plates. Cells were washed then transfected with 24 HT 2 rece ptor subtype cDNA mixed with 40 Lipofectamine 2000 reagent in Opti MEM and placed in an incubator f or 24 to 48 hr. Memb ranes were then collected in 50mM Tris, 10 mM MgCl 2 6H 2 O, and 0.1mM EDTA (assay buffer) using previous methods 123 and stored at 80 Celsius until binding assays were performed. Radiorecept or Competition Binding A ssays Radioligand competiti ve displacement binding assays were perform ed in 96 well plates, using 3 5 methods used previously 123 Radioligands were included in assay mixtures at ~ K d concentration, i.e., 2.0 nM [ 3 H] ketanserin (5 HT 2A receptors), 1.95 nM [ 3 H] mesulergine (5 HT 2B receptors), or 1.4 nM [ 3 H] mesulergine (5 HT 2C receptors). Non specific binding was determined in the presence of 10 M mianserin for all 5 HT 2 receptors. Incubation

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45 of radioreceptor binding assay mixtures was for 1.0 h at 37C, with termination by rapid filtration through Whatman GF/B filters using a 96 well cell harvester (Tomtec, Hamden, CT) and subsequ ently washed five times with 50 mM Tris HCl at room temperature. Filters containing bound [ 3 H] radioligand were drie d, placed in vials containing 2 mL scintillation cocktail (ScintiVerse), allowed to eq uilibrate overnight, and then were counted for 3 H induced scintillation using a Beckman Coulter LS6500 counter. Each binding experiment had concentrations of ligands in triplicates, and each experiment was performed a minimum of three times. Data were anal yzed using nonlinear regression curve fitting algorithms in GraphPad Prism, 5.03 for Windows (San Diego, CA). Data points were limited to eight, thus Hill slopes were not calculated 124 ; data were Ki expressed as K i values by conversion of the IC50 data using the equation K i = IC 50 /1 + L / K D where L is the concentration of radioligand 125 Results and D iscussion for Competition Binding Assays Affinity of U nsubstituted PAT s at the 5 HT 2A R eceptor Each PAT analog synthesized in the lab has 4 diastereomers, two pairs of enantiomers for each cis and trans stereoisomer (Figure 2 1) Out of the 4 diastereomers the trans PAT enantiomers showed the best binding profile at the 5 HT 2A receptors 131 T rans PAT had a Ki value of 100 10 nM and (+) trans PAT showed a binding affinity of 470 46 nM (Table 2 2) T rans PAT showed a higher affinity for the 5 HT 2A receptor s as compared to the (+) tran s PAT enantiomer ( p =0.0098). Affinity of U nsubst ituted PAT s at the 5 HT 2C R eceptor Binding studies of the PAT stereoisomers followed a similar trend at 5 HT 2C receptors like 5 HT 2A receptors. The (+) trans PAT enantiomer had an affinity of 430

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46 86 nM and t he trans PAT analog binds at the 5 HT 2C receptors with a Ki of 30 3 nM (Table 2 2) trans PAT had a 1 3 fold higher affinity at 5 HT 2C receptor as compared to (+) trans PAT and this difference was statistically significant ( p =0.0021). 5 HT 2C receptor affinity of trans PAT analog was significantly higher than 5 HT 2A receptor affinity (100 nM ) ( p =0.0016). This difference in affinity indicates that trans PAT has stronger interactions or interacts with additional amino acids in 5 HT 2C receptor bin ding pocket. Affinity of 4 ( ortho S ubstituted ) PAT s at the 5 HT 2A R eceptor (+) and T rans o Br PAT enantiomers had an affinity of 760 73 nM and 1100 130nM respectively ( p >0.05). Both (+) and t rans PAT enantiomer s had a significantly higher affinity relative to (+) and trans o Br PAT enantiomer s ( p =0.009 and P<0.0001 ) respectively Introduction of sterically large halogen substituent at the ortho position could hinder the free rotation of the pendant phenyl rin g attached to sp 3 hybridized carbon of tetrahydronaphthalene ring Thereby inducing a conformation of t rans o Br PAT that resulted in decreased affinity of both (+) and t rans o Br PAT enantiomers at 5 HT 2A receptors. (+) and T rans o Cl PAT enantiomers had an affinity of 2800 110 nM and 2400 280 nM respectively ( p >0.05). The affinity of (+) and t rans o Cl PAT enantiomers was significantly lower than both (+) and trans PAT (P<0.0001) and (+) and t rans o Br PAT enantiomers ( p <0.005). These data indicate that substitutions at ortho position of pendant phenyl ring did not favor binding at 5 HT 2A receptors. Both ortho substituted analogs demonstrated a loss in stereoselectivity at 5 HT 2A receptors relative to unsubs tituted PAT enantiomers.

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47 Affinity of 4 ( ortho S ubstituted ) PAT s at the 5 HT 2 C R eceptor (+) and t rans o Br PAT enantiomers had an affinity of 78 16 nM and 70 8 nM respectively ( p >0.05). Both these enantiomers had 10 fold and 15 fold higher affinity at 5 HT 2C receptors relative to 5 HT 2A receptors ( p <0.0001). T rans PAT had a 2 fold higher affinity relative to t rans o Br PAT enantiomers at 5 HT 2C receptors ( p <0.0001). In contrast, (+) t rans o Br PAT enantiomer had a 5 fold higher affinity relative to (+) trans PAT enantiomer ( p =0.0095). These data indicate that the conformation induced by restricted rotation of the pendant phenyl ring favors selective binding at 5 HT 2C receptors without any st ereoselectivity. (+) and t rans o Cl PAT enantiomers had an affinity of 50 10 nM and 290 47nM respectively ( p = 0.009 ) These enantiomers demonstrated reversed stereoselectivity relative to unsubstituted PAT enantiomers There was no significant difference between the affinity of (+) t rans o Cl PAT and (+) t rans o Br PAT enantiomers at 5 HT 2C receptors. In contrast, the affinity of t rans o Cl PAT was significantly lower than t rans o Br PAT affinity at 5 HT 2C recept ors. 4 ( Meta substituted ) PAT analogs. Meta substituted PATs were synthesized with the intention of perturbing the chemical space surrounding the pendant phenyl ring and also explore the differences in binding pocket interactions between 5 HT 2A and 5 HT 2C receptors. In silico docking studies of trans PAT revealed that the pendant phenyl ring is surrounded by aromatic amino acids (W6.48 and F6.51) that assist in fun ctional group results in changes in affinity that need to be analyzed keeping in mind the electronic and the steric nature of the substituent. Different substituents like fluorine, chlorine, bromine, nitro and trifluoromethyl were added to the meta positio n (Figure 2

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48 2 ) and the resulting changes in the binding affinity were analyzed. The steric size of these substituents changed in the order: fluorine < chlorine < bromine = nitro < trifluoromethyl ( Courtesy Dr Cordova Sintjago Tania). Addition of differen t halogen atoms to the pendant phenyl ring also results in a change in the bond length between the aromatic carbon and the halogen substituent added. The bond length increases as the substituent changes from a fluorine to a chlorine to a bromine substituen t (Table 2 1). Fluorine had the highest electronegativity among the group hence a strong electron withdrawing inductive ( I) effect 126 The inductive effect of fluorine is greater than chlorine that in turn is greater than bromine. However halogens, when attached to an aromatic system (in this case, the C(4) phenyl moiety of PATs) are unique in their propensity to engage in resonance with the aromatic system by donating a lone pair of electrons (+R). If the halogen is attached at the ortho or para position of the C(4) phenyl moiety, the result is an increase in electron density at these positions. Hence both inductive ( I) and resonance (+R) effects play a role in determining the interactions of the C(4) phenyl moiety of PATs with the nearby amino acids of the binding pocket of 5 HT 2 receptor 127 Affinity of 4 ( m eta S ubstituted ) PAT s at the 5 HT 2A R eceptor (+) and Trans m F PAT were synthesized to examine the effects caused by changing a hydrogen atom to a fluorine at the meta position on pendant phenyl ring The Ki value of trans m F PAT at 5 HT 2A was 110 10 nM and the affinity of the (+) trans m F PAT was 320 26 nM (Table 2 2) T rans m F PAT had a higher affinity than (+) trans m F PAT ( p =0.0357). T rans m F PAT affinity was not significantly different from trans PAT (100 nM ) affinity at the 5 HT 2A receptor.

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49 benzene ring because of the high electronegativity of fluorine 128 Since the pendant phenyl ring of trans PAT binds in a region where there is a high density of aromatic amino acids this could lead to with surrounding aromatic amino acids. Sterically, fluorine substituent is generally considered a bioisoster of hydrogen or methyl group 129 However, t rans m F PAT has the same affinity as trans PAT indicating that there was no significant effect of introduction of fluorine at the meta position. (+) and T rans m Cl PAT enantiomers were synthesized with an intent to gain further insight into the milieu of the pendant phenyl ring in the binding pock et. Trans m Cl PAT enantiomers followed a similar trend in stereoselectivity that is, the trans m Cl PAT ha d a higher affinity than (+) trans m Cl PAT (P=0.0294). The Ki value of trans m Cl PAT was 40 5 nM and the affinity of (+) trans m Cl PAT was 130 8.7 nM (Table 2 2). T he affinities of trans m F PAT (110 nM ) and trans m Cl PAT were found to be significantly different ( p =0.0223). Addition of a chlorine atom resulted in significant changes in the electronics and sterics of the pendant phenyl ring. Chlorine as compared to fluorine has a larger size and a smaller electronegativity 130 Hence both I inductive effect and +R resonance effect are seen to a lower extent with chlorine than fluorine 127 The decrease in the resonance is because of the disproportionate size of the p orbitals of chlorine c ompared to carbon. This leads to a decreased propensity of the meta chloro PAT analog to form resonance hybrid structures. Introduction of a chlorine at the meta position also resulted in a significant higher ( p =0.0002) affinity of trans m Cl PAT (40 n M ) relative to affinity of trans PAT (100 nM ) at the 5 HT 2A receptor s (Figure 2 3 )

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50 The series was further expanded with the synthesis of (+) and trans m Br PAT. The stereoselectivity trend was again confirmed as (+) trans m Br PAT had a lowe r affinity of 260 22 nM relative to trans m Br PAT enantiomer, Ki of 20 3 nM (Table 2 2) This higher affinity of trans m Br PAT enanti omer was slightly significant ( p =0.0459) when compared to trans m Cl PAT (40 nM ) enantiomer However, whe n compared to trans m F PAT (110 nM ) the affinity of trans m Br PAT was significantly higher ( p =0.0229). The bromine substituent has the largest size and the lowest electronegativity compared to the fluorine and chlorine atoms 130 Comparatively, then, displacement of C(4) least with the bromine substituent because the probability of resonance effect is low due orbital. The ( trans m Br PAT enantiomer has the highest affinity among the meta substituted PAT analogs at 5 HT 2A receptors. Hence, the characteristics of a substituent at the meta position that favored high affinity binding at 5 HT 2A receptors are : large steric effe ct and a low electronegativity. The order of affinity of the levorotatory enantiomers is as follows: trans m Br PAT > trans m Cl PAT > trans m F PAT = trans PAT (Table 2 2) Since more bulky atoms at meta position increased the affinity at 5 HT 2A receptors, more analogs with large sterics were synthesized in this series: (+) and trans m NO 2 PAT and (+) and trans m CF 3 PAT. These derivatives differed from previous meta halogenated derivatives in being predominantly electron withdrawin g and deactivating with respect to the phenyl ring. Trifluoromethyl substituent decreases the electron density in the phenyl ring by inductive effect ( I) and nitro substituent in addition

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51 to having inductive effect ( I) also possessed resonance effect ( R ) 131 Hence the nitro group was a more potent ring deactivator than the trifluoromethyl substituent. The sterics of the trifluoromethyl substituent were slightly larger than t he nitro group by about 20 3 (Data courtesy Dr. Tania Cordova Sintjago). The (+) trans m NO 2 PAT enantiomer had an affinity of 500 20 nM and the trans m Nitro PAT had an affinity of 74 18 nM ( p =0.0001) (Table 2 2) The affinity of both (+) and trans m NO 2 PAT was not significantly different from the affinity of (+) trans PAT (470 nM ) and trans PAT (100 nM ) enantiomers respectively Comparing the affinities of other meta substituted PAT derivatives showed that: trans m Br PAT (20 nM ) enantiomer had significantly higher affinity than trans m NO 2 PAT enantiomer ( p =0.0150). T he affinity of trans m F PAT (110 nM ) and trans m Cl PAT (40 nM ) enantiomers were not significantly different relative to trans m NO 2 PAT enantiomer affinity at 5 HT 2A receptors ( p >0.05) The (+) tran s m CF 3 PAT enantiomer had an affinity of 1300 67 nM and the trans m CF 3 PAT enantiomer had an affinity of 80 10 nM ( p =0.0272) (Table 2 2) The affinity of trans m CF 3 PAT was not significantly different from trans PAT, trans m F PAT and trans m NO 2 PAT affinity. However, the affinity was significantly lower when compared to trans m Cl PAT (P=0.004) and trans m Br PAT ( p =0.0023) affinity at 5 HT 2A receptors Meta trifluoromethyl substituent possessed the largest ster ics of all the meta substituted PAT derivatives 126 132 The literature values of electronegativity of this substituent varied diversely with some pape r reporting the electronegativity to be between fluorine and chlorine and other papers reporting electronegativity similar to

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52 bromine 133 134 Trifluoromet hyl substituent is unique; unlike other substituents at meta position it possesses no resonance effect. Hence, it is only capable of reducing the electron density of the phenyl ring. The newer meta trifluoromethyl and nitro PAT analogs possess larger ste rics than bromine but resulted in lower affinity at 5 HT 2A receptors This drop in affinity when the substituent changes from bromine to nitro or trifluoromethyl substituents could be because the sterics of these newer substituents are too large to fit in to the binding pocket (Figure 2 3 ) The affinity of meta substituted derivatives increased steadily as the size of the substituents increases until nitro and the trifluoromethyl substituent were added. Summary of 4 ( meta substituted ) PAT analogs at 5 HT 2A receptor Fluorine, chlorine and bromine possessed I inductive effect and +R resonance effect. Nitro substituent possessed I inductive and R resonance effect and trifluoromethyl substituent possessed I inductive effect only. The order of affinity of t he levorotatory meta substituted PAT analogs is as follows: trans m Br PAT > trans m Cl PAT = trans m NO 2 PAT = trans m CF 3 PAT = trans m F PAT = trans PAT (Figure 2 3 ) Affinity of 4 ( m eta S ubstituted ) PAT s at the 5 HT 2C R eceptor (+) Trans m F PAT binds to 5 HT 2C receptors with an affinity of 200 30 nM and trans m F PAT has a Ki value of 43 14 nM ( p =0.0076) (Table 2 2) The introduction of a fluorine group did not significantly change the affinity of either enantiomer relat ive to the corresponding trans PAT enantiomers at 5 HT 2C receptors. Comparing trans m F PAT affinity at 5 HT 2A with its affinity at 5 HT 2C receptors revealed no significant difference. Introduction of a fluorine group had no affect o n its selectivity b etween the two receptors.

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53 (+) Trans m Cl PAT binds to the 5 HT 2C receptors with an affinity of 170 43 nM and trans m Cl PAT has a Ki value 8 3 nM ( p =0.0098) (Table 2 2) Introduction of chlorine at the meta position increases the affinity of the trans enantiomer 3 fold relative to trans PAT affinity ( p = 0.0262 ) Comparing the affinities of meta fluorine (43 nM ) and chlorine substituted PATs at 5 HT 2C receptors showed that trans m Cl PAT has a significantly ( p =0.0139) higher affinity. Th ere was no significant difference in the affinity of (+) trans PAT (430 nM ) (+) trans m F PAT (200 nM ) and (+) trans m Cl PAT (170 nM ) A ffinity of (+) t rans m Cl PAT did not significantly differ between the 5 HT 2A and 5 HT 2C receptor s On the other hand the affinity of trans m Cl PAT was significantly higher at 5 HT 2C receptors relative to its affinity at 5 HT 2A receptors ( p =0.0350). Enantiomers of trans m Br PAT were tested for their binding at 5 HT 2C receptors and their affinities compared to other meta substituted analogs. (+) Trans m Br PAT had an affinity of 200 30 nM and the trans m Br PAT enantiomer had a Ki of 4 1 nM ( p <0.0001) (Table 2 2) Comparing trans m Br PAT affinity with trans PAT (30 nM ) affinity showed that trans m Br PAT bound with a significantly higher affinity at 5 HT 2C receptors ( p =0.0229). The affinity of the (+) trans m Br PAT enantiomer was not significantly different from (+) trans PAT (430 nM ) The affinities of trans m F PAT (43 nM ) and trans m B r PAT were found to be significantly different ( p = 0.0425 ) Comparing the affinities of trans m Cl PAT (8 nM ) and trans m Br PAT revealed that their affinities were not significantly different. This indicates that both chlorine and bromine substitu ents formed stronger interactions with the amino acids in the binding pocket as compared to fluorine. The trans m Br PAT analog had 6 fold

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54 higher affinity at 5 HT 2C receptors than 5 HT 2A receptors ( p =0.0218) similar to trend observed with trans m Cl PAT enantiomer The (+) trans m NO 2 PAT and trans m NO 2 PAT enantiomers had an affinity of 120 13 nM and 10 1 nM respectively ( p =0.0002) (Table 2 2) Comparing these enantiomers with the parent (+) trans PAT (430 nM ) and trans PAT (30 nM ) s howed that there was a significant difference between trans PAT and trans m NO 2 PAT affinities at 5 HT 2C receptor ( p =0.0159). T he affinity of (+) trans m NO 2 PAT was also higher compared to (+) trans PAT enantiomer ( p =0.0291). The affinity of trans m Br PAT (4 nM ) was significantly higher than trans m NO 2 PAT ( p =0.0002). Affinities of other meta substituted PAT analogs (fluorine (43 nM ) and chlorine (8 nM ) ) were not significantly different at the 5 HT 2C receptors. The affinity of trans m NO 2 PAT was significantly higher at 5 HT 2C receptors compared to 5 HT 2A receptors ( p =0.0004). The trans m NO 2 PAT enantiomer had a 4 fold higher selectivity for 5 HT 2C receptors as compared to 5 HT 2A receptors. The next pair of enantiomers assessed at 5 HT 2C receptors had a trifluoromethyl substituent at the meta position. (+) Trans m CF 3 PAT had an affinity of 230 20 nM and trans m CF 3 PAT had a Ki value of 10 2 nM at 5 HT 2C receptors ( p <0.0001) (Table 2 2) The affinities of both the enantiomers were not significantly different from the corresponding parent unsubstituted PAT analogs. Comparing the affinity with other meta substituted analogs showed that there was no significant difference between trans m CF 3 PAT and any of the other meta substituted analogs. The selectivity for 5 HT 2C receptors versus 5 HT 2A receptors was the highest for this enantiomer. This

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55 enantiomer is about 9 fold more selective for 5 HT 2C receptors as compared to 5 HT 2A receptors ( p =0.0055). Summary of 4 ( meta substituted ) PAT analogs at 5 HT 2C receptor Trans m F PAT did not show selectivity between 5 HT 2A and 5 HT 2C receptors. However as the sterics of the substituent increased, the selectivity for 5 HT 2C receptors increased. Trans m Cl PAT had 7 fold higher affinity at the 5 HT 2C receptors. When the size of substituent further increased in trans m Br PAT this difference was 6 fold. Further increase in sterics with trans m NO 2 PAT revealed a 4 fold increase in affin ity for 5 HT 2C receptors. The largest substituent trifluoromethyl at the meta position showed the highest selectivity for the 5 HT 2C receptor, trans m CF 3 PAT has 9 fold higher selectivity for the 5 HT 2C receptors (Table 2 2) This indicates that 5 HT 2 C receptor is better able to accommodate the sterics of the larger substituents as compared to the 5 HT 2A receptor leading to higher affinities of these enantiomers at 5 HT 2C receptor. Similar to the trend observed at 5 HT 2A receptors, the affinities of me ta substituted PAT at 5 HT 2C receptors increases with the size of the substituents until the sterics size of nitro and trifluoromethyl substituents. However, the drop in affinities is steeper at 5HT 2A receptors as compared to 5HT 2 C receptors. A ffinity of 4 ( para S ubstituted ) PAT s at the 5 HT 2A R eceptor (+) Trans p F PAT had an affinity of 71 12 nM at 5 HT 2A receptors and trans p F PAT bound to 5 HT 2A with a Ki of 140 24 nM ( p = 0.0345 ) (Table 2 3) Comparing the affinity of the para fluorine enantiomers with the parent unsubstituted PAT shows that (+) trans p F PAT had 6 fold higher affinity than (+) trans PAT (470 nM ) enantiomer ( p =0.0015). In contrast, addition of fluorine at para position did not change the affinity of trans p F PAT rel ative to trans PAT (100 nM ) I ntroduction

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56 of a fluorine group at the para position enhanced the affinity of (+) trans p F PAT enantiomer, resulting in a reversal of stereoselectivity. This pronounced increase in the affinity (+) trans p F PAT enantiome r probably resulted from the formation of a favorable interaction (hydrogen bonding) with the surrounding amino acids in the binding pocket. The next p air of enantiomers synthesized were (+) and trans p Cl PAT (Figure 2 4 ) Changing the halogen substi tuent at the para position from fluorine to chlorine resulted in a more pronounced reversal of stereoselectivity. The affinity of (+) trans p Cl PAT was 50 8 nM and the other enantiomer trans p Cl PAT binds with a Ki of 250 37 nM (Table 2 3) Similar to para fluoro PAT analog s introduction of the chlorine group at the para position resulted in reversed stereoselectivity The affinity of the (+) trans p Cl PAT enantiomer increased 9 fold as compared to (+) trans PAT (470 nM ) ( p =0.0001) but th e affinity of trans p Cl PAT enant iomer decreased 2 fold as compared to trans PAT (100 nM ) ( p =0.0280). This suggests that the (+) trans p Cl PAT is able to better fit into the binding pocket than the trans p Cl PAT. (+) Trans p F PAT (71 nM ) had a 2 fold higher affinity than trans p F PAT (140 nM ) in contrast (+) trans p Cl PAT shows a 5 fold higher affinity than trans p Cl PAT. Comparing the affinity of (+) trans p F PAT and (+) trans p Cl PAT revealed that these values were not stat istically significant. The reversal in stereoselectivity was further confirmed by the synthesis of (+) and trans p Br PAT. Introduction o f a bulkier bromine atom retained the reversal of stereoselectivity observed in previous analogs. (+) Trans p Br PA T binds with a Ki of 60 10 nM at 5 HT 2A receptors and trans p Br PAT has an affinity of 220 29 nM ( p =0.0025) (Table 2 3). There wa s no significant difference between the affinity of (+)

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57 trans p Br PAT when compared to (+) trans p Cl PAT (50 nM ) and (+) trans p F PAT (71 nM ) However, comparing the affinities of (+) trans p Br PAT and (+) trans PAT revealed that there was a 8 fold increased affinity for (+) trans p Br PAT ( p =0.0003). This provides further evidence to the hypothesis that (+) trans pa ra substituted PAT enantiomers ha d higher affinity than parent PAT because they form ed favorable interactions with amino acids in the binding pocket. The next pair of compounds synthesized in this series showed the most pronounced difference in the affinit y between (+) and trans enantiomer s relative to any other para substituted PAT analogs. The affinity of (+) trans p CF 3 PAT at 5 HT 2A receptors was 210 16 nM and the affinity of trans p CF 3 PAT was 2000 200 nM ( p =0.001) (Table 2 3) Trans p CF 3 PAT has 20 fold lower affinity as compared to trans PAT (100 nM ) ( p =0.0008) and (+) trans p CF 3 PAT has 2 fold higher affinity as compared to (+) trans PAT ( p =0.0258). This loss in affinity could be because the sterics of the trifluoromethyl subst ituent result in a clash with amino acids in the binding pocket. This is reflected when the statistical analysis was performed to compare the trifluoromethyl subst ituent affinity with other para halogenated substituents. (+) Trans p CF 3 PAT substituent had significantly lower affinity compared to (+) trans p F PAT (71 nM ) ( p =0.0009), (+) trans p Cl PAT (50 nM ) ( p =0.0003) and (+) trans p Br PAT (60 nM ) ( p =0.0002). Summary of 4 ( para substituted ) PATs at the 5 HT 2A receptor Para substituted PAT analogs were unique as they demonstrated reversed stereoselectivity of PAT enantiomers (Table 2 3). This reversal in stereoselectivity became more pronounced as the size of the substituents increased. The mean affinities of para

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58 substitute d enantiomers followed a trend where: (+) trans p Cl PAT > (+) trans p Br PAT > (+) trans p F PAT > (+) trans p CF3 PAT (Table 2 3), this trend was not stati stically significant (Figure 2 5 ). The absolute conformation of (2 S 4 R ) trans 4 (4 chloro phen yl) N N dimethyl 2 aminotetralin or trans p Cl PAT was confirmed (by Dr Khalil A. Abboud) using X ray crystallography (Figure 2 6 ). Affinity of racemate () trans p NO2 PAT was also determined ( Ki = 8100 180 nM ) This affinity of racemate further confirms the influence of substituent sterics at the para position. Affinity of 4 ( para S ubstituted ) PAT s at the 5 HT 2C R eceptor All the para substituted PAT analogs synthesized were also tested at 5 HT 2C receptor s to check whether they show any selectivity between 5 HT 2C and 5 HT 2A receptors. These results provided more information about the binding pocket milieu differences between the 5 HT 2A and 5 HT 2C receptors. (+) and Trans p F PAT enantiomers were analyze d for their affinity at 5 HT 2C receptors. The affinity of these enantiomers revealed an interesting facet; they showed a loss of stereoselectivity instead of a reversal (Table 2 3) The affinity of the (+) trans p F PAT at 5 HT 2C receptors was 75 12 nM and the Ki of trans p F PAT was 68 11 nM ( p =0.5665). Comparing these Ki with the corresponding 5 HT 2A affinities showed that there was no statistical difference in the affinities of (+) trans p F PAT. However trans p F PAT had a significantly h igher affinity at 5 HT 2 C receptors compared to 5 HT 2A receptors ( p =0.0339) This indicated that trans p F PAT binds differently with the two receptors and it was able to make stronger interactions in the binding pocket of 5 HT 2C receptors. Comparing th ese affinities with the parent PAT enantiomers showed that trans p F PAT affinity was not significantly different from trans PAT (30 nM ).

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59 In contrast, (+) trans p F PAT enantiomer had a significantly higher affinity compared to (+) trans PAT (430 nM ) ( p =0.0045). The next pair of enantiomers tested at 5 HT 2C receptors were (+) and trans p Cl PAT. (+) Trans p Cl PAT had a Ki of 55 17 nM and trans p Cl PAT had an affinity of 120 9.0 nM (Table 2 3) This difference in affinity was st atisti cally significant with a p value of 0.0035. Substituting a fluorine group with a chlorine group at the para position did not result in statistically significant ch ange in the affinity of the (+) and trans p Cl PAT relative to (+) and trans p F PAT respectively Comparing the affinities of (+) and trans p Cl PAT at 5 HT 2C receptor with their affinities at 5 HT 2A receptor s howed no significant difference for (+ ) trans p Cl PAT and significantly higher affinity for trans p Cl PAT at 5 HT 2C rec eptors ( p =0.0441) Thi s indicated that trans p Cl PAT had 2 fold selectively for 5 HT 2C receptors over 5 HT 2A receptors. Introduction of a chlorine group at the para position produced a pronounced reversal in stereoselectivity of (+) and trans p Cl PAT relative to (+) trans PAT enantiomer (430 nM ) ( p =0.0011) and trans PAT (30 nM ) enantiomer ( p =0.0088) respectively The next pair of enantiomers analyzed at 5 HT 2C receptors were (+) and trans p Br PAT. (+) Trans p Br PAT bound to 5 HT 2C receptors with a Ki of 7 0 8 nM and the other enantiomer trans p Br PAT had an affinity of 100 3 nM ( p =0.0214). Comparing the affinities of para bromo enantiomers with other para substituted enantiomers revealed no significant difference in the ir b inding affinities at 5 HT 2C receptor s. In contrast to this, (+) trans p Br PAT has significantly higher affinity compared to (+) trans PAT ( p =0.0038) and trans p Br PAT has significantly lower affinity as compared to trans PAT ( p =0.0001). C omparing the affinities of trans

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60 p Br PAT between 5 HT 2A and 5 HT 2C receptor s revealed that its affinity is significantly higher at 5 HT 2C receptors ( p = 0.0217 ) The other enantiomer (+) trans p Br PAT did not show any difference in affinities between 5 HT 2A and 5 HT 2C receptors. This 2 fold higher affinity trans p Br PAT at 5 HT 2C receptor revealed that 5 HT 2C receptor is better at accommodating the large sterics of bromine substituent The last pair of enantiomers to be tested in this series were (+) and trans p CF 3 PAT. Similar to their affinities at 5 HT 2A receptor s, these enantiomers showed the lowest affinity for para substituted analogs at 5 HT 2C receptors (+) T rans p CF 3 PAT b ound to 5 HT 2C receptors with a Ki of 220 27 nM and the trans p CF 3 PAT enantiomers had an affinity of 520 51 nM ( p =0.0009) (Table 2 3) This difference in affinity between enantiomers was only 2 fold at 5 HT 2C receptors as compared to 8 fold difference in affinity between the se enantiomers at 5 HT 2A receptors (Table 2 3) Comparing the ir affinities at 5 HT 2A versus 5 HT 2C receptors revealed that the trans p CF 3 PAT bound better at 5 HT 2C receptor s as compared to 5 HT 2A receptor s ( p =0.0005 ). In contrast, the other enantiomer (+) trans p CF 3 PAT sh owed no significant difference between its affinities at 5 HT 2A and 5 HT 2C receptors. This lower affinity of the trans p CF 3 PAT at 5 HT 2A receptor s again indicated that 5 HT 2C receptor s were better able to accommodate the size and charge on the large trifluoromethyl substituent as compared to 5 HT 2A receptor s T rans p CF 3 PAT enantiomer showed 4 fold selectivity for the 5 HT 2C receptor s as compared to the 5 HT 2A receptor s, but this select ivity wa s accompanied by a loss of affinit y Comparing these affinities with parent unsubstituted PAT showed that (+) trans p CF 3 PAT did no t

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61 bind significantly different from (+) trans PAT (430 nM ) T rans p CF 3 PAT in contrast, had a significantly lo wer affinity relative to trans PAT ( p <0.0001). Summary of 4 ( para substituted ) PATs at 5 HT 2C receptor (+) and T rans p F PAT showed a loss of stereoselectivity at the 5 HT 2C receptor s However, both (+) and t rans p Cl PAT and (+) and trans p CF 3 PAT showed a 2 fold reversal in stereoselectivity of corresponding enantiomers at 5 HT 2C receptor s T rans p Br PAT showed a 2 fold selectivity for binding at 5 HT 2C receptor s over 5 HT 2A receptor s (Table 2 3 ). This selectivity was further increa sed when the trifluoromethyl group was introduced at para position. T rans p CF 3 PAT showed a 4 fold selectivity for 5 HT 2C receptor s over 5 HT 2A receptor s These data indicate that 5 HT 2C receptor s were better able to accommodate the larger sterics of the substituents as compared to 5 HT 2A receptor s (Figure 2 5 ). Affinity of racemate () trans p NO 2 PAT was also determined as was found to be 1 000 10 0 nM significantly lower than its affinity at 5 HT 2C receptors A ffinity of 4 ( meta Substituted Phenyl ) and /or (6, 7 substituted Tetrahydronaphthalene) PAT s at the 5 HT 2A R eceptor (+) and T rans 6 OMe m Cl PAT enantiomers were synthesized to evaluate the effects of the methoxy substituent on binding profile at 5 HT 2A receptor s These affinities wer e compared to (+) and trans m Cl PAT as the ir structures differed only at 6 methoxy position The (+) trans 6 OMe m Cl PAT had a Ki of 350 16 nM and the trans 6 OMe m Cl PAT bound with an affinity of 35 3 nM ( p =0.0015) (Table 2 4) These compou nds followed a similar trend in stereoselectivity as the parent PAT and meta substituted analogs Comparing these affinities with meta chlorine substituted PATs revealed that trans m Cl PAT (40nM) and trans 6 OMe m Cl PAT had no significant differe nce in their affinities B ut (+) trans 6 OMe m Cl PAT enantiomer had a

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62 significantly lower affinity relative to (+) trans m Cl PAT (130 nM ) ( p =0.0375). This 2 fold reduction in affinity could be because of a steric hindrance caused by the i ntroduction of a methoxy group. The next pair of enantiomers synthesized in this series had a methoxy substitution at the 6 position and a chlorine substituent at the 7 position of tetrahydronaphthalene ring. Both the cis and the trans stereoisom ers of this substituent were separated using a chiral HPLC. The affinities of (+) and trans 6 OMe 7 Cl PAT were found to be 240 23 nM and 160 24 nM respectively. T he affinities of the other pair of enantiomers (+) and cis 6 OMe 7 Cl PAT were fou nd to be 2300 130 an d 320 4 2 n M ( p =0.0065) respectively (Table 2 4). T he cis enanti omers followed the trend of levorotatory enantiomer having a higher affinity than the dextrorotato ry enantiomer The (+) cis 6 OMe 7 Cl PAT enantiomer had a 10 fold lower affinity relative to (+) trans 6 OMe 7 Cl PAT ( p =0.0141). T rans 6 OMe m Cl PAT had 4 fold higher affinity relative to trans 6 OMe 7 Cl PAT (160 nM ) enantiomer at 5 HT 2A receptors ( p <0.0001). This loss in affinity could be because of either introduction of chloro su bstituent at 7 position or removal of the chlorine atom from the meta position. The next set of compounds synthesized in this series had substituents at 6, 7 position of tetrahydronaphthalene ring and halogen at meta position of pendant phenyl ring. A ll th e four stereoisomers of 6 OMe 7 Cl m Cl PAT analog were synthesized and isolated. (+) and trans 6 OMe 7 Cl m Cl PAT bound with an affinity of 95 18 nM and 50 20 nM respectively ( p >0.05). (+) and cis 6 OMe 7 Cl m Cl PAT bound with a Ki of 230 64 nM and 3500 390 nM ( p =0.0414) respectively (Table 2 4) It is

PAGE 63

63 interesting to note that there was a loss of stereoselectivity for the trans enantiomers and that there was a reversal of stereoselectivity for the cis enantiomers. (+) Trans 6 OM e 7 Cl m Cl PAT had a 3 fold higher affinity relative to (+) trans 6 OMe 7 Cl PAT ( p =0.0002). The other enantiomer trans 6 OMe 7 Cl m Cl PAT also had a 3 fold higher affinity compared to trans 6 OMe 7 Cl PAT (160 nM ) ( p =0.0002). The affinity of trans 6 OMe 7 Cl m Cl PAT enantiomer was not significantly different from affinity of trans m Cl PAT (40 nM ) ( p >0.05) The addition of 6 methoxy and 7 chloro substituents on the naphthalene ring resulted in a significant increase in affinity of (+) trans 6 OMe 7 Cl m Cl PAT as compared to (+) trans m Cl PAT (130 nM ) ( p =0.0091). This higher affinity of (+) trans 6 OMe 7 Cl m Cl PAT indicates that this enantiomer could bind at a different region in the binding pocket compared to other tetrahydronaphth alene ring substituted analogs. The next pair of enantiomers synthesized in this series were (+) and trans 6 OMe 7 Cl m Br PAT. These enantiomers bound to 5 HT 2A receptors with affinities similar to trans 6 OMe 7 Cl m Cl PAT enantiomers. The affinity of the (+) trans 6 OMe 7 Cl m Br PAT was found to be 79 14 nM. The other enantiomer trans 6 OMe 7 Cl m Br PAT bound with a Ki of 61 15 nM ( p >0.05). However, the affinities of (+) trans m Br PAT was significantly lower compared to (+) trans 6 OMe 7 Cl m Br PAT ( p =0.0043), indicating again that the presence of the 6 methoxy and 7 chloro groups in the tetrahydronaphthal ene ring significantly increased the affinity of only the (+) trans enantiomers. Another important point to note is that both tra ns m Cl PAT (40 nM ) and trans m Br PAT (20 nM ) enantiomers, without the 6,7 substitutions were capable of binding with a similar affinity as trans 6 OMe 7 Cl m Cl PAT (50 nM ) and

PAGE 64

64 trans 6 OMe 7 Cl m Br PAT (61 nM ) respectively Therefore trans met a halogen PAT moiety seems to be the core pharmacophore and the other substituents on the tetrahydronaphthalene ring did not significantly influence the binding of the dextrorotatory enantiomers in the binding pocket. Summary of affinity of tetrah ydronaphthalene and/or pendant phenyl ring substituted PAT analogs at 5 HT 2A receptors Comparing the affinities of analogs that have substitutions on both phenyl and tetrahydronaphthalene ring systems with meta substituted analogs revealed that both these analogs had similar affinities at 5 HT 2A receptors. The tetrahydronaphthalene substituents with halogen at meta position of phenyl ring showed a significant increase in affinity for only the (2 R ,4 S ) (+) trans enantiomers The (2 S ,4 R trans enantiomers that have both tetrahydronaphthalene and pendant phenyl ring substituted showed no significant difference in affinity when compared to trans meta halogenated analogs. This indicates that the core pharmacophore required for the activity of the trans enantiomer was meta substitution of trans PAT enantiomer with halogen group. This series of compounds are difficult to synthesize and separate, hence these compounds were not selected as candidates for in vivo screening in mice. A ffinity of 4 ( meta Substituted Phenyl) and /or (6, 7 substituted Tetrahydronaphthalene) PATs at the 5 HT 2C R eceptor The same series of tetrahydronaphthalene and/or pendant phenyl ring substituted compounds were again tested for their affinity at 5 HT 2C receptor s (Figure 2 7 ) (+) Trans 6 OMe m Cl PAT had an affinity of 280 3 0 nM and trans 6 OMe m Cl PAT had an affinity of 17 1 nM ( p =0.0155) (Table 2 4) (Data for trans 6 OMe m Cl PAT courtesy Dr Clinton Canal). The affinity of (+) trans 6 OMe m Cl PAT at 5

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65 HT 2C receptors was not significant ly different from their affinity at 5 HT 2A receptors. In contrast, trans 6 OMe m Cl PAT affinity was higher at 5 HT 2C receptor s compared to their affinity at 5 HT 2A receptor s ( p =0.0417). Comparing trans 6 OMe m Cl PAT and trans m Cl PAT revealed that trans m Cl P AT (8 nM ) had a 2 fold higher affinity ( p =0.0177) The next series of analog s that were assayed at the 5 HT 2C receptor s are (+) and trans 6 OMe 7 Cl PAT and (+) and cis 6 OMe 7 Cl PAT. (+) and T rans 6 OMe 7 Cl PAT en antiomers had an affinity of 37 5.1 nM and 24 2.5 nM respectively. T he other set of enantiomers (+) and cis 6 OMe 7 Cl PAT, had an affinity of 1600 6 4 n M and 96 18 nM respectively ( p <0.0001) (Table 2 4) Comparing th e affinities of (+) cis 6 OMe 7 Cl PAT at 5 HT 2A and 5 HT 2C receptor s revealed no significant dif ference The other enantiomer cis 6 OMe 7 Cl PAT however had higher affinity at 5 HT 2C receptor s compared to 5 HT 2A receptor s ( p =0.0194). Comparing the affinities of (+) trans 6 OMe 7 Cl PAT at 5 HT 2A receptor s with 5 HT 2C receptor s revealed that it binds with a significantly higher affinity at 5 HT 2C receptor s ( p =0.0003). The other enantiomer trans 6 OMe 7 Cl PAT also binds with a significantly highe r affinity at 5 HT 2C receptor s compared to 5 HT 2A receptor s ( p <0.0001). Comparing the affinities of trans 6 OMe m Cl PAT with trans 6 OMe 7 Cl PAT revealed no significant difference The next analog to be assayed at the 5 HT 2C receptor s was 6 OMe 7 Cl m Cl PAT. All the four stereoisomers of this analog were also isolated and tested at this receptor. (+) and trans 6 OMe 7 Cl m Cl PAT enantiomers had an affinity of 7 3 nM and 9 2 nM respectively. These values indicate a loss of stereose lectivity at 5

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66 HT 2C receptor s similar to 5 HT 2A receptor s. The affinity of (+) trans 6 OMe 7 Cl m Cl PAT was significantly higher at the 5 HT 2C receptor s compared to 5 HT 2A receptor s ( p =0.0002). Trans 6 OMe 7 Cl m Cl PAT had a 5 fold higher affinity at 5 HT 2C receptor s compared to 5 HT 2A receptor s (not significantly different) The cis enantiomers of these compounds follow ed the same trend at both 5 HT 2A and 5 HT 2C receptor s, that is, a reversal in their stereoselectivity. Comparing the affinities of both (+) and cis 6 OMe 7 Cl m Cl PAT at 5 HT 2A versus 5 HT 2C receptor s rev ealed no significant difference There was no difference in the affinity of trans 6 OMe 7 Cl PAT enantiomer when compared to trans 6 OMe 7 Cl m Cl PAT. Addition of chlori ne substituent at the meta position of pendant phenyl ring did not significantly change the affinity of (2 S ,4 R ) trans enantiomer. The last pair of enantiomers that were tested at 5 HT 2C receptors were (+) and trans 6 OMe 7 Cl m Br PAT. (+) T rans 6 OMe 7 Cl m Br PAT had an affinity of 350 130 nM and t rans 6 OMe 7 Cl m Br PAT had a Ki of 130 43 nM at 5 HT 2C receptors (Data courtesy Dr. Clinton Canal) (Table 2 4) These affinities were not significantly different from each other. These enantiomers demonstrated a loss of stereoselectivity at both 5 HT 2A and 5 HT 2C receptors. Summary of affinity of tetrahydronaphthalene ring and/or pendant phenyl ring substituted PAT analogs at 5 HT 2C receptors The substitution at the 6 and 7 position of tetrahydronaphthalene ring with a concomitant substitution at the meta position revealed a loss in stereoselectivity for the stereoisomers. (+) Trans 6 OMe 7 Cl PAT had 7 fold selectivity in binding at 5 HT 2C receptors by exploiting the subtle differences in the binding pocket of 5 HT 2C receptors compared to 5 HT 2A receptors.

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67 Similar to the trend observed at 5 HT 2A receptors, comparison of affinities of trans meta halogenated PAT with analogs that have both tetrahydronaphthalene and pendant phenyl ring substituents revealed no significant difference at 5 HT 2 C receptors. Computational Chemistry and Molecular Modeling Studies: In silico Docking of PAT s at 5 HT 2 receptors The data presented in this section is from work done by our collaborator Dr. Cordova Sintjago Tania in the Department of Medicinal Chemistry at the University of Florida (2009 2012). The ligand docking studies aim to probe PAT interaction with amino acids in the binding pocket of 5 HT 2 GPCRs. Delineation of these molecular interact ions will assist in explaining the PAT ligand affinity results and also assist in the design of future ligand structure optimized to exploit hypothesized PAT 5 HT 2 interactions. In silico docking studies revealed that trans PAT, protonated at physiolog ical pH, forms an i onic interaction with D3.32 (2 ) at 5 HT 2A receptors The amine group was also able to form a hydrogen bond with para hydroxy group of Y7.43 (3.3). The methyl groups on the amines were in close proximi ty to F6.51 ( 4 .2 ). The pendant phe nyl ring interacts with W6.48 (3.8 ) and F6.52 (4.2 ) amino acids (Figure 2 8 ) These interactions assist in stabilizing the trans PAT molecule in the binding pocket. In contrast, phenyl and tetrahydronaphthalene rings of (+) trans PAT did not form sta bilizing interaction with aromatic amino acids W6.48, F6.51 and F6.52. This explains the lower affinity of (+) trans PAT despite interacting with D3.32 (2) of 5 HT 2A receptors 134 In silico docking of trans PAT at 5 HT 2C receptors revealed that amino acids D3.32 (1.84), S3.36 (1.58) and Y7.43 (1.56 ) interact ed with the protonated am ine of trans PAT similar to interactions at 5 HT 2A receptors 30 H owever the pendant phenyl ring interacted with W6.48 (5) F6.51 (2.2) and F6.52 amino acids of

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68 5 HT 2C receptors In contrast to 5 HT 2A receptors, tetrahydronaphthalene rin g was oriented parallel to Y7.32 (3.7 ) and in close proximity to S3.36 (3.5) (Figure 2 9 ) The closer proximity of terminal amine of trans PAT to D3.32 and Y7.43 and its added interaction with S3.36 help explain its higher affinity at 5 HT 2C receptors in contrast to 5 HT 2A receptors In silico docking studies on trans m Cl PAT enantiom er at 5 HT 2C receptors revealed that some of its amino acid interaction trans PAT at the 5 HT 2C receptors. The common interactions include D3.32 (3.3 ) S3.36 (2 9 ) W6.48 ( 5 ) Y7.43 ( 3. ) and F6.51 (6 ) However, different po trans m Cl PAT were possible as the pendant phenyl ring was freely rotatable around the sp 3 hybridized carbon of the tetrahydronaphthalene ring. The chlorine at the meta position when present in one of the poses is at a distance of 3 from asparagine N6.55 amino acid thereby assisting in binding in the binding pocket. This proximity could lead to a stabilizing interaction between the hydrogen on amine of asparagine and chlorine This additional interaction could explain the significantly hig her affinity of trans m Cl PAT enantiomer at 5 HT 2C receptor relative to trans PAT enantiomer. In silico trans m Br PAT at 5 HT 2C receptors showed that protonated amine interacts with D3.32 (1.7 ), S3.36 (2.3 ) and pendant phenyl ring was in close proximity to F6.44 (4.5 ). The bromine on the pendant phenyl ring was oriented towards transmembrane 6 and was present in close proximity, with in 5 to F6.44, M6.47, W6.48 and C7.45. In an alternate pose, due to rotation of the p endant trans m Br PAT again interacted with D3.32 (1.6 ), S3.36 (2.8 ). The tetrahydronaphthalene ring was present parallel to Y7.43 (4 )

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69 and bromine was oriented towards transmembrane 2 and 7. Bromine substituent w as present in close proximity, with in 5 to V2.53, F6.44, C7.45 and S7.46 (Figure 2 10 ) Presence of these aromatic and polar amino acids in close proximity to the meta bromine could explain the significantly higher affinity trans m Br PAT e nantiomer at 5 HT 2C trans PAT. In silico docking studies revealed that protonated amine of (+) trans p Cl PAT form ed a hydrogen bond with D3.32 (1.75 ) and Y7.43 (1.49 ) at 5 HT 2 A receptors In addition to this the chlorine at para position could also interact with the hydroxyl group of S5.43 (2.50 ) In contrast, trans p Cl PAT formed a hydrogen bond with D3.32 (1.83 1.96 ) and indole side chain of W3.28 (1.78 ) at 5 HT 2 A receptors The lack of interaction with Y7.43 and S5 .43 could explain the decreased affinity trans p Cl PAT enantiomer at 5 HT 2A receptors ( Figure 2 11 ) 134 D ocking studies at 5 HT 2C receptors t rans p Cl PAT forms a hydrogen bond with carboxylate moiety of D3.32 (1.78 ) indole side chain of W3.28 (1.78 ) and hydroxyl side chain of Y7.43 (1.36 ) The additional interaction with hydroxyl side chain of Y7.43 trans p Cl PAT at 5 HT 2C receptors compared to 5 HT 2A receptors. The (+ ) trans p Cl PAT enantiomer forms similar hydrogen bonds with D3.32 (1.60 ) and Y7.43 (1.48 ) In addition to this, the chlorine on pendant phenyl ring also forms a hydrogen bond with S5.43 (2.50 ) this interaction could explain the higher affinity of (+ ) trans p Cl PAT trans p Cl PAT (Figure 2 12 ) 30 Preliminary i n silico studies at 5 HT 2C receptors revealed the amino acid residues that are present in a 6 trans p CF 3 PAT docked at 5 HT 2C receptors.

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70 Similar interactions as were observed in other PAT enantiomers were also observed here (D3.32, S3.36, S5.43 and N6.55). Fluorine atoms i n t rifluoromethyl substituent at the para position were found to interact with hydrogen on amide of C7.45 (4 ) and Y7.43 (2 ) 135 This C7.45 amino acid is not conserved at 5 HT 2A and 5 HT 2B receptors trans p CF 3 PAT at 5 HT 2C receptors. Preliminary i n silico studies at 5 HT 2C receptors revealed the amino acid residues that are present at a 6 trans m CF 3 PAT docked at 5 HT 2C trans PAT include D3.32, S3.36, F6.51, F6.52 and Y7.43. Similar trans p CF 3 PAT C7.45 was present in close proximity with the trifluoromethyl substituent. This could result in hydrogen bonds between fluorine and hydrogen on amide of C7.45. In silico docking studies explained the higher affinity of (+) trans 6 OMe 7 Cl PAT enantiomer at 5 HT 2C receptors relative to 5 HT 2A receptors. The 6 methoxy group of (+) trans 6 OMe 7 Cl PAT interacted with S5.46 at 5 HT 2A receptors and the ensuing steric clash explained the 7 fold lower affinity of the enantiomer at 5 HT 2A receptors. The same position 5.46, at 5 HT 2C receptors has a n alanine amino acid resulting in less steric hindrance at this position relative to 5 HT 2 A receptors. Hence, the higher affinity of (+) trans 6 OMe 7 Cl PAT enantiomer at 5 HT 2C receptors com pared to 5 HT 2A receptors. Th is steric clash at 5 HT 2A receptor between amino acid S5.46 and methoxy substituent of (+) trans 6 OMe 7 Cl PAT was further corroborated by the significant higher affinity of (+) trans 6 OH 7 Cl PAT at 5 HT 2A receptors. This high affinity of (+) trans 6 OH 7 Cl PAT at 5 HT 2A receptors was due to favorable hydrogen bond between the 6 hydroxy substituent and S5.46 amino acid (Figure 2 13 ) The change in amino

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71 acid to alanine at 5 HT 2B and 5 HT 2C receptors result s in a loss of this hydrogen bond seen as a significant drop in affinity of (+) trans 6 OH 7 Cl PAT at these receptors (Figure 2 1 4) (Data for trans 6 OH 7 Cl PAT courtesy Dr Clinton Canal).

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72 Figure 2 1. Stereochemical relationship between PAT diastereomers and enantiomers.

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73 Figure 2 2 Structure of meta substituted PAT analogs. A) Structure of different (+) trans m substituted PAT analogs. B) Structure of different trans m substi tuted PAT analogs. Figure 2 3 Change in Ki value at 5 HT 2A receptors with increase in the size of substituent at meta position.

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74 Figure 2 4 Structure of para substituted PAT analogs. A) Structure and stereochemistry of (+) trans para substituted PAT analogs. B) Structure and stereochemistry of trans para substituted PAT analogs. Figure 2 5 Effect of sterics on the affinity of para substituted PAT enantiomers at 5 HT 2A and 5 HT 2C receptors.

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75 Figure 2 6 Single X ray crystallographic structure of (2 S ,4 R ) trans p Cl PAT. HCl Figure 2 7 T etrahydronaphthalene and/or pendant phenyl ring substituted PAT analogs.

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76 Figure 2 8 t rans PAT enantiomer docked at 5 HT 2A receptor s with the corresponding amino acid interactions in binding pocket Figure 2 9 t rans PAT docked at 5 HT 2 C receptor s with the corresponding amino acid interactions in binding pocket

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77 A B Figure 2 10. t rans m Br PAT enantiomer docked at 5 HT 2C receptors. A) Shows the bromine substituent at the meta position orie nted towards transmembrane 6. B) Shows the bromine substituent at meta position oriented towards transmembrane 2 and 7. A B Figure 2 11. (2 R ,4 S ) (+) trans and ( 2 S ,4 R ) ( ) trans p Cl PAT enantiomer docked at 5 HT 2A receptors. A) R epresents the amino acid interactions of (2 R ,4 S ) (+) trans p Cl PAT at 5 HT 2A receptors. B) R epresents the amino acid interactions of (2 S ,4R) ( ) trans p Cl PAT at 5 HT 2A receptors.

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78 A B Figure 2 12. (2 R ,4 S ) (+) trans and ( 2 S ,4 R ) ( ) trans p Cl PAT enantiomer docked at 5 HT 2 C receptors. A) R epresents the amino acid interactions of (2 R ,4 S ) (+) trans p Cl PAT at 5 HT 2 C receptors. B) Represents the amino acid interactions of (2 S ,4R) ( ) trans p Cl PAT at 5 HT 2 C receptors.

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79 Figure 2 13. trans 6 OH 7 Cl PAT at 5 HT 2A 5 HT 2B and 5 HT 2C receptors. The affinity of (+) trans 6 OH 7 Cl PAT was significantly higher at 5 HT 2A receptors relative to other receptors.

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80 A B Figure 2 14. Trans 6 OH 7 Cl PAT and (+) trans 6 OH 7 Cl PAT docked at 5 HT 2A r eceptors. D epicts the v i e w of the binding pocket looking from the extracellular region into the intracellular region. trans 6 OH 7 Cl PAT enantiomer is docked away from transmembrane 5 in binding pocket. B) The (+) trans 6 OH 7 Cl PAT enantiomer is d ock ed closer to transmembrane 5.

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81 Table 2 1. Shows changes in the bond length relative to changes in substituent. Table 2 2. Binding affinities of parent PAT enantiomers and meta substituted PAT enantiomers at 5 HT 2A and 5 HT 2C receptors. R Configuration 5 HT 2A 5 HT 2C Affinity ( Ki SEM; nM) C 6 H 5 (+) trans 470(46) 430(86) trans 100(10) 30(3) m F C 6 H 4 (+) trans 320(26) 200(30) trans 110(26) 43(14) m Cl C 6 H 4 (+) trans 130(8.7) 170(43) trans 40(5) 8(3) m Br C 6 H 4 (+) trans 260(22) 200(30) trans 20(3) 4(1) m CF 3 C 6 H 4 (+) trans 1300(67) 230(20) trans 80(10) 10(2) m NO 2 C 6 H 4 (+) trans 500(20) 120(13) trans 74(18) 10(1) Bond type Bond distance ( ) C ar H 1.09766 C ar F 1.34015 C ar Cl 1.67760 C ar Br 1.86251 C ar C 1.53114 C sp3 F 1.35127

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82 Table 2 3. Binding affinities of para substituted PAT enantiomers at 5 HT 2A and 5 HT 2C receptors. R Configuration 5 HT 2A 5 HT 2C Affinity ( Ki SEM; nM) p F C 6 H 4 (+) trans 71(12) 75(12) trans 140(24) 68(11) p Cl C 6 H 4 (+) trans 50(8) 55(17) trans 250(37) 120(9.0) p Br C 6 H 4 (+) trans 60(10) 70(8) trans 220(29) 100(3) p CF 3 C 6 H 4 (+) trans 210(16) 220(27) trans 2000(200) 520(51) p NO 2 C 6 H 4 racemic 8100(180) 1000 (100)

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83 Table 2 4. Binding affinities of tetrahydronaphthalene ring with (or without) pendant phenyl ring substituted PAT analogs. R Configuration 5 HT 2A 5 HT 2C Affinity ( Ki SEM; nM) 6 OMe 7 Cl PAT (+) trans 240(23) 37(5.1) trans 160(24) 24(2.5) 6 OMe 7 Cl PAT (+) cis 2300(130) 1600(60) cis 320(42) 96(18) 6 OMe 7 Cl m Cl PAT (+) trans 95(18) 7(3) trans 50(20) 9(2) 6 OMe 7 Cl m Cl PAT (+) cis 230(64) 320(41) cis 3500(390) 1560(390) 6 OMe 7 Cl m Br PAT (+) trans 79(14) 350(130) trans 61(15) 130(43) 6 OMe m Cl PAT (+) trans 350(16) 280(30) trans 35(3) 17(1.0) 6 OH 7 Cl PAT (+) trans 70(2.6) 1000(80) trans 60(5.4) 23(3.0)

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84 C HAPTER 3 CHARACTERIZATION OF THE 5 HT 2 FUNCTIONAL PHARMACOLOGY OF PAT ANALOGS Specific A im 2 The ideal pharmacological profile of a ligand targeting the 5 HT 2 system is: antagonist/inverse agonist at 5 HT 2A/2B receptors and pote nt agonist at 5 HT 2C receptors. Analogs that had high affinity at therapeutically relevant 5 HT 2A and 5 HT 2C receptors were selected from both pendant phenyl ring substituted and tetrahydronaphthalene ring substituted group of PAT analogs. Trans m Cl PAT and trans m Br PAT analogs were selected because these anal ogs had significantly higher affinity at both 5 HT 2A and 5 HT 2C receptors among all the meta substituted PAT analogs tested (+) Trans p Cl PAT analog had the highest affinity among all the para substituted PAT analogs (not statistically significant) and w as chosen from the para substituted group of analogs. Trans 6 OMe m Cl PAT was chosen from the tetrahydronaphthalene ring series of analogs as this analog had high affinity at both 5 HT 2A and 5 HT 2C receptors. Analogs that possessed potency values < 500nM are considered for translational studies of AIM 3. Methodology Functional assays were performed to measure the ability of the ligands to modulate phospholipase C (PLC) pathway. These assays measured the level of [ 3 H] IP formation in the presence of d ifferent concentrations of ligand. HEK cells grown to approximately 80% confluency in DMEM containing 10% fetal bovine serum and 1% antibiotic in 10 cm plates at 37C, 5% CO 2 (incubator) were washed one time with PBS, then transfected with 20 g 5 HT 2 pcDN A (5 HT 2C INI, 5 HT 2B or 5 HT 2A ), and 30 l lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in 5 mL DMEM containing 5%

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85 dialyzed fetal bovine serum and 5 mL Opti MEM (transfection media). Cells were placed in an incubator, and approximately 16 h ou r s later, transfection media was removed and replaced with 16 mL inositol free DMEM containing 2.5% dialyzed fetal bovine serum. Cells were then detached by vigorous pipetting, 0.1 Ci/mL [ 3 H] myo inositol was added to the mixture (labeling media), and cells were seeded 300 l per well into 48 well CellBind plates (Corning, Lowell, MA), and placed in an incubator. 24 hr. later, plates were centrifuged at 2500 r.p.m. for 10 min. at room temperature, labeling media was discarded, and 450 L inositol free, serum free DMEM was added to each well. Cells were placed in an incubator for one hour. Cells were then incubated for 30 min. with increasing concentrations of ligands diluted in inositol free, serum free DMEM containing a final concentration of 50 mM LiCl and 10M pargyline per well. Plates were again centrifuged at 2500 r.p.m. for 10 min. at room temperature, drug incubation media was discarded, and 400 l of 50 mM formic acid was add ed to each well to lyse cells. O ne hour later, 200 l of 150 mM NH 4 OH was add ed to each well to neutralize cells, well was added to individual anion exchange columns to separate [ 3 H] inositol phosphates formed from [ 3 H] myo inositol. Following a 10 mL wash with dH 2 O, bound [ 3 H] inositol phosphates were eluted with 4 mL 800 mM ammonium formate into vials. 1 mL of eluate was added to 10 mL scintillation fluid (ScintiVerse Cocktail, Fisher). After mixing, 3 H induced scintillations were counted with a Beckman Coulter LS6500 counter. Each independent experiment was performed a minimum of three times.

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86 Competitive functional antagonism assays were performed using a similar methodology, except the antagonist was incubated in 48 well plates for 15 minutes, prior to addition of serotonin. Results and D iscussion for Functional Assays Functional A ctivity of trans PAT and trans 4 ( meta h alogenated ) PAT s at 5 HT 2 R eceptors T rans PAT enantiomer was a partial agonist at 5 HT 2C receptors and an inverse a gonist at both 5 HT 2A and 5 HT 2B receptors 123 Trans PAT had an EC 50 of 61.1 18.2 nM at the 5 HT 2C receptors (Table 3 1) 84 The IC 50 of trans PAT at 5 HT 2A and 5 HT 2B receptors was found to be 490 96 nM and 1000 0.5 nM respectively. These data corroborate the in silico dock ing results showing trans PAT interacting with differently amino acids in binding pocket at 5 HT 2A versus 5 HT 2C receptors and potentially leading to stabilization of different conformations. M eta substituted analogs of PATs showed the best binding profile at both 5 HT 2A and 5 HT 2C receptors. Similar to trans PAT, trans m Cl PAT enantiomer did not activate both 5 HT 2A and 5 HT 2B receptors (Figure 3 1 and 3 2 ). T rans m Cl PAT was a selective agonist at 5 HT 2C receptor and had an EC 50 value of 40.1 7 60 nM (Figure 3 3) (Data at 5 HT 2C receptors courtesy Daniel Felsing). Since trans m Cl PAT was an antagonist at 5 HT 2A receptor, the potency of the ligand at 5 HT 2A receptor can be measured using functional antagonism assays. These assays involved performing functional assays where trans m Cl PAT to antagonize 5 HT induced activation of PLC mediated by 5 HT 2A receptor is measured in the presence of different concentrations of ant ago nist These assays reveal the nature of the antagonist : competitive or non competitive antagonist.

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87 Competitive antagonists displace the curve to the right with no change in the maximal response of serotonin. Non competitive antagonist, in contrast, decrease the maximal response produced by the highest concentration of serotonin 136 These assays also 2 values the negative log arithm of the concentration of antagonist required to shift the EC 50 by 2 fold. The pA 2 value for trans m Cl PAT was calculated to be 6.4 0.092 (Figure 3 4 ). T rans m Cl PAT was a competitive antagonist of serotonin at 5 HT 2A receptors as there was no decrease in the maximal serotonin response Similar to trans PAT and trans m Cl PAT enantiomers, trans m Br PAT enantiomer was also an antagonist at both 5 HT 2A and 5 HT 2B receptors The functional potency of trans m Br PAT enantiomer at 5 HT 2C receptors was 17 3.2 nM (Table 3 1) Functional curves for all 5 HT 2 receptors are represented in figure 3 5. ( trans m Br PAT analog was done by Dr. Clinton Canal. ) Functional experiments revealed that trans m CF 3 PAT was an inverse agonist at the 5 HT 2C receptors with an efficacy of 40 0.7 nM (Figure 3 11) This revealed an interesting shift from agonism to inverse agonism when the substituent at meta position of pendant phenyl ring was changed from a halogen to a trifluoromethyl substituent. The steric and the electronic nature of the trifluoromethyl substituent are significantly different from other halogen substituents at meta position. This indicated that the trifluor omethyl substituent bound differently and hence interacted with different amino acids in the binding pocket thereby stabilizing a different conformation of the receptor. This result warrants more investigation to characterize the molecular

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88 determinants tha trans m CF 3 PAT and mediate the inverse agonist response. Functional A ctivity of (+) and ( ) t rans 4 ( p Cl ) PAT s at 5 HT 2 R eceptors Both (+) and ( ) t rans p Cl PAT enantiomers did not activate the PLC pathway at 5 HT 2A and 5 HT 2B receptors even at 10 M concentration (Figure 3 6 and 3 7). In trans p Cl PAT were agonist at 5 HT 2C receptors. The EC 50 of (+) trans p Cl PAT was 140 20 nM and EC 50 of trans p Cl PAT was 1650 149 nM (Table 3 1) (+) Tran s p Cl PAT was 12 times more potent than trans p Cl PAT at activating PLC pathway ( p <0.0001 ) (Figure 3 8) This reversal in stereoselectivity corroborated the affinity of (+) and ( ) t rans p Cl PAT enantiomers observed at 5 HT 2C receptors Schild analysis of this enantiomer at 5 HT 2A receptor s revealed a pA 2 value of 6.21 0.545 (Figure 3 9 ) The pA 2 values of (+) t rans p Cl PAT trans m Cl PAT were not significantly different. These values correspond with the observed lack of significant difference in the affinities of (+) trans p Cl PAT and trans m Cl PAT at 5 HT 2A receptors. However, comparing the EC 50 values of (+) trans p Cl PAT and trans m Cl PAT at 5 HT 2 C receptors trans m Cl PAT enantiomer had significantly higher potency at 5 HT 2C receptors ( p = 0.0014). Functional A ctivity of ( ) t rans 4 ( m Cl ) (6 OMe Tetrahydronaphthalene) PAT Analog at 5 HT 2 A and 5 HT 2C R eceptors Funct ional activity of ( ) t rans 6 OMe m Cl PAT enantiomer at 5 HT 2 A and 5 HT 2C receptors was assessed. Similar to other PAT analogs it was found to be an antagonist at 5 HT 2A receptor and an agonist at 5 HT 2C receptors (Figure 3 10) The EC 50 of ( ) t rans 6 OMe m Cl PAT enantiomer at 5 HT 2C receptors was found to be 43 14nM (Table 3 1) Comparing the EC 50 of ( ) t rans 6 OMe m Cl PAT enantiomer with

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89 ( ) t rans m Cl PAT enantiomer showed no significant diffe rence between their potencies ( p >0.05).

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90 Figure 3 1 Functional assay curve representing the action of trans m Cl PAT enantiomer at 5 HT 2A receptor. Figure 3 2 Functional assay curve representing the action of trans m Cl PAT enantiomer at 5 HT 2B receptor.

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91 Figure 3 3. Functional assay curve representing the action of trans m Cl PAT enantiomer at 5 HT 2 C receptor. Figure 3 4 Schild plot of trans m Cl PAT at 5 HT 2A receptors. T rans m Cl PAT enantiomer was incubated at 100 nM 1000 nM and 10000 nM concentrations.

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92 Figure 3 5. trans m Br PAT at 5 HT 2A 5 HT 2B and 5 HT 2C receptors. Figure 3 6. trans p Cl PAT at 5 HT 2A receptors.

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93 Figure 3 7. trans p Cl PAT at 5 HT 2 B receptors. Figure 3 8. trans p Cl PAT demonstrating partial agonism at 5 HT 2 C receptors.

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94 Figure 3 9. Schild plot of (+) trans p Cl PAT at 5 HT 2A receptors. (+) trans p Cl PAT enantiomer was incubated at 100 nM 1000 nM and 10000 nM concentrations. Figure 3 10. Functional activity of trans 6 OMe m Cl PAT enantiomer at 5 HT 2A and 5 HT 2C receptors.

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95 Figure 3 11. Functional response of trans m CF 3 PAT enantiomer at 5 HT 2C receptors.

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96 Table 3 1. Functional activity of PAT analogs at 5 HT 2 receptors. PAT analog 5 HT 2A 5 HT 2B 5 HT 2C trans PAT Inverse agonist; IC 50 = 490 96 nM Inverse agonist; IC 50 = 1000 .5 nM Agonist; EC 50 = 61.1 18.2 nM trans m Br PAT Not activated Not activated Agonist; EC 50 of 17 3 .2 nM trans m Cl PAT Not activated ; pA 2 = 6.4 0.092 Not activated Agonist; EC 50 of 40.1 7 60 nM (+) trans p Cl PAT Not activated Not activated Agonist; EC 50 of 140 20 nM trans p Cl PAT Not activated ; pA 2 = 6.21 0.545 Not activated Agonist; EC 50 of 1650 149 nM trans 6 OMe m Cl PAT Not activated Agonist; EC 50 of 43 1 4 nM

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97 CH APTER 4 TRANSLATIONAL STUDIES: ASSESSMENT OF PAT ANALOGS EFFICACY IN RODENT MODELS OF PSYCHOSIS Specific A im 3 Analogs of trans PAT that had high affinity and potency at 5 HT 2A and 5 HT 2C receptors were screened in different mice model s each manipulating a different neurotransmitter implicated in s chizophrenia. (+) and T rans PAT, (+) and trans p Cl PAT, (+) and trans m Cl PAT and trans m Br PAT analogs were selected for in vivo screening. These meta and para substituted analogs were chosen because they had the highest affinity and potency at both 5 HT 2A and 5 HT 2C receptors among all the PAT analogs. Another objective was to confirm the effectiveness of these models for screening antipsychotics in our laboratory conditions. This was ascertained by screening clinically approved antip sychotics in these models. These PAT analogs were then screened to test their efficacy in HTR, MK 801 and amphetamine induced hyperlocomotion models. These experiments also provide d a preliminary insi ght into the pharmacokinetics of the analog s being teste d. Methodology In vivo Behavioral P harmacology C57B l /6J male mice were obtained from Jackson Laboratories at approximately 8 weeks of age and allowed to acclimate to the temperature and humidity controlled colony room for at least 1 week prior to testing Mice were housed in pairs, in standard cages and allowed unlimited access to laboratory chow and water. Experiments were conducted at approximately the middle of the light phase (lights on at 6 am, and light s off at 6 pm). All compounds were dissolved in sterile saline prior to behavioral testing, and administered in a volume of 0.01 mL /g body weight. All experimental procedures

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98 were performed in accordance with the Guide for the Care and Use of Laboratory Animals, as promulgate d by the National Institutes of Health, and were approved by the Psycholocomotor Activity (Head Twitch Response; HTR) Elicited by the Serotonin 5 HT 2 Agonist 2,5 dimethoxy 4 iodoamphetami ne (DOI) On the day of testing, mice were habituated to the testing room for approximately 30 minutes. Testing typically consisted of administration (i.p.) of sterile saline or a particular dose of (+) or ( ) trans PAT analog s Ten minutes later, mice were administered ( ) DOI or sterile saline. Ten minutes later, mice were individually placed ( ) DOI was administered 20 minutes after oral administration of PAT analogs or saline Head twitch responses (HTRs) defined as a clear, rapid, and discrete, back and forth rotation of the head. HTRs during a 10 minute session were coun ted by a trained observer who was bli nd to drug treatment conditions An overhead camera videotaped the session, and activity (distance travelled in cm) was analyzed and calculated by Ethovision software (Noldus I nformation Technology Inc. ). Psycholocomotor Activity Elicited by the Glutamate Antagonist MK 801 Experimentally naive mice were habituated to the testing room for approximately 30 minutes. Locomotor activity testing consisted of administration (i.p.) of saline, clozapine, or PAT analogs (3.0, 5.6, and 10.0 mg/kg), followed 10 minutes later by an injection of the NMDA antagonist MK 801 (0.3 mg/kg). Mice were immediately placed int o a plexiglas chamber (43 x 43 cm, Med Assoc iates, Inc.) for a 60 minute session. An overhead camera videotaped the session and activity (distance travelled in cm) was calculated by Ethovision software (Noldus Information Technology Inc.).

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99 Psycholocomotor Activity Elicited by the Dopamine/Serotonin Agonist Amphetamine Experimentally naive mice were habituated to the testing room for approximately 30 minutes. Locomotor activity testing consisted of administration (i.p.) of saline or (+) and trans PAT ana logs (3.0, 5.6, and 10.0 mg/kg), followed (10 minutes later) by an injection of the indirect dopamine agonist amphetamine (3.0 mg/kg). Locomotor activity was assessed exactly as noted in the MK 801 experiment. Results for PAT Efficacy in Rodent Models of P sychosis Clozapine is a prototypical atypical antipsychotic used to treat schizophrenia. Clozapine was used as a control to test the efficacy of these models to screen for antipsychotics. Clozapine was administered in 0.1 and 1 mg/kg doses 10 minutes prior to DOI. Both these doses significantly decreased the HTR produced by 1 mg/kg of DOI (F 3,31 =138.7; p <0.0001) (Figure 4 1) The ED 50 (95% CL) for clozapine was found to be 0.23 (0.18 0.28) mg/kg ( Table 4 1). The same doses of clozapine were then tested in MK 801 and amphetamine stimulated locomotion models. The 1 mg/kg dose of clozapine was found to significantly lower both MK 801 and amphetamine induced locomotion (F 4,61 =33.02 and F 4,49 =35.26 respectively; p <0.0001 in both cases). The ED 50 (95% CL) of c lozapine was found to be 0.25 (0.10 0.64) mg/kg for MK 801 induced hyperactivity model and 0.27 (0.16 0.44) mg/kg for amphetamine induced locomotion (Figure 4.2). The 1 mg/kg dose of clozapine alone resulted in significant lower locomotor activity as compa red to locomotor activity after saline administration (Figure 4 5). This data is concordant with published literature that show similar effect of clozapine on spontaneous locomotor activity 137 This data indicates that these models are effective for screening antipsychotics.

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100 (+) and Trans PAT enantiomers significantly attenuated the HTR elicited by 1 mg/kg dose of DOI (F 6,57 =86.03; p <0.001) 138 Trans PAT enant iomer was significantly more potent than the other PAT enantiomer ( p <0.05). Locomotor activity was also recorded during these 10 minute observation sessions. T here was no significant difference between locomotor activity for DOI alone dose and PAT enantiom er pretreated DOI dose (HTR d ata of PAT courtesy Dr. Clinton Canal). These enantiomers were then tested in MK 801 and amphetamine induced hyperlocomotor experiments. Administration of 3, 5.6 and 10 mg/kg doses of (+) trans PAT did not result in any attenua tion of MK 801 induced locomotor activity. In contrast, both 5.6 mg/kg and 10 mg/kg doses of trans PAT resulted in significant reduction in locomotor activity compared to MK 801 induced hyperactivity (F 5,67 =28.77; p <0.0001) (Figure 4 3). Similar to MK 801 results, (+) trans PAT did not modulate amphetamine induced locomotion T he 10 mg/kg dose of trans PAT significantly decreased amphetamine induced hyperlocomotion (F 5, 55 =17.73; p <0.0001) (Figure 4 4). However, administration of 10 mg/kg dose of (+) trans PAT alone resulted in a significantly lower locomotor activity relative to saline locomotor activity ( p <0.05) (Figure 4 5). Activity of ligands that modulate both saline and drug stimulated activity could be attributed to non specific ac tion on locom otion in general 109 However, (+) trans PAT did not have any action on NMDA or amphetamine stimulated activity hence these effects on saline activity were not relevant The ED 50 (95% CL) of trans PAT in MK 801 induced locomotor activity model was found to be 4.02 (3.18 5.08) mg/kg. The ED 50 (95% CL) of trans PAT in amphetamine induced locomotor activity was found to be 6.44 ( 4.57 9.08)

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101 mg/kg. There was no significant difference between the ED 50 s of trans PAT in amphetamine and MK 801 induced hyperlocomotor assays. The next enantiomer to be tested in these models was trans m Br PAT. This enantiomer when administered prior to DOI demonstrated the ability to attenuate DOI induced HTR. Doses of 3, 5.6 and10 mg/kg of trans m Br PAT enantiomers resulted in a statistically significant lowering of DOI induced HTR (F 6,30 =28.2; p <0.0001). The ED 50 (95% CL) of trans m Br PAT was found to be 2.67 (1.60 4.20) mg/kg (Data courtesy Dr. Clinton Canal). Higher doses of trans m Br PAT, 10 mg/kg and 5.6 mg/kg resulted in a significant decrease in MK 801 induced hyperactivity (F 5,69 =16.93; p <0.0001). The ED 50 (95% CL) of trans m Br PAT at MK 801 hyperactivity model was found to be 4.41 (2.96 6.57) mg/kg. In contrast, only the highest dose of 10 mg/kg of trans m Br PAT resulted in a decrease in amphetamine induced hyperactivity (F 5,65 = 8.316; p <0.0001). The ED 50 (9 5% CL) of trans m Br PAT at amphetamine hyperactivity model was found to be 6.13 (1.94 19.40). There was again no significant difference between the ED 50 values in MK 801 and amphetamine hyperactivity models (Figure 4 6). There was no significant dif ference between locomotor activity of 10 mg/kg dose of trans m Br PAT alone and saline locomotor activity (Figure 4 5). Hence, trans m Br PAT demonstrated ability to selectively modulate MK 801 and amphetamine hyperactivity and did not affect locomotor activity when administered alone Both (+) and t rans m Cl PAT enantiomers were tested for their potency to attenuate DOI induced HTR. Both these enantiomers were effective in attenuating DOI induced HTR. The t rans m Cl PAT enantiomer sign ificantly decreased DOI induced

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102 HTR at doses of 10, 5.6, 3 and 1 mg/kg (F 5,41 =40.62; p <0.0001). In contrast, only the 10 and 5.6 mg/kg doses of (+) t rans m Cl PAT enantiomer attenuated DOI induced HTR (F 4,38 =35.31; p <0.0001) (Figure 4 7). The ED 50 (95% C L) of both (+) and t rans m Cl PAT enantiomers was found to be 7.23 (5.51 9.49) mg/kg and 2.19 (1.39 3.42) mg/kg respectively. The ED 50 value of t rans m Cl PAT was significantly lower than ED 50 value of (+) t rans m Cl PAT enantiomer in HTR model. Only the t rans m Cl PAT enantiomer was effective at attenuating both MK 801 and amphetamine induced hyperactivity models. The (+) t rans m Cl PAT was not effective at either MK 801 or amphetamine induced hyperactivity mo dels. Both the 10 and 5.6 mg/kg doses of t rans m Cl PAT enantiomer significantly reduced MK 801 induced hyperactivity (F 5,73 =18.82; p <0.0001). The ED 50 (95% CL) of t rans m Cl PAT in the MK 801 hyperactivity model was found to be 5.58 (4.43 7.04) mg/kg. In contrast, only the 10 mg/kg dose of t rans m Cl PAT decreased amphetamine induced hyperactivity (F 5,55 =13.14; p <0.0001). The ED 50 (95% CL) of t rans m Cl PAT in the amphetamine hyperactivity model was found to be 4.86 (3.50 6.74) mg/kg (Figure 4 8). There was no significant difference between the ED 50 values of t rans m Cl PAT in amphetamine and MK 801 hyperactivity assays. There was a significant increase in trans m Cl PAT locomotor activity as compared to saline locomotor activi ty ( p <0.005) (Figure 4 5). Hence trans m Cl PAT was not a selective modulator of amphetamine or MK 801 locomotor activity. Both (+) and trans p Cl PAT enantiomers were administered 10 minutes before DOI administration and resulted in a dose depend ent a ttenuation of HTR. Both 10 and 30 mg/kg doses of (+) trans p Cl PAT resulted in a significantly lower HTR

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103 relative to DOI alone (F 6,27 =15.66; p <0.001) In contrast, only the 30 mg/kg dose of trans p Cl PAT resulted in a sig nificantly lower HTR (Figure 4 9 right panel). The ED 50 95% CL of these enantiomers was found to be 8.2 (5.4 12.4) and 20.1 (14.0 28.8) mg/kg for (+) and trans p Cl PAT respectively. These ED 50 values for (+) and trans p Cl PAT enantiomers were significantly differe nt. These results were in conformation with the affinity and the potency results of these enantiomers observed in in vitro binding and functional studies A dose of 30 mg/kg of (+) and trans p Cl PAT enantiomer and th e racemate of these compounds were administered orally (Figure 4 10). All the three dose combinations resulted in a significantly lower HTR as compared to DOI alone (F 3,16 =31.1; p <0.001) The attenuated HTR response of (+) trans p Cl PAT enantiomer was significantly different relative to ) trans p Cl PAT enantiomer ( p =0.038). (Data of trans p Cl PAT at HTR courtesy Dr. Drake Morgan) Both (+) and trans p Cl PAT were then screened in MK 801 and amphetamine locomotor models. Both these enantiomers were tested at 3 mg/kg, 5.6 mg/kg and 10 mg/kg doses. None of these doses for both enantiomers resulted in a significant decrease in locomotion induced by administ ration of 0.3 mg/kg of MK 801 ( p >0.05) (Figure 4 11). Similar results were obtained for both enantiomers when tested for their ability to attenuate amphetamine induced (3 mg/kg) locomotion ( p >0.05) (Figure 4 12). When administered alone 10 mg/kg doses of each enantiomer did not result in any significant difference in locomotor activity from saline (Figure 4 5). This data is in concordanc e with HTR response observed for (+) trans p Cl PAT enantiomer. Both 5.6 mg/kg and 10 mg/kg doses did not result in attenuation of HTR MK 801 and amphetamine induced locomotor activity. One explanation for this could be the

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104 significantly lower potency of (+) trans p Cl PAT enantiomer at serotonin 5 HT 2C receptors as compared to parent PAT and trans m Cl PAT analogs. Alternatively, a HT 2A receptors could be a reason for the lack of effect in MK 801 and amphetamine mod els. But comparison of pA 2 valued of (+) trans p Cl PAT and trans m Cl PAT did not reveal any significant differen ce. Hence the lack of response to (+) trans p Cl PAT enantiomer at both MK 801 and amphetamine hyperactivity assays could be attributed to lower potency at 5 HT 2C receptors. Summary of in vivo results These results indicate that both (+) and trans p Cl PAT enantiomers are not effective at either MK 801 or amphetamine hyperactivity model (Table 4 1) trans m Cl trans m Br PAT enantiomers demonstrated significantly higher potency at HTR model compared to MK 801 and amphetamine hyperlocomotor model. All the trans enantiomers had a higher potency at MK 801 hyperactivity model than amphetamine hyperactivity model (not significantly different). The enantiomers trans PAT, trans m Cl PAT and trans m Br PAT demonstrated the highest potency in the in vivo models. All of these enantiomers were also tested for their anorecti c potential by our collaborator, Dr. Neil Rowland, in a mice model of compulsive or bi nge eating. These ligands dose dependently decreased the amount of treats consumed in a 30 minute session. Hence, these analogs do not exhibit the weight gain side effects showed by clinically approved antip sychotics and also demonstrated a novel mechanism of action for modulating dopaminergic and glutamatergic neurotransmission disrupted in schizophrenia.

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105 Figure 4 1. Clozapine decreases DOI induced HTR in a dose dependent manne r. X indicates that the respective doses of clozapine attenuate DOI (1 mg/kg ) HTR in a sta tistically significant manner ( p <0.05).

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106 A B Figure 4 2. Clozapine 1 mg/kg dose attenuates both MK 801 and amphetamine (Amp) induced locomotion. A) S hows the effect of clozapine on M K 801 induced locomotion. B) S hows effect of clozapine on amphetamine induced locomotion. X indicates statistically significant reduction in stimula ted locomotion of MK 801 or amphetamine alone compared to pretreated clozapine

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107 Figure 4 3. Attenuation of MK 801 induced locomotor activity by (+) and trans PAT analogs. PAT analogs and their respective doses are represented on t he abscissa X indicates statistically significant difference between pretreated PAT relative to MK 801 alone induced locomotor activity.

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108 Figure 4 4. Attenuation of amphetamine stimulated locomotor activity b y PAT enantiomers. X indicates significant difference between amphetamine (3 mg/kg ) dose and the corresponding pretreated PAT dose response.

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109 Figure 4 5. Effect of highest dose of various ligands on locomotor activity. X indi cates statistically significant changes in locomotor activity of drug alone compared to saline locomotor activity

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110 Figure 4 6. Attenuation of MK 801 and amphetamine hyperactivity by trans m Br PAT. X indicates significant difference in locomotor activity at that pretreated dose compared to the corresponding MK 801 or amphetamine induced hyperactivity. Figure 4 7. Demonstrates the attenuation of DOI induced HTR b y (+) and trans m Cl PAT. X indicates significant decrease in the number of HTR by the respective dose of (+) or trans m Cl PAT relative to DOI 1mg/kg dose.

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111 Figure 4 8. Attenuation of MK 801 and amphetamine hyperactivi ty by trans m Cl PAT. X indicates significant difference in locomotor activity at that dose compared to the corresponding MK 801 or amphetamine induced hyperactivity. Figure 4 9. Attenuation of HTR by (+) and trans p Cl PAT analogs. D emonstrates the dose dependent decrease of DOI induced HTR by both (+) and trans p Cl PAT. Head twitch response (10 minute)

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112 Figure 4 10. Effect of oral administration of (+) and trans p Cl PAT analogs 20 minutes prior to DOI. Indicates differences from saline treatment and indicated differences from saline and each other.

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113 Figure 4 11. Effect of (+) a nd trans p Cl PAT on MK 801 (0.3 mg/kg ) induced locomotor activity. Both enantiomers did not significantly affect MK 801 induced locomotor activity. MK 801 induced locomotor activity was significantly higher than saline and (+) and trans p Cl PAT a lone locomotor activity.

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114 Figure 4 12. Effect of (+) and trans p Cl PAT on amphetamine (3 mg/kg ) induced locomotor activity. Both enantiomers did not significantly affect MK 801 induced locomotor activity. MK 801 induced locomotor activity was significantly higher than saline and (+) and trans p Cl PAT alone locomotor activity.

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115 Table 4 1. Ef ficacy of PAT analogs ED 50 (95% CL) mg/kg at in vivo models of schizophrenia. PAT analog HTR MK 801 induced hyperlocomotion Amphetamine induced hyperlocomotion Clozapine 0.23 (0.18 0.28) 0.25 (0.1 0.64) 0.27 (0.16 0.44) trans PAT 4.02 (3.18 5.08) 6.44 (4.57 9.08) trans m Br PAT 2.67 (1.6 4.2) 4.41 (2.96 6.57) 6.13 (1.94 19.40) trans m Cl PAT 2.19 (1.39 3.42) 5.58 (4.43 7.04) 4.86 (3.50 6.74) (+) trans p Cl PAT 8.2 (5.4 12.4) Not active Not active trans p Cl PAT 20.1 (14.0 28.8) Not active Not active

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116 CHAPTER 5 CONCLUSION In this dissertation, we evaluated the structure activity relationship of various analogs of 4 phenyl 2 N,N dimethyl 1,2,3,4 tetrahydronaphthalene 2 amines These analogs targeted the 5 HT 2 receptors selective ly activating 5 HT 2C receptors and were antagonists /inverse agonists at 5 HT 2A and 5 HT 2B receptors. Substitutions were made to this analog to enhance the affinity, potency and selectivity at 5 HT 2 receptors. In silico docking studies assisted in identifying the molecular determinants that assist ed in binding of these ligands at individual 5 HT 2 receptors. N N dimethyl 4 ( 2 or 3 or 4 substituted phenyl) 1,2,3,4 tetahydro 2 naphthalen e amines analogs had subs titutions at the ortho meta and para position of the pendant phenyl ring. The general trend that was observed was a relatively higher affinity of sterically larger substituents at 5 HT 2C receptors compared to 5 HT 2A receptors. Hence larger sterics favo red preferential binding to 5 HT 2C receptors. Reversed stereoselectivity of para substituted enantiomers was due to favorable amino acid interactions in binding pocket (S5.43) assisting the (2 R ,4 S ) (+) trans p Cl PAT configuration. (2 S ,4 R ) trans p Cl PAT configuration lacked this interaction with S5.43 and hence showed decreased affinity at both 5 HT 2A and 5 HT 2C receptors N,N dimethyl 4 (3 substituted phenyl) 6,7 substituted 1,2,3,4 tetrahydro 2 naphthalene amines had substituents at both tetrahy dronaphthalene ring and pendant phenyl ring. These enantiomers did n o t result in significant enhanced affinity or potency at 5 HT 2 receptors relative to meta substituted PAT analogs But the tetrahydronaphthalene substituted analogs identified a key region of the binding pocket that was different between the 5 HT 2A and 5 HT 2C receptors. There is an alanine amino acid at position

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117 5.46 in 5 HT 2A receptors and a serine (S5.46) at the same position in 5 HT 2C receptors. Th is difference resulted in 14 fold selectivity for binding at 5 HT 2A receptors over 5 HT 2C receptors for (+) trans 6 OH 7 Cl PAT enantiomer The chemical space occupied by the ligand can be exploited to further synthesize ligands that are more selective between these receptors. In vivo stu dies corroborated the in vitro studies and confirmed the stereoselectivity of unsubstituted and trans 4 ( meta Cl and meta Br) PAT analogs They demonstrated revers ed stereoselectivity when trans 4 ( para substituted) PAT analogs were evaluated. These result s demonstrated that in vitro pharmacology can predict the preclinical potential of PAT ligands as antipsychotics. The higher affinity and more potent (2 S ,4 R ) trans 4 ( meta Cl and meta Br) PAT analogs demonstrated highest potency in these models. These results confirm the efficacy of selective 5 HT 2C agonists as potential therapeutic ligands for schizophrenia.

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118 APPENDIX A AFFINITY OF 4 ( META OR PARA SUBSTITUTED ) PATs at HISTAMINE H1 GPCRs Histamine H 1 receptors share a 44% transmembrane domain se quence similarity with 5 HT 2 receptors 139 140 This similarity in sequence results in ligands targeting 5 HT 2 receptors also binding to H 1 receptors. Some meta and para PAT analogs that were analyzed at 5 HT 2 receptor s were also screened for affinity at H 1 receptors. (+) and T rans m CF 3 PAT enantiomers had an affinity of 4100 160 nM and 230 27 nM respectively at H 1 receptors ( p <0.0001). Similar to the trend at 5 HT 2 receptors the t rans m CF 3 PAT enantiomer had higher affinity at H 1 receptors. The next set of enantiomers (+) and trans m NO 2 PAT had an affinity of 1000 80 nM and 800 100 nM respectively at H 1 receptors. There was no significant difference between the affinities of these enantiomers. Com paring affinity of (+) t rans m NO 2 PAT with (+) t rans m CF 3 PAT revealed that (+) t rans m NO 2 PAT enantiomer had a 4 fold higher affinity ( p <0.0001). In contrast, t rans m CF 3 PAT enantiomer had a 3 fold higher affinity relative to trans m NO 2 PAT ( p =0.007). The affinities of both (+) and trans m NO 2 and CF 3 PAT enantiomers are significantly higher at all the 5 HT 2 receptors. Hence these enantiomers bind preferentially at 5 HT 2 receptors decreasing off target binding. The next series of enantiom ers that were tested at H 1 receptors had substituents at para position. (+) and T rans p CF 3 PAT enantiomers had an affinity of 230 35 nM and 5100 360 nM re spectively ( p <0.0001). These enantiomers had the same trend as other para substituted enantiomers at 5 HT 2 receptors, in that they demonstrate reversed stereoselectivity. Comparing the meta versus para trifluoromethyl substituted analogs revealed significant higher affinity for (+) t rans p CF 3 PAT and t rans m CF 3

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119 PAT enantiomers relati ve to (+) t rans m CF 3 PAT and t rans p CF 3 PAT enantiomers respectively at H 1 receptors ( p <0.0001). There was no significant difference in affinity of (+) t rans p CF 3 PAT at H 1 receptors relative to its affinity at all other 5 HT 2 receptors. The last analog that was analyzed at H 1 receptors was a racemate of ( ) trans p NO 2 PAT. The affinity of this enantiomer at H 1 receptors was found to be 680 31 nM. This racemate had a significantly higher affinity at H 1 receptors relative to other 5 HT 2 receptors (F3,9=93.61; p <0.0001).

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120 APPENDIX B LIST OF PUBLICATIONS RESULTING FROM THE WORK IN THIS DISSERTATION Morgan D, Canal CE, Kondabolu K, Sakhuja R, Robertson K, Rowland NE, Booth RG. A Novel Serotonin 2 (5 HT2) Modulator as a Can didate Drug to Treat Impulsive Behavioral Disorders and Psychoses without Weight Gain as a Side Effect. Neuropsychopharmacology 2012;38:S104 105. Cordova Sintjago T, Sakhuja R, Kondabolu K, Canal CE, Booth RG. Molecular determinants for ligand binding at serotonin 5 HT2A and 5 HT2C GPCRs: Experimental affinity results analyzed by molecular modeling and ligand docking studies. Int. Journal Quantum Chemistry 2012 ; 112: 3807 3814. Morgan D, Kodabolu K, Kuipers, A, Sakhuja R, Robertson K, Rowland NE, Booth RG Molecular and behavioral pharmacology of two novel orally active 5HT2 modulators: potential utility as antipsychotic medications. Neuropharmacology. 2013, submitted. Zhuming S and Kondabo lu K (equal), Cordova Sintjago T Canal C, Travers S, Kim MS, Fels ing D, Booth RG. Novel 4 Aryl 6,7 Substituted N,N dimethyl 2 aminotetralins: Synthesis and Binding at Serotonin 5 HT2 type and Histamine H1 G Protein Coupled Receptors with In Silico Docking Studies Journal of Medicinal Chemistry, 2013, in preparation. Sakhuja R and Kondabo lu K (equal), Cordova Sintjago T Zhuming S, Canal C, Travers S, Fang L, Kim MS, Vincek A, Abboud KA, Booth RG. Novel 4 substituted phenyl and cycloalkyl 2 dimethylaminotetralins: Synthesis and Binding Affinity at Serotonin 5 HT2 type and Histamine H1 G Protein Coupled Receptors with In Silico Docking Studies Journal of Medicinal Chemistry, 2013, in preparation.

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121 Kondabolu K, Morgan D Felsing D, Sakhuja, R, Booth RG. A novel serotonin 5 HT2C agonist with 5 HT2A/2B antagonist/inverse ag onist activity for schizophrenia: Characterization of the molecular pharmacology of 4 ( meta chloro) phenyl N,N dimethyl 1,2,3,4 tetrahydronaphthalene 2 amine (meta Cl PAT) and its efficacy in three rodent models of psychosis. Neuopharmacology. 2013, in pr eparation. Kondabolu K, Sakhuja R, Morgan D, Booth RG. Preclinical antipsychotic efficacy of novel p henyl a mino t etralin (PAT) analogs acting at serotonin 5 HT 2 receptors. At College of Pharmacy Research Day, University of Florida, 2012. Booth RG, Canal CE, Kondabolu K, Strehler KE, Cordova Sintjago T, Kim MS, Morgan D. Development of Serotonin 5 HT 2C agonists with 5 HT 2A/2B antagonist activity as antipsychotics without weight gain liability. At 47th International Conference on Medicinal Chemistry. Lyon, Fran ce 2011. Morgan D, Kondabolu K, Kuipers A, Canal CE, Booth RG. Behavioral and pharmacological effects of two novel serotonin 5 HT modulators. At annual meeting of the College on the Problems of Drug Dependence: Palm Springs, CA, 2012. Sakhuja R, Kondabolu K, Canal CE, Cordova Sintj ago T, Booth RG. Synt hesis and binding affinities of trans m substituted p henyl a mino t etralins (PAT) against 5 HT 2A and 5 HT 2C serotonin receptors. Morgan D, Kondabolu K, Sakhuja R, Kuipers A, Booth RG. Development and preclinical evaluation of the antipsychotic efficacy of novel 5 HT 2 modulators. At University of Florida College of Medicine Celebration of Research Day: Gainesville, 2012.

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122 Cordova Sintjago T, Sakhuja R, Kondabolu K, Villa N, Canal CE, Booth RG. Molecular determinan t s for binding and function at s erotonin 5 HT 2A and 5 HT 2C GPCRs: Ligand docking, molecular dynamic and QM studies. 52nd Sanibel symposium, 2012. Canal CE Morgan D Kondabolu K Rowland N and Bo oth RG. A new potent and specific 5 HT2C receptor agonist wit h preclinical anti psychotic and anti obesity properties. Experimental Biology, Boston, 2013.

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135 BIOGRAPHICAL SKETCH Krishnakanth Kondabolu was born in 1987, to Harischandra Prasad and Durga Kondabolu, in Andhra Pradesh, India and has a younger brother Srikanth. He attended Smt. SulochanaDevi Singhania School in Mumbai, Maharastra. After graduation, he was accepted in to one of the premier universities of India, Manipal University. He graduated in 2008 with a Bachelor of Pharmacy degree from Manipal College of Pharmaceutical Sciences. As an undergraduate he took interest in the drug discov ery process, which prompted him to pursue doctoral degree in the Department of Medicinal Chemistry at University of Florida in 2008. He was mentored by Dr. Raymond Booth and completed his Doctor of Philosophy from University of Florida in May 2013.