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Novel Phenylaminotetralin (PAT) Analogs

Permanent Link: http://ufdc.ufl.edu/UFE0042056/00001

Material Information

Title: Novel Phenylaminotetralin (PAT) Analogs Multifunctional Serotonin 5HT2 Receptor Drugs for Neuropsychiatric Disorders
Physical Description: 1 online resource (118 p.)
Language: english
Creator: Sun, Zhuming
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 5ht2c, agonist, neurodisorders
Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: NOVEL PHENYLAMINOTETRALIN (PAT) ANALOGS: MULTIFUNCTIONAL SEROTONIN 5HT2 RECEPTOR DRUGS FOR NEUROPSYCHIATRIC DISORDERS By Zhuming Sun August 2010 Chair: Raymond Booth Major: Pharmaceutical Science Medicinal Chemistry This Ph.D. thesis research describes drug discovery targeting serotonin 5HT2 G protein-coupled receptor (GPCR) subtypes. Brain 5HT2C receptor activation in humans leads to anti-obesity effects, antipsychotic effects, attenuation of psychostimulant addiction, and other psychotherapeutic effects. Meanwhile, brain 5HT2A receptor activation produces hallucinogenic effects and activation of peripheral 5HT2B receptors produces cardiac valvulopathy and pulmonary hypertension. Until our recent publication, there was no report of a 5HT2C receptor agonist that does not also activate 5HT2A and/or 5HT2B receptors. Thus, clinical 5HT2C receptor-based pharmacotherapy was hampered. We reported (-)-trans-N,N-dimethyl-4-phenyl-1,2,3,4-tetrahydro-2-naphthalenamine (phenylaminotetralin; PAT) as a full-efficacy agonist at human 5HT2C receptors and inverse agonist at 5HT2A and 5HT2B receptors. In addition to selective activation of the 5HT2C receptor, the pharmacotherapeutic potential of (-)-trans-PAT is promising given that 5HT2A inverse agonists are used clinically as antipsychotic drugs. Thus, the general goals of these Ph.D. thesis studies included facile routes for scale-up synthesis of ( )-trans-PAT for preclinical in vivo studies in rodents, as well as, design and synthesis of PAT analogs with enhanced selective 5HT2C agonist potency and/or more potent 5HT2A and/or 5HT2B inverse agonist activity. In addition to development of pharmacotherapy for neuropsychiatric disorders and obesity, results are expected to help delineate 5HT2 GPCR structure and molecular requirements for activation for drug design purposes
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 Zhuming Sun.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Booth, Raymond.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042056:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042056/00001

Material Information

Title: Novel Phenylaminotetralin (PAT) Analogs Multifunctional Serotonin 5HT2 Receptor Drugs for Neuropsychiatric Disorders
Physical Description: 1 online resource (118 p.)
Language: english
Creator: Sun, Zhuming
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 5ht2c, agonist, neurodisorders
Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: NOVEL PHENYLAMINOTETRALIN (PAT) ANALOGS: MULTIFUNCTIONAL SEROTONIN 5HT2 RECEPTOR DRUGS FOR NEUROPSYCHIATRIC DISORDERS By Zhuming Sun August 2010 Chair: Raymond Booth Major: Pharmaceutical Science Medicinal Chemistry This Ph.D. thesis research describes drug discovery targeting serotonin 5HT2 G protein-coupled receptor (GPCR) subtypes. Brain 5HT2C receptor activation in humans leads to anti-obesity effects, antipsychotic effects, attenuation of psychostimulant addiction, and other psychotherapeutic effects. Meanwhile, brain 5HT2A receptor activation produces hallucinogenic effects and activation of peripheral 5HT2B receptors produces cardiac valvulopathy and pulmonary hypertension. Until our recent publication, there was no report of a 5HT2C receptor agonist that does not also activate 5HT2A and/or 5HT2B receptors. Thus, clinical 5HT2C receptor-based pharmacotherapy was hampered. We reported (-)-trans-N,N-dimethyl-4-phenyl-1,2,3,4-tetrahydro-2-naphthalenamine (phenylaminotetralin; PAT) as a full-efficacy agonist at human 5HT2C receptors and inverse agonist at 5HT2A and 5HT2B receptors. In addition to selective activation of the 5HT2C receptor, the pharmacotherapeutic potential of (-)-trans-PAT is promising given that 5HT2A inverse agonists are used clinically as antipsychotic drugs. Thus, the general goals of these Ph.D. thesis studies included facile routes for scale-up synthesis of ( )-trans-PAT for preclinical in vivo studies in rodents, as well as, design and synthesis of PAT analogs with enhanced selective 5HT2C agonist potency and/or more potent 5HT2A and/or 5HT2B inverse agonist activity. In addition to development of pharmacotherapy for neuropsychiatric disorders and obesity, results are expected to help delineate 5HT2 GPCR structure and molecular requirements for activation for drug design purposes
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 Zhuming Sun.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Booth, Raymond.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042056:00001


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1 NOVEL P HENYL A MINO T ETRA LIN (PAT) ANALOGS : MULTIFUNCTIONAL SEROTONIN 5HT 2 RECEPTOR DRUGS FOR NEUROPSYCHIATRIC DISORDERS By ZHUMING SUN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FUL FILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 Zhuming Sun

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3 To my mom and d ad

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4 ACKNOWLEDGMENTS I thank Dr. Raymond Booth, as my mentor, who directed me through my graduate study. I thank Dr Margaret James, Dr. Kenneth Sloan and Dr. Drake Morgan, for their effort as my committee members. I thank Dr. Neil Rowland and Dr. Joanna Peris for their collaboration on my research projects. I thank my colleagues who taught me bench work skills, gave m e helpful suggestions and valuable information, provided me with necessary material for my experiments, and worked with me during my graduate study: Dr. Lijuan Fang, Dr. Adam Vincek, Dr. Myong Sang Kim, Dr. Clint Canal, Dr. Tania Cordova Sintjago, Dr. Nanc y Villa, Dr. Sashi Sivendren, Dr. Andrzej Wilczynski Sean Travis, and K ondabolu Krishnakanth

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 BACKGROUND AND SIGNIFICANCE ................................ ................................ ... 14 5HT2 Receptors as Drug Targets ................................ ................................ ........... 14 5HT2A, 5HT2B and 5HT2C Receptors Physiological Roles ................................ ... 14 5HT2C Receptors in Obesity ................................ ................................ .................. 15 Mechanistic Model for Serotonergic Regulat ion of Food Intake .............................. 16 5HT2A and 5HT2C Receptors in Psychiatric Disorders ................................ .......... 16 Ligands with 5HT2A Inverse Agonism and/or 5HT2C Agon ism for Psychoses, Depression, and Psychostimulant Abuse ................................ ............................ 17 Targeting the 5HT2C Receptor i n Drug Discovery ................................ .................. 19 Design of Selectiv e 5HT2C Agonists: A Brief Review of Ligand Structures and Their 5HT2 Type Activity ................................ ................................ ..................... 20 Classic nonselective 5 HT2 agonists ................................ ................................ 20 3 Su bstituted indole analogues ................................ ................................ ........ 21 N S ubstituted indole ana logues ................................ ................................ ........ 21 M CPP and piperazine analogues ................................ ................................ .... 23 Benzodiazepinoindole analogues ................................ ................................ ..... 24 Benzazepines ................................ ................................ ................................ ... 25 Design of Selective 5HT2C Agonists: ( ) Tran s PAT as a Lead Molecule .............. 27 2 SYNTHESIS OF ( ) TRANS N,N DIMETHYL 4 PHENYL 1,2,3,4 TETRAHYDRO 2 NAPHTHALENAMINE ( 30 ) ................................ ........................ 34 Rational e ................................ ................................ ................................ ................. 34 Synthesis Results and Discussion ................................ ................................ .......... 35 In vitro Pharmacological Characterization Results ................................ .................. 42 In vivo Pharmacological Characterization Results ................................ .................. 42 Discussion: ( ) Trans PAT is a 5HT2C Full Agonist with 5HT2A/2B Inverse Agonism that Shows Promise for Treatin g Obesity, Drug Abuse and Psychoses ................................ ................................ ................................ ........... 43

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6 3 SYNTHESIS OF N,N DIMETHYL 4 (4 METHYLPHENYL) 1,2,3,4 TETRAHYDRO 2 NAPHTHALENAMINE ANALOGS OF PAT ............................... 53 Rationale ................................ ................................ ................................ ................. 53 Synthesis Results and Discussion ................................ ................................ .......... 54 In vitro Pharmacological Characterization Results ................................ .................. 57 In vivo Anti Stimulant Effects and Discussion of ( ) Trans PAT and ( ) Trans p CH 3 PAT: Indication for Drug Abuse Pharmacotherapy ................................ ...... 58 In vivo Anorexia effect and Disscussion of ( ) Trans p CH 3 PAT ............................ 59 4 SYNTHESIS OF PAT ANALOGS WITH SUBSTITUTIONS ON THE TETRAHYDRONAPHTHYL AND PENDANT PHENYL. ................................ ......... 68 Rationale ................................ ................................ ................................ ................. 68 Synthesis Results and Discussion ................................ ................................ .......... 72 In vitro Pharmacological Characterization Results ................................ .................. 75 Disscussion and Future Studies ................................ ................................ ............. 7 6 A PPENDIX ................................ ................................ ................................ .................... 83 A EXPERIMENTAL PROC EDURES : SYNTHETIC CHEMISTRY .............................. 83 B EXPERIMENTAL PROCEDURES : PHARMACOLOGICAL ASSAYS .................. 101 LIST OF REFERENCES ................................ ................................ ............................. 105

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7 LIST OF TABLES Table page 1 1 List of in vitro biological data of published compounds (1) ................................ 28 1 2 List of in vitro biological data of published compounds (2) ................................ 29 2 1 ( ) Trans PAT and isomers 5 HT 2 receptors affinity. ................................ ........... 45 2 2 Functional activities of ( ) trans PAT at 5 HT 2 receptors.. ................................ ... 45 3 1 ( ) Trans p methyl PAT i somers 5HT2 receptors affinity ................................ .... 61 3 2 Functional ac tivities of ( ) t rans p methyl PAT at 5HT2 r eceptors ...................... 61 4 1 Preliminary binding affinities of PAT a nalogs 66,67,68,69 at 5HT2C r eceptor s 79

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8 LIST OF FIGURE S Figure page 1 1 Structures of some published 5HT2 agonists ................................ ..................... 30 1 2 S (+) Fenfluramine triggers 5HT release, leads to 5HT2C receptors activation in arcuate hypothalamic nucleus, regulates downstream melanocortinergic signaling ................................ ................................ ................ 30 1 3 Classic nonselective 5HT2 agonists ................................ ................................ ... 31 1 4 3 Substituted indole analogues ................................ ................................ .......... 31 1 5 Molecular modeling comparing structural similarities between ( ) trans PAT and 1 methylpsilocin ................................ ................................ ........................... 31 1 6 N substituted indole analogues ................................ ................................ .......... 32 1 7 Molecular modeling comparing structural similarities between ( ) trans PAT, and Ver 2692 ................................ ................................ ................................ ...... 32 1 8 M CPP and piperazine analogues ................................ ................................ ...... 32 1 9 Molecular modeling comparing structural similarities between ( ) trans PAT and WAY 161503 ................................ ................................ ............................... 33 1 10 Benzodiazepinoindole analogues ................................ ................................ ....... 33 1 11 molecular modeling comparing structural similarities between ( ) trans PAT and WAY 163909 ................................ ................................ ............................... 33 1 12 Benzazepines ................................ ................................ ................................ ..... 33 2 1 Summary of ( ) trans PAT synthetic routes ................................ ........................ 45 2 2 Retrosynthetic analysis of diastereomer recrystallization strategy ...................... 45 2 3 Synthesis of () trans 2 amino 4 phenyl 1,2,3,4 tetrahydronaphthalene 28 ...... 46 2 4 Formation of 2 tetralone 23 ................................ ................................ ................ 46 2 5 NaBH 4 reduction to prepare 2 tetralol 24 ................................ ............................ 46 2 6 Resolution o f () trans 1 phenyl 3 amino 1,2,3,4 tetrahydronaphthalene 28 .... 47 2 7 ) trans pat resolution ................................ ............. 47 2 8 Conversion of pure salts to final product 30 ................................ ....................... 48

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9 2 9 (+) DIP chloride failed to afford (2 R ,4 R ) cis tetralol 35 ................................ ....... 48 2 10 Brominatio n Suzuki coupling failed to introduce phenyl group to C4 of tetralol 38 ................................ ................................ ................................ ....................... 48 2 11 Jacobsen epoxidation yielded mainly trans tetralol ................................ ............ 49 2 12 Stereochemistry of Jacobsen epoxidation on dihydronaphthalene 41 ................ 49 2 13 Representative concentration response curve for serotonin and ( ) trans PAT activation of PLC/ [ 3 H] IP form ation in HEK cells expressed cloned human 5HT2C receptors.. ................................ ................................ .................. 50 2 14 Representative data for ( ) trans PAT inverse agonist activity at cloned human 5HT2A receptors ................................ ................................ .................... 50 2 16 Dose effect curve after i.p. administration of ( ) trans PAT vs. the non selective 5HT2A/2B/2C agonist WAY161503 on 30 min intake of platatable food by mice ................................ ................................ ................................ ....... 51 2 17 No tolerance to ( ) trans PAT anorectic effect with chronic administration ......... 52 2 18 ( ) Trans PAT in modulating amphetamine induced locomotion ......................... 52 3 1 Synthesis of ( ) trans p CH 3 PAT 48 ................................ ................................ .. 61 3 2 Asymmetric reduction with RuCl 2 Ph 2 and Noyori ligand NAPT to prepare 2 tetralol 44 ................................ ................................ ................................ ............ 62 3 3. ) trans p CH 3 PAT resolution ................................ 62 3 4 Synthesis of (+) cis and ( ) cis p CH 3 PAT(54 and 55) ................................ ....... 63 3 5 Synthesis of (+) trans p CH 3 PAT 57 ................................ ................................ 63 3 6 Representative concentration response curves for p CH 3 PAT isomers displacement of [ 3 H] ketanserin from 5HT2A receptor ................................ ....... 64 3 7 Representative concentration response curves for p CH 3 PAT isomers displacement of [ 3 H] mesulergine from 5HT2B receptor ................................ .... 64 3 8 Representative concentration response curves for p CH 3 PAT isomers displacement of [ 3 H] mesulergine from 5HT2C receptor ................................ .... 65 3 9 Representative data for ( ) trans p CH 3 PAT compa red to ( ) trans PAT inverse agonist activities at cloned human 5HT2A receptors expressed in HEK Cells ................................ ................................ ................................ ........... 65

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10 3 10 Representative data for ( ) trans p CH 3 PAT compared to ( ) trans PAT inverse agonist activities at cloned human 5HT2B receptors expressed in HEK Cells ................................ ................................ ................................ ........... 66 3 11 Representative data for ( ) trans p CH 3 PAT compared to ( ) trans PAT agonist activities at cloned human 5HT2 C receptors expressed in HEK cells .... 66 3 12 ( ) Trans p CH 3 PAT compared to ( ) trans PAT in modulating amphetamine induced locomotion ................................ ................................ ............................. 67 3 13 Single dosage ( ) trans p CH 3 PAT in modulating amphetamine and methamphetamine induced locomotion ................................ .............................. 67 4 1 Preliminary in vitro characterization results of 6 OH,7 Cl PATs and 6 OH 7 OH PATs ................................ ................................ ................................ ............ 79 4 2 Synthesis of N,N dimethyl 4 phenyl 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine 58,59,60,61 ................................ ................................ ......... 79 4 3 Synthesis of N,N dimethyl 4 (3 chlorophenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine 62,63,64,65 ................................ .................... 80 4 4 Synthesis of trans N,N dimethyl 4 (3 bromophenyl) 6 methoxy 7 chlor o 1,2,3,4 tetrahydro 2 naphthalene amine 66,67 ................................ .................. 80 4 5 Synthesis of Trans N,N dimethyl 4 (3 chlorophenyl) 6 methoxy 1,2,3,4 tetrahydro 2 naphthalene amine 68,69 ................................ .............................. 81 4 6. Representative concentration response curves for trans 6 OMe 7 Cl Br PAT isomer (66, 67) displacement of [ 3 H] mesulergine from 5HT2C receptors ......... 81 4 7. Representative concentration response curves for trans 6 OMe Cl PAT isomer (68, 69) displacement of [ 3 H] mesulergine from 5HT2C receptors ......... 82 4 8 Representative concentration response cu rve for serotonin and ( ) trans 6 OMe Cl PAT 68 activation of PLC/ [ 3 H] IP Formation in clonal cells expressed cloned Human 5HT2C receptors ................................ ....................... 82

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11 LIST OF ABBREVIATION S 5HT 5 hydroxytryptamine serotonin M elanocorti n stimulating hormone BAC bovine adrenal chromaffin cAMP cyclic monophosphate CAR C onditioned avoidance response CMTB 8,9 dichloro 1 methyl 2,3,4,5 tetrahy dro 1H benzo[d]azepine DAG D iacylglycerol DOI 2,5 dimethoxy 4 iodoamphetamine EE enan tiomeric excess GPCR G protein coupled receptor IP3 Inositol trisphosphate LSD D lysergic acid diethylamide MCR M elanocortin receptors M CPP M chlorophenyl piperazine NAPT ( R R ) N (2 amino 1,2 diphenylethyl) p toluenesulfonamide OCD Obsessive compulsive di sorder PAT Phenylaminotrtralin; ( ) T rans N,N dimethyl 4 phenyl 1,2,3,4 t etrahydro 2 naphth alenamine ( ) trans PAT PCC pyridinium chlorochromate PLC P hospholipase C PPA polyphosphoric acid R.T. room temperature TBDMS t ert butyldimethylsilyl chloride TM D Transmembrane domain

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12 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 NOVEL P HENYL A MINO T ETRA LIN (PAT) ANALOGS : MULT IFUNCTIONAL SEROTONIN 5HT 2 RECEPTOR DRUGS FOR NEUROPSYCHIATRIC DISORDERS By Zhuming Sun August 2010 Chair: Raymond Booth Major: Pharmaceutical Science Medicinal Chemistry This Ph.D. thesis research describes drug discovery targeting serotonin 5HT2 G p rotein coupled receptor (GPCR) subtypes. Brain 5HT2C receptor activation in humans leads to anti obesity effects, antipsychotic effects, attenuation of psychostimulant addiction, and other psychotherapeutic effects. Meanwhile, brain 5HT2A receptor activati on produces hallucinogenic effects and activation of peripheral 5HT2B receptors produces cardiac valvulopathy and pulmonary hypertension. Until our recent publication there was no report of a 5HT2C receptor agonist that does not also activa te 5HT2A and/or 5HT2B receptors. T hus, clinical 5HT2C receptor based pharmacother apy was hampered. We reported ( ) trans N,N dimethyl 4 phenyl 1,2,3,4 t etrahydro 2 naphthalenamine ( p henyl aminotetralin ; PAT) as a full efficacy agonist at human 5HT 2C receptors and inverse agonist at 5HT2A and 5HT2B receptors. In addition to selective activation of the 5HT2C receptor, the pha rmacotherapeutic potential of ( ) t rans PAT is promising given that 5HT2A inverse agonists are used clinically as antipsychotic drugs. Thus, the general goals of these Ph.D. thesis studies included facile routes for scale up synthesis of ( ) t rans PAT for preclinical in vivo studies in rodents, as well as, design

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13 and synthesis of PAT analogs with enhanced selective 5HT2C agonist potency and/or more potent 5HT2A and/or 5HT2B inverse agonist activity. In addition to development of pharmacotherapy for neuropsychiatric disorders and obesity, results are expected to help delineate 5HT2 GPCR structure and molecular requirements for activation for drug design pur poses.

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14 CHAPTER 1 BACKGROUND AND SIGNI FICANCE 5HT2 R eceptors as D rug T arget s The biogenic amine Serot onin (5 hydroxytryptamine, 5HT, Fig. 1 1 ) regulates a wide range of central and peripheral psychological and physiological effects through activation of f ourteen mammalian 5HT receptor subtypes that are grouped into the 5HT 1 5HT 7 families. The 5HT2 receptor class has three subtypes: 5HT2A, 5HT2B and 5HT2C (Roth et a l.,1998; Sanders Bush et al. ,2006). They associate with q before activate phospholipase (PL ) C This enzyme hydrolyses phospholipids, yielding inositol phosphates and diacylglycerol (DAG). Inositol trisphosphate (IP3) acts to liberate Ca 2+ from intracellular stores, resulting in depolarization of the neuron. In addition, DAG activates protein ki nase C, which can indirectly modulate the activity of ion channels (Raymond et al., 2001; Turner & Raymond, 2006, Lam et al., 2007 ). 5HT2A 5HT2B and 5HT2C R eceptors P hysiological R oles 5HT2A receptors are broadly distributed in CNS. Hallucinogens s uch as D lysergic acid diethyla mide (LSD) exert their psychedelic effect mainly by activation of 5HT2A receptors (Glennon, 1990; Sanders Bush et al., 2006). Meanwhile, 5HT 2A antagonist/inverse agonist activity is shared by most atypical antipsychotics (e.g ., clozapine, olanzapine, ziprazidone) and is thought to partially explain their therapeutic properties in schizo phrenia (Weiner et al., 2001; S hapiro et al., 2003; Roth et al., 2004; Davies et al., 2004). 5HT2B receptor mRNA and protein are also present i n human brain ( Kursar et al., 1994) although its role in the CNS remains unclear. Peripherally 5HT 2 B activation can lead to valvular heart disease (Fitzgerald et al., 2000; Rothman et al., 2000; Roth, 2007) and pulmonary hypertension ( Launay et al., 2002) 5HT2B

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15 receptor was first identified in rat stomach fundus (Foguet et al., 1992). In the 5HT2B selective antagonist s were regarded as tool for the treatment of irritable bowel syndrome (IBS) but recent publications on this field are limited (Wa insc ott et al., 2004, Giorgioni et al., 2005). The human 5HT 2C receptor ( Lubbert et al., 1987 Saltzman et al., 1991) is found exclusively in the central nervous system where it is widely expressed and putatively involved in several (patho) physiologic al and p sychological processes i.e. ingestive behavior (Tecott et al., 1995), psychosis and response to schizophrenia pharmacotherapy (Reynolds et al., 2005 ; Siuciak et al., 2007; Marquis et al., 2007 ), motor function (Heisler and Tecott, 2000; Segman et al., 2000 ), cocaine addiction ( Bubar and Cunningham, 2006; Muller and Huston, 2006), anxiety (Sard et al., 2005 Heisler et al., 2007), depression (Palvimaki et al., 1996 ; Rosenzweig Lipson et al., 2007 ), epilepsy (Heisler et al., 1998 ), and sleep homeostasis (Frank et al., 2002). Contemporary receptor theory classifies ligands as agonists, inverse agonists and antagonists. Constitutive activity of a receptor is defined as its ability to activate cellular signaling pathways in the absence of an agonist (L eff et al. 1997). In several in vitro systems 5HT2A and 5HT2C receptors dem onstrate constitutive activities and inverse agonism (Aloyo et al., 2009), but the role of constitutive activity in vivo is not clear (Li et al., 2009). 5 HT2C R eceptors in O besity There are many studies documenting the importance of 5HT2C receptor regulation of body weight in rodents and humans (Bickerdike et al., 1999). N on selective 5HT 2 agonists ( Fig 1 1 ) such as m CPP and RO 60 0175 are known to reduce food intake and lead to weight loss in rodents ( Halford et al., 2005) and t he anti obesity effects are diminished if a 5HT2C antagonist is pre administered (Schreiber et al., 2002) The

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16 5 HT2C knockout mouse demonstrate s increased feeding and obesity, and, resistance to the anorectic effects of S (+) fenfluramine (Tecott et al., 1995; Vickers et al., 1999; 2001; Heisler et al., 2002). T he now banned weight loss drug S (+) fenfluramine ( d fenfluramine), produces sustained weight loss of about 10% in humans (Tecott et al., 1995; Mccann et al., 19 97; Vickers et al., 2001). S (+) and () F enfluramine promote serotonin release (Rothman et al., 1999) and both were banned by the US Food and Drug Administration in 1997 because fenfluramine s and the metabolite S (+) norfen fluramine cause activation of 5 HT 2B receptors that can lead to valvular heart disease (Fitzgerald et al., 2000; Setola et al., 2005) and/or pulmonary hypertension (Launay et al., 2002) fatalities have resulted. Other 5HT2C agonists continue to be developed as weight loss drugs, includ ing l orcaserin ( Smith et al., 2008; Thomsen et al., 2008) M echanistic M odel for S erotonergic R egulation of F ood I ntake The 5HT 2C receptor is high ly express ed in the arcuate nucleus of the hypothalamus, an area known to be important for appetite and feedi ng. The 5HT2C receptor exerts regulatory control of melanocor tin signaling. Stimulation of 5 HT 2C receptors by indirect agonists such as S (+) fenfluramine ind melanocortin (MCR) 3 and 4 to alter energy homeostasis. This circuit is modeled in Fig 1 2 (Heisler et al., 2002, 2007 ). 5HT2 A and 5HT2C R eceptors in P sychiatric D isorder s Serotonergic neurons innervate virtually all parts of the central nervous system. In the v entral tegmental area and substantia nigra, dopamine (DA) neurons receive projections from serotonin containing cell bodies (Herve et al, 1987; Hoyer et, al., 1994)

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17 The precise elucidation of the interaction between 5HT and DA systems, as well as the pharmacological evaluation is an ongoing task, some recent reviews are listed here ( Esposito 2006; Fink et al., 2007; Gruender et al, 2009). A ntagonist/inverse agonist activity at 5H T2A receptor is shared by most atypical a ntipsychotics (e.g., cloza pine, olanzapine, ziprazidone) and partially contributes to their therapeutic pro perties in schizophrenia ( Weiner et al., 2001; Schapiro et al., 2003; Roth et al., 2004; Davie s et al., 2004). In contrast, agonist activity at 5HT2A receptors is displayed by hallucinogenic drugs such as lysergic diethylamide (LSD), psilocybin and mescaline .The 5HT 2A receptor signaling is necessary for their psychotomimetic properties (Nichols, 20 04). It is proposed that antipsychotic drugs with enhanced 5HT2A receptor antagonist/inverse agonist activity compared to dopamine D2 antagonist activity may cause less extrapyramidal movement disorder side effects ( Horacek et al., 2006). Historically, li ttle attention was paid to specific interactions of 5HT2C receptors and antipsychotic clinical agents. It was revealed later that some atypical as well as some conventional antipsychotics in fact, have high affinity at 5HT2C receptors ( Horacek et al., 200 6). Research on 5HT2A/2C receptors as potential antipsychotic drug targets currently is focused on 5HT2A inverse agonist s, 5HT2C agonist s and ligands with both 5 HT2A/2C inverse agonist activities Ligands w ith 5HT2A I nverse A gonism and/or 5HT2C A gonis m fo r Psychoses Depression, and Psychostimulant Abuse The mRNA for 5HT2C receptors is abundant in the nucleus accumbens and ventral tegmentum which are limbic system structures that integrate emotional function.

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18 Recent literature suggests 5HT2C receptor s in the limbic system may be involved in symptoms of psy chosis, depression and psychostimulant addiction ( Eltayb et al., 2007; Marquis et al., 2007). For example, non selective 5HT2C receptor agonists such as m chlorophenyl piperazine ( m CPP, 10 ) and RO 60 0 175 ( 7 ) have been reported to show antipsychotic like effects in animal models of schizophrenia (Browning et al.,1999; Grauer et al.,2004). Recently, WAY163909 ( 16 ), a 5HT2C agonist, showed antipsychotic and anti depressant activity in several rodent model s (Dunlop et al., 2006; Marquis et al., 2007 ). Another 5HT2C agonist CP809 101 ( 12 ) also was reported to improve cognitive function associated with schizophrenia in animal models (Siuciak et al., 2007 ). Some conventional antipsychotics (chloropromazine, me soridazine and loxapine) have high affinities for 5HT2C receptors (Horacek et al., 2006). Blockade of 5HT2 receptors along with dopamine D2 receptors has been proposed as a strategy for antipsychotic drug design (Meltzer et al., 2003, 2004). However, studi es have revealed that 5HT2C antagonism or inverse agonism elevates limibic dopamine levels. In animal models (Di Matteo et al., 2001), 5HT2C antagonism that increases dopamine concentration in brain produces hyperlocomotion that correlates with psychotic t ype activity. Also, animals receiving 5HT2C antagonis t s show disfunction in information processing (Hutson et al., 2000). Predictably, 5HT2C blockade also is directly associated with weight gain as an adverse effect of some antipsychotics (Ellingrod et al. 2005; Miller et al., 2005; 2009). Nevertheless, some drug s with inverse activity at both 5HT2A and 2C receptors have demonstrated efficacy for certain psychoses. For example, the 5HT2A/2C inverse

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19 agonist ACP 103 (pimavanserin) entered Phase III trials f or the treatment of psychoses associated w ith Parkinson's disease. H owever, results did not meet expectations (not specified) and the study was discontinued (Clinical trials, 2009). There is also preclinical evidence that 5HT 2C receptors modulate psychosti mulant effects. For example, cocaine and amphetamine induced locomotor activity in rats is blocked b y 5HT 2C agonists (Grottick et al, 2000 ). In contrast, 5HT 2C antagonists enhance locomotor stimulant effects induced by amphetamine and other drugs that release and/or inhibit reuptake of dopamine (Fletcher, 2006 ) The effect of 5HT 2C antagonists on psychostimulant effects and baseline locomotion correlates with th eir effect on dopamine efflux and dopamine neuronal firing. On the other hand, 5HT 2C receptor agonists produce the opposite effect suppressing dopamine release and dopamine neuronal firing (Weiner, 2001) In other studies, the non selective 5HT2 agonist RO 60175 (Porter et al., 1999), reduc es the rate of cocaine self administration in rats and this effect is blocked selectivel y by the 5HT 2C antagonist SB2420 84 (Bromidge et al., 1997). Correspondingly SB2420 84 increases the rate of cocaine self administratio n in rats in a dose dependent manner (Fletcher et al., 2002). Targeting t he 5HT2C Receptor i n Drug Discovery As indicated above, it has been recognized for about 10 years that the 5HT2C receptor holds great promise as a pharmacotherapeutic target for neur ops ychiatric disorders and obesity. H owever, 5 HT2C receptor selective drugs still are not available T he biggest challenge regarding drug discovery targeting the 5HT 2C receptor is that this GPCR shares a transmembrane domain (TMD) sequence identity of abou t 80% with the 5HT 2A receptor and about 70% with the 5HT 2B receptor ( Julius et al., 1988;

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20 1990). The highly conserved TMDs and similar second messenger coupling has made development of ligands selective for the 5HT 2C receptor especially difficult As menti oned activation of 5HT2A receptors induce s LSD (d lysergic acid diethylamide) like hallucination and stimulant effects. Activation of peripheral 5HT2B receptors leads to valvular heart disease and pulmonary hypertension, as is the case of the indire ct and nonselective 5HT agonist S (+) fenfluramine. Thus, for clinical purposes, there is no tolerance for activation of 5HT2A and/or 5HT2B receptors absolutely selective activation of 5HT2C is required. Design o f Selective 5HT2C Agonists: A Brief Review o f Lig and Structures a nd Their 5HT2 Type Activity In the absence of X ray crystal structure data for any of the serotonin 5HT2 GPCRs, 5HT2C agonist drug design has focused on a ligand based approach. This section includes a brief summary of the compounds reporte d in the literature as the candidate selective 5HT2C agonists over the last 10 years. Structures are shown in Fig 1 3 1 10 and in vitro pharmacological data are summarized in Table 1 1 1 2. The discussion focuses on comparing the structures of putativ e 5HT2C agonist s described in the literature with ( ) trans N,N dimethyl 4 phenyl 1,2,3,4 t etrahydro 2 naphthalenamine (1 p henyl 3 dimethyl a mino t etralin; PAT; 30 ), the only ligand reported so far that demonstrates full efficacy agonist activity at human 5H T2C receptors while showing inverse agonist activity at 5HT2A and 5HT2B receptors i.e., ( ) trans PAT is an absolutely selective 5HT2C agonist (Booth et al., 2009) Classic N onselective 5HT2 A gonists The e ndogenous agonist 5HT ( 1 ), of course, is non selec tive and activates all 5HT receptors. Meanwhile, DOI (2,5 dimethoxy 4 iodoamphetamine, 2 ) i s the archetype of

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21 agonist molecules that target 5HT2 receptors, albeit, nonselectively. As mentioned, S (+) norfenfluramine ( 3 ) is the major metabolite of the indir ect 5HT agonist S (+) fenfluramine and, shows highest agonist potency at 5HT2B compared to 5HT2A and 5HT2C receptors These agonist ligands all possess the highly flexible arylethylamine motif T he amine moiety of ( ) trans PAT ( 30 ) and its analogs, h owev er, is attached equatorially to the tetrahydronaphthalene (Wyrick et al 1993), that greatly restricts flexibility. 3 Substituted I ndole A nalogues Tryptamine ( 4 ) i s a non selective 5HT2 agonist and the structural analog BW723C86 ( 5 ) also activates all three 5HT2 receptor types ( Porter et al; 1999). The tryptamine analog 1 methylpsilocin ( 6 ) is a member of a group of derivatives called psiloci n s I t i s a partial agonist at 5HT2A and 5HT2C receptors In a mouse model of obsessive compulsive disorder ( OCD ), 1 methylpsilocin reduced scra tching after IP administration, an effect attributed t o in vivo 5HT2C agonism (Sard et al.,2005). Molecular modeling was performed to closely compare the structural simila rities between 1 methylpsilocin and ( ) trans PAT ( Fig 1 5 Wilczynski and Booth, unpublished data 2009). 1 Methylpsilocin and ( ) trans PAT share structural overlap when the flexible amine side chain of 1 methylpsilocin is in the energy minimized conformation. The absence of a pendant phenyl group in the 1 methylpsilocin compound may explain its partial agonist activity at 5HT2C receptors in comparison to ( ) trans PAT, which, is a full efficacy agonist at 5HT2C receptors (Booth et al., 2009). N substituted I ndole A nalogues RO600175 ( 7 ) was originally report ed as a selective 5HT2C agonist (Martin et al., 1998). Subsequently, it has been determined that RO600175 also is a 5HT2B full

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22 efficacy agonist ( Porter et al., 1999). P yrroloisoquinoline type derivatives of RO600175 having an N propylamine substituted ind ole core structure recently were synthesized and reported (Adams et al., 2006) Ver 2692 ( 8 ) was reported as a potent 5HT2C agonist However, it also activates 5HT2A and 5HT2B receptors In the original report, Ver 2692 was orally administered to rats an d the author observed significant food intake reduction but, no data was presented (Adams et al., 2006). Th e amine moiety of Ver 2692 in the energy minimized conformation and the dimethylamine moiety of ( ) trans PAT superimposed very closely ( Fig 1 7 ; Wilczynski and Booth, unpublished data 2009) Given the very high affinity of Ver 2692 for the 5HT2C receptor (Ki=2nM ), the preliminary molecular modeling data suggests the PAT amine moiety already is at t he optimal orientation held fixed by the tetrahydr onaphthyl scaffold. Although Ver 2692 has high 5HT2C affinity and efficacy, it does not have 5HT2 subtype selectivity the notable absence of a pendant phenyl ring in the Ver 2692 molecule that is equivalent to the PAT phenyl ring may account for the lack o f 5HT2 selecti vity of Ver 2692. Also, the Ver 2692 flexible propylamino sidechain may interact with 5HT2 receptors in a conformation different than the global energy minimum conformation, perhaps, contributing to relatively poor 5HT2 selectivity profile in comparison to the more rigid ( ) trans PAT. Another series of indole derivates related to RO600175 includes YM348 ( 9 ) where the indole core i s replaced by bioisostere indazole ring system (Shimada et al., 2008). YM348 is a 5HT2A and 5HT2B agonist and a 5H T2C partial agonist. After oral administration in rats, it produces penile erection, hypo locomotion, and a tra nsient decrease in food intake these effects are blocked by a 5HT2C antagonist (Kimura et

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23 al., 2004; Hayashi et al., 2005). YM348 is not selectiv e and activates 5HT2A and 5HT2B receptors as well as 5HT2C receptors. In summary, the N substituted indole ring and its bioisostere systems are not suitable as scaffolds for development of selective 5HT2C agonists. M CPP and P iperazine A nalogues Meta chlo rophenylpiperazine ( m CPP 10 ) i s a classic non selective 5HT2 agonist A new series of 5HT2C agonist s based on the m CPP scaffold was synthesized by fusing the piperazine and aryl rings using ethylene as bridge. It was reported that compound 11 red uced foo d intake in Wistar rats (Rover et al., 2005). Other compounds structura l l y related to m CPP that recently emerged from a high throughput screening study include CP 809, 901 ( 12 ). CP 809, 901 i s a potent 5HT2C full agonist (Siuciak et al., 2007). CP 809,901 i s active in an animal model of cognitive function, but i s inactive in two animal models of antidepressant like activity. In addition to 5HT2C receptors, CP 809, 901 also activates 5HT2A and 5H 2B receptors. T hus hallucination and valvular heart disease sid e effects are predicted to occur with in vivo administration in humans accordingly drug development of CP 809,901 was discontinued (Liu et al., 2010). Another m CPP derivative WAY161503 ( 13 ) was reported in 2006 ( Rosenzweig et al). The tricyclic core s tructure could be viewed as an amide bridge fusing t he aryl and piperazine groups WAY161503 is an agonist both at 5HT2A and 5HT2B receptors. The affinity of WAY16150 3 for 5HT2A, 5HT2B, and 5HT2C receptors (Ki ~20, 60, and 30 nM) in comparison to ( ) t rans PAT (Ki ~400, 1,000, and 40 nM) indicates the WAY compound ha s much higher affinity at 5HT2A and 5HT2B receptors, and about equivalent affinity at 5HT2C receptors. The S enantiomer, WAY 16150 4 i s about 100

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24 fold less potent at activating 5HT2C receptors, with no improvement in receptor subtype selectivity. Significant overlap can be seen in alignment of ( ) trans PAT and WA Y161503 ( Fig 1 9 ; Wilczynski and Booth, unpublished data 2009) especially with regard to the PAT tetrahydronaphthyl dimethylamine an d WAY d ichlorotetrahydropyrazinoquin oxalinone moieties. Molecular modeling indicate s the NH moiety of WAY 161503 superimposes closely with N(CH 3 ) 2 group of ( ) trans PAT, so they might occupy similar 3D space in the 5HT2C active site. The most significant difference between WAY 161503 and ( ) trans PAT is the absence of a pendant phenyl m oiety in the WAY molecule. It is proposed that the PAT pendant phenyl ring provides for its selective 5HT2C agonism and 5HT2A/5HT2B antagonism (Booth et al., 2009). Benzodi azepinoindole A nalogues WAY629 ( 14 ) was identified as a 5HT2C agonist structure in high throughput screening studies (Sabb et al 2004). WAY629 contains an N ethylamine subs tituted indole motif. WAY629 has 45 fold selectivity regarding binding at th e 5HT2C versus 5HT2A receptors. In functional assays, WAY629 is an agonist at 5HT2A and 5HT2C receptor s however, it is 610 fold more potent at the 5HT2C receptor. Unfortunately, affinity and function data for WAY629 at the 5HT2B receptor s were not reported. The d i hydrogenated compound WAY162545 ( 15 ) ( racemate) and the ( R,R ) enantiomer WAY 163909 ( 16 ) were reported as 5HT2C agonists (Dunlop et al ., 2005 ). These compounds do not activate 5HT2A receptors, h owever they are 5HT2B partial agonists. WAY 163909 reduces f ood intake in normal and obese rats and this effect is blocked by 5HT 2C antagonist. In rodent models, WAY 163909 has antidepressant activity and reduces impulsivity ( Rosenzwig Lipson et al., 2007 ; Navarra et al., 2008 ). WAY 163909

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25 also activates 5HT2B rece ptors suggesting that it may cause cardiotoxicity thus, it is not suitable for development as a human therapeutic. There is not a high degree of structural similarity between ( ) trans PAT and WAY 163909 (RMS=0.520.35 ) ( Fig 1 1 1 ; Wilczynski and Booth, unpublished data 2009). The pendantcyclopentyl group of WAY 163909, held in a nearly co planar fixed conformation relative to the octahydrocyclopenta diazepinoindole nucleus, shares no structural counterpart in the ( ) trans PAT molecule. Importantly, th e cyclopentyl group, unlike the phenyl moiety of ( ) trans binding interactions with protein aromatic amino acids. Thus, the WAY 163909 pendant cyclopentyl group likely provides only steric bulk in the binding po cket of 5HT2 selectivity. Mutational analysis and molecular modeling studies of the 5HT2A receptor eties of ligands and receptor amino acids in TMDs 5 & 6 (Choudhary et al., 1993; Shapiro et al., 2000). Benzazepines Benzazepines derivatives w ere first reported in 2005 (Smith et al., 2005). The ( S ) enantiomer of 8,9 dichloro 1 methyl 2,3,4,5 tetrahy dro 1H benzo[d]azepine (CMTB ) ( 17 ) and lorcaserin [(1 R (+) 8 chloro 2,3,4,5 tetrahydro 1 methyl 1H 3 benzazepine] ( 18 ) are underwent development as 5HT2C agonists. ( S ) CMTB i s a partial agonist at 5HT2A receptors and nearly a full agonist at 5HT2C receptors. ( S ) CMTB ha s low potency partial agonism at 5HT2B receptors After oral administration to rats, ( S ) CMTB reduces food intake over a 6 hour period with an EC 50 value reported as 40 mg/kg.

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26 The benzazepine lorcaserin is a nonselective 5HT2C agonist with signif icant activation of 5HT2A receptors (75% efficacy) and 5HT2B receptors (100% efficacy) (Jensen, 2006; Smith et al., 2006; Thomsen et al., 2008). Affinity of lorcaserin for 5HT2C receptors (Ki~15 nM) is only about 7.5 times higher than at 5HT2A receptors (K i~112 nM), thus, activation of both receptors is likely in vivo with possible 5HT2A receptor mediated psychiatric and cardiovascular effects. Affinity of lorcaserin for human 5HT2C over 5HT2B receptors (Ki~174 nM) is a modest 12 times, suggesting, cardiop ulmonary problems could occur as a result of in vivo 5HT2B receptor activation. Nevertheless, lorcaserin underwent a large 2 year phase 3 clinical trial (Olmos, 2009). Except for headache/dizziness (18% for lorcaserin, 11% for placebo), incidence of other central nervous system or psychiatric side effects (e.g., 5HT2A mediated) has not been disclosed. Echocardiograms performed at baseline and at the end of the 2 year trial, however, suggested no drug effect on heart valves or pulmonary artery pressure. The main problem with the lorcaserin clinical trial is efficacy placebo adjusted weight loss was just 3.6% and this was judged to be not impressive (Goldstein, 2009). Increasing the dose of lorcaserin to boost anorexia/weight loss efficacy likely will result i n 5HT2A mediated psychiatric and cardiovascular side effects, as well as, 5HT2B mediated cardiopulmonary toxicity. Recently, another series of benzazepine analogs was reported as selective 5HT2C agonists (Shimadaet al., 2008). These compounds have the same benzazepine ring as locarserin without the 1 methyl group. Compound 19 was regarded as the most promising one with a 10 fold of selectivity to 5HT2C over 5HT2A. Compound 19 was a 5HT2A partial agonist, its 5HT2B functional data has not been reported yet.

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27 D esign o f Selective 5HT2C Agonists: ( ) Trans PAT as a Lead Molecule As suggested by information summarized in the preceding section, with the exception of ( ) trans PAT 30 (Booth et al., 2009), there are no compounds reported in the literature that activat e 5HT2C receptors without also activating 5HT2A and/or 5HT2B receptors. Thus, a marketable 5HT2C agonist drug has not come forth due to liability associated with activation of 5HT2A receptors that can lead to hallucinations and frank psychosis (Nichols, 20 04) and/or activation of 5HT2B receptors that can lead to cardio pulmonary toxicity (Fitzgerald et al., 2000; Launay et al., 2002 ; Setola et al., 2005) ( ) T ra ns PAT, 30 is a stereoselective, high affinity (Ki=40nM), high potency (EC 50 =20nM) full efficacy agonist at the human 5HT2C receptor A t 5HT2A and 5HT2B receptors ( ) trans PAT is an inverse agonist (IC 50 =490 and 1 000 nM, respectively) and competitive antagonist (K B =460 and 1400 nM, respectively) of serotonin (Booth et al., 2009) Thus, drug discover y using ( ) trans PAT as a lead molecular chemical scaffold may provide high potency, tru ly selective 5HT2C agonist s

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28 Tabl e 1 1 List of i n v itro biological data of published compounds (1) Section name 5HT2A 5HT2B 5HT2C 5HT 1 pEC 50 =7.51 pEC 50 = 8.68 pEC 50 =8.24 DOI 2 pEC 50 =9.05 pEC 50 =8.85 pEC 50 =8.10 E max =61% E max =65% E max =57% Nor d fenfluramine 3 pEC 50 =5.98 pEC 50 =8.06 pEC 50 =6.77 E max =54% E max =66% E max =77% Tryptamine 4 pEC 50 =6.59 pEC 50 =7.53 pEC 50 =7.34 E max =71% E max =92% E max =71% BW723C86 5 pEC 50 =6.66 pEC 50 =8.97 pEC 50 =7.03 E max =43% E max =83% E max =51% 1 M ethylpsilocin 6 E max =31% E max =12% EC 50 =633 nM EC 50 =12 nM RO600175 7 pEC 50 =6.35 pEC 50 =9.05 pEC 50 = 7.49 E max = 69 % E max =79 % E max =84 % Ver 2692 (PIP) 8 K i=31nM K i=12nM K i=1.6nM (Displace [ 125 I]DOI) (Disp lace [ 3 H]5HT) ( Disp lace [ 3 H]5HT ) EC 50 = 32 nM EC 50 = 1.1 nM EC 50 = 2.9 nM E max = 88 % E max = 65 % E max = 99 % YM 348 9 EC 50 = 93 nM EC 50 = 3.2 nM EC 50 = 1 nM E max = 97 % E max = 110 % E max = 76 % m CPP 10 pEC 50 =6.65 pEC 50 =7.2 pEC 50 = 7.09 E max = 22 % E max = 24 % E max = 65 % (4R,10aS) 7 Chloro 4,6 dimethyl 1,2,3,4,10,10a hexahydropyrazino[1,2 a]indole 11 K i= 40 nM K i=1 9 nM K i=1.9 nM (Displace [ 125 I]DOI) (Disp lace [ 3 H]5HT) (Disp lace [ 3 H]5HT ) E max = 97 % CP 809,101 1 2 K i= 6 nM K i= 64 nM K i=1.6 nM (Displace [ 125 I]DOI) (Disp lace [ 3 H]5HT) (Disp lace [ 3 H]5HT ) EC 50 = 153 nM EC 50 = 65.3 nM EC 50 = 0.11 nM E max = 67 % E max = 57 % E max = 93 %

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29 Table 1 2 List of in v itro b iological data of published c ompounds (2) Section name 5HT2A 5HT2B 5HT2C WAY 161503 13 K i= 18 nM K i= 60 nM K i=32 nM (Displace [ 125 I]DOI) (Disp lace [ 3 H]5HT) Disp lace [ 3 H] mesulergine EC 50 = 501 nM EC 50 = 19.5 nM EC 50 = 39.8 nM Partial agonist full agonist full agonist WAY629 14 K i=2530nM K i=56nM (Displace [ 125 I]DOI) (Displace [ 125 I]DOI) EC 50 =260,000nM EC 50 =426nM E max =60% E max =90% WAY16 2545 1 5 K i= 136 nM K i= 2101 nM K i=385 nM (Displace [ 125 I]DOI) (Disp lace [[ 125 I]DOI) Disp lace [ 3 H] mesulergine No activity EC 50 = 563 nM EC 50 = 39 nM E max =4 0% E max =85 % WAY163909 16 K i= 212 nM K i= 485 nM K i=221 nM (Displace [ 125 I]DOI) (Disp lace [[ 125 I]DOI) Disp lace [ 3 H] mesulergine No a ctivity EC 50 = 185 nM EC 50 = 8 nM E max =4 0% E max =90 % ( S ) CMTB 17 EC 50 = 135 nM EC 50 = EC 50 = 3 nM E max = 35 % E max = 25 % E max = 90 % Lorcaserin 18 K i= 112 nM K i= 174 nM K i=15 nM (Displace [ 125 I]DOI) (Disp lace [ 125 I]DOI) (Disp lace [ 1 25 I]DOI ) EC 50 = 168 nM EC 50 = 943 nM EC 50 = 9 nM E max = 75 % E max = 100 % E max = 100 % 6,7 dichloro 2,3,4,5 tetrahydro 1 H 3 benzazepine 19 K i= 93 nM K i= 100 nM K i=8.8 nM (Displace [ 3 H]5HT) (Disp lace [ 3 H]5HT) (Disp lace [ 3 H]5HT ) E max = 27 % E max = 87 %

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30 Fig ure 1 1 Structure s of some published 5HT2 a gonists F ig ure 1 2 S (+) F enfluramine triggers 5 HT release, leads to 5HT2C r eceptors activation in a rcuate h ypothalamic n ucleus, r egulate s d ownstream m elanocortinergic s ignaling

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31 Fig ure 1 3 Classic n onselective 5HT2 a gonists Fig ure 1 4 3 Substituted i ndole a nalogues Fig ure 1 5 Mol ecular modeling comparing structural similarities between ( ) t rans PAT and 1 methylpsilocin

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32 Fig ure 1 6 N substituted indole a nalogues Fig ure 1 7 Mol ecular modeling comparing structural similarities between ( ) t rans PAT, and Ver 2692 Figure 1 8 M CPP and p iperazine a nalogues

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33 Fig ure 1 9 Mol ecular modeling comparing structural similarities between ( ) t rans PAT and WAY 161503 Fig ure 1 1 0 Benzodiazepinoindole a nalogues Fig ure 1 1 1 molecular modeling comparing structural similarities between ( ) t rans PAT and WAY 163909 Fig ure 1 1 2 Benzazepines

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34 CHAPTER 2 SYNTHESIS OF ( ) TRANS N,N DIMETHYL 4 PHENYL 1,2,3,4 TETRAHYDRO 2 NAPHTHALENAMINE 30 Rationale As indicated above, ( ) trans PAT 30 is a 5HT2C agonist with 5HT2A and 5HT2B inverse agonist activity. This type of multi functional activity at 5HT2 receptors is consistent with literature th at suggests 5HT2C agonists and/or 5HT2A inverse agonists may be beneficial in treating neuropsychiatric disorders such as psychoses and psychostimulant (cocaine, amphetamines) abuse. Moreover, a compound that demonstrates trul y selective activation of 5HT2 C receptors (i.e., no activation of 5HT2A and 5HT2B receptors) is predicted to show clinical activity for obesity (5HT2C) without troubling psychiatric (5HT2A) and/or cardio pulmonary (5HT2B) side effects. T o evaluate the in vivo pharmacotherapeutic efficac y of ( ) trans PAT in psychoses, psychostimulant drug addiction, and obesity psychosis p reclinical study in several rodent models was planned. Thus, a large enough quantity (500 mg) of ( ) trans PAT was needed. Scale up synthesis of ( ) trans PAT was acco mplished through m odification and optimization of a published synthesis route

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35 Synthesis Results and D iscussion The scale up preparation of ( ) t rans PAT was based on a published diastereomer recrystallization strategy ( Wyrick et al., 1992, 1993, 1995) Me anwhile, s everal other synthetic routes ( Fig 2 1 ) were proposed and tested This chapter includes a detailed discussion of the formal synth e sis of ( ) trans PAT in multi gram scale through diastereomer recrystallization and summ a rizations of other investi gated routes Retrosynthetic analysis ( Fig 2 2 ) indicates ( ) trans PAT 30 i s derived from 2 tetralol 24 which i s prepared by reduction of 2 tetralone 23 C ompound 23 can be prepared straightforwardly through unsatuated ketone 22 The synthesis schemes are shown in Fig 2 3 2 9 The synthesis started with oxidation of 1 phenyl 2 propanol 20 with pyridinium chlorochromate (PCC) and Al 2 O 3 for 5 h to provide product 1 phenyl 2 propanone 21 in 75% yields ( maximum loading amount should not exceed 20 g). The next step is a kinetic controlled aldol condensation of compound 21 and benzaldehyde. The original method (Wyrick et al 1993, 1995, 1999) indicated the condensation was complete under aqueous KOH environment at 55C over a period of 16 h. The pre cyclizing intermediate 22 c ould be obtained in 60% yield However, we found over condensation occur ed given this long reaction time. Large amount of side products made recrystallization much less producti ve. I t was found that the optimal reaction period is 6.5 hours. After acidic workup and consequent r e crystallization from methanol, the majority of pure product was collecet e d To achieve the highest yield, all the leftover mother solutions were combined a nd impurities were removed as much as possible by chromatography. The enriched leftover crude product was allowed to r ecrytallizing for the second time in hexane methanol (2:1) solvent system. The combin e d yield wa s ~ 62% on a 15 g scale

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36 Other procedure s e .g. using lithium diisopropyamide (LDA) as base, THF as aprotic solvent, conducting the reaction at 78C, were not used d ue to facility limitation and workup complexity. The next step was a polyphosphoric acid (PPA) mediated Frediel Craft type alkylation to get 2 tetralone 23 The previous procedure (Wyrick et al 1993, 1995, 1999) employed xylene as solvent. We used toluene instead for cleaner workup. The yield was reporte d as 80% (Wyrick et al 1993), in contrast to all the other reported yields (all belo w 50%) for similar ring closure (Vincek & Booth 2009). T he highest yield we achieved was ar ound 40%. The discrepancy might be explained in Fig 2 4 Compound 22 exists as (Z) and (E) isomers. (E) isomer would undergo intra molecule cyclization however, steric effect could impede the ring closure for the (Z) isomer. The yield would be in agreement with (80%) only by assuming all the reactant 22 was the (E) isomer By monitoring the reaction conditions, we discovered the reaction was signifi cantly impacted by stirring condition and stoichiometry between the reactant s and solvent: (1) V igorously stirring by a mechanic stirrer would afford the product 23 in 40% yield in 10 g scale over a period of 3.5 h, while magnetic bar stirring would afford product in 300 mg scale after 12 h reaction period, with total yield around 20%. (2 ) The stoichiometry between toluene and reactant 22 should be no less than 14 L: 1 mol. D iluted solution enviroment favor s intra molecule cyclization over inter molecule co ndensation. In fact, the ratio of toluene v s reactant 22 as 2.2 L: 1 mol reduced the yield to 30%. The major side product was un characterizable, 1 H NMR indicated it might be polymers resulting from (Z) 22 inter molecule condensation.

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37 Several other Lewis acids e.g. AlCl 3 AlBr 3 etc, were tested as cyclization catalysts in thi s stage of synthesis. However, none of them proved to be productive. In the next step 2 tetralone 23 was converted to 2 tetralol 24 by NaBH 4 reduction In this way 2 tetralol 24 was s ynthesized in 10 g scale. P roduct 2 4 contains four stereoisomers ( Fig 2 5 ) with a ratio of approximately 75% cis v s 25% tran s The result is in agreement with the previous publication ( Wyrick et al., 1993 ). In the original paper (Wyrick et al., 1993), th e reaction period and the condition s to separate racemic cis 2 tetralol 24 were not reported. After a detailed investigation for the optimal reaction condition s we concluded ; (1) 10 h was the optimal reaction period (2) s ilica column method alone was inef ficient in complete ly separating racemic cis 2 tetralol 24 on a 10 g scale as was indicated by the fact that the difference of the retention time (R f ) of cis 24 and trans 24 was minimal in all of the TLC developing systems we tested. Instead, a robust rec rystallization method was developed. In short, a fter workup, in 10 gram scale, a medium sized silica gel chromatog raphy remove d most of impurities in the crude product 24 Subsequently the product was allowed to recrytallize in a solvent system ( 1% ethyl acetate in 99% hexan e ) following the stoichiometry of 1 g compound : 700 ml solvent After the crude product 24 was dissolved by rotation in 85C water bath the solution was allowed to cool smoothly. The recrystallization was completed after the mixture wa s kept in 20C freezer for 2 days. During this period the desired product cis 25 slowly solidified and attached to the wall of the flask. Repeated recrystllizing (u p to 4 times ) afford ed pure cis tetralols 25 (>97%) in 7g scale.

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38 It was found that the lef tover solution contained approximately 30%~40% trans 24 ~40% cis 24 and impurities. Purification and r ecrystalliz ation of the combined mother solution provided the final batch of desired cis 24 The combined yield of reduction and separation was 62%. The cis 24 and trans 24 are diastereomers, thus their 1 H NMR spectrums are different. The percentage of trans 24 was monitored by 1 H NMR peak integration of characteristic 1 H NMR signals ( cis 24 : dd v s trans 24 C4 proton t ) (Wyrick et al., 1993; Gatti et al., 2003). In the next step, pure racemic cis 24 (also shown as cis 25 in Fig 2 3 ) was tosylated by p toluenesulfonyl chloride in pyridine o n a 5 g scale over a period of 2 days. The pure product () cis 2 6 w as obtained a s a solid (yield 81% ) The S N 2 type transformation of () cis 26 into azide () trans 27 was achieved by using N 3 anion as nucleophilic attacking group. The yield was around 75% when DMF H 2 O was used as s olvent and the reaction mixture was refux ed for 4 h (Wyrick et al., 1993, 1999). It was foun d higher yield (~90%) was achieved when DMF alone was used as solvent and the mixture was stirred at R.T. for 3 days N o cis azide 1 H NMR signals was detected in pr oduct () trans 2 7 indicating t he chiral convension was complete The azide () trans 27 was reduced by H 2 using Pd/charcoal as catalyst ( yield 95 %) following the previous procedure (Wyrick et al., 1993). Starting with more than 350 g of 1 phenyl 2 propan ol 20 we obtained 11g racemic free base () trans 28 a fter the above process. The key diastereomeric recrystallization of () trans 28 is shown in Fig 2 6 A p revious publication employed ( 1R ) ( ) camphor 10 sulfonic acid b ut a poor yield (4%)

PAGE 39

39 was repor ted (Wyrick et al., 1993). Recrystallization using ( ) dibenzenyl L tartaric acid was tested but no crystals formed. We decided to proceed following the aforementioned camphorsulfonic acid strategy with modification s In the solvent system of acetonitrile and methanol (2:1), () trans 28 was treated with1.3 eq. ( 1R ) ( ) camphor 10 sulfonic acid. The mixture wa s first vigorously refluxed for 1.5 h, then cooled and stirred at R.T. overnight. Subsequent workup removed dark red impurities from the crude product Recrystallization was carried out in a modified solvent system (acetonitrile:methanol = 4:1). The salt t was first dissolve d ( 1g compound : 650 ml solvent) by rotating in 75C water bath, then cooled smoothly finally kept at 0C for up to 2 days. During the first several rounds of recrystallization w e noticed it was t he undesired isomer (+) trans amine that formed the needle shaped crystals (optically dextrorotatory) with camphorsulfonic acid However, by separating the undesired salts the desired ( ) tra ns 28 was enriched in the mother solution. S ubsequent rounds of recrystallization afford ed crystals 29 as prisms containing the single enantiomer ( ) trans 28 in the end T o ensure the purity of the final products t he percentage of (+) trans 28 ( Fig 2 6 component I ) v s ( ) trans 28 ( Fig 2 6 component II ) was monitored carefully. To this purpose, optical rotation measurement and Mosher reagent [ ( R ) ( ) methoxy [(trifluoromethyl)phenyl]acetamide ] derivatization assay s were performed in ea ch round of recrystallization. T he optical rotation test gave levorotory value of salts 29 (10 mg) in 1 ml absolute methanol High levorotory value s indicate hi gh percentage s of component I in the salts The end point (possible 100% component I) is in 25 D 68.5 ~ 70. However, t he optical rotation results were significantly influenced by experimental

PAGE 40

40 errors. Thus, was conduc ted by converting recrystallized salts in to ( R ) ( ) methoxy [(trifluoromethyl)phenyl]acetamide diastereomers ( Fig 2 7 ; Dale et al., 1969, 1972). Recrystallized salts 29 (3~5 mg) was converted to free amine 28 The free amine was transformed to the di astereomeric salts with ( R ) Mosher acid chloride ( Fig 2 7 ) T he sample was directly analysed by 1 H NMR in CDCl 3 without purification. The percentage of 31 vs 32 was monitored by 1 H NMR peak integration of the characteristic 1 H NMR signals ( 31 : C2 proton vs 32 C 2 3,t ) (Wyrick et al., 1993; Gatti et al., 2003) Several batches of pure 2 S ,4 R R salt 29 (also shown as compound 33 in Fig 2 8 ) were collected Compound 33 was first converted to free base 34 then dimethylated by being refluxed with formaldehyde and formic acid. The products were converted to final HCl salt 30 ( Fig 2 8 Wyrick et al.,1993). Collectively 500 mg of enantiometically pure final compound 30 was obtained through the resolution of 4 g racemic () trans amine 28 The diastereomer recrystallization ( yield 10% ) proved to be very labor intensive and time consuming To achieve better overall yield, s everal alternative approaches were investigated along with the scale up. As shown in Fig. 2 9 chiral organoboron re agent (+) DIP chloride was proposed to asymmetric ally reduce tetralone 23 to (2 R, 4 R ) tetralol 35 DIP chloride has been sucessful l y used for the reduction of many acyclic substrates H owever, it did not afford the desired (2 R, 4 R ) cis tetralol 3 5 Another proposed asymmetric synthesis route ( Fig 2 10 ) started with conversion of 2 tetralone 36 to (2 R ) 2 tetralol 37 (88% ee ) using benzene ruthenium (II) chloride

PAGE 41

41 dimer and ( R R ) N (2 amino 1,2 diphenylethyl) p toluenesulfonamide (NAPT ). Subsequently t he hydro xyl group in compound 37 was protected by t ert butyldimethylsilyl chloride (TBDMS) A strategy included (1) N bromosuccinimide (NBS) mediated bromination at the benzylic position (2) nickle (Ni ) catalzyed Suzuki coupling a phenyl group at this position w as tested It was revealed the product from the bromination reaction was unstable even in neutral CS 2 environment, let alone sustaining the strong basic environment that is ne cessary for Suzuki coupling. An alternative synthetic strategy based on Jacobsen asymmetric epoxidation on 4 phenyl 3,4 dihydronaphthalene 41 was proposed ( Fig 2 1 1 ; Palucki, et al., 1994. Boger et al.,1997 ) This process began with conversion of naphthol 39 to 4 phenyl 1 tetralone 40 using aluminum chloride as the Lewis acid and benzene as the solvent. After (1) NaBH 4 reduction, (2) azotropic distillation (3) e poxidation catalysed by and using either bleach (NaOCl) or 3 ch loroperoxybenzoic acid ( m CPBA) as oxydant p roduct 4 0 was syntehsized The epoxide 4 0 was verified by high definition MS and was reduced to tetralols 4 1 either by H 2 /Pd or by diisobutylaluminum hydride (DI BAL H). Unfortunately, 1 H NMR revealed the major tetralols obtained was undesired trans configuration product (Most likely 2R,4S enantiomer, the absolute stereochemistry assignment was not conducted ) with trace amount of desired cis tetralol. T he 4 phenyl group might block the bulky catalyst resulting in the oxidative reagent complex approaching the olefin from the same side ( Fig 2 1 2 ) thus most of the epoxdation occurred on the opposite side of the 4 phenyl group and formed trans product (Martinelli et al., 1994; Lucero et al., 1994; Maeda et al., 2002).

PAGE 42

42 These alternative synthetic projects although being unsuc c essful, did advance our synt hetic knowledge of the PAT structure, as well as, providing valuable procedures for PAT analog preparation that are described in the following chapters. In vitro Pharmacological C haracterization R esults The detailed in vitro characterization of ( ) trans PAT and its stereo isomers was published ( Booth et al., 2009 ). In Table 2 1 i n vitro competitive binding assay data (measuring displacement of [ 3 H] r adioligands from human 5HT2 receptors) are summarized. In Table 2 2 I n vitro f unctional activity assay data (measuring activation of PLC/ [ 3 H] IP formation i n c lonal c ells e xpress ing h uman 5HT2C r eceptors ) are summarized. Fig 2 1 3 2 1 4 2 1 5 shows representative curves for ( ) trans PAT activity in functional assays ( ) Trans PAT is a full efficacy agonist, c omparable to the endogenous agonist serotonin, at human 5HT2C re ceptors, ( ) t rans PAT is an inverse agonist at human 5HT2A and 5HT2B receptors. In vivo Pharmacol ogical C haracterization R esults Results of an in vivo study evaluating anti obesity efficacy of ( ) t rans PAT in mice were published (Rowland NE, Zhuming Sun, et al., 2008). In summary, ( ) trans PAT produces a dose dependent inhibition of food intake with a 50% inhibitory dose (ID 50 ) of 4.2 mg/kg in C 57BL/6 mice that are not food deprived. The do se effect curve was similar to that obtained using a published 5HT2 non selective agonist WAY 161503 ( Fig 2 1 6 ) After 4 days consequent ly administration, the anorectic effect of ( ) t rans PAT is maintained ( Fig 2 1 7 ). In a nother study the ability of ( ) trans PAT to counteract the effec t s of the psychostimulant amphetamine were measured in rats ( data from Dr. Drake Morgan UF Department of Psychiatry) As shown in Fig 2 18 ( ) trans PAT f ully blocks the

PAGE 43

43 amphetamine induced locomotor activating effects in rats ( ED 50 ~ 5mg/kg ). This effect is not simply due to a generalized sedative effect as a dose of 10 mg/kg ( ) t rans PAT failed to decrease loco motor activity when given alone. In addition to the overt anti amphetamine behavioral effects of ( ) trans P AT demonstrated here, it is noted that amphetamine induced locomotion is a widely used model to mimic psychosis (schizophrenia) in rodents ( Powell et al., 2006) Thus, the current results suggest antipsychotic activity of ( ) trans PAT Discussion : ( ) Tra ns PAT is a 5HT2C Full Agonist with 5HT2A/2B Inverse Agonism that Shows Promise for Treating Obesity, Drug Abuse and Psycho ses The s cale up synthesis based on the diastereomeric recrystallization strategy afforded 500mg enantiomerically pure ( ) trans PAT In re searching an alternative high efficient route, new synthetic reactions were investigated. Some of the new reactions were applied in preparation PAT analogs described in the following chapters. 2 and 3. ( ) Trans PAT is the first reported 5HT2C full agonist with 5HT2A/2B inverse agonism. I n vitro characterization data of ( ) trans PAT and its stereoisomers and results of molecular modeling studies were published (Booth et al., 2009). Subsequent mutagenesis studies in our lab ( Fang et al. 2010; unpu blished data ) confirmed ( ) trans PAT protonated amine can for m an ionic bond with D3.32 of 5 HT 2A and 5 HT 2C receptors, but, not with 5 HT 2B receptor. Analogs synthesis and characterization focusing on the substitution on the fused phenyl of tetrahydronapht halene ring and the pending phenyl of PAT are discussed in the following chapters. A new molecular model based on crystallographic structure of adrenergic receptor has been developed and advanced SAR study is initiated.

PAGE 44

44 Precilnical in vivo stud ies in rodents suggest ( ) trans PAT is a suitable lead compound for development as a drug to treat obesity, psychostimulant addiction, and psychoses. No tably, no overt toxicity was observed in any of the dozens of animals that received peripheral (intraperitoneal) injections of ( ) trans PAT Moreover, the in vivo neurobehavioral studies showing ( ) trans PAT efficacy to modulate amphetamine induced locom otion in rats confirms that ( ) trans PAT enters the brain after peripheral administration, where is presumably acts as a 5HT2C agonist with 5HT2A/5HT2B inverse activity.

PAGE 45

45 Table 2 1 ( ) Trans PAT and isomers 5HT2 receptors affinity. All t he data are pre sented as Ki SEM (nM) 5HT2A Ki (nM) 5HT2B Ki (nM) 5HT2C Ki (nM) H1 Ki (nM) ( ) trans PAT 410 38 1200 6.8 37.6 3 1.95 0.5 (+) trans PAT 520 3 ~ 2500 1300 80 29.8 3.5 ( ) cis PAT (+ ) cis PAT 780 2 ~ 5000 980 7.8 13.7 2 1500 2 ~ 10000 430 4.8 177.2 9.4 Tabl e 2 2 Functional activities of ( ) tran s PAT at 5HT2 r eceptors. E max was e xpressed as % 5HT, I max was e xpressed as % b asal i nhibition Section name 5HT2A 5HT2B 5HT2C ( ) trans PAT IC 50 =490 96 nM IC 50 = 1 00 0 5 n M EC 5 0 = 20 2.2 nM I max = 6 0 5 % I max = 35 2.0 % E max =100 2 % Fig ure 2 1 Summary of ( ) tr ans PAT s ynthetic r outes Fig ure 2 2 Retrosynthetic a nalysis of d iaster eomer r ecrystallization strategy

PAGE 46

46 Fig ure 2 3 Synthesis of () t rans 2 amino 4 phenyl 1,2,3,4 tetrahydronaphthalene 28 Fig ure 2 4 Formation of 2 t etralone 23 Fig ure 2 5 NaBH 4 r eduction to p repare 2 t etralol 24

PAGE 47

47 Fig ure 2 6. Resolution of () t rans 1 p henyl 3 amino 1,2,3,4 tetrahydronaphthalene 28 Fig ure 2 7. r eagent a ssay of ( ) trans pat r esolution

PAGE 48

48 Fig ure 2 8. Conversion of p ure salts to f inal p roduct 30 Fig ure 2 9. (+) DIP chloride f ailed to a fford (2 R ,4 R ) cis t e tralol 35 Fig ure 2 1 0. Bromination Suzuki c oupling f ailed to i ntroduce p henyl g roup to C4 of t etralol 38

PAGE 49

49 Figure 2 11. Jacobsen e poxidation y ielded m ainly t rans tetralol Fig ure 2 1 2. Stereochemistry o f J acobsen e poxidation o n d ihydronaphthalene 41

PAGE 50

50 Fig ure 2 1 3 Representative concentration r esponse curve for serotonin (closed squares) and ( ) trans PAT ( close d circles ) activation of PLC / [ 3 H ] IP formation i n HEK c ells e xpressed c loned h uman 5HT2C r eceptors Single concentration effect of ( ) trans PAT at 5HT2A ( open triangles ) and 5HT2B ( open squares ) receptors also is shown ( Booth e t al., 2009 ). Fig ure 2 1 4 Representative data for ( ) trans PAT inverse a gonist activity at cloned human 5HT2A r eceptors (Booth et al., 2009)

PAGE 51

51 Fig ure 2 1 5. Representative data for ( ) trans PAT i nverse a gonist a ctivity at c loned h uman 5 HT2B r eceptors (Booth et al., 2009) Figure 2 1 6. Dose e ffect c urve after i.p. a dministration of ( ) trans PAT vs. the no n s elective 5HT2A/2B/2C agonist WAY161503 on 30 min i ntake of p latatable f ood by m ice D ose r elated i nhibition of f ood i ntake (DI 50 ): PAT 9.2mg/kg WAY 8.4mg/kg (Rowland, Sun et al., 2008)

PAGE 52

52 Fig ure 2 1 7. No tolerance to ( ) trans PAT a norectic e ffect with c hronic a dmini stration. anorectic effect of ( ) trans PAT is m aintained after c hronic a dministration ( d aily i.p. i n jection for 4 days) (Rowland, Sun et al., 2008) Fig ure 2 18. ( ) Trans PAT in m odulat ing a mphetamine i nduced l ocomotion ( d ata from Dr. Drake Morgan UF Department of Psychiatry). Left P anel: a mphetamine d ose d ependent ly i ncrease d l ocomotor a ctivity R ight P anel : ( ) t rans PAT d ose d ependently i nhibit p sycho l ocomotor b ehavioral e ffect r espectively. Agents a dministered i ntraperitoneally a lone or i n c ombination i mmediately b efore the session

PAGE 53

53 CHAPTER 3 SYNTHESIS OF N,N DIMETHYL 4 (4 METHYLPHENYL) 1,2 ,3,4 TETRAHYDRO 2 NAPHTHALENAMINE ANALOGS OF PAT Rational e Often, the addition of an akyl moiety to a lead molecule can enhance lipophilicity to improve penetration into brain tissue. If the alkyl moiet y is relatively small with regard to steric bulk, then, affinity for the target receptor may not be adversely affected in fact, affinity may improve due to enhanced van der Waals interactions between ligand and receptor. Given that the lead molecule ( ) tr ans PAT 30 demonstrated preclinical efficacy to treat obesity, psychostimulant abuse, and psychoses after peripheral administration in laboratory animals (see Fig. 2 17 2 18 2 19 ), it was hypothesized that or para methyl analog ( p C H 3 PAT 4 8 ) might achieve faster and/or great brain penetration after in vivo administration and perhaps demonstrate higher potency and efficacy for obesity and neuropsychiatric disorders compared to the parent compound. Implicit in this hypothesis is the assumption that the addition of the CH 3 moiety to ( ) trans PAT 30 would not adversely impact 5HT2C agonist and 5HT2A/2B inverse agonist activity. For ( ) trans p CH 3 PAT logP = 4.6, making it nearly

PAGE 54

54 half log unit more lipophilic compared to ( ) t rans P AT (logP = 4.2), suggesting the possibility of improved brain penetration after peripheral administration by intraperitoneal injection to laboratory animal In a preliminary screen, racemic () trans p CH 3 PAT demonstrated higher affinity at 5HT2 type recep tors when compared to ( ) trans PAT Thus, 5 mg of () trans p CH 3 PAT was resolved to the (+) and ( ) trans p CH 3 PAT enantiomers using a chiral HPLC system (see Methods). Like the lead ( ) trans PAT, preliminary functional screening indicated that ( ) t rans p CH 3 PAT is a 5HT2A and 5HT2B inverse agonist and a 5HT2C agonist. Thus, scale up synthesis of ( ) trans p CH 3 PAT was undertaken for complete in vitro pharmacological characterization, as well as, to obtain enough compound for preclinical in vivo s tudies. Also, the trans p CH 3 PAT analog is a useful initial molecule to probe the role of the PAT (C2) pendant phenyl moiety for binding and function at 5HT 2 receptors and to characterize the 3 dimensional structure of the binding pocket of 5HT2 recepto rs. Likewise, synthesis of other p CH 3 PAT isomers, (+) trans ( ) cis and (+) cis was undertaken for pharmacological studies, SAR studies, and, to characterize the 3 dimensional structure of 5HT2 receptor subtypes. Synthesis R esults and D iscussion Prep aration of ( ) trans p methyl PAT and stereoisomers ( Fig 3 1 3 5 ) followed methods used to synthesize ( ) t rans PAT described in chapter 2 Two major improvements ; (1) asymmetric hydrogenation (2) enantiomeric separation by c hiral HPLC system (Mongi et al ., 2004) afford ed the desired p CH 3 PAT s ( 54, 55, 48, and 5 7 respectively) in good yield s and excellent enantiomer excess The synthesis ( Fig 3 1 ) started with condensation of 1 phenyl 2 propanone 21 and toluadehyde ( reflux at 55C in aqueous KOH for 14 h ) to provide product 42

PAGE 55

55 Compared to the analogous synthesis in chapter 2 the reaction time was doubled; however, the total y ield (25%) was much worse. The reduced electrophillic reactivity of toluadehyde compared to benzaldehyde makes the condensation l argely incomplete. Due to limited time w e did not ex plore other alternative methods Started with 30 g compound 21 13.6 g product 42 was synthesized. In the next step, intermediate 42 was cyclized in toluene using polyphosphori c acid as the catalyst (refl ux for 4.5 h, y iel d 40%) to afford 2 tetralone 43 ( 4.6 g ) as the product The next step was b ased on a modified Noyori type asymmetric hydrogen transferring procedure (Muneto et al., 2004). The intermediate 2 t etralone 43 was converted to ( 2R,4R ) cis tetra lol 44 in several batches with good yield and 92 % ee ( Fig 3 2 ) In this reaction, 2 propanol served as the solvent and the hydrogen source. Compound 43 was first treated by a catalytic complex ( be nzene ruthenium (II) chloride and R,R NAPTS solution ) and then by KOH solution. The mixture was allowed to react over 1.5h. The workup should be conducted directly after the reaction by thoroughly filtering through silica gel/ celite pad based on our observation that ruthenium containing impurities might slowly d ecompose 2 tetralol products The percentage of cis v s trans 4 4 ( Fig 3 2 ) in the product 44 was monitored by 1 H NMR peak intergation of characteristic 1 H NMR signals ( cis 4 4 : 2 ,dd v s trans 4 4 4 ,t ; Wyrick et al., 1993; Gatt i et al., 2003) The enantiomeric excess ( 92 %) was determined by comparing the percentage of each enantiomer after the final product was separated using chiral HPLC system discussed later.

PAGE 56

56 To separate 2 g racemic cis 44 and trans 44 ( similar R f in most TLC systems) A s ilica gel chromatography procedure was developed and used 7%ethyl acetate in hexane as eluent The separation was monitored by 1 H NMR spectrum In this way cis 44 (>9 8 % 1 g ), trans 44 ( 20 mg) and 450 mg mixture were collected. The conversion from cis 44 ( 1 g ) to final product dimethylated HCl salt 48 ( 500 mg ) was completed following analogous procedures described in chapter 2. O ptical rotation assay indicated compound 48 25 D 67.5 in absolute methanol) contains mostly the desired ( 2 S,4R ) trans isomer indicates the undesired ( 2R,4S ) enantiomer exists in trace amount ( <2%, Fig 3 3 ). Prior to in vitro af finity and functional experiment s, a chiral stationary phase HPLC (CSP HPLC) using a Kromasil CelluCoat TM c olumn was employed to remove the trace amount of ( 2R,4S ) trans p CH 3 PAT impurity from the product ( 2S ,4 R ) trans p CH 3 PAT 48 The HPLC system used a mobile phase system consisting of 15% ethanol in 85% hexane with 0.1% diethyl amine as modifier and the fl ow rate 1.5ml/min to resolve 1 mg compound dis solved in 200l mobile phase in a single run Further purified amine 48 (40 mg) was collected in this manner Later a more efficient mobile phase system ( 5% methanol, 5% ethanol, 90% hexane and 0.2% diethyl ami ne as modifier ) was developed and applied to (+) cis ( ) cis and (+) trans p CH 3 PAT isomers ( 54 55 48 and 57 ) separation. Preparation of the (+) cis ( ) cis and (+) trans p CH 3 PAT isomers 54 and 55 are summarized in Fig 3 4 and 3 5 The previously s ynthesized 2 tetralone 43 was reduced by NaBH 4 and the resulting () cis tetralol 49 was converted to () trans tetralol 50 using a modified Mitsunobu conditions ( Wyrick et al., 1993 ). The trans 2 tetralol 50

PAGE 57

57 was converted to racemic cis p CH 3 PAT 53 follo wing previously described procedures. Chiral HPLC separation afforded enantiomerically pure products (+) cis 54 and ( ) cis 55 R f for ( ) cis 54 : 11 8 min; R f for (+) cis 55 : 11.2 min Assignment of the a bsolute configuration was based on analog y to the X ray crystal structure of ( ) cis N,N dimethyl 1,2,3,4 tetrahydro 2 naphthalenamine 1 ( R ) ( ) camphor 10 sulfonic acid salt ( Bucholtz et al., 199 8 ) To synthesize the (+) trans product 5 7 the () cis tetralol 49 was converted to () trans p CH3 PAT follow ing the routine synthesis ( Fig 3 7 ). The final product was collected after chiral HPLC separation. R f for ( ) trans 48 : 1 1 8 min; R f for (+) trans 57 : 11.3 min; Structural elucidation was confirmed by polarimetry In vitro Pharmacological C haracterization R esults To determine 5HT2 receptor affinity for the p CH 3 PAT isomer I n vitro competitive binding assay measured p CH 3 PAT isomers ability to displace of [ 3 H] radioligands from human 5HT2 receptors expressed in HEK cell membranes (Booth et al., 2009) wa s conducted Affinity r esults are summarized in T able 3 1 and r epresentative competition displacement are shown in Fig 3 6 3 7 and 3 8 I n vitro functional activity was measured as ( ) trans p CH 3 PAT activation of PLC/ [ 3 H] IP formation i n HEK c ells e xp ressed h uman 5HT2C r eceptors (Booth et al., 2009) Functional activity r esults are summarized in Table 3 2 and r epresentative potency efficacy curves are listed in Fig 3 9 3 11 Overall, results indicate ( ) t rans p CH 3 PAT is a near full efficacy agonis t at human 5HT2C receptors and an inverse agonist at human 5HT2A and 5HT2B receptors ( ) T rans p CH 3 PAT is 3 times more potent regarding inverse agonism at 5HT2A receptor s compared to ( ) trans PAT (2 times more potent at 5HT2B). However, ( ) t rans p CH 3 PAT is 10 times less potent regarding agonism at 5HT2C receptors

PAGE 58

58 I n vivo Anti Stimulant Effects and Discussion of ( ) Trans PAT and ( ) T rans p CH 3 PAT: Indication for Drug A buse Pharmacotherapy Amphetamine induced locomotion in rodents is a widely used rodent behavioral model for schizophrenia In our studies ( Fig 3 12 ), m ale, Sprague Dawley rats (n=8) were tested during 1 hour locomotor activity sessions, with s aline, PATs, or amphetamine administered intraperitoneally alone or in combination immediate ly before the session. A mphetamine dose dependent ly increase d locomotor activity that is taken as psychotomimetic activity When combined with the highest dose of amphetamine (2 mg/kg), b oth ( ) t rans PAT and ( ) trans p C H 3 PAT dose dependently inhibit the psy cho locomotor behavioral effects Time course analyses suggest that the PATs are active within 15 minutes and effects are maintained through out the 1 hr session Preliminary studies with methamphetamine suggest that at the dosage of 1.0 mg/kg, ( ) tran s p C H 3 PAT partially blocks the stimulant effects of methamphetamine ( Fig 3 13 ) In all cases, t he rats displayed no overt signs of toxicity According to the results, ( ) t rans PAT and ( ) trans p CH 3 PAT have equal efficacy. However, the p CH 3 analog i s 3 times more potent T he results suggest ( ) t rans PAT and ( ) trans p CH 3 PAT may show efficacy for treatment of psychoses, as well as, amphetamine and methamphetamine abuse Disscussion As mentioned above, ( ) t rans p CH 3 PAT like the leading compound ( ) trans PAT is a 5 HT2C agonist with 5HT2A/2B inverse agonist activity. It is quite surprising that at 5HT2C receptors, ( ) trans p CH 3 PAT has about 1/ 9 affinity and agonist functional potency (PLC/IP signaling) compared to ( ) trans PAT However, at 5HT2A receptors, ( ) trans p CH 3 PAT is 3 times more potent than ( ) t rans PAT as an inverse

PAGE 59

59 agonist. I t is intriguing that the pending toluyl v s. phenyl make such activity differences. In vitro results above indicate ( ) trans p CH 3 PAT is 3 times more pot ent regarding 5HT2A inverse agonist activity compared to ( ) trans PAT. In the a mphetamine induced locomotion model, ( ) trans p CH 3 PAT is 3 times more potent to inhibit the psycho locomotor behavioral effect This might due to the fact that 5HT2A inverse agonism activity of PATs may be at least as important as 5HT2C agonist activity regarding PAT type pharmacotherapeutic potential to treat psychostimulant drug abuse (Fletcher et al., 2002; Bubar and Cunningham, 2006) Also, ( ) trans p CH 3 PAT (LogP=4.6) is more lipophilic than ( ) t rans PAT (LogP=4.2), thus, superior brain penetration may als o contribute to higher potency of Me PAT vs. PAT regarding psychotherapeutic activity Future studies, especially in silica molecule modeling will greatly help the st ructure activity relation understanding here. I n vivo A norexia effect and Disscussion of ( ) T rans p CH 3 PAT I n vivo study evaluating anti obesity efficacy of ( ) trans p CH 3 PAT was conducted (data from Dr. Neil Rowland, UF Department of Psychology) usin g a published rodent model (Rowland, Zhuming et al., 2008) P reliminary results are summarized below ( as % untreated mice food consumption for vehicle treated and ( ) trans p CH 3 PAT treated mice ) % untreated mice food consumption Vehicle: 112.8 +/ 6.3 Dose of ( ) trans p methyl PAT 1 mg/kg: 89.0 +/ 4.0 3 mg/kg: 98.5 +/ 5.7 9 mg/kg: 83.0 +/ 10.6

PAGE 60

60 Discussion The above in vivo results closely relate to the In v itro binding and function activities data. ( ) trans p CH 3 PAT produces no anorexia effects at 10mg/kg, whereas, the ED 80 for ( ) t rans PAT anorexia effect occurs at 10mg/kg and ED 50 is 5mg/kg. It correlates well with the fact that ( ) trans p CH 3 PAT has about 1/10 affinity and agonist functional potency (PLC/IP signaling) compared to ( ) trans PAT Overall, It is highly possible that 5HT2C agonism might translate more closely to anorexia activity and 5HT2A inverse agonism might translate more closely to anti amphetamine effects (anti addiction for psychostimulant s, antipsychotic).

PAGE 61

61 Table 3 1 ( ) T rans p methyl PAT i somers 5HT2 receptors affinity 5HT2A Ki (nM) 5HT2B Ki (nM) 5HT2C Ki (nM) H1 Ki (nM) ( ) trans p methyl PAT 210 40 250 27 330 50 18 2.0 (+) trans p methyl PAT 70 3.8 52 6.6 3 81 76.7 12 0.9 ( ) cis p methyl PAT (+ ) cis p methyl PAT 1500 10 9000 3 000 1 0 3000 520 17 500 20 1500 12 19.6 3.30 Table 3 2 Functional a ctivities of ( ) trans p methyl PAT at 5HT2 receptors (in comparis on to ( ) t rans PAT) Section name 5HT2A 5HT2B 5HT2C ( ) trans PAT IC 50 =490 96 nM IC 50 = 1 00 0 5 n M EC 5 0 = 20 2.2 nM I max = 60 5 % I max = 35 2.0 % E max =100 2 % ( ) trans p methyl PAT IC 50 =140 15 nM IC 50 = 420 12 n M EC 5 0 ~ 2 0 0 nM I max = 50 4 % I max = 50 3 % E max ~100 % Fig ure 3 1 Synthesis of ( ) t rans p CH 3 PAT 48

PAGE 62

62 Fig ure 3 2 A symmetric r eduction with RuCl 2 Ph 2 and Noyori l igand NAPT to p repare 2 t etralol 44 Fig ure 3 3 r eagent a ssay of ( ) t rans p CH 3 PAT r esolution

PAGE 63

63 Fig ure 3 4 S ynthesis of (+) c is and ( ) c is p CH 3 PAT (54 and 55) Fig ure 3 5 S ynthesis of (+) t rans p CH 3 PAT 57

PAGE 64

64 Fig ure 3 6 Representative concentration response curves for p CH 3 PAT isomers displacement of [ 3 H] k etanserin from 5HT2A r eceptor Ki values summarize d in Table 3 1 Fig ure 3 7 Representative concentration response curves for p CH 3 PAT i somers d isplacement of [ 3 H] m esulergine from 5HT2B r eceptor. Ki Values s ummarized in Table 3 1

PAGE 65

65 Fig ure 3 8 Representative concentration response curves for p CH 3 PAT i somers d isplacement of [ 3 H] m esulergine from 5HT2C r eceptor. Ki Values s ummarized in Table 3 1 Fig ure 3 9 Representative data for ( ) t rans p CH 3 PAT ( closed cubic ) compared to ( ) t rans PAT ( closed ci rcles) i nverse a gonist activities at c loned human 5HT2A r eceptor s e xpressed in HEK C ells IC 50 D ata summarized i n Table 3 2 ( d ata from Dr. Lijuan Fang)

PAGE 66

66 Fig ure 3 10 R epresentative data for ( ) t rans p CH 3 PAT ( closed cubic ) c ompared to ( ) trans PAT (closed circles) i nverse a gonist activities at c loned human 5HT2B receptors e xpressed in HEK C ells IC 50 data summarized in Table 3 2 ( d ata from Dr. Lijuan Fang) Fig ure 3 11 Representative data for ( ) t rans p CH 3 PAT (closed cubic ) compared to ( ) t rans PAT (closed circles) a gonist activities at c loned human 5HT2C receptors e xpressed in HEK c ells IC 50 d ata summarized in Table 3 2 ( d ata from Dr. Lijuan Fang)

PAGE 67

67 Fig ure 3 12 ( ) T rans p CH 3 PAT compared to ( ) trans PAT in m odulat ing amphetamine induced locomotion ( d ata from Dr. Drake Morgan UF Department of Psychiatry). Left panel: a mphetamine d ose d ependent ly i ncrease d l ocomotor a ctivity Middle and right panels : ( ) Trans PA T and ( ) tr ans p C H 3 PAT d ose d ependently i nhibit the p sycho l ocomotor b ehavioral e ffect respectively PATs, or amphetamine a dministered i ntraperitoneally a lone or i n c ombination i mmediately before the session Fig ure 3 1 3 Single dosage ( ) t rans p CH 3 PAT in m odulat ing amphetamine and methamphetamine induced locomotion ( d ata from Dr. Drake Morgan UF Department of Psychiatry). Left panel: ( ) t rans p CH 3 PAT blocks the stimulant effects of a mphetamine at 10mg/ k g. R ght p anels: ( ) t rans p C H 3 PAT partially block methamphetamine induced locomotion at 10mg/kg. Agents a dministered i ntraperitoneally i n c ombination i mmediately before the 12 min experiment sessions

PAGE 68

68 CHARPTER 4 SYNTHESIS OF PAT ANA LOGS WITH SUBSTITUTI ONS ON THE TETRAHYDRONAPHTHY L AND PENDANT PHENYL Rational e Preliminary results ( Fig 4 1 ) from our lab indicats the racemic trans PAT analog () trans 6 OH,7 Cl PAT has relatively high affinity (Ki~23 nM) for 5HT2C receptors (Fan g and Booth, unpublished data, 2008) In comparison to 5HT2C affinity of the lead analog ( ) t rans PAT (Ki~40 nM), racemic () trans 6 Cl,7 OH PAT appears to have about 2 times higher affinity Using a 5HT2C molecular model built by homology to the crystal structure of bovine rhodopsin ligand docking studies suggested the 7 Cl and/or or 6 OH substituent may form a hydrogen bond with the 5HT2C amino acid residue S3.36 (Wilczynski and Booth, unpublished data, 2009) Likewise, several other residues in 5HT2C TMD helices 3, 6, and 7 may form hydrogen bond interactions with the substituted tetrahydronapthyl moiety of PAT Interestingly preliminary functional activity results indicate d that () trans 6 OH,7 Cl PAT is a 5HT2C inverse agonist with IC 50 ~15 nM. Thus 6 OH,7 Cl PAT and related molecules, in comparison to the 5HT2C agonist ( ) trans PAT may provide useful drug design i nformation regarding the structural requirements for agonist vs. inverse agonist activity at 5HT2C receptors. The preparation of enan tiomerically pure (+) and ( ) trans 6 OMe,7 Cl PAT ( 58 and 59 ) as the precursors t o obtain (+) and ( ) trans 6 OH,7 Cl PAT was proposed. It is noteworthy that the 6 OMe,7 Cl PATs (LogP=4.6) are more lipophilic than 6 OH,7 Cl PATs (LogP=4.3), suggesting su perior brain penetration may be apparent for the O methylated derviatives. To fully characterize the 3 dimensional structure activity requirements governing agonist vs. inverse agonist activity, the corresponding cis 6 OMe,7 Cl PATs ( 60 and 61 ) also were p roposed for synthesis

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69 In addition to the 6 OMe,7 Cl PAT compounds, above, PAT analogs 62 63 64 65 66 and 67 with a chlorine or bromine moiety at the meta position of the (C4) pendant phenyl were proposed, based on results of on going studies in our lab that indicate halogen substitution at the meta position enhances 5HT2 receptors affinity of PAT type structures. It is noted, too, that a halogen moiety enhances lipophilicity (LogP>5 for analogs proposed ) .that may allow for superior brain penetration To establish the relative contribution of the 7 Cl substituent (analogs 58 67 ) with regard to binding and function, t he n ovel analogs (+) and ( ) trans 6 Cl PAT 68 and 69 that have no hydrogen at position 7 also were proposed. for synthesis.

PAGE 70

70

PAGE 71

71

PAGE 72

72 Synthesis R esults and D iscussion To synthesi ze 6 OMe, 7 Cl PAT analogs 58 59 60 a nd 61 ( Fig 4 2 ) commercially available 2 (4 methoxyphenyl) acetic acid was chlorinated (20 g scale ) based on a published procedure (Mai et al., 2006). Recrystal l ization ( 10% toluene in 90% hexane as solvent) afforded p ure product 70 as colorless needle l ike crystals ( yield 50% ). In the next step a n ovel trifluoroacetic acid anhydride (TFAA) mediated Friedel Craft type condensation reaction developed in our lab ( Vincek and Booth, 2009 ) successfully converted compound 70 to phenyltetralen 2 ol phenylaceta te 71 with m eta bromostyrene. Brief ly pre cooled compound 70 was dissolved in TFAA under nitrogen protection Once the mixture turned an orange color ed liquid, it was transferred (via a double end needle ) to a bottle containing s tyrene and allowed to sit overnight. W orkup involved ethyl acetate extraction followed by multiple washing the organic layer using saturated NaHCO 3 in water After silica gel chromatography using 5% ethyl acetate in hexane as solvent, product 71 was obtained as thick oil (yield 70% calculated based on starting bromo styrene). The phenyltetralen 2 ol phenylacetate 71 was reduced by NaBH 4 (yield 50%) using the procedure in the analogous sy n thesis described in chaptor 2 Racemic ( ) cis hydroxy intermediate 72 was obtained. It was found that ~ 2% of trans 75 co exists based on 1 H NMR peak integration of the characteristic 1 H NMR signals ( cis 72 : C4 Vs trans 75 3,t; Wyrick et al., 1993; Gatti et al., 2003). T he product collected was used in the next synthetic st ep without further separation T o synthesize cis product 60 and 61 t he racemic t rans ol 75 was prepared from racemic cis 72 based on a modified Mitsunobu reaction procedure (Wyrick et al., 1993)

PAGE 73

73 In the next step, b oth intermediate 72 and 75 were treate d with platinum/charcoal and triethyl amine (TEA) in MeOH solution under a H 2 baloon (Monguchi et al., 2006). In this way the m bromo on the pendant phenyl was de coupled This mild reaction conditio n proved selective i.e., no de coupling of the 7 chloro w as observed. The products ( 73 and 76 ) were quantitatively collected through simple filtration and concentration. In the next step, c ompound 73 or 76 was tosylated by TsCl and pyridine respectively Compared to the analogous reaction in chapter 2 and 3, t he reaction time in this case was adjusted to 8 hours The crude tosylated product was dire ctly used in the amination step to avoid possible decomposing. A facile amination procedure was developed to obtain intermediates 74 and 77 Brieftly, the tosylated ve rsions of 73 and 76 were transferred to a thick wall flask containing dimethyl amine (40% in H 2 O). The bottle was sealed and the mixture was stirred at 80 C overnight. After workup and silica gel chromatography racemic dimethylated product 74 or 77 w as col lected This procedure replaced the NaN 3 mediated azide formation and Pd/ch arcoal catalysed hydrogenation used previously (Wyrick et al., 1993), wherein Pd/C mediated hydrogenation at 45psi could result in de coupling of the 7 chlorine. It should be noted the yields for the tosylation, Mitsunobu reaction and amination reactions were low (around 30 40%). however, quantities of products obtain ed were suitable to immediately undertake in vitro pharmacological experiments. In the final step, a chiral stationary phase HPLC (CSP HPLC) using a Kromasil CelluCoat TM column was employed to enantiomerically separate t he racemic N,N dimethyl PAT analogs 74 and 77 The HPLC system used a mobile phase system

PAGE 74

7 4 consisting of 8% ethanol in 92% hexane with 0.1% diethyl amine and 0.1% trifluoroacetic acid added in as modifiers and the flow rate 4ml/min to resolve 1 mg compound dis solved in 200 l mobile phase in a single run The collected eluents w ere partitioned between CH 2 Cl 2 and water (adding H 2 O in first), extracted by CH 2 Cl 2 and dried by Na 2 SO 4 to completely remove the salt formed by diethyl amine and trifluoroacetic acid in the HPLC mobile phase Repeated separat ion and purification using the CSP HPLC system described above afforded each enantiomerically pure final produc t in 10 mg scale as white highly hydroscopic solids R f f or ( ) trans 58 : 15.5 min; R f f or (+) trans 59 : 12.0 min; R f f or ( ) cis 60 : 14.7 min; R f f or (+) cis 61 : 12.8 min Assignment of a bsolute configuration was based on analog y to the X ray crystal stru ctures of ( ) trans N,N dimethyl 1,2,3,4 tetrahydro 2 naphthalenamine 1 ( R ) ( ) camphor 10 sulfonic acid salt (Wyrick et al., 1993) and ( ) cis N,N dimethyl 1,2,3,4 tetrahydro 2 naphthalenamine 1 ( R ) ( ) camphor 10 sulfonic acid salt ( Bucholtz et al., 199 8 ) To synthesize 6 OMe,7 Cl PAT analogs 62 63 64 and 65 ( Fig 4 3 ) c ompound 70 and commercially available meta chloro styrene yielded substituted phenyltetralen 2 ol phenylacetates 78 using TFAA procedure described above (Vincek & Booth, 2009). Com pound 78 was reduced by NaBH 4 to afford racemic cis ol 79 Trans ol 81 was prepared from compound 7 9 using Mitsunobu conditions. Both cis 79 and trans 81 were co nverted to final enantiomers 62 63 64 and 6 5 using analogous methods described above CSP HPL C separation results: R f for ( ) trans 62 : 14.8 min. R f for (+) trans 6 3 : 12.2 min; R f for ( ) cis 64 : 12.7 min, R f for (+) cis 6 5 : 19.4 min.

PAGE 75

75 To synt h e size trans 6 OMe,7 Br PAT analogs 66 and 67 ( Fig 4 4 ) the p h enyltetralen 2 ol phenylacetate inter mediate 71 was reduced by NaBH 4 followed by tosylation and amination to give () trans 83 The CSP HPLC system described above was used to separate the entantiomers of () trans 83 R f for ( ) trans 66 : 14.6 min; R f for (+) trans 6 7 : 13.1 min. To synthesi ze trans 6 Cl PAT analogs 68 and 69 ( Fig 4 5 ) c ommercially available 2 (4 methoxyphenyl) acetic acid and and meta chlorostyrene yielded intermediate 84 using TFAA procedure described above (Vincek & Booth, 2009). After NaBH 4 reduction, product ci s 85 was subjected to tosylation and amination. The racemic ( ) trans 86 was separated by CSP HPLC. R f for ( ) trans 68 : 14.5 min; R f for (+) trans 6 9 : 12.7 min In vitro Pharmacological C haracterization R esults P reliminary i n vitro competitive binding assay measured the ability of PAT analog 66 67 68 and 69 to displace [ 3 H] radioligands from human 5HT2 C receptors expressed in HEK cell membranes (Booth et al., 2009). A ffinit y values (Ki) are summarized in Table 4 1 and representative radioligand compe tition displacement curves are shown in Fig 4 6 and 4 7 In addition, f unctional activity of PAT analog 68 was measured as activation of PLC/ I P signaling i n HEK c ells e xpressing h uman 5HT2C r eceptors (Booth et al., 2009). Preliminary results are that co mpound 68 is a potent partial agonist at 5HT2C receptor s compared to the endogenous agonist 5HT. Representative potency efficacy curves are shown in Fig 4 8 (Canal and Booth unpublished data, 2010) Comprehensive in vitro characterization of all the nove l PAT analogs described in this chapter has been

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76 initiated by pharmacologists in the medicinal chemistry laboratories of Dr. Booth at University of Florida Disscussion and F uture S tudies Novel Friedel Craft type cycli acylalkylation and enolized O acylati on reaction ( Vincet Booth 2009) facilitated preparation of stable phenylacetate intermediates 71 78 and 84 avoided the need of low yiel ding claisen condensation and polyphosphoric acid ( PPA ) mediated cyclization that used in previous PAT analogs synthe sis. All the novel analogs were obtained in 10 40mg scale ; enantiomeric purity is 100% as measured by the sensitive CSP HPLC methods described above. In v itro 5HT2C r adioligand competitive displacement assay results for analogs 66 67 68 a nd 69 revealed ( ) t rans 6 OMe 7 Cl Br PAT 66 (Ki=~10nM) has 5 fold higher binding affinity than its (+) trans isomer 67 (Ki=~ 5 0 nM) whilst ( ) trans 6 OMe Cl PAT 68 ( Ki=~ 1 0nM ) has 10 fold higher binding affinity than its (+) trans isomer 69 (Ki=~100 nM ). In both c ases, ( ) trans enantiomers demonstrate higher binding affinity on 5HT2C receptor s consiste nt with our observation that most ( ) trans configuration PAT analogs show higher binding affinity and functional potency than their corresponding (+) trans enantio mers (Booth et al., unpublished data, 2010) para subs tituted analogs are exceptions (e.g., see Chapter 3, in vitro affinity results ) And c omputer aided 5HT2C molecular modeling and ligand docking studies have been initiated by Dr. Cordova in the Booth lab to probe binding modes of PATs. Preliminary results support the hypothesis tested that ( ) trans PAT analogs with halogen substituted on the meta position of the pending phenyl ha ve high binding affinity at 5HT2C receptors than (+ ) trans isomers In fact, analogs 66 and 68 are among the highest affinity of PAT analogs (n=80) that the Booth labs have synthesized

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77 T he shallow Hill slopes (n H =0.6) of the radioligand competitive displacement curves for analogs 66 67 68 and 69 are characteristic of ligands wi th agonist functional activity at aminergic GPCRs (Knight et al., 2004). Encouragingly, analog 68 showed potent partial agonis m (EC 50 =20 nM) in compari son to the endogenous agonist 5 HT i n preliminary study suggesting, one or more of the other analogs 58 69 may also be potent 5HT2C agonists. T he results mentioned above indicate novel PAT analogs described in this chapter significantly expand the biochemical space probed in the putative orthosteric binding pocket of 5HT2 type receptors The analogs synthes ized and pharmacotherapeutical information that has/ will be obtained greatly contribute to our research for high potency, hi gh selectivity 5HT2C agonists. P revious 5HT2C molecular modeling studies based on bovine rhodopsin (Booth et al., 2009) indicated t he ( ) t rans PAT protonated amine can form an ionic bond with D3.32 of 5HT2A and 5HT2C receptors (distance 1.7), but, not with 5HT2B receptors. Experimental result from mutagenesis studies confirmed PAT ligand interaction with 5HT2C residue D3.32 ( Li and Booth; unpublished data, 2010). The bovine rhodopsin based model also suggested 5HT2C amino acid residue S3.36 might form hydrogen bonds with the 7 Cl and/or or 6 OH substituent. Recently, however, a new 5HT2C molecular model based on crystallographic str ucture of the human adrenergic receptor ( ADRB2, which is closer to 5HT2 receptors concerning sequence similarity than bovine rhodopsin ) revealed the PAT tetrahydronaphthalene moiety does not bind in close proximity to 5HT2C S 3.36 (Cordova and Booth, unp ublished results, 2010) this was experimentally confirmed by m utagenesis studies results that indicate ( ) trans

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78 PAT binds to the S3.36A 5HT2C point mutated receptor with affinity similar to the wild type receptor (Canal and Booth, unpublished data, 2010) New molecular modeling results (Cordova and Booth, unpublished data, 2010) suggest PAT t etrahydronaphthyl ring substituents (6 and/or 7 positions) might be capable of hydrogen bonding with 5HT2C residues in TMD 5 (e.g., S5.43) similar to the putative 5HT 2C interaction with the 5 OH moiety of 5HT (Kroeze et al., 2002). In addition to mutagenesis studies, the analogs synthesized here ( 58 69 ) will help determine the 3 dimensional interactions between the 5HT2C receptor and the PAT tetrahydronaphthyl and (C 4) pendant phenyl moieties. To further investigate these new 6,7 substituted PAT analogs as neurobiochemical probes and drug discovery leads more than one gram of versatile intermediate 72 was synthesized and purified, provid ing sufficient agent to get t rans 6 OMe ,7 Cl PAT analogs as well as trans 6 OH,7 Cl analogs in the near future. It is intriguing to verify t he previous notion that trans 6 OH,7 Cl PAT is an inverse agonist on 5HT2C receptor, and, if it is the case, to probe the possible mechanisms of the functional activity switch i.e. trans 6 OMe ,7 Cl PAT agonism v s. trans 6 OH,7 Cl PAT inverse agonism. With regard to drug discovery, a nalogs with highest 5HT2C agonist activity and 2A/2B inverse agonism will be assessed as pharmacotherapeutic candida te s in rodent models of obesity psychostimulant abuse and psychotic disorders e.g., see Rowland et al., 2008.

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79 Table 4 1 Preliminary binding affinities of PAT a nalogs 66,67,68,69 at 5HT2C r eceptor s 5HT2C binding affinity (nM) ( ) trans 6 OMe 7 Cl Br PAT 66 ~ 10 (+) trans 6 OMe 7 Cl Br PAT 67 ~ 50 ( ) trans 6 OMe Cl PAT 68 (+) trans 6 OMe Cl PAT 69 ~ 1 0 ~ 100 Fig ure 4 1 Preliminary in vitro c haracterization results of 6 O H ,7 Cl PATs and 6 OH, 7 OH PATs Fig ure 4 2 S ynthesi s of N,N dimethyl 4 phenyl 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine 58,59,60,61

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80 Fig ure 4 3 Synthesis of N,N dimethyl 4 (3 chlorophenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine 62,63,64 ,65 Fig ure 4 4 Synthesis of t rans N,N dimethyl 4 ( 3 bromo phenyl ) 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine 66,67

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81 Fig ure 4 5 S ynthesis of T rans N,N dimethyl 4 (3 ch lorophenyl) 6 methoxy 1,2,3,4 tetrahydro 2 naphthalene amine 68,69 Fig ure 4 6 Representative concentration response curves for t rans 6 OMe 7 Cl Br PAT i somer ( 66, 67 ) displacement o f [ 3 H] m esulergine from 5HT2C r eceptors Ki v alues s ummarized in Table 4 1

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82 Fig ure 4 7 Representative concentration response curves for t rans 6 OMe Cl PAT i somer (68, 69 ) displacement of [ 3 H] m esulergine from 5HT2C r eceptors. Ki v alues s ummarized in Table 4 1 Figgure 4 8 R epresentative c oncentra tion r esponse c urve for s erotonin ( c ircles ) and ( ) t rans 6 OMe Cl PAT 68 ( s quares ) a ctivation of PLC/ [ 3 H] IP F ormation in c lonal c ells e xpressed c loned H uman 5HT2C r eceptors

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83 APPEND IX : EXPERIMENTAL P ROCEDURES All chemicals were used as received from the manufacture r s Proton NMR spectra were obtained on an Oxford NMR AS400 spectrometer using CDCl 3 as solvent (TMS). Melting points were determined on a Meltemp apparatus and are correc ted. Thin layer chromatography was performed using Silica gel 60 precoated plates (EMD Chemicals Inc.). Column chromatography was performed using silica gel (230 400 mesh, Fisher Scientific). [ 3 H] Ketanserin (specific activity 72.2 Ci/mmol) and myo [2 3 H(N )] inositol (specific activity 18.5 Ci/mmol) were purchased from Perkin Elmer Life Science (Boston, MA) and [N 6 methyl 3 H] mesulergine (specific activity 72.0 Ci/mmol) was purchased from Amersham Biosciences (GE healthcare, Piscataway, NJ). Unless otherwis e noted, all other compounds were obtained in highest purity from Sigma Aldrich (St. Louis, MO). A: SYNTHETIC CHEMISTRY 1, Phenyl 2 propanone ( 2 1) P yridinium chlorochromate (40.2g, 0.186mol) and Al 2 O 3 (45g, 0.44mol) were dissolved in CH 2 Cl 2 200ml in a 500ml flask. The flask was connected with a mechanic stirrer, then was put into ice/salt bath and stirred for 20min. To this cooled solution, 1 phenyl 2 propanol 20 (21ml, 0.156mol) was added dropwise. The mixture was stirred at 0~4C for 30 min, then was allowed to recover to R.T. and proceeded for another 8 hours. TLC showed no starting material 21 could be detected. F or the workup, diethyl ether was added (150mlx4) to extract the product, the ether solution was filtered through a thick pad of celite/flor isil. The solvent was concentrate to afford dark green liquid as crude product. After silica column (3.5 % ethyl acetate in hexane as eluent), 15.6g pure product 21 was obtained as colorless liquid with the yield of 75%. 1 H 7.08 (m, 5H ), 3.65 (s, 2H), 2.08 (s, 3H)

PAGE 84

84 1,4 Dipheny 3 buten 2 one (22) and 4 (4 methylphenyl) 1 phenyl 3 buten 2 one (42 ) Benzaldehyde (23.85g, 0.225mol) was mixed with 850ml water containing KOH ( 3.5g, 85%), Compound 2 1 (15.1g,0.113mol) was added into this mixture. The ba tch was stirred and reflux ed at 55 C for 6.5 hours. For the workup, hydrochloric acid (37%) was added to monitor the PH of the mixture turn to 4, CH 2 Cl 2 was used (200mlx3) to extract the product The organic layer was washed with water, dried with anhydro us Na 2 SO 4 The crude product was concentrated and recrystalized by adding 20ml methanol and put in 20C freezer overnight. The light yellow solid formed over this period was filtered out for the next step. After several rounds of the same process, all the leftover mother solutions were combined and concentrated. The mixture was silica chromatographed using 2.5% ethyl acetate in hexane as eluent. The crude product was concentrated and recrystallized time in hexane methanol (2:1) solvent system to afford the last batch. MP 66 69 C ; 1 H 3 (d, J =12 H z 1H), 7.52 7.25 (m, 10H ), 6.78 (d d J =12, 1.8Hz, 1H), 3.9 5 (s, 2H). In preparation of compound 42 the reaction time was adjusted to13 h. 1 H J =12 H z 1H ), 7.4 3 7.1 7 (m, 10H), 6.74 (d, J = 12 H z 1H ), 3.97 (s, 2H ) 2.37 (s,3H ). 4 Phenyl 2 tetralone (23) and 3,4 dihydro 4 (4 methylphenyl) 2(1H) naphthalenone (43) In a 1L multi neck flask contained 470ml toluene and 36g polyphosphoric (110%), Compound 3 (7.5g, 0.0339mol) was added. T he mixtur e was allowed to be agitated vigorously by a mechanic stirrer and refluxed at 110 12 0 C for 3 h with nitrogen protection. After TLC showed no starting material existed in the reaction

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85 mixture, the batch was cooled to R.T. CH 2 Cl 2 was added for extraction ( 200mlx 5 ). T he organic layer was partitioned with water, washed with brine. The solution was concentrated off half the solvent, and then dried with anhydrous Na 2 SO 4 After being evaporated in vacuo the crude product was collected as thick dark orange oil. S ilica column was performed using 3% ethyl acetate in hexane to get the pure compound 23 as orange yellow oil. The yield was 41%. 1 H 7.14 (m,8 H), 7.15 (d, J =5.1 Hz, 1H ), 4.48 (t J =4.2 H z, 1H ), 3.62 (dd, J =24.8 15Hz, 2H), 2.9 (m, 2H ). For the preparation of compound 43 the reaction time was elongated to 4.5h. The purified product was solidified slowly in 4C environment. 1 H 6.99 (m, 8H ), 4.4 3(t J =4.8 Hz, 1H ), 3.70 3.56 (dd, J =25.5 15.6Hz, 2H), 2.90 (m, 2H ), 2.36 (s, 3H ) 1,2,3,4 T etrahydro 4 pheynl 2 naphthol (24) Compound 23 (10.5g, 0.047mol) was dis solved in 345 ml MeOH The solution was cooled to 0 C by ice/salt bath NaBH 4 (6.23g, 0.16mol) was added in portion over a period of 5min. The mixture was stirred at 0 C for 30min, then was s lowly recover ed to R T Till no more H 2 bubble was detectible, The mixture was allowed to reflux at 55 C for 10 hrs. Fo r the workup, 65ml water was added The mixture was evaporated to nearly dryness the gem was extracted by CH 2 Cl 2 the solution was partitioned with water, dried with anhydrous Na 2 SO 4 After evaporation in vacuo crude product 11.2g was obtained as orange yellow solid. A mediun size silica gel column (80ml) was performed to remove the majority of impurities using 6.5% ethyl acetate in hexane as eluent. The purified crude product 24 9.1g was afforded as yellow solid. 1 H NMR showd cis:trans = 75%:25%

PAGE 86

86 () C is 1,2,3,4 tetrahydro 4 pheynl 2 naphthol (25) and () cis 1,2,3,4 tetrahydro 4 (4 methylphenyl) 2 naphthol (49) The purified crude product 24 was treated with 650ml solvent (1% ethyl acetate in hexane). The mixture was heated and rotated in 85C water bath till the compound was totally dissolved. The solution was allowed to cool down smoothly. The recrystallization was completed after the mixture was kept in 20C freezer for 2 days. During this period the desired product cis 24 was slowly solidified and at tached to the wall of the flask. Up to 4 times of repeatedly recrystllizing would afford pure cis tetralols 25 (>97%) in 6g scale After several rounds of the same process, all the remaining mother solvent was combined and concentrated. The obtained compoun d was purified and recrystallized follow the same procedure again to afford the final batch of desired product 25. The total yield of this reduction and separation step was 62%. MP 110 112 C; 1 H 7.02 (m, 8H) 6.76 (d, J =6, 1H ), 4.21 4.13 (m, 2H ), 3.2 (dd J =11.7, 2.7 H z 1H), 2.92 (dd J = 1 1.1, 7.8 H z, 1H ), 2.39 (m, 1H). 1.89 ( dd, J =17.7, 9Hz 1H). Compound 49 was prepared through the same process (70% yield). 1 H NMR: 7.3 7.0 (m, 7H), 6.77 (d, J =5.7 Hz,1H), 4.42 (m, 1H), 4.40 (dd, J =9, 4.2 H z, 1H), 3.22 3.17 (dd, J =11.6, 2.6Hz, 1H), 2.95 2.88 (dd, J =11.7, 7.2Hz, 1H), 2.38 (m, 1H), 2.36 (s,3H), 1.9 (dd, J =17.7, 9 Hz, 1H). ( 2R,4R) cis 1,2,3,4 tetrahydro 4 (4 methylpheynl) 2 naphthalenol (44) A mixture of benzene ruthenium (II) chloride dime r (86mg, 0.17mmol) and ( R R ) N (2 amino 1,2 diphenylethyl) p toluenesulfonamide ( 125mg, 0.34mmol) in 2 propanol 60ml was stirred at 80C for 30 mim under argon. In a separate flask, a solution of KOH (96

PAGE 87

87 mg 1.7mmol) in 2 propanol 45ml was stirred and pre h eated to 50C. The starting material 43 (2.0g, 8.47mmol) in 2 propanol 150ml was pre heated to 50C, and was added the catalyst mixture followed by KOH solution and stirred at 50C for 1.5h. After the reaction, the mixture was immediately filtered through a thick pad of silica gel/ celite using ethanol as solvent. The solution was concentrated. The crude product existed as dark green solid. Medium size silica gel column was performed using 7% ethyl acetate in hexane as eluent. The separation was monitored by TLC and 1 H NMR. Up to 3 times of column chromatography afforded 500 mg pure ( 2R,4R) cis 44 (yield 25%). 1 H NMR: 7.32 7.0 (m, 7H), 6.77 (d, J =5.7 Hz,1H), 4.42 (m, 1H), 4.40 (dd, J =9, 4.2 Hz, 1H), 3.22 3.17 (dd, J =11.6, 2.6Hz, 1H), 2.95 2.88 (dd, J = 11.7, 7.2Hz, 1H), 2.38 (m, 1H), 2.36 (s,3H), 1.9 (dd, J =17.7, 9 Hz, 1H). [ ] 25 D 18 (MeOH, c=1). () Trans 1,2,3,4 tetrahydro 4 (4 methylpheynl) 2 naphthol (50) Tetralol 49 (100mg, 0.42mmol) was dissolved in 5.5ml THF. Triphenylphosphine (216mg, 0.82mmo l) and benzoic acid (103mg, 0.83mmol) was added into this solution. Diisopropyl azodicarboxylate (163l, 0.83mmol) was added dropwise. The mixture was stirred at R.T. overnight. The solvent was evaporated in vacuo The remaining gum was silica gel purifie d using toluene as eluent. The intermediate was dissolved in ethanol. 800l 1N NaOH in methanol was added. The mixture was stirred at R.T. overnight. The mixture was then evaporated in vacuo The final product was obtained as colorless thick oil through si lica gel column using 10% ethyl acetate in hexane as eluent. Total yield was 52%.

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88 1 H NMR: 7.4 7.0 (m, 8H, ArH), 4.42 (t, J =5.1 Hz, 1H), 4.40 (m, 1 H), 3.37 (dd, J = 12.5, 3.8Hz, 1H), 2.96 (dd, J =12.5, 5.3Hz, 1 H), 2.42 (s, 3 H ), 2.35(m, 1H), 2.18 (m, 1 H) () Ci s 2 tosyl 1,2,3,4 tetrahydro 4 phenyl naphthalene (26) and (2R,4R) 2 tosyl 1,2,3,4 tetrahydro 4 (4 metylphenyl) naphthalene (45) Compound 25 (5g, 22.3mmol) was dissolved in pyridine 120ml. To this solution p toluenesulfonyl chloride (8.5g, 44.5mmol) wa s added in portion. The mixture was stirred at R.T. for 2 days. The reaction was quenched by adding ice/water. The crude compound was extracted with ethyl acetate and dried with anhydrous Na 2 SO 4 It was further purified by silica column using first 4% ethy l acetate in hexane 500ml then 10% ethyl acetate in hexane to the end as eluent. Pure compound 26 was collected as light yellow solid (81% yield) 1 H NMR: 7.81(d, J =6.3 Hz, 2H), 7.32 7.01 (m, 10H ) 6.72 (d, J =6 Hz, 1H), 4.9 (m, 1H), 4.1(dd, J =8.7, 3.6 H z, 1H), 3.14(d, J =6.3 Hz, 2H), 2.45 (m, 1H), 2.43 (s, 3H), 2.1(dd, J =18, 9 Hz, 1H). Compound 45 was prepared from 44 through the same process as a white solid (81% yield). 1 H NMR: 7.81 (d, J =6.2 Hz, 2H), 7.35 7.0 (m, 9H), 6.73 (d, J =5.7 Hz, 1H) 4.90 (m 1H), 4.1 (m, 1H), 3.13 (d, J =6 Hz, 2H ), 2.45 (s, 3H), 2.37 (m, 1H), 2.33 (s, 3H), 2.05 (dd, J =18, 9Hz 1H). () T rans 2 azido 1,2,3,4 tetrahydro 4 phenyl naphthalene (27) and (2 R, 4 R ) 2 azido 1,2,3,4 tetrahydro 4 (4 metylphenyl) naphthalene (46) and ( ) cis 2 azido 1,2,3,4 tetrahydro 4 (4 methylphenyl) naphthalene (51) Compound 26 (4.5g, 11.3mmol) was dissolved in DMF 41ml The solution was treated with NaN 3 (1.85g,

PAGE 89

89 28.5mmol). The mixture was stirred at R.T. for up to 3 days. The reaction was quenched by adding ice/wa t er. The product was extracted by CH 2 Cl 2 The organic layer was washed by water, dried with anhydrous Na 2 SO 4 The concentrated crude product was purified by silica gel column using hexane as eluent first, then swiched to 10% ethyl acetate i n hexane. The pure product 27 was collected as thick colorless oil (yield 89%). 1 H 7.04 (m, 8H), 6.91 (d, J = 5.7Hz, 1H), 4,36 (t, J =4.5 Hz, 1H), 3.98 (m, 1H ), 3.24 (dd J =12.3, 3.6 Hz 1H), 2.93 (dd J =12.6, 5.7 Hz, 1H), 2.29 2.12 (m, 2 H). Compound 4 6 was prepared from 45 through the same process as thick colorless oil 1 H NM R: 7. 29 6.90 (m, 8 H ), 4,32 (t J =4.7 Hz, 1H), 3.98 (m, 1H), 3. 16 (dd, J =12.8, 3.7Hz, 1H), 2.91 ( dd J =12.6, 5.4Hz 1H) 2.35 (s, 3H), 2.32 2.1 (m, 2H) Compound 51 was prepared from 50 through the same process as thick colorless oil which solidified at 4C overnight. 1 H NMR: 7.26 7.0 (m, 7 H), 6.78 (d, J =6 Hz, 1H), 4.01 (m, 1H), 3.86 (m, 1H), 3. 17 (m, 1H), 2.97 (m, 1 H ), 2.43 (m, 1H), 2.35 (s, 3H ), 1.91 (dd, J =18, 9Hz 1H ) () T rans 2 amino 1,2,3,4 tetrahydro 4 phenyl naphthalene (28) and ( 2R,4 S ) 2 amin o 1,2,3,4 tetrahydro 4 (4 metylphenyl) naphthalene (47) and () cis 2 amino 1,2,3,4 tetrahydro 4 (4 metylphenyl) naphthalene (52) Compound 27 (2.4g, 9.6 mmol) was dissolved in 2 propanol 110ml and CH 2 Cl 2 10ml. The mixture was treated with 10% Pd on carbon (0.1g) and shaken on a Parr hydrogenation apparatus (45 psi) overnight. The catalyst was filtered off and the filtrate was evaporated in vacuo to afford the crude amine as a solid. For further purification, the crude amine was column chromatographed on sil ica gel using CH 2 Cl 2 as the eluent first then switched to 10%

PAGE 90

90 methanol in CH 2 Cl 2 The purified () trans 28 was obtained as slight green solid (yield 93%). MP 62 63 C 1 H 7.0 1 (m, 8H ), 6.94 (d, J =12, 6.3Hz 1H), 4,35 (t J =3.9 Hz 1H ), 3.28 (m 1H ) 3.1 4 (dd, J =6.2, 3.9Hz, 1H), 2.6 3 (dd J =12.3, 6.3Hz, 1H ), 2.0 3 (m, 2H). Compound 47 was prepared from 46 through the same process as dark green thick oil 1 H NMR: 7.17 6.91 (m, 8H), 4.32 ( t J =3.9 Hz, 1H ), 3.3 (m, 1H ), 3.16 (dd, J =12.3, 3.6Hz, 1H), 2.62 (dd, J =12, 6.3Hz, 1H) 2.32 (s, 3H ), 2.02 (m, 2H). Compound 52 was prepared from 51 through the same process as thick green oil 1 H NMR: 7.26 7.0 (m, 7 H), 6.77 (d, J =5.7 Hz 1H), 4.07 (m, 1H), 3.27 (m, 1H ), 3.06(m, 1H), 2.73 (m, 1 H ), 2.34 (s, 3H ), 2 .27 (m, 1H), 1.70 (dd, J =18, 9Hz 1 H) Resolution of () trans 2 amino 1,2,3,4 tetrahydro 4 phenyl naphthalene (28) Compound 28 (4.3g, 19.2m mol) and (1R) ( ) camphorsulfonic acid (5.81g, 25m mol) were dissolved in 160ml of acetonitrile/methanol 2:1. The sol ution was stirred and heated to 110C and kept under ref lux for 1.5hrs, then was cooled smoothly to R.T. and stirred overnight. The solvent were evaporated in vacuo to afford the crude salt as dark orange solid. The crude salt was washed by hexane and sma ll amount of methanol. The remaining solid was treated with acetonitrile/methanol 4:1 at the approximate ratio of 1g: 650ml The mixture was rotated in 75C water bath till the compound was dissolved completely. T he solution was cooled down smoothly and kep t at 0C for up to 2 days. I n the first several rounds of processing the crystals falling off were needle shaped and optically dextrorotatory. Mosher reagent derivatization assay

PAGE 91

91 revealed it contains more of the undesired (+) trans amine than ( ) trans iso mer. The needle like crystals were separated and the mother solution was concentrated. Several subsequent recrystallizations using the same solvent system afforded pure ( ) trans 28 camphorsulfonic salt as colorless prisms ~650mg in 5 batches. MP 218 220 C 25 D 68.5 (MeOH). Each batch of the pure crystals was recovered to free amine ( ) trans 28 by stirring in a mixture of 10ml CH 2 Cl 2 and 10ml of saturated aqueous NaHCO 3 10ml overnight. The organic layer was separated and dried with anhydrous Na 2 SO 4 The solvent was evaporated in vacu e The pure amine was a slight green solid. (Total resolution yield 10%) Proc edures for Mosher reagent assay. 3~5mg previously obtained crystals was first converted back to free amine 28 by being partitioned between dichl oromethane and aqueous NaHCO 3 The amine was then dissolved in 200l dichloromethane, treated by 50l pyridine and 5l (R) Mosher acid chloride. The mixture was stirred at R.T. for 2 h, After evaporation in vacu e the mixture was directly tested to get 1 H NMR spectrum in CDCl 3 without purification. ( ) T rans N,N dimethyl 4 pheynl 1,2,3,4 t etrahydro 2 naphthalenamine (30); ( ) trans N,N dimethyl 4 (4 methyl pheynl ) 1,2,3,4 t etrahydro 2 naphthalenamine (48) and () cis N,N dimethyl 4 (4 methyl pheynl ) 1,2,3,4 t etrahydro 2 naphthalenamine (53) F ree amine ( ) trans 28 (280mg, 1.26mmol) of amine was added in 5.1 ml of 95% formic acid and 3.4ml of 38% formaldehyde The mixture was stirred at 100 C for 7 hrs. The volatiles was evaporated in vacuo and the residue wa s dissolved in CH 2 Cl 2 and partitioned with saturated aqueous NaHCO 3 The organic layer

PAGE 92

92 was dried with anhydrous Na 2 SO 4 After evaporation the crude product was collected as a light yellow gum which solidified upon standing. The amine was dissolved in ethy l acetate. 3ml of 1N HCl in ether was added dropwise. The solvent was evaporated in vacue The salt was washed by ethyl acetate and hexane, dried in vacue 30 free base 25 D 57.1 (MeOH ) 1 H 7.0 (m, 8H), 6.95 (d, J =5.4 Hz, 1H), 4.36 (d, J =3.3 Hz, 1H), 3.03 (dd, J =12, 3Hz, 1H), 2.85 (dd, J =12.2, 7.3Hz, 1 H) 2.64 (m, 1 H ), 2.23 (s, 6 H ) 2.13 (m, 2H ). 30 HCl salt MP 208 210 25 D 6 6 .5 (MeOH ) 1 H NMR (CD 3 OD) 7.36 6.98 (m, 8H), 4.58 (m,1H), 3.47 (m, 1H), 3.1 2.92 (m, 2 H), 2.9 (s, 6H), 2.21 (m, 2H ) Compound 48 was prepared from 47 through the same process as yellow thick oil 48 free base 25 D 64.2 (MeOH). 1 H NMR 7.26 6.89 (m, 8H), 4.32 (t, J =3.7 Hz, 1H), 3.0 2 (dd, J =12.2, 3.8Hz, 1H ), 2.85 (dd, J =12.2, 7.4Hz, 1H ) 2.64 (m, 1H), 2.3 (s, 3H), 2.25 (s, 6H), 2.1(m, 2H) 48 HCl salt MP 220 222 25 D 67 (MeOH). 1 H NMR (CD 3 OD) 7.35 6.91 (m, 8H), 4.55 (m,1H), 3.57 (m,1H), 3.16 2.91 (m, 2H), 2.88(s, 6H), 2.4 (m, 2H). 2.34 (s, 3H) Compound 53 was prepared from 52 through the same process as yellow thick oil 1 H NMR 7.39 (s,1H), 7.28 7.2 (m,5H), 7.14 (t, J =5.4Hz,1H), 6.90 (d, J =5.7Hz,1H), 4.2 (dd, J =9, 3.9Hz,1H), 3.18 3.03 (m,2H ), 2.96 (m,1H), 2.51 (s,6H ), 2.48 (s,3 H), 2.46 (m,1H), 1.87 (dd, J =18.2, 9.2Hz 1H ). 1 H NMR spectra of 54 are the same as 53 (+) C is N,N dimethyl 4 (4 methyl pheynl ) 1,2,3,4 t etrahydro 2 naphthalenamine ((+) cis 54) Free amine 25 D + 28.6 HCl salt 25 D + 17.0

PAGE 93

93 ( ) C is N,N dimethyl 4 (4 methyl pheynl ) 1,2,3,4 t etrahydro 2 naphthalenamine (( ) cis 5 5 ) Free amine 25 D 28.9 HCl salt 25 D 18.2 (+) Trans N,N dimethyl 4 pheynl 1,2,3,4 t etrahydro 2 naphthalenamine ((+ ) trans 57 ) () T rans 57 was prepared from 49 through tosylation, azidition, reduction NMR are the same as preparation and spectra of 45 48. () T rans 57 was collected from HPLC separation Free amine ] 25 D + 65.4 HCl salt 25 D + 61.3 2 (3 chloro 4 methoxyphenyl)acetic acid ( 70 ) 2 (4 methoxyphenyl)acetic acid (1g, 6.0mmol) was dissolved in 10ml acetone, Oxone (3.7g, 6.0mmol) was added in, the suspension was stirred at R.T. for 15min, NaCl aqueous solution (1.4g in 10ml H 2 O) was added, the mixture was stirred for 6hrs. For the workup, the mixture was evaporated in vacuo, the residue was diluted with water and extracted with ethyl acetate, the organic layer was washed with brine, dryed by Na 2 SO 4 co ncentrated in vacuo The crude product was recrystalized using 10% toluene in hexane as solvent. The final product was collected as white needle like solid (yield 50%). 1 H NMR (CD 3 OD) 7.3 (m, 1H), 7.1 (m,1H) 6.9 (m, 1H) 3.9 (s,3H) 3.6 (S,2H) 4 (3 bromo phen yl) 6 methoxy 7 chloro 3,4 dihydronaphthalen 2 yl 2 (3 chloro 4 methoxyphenyl)acetate ( 71 ), 4 (3 chloro phenyl) 6 methoxy 7 chloro 3,4 dihydronaphthalen 2 yl 2 (3 chloro 4 methoxyphenyl)acetate ( 78), and 4 (3 chlorophenyl) 6 methoxy 3,4 dihydronaphthalen 2 yl 2 (4 methoxyphenyl)acetate ( 84 ) A flask containing meta chlorostyrene(0.4ml, 3mmol) was pre cooled in ice/water. In a separated flask, under nitrogen protection, Compound 70 (1.8g, 9mmol) was dissolved in trifluoroacetic acid anhydride (1.3ml, 9.5mmol). Once the mixture turned to

PAGE 94

94 an orange color liquid, a double end needle was used to transfer the liquid into the bottle contains styrene. The mixture was stirred overnight. For the workup, ethyl acetate was used for extraction for three times. Large amount of saturated NaHCO 3 water solvent was used washing the crude product for four times. The organic layer was combined and dried by Na 2 SO 4 After medium silica gel column using 5% ethyl acetate in hexane as solvent, crude product 78 was obtained as colorless thick oil. 70% yield calculated based on meta chlorostyrene consumption. 78 1 H NMR ( CDCl 3 ) 6.2 (m,9H), 6.20 (s, 1H), 4.21 (t, J = 3.7 Hz, 1H), 3.9 (s,3H), 3.8 (s,3H), 3.69 (s, 2H), 2. 0 5 ( m 2 H ) Compound 71 was prepared using meta bromostyrene following the same procedure. 71 1 H NMR ( CDCl 3 ) 6.2 (m,9H), 6.20 (s, 1H), 4.21 (t, J = 3. 6 Hz, 1H), 3. 8 (s,3H), 3. 7 (s,3H), 3.6 5 (s, 2H), 2. 0 5 ( m 2 H) Compound 84 was prepared using 2 (4 methoxyphenyl)acetic acid and meta chlorostyrene following the same procedure. 84 1 H NMR ( CDCl 3 ) 0 6.3 4 (m,11H), 6.2 5 (s, 1H), 4.2 4 (t, J = 3.7 Hz, 1H), 3.8 (s,3H), 3.72 (s,3H), 3.69 (s, 2H), 2.05 (m,2H) () C is 4 (3 bromo phenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydronaphthalen 2 ol ( 72 ) () cis 4 (3 chlorophenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydronaphthalen 2 ol ( 79 ), and () cis 4 (3 chlor ophenyl) 6 methoxy 1,2,3,4 tetrahydronaphthalen 2 ol ( 85 ) NaBH 4 (240mg,6.34mmol) was dissolved in 17ml MeOH. The solution was cooled to 0 C by ice/salt bath. Compound 78 (1.1g, 2.17mmol) was dissolved in 4ml toluene/methanol (1:1). The solution was injec ted into

PAGE 95

95 the flask containing NaBH 4 solution. The mixture was stirred at 0 C for 15min, then was slowly recovered to R.T. Till no more H 2 bubble was detectible, The mixture was allowed to reflux at 55 C overnight. For the workup, 10ml water was added. T he mixture was evaporated to nearly dryness, the gem was extracted by CH 2 Cl 2 the solution was partitioned with water, dried with anhydrous Na 2 SO 4 After evaporation in vacuo crude product was obtained as orange yellow solid. A mediun size silica gel colu mn (80ml) was performed first using pure CH 2 Cl 2 as eluent, then switched to 3% MeOH in CH 2 Cl 2 The purified crude product 79 was afforded as yellow thick oil (yield75%). 79 1 H NMR ( CDCl 3 ) 7. 01(m, 5H), 6.27 (s,1H), 4.1 6 4.0 7 (m,2H), 3.6 2 (s,3H) 3.08 ( dd J = 10.8, 3.9 Hz, 1H ), 2.79 ( dd J = 11.1, 6 Hz, 1H), 2.38 (m, 1H), 1.8 (dd J = 19, 9 Hz, 1H ). 72 was prepared through reduction of 71 following the dame procedure. 72 1 H NMR ( CDCl 3 ) 7.2 9 7. 0 (m, 5H), 6.2 4 (s,1H), 4.1 3 4.0 8 (m,2H), 3.6 (s,3H) 3. 1 ( dd J = 1 1.3 3. 5 Hz, 1H ), 2. 81 ( dd J = 1 2.3 6 Hz, 1H), 2. 41 (m, 1H), 1. 78 (dd J = 1 8.5 8.7 Hz, 1H ). 85 was prepared through reduction of 84 following the dame procedure. 85 1 H NMR ( CDCl 3 ) 7.1 (m, 4H), 6.73 (m,1H), 6.26 (s,1H), 4.14 4.0 7 (m,2H), 3.6 7 (s,3H) 3.13 (dd J = 11.6, 3.6 Hz, 1H), 2.82(dd, J = 11.3, 7.8 Hz, 1H), 2 .37(m, 1H), 1.83 (dd J = 18.2, 8.7 Hz, 1H). () T rans 4 (3 bromo phenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydronaphthalen 2 ol ( 75 ) () trans 4 (3 chloro phenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydronaphthalen 2 ol ( 81) Compound 79 (616mg, 1.9mmol) was

PAGE 96

96 dissolved in 25ml THF. Triphenylphosphine (998mg, 3.8mmol) and benzoic acid (465mg, 3.8mmol) was added into this solution. Diisopropyl azodicarboxylate (749l, 3.8mmol) was added drop wise. The mixture was stirred at R .T. overnight. The solvent was evaporated in vacuo The remaining gum was silica gel purified using toluene as eluent. The intermediate was dissolved in ethanol 45ml. 1N NaOH in methanol (3.6ml) was added. The mixture was stirred at R.T. overnight. The mix ture was then evaporated in vacuo The final product was obtained as colorless thick oil through silica gel column using 3% MeOH in CH 2 Cl 2 as eluent. Total yield was 35%. 81 1 H NMR ( CDCl 3 ) 7.0 (m, 5H), 6.41 (s,1H), 4.3 ( m 1H), 4.21 (m,1H), 3.6 (s,3H), 3.14 (dd J = 12.2, 3.6 Hz, 1H), 2.79 ( J = 12.4, 4.5 Hz, 1H ), 2.21 2.17 (m, 1H), 2.04 1.98 (m, 1H). Compound 75 was prepared from 72 following the same procedure 75 1 H NMR ( CDCl 3 ) 6 .98 (m, 5H), 6.40 (s,1H), 4.2 (m,2H), 3.7 (s,3H) 3.14 ( dd J = 12.3, 3.6 Hz, 1H), 2.75 ( J = 12.2, 4.6 Hz, 1H), 2.24 2.17 (m, 1H), 2.05 1.97 (m, 1H). () C is 4 phenyl 6 methoxy 7 chloro 1,2, 3,4 tetrahydronaphthalen 2 ol (73 ) and () trans 4 phenyl 6 methoxy 7 chloro 1,2,3,4 tetrahydronaphthalen 2 ol ( 76 ) Hydroxyl intermediates cis 72 (170mg, 0.46mmol) was dissolved in 5ml MeOH, platinum/charcoal (5.1mg), and triethyl amine 80l were added in consequently. The mixture was stirred at RT under a hydrogen bulb for 2hs. The products were quantitatively obtained through simple filtration and concentration. 73 1 H NMR ( CDCl 3 ) 7.40 6.82 (m, 6H), 6.21 (s,1H), 4.2 4.02 (m,2H), 3.6 (s,3H) 3.14 3.04 (m, 1H), 2.82 2.77 (m,1H), 2.39 (m, 1H), 1. 8 (q, J = 12.1 Hz, 1H)

PAGE 97

97 76 1 H NMR ( CDCl 3 ) 7.35 7.02 (m, 6H), 6.41 (s,1H), 4.3 (t, 1H), 4.21 (m,1H), 3.6 (s,3H), 3.14 3.10 (m, 1H), 2.79 2.72 (m,1H), 2.21 2.17 (m, 1H), 2.04 1.98 ( m, 1H) ( ) T rans N,N dimethyl 4 (3 chlorophenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthale ne amine (6 2), (+ ) trans N,N dimethyl 4 (3 chlorophenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine ( 63 ), ( ) Cis N,N dimethyl 4 (3 chlorophenyl) 6 methoxy 7 chloro 1,2,3,4 tet rahydro 2 naphthalene amine (64 ), and (+) Cis N,N dimethyl 4 (3 c hlorophenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine ( 65 ) Compound 80 (or 82 for cis analogs preparation) (337mg, 1.04mmol) was dissolved in pyridine 7ml. To this solution p toluenesulfonyl chloride (397mg, 2.08mmol) was added in by port ion. The mixture was stirred at R.T. overnight. The reaction was quenched by adding ice/water. The crude compound was extracted with ethyl acetate and dried with anhydrous Na 2 SO 4 It was further purified by silica column using 10% ethyl acetate in hexane t o get the crude product.Without further purification, the crude product was transferred into a thick wall flask, dimethyl amine 4ml(40% in H 2 O) was added. The bottle was sealed and the mixture was stirred at 80 C overnight. After extraction using CH 2 Cl 2 t he crude product was washed by water, dried by Na 2 SO 4 Small silica gel column and 5% MeOH in dichloromethane as solvent was used to purify the product. Shimadzu HPLC system with Kromasil CelluCoat TM column was used to separate the racemic products. The m obile phase system contained 8% ethanol in 92% hexane with 0.1% diethyl amine and 0.1% trifluoroacetic acid added as modifiers. The flow rate was 4ml/min. loading amount was 1~2mg compound solved in 200l mobile phase. The eluents containing the desired pr oduct were combined and concentrated. The salt from

PAGE 98

98 DEA and TFA made the product a colorless liquid. The product needed to be partitioned between CH 2 Cl 2 and water (adding H 2 O first), extracted by CH 2 Cl 2 and dried by Na 2 SO 4 to totally get rid of the salt. Several rounds of HPLC separation and purification afforded us pure final products. The compounds were converted to HCl salts by being dissolved in HCl/ether solution and evaporated in vacuo 6 2 Highly hygroscopic solid 25 D = ( )14 ( c 1 00 CH 2 Cl 2 ) R f of ( ) trans 6 2 : 14.8min in HPLC separation 1 H NMR ( CDCl 3 ) 7.38 7.20 (m, 3H), 6.92 (s,1H), 6.85 (m, 1H), 6.54 (s, 1H), 4.42 (m, 1H), 3.79 (s,3H), 3.34 3.21 (m,2H), 3.10 2.98 (m,1H), 2.75 (s,6H), 2.40 2.35 (m,2H), 13 C NMR (CDCl 3 ) 29.1, 31.8, 42.2, 43.4, 56.2, 57.9, 112.8, 122.1, 125.5, 126.4, 127.4, 128.2, 130.1, 130.7, 134.3, 134.8, 146.0, 154.1. HRMS m/z Calcd for C 19 H 2 2 Cl 2 N O 350.108, 352.105 [M+H] + Found 350.1085, 352.1055 [ C 19 H 2 1 Cl 2 N O +H] + isotope pattern confi rmed. 63 Highly hygroscopic solid 25 D = (+)14 ( c 1 00 CH 2 Cl 2 ) R f of (+) trans 6 3 : 12.2min in HPLC separation. 1 H NMR 13 C NMR and HRMS spectra are the same with 62 64 Highly hygroscopic solid 25 D = ( )272 ( c 1 00 CH 2 Cl 2 ) R f of ( ) ci s 6 4 : 12. 7min in HPLC separation 1 H NMR ( CDCl 3 ) 7.05 (m, 5H), 6.26 (s, 1H), 4.15 (dd, J =9.15, 3.45 Hz,1H), 3.63 (m,1H), 3.62 (s,3H), 3.17 2.99 (m,2H), 2.85 (s,6H), 2.54 2.50 (m,1H), 1.89 (dd, J =18.6, 9 Hz, 1H), 13 C NMR (CDCl 3 ) 28.7, 29.7, 34.4, 45.7, 56. 1, 60.9, 112.4, 121.7, 125.0, 126.8, 127.7, 128.5, 130.3, 130.6, 134.8, 136.7, 146.0, 154.0. HRMS m/z Calcd for C 19 H 2 2 Cl 2 N O 350.108, 352.105 [M+H] + Found 350.1086, 352.1057 [ C 19 H 2 1 Cl 2 N O +H] + isotope pattern confirmed.

PAGE 99

99 6 5 Highly hygroscopic solid 25 D = (+)282 ( c 1 00 CH 2 Cl 2 ) R f of (+) ci s 6 5 : 19.4min in HPLC separation. 1 H NMR 13 C NMR and HRMS spectra are the same with 6 4 ( ) T rans N,N dimethyl 4 phenyl 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine ( 58), (+ ) trans N,N dimethyl 4 phenyl 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine ( 59 ), ( ) c is N,N dimethyl 4 phenyl 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine (6 0 ), (+) c is N,N dimethyl 4 phenyl 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine (6 1 ) Com pounds were prepared began with intermediate 74 or 77 following the same procedure described above. 58 Highly hygroscopic 25 D = ( )46 ( c 1 .00, CH 2 Cl 2 ), R f of ( ) trans 58 : 14.6min in HPLC separation. 1 H NMR ( CDCl 3 ) 7.14 (m, 4H), 6.93 (d, J =6 Hz, 2H), 6.54 (s,1H), 4.49 (m,1H), 3.76(s,3H), 3.27 3.21 (m,1H), 3.08 2.98 (m,2H), 2.72 (s,6H), 2.41 2.30 (m,2H), 13 C NMR (CDCl 3 ) 29.3, 31.8, 42.2, 43.7, 56.1, 58.0, 112.9, 121.8, 125.6, 127.1, 128.1, 128.5, 128.8, 130.6, 135.2, 143.9, 154.0, 162.6. H RMS m/z Calcd for C 19 H 23 ClNO 316.147, 318 .144 [M+H] + Found 316.146, 318.145 [ C 19 H 2 1 ClN O +H] + isotope pattern confirmed. 59 Highly hygroscopic solid 25 D = (+)42 ( c 1 00 CH 2 Cl 2 ) R f of (+) trans 59 : 13.06min in HPLC separation. 1 H NMR 13 C NMR and HRM S spectra are the same with 58. 60 Highly hygroscopic solid 25 D = ( )98 ( c 1 00 CH 2 Cl 2 ) R f of ( ) ci s 66b : 14.6min in HPLC separation. 1 H NMR ( CDCl 3 ) 7.15 (m,6H), 6.28 (s,1H), 4.18 4.14 (dd, J =8.7, 3.6 Hz, 1H), 3.59 (s,3H), 3.19 3.07 (m,2H), 2 .85 (s,6H), 2.52 2.50 (dd,

PAGE 100

100 J =7, 2 Hz, 1H), 2.34 2.30 (t, J = 5.5 Hz, 1H), 1.96 1.87 (dd, J = 18.5, 9.1 Hz, 1H). 13 C NMR (CDCl 3 ) 34.3, 39.2, 40.3, 46.0, 56.0, 61.1, 112.6, 121.3, 124.9, 127.4, 128.5, 129.0, 130.4, 137.5, 143.8, 153.8, 162.7. 163.1.HRMS m/ z C alcd for C 19 H 2 3 ClN O 316.147, 318.144 [M+H] + Found 316.146, 318.145 [ C 19 H 2 1 ClN O +H] + isotope pattern confirmed. 6 1 25 D = (+)111 ( c 1 .00, CH 2 Cl 2 ), R f of (+) cis 67b : 12.8min in HPLC separation. 1 H NMR 13 C NMR and HRMS spect ra are the same with 60. ( ) T rans N,N dimethyl 4 (3 bromophenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine (6 6 ) and ( + ) trans N,N dimethyl 4 (3 bromophenyl) 6 methoxy 7 chloro 1,2,3,4 tetrahydro 2 naphthalene amine (6 7 ), ( ) t rans N,N dim ethyl 4 (3 chlorophenyl) 6 methoxy 1,2,3,4 tetrahydro 2 naphthalene amine (6 8 ), and ( ) Trans N,N dimethyl 4 (3 chlorophenyl) 6 methoxy 1,2,3,4 tetrahydro 2 naphthalene amine ( 69) Compounds were prepared began with intermediate 71 or 85 respectively follo wing the same procedure described above. 6 6 25 D = ( )14 ( c 1 .00, CH 2 Cl 2 ), R f of ( ) trans 6 6 : 14.6min in HPLC separation. 1 H NMR ( CDCl 3 ) J =5.7 Hz, 1H), 7.26 (d, J =3.6Hz,1H), 7.19 (t, J =5.7 Hz, 1H), 7.09 (s, 1H), 6.87 (d, J =6 Hz, 1H), 6.50 (s,1H), 4.45 (m,1H), 3.78(s,3H), 3.29 3.23 (m,2H), 3.9 3.02 (m,3H), 2.76 (s,6H), 2.38 2.34 (m,2H), 13 C NMR (CDCl 3 ) 29.1, 31.8, 42.3, 43.4, 56.2, 57.9, 112.8, 122.2, 123.1, 125.5, 126.9, 130.3, 130.8, 131.1, 134.3, 146.3, 154.2, 162. 7. HRMS m/z Calcd for C19H 22 BrClNO

PAGE 101

101 394.057, 396.055 [M+H] + Found 394.058, 396.056 [ C 19 H 21 Br ClNO +H] + isotope pattern confirmed. 6 7 25 D = (+)14 ( c 1 .00, CH 2 Cl 2 ), R f of (+) trans 6 7 : 13.1min in HPLC separation 1 H NMR 13 C NMR and HRMS spectra are the same with 66. 6 8 25 D = ( ) 10 ( c 1 .00, CH 2 Cl 2 ), R f of ( ) trans 6 8 : 14.5 min in HPLC separation. 1 H NMR ( CDCl 3 ) J =1.2 Hz, 1H), 7.260 7.15 (m,1H), 7.10 (d, J =6.3 Hz, 1H), 7.09 (s, 1H), 6.91 (d d, J =4.8, 1.5 Hz,1H), 6.78 (dd, J =5.4, 1.8 Hz, 1H), 6.44 (d, J =1.5 Hz, 1H), 4.32 (t, J =3.6 Hz, 1H), 3.69 (s,3H), 3.04 2.99 (dd, J = 11.9, 3.8 Hz, 1H), 2.85 2.79 (m,1H), 2.69, (m,1H), 2.34 (s,6H), 2.16 2.11 (m,2H), 13 C NMR (CDCl 3 ) 31.1, 34.4, 41.5, 44.0, 55.2, 56.6, 113.4, 114.3, 126.4, 126.8, 127.8, 128.7, 129.5, 130.3, 134.1, 137.7, 148.4, 157.9. HRMS m/z Calcd for C19H 23 ClNO 316.147, 318.144 [M+H] + Found 316.417, 318.144, [C 19 H 23 ClNO +H] + isotope pattern confirmed 6 9 25 D = (+)1 2 ( c 1 .00, CH 2 Cl 2 ), R f of (+) trans 6 9 : 12.7 min in HPLC separation 1 H NMR 13 C NMR and HRMS spectra are the same with 68. B: PHARMACOLOGICAL ASSA YS Clonal cell culture and transfection (Booth et al., 2009) Chinese hamster ovary K1 cells (CHO, ATCC CCL 12 medium supplemented with 10% fetal bovine serum and 1% sodium bicarbonate (Mediatech 25 035 CI), 10 IU/ml penicillin and 10 ug/ml streptomycin. Human embryonic kidney 293 cells (HEK, ATCC CRL 1573) were maintained in Eagle minimum essential medium with 10% fetal

PAGE 102

102 bovine serum and 2 mM L glutamine, 0.1 mM non essential amino acids, 1.5 g/L sodium bicarbonate, 1.0 mM sodium pyruvate, 10 IU/ml penicillin, and 10 ug/ml streptomycin. Cells were grown in a humidified incubato r at 37 C with 5% carbon dioxide. The cDNAs encoding the human serotonin 5 HT 2A 5 HT 2B and 5HT 2C (unedited isoform) receptors were obtained fr om UMR (Rolla, MO). Serotonin 5 HT 2A and 5 HT 2B receptors were transiently expressed in HEK ce lls (Setola et al., 2005) and 5 HT 2C receptors were transiently expressed in CHO cells (Porter et al., 1999). HEK cells were grown to 90 95% confluence in 100 mm dishes and transfected with 24 g of plasmid DNA for the wild type 5 HT 2A or 5 HT 2B receptor sequences using 40 l of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per dish. Transfection proceeded for 24 hrs, then, medium was replaced by fresh growth medium and cells were allowed to express 5 HT 2A or 5 HT 2B receptors for another 24 hrs. CHO cells were grown to 40% conflu ence in 100 mm dishes and transfected with 12 g of plasmid DNA for the wild type 5HT 2C receptor sequence and 32 l of Lipofectamine 2000, then, transfection and expression proceeded as above. Radioreceptor assays (Booth et al., 2009) Radioreceptor satu ration and competition binding assays were performed using membrane homogenates prepared from transfected CHO cells cells, similar to methods reported previously (Booth et al., 2002; Moniri et al., 2004). [ 3 H] Ketanserin was used to radiolabel serotonin 5 H T 2A receptors and [ 3 H] mesulergin e was used to label serotonin 5 HT 2B and 5 HT 2C receptors (Knight et al., 2004). Forty eight hours following transfection, cells were harvested and homogenized in 50 mM Tris HCl containing 0.1 % ascorbic acid and 4.0

PAGE 103

103 mM calci um chloride at pH 7.4 (assay buffer). The homogenate was centrifuged at 35,000 x g for 25 min and the resulting membrane pellet was re suspended in assay buffer. Protein concentration was determined by the method of Lowry et al. (Lowry, 1951). For saturatio n binding assays, membrane suspension containing 20 g (for 5 HT 2A receptor), 50 g (for 5 HT 2B receptor) or 100 g (for 5 HT 2C receptor) protein was incubated with 0.1 5.0 nM [ 3 H] ketanserin (5 HT 2A receptors) or 0.1 20 nM [ 3 H] mesulergine (5 HT 2B and 5 H T 2C receptors) in a total assay buffer volume of 250 l. Non specific binding was determined in the presence of 10 M methysergide (5 HT 2A receptors) or 1.0 M mianserin (5 HT 2B and 5 H T2C receptors). Competition binding assays were conducted under the same c onditions using 1.0 nM [ 3 H] ketanserin (5 HT 2A receptors), 5.0 nM [ 3 H] mesulergine (5 HT 2B receptors), or 1.0 nM [ 3 H] mesulergine (5 HT 2C receptors) (~ K d concentration). Incubation of radioreceptor binding assay mixtures was for 1.0 h at 37C, with terminatio n by rapid filtration through Whatman GF/B filters using a 96 well cell harvester (Tomtec, Hamden, CT). The membrane bound [ 3 H] radioligand retained on the filter discs was quantified by liquid scintillation spectrometry. Data were analyzed by nonlinear re gression using the sigmoidal curve fitting algorithms in Prism 4.03 (GraphPad Software Inc., San Diego, CA). Ligand affinity is expressed as an approximation of K i values by conversion of the IC 50 data to K i values using the equation K i = IC 50 /1 + L / K d whe re L is the concentration of radioligand having affinity K d (Cheng, 1973). Each experimental condition was performed in triplicate and each experiment was performed a minimum of three times to determine S.E.M.

PAGE 104

104 Assay for activation of PLC and [ 3 H] IP forma tion Functional activation of PLC was measured as [ 3 H] IP formation in HEK cells tr ansiently expressing serotonin HT 2A or 5 HT 2B receptors or CHO cells transiently expressing serotonin 5 HT 2C receptors, as previously reported (Moniri et al., 2004). HEK cel ls expressing serotonin 5 HT 2A or 5 HT 2B recept ors, or, CHO cells expressing 5 HT 2C receptors were seeded at 10 5 cells per well in 12 well plates in inositol free Dulbecco's modified Eagle's medium (DMEM) for 12 24 hours with 1.0 Ci/ml myo [2 3 H] inosito l, the radiolabeled precursor of the PLC DMEM for HEK cells). Cells then were washed and incubated in inositol free DMEM containing 10 mM lithium chloride, 10 M pargyline and various concentrations of test ligand for 45 60 min at 37 C and 5% CO 2 After aspiration of media, wells were lysed by incubation with 50 mM formic acid (15 60 min). Formic acid was neutralized with ammonium hydroxide and contents from each well were added to individual AG1 X8 200 400 formate resin anion exchange columns. Ammonium formate/formic acid (1.2 M/0.1 M) was used to elute [ 3 H] IP directly into scintillation vials for counting of tritium by liquid scintillation spectrometry. Resulting da ta were analyzed using the nonlinear regression algorithms in Prism 4.03 and are expressed as mean percentage of basal control [ 3 H] IP formation, with potency expressed as concentration required to stimulate (EC 50 ) or inhibit (IC 50 ) maximal basal (constitu tive) [ 3 H] IP formation by 50% S.E.M. (n

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105 LIST OF REFERENCES Adams DR, Bentley JM, Benwell KR, Bickerdike MJ, Bodkin CD, Cliffe IA, Dourish CT, George AR, Kennett GA, Knight AR, Malcolm CS, Mansell HL, Misra A, Quirk K, Roffey JR, Vickers SP. Pyrrolo(iso)quinoline deri vatives as 5 HT(2C) receptor agonists. Bioorg Med Chem Lett. 2006 ; 16:677 80. Adrian, GP. Givors. Preparation of 4 (disubstituted aryl) 1 tetralones as intermediates for serotonin antagonists. Eur Pat Appl 1989; US5019655 Allerton CMN, Andrews MD, Blag g J, Ellis D, Evrard E, Green MP, Liu KKC, McMurray G, Ralph M, Sanderson V, Ward R, Watson L. Design and synthesis of pyridazinone based 5 HT2C agonists Bioorg Med Chem Lett 2009; 19: 5791 95. Andersson PG, Schink HE, sterlund K Asymmetric total synth esis of (+) tolterodine, a new muscarinic receptor antagonist, via copper assisted asymmetric conjugate addition of aryl grignard reagents to 3 phenyl prop 2 enoyl oxazolidinones J Org Chem 1998; 8067 70. Berg KA, Harvey JA, Spampinato U, Clarke WP. Phys iological and therapeutic relevance o f constitutive activity of 5 HT2A and 5 HT 2C receptors for the treatmen t of depression. Prog Brain Res 2008; 172:287 305. Bickerdike M J Vickers SP, Dourish CT. 5 HT2C receptor modulation and the treatment of obesity. Diabetes Obes Metab 1999 ; 1 ( 4 ): 207 14 Browning J, Young K, Hicks P. The antipsychotic like action of a 5 HT 2C agonist on conditioned avoidance response behavior i n rats. Abstr Soc Neurosci 1999; 25 : 2075 87 Boger DL, Mckie JA, Boyce CW. Asymmetric syn thesis of the CBI alkylation subunit of the CC 1065 and du ocarmycin analogs. Synlett 1997; 515 517. Boger DL. Morden organic synthesis. TSRI Press 1999 P114. Booth RG, Fang L, Wilczynski A, Sivendren S, Sun Z, Bruysters M, Sansuk K, and Leurs R. Molecul ar determinants of ligand directed Gq versus Gs signaling for the histamine H 1 receptor. Abstracts of the Histamine Research Society 2007; 36:32, submitted for publication to Inflamm Res. June, 2007. Booth RG, Baldessarini RJ. (+) 6,7 benzomorphan sigma ligands stimulate dopamine synthesis in rat corpus striatum tiss ue. Brain Res 1991; 5 57:349 52.

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110 Itoh T, Mase T, Nishikata T, Iyama T, Tachikawa H, Kobayashi Y, Yamamoto Y, Miyaura N. 1,4 aryl unsaturated ketones and esters catalyzed by a rhodium(I) chiraphos complex for catalytic and enantioselective synthesis of selective endothelin A recepto r antagonists. Te trahedron 2006; 9610 21 Julius D, Huang KN, Livelli TJ, Axel R, Jessel TM. The 5HT2 receptor defines a family of structurally distinct but functionally conserved serotonin receptors. Proc Natl Acad Sci 1990 ; 87 :928 32. Julius D, MacDermott AB, Axel R, J essell TM. Molecular Characterization of a functional cDNA encoding the serotonin 1c receptor. Science 1988 ; 241 :558 64. Jun Xu L, Unzeitig A, Javors MA, Rice KC, Koek W, France CP. Discriminative stimulus effects of 1 (2,5 dimethoxy 4 methylphenyl) 2 aminopropane (DOM), ketanserin, and (R) (+) .alpha. (2,3 dimethoxyphenyl) 1 [2 (4 fluorophenyl)ethyl] 4 pipidinemethanol (MDL100907) in rats. J Pharmacol Exp Ther 2009; 331: 671 79. Kehne JH, Moser PC, Moran PM, Frank RA. Reversal of amphetamine induc ed behaviours by MDL 100,907, a selective 5 HT2A antagonist. Behav Brain Res 1996; 73 :163 7. Kimura Y, H atanaka K, Naitou Y, Maeno K, Shimada I, Koakutsu A, Wanibuchi F, Yamaguchi T. Pharmacological profile of YM348, a novel, potent and orally active 5 HT2C rec eptor agonist. Eur J Pharmacol 2004 ; 483:37 43. Knight AR, Misra A, Quirk K, Benwell K, Revell D, Kennett G, Bickerdike M. Pharmacological characterisation of the agonist radioligand binding site of 5 HT(2A), 5 HT(2B) and 5 HT(2C) receptors. Nauny n Schmiedebergs Arch Pharmacol 2004 ; 370:114 23. Kroeze WK, Hufeisen SJ, Popadak B A, Renock S M Steinbe rg S, Ernsberger P, Jayathilake K Mel tzer H Y Roth BL. 2003. H1 histamine receptor affinity predicts short term weight gain for typical and atypical antipsychotic drugs. Neuropsychopharmacology 28, 519 526. Kroeze WK, Kristiansen K, Roth B L. Molecular Bi ology of Serotonin Receptors Structure and Function at the Molecular Level Curr Top Med Chem 2002; 22: 507 28. Kursar JD, N elson DL, Wainscott DB, Baez M. Molecular cloning, functional expression, and mRNA tissue distribution of the human 5 hydroxytryp tamine2B receptor. Mol. Pharmacol 1994; 46: 227 34.

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117 BIOGRAPHICAL SKETCH Mr. Zhuming Sun was born in Yangzhou, China. He received his bachelor degree in China Pha rmaceutical Univer sity. He complete d his graduate research in the Dept ment of Medicinal Chemistry Univ ersity of Florida in the summer of 2010.

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