Curriculum Vitae

Home , Alvameline, Himbacine, Milameline, Muscarinic toxin 7, Sabcomeline, Talsaclidine

Novel Cholinergics for Treatment of Central Nervous System Disorders

Item Type dissertation

Authors Johnson, Chad

Publication Date 2019

Abstract Approximately 16% of Americans are diagnosed with major depressive disorder, a mental disorder thought be caused by a combination of characterized by genetic, biological, environmental, and psychological factors. It can be accompanied by low self-est...

Keywords Pharmaceutical sciences; behavioral pharmacology; medicinal chemistry; Antidepressive Agents; Chemistry, Pharmaceutical; Drug Design; Pharmacology; Receptors, Muscarinic

Download date 07/10/2021 09:00:20

Link to Item http://hdl.handle.net/10713/11602 Curriculum Vitae

NAME: Chad Johnson

TITLE: Ph.D. Candidate Department of Pharmaceutical Sciences University of Maryland School of Pharmacy 20 N Pine St, Room N706 Baltimore, MD 21201 Phone: 410-706-1578 Email: [email protected]

RESIDENT STATUS: USA Permanent Resident

EDUCATION: B.S. Chemistry University of Virginia Charlottesville, VA 2005-2009

M.A. Chemistry (Organic/Bio-Organic) Johns Hopkins University Baltimore, MD 2009-2013

Ph.D. Candidate (Pharmaceutical Sciences) University of Maryland, Baltimore Baltimore, MD 2015-2019

TEACHING EXPERIENCE:

UNIVERSITY OF MARYLAND, BALTIMORE Training course for departmental hydrogenator Training course for Advion Expression L benchtop Mass Spectrometer Weekly synthetic Chemistry group meetings Teaching Assistant for Medicinal Chemistry 1/2 and Infectious Disease Therapeutics 1/2 PHMY5008-Professional Communication Strategies MS in Medical Cannabis and Therapeutics-Instructor for MSMC 602 (Principles of Drug Action and Cannabinoid Pharmacology, Teaching Assistant for MSMC 601 (Introduction to Medical Cannabis History, Culture, and Policy) JOHNS HOPKINS UNIVERSITY Teaching Assistant Organic Chemistry and Organic Chemistry Laboratory 1/2 (August 2009-July 2014)

UNIVERSITY OF VIRGINIA Teaching Assistant Organic Chemistry 1/2 Laboratory (August 2008-May 2009) PRIVATE TUTOR GRE, MCAT, and Organic Chemistry (August 2009- Current)

HONORS/AWARDS: Finalist for Best Teaching Assistant Award (JHU, 2014) Finalist for Hertz Fellowship Award (UMB, 2016) Inducted into Rho Chi Honor Society (UMB, 2016) Travel Award for Behavior, Biology, and Chemistry (BBC) Translation Research in Addiction Conference (2017,2018, and 2019) Wal-Mart Scholar (UMB, American Association of Colleges of Pharmacy, 2019)

PROFESSIONAL MEMBERSHIPS: American Chemical Society: Student Member 2009- Current American Association of Pharmaceutical Scientists: Student Member 2016-Current American Association of Colleges of Pharmacy: Student Member 2017-Current American Association for the Advancement of Science: Student Member: 2016-Current Rho Chi Honor Society: 2016-Current (Serve on Academic Committee) PUBLICATIONS: 1) Ansari, M. I.; Johnson, C.; Coop, A. PARP Inhibitors: A Breakthrough in Cancer Chemotherapy. Modern Approaches in Drug Design , 2(1), 2018.

2) Johnson, C.; Ansari, M. I.; Coop, A. Tetrabutylammonium Bromide Promoted Metal-Free, Efficient, Rapid, and Scalable Synthesis of N-Arylated Amines. ACS Omega. 2018, 3(9), 10886- 10890.

3) Saquib, M., Ansari, M. I., Johnson, C., Khatoon, S., Hussain, M. K., and Coop. A. Recent Advances in the Targeting of Human DNA Ligase 1 as a Potential New Strategy for Cancer Treatment. Eur. J. Med. Chem. 2019 , 182, 111657.

PRESENTATIONS:

1. ACS National Meeting: Chemistry of the People, by the People, for the People . Philadelphia, PA. "Inhibitors of LHR-1 as Novel Anti-Parasitic Drugs." Poster Presentation, August 2016 .

2. UMD-JHU Joint Symposium on Drug Discovery : Baltimore, MD. Reinforcing Activity of Meta-Nicotine, Nicotine, and A8530. Poster Presentation. 2017 3. Behavior, Biology, and Chemistry: Translational Research in Addiction , San Antonio, TX. "Reinforcing Properties of Meta-Nicotine." Poster Presentation, Travel Awardee , 2017 . 4. School of Pharmacy Research Day : Baltimore, MD. "Comparisons of the Reinforcing Activity of Meta-Nicotine, Nicotine, and A8530." Poster Presentation, 2017 . 5. Frontiers in Chemistry and Biology Interface Symposium : Newark, DE. "Comparisons of the Reinforcing Activity of Meta-Nicotine, Nicotine, and A8530." Poster Presentation, May 2017. 6. Ph.D. Candidacy Public Seminar : Baltimore, MD. "Novel Cholinergics for Treatment of CNS Disorders." Oral Presentation, 2017 . 7. Behavior, Biology, and Chemistry: Translational Research in Addiction, San Antonio, TX. "Muscarinic Antagonists and Anti-depressant-like Effects in Rodents: Some Chemical Forays Toward New Compounds." Poster Presentation, Travel Awardee, 2018. 8. Graduate Research Conference: Baltimore, MD. "Muscarinic Antagonists and Anti- depressant-like Effects in Rodents: Some Chemical Forays Toward New Compounds." Poster Presentation, 2018 . 9. School of Pharmacy Research Day : Baltimore, MD. "Muscarinic Antagonists and Anti- depressant-like Effects in Rodents: Some Chemical Forays Toward New Compounds." Poster Presentation, 2018 . 10. Frontiers in Chemistry and Biology Interface Symposium : Philadelphia, PA. "Novel Muscarinic Antagonists: Design, Synthesis and Pharmacological Evaluation in Rodents." Poster Presentation, 2018 . 11. Computer Aided Drug Design Symposium , Baltimore, MD. "Novel Muscarinic Antagonists: Design, Synthesis and Pharmacological Evaluation in Rodents." Poster Presentation, 2018 . 12. American Association of Colleges of Pharmacy National Meeting , Boston, MA. "Mentoring Undergraduate Students in an Academic Research Laboratory: What to Know, What to Do, and What to Expect as a Graduate Student and Faculty Mentor." Poster Presentation. 2018 . 13. Behavior, Biology, and Chemistry: Translational Research in Addiction , San Antonio, TX. "Muscarinic Antagonists and Anti-depressant-like Effects in Rodents: Some Chemical Forays Toward New Compounds." Poster Presentation, Travel Awardee, 2018 . 14. School of Pharmacy Research Day : Baltimore, MD. "Muscarinic Antagonists and Anti- depressant-like Effects in Rodents: Methyl to Cyclopropyl." Poster Presentation, 2018 . 15. American Chemical Society Mid-Atlantic Regional Meeting : Baltimore, MD. "Novel Muscarinic Antagonists with Anti-Depressant-Like Effects in Rodents." Poster Presentation, 2019 .

RESEARCH:

UNIVERSITY OF MARYLAND, BALTIMORE

Research Topic : Novel Cholinergics for Treatment of CNS Disorders

Major depression is a widespread psychiatric disorder demonstrating severe symptoms in how a person feels, thinks, and handles daily activities (nih.gov). Furthermore, it is linked to diminished quality of life, medical morbidity, and mortality. Depression has a lifetime prevalence of 16% in the United States and appears to be caused by a combination of genetic, biological, environmental, and psychological factors. Current antidepression medications possess significant problems, including a delayed onset of action and different therapeutic effects in differing patients. As such, rapid acting fully efficacious antidepressants are urgently needed. Non- selective muscarinic antagonists have been shown to display antidepressant effects, but this is accompanied by undesired cognitive deficits. The hypothesis of this project is that antagonism of one or more muscarinic subtypes leads to an antidepressant effect, and antagonism at others lead to the cognitive deficits. As selective agonists and antagonists are not available for the 5 muscarinic receptor subtypes, we aim to design and synthesize-- using the scaffolds of L-670548, L-687306, WAY-132983, and L-689660 as model scaffolds-- muscarinic ligands to allow a delineation of the structure activity relationship (SAR) for both selectivity and efficacy, with the ultimate goal of a muscarinic antagonist lacking cognitive deficits.

JOHNS HOPKINS UNIVERSITY (Graduate Research Assistant):

• Constructed two total syntheses with optimization of all reactions to obtain novel β-methyl carbapenem antibiotics and various derivatives thereof. • Purified each novel compound via column chromatography or preparative HPLC Ran biological assays using these compounds and purified ThnQ/SarQ grown from bacterial cells over-expressing these proteins. • Constructed synthetic standards to compare the compounds produced from the above assays (2009-2013).

UNIVERSITY OF VIRGINIA (Undergraduate Research Assistant):

• Designed and successfully synthesized various derivatives of FTY720 (2008-2009)

Abstract

Title of Dissertation: Novel Cholinergics for Treatment of Central Nervous System Disorders Chad R. Johnson, Ph.D., 2019 Dissertation Directed by: Dr. Andrew Coop, Professor and Associate Dean for Academic Affairs, Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore Approximately 16% of Americans are diagnosed with major depressive disorder, a mental disorder thought be caused by a combination of characterized by genetic, biological, environmental, and psychological factors. It can be accompanied by low self-esteem, loss of interest in normally enjoyable activities, low energy, and diminished quality of life. Between 2-

7% of adults with this disorder die by suicide. In addition, almost half of patients who are treated initially with an SSRI do not achieve complete remission, and nearly a third after four different treatment regimens (nimh.nih.gov). While counseling and antidepressant medication can be effective treatments, current selective serotonin re-uptake inhibitors (SSRI's) take weeks before therapeutic effects are observed. This "delay" period of action is not well understood and presents a significant challenge for medical professionals in the management of major depression.

Mechanisms of anti-depressants have been a major focus of both current/past research in hopes of developing more effective and faster acting drugs. Directly related to this, clinical data

(nimh.nih.gov) that oral and intravenous treatment with the muscarinic cholinergic antagonist scopolamine had rapid anti-depressant effects in humans--likely mediated through an antimuscarinic effect. Unfortunately, scopolamine can produce cognitive impairment including memory disturbances due to its anticholinergic properties. Since major depressive disorder is

associated with deficits in cognition, this would produce an undesired additive effect that would only exacerbate the problem.

It is our goal to identify a muscarinic antagonist that may be able to relieve depression and have little to no effect on memory or cognition. The 3-exo -1-azabicyclo[2.2.1]heptane, 1- azabicyclo[2.2.2]octane, 1-azabicyclo[3.2.1]octane, and N-methyltetrahydropyidine 3 (and 4)- substituted-1,2,4-oxadiazoles appear to be excellent chemical scaffolds for the generation of potent muscarinic agonists/antagonists. In order to probe the orthosteric site of the mAChRs we designed a large library of compounds and evaluated them via a battery of pharmacological assays to confirm both their antidepressant and cognitive effects. This resulted in the identification of lead compound (CJ2100) that showed potent antidepressant activity without cognitive impairment. (Supported by NIMH Grant 107499)

Novel Cholinergics for Treatments of Central Nervous System Disorders

by Chad R. Johnson

Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2019

Dedicated to my mother, Shelia Johnson, for her love, support, and encouragement over the many years of my education. This dissertation would not be possible without her.

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Acknowledgments

I would like to express my appreciation for my advisor, Dr. Andrew Coop. His support, guidance, and encouragement have fostered my development as a scientist. His encouragement to explore areas that would best fit my future career goals shows how dedicated he is to seeing his students succeed. Andy taught me what it means to be an interdisciplinary scientist, and how to bridge my work with other disciplines. Because of this I am a better scientist and believe I will be more effective in an academic setting. Andy has been a great mentor, a good listening ear, and a great friend. I've thoroughly enjoyed my time in his laboratory.

I would also like to thank my committee members: Dr. Steven Fletcher, Dr. Alexander MacKerell, Dr. Edward Moreton, and Dr. James Woods for their instruction and constructive comments regarding my project. This motivated me to work harder and make my project the best it could be.

I am thankful for my collaborators: Dr. James Woods, Dr. Gail Winger, Dr. Emily Jutkiewicz, Dr. Jack Bergman, and Dr. Brian Kangas for their patience in teaching me pharmacology, advice for my future career path, and for their support with this project. I would also like to thank Dr. Kellie Hom for all her help with setting up and analyzing NMR experiments.

My time here would not have been nearly as enjoyable if not for my labmates/colleagues/classmates. I enjoyed our volleyball/intramural games and outings together. I am also grateful for the friends that I have outside of the department that made the weekends fun when I could take a break from working in the laboratory.

I would never have reached this point if not for my mother, Shelia Johnson. She is a wonderful mother who has supported me through everything, and loved me unconditionally. She established within me a hard work ethic, taught me to always strive for the best and love others, and to never take any blessings for granted. My mother has always been my source of encouragement and strength. I am truly grateful that she is my best friend.

My extended family has always been there for me as well. I am grateful for their support and love over the years.

Table of Contents

Chapter Page

List of Tables viii

List of Figures x

List of Schemes xv

List of Abbreviations xvi

1. The Muscarinic Receptors: History, Mechanism of Action, Location, and Functions within the Brain and Periphery 1

1.1 History 1

1.2 Cholinergic Biosynthesis and Effects on Biological Systems 3

1.3 G-Protein-Coupled Receptors 5

1.4 Muscarinic Receptor Location and Function 14

1.5 References 23

2. Drug Design Targeting the Muscarinic Receptors: The Cholinergic Hypothesis, Muscarinic Agonists/Antagonists, and Design of Novel Cholinergics as Rapid Acting Antidepressants 33

2.1 Drug Design Targeting the Muscarinic Receptors 33

2.2 The Cholinergic Hypothesis: Merck/Smith Kline Beecham/Eli Lilly and Alzheimer's Disease 35

2.3 The Design of Functionally Selective Muscarinic Agonists Targeting the M1 Receptor 52

2.4 Muscarinic Antagonists and Depression 58

2.5 Rational of Design for Novel Muscarinic Antagonists 69

2.6 Synthesis of Muscarinic Antagonists for Comparison of Structure- Activity Relationships 75

2.7 Experimental Section 87

2.8 References 135

3. Pharmacology of Muscarinic Antagonists: Studies on Reference Compounds and Novel Muscarinic Antagonists as Rapid Acting Antidepressants that Lack Cognitive Deficits 148

3.1 Pharmacological Assays Utilized in Evaluation of Compounds 148

3.2 Pharmacological Evaluation of Known Compounds 155

3.3 Pharmacological Evaluation of Novel Anticholinergics and Rational of Design 166

3.4 Methyl vs. Cyclopropyl: Agonist/Antagonist Pairs 169

3.5 Evaluation of Novel Muscarinic Antagonists 183

3.6 References 196

Supplementary Data/Figures 199

APPENDIX A. Current Trends in Drug Design Targeting the Muscarinic Receptors 201

A.1 Background and Crystal Structure of the mAChR Allosteric Site 201

A.2 Allosteric Modulator Design for the Muscarinic Receptors 210

A.3 References 225

APPENDIX B. Site Identification by Ligand Competitive Saturation (SILCS) Methodology on Novel Muscarinic Antagonists 235

B.1 Background 235

B.2 Results 237

B.3 Discussion 246

B.4 References 249

Cumulative List of References 250

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List of Tables

1. The Muscarinic Receptors: History, Mechanism of Action, Location, and Functions within the Brain and Periphery 1

2. Drug Design Targeting the Muscarinic Receptors: The Cholinergic Hypothesis, Muscarinic Agonists/Antagonists, and Design of Novel Cholinergics as Rapid Acting Antidepressants 33

Table 1 38

Table 2 40

Table 3 42

Table 4 44

Table 5 45

Table 6 47

Table 7 47

Table 8 49

Table 9 51

Table 10 53

Table 11 56

Table 12 73

Table 13 81

3. Pharmacology of Muscarinic Antagonists: Studies on Reference Compounds and Novel Muscarinic Antagonists as Rapid Acting Antidepressants that Lack Cognitive Deficits 148

Table 14 164 Table 15 177

Table 16 189

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APPENDIX A. Current Trends in Drug Design Targeting the Muscarinic Receptors 201

Table 17 220

APPENDIX B. Site Identification by Ligand Competitive Saturation (SILCS) Methodology on Novel Muscarinic Antagonists 235

Table 18 238

Table 19 238

Table 20 245

List of Figures

1. The Muscarinic Receptors: History, Mechanism of Action, Location, and Functions within the Brain and Periphery 1

Figure 1: Muscimol and Ibotenic Acid 1

Figure 2: Neurotransmission of ACh 4

Figure 3: A 2A AR Structure-General Structure of GPCRs 7

Figure 4: Classes of GPCRs 8

Figure 5: GPCRs Coupling to Different Signaling Pathways 11

Figure 6: Regulation of GPCR Signaling 13

Figure 7: Signaling Transduction Pathways Regulated by CHRM1-5 15

Figure 8: Locations of Cholinergic Neurons in the Brain 16

Figure 9: Distribution of Muscarinic Receptors in the Brain 17

2. Drug Design Targeting the Muscarinic Receptors: The Cholinergic Hypothesis, Muscarinic Agonists/Antagonists, and Design of Novel Cholinergics as Rapid Acting Antidepressants 33

Figure 10: Early Examples of Muscarinic Agonists and Antagonists 33

Figure 11: Main Events of Alzheimer's Disease in the Brain 36

Figure 12: Examples of AChE Inhibitors and Agonists 37

Figure 13: 2+3 Cycloaddition with a Chiral Azomethine Ylide to Afford Chiral 1-azanorbornanes 39

Figure 14: E vs. Z Rotamers of Substituted Tetrahydropyridines 46

Figure 15: Structure of QNB 48

Figure 16: 6-membered Heteroaromatic Diazine Rings as mAChR pharmacophores 48

Figure 17: Xanomeline 54

Figure 18: Early M 1 Preferring Agonists 57

Figure 19: Pathological States of Chronic Stress/Depression 62

Figure 20: Infusion Blocks of Scopolamine in Clinical Trials 63

Figure 21: MADRS Scores of Scopolamine Clinical Trial 65

Figure 22: Ketamine 66

Figure 23: General Design Scheme for Novel Muscarinic Antagonists 70

Figure 24: General Structure of Quinuclidine-3-Oxadiazoles and QNB Oxadiazole 71

Figure 25: C2/C3 Unsaturated Quinuclidine Oxadiazole 71

Figure 26: L689660 and L687306 72

Figure 27: Antidepressant-like Effects of Scopolamine are Blocked in M 1/M 2 Knockout Mice 74

3. Pharmacology of Muscarinic Antagonists: Studies on Reference Compounds and Novel Muscarinic Antagonists as Rapid Acting Antidepressants that Lack Cognitive Deficits 148

Figure 28: Bradycardia/Drug Discrimination of Scopolamine 156

Figure 29: Scopolamine vs. Arecoline (Rates of Responding and Forced Swim Test 157

Figure 30: Psychomotor Vigilance Task and Delayed Matching to Sample of Scopolamine 158

Figure 31: L687306 Blocks Arecoline Induced Bradycardia 159

Figure 32: L687306 vs. Arecoline (Discrimination, Rates of Responding 160

Figure 33: L687306 in the Forced Swim Test, Psychomotor Vigilance Task, and Delayed Matching to Placement Task 161

Figure 34: L689660 Blocks Arecoline Induced Bradycardia 162

Figure 35: L68 9660 vs. Arecoline in Drug Discrimination/Rates of Responding 163

Figure 36: L689660 in the Forced Swim Test 164

Figure 37: L689660 Evaluation in the Delayed Matching to Sample and Psychomotor Vigilance Task 165

Figure 38: L670548 is Antagonized by L687306 in Rates of Responding and Drug Discrimination 166

Figure 39: L687306 Antagonizes L670548 in the Forced Swim Test 166

Figure 40: CJ2100 167

Figure 41: CJ2100 Blocks Arecoline Induced Bradycardia/Rate Suppressing Effects, but does not Block the Discriminative Stimulus of Arecoline 167

Figure 42: CJ2100 is Active in the Forced Swim Test 168

Figure 43: CJ2100 is Silent Up to 10mg/kg in Both DMTS/PVT Tasks 168

Figure 44: Methyl and (Methylene) Cyclopropyl Pairs in Opioid Agonist/ Antagonist Design 169

Figure 45: Scopolamine, L670548, and L687306 170

Figure 46: Methyl/Cyclopropyl Pairs 171

Figure 47: CJ2100 Directly Antagonizes CJ2099 Induced Bradycardia 172

Figure 48: CJ2100 Acts as an Antagonist of CJ2099 in Rates of Responding and Drug Discrimination 172

Figure 49: CJ2126 Antagonizes CJ2051 in Rates of Responding and Drug Discrimination 173

Figure 50: CJ2126 Shows Increase in Immobility in the FST but Shows Detrimental Effects on Cognition 175

Figure 51: CJ2126 Blocks Arecoline Induced Bradycardia and Rate Suppressing Effects 176

Figure 52: Comparison of CJ2139/2150 vs. Arecoline in Discrimination and Rates of Responding 178

xii

Figure 53: CJ2139 vs. CJ2150 in Discrimination and Rates of Responding 179

Figure 54: CJ3094 Produces a Strong Bradycardia Effect that can be Antagonized by CJ3095 181

Figure 55: CJ3095 Antagonizes the Effects of CJ3094 in Both Discrimination and Rates of Responding 181

Figure 56: CJ3120/3120.1 and CJ3125.1/3125.2 vs. Arecoline in Drug Discrimination and Rates of Responding 182

Figure 57: 4-Substituted Arecoline Oxadiazoles Cannot Antagonize the Rate Suppressing or Discriminative Stimulus Effects of Arecoline 183

Figure 58: First Series of Compounds Evaluated for Activity in the FST 186

Figure 59: Second Series of Compounds Evaluated in the FST 187

Figure 60: CJ2165.1/2173.1/3018 Evaluation in the PVT 190

Figure 61: CJ2165.1/2173.1/3018 Evaluation in the DMTS Task 191

Figure 62: CJ2150/2174/3095/3100 Evaluation in the PVT 192

Figure 63: CJ2150/2174/3095/3100 Evaluation in the DMTS Task 193 Supplementary Data/Figures 199

Figure 64: QNB Causes Detrimental Effects in both PVT/DMTP Tasks 199

Figure 65: CJ3095 Discriminates to Arecoline 200

APPENDIX A. Current Trends in Drug Design Targeting the Muscarinic Receptors 201

Figure 66: Antagonists Utilized to Construct a Receptor Profile 201

Figure 67: Crystal Structure of M 2 Receptor 203

Figure 68: QNB Binding Pocket in the M 2 Receptor 204

Figure 69: M 2 and M 3 Receptor Comparison 205

Figure 70: Tiotropium and QNB Binding Comparison in M2 and M 3 Receptors 206

Figure 71: Simulations of Tiotropium in the M 2 and M 3 Receptors 207

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Figure 72: Comparisons of M 1-4 Receptors with Tiotropium or QNB Bound in the Orthosteric Site 209

Figure 73: Allosteric Ternary Complex Model 211

Figure 74: Allosteric Binding in the M 2 Receptor 213

Figure 75: Neuromuscular Blocking Agents as Allosteric Modulators of the mAChRs 214

Figure 76: Staurosporine 215

Figure 77: Early Examples of mAChR Allosteric Agonists 215

Figure 78: Binding Simulations of Diverse Allosteric Modulators to the M 2 Receptor 217

Figure 79: 40 Snapshot Simulation of C 7/3-phth Binding to the M 2 Allosteric Site 218

Figure 80: Crystallization of Inactive/Active Sites of the M 2 Receptor 221

Figure 81: Structure of M 2 Receptor Occupied by Agonist Iperoxo in Complex with the Positive Allosteric Modulator LY2119620 222

Figure 82: Dualsteric Ligands as Allosteric Modulators 223

APPENDIX B. Site Identification by Ligand Competitive Saturation (SILCS) Methodology on Novel Muscarinic Antagonists 235

Figure 83: Sequence Homology of the Muscarinic Receptors 236

Figure 84: Compounds to Which SILCS-MC was Applied to Obtain LGFE Scores 239

xiv

List of Schemes

1. The Muscarinic Receptors: History, Mechanism of Action, Location, and Functions within the Brain and Periphery 1

2. Drug Design Targeting the Muscarinic Receptors: The Cholinergic Hypothesis, Muscarinic Agonists/Antagonists, and Design of Novel Cholinergics as Rapid Acting Antidepressants 33

Scheme 1: Synthesis of L670548 and L687306 76

Scheme 2: Synthesis of WAY-132983 Derivatives 77

Scheme 3: Synthesis of L689660 and Derivatives 78

Scheme 4: Synthesis of 3-Substituted Arecoline and Norarecoline Oxadiazole Series 80

Scheme 5: Synthesis of 3-Quinuclidinyl Oxadiazole Series 82

Scheme 6: Synthesis of 1-Azabicyclo[3.2.1]octane Oxadiazole Series 83

Scheme 7: Synthesis of 4-Substituted Arecoline/Quinuclidinyl/1-Azanorbornane Oxadiazole Series 84

Scheme 8: Synthesis of Arecoline and Quinuclidinyl Amides/Sulfonamides 85

3. Pharmacology of Muscarinic Antagonists: Studies on Reference Compounds and Novel Muscarinic Antagonists as Rapid Acting Antidepressants that Lack Cognitive Deficits 148

APPENDIX A. Current Trends in Drug Design Targeting the Muscarinic Receptors 201

APPENDIX B. Site Identification by Ligand Competitive Saturation (SILCS) Methodology on Novel Muscarinic Antagonists 235

List of Abbreviations

GABA γ-aminobutyric acid

NMDA N-methyl-D-aspartic acid

ACh Acetylcholine

ChAT Cholineacetyl-transferase

CoA Coenzyme A mAChR Muscarinic Acetylcholine Receptor

GPCR G-protein-coupled receptor

CNS Central nervous system

VAChT Vesicular acetylcholine transporter nAChR Nicotinic Acetylcholine receptor

AChE Acetylcholinesterase

Cht1 Choline transporter

NME New molecular entities

FDA US Food and Drug Administration

TM Transmembrane

ICL Intracellular loops

ECL Extracellular loops

GRK GPCR kinase

LCP Liquid cubic phase cAMP cyclic AMP mGlu Metabotropic glutamate receptors

VFT Venus fly trap

GTP Guanosine-5'-triphosphate

GDP Guanosine-5'-diphosphate

xvi

PLCβ Phospholipase Cβ

GIRK G-protein-regulated inwardly rectifying potassium channels

AC Adenylyl cyclase

GEF Guanine nucleotide exchange factors

PKA Protein kinase A

PIP2 Phosphatidylinositol 4,5-bisphosphate

DAG Diacylglycerol

IP3 Inositol-1,4,5-trisphosphate

ER Endoplasmic reticulum

CHRM1-5 Cholinergic Receptor Muscarinic 1-5

AD Alzheimer's disease

COPD Chronic obstructive pulmonary disease

BF Basal forebrain

VTA Ventral tegmental area

SN Substantia nigra

KO Knock-out (mice)

ERK Extracellular signal-regulated kinase

GI Gastrointestinal tract

PPI Prepulse inhibition

OXO-M Oxotremorine

QNB Quinuclidinyl-3-benzilate

MDD Major Depressive Disorder

Aβ Amyloid-β peptide

APP Amyloid precursor protein

NMS N-methyl scopolamine

xvii

LDA Lithium diisopropylamide

PI Phosphoinositol

DFP Diisopropylfluorophosphate

BPD Bipolar disorder

REM Rapid eye movement

SNP Single nucleotide polymorphism

FSL Flinders Sensitive Line (rats)

FST Forced swim test

MAOI Monoamine oxidase inhibitors

TCAs Tricyclic antidepressants

SSRI Selective serotonin reuptake inhibitors

SNRI Serotonin-norepinephrine reuptake inhibitors

NRI Norepinephrine reuptake inhibitors

DG Dentate gyrus

PFC Pre-frontal cortex

STAR*D Sequenced Treatment Alternative to Relieve Depression

MADRS Montgomery-Asberg Depression Scale

HARS Hamilton Anxiety Rating Scale

CGI Clinical Global Impressions

VAS Visual Analogue Scale

POMS Profile of Mood State mTor Mammalian target of rapamycin

ED 50 Median effective dose

DMTS Delayed Matching to Sample

PAM Positive allosteric modulator

xviii

TFA Trifluoroacetic acid

NaHCO 3 Sodium bicarbonate

HBr Hydrogen bromide

EtOH Ethanol

Na 2CO 3 Sodium carbonate

EtOAc Ethyl Acetate

CHCl 3 Chloroform

H2 Hydrogen (gas)

Pd/C Palladium on carbon

THF Tetrahydrofuran

NaH Sodium hydride iPrOH Isopropanol

Ki Inhibition constant

SAR Structure activity relationship

KOtBu Potassium tert-butoxide

DMF Dimethylformamide

K2CO 3 Potassium carbonate

NH 4OH Ammonium hydroxide

NaN(TMS) 2 Sodium bistrimethylsilylamide

NaOH Sodium hydroxide

BH 3-THF Borane-tetrahydrofuran complex

MsCl Methanesulfonylchloride

TEA Triethylamine

HCl Hydrochloric acid

CH 3I Methyl iodide

xix

NaBH 4 Sodium borohydride

MeOH Methanol

ACE-Cl 1-Chloroethyl chloroformate

DCE Dichloroethane

Boc 2O t-butoxycarbonic anhydride

DCM Dichloromethane

KB Equilibrium dissociation constant of antagonist

DME Dimethoxyethane

Ac-Cl Acetyl chloride

RT Room temperature

Cs 2CO 3 Cesium Carbonate

Et 2O Diethyl ether

DCC Dicyclohexyl carbodiimide pA 2 Measure for equilibrium dissociation constant of antagonist

AUC Area under curve

TVT/PVT Titrating Vigilance (or Psychomotor Vigilance) Task

DMP Delayed-Matching-to-Place Task

Chapter 1: The Muscarinic Receptors: History, Mechanism of Action, Location, and Functions within the Brain and Periphery

History (1.1): In traditional medicine mushrooms have been used over generations for both religious, culinary, and magical purposes. 1 Amanita muscaria, commonly known as the fly agaric or fly amnita, is one of these. It was shown by Schmiedeberg and Koppe in 1869 that extracts from this mushroom could slow, or at higher concentrations, stop the beat of the frog heart. Concluding that it was likely an alkaloid they used this as a guide to purify the extract with the methods known at the time. While no compound characterization was provided, they coined the name muscarine (Figure 1). Later evidence showed that the extract was likely ~25% pure, 2 but it was sufficient enough to conduct pharmacological experiments. For example, they showed that it contracted smooth muscle in the stomach, intestine, stimulated secretion of tears, saliva and mucus, and constricted the pupil, among other periphery effects.

The chemistry behind muscarine remained vague until Schmiedberg and Harnack 3 found that when choline was treated with nitric acid, a substance was produced that had the same effects as muscarine. It was later shown that this product was simply the nitrite ester of choline by Ewins 4 and Dale 5 attributed its effects to its strong activity on the nicotinic receptors. It was not until the late 1950s when the X-ray crystallographic structure 6 of muscarine was completed, and further confirmed by total synthesis. 7

O COOH The interest in the Amanita muscaria was due to its N N O NH HO 2 HO NH 2 psychopharmacological actions, but it is not likely that Muscimol Ibotenic acid muscarine contributes to this. The major toxins involved Figure 1 : Muscimol and Ibotenic Acid

1 in A. muscaria poisoning are muscimol -both discovered in the mid-20th century. A simple decarboxylation of ibotenic acid yields muscimol, and both are potent agonists of the gamma-

8 amino butyric acid (GABA A) and N-methyl-D-asparatic (NMDA) receptors respectively.

Acetylcholine (ACh) was first synthesized in 1867 by von Baeyer and was found to be

~10000 times as active as choline in lowering blood pressure, and could be effectively blocked by atropine like choline. But it was not until 1929 that ACh was found to be a normal part of animal tissue by Dale and Dudley. 9 As they were extracting histamine from ox spleen they found the presence of a substance that lowered blood pressure of a rabbit, and subsequently led to contraction of the ileum. It was eventually shown to be ACh after purification. Later it was confirmed as a neurotransmitter by Otto Loewi, who coined the name "Vagusstof" since it was released from the vagus nerve. 10 Both Dale and Loewi received the 1936 Nobel Prize in

Physiology or Medicine for their work.

In 1943, David Nachmansohn and A. L. Machado 11 described the synthetic enzyme cholineacetyl-transferase (ChAT), but its exact action was unknown at the time. It was not until

1945 that Coenzyme A (CoA) was discovered and subsequently Acetyl-CoA in 1951. The X-ray structure of rat-derived ChAT was not solved until 2004 and was found to have a high sequence homology across the animal genome. 12

The first evidence of muscarinic receptor subtypes were the cardioselective actions of gallamine 13 along with the sympathetic ganglionic stimulant behavior of (McN-A-343) as shown by Roszkowski in 1961. 14 Shortly thereafter, Barlow et al showed differences in the pharmacological properties of ileal and atrial muscarinic receptors. 15 Pirenzepine, a drug originally used for the treatment of peptic ulcer disease, also played a major role in clarifying the perplexing question of receptor subtypes. Hammer et al 16 performed binding studies and Brown

2 et al 17 performed functional studies to elucidate it's in vivo selectivity. However, the knowledge of the potential functions and roles of the muscarinic receptors (mAChRs) and subtypes was advanced significantly when the cloning of the five mammalian genes that encoded them were completed. The cloning of the complementary DNA for muscarinic receptor genes was performed by Kubo et al ,18a,b further extended by Bonner et al ,19a,b Peralta et al ,20 Braun et al. ,21 and Akiba et al. 22 which confirmed that there were at least five subtypes of mAChRs. Each of these five genes encode the muscarinic receptor proteins, which are classified as members of the

G-protein-coupled receptor (GPCR) superfamily.

Cholinergic Biosynthesis and effects on biological systems (1.2):

Acetylcholine was the first neurotransmitter to be identified and is used by all cholinergic neurons, playing important roles in both peripheral and central nervous systems (CNS). 23 In the

CNS cholinergic neurons are widely distributed, hence it is not surprising that cholinergic neurotransmission is responsible for modulating important neural functions such as: attention, learning, memory, stress response, wakefulness/sleep, and sensory information. Additionally, evidence points to the cholinergic system in the control of memory and learning. 24a,b

Neurotransmission of ACh relies on proteins implicated in its synthesis, storage, transportation, and breakdown as shown in Figure 2. In the presynaptic terminal, ACh is synthesized from its precursors acetyl coenzyme A and choline by the enzyme choline acetyltransferase (ChAT) and then loaded into synaptic vesicles by the vesicular acetylcholine transporter, or VAChT.

Depolarization of the presynaptic neuron promotes exocytosis of ACh from the synaptic vesicles and subsequently released into the synaptic cleft where it can activate both the muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs). The

3 activation occurs at the pre- and post-synaptic acetylcholine receptors only for a brief time until it is rapidly terminated by hydrolysis of ACh by acetylcholinesterase (AChE), releasing choline and acetate. 25 AChE is secreted by cholinergic neurons into the synaptic cleft where it is

Figure 2 Figure adopted from Ferreira-Vieira et al. Curr. Neuropharmacol. 2016, 14(1), 101-115)

normally associated with the plasma. 26a,b Each AChE can hydrolyze approximately 5000 molecules of ACh per second, making the enzyme one of the most efficient enzymes known to

4 date. 27 Following degradation, choline is taken up into the presynaptic axon terminal by a choline transporter (Cht1)--the main transporter found in cholinergic neurons and the transporter responsible for supplying choline for regeneration of ACh. 28a,b Disruption of Cht1gene expression in mice experience death within an hour of birth due to symptoms related to ACh deficits. 29

ACh activates both muscarinic and nicotinic receptors, which originally derived their names due to their additional activation by muscarine, a natural product of certain mushrooms, and nicotine, an alkaloid found in tobacco. The nicotinic receptors are part of the ligand-gated ion channel superfamily that form homo and heteropentamers whereas the mAChRs are GPCRs that are subdivided into two families depending on the type of G-protein to which they couple.

G-Protein-Coupled Receptors (GPCRs) (1.3): G protein-coupled receptors (GPCRs) are a large family of plasma membrane proteins highly conserved throughout evolution with nearly 800 different human genes (>2% of the human genome) coding for them. 30 The evolution of multicellular organisms was partly dependent on the successful evolution of GPCRs, which translate extracellular signals to intracellular functions thus enabling cells to communicate with each other and with their environment. 31 GPCRs are widely present in most life forms, from bacteria, fungi and animals, and are involved in nearly all aspects of animal physiology.32 GPCRs are recognized by a vast variety of ligands such as: ions, odorants, lipids, photons, amino acids, hormones and polypeptides. 33 Additionally, they are involved in several cell signaling transduction cascades that regulate numerous important cellular processes.34 Thus they have been implicated in a wide range of biological functions (including behavior, cognition, immune response, mood, olfaction, blood pressure regulation, and taste among others). 35

GPCR function is implicated in the regulation of various physiological systems and disease states such as: central nervous system disorders, inflammatory diseases, metabolic imbalances, cardiovascular dysfunction, cancer, gastrointestinal disorders, and others. 36 The cell membrane location of GPCRs and the diversity of tissue expression make GPCRs ideal targets for drug discovery. Out of 219 new molecular entities (NMEs) approved the US Food and Drug

Administration (FDA) from 2005-2014), 54 (~25%) target GPCRs. 37 GPCRs continue to be one of the most popular targets for drug design since current therapies only target approximately 80 of the known GPCRs (~10% of the known coded receptors) leaving much room for discovery of novel therapies. This task has not been easily accomplished due to complicating factors such as commonality of ligands targeting subfamilies of GPCRs, homology among ligand binding sites, and difficulty in obtaining 3-dimensional structures receptors in both active/inactive states

(although several advances have been made in this area).

Despite the GPCR superfamily being so diverse, very little sequence homology exists among families. Nevertheless, the superfamily does possess a characteristic seven trans-membrane (7-

TM) structure connected by three intracellular (ICLs) and three extracellular loops (ECLs). The

N-terminus, located on the extracellular side, and the ECLs are responsible for the recognition of a wide variety of ligands and also controlling ligand entry into the orthosteric binding pocket.

The C-terminus and ICLs interact with G-proteins, arrestins, GPCR kinases (GRKs), and other effectors needed for signal transduction or other modulator-like functions. 39 Each TM helix is composed of 25-30 amino acid residues with a relatively high degree of hydrophobicity.

Different types of GPCRs demonstrate sequence variations in the length/function of their N- terminal/C-terminal domains and their intracellular loops (Figure 3).31

Figure 3: Figure from Lee et al. J. Med. Chem. 2018. 61, 1, 1-46) A2A AR structure shown above as an example (PDB ID: 4EIY bound to ZM2411385, a known inverse agonist. Highlighted portions on transmembrane (TM) module indicate highly conserved/functional sequence motifs.

The majority of the information on GPCR three dimensional (3D) structure was initially based on the high resolution structures of the inactive state of rhodopsin. Rhodopsin is a visual pigment located in the photoreceptor cells of retina and is responsible for converting photons into chemical signals. 40 This protein was well suited for structural studies due to the possibility of obtaining large quantities of high purity protein from bovine retina. 41 Several groups have since obtained 3D crystal structures of the protein (Okada et al. 2000, Palczewski et al., 2000, Teller et al. 2001, among others) which served as a template for other GPCRs (for modeling studies) until

7 other structures of GPCRs were subsequently discovered. Due to the developments in protein engineering (site directed mutagenesis, introduction of fusion partners, truncation of loops/termini to stabilize tertiary structure, etc) and new crystallographic techniques (vapor diffusion and lipidic cubic phase--LCP methods) over 180 GPCR structures are now known. This is far from complete, but these new methodologies along with advanced molecular dynamics simulation techniques suggest that an unprecedented amount of information about GPCRs and drug discovery will be available in the coming years.

GPCRs are classified into six groups (A to F, gpcrdb.org) based on sequence homology and

Figure 4 from B. Moran and P. Flatt. Acta. Diabetologica. 2016, 53(2), 177-188. Different classes of GPCRs.

8 endogenous ligands that bind to them (Figure 4). These include: class A (rhodopsin-like, class B

(secretin-like), class C (metabotropic glutamate), class D (pheromone), class E (cAMP), and class F (Frizzled). 42a,b Each of these classes can be further divided into subclasses based upon agonist selectivity and downstream signaling events. 42b The rhodopsin-like (class A) family accounts for approximately 80% of all GPCRs that share high sequence similarity 43 and share several structural characteristics with rhodopsin. 44 In general, ligand binding for most rhodopsin- like receptors occurs within a pocket inside the TM cavity involing residues in TM3/5/6. 45

Notable members of this class include: muscarinic acetylcholine receptors, dopamine receptors, adrenergic receptors, opioid receptors, adenosine receptors and histamine receptors.

The secretin-like (class B) family consists of: calcitonin receptor, corticotropin releasing hormone receptor, glucagon and glucagon-like receptor, growth hormone releasing receptor, and several others. This receptor family is characterized by binding to large peptide hormones. 43 The secretin receptor and other members of this family play a key role in hormonal homeostasis.

Novel insights into the structure and function of the class B receptor family was offered by determination of the 3D crystal structures of the glucagon receptor and the corticotropin releasing factor receptor 1. 46 Class B represents an important family of receptors with high potential targets for drug development due to their role in central homeostasis. 45

The glutamate-like receptor (class C) family consists of metabotropic glutamate receptors

+2 (mGlu receptors), Ca sensing receptors, γ-aminobutyric acid B receptors (GABA B), sweet and amino acid taste receptors, pheromone receptors, odorant receptors (fish) and some orphan receptors. 47 The N-terminus of glutamate receptor family members is 280-580 amino acids long and contains the ligand recognition domain 30 generated by forming two lobes separated by a cavity in which glutamate binds to produce the so called "Venus fly trap" or VFT domain. 34,48

This extracellular domain is common to class C receptors and it is responsible for ligand recognition. The VFT domain may also accommodate allosteric binding sites and it is connected with the 7-TM core through a cysteine rich domain, which also plays a role in receptor activation. 49 Class D/E are unique to Fungi and Amoeba respectively.

GPCRs couple to and activate guanine nucleotide binding proteins; hetero-trimeric proteins that consist of three subunits (α, β and γ) that exist in an inactive complex. Gα is the largest subunit (40-45 kDa) in the heterotrimeric complex. It is composed of two domains; a GTPase

(guanosine-5'-triphosphate) domain and an α-helical domain) and is capable of forming a complex with one β (35 kDa) and one γ subunit (15 kDa), when in GDP-bound (guanosine-5'- diphosphate) form. 50 Upon ligand mediated activation the receptor undergoes a conformational change leading to the exchange of GDP for GTP on the Gα subunit, which then dissociates from the Gβγ complex. The G-protein subunits then act on downstream effectors and initiate signaling pathways (Figure 5). The GTPase activity of the Gα subunit hydrolyses GTP into GDP, leading to the termination of the G protein mediated downstream signaling and the different subunits reassociate back into the hetero-trimeric complex.32 While initially thought to be a passive partner to the G α subunit, Gβγ complexes are now understood to regulate a myriad of effectors initiating signal transduction pathways. 32 The Gβγ complex can interact with phospholipase Cβ

(PLCβ), 51 G protein receptor kinase 2 (GRK2), 52 G protein-regulated inwardly rectifying potassium channels (GIRK), 53 N-type calcium channels, 54 and adenylyl cyclase (AC) isoforms

(Figure 5).55

There are four main classes of Gα subunits that have been identified based on sequence similarity including: Gα s, which stimulates adenylyl cyclase (AC) activity, Gα i-o, which inhibits

10 adenylyl cyclase activity (leading to decrease in intracellular cAMP levels), Gα q, which activates

+2 phospholipase Cβ (leads to increase in intracellular Ca ), and Gα 12/13 , which promotes Rho

Figure 5: Adopted from N. Wettschureck and S. Offermanns. Phys. Rev. 2005, 85, 1159- 1204. Representative GPCRs coupling to different signaling pathways. Although there are exceptions, three basic patterns of GPCR coupling largely dominate cellular responses.

activation (regulation of Rho guanine nucleotide exchange factors, RhoGEF proteins).45 In the activated heterotrimeric G protein complex, the Gα-GTP bound subunit dissociates from the βγ subunit resulting in propagation of signaling cascades.

Gα s subunit activates the enzyme AC, which in turn catalyses the conversion of ATP into cAMP, resulting in increased cytosolic cAMP levels. Four molecules of cAMP are required for binding to the 2 R-subunits (each having 2 cAMP binding sites) of PKA. When activated, the regulatory units detach, revealing two catalytic subunites which phosphorylate several specific

56 serine and/or threonine residues on multiple proteins. The Gα i/o subunit has the opposite effect, leading to the inhibition of AC activity and thus reduce cAMP production (therefore decreasing

PKA activity). The Gα q/11 subunit activates the enzyme PLC which hydrolyses phosphatidylinositol 4, 5 -bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5- trisphosphate (IP3). 57 DAG remains bound to the membrane and acts as a second messenger by activating protein kinase C (PKC) which phosphorylates proteins. IP3 diffuses in the cytosol and acts on IP3 receptors located in the endoplasmic reticulum (ER), resulting in an increase in intracellular calcium ion levels. 58 Calcium ions together with DAG work towards the activation of PKC. The Gα 12/13 subunits act by activating Rho guanine nucleotide exchange factors

(RhoGEFs) which in turn activate the small cytosolic Rho-GTPase. The Rho-GTPase can activate numerous proteins such as Rho-kinase that are responsible for regulation of actin cytoskeletal remodeling in cells. 59

Regulation of GPCR signaling occurs through desensitization, internalization (into clathrin coated vesclies), and down-regulation (recycling) with β-arrestin playing the critical role in uncoupling GPCRs from their G-proteins (Figure 6).60 The family of arrestins contains four members including two visual arrestins (arrestin 1 and 4) and two non-visual arrestins (arrestin 2 and 3). The visual arrestins are restricted to binding to photoreceptors such as rhodopsin. 61 β- arrestins (1 and 2) are of broader importance in terms of GPCR signaling regulation. 62 Arrestins bind to the ligand (agonist) activated receptor after GRK has phosphorylated several

12 serine/threonine residues within the C-terminus or the third ICL, creating a high-affinity binding site.

Figure 6: Figure from L. Luttrell and R. Lefkowitz. J. Cell Science. 2002, 115, 455- 465. Regulation of GPCR signaling. Arrestins facilitate desensitization, sequestration, and trafficking of GPCRs.

Following receptor stimulation for a prolonged period of time, receptor mediated signaling is attenuated (i.e. desensitization). The process begins with exposure of the receptor to agonist and is initiated by the phosphorylation of key residues in the C tail and cytoplasmic loops of the receptor by protein kinases (GRKs). The interaction between the receptor and β-arrestin confers conformational changes upon the receptor to dissociation of the receptor-G protein complex and the termination of signaling cascades. 60 Ligand binding is not required for this process, as

13 unbound receptors can be desensitized by via receptor phosphorylation by second messenger- dependent protein kinases. 60

Arrestins have the ability to act as adaptor proteins by binding to components of the endocytic machinery. The high affinity binding of β-arrestin to clathrin, dynamin and β2- adaptin subunit of the AP-2 adaptor protein results in a multi-complex formation linking the receptor-bound arrestin to the clathrin endocytic machinery. This process initiates receptor internalization in clathrin coated pits, and subsequently in vesicles.63a,b This process of regulation/recycling of

GPCRs via the arrestin pathway has been observed in real time via immuno-fluorescence microscopy in live cells. 64 β-arrestins have been found to bind a plethora of proteins, hence serving as critical regulators of GPCR signal transduction and permitting them to integrate

GPCR-mediated signals with other inputs. 65

Muscarinic Receptor Location and Function (1.4):

Muscarinic acetylcholine receptors (mAChRs) are a family of five closely related class A

GPCRs (M 1-M5), encoded by the CHRM1-CHRM5 genes, and are highly conserved throughout evolution. 66 The muscarinic acetylcholine system is thought to regulate several important central and peripheral functions including: cognitive, behavioural, sensory, motor, and autononic phenomena. Dysfunction of this system has been implicated in various pathologies such as

Alzheimer's disease (AD), Parkinson's disease, depression, schizophrenia, urinary incontinence, and chronic obstructive pulmonary disease (COPD). Acetylcholine-mediated activation of the receptors is responsible for G protein activation and the downstream signaling pathway that follows activation. 67

The mAChRs are sub-divided into two groups based on the preference for G protein coupling efficiency. M 1, M 3 and M 5 couple to G q/11 proteins upon agonist activation of the receptor.

Activation of the G q/11 protein is followed by activation of PLC, which hydrolyses PIP 2 into two

68 second messengers (IP 3)/(DAG) (Figure 7). IP 3 binds to receptors on the ER and induces their opening causing an influx of Ca 2+ into the cytoplasm 69 DAG activates several isoforms of

Figure 7: Figure from Jeon et al. Curr. Neuropharmacol. 2015 , 13, 739-749 Signal transduction pathways regulated by CHRM1-5.

PKC that have various functions such as participating in negative feedback of PLC-β signal

70 transduction, and activating protein kinases/transcription factors. The βγ subunits of the Gα q protein can regulate the function of certain ion channels including potassium or calcium

71a,b channels. M2 and M 4 muscarinic acetylcholine receptors show a preference for G i/o protein coupling that leads to direct inhibition of AC, which is responsible for catalyzing the conversion

15 of ATP to cAMP. 72 Lower levels of intracellular cAMP in the cytoplasm leads to inactivation of

PKA (which regulates ion channel activity), either opening Ca 2+ /K + or closing Na + channels in the plasma membrane. Therefore, inactivated PKA decreases the rate of internalization of Ca 2+

+ 73a,b into the cell yet increases efflux of K . This is a key pathway that follows activation of M 2

74 receptors in the heart muscle. The M 2 receptor has also demonstrated the ability to activate alternate G proteins such as G s resulting in a stimulatory effect on cAMP synthesis (Figure below).

The mAChRs play an important role in the regulation of numerous essential functions of the central and peripheral nervous systems. The expression of these receptors is widespread in numerous tissues and organs as observed by immunological and immunocytochemical methods, 75a,b in situ hybridization histochemistry 76 and generation and analysis of muscarinic

CHRM1-CHRM5 knock-out

(KO) mice. 66 Because of

advanced imaging techniques

researchers have been able to

define the regions of the

brain in which cholinergic

projection neurons are

confined typically defined by

Ch1-Ch8 (Figure 8). 77

Figure 8 : Figure from Thiele, A. Annu. Rev. Neurosci. 2013 , 36, 271-294. Locations of Cholinergic Neurons in the Brain.

Their contribution to cognition is partially linked to the locations of the cholinergic neurons.

For example, Ch1-4 are in the basal forebrain (BF), which has been shown to support attention, learning, and memory. Ch5 (pedunculopontine tegmental nucleus) and Ch6 (laterodorsal tegmental nucleus) reside in the caudal midbrain. 78 They supply the thalamus, the pontine reticular formation, the ventral midbrain, the ventral tegmental area, and the substantia nigra (the distinct location of CHRM5). Neurons in Ch5 and Ch6 contribute to arousal, sleep, and the control of dopaminergic cell groups. 77 The mAChRs are found abundantly throughout the CNS and periphery as shown in the following figure. M 1,2,4 are abundant in the cortex and striatum,

75b, 79 but M 2 shows greater expression in the striatum. M1-4 are predominantly expressed in the hippocampus whereas the BF is mainly composed of M 2 receptors (Figure 9). However, linking the contribution of individual mAChR subtypes to performance in specific domains in the brain

has been challenging largely because each

individual subtype does not perform any

singular cognitive function. Instead, they

shape neuronal and local network

processing abilities. Depending on the

network/function needed, a particular

cognitive function will be supported.

However, the mAChRs are not distributed

homogenously throughout brain, so some

specificity must exist in order for proper Figure 9: Figure from Thiele, A. Annu. Rev. Neurosci. 2013 , 36, 271-294. Distribution of mAChRs in the brain. physiological function to occur.

Historically ACh signaling in attentional processes has been met with skepticism as ACh was thought to contribute largely to unspecific arousal versus more selective processes of attention.

Nevertheless, there has been key findings that dispute the notion that cholingeric signaling does not have good resolution. Zaborsky et al .80 found that cholinergic input to the cortex consists of specific clusters of projected neurons in the basal forebrain together with specific prefrontal and posterior cortical regions that target selected areas in a task/context dependent manner.

Robbins et al .81 found that the functioning of a particular area of the brain in a cognitive task determines the extent of muscarinic signaling, not simply the fact that ACh is released within it.

Several others 82a,b,c found that densities and subtypes of mAChRs differ among areas, layers, and cell types within areas of the brain. These findings lend credence that despite the relatively global nature acetylcholine, a great level of signaling specificity can be achieved.

mAChR (CHRM1-5) knock-out (KO) mice 66 have provided new insights into the physiological and pathophysiological roles of the individual subtypes. All transgenic KO mice were viable, fertile, and without signs of major physiological defects. Phenotypic analysis of mutant mouse strains deficient in each of the five mAChRs offered insight into the numerous physiological functions with which they are involved. 83

M1 (G q/11 ) receptor is widely expressed in the major forebrain areas such as the cerebral cortex, striatum, and hippocampus--but is also abundant in the periphery (salivary glands/sympathetic ganglia). The M 1 receptor has been implicated in AD and related cognitive disorders--hence the initial impetus by Merck and others to develop M 1 selective ligands

(detailed later in this thesis). Studies in mouse models for AD have demonstrated that M 1 deficiency increased the risk for development of the disease 84 and further enhanced the development of amyloid plaques that are characteristic of the disease. Studies using knockout

18 mice have suggested a role for M 1 in learning and memory, and it has been demonstrated that the receptor potentiates NMDA receptor currents. 85 Additionally, it is the only mAChR responsible for activation of extracellular signal-regulated kinase (ERK 1/2) in the hippocampus, a protein involved in synaptic plasticity. 86

Behaviorally, M 1 knockout mice perform as well as wild-type in the Morris water maze, a paradigm used to assess hippocampal-dependent spatial memory. However, performance was impaired under certain experimental conditions in the eight-arm radial maze and in fear conditioning studies (animals learn to predict aversive events). 87 The drastic increase in hyperactivity of these animals (resembling attention-deficit/hyperactivity disorder in humans) 88 makes it unclear whether some of the behavioral impairments are actually due to cognitive

-/- impairments. M 1 mice display significant impairment in non-matching-to-sample working

89 memory and consolidation, implicating M 1 in cortical memory function and tasks requiring prefrontal cortical signaling. 90 A critical finding by Paule et al .88 directly related to our work was that scopolamine/atropine (non-selective mAChR antagonists) produced severe deficits in

-/- cognition in both learning/memory tasks, but M 1 mice only displayed modest effects. This suggests other mAChRs subtypes are also involved in the cognition-enhancing effects of ACh. 66

M2 (G i/o ) )is the most widespread mAChR subtype in brain (nucleus basalis, hippocampus, and basal ganglia) that is expressed predominantly on presynaptic terminals and functions as an autoreceptor controlling ACh release in the hippocampus/cerebral cortex. 91 It is expressed in the periphery and smooth muscle organs (GI tract) but is most predominant in the heart. There it mediates parasympathetic decreases in the force of contraction and in the rate of cardiac contraction by inhibiting voltage-gated calcium channels and activating inwardly rectifying potassium channels. 92

Mice deficient in M 2 receptor showed significant deficits in behavioral flexibility and working memory 93 and demonstrated performance deficits in passive avoidance tests (mice learn to avoid environment in which aversive stimulus was previously delivered). 94 Short term and

-/- long term potentiation were drastically reduced following high frequency stimulation of M 2 mice hippocampi--likely due to increased GABAergic inhibition. These results in KO mice along with several in vitro studies suggested that M 2 is the key presynaptic mAChR autoreceptor mediating inhibition of cortical/hippocampal ACh release 91,94 and prompted the proposal that pharmacological blockade of M 2 autoreceptors may be a useful approach to enhance cognition

(through enhancing synaptic ACh levels). 95a,b This hypothesis was soon rejected after further studies were conducted with selective M 2 antagonists, which showed cognition-enhancing effects, suggesting only a partial blockade is necessary. Other studies on M 2 KO mice revealed the receptor has a role in the regulation of body temperature 96 and also in controlling cardiac myocyte contraction. 97

The M 3 receptor (G q/11 ) is expressed at relatively low levels in brain, accounting for only 5-

10% of total mAChRs in various brain regions. 98 It is mainly found in smooth muscle of the GI tract, urinary tract, exocrine glands, and eyes. Activation of M 3 promotes smooth muscle contraction, salivary gland secretion and is involved in the stimulation of the parasympathetic

79 system. The study of the role of M 3 receptor was assisted by the generation of whole body M 3

KO mice and it was proposed that activation of M 3 is implicated in stimulation of smooth muscle contraction, promotion of glandular secretion, and dilation of peripheral blood vessels. The mice demonstrated reduced food intake and hence, reduced body fat mass.99

It was subsequently shown that M 3 receptor activation in β-pancreatic cells correlated with

100 glucose tolerance and promotion of insulin release. To confirm this, M 3 KO mice that lacked

20 the receptor selectively in pancreatic β-cells were generated and were observed to have impaired

glucose tolerance and reduced insulin secretion. Conversely, transgenic mice over-expressing M 3 in β-cells displayed enhanced glucose tolerance and increased insulin release 101 potentially offering an approach in the treatment of type 2 diabetes through selective activation of M 3

99 receptors. It is believed that activation of peripheral M 3 receptors leads to side effects such as excess salivation and GI distress induced by cholinergic agonists and AChE inhibitors used to treat AD.

M4 receptor (G i/o ) shows similar expression levels as M 1 in brain regions (hippocampus, cerebral cortex, striatum). In the striatum where M4 is most highly expressed along with M 1, dense patches of receptor expression are observed that correspond to postsynaptic sites on medium spiny neurons (GABAergic inhibitory cells representing 95% of neurons with the human striatum). Analogous to the autoinhibitory role that M 2 plays in the hippocampus/cortex,

M4 is the major autoreceptor in the striatum responsible for feedback regulation of ACh release

102 from the presynaptic terminal. M4 proposed actions include regulation of locomotor activity, analgesia, auto-inhibition of acetylcholine and balancing dopaminergic activity. 103

Regulation of dopaminergic signaling and release appears to be mediated by M 4, as KO mice

104 show increased basal and dopamine-regulated locomotor responses. This result suggests M 4 receptors exert inhibitory control on D 1 receptor mediated locomotor stimulation, likely at striatal

105a,b projection neuron where both D 1 and M 4 receptors are coexpressed. Double KO (M 1 and

M4) animals are hypersensitive to agents that disrupt prepulse inhibition (PPI) of the acoustic startle response, a measure of sensorimotor gating which is also disrupted in schizophrenic patients, but can restored through administration of xanomeline. 106 In vivo microdialysis studies revealed that M 4 knockout mice also have elevated basal dopamine levels in the nucleus

21 accumbens and show heightened dopamine efflux in response to psychostimulants (e.g.

94, 107 amphetamine and phencylidine). It should be noted that overlapping functions of M1 and M 4 occur as deletion of either subtype can be compensated for in mice to maintain relatively normal function--suggesting lack of both M 1 and M 4 generates a phenotype similar to that of schizophrenia. This is further confirmed by multiple studies reporting decreased densities of

108a,b,c,d M1/M 4 receptors in the brains of schizophrenic patients.

Expression levels of the M 5 receptor are quite low, but are found in the hippocampus,

109 substania nigra, ventral tegmental area, and in peripheral and cerebral blood vessels. M5 knockout mice have revealed roles for M 5 in dilation of cerebral blood vessels and in reward and reinforcement behaviors, specifically in response to drugs of abuse such as morphine and cocaine. Further support for the role of M 5 in drug addiction came after observing less severe withdrawal symptoms after chronic morphine exposure as well as decreased cocaine conditioned place preference and reduced acute cocaine self-administration. 110a,b

In conclusion, the KO mice studies offered insights into the physiological and behavioral functions of the mAChRs which allowed us to create a hypothesis about the desired pharmacological profile of potential muscarinic antagonists. We knew that antagonism at M 2 and

M3 were especially important to prevent both cardiovascular and peripherally mediated side effects. However, we were uncertain about the desired pharmacological profile regarding M 1 and

M4. We did not focus our efforts on delineating the effects of M 5 since the expression levels were shown to be quite low, and there is no validated behavioral pharmacological test to assay for M 5 activity. A structure-activity approach was taken to construct a large library of potential mAChR antagonists and evaluate them in vivo /in vitro with the goal of elucidating the structural features for a muscarinic antidepressant that lacked cognitive deficits.

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Chapter 2: Drug Design Targeting the Muscarinic Receptors: The Cholinergic Hypothesis, Muscarinic Agonists/Antagonists, and Design of Novel Cholinergics as Rapid Acting Antidepressants

Drug Design Targeting the Muscarinic Receptors (2.1): Promptly after it was discovered that acetylcholine was the natural ligand (agonist) for the mAChRs, almost every simple analog of it was synthesized. In general, it was found that

Figure 10: Early examples of muscarinic agonists and antagonists

33 increasing the size of the acyl group leads to a decrease in agonist activity--as exemplified by butyrylcholine (Figure 10), which has only weak agonist activity. 1 Removing the quaternary nitrogen (i.e. reduced methylation) also reduces activity, as the replacement of the nitrogen by phosphorus/arsenic/sulfur. If replaced by carbon there is essentially no activity since the charge is removed. Figure 2 illustrates some early muscarinic agonists. Muscarine has three asymmetric centers, meaning eight stereoisomers, but only one of the eight has high activity. It was found that the (2S, 3R, 5S) enantiomer possessed the greatest activity through asymmetric total synthesis by Whiting et al in 1972. 2 Muscarine itself has no clinical applications.

F2268 is structurally similar to muscarine with a dioxolane moiety, aceclidine incorporates a hindered bicyclic amine (1-azabicyclo[2.2.2]octane scaffold), and arecoline, an alkaloid found in the areca nut, is widely abused around the world for its effects similar to nicotine. 3 Some of the less obviously related are oxotremorine (known as OXO-M) which became quite useful for its selectivity when incorporated into radiolabeled binding assays. Pilocarpine, first isolated in 1874 from plants from the genus pilocarpus ,4 is a nonpolar tertiary ammonium compounds readily absorbed via mucous membranes. It has since been named to the World Health Organization's

(WHO) list of Essential Medicines for treatment of pressure inside the eye and dry mouth. 5a,b

Although many compounds have muscarinic actions, they tend to be fairly small molecules with the common structural features: (1) a tertiary/quaternary ammonium group, (2) an ester/ether/similar moiety that is electron rich, and (3) a methyl/methylene group to mimic the acetyl methyl group on ACh (Figure 10). However, these requirements do not apply to many of the future agonists that were subsequently designed by Merck, Smith Kline Beecham, Ferrosin

A/S, and Lilly pharmaceutical companies as described in the following section.

The characteristics features of mAChR antagonists include the introduction of a bulky aromatic, heterocyclic, polycylic, or even large aliphatic groups on the acyl end of an acetylcholine-like derivative. 6 Atropine, now on the WHO's list of Essential Medicines, is used to treat a multitude of symptoms, most notably nerve agent/pesticide poisoning. 7a,b It is thought to be slightly selective for the M 1 receptor, but its selectivity is modest at best. Quinuclidinyl benzilate (QNB) was originally developed by Hoffman-LaRoche in 1951 8 while looking for antispasmodic agents (similar to tropine), for treating gastrointestinal ailments 9 and as a chemical warfare agent to incapacitate enemy soldiers. 8 It later proved to be instrumental in delineating the first crystal structures of the muscarinic receptors due to its extremely high affinity for the mAChRs. 10a,b,c Hyoscine, originally isolated from the nightshade family of plants as early as the late 1800s 11 has many current uses in medicine such as: Postoperative nausea/vomiting/sea sickness, motion sickness, GI spasms, irritable bowel syndrome, eye inflammation, among others

(National Library of Medicine, NIH. toxnet.nlm.nih.gov) (Figure 10). More recently (2010), a clinical trial was conducted by WC Drevets and ML Furey 12 to assess scopolamine's antidepressant efficacy in major depressive disorder (MDD). This will be detailed in a later chapter in this thesis.

The Cholinergic Hypothesis: Merck/Smith Kline Beecham/Eli Lilly (among others) and

Alzheimer's Disease (2.2):

From the 1960s-80s much was discovered regarding the physiological roles of neurotransmitters. Evidence that stemmed from this worldwide research effort contributed to the idea that altered function of neurotransmitter pathways was associated with several CNS disorders. 13 This was further confirmed by tissue samples from the brains of post-mortem

Alzheimer's disease patients. It was discovered that cholinergic projection from a basal forebrain neuronal population, the nucleus basalis magnocellularis of Meynert, to the cortex and hippocampus was present in all tissue samples collected. In addition, the activity of choline acetyltransferase, the enzyme primarily responsible for the synthesis of acetylcholine and a marker of cholinergic neurons/synapses, was found to be significantly decreased in the cortex/hippocampus of AD patients. 14a,b Finally, depolarization-induced acetylcholine release and choline uptake in nerve terminals were reduced in these same tissue specimens. 15a,b Thus the cholinergic model of impairment was born in a groundbreaking review published by Bartus et al 16 that summarized that the major event in the pathology of Alzheimer's disease was the degeneration of the cholinergic connection from the nucleus of Meynert to the cortex and hippocampus. 17

Alzheimer's disease (AD) is the most common neurodegenerative disorder that affects the

Figure 11: Main events of Alzheimer's Disease. Adopted from Langmead et al. Pharmacol. and Therapeutics. 2008, 117(2), 232-243

36 elderly, resulting in memory loss and severe cognitive dysfunction. 18 While the disease itself is complex it is characterized by two main events in the brain: 1) aggregates of amyloid plaques

(largely composed of amyloid-β peptide (Aβ)), and neurofibrillary tangles formed via hyperphosphorylated tau proteins. 19 Aβ is generated by proteolytic cleavage of amyloid precursor protein (APP), which can be processed in two different ways. Cleavage of APP by α- sectretase yields soluble APPα, in which the proteolytic site is with the Aβ sequence, preventing its formation. However, when APP is cleaved by β-secretase to yield soluble APPβ and Aβ

(Figure 11 ).20

Several approaches to improve cholinergic transmission and enhance cognitive function were utilized to treat Alzheimer's disease patients including the design of Acetylcholinesterase inhibitors such as: tacrine and physostigmine and directly acting agonists at post- synaptic muscarinic receptors in the cerebral cortex (i.e. Arecoline), RS-86, 21 and pilocarpine (Figure

12). 22

Of these, the AChE inhibitors, which function through enzyme inactivation leading to ACh

accumulation, showed the greatest early success

in clinical trials. Clinical trials with direct acting

agonists were generally disappointing, partly due

to the side effects associated with mAChR

stimulation. Merck originally suggested that the

lack of clinical value of directly acting agonists

could potentially be attributed to their low

Figure 12: Early examples of AChE inhibitors efficacy at the cortical mAChRs as and direct acting agonists compared to acetylcholine. 23

In order to improve the clinical profile of Arecoline, which behaves as only a weak partial agonist in the cortex compared to ACh, the metabolically labile ester group was replaced with the 3-methyl-1,2,4-oxadiazole moiety. The arecoline-based oxadiazoles showed greater potency than arecoline itself but showed marginally less efficacy, as assessed by a two stage binding assay that had been previously established. 24a,b Nevertheless, it warranted the synthesis of related molecules with functionality that was predicted via ab initio Gaussian calculations. This led

Table 1

Merck scientists to develop the methyl and amino oxadiazoles in the quinuclidine series, that were prepared from methyl quinuclidine-3-carboxylate and showed a marked increase in activity over arecoline (Table 1). 25

The binding assays used in this paper which were previously described,24a,b measure affinity and also predict the cortical efficacy of the compounds via the antagonist/agonist binding ratio in rat cortical membranes. Merck used [3H]Oxotremorine-M (OXO-M) to label the high affinity state of the cerebral cortex muscarinic receptors and [3H]-N-methyl-scopolamine (NMS) to label predominantly the low affinity state. By measuring displacement of the desired radioligand it is possible to get an approximate measure of affinity for either state of the GPCR. The log of the

NMS/OXO-M ratio obtained has been shown to be an indicator of the ability of the compound to stimulate cortical phosphoinositol hydrolysis. 24a,b Antagonists, such as atropine, show equal affinity in both binding assays and have ratios close to unity, while weak partial agonists (i.e. arecoline) have ratios between 10-200, partial agonists (i.e. arecoline again) have ratios between

200-800, and full agonists (i.e. carbachol) have ratios greater than 800. In order to probe the stereochemical features of the agonist binding site of the mAChRs,the 1- azabicyclo[2.2.1]heptane (i.e. azanorbornane) scaffold was prepared in place of quinuclidine (3-6 above). A stereoselective route to both diastereomers was developed by Saunders et al .26 Later, a scalable, enantioselective route was developed by Cottrell et al 27 utilizing a 2+3 cycloaddition with a chiral azomethine ylide precursor that affords two separable diastereomers in a 1:1 ratio

(Figure 13).

Ph 1. TFA, EtOAc O O O 2. aq NaHCO 3 O O + O + Me N SiMe 3 H H H H OMe N 1:1 N

Ph Me Ph Me Figure 13: 2+3 Cycloaddition of 5.6-dihydro-2H-pyran-2-one and chiral azomethine ylide precursor

The diastereomers are resolved via crystallization and the desired enantiomer ( R,R,R ) can be obtained in high enantiomeric purity. The synthetic route is detailed later in this chapter. As seen from Table 2, reductions in the bulk size and pKa of the quinuclidine ring (1- azabicyclo[2.2.2]octane core) showed an enhancement in both affinity and predicted cortical efficacy between quinuclidine and the exo -1-azanorbornane (1-azabicyclo[2.2.1] core). The endo diastereomer showed reduced affinity for both the high and low affinity states of the receptor, but is the not thermodynamically favored product. The endo product can be captured under kinetic conditions (LDA at -78 oC) from the exo product in a 31 ratio. 23 The same group synthesized

Table 2

40 isotropane, isoquinuclidine, piperizine, and diazabicyclic oxadiazoles in order to evaluate the viability of other hindered amine core structures. They were then tested using the same assays above (Table 2).

As seen from the binding assay results, increasing the conformational flexibility and bulk of the azabicyclic ring in the isotropane series reduces affinity compared to the azanorbornane. In addition, the exo diastereomer is once again most active which Merck resumes to be due to less steric demand of the smaller ring face in the active site of the receptor. Increasing ring size and conformational flexibility (i.e. the saturated series, 7a-7f ) causes a reduction in affinity, whereas both efficacy and affinity are enhanced in the tetrahydropyridine series ( 8a-8d ). With over 100- fold higher affinity, this provides a semi-rigid template which appears to be favored when targeting the mAChRs. As seen from the isoquinuclidine series, demethylation of the azacyclic nitrogen results in improvement in binding affinity, and in general replacement of methyl with amino (hence increasing hydrogen bonding capabilities of the oxadiazole) also results in enhancement of affinity/efficacy.

Finally, as the pKa of the hindered amine increases, so does the affinity for the mAChRs. The tertiary amine has a pKa~5.5 and would not be significantly protonated at physiological pH and, hence, binds with lower affinity. The reduction in affinity in switching from the azabicycle ( 1 or

2) to the diazabicycle ( 10 ) can be explained in terms of pKa, since it drops from 8.6 to ~6, once again leading to a lower amount of compound that would be protonated at physiological pH.

Since it is the protonated form that binds the mAChRs, tailoring the design of new compounds must account for pKa. According to the data presented above, the 1-azanorbornane scaffold appears superior to achieve high efficacy/affinity at cortical mAChRs.

Shortly thereafter, Sauerberg et al. 28 published a short structure-activity relationship on the arecoline series of compounds exploring the R-group (3-position) substitution on the 1,2,4- oxadiazole moiety. They included multiple lipophilic substituents and measured their in vitro affinity to central mAChRs and their effects on peripheral ileal mAChRs. The results are listed in

Table 3 below. Shifting the alkyl substituent from 3 to 5 in the oxadiazole (i.e. 14a-c) ring diminished both binding affinity and predicted efficacy. Each compound had only very low

Table 3

affinity to central muscarinic receptors and acted as weak antagonists in the ileum assay. A possible explanation for this could be the differences in electron distribution in the oxadiazoles--

42 as they are non-superimposable on each other (double bonds cannot be aligned) and have different orientations in space. In the arecoline series, all compounds with unbranched alkyl chains (i.e. 13a-13g ) had good afinity to central mAChRs and were full agonists in thon the guinea pig ileum, except 13e (pentyl), which acted as a partial agonist.

All derivatives were partial agonist according the agonist index values (i.e. the ratio) with

13h-l all acting as antagonists in the functional assay. Substitution of carbon with oxygen in the alkyl substituent (i.e. 13o-q) caused a decrease in receptor affinity and in ileum agonist potency.

In an attempt to demonstrate that lipophilicity of the oxadiazole substituent contributed to the highest binding affinity, variations to the hindered amine core were explored (Table 4). The butyl oxadiazole was chosen by this group because it was a substituent of intermediate length and lipophilicity. The N-desmethyl analogue ( 15 ) is an agonist just like its arecoline counterpart ( 16 ) but showed a higher agonist index value. This same trend holds true for compound 13a (previous table) and its desmethyl analogue. 29 Substitution of the N-methyl for N-ethyl ( 17 ) or 5 /6-methyl substitution in the arecoline ring ( 18 and 19 respectively) produced compounds with lower affinity for the OXO-M binding site and lower agonist index values. This will be illustrated shortly hereafter in a subsequent study done by G. Showell et al. 30 Compounds ( 17-21 ) were all antagonists on the ileum and displaced QNB at lower concentrations than OXO-M, which resulted in low agonist index values. It should be noted that the index scale is based off of central muscarinic affinity and efficacy, so the scale value is only an indication of the efficacy on the mAChRs in the ileum. Since the ileum has a large muscarinic receptor reserve most agonists will show as "full agonists" even when they could be partial.

Table 4

which resulted in low agonist index values. It should be noted that the index scale is based off of central muscarinic affinity and efficacy, so the scale value is only an indication of the efficacy on the mAChRs in the ileum. Since the ileum has a large muscarinic receptor reserve mosts agonists will show as "full agonists" when they could be partial.

The arecoline scaffold was further explored by Showell et al 30 in attempt to design novel non- quaternary muscarinic agonists related to arecoline but with higher cortical efficacy while

44 successfully penetrating the CNS. They prepared a series of novel analogues and analyzed their affinity to the mAChRs. The results are shown in the table below.

Table 5

Oxadiazole ( 22b, also 8a in Table 2) once again showed improved affinity over arecoline, but

(22f, 8d in Table 2) once again retained the highest efficacy. This once again follows the trend of the quinuclidine series in which small electron donating substituents on the oxadiazole ring enhance its hydrogen bonding acceptor properties. In general, the NH analogues showed higher predicted efficacy than their corresponding N-methyl counterparts. The vinyl oxadiazole ( 22i ) was chosen by Showell to introduce extra rigidity into the system but showed lower efficacy

(NMS/OXO-M ratio=73) than the saturated alkyl analogues (i.e. 22c and d). In the effort to

probe further into the acetyl methyl group (of ACh) binding pocket of the mAChR and study the

hydrogen bond donating/acceptor characteristics analogues 22j/22k were synthesized. The

presence of the hydroxyl groups as H-bond donors reduced affinity for both agonist/antagonist

states of the receptor by ~10 fold, but the fluoroethyl analogue ( 22l , an H-bond acceptor)

retained modest affinity.

O N N N Molecular mechanics calculations were R R 3 N ~2.3 kcal/mol 3 O performed on the protonated forms to R4 N R4 N R1 R1 determine which rotamer (E or Z, Figure

E-rotamer Z-rotamer 14) would be favored. The E rotamer is by

Figure 14: E and Z rotamers of 5,6- approximately ~2.3 kcal/mol, and was substituted arecoline-3-oxadiazoles further confirmed by crystal structure. 30

The nitrogen of the tetrohydropyridine ring

can flip from one conformer to another, and alkylation at C5 or 6 results in decreased mobility

of the ring. This corresponds to reduction in predicted cortical efficacy (comparing 22c to

m/p/q ).

As shown in the above tables, the quinuclidine core showed higher affinity and efficacy than

the corresponding arecoline derivatives. This was further expanded upon by Saunders et al. 31 in

their design of novel quinuclidine-based derivatives. The series they designed included fully

efficacious agonists, partial agonists, and antagonists that were comparable or superior to

established quaternary ammonium ligands. It was well understood that the 1,2,4-oxadiazole was

a suitable replacement for the ester moiety, but the group sought to understand which features of

the oxadiazole ring contributed to the improvement in binding affinity and whether some of the

46 features could be manipulated to generate structure activity relationships. The series was evaluated for binding affinity/efficacy as previously shown (Tables 6 and 7).

Removal of the oxadiazole ring heteroatoms caused a reduction in predicted efficacy although binding to the low affinity was not altered. These compounds confirmed earlier results that showed ligand efficacy was largely dependent upon hydrogen bonding capabilities of the heterocyclic ring. At least two hydrogen bond acceptor sites are required for high affinity at the agonist binding site and the exact position of the acceptors is crucial to obtain a high NMS/OXO-

Table 7

Table 6 47

M ratio.

The group also designed a series of oxadiazoles differing only in the nature of the 3- substituent and found that lipophilicity and electronic factors have a major influence on both affinity and efficacy (Table 7). As the size and lipophilicity of the 3-substituent increased, the low affinity state of the receptor was favored, resulting in compounds with antagonist profiles.

Interestingly, a simple addition of a methylene group (i.e. methyl to ethyl, 33/34 ) resulted in a partial agonist/antagonist profile. Most notably was compound 35 , a mimic of QNB (Figure 15),

had sub nanomolar affinity at both high and low affinity states of the

OH receptor making it one of the most potent antagonists known to O

O 31 N date. As shown earlier the amino oxadiazole proved to be the most Quinuclidine-3-benzilate (QNB) potent and efficacious, confirming the role of hydrogen bonding Figure 15: Structure of QNB stabilizing the agonist state of the receptor. However, N-alkylation

(37 and 38 ) rapidly eliminated the affinity/efficacy for the agonist binding site. Surprisingly, N-acylation did not have as drastic an effect.

Since it had been previously established that five membered heteroaromatic rings such as the oxadiazole and thiadiazole were stable ester bioisosteres, and that 1-azanorbornane represented the optimum azabicyclic ring for mAChR activity, Street et al .32 concluded that high potency/efficacy correlates to two hydrogen bond acceptors in an exact location of the

O N heterocycle. As a result, the N N N N N N NN N N N group prepared a series of 6-

Figure from Street et al J.Med.Chem. 1992, 35, 295-305 membered heteroaromatic

Figure 16: Diazine rings as mAChR diazine rings as potential pharmacophores mAChR pharmacophores

(Figure 16). It was proposed that the diazine ring system should have two H-bond acceptor sites in a comparable location to N2/N4 of the 1,2,4-oxadiazole ring. They validated this proposal via the calculation of two electron potential maps for dimethyl-substituted diazine rings (Figure 16) and concluded that the pyrazine's negative potential was sufficiently comparable to the oxadiazole. However, this was not the case for the pyrimidine and pyridazine rings. The group prepared a series of pyrazine based analogues and evaluated them based on their affinity for cortical mAChRs as above (Table 8).

R N R N R N N 1 X N O N R N exo N 2 N 1) X=O 3) R=Me O N N 24 23) X=S 5) R=NH 2 N endo R 4) R=Me 6) R=NH 2 K , µM app 3 3 Compound R R1 R2 [ H]NMS[ H]OXO-M Ratio

arecoline 6.2 0.011 560 RS-86 5.0 0.040 130 pilocarpine 4.0 0.040 100 atropine 0.0010 0.00048 2.1 carbachol 22 0.0049 4500 1 0.44 0.00096 460 23 0.36 0.00078 460 3 (exo) 0.10 0.00009 1110 5 (exo) 3.6 0.0021 1700 4 (endo) 0.031 <0.000043 >1000 0.0051 1060 6 (endo) 1.6 24a H H H 0.27 0.00068 400 31 24b H H Me 0.066 0.0021 24c Me H H 1.51 0.010 151 24d H H OMe 0.14 0.0076 18 24e H Me Me 0.078 0.0095 8 24f Me H Me 0.26 0.026 10 24g H H Cl 0.051 0.00087 59 24h H H Et 0.020 0.0017 12 24i H H OEt 0.019 0.0021 9 24j H H Br 0.033 0.0010 33 24k H H OH 27 0.28 96 Table adopted and modified from Street et al. J.Med.Chem. 1992, 35, 295-305 Table 8

In addition the group designed a series of 3-substituted-1-azanorbornane analogues that proved to be both the highest affinity agonists ever created (Table 9). The binding data for unsubstituted quinuclidinylpyrazine ( 24a ) shows affinity and predicted cortical efficacy comparable to that of ( 1) and ( 23 ). The electron withdrawing effect (see C. A. Grob 1985) 33 of

31,34 the pyrazine on the base is reduced when compared to the methyloxadiazole (1) (pK a=8.6)

32 increasing its pK a value to 9.8. Previous studies from this group and others showed that when that pK a of the ligand can be reduced, the mAChR activity generally enhanced.

The endo diastereomer ( 25a ) had reduced affinity to the antagonist binding state of the receptor when compared to ( 1), but agonist binding is unchanged resulting in one of the highest ratios ever reported for a nonquaternary muscarinic agonist. Increasing the conformational flexibility of the ring in going from the azanorbornane ring to isotropane resulted in a loss of binding to both the high/low affinity states (data not shown). The 6'-methyl-substituted pyrazine

(24b ) showed a higher affinity for the the NMS-labeled state of the receptor compared to ( 24a ) and hence reduced predicted efficacy. Methyl substitution at C3' resulted in reduced affinity at both states of the receptor, and similar activity was shown for the C3'/C6' dimethyl pyrazine

(24f ). Introduction of larger substituents (i.e. ethyl, 24h , methoxy, 24d , and ethoxy, 24i ) all favored the low-affinity state binding--potentially useful for designing muscarinic antagonists.

The trend for the 1-azanorbornanes was harder to predict. However, this series showed a greater tolerance to steric bulk at C3'/C5'/C6'. As shown by the drastically different profiles of

25/26a (endo and exo isomers), affinity was dependent upon both the nature of the substitution and the position. Increasing the size of the substitution at C6' (i.e. 25/26l ) generally led to reduction in predicted efficacy since the compounds had increased affinity for the low-affinity

50 binding state. Nevertheless, the examples show that the pyrazine is a suitable bioisostere for the ester group of the endogenous ligand, ACh. However, unlike the endogenous ligand and the

R N R1 endo N R2 N N N exo 25 R R2 26 N

R1 Kapp , µM 3 3 Compound R R1 R2 Stereochemistry [ H]NMS[ H]OXO-M Ratio arecoline 6.2 0.011 560 RS-86 5.0 0.040 130 pilocarpine 4.0 0.040 100 atropine 0.0010 0.00048 2.1 carbachol 22 0.0049 4500 25a H H H endo 1.6 0.00070 2286 26a H H H exo 0.059 <0.000086 >686 25b endo 0.47 0.0015 313 H H Me 26b H H Me exo 0.18 0.0014 129 25c 0.0016 325 Me H H endo 5.2 26c Me H H exo 0.21 0.00026 800 25d endo 0.53 0.013 41 26d H Me Me 0.015 19 25e H Me Me exo 0.28 23 26e Me H Me endo 3.0 0.13 25f Me H Me exo 0.19 0.011 17 26f Me Me H endo 6.2 0.66 9 25g Me Me H exo 0.75 0.017 44 26g endo 1.6 0.25 25h Me Me Me 6 exo 26h Me Me Me 0.35 0.53 7 26i Et H H endo 2.2 0.14 16 26j Et H H exo 0.69 0.020 35 25k nPr H H exo 0.35 0.015 23 26k iPr H H exo 0.48 0.022 22 25l H H Et endo 0.15 0.0021 71 26l exo 0.13 0.0095 14 25m H H Et endo 47 26m H H OMe 0.46 0.0098 exo 0.038 25 25n H H OMe 0.96 26n H H OEt endo 0.12 0.012 10 25o H H OEt exo 0.53 0.055 10 26o H H OiPr endo 0.049 0.012 4 25p H H OiPr exo 0.13 0.031 4 26p endo 0.12 0.021 6 25q H H O-allyl exo 0.030 8 26q H H O-allyl 0.23 endo 0.070 13 25r H H NMe 2 0.93 exo 0.86 0.057 15 26s H H NMe 2 25t H H Cl endo 0.21 0.00056 375 25u H H O-propargyl endo 0.15 0.0088 17 Table adopted and modified from Street et al. J.Med.Chem. 1992, 35, 295-305

Table 9

51 oxadiazole series, hydrogen is the preferred size of the substituent for binding to the high affinity state. Any substitutions on the pyrazine ring (methyl or larger) result in reduced cortical efficacy and show enhanced binding to the antagonist state of the mAChR (Table 9).

The Design of Functionally Selective Muscarinic Agonists Targeting the M 1 Receptor (2.3):

While successful development of some of the most potent non-quaternary agonists ever known was achieved, the compounds provided little therapeutic benefit due to their lack specificity. This led to dose-limiting cholingeric side effects. Therefore, effort shifted in the early

90's to generate functionally selective agonists, generally targeting the M 1 receptor.

Neurochemical examination of brain material from Alzheimer's patients showed loss of the presynaptic marker enzyme (i.e. loss of synaptic terminals), choline acetyltransferase, and of

35a,b,c mAChRs of the M 2 subtype. The M 2 receptors appear to be located presynaptically, where they function as autoreceptors regulating the release of ACh. 36 The post-synaptic mAChRs, primarily M 1, appear to survive the loss of cholinergic nerve endings in different brain regions.

Those findings led to clinical studies with the agonists arecoline, RS-86, and pilocarpine but produced disappointing results likely due to lack of specificity and side effects associated with

37 over stimulation of M 2/M 3 receptors. Therefore, Sauerberg et al. , and a few months later Ward

38 et al . set out to design M 1 selective compounds of the 3-(1,2,5-thiadiazolyl)-1,2,5,6-tetro1- methylpyridine and 3-pyrazinyl-1,2,5,6-tetrahydro-1-methylpyridine types (Table 10).

Where comparisons were possible, the alkoxythiadiazoles were more active than the alkoxypyrazines of the same chain length in all biological assays. 38 This same trend held when comparing alkylthio and chloro substitutions as well. Additionally, adjusting the position of the

52 nitrogens/oxygen atoms in the 1,2,4-oxadiazole to generate 1,2,5-oxadiazole significantly reduced affinity and efficacy in all cases. In short, the length of the alkyl chain attached to the

Table 10

thiadiazole or pyrazine had a profound effect on M 1 efficacy, where 5-6 carbons appeared to confer maximum efficacy. It was proposed that the side chain likely fits into a hydrophobic pocket in the receptor or lies along a lipophilic section on one of the α-helices in a

53 transmembrane segment of the receptor--stabilizing the active conformer of the protein. 37,38 The proposal was further extended to include that the length of the alkyl side chain is likely responsible for the separation of the M 1-M3 functional agonist activity. Generally, the shorter the alkyl chain were effective in suppressing the force of contraction in the guinea pig atria, an M 2 agonist response, while longer chain derivatives were poor M 2 agonists, suggesting that the longer alkyl chains either interfere with binding to the receptor of prevent its activation. As shown in previous studies (above tables) containing 3-substituted 1,2,4-oxadiazoles attached to quinuclidinyl or arecoline-like rings, increading the lipohilicity and size of the 3-substituent diminished agonist activity. Similarly, substitutions at the 5- or 6-position of the pyrazine ring diminished agonist activity. It can be inferred from this data that this particular region of the

receptor is sterically demanding.

In fact, the hexyloxythiadiazole derivative, also known as

Xanomeline (Figure 17), showed early promise as a potential reatmentFigure 17: for Xanomeline AD. Binding studies conducted showed it was a subtype selective M 1

receptor agonist, although other in vitro/in vivo functional studies suggest that the compound is better classified as a subtype selective M 1/M 4 agonist. Xanomeline was shown to improve psychosis and behavioral disturbances in AD patients 39 and also found to produce both significant psychiatric improvements along with improvements in learning/memory in schizophrenia patients. 40 Unfortunately, the clinical use of the drug was limited by its side effect profile that included: salivation, sweating, and gastrointestinal distress, all of which are

41 classical cholinergic side effects due to stimulation of M 2 and M 3 receptors. The GI side effects were the main culprit of the high drop-out rate in clinical trials. 42a,b The method of

54 delivery played a major role in the side effect profile, as the oral formulation which caused the high dropout rate in the clinical trials has now been substituted with a transdermal patch. 42b

43 In attempts to improve upon the M 1 agonist activity of Xanomeline, Ward et al designed a series of 3- and 6-substituted pyrazinylazacycles to assess for M 1 activity/selectivity. Although

Street et al 32 had described several pyrazinyl azacycles in a previous publication (Table 9 above), those studies focused on 6-substituted pyrazines, relatively small substituents, and the few 3- substituted pyrazines that were made did not utilize the alkoxy/alkylthio moieties previously employed in the design/synthesis of the arecoline-like series (i.e. 27, 28 a-l). Those studies showed enhanced selectivity for M 1 was largely related to the side chain and length of the alkyl substituent (alkoxy/alkylthio with 5-6 carbons), which were never explored by Street et al . In addition, M 1 selectivity was not evaluated, rather, the ratio of high/low affinity states of the receptor. This is correlated to cortical efficacy, the ability of a compound to stimulate phosphoinositol (PI) hydrolysis in rat cortical slices. Unfortunately, because the rat cortex contains both M 1 and M 3 receptors that are coupled to PI hydrolysis, it does not reliably distinguish between M 1 and M 3 agonist activity. Therefore, the group's strategy was to evaluate

M1 agonist activity by measuring the compound's ability to stimulate PI hydrolysis in a cell line stably expressing the cloned M 1 receptor. Results are shown in Table 11 below.

For the quinuclidinylpyrazines containing small 3-substituents the order of affinity for the

OXO-M binding site was H>Me>Cl>OCH 3. Among the alkoxypyrazines ( 30d-j) the affinity for the agonist binding state of the receptor increased until reaching the butyloxy side chain, where it plateaued between butyl- and hexyloxy, then decreased for the longer side chains. The enantiomers of 30i were separated, although there was only a modest improvement for the ( S) over the ( R) (5x higher affinity). Replacing the oxygen with sulfur ( 30k-m) significantly

55 enhanced binding to the OXO-M site, with maximum affinity reached with the butythio derivative. All had higher affinity for the agonist binding state than arecoline. Because the hexyloxy side chain had the highest affinity, the group designed other azacyclic(hexyloxy)pyrazines with the following hindered amine cores: arecoline,

Table 11

tetrahydroazapine, endo -isotropane, and exo -azanorbornane (data not shown). Surprisingly the exo -azanorbornane did not show the highest affinity, but rather the quinuclidine ( 30i ) and the isotropane derivative. All alkoxypyrazines (or alkylthio) did not produce salivation in mice at the

10mg/kg ip screening dose lending support that the group was successful at developing functionally selective ligands for the M 1 receptor. The data shows that affinity and efficacy are

56 highly dependent on the lipophilic binding site occupied by the alkoxy and alkylthio side chains.

While the alkylthio derivatives have higher affinity than their alkoxy counterparts, the efficacy of the alkoxypyrazines is greater in magnitude when similar carbon side chains are compared.

Efforts continued to develop M 1 selective ligands along with lower efficacy partial agonists in hopes of avoiding cholinergic side effects. In fact, the development of M 1 preferring receptor agonists has been pursued for over two decades with limited success. 42b Tetrazole/1,2,3-triazole scaffolds, 44 oxime ether functionality, 45,46 and ether linkages directly to functionalized pyrazine rings were designed in attempt to replace the 1,2,4-oxadiazole ring. This led to the development of the "first generation" of compounds that advanced to clinical trials such as Tazomeline, 47

Alvameline, 48 Cevimeline, 49 Milameline, 50 NGX-267, 51 Talsaclidine, 52 Sabcomeline, 53 and

WAY-132983 (Figure 18).54 While the majority of the compounds failed at some stage clinical development, Xanomeline demonstrated efficacy in improving cognitive deficits and behavioral disturbance in

O O N N N Et N S N N N S NH

N S N N N Tazomeline Milameline Alvameline LY-287041 CI-979, PD129409 LU25109 NGX-267 RU35926 O N N O S O N CN N N N S N O

Sabcomeline Talsaclidine Cevimeline (Evoxac) SB202026 WAL2014 WAY-132983

Figure 18: Early M 1 preferring agonists

AD patients 39 and with schizophrenia patients to sustain efforts to continue to orthosteric ligands for the mAChRs.

In conclusion, pharmaceutical companies spent great time, effort, and finances in designing mAChR ligands with the hopes of treating AD patients. Unfortunately, while successful in generating some the most potent agonists known to date, they were met with little success due to cholinergic side effects--primarily due to lack of specificity for a specific mAChR subtype. 42b

Nevertheless, their efforts were crucial for this current project as they were successful in designing a small number of potent mAChR antagonists. Although Merck/Lilly/and others were not interested in the potential of muscarinic antagonists, we have used their modeling studies and design of compounds to serve as starting points for designing a library of muscarinic antagonists that were evaluated for both binding/efficacy in vitro and pharmacologically in a battery of tests in rats. More recently designed second generations of muscarinic-M1-selective agonists have emerged from high throughput screening (HTS) followed by medicinal chemistry along with the synthesis of allosteric modulators. This will be detailed in a later chapter of this thesis.

Muscarinic Antagonists and Depression (2.4): Interest in the mAChRs in depression and mood disorders initially stemmed from a study conducted by Rowntree (1950) that showed the organophosphorus insecticide diisopropylfluorophosphate (DFP), a cholinesterase inhibitor that increases synaptic ACh levels, caused a characteristic depressive effect in healthy people but decreased manic symptoms in patients with mania. 55 It was later shown that increasing cholinergic activity using the anticholinesterase inhibitor, physostigmine, not only exacerbated depressive symptoms in currently depressed subjects with major depressive disorder (MDD) but also induced depressive symptoms while reversing manic symptoms in patients with bipolar disorder (BPD). Neuroendocrine and

58 pupillary responses to physostigmine were also abnormally increased among depressed individuals. Janowsky and colleagues concluded that an imbalance between central cholinergic and adrenergic neurotransmitter activity could induce mania and depression. 56 More specifically, they suggested that a hypocholinergic-hyperadrenergic state could cause mania, and vice-versa could contribute to symptoms of depression.

The mAChRs were specifically implicated in these effects by evidence showing that polysomnographic responses to selective mAChR agonists were much greater in depressed patients versus control samples, implying that muscarinic receptor hypersensitivity existed in depressed individuals. The cholinergic system in the brain stem plays a significant role in the activation of the rapid eye movement (REM) and the non-REM sleep cycle. When cholinergic agonists were given such as RS86, arecoline, and physostigmine to patients with major depression, the sensitivity of REM sleep response in increased. 57 In contrast, scopolamine, a mAChR antagonist has been shown to decrease REM sleep and increase sleep latency in patients with depression. 58a,b Furthermore, Cannon et al .59 and others 60a,b showed that variation in the muscarinic M 2 cholinergic receptor gene ( CHRM2 ) was associated with a higher frequency and severity of unipolar depression and with abnormal reductions in M 2 receptor binding in bipolar depression.

Mood disorders related to abnormal cholinergic receptor function showed sex effects. In premenopausal females with MDD it was shown that there was a higher frequency of heightened cholinergic sensitivity. 61a,b Comings et al 60 found that genetic variants in the CHRM2 gene (A/T

1890 polymorphism in 3' UTR of CHRM2 gene, homozygotes) was directly associated with

MDD in female subjects. Several other studies followed about the CHRM2 gene to reveal that modest associations existed between variation in gene sequence and populations of European-

Americans/African Americans with affective disorders. 62 Furthermore, a combination of SNPs, which has a 40% frequency in patients with alcohol dependence and MDD, at the 5' end of the

CHMR2 gene was found to be associated with these two diseases based on the collaborative study on the genetics of alcoholism 60b although there have been conflicting results by others in clinical cases. 63a,b Although much remains to be known about the genetic associations underlying depression, a few general hypotheses have been suggested: 1) M 1 receptors provide a reliable

64 target for disorder associated cognitive deficits as described by Merck and others, 2) M2 has been associated with MDD based on single nucleotide polymorphism (SNP) studies (above) and

65 66 KO mice, and 3) Gibbons et al . reported decreased binding of M 2/M 4 mAChRs in the dorsolateral prefrontal cortex of depressed subjects.

This hypothesis that the cholinergic system is involved in mood disorders was further supported by animal studies that investigated the psychopharmacological effects of antidepressants on the CNS. Flinders Sensitive Line rats (FSL), bred selectively for increased sensitivity of mAChRs, are thought to model certain aspects of depression such as: loss of appetite, increases in REM sleep (lethargy), reductions in self stimulation, learning difficulties, and decreased mobility in the forced swim test (FST) in response to agents that stimulate cholinergic function. However, it does not mimic all of the biochemical aspects of depression. 67

Several classes of drugs exist today for treatment of depression. Although they differ from each other with respect to their selectivity for certain receptors/transporters, most function to enhance serotoneric and/or noradrenergic neutrotransmission. Monoamine oxidase inhibitors

(MAOIs) and tricylic antidepressants (TCAs) are the oldest drugs. MAOIs function by inhibiting the enzymatic degradation of serotonin, noradrenaline, and dopamine therefore increasing their availability. TCAs increase synaptic concentrations of serotonin and/or noradrenaline by

60 inhibiting re-uptake by serotonin/noradrenaline transporters. Unfortunately TCAs also have high affinity for other receptors resulting in undesired side-effect profile and MAOIs can cause high blood pressure when taken with certain foods/medications. 68 Because of this, selective serotonin reuptake inhibitors are typically the first line of treatment followed by serotonin and noradrenaline reuptake inhibitors (SNRIs). While these have been designed to be more selective for their respective transporters (hence more favorable side effect profile) they are not more efficacious than the older drugs. 69a,b

Low mood is perhaps the most prominent clinical symptom of MDD but is also accompanied by impairments in cognitive function. However, these cognitive symptoms could be independent of mood. 70a,b,c The cognitive symptoms of depression such as poor attention/concentration in addition to detriments in information processing point to cholinergic function--since ACh is largely responsible for the regulation of these processes. 71 It is well accepted that depression is a multi-faceted disease in which an imbalance of multiple neurobiological systems underlying depression result in cholinergic dysfunction (figure below).

Depressive disorder is thought arise from genetic/environmental factors which in turn may lead to higher susceptibility to stress. Chronic stress and depression are associated with cholinergic dysfunction along with structural/functional deviations in multiple brain areas

(hippocampus, dentate gyrus [DG], pre-frontal cortex[PFC], and amygdala). In fact, cholinergic projections from the basal forebrain (BF) modulate functions areas of the brain that have proven to play a role in depression. 72 Combined, these alterations likely account for the development of the cognitive symptoms of depression (Figure 19). 73

Despite over 50 years of research, little has been produced that is more effective than the original TCAs or MAOIs. Although the antidepressant drugs have been beneficial to many

61 patients, only one third of depressed patients who are treated with a single antidepressant achieve remission after three months. According to the NIMH-funded STAR*D (Sequenced Treatment

Alternatives to Relieve Depression) practical clinical trials, 47% of patient's symptoms are not treated with the first anti-depressant regimen, usually an SSRI, and 30% after four different treatments. 74a,b,c These current approved therapies require a four to six week period of chronic

Figure 19: Figure adopted from Dagyte, G. et al. Behavioural Brain Research. 2011, 221, 574-582. Solid arrows indicate consequences arising from pathological states of chronic stress/depression (hence causing further disruptions leading to depressive disorder), and dashed arrows represent predisposing factors to depression.

62 administration before a therapeutic effect is achieved. This "delay" period of action is not well understood and presents a significant challenge for medical professionals in the management of major depression.

To properly treat patients suffering from mood disorders, a great need persists for rapid antidepressant therapies. Mechanisms of anti-depressants have been a major focus of both current/past research in hopes of developing more effective and faster acting drugs. In studies

Figure 20: Figure adopted from Drevets, W. C. and Furey, M. L. Biol. Psychiatry . 2010 . 67, 432-438. Blocked experimental design showing infusion series and assessment sessions for each of the two randomized patient groups. P/S=placebo then scopolamine and S/P=scopolamine then placebo.

aimed at assessing whether a reduction in mAChR function would alleviated depressive symptoms, clinical data showed that mAChR antagonist scopoloamine had antidepressant effects in depressed patients (N=18) with either MDD (n=9) or BPD (n=9). 75a,b The experiments involved a series of randomized, double-blind, placebo-controlled studies (shown below) in

63 which subjects were administered the muscarinic cholinergic antagonist scoploamine intravenously (4.0 μg/kg). Before each infusion, depression severity was rated via Montgomery-

Asberg Depression Scale (MADRS), 76 anxiety symptoms rated using Hamilton Anxiety Rating

Scale (HARS), 77 and the Clinical Global Impressions (CGI) (Khan et al . Int. Clin.

Psychopharmacol. 2002, 17, 281-285) was applied as a total assessment of illness severity. To evaluate changes in mood during the sessions visual analogue scale (VAS) were administered at baseline, 20, 60, 120, and 150 minutes following the start of infusion, and Profile of Mood State

(POMS) 78 at the same times. In both the placebo/Scop and Scop/placebo subgroups, all showed improvement in the first evaluation that followed administration of Scopolamine (3-5 days). This result was repeated in 2010 by Drevets and Furey (Figure 20).79

Antidepressant response was assessed by reviewing changes in MADRS scores (Figure 21).

Using already established scoring criteria 80 subjects were rated as achieving full response (≥50% reduction in MADRS score), partial response (≤50% but ≥25% reduction), or nonresponsive

(<25% reduction). Remission was classified as a MADRS ≤10. MADRS data were separated into a baseline block (Assessments 1 and 2, Figure 20), the first and last measures of block 1 (3 and 5), and block 2 (6 and 8). Results are shown below. A clear reduction in MADRS scores was seen within each scopolamine block with further reduction in severity following repeated infusions. By the end of the study 14 of the 22 participants achieved a full response with 11 achieving remission. 79 The authors note two significant findings from this study: 1) subjects showed improvement across the scopolamine block potentially suggesting that repeated administrations provided further benefit; 2) the improvement seen in subjects receiving scopolamine in block 1 persisted during block 2, which indicates persistent action of the drug.

It should be noted that in the study conducted that no participants dropped out due to adverse side effects with the drug. When compared with placebo, scopolamine resulted in higher rates of drowsiness, blurred vision, dry mouth, and light-headedness, but these effects were transient

(resolving within 2-4 hrs). However, no serious side effects were observed such as: psychosis, delirium, overt confusion, cardiovascular effects, or suicidal ideations. 79 Heart rate,

Figure 21: Figure adopted from Drevets and Furey. Biol. Psychiatry. 2010, 67, 432-438. Mean Montgomery-Asberg Depression Rating Scale (MADRS) scores for the P/S group (yellow) and S/P group (Red) across eight assessments. P indicates placebo session and includes block of three assessments of placebo infusions. S indicates the scopolamine sessions and includes a block of three assessments of infusions.

systolic/diastolic BP did decrease upon administration which is expected due to the drug's central effects on parasympathetic autonomic function. While side effects were not prominent in this trial, perhaps due to the very low dose of 4μg/kg, scopolamine and other known anti-cholinergic drugs do have known side effects, especially with long term use, such as double vision, increased

65 heart rate, confusion, disorientation, euphoria/dysphoria, memory problems, mental confusion, or other CNS effects that resemble delirium. In fact, one of the major downsides of scopolamine being used as an anti-depressant is cognitive impairment. Since major depressive disorder is associated with deficits in cognition, this would produce an undesired additive effect that would only exacerbate the problem.

The precise mechanism of scopolamine's antidepressant effects is still not well understood, especially its persistence of antidepressant action due to its relatively short expected clearance from plasma (t 1/2 = 2-4 hrs). This alone suggests a mechanism other than direct action of the mAChR receptors. Additionally, the delay of onset of the antidepressant response until far after the anticholingeric side effects (drowsiness, dry mouth, blurred vision, lightheadedness, dizziness, etc. all reported to have dissipated within 5 hours) lends to the hypothesis that the drug may alter synaptic plasticity or gene expression through a variety of direct/indirect mechanisms. 81

Additionally, while a potent antagonist at the mAChRs, scopolamine drastically increases

ACh release via inhibition of release-controlling mAChR autoreceptors (i.e. M 2) and therefore

increases cholinergic effects on the neighboring nicotinic receptors. 82a,b

Scopolamine also shares the ability to modulate N-methyl-D-aspartate receptor

(NMDAR) function, as does ketamine which is a recently FDA approved nasal Figure 22: Ketamine spray, esketamine, earlier in 2019 (Figure 22. In fact , it has been shown that

mAChR activation enhances NMDAR gene expression, 83 and since elevated mAChR sensitivity has been implicated in mood disorders (see source above) the receptors may contribute to subsequent elevation in NMDAR transmission. Blocking mAChRs with scopolamine reduces mRNA concentrations of NMDAR types 1 and 2A in the rat brain in vivo

66 and serves as a neuroprotectant to hippocampal neurons from glutamate mediated neurotoxicity in vitro .83,84 Hence, it cannot be ruled out that scopolamine's antidepressant action could be partially mediated through the reduction in gene expression of NMDAR.

Another similarity between ketamine and scopolamine was reported by Li et al .85 in which it was reported that ketamine activated the mammalian target of rapamycin (mTor) pathway, leading to increased protein expression for synaptic signaling along with increases in the number of spine synapses in rat cortex. Blocking this pathway rendered ketamine's antidepressant-like behavioral responses ineffective. In the same manner, the same group demonstrated scopolamine can also activate the mTor pathway almost identical to ketamine. 86

Other studies have been conducted on scopolamine involving its effects on treatment resistant patients vs. naive. While MADRS scores dropped for both after the first block, the treatment resistant patient did not show further improvement after this--whereas treatment naive patients continued to improve. 87 More studies are needed to understand the mechanism of action and whether other methods of administration (other than IV) are feasible. It is poorly absorbed through the oral route. 88 While it is well proven that it is readily absorbed transdermally, gene expression changes are concentration dependent 89 and it is uncertain whether desired concentrations can be achieved via this method. 90 Intranasal methods have been explored but it is still unclear whether this route will increase risk of adverse side effects due to greater variability of absorption. 88

In conclusion, many therapeutic options currently exist to treat depression such as: MAOIs,

TCAs, SSRIs, SNRIs, and NRIs. Yet although today's antidepressant drugs are beneficial for many patients, up to 50% of patients fail to achieve remission with the first line treatment . 74

Even more concerning, according to the Sequenced Treatment Alternatives to relieve Depression

(STAR*D) clinical trial confirmed that approximately 47% of patients did not respond to the first line antidepressant treatment, and 1 in 3 did not achieve remission after 4 different consecutive treatments. 74b,c As a result, fully efficacious and faster-onset anti-depressant drugs are urgently required.

While ketamine was a major breakthrough in the field of rapid acting antidepressants for treatment resistant MDD patients, it has potential for addiction due to activation of opioid receptors. 91a,b There have been no such findings for any muscarinic compounds produced, agonist/antagonist alike. In fact, it has been found that the rewarding effects of morphine were

92 significantly reduced in M 5 KO mice, as studied in the conditioned place preference test. In a

93 related study, Fink-Jensen et al. showed that M 5 KO mice displayed decreased cocaine conditioned place preference and reduced cocaine self administration in a single session procedure. Shortly thereafter, M. Thomsen et al. 94 found that chronic cocaine self-administration was decreased (for low to moderate doses) in M 5 KO mice. This lends credence that a centrally

95 acting M 5 antagonist could be potentially useful in the treatment of drug addiction.

The clinical trials with scopolamine have raised the interesting possibility that targeting the muscarinic cholinergic pathway could yield novel and effective anti-depressants. The significance of this thesis is that, based on the hypothesis and preliminary evidence that the antidepressant effect of cholinergics can be separated from the anti-cognitive effects, our goal is to create selective novel antimuscarinic compounds or mixed agonist/antagonists that have little to no effect on memory or cognition. No one has yet concentrated on measuring both the antidepressant and anti-cognitive aspects of muscarinic compounds and attempted to separate them.

Rationale of Design for Novel Muscarinic Antagonists (2.5):

There are five muscarinic receptors, and all are G-protein coupled receptors (GPCRs). They differ markedly in their abundance and location within the CNS and heart. 96 Clinical data (and repeat trial data in 2010, nimh.nih.gov) 75b,c; 95a,b showed that oral and intravenous treatment with the muscarinic cholinergic antagonist scopolamine (previous section) had rapid anti-depressant effects in humans. This raises the interesting possibility that targeting the muscarinic cholinergic pathway has the potential to yield novel and effective anti-depressants.

Approximately 16% of Americans are diagnosed with major depressive disorder, a mental disorder characterized by at least two weeks of low mood that is present across most situations. 75a,b,c It can be accompanied by low self-esteem, loss of interest in normally enjoyable activities, low energy, and diminished quality of life. Furthermore, it is linked to diminished quality of life, medical morbidity, and mortality. Depression has a lifetime prevalence of 16% in the United States and appears to be caused by a combination of genetic, biological, environmental, and psychological factors. Between 2-7% of adults with this disorder die by suicide. 12,75a,b While counseling and antidepressant medication can be effective treatments, current selective serotonin re-uptake inhibitors (SSRI's) take weeks before therapeutic effects are observed. In addition, only one third of patients who are treated initially with an SSRI achieve complete remission. 97,98 As a result, fully efficacious and faster-onset anti-depressant drugs are urgently required.

Scopolamine is an antagonist at all five muscarinic receptors and disrupts memory and cognitive function. This effect is thought to be due to its anticholinergic properties through a muscarinic effect. Since these findings significant effort has been placed on finding novel compounds that reverse these effects of scopolamine. Muscarinic agonists at one or more

69 receptors have been considered potential treatments in both schizophrenia and Alzheimer's disease. This led to many novel compounds being produced beginning in the late 1980s-1990s by

Figure 23: General design scheme for novel mAChR antagonists .

pharmaceutical companies that targeted one of more of the mAChRs as detailed in a previous section of this thesis. 25-32, 34, 37-38, 43, 45-46 The current research focuses on the 3-heteroaryl substituted azabicyclo[2.2.1]heptanes, azabicyclo[2.2.2]octanes, and other substituted bicycles-- initially pioneered by Merck researchers--as support for our hypothesis that scopolamine's antidepressant effects can be separated from the anticognitive effects (Figure 23). As there has been very little work completed on the rationale for design of mAChR antagonists as antidepressants, we were forced to rely upon previously used scaffolds that were shown to effectively bind the muscarinic receptors. However, thanks to past work completed by both

70 pharmaceutical companies and academics, we had a hypothesis of what an effective muscarinic antidepressant's ideal pharmacological profile should resemble.

From previous work 31 it was shown that introduction of substituents larger than a methyl group in the 3-position (in red, left) of the oxadiazole ring (shown right) produced compounds with weak partial agonist (i.e. ethyl, shown in previous table) or antagonist (i.e. benzyl) profiles. 3-Quinuclidine benzilate (QNB, right) has long been regarded as one of the most potent mAChR antagonists known aside Figure 24: (top) General structure of quinuclidine- [3-alkyl-1,2,4-oxadiazole]. (bottom) QNB (left) and from atropine. QNB was a compound originally QNB oxadiazole (right). Adopted from Saunders et al . J. Med. Chem. 1990, 33, 1128-1138). developed to be an antispasmodic agent or to treat GI spasms 99 although eventually eventually developed as a non-lethal incapacitating agent to be used in battle. 100 The oxadiazole derivative of QNB was shown to be one of the most mAChR antagonists to date, displaying ~8 fold higher affinity to the cortical mAChRs than atropine (Figure 24).31

The introduction of a double bond (Figure 25) between C2/C3 in the quinuclidine ring (left)

O N was detrimental to efficacy/affinity (verified by our group as well) and

N therefore was not considered for further study. Hydrogen bonding N

Figure 25: C2/C3 interactions between the muscarinic receptor and the heteroaromatic ring unsaturated quinuclidine oxadiazole. of the ligand seem to be of minor importance, but rather lipophilicity and

size are the major factors for binding to the low-affinity (antagonist) state. 101 In contrast, optimal binding at the agonist site was found with substituents containing both hydrophilic and electron donating properties (i.e. Me, NH 2)

Freedman et al .102 attempted to pursue the design of receptor-selective ligands by manipulating efficacy to reduce side effects and enhance selectivity. In their efforts L689660 and

L687306 were discovered, each with low NMS/OXO-M ratios (28 and 15 respectively),

indicative of partial agonist activity. However, due

N N O to L689660's agonist activity at M 1/M 3, it began to N Cl N N N show cholinergic side effects (both peripheral and L689660 L687306 central). It produced salivation (ED 100 0.4 mg/kg ip) Figure 26: L689660 (left) and L687306 (right) and hypothermia (ED 50 0.34 mg/kg ip) although less

noticeable than those produced by L670548. In a rat conditioned suppression of drinking test, doses of 0.3 and 1.0 mg/kg of the drug reversed a scopolamine induced (0.6 mg/kg) deficit. However, in cognitive tasks L689660 performed poorly (detailed later in this thesis) causing a significant delay in delayed-matching-to-sample

(DMTS). L687306, even lower on the efficacy scale, was a competitive antagonist at both M 2 and M 3 but retained partial agonist (0.55 efficacy relative to muscarine) activity in the ganglion.

Doses up to 30mg/kg produced no salivation, diarrhea, tremors, hypothermia, and also had no effect in the heart (i.e. no bradychardia or tachychardia). 102-103

While selectivity at M 1 was a priority for Merck and others in their design of mAChR agonists for treatment of AD patients, two studies conducted by Alt et al .104 and Witkin et al .65 focused on cholinergic toxicity associated with M 1 selective activation and M 1/M 2 receptor effects in regulating antidepressant like effects of scopolamine respectively.

Alt et al .104 used three different compounds, all with mixed agonist/positive allosteric modulator activities that were functionally selective for the M 1 receptor, and tested them in rats, dogs, and cynomologous monkeys. As seen in the Table 12, despite the high selectivity of each

72 of the PAMs used, they were surprised to observe that all three produced in vivo effects assocaited with cholinergic toxicity., These effects are similar to thos produced by Xanomeline in humans during clinical trials 39,40 which suggests the side effects could be mediated by stimulation of the M 1 receptor instead of the other mAChRs. The group also rigorously screened the PAMs utilized in the study for activity at 43 GPCRs, ion channels, nuclear hormone receptors, and enzymes. While some showed binding affinity to other GPCRs, none showed any significant efficacy at the receptors further lending credence (when combined with the in vivo toxicity profile) that the effects observed were likely mediated by the M 1 receptor. However, the authors noted that the high plasma concentration achieved in exposed animals could be the main

O O N N CO H HN OH N 2 O N

N N CN N N N A N B C O O

Compound Species Route Dose (mg/kg) Observed Effect

A Rat i.v. 1 No Effects A Rat By mouth 2.5 Diarrhea, bright yellow urine A Cyno i.v. 0.3 Hypersalivation, licking, emesis, urination, mioses A Dog i.v. 1.5 Hypersalivation, licking A Dog i.v. 3 Hypersalivation, licking, emesis B Rat i.v. 1 No Effects B Rat By mouth 2.5 Bright yellow urine B Dog i.v. 1 Slight drooling, runny nose B Dog i.v. 1.5 Mild salivation, licking lips B Dog i.v. 3 Nose running, licking lips, salivation, vomiting, diarrhea, ataxia C Dog i.v. 1 Nasal discharge, hypersalivation, licking, decreased activity C Dog i.v. 5 Nasal discharge, hypersalivation, licking, decreased activity, ataxia Table adopted and modified from Alt et al. J.Pharmacol. Exp. Ther. 2016, 356, 293-304

Table 12

73 culprit for the adverse events in vivo. Additionally, the fact that the side effects were transiently observed suggests that the side effects could have resulted from the high concentrations of the compounds hence producing direct agonism at the receptor. Therefore, it is likely that "direct" activation of M 1 is required to produce the unwanted cholinergic side effects, and that PAMs administered at doses below that could potentially be safe. 104

Witkin et al. 65 utilized the forced swim test (FST, described later in this thesis) as an antidepressant detecting assay, in wild type and transgenic mice in which each mAChR had been genetically deleted in order to elucidate the subtypes responsible for the antidepressant effects of scopolamine. Only M 1 and M 2 knockout (KO) mice showed a decrease in response to

Figure 27: Figure adopted from JM Witkin et al. JPET. 2014, 351, 448-456. Antidepressant - like effects of scopolamine are blocked in M 1 and M 2 KO mice (but not imipramine). Both compounds were administered 30 minutes before testing. n=8 either mAChR KO mice or C57BL/6 WT mice. Imipramine (imi) was used as a positive control (15 mg/kg).

scopolamine in the FST assay (Figure 27). The authors acknowledged the limitations of the study in that further experimentation with the KO mice strains in a broader range of behavioral, neurochemical, and electrophysiological studies is necessary to further solidify the conclusions they generated.

From the above information we concluded that an mAChR antidepressant lacking cognitive deficits should have the following pharmacological profile: 1) low efficacy partial agonist or antagonist at M 1, 2) antagonist at M 2 (to prevent bradychardia) 3) and antagonist at M 3 (to reduce peripheral cholinergic side effects). The desired profile for M 4 and M 5 was not clear, as there has been few examples of selective M 4 or M 5 ligands designed to date (mainly PAMs that show enhanced selectivity for these particular receptors). However, according to Langmead et al .20 the

M4 receptors are found in the cortex and hippocampus, but are most prominent in the striatum, where they are thought to play a role in controlling dopamine release and locomotor activity-- potentially important in the treatment of schizophrenia symptoms. M 5 has a discrete localisation in the ventral tegmental area (VTA), a brain region known to be involved in reward and

105 addiction. In fact, infusion of M 5 antisense mRNA was shown to reduce hypthamlamic stimulation-induced reward 106 and intra-VTA administration of mAChR antagonists reduced dopamine release in the nucleus accumbens. 107 The above effect and the acquisition of self-

108a,b administration of cocaine and morphine were reduced in M 5 KO mice. Taking these conclusions into account it would appear that a low efficacy partial agonist/antagonist would be therapeutically useful at M 4-5.

Synthesis of Muscarinic Antagonists for Comparison of Structure-Activity Relationships (2.6): In order to prepare standards to which the novel compounds could be compared, it was necessary to first prepare L670548/687306/689660 as previously described by Merck with slight adjustments (Scheme 1). 27 The synthesis of L670548/687306 begins with an acid catalyzed

[2+3]cycloaddition with azomethine ylide precursor ( 2) and 5,6-dihydropyranone to afford the pyrrolidine ring system as a 1:1 mixture of diastereomers that can be separated by

75 chromatography (optional). The mixture is then subjected to gaseous HBr which results in fracture of the pyranone ring to yield (5) and ( 6). Subsequent neutralization with sodium carbonate yields the azabicycloheptane core. Recrystallization in Acetone/Ether affords the pure

(R) diastereomer as the quaternary ammonium salt (7) . Transfer hydrogenation of the chiral auxiliary affords (10) as the hydrobromide salt followed by subsequent oxadiazole formation under refluxing sodium hydride to yield the respective L-compounds. It is a brute force, expensive, and low-yielding synthesis and has yet to be optimized.

Scheme 1- Synthesis of L670548 and L687306

As a further extension, it has been shown that WAY-132983 designed by Wyeth Ayerst

Pharma showed excellent in vitro /in vivo activity (K i=2 nM) along with selectivity for the M 1 receptor. 109 They accomplished this by building a homology model based on the X-ray crystal structure of bacteriorhodopsin and the amino acid sequence of the human M 1 muscarinic receptor. Note the 2,3-disubstitution on the pyrazine ring and either butyl/hexyl oxy/thiol substituent at the 3 position. The hexyloxy side chain was attributed to the high M 1 affinity via

76 extension between the fifth and sixth transmembrane loops of the receptor model as in the case of Xanomeline (Eli Lilly, a potent M 1 agonist currently prescribed, shown above). It is also important to note the ether linkage is a novelty not yet applied for muscarinic agonists/antagonists until pioneered by this group. We utilized this scaffold as a model to create new libraries with different substitution patterns along with the evaluation of the individual enantiomers of the quinucidine system to develop SAR.

The synthesis of the 1-azabicyclooctanes to mimic WAY-132983 begins with the readily available 3-quinuclidinols ( 11, 11a-b) with a/b being the R/S enantiomers respectively.

Stoichiometric potassium tert-butoxide followed by addition of the desired heterocycle affords the SnAr products ( 12, 12a-b) in moderate yield. 110 This is followed by a subsequent substitution reaction via tert-butoxide and the desired thiol or sodium hydride with the desired alcohol to afford ( 13, 13a-b) in low yield. This is explained by the newly acquired electron donating group

(quinuclidinol) of ( 12, 12a-b) making a subsequent SnAr reaction challenging. As isomers of the original 2,3-dichloropyrazine (from WAY-132983) are readily available we have chose the

Scheme 2- Synthesis of WAY-132983

(2,6)/(3,6)-pyrazines, and (2,6)-dichloropyridazine with an ether linkage attaching the bicycle to the alkyloxy or alkylthio substituted heterocylic ring (Scheme 2).

Finally, synthesis of L-689660 (Figure 5) begins with the commercially available Boc-4- piperidinone ( 14 ) undergoing a Horner Wadsworth Emmons reaction followed by subsequent transfer hydrogenation to afford ( 15 ) in 85% combined yield. Subsequent α-deprotonation of the ester via sodium bistrimethylsilylamide followed by addition of 2,6-dichloropyrazine, followed by saponification of the ester yields the racemic free acid. Salt formation via one half equivalent addition of (S)-1-phenylethan-1-amine and three subsequent recrystallizations in boiling ethyl acetate affords ( 16 ) enriched in the desired enantiomer. This was confirmed by measuring the optical rotation and compared to literature value provided by Merck. 111 Borane reduction of the acid affords the alcohol in moderate yield (~50%) which is mesylated under basic conditions.

Scheme 3- Synthesis of L689660

2M hydrochloric acid in ether allows for a facile deprotection of the BOC protecting group followed by ring closure under basic conditions to produce L-689660.

Separation of enantiomers can also be performed at the final step via salt formation and two recrystallizations utilizing di-O,O'-toluyl-D-tartaric acid (EP 0416754 A2). Free basing under slightly acidic conditions followed by the addition of stoichiometric maleic acid affords L-

689660 maleate in ~55% yield after recrystallization. 112 We were hopeful to utilize the above synthetic scheme with the isomers of the 2,6-dichloropyrazine to generate novel, chiral L689660 derivatives. Unfortunately, this scheme was unsuccessful in generating the desired derivatives-- likely due to the borane reduction step (over-reduction of pyrazine/pyridazine ring). These derivatives were successfully made by Street et al. 32 but were tested as a racemic mixture with no proposed method of separating the individual enantiomers.

Merck, Lilly, and others published several manuscripts detailing the synthesis of potent muscarinic agonists utilizing the arecoline (tetrahydropyridine), quinuclidine (1- azabicyclo[2.2.2]octane), azanorbornane (1-azabicyclo[2.2.1]heptane]. As seen from the reference compounds (Tables 1-3), all bicycles contained a 3-[3-substituted-1,2,4-oxadiazole] moiety which was utilized in place of the metabolically labile ester. Increasing the size of the substituent on the 1,2,4-oxadiazole ring decreased the affinity for the agonist state of the receptor

(OXO-M) (Table 3 from Sauerberg et al ).28 The arecoline series is an example of this phenomenon.

Noticing this trend, we sought to explore the structure activity relationship across several series of hindered amines. We began with the arecoline series as shown above. While it is acknowledged this series is not novel, it was desirable to study these antagonist's pharmacological effects in vivo due to the ease of preparation and lack of chirality. The synthesis of the arecoline series (shown below) begins by generating the methyl iodide salt of

OH O N N O 1) O 1) CH 3I, Acetone, R Reflux O R NH O 2 N

R=Me, ethyl, propyl,isopropyl, 2) NaBH 4, MeOH/H 2O N 4A Sieves, Dioxane N N o cyclopropyl, 0 C-->Reflux 19 then NaH, 95 C butyl, cyclobutyl, pentyl, 18 27% over 2 steps 2) Oxalate salt cyclopentyl, hexyl, cyclohexyl, heptyl,cycloheptyl, phenyl, 1) ACE-Cl, DCE, formation and benzyl, t-buyl recrystallization reflux, 50-60% 29-58% yield

2) Boc 2O, NaHCO 3, o DCM, 0 C to RT, 95% OH N N O 1) O R R NH O 2 N 4A Sieves, Dioxane N N R=Me, cyclopropyl, pentyl, then NaH, 95 oC H cyclohexyl, benzyl Boc 2) HCl, Et 2O 20 3) HCl salt formation and recrystallization (19-31% overall) Scheme 4-Synthesis of 3-Substituted Arecoline and Norarecoline Oxadiazoles.

methyl nicotinate ( 18) followed by subsequent reduction to afford the arecoline free base as an oil ( 19) . Unfortunately the oil cannot be stored for long term (noticeable color change within a matter of days) so it is converted to an appropriate salt (HCl, HBr, or oxalate). As arecoline hydrobromide is readily available, we opted to purchase from AK Scientific and free base using standard procedures. Standard oxadiazole formation conditions with the appropriate oxime (see

J. Saunders et al. 31 for example procedure) under basic conditions afforded the desired products in moderate yields. After extraction, precipitation of leftover oxime (if possible), and filtration through neutral alumina, allowed for a sufficiently pure enough crude mixture to generate the oxalate salt--thereby avoiding column chromatography. In a few select cases column chromatography was necessary to obtain a sufficiently pure enough product to generate the salt.

Several derivatives were prepared and evaluated both in vitro /in vivo pharmacologically as detailed in Chapter 3 of this thesis. The norarecoline series was prepared similarly except for N-

80 demethylation, which was accomplished via chloroethylchloroformate (ACE-Cl) under reflux followed by hydrolysis in methanol. The arecoline nitrogen was then protected ( 20 ) and oxadiazole formation under standard conditions afforded the protected final products. Stirring in ethereal hydrochloric acid provided the HCl salts of the norarecoline derivatives.

The 3-substituted-1-azabicyclo[2.2.2]octanes were then explored. Merck and others had already shown this hindered amine scaffold to be superior to arecoline in developing potent agonists. In fact, Sauerberg et al . (Table 4)28 showed that 3-[(3-butyl-1,2,4-oxadiazole)]-1- azabicyclo[2.2.2]octane showed low μM affinity for the antagonist state of the receptor (shown below). Encouraged by this result, we sought to design the full series of 3-quinuclidine-1,2,4- oxadiazoles for pharmacological and in vitro evaluation.

Table 13

3-methylquinuclidine carboxylate was prepared via known literature procedures (Orlek et al .

1991, 34(9), 2726-35) from commercially available 3-quinuclidinone hydrochloride ( 21 ). After free basing via standard procedures the Van Leusen reaction 113 with tosylmethyl isocyanide

(TosMIC) under basic condition afforded the cyano-quinuclidine in moderate yield after flash chromatography. Acidic ethanolysis provided the desired ester in high yield ( 22 ) which was subsequently reacted under standard oxadiazole forming conditions as described previously. The yields were significantly lower for the quinuclidine series presumably due to the increased steric

81 hindrance from the bridgehead methylene groups. As the steric bulk of the oxime increased the

Scheme 5- Synthesis of 3-Quinuclidinyl Oxadiazole Series.

reaction proceeded sluggishly and afforded very poor yields (or the reaction did not proceed).

This was especially true of the aromatic oximes, including phenyl/benzyl oxime.

Seeking to further explore the potential of other hindered amine scaffolds we attempted to alter the positions of the bridgehead carbons. We were also curious if altering the position of the oxadiazole ring would lead to either enhancement/decrease in binding affinity/efficacy to the mAChRs. This led us to design a full series of 5-[3-substituted-1,2,4-oxadiazole)]-1- azabicyclo[3.2.1]octanes as detailed below.

The precursor ester was synthesized as previously reported by Laine et al. 114 starting from commercially available ethyl nipecotate ( 23 ) and alkylating with 1-bromo-2-chloro-ethane.

Column chromatography afforded the alkylated product in low yield ( 24 ) which was subsequently cyclized with LDA to provide the desired ester ( 25 ). As before, the ester was reacted under oxadiazole forming conditions to generate the desired final products. Generation of the oxalate salt directly furnished pure products (purirty assessed by NMR) without the need for flash chromatography. Much to our delight the 1-azabicyclo[3.2.1]octanes proved to be quite potent/efficacious compounds, which is detailed in the proceeding chapter.

While we introduced a new complexity into the discussion by creating a new chiral center, there is currently no method in the literature to separate the enantiomers. This is something we are exploring both for the 3-substituted quinuclidines and the [3.2.1]octanes for future efforts should it be appropriate to correlate potency/efficacy with a particular enantiomer.

Scheme 6- Synthesis of 1-Azabicyclo[3.2.1]octane Oxadiazole Series.

The final question in our SAR that remained to be answered was the consequences of shifting the oxadiazole ring one position over (3 to 4) in the arecoline, quinuclidine, and azanorbornane series.

Standard procedures were used to generate the isoarecoline derivatives (as detailed previously) in fair yields as the oxalate salts. The procedure of Laine et al. 114 was utilized to synthesize the 4-substituted quinuclidine ester. Beginning from ethyl isonipecotate ( 28 ), alkylation, identical to the 3.2.1 series on the previous page, with 1-bromo-2-chloro ethane provided the ethyl chloro derivative ( 29 ) in low yield as before, followed by subsequent cyclization under basic conditions to afford the desired ester in high yield. Oxadiazole formation

83 under standard conditions provided the 4-(3-alkyl-1,2,4-oxadiazole)-1-azabicyclo[2.2.2]octanes which were made pure by formation of the oxalate salts (purity assessed by NMR, Scheme 6).

Scheme 7- Synthesis of 4-Substituted Arecoline/Quinuclidinyl/1-Azanorbornane Oxadiazole Series

The procedure of Jenkins et al. and Orleck et al. 115a,b was followed to generate the precursor ester for the 4-substituted-1-azanorbornane series. This begins with a 2+3 cyclization of azomethine ylide precursor ( 31 ) and α-methylene butyrolactone ( 32 ) to afford the spirolactone

(33 ) in good yield. The fracturing of the lactone with gaseous HBr (generated from acetyl bromide) in ethanol followed by basic work up provides the quaternized bicycle, which is

84 subsequently hydrogenated under palladium to afford the hydrobromide salt of the desired ester

(34 ). After free-basing, it undergoes oxadiazole formation under standard conditions as before to generate the 4-(3-alkyl-1,2,4-oxadiazole)-1-azabicyclo[2.2.1]heptane series.

Finally, in following with the general theory that mAChR antagonists require a hindered amine core linked to a bulky hydrophobic/aromatic moiety and the relative ease of preparation we designed a small library of arecoline amides and quinuclidine amides/esters (Scheme 8 ).

R1 R O 2 O O O Cl Cl O 1) Na 2CO 3,H2O OH 1) Toluene, O N R3 N 2) H O, reflux N 2) DCM, TEA, N HBr 2 Various Anilines 82% 32-50% over two steps Arecoline 35 R1 R2 O O 1) DCC, DMAP, R =H, Cl, F OH N 1 R2=H, Cl, OCH 3,F Various Anilines, THF R3 R =H, Phenyl N N 3 2) HCl, Et 2O H Boc 22-46% over two steps 20

R1 R2 1) Cs CO , DMF H H O NH 2 2 3 N or N S R N 1 N N O 2HCl 2) Various acyl O chlorides or sulfonyl (+ R/S (+ R/S (+ R/S R2 enantiomers) chlorides enantiomers) enantiomers) 35 46-62% Scheme 8- Synthesis of Arecoline and Quinuclidinyl Amides/Sulfonamides

Hydrolysis of the ester of arecoline was accomplished via refluxing in water to afford to free acid ( 35 ) followed by standard acid chloride/amine coupling to generate the arecoline amides.

The desmethyl arecoline amides were synthesized starting from ( 20 ) (as noted in Scheme 4)

85 using DCC coupling conditions followed by subsequent acidic deprotection, HCl salt formation, and recrystallization to afford pure (purity assessed by NMR) products. Finally, the quinuclidine amides/sulfonamides were generated from quinuclidine-3-amine dihydrochloride salt ( 35 ) and cesium carbonate, followed by subsequent addition of the desired acid chloride or sulfonyl chloride. As both enantiomers of quinuclidine-3-amine are commercially available the R/S enantiomers were also prepared for each compound. Notably, no products from this series required column chromatography for purification; instead purification via extraction methods and salt formation was sufficient. This expedited the production of a small library of compounds, which unfortunately showed no (or very weak) activity in pharmacological assays. Therefore, the compounds were not explored further and will not be further discussed in this thesis.

In summary, we were successful in generating a large library (and wide variety) of compounds to probe the orthosteric site of the mAChRs. Currently there has been little work completed on the design rationale for mAChR antagonists as antidepressants, hence we relied on previously used scaffolds originally pioneered by Merck and others. Our goal was to explore structure activity relationships in attempt to identify the factors that could turn previously produced agonists into antagonists in vivo . We sought to evaluate these compounds pharmacologically to evaluate their antidepressant potential. This will be detailed in the following chapter of this thesis.

Experimental Section (2.7)

General information. Reagents and solvents were ACS grade, and purchased from Sigma-

Aldrich/Fischer Scientific. Reactions were performed under inert atmosphere (N 2 or Argon) unless otherwise noted. Anhydrous solvents were used as provided without further purification.

Reactions were monitored by thin-layer chromatography (TLC), visualizing with a UV lamp

® and/or I 2. Flash column chromatography was performed on RediSep Rf Gold columns with

Whatman Purisil ® 60A silica gel (230-400 mesh) loading column on a Teledyne Isco

CombiFlash ® Rf. 1H/C 13 NMR spectra were recorded on a Varian INOVA 400 MHz NMR spectrometer at 25 °C. Chemical shifts are reported in parts per million. The residual solvent peak was used as an internal reference: CDCl 3/DMSO-d6/CD 3OD. Mass spectra were obtained on an Electrospray TOF (ESI-TOF) mass spectrometer (Bruker AmaZon X) or Advion CMS-L mass spectrometer.

General Procedure for Synthesis of 3-(3-alkyl-1,2,4-oxadiazol-5-yl)-1,2,5,6-tetrahydro-1-

methylpyridine oxalates: Procedure of Street et al and Showell et al was

O N modified. Arecoline Hydrobromide (750 mg, 3.18 mmol) was dissolved in R N minimal amount of water (~5 mL) and a saturated solution of potassium N carbonate was added (~10 mL). The solution was stirred 30 minutes at room temperature. Diethyl ether (15 ml) was added, and the layers were separated. The aqueous layer was extracted three more times with ether. The combined organic layers were dried with magnesium sulfate, filtered, and concentrated under reduced pressure. Free base of arecoline as a light yellow oil.

Desired alkylcarboxamide oxime (2.5 eq) was dissolved in dry dioxane (~15 mL) and activated 4A sieves were added. The mixture was allowed to stir at room temperature for 30 minutes. Then sodium hydride (dry, 95%, 2.5 eq) was added in one portion and mixture heated at

50 C for 1 hour. Arecoline free base above was dissolved in 10 ml dioxane and activated 4A sieves were added. The mixture was added in one portion to the solution of carboxamide and sodium hydride and heated at 90 C overnight.

The reaction was cooled to room temperature, filtered through celite, and concentrated under reduced pressure. Water was added followed by diethyl ether, and the layers were separated.

Aqueous layer was extracted three more times with ether. The organic layers were combined, dried with magnesium sulfate, filtered, and concentrated under reduced pressure to yield crude product. Residue was taken up into minimum amount of ethanol/acetone, warmed to approx 40

C, and approximately 0.85 eq oxalic acid added. The oxalate salt crystallized as the solution cooled and drops of diethyl ether were added. The pure oxalate salts were furnished in 30-60% yield.

3-methyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-oxadiazole O N 1 N (CJ2099): hydrochloride salt, white solid. H NMR (400MHz, CD 3OD,

N hydrochloride salt): 2.36 (s, 3H), 2.72-2.78 (m, 2H), 3.06 (s, 3H), 3.45-

3.48 (m, 2H), 4.13-4.15 (m, 2H), 7.22-7.24 (m, 1H). 13 C NMR (100 MHz,

CD 3OD, hydrochloride salt): 11.47, 24.44, 43.41, 50.94, 51.73, 119.37,

+ 135.83, 169.12, 173.46. MS (ESI ): C9H14 N3OCl, requires 179.1, found

180.1 (M+H +).

3-cyclopropyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-

oxadiazole (CJ2100): oxalate salt, white solid. 1H NMR (400MHz, O N CD 3OD, oxalate salt): 0.84-0.88 (m, 2H), 0.94-0.98 (m, 2H), 1.96-2.00 (m, N

N 1H), 2.64-2.65 (m, 2H), 2.89 (s, 3H), 3.29-3.32 (m, 2H), 4.03-4.04 (m,

13 2H), 7.05-7.07 (m, 1H). C NMR (100 MHz, CD 3OD, oxalate salt): 8.31,

9.05, 25.19, 44.19, 51.62, 52.47, 120.35, 136.58, 166.45, 174.18, 175.03.

+ + MS (ESI ): C13 H17 N3O5 requires 205.1, found 206.1 (M+H ).

3-ethyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-oxadiazole O N 1 N (CJ2162.2): oxalate salt, white solid. H NMR (400MHz, CD 3OD, oxalate

N salt): 1.28-1.32 (t, 3H, J=7.6Hz), 2.72-2.78 (m, 4H, triplet overlap with

arecoline 5-CH 2), 3.03 (s, 3H), 3.46 (m, 2H), 4.21 (m, 2H), 7.22 (m, 1H).

13 C NMR (100 MHz, CD 3OD, oxalate salt): 11.80, 20.58, 24.36, 43.39,

50.79, 51.64, 119.53, 135.86, 166.52, 173.41, 173.59. MS (ESI +):

+ C12 H17 N3O5, requires 193.1, found 194.1 (M+H ).

3-isopropyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4- O N oxadiazole (CJ2162.1): oxalate salt, white solid. 1H NMR (400MHz, N

N CD 3OD, oxalate salt): 1.31-1.33 (d, 6H, J=7.2 Hz), 2.78-2.80 (m, 2H),

3.03 (s, 3H), 3.05-3.14 (septet, 1H, J=6.8, 7.2, 13.6, 14 Hz), 3.44-3.47 (m,

13 2H), 4.21 (m, 2H), 7.22 (m, 1H). C NMR (100 MHz, CD 3OD, oxalate

salt): 20.92, 24.38, 43.40, 50.82, 51.71, 119.62, 135.75, 166.24, 173.54,

+ + 176.80. MS (ESI ): C13 H19 N3O5, requires 207.1, found 208.1 (M+H ).

5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-3-propyl-1,2,4-oxadiazole O N 1 N (CJ2159.1): oxalate salt, white solid. H NMR (400MHz, CD 3OD oxalate N salt): 0.96-1.00 (t, J=7.6, 7.2 Hz), 1.72-1.81 (sextet, 2H, J=7.2, 14.8 Hz),

2.69-2.72 (t, 2H, 6.8, 7.6 Hz), 2.76-2.79 (m, 2H), 3.02 (s, 3H), 3.42-3.46

13 (m, 2H), 4.16-4.19 (m, 2H), 7.22 (m, 1H). C NMR (100 MHz, CD 3OD,

oxalate salt): 13.97, 21.47, 24.44, 28.73, 43.12, 50.83, 51.72, 119.69,

+ 135.87, 172.24, 173.59. MS (ESI ): C13 H19 N3O5, requires 207.1, found

208.1 (M+H +).

3-butyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-oxadiazole

N O 1 (CJ2159.2): oxalate salt, white solid. H NMR (400MHz, CD 3OD, oxalate N

N salt): 0.93-0.97 (t, 3H, J=7.2Hz, 7.6Hz), 1.34-1.44 (sextet, 2H, J= 7.2, 7.6,

14.4, 14.8 Hz), 1.68-1.75 (pent., 2H, 7.2, 14.8 Hz), 2.71-2.75 (t, 2H, J=7.2,

7.6 Hz), 2.76-2.79 (m, 2H), 3.03 (s, 3H), 3.44-3.47 (m, 2H), 4.20 (m, 2H),

13 7.23 (m, 1H). C NMR (100 MHz, CD 3OD oxalate salt): 14.12, 23.26,

24.41, 26.49, 30.21, 43.41, 50.82, 51.69, 119.58, 135.88, 166.71, 172.41,

+ 173.56. MS: C14 H21 N3O5, requires 221.1, found 222.1 (M+H ).

3-cyclobutyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4- O N 1 N oxadiazole (CJ2164.1): oxalate salt, white solid. H NMR (400MHz,

N CD 3OD, oxalate salt): 1.68-1.84 (m, 4H), 2.01-2.04 (m, 2H), 2.76-2.77 (m,

2H), 3.01 (s, 3H), 3.20-3.24 (pent. 1H, J=8Hz), 3.42-3.45 (m, 2H), 4.19

13 (m, 2H), 7.19-7.21 (m, 1H). C NMR (100 MHz, CD 3OD, oxalate salt):

24.24, 26.44, 32.29, 37.60, 43.24, 50.68, 51.59, 119.47, 135.50, 166.67,

+ 173.34, 175.61. MS (ESI ): C14H19N3O5, requires 219.1, found 220.1

(M+H +).

5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-3-pentyl-1,2,4-oxadiazole

1 (CJ2161.2): oxalate salt, white solid. H NMR (400MHz, CD 3OD, oxalate

salt): 0.89-0.93 (t, 3H, J=6.4Hz), 1.20-1.24 (m, overlapping pentets, 4H),

1.72-1.78 (sextet, 2H, J=7.2, 14.4, 21.6 Hz), 2.70-2.74 (t, 2H, J=7.2,

7.6Hz), 2.79 (m, 2H), 3.03 (s, 3H), 3.42-3.47 (m, 2H), 4.20 (m, 2H), 7.23

13 (m, 1H). C NMR (100 MHz, CD 3OD, oxalate salt): 13.99, 21.49, 24.53,

26.21, 28.74, 31.26, 43.50, 50.90, 51.82, 119.72, 135.89, 166.48, 172.27,

+ + 173.54. MS (ESI ): C15H23 N3O5, requires 235.2, found 236.2 (M+H ).

3-cyclopentyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4- O N 1 N oxadiazole (CJ2164.2): oxalate salt, white solid. H NMR (400MHz,

N CD 3OD, oxalate salt): 1.97-2.01 (m, 1H), 2.07-2.15 (m, 1H), 2.32-2.38 (m,

4H), 2.78 (m, 2H), 3.03, (s, 3H), 3.45 (m, 2H), 3.62-3.70 (pentet, J=8.4,

13 16.8 Hz), 4.21 (m, 2H), 7.22 (m, 1H). C NMR (100 MHz, CD 3OD,

oxalate salt): 19.93, 24.38, 28.04, 32.54, 43.47, 50.84, 51.69, 119.54,

+ 135.89, 166.56, 173.71, 174.77. MS (ESI ): C15H21 N3O5, requires 233.1,

found 234.1 (M+H +).

3-cyclohexyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-

oxadiazole (CJ2159.3): oxalate salt, white solid. 1H NMR (400MHz, O N

N CD 3OD, oxalate salt): 1.12-1.17 (m, 2H), 1.28-1.56 (m, 6H), 1.71-1.83 (m, N 2H), 1.96-1.99 (m, 1H), 2.76-2.79 (m, 2H), 3.02 (s, 3H), 3.41-3.49 (m,

13 3H), 4.18 (m, 2H), 7.21 (m, 1H). C NMR (100 MHz, CD 3OD, oxalate

salt): 24.37, 26.80, 27.03, 31.86, 37.15, 43.37, 50.80, 51.68, 119.60,

+ 166.66, 173.39, 175.77. MS (ESI ): C16H23 N3O5, requires 247.1 found

248.1 (M+H +).

3-heptyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-oxadiazole O N

N 1 (CJ3006): oxalate salt, white solid. H NMR (400MHz, CD 3OD, oxalate

N salt): 0.88- 0.90 (t, 3H, J=6.4 Hz), 1.30-1.34 (m, 6H), 1.67-1.72 (m, 2H),

2.70-2.74 (t, 2H, J=7.6Hz), 2.79 (m, 2H), 3.03 (s, 3H), 3.45-3.47 (m, 2H),

13 4.21 (m, 2H), 7.22 (m, 1H). C NMR (100 MHz, CD 3OD, oxalate salt):

14.53, 23.77, 24.35, 26.75, 28.05, 29.93, 32.95, 43.37, 50.72, 51.58,

+ 119.51, 135.88, 166.64, 172.36, 173.53. MS (ESI ): C17H27N3O5, requires

263.2 found 264.2 (M+H +).

3-cycloheptyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4- O N N oxadiazole (CJ3007): oxalate salt, white solid. 1H NMR (400MHz, N CD 3OD): 1.70-1.83 (m, 11H), 1.99-2.05 (m, 2H), 2.77-2.78 (m, 2H), 3.01

(s, 3H), 3.44-3.47 (m, 2H), 4.20 (m, 2H), 7.22 (m, 1H). 13 C NMR (100

MHz, CD 3OD): 24.39, 27.41, 29.54, 33.69, 38.93, 43.39, 50.82, 51.71,

+ 119.65, 135.68, 166.74, 173.38, 176.73. MS (ESI ): C12 H17 N3O5, requires

261.2 found 262.2 (M+H +).

3-(tert-butyl)-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4- O N N oxadiazole (CJ2164.3): oxalate salt, white solid. 1H NMR (400MHz,

N CD 3OD): 1.34 (s, 9H), 2.76-2.77 (m, 2H), 3.02 (s, 3H), 3.43-3.46 (m, 2H),

13 4.2 (m, 2H), 7.2 (m, 1H). C NMR (100 MHz, CD 3OD): 24.35, 28.84,

33.60, 43.38, 50.78, 51.66, 119.61, 135.65, 166.65, 173.43, 179.33. MS

+ + (ESI ): C14H21 N3O5, requires 221.1, found 222.1 (M+H ).

5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-3-phenyl-1,2,4-oxadiazole

1 (CJ2101): oxalate salt, white solid. H NMR (400MHz, CD 3OD, oxalate

O N salt): 2.80-2.82 (m, 2H), 3.06 (s, 3H), 3.44-3.49 (m, 2H), 4.28 (m, 2H), N 7.32-7.34 (m, 1H), 7.49-7.55 (m, 3H), 8.05-8.08 (m, 2H). 13 C NMR (100 N MHz, CD 3OD, oxalate salt): 24.91, 43.75, 50.99, 52.09, 52.71, 120.17,

128.52, 130.26, 132.80, 136.44, 166.24, 167.26, 170.02. MS (ESI +):

+ C16H17 N3O5, requires 241.1, found 242.1 (M+H ).

3-benzyl-5-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-oxadiazole O N 1 N (CJ3022): oxalate salt, white solid. H NMR (400MHz, CD 3OD): 2.72-

N 2.73 (m, 2H), 2.96 (s, 3H), 3.40-3.41 (m, 2H), 4.05 (s, 3H), 4.13 (m, 2H),

13 7.22-7.28 (m, 5H). C NMR (100 MHz, CD 3OD): 24.37, 32.96, 43.34,

50.74, 51.57, 119.45, 128.28, 129.84, 130.17, 136.12, 137.07, 171.42,

+ + 173.88. MS (ESI ): C17H19N3O5, requires 255.1, found 256.1 (M+H ).

General Procedure for Synthesis of 5-(1,6-dimethyl-1,2,5,6-

tetrahydropyridin-3-yl)-3-alkyl-1,2,4-oxadiazoles (i.e. 6-Methyl-

arecoline oxadiazoles) and N-ethyl arecoline analogues:

Methyl 6-methylnicotinate methyl iodide salt: Methyl 6- O

O methylnicotinate (10g, 66 mmol) was dissolved in acetone (100mL) and

N methyl iodide (12.4 mL, 0.198mol, 3eq) added in one portion. The mixture I- was refluxed overnight. It was allowed to cool to room temperature upon

which the methyl iodide salt precipitated out of solution. The solid was

filtered and washed with cold acetone to afford the methyl iodide salt as a

light yellow solid (19.34 g, 98%).

Methyl 1,6-dimethyl-1,2,5,6-tetrahydropyridine-3-carboxylate: The

above solid (17.8 g, 61 mmol) was dissolved in ethanol (150 mL) and

water (50 mL) and cooled via ice bath. Then sodium borohydride was O O added in small portions (2.3 g, 61 mmol, 1 eq). The solution turned bright

N orange upon addition. The reaction was monitored by TLC (1:99

MeOH/DCM). After allowing to warm to room temperature over 3 hrs the

reaction was concentrated in vacuo , taken up into water, and extracted 3x

with DCM. The organic layers were combined, washed with brine, dried

with sodium sulfate, filtered, and concentrated in vacuo . The residue was

purified via flash chromatography to afford the tetrahydropyridyl ester as a

yellow oil (4.4 g, 43%) which was used directly in the oxadiazole forming

step without further purification. Spectra matched that already in the

literature (G. Showell et al . J. Med. Chem. 1991, 354, 1086-1094). MS:

+ C9H15 NO 2, requires 169.1, found 170.1 (M+H ).

O Ethyl-6-methylnicotinate ethyl iodide salt: The same procedure for the

O methyl iodide salt was followed. The salt was precipitated out of solution

N - I via dropwise addition of diethyl ether (dark yellow solid, 6.2g, 31%).

Methyl 1-ethyl-6-methyl-1,2,5,6-tetrahydropyridine-3-carboxylate: O

O The same porcedure above was followed for the reduction with NaBH 4

N (1eq) to afford the desired ester (2.2g, 62%) as a yellow/orange oil that

was carried onto the oxadiazole forming step. A crude NMR verified the

product. This was immediately used in the oxadiazole forming reaction.

+ + MS (ESI ): C10H17 NO 2, requires 183.1 found 184.1 (M+H ). O N

N 5-(1-ethyl-1,2,5,6-tetrahydropyridin-3-yl)-3-methyl-1,2,4-oxadiazole

N 1 (CJ2182.1): oxalate salt, white solid. H NMR (400MHz, CD 3OD, oxalate

salt): 1.38- 1.42 (t, 3H, J=7.2, 7.6 Hz), 2.36 (s, 3H), 2.77 (m, 2H), 3.36 (m,

+ 2H), 3.46 (m, 2H), 4.18 (m, 2H). 7.21 (m, 1H). MS (ESI ): C10H15NO 3,

requires 193.1 found 194.1 (M+H +).

3-cyclopropyl-5-(1-ethyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,4- O N oxadiazole (CJ2182.2): oxalate salt, white solid. 1H NMR (400MHz, N

N CD 3OD, oxalate salt): 0.96-0.99 (m, 2H), 1.04-1.08 (m, 2H), 1.37-1.41 (t,

3H, J=6.8, 7.6 Hz), 2.07-2.11 (m, 1H), 2.74-2.76 (m, 2H), 3.31-3.36 (q,

+ 2H, J=7.2 Hz), 3.44-3.46 (m, 2H), 7.18 (m, 1H). MS (ESI ): C14H19N3O5,

requires 219.1 found 220.1 (M+H +).

O Ethyl nicotinate ethyl iodide salt: The same general procedure above

O was used but unable to precipitate out the salt via conventional methods. N Therefore, the crude was carried on immediately to reduction with sodium

borohydride to afford the crude ester. NMR of crude material verified the

product. This was subsequently carried on the same manner (as above) to

+ generate the desired oxadiazole. MS (ESI ): C10H17 NO 2, requires 183.1

found 184.1 (M+H +).

5-(1-ethyl-1,2,5,6-tetrahydropyridin-3-yl)-3-isopropyl-1,2,4-oxadiazole O N 1 N (CJ3012): oxalate salt, light yellow solid. H NMR (400MHz, CD 3OD,

N oxalate salt): 0.95-1.10 (m, 6H), 1.37-1.41 (t, 3H, 7.2, 7.6 Hz), 2.06-2.11

(septet, 1H), 2.75-2.76 (m, 2H), 3.31-3.37 (qt, 2H, J=7.6 Hz), 3.45-3.48

+ (m, 2H), 4.15 (m, 2H). MS (ESI ): C14H21 N3O5, requires 221.1 found

222.1 (M+H +).

General Procedure for Synthesis of 3-methyl-5-(1,2,5,6-

tetrahydropyridin-3-yl)-1,2,4-oxadiazoles (demethylated Arecoline

analogues):

1-(1-Chloroethyl) 3-methyl 5,6-dihydropyridine-1,3(2 H)-

Cl O O dicarboxylate: The procedure of Song et al (Ang. Chem. Int. Ed. 2013,

O N O 52(23), 6022-5) was modified. 10g (51 mmol) of Arecoline Hydrobromide

was dissolved in water (~50 mL) and a saturated solution of potassium

carbonate was added (~50 mL). The solution was stirred 60 minutes at

room temperature. Diethyl ether (50 ml) was added, and the layers were

separated. The aqueous layer was extracted three more times with ether.

The combined organic layers were dried with magnesium sulfate, filtered,

and concentrated under reduced pressure. Free base of arecoline as a light

yellow oil. The oil was taken up into 50 mL 1,2-dichloroethane, cooled to

0oC, and 1-chloroethyl chloroformate (1 eq, 55 mmol, 7.87g, 5.94mL) was

added dropwise. The reaction was allowed to warm to room temperature

then refluxed overnight. The reaction was concentrated under reduced

pressure to yield a dark brown oil that was subjected to flash

chromatography in EtOAc/Hex (0-50% EtOAc) to afford the amide as a

dark yellow/orange oil (spectra data matched that reported in the literature,

1 H NMR (400 MHz, CDCl 3): 7.10 (brs, 1H), 6.67–6.59 (m, 1H), 4.22–

4.09 (m, 2H), 3.75 (s, 3H), 3.61–3.56 (m, 2H), 2.36 (s, 2H), 1.84 (d, J =

+ + 5.7 Hz, 3H). MS (ESI ): C10H14NClO4, requires 248.1 249.1 (M+H ).

O Norarecoline: The above oil was taken up into methanol and refluxed

HN O again overnight. The mixture was concentrated under reduced pressure,

dissolved in sat. aqueous potassium carbonate, and extracted 5x with

diethyl ether. Organic layers were combined, dried with magnesium

sulfate, filtered, and concentrated in vacuo yielding the desired

+ + norarecoline is a red/orange oil. MS (ESI ): MS (ESI ): C7H11NO 2,

requires 141.1, found 142.1 (M+H +).Spectra matched that previously

published in the literature (Song et al (Ang. Chem. Int. Ed. 2013, 52(23),

6022-5).

1-(tert-butyl) 3-methyl 5,6-dihydropyridine-1,3(2H)-dicarboxylate (N- O Boc-norarecoline): 2.5 g (17.71mmol) of crude norarecoline was O dissolved in dichloromethane (35 mL) and cooled to 0 C. 2 eq. NaHCO 3 N Boc (3g, 34.34 mmol), and 1.5 eq BOC 2O (5.8 g, 26.57 mmol) were added

portionwise and allowed to warm to room temperature overnight. The

mixture was washed with brine, and the aqueous layer back extracted 2x

with dichloromethane. Organic layers were combined, dried with sodium

sulfate, filtered, and concentrated in vacuo to yield a dark orange oil. This

was subjected to flash chromatography (EtOAc/Hex, 5:95) to afford the

Boc-protected product as a yellow oil that matched literature data (P.

Jakobsen et al. Bioorg. Med. Chem. 2001, 9, 733).

3-methyl-5-(1,2,5,6-tetrahydropyridin-3-yl)-1,2,4- O N N oxadiazole (CJ2139): hydrochloride salt; Off white solid. 1H NMR (400

N H MHz, CD 3OD, hydrochloride salt): 2.36 (s, 3H), 2.72-2.74 (m, 2H), 3.37-

+ 3.39 (m, 2H), 4.19-4.22 (m, 2H), 7.24 (m, 1H). MS (ESI ): C8H11 N3O,

requires 165.1, found 166.1 (M+H +).

3-cyclopropyl-5-(1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-oxadiazole

N O (CJ2150): hydrochloride salt; Off white solid. 1H NMR (400 MHz, N CD 3OD, hydrochloride salt): 0.96-0.98 (m, 2H), 1.04-1.08 (m, 2H), 2.07- N H 2.10 (m, 1H), 2.68-2.69 (m, 2H), 3.38-3.41 (m, 2H), 4.05-4.06 (m, 2H),

13 7.19 (m, 1H). C NMR (100 MHz, CD 3OD, hydrochloride salt): 7.48,

8.23, 23.44, 41.13, 42.07, 119.50, 136.22, 173.53, 174.18. MS (ESI +):

+ C10H14N3ClO, requires 191.1, found 192.1 (M+H ).

3-pentyl-5-(1,2,5,6-tetrahydropyridin-3-yl)-1,2,4- O N oxadiazole (CJ3119.1): hydrochloride salt; white solid. 1H NMR (400 N

N MHz, CD 3OD): 0.91-0.94 (t, 3H, J=5.6, 6.8 Hz), 1.35-1.41 (m, H pentet/sextet overlap, 4H), 1.74-1.78 (pent, 2H, J=6.8, 7.2 Hz), 2.72-2.76

(m, triplet/multiplet overlap, 4H), 3.42-3.45 (m, 2H), 4.14 (m, 2H), 7.27

+ + (m, 1H). MS (ESI ): C16H25 N3O5, requires 221.1, found 222.1 (M+H ).

3-cyclohexyl-5-(1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-

oxadiazole (CJ3119.2): hydrochloride salt; white solid. 1H NMR O N

N (400MHz, CD 3OD, hydrochloride salt): 1.29-1.61 (m, 5H), 1.73-1.81 (m, N H 3H), 1.98-2.01 (m, 2H), 2.70-2.71 (m, 2H), 2.78-2.84 (m, 1H), 3.40-3.46

13 (m, 2H), 4.12 (m, 2H), 7.24 (m, 1H). C NMR (100 MHz, CD 3OD,

hydrochloride salt): 23.49, 26.78, 27.01, 31.85, 37.16, 41.18, 42.19,

+ 119.69, 136.15, 173.56, 175.80.MS (ESI ): C13H20 N3ClO, requires 233.1,

found 234.1 (M+H +).

3-(tert-butyl)-5-(1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-

1 O N oxadiazole (CJ3018): hydrochloride salt, white solid. H NMR (400MHz,

N CD 3OD, hydrochloride salt): 1.34 (s, 9H), 2.69-2.71 (m, 2H), 3.39-3.43 N H (m, 2H), 4.12-4.13 (m, 2H), 7.11-7.13 (m, 1H). 13 C NMR (100 MHz,

CD 3OD, hydrochloride salt): 23.48, 28.83, 33.61, 41.19, 42.18, 119.68,

+ 136.06, 173.60, 179.37. MS (ESI ): C11H18N3ClO, requires 207.1, found

208.1 (M+H +).

O N 3-benzyl-5-(1,2,5,6-tetrahydropyridin-3-yl)-1,2,4-oxadiazole (CJ3029):

N 1 hydrochloride salt, white solid. H NMR (400MHz, CD 3OD, N H hydrochloride salt): 2.67-2.69 (m, 2H), 3.37-3.40 (dd, 2H), (m, 4H), 7.21-

13 7.28 (m, 5H). C NMR (100 MHz, CD 3OD, hydrochloride salt): 23.49,

32.94, 41.14, 42.09, 119.52, 128.28, 129.82, 130.14, 136.55, 137.06,

+ 171.42, 174.07. MS (ESI ): C14H16N3ClO, requires 241.1, found 242.1

(M+H +).

General Procedure for Synthesis of Arecoline Amides: Cl O O 1-(1-Chloroethyl) 3-methyl 5,6-dihydropyridine-1,3(2 H)- O N O dicarboxylate: The procedure from Song et al (Ang. Chem. Int. Ed.

2013, 52(23), 6022-5) as listed above was followed.

1-(tert -Butoxycarbonyl)-1,2,5,6-tetrahydropyridine-3-carboxylic acid O O (N-Boc Guvecine): The amide (4.2 g, 17 mmol) was dissolved in MeOH O N OH (30 mL), and the solution was heated under reflux conditions for 2 hrs.

The mixture was cooled to room temperature, and the solvent was

removed in vacuo . The residue was dissolved in aqueous HBr (40%, 30

99 mL), and the mixture was heated under reflux conditions for 2 hrs, then cooled to room temperature, and the solvent was removed in vacuo to afford crude product which was purified by recrystallization with water to afford the hydrobromide salt. The salt was dissolved in NaOH solution (4

N, 20 mL) and tBuOH (18 ml), added Boc 2O (4.5 g, 20.4 mmol, 1.2 eq) and vigorously stirred at 45–50 °C for 2 h. The mixture was washed with hexane (2 × 20 mL), acidified with 6 N HCl and extracted with ethyl acetate (3 × 20 mL). The combined extracts were washed with brine, dried over MgSO 4, and evaporated in vacuo . The residue was purified by recrystallization with petroleum ether/ethyl acetate to give the product (2

o 1 g, 49%) as a white solid. m.p.: 159–161 C. H NMR (400 MHz, CDCl 3):

1.48 (s, 9H), 2.35 (s, 2H), 3.53 (s, 2H), 4.11 (s, 2H), 7.20 (brs, 1H).

General Coupling Procedure(s) for Arecoline Amides: a) 1-methyl-N-phenyl-1,2,5,6-tetrahydropyridine-3- carboxamide (CJ2137): 1g (7.1 mmol) of arecaidine (prepared via known literature procedures--see Song et al (Ang. Chem. Int. Ed. 2013,

52(23), 6022-5)) in toluene (10 mL) is added excess thionyl chloride (5 eq) dropwise at 0 C. It is gradually allowed to warm to room temperature and refluxed overnight. The mixture is cooled to room temperature, concentrated in vacuo , and crystallized as the HCl salt via iPrOH/Et 2O

(1.14g, 68%). This is taken up into THF (20mL), cooled to 0 C, added 1.5 eq aniline and allowed to warm to room temperature overnight. The mixture is concentrated in vacuo and columned in DCM/MeOH (99:1-

100

95:5) to afford the crude product as an oil. The hydrochloride salt is

1 generated from iPrOH/Et 2O to afford the pure product (269 mg, 19%). H

NMR (400 MHz, free base): 2.25-2.27 (m, 2H), 2.32 (s, 3H), 2.40-2.43

(m, 2H), 3.14-3.16 (m, 2H), 6.56-6.59 (m, 1H), 6.99-7.03 (m, 1H), 7.19-

7.23 (m, 2H), 7.48-7.50 (m, 2H), 7.95 (s, amide NH, 1H). MS (ESI +):

+ C13H16 N2O, requires 216.1, found 217.1 (M+H ). b) N-phenyl-1,2,5,6-tetrahydropyridine-3-carboxamide (CJ2142):

300mg (1.32 mmol) of N-Boc-guvecine was dissolved in DCM (10 mL) and 1.5 eq DCC (410mg, 1.98 mmol) then stir for 15 minutes at room temperature. Then 1.5 eq HOBt (303 mg, 1.98 mmol) is added and the mixture is stirred for another 30 minutes. Finally, excess aniline (0.4 mL,

3.3 eq) added slowly and allow reaction to stir overnight. The mixture is filtered through celite (celite washed with DCM), the filtrate is concentrated in vacuo , and the residue is purified by flash chromatography

(EtOAc:Hex, 0:100-60:40) to afford the Boc-protected intermediate as an oil (crude confirmed by NMR/mass spec (m/z requires 202.1, found 203.1,

M+H +, Boc deprotection observed under MeOH/0.1% formic acid MS solvent). The oil is dissolved in minimal amount of iPrOH, diluted with

Et 2O, and 2M HCl in Et 2O is added. Reaction is stirred overnight to afford the deprotected product after recrystallization in iPrOH/Et 2O to afford the pure hydrochloride salt (90 mg, 34% over 2 steps). 1H NMR (400 MHz):

2.63-2.64 (m, 2H), 3.35-3.37 (m, 2H), 3.97 (m, 2H), 6.94 (m, 1H), 7.12-

101

7.14 (m, 1H), 7.32-7.34 (m, 3H), 7.57-7.59 (m, 2H). MS (ESI +):

+ C12 H17 N3O5, requires 202.1, found 203.1 (M+H ).

c) 1-methyl-N,N-diphenyl-1,2,5,6-tetrahydropyridine-3-carboxamide O (CJ3077): white solid, oxalate salt. 500mg (3.52 mmol) arecaidine was N N taken up into DCM (20 mL), cooled to 0 C, and slowly added oxalyl

chloride (1.2 eq, 0.36 mL) followed by a drop of DMF. The reaction was

allowed to warm to room temperature and stirred for 2 hrs. The mixture

was concentrated in vacuo , re-dissolved, in DCM (20mL), diphenylamine

(1.1 eq, 655mg, 3.87 mmol) was added in one portion, and the reaction

was stirred at room temperature overnight. The reaction was diluted with

DCM, washed brine, and purified by flash chromatography (gradient

MeOH/DCM 0:100-15:85). The oxalate salt was generated as described

1 earlier. H NMR (400 MHz, CD 3OD, oxalate salt): 2.55-2.57 (m, 2H),

3.28-3.32 (m, 5H), 3.88-3.90 (m, 2H), 6.85 (m, 1H), 7.23-7.33 (m, 10H).

+ + MS (ESI ): C21 H22 N2O5, requires 292.1, found 293.1 (M+H ).

N-benzhydryl-1-methyl-1,2,5,6-tetrahydropyridine-3- O Ph carboxamide (CJ3071): white solid, oxalate salt. General procedure (c, N Ph H 1 N as above) was followed. H NMR (400 MHz, CD 3OD, oxalate salt): 2.58-

2.59 (m, 2H), 2.89 (s, 3H), 3.24-3.29 (m, 2H), 3.87 (m, 2H), 6.25 (s, 1H),

6.78 (m, 1H), 7.23-7.32 (m, 10H). MS (ESI +): requires 306.2, found 307.2

(M+H +).

O Ph N-benzhydryl-1,2,5,6-tetrahydropyridine-3-carboxamide (CJ3082): N Ph H hydrochloride salt, white solid. General procedure (b, as above) was N H

102

1 followed. H NMR (400 MHz, CD 3OD, hydrochloride salt): 2.55-2.56 (m,

2H), 3.28-3.31 (m, 2H), 3.89 (m, 2H), 6.26 (s, 1H), 6.85 (s, 1H), 7.23-7.33

13 (m, 10H). C NMR (100 MHz, CD 3OD, hydrochloride salt): 23.21, 41.47,

42.73, 58.60, 128.86, 129.17, 129.93, 132.22, 143.04, 167.29. MS (ESI +):

+ C19H21 N2ClO, requires 292.1, found 293.1 (M+H ).

N-(4-methoxyphenyl)-1,2,5,6-tetrahydropyridine-3-carboxamide OMe O

N (CJ2145.1): Hydrochloride salt, white solid, 252 mg (43% over two H N 1 H steps). General procedure (b, as above) was followed. H NMR (400

MHz, CD 3OD, hydrochloride salt): 2.61-2.63 (m, 2H), 3.33-3.36 (m, 2H),

3.77 (s, 3H), 3.95-3.96 (m, 2H), 6.87 (m, 1H), 6.90 (d, 2H, J=4 Hz), 7.45-

+ 7.48 (d, 2H, 8 Hz). MS (ESI ): C13 H17 N2ClO, requires 232.1, found 233.1

(M+H +).

N-(4-fluorophenyl)-1,2,5,6-tetrahydropyridine-3-carboxamide F O (CJ2145.3): Hydrochloride salt, white solid, 196 mg (40% over two N H N steps). General procedure (b, as above) was followed. 1H NMR (400 H

MHz, CD 3OD, hydrochloride salt): 2.60-2.63 (m, 2H), 3.31-3.34 (m, 2H),

3.93-3.94 (m, 2H), 6.90-6.93 (m, 1H), 6.90-6.93 (m, 2H), 7.55-7.59 (m,

+ + 2H). MS (ESI ): C12 H14 FN2ClO , requires 220.1, found 221.1 (M+H ).

N-(4-methoxyphenyl)-1-methyl-1,2,5,6-tetrahydropyridine-3- OMe O carboxamide (CJ2152): free base, oil, 148mg (33% over two steps). N H 1 N General procedure (a, as above) was followed. H NMR (400 MHz): 2.30-

2.31 (m, 2H), 2.37 (s, 3H), 2.46-2.49 (m, 2H), 3.18-3.19 (m, 2H), 3.73 (s,

3H), 6.55 (m, 1H), 6.78-6.80 (d, 2H, J=8Hz), 7.37-7.39 (d, 2H, J=8Hz),

103

+ 7.65 (s, 1H, amide NH). MS (ESI ): C14H18N2O2 requires 246.1, found

247.1 (M+H +).

General Procedure for Synthesis of 3-(3-alkyl-1,2,4-oxadiazol-5-yl)-[1-

azabicyclo[2.2.2]octane] oxalates: Desired alkylcarboxamide oxime (2.5 O N R eq) was dissolved in dry dioxane (~15 mL) and activated 4A sieves were N N added. The mixture was allowed to stir at room temperature for 30

minutes. Then sodium hydride (dry, 95%, 2.5 eq) was added in one

portion and mixture heated at 50 C for 1 hour. Ethyl-3-quinuclidine

carboxylate (prepared according to literature procedures or purchased

from ChemDiv, Inc) (1 eq) was dissolved in 10 ml dioxane and activated

4A sieves were added. The mixture was added in one portion to the

solution of carboxamide and sodium hydride and heated at 90 C

overnight. The reaction was cooled to room temperature, filtered through

celite, and concentrated under reduced pressure. Water was added

followed by diethyl ether, and the layers were separated. Aqueous layer

was extracted three more times with ether. The organic layers were

combined, dried with magnesium sulfate, filtered, and concentrated under

reduced pressure to yield crude product. Residue was taken up into

minimum amount of ethanol/acetone, warmed to approx 40 C, and

approximately 0.85 eq oxalic acid added. The oxalate salt crystallized as

the solution cooled and drops of diethyl ether were added. The pure

oxalate salts were furnished in 15-45% yield.

104

3-methyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2051): O N 1 N Hydrochloride salt, white solid. H NMR (400MHz, CD 3OD, N hydrochloride salt): 1.81-1.93 (m, 2H), 2.12-2.19 (m, 2H), 2.38 (s, 3H),

2.55-2.57 (m, 1H), 3.37-3.46 (m, 4H), 3.83-3.85 (m, 3H). 13 C NMR (100

MHz, CD 3OD, hydrochloride salt): 11.53, 20.03, 24.01, 25.89, 33.89,

+ 47.42, 48.55, 50.39, 168.72, 180.21. MS (ESI ): C10H16N3ClO, requires

193.1, found 194.1 (M+H +).

3-ethyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ3063): oxalate salt, O N 1 N white solid. H NMR (400MHz, CD 3OD, oxalate salt): 1.81-1.93 (m, 2H), N 2.12-2.19 (m, 2H), 2.38 (s, 3H), 2.55-2.57 (m, 1H), 3.37-3.46 (m, 4H),

13 3.83-3.85 (m, 3H). C NMR (100 MHz, CD 3OD, oxalate salt): 11.70,

20.08, 20.59, 24.06, 25.98, 33.95, 47.27, 47.63, 50.32, 173.00, 180.26. MS

+ + (ESI ): C13H19N3O5, requires 207.1, found 208.1 (M+H ).

3-isopropyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2169.1): oxalate O N 1 N salt, white solid. H NMR (400MHz, CD 3OD): 1.30-1.31 (d, 6H, N J=6.8Hz), 1.76-1.84 (m, 2H), 2.04-2.11 (m, 2H), 2.48-2.49 (m, 1H), 3.05-

3.08 (septet, 1H, J=6.4, 7.2 Hz), 3.31-3.35 (m, 5H), 3.69-3.80 (m, 3H).

+ + MS (ESI ): C14H21 N3O5, requires 221.1, found 222.1 (M+H ).

3-propyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2165.3): oxalate salt, O N 1 N white solid. oxalate salt, white solid. H NMR (400MHz, CD 3OD): 0.95- N 0.98 (t, 3H, J=7.6Hz), 1.73-1.83 (sextet overlapping with multiplet, 4H,

2.03-2.09 (m, 2H), 2.48 (m, 1H), 2.67-2.71 (t, 2H, J=7.6Hz), 3.29-3.34 (m,

13 4H), 3.69-3.76 (m, 3H). C NMR (100 MHz, CD 3OD): 14.39, 20.76,

105

21.83, 24.77, 26.58, 29.12, 34.55, 47.65, 48.01, 50.82, 166.93, 172.22,

+ + 180.99. MS (ESI ): C14H21 N3O5, requires 221.1, found 222.1 (M+H ).

3-cyclopropyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2126): oxalate O N 1 N salt, white solid. H NMR (400MHz, CD 3OD, oxalate salt): 0.96-1.00 (m, N 2H), 1.05-1.10 (m, 2H), 1.75-1.80 (m, 1H), 1.87-1.90 (m, 1H), 2.06-2.16

(m, 4H, 3.34-3.42 (m, 4H), 3.75-3.78 (m, 3H). 13 C NMR (100 MHz,

CD 3OD, oxalate salt): 7.53, 8.30, 20.06, 23.97, 25.89, 33.91, 47.32, 47.67,

+ 50.30, 166.87, 173.82, 180.03. MS (ESI ): C14H19N3O5, requires 219.1,

found 220.1 (M+H +).

3-butyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2165.3): oxalate salt,

1 white solid. H NMR (400MHz, CD 3OD): 0.92-0.95 (t, 3H, J=7.6Hz),

1.33-1.43 (sextet, 2H, J=7.6Hz, ), 1.65-1.79 (multiplet, 3H), 1.05-1.10 (m,

2H), 1.75-1.80 (m, 1H), 1.87-1.90 (m, 1H), 2.06-2.16 (m, 4H, 3.34-3.42

13 (m, 4H), 3.75-3.78 (m, 3H). C NMR (100 MHz, CD 3OD, oxalate salt):

14.95, 24.10, 20.79, 22.75, 27.32, 30.98, 34.74, 48.17, 48.51, 51.16,

+ 166.69, 172.84, 180.99. MS (ESI ): C15 H23 N3O5, requires 235.2, found

236.2 (M+H +).

3-cyclobutyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2165.1): oxalate O N 1 N salt, white solid. H NMR (400MHz, CD 3OD, oxalate salt): 1.68-1.89 (m, N 7H), 2.03-2.15 (m, 4H), 2.53 (m, 1H), 3.21-3.25 (pent. 1H, J=7.2, 7.6,

+ 13.2Hz), 3.34-3.42 (m, 3H), 3.76-3.86 (m, 3H). MS (ESI ): C15 H21 N3O5,

requires 233.1, found 234.1 (M+H +). N O 3-pentyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2173.1): oxalate salt, N N

106

1 white solid. H NMR (400MHz, CD 3OD, oxalate salt): 0.89-0.93 (t, 3H,

J=6.4, 7.6 Hz), 1.35-1.37 (m, 4H), 1.72-1.84 (m, 4H), 2.03-2.08 (m, 1H),

2.71-2.74 (t, 2H, J=7.6 Hz), 3.30-3.36 (m, 6H), 3.69-3.76 (m, 3H). MS

+ + (ESI ): C16 H25 N3O5, requires 249.2, found 250.2 (M+H ).

3-cyclopentyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2165.2): oxalate O N 1 salt, white solid. H NMR (400MHz, CD 3OD oxalate salt): 1.56-1.77 (m, N N 8H), 1.89-2.03 (m, 4H), 2.40-2.41 (m, 1H), 3.06-3.14 (pent, 1H, J=7.6,

8.0, 15.2 Hz), 3.22-3.30 (m, 4H), 3.64-3.74 (m, 3H). 13 C NMR (100 MHz,

CD 3OD, oxalate salt): 20.29, 24.19, 26.14, 26.86, 32.73, 34.18, 38.05,

+ 47.57, 47.91, 50.58, 166.98, 175.61, 180.38. MS (ESI ): C16 H23 N3O5,

requires 247.2, found 248.2 (M+H +).

3-cyclohexyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2166.2): oxalate O N 1 salt, white solid. H NMR (400MHz, CD 3OD, oxalate salt): 1.01-1.04 (t, N N 1H, J=7.2Hz), 1.15-1.44 (m, 5H), 1.58-1.75 (m, 5H), 1.85-1.87 (m, 2H),

1.97-2.02 (m, 2H), 2.39-2.40 (m, 1H), 2.66 (m, 1H), 3.16-3.34 (m, 4H),

+ 3.62-3.73 (m, 3H). MS (ESI ): C17 H25 N3O5, requires 261.2, found 262.2

(M+H +).

O N 3-heptyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ3165): oxalate salt, N N 1 white solid. H NMR (400MHz, CD 3OD, oxalate): 0.87-0.91 (t, 3H, J=6.8

Hz), 1.28-1.35 (m, 8H), 1.71-1.86 (m, 4H), 2.03-2.19 (m, 3H), 2.50 (m,

1H), 2.71-2.74 (t, 2H, J=7.2, 7.6 Hz), 3.29-3.37 (m, 4H), 3.70-3.81 (m,

13 2H). C NMR (100 MHz, CD 3OD, oxalate): 14.58, 20.39, 23.82, 24.40,

26.23, 26.83, 28.09, 30.21, 33.02, 34.19, 47.28, 47.62, 50.45, 171.71,

107

+ 172.02, 180.61. MS (ESI ): C18 H29 N3O5, requires 277.2, found 278.2

(M+H +).

3-(tert-butyl)-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ2174): oxalate N O 1 salt, white solid. H NMR (400MHz, CD 3OD, oxalate salt): 1.35 (s, 9H), N N 1.77-1.79 (m, 1H), 1.87-1.89 (m, 1H), 2.09-2.16 (m, 2H), 2.53-2.54 (m,

13 1H), 3.34-3.46 (m, 4H), 3.76-3.87 (m, 3H). C NMR (100 MHz, CD 3OD,

oxalate salt): 20.56, 24.55, 26.41, 29.03, 33.79, 34.37, 47.47, 47.82, 50.67,

+ 174.08, 179.15, 180.58. MS (ESI ): C15 H23 N3O5, requires 235.2, found

236.2 (M+H +).

N,N-dimethyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazol-3-amine (CJ3017): O N N 1 N off white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): N 1.81-1.92 (m, 2H), 2.08-2.15 (m, 2H), 2.53 (m, 1H), 2.99 (s, 6H), 3.36-

+ 3.41 (m, 4H), 3.70-3.82 (m, 3H). MS (ESI ): C13 H20 N4O5, requires 222.1,

found 223.1 (M+H +).

3-phenyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole (CJ3173) : oxalate salt, O N 1 white solid. H NMR (400MHz, CD 3OD, oxalate salt): 1.92-1.94 (m, 2H), N N 2.10-2.20 (m, 2H), 2.63-2.64 (m, 1H), 3.36-3.54 (m, 4H), 3.83-4.00 (m,

13 3H), 7.50-7.57 (m, 3H), 8.07-8.09 (m, 2H). C NMR (100 MHz, CD 3OD,

oxalate salt): 20.08, 23.99, 25.93, 34.04, 47.36, 47.75, 50.40, 127.89,

128.53, 130.23, 132.77, 166.43, 169.61, 180.70. MS (ESI +): requires

255.1, found 256.1 (M+H +).

General Procedure for the Synthesis of Quinuclidinyl-3-amides: H N R N 500 mg (2.51 mmol) of quinuclidine-3-amine dihydrochloride salt (Sigma O

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Aldrich or Combi-Blocks inc for (R) or (S) enantiomers) was dissolved in

dry DMF (10 mL), 3 eq cesium carbonate (2.45 g, 7.45 mmol) was added

and mixture was allowed to stir for 1 hr at room temperature. Then 1.2 eq

(3.01 mmol) of the desired acid chloride was added and mixture was

allowed to stir overnight. The mixture was diluted with water/brine,

acidified with 2N HCl, extracted 2x diethyl ether (discard), basified with

sat. K 2CO 3 (pH>8), then extracted 3x EtOAc. The organic layers were

combined, washed brine, dried with sodium sulfate, filtered, and

concentrated in vacuo . The hydrochloride salt was directly prepared via

iPrOH/Et 2O.

4-fluoro-N-quinuclidin-3-yl)benzamide (CJ2029): hydrochloride salt, F H 1 N white crystalline solid. H NMR (400MHz, free base, CDCl 3): 1.39-1.41 N O (m, 1H), 1.51-1.53 (m, 2H), 1.63-1.65 (m, 1H), 1.78-1.82 (m, 3H), 2.57-

2.90 (m, 4H), 2.95-3.11 (m, 2H), 3.32-3.34 (m, 2H), 7.16-7.20 (m, 2H),

+ 7.87-7.91 (m, 2H), 8.00 (s, 1H, NH). MS (ESI ): C14 H18 FN2O, requires

248.1, found 249.1 (M+H +). The R and S enantiomers matched the above

listed values.

Synthesis of Ethyl 1-azabicyclo[3.2.1]octane-5-carboxylate: The O O procedure of Laine et al was followed. To a solution of ethyl nipecotate

(20.0 mL, 130 mmol) in acetone (180 mL) was added 1-bromo-2- N chloroethane (21.6 mL, 260 mmol) followed by anhydrous K 2CO 3 (27.12

g, 196 mmol). The reaction mixture was stirred overnight then

concentrated under reduced pressure. The resulting residue was treated

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with H 2O and extracted 3x Et 2O. The combined organic layers were dried

with MgSO4, filtered, and concentrated under reduced pressure to give an

oil. Purification by flash chromatography (gradient 10-30 % ethyl acetate

in hexanes) on silica gel afforded ethyl 1-(2-chloroethyl)-4-

piperidinecarboxylate (10.99 g, 38.6%). MS (ESI +): requires 220 (M +

H)+. The product was dissolved in THF (250 mL) and was cooled to -50

°C under N 2. LDA (2.0 M in heptane/ THF/ethyl benzene, 35mL, 70

mmol) was slowly added to the solution at -50 °C over 25 min. The

reaction was warmed to room temperature overnight and quenched with

sat. aq. K 2CO 3. The mixture was extracted with Et 2O (3 × 100 mL). The

combined organic layers were dried over MgSO 4, filtered, and

concentrated under reduced pressure. The resulting orange oil was

coevaporated three times with DCM to remove excess ethyl benzene to

give the title compound (8.29 g, 94%). The product was used without

further purification and kept at 0 C for storage.

N R General Procedure for Synthesis of 3-(3-alkyl-1,2,4-oxadiazol-5-yl)-[1- O N azabicyclo[3.2.1]octane] oxalates: Desired alkylcarboxamide oxime (2.5

eq) was dissolved in dry dioxane (~15 mL) and activated 4A sieves were N added. The mixture was allowed to stir at room temperature for 30

minutes. Then sodium hydride (dry, 95%, 2.5 eq) was added in one

portion and mixture heated at 50 C for 1 hour. Ethyl-1-

azabicyclo[3.2.1]octane-5-carboxylate (prepared according to literature

procedures above) (1 eq) was dissolved in 10 ml dioxane and activated 4A

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sieves were added. The mixture was added in one portion to the solution

of carboxamide and sodium hydride and heated at 90 C overnight.

The reaction was cooled to room temperature, filtered through celite,

and concentrated under reduced pressure. Water was added followed by

diethyl ether, and the layers were separated. Aqueous layer was extracted

three more times with ether. The organic layers were combined, dried with

magnesium sulfate, filtered, and concentrated under reduced pressure to

yield crude product. Residue was taken up into minimum amount of

ethanol/acetone, warmed to approx 40 C, and approximately 0.85 eq

oxalic acid added. The oxalate salt crystallized as the solution cooled and

drops of diethyl ether were added. The pure oxalate salts were furnished in

27-44% yields.

N 5-(1-azabicyclo[3.2.1]octan-5-yl)-3-methyl-1,2,4-oxadiazole (CJ3094): O N 1 off-white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt):

0.90-0.93 (t, 3H, J=7.2Hz), 1.34-1.37 (m, 4H), 1.73-1.91 (m, 4H), 2.08- N 2.11 (m, 2H), 2.71-2.74 (t, 2H, J=7.2, 7.6Hz), 3.30-3.36 (m, 3H), 3.71-

13 3.79 (m, 3H). C NMR (100 MHz, CD 3OD, oxalate salt): 11.23, 17.70,

32.58, 33.15, 44.24, 51.65, 53.49, 60.61, 166.85, 168.61, 180.16. MS

+ + (ESI ): C12 H17 N3O5, requires 193.1, found 194.1 (M+H ).

N 5-(1-azabicyclo[3.2.1]octan-5-yl)-3-cyclopropyl-1,2,4-oxadiazole O N 1 (CJ3095): off-white solid, oxalate salt. H NMR (400MHz, CD 3OD,

oxalate salt): 0.94-0.98 (m, 2H), 1.05-1.09 (m, 2H), 1.93-1.97 (m, 1H), N 2.05-2.20 (m, 4H), 2.37-2.49 (m, 2H), 3.27-3.31 (m, 2H), 3.45-3.48 (m,

111

13 2H), 3.62-3.67 (m, 2H). C NMR (100 MHz, CD 3OD, oxalate salt): 7.47,

8.22, 17.91, 32.49, 32.76, 33.32, 44.50, 51.84, 53.67, 60.81, 173.95,

+ + 180.14. MS (ESI ): C14 H19 N3O5, requires 219.1, found 220.1 (M+H ).

5-(1-azabicyclo[3.2.1]octan-5-yl)-3-isopropyl-1,2,4-oxadiazole N O 1 N (CJ3129): off-white solid, oxalate salt. H NMR (400MHz, CD 3OD):

1.30-1.32 (d, 6H, J=7.2 Hz), 2.03-2.06 (m, 1H), 2.13-2.27 (m, 3H), 3.02- N 3.11 (septet, 1H, J=6.8, 7.2Hz), 3.38-3.42 (m, 2H), 3.59-3.62 (m, 2H),

13 3.73-3.83 (m, 2H). C NMR (100 MHz, CD 3OD): 17.72, 20.63, 27.853,

32.60, 33.18, 44.37, 51.69, 53.51, 60.65, 176.34, 180.04. MS (ESI +):

+ C14 H21 N3O5, requires 221.1, found 222.1 (M+H ). N O 5-(1-azabicyclo[3.2.1]octan-5-yl)-3-propyl-1,2,4-oxadiazole(CJ3139.1): N

1 off-white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt):

N 0.96-1.00 (t, 3H, J=7.2, 7.6 Hz), 1.73-1.80 (sextet, 2H, J=7.2 Hz), 2.03-

2.06 (m, 1H), 2.10-2.30 (m, 3H), 2.44-2.61 (m, 2H), 2.68-2.72 (t, 2H,

J=7.2, 7.6 Hz), 3.35-3.42 (m, 2H), 3.57-3.63 (m, 2H), 3.73-3.83 (m, 2H).

13 C NMR (100 MHz, CD 3OD, oxalate salt): 13.99, 17.93, 21.41, 28.73,

32.81, 33.39, 44.55, 51.92, 53.73, 60.85, 166.81, 172.03, 180.29. MS

+ + (ESI ): C14 H21 N3O5, requires 221.1, found 222.1 (M+H ).

5-(1-azabicyclo[3.2.1]octan-5-yl)-3-butyl-1,2,4-oxadiazole (CJ3097): N O 1 N white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 0.90-

0.93 (t, 3H, J=7.2Hz), 1.34-1.37 (m, 4H), 1.73-1.91 (m, 4H), 2.08-2.11 (m, N 2H), 2.71-2.74 (t, 2H, J=7.2, 7.6Hz), 3.30-3.36 (m, 3H), 3.71-3.79 (m,

+ + 3H). MS (ESI ): C15 H23 N3O5, requires 235.2, found 236.2 (M+H ).

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5-(1-azabicyclo[3.2.1]octan-5-yl)-3-pentyl-1,2,4-oxadiazole (CJ3122):

N 1 O white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 0.89- N 0.93 (t, 3H, 6.4Hz), 1.35-1.37 (m, 4H), 1.69-1.77 (pent. 2H, J=7.6, 7.2

N Hz), 2.03-2.27 (m, 3H), 2.49-2.61 (m, 2H), 2.69-2.73 (t, 2H, J=7.6 Hz),

3.38-3.42 (m, 2H), 3.56-3.62 (m, 2H), 3.74-3.83 (m, 2H). 13 C NMR (100

MHz, CD 3OD, oxalate salt): 16.77, 20.33, 25.82, 29.14, 30.11, 35.21,

35.79, 46.94, 52.18, 54.25, 56.08, 63.22, 166.69, 174.57, 182.72. MS

+ + (ESI ): C15 H25 N3O5, requires 249.2, found 250.2 (M+H ).

5-(1-azabicyclo[3.2.1]octan-5-yl)-3-cyclopentyl-1,2,4-oxadiazole N O 1 N (CJ3132): white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate

salt): 1.70-1.80 (m, 6H), 2.03-2.07 (m, 3H), 2.10-2.30 (m, 3H), 2.48-2.61 N (m, 2H), 3.18-3.26 (pent. 1H, J= 6.8, 7.6 Hz), 3.56-3.62 (m, 2H), 3.74-

13 3.83 (m, 2H). C NMR (100 MHz, CD 3OD, oxalate salt): 17.77, 26.45,

32.26, 32.68, 33.25, 37.64, 44.41, 51.70, 53.53, 60.69, 166.85, 175.35,

180.19. MS (ESI +): requires 247.2, found 248.2 (M+H +).

5-(1-azabicyclo[3.2.1]octan-5-yl)-3-cyclobutyl-1,2,4-oxadiazole

1 N (CJ3148): white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate O N salt): 1.97-2.33 (m, 6H), 2.32-2.39 (m, 4H), 2.45-2.52 (m, 1H), 2.55-2.62

(m, 1H), 3.36-3.42 (m, 2H), 3.57-3.69 (m, 3H), 3.72-3.84 (m, 2H). 13 C N

NMR (100 MHz, CD 3OD, oxalate salt): 17.92, 19.89, 27.98, 32.48, 32.81,

33.39, 44.61, 51.92, 53.73, 60.86, 166.88, 174.46, 180.41. MS (ESI +):

+ C15 H21 N3O5, requires 233.1, found 234.1 (M+H ).

5-(1-azabicyclo[3.2.1]octan-5-yl)-3-cyclohexyl-1,2,4-oxadiazole

113

1 (CJ3098): white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate

N salt): 1.28-1.60 (m, 5H), 1.72-1.84 (m, 3H), 1.97-2.10 (m, 3H), 2.13-2.32 O N (m, 3H), 2.43-2.60 (m, 2H), 2.76-2.82 (m, 1H), 3.38-3.42 (m, 2H), 3.56-

13 3.59 (m, 2H), 3.62-3.82 (m, 2H). C NMR (100 MHz, CD 3OD, oxalate N salt): 17.73, 26.59, 26.81, 31.60, 32.63, 33.19, 36.96, 44.37, 51.68, 53.51,

+ 60.65, 166.69, 175.34, 179.92. MS (ESI ): C17 H25 N3O5, requires 261.2,

found 262.2 (M+H +).

5-(1-azabicyclo[3.2.1]octan-5-yl)-3-cycloheptyl-1,2,4-oxadiazole

1 (CJ3157): white solid, oxalate salt. H NMR (400 MHz, CD 3OD, oxalate N O N salt): 1.55-1.81 (m, 10H), 1.98-2.05 (m, 3H), 2.13-2.27 (m, 3H), 2.45-2.60

(m, 2H), 2.96-3.02 (m, 1H), 3.37-3.42 (m, 2H), 3.55-3.62 (m, 2H), 3.72- N 13 3.82 (m, 2H). C NMR (100 MHz, CD 3OD, oxalate salt): 17.94, 27.44,

29.55, 32.84, 33.42, 33.64, 38.99, 44.59, 51.91, 53.74, 60.88, 166.83,

+ 176.50, 180.13. MS (ESI ): C18 H27 N3O5, requires 275.2, found 275.2

(M+H +).

5-(1-azabicyclo[3.2.1]octan-5-yl)-3-(tert-butyl)-1,2,4-oxadiazole N 1 O (CJ3100): oxalate salt, white solid. H NMR (400MHz, CD 3OD, oxalate N salt): 1.35 (s, 9H), 2.03-2.06 (m, 1H), 2.14-2.27 (m, 3H), 2.43-2.58 (2H),

N 3.38-3.42 (m, 2H), 3.60-3.64 (m, 2H), 3.73-3.83 (m, 2H). 13 C NMR (100

MHz, CD 3OD, oxalate salt): 17.91, 28.79, 32.81, 33.37, 33.58, 44.55,

+ 51.90, 53.70, 60.86, 166.86, 179.10, 180.09. MS (ESI ): C15 H23 N3O5,

requires 235.2, found 236.2 (M+H +).

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5-(1-azabicyclo[3.2.1]octan-5-yl)-3-phenyl-1,2,4-oxadiazole (CJ3166): N O 1 N oxalate salt, white solid. H NMR (400MHz, CD 3OD): 2.06-2.09 (m, 1H),

2.22-2.31 (m, 3H), 2.51-2.58 (m, 1H), 2.63-2.70 (m, 1H), 3.41-3.45 (2H), N 3.61-3.71 (m, 2H), 3.77-3.85 (m, 2H), 3.89-3.92 (m, 1H), 7.49-7.55 (m,

+ 3H), 8.04-8.06 (m, 2H). MS (ESI ): C17 H19 N3O5, requires 255.1, found

256.2 (M+H +).

Synthesis of ethyl 1-azabicyclo[2.2.1]heptane-4-carboxylate:

Benzyl-7-aza-2-oxaspiro[4.4]nonan-l-one: A stirred solution of N-

O benzyl-N- (methoxymethyl)-N-[(trimethylsily1)-methylamine (80% pure,

O N AK Scientific) (32 g, 0.102 mol) and α-methylene-γ-butyrolactone (10 g,

0.51 mol) in DCM (200 mL) was cooled to 0 °C and treated with

trifluoroacetic acid in DCM (3.9 mL in 10mL DCM)) below 5 °C, and the

reaction was allowed to stir at room temperature for 2 hrs. The reaction

was then washed with saturated NaHCO 3 solution, washed with brine,

dried, and evaporated to dryness. Purification by flash chromatography in

DCM/MeOH (100:0-95:5) afforded the spiro-lactone as a single main

fraction (18.64g, 79%). Spectra matched that currently in the literature (S.

Jenkins e al. J. Med. Chem. 1992, 35, 2392-2406 and B. Orlek et al. J.

Med. Chem. 1991, 34, 2726-2735). MS: requires 231.1, found 232.1

(M+H +).

Ethyl-1-Azabicyclo[2.2.1]heptane-4-carboxylate Hydrobromide (4a). O O A stirred solution of spiro-lactone (18 g, 77.8 mmol) in EtOH (150 mL)

N was saturated with HBr gas (via acetyl bromide in ethanol) at 0 °C and

115

allowed to stir at room temperature overnight. The solution was

evaporated to dryness and the residue partitioned between saturated

aqueous K 2C0 3 and DCM. The organic extracts were dried and evaporated

to dryness, and the residue was treated with Et 2O. The resulting solid was

filtered off, washed with Et2O, and dried to give a white solid (24.2 g).

This was suspended in EtOH (150 mL) and hydrogenated over 10% Pd-C

(5g, 20 mol% Pd/C) at 45 psi for 24 hrs. The mixture was filtered through

Celite, the solid washed several times with hot EtOH, and the combined

filtrate evaporated to dryness to give 4a (11.9 g, 61% over two steps).

Spectra matched that currently in the literature (S. Jenkins e al. J. Med.

Chem. 1992, 35, 2392-2406 and B. Orlek et al . Tet. Lett. 1991, 32(9),

1245-1246). MS: requires 169.1, found 170.1.

General Procedure for Synthesis of 5-(1-azabicyclo[2.2.1]heptan-4-yl)- R N O N 3-alkyl-1,2,4-oxadiazole oxalates: Desired alkylcarboxamide oxime (2.5 eq) was dissolved in dry dioxane (~15 mL) and activated 4A sieves were

N added. The mixture was allowed to stir at room temperature for 30

minutes. Then sodium hydride (dry, 95%, 2.5 eq) was added in one

portion and mixture heated at 50 C for 1 hour. Ethyl-1-

Azabicyclo[2.2.1]heptane-4-carboxylate Hydrobromide (prepared

according to literature procedures above) (1 eq) was free based via

standard methods, dissolved in 10 ml dioxane, and activated 4A sieves

were added. The mixture was added in one portion to the solution of

carboxamide and sodium hydride and heated at 90 C overnight. The

116

reaction was cooled to room temperature, filtered through celite, and

concentrated under reduced pressure. Water was added followed by

diethyl ether, and the layers were separated. Aqueous layer was extracted

three more times with ether. The organic layers were combined, dried with

magnesium sulfate, filtered, and concentrated under reduced pressure to

yield crude product. Residue was taken up into minimum amount of

ethanol/acetone, warmed to approx 40 C, and approximately 0.85 eq

oxalic acid added. The oxalate salt crystallized as the solution cooled and

drops of diethyl ether were added. The pure oxalate salts were furnished in

27-62% yields.

5-(1-azabicyclo[2.2.1]heptan-4-yl)-3-methyl-1,2,4-oxadiazole N 1 O N (CJ3109): white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate

salt): 2.29-2.33 (m, 2H), 2.37 (s, 3H), 2.53-2.56 (m, 2H), 3.48 (m, 2H),

N 13 3.64-3.69 (m, 2H), 4.88-4.90 (m, 2H). C NMR (100 MHz, CD 3OD,

oxalate salt): 11.42, 32.96, 46.95, 54.34, 61.95, 166.67, 169.03, 177.62.

+ + MS (ESI ): C11 H15 N3O5, requires 179.1, found 180.1 (M+H ).

5-(1-azabicyclo[2.2.1]heptan-4-yl)-3-propyl-1,2,4-oxadiazole (CJ3153):

1 N white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 0.97- O N 1.01 (t, 3H, J=7.2, 7.6 Hz), 1.72-1.82 (sextet, 2H, J=7.2, 7.6, 8.0 Hz),

2.30-2.33 (m, 2H), 2.54-2.56 (m, 2H), 2.70-2.74 (t, 2H, J=7.2 Hz), 3.42- N

13 3.61 (m, 2H), 3.66-3.71 (m, 4H). C NMR (100 MHz, CD 3OD, oxalate

salt): 13.99, 21.41, 28.72, 32.94, 47.02, 54.42, 61.96, 166.60, 172.21,

+ + 177.51. MS (ESI ): C13 H19 N3O5, requires 221.1, found 222.1 (M+H ).

117

5-(1-azabicyclo[2.2.1]heptan-4-yl)-3-cyclopropyl-1,2,4-oxadiazole

1 N (CJ3110): white solid, oxalate salt. H NMR (400MHz, CD 3OD): 0.97- O N 0.98 (m, 2H), 1.06-1.08 (m, 2H), 2.08-2.12 (m, 1H), 2.25-2.31 (m, 2H),

2.47-2.54 (m, 2H), 3.44-3.50 (m, 2H), 3.65-3.71 (m, 2H), 4.96-4.99 (m, N 13 2H). C NMR (100 MHz, CD 3OD): 7.45, 8.43, 32.90, 46.89, 54.20,

61.86, 174.10, 177.45. MS (ESI +): requires 205.1, found 206.1 (M+H +).

5-(1-azabicyclo[2.2.1]heptan-4-yl)-3-butyl-1,2,4-oxadiazole (CJ3118):

1 white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 0.93- N O N 0.98 (t, 3H, J=7.6 Hz), 1.34-1.44 (sextet, 2H, J=7.2, 7.6, 14.4, 15.2 Hz),

1.66-1.74 (pentet, 2H, J=7.2, 7.6, 15.2 Hz), 2.11-2.15 (m, 1H), 2.32-2.38

N (m, 5H), 2.69-2.73 (t, 2H, J=7.6 Hz), 3.36-3.39 (m, 1H), 3.48-3.52 (m,

+ + 5H). MS (ESI ): C14 H21 N3O5, requires 221.1, found 222.1 (M+H ).

5-(1-azabicyclo[2.2.1]heptan-4-yl)-3-cyclobutyl-1,2,4-oxadiazole

1 (CJ3150): white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate

N salt): 0.97- 1.01 (t, 3H, J=7.6 Hz), 1.72-1.82 (sextet, 2H, J=7.2, 7.6, 14.4 O N Hz), 2.29-2.35 (m 2H), 2.52-2.56 (m, 2H), 2.70-2.74 (t, 2H, J=7.2 Hz),

+ 3.47-3.53 (m, 2H), 3.66-3.70 (m, 4H). MS (ESI ): C14 H19 N3O5, requires N 219.1, found 220.1 (M+H +).

5-(1-azabicyclo[2.2.1]heptan-4-yl)-3-cyclopentyl-1,2,4-oxadiazole

N 1 O N (CJ3142): white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 1.71-1.86 (m, 6H), 2.06-2.08 (m, 2H), 2.29-2.37 (m, 2H), 2.51-2.58

N (m, 2H), 2.74-2.82 (pent. 1H, J=8.4 Hz), 3.22-3.27 (m, 1H), 3.46-3.49 (m,

13 2H), 3.65-3.71 (m, 4H). C NMR (100 MHz, CD 3OD, oxalate salt).

118

26.84, 32.12, 32.67, 33.30, 38.03, 41.45, 47.30, 54.55, 62.23, 167.87,

+ 175.90, 177.96. MS (ESI ): C15 H21 N3O5, requires 233.1, found 234.1

(M+H +).

5-(1-azabicyclo[2.2.1]heptan-4-yl)-3-cyclohexyl-1,2,4-oxadiazole

1 (CJ3143): white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate N O N salt): 1.29-1.61 (m, 5H), 1.72-1.75 (m, 1H), 1.81-1.84 (m, 2H), 1.98-2.01

(m, 2H), 2.29-2.35 (m, 2H), 2.52-2.58 (m, 2H), 2.79-2.84 (m, 1H), 3.47- N 3.53 (m, 2H), 3.66-3.74 (overalapping singlet/multiplet, 4H). 13 C NMR

(100 MHz, CD 3OD, oxalate salt): 27.02, 27.24, 31.05, 32.03, 37.38, 47.26,

+ 54.54, 62.17, 167.02, 175.92, 177.62. MS (ESI ): C16H23 N3O5, requires

247.2, found 248.2 (M+H +).

5-(1-azabicyclo[2.2.1]heptan-4-yl)-3-cycloheptyl-1,2,4-oxadiazole

1 (CJ3156): white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate N O N salt): 1.57-1.68 (m, 6H), 1.75-1.83 (m, 4H), 2.00-2.05 (m, 2H), 2.29-2.34

(m, 2H), 2.50-2.58 (m, 2H), 2.99-3.06 (m, 1H), 3.48-3.52 (m, 2H), 3.66- N 13 3.71 (m, 4H). C NMR (100 MHz, CD 3OD, oxalate salt). 27.45, 29.57,

33.00, 33.67, 38.99, 47.07, 54.36, 61.98, 166.62, 176.69, 177.40. MS

+ + (ESI ): C17 H25 N3O5, requires 261.2, found 262.2 (M+H ).

N 5-(1-azabicyclo[2.2.1]heptan-4-yl)-3-(tert-butyl)-1,2,4-oxadiazole O N

1 (CJ3114): White solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate

N salt): 1.34 (s, 9H), 2.27-2.34 (m, 2H), 2.51-2.57 (m, 2H), 3.45-3.51 (m,

119

13 2H), 3.65-3.72 (m, 4H). C NMR (100 MHz, CD 3OD, oxalate salt):

27.59, 28.75, 32.91, 46.95, 54.29, 61.89, 167.02, 177.36, 179.23. MS

+ + (ESI ): C14 H21 N3O5, requires 221.1, found 222.1 (M+H ).

5-(1-azabicyclo[2.2.1]heptan-4-yl)-N,N-dimethyl-1,2,4-oxadiazol-3- N N 1 amine (CJ3145): white solid, oxalate salt. H NMR (400MHz, CD 3OD, O N oxalate salt): 2.27-2.30 (m, 2H), 2.51-2.55 (m, 2H), 3.01 (s, 6H), 3.48-3.52

+ N (m, 2H), 3.64-3.71 (m, 4H). MS (ESI ): C12 H18 N5O5, requires 208.1, found

209.1 (M+H +).

Ethyl 1-(2-chloroethyl)-4-piperidinecarboxylate: The procedure of O O Laine et al (J. Med. Chem . 2009 , 52(8), 2493-2505) was followed. To a

N solution of ethyl isonipecotate (20.0 mL, 130 mmol) in acetone (180 mL) Cl was added 1-bromo-2-chloroethane (21.6 mL, 260 mmol) followed by

anhydrous K2CO 3 (27.12 g, 196 mmol). The reaction mixture was stirred

for 24 h and then concentrated under vacuum. The resulting residue was

treated with H 2O (75 mL) and extracted with Et 2O. The combined organic

layers were dried with MgSO 4, filtered, and concentrated under vacuum.

Purification of the crude residue by flash chromatography (50% Hex/50%

hexane) on silica gel gave ethyl 1-(2-chloroethyl)-4-piperidinecarboxylate

(9.33 g, 33%) as a clear oil. MS (ESI +): requires 219.1, found 220.1 (M +

H) +. Spectra matched that already in the literature (Laine, D. et al . J.Med.

Chem. 2009 , 52(8), 2493-2505)

Ethyl 1-Azabicyclo[2.2.2]octane-4-carboxylate: A solution of ethyl 1-

(2- chloroethyl)-4-piperidinecarboxylate (9.33g, 42.46 mmol) in THF (200

120

mL) was cooled to -50 °C. LDA (2.0 M in heptane/THF/ethyl benzene, 35 O O mL, 70 mmol) was slowly added to the solution at -50 °C over 15 min.

The reaction was warmed up to room temperature overnight. The reaction N was quenched with sat. aq. K 2CO 3 and extracted with Et 2O (3x). The

combined organic layers were dried over MgSO 4, filtered, and

concentrated under vacuum. The resulting orange oil was co-evaporated

three times with DCM to remove excess ethyl benzene to give the title

compound (16.29 g, 95.7%) as a dark orange oil. It was used without

further purification. Spectra matched that currently in the literature (Laine

et al . J. Med. Chem. 2009, 52(8), 2493-2505). MS: requires 183.1, found

184.1 (M+H +).

3-methyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3125.1): off-white

N 1 solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 2.32-2.35 O N 13 (m, 9H), 3.48-3.52 (m, 6H). C NMR (100 MHz, CD 3OD, oxalate salt):

+ N 11.45, 25.72, 27.53, 31.36, 166.82, 168.80, 182.56. MS (ESI ):

+ C12 H17 N3O5, requires 193.1, found 194.1 (M+H ).

3-ethyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3151): white solid, N 1 O N oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 1.27-1.31 (t, 3H, J=7.2, 7.6 Hz), 2.32-2.36 (m, 6H), 2.71-2.77 (qt, 2H, J=7.6), 3.49-3.53 (m,

13 N 6H). C NMR (100 MHz, CD 3OD, oxalate salt): 11.93, 20.81, 27.41,

27.75, 31.65, 44.27, 47.45, 166.72, 173.36, 182.75. MS (ESI +):

+ C13 H19 N3O5, requires 207.1, found 208.1 (M+H ).

121

3-propyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3139.2): white solid, N 1 O N oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 0.96-1.00 (t, 3H,

J=7.2, 7.6 Hz), 1.71-1.80 (sextet, 2H, J=7.6 Hz), 2.32-2.36 (m, 6H), 2.67- N 13 2.71 (t, 2H, J=7.6 Hz), 3.49-3.52 (m, 6H). C NMR (100 MHz, CD 3OD,

oxalate salt): 14.04, 21.46, 27.56, 28.80, 31.43, 44.13, 47.31, 166.92,

+ 172.04, 182.54. MS (ESI ): C14 H21 N3O5, requires 221.1, found 222.1

(M+H +).

3-cyclopropyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3125.2): white N 1 O N solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 0.94-0.98

(m, 2H), 1.05-1.09 (m, 2H), 2.04-2.10 (m, 1H), 2.29-2.33 (m, 6H), 3.47- N 13 3.51 (m, 6H). C NMR (100 MHz, CD 3OD, oxalate salt): 7.49, 8.17,

+ 27.48, 31.38, 47.23, 173.94, 182.31. MS (ESI ): C14 H19 N3O5, requires

219.1, found 220.1 (M+H +).

3-isopropyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3130): white

N 1 solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 1.30-1.32 (d, O N 6H, J=6.8Hz), 2.32-2.36 (m, 6H), 3.01-3.10 (septet, 1H, J=6.8, 7.2 Hz),

13 N 3.49-3.53 (m, 6H). C NMR (100 MHz, CD 3OD, oxalate salt): 20.69,

27.38, 27.90, 31.30, 43.89, 47.06, 166.68, 176.36, 182.31. MS (ESI +):

+ C14 H21 N3O5, requires 247.2, found 248.2 (M+H ).

3-cyclobutyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3149): white

N 1 solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 1.96-2.03 O N (m, 1H, J=5.2, 6 Hz), 2.07-2.19 (sextet, 1H, J=8.8, 9.2 Hz), 2.33-2.38 (m,

13 N 10H), 3.49-3.51 (m, 6H), 3.61-3.70 (pent. 1H, J=8.4, 8.8 Hz). C NMR

122

(100 MHz, CD 3OD, oxalate salt): 19.93, 27.25, 27.60, 28.03, 31.52, 32.60,

+ 44.12, 47.30, 166.90, 174.49, 182.69. MS (ESI ): C15 H21 N3O5, requires

247.2, found 248.2 (M+H +).

3-cyclopentyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3133): white

1 solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 1.72-1.82 N O N (m, 5H), 2.05-2.09 (m, 2H), 2.34-2.38 (m, 6H), 3.19-3.27 (pent. 1H, J=7.2,

13 7.6 Hz), 3.50-3.54 (m, 6H). C NMR (100 MHz, CD 3OD, oxalate salt): N 26.61, 27.55, 31.45, 32.44, 37.91, 44.13, 47.29, 167.04, 175.54, 182.52.

+ + MS (ESI ): C16 H23 N3O5, requires 233.1, found 234.1 (M+H ).

3-cyclohexyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3135): white

1 N solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 1.29-1.59 (m, O N 5H), 1.72-1.75 (m, 1H), 1.80-1.84 (m, 2H), 1.97-2.00 (m, 2H), 2.32-2.36

(m, 6H), 2.75-2.81 (m, 1H), 3.48-3.52 (m, 6H). 13 C NMR (100 MHz, N

CD 3OD, oxalate salt): 26.80, 27.03, 27.53, 31.42, 31.82, 37.22, 44.09,

+ 47.27, 166.86, 175.54, 182.27. MS (ESI ): C17 H25 N3O5, requires 261.2,

found 262.2 (M+H +).

3-cycloheptyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3158): white

1 N solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 1.58-1.81 (m, O N 10H), 1.97-2.07 (m, 2H), 2.33-2.38 (m, 6H), 2.99-3.03 (m, 1H), 3.49-3.55

(m, 6H). 13 C NMR (100 MHz, CD OD, oxalate salt): 27.46, 27.58, 29.55, N 3 33.67, 39.05, 44.1, 47.28, 166.84, 176.51, 182.34. MS (ESI +):

+ C18 H27 N3O5, requires 275.2, found 276.2 (M+H ).

123

3-heptyl-5-(quinuclidin-4-yl)-1,2,4-oxadiazole (CJ3152): white solid, N

O N 1 oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 0.87-0.91 (m, 3H),

1.27-1.35 (m, 7H), 2.31-2.41 (m, 6H), 2.68-2.72 (m, 2H), 3.48-3.54 (m, N 13 5H). C NMR (100 MHz, CD 3OD, oxalate salt): 14.52, 23.68, 26.82,

27.57, 28.01, 29.61, 29.91, 30.11, 31.47, 32.69, 47.24, 166.81, 172.17,

+ + 182.56. MS (ESI ): C18 H29 N3O5, requires 277.2, found 278.2 (M+H ).

N O 3-cyclopropyl-5-(8-methyl-8-azabicyclo[3.2.1]oct-2-en-2-yl)-1,2,4- N oxadiazole (CJ3120.1) : white solid, oxalate salt. 1H NMR (400MHz, N CD 3OD, oxalate salt): 1.07-1.18 (m, 4H), 2.05-2.18 (m, 5H), 2.35-2.48 (m,

2H), 2.85 (s, 3H), 3.89-3.91 (m, 1H), 3.97-3.99 (m, 1H), 4.12-4.14 (m,

+ 1H), 4.45-4.49 (m, 1H). MS (ESI ): C15 H19 N3O5, requires 231.1, found

232.1 (M+H +).

Quinuclidine-3-benzilate (QNB): Procedure adopted from US. Patent

App. US10034867. Benzilic acid (1g, 4.38mmol) dissolved in 20 mL OH O THF. To this solution was added 1.1 eq CDI (4.82 mmol, 781mg) and the N O mixture was refluxed for 1 hr. The formation of the imidazolide was

followed by TLC (50/50 EtOAc/Hex). Then quinuclidinol (1.1 eq, 4.82

mmol, 608 mg) was added and mixture refluxed overnight. The reaction

was cooled to room temperature, diluted with diethyl ether, and washed

with water. The organic layer was extracted with 2N HCl (2x) and org

layer was discarded. Acidic aqueous layer was basified with sat. aqueous

potassium carbonate (monitored via pH strip) and extracted (3x) DCM.

Organic layers were combined, washed brine, dried with sodium sulfate,

124

filtered, and concentrated under reduced pressure to afford a white solid.

The HCl salt was generated via iPrOH/Et 2O. Spectral data matched that

+ previously reported in the literature. MS (ESI ): C21 H24 NClO3, requires

337.2, found 338.2 (M+H +).

(R)-Quinuclidine-3-benzilate (R-QNB): As above but with (R)-

OH O quinuclidinol (purchased from Combi-Blocks, Inc). Spectral data were N O identical to that already published in the literature (M. Prat et al. J. Med.

Chem. 2009, 52(16), 5076-5092).

(S)-Quinuclidine-3-benzilate (S-QNB): As above but with (S)-

OH quinuclidinol (purchased from Combi-Blocks, Inc). Spectral data were O N O identical to that already published in the literature (W. Rzeszotarski et al.

J. Med. Chem. 1988, 31(7), 1463-1466).

Quinuclidin-3-yl 2,2-diphenylacetate (CJ3056): White solid,

1 hydrochloride salt. H NMR (400MHz, CD 3OD): 1.73-2.00 (m, 4H), 2.30 O N O (m, 1H), 3.02-3.10 (m, 1H), 3.15-3.23 (m, 4H), 3.67-3.70 (m, likely dt.,

1H), 5.17 (m, singlet overlap with qt. 2H), 7.24-7.32 (m, 10H). MS (ESI +):

requires 321.2, found 322.2.

(R)-Quinuclidin-3-yl 2,2-diphenylacetate (CJ3056R): white solid,

oxalate salt. Procedure for 3056 was repeated except with R-quinuclidinol O N O (purchased from Combi-Blocks, Inc.). Proton spectra and MS matched

that of CJ3056.

(S)-Quinuclidin-3-yl 2,2-diphenylacetate (CJ3056S): white solid,

O oxalate salt. Procedure for 3056 was repeated except with R-quinuclidinol N O

125

(purchased from Combi-Blocks, Inc.). Proton spectra and MS matched

that of CJ3056.

2,2-diphenyl-N-(-quinuclidin-3-yl)acetamide (CJ3046): Quinuclidine-

3-amine dihydrochloride salt (750mg, 3.77 mmol, 1 eq) was dissolved in

10 mL DMF and cesium carbonate (3.77g, 11.57 mmol, 3 eq) added. The H N N mixture was stirred for 2 hrs at room temperature. Diphenylacetyl chloride O was then added in one portion and mixture stirred overnight. The reaction

was diluted with water/brine and acidified with 1N HCl. The mixture was

extracted 2x Et 2O, and the organic layer was discarded. Sat. aq. K 2CO 3

was added to pH~9 then mixture extracted 3x EtOAc. Organic layers were

combined, washed with brine, dried sodium sulfate, filtered, and

1 concentrated in vacuo . The HCl salt was generated from iPrOH/Et 2O. H

NMR (400MHz, CD 3OD): 1.83-1.85 (m, 1H), 1.99-2.05 (m, 3H), 2.19-

2.20 (m, 1H), 3.02-3.07 (m, 1H), 3.24-3.29 (m, 5H), 3.69-3.75 (t, 1H),

4.23-4.25 (m, 1H), 5.05 (s, 1H), 7.21-7.29 (m, 10H), 8.79-8.80 (m, 1H,

amide NH). MS (ESI +): requires 320.2, found 321.2 (M+H +).

2-hydroxy-2,2-diphenyl-N-((1s,4s)-quinuclidin-3-yl)acetamide

(CJ3053): Benzilic acid (1g, 4.38mmol) dissolved in 20 mL DMF. To this

solution was added 1.1 eq CDI (4.82 mmol, 781mg) and the mixture. The H OH N N formation of the imidazolide was followed by TLC (50/50 EtOAc/Hex). O Then the free base of quinuclidine-3-amine (1.05 eq, 4.82 mmol, 608 mg)

was added and mixture heated at 90 oC overnight. The reaction was cooled

to room temperature, diluted with mixture of water/brine. The aqueous

126

layer was made acidic with 2N HCl (2x) and extracted with diethyl ether

(2x, discarded). Acidic aqueous layer was basified with sat. aqueous

potassium carbonate (monitored via pH strip) and extracted (3x) with

diethyl ether. Organic layers were combined, washed brine, dried with

magnesium sulfate, filtered, and concentrated under reduced pressure to

afford the crude product as an oil. The HCl salt was generated via

iPrOH/Et 2O.

General Procedure for Synthesis of Synthesis of L689660: Procedure

detailed in Ashwood et al . (J.Chem.Soc.Perkin.Trans 1, 1995, 641-645)

and Baker, R., Saunders, J., and Leslie Street. (EP0416754 A2, 1991) was

followed and modified. O Ethyl 2-(N-tert-Butoxycarbonylpiperidin-4-ylidene)acetate: Diethyl O ethoxycarbonylmethylphosphonate (142g, 0.635 mol) was added to a

N stirred slurry of anhydrous potassium carbonate (202g, 1.466 mol) in Boc DMF (970 mL). The piperidone (97.2 g, 0.488 mol) was added to the

mixture which was then heated at 70 C under a nitrogen atmosphere for 22

h. The reaction mixture was cooled to room temperature and water was

added. The slurry stirred at 0 C and a precipitate formed. The product was

collected by filtration, washed with water and dried at room temperature

under vacuum to give the ester as a crystalline solid (128g, 97%). The

spectra were identical to that already reported in the literature (M.

Ashwood et al. J. Chem. Soc. Perkin Trans. 1. 1995, 641-644).

127

Ethyl 2-(N-tert-Butoxycarbonylpiperidin-4-yl)acetate: Ammonium O formate in water was added over 30 min to a slurry of the above ester and O palladium on carbon (10%) in ethanol and water at room temperature. The

N mixture was stirred for 3 hrs and then the catalyst removed by filtration. Boc The filtrate was evaporated under reduced pressure and the residue

partitioned between hexane and water. The organic layer was separated,

washed with water (2x) and evaporated under reduced pressure to give the

ester as an oil (98%). The spectra were identical to that already reported in

the literature (M. Ashwood et al. J. Chem. Soc. Perkin Trans. 1. 1995,

+ 641-644). MS (ESI ): C 14 H25 NO 4, requires 271.2, found 171.1 (loss of

Boc group).

(S)-2-(N-tert-Butoxycarbonylpiperidin-4-yl)-2-(6-chloro-pyrazin-2- enriched N O H y1)acetic acid (16): To a stirred solution of sodium Cl N OH bis(trimethylsily1)amide in THF (100 mL of 1M solution) was slowly

N added (over 30-40 mins) a solution of 2,6-dichloropyrazine (43.05 g, Boc 0.289 mol) and ester (74.5 g, 0.274 mol) in THF (300mL) maintaining the

temperature < -15 C. The mixture was stirred at -10 C for 1.5 h (monitored

by TLC, EtOAc/Hex 30/70) and then added to a mixture of aqueous

hydrochloric acid (200 mL; 7.5 dm3) and hexane (400 mL). The organic

phase was separated, washed with aqueous hydrochloric acid (200 mL)

and then water (2 x 250 mL). The hexane solution was evaporated under

reduced pressure to give the crude ester as an oil, which was dissolved in

ethanol (1 L). Sodium hydroxide (34.1 g, 0.85 mol) in water was added to

128 the solution of the ester and the mixture stirred at 25 C for 2 h. The solution was concentrated under reduced pressure at <25 C to remove most of the ethanol and the aqueous residue extracted with ethyl acetate

(3x) to remove non-acidic material. The aqueous solution was acidified with conc. hydrochloric acid (monitored by pH strips) and extracted with ethyl acetate (3x). The solution of the acid treated with a solution of (S)-(-

)-1-phenylethylamine in ethyl acetate. The slurry of the salt was stirred at

25 oC for 30 min and heated to 60 oC for 1 h and cooled to room temperature. This process was repeated 3x to give the enriched (S)- enantiomer. The optical rotation reported in the literature was not able to be duplicated using this method, but the salt was carried on as is. The salt was partitioned between ethyl acetate (300 mL) and aqueous hydrochloric acid (200 mL). The aqueous layer was separated and extracted with ethyl acetate. The combined extract was washed with aqueous hydrochloric acid then with water (2x), dried with magnesium sulfate, filtered, and concentration under reduced pressure to provide the enriched-(S)-acid as a crystalline solid (~36%), mp 148 C. Spectra matched that currently published in the literature. M. Ashwood et al. J. Chem. Soc. Perkin Trans.

1. 1995, 641-644).

(R)-( + )-2-(N-tert-Butoxycarbonylpiperidin4yl)-2-(6-chloropyrazin-2- yI) ethanol (17): A solution of the acid 9 (14 g, 39.4 mmol) in dry THF

(280 mL) was cooled to ice bath temperature and a solution of borane in

THF (1M in THF; 118.2 mL, 118.2 mmol, 3 eq) was slowly added

129

maintaining the temperature <15 C. The solution was allowed to warm to

room temperature and stirred for 1.5 h (stirring for longer periods of time

results in reduced yields). The solution was cooled back to 0 C and water

was slowly added (exothermic) with vigorous stirring. The mixture was

allowed to warm to room temperature and stirred for 2 h to complete

hydrolysis of the borate esters. The solution was concentrated under

reduced pressure at <24 C and the aqueous residue extracted with ethyl

acetate (3x). The organic layers were combined and washed with water

(1x), brine (1x), dried with magnesium sulfate, filtered, and then

evaporated under reduced pressure (<24 C). The residue was columned in

50:50 EtOAc/Hex to afford the alcohol as an oil (7.67 g, 57%). The

spectra were identical to that already reported in the literature (M.

Ashwood et al. J. Chem. Soc. Perkin Trans. 1. 1995, 641-644). MS (ESI +):

C16 H24 ClN 3O3, requires 341.1, found 241.1 (loss of Boc group).

N (R)-( + )-2-(N-tert-Butoxycarbonylpiperidin4yl)-2-(6-chloropyrazin-2- H yl)ethyl Methanesulfonate. A solution of the alcohol above (7.67g, 22.48 Cl N OMs mmol) and triethylamine (6.3 mL, 45 mmol) in dichloromethane (75 mL)

N was cooled to ice bath temperature and treated with methanesulfonyl Boc chloride (2.61 mL, 33.72 mmol) added dropwise over 10 min. The mixture

was stirred for another 2 hrs at zero degrees then 1N HCl was slowly

added maintaining the temperature at or slightly above 0 C. The organic

layer was separated, washed with 1N HCl, brine, and dried with MgSO 4.

The solution was concentrated in vacuo to afford the methanesulfonate as

130

an oil (9.34g, 99%). The sample was purified by flash chromatography

(EtOAc/Hex 1:1). The spectra were identical to that already reported in the

literature (M. Ashwood et al. J. Chem. Soc. Perkin Trans. 1. 1995, 641-

644).

(1S,3R,4S)-3-(6-chloropyrazin-2-yl)quinuclidine (L689660): The

methanesulfonate (5g, 11.91 mmol) was dissolved in diethyl ether (75

mL), and 2M HCl in ether (excess) was added at room temperature. The

mixture was stirred for 2 hrs to complete deprotection. Water was added to

the mixture followed by careful addition of potassium carbonate (excess).

The two phase mixture was heated at 60 C for 2 hrs then cooled to room

temperature. The aqueous layer was separated, back extracted 2x with

EtOAc, the organic layers were combined, and the mixture was

concentrated in vacuo to afford the crude base as an oil (2.66g, 85%). The

L-tartrate salt was generated in iPrOH and recrystallized in MeOH to yield

pure L689660 (2.53g, 56%). In order to match the optical rotation

presented in the literature further enantiomeric enrichment was performed

22 following the procedure of EP0416754 A2, [α D] = -20.9 (c=1 in MeOH)

followed by maleate salt formation.

(1S,3R,4S)-3-(6-ethoxypyrazin-2-yl)quinuclidine (CJ3101): white N 1 solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 1.41-1.44 (t, N O N 3H, J=6.8, 7.2 Hz), 1.74-1.77 (m, 1H), 1.98-2.10 (m, 3H), 2.27 (m, 1H),

3.34-3.40 (m, 2H), 3.46-3.49 (m, 2H), 3.54-3.59 (m, 2H), 3.89-3.95 (m,

1H), 4.41-4.51 (m, 2H), 8.08 (s, 1H), 8.14 (s, 1H). 13 C NMR (100 MHz,

131

CD 3OD, oxalate salt): 14.90, 19.74, 25.35, 28.43, 39.12, 47.41, 48.02,

+ 50.55, 63.73, 134.72, 136.78, 153.94, 161.30. MS (ESI ): C13 H29 N3O,

requires 233.1, found 234.1 (M+H +).

(1S,3R,4S)-3-(6-isopropoxypyrazin-2-yl)quinuclidine (CJ3101.1): N 1 white solid, oxalate salt. H NMR (400MHz, CD 3OD, oxalate salt): 1.37- N O N 1.39 (m, doublet overlap with multiplet, 7H), 1.70-1.80 (m, 2H), 2.07-2.11

(m, 2H), 3.09-3.20 (m, 3H), 3.47-3.51 (m, 4H), 5.27-5.34 (septet, 1H),

13 8.01 (s, 1H), 8.32 (s, 1H). C NMR (100 MHz, CD 3OD, oxalate salt): MS

+ + (ESI ): C14 H21 N3O, requires 247.2.1, found 248.2 (M+H ).

Synthesis of L670548/687306: The procedure detailed in Cottrell et al . (J.

Chem. Soc. Perkin. Trans 1, 1991, 1091-1097) was modified.

2-[(R)-1-Phenylethyl]-perhydropyrano[3,4-c]pyrrol-4-one (3/4) : Crude

amine ( 2, 53 g, 1 eq), was added over 15 mins to a cooled, stirred solution

of 5,6-dihydropyran-2-one ( 1, 20g, 0.20 mol, 1.1 eq) in dry ethyl acetate

(1.13 L). Trifluoroacetic acid (cat. 0.1 eq) was dropwise added and the

mixture was allowed to warm to room temperature over 3 hrs. After

verifying m/z product peak (ESI +: 245.1 required, found 246.1 (M+H +))

reaction was quenched with sat. aqueous sodium bicarbonate and the

layers were separated. The aqueous phase was extracted 2x with ethyl

acetate, the organic layers were combined, washed with brine, dried over

sodium sulfate, and concentrated in vacuo . The residue was columned on

silica gel in 0-5% MeOH/DCM to afford 2-[(R)-1-Phenylethyl]-

perhydropyrano[3,4-c]pyrrol-4-one as a 1:1 mixture of diastereomers in

132

~72 % yield as a yellow oil (49.06 g). Spectra matched that as previously

described in the literature (see Cottrell , I. F., et al . J. Chem. Soc. Perkin

Trans. 1, 1991 , 1091-1097).

(3S,4R(-3-Ethoxycarbonyl-1-[(R)-1-phenylethyl]-1-

azoniabicyclo[2.2.1]heptane bromide (7): Hydrogen bromide gas was O

O generated via acetyl bromide (80 mL) and ethanol (500 mL) at 0 C and N Br - allowed to stir for 10-15 minutes. The mixture of diastereomers (3/4 )

were dissolved in ethanol (100 mL) and slowly added (via additional

funnel) at 0 C over 30 minutes. The reaction was allowed to warm to room

temperature and stirred overnight. The solvent was removed in vacuo

followed by neutralization via sat. aqueous sodium bicarbonate until

pH~9. Dichloromethane was added to the residue and the biphasic mixture

was stirred 3 hrs at room temperature. The layers were separated and the

aqueous layer back extracted 2x dichloromethane. Organic layers were

combined, washed with brine, and dried with sodium sulfate. After

removal of solvent under reduced pressure the desired diastereomer was

crystallized via ethanol/acetone/diethyl ether to afford the 3S,4R

quaternary salt (22.58g, 30%). Spectra matched that as previously

described in the literature (see Cottrell , I. F., et al . J. Chem. Soc. Perkin

Trans. 1, 1991 , 1091-1097).

Ethyl (3S, 4R)-1-azabicyclo[2.2.1]heptane-3-carboxylate (8): The O

O quaternary salt (7, 1g, 2.82 mmol) above was dissolved in ethanol (30 ml) N HBr and 50% by weight Pd/C was added at room temperature. Then 1,4-

133

cyclohexadiene (~8 mL) was added and the mixture refluxed until

completion (usually 5-6 hrs). The reaction was monitored by TLC (95%

MeOH/5% NH 4OH, stained with I 2). After cooling to room temperature

the mixture was filtered through celite and concentrated under reduced

pressure to give the crude ester. This was recrystallized via acetone/diethyl

ether to afford the pure ester as the hydrobromide salt (705 mg, 90%).

Spectra matched that as previously described in the literature (see Cottrell,

I. F., et al . J. Chem. Soc. Perkin Trans. 1, 1991 , 1091-1097).

5-((1R,3R,4R)-1-azabicyclo[2.2.1]heptan-3-yl)-3-methyl-1,2,4- O N oxadiazole (L670548): See representative oxadiazole formation procedure N N above. The HCl salt was generated according to literature procedures

(Street et al . J.Med.Chem. 1990, 33, 2690-2697). Spectra matched that

+ currently published in the literature. MS (ESI ): C 9H14 N3OCl, requires

179.1, found 180.1 (M+H +).

5-((1R,3R,4R)-1-azabicyclo[2.2.1]heptan-3-yl)-3-cyclopropyl-1,2,4-

oxadiazole (L687306): See representative oxadiazole formation procedure

above. The p-tosyl salt was generated according to literature procedures

(Houghton et al . J.Chem.Perkin.Trans 1, 1993, 1421-1424). Spectra

matched that currently published in the literature. MS (ESI +):

+ C18 H23 N3O4S, requires 205.1, found 206.1 (M+H ).

134

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Chapter 3: Pharmacology of Muscarinic Antagonists: Studies on Reference Compounds and Novel Muscarinic Antagonists as Rapid Acting Antidepressants that Lack Cognitive Deficits

Pharmacological Assays Utilized in Evaluation of Compounds (3.1) : The role of acetylcholine in normal and abnormal mental functions has been guided by the observation of cholinergic drugs on behavior. 1a,b As stated above, the muscarinic antagonist scopolamine produced memory deficits that mimicked that of dementia. 2 Taken together it can be concluded that muscarinic antagonists may cause a decrease in cognitive behavior. In addition, muscarinic antagonists appear to be effective antidepressants in animals, whereas agonists are pro-depressants.

The hypothesis we will examine is the ability to identify an antimuscarinic drug(s) that have the desired anti-depressant activity, but have less ability to impair cognitive tasks in animal behavioral models. In order to evaluate the drug candidates we operate under the criterion that significantly smaller doses of the produced compounds will be required to produce the desired anti-depressant effects than are necessary to elicit cognitive deficits. In vivo assays will be used initially to confirm if novel compounds have antimuscarinic behavior effects in intact rats, and whether they have M 2 antagonistic activity in the peripheral nervous system (bradycardia test).

O Central muscarinic effects will be elucidated by testing whether the novel N O compounds can block the discriminative stimulus effects of arecoline (left), and

Arecoline whether (in a small number of examples) it can mimic the discriminative effects of scopolamine. Should the compounds be able to antagonize the bradycardia induced by arecoline, it will serve as verification of the compound as an M 2 antagonist in the various models.

The M 2 receptor is responsible for parasympathetic cardiac control, with agonists (i.e. arecoline)

148 producing bradychardia and antagonists blocking it.3 The antidepressant effects will be assayed using the forced swim test (FST) primarily, with novelty-induced hypophagia (under-eating) and sucrose preference test (indication of anhedonia--lack of interest in rewarding stimuli) as supportive measures. 4,5,6,7,8 Each assay has been verified and are considered reliable predictors of antidepressant action in humans and rats alike.

Drug Discrimination: These assays will indicate 1) whether the antagonist blocks the discriminative stimulus effects of arecoline in a surmountable fashion, and at what doses; 2) whether the antagonist blocks the rate-decreasing effects of arecoline in a competitive manner, and at what doses; and 3) whether it generalizes to or antagonizes the discriminative stimulus effects of scopolamine. We cannot be certain that drugs that block arecoline’s discriminative stimulus effects will necessarily generalize to scopolamine, or what the nature of the difference would be if a discrepancy arises. The scopolamine discrimination assay will be very helpful in establishing whether some drugs are antimuscarinic antidepressants but lack the ability to produce a scopolamine discriminative stimulus, suggesting that these two behavioral outcomes are differentially mediated.

Male and female Sprague-Dawley rats are trained in standard operant conditioning chambers that contain two (scopolamine vs saline or arecoline vs saline discrimination) nose-poke devices with apertures that contain yellow LED lights on either side of a dipper that can deliver 50 µl of vanilla-flavored Ensure. Rats are initially trained to nose-poke 10 times in any aperture to receive a dipper of Ensure (fixed ratio 10

– FR 10 schedule of reinforcement). Nose-poke biases are noted, and the following assignments are made for the 2- nose-poke condition: 1) approximately half of the rats in each group will have the drug-associated aperture on the left, and the remaining rats will

149 have the drug-associated aperture on the right; 2) the drug-associated aperture is assigned to the non-preferred side in half of the rats.

During training, the rats are injected with saline or their training drug and immediately placed in the chamber for a 5-min blackout, followed by a 20-min response period. If they received drug, they must respond at the drug-appropriate aperture in order to receive access to

Ensure. If they received saline, they must respond at the other, saline-appropriate aperture in order to receive Ensure. Inappropriate responses result in a 1 min blackout period. Training continues until the following criteria are met: 1) responding on the first FR of the session is completed on the appropriate aperture and 2) 85% of the total responses of the session are made on the appropriate aperture.

When these criteria have been met on 3 consecutive days, testing can begin. During test sessions, the animals are injected with a dose of the test compound (or a non-training dose of the trained compound) and placed in the chambers as usual. However, during the 20-min session, 10 consecutive responses in either aperture results in delivery of Ensure. When the effects of putative antagonists are to be evaluated, these drugs are given 15 min before the start of the session, and the training drug is injected immediately before the rat is placed in the chamber. It is required that the rat meet the training criteria on two consecutive sessions between test sessions. Testing is carried out no more frequently than twice per week.

Data analysis : Full generalization to a test compound is defined as >85% responding on the drug-appropriate aperture, and completing at least one ratio. Discrimination data are expressed as the percent responses on the drug-appropriate aperture, out of the total number of responses made at both apertures. Rates of responding are calculated as well: total number of responses/time during the light-on parts of the session.

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Quantitative assessment of agonist-antagonist interactions are made using individual dose-response curves and ED 50 values as determined using Graph-Pad Prism

(6.0). Measurement of apparent pA 2 values (the dose of antagonist required to shift the agonist dose effect 2 fold to the right) or pK B values can be calculated from drug discrimination data. These values reflect the affinity of the antagonist, and when they are the same using the same antagonist in different assays, it strongly suggests that the agonist under study is acting at the same receptor in the assays. 9a,b,c To obtain these measures, Schild plots are determined by expressing the average logarithm of the dose ratio -1 as a function of the negative logarithm of the molar dose of the antagonist. 10

Dose ratios [ratio of the ED 50 dose of the agonist in the presence of the antagonist (A’) to the ED 50 dose of the agonist in the absence of the antagonist (A)] are calculated for individual rats. A straight line model of nonlinear regression is fit to the equation:

log(dose ratio-1) = -log (antagonist) X slope + x-intercept at y=0.

Apparent pA 2 values are calculated with unconstrained slopes as well as with slopes constrained to -1. If a limited number of antagonist doses can be administered, precluding the calculation of a pA 2, a pK B will be calculated which is an apparent affinity estimate for a single dose of the antagonist.

Cardiovascular Assay (Bradycardia): To measure changes in heart rate and mean arterial pressure, rats are implanted with telemetric transmitters (TA11PA-C40 or TL11M2-C50-PXT,

Data Sciences International, Transoma Medical Inc., St. Paul, MN, USA) under ketamine (90 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia. Each transmitter is placed into a subcutaneous pocket on the side of the abdomen, and the catheter extending from the base of the transmitter is inserted 2–3 cm into the femoral artery and secured with a suture.

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Following surgery, rats are singly-housed and allowed to recover for at least 7 days prior to experimentation. All rats continued to have free access to food and water at all times. The telemetry system consists of battery-operated subcutaneous transmitters, Physiotel receivers, the

DSI Data Exchange Matrix, and the Dataquest A.R.T. system. These transmit, collect and store the digital data on blood pressure and heart rate to a computer (Data Sciences International,

Transoma Medical Inc., St. Paul, MN, USA). Blood pressure and heart rate data are compiled by the Dataquest A.R.T. Gold Analysis 3.01 software.

The analysis program calculates an average heart rate and mean arterial pressure (MAP) every 10 s. These 10 s epochs are subsequently averaged over 1 min per rat, and data from at least 6 rats are averaged for each treatment group with standard error of the mean as a measure of variability. Data are also expressed as area under curve (AUC) over the ten min period following the last injection, when the most robust arecoline effects are observed. AUCs are analyzed using one-way ANOVA with Tukey's post hoc tests; saline control conditions are compared with agonist alone and with antagonist + agonist combinations. If we look at antagonists alone, these are analyzed by two-way ANOVA with Bonferroni's post hoc tests.

Forced Swim Test: Rats of both sexes as well will be used. They are injected with a test compound and 30 min later placed individually a cylindrical container (46 cm tall X cm diameter) filled to 30 cm with 25 oC water. We have found that a single 15-min forced swim test is sufficient to observe antidepressant effects of both known antidepressant compounds and novel drugs. 11a,b The rats’ behavior is videotaped from above and is later scored by a trained observer who is blind to the drug conditions. Behavior is classified every 5 seconds over the 15 minute swim period as the following: immobility, the rat is hanging quietly in the water, making only enough movement to keep its nose out of the water; swimming, moving limbs in an active

152 manner, climbing, or actively moving its forepaws against the side of the chamber. 7,8 Based on the experimental variance and expected effect sizes of 1.5-3 (Cohen's d), power analyses determine that 6-8 mice per condition is sufficient to observe a significant effect, if one exists. A one-way ANOVA with Dunnett’s post-hoc test is used when comparing more than one dose to control.

Sucrose Preference Test: In the rat sucrose preference test (used occasionally), rats are trained to drink a 1% sucrose (or Ensure ®) solution. A baseline sucrose preference is then established prior to exposure to a chronic mild stress situation for 6 weeks--in which the rats are exposed to different stressors on a daily basis. Following this exposure, sucrose preference will be re-determined in rats treated with either vehicle or muscarinic compounds. Mild stressors include 10-14 hours exposure to cat odors (kitty litter), switching the rats from their home cage to another rat's cage, a succession of light/dark cycles (30 minutes each), a 45 degree cage tilt, and food deprivation. Control animals have unlimited access to food/water and will be housed in a room separate from that of the stressed animals. 5

The effect of the drug on sucrose consumption is recorded and graphed as area under the curve (AUC) using known software (PRISM). The AUC (area under curve) of the stressed group will be subtracted from the AUC of nonstressed to obtain a measure of the effect of the repeated stress. A measure of the effect of drug treatment will be obtained by subtracting the AUC of stressed group from AUC of the stressed+treated group. Treatment is the between groups factor and time is the repeated measures factor.

Novelty Induced Hypophagia Test: In the novelty induced hypophagia test 4 rats are trained to consume a "pleasurable" novel food (i.e. high in sugar) in their home cages for 4 days.

Latency to consume and volume consumed are measured. Following these measurements rats are

153 placed into new cages with the same food source. Latency to consume and volume are measured and compared again. Vehicle (saline), known antidepressants, and novel muscarinic compounds that we have designed are administered prior to switching the rats to their new cages. Under control treatments latency to consume increases and volume decreases (as expected) when compared to their home cage environment. Antidepressants decrease latency to consume and volume consumed. This is only observed with chronic antidepressant treatment. We expect our novel compounds to be much faster acting and reverse novelty-induced hypophagia following acute treatment--similar to that observed in humans.

Cognitive Evaluation : This involves a novel touch-screen procedure that evaluates memory

(delayed-matching-to-sample, DMTS) and attention (psychomotor vigilance). In the DMTS, a commonly used recognition task to test short-term memory, the subject (a rat) is presented with a sample stimulus. When the rats touch the sample stimulus it terminates on the screen. After a pre-set delay two comparison stimuli appear. A touch response on the comparison stimulus that matches some physical property of the sample stimulus (i.e. color) results in reward delivery.

Touching the incorrect comparison stimulus results in a

timeout. Accuracy is then plotted vs. different delay values

to determine a "forgetting function." This same procedure

has been utilized by many others to assess the deleterious

effects of various classes of psychoactive drugs. 12,13

Psychomotor vigilance is used to evaluate drug effects on

attention and vigilance over a period of time. Championed Photo courtesy of Brian Kangas (Harvard Medical School) by David Dinges of UPenn School of Medicine 14 it is a

reaction-timed task to measure the speed with which

154 subjects respond to a visual stimulus. In the task, stimuli of varying intensity are presented to the subject (again, a rat) in randomized locations on the screen following unpredictable intervals.

The reaction time of the rat to the stimuli will provide a reliable attention threshold in each individual subject. Attention will decrease over time which serves as a baseline for degradation of sustained vigilance. For these studies, a stimulus is presented on a screen in a random location following a variable time interval (5, 15, 30 seconds). Duration of presentation of the stimulus is

2 seconds on first trial. If the correct stimulus is touched by the rat within 2 seconds, a milk reward is delivered and duration of stimulus presentation is increased by 0.25 seconds for subsequent trials (across 500 trials).

In Vitro pharmacology will be used to determine the selectivity (or lack thereof) of compounds for the orthosteric site on the various human muscarinic receptors subtypes. Since we are primarily seeking antagonists, the degree of intrinsic activity of the compounds at each muscarinic receptor subtype (whether they act as agonists, partial agonists, antagonists, or inverse agonist) will also be determined via functional assays (described below).

Ligand affinity will be determined by competitive displacement of the non-selective muscarinic antagonist 3H-N-methyl-scopolamine (NMS). In this assay ligand receptor is separated from unbound ligand via filtration and the level of bound receptors determined by scintillation counting. Each of the above mentioned assays will be performed by Dr. Brian Roth at UNC Chapel Hill, Director of the Psychoactive Drug Screening Program (PDSP). 15a,b

Pharmacological Evaluation of Known Compounds (3.2):

The first step in designing novel antimuscarinics with reduced side effect profiles is to understand the mechanism of current muscarinic agonists/antagonist's function in the CNS and

155 the periphery. To accomplish this we use three in vivo assays in male Sprague-Dawley rats: (1) telemetered changes in heart rate, (2) drug discrimination, and (3) the forced swim assay of antidepressant activity. Drugs were given by the same route of administration, and over similar time courses in all cases. We began with the most studied and well known antimuscarinic--

Scopolamine.

Scopolamine appears to have antimuscarinic activity in the periphery as shown by its ability to antagonize arecoline-induced bradychardia. In addition, it has limited ability to antagonize arecoline's discriminative stimulus effects and has no ability to block its rate-suppression effects

(Figures 28/29). It was especially active in the forced swim test, a measure of anti-depressant activity at doses as low as 0.3 mg/kg. However, its limited ability to antagonize the central effects of arecoline, as well as its well-recognized disruptive effects on cognition, suggest that it

Arecoline

Figure 28-(Top) Bradychardia induced by a 10mg/kg IV of arecoline is blocked by Scopolamine. (Bottom ) Scopolamine has little ability to antagonize discriminative effects of arecoline

156

Figure 29-(Top) Scopolamine does not block arecoline's rate suppressing effects. (Bottom) Scopolamine shows potent activity in the forced swim test (FST) indicative of antidepressant activity at doses as low as 0.1 mg/kg (n=6 for each dose in FST).

will have undesirable side effects. In fact, as shown by the Delayed-Matching-To-Sample

(DMTS) task and Psychomotor Vigilance task (PVT) scopolamine produced severe impairments.

In the DMTS, which is thought to assay short-term spatial memory, rats have to remember the position on the touchscreen of a previously presented geometric shape. If they respond correctly, the delay value increases on the subsequent trial; if they respond incorrectly, the delay value decreases on the subsequent trial (in this task smaller numbers are negative effects). As shown by

157 the control and saline data points, under these conditions, rats can remember the position for ~7 seconds. Scopolamine produces dose-related impairments on task performance (Figure 30).

Figure 30-PVT (Top), DMTS (Bottom) of scopolamine

The alternative antagonist, L687306, was developed some time ago in an attempt to identify cholinergic cognitive enhancers that had a reduced side-effects profile by virtue of having reduced efficacy at any of the (then) three identified muscarinic receptors, and more selective

158 subtype affinity. 16a,b In these studies, whereas scopolamine had affinity but no efficacy at any muscarinic receptor (i.e, it was an antagonist at all receptor subtypes), L 687306 had affinity but no efficacy at M 2 and M 3 receptor subtypes, and affinity with very limited efficacy at the M 1 receptor subtype as measured in peripheral nervous system functional assays. L687306 produced no agonist action in autonomic measures (no salivation, hyperthermia, diarrhea, or heart rate changes) 16b and no suppression of operant food-reinforced responding in rodents 17 at doses up to

30 mg/kg. Interestingly, L687306 was able to reverse much of scopolamine’s disruptive effects in several memory tasks. 16b,17

The authors would likely

ascribe this antagonism to the

limited efficacy of L-687,306 at

M1 receptor subtypes, a

supposition that should be

evaluated if other M1 selective

agonists can be identified.

Figure 31-L687306 effectively blocks arecoline induced bradycardia As seen in the graph on the

left, L687306 was as capable as scopolamine to antagonize arecoline-induced bradycardia even at rates as low as 0.1mg/kg

(Figure 31). In addition, the compound was far superior to scopolamine in its ability to antagonizing both arecoline discrimination and rate suppression (Figure 32). The forced swim test assay was used once again to determine antidepressant activity, and hence clinical usefulness. As shown by the far right graph directly below L687306 is just as active as scopolamine. The data on the superior ability of L687306 to antagonize the central effects of

159 arecoline and its activity in the forced swim assay suggest that L687306 may be an equally effective antidepressant with a reduced side-effect profile. Additionally, the drug was relatively silent across both DMTS and PVT tasks well above doses that were effective in the FST (white hexagon indicates lowest effective dose in the FST, Figure 33).

L689660, developed by Merck in the early 1990s was shown to bind with high affinity to rat cerebral cortex muscarinic receptors with a low ratio of displacement of NMS compared with

displacement of oxotremorine, consistent 1.0 Arec + L 687,306 with low efficacy agonists. There was no 100 muscarinic receptor subtype specificity in N=8 radioligand binding assays, but it did have 50

N=8 functional selectivity in pharmacological

assays. It was a full agonist at M in rat 0 1 1.0 A Sal 0.03 0.1 superior cervical ganglion. At M 3 in

% Arecoline-Appropriate Responding Arecoline-Appropriate % Mg/Kg L 687,306 Rate guinea-pig ileum it was a potent agonist 2.5 with lower maximum response. It was an 2.0 antagonist at M 2 (guinea-pig atrium and at 1.5 muscarinic autoreceptors in hippocampal 1.0 18 16b

Responses/Sec slices. Data from Freedman et al. 0.5

0.0 confirm this data from Hargreaves. 1.0A Sal 0.03 0.1 Mg/Kg L 687,306 Aagaard and McKinney 20 confirmed Figure 32: L687606 antagonizes both the discriminative stimulus and rate suppression effects of arecoline selectivity for M 1 and M 3 receptors in

functional assays of brain muscarinic receptors and cloned human receptors transfected in CHO cells. It was a partial agonist at M 1

160

(24%) and M3 (26%) relative to oxotremorine-M and a competitive antagonist at M 4 in rat striatum. In addition it was shown to be an antagonist at putative M 2/M 4 autoreceptors in the hippocampus.

Figure 33-(Top): Directly above: L687306 significantly increases immobility in the FST. Bottom left= DMTS . Bottom right= PVT : L687306 does not produce a dose related effect on performance in either task. Note: white hexagon/circle indicates lowest effective dose in the FST (n=6).

It was only a partial agonist at transfected hm1 and hm3 receptors and inactive at M 5 receptors. Therefore, it was concluded that either with brain tissue or with transfected cell lines,

161

L689,660 was shown to be an agonist for the M 1 and M 3 receptors, but not for M 4 or M 5 receptors.

500

450 10 Mg/Kg Arecoline +1 Mg/Kg L689,660 400 350 300 250 10 mg/kg Arecoline

0 5 10 Time (min)

Figure 34-L689660 blocks arecoline induced bradychardia.

Dawson and Iversen 17 conducted a battery of assays in rodents: mouse tail-flick, rat response sensitivity (RS) test, rat reference memory, passive avoidance, conditioned suppression of drinking, amd working memory (delayed-matching-to-position) task. L689,660 induced antinociception at 0.03 mg/kg, reduced response rates at 0.1 mg/kg, reversed the effects of scopolamine in the reference memory task at 0.01 mg/kg, but did not block with scopolamine in the DMP task.

Rupniak, Tye, Iversen, 19 interested in treating Alzheimer’s disease, reported that L-689,660 partially reversed “the disruptive effects of scopolamine on cognition” in rhesus monkeys using a visuospatial memory task similar to delayed-matching-to-position. But reversal was far from complete, and adverse effects (salivation at doses of 0.1 mg/kg and higher) were present at larger doses, and two monkeys “refused to be tested after treatment with 0.4 mg/kg i.m.”

162

In our hands, L689660 was an antagonist at M 2 in the bradychardia assay at 1 mg/kg (Figure

34). For food-reinforced responding, it fails to suppress responding at small doses (data not shown) but at larger doses it suppresses responding on its own (Figure 35). At higher doses, it adds to the suppression of responding produced by arecoline. The higher the dose, the greater the interaction with arecoline, hence a longer duration of action.

1.0 Mg/Kg Arecoline alone 2

+ 0.032 Mg/Kg L 689,660

0 1 2 3 4 Time (15 Min Components)

100

0 1.0 ASal 0.003 0.03 Mg/Kg L 689,660

Figure 35-Top: L689660 adds to the rate suppression effect of arec oline . Bottom: L689660 does not generalize to arecoline .

163

In the FST, the drug decreases immobility at 0.1, 1.0, and 3.2 mg/kg using 1 hour pretreatment time (indicative of L689660's persistance as an antidepressant, Figure 36).

In Bryan Roth’s binding assays (UNC Chapel Hill Psychoactive Drug Screening Program-

Ki(nM) PDSP), L689660 is unusually selective for muscarinic receptors, but has M1 77.59 M2 21.42 nearly equal affinity at M1-M4 (Table 14). There is some attachment to D 3 M3 60.72 (dopamine receptor) as well. A dose of 0.1 mg/kg L689660 suppressed M4 25.13 M5 4340.82 food-reinforced responding in rats, and this was antagonized completely D3 14.76 Table 14 -All values are by 1.0 mg/kg L687306 (data not shown). Despite these "agonist-like" an average of K i calculated in triplicate. effects, 1 mg/kg L689,660 antagonized the bradycardia produced by 10

mg/kg arecoline. It did not modify the rate-suppressing effects of arecoline at doses that partially suppressed behavior alone (0.01 and 0.032 mg/kg respectively). Rats given

0.3 mg/kg showed over 80% responding on the arecoline-appropriate lever in the arecoline discrimination assay (not shown). Rates of responding were low at 0.1 and 0.3 mg/kg (drastic drop was shown even at 0.032 mg/kg as shown in Figure 35).

In the DMTS and PVT, while to a lesser extent than scopolamine, L689660 produces dose related impairments on task performance white hexagon indicates lowest effective dose in the

Figure 36 -L689660 is an effective (and persistent) antidepressant in the FST at doses of 0.1, 1.0, and 3.2 mg/kg with 1 hr pretreatment time (n=6 at each dose).

164

FST, Figure 37).

Figure 37 -Performance of L689660 in the DMTS and PVT

If we assume that the agonist effects of L689,660 are mediated primarily through M 1 or M 3, then the discriminative stimulus effect could be postulated to occur through one or both of these receptors. We concur that it is an M 2 antagonist, which means that the stimulus effects are

20 unlikely to be mediated there. If Aagaard and McKinney are correct, and it is inactive at M 5 and a competitive antagonist at M 4, then M 1/M 3 appear to show up as the likely candidate for mediating discrimination.

It could also be concluded that L689,660 produces decreased immobility in the FST through competitive antagonism at M 2 (recall that it blocks the cardiovascular effects of arecoline), or perhaps M 4 (Aangaard and McKinney found competitive antagonism at both of these receptors). 20 However, this hypothesis requires that we determine if there are drugs that are antagonists in the heart but are ineffective in the FST, or vice versa. This will not be explored in this thesis. High doses of L687306 antagonize L689660 suppression of responding (data not shown), lending evidence that L689660 is a muscarinic agonist at these high doses.

165

Pharmacological Evaluation of Novel Anticholingerics and Rational of Design (3.3): It was shown by our group that the pharmacological effects of the potent agonist L670548 could be directly antagonized by L687306. As shown in the arecoline discrimination task, a dose

Figure 38 -L687306 directly antagonizes L670548

of 0.1 mg/kg produces a rightward shift indicative of in vivo antagonism. Addtionally, L687306 produces a rightward shift in the dose response curve in rates of responding (Figure 38). Finally, an increase in immobility is observed when 0.1 mg/kg L687306 is given along with L670548 in the FST (Figure 39).

Figure 39 -L687306 directly antagonizes L670548 in the FST (water depth=30 cm, n=6 at each dose)

166

The only factor that differentiates these compounds is substitution of methyl for cyclopropyl

group on the 3-position of the 1,2,4-oxadiazole ring. Previous papers

published by Merck 21 showed that the methyl oxadiazole of arecoline was

also a potent agonist--albeit not as potent as L670548. Knowing this, we Figure 40-CJ2100 sought to investigate whether the cyclopropyl oxadiazole arecoline would

be the antagonist pair of the methyl. We were pleased to find that it was

(detailed below). In fact, CJ2100 proved to have a similar pharmacological profile to L687306

(Figure 41). It showed antagonism at M 2 by blocking arecoline induced bradychardia. It also

blocked the rate-suppressing effects of arecoline, but not its discriminative stimulus effects.

CJ2100 also showed modest antidepressant-like activity in the forced swim test (Figure 42).

3 1.0 Mg/Kg Arecoline + 1 Mg/Kg CJ 2100 500

450 10 Mg/Kg Arecoline + 1.0 Mg/Kg CJ 2100 2 400

350 1 300 1.0 Mg/Kg Arecoline alone

250 10 Mg/Kg Arecoline

0 0 5 10 1 2 3 4 Time (min) Time (15 Min Components)

100

Figure 41- (Top Left) CJ2100 antagonizes arecoline induced bradycardia and (Top Right) rate suppressing effects, but does not block arecoline's discriminative 0 stimulus effects 1.0 ASal0.1 1 3.2 Mg/kg CJ 2100 167

Figure 42-CJ2100 is active in the FST producing a notable decrease in immobility.

We were even more encouraged by the results of the DMTS and PVT tasks to evaluate the cognitive effects of CJ2100. As noted above one of the limitations of scopolamine is the severe cognitive deficits it produces (effects on both attention and spatial memory). As shown below

CJ2100 was silent (up to 10mg/kg, white hexagon indicates lowest effective dose in the FST)

Figure 43-CJ2100 is silent up to 10mg/kg in both cognitive tasks (DMTS, left) or sustained attention (PVT, right). White hexagon indicates lowest effective dose in the FST.

168

across both tasks at doses well above those therapeutically effective in the FST.

The above preliminary results allow us to draw the following conclusions: 1) that orthosteric

ligands can potentially have selective activity at muscarinic receptors (which can provide

information about the mechanism of action). 2) The discriminative stimulus effect of arecoline

appears to be mediated through activity at a receptor other than M 2 (likely M 1 or M 3). 3) CJ2100

was our first lead compound to act as an antidepressant that did not cause cognitive deficits

(aside from L687306). 4) Switching from methyl (L670548 and CJ2099) to cyclopropyl

oxadiazole analogues (L687306 and CJ2100) appears to confer antagonist activity indicating that

either sterics or electronics could potentially be correlated to muscarinic antagonist activity.

More work has been completed on this hypothesis, and it is detailed below.

Methyl vs. Cyclopropyl: Agonist/Antagonist pairs (3.4):

Since its introduction in the 1960s for the reversal of opioid overdose, naloxone has earned its

place on the World Health Organization’s List of Essential Medincines as an opioid receptor

antagonist. It is a synthetic morphinan derivative derived from oxymorphone, albeit with a

relatively simple chemical alteration; replacing the N-methyl group on the oxymorphone with an

HO allyl group appears to confer opioid

HO HO HO 22 O antagonism. In 1981,

N O OH O OH OO buprenorphrine, a semi-synthetic O H N N N OH O O analogue of thebaine, was approved

oxymorphone naltrexone nalmefene Buprenorphrine for medical use. 23 It has been shown to Figure 44-AF Casy and RT Parfitt, Opioid Analgesics: Chemistry and Receptors, 1986. Methyl vs. (methylen e) be a partial agonist at the mu receptor, Cyclopropyl examples in opioid agonist/antagonist design.

169 an antagonist at kappa, antagonist at delta, and possess weak affinity for the nociceptin receptor. 24 Again the N-methyl group has been modified to include a methylene cyclopropyl moiety that affords antagonist characteristics. This same methylene cyclopropyl group is seen in multiple other opioid analogues and appears to produce opioid antagonists (Figure 44). Our hypothesis was that this same chemical change would yield antagonists for other receptors of interest.

Clinical data showed that oral and intravenous treatment with the muscarinic cholinergic antagonist scopolamine has rapid anti-depressant effects in humans 25 and was quite potent in the forced swim assay in rats. 26 This raises the interesting possibility that targeting the muscarinic cholinergic pathway has the potential to yield novel and effective anti-depressants. Scopolamine and other anticholinergics can produce a wide array of adverse effects, ranging from visual disturbances, and lead to significant impairment of cognitive function. Impairment of cognitive function is of upmost concern as depression itself is directly associated with cognitive deficits.

Merck published an interesting lead N O N O N O O N N N N compound, L687306, as an M 2 and M 3 O OH L687306 Scopolamine L670548 27 antagonist, with low efficacy at M 1. In

Figure 4 5 - Scopolamine, L670548, and L687306 fact, L687306 is able to alleviate the

effects of scopolamine in cognitive tests.

In our assays, it blocks the cardiovascular effect of arecoline, and is as active as scopolamine in the forced swim test—a measure for antidepressant activity. We failed to find scopolamine to be an effective antagonist of the discriminative effects of arecoline (shown earlier), a muscarinic agonist. Its counterpart, L670548, also designed by Merck, 21 is still one of the most potent muscarinic agonists known. Interestingly, a switch from methyl to cyclopropyl on the 1,2,4-

170 oxadiazole ring reduces the agonist effect of the molecule. We sought to investigate whether a shift from methyl to cyclopropyl on the oxadiazole ring would confer antagonist character over other scaffolds that have been used as potential muscarinic agonists/antagonists such as the arecoline (tetrahydropyridine), 1-azanorbornane (1-azabicyclo[2.2.1]heptane), 1- azabicyclo[2.2.2]octane (quinuclidine), and 1-azabicyclo[3.2.1]octane cores. We also varied the substitution pattern of the arecolines, 1-azanorbornanes, and quinuclidines to validate whether

Figure 46 : Methyl/Cyclopropyl pairs.

the placement of the oxadiazole ring was crucial for binding/efficacy to the mAChRs. The nine pairs are listed above (Figure 46).

171

As shown from the data below, CJ2099 and CJ2100 follow the same trend as the L-series.

They were unable to match the binding affinity or potency of the L-compounds (usually decreased by 10 fold), but nevertheless proved to be an interesting agonist/antagonist pair.

500 C9-C16 July 25,2018 Saline 3 mg/kg CJ 2099 400

300

sem) ± ± ± ± 200

HR ( HR all rats in severe respiratory distress Gave C12 1 mg/kg CJ 2100 at this point and 100 respiratory distress relieved.

0 0 20 40 60 Time (min) Figure 47-CJ2100 directly antagonizes severe CJ2099 induced bradycardia.

CJ2099 induces a strong bradycardia effect at 3 mg/kg, nearly fatal to the animals, but respiratory distress can be relieved by administration of 1 mg/kg of CJ2100, a direct indication of

M2 antagonism (Figure 47). Additionally, CJ2099 discriminates to arecoline at doses ~0.3

Figure 48- CJ2100 antagonizes rate suppression effects of CJ2099

172 mg/kg (interestingly, CJ2100 does not discriminate to arecoline, despite being an arecoline derivative) but is antagonized directly by 1 mg/kg CJ2100 until reaching doses of 1 mg/kg.

While the agonist effects predominate at this dose, arecoline response rates still do not match those of arecoline alone (Figure 48).

Figure 48 (continued)-CJ2100 antagonizes discriminative stimulus of 2099 (2099 discriminates to arecoline) .

The quinuclidine series was shown by Merck and others to be one of the most successful scaffolds in their early design of mAChR agonists, and indeed this hindered amine is far superior

[CJ2051] Figure 49-CJ2126 antagonizes the agonist effects of CJ2051 in drug discrimination.

173 to that of arecoline in both binding affinity and potency.

[CJ2051] Figure 49 (continued)-CJ2126 antagonizes the agonist effects of CJ2051 in rates of responding.

CJ2051/CJ2126 (3-methyl-5-(quinuclidin-3-yl)-1,2,4-oxadiazole) were synthesized as previously described above and proved to be another agonist/antagonist pair in the three pharmacological assays.

CJ2051 was a potent agonist in our hands; ~10 fold more potent than CJ2099 and ~100 fold more than arecoline. It rapidly suppressed the rats rates of responding at doses as low as 0.01 mg/kg but did discriminate to arecoline. CJ2126, the cyclopropyl derivative, readily antagonized

CJ2051's effects on both rates of responding and discrimination once again (at doses as low as

0.1 mg/kg) indicating in vivo antagonism (rightward shift of dose-response curve) (Figure 49).

We became interested in CJ2126 as a potent antagonist and evaluated it further in the FST and in

DMTS/PVT.

In the FST, CJ2051 proved to be a potent pro-depressant as the immobility score drastically increased at doses as low as 0.03 mg/kg. In contrast, CJ2126 showed a dose dependent decrease

174 in immobility, and hence an antidepressant effect. Cognitively, CJ2126 proved disappointing

(Figure 52).

DMTS TVT

Figure 50-CJ2126 shows substantial decrease in immobility (unlike CJ2051) in the FST (top two graphs, water depth=30 cm with n=6 at each dose) but had severe cognitive effects on both spatial memory (lower left)/sustained attention (lower right)

The drug produced a dose dependent impairment on both attentional and spatial memory tasks

(Figure 50) at doses as low as 0.32 mg/kg (below the minimum dose necessary to show response in the FST) perhaps indicative of a cholingeric blockade, which is detrimental to cognitive

175 function as detailed above (Chapter 2) likely due to the blocking of the M 2 autoreceptors. More experiments must be performed to confirm this phenomenon. Other interesting results were found when studying CJ2126 by itself (i.e. without the presence of any agonist). Antagonism of bradychardia was first performed to test for antagonism of the M 2 receptor. Surprisingly, a 1.0 mg/kg administration of the drug produced a 50 bps tachychardia, resembling that of a classic anticholinergic effect. Additionally, 0.1 mg/kg CJ2126 was readily able to antagonize the rate suppressing efforts of 1.0 mg/kg arecoline. However, when CJ2126 was administered at a dose

Figure 51-CJ2126 readily blocks arecoline induced bradycardia (top) but produces a 50bps tachycardia. (Bottom) 0.1 mg/kg CJ2126 can antagonize rate suppressing effects of 1.0 mg/kg arecoline, but higher doses played an additive role in rate suppression.

of 1.0 mg/kg it played an additive role in rate suppression (Figure 51).

176

The molecule is quite specific for the mAChRs according to off target screening performed

15 on 45 different receptors in the CNS and binds to M1-4 with low nanomolar affinity (PDSP).

However, it does show high affinity for the H 1 receptor (histamine). Efficacy screens are currently being conducted to further clarify the mechanism of action of CJ2126 (Table 15).

Receptor Ki (nM) It should be noted that both CJ2051 and CJ2126 are chiral molecules, H1 42.17 but the racemates were used for the pharmacological evaluation. There is M1 32.08 M2 5.77 a possibility that the two enantiomers elicit different responses, but due M3 17.77 M4 7.84 to technology limitations and cost of separation (as no scientific M5 6708.8 Table 15-All values are an literature has reported any methods to complete this task) the average of K i calculated in triplicate. Assay conducted enantiomers have not yet been separated and tested individualy. by Roth Laboratory PDSP (Psychoactive Drug However, SILCS modeling of both enantiomers indicates that the Screening Program) predicted ligand free energy scores of binding are approximately equal

(Appendix B). 2139 and 2150 are the most similar to 2099/2100 with the exception of N-methylation on the tetrohydropyridine ring. Therefore, it was expected we would find similar results from the battery of pharmacological tests. However, this was not the case.

2139 proved to be a dangerously potent agonist in our hands, killing several animals at doses as low as 1 mg/kg. As seen from the graphs below, 2139 generalized to arecoline, but only at doses that rapidly suppressed responding. CJ2150 was able to antagonize both arecoline's rate suppression and discriminative effects, albeit mildly (at 1mg/kg). When tested together as an agonist/antagonist pair CJ2150 was unable to antagonize either the discriminative or rate suppressing effects of CJ2139. Agonist effects predominated at these doses. More work is currently being done at lower doses to see if CJ2150 can indeed antagonize CJ2139.

177

Arecoline Group II 100 N=2 75

50 N=7

25 N=7 N=7 0 0.0001 0.001 0.01 0.1 1 CJ 2139 (mg/kg)

% Arecoline-Appropriate Responding Rate 4

1 Responses/Sec

0 0.0001 0.001 0.01 0.1 1

CJ 2139 (mg/kg)

Figure 52-CJ2139 discriminates to arecoline ( top left ) but rapidly supresses rates of responding ( bottom left ) indicative of potent mAChR agonist activity. CJ2150 moderately antagonizes discriminative/rate supressing effects of arecoline.

CJ2150 was able to antagonize both arecoline's rate suppression and discriminative effects,

albeit mildly (at 1mg/kg) (Figure 52). When tested together as an agonist/antagonist pair CJ2150

was unable to antagonize either the discriminative or rate suppressing effects of CJ2139. Agonist

effects predominated at these doses (Figure 53). More work is currently being done at lower

doses to see if CJ2150 can indeed antagonize CJ2139.

178

Figure 53-(Left) 1mg/kg CJ2150 cannot effectively antagonize discrimination of CJ2139. (Right) CJ2150 (1mg/kg) is unable to antagonize rate suppression induced by CJ2139.

CJ3094 and 3095 are novel compounds that incorporate the 1-azabicyclo[3.2.1]octane scaffold, which is a structural isomer to the quinuclidines (1-azabicyclo[2.2.2]octane) and

(should) behave similarly chemically due to closeness their pKa values. As shown from the graphs below, CJ3094 is a potent agonist (resembling CJ2051). It induces a strong bradycardia

Figure 54-CJ3094 produces a strong and long-lasting bradycardia effect

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Figure 54 (continued)-CJ3094 produces a strong and long- lasting bradycardia effect that can be antagonized by CJ3095

effect as low as 0.056 mg/kg which persists for well over an hour. However, it is completely blocked by 1 mg/kg CJ3095, following with the hypothesis that the cyclopropyl derivative is an

M2 antagonist (Figure 54).

By itself it completely suppresses rates of responding (indicative of agonist character) and generalizes to arecoline. CJ3095 is a potent antagonist of arecoline's rate suppression/discriminative effects at doses as low as 0.3 mg/kg. Both rates of responding and discrimination of CJ3094 are directly antagonized after administration of 1 mg/kg CJ3095

(Figure 55) as indicated in the rightward shift of the dose response curve. However, by itself

CJ3095 produced substantial discrimination to arecoline in 5/6 rats, indicative of agonist character in at least one (or more) receptors (Supplementary Figure 68).

It has been shown in earlier studies by Merck (see above section) that the 3-position of the oxadiazole ring was crucial for activity due to its ability to participate in hydrogen bonding with key residues in the orthosteric pocket. We wanted to evaluate the pharmacological effects of

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Figure 55-CJ3094 discriminates to arecoline at doses that markedly suppress rates of responding (top and bottom left). CJ3095 antagonizes the agonist effects of CJ3094 (top and bottom right). n=6

switching the position of the azacyclic nitrogen (CJ3120/3120.1) and the oxadiazole ring from the 3 to 4 position (CJ3109/3110, 3125/3125.1, MA418/430B). Unfortunately, tropane-like derivatives 3120/3120.1, isoarecolines (418/430B), and 4-substituted quinuclidines 3125/3125.1 were not active in any of our pharmacological tests (up to 3.2 mg/kg, 32 mg/kg, and 3.2mg/kg respectively (Figures 56 and 57).

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Figure 56 - 3120 (top), 3120.1 (middle) cannot antagonize either rate suppressing or discriminative effects of arecoline up to 3.2 mg/kg. (bottom) CJ3125.1 and .2 show no ability to affect rates of responded up to 3.2 mg/kg

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Figure 57 -MA418/430 cannot antagonize either rate suppressing or discriminative effects of arecoline up to 3.2 or 32 mg/kg respectively

It is highly likely that the chemical shifts in these scaffolds diminished affinity and efficacy for the mAChRs although this must be confirmed in vitro testing (affinity and efficacy assays). This lends credence to the original hypothesis by Merck that a rigid scaffold with precise placement of H-bond acceptors is key for affinity to the muscarinic receptors.

Evaluation of Novel Muscarinic Antagonists (3.5):

We were pleased to see that L687306 and CJ2100 showed antidepressant activity (as indicated by decrease in immobility in the FST) yet lacked cognitive deficits (as indicated in both PVT and DMTS). Each of these shared the cyclopropyl oxadiazole ring in common, but we sought to further optimize the scaffold if possible. The simplest method was to generate a series of various 3-alkyl substituted oxadiazoles with different hindered amine cores (i.e. arecoline, quinuclidine, 1-azabicyclo[3.2.1]octane, etc. as described above). Due to the large number of compounds produced it was necessary to develop a faster pharmacological method for screening.

To accomplish this the compounds were first screened at 3 mg/kg in the FST by our collaborator

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Dr. Emily Jutkiewicz (University of Michigan, Department of Pharmacology) to assess for antidepressant activity. If the compounds did not produce a significant decrease in immobility they were not further evaluated (Figures 58/59). If they were successful in decreasing immobility a dose response curve was generated to establish the lowest effective dose applicable to the FST.

This was used as a reference for cognitive evaluation in both the DMTS and PVT tasks. These compounds were further evaluated in their ability to block arecoline-induced bradycardia, arecoline's rate suppressing effects, and drug discrimination of arecoline (as detailed above).

Finally, in vitro analysis by the Roth lab, binding and functional assays, were performed to assess subtype selectivity and efficacy (work is on-going). 15a,b

Below (Table 15) is a representative analysis of several compounds in the FST along with all corresponding structures (work is on-going). The values highlighted in green represent those compounds that significantly decreased immobility in the FST. As seen from the table, CJ2099 severely increased immobility (indicative of its agonist character) hence acting as a prodepressant. CJ2051 and 2139 proved to be very potent agonists at 3 mg/kg and induced severe cholingeric side effects on the rats (unable to swim, porphyrin staining, and convulsions, hence, euthanized). Despite Sauerberg et al .28 showing that the 3-alkyl-substituted arecoline oxadiazoles were agonists up to the pentyl derivative (CJ2161.2), and all branched/cyclic substitutions produced antagonists in vitro --none of the compounds showed efficacy in vivo in the FST up to 3 mg/kg. It raises an interesting question as to why the methyl and cyclopropyl arecolines perform so well (both in vitro/in vivo but their other derivatives do not). More work is needed to delineate why this is the case. Interestingly, CJ3018 produced a modest effect at 3 mg/kg and only produced detrimental performance in the DMTS and TVT cognitive tasks at high

184 doses (i.e. 10 mg/kg, shown on following pages). In contrast, the N-demethylated cyclopropyl derivative (CJ2150) produced no change in behavior in the FST (Table 16).

In following the general hypothesis that mAChR antagonists require a basic amine attached to a bulky hydrophobic/aromatic moiety, we designed several quinuclidine amides/sulfonamdes (a few are represented below, CJ2009/2029/2070/3046). Disappointingly, neither showed any efficacy in vivo in the FST, despite showing modest affinity for the mAChRs in vitro . Perhaps more surprising is that CJ3046, a close mimic of QNB (thought to be the prototypical mAChR antagonist, but was later shown to be an inverse agonist), showed no activity in the FST.

However, CJ3046 was able to block the effects of succinylcholine poisoning and so proves that it functions as an anticholinergic (data not shown). The best arecoline-derivative by far was

CJ2100, which produced a clear antidepressant in the FST along with no detrimental effects on cognition/attention (up to 10mg/kg). However, the isopropyl, cyclohexyl, heptyl, and cycloheptyl arecoline derivatives also produced a mild antidepressant effect. These are currently being tested in the PVT/DMTS to assess their effects on sustained attention/memory. Overall, the arecoline series performed moderately well, but while many appeared to be good antagonists in vitro (as detailed by Sauerberg et al , Table 3, Chapter 2), it was not the case when evaluated in vivo. The quinuclidine series proved to be a much more promising chemical scaffold.

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N O N O N O N O N O O N N N N N N N CJ2164.1 N CJ2161.2 N N CJ2162.1 N CJ2159.2 N N CJ2099 CJ2162.2

O N N N O N O N O O N N N N N CJ2164.3 N CJ2101 CJ3006 N CJ2164.2 N CJ2159.3 N N

N N O N O O N O N O N N N N N N N N N CJ2139 H N CJ3007 CJ3022 H H CJ2150 CJ3018

O N F O N O O N O N N H N N N CJ2189.1 N N N N N H CJ2145.3 CJ2126 CJ2165.1 CJ2166.2

O N O N F H O H H N N O N S N N N Cl S N O N N N O O CJ2173.1 CJ2174 CJ2009 Cl CJ2029 CJ2070

H N N O CJ3046

Figure 58 -First series of compounds evaluated for FST activity

186

N O O O N N O O N N N N N N N N N N N CJ2165.3 CJ2169.3 CJ3017 CJ3061 CJ3063

OH O N Ph Ph Ph Ph O N O O N O O O O N N N H H N N N N N H CJ3071 CJ3077 CJ3082 CJ3094 CJ3070 CJ3065 N N N N N N N O O O O N O N O N O N N N N

N N N N N N N CJ3095 CJ3098 CJ3100 CJ3109 CJ3110 CJ3114 CJ3120

N N N N N N O O O N O N O O N N N N

N N N N N N CJ3120.1 CJ3125.1 CJ3125.2 CJ3129 CJ3130 CJ3132

N N N N N O O N O N O N O N N O NH

N N N N N N CJ3133 CJ3137 CJ3139.1 CJ3139.2 CJ3141 CJ3138

N N N N N N O O N O N O N O N N

N N N N N CJ3142 CJ3145 CJ3148 CJ3149 CJ3153

Figure 59 -Second series of compounds evaluated for FST activity

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Compound N Raw values % Ctrl Average SEM Control 5 138 2.72

2029 2 142 6 102.9 2051 2 euthanized 2070 2 141.5 0.5 102.5 2099 2 156.5 19 113.4 2100 2 100.5 0.5 72.8 2101 2 133.5 0.5 96.7 2126 2 75.5 6.5 54.7 2139 2 euthanized-convulsions 2145.3 2 141.5 1.5 102.5 2150 4 128 4.8 92.8 2159.2 2 137.5 1.5 99.6 2159.3 2 121 4 87.7 2161.2 2 137 0 99.3 2162.1 2 118.5 3.5 85.9 2162.2 2 131.5 0.5 95.3 2164.1 2 130.5 1.5 94.6 2164.2 2 132 3 95.7 2164.3 2 138 3 100.0 2165.1 2 91 14 65.9 2166.2 2 127 12 92.0 2173.1 2 119 3 86.2 2174 2 79.5 7.5 57.6 2189.1 2 145 1 105.1 3006 2 139.5 3.5 101.1 3007 2 117.5 3.5 85.1 3018 2 121.5 16 88.0 3022 2 145 2 105.1 3046 2 134.5 4.5 97.5 2164.3 4 136.5 2.52 98.9 2165.3 2 118 5 85.5 3063 2 125 5 90.6 3065 2 126 9 91.3 3070 2 135.5 3.5 98.2 3077 2 143 7 103.6 3082 2 138.5 7.5 100.4 3094 2 convulsions 3095 2 108.5 4.5 78.6 3098 2 145 2 105.1 3100 2 122 2 88.4

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3109 2 convulsions 3110 2 142 5 102.9 3114 2 132.5 12.5 96.0 3120.1 2 132 6 95.7 3125.1 2 136.5 9.5 98.9 3125.2 2 140.5 0.5 101.8 3130 2 134 1 97.1 3132 2 131 9 94.9 3133 2 142.5 1.5 103.3 3137 2 134.5 3.5 97.5 3138 2 132 2 95.7 3139.1 2 138.5 0.5 100.4 3139.2 2 127.5 10.5 92.4 3141 2 147 3 106.5 3142 2 133 9 96.4 3145 2 150 1 108.7 3148 2 140 8 101.4 3149 2 142.5 0.5 103.3 3153 2 146.5 0.5 106.2

Table 16: FST results for several novel compounds. Red (or euthanized)=increased immobility (agonist), light green =partial, but significant response, green =significant increase in immobility, no color=no significant change in immobility

In fact, aside from CJ2100, all other compounds highlighted in green, meaning they were most effective at reducing immobility, are quinuclidines. CJ2126 has already been described in the above section, and as expected performed well in the FST task decreased immobility starting at 0.1-0.3 mg/kg). However, it produces cognitive deficits as low as 0.32 mg/kg making the therapeutic window very narrow for this drug. CJ2165.1 (cyclobutyl), CJ2173.1 (pentyl), and

CJ2174 (t-butyl) all produced signficant decreases in immobility (2173.1~borderline value) at 3 mg/kg and were further evaluated. CJ2165.1 decreased immobility starting at 1 mg/kg, had mild effects on attention (PVT) at 3.0 mg/kg (Figure 60), but severe effects on memory (DMTS,

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Figure 61). CJ2173.1, like 2165.1, had mild effects on attention at 3 mg/kg, but once again produced detrimental effects on memory (although not as severe as 2165.1).

Titrating Psychomotor Vigilance Task (Sustained Attention)

Minimum effective dose in the FST Minimum effective dose in the FST

Minimum effective dose in the FST

Figure 60 -CJ2165.1/2173.1/3018 in the PVT. All produce impairments in sustained attention at higher doses, but CJ3018 produces the mildest effects at 3.0mg/kg (dose utilized in the FST)

Interestingly, CJ3018 (a norarecoline derivative) showed moderate antidepressant activity in the FST, produced only a mild effect in the PVT for sustained attention (at the relevant FST dose of 3 mg/kg), and was silent in the spatial memory task (up to 3.2 mg/kg). After reviewing the data it has a strikingly similar profile to CJ2100, and can therefore be considered another lead compound found from this study. Surprisingly, the N-methyl derivative (i.e. 2164.3) showed no activity in the FST at 3 mg/kg and was therefore not further evaluated.

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Aside from being more potent than its arecoline couterparts, an interesting trend in the quinuclidine series is the wider toleration of the R-group of the oxadiazole. For example, 2164.1, the cyclobutyl arecoline derivative, shows no appreciable activity in the FST, yet 2165.1 does.

CJ2161.2 and CJ2173.1, the pentyl derivatives, show a similar trend.

Titrating Matching-to-Position (Spatial Memory)

Minimum effective dose in the FST Minimum effective dose in the FST

Minimum effective dose in the FST

Figure 61-CJ2165.1/2173.1/3018 in DMTS. Note 3018 produces the mildest effects on spatial memory, whereas CJ2165.1 (worst effects, but only at higher doses) and 2173.1 produce significant impairments.

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Titrating Psychomotor Vigilence Task (Sustained Attention)

Minimum effective dose in the FST

Figure 62-CJ2150/2174/3095/3100 in PVT. All produce relatively mild impairment when compared to active dose in the FST. Minimum doses active in the FST had not yet been established when this data was generated. However, it was known that CJ2174 was active at 1 mg/kg and that CJ3095 was active at 3 mg/kg.

The norarecoline derivative of CJ2100 (i.e. CJ2150) disappointingly had the reverse trend that was seen with CJ2164.3/CJ3018. Not only did CJ2150 display very weak activity in the FST (see

Table 1 , page 38), it surprisingly produced small (but noticeable) impairments in both sustained attention/cognition (completely opposite of CJ2100) (Figure 62 and 63). The t-butyl quinuclidine derivative of CJ3018 (i.e. CJ2174) was a potent antidepressant in the FST (1 mg/kg), produced little impairment in the PVT yet had a severe impact in performance in the spatial memory task.

The same analysis applies to CJ3095 (1-azabicyclo[3.2.1]octane derivative of CJ2100), which

192 performed almost identically to CJ2126 (Figures 62 and 63). This shows that task sensitivity and muscarinic antagonism proves to be a highly reliable and robust effect. Recall that scopolamine adversely affects both tasks but, as with most antagonists we have shown here, impacts short- term spatial memory at smaller doses needed to impact attention.

Titrating Matching-to-Position (Spatial Memory)

Minimum effective dose in the FST

Figure 63-CJ2150/2174/3095/3100 in DMTS. All produce a significant impairment illustrating the differences/subtleties in task sensitivity. Minimum doses active in the FST had not yet been established when this data was generated.

However, it was known that CJ2174 was active at 1 mg/kg and that CJ3095 was active at 3 mg/kg.

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In conclusion, much work is still left to be done to establish clear trends (if they exist) in structure-activity relationships between our series of muscarinic antagonists. CJ2100 readily antagonized arecoline induced bradychardia, was active in the FST (i.e. increased immobility in the rats), and was silent in both PVT/DMTS tasks (had no effect on spatial memory or sustained attention of to 10mg/kg). This represents the ideal compound that we initially set out to discover.

L687306, while not a novel compound (synthesized originally by Merck for treatment of

Alzheimer's disease) displayed an almost identical pharmacological profile to CJ2100, albeit more potent. Further work in higher animals (preferably primates) is needed to observe if these therapeutic effects are maintained.

The quinuclidine and 1-azabicyclo[3.2.1]octane oxadiazole derivatives showed overall higher potency and activity in the FST than their arecoline counterparts, however generally contributed to detrimental performance in either sustained attention or spatial memory (pharmacological testing is still underway, and therefore all derivatives have not been completely evaluated). One of the most interesting findings was observed when the oxadiazole ring was shifted to the 4- position, which served to essentially abolish all mAChR activity (represented in the FST). This is a clear indication that the precise position of the hydrogen bond/donor atoms (from the oxadiazole ring) is critical for binding (and efficacy) to the muscarinic receptors.

It should be noted that none of the above compounds showed any appreciable subtype selectivity, and therefore can all be considered (for now) nonspecific muscarinic antagonists.

Due to the high potency of the quinuclidine and 1-azabicyclo[3.2.1]octane compounds the poor cognitive performance is likely due to a M 2 (key presynaptic autoreceptor mediating the inhibition of hippocampal and cortical ACh release) blockade. Although this hypothesis will need to be validated after more in vitro data (affinity/efficacy) are generated by the Roth

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Laboratory (PDSP). 15 This represents the primary challenge of this project when attempting to target the orthosteric site of the mAChRs. There is a delicate balance between mAChR potency/efficacy and antidepressant activity/cognitive load.

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Supplementary Figures

Figure 64: QNB causes severe detrimental effects in both cognition and sustained attention in the PVT and DMTP tasks.

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Figure 65 -CJ3095 discriminated to arecoline by itself, indicative of agonist character.

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Appendix A. Current Trends in Drug Design Targeting the Muscarinic Receptors

Background and Targeting of the Allosteric Site (A.1): The mAChRs are a subfamily of GPCRs that have been shown to play a central role in human physiology, regulating heart rate, smooth muscle contraction, glandular secretion, and fundamental functions of the CNS and peripheral nervous system. 1 The pharmacological characterization of mAChRs is not a straightforward task due to the high level of sequence conservation within the orthosteric binding site across all five mAChR subtypes. As a consequence, there are very few orthosteric agonists and antagonists that exhibit high selectivity for one subtype to the relative exclusion of others.

The traditional approach to pharmacological delineation of which mAChR governs a given

O response has thus been to use a NH combination of compounds, generally O N N NH O N antagonists, to build up a receptor N N O NH 2 profile. The M 1 mAChR is typically

O N N + defined as having high affinity for O N N I- 4-DAMP AQRA-741 AF-DX 384 pirenzepine and 4-DAMP- (4-

O H H N N diphenylacetoxy-N-methyl-piperidine N N H H Methoctramine O methiodide), 3 but low affinity for O HN 2 O methoctramine and himbacine. N N N O O NH M2 mAChRs have high affinity for H H N N O O N methoctramine, himbacine, AF-DX H H O O Pirenzepine Himbacine PD102807 116 and AF-DX 384 3 and have low Figure 66 : Antagonists utilized to construct a receptor profile for the mAChRs. 201 affinity for pirenzepine and 4-DAMP. 2 A high affinity for 4-DAMP, but low affinity for

2 pirenzipine, methoctramine and himbacine suggests involvement of the M 3 receptor. The

4 presence of the M 4 mAChR can be determined using PD102807 and the toxin, Muscarinic

Toxin 3 (MT3, one of the various toxins isolated from the venom of Dendroaspis snakes). 5 The

M5 mAChR has been quite difficult to identify pharmacologically, however both AF-DX 384 and AQRA741 have the lowest affinity (~10 fold lower) for this subtype than any other (Figure

66).2

A major breakthrough in 2012 occurred when the first mAChR structures (M 2 and M 3) was determined. 6a,b It provided the first molecular view of mAChRs in their inactive conformations

(antagonist-bound), giving insight into the molecular nature of the binding sites for orthosteric and allosteric ligands. To accomplish this Haga et al. 6a first replaced the third intracellular loop

7 with T4 lysozyme (T4L) as initially described for the β 2 adrenergic receptor. The same method was utilized to obtain crystal structures of four other major subclasses of GPCRs such as:

8 9 10 adenosine A 2A receptor, the CXCR4 receptor, the dopamine receptor, and the histamine H 1 receptor. 11 The receptor was crystallized with the inverse agonist R-(-)-3-QNB, and binding properties were shown to be essentially the same as for wild type M 2 indicating the overall architecture was minimally affected by introduction of the T4L domain.

The overall structure of M 2 receptor closely resembles that of rhodopsin. The structure shown below in different orientations with QNB in orange (Figure 67). The receptor has a relatively

6a simple and open extracellular surface resembling that of the D 3 receptor (data not shown.

Interestingly the receptor has network of hydrogen bonds that extends from the binding pocket to the cytoplasmic surface (e in figure). The network appears to be a part of a long aqueous channel extending from the extracellular surface to a depth of ~33A o (from ECL2) (Figure 68, (e)). The

202 channel contains the ligand binding pocket and extends beyond the orthosteric pocket until it reaches a hydrophobic layer that blocks the channel from the cytoplasmic surface (Leu65,

Leu114, and Ile392). Each of these residues is conserved among all mAChRs.

Figure 67 - (a) Overall structure of M receptor. (b) Cytoplasmic surface/QNB binding pocket. 2 (c) Extracellular view into QNB binding pocket. (d) Extracellular view with solvent-accessible surface showing buried QNB binding pocket. (e) Aqueous channel (green) extending from extracellular surface to TM core. Ordered water molecules shown in red. Adopted from Haga et al . Nature . 2012, 482(7386), 547-551

QNB binds within a buried pocket surrounded by side chains of TM3-7 (a-c Figure 68) with aromatic residues encasing the amine; essentially forming a lid over the ligand. Asn 404 and

Asp103 contribute to the orientation of the ligand in the hydrophobic pocket by forming key hydrogen bond interactions with the carbonyl/ hydroxyl moieties or a charge-charge interaction

203 with the 1-azabicyclo[2.2.2]octane core (pKa~9.6), protonated form binds to the receptor) respectively. The amino acids that form the binding pocket are identical in all five mAChRs except for Phe181, which extends downward from ECL2 and interacts with a phenyl ring on

QNB. The other mAChRs have a leucine in the same position--potentially a subtle difference to

Figure 68 - (a), (b) Two views of QNB binding pocket. Amino acids within 4A o=blue sticks. (c) A representation of QNB binding interactions. Blue dashed lines indicate potential hydrophobic interactions, red lines indicate polar interactions Adopted from Haga et al . Nature . 2012, 482(7386), 547-551

exploit in drug design. It remains to be determined in which pose ACh binds to the M 2 receptor or to the G protein complex.

6b Shortly thereafter the M 3 receptor structure was published by Kruse et al. . The M3 receptor has been shown to control many physiological processes such as: smooth muscle contraction,

204

Figure 69 - (b) M 2 (orange) and M 3 (green) are similar in overall structure. Tiotropium is shown as yellow/red spheres. (c) Intracellular surfaces shows slight divergence in the cytoplasmic end of TM5. (d) Extracellular surfaces show less deviation. (e) Solvent accessible surface bound to tiotropium (tyrosine lid outlined in red). (f) M 3 receptor structure overlay on other four mAChR subtypes. Poorly conserved regions are shown larger backbone diameter. Allosteric and orthosteric sites are indicated in red/blue respectively. Figure adopted from Kruse, A. C. et al . Nature . 2012 , 482(7386), 552-556

glandular secretion, regulation of food intake, learning and memory, and proper development of

the anterior pituitary gland. 12a,b,c,d,e

As seen from Figure 69 (b), the overall structure of M 3 is quite similar to that of M 2, even the intracellular loops (ICL) 1/2, and extracellular loops (ECL) 1/2/3--which share similar folds despite lacking similar amino acid sequences (b/f). Similar to the M 2 receptor, the long

hydrophilic channel exists in which the orthosteric binding site is located. The M 3 structure was crystallized in the presence of tiotropium (brand name Spiriva, for treatment of asthma/COPD), a

205

6a, 13 potent mAChR inverse agonist similar to QNB utilized in the M 2 receptor .

Since QNB and tiotriopium are similar in structure it is not surprising to observe that they both bind in almost identical poses within the orthosteric site (b, Figure 70) and is once again covered by an aromatic "lid" as in the M 2 receptor (Y148, Y506, and Y529) (e). The ligand has ample hydrophobic contacts with the receptor and is blocked from exposure to solvent. Like M 2,

N507 (N404 in M 2) appears to form hydrogen bonds with both the carbonyl and hydroxyl of tiotropium, while D147 (D103 in M 2) forms the charge-charge interaction with the protonated quinuclidine. The group notes the subtle difference mentioned above, Phe181 in M 2 vs. Leu225, which could potentially create a pocket in M 3 not found in M 2. The second subtlety is a 2.8A

Figure 70- M2 is shown in orange and M 3 green with tiotropium and QNB shown in yellow/cyan respectively. (a) Tiotropium binding site in the M 3 receptor. (b) Structures of tiotropium and QNB. Both ligands adopt a similar binding pose within the receptor. (c,d) There is a sequence difference between M (Phe)/M (Leu) near the binding site 2 3 producing a larger binding pocket in M 3. (e,f) A shift in Tyr529 is observed in M 3

potentially arising from a sequence difference in TM2 (Tyr80 in M 2 and Phe124 in M 3). Figure adopted from Kruse, A. C. et al . Nature . 2012 , 482(7386), 552-556

206 shift of Tyr529 (e, below) relative to the M 2 position which they attribute to the difference in

6b TM2 (Phe124 in M 3 vs. Tyr80 in M 2) (f, below). It should be noted that while this position on

TM2 is not part of the orthosteric pocket but is relatively close in position to a possible allosteric binding site. 6a

Kruse et al .6b then performed molecular dynamics simulations in attempt to elucidate the method in which tiotropium binds/dissociates from the M 2 and M 3 receptors, as had previously been accomplished with the β-adrenergic receptor. 14 The simulations indicate that tiotropium pauses at an alternative binding site in the extracellular space, which corresponds to an allosteric site previously identified (Figure 71). 6a This finding is consistent with other studies showing that

15 orthosteric ligands can also act as allosteric modulators at the M 2 receptor. It was also

Figure 71 -Simulations suggest that binding/dissociation of tiotropium pathway for both receptors involves a "metastable" state in which the compound pauses in the extracellular vestibule. (a) As tiotropium dissociates it pauses in the extracellular vestibule--spheres represent the ligand's tropane atom at successive points in time. (b) The tiotropium placed in solvent it binds the same site. (c) Free energy coordinates for binding/dissociation. (d) Common binding poses for tiotropium in the extracellular vestibule. Figure adopted from Kruse, A. C. et al . Nature . 2012 , 482(7386), 552-556

207

13 found by Barnes et al . that tiotropium dissociates more slowly from M 3 than M 2--named

"kinetic selectivity"--for M 3 despite similar in vitro binding affinities for both subtypes.

Simulations with tiotropium bound showed a portion of the extracellular loop 2 (ECL 2) was more flexible in M 2 than M 3, and the extra mobility apparently disrupts a hydrophobic cluster surrounding one of the thiophene ring, facilitating movement of the aromatic "lid" over the orthosteric site and clearing a way for the molecule's exit of the binding pocket.

16 It was not until 2016 that the M 1,4 crystal structures were published that revealed differences in the orthosteric and allosteric binding sites that could contribute to drug selectivity at the mAChRs. Once again both structures were crystallized with the same methodology employed above (T4L fusion) in the presence of tiotropium, to stabilize the inactive state of the receptors.

Certain mutations were necessary to improve diffraction but the authors confirmed that binding affinities of the fusion construct vs. wild type to QNB (for M 1), ACh, N-methylscopoalmine

(NMS, for M 4), or tiotropium were not altered.

As expected due to the high sequence homology, the inactive states for M 1,4 were similar to that of previously published M 2,3 . However, subtle differences existed on the extracellular and intracellular sides. The authors note that the areas where the most noticeable difference were observed are solvent accessible or involved in crystal packing interactions. 16

As seen from the figure, M 1-M4 are structurally very similar (Figure 72, a), and the ligands

(QNB/tiotropium) are oriented almost identical to each other in the orthosteric binding site (e).

While the binding mode similarity is not a surprise due to the conservation of residues in the orthosteric site, the authors note that this is not a predictor of differences in tertiary structure. In fact, the group identified a change in the rotamer of D112(3.32) of the M 4 receptor (f and g) that is conserved throughout the mAChRs and serves as partner to the charge-charge interaction with

208

Figure 72 -(a) Overall view of M 1-M4 receptors aligned to the M 3 receptor (M 1 in green, M2 in yellow, M 3 in orange, and M 4 in blue). Tiotropium is colored according to element. (b,c) Comparison of views from extracellular (b) vs. intracellular (c) side. (d) M 1/M 4 residues involved in tiotropium binding, black dashed lines indicate a bidentate hydrogen bond between Asn(6.52) and tiotropium. (e) Tiotropium adopts an almost identical pose

in the M 1,2,3 receptors and similar to that of QNB in M 2 receptor. (f) Comparison of

orthosteric site in the M 1-M4 receptors. Rotameric change of Asp112 (3.32) is stabilized by H-bonds. Figure adopted from Thal, D. M., et al . Nature , 2016 , 531, 335-340.

the protonated quinuclidine. 17 This shift points the residue away from tiotropium and causes

shifts of Y439 (7.39), Y443 (7.43), facilitating the formation of a network of hydrogen bonds between D112 and S85(2.57)/W108(3.28)/Y439(7.39), and Y443(7.43) which is distinct from all other mAChRs (g).

209

Allosteric Modulator Design for the mAChRs (A.2):

Given the high degree of sequence homology within the mAChR orthosteric site, it is no surprise that many ligands designed for the orthosteric site have largely been unsuccessful in the clinic due to unwanted cholinergic side effects--especially due to over stimulation of M 2 and M 3 in the periphery. This has led to an increase in interest in development of allosteric ligands for mAChRs to create more subtype selective ligands targeting less conserved regions of the receptor. The approach is advantageous since it offers the potential for achieving specific modulation of GPCRs that (as of now) can still not be achieved through targeting of the orthosteric site. Mechanistically this can be explained in two ways: 1) because of lower sequence conservation in allosteric sites, modulators can be identified with higher affinity for one subtype over others and 2) subtype selectivity can also arise from cooperativity instead of affinity

(termed "absolute subtype selectivity,"). 18 In addition, an allosteric effect only occurs when the endogenous ligands are present (ACh in the case of mAChRs) therefore better mimicking physiological regulation of receptor activity. 6a Finally, allosteric effects are saturable (they exhibit a "ceiling" effect,19 which is particularly useful in situations where there is a danger of overdose.

All five mAChRs possess at least one, 20 and likely two 21 extracellular allosteric binding sites for small molecules, and numerous efforts have been made since the 1990s to try and understand the nature of these sites. The most important challenge in this effort remains the ability to detect/quantify the diverse number of possible allosteric effects that can arise when two ligands occupy a receptor simultaneously. 2 The binding of an allosteric ligand to its site will change the conformation of the receptor, which means the positioning of the orthosteric sites and other potential receptor-ligand/protein interfaces could also change.

210

The allosteric concept was coined by Monod, Wyman, Changeux, and colleagues 22a,b with the first model, the Monod/Wyman/Changeux (MWC) model, built on the premise of a conformational selection mechanism. In this hypothesis, the protein is proposed to exist in a spontaneous equilibrium between active/inactive states in the absence of ligand. Upon ligand binding (orthosteric or allosteric modulator) it would stabilize one conformation at the expense of another. In other words, the binding of an allosteric ligand is dependent upon the topology of the orthosteric site and other potential receptor-ligand/protein interfaces that can alter the binding affinity and/or signaling efficacy of the orthosteric ligand either positively or negatively. The effect that the presence of one ligand has on the other is known as allosteric interaction. 23

The first considerations of allosteric mechanisms between ligands and GPCRs dates back to the development of the "ternary complex" model in attempt to explain reciprocal effects that orthosteric ligands/G-proteins had on each other. 24a,b Around this same time studies were being conducted on ACh with mAChRs and gallamine,25a,b where it was found that gallamine produced a greater degree of antagonism toward carbachol vs. ACh. It was concluded that an antagonist of this type allosterically altered the affinity of the agonist for its binding site rather than effecting the agonist-receptor interaction. 25a

The simplest allosteric model makes the assumption that

binding of an allosteric modulator only affects only affinity of

the orthosteric ligand (called the allosteric ternary complex

model -ATCM, in red, Figure 73). The interaction is governed

by concentration of each ligand (orthosteric/allosteric), the

equilibrium dissociation constants (K A and K B), and the Figure 73 from P. Keov et al. Neuropharmacol. 2011, 60(1), cooperativity factor (α), a measure of both magnitude and 24 -35 .

211 direction of the allosteric interaction. 24b, 25b This can be determined empirically through isothermal titration calorimetry (ITC). 26 Values of α >1 are indicative of positive cooperativity

(i.e. allosteric modulator promotes binding of the orthosteric ligand); values <1 (but greater than zero) indicate negative cooperativity (i.e. the modulator inhibits binding of the orthosteric ligand); a value of 1 indicates neutral cooperativity.

However, functional assays, to determine efficacy, have uncovered certain modulators that cannot be explained by the simple ATCM as they can affect signaling capacity of orthosteric agonists 27 and act as orthosteric agonists themselves. 28 Additionally, the model is limited since it does not account for the isomerization of a GPCR between active/inactive states. As a result the two state model 29 was generated to explicitly incorporate the isomerization of a receptor between active (R*) and inactive (R) states (determined by an isomerization constant (L)), and introduces additional coupling constants to describe selective stabilization of these states by either orthosteric or allosteric ligands (i.e. binding cooperativity parameter γ and activation cooperativity parameter δ). 29,30

Allosteric regulation of GPCRs was first observed for the M 2 receptor which is one of the first and most extensively characterized model systems for the mAChRs. 6a As seen from Figure 74

(a), the difference between M 2,4 are shown as green residues mapped onto the inner surface of the M 2 receptor (blue) while QNB is represented by orange. The orthosteric pocket and transmembrance core are highly conserved whereas greater diversity is observed among the

ECLs and extracellular end of transmembrane segments. In fact, mutagenesis studies and chimaeric receptor studies have confirmed the involvement of these residues in the binding of allosteric modulators some of which are listed below. In (b,d) the residues are primarily located

212 at the ECL 2 and the amino terminus of TM7 at the entrance to the orthosteric site. Trp422(7.35) appears to form an edge-to face π-π interaction with Tyr403, which is part of the aromatic cage

Figure 74 -Allosteric binding in the M 2 receptor. (a) Differences between M 2/M 4 shown as green residues mapped onto M surface. (b) Yellow color indicates mutations that alter 2 allosteric binding. (c) Other view of possible allosteric sites on the M 2 receptor--notice the locations are near the path to the QNB binding pocket. (d) Trp422 (yellow), implicated in the binding of allosteric modulators, forms an edge-to-face interaction with Tyr403 (part of the aromatic cage). Figure adopted from Haga et al . Nature . 2012, 482(7386), 547 -551 .

implicated in orthosteric ligand binding (d). Therefore it is feasible to conclude that allosteric ligands binding this site affect rates of association and dissociation of orthosteric ligands.2, 6a

213

The eldest and most well studied allosteric modulators of the mAChRs are the neuromuscular blocking agents (gallamine and alcuronium), and a series alkyl-bis-quaternary ammonium compounds such as (Obidoxime, C7/3-phth, and W84) (Figure 75). Early studies done at

N+ N N

O N N O

N + O N gallamine OH alcuronium

HON NOH N O N

Obidoxime N N N N + O O O O N N N N C7/3-phth Diemethyl-W84 O O O O

Figure 75-Neuromuscular blocking agents as allosteric modulators of the mAChRs

native guinea pig atrial M 2 mAChRs with gallamine revealed that antagonism of orthosteric agonists responses approached a limit at the highest modulator concentrations resulting in curvilinear Schild regressions (for curvilinear Schild regressions see TP Kenakin and D.

Beek). 25a,31 Alcuronium was found to be the first allosteric enhancer of the binding of an orthosteric mAChR ligand 32a,b even though it was shown to bind at the same site as gallamine and the alkyl-bis-quaternary ammonium compounds. 33 Interestingly alcuronium enhances NMS

34 binding at the M 2,4 receptors, but inhibits it in the M 1,3,5 subtypes. Studies have also been done with regards to the native agonist, ACh, and it has been shown that positive, negative, and

214 neutral cooperativity is possible depending upon the mAChR subtype and the modulator

18, 35a,b chosen. W84 was the first radiolabeled allosteric modulator of the M 2 receptor designed and has proven useful for validation of the ATCM and allowing for screening of other common site modulators via competition binding assays (Figure 75).2

Shortly thereafter a second allosteric site was found by Lazareno et al. 36 and a number of indolocarbazole derivatives of staurosporine (a potent non-selective inhibitor of protein kinases, Figure 76) were shown to exhibit positive, negative, or neutral cooperativity, yet did not bind to the common allosteric site (e.g. gallamine and brucine). Other Figure 76 : Staurosporine classes of molecules such as the commercially available neurokinin receptor antagonists were also shown to interact with gallamine/strychnine in a non-competitve manner, while competing with staurosporine and its derivatives.

After the identification of the first allosteric modulators, reports of other putative allosteric agonists of GPCRs began to increase. McN-A-343, originally designed as an orthosteric ligand

37 back in the 1980s (functionally selective for M 1), was found by Birdsall et al. to have allosteric

O O O N N NH

AC-42 Cl McN-A-343 NH

N O Cl N O N N N H F AC-260584 N-desmethylclozapine

Figure 77-Early examples of mAChR allosteric agonists

215 interactions with scopolamine in a radioligand binding assay on rat atrial M 2. It was later shown to bind both orthosteric and allosteric sites. 38 AC-42 was shown to have high functional selectivity for M 1, despite showing almost equal binding affinity for all five mAChRs leading to the assumption that it was binding allosterically (confirmed by Landmead et al. ). 28 AC-260584, a close but more potent analogue of AC-42, was also shown to act allosterically at M 1 perhaps indicating a potential structure activity relationship. Spalding et al. 39 and Sur et al. 40 demonstrated that N-desmethylclozapine, a metabolite of the antipsychotic clozapine, was a functionally selective M 1 agonist that potentially acted through an allosteric mechanism (Figure

77).

Despite advances in GPCR crystallography in solving the structures of the inactive forms of

6a,b M1-M4 (Haga et al ., Kruse et al . as above) no structure has currently been determined for an allosteric modulator bound to a GPCR, perhaps due to the face that underlying molecular mechanisms of how such modulators affect signaling may not be evident from a single static structure. 41 Nevertheless, atomic-level molecular dynamics simulations of several modulators bound to the M 2 receptors have been performed (used in the past as a model for allosteric modulation, see Lazareno et al. ). 42 Dror and colleagues included water, lipids, and ions surrounding the receptor, allowed conformational changes in both receptor and ligands, and made no previous assumptions about where the modulators would bind. (Figure 78, a) shows the simulated binding process of C7/3-phth to a naked M2 receptor (i.e. no endogenous ligand). The modulator binds in an area ~15A 0 from the orthosteric site according to Dror et al . simulation

(Figure 78, b), and the two quaternary ammonium groups/heptyl linker remained tightly bound to the receptor (c,e). 41 The phthalimide groups were highly mobile as evidenced by a 40 snapshot

216 simulation (Figure 79). W84 bound in almost identical fashion (Figure 82, c) and verified already existing mutagenesis data (see Prilla et al . and Huang et al. among others)43a,b for C7/3-phth and

Figure 78 -Binding simulations of diverse allosteric modulators. (a) C 7/3-phth diffuses freely before binding the M 2 extracellular vestibule. (b) Binding pose of C 7/3-phth (purple). Residues in cyan have been shown to reduce binding greater than five-fold all make contact with the ligand. (c) Binding poses for dimethyl-W84, gallamine, strychnine, and alcuronium. (d)

Representation of the ammonium bindign centres. (e) Bound locations of ammonium groups--all occupy at least one of the centres. However, the positon of the centre varies slightly as the adjacent residues reposition to accomodate the ligand. Figure adopted from Dror, R. O. et al . Nature . 2013, 503, 295-299

W84. Binding simulations were extended to other well known modulators including: gallamine, alcuronium, and strychnine, and all were found to bind to the extracellular vestibule.

Surprisingly, despite their vast structural diversity, the modulators also had similar binding interactions in one (or both) of the common allosteric binding sites." 41

217

C7/3-phth mobile phthalimide arms

O O N N O O

N N

Figure 79 -40 snapshot simulation of C 7/3-phth binding with a stable core while the phthalamide arms are highly mobile. Figure adopted from Dror, R. O. et al . Nature . 2013, 503, 295 -299

Surprisingly, despite their vast structural diversity, the modulators also had similar binding interactions in one (or both) of the common "binding centers." 41 The centers are defined by a pair of aromatic amino acids (Tyr 177-ECL 2 and Trp 422-7.35) that form cation- interactions with the quaternary ammonium (Figure 78, d and e). The group acknowledges that this finding is in disagreement with previous studies that suggest that the aromatic residues interact with the aromatic group of the modulator.

Over the past decade there have been monumental advances in crystallographic information of GPCRs due to improved crystallization methods. Now more than 100 X-ray structures are available. 44 Because of this efforts have largely shifted from traditional HTS--requiring large number of chemical compound libraries, and hence requiring significant resources--to less expensive virtual screening (VS) or fragment-based drug design (FBDD). 45 Structure-based virtual screening involves the docking of chemical libraries into an already published X-ray structure or homology model followed by a ranking score of the docked molecules. This serves

218 as a template for either the selection of potential lead molecules or leads that can be evaluated via other methods. 46 This has led to the identification of numerous allosteric modulators summarized in Table 17.

Receptor Modulator Binding Functional Reference M1 brucine + + Birdsall et al . 1997 47 KT5720 + nd Lazareno et al . 2000 36 BQCA + + Ma et al . 2009 48 VU0119498 nd + Bridges et al . 2009 49 VU0027414 nd + Marlo et al . 2009 50 VU0090157 + + Marlo et al . 2009 50 VU0029767 + + Marlo et al . 2009 50 VU0366369 nd + Bridges et al . 2010 (a and b) 51a,b Lu AE51090 nd + Sams et al. 2010 52 MK7622 (=PQCA) nd + Kuduk et al . J. Med. Chem. 2011 53 VU0405652 nd + Reid et al . 2011 54 VU0456940 nd + Tarr et al . 2012 55 VU0413162 nd + Poslusney et al . 2013 56 VU0448350 nd + Melancon et al . 2013 57 4-phenylpyridin-2-one derivatives + + Mistry et al . 2016 58 MT3 nd - Jolkkonen et al . 1994 and Olianas et al. 1999 59a,b MT7 - - Olianas et al . 2000 and Onali et al . 2005 60a,b Staurosporine - - Lazareno et al . 2000 36 Tacrine - nd Fang et al . 2010 and Potter et al . 1989 61a,b M2 (-)eburnamonine + nd Jakubik et al . 1997 62 LY2033298 + + Valant et al . 2012 63 LY2119620 + + Croy et al . 2014 and Kruse et al . 2013 64a,b W84 nd - Lullman et al . 1969 65 Gallamine nd - Clark and Mitchelson. 1976 66 C7/3-phth nd - Lanzafame et al. 1996 67 Alcuronium - nd Jakubik et al .1997 62 Strychnine - - Jakubik et al . 1997 and Birdsall et al . 1995 62,42 Brucine - nd Jakubik et al . 1997 62 WIN-51708 - nd Lazareno et al . 2002 68 WIN-62577 - nd Lazareno et al . 2002 68 dimethyl-W84 nd - Maier-Peuschel et al . 2010 69 M3 Brucine + nd Jakubik et al . 1997 and Lazareno et al . 1998 62,21 N-chloromethyl-brucine + nd Lazareno et al. 1998 21 N-benzyl-brucine + nd Lazareno et al. 1998 21 Brucine-N-oxide + nd Lazareno et al. 1998 21 WIN-62577 + nd Lazareno et al . 2002 68

219

VU0119498 nd + Bridges et al . 2009 49 Alcuronium - nd Jakubik et al . 1997 62 Brucine - nd Jakubik et al. 1997 and Lazareno et al. 1998 62,21 WIN-51708 - nd Lazareno et al . 2002 68 M4 Thiochrome + + Lazareno et al . 2004 18 LY2033298 + + Chan et al . 2008 70 VU0010010 + + Shirey et al . 2008 71 VU0152099 + + Brady et al . 2008 72 VU0152100 + + Brady et al . 2008 72 ML293 nd + Salovich et al . 2012 73 ML253 nd + Le et al . 2013 74 LY2119620 + + Croy et al. 2014 and Kruse et al. 2013 64a,b VU0467154 + + Bubser et al. 2014 75 MT3 nd - Jolkkonen et al . 1994 and Olianas et al. 1999 59a,b Alcuronium - nd Jakubik et al . 1997 62 M5 VU0119498 nd + Bridges et al . 2009 49 VU0238429 + + Bridges et al . 2009 49 VU0365114 nd + Bridges et al . 2010 51a VU0400265 nd + Bridges et al . 2010 51a VU0467903 nd + Gentry et al . 2013 76 ML380 + + Gentry et al . 2014 77 VU0483253 nd - Gentry et al . 2013 76 VU6000181 nd - Kurata et al . 2015 79

Table 17-adopted and modified from Bock, A. et al . Neuropharmacol. 2018, 136(C), 427-437

While much is known about the structure and binding affinities for some groups of allosteric modulators, very little is known about the conformational changes that the compounds induce on

80 the receptor. Kruse et al. was able to successfully obtain a crystal structure of the human M 2 receptor with an agonist bound (iperoxo) stabilized by a G-protein mimetic camelid antibody fragment simultaneously bound to the positive allosteric modulator LY2119620 (Figures 80/81 below). 64b The group once again confirmed that the allosteric modulator binds to the extracellular vestibule. 2 Notice in Figure 80 (b) how the receptor partially encloses the antagonist QNB, while the active form closes the agonist almost entirely so it becomes buried in the receptor. Iperoxo

220

adopts a bent conformation ( c) and TM6 has a large inward movement (~2A, e) that allows for

formation of a hydrogen bond between iperoxo and Asn404 (d). This inward shift of TM6 allows

for Tyr403(6.51) to form a hydrogen bond with 104(3.33), which then forms another hydrogen bond with Tyr426(7.39) (f) generating the tyrosine "lid" that closes over the agonist (Kruse et al .). 64b

Figure 80-(a) Ligands used for crystallization of inactive/active sites of M 2 receptor. (b) In active conformation, the receptor partially encloses QNB (blue, left), while active conformation encloses the agonist entirely (orange, right). (c) Conformational changes in binding pocket, with changes denoted with red arrows. (d) Inward motion of TM6, which is responsible for formation of hydrogen bond between Asn404(6.52) and iperoxo. (e) TM6 pivots to activate the receptor, which moves inward in the orthosteric site and outward at the intracellular side. (f) Closure of

the binding pocket allows formation of hydrogen bonded Tyrosine lid. Figure adopted from Kruse, A. C., et al . Nature , 2013 , 504(7478), 101-106.

221

LY2119620 was bound directly above the agonist (Figure 81, b) with its aromatic rings stacked between Tyr 177 (ECL2) and Trp 422(7.35). The group found that M 2-iperoxo-

LY2119620 complex was quite similar to that of the agonist-M2 (hinting at a pre-formed allosteric site when an agonist is present) despite the significant closure of the extracellular vestibule (d).

Other strategies for designing selective muscarinic ligands have included hybrid or dualsteric compounds. Since it was demonstrated that the less conserved allosteric site (located in the extracellular vestibule) was relatively close to the conserved orthosteric site, the possibility of a

Figure 81 -(a) Structure of M 2 receptor occupied by the agonist iperoxo in complex with the PAM LY2119620. (b) The allosteric ligand binds to extracellular vestibule above the orthosteric agonist. (c) Polar contacts along with aromatic stacking (with Trp422(7.35) and Tyr177 (ECL2))

play a major role in binding of LY2119620. (d) The M 2 receptor undergoes substantial conformational changes on the extracellular surface resulting in contraction of the extracellular vestibule and allowing the creation of a binding site that fits tighly around the allosteric modulator. Figure adopted from Kruse, A. C., et al . Nature , 2013 , 504(7478), 101 -106.

molecule simulataneously interacting with both binding sites became a reality.

This was first investigated with Iper-8-napth, synthesized by Matera et al ,81 and was found to be a partial agonist at the M 2 receptor. In the "dual-steric" binding pose, the iperoxo moiety is

222 located in the orthosteric site and the naph-group in the allosteric which allows for functional activation of the G-protein and, hence, cellular signaling. However, in the allosteric binding pose the entire ligand is in the extracellular vestibule region and stabilizes the receptor in the inactive conformation. 82 The bitopic binding pose was validated through radioligand binding assays (WT and mutant receptors in which Tyrosine of the aromatic lid was replaced with Ala), downstream signaling assays, and receptor docking simulations.

- - Br Br N O N N O Iper-8-naph O O N

O O

R O O - 1 OH Br R 1 NH N N O N n N O n=4,6,8,10

BQCA R1=H, R2=OCH 3 BQCAd-Iper BQCAd R1=F, R2= H

O O O O Br - F F O N NH N H n O N N n=4,6,8,10

N O N BrH - O BQCAd-ACh BQCA-rigid spacer-Iper Figure 82-Dualsteric ligands as allosteric modulators

Merck used this same methodology when exploring the BQCA (N-benzyl quinolone

83 carboxylic acids) when designing M 1 dualsteric hybrids first synthesized by Chen et al. and

84 Messerer et al . They were able to achieve unprecedented M 1 selectivity in which there was no agonism, antagonism, or even potentiation activity for any of the other subtypes up to 100μM.

223

The allosteric modulation was found to be through Tyr179 and Trp400 (common residues of the aromatic "lid") suggesting that the BQCA (and derivatives) were likely binding to the already identified site (Figure 82).48,85

Replacing the flexible alkyl chain with a rigid alkynylbenzene linker did not allow the molecule to bind in a purely allosteric mode such as Iper-6-naph, similar to Iper-8-naph above but 2-carbons short in the linker region, since the linker was shown to be unable to enter the allosteric site according to molecular simulations. Rather, it could only bind in the dualsteric pose and functioned as a full agonist. 86 Other dualsteric ligands have been designed such as: bis tacrine homodimers 87,88 as AChE inhibitors with reduced hepatotoxicity, tacrine-Iperoxo

81 61a hybrids as M 2 selective ligands, and Tacrine-Xanomeline hybrids as activation of M1 receptor (via Xanomeline), PAM via tacrine, all while increasing ACh levels due to the AChE ability of tacrine.

There is still much to be learned about the allosteric modulation of muscarinic receptors. This is likely due to the complex nature of allosteric interactions that require specific interactions of the receptor, orthosteric ligand, and allosteric modulator. Alteration of one of these could potentially lead to a different cooperativity and subsequent skew the interpretation of the drug's pharmacological profile. Additionally, unlike affinity for the orthosteric site that can likely be narrowed to certain key residues responsible for high affinity binding, single residues cannot be identified that determine the type and strength of cooperativity. This is likely due to many domains playing a crucial role in cooperativity. Nevertheless, a vast amount of effort has been put into solving this important problem with some success, and the field remains largely open for new discoveries.

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74) Le. U., Melancon, B. J., Bridges, T. M., Vinson, P. N., Utley, T. J., Lamsal, A., Rodriguez, A. L., Venable, D., Sheffler, D. J., Jones, C. K., Blobaum, A. L., Wood, M. R., Daniels, J. S., Conn, P. J., Niswender, C. M., Lindsley, C. W., and Hopkins, C. R. Discovery of a selective M(4) positive allosteric modulator based on the 3-amino-thieno[2,3-b]pyridine-2-carboxamide scaffold: development of ML253, a potent and brain penetrant compound that is active in a preclinical model of schizophrenia. Bioorg. Med. Chem. Lett. 2013 , 23, 346-350.

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77) Gentry, P. R., Kokubo, M., Bridges, T. M., Noetzel, M. J., Cho, H. P., Lamsal, A., Smith, E., Chase, P., Hodder, P. S., Niswender, C. M., Daniels, J. S., Conn, P. J., Lindsley, C. W. and Wood, M. R. Development of a highly potent, novel M5 positive allosteric modulator (PAM) demonstrating CNS exposure: 1-((1H-indazol-5-yl)sulfoneyl)-N-ethyl-N-(2- trifluoromethyl)benzyl)piperidine-4-carboxamide (ML380). J. Med. Chem. 2014 , 57, 7804-7810.

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Appendix B. Site Identification by Ligand Competitive Saturation (SILCS) Methodology on Novel Muscarinic Antagonists

Background (B.1):

GPCRs are seven transmembrane receptors that represent largest and most physiologically important protein family with over 800 different human genes (>2% of the human genome) coding for them. 1 Crystallization of GPCRs proved to be a daunting task for structural biology for decades but recent technological (GPCR biochemistry/X-Ray crystallography) and experimental techniques (lipidic cubic phase crystallization, protein engineering, new detergents, etc.) over the past ten years have since increased the success rate. 2 The mAChR are subdivided into five subtypes (M 1-M5) and are classified into two classes based on the G-protein coupling:

M1,3,5 to G q/11 and M 2,4 to G i/o . As noted in Appendix A, except for M 5, all three dimensional structures of the other mAChRs have been solved to date. The M 2 structure was obtained for both active and inactive states whereas the others were obtained only in the inactive state.

Nevertheless, it has provided vast structural details that have been exploited to design subtype selective ligands/allosteric modulators. 3

Like other GPCR families the mAChRs showed high sequence homology in their ACh binding sites, especially between M 2,3 , making designing selective orthosteric ligands a challenge

(Figure 83). For instance, the only significant difference between M 2,3 is F181 of M 2 and L225 in

3 M3 in ECL2, which creates a larger binding pocket in M 3 (Figure 83). The tyrosine "lid" is located directly above the ligand binding pocket and divides the solvent-accessible binding pocket into two distinct sites (antagonists were to found to only bind one site). The upper portion of the pocket has been implicated in the binding of allosteric modulators. 4

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Since the 1980s, molecular docking--which predicts the most likely binding conformations of small molecules at the target binding site--is considered one of the most common applications for structure based drug design. 3 Docking calculations typically consist of the following steps:

Figure 83 -adopted from Lee, Y., et al . J. Med. Chem. 2018 , 61(1), 1-46. Key residues are represented as sticks, and the H-bonds denoted by black dashed lines. The

displayed receptor–ligand complexes correspond to (A) human M 2R in complex

with QNB, (B) rat M 3R with tiotropium, (C) human M 1R with tiotropium, (D) human

M4R with tiotropium, and (E) human M 2R.

1) extensive sampling of ligand conformations representing various binding modes at the target binding site; 2) accurate prediction of the interaction energy of the predicted binding conformation; 5 and 3) ranking of the ligand conformation by specific scoring function(s) via a recurrent process until convergence to a solution of minimum energy. 5,6a,b

The Site Identification by ligand competitive sampling SILCS methodology is a drug design technique that maps free energy affinity patterns of specific functional groups at protein surfaces as FragMaps (SILCS-MC). 7 In order to probe occluded ligand binding pockets, small solutes

236 representative of the functional groups, are then subjected to an iterative Grand Canonical Monte

Carlo and Molecular Dynamics (MD) methodology in the presence of water to predict the functional group affinity pattern, termed FragMap. 8,9 The inclusion of MD allows for additional conformational sampling of the probe molecules/water and incorporates protein flexibility into the sampling regimen. This allows the probe molecules and water to sample regions "under" the traditional solvent accessible surface (i.e. occluded LBPs). 9,10 Eight representative solutes with different chemical functionalities: benzene/propane (non-polar), acetaldehyde/ methanol/formamide/imidazole (neutral molecules that participate in hydrogen bonding), and methylammonium/acetate (probes for charged donor/acceptors, respectively) were used to calculate the FragMaps for the entire protein including the orthosteric site. Binding affinities of the ligands of interest are estimated using Ligand Grid Free Energy (LGFE) in conjunction with

SILCS Monto Carlo (SILCS-MC) conformational sampling of the ligands in the field of the

FragMaps. The most favorable LGFEs from the SILCS-MC sampling are compared to experimental binding affinities ( in vitro radiolabeled affinity assays).

Results:

SILCS simulations and SILCS-MC analysis was applied to Muscarinic acetylcholine receptors, which have four have crystal structures in the protein database (M 1-M4) (Table 18).

Representative inactive crystal structure for the four muscarinic receptor (M1-M4) given in Table

17, were selected for analysis. Previously calculated SILCS FragMaps for each protein were used for SILCS MC on a collection of compounds supplied by Chad Johnson from the Andy

Coop laboratory. Exhaustive SILCS-MC docking was performed on each ligand from which the

LGFE scores were obtained.

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Table 18) Information on the muscarinic GPCR structures subjected to SILCS-MC docking.

Receptor PDB ID STATE

M1 5CXV Inactive

M2 3UON Inactive

M3 5ZHP Inactive

M4 5DSG Inactive

SILCS-MC docking was performed using the exhaustive sampling method targeting the orthosteric site of the receptors. The location of the 10 Å radius sphere into which the ligands are initially placed was based on selected atoms of the ligands found in the PDB files (Table 19).

Note that the ligand placement sphere of each mAChR was near a key Tyr residue that has been observed to act as a "lid" to allow access to the ligand binding pocket. SILCS-MC was performed using the published protocol from which the LGFE and LE scores were obtained along with the minimum energy docked conformations. The 133 compounds that were analyzed are shown below followed by the LGFE scores (i.e. predicted binding affinity).

Table 19) Information on the ligand atoms used to define the ligand-placement sphere for SILCS MC.

PDB Ligand Centroid atom X,Y,Z coordinates ID

5CXV [(1R,2S,4R,5R)-9,9-Dimethyl-3-oxa-9- HH of TYR 259 97, 98.2, 23.3 azoniatricyclo[3.3.1.02,4]nonan-7-yl] 2- hydroxy-2,2-dithiophen-2-ylacetate

3UON Quinuclidinyl benzilate HH of TYR 248 97.7, 95.4, 50.9

5ZHP (1R,2R,4S,5S,7s)-7-({[4-fluoro-2-(thiophen- HH of TYR 228 0.927, -5.369, -5.14 2-yl)phenyl]carbamoyl}oxy)-9,9-dimethyl-3- oxa-9-azatricyclo[3.3.1.0~2,4~]nonan-9-ium

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5DSG Tiotropium HH of TYR 245 -3.8, 4.1, -8.9

It should be noted that these values should be correlated experimentally via radiolabeled binding assays, which are currently in progress in collaboration with Bryan Roth (UNC Chapel Hill

Psychoactive Drug Screening Program, PDSP).

H O N O O N C l O O N B r N S C l N N N O O H N N O N N C l Atropine C l C J1193.1 C J1193.2 C J11 9 4 C J2009 S u lfa te F O N C l O H O N B r N O N C l N N N N N N N N C l O C J2013 C J2013.1 C J2013.3 C J2029 C J2046

H O H N N O O N S O O N S N S O N N N O N N F N N S C J2054 C J2070 C J2087 N C J2090.1 C J2091

O N O N O N O N N H 2 N N N N N O S N N N N N N C J2094 C J2099 C J2101 C J2108 C J2126 N O M e O M e O O O O O

N N N N N H H H H N N N N N H H H H C J2137 C J2139 C J2142 C J2145.1 C J2145.3

O N O N O N O N O N N N N N N

N N N N N H

C J2150 C J2159.2 C J2159.3 C J2161.2 C J2162.1 O N O N O N O N

N N N N O N

N N N N N N C J2162.2 C J2164.1 C J2164.2 C J2164.3 C J2165.1 O N O N N O N O N O N N N N N N N N N N C J2165.3 C J2166.2 C J2099 CJ2173.1 CJ2174 O N O N O N N S N S N N N N N O N O N N N N

C J2181 C J2182.1 C J2182.2 C J2184 C J2184.3 O M e N S N O N N O N P h N O O N N N N N N C J2186.1 C J2186.1 C J2200 C J3006 C J3007

239

N N O N O N O N O O O N N N N N N N N N N N N N N N H CJ3009 CJ3011 CJ3012 CJ3016 CJ3017 CJ3018

N O O N N H H OH N N N O N N N N N O O O CJ3022 H CJ3029 CJ3046 CJ3053 CJ3056

OH Ph Ph O N Ph Ph O O O O N O O N N O N N N CJ3063 H H N N N N H CJ3071 CJ3077 CJ3082 CJ3065 CJ3070

N N N N N N N N O O O O N O N O N O N O N N N N

N N N N N N N N CJ3094 CJ3095 CJ3098 CJ3100 CJ3109 CJ3110 CJ3114 CJ3116

Ph N N N N N N O O O N N O N O N O N O N O N N N N

N N N N N N N N CJ3120 CJ3120.1 CJ3117 CJ3118 CJ3122 CJ3123 CJ3125.1 CJ3125.2

N N N N O N N N O N O O O N O N N N O N N

N N N N N N N CJ3127 CJ3128 CJ3129 CJ3130 CJ3132 CJ3133 CJ3134

N N N N N N O O O N O N O N N O N N O NH

N N N N N N N CJ3135 CJ3136 CJ3137 CJ3138 CJ3139.1 CJ3139.2 CJ3141

N N N N N N N N N N N N O O O N O N O N O N O N O N N N O N

N N N N N N N N N CJ3142 CJ3143 CJ3144 CJ3145 CJ3146 CJ3147 CJ3148 CJ3149 CJ3150 240

Figure 84 -List of compounds to which SILCS-MC was applied to generate LGFE scores

M1 M2 M3 M4 Ligand LGFE(kcal/mol) LGFE(kcal/mol) LGFE(kcal/mol) LGFE(kcal/mol) atropine -8.259 -8.354 -8.304 -9.593 atropineprot -8.166 -7.517 -9.35 -9.474 CJ1193_1 -9.172 -7.318 -7.051 -8.274 CJ1193_2 -8.962 -7.225 -7.03 -8.315 CJ1193 -9.172 -7.318 -7.051 -8.274 CJ1194 -8.057 -6.402 -6.598 -8.194 CJ2009prot -7.858 -6.36 -8.381 -7.892 CJ2013_1 -7.481 -6.385 -7.042 -7.647

241

CJ2013_2 -7.27 -6.567 -7.159 -7.862 CJ2013 -7.481 -6.385 -7.042 -7.647 CJ2029 -6.359 -6.243 -7.61 -7.181 CJ2029prot -6.374 -6.144 -7.249 -7.279 CJ2046 -8.092 -6.502 -6.724 -8.573 CJ2046prot -7.432 -6.576 -6.722 -8.353 CJ2054 -7.895 -6.267 -7.773 -7.882 CJ2054prot -7.746 -6.337 -8.342 -8.028 CJ2070 -7.465 -6.064 -8.113 -7.765 CJ2070prot -7.678 -6.089 -7.565 -7.787 CJ2087 -7.658 -7.164 -6.915 -8.189 CJ2090_1 -7.96 -8.552 -10.082 -9.208 CJ2091 -9.618 -8.851 -9.861 -10.34 CJ2094 -9.354 -8.685 -9.515 -8.958 CJ2099 -6.475 -5.935 -6.354 -7.537 CJ2099prot -6.859 -5.886 -6.436 -7.24 CJ2101 -7.441 -6.517 -7.681 -7.505 CJ2108 -7.207 -5.79 -6.553 -7.815 CJ2126_R_prot -7.52 -6.523 -7.709 -8.606 CJ2126_S_prot -7.859 -6.907 -7.686 -8.743 CJ2126 -8.002 -7.144 -7.724 -8.572 CJ2137 -7.073 -5.536 -6.945 -6.792 CJ2139 -6.682 -5.448 -6.063 -7.275 CJ2142 -7.44 -5.558 -6.641 -7.127 CJ2145_1 -6.048 -5.42 -6.815 -7.306 CJ2145_3 -7.319 -5.255 -6.848 -7.359 CJ2150 -8.144 -6.563 -6.942 -8.466 CJ2159_2 -8.369 -7.125 -7.78 -8.415 CJ2159_3 -9.019 -7.51 -9.022 -8.669 CJ2161_2 -9.032 -7.452 -8.274 -8.771 CJ2162_1 -8.102 -6.25 -7.206 -8.335 CJ2162_1prot -7.503 -6.16 -7.025 -8.29 CJ2162_2 -7.45 -5.875 -6.971 -7.936 CJ2162_2prot -7.201 -5.871 -6.785 -7.635 CJ2164_1 -8.308 -7.176 -7.695 -8.039 CJ2164_2 -9.383 -7.646 -8.297 -7.739 CJ2164_3 -8.481 -6.528 -7.622 -8.646 CJ2165_1_R_prot -7.795 -6.777 -8.143 -8.884 CJ2165_1_S_prot -8.636 -7.675 -8.227 -8.382 CJ2165_1 -7.522 -7.151 -8.129 -8.938 CJ2165_3 -8.255 -7.364 -8.064 -8.834 CJ2166_2_R_prot -8.997 -8.405 -9.143 -9.205 CJ2166_2_S_prot -9.288 -8.331 -9.226 -9.449

242

CJ2166_2 -9.264 -7.996 -9.003 -9.174 CJ2168_1 -6.512 -6.092 -6.412 -7.867 CJ2168_2 -7.991 -6.612 -7.341 -8.871 CJ2169_1_R_prot -7.067 -6.495 -7.653 -8.537 CJ2169_1_S_prot -7.785 -6.883 -7.739 -8.203 CJ2169_1 -8.097 -6.516 -7.555 -8.578 CJ2169_3 -7.138 -6.543 -7.625 -8.379 CJ2173_1_R_prot -8.526 -7.863 -8.7 -9.301 CJ2173_1_S_prot -8.288 -8.436 -8.629 -8.308 CJ2173_1 -8.527 -8.213 -8.767 -8.601 CJ2174_R_prot -7.263 -6.423 -8.041 -8.6 CJ2174_S_prot -8.295 -7.108 -7.976 -8.643 CJ2174 -8.755 -6.435 -7.957 -8.887 CJ2181 -7.148 -6.959 -7.759 -7.858 CJ2182_1 -6.814 -5.938 -6.892 -7.765 CJ2182_2 -7.72 -6.726 -7.658 -8.185 CJ2184_3 -7.172 -5.827 -6.971 -7.767 CJ2184 -6.619 -5.523 -6.182 -7.903 CJ2186_1 -7.53 -6.656 -7.291 -8.277 CJ2189_1 -6.28 -6.152 -6.231 -6.355 CJ2200 -7.807 -6.967 -7.985 -8.064 CJ3006 -9.047 -8.435 -8.899 -9.349 CJ3007 -9.317 -8.89 -9.385 -8.124 CJ3009 -6.949 -6.379 -7.937 -8.887 CJ3011 -7.133 -6.078 -6.672 -7.413 CJ3012 -7.248 -6.694 -7.565 -8.468 CJ3016 -6.938 -6.406 -7.334 -7.892 CJ3017_R_prot -7.642 -6.383 -7.497 -8.595 CJ3017_S_prot -7.642 -6.383 -7.497 -8.595 CJ3017 -6.927 -6.386 -7.465 -8.514 CJ3018 -8.317 -6.832 -7.22 -8.568 CJ3022 -7.721 -6.928 -7.755 -7.294 CJ3029 -8.093 -6.473 -7.442 -8.122 CJ3046_R_prot -8.096 -6.645 -8.109 -7.949 CJ3046_S_prot -8.001 -6.955 -8.425 -7.927 CJ3046 -8.096 -6.661 -8.036 -8.054 CJ3053_R_prot -6.871 -7.241 -8.398 -9.869 CJ3053_Race_prot -8.338 -7.648 -9.132 -8.586 CJ3053_S_prot -8.338 -7.648 -9.132 -8.586 CJ3056_RACE -6.71 -6.968 -8.041 -7.889 CJ3056_R -6.72 -6.602 -8.276 -8.181 CJ3056_S -6.71 -6.968 -8.041 -7.889 CJ3063_R_prot -6.738 -6.529 -7.17 -8.136

243

CJ3063_S_prot -7.112 -6.478 -7.128 -8.002 CJ3063 -7.625 -6.409 -7.293 -7.51 CJ3065 -7.437 -7.601 -8.328 -7.684 CJ3070 -6.614 -6.865 -7.679 -6.939 CJ3071 -6.167 -5.083 -6.458 -6.669 CJ3077 -6.097 -5.198 -7.52 -8.011 CJ3082 -7.378 -5.932 -7.298 -7.109 CJ3094 -6.078 -6.022 -6.12 -7.301 CJ3095 -7.304 -6.563 -7.408 -8.226 CJ3098 -7.826 -7.752 -8.601 -9.324 CJ3100 -6.774 -6.108 -7.896 -8.649 CJ3109 -6.339 -5.937 -5.95 -6.903 CJ3110 -6.877 -6.162 -7.17 -7.666 CJ3114 -6.999 -6.326 -7.347 -8.306 CJ3116 -7.522 -7.771 -8.102 -8.97 CJ3117 -7.977 -7.012 -7.558 -7.622 CJ3118 -7.46 -6.829 -7.409 -8.583 CJ3120_1 -7.396 -7.387 -7.882 -8.409 CJ3120 -6.787 -6.787 -7.442 -7.939 CJ3122 -8.195 -7.747 -8.237 -8.866 CJ3123 -7.626 -8.028 -8.305 -9.228 CJ3125_1 -6.698 -6.076 -6.319 -7.298 CJ3125_2 -7.172 -6.954 -7.292 -8.303 CJ3127 -7.797 -7.23 -7.823 -8.439 CJ3128 -7.36 -7.737 -7.733 -8.583 CJ3129 -6.814 -6.061 -7.389 -8.306 CJ3130 -6.89 -6.702 -7.21 -8.144 CJ3132 -7.89 -7.456 -8.212 -8.756 CJ3133 -7.736 -7.909 -7.95 -8.03 CJ3134 -8.317 -7.461 -8.807 -9.125 CJ3135 -7.674 -8.24 -8.645 -9.157 CJ3136 -8.236 -8.528 -9.064 -8.999 CJ3137 -8.217 -8.531 -8.923 -8.862 CJ3138 -7.157 -6.388 -8.699 -7.998 CJ3139_1 -6.955 -6.349 -7.274 -8.361 CJ3139_2 -7.147 -7.092 -7.183 -8.336 CJ3141 -7.288 -5.999 -7.042 -7.92 CJ3142 -7.202 -7.071 -7.757 -7.478 CJ3143 -7.623 -7.393 -8.59 -8.834 CJ3144 -6.816 -6.303 -7.232 -7.888 CJ3145 -6.668 -6.123 -6.694 -7.229 CJ3146 -7.049 -6.587 -6.827 -8.348 CJ3147 -7.348 -6.474 -6.774 -8.108

244

CJ3148 -7.534 -6.974 -7.619 -8.595 CJ3149 -7.371 -7.362 -7.807 -8.446 CJ3150 -7.228 -6.743 -7.367 -7.871 CJ3151 -7.107 -5.954 -6.754 -7.633 CJ3152 -8.46 -8.489 -9.32 -9.233 CJ3153 -6.823 -6.44 -6.943 -7.94 CJ3155 -8.371 -8.366 -8.963 -8.882 CJ3156 -7.794 -8.458 -8.819 -8.181 CJ3157 -9.01 -8.213 -9.146 -9.148 CJ3158 -8.257 -9.095 -9.172 -8.578 CJ3159 -7.358 -6.755 -7.726 -8.176 CJ3160 -9.091 -8.733 -9.045 -9.328 CJ3161 -7.268 -7.613 -7.514 -8.016 CJ3162 -7.71 -7.892 -8.61 -9.138 CJ3163 -8.744 -8.146 -8.815 -8.774 CJ3164 -8.656 -8.716 -8.797 -8.872 CJ3165 -8.976 -8.796 -9.29 -9.341 CJ3166 -6.542 -6.761 -7.152 -8.262 CJ3167 -7.027 -6.992 -7.269 -7.703 CJ3168 -9.912 -9.161 -9.724 -8.255 CJ3169 -6.648 -6.434 -6.716 -7.877 L670548 -7.017 -6.1 -6.231 -7.02 L687306 -7.562 -6.585 -7.469 -7.815 L689660_ethoxy -7.998 -7.187 -7.533 -8.507 L689660_isopropoxy -8.235 -7.036 -7.573 -8.893 L689660_RACE -8.386 -6.453 -7.071 -8.297 L689660_S_prot -8.121 -6.429 -7.123 -8.291 L689660 -8.05 -6.583 -6.726 -8.019 MA418 -6.619 -5.73 -5.602 -7.271 MA430 -7.01 -6.804 -6.832 -8.011 R_QNB -7.549 -7.012 -8.713 -7.986 RACE_QNB -6.92 -6.551 -8.581 -9.454 S_QNB_prot -6.798 -6.248 -8.803 -8.959 Tasclidine -5.935 -6.493 -6.669 -6.891

Table 20 -LGFE scores of above list of compounds to which SILCS-MC was applied.

245

Discussion (B.3):

CJ1193.1, 1193.2, 1194, 2013, 2013.1, 2013.3, 2046, and 2087 (and others similar) were some of the original compounds synthesized to explore structure activity relationships for mAChR antagonism. These scaffolds were based off of the parent scaffold (WAY132983) which

2+ showed (slightly) selective M 1 (Ca mobilization studies) and M 4 (fully inhibiting forskoin- induced increased in cAMP levels) agonism, as it was originally designed for the treatment of

AD. The LGFE scores show these compounds have a slightly higher predicted affinity for M 1,4 .

Unfortunately, these compounds showed no activity in vivo and were not further evaluated (not detailed in this thesis). The WAY-derivatives (CJ2090.1, 2091, 2094) appear to show very similar affinities to all mAChRs (as did WAY132983), however these compounds were shown bind to multiple other receptors through in vitro binding studies by the Roth laboratory (Sigma 1 and 2, 5HT7A, α2A, α2C, D5, DAT, H2) while only possessing moderate affinity for the mAChRs (>100 nm). A significant difference between our compounds and WAY132983 is the use of a quinuclidine vs. 1-azanorbornane ring, which has proven to be a potent amine core for the mAChRs. Because our goal was to identify a compound that functioned via a cholinergic effect these compounds were not explored further due to their lack of specificity.

Recall from Chapter 3 that the arecoline compounds were largely inactive in vivo in the FST

(exceptions were CJ2099, 2100, 2159.3, 2162.1, 3007, and 3018). The LGFE scores show an interesting trend--in that the longer, bulkier, and more flexible alkyl chains (and rings) substituted at the 3-position of the oxadiazole in general show a larger negative value (i.e. a more stable complex). While many of these compounds are currently undergoing in vitro evaluation by the Roth Laboratory (for both affinity an efficacy) this trend could perhaps be explained similarly to that seen by WAY132983 and to other M 1 preferring compounds (see Ward et al , J.

Med. Chem. 1992). The side chain could potentially fit into a hydrophobic pocket in the receptor

246 or lie along a lipophilic section of one of the α-helices in a transmembrane segment of the receptor, stabilizing the particular conformation of the protein. In general, Sauerberg et al. (see

Table 3, Chapter 2) showed that any substitution at the 3-position of the oxadiazole ring larger than butyl began to show an NMS/OXO-M ratio indicative to that of a partial agonist/antagonist.

The SILCS-MC data agree well with this experimental observation (i.e. more negative LGFE values for larger substituents on the arecoline-oxadiazole derivatives). Notice CJ2099 (a potent agonist) has one of the lowest LGFE scores of the arecoline group. This correlates well with it being an agonist (this was proven experimentally), and hence would be expected to have weak affinity for the inactive state.

The quinuclidines represented a successful and potent scaffold for designing compounds that selectively targeted the mAChRs (CJ2051, 2126, 2165.1, 2165.3, 2166.2, 2173.1, 2174, 3017,

3063, 3160, 3165, 3168). While the trends match the arecolines, the change in LGFE scores is smaller. For example CJ2126 (cyclopropyl) has LGFE scores of ~-8, -7, -7.7, and -8.5 whereas

CJ2173.1 (pentyl) has ~-8.5, -8.2, -8.7, -9.3. However, this small a change in LGFE does not correlate to the in vivo efficacy observed by the quinuclidine vs. the arecoline series. The quinuclidine compounds were shown to have low μM binding affinities (Roth Lab) and on average a log (or more) potent than their arecoline counterparts (in the bradychardia assay, FST,

PVT/TVT, etc.). Interestingly, CJ3165 and 3168 possessed some of the highest LGFE scores in the entire set of compounds (heptyl/cycloheptyl, respectively). We shall soon observe whether this will correlate to in vitro/in vivo efficacy. The 1-azabicyclo[3.2.1]octane was a novel scaffold for the mAChRs--of which several derivatives were shown to be equi-potent to the quinuclidine series (CJ3094, 3095, 3098, 3100, 3122, 3127, 3129, 3132, 3134, 3136, 3139.1, 3151, 3157,

3159, 3162, 3166) and reflected the same trends in LGFE scores. SILCS-MS was also performed

247 on several of the stereoisomers (R or S enantiomers) but little difference was observed in LGFE scores.

The 4-substituted compounds showed significant decrease in activity across all pharmacological assays, although only the methyl/cyclopropyl derivatives have been evaluated at the moment. The 4-substituted 1-azanorbornanes, quinuclidines, and arecolines all show lower

LGFE scores which is in agreement that shifting the oxadiazole one position over is detrimental to mAChR activity. However, with the azanorbornane/quinuclidine series a similar trend to the

3-substituted counterparts arises. As the substitution on the oxadiazole ring is 4-5 carbons or larger, the LGFE scores start to match those of the 3-substituted group. More work is needed to obtain (and compare) the experimental binding affinities and subsequent efficacy screens before further conclusions can be made. The arecoline amides and sulfonamides overall showed no efficacy in vivo and produced poor LGFE scores and were not further explored.

Overall the SILCS-MC method has proven to be a useful tool to predict (or validate) whether a particular scaffold is viable for the mAChRs. The only discrepancy can be seen with QNB, which had lower LGFE scores than the arecoline series. However, it showed low nM binding affinity (<20 nm at each receptor) and was dangerously potent in vivo. However, the LGFE scores must be used in tandem with in vitro/in vivo testing as a direct correlation between affinity and efficacy can only be made by empirical evidence.

248

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