Stereochemical Synthesis of Ring E Analogs of Methyllycaconitine and 4,5-Disubstituted

Oxazolidinones

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Crina M. Orac

November 2009

© 2009 Crina M. Orac. All Rights Reserved. 2

This dissertation titled

Stereochemical Synthesis of Ring E Analogs of Methyllycaconitine and 4,5-Disubstituted

Oxazolidinones

by

CRINA M. ORAC

has been approved for

the Department of Chemistry and Biochemistry

and the College of Arts and Sciences by

Stephen C. Bergmeier

Associate Professor of Chemistry and Biochemistry

Benjamin M. Ogles

Dean, College of Arts and Sciences 3

ABSTRACT

ORAC, CRINA M., Ph.D., November 2009, Chemistry and Biochemistry

Stereochemical Synthesis of Ring E Analogs of Methyllycaconitine and 4,5-Disubstituted

Oxazolidinones (320 pp.)

Director of Dissertation: Stephen C. Bergmeier

The pharmacological activity of enantiomers of a chiral compound can be

influenced to a great extent by molecular chiral recognition, the ability of a chiral

biological system to discriminate between enantiomers. Typically one enantiomer will

have better activity than its enantiomer. This difference in biological response generated

by enantiomers upon binding to a biological target is a consequence of their differential

interaction with the binding site. When designing new ligands for biological targets it is

important to examine their constituent enantiomers. This dissertation is concerned with

the synthesis of enantiomerically pure ring E analogs of methyllycaconitine and trans

4,5-disubstituted oxazolidinones in an effort to understand their interaction with their

biological targets in a three-dimensional sense.

Due to their involvement in many physiological functions, nicotinic acetylcholine

receptors (nAChRs) are important potential therapeutic targets for a large variety of

neurodegenerative and psychiatric disorders. Ring E analogs of methyllycaconitine have

been previously reported to act as noncompetitive antagonists of a subtype of nAChRs. In

this dissertation, a new set of 20 compounds was prepared, consisting of enantiomers and

various diastereomeric mixtures of three lead compounds from previously studied ring E 4 analogs. The examination of the 3-D interaction of this set of compounds with the nAChRs leads to a better understanding of the formation of ligand-receptor complex and the structural demands required for a good binding.

A new class of antibacterial agents, the 4,5-disubstituted oxazolidinones, has been developed that exhibit inhibitory activity at the T box transcription antitermination system that controls the gene expression in many bacteria. In this dissertation, the enantiomers and the cis isomers of two lead compounds were prepared and used to study the effect of their chirality on the binding to the T box antiterminator. The biological results indicated that there is chiral recognition associated with the binding of 4,5- disubstituted oxazolidinones to the T box antiterminator.

Approved: ______

Stephen C. Bergmeier

Associate Professor of Chemistry and Biochemistry 5

ACKNOWLEDGMENTS

First and foremost I want to express my deepest appreciation to my advisor Prof.

Stephen Bergmeier. His mentorship and expertise considerably extended my graduate experience allowing me to grow as an experimentalist and as a chemist but, more importantly, as an independent thinker. Without his constant help and understanding this dissertation would not have been possible.

I would also like to acknowledge my committee members Prof. Jennifer Hines,

Prof. Jeff Rack and Prof. Peter Jung for their insightful suggestions and guidance. In addition, I thank my collaborators: Dr. John Means, Dr. Raj Anupam and Shu Zhou from

Prof. Hines group for performing the biological tests for the oxazolidinones project and to

Prof. McKay from Ohio State University and his graduate student Brandon Henderson for performing the biological tests for the methyllycaconitine project.

Lastly but not the least my thanks and appreciation go to Daniel Sayre for his emotional support, encouragement, and patience throughout the course of my graduate studies and to my parents and my dear brother for their immense love, support and faith in me that made it possible for me to reach this stage of my professional life.

6

TABLE OF CONTENTS

Page

Abstract ...... 3

Acknowledgments...... 5

List of Tables ...... 12

List of Figures ...... 13

List of Scheme ...... 17

CHAPTER 1: NICOTINIC ACETYLCHOLINE RECEPTORS ...... 20

1.1. Introduction ...... 20

1.2. Receptor types ...... 23

1.2.1. Muscle-type nAChR ...... 23

1.2.2. Neuronal-type nAChRs ...... 24

1.2.2.1. Subtype α4β2 ...... 28

1.2.2.2. Subtype α7 ...... 28

1.2.2.3. Subtype α3β4* ...... 29

1.2.2.4. Other subtypes ...... 30

1.3. Structure of nicotinic acetylcholine receptors ...... 30

1.3.1. Topology of nAChR subunit ...... 31

1.3.2. Ion channel domain of nAChRs ...... 34

1.3.3. N-Terminal domain of nAChRs ...... 38

1.3.3.1. Orthosteric binding site ...... 39

1.3.3.2. Allosteric binding sites ...... 41 7

1.4. Therapeutic potential of nicotinic acetylcholine receptors ...... 44

1.4.1. Disorders involving nAChRs ...... 45

1.4.1.1. Alzheimer’s disease ...... 45

1.4.1.2. Parkinson’s disease ...... 45

1.4.1.3. Tourette’s syndrome ...... 46

1.4.1.4. Schizophrenia ...... 47

1.4.1.5. Depression...... 47

1.4.2. Drug discovery of nAChR modulators ...... 48

1.4.2.1. Agonists ...... 50

1.4.2.2. Antagonists ...... 55

1.4.2.3. Allosteric modulators ...... 57

1.4.3. Methyllycaconitine ...... 60

1.5. Chiral recognition ...... 64

1.5.1. Receptor-enantiomers interactions ...... 66

1.5.2. Biological activities of enantiomers ...... 69

1.6. Summary ...... 74

CHAPTER 2: SYNTHESIS OF RING-E ANALOGS OF MLA ...... 75

2.1. Analogs of IB-10 ...... 75

2.1.1. Significance ...... 75

2.1.1.1. Lead compound’s chirality ...... 77

2.1.1.2. Target compounds ...... 80

2.1.1.3. Retrosynthesis ...... 84 8

2.1.2. Synthesis of benzylsuccinimidobenzoic acid precursor ...... 85

2.1.2.1. Synthesis of racemic benzylsuccinimidobenzoic acid ...... 85

2.1.2.2. Synthesis of enantiomerically pure benzylsuccinimidobenzoic acid ...... 87

2.1.2.3. Determination of enantiomeric excess...... 100

2.1.3. Synthesis of N-(3-phenylpropyl)-3-piperidinemethanol precursor...... 108

2.1.3.1. Synthesis of racemic N-(3-phenylpropyl)-3-piperidinemethanol ...... 108

2.1.3.2. Synthesis of enantiomerically pure N-(3-phenylpropyl)-3-

piperidinemethanol ...... 109

2.1.3.3. Determination of enantiomeric excess ...... 114

2.1.4. Synthesis of enantiomers of IB-10 ...... 119

2.1.5. Synthesis of diastereomers of IB-10 ...... 120

2.1.6. Synthesis of other analogs of IB-10 ...... 121

2.1.6.1. Synthesis of unsaturated precursor ...... 121

2.1.6.2. Coupling ...... 122

2.2. Analogs of APB-12 ...... 126

2.2.1. Significance ...... 126

2.2.1.1. Target compounds ...... 128

2.2.2. Synthesis of methylsuccinimidobenzoic acid precursor ...... 129

2.2.3. Synthesis of N-(3-phenylpropyl)-2-pyrrolidinemethanol precursor ...... 131

2.2.4. Coupling reaction ...... 132

2.3. Analogs of COB-1 ...... 134

2.3.1. Significance ...... 134 9

2.3.2. Synthesis of COB-1, COB-2 and COB-3 ...... 135

2.3.3. Synthesis of enantiomers of COB-1 ...... 136

2.4. Summary ...... 140

CHAPTER 3: OXAZOLIDINONES AS ANTIBIOTICS ...... 141

3.1 Resistance to antibiotics ...... 145

3.2. Targets of antibiotics ...... 149

3.2.1. Inhibitors of bacterial cell wall biosynthesis...... 150

3.2.2. Inhibitors of nucleic acid synthesis ...... 154

3.2.3. Inhibitors of protein synthesis ...... 156

3.3. New mechanism/structural class of antibiotics ...... 161

3.3.1. New class of oxazolidinones ...... 165

3.4. Summary ...... 170

CHAPTER 4: STEREOCHEMICAL SYNTHESIS OF 4,5-DISUBSTITUTED

OXAZOLIDINONES ...... 171

4.1. Enantiomers of ANB-22 and ANB-40 ...... 171

4.1.1. Significance ...... 171

4.1.1.1. Lead compound’s chirality ...... 171

4.1.1.2. Target compounds ...... 175

4.1.1.3. Plan of synthesis ...... 176

4.1.2. Studies on the intramolecular aziridination ...... 178

4.1.2.1. Intramolecular aziridination of allyl-substituted carbamate ...... 179

4.1.2.2. Intramolecular aziridination of allyl-substituted N-tosyloxy carbamate 182 10

4.1.2.3. Intramolecular aziridination of allyl-substituted azidoformate ...... 185

4.1.3. Enantiomerically pure 3-butene-1,2-diol ...... 188

4.1.3.1. Sharpless asymmetric epoxidation ...... 191

4.1.3.2. Determination of enantiomeric excess ...... 193

4.1.3.3. Hydrolytic kinetic resolution ...... 198

4.1.3.4. Enantiomeric excess determination of (R)-4.1 and (S)-4.1 ...... 199

4.1.4. Synthesis of enantiomerically pure precursors ...... 202

4.1.5. Synthesis of target compounds ...... 204

4.2. The cis isomer of ANB-22 and ANB-40 ...... 206

4.2.1. Rational ...... 206

4.2.2. Plan of synthesis for cis 4,5-disubstituted oxazolidinones ...... 207

4.2.2.1. First plan ...... 207

4.2.2.2. Execution and discussions for first plan ...... 210

4.2.2.3. Second plan ...... 211

4.2.2.4 Execution and discussions for second plan ...... 213

4.2.3. Synthesis of target compounds ...... 217

4.3. Summary ...... 221

CHAPTER 5: EXPERIMENTAL...... 223

References ...... 288

Appendix A: Expanded 1H NMR spectrum of (S,S)-2.20 ...... 315

Appendix B: Expanded 1H NMR spectrum of (S,S)-2.21 ...... 316

Appendix C: Expanded 1H NMR spectrum of (R,R)-2.29 ...... 317 11

Appendix D: Expanded 1H NMR spectrum of (S,S)-4.18 ...... 318

Appendix E: 19F NMR spectrum of (R,S)-4.18 ...... 319

Appendix F: 19F NMR spectrum of (S,S)-4.18 ...... 320 12

LIST OF TABLES

Page

Table 2.1: Reaction optimization for nipecotic acid acylation ...... 110

Table 2.2: Biological activity of IB-10 analogs ...... 123

Table 2.3: Biological activity of APB-12 analogs ...... 132

Table 2.4: Biological activity for COB-1 analogs ...... 138

Table 4.1: Aziridination of allyl-substituted N-tosyloxycarbamate ...... 183

Table 4.2: Comparison of intramolecular aziridination reactions ...... 186

Table 4.3: Binding to T box antiterminator model AM1A ...... 219 13

LIST OF FIGURES

Page

Figure 1.1: Ligands for acetylcholine receptors ...... 20

Figure 1.2: Schematic representation of nAChRs...... 21

Figure 1.3: Subunit composition of muscle-type nAChRs ...... 23

Figure 1.4: Subunit composition of neuronal-type nAChRs ...... 25

Figure 1.5: Topology of the nAChR subunit ...... 31

Figure 1.6: Pentameric structure of nAChRs ...... 33

Figure 1.7: Ion pore of nAChRs ...... 34

Figure 1.8: Residues lining the ion pore of nAChRs ...... 34

Figure 1.9: Proposed gate opening mechanism of nAChRs ...... 36

Figure 1.10: Pentameric structure of AChBP ...... 38

Figure 1.11: Orthosteric binding site of AChBP ...... 39

Figure 1.12: Allosteric binding sites of nAChRs ...... 40

Figure 1.13: Allosteric nature of nAChRs ...... 42

Figure 1.14: Naturally occurring nicotinic ligands ...... 48

Figure 1.15: Agonists of nicotinic receptors ...... 50

Figure 1.16: Antagonist for nicotinic receptors ...... 55

Figure 1.17: Allosteric modulators of nAChRs ...... 57

Figure 1.18: Modifications of methyllycaconitine ...... 60

Figure 1.19: General structure of ring E analogs of methyllycaconitine ...... 61

Figure 1.20: Ring E analogs of methyllycaconitine ...... 63 14

Figure 1.21: Interaction of enantiomers of a chiral ligand with the receptor ...... 66

Figure 1.22: The active enantiomer of citalopram ...... 69

Figure 1.23: Structures of chiral drug molecules ...... 70

Figure 1.24: Enantiomers of picenadol ...... 71

Figure 1.25: Enantiomers of albuterol ...... 72

Figure 1.26: Enantiomers of thalidomide ...... 72

Figure 2.1: Lead compound IB-10...... 75

Figure 2.2: Three-dimensional representation of piperidine ring enantiomers ...... 77

Figure 2.3: Three-dimensional representation of succinimide ring enantiomers ...... 78

Figure 2.4: Stereochemical features of ring E analogs ...... 79

Figure 2.5: Enantiomers of IB-10 ...... 80

Figure 2.6: Diastereomers of IB-10 ...... 81

Figure 2.7: Unsaturated analogs of IB-10 ...... 82

Figure 2.8: Retrosynthesis ...... 83

Figure 2.9: Sodium chelated Z-enolates of 2.14 and 2.16 ...... 91

Figure 2.10: Chiral HPLC chromatograms for (S)-2.18 and (R)-2.18 ...... 94

Figure 2.11: 1H NMR spectra of mixture of diastereomers and (S,R)-2.20 ...... 97

Figure 2.12: Chiral HPLC chromatogram of racemic succinimidobenzoic acid 2.2 ....101

Figure 2.13: Structure of Eu(hfc)3 ...... 102

1 Figure 2.14: H NMR spectrum of CDCl3 solution of Eu(hfc)3 and 2.2 ...... 103

Figure 2.15: 1H NMR spectra of mixture of diastereomers and (R,S)-2.21 ...... 105

Figure 2.16: Possible racemization mechanism ...... 106 15

Figure 2.17: 1H NMR spectra of mixture of diastereomers and (R,R)-2.29 ...... 117

Figure 2.18: Lead compound APB-12 ...... 126

Figure 2.19: Three-dimensional representation of pyrrolidine ring enantiomers ...... 127

Figure 2.20: Enantiomers of APB-12 ...... 127

Figure 2.21: Retrosynthesis ...... 128

Figure 2.22: Target compounds ...... 133

Figure 3.1: Representatives of old classes of antibiotics ...... 142

Figure 3.2: Representatives of new classes of antibiotics...... 143

Figure 3.3: Mechanisms of antibiotic resistance ...... 145

Figure 3.4: Mechanisms of genetic transfer ...... 147

Figure 3.5: Peptidoglycan structure of Gram-positive bacteria ...... 150

Figure 3.6: Site of action for penicillins ...... 151

Figure 3.7: Bacterial resistance to β-lactams ...... 152

Figure 3.8: Vancomycin mechanism of action and resistance ...... 153

Figure 3.9: Structure of folic acid, PABA and prontosil ...... 154

Figure 3.10: Schematic mechanism of bacterial protein synthesis ...... 156

Figure 3.11: T box transcription antitermination system ...... 162

Figure 3.12: Aminoacyl-tRNA formation ...... 163

Figure 3.13: A) T box antiterminator models; B) FRET-labeled AM1A model ...... 165

Figure 3.14: Substituted oxazolidinones ...... 167

Figure 3.15: Linezolid and representatives of the new class of oxazolidinones ...... 168

Figure 4.1: Enantiomers of trans 4,5-disubstituted oxazolidinones ...... 171 16

Figure 4.2: Possible binding interactions ...... 173

Figure 4.3: Enantiomers of lead compounds ANB-22 and ANB-40 ...... 174

Figure 4.4: Kinetic resolution ...... 188

Figure 4.5: Chiral HPLC chromatogram for racemic 4.18 ...... 194

Figure 4.6: 1H NMR spectra of racemic 4.18 and diastereomer (R,S)-4.18 ...... 196

Figure 4.7: Jacobsen’s catalyst ...... 197

Figure 4.8: 1H NMR spectrum of (R,S)-4.18 ...... 200

Figure 4.9: The trans and cis isomers of 4,5-disubstituted oxazolidinones ...... 205

Figure 4.10: Target compounds ...... 206 17

LIST OF SCHEME

Page

Scheme 2.1: Synthesis of succinic acid derivative ...... 85

Scheme 2.2: Synthesis of succinic anhydride derivative ...... 86

Scheme 2.3: Synthesis of precursor 2.2 ...... 86

Scheme 2.4: Synthesis of chiral auxiliary ...... 90

Scheme 2.5: Chiral auxiliary assisted asymmetric alkylation ...... 91

Scheme 2.6: Chiral auxiliary removal ...... 93

Scheme 2.7: Synthesis of diastereomers of 2.18...... 96

Scheme 2.8: Synthesis of enantiomers of succinic anhydride derivative ...... 99

Scheme 2.9: Synthesis of enantiomers of precursor 2.2 ...... 100

Scheme 2.10: Synthesis of diastereomers of 2.21 ...... 105

Scheme 2.11: Synthesis of racemic N-(3-phenylpropyl)-3-piperidinemethanol ...... 108

Scheme 2.12: Nipecotic acid resolution with camphorsulfonic acid ...... 109

Scheme 2.13: Nipecotic acid acylation ...... 110

Scheme 2.14: Reduction of nipecotic acid derivative ...... 112

Scheme 2.15: Ethyl nipecotate resolution with tartaric acid ...... 112

Scheme 2.16: Synthesis of enantiomers of precursor 2.3 ...... 114

Scheme 2.17: Mosher ester synthesis ...... 116

Scheme 2.18: Synthesis of enantiomers of IB-10 ...... 119

Scheme 2.19: Synthesis of diastereomers of IB-10 ...... 120

Scheme 2.20: Synthesis of unsaturated piperidine precursor ...... 121 18

Scheme 2.21: Synthesis of compound 2.30 ...... 122

Scheme 2.22: Synthesis of unsaturated analogs of IB-10 ...... 123

Scheme 2.23: Synthesis of enantiomers of methyl succinimidobenzoic acid ...... 130

Scheme 2.24: Synthesis of enantiomers of prolinol derivative ...... 131

Scheme 2.25: Synthesis of enantiomers of APB-12 ...... 132

Scheme 2.26: Synthesis of COB-1 ...... 135

Scheme 2.27: Synthesis of COB-2 and COB-3 ...... 136

Scheme 2.28: Synthesis of enantiomers of precursor 2.42 ...... 137

Scheme 2.29: Synthesis of enantiomers of COB-1 ...... 138

Scheme 4.1: Parallel synthesis of trans 4,5-disubstituted oxazolidinones ...... 176

Scheme 4.2: Iodine(III)-mediated aziridination reaction ...... 179

Scheme 4.3: Synthesis of carbamic acid allyl-substituted ester ...... 180

Scheme 4.4: Intramolecular aziridination of carbamate ...... 181

Scheme 4.5: Copper-catalyzed aziridination reaction ...... 182

Scheme 4.6: Synthesis of allyl-substituted N-tosyloxycarbamate ...... 183

Scheme 4.7: Aziridination of allyl-substituted azidoformate ...... 186

Scheme 4.8: Synthesis of monotritylated 3-butene-1,2-diol ...... 192

Scheme 4.9: Sharpless asymmetric epoxidation of 4.2 ...... 192

Scheme 4.10: Preparation of Mosher’s esters (R,S)-4.18 and 4.18 ...... 194

Scheme 4.11: Hydrolytic kinetic resolution of butadiene monoepoxide ...... 199

Scheme 4.12: Preparation of Mosher’s esters (R,S)-4.18 and (S,S)-4.18 ...... 200

Scheme 4.13: Synthesis of enantiomers of bicyclic aziridine ...... 202 19

Scheme 4.14: Synthesis of enantiomers of alcohol precursor ...... 203

Scheme 4.15: Synthesis of target compounds ...... 205

Scheme 4.16: First proposed synthesis for cis oxazolidinones ...... 208

Scheme 4.17: Regioselective azide opening of 2,3-epoxy alcohols ...... 209

Scheme 4.18: Synthesis of monoprotected 2-butyne-1,4-diol ...... 210

Scheme 4.19: Synthesis of monoprotected 2-butene-1,4-diol ...... 211

Scheme 4.20: Second proposed synthesis for cis oxazolidinones ...... 212

Scheme 4.21: Synthesis of 3-azido-butane-1,2,4-triol ...... 213

Scheme 4.22: Synthesis of intermediate 4.37 ...... 214

Scheme 4.23: Synthesis of carbamate ...... 215

Scheme 4.24: Two steps synthesis of carbamate ...... 215

Scheme 4.25: Synthesis of precursor cis-4.23 ...... 216

Scheme 4.26: Synthesis of final compounds ...... 218 20

CHAPTER 1: NICOTINIC ACETYLCHOLINE RECEPTORS

1.1. Introduction

Nerve cells communicate between themselves and with their non-neuronal cell targets, such as those found in muscles and glands, through synapses. A presynaptic cell

(neuron) and a postsynaptic cell (neuron or a target cell) are separated by a specialized gap called synaptic cleft that allows an electrical signal (nerve impulse) to be transmitted from one cell to the other. The electrical impulses that arrive at the presynaptic terminal trigger the release of a chemical messenger called neurotransmitter that diffuses into the synaptic cleft. Immediately opposite to the presynaptic terminal is a region of the postsynaptic cell that contains receptors that bind the neurotransmitter molecules. Upon binding of the neurotransmitter, the receptors change conformation and open a gate causing ions to flow selectively in or out. This ion flow changes the ionic permeability of the postsynaptic cell. In this way the receptors mediate the propagation of the electrical signals from one neuron to other neurons or to its targets.1

One of the most widely distributed neurotransmitter in the body is acetylcholine

(ACh) (Fig. 1.1). The effect of this intercellular signaling molecule on the central nervous

system and peripheral nervous system is exerted through a large group of receptors called

acetylcholine receptors (AChR). These receptors that mediate ACh signaling were

originally divided into two classes of receptors called muscarinic (mAChRs)2 and

nicotinic acetylcholine receptors (nAChRs)3. The nomenclature is based on their relative affinities and differential responsiveness to muscarine (a mushroom toxin) and nicotine, respectively (Fig. 1.1). The mAChRs are known to be members of the G-protein-coupled 21 receptors type and mediate biological effects such as heart rate and glandular secretion among others.2, 4 The nAChRs are members of the superfamily of ligand-gated ion

channels and play a central role in modulating the fast synaptic neurotransmission and

fundamental intracellular signaling.5-7 The research presented in this dissertation has

application in the study of nAChRs, therefore the mAChRs will be excluded from the

following discussion.

O O N N N O N HO Acetylcholine (S)-Nicotine Muscarine

Figure 1.1. Ligands for acetylcholine receptors.

Nicotinic acetylcholine receptors, as mentioned before, are the prototypic

members of ligand-gated ion channels superfamily. As illustrated in Fig. 1.2 they are

integral membrane proteins composed of five separate transmembrane subunits that are arranged around a central aqueous pore constituting the ion channel.8, 9 Access to the pore

is controlled by a gate that is opened by the binding of a chemical messenger (e.g.

acetylcholine) that allows the ions to move down their electrical gradients. Mammalian

nicotinic acetylcholine receptors are cation selective, being permeable to small

monovalent and divalent cations (Ca2+, Na+ and K+). Because of their oligomeric

structure, this type of receptor poses more than one ligand-binding site.

22

Na+ Ca2+

Synaptic side

Cytosolic side

Figure 1.2. Schematic representation of nAChRs.1

Nicotinic acetylcholine receptors are widely expressed at several different regions

in brain and spinal cord, on sensory nerves and some peripheral nerve terminals, in

autonomic ganglia of the peripheral nervous system and on the skeletal muscle at the

neuromuscular junction. They are localized in clusters at presynaptic, preterminal and

postsynaptic positions where they exert a broad variety of functions.10, 11 They are the

major players in the rapid synaptic transmission, influence the neuronal excitability and

have roles in development and synaptic plasticity. A very important role for the overall

contributions of nAChRs to cholinergic neurotransmission is that presynaptic and preterminal receptors act as synaptic modulators by regulating the release of acetylcholine and other important neurotransmitters such as dopamine, serotonin, γ- aminobutyric acid, norepinephrine, and glutamate.11-13 They are involved in cognitive

processes such as attention, memory, and learning. Also, nAChRs are known to play an

important role in mood, neuroprotection, and regulation of inflammation processes among others. It is because of their involvement in many physiological functions and their modulatory input on the neurotransmitter system that nAChR have been proposed as 23 therapeutic targets for a large variety of psychiatric disorders such as Alzheimer’s disease, Parkinson’s disease, Tourette’s syndrome, autism, depression, anxiety and schizophrenia.12, 14-16 They have also been suggested as targets for the treatment of pain,

some forms of epilepsy and drug addiction.

1.2. Receptor types

In a broad classification based on their primary site of expression, the nAChRs are divided into two major types: the muscle-type nAChR that is located at the postsynaptic membrane of the muscle fibers and the neuronal-type nAChR that is located throughout the nervous system.

1.2.1. Muscle-type nAChR

The muscle-type nAChR is present at the neuromuscular junction in the postsynaptic membrane of the mature muscles in high concentration and is absent from the rest of the muscle fiber surface.17 The receptor has a heteropentameric structure composed of α1, β1, γ or ε, and δ subunits organized in a clockwise (α1)2β1γ/εδ arrangement as illustrated in Fig. 1.3. The subunit γ is present only in the fetal muscles

and it is replaced in the adult muscle by the ε subunit.18, 19 The endogenous neurotransmitter acetylcholine and other exogenous ligands bind to two domains located at the α1-γ/ε and α1-δ interfaces of the receptor complex.20, 21 These sites are indicated in

Fig. 1.3 by the white marks.

24

Heteromeric Muscle AChRs

(α1)2β1γδ (α1)2β1εδ β1 β1 α1 δ α1 δ α1 ε α1 γ Orthosteric binding sites Adult Form Fetal Form ACh binding site

Figure 1.3. Subunit composition of muscle-type nAChRs.22

Being located at the neuromuscular junction, the muscle-type nAChRs are key

mediators of the electrical transmission creating the skeletal muscle tone. It is for this

physiological role that they have been the target for muscle relaxants.23 The alteration or

disruption of their function leads to neuromuscular diseases characterized by weakness

and muscle fatigue. An example is myasthenia gravis that is caused by the development

of antibodies to muscular nAChRs. The antibodies sterically hinder the binding sites for

neurotransmitter acetylcholine and also induce the degradation of the muscular

nAChRs. As a result, the number of available nAChRs at the neuromuscular junction is

reduced, which in turn leads to decreased cholinergic signaling and severe muscle

weakness.

1.2.2. Neuronal-type nAChRs

The neuronal-type nAChRs are widely present throughout the nervous system and in the nonexcitable cells, such as cells of the immune system and epithelial cells. They have a similar pentameric structure but they present a considerable diversity in terms of subunit composition. The various subunits of neuronal nAChRs that have been isolated and cloned to date are divided into two groups: α and β subunits. There are nine α 25 neuronal subunits (α2-α10) and three β neuronal subunits (β2-β4). With such a variety of

subunits the number of pentameric combinations that could form subtypes of nAChRs is

quite large. The diversity in subunit composition leads to subtypes of nAChR that are

characterized by dramatically different characteristics such as ligand pharmacology,

cation permeability, and activation and desensitization kinetics.24

The lower-order nAChR subunits α2-α6 and β2-β4 are involved in the formation

of heteromeric receptors usually with a (αx)2(βy)3 stoichiometry, arranged as αβαββ

(Fig. 1.4), although (αx)3(βy)2 and other possible stoichiometries have been demonstrated

in vitro.25 The α5 and β3 subunits are termed “orphan” or “auxiliary” subunits because

they cannot form operational channels when express alone or paired with other α or β subunits but they can participate in the formation of functional receptors when coexpressed with combinations of other functional subunits such as α4β2 and α3β4 (Fig

1.4). This does not rank them as less important subunits because it had been shown that the presence of either of these two subunits in a nAChR has a drastic effect on its pharmacology, calcium ions permeability, and desensitization kinetics.26-28 In Fig. 1.4 are

illustrated only the likely variants of nAChR subtypes that contain the α5 subunit but

combinations with β3 subunit instead of α5 subunit are formed as well. In fact, it is

believed that most heteromeric nAChRs in native tissues are more structurally complex

29 being constituted by three, four or five different subunits (e.g. (α6)2β4β3α5).

26

Homomeric Heteromeric Neuronal AChRs Neuronal AChRs

(α4)2(β2)2α5 (α4)3(β2)2 (α7)5 (α4)2(β2)3 α7 β2 α5 α4 α7 α7 α4 β2 Likely α4 β2 α4 β2 variants α7 α7 β2 α4 β2 α4 β2 α4

(α3)2(β4)3 (α3)2β2β4α5 (α3)2(β4)2α5 β4 α5 α5 α3 β4 Likely α3 β2 α3 β4 variants α3 β4 α3 β4 α3 β4

Figure 1.4. Subunit composition of neuronal-type nAChRs.22

The higher-order α7-α9 subunits form homomeric receptors (Fig. 1.4) that are characterized by larger Ca2+ permeability. Combined with other subunits, the α7 nAChR subunit is also capable of forming heteromeric receptors in vitro. These receptors display significantly different pharmacological properties than those of the homomeric receptors.30, 31 The α10 nAChR subunit is not capable of forming functional homomeric receptors but it can form a functional receptor in combination with α9 subunit.32 The homomeric α9 and the heteromeric α9α10 subtypes of nAChRs have significantly different characteristics than those of other nAChRs because they present mixed muscarinic/nicotinic pharmacology. The α9 and α10 subunits are not expressed in the central nervous system and the α8-containing receptors have been exclusively found in the chick nervous system.33, 34

The nicotinic acetylcholine receptors contain multiple-binding domains, usually referred as binding sites or orthosteric sites. These domains accommodate different endogenous neurotransmitters (e.g. acetylcholine) and exogenous ligands (e.g. nicotine). 27

They are small pockets located at the interface between two adjacent α and β subunits for

heteromeric nAChR subtypes while for homomeric subtypes the binding sites are defined

by adjacent α subunits as indicated in Fig. 1.4 by the white marks.35, 36 Therefore, in the

heteromeric nAChR subtypes two such orthosteric sites are present, whereas for the

homomeric nAChR subtypes there are potentially five orthotopic binding sites.

The identity and subsequently, the subunit composition of several native neuronal

nAChR subtypes were revealed in studies combining electrophysiological recording on

neurons, single-cell reverse transcription-polymerase chain reaction techniques, nAChR

knock-out mice, and few available ligands that are subtype-selective.24, 37 Despite the fact

that the individual neuron contains the transcripts for most of the neuronal subunits (α2-

α7 and β2-β4) providing the potential to express a very large number of distinct nAChR

subtypes, preferential association of subunits has been demonstrated.38, 39 However, in a

specific region of the central nervous system, different sets of neurons usually express

completely different nAChR subtypes leading to a diverse nAChR population in that

particular region. It is believed that these interneuronal differences in nicotinic receptors

expression is very important for the role of these receptors in modulating the

neurotransmitter systems.24 Although an enormous amount of data has been accumulated

so far, the complete identification of subunit composition and the full understanding of

the physiological functions of specific nAChR subtypes is an ongoing research that was

and still is slowed down due to the diversity of native nAChR populations, the

differences in distribution patterns40 of nicotinic subunits among species and the lack of

truly subtype-selective ligands. Several native neuronal nAChR subtypes will be 28 presented in the following sections without the pretension to an exhaustive discussion on the subject.

1.2.2.1. Subtype α4β2

The nicotinic acetylcholine receptor subunits most abundantly expressed in brain

are the α4 and β2 subunits.12, 41 Therefore it is with no surprise that the majority of

nAChRs in the central nervous system are α4β2 receptors containing two α4 subunits

and three β2 subunits.42, 43 The α4β2 receptors are present in many areas of the brain but in particular they are localized in hippocampus, cerebral cortex and thalamus.41, 44 They are the primary nAChR subtype involved in neuromodulation11 and are known to have a

role in aspects of cognition and associative memory and to participate in sensory gating

including anxiety, depression and pain.3, 45

In addition to the predominant α4β2 nAChR subtype, the presence of minor

populations of receptors containing α4 and β2 subunits combined with other subunits

(Fig. 1.4) is also possible. These subtypes are generally referred to as α4β2* receptors

where the asterisk indicates the presence of one or more additional neuronal nAChR

subunits in the receptor complex.24 The α4β2 receptors and α4β2* in general, have high- affinity for endogenous neurotransmitter acetylcholine and exogenous nicotine.3

1.2.2.2. Subtype α7

Besides α4 and β2 subunits, there is one more subunit abundantly expressed in

the central nervous system and that is the α7 subunit.12 As it was previously mentioned,

the α7 subunit forms homopentameric receptors (Fig. 1.4). The heteromeric α7* combinations have been observed in vitro studies but their presence in vivo has not been 29 indubitably proven.30, 31 The homomeric α7 receptors are highly expressed in cerebral

cortex, hippocampus and hypothalamus.46, 47 They play a role in stimulating release of the

excitatory neurotransmitter glutamate,48 in neuronal development,49 in cognition,

memory, neuroprotection and auditory processing.3, 13 Moreover, these receptors were also identified on the surface of human macrophages suggesting their involvement in regulation of inflammation.50

The α7 nAChR subtype are characterized by a lower affinity for acetylcholine

and nicotine than for α-bungarotoxin, a snake toxin. As a matter of fact, historically, the

α4β2* and α7 subtypes of nAChRs have been distinguished in binding assays using

radiolabeled nicotine, cytisine or epibatidine that identified the α4β2* subtype and with

radiolabeled α-bungarotoxin that binds to α7 but not to α4β2* subtype. Another

characteristic of α7 receptors is their calcium permeability that is the highest among all

nAChR subtypes.51

1.2.2.3. Subtype α3β4*

The nAChR subunits α3 and β4 are also expressed in few regions of the central

nervous system although they have a much more limited distribution in brain than in the

periphery.12 These subunits are the predominant subunits expressed in autonomic ganglia presenting a high degree of colocalization with α5 and β2 subunits. This colocalization

leads to a heterologous population of α3β4* receptors that includes α3β4, α3β4α5,

α3β2β4 and α3β2β4α5 subtypes with α3β4α5 as the major subtype.38, 39 The α3β4* receptors play a role in norepinephrine release and in autonomic control of peripheral organs.52 30

1.2.2.4. Other subtypes

The other neuronal subunits, α2, α6 and β3 show a much more limited expression

and distribution in central nervous system. The α2 subunit has not been studied in great

detail and the α6 and β3 subunits are highly colocalized and expressed only by

catecholaminergic regions.53-55 The presence of the heteromeric α6β3 subtype has not

been proven but is strongly suggested by the similar distribution of the two subunits.56, 57

The α9 and α10 nAChR subunits are not expressed in the central nervous system

and they are unique in that they are specifically expressed by cochlear outer hair cells.33,

34 They present mixed muscarinic/nicotinic properties and are characterized by high

calcium permeability.33, 58

1.3. Structure of nicotinic acetylcholine receptors

Most of the early physiological and biochemical studies on nAChR structure and

function were performed on nicotinic receptors isolated from the electric organ of

Torpedo electric ray.6 The Torpedo electric organ is a valuable source of tissue for studies because it is highly enriched in nAChRs that have been shown to have a high degree of homology with the vertebrate muscle-type nAChRs.6, 59 The isolated receptor-

containing membranes were amenable to structure analysis by cryoelectron

microscopy.60, 61 These studies conducted by Unwin et. al., provided atomic-scale 2D images of the nAChRs revealing the dimension and the shape of the molecule, the subunit arrangement and the boundaries between them and also gave insights into the location of the orthosteric sites and the constitution of the ion pore.6, 61 31

More recently, a breakthrough in the understanding of the nAChRs structure came from the elucidation of the 3-dimmensional X-ray structure at 2.7 Å resolution of the acetylcholine-binding protein (AChBP).62 The AChBP was isolated from the glial cells of

the mollusk Lymnaea stagnalis and is a naturally occurring protein that is a structural and functional homolog of the extracellular domain of the human nAChRs.6, 62, 63 A detailed

description of the whole receptor in near-physiological conditions was possible by combining the information provided by the crystal structure of AChBP with the 4 Å resolution electron microscopy structure of the Torpedo.24 No 3D X-ray structure of any nAChR or any other ligand-gated ion channel is available yet, although a few attempts have been reported.6, 64, 65

Although the 17 various nAChR subunits (α1-α10, β1-β4, δ, γ, and ε) show

differences in their protein sequence they all share the same structural features therefore

the structural information obtained from studying the Torpedo nAChR is true for all

nAChRs and as a matter of fact for all ligand-gate ion channel receptors. The structural

information derived from the aforementioned studies will be discussed in the following

sections.

1.3.1. Topology of nAChR subunit

As it was mentioned before, the nicotinic receptors are membrane proteins

composed of five subunits. In Fig. 1.5 is illustrated the topology of a single nAChR α subunit in a schematic linear representation (left image) and a ribbon diagram (right image). Each nAChR subunit is 500-600 amino acids residues long and consists of two regions: the N-terminal domain of ~210 amino acids positioned in the extracellular 32 membrane space and a transmembrane domain that is composed of four α-helical segments of ~20 amino acids each.24 The transmembrane α-helices M1-M4, shown in yellow in the ribbon diagram, are linked by alternating two intracellular and one extracellular loops. The intracellular loop is a large segment varying in size (100-150 residues) that includes a curved α-helix MA also shown in yellow (the rest of the loop is not seen in the picture). The C-terminal is a small segment of 4-28 amino acids that is oriented in the extracellular space.24

L4 N α-helix L1 L3 L8 L6 L10

β10 β3 β5 β6 β2 β1 β8 β9 β7 β4

C C N L2 L9 L7 C L5

Extracellular M4 M3 M2 M1

Intracellular o I

Figure 1.5. Topology of the nAChR subunit, linear representation (left)24 and ribbon

diagram (right).66

33

The N-terminal portion is composed of a small α-helix and ten β-strands termed

β1-β10, linked by loops.24 The β-strands are organized in a curled β-sandwich as

illustrated in the ribbon diagram (Fig. 1.5) by the blue color for the inner sheet composed

of β1-3, β5, β6 and β8 and red color for the outer sheet composed of β4, β7, β9 and β10.

The two sheets are joined through a disulfide bridge between a cysteine residue from β6

and a cysteine residue from β7. This disulfide bridge named “Cys-loop” shown in light

blue in the linear representation from Fig. 1.5, is a unique common characteristic feature

of all ligand-gated ion channels that are often referred to as the Cys-loop superfamily.6, 24

The numbering of the β-strands and the loops in the N-terminal domain are according to the AChBP X-ray structure.62

The ensemble of five subunits forms a pentameric nAChR complex shown in Fig.

1.6 where one subunit is represented in red, the subunit adjacent to it is represented in

blue and the other three subunits are represented in gray. This pentameric structure of the

receptor consists of two domains: the N-terminal domain also called ligand binding

domain that is created by the N-terminal regions of the subunits and lies in the

extracellular space (E) and the ion channel domain created by the transmembrane regions

of the subunits that is spanning the membrane and continues into the intracellular space

(I).

34

Figure 1.6. Pentameric structure of nAChRs.66

1.3.2. Ion channel domain of nAChRs

The electron microscopy studies on the Torpedo nAChRs revealed that the ion channel of nicotinic receptors is 40 Å long and has an outer diameter of 80 Å.9 The aqueous pore is encircled by a near perfect five-fold arrangement of α-helical segments as illustrated by the cross section in Fig. 1.7. The five M2 helical segments of all subunits line the pore and form an inner ring shown in white. The ten M1 and M3 helical segments of all subunits form an outer ring behind the inner ring while the remaining five M4 segments are positioned at the periphery.9, 24 This outer ring, shown in red in the cross section (Fig. 1.7) completely shields the inner ring (white) from the surrounding membrane lipids. It is notable that there is only minimal contact between the inner helices and the outer helices, an essential feature for the proper receptor function. The cavities between the two rings are filled with water, shown in black, and provide the space needed for the inner part to move relative to the outer part when the receptor is activated.

35

Figure 1.7. Ion pore of nAChRs, cross section.67

The five M2 helical segments are kinked at two positions such that the ion

channel has a narrow section in the middle as shown in Fig. 1.8 that illustrates the

residues lining the ion pore.24 This narrow segment of the ion channel contains rows of

predominantly aliphatic residues of all five subunits that are exposed to the central pore

forming a hydrophobic girdle. This girdle is considered the gate of the channel, which is

marked with red.9 This nonpolar gate acts as a molecular barrier preventing the ions from

passing through the channel when the receptor is in its inactive state.

Ala270 Ala270 Proposed resting gate Ser266 Ser266 (Unwin) Glu262 Glu262 Val259 Val259

Val255 Val255 Leu251 Leu251 Ser248 Ser248 Thr244 Thr244 Glu241 Glu241

Asp238 Asp238 Ion charge selectivity filter

Figure 1.8. Residues lining the ion pore of nAChRs.24 36

The ion pore presents two more rings formed by the residues of all five M2 helical segments. One ring is situated at the extracellular end and the second is positioned at the intracellular end of segments M2 being marked in gray (Fig. 1.8). These rings are formed by negatively charged residues such as serine (Ser266) and glutamic acid (Glu262,

241) and facilitate the flow of cations through the receptor.9, 68, 69 The small peptide

sequence containing the intracellular charged ring is also called “charge selectivity filter”

because it is the receptor region that determines the characteristics and magnitude of ion-

charge discrimination.69 The residues involved in the formation of the rings are conserved

in most of the neuronal nAChR subunits.24

The gate blocks the ion flow through the ion channel when the receptor is in its

inactive state but when the receptor is activated by the agonist binding, the gate is opened

and the ion flow is allowed. The proposed mechanism through which the gate is opened

is schematically illustrated in Fig. 1.9.9 On the left side of Fig. 1.9 is shown the region of

an α subunit that contains the parts involved in communicating the conformational changes induced by ligand binding in the extracellular domain down through the M2 segments and to the gate causing it to open. The blue and red colors of the helices mark the portions that are located outside (blue) and inside (red) the membrane. The yellow region indicates part of the inner β-sheet of the N-terminal domain of the subunit and the rest of the structure is not shown (for a complete picture of the subunit structure refer back to Fig. 1.5). Also indicated are two loops β1-β2 and M2-M3 that are significant for the gating mechanism.9 In the image from the right side of Fig. 1.9, the inner sheets and 37 the M2 helices of the α subunits are marked in gray while the outer sheet and M1, M3,

M4 segments are shown in white. This image also shows the binding site for agonists

ACh.

Figure 1.9. Proposed gate opening mechanism of nAChRs.9

Some residues located on the inner β-sheet of the α subunit are components of the

binding site, therefore, upon ligand binding, conformational changes occur at this

position that lead to a rotation of the inner sheet by 15° (indicated in Fig. 1.9 by the black

curved arrows) about an axis oriented perpendicular to the membrane plane.9 The rotational movements of the inner sheet are transmitted to the M2 segment through a pin- into-socket interaction between the β1-β2 and M2-M3 loops that is translated into a twist of M2 segment in the same sense since there is only minimal contact of M2 with the other segments.9, 60, 70 Similarly, the same rotational movements are produced by the

ligand binding at the second binding site as illustrated in the right image from Fig. 1.9. 38

The same sense twisting movements of the M2 helices weaken the hydrophobic interactions that hold the girdle together and as a result the gate is widened thereby allowing the ions to pass through the channel.

1.3.3. N-Terminal domain of nAChRs

Remarkable insights in the understanding of the three-dimensional organization of the N-terminal domain (NTD) of nAChRs were obtained from the elucidation of the crystal structure of acetylcholine-binding protein (AChBP).62 As illustrated in Fig. 1.10

this protein lacks the transmembrane and intracellular domains present in the nAChRs.

Although the subunits are marked in different colors (yellow, blue and gray) for a better

clarity, the AChBP is a homopentamer. This protein is applicable as a model for the NTD

of the nAChRs because displays low but significant amino acid sequence homology with

this region of all members of ligand-gated ion channels.24 Almost all residues that are

conserved within the nAChR family are found in AChBP, including those that are

relevant for ligand binding and the subunit most closely relates to the α-subunit of

nAChRs.24, 62 For example, AChBP shows 24% sequence identity with the corresponding

region of the homomeric α7 nAChR.24 The crystal structure of AChBP revealed that the

pentameric complex has an outer diameter of 80 Å, an inner diameter of 18 Å, and 62 Å

height, which are in concordance with the findings in studies of the NTD of the Torpedo

nAChR structures.24, 62, 71

39

Figure 1.10. Pentameric structure of AChBP.62

1.3.3.1. Orthosteric binding site

The NTD contains the ligand binding sites for acetylcholine, also called

orthosteric binding sites, located at the interface between two subunits as indicated in Fig.

1.10 by the residues in ball-and-stick representation. The binding site, as shown in more

detail in Fig. 1.11, is a cleft formed by a series of residues from a region of one subunit

called principal or positive face and a series of residues from a region of the second

subunit called complementary or negative face.62 All residues participating in the ligand

binding were previously identified by photo-affinity and mutagenesis studies but only the

AChBP model revealed the spatial orientation of these residues with respect to each other.62, 72-76 The majority of the residues are aromatic residues grouped in six loops: loop

A, B, C positioned on the principal face and loop D, E, F positioned on the complementary face. The term loop used here does not reflect fully its true definition because the residues constituting the loop D-F are actually positioned on β-strands.24

40

Figure 1.11. Orthosteric binding site of AChBP.62

Often, the binding site is called the “aromatic box” because the bottom half and the walls of the cavity are formed by aromatic residues (Tyr and Trp) and a disulfide bridge from loop A, B, C, D and F.24 The hydrophobic residues from loop E form the top part of the binding site. The aromatic residues and the vicinal cysteines from the principal face are conserved in all nAChR α subunits except in α5 while the residues from the complementary face are much less conserved with the exception of a Trp from loop D.24

Although in Fig. 1.10 is shown only one binding site, the AChBP has five such orthosteric sites located at each of the five subunit interfaces. Analogously, the homomeric neuronal nAChRs have five orthosteric sites composed of the principal and complementary faces of the α subunits. On the other hand the muscle type nAChRs have two binding sites composed by the principal face represented by the α subunit and the complementary binding component represented by the γ/δ subunit. Similarly, the heteromeric neuronal nAChRs have two binding sites formed by residues from α subunit 41 as the principal components and residues from β subunit as the complementary

components.24

1.3.3.2. Allosteric binding sites

As mentioned before, the orthosteric sites are the positions where the endogenous

and the exogenous agonists as well as the competitive antagonists (inhibitors) bind. There

are other ligands known to influence the activity of the receptor without binding to the

orthosteric sites. These ligands are called positive allosteric modulators if their binding

activates the receptor and negative allosteric modulators if the receptor is inactivated as a

result of their binding. The allosteric modulators bind to the receptor at different sites

known as allosteric binding sites or noncompetitive binding sites. Several such binding

sites have been identified to date but three major ones have been show to exist on all

subtypes of nAChRs, specifically, the luminal, ethidium, and quinacrine-binding sites.45,

77-80 These three binding sites, two of them shown in the schematic representation of Fig.

1.12, have been characterized as noncompetitive inhibitor-binding sites because the

ligands bound at this positions inhibit the gate opening mechanism of the receptors.

β α δ

Ethidium γ α ACh Site 60 Å 46 Å 7 Å

Quinacrine

Figure 1.12. Allosteric binding sites of nAChRs.45

42

1.3.3.2.1. Luminal-binding site

The luminal-binding site is reported to be located on the surface of the internal lumen that forms the ion pore close to the gate and under the extracellular negatively charged ring.45, 77 It was initially assumed that the noncompetitive antagonists bind at this

site and exercise their inhibitory action by sterically blocking the channel thereby

preventing the ion flow.81 Further studies suggested an alternative inhibitory mechanism of these ligands, at least for one nAChR subtype that is the freezing of the receptor in its closed conformation by acting as a wedge and prohibiting the necessary movements for opening the gate to be finalized.45, 82

1.3.3.2.2. Ethidium-binding site

The high-affinity ethidium-binding site has been identified using the quaternary

amine ethidium, hence the name.83 The site has been described as a polyamine-binding

site and it has been indicated to be located in the NTD on the vestibule wall of the

channel, around 46 Å above the transmembrane region and slightly above the level of the

two orthosteric sites as indicated in Fig. 1.12.45, 77 For the muscle-type nAChRs that were

the most extensively studied, this binding site can include either a region near the αγ interface or a portion of the β subunit.77

1.3.3.2.3. Quinacrine-binding site

The quinacrine-binding site has been indicated to be located in the transmembrane

domain at the border between the nAChR and the membrane, around 7 Å below the

aqueous-lipid interface as illustrated in Fig. 1.12.77 This site is the primary-binding site

for quinacrine, an antimalarial agent.45 43

The function of the nicotinic acetylcholine receptors can be modulated through the multiple orthosteric and allosteric binding sites. In Fig. 1.13 is shown a schematic representation that describes the allosteric nature of nAChRs. It is believed that in the absence of the agonist or antagonist the receptor fluctuates between at least three functional states, namely, a resting state (gray complex in Fig. 1.13), an active state (red) and a desensitized state (blue).24, 84 The energy barrier between two states determines the

kinetic rate for the transition from one state to another. Activation is a rapid transient process while desensitization occurs slowly. The rates of these two processes vary among different nAChR subtypes and have a great contribution to the different pharmacological properties of this class of receptors.24 The channel is inactive in the resting and

desensitized states and is only open in the active state.

+ 2+ + 2+ Na Ca Na Ca Na+ Ca2+

Resting Active State State + 2+ Na Ca Na+ Ca2+

Desensitized Desensitized State State

Figure 1.13. Allosteric nature of nAChRs.24

The resting and the active states are stabilized upon binding of an agonist (green

square in Fig. 1.13) or a competitive antagonist at the orthosteric sites. Typically the 44 agonists have a higher affinity for the active state than for the resting state leading to channel opening and ion flow. On the other hand, the competitive antagonists have a higher affinity for the resting state than for the active state, which prevents the transition to an active state thereby preventing the channel opening. An antagonist could also have a higher affinity for the desensitized state leading to an increase in the recovery from this state that can be translated to an essential blockage of the receptor. The binding of the agonist to one of the binding sites increases the probability of channel opening, which is shown by the gray arrow passing through the magenta complex in Fig. 1.13, and it is even more increased by the binding of agonist at both orthosteric sites.24 The noncompetitive ligands that bind to the allosteric sites can also modulate the function of the receptor by having an effect on the desensitization kinetics or by influencing the equilibrium between the resting and the active states.24, 84

1.4. Therapeutic potential of nicotinic acetylcholine receptors

Because of their important functional roles as mediators of synaptic transmission

and modulators of many important neurotransmitter systems, their membrane location

and structural heterogeneity, nAChRs are ideal as targets for the manipulation of brain

and body functions to reestablish homeostasis when it goes away.6 The most compelling

arguments in the rational for targeting nAChRs to treat a variety of diseases derive from the beneficial effects observed after administration of nicotine to humans.3 Such effects

of nicotine include increased alertness, improved memory, enhanced learning, muscle

relaxation, reduced anxiety and analgesia.45, 46, 85 Clinical and laboratory studies shown

that neuronal nAChRs are involved in complex brain functions such memory, attention 45 and cognition, therefore the observed beneficial effects of nicotine lead to the assumption that the therapeutic responses associated with this compound are primarily due to its interaction with these receptors.45 Nicotinic acetylcholine receptors are involved in a

large number of neurodegenerative and psychiatric disorders for which nicotine proved to

have therapeutic applications.3 Some of the major disorders are briefly mentioned in the

following discussion with emphasis on nicotine effects and nAChRs involvement

although the exact mechanisms of their involvement remain unclear.

1.4.1. Disorders involving nAChRs

1.4.1.1. Alzheimer’s disease

One of the most studied neurodegenerative disease is Alzheimer’s disease (AD)

that is the most common cause of dementia characterized by undisturbed motor and

sensory abilities while cognitive functions, particularly memory, attention and orientation are progressively declining.6, 86 Studies on the AD patients indicated a consistent loss of

subtypes of nAChRs in their brain, proving the involvement of nAChRs in this disease.87,

88 It is believed that the decrease in receptor density starts early in the course of the

disease and is correlated with cognitive impairment.89 Some studies revealed that

smoking reduces the likelihood of developing AD.90 Moreover, nicotine was shown to

improve cognitive function in AD patients by a direct cholinergic stimulation and/or by

increasing the release of other neurotransmitters.89, 91-94

1.4.1.2. Parkinson’s disease

Another disorder that received much attention is Parkinson’s disease (PD), a

neurodegenerative movement disorder characterized by muscular rigidity and tremor. In a 46 recent study was found that there is a decrease by 50 to 70% of high-affinity nAChR density in specific regions of the brain of patients with PD.95 Epidemiological studies

indicated that like in the case of Alzheimer’s disease, there is also a decrease incidence of

PD among smokers compared to nonsmokers proving the protective effect of nicotine

against this disorder.89, 96 Other studies shown that the administration of nicotine as a

combination of patch and gum to nonsmoking patients with PD, significantly reduces

rigidity, tremor, disorganized thinking and depression in these patients.89, 97 It appears

that nicotine exerts its beneficent action on PD by increasing the release of a

neurotransmitter called dopamine98 and by inhibiting the enzyme responsible for dopamine breakdown.99

1.4.1.3. Tourette’s syndrome

Tourette’s syndrome (TS) is a hyperkinetic disorder of genetic origin that is

characterized by brief, rapid and sudden vocal and motor tics typically experienced as irresistible. It is commonly associated with obsessive-compulsive behavior, extreme temper or aggressive behavior, attention deficit/hyperactivity disorder and learning disabilities.99 It was proposed that the TS symptoms are produced by excessive dopamine

release which is supported by the therapeutic effectiveness of antagonists for dopamine

receptors.100 Although nAChRs are not directly affected in this disorder, they can be used as therapeutic targets based on the fact that some studies shown that stimulating the brain nAChR reduces the tics.99 Thus, tics and other symptoms associated with TS that were

not optimally controlled by the dopamine receptor antagonist haloperidol alone were

rapidly decreased when nicotine was associated with this compound.101, 102 Other 47 observed beneficial effects of nicotine in TS are improvements in attention span and concentration, effects that were attributed to nicotine’s properties to enhance cognition.103

1.4.1.4. Schizophrenia

Schizophrenia is a chronic psychiatric complex disorder with a strong genetic

predisposition.89 The involvement of nAChRs in this disorder was confirmed by the post-

mortem studies on schizophrenic patients that revealed a decrease in nicotinic receptors

expression affecting the α7 and α4β2 subunits in various cerebral areas.104 High levels of

nAChR antibodies were also observed in schizophrenic patients, which were suggested to

contribute to nicotinic receptors deficit.89, 105 An extremely high incidence of smoking in

schizophrenic patients was observed and smoking withdrawal resulted in worsening of

the disease symptoms.106 These observations suggested that smoking represents an

attempt at self-medication in schizophrenic patients. Studies shown that nicotine also

normalizes an auditory gating deficit found in schizophrenia.107

1.4.1.5. Depression

Depression is a disease characterized by affective, cognitive and behavioral

deficits. Although not fully understood, it is hypothesized that the cause of depression is

due to a deficit of dopamine and serotonin (another neurotransmitter) release in brain.99

Lesion of dopaminergic system induces the incapacity of experiencing pleasure.108, 109

There are studies suggesting that smoking is also a sort of self-medication among patients with depression.99, 110

Nicotinic acetylcholine receptors have also been suggested for the treatment of

other disorders such as attention-deficit hyperactivity disorder, autism, hereditary 48 epilepsy as well as anxiety, pain and smoking cessation.3, 6, 24, 99, 110 Nevertheless, the above discussion brings enough evidence to emphasis that nAChRs are an important class of targets with high potential to impact numerous therapeutic areas.

1.4.2. Drug discovery of nAChR modulators

Despite the existence of proof-of-principle findings for the effectiveness of nicotine in previously mentioned diseases among others, issues remain with respect to minimizing the complications observed in using nicotine. These include adverse effects on health such as action on the cardiovascular and gastrointestinal systems, sleep disturbance, elevated blood pressure, and at high doses neuromuscular effects and seizures.3, 85 Also, the use of nicotine creates addiction. Given these side effects

associated with nicotine administration, the main goal for drug discovery in the nAChRs

field was to find new compounds that would retain the beneficial effects of nicotine and

would not exhibit its side effects.

Most of the efforts in medicinal chemistry targeting nAChRs have been focused on the search for analogs of nicotine. However, besides nicotine other compounds are also known to have high affinity and selectivity for nAChRs and were also used as sources for designing and synthesizing new ligands for these receptors.3, 24, 89 Some of these compounds are presented in Fig. 1.14 along with the endogenous neurotransmitter acetylcholine 1.1. These compounds are isolated from natural sources and since they show selectivity for nAChRs, their use as leads ascertains that the new generations of ligands will not show cross-activities with muscarinic acetylcholine receptors or other classes of receptors. However, except methyllycaconitine (MLA) 1.6 none of these 49 ligands display any significant functional preference between the very diverse neuronal nAChR subtypes.24

O H Cl N N N O N N OMe OMe Acetylcholine (1.1) (S)-Nicotine (1.2) (± )-Epibatidine (1.3) OH MeO OH OMe N O O N HN O O N N

O N (-)-Cytisine (1.4) Anabaseine (1.5) Methyllycaconitine (1.6)

Figure 1.14. Naturally occurring nicotinic ligands.

The subtype-selectivity is a key characteristic for a successful potential drug. For example, a good candidate would be a compound that has selectivity for the nAChR subtypes (i.e. α4β2*) through which nicotine elicits its benefic effects and would show

no binding to those subtypes (i.e. α3β4*) that are mediating the adverse effects of

nicotine. The subtype-selectivity remains the biggest challenge for medicinal chemistry efforts in the nAChRs field, hampered by the high degree of homology between the orthosteric sites of the nAChR subtypes. As it was mentioned before (recall 1.3.3.1) the residues constituting the principal component located on the α-subunits that participate in

the orthosteric ligand binding are highly conserved and only the residues from the

complementary component are more diverse. Therefore, it is expected that the distinction

between α subunits, for example α2 and α4, to be a more difficult task than the 50 differentiation between β2 and β4 subunits. An important aspect about selectivity that needs to be noted is that this term is usually used in a broad sense, namely, when a ligand

is reported to be selective for one subtype of receptors it does not mean that only binds to

this specific receptor, it just means that the selectivity toward other receptor subtypes is

much lower. No truly subtype-selective ligand for nAChRs has been identified yet.24

Despite all of the challenges associated with the rational ligand design, a very large number of nAChR ligands have been published to date.24, 89, 111 The following

discussion will not be a systematic review of the plethora of these compounds, instead, a

selection of the key nAChRs ligands and their characteristics as agonists, competitive

antagonists and allosteric modulator will be presented.

1.4.2.1. Agonists

An agonist for nicotinic receptors is a compound (ligand) that binds at the

orthosteric sites and triggers the same activity produced by the binding of endogenous

neurotransmitter acetylcholine. When the agonist elicits full efficacy at the receptor it is

called full agonist and when only partial efficacy is displayed at that particular receptor it

is called partial agonist.

Since early 1990s, when rational approach to the design of new nicotinic receptor

agonists began, a large number of compounds were reported to exhibit more favorable

side effects and toxicity profiles compared to nicotine in animal models. Many of them

were also reported to exhibit therapeutic potential in preclinical studies.46 One of the first in a series of ligands developed by Abbot scientists was ABT-594 also called tebanicline

(Fig. 1.15), an analog of epibatidine (1.3 in Fig. 1.14) with full agonist potency at α4β2 51 and α3β4* nAChRs.112, 113 In animal models, this compound was found to be an effective

broad-spectrum analgesic for acute, persistent pain with a potency of 200-times greater

than that of morphine. Moreover, the compound did not evoke the withdrawal symptoms

or physical dependence associated with opioids.114, 115 This discovery had a full media

coverage, print, radio and TV that led even a songwriter (Paul Simon) to memorialize the

event in a song.116 It was shown that the analgesic activity of tebanicline was due to its

potency at α4β2 nAChR subtype and it was in good agreement with the well-documented

antinociceptive properties of nicotine and epibatidine.114, 115, 117 Moreover, due to its lack

of activity at the muscle-type nAChRs, tebanicline did not evoke side effects such as

hypertension and neuromuscular paralysis observed for epibatidine at the same level of

severity and entered the clinical trials.24, 114 However, its development has been

discontinued after phase II trials as a consequence of its gastrointestinal adverse effects

generated by its activity at α3β4* nAChRs.24

OMe

OMe

(R) (S) (S) O O N N N H H N N Cl N N N

Tebanicline (ABT-594) ABT-089 Altinicline (SIB-1508Y) GTS-21

H N H2N O N HN N N N

TC-2403 TC-2559 Varenicline

Figure 1.15. Agonists of nicotinic receptors. 52

Another compound developed by Abbot is ABT-089 (Fig 1.15), an analog of nicotine. Opposite to tebanicline that is a full agonist of α4β2 and α3β4* nAChRs, ABT-

089 displayed remarkable low potencies at these receptors as well as at α7 nAChR.118, 119

Nevertheless, it exhibited very good binding affinity for native α4β2* receptors

suggested to be a consequence of a possible potency at other subunits incorporated in

α4β2* receptors such as α3 and/or α5 subunits.24, 119 ABT-089 was found to be as potent

and efficacious as nicotine in stimulating the release of acetylcholine and exhibited

significant positive effects for anxiety and cognitive functions. Although the molecular

basis of its effects have not been completely elucidated, the compound is currently in

phase II clinical trials for cognitive disorders such as Alzheimer’s disease and attention-

deficit hyperactivity disorder. Furthermore, the compound has a dramatically improved

safety profile compared with nicotine with regard to cardiovascular and gastrointestinal

side effects.24, 118-121

A lead compound also targeting central nervous system disorders is SIB-1508Y

termed altinicline (Fig 1.15), a nicotine analog developed by SIBIA/Merck Company.

This compound is a partial agonist characterized by a modest functional preference for

α4β2 nAChR over β4-containing subtypes, while displaying no activity at α7 or muscle- type nAChRs.24, 122, 123 The low activity at α3β4-containing nAChR subtypes indicates the potential low cardiovascular effects of this drug. Altinicline was found to be as

efficacious as nicotine in inducing the release of the neurotransmitter norepinephrine in

thalamus and cortical regions and more efficacious than nicotine in stimulating the 53 release of striatal dopamine.123 Since the Parkinson’s disease manifests brain cholinergic and dopamine deficits, altinicline was suggested for the treatment of this disease. It was also found that the compound enhanced vigilance, reversed the attention deficit and alleviated a learned helplessness task in animal models.46, 124, 125 Therefore, it entered

phase II clinical trials for early-stage Parkinson’s disease.

GTS-21 (Fig. 1.15) was patterned after the invertebrate toxin anabaseine (1.5 in

Fig. 1.14) and has been characterized pharmaceutically as partial agonist with preference

for the α7 subtype nicotinic receptors.46, 126 This compound was shown to have cognitive

enhancing action with a low side effect profile relative to nicotine.127 It also has

neuroprotective effects in the experimental models of Alzheimer’s disease and improves

attention and working memory.128 GTS-21 was reported to improve auditory gating

deficits in animal models and therefore was advanced for clinical trials for schizophrenia and also AD and ADHD based on preclinical profile in animal models.129

The company Targacept has published several interesting subtype-selective

nAChR agonists.24 In addition to the following examples, other nAChR agonists have entered the phase II clinical trials for pain and depression among others.130 One example

is compound TC-2403 (Fig. 1.15) derived from nicotine by opening the pyrrolidine ring.

In fact TC-2403 is a metabolite of nicotine. This compound is a partial agonist exhibiting

some functional preference for α4β2 over other α/β, α7 and muscle-type nAChRs.131, 132

In microdialysis studies of cortical neurotransmitter release, TC-2403 has been demonstrated as efficacious as nicotine in increasing the extracellular levels of acetylcholine, dopamine, norepinephrine and serotonin and it was also reported to display 54 analgesic effects in several animal models.24, 133 This compound is currently in phase II

clinical trials for ulcerative colitis.134

Another example of agonist developed by Targacept is TC-2559 (Fig. 1.15) that is not very different in structure compared to TC-2403. The two minor modifications though, resulted in a very different pharmacological characteristics for TC-2559 that is a potent and selective agonist for α4β2 receptor.135 This compound has shown at least 70-

fold higher potencies at α4β2 than at α3β2, α2β4, α3β4, α4β4, and α7 nAChRs.136 It has shown promising performance in the models of cognitive functions and exhibited significantly reduced degrees of side effects such as changes in blood pressure and heart rate and hypothermia compared to nicotine.110, 135

The only approved drug active at central nervous system nAChRs that was

derived from rational medicinal chemistry efforts is varenicline (ChantixTM; Fig. 1.15)

that was lunched in 2006 by Pfizer for use in treatment of smoking cessation.130 This

compound is an analog of cytisine (1.4 in Fig. 1.14) and has been shown to be a partial

agonist of α4β2 nAChR subtype.137 It was believed that the partial agonism of this

compound may reduce the nicotine craving without itself being rewarding or addictive.

This hypothesis was supported by the preclinical studies and consequently varenicline is

the best of any approved smoking cessation drugs. The introduction of varenicline on the

market solidifies the proof-of-concept for a “safe” and non-addictive nicotine-like

compound and also lends credibility to the use of synthetic nicotinic receptor agonists for

other indications.130 55

1.4.2.2. Antagonists

An antagonist for nicotinic receptors is a ligand that binds at the orthosteric sites and inhibits the activity of the receptors, more exactly, prevents the gate opening of the channel. This type of ligand is commonly named competitive antagonist because it competes with the endogenous ligand and the agonist for the same binding sites. There are also noncompetitive antagonists but they will be discussed in a separate section.

The nAChR antagonist development has received limited attention compared to agonists possibly because the potential therapeutic application of the agonists is clearer than that of antagonists. However, antagonists can be valuable if used as a tool in understanding the physiological role of nAChRs, especially if they show subtype selectivity.89 For example, considerably insights into the molecular composition of

heterologous native nAChR population were obtained using α-conotoxins that act as

inhibitors of these receptors. The α-conotoxins are a group of small peptides of 12-20

amino acid residues that were isolated from the venom of a snail.138, 139 One of these α- conotoxins, called ImI has been an important tool in studies of α7 subtype for which it is

a selective antagonist.139-141 Other important α-conotoxins are MII that was shown to interact with high-affinity with α3β2 and probably with α6* receptors142, 143 and AuIB that was reported to be the only α-conotoxin that is only selective for α3β4 nAChR subtype although it remains to be characterized at α6β4 nAChRs.144

Other competitive antagonist for nAChRs are α-bungarotoxin, a peptide toxin isolated from the venom of Taiwan banded krait and methyllycaconitine 1.6 (Fig. 1.14), an alkaloid isolated from Delphinium species of plants. These two compounds have 56 shown high competitive selectivity for the homomeric α7 nAChR subtype.24

Methyllycaconitine 1.6 is the main lead compound of the research presented in this dissertation therefore a more detailed discussion on this compound and its role in drug development will be presented in a separate section (Section 1.4.3)

The peptide α-bungarotoxin as mentioned previously (recall section 1.2.2.2) made it possible to identify the α7 nAChR subtype in the central nervous system. Its selectivity

though, only applies to the central nervous system because it is a potent antagonist for the

muscle-type nAChRs and binds to the α9 and α9α10 nAChR subtypes as well. These

subtypes are not located in the central nervous system hence the affinity shown by α-

bungarotoxin to these receptors does not diminishes its utility for studies targeting the

central nervous system.24

An example of synthetic antagonist with therapeutic potential is compound 1.7

(Fig. 1.16), an analog of epibatidine. This compound is the most potent competitive nAChR antagonist published to date with a binding affinity of Ki = 1 pM for rat brain

α4β2* nAChRs which is 26 times greater than that of epibatidine.145 The compound

showed antinociceptive activity in models of acute pain. However, it exhibited a dual

agonist/antagonist profile that could be caused by activity at different subtypes, which

remains to be verified.145

NH2 H Cl N N

1.7

Figure 1.16. Antagonist for nicotinic receptors. 57

1.4.2.3. Allosteric modulators

Although allosteric modulators can influence the nAChRs signaling by acting as agonists or antagonists they are discussed separately from the previous sections because they exerts their modulatory action by binding at different sites than the orthosteric sites.

For the same reason they are also called noncompetitive ligands and they can act by potentiating the effect of agonist hence are called positive allosteric modulators or can act as inhibitors and therefore are called negative allosteric modulators or noncompetitive antagonists.

Many endogenous allosteric modulators are known for nAChRs and for ligand- gated ion channels in general such as ions, proteins and fatty lipids.24 An interesting

example of such modulator is the divalent cation Zn2+ that modulates a wide range of

neurotransmitter receptors and transporters. In the case of nAChRs, the ion metal exerts

its action on α2β2, α4β2, α2β4, α3β4, and α4β4 subtypes displaying a biphasic

modulation. At concentrations up to 100 μM it acts by increasing the agonist responses

while at higher concentrations acts as inhibitor of the receptor.146

Considering the known susceptibility of other ligand-gated ion channels to allosteric modulation, surprisingly few exogenous allosteric ligands for nAChRs have

been published.24 Nonetheless, a few examples are worth mentioning. Galantamine (Fig.

1.17) is the most frequently prescribed treatment for Alzheimer’s disease that acts as an inhibitor of acetylcholinesterase, the enzyme that breaks down the neurotransmitter acetylcholine after the activation of the receptors. It was shown that this compound also 58 acts as allosteric potentiator of nAChRs.147 It is believed that it binds to an allosteric site

in the N-terminal domain increasing the receptor’s affinity for the orthosteric agonist

and/or the probability of ion channel opening.147, 148 It is not selective for a specific

subtype of nAChRs and the significance of its allosteric modulation for its beneficial effects in Alzheimer treatment is still to be established.

MeO Cl HO HN O O N H N Cl N H NH H O

HO Galantamine 5-Hydroxyindole (1.8) Mecamylamine Bupropion Ketamine

Figure 1.17. Allosteric modulators of nAChRs.

Another example of positive allosteric modulators is 5-hydroxyindole 1.8 (Fig.

1.17). This compound has been reported to enhance the release of neurotransmitter glutamate mediated by α7* receptors and also acts as allosteric potentiator at native homomeric α7 nAChRs.149 A 4-fold increase of the potency and the maximal response of

ACh response at the respective receptors was observed at a concentration of 1 mM of 1.8

while at a concentration of 10 mM the response was enhanced by 12-fold.24, 149

An interesting example of noncompetitive antagonist for the nAChRs is

mecamylamine also shown in Fig. 1.17. In 1950s, this compound was marketed in US as

an antihypertensive drug (Inversine) but its use was limited because at doses required for its antihypertensive effects, side effects were observed arising from the inhibition of α3* 59 receptors of the peripheral system.24, 150 However, it is for this inhibitory activity that this

compound was reintroduced on the market in recent years for use in treatment of

symptoms of Tourette’s syndrome. Mecamylamine is only slightly selective for α3-

containing subtypes and it was reported to bind to the luminal-binding site.151 It was also

proposed as potential therapeutic for treatment of drug abuse and at doses below the one required for antihypertensive effects is currently undergoing phase II clinical trials for attention-deficit hyperactivity disorder.24, 150

Another example of noncompetitive antagonist is bupropion (Fig. 1.17). This compound is also a marketed drug (Zyban) that is used clinically as an antidepressant and as a smoking cessation aid.24 When the compound was marketed its pharmacological

effect was ascribed to its inhibitory activity on other type of receptors, namely the

norepinephrine and dopamine transporters, but recently it has been shown to also have

moderate antagonistic effects on nAChRs.152-154 This finding led to an increased interest

in nAChRs antagonists as potential antidepressants and smoking cessation aids.16 For

most of the compounds reported as noncompetitive antagonists, their inhibitory activity

to nAChRs was a secondary discovery since they were drugs originally directed at other

receptors. An example is ketamine (Fig. 1.17), a drug used in anesthesia that shows

impairment of cardiovascular function. This side effect was recently attributed to its

inhibitory action on the ganglionic nAChRs.45, 155

The allosteric modulators present an opportunity for new drug development

because of their advantage over the ligands that bind to orthosteric sites. This advantage

comes from the fact that the allosteric binding sites are typically less conserved than the 60 orthosteric sites therefore the development of allosteric ligands could be a way to circumvent the subtype-selectivity problems associated with orthosteric ligands. More efforts in this promising approach for drug design targeting nicotinic receptors are illustrated by the structurally simplified analogs of methyllycaconitine in the following discussion.

1.4.3. Methyllycaconitine

Methyllycaconitine (MLA) 1.6 (Fig. 1.18) is one member of a larger family of diterpene alkaloids, isolated from Delphinium and Aconitum species of plants that have been known as sources of poisons and medicinal agents for a long time.156, 157 As it was

mentioned before, methyllycaconitine is the most potent nonpeptide competitive

antagonist of nicotinic acetylcholine receptors known with selectivity for α7 nAChR subtype at low nanomolar concentrations (1.7 nM).156, 158-163 It is for this characteristic

that MLA received much attention and it was subjected to derivatization and structure-

activity relationship (SAR) studies. Small modifications of its structure as illustrated in

Fig. 1.18 by red marks has resulted in elatine (1.9) and nudicauline (1.10) that are

equipotent to and slightly more potent than MLA as α7 antagonist.164 61

OMe OMe OAc OMe OMe OMe OH O OH MeO MeO MeO OH O OH OMe OMe OMe N N N O O O O O O N N N O O O O O O

Methyllycaconitine (1.6) Elatine (1.9) Nudicauline (1.10)

Figure 1.18. Modifications of methyllycaconitine.

The structure-activity relationship studies on the MLA164, 165 revealed the

importance of the methylsuccinimidobenzoyl portion of the molecule for the high-affinity

at α7 receptors since a 2000-fold decrease in affinity was observed when this moiety was

removed.166 The removal of the methyl group from the succinimide ring decreased the

binding affinity 20 times and the removal of the methylsuccinimide ring resulted in more

than 1000-fold decrease in affinity.167

Later was reported that besides its α7 affinity, MLA also competitively antagonizes heteromeric nAChRs such as bovine adrenal α3β4* nAChRs, although with

potencies ~1000-fold lower than at α7.168 The α3β4* nAChRs are reported to be the

principal receptors that mediate the adrenal catecholamine secretion.169-171 A series of

simplified analogs of MLA, so-called ring E analogs with general structure shown in Fig.

1.19 were reported to inhibit nAChR-stimulated adrenal catecholamine secretion thus proving the activity of MLA on α3β4* nAChRs.156 Moreover, these analogs displayed

negligible activities at α7 indicating that ring E analogs are moderately selective for

α3β4* receptors.168 62

R2

O O O O O N O N O O N N R1 Ph

Ring E analogs LB-8

Figure 1.19. General structure of ring E analogs of methyllycaconitine.

The first set of ring E analogs was constituted of compounds with various

substituents (R1) on the nitrogen atom of the piperidine ring with or without the methyl

group on the succinimide ring.168 For these compounds it was reported that the optimal

potency at α3β4* nAChRs was observed when the substituent at the nitrogen of the piperidine ring was 3-phenylpropyl chain. Also the study revealed that the potency decreases if the succinimide ring is not substituted. Therefore, the most potent of this set of analogs was compound LB-8 shown in Fig. 1.19 that proved to be almost as efficacious as MLA at inhibiting the adrenal secretion.156 Competitive binding studies

revealed that ring E analogs interact with α3β4* nAChRs in a different manner than

MLA. Methyllycaconitine binds to the orthosteric binding sites hence is a competitive

antagonist for α3β4* receptors while ring E analogs displayed a noncompetitive

interaction with these receptors acting as noncompetitive antagonists.168 Furthermore,

information about the stereospecificity on binding of ring E analogs at nAChRs were obtained by preparing all four diastereomers of LB-8 in enantiopure form and assayed for

potency at α3β4* and α7 receptors. The results revealed that all four diastereomers 63 showed the same potency for both types of receptor indicating that the binding at nAChRs is probably non-stereospecific or that the difference in how the enantiomers bind to the receptors is quite small.172

A second study was initiated using LB-8 as lead compound, aimed to optimize the

α3β4* functional activity of ring E analogs through modifications at the succinimide

moiety while retaining the 3-phenylpropyl substituent on the nitrogen.158 These modifications involved the replacement of the methyl group on the succinimide ring with various alkyl chains and also involved the complete replacement of the succinimide ring with other substituents. All new analogs have displayed improved potency compared with the lead LB-8 and none of them have shown affinity for the orthosteric sites of either α7,

α4β2 and α3β4* nAChRs indicating that they also act as noncompetitive antagonist.

Extensive studies based on the pharmacological activities of all ring E analogs mentioned

above have led to the generation of 3D-QSAR computational models that could qualitatively identify new allosteric nAChR antagonists.173 In addition, a pharmacophore

model was also developed that delineated the binding requirements to the allosteric

binding site. Furthermore, the position of the allosteric binding site for ring E analogs

was identified as located within the pore at the α/β interface at ~7 Å from the orthosteric

binding site.174

Although previous studies mentioned above indicated that the binding of ring E

analogs to the allosteric site is probably non-stereospecific, two compounds from the

latest series of analogs, compounds 1.11 and 1.12 (Fig. 1.20) with disubtitution on the succinimide ring, due to their increased potency, suggested that the stereochemistry of the substitution on this ring is an important consideration in the development of future 64 analogs. To address this issue and to better understand the structural features of the nAChR-small molecules interactions more studies have been initiated that constitute part of my research project presented in this dissertation.

Ph

O N O O O O N O O O N O

O O O

N N N

Ph Ph Ph

1.11 1.12 IB-10

Figure 1.20. Ring E analogs of methyllycaconitine.

For this new project a new lead compound, IB-10 shown in Fig. 1.20, was chosen from the second set of analogs because of its increased potency compared to LB-8 analog. The enantiomers, various diastereomers and some analogs of this new lead compound were synthesized and analyzed to probe the stereospecific character of the interaction with the receptors. Before giving a detailed description of the work involved in this project, a discussion intended to explain the importance of chirality of a small molecule when considering its interaction with a chiral biomolecule will be presented in the following section.

1.5. Chiral recognition

The biological systems are biopolymers made of various constituents such as amino acids and carbohydrates, with the same sense of chirality. The essential amino acids in the case of mammalian proteins are all L-enantiomers, while most of the 65 carbohydrates have the D-configuration. This defined configuration of their building blocks and the resulting secondary and tertiary structures confer to biological systems the characteristics of chiral macromolecules.

When dealing with relative small molecules, ‘chirality’, in the broadest meaning of the term is a molecular property of nonsuperimposable molecules that are related as mirror image, called enantiomers. Enantiomers have identical chemical and physical properties but differ in the position of atoms in space and the sense of which they rotate the plan-polarized light. The enantiomers may be seen as identical or different depending on the environment in which they are studied. In an achiral environment the enantiomers will show identical behavior and it will be impossible to differentiate between them.

When placed in a chiral environment, the enantiomers will show different behavior and they can be differentiated. Therefore, the enantiomers of a chiral ligand introduced into the body will show differential interaction with chiral targets such as receptors, enzymes and carrier proteins. A receptor will be able to recognize certain geometrical features of a chiral molecule such as crucial distances, angles and optical handedness. Upon binding of a ligand to a receptor, the receptor emits a biological message also called signal that will be transmitted to some target in the organism. As a consequence of their differential interaction with chiral receptors, the enantiomers of a chiral compound will effect different changes in the organism.175 Therefore, when studying chiral biologically active

compounds it is very important to study their individual enantiomers too. Such a study

will lead to a better understanding of their biological activity and their mechanism of

action but in the same time will lead to a better understanding of the structural features of 66 their biological targets. The molecular aspects and the difference in the biological response generated by a chiral molecule upon binding to a receptor will be further discussed in more detail.

1.5.1. Receptor-enantiomers interactions

In binding to a receptor, a ligand will form a complex with the binding site of the receptor. This binding is usually not achieved covalently but by noncovalent interactions.

The major noncovalent binding forces are hydrogen bonding, ionic interactions, ion- dipole and dipole-dipole interactions, π donor and π acceptor association, and hydrophobic interactions.176 These noncovalent interactions are weak forces that are

worth only between –1 and –7 kcal/mol in stability.177 Usually the cooperation of several

noncovalent interactions are required for the receptor to deliver the signal. After the

signal was transmitted, the noncovalent bonds are broken and the ligand is released

unchanged.

In the case of a chiral ligand binding to a receptor, with the assumption that both

enantiomers bind to the same type of receptor, two diastereomeric complexes are possible

since the receptor as a protein is a chiral molecule itself. Diastereomers have different

chemical and physical properties and in this case they also have different biological

properties. The ability of a receptor to discriminate between enantiomers, also called

molecular chiral recognition, is a result of these properties and energy differences

between the diastereomeric complexes formed upon binding of the enantiomers to the

receptor.175 For chiral recognition to be achieved, the 3-dimensional spatial arrangement

of the ligand requires a complementary 3-dimensional structure on the receptor with 67 which to form the necessary interactions (Fig 1.21).178 There are at least three different

interactions required for a receptor to differentiate enantiomers, according to a model first

proposed by Easson & Stedman in 1933.179 This model is known as the “three-point

contact” model that is used to characterize the requirements for chiral recognition.

Figure 1.21. Interaction of enantiomers of a chiral ligand with the receptor.178

According to the “three-point” interaction model, the enantiomer on the left in Fig 1.21 shows a higher affinity for the receptor than its paired enantiomer on the right. A high affinity means a more stable ligand-receptor complex. The enantiomer on the left is more tightly bound to the receptor because three of its substituents match the complementary triad of binding sites on receptor. This will lead to a stable diastereomeric complex. The interactions between the ligand and the receptor are symbolized by the dashed lines in Fig

1.21. For the enantiomer on the right, only two out of three possible interactions are formed with the receptor producing a less stable diastereomeric complex. The biological activity of a compound is directly related to its affinity for the receptor. Therefore, the 68 enantiomer on the right is expected to show lower biological activity, lower intensity of the signal transmitted by the receptor to the organism.175

The “three-point” contact model successfully explains the interaction between

relatively rigid small molecules and biomolecules. However, it does not wholly describe

the chiral recognition process. For more complex ligands or drug molecules, an important

aspect in chiral recognition is the conformational flexibility. When a ligand approaches a

receptor various attractive and repulsive forces arose. As a result, both the ligand and the

receptor will undergo slight conformational changes in order to reach a point where these

forces balance each other.180 To maximize all the positive interactions, a high degree of

complementarity is required because the greater the fit of the ligand with the receptor the higher the binding activity. For a better understanding of the recognition between a chiral

ligand and a receptor it is very useful to use the analogy of a hand and a glove.180

If a chiral molecule is viewed as a hand then, in the simplest form of chiral

recognition, there are few possibilities for interaction. If there were to be no chiral

recognition then the receptor could be regarded as a bag, with no front or back and no

fingers. In this case a loose fit would be achieved and either hand could fit in the bag.

However this is an uncommon situation because it implies that there is no asymmetry associated with the receptor site. A more probable case is that there is chirality associated with the receptor site but is not very discriminating. This would be a case where the receptor could be regarded as a mitten, which beside a front and a back has also a thumb.

In this situation a moderately good interaction will be allowed for one hand but a poor interaction for the other one. Starting from this last situation and taking in consideration 69 the conformational flexibility, a more likely situation could be imagined. After the interaction between the hand and the mitten, the fit for the receptor could improve and the mitten could grow fingers through conformational changes and turn into a glove. If the glove is very flexible then both hands could be accommodated but only the one that fits the best would be able to produce the desired intensity of the response. The other hand would be much less effective. There is also an extreme to this case where the glove doesn’t have much flexibility and therefore only the correct hand could fit and will be able to interact with the receptor.180 To conclude, the better the extent of the fit with the

receptor the better the differentiation between the enantiomers of a chiral ligand. This observation was first made by Carl Pfeiffer in 1956 and is known as Pfeiffer’s rule.181

1.5.2. Biological activities of enantiomers

As it was mentioned before, a better interaction with the receptor of one enantiomer of a chiral ligand over the other leads to a more stable complex that is translated into a better binding affinity. Since the binding affinity is closely related to the biological activity of the ligands, their chirality will have a direct effect on the message generated by their two component enantiomers leading to difference in their biological behavior. Typically one enantiomer will have a better activity then its paired enantiomer.

In the modern nomenclature of Ariëns the more potent enantiomer is referred to as eutomer and the less potent isomer is referred to as distomer.182, 183 The ratio of their

potency (receptor affinity) is referred to as eudismic ratio.

The effect of chirality on the pharmacological activity of enantiomers can be

divided in few types.175, 178, 180 One case is when both enantiomers have activity but they 70 differ in the intensity of the biological response generated. This is the case of the majority of biologically active compounds that occur as stereoisomers. An example is citalopram

(Celexa, Fig 1.22). Citalopram is an antidepressant medication that was marketed as the racemic mixture. It acts as a selective serotonin reuptake inhibitor. Clinical studies revealed that in fact the S enantiomer is about 150 times more potent then the R enantiomer and now it is also sold as the single active enantiomer.184

NC O N

F (S)-citalopram

Figure 1.22. The active enantiomer of citalopram.

The extreme of the above case is that one enantiomer is the eutomer while the second enantiomer is devoid of activity within the error limits of the measurement.

Actually the distomer is considered as an impurity in the mixture and in the modern nomenclature of Ariëns is term as isomeric ballast.182

Another case is when both enantiomers are biologically active and generate equal

responses. This means that there is no stereoselectivity of their activity. Referring to the

hand-glove analogy, this is the case where the receptor could be viewed as a bag. It is

also possible that the reason for equal activities to be the fact that the stereocenter of the

chiral compound lies outside the region critically involved in ligand binding. Either way,

this is a very unusual situation. An example for this type of behavior is promethazine (Fig 71

1.23 - the asterisks denote the chiral centers). Both enantiomers of promethazine

(Phenergam) are almost identical in their antagonistic action at the histamine H1 receptors and also in their toxicity.175

N N * N(CH3)2 * * * O O * N O O

S

promethazine dextropropoxyphene levopropoxyphene

Figure 1.23. Structures of chiral drug molecules.

It is also possible for both enantiomers to be active but to have different therapeutic activities. The unequal character of the messages generated by two enantiomers could be explained by their association with different receptor subtypes. An

example for such a case is propoxyphene for which the enantiomers are shown in Fig.

1.23. The 2R,3S-(+) enantiomer called dextropropoxyphene is a morphine like analgesic

while its 2S,3R-(-) enantiomer called levopropoxyphene is a therapeutically effective

antitusive agent.185 Therefore, the two enantiomers are marketed separately and even

their trade names are enantiomers. The dextropropoxyphene is called Darvon while the

levopropoxyphene is called Novrad (the mirror image of Darvon).

Another situation illustrating the difference in biological activity generated by the

stereochemistry of two enantiomers is when both enantiomers are active but have

opposite therapeutic effects. This is the case of picenadol (Fig 1.24) for which the 3R,4R

enantiomer is a μ-opiate agonist while the 3S,4S enantiomer is an antagonist.186 This case 72 could be used as an example of a potential danger in studying only the racemate of a chiral active compound. Studying only the racemate could lead to the wrong conclusion because the action of one enantiomer cancels the action of the second one while in fact, it is possible that the single enantiomers to show quite good binding affinities for a particular receptor.

HO HO

N N

(3R,4R)-(+)-picenadol (3S,4S)-(-)-picenadol

Figure 1.24. Enantiomers of picenadol.

A final possible case is when one enantiomer has therapeutic activity and the second is toxic. In Fig. 1.25 are presented the enantiomers of albuterol (Proventil) as an

example for this case. The compound is an antiasthma drug that acts as an agonist for β2- adrenergic receptors leading to bronchodilation that relaxes muscles in the airways and increases air flow to the lungs.187 The eutomer is the R enantiomer that appears to be solely responsible for the therapeutic effects. The distomer is the S enantiomer that is responsible for the side effects such as tremor, increase in pulse rate, and decrease in

blood glucose and potassium levels.188

73

OH OH H H N N HO HO

HO HO

(R)-(+)-albuterol (S)-(-)-albuterol

Figure 1.25. Enantiomers of albuterol.

The toxicity of biologically active compounds is most of the times related to the

distomer of a racemate and it should be considered as an impurity in the mixture.

Probably one of the most atrocious examples of drug toxicity is the well known case of

thalidomide (Contergan) (Fig. 1.26) that was used in early 1960s to prevent nausea during the pregnancy.175 Unfortunately if the drug was taken during the first trimester of

pregnancy, the side effects of this drug were shown to cause severe fetal limb

abnormalities. This teratogenicity (birth defect) of thalidomide was later attributed to the

S enantiomer.175

O O

N (S) O N (R) O NH NH O O O O (S)-(-)-thalidomide (R)-(+)-thalidomide

Figure 1.26. Enantiomers of thalidomide.

In 1999 almost a third of marketed drugs were single enantiomers and in only one

year the percentage increased to 40%.189 Lately, the availability of single enantiomer

drugs is rapidly increasing worldwide. The above discussion and the examples used to

illustrate the various cases, clearly underline the importance of stereochemistry in the 74 development and application of bioactive compounds. From a biological point of view, the enantiomers of a chiral molecule and a drug in particular, must be considered as different substances.

1.6. Summary

In this chapter was presented a discussion about the localization, structure and function of nicotinic acetylcholine receptors. These topics clearly illustrate that nAChRs are an important class of targets with high potential to impact numerous therapeutic areas.

Medicinal chemistry exploration into nAChRs led to a large variety of ligands that are used in the treatment of various phatophysiological conditions involving these receptors or are used as important tools for elucidating their physiological functions. Further elucidation of the composition and functioning of the subtype of these receptors and development of subtype-selective compounds are needed. Based on its unique interaction with nAChRs, methyllycaconitine is an inspiration source for medicinal chemistry. A series of smaller analogs of methyllycaconitine have shown promises as negative allosteric modulators that could provide important information for a further rational design of novel better and subtype-selective ligands. When designing new ligands for biological targets, an important characteristic that needs to be considered is the chirality of these ligands. A discussion on the importance and the effect of chiral recognition on biological activity of drugs was presented at the end of this chapter. 75

CHAPTER 2: SYNTHESIS OF RING-E ANALOGS OF MLA

2.1. Analogs of IB-10

The focus of this research project was to investigate the stereospecific character of the interaction of small analogs of methyllycaconitine with nicotinic acetylcholine receptors. As it was mentioned before, these analogs act as noncompetitive antagonists by binding to an allosteric binding site. This allosteric site was identified by computational modeling and blind docking which means that its characterization is yet to be elucidated.

Studying and understanding the structural features of the complex formed upon binding of the small molecules to the nicotinic receptors could provide valuable information about the structural feature of the binding site that could be further used for a rational design of new more potent and selective ligands.

2.1.1. Significance

The results obtained from the previous studies on ring E analogs of methyllycaconitine, conducted in the Bergmeier and McKay laboratories, provided a new lead compound IB-10 shown in Fig. 2.1. The choice of this new lead compound was based on its increased potency compared to the previous lead compound LB-8 and on the presence in its structure of the required structural features identified with SAR studies, namely the 3-phenylpropyl substituent on the piperidine nitrogen and substituent larger than methyl on the succinimide ring. The enantiomers and diastereomers and also some analogs of this new lead compound were prepared and assayed for their biological activity.

76

3' Ph

O N O O 3 O

N

Ph IB-10

Figure 2.1. Lead compound IB-10.

As it can be seen in Fig. 2.1, the structure of IB-10 presents two chiral centers:

one on the piperidine ring at position 3 and the second one on the succinimide ring at

position 3’. For reasons discussed in detail in Section 1.5 “Chiral recognition”, when

dealing with chiral compounds, the interaction of their enantiomers with the receptor is usually affected by the chiral recognition. If a chiral ligand is selective for a particular

subtype of a receptor this means that there is structural complementarity between the

ligand and the binding site of that receptor. This could be used to identify to some extent,

the structural features of the binding site. Understanding the interactions involved in the

formation of the ligand-receptor complex and the structural demands of a receptor for

good binding will be of a great help in developing more potent and selective bioactive

agents. Therefore, the long term goal of this research project is to better understand the

interaction of IB-10 with the receptor in a 3-dimensional (3D) sense. This can be

achieved by studying the individual enantiomers and diastereomers of the lead compound

separately. The next section is focused on a discussion about how the chirality of the lead

compound could affect its binding to the receptor. 77

2.1.1.1. Lead compound’s chirality

Because of the presence of two chiral centers in the structure of IB-10 and based on the chiral recognition abilities of a receptor, it is only fair to expect that one enantiomer will have a better fit with the receptor in comparison with its paired enantiomer. This better fit usually is translated into a better binding affinity. A closer look at the 3-D structure of IB-10 reveals characteristics that pose two questions about the stereospecificity of binding to the receptor. How would the stereocenter on the piperidine ring influence the interaction with the receptor? And what effect the stereocenter on the succinimide ring could have on the binding to the receptor?

Fig. 2.2 depicts the R and S enantiomers of the piperidine moiety of IB-10 (the remainder of the structure was omitted for clarity) in a chair conformation with the substituent at position 3 in an equatorial orientation. Only the chair conformations with substituents in equatorial positions are considered here because this conformations are the most abundant at equilibrium. The piperidine ring presents two chair conformations A and B (Fig. 2.2) that are interconvertable through inversion. When there is no substitution on the ring, the two chair conformations are equivalent and in a rapid equilibrium.

However, when the ring is substituted it will preferentially adopt conformations in which the substituent assumes an equatorial orientation. This can be exemplified on the two enantiomers of the piperidine ring. The spatial structure of the two enantiomers differs only in the orientation of the substituent at the chiral center. Therefore, for the enantiomers of a six-membered ring in a chair conformation, the substituent will be equatorial for one enantiomer and axial for the other enantiomer as illustrated in Fig. 2.2. 78

The inversion of conformation A of the R enantiomer to conformation B will change the position of the substituent from equatorial to axial. This inversion is energetically disfavored because of the axial steric interaction. Therefore, the preferential conformation of the R enantiomer will be A equatorial. On the other hand, the inversion of conformation A of the S enantiomer to conformation B will bring the substituent from an axial to an equatorial orientation that is devoid of steric interactions. As a consequence, the more favorable conformation for the S enantiomer will be B equatorial.

OR N N (R) OR A B eq ax

N N (S) OR A OR B ax eq

O (R) (S) O N N

Figure 2.2. Three-dimensional representation of piperidine ring enantiomers.

The preference of the substituted piperidine ring for one chair conformation over

the other was also supported by the conformational analysis carried out using Spartan.190

These calculations shown that the structure with the substituent in an axial orientation is less stable than the equatorial conformation by 2.223 kJ/mol (0.531 kcal/mol). Therefore, at any instant at room temperature 75% molecules have the substituent in an equatorial 79 position and only 25% in an axial position. Of course this ratio is dependent on the temperature, but even at physiological conditions (37 °C) the majority of the molecules will have the conformation with the substituent in the equatorial position 70%, and only

30% in the axial position, according to the same theoretical calculations.

A closer look at the spatial orientation of the substituent for the enantiomers of piperidine ring (ball and stick structure in Fig. 2.2) reveals that the substituent for both enantiomers is pointing in the same area in space. It is possible that for both conformations the interaction of the substituent with the binding site is the same. This might be translated as a non-detrimental effect from this chiral center on the binding affinity of the two enantiomers of the lead compound to the receptor.

On the other hand, the expectations are a little bit different when considering the

R and S enantiomers of the succinimide ring because this ring cannot flip to lead to different conformations. As a matter of fact the 5-membered ring is almost planar. As it can be seen from the 3D structures depicted in Fig. 2.3 (the remainder of the structure was omitted for clarity), the spatial orientation of the substituent is clearly different.

(R) (S)

O N O O N O

Figure 2.3. Three-dimensional representation of succinimide ring enantiomers.

For each enantiomer the substituent is pointing in different areas in space; coming out of the plane of the paper for the R enantiomer (left) and going into the plane of the paper for 80 the S enantiomer (right). Such a 3D structure could lead to different interactions of the substituent with the receptor. The substituent of one enantiomer can be associated with a complementary fragment of the binding site while the substituent of the second enantiomer may or may not interact with a different fragment of the receptor. This high possibility for different interactions offered by this stereocenter may have a great effect on the binding of IB-10 enantiomers to the receptor.

The hypothesis is that the two chiral centers will not have equal contributions in the stereoselective binding to the receptor. It is expected that the structural differentiation sensed by the receptor arose from the stereocenter on the succinimide ring (position 3’ on the general structure 2.1 in Fig. 2.4) to be more significant than the one from the piperidine ring (position 3 on structure 2.1). The stereochemistry of the piperidine ring may not have a significant contribution to the chiral recognition, due to equatorial orientation of the substituent for both enantiomers.

stereochemistry may R2 3' not be significant stereochemistry may be significant O N O O 3 O

N R1 2.1

Figure 2.4. Stereochemical features of ring E analogs.

2.1.1.2. Target compounds

With the structural differences and their possible effects on binding affinity,

generated by the presence of the two chiral centers in mind, a few sets of target 81 compounds were proposed for synthesis. The first set of compounds, shown in Fig. 2.5, consists of the two pairs of enantiomers of IB-10. A compound with ‘n’ stereocenters has

2n enantiomers, hence IB-10 has four enantiomers. Compounds COB-18 and COB-21

are one pair of enantiomers and compounds COB-19 and COB-20 are the other pair of enantiomers. The two pairs of enantiomers are related to each other as diastereomers.

(R) Ph (R) Ph (S) Ph (S) Ph

O O O O O N O N O N O O N O O O

(R) O (S) O (R) O (S) O

N N N N

Ph Ph Ph Ph (3R),(3'R)-COB-18 (3S),(3'R)-COB-20 (3R),(3'S)-COB-19 (3S),(3'S)-COB-21

Figure 2.5. Enantiomers of IB-10.

Depending on how stereospecific the binding to the receptor is, differences in the binding affinities are expected for the enantiomers from each pair. Small differences will suggest a less stereospecific binding while a large difference will indicate a great deal of chiral recognition. Similar or small variations in potency are expected between compounds COB-18 and COB-20 and also between compounds COB-19 and COB-21 because they only vary at the stereochemistry of the piperidine ring. Also difference in potency is expected between the two pairs.

A second set of desired compounds shown in Fig. 2.6, consists of diastereomeric mixtures of IB-10. This diastereomers include a single enantiomeric form for only one stereocenter while the second stereocenter is in the racemic form. The combination with 82 racemic form at position 3’ and enantiomeric form at position 3 provides diastereomers

(3S)-COB-6 and (3R)-COB-7. The reverse of this combination, racemic form at position

3 and enantiomeric form at position 3’ is illustrated by diastereomers (3’R)-COB-22 and

(3’S)-COB-23 (Fig. 2.6). These four compounds will probe the validity of the hypothesis by providing information about the importance of each chiral center individually.

racemic racemic Ph Ph 3' Ph 3' Ph 3' 3' (R) (S) O O O O O O O O O N O N O N O N 3 3 3 3 (S) O (R) O O O

N N N N

Ph Ph Ph Ph racemic racemic (3S)-COB-6 (3R)-COB-7 (3'R)-COB-22 (3'S)-COB-23

Figure 2.6. Diastereomers of IB-10.

It is expected that the two diastereomers with the racemic form at the succinimide ring, COB-6 and COB-7, will be equally potent due to similarities in spatial arrangement of the piperidine ring enantiomers. This will prove that the stereocenter at the piperidine ring doesn’t have a large contribution to chiral recognition as it was postulated in the hypothesis. On the other hand, for compounds COB-22 and COB-23 the binding affinities are expected to be different based on the structural differences of the two enantiomers derived from the substituent on the succinimide ring (recall section 2.1.1.1).

In addition, a difference in potency between the two pairs COB-6 and COB-7 vs. COB-

22 and COB-23 is also expected. 83

More information about the importance of the two chiral centers of IB-10 will be obtained from the examination of the third set of compounds presented in Fig. 2.7. For these compounds the chiral center from the piperidine ring is completely removed by including a double bond at this position, which only induces a small change in the 3-D structure of the ring as illustrated by the ball-and-stick structure in Fig. 2.7. Compound

COB-5, which is an unsaturated analog of IB-10, will be prepared in its racemic form as well as in its individual enantiomers (3’R)-COB-24 and (3’S)-COB-25 (Fig. 2.7).

racemic Ph 3' Ph 3' Ph 3' (R) (S) O O O O O N O O N O N O 3 3 3 O O O

N N N

Ph Ph Ph COB-5 (3'R)-COB-24 (3'S)-COB-25

Figure 2.7. Unsaturated analogs of IB-10.

For this set of compounds, if the racemic COB-5 shows similar binding affinity as

the lead compound IB-10 will prove again that the chiral center at the piperidine ring is

not a structural feature required for a proper interaction with the receptor. In this case,

one of the enantiomers, COB-24 and COB-25, is expected to have a better affinity than

the racemic COB-5 while the second enantiomer will be less potent. It is also expected

that the two enantiomers COB-24 and COB-25 to display similar potencies with the two

diastereomers COB-22 and COB-23 for which the piperidine ring chiral center is in

racemic form. Of course, there is also the case where the racemic COB-5 will be less 84 potent compared to the lead IB-10 which would indicate that the presence of the chiral center at the piperidine ring is a requirement for a proper interaction with the receptor.

Now that the target compounds were identified, the next step was to find a plan to synthesize these analogs. The proposed retrosynthesis and the results from the synthesis are discussed in detailed in the following sections.

2.1.1.3. Retrosynthesis

The synthesis of the desired compounds was envisioned as an esterification reaction of benzoic acid derivative 2.2 with piperidine alcohol 2.3 as shown in the retrosynthesis in Fig. 2.8. This method was developed by Bergmeier et al. for the preparation of racemic previous ring E analogs of MLA.168

3' Ph Ph 3' O O O N 3 O O OH 3 N O + COOH N N Ph Ph IB-10 2.2 2.3

Figure 2.8. Retrosynthesis.

The same method can be used for the synthesis of stereoisomers of lead compound if two

key requirements are fulfilled that is the control of chirality at position 3 and control of

chirality at position 3’. This can be accomplished by preparing the precursors succinimidobenzoic acid 2.2 and piperidine alcohol 2.3 in enantiomerically pure form as 85 well as in a racemic mixture. The synthesis of the precursors is presented in detail in the following sections.

2.1.2. Synthesis of benzylsuccinimidobenzoic acid precursor

2.1.2.1. Synthesis of racemic benzylsuccinimidobenzoic acid

The synthesis of racemic benzylsuccinimidobenzoic acid 2.2 has been previously reported for the synthesis of IB-10.158, 191 Therefore, for the preparation of racemic

precursor 2.2, the reported sequence of reactions was followed. The synthesis started from succinic anhydride as shown in Scheme 2.1. Refluxing succinic anhydride with tert- butanol in toluene with catalytic amounts of N-hydroxysuccinimide (NHS) and 4-

(dimethylamino)pyridine (DMAP) afforded the monoester 2.5 in 54% yield. Treating the monoester 2.5 with lithium diisopropylamine (LDA) formed in situ a dianion that was then alkylated with benzyl bromide to provide the monoester succinic acid derivative 2.6 in 55% yield.

tBuOH Ph

DMAP, Et3N LDA, THF O HOOC HOOC O O COOtBu COOtBu NHS, Toluene Br Ph 2.454% 2.555% 2.6

Scheme 2.1. Synthesis of succinic acid derivative.

As shown in Scheme 2.2, the monoester 2.6 was next hydrolyzed using trifluoroacetic acid to provide the benzyl substituted succinic acid 2.7 in very good yield

(95%). The diacid 2.7 was converted to the corresponding anhydride 2.8 by using acetyl 86 chloride as the dehydrating agent. The reaction proceeded in excellent yield (97%). This last reaction was also performed under microwave irradiation providing similar results.

Ph Ph Ph CF COOH CH COCl HOOC 3 HOOC 3 COOtBu COOH O O DCM 97% O 2.6 95% 2.7 2.8

Scheme 2.2. Synthesis of succinic anhydride derivative.

With the substituted succinic anhydride 2.8 in hand, only one more step was left to complete the synthesis of the benzylsuccinimidobenzoic acid precursor. As illustrated in Scheme 2.3, this was achieved by fusing the anhydride 2.8 with antranilic acid 2.9 at

high temperature. In this way the desired precursor 2.2 was obtained in 34% yield.

Ph

NH2 Ph O COOH neat, Δ N O + O COOH O O 34%

2.8 2.9 2.2

Scheme 2.3. Synthesis of precursor 2.2.

At this point, the synthesis of the racemic precursor benzylsuccinimidobenzoic

acid 2.2 has been successfully completed following the reported method. Next, all the

efforts were concentrated on the preparation of the individual enantiomers of this

precursor that is discussed in the following section. 87

2.1.2.2. Synthesis of enantiomerically pure benzylsuccinimidobenzoic acid

When using conventional chemical transformations, compounds that have at least a chiral center are obtained as a racemic mixture. A racemic mixture is an equimolar mixture of a pair of enantiomers that are related as object and mirror images. The enantiomers have identical chemical and physical properties and therefore it is difficult to separate them. However, different methods have been developed to prepare pure enantiomers. In a broad classification, these methods have been divided into resolution and stereoselective or asymmetric syntheses.

In resolution methods, the most common situation is that a racemic compound is separated into its two enantiomeric constituents by converting it into a diastereomeric mixture. The racemic compound is treated with an enantiopure compound (resolving agent) and diastereomers are formed either by a physical process or a chemical reaction.

The diastereomers differ in their properties and therefore they can be separated. For example by treating a racemic acid with one enantiomer of an amine, the resulting salt is a mixture of diastereomers that have different solubilities and so are separable through recrystallization. Afterwards, the interaction that led to diastereomer formation could be reversed and the enantiomer of the starting material released in its enantiopure form. For the above mentioned example, after separation by recrystallization, the diastereomeric salt could be treated with an acid solution for example, and the initial acid would be isolated as single enantiomer. The yield of a resolution is necessary limited to 50% because only one enantiomer of the pair is being resolved at a time. 88

An asymmetric synthesis in its most common form is the introduction of a chiral center into an achiral starting material by stereoselective reaction with a chiral reagent or a chiral catalyst. In this type of reactions one stereoisomer is formed in a substantially greater proportion than the other one(s). This proportion will be influenced to a significant degree by stereoelectronic factors and steric hindrances. In a broad classification, there are two fundamentally distinct processes used for the introduction of a new stereogenic center into a target molecule. One process is through addition to one or the other stereoheterotopic faces of a double bond and the second process is through modification or replacement (substitution) of stereoheterotopic ligands. As already mentioned, the substrate is achiral and the formation of the new stereocenter requires reagents or catalysts that are chiral. But the most prevalent situation used is to attach temporarily a chiral auxiliary to the substrate. Then achiral reagents could be used to introduce the new stereocenter in a diastereomeric relationship dictated by the stereocenter on the chiral auxiliary. Subsequently, the chiral auxiliary is cleaved and the product containing the new stereocenter is released in enantiomeric pure form.

The two above mentioned methods were considered when planning the synthesis of the enantiomeric pure benzylsuccinimidobenzoic acid 2.2. One option was to prepare the desired compound in racemic form and then use resolution to isolate one enantiomer at a time. This strategy is most efficient if it can be performed at the early steps of the synthesis with cheap starting materials because the theoretical yield of a resolution is only 50%. The starting material, succinic anhydride (recall Scheme 2.1) is not a candidate for resolution because of its lack of chiral center, therefore the resolution can only be 89 applied to one of the intermediates 2.6 or to the final product 2.2. Given the moderate yield of the first two steps of the synthesis and the lost of half of substrate in resolution, this strategy would lead to large amounts of material wasted. In other words, in theory this method did not show very promising results. Therefore, the strategy of using resolution was disregarded.

The other alternative was to use a stereoselective reaction somewhere in the synthetic pathway. Since the desired chiral center is directly adjacent to a carbonyl group, the use of a chiral auxiliary would be a good choice. The presence of the carbonyl group, in the form of acid or acid chloride for example, offers an easy way to attach the substrate to and to cleave the substrate from the auxiliary. Evans oxazolidinones are known compounds used as chiral auxiliary in various transformations.192, 193 Therefore, the plan

was to prepare both enantiomers of succinic acid derivative 2.7 by attaching the proper

substrate to a chiral oxazolidinone followed by the introduction of the proper substituent

to create the new chiral center and then remove the auxiliary.

2.1.2.2.1. Synthesis of chiral auxiliary

Before proceeding with the synthesis, the chiral auxiliary was prepared. Evans

oxazolidinones are readily available from commercially available chiral amino

alcohols.194 Since both enantiomers of the target compound are desired, two auxiliary

compounds were prepared with different stereochemistry. As shown in Scheme 2.4,

enantiomerically pure (R)-(-)-2-phenylglycinol 2.10 and (S)-(+)-2-amino-3-methyl-1-

butanol 2.12 were used as starting materials for the preparation of oxazolidinones.

90

O HO NH 2 Et2CO3 O NH (R) EtONa Ph 91% (R) Ph 2.10 2.11

O HO NH2 Et2CO3 O NH (S) EtONa (S) 86% 2.12 2.13

Scheme 2.4. Synthesis of chiral auxiliary.

Refluxing the amino acid with diethyl carbonate in the presence of catalytic amounts of

sodium ethoxide while removing the side product, ethanol, afforded the desired (4R)-4-

phenyl-2-oxazolidinone 2.11 in 91% yield and (4S)-4-isopropyl-2-oxazolidinone 2.13 in

86% yield respectively. The oxazolidinone 2.11 will be used to prepare the S enantiomer

of the desired substrate while oxazolidinone 2.13 will provide the R enantiomer. This preferential stereochemistry will be explained in the next section.

2.1.2.2.2. Asymmetric alkylation

With both chiral auxiliaries in hand, I proceeded with the asymmetric synthesis.

Initially, hydrocinnamoyl chloride was attached to the chiral auxiliary, as shown in

Scheme 2.5. The nBuLi removes the proton from the NH of the oxazolidinone, which then is acylated with the acyl chloride.195 The reactions proceeded in good yield affording

the oxazolidinones 2.14 in 79% yield and 2.16 in 75% yield respectively.

91

O O O O O (S) nBuLi, THF NaHMDS N O NH O N Ph O Ph O Br CO2tBu (R) (R) Ph (R) Ph Ph CO2tBu Cl Ph 68% 2.11 79% 2.14 2.15

O O O O O nBuLi, THF NaHMDS (R) O NH O N Ph O N Ph O Br CO tBu (S) (S)2 (S) CO2tBu Cl Ph 70% 2.13 75% 2.16 2.17

Scheme 2.5. Chiral auxiliary assisted asymmetric alkylation.

The next step was to introduce the new chiral center. This was accomplished by first deprotonation of the oxazolidinones using sodium hexamethyldisilylamide

(NaHMDS) that leads to an enolate that acts as nucleophile and reacts with tert-butyl bromoacetate to provide 2.15 in 68% yield and 2.17 in 70% yield respectively.196 The

new stereocenter was introduced in a diastereomeric fashion dictated by the substituent

on the oxazolidinone ring. The substituent is obstructing one face of the molecule forcing

the incoming alkylation group to be attached from the other face of the molecule.

Therefore, compound 2.14 with R stereochemistry will lead to the formation of R,S

diastereomer 2.15 while 2.16 with S stereochemistry will form the S,R diastereomer 2.17

(Scheme 2.5). This high selectivity observed for this type of alkylation is in part

attributed to the high degree of stereoselectivity observed during enolate formation. As

shown in Fig 2.9, the sodium amide base (NaHMDS) transforms the imides of 2.14 and

2.16 in their respective sodium chelated enolates. Under these strong basic conditions the

process is highly selective and only the Z-enolate is obtained.

92

Na Na O O O O O O O O NaHMDS NaHMDS O N O N O N O N Ph Ph Ph Ph Ph Ph 2.16 2.14 Z-sodium enolate

Figure 2.9. Sodium chelated Z-enolates of 2.14 (left) and 2.16 (right).

The structure of the Z-sodium enolate is planar creating a diastereofacial bias in the alkylation process. The only thing that deviates from this planarity is the substituent on the oxazolidinone ring. The substituent on the oxazolidinone ring of the enolate of

2.14 (left structure, Fig 2.9) is obstructing the bottom face of the molecule therefore the alkylation will occur on the top face. Opposite configuration of the alkylation product is observed for the enolate of 2.16 (right structure, Fig 2.9) because the substituent in this case is obstructing the top face forcing the alkylation to occur on the bottom face of the molecule.

Now that the new chiral center was introduced in a stereospecific fashion, the final step in the asymmetric synthesis is to remove the chiral auxiliary. Depending on the reaction conditions used, the product could be obtained as an alcohol or an acid/ester.193

For the scope of my synthesis, the substrate cleaved from chiral auxiliary should be an acid. Therefore, as shown in Scheme 2.6 hydrolysis was chosen to remove the auxiliary. 93

O O O (S) LiOH, H O (S) N 2 2 O Ph HO Ph 86% (R) Ph CO tBu 2 CO2tBu 2.15 (S)-2.18

O O O (R) LiOH, H O N Ph 2 2 (R) O HO Ph 92% (S) CO2tBu CO2tBu 2.17 (R)-2.18

Scheme 2.6. Chiral auxiliary removal.

The reaction conditions for the hydrolysis should be mild enough so that the

racemization can be avoided. Therefore, for a basic hydrolysis, instead of using OH- as a

- base the hydroperoxide HOO is used. Under LiOH/H2O2 conditions the desired

enantiomers of 2.18 were obtained in very good yield. The S enantiomer was obtained

from diastereomer 2.15 in 86% yield and the R enantiomer was obtained from

diastereomer 2.17 in 92% yield.

2.1.2.2.3. Enantiomeric excess determination for enantiomers of 2.18

In order to use the enantiomers (S)-2.18 and (R)-2.18 further in the synthesis it

was important to first check their enantiomeric purity. Methods currently available for the

determination of enantiomeric excess include chiral chromatographic techniques such as

chiral high-performance liquid chromatography (HPLC) and spectroscopic techniques

such as nuclear magnetic resonance (NMR).

The HPLC technique involves the separation of a racemic mixture or a mixture of

enantiomers by passing it through a chiral chromatographic column. A chiral column is 94 filled with a chiral stationary phase (CSP), usually silica gel that has chiral molecules bound to it. This creates a chiral environment. When a mixture of enantiomers is passed through this chiral environment, different interactions occur between each enantiomers and the CSP, which lead to diastereomeric species whose different stabilities, solubilities or absorption characteristics are responsible for the separation of the stereoisomers. As a consequence, chiral HPLC offers a fast and direct method to analyze chiral substrates.

Based on these considerations, I chose to analyze the enantiomeric composition of both

(S)-2.18 and (R)-2.18 by chiral HPLC. Both enantiomers were passed through a (R,R)-

Whelk-01 chiral HPLC column, eluting with 10% isopropanol in hexane. The two recorded chromatograms are presented in Fig. 2.10. It was expected that the different interactions of (S)-2.18 and (R)-2.18 that might occur with the CSP would lead to different retention time values. As can be seen from chromatograms, there is no difference in the retention time (tR) of the two enantiomers; the tR for the S enantiomer is

5.52 min while the tR for the R enantiomer is 5.56 min. This suggests that this particular

chiral column cannot resolve the two enantiomers and as a consequence the enantiomeric

excess cannot be determined with this method.

95

30 30 O Retention Time Area Percent (S) HO Ph

20 CO2tBu 20 (S)-2.18 mV m olts o

10 10

0 0

0 2 4 6 8 10 12 14 16 18 20 22 24 26 Minute

30 O 30 Retention Time Area Percent (R) HO Ph

CO2tBu 20 20 (R)-2.18 mV m olts o

10 10

0 0

2 4 6 8 10 12 14 16 18 20 22 24 Minute

Figure 2.10. Chiral HPLC chromatograms for (S)-2.18 (top) and (R)-2.18 (bottom).

As I mentioned earlier, another technique commonly used to determine the enantiomeric excess of a chiral substrate is NMR spectroscopy. The spectroscopic techniques are by default non-chiral thus they require a prior quantitative conversion of the enantiomeric mixtures into diastereomeric mixtures using a chiral reagent also called chiral derivatizing agent. Certain diastereotopic nuclei of the diastereomers exhibit chemical shift nonequivalence that is distinguishable in an achiral medium. The 96 integration of the appropriate signal gives a direct measure of diastereomeric composition that can then be related directly to the enantiomeric composition of the original mixture.

In order to use the NMR spectroscopy to determine the enantiomeric excess of (S)-2.18 and (R)-2.18, both enantiomers were converted to their corresponding amide diastereomers, (S,S)-2.20 and (R,S)-2.20, under standard amidation conditions using the S enantiomer of methyl benzylamine 2.19 as shown in Scheme 2.7.

(S) O O NH2 (S) Si-DCT, NMM (S) Ph (S) N HO Ph + Ph DCM H CO tBu CO2tBu 2 (S)-2.18 (S)-2.19 (S,S)-2.20

(S) O O NH2 (R) Si-DCT, NMM (R) Ph (S) N HO Ph + Ph DCM H CO tBu CO2tBu 2 (R)-2.18 (S)-2.19 (S,R)-2.20

Scheme 2.7. Synthesis of diastereomers of 2.18.

The crude products were then analyzed using 1H NMR. Due to diastereomeric

relationship between (S,S)-2.20 and (R,S)-2.20 it was expected to observe chemical shift

nonequivalence for at least one pair of protons. A closer look at the proton spectra of the

two diastereomers revealed that the amidic (NH) proton exhibited nonequivalence in

chemical shift. Usually, the racemate of the substrate analyzed is also converted into a

diastereomeric mixture and used as a control. Since the substrate 2.18 was not prepared in

a racemic form, known amounts of (S)-2.18 (78%) and (R)-2.18 (22%) enantiomers were

combined and the resulting mixture was converted into a diastereomeric mixture by 97 reaction with (S)-2.19. The crude product was also analyzed by 1H NMR. Fig. 2.11

presents the expanded regions of the 1H NMR spectra for the mixture of diastereomers

(top spectrum) and for the single diastereomer (R,S)-2.20 (bottom spectrum). A spectrum

corresponding to the second diastereomer (S,S)-2.20 can be found in Appendix A. For

more clarity, the expansion shows the signal corresponding to amidic proton NH

(doublet) and the benzylic proton PhCHCH3 (quintet). In the spectrum corresponding to

the diastereomeric mixture (top spectrum Fig. 2.11) it is observed that the amidic protons

show chemical shift nonequivalence since two distinct doublets are observed.

By integrating the peaks corresponding to the proton of interest, the mole fraction

(x) of the dominant enantiomer can be obtained and then used to calculate the percentage

enantiomeric excess using the following equation: ee = 100(2x – 1). For example, in the

spectrum corresponding to (R,S)-2.20, the NH signal for the major enantiomer is

integrated for 1.000 while for the second is 0.0482. Therefore, the mole fraction is x =

0.954 that corresponds to an enantiomeric excess of ee = 90.8 % for the R enantiomer of

2.18. Similarly, the enantiomeric excess of the S enantiomer of 2.18 was calculated to be

93.7 %. To be clear, an enantiomeric excess of 90% means that there is 90% excess of the

dominant enantiomer over the racemic, which for a mixture of 100 molecules means that

there are 95 molecules of the dominant enantiomer and 5 molecules of the other

enantiomer. 98

(S,S)-2.20 : (S,R)-2.20 78 : 22

O (R) Ph (S) N Ph H

CO2tBu (S,R)-2.20

Figure 2.11. 1H NMR spectra of mixture of diastereomers (top) and (S,R)-2.20 (bottom).

99

2.1.2.2.4. Individual preparation of both enantiomers of 2.2

The enantiomeric excess of both enantiomers of 2.18 was satisfactory enough to continue with the synthesis. From this point the synthesis follows the same steps as in the synthesis of racemic precursor benzylsuccinimidobenzoic acid 2.2. Both enantiomers of succinic anhydride derivative 2.8 were prepared starting from (S)-2.18 and (R)-2.18, respectively, as illustrated in Scheme 2.8.

Ph O O (S) CF3COOH CH COCl (S) (S) 3 HO Ph DCM HO Ph O 97% O O 98% CO2tBu COOH (S)-2.18 (S)-2.7 (S)-2.8

Ph O O (R) CF3COOH CH COCl (R) (R) 3 HO Ph DCM HO Ph O 96% O O 98% CO2tBu COOH (R)-2.18 (R)-2.7 (R)-2.8

Scheme 2.8. Synthesis of enantiomers of succinic anhydride derivative.

The esters (S)-2.18 and (R)-2.18 were hydrolyzed under acidic conditions using

trifluoroacetic acid to provide the enantiomers of benzyl substituted succinic acid (S)-2.7

and (R)-2.7 in almost quantitatively yields. Very good yields were also obtained for the

dehydration of the succinic acid using acetyl chloride that afforded the enantiomers of

benzyl substituted succinic anhydride (S)-2.8 and (R)-2.8 that were used in the final step

as illustrated in Scheme 2.9.

100

Ph (S) Ph NH2 O O (S) COOH N neat, Δ O + COOH O O 36%

(S)-2.8 2.9 (S)-2.2

Ph (R) Ph NH 2 O O (R) COOH N neat, Δ O + COOH O O 48%

(R)-2.8 2.9 (R)-2.2

Scheme 2.9. Synthesis of enantiomers of precursor 2.2.

The final step in the preparation of the enantiomers of the desired precursor benzylsuccinimidobenzoic acid (S)-2.2 and (R)-2.2 was the condensation of succinic anhydride (S)-2.8 and (R)-2.8 with antranilic acid 2.9 at high temperature. The reactions occurred in moderate yields (36 % and 48 % respectively). In an attempt to increase the reaction yield, the condensation was also carried out under microwave irradiation but with no improvement. Before using the precursors (S)-2.2 and (R)-2.2 for the synthesis of the target compounds, the enantiomeric excess was determined as presented in detail in the next section.

2.1.2.3. Determination of enantiomeric excess.

When dealing with enantiopure compounds a possible problem is the racemization during the synthesis. Racemization is an irreversible process in which an enantiopure sample becomes racemic. This process is arising from the reversible interconversion of enantiomers triggered by various conditions. For example thermal conditions could facilitate rotations around bonds or acidic and basic conditions could 101 lead to carbocation or carbanion intermediates that are planar (or easily attain a planar geometry). If the stereocenter is involved in these processes than the chirality is lost and racemic or partial racemic mixture would be obtained. Therefore, even if the enantiomeric excess of the initial substrate is known, at the end of a synthesis it is important to determine if the ee was retained. During the synthesis of both R and S enantiomers of precursor succinimidobenzoic acid 2.2 the enantiomeric excess of the initial chiral substrate 2.18 was determined (recall section 2.1.2.2.3) and now, at the end of the synthesis, it was necessary to determine the ee of the final products (S)-2.2 and (R)-

2.2. Different methods were examined such as chiral HPLC and 1H NMR that are in

detail discussed in the next section.

2.1.2.3.1. Chiral HPLC

The succinimidobenzoic acid 2.2 in its racemic form was passed through a (R,R)-

Whelk-01 chiral HPLC column eluting with 10-50% isopropanol in hexane.

Unfortunately no separation was observed as shown by the spectrum in Fig. 2.12. When a

racemic mixture is resolved on a chiral column, two distinct peaks of same intensity are

observed. In the chromatogram shown in Fig 2.12 there is only one peak (tR = 8.6 min) observed that corresponds to the sample analyzed. The smaller peak tR = 6.5 min,

corresponds to an impurity.

102

15 15 Ph Retention Time Area Percent O N O 10 COOH 10

mV m olts o 2.2 5 5

0 0

2 4 6 8 10 12 14 16 18 20 22 24 Minute

Figure 2.12. Chiral HPLC chromatogram of racemic succinimidobenzoic acid 2.2.

The separation of two enantiomers using HPLC is dependent on the chiral stationary phase of the column used. Usually, a specific type of chiral stationary phase will successfully separate the enantiomers of a particular class of organic compounds but no CSP will be able to achieve separation for all organic compounds. Since with the chiral column available in our laboratory I obtained unsatisfactory results, a sample of racemic 2.2 was send to Registech, a chiral HPLC columns provider, for a chiral screen through all their available columns. There were 8 different chiral columns tested and unfortunately none of the columns gave any separation. Therefore, a different method to determine the ee of precursors (S)-2.2 and (R)-2.2 was sought.

2.1.2.3.2. Chiral lanthanide shift reagent

The second method used involved a chiral lanthanide shift reagent (CLSR).

CLSRs are compounds containing a certain metal (Eu, Pr, or Yb) and behave as Lewis acids. When such a CLSR is added to an organic Lewis base solute, it will undergo rapid 103 and reversible coordination with the Lewis base forming a diastereomeric complex. In this complex, the paramagnetic ion of the transition metal is brought into close proximity to the chiral organic substrate and as a result the individual groups of substrate will experience different magnetic environments and will be chemically shifted to an extent that depends on the strength of the complex and how far the nuclei are from the paramagnetic metal atom.197

For the analysis of compound 2.2 with this method, the CLSR chosen was

Eu(hfc)3 (tris[3-heptafluoropropylhydroxymethylene-d-camphorato]europium III) shown

in Figure 2.13. A solution of an equimolar mixture of racemic 2.2 and Eu(hfc)3 in deuterium chloroform was analyzed by proton NMR. The resulting spectrum is presented in Fig. 2.14.

CF2CF2CF3

O

O Eu 3

Eu(hfc)3

Figure 2.13. Structure of Eu(hfc)3.

Unfortunately no information was achieved using this method. No induced chemical

shifts were observed in the NMR spectrum but only line broadening of the peaks.

Therefore this method was also abandoned.

104

1 Figure 2.14. H NMR spectrum of CDCl3 solution of Eu(hfc)3 and 2.2.

2.1.2.3.3. 1H NMR using chiral derivatizing agent

As the above mentioned methods for the ee determination failed to deliver a

conclusive result, discrete diastereomers from the precursor 2.2 in its racemic form as

well as its individual enantiomers were prepared as shown in Scheme 2.10. The acid was

activated by silica supported dichlorotriazine (Si-DCT) and N-methylmorpholine toward the reaction with the S enantiomer of methyl benzylamine 2.19 to form amide 2.21. The

reaction mixture was then analyzed by proton NMR.

105

Ph Ph

H N (S) O O O N O 2 CH3 N Si-DCT, NMM O COOH + DCM, rt N (S) Ph H

2.2 2.19 2.21

1 R1 R R2 R2 H N (S) 2 CH3 O O O O Si-DCT, NMM N N + O COOH DCM, rt N (S) Ph H

1 2 1 2 (R)-2.2 : R = H, R = Bn 2.19 (R,S)-2.21 : R = H, R = Bn (S)-2.2 : R1 = Bn, R2 = H (S,S)-2.21 :R1 = Bn, R2 = H

Scheme 2.10. Synthesis of diastereomers of 2.21.

In Fig. 2.15 are presented the expanded regions of the spectra for racemic 2.21

(top spectrum) and for diastereomer (R,S)-2.21 (bottom spectrum). The spectrum

corresponding to the second diastereomer (S,S)-2.21 can be found in Appendix B. From

the proton NMR analyses of the diastereomers (R,S)-2.21 and (S,S)-2.21 it was concluded

that the ee of the R enantiomer of 2.2 is 74% and the ee of the S enantiomer is 68%. For a

better clarity, an enantiomeric excess of 74% for the R enantiomer is translated into a

6.7:1 ratio of R:S enantiomers.

106

Ph

O N O O

N (S) Ph H

2.21

Ph (R)

O N O O

N (S) Ph H

(R,S)-2.21

Figure 2.15. 1H NMR spectra of mixture of diastereomers (top) and (R,S)-2.21 (bottom). 107

The loss in enantiomeric purity that most probably occurred during the condensation reaction of enantiomers of succinic anhydride derivative 2.8 with the antranilic acid could be possibly attributed to a base induced racemization. For both the succinic anhydride derivative 2.8 and the succinimide derivative 2.2, the chiral center C3 is adjacent to a carbonyl group leading to an increased acidity for the hydrogen atom at that position. It is possible that under the high temperature conditions of the reaction, the antranilic acid, although a weak base, to deprotonate the chiral center leading to racemization. In Fig. 2.16 is presented a possible mechanism for this reversible process exemplified on the R enantiomer.

B

H Ph Ph Ph 4 3 slow O O O O X O X O X (R)-2.8, X = O 2.8a 2.8b (R)-2.2, X = N-Ar 2.2a 2.2b

Figure 2.16. Possible racemization mechanism.

The deprotonation is a slow process that gives rise to a carbanion 2.8a that is stabilized by the adjacent carbonyl leading to a planar intermediate 2.8b. The reverse

process, the reprotonation at position C3, can occur at either side of the double bond

leading to racemization. In an attempt to increase the reaction yield and to minimize the

racemization, the condensation reaction of succinic anhydride derivative with antranilic

acid was performed under microwave irradiation. The expectations were that a lower

reaction temperature and a shorter reaction time would significantly decrease the rate of 108 racemization. However, no improvements in the enantiomeric excess or the yield were observed under these reaction conditions.

2.1.3. Synthesis of N-(3-phenylpropyl)-3-piperidinemethanol precursor

As it was presented in the retrosynthesis in section 2.1.1.3, the strategy for the preparation of the target compounds was to separately synthesize two precursors in their racemic and enantiopure forms and then combine them to form the final desired compounds. The synthesis for one of the precursors was presented above therefore the following section will discuss the synthesis of the second precursor N-(3-phenylpropyl)-

3-piperidinemethanol 2.3 as a racemate and as individual enantiomers as well.

2.1.3.1. Synthesis of racemic N-(3-phenylpropyl)-3-piperidinemethanol

The synthesis of the racemic compound 2.3 presented in Scheme 2.11 had been previously reported for the preparation of lead compound IB-10.158 The synthesis is

straight forward starting from commercially available piperidine-3-methanol 2.22

providing the desired product in 69% yield through a standard alkylation reaction using

3-bromo-1-phenyl-propyl.

OH Br Ph OH K CO , EtOH N 2 3 N H 69% Ph 2.22 2.3

Scheme 2.11. Synthesis of racemic N-(3-phenylpropyl)-3-piperidinemethanol. 109

2.1.3.2. Synthesis of enantiomerically pure N-(3-phenylpropyl)-3-piperidinemethanol

The individual synthesis of R and S enantiomers of 2.3 was a bit more challenging. The racemic 2.3 is not a very good candidate for a resolution by means of diastereomeric salts formation followed by separation using fractional crystallization.

Therefore, the enantiomers should be prepared starting from chiral starting materials.

However, the synthesis of both enantiomers (R)-2.3 and (S)-2.3 had been previously reported by Bergmeier et. all.172 Their method involved a resolution of the piperidine ring as the first step in the synthesis starting from commercially available racemic nipecotic acid. I decided to follow the reported procedure for the preparation of (R)-2.3 and (S)-2.3.

2.1.3.2.1. Resolution using camphorsulfonic acid

As mentioned before, the first step in the synthesis involves the resolution of

nipecotic acid. This was achieved by preparing diastereomeric salts from racemic

nipecotic acid 2.23 using enantiomerically pure camphorsulfonic acid (CSA) as

illustrated in Scheme 2.12. By mixing equimolar amounts of 2.23 with either of the

enantiomers of CSA in refluxing acetone followed by addition of water, a precipitate is

formed that contains one of the enantiomers of nipecotic acid as camphorsulfonate salt.

SO3H O HO S O COOH COOH 3 COOH (S)-(+)-CSA (R)-(-)-CSA (S) (R) resolution resolution N CSA N N CSA H 7%H 10% H (S)-2.23 2.23 (R)-2.23

Scheme 2.12. Nipecotic acid resolution with camphorsulfonic acid. 110

When (R)-CSA was used the R enantiomer of nipecotic acid was separated as salt

(R)-2.23 in 10% yield and when the (S)-CSA was involved in the resolution the S enantiomer of nipecotic acid was provided as salt (S)-2.23 in 7% yield. Although the maximum theoretical yield of a resolution is 50% since only one enantiomer is resolved at a time, the results obtained from nipecotic acid resolution were not very satisfactory.

Despite these results I decided to carry on with the synthesis expecting good yields from the next steps that would compensate for the low yield of the resolution. The reactions for this trial were performed only on the R enantiomer of nipecotic acid.

The next step in the reported procedure was to acylate the piperidinic nitrogen as shown in Scheme 2.13. Acylation was performed with hydrocinnamoyl chloride 2.24 in the presence of excess of triethyl amine with catalytic amounts of DMAP in methylene chloride. In my hands, this procedure afforded only 11% yield (entry 1, Table 2.1) of the desired compound (R)-2.25, that prompted me to search for different reaction conditions that would lead to better results.

COOH COOH base, solvent (R) O N N CSA H Cl Ph O Ph (R)-2.23 2.24 (R)-2.25

Scheme 2.13. Nipecotic acid acylation.

In Table 2.1 are summarized the various changes applied to the acylation reaction

of camphorsulfonate salt of (R)-nipecotic acid with hydrocinnamoyl chloride. In each

case, excess of base was used because prior acylation, the NH should be freed from the 111 diastereomeric salt. No much improvement in terms of the yield was observed when iPr2EtN (entry 2) was used. The same conclusion could be drawn for the reaction using

pyridine (entry 3) for which the yield only increased to 15%. A slight increase in the

yield was observed when pyridine (entry 4) was used as a base and as a solvent as well.

None of these conditions succeeded in providing significantly better results. However,

sodium hydroxide in a mixture of dioxane and water (entry 5) afforded the desired

product (R)-2.25 in 72% yield.

Table 2.1

Reaction optimization for nipecotic acid acylation

Entry Base Solvent Yield (%)

1 Et3N / DMAP methylene chloride 11

2 iPr2EtN / DMAP methylene chloride 12

3 pyridine methylene chloride 15

4 pyridine pyridine 17

5 NaOH dioxane:water 72

The final step in the synthesis was to reduce the amide and carboxylate groups at the same time using lithium aluminum hydride as shown in Scheme 2.14. Unfortunately the reaction afforded the desired product (R)-2.3 in only 19-25% yield. The low yield of this final step combined with the low yield of the resolution prompted me to look for an

alternative synthesis. 112

COOH (R) LiAlH4 (R) OH N THF, Δ N

O Ph Ph (R)-2.25 (R)-2.3

Scheme 2.14. Reduction of nipecotic acid derivative.

2.1.3.2.1. Resolution using tartaric acid

Racemic ethyl nipecotate 2.26 (Scheme 2.15) can also undergo separation into its two enantiomers by means of resolution. As a matter of fact, a SciFinder search revealed that both enantiomers of 2.26 are commercially available as free base and as tartarate salts as well. However, it is more expensive to buy the enantiomers than to prepare them by resolution. Aldrich supplies (S)-ethyl nipecotate•D-TA salt at a price of $772 per 50g while all starting materials theoretically required to prepare this quantity are five times less expensive ($100/100g ethyl nipecotate and $60/100g D-TA). Therefore, the diastereomeric salts of ethyl nipecotate with tartaric acid were prepared as shown in

Scheme 2.15 following a known procedure.198

O O H OH H OH OH OH HO HO H OH O H OH O COOEt COOEt L-(+)-TA COOEt D-(-)-TA (R) (S) resolution N TA resolution N N TA H H H 27% 25% (R)-2.26 2.26 (S)-2.26

Scheme 2.15. Ethyl nipecotate resolution with tartaric acid.

113

To a hot solution of tartaric acid in iPrOH and water was added racemic ethyl nipecotate and after 30 minutes the mixture was allowed to cool down which led to crystals of salt formation. After the specific rotation ([α]D) of the precipitate was

recorded, the precipitate was recrystallized in order to increase the diastereomeric excess.

After each recrystallization the [α]D was recorded again and this process was continued until no variation in specific rotation was observed. When L-tartaric acid was used the diastereomeric salt (R)-2.26 obtained in 27% yield contained the R enantiomer of ethyl nipecotate. The specific rotation of the salt (R)-2.26 after the final recrystallization was

[α]D +10.7° (c 1, H2O). When D-tartaric acid was used the diastereomer (S)-2.26 was

obtained in 25% yield affording the S enantiomer of ethyl nipecotate. The specific

rotation of the salt (S)-2.26 after the final recrystallization was [α]D -10.7° (c 1, H2O).

With both enantiomers of 2.26 in hand I proceeded with the synthesis of precursor 2.3.

2.1.3.2.2. Individual preparation of both enantiomers of 2.3

For the synthesis of both enantiomers of precursor 2.3 was followed the same strategy as the one reported for nipecotic acid and presented in previous section. This strategy involved the introduction of the proper substituent at the piperidinic nitrogen followed by reduction of the ester group into an alcohol group as illustrated in Scheme

2.16.

114

O

Cl Ph COOEt COOEt 2.24 (R) LiAlH4 (R) (R) OH 15% Na CO N THF, Δ N TA 2 3 N H 77% (2 steps) O Ph Ph (R)-2.26 (R)-2.27 (R)-2.3

O

COOEt Cl Ph COOEt 2.24 LiAlH4 (S) (S) (S) OH THF, Δ N TA 15% Na2CO3 N N H 75% (2 steps) O Ph Ph (S)-2.26 (S)-2.27 (S)-2.3

Scheme 2.16. Synthesis of enantiomers of precursor 2.3.

The required piperidine substituent was introduced by an acylation reaction following a reported procedure.199 The diastereomer (R)-2.26 was treated first with an

aqueous solution of sodium carbonate and then with hydrocinnamoyl chloride to afford the amide (R)-2.27 that was immediately subjected to reduction with lithium aluminum hydride to provide the desired R enantiomer of precursor 2.3 in 77% two steps yield

(Scheme 2.16). The S enantiomer of 2.3 was obtained in the same manner in 75% two

steps yield. Before enantiomers (R)-2.3 and (S)-2.3 could have be used for the synthesis

of the final compounds, their enantiomeric excess needed to be determined.

2.1.3.3. Determination of enantiomeric excess

In previous cases where the enantiomeric excess was determined, the NMR

spectroscopy method proved to be most reliable method, therefore for ee determination of

enantiomers of 2.3 the first choice was to use this method. Since the substrate to be

analyzed contains a hydroxyl group adjacent to the chiral center, the chiral derivatizing agent of choice should react with this functional group. Mosher’s chloride 2.28 is a very 115 well known chiral derivatizing agent used for the determination of ee of alcohols and amines.200 As illustrated in Scheme 2.17, the R enantiomer of Mosher’s chloride was used to convert the racemate 2.3 into a mixture of diastereomers 2.29 using triethyl amine and catalytic amount of DMAP. This mixture of diastereomers was prepared as the control with which the individual diastereomers obtained from (R)-2.3 and (S)-2.3 would be compared. Each enantiomer of 2.3 was also converted into its corresponding diastereomer following the same procedure as for the racemate. The crude of each reaction from Scheme 2.17 was then analyzed using NMR spectroscopy. The first choice

19 was to used F NMR since the molecules of the diastereomers contain a CF3 group and

because of the simplicity of the spectrum since only one signal would be generated.

Therefore, the mixture of diastereomers 2.29 was first analyzed with the expectation that

two singlets will be observed each corresponding to the CF3 group of each diastereomer

from the mixture. Unfortunately, only one signal was observed which means that there is

no chemical shift difference between the CF3 of the diastereomers and the method cannot

be used to determine the ee. This is probably because the CF3 group is too far from the

second chiral center to experience different magnetic environment.

116

O O (R) OCH (R) 3 OH OCH Et3N, DMAP O + Cl 3 DCM Ph CF3 N Ph CF3 N

Ph Ph 2.3 (R)-2.28 2.29 O (R) O OCH3 OH (R) Et3N, DMAP (R) O (R) OCH3 + Cl DCM Ph CF3 N N Ph CF3 Ph Ph (R)-2.3 (R)-2.28 (R,R)-2.29 O O (R) OCH Et N, DMAP 3 OH (R) 3 (S) O (S) + OCH3 Cl DCM Ph CF3 N N Ph CF3 Ph Ph (S)-2.3 (R)-2.28 (S,R)-2.29

Scheme 2.17. Mosher ester synthesis.

Next, the diastereomeric mixture 2.29 was analyzed using 1H NMR. It was gratifying to see that methylene protons immediately adjacent to ester group presented chemical shift differences probably generated by the phenyl ring anisotropy. For one of the diastereomers, the two protons generate a doublet while for the other diastereomer each of the two protons generates a doublet of doublets. This can be clearly seen in the spectrum corresponding to the mixture of diastereomers 2.29 (Fig. 2.17, top). Both diastereomers (R,R)-2.29 and (S,R)-2.29 were then also analyzed. In Fig. 2.17 is also presented the spectrum corresponding to diastereomer (S,R)-2.29 (bottom). The spectrum for (R,R)-2.29 can be found in Appendix C.

In the spectrum corresponding to (S,R)-2.29 can be observed a double of doublets

(dd) around 4.02 ppm that was integrated to one proton. The second dd generated by the other proton of CH2 group is not clearly observed because of an overlap with a multiplate 117 corresponding to an impurity. It is also observed a small doublet around 4.11 ppm, integrated for 0.0698 that corresponds to CH2 group in the other diastereomer. From the integration of these signals the ee was calculated to be 93.3% for (S)-2.3. For the second enantiomer, (R)-2.3 the ee was determined to be >98% because in the spectrum corresponding to diastereomer (R,R)-2.29 only one signal for CH2 group was observed.

The absence of signal for the other diastereomer does not mean it is completely missing from the mixture but it may be in such a small amount that falls below the detection limit of the NMR spectroscopy. Therefore, in such cases the ee is not reported as 100%. 118

O (R) OCH O 3 Ph CF3 N

Ph 2.29

O (R) OCH3 (S) O Ph CF3 N

Ph (S,R)-2.29

Figure 2.17. 1H NMR spectra of mixture of diastereomers (top) and (R,R)-2.29 (bottom). 119

2.1.4. Synthesis of enantiomers of IB-10

With the synthesis of both precursors 2.2 and 2.3, in racemic and individual enantiomer forms completed, the final step toward the target compounds presented in introduction was the coupling of the two precursors. The two pairs of enantiomers of IB-

10 were first prepared as presented in Scheme 2.18. Each enantiomer of precursor 2.3 was coupled with each enantiomer of precursor 2.2 such that at the end, four enantiomers were obtained. The coupling reaction involved the activation of the acid with polystyrylsulfonyl chloride resin (PS-TsCl) and N-methyl imidazole followed by a nucleophilic attack by the alcohol. The resin bound reagent had a second advantage that is at the end of the reaction part of the byproducts were removed from the mixture by filtration. The coupling reaction afforded the final enantiomers in relatively good yields:

56% yield for COB-18, 60% yield for COB-19, 62% yield for COB-20, and 68% yield for COB-21 respectively.

3' Ph

R1 R2 O N O O R1 R2 3 O N O PS-TsCl, DCM O + COOH N N-Methyl imidazole N

Ph Ph (3R),(3'R)-COB-18 (R)-2.3 : R1 = H, R2 = CH OH (R)-2.2 : R1 = H, R2 = Bn (3R),(3'S)-COB-19 2 (3S),(3'R)-COB-20 1 2 (S)-2.2 : R1 = Bn, R2 = H (S)-2.3 : R = CH2OH, R = H (3S),(3'S)-COB-21

Scheme 2.18. Synthesis of enantiomers of IB-10.

120

2.1.5. Synthesis of diastereomers of IB-10

A second group of target compounds contains the various diastereomers of IB-10.

These compounds were prepared following the same procedure as the one used for the preparation of enantiomers of IB-10 as illustrated in Scheme 2.19. Each enantiomer of precursor 2.3 was coupled with the racemate of precursor 2.2 to afford the diastereomeric mixtures COB-6 and COB-7 in 68% yield and 66% yield respectively. Also, each enantiomer of 2.2 was coupled with the racemate of precursor 2.3 to afford two more diastereomers COB-22 and COB-23 in 69% yield and 71% yield respectively.

Ph

Ph O N O O R1 R2 3 O N O PS-TsCl, DCM O + COOH N N-Methyl imidazole N

Ph Ph

1 2 (3S)-COB-6 (R)-2.3 : R = H, R = CH2OH 1 2 2.2 (3R)-COB-7 (S)-2.3 : R = CH2OH, R = H 3' Ph

R1 R2 O N O O

OH O N O PS-TsCl, DCM O + COOH N N-Methyl imidazole N

Ph Ph (R)-2.2 : R1 = H, R2 = Bn (3'R)-COB-22 2.3 (S)-2.2 :R1 =Bn,R2 =H (3'S)-COB-23

Scheme 2.19. Synthesis of diastereomers of IB-10.

121

2.1.6. Synthesis of other analogs of IB-10

As was presented in the introduction of this chapter, three other analogs of IB-10 were proposed to be prepared that would lack the chiral center on the piperidine ring by introducing an unsaturation at that position. For this purpose a derivative of piperidine alcohol precursor 2.3 that would present a double bond at position 3 was required.

Therefore, before discussing the preparation of the unsaturated analogs of IB-10, the synthesis of the required unsaturated precursor will be presented.

2.1.6.1. Synthesis of unsaturated precursor

The unsaturated piperidine alcohol 2.32 was prepared as shown in Scheme 2.20 from compound 2.30 that was first converted to its corresponding secondary amine 2.31 under acidic conditions in almost quantitatively yield. Using the same alkylation procedure as the one used for the preparation of precursor 2.3, discussed in previous sections, the unsaturated precursor 2.32 was obtained in 75% yield.

OH AcCl OH Br Ph OH MeOH K CO N N 2 3 N HCl EtOH Boc 92% H 75% Ph 2.30 2.31 2.32

Scheme 2.20. Synthesis of unsaturated piperidine precursor.

Compound 2.30 used for the synthesis of precursor 2.32 is not commercially

available. However it is a known compound and its synthesis has been previously

reported.201 Therefore, compound 2.30 was prepared following the reported procedure as

illustrated in Scheme 2.21. The synthesis started from arecoline 2.33 that was obtained by 122 base-extraction form commercially available arecoline hydrobromide. The removal of N- methyl was achieved by treating 2.33 with α-chloroethyl chloroformate in refluxing toluene that formed a carbamate intermediate which was not isolated by purification instead was heated in methanol to give guvacoline hydrochloride 2.34. Protection of amine 2.34 with di-tert-butyl dicarbonate afforded N-Boc protected compound 2.35 in

65% yield. The reduction of the ester group with diisobutylaluminum hydride (DIBAL-

H) provided the desired unsaturated alcohol 2.30 in 69 % yield, that was used further to prepare precursor 2.32 as already presented in the beginning of this section.

O Cl

Cl O COOMe COOMe COOMe Toluene, Δ (Boc)2O 2) MeOH, Δ Et3N, DCM N N HCl N H 90% (2 steps) 65% Boc 2.33 2.34 2.35

COOMe DIBAL-H OH Et O N 2 N Boc 69% Boc

2.35 2.30

Scheme 2.21. Synthesis of compound 2.30.

2.1.6.2. Coupling

With the precursor 2.32 in hands, the unsaturated analogs of IB-10 were prepared

using the same coupling conditions as for the already presented target compounds as

illustrated in Scheme 2.22. Compound COB-5 was obtained in racemic form from

alcohol 2.32 and racemic precursor 2.2 in 78% yield. Similar results were obtained for 123 the individual enantiomers of COB-5. The coupling reaction of 2.32 with R enantiomer of acid 2.2 afforded COB-24 in 80% yield while its paired enantiomer COB-25 was obtained in 76% yield from the S enantiomer of precursor 2.2.

3' Ph

Ph O N O O

OH O N O PS-TsCl, DCM O + COOH N-Methyl imidazole N N 78% Ph Ph

2.32 2.2 COB-5

3' Ph

R1 R2 O N O O

OH O N O PS-TsCl, DCM O + COOH N N-Methyl imidazole N

Ph Ph (R)-2.2 : R1 = H, R2 = Bn (3'R)-COB-24 2.32 (S)-2.2 :R1 =Bn,R2 =H (3'S)-COB-25

Scheme 2.22. Synthesis of unsaturated analogs of IB-10.

All eleven final compounds presented so far were converted to their oxalic salts that were used to prepare 10 mM solutions in DMSO:H2O (1:1) and then sent for

biological assays that were performed in Prof. McKay’s laboratory at Ohio State

University. They investigated our compounds by performing calcium accumulation assays using a cell line stably expressing rat α3β4 nAChR subtypes. The functional

responses of the 11 molecules on the nAChR-stimulated calcium accumulation in these

cells are found in Table 2.2.

124

Table 2.2

Biological activity of IB-10 analogs

Entry Structure Stereochemistry Name Rα3β4 (IC50 μM)

Ph

O 1 O -- IB-10 7.6 (6.8-8.6) O N

O

N Ph

Ph

O 2 O -- COB-5 6.3 (5.3-7.7) O N

O

N Ph

Ph

O 3 O 3S COB-6 8.4 (5.5-12.8) O N

(S) O

N Ph

Ph

O 4 O 3R COB-7 6.2 (5.3-7.3) O N

(R) O

N Ph

(R) Ph

O 5 O 3R,3’R COB-18 13.8 (0.9-213) O N

(R) O

N Ph

(S) Ph

O 6 O 3R,3’S COB-19 9.54 (1.4-64.5) O N

(R) O

N Ph

(R) Ph

O 7 O 3S,3’R COB-20 17.1 (2.6-110) O N

(S) O

N Ph

(S) Ph

O 8 O 3S,3’S COB-21 8.92 (1.5-52.8) O N

(S) O

N Ph

(R) Ph

O 9 O 3’R COB-22 10.5 (3.6-30.5) O N

O

N Ph 125

(S) Ph

O 10 O 3’S COB-23 11.8 (3.1-45.5) O N

O

N Ph

Ph (R) O 11 O 3’R COB-24 7.4 (0.8-72.2) O N

O

N Ph

Ph (S) O 12 O 3’S COB-25 9.9 (1.7-57.8) O N

O

N Ph

At a first glance at the data shown in Table 2.2 two aspects stand out. First, the

results indicate that all eleven compounds display inhibitory activity at micromolar

concentrations demonstrating that they are active antagonists of the α3β4 receptors. The

smaller the IC50 values, the better the inhibitory activities. Second, with two exceptions

(entries 2 and 4), all analogs are less potent than the lead compound, a situation that was

not expected. Important information about the stereospecificity of the interaction of our

molecules with the receptors comes from the two pairs of enantiomers (entries 5-8). The

enantiomers COB-18 and COB-21 exhibited differences in potency (13.8 μM vs 8.92

μM), which indicates that there is chiral recognition involved in binding of ring E analogs of MLA to the nicotinic receptors. This is supported by the activity data of the other pair

of enantiomers, COB-19 and COB-20, which also displayed differences in potencies.

The small difference in potencies between diastereomers COB-6 and COB-7 that

have enantiomeric forms at the piperidine ring indicates that, as expected, the chirality at

piperidine ring doesn’t have a large contribution to the stereospecific interaction with the

binding site. On the other hand, unexpected results were observed for the succinimide 126 chiral center. The small difference in potencies between compounds COB-22 and COB-

23 (or COB-24 and COB-25) indicates a non-significant contribution of this chiral center for stereospecific interaction suggesting that that the presence of both chiral centers is required for the chiral recognition. Another interesting observation is that the unsaturated analogs COB-5, COB-24 and COB-25 display better activity than their corresponding saturated analogs IB-10, COB-22 and COB-23 respectively, which may suggest that the double bond is an additional point of interaction with the binding site of the receptors.

To conclude, the hypothesis postulating that chiral center on the succinimide ring

(position 3’) would have a larger effect on the biological activity than the one on the piperidine ring (position 3) is not supported by the results obtained in this study. Instead, it is clear that the combined contributions of the two chiral centers lead to chiral recognition at the interaction with the receptors. The decrease in potency compared with the lead compound observed for the majority of analogs indicates that more about the mechanism of action for these molecules needs to be elucidated.

2.2. Analogs of APB-12

2.2.1. Significance

As it was mentioned before, a large number of compounds structurally related to methyllycaconitine were synthesized in our laboratory and were preliminarily characterized in Prof. McKay’s laboratory (Section 1.4.3). The compounds act as noncompetitive antagonists of nAChR by binding to an allosteric binging site. From these studies another compound that showed good pharmacological properties was identified,

APB-12 shown in Fig. 2.18. This compound also became a lead compound for my 127 project because of its structural differences compared to all the other compounds studied.

Structurally, all studied compounds contain a piperidine ring while for APB-12 the piperidine ring was changed to a pyrrolidine ring.

3'

O N O O 2 O N

Ph APB-12

Figure 2.18. Lead compound APB-12.

The replacement of piperidine ring with a pyrrolidine ring leads to structural differences that are worth being studied. One of the differences is the position of the hydroxymethyl linker to the benzoyl group that is closer to the nitrogen atom as it can be seen in Fig. 2.19 (the remainder of the structure was omitted for clarity). Another structural difference is the spatial orientation of the linker when the enantiomers of pyrrolidine ring are considered. For piperidine ring enantiomers there was no significant difference in the spatial orientation of the substituent (recall Fig. 2.2). On the other hand, as illustrated in Fig. 2.18, for pyrrolidine ring enantiomers hydroxymethyl substituents are pointing in opposite areas of the space. For the R enantiomer the substituent is pointing down while for the S enantiomer is pointing up, on the other side of the ring.

These spatial differences might lead to different interactions with the receptor. Therefore the hypothesis is that both chiral centers will contribute to the chiral recognition between 128 the receptor and the analogs. This chiral recognition will be translated into differences in potency between enantiomers.

O O (R) (S) N N

Figure 2.19. Three-dimensional representation of pyrrolidine ring enantiomers.

2.2.1.1. Target compounds

The lead compound APB-12 also presents two chiral centers therefore has two pairs of enantiomers; COB-13 is the enantiomer of COB-17 while COB-14 is the

enantiomer of COB-15. All four of these stereoisomers were synthesized and assayed for

biological activity. Their structures are presented in Fig. 2.20.

(R) (S) (R) (S)

O O O O O N O O N O N N O O O O (S) (S) (R) (R) O O O O N N N N

Ph Ph Ph Ph (2S),(3'R)-COB-13 (2S),(3'S)-COB-14 (2R),(3'R)-COB-15 (2R),(3'S)-COB-17

Figure 2.20. Enantiomers of APB-12.

The synthesis of the above-mentioned target compounds was envisioned as shown by the retrosynthesis in Fig. 2.21. The strategy was the same as the one used for the 129 preparation of IB-10 analogs that is to separately synthesis two precursors that at the end will be coupled to provide the target compounds. The two required precursor are methylsuccinimidobenzoic acid 2.36 and N-(3-phenylpropyl)-2-pyrrolidinemethanol 2.37 that were synthesized in their enantiomeric pure form as discussed in detail in the next section.

3'

O O 3' N 2 O OH 2 O N O N O + N COOH

Ph

Ph APB-12 2.36 2.37

Figure 2.21. Retrosynthesis.

2.2.2. Synthesis of methylsuccinimidobenzoic acid precursor

The synthesis of enantiomers of methylsuccinimidobenzoic acid 2.36 had been previously reported.172 Therefore, the compounds (R)-2.36 and (S)-2.36 were prepared

following the reported procedure. The synthesis started from the enantiomerically pure

commercially available methyl succinic acids (R)-2.38 and (S)-2.38 as illustrated in

Scheme 2.23. The dehydration of the succinic acid under microwave irradiation afforded

the enantiomers of succinic anhydride (R)-2.39 and (S)-2.39 in quantitatively yields. The

condensation of the succinic anhydride (R)-2.39 and (S)-2.39 with antranilic acid under

microwave irradiation provided the enantiomers (R)-2.36 and (S)-2.36 in good yields.

130

NH2 (R) COOH (R) O N O O AcCl COOH O HO (R) COOH MW 100° C, 7min O O MW 170°C, 40min 100% 73% (R)-2.38 (R)-2.39 (R)-2.36

NH2 (S) COOH (S) O O O AcCl N O COOH MW 100° C, 7min O HO (S) COOH O MW 170°C, 40min 100% 72% (S)-2.38 (S)-2.39 (S)-2.36

Scheme 2.23. Synthesis of enantiomers of methyl succinimidobenzoic acid.

With both (R)-2.36 and (S)-2.36 prepared, the next step was to determine their enantiomeric excess. The methods used for the ee determination of the enantiomers of benzylsuccinimidobenzoic acid 2.2 were also tried for the enantiomers of methylsuccinimidobenzoic acid 2.36 only to find out that none of the methods were successful in providing the desired result. Both enantiomers of 2.36 were converted to their corresponding diastereomers using the same chiral derivatizing agent, (S)-methyl benzylamine and analyzed using 1H NMR. Different deuterated solvents were used such

as CDCl3, CD3CN, and C6D6 in the attempt to visualize chemical shift nonequivalence

for any of the signals but this approach failed to simplify the spectra. A solution of the

diastereomer that contained the chiral lanthanide shift reagent Eu(hfc)3 was also

analyzed, but it provided similar unsatisfying results. Since the 1H NMR spectroscopy

analyses failed in providing the necessary tool for the ee determination I turned my

attention to chiral HPLC. However, both diastereomers of 2.36 showed the same

retention times when passed through the chiral column. At this junction, the decision was 131 made to use both (R)-2.36 and (S)-2.36 further in the synthesis even if their ee could not be determined.

2.2.3. Synthesis of N-(3-phenylpropyl)-2-pyrrolidinemethanol precursor

The second precursor required for the synthesis of enantiomerically pure diastereomers of APB-12 is the N-phenylpropyl substituted pyrrolidine methanol 2.37.

Both enantiomers of precursor 2.37 were prepared in only one step as shown in Scheme

2.24. Both commercially available enantiomers of prolinol 2.40 were converted to the desired enantiomers (S)-2.37 and (R)-2.37 by a standard alkylation with 1-bromo-3- phenyl-propyl in 67% yield and 36% yield respectively under microwave irradiation.

HO HO Ph (S) (S) Br Ph NH N K2CO3, EtOH MW, 130° C, 15 min. (S)-2.40 (S)-2.37 67%

HO HO Ph (R) (R) Br Ph NH N K2CO3, EtOH MW, 130° C, 15 min. (R)-2.40 36% (R)-2.37

Scheme 2.24. Synthesis of enantiomers of prolinol derivative.

Since the alkylation reaction at the nitrogen atom involves relatively mild

conditions, there is no danger of racemization to occur. Therefore, the enantiomeric

purity of enantiomers (S)-2.37 and (R)-2.37 was not determined. It was assumed that the 132 enantiomeric purity of the starting materials (S)-2.40 and (R)-2.40 was retained during the transformation.

2.2.4. Coupling reaction

With both precursors, methyl succinimidobenzoic acid 2.36 and prolinol derivative 2.37, in their enantiomeric form in hand, the next step toward the synthesis of the target diastereomers of APB-12 was the coupling of the two precursors. As shown in

Scheme 2.25, each enantiomer of prolinol derivative 2.37 was reacted with each enantiomer of methyl succinimidobenzoic acid 2.36 such that four diastereomers were formed. The reaction involved the same reaction conditions as those used for the preparation of analogs of IB-10. The coupling reaction afforded the final enantiomers in relatively good yields: 75% yield for COB-13, 74% yield for COB-14, 55% yield for

COB-15, and 50% yield for COB-17 respectively.

3'

R1 R2 O N O 1 O R 2 R 2 O N O PS-TsCl, DCM O N + COOH N-Methyl imidazole N

Ph Ph

1 2 1 2 (S)-2.37 : R = H, R = CH2OH (R)-2.36 : R = H, R = Me (2S),(3'R)-COB-13 (R)-2.37 : R1 = CH OH, R2 = H 1 2 (2S),(3'S)-COB-14 2 (S)-2.36 : R = Me, R = H (2R),(3'R)-COB-15 (2R),(3'S)-COB-17

Scheme 2.25. Synthesis of enantiomers of APB-12.

The target compounds were converted to their water-soluble oxalate salts from which 10 mM solutions in DMSO:H2O (1:1) were prepared and sent for biological

evaluation at Prof. McKay’s laboratory. Calcium accumulation assays were performed 133 using a cell line stably expressing rat α3β4 nAChR subtypes. The functional responses of

the four enantiomers on the nAChR-stimulated calcium accumulation in these cells are

presented in Table 2.3.

Table 2.3

Biological activity of APB-12 analogs

Entry Structure Stereochemistry Name Rα3β4 (IC50 μM)

1 racemic APB-12 6.6 (6.0-7.2) O N O O

O N Ph

2 (R) 2S,3’R COB-13 9.9 (7.6-12.9) O N O O (S) O N Ph

3 (S) 2R,3’S COB-17 11.0 (7.9-15.4) O N O O (R) O N Ph

4 (S) 2S,3’S COB-14 12.3 (9.1-16.6) O N O O (S) O N Ph

5 (R) 2R,3’R COB-15 8.6 (4.7-15.7) O N O O (R) O N Ph

The results presented in Table 2.3 follow the same pattern as for the analogs of

IB-10. It can be observed that all compounds show inhibitory activity on nAChR-

stimulated increases in intracellular calcium. And also all diastereomers have decreased

potency compared to the lead compound APB-12 that is an unexpected situation. Once

again, these results suggest that more investigations need to be made. However, the 134 differences in potency, although small, between enantiomers COB-13 and COB-17 indicate that there is stereochemical recognition involved in the interaction with the receptors. This is supported by the difference in potency of the other pair of enantiomers

COB-14 and COB-15. Although the differences are small, they are significant enough to indicate the chiral recognition and to affirm the hypothesis.

2.3. Analogs of COB-1

2.3.1. Significance

The analogs of methyllycaconitine synthesized and studied in Bergmeier’s and

McKay’s laboratories fall into a structural pattern that is their molecules contain large alkyl groups on the piperidine nitrogen and large benzoyl ester or heterocyclic ester. It is also interesting to look into what effect would have the small alkyl groups on the piperidine ring on the potency and binding of these type of compounds. Therefore, a group of few compounds are synthesized and biologically evaluated, shown in Fig. 2.22, that contain no alkyl group on the nitrogen (COB-1 and its enantiomers COB-11 and

COB-12) or contain one and two methyl groups on the nitrogen as represented by COB-2 and COB-3. The synthesis of these target compounds is discussed in detail in the next section.

O Ph O Ph O Ph O Ph O Ph (R) (S) O O O O O

N N N N N H H H COB-1 COB-2 COB-3 (R)-COB-11 (S)-COB-12

Figure 2.22. Target compounds.

135

2.3.2. Synthesis of COB-1, COB-2 and COB-3

Compound COB-1 could be prepared by a coupling of 3-piperidinemethanol 2.41

(Scheme 2.26) with 2-phenylbenzoic acid. Prior to this esterification reaction the nitrogen at the piperidine ring needed to be protected. There are various protecting groups known for amines. For the purpose of my synthesis the protective group of choice was benzyl chloroformate (CbzCl). The choice was based on the reaction conditions required for the subsequent cleavage of the protecting group that would not affect the newly formed ester since the Cbz is removed by catalytic hydrogenation.

Ph COOH O Ph O Ph

OH CbzCl OH 2.43 O H2, Pd/C O Et3N, DCM DCC, DMAP EtOH N N N N H DCM H 75% Cbz 59% Cbz 82% 2.41 2.42 2.44 COB-1

Scheme 2.26. Synthesis of COB-1.

The protection of nitrogen occurred in good yield (75%) as shown in Scheme 2.26 using triethylamine and CbzCl. The coupling reaction of Cbz protected 2.42 with 2- phenylbenzoic acid 2.43 activated with dicyclohexylcarbodiimide (DCC) and catalytic amounts of dimethylaminopyridine (DMAP) afforded the Cbz protected desired compound 2.44 in 59% yield. The final step toward the target compound COB-1 was to remove the protecting group of piperidinic nitrogen by hydrogenation on palladium on charcoal. The hydrogenation afforded the target compound in 82 % yield. 136

Compounds COB-2 and COB-3 were prepared in the same fashion as illustrated in Scheme 2.27. The commercially available N-methyl-3-piperidinemethanol 2.45 was coupled with 2-phenylbenzoic acid 2.43 using the same conditions as for COB-1 that is

DCC/DMAP to provide the desired compound COB-2 in 70% yield. Its corresponding dimethylated compound COB-3 was prepared from COB-2 by alkylation with iodomethyl in quantitatively yield.

O Ph O Ph Ph OH COOH DCC, DMAP O CH3I O + N DCM N CH3CN N 70% 2.45 2.43 COB-2 COB-3

Scheme 2.27. Synthesis of COB-2 and COB-3.

2.3.3. Synthesis of enantiomers of COB-1

The synthesis of enantiomers of COB-1 could be carried out following the same pathway as for the synthesis of the racemic with the condition to start from individual enantiomers of the Cbz protected piperidinemethanol 2.42. The preparation of enantiomers of piperidinemethanol derivatives from ethyl nipecotate resolution with tartaric acid was presented in section 2.1.3.2.1 of this dissertation. Therefore, the tartaric salts of both enantiomers of ethyl nipecotate 2.26 were used as starting materials for the synthesis of enantiomers of Cbz protected precursor 2.42 as shown in Scheme 2.28.

137

(R) (R) COOEt (R) COOEt 15 % Na2CO3 DIBAL OH CbzCl Et2O, -20°C N N TA EA-H2O N H 95% Cbz 35% Cbz (R)-2.26 (R)-2.45 (R)-2.42

(S) (S) COOEt (S) COOEt 15 % Na2CO3 DIBAL OH CbzCl Et O, -20°C N 2 N N TA EA-H2O H 90% Cbz 37% Cbz (S)-2.26 (S)-2.45 (S)-2.42

Scheme 2.28. Synthesis of enantiomers of precursor 2.42.

The diastereomeric salt (R)-2.26 was treated with base to free the basic nitrogen of piperidine ring that was then protected with CbzCl to afford the Cbz protected R enantiomer of ethyl nipecotate in 95% yield. The same treatment was applied to the second enantiomer (S)-2.26 that provided the Cbz protected S enantiomer of ethyl nipecotate in 90% yield. The next step in the synthesis was to reduce the ester group to an alcohol group that was achieved using diisobutylaluminum hydride. The reduction reaction afforded the desired compounds (R)-2.42 and (S)-2.42 in moderate yields 35% and 37% respectively, that were further used for the preparation of enantiomers of COB-

1 as illustrated in Scheme 2.29.

138

Ph O Ph O Ph (R) COOH (R) (R) OH PS-TsCl, DCM O H2, Pd/C O + N-methyl imidazole EtOH N N N H Cbz 73%Cbz 85% (R)-COB-11 (R)-2.42 2.43 (R)-2.44

Ph O Ph O Ph (S) OH COOH (S) (S) PS-TsCl, DCM O H2, Pd/C O + N N-methyl imidazole EtOH N N Cbz H 80%Cbz 88% (S)-2.42 2.43 (S)-2.44 (S)-COB-12

Scheme 2.29. Synthesis of enantiomers of COB-1.

The coupling reaction of racemic 2.42 with 2.43 to prepare the racemic COB-1 afforded the racemic 2.44 in 59% yield by using DCC/DMAP conditions (recall Scheme

2.26). In an attempt to increase the yield of this transformation for the enantiomers of

2.42 and subsequently to compensate for the moderate yield obtained for the reduction, the coupling reaction conditions were changed by using the resin bound reagent PS-TsCl.

Each enantiomer of protected piperidinemethanol 2.42 was converted to enantiomers of ester 2.44 by coupling reaction with 2-phenylbenzoic acid 2.43 using these reaction conditions. The yields of the transformations improved, affording the enantiomers (R)-

2.44 and (S)-2.44 in 73% yield and 80% yield respectively. The protective group was removed by catalytic hydrogenation in good yields, 85% and 88%, to afford the final desired compounds COB-11 and COB-12. All final compounds were converted to their oxalic salts, which were used to prepare 10 mM solution in DMSO:H2O (1:1) and then

sent for biological evaluation at Prof. McKay’s laboratory. Calcium accumulation assays

were performed using a cell line stably expressing rat α3β4 nAChR subtypes. The 139 functional responses of these molecules on the nAChR-stimulated calcium accumulation in these cells are presented in Table 2.4.

Table 2.4

Biological activity for COB-1 analogs

Entry Structure Stereochemistry Name Rα3β4 (IC50 μM)

O Ph 1 O -- COB-1 1.2 (1.0-1.4)* N H

O Ph 2 O -- COB-2 1.1 (1.0-1.1)* N

O Ph 3 O -- COB-3 0.7 (0.6-0.9)* N

O Ph (R) 4 O 3R COB-11 6.7 (5.1-8.7) N H

O Ph (S) 5 O 3S COB-12 4.0 (3.9-4.1) N H

* Data are from McKay et. al., Mol. Pharmacol. 2007, 71, 1288-1297.

The biological results from Table 2.4 indicate that compounds COB1 to COB-3

are inhibitors of functional activation of recombined nAChRs with high potency. Once again, an unexpected decrease in potency is observed for the enantiomers COB-11 and

COB-12 compared to the racemic compound COB-1. The small difference in potency between enantiomers is in agreement with the results observed for analogs of IB-10 and

APB-12 indicating some degree of stereospecificity for the interaction with the receptors.

140

2.4. Summary

In this chapter was presented a discussion about the synthesis of some ring E analogs of MLA in enantiomeric forms and diastereomeric forms as well. The synthesis proved to be strait forward with complications only arising from the enantiomeric excess determination of the desired chiral precursors. Except for the enantiomers of precursor

2.2 that were prepared in moderate enantiomeric purity, the enantiomers of all other precursors were obtained with very good enantiomeric purity. The desired compounds were used to study the three-dimensional interaction of small ligands with nicotinic acetylcholine receptors. Although the majority of compounds shown unexpected small decreases in potency compared with the lead compounds, they all suggest that there is chiral recognition involved in their interaction with the receptors. 141

CHAPTER 3: OXAZOLIDINONES AS ANTIBIOTICS

Oxazolidinones have been mentioned in this dissertation in section 2.1.2.2.1 where it was discussed their use as chiral auxiliary in asymmetric transformations for the introduction of a new stereocenter. This chiral oxazolidinone methodology was particularly applied in the synthesis of the enantiomers of benzylsuccinimidobenzoic acid precursor. In addition to their efficiency in asymmetric syntheses, an even more valuable characteristic of oxazolidinone class of compounds is their biological activity as antibacterial agents. It is for this characteristic that oxazolidinones received much attention and became the first new class of synthetic antimicrobial agents implemented in the clinic after more then three decades of pause in the development of new antibiotics.202

The research project presented in this second part of my dissertation is part of a larger study focused on the rational design of novel oxazolidinones as antimicrobial agents with new mechanism of action. Before giving a detailed description of the work involved in this project, a brief discussion on how antibiotics work and the problems encountered in their development will be presented in the following sections. The discussion is intended to emphasize the continuous need for novel more powerful antibacterial agents with improved mechanisms of action.

In its general definition, an antibiotic is an agent that interferes with the growth and the proliferation of pathogenic microorganisms. Antibiotics are among the most frequently prescribed drugs in modern medicine, used to treat various infectious diseases caused by bacteria. They can act primarily by preventing the growth of bacteria in which case they are called bacteriostatic agents or can act by killing the bacteria therefore are 142 termed bactericidal agents. Some antibiotics are narrow spectrum drugs, exerting their effect on a specific type of bacteria, while others are broad-spectrum antibiotics being effective against a wide range of bacteria. The broad-spectrum activity is a very desirable characteristic for a drug because it offers the opportunity to treat the infectious disease at an early stage, eliminating the time consuming need to identify the pathogen that is required before a narrow-spectrum antibiotic could be used.

The vast majority of antibiotics in use to date have been discovered and developed during the so-called ‘gold’ period of antibiotics between 1940s and 1960s.203

In Figure 3.1 are illustrated few representative antibiotics from this period. The only class of antibiotics used to treat infectious diseases in mid 1930s was sulfonamides class of compounds with prontosil (Fig. 3.1) as its first member.204, 205 However, the discovery

and the introduction of penicillins in the clinical practice in the early 1940, was shortly

followed by the development of other classes of antibiotics such as aminoglycosides and

phenylpropanoids (Fig. 3.1). The efforts in antibiotics field continued in the 1950s and

1960s with the characterization and approval of six more new classes of antibacterial

agents: tetracyclines, macrolides, quinolones, glycopeptides, rifamycins, and

trimethoprim (Fig 3.1).206 After such an explosive growth in the development of

antimicrobial agents it was believed that infectious diseases did not pose a threat for

public health anymore being kept under control by the existing therapies.203, 205, 207 As a result the pharmaceutical industry support for antibiotic research had declined from 1970 leading to a complete absence of novel classes of compounds from the antibiotic market.208

143

NH 2 H2N NH HO NO2 HO NH H H OH Ph N H2N S O HN NH2 N O O N CHO N NH H O Cl2HC N O OH CO2H OH NHMe OH O O OH OH SO2NH2 HO Penicillin G Prontosil Streptomycin Chloramphenicol (β-lactams) (sulfonamides) (aminoglycosides) (phenylpropanoids)

O O HO N OH F COOH HO N OH OH HO O NH2 N N O O OH HN OH O OH O O O O OMe OH O Tetracycline Ciprofloxacin Erythromycin (tetracyclines) (quinolones) (macrolides)

HO OMe HO MeO H2N N NH2 O OH N OH O O MeO NH Trimethoprim HO 2 O Cl O O O OH O O OH OH Cl NH HO OH O O O H H MeO N N NHMe O O N N N H H H OH N O O O N HN H NOC O 2 N HOOC OH OH Vancomycin Rifampicin HO (glycopeptides) (rifamycins)

Figure 3.1. Representatives of old classes of antibiotics.

The only new active compounds introduced in the clinical practice after 1970 were revised versions of the members of already existing classes. It wasn’t until recently that few structurally new classes of antibiotics were developed and approved for clinical use, which are oxazolidinones, lipopeptides and glycylcyclines. In Figure 3.2 are illustrated 144 representatives of these new classes of antibiotics with the exception of lipopeptides class because of their more complex architectural scaffold.

F O O N N N H H OH O N O H N NH2 NH N H OH O OH O OH O O

Linezolid Tigecycline (oxazolidinones) (glycylcyclines)

Figure 3.2. Representatives of new classes of antibiotics.

The major source for discovering compounds with antimicrobial activity was the big pool of natural products.205 As a defensive strategy, one species of microorganisms

produces a compound that is used as a chemical weapon against co-existing life forms or

predators from their neighboring microenvironment.209 The identification and isolation of

these natural products by screening of soil samples led to the discovery of the majority of

antibiotics used today. For example, penicillin was isolated from fungi while species of soil bacteria such as Streptomyces and Actinomyces offered a variety of antibiotics including streptomycin, erythromycin, tetracycline and vancomycin (Fig 3.1).210

Furthermore, semisynthetic modification of these antibacterial natural compounds produced next generation drugs with incremental improvements in properties. Only a small contribution to the larger number of antibiotics came from the synthetic chemical collections. In fact only three classes: sulfonamides, quinolones (Fig 3.1) and oxazolidinones (Fig 3.2) represent totally synthetic classes of antibiotics.205 145

The above mentioned microbial origin of the antibiotics indicates the intrinsic capability of bacteria to fight against these compounds. It was only fair to infer that the microorganisms producing a particular antibacterial compound possess some sort of self- defensive mechanism that protects them from their own chemical weapon.211 Indeed, the

major problem in antibiotic drug discovery was and still is the adaptability of bacteria in the presence of antibacterial agents by developing or acquiring mechanisms of resistance toward these agents. A brief discussion on the various strategies developed by bacteria to fight against antibiotics is presented in the following section.

3.1 Resistance to antibiotics

The emergence of bacterial resistance to antibacterial agents had been recognized as a biological phenomenon212 since resistant strains of bacteria have been identified for

every main class of antibiotics.213 Moreover, the existence of multidrug resistant bacteria

led to untreatable cases of infectious diseases that continue to pose a public health

threat.212, 214, 215 The antimicrobial resistance is a consequence of the natural selection

viewed as the Darwinian principle of “survival of the fittest” combined with the bacterial

natural tendency to share acquired resistance mechanisms between them and with other

microorganisms.

As I mentioned earlier, certain microorganisms that produce antibacterial agents

are equipped with protective mechanisms against these agents. Such a mechanism

involves the use of membrane proteins to immediately pump outside the cell the

antibiotic compounds produced.210 These membrane proteins are essentially channels

called efflux pumps present in all bacteria and are used to actively transport lipophilic

molecules and other compounds in and out of the cells. Besides the antibiotic-producing 146 microbes, it was observed that other bacteria also gained resistance to antibiotics by overproducing related efflux pumps that were used to export the drugs back out the cell.210 This process is illustrated in the schematic representation of a bacterium cell in

Fig. 3.3 where the antibiotic is the blue circle and the efflux pump is the gray membrane

imbedded protein. As a consequence, the efflux pumps prevent the intracellular

accumulation of the antibiotic at the therapeutic concentration required for its lethal

activity.216

Figure 3.3. Mechanisms of antibiotic resistance.217

Inactivation and destruction of antibiotics before they could reach their target sites

are two other strategies developed by bacteria to become resistant, also illustrated in Fig.

3.3.210 Inactivation of the antibiotic is one of the most common resistance mechanism.218

Some bacteria acquire enzymes, exemplified by the green protein in Fig. 3.3, that chemically modify the structure of the drugs so that they could no longer bind to their 147 target site or just simply rendering them inactive. Other enzymes, exemplified by the orange protein in Fig. 3.3, chemically degrade the drugs before having the chance to exert their toxic activity.

Another resistance strategy adopted by bacteria is to induce modifications at the target site of antibiotics by mutations.210 Mutations are changes in the DNA that can

cause changes in the gene products (proteins) and are typically rare events.219 However,

due to the rapid growth rate and the large number of bacteria in an infection cycle, it

doesn’t take that long for spontaneous mutation to occur.210 Such spontaneous mutations

can lead to resistance by modifying or camouflaging the binding site for an antibiotic that

in turn lowers the affinity of the antibiotic for that particular target site preventing it from

damaging the cell.216, 219 Resistance can also be caused by downregulating an outer membrane protein port that is required for the entry of an antibiotic.217

The resistance genes can be integrated into the bacterial chromosome and

transmitted from generation to generation or, more typically, they can be collected on a

particular kind of circular DNA called plasmid that is an autonomously replicating

transferable element.218, 219 The rapid spread of the antibiotic resistance is highly

facilitated by the transfer of the resistance genes between bacterium cells of the same species and even between genetically unrelated microorganisms. This type of genetic exchange occurs through three prevalent mechanisms: transformation, transduction, and conjugation that are schematic illustrated in Fig. 3.4.217

Transformation is a process through which pieces of DNA are picked up from

surrounding environment.216, 218, 219 When a bacterium cell dies, it brakes apart or lyses 148 releasing its content into the environment. Other bacteria from its vicinity can pick up liberated resistance genes and incorporate them into their on DNA. This process is limited by the somewhat narrow compatibility range between donor and recipient cells.219

Figure 3.4. Mechanisms of genetic transfer.217

Transduction is a process through which DNA is transferred from one bacterium

to another by a virus called bacteriophage.216, 218, 219 These bacteriophages infect bacteria

and use their hosts DNA replication machinery to make more viruses, which

inadvertently leads to the incorporation of the bacterial DNA into the new virus DNA.

When the infected bacterium dies, the virus moves and infects another bacterium. If the

virus carries resistance genes from the first host, these genes will be transferred to the

new host. This process is also limited by the narrow range of hosts that a virus could

infect.219 149

The most important mechanism for spreading resistance through gene transfer is believed to be the conjugation.216, 218, 219 During this process the genetic material is transferred from one cell to another through an elongated bridge-like structure called

pilus.216 A pilus is formed between two cells when they are in close proximity and allows

a copy of plasmid as it is duplicated to be exchanged between them. Moreover this process is very effective because it is not dependent on compatibility between the donor and the receiver cells. Conjugation can occur in a broad range of bacterial species therefore resistance to multiple drugs can be easily transferred.219

Given that in the presence of antibiotics bacteria can easily adapt by modifying

their existing genetic material or by acquiring new genetic material, the emergence of

resistance to antibacterial agents is an inevitable response to the widespread use of

antibiotics. Moreover, a resistant strain can possess combinations of different resistance

strategies. Selective examples of above mentioned antibacterial resistance mechanisms

applied on particular classes of antibiotics will be presented in the following section

along with brief discussion on the mechanisms of action of antibiotics.

3.2. Targets of antibiotics

The large number of antibacterial agents has been classified in antibiotic classes

according to their structures. These classes can be further classified based on their site of

action. The antibiotics typically attack targets that are unique for bacterial cells or are

sufficiently different from their mammalian counterparts such that the toxic effect of

these drugs does not affect the mammalian cells. Based on their target sites, the majority

of contemporary antibiotics fall into three large categories: inhibitors of cell wall 150 biosynthesis, inhibitors of DNA replication and repair and inhibitors of protein synthesis.203, 212

3.2.1. Inhibitors of bacterial cell wall biosynthesis

One structural characteristic of bacteria cells that is not found in mammalian cell

is the cell wall located directly outside the cytoplasmic membrane. The main component

of bacterial cell wall is the peptidoglycan, a polymer consisting of repeating disaccharide subunits of N-acetylglucosamine (NAG, marked in blue in Fig. 3.5) and N-acetylmuramic acid (NAM, marked in green in Fig. 3.5). A short peptide chain, typically composed of four alternating L- and D- amino acids, is attached to the NAM subunits. The peptide chains from adjacent strands are most often cross-linked through pentaglycine chains

(Gly5). These interpeptide bridges confer mechanical strength to the cell wall. The amino

acid composition of the peptide chains and interpeptide bridges as well as the length of

the interpeptide bridges vary among different bacteria species.220 In Fig. 3.5 is presented the peptidoglycan of gram-positive bacteria.

The peptidoglycan is essential for the survival of bacteria providing mechanical support by counteracting their high internal osmotic pressure. The loss of peptidoglycan integrity is immediate followed by bacterial death.203 Therefore, it is with no surprise that

there are many antibacterial agents used in clinical practice targeting the biosynthesis of

peptidoglycan. The most commonly used inhibitors of cell wall synthesis are

glycopeptides and β-lactams such as penicillins and cephalosporins classes of antibiotics

(recall Fig. 3.1).212

151

NAG NAM OH OH O O O HO O NH O CO NH HC(CH3) CH CO 3 peptide bond formation L-Ala CH3 n inhibited by penicillins D-iGln

L-Lys (Gly)5 D-Ala

D-Ala L-Lys

D-iGln

L-Ala

NAG - NAM n

Figure 3.5. Peptidoglycan structure of Gram-positive bacteria.220

The well known penicillins, and β-lactams in general, interfere with the last step

in bacterial cell wall synthesis, namely the cross-linking of different peptidoglycan

strands. This cross-linking reaction forms the peptide bond indicated by red in Fig. 3.5

(right structure) and is catalyzed by a family of enzymes called transpeptidases

(TPase).212, 220 The structure of penicillins mimics the normal substrate of TPase and

binds to the active site forming a covalent bond with a serine residue as indicated in Fig.

3.6. This penicilloyl-enzyme complex formed does not react further therefore preventing the cross-linking of peptide chains leading to a mechanically weak peptidoglycan that is

susceptible to osmotic lysis.210

152

R R S O S N O O HN COO- COO- HO Ser Ser TPase TPase Penicilloyl-enzyme intermediate (enzymatically inactive)

Figure 3.6. Site of action for penicillins.210

The therapeutic use of β-lactams was quickly followed by the emergence of

resistance to these agents in some species of bacteria.221 Almost all resistance strategies

discussed in the previous section 3.1, have been reported to have a contribution in the appearance of β-lactam resistant bacteria strains.212 Among these, the changes in the cell

wall porins leading to reduced uptake of the drugs and the active efflux of the drugs are

less frequently used by bacteria and therefore have less contribution. Typically the β-

lactam resistance is caused by acquiring new enzymes with decrease affinity for these

drugs or by mutations in the active site of TPase that lead to a lower affinity for these

drugs.222 But the most effective strategy used, is the elaboration of enzymes that inactivate the β-lactams by chemical modifications.221 These enzymes are called β-

lactamases because they catalyze the hydrolysis of lactam ring as indicated in Fig. 3.7.

The hydrolyzed product, penicilloic acid does not mimic the natural substrate for TPase

anymore therefore doesn’t function as an antibiotic.210

153

R R S β−lactamase O S N O- HN O - COO COO- penicillins penicilloic acid active inactive

Figure 3.7. Bacterial resistance to β-lactams.210

Another example of inhibitors of cell wall biosynthesis is vancomycin (recall Fig.

3.1), a representative of glycopeptide class of antibiotics. This compound also interferes

with the interpeptide cross-linking step of the peptidoglycan synthesis but in a different way than the antibiotic class mentioned earlier. Rather than binding to the enzymes, vancomycin interacts with the substrate for these enzymes, namely the peptide chain attached to the NAM sugar.223 As shown in Fig. 3.8 (left structure), vancomycin interacts

with the terminal alanine (Ala) residues (D-Ala-D-Ala, the rest of the molecule was omitted for clarity) of the uncrosslinked peptide chain through five hydrogen bonds indicated by the red and blue lines. This interaction prevents the enzyme TPase from

binding its substrate therefore leading to a weak peptidoglycan structure deprived of

crosslinks and susceptible to osmotic lysis.210

154

HO HO HO HO O O OH OH O O O O

NH2 O NH2 HO Cl HO O Cl O O O O Cl Vancomycin Cl HO OH HO OH O H O O O O H O O O N NHMe N NHMe O N N N N O N N N N H O H H H H O H H H N H O N H O - - OOC NH2 OOC NH2

OH OH OH OH HO HO O H O- O O- N O R N O R N O H H O O N-acyl-D-Ala-D-Ala N-acyl-D-Ala-D-Lac

Figure 3.8. Vancomycin mechanism of action and resistance.210

Strains of bacteria resistant to glycopeptides have also been identified.213, 224, 225

The strategy used by these bacteria to gain resistance to vancomycin was to modify its target site by replacing the terminal D-alanine in the peptide chain of peptidoglycan with a D-lactate (D-Lac) residue as shown in Fig 3.8 (right structure). This replacement of the amide linkage, as in D-Ala-D-Ala, with an ester linkage, as in D-Ala-D-Lac, disrupts one hydrogen bond in the interaction of vancomycin with the peptide chain while has no effect on the cross-linking reaction carried out by TPase.210 As a consequence, the

binding affinity of vancomycin for its target decreases by 1000-fold, which renders the

compound inefficient as an antibiotic.226

3.2.2. Inhibitors of nucleic acid synthesis

Only few antibacterial agents are known to interfere with the synthesis of nucleic

acids and these are sulfonamides and fluoroquinolones classes of antibiotics (recall Fig.

3.1). Sulfonamides inhibit the synthesis of folic acid (Fig. 3.9), a highly versatile carrier 155 of activated one-carbon units. For this role, folic acid is utilized in many biosyntheses including the synthesis of purine ring system required for the nucleic acid synthesis.1

While all cells require folic acid, the mammals do not synthesis it, instead they obtain the

1 folic acid from diets as vitamin B9 or from microorganisms in their intestinal tracts.

Therefore, the synthesis of folic acid is an ideal antibacterial target.

H2N N N NH2 N N OH HN H2N N H2N O O H N S N NH2 O O O OH O O O Para-aminobenzoic Prontosil Folic acid acid (PABA) (sulfonamides)

Figure 3.9. Structure of folic acid, PABA and prontosil.

Bacteria synthesize folic acid starting from para-aminobenzoic acid (PABA, Fig.

3.9). Sulfonamides, such as prontosil (Fig. 3.9), are analogs of PABA and therefore compete with PABA for the same enzyme active site and by doing so they inhibit the synthesis of folic acid and subsequently the synthesis of nucleotide precursors.212

Bacterial resistance to sulfonamides is caused by the acquisition of new enzymes that do not recognize the drugs as substrates while the binding of PABA is not affected.212, 213

The target site for fluoroquinolones such as ciprofloxacin (recall Fig. 3.1) is an enzyme involved in the DNA replication called DNA gyrase.210 This enzyme is an ideal

drug target because has no direct counterpart in mammalian cell and is indispensable for

the replication and therefore for the survival of bacterial cell.203 The DNA gyrase is 156 responsible for introducing negative supercoils into the intertwined circles of double- stranded bacterial DNA during the replication. This process occurs in three steps: the first step involves the cleavage of both strands of DNA by the DNA gyrase, this is then followed by the passage of a segment of DNA through the break and in the final step, the enzyme reseals the cleaved DNA. In the first step, the enzyme covalently binds the 5’ ends of cleaved DNA using tyrosyl residues forming a gyrase-DNA complex.1 The

antibiotic ciprofloxacin inhibits the DNA gyrase by binding to this complex and

preventing the enzyme from catalyzing the resealing of the DNA chains. As a

consequence, the double-strand breaks accumulate which in turn triggers the SOS repair

system that leads to cell death.210

Low-level resistance to fluoroquinolones is typically caused by reduce uptake of

the drugs by reducing the permeability of the cell wall and by active efflux. The main

mechanism involved in resistance to these drugs is the reprogramming of the target

through mutations. Mutations in the genes encoding the DNA gyrase lead to decrease in

the binding affinity of the ciprofloxacin such that the DNA replication continues

undisturbed in its presence.227 More often the resistance to fluoroquinolones is a

consequence of the combination of two or all of the three mentioned mechanisms.

3.2.3. Inhibitors of protein synthesis

Another major target site for antibacterial agents is the protein synthesis. Protein

synthesis, called translation, takes place on an enormous complex containing RNA

molecules and proteins, called ribosome. Although bacterial and mammalian ribosomes

show many similarities, they differ in composition and size. The bacterial ribosome has a 157 sedimentation coefficient of 70S and can be dissociated into a small (30S) subunit and a large (50S) subunit while its mammalian counterpart has a sedimentation coefficient of

80S and is made of a 40S subunit and a 60S subunit.1 The protein synthesis requires the

translation of nucleotide sequences (mRNA) into amino acid sequences (proteins) that

starts with an initiation complex as shown in Fig. 3.10. The initiation complex is formed

by the interaction of the 30S ribosomal subunit with a specific segment of the mRNA

which is followed by the binding of a special tRNA (marked in purple in Fig. 3.10) that carries the initiating amino acid. The fully assembled 70S initiation complex is formed by the binding of 50S ribosomal subunit to the 30S subunits.1 The initiating aminoacyl-

tRNA is located at the P (for peptidyl) site and bridges between the 30S and 50S subunits

of the ribosome.

50S tunel E A P aa-tRNA Peptide-bond E P A binding formation

30S

GTP

Translocation Elongation factor G

GDP + Pi

tRNA dissociation

Figure 3.10. Schematic mechanism of bacterial protein synthesis.1

158

As indicated in Fig. 3.10, the ribosome has two more binding sites, the A site (for aminoacyl) and the E site (for exit). An aminoacyl-tRNA (marked in brown in Fig. 3.10) enters the A site and binds to the mRNA by base pairing its complementary anticodon with the codon (brown segment) on the mRNA. There is a unique codon, a sequence of three bases, and a specific tRNA, which contains an anticodon, corresponding to each amino acid. The codon on the mRNA dictates which amino acid will be next inserted in the A site. Only the correct aminoacyl-tRNA with the complementary anticodon will base pair with the codon in the A site ensuring a high fidelity of protein synthesis. With the two amino acids located in the P and A site respectively, the peptide bond is formed, shifting the elongated peptide chain to the A site while the entire ribosome moves three nucleotides along the mRNA. This translocation brings the tRNA carrying the peptide chain in the P site while the uncharged tRNA leaves the ribosome through the E site and the new mRNA codon becomes accessible in the A site (Fig. 3.10). The next required aminoacyl-tRNA enters the A site and the above described process is repeated, creating a polypeptide chain in the P site. The synthesis ends when a stop codon that is not recognized by any charged tRNA, enters the A site and triggers the dissociation of the entire ribosomal complex.1

The protein synthesis shows sufficient differences in bacterial cell compared to

mammalian cell especially in terms of ribosomal composition so that it presents an ideal

target for antibacterial agents. Many classes of antibiotics used in clinical practice today

are inhibitors of protein synthesis acting at various sites of ribosomal machinery. These

include important classes such as aminoglycosides, tetracyclines, macrolides, 159 phenylpropanoids (recall Fig. 3.1) and the newly introduced class of oxazolidinones (Fig.

3.2). Given the large number of such antibiotics only selective examples will be mentioned here.

The mechanism of action for aminoglycosides is not fully understood but it was reported that some of them might inhibit the protein synthesis by interfering with the proper assembly of the initiation complex while others by blocking the translocation of the ribosome along the mRNA.212 However, the most common and studied mechanism of action of these antibiotics is the interference with the proofreading of the required amino acid during synthesis. The drugs bind in the proximity of the ribosomal decoding center at the A site and trigger conformational changes of the site that diminish the mRNA- tRNA (codon-anticodon) interaction leading to the incorporation of the wrong amino acid. The accumulation of inaccurate polypeptide chains leads to truncated or incorrectly folded proteins which in turn leads to cell death.228 Bacterial resistance to

aminoglycosides is caused, at a smaller extend, by decreased uptake and active efflux of the drugs.228, 229 However, the most frequently used strategy for resistance to

aminoglycosides is the inactivation of the drugs by chemical modifications. The structure

of these antibiotics contains many functional groups that are targets for enzymes that

could attach various substituents to them. For example, specific hydroxyl groups of the

aminoglycosides can by phosphorylated by phosphorylyltransferases or adenylated by

nucleotidyltransferases.210 Such structural decorations will interfere with the binding of

the antibiotics to the target site preventing them from exerting their lethal effect. 160

Tetracyclines are another examples of inhibitors of bacterial protein synthesis.

This class of broad-spectrum bacteriostatic agents act by binging to the 30S ribosomal subunit in the A site preventing the association of aminoacyl-tRNA with the ribosomal complex.230 The required amino acids cannot be attached to the growing polypeptide

chain thus the proteins synthesis is stopped. Strains of bacteria resistant to tetracyclines

have also been observed. The main mechanism responsible for tetracycline resistance is

the active efflux. Resistant bacteria overproduce efflux pumps that expel the drugs faster

than they can diffuse in, so that the therapeutic concentrations are not reached and the

drugs are ineffective.210, 230

Another class of antibiotics that act as inhibitors of protein synthesis are

oxazolidinones. Oxazolidinones are the first structurally new class of antibiotics

developed in the past forty years in response to the alarming emergence of bacterial resistance for all other existing antibacterial agents.202 The only member of this class of

compounds approved by FDA in 2000 for use in clinical practice is linezolid,231-233 that was observed to be active against many important resistant pathogens.234 Linezolid,

interferes with the bacterial protein synthesis through a unique mechanism that inhibits

the proper assembly of the functional initiation complex. It was reported that before the

binding of the 50S subunit to the complex formed by the 30S subunit, the initiating

aminoacyl-tRNA and the mRNA, which would form the functional 70S ribosome,

linezolid binds to the P site (recall Fig. 3.10) at the 50S ribosomal subunit. By doing so,

the drug blocks the required interaction of the aminoacyl-tRNA with the P site at the 50S

subunit, which in turn blocks the formation of the ribosome. As a consequence, the 161 initiation complex is not formed and the synthesis is stopped.235-237 Unfortunately, strains

of bacteria resistant to linezolid have already been identified.238 It was reported that

resistance to oxazolidinones is associated with specific mutations that changed the ribosomal target site of the antibiotic, lowering its binding affinity without affecting the translation machinery.234

The above discussion indicates that antibiotics are unique in that their extensive

uses in clinical therapy will inevitable facilitate the development of resistant bacteria. The

acquired resistance to a particular antibiotic will significantly compromise its therapeutic

utility. Not only that resistant bacteria will no longer be susceptible to that individual

drug but they will also be resistant to the entire class of antibiotics that act through the

same mechanism. Although a strain of bacteria resistant to one class of antibiotics is still

susceptible to other classes of antibiotics, the risks of becoming a multiple drug resistant

strain is highly increased by the propensity of bacteria to share acquired resistance even

with genetically unrelated microorganisms. For all these reasons, there is a continuous

need for new antibacterial agents with improved or new mechanisms of action.

3.3. New mechanism/structural class of antibiotics

Various strategies were developed to overcome the emergence of resistance to

antibiotics such as the introduction of incremental improvements in the structure of the

already existing drugs to increase their efficiency and the development of compounds that

would target and neutralize the resistance mechanisms that destroy the antibiotics.210

While these strategies successfully delivered new generations of antibiotics, the effectiveness and the shelf life of these compounds will have a relatively short duration. 162

A presumably better strategy is to identify new antibacterial agents and new biological processes or pathways unique for bacteria that could potentially became targets for antibiotics.

The approach of identifying new potential targets for antibacterial agents, led

Henkin’s group to the discovery of an unique and essential regulatory mechanisms with no mammalian correspondent, the T box transcription antitermination system that controls the gene expression in many bacteria.239 The genetic information required to

synthesize the all kinds of proteins in cells is store by DNA. But DNA is not the template

for the protein synthesis. Rather, the instructions for protein synthesis are given by the

mRNA templates. Therefore, the genetic information is first transferred from DNA

templates to mRNA templates, through a process called transcription, which then is

translated into proteins.1 This flow of genetic information from DNA to mRNA and into proteins constitutes the gene expression.

The T box transcription antitermination system, present in a variety of bacterial genes, is a mechanism that controls the gene expression by regulating the transcription, which ultimately regulates the translation of the proteins encoded in those genes. This regulatory mechanism is located at the 5’-untranslated leader region of the mRNA transcript.240 The leader region is the first segment of mRNA transcribed in the initiation

step of the transcription and its structural elements dictate if the transcription will be

continued or it will be stopped (Fig. 3.11). The transcription is continued when a

molecule of uncharged tRNA, as indicated in Fig. 3.11A, interacts with the nascent

mRNA at two positions. This two point interaction stabilizes a structural element called 163 the T box antiterminator that allows the transcription to continue.241 The lateral loop of

the antiterminator contains nucleotides of a highly conserved sequence called the T box, marked by the heavy black line in Fig 3.11.240 In the absence of the second mRNA-tRNA

interaction, that is the base pairing of the tRNA acceptor end with four complementary nucleotides in the T box, the antiterminator cannot be stabilized. Instead, a competing secondary structure, called the terminator is formed that signals the stop of the transcription (Fig. 3.11B).239, 241 Therefore, the leader mRNA controls the gene

expression through this mechanism of formation of the terminator or the antiterminator

determined by the tRNA that acts as a molecular effector.242 A specific example is

presented in the followings.

Figure 3.11. T box transcription antitermination system.241

Examples of proteins regulated by this T box transcription antiterminator

mechanism are the aminoacyl-tRNA synthetases, enzymes that are essential for the

protein synthesis. One role of these enzymes is to attach the amino acids to their

particular tRNA in order to be incorporated into the growing polypeptide chain during the 164 protein synthesis. For each amino acid (aa) there is one such enzyme that catalyzes the process depicted in Fig 3.12.1 The tRNA molecule without an amino acid attached to it is

termed uncharged tRNA and is termed charged tRNA (aa-tRNA) when an amino acid is

attached to it.

aminoacyl-tRNA synthetase aa + tRNA aa-tRNA

Figure 3.12. Aminoacyl-tRNA formation.

When bacteria cell is in need of a given aminoacyl-tRNA synthetase, the level of

uncharged tRNA is high since there is not enough enzyme around to attach an amino acid

to it. This means that the uncharged tRNA can interact with the nascent mRNA to stabilize the antiterminator structure, which results in the synthesis of full-length mRNA that is further used for the synthesis of that particular aminoacyl-tRNA synthetase. When the cell has enough of that enzyme the level of its product, the charged tRNA (aa-tRNA), increases. Because the amino acid is attached to it, the acceptor end of the charged aa- tRNA is unable to form the second interaction with the T box of leader mRNA.

Therefore, the terminator element is formed and the transcription is stopped, which in

turn stops the production of that particular protein.239

This regulatory mechanism is an ideal target for antibacterial agents. It regulates a

process essential for cell survival and is absent from the mammalian cells, therefore

inhibitors of the T box antiterminator system will prevent the bacterial growth with low

probability of affecting the host cells. Further more, the T box transcription 165 antitermination system is found in all groups of Gram-positive bacteria and each microorganism has multiple essential genes that are regulated by this T box system.239

Therefore, inhibitors of the T box system will be very efficient as antibacterial agents since they would have multiple target sites in one microorganism. Another advantage of targeting multiple sites in one organism is that the probability of developing resistance through mutations is highly reduced because it would require that all T box-regulated genes to mutate simultaneously. In view of these, the prospects for developing novel antibacterial agents with a new mechanism of action targeting the T box system are good.

3.3.1. New class of oxazolidinones

A study focused on the rational design of ligands with potential high affinity and specificity for the T box antiterminator has already been initiated. Hines’ group investigated the interactions between the tRNA and the antiterminator element in order to determine the structural characteristics that affect the function of the antiterminator. They developed two functionally relevant models of the T box antiterminator, named AM1A and AM1A(C11U) illustrated in Fig. 3.13A.243 The nucleotide sequences of the two

models differ by one nucleotide. As presented in Fig. 3.13A, at position 11, model

AM1A has a C while this C is replaced with a U in model AM1A(C11U). This

replacement, not only that induces structural changes in that part of the molecule but it

also affects the function, significantly decreasing the antitermination efficiency of the

AM1A(C11U) model compared with AM1A that is fully functional.244, 245 For these

reasons the AM1A(C11U) model was used as a specificity control in their studies. The

detailed structural-function studies revealed that for a high affinity binding of the tRNA 166 acceptor end to the first four nucleotides (U6GGA9) in the lateral loop of AM1A, a full

base pair complementarity is required. Only high affinity binding stabilizes the

antiterminator to promote expression.243 The studies also indicated that the tertiary

structure of the antiterminator influences the binding of the ligand. This specific ligand

recognition dictated by the structural features of the antiterminator was indicated by the reduced binding affinity of the tRNA for the AM1A(C11U) model.244

ABRhd U U U U U C C C C 11 G G G 11 G G C C G 9 A C C C 9 A C C C A G G A G G G U G U G G U GC U GC GC GC GC GC AU AU GC GC 5' 5' Fl AM1A AM1A(C11U)

Figure 3.13. A) T box antiterminator models; B) FRET-labeled AM1A model.241

Aminoglycosides, are well known compounds that bind to the RNA, particularly

the ribosomal RNA.246 For this characteristic, aminoglycosides were chosen to investigate the binding of small molecules to the AM1A model. The results established

that the T box antiterminator is a viable target for binding small molecules.247 It was observed that the binding of aminoglycosides induces conformational changes in the antiterminator that could be monitored with FRET (fluorescent resonance energy transfer) technique by using a fluorescent-labeled analog of AM1A, shown in Fig. 3.13B, 167 that has rhodamine (Rhd) attached at U18 position and fluorescein (Fl) attached at C29 position.247

The FRET antiterminator model system implemented by Hines’ group, was further used for screening of small molecules that would interfere with the binding of tRNA to the antiterminator. The binding of a ligand would induce conformational changes within the antiterminator that would lead to changes in fluorescence intensity

242 that can be further used to determine the binding constant, Kd, of that ligand. In addition, both AM1A and AM1A(C11U) models were used in the binding assays because of their differences in functionality and binding of the tRNA, that could be used to determine the specificity in binding of the small molecules. A better binding affinity for the AM1A than for AM1A(C11U) means that the ligand is able to distinguish between closely related tertiary structural features of the two antiterminator models which is indicative of binding selectivity.

The above mentioned binding assay was used to test a new group of oxazolidinones for their ability to bind to the two antiterminator models. Bergmeier’s group developed and synthesized a library of 3,4,5-trisubstituted and 4,5-disubstituted oxazolidinones (Fig. 3.14) that were used in preliminary SAR (structure-activity relationship) studies.242, 248 It was found that while all compounds bind to the

antiterminator, the 4,5-disubstituted compounds have a higher affinity, in the nanomolar

range, and also are able to differentiate between the two functionally different model

preferentially binding to the more functional AM1A model. The results indicate that the 168

4,5-disubstituted oxazolidinones are compounds that can potentially be developed into antibacterial agents targeting the T box antitermination system.242

O O 1 3 3 ON2 R ONH

5 4 R1 R2 R1 R2

3,4,5-trisubstituted 4,5-disubstituted oxazolidinones oxazolidinones

Figure 3.14. Substituted oxazolidinones.

Two of the 4,5-disubstituted oxazolidinones investigated so far showed interesting characteristics and results. As illustrated in Fig. 3.15, these compounds, ANB-22 and

ANB-40, are structurally different than the established oxazolidinone antibiotics represented here by linezolid. The main differences come from the substitution at N3 and

C4 positions. Compounds ANB-22 and ANB-40 have no substituents at the nitrogen N3 while at carbon C4 there is a basic substituent, which is in contrast with the structure of

linezolid that has substitution at the nitrogen while there is no substitution at C4 position.

Moreover, at position C5 the 4,5-disubstituted oxazolidinones have an ester group

compared to an acetamidomethyl group present in linezolid, that is in a trans relative

configuration with the substituent at C4 position.

169

F O O O O O N O N O NH O NH N Ph N Ph HN (S) O N O N HN O Ph O O Linezolid ANB-22 ANB-40

Figure 3.15. Linezolid and representatives of the new class of oxazolidinones.

The binding assays involving oxazolidinones ANB-22 and ANB-40 indicate that

both compounds bind the T box antiterminator with good affinity, ANB-40 showing a

better binding affinity, at nanomolar concentration (Kd = 0.9 μM) compared with ANB-

22 that binds at micromolar concentration (Kd = 13 μM). However, the compounds

showed difference in the binding selectivity, ANB-22 binding with 8-fold higher affinity

at AM1A than at AM1A(C11U) model while ANB-40 showed relative similar binding affinities for the two models.249 It was also observed that the two compounds interact

differently with the antiterminator. The difference in interaction was indicated by the

different maximal relative fluorescence (Frel) value that was 1.5-fold higher for ANB-40

then for ANB-22.249 The study also showed that they have different effect on the antitermination in vitro, ANB-22 competes with the tRNA and acts as an inhibitor of the antitermination while ANB-40 stabilizes the antiterminator acting as an enhancer of antitermination.

Due to their differences in the way they interact with the antiterminator and in the way they affect the antitermination, the two compounds ANB-22 and ANB-40 became the lead compounds for my research project intended to study their interaction with the

RNA structure from a three-dimensional point of view. Such a study will provide 170 valuable information that can be further applied for rational drug design strategies to develop compounds that would exhibit the high affinity of ANB-40 while retaining the inhibitory activity of ANB-22. A detailed discussion of this project is presented in the next chapter.

3.4. Summary

The discussion presented in this chapter was intended to emphasis the continuous need for new antibacterial agents with improved or novel mechanism of action as a consequence of the wide spread resistance to existing antibiotics. During their evolution, bacteria developed various strategies to fight against these compounds. Despite the large number of antibiotics used in clinical practice, there are only three bacterial targets attacked by these compounds: the cell wall, the DNA replication and repair and the protein synthesis. Resistant strains of bacteria have been identified for every main class of antibiotics. Therefore, a good strategy to overcome the emergence of resistance to antibiotics is to identify new biological processes that are unique for bacterial cell that could be potential targets for antibiotics. The T box transcription antitermination system was discovered to be an ideal target for antibiotics leading to the development of a novel class of antibacterial agents, the 4,5-disubstituted oxazolidinones.

171

CHAPTER 4: STEREOCHEMICAL SYNTHESIS OF 4,5-DISUBSTITUTED

OXAZOLIDINONES

4.1. Enantiomers of ANB-22 and ANB-40

4.1.1. Significance

Although it was established that our oxazolidinones bind to the T box antiterminator system, disrupting its interaction with tRNA, nevertheless there are intriguing gaps in our understanding of the three-dimensional interaction of these small molecules with the RNA structure. Therefore, a new study was initiated to investigate the stereospecificity of binding of the two lead compounds, ANB-22 and ANB-40, to the T box antiterminator. The ultimate goal is to develop the 4,5-disubstituted oxazolidinones into a new class of antibacterial agents. Therefore, for the complete understanding of their interaction with their biological target it is very important to take into account the chiral recognition. As I mentioned in Section 1.5, chiral recognition is the ability of a biological system to discriminate between enantiomers of a chiral drug. One of the main feature of the biological systems is their chirality which leads to differential interactions with chiral ligands. When enantiomers are introduced in a chiral environment, their behavior is different as a matter of principal. The enantiomers of a chiral ligand will interact differently with the chiral biomolecule, which in turn can lead to different binding affinities and consequently different biological activities.

4.1.1.1. Lead compound’s chirality

The new class of antibacterial agents, trans 4,5-disubstituted oxazolidinones, studied in this project exhibit chirality generated by the presence of two chiral centers on 172 the oxazolidinone ring, one at position C4 and the second at position C5 (Fig. 4.1). In

order to study the effect of their chirality on their binding affinity and biological activity,

it is necessary to know the exact 3-D structure of the compounds tested. This can be

accomplished by synthesizing individually both enantiomers of each lead compound.

Then, each enantiomer separately will be assayed for binding affinity and biological

activity. Any observed variation in the biological properties of two enantiomers will be

indicative of differences in their interaction with the biological target which can only be caused by their distinct spatial arrangements.

O O 1 3 O NH O NH 5 4 (R) (S) (S) (R) RO NR2 RO NR2

Figure 4.1. Enantiomers of trans 4,5-disubstituted oxazolidinones: 4R,5S (left) and 4S,5R

(right).

As shown in Fig. 4.1, one enantiomer of a trans 4,5-disubstituted oxazolidinone presents a 4R,5S configuration (left structure) while the second enantiomer has a 4S,5R configuration (right structure). The 3-D structures, presented in Fig. 4.1, illustrate the differences in the spatial arrangement of the two enantiomers. The substituent at position

C4 for the 4S,5R enantiomer is pointing up relative to oxazolidinone ring while for the

4R,5S enantiomer, the same substituent is pointing down. As the ligand approaches the

binding site of the T box antiterminator, various attractive and repulsive forces will be 173 generated until they will arrive at a point where these forces will balance each other. The different orientation in space of the same substituent will lead to different attractive or repulsive forces involved in the binding to the T box antiterminator of one enantiomer compared to its paired enantiomer. For example, a possible scenario could be that the binding site features a cationic binding pocket and a hydrogen-bond pocket as illustrated in Fig. 4.2. In this case, for 4S,5R enantiomer, the piperazine ring (the substituent at position C4) may be involved in ionic interactions with the cationic binding pocket as

indicated by the red lines in Fig. 4.2A. The piperazinic nitrogen atoms are shown in the

protonated form because prior testing, the oxazolidinones are converted to their

corresponding hydrochloric salts to improve their solubility. As illustrated in Fig. 4.2B,

for 4R,5S enantiomer, the substituent at position C4 is pointing in a different area in

space, which may prevent the ionic interactions to occur and also may lead to a possible

interaction with a different part of the binding site. Either way, the different orientation in space of this substituent could give rise to different interactions that could result in binding affinity variations.

174

A B

O O cationic cationic binding O binding O pocket pocket

Ph NH R O NH H H O O N 4 O N O 5 4S,5R O 4R,5S enantiomer NH enantiomer O NH Ph O R H-bond H-bond pocket pocket H H

Figure 4.2. Possible binding interactions.

The above reasoning could be also applied to the second substituent, at C5 position. The ester group could be involved in a H-bond interaction with the binding site as indicated in Fig. 4.2A for the 4S,5R enantiomer. For the 4R,5S enantiomer, this substituent has a significantly different orientation in space (Fig. 4.2B) that could interfere with the H-bond formation. As a consequence, the substituent will probe different parts of the binding site leading to different interactions. In conclusion, for the scenario exemplified in Fig. 4.2, the enantiomer with 4S,5R configuration will bind the T box antiterminator while the enantiomer with 4R,5S configuration will show no binding.

Based on the above explanation, the hypothesis is that, as a consequence of chiral recognition generated by the different interactions with the T box antiterminator, the enantiomers of each lead compound will exhibit different binding affinities. In addition, the more active enantiomer of both lead compounds, ANB-22 and ANB-40, will have the same configuration at the two chiral centers. 175

4.1.1.2. Target compounds

As I mentioned before, for investigating the interaction of trans 4,5-disubstituted oxazolidinones with the T box antiterminator from a 3-D stand point and for studying the effect of chirality on their binding affinity, it is necessary to analyze the enantiomers of the lead compounds individually. Therefore, the project discussed in this chapter is specifically focused on the synthesis of the enantiomerically pure compounds shown in

Fig. 4.3. Compounds COB-30 and COB-31 are the two enantiomers of lead compound

ANB-22 while compounds COB-32 and COB-33 are the enantiomers of lead compound

ANB-40.

O O

O NH O NH (R) (S) (S) N Ph (R) N Ph O N O N

Ph O Ph O (4R),(5S)-COB-30 (4S),(5R)-COB-31

O O O O

O NH O NH (R) (S) (S) N Ph (R) N Ph O N O N HN HN O O (4R),(5S)-COB-32 (4S),(5R)-COB-33

Figure 4.3. Enantiomers of lead compounds ANB-22 and ANB-40.

Based on the hypothesis presented earlier, upon biological evaluation of each enantiomer separately it is expected that one of the enantiomers COB-30 and COB-31 to have a better binding affinity then the racemic ANB-22 while the other enantiomer will exhibit a lower binding affinity or it will not bind at all. The same holds true for the 176 enantiomers COB-32 and COB-33 of the second lead compound ANB-40. The synthesis of the target compounds is presented in detail in the following sections.

4.1.1.3. Plan of synthesis

For the preparation of the target compounds I chose to follow the synthetic plan employed in the preparation of the racemic lead compounds ANB-22 and ANB-40. This synthetic plan involves a method developed by Bergmeier’s group for parallel synthesis of racemic trans 4,5-disubstituted oxazolidinones.248 The synthesis, presented in Scheme

4.1, starts from the commercially available racemic 3-butene-1,2-diol 4.1 which is

converted to the allylic alcohol 4.2 by a selective protection of primary hydroxyl with

trityl (Tr) group. The allylic alcohol is then converted to an azidoformate 4.3 that

undergoes thermolysis to provide bicyclic compound 4.4 as a single diastereomer. The

aziridine ring of compound 4.4 can then be opened with a large variety of nucleophiles to

afford the oxazolidinones 4.5. After removing the trityl group, the resulting

oxazolidinones can be readily acylated with a variety of acid chlorides or isocyanates to

provide the desired compounds 4.6.

O

OH OH O N TrCl 1)ClC(O)O(4-NO2Ph) 3 HO TrO TrO 2) NaN3 4.1 4.2 4.3 Δ O O O O R Cl Nu O NH ON or O NH 5 4 O Nu NCO TrO R R TrO Nu H 4.6 O 4.5 4.4

Scheme 4.1. Parallel synthesis of trans 4,5-disubstituted oxazolidinones.

177

In order for this sequence of reactions to be successfully applied for the preparation of the desired enantiomers, a key requirement needed to be addressed, that is the control of the absolute configuration of the two chiral centers (C4 and C5 positions) in

the oxazolidinone ring during the synthesis. As it was discussed in Section 2.1.2.2 of this

dissertation, there are two strategies that could be used to prepare enantiomerically pure

compounds from racemic starting materials. The first option would be to prepare the

desired compound in racemic form and then to separate it in its enantiomer constituents

by resolution. Resolution involves the conversion of a racemic mixture into a

diastereomeric mixture using an enantiopure resolving agent. The best candidates for

resolution are substrates such as acids and amines that could form diastereomeric salts

separable through recrystallization. Compounds like 4.6 as well as the intermediates in

the synthesis presented in Scheme 4.1, are lacking the mentioned structural feature

required for a successful resolution. Therefore, resolution was not the best option for the

preparation of the desired enantiomers.

The second option would be to introduce the chiral centers in a stereoselective

fashion by using enantioselective reactions somewhere down the synthetic pathway. This

strategy is only partially applicable for this case because only one of the chiral center (at

C4 position) is introduced during the synthesis while the other chiral center (at C5 position), marked in red (Scheme 4.1), is actually present from the beginning in the starting material 4.1. However, this observation suggested an additional option that is to carry out the synthesis using enantiomerically pure starting materials. By doing so, the absolute configuration of the chiral center at C5 position would be set at the initial stage 178 of the synthesis. Then, the second chiral center would be introduced during the intramolecular conversion of compound 4.3 into bicyclic compound 4.4 when the fussed oxazolidinone and aziridine rings are assembled. This conversion is diastereoselective since only the trans diastereomer is obtained. Therefore, by using enantiomerically pure

4.3 that can be readily available from enantiomerically pure starting material 4.1, the configuration at C5 stereocenter will dictate the configuration at the newly formed C4 stereocenter. This last option was chosen for the synthesis of the desired enantiomers of the lead compounds.

Before starting the preparation of the target compounds, there was one more point about the synthesis presented in Scheme 4.1 that I needed to address. When the synthesis was used to prepare racemic products all the reactions proceeded in excellent yields with the exception of the diastereoselective intramolecular formation of bicyclic aziridine 4.4 that afforded the product in 50 % yield. Although this is a satisfactory result, an attempt to improve even more the outcome of the intramolecular aziridination reaction was initiated by evaluating few other methods to prepare the bicyclic aziridine 4.4, which is presented in the following section.

4.1.2. Studies on the intramolecular aziridination

The examination of several other methods to prepare the bicyclic aziridine 4.4 was performed using racemic starting materials, as they are easily available and less expensive than their enantiomerically pure versions. I was looking for a method that would be high yielding but in the same time would retain the diastereoselectivity, since 179 the product of interest should posses a trans relative configuration at C4 and C5 positions for the ensuing oxazolidinone.

4.1.2.1. Intramolecular aziridination of allyl-substituted carbamate

The first method examined was the iodine(III)-mediated intramolecular aziridination of unsaturated sulfonamides (Scheme 4.2, eq1), a method reported by

Padwa et al. in 2002.250 Their study shown that by treating unsaturated sulfonamides 4.7

with PhI(OAc)2, MgO and catalytic amounts of Rh2(OAc)4, bicyclic aziridines 4.8 are obtained in very good yields.251 They extended the initial study by exploring various

reaction conditions and including other substrates such as cycloalkenyl carbamates

(Scheme 4.2, eq2). They observed that the intramolecular aziridination reaction of cyloakenyl carbamates 4.9 proceeds smoothly in the presence of iodosobenzene (PhIO) with or without the rhodium (II) catalyst affording tricyclic aziridines 4.10 in good

yields.250, 251

PhI(OAc)2 N SO NH O2S eq 1 n 2 2 Rh(II)Ln n = 1, 2 n MgO, CH2Cl2 4.7 85-91 % 4.8

O O O O PhIO R NH2 N eq 2 with or without R R Rh2(OAc)4 R 71-75 % 4.9 4.10

Scheme 4.2. Iodine(III)-mediated aziridination reaction.

180

Inspired by the above mentioned study involving carbamates, the iodine(II)- mediated aziridination reaction was applied to the synthesis of bicyclic aziridine 4.4.

Initially, the substrate carbamate was prepared starting from tritylether 4.2 as presented in

Scheme 4.3. Treating 4.2 with trichloroacetyl isocyanate (CCl3C(O)NCO) followed by

251 K2CO3/MeOH provided carbamate 4.11 in almost quantitative yield.

O OH 1) CCl3C(O)NCO O NH2 TrO TrO 2) K2CO3, MeOH 4.2 98% 4.11

Scheme 4.3. Synthesis of carbamic acid allyl-substituted ester.

With the carbamate 4.11 in hand, the intramolecular cyclization reaction was

initially performed following the reported reaction conditions that involved the treatment

of 4.11 with 2 equivalents of iodosobenzene in the presence of 0.025 equivalents of

251 Rh2(OAc)4 catalyst. When the crude product of this reaction was analyzed by NMR it

was observed that along with the desired product 4.4, its diastereomer cis-4.4 and an aziridine ring-opened byproduct were also obtained while large amount of starting material was present in the mixture in a 2:1.2:1:5 ratio as shown in Scheme 4.4.

Although, the aziridine ring-opened byproduct was not unequivocally identified, I strongly believe that is compound 4.12 based on the presence of an amidic NH peak in

the proton NMR and based on the good reactivity of aziridine ring toward nucleophilic

attack, which indicates the possibility that the presence of water in the reaction mixture

could lead to such a product. 181

O O O O PhIO, MS ON ON O NH2 + + O NH TrO DCM, 50 °C, 12h TrO H TrO H TrO OH 4.11 4.4 cis-4.4 4.12

Conditions Crude product ratio Isolated Isolated product ratio 4.4 : cis-4.4 : 4.12 4.4 : 4.12 PhIO Rh2(OAc)4 yield

2 eq 0.025 eq 2 : 1.2 : 1 : 5 (4.11) -- --

3 eq -- 2 : 1 : 1.25 17 % 2 : 1

5 eq -- 2.5 : 1 : 1.25 47 % 2 : 1

Scheme 4.4. Intramolecular aziridination of carbamate.

In the next attempt, the reaction was carried out in the absence of rhodium (II) catalyst while increasing the amount of PhIO to 3 equivalents. The outcome of the reaction was somewhat improved compared with the previous reaction as no starting material was observed in the crude reaction mixture. However, no improvement was observed regarding the selectivity since the reaction provided all three mentioned

products in similar ratio 2:1:1.25 (Scheme 4.4). The effort of isolating the bicyclic

aziridine 4.4 by flash chromatography only resulted in a successful separation of the diastereomer cis-4.4. The desired product was obtained in 17 % yield as a mixture with compound 4.12 in a 2:1 ratio. As shown in Scheme 4.4, further increasing the amount of

PhIO to 5 equivalents provided again a mixture of the three products in 2.5:1:1.25 ratio.

The only improvement observed was a significant increase in the reaction yield. Once again the desired product could not be separated from the byproduct 4.12 and it was isolated in 47 % yield as 2:1 mixture. 182

The moderate yield, the lack of diastereoselectivity along with the presence of an undesired byproduct, rendered this intramolecular aziridination of carbamates as an inefficient method for the purpose of preparing bicyclic aziridines such as 4.4. Therefore,

I discarded this method and proceeded to examine other option.

4.1.2.2. Intramolecular aziridination of allyl-substituted N-tosyloxy carbamate

In a recent study, S. A. Fleming and coworkers reported the formation of bicyclic aziridines 4.14 by a copper-catalyzed intramolecular aziridination of allylic N- tosyloxycarbamates 4.13 as illustrated in Scheme 4.5.252 Their study included

examination of various substituted allylic N-tosyloxycarbamates, different copper

complexes and various bases and solvents. They concluded that for an efficient

aziridination the reaction conditions should involve large amounts of potassium base in

the presence of catalytic amounts of (CF3SO3Cu)2•C6H6 complex (Scheme 4.5).

O 3 R 2 H (CF SO Cu) C H (5 mol%) R N R1 O N 3 3 2 6 6 OTs 1 O K2CO3 (7 eq) R R2 O 3 CH3CN, rt R 4.13 42-90 % 4.14

Scheme 4.5. Copper-catalyzed aziridination reaction.

The experimental results reported in the above mentioned study, indicated that the

copper-catalyzed aziridination might be a useful method for the synthesis of my desired

bicyclic aziridine 4.4. In order to proceed with the examination of this method, the

substrate N-tosyloxycarbamate 4.16 needed to be synthesized first. As shown in Scheme

4.6, following a reported one pot procedure to prepare N-hydroxycarbamates, the 183 tritylether 4.2 was treated with 1,1’-carbodiimidazole (CDI) followed by treatment with hydroxylamine hydrochloride and imidazole to provide compound 4.15 in 79% yield.253

Using standard tosylation reaction, the desired tosyloxycarbamate 4.16 was prepared in

65% yield. The attempt to increase the yield of tosylation reaction by increasing the amount of TsCl proved to be detrimental for the reaction affording a complex of unidentified products.

O O OH OH OTs 1) CDI, rt, 2 h N TsCl, Et3N N TrO O H O H 2) NH OH HCl rt, 12 h 2 TrO TrO Imidazole, rt, 5 h 65% 79% (2 steps) 4.2 4.15 4.16

Scheme 4.6. Synthesis of allyl-substituted N-tosyloxycarbamate.

With the N-tosyloxycarbamate 4.16 in hand, I proceeded with the examination of its intramolecular aziridination reaction. Following the reported procedure mentioned earlier, that consisted of treating compound 4.16 with large excess of potassium carbonate in the presence of 5 mol% (CF3SO3Cu)2•C6H6 complex, the starting material

was recovered while no aziridination product was obtained. Therefore a study of different

catalysts and solvents was initiated and the results are summarized in Table 4.1 where

entry 1 corresponds to the already mentioned reaction carried out under the reported

procedure.

184

Table 4.1

O O OTs N Catalyst (5 mol%) O N O H TrO K2CO3 (7 eq) TrO Solvent, rt, 16 h 4.16 4.4

Aziridination of allyl-substituted N-tosyloxycarbamate

Entry Catalyst Solvent Isolated yield of 4.4

1 (CuOTf)2⋅C6H6 CH3CN no reaction

2 Rh2(OAc)4 acetone no reaction

3 Cu(Py)4(OTf)2 acetone no reaction

4 Cu(OTf)2⋅C6H6 CH2Cl2 40 %

5 Cu(OTf)2 CH2Cl2 40 %

6 Cu(Py)4(OTf)2 CH2Cl2 47 %

The rhodium (II) catalyzed version of the intramolecular aziridination of allylic

N-tosyloxycarbamates has also been reported.254 Therefore, for my second trial I decided

to follow this procedure which consisted of using Rh2(OAc)4 as the catalyst, an excess of

potassium carbonate and acetone as the solvent (entry 2, Table 4.1). Unfortunately, the

reaction only afforded an unidentified complex mixture of compounds. Copper (II)

complexes, especially pyridine (Py) copper complexes, have also been reported to be

active catalysts in this type of intramolecular aziridination reaction.255 Based on this

information, I chose to examine three such copper (II) catalysts, namely Cu(OTf)2•C6H6 and Cu(OTf)2 which are commercially available and Cu(Py)4(OTf)2 that was prepared following a known procedure, by treating Cu(OTf)2 with excess of pyridine in boiling 185

256 methanol. The aziridination reaction employing Cu(Py)4(OTf)2 was performed in

acetone (entry 3) and provided once again an unidentified complex mixture of compounds. However, the reactions performed in methylene chloride with either of

Cu(OTf)2•C6H6 and Cu(OTf)2 catalysts (entry 4 and 5) afforded exclusively the desired

bicyclic aziridine 4.4 in 40 % yield. These results indicated that methylene chloride might

be a better solvent for aziridination than acetone. Therefore, in an attempt to improve the yield, the intramolecular aziridination 4.16 was performed using Cu(Py)4(OTf)2 in methylene chloride (entry 6). This procedure afforded the desired product 4.4 in 47 % yield.

In order to be able to choose the most suitable method for the preparation of bicyclic aziridine 4.4, it was important to also perform the aziridination reaction by thermal decomposition of allyl-substituted azidoformate used in the parallel synthesis reported by Bergmeier. The comparison between the results obtained with this method and the results provided by the examined methods discussed earlier, will indicate the most efficient path to the desired compound.

4.1.2.3. Intramolecular aziridination of allyl-substituted azidoformate

The studies on the thermal intramolecular reaction of azidoformates with olefins, conducted in Bergmeier’s laboratory, indicated that by heating an allylic azidoformate in a sealed tube generates a bicyclic aziridine in good yields and excellent diastereoselectivity.257-259 As mentioned in Section 4.1.1.3, this method was successfully

applied for the synthesis of a library of compounds.248 I reproduced the synthesis of

bicyclic aziridine 4.4 by following the reported procedures as illustrated in Scheme 4.7. 186

1)ClC(O)O(4-NO2Ph) O O Py, DCM OH rt, 4h O N3 DCM ON TrO 2) NaN , DMF TrO 109°C, 13h 3 TrO 50% H 35°C, 18h 4.2 4.3 4.4 80% (2steps)

Scheme 4.7. Aziridination of allyl-substituted azidoformate.

The allylic azidoformate 4.3 is readily available from tritylether 4.2 in two steps.

Treating 4.2 with p-nitrophenyl chloroformate followed by sodium azide afforded 4.3 in

80 % yield (Scheme 4.7). The thermal intramolecular aziridination was then performed by heating compound 4.3 in a seal tube at 109 °C for 13 hours to provide the desired bicyclic aziridine 4.4 in 50% yield as single trans diastereomer. With this result in hand I proceeded with the comparison of the three studied methods for the preparation of compound 4.4 in order to decide which one would be the most efficient for the synthesis of its enantiomers.

The decision was made based on the following criteria: substrate accessibility, diastereoselectivity, product separation and yield. In Table 4.2 are summarized the important observations for each method. For the first aziridination method, the advantage was that the substrate carbamate 4.11, was obtained in almost quantitative yield.

However, the drawback of the intramolecular aziridination reaction was the lack of diastereoselectivity since besides the trans desired compound, the cis diastereomer was also obtained. Another negative point of this method was that provided a byproduct that significantly complicated the purification of the desired product. As a consequence it was easy to render this method as highly inefficient. Thus the real comparison was between 187 the second and the third method. Both these methods exclusively provided the desired product in moderate yield and high diastereoselectivity. However, the third method has an advantage over the second method in terms of substrate accessibility. The azidoformate 4.3 was prepared in two steps and good yield (80 %) compared with the substrate for the second method, N-tosyloxycarbamate 4.16 that was obtained in three steps and moderate yield (52 %). Moreover, in the aziridination of azidoformate, there are no other reagents required besides the substrate, which significantly simplifies the purification of the product.

Table 4.2

Comparison of intramolecular aziridination reactions

Method Substrate (yield %) Yield (%) Problem

1 carbamate 4.11 (98) 47 diastereoselectivity

byproduct

yield

2 N-tosyloxycarbamate 4.16 (52) 47 substrate preparation

yield

3 azidoformate 4.3 (80) 50 yield

Based on the above evaluation, I concluded that the intramolecular aziridination

of the azidoformate 4.3, is the most efficient method for the preparation of bicyclic

aziridine 4.4 and I proceeded with the synthesis of the enantiomerically pure target 188 compounds by starting with the individual preparation of each enantiomer of the starting material as presented in the following section.

4.1.3. Enantiomerically pure 3-butene-1,2-diol

As I mentioned earlier, the synthesis of the enantiomerically pure lead compounds

ANB-22 and ANB-40 needs to start from the enantiomerically pure starting material 3- butene-1,2-diol 4.1 (recall section 4.1.1.3). Therefore, an evaluation of the existing methodologies for the preparation of both enantiomers of 4.1 had been initiated with the desire to find the most efficient and suitable method. I was specifically interested in methods that would meet the following criteria: the desired enantiomers should be obtained in a single step and very high enantiomeric excess, the commercially available materials should be inexpensive, and if catalysts are used then they should be readily available in both enantiomeric forms and optimally would be used in small amounts.

The early preparations of enantiomerically enriched 3-butene-1,2-diol made use of naturally occurring chiral materials such as D-mannitol or tartaric acid. D-mannitol, an inexpensive chiral pool hexitol with four chiral centers, has been used as a chiral source in our laboratory for the synthesis of S enantiomer of 3-butene-1,2-diol.259, 260 This method involves a multi-step synthesis that provides only the S enantiomer of diol 4.1 in good yields. Such an approach is not a viable choice for the synthesis of my desired target compounds (recall section 4.1.1.2) since both enantiomers of 4.1 are required and because it would significantly increase the number of steps in the synthesis. The conversion of enantiomers of tartaric acid into enantiomers of diol 4.1 also requires 189 multi-step chemical transformations, therefore presenting no interest for the purpose of my synthetic plan.261

More recent reported methods for the preparation of enantiomerically enriched 3-

butene-1,2-diol include kinetic resolutions such as enzymatic resolution262, 263 and

chemical kinetic resolution.264, 265 A kinetic resolution, as illustrated in Fig. 4.4, is

actually a chemical reaction of a racemate (A) in which the reaction rates of the

enantiomers are different such that one enantiomer is converted into the product (B) more

rapidly than the other.197 In order for this process to take place, a chiral reagent is

required that is typically not used in stoichiometric amounts to ensure that only the most

reactive enantiomer will undergo the transformation leaving behind the unreactive

enantiomer that would be essentially enantiomerically pure. As a consequence, the

theoretical yield of a kinetic resolution can only be 50 %.

kR > kS A B + (S)-A chiral reagent

Figure 4.4. Kinetic resolution.

In enzymatic resolutions, the enzymes are the chiral catalysts that promote the

transformations. The advantage of using enzymes is that they are highly effective and

very selective in their activity but they also have the disadvantage of being easily

denatured and most often require stoichiometric amounts of cofactors (coenzymes) such

as ATP. Nevertheless, the enzymatic resolutions have been extensively exploited.197 The enzymatic resolution of racemic 3-butene-1,2-diol has been accomplished by using 190 glycerol kinase that catalyzes the phosphorylation of primary hydroxyl group in a highly regiospecific and stereoselective manner.263 The enzyme catalyzes the phosphorylation of

the substrate at the expense of an ATP (adenosine triphosphate) molecule. To minimize

the cost of using ATP and to prevent the inhibition of glycerol kinase by the

accumulation of ADP (adenosine diphosphate), in the reported procedure the ATP is

regenerated from ADP over the course of reaction by using PEP (phosphoenolpyruvate)

and pyruvate kinase. Despite of this tactic, the method can still be rendered as expensive.

Although this reported enzymatic resolution of 3-butene-1,2-diol afforded the desired

enantiomers in excellent enantiomeric excess and moderate yield, due to high cost, I will

only consider it for the synthesis of the enantiomers of the lead compounds ANB-22 and

ANB-40 as a last resort .

The chemical kinetic resolutions used for the preparation of enantiomers of 3-

butene-1,2-diol or its simple derivatives include Sharpless asymmetric epoxidation of

allylic alcohols and hydrolytic kinetic resolution of terminal epoxides.264-266 Both of these

methods offered the desired enantiomers in high enantiomeric excess and relative good

yields. Moreover, the enantiomerically pure products were obtained in one step from

fairly inexpensive racemic materials and chiral catalysts thus meeting the required criteria

mentioned in the begging of this section. For these considerations I decided that both

methods could be applied to my synthetic plan for the preparation of target compounds

and I chose to examine the Sharpless epoxidation first only because it is a well known

reaction that proved to have wide applicability. In the following section is in detailed

discussed the work involved and the results obtained with this method. 191

4.1.3.1. Sharpless asymmetric epoxidation

Sharpless asymmetric epoxidation is the reaction of an allylic alcohol with stoichiometric amounts of t-butyl hydroperoxide (TBHP) in the presence of titanium alkoxide tartrate chiral catalyst that forms an epoxy alcohol in high enantiomeric excess.

With a slight modification of this asymmetric epoxidation procedure that involves the use of only 0.6 equivalents of the oxidant TBHP, the kinetic resolution of secondary allylic alcohols can also be effected.267 This Sharpless kinetic resolution was successfully applied for the synthesis of two derivatives of 3-buten-1,2-diol in their enantiomerically

pure forms. One of them was 1-benzyloxy-3-buten-2-ol that underwent kinetic resolution to provide the R enantiomer in good yield and good enantiomeric composition, that was further used to prepare the S enantiomer under Mitsunobu conditions.264 The second

reported derivative of 3-buten-1,2-diol to undergo kinetic resolution was 1-tosyloxy-3- buten-2-ol.265 In this case, the Sharpless procedure provided a direct access to either enantiomer of 1-tosyloxy-3-buten-2-ol in good yield and moderate enantiomeric excess.

Further recrystallizations of the products afforded high enantiomeric composition for the desired compounds.

Based on these two reported examples, I decided to prepared in the racemic form the primary monotritylated 3-butene-1,2-diol 4.2, which is the first intermediate in the synthesis of target compounds and then use Sharpless kinetic resolution to prepare its enantiomers. Compound 4.2 is readily available from 3-butene-1,2-diol 4.1 in 87 % yield as shown in Scheme 4.8, by selectively protecting the primary hydroxyl with trityl group.

192

OH OH TrCl, Et3N HO TrO DMAP, CH2Cl2 4.1 87 % 4.2

Scheme 4.8. Synthesis of monotritylated 3-butene-1,2-diol.

The kinetic resolution of compound 4.2 by means of Sharpless asymmetric

epoxidation was performed following the reported procedure as illustrated in Scheme

4.9.265, 268 By using only 0.6 eq of oxidant TBHP relative to 1 eq of substrate only one enantiomer of the racemate will undergo epoxidation while the less reactive enantiomer will be recovered in enantiomerically enriched form. The chiral catalyst used is a titanium complex formed between titanium tetraisopropoxide Ti(OiPr)4 and the enantiomers of

diisopropyltartrate (DIPT) as chiral ligands. When the D-(-)-DIPT is employed for this

kinetic resolution the S enantiomer of allylic alcohol 4.2 would be obtained while L-(+)-

DIPT will afford the R enantiomer. In the initial reaction performed, a methylene chloride

solution of racemic allylic alcohol 4.2 was treated with L-(+)-DIPT, Ti(OiPr)4 and 0.6 equivalents of TBHP and kept at –10 °C for 72 h.

OH L-(+)-DIPT OH OH TBHP (0.5 eq) TrO TrO + TrO (S) Ti(OiPr) (R) (R) 4 O -t °C, CH2Cl2, MS 4.2 (R)-4.2 4.17

Scheme 4.9. Sharpless asymmetric epoxidation of 4.2.

After the product separation by column chromatography it was observed that the

enantiomerically enriched allylic alcohol (R)-4.2 was recovered in 87% yield indicating 193 that the epoxidation proceeded in only 13% yield. This was an undesired result since in order to obtain high enantiomeric excess (ee) for the unreacted allylic alcohol the epoxidation should proceed in at least 50% yield. Therefore, the recovered R enriched allylic alcohol from the initial kinetic resolution was subjected to a second epoxidation reaction performed at a higher temperature (–5 °C) and for a longer period of time (89 h).

This time the R enriched allylic alcohol (R)-4.2 was isolated in 49% yield. The specific rotation of (R)-4.2 obtained in this reaction was [α]D = - 15.9 (c 1.05, MeOH). In order to

prove the efficacy of this kinetic resolution by means of Sharpless epoxidation it was

required to determine the enantiomeric composition of the allylic alcohol (R)-4.2. The

method of choice and the reasoning behind it are presented in the next section.

4.1.3.2. Determination of enantiomeric excess

It was mentioned before (recall section 2.1.2.2.3) that the most common

techniques used to determine the enantiomeric excess of a chiral substrate are chiral

chromatography and NMR spectroscopy. In the chiral chromatography, the enantiomeric

separations are usually achieved by employing chiral stationary phases. The enantiomeric

mixtures are separated based on the differential interactions that can occur as a result of

their unique spatial orientation with the chiral stationary phase. The non-chiral nature of

the spectroscopic techniques requires that the enantiomeric mixtures to be converted into

their corresponding diastereomeric mixtures prior to analysis. Diastereomers can be distinguished in the NMR based on the chemical shift nonequivalence exhibited by specific diastereotopic nuclei. The diastereomeric composition is directly related to the enantiomeric composition of the original mixture. 194

Since chromatographic methods seem to offer a simple and direct route to the assessment of enantiomeric composition of chiral substrates the first method I chose for the ee determination of compound (R)-4.2 was HPLC by using a (R,R)-Welck 01 chiral column. My plan was to initially analyze the racemic 4.2 with the expectation that two distinct peaks, one for each enantiomer, of same intensity would be observed. Then, this observation would be used to unequivocally identify the peaks that would indicate the enantiomeric composition when (R)-4.2 would be analyzed. Unfortunately, when the racemate 4.2 was passed through the chiral column, no enantiomeric separation was achieved. This result indicates that the chiral recognition abilities of the column used are not suitable for this particular analyte. To enhance the differences in spatial orientation of the enantiomers encompassed in the racemic mixture that would ultimately increase their difference in interaction with the column, the racemate 4.2 was converted to a diastereomeric mixture as presented in Scheme 4.10. The ester 4.18 was prepared by standard esterification procedure200 using (S)-(-)-α-methoxy-α-trifluoromethyl

phenylacetyl chloride (Mosher’s chloride; MTPA-Cl) as chiral derivatizing agent.

O CF3 O OMe OH (S) MTPA-Cl Ph TrO TrO DMAP, DCM Et N 4.2 3 4.18

O CF3 OH O OMe MTPA-Cl (S) TrO Ph (R) DMAP, DCM TrO (R) Et3N (R)-4.2 (R,S)-4.18

Scheme 4.10. Preparation of Mosher’s esters (R,S)-4.18 and 4.18. 195

I was expecting that this additional spatial differentiation would facilitate the separation on the chiral HPLC column but, on the other hand, in the event that the chiral

HPLC would fail to achieve the desired separation, the diastereomers could be used to determine the diastereomeric composition by NMR analysis. Therefore, the diastereomer

(R,S)-4.18 was also prepared (Scheme 4.10). When the mixture of diastereomers 4.18 was analyzed by chiral HPLC only slight separation was observed but no baseline resolution was achieved as illustrated in Fig. 4.5. This result could not be used to determine the ee of (R,S)-4.18, therefore, the chiral chromatographic method was abandoned.

O Retent ion Time CF3 O OMe 30 Area Percent (S) 30 Ph TrO

4.18 20 20

10 10

0 0

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 Minute

Figure 4.5. Chiral HPLC chromatogram for racemic 4.18.

The second method that could be used to indirectly determine enantiomeric composition is by analyzing the corresponding diastereomeric mixture by proton NMR. 196

When the 1H NMR of the diastereomeric mixture 4.18 was taken in deuterium chloroform, no chemical shift differences were clearly observed for any proton. However, it was gratifying to find out that when deuterated benzene (C6D6) was used, a clear

diastereomeric pair, one peak at 3.64 ppm and the second at 3.57 ppm, was observed for

the methoxyl protons (OMe) as can be seen in the spectrum expansion presented in Fig.

4.6 (top spectrum). The proton spectrum of (R,S)-4.18 was also recorded and an

expansion of the region of interest is presented in Fig. 4.6 (bottom spectrum). The mole

fraction of the dominant enantiomer in (R,S)-4.18 was obtained by integrating the

mentioned diastereomeric pair of signals and it was further used to calculate the ee. The

enantiomeric excess was determined to be 45%. This peculiar result is possibly due to a

highly steric hindrance caused by trityl group. Given this result, the kinetic resolution by

means of Sharpless epoxidation was rendered inefficient for the preparation of R and S

enantiomers of allylic alcohol 4.2 thus I turned my attention toward hydrolytic kinetic

resolution. 197

O CF3 O OMe (S) Ph TrO

4.18

O CF3 O (S) OMe Ph TrO (R) (R,S)-4.18

Figure 4.6. 1H NMR spectra of racemic 4.18 (top) and diastereomer (R,S)-4.18 (bottom).

198

4.1.3.3. Hydrolytic kinetic resolution

The unsatisfying results obtained with Sharpless kinetic resolution led me to examine the second possible approach for the synthesis of enantiomerically pure starting material, that is hydrolytic kinetic resolution (HKR) of terminal epoxides. The HKR was developed by Jacobsen in 1997 as a method to kinetically resolve terminal epoxides.269 It was reported that kinetic resolution of butadiene monoepoxide provides a direct access to both the 3-butene-1,2-diol product and the unreacted epoxide in high ee.266 The racemic

terminal epoxide is treated with approximately half equivalent of water and low loading

of a recyclable chiral catalyst (< 0.5 mol%) to provide the 1,2-diol product while the

unreacted enantiomer of the epoxide is recovered. The chiral catalyst is a (salen)-Co(III)

complex (R,R)-4.20, called Jacobsen’s catalyst (Fig. 4.7). This active form of the catalyst is prepared in situ from the Co(II)-salen 4.19 which is catalytically inactive. Both enantiomers of 4.19 are commercially available and by direct exposure to air in the presence of acetic acid can easily be converted to the catalytically active form 4.20.

H H H H N N N N Co Co t-Bu O O t-Bu t-Bu O O t-Bu OAc t-Bu t-Bu t-Bu t-Bu (R,R)-SalenCoc(II) complex (R,R)-SalenCoc(III) OAc complex inactive form active form (R,R)-4.19 (R,R)-4.20

Figure 4.7. Jacobsen’s catalyst.

199

Using the hydrolytic kinetic resolution starting from commercially available racemic butadiene monoepoxide 4.21 and using the appropriate Jacobsen catalyst, I prepared the R and S enantiomers of allylic diol 4.1 as shown in Scheme 4.11 following the reported procedure. When the (R,R)-4.20 catalyst was used the (S)-4.1 was obtained in 20% yield while the other enantiomer of the catalyst afforded the (R)-4.1 diol in 20% yield. It is important to note that to ascertain a high enantiomeric excess of the allylic diol, only 0.45 eq of water were used therefore the theoretical yield for this reaction is necessary limited to 45%. With both enantiomers of the starting material 4.1 in hand, the next required step was to determine their enantiomeric excess which is presented in the next section.

OH OH (S,S)-4.19 O (R,R)-4.19 HO HO (R) AcOH, H2O AcOH, H2O (S) (R)-4.1 20% 4.21 20% (S)-4.1

Scheme 4.11. Hydrolytic kinetic resolution of butadiene monoepoxide.

4.1.3.4. Enantiomeric excess determination of (R)-4.1 and (S)-4.1

Before the enantiomers of 3-butene-1,2-diol could be used further in the

synthesis, their enantiomeric excess needed to be determined. Again, my first attempt to

determine the ee employed chiral HPLC by passing each enantiomers of 4.1 through the

(R,R)-Welck 01 chiral column. Unfortunately, this direct method failed again to provide

separation. Because the ee of monotritylated derivative of 4.1 obtained from the

Sharpless kinetic resolution, discussed earlier, was successfully determined using NMR

spectroscopy, I proceeded with the conversion of both enantiomers (R)-4.1 and (S)-4.1 to 200 their corresponding monoprotected derivatives (R)-4.2 and (S)-4.2 as shown in Scheme

4.12. Then each enantiomer was further derivatized with optically active Mosher’s chloride following a standard esterification procedure.200

O CF3 OH OH O OMe TrCl, Et N MTPA-Cl (S) HO 3 TrO Ph (R) TrO DMAP, DCM (R) Et3N (R) DMAP, DCM (R)-4.1 64% (R)-4.2 (R,S)-4.18

O CF3 O OMe OH OH (S) TrCl, Et3N MTPA-Cl Ph HO TrO DMAP, DCM Et3N TrO (S) (S) (S) 60% DMAP, DCM (S)-4.1 (S)-4.2 (S,S)-4.18

Scheme 4.12. Preparation of Mosher’s esters (R,S)-4.18 and (S,S)-4.18.

As it was mentioned in section 4.1.3.2, when the proton NMR spectra of

diastereomers 4.18 and (R,S)-4.18 are recorded in C6D6, the methyl protons of methoxy

groups (OMe) experience chemical shift nonequivalence. For one enantiomer these

protons generate a signal at 3.51 ppm and for the other enantiomer at 3.44 ppm. The

proton NMR spectra for both diastereomers (R,S)-4.18 and (S,S)-4.18 prepared as

mentioned before (Scheme 4.12) were therefore recorded in C6D6. As expected, the

diastereotopic Me groups generated a pair of signals of different intensities in each

spectrum. An expansion of the region showing the peaks of interest in the spectrum corresponding to (R,S)-4.18 diastereomer is presented in Fig. 4.8 (the corresponding expansion for (S,S)-4.18 diastereomer can be found in Appendix D). The integration of this pair of peaks provided the diastereomeric mole fraction which was further used to 201 calculated the enantiomeric excess. The ee for (R)-4.2 enantiomer was calculated to be

97.9% while the ee for the second enantiomer, (S)-4.2, was calculated to be 96%.

O CF3 O OMe (S) Ph TrO (R) (R,S)-4.18

Figure 4.8. 1H NMR spectrum of (R,S)-4.18.

The diastereomers (R,S)-4.18 and (S,S)-4.18 also have a CF3 group which offers

the alternative of recording 19F NMR spectra. As expected, when the two 19F NMR spectra (Appendix E and F) were recorded, a chemical shift nonequivalence was observed for the CF3 group. The signal corresponding to S,S diastereomer was observed

at –135.45 ppm and for the R,S diastereomer at –135.75 pmm. The integration of these

signals were again used to calculate the ee. The values matched those obtained from 202 proton NMR ascertaining the accuracy and validity of the method used to determine the enantiomeric excess.

4.1.4. Synthesis of enantiomerically pure precursors

With the certainty that both enantiomers of starting compound 4.2 were obtained in high enantiomeric excess I proceeded with the preparation of the enantiomers of the lead compounds ANB-22 and ANB-40 following the synthetic pathway presented in section 4.1.1.3. First, the enantiomers of bicyclic aziridine 4.4 were prepared as indicated in Scheme 4.13. The enantiomers of primary monotritylated diol 4.2 were converted to their corresponding azidoformates (R)-4.3 and (S)-4.3 by using p-nitrophenyl chloroformate followed by sodium azide.248 The (R)-4.3 enantiomer was obtained in 80%

yield starting from the (R)-4.2 compound. The same reaction efficiency was observed for enantiomer (S)-4.3 that was prepared in 80% starting from (S)-4.2. The azidoformates

(R)-4.3 and (S)-4.3 were further subjected to the thermal intramolecular aziridination at high temperature in a sealed tube.258 Both reactions afforded the desired bicyclic

aziridines (R)-4.4 and (S)-4.4 in 55% yield and with very good diastereoselectivity since only the trans diastereomer was formed.

O O

OH 1) p-NO2PhOC(O)Cl Py, DCM, rt, 2h O N3 DCM O N TrO TrO (R) sealed tube (S) 2) NaN3 (R) (R) TrO H Acetone:H2O, 4d 109°C (R)-4.2 80% (R)-4.3 55% (R)-4.4

O O

OH 1) p-NO2PhOC(O)Cl O N3 DCM O N TrO Py, DCM, rt, 2h (S) TrO sealed tube (R) 2) NaN (S) (S) 3 TrO H Acetone:H2O, 4d 109°C (S)-4.2 80% (S)-4.3 55% (S)-4.4

Scheme 4.13. Synthesis of enantiomers of bicyclic aziridine. 203

The next step in the synthesis was to prepare the enantiomers of the 4,5- disubstituted oxazolidinone precursor 4.23 as presented in Scheme 4.14. The aziridine ring of the bicyclic compound (R)-4.4 was opened by a nucleophilic attack of the secondary amine N-phenyl piperazine that provided the oxazolidinone (4S),(5R)-4.22 in very good 91% yield.248 The other enantiomer, (4R),(5S)-4.22, was also readily available

from (S)-4.4 in 86% yield by using the same aziridine ring-opening reaction. The

enantiomers of the desired precursor were then obtained by removing the trityl protecting

group using a hydrochloric acid solution in ethyl acetate. This deprotection reaction

afforded the enantiomers (4S),(5R)-4.23 and (4R),(5S)-4.23 in 93% and 92% yield

respectively (Scheme 4.14).

O O O HN N Ph O N NH NH O HCl/EtOAc O (S) (S) (R) (S) DCM, rt, 2 h (R) N Ph rt, 1/2 h (R) N Ph TrO H TrO N HO N 91% 93% (R)-4.4 (4S),(5R)-4.22 (4S),(5R)-4.23

O O O HN N Ph O N O NH O NH (R) HCl/EtOAc (R) (R) (S) N Ph rt, 1/2 h (S) N Ph TrO (S) DCM, rt, 2 h TrO N HO N H 86% 92% (S)-4.4 (4R),(5S)-4.22 (4R),(5S)-4.23

Scheme 4.14. Synthesis of enantiomers of alcohol precursor.

204

With both enantiomers of precursor 4.23 in hand I was able to proceed to the final step of the synthesis of the enantiomers of the lead compounds ANB-22 and ANB-40 which is discussed in the following section.

4.1.5. Synthesis of target compounds

The four desired final compounds were readily available in one single step from precursors (4S),(5R)-4.23 and (4R),(5S)-4.23 as indicated in Scheme 4.15. In order to prepare the enantiomers of ANB-22, namely COB-30 and COB-31, the hydroxyl group of precursor 4.23 was acylated using the phenyl acetyl chloride. This transformation afforded the final compound COB-30 in 77% yield from (4R),(5S)-4.23 enantiomer while the compound COB-31 was obtained from the second enantiomer, (4S),(5R)-4.23, in

81% yield. The other two final compounds COB-32 and COB-33 which are the enantiomers of the lead compound ANB-40, were prepared in a similar fashion by using

4-acetyl-phenyl isocyanate. The reactions proceeded in very good yields affording COB-

32 in 82% and COB-33 in 80% yield respectively. This concluded the synthesis of the target compounds.

205

O O

O NH O NH (R) (S) (S) N Ph (R) N Ph O N O N

Ph O Ph O COB-30 COB-31

O O Ph Ph Cl Cl Et3N, DMAP Et3N, DMAP DCM, rt, 5 h DCM, rt, 5 h 77% 81%

O O NH O NH O (S) (R) Ph (S) N Ph (R) N N HO N HO

(4S),(5R)-4.23 (4R),(5S)-4.23

NCO NCO

O O

Et3N, DMAP Et3N, DMAP DCM, rt, 5 h DCM, rt, 5 h 82% 80%

O O O O

O NH O NH (R) (S) (S) N Ph (R) N Ph O N O N HN HN O O COB-32 COB-33

Scheme 4.15. Synthesis of target compounds.

The final compounds and all intermediates prepared in the synthesis were fully characterized and their specific rotations were recorded. This information is presented in the experimental part of this dissertation. The four final compounds COB-30, COB-31,

COB-32, and COB-33 were converted to their corresponding hydrochloric salts which were then used to prepare the 0.05 M solutions in DMSO for biological tests. The 206 biological assays were performed in prof. Hines laboratory at Ohio University by Shu

Zhou and are presented at the end of this chapter.

4.2. The cis isomer of ANB-22 and ANB-40

4.2.1. Rational

All 4,5-disubstituted oxazolidinones synthesized and studied so far in Bergmeier and Hines laboratories have a trans disposition of the two substituents on the oxazolidinone ring. The 3-dimensional structure of a small molecule has an important effect on its interaction with a receptor or other targets such as RNA as it was emphasized earlier in this dissertation. Therefore it is expected that trans 4,5-disubstituted oxazolidinones to interact differently with mRNA than cis 4,5-disubstituted oxazolidinones would. The difference in their spatial arrangement can be clearly seen illustrated in Figure 4.9. For the trans conformation (left structure) the two substituents are orientated on separate sides of the oxazolidinone ring while the substituents for the cis conformation (right structure) both are orientated on the same side of the ring.

O O

O NH O NH

R R R R trans cis

Figure 4.9. The trans (left) and cis (right) isomers of 4,5-disubstituted oxazolidinones.

It is possible that one substituent of both the trans and the cis conformations would interact with the same part of the binding site while the second substituent would be involved in interactions with different parts of the binding site. With the desire to 207 understand more about the structural demands and structural features of 4,5-disubstituted oxazolidinones that confers them the characteristic ability to specifically bind the T box antiterminator with high affinity, the cis isomer of both lead compounds (Fig. 4.10) were synthesized and evaluated. Compound COB-34 is the cis isomer of ANB-22 while compound COB-35 is the cis isomer of ANB-40. The progression of their syntheses is presented in the following sections.

O O O

O NH O NH N Ph O N O N N Ph HN Ph O O COB-34 COB-35

Figure 4.10. Target compounds.

4.2.2. Plan of synthesis for cis 4,5-disubstituted oxazolidinones

4.2.2.1. First plan

The thinking process for a synthetic plan to prepare cis 4,5-disubstituted

oxazolidinones was build around compound 4.22. By preparing the cis isomer of 4.22,

the final desired compounds COB-34 and COB-35 could be easily obtained in two steps

as previously shown for the trans isomer. The initial proposed sequence of reactions for

the synthesis of cis-4.22 is presented in Scheme 4.16. The synthesis would start from

commercially available 2-butyne-1,4-diol 4.24. The protection of one hydroxyl group

with t-butyldimethylsilyl (TBS) followed by the reduction of the triple bound to a trans

double bond that would undergo epoxidation, would afford compound 4.25. 208

OH OH 1) NaH, TBSCl O 1) Ti(OiPr)2(N3)2 TrO HO OTBS OTBS 2) LiAlH 2) TrCl, Et3N 4 NH 3) Ph P, H O 2 OH 3) VO(acac)2 3 2 4.24tBuOOH 4.25 4.26

CDI

O O

O NH 1) nBu4NF O NH Ph 2) TsCl TrO N N TrO OTBS 3) N-Phenylpiperazine cis-4.22 4.27

Scheme 4.16. First proposed synthesis for cis oxazolidinones.

A regioselective epoxide ring opening induced by titanium diazidodiisopropoxide

(Ti(OiPr)2(N3)2) would introduce an azido (N3) group in the proper position and

conformation which would be further reduced to an amino group (NH2) after the primary

hydroxyl would have been protected with trityl providing compound 4.26. The trans

relative configuration of the OH and NH2 groups in compound 4.26 will lead to the

desired cis conformation of the substituents in the oxazolidinone ring closing reaction upon treating 4.26 with carbonyldiimidazole (CDI). The TBS protecting group of compound 4.27 would be removed and the resulting hydroxyl would be converted to its corresponding tosylate that would be further displaced by N-phenylpiperazine in a SN2 reaction to provide the desired precursor cis-4.22

This proposed synthesis has two delicate key points. The first one is the monoprotection of starting material 4.24 which is a symmetrical molecule. This first step is important because induces structural dissymmetry in the molecule that allows for a 209 differentiation of the two hydroxyl groups facilitating the other transformations required to introduce the needed moieties for the construction of the desired product. The selective monoprotection of symmetric diols can be a difficult task and in general the yields are low because a statistical mixture of unprotected, monoprotected and diprotected products is obtained. However, by carefully choosing the reagents and the reaction conditions the selective protection could be performed in acceptable yields. The second sensitive point of the synthesis that could pose difficulties is the epoxide ring opening of compound 4.25 because the reaction requires a rigorous control of the nucleophilic attack in order to provide the desired compound. Such a regioselectivity had been reported for similar substrates like epoxide 4.28 as presented in Scheme 4.17.

OH N3 1 3 2 OBn Ti(OiPr)2(N3)2 OBn OBn HO HO + HO O benzene, 70 °C OH N3 93 % 4.28 4.29a 4.29b 6:1

Scheme 4.17. Regioselective azide opening of 2,3-epoxy alcohols.

In 1988 Sharpless et al. reported that Ti(O-i-Pr)2(N3)2 is a mild reagent for azide

ring opening of 2,3-epoxy alcohols.270 The reaction is regioselective with C3 selectivity

that is strongly dependent on a delicate balance between electronic and steric effects. In

their study the substrate 4.28 underwent ring opening in 93% yield with a 6:1 ratio of

4.29a : 4.29b products (Scheme 4.17). If similar results would be obtained by applying

this procedure to substrate 4.25, the method would be acceptable provided that no other

drawbacks would be encounter during the synthesis. 210

4.2.2.2. Execution and discussions for first plan

With a plan in hand I proceeded with the synthesis of the desired precursor cis-

4.22. As I mentioned before, the initial step involves the monoprotection of 2-butyne-1,4- diol 4.24 that could be accomplished by proper choice of reagents. The selective monoprotection of 2-butyne-1,4-diol had been reported to provide the product 4.30 in

30% yield by using sodium hydride and tert-butyldimethylsilyl chloride (TBSCl).271 As

illustrated in Scheme 4.18, my attempt to reproduce this procedure resulted in only 15%

yield of the desired product.

OH OTBS NaH, TBSCl THF, rt, 6 h OH 15% OH 4.24 4.30

Scheme 4.18. Synthesis of monoprotected 2-butyne-1,4-diol.

Given this unfortunate outcome of the reaction, a second attempt to prepare 4.30

was made by following a different known procedure.272 It was reported that by using

imidazole as the base, the silylation reaction afforded monoprotected 2-butyne-1,4-diol in

68% yield. In my hands this procedure provided the product in 45% yield (Scheme 4.19).

Although this was not the expected result, I proceeded with the next step, the triple bond reduction of compound 4.30 to a double bond by using lithium aluminum hydride

(LiAlH4) which afforded the product 4.31 in only 5% yield. I observed that large amounts

of TBS-OH have also been produced which indicated that the preferred transformation

was the deprotection of hydroxyl group rather than the reduction of the double bond. 211

OH OTBS LiAlH Im, TBSCl 4 OTBS HO DMF, rt, 24 h THF, Δ, 18 h OH 45% OH 5% 4.24 4.30 4.31

OH LiAlH 4 OH Im, TBSCl OTBS HO HO THF, Δ, 18 h DMF, rt, 24 h OH 68% 24% 4.24 4.32 4.31

Scheme 4.19. Synthesis of monoprotected 2-butene-1,4-diol.

I thought that the solution to overcome this undesired result would be to switch

the order in which the two transformations, monoprotection and reduction, are performed.

Therefore, as shown in Scheme 4.19, the starting material 4.24 was converted to diol 4.32

prior to selectively protecting one of the hydroxyl groups. With this strategy, compound

4.32 was prepared in 68% yield upon treating diol 4.24 with lithium aluminum hydride.

However, the monoprotection of diol 4.32 with imidazole and TBS-Cl afforded compound 4.31 in only 24% yield. This also represented an unsatisfactory result. In the context of an inefficient first step and with the anticipation that the second mentioned key point of the synthesis, the regioselective ring opening reaction, would weaken the overall synthesis even more, I started thinking of an alternative synthetic plan for the preparation of cis oxazolidinones. The goal was to avoid the necessity of introducing dissymmetry in the starting materials and if possible to avoid a regioselective reaction.

4.2.2.3. Second plan

Inspired by the successful preparation of unsaturated diol 4.32 (Scheme 4.19), I decided to design a synthetic plan that would use this compound as the starting material. 212

Moreover, with the desire to reduce the number of steps in the synthesis, I chose compound cis-4.23 as the target molecule rather than its tritylated derivative cis-4.22. By preparing precursor cis-4.23 the final desired compounds would be available in only one more step. The retrosynthesis for this target molecule is presented in Scheme 4.20. I envisioned that oxazolidinone cis-4.23 could be prepared from compound 4.33 by cleaving the acetonide and then engaging the secondary hydroxyl in the oxazolidinone ring closing with the amino group. Compound 4.33 could be easily obtained from 4.34 by replacing the primary hydroxyl with phenylpiperazine and then reducing the azide group to an amino group. An epoxide ring opening reaction with an azide followed by acetonide formation would provide intermediate 4.34 from epoxide 4.35 that could be readily available from unsaturated diol 4.32 in a simple epoxidation reaction.

O Ph N3 NH2 N OH O NH N O O N Ph O HO N O cis-4.23 4.33 4.34

O OH OH HO HO 4.32 4.35

Scheme 4.20. Second proposed synthesis for cis oxazolidinones.

Compared with the first proposed synthesis of the cis oxazolidinones, this second

synthetic plan has the advantage of being three steps shorter and also has only one

reaction that could require careful execution. This step is the formation of acetonide 4.34

that will be discussed in detail in the next section. 213

4.2.2.4 Execution and discussions for second plan

The synthesis, following the second proposed plan, started from the trans unsaturated diol 4.32 as presented in Scheme 4.21. This starting material underwent a standard epoxidation reaction with m-chloroperbenzoic acid (mCPBA) in acetonitrile to afford compound 4.35 in very good 90% yield.273 The next step in the synthesis was the

nucleophilic ring opening of the epoxide. It was very convenient to use compound 4.35 as a substrate for this ring opening reaction because I was able to take advantage of its symmetry avoiding the need for a regioselective reaction. Regardless of what position on the epoxide ring would be attacked by the nucleophile, due to epoxide symmetry there will be only one product formed. Therefore, by treating compound 4.35 with sodium azide (NaN3) the azido triol 4.36 was prepared in 90% yield that has the desired trans

relative stereochemistry between the secondary hydroxyl and azide groups.

N3 OH mCPBA O NaN3, NH4Cl OH HO OH HO CH3CN HO EtOH 4 °C, 4 d reflux, 17 h OH 4.36 4.32 90% 4.35 90%

Scheme 4.21. Synthesis of 3-azido-butane-1,2,4-triol.

With compound 4.36 in hand I reached the delicate point of the synthesis

mentioned before that is the differentiation between its three hydroxyl groups. I planed to

do that by protecting the two adjacent hydroxyl groups leaving one primary hydroxyl

available for further transformation. One of the most commonly used method for the

protection of two hydroxyl groups is the formation of isopropyliden acetal using p- 214 toluenesulfonic acid (p-TsOH) in acetone.274 Applying this method to compound 4.36 is a

delicate matter because the presence of the third hydroxyl group could lead to three

possible acetals. Sánchez-Sancho et al. studied the influence of experimental conditions on the reaction selectivity and yield for this type of triol substrates.275 They found that

good selectivity of acetonization could be achieved by using high amount of p-

toluenesulfonic acid in high excess of acetone at room temperature. Adapting this

method, I prepared the acetonide 4.34 as single product in 67% yield as indicated in

Scheme 4.22. The product was identified as the desired acetonide by using NMR

spectroscopy. Previous spectroscopy studies on acetonides indicated that the peak

corresponding to the acetalic carbon in the 13C NMR spectrum is located in the region of

107-110 ppm for a five-membered ring acetonide and 98.5-100.6 ppm for a six-

membered ring acetonide.276-278 When I recorded the 13C NMR spectrum of compound

4.34 I observed that the acetalic carbon generated a signal at 109.9 ppm clearly

identifying the five-membered ring acetonide that was the desired compound.

N N3 N3 p-TsOH 3 OH acetone OH TsCl, Py OTs HO O O rt, 12 h DCM O rt, 12 h O OH 67% 4.36 4.3470% 4.37

Scheme 4.22. Synthesis of intermediate 4.37.

I proceeded with the synthesis by converting the free primary hydroxyl group of

compound 4.34 into a leaving group using a standard tosylation reaction that provided the

product 4.37 in good yields (Scheme 4.22). The nucleophilic displacement of tosyloxy

group by N-phenylpiperazine afforded compound 4.38 in 60% yield as illustrated in 215

Scheme 4.23. The plan for the next step in the synthesis was to reduce the azide group of

4.38 to an amino group in the presence of an acylating agent to form a carbamate in one pot as presented in Scheme 4.23. Unfortunately, this one pot reaction did not afford the desired carbamate 4.39 thus the conversion was performed in two separate steps.

O N Ph H , Pd/C Ph 3 N3 N 2 PhO NH N OTs N-Ph-piperazine N PhOCOCl N O O O K CO , EtOH EtOAc O 2 3 O O Δ, 12 h 4.37 4.38 4.39 60% Scheme 4.23. Synthesis of carbamate.

The catalytic hydrogenation of 4.38 using palladium on charcoal afforded the amine 4.33 in quantitative yield (Scheme 4.24). This amine was then further acylated with phenyl chloroformate in the presence of pyridine to provide 4.39 in low yields (25-

30%). Using excess of triethyl amine and catalytic amount of dimethylaminopyridine

(DMAP), the desired carbamate 4.39 was obtained in very good yield (89%).

O Ph Ph N3 N NH N Ph 2 PhO NH N N H2, Pd/C N PhOCOCl O O N EtOAc Et3N, DMAP O O O 100% DCM, rt, 15 min O 4.38 4.33 89% 4.39

Scheme 4.24. Two steps synthesis of carbamate.

In order to finalize the synthesis by converting compound 4.39 into the desired precursor cis-4.23, it is required to deprotect the hydroxyl groups. Several methods to 216 cleave acetonides are available and few of them were used for compound 4.39.274 The

first method, involved the treatment of acetonide 4.39 with 80% trifluoroacetic acid in

water which afforded the desired diol in only 10% yield.279 Using pyridinium

paratoluenesulfonate in methanol resulted in recovering the starting material along with

trace amounts of product.280 A more suitable method proved to be the use of 10%

aqueous oxalic acid in tetrahydrofuran with catalytic amounts of hydrochloric acid.281

(Scheme 4.25) It was gratifying to see that this method deprotected the diol in 65% yield.

However, two products in 1:2 ratio were observed. The two products were separated by means of flash chromatography and NMR spectroscopy characterization showed that along with the desired diol product 4.40, the oxazolidinone cis-4.23 was also formed.

This was a fortunate discovery since the next step in the synthesis towards the final compounds was to close the oxazolidinone ring. This observation suggested the possibility of cleaving the acetonide and closing the oxazolidinone ring in one pot reaction.

O O O Ph Ph PhO NH N PhO NH N O NH oxalic acid N N + HO O THF HO N N Ph O 65% OH 4.39 4.40 cis-4.23 1 : 2

oxalic acid

THF: H2O 60 °C, 8h basic work up 66%

Scheme 4.25. Synthesis of precursor cis-4.23.

217

I believed that the oxazolidinone product was formed during the basic work up of the reaction. Therefore when I repeated the reaction and at the end I poured the reaction mixture in a 5% aqueous K2CO3 solution and allowed to stir for 30 to 60 minutes. In this

way the desired precursor oxazolidinone cis-4.23 was isolated in 66% yield as the only

product. The precursor cis-4.23 was then further used to synthesize the cis isomer of both

lead compounds ANB-22 and ANB-40.

4.2.3. Synthesis of target compounds

The final two compounds COB-34, the cis isomer of ANB-22, and COB-35, the

cis isomer of ANB-40, were prepared from precursor cis-4.23 in one step following the

same procedure as for their corresponding trans isomers. As shown in Scheme 4.26,

compound cis-4.23 was treated with phenyl acetyl chloride in the presence of dimethyl

amino pyridine to afford the desired product, COB-34 in 36% yield and with 4-acetyl-

phenyl isocyanate to afford the final product COB-35 in 50% yield. Comparing the

acylation reaction results of cis-4.23 (36% and 50%) with the results of the same

reactions obtained for its corresponding trans isomer (77-82%, recall section 4.1.5) it is

clearly seen a decrease in the outcome of the reaction. This discrepancy can be attributed

to steric hindrance caused by the proximity of the two substituents as a result of their cis

orientation. The hydroxyl group of cis-4.23 is more hindered and therefore less accessible

for acylation than the hydroxyl group of the trans isomer.

218

O

O NH

O N N Ph

Ph O COB-34

O Ph Cl

Et3N, DMAP DCM, rt, 5 h 36% O

O NH

HO N N Ph

cis-4.23 NCO

O

Et3N, DMAP DCM, rt, 5 h 50%

O O

O NH

O N N Ph HN O COB-35

Scheme 4.26. Synthesis of final compounds.

The final compounds COB-34 and COB-35 were converted to their corresponding hydrochloric salts, which were then used to prepare 50 mM solutions in

DMSO for biological tests. These two cis isomers were sent, along with the tans enantiomers COB-30, COB-31, COB-32, and COB-33, to Prof. Hines laboratory at Ohio

University where Shu Zhou performed the biological assays. The binding affinities of these compounds were determined using the FRET-label antiterminator model 3’Fl- 219

AM1A-Rhd and the data are summarized in Table 4.3. In the case of lead compound

ANB-22, the results indicate that one of its enantiomers, the 4R,5S enantiomer COB-30

(entry 2), binds the antiterminator while the second enantiomer, the 4S,5R enantiomer

COB-31, shown no binding at all. Similar results have been observed for the enantiomers of the lead compound ANB-40: the 4R,5S enantiomer COB-32 (entry 6) binds the antiterminator while the 4S,5R enantiomer COB-33 (entry 7), shows no binding. The finding that only one enantiomer binds the antiterminator supports the hypothesis that there is chiral recognition involved in the interaction of 4,5-disubstituted oxazolidinones with the T box antiterminator. The hypothesis is supported further more by the fact that the eutomer of each lead compound, COB-30 and COB-32, have the same 4R,5S configuration.

220

Table 4.3

Binding to T box antiterminator model AM1A

Entry Structure Stereochemistry Compound 3’Fl-AM1A-Rhd

name Kd (μM)

1 O trans racemic ANB-22 O NH 13 ± 4 N Ph O N

Ph O

2 O trans 4R,5S COB-30 O NH 0.66 ± 0.56 (R) (S) N Ph O N

Ph O

3 O trans 4S,5R COB-31 nb O NH (S) (R) N Ph O N

Ph O

4 O cis COB-34 O NH 0.81 ± 0.67

O N N Ph

Ph O

O 5 O trans racemic ANB-40 0.9 ± 0.4 O NH N Ph O N HN O

O 6 O trans 4R,5S COB-32 1.76 ± 0.46 O NH (R) (S) N Ph O N HN O

O 7 O trans 4S,5R COB-33 nb O NH (S) (R) N Ph O N HN O

8 O O cis COB-35 0.99 ± 0.29 O NH

O N N Ph HN O

221

As it was expected, due to chiral recognition, the binding affinity of eutomer

COB-30 is much stronger than that of its corresponding racemate, ANB-22, indicated by the dissociation constant Kd of 0.66 μM for COB-30 (entry 2) vs. 13 μM for ANB-22

(entry 1). The lower the Kd value, the stronger the binding. However, in the case of

eutomer COB-32, the Kd value (1.76 μM , entry 6) is slightly larger than that of ANB-40

(0.9 μM , entry 5), indicating that the enantiomer binds more weakly than the racemic.

This is an unexpected result since the second enantiomer, COB-33, is devoid of affinity.

Regarding the cis or trans relative configuration for the substituents at the

oxazolidinone ring, the binding affinity data indicate that there is no difference in binding

to the antiterminator of the cis isomer COB-35 (entry 8) compared to the trans isomer

ANB-40 (entry 5). On the other hand, the better binding affinity of COB-34 (entry 4)

compared to its trans isomer ANB-22, indicates that the cis conformation is more

favorable.

4.3. Summary

This chapter describes the synthesis of enantiomerically pure lead compounds

ANB-22 and ANB-40, representatives of a novel class of antibacterial agents. The

synthesis started from enantiomerically pure materials that were prepared in very high

enantiomeric excess. The synthesis of the cis isomer of both lead compounds was also

presented. All desired final compounds were prepared in good overall yields and were

further used to study the effect of their chirality on the binding to the T box

antiterminator. The biological results were consisted with the hypothesis that there is 222 chiral recognition associated with the binding of 4,5-disubstituted oxazolidinones to the T box antiterminator. 223

CHAPTER 5: EXPERIMENTAL

General. Reagents and starting materials were obtained form Aldrich unless otherwise stated. Racemic butadiene monoepoxide 4.21 was purchased from Alfa Aesar. Both enantiomers of the (salen)Co(II) complex 4.19 are commercially available from Aldrich.

The polystyrene sulfonyl chloride (PS-TsCl) was purchased from Aldrich. CH2Cl2 and

THF were dried using SOLVETECH solvent purification system. Acetone was dried over

4 Å molecular sieves and distilled immediately before use. Et3N was dried over calcium

hydride and distilled immediately before use. DMF was stirred over MgSO4 overnight

and then distilled under vacuum. Microwave-assisted reactions were performed using

INITIATOR Biotage Microwave Synthesizer (400W, 2.45 GHz). Melting points were

determined with a MEL-TEMP II melting point apparatus and are reported uncorrected.

Specific rotations were measured on a AUTOPOL ® IV (Rudolph Research Analytical)

T °C polarimeter with a sodium (λ = 589 nm) lamp, and are reported as follows: [α] λ (c g/100 mL, solvent). The capillary GC analyses were performed on a Shimadzu GC-17A

Gas Chromatograph employing the Rtx®-5 (15 m x 0.25 mm id x 0.25 μm df; Restek) column. The times of retention are reported in minute as followed: initial T °C to final T

°C, rate, duration of run. HPLC analyses were performed with a Shimadzu LC-10AT machine equipped with a UV detector by employing a chiral (R,R) Whelk O1 (25 cm x

4.6 mm x 5 μm; Regis) column eluting with IPA in hexane at 1 mL/min flow or a reverse phase Discovery–C8 (15 cm x 4.6 mm x 5 μm; Supelco) column eluting with acetonitrile

1 13 in H2O at 1 mL/min flow. H NMR and C NMR spectra were recoded with a Brüker

AVANCE (300 MHz) or a VARIAN (500 MHz) spectrometer. Chemical shifts are 224 reported in ppm on the δ scale relative to deuterated chloroform as an internal standard.

Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, qn = quintet, m = multiplet), coupling constant in Hz, integration.

HOOC COOtBu 2.5

Succinic acid mono-tert-butyl ester (2.5). Succinic anhydride (5 g, 50 mmol) was suspended in toluene (100 mL). At this mixture was added N-hydroxysuccinimide (1.72 g, 15 mmol), DMAP (0.61 g, 5 mmol), t-butanol (6.2 mL, 65 mmol) and freshly distilled

Et3N (2.1 mL, 15 mmol). The reaction mixture was heated to reflux for 24 h when

complete dissolution occurred. The reaction was allowed to cool down to room

temperature, diluted with EtOAc (30 mL) and washed with 10% citric acid (3 x 20 mL)

and brine. The organic phase was dried over MgSO4 and the solvent removed by rotary

evaporation. The residue was then crystallized from Et2O to afford 4.7 g (54%) of 2.5 as

1 a white solid (mp 47.1-50 °C). Rf 0.35 (30% EtOAc, 1% AcOH in hexanes). H NMR

(CDCl3, 500 MHz) δ 2.63 (t, J = 6.6 Hz, 2H, CH2COOH), 2.54 (t, J = 6.6 Hz, 2H,

CH2COOtBu), 1.45 (s, 9H, (CH3)3). Additional analytical data have been previously reported.282

Ph

HOOC COOtBu 2.6 225

2-Benzyl-succinic acid 4-tert-butyl ester (2.6). To a cold (-78 °C) solution of freshly distilled i-Pr2NH (2.8 mL, 22.3 mmol) in THF (20 mL) was added dropwise a solution of

n-BuLi (11.5 mL of 1.85 M solution in hexanes, 26.4 mmol). After stirring for 30

minutes a solution of 2.5 (1.76 g, 10.14 mmol) in THF (6 mL) was added through

cannula and the reaction was warmed to 0 °C and stirred for 2 h. The reaction was cooled

again to -78 °C and benzyl bromide (1.7 mL, 14.2 mmol) was added neat to the reaction

flask. The reaction mixture was warmed up to room temperature and allowed to stir for

24 h. The reaction was quenched with H2O (6 mL) and diluted with EtOAc (60 mL) and

washed with a cold solution of 1M HCl (2 x 20 mL), brine, dried over MgSO4, filtered and concentrated. The residue was purified by flash chromatography (30% EtOAc, 1%

AcOH in hexanes) to provide 1.46 g (55%) of 2.6 as colorless oil. Rf 0.14 (10% EtOAc in

1 hexane). H NMR (CDCl3, 500 MHz) δ 7.31-7.17 (m, 5H, Ph), 3.04-2.99 (m, 2H,

CH2Ph), 2.79-2.73 (m, 1H, CHCOOtBu), 2.68-2.62 (m, 1H, CHHCOOH), 2.41 (dd, J =

4.5, 17 Hz, 1H, CHHCOOH), 1.38 (s, 9H, (CH3)3). Additional analytical data have been

previously reported.191

Ph

HOOC COOH 2.7

226

2-Benzyl-succinic acid (2.7). Trifluoroacetic acid (4.5 mL, 60.5 mmol) was added to a solution of 2.6 (1.6 g, 6.05 mmol) in CH2Cl2 (6 mL) and the reaction mixture was stirred for 24 h at room temperature. The solvent was removed by rotary evaporation and the residue chromatographed (35% EtOAc, 1% AcOH in hexanes) to afford 1.2 g (95%) of

1 2.7 as white solid. Rf 0.18 (20% EtOAc, 1% AcOH in hexanes). H NMR (DMSO-d6,

500 MHz) δ 7.30-7.17 (m, 5H, Ph), 2.93-2.86 (m, 2H, CH2Ph), 2.72 (m, 1H, CHCOOH),

2.40 (dd, J = 9, 17 Hz, 1H, CHHCOOH), 2.25 (dd, J = 4, 16.5 Hz, 1H, CHHCOOH).

Additional analytical data have been previously reported.191

Ph

O O O 2.8

3-Benzyl-dihydro-furan-2,5-dione (2.8). A flask equipped with a condenser was loaded with 2.7 (0.33 g, 1.6 mmol) and acetyl chloride (0.68 mL, 9.6 mmol). The flask was heated at 52 °C for 3 h and then allowed to cool to room temperature. The reaction mixture was concentrated down by rotary evaporation and the residue chromatographed

(30% EtOAc, 1%AcOH in hexanes) to afford 0.29 g (97%) of 2.8 as a white solid. Rf

1 0.33 (30% EtOAc, 1% AcOH in hexanes). H NMR (DMSO-d6, 500 MHz) δ 7.33-7.23

(m, 5H, Ph), 3.59-3.53 (m, 1H, C(O)CH), 3.12 (dd, J = 5, 13.5 Hz, 1H, PhCHH), 2.95

(dd, J = 10, 13.5 Hz, 1H, PhCHH), 2.89 (dd, J = 10, 18 Hz, 1H, C(O)CHH), 2.76 (dd, J =

6.5, 18.5 Hz, 1H, C(O)CHH). Additional analytical data have been previously reported.191 This reaction was also carried out under microwave conditions. The reaction 227 mixture containing the same amounts of reactants as above was placed in a microwave vial and heated at 100 °C for 7 minutes. Same results were obtained.

Ph

O N O COOH

2.2

2-(3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid (2.2). Succinic anhydride 2.8 (1.15

g, 6 mmol) was loaded in a flask followed by antranilic acid (0.83 g, 6 mmol). The flask

was lowered into an oil bath, preheated to 145 °C and stirred under vacuum for 4 h. After

this time the reaction mixture was allowed to cool and the resulting solid was

chromatographed (5% EtOAc, 5% AcOH in toluene) to provide 0.64 g (34%) of 2.2 as a

1 white solid. Rf 0.24 (5% EtOAc, 5% AcOH in toluene). H NMR (CDCl3, 300 MHz) δ

9.0 (br s, 1H, COOH), 8.12-8.09 (m, 1H, Ar), 7.61-7.57 (m, 1H, Ar), 7.48-7.44 (m, 1H,

Ar), 7.28-7.15 (m, 6H, Ar), 3.25 (br, 1H, 3-CH), 3.16-2.97 (m, 2H, PhCH2), 2.77-2.69

13 (m, 1H, 4-CHH), 2.60 (m, 1H, 4-CHH); C-NMR (CDCl3, 75 MHz) δ 178.8, 178.6*,

176.1*, 176.0, 169.3, 138.1, 137.0, 134.4, 132.9, 132.7, 130.0*, 129.9, 129.7, 129.5,

129.1, 127.4, 127.2*, 126.7,42.3*, 42.0, 37.0*, 36.5, 34.7*, 33.8. (* denotes the signals of

rotamer)

O

O NH

(R) Ph 2.11 228

(R)-4-Phenyl-oxazolidin-2-one (2.11). A 25 mL flask was equipped with a Vigreux column, a distillation head and a receiver flask. The receiver was cooled in an ice bath.

The reaction flask was loaded with (R)-(-)-2-phenylglycinol (5 g, 36.5 mmol), diethyl carbonate (5.53 mL, 45.6 mmol) and NaOEt (0.03 mL, 0.44 mmol, generated from Na and 0.012 eq of EtOH) and connected to a nitrogen source. The reaction flask was lowered in an oil bath preheated at 135 °C. EtOH started to distill and was collected in the receiver flask. When EtOH ceased to distill the reaction mixture was cooled to 65 °C and then transferred to a flask containing cold (0 °C) Et2O. The product precipitated as a

white solid and 5.4 g (91%) of 2.11 were isolated by filtration. No further purification

1 was necessary. Rf 0.14 (30% EtOAc in hexanes). H NMR (CDCl3, 500 MHz) δ 7.43-

7.35 (m, 5H, Ph), 5.25 (br s, 1H, NH), 4.96 (t, J = 8 Hz, 1H, CHPh), 4.75 (t, J = 8.5, 1H,

CHHO), 4.21 (dd, J = 7, 8.5, 1H, CHHO).

O

O NH

(S)

2.13

(S)-4-Isopropyl-oxazolidin-2-one (2.13). A 25 mL flask was equipped with a Vigreux

column, a distillation head and a receiver flask. The receiver was cooled in an ice bath.

The reaction flask was loaded with (S)-(+)-2-amino-3-methyl-1-butanol (5 g, 48.5 mmol),

diethyl carbonate (7.34 mL, 60.6 mmol) and NaOEt (0.037 mL, 0.58 mmol, generated

from Na and 0.012 eq of EtOH) and connected to a nitrogen source. The reaction flask 229 was lowered in an oil bath preheated at 135 °C. EtOH started to distill and was collected in the receiver flask. When EtOH ceased to distill the reaction mixture was cooled to 65

°C and then transferred to a flask containing cold (0 °C) Et2O. The product precipitated

very slowly as a white solid and 3.55 g of 2.13 were isolated by filtration. Hexanes were

added to the filtrate and more precipitate was formed (1.85 g, 86% combined yield). No

1 further purification was necessary. Rf 0.13 (30% EtOAc in hexanes). H NMR (CDCl3,

500 MHz) δ 5.89 (br s, 1H, NH), 4.45 (t, J = 8.5 Hz, 1H, CHHO), 4.11 (dd, J = 6, 8.5 Hz,

1H, CHHO), 3.61 (dd, J = 7, 15 Hz, 1H, CHNH), 1.74 (st, J = 6.5 Hz, 1H, CH(CH3)2),

0.97 (d, J = 6.5 Hz, 3H, CH3), 0.91 (d, J = 6.5 Hz, 3H, CH3).

O O

O N Ph

(R) Ph 2.14

(4R)-4-Phenyl-3-(3-phenyl-propionyl)-oxazolidin-2-one (2.14). To a cold (-78 °C)

solution of oxazolidinone 2.11 (2 g, 12.3 mmol) in THF (40 mL) was added nBuLi (5.6

mL of 2.38 M sol in THF, 13.5 mmol) over a period of 5 minutes. The mixture was

stirred at -78 °C for 30 minutes when freshly distilled hydrocinnamoyl chloride (2.7 mL,

18.45 mmol) was added in one portion. The reaction mixture was allowed to warm

slowly to room temperature and stirred until the starting material disappeared (usually 2

h). The reaction was quenched with saturated aqueous solution of NH4Cl (20 mL) and the

volatiles were removed by rotary evaporation. The aqueous residue was extracted with

Et2O (3x50 mL) and the combined organic layers washed with brine, dried over MgSO4, 230 filtered and concentrated. The oily residue was refrigerated overnight to form a yellowish solid that was crystallized from hexanes/EtOAc to provide 2.83 g (79%) of 2.14 as a

1 white solid. Rf 0.3 (20% EtOAc in hexanes). H NMR (CDCl3, 300 MHz) δ 7.22-7.3 (m,

10H, Ph), 5.43 (dd, J = 3.6, 8.8 Hz, 1H, PhCHNH), 4.68 (t, J = 8.8 Hz, 1H, OCHtransH),

4.29 (dd, J = 3.6, 8.8 Hz, 1H, OCHHcis), 3.29 (t, J = 7.5 Hz, 2H, C(O)CH2), 2.95 (t, J =

7.5 Hz, 2H, CH2Ph).

O O

O N Ph

(S) 2.16

(4S)-4-Isopropyl-3-(3-phenyl-propionyl)-oxazolidin-2-one (2.16). To a cold (-78 °C) solution of oxazolidinone 2.13 (2 g, 15.5 mmol) in THF (40 mL) was added nBuLi (7.1 mL of 2.4 M sol in THF, 17.03 mmol) over a period of 5 minutes. The mixture was stirred at -78 °C for 30 minutes when freshly distilled hydrocinnamoyl chloride (2.7 mL,

18.6 mmol) was added in one portion. The reaction mixture was allowed to warm slowly to room temperature and stirred until the starting material disappeared (usually 2 h). The reaction was quenched with saturated aqueous solution of NH4Cl (20 mL) and the

volatiles were removed by rotary evaporation. The aqueous residue was extracted with

Et2O (3x50 mL) and the combined organic layers washed with brine, dried over MgSO4, filtered and concentrated. The oily residue was refrigerated overnight to form crystals that were washed with cold hexanes and isolated by filtration to provide 3.04 g (75%) of

1 2.16 as a white solid. Rf 0.21 (10% EtOAc in hexanes). H NMR (CDCl3, 500 MHz) δ 231

7.30-7.21 (m, 5H, Ph), 4.44-4.41 (m, 1H, CHN), 4.24 (t, J = 9 Hz, 1H, OCHH), 4.20 (dd,

J = 3.5, 9 Hz, 1H, OCHH), 3.33 (ddd, J = 6.5, 8, 15 Hz, 1H, C(O)CHH), 3.23 (ddd, J =

6.5, 8, 15 Hz, 1H, C(O)CHH), 3.00 (m, 2H, PhCH2), 2.36 (m, 1H, CH(CH3)2), 0.91 (d, J

= 7 Hz, 3H, CH3), 0.84 (d, J = 7 Hz, 3H, CH3).

O O (S) O N Ph

(R) Ph CO2tBu 2.15

(4R)-3-[(2S)-2-[(tert-butyloxycarbonyl)methyl]-3-phenyl-propionyl]-4-phenyl-

oxazolidin-2-one (2.15). To a cold (-78 °C) solution of 2.14 (2 g, 6.8 mmol) in THF (23 mL) under argon atmosphere was added NaHMDS (7.5 mL of 1M sol in hexane, 7.5 mmol). The mixture was stirred at -78 °C for 30 minutes and then t-butyl bromoacetate

(1.3 mL, 8.8 mmol) was added neat. The reaction mixture was stirred for another 1 h when it was quenched with saturated aqueous solution of NH4Cl (14 mL). The reaction

mixture was partitioned between Et2O and H2O and extracted with Et2O (3 x 25mL). The

combined organic layers were washed with 10% aqueous solution of HCl, saturated

solution of NaHCO3 and brine, then dried over anhydrous MgSO4, filtered and the

solvent removed by rotary evaporation. The residue was purified by flash

chromatography (10% EtOAc in hexanes) to provide 1.9 g (68%) of 2.15 as a white solid.

1 Rf 0.4 (20% EtOAc in hexanes). H NMR (CDCl3, 500 MHz) δ 7.31-7.20 (m, 10H, Ph),

5.27 (dd, J = 3, 8.5 Hz, 1H, PhCHN), 4.55-4.49 (m, 1H, C(O)CH), 4.42 (t, J = 8.5 Hz,

1H, OCHtransH), 4.15 (dd, J = 3, 8.5 Hz, 1H, OCHHcis), 2.99 (dd, J = 6.5, 13 Hz, 1H, 232

PhCHH), 2.68 (dd, J = 10.5, 16 Hz, 1H, C(O)CHH), 2.59 (dd, J = 9, 13 Hz, 1H, PhCHH),

13 2.26 (dd, J = 3, 16 Hz, 1H, C(O)CHH), 1.23 (s, 9H, (CH3)3). C NMR (CDCl3, 75 MHz)

δ 175.0, 171.0, 153.4, 138.9, 138.3, 129.5, 129.1, 128.6, 128.5, 126.9, 126.0, 80.8, 70.0,

58.1, 41.4, 38.4, 36.8, 28.1.

O O (R) O N Ph

(S) CO2tBu 2.17

(4S)-3-[(2R)-2-[(tert-butyloxycarbonyl)methyl]-3-phenyl-propionyl]-4-isopropyl-

oxazolidin-2-one (2.17). To a cold (-78 °C) solution of 2.16 (2 g, 7.6 mmol) in THF (23 mL) under argon atmosphere was added NaHMDS (8.4 mL of 1M sol in hexane, 8.4 mmol). The mixture was stirred at -78 °C for 30 minutes and then t-butyl bromoacetate

(1.5 mL, 9.9 mmol) was added neat. The reaction mixture was stirred for another 1 h when it was quenched with saturated aqueous solution of NH4Cl (14 mL). The reaction

mixture was partitioned between Et2O and H2O and extracted with Et2O (3 x 25mL). The

combined organic layers were washed with 10% aqueous solution of HCl, saturated

solution of NaHCO3 and brine, then dried over anhydrous MgSO4, filtered and the

solvent removed by rotary evaporation. The solid residue was crystallized from 10%

EtOAc in hexanes to provide 2 g (70%) of 2.17 as a white solid. Rf 0.23 (10% EtOAc in

1 hexanes). H NMR (CDCl3, 500 MHz) δ 7.31-7.21 (m, 5H, Ph), 4.53-4.48 (m, 1H,

C(O)CH), 4.31-4.28 (m,1H, NCH), 4.14 (dd, J = 2, 8.5 Hz, 1H, OCHcisH), 4.03 (t, J =

8.5 Hz, 1H, OCHHtrans), 2.99 (dd, J = 6.5, 13.5 Hz, 1H, PhCHH), 2.78 (dd, J = 10.5, 16.5 233

Hz, 1H, C(O)CHH), 2.61 (dd, J = 9, 13 Hz, 1H, PhCHH), 2.36-2.32 (m, 2H, C(O)CHH and CH(CH3)2), 1.39 (s, 9H, (CH3)3), 0.92 (d, J = 6.5 Hz, 3H, CH3), 0.90 (d, J = 7 Hz,

13 3H, CH3). C NMR (CDCl3, 75 MHz) δ 175.5, 171.3, 153.8, 138.4, 129.5, 128.6, 126.8,

80.8, 63.4, 59.0, 41.4, 38.3, 37.0, 28.6, 28.2, 18.2, 14.9.

O (S) HO Ph

CO2tBu (S)-2.18

(2S)-Benzyl-succinic acid-4-tert-butyl ester ((S)-2.18). The oxazolidinone adduct 2.15

(0.9 g, 2.2 mmol) was dissolved in THF and H2O (4:1, 22 mL) and the resulting solution cooled to 0 °C. To this solution, H2O2 (30% aqueous, 1.23 mL, 11 mmol) was added

followed by aqueous LiOH solution (5.3 mL, 0.1 g/5 mL, 4.4 mmol) and the reaction

mixture was stirred for 1 h at 0 °C. An aqueous solution of Na2SO3 (8.25 mL, 1.3M, 11

mmol) was added to the reaction mixture and stirred for an additional 20 minutes. After

this time the THF was evaporated and the residue was dissolved in CH2Cl2 (100 mL) and

H2O and the layers separated. The organic layer contains the oxazolidinone. The aqueous

layer was acidified with a solution of 3M HCl and extracted with CH2Cl2 (3 x 20 mL).

The combined organic layers were washed with H2O and brine, dried over MgSO4, filtered and the solvent removed by rotary evaporation to provide 0.5 g (86%) of acid (S)-

25.2 2.18. No further purification was necessary. Rf 0.14 (10% EtOAc in hexanes). [α]D -

1 8.1° (c 1.0, CHCl3). H NMR (CDCl3, 300 MHz) δ 7.35-7.18 (m, 5H, Ph), 3.12-3.09 (m,

2H, PhCHH and C(O)CH), 2.78 (dd, J = 10.5, 15 Hz, 1H, PhCHH), 2.58 (dd, J = 8.7, 234

16.8 Hz, 1H, C(O)CHH), 2.36 (dd, J = 4.5, 16.8 Hz, 1H, C(O)CHH), 1.42 (s, 9H,

13 C(CH3)3). C NMR (CDCl3, 75 MHz) δ 177.3, 168.6, 135.7, 126.7, 126.2, 124.4, 78.8,

40.7, 34.9, 33.7, 25.6.

O (R) HO Ph

CO2tBu (R)-2.18

(2R)-Benzyl-succinic acid-4-tert-butyl ester ((R)-2.18). Compound (R)-2.18 was

prepared from 2.17 on a 1.9 mmol scale following the procedure described for (S)-2.18,

to afford 0.46 g (92%) of acid (R)-2.18. All analytical data were identical with those

25.5 reported for (S)-2.18 except for [α]D +8.7° (c 1.05, CHCl3).

O (S) HO Ph

COOH (S)-2.7

(S)-2-Benzyl-succinic acid ((S)-2.7). Trifluoroacetic acid (2.5 mL, 32 mmol) was added

to a solution of ester (S)-2.18 (0.85 g, 3.2 mmol) in CH2Cl2 (3.5 mL) and the reaction

mixture was stirred for 5 h at room temperature. The solvent was removed by rotary

evaporation to afford 0.65 g (98%) of (S)-2.7 as white solid. No further purification was

25.3 necessary. Rf 0.18 (20% EtOAc, 2% AcOH in hexanes). [α]D -30.5° (c 1.03, DMSO).

1 H NMR (DMSO-d6, 500 MHz) δ 7.35-7.17 (m, 5H, Ph), 2.93-2.88 (m, 2H, PhCHH and 235

C(O)CH), 2.74 (dd, J = 10, 15 Hz, 1H, PhCHH), 2.43 (dd, J = 9, 17 Hz, 1H,

13 CHHCOOH), 2.25 (dd, J = 4.5, 17 Hz, 1H, CHHCOOH). C NMR (DMSO-d6, 75 MHz)

δ 175.1, 172.9, 138.7, 128.9, 128.3, 126.3, 42.4, 36.9, 34.8.

O (R) HO Ph

COOH (R)-2.7

(R)-2-Benzyl-succinic acid ((R)-2.7). Compound (R)-2.7 was prepared from (R)-2.18 on

a 2.65 mmol scale following the procedure described for (S)-2.7, to afford 0.54 g (98%)

of (R)-2.7. All analytical data were identical with those reported for (S)-2.7 except for

25.3 [α]D +30.5° (c 1.05, DMSO).

Ph (S) O O O

(S)-2.8

(S)-3-Benzyl-dihydro-furan-2,5-dione ((S)-2.8). A flask equipped with a condenser was

loaded with (S)-2.7 (0.24g, 1.1 mmol) and acetyl chloride (0.5 mL, 6.8 mmol). The flask was heated to 52 °C for 3 h and then allowed to cool to room temperature. The reaction mixture was concentrated down by rotary evaporation to afford 0.21 g (97%) of (S)-2.8 as

26 a white solid. Rf 0.33 (30% EtOAc, 1% AcOH in hexanes). [α]D +45.3° (c 1.01,

1 DMSO). H NMR (DMSO-d6, 500 MHz) δ 7.42-7.15 (m, 5H, Ph), 3.60-3.54 (m, 1H, 236

C(O)CH), 3.12 (dd, J = 5, 14 Hz, 1H, PhCHH), 2.95 (dd, J = 9.5, 14 Hz, 1H, PhCHH),

2.99 (dd, J = 9.5, 18 Hz, 1H, C(O)CHH), 2.76 (dd, J = 6.7, 18 Hz, 1H, C(O)CHH).

Ph (R) O O O

(R)-2.8

(R)-3-Benzyl-dihydro-furan-2,5-dione ((R)-2.8). Compound (R)-2.8 was prepared from

(R)-2.7 on a 2 mmol scale following the procedure described for (S)-2.8, to afford 0.365 g

(96%) of (R)-2.8. All analytical data were identical with those reported for (S)-2.8 except

26 for [α]D -46.5° (c 1.01, DMSO).

Ph (S)

O N O COOH

(S)-2.2

2-((S)-3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid ((S)-2.2). Succinic anhydride

(S)-2.8 (0.51 g, 2.6 mmol) was loaded in a flask followed by antranilic acid (0.37 g, 2.6 mmol). The flask was lowered into an oil bath, preheated to 145 °C and stirred under vacuum for 7 h. After this time the reaction mixture was allowed to cool and the resulting solid was chromatographed (5% EtOAc, 5% AcOH in toluene) to provide 0.29 g (36%)

24.5 of (S)-2.2 as a white solid. Rf 0.24 (5% EtOAc, 5% AcOH in toluene). [α]D + 63.4° (c 237

1 1.07, CHCl3). H NMR (CDCl3, 300 MHz) δ 9.0 (br s, 1H, COOH), 8.12-8.09 (m, 1H,

Ar), 7.61-7.57 (m, 1H, Ar), 7.48-7.44 (m, 1H, Ar), 7.28-7.15 (m, 6H, Ar), 3.25 (br, 1H,

13 3-CH), 3.16-2.97 (m, 2H, PhCH2), 2.77-2.69 (m, 1H, 4-CHH), 2.60 (m, 1H, 4-CHH). C

NMR (CDCl3, 75 MHz) δ 178.8, 178.6*, 176.1*, 176.0, 169.3, 138.1, 137.0, 134.4,

132.9, 132.7, 130.0*, 129.9, 129.7, 129.5, 129.1, 127.4, 127.2*, 126.7,42.3*, 42.0, 37.0*,

36.5, 34.7*, 33.8. (* denotes signals of rotamer)

Ph (R)

O N O COOH

(R)-2.2

2-((R)-3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid ((R)-2.2). Compound (R)-2.2

was prepared from (R)-2.8 on a 1.95 mmol scale following the procedure described for

(S)-2.2 to afford 0.29 g (48%) of (R)-2.2. All analytical data were identical with those

24.6 reported for (S)-2.2 except for [α]D - 70.6° (c 1.08, CHCl3).

COOEt (R)

N TA H (R)-2.26

(R)-(-)-Ethyl Nipecotate (+)-Tartrate ((R)-2.26). L-(+)-Tartaric acid (4.8 g, 32 mmol) was suspended in i-PrOH (34 mL) and H2O (1.5 mL) and the mixture was heated at 60- 238

65 °C until the dissolution was complete. Racemic ethyl nipecotate (5g, 32 mmol) was added and the temperature was increased to 70-75 °C. After 30 minutes the mixture was allowed to cool at room temperature. A white precipitate was formed that was isolated by filtration and washed twice with a solution of i-PrOH (2.7 mL) and H2O (0.13 mL) to

afford 4.1 g of white crystals ([α]D = +11.5° (c 1.02, H2O); mp = 151-152 °C). The crude

was then suspended in a mixture of i-PrOH and H2O (17 mL, 23:1) and heated to reflux for 15 minutes and allowed to cool to room temperature. This process was repeated until no change in the melting point and the [α]D was observed (usually three times). The last

recrystallization afforded 2.75 g (28%) of salt (R)-2.26 as white crystals: mp = 154.8-156

24.6 °C, [α]D = +10.7° (c 1.0, H2O). Additional analytical data have been previously

reported.198

COOEt (S)

N TA H (S)-2.26

(S)-(+)-Ethyl Nipecotate (-)-Tartrate ((S)-2.26). D-(-)-Tartaric acid (4.8 g, 32 mmol)

was suspended in i-PrOH (34 mL) and H2O (1.5 mL) and the mixture was heated at 60-

65 °C until the dissolution was complete. The combined mother liquors resulted from the

preparation of (R)-ethyl nipecotate were treated with Na2CO3 (3.3g, 31 mmol) and

extracted with EtOAc to afford 2.6 g of (S)-enriched ethyl nipecotate. The (S)-enriched

ethyl nipecotate was added to 2.4 g of racemic ethyl nipecotate and the resulting amount

(5g, 32 mmol) was added to the tartaric acid solution and the temperature was increased 239 to 70-75 °C. After 30 minutes the mixture was allowed to cool at room temperature. A white precipitate was formed that was isolated by filtration and washed twice with a solution of i-PrOH (2.7 mL) and H2O (0.13 mL) to afford 5.1 g of white crystals ([α]D = -

11.2° (c 1.02, H2O); mp = 152-153 °C). The crude was then suspended in a mixture of i-

PrOH and H2O (17 mL, 23:1) and heated to reflux for 15 minutes and allowed to cool to room temperature. This process was repeated until no change in the melting point and the

[α]D was observed (usually three times). The last recrystallization afforded 4 g (41%) of

24.9 salt (S)-2.26 as white crystals: mp = 155.1-156.3 °C, [α]D = -10.7° (c 1.02, H2O).

(R) OH N

Ph (R)-2.3

[(R)-1-(3-Phenyl-propyl)-piperidin-3-yl]-methanol ((R)-2.3) To a solution of (R)-2.26

(1 g, 3.25 mmol) in EtOAc (7 mL) and H2O (8.4 mL) was added an aqueous 15%

Na2CO3 solution (7 mL, 9.75 mmol) over 15 minutes. A solution of hydrocinnamoil

chloride (1.26 mL, 8.45 mmol) in EtOAc (1.7 mL) was then added to the reaction

mixture and stirred for 30 minutes. At this time, the layers were separated and the

aqueous layer was washed with EtOAc (2 x 10 mL). The combined organic layers were

dried over MgSO4, filtered and concentrated under reduced pressure to provide 0.9 g

(97%) of (R)-2.27 that was used in the next step without purification. LiAlH4 (0.47 g, 12

mmol) was suspended in THF (5.4 mL) and cooled to 0 °C. The ester (R)-2.27 (0.9 g, 3.1

mmol) was dissolved in THF (8 mL) and the solution was added to the reaction flask 240 through cannula. The reaction mixture was then allowed to warm to room temperature and stirred for 24 h. The reaction mixture was then cooled again at 0 °C and diluted with

Et2O (10 mL). H2O was added (40 μL/mmol LiAlH4) and stirred for 15 minutes followed

by addition of 15% aqueous solution of NaOH (40 μL/mmol LiAlH4) and stirred another

15 minutes. H2O was again added (100 μL/mmol LiAlH4) and allowed to stir for 2 h

when a white precipitate was formed which was removed by filtration. The filtrate was

concentrated and dried under vacuum to provide 0.58 g (81%) of (R)-2.3 as yellow oil.

25.9 No further purification was necessary. Rf 0.14 (5% MeOH in CHCl3). [α]D = + 5.6° (c

1 1.01, CHCl3). H NMR (CDCl3, 500 MHz) δ 7.35-7.17 (m, 5H, Ph), 3.62-3.60 (m, 1H,

HOCHH), 3.51-3.49 (m, 1H, HOCHH), 2.73 (bd, J = 10 Hz, 1H, PhCHH), 2.56 (t, J = 8

Hz, 3H, PhCHH and NCH2), 2.28 (t, J = 7 Hz, 2H, NCH2), 2.11 (bt, 1H), 2.03 (bt, 1H),

13 1.78-1.51 (m, 6H), 1.19-1.14 (m, 1H). C NMR (CDCl3, 75 MHz) δ 141.3, 127.6, 125.3,

66.3, 58.0, 56.8, 53.7, 37.1, 33.3, 28.1, 27.3, 24.3. Additional analytical data have been

previously reported.172

(S) OH N

Ph (S)-2.3

[(S)-1-(3-Phenyl-propyl)-piperidin-3-yl]-methanol ((S)-2.3). Compound (S)-2.3 was

prepared from (S)-2.26 on a 2.6 mmol scale following the procedure described for (R)-2.3 241 to provided 0.46 g (75% two steps) of (S)-2.3. All analytical data were identical with

25.9 those reported for (R)-2.3 except for [α]D = - 6.0° (c 1.04, CHCl3).

COOMe

N Boc 2.35

5,6-Dihydro-2H-pyridine-1,3-dicarboxylic acid 1-tert-butyl ester 3-methyl ester

(2.35) To a solution of arecoline hydrobromide (4.5 g, 19.1 mmol) in H2O (20 mL) was

added K2CO3 (3.5 g, 23.9 mmol) and allowed to stir at room temperature. After 1/2 h the

mixture was extracted with Et2O (4 x 20 mL) and the combined organic layers were dried

over MgSO4 and filtered. The solvent was removed by rotary evaporation and the residue

was dissolved in toluene (30 mL) followed by the slowly addition of 1-chloroethyl

chloroformate (2.4 mL, 23 mmol). The reaction mixture was heated under reflux for 24 h.

After this time, the reaction mixture was allowed to cool down to room temperature and

HCl solution (20 mL, 0.1M) was added. Then it was extracted with Et2O (3 x 20 mL) and

the combined organic phases were dried over MgSO4, filtered and the solvent removed

by rotary evaporation. The residue was dissolved in MeOH (25 mL) and the resulting

solution was heated under reflux for 3 h. The solvent was then evaporated and the residue

was dissolved in CH2Cl2 (25 mL) and cooled to 0 °C. To this solution, Et3N (2.9 mL, 21 mmol) was added followed by the addition of di-t-butyl dicarbonate (5.8 g, 26.7 mmol).

The reaction mixture was stirred for 24 h at room temperature. Aqueous solution of HCl

(20 mL, 1M) was added to the reaction mixture and extracted with CH2Cl2 (3 x 30 mL). 242

The combined organic layers were washed with saturated solution of NaHCO3 (30 mL), dried over MgSO4 and filtered. The solvent was removed by rotary evaporation and the

residue was purified by means of flash chromatography (10% EtOAc in hexanes) to

1 afford 2.97 g (65%) of 2.35 as an oil. Rf 0.3 (10% EtOAc in hexanes). H NMR (CDCl3,

500 MHz) δ 7.04 (s, 1H, 4-CH), 4.08 (s, 2H, 2-CH2), 3.72 (s, 3H, OCH3), 3.44 (t, J = 5.5

Hz, 2H, 6-CH2), 2.27 (s, 2H, 5-CH2), 1.44 (s, 9H, C(CH3)3).

OH

N Boc 2.30

5-Hydroxymethyl-3,6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester (2.30).

A solution of 2.35 (2.9 g, 12.3 mmol) in dry Et2O (50 mL) under argon atmosphere was

cooled to – 78 °C and DIBAL-H (49 mL, 1M solution in CH2Cl2, 49 mmol) was added.

After 1 h of stirring at – 78 °C, MeOH (3 mL) was added and the reaction mixture was

allowed to warm to room temperature. Aqueous solution of potassium tartrate (30 mL,

1M) was added and extracted with EtOAc (3 x 20 mL). The combined organic layers

were dried over MgSO4 and filtered. The solvent was removed by means of rotary

evaporation and the residue was purified by flash chromatography (40 % EtOAc in

1 hexanes) to afford 1.85 g (70%) of 2.30 as an oil. Rf 0.12 (20% EtOAc in hexanes). H

NMR (CDCl3, 500 MHz) δ 5.80 (br s, 1H, 4-CH), 4.04 (br s, 2H, CH2OH), 3.89 (br s,

2H, 2-CH2), 3.44 (t, J = 5.5 Hz, 2H, 6-CH2), 2.13 (br s, 2H, 5-CH2), 1.44 (s, 9H,

C(CH3)3). 243

OH

N HCl H

2.31

(1,2,5,6-Tetrahydro-pyridin-3-yl)-methanol (2.31). To a cold (0 °C) solution of 2.30

(0.84 g, 3.9 mmol) in dry MeOH (7.8 mL) was added acetyl chloride (1.4 mL, 19.5

mmol). After 15 minutes, the reaction mixture was allowed to warm to room temperature

and stirred for 18 h. The solvent was removed by rotary evaporation and to afford 0.55 g

1 (92%) of 2.31 as a hydrochloric salt. H NMR (D2O, 500 MHz) δ 5.94 (s, 1H, 4-CH),

4.04 (s, 2H, CH2OH), 3.67 (s, 2H, 2-CH2), 3.29 (t, J = 6.5 Hz, 2H, 6-CH2), 2.39 (m, 2H,

5-CH2).

OH

N

Ph 2.32

[1-(3-Phenyl-propyl)-1,2,5,6-tetrahydro-pyridin-3-yl]-methanol (2.32). To a solution

of 2.31 (0.3 g, 2 mmol) in EtOH (2 mL) was added K2CO3 (0.83 g, 6 mmol) followed by

the addition of 1-bromo-3-phenylpropane (0.36 mL, 2.4 mmol). The reaction mixture was

heated under reflux for 8 h then allowed to cool to room temperature. H2O was added and

extracted with EtOAc (3 x 10 mL). The combined organic phases were dried over MgSO4 and filtered. The solvent was removed by rotary evaporation and the residue was purified by flash chromatography (5% MeOH in CHCl3) to provide 0.34 g (75%) of 2.32 as 244

1 colorless oil. Rf 0.26 (5% MeOH in CHCl3). H NMR (CDCl3, 500 MHz) δ 7.2-7.01 (m,

5H, Ph), 5.75 (s, 1H, 4-CH), 4.08 (s, 2H, CH2OH), 3.08 (s, 2H, 2-CH2), 2.69 (t, J = 7.5

Hz, 2H, 3’-CH2), 2.62 (t, J = 5.5 Hz, 2H, 6-CH2), 2.56 (t, J = 7.5 Hz, 2H, 1’-CH2), 2.27

(m, 2H, 5-CH2), 1.96 (qv, J = 7.5 Hz, 2H, 2’-CH2).

(R)

O O O (R)-2.39

(R)-3-Methyl-dihydro-furan-2,5-dione ((R)-2.39). A solution of (R)-methylsuccinic

acid (1g, 7.5 mmol) in acetyl chloride (3.2 mL, 45 mmol) was heated under microwave

irradiation at 100 °C for 7 minutes. The reaction mixture was then concentrated on the

rotary evaporator to give white crystals that were washed with cold Et2O and then

vacuum dried to afford 0.85 g (100%) of (R)-2.39. Rf 0.25 (10% EtOAc, 1% AcOH in

25.2 hexanes). Mp 45.3-46.5 °C. [α]D = + 12.0° (c 1.0, H2O) and + 33.7° (c 1.0, CHCl3),

172 25.3 1 lit. [α]D = + 34.4° (c 1.0, CHCl3). H NMR (DMSO-d6, 300 MHz) δ 3.22-3.14 (m,

1H, 3-CH), 2.97 (dd, J = 10, 18 Hz, 1H, 4- CHH), 2.60 (dd, J = 7, 18 Hz, 1H, 4-CHH),

13 1.17 (d, J = 7.4 Hz, 3H, CH3). C NMR (DMSO-d6, 75 MHz) δ 176.0, 171.6, 35.6, 35.1,

14.6.

(S)

O O O (S)-2.39

245

(S)-3-Methyl-dihydro-furan-2,5-dione ((S)-2.39). Compound (S)-2.39 was prepared from (S)-methylsuccinic acid on a 7.5 mmol scale following the procedure described for

(R)-2.39 to provide 0.85 g (100%) of (S)-2.39. All analytical data were identical with

25.3 those reported for (R)-2.39 except for [α]D = - 12.6° (c 1.0, H2O) and - 34.3° (c 1.0,

CHCl3).

(R)

O N O COOH

(R)-2.36

2-((R)-3-Methyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid ((R)-2.36). A mixture of antranilic acid (0.94 g, 6.9 mmol) and (R)-2.39 (0.78 g. 6.9 mmol) was heated under microwave irradiation at 170 °C for 45 minutes. The residue was purified by means of

flash chromatography (1.5% EtOAc, 0.5% AcOH in toluene) to afford 1.17 g (73%) of

25.5 (R)-2.36 as yellowish crystals. Rf 0.125 (5% EtOAc, 5% AcOH in toluene). [α]D = +

172 1 11.2° (c 1.0, CDCl3), lit. [α]D = + 14.1° (c 1.0, CHCl3). H NMR (CDCl3, 300 MHz) δ

9.0 (br s, 1H, COOH), 8.20 (d, J = 7.5 Hz, 1H, Ar), 7.71 (t, 1H, Ar), 7.56 (t, 1H, Ar),

7.25 (d, J = 7.5 Hz, 1H, Ar), 3.16-3.08 (m, 2H, 3-CH and 4-CHH), 2.52 (d, J = 14.5 Hz,

13 1H, 4- CHH), 1.43 (d, J = 7.4 Hz, 3H, CH3). C NMR (CDCl3, 75 MHz) δ 180.3,

180.2*, 176.4, 176.3*, 169.5, 169.3*, 134.4, 133.0, 132.7, 130.0, 129.7, 126.7, 37.2*,

37.1, 35.5*, 35.3, 16.6. (* denotes signals of rotamer) Additional analytical data have

been previously reported.172 246

(S)

O N O COOH

(S)-2.36

2-((S)-3-Methyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid ((S)-2.36). Compound (S)-

2.36 was prepared from (S)-2.39 on a 6.9 mmol scale following the procedure described for (R)-2.36 to provide 1.15 g (72%) of (S)-2.36. All analytical data were identical with

25.6 those reported for (R)-2.36 except for [α]D = - 10.6° (c 1.0, CDCl3).

HO Ph (R) N

(R)-2.37

(R)-[1-(3-Phenyl-propyl)-pyrrolidin-2-yl]-methanol ((R)-2.37). To a solution of (R)-(-

)-2-pyrrolidinemethanol (0.5 g, 5 mmol) in EtOH (4 mL) was added K2CO3 (1.36 g, 9 mmol) followed by the addition of 1-bromo-3-phenylpropane (0.9 mL, 6 mmol). The reaction mixture was heated under microwave irradiation at 130 °C for 15 minutes. The reaction mixture was then concentrated by means of rotary evaporation and the residue dissolved in EtOAc followed by a filtration to remove the precipitate formed. The filtrate was concentrated by rotary evaporation and the residue was purified by flash chromatography (5% MeOH in CHCl2) to afford 0.7 g (65%) of (R)-2.37 as colorless oil. 247

25.3 1 Rf 0.19 (5% MeOH in CHCl3). [α]D = - 33.6° (c 1.0, toluene). H NMR (CDCl3, 300

MHz) δ 7.24-7.16 (m, 5H, Ph), 3.58 (dd, J = 3, 11 Hz, 1H, HOCHH), 3.39 (dd, J = 3, 11

Hz, 1H, HOCHH), 3.19-3.16 (m, 1H, 2-CH), 2.78-2.53 (m, 4H, 1’-CH2 and 3’-CH2),

13 2.34-2.21 (m, 2H, 5-CH2), 1.93-1.78 (m, 6H, 3-CH2, 4-CH2 and 2’-CH2). C NMR

(CDCl3, 75 MHz) δ 141.6, 128.4, 128.3, 125.9, 65.9, 61.5, 54.4, 54.2, 33.4, 29.8, 27.4,

23.7.

HO Ph (S) N

(S)-2.37

(S)-[1-(3-Phenyl-propyl)-pyrrolidin-2-yl]-methanol ((S)-2.37). Compound (S)-2.37

was prepared from (S)-(+)-2-pyrrolidinemethanol on a 16.2 mmol scale following the

procedure described for (R)-2.37 to provide 2.5 g (70%) of (S)-2.37. All analytical data

25.2 were identical with those reported for (R)-2.37 except for [α]D = + 33.9° (c 1.0,

toluene).

OH

N Cbz 2.42

3-Hydroxymethyl-piperidine-1-carboxylic acid benzyl ester (2.42). To a solution of 3-

piperidinemethanol (1 g, 7.7 mmol) in CH2Cl2 (15.5 mL) was added Et3N (2.15 mL, 15.5 248 mmol) and the mixture was cooled to 0 °C. A solution of benzyl chloroformate (1.2 mL,

8.5 mmol) in CH2Cl2 (8.5 mL) was then added to the reaction mixture and allowed to stir

at room temperature for 12 h. After this time, the reaction mixture was washed with H2O

(10 mL), 1M HCl (10 mL), H2O (10 mL), saturated aqueous NaHCO3 (10 mL) and brine.

The organic phase was then dried over MgSO4, filtered and the solvent removed by rotary evaporation. The residue was purified by flash chromatography (40% EtOAc in hexanes) to afford 1.44 g (75%) of 2.42 as colorless oil. Rf 0.19 (40% EtOAc in hexanes).

1 H NMR (CDCl3, 500 MHz) δ 7.28-7.38 (m, 5H, Ph), 5.15 (s, 2H, PhCH2), 4.05-3.62 (m,

2H, 2-CH2), 3.51 (d, J = 6.2 Hz, 2H, HOCH2), 3.20-2.80 (m, 2H, 6-CH2), 1.79-1.27 (m,

5H, 3-CH, 4-CH2, 5-CH2).

O Ph

O

N Cbz 2.44

3-(Biphenyl-2-carbonyloxymethyl)-piperidine-1-carboxylic acid benzyl ester (2.44).

To a solution of 2-phenyl benzoic acid (0.2 g, 1 mmol) in CH2Cl2 (1 mL) was added

DMAP (0.02 g, 0.1 mmol) and a solution of alcohol 2.42 (0.25 g, 1 mmol) in CH2Cl2 (1 mL). The mixture was cooled to 0 °C and DCC (0.2 g, 1 mmol) was added in one portion. The reaction mixture was stirred for 24 h at room temperature. After this time, the reaction was diluted with Et2O and filtered to remove the precipitate. The solvent was

then removed by rotary evaporation and the residue purified by flash chromatography

(20% EtOAc in hexanes) to provide 0.25 g (59%) of 2.44. Rf 0.24 (20% EtOAc in 249

1 hexanes). H NMR (CDCl3, 500 MHz) δ 7.85 (d, 1H, Ar), 7.58 (m, 1H, Ar), 7.40-7.18

(m, 12H, Ar), 5.15 (s, 2H, PhCH2), 3.95-3.82 (m, 4H, OCH2 and 2-CH2), 2.74 (t, 1H, 6-

CHH), 2.38 (t, 1H, 6-CHH), 1.59-0.97 (m, 5H, 3-CH, 4-CH2, 5-CH2).

O Ph

O

N H

COB-1

Biphenyl-2-carboxylic acid piperidin-3-ylmethyl ester (COB-1). To a suspension of

palladium on charcoal (20 mg, 10% Pd/C) in dry EtOAc (0.25 mL) was added a solution

of 2.44 (0.2 g, 0.48 mmol) in EtOAc (0.25 mL). The flask was equipped with a 3-way

adapter to which a line for argon and a vacuum aspirator were attached. The gases from

the flask were evacuated by alternating (3x) aspiration with positive argon atmosphere.

The argon line was replaced with a balloon filled with H2 and the evacuation was repeated again. Keeping the flask opened for H2, the vacuum line was removed and the

reaction mixture was allowed to stir at room temperature under H2 atmosphere for 24 h.

The reaction mixture was then filtered through a short pad of celite and washed

thoroughly with EtOAc. The solvent was removed by rotary evaporation to provide 117

mg (82%) of COB-1 as colorless liquid. No further purification was necessary. 1H NMR

(CDCl3, 500 MHz) δ 7.76-7.73 (d, 1H, Ar), 7.35-7.19 (m, 8H, Ar), 4.7 (brs, 1H, NH),

3.83 (dq, 2H, OCH2), 3.11 (d, J = 12 Hz, 1H, 2-CHH), 2.77 (d, J = 8 Hz, 1H, 6-CHH),

2.43-2.38 (m, 1H, 2-CHH), 1.97-1.88 (m, 1H, 6- CHH), 1.60 (m, 2H, 4-CHH and 5- 250

CHH), 1.43-1.40 (m, 1H, 3-CH), 0.82-0.75 (m, 2H, 4-CHH and 5-CHH). 13C NMR

(CDCl3, 125 MHz) δ 168.8, 142.2, 141.6, 131.3, 130.7, 130.0, 128.3, 127.3, 127.2,

127.18, 66.4, 46.0, 44.0, 33.1, 25.4, 22.2.

O Ph

O

N

COB-2

Biphenyl-2-carboxylic acid N-methyl-piperidin-3-ylmethyl ester (COB-2). To a solution of 2-phenyl benzoic acid (0.4 g, 2 mmol) in CH2Cl2 (2 mL) was added DMAP

(0.02 g, 0.2 mmol) and a solution of N-methyl-piperidine-3-methanol (0.25 g, 2 mmol) in

CH2Cl2 (6 mL). The mixture was cooled to 0 °C and DCC (0.41 g, 2 mmol) was added in

one portion. The reaction mixture was stirred for 24 h at room temperature. After this

time, the reaction was diluted with Et2O and filtered to remove the urea. The solvent was

then removed by rotary evaporation and the residue purified by flash chromatography

(20% EtOAc in hexanes) to provide 0.43 g (70%) of COB-2. Rf 0.5 (20% EtOAc in

1 hexanes). H NMR (CDCl3, 500 MHz) δ 7.76-7.73 (d, 1H, Ar), 7.45 (t, 1H, Ar), 7.40-

7.27 (m, 7H, Ar), 3.84 (dq, 2H, OCH2), 2.71 (d, J = 12 Hz, 1H, 2-CHH), 2.41 (d, J = 8

Hz, 1H, 2-CHH), 2.19 (s, 3H, CH3), 1.80-1.35 (m, 7H, 6-CH2, 4-CH2, 5-CH2, 3-CH).

251

O Ph

O

N

COB-3

Biphenyl-2-carboxylic acid N-dimethyl-piperidin-3-ylmethyl ester (COB-3). To a

solution of COB-2 (0.5g, 1.6 mmol) in CH3CN (4 mL) was added CH3I (0.4 mL, 6.4

mmol). The reaction mixture was stirred and refluxed for 20 h. After this time, the

solvent was removed by rotary evaporation to provide 0.51 g (98.5%) of COB-3. 1H

NMR (CDCl3, 500 MHz) δ 7.76-7.73 (d, 1H, Ar), 7.45 (t, 1H, Ar), 7.40-7.34 (m, 7H, Ar),

4.12 (brs, 1H, OCHH), 3.95 (brs, 1H, OCHH), 3.52-3.39 (m, 4H, CH3 and 2-CHH), 3.29-

3.15 (m, 4H, CH3 and 2-CHH), 2.85 (m, 1H, 6-CHH), 2.65 (m, 1H, 6-CHH), 2.12-1.54

(m, 5H, 4-CH2, 5-CH2, 3-CH).

(R) COOEt

N Cbz (R)-2.45

(R)-Piperidine-1,3-dicarboxylic acid 1-benzyl ester 3-ethyl ester ((R)-2.45). To a

suspension of (R)-2.26 (1.38 g, 4.5 mmol) in EtOAc (9 mL) was added H2O (9.4 mL)

followed by the addition of a solution of 15% Na2CO3 (9.54 mL) over a 15 minutes

period. A solution of benzyl chloroformate (1.3 mL, 9 mmol) in EtOAc (1.6 mL) was

added to the reaction flask over a period of 10 minutes and the reaction mixture was stirred at room temperature for 2 h. The layers were separated and the aqueous layer was 252 extracted with EtOAc (3 x 10 mL). The combined organic layers were dried over MgSO4 and filtered. The solvent was removed by rotary evaporation and the residue was purified by means of flash chromatography (20% EtOAc in hexanes) to afford 1.2 g (92%) of (R)-

25.8 1 2.45 as colorless oil. Rf 0.2 (20% EtOAc in hexanes). [α]D = - 44.2° (c 1, CHCl3). H

NMR (CDCl3, 300 MHz) δ 7.38-7.27 (m, 5H, Ph), 5.15 (s, 2H, PhCH2), 4.15 (q, J = 7.1

Hz, 2H, CH2CH3), 4.03-3.98 (m, 1H, 2-CHH), 2.94-2.85 (m, 1H, 2-CHH), 2.45 (m, 1H,

3-CH), 2.08-2.02 (m, 1H, 6-CHH), 1.70-1.55 (m, 5H, 4- CH2, 6-CHH and 5-CH2), 1.20

(t, J = 7.1 Hz, 3H, CH2CH3).

(S) COOEt

N Cbz (S)-2.45

(S)-Piperidine-1,3-dicarboxylic acid 1-benzyl ester 3-ethyl ester ((S)-2.45). Compound

(S)-2.45 was prepared from (S)-2.26 on 4.5 mmol scale following the procedure

described for (R)-2.45 to provide 1.16 g (89%) of (S)-2.45 as colorless oil. All analytical

25.9 data were identical with those reported for (R)-2.45 except for [α]D = + 45.2° (c 1,

CHCl3).

(R) OH

N Cbz (R)-2.42

253

(R)-3-Hydroxymethyl-piperidine-1-carboxylic acid benzyl ester ((R)-2.42). To a cold

(-78 °C) solution of (R)-2.45 (1 g, 3.43 mmol) in dry Et2O (3.5 mL) was added DIBAL-H

(13.8 mL, 1M solution in CH2Cl2, 13.8 mmol) and the reaction mixture was allowed to

warm to –20 °C. After stirring 2 h at –20 °C, the reaction was quenched by the addition

of an aqueous solution of sodium tartrate (1M, 14 mL). The aqueous layer was extracted

with EtOAc (3 x 10 mL) and the combined organic layers were dried over MgSO4 and

filtered. The solvent was removed by rotary evaporation and the residue was purified by

means of flash chromatography (40% EtOAc in hexanes) to afford 0.3 g (35%) of (R)-

25.1 2.42 as colorless oil. Rf 0.19 (40% EtOAc in hexanes). [α]D = - 7.4° (c 1.315, CHCl3).

1 tR 8.88 min. (55 % IPA/hexane in 22 min). H NMR (CDCl3, 300 MHz) δ 7.28-7.18 (m,

5H, Ph), 5.05 (s, 2H, PhCH2), 3.85-3.72 (m, 2H, 2-CH2), 3.41 (d, J = 6.2 Hz, 2H,

HOCH2), 2.99-2.84 (m, 2H, 6-CH2), 1.69-1.37 (m, 5H, 3-CH, 4- CH2, 5-CH2).

(S) OH

N Cbz (S)-2.42

(S)-3-Hydroxymethyl-piperidine-1-carboxylic acid benzyl ester ((S)-2.42). Compound

(S)-2.42 was prepared from (S)-2.45 on 3.43 mmol scale following the procedure described for (R)-2.42 to provide 0.32 g (37%) of (S)-2.42 as colorless oil. All analytical

25.1 data were identical with those reported for (R)-2.42 except for [α]D = + 8.3° (c 1.34,

CHCl3).

254

O Ph (R) O

N Cbz (R)-2.44

(R)-3-(Biphenyl-2-carbonyloxymethyl)-piperidine-1-carboxylic acid benzyl ester

((R)-2.44). To a solution of (R)-2.42 (50 mg, 0.2 mmol) in CH2Cl2 (2 mL) was added 2-

phenylbenzoic acid 2.43 (48 mg, 0.24 mmol) followed by the addition of PS-TsCl (0.2 g,

0.4 mmol, 1.97 mmol/g). The reaction mixture was shaken for 5 minutes. N-methyl

imidazole (63 μL, 0.8 mmol) was added and the reaction mixture was shaken for another

2 h. The reaction mixture was then filtered and the polystyrene beads were washed with

CH2Cl2 (5 mL). The solvent was removed by rotary evaporation and the residue was

purified by flash chromatography (20% EtOAc in hexanes) to afford 63 mg (73%) of (R)-

24.2 2.44 as colorless oil. Rf 0.24 (20% EtOAc in hexanes). [α]D = - 19.1° (c 0.805,

1 CHCl3). H NMR (CDCl3, 300 MHz) δ 7.82 (d, 1H, Ar), 7.53 (d, 1H, Ar), 7.33-7.26 (m,

12H, Ph), 5.11 (s, 2H, PhCH2), 3.92-3.90 (m, 4H, OCH2 and 2-CH2), 2.75 (m, 1H, 6-

CHH), 2.36 (m, 1H, 6-CHH), 1.58-1.28 (m, 3H, 3-CH, 5-CH2) 0.95 (m, 2H, 4-CH2).

O Ph (S) O

N Cbz (S)-2.44

(S)-3-(Biphenyl-2-carbonyloxymethyl)-piperidine-1-carboxylic acid benzyl ester

((S)-2.44). Compound (S)-2.44 was prepared from (S)-2.42 on 0.2 mmol scale following 255 the procedure described for (R)-2.44 to provide 70 mg (81%) of (S)-2.44 as colorless oil.

24.3 All analytical data were identical with those reported for (R)-2.44 except for [α]D = +

17.6° (c 0.825, CHCl3).

O Ph (R) O

N H COB-11

(R)-Biphenyl-2-carboxylic acid piperidin-3-ylmethyl ester (COB-11). To a suspension

of 10% Pd/C (w/w) (16 mg) in dry EtOAc (0.25 mL) was added a solution of (R)-2.44

(63 mg, 0.15 mmol) in EtOAc (0.25 mL). The flask was equipped with a 3-way adapter to which a line for argon and a vacuum aspirator were attached. The gases from the flask were evacuated by alternating (3x) aspiration with positive argon atmosphere. The argon

line was replaced with a balloon filled with H2 and the evacuation was repeated again.

Keeping the flask opened for H2, the vacuum line was removed and the reaction mixture

was allowed to stir at room temperature under H2 atmosphere for 24 h. The reaction

mixture was then filtered through a short pad of celite and washed thoroughly with

EtOAc. The solvent was removed by rotary evaporation to provide 38 mg (88%) of

24.2 COB-11 as colorless liquid. No further purification was necessary. [α]D = - 4.1° (c 1,

1 CHCl3). H NMR (CDCl3, 300 MHz) δ 7.76-7.73 (d, 1H, Ar), 7.35-7.19 (m, 8H, Ar), 3.83

(m, 2H, OCH2), 3.11 (d, J = 12 Hz, 1H, 2- CHH), 2.77 (d, J = 8 Hz, 1H, 6-CHH), 2.43-

2.38 (m, 1H, 2-CHH), 1.97-1.88 (m, 1H, 6- CHH), 1.60 (m, 2H, 4- CHH and 5-CHH),

13 1.43-1.40 (m, 1H, 3-CH), 0.82-0.75 (m, 2H, 4- CHH and 5- CHH). C NMR (CDCl3, 75 256

MHz) δ 168.8, 142.2, 141.7, 131.5, 130.8, 130.5, 130.1, 128.4, 128.3, 127.6, 127.0, 66.4,

46.0, 44.0, 33.1, 25.4, 22.2.

O Ph (S) O

N H COB-12

(S)-Biphenyl-2-carboxylic acid piperidin-3-ylmethyl ester (COB-12). Compound

COB-12 was prepared from (S)-2.44 on 0.16 mmol scale following the procedure described for COB-11 to provide 42 mg (87%) of COB-12 as colorless oil. All analytical

25.2 data were identical with those reported for COB-11 except for [α]D = + 4.7° (c 1.01,

CHCl3).

General procedure for coupling reaction. To a solution of alcohol (0.2 mmol) in

CH2Cl2 (2 mL) was added acid (0.24 mmol) followed by the addition of PS-TsCl (0.4

mmol, 1.97 mmol/g). The reaction mixture was shaken for 5 minutes. N-methyl

imidazole (0.8 mmol) was added and the reaction mixture was shaken for another 2 h.

The reaction mixture was then filtered and the polystyrene beads were washed with

CH2Cl2 (5 mL). The solvent was removed by rotary evaporation and the residue was

purified by flash chromatography. The following compounds were prepared using this

procedure.

257

(R)

O N O O (S) O N

Ph COB-13

2-((R)-3-Methyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-

pyrrolidin-(S)-2-ylmethyl ester (COB-13). Compound COB-13 was prepared from acid

(R)-2.36 and alcohol (S)-2.37 on a 0.214 mmol scale to provide 68.5 mg (75%) of COB-

13. Rf 0. (% EtOAc in hexane). Reverse phase HPLC for its corresponding hydrochloric

1 salt: tR 12.0 min. (50% CH3CN/H2O, 22 min.). H NMR (CD3CN, 300 MHz) δ 7.94 (d, J

= 7.41 Hz, 1H, Ar), 7.62 (t, J = 6.24 Hz, 1H, Ar), 7.47 (t, J = 6.6 Hz, 1H, Ar), 7.23-7.06

(m, 6H, Ar), 4.19-4.14 (m, 1H, OCHH), 4.06-4.00 (m, 1H, OCHH), 3.12-3.08 (m, 1H),

2.97-2.93 (m, 2H), 2.81-2.74 (m, 2H), 2.57-2.48 (m, 2H), 2.38-2.24 (m, 2H), 1.87-1.84

13 (m, 2H), 1.73-1.67 (m, 5H), 1.27 (d, J = 6.75 Hz, 3H, CH3). C NMR (CD3CN, 75 MHz)

δ 179.8, 175.9, 164.2, 142.2, 133.3, 133.0. 131.4, 130.1, 129.8, 129.3, 128.4, 128.3,

125.7, 68.0, 62.4, 55.0, 54.2, 37.0, 33.6, 30.5, 28.5, 23.1.

258

(S)

O N O O (R) O N

Ph COB-17

2-((S)-3-Methyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-

pyrrolidin-(R)-2-ylmethyl ester (COB-17). Compound COB-17 was prepared from

acid (S)-2.36 and alcohol (R)-2.37 on a 0.214 mmol scale to provide 36 mg (50%) of

COB-17. All analytical data were identical with those reported for COB-13.

(S)

O N O O (S) O N

Ph COB-14

2-((S)-3-Methyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-

pyrrolidin-(S)-2-ylmethyl ester (COB-14). Compound COB-14 was prepared from acid

(S)-2.36 and alcohol (S)-2.37 on a 0.214 mmol scale to provide 69.2 mg (74%) of COB-

1 14. H NMR (CD3CN, 300 MHz) δ 7.94 (d, J = 7.41 Hz, 1H, Ar), 7.62 (t, J = 6.24 Hz,

1H, Ar), 7.47 (t, J = 6.6 Hz, 1H, Ar), 7.23-7.06 (m, 6H, Ar), 4.18-4.13 (m, 1H, OCHH),

4.07-4.00 (m, 1H, OCHH), 3.12-3.08 (m, 1H), 2.96-2.93 (m, 2H), 2.81-2.74 (m, 2H), 259

2.50-2.37 (m, 4H), 1.86-1.84 (m, 2H), 1.72-1.67 (m, 5H), 1.26 (d, J = 6.75 Hz, 3H, CH3).

All other analytical data were identical with those reported for diastereomer COB-13.

(R)

O N O O (R) O N

Ph COB-15

2-((R)-3-Methyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-

pyrrolidin-(R)-2-ylmethyl ester (COB-15). Compound COB-15 was prepared from

acid (R)-2.36 and alcohol (R)-2.37 on a 0.214 mmol scale to provide 32.4 mg (55%) of

COB-15. All other analytical data were identical with those reported for COB-14.

Ph 3' O O O N 3 O

N

Ph COB-5

2-(3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-1,2,5,6-

tetrahydro-pyridin-3-ylmethyl ester (COB-5). Compound COB-5 was prepared from

acid 2.2 and alcohol 2.32 on a 0.181 mmol scale to provide 63.3 mg (78%) of COB-5. Rf

1 0.54 (5% MeOH in CHCl3). H NMR (CDCl3, 300 MHz) δ 8.05 (dd, 1H), 7.55-7.52 (m, 260

1H), 7.45-7.40 (m, 1H), 7.26-7.09 (m, 11H), 5.79 (s, 1H), 4.51 (m, 2H), 3.36 (m, 1H),

3.17-3.01 (m, 1H), 2.98-2.82 (m, 4H), 2.60-2.55 (m, 3H), 2.46-2.38 (m, 4H), 2.12 (m,

13 2H), 1.86-1.76 (m, 2H). C NMR (CDCl3, 125 MHz) δ (* denotes signals of rotamer)

178.6, 175.7, 170.9, 164.3, 141.9, 133.5, 131.9, 129.7, 129.4, 129.2, 128.8, 128.4, 128.3,

127.1, 126.0, 125.8, 125.8, 125.1, 67.6, 66.7*, 57.6, 57.5*, 53.15, 53.10*, 49.3, 42.3*,

41.6, 36.7*, 36.3, 34.7*, 33.7, 28.4, 25.7, 25.5*, 20.9.

3' Ph (R) O O O N 3 O

N

Ph COB-24

2-((R)-3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-1,2,5,6- tetrahydro-pyridin-3-ylmethyl ester (COB-24). Compound COB-24 was prepared from acid (R)-2.2 and alcohol 2.32 on a 0.181 mmol scale to provide 67.5 mg (80%) of

COB-24. All analytical data were identical with those reported for COB-5 except for

27 [α]D = - 7.5° (c 1.0, CHCl3).

3' Ph (S) O O O N 3 O

N

Ph COB-25 261

2-((S)-3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-1,2,5,6- tetrahydro-pyridin-3-ylmethyl ester (COB-25). Compound COB-25 was prepared from acid (S)-2.2 and alcohol 2.32 on a 0.181 mmol scale to provide 64.2 mg (76%) of

COB-25. All analytical data were identical with those reported for COB-5 except for

27.1 [α]D = + 3.2° (c 0.79, CHCl3).

Ph 3' O O O N 3 (S) O

N

Ph COB-6

2-(3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-piperidin-

(S)-3-ylmethyl ester (COB-6). Compound COB-6 was prepared from acid 2.2 and

alcohol (S)-2.32 on a 0.181 mmol scale to provide 57.3 mg (68%) of COB-6. Rf 0.3 (5%

1 MeOH in CHCl3). H NMR (CDCl3, 300 MHz) δ 8.11 (dd, 1H), 7.65-7.62 (m, 1H), 7.54-

7.49 (m, 1H), 7.38-7.16 (m, 11H), 4.12 (m, 2H), 3.47-2.83 (m, 6H), 2.67-2.62 (m, 3H),

2.40 (m, 2H), 2.12-1.72 (m, 8H), 1.27-1.11 (m, 1H).

262

Ph 3' O O O N 3 (R) O

N

Ph COB-7

2-(3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-piperidin-

(R)-3-ylmethyl ester (COB-7). Compound COB-7 was prepared from acid 2.2 and alcohol (R)-2.3 on a 0.181 mmol scale to provide 56.1 mg (66%) of COB-7. All analytical data were identical with those reported for COB-6.

(R) Ph

O O O N

O

N

Ph COB-22

2-(3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-piperidin-

(R)-3-ylmethyl ester (COB-22). Compound COB-22 was prepared from acid (R)-2.2 and alcohol 2.3 on a 0.15 mmol scale to provide 54 mg (69%) of COB-22. All analytical data were identical with those reported for COB-6.

263

(S) Ph

O O O N

O

N

Ph COB-23

2-(3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-piperidin-

(S)-3-ylmethyl ester (COB-23). Compound COB-23 was prepared from acid (S)-2.2 and

alcohol 2.3 on a 0.15 mmol scale to provide 59 mg (71%) of COB-23. All analytical data

were identical with those reported for COB-6.

(R) Ph

O O O N

(R) O

N

Ph COB-18

2-((R)-3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-

piperidin-(R)-3-ylmethyl ester (COB-18). Compound COB-18 was prepared from acid

(R)-2.2 and alcohol (R)-2.3 on a 0.21 mmol scale to provide 62 mg (56%) of COB-18.

All analytical data were identical with those reported for COB-6.

264

(S) Ph

O O O N

(R) O

N

Ph COB-19

2-((S)-3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-

piperidin-(R)-3-ylmethyl ester (COB-19). Compound COB-19 was prepared from acid

(S)-2.2 and alcohol (R)-2.3 on a 0.21 mmol scale to provide 67.2 mg (60%) of COB-19.

All analytical data were identical with those reported for COB-6.

(R) Ph

O O O N

(S) O

N

Ph COB-20

2-((R)-3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-

piperidin-(S)-3-ylmethyl ester (COB-20). Compound COB-20 was prepared from acid

(R)-2.2 and alcohol (S)-2.3 on a 0.15 mmol scale to provide 51 mg (62%) of COB-20. All analytical data were identical with those reported for COB-6.

265

(S) Ph

O O O N

(S) O

N

Ph COB-21

2-((S)-3-Benzyl-2,5-dioxo-pyrrolidin-1-yl)-benzoic acid 1-(3-phenyl-propyl)-

piperidin-(S)-3-ylmethyl ester (COB-21). Compound COB-21 was prepared from acid

(S)-2.2 and alcohol (S)-2.3 on a 0.15 mmol scale to provide 56 mg (68%) of COB-21. All

analytical data were identical with those reported for COB-6.

O

O NH2 TrO 4.11

Carbamic Acid 1-(Triphenylmethoxy)-allyl ester (4.11). To a cold solution (0 °C) of

allylic alcohol 4.2 (2.36 g, 7.14 mmol) in CH2Cl2 (40 mL) was slowly added

trichloroacetyl isocyanate (0.93 mL, 7.85 mmol). The solution was stirred at room

temperature for 3 h. The solvent was removed under reduced pressure and the residue

was dissolved in MeOH (40 mL). K2CO3 (0.12 g, 0.86 mmol) was added and the solution

was stirred at room temperature for 2 h. The solvent was removed under reduced pressure

and the residue was purified by flash chromatography (40% EtOAc in hexanes) to

1 provide 2.6 g (98%) of 4.11. Rf 0.25 (40% EtOAc in hexanes). H NMR (CDCl3, 300

MHz) δ 7.25-7.48 (m, 15H), 5.86 (ddd, J = 5.9, 10.6, 16.6 Hz, 1H), 5.38-5.44 (m, 1H), 266

5.31 (dt, J = 1.4, 17.3 Hz, 1H), 5.22 (dt, J = 1.4, 10.6 Hz, 1H), 4.68 (s, 2H), 3.23 (dd, J =

1.4, 6.0 Hz, 2H).

O OH N O H TrO 4.15

N-Hydroxy Carbamate (4.15). To a solution of allylic alcohol 4.2 (4 g, 12.1 mmol) in

CH2Cl2 (24 mL) was added 1,1’-carbodiimidazole (4 g, 24.2 mmol). The reaction mixture

was stirred at room temperature until complete consumption of alcohol 4.2 shown by

TLC (typically 1 h). Imidazole (3.3 g, 48.4 mmol) and NH2OH•HCl (4 g, 60.5 mmol) were added and the resulting mixture was stirred at room temperature for another 4 h or until the TLC showed complete consumption of the initial alcohol adduct. The reaction mixture was washed with 1M HCl (25 mL) and the aqueous phase extracted with CH2Cl2

(2 x 20 mL). The combined organic phases were washed with brine, dried over MgSO4, filtered and the solvent removed under reduced pressure. The residue was purified by flash chromatography (30% EtOAc in hexanes) to provide 3.7 g (79%) of 4.15. Rf 0.3

1 (30% EtOAc in hexanes). H NMR (CDCl3, 300 MHz) δ 7.27-7.46 (m, 15H), 5.86 (m,

1H), 5.46 (m, 1H), 5.31 (d, J = 17.28 Hz, 1H), 5.23 (d, J = 10.59 Hz, 1H), 3.23-3.27 (m,

2H).

267

O OTs N O H TrO 4.16

N-Tosyloxy Carbamate (4.16). To a cold (0 °C) solution of 4.15 (1.5 g, 3.85 mmol) in

Et2O (15 mL) was added through cannula a solution of tosylchloride (0.807 g, 4.23

mmol) in Et2O (5 mL). Et3N (0.643 mL, 4.62 mmol) was then added neat. The reaction

mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was

washed with H2O and brine, then dried over MgSO4, filtered and the solvent removed

under reduced pressure. The residue was purified by flash chromatography (20% EtOAc

1 in hexanes) to provide 1.35 g (65%) of 4.16. Rf 0.23 (20% EtOAc in hexanes). H NMR

(CDCl3, 300 MHz) δ 7.88 (m, 3H), 7.33-7.41 (m, 22H), 5.50-5.62 (m, 1H), 5.24-5.30 (m,

1H), 5.07-5.13 (m, 2H), 3.09-3.24 (m, 2H).

OH HO (S) (S)-4.1

(S)-3-Butene-1,2-diol ((S)-4.1). The catalyst (R,R)-4.19 (30 mg, 0.05 mmol) was

dissolved in butadiene monoepoxide (0.7 g, 10 mmol) and AcOH was added (11 μL, 0.2 mmol). The solution was cooled to 0 °C and then H2O (81 μL, 4.5 mmol) was added. The

reaction mixture was then stirred at 0 °C for 18 h when the residual epoxide was removed

by rotary evaporation. The diol product was isolated by vacuum distillation to provide

266 0.18 g (20%) of (S)-4.1. [α]D –44.6° (c 1.04, iPrOH); lit. [α]D –43.6° (c = 4.62, 268

1 iPrOH). H NMR (CDCl3, 300 MHz) δ 5.86 (ddd, J = 5.6, 10.6, 17 Hz, 1H), 5.35 (d, J =

17 Hz, 1H), 5.25 (d, J = 10.6 Hz, 1H), 4.27-4.26 (m, 1H), 3.68 (dd, J = 3.3, 10.6 Hz, 1H),

3.51 (dd, J = 7.7, 10.6 Hz, 1H), 2.57 (br s, 2H).

OH HO (R) (R)-4.1

(R)-3-Butene-1,2-diol ((R)-4.1). Compound (R)-4.1 was prepared following the

procedure described for (S)-4.1 using the (S,S)-4.19 catalyst to provide 1.8 g (20%) of

(R)-4.1. All analytical data were identical with those reported for (S)-4.1 except for [α]D

+43.5° (c 1.01, iPrOH).

OH TrO (S) (S)-4.2

(S)-1-(Triphenylmethoxy)-3-buten-2-ol ((S)-4.2). To a solution of (S)-4.1 (0.24 g, 2.72

mmol) in CH2Cl2 (5 mL) was added trityl chloride (0.909 g, 3.26 mmol) and DMAP (40

mg, 0.331 mmol) followed by Et3N (0.76 mL, 0.726 mmol). The reaction mixture was

stirred under argon atmosphere at room temperature for 24 h. The reaction mixture was

diluted with Et2O (20 mL) and washed with H2O (10 mL), cold 1M HCl (10 mL),

saturated NaHCO3 (10 mL), H2O (10 mL) and brine. The organic phase was dried over

MgSO4, filtered, and concentrated. The residue was purified by flash chromatography 269

(10% EtOAc in hexanes) to provide 0.5377 g (60%) of (S)-4.2 as a colorless liquid. Rf

1 0.25 (10% EtOAc in hexanes). [α]D –28.0° (c 1.01, iPrOH). H NMR (CDCl3, 300 MHz)

δ 7.45 (m, 15H), 5.82 (ddd, J = 5.6, 10.6, 16.2 Hz, 1H), 5.35 (d, J = 17.2 Hz, 1H), 5.20

(d, J = 10.6 Hz, 1H), 4.27-4.32 (m, 1H), 3.25 (dd, J = 3.8, 9.4 Hz, 1H), 3.15 (dd, J = 7.4,

13 9.4 Hz, 1H), 2.4 (d, J = 3.9, 2H). C NMR (CDCl3, 75 MHz) δ 143.8, 137.0, 128.7,

127.9, 127.2, 116.4, 86.8, 72.1, 67.5. Additional analytical data have been previously

reported.248

OH TrO (R) (R)-4.2

(R)-1-(Triphenylmethoxy)-3-buten-2-ol ((R)-4.2). Compound (R)-4.2 was prepared from (R)-4.1 following the procedure described for (S)-4.2 that provided 0.574 g (64%) of (R)-4.2 as a colorless liquid. All analytical data were identical with those reported for

(S)-4.2 except for [α]D +27.8° (c 1.0, iPrOH).

O

O N3 TrO (S)

(S)-4.3

1-(Triphenylmethoxy)-(S)-2-[(azidocarbonyl)oxy]-3-butene ((S)-4.3). CAUTION:

Azides are potentially explosive especially when heated. While we did not experience any 270 problems precaution should be taken when running reactions involving azides. p-

Nitrophenyl chloroformate (2.4 g, 12 mmol, 2 eq) was added to a solution of allylic alcohol (S)-4.2 (2 g, 6 mmol) in CH2Cl2 (20 mL). The solution was cooled to 0 °C and

pyridine (1.46 mL, 18 mmol, 3 eq) was added dropwise. The reaction mixture was

allowed to warm to room temperature and stirred for 1 h. Then it was washed with

saturated aqueous NaHCO3 solution (2 x 20 mL) and brine (2 x 20 mL). The organic

layer was dried over MgSO4, filtered, concentrated to dryness and the crude was

dissolved in acetone (22 mL). A solution of NaN3 (2.73 g, 42 mmol, 7 eq) in H2O (13 mL) was then added to this solution and the reaction mixture was stirred at room temperature for 72 h when was diluted with H2O and extracted with EtOAc (3 x 20 mL).

The combined organic layers were washed with 10% K2CO3 aqueous solution (2 x 20

mL) and then dried over MgSO4, filtered and concentrated by rotary evaporation. The

residue was purified by flash chromatography (5% EtOAc in hexanes) to provide 1.9 g

24.3 (80%) product as colorless oil. Rf 0.36 (5% EtOAc in hexanes). [α]D -20.8 (c 1.0,

1 CHCl3). H NMR (CDCl3, 300 MHz) δ 7.45-7.26 (m, 15H, Ar), 5.82 (ddd, J = 6.5, 10.6,

17.2 Hz, 1H, HC=CHH), 5.47-5.42 (m, 1H, OCH), 5.37 (d, J = 17.2 Hz, 1H, HC=CHH),

5.29 (d, J = 10.6 Hz, 1H, HC=CHH), 3.32 (dd, J = 7.2, 10.2 Hz, 1H, TrOCHH), 3.23 (dd,

13 J = 3.9, 10.2 Hz, 1H, TrOCHH). C NMR (CDCl3, 75 MHz) δ 156.9, 143.5, 132.1,

128.6, 127.9, 127.2, 119.4, 86.8, 78.5, 64.8. Additional analytical data have been

previously reported.248

271

O

O N3 TrO (R)

(R)-4.3

1-(Triphenylmethoxy)-(R)-2-[(azidocarbonyl)oxy]-3-butene ((R)-4.3). Compound (R)-

4.3 was prepared from (R)-4.2 on a 5.75 mmol scale following the procedure described

for (S)-4.3 to provide 1.85 g (80%) of (R)-4.3 as colorless oil. All analytical data were the

24.6 same as those reported for (S)-4.3 except for [α]D +21.1 (c 1.04, CHCl3).

O

O N

TrO 4.4

4-Trityloxymethyl-3-oxa-1-aza-bicyclo[3.1.0]hexan-2-one (4.4). CAUTION: Reactions

carried out in pressure tubes are potentially explosive. While we did not experience any

problems the reactions should be carried out behind a protecting shield. A solution of the

azidoformate 4.3 (1.8 g, 4.5 mmol) in CH2Cl2 (70 mL) was placed in an Ace sealed tube

(catalog no. 8648-79). The tube was cooled to –78 °C, evacuated, sealed and heated to

110 °C for 14 h. Then the tube was allowed to cool to room temperature and the solvent

removed by rotary evaporation. The residue was washed with 5% EtOAc in hexanes and

a precipitate was formed that was isolated by filtration to afford 0.93 g of 4.4 (55%). Rf

1 0.24 (30% EtOAc in hexanes). H NMR (CDCl3, 300 MHz) δ 7.36-7.25 (m, 15H, Ar),

4.68 (t, J = 3.2 Hz, 1H, C(O)OCH), 3.56 (dd, J = 3.8, 10.5 Hz, 1H, TrOCHH), 3.25 (dd, J

= 3.4, 10.5 Hz, 1H, TrOCHH), 3.05 (t, J = 4.1 Hz, 1H, NCH), 2.56 (d, J = 4.2 Hz, 1H, 272

NCHH), 2.18 (d, J = 4.2, Hz, 1H, NCHH). Additional analytical data have been previously reported.248

O

O N

(S) (R) TrO H

(4R,5S)-4.4

(S)-4-Trityloxymethyl-3-oxa-1-aza-bicyclo[3.1.0]hexan-2-one ((4R,5S)-4.4).

Compound (4R,5S)-4.4 was prepared from (S)-4.3 on a 4.25 mmol scale following the procedure described for 4.4 to afford 0.87 g (55%) of (4R,5S)-4.4. Rf 0.24 (30% EtOAc

23.5 1 in hexanes). [α]D +20.9 (c 1.04, CHCl3). H NMR (CDCl3, 300 MHz) δ 7.46-7.25 (m,

15H, Ar), 4.68 (t, J = 3.2 Hz, 1H, C(O)OCH), 3.57 (dd, J = 3.7, 10.5 Hz, 1H, TrOCHH),

3.25 (dd, J = 3.4, 10.5 Hz, 1H, TrOCHH), 3.05 (t, J = 4.1 Hz, 1H, NCH), 2.56 (d, J = 4.3

13 Hz, 1H, NCHH), 2.18 (d, J = 4.1, Hz, 1H, NCHH). C NMR (CDCl3, 75 MHz) δ 166.9,

143.2, 128.5, 128.05, 127.3, 87.1, 63.9, 40.0, 34.7.

O

O N

(R) (S) TrO H (4S,5R)-4.4

(R)-4-Trityloxymethyl-3-oxa-1-aza-bicyclo[3.1.0]hexan-2-one ((4S,5R)-4.4).

Compound (4S,5R)-4.4 was prepared from (R)-4.3 on a 4.25 mmol scale following the procedure reported for (4R,5S)-4.4 to provide 0.86 g (55%) of (4S,5R)-4.4. All analytical 273

23.5 data were identical with those reported for (4R,5S)-4.4 except for [α]D -21.1 (c 1.04,

CHCl3).

O

O NH (R) (S) N Ph TrO N

(4R,5S)-4.22

(R)-4-(4-Phenyl-piperazin-1-ylmethyl)-(S)-5-trityloxymethyl-oxazolidin-2-one

((4R,5S)-4.22). To a solution of (4R,5S)-4.4 (0.78 g, 2.09 mmol) in CH2Cl2 (4 mL) was

added freshly distilled 4-phenyl-piperazine (0.35 mL, 2.3 mmol) neat. The reaction was

stirred at room temperature until complete conversion shown by TLC (typically 2 h). The solvent was removed under reduced pressure and the residue was purified by flash chromatography (30% EtOAc in CH2Cl2) to provide 0.96 g (86%) of (4R,5S)-4.22 as

23.3 white solid (mp 96.1-97.9). Rf 0.30 (30% EtOAc in CH2Cl2). [α]D +33.0 (c 1.02,

1 CHCl3). H NMR (CDCl3, 300 MHz) δ 7.49 (d, 6H, Ar), 7.36-7.24 (m, 11H, Ar), 6.91 (d,

3H, Ar), 5.69 (s, 1H, NH), 4.28 (q, J = 4.5, 9.3 Hz, 1H, C(O)OCH), 3.87 (q, J = 6, 13.3

Hz, 1H, NHCH), 3.49 (dd, J = 4.5, 10.3 Hz, 1H, TrOCHH), 3.25 (dd, J = 4.2, 10.3 Hz,

13 1H, TrOCHH), 3.12-3.02 (m, 4H, PhNCH2), 2.64-2.42 (m, 6H, NCH2). C NMR

(CDCl3, 75 MHz) δ 158.7, 151.1, 143.4, 129.1, 128.6, 128.0, 127.2, 119.9, 116.1, 86.9,

79.3, 63.8, 62.4, 53.4, 51.5, 49.0. Anal. Calcd for C34H35N3O3: C,76.52; H, 6.61; N, 7.87.

Found: C, 76.54; H, 6.21; N,7.48.

274

O

O NH (S) (R) N Ph TrO N

(4S,5R)-4.22

(S)-4-(4-Phenyl-piperazin-1-ylmethyl)-(R)-5-trityloxymethyl-oxazolidin-2-one

((4S,5R)-4.22). Compound (4S,5R)-4.22 was prepared from (4S,5R)-4.4 on a 1.99 mmol

scale following the procedure described for (4R,5S)-4.22 to provide 0.965 g (91%) of

(4S,5R)-4.22 as white solid. All analytical data were identical with those reported for

23.3 (4R,5S)-4.22 except for [α]D -32.1 (c 1.04, CHCl3).

O

O NH (R) (S) N Ph HO N

(4R,5S)-4.23

(S)-5-Hydroxymethyl-(R)-4-(4-phenyl-piperazin-1-ylmethyl)-oxazolidin-2-one

((4R,5S)-4.23). To a solution of (4R,5S)-4.22 (0.69 g, 1.3 mmol) in EtOAc (5 mL) was

added a solution of HCl in EtOAc (1.5M, 4.5 mL, 6.5 mmol). The mixture was allowed

to stir at room temperature for 10 min when was diluted with EtOAc (20 mL) and H2O

(30 mL) and the layers separated. Saturated aqueous NaHCO3 solution (10 mL) was

added to the aqueous layer and extracted with EtOAc (4 x 10 mL). The combined organic

layers were washed with H2O (10 mL), dried over MgSO4, filtered and concentrated by

rotary evaporation to afford 0.35 g (92%) of (4R,5S)-4.23 as white solid (mp 140.5-

28.2 141.9). No further purification was necessary. Rf = 0.16 (EtOAc). [α]D +54.5 (c 1.05, 275

1 CHCl3). H NMR (CDCl3, 300 MHz) δ 7.31-7.26 (m, 2H, Ar), 6.94-6.87 (m, 3H, Ar),

5.98 (s, 1H, NH), 4.33 (q, J = 4.3, 10.3 Hz, 1H, C(O)OCH), 3.96 (q, J = 6.7, 13.3 Hz, 1H,

NHCH), 3.87 (dd, J = 4.6, 12.0 Hz, 1H, HOCHH), 3.76 (dd, J = 4.0, 12.0 Hz, 1H,

HOCHH), 3.21 (m, 4H, PhNCH2), 2.70 (m, 4H, NCH2), 2.66 (dd, J = 7.2, 12.5 Hz, 1H,

13 NCHH), 2.53 (dd, J = 6.7, 12.5 Hz, 1H, NCHH). C NMR (CDCl3, 75 MHz) δ 157.3,

149.5, 127.7, 118.7, 114.8, 79.9, 61.3, 60.7, 52.3, 50.6, 47.7.

O

O NH (S) (R) N Ph HO N

(4S,5R)-4.23

(R)-5-Hydroxymethyl-(S)-4-(4-phenyl-piperazin-1-ylmethyl)-oxazolidin-2-one

((4S,5R)-4.23). Compound (4S,5R)-4.23 was prepared from (4S,5R)-4.22 on a 1.65 mmol

scale following the procedure described for (4R,5S)-4.23 to afford 0.445 g (93%) of

(4S,5R)-4.23. All analytical data were identical with those reported for (4R,5S)-4.23

28.1 except for [α]D -55.4 (c 1.05, CHCl3).

O

O NH (R) (S) N Ph O N

Ph O COB-30

276

Phenyl-acetic acid 2-oxo-(R)-4-(4-phenyl-piperazin-1-ylmethyl)-oxazolidin-(S)-5- ylmethyl ester (COB-30). To a solution of (4R,5S)-4.23 (64 mg, 0.22 mmol) in CH2Cl2

(2 mL) was added DMAP (3.3 mg, 0.027 mmol), Et3N (62 μL, 0.44 mmol) and

phenylacetyl chloride (36 μL, 0.27 mmol). The mixture was stirred at room temperature

for 5 h. The solvent was removed by rotary evaporation and the residue purified by flash

chromatography to provide 70 mg (77%) of COB-30 as colorless oil. Rf = 0.21 (50%

24.6 1 EtOAc in hexanes). [α]D +52.8 (c 1.04, CHCl3). H NMR (CDCl3, 300 MHz) δ7.30-

7.17 (m, 7H, Ar), 6.85-6.78 (m, 3H, Ar), 5.34 (s, 1H, NH), 4.31 (q, J = 4.6, 9.1 Hz, 1H,

C(O)OCH), 4.22 (d, J = 4.3 Hz, 2H, C(O)OCH2), 3.60 (s, 2H, PhCH2), 3.58-3.51 (m, 1H,

HNCH), 3.07 (t, J = 4.8 Hz, 4H, PhNCH2), 2.56-2.41 (m, 5H, NCH2 and NCHH), 2.30

13 (dd, J = 5.5, 12.5 Hz, 1H, NCHH). C NMR (CDCl3, 75 MHz) δ 171.2, 158.0, 151.3,

133.7, 129.5, 129.4, 128.9, 127.5, 120.2, 116.4, 77.6, 64.4, 62.3, 53.7, 51.7, 49.4, 41.4.

O

O NH (S) (R) N Ph O N

Ph O COB-31

Phenyl-acetic acid 2-oxo-(S)-4-(4-phenyl-piperazin-1-ylmethyl)-oxazolidin-(R)-5-

ylmethyl ester (COB-31). Compound COB-31 was prepared from (4S,5R)-4.23 on a 022

mmol scale following the procedure described for COB-30 to afford 74 mg (81%) of

COB-31. All analytical data were identical with those reported for COB-30 except for

24.6 [α]D -51.7 (c 1.00, CHCl3). 277

O O

O NH (R) (S) N Ph O N HN O COB-32

(4-Acetyl-phenyl)-carbamic acid 2-oxo-(R)-4-(4-phenyl-piperazin-1-ylmethyl)-

oxazolidin-(S)-5-ylmethyl ester (COB-32). To a solution of (4R,5S)-4.23 (0.12 g, 0.41 mmol) in CH2Cl2 (4 mL) were added DMAP (6.1 mg, 0.049 mmol), Et3N (115 μL, 0.82

mmol) and 4-acetyl-phenyl-isocyanate (82 mg, 0.49 mmol). The mixture was stirred at

room temperature for 2 h. The solvent was removed by rotary evaporation and the residue

purified by flash chromatography to provide 0.153 g (82%) of COB-32 as white solid

21.8 1 (mp 102.3-103.9). Rf 0.3 (95% EtOAc in hexanes). [α]D +42.4 (c 1.02, CHCl3). H

NMR (CDCl3, 300 MHz) δ7.74 (d, J = 8.5 Hz, 2H, Ar), 7.29 (d, J = 8.5 Hz, 2H, Ar),

7.09-7.04 (m, 2H, Ar), 6.96 (s, 1H, NH), 6.72-6.65 (m, 3H, Ar), 5.26 (s, 1H, NH), 4.32

(q, J = 4.5, 8.0 Hz, 1H, C(O)OCH), 4.20 (m, 2H, C(O)OCH2), 3.66 (q, J = 5.6, 13.0 Hz,

1H, HNCH), 2.98 (t, J = 4.6 Hz, 4H, PhNCH2), 2.54-2.30 (m, 6H, NCH2), 2.37 (s, 3H,

13 COCH3). C NMR (CDCl3, 75 MHz) δ 196.9, 158.4, 152.7, 151.3, 142.2, 132.9, 130.1,

129.4, 120.3, 118.1, 116.4, 78.3, 65.2, 62.4, 53.9, 51.7, 49.4, 26.5. Anal. Calcd for

C24H28N4O5: C,63.70; H, 6.24; N, 12.38. Found: C, 63.71; H, 6.23; N,12.21.

278

O O

O NH (S) (R) N Ph O N HN O COB-33

(4-Acetyl-phenyl)-carbamic acid 2-oxo-(S)-4-(4-phenyl-piperazin-1-ylmethyl)-

oxazolidin-(R)-5-ylmethyl ester (COB-33). Compound COB-33 was prepared from

(4S,5R)-4.23 on a 0.41 mmol scale following the procedure described for COB-32 to

provide 0.15 g (80%) of COB-33 as white solid. All analytical data were identical with

22.1 those reported for COB-32 except for [α]D –41.7 (c 1.05, CHCl3).

OH HO

4.32

trans-But-2-ene-1,4-diol (4.32). To a cold (-78 °C) suspension of LiAlH4 (6 g, 160

mmol) in THF (150 mL) was cannulated a solution of recrystalized 1,4-dihydroxy-3- butyn (6.9 g, 80 mmol) in THF (50 mL) with vigorous stirring. The reaction mixture was allowed to warm to room temperature and then was heated to reflux for 18 h. After this time the reaction mixture was cooled (0 °C) and quenched with saturated aqueous solution of NH4Cl (30 mL). The mixture was filtered through a short pad of celite and

washed thoroughly with Et2O. The filtrate was concentrated by rotary evaporation. The

residue was purified by flash chromatography (95% EtOAc in hexanes) to afford 4.7 g

(67%) of 4.32 as colorless oil. Rf 0.24 (95% EtOAc in hexanes). tR = 2.3 min (40 °C to 279

1 290 °C at 25 °/min, 12 min). H NMR (Acetone-d6, 300 MHz) δ5.80-5.78 (m, 2H, =CH),

4.07-4.04 (m, 4H, OCH2), 3.66 (t, J = 5.5 Hz, 2H, OH). Additional analytical data have been previously reported.283

O OH HO

4.35

(3-Hydroxymethyl-oxiranyl)-methanol (4.35). To a cold (0 °C) solution of diol 4.32 (1 g, 11.3 mmol) in CH3CN (25 mL) was added m-chloroperbenzoic acid (3 g, 14.66 mmol).

The reaction was allowed to set in the refrigerator (0-4 °C) for 4 days. The benzoic acid

formed in this time was removed by filtration and the filtrate was washed with CH2Cl2 (2 x 20 mL) to remove the unreacted m-chloroperbenzoic acid and the remaining chlorobenzoic acid. The product was isolated from the aqueous solution by lyophilization to provide 1.06 g (90%) of 4.35. Rf 0.24 (95% EtOAc in hexanes). tR = 2.5 min (40 °C to

1 100 °C at 10 °/min then to 250 °C at 25 °/min, 12 min). H NMR (Acetone-d6, 300 MHz)

13 δ3.82-3.72 (m, 4H, OCH2), 3.54-3.48 (m, 2H, OCH), 2.99-2.96 (m, 2H, OH). C NMR

(Acetone-d6, 75 MHz) δ 62.9, 56.9.

N3 OH HO OH 4.36

280

2-Azido-2-(2,2-dimethyl-[1,3]dioxolan-4-yl)-ethanol (4.36). To a solution of epoxide

4.35 (0.52 g, 5 mmol) in EtOH (8 mL) at room temperature were added successively

NH4Cl (0.615 g, 11.5 mmol) and NaN3 (0.747 g, 11.5 mmol). The mixture was heated to

reflux for 17 h. After cooling, the reaction mixture was diluted with EtOAc (20 mL) and

then filtered. The filtrate was concentrated by rotary evaporation to provide 0.66 g (90%)

of 4.36 as yellowish oil. Rf 0.3 (EtOAc). tR = 1.1 min (40 °C to 100 °C at 10 °/min then to

1 250 °C at 25 °/min, 12 min). H NMR (D2O, 300 MHz) δ3.93 (dd, J = 3, 11 Hz, 1H),

13 3.81-3.62 (m, 5H). C NMR (D2O, 75 MHz) δ 70.4, 64.2, 61.9, 60.5.

N3 OH O O

4.34

2-Azido-2-(2,2-dimethyl-[1,3]dioxolan-4-yl)-ethanol (4.34). To a solution of triol 4.36

(0.6 g, 4 mmol) in dry acetone (110 mL/ mmol) was added p-toluenesulfonic acid (0.3 g,

1.6 mmol). The reaction mixture was stirred at room temperature for 18 h. K2CO3 (0.77 g, 5.6 mmol) was added to the reaction flask and stirred another 2 h. Then the reaction mixture was filtered and the filtrate was concentrated by rotary evaporation. The residue was purified by flash chromatography (20% EtOAc in hexanes) to provide 0.5 g (67%) of

4.34 as colorless oil. Rf 0.18 (20% EtOAc in hexanes). tR = 3.13 min (75 °C to 300 °C at

1 25 °/min, 12 min). H NMR (CDCl3, 300 MHz) δ 4.14-4.04 (m, 2H, OCH and OCHH),

3.92 (dd, J = 5, 8 Hz, 1H, OCHH), 3.89-3.81 (m, 1H, HOCHH), 3.68 (dd, J = 6, 12 Hz, 281

1H, HOCHH), 3.63-3.57 (m, 1H, N3CH), 2.61 (t, 1H, OH), 1.44 (s, 3H, CH3), 1.34 (s,

13 3H, CH3). C NMR (CDCl3, 75 MHz) δ 109.9, 75.5, 66.6, 64.8, 62.7, 26.5, 25.2.

N3 OTs O O

4.37

2-Azido-2-(2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl-4-methylbenzenesulfonate (4.37).

To a solution of acetonide 4.34 (0.5 g, 2.67 mmol) in CH2Cl2 (5 mL) was added

toluenesulfonyl chloride (0.61 g, 3.2 mmol). The mixture was cooled to 0 °C and

pyridine (0.65 mL, 8.01 mmol) was added. The reaction mixture was allowed to warm up

to room temperature and stirred overnight. Then it was diluted with EtOAc and washed

with saturated aqueous NaHCO3 and brine. The organic phase was dried over MgSO4, filtered and the solvent removed by rotary evaporation. The residue was purified by flash chromatography (10% EtOAc in hexanes) to afford 0.64 g (70%) of 4.37 as colorless oil.

1 Rf 0.35 (20% EtOAc in hexanes). tR = 7.57 min (75 °C to 300 °C at 25 °/min, 10 min). H

NMR (CDCl3, 300 MHz) δ 7.83 (d, J = 8 Hz, 2H, Ph), 7.38 (d, J = 8 Hz, 2H, Ph), 4.33

(dd, J = 3, 10.7 Hz, 1H, OCH), 4.08-4.02 (m, 2H, OCH2), 3.99-3.87 (m, 2H, TsOCH2),

13 3.71-3.65 (m, 1H, N3CH), 2.47 (s, 3H, PhCH3), 1.40 (s, 3H, CH3), 1.31 (s, 3H, CH3). C

NMR (CDCl3, 75 MHz) δ 145.5, 132.6, 130.2, 128.2, 110.4, 74.4, 69.4, 66.7, 62.2, 26.7,

25.1, 21.9.

282

Ph N3 N N O O 4.38

1-Azido-1-(2,2-dimethyl-[1,3]dioxolan-4-yl)-2-(4-phenyl-piperazine-1-yl)-ethane

(4.38). To a solution of 4.37 (0.51 g, 1.5 mmol) in EtOH (5 mL) was added K2CO3 (0.41 g, 3 mmol) followed by 1-phenyl-piperazine (0.25 mL, 1.65 mmol). The reaction mixture was heated to reflux overnight. After cooling to room temperature the reaction mixture was transferred in a separatory funnel, diluted with EtOAc and water. The aqueous layer was extracted with EtOAc (3 x 10 mL) and the combined organic layers were dried over

MgSO4, filtered and the solvent removed by rotary evaporation. The residue was purified

by means of flash chromatography (10% EtOAc in toluene) to provide 0.29 g (60%) of

4.38 as colorless oil. Rf 0.25 (10% EtOAc in toluene). tR = 8.09 min (75 °C to 300 °C at

-1 1 25 °/min, 10 min). IR 2270 cm (s, N3). H NMR (CDCl3, 300 MHz) δ 7.31-7.26 (m, 2H,

Ph), 6.96-6.85 (m, 3H, Ph), 4.11-4.04 (m, 2H, OCH2), 3.97-3.91 (m, 1H, OCH), 3.77-

3.71 (m, 1H, N3CH), 3.23-3.18 (m, 4H, PhN(CH2)2), 2.79-2.72 (m, 2H, NCH2), 2.70-2.63

(m, 3H, NCH2 and NCHH), 2.56 (dd, J = 9, 13.3 Hz, 1H, NCHH), 1.49 (s, 3H, CH3),

13 1.38 (s, 3H, CH3). C NMR (CDCl3, 75 MHz) δ 151.4, 129.3, 119.9, 116.3, 109.9, 76.4,

66.5, 61.0, 59.7, 53.8, 49.4, 26.6, 25.4.

Ph NH2 N N O O 4.33 283

1-Amino-1-(2,2-dimethyl-[1,3]dioxolan-4yl)-2-(4-phenyl-piperazine-1-yl)-ethane

(4.33). To a solution of 4.38 (0.7 g, 2.11 mmol) in EtOAc (4 mL) was added 10% Pd/C

(w/w) (0.225 g, 0.211 mmol). The reaction flask was equipped with a three-way stopper and a balloon with H2 was attached. The air from the flask was evacuated three times

using and aspirator and then the reaction mixture was stirred under hydrogen for 24 h.

The reaction mixture was passed through a short pad of celite. The solvent was then

removed by rotary evaporation to provide 0.614 g (95%) of 4.33 as a colorless liquid that

solidified in the refrigerator. tR = 7.94 min (75 °C to 300 °C at 25 °/min, 10 min). IR

-1 -1 1 3300 cm (2 bands, N-H stretch for primary amines), 1600 cm . H NMR (CDCl3, 300

MHz) δ 7.21-7.16 (m, 2H, Ph), 6.86-6.75 (m, 3H, Ph), 3.98-3.80 (m, 3H, OCH and

OCH2), 3.14-3.05 (m, 5H, NH2CH and PhN(CH2)2), 2.68-2.61 (m, 2H, NCH2), 2.50-2.44

(m, 2H, NCH2), 2.41 (dd, J = 4, 12,4 Hz, 1H, NH2CHCHHN), 2.22 (dd, J = 10, 12.4 Hz,

13 1H, NH2CHCHHN), 1.53 (br, 2H, NH2), 1.36 (s, 3H, CH3), 1.29 (s, 3H, CH3). C NMR

(CDCl3, 75 MHz) δ 151.5, 129.3, 119.9, 116.2, 109.1, 78.7, 66.1, 61.6, 53.8, 50.0, 49.4,

26.8, 25.5.

O Ph PhO NH N N O O 4.39

284

N-[1-(2,2-Dimethyl-[1,3]dioxolan-4-yl)-2-(4-phenyl-piperazine-1-yl)-ethyl]- benzamide (4.39). To a solution of 4.33 (0.1 g, 0.33 mmol) in CH2Cl2 (0.5 mL) was added DMAP (4 mg, 0.033 mmol) followed by Et3N (0.276 mL, 1.98 mmol). The

mixture was cooled to 0 °C and phenyl chloroformate (63 µL, 0.5 mmol) was added neat.

The reaction mixture was allowed to warm to room temperature. After 15 minutes of

stirring the reaction mixture was diluted with EtOAc and transferred in a separatory

funnel. The reaction mixture was washed with H2O, saturated aqueous NaHCO3 and

brine. The organic layer was dried over MgSO4, filtered and concentrated down. The

solid residue was washed with hexanes and filtered to afford 0.11 g (78%) of 4.39. No

further purification was necessary. Rf 0.3 (30% EtOAc in hexanes). tR = 8.1 min (75 °C

1 to 300 °C at 25 °/min, 12 min). H NMR (CDCl3, 300 MHz) δ 7.30-7.02 (m, 7H, Ph),

6.86-6.75 (m, 3H, Ph), 5.38 (d, 1H, NH), 4.22-4.16 (m, 1H, OCH), 4.03 (dd, J = 6, 8.7

Hz, 1H, OCHH), 3.92 (dd, J = 6, 8.7 Hz, 1H, OCHH), 3.87-3.78 (m, 1H, NHCH), 3.13-

3.10 (m, 4H, PhN(CH2)2), 2.69-2.54 (m, 6H, N(CH2)3), 1.38 (s, 3H, CH3), 1.29 (s, 3H,

13 CH3). C NMR (CDCl3, 75 MHz) δ 154.8, 151.2, 150.9, 129.3, 129.1, 125.4, 121.6,

119.7, 116.0, 109.6, 76.8, 66.8, 57.7, 53.7, 51.2, 49.3, 29.7, 26.4, 25.1.

O

O NH

HO N N Ph cis-4.23

5-Hydroxymethyl-4-(4-phenyl-piperazin-1-ylmethyl)-oxazolidin-2-one (cis-4.23). The

acetonide 4.39 (1.4 g, 2.62 mmol) was placed in a flask followed by oxalic acid (1.65 g, 285

18.34 mmol). H2O (16.5 mL), THF (33 mL) and 12N HCl (0.2 mL) were added and the

reaction mixture was stirred at 60 °C for 8 h. The reaction mixture was poured into a 5%

aqueous K2CO3 and stirred for 30 minutes and then was extracted with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and the solvent removed by

rotary evaporation to provide 0.5 g (66%) of cis-4.23. No further purification was

1 necessary. Rf 0.22 (95% EtOAc in hexanes). H NMR (CDCl3, 300 MHz) δ 7.27-7.22 (m,

2H, Ph), 6.90-6.84 (m, 3H, Ph), 5.73 (s, 1H, NH), 5.36 (br s, 1H, OH), 4.81 (ddd, J = 5,

7.7, 9 Hz, 1H, OCH), 4.16 (ddd, J = 4, 7.7, 11 Hz, 1H, NHCH), 3.90-3.77 (m, 2H,

HOCH2), 3.25-3.12 (m, 4H, PhN(CH2)2), 2.90 (dd, J = 11, 13 Hz, 1H, NCHH), 2.83-2.76

13 (m, 2H, NCH2), 2.69-2.62 (m, 2H, NCH2), 2.42 (dd, J = 4, 13 Hz, 1H, NCHH). C NMR

(CDCl3, 75 MHz) δ 158.7, 150.9, 129.4, 120.7, 116.6, 78.3, 59.3, 57.8, 53.7, 51.2, 49.2.

O

O NH

O N N Ph

Ph O COB-34

Phenyl-acetic acid 2-oxo-4-(4-phenyl-piperazin-1-ylmethyl)-oxazolidin-5-ylmethyl

ester (COB-34). To a solution of cis-4.23 (0.1 g, 0.34 mmol) in CH2Cl2 (1.5 mL) was added DMAP (5 mg, 0.04 mmol), Et3N (0.2 mL, 1.37 mmol) and phenylacetyl chloride

(51 μL, 0.41 mmol). The mixture was stirred at room temperature for 5 h. The solvent was removed by rotary evaporation and the residue purified by flash chromatography

(reverse phase column, 45% MeCN in H2O) to provide 50 mg (36%) of COB-34 as 286

1 colorless oil. Rf 0.21 (50% EtOAc in hexanes). H NMR (CDCl3, 300 MHz) δ 7.29-7.16

(m, 7H, Ar), 6.84-6.77 (m, 3H, Ar), 5.56 (s, 1H, NH), 4.73 (ddd, J = 4.5, 6.5, 8 Hz, 1H,

C(O)OCH), 4.35 (dd, J = 4.5, 12 Hz, 1H, C(O)OCHH), 4.19 (dd, J = 6.5, 12 Hz, 1H,

C(O)OCHH), 3.94 (ddd, J = 5, 8, 9.5 Hz, 1H, NHCH), 3.60 (s, 2H, PhCH2), 3.08-3.05

(m, 4H, PhN(CH2)), 2.58-2.51 (m, 2H, NCH2), 2.41-2.34 (m, 3H, NCH2 and NCHH),

13 2.25 (dd, J = 5, 12 Hz, 1H, NCHH). C NMR (CDCl3, 75 MHz) δ 171.1, 158.3, 151.2,

133.6, 129.5, 129.3, 128.9, 127.5, 120.2, 116.3, 75.4, 61.9, 57.5, 53.6, 51.2, 49.3, 41.4.

O O

O NH

O N N Ph HN O COB-35

(4-Acetyl-phenyl)-carbamic acid 2-oxo-4-(4-phenyl-piperazin-1-ylmethyl)-

oxazolidin-5-ylmethyl ester (COB-35). To a solution of cis-4.23 (0.2 g, 0.68 mmol) in

CH2Cl2 (3 mL) were added DMAP (10 mg, 0.08 mmol), Et3N (0.4 mL, 2.74 mmol) and

4-acetyl-phenyl-isocyanate (0.133 g, 0.82 mmol). The mixture was stirred at room

temperature for 2 h. The solvent was removed by rotary evaporation and the residue

purified by flash chromatography to provide 0.14 g (48%) of COB-35 as a white solid

1 (mp 102.5-104.0). Rf 0.23 (80% EtOAc in hexanes). H NMR (CD2Cl2, 300 MHz) δ7.92

(dt, J = 8.8 Hz, 2H, Ar), 7.50 (dt, J = 8.8 Hz, 2H, Ar), 7.26-7.21 (m, 3H, Ar and NH),

6.92-6.89 (dd, 2H, Ar), 6.84 (t, 1H, Ar), 5.54 (s, 1H, NH), 4.90 (ddd, J = 3.5, 7.5, 11.5

Hz, 1H, C(O)OCH), 4.56 (dd, J = 3.5, 12 Hz, 1H, OCHH), 4.35 (dd, J = 7.5, 12.0 Hz, 287

1H, OCHH), 4.16 (ddd, J = 4.8, 10.0, 12.0 Hz, 1H, C(O)NHCH), 3.22-3.12 (m, 4H,

13 PhNCH2), 2.77-2.70 (m, 2H, NCH2), 2.67-2.50 (m, 7H, C(O)CH3 and N(CH2)2). C

NMR (CD2Cl2, 75 MHz) δ 197.0, 158.5, 152.9, 151.8, 142.6, 133.1, 130.2, 129.6, 120.2,

118.3, 116.5, 76.2, 63.2, 57.9, 51.5, 49.7, 30.3, 26.8. 288

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APPENDIX A: EXPANDED 1H NMR SPECTRUM OF (S,S)-2.20

O (S) Ph (S) N Ph H

CO2tBu (S,S)-2.20

316

APPENDIX B: EXPANDED 1H NMR SPECTRUM OF (S,S)-2.21

(S) Ph

O N O O

N (S) Ph H

(S,S)-2.21

317

APPENDIX C: EXPANDED 1H NMR SPECTRUM OF (R,R)-2.29

O (R) OCH3 (R) O Ph CF3 N

Ph (R,R)-2.29

318

APPENDIX D: EXPANDED 1H NMR SPECTRUM OF (S,S)-4.18

O CF3 O (S) OMe Ph TrO (S) (S,S)-4.18

319

APPENDIX E: 19F NMR SPECTRUM OF (R,S)-4.18

O CF3 O OMe (S) Ph TrO (R) (R,S)-4.18

320

APPENDIX F: 19F NMR SPECTRUM OF (S,S)-4.18

O CF3 O (S) OMe Ph TrO (S) (S,S)-4.18