Chapter 1: Synthesis of Sidechain Functionalized Polyamines and Study of Their RNA-Binding Properties

NORTHWESTERN UNIVERSITY

Part 1: Synthesis of Side-Chain Functionalized Polyamines and Study of their RNA-Binding Properties

Part 2: Synthesis and Evaluation of Heterocycle-Based Selective Inhibitors of Neuronal Nitric Oxide Synthase with Improved Bioavailability

A DISSERTATION

SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

for the degree

DOCTOR OF PHILOSOPHY

Field of Chemistry

Graham R. Lawton

EVANSTON, ILLINOIS

JUNE 2007

ABSTRACT

Part 1: Synthesis of Side-Chain Functionalized Polyamines and Study of their RNA-Binding Properties

Part 2: Synthesis and Evaluation of Heterocycle-Based Selective Inhibitors of Neuronal Nitric Oxide Synthase with Improved Bioavailability

Graham R. Lawton

Part 1. The complex folded structures associated with RNA allow for specific protein-RNA interactions and also create binding sites for small molecules. Developing organic molecules that can bind RNA with high affinity and specificity is a challenge that must be overcome for RNA to be considered a viable drug target. Polyamines with different side chains were synthesized to test for binding affinity and specificity to TAR and RRE of HIV. Binding interactions between polyamines and RNAs were examined using fluorescence assays and two foot-printing assays based on terbium-induced cleavage and magnesium-catalyzed cleavage at high pH. Binding constants and specificity were highly dependent on the side chains of the polyamines, demonstrating that this class of molecules is a promising starting point for the development of highly selective RNA binding ligands.

Part 2. The overproduction of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS) has been implicated in a variety of neurological diseases, including Parkinson’s and neuronal damage due to hypoxic conditions such as stroke. Inhibition of nNOS could have therapeutic benefit, but must be achieved without inhibition of the endothelial isoform (eNOS), as that would

lead to hypertension. Using computer modeling of drug-like fragments in the crystal structures of

nNOS and eNOS, a class of highly selective and potent nNOS inhibitors was discovered.

Modification of the structures of these lead compounds to optimize for pharmacological properties without sacrificing potency and selectivity was carried out. Replacing a 2- aminopyridine group in the leads with aminothiazoles resulted in a dramatic loss in potency. The replacement of secondary amines with ether and amide linkages generally reduced potency; however, in one case an ether-containing compound was as potent and selective as the best lead compound. Brain uptake studies in mice proved that the modification did have a beneficial effect

on the amount of compound that crosses the blood brain barrier. The neutralization of other

secondary amines in the molecule with amides and carbamates was carried out to investigate whether a prodrug approach could further increase brain uptake. In all cases, however, the capping of the secondary amine had no beneficial effect on brain uptake.

ACKNOWLEDEMENTS

I would like to express my deepest gratitude to all those who helped me throughout my graduate career. I have been extremely lucky in my colleagues, mentors and collaborators without whom this work would not have been possible.

I am grateful to Professor Richard B. Silverman for taking me into his group in 2004 and giving me the chance to carry out interesting chemistry on an exciting project. I appreciate the freedom he gave me to design my own syntheses, explore new avenues in the project, and develop into an independent researcher. I wish to thank all of the members of the Silverman group, past and present, for creating an enjoyable atmosphere in which to carry out research and for their assistance and ideas. I especially need to thank Dr. Haitao Ji for the use of his molecular modeling diagrams and for his assistance with my syntheses, Michael Clift for the work he did on ether formation reactions, and Ian Pulford for help with the aminoimidazole synthesis.

I would like to thank Dr. Daniel H. Appella for his enthusiasm, encouragement and guidance on the polyamine project. I want to thank all of the members of the Appella lab from

2002-2004, in particular Dr. Michael C. Myers, who gave me many practical tips on organic synthesis.

I wish to thank Professor Olke Uhlenbeck for allowing me to carry out the RNA binding assays in his laboratory, providing essential equipment and supplies, and for offering advice and encouragement. I am grateful to all the members of his laboratory who went out of their way to help me with my project, especially Dr. Richard P. Fahlman and Dr. Fedor (Ted) V. Karginov, whose ideas and critiques were invaluable in the design of my RNA assays, interpretation of the results and preparation of the corresponding manuscript.

My warmest thanks go to Professor D. Martin Watterson for providing me with a place to

carry out research essential to my project. In addition, he offered helpful advice and taught me a

great deal about pharmacology and drug discovery. I apologize to his group members for any

inconvenience I may have caused them while conducting research in their lab, and am grateful to them all for their assistance, especially Dr. Hantamalala Ralay Ranaivo, who carried out the majority of the animal administrations, and Laura K. Wing, who helped me design and interpret the in vitro metabolism assays.

I am grateful to Professor Michael J. Avram and Lynn Luong for carrying out the LCMS analysis of my biological samples. They were both extremely helpful and accommodating and I enjoyed working with them and learning from them.

My deepest thanks go to all those who have offered me emotional support and have encouraged me and made me the person I am today. I want to thank my Mum and Dad and all my family for their love and encouragement over the years, and for supporting my decision to come to Chicago. I want to thank my wife’s family, particularly Howard and Beverly, for providing me with a loving support network here in the U.S. I want to thank Dr. Benjamin

Brooks for igniting my interest in chemistry, all my friends, Ozzie and Kenny, and all others who have been a part of my life over the last 5 years. Finally, and most importantly, I need to thank my beloved wife, Victoria Lawton, for her love, friendship and support, for helping to keep me sane, and for being the best companion a man could want.

TABLE OF CONTENTS

Abstract...... 3

Acknowledgements...... 5

Table of Contents...... 7

List of Figures...... 10

List of Schemes...... 15

List of Tables ...... 17

Chapter 1: Synthesis of Sidechain Functionalized Polyamines and Study of their RNA-Binding Properties...... 18

1.1 RNA as a drug target ...... 19

1.2 Antibiotics that bind ribosomal RNA ...... 22

1.3 Binding of small molecules to other RNAs...... 23

1.4 Polyamines as RNA-binding small molecules...... 29

1.5 Analysis of RNA binding ...... 30

1.6 Synthesis ...... 34 a) Reduction of resin-bound tripeptides using borane ...... 34 b) Reductive amination...... 37

1.7 Fluorescence-based assay ...... 44

1.8 Footprinting assays ...... 48

1.9 Test for epimerization during reductive amination...... 53

1.10 Synthesis of alternative aldehydes...... 57 a) Fmoc protected γ-amino aldehydes ...... 57 b) Trityl protection of glutamine and citrullene sidechains...... 59

1.11 Summary...... 61

1.12 Experimental procedures ...... 62

Chapter 2: Synthesis and Evaluation of 2- and 4-Aminothiazole-based Inhibitors of Neuronal Nitric Oxide Synthase ...... 89

2.1 Biological role of nitric oxide...... 90

2.2 The nitric oxide synthase family...... 91

2.3 NO and disease ...... 92

2.4 Selective inhibitors of nNOS ...... 94

2.5 Rationale for the synthesis of aminothiazole-based inhibitors ...... 103

2.6 Synthesis of aminothiazoles...... 104

2.7 Results and discussion ...... 114

2.8 Synthesis of aminoimidazoles...... 116

2.9 Summary...... 118

2.10 Experimental procedures ...... 118

Chapter 3: Synthesis and Evaluation of Ether and Amide Analogues of 2-Aminopyridine-based nNOS Inhibitors...... 157

3.1 Introduction...... 158

3.2 Synthesis of trans-ether, III-1...... 161

3.3 Solving the Mitsunobu problem ...... 164

3.4 Synthesis of ethers and amides ...... 167

3.5 Results and discussion ...... 171

3.6 Summary...... 173

3.7 Experimental procedures ...... 174

Chapter 4: Increasing the Bioavailability of nNOS Inhibitors using a Prodrug Strategy ...... 203

4.1 Introduction...... 204

4.2 Azides as prodrugs for primary amines ...... 213

4.3 Microsomal stability of ether III-2...... 224

4.4 Brain uptake studies on III-2 and II-6...... 226

4.5 The effect of removing a charge on brain uptake ...... 237

4.6 Summary, conclusions and future directions...... 254

4.7 Experimental procedures ...... 258

References for Chapter 1 ...... 278

References for Chapter 2 ...... 286

References for Chapter 3 ...... 294

References for Chapter 4 ...... 294

Appendix 1...... 300

LIST OF FIGURES

1.1 Thiamine pyrophosphate, I-1, and flavine mononucleotide, I-2 ...... 19

1.2 Secondary structures of (A) TAR, and (B) RRE-IIB of HIV. Numbering refers to the relative position of the base in the intact RNA...... 21

1.3 Examples of antibiotics that are known to target the ribosome...... 22

1.4 Modifications to neomycin that improve its affinity for RNA ...... 25

1.5 Examples of synthetically designed ligands that combine intercalating ability with positive charge...... 26

2+ 1.6 Λ-[Ru(bpy)2Eilatin] , a metal-ligand complex that was found to bind to RRE and displace Rev ...... 26

1.7 A peptide that binds to TAR, selected via a combinatorial approach...... 28

1.8 A cyclic peptide, I-19, and a peptide-acridine conjugate, I-20. Both were selected from libraries for their RNA binding properties ...... 28

1.9 General structure of a polyamine trimer...... 29

1.10 Secondary structures of (A) TAR and (B) RRE showing the positions of the 2-aminopurine modifications. (C) Wobble base pairing between 2AP and uracil...... 45

1.11 Graphs of fluorescence against polyamine concentration ...... 46

1.12 Mechanism of base- / metal ion-catalyzed intramolecular strand cleavage ...... 50

1.13 Example of PAGE displaying magnesium catalyzed cleavage of TAR in the presence of increasing concentrations of YYY, and graph of normalized fraction cleaved at the bulge vs. polyamine concentration...... 52

1.14 Graphs of normalized fraction cleaved vs. polyamine concentration for (A) YYY vs. TAR, bulge region (♦), and loop region (), and (B) YYY vs TAR bulge region (♦), RRE bulge region (), and RRE loop region (σ)...... 53

1.15 Mechanism of epimerization of resin-bound imine...... 55

1.16 PAGE displaying cleavage products from (A) “In-line” cleavage of RRE, and (B) terbium catalyzed cleavage of TAR, with increasing concentrations of YYY...... 83

1.17 Graphs of normalized fraction of RNA cleaved vs polyamine concentration, displaying curve fitting ...... 83

2.1 NO produced by nNOS triggers the formation of cGMP, a secondary messenger...... 90

2.2 The formation of NO from L-arginine...... 92

2.3 Strucutres of L-nitroarginine-based inhibitors of nNOS with their inhibition constants against nNOS. “n/e” and “n/i” are the selectivities for nNOS over eNOS and iNOS, respectively ...... 95

2.4 Overlay of the conformations of II-1 bound to the active sites of nNOS (blue) and eNOS (purple). Key residues are highlighted for nNOS (pink) and eNOS (green)...... 96

2.5 Overlay of the conformations of II-1 bound to the active sites of nNOS wild type (blue) and nNOS D597N (purple)...... 97

2.6 Overlay of the conformations of II-1 bound to the active sites of nNOS wild type (blue) and eNOS N368D (purple)...... 98

2.7 Molecular modeling diagrams of II-2 and II-4 bound to the active site of nNOS, and the structure of II-4 ...... 99

2.8 Structures of potent and selective aminopyridine-based nNOS inhibitors...... 100

2.9 (A) Crystal structure of II-5 bound to the active site of nNOS showing the important interactions with key residues. (B) Predicted conformation of II-5 in the active site of nNOS (gold) versus the actual conformation (blue) ...... 102

2.10 Structures of desired 2- and 4-aminothiazole-based nNOS inhibitors ...... 104

2.11 Tautomerization, hydrolysis and decomposition of 4-aminothiazoles in water ...... 111

2.12 Protonation state of 2-aminoimidazoles at pH 7 and the structure of a potential aminoimidazole-based nNOS inhibitor...... 116

3.1 Structures of potent nNOS inhibitor, II-6, lead compound II-4, and its trans ether analog III-1, a proposed nNOS inhibitor...... 158

3.2 Modeling diagram of III-1 (green) in the active site of nNOS ...... 159

3.3 Structures of potential nNOS inhibitors that have fewer basic groups than the lead compound, II-6...... 160

4.1 Prodrugs of the anti-HIV therapeutic AZT, designed for increased brain uptake...... 208

4.2 The conversion of brain-penetrating prodrugs to the active drugs, 2’F-ara-ddI (IV-4) and ddI (IV-6)...... 208

4.3 Dihydropyridine-based chemical delivery system...... 209

4.4 Structure of IV-9, a pivaloyloxyethylcarbamate prodrug of the potent and selective nNOS inhibitor II-3 ...... 210

4.5 Gabapentin (IV-10) and an (acyloxy)alkyl carbamate prodrug of gabapentin...... 211

4.6 Structures of potent and selective nNOS inhibitors, III-2 and II-6...... 212

4.7 Structures of AZT (IV-12) and its metabolite (IV-13)...... 213

4.8 Structures of azide prodrug IV-14, and its primary amine analog IV-15...... 214

4.9 Structures of prodrugs IV-9 and IV-14 ...... 217

4.10 Incubation of IV-9 and IV-14 in fresh mouse plasma. The area under the peak was integrated and the value was normalized based on the integration at 0 min. Two diastereoisomers of IV-9 are shown separately (IV-9 A and IV-9 B)...... 218

4.11 Formation of active drug II-3 from prodrug IV-9 during incubation in fresh mouse plasma...... 219

4.12 Metabolism of IV-9 and IV-14 in brain homogenate...... 220

4.13 Metabolism of IV-9, IV-14 and the prescription CNS drug minaprine by liver microsomes at 37 °C...... 222

4.14 Structures of 2’-F-araddA (IV-20) and a potential azide prodrug (IV-19) ...... 223

4.15 Structure of potent and selective nNOS inhibitor, III-2...... 224

4.16 Metabolic stability of III-2 with microsomes at 37 °C. The experiment was performed in the presence (III-2 red) and absence (III-2 ox) of additional NADPH. Minaprine concentration at 20 min was >0 but was not quantifiable ...... 225

4.17 Standard curve for the quantification of III-2 in plasma...... 228

4.18 Standard curve for the quantification of III-2 in brain tissue...... 229

4.19 Concentration of III-2 in plasma after an i.p. dose of 3.7 mg / kg. Error bars show standard error mean ...... 230

4.20 Concentration of III-2 in the brain after an i.p. dose of 3.7 mg / kg. Error bars show standard error mean ...... 231

4.21 Concentration of II-6 in plasma after a dose of 3.7 mg / kg in mice. Error bars show standard error mean ...... 234

4.22 Concentration of II-6 in the brain after a dose of 3.7 mg / kg in mice. Error bars show standard error mean...... 235

4.23 Comparison of the averaged brain concentrations of III-2 and II-6 when both compounds were administered at 3.7 mg / kg. Error bars show standard error mean ...... 236

4.24 Structure of IV-22, an acetylated version of III-2...... 240

4.25 Concentration of IV-22 in plasma after a dose of 4.1 mg / kg. Error bars show standard error mean ...... 242

4.26 Concentration of IV-22 in brain after a dose of 4.1 mg / kg. Error bars show standard error mean ...... 243

4.27 Comparison of the brain concentrations of III-2 and IV-22 after administration of 3.7 mg / kg and 4.1 mg / kg respectively. Error bars show standard error mean ...... 244

4.28 Structures of carbamates IV-26 and IV-27...... 245

4.29 Plasma concentration of IV-26 after a dose of 4.3 mg / kg. Error bars show standard error mean...... 246

4.30 Concentration of IV-26 in the brain after a dose of 4.3 mg / kg. Error bars show standard error mean...... 247

4.31 Comparison of the brain concentrations of III-2 and IV-26 after a dose of 3.7 and 4.3 mg / kg, respectively. Error bars show the standard error mean...... 248

4.32 Plasma concentration of IV-27 after a dose of 5.1 mg / kg. Error bars show standard error mean...... 249

4.33 Brain concentration of IV-27 after a dose of 5.1 mg / kg. Error bars show standard error mean...... 250

4.34 Comparison of the brain concentrations of III-2 and IV-27 after a dose of 3.7 and 5.1 mg / kg, respectively. Error bars show the standard error mean...... 251

4.35 Comparison of plasma concentrations after i.p. injection ...... 252

4.36 Comparison of brain concentrations after i.p. injection...... 253

4.37 Results of standard rat liver microsome assay performed on compounds used in brain uptake studies. The prescription CNS drug minaprine was used as a control ...... 272

LIST OF SCHEMES

1.1 Functionalization of trityl chloride resin ...... 35

1.2 Synthesis of polyamine trimer via exhaustive reduction of the resin-bound Tripeptide..... 36

1.3 Synthesis of Boc protected α-amino aldehydes...... 37

1.4 Solution phase reductive amination...... 38

1.5 Test of solid phase reductive amination/alkylation methodology ...... 39

1.6 Synthesis of Fmoc protected α-amino aldehydes...... 40

1.7 Solid phase reductive alkylation strategy that resulted in branching...... 41

1.8 Modified synthesis used in the formation of polyamine trimers ...... 42

1.9 Synthesis of Fmoc-γ-Phe Weinreb. Reduction to the aldehyde was unsuccessful...... 58

1.10 Trityl protection of the urea sidechain of Fmoc citrullene Weinreb...... 60

2.1 Synthetic route to trans-alcohol II-11...... 105

2.2 Attempts to synthesize racemic II-13 and II-16 using a route analogous to that used to prepare II-11...... 106

2.3 Synthesis of Boc-protected 4-aminothiazoles...... 107

2.4 Alternative approach to 4-aminothiazole synthesis...... 109

2.5 Completion of 4-aminothiazole inhibitor ...... 110

2.6 First attempt to synthesize α-bromoketone II-34 ...... 112

2.7 Route to 2-aminothiazoles ...... 113

2.8 Completion of 2-aminothiazole inhibitor II-7...... 113

2.9 Alternative 2-aminothiazole inhibitor...... 114

2.10 Synthesis of 2-aminoimidazoles ...... 117

3.1 Ether formation...... 162

3.2 Alternative route to III-1 ...... 163

3.3 Diprotection of aminopyridine allows a Mitsunobu reaction to occur ...... 165

3.4 Mitsunobu reaction using a dimethylpyrrole protected aminopyridine...... 166

3.5 Synthetic route to aldehyde III-31...... 167

3.6 Synthesis of inhibitor III-2...... 167

3.7 Synthesis of amine III-35...... 168

3.8 Synthesis of acid II-51 and aldehyde III-37 ...... 168

3.9 Completion of inhibitors III-3 and III-4 ...... 169

3.10 Synthesis of inhibitor III-5...... 170

3.11 Synthesis of inhibitor III-6...... 170

4.1 Synthetic route to IV-14 and IV-15...... 215

4.2 Synthesis of II-6 ...... 233

4.3 Synthesis of IV-22...... 241

4.4 Synthesis of IV-26 and IV-27 ...... 245

LIST OF TABLES

1.1 The eight polyamines that were synthesized, characterized, and tested for their RNA binding properties...... 43

1.2 Dissociation constants for polyamines to TAR and RRE, as determined by 2AP fluorescence assay...... 47

1.3 The dissociation constants for three polyamines to TAR and RRE, as determined by the terbium cleavage and in-line probing footprinting assays. (NM = not measurable) ...... 55

1.4 Calculated extinction coefficients for six polyamines...... 77

2.1 Inhibition constants of 2-aminothiazoles against the three NOS isoforms...... 115

3.1 Inhibition constants against the three NOS isoforms...... 171

4.1 Inhibition constants for IV-14 and IV-15...... 216

4.2 A comparison of various carbamate and amide based prodrug moieties ...... 238

4.3 Retention times of various compounds...... 269

Chapter 1

Synthesis of Sidechain Functionalized Polyamines and Study of their RNA-Binding Properties

Advisor: Daniel H. Appella

1.1. RNA as a drug target

Once thought to be a simple carrier of genetic information, RNA has, in recent years,

been recognized as a molecule that shows vast structural and functional diversity involved in

many key cellular processes.1-3 RNA plays several critical roles in the expression of proteins.

Messenger RNA (mRNA) carries genetic information from DNA in the nucleus to the ribosomes in the cytoplasm. The ribosomes themselves are complex molecules composed of RNA and proteins, and it has been shown that the RNA part of the complex, rRNA, is critical for function.

Amino acids are delivered to the ribosome by tRNAs, which are highly structured and exhibit remarkable recognition properties. Although single stranded, mRNAs can have 5’-UTRs

(untranslated regions) that can fold to form complex structures capable of recognizing specific

ligands, such as thiamine pyrophosphate (I-1)4 and flavine mononucleotide (Fig. 1.1, I-2).5

Binding of the ligand changes the conformation of the RNA, which causes an effect on the rate of its translation. RNA is also capable of catalytic activity,6, 7 providing evidence for the hypothesis that life began with self-replicating RNAs,8 and plays an important role in many pathogen life cycles.9 With such a diverse range of functionality it is obvious why RNA would make an attractive target for drug discovery.

H O N O

N N OH OH NH2 Me - O O OH N OPO3H N N+ P P -O O O -O OH Me N S Me Me I-1 I-2

Figure 1.1 Thiamine pyrophosphate, I-1, and flavine mononucleotide, I-2.

Targeting mRNA could be an option when a protein target is not available or is highly mutagenic. Binding small molecules to 5’-UTRs of mRNAs could allow either up- or down- regulation of protein translation resulting in a therapeutic response.10 Also, quantities of mRNA differ considerably between tissues, so there is the possibility of targeting specific tissues. This is not possible with drugs that bind to DNA, as DNA has uniform distribution throughout the body.

Yet, except for the case of antibiotics that bind bacterial rRNA and disrupt translation,11 RNA remains underutilized in drug therapies.12 This is largely because approaching RNA as a target requires new strategies and novel ideas. Unlike DNA, which exists almost exclusively as a double helix, RNA is capable of adopting a broad range of three-dimensional structures. Proteins and ligands that bind to RNA typically recognize the RNA’s spatial positioning of groups, rather than its base sequence.13, 14 In this respect, binding of small molecules to structured RNA is more analogous to binding to protein active sites than to DNA. However, electrostatic forces

inevitably play a major role in interactions because of the polyanionic nature of the sugar-

phosphate backbone. The majority of small molecules that bind with high affinity to a specific

RNA are polycationic, and so tend to bind well to other RNA structures. Selectivity for a specific

RNA over all others is the greatest challenge that must be faced before RNA-based therapies are

possible.

In our research, we chose to investigate the binding of small molecules to two structurally

similar RNAs, TAR15, 16 and RRE17, 18 (Fig. 1.2) of the human immunodefeciency virus (HIV).

The HIV life cycle involves two specific RNA-protein binding events. The regulatory proteins

Tat and Rev bind to short stem-loop RNA motifs labeled TAR (trans-activator region) and RRE

(Rev response element).

A. B. CA G G G A U G C G64 30 C A 35 G C C G AU G C C GA A U 50G C G C U C G 70 24C G G U G U A U40 A G C G C 20AU 45UA75 C G C G C G U G G C45 G C G C G C 5' 3' 5' 3'

Figure 1.2 Secondary structures of (A) TAR, and (B) RRE-IIB of HIV. Numbering refers to the

relative position of the base in the intact RNA.

The binding of Tat to TAR, which is found at the 5’ end of the HIV transcript, greatly

enhances the rate of transcription of the RNA genome. Inhibiting this interaction should cause

complete viral latency.19 The Rev-RRE interaction initiates the late replication phase, facilitates export of HIV RNA out of the nucleus, and protects the RNA from being degraded by cellular machinery. Inhibiting this interaction should prevent the proteins necessary for viroid formation from being assembled and will allow the RNA to become highly spliced.

The mortality rate of HIV positive people has dropped in western countries owing to the availability of 16 FDA approved drugs, all of which are reverse transcriptase or protease

inhibitors.20 One of the main problems with treating AIDS is the emergence of multi-drug

resistant strains of HIV that arise through mutation. TAR and RRE are highly conserved across

different HIV strains, so it has been hypothesized that mutation at these sites to prevent drugs

from binding may not occur.21 Anti-TAR or anti-RRE drugs may be a way around the resistance

problem. For this to become a reality, an understanding of RNA-ligand interactions and development of new types of RNA-binding molecules are critical.

1.2 Antibiotics that bind to ribosomal RNA

The paradigm for the binding of small molecules to an RNA to produce a therapeutic

response is provided by certain classes of natural antibiotics (Fig. 1.3).22 These can be extremely varied in structure and can bind to bacterial rRNA at different sites and in different ways, but all inhibit bacterial protein synthesis.

Me HO NMe2 H H O NH2 OH Me Me 1 Me HO O R

H N O

NH2 HO OH HO 2

O H NH 2 Me OH H2N O 2 R N OH O O O O HO Me HO O O O NMe2 OH OH NH O O 2 HN I-3 O OH Me O O H N OH OH Cl 2 H OMe N Me HO Cl Me I-7 OH OH O O2N I-5 I-6 I-4

Figure 1.3 Examples of antibiotics that are known to target the ribosome. Charges at physiological pH are not shown, for clarity.

About 40% of known antibiotics inhibit ribosome function.3 Tetracycline (I-3) generally blocks the A-site of the ribosome preventing incoming tRNAs from binding.23 Chloramphenicol

(I-4) occupies the position that the incoming amino acid should occupy, and so interferes with A- site binding.24 Macrolides (e.g. erythromycin, I-5) bind at the exit tunnel to prevent the nascent

peptide chain from leaving the ribosome; this leads to stalling of elongation.25 Aminoglycosides

(e.g. neomycin, I-6) are perhaps the most interesting antibiotics. These bind to rRNA at a position that induces a conformational change that increases the affinity of near-cognate tRNAs to the A-site. This leads to mis-translation of mRNA, producing nonsense proteins.26, 27

To date, there is only one class of fully synthetic antibiotics that bind rRNA, the oxazolidinones (I-7, R1 = morpholino, R2 = F, linezolid).28, 29 Although their precise binding site is still unknown, they inhibit the peptidyl transferase loop to prevent formation of peptide bonds.

Only one enantiomer is active, but their relative structural simplicity has allowed for numerous sidechain modifications. Oxazolidinones demonstrate that potent RNA-binding molecules can be produced synthetically, and their mechanism of action should give new insights into RNA interactions.

1.3 Binding of small molecules to other RNAs

In 1993, Zapp et al. reported the discovery that neomycin (I-6) could inhibit the interaction between RRE and Rev, and that in vivo HIV replication was arrested by addition of

neomycin.30 Since then, numerous studies have been carried out on the binding of neomycin and

other aminoglycosides to various RNAs.31 Aminoglycosides display amine groups on a rigid scaffold. At physiological pH, most of the amines are protonated, and these cationic groups interact favorably with anionic phosphate groups. Folded RNAs typically have sites for the binding of divalent magnesium ions that help support the folded structure. Aminoglycosides bind well to structured RNAs that have magnesium ion binding sites in the same spatial orientation as the amines on the aminoglycoside, as the protonated amines mimic magnesium ions fairly

effectively.32, 33 However, aminoglycosides can also bind to RNA structures that have no defined

metal binding sites, through electrostatic interactions, hydrogen bonding to phosphates, bases

and structural water molecules, and hydrophobic interactions between the carbon skeleton and

the bases. Binding affinity can be reasonably high (low micromolar dissociation constants for

TAR and RRE),34 and protein-RNA interactions can be inhibited. Neomycin binds TAR just below the bulge causing the structure to be locked in a conformer that no longer binds Tat.35

Although this class of molecules is selective for RNA over DNA, selectivity for a particular

RNA is poor. Also, increasing the ionic strength of the buffer decreases specific binding at the same rate as non-specific binding.36

To decrease the promiscuity of aminoglycosides, several research groups have modified their structures. Aminoglycosides are difficult to synthesize de novo, but modifications to naturally occurring molecules have been successful. A significant increase in affinity for TAR resulted when neomycin was perguanylated to give I-8,37 though the aminoglycoside no longer bound to the same site.38 Generally, increasing basicity does lead to an increase in affinity, though not necessarily selectivity.39 An acridine group was attached to neomycin to give I-9 (Fig.

1.4), causing an increase in potency against RRE, though selectivity was not significantly improved.40

H2N NH NH2

O H2N N O HO HO H N HO NH HO 2 NH HN H H N O NH2 H2N NH2 2 O N N S O NH OH OH NH H O HO NH2 H O OH N NH OH O OH H2N HN H NH N OH 2 HO

H2N HO I-8 I-9

Figure 1.4 Modifications to neomycin that improve its affinity for RNA.

Several other groups have adopted a similar strategy of combining positively charged

groups with intercalators (Fig. 1.5). Various molecules that have combinations of known RNA-

binding functionality have been synthesized: acridine-polyamine conjugates (I-10),41 phenothiazine derivatives (I-11),42 cationic diphenylfurans (I-12),43, 44 ethidium-arginine conjugates (I-13),45 quinoxaline-2,3-diones (I-14),46 2-arylquinolines (I-15),47 and metal-ligand complexes (I-16, Figure 1.6).21 These molecules can exhibit high affinity for a particular RNA motif, such as the bulge of TAR, but typically, selectivity is still not sufficient for in vivo use.

Cl N S NHR1 R2HN

OMe N HN NH NH Me O NH2 N N Me NH2

I-10 I-11 I-12

NH2 N H HN Me O2N N O

N O OH N O H2N Me H N

H2N NH HN N N NH N NH N N O

I-13 I-14 I-15

Figure 1.5 Examples of synthetically designed ligands that combine intercalating ability with positive charge.

N N N N Ru N N N N

I-16

2+ Figure 1.6 Λ-[Ru(bpy)2Eilatin] , a metal-ligand complex that was found to bind to RRE and displace Rev.

The portions of the natural proteins, Tat and Rev, that bind TAR and RRE, respectively, are rich in basic residues such as arginine and lysine. In isolation, these regions bind to the RNA with an affinity comparable to that of the intact protein.48-51 Although the peptides themselves are not ideal drug candidates, several peptide analogues have been synthesized, including peptoids,52 D-peptides,53 β-peptides,54 and cyclic peptides.55 These molecules bind with high affinity and reasonable selectivity to their target RNA, but their high molecular weight gives

them poor pharmacological properties, and the abundance of cationic charge leads to binding of

non-target RNA.

Ultimately, the best way to obtain high affinity, highly selective binding is through

iterative modifications of a lead compound based on structural data. Combinatorial libraries are a

promising source of lead compounds. RNA aptamers have been evolved that bind their target

with high affinity and remarkable selectivity.56-59 A target molecule, such as tobramycin (I-17),

OH is attached to a solid support, then random sequences of RNA are HO O H2N O OH H N HO 2 O NH2 HO O NH2 passed over it under conditions that allow the RNA to fold. Those H2N

I-17 sequences that bind to the target are amplified by PCR, and the procedure is repeated under more stringent conditions. Several repetitions produce an RNA aptamer. It seems logical that the reverse should be possible: that by screening a large enough library of compounds a molecule can be found that binds a target RNA with high affinity and selectivity. Reported attempts to identify such molecules by using a combinatorial library have typically focused on amino acid monomers due to the ease of peptide synthesis on solid support.

Not surprisingly, screens reveal short peptides that have arginine and lysine side chains as being

potent RNA binders.20, 60 Aromatic residues, such as tyrosine, also often emerge as being important (Fig. 1.7, I-18).61

HO NH2 NH2

HS O O O H H H N N N NH2 H2N N N N O H O H O H O Me

OH Me

NH2 I-18

Figure 1.7 A peptide that binds to TAR, selected via a combinatorial approach.

Other RNA-binding molecules that were found by a combinatorial approach include

cyclic peptides (I-19)62 and peptide conjugates (I-20).63

NH2 H2N O Me O O O H N Me N N OH H H O NH O NH O H H N N NH NH 2 NH 2 O OH NH O N O H N H NH NH 2 O N 2 2 H O I-19 I-20

Figure 1.8 A cyclic peptide, I-19, and a peptide-acridine conjugate, I-20. Both were selected from libraries for their RNA binding properties.

1.4 Polyamines as RNA-binding small molecules

Although a wide variety of molecules have been synthesized to target RNA, so far very few have exhibited promising characteristics for use as drugs. The primary reason for this is insufficient selectivity for the target RNA over other RNAs. Most of the desired targets (TAR,

RRE, mRNA) are present in very low copy number, and are dwarfed by the quantities of

ribosomal RNA, tRNA, and other short RNAs in the cell. The drug must have an affinity for its

target that is several orders of magnitude higher than for other RNAs, as unintentional binding

will not only reduce the effective concentration of the drug, but might also result in toxic side

effects.

We hypothesize that the problem with most of the compounds mentioned previously is

that cationic charge is displayed on the periphery of the molecule. Although important for high

affinity binding, this charge leads to non-specific electrostatic interactions with the negatively

charged backbone. A functionalized polyamine (I-21), analogous to a reduced tripeptide, shields a cationically charged backbone with neutral functional groups.

O R2 H R1-R3 = F Y N NH2 H2N N N H H OH R1 R3 Me I-21 S V OH Me

Figure 1.9 General structure of a polyamine trimer. Letters refer to the one letter code of the

amino acid from which the side chain is derived.

We predict that this situation will limit the extent of undesired electrostatic binding, and that only if the side chains are interacting favorably with the RNA will the backbone charge play a role. Groups capable of intercalation and hydrogen bonding were chosen, along with other groups that would provide steric bulk. The purpose of this investigation was to (a) develop a practical synthetic route to the polyamines, (b) determine whether side chains affect binding properties, and (c) determine whether selectivity for one RNA structure is possible.

1.5 Analysis of RNA binding

From the standpoint of pharmaceutical testing, RNA is an attractive biomolecule to work with.64 Once the sequence is known, an RNA can be synthesized in vitro and therefore does not require purification from cells. In addition, RNA secondary structures show conserved three- dimensional structure even when taken in isolation, and show binding properties similar to those of the intact RNA. There is not a problem with aqueous solubility, and modifications, such as the addition of fluorescent groups, can frequently be achieved without altering the RNA structure.

There are many ways of monitoring interactions between small molecules and an RNA.

A brief overview of some of the more common techniques is given below.

Poly-Acrylamide Gel Electrophoresis (PAGE) is a technique that has become standard in most RNA labs. The basic premise is that a hydrophilic polymer gel matrix is created, the

RNA is loaded at one end, and a current is applied across the gel. The RNA migrates through the

gel to varying degrees determined primarily by its size. In native PAGE, the RNA remains

folded, and, under the right conditions, non-covalent interactions can be preserved. If the ligand

has a large molecular weight, the difference in size between the bound and unbound species will

cause a difference in the distance migrated, leading to a “gel shift”. The most common way of visualizing this is by labeling the RNA with a radioactive isotope of phosphorus, 32P, then measuring the radioactivity in the gel. Denaturing PAGE causes the RNA to unfold so that migration is based only on size. This is useful for separating out fragments of RNA resulting from strand cleavage. Monitoring the extent of cleavage by enzymes or chemicals in the presence or absence of a ligand is called “footprinting”. The advantage of footprinting is that it gives specific information on the exact binding site, which most other methods do not.

Disadvantages of PAGE include the length of time it takes to determine ligand/RNA interactions, and the poor reproducibility of results.

Nuclear Magnetic Resonance (NMR) Spectroscopy can be used to monitor changes in

the local environment of specific atoms that might result from a ligand + H2N N NH2 binding. There are many different NMR experiments that can be H adapted to high-throughput ligand screening. Changes in the 1H NMR I-22 spectrum of the RNA, such as the effect on the imino resonances of RRE when neomycin (I-6)65 and proflavin (I-22)66 were added, have been observed. Although binding interactions could only be measured qualitatively, stoichiometry was determined along with some structural information.

Another technique for monitoring interactions makes use of the sign change of ligand NOEs that occurs when the small molecule binds to a molecule of much higher molecular weight.67 It is also possible to isotopically enrich either ligand or RNA, e.g. with 13C or 15N, to monitor changes in chemical shift on binding. RNA that has been substituted with 19F at either the 5-position on pyrimidines or the 2’-position of the ribose has the same structure as the unsubstituted compound.68, 69 F-19 NMR is highly sensitive to local environment, and has a large chemical

shift range (900 ppm). For detailed structural information on RNA-ligand interactions, an NMR

structure of the complex can be solved to give precise atomic coordinates.55 The main drawbacks of NMR-based ligand screening are the cost, both of isotopically labeled material and of instrument use, and the large amounts of material that are needed.

Mass Spectrometry employing soft ionization techniques, e.g. electrospray, can be used to monitor binding of ligands to RNA.70, 71 The ligand-RNA complex is preserved in the spectrometer, so the ratio of bound to unbound RNA can be calculated. This technique is fairly direct, gives accurate stoichiometry information, and requires very little material so it is amenable to high throughput screening. However, rigorous desalting of samples is required, no structural information is produced, and acquiring a good signal can be difficult.

Surface Plasmon Resonance (SPR) is another technique that lends itself to high throughput screening. Typically, an RNA is fixed to a sensorchip surface. The surface plasmon resonance, an optical effect, changes if the mass of the RNA changes, which occurs if the ligand binds. The binding of aminoglycosides to RRE was studied by this technique, and proved to give accurate binding constants.39, 72 The major disadvantages of this method are its initial set up cost and the lack of structural information obtained.

Electron Paramagnetic Resonance (EPR) of spin labeled uracil residues incorporated into TAR RNA gave accurate information about the binding of several ligands.38 In this study, the change in signal of each modified base could be monitored, which gave information about the change in RNA internal dynamics upon binding. Compounds that bound to TAR in a similar manner gave similar signals. Structural information could be determined from a comparative perspective, but binding constants were not obtained.

Equilibrium Dialysis is a classical biochemical technique that can be applied to RNA- binding ligands. A known concentration of RNA and a known concentration of ligand are separated by a semipermeable membrane through which the ligand can pass but the RNA cannot.

After a suitable length of time, the ligand concentration on each side of O NH2 H the membrane is measured. If no binding occurs, the two N NH2 H2N NH concentrations should be the same, and equal to half the original

I-23 ligand concentration. If binding occurs, the concentration on the side of the membrane without the RNA will be less than half the original value. The difference can be used to calculate binding constants and stoichiometry. The main drawback with this method is that there must be a way of measuring ligand concentrations very accurately. The binding of 3H- labeled argininamide (I-23) to an RNA aptamer was measured by this technique.73

Fluorescence based assays provide rapid information and are frequently used in high throughput screens. There are several methods available, and numerous fluorescent groups to choose from. One approach uses RNA that has been modified with a fluorescent group, e.g. a 2- aminopurine base. This unusual nucleotide can be incorporated into single stranded regions in the place of any nucleotide provided the structure remains the same, or can be used in place of adenine in helical regions as it can form a wobble base pair with uracil. Binding of ligands causes a change in the conformation of the RNA, which is observed by a change in fluorescence.74, 75 Fluorescence Resonance Energy Transfer (FRET) can be used to observe displacement of peptide from RNA.76, 77 Typically, a donor fluorescent group, such as fluorescein, on the RNA transfers energy via a non-radiative dipole-dipole interaction to an acceptor group, such as rhodamine, on the peptide when the two are bound. If the peptide is

displaced, quenching no longer occurs and the fluorescence of the donor increases. Another

phenomenon that can be measured is fluorescence polarization (FP, also known as anisotropy), which is inversely proportional to the rate at which the fluorescent group tumbles through

solution. High molecular weight molecules tumble more slowly than smaller ones, so if a

fluorescently tagged ligand binds to a much larger RNA, there is a dramatic increase in its

fluorescence polarization.78 Alternatively, Tor and co-workers fixed RRE to a solid support and added fluorescently tagged Rev.21 The bound Rev had a very slow tumbling time and so had a high FP. On titrating in RRE-binding ligand, Rev was displaced into solution, resulting in a large decrease in FP. Fluorescence-based assays can be extremely useful tools for rapid, accurate measurement of binding, but do have some drawbacks. If the ligand is fluorescent itself, or quenches fluorescence, the assay will not give reliable data.79 Fluorescence polarization changes only occur when there are large changes in molecular weight, so binding of small ligands to

fluorescent RNAs cannot be measured. Although fluorescence data can give limited information

as to where a ligand is binding, it is not an accurate structural probe, as allosteric binding can

affect fluorescence.

1.6 Synthesis

a) Reduction of tripeptides using borane

Initial attempts to synthesize functionalized polyamines followed a procedure that

involved the reduction of amide bonds with diborane, followed by oxidative cleavage of the

borane amine adducts using iodine.80-82 This procedure is reportedly racemization free and gives

high yields of the desired amine. A polystyrene trityl chloride resin (I-24) was functionalized with 1,3-diamino propane to provide a free primary amine (I-25).

i Cl N NH2 H

I-24 I-25

i) diaminopropane, CH2Cl2,3x1h

Scheme 1.1 Functionalization of trityl chloride resin.

A tripeptide was assembled on the resin using standard Fmoc chemistry with HATU as the coupling reagent. The synthesis of the tripeptides proceeded with high yield and purity, as shown by cleavage of a small portion from the resin and analysis by HPLC and ESMS. The resin-bound peptide (I-28) was then transferred to a silanized Schlenk tube and 40 equivalents of

BH3 in THF were added. The Schlenk tube was sealed, suspended in an oil bath at 65 ºC, and shaken for two days. The resin was then returned to a filter vessel and the borane solution was removed. After washing, the resin was shaken with iodine in THF with catalytic acetic acid and

DIEA. These buffered conditions were necessary to trap the hydroiodic acid formed in the reaction. Aliquots of the iodine mixture were added to the resin with shaking. The solution turned from brown to colorless as the iodine was consumed. Aliquots were added until the solution maintained its brown color. The polyamine was then cleaved from the resin under acidic

conditions, simultaneously deprotecting the side chains, to give a salt, which was analyzed by

HPLC and ESMS.

O O i ii NHFmoc NH2 N NH2 N N N N H H H H H R1 R1 I-26 I-27

O R2 O R2 H H N NH iii N NH N N N 2 N N N 2 H H 1 H H H 1 H R O R3 R R3

I-28 I-29

1 3 R -R = CH3 OtBu Ph

N OtBu Boc

o i) Fmoc-AA-OH, EDC, HOBt, DIEA, DMF; ii) piperidine, DMF; BH3,THF,65 C, 2 d, then I2, DIEA, AcOH, 1 h.

Scheme 1.2 Synthesis of polyamine trimer via exhaustive reduction of the resin-bound tripeptide.

Initial attempts to create polyamine trimers containing tryptophan (W), tyrosine (Y) and serine (S) residues did not give the desired products. The procedure was repeated on a triple alanine (AAA) tripeptide, as it was hypothesized that the complex sidechains were responsible for inhibiting the reaction. Some of the desired AAA polyamine was formed (~ 50% by ESMS), but the incompletely reduced product was seen in the mass spectrum, along with other unidentified impurities of higher mass. Reduction of peptide monomers proceeded to completion in high purity (75-100%), but reduction of dimers led to increased amounts of impurities (50–

70% fully reduced by ESMS), and reduction of trimers continued to produce low yields (10-

30%) of the desired product, regardless of attempts to optimize conditions. The quantity of

borane solution, concentration of borane, reduction time, and temperature were all increased with

little increase in desired product resulting. The HPLC spectra were complex, as presumably there

were multiple isomers of the partially reduced tripeptides. Pure polyamine trimers with complex

sidechains, e.g. WSY, were never isolated via this route.

(b) Reductive Amination

A second method of building up the polyamine backbone involved condensing aldehydes with amines, followed by reduction of the imine formed.83-85 This was first tested in solution using Boc protected amino aldehydes and amino esters. The N-Boc amino aldehydes (I-31) were prepared via reduction of the corresponding Weinreb amide (I-30) using lithium aluminum

86 hydride (LiAlH4). The Weinreb amides were prepared in high yield (85-95%) and purity from the commercially available N-Boc amino acid. The Weinreb was reduced with LiAlH4 in THF to give the aldehyde, also in high yield (80-90%) and purity.

O O O i ii NHBoc MeO NHBoc NHBoc HO N H

R1 Me R1 R1 I-30a,R1 =Me I-31a,R1 =Me 1 1 I-30b,R =CH2(C6H4)OBn I-31b,R = CH2(C6H4)OBn

o i) HN(OMe)Me.HCl, EDC, HOBt, TEA, CH2Cl2; ii) LAH, THF, 0 C, 1 h.

Scheme 1.3 Synthesis of Boc protected α-amino aldehydes.

I-31 was mixed with an equal amount of the hydrochloride salt of an amino ester and one equivalent of base, to form the imine (I-32). Sodium triacetoxyborohydride, a mild, efficient reducing agent that produces no toxic byproducts, was then added to reduce the imine to a secondary amine (I-33).87 This was then protected with a carboxybenzyl protecting group (Z) to give I-34, and the Boc group was removed by the addition of trifluoroacetic acid (TFA). The

TFA was evaporated under a stream of nitrogen to yield the dimer salt (I-35). Condensation with another amino aldehyde using one equivalent of base, followed by reduction to give the secondary amine, gave the protected polyamine trimer (I-36). Deprotection of the Z group using palladium on carbon in a hydrogen atmosphere, followed by treatment with TFA to remove the

Boc group gave the polyamine trimer ester (I-37).

O O R1 O R1 i ii H iii NH2 N N MeO MeO NHBoc MeO NHBoc 2 R R2 R2 I-32 I-33

O R1 O R1 O R1 Z iv Z i, ii Z N N N NHBoc MeO NHBoc MeO NH2 MeO N H R2 R2 R2 R1 I-34 I-35 I-36

O R3 O R3 v H iv H N NHBoc N NH .3 CF COOH MeO N MeO N 2 3 H H R2 R3 R2 R3

I-37

1 2 3 R =Me,CH2(C6H4)OBn R =Me,CH2C6H5 R =Me,CH2(C6H4)OH

i) I-31 TEA, CH2Cl2;ii)NaHB(OAc)3, 1 h; iii) benzyl chloroformate, TEA, MeOH, 1 h; iv) TFA, 3 h; v) H2,Pd/C,EtOH,16h.

Scheme 1.4 Solution phase reductive amination.

This route demonstrated that a reductive amination strategy could be employed to

successfully form the desired trimer products. However, the synthesis was lengthy and low

yielding, and intermediates were difficult to purify. Instead of optimizing this route, the

methodology was adapted to a solid-phase synthesis. This had the advantage of removing the

need to purify intermediates, and would ultimately allow a library of trimers to be readily

synthesized.

The first test of the solid-phase methodology involved coupling Boc protected alanine

aldehyde to alanine functionalized trityl resin (Scheme 1.5). Three equivalents of aldehyde, the

resin, and one equivalent of base were shaken together to preform the imine then sodium

triacetoxyborohydride was added. Deprotection of the Boc group and cleavage from the resin

were done simultaneously under acidic conditions, and the product dimer (I-38) was obtained in high purity.

O Me H O O Me N i H ii HN NH2 NH N N N 2 N N NHBoc Me H H H H Me Me

NH2 I-38

i) I-31,NaHB(OAc)3,CH2Cl2; ii) TFA, 3 h

Scheme 1.5 Test of solid phase reductive amination / alkylation methodology.

Following this success, attempts were made to make polyamine trimers using Fmoc chemistry, so that acid sensitive trityl and Rink resins could be used to make trimers.

Commercially available Fmoc protected amino acids were converted to the corresponding

Weinreb amide using a procedure similar to that employed above, the only difference being that

DIEA was used as a base instead of TEA to minimize cleavage of the Fmoc group. The Fmoc amino-Weinrebs (I-39) were obtained in high yield and purity as white crystalline solids. These

were stored at –20 ºC and used as needed. Aldehydes (I-40) were made immediately prior to reductive alkylation using LiAlH4 in THF. Reactions proceeded in high yields (80-90%) and afforded the aldehyde in sufficient purity that it could be used directly. In contrast to the literature procedure,88 we performed the reduction at 0 ºC, and no significant loss of the Fmoc

group was observed.

O O O i ii NHFmoc MeO NHFmoc NHFmoc HO N H R1 Me R1 R1 I-39a-f I-40a-f

I-39a, I-40a:R1 =Me I-39d, I-40d:R1 =Bn 1 1 I-39b, I-40b:R =CH2Ot-Bu I-39e, I-40e:R =CH(CH3)2 1 1 I-39c, I-40c:R =CH2(C6H4)Ot-Bu I-39f, I-40f:R =

N Boc o i) HN(OMe)Me.HCl, EDC, HOBt, TEA, CH2Cl2,16h;ii)LAH,THF,0 C, 1 h.

Scheme 1.6 Synthesis of Fmoc protected α-amino aldehydes.

Both trityl resin functionalized with diaminopropane linker, and Rink resin functionalized

with β-alanine linker were used. From analysis of the first trimers it was apparent that significant branching was occurring (Scheme 1.7). Three equivalents of aldehyde were used, and once the secondary amine had been formed a second aldehyde equivalent added to it to make a tertiary amine (I-41). Further study revealed that the main point of branching was the first, relatively

unhindered secondary amine. Branching had not been seen in the test case I-38, as the methyl group of the first alanine likely hindered further attack at the secondary amine that was formed.

O O O i ii NHFmoc NH2 N NH2 N N N N H H H R1 R1 R1 R1

NHFmoc NH2 I-41

i) I-40 (3 equiv),NaHB(OAc)3,CH2Cl2, 1 h; ii) piperidine, DMF, 20 min.

Scheme 1.7 Solid phase reductive alkylation strategy that resulted in branching.

The synthesis was modified to prevent branching (Scheme 1.8). First, the aldehyde and

resin were shaken together to form the imine. Next, any unreacted aldehyde was drained away,

the resin was washed, and then the reducing agent was added. Once the secondary amine had

been formed, it was protected using a Boc group. The terminal Fmoc group was then removed to

reveal a primary amine, which could be coupled with a second aldehyde. Although branching

was less prevalent in later couplings due to steric effects, the above method was repeated to add

the second and third residues. Five equivalents of the second and third aldehydes were needed to

maximize coupling and prevent truncated chains.

O O O i NHFmoc ii NHFmoc N NH N N N N H 2 H H H Boc R1 R1

O O R2 Boc iii NH i, ii, iii, i, iii N NH N N 2 N N N 2 H Boc H Boc H R1 R1 R3

O R2 iv H N NH 4CF COOH H N N N 2 3 2 H H R1 R3 I-42

i) I-40 (3 equiv),NaHB(OAc)3,CH2Cl2, 1 h; ii) Boc2O, TEA, CH2Cl2,1h; iii) piperidine, DMF, 20 min; iv) TFA, Et3SiH, CH2Cl2,16h.

Scheme 1.8 Modified synthesis used in the formation of polyamine trimers.

The polyamine trimer was cleaved from the resin using 10% TFA in CH2Cl2, with 0.5%

Et3SiH added to scavenge any carbocations formed. This cleavage solution did not fully

deprotect the sidechains and secondary amines. Increasing cleavage time and acid concentration

caused an increase in the amount of an unidentifiable, colored impurity in the product. The

optimal conditions for obtaining relatively pure product were found to be cleavage under milder

conditions, followed by deprotection under harsher conditions. Cleavage was performed in 10%

TFA for 5 minutes, with no scavenger added. The resin was removed by filtration, and the

solvent was evaporated under a stream of nitrogen. The residue was dissolved in 50%

TFA/CH2Cl2, and stirred for 2 hours. This was sufficient to remove all Boc groups and deprotect the tyrosine tert-butyl ether. However, to deprotect the serine tert-butyl ether, the residue had to

be stirred with 99.5% TFA, 0.5% Et3SiH for 16 hours. The solvent was then evaporated under a

stream of nitrogen, and the product was precipitated out with ether. The ether was decanted off

43 and the residue was dissolved in water. The desired trimer (I-42) was purified by reverse phase

HPLC, and characterized by ESMS.

The polyamines were labeled by their sidechains using the one letter code that is used for amino acids. For example, a polyamine composed of tyrosine, serine and tryptophan would be named YSW. To be consistent with peptide nomenclature, the polyamines were labeled from the

N-terminus to the C-terminus, therefore the sidechain order was R3-R1 (Table 1.1).

Polyamine R1 R2 R3

YSW OH N OH H

WYS OH OH N H

FFF

FSF OH

FSS OH OH

YYY OH OH OH

YSY OH OH OH Me Me YVV Me Me OH

Table 1.1 The eight polyamines that were synthesized, characterized, and tested for their RNA binding properties. For general structure see Fig. 1.9.

1.7 Fluorescence-based assay

The unusual nucleo-base 2-aminopurine (2AP) is excited by light at 310 nm and

fluoresces at 372 nm. The fluorescence of the base is easily quenched, such that the local

environment has a large effect on the emission intensity. The base has been incorporated into

structured RNAs to monitor for changes in conformation caused by ligand binding. Binding

events result in a change in the emission intensity of 2AP as its exposure to the solvent

changes.65, 66 If this change is large, the ligand can be titrated in to generate a binding curve, from which a KD can be extracted.

To test the binding properties of the polyamines to structured RNAs, interactions with

TAR and RRE were investigated. These RNAs are structurally similar and are both biologically relevant. An established assay exists for studying interactions with RRE that incorporates 2AP at the 72-position (Fig 1.10 B). An analogous assay for TAR has not yet been published, so we designed TAR with a 2AP replacing an adenine at position 27 (Fig 1.10 B). 2AP forms a wobble base pair with uracil (Fig. 1.10 C) so there should not have been a great change in the structure of the RNA from the wild type.

A. B.CA C. G G G A U G C G64 30 C A 35 G C C G AU U G C C GA O 2AP U 50G C G C U C G 70 NN 24C G G H 2AP N U G O N 2AP A U40 A G C G C H N 20AU 45UA75 N N C G C G H C G U G G C45 G C G C G C 5' 3' 5' 3'

Figure 1.10 Secondary structures of (A) TAR and (B) RRE showing the positions of the 2- aminopurine modifications. (C) Wobble base pairing between 2AP and uracil.

Two of the first polyamines tested were WYS and YSW against RRE 2AP72. Both polyamines caused a large increase in fluorescence (50-fold) that appeared to saturate at millimolar polyamine concentrations. However, titrating in polyamine in the absence of RNA caused the same increase in fluorescence, indicating that the increase must have been due to fluorescence of the tryptophan, not from the 2AP. Other polyamines caused a decrease in fluorescence as polyamine concentration was increased (Fig. 1.11), and in some cases the curve saturated. From these curves, a KD was extracted.

YSY vs RRE 2AP7 YYY vs TAR 2AP27 620 250 600 580 200 560 540 150 520 500 100 0 20406080100 0 5 10 15 20 [YSY] / uM [YYY] / uM

YVV vs TAR 2AP27 YYY vs RRE 2AP72 220 800 200 600 180 400 160 200 0 140 0 50 100 150 200 0 200 400 600 800 [YVV] / uM [YYY] / uM

Figure 1.11 Graphs of fluorescence against polyamine concentration.

The polyamines were also titrated against TAR 2AP27, and the fluorescence intensity showed a similar decrease. The apparent KD values for each RNA with 6 polyamines are shown in Table 1.2.

Polyamine KD TAR KD RRE

FSS > 1 mM > 1 mM

FSF 0.9 µM ± 0.2 5.6 µM ± 0.6

FFF 1.6 µM ± 0.4 6 µM ± 1

YVV 16 µM ± 3 840 µM ± 140

YSY 10 µM ± 3 18 µM ± 2

YYY 2.8 µM ± 0.3 335 µM ± 35 Table 1.2 Dissociation constants for polyamines to TAR and RRE, as determined by 2AP fluorescence assay.

If the results are the actual KDs, some interesting trends are observed. It appears that changing the sidechains by just one residue can have a dramatic effect on the binding affinity.

Also, simply having more aromatic groups does not necessarily increase binding. Most importantly, two of the polyamines, YYY and YVV, demonstrate significantly different binding to the two RNAs, implying that these polyamines are recognizing the structure of the RNA.

The drawback of fluorescence-based assays is that they do not give specific information as to where exactly the ligand is binding. The fitting of the curve can give some information about the stoichiometry of the binding event that is being measured, but there is no way to tell whether the ligands are binding elsewhere on the RNA. We decided that an assay that would give information about the exact nature of the binding events was needed.

1.8 Footprinting assay

Footprinting assays in general rely on the rate of cleavage of the RNA studied being

affected by binding of a ligand. They are extremely useful for visualizing specific binding sites

on an RNA, and a quantitative analysis hypothetically allows a KD to be determined for each

base in the RNA strand. The RNA is labeled with radioactive phosphorus (32P) at one end, and

incubated under conditions that promote cleavage in the presence of varying concentrations of

the ligand that is being analyzed. The cleavage products are separated based on their size by

denaturing PAGE. When cleavage occurs, only the part with the label will be visible on the gel.

The amount of RNA that has been cleaved at any particular point is typically much less than the

amount of uncleaved RNA.

The majority of footprinting assays performed use RNAses (RNA-cleaving enzymes) to

promote cleavage of the RNA. Ligands that bind to the RNA shield certain regions from the

enzyme, so the rate of cleavage is reduced, creating a “footprint” in the image of the gel. Other

parts of the RNA remain exposed, and are cleaved at the same rate as when the ligand is not

bound. In some cases, the binding of a ligand causes a conformational change in another part of

the RNA that exposes it to cleavage, so the rate is increased. There are several RNAses that can

be used to promote cleavage of the phosphoester bonds. RNAse V1 cleaves double stranded or

strongly stacked regions preferentially. This enzyme is typically used for footprinting the binding

of neomycin89 and neomycin analogues78 to TAR, as the aminoglycoside binds to the stem

region below the bulge. RNAse V1 has also been used to footprint peptide-quinoline

conjugates.63 RNAse A and RNAse T2 cleave single stranded regions, such as loops and bulges, and so have been used for mapping binding to the bulge of TAR with several ligands, including

aminoglycoside-acridine conjugates.19 RNAse T1 cleaves the phosphoester linkage on the 3’ side

of guanosine residues, and so is commonly used for determining base sequence. However, if the

binding site is G-rich, T1 can be used for footprinting as effectively as the less specific

RNAses.34 One drawback of using enzymes is that in many cases the RNAse can cleave the ligand-bound RNA just as effectively as the unbound, so no footprint is seen.

In addition to enzymatic methods, there are numerous chemical methods of footprinting.

These generally fall into two categories: chemicals that cause strand cleavage directly, and chemicals that modify the bases without causing cleavage. To use the latter category, “cold”

(unlabeled) RNA is incubated with the chemical in a buffer containing various concentrations of a ligand. Binding of a ligand often affects the rate of chemical modification. The RNA is then reverse transcribed using a DNA primer that has been labeled with 32P. Reverse transcriptase will

stall at modified bases resulting in truncated DNA sequences. These are separated by denaturing

PAGE. Typical chemicals include DMS, which selectively methylates cytosines, and kethoxal,

which forms adducts to guanines. The main problem with this technique is that there are so many

steps that errors are large; therefore it is not ideal for quantitative titration. It can be useful for a

qualitative result.

Chemicals that cleave the phosphoester linkages in the RNA backbone include heavy

metal cations and radicals. Uranyl nitrate, which causes photoinduced cleavage (420 nm), and

lead II acetate were both used to footprint binding of aminoglycoside-acridine conjugates to

TAR. Fe-EDTA can be used to generate hydroxyl radicals that cleave the phosphodiester

linkages.89 Terbium cations also catalyze hydrolysis of the backbone. Their interactions with

RNA are similar to those of magnesium. At low concentrations they bind to magnesium-binding

sites and cleave at those positions selectively, but at higher concentrations they cleave single-

stranded regions.90, 91 Terbium cleavage is often used to monitor structural changes in large

RNAs, such as ribozymes, or folding in tRNAs,92 but has also been used to study binding of HIV

nucleocapsid to tRNAlys.93

Another footprinting method relies on the inherent instability of RNA, which is due to an

intramolecular reaction between the 2’ hydroxyl and the 3’ phosphate that forms a cyclic

phosphate and cleaves the strand (Figure 1.12). This reaction is catalyzed by magnesium ions

and by high pH (> 8).

O O B O B O

- O 3' 2' O O O P O P O H - O O B O + 5' B H OH

- O Optimal OPO bond angle O OR1 =180o for "in-line" cleavage P R2O O

Figure 1.12 Mechanism of base / metal ion catalyzed intramolecular strand cleavage (B =

general base, such as ligand in metal coordination sphere).

The rate of the reaction depends on the ability of the RNA to line up the 2’ hydroxyl, the

phosphorus, and the 5’ oxygen of the leaving group, leading to the optimal bond angle for

cleavage, i.e. 180º.94 This “in-line” conformation cannot be accessed by regions of RNA that are

double stranded helices, as in helices the angle is confined to about 45º. Single stranded regions

of RNA are more flexible and an in-line conformation can be accessed more readily, resulting in

higher rates of cleavage. Soukup and Breaker showed that binding of ligands to RNA can cause a

change in the RNA conformation leading to a change in cleavage rates. Typically, binding of ligands to loops and bulges causes a rigidification of the RNA producing a reduction in cleavage.95 A comparison of the binding of guanine and other nucleotides to a guanine aptamer,

and the binding of FMN (I-2) to RFN (found in the 5’-UTR of prokaryotic mRNAs that encode

for FMN biosynthesis) were studied using this technique.96

Several of the techniques described above were used to analyze binding of polyamines to

TAR and RRE, but the best results came from in-line cleavage and terbium cleavage.

TAR and RRE-IIB were created via in vitro transcription,97 then 5’-end labeled with 32P.

In the first assay, small quantities of RNA were incubated with increasing polyamine

concentrations in the high magnesium pH 8 buffer used for probing effects on in-line cleavage.

The samples were incubated at room temperature for 48 hours, then quenched with denaturing

formamide buffer that contained EDTA. The reaction products were then separated using a 20%

polyacrylamide gel. The gels were dried and viewed using a Phosphorimager.

A second assay involved the use of terbium cations as the catalyst for intramolecular

strand cleavage. The advantage of this assay is that the metal ion catalyzes the reaction even at

pHs as low as 6.5. We envisioned that the polyamines would shield the backbone from the

terbium ions at any binding sites on the RNA. The RNAs were incubated in the presence of 100

µM TbCl3, in the presence of varying concentrations of polyamine, at room temperature for 4 hours. The cleavage products were again separated by PAGE, and quantified to give binding curves. Both of these techniques allowed the study of individual bases. The cleavage at points of interest was quantified and divided by the total amount of radiation in the lane. This correction accounted for differences in loading, and gave the value of the fraction cleaved. These values

were normalized to between 0 and 1, 0 being the minimum amount of cleavage, 1 the maximum.

The normalized fraction cleaved against polyamine concentration (on a logarithmic scale) was

plotted, and, if the data fit a single-site binding curve, a KD could be extracted.

The first polyamine tested was YYY. In the in-line assay, YYY caused an increase in the rate of cleavage at the bulge of TAR, rather than the expected decrease. Encouragingly, YYY appeared to have no effect on cleavage at the loop of TAR, or at either helical region. The increase in cleavage at the bulge saturated at higher concentrations of YYY, and the data fit a simple single-site binding curve (Figure 1.13).

YYY vs TAR, Bulge Region, In-line Cleavage 1

0.8

0.6

0.4

0.2

0 10-1 100 101 102 103

Figure 1.13 Example of PAGE displaying magnesium-catalyzed cleavage of TAR in the

presence of increasing concentrations of YYY, and graph of normalized fraction cleaved at the

bulge vs polyamine concentration. The data accumulated from several gels were fit to a single-

site binding curve, from which the KD was extracted.

The KD obtained was 4 µM, which was in good agreement with that obtained from the fluorescence experiments. In the terbium cleavage assay, increasing YYY concentration caused a decrease in cleavage as the polyamine competed with the metal ion. Although competition was observed over the whole RNA, it was particularly apparent at the bulge, and the reduction in cleavage could be fit to a single-site binding curve. From this curve a KD of 4 µM was obtained,

which agreed with that obtained above. Attempts to fit the reduction in cleavage at other sites

yielded KDs at least one order of magnitude higher than that obtained at the bulge (Fig 1.14A).

Figure 1.14 Graphs of normalized fraction cleaved vs. polyamine concentration for (A) YYY vs.

TAR, bulge region (♦), and loop region (), and (B) YYY vs TAR bulge region (♦), RRE bulge

region (), and RRE loop region (σ).

The same two assays were performed on 32P-RRE in the presence of increasing YYY

concentration. As with TAR, an amplification in cleavage was seen in the in-line assay, though

the increase was not as great as that seen in the TAR case, and was observed over the whole

RNA. Fitting the increase in cleavage to a binding curve gave a KD of about 50 µM, significantly

54 higher than that obtained for TAR. The terbium assay was also performed, and the reduction in cleavage at the bulge was again plotted. The binding curve obtained gave a KD of about 50 µM, which was consistent with the in-line assay.

Of the other polyamines tested with the in-line assay, only YSY showed the same increase in cleavage as seen with YYY, and only at much higher concentrations. YVV showed a general decrease in cleavage, suggesting that it binds differently or has a different effect on the

RNA. All the other polyamines tested had no effect on the rate of cleavage, implying that they didn’t cause any change in the conformation of the RNA.

The terbium assay was performed on all six polyamines with both RNAs. In the cases of

FFF, FSF and FSS, only weak, non-specific competition was observed, and no accurate KD could be obtained. With YVV, binding to both RNAs was measurable, but not specific to any region.

The binding of YSY seemed to be specific to the TAR bulge but bound much more weakly than

YYY.

Two controls were also tested. The tripeptide version of YYY was made by standard peptide chemistry, to test whether the charged backbone is necessary for binding, or whether the sidechains alone dominate interactions. Spermine, a straight chain polyamine with no sidechains, was also tested for its ability to compete out terbium. Neither control caused any change in the rate of terbium-induced cleavage of TAR or RRE. This indicates that a combination of side chains with a polycationic backbone is necessary for binding.

Polyamine RNA Tb KD (µM) In-line KD (µM)

YYY TAR 4.8 ± 0.6 4.1 ± 0.6 YYY RRE 50 ± 7 54 ± 7 YSY TAR >300 >150 YSY RRE 250 ± 50 NM YVV TAR 100 ± 23 NM YVV RRE 85 ± 18 NM Table 1.3 The dissociation constants for three polyamines to TAR and RRE, as determined by

the terbium cleavage and in-line probing footprinting assays. (NM = not measurable).

1.9 Test for epimerization during reductive alkylation

Although it has not been determined whether the stereochemistry of the polyamines has

any effect on binding, the extent of epimerization of the stereocenters during reductive alkylation was an issue that needed to be addressed. The reduction of the Weinreb amides to the aldehydes is reportedly racemization free, but it is possible that the imine formed during condensation could undergo tautomerization to the enamine resulting in a loss of stereochemistry (Fig. 1.15).

B + H O O O NHFmoc NHFmoc NHFmoc N N N N N 1 N 1 H R H H R1 H H H R B + racemic B B

Figure 1.15 Mechanism of epimerization of resin-bound imine.

We deliberately selected an anhydrous, aprotic solvent and used no acid or base during imine formation to try to prevent enamine formation, but the ten minute pre-formation of the imine may have allowed sufficient time for racemization to occur. Recent studies into rates of epimerization during reductive amination when NaH3BCN is used as the reducing agent show

that significant loss of stereochemistry can occur.98 However, this reducing agent typically

requires a protic solvent, such as methanol, and catalytic amounts of acetic acid, both of which

would promote racemization.

To determine the extent of epimerization two experiments were performed. The first

involved condensing Fmoc protected L-alanine aldehyde with the β-alanine linker on solid

support, followed by reduction of the imine to the secondary amine. This was Boc protected

before the Fmoc group was removed. A phenylalanine residue was

O H N then added via standard peptide coupling, the Fmoc group was H2N N NH2 H Me O removed, and the dimer was cleaved from the resin to give I-43. I-43 1 Analysis of the product by H NMR in D2O showed a doublet corresponding to the alanine

methyl group at δ 1.02, and a second smaller doublet at δ 0.79. This integrated out to being 8%

of the major peak. The D-enantiomer of alanine was then used to make the diastereomer of the

dimer. Analysis of this NMR showed a major doublet at δ 0.79, and a minor doublet at δ 1.02

that integrated out to be about 10% of the major peak. Assuming that the peptide bond forming

reaction occurs with 100% retention of stereochemistry, this means that the extent of

racemization at the first residue is 8-10%.

FmocHN In a second experiment, racemic Fmoc phenylalanine aldehyde was N H condensed with benzylamine in solution. The mixture was stirred for 10 min

I-44 then NaBH(OAc)3 was added. The product I-44 was purified by flash

column chromotography and analyzed by chiral HPLC. The compound, however, was not stable

in solution, as the secondary amine deprotected the Fmoc group. In spite of this, conditions were

obtained to separate the two enantiomers by analytical chiral HPLC. The reaction was repeated

with L-phenylalanine aldehyde, and with D-phenylalanine aldehyde. The D-enantiomer gave two

peaks that integrated out to being about 96:4 (92% e.e.). The L-enantiomer gave only one peak,

as presumably the peaks did not separate.

These results indicate that epimerization does occur to some degree, and that the extent

depends upon the conditions of the assay.

1.10 Syntheses of alternative aldehydes

a) Fmoc protected γ-amino aldehydes

The spacing between the sidechains in the polyamine backbone could have a significant

effect on its ability to recognize a three-dimensional RNA structure. To test this, an attempt was

made to synthesize γ-amino analogues of the Fmoc α-amino aldehydes described previously.

Phenylalanine was chosen as a model for the synthesis. First, Fmoc phenylalanine aldehyde (I-

40d) was made by reduction of the Weinreb I-39d as described above. The aldehyde was then condensed with benzyl-(triphenylphosphoranylidene)acetate in a Wittig reaction.99 The product

I-45 was purified by column chromatography then reduced under one atmosphere of hydrogen in

the presence of palladium on carbon. These conditions also cleaved the benzyl ester to give the

Fmoc protected γ-amino acid (I-46).

Ph Ph Ph OBn i ii H + Ph3P OBn OH FmocHN O FmocHN FmocHN O O O I-45 I-46

Ph Ph Me iii iv N H FmocHN OMe FmocHN O O I-47 I-48

o i) THF, 50 C, 90 min; ii) H2, Pd / C, EtOH, 16 h; iii) EDC, HOBt, HN(OMe)Me.HCl, o DIEA, CH2Cl2,16h;iv)LAH,THF,0 C, 1 h.

Scheme 1.9 Synthesis of Fmoc-γ-Phe Weinreb. Reduction to the aldehyde was unsuccessful.

I-46 was converted to the Weinreb amide (I-47) under standard conditions, then purified

by column chromatography. Attempts to reduce I-47 with LiAlH4 did not give the aldehyde I-48.

The 1H NMR showed very different peaks compared to the Weinreb, and no peak corresponding

to the aldehyde proton. Although the structure of the reduction product was not fully elucidated,

it is likely that the aldehyde condensed with the nitrogen of the carbamate to form a five- membered ring. Analogous reactions have been witnessed in the formation of ornithine aldehydes, where the nitrogen in the sidechain is five atoms away from the aldehyde carbonyl.100

Regardless of the exact nature of the product, it was clear that this route was unsuitable for the

formation of γ-amino aldehydes without additional protection of the nitrogen.

b) Trityl protected glutamine and citrullene sidechains

The amide and urea functionality present on the side chains of glutamine and citrullene

could have interesting RNA binding properties. However, in order to introduce these functional

groups into a polyamine by the reductive alkylation method, they must survive conditions to

make the corresponding aldehyde. Commercially available trityl protected Fmoc glutamine was converted to the Weinreb by the standard route. Problems were encountered during the standard

base wash that is done to remove any unreacted amino acid starting material. At this stage an

emulsion formed between the aqueous and organic layers. It is likely that significant amounts of

the amino acid remained in solution, and the deprotonated form acted like a detergent because it

had hydrophobic aromatic groups and an anionic portion. The emulsion was acidified causing the

layers to separate. Column chromatography was performed on the crude product to give the pure

Weinreb (I-49). Only 1.25 equivalents of LiAlH4 were used to reduce the

Weinreb to the aldehyde, but there was still the possibility that the side chain

HN O amide would be reduced. However, the reduction appeared to proceed smoothly OMe N 1 FmocHN Me as shown by H NMR, confirming loss of the Weinreb to give an aldehyde, and O I-49 by 13C NMR, which showed retention of the sidechain amide carbonyl. The

glutamine aldehyde was successfully added to solid support by reductive alkylation, although

removal of the trityl group required conditions similar to those used to remove a serine t-Butyl

ether.

Fmoc protected citrullene is commercially available without protection of the urea group

on the side chain. This was easily converted to the Weinreb amide, however, reduction with

LiAlH4 did not give the aldehyde, but an unidentifiable white solid. Following the success of the

60 trityl protected glutamine, it was possible that a similar approach would allow formation of the citrullene aldehyde. The Weinreb was trityl protected using triphenylmethanol and catalytic p- toluenesulfonic acid in benzene under Dean-Stark conditions.101

H Ph O NH2 O N Ph HN HN Ph i Me Me N FmocHN OMe N FmocHN OMe O O I-50 I-51

Scheme 1.10 Trityl protection of the urea sidechain of Fmoc citrullene Weinreb.

After column chromatography, the protected Weinreb was subjected to the standard

1 LiAlH4 reduction conditions. However, no aldehyde peak was present in the H NMR of the product, suggesting that the trityl protecting group had not solved the problems encountered in the reduction step. Reports on the reduction of Boc-Arg Weinreb amides illustrate that the guanidino group must be diprotected, otherwise cyclization to the hemi-aminal occurs.

Monoprotected Fmoc-Arg aldehyde could not be synthesized.102 It is likely that the urea group of citrullene is behaving in a similar manner. ESMS of the crude product showed a peak corresponding to the mass of the aldehyde, which could also be the hemiaminal, but the major peaks in the spectrum were of much lower molecular weight, suggesting that degradation also occurs.

1.11 Summary

Polyamines with a variety of sidechains were successfully synthesized via a solid-phase reductive amination strategy. Initial testing of their RNA-binding properties using a fluorescence-based assay revealed some promising results. Binding affinities depended strongly on the nature of the sidechains, and moderate selectivity for TAR over RRE was seen in the case

of YYY. However, when the binding properties were tested by a footprinting method, the results

did not agree with those obtained from fluorescence. This discrepancy could exist for several reasons. The fluorescence experiments gave much tighter binding constants for most of the polyamines than the footprinting experiments; therefore the fluorescence assay could have detected binding that was not evident from the footprinting assay. Alternatively, the 2AP modification may have caused a structural change in the RNA that was significant enough to alter the binding properties of the polyamines. It is more likely, however, that the ligands were causing a non-specific quenching of the 2AP, without necessarily binding to the RNA. All of the curves showed quenching of fluorescence with increasing polyamine concentration. The footprinting data indicated that the majority of the polyamines tested bound very poorly to the

RNA. This supports the hypothesis that the neutral side chains shield the charged backbone. The extent of binding does depend on which functional groups are present, but having more aromatic groups does not necessarily translate to tighter binding. In the case of YYY, binding is specific to the bulge of TAR over the rest of TAR, and over any part of RRE, but only by about one order of magnitude. Although the polyamines are a long way from being useful in vivo, there are signs that they may be tailored to the structure of a particular RNA. Polyamines bearing amino acid

sidechains are a new class of RNA-binding ligand, and further study may reveal important information about RNA-small molecule interactions.

1.12 Experimental procedures

General Methods. Proton nuclear magnetic resonances (1H NMR) were recorded in deuterated

solvents on a Mercury 400 (400 MHz) or a Varian Inova 500 (500 MHz) spectrometer. Chemical

shifts are reported in parts per million (ppm, δ) relative to tetramethylsilane (δ 0.00). 1H NMR

splitting patterns are designated as singlet (s), doublet (d), triplet (t), quartet (q). Splitting

patterns that could not be interpreted or easily visualized were recorded as multiplet (m) or broad

(br). Coupling constants are reported in Hertz (Hz). Proton-decoupled carbon (13C-NMR)

spectra were recorded on a Mercury 400 (100 MHz) or a Varian Inova 500 (125 MHz)

spectrometer and are reported in ppm using the solvent as an internal standard (CDCl3, δ 77.23;

DMSO, δ 39.52). Electrospray mass spectra (ESMS) were obtained using an LCQ-Advantage.

Tetrahydrofuran (THF) was distilled from sodium and benzophenone prior to use. Methylene chloride (CH2Cl2) was distilled from calcium hydride prior to use, if dry solvent was required.

Nitrogen was bubbled through dimethylformamide (DMF) for 16 hours prior to its use and the

DMF was stored at 5 ºC. All solution phase reactions were performed in oven dry glassware under a positive pressure of nitrogen. Where indicated, glassware was silanized using Sigmacote

(Sigma-Aldrich). The internal surface of the glass was coated with Sigmacote then allowed to air-dry. The vessel was then washed with dH2O and acetone and oven-dried. All protected amino

acids, Rink resin and EDC were purchased from Advanced ChemTech. HATU was purchased

from Applied Biosystems. The PNK, T1 RNAse, and T7 RNA polymerase used were generous

gifts from the Uhlenbeck lab. RQ1 DNAse, boric acid, ammonium peroxydisulfate (APS) were

purchased from Fisher. Urea, TRIS, and formamide were purchased from Baker, Inc. Shrimp

alkaline phosphatase (SAP) was purchased from Roche. Xylene cyanol was purchased from

Eastman. Unless otherwise noted, all other reagents were purchased from Sigma-Aldrich and

used without further purification. All polyamines were purified on reverse-phase HPLC using a

Varian ProStar with UV detection at 260 nm for compounds containing phenylalanine, or at 270 nm for compounds containing tyrosine. Both MetaChem Polaris C18 (d = 21.2 mm, l = 250 mm,

10 microns) and VYDEK C18 (d = 10 mm, l = 250 mm, 5 microns) semi-prep columns were utilized. The column was kept at room temperature. Solution A was 0.05% TFA in water and solution B was 0.05% TFA in acetonitrile (ACN). A typical elution was 100% A for 5 min, followed by a gradient of 100% of A to 100% of B over 40 minutes at flow rate 5.05 mL/min for

MetaChem column and 2.2 mL/min for VYDAC column. Analytical HPLC on purified material used a VYDAC C18 column (d = 4.6 mm, l = 250 mm, 5 microns) with detection at 215 nm.

Elution was achieved using 100% A for 8 min, then a gradient of 100% of A to 100% of B over

32 minutes at flow rate 0.5 mL/min. UV quantification was performed using either a Nanodrop

ND 1000, or an Agilent 8453 UV-Vis Spectrophotometer. Denaturing polyacrylamide gels were made from gel mix that contained 19:1 acrylamide:bisacrylamide, 7 M urea, 1 x TBE (TRIS,

0.09 M, boric acid, 0.09 M, disodium EDTA, 2.5 mM), which, before pouring, was mixed with

10% APS in water, and TEMED (350 µL APS, 35 µL TEMED per 45 mL gel mix) to initiate

polymerization. Gel percentages refer to the ratio of weight of acrylamide to total volume.

Trityl-Diaminopropane PS Resin (I-25).86 PS-Trityl Chloride resin

NH N 2 H (I-24, 300 mg, ~ 0.24 mmol loading) was washed with CH2Cl2 (2 x 5

mL) in a clean, silanized SPPS filter vessel. 1,3- diaminopropane (1 mL, 12 mmol) dissolved in CH2Cl2 (1 mL) was added to the resin, the vessel was sealed, and the mixture was shaken for one hour. This process was then repeated. After the second hour MeOH

(1 mL) was slowly added to the resin to cap any unreacted sites, and the mixture was shaken a further 20 min. The resin was then washed with MeOH, CH2Cl2, and 4:1 TEA/DMF (3x 2 mL of each) and finally with CH2Cl2 (5 mL). The functionalized resin was dried under vacuum overnight, then transferred to a glass vial for storage. The success of the reaction was demonstrated by a positive Kaiser test.103

O General Procedure for Peptide Coupling (I-26). I-25 (200 mg, ~ NHFmoc N N H 1 H R 0.14 mmol loading) was allowed to swell for 20 min in DMF (5 mL) in a silanized filter vessel. Fmoc protected amino acid (0.42 mmol) was dissolved in DMF (2 mL) and added to the resin. HATU (157 mg, 0.42 mmol), HOBT (56 mg, 0.42 mmol), and DIEA

(55 µL, 0.42 mmol) were dissolved in DMF (5 mL) and also added to the resin. The mixture was shaken for 75 min, then the reagents were drained away. The resin was washed with MeOH,

DMF, and CH2Cl2 (3 x 5 mL of each). This general procedure was used for all peptide-coupling reactions: only the quantities of reagents were changed to maintain a ratio of three equivalents of coupling reagents to each mole of loading. A Kaiser test after this step showed negative if coupling was achieved.

O General Procedure for Fmoc Deprotection (I-27). The resin was NH2 N N H 1 H R washed with DMF, then treated with 20% piperidine in DMF (5 mL for 5 min, DMF wash, 5 mL for 10 min). Following this, the resin was washed with DMF and CH2Cl2

(3 x 5 mL of each). This procedure was followed for all Fmoc deprotections on 200-300 mg of

resin. For significantly more or less resin, appropriate scaling was performed. A Kaiser test after

this step showed blue indicating a free amine.

O R2 O H Peptide trimers (I-28). The general steps outlined above N NH2 N N N H 1 H 3 H R O R were repeated until a peptide trimer had been formed. The

final Fmoc group was removed. In some cases the terminal amine was capped with acetic

anhydride (10 equivalents) with DIEA (10 equivalents) in dry CH2Cl2 before the reduction step.

In this case the terminal acetyl group would be reduced to an ethyl group. Generally, however,

the primary amine was left uncapped.

2 H R Reduction of peptide trimers (I-29). I-28 was washed with N NH2 N N N H 1 H 3 H R R dry THF and transferred with a silanized pipette to a silanized

Schlenk tube. The tube was flushed with nitrogen, then borane in THF (1 M, 40 equivalents) was

added. The Schlenk tube was sealed then suspended in a water bath at 65 ºC and shaken for 48

hours. After this time the resin was transferred back to the filter vessel and the borane mixture

was drained away. The resin was washed with THF (3 x 5 mL) and CH2Cl2 (3 x 5 mL). The resin

was suspended in dry THF with DIEA (200 µL) and acetic acid (400 µL) added as a buffer. A

concentrated solution of iodine (6 equivalents) in dry THF (2 mL) was added in 0.5 mL portions

with shaking for 30 min between additions, until the solution maintained its color. The mixture was then shaken for one hour. The resin was washed with THF, 3:1 DMF / TEA, MeOH, and

CH2Cl2 (3 x 5 mL of each). See below for optimum cleavage conditions.

O Boc-Ala-Weinreb (I-30a). Boc-Ala-OH (1.89 g, 10.0 mmol) was dissolved in Me NHBoc N

OMe CH3 dry CH2Cl2 (45 mL) with TEA (1.4 mL, 10.0 mmol) and chilled to 0 ºC. EDC

(2.3 g, 12.0 mmol) and HOBT (1.6 g, 12.0 mmol) were added to the flask, and the mixture was

stirred for 10 min at 0 ºC. N,O- dimethylhydroxylammonium chloride (1.45 g, 15.0 mmol) and

TEA (2 mL, 15.0 mmol) were added and the mixture was allowed to warm to room temperature,

then stirred for 16 h. The mixture was diluted with CH2Cl2 (50 mL) and washed with 2 N HCl (3

x 60 mL), sat NaHCO3 (3 x 40 mL), and sat NaCl (2 x 40 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give I-30a (2.03 g, 87%) as a white crystalline solid. 1H

NMR (400 MHz, CDCl3): δ 5.25 (br, 1H, NH), 4.65 (m, 1H, α-CH), 3.74 (s, 3H, OCH3), 3.18 (s,

3H, NCH3), 1.41 (s, 9H, C(CH3)3), 1.28 (d, J = 6.4 Hz, 3H, CHCH3).

O Boc-Tyr-(OBn)-Weinreb (I-30b). Same procedure as for I-30a. Yield MeO NHBoc N 1 Me 86%. H NMR (400 MHz, CDCl3): δ 7.45-7.32 (m, 5H, C6H5), 7.08 (d, J

OBn = 8.8 Hz, 2H, C6H4), 6.90 (d, J = 8.4 Hz, 2H, C6H4), 5.16 (m, 1H, NH),

5.05 (s, 2H, OCH2C6H5), 4.92 (m, 1H, αCH), 3.65 (s, 3H, OCH3), 3.17 (s, 3H, NCH3), 2.99, 2.84

+ (m, 2H, CHCH2), 1.40 (s, 9H, C(CH3)3); ESMS m/z = 415 (M + H) .

O Boc-Ala-aldehyde (I-31a). I-30a (580 mg, 2.5 mmol) was dissolved in dry THF NHBoc H

CH3 (22 mL) and cooled to 0 ºC. LiAlH4 (119 mg, 3.1 mmol) was added in portions to the solution, and the mixture was stirred at 0 ºC for one hour. A solution of NaHSO4 (604 mg,

4.4 mmol) in water (11.2 mL) was cooled to 0 ºC and added dropwise to the reaction mixture.

The product was extracted from the mixture with diethyl ether (3 x 50 mL), then washed with 1.5

M HCl (3 x 50 mL), sat NaHCO3 (3 x 30 mL) and sat NaCl (3 x 30 mL), dried over anhydrous

sodium sulfate, and concentrated in vacuo to give I-31a (310 mg, 85%) as a white powder. This

1 was stored under vacuum until needed. H NMR (400 MHz, CDCl3): δ 9.56 (s, 1H, CHO), 5.08

(bs, 1H, NH), 4.24 (m, 1H, α-CH), 1.46 (s, 9H, C(CH3)3), 1.34 (d, J = 7.6 Hz, 3H, CHCH3).

O Boc-Tyr(OBn)-aldehyde (I-31b). I-30b (333 mg, 0.80 mmol) was reduced NHBoc H following the same procedure as for the formation of I-31a to give I-31b (244

OBn 1 mg, 82%) as an oily solid. H NMR (400 MHz, CDCl3): δ 9.62 (s, 1H, CHO),

7.43-7.32 (m, 5H, C6H5), 7.08 (d, J = 8 Hz, 2H, C6H4), 6.91 (d, J = 8.4 Hz, 2H, C6H4), 5.04 (s,

2H, OCH2C6H5), 4.39 (d, J = 6.8 Hz, 1H, NH), 3.75 (m, 1H, αCH), 3.06 (m, 2H, CHCH2), 1.44

(s, 9H, C(CH3)3).

1 R H O Boc-Ala-Ala-OMe (I-33). H-Ala-OMe.HCl (131 mg, 0.94 mmol) dissolved N BocHN OMe R2 in dry CH2Cl2 (15 mL) with TEA (131 µL, 0.94 mmol) was added to a

solution of I-31a (150 mg, 0.87 mmol) in dry CH2Cl2 (15 mL). The mixture was stirred for 10

min, then NaHB(OAc)3 (257 mg, 1.21 mmol) was added. After 2.5 h the reaction was quenched by the addition of sat NaHCO3 (60 mL). The product was extracted with CH2Cl2 (3 x 60 mL),

dried over anhydrous sodium sulfate, and concentrated in vacuo to give I-33 as a colorless oil. 1H

NMR (500 MHz, CDCl3): δ 5.30 (m, 1H, NH), 4.72 (br, 1H,NH), 3.72 (s, 3H, OCH3), 3.67 (m,

1H, COCHCH3), 3.36 (q, J = 7 Hz,1H, CH2CHCH3), 2.66 (dd, J = 11.5, 5 Hz, 1H, CHCH2NH),

2.47 (dd, J = 12, 7 Hz, 1H, CHCH2NH), 1.45 (s, 9H, C(CH3)3), 1.29 (d, J = 7 Hz, 3H,

+ + COCHCH3), 1.14 (d, J = 6.5 Hz, 3H, CH2CHCH3); ESMS m/z = 261 (M + H) , 521 (2M + H) ,

543 (2M + Na)+.

O Me O H I-38. Fmoc protected alanine (123 mg, 0.37 mmol) was coupled to I- N N NH2 H2N H Me 25 (125 mg, ~ 0.20 mmol) in a silanized filter vessel following the same procedure as that used to make I-26. The Fmoc group was removed as described above. I-

31a (150 mg, 0.87 mmol) and TEA (0.1 mL) were dissolved in dry CH2Cl2 (10 mL) and added to

the resin. The mixture was shaken for 10 min, then NaHB(OAc)3 (100 mg, 0.47 mmol) was

added as a solid. The mixture was shaken for a further 3 h. The reagents were drained away and

the resin was washed with MeOH, DMF, and CH2Cl2 (3 x 5 mL of each). A solution of 20%

TFA in dry CH2Cl2 was added to the resin, and the mixture was shaken for 10 min. The resin was filtered off with CH2Cl2 washings, and the solvent was reduced under a stream of nitrogen

until 2 mL remained. TFA (2 mL) and Et3SiH (0.25 mL) were added and the mixture was stirred

for 2 h. The solvent was evaporated under a stream of nitrogen and the residue was precipitated

with ether. The ether was decanted off to leave I-38 as a white solid. ESMS m/z = 217 (M + H)+.

General Procedure for synthesis of N-Fmoc-α-amino-Weinreb Amides (I-39). An Fmoc- protected amino acid (2.5 mmol) was dissolved in dry CH2Cl2 (10 mL) with DIEA (0.4 mL) and

69 cooled to 0 ºC. EDC (575 mg, 3.0 mmol) and HOBT (400 mg, 3.0 mmol) were added as solids.

The mixture was stirred at 0 ºC for 10 min, then N,O-dimethylhydroxylammonium chloride (300 mg, 3.0 mmol) and DIEA (0.5 mL) were added. The mixture was stirred at 0 ºC for an additional hour, allowed to warm to room temp, and stirred for 16 h. The reaction mixture was transferred with CH2Cl2 rinses (50 mL) to a separatory funnel, then washed with 2N aqueous HCl (3 x 30 mL), sat NaHCO3 solution (2 x 30 mL) and sat NaCl solution (2 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, and concentrated in vacuo to give a white crystalline solid. If necessary, the residue was purified by flash column chromatography (SiO2, EtOAc / hexanes,1:3). In the case of serine, the product appeared to have a low melting point, but could be made brittle by cooling to –20 ºC. The Weinreb amide was stored at –20 ºC until needed.

There did not appear to be any degradation even after several months of storage. All Weinreb amides were made using the same procedure as that outlined above. Scaling the reaction down to

1 mmol scale did not have any significant effect on yield or purity.

O 1 FmocHN OMe Fmoc-Ala-Weinreb (I-39a). H NMR (CDCl3-d, 400 MHz): δ 7.76 (d, J = 7.4 N CH Me 3 Hz, 2H, Fmoc aromatic CH), 7.61 (d, J = 6.8 Hz, 2H, Fmoc aromatic CH),

7.40 (t, J = 7 Hz, 2H, Fmoc aromatic CH), 7.31 (t, J = 7.5 Hz, 2H, Fmoc aromatic CH), 5.59 (d,

J = 8 Hz 1H, NH), 4.75 (m, 1H, α-CH), 4.36 (d, J = 6.4 Hz, 2H, Fmoc CH2), 4.22 (t, J = 7 Hz,

1H, Fmoc CH), 3.78 (s, 3H, OCH3), 3.23 (s, 3H, NCH3), 1.37 (d, J = 6.8 Hz, 3H, CHCH3).

O 1 Fmoc-Ser(t-Bu)-Weinreb (I-39b). Yield 90%. H NMR (CDCl3-d, 500 FmocHN OMe N Me OtBu MHz): δ 7.76 (d, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.62 (t, J = 8 Hz, 2H,

Fmoc aromatic CH), 7.40 (t, J = 8, 7.5 Hz, 2H, Fmoc aromatic CH), 7.32 (d, J = 7.5 Hz, 2H,

Fmoc aromatic CH), 5.69 (d, J = 8.5 Hz, 1H, carbamate NH), 4.88 (m, 1H, α-CH), 4.36 (d, J =

7.5 Hz, 2H, Fmoc CH2), 4.24 (t, J = 7.5 Hz, 1H, Fmoc CH), 3.78 (s 3H, OCH3), 3.66, 3.61 (m

13 1H, 1H, CHCH2O), 3.24 (s, 3H, NCH3), 1.17 (s, 9H, C(CH3)3); C NMR (CDCl3-d, 125 MHz):

δ 156.3, 144.2, 144.1, 141.5, 127.9, 127.3, 125.5, 120.2, 73.8, 67.3, 62.2, 61.7, 52.0, 47.4, 32.4,

27.6; ESMS m/z = 427 (M + H)+.

O 1 Fmoc-Tyr(t-Bu)-Weinreb (I-39c) Yield 90%. H NMR (CDCl3-d, 500 FmocHN OMe N Me MHz): δ 7.76 (d, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.57 (t, J = 8 Hz, 2H,

OtBu Fmoc aromatic CH), 7.40 (t, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.31 (t, J =

7.5 Hz, 2H, Fmoc aromatic CH), 7.08 (d, J = 7.5 Hz, 2H, C6H4), 6.90 (d, J = 8 Hz, 2H, C6H4),

5.54 (d, J = 8.5 Hz, 1H, NH), 5.01 (m, 1H, α-CH), 4.31 (m, 2H, Fmoc CH2), 4.18 (t, J = 7 Hz,

1H, Fmoc CH), 3.62 (s, 3H, OCH3), 3.16 (s, 3H, NCH3), 3.05, 2.91 (m, m, 1H, 1H, CHCH2C6H4),

13 1.30 (s, 9H, C(CH3)3); C NMR (CDCl3-d, 125 MHz): δ 172.2, 156.0, 154.5, 144.1, 141.5,

131.4, 130.1, 127.9, 127.3, 125.4, 124.4, 120.2, 78.6, 67.2, 61.7, 52.3, 47.4, 38.3, 32.3, 29.0;

ESMS m/z = 503 (M + H)+.

O 1 FmocHN OMe Fmoc-Phe-Weinreb (I-39d) Yield 90%. H NMR (CDCl3-d, 500 MHz): δ N Me 7.77 (d, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.58 (t, J = 8.5, 2H, Fmoc

aromatic CH), 7.41 (t, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.30 (m, 5H, C6H5), 7.24 (d, J = 8.5

Hz, 2H, Fmoc aromatic CH), 5.60 (d, J = 4 Hz, 1H, NH), 5.06 (m, 1H, α-CH), 4.38 (d, J = 8 Hz,

2H, Fmoc CH2), 4.26 (t, J = 8, 1H, Fmoc CH), 3.97, 3.11 (m, m, 1H, 1H, CHCH2C6H5), 3.69 (s,

13 3H, OCH3), 3.20 (s, 3H, NCH3); C NMR (CDCl3-d, 125 MHz): δ 172.1, 156.0, 144.1, 144.1,

141.5, 136.6, 129.7, 128.7, 127.9, 127.3, 127.2, 125.5, 125.4, 120.2, 67.2, 61.8, 52.3, 47.4, 38.9,

32.3; ESMS m/z = 431 (M + H)+.

O 1 FmocHN OMe Fmoc-Val-Weinreb (I-39e) Yield 94% H NMR (CDCl3-d, 400 MHz): δ 7.76 N Me Me Me (d, J = 8 Hz, 2H, Fmoc aromatic CH), 7.61 (t, J = 7 Hz, 2H, Fmoc aromatic

CH), 7.40 (t, J = 8 Hz, 2H, Fmoc aromatic CH), 7.31 (t, J = 7.5 Hz, 2H, Fmoc aromatic CH),

5.55 (d, J = 9 Hz, 1H, NH), 4.67 (m, 1H, α-CH), 4.37 (m, 2H, Fmoc CH2), 4.23 (t, J = 7.5 Hz,

1H, Fmoc CH), 3.77 (s, 3H, OCH3), 3.23 (s, 3H, NCH3), 2.05 (m, 1H, CHCH(CH3)2), 0.99 (d, J

13 = 7 Hz, 3H, CHCH(CH3)2), 0.96 (d, J = 7 Hz, 3H, CHCH(CH3)2); C NMR (CDCl3-d, 100

MHz): δ 172.7, 156.7, 144.0, 141.5, 127.9, 127.3, 125.4, 120.2, 67.2, 61.9, 55.9, 47.5, 32.2, 31.7,

19.7, 18.0; ESMS m/z = 383 (M + H)+.

O Fmoc-Trp(Boc)-Weinreb (I-39f). 1H NMR (CDCl -d, 500 MHz): δ 8.10 (s, FmocHN OMe 3 N Me 1H, indole CH), 7.75 (d, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.67- δ 7.3 (m,

N Boc 10H, Fmoc aromatic CHs, indole aromatic CHs), 6.29 (d, J = 8.5 Hz 1H, NH),

5.22 (m, 1H, α-CH), 4.39 (m, 1H, Fmoc CH2), 4.31 (m, 1H, Fmoc CH2), 4.22 (m, 1H, Fmoc CH),

3.73 (s, 3H, OCH3), 3.29 (m, 2H, CH2, overlaps with NCH3), 3.23 (s, 3H, NCH3), δ 1.67 (s, 9H,

13 C(CH3)3); C NMR (CDCl3-d, 100 MHz): δ 172.4, 156.4, 149.9, 144.2, 141.5, 135.7, 131.0,

129.0, 127.9, 127.4, 125.5, 124.7, 122.9, 120.2, 119.1, 115.9, 115.6, 83.8, 67.4, 61.9, 51.4, 47.4,

32.4, 28.5; ESMS m/z = 570 (M + H)+.

General procedure for the synthesis of N-Fmoc-α-amino-aldehydes (I-40).92 I-39 (0.12

mmol) was dissolved in dry THF (5 mL) and cooled to 0 ºC. LiAlH4 (6 mg 0.15 mmol) was

added as a solid, and the mixture was stirred at 0 ºC for 60 min. After this time the reaction

mixture was quenched by dropwise addition of 20% aqueous NaHSO4 (5 mL). The mixture was

stirred for an additional 10 min before being transferred to a separatory funnel. The mixture was

diluted with sat NaCl solution (30 mL) and EtOAc (30 mL). The layers were separated and the

aqueous layer extracted with EtOAc (30 mL). The organic layers were combined and washed

with 1.5 N HCl (3 x 30 mL), sat NaHCO3 solution (2 x 30 mL), and sat NaCl solution (2 x 30

mL). The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to

give I-40. It was difficult to remove all the EtOAc under vacuum, leaving the product with an

oily appearance. However, re-dissolving the product in a small amount of ether, followed by

removal of the solvent under vacuum usually gave the product as a fine white powder. All

aldehydes were prepared as needed using the above method and were stored under vacuum until

used, which was not more than 16 hrs after isolation. Scaling the reaction up to 1 mmol did not

have a significant effect on yield or purity.

1 O Fmoc-Ala-aldehyde (I-40a). Yield 86%. H NMR (CDCl3-d, 400 MHz): δ 9.56 (s, FmocHN H CH3 1H, CHO), 7.77 (d, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.60 (d, J = 7 Hz, 2H,

Fmoc aromatic CH), 7.41 (t, J = 7 Hz, 2H, Fmoc aromatic CH), 7.32 (t, J = 7.5 Hz, 2H, Fmoc

aromatic CH), 5.43 (b, 1H, NH), 4.44 (d, J = 5 Hz, 2H, Fmoc CH2), 4.32 (t, J = 7 Hz, 1H, Fmoc

13 CH), 4.23 (m, 1H, α-CH), 1.38 (d, J = 6.5 Hz, 3H, CH3); C NMR (CDCl3-d, 100 MHz): δ

199.1, 143.9, 141.5, 128.0, 127.3, 125.2, 123.5, 120.2, 67.3, 56.2, 47.4, 15.2.

O 1 FmocHN Fmoc-Ser(t-Bu)-aldehyde (I-40b). Yield 85%. H NMR (CDCl3-d, 500 MHz): δ H

OtBu 9.63 (s, 1H, CHO), 7.7 (d, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.5 (t, J = 7.5 Hz,

2H, Fmoc aromatic CH), 7.4 (t, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.2 (d, J = Hz, 2H, Fmoc aromatic CH), 5.67 (d, J = 8.5 Hz, 1H, NH), 4.43 (d, J = 8.5, 2H, Fmoc CH2), 4.36 (m, 1H, α-

CH), 4.25 (t, J = 8.5 Hz, 1H, Fmoc CH), 3.96, 3.64 (m, m, 1H, 1H, CHCH2O), 1.17 (s, 9H,

13 C(CH3)3); C NMR (CDCl3-d, 125 MHz): δ 199.3, 156.4, 143.9, 141.5, 128.0, 127.3, 125.3,

120.2, 73.5, 67.4, 60.7, 60.3, 47.4, 27.5.

O 1 Fmoc-Tyr(t-Bu)-aldehyde (I-40c). Yield 90%. H NMR (CDCl3-d, 400 FmocHN H MHz): δ 9.62 (s, 1H, CHO), 7.77 (d, J = 7.5 Hz, 2H, Fmoc aromatic CH), OtBu 7.57 (m, 2H, Fmoc aromatic CH), 7.41 (t, J = 7 Hz, 2H, Fmoc aromatic CH), 7.32 (t, J = 7.5 Hz,

2H, Fmoc aromatic CH), 7.02 (d, J = 8 Hz, 2H, C6H4), 6.92 (d, J = 8 Hz, 2H, C6H4), 5.37 (d, J =

6.4 Hz, 1H, NH), 4.44 (m, 3H, α-CH and Fmoc CH2), 4.22 (t, J = 7 Hz, 1H, Fmoc CH), 3.10 (d,

13 J = 7 Hz, 2H, CHCH2C6H4), 1.33 (s, 9H, C(CH3)3); C NMR (CDCl3-d, 100 MHz): δ 199.1,

156.0, 154.7, 143.9, 141.5, 130.3, 130.0, 128.0, 127.3, 125.2, 124.6, 120.2, 78.8, 67.2, 61.4, 47.5,

35.0, 29.1.

O Fmoc-Phe-aldehyde (I-40d). Yield 85%. 1H NMR (CDCl -d, 400 MHz): δ 9.64 FmocHN 3 H (s, 1H, CHO), 7.79 (d, J = 7 Hz, 2H, Fmoc aromatic CH), 7.59 (m, 2H, Fmoc aromatic CH), 7.43 (m, 2H, Fmoc aromatic CH), 7.33 (m, 5H, Phe aromatic CH), 7.14 (d, J = 6.5

Hz, 2H, Fmoc aromatic CH), 5.41 (d, J = 6 Hz, 1H, NH), 4.51 (m, 1H, α-CH), 4.42 (m, 2H,

13 Fmoc CH2), 4.23 (t, J = 6.5 Hz, 1H, Fmoc CH), 3.16 (m, 2H, CHCH2C6H5); C NMR (CDCl3-d,

125 MHz): δ 199.0, 156.1, 144.0, 141.6, 135.8, 129.6, 129.5, 129.1, 128.0, 127.4, 127.4, 125.4,

125.3, 120.3, 67.2, 61.4, 47.4, 35.6.

O 1 FmocHN Fmoc-Val-Aldehyde (I-40e). Yield 85% H NMR (CDCl3-d, 400 MHz): δ 9.65 (s, H Me Me 1H, CHO), 7.77 (d, J = 7.5 Hz, 2H, Fmoc aromatic CH), 7.61 (t, J = 7 Hz, 2H,

Fmoc aromatic CH), 7.41 (t, J = 7 Hz, 2H, Fmoc aromatic CH), 7.32 (t, J = 7.5 Hz, 2H, Fmoc

aromatic CH), 5.41 (d, J = 7 Hz, 1H, NH), 4.42 (d, J = 7 Hz, 2H, Fmoc CH2), 4.35 (m, 1H, α-

CH), 4.23 (t, J = 6 Hz, 1H, Fmoc CH), 2.32 (m, 1H, CHCH(CH3)2), 1.04 (d, J = 7 Hz, 3H,

13 CHCH(CH3)2) 0.97 (d, J = 7 Hz, 3H, CHCH(CH3)2); C NMR (CDCl3-d, 100 MHz): δ 199.9,

156.6, 143.9, 141.5, 128.0, 127.3, 125.3, 125.0, 120.2, 67.3, 65.3, 47.5, 29.4, 19.3, 17.9.

O Fmoc-Trp (Boc)-aldehyde (I-40f) 1H NMR (CDCl -d, 400 MHz): δ 9.66 (s, FmocHN 3 H 1H, CHO), 8.13 (s, 1H, indole CH), 7.76 (d, J = 6.4 Hz, 2H, Fmoc aromatic

N Boc CH), 7.60-7.26 (m, 10H, Fmoc aromatic CHs, indole CHs), 5.50 (d, J = 6.8 Hz,

1H, NH), 4.61 (m, 1H, Fmoc CH2), 4.41 (m, 1H, Fmoc CH2), 4.22 (m, 1H, Fmoc CH), 3.74 (m,

1H, α-CH), 3.32 (m, 1H, CH2), 3.23 (m, 1H, CH2), 1.65 (s, 9H, C(CH3)3).

O Rink resin-β-alanine(Fmoc). Rink amide resin (300 mg, ~ 0.21 mmol of N NHFmoc H free sites) was allowed to swell in DMF in a clean, silanized SPPS filter

vessel. The Fmoc protecting group was cleaved with the standard method described above, 20%

piperidine in DMF (5 mL for 5 min, DMF wash, 5 mL for 20 min). The resin was washed with

DMF and CH2Cl2 (3 x 10 mL of each), then with DMF. Fmoc-β-alanine (196 mg, 0.63 mmol)

was dissolved in DMF (2 mL) and added to the resin. HATU (236 mg, 0.63 mmol), HOBT (84 mg, 0.63 mmol) and DIEA (0.25 mL) were dissolved in DMF (5 mL) and added to the resin. The mixture was shaken for 1 h. The reagents were drained away and the resin was washed with

MeOH, DMF, and CH2Cl2 (3 x 10 mL of each). The resin was dried under vacuum and

transferred to a silanized glass vial for storage.

General Procedure for Polyamine Formation. Rink resin-β-alanine(Fmoc) (56 mg, ~ 0.035

mmol loading) was allowed to swell in a clean silanized filter vessel. The Fmoc group was

deprotected and the resin was washed with DMF and CH2Cl2 (3 x 10 mL of each). A Kaiser test

on the beads gave a blue result indicating the presence of a primary amine. The resin was washed

with dry CH2Cl2 (2 x 10 mL). The aldehyde, e.g. Fmoc-Ser(t-Bu)-aldehyde (39 mg, 0.11 mmol),

was added to the resin as a solution in dry CH2Cl2 (5mL). The mixture was shaken for 10 min,

the aldehyde was drained away and the resin was rinsed with dry CH2Cl2 (3 x 5 mL). Dry

CH2Cl2 (5 mL) was added, followed by NaHB(OAc)3 (23 mg, 0.11 mmol). The mixture was

shaken vigorously to dissolve the solid, then shaken for 45 min. The reagents were drained away

and the resin was washed with MeOH, DMF, and CH2Cl2 (3 x 10 mL of each). A Kaiser test on a

few beads showed rust brown, indicating the formation of a secondary amine.

The resin was washed with dry CH2Cl2 (2 x 5 mL). Di-tert-butyl dicarbonate (76.3 mg,

0.35 mmol) and DIEA (24 µL) were added to the resin as a solution in dry CH2Cl2 (3 mL). The

mixture was shaken for 2 h. The reagents were drained away and the resin was washed with

CH2Cl2 and DMF (3 x 10 mL of each). A Kaiser test on a couple of beads gave a negative result

indicating that the secondary amine had been protected. The Fmoc group was removed as

described above. A Kaiser test on a couple of beads showed deep blue, indicating the presence of a primary amine again.

The above steps were repeated to add the second and third residues. In these cases, higher quantities of aldehyde were needed to ensure complete coupling (5 equiv, 0.18 mmol). In some cases a Kaiser test after the reductive amination step gave a negative result instead of a rust brown color, presumably because the secondary amine was too hindered. Boc protection followed by Fmoc deprotection produced a primary amine. In these cases the reductive amination was assumed to have been successful.

Cleavage from the resin. The resin was shaken in 20% TFA, 1% Et3SiH, in CH2Cl2 for 10 min.

This was found to cleave most of the polyamine. Note: longer times gave higher amounts of

impurity from resin degradation. The resin was removed by filtration and the solution was

concentrated under a stream of nitrogen. In order to remove all of the Boc groups, the residue

was dissolved in CH2Cl2, then TFA and Et3SiH were added to obtain a final concentration of

50% TFA, 5% Et3SiH. This mixture was stirred for 1 h. The solution was concentrated under a

stream of nitrogen and the residue was dissolved in 95% TFA, 5% Et3SiH. This step was

necessary to completely deprotect the serine t-butyl ether. This mixture was stirred for 16 h. The

solution was evaporated under a stream of nitrogen and the residue was precipitated with diethyl

ether (20 mL). The solution was decanted off; the product was dried briefly under nitrogen, and

then dissolved in H2O (5 mL) and purified by HPLC (See General Procedures).

Fractions collected from the HPLC were reduced to dryness using a vacuum centrifuge

(speed vac) to give a white powder, and immediately redissolved in autoclaved water. In our

77 experience the polyamines soon turned brown if left as a completely dry solid, even though the molecule should be present as the tetratrifluoroacetate salt. It is unclear why this occurs, but evidence from mass spectrometry data suggests oxidation, perhaps of the primary amine. The polyamines were stored in water at –20 °C until needed.

Quantification of Polyamines. The concentrations of polyamine solutions were calculated by

UV absorption using the extinction co-efficients given below. These were based on the known extinction coefficients of the bases tyrosine and phenylalanine when found in solution.104 It was assumed that extinction co-efficients were cumulative and were not affected by the substitution of a peptide backbone for a polyamine backbone. The calculated concentrations were found to be in good agreement with those calculated from the mass of dried product, so these extinction coefficients were used in further concentration calculations.

Polyamine E(270 nm) / mol-1cm-1L E(261 nm) / mol-1cm-1L

YYY 4336 YSY 2891 YVV 1445 FFF 672 FSF 448 FSS 224 Table 1.4 Calculated extinction coefficients for six polyamines.

Binding assays based on 2-aminopurine fluorescence. The fluorescence of the samples was recorded using a fluorescence plate reader. This allowed rapid detection of multiple samples using very small volumes. However, there is noise inherent in using this technique, so all

samples were prepared in duplicate and the results were averaged. The modified RNAs used

were 2AP 72 RRE and 2AP 27 TAR. Both RNAs were prepared in 10 µM batches in autoclaved

water that were heated to 100 ºC for 2 min, then cooled rapidly in a dry ice / isopropanol bath.

This snap-cooling causes the RNA to adopt the kinetically favored hairpin rather than

thermodynamically favored duplexes. Polyamine stock solutions were prepared in pure

autoclaved water. Samples were prepared to have a final concentration of 400 nM RRE 2AP72

or 800 nM TAR 2AP27, 100 mM NaCl, 20 mM HEPES, 1 mM MgCl2 in 80 µL. Concentrations

of polyamine were varied between 0 and 1 mM. The samples were incubated at room

temperature for 2 h, then 75 µl was transferred to a well in a 384 well opaque plate (Corning).

Fluorescence at 372 nm following excitation at 310 nm was recorded. Fluorescence was plotted

against concentration of polyamine to give a binding curve, which was then fitted in Origin 6.1

(Origin Lab Corporation) using the equation for single-site binding created by Lacouciere et al.65

The KDs were calculated from the fit, and errors were based on deviancy from the curve.

2 1/2 F = Fo - {(Fo – Ff)/2[RNA]tot}.{b – (b – 4[L]tot[RNA]tot) }

Where: b = KD + [L]tot + [RNA]tot

Equation 1.1 Single-site binding equation, adapted from Lacourciere et al. Fo and Ff are initial

and final fluorescence intensities, [RNA]tot is the total concentration of RNA, [L]tot is the total

concentration of ligand, which is the independent variable.

The data were also fit to a second equation reported by Blount and Tor.105

n n F = {Fo + Ff.([L] /KD)} / {1 + ([L] /KD)}

Equation 1.2 Multi-site binding equation, adapted from Blount and Tor. Fo and Ff are initial and final fluorescence intensities, [L] is the ligand concentration, n is the number of ligands per RNA molecule.

This equation had the advantage that it did not assume a 1:1 stoichiometry, and the number of ligands binding, n, could be determined. In all cases the n value for the best fit was approximately 1, and fixing n as 1 generated KDs that were very similar to those obtained from the Lacourciere equation. The equation does suffer from the fact that it assumes that the RNA concentration is very much smaller than the KD, which might not be the case.

Preparation of RNA for footprinting assay. The two RNAs were prepared by in vitro transcription of DNA templates with T7 RNA polymerase. The DNA strands (see below) were purchased from IDT and were premixed to a final concentration of 10 µM, heated to denature the strands and allowed to re-anneal. Transcription reactions were carried out in 40 mM Tris HCl, pH 8.0, 10 mM DTT, 0.05 mg / mL BSA, 20 mM Mg(OAc)2, 2.5 mM ATP, 2.5 mM GTP, 2.5 mM UTP, 2.5 mM CTP, 2 mM spermidine, 25 mM NaCl, 0.2 mg / mL T7 RNA polymerase.

The reaction mixture was incubated for 4 h at 37 ºC. 0.5 U Promega RQ1 DNAse were added and the mixture was incubated for 15 min at 37 ºC. Pyrophosphate that had precipitated out was

removed by centrifugation. The supernatant was removed, EDTA was added (final concentration

20 mM), and the RNA was ethanol precipitated: first 3M NaOAc was added to give a final concentration of 300 mM, then 2.5 volumes of ethanol were added, and finally the tube was vortexed and incubated 1 h at –20 ºC. The RNA was precipitated by centrifugation (30 min, 14

000 rpm) and the supernatant was removed. The RNA was washed with 70% ethanol, allowed to dry and redissolved in 300 µL 20 mM HEPES. 100 µL formamide loading dye (80% formamide,

10 mM EDTA, 0.5 mg / mL bromophenol blue, 0.5 mg / mL xylene cyanol) was added.

Transcription products were purified using a 12% denaturing polyacrylamide gel. The portion of gel containing the RNA was identified by UV shadowing over a silica TLC plate, then excised with a sterile razor. The RNA was crush-soak eluted out of the gel (300 mM NaOAc, 0.5 µM

EDTA, 4 ºC overnight) then ethanol precipitated (as above) and redissolved in 5 mM HEPES,

pH 7.5, to a final concentration of 15 µM.

Prior to analysis, RNA was dephosphorylated using shrimp alkaline phosphatase (SAP) and 5’ end-labeled using T4 polynucleotide kinase (T4 PNK). Phosphatase reactions (50 µL)

containing ≈ 400 pmol RNA, 50 mM Tris-HCl (pH 8.5 at 20 ºC), 5 mM MgCl2 and 5U SAP,

were incubated at 37 ºC for 1 h. The enzyme was denatured by heating to 65 ºC for 15 min, and

the RNA was precipitated with ethanol and redissolved in dH2O. Kinase reactions (10 µL)

containing ≈ 10 pmol dephosphorylated RNA, 70 mM Tris-HCl (pH 7.6 at 25 ºC), 10 mM MgCl2,

5 mM DTT, ≈ 10 pmol [γ-32P]-ATP, and 20 U T4 PNK were incubated at 37 ºC for 3 h. End-

labeled RNAs were purified by 15% PAGE, isolated by crush-soak elution, precipitated with

ethanol, redissolved in water, and quantified by liquid scintillation counting. The labeled RNA

was separated into several small batches (50 µL), which were heated to 95 ºC for 2 min then

81 immersed in a dry ice / ethanol bath for 5 min. This procedure was performed to encourage correct folding of the RNA. RNA batches were stored at –20 ºC until needed, whereupon they were transferred to ice to allow to thaw.

DNA Templates

TAR(+): 5’-TAA TAC GAC TCA CTA TAG GCC AGA TCT GAG CCT GGG AGC TCT

CTG GCC- 3’

TAR(-): 5’- GGC CAG AGA GCT CCC AGG CTC AGA TCT GGC CTA TAG TGA GTC

GTA TTA- 3’

RRE(+): 5’- TAA TAC GAC TCA CTA TAG GTC TGG GCG CAG CGC AAG CTG ACG

GAA CAG GCC- 3’

RRE(-): 5’- GGC CTG TTC CGT CAG CTT GCG CTG CGC CCA GAC CTA TAG TGA GTC

GTA TTA- 3’

Terbium Cleavage Assay. RNA cleavage was determined using 5’ 32P-labeled RNAs. Labeled

TAR or RRE hairpins were incubated in the presence of increasing concentrations of polyamines

FSS, FSF, FFF, YVV, YSY or YYY, in reactions (10 µL) containing ≈ 100 fmol 5’ 32P-labeled

RNA, 50 mM Tris-HCl (pH 6.5 at 20 ºC), 100 µM TbCl3, 10 mM MgCl2 and 100 mM NaCl.

Reactions were incubated at room temperature for 4 h, then terminated by the addition of an equal volume of gel loading buffer containing 80% formamide, 10 mM EDTA, 0.5 mg / mL bromophenol blue, 0.5 mg / mL xylene cyanol.

“In-Line Attack” Assay.101-103 RNA cleavage was determined using 5’ 32P-labeled RNAs.

Labeled TAR or RRE hairpins were incubated in the presence of increasing concentrations of

polyamines FSS, FSF, FFF, YVV, YSY or YYY, in reactions (10 µL) containing ≈ 100 fmol 5’

32 P-labeled RNA, 50 mM Tris-HCl (pH 8.0 at 20 ºC), 20 mM MgCl2 and 100 mM NaCl.

Reactions were incubated at room temperature for 44 h, then quenched with formamide loading

buffer.

Analysis of RNA Cleavage. RNA partial hydrolysis cleavage ladders were generated by

32 incubating ≈ 300 fmol 5’ P-labeled RNA in reactions (30 µL) containing 50 mM NaHCO3 (pH

9.0) for 8 min at 95 ºC. G-specific sequencing ladders were created by incubating ≈ 100 fmol 5’

32P-labeled RNA in reactions (10 µL) containing 4 mM sodium citrate pH 5, 0.2 mM EDTA, 840

mM urea, 0.12 µg / µL Torula Yeast RNA, 0.05 U T1 RNAse for 15 min at 55 ºC. Reactions

were quenched with equal volumes of loading buffer. Reaction products (3 µL) were separated

using a 20% denaturing polyacrylamide gel, and visualized using a PhosphorImager with

Imagequant software. The intensities of bands of interest were quantitated, and differences in sample loading were corrected for by dividing by the amount of unreacted RNA. Values were normalized to be a fraction of the RNA cleaved at each site, 0 being the minimum amount of cleavage, 1 being the maximum amount of cleavage. The apparent KD for each ligand was

determined by plotting the normalized fraction cleaved against the logarithm of ligand

concentration. Curve-fitting was performed using Kaleidagraph software. For fitting to the

terbium competition data, the equation y = 1 – (x/(x + KD)) was used. For the in-line attack assay

the equation y = x/(x + KD) was used.

A. B.

Figure 1.16 PAGE displaying cleavage products from (A) “In-line” cleavage of RRE, and (B) terbium catalyzed cleavage of TAR, with increasing concentrations of YYY.

YYY vs TAR, Bulge Region, In-line Cleavage YYY vs TAR, Bulge Region, Tb Cleavage 1 1

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0 0 10-1 100 101 102 103 10-1 100 101 102 103 [YYY] / µM

Figure 1.17 Graphs of normalized fraction of RNA cleaved vs polyamine concentration, displaying curve fitting.

FmocHN N Fmoc-Phe-Benzylamine (I-44). I-40d (55 mg, 0.15 mmol) was dissolved H

in dry CH2Cl2 (5 mL) in a clean RB flask equipped with a stir bar. While

stirring, benzylamine (17 µL, 0.15 mmol) was added to the flask. After ten minutes,

NaHB(OAc)3 was added, and the mixture was stirred for a further 45 min. The mixture was

diluted with CH2Cl2 (25 mL) and washed with saturated K2CO3 (3 x 30 mL), then dried over

anhydrous sodium sulfate. The solvent was removed in vacuo, and the product was purified by

1 flash column chromotography (SiO2, 75% EtOAc/hexanes). H NMR (400 MHz, CDCl3) δ 7.77

(d, J = 6.8 Hz, 2H, Fmoc aromatic CH), 7.58 (t, J = 6 Hz, 2H, Fmoc aromatic CH), 7.41 (m, 2H,

Fmoc aromatic CH), 7.33-7.17 (m, 7H, C6H5, Fmoc aromatic CH), 5.01 (m, 1H, OCONH), 4.39

(m, 2H, Fmoc CH2), 4.19 (m, 1H, Fmoc CH), 3.99 (m, 1H, α-CH), 3.71 (m, 2H, NHCH2C6H5),

+ 2.88-2.65 (m, 4H, CHCH2NH, CHCH2C6H5); ESMS m/z = 463 (M + H) .

Ph Benzyl (2E,4S)-4-[(9H-fluoren-9-ylmethoxy)carbonyl]amino-5-phenyl-

OBn FmocHN pent-2-enoate (I-45). I-40d (292 mg, 0.79 mmol) and benzyl- O (triphenylphosphoranylidene)acetate (355 mg, 0.87 mmol) were dissolved in dry THF (10 mL).

The mixture was heated under reflux at 50 ºC for 90 min. The reaction mixture was allowed to

cool, then quenched with saturated NH4Cl solution (20 mL). The product was extracted with

EtOAc (3 x 30 mL), washed with brine (3 x 30 mL), dried with anhydrous sodium sulfate and

concentrated in vacuo. The crude product formed a white foam solid under reduced pressure.

Pure I-45 (266 mg, 67%) was isolated by flash column chromatography (SiO2, EtOAc/hexanes,

1 1:4, Rf = 0.30). H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 6.8 Hz, 2H, Fmoc aromatic CH), 7.5

(m, 2H, Fmoc aromatic CH), 7.4–7.26 (m, 12H, C6H5 (Phe), C6H5 (Bn), Fmoc aromatic CH),

6.96 (m, 1H, CH=CHCO), δ 5.89 (d, J = 16.4 Hz, 1H, CH=CHCO), 5.23–5.18 (m, 3H,

OCH2C6H5, NH), 4.70 (m, 2H, CH), 4.45 – 4.35 (m, 2H, Fmoc CH2), 4.16 (t, J = 6 Hz, 1H,

Fmoc CH), 2.90 (m, 2H, CHCH2C6H5).

Ph (4R)-4-[(9H-Fluoren-9-ylmethoxy)carbonyl]amino-5-phenyl-pentanoic OH FmocHN O acid (Fmoc-γ-PheOH, I-46). I-45 (266 mg, 0.52 mmol) was dissolved in

ethanol (10 mL) by heating to 75 ºC with stirring. The solution was cooled to room temperature

and 10% Pd / C (6 mg) was added. The flask was flushed with hydrogen gas and the mixture was

stirred under atmospheric pressure of hydrogen for 16 h. The mixture was filtered through

diatomaceous earth with CH2Cl2 washings, then concentrated to give I-41 (quantitative) as a white solid. Spectroscopic data were consistent with the literature.

Ph Me Fmoc-γ-Phe-Weinreb (I-47). I-46 (110 mg, 0.27 mmol) was dissolved in N FmocHN OMe O dry CH2Cl2 (5 mL) in a clean, dry flask, put under nitrogen, and cooled to

0 ºC. EDC (63 mg, 0.33 mmol) and DIEA (50 µL, 0.38 mmol) were added, and the mixture was

stirred for 10 min. N, O-dimethylhydroxylammonium chloride (40 mg, 0.41 mmol) and DIEA

(50 µL, 0.38 mmol) were then added to the flask. The solution was allowed to come to room temperature and stirred for 16 h. The reaction mixture was transferred with CH2Cl2 rinses (50

mL) to a separatory funnel, then washed with 2N HCl (3 x 30 mL), saturated NaHCO3 solution

(2 x 30 mL) and saturated NaCl solution (2 x 30 mL). The organic layer was dried over

anhydrous sodium sulfate, concentrated in vacuo, and dried under vacuum to give a white, sticky

solid. This was purified by flash column chromotography (SiO2, EtOAc / hexanes, 1:1) to give I-

1 42 (95 mg, 0.207 mmol, 78%). H NMR (CDCl3-d, 400 MHz): δ 7.77 (d, J = 7.2 Hz, 2H, Fmoc

aromatic CH), 7.56 (m, 2H, Fmoc aromatic CH), 7.38 (t, J = 6.8 Hz, 2H, Fmoc aromatic CH),

7.33–7.17 (m, 7H, Fmoc aromatic CH, C6H5), 4.95 (d, J = 8.4, 1H, NH), 4.41 (m, 1H, Fmoc

CH2), 4.30 (m, 1H, Fmoc CH2), 4.20 (m, 1H, Fmoc CH), 3.89 (m, 1H, NHCH), 3.60 (s, 3H,

OCH3), 3.15 (s, 3H, NCH3), 2.92–2.77 (m, 2H, CH2C6H5), 2.49 (m, 2H, CH2CH2CO), 1.81 (m,

+ 2H, CHCH2CH2); ESMS m/z = 458 (M + H) .

Reduction of I-47. I-47 (95 mg, 0.21 mmol) was dissolved in dry THF (5 mL) and cooled to 0

ºC under nitrogen. LiAlH4 (10 mg, 0.26 mmol) was added with stirring, the flask was put under nitrogen and stirred for 45 min at 0 ºC. After this time, the reaction was quenched by the addition of NaHSO4 (70 mg, 0.5 mmol) in H2O (5 mL), which was added dropwise. The mixture was

stirred for an additional 10 min before being transferred to a separatory funnel. Sat NaCl solution

(30 mL) and EtOAc (30 mL) were added to the mixture. The layers were separated and the

aqueous layer extracted with EtOAc (30 mL). The organic layers were combined and washed

with 1.5 N HCl (3 x 30 mL), saturated NaHCO3 solution (2 x 30 mL) and saturated NaCl

solution. (2 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated

on a rotary evaporator, and dried in vacuo to give a white powder.

Fmoc-Gln(Trt)Weinreb (I-49). Prepared by same method as I-39. 1H NMR

(CDCl3-d, 400 MHz): δ 7.76 (d, J = 7.6 Hz, 2H, Fmoc aromatic CH), 7.59 (t, J HN O

OMe = 7.6 Hz, 2H, Fmoc aromatic CH), 7.38 (m, 2H, Fmoc aromatic CH), 7.33 – N FmocHN Me O 7.23 (m, 17H, Fmoc aromatic CH, C(C6H5)3), 6.98 (s, 1H, CH2CONH), 5.76 (d,

J = 7.6 Hz, 1H, OCONH), 4.78 (m, 1H, αCH), 4.38 (d, J = 6 Hz, 2H, Fmoc CH2), 4.20 (t, J = 6.8

Hz, 1H, Fmoc CH), 3.65 (s, 3H, OCH3), 3.18 (s, 3H, NCH3), 2.36 (m, 2H, CH2CH2CO), 2.16,

13 1.82 (bm, 2H, CHCH2CH2); C NMR (CDCl3-d, 100 MHz): δ 172.2, 171.2, 156.8, 145.0, 144.2,

144.0, 141.6, 141.5, 128.9, 128.1, 127.9, 127.4, 127.1, 125.3, 120.2, 70.8, 67.1, 61.9, 50.8, 47.5,

32.4, 29.2; ESMS m/z = 654 (M + H)+.

Fmoc Gln Aldehyde. Prepared from I-49 by same method as I-40. 1H NMR

(CDCl3-d, 400 MHz): δ 9.43 (s, 1H, CHO), 7.69 (t, J = 7.6, 2H, Fmoc aromatic

CH), 7.57 (d, J = 6.8 Hz, 2H, Fmoc aromatic CH), 7.35 (t, J = 6.8 Hz, 2H, Fmoc HN O

aromatic CH), 7.29 – 7.18 (m, 17H, Fmoc aromatic CH, C(C6H5)3), 6.78 (s, 1H, H FmocHN O CH2CONH), 5.54 (d, J = 4.8, 1H, OCONH), 4.45 (m, 1H, αCH), 4.21 – 4.05 (m,

3H, Fmoc CH2 CH), 2.39 – 2.21 (m, 3H, CH2CH2CO, CHCH2CH2), 1.82 (m, 1H, CHCH2CH2);

13 C NMR (CDCl3-d, 100 MHz): δ 199.0, 171.2, 156.7, 144.8, 144.0, 141.7, 128.9, 128.3, 128.0,

127.4, 127.2, 125.3, 124.3, 120.3, 120.1, 71.0, 67.0, 59.7, 47.6, 30.0.

Fmoc Cit Weinreb (I-50). Prepared following same procedure as I-39. 1H O NH2 HN NMR (CDCl3-d, 400 MHz): δ 7.75 (d, J = 6 Hz, 2H, Fmoc aromatic CH), 7.62 OMe N FmocHN Me (m, 2H, Fmoc aromatic CH), 7.39 (m, 2H, Fmoc aromatic CH), 7.30 (m, 2H, O

Fmoc aromatic CH), 6.32 (d, J = 6 Hz, 1H, OCONH), 5.58 (s, 1H, CH2NHCO), 4.98 (s, 2H,

CONH2), 4.75 (br, 1H, αCH), 4.35 (d, J = 5.6 Hz, 2H, Fmoc CH2), 4.20 (t, J = 5.6 Hz, 1H, Fmoc

CH), 3.76 (s, 3H, OCH3), 3.26 – 3.14 (m, 5H, NCH3, CH2CH2NH), 1.82 – 1.28 (m, 4H,

13 CHCH2CH2, CH2CH2CH2); C NMR (CDCl3-d, 100 MHz): δ 173.0, 159.8, 156.8, 144.2, 144.0,

141.5, 129.0, 128.0, 127.4, 125.5, 120.2, 67.2, 61.9, 53.7, 51.1, 47.4, 40.3, 32.3, 30.2, 26.2.

Fmoc-Cit(Trt)-Weinreb (I-51). I-50 (109 mg, 0.25 mmol) and H O N triphenylmethanol (42 mg, 0.17 mmol) were dissolved in benzene (5 ml) HN

OMe that had been dried over 4 Å molecular seives in a dry three-necked flask N FmocHN Me O equipped with a stir bar. The apparatus was set up under Dean-Stark

conditions, with molecular seives in the benzene in the trap, as the quantities of water driven off

were small. The system was flushed with nitrogen, then p-toluene sulfonic acid (16 mg, 0.06

mmol) was added with stirring. The solution was heated to 98 ºC producing a yellow color in the

solution as the trityl cation formed. The mixture was heated for 6 h, then allowed to cool and

quenched with 2% aqueous sodium bicarbonate solution (10 mL). The product was extracted

with ethyl acetate (2 x 20 mL), washed with brine (2 x 20 mL) and dried over anhydrous sodium

sulfate. The crude product was purified by flash column chromotography (SiO2, EtOAc) to give

1 I-51 (50 mg, 43 % based on triphenylmethanol) as a white foam solid. H NMR (CDCl3-d, 400

MHz): δ 7.74 (m, 2H, Fmoc aromatic CH), 7.60 (m, 2H, Fmoc aromatic CH), 7.38-7.1 (m, 19H,

Fmoc aromatic CH, C(C6H6)3), 5.89 (m, <1H, OCONH), 5.71 (s, 1H, CH2NHCO), 5.33 (bs, 1H,

CONHC(C6H5)3), 4.55 (m, 1H, αCH), 4.37 (m, 2H, Fmoc CH2), 4.20 (m, 1H, Fmoc CH), 3.71 (s,

3H, OCH3), 3.18 (s, 3H, NCH3), 2.99 (m, 2H, CH2CH2NH), 1.44-1.12 (m, 4H, CHCH2CH2,

+ + CH2CH2CH2); ESMS m/z = 705 (50%, (M + Na) ), 1365 (20%, (2M + H) ), 1387 (100%, (2M +

Na)+).

Chapter 2

Synthesis and Evaluation of 2- and 4-Aminothiazole-based Inhibitors of Neuronal Nitric Oxide Synthase

Advisor: Richard B. Silverman 90

2.1. Biological role of nitric oxide

Nitric oxide (NO) is an uncharged gas with one unpaired electron. It reacts readily with other paramagnetic species and binds to metal centers.1 Its reactive free radical properties make it an unlikely molecule for use in biological systems, but in actual fact it is critical for a variety of physiological processes, and is a key player in cardiovascular systems, immunology and neural biology.2 The half life of NO in mammalian cells is approximately 5-10 seconds.3 In the case of NO produced in neuronal and vascular tissue, the NO diffuses through cell membranes and binds to the heme of soluble guanylate cyclase (sGC), which is then stimulated to convert

GTP into guanine 3’, 5’-cyclic monophosphate (cGMP).4 This in turn is a secondary messenger capable of triggering a wide variety of enzyme cascades, resulting in responses such as smooth muscle relaxation and activation of protein kinases (Fig 2.1).5

Figure 2.1 NO produced by nNOS triggers the formation of cGMP, a secondary messenger.

NO produced by endothelial cells is critical for blood pressure homeostasis and inhibits

platelet aggregation and leukocyte adhesion.6 In the central nervous system, NO is responsible

for memory formation, brain development and neurotransmission, and for long term

potentiation.7 NO produced by the immune system in response to proinflammatory signals, such as cytokines and bacterial lipopolysaccharides, has antimicrobial and cytotoxic properties.8 NO

.- itself can disrupt iron containing proteins, and when it is combined with superoxide (O2 ), also

produced by macrophages, it forms the highly oxidative peroxynitrite anion (ONOO-).9

2.2 The nitric oxide synthase family

The class of enzymes responsible for NO production, the nitric oxide synthases (NOS),

comprises three main isoforms; neuronal (nNOS),10 endothelial (eNOS)11 and inducible (iNOS).8

The three isoforms share only 50% sequence homology,2 but are structurally very similar.12 They

each consist of a C-terminal reductase domain and an N-terminal heme-containing oxygenase

domain joined by a calmodulin-binding linker. The cofactors NADPH, FAD, FMN and

tetrahydrobiopterin (H4B) are required for enzyme activity. The active enzyme exists as a homodimer, with electrons flowing from NADPH in the reductase domain of one monomer to the oxygenase domain of the other.13 Electron flow is prevented by an autoinhibitory loop in the calmodulin binding linker. When calmodulin is bound, the loop is removed, and the electrons can flow. The binding of calmodulin to nNOS and eNOS is Ca2+ dependent. These isoforms are

named the constitutive forms of NOS, as they are expressed at a low level at all times. They are

activated by increased cellular Ca2+ levels, which can occur as a result of both endocrine and

nervous stimulation. The binding of calmodulin to iNOS is very tight, so iNOS activity is not

Ca2+ dependent and regulation of iNOS is achieved at the transcriptional level.

The three isoforms catalyze the stepwise oxidation of L-arginine to L-citrullene using molecular oxygen, producing a molecule of NO (Fig 2.2).14 The oxygen in the NO and the

oxygen in the urea group of citrullene are derived from the molecular oxygen. The active sites of

the isoforms are very similar, and the Km for L-arginine is similar for each.

OH + H2N NH2 HN NH H2N O

NH NH NH O2,(NOS) O2,(NOS) + NO NADPH 0.5 NADPH

+ - + - + - H3N COO H3N COO H3N COO L-arginine L-citrullene

Figure 2.2 The formation of NO from L-arginine.

2.3 NO and disease

Although it plays an essential role in the regulation of many biological processes, NO is a

reactive molecule, and its over-production is detrimental and has been linked to a variety of

diseases. Acute over-expression of iNOS, leading to increased NO levels in the blood, can result

in septic shock.15 The death of pancreatic cells that ultimately leads to Type I diabetes has also

been linked to elevated iNOS levels.16, 17 Chronic overproduction of iNOS is believed to cause

joint damage leading to arthritis,18, 19 can result in destruction of photoreceptors in the retina,20

and can cause cerebral inflammation21 and inflammatory bowel disease. Increased glucosamine

in the diet inhibits iNOS expression and therefore helps to alleviate some of the problems

associated with chronic high iNOS levels.22

NO produced by eNOS causes vascular dilation resulting in a lowering of blood pressure.

As a result of this, overproduction of NO by eNOS could cause hypotension. In general, however, insufficient eNOS activity is much more harmful, as it can cause endothelial dysfunction,

hypertension and atherosclerosis, and can even lead to organ perfusion.23, 24

Although critical for proper neuronal signaling, overproduction of NO by nNOS has been

implicated in various neurological diseases, including Parkinson’s and neuronal damage due to

hypoxic conditions such as stroke.25-28 It has also been linked to Alzheimer’s29, 30 and

Huntington’s31 diseases, migraines,32 schizophrenia,33 amyotrophic lateral sclerosis34 and

seizures.35 Studies have shown that nNOS knock-out mice are protected from both cerebral

damage due to neonatal hypoxia-ischemia (HI),36 a condition caused by a lack of blood supply to

the fetus that results in cerebral palsy symptoms in the new born, and from the neurodegenerative

effects of MPTP, an agent that causes parkinsonian symptoms.27, 37-39 The administration of

nNOS inhibitors such as 2-aminobiotin,40 7-nitroindazole41 and aminoguanidine42 reduced brain

damage caused by HI, but also produced a sharp rise in blood pressure because of the fact that

these compounds are only moderately selective for nNOS over eNOS.43 Inhibition of nNOS

could have therapeutic benefit in many neurodegenerative disease states, but must be achieved

without significant inhibition of the other isoforms, as this could result in hypertension and other

side effects.44 Indeed it has been shown that an inhibitor of nNOS increased recovery after a

stroke, whereas a non-selective NOS inhibitor had a damaging effect because it decreased blood

flow to the site of the damage.45

The similarity of the three isoform active sites makes the design of selective inhibitors

extremely challenging.46 However, selective inhibitors will not only have therapeutic potential,

but will be useful tools for understanding the role NOS isoforms play in biological systems and

in diseases.

2.4 Selective inhibitors of nNOS

There has been an enormous effort by academic and industrial laboratories to identify

selective inhibitors of both nNOS and iNOS.47, 48 A variety of inhibitors based on modified L-

arginine were discovered,48, 49 but the selectivity for one isoform over the others was modest and

insufficient for the compounds to have therapeutic value. Other types of inhibitors include S-

alkyl-L-thiocitrullenes,50 amidines,51 guanidines,52 isothioureas,53 and heterocycles such as

aminopyridines54, 55 and imidazoles.56 However, in general, low selectivity has prevented these

compounds from entering clinical trials.57 For example, 1400W, an iNOS inhibitor with modest

selectivity,58 is toxic when administered in vivo due to its activity against eNOS.59 Clearly, for an

inhibitor to be of any value as a therapeutic, it must be extremely selective for the isoform of

interest. An understanding of the subtle differences among the isoform active sites has led to the

discovery of nNOS inhibitors with the level of selectivity needed.

One of the best non-selective inhibitors of NOS, L-nitroarginine, was used as the base for

a library of dipeptide inhibitors.60 Initially natural amino acids were coupled to nitroarginine, but using non-natural amino acids and then various amines allowed several potent and highly selective inhibitors of nNOS to be discovered (II-1 and II-2, Figure 2.3).48, 60-63 Removing the

internal amide and replacing it with an amine improved the potency further (II-3).64, 65 Despite

their high potency and selectivity in in vitro assays, these compounds showed poor activity in

vivo due to low bioavailability caused by their high polar surface area and the number of charges

and hydrogen bond donors. However, crystal structures of the inhibitors in the active sites of

nNOS and eNOS, along with single site mutations of the enzyme, gave valuable information about what characteristics an inhibitor needs to achieve high potency and selectivity.66

HN NHNO2 HN NHNO2 HN NHNO2 NH NH NH NH2 O H N H H H2N NH2 N N H N H N O 2 CONH2 2 O NH NH2 II-1 II-2 II-3

Ki (nNOS) = 130 nM Ki (nNOS) = 100 nM Ki (nNOS) = 50 nM n/e=1500 n / e = 1300 n/e=1300 n/i=200 n/i=300 n/i=300

Figure 2.3 Strucutres of L-nitroarginine-based inhibitors of nNOS with their inhibition constants

against nNOS. “n/e” and “n/i” are the selectivities for nNOS over eNOS and iNOS, respectively.

The two crystal structure of II-1 bound to the active sites of nNOS and eNOS were

compared (Fig. 2.4). The inhibitor adopts a different conformation in each active site, leading to

the difference in affinities. In rat nNOS, the α-amino group forms hydrogen bonds with Asp597,

leading to a strong charge-charge interaction. The analogous residue in bovine eNOS is Asn368,

which is uncharged. The α-amino group twists around to interact with a heme propionate group instead, leading to the different conformations.

Figure 2.4 Overlay of the conformations of II-1 bound to the active sites of nNOS (blue) and eNOS (purple). Key residues are highlighted for nNOS (pink) and eNOS (green).

It appears that one amino acid difference results in greater than a thousand fold difference in binding affinities. This hypothesis was confirmed using single-site mutation studies. The

Asp597 residue of nNOS was replaced with an asparagine. As expected, this resulted in a significant loss in binding affinity, and a crystal structure of II-1 in the nNOS (D597N) mutant showed that it now adopted a conformation similar to that seen in eNOS (Fig. 2.5).

Figure 2.5 Overlay of the conformations of II-1 bound to the active sites of nNOS wild type

(blue) and nNOS D597N (purple).

In addition, a mutant eNOS was created in which the Asn368 was replaced with an aspartic acid residue. A significant increase in binding affinity was observed, and the crystal structure of II-1 in eNOS (N368D) showed that the inhibitor had adopted a conformation similar to that seen in nNOS (Fig. 2.6). Although complete activity was not recovered, it is clear that this one residue is the key to the selectivity of these compounds.

Figure 2.6 Overlay of the conformations of II-1 bound to the active sites of nNOS wild type

(blue) and eNOS N368D (purple).

Using computer modeling,67 molecular fragments with the desired properties for binding but with more favorable pharmacokinetic properties68 were docked in the NOS active sites. For example, the nitroguanidine moiety was replaced with an aminopyridine ring. When the pyridine ring nitrogen is protonated, the group can form hydrogen bonds with Glu592 (nNOS) in the same way as a guanidine group can, but it is more lipophilic. A pyrrolidine ring formed charge-charge interactions with Asp 597 providing the required nNOS selectivity, and locked the molecule in a favorable conformation for binding. In this way, a new class of inhibitors was designed (Fig. 2.7).

H N

NH2 H2N N N H II-4

Figure 2.7 Molecular modeling diagrams of II-2 and II-4 bound to the active site of nNOS, and the structure of II-4.

Those compounds that bound tightly in silico were synthesized and found to possess good potency and selectivity for nNOS over the other isoforms. Modifications to the lead

100

structure discovered using a combination of structure-based and a more traditional SAR

approach gave II-5 and later II-6, both highly potent and selective inhibitors.

H H N N Cl H H H N N H2N N N N 2 N N F H H II-5 II-6

Figure 2.8 Structures of potent and selective aminopyridine-based nNOS inhibitors.

The addition of a methyl group at the 4-position of the aminopyridine ring resulted in almost a ten-fold increase in potency. It is believed that this group interacts favorably with a hydrophobic pocket in the active site.

The halogenated phenyl rings at the termini of these molecules were added for several reasons. On binding, the phenyl rings form hydrophobic interactions with a shallow hydrophobic binding pocket in nNOS (Fig. 2.9A). This pocket is not present in iNOS, and so, in addition to increasing the binding affinity for nNOS, the groups also increase selectivity over iNOS. In addition, a primary amine is converted to a secondary amine, thus decreasing its polar surface

area and number of hydrogen bond donors, and the overall lipophilicity of the molecule is

increased by the hydrophobic aromatic ring. These factors will increase the molecule’s ability to

diffuse through biomembranes. The halogens also decrease the potential for metabolic

degradation of the phenyl ring.69 As well as showing high in vitro activity, these compounds also

showed in vivo activity toward protection of newborn rabbit kits from perinatal cerebral injury,

101 and had no effect on systolic or diastolic blood pressure when administered via intra-aorta injection.70

Compounds II-5 and II-6 were cocrystallized with rat nNOS. When racemic II-6 was crystallized with nNOS (Fig 2.9A), only the 3’S,4’S enantiomer (configuration shown in Fig.

2.8) was seen in the active site, as predicted. Enantiomerically pure II-6 showed an even higher potency towards nNOS. Figure 2.9B shows how the actual binding conformation of II-5 seen in the crystal structure closely matches the conformation predicted by molecular modeling. This validates the molecular modeling approach and is encouraging for future predictions based on this method.

102

Figure 2.9 (A) Crystal structure of II-5 bound to the active site of nNOS showing the important interactions with key residues. (B) Predicted conformation of II-5 in the active site of nNOS

(gold) versus the actual conformation (blue).

103

2.5 Rationale for the synthesis of aminothiazole-based inhibitors

Although the aminopyridine-based inhibitors were designed with bioavailability in mind,

they still possess four basic functional groups. The active sites of the NOS isoforms contain

multiple acidic groups, and these interact strongly with the basic parts of the inhibitor, leading to

high potency. However, it was feared that multiple cationic charges and hydrogen bond donating

groups could be detrimental for crossing the blood brain barrier, as it was in the case of the L- nitroarginine-based inhibitors.71, 72 Replacing one of these basic groups with a non-basic or less

basic moiety may improve the pharmacokinetic properties of the molecule. The first

modification will be described in this chapter; further modifications will be covered in Chapters

3 and 4.

The conjugate acid of the aminopyridine ring nitrogen has a pKa of approximately 7, so it exists as both the protonated and neutral forms at physiological pH.73 In the active site, the group

interacts through hydrogen bonding and a charge-charge interaction with Glu592, and so the ring

nitrogen must be protonated for tight binding. The ring nitrogen of an aminothiazole has a lower

pKa (approximately 6), and therefore only a small fraction is protonated at pH 7.4. However, in

the highly acidic environment of the NOS active site it could be sufficiently protonated to allow

a strong interaction with Glu592. To test this hypothesis, 2-aminothiazole II-7 and 4-

aminothiazoles II-8a-c were synthesized. It was believed that the 5-position of the 4-

aminothiazole could be substituted with a variety of alkyl groups to probe interactions with the

hydrophobic pocket present in the active site, further increasing the potency of these compounds.

The synthetic routes gave racemic final products, but the 3’S,4’S-enantiomers shown in Fig. 2.10

104 were expected to be the more active enantiomer, as was the case with the aminopyridine-based analogs.

H R H N N S S Cl H N Cl H2N 2 H N H N N N N N H H II-7 II-8a,R=H II-8b,R=Me II-8c,R=iPr

Figure 2.10 Structures of desired 2- and 4-aminothiazole-based nNOS inhibitors.

2.6 Synthesis of aminothiazoles

Precursors to inhibitors II-5 and II-6 were prepared using the route outlined in Scheme

2.1. 2-Amino-4,6-dimethylpyridine was protected with a tert-butoxycarbamate (Boc) group to form II-9. 3-pyrroline was protected with a Boc group and converted to epoxide II-10. The methyl groups of II-9 were deprotonated with butyllithium, and the resulting anions were treated with II-10. A mixture of trans-alcohols, resulting from attack on the epoxide by either methyl group, was then separated to give the desired trans-alcohol II-11.

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Boc i iii N

H N N 2 BocHN N BocHN N OH II-9 II-11

H Boc N ii N

O II-10

o i) Boc2O, tBuOH, 55 C, 48h; ii) Boc2O, MeOH, rt, 16h, then mCPBA, CH2Cl2,rt,48h; iii) nBuLi, THF, -78 oC-rt,30min,thenII-10,THF,-78oC-rt,4h.

Scheme 2.1 Synthetic route to racemic trans-alcohol II-11.

It was first believed that thiazole trans-alcohol II-13 could be prepared using an

analogous step. Commercially available 2-amino-4-methylthiazole was Boc protected to give II-

12. II-12 was treated with butyllithium followed by epoxide II-10 under conditions similar to

those used to generate II-11. However, none of the desired product was formed. The conditions

were varied, including longer reaction times and different temperatures, but there was no desired

product formed. To determine whether the desired dianion was being formed, II-12 was treated

with butyllithium and the reaction was quenched with D2O. Analysis of the NMR revealed that

the deuterium had been incorporated into the ring at the 5-position (II-14), not into the methyl

group. It was hoped that the 5-position could be protected.74 Following treatment of II-12 with

butyllithium, chlorotrimethylsilane was added, resulting in the formation of II-15. However,

when II-15 was treated with nBuLi and epoxide II-10, there was still no formation of the desired

product. As well as the lack of acidity of the protons in the 4-methyl group, there was also a

problem with the nucleophilicity of the carbanion.

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Boc S i S ii S N H N BocHN BocHN 2 N N N OH II-12 II-13 D iii S S BocHN N BocHN N II-12 II-14 Boc TMS TMS iv N S S ii S BocHN BocHN N BocHN N N OH II-12 II-15 II-16

o o o i) Boc2O, tBuOH, 55 C, 48h; ii) nBuLi, THF, -78 C-rt,30min,thenII-10,THF,-78 C-rt, o o 4h; iii) nBuLi, THF, -78 C-rt,30min,thenD2O; iv) nBuLi, THF, -78 C-rt,30min,then TMSCl, THF, -78 oC-rt,4h.

Scheme 2.2 Attempts to synthesize racemic II-13 and II-16 using a route analogous to that used

to prepare II-11.

As the anions of 4-amino-2-methylthiazoles might have different electronic properties,

they may have been more capable of opening up epoxides. However, 4-amino-2-methylthiazole

is not commercially available and 4-aminothiazoles are not well known in the literature. It was

envisioned that the appropriate Boc protected compounds could be synthesized using the route outlined in Scheme 2.3. Thioacetamide was condensed with either ethyl bromopyruvate to give thiazole II-18a, or with epoxides II-17a-c to give thiazoles II-18b-d.75 The esters were

hydrolyzed to give II-19a-d, and a Curtius rearrangement was carried out in tert-butanol to give

Boc protected 4-aminothiazoles II-20a-d.76 Thiazoles II-20a-d were treated with nBuLi and

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epoxide II-10, but none of the desired product was formed, despite numerous attempts and the

use of a variety of conditions.

O O R i Cl EtO EtO O Cl Cl

II-17a R=Me II-17b R=Et II-17c R=iPr

R R R S ii S iii S iv S NH 2 EtOOC N HOOC N BocHN N

II-18a R=H II-19a R=H II-20a R=H II-18b R=Me II-19b R=Me II-20b R=Me II-18c R=Et II-19c R=Et II-20c R=Et II-18d R=iPr II-19d R=iPr II-20d R=iPr

R Boc R S N v S BocHN BocHN N N OH II-20a-d II-21a-d

o i) NaOMe, RCHO, MeOH or EtOH, Et2O, 0 C, 2h; ii) ethyl brompyruvate or II-17a-c, EtOH, reflux, 5h; iii) 1N NaOH(aq), MeOH, 16h; iv) TEA, DPPA, 3Ao mol. sieves, tBuOH, 50 oC, 16h; v) nBuLi, THF, -78 oC-rt,30min,thenII-10,THF,-78oC-rt,4h.

Scheme 2.3 Synthesis of Boc-protected 4-aminothiazoles.

A different synthetic route to the intermediates II-21a-d proved more successful.

Acetonitrile was deprotonated with LDA, and the resulting anion was used to open epoxide II-10

to give nitrile II-22. This was refluxed with ammonium sulfide to give thioamide II-23 in low

yields.77 Unreacted II-22 could be recovered, thus improving the efficiency of the step. An alternative route to the thioamide involved oxidation of the nitrile group to a primary amide with

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hydrogen peroxide, followed by conversion to the thioamide with Lawesson’s reagent. However, the ammonium sulfide route was simpler and more efficient. Thioamide II-23 was condensed

with either ethyl bromopyruvate or epoxides II-17a or II-17c. This step produced an equivalent

of HBr or HCl, which was acidic enough to deprotect the Boc group. Buffering the reaction with

triethylamine resulted in incomplete thiazole formation. Apparently, an acidic environment is

needed for the final dehydration step of the reaction.78 The problem could be solved by simply

neutralizing the reaction mixture upon completion and adding tert-butyl dicarbonate to replace

the Boc group. The esters of II-24a-c were hydrolyzed and a Curtius rearrangement was carried

out to form 4-aminothiazoles II-21a,b,d. The trans-alcohols were converted to cis-amines II-

27a-c using a Mitsunobu reaction followed by appropriate deprotection. In the case of aminopyridine II-11, a Mitsunobu reaction cannot be carried out without further protection due

to the interference of an unwanted intramolecular cyclization (See Chapter 3). However, there

was no such problem in the case of the aminothiazoles, presumably because the thiazole ring

nitrogen is less nucleophilic than the pyridine one.

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Boc Boc Boc N i N N ii S iii NC H N O OH 2 OH II-10 II-22 II-23

R Boc R Boc R Boc N N N S v S vi S R'OOC BocHN BocHN N N N OH OH R' II-24a R=H,R'=Et II-21a R=H II-26a R=H,R'=NPhth II-24b R = Me, R' = Me II-21b R=Me II-26b R=Me,R'=NPhth II-24c R=iPr,R'=Me II-21d R=iPr II-26c R=iPr,R'=NPhth iv vii II-25a R=H,R'=H II-27a R=H,R'=NH2 II-25b R=Me,R'=H II-27b R=Me,R'=NH2 II-25c R=iPr,R'=H II-27c R=iPr,R'=NH2

o i) LiCH2CN, THF, 0 C, 4h; ii) (NH4)2S (aq), MeOH, 16h; iii) ethyl brompyruvate (for R = H) or II-17a,c, o MeOH, reflux, 5h, then DIEA, Boc2O, rt 16h; iv) 1N NaOH (aq), MeOH, rt, 16h; v) DPPA, TEA, 3A mol. sieves, tBuOH, reflux, 16h; vi) PPh3, DIAD, phthalimide, THF, rt 16h; vii) H2NNH2 (aq), MeOH, rt, 16h, then 2N HCl, rt, 30 min.

Scheme 2.4 Alternative approach to 4-aminothiazole synthesis.

Ethyl glycinate was alkylated with 4-chlorobenzyl chloride to give II-28. The amine was

Boc protected (II-29) and the ester was hydrolyzed (II-30). The resulting acid was converted to a

Weinreb amide, which was then reduced to aldehyde II-32. Aldehyde II-32 and amines II-27a-c were condensed to form imines, which were reduced with sodium triacetoxyborohydride to give amines II-33a-c. Deprotection of the Boc groups with 4N HCl gave II-8a-c as salts.

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Cl O Cl Cl i O iii O v R Boc EtO N N EtO R NH2 Cl II-28,R=H II-30,R=OH iiII-29,R=Boc iv II-31, R = N(OMe)Me

R Boc Cl N O vi S Cl vii Boc BocHN Boc II-8a-c N N N H N H II-32 II-33a R=H II-33b R=Me II-33c R=iPr

i) DIEA, MeOH, rt, 16h; ii) Boc2O, MeOH, 2h; iii) 1N NaOH (aq), MeOH; iv) EDC, HOBT, HN(OMe)Me, DIEA, rt, o 16h; v) LiAlH4,THF,0 C, 1h; vi) II-27a-c, MeOH, 30 min., then Na HB(OAc)3, rt, 1h; vii) 4N HCl, dioxanes, rt, 16h.

Scheme 2.5 Completion of 4-aminothiazole inhibitors.

If the residue was dissolved in an organic solvent such as methanol, a mass spectrum could be obtained showing that the desired compound had been formed. However, once the compound was dissolved in water, it decomposed, such that the desired peak in the MS was no longer present. Analysis of the 1H NMR revealed that the thiazole was present as the thiazoline tautomer. This was particularly apparent in the case of the 5-methylthiazole, II-8b. In the 1H

NMR, the methyl peak was present as a doublet at 1.44 ppm, instead of a singlet at about 2.3

ppm. In the 13C NMR, the peak from the methyl group was present at 20.8 ppm instead of about

11 ppm, and there were no peaks at about 140 or 130 which would be present in an intact

thiazole. A thorough investigation of the literature revealed that 4-aminothiazoles are known to

undergo tautomerization followed by hydrolysis to give thiazolines (Fig. 2.11).79 The data were

consistent with this mechanism of degradation. However, no conclusive proof could be found, as

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the compounds appeared to degrade further in water, ultimately turning brown and forming a

precipitate. The MS gave peaks at 276 / 278 (3:1) for all three compounds, suggesting a common

degradation mechanism for all three that eliminated the thiazole portion containing the R group.

Due to the degradation, no mass of the compounds and no elemental analyses could be obtained,

but it is likely that the thiazolone was formed prior to decomposition.

R R H R In water: S S S Decomposition H N HN O 2 N N N

Figure 2.11 Tautomerization, hydrolysis and decomposition of 4-aminothiazoles in water

2-Aminothiazoles are stable in water, and so a route to II-7 was still sought. It was

believed that a 2-aminothiazole could be constructed from the appropriate α-bromoketone, II-34.

An attempt to synthesize II-34 is shown in Scheme 2.6. The hydroxyl group of nitrile II-22 was

protected as a TBS ether (II-35). The nitrile group was reduced to an aldehyde (II-36), and a

Grignard reaction was used to add a methyl group to give II-37. Swern oxidation of the alcohol

gave ketone II-38.80-82 Nitrile II-35 could be converted to ketone II-38 directly by refluxing with

excess methyl Grignard, but the reaction was low yielding. Attempts to selectively brominate the

α-position of II-38 resulted in a mixture of regioisomers of the mono- and di-brominated compounds that could not be separated. An alternative route to II-34 was needed.

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Boc Boc Boc N N N i ii O iii NC NC H OH OTBS OTBS II-22II-35 II-36

Boc Boc Boc N N N HO iv O O Br OTBS OTBS OTBS II-37 II-38 II-34

o o o i) TBSCl, imidazole, DMF, 35 C, 16 h; ii) LAH, THF, 0 C, 1h; iii) CH3MgBr, THF, 0 C, 1h; o iv) (COCl)2,DMSO,TEA,CH2Cl2,-78 C, 1h;

Scheme 2.6 First attempt to synthesize α-bromoketone II-34.

Epoxide II-10 was opened with allyl Grignard to give II-39. A variety of conditions were used before the discovery that addition of the epoxide to the Grignard reagent in ether at 0 °C gave high yields of the desired product almost instantly. The hydroxyl group was protected as a tert-butyldimethylsilyl (TBS) ether (II-40), and the alkene was converted to an epoxide (II-41).

The epoxide was opened with bromide under acidic conditions83 to give a mixture of two diastereomeric bromohydrins, but both could be oxidized to bromoketone II-34.82 Bromoketone

II-34 was condensed with thiourea to give 2-aminothiazole II-43. The amino group was di-Boc protected, and the TBS ether was removed. A phthalimide group was installed with cis stereochemistry to give II-46. Cleavage of the phthalimide group with hydrazine also resulted in loss of one of the Boc groups protecting the aminothiazole, to give cis-amine II-47.

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Boc Boc Boc Boc N N N N i iii iv HO v O

OTBS O OR OTBS Br II-10 II-41 ii II-39,R=H II-42 II-40,R=TBS

Boc Boc Boc N N N O vi S ix S R'2N BocN N N OTBS OR'' Br R' R'' II-43,R'=H,R''=TBS II-34 vii II-46, R' = Boc, R'' = NPhth II-44, R' = Boc, R' = TBS x viii II-45,R=Boc,R''=H II-47,R'=H,R''=NH2 i) AllylMgBr, ether, 0 oC, 15 min.; ii) TBSCl, imidazole, DMF, 35 oC, 16h; iii) m-CPBA, rt, 48h; iv) LiBr, AcOH, o THF,rt,16h;v)(COCl)2,DMSO,TEA,CH2Cl2,-78 C, 1h; vi) thiourea, EtOH, reflux, 5h; vii) Boc2O(2.5eq), DMAP, THF, rt, 16h; viii) TBAF, THF, rt 16h; ix) PPh3, DIAD, phthalimide, THF, rt 16h; x) H2NNH2 (aq), MeOH, rt, 16h, then 2N HCl, rt, 30 min

Scheme 2.7 Route to 2-aminothiazoles.

II-47 was coupled with aldehyde II-32 in a reductive amination reaction to give II-48.

The Boc groups were removed with 4N HCl to give II-7 as a salt.

Boc N i S Cl ii II-47+ II-32 BocHN II-7 N Boc N N H II-48

i) MeOH, 30 min., then Na HB(OAc)3, rt, 1h; ii) 4N HCl, dioxanes, rt, 16h.

Scheme 2.8 Completion of 2-aminothiazole inhibitor II-7.

While this synthesis was being carried out, it was found that replacing the 4-chlorobenzyl group with a 3-fluorophenethyl group (compare II-5 and II-6) resulted in a modest increase in

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affinity for nNOS. Amine II-47 was coupled with aldehyde II-53, which had been prepared by a

route analogous to that used to prepare II-32, to give II-54. This was deprotected to give II-55.

NH2 O O O i R iii Boc N F N F EtO + EtO R Br F II-49,R=H II-51,R=OH ii II-50,R=Boc iv II-52, R = N(OMe)Me

Boc N O S v Boc vi N F BocHN Boc H N N F N H II-53 II-54

H N S vii H N H .4HCl 2 N N F N H II-55

i) DIEA, MeOH, rt, 16h; ii) Boc2O, MeOH, 2h; iii) 1N NaOH (aq), MeOH; iv) EDC, HOBT, HN(OMe)Me, o DIEA, rt, 16h; v) LiAlH4,THF,0 C, 1h; vi) II-47, MeOH, 30 min., then Na HB(OAc)3, rt, 1h; vii) 4N HCl, dioxanes, rt, 16h.

Scheme 2.9 Alternative 2-aminothiazole inhibitor.

2.7 Results and discussion

nNOS and iNOS were overexpressed in E.coli and purified following literature procedures.84, 85

eNOS was obtained in an impure form and washed and concentrated before use.86 Compounds

II-7 and II-55 were tested for their ability to inhibit the three isoforms using the standard hemoglobin capture assay (See Experimental Procedures for details).87 The results are shown in

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Table 2.1 along with the inhibition data for the corresponding 2-aminopyridine analogs, II-5 and

II-6.

K (nNOS) / K (eNOS) / K (iNOS) / n/e n/i Compound i i i µM µM µM selectivity selectivity

II-5 0.085 85 9 1000 100 II-6 0.014 28 4 2000 300 II-7 10 760 12 76 1.2 II-55 1.7 28 12 16 7 Table 2.1 Inhibition constants of 2-aminothiazoles against the three NOS isoforms.

Replacement of the aminopyridine group with an aminothiazole results in a 100-fold loss in potency against nNOS. A large portion of the loss comes from the fact that the pKa of the ring nitrogen is an order of magnitude lower for the aminothiazole than for the aminopyridine. It has been shown that different substituents on an aminopyridine ring affect the compound’s affinity for NOS.55 It was hypothesized that electron withdrawing groups, such as halogens, lower the pKa of the ring nitrogen to the point where it is no longer significantly charged at physiological pH. This means that it does not engage in a charge-charge interaction with Glu592 of nNOS, nor can it hydrogen bond to that residue. In that study, 2-aminopyridine was found to have an IC50 of

1.9 µM against iNOS, and 2-amino-4-methylpyridine an IC50 of 0.17 µM. However, replacing

the methyl group with a chlorine atom, to give 2-amino-4-chloropyridine, resulted in almost a

total loss of affinity (IC50 >50 µM). Sterically, a methyl group and a chlorine atom are very similar, suggesting that this effect is mostly electronic. The methyl group at the 4 position of the aminopyridine analogs II-5 and II-6 contributes a significant amount to binding due to an

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interaction with the hydrophobic pocket of the active site. This interaction is not present in the 2-

aminothiazoles II-7 and II-55.

2.8 Synthesis of aminoimidazoles

The ring nitrogens of 2-aminoimidazoles are basic (pKa ~9),73 such that at pH 7 both will

be protonated and the ring will carry a positive charge (Fig. 2.12).

H N HN RN + N H2N H H2N N H N N H F II-56

Figure 2.12 Protonation state of 2-aminoimidazoles at pH 7 and the structure of a potential

aminoimidazole-based nNOS inhibitor.

If the 2-aminopyridine ring of II-5 or II-6 is replaced with a 2-aminoimidazole (II-56), the

resulting compound should interact strongly with Glu 592 of nNOS. It is possible that the ring

nitrogen furthest from Glu 592 could be alkylated (II-56, R = Me, Et, iPr) to allow hydrophobic

interactions with the binding site pocket, just as the methyl group of II-5 and II-6 do. A route to

II-56 (R = H) was envisioned that made use of intermediates already in hand (Scheme 2.9).

Bromoketone II-34 and Boc protected guanidine were condensed to give the 2- aminoimidazole.88-90 Surprisingly, the major product was II-57, which has the ring nitrogen Boc

protected not the external amine. This suggests a potential route for making selectively alkylated

versions of II-56. The external amine of II-57 was di-Boc protected to give II-58, and the TBS

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group was removed. A Mitsunobu reaction using phthalimide as the nucleophile was attempted,

but none of the desired product was formed.

Boc Boc Boc N N N O i BocN ii BocN Br H N Boc N 2 N 2 N OTBS OTBS OTBS II-34 II-57 II-58

Boc Boc N N iii BocN BocN Boc N Boc N 2 N 2 N OH NPhth II-59

o i) Boc-guanidine, EtOH, 55 C, 16 h; ii) Boc2O, DMAP, THF, 16 h; iii) TBAF, THF, 3 h.

Scheme 2.10 Synthesis of 2-aminoimidazoles.

Whereas the monoprotected aminothiazole (e.g. II-21) can undergo the Mitsunobu

reaction, the monoprotected aminopyridine cannot, due to an undesired intramolecular

cyclization (See Chapter 3). Diprotecting the 2-amino group of the aminopyridine allows the

Mitsunobu to proceed, but in the case of the aminoimidazole, even the diprotected compound undergoes intramolecular cyclization. This problem could easily be circumvented by performing the Mitsunobu reaction earlier in the synthesis, for example on II-39, inverting the

stereochemistry and leaving the group in a protected form, e.g. as an azide. However, the

synthesis was aborted at this stage. Although the 2-aminoimidazoles might have interesting

properties and be potent nNOS inhibitors, they will carry a full positive charge at physiological

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pH. This defeats the purpose of this project, which is to find inhibitors that carry less positive

charge and so should show improved bioavailability.

2.9 Summary

Synthetic routes to 2- and 4-aminothiazole based inhibitors of nNOS were developed.

The 4-aminothiazoles could be synthesized, but on addition of water, the thiazoles tautomerized

and were then hydrolyzed to give thiazolones. These degraded further to give unidentifiable products. Replacement of the 2-amino-4-methylpyridine fragment of II-5 or II-6 with a 2-

aminothiazole resulted in a dramatic loss of potency. A modest gain in bioavailability will not

justify such a sacrifice in potency. The low potency against nNOS also hurts the isoform

selectivity, minimizing one of the most beneficial attributes of this class of compounds.

2.10 Experimental Procedures

General Methods. Proton nuclear magnetic resonances (1H NMR) were recorded in deuterated

solvents on a Mercury 400 (400 MHz) or a Varian Inova 500 (500 MHz) spectrometer. Chemical

shifts are reported in parts per million (ppm, δ) relative to tetramethylsilane (δ 0.00). 1H NMR

splitting patterns are designated as singlet (s), doublet (d), triplet (t), quartet (q). Splitting

patterns that could not be interpreted or easily visualized were recorded as multiplet (m) or broad

(br). Coupling constants are reported in Hertz (Hz). Proton-decoupled carbon (13C-NMR) spectra

were recorded on a Mercury 400 (100 MHz) or a Varian Inova 500 (125 MHz) spectrometer and

are reported in ppm using the solvent as an internal standard (CDCl3, δ 77.23). NMR spectra

recorded in D2O were not normalized. In many cases, the presence of rotamers made the NMR

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spectra complex. In the case of two peaks that are clearly a pair of rotamers, but are too far apart

for an average to accurately represent the spectrum, the pair is written enclosed in parentheses.

Electrospray mass spectra (ESMS) were obtained using an LCQ-Advantage with methanol as the

solvent in positive ion mode, unless otherwise stated. For most compounds, 1H and 13C NMR

and ESMS data are presented.

All chemical reagents were purchased from Aldrich and were used without further

purification unless stated otherwise. NADPH, calmodulin, and human ferrous hemoglobin were

also obtained from Sigma-Aldrich. Tetrahydrobiopterin (H4B) was purchased from Alexis

Biochemicals. HEPES, DTT, and some conventional organic solvents were purchased from

Fisher Scientific.

Tetrahydrofuran (THF) was distilled from sodium and benzophenone prior to use.

Methylene chloride (CH2Cl2) was distilled from calcium hydride prior to use, if dry solvent was required. Dimethylformamide (DMF) was purchased as an anhydrous solvent and used directly.

Boc tert-Butyl 6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate (II-10). To a solution of 3- N pyrroline (765 µL, 10 mmol, 65% pure, Fluka) in methanol (30 mL) at 0 °C was added O

di-tert-butyldicarbonate (Boc2O) (2.4 g, 11 mmol). The mixture was stirred for 20 h. The solvent

was removed in vacuo, and the residue was dissolved in CH2Cl2 (30 mL) and cooled to 0 °C.

mCPBA (1.9 g, 11 mmol, 70% pure) was added, and the mixture was stirred for 44 h. 20%

sodium thiosulfate (20 mL) was added, and the mixture was stirred vigorously for 30 min. The

mixture was separated, and the organic layer was washed with sat NaHCO3 (20 mL), 20 %

NaS2O3 (20 mL), sat NaHCO3 (20 mL), and brine (20 mL), dried over Na2SO4 and concentrated

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in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl

acetate / hexanes, 2:3) to afford II-10 as a colorless oil (870 mg, 4.7 mmol, 72%, based on

1 maximum possible yield). H NMR (500 MHz, CDCl3) δ 3.86 – 3.67 (m, 2H), 3.32 (dd, J = 6, 13

13 Hz, 4H), 1.45 (s, 9H); C NMR (125 MHz, CDCl3) δ 155.0, 80.0, (55.9 + 55.4), (47.6 + 47.2),

28.7.

S tert-Butyl 4-methylthiazol-2-ylcarbamate (II-12). To a solution of 2-amino-

BocHN N 4-methylthiazole (1.71 g, 15 mmol) in tert-butanol (20 mL) was added Boc2O

(3.6 g, 16 mmol). The mixture was heated to 55 °C for 40 h. The solvent was removed in vacuo.

The residue was dissolved in ethyl acetate, washed with sat NaHCO3 (aq) and brine, dried over

anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash

column chromatography (silica gel, ethyl acetate / hexanes, 1:3) to afford II-12 as a white solid

1 (2.1 g, 10 mmol, 67%). H NMR (500 MHz, CDCl3) δ 11.01 (s, 1H), 6.42 (s, 1H), 2.36 (s, 3H),

1.51 (s, 9H); mp = 114 – 116 °C.

D tert-Butyl 5-deutero-4-methylthiazol-2-ylcarbamate (II-14). To a solution S

BocHN N of tert-butyl 4-methylthiazol-2-ylcarbamate (43 mg, 0.2 mmol) in anhydrous THF (8 mL) at -78 °C was added nBuLi (315 µL, 1.6M in hexanes, 0.5 mmol) dropwise. After

addition the solution was allowed to warm to room temperature and stirred for 30 min. The

reaction was quenched by the addition of D2O. The product was extracted with ethyl acetate. The

organic layer was washed with brine dried over anhydrous Na2SO4 and concentrated in vacuo.

The crude product was purified using flash column chromatography (silica gel, ethyl acetate /

121 hexanes, 1:3) to afford II-14 as a white solid (39 mg, 0.18 mmol, 99%). 1H NMR (500 MHz,

+ CDCl3) δ 2.36 (s, 3H), 1.52 (s, 9H); ESMS m/z = 216 (M + H) .

TMS tert-Butyl 4-methyl-5-(trimethylsilyl)thiazol-2-ylcarbamate (II-15). To a S solution of tert-butyl 4-methylthiazol-2-ylcarbamate (214 mg, 1 mmol) in BocHN N anhydrous THF (8 mL) at -78 °C was added nBuLi (1.6 mL, 1.6M in hexanes, 2.5 mmol) dropwise. After addition the solution was allowed to warm to room temperature and stirred for

30 min. The reaction was quenched by the addition of TMSCl (2.5 mL, 1 M in THF, 2.5 mmol).

The product was extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:9) to afford II-15 as a white solid

1 (220 mg, 0.77 mmol, 77%). H NMR (500 MHz, CDCl3) δ 2.39 (s, 3H), 1.52 (s, 9H), 0.31 (s,

13 9H); C NMR (125 MHz, CDCl3) δ 163.8, 153.4, 117.0, 94.6, 82.4, 28.5, 18.2, 0.4; ESMS m/z =

287 (M + H)+.

O Me Ethyl 2-chloro-3-methyloxirane-2-carboxylate (II-17a). A fresh solution of EtO O sodium ethoxide was prepared by addition of small pieces of sodium metal (0.3 Cl g, 13 mmol) to ethanol (5 mL) at 0 °C. Once the sodium had reacted, the solution was added via cannula to a solution of ethyl dichloroacetate (1.6 mL, 12.7 mmol) and acetaldehyde (840 µL, 15 mmol) in anhydrous ether (10 mL) at 0 °C. The mixture was stirred at 0 °C for 1 h. Ether (10 mL) and sat NH4Cl (10 mL) were added to the mixture and the layers were separated. The aqueous layer was extracted with ether (2 x 10 mL). The organic layers were combined, dried

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over MgSO4 and concentrated to a colorless oil. A large portion of the product was lost when put

under reduced pressure to remove solvent; therefore, no accurate yield was obtained. When

sodium methoxide was used as the base, the major product was the methyl ester. Mixture of cis

1 and trans stereoisomers. Stereoisomer A: H NMR (500 MHz, CDCl3) δ 4.04 (s, 3H), 3.74 (q, J

1 = 8 Hz, 1H), 1.56 (d, J = 7 Hz, 3H); Stereoisomer B: H NMR (500 MHz, CDCl3) δ 4.04 (s, 3H),

3.54 (q, J = 6 Hz, 1H), 1.46 (d, J = 7 Hz, 3H).

O Et Ethyl 2-chloro-3-ethyloxirane-2-carboxylate (II-17b). The procedure used to EtO O create II-17a was repeated, except that propanal was used instead of Cl 1 acetaldehyde. Major diastereoisomer: H NMR (500 MHz, CDCl3) δ 4.30 (q, J = 7 Hz, 2H), 3.36

(t, J = 7 Hz, 1H), 1.85 (m, 2H), 1.34 (t, J = 7 Hz, 3H), 1.13 (t, J = 7 Hz, 3H); 13C NMR (125

MHz, CDCl3) δ 165.8, 79.0, 64.3, 63.7, 21.9, 14.1, 9.9.

Methyl 2-chloro-3-isopropyloxirane-2-carboxylate (II-17c). The procedure O

MeO used to create II-17a was repeated, except that isobutyraldehyde was used O Cl instead of acetaldehyde. As sodium methoxide was used as the base, a mixture of the methyl and ethyl esters of II-17c were formed, with the methyl ester being the major product (9.0 mmol,

1 71%). H NMR (500 MHz, CDCl3) δ 3.83 (s, 3H), 3.08 (d, J = 11 Hz, 1H), 1.88 (m, 1H), 1.17 (d,

J = 8.5 Hz, 3H), 1.06 (d, J = 8 Hz, 3H).

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H S Ethyl 2-methylthiazole-4-carboxylate (II-18a). Thioacetamide (670 mg, 8.9

EtOOC N mmol) and ethyl bromopyruvate (1.74 g, 8.9 mmol) were refluxed in ethanol

(20 mL) for 5 h. The solvent was removed in vacuo, and the residue was redissolved in ethyl acetate, washed with sat NaHCO3 (10 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:3) to afford II-18a as a white solid (855 mg, 5.0 mmol, 56%). 1H NMR (500

MHz, CDCl3) δ 8.05 (s, 1H), 4.42 (q, J = 7 Hz, 2H), 2.78 (s, 3H), 1.41 (t, J = 7 Hz, 3H).

Me S Ethyl 2,5-dimethylthiazole-4-carboxylate (II-18b). To a solution of

EtOOC N thioacetamide (83 mg, 1.1 mmol) in ethanol (10 mL) was added II-17a (165 mg, 1 mmol). The mixture was refluxed for 5 h. The solvent was removed in vacuo and the residue was redissolved in ethyl acetate, washed with sat NaHCO3 (10 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:2) to afford II-18b as a white solid

1 (154 mg, 0.83 mmol, 83%). H NMR (500 MHz, CDCl3) δ 4.41 (q, J = 7 Hz, 2H), 2.72 (s, 3H),

13 2.67 (s, 3H), 1.41 (t, J = 7 Hz, 3H); C NMR (125 MHz, CDCl3) δ 162.7, 162.1, 144.8, 141.0,

61.3, 19.4, 14.7, 13.3; ESMS m/z = 186 (M + H)+.

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Et S Ethyl 5-ethyl-2-methylthiazole-4-carboxylate (II-18c). The procedure Me EtOOC N used to create II-18b was repeated, except that II-17b was used instead of

1 II-17a. H NMR (500 MHz, CDCl3) δ 4.42 (q, J = 7 Hz, 2H), 3.22 (q, J = 7.5 Hz, 2H), 2.68 (s,

13 3H), 1.41 (t, J = 7 Hz, 3H), 1.31 (t, J = 7.5 Hz, 3H); C NMR (125 MHz, CDCl3) δ 162.6, 162.2,

152.7, 140.0, 61.2, 21.4, 19.5, 16.2, 14.6; ESMS m/z = 200 (M + H)+.

Ethyl 5-isopropyl-2-methylthiazole-4-carboxylate (II-18d). The S Me procedure used to create II-18b was repeated, except that II-17c was used EtOOC N instead of II-17a. A mixture of the methyl and ethyl esters of II-18d was formed. Ethyl ester: 1H

NMR (500 MHz, CDCl3) δ 4.39 (q, J = 7 Hz, 2H) 4.09 (sept, J = 6 Hz, 1H), 2.66 (s, 3H), 1.39 (t,

13 J = 7 Hz, 3H), 1.28 (d, J = 6 Hz, 6H); C NMR (125 MHz, CDCl3) δ 162.9, 162.2, 159.4, 138.8,

60.9, 28.1, 25.4, 19.6.

S 2-Methylthiazole-4-carboxylic acid (II-19a). To a solution of II-18a (855 mg,

HOOC N 5 mmol) in methanol (10 mL) was added 1 N NaOH (aq. 11 mL). The solution was stirred for 16 h. The mixture was acidified to pH 3 by dropwise addition of 2N HCl. The product was extracted with ethyl acetate (5 x 25 mL). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo to afford II-19a as a white solid (663 mg,

1 13 4.64 mmol, 93%). H NMR (500 MHz, CDCl3) δ 10.4 (br, 1H), 8.17 (s, 1H), 2.79 (s, 3H); C

NMR (125 MHz, CDCl3) δ 167.7, 165.0, 146.1, 129.0, 19.5.

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S 2,5-Dimethylthiazole-4-carboxylic acid (II-19b). The procedure used to

HOOC N create II-19a was carried out on II-18b to give II-19b. 1H NMR (500 MHz,

13 CDCl3) δ 2.70 (s, 3H), 2.65 (s, 3H); C NMR (125 MHz, CDCl3) δ 165.3, 162.9, 146.1, 140.3,

19.0, 13.3.

S 5-Ethyl-2-methylthiazole-4-carboxylic acid (II-19c). The procedure used to

HOOC N create II-19a was carried out on II-18c to give II-19c. 1H NMR (500 MHz,

13 CDCl3) δ 3.15 (q, J = 7.5 Hz, 2H), 2.63 (s, 3H), 1.22 (t, J = 7.5 Hz, 3H); C NMR (125 MHz,

CDCl3) δ 165.1, 163.0, 153.7, 139.4, 21.3, 19.1, 16.1.

5-Isopropyl-2-methylthiazole-4-carboxylic acid (II-19d). The procedure S used to create II-19a was carried out on II-18d to give II-19d. 1H NMR (500 HOOC N MHz, CDCl3) δ 10.62 (br, 1H), 4. 17 (sept., J = 8 Hz, 1H), 2.70 (s, 3H), 1.32 (d, J = 8 Hz, 6H);

13 C NMR (125 MHz, CDCl3) δ 164.4, 162.7, 159.9, 138.5, 28.1, 25.3, 19.3.

S tert-Butyl 2-methylthiazol-4-ylcarbamate (II-20a). Molecular sieves (3 A°,

BocHN N 50 mg) were flame dried under vacuum in a three necked flask equipped with

stir bar and condenser. The sieves were allowed to cool under a dry nitrogen atmosphere. A solution of II-19a (315 mg, 2.2 mmol) in tert-butanol (20 mL) was added via cannula, followed

by TEA (337 µL, 2.4 mmol). The solution was refluxed for 30 min, then allowed to cool. DPPA

(474 µL, 2.2 mmol) was added, and the solution was stirred at 50 °C for 30 min. The solution

was then refluxed for 16 h. The sieves were removed by filtration, and the solvent was removed

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in vacuo. The residue was redissolved in ethyl acetate, washed with sat NaHCO3 (10 mL), dried

over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash

column chromatography (silica gel, ethyl acetate / hexanes, 1:9) to afford II-20a as a white solid

1 (267 mg, 1.3 mmol, 57%). H NMR (500 MHz, CDCl3) δ 8.39 (s, 1H), 7.04 (s, 1H), 2.64 (s, 3H),

13 1.50 (s, 9H); C NMR (125 MHz, CDCl3) δ 164.2, 153.0, 147.6, 97.7, 80.8, 28.6, 19.3; ESMS

m/z = 215 (M + H)+; mp = 123 – 124 °C.

S tert-Butyl 2,5-dimethylthiazol-4-ylcarbamate (II-20b). The procedure used

BocHN N to create II-20a was carried out on II-19b to give II-20b. 1H NMR (500 MHz,

13 CDCl3) δ 7.24 (s, 1H), 2.58 (s, 3H), 2.27 (s, 3H), 1.49 (s, 9H); C NMR (125 MHz, CDCl3) δ

160.8, 153.7, 142.0, 122.4, 80.5, 28.5, 19.4, 11.5; ESMS m/z = 229 (M + H)+.

S tert-Butyl 5-ethyl-2-methylthiazol-4-ylcarbamate (II-20c). The procedure

BocHN N used to create II-20a was carried out on II-19c to give II-20c. 1H NMR (500

MHz, CDCl3) δ 6.81 (br, 1H), 2.71 (q, J = 7.5 Hz, 2H), 2.59 (s, 3H), 1.48 (s, 9H), 1.23 (t, J = 7

13 Hz, 3H); C NMR (125 MHz, CDCl3) δ 160.8, 153.8, 140.6, 130.9, 80.6, 28.5, 20.0, 19.6, 15.7;

ESMS m/z = 243 (M + H)+.

tert-Butyl 5-isopropyl-2-methylthiazol-4-ylcarbamate (II-20d). The S procedure used to create II-20a was carried out on II-19d to give II-20d. 1H BocHN N

127

NMR (500 MHz, CDCl3) δ 6.31 (s, 1H), 3.21 (sept., J = 8 Hz, 1H), 2.60 (s, 3H), 1.48 (s, 9H),

13 1.25 (d, J = 8 Hz, 6H); C NMR (125 MHz, CDCl3) δ 160.5, 154.0, 139.6, 137.3, 80.5, 28.5,

27.1, 24.9, 19.7; ESMS m/z = 279 (M + Na)+; mp = 125 – 126 °C.

Boc tert-Butyl 3-(cyanomethyl)-4-hydroxypyrrolidine-1-carboxylate (II-22). A 3- N

NC necked flask equipped with stir bar and addition funnel was flame dried, sealed OH and allowed to cool under a dry N2 atmosphere. The flask was charged with dry

THF (10 mL) and diisopropylamine (280 µL, 2 mmol), and the mixture was cooled to -78 °C. n-

BuLi (2.3 mL, 1.4 M in hexanes, 1.8 mmol) was added dropwise via the addition funnel. The mixture was allowed to warm to room temperature and stirred for 30 min, before being cooled down to -78 °C. Anhydrous acetonitrile (104 µL, 2 mmol) was added dropwise, and the mixture was allowed to warm to room temperature. After 15 min of stirring the mixture was cooled to -

5 °C. A solution of II-10 (370 mg, 2 mmol) in anhydrous THF (10 mL) was added dropwise.

The mixture was stirred for 2 h then quenched with sat NH4Cl (aq.). The product was extracted with ethyl acetate (3 x 15 mL), dried over Na2SO4 and concentrated. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 3:1) to afford II-

1 22 as a pale yellow oil (294 mg, 1.30 mmol, 72%). H NMR (500 MHz, CDCl3) δ 4.14 (m, 1H),

13 3.70 (m, 2H), 3.24 (m, 2H), 2.53 – 2.41 (m, 3H), 1.46 (s, 9H); C NMR (125 MHz, CDCl3) δ

154.7, 118.0, 80.4, (73.5 + 72.8), (52.4 + 52.0), (48.6 + 48.2), (42.5 + 41.9), 28.7, 18.7; ESMS m/z = 249 (M + Na)+, 475 (2M + Na)+.

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Boc tert-Butyl 3-(2-amino-2-thioxoethyl)-4-hydroxypyrrolidine-1-carboxylate N S (II-23). To a solution of II-22 (226 mg, 1 mmol) in ethanol (3 mL) was added H N 2 OH 50% (NH4)2S (aq., 0.3 mL, 4.5 mmol). The mixture was stirred for 44 h. The

mixture was added to sat NaCl solution (15 mL) and extracted with ethyl acetate (3 x 15 mL).

The organic layers were combined, dried over Na2SO4 and concentrated in vacuo. The crude

product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 9:1)

to afford II-22 as a colorless, viscous oil (0.34 mmol, 34%). Unreacted II-22 was also recovered

and could be submitted to the same conditions to generate more thioamide. 1H NMR (500 MHz,

CDCl3) δ 8.82 (br, 2H), 4.13 (m, 1H), 3.66 (m, 2H), 3.25 (m, 2H), 2.89 – 2.65 (m, 3H), 1.50 (s,

13 9H); C NMR (125 MHz, CDCl3) δ 206.3, 154.5, 78.8, (74.0 + 73.4), (52.9 + 52.6), (49.2 +

48.9), 46.3, 45.6, 28.2; ESMS m/z = 261 (M + H)+, 283 (M + Na)+.

General procedure for synthesis of II-24a-c.

To a solution of II-23 in ethanol (10 mL) was added ethylbromopyruvate, II-17a or II-17c (1.1

eq.). The mixture was refluxed for 5 h. The solution was cooled to room temperature and

neutralized with diisopropylethylamine (1.25 eq.). Boc2O (1.25 eq.) was added and the mixture

was stirred overnight. The solvent was removed in vacuo and the residue was dissolved in brine

(15 mL) and extracted with ethyl acetate (3 x 15 mL). The organic layers were combined, dried

over Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:1) to afford II-24a-c as a white solid.

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Boc Ethyl 2-((1-(tert-butoxycarbonyl)-4-hydroxypyrrolidin-3- N S EtOOC yl)methyl)thiazole-4-carboxylate (II-24a). II-23 (57 mg, 0.22 N OH mmol) and ethylbromopyruvate (44 µL, 0.23 mmol) were combined

using the general procedure above to give II-24a (68 mg, 0.19 mmol, 85%). 1H NMR (500 MHz,

CDCl3) δ 8.06 (s, 1H), 4.38 (q, J = 7 Hz, 2 H), 4.22 (m, 1H), 3.72 (m, 2H), 3.26 – 3.09 (m, 4H),

13 2.56 (m, 1H), 1.43 (s, 9H), 1.37 (t, J = 6.5 Hz, 3H); C NMR (125 MHz, CDCl3) δ 169.6, 161.2,

154.7, 146.9, 127.4, 79.8, (75.0 + 74.3), 61.8, 52.7, (49.7 + 49.5), (45.6 + 44.7), 35.4, 28.7, 14.5;

ESMS m/z = 357 (M + H)+.

Boc Methyl 2-((1-(tert-butoxycarbonyl)-4-hydroxypyrrolidin-3- N S MeOOC yl)methyl)-5-methylthiazole-4-carboxylate (II-24b). II-23 (182 N OH mg, 0.7 mmol) and II-17a (295 mg, methyl ester, 1.8 mmol,

contains solvent), were combined using the general procedure above to give II-24b (196 mg,

1 0.55 mmol, 79%). H NMR (500 MHz, CDCl3) δ 4.15 (m, 1H), 3.92 (s, 3H), 3.71 (m, 2H), 3.23

(m, 1H), 3.08 (m, 2H), 2.74 (s, 3H), 2.83 – 2.45 (m, 2H), 1.45 (s, 9H); 13C NMR (125 MHz,

CDCl3) δ 207.7, 164.8, 162.7, 154.9, 145.3, 140.3, 94.6, 80.0, (74.8 + 74.1), (52.9 + 52.7), 52.5,

(49.7 + 49.1), (46.6 + 45.5), 35.0, 28.7, 13.3; ESMS m/z = 357 (M + H)+.

Methyl 2-((1-(tert-butoxycarbonyl)-4-hydroxypyrrolidin-3- Boc N S yl)methyl)-5-isopropylthiazole-4-carboxylate (II-24c). II-23 (364 MeOOC N OH mg, 1.4 mmol) and II-17c (346 mg, methyl ester, 1.8 mmol) were

combined to give II-24c as a white solid (614 mg, 1.06 mmol, 76%). 1H NMR (500 MHz,

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CDCl3) δ 4.07 (m, 2H), 3.82 (s, 3H), 3.64 (m, 2H), 3.17 (m, 1H), 3.09 – 2.92 (m, 3 H), 1.36 (s,

13 9H), 1.22 (d, J = 8.5 Hz, 6H); C NMR (125 MHz, CDCl3) δ 164.7, 162.4, 159.3, 154.5, 138.5,

94.6, 79.7, (75.0 + 74.3), (53.0 + 52.5), 52.4, (49.9 + 49.5), (45.3 + 44.5), 35.3, 28.7, 28.0, 25.3;

ESMS m/z = 385 (M + H)+.

Boc 2-((1-(tert-Butoxycarbonyl)-4-hydroxypyrrolidin-3-yl)- N S HOOC methyl)thiazole-4-carboxylic acid (II-25a). To a solution of II-24a N OH (68 mg, 0.19 mmol) in methanol (3 mL) was added 1 N NaOH (3 mL)

dropwise. The mixture was stirred for 14 h. The mixture was acidified to pH 2 using 2N HCl,

and the product was extracted with ethyl acetate (3 x 15 mL). The organic layers were combined,

dried over Na2SO4 and concentrated in vacuo to afford II-25a as a white solid (59 mg, 0.18

1 mmol, 95%). H NMR (500 MHz, CDCl3) δ 8.16 (s, 1H), 4.25 (m, 1H), 3.70 (m, 2H), 3.32 –

13 3.17 (m, 4H), 2.64 (m, 1H), 1.45 (s, 9H); C NMR (125 MHz, CDCl3) δ 169.7, 163.3, 155.0,

146.5, 128.5, 80.3, (74.5 + 73.8), (52.6 + 52.3), (49.6 + 49.2), (45.7 + 44.9), 34.9, 28.7; ESMS (-

ve mode) m/z = 327 (M-H)-.

Boc 2-((1-(tert-Butoxycarbonyl)-4-hydroxypyrrolidin-3-yl)-methyl)-5- N S HOOC methylthiazole-4-carboxylic acid (II-25b). II-25b was prepared N OH from II-24b following the same method as that used to prepare II-25a.

1 H NMR (500 MHz, CDCl3) δ 4.20 (m, 1H), 3.71 (m, 2H), 3.25 (m, 1H), 3.11 (m, 2H), 2.73 (s,

13 3H), 2.55 (m, 2H), 1.45 (s, 9H); C NMR (125 MHz, CDCl3) δ 175.5, 164.4, 154.9, 145.6, 140.6,

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80.1, (74.5 + 73.8), (52.6 + 52.3), (49.6 + 49.3), (45.4 + 44.6), 34.6, 28.7, 13.3. ESMS m/z = 343

(M + H)+, 343 (M + Na)+, 685 (2M + H)+, 685 (2M + Na)+.

2-((1-(tert-Butoxycarbonyl)-4-hydroxypyrrolidin-3-yl)methyl)-5- Boc N S isopropylthiazole-4-carboxylic acid (II-25c). II-25c was prepared HOOC N OH from II-24c following the same method as that used to prepare II-25a.

1 H NMR (500 MHz, CDCl3) δ 4.30 (m, 1H), 4.14 (m, 1H), 3.68 (m, 2H), 3.36 – 2.99 (m, 4H),

- 2.55 (m, 1H), 1.46 (s, 9H), 1.32 (d, J = 8 Hz, 6H); ESMS m/z = 369 (M - H) .

Boc tert-Butyl 3-((4-(tert-butoxycarbonylamino)thiazol-2-yl)methyl)-4- N S BocHN hydroxypyrrolidine-1-carboxylate (II-21a). A 3-necked flask with N OH stir bar, condenser, and 3 Å molecular sieves was flame dried under vacuum and allowed to cool under a dry nitrogen atmosphere. A solution of II-25a (400 mg,

1.22 mmol) in warm t-BuOH (20 mL) was added via cannula, followed by triethylamine (186 µL,

1.4 mmol). The mixture was refluxed for 30 min then allowed to cool. Diphenylphosphoryl azide

(270 µL, 1.25 mmol) was added, and the mixture was stirred at 50 °C for 30 min. The system was heated to reflux for 14 h. The sieves were removed by filtration and the solvent was removed in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:1) to afford II-21a as a white solid (260 mg, 0.65 mmol, 53%). 1H

NMR (500 MHz, CDCl3) δ 8.26 (br, 1H), 7.11 (s, 1H), 4.90 (m, 1H), 4.17 (m, 1H), 3.72 (m, 2H),

3.28 (m, 1H), 3.15 – 3.02 (m, 3H), 2.51 (m, 1H), 1.51 b(s, 9H), 1.45 (s, 9H); 13C NMR (125

132

MHz, CDCl3) δ 166.2, 154.6, 152,7, 147.5, 97.9, 81.1, 79.8, (75.0 + 74.3), (52.8 + 52.4), (49.7 +

49.4), (45.6 + 44.9), 34.9, 28.8, 28.6; ESMS m/z = 400 (M + H)+.

Boc tert-Butyl 3-((4-(tert-butoxycarbonylamino)-5-methyl-thiazol-2- N S BocHN yl)methyl)-4-hydroxypyrrolidine-1-carboxylate (II-21b). II-21b N OH was prepared from II-25b following the same method as that used to

1 prepare II-21a. H NMR (500 MHz, CDCl3) δ 6.73 (s, 1H), 4.18 (m, 1H), 3.72 (m, 2H), 3.23 (m,

1H), 3.07 – 2.89 (m, 2H), 2.48 (m, 2H), 2.29 (s, 3H), 1.49 (s, 9H), 1.45 (s, 9H); 13C NMR (125

MHz, CDCl3) δ 163.2, 153.4, 142.4, 130.1, 81.1, 79.8, (75.1 + 74.5), (53.0 + 52.5), (49.9 + 49.6),

(45.3 + 44.5), 35.4, 28.8, 28.5, 11.4; ESMS m/z = 414 (M + H)+, 436 (M + Na)+, 827 (2M + H)+,

849 (2M + Na)+.

tert-Butyl 3-((4-(tert-butoxycarbonylamino)-5-isoprop-ylthiazol-2- Boc N S yl)methyl)-4-hydroxypyrrolidine-1-carboxylate (II-21d). II-21d BocHN N OH was prepared from II-25c following the same method as that used to

1 prepare II-21a. H NMR (500 MHz, CDCl3) δ 6.92 (m, 1H), 4.12 (m, 1H), 3.71 (m, 2H), 3.21 (m,

2H), 3.07 (m, 1H), 2.98 (m, 2H), 2.48 (m, 1H), 1.48 (s, 9H), 1.45 (s, 9H), 1.25 (d, 6H); 13C NMR

(125 MHz, CDCl3) δ 162.9, 154.5, 153.9, 139.7, 137.0, 94.6, 80.8, 79.6, (74.8 + 74.2), (52.8 +

52.4), (45.4 + 44.6), 35.5, 31.1, 28.7, 28.5, 27.0, 24.8. ESMS m/z = 442 (M + H)+, 464 (M +

Na)+.

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Boc tert-Butyl 3-((4-(tert-butoxycarbonylamino)thiazol-2- N S BocHN yl)methyl)-4-(1,3-dioxoisoindolin-2-yl)pyrrolidine-1- N N O O carboxylate (II-26a). To a flame-dried flask was added

triphenylphosphine (210 mg, 0.8 mmol) and phthalimide (118 mg,

0.8 mmol). A solution of II-21a (260 mg, 0.65 mmol) in anhydrous THF (10 mL) was added via cannula. Diisopropylazodicarboxylate (141 µL, 0.75 mmol) was added dropwise, and the mixture was stirred for 14 h. The mixture was concentrated in vacuo and purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:2) to afford II-26a as a white solid (327 mg,

1 0.62 mmol, 95%). H NMR (500 MHz, CDCl3) δ 8.04 (br, 1H), 7.82 (d, J = 3 Hz, 2H), 7.74 (d, J

= 3 Hz, 2H), 7.01 (br, 1H), 5.00 (m, 1H), 3.88 – 3.69 (m, 3H), 3.50 (m, 1H), 3.13 – 2.99 (m, 2H),

13 2.85 (m, 1H), 1.50 (s, 18H); C NMR (125 MHz, CDCl3) δ 168.3, 165.1, 154.2, 152.5, 147.5,

134.4, 131.6, 123.5, 97.6, 80.8, 79.6, (53.8 + 51.9), (51.0 + 50.6), (49.7 + 49.3), (42.8 + 42.0),

32.0, 28.7, 28.5; ESMS m/z = 551 (M + Na)+.

Boc tert-Butyl 3-((4-(tert-butoxycarbonylamino)-5-methyl-thiazol- N S BocHN 2-yl)methyl)-4-(1,3-dioxoisoindolin-2-yl)pyrrol-idine-1- N N O O carboxylate (II-26b). II-26b was prepared from II-21b

following the same method as that used to prepare II-26a. 1H

NMR (500 MHz, CDCl3) δ 7.84 (s, 2H), 7.76 (s, 2H), (6.96 + 6.87) (s, rotamers, 1H), 4.98 (m,

1H), 3.88 – 3.71 (m, 3H), 3.46 (m, 1H), 3.10 – 2.74 (m, 3H), 2.25 (s, 3H), 1.50 (s, 9H), 1.48 (s,

13 9H); C NMR (125 MHz, CDCl3) δ 168.4, 161.7, 154.5, 154.2, 142.3, 134.5, 131.7, 123.6, 80.5,

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79.6, (51.9 + 51.1), 50.5, (49.7 + 49.3), (42.7 + 42.0), 32.3, 28.7, 28.4, 11.5; ESMS m/z = 565

(M + Na)+.

tert-Butyl 3-((4-(tert-butoxycarbonylamino)-5-isoprop-ylthiaz- Boc N S ol-2-yl)methyl)-4-(1,3-dioxoisoindolin-2-yl)pyr-rolidine-1- BocHN N O N carboxylate (II-26c). II-26c was prepared from II-21d following O the same method as that used to prepare II-26a. 1H NMR (500

MHz, CDCl3) δ 7.83 (m, 2H), 7.76 (m, 2H), 6.54 (m, 1H), 4.99 (m, 1H), 3.88 – 3.74 (m, 3H),

3.49 (t, 1H), 3.23 – 3.05 (m, 2H), 2.92 (m, 1H), 2.80 (m, 1H), 1.47 (m, 18H), 1.20 (m, 6H); 13C

NMR (125 MHz, CDCl3) δ 168.3, 161.5, 154.5, 154.2, 153.7, 139.9, 137.5, 134.4, 131.7, 123.5,

80.5, 79.7, 51.8, 51.1, 51.0, 50.6, 49.7, 49.3, (42.6 + 41.9), 32.5, 28.7, 28.5, 27.0, 24.8; ESMS

m/z = 571 (M + H)+, 593 (M + Na)+.

Boc tert-Butyl 3-amino-4-((4-(tert-butoxycarbonyl-amino)-thiazol-2- N S BocHN yl)methyl)pyrrolidine-1-carboxylate (II-26a). To a solution of II- N NH2 26a (325 mg, 0.62 mmol) in methanol (3 mL) was added 50% aq. hydrazine (3 mL) dropwise. The solution was stirred at room temperature for 14 h. HCl (2N, 15 mL) was added dropwise until the pH reached approximately 5. The mixture was stirred a further

2 h. The solution was poured into sat K2CO3 (20 mL, final pH ~10) and extracted with CH2Cl2 (5

x 15 mL). The organic layers were combined, dried over Na2SO4 and concentrated in vacuo to

1 afford II-27a as a white solid (235 mg, 0.59 mmol, 95%). H NMR (500 MHz, CDCl3) δ 8.22 (s,

1H), 7.02 (s, 1H), 3.52 (m, 1H), 3.40 (m, 2H), 3.23 – 2.88 (m, 4H), 2.45 (m, 1H), 1.45 (s, 9H),

135

13 1.37 (s, 9H); C NMR (125 MHz, CDCl3) δ 166.2, 154.7, 152.7, 147.6, 97.6, 80.9, 79.5, (55.0 +

54.6), (52.5 + 51.5), (48.7 + 48.3), (44.2 + 43.5), 31.0, 28.7, 28.6; ESMS m/z = 399 (M + H)+.

Boc tert-Butyl 3-amino-4-((4-(tert-butoxycarbonylamino)-5- N S BocHN methylthiazol-2-yl)methyl)pyrrolidine-1-carboxylate (II-27b). II- N NH2 27b was prepared from II-26b following the same method as that

1 used to prepare II-27a. H NMR (500 MHz, CDCl3) δ 7.01 (s, 1H), 3.44 (m, 2H), 3.20 – 2.99 (m,

3H), 2.83 (m, 1H), 2.43 (m, 2H), 2.21 (s, 3H), 1.41 (s, 9H), 1.37 (s, 9H); 13C NMR (125 MHz,

CDCl3) δ 162.8, 154.8, 153.6, 142.3, 122.7, 80.6, 79.4, 54.8, (52.4 + 51.5), (48.7 + 48.3), (43.8 +

43.1), 31.2, 28.7, 28.4, 11.5; ESMS m/z = 413 (M + H)+.

tert-Butyl 3-amino-4-((4-(tert-butoxycarbonylamino)-5- Boc N S isopropylthiazol-2-yl)methyl)pyrrolidine-1-carboxylate (II-27c). BocHN N NH2 II-27c was prepared from II-26c following the same method as that

1 used to prepare II-27a. H NMR (500 MHz, CDCl3) δ 6.76 (s, 1H), 3.73 – 3.46 (m, 3H), 3.29 –

3.07 (m, 4H), 2.93 (m, 1H), 2.54 (m, 1H), 1.48 (s, 9H), 1.45 (s, 9H), 1.26 (d, J = 5 Hz, 6H); 13C

NMR (125 MHz, CDCl3) δ 162.6, 154.7, 153.9, 139.9, 137.6, 80.5, 79.5, (55.0 + 54.6), (52.5 +

51.5), (48.8 + 48.3), (43.8 + 43.1), 31.6, 28.7, 28.5, 27.1, 24.9; ESMS m/z = 441 (M + H)+, 881

(2M + H)+.

136

Cl Ethyl 2-(4-chlorobenzylamino)acetate (II-28). Ethyl glycinate (700 O H N EtO mg, 5 mmol) and 4-chlorobenzyl chloride (480 mg, 3 mmol) were dissolved in methanol (10 mL). DIEA (872 µL, 5 mmol) was added and the mixture was refluxed for 14 h. The solvent was removed in vacuo and the residue was purified using flash column chromatography (silica gel, ethyl acetate / methanol, 9:1) to afford II-28 as a colorless oil (256 mg, ~1.15 mmol, 38%). A mixture of methyl and ethyl esters was formed. 1H NMR (500 MHz,

CDCl3) δ 7.28 (s, 4H), 4.18 (q, J = 9 Hz, 2H), 3.77 (s, 2H), 3.38 (s, 2H), 1.94 (br, 1H), 1.27 (t, J

13 = 9 Hz, 3H); C NMR (125 MHz, CDCl3) δ 172.5, 138.2, 133.0, 129.8, 128.7, 61.0, 52.8, 50.2,

14.5; ESMS m/z = 228 / 230 (3:1) (M + H)+.

Cl Ethyl 2-(tert-butoxycarbonyl(4-chlorobenzyl)amino)-acetate (II- O Boc N EtO 29). To a solution of II-28 (256 mg, 1.15 mmol) in MeOH (10 mL) was added DIEA (280 µL, 1.5 mmol) and Boc2O (327 mg, 1.5 mmol). The mixture was stirred for 4 h. The solvent was removed in vacuo, and the crude residue was dissolved in sat NH4Cl solution. The product was extracted with ethyl acetate (3 x 15 mL). The organic layers were combined, dried over Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:4) to afford II-28 as a white

1 solid (406 mg, 1.14 mmol, 99%). H NMR (500 MHz, CDCl3) δ 7.28 (m, 2H), 7.12 (m, 2H),

(4.50 + 4.47) (s, rotamers, 2H), 4.16 (m, 2H), 3.91 (s, 1H), 3.77 (s, 1H), 1.46 (s, 9H), 1.25 (t, J =

13 8.5 Hz, 3H); C NMR (125 MHz, CDCl3) δ 169.9, 155.7, (136.4 + 136.1), 133.4, 129.6, 128.9,

80.9, 61.3, (51.3 + 50.8), (48.6 + 48.2), 28.5, 14.4; ESMS m/z = 328 / 330 (3:1) (M + H)+.

137

Cl O 2-(tert-Butoxycarbonyl(4-chlorobenzyl)amino)acetic acid (II-30). Boc N HO To a solution of II-29 (406 mg, 1.14 mmol) in methanol (3 mL) was

added 1 N NaOH (3 mL) dropwise. The mixture was stirred for 14 h. The mixture was acidified

to pH 2 using 2N HCl and the product was extracted with ethyl acetate (3 x 15 mL). The organic

layers were combined, dried over Na2SO4 and concentrated in vacuo to afford II-30 as a white

1 solid (322 mg, 1.08 mmol, 95%). H NMR (500 MHz, CDCl3) δ 10.84 (br, 1H), 7.30 (m, 2H),

7.20 (m, 2H), 4.48 (d, rotamers, 2H), 3.97 (s, 1H), 3.82 (s, 1H), 1.47 (s, 9H); 13C NMR (125

MHz, CDCl3) δ 175.5, 155.9, 136.0, 133.6, 129.7, 129.0, 81.5, (51.4 + 50.7), 48.1, 28.5; ESMS

(-ve mode) m/z = 298 / 300 (3:1) (M-H)-.

Cl O tert-Butyl 4-chlorobenzyl(2-(methoxy(methyl)amino)-2- Boc MeO N N oxoethyl)carbamate (II-31). To a solution of II-30 (322 mg, 1.1 Me mmol) in dry CH2Cl2 (5 mL) was added DIEA (262 µL, 1.5 mmol). The solution was cooled to

0 °C and EDC (288 mg, 1.5 mmol) and HOBt (203 mg, 1.5 mmol) were added. After 5 min,

HN(OMe)Me.HCl (147 mg, 1.5 mmol) and DIEA (262 µL, 1.5 mmol) were added. The mixture

was stirred at room temperature for 14 h. The mixture was diluted with CH2Cl2 (15 mL) and

washed with 1N HCl (2 x 20 mL), sat NaHCO3 (2 x 20 mL) and brine (1 x 20 mL), dried over

Na2SO4 and concentrated in vacuo. The crude product was purified using flash column

chromatography (silica gel, ethyl acetate / hexanes, 1:3) to afford II-31 as a white solid (360 mg,

1 1.05 mmol, 97%). H NMR (500 MHz, CDCl3) δ 7.31 – 7.18 (m, 4H), (4.54 + 4.50) (s, rotamers,

2H), 4.10 (s, 1H), 3.96 (s, 1H), (3.66 + 3.62) (s, rotamers, 3H), 3.18 (s, 3H), 1.46 (s, 9H); 13C

138

NMR (125 MHz, CDCl3) δ 169.9, 156.1, 136.8, 133.1, 129.6, 128.9, 80.6, 61.5, (51.3 + 50.6),

47.3, 32.6, 28.6; ESMS m/z = 343 / 345 (3:1) (M + H)+.

Cl tert-Butyl 4-chlorobenzyl(2-oxoethyl)carbamate (II-32). A solution O Boc N H of II-31 (137 mg, 0.4 mmol) in anhydrous THF (3 mL) was cooled to

0 °C. A solution of lithium aluminum hydride (0.5 mL, 0.5 mmol, 1 M in THF) was added dropwise and the mixture was stirred at 0 °C for 1 h. The reaction was quenched by the addition of 20% sodium bisulfate solution (15 mL). The product was extracted with ethyl acetate (2 x 15 mL) and the combined organic layers were washed with 1N HCl (2 x 20 mL), sat NaHCO3 (2 x

20 mL) and brine (1 x 20 mL), dried over Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:3) to

1 afford II-32 as a white solid (108 mg, 0.38 mmol, 94 %). H NMR (500 MHz, CDCl3) δ (9.51 +

9.44) (s, rotamers, 1H), 7.31 – 7.15 (m, 4H), (4.50 + 4.56) (s, rotamers, 2H), 3.95 (s, 1H), 3.80 (s,

13 1H), 1.48 (9H); C NMR (125 MHz, CDCl3) δ 198.4, 155.5, 136.0, 133.7, 129.6, 129.1, 81.5,

56.8, (51.7 + 51.2), 28.5.

Boc tert-Butyl 3-(2-(tert-butoxycarbonyl(4- N S Cl BocHN Boc chlorobenzyl)amino)ethylamino)-4-((4-(tert- N N N H butoxycarbonylamino)thiazol-2- yl)methyl)pyrrolidine-1-carboxylate (II-33a). To a solution of II-27a (97 mg, 0.24 mmol) in methanol (3 mL) was added a solution of II-32 (112 mg, 0.24 mmol) in CH2Cl2 (1 mL). The mixture was stirred for 15 min at room temperature, then NaHB(OAc)3 (64 mg, 0.3 mmol) was

139

added. The mixture was stirred for 90 min then poured into K2CO3 solution (15 mL). The

product was extracted with EtOAc (3 x 15 mL), dried over Na2SO4 and concentrated in vacuo.

The crude product was purified using flash column chromatography (silica gel, ethyl acetate /

hexanes, 3:1) to afford II-33a as an oily solid (60 mg, 0.09 mmol, 38%). 1H NMR (500 MHz,

CDCl3) δ 7.91 (br, 1H), 7.33 – 7.08 (m, 5H), 4.45 (m, 2H), 3.46 – 3.10 (m, 8H), 2.88 – 2.77 (m,

2H), 2.58 (m, 2H), 1.45 (m, 27H); ESMS m/z = 666 / 668 (3:1) (M + H)+.

Boc tert-Butyl 3-(2-(tert-butoxycarbonyl(4- N S Cl BocHN Boc chlorobenzyl)amino)ethylamino)-4-((4-(tert- N N N H butoxycarbonylamino)-5-methyl-thiazol-2-

1 yl)methyl)pyrrolidine-1-carboxylate (II-33b). H NMR (500 MHz, CDCl3) δ 7.29 (m, 2H),

7.17 (m, 2H), 6.86 (br, 1H), 4.49 (m, 2H), 3.53 – 3.04 (m, 8H), 2.80 (m, 2H), 2.57 (m, 2H), 2.90

13 (m, 3H), 1,46 (m, 27H); C NMR (125 MHz, CDCl3) δ 163.2, 154.9, 153.6, 142.1, 137.0, 133.2,

129.3, 128.9, 122.9, 80.6, 80.4, 79.5; ESMS m/z = 680 / 682 (3:1) (M + H)+.

tert-Butyl 3-(2-(tert-butoxycarbonyl(4- Boc N Cl S chlorobenzyl)amino)ethylamino)-4-((4-(tert- BocHN Boc N N N H butoxycarbonylamino)-5-isoprop-ylthiazol-2-

1 yl)methyl)-pyrrolidine-1-carboxylate (II-33c). H NMR (500 MHz, CDCl3) δ 7.30 (m, 2H),

7.17 (m, 2H), 6.46 (m, 1H), 4.42 (m, 2H), 3.52 – 3.04 (m, 9H), 2.82 (m, 2H), 2.61 (m, 2H), 1.47

(m, 27H), 1.25 (m, 6H); ESMS m/z = 708 / 710 (3:1) (M + H)+, 730 / 732 (3:1) (M + Na)+.

140

H 2-((4-(2-(4-Chlorobenzylamino)ethylamino)- N S Cl O H pyrrolidin-3-yl)methyl)-5-methylthiazol-4(5H)-one N N N H (II-8b, hypothesized actual structure in water). A

solution of II-33b (136 mg, 0.2 mmol) in 4N HCl in dioxanes (3 mL) was stirred for 16h. The

solvent was removed under a stream of N2, and the crude residue was dissolved in methanol.

ESMS 380 (M + H)+ for II-8b, desired structure (aminothiazole). The solvent was removed in

vacuo and the residue was dissolved in water. The solution was washed with ethyl acetate and

the solvent was removed in vacuo to give a white solid. The aqueous solution turned brown and

1 an unknown precipitate formed. H NMR (500 MHz, D2O) δ 7.29 (d, J = 9 Hz, 2H), 7.21 (d, J =

9Hz, 2H), 4.53 (t, J = 6.5 Hz, 1H), 4.41 (q, J = 9.5 Hz, 2H), 4.00 (t, J = 9.5 Hz, 2H), 3.76 (m,

3H), 3.62 (m, 2H), 3.36 (m, 2H), 3.16 (m, 1H), 3.10 (m, 1H), 2.73 (d, J = 19 Hz, 1H), 1.44 (d, J

13 = 7 Hz, 3H); C NMR (125 MHz, CDCl3) δ 174.4, 173.4, 134.3, 131.5, 130.5, 129.2, 62.5, 54.5,

51.3, 50.4, 47.9, 43.9, 42.3, 34.3, 29.6, 20.8; ESMS m/z = 276 / 278 (3:1) unknown

decomposition product.

Boc tert-Butyl 3-(tert-butyldimethylsilyloxy)-4-(cyanomethyl)pyrrol-idine-1- N

NC carboxylate (II-35). A solution of II-22 (751 mg, 3.32 mmol), TBSCl (525 OTBS mg, 3.5 mmol) and imidazole (408 mg, 6.0 mmol) in anhydrous DMF (5 mL)

was stirred at 35 °C for 16 h. The solvent was removed in vacuo, and the crude product was

purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:4) to afford II-

1 35 as a colorless solid (942 mg, 2.77 mmol, 83%). H NMR (500 MHz, CDCl3) δ 3.96 (m, 1H),

3.53 (m, 2H), 3.02 (m, 2H), 2.32 (m, 2H), 2.20 (m, 1H), 1.33 (s, 9H), 0.77 (s, 9H), -0.03 (s, 6H);

141

13 C NMR (125 MHz, CDCl3) δ 154.3, 117.6, 79.8, (74.0 + 73.3), (52.5 + 52.0), (48.0 + 47.6),

(42.9 + 42.3), 28.6, 25.9, 18.3, 18.0, -4.5; ESMS m/z = 341 (M + H)+.

Boc tert-Butyl 3-(tert-butyldimethylsilyloxy)-4-(2-oxoethyl)pyrrol-idine-1- N O carboxylate (II-36). A solution of II-35 (942 mg, 2.77 mmol) in anhydrous H OTBS CH2Cl2 (5 mL) was cooled to 0 °C. DIBAL (8.31 mL, 8.31 mmol, 1M in

THF) was added dropwise. The solution was stirred for 1 h. Sodium bisulfate solution (20%, 5 mL) was added dropwise to quench the reaction. The product was extracted with ethyl acetate (3

x 15 mL). The organic layers were combined, dried over Na2SO4 and concentrated in vacuo. The

crude product was purified using flash column chromatography (silica gel, ethyl acetate /

hexanes, 1:4) to afford II-36 as a white solid (912 mg, 2.66 mmol, 96%). 1H NMR (500 MHz,

CDCl3) δ 9.77 (s, 1H), 3.91 (m, 1H), 3.59 (m, 2H), 3.01 (m, 2H), 2.66 – 2.32 (m, 3H), 1.43 (s,

13 9H), 0.86 (s, 9H), 0.05 (s, 6H); C NMR (125 MHz, CDCl3) δ 200.7, 154.8, 79.7, (75.2 + 74.5),

(52.7 + 52.1), (49.0 + 48.6), 45.6, (41.1 + 40.4), 28.7, 26.0, 18.2, -4.4.

Boc tert-Butyl 3-(tert-butyldimethylsilyloxy)-4-(2-hydroxypropyl)-pyrrolidine- N HO 1-carboxylate (II-37). To a solution of II-36 (345 mg, 1.0 mmol) in OTBS anhydrous THF (5 mL) at -78 °C was added CH3MgBr (0.66 mL, 2.0 mmol, 3

M in THF). The solution was stirred for 1 h, allowed to warm to room temperature, and stirred a

further 15 min. Sodium bisulfate solution (20%, 5 mL) was added dropwise to quench the

reaction. The product was extracted with ethyl acetate (3 x 15 mL). The organic layers were

combined, dried over Na2SO4 and concentrated in vacuo. The crude product was purified using

142

flash column chromatography (silica gel, ethyl acetate / hexanes, 1:2) to afford II-37 as a white

1 solid (237 mg, 0.66 mmol, 66%). H NMR (500 MHz, CDCl3) δ 3.84 (m, 1H), 3.75 (m, 1H),

3.47 (m, 2H), 2.93 (m, 2H), 2.10 (m, 2H), 1.49 (m, 1H), 1.35 (s, 9H), 1.11 (m, 3H), 0.79 (s, 9H),

13 -0.02 (m, 6H); C NMR (125 MHz, CDCl3, average of diastereomers) δ 154.7, 79.4, 75.8, 66.4,

52.6, 49.4, 43.5, 40.9, 28.7, 26.0, 24.0, 18.1, -4.3; ESMS m/z = 382 (M + Na)+, 741 (2M + Na)+.

Boc tert-Butyl 3-(tert-butyldimethylsilyloxy)-4-(2-oxopropyl)pyrrol-idine-1- N O carboxylate (II-38). A 3-necked flask equipped with stir bar and addition OTBS funnel was flame dried, sealed and allowed to cool under dry N2. Dry CH2Cl2

(15 mL) and DMSO (184 µL, 2.6 mmol) were added, and the mixture was cooled to -78 °C.

Oxalyl chloride (0.9 mL, 1.8 mmol, 2 M in CH2Cl2) was added, and the mixture was stirred for 5

min. A solution of II-37 (472 mg, 1.31 mmol) in dry CH2Cl2 (5 mL) was added dropwise via the

addition funnel. The mixture was stirred at -78 °C for 1 h. Triethylamine (375 µL, 2.6 mmol)

was added, and the mixture was allowed to warm to room temperature. The reaction was

quenched with brine (10 mL), and the product was extracted with CH2Cl2. The organic layers

were combined, dried over Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:5) to afford II-38 (412

1 mg, 1.15 mmol, 88%). H NMR (500 MHz, CDCl3) δ 3.83 (m, 1H), 3.60 (m, 1H), 3.51 (m, 0.5H),

3.40 (m, 0.5H), 3.01 (m, 1H), 2.90 (m, 0.5H), 2.81 (m, 0.5H), 2.60 – 2.22 (m, 3H), 2.08 (s, 3H),

13 1.37 (s, 9H), 0.80 (s, 9H), 0.00 (s, 6H); C NMR (125 MHz, CDCl3) δ 206.8, 154.7, 79.5, (75.1

+ 74.4), (52.8 + 52.2), (49.2 + 48.9), 45.2, (42.2 + 41.5), 30.4, 28.7, 25.9, 18.2, -4.4; ESMS m/z

= 380 (M + Na)+.

143

Boc tert-Butyl 3-allyl-4-hydroxypyrrolidine-1-carboxylate (II-39). A flame-dried N 3-necked flask equipped with stir bar and addition funnel was charged with dry OH ether (20 mL) and allyl magnesium bromide (11 mL, 1 M solution in ether, 11

mmol). The mixture was cooled to 0 °C. A solution of II-10 (920 mg, 5 mmol) in dry ether (20

mL) was added dropwise via the addition funnel. A white precipitate was formed immediately on

addition. After the addition had been completed, the mixture was stirred for a further 15 min at

0 °C then quenched by dropwise addition of sat NH4Cl solution (25 mL). The layers were

separated, and the aqueous layer was further extracted with ether (2 x 10 mL). The organic layers

were combined, dried over Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:2) to afford II-39 as a

1 colorless oil (1.09 g, 4.8 mmol, 96%). H NMR (500 MHz, CDCl3) δ 5.80 (m, 1H), 5.07 (m, 2H),

4.06 (m, 1H), 3.57 (m, 2H), 3.23 (m, 1H), 3.06 (m, 1H), 2.28 – 2.04 (m, 3H), 1.46 (s, 9H); 13C

NMR (125 MHz, CDCl3) δ 155.0, 135.8, 116.7, 79.7, (74.5 + 73.8), 52.7, 49.2, (45.6 + 45.0),

35.8, 28.7; ESMS 228 (M + H)+.

Boc tert-Butyl 3-allyl-4-(tert-butyldimethylsilyloxy)pyrrolidine-1-carboxylate N (II-40). A solution of II-39 (130 mg, 0.57 mmol), TBSCl (107 mg, 0.72 OTBS mmol) and imidazole (95 mg, 1.4 mmol) in anhydrous DMF (5 mL) was

stirred at 40 °C for 16 h. The solvent was removed in vacuo, and the crude product was purified

using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:9) to afford II-40 as a

1 colorless solid (181 mg, 0.53 mmol, 93%). H NMR (500 MHz, CDCl3) δ 5.71 (m, 1H), 4.99 (m,

2H), 3.89 (m, 1H), 3.58 – 3.44 (m, 2H), 3.10 – 2.93 (m, 2H), 2.19 (m, 1H), 2.03 (m, 1H), 1.91 (m,

144

13 1H), 1.42 (s, 9H), 0.84 (s, 9H), 0.02 (s, 6H); C NMR (125 MHz, CDCl3) δ 154.9, 136.1, 116.7,

79.4, (75.2 + 74.5), (53.2 + 52.7), (49.0 + 48.6), (46.3 + 45.6), 35.6, 28.7, 25.9, 18.2, -4.4; ESMS

m/z = 342 (M + H)+.

Boc tert-Butyl 3-(tert-butyldimethylsilyloxy)-4-(oxiran-2-ylmethyl)- N O pyrrolidine-1-carboxylate (II-41). A solution of II-40 (181 mg, 0.53 mmol) OTBS in CH2Cl2 (10 mL) was cooled to 0 °C. m-CPBA (149 mg, 0.86 mmol, 77% pure, 1.3 eq.) was added and the mixture was stirred for 40 h. 20% NaHSO4 solution (10 mL)

was added, the mixture was stirred for 15 min and the layers were separated. The organic layer

was washed with NaHCO3 (2 x 10 mL), sat NH4Cl (10 mL) and brine (10 mL), dried over

Na2SO4 and concentrated in vacuo. The crude product was purified using flash column

chromatography (silica gel, ethyl acetate / hexanes, 1:5) to afford II-41 as an inseparable mixture

1 of diastereomers (141 mg, 0.39 mmol, 74%). H NMR (500 MHz, CDCl3) δ 3.95 (m, 1H), 3.84

(m, 1H), 3.66 – 3.38 (m, 4H), 3.12 – 2.94 (m, 2H), 2.75 (dd, J = 4.5, 26.5 Hz, 1H), 2.25 (m, 1H),

13 1.71 (m, 1H), 1.45 (s, 9H), 0.88 (s, 9H), 0.07 (d, 6H); C NMR (125 MHz, CDCl3) δ 154.8, 79.6,

(75.6 + 74.9), (53.0 + 52.5), (51.4 + 50.9), (49.3 + 48.9), 47.2, 45.1, (44.2 + 43.8), 28.8, 26.0,

18.2, -4.4; ESMS m/z = 358 (M + H)+, 380 (M + Na)+.

Boc tert-Butyl 3-(3-bromo-2-hydroxypropyl)-4-(tert-butyldimethyl- N HO silyloxy)pyrrolidine-1-carboxylate (II-42). To a flame dried flask OTBS Br containing lithium bromide (55 mg, 0.62 mmol) under dry N2 was added a

solution of II-41 (141 mg, 0.39 mmol) in dry THF (5 mL). Acetic acid (57 µL, 1 mmol) was

145 added dropwise and the mixture was stirred for 16 h. NaHCO3 solution (10 mL) was added, and the product was extracted with ethyl acetate (3 x 10 mL). The organic layers were combined, dried over Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:4) to afford II-42 as two diastereomers. The diastereomers could be separated, but were combined for further reactions

1 (combined: 146 mg, 0.33 mmol, 85%). Diastereoisomer A: H NMR (500 MHz, CDCl3) δ 3.92

(m, 2H), 3.67 (m, 1H), 3.52 (m, 1H), 3.38 (m, 1H), 3.09 – 2.96 (m, 2H), 2.56 (m, 1H), 2.20 –

2.12 (m, 1H), 1.76 – 1.69 (m, 1H), 1.58 – 1.49 (m, 1H), 1.46 (s, 9H), 0.89 (s, 9H), 0.08 (m, 6H);

13 C NMR (125 MHz, CDCl3) δ 154.8, 79.8, (76.2 + 75.6), 69.8, (53.1 + 52.5), (49.3 + 48.8),

1 (43.5 + 42.8), 40.4, 36.8, 28.8, 26.1, 18.2, -4.3). Diastereoisomer B: H NMR (500 MHz, CDCl3)

δ 3.94 (m, 1H), 3.83 (m, 1H), 3.67 – 3.58 (m, 1H), 3.52 (m, 1H), 3.41 (m, 1H), 3.11 – 2.96 (m,

2H), 2.54 + 2.47 (dd, J = 4.5, 38 Hz, 1H), 2.24 (m, 1H), 1.71 (m, 1H), 1.61 (s, 1H), 1.46 (s, 9H),

13 0.89 (s, 9H), 0.08 (m, 6H); C NMR (125 MHz, CDCl3) δ 154.8, 79.7, (76.2 + 75.3), 70.1, (52.7

+ 52.3), (49.9 + 48.8), (43.8 + 43.1), 39.8, 36.8, 28.8, 26.0, 18.2, -4.3); ESMS m/z = 460, 462

(1:1) (M + Na)+, 897, 899, 901 (1:2:1) (2M + Na)+.

Boc tert-Butyl 3-(3-bromo-2-oxopropyl)-4-(tert-butyldimethyl-silyl- N O oxy)pyrrolidine-1-carboxylate (II-34). A 3-necked flask equipped with stir OTBS Br bar and addition funnel was flame dried, sealed and allowed to cool under dry N2. Dry CH2Cl2 (15 mL) and DMSO (45 µL, 0.66 mmol) were added, and the mixture was cooled to -78 °C. Oxalyl chloride (250 µL, 0.5 mmol, 2M in CH2Cl2) was added, and the mixture was stirred for 5 min. A solution of II-42 (146 mg, 0.33 mmol) in dry CH2Cl2 (5 mL) was added

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dropwise via the addition funnel. The mixture was stirred at -78 °C for 1 h. Triethylamine (91 µL,

0.63 mmol) was added, and the mixture was allowed to warm to room temperature. The reaction was quenched with brine (10 mL), and the product was extracted with CH2Cl2. The organic

layers were combined, dried over Na2SO4 and concentrated in vacuo. The crude product was

purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:5) to afford II-

1 34 (84 mg, 0.19 mmol, 58%). H NMR (500 MHz, CDCl3) δ 4.07 (m, 1H), 3.90 – 3.86 (m, 2H),

3.72 – 3.46 (m, 2H), 3.10 (m, 1H), 2.98 – 2.77 (m, 2H), 2.61 – 2.44 (m, 2H), 1.45 (s, 9H), 0.87 (s,

13 9H), 0.06 (m, 6H); C NMR (125 MHz, CDCl3) δ 201.0, 154.8, 79.8, (75.0 + 74.4), (52.8 +

52.2), (49.1 + 48.7), 48.4, (42.3 + 42.1), (41.6 + 41.3), 34.3, 28.8, 26.0, 18.2, -4.4; ESMS m/z =

458, 460 (1:1) (M + Na)+, 893, 895, 897 (1:2:1) (2M + Na)+.

Boc tert-Butyl 3-((2-aminothiazol-4-yl)methyl)-4-(tert-butyldi- N S H N methylsilyloxy)pyrrolidine-1-carboxylate (II-43). A solution of II- 2 N OTBS 34 (84 mg, 0.19 mmol) and thiourea (15 mg, 0.2 mmol) in ethanol (10

mL) was refluxed for 5 h. The mixture was poured into brine (20 mL) and extracted with ethyl

acetate. The organic layers were combined, dried over Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:1) to afford II-43 (62 mg, 0.15 mmol, 79%) as a white solid. 1H NMR (500 MHz,

CDCl3) δ 6.06 (s, 1H), 5.52 (s, 2H), 3.98 (m, 1H), 3.62 – 3.50 (m, 2H), 3.18 – 3.04 (m, 2H), 2.62

13 (m, 1H), 2.35 (m, 1H), 1.45 (s, 9H), 0.86 (s, 6H), 0.02 (s, 6H); C NMR (125 MHz, CDCl3) δ

168.5, 155.1, 150.4, 103.3, 79.6, (75.4 + 74.6), (53.3 + 52.8), (49.3 + 48.9), (46.3 + 45.7), 33.3,

+ 28.8, 26.1, 18.3, -4.5; ESMS m/z = 414 (M + H) .

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Boc tert-Butyl 3-((2-(bis(tert-butoxycarbonyl)amino)thiazol-4-yl)- N S Boc N methyl)-4-(tert-butyldimethylsilyloxy)pyrrolidine-1-carboxylate 2 N OTBS (II-44). To a solution of II-43 (62 mg, 0.15 mmol) in dry THF (5

mL) were added Boc2O (82 mg, 0.37 mmol) and DMAP (10 mg). The mixture was stirred under

N2 for 16 h. The solvent was removed in vacuo, and the crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:6) to afford II-44 (83 mg, 0.135

1 mmol, 91%) as a white solid. H NMR (500 MHz, CDCl3) δ 6.71 (s, 1H), 4.00 (m, 1H), 3.64 –

3.43 (m, 2H), 3.17 – 3.00 (m, 2H), 2.88 – 2.77 (m, 1H), 2.51 – 2.41 (m, 2H), 1.51 (s, 18H), 1.43

(s, 9H), 0.86 (s, 9H), 0.03 (s, 6H); ESMS m/z = 614 (M + H)+.

Boc tert-Butyl 3-((2-(bis(tert-butoxycarbonyl)amino)thiazol-4-yl)- N S Boc N methyl)-4-hydroxypyrrolidine-1-carboxylate (II-45). To a solution 2 N OH of II-44 (375 mg, 0.61 mmol) in anhydrous THF (5 mL) was added

TBAF (780 µL, 0.78 mmol, 1M solution in THF) dropwise, and the mixture was stirred

overnight. The reaction mixture was poured into brine and extracted with ethyl acetate (3 x 25

mL). The organic layers were combined, dried over Na2SO4 and concentrated in vacuo. The

crude product was purified using flash column chromatography (silica gel, ethyl acetate /

hexanes, 1:1) to afford II-45 (300 mg, 0.60 mmol, 98%) as a white solid. 1H NMR (500 MHz,

CDCl3) δ 6.80 (s, 1H), 4.16 (m, 1H), 3.75 – 3.59 (m, 2H), 3.20 (m, 1H), 3.06 (m, 1H), 2.81 –

13 2.72 (m, 2H), 2.40 – 2.30 (m, 1H), 1.53 (s, 18H), 1.45 (s, 9H); C NMR (125 MHz, CDCl3) δ

148

158.9, 154.7, 150.6, 149.8, 112.7, 85.1, 79.5, (75.1 + 74.3), 64.5, (52.7 + 52.3), 49.6, (45.9 +

45.3), 33.4, 28.7, 27.9; ESMS m/z = 500 (M + H)+, 522 (M + Na)+.

Boc tert-Butyl 3-((2-(bis(tert-butoxycarbonyl)amino)thiazol-4-yl)- N S Boc N methyl)-4-(1,3-dioxoisoindolin-2-yl)pyrrolidine-1-carboxylate 2 N N O O (II-46). To a solution of PPh3 (170 mg, 0.65 mmol) and

phthalimide (110 mg, 0.65 mmol) in anhydrous THF (5mL) was

added II-45 (300 mg, 0.60 mmol) as a solution in anhydrous THF (5 mL). DIAD (122 µL, 0.65

mmol) was added dropwise, and the solution was stirred overnight. The reaction mixture was

poured into sat NaHCO3 (aq) and extracted with ethyl acetate (3 x 25 mL). The organic layers

were combined, dried over Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:3) to afford II-46 (333

1 mg, 0.53 mmol, 88%) as a white solid. H NMR (500 MHz, CDCl3) δ 7.86 (s, 2H), 7.77 (s, 2H),

6.65 (s, 1H), 4.98 (m, 1H), 3.91 (m, 2H), 3.63 (m, 1H), 3.43 (m, 1H), 3.08 (m, 1H), 2.78 (m, 1H),

13 2.55 (m, 1H), 1.52 (m, 27H); C NMR (125 MHz, CDCl3) δ 168.5, 158.4, 154.6, 150.2, 149.7,

134.5, 131.7, 123.6, 112.5, 84.6, 79.5, 64.5, (52.3 + 51.5), (49.8 + 49.1), (42.5 + 41.5), 30.5, 28.7,

27.9; ESMS m/z = 629 (M + H)+, 651 (M + Na)+.

Boc tert-Butyl 3-amino-4-((2-(tert-butoxycarbonylamino)-thiazol-4- N S BocHN yl)methyl)pyrrolidine-1-carboxylate (II-47). To a solution of II-46 N NH2 (126 mg, 0.2 mmol) in methanol (3 mL) was added 50% aqueous

hydrazine (3 mL) dropwise. The solution was stirred at room temperature for 14 h. 2N HCl (15

149

mL) was added dropwise until the pH reached approximately 5. The mixture was stirred a further

2 h. The solution was poured into sat K2CO3 (20 mL, final pH ~10) and extracted with CH2Cl2 (5

x 15 mL). The organic layers were combined, dried over Na2SO4 and concentrated in vacuo to

afford II-47 as a white solid (76 mg, 0.19 mmol, 95%). Note: one of the Boc groups protecting

1 the aminothiazole was removed during this procedure. H NMR (500 MHz, CDCl3) δ 6.53 (s,

1H), 3.57 – 3.40 (m, 2H), 3.36 – 3.16 (m, 2H), 2.86 (m, 1H), 2.75 (m, 1H), 2.64 (m, 1H), 2.46 (m,

13 1H), 1.54 (s, 9H), 1.44 (s, 9H); C NMR (125 MHz, CDCl3) δ 161.8, 160.6, 154.9, 152.7, 149.7,

107.6, 82.6, 79.5, (54.6 + 54.3), (52.7 + 51.8), (48.9 + 48.5), (43.6 + 43.0), 29.2, 28.7, 28.4;

ESMS m/z = 399 (M + H)+.

Boc tert-Butyl 3-(2-(tert-butoxycarbonyl(4-chloro- N S Cl BocHN benzyl)amino)ethylamino)-4-((2-(tert-butoxy- N Boc N N H carbonylamino)thiazol-4-yl)methyl)pyrrolidine-

1-carboxylate (II-48). To a solution of II-47 (76 mg, 0.19 mmol) in MeOH (3 mL) was added a

solution of II-32 (54 mg, 0.19 mmol) in CH2Cl2 (2 mL). The solution was stirred for 10 min

before NaHB(OAc)3 (49 mg, 0.25 mmol) was added. The mixture was stirred for 1 h. The solution was poured into sat NaHCO3 and extracted with EtOAc. The organic layers were

combined, dried over Na2SO4 and concentrated in vacuo. The crude product was purified using

flash column chromatography (silica gel, ethyl acetate / hexanes, 3:1) to afford II-48 (100 mg,

1 0.15 mmol, 88%) as a white solid. H NMR (500 MHz, CDCl3) δ 7.28 (s, 2H), 7.17 (s, 2H), 6.51

(s, 1H), 4.47 (m, 2H), 3.48 – 3.06 (m, 6H), 2.77 – 2.62 (m, 4H), 2.45 (m, 1H), 2.01 (m, 1H), 1.54

13 – 1.44 (m, 27H); C NMR (125 MHz, CDCl3) δ 159.9, 155.0, 152.9, 149.9, 133.2, 128.9, 108.1,

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82.4, 80.6, 79.4, 58.6, 53.9, 51.3, 50.9, 49.7, 49.4, 47.1, 29.2, 28.8, 28.5; ESMS m/z = 666 / 668

(3:1) (M + H)+.

H N1-(4-((2-Aminothiazol-4-yl)methyl)-pyrrolidin-3- N S Cl 2 H N yl)-N -(4-chlorobenzyl)ethane-1,2-di-amine (II-7). 2 N H N N H HCl in dioxanes (4 N, 3mL) was added to II-48 (100

mg, 0.15 mmol), and the mixture was stirred overnight. The deprotection was monitored by

removing small aliquots, quenching and analyzing by ESMS. Once the deprotection was

complete, the excess solvent and HCl were removed under a stream of N2. The residue was

dissolved in H2O (10 mL) and washed with ethyl acetate (2 x 10 mL), and the water was removed to give II-7 as an off-white solid (42 mg, 0.083 mmol, 55 %). 1H NMR (500 MHz,

D2O) δ 7.32 (s, 4H), 6.50 (s, 1H), 4.16 (m, 3H), 3.78 (m, 1H), 3.65 – 3.37 (m, 6H), 3.23 (m, 1H),

3.01 (m, 1H), 2.93 – 2.89 (m, 1H), 2.65 (m, 1H); ESMS m/z = 366 / 368 (3:1) (M + H)+; mp =

175 °C.

O Boc Ethyl 2-(tert-butoxycarbonyl(3-fluorophenethyl)-amino)- N F EtO acetate (II-50). A solution of ethyl bromoacetate (119 µL, 1

mmol) and 3-fluorophenethylamine (130 µL, 1.5 mmol) in ethanol was stirred for 16 h.

Boc2O (327 mg, 1.5 mmol) was added and the solution was stirred a further 2 h. The mixture

was poured into brine and extracted with ethyl acetate (3 x 15 mL). The organic layers were

combined, dried over Na2SO4 and concentrated in vacuo. The crude product was purified

using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:4) to afford II-50 as

151

a colorless oil (146 mg, 0.45 mmol, 45%). 1H NMR (500 MHz, CDCl3) δ 7.24 (m, 1H), 6.99

– 6.88 (m, 3H), 4.18 (q, J = 7 Hz, 2H), 3.88 (s, 1H), 3.76 (s, 1H), 3.49 (m, 2H), 2.84 (m, 2H),

13 1.44 (s, 9H), 1.27 (t, J = 6.5 Hz, 3H); C NMR (125 MHz, CDCl3) δ 170.2, (164.7 + 162.8),

155.8, 141.9, 130.2, 124.8, 115.9, 113.5, 80.6, 61.3, 50.4, 49.4, 35.0, 28.5, 14.5; ESMS m/z =

326 (M + H)+.

O Boc 2-(tert-Butoxycarbonyl(3-fluorophenethyl)amino)acetic acid (II- N F HO 51). To a solution of II-50 (146 mg, 0.45 mmol) in MeOH (3 mL) was added aqueous NaOH (1 N, 3 mL) dropwise. The solution was stirred for 16 h. Aqueous

HCl (1N, 4 mL) was added dropwise. The mixture was diluted with brine (5 mL) and extracted with ethyl acetate (3 x 15 mL). The organic layers were combined, dried over Na2SO4 and concentrated in vacuo to afford II-51 as a white solid (133 mg, 0.45 mmol, quant.). 1H NMR

(500 MHz, CDCl3) δ 10.22 (br, 1H), 7.24 (m, 1H), 6.98 – 6.86 (m, 3H), 3.93 (s, 1H), 3.81 (s,

13 1H), 3.49 (m, 2H), 2.84 (m, 2H), 1.43 (s, 9H); C NMR (125 MHz, CDCl3) δ (175.9 + 175.4),

(164.1 + 162.2), 156.1, 141.7, 130.3, 124.8, 116.0, 113.5, 81.2, 50.5, 49.3, 34.8, 28.5; ESMS

(negative ion mode) m/z = 296 (M - H)-.

O Boc tert-Butyl 3-fluorophenethyl(2-(methoxy(methyl)-amino)-2- MeO N F N oxoethyl)carbamate (II-52). To a solution of II-51 (66 mg,

0.22 mmol) in dry CH2Cl2 (5 mL) was added DIEA (46 µL, 0.25 mmol). The solution was cooled to 0 °C, and EDC (48 mg, 0.25 mmol) and HOBT (34 mg, 0.25 mmol) were added. After

5 min, HN(OMe)Me.HCl (25 mg, 0.25 mmol) and DIEA (46 µL, 0.25 mmol) were added. The

152

mixture was stirred at room temperature for 14 h. The mixture was diluted with CH2Cl2 (15 mL)

and washed with 1N HCl (2 x 20 mL), sat NaHCO3 (2 x 20 mL) and brine (1 x 20 mL), dried

over Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:3) to afford II-52 as a colorless oil (66 mg,

1 0.19 mmol, 86%). H NMR (500 MHz, CDCl3) δ 7.20 (m, 1H), 6.88 (m, 3H), (4.04 + 3.93) (s,

rotamers, 2H), (3.67 + 3.02) (s, rotamers, 3H), 3.47 (m, 2H), 3.14 (s, 3H), 2.83 (m, 2H), 1.41 (s,

13 9H); C NMR (125 MHz, CDCl3) δ 164.0, 162.1, 156.0, 142.3, 130.1, 124.8, 115.9, 113.3, 80.2,

61.5, 50.4, (49.3 + 48.5), (35.0 + 34.6), 32.5, 28.5; ESMS m/z = 363 (M + Na)+.

O tert-Butyl 3-fluorophenethyl(2-oxoethyl)carbamate (II-53). A Boc N F H solution of II-52 (66 mg, 0.19 mmol) in anhydrous THF (5 mL) was

cooled to 0 °C. Lithium aluminum hydride (0.25 mL, 0.25 mmol, 1M in THF) was added

dropwise. The mixture was stirred for 1 h at 0 °C. Sodium bisulfate (20% in water, 5 mL) was

added dropwise. The mixture was poured into brine (15 mL) and extracted with ethyl acetate (3 x

15 mL). The organic layers were combined, dried over Na2SO4 and concentrated in vacuo. The

crude product was purified using flash column chromatography (silica gel, ethyl acetate /

hexanes, 1:2) to afford II-53 as a colorless oil (54 mg, 0.19 mmol, quant). 1H NMR (500 MHz,

CDCl3) δ (9.47 + 9.38) (s, rotamers, 1H), 7.21 (m, 1H), 6.88 (m, 3H), (3.84 + 3.69) (s, rotamers,

13 2H), 3.48 (m, 2H), 2.80 (m, 2H), 1.39 (m, 9H); C NMR (125 MHz, CDCl3) δ 199.1, (164.1 +

162.2), 155.5, 141.5, 130.4, 124.8, 116.0, 113.7, 81.1, (58.6 + 57.9), 50.6, 35.0, 28.4; ESMS m/z

= 315 (M + MeOH + H)+.

153

Boc tert-Butyl 3-(2-(tert-butoxycarbonyl-(3- N S BocHN Boc fluorophenethyl)amino)ethyl-am-ino)-4-((2- N N F N H (tert-butoxycarbonyl-ami-no)thiazol-4- yl)methyl)-pyrrolidine-1-carboxylate (II-54). To a solution of II-47 (89 mg, 0.22 mmol) in

CH2Cl2 (2 mL) was added II-53 (54 mg, 0.19 mmol) as a solution in CH2Cl2 (3 mL). The

mixture was stirred for 10 min before NaHB(OAc)3 (49 mg, 0.23 mmol) and MeOH (1 mL) were

added. The solution was stirred for 1 h. The mixture was poured into NaHCO3 (aq., 20 mL) and

extracted with CH2Cl2 (3 x 20 mL). The organic layers were combined, dried over anhydrous

sodium sulfate and concentrated in vacuo. The crude product was purified using flash column

chromatography (silica gel, ethyl acetate / hexanes, 4:1) to afford II-54 as an oily solid (20 mg,

1 0.03 mmol, 16%). H NMR (500 MHz, CDCl3) δ 7.25 (m, 1H), 6.90 (m, 3H), 6.50 (s, 1H), 3.47

(m, 3H), 3.35 – 3.16 (m, 5H), 3.05 (m, 1H), 2.85 – 2.56 (m, 6H), 2.42 (m, 1H), 1.54 (s, 9H), 1.45

(s, 18H); ESMS m/z = 664 (M + H)+.

H N1-(4-((2-aminothiazol-4-yl)methyl)-pyrrolidin- N S 2 H N H 3-yl)-N -(3-fluorophenethyl)-ethane-1,2- 2 N N F N H diamine (II-55). HCl in dioxanes (4 N, 3 mL) was

added to II-54 (20 mg, 0.03 mmol), and the mixture was stirred overnight. The deprotection was monitored by removing small aliquots, quenching and analyzing by ESMS. Once the deprotection was complete, the excess solvent and HCl were removed under a stream of N2. The

residue was dissolved in H2O (10 mL) and washed with ethyl acetate (2 x 10 mL), and the water

was removed to give II-55 as an off-white solid (13.8 mg, 0.027 mmol, 90%). 1H NMR (500

154

MHz, D2O) δ 7.28 (m, 1H), 6.96 (m, 3H), 6.45 (s, 1H), 3.78 (m, 1H), 3.61 (m, 2H), 3.43 (m, 1H),

3.30 – 3.07 (m, 7H), 2.95 (m, 3H), 2.74 (m, 1H), 2.63 – 2.49 (m, 1H); ESMS m/z = 364 (M +

H)+; mp = 163 °C.

Boc tert-Butyl 2-amino-4-((1-(tert-butoxycarbonyl)-4-(tert-butyldi- N BocN H N methylsilyloxy)pyrrolidin-3-yl)methyl)-1H-imid-azole-1- 2 N OTBS carboxylate (II-57). A solution of II-34 (218 mg, 0.5 mmol) and

Boc-guanidine (160 mg, 1 mmol) in DMF (10 mL) was heated to 50 °C for 42 h. The solvent

was removed in vacuo. The crude product was purified using flash column chromatography

(silica gel, ethyl acetate / hexanes, 1:2) to afford II-57 as a white solid (248 mg, 0.5 mmol,

1 quant). H NMR (500 MHz, CDCl3) δ 6.48 (s, 1H), 6.08 (s, rotamers, 2H), 3.96 (m, 1H), 3.52 (m,

2H), 3.07 (m, 2H), 2.48 (m, 1H), 2.30 (m, 1H), 2.15 (m, 1H), 1.55 (s, 9H), 1.42 (s, 9H), 0.83 (s,

13 9H), 0.01 (s, 6H); C NMR (125 MHz, CDCl3) δ 155.0, 150.7, 149.5, 136.8, 107.3, 84.8, 79.3,

(75.5 + 74.6), (53.2 + 52.7), (49.2 + 48.9), (45.9 + 45.1), 30.0, 28.7, 28.2, 26.0, 18.2, -4.5; ESMS

m/z = 497 (M + H)+, 993 (2M + H)+.

Boc tert-Butyl 2-(bis(tert-butoxycarbonyl)amino)-4-((1-(tert- N BocN Boc N butoxycarbonyl)-4-(tert-butyldimethylsilyloxy)pyrrol-idin-3- 2 N OTBS yl)methyl)-1H-imidazole-1-carboxylate (II-58). To a solution of

II-57 (248 mg, 0.5 mmol) in anhydrous THF (5 mL) was added DMAP (10 mg) and Boc2O (240

mg, 1.1 mmol). The solution was stirred overnight. The solvent was removed in vacuo and the

crude product was purified using flash column chromatography (silica gel, ethyl acetate /

155

hexanes, 1:4) to afford II-58 as a white solid (300 mg, 0.43 mmol, 86%). 1H NMR (500 MHz,

CDCl3) δ 7.03 (s, 1H), 3.92 (m, 1H), 3.55-3.34 (m, 2H), 3.06 – 2.93 (m, 2H), 2.66 (m, 1H), 2.25

13 (m, 2H), 1.49 (s, 9H), 1.33 (s, 27H), 0.79 (s, 9H), -0.02 (s, 6H); C NMR (125 MHz, CDCl3) δ

154.8, 149.5, 146.4, 137.9, 115.0, 85.9, 83.6, 79.4, (75.2 + 74.3), (53.1 + 52.5), 48.4, (46.2 +

45.3), 29.7, 28.7, 28.0, 25.9, 18.1, -4.5; ESMS m/z = 697 (M + H)+.

Boc tert-Butyl 2-(bis(tert-butoxycarbonyl)amino)-4-((1-(tert- N BocN Boc N butoxycarbonyl)-4-hydroxypyrrolidin-3-yl)methyl)-1H-imidazole- 2 N OH 1-carboxylate (II-59). To a solution of II-58 (300 mg, 0.43 mmol) in

anhydrous THF (5 mL) was added TBAF (141 mg, 0.54 mmol). The mixture was stirred for 3 h.

The solvent was removed in vacuo and the crude product was purified using flash column

chromatography (silica gel, ethyl acetate / hexanes, 1:2) to afford II-59 as a white solid (204 mg,

1 0.35 mmol, 81%). H NMR (500 MHz, CDCl3) δ 7.17 (s, 1H), 4.19 (m, 1H), 3.70 (m, 2H), 3.19

(m, 1H), 3.04 (t, J = 9 Hz, 1H), 2.61 (m, 2H), 2.21 (m, 1H), 1.58 (s, 9H), 1.44 (m, 27H); ESMS

m/z = 583 (M + H)+.

Enzyme Assay. The NOS isoforms used were recombinant enzymes overexpressed in E. coli.

Murine macrophage iNOS, rat nNOS, and bovine eNOS were overexpressed and isolated as

reported. The formation of nitric oxide was monitored using a hemoglobin capture assay as

described previously. Briefly, a solution of nNOS or eNOS containing 10 µM L-arginine, 1.6

mM CaCl2, 11.6 µg /mL calmodulin, 100 µM DTT, 100 µM NADPH, 6.5 µM H4B, 3 mM

oxyhemoglobin, and varying concentrations of inhibitor in 100 mM Hepes (pH 7.4) was

156

monitored at 30 °C. For the determination of inhibition of iNOS, no additional Ca2+ or

calmodulin were added. The assay was initiated by the addition of enzyme, and the absorption of

UV light at 400 nm was recorded over one minute. As NO was evolved and coordinated to the

hemoglobin, the absorption at 400 nm increased, producing a value for the enzyme velocity

under these conditions. A value for the initial rate was obtained when no inhibitor was added (v0).

The velocity of the enzyme (v) was then determined in the presence of varying concentrations of

inhibitor, until a concentration of inhibitor that reduced the enzyme velocity to half its initial

value (v / v0 ~ 0.5) was discovered. Concentrations of inhibitor above and below this value were tested and a graph of v / v0 versus inhibitor concentration ([I]) was plotted. Extrapolation of this

graph allowed the determination of an IC50 value. This is defined as the inhibitor concentration

that produces 50% inhibition in the presence of a constant substrate concentration. The Ki of an

inhibitor is an equilibrium constant that relates the concentration of an inhibitor to enzyme

velocity in the presence of varying substrate concentrations. The best way to determine Ki is

from Dixon plots. However, the Ki can be estimated from the IC50 if the Km for the substrate is

known, using the equations below. The Km values used were: 1.3 µM (nNOS), 8.3 µM (iNOS)

and 1.7 µM (eNOS).

% inhibition = 100 [I] / ([I] + Ki {1 + [S] / Km})

Ki = IC50 / (1 + [S] / Km)

Equation 2.1 Calculation of Ki from IC50

157

Chapter 3

Synthesis and Evaluation of Ether and Amide Analogues of 2-Aminopyridine-Based nNOS Inhibitors 158

3.1 Introduction

In Chapter 2, compound II-6 (Fig. 3.1) was introduced as a potent and highly selective inhibitor of nNOS. As mentioned previously, the high number of basic groups in the molecule could cause problems with bioavailability. Replacing the aminopyridine with an aminothiazole resulted in a dramatic loss in potency, indicating that the aminopyridine is necessary for tight binding. However, replacing other basic groups in the molecule with non-basic groups could have more success. The secondary amine of the pyrrolidine ring is necessary for selectivity for nNOS over eNOS and so was not modified. The other two secondary amines could potentially be swapped with non-basic functional groups. Even if that resulted in a moderate or small loss in potency, the gain in bioavailability from losing a positive charge could potentially make up for any loss.

H H N N H H2N N N F H2N N NH N N 2 H H II-6 II-4

H N

H2N N NH O 2

III-1

Figure 3.1 Structures of potent nNOS inhibitor, II-6, lead compound II-4, and its trans ether analog III-1, a proposed nNOS inhibitor.

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Molecular modeling was performed on an ether analog of II-4. It should be noted that

although II-4 has a cis configuration, the trans ether, III-1, was predicted to be as potent and

selective as the lead compound II-4. The modeling predicted that the aminopyridine would

interact with Glu592 of nNOS to give tight binding and that the pyrrolidine nitrogen would form

a charge-charge interaction with Asp597 to provide isoform selectivity, just as they had with the lead. However, whereas the secondary amine of II-4 forms a long range interaction with a heme

propionate, the ether oxygen of III-1 was predicted to form a hydrogen bonding interaction with

Gln478 (Fig. 3.2). As III-1 has one less secondary amine than II-4, it was predicted to show

better pharmacokinetics. The synthesis of III-1 and its evaluation as an nNOS inhibitor will be

described below.

Figure 3.2 Modeling diagram of III-1 (green) in the active site of nNOS.

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The secondary amine next to the pyrrolidine ring in II-6 interacts weakly through long-

range hydrogen bonds with an active site heme propionate. It was hypothesized that this nitrogen

contributed only a small amount to the overall binding of the molecule, and could be replaced

with an ether, to form III-2, or an amide, to form III-3, with little loss in potency. It should be

noted that unlike III-1, this replacement maintains the cis-configuration found in II-6. Also, the

importance of the secondary amine in the chain was investigated by replacing it with an ether to

give III-4. The two ether-amide analogs, III-5 and III-6, would reduce the overall number of

basic groups in the molecule to two, with one of those being the aminopyridine ring. If they

proved to be potent nNOS inhibitors, this could result in a significant increase in bioavailability.

H H N N

H H H2N N N F H2N N N F N O H II-6 III-2

H H N N O H H2N N N F H2N N O F N N H H III-3 III-4

H H N N

H H H2N N N F H2N N N F O O O O III-5 III-6

Figure 3.3 Structures of potential nNOS inhibitors that have fewer basic groups than the lead compound, II-6.

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3.2 Synthesis of trans-ether, III-1

Alcohol III-8 was synthesized by the method described in Chapter 2 used to prepare II-

11. Commercially available 2-amino-6-methylpyridine was Boc protected, and the resulting compound was treated with two equivalents of butyl lithium to remove a proton from the methyl group (Scheme 3.1). The resulting anion was used as a nucleophile to open epoxide II-10 to give

the trans-alcohol. Sodium hydride was added to alcohol III-8 in an attempt to form the alkoxide.

Effervescence occurred, indicating that the sodium hydride was being consumed. The mixture

was then treated with a variety of electrophiles to form an ether linkage. Addition of bromide

III-9 and tosylate III-10 returned only the unreacted alcohol. Various conditions were used,

including different bases, solvents and temperatures, and the use of additives such as sodium

iodide and TBAI, but none of the desired product was formed. The use of bromoacetonitrile as

the electrophile gave a small amount of a new product with the desired mass, but analysis of the

NMR showed that the nitrogen of the amino group at the 2-position of the pyridine ring had been

alkylated, instead of the oxygen of the hydroxyl group, to give III-11. Apparently, the proton of

the carbamate nitrogen is more acidic than the hydroxyl proton.

This difference in acidity was used to selectively protect the carbamate nitrogen. Alcohol

III-8 was treated with one equivalent of sodium hydride in DMF. After 30 min, benzyl bromide

was added. This selectively added a benzyl protecting group to the nitrogen to form III-12.

Treating III-12 with sodium hydride resulted in effervescence, but adding III-9 or III-10 gave

back the alcohol. Use of bromoacetonitrile as the electrophile gave a low yield of the desired

ether. Nitrile III-13 was reduced under hydrogenation conditions to give amine III-14, with

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concurrent removal of the benzyl group. Removal of the Boc groups under acidic conditions gave compound III-1 as a salt.

Boc N NHBoc i Br

BocHN N N III-9 BocHN OH NHBoc III-7 III-8 TsO III-10 Boc N ii III-8 N BocN OH CN III-11

Boc Boc N N iii ii iv III-8 N N BocN OH BocN O CN Ph Ph III-12 III-13

Boc H N v N

N NH N NH BocHN O 2 H2N O 2 III-14 III-1

o o i) nBuLi, THF, -78 C - rt, 30 min, then II-9,THF,,-78 C - rt, 4h; ii) NaH, DMF, rt, 30 min, then BrCH2CN, o rt, 16h; iii) NaH, DMF, rt, 30 min, then BnBr, rt, 16h; iv) H2,Pd(OH)2 /C,1:1MeOH/CH3COOH, 60 C, 20h; v) 4N HCl, dioxanes, rt, 16h

Scheme 3.1 Ether formation.

Allyl bromide proved to be a much more efficient alkylating agent, giving high yields of

ether III-15 when used as the electrophile. Allyl ether III-15 could be converted to alcohol III-

163

16 under ozonolysis conditions, using sodium borohydride to reduce the ozonide intermediate.

The benzyl group was removed under hydrogenation conditions, and a Mitsunobu reaction using ethyl N-Boc oxamate converted the alcohol to the Boc protected primary amine.1 The Boc groups were cleaved with acid to give trans-ether III-1.

Boc Boc N N i ii III-12 BocN N BocN N OH O O R Ph III-15 iii III-16 R=Bn III-17 R=H

Boc N H N iv v BocHN N NHBoc O H N N NH2 2 O III-18 III-1

o i) NaH, DMF, rt, 30 min, then allyl bromide, rt, 16h; ii) O3,MeOH,-78 C, then NaBH4,rt1h;iii)H2, o Pd(OH)2 /C,MeOH,60 C, 20h; iv) PPh3, DIAD, BocNHCOOEt, THF, rt, 16h; v) 4N HCl, dioxanes, rt, 16h

Scheme 3.2 Alternative route to III-1.

Enzyme inhibition assays were performed using nNOS and eNOS. Although the computer modeling had predicted that III-1 should be a potent and selective inhibitor of nNOS, it was discovered that its affinity for nNOS was lower than expected (Ki = 4.5 µM) and that the affinity for eNOS was much higher than expected (Ki = 3.8 µM). This surprising result indicated that the modification from cis amine to trans ether resulted in a complete loss of isoform selectivity. The trans-amine version of II-4 was less potent than the cis-analog, but was still

164

highly selective for nNOS. Without crystal structures of III-1 in the active sites of nNOS and

eNOS, the binding conformations cannot be predicted, but it appears that III-1 must bind to the

two isoforms in a similar way. It is likely that the pyrrolidine ring of III-1 is twisted around so

that it no longer interacts with Asp597, and instead it is interacting with the heme propionate.

This is the case for II-4 bound to eNOS. If so, the major difference between the two active sites, an aspartate residue in nNOS and an asparagine in eNOS, is no longer interacting with the inhibitor, hence there is no selectivity.

Although III-1 will clearly not be useful as an nNOS inhibitor, the chemistry used to

make it can be applied to the syntheses of III-2, III-5 and III-6. Most importantly, the need for

diprotection of the 2-aminopyridine group before ether formation can occur has been discovered.

Secondly, it appears that the secondary alkoxide is not very nucleophilic and would rather act as

a base to deprotonate a potential electrophile. This makes the selection of electrophile critical to

the success of the ether forming reaction.

3.3 Solving the Mitsunobu Problem

It was envisioned that ether III-1 could be derived from a cis-alcohol, and that amide III-

2 and III-3 could be formed from a cis-amine. However, there was no easy way to invert the

stereochemistry at that position, as submitting trans-alcohol III-8 (or II-11) to Mitsunobu

conditions resulted in intramolecular cyclization (Scheme 3.3). The aminopyridine ring nitrogen

was sufficiently nucleophilic to displace the activated Mitsunobu intermediate and form a 5-

membered ring. Also, converting the trans-alcohol to a tosylate or triflate gave the same

cyclization product. The cis-alcohol could be formed by oxidation of the alcohol to the ketone,

165

then non-selective reduction back to a separable mixture of cis- and trans-alcohols, but this was

clearly a poor route to take.

It was hypothesized that adding steric hindrance to the aminopyridine could minimize the level of intramolecular cyclization. In fact, submitting benzyl protected III-12 to Mitsunobu

conditions, using phthalimide as the nucleophile, gave high yields of the desired product III-19

and no trace of the cyclization product.

Boc N i III-8 N+ NHBoc

Boc Boc N N ii i III-12 III-12 BocN N BocN N NPhth N3 Ph Ph III-19 III-20

Boc Boc N N iii iv v III-8 BocHN N Boc N N OTBS 2 OTBS III-21 III-22

Boc Boc N N i

N N Boc2N Boc2N OH N3 III-23 III-24

i) PPh3, DIAD, DPPA, THF, rt, 16h; ii) PPh3, DIAD, phthalimide, THF, rt, 16h; iii) TBSCl, imidazole, o DMF, 40 C, 16h; iv) Boc2O, DMAP, THF, 16h; v) TBAF, THF, rt, 16h

Scheme 3.3 Diprotection of aminopyridine allows a Mitsunobu reaction to occur.

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The generality of this phenomenon was investigated. As expected, the Boc and benzyl

protected III-12 formed azide III-20 in high yield. Next, alcohol III-8 was protected with

another Boc group, to see whether two carbamate groups would prevent cyclization as

effectively as a Boc and benzyl group had. The hydroxyl group of III-8 needed to be protected as

a TBS ether before the second Boc group could be added. Removal of the TBS group gave III-

23, which underwent the Mitsunobu reaction to give azide III-24 in high yield.

Dimethylpyrrole protected III-25, which could be formed by attack of the

dimethylpyrrole protected aminopyridine on ether II-10, gave the desired Mitsunobu product III-

26 in low yield; the remainder of the material was lost to general decomposition, not to

intramolecular cyclization (Scheme 3.4).

Boc Boc N N i

N N N N OH N3

III-25 III-26

i) PPh 3, DIAD, DPPA, THF, rt, 16h;

Scheme 3.4 Mitsunobu reaction using a dimethylpyrrole protected aminopyridine.

Using one of these diprotection strategies, a direct route to converting the trans-alcohol to

a cis-alcohol or amine was now available. As the aminopyridine needed to be diprotected for

ether formation, this method provided a high yielding stereoselective route to the desired

intermediates without too many additional steps, which would prove invaluable in the syntheses

of III-2 to III-6.

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3.4 Synthesis of ethers and amides

Alcohol II-11 could be benzyl protected in the same way as III-8, to give III-27 (Scheme

3.5). A Mitsunobu reaction using acetic acid as the nucleophile gave acetate III-28, which was then hydrolyzed to cis-alcohol III-29. The hydroxyl group was alkylated with an allyl group

(III-30), which was converted to an aldehyde (III-31) by ozonolysis.2

Boc Boc Boc N N N i ii iii BocN N BocN N BocHN N OH OAc OH Ph Ph II-11 III-27 III-28

Boc Boc Boc N N N iv v BocN N BocN N BocN N O OH O O Ph Ph Ph H III-29 III-30 III-31

o i) NaH, DMF, 0 C, 30 min, then BnBr, rt, 16h; ii) PPh3, DIAD, AcOH, THF, rt, 16h; iii) 1N NaOH (aq), MeOH, o o rt, 16h; iv) NaH, DMF, 0 C, 30 min, then allyl bromide; v) O3,CH2Cl2,-78 C, 1h, then Zn, AcOH (aq), rt, 3h

Scheme 3.5 Synthetic route to aldehyde III-31.

A reductive amination reaction with 3-fluorophenethylamine and Boc protection of the resulting secondary amine gave III-32. Removal of the benzyl group under hydrogenation conditions and the Boc groups under acidic conditions gave III-2 as a salt (Scheme 3.6).

H2N Boc N i iii III-31 + Boc III-2 BocN N N F O R F III-32, R=Bn ii III-33, R=H i) Na HB(OAc)3, MeOH, rt, 1h, TEA, Boc2O, 2 h; ii) H2,Pd(OH)2 / C, MeOH, rt, 1-2 d; iii) 4N HCl, dioxanes, 16 h

Scheme 3.6 Synthesis of inhibitor III-2.

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Alcohol III-27 was converted to azide III-34. The azide was reduced to the amine with concurrent removal of the benzyl group to give III-35 (Scheme 3.7).

Boc Boc N N Boc N i ii BocN N BocN N OH N BocHN N 3 NH Ph Ph 2 III-27 III-34 III-35

o i) PPh3, DIAD, DPPA, THF, rt, 16h; ii) H2,Pd(OH)2 /C,MeOH,60 C, 1-2 d

Scheme 3.7 Synthesis of amine III-35.

Ethyl bromoacetate and 3-fluorophenethylamine reacted to give the secondary amine, which was then Boc protected. The ester was hydrolyzed to give acid II-51 (Scheme 3.8). 3-

Fluorophenethanol was deprotonated with sodium hydride and alkylated with allyl bromide to form III-36. The allyl ether was oxidized to aldehyde III-37 under ozonolysis conditions.

O O F NH i Boc ii Boc 2 F N F N OEt OH

II-50 II-51

H F OH i F O ii F O O

III-36 III-37

i) Ethyl bromoacetate, MeOH, rt, 4h, then DIEA, Boc2O, rt, 2h; ii) 1N NaOH (aq), MeOH, rt, 16h; o o iii) NaH, THF, 0 C, then allyl bromide, rt, 16h; iv) O3,CH2Cl2,-78 C, 1h, then Zn, AcOH (aq), rt, 3h

Scheme 3.8 Synthesis of acid II-51 and aldehyde III-37.

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Amine III-35 and acid II-51 were coupled under standard peptide bond formation conditions to give amide III-38 (Scheme 3.9). The Boc groups were removed under acidic conditions to give III-3. Amine III-35 and aldehyde III-37 were coupled under reductive amination conditions to give III-39. Removal of the Boc groups gave III-4.

Boc N i O ii III-35 + II-51 H III-3 BocHN N N F N H III-38

Boc N iii ii III-35 + III-37 III-4 BocHN N O F N H III-39

i) EDC, HOBt, DIEA, CH2Cl2, rt, 16h; ii) 4N HCl, dioxanes, 16h; iii) Na HB(OAc)3,MeOH,rt,1h

Scheme 3.9 Completion of inhibitors III-3 and III-4.

Attempts to oxidize allyl ether III-30 to acid III-40 proved unsuccessful, but aldehyde

III-31 could be oxidized to the acid using oxone (Scheme 3.10).3 Acid III-40 and 3- fluorophenethylamine were coupled to give amide III-41. The benzyl group and Boc groups were removed to give III-5.

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Boc Boc N N i ii III-31 H BocN N OH BocN N N F O O Ph O Ph O III-40 III-41

Boc N iii iv H III-5 BocHN N N F O O III-42

i) Oxone, DMF, rt, 16h; ii) 3-fluorophenethylamine, EDC, HOBt, DIEA, CH2Cl2, rt, 16h; iii) H2,Pd(OH)2 /C, MeOH, 60 oC, 1-2d; iv) 4N HCl, dioxanes, 16h;

Scheme 3.10 Synthesis of inhibitor III-5.

Allyl ether III-30 could be converted to aldehyde III-31, which underwent reductive amination with benzylamine to give III-43 (Scheme 3.11). Removal of the benzyl groups gave

III-44. Amine III-44 and 3-fluorophenylacetic acid were coupled to give amide III-45. The Boc groups were removed to give III-6.

Boc N Boc N i ii III-31 BocN N NHBn O BocHN N NH O 2 Ph III-43 III-44

Boc N iii iv H III-6 BocHN N N F O O III-45

o i) Benzylamine, NaHB(OAc)3, rt, 1h; ii) H2,Pd(OH)2 /C,MeOH,60 C, 1-2d; iii) 3-fluorophenylacetic acid, EDC, HOBT, TEA, CH2Cl2, rt, 16h; iv) 4N HCl, dioxanes, 16h.

Scheme 3.11 Synthesis of inhibitor III-6.

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3.5 Results and discussion

Compounds III-2 through III-6 were tested for potency against the three isoforms of

NOS using the standard hemoglobin capture assay. The results are shown in Table 3.1.

H H N N

H H H2N N N F H2N N N F N O H II-6 III-2

H H N N O H H2N N N F H2N N O F N N H H III-3 III-4

H H N N

H H H2N N N F H2N N N F O O O O III-5 III-6

Figure 3.3 Structures of potential nNOS inhibitors that have fewer basic groups than the lead compound, II-6.

II-6 III-2 III-3 III-4 III-5 III-6

Ki (nNOS) / µM 0.014 0.015 0.053 0.4 2.3 5.0

Ki (eNOS) / µM 28 31 27 33 145 107

Ki (iNOS) / µM 4 9.5 5.4 4 52 77

Table 3.1 Inhibition constants against the three NOS isoforms.

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Ether III-2 has a potency against nNOS that is almost as good as the analogous amine

compound II-6. The potencies against eNOS and iNOS are also comparable, so the selectivity

for nNOS over the other isoforms is excellent. This indicates that, as suspected, the secondary

amine adjacent to the pyrrolidine ring is not contributing a significant amount to the overall

binding of II-6. Replacing the amine with an amide resulted in a slight loss of binding affinity, perhaps due to increased sterics or hindered rotation about the amide bond. However, replacing the amine in the chain with an ether linkage as in III-4, or with an amide as in III-5 and III-6,

resulted in a dramatic loss in potency, suggesting that this amine is critical for tight binding. Not

only is the ether linkage in III-4 neutral, but a hydrogen bond donor has been replaced with a

hydrogen bond acceptor. In the case of the amides, the NH is a potential H-bond donor, but the

potency is not recovered and is actually worse. This indicates that this amine is involved in a

charge – charge interaction that is important for binding, not a hydrogen bonding interaction.

The replacement of an aliphatic secondary amine with an ether linkage would normally

remove a charge from the molecule, as secondary amines usually have pKa’s of 9 or 10, and so

are fully protonated at physiological pH. However, when a secondary amine is two atoms away

from a strongly electron withdrawing group, its pKa can be lowered. If the pyrrolidine ring

nitrogen and the amine closest to the phenethyl group in II-6 are protonated, the central amine

now has two positively charged groups two atoms away. This could lower the pKa of the amine

to less than 7, so that it is not charged at physiological pH. This applies in other polyamine

molecules such as spermine. When a secondary amine is two carbons away from two other

amines, its pKa can be as low as 4.4 If this is the case in II-6, the replacement of the central

secondary amine in the molecule with an ether linkage will have no effect on the overall charge

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of the molecule. The only effect it will have will be to reduce the number of hydrogen bond donors by one. Lipinski has recently suggested that the number of H-bond donors is critical to

blood-brain barrier penetration, showing that the vast majority of CNS drugs have three or fewer hydrogen bond donors (see Chapter 4 for further discussion). If so, III-2 could cross the blood

brain barrier more effectively than II-6, as it has 4 H-bond donors (6-7 if the extra protons that

will bind at physiological pH are included) instead of 5 (7-8). Whether this is true will be

investigated in Chapter 4 using in vivo brain uptake experiments. However, it is unlikely that

such a subtle change will have a great impact on brain uptake, so further methods to improve the

pharmacokinetics of these molecules will be investigated.

3.6 Summary

Diprotection of the amino group of the aminopyridine was found to be necessary before

ether formation could be carried out. The replacement of a cis-amine linkage in lead compound

II-4 with a trans-ether linkage gave compound III-1. III-1 was only a weak inhibitor of nNOS,

and all isoform selectivity was lost in making this modification. Diprotection of the

aminopyridine also prevented an unwanted intramolecular cyclization reaction from occurring

during a Mitsunobu reaction. The replacement of secondary amines of inhibitor II-6 with ethers

and amides generally resulted in a loss of potency against nNOS. However, in one case,

substituting the secondary amine adjacent to the pyrrolidine ring with an ether linkage to give

III-2 resulted in no loss of potency or isoform selectivity. Whether this modification will

produce a significant increase in the bioavailability of the inhibitor will be investigated in the

next chapter.

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3.7 Experimental procedures

General Procedures: See Chapter 2 for general procedures and procedure for enzyme inhibition

assay.

General alkylation procedure: To a solution of the compound in anhydrous DMF (3 - 10 mL)

at 0 °C was added sodium hydride (1.1 equiv). The mixture was allowed to warm to room

temperature and stirred for 30 min. The alkylating agent (1.1 equiv) was added dropwise, and the solution was stirred for 16 h. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate, washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The

crude product was purified using flash column chromatography (silica gel, ethyl acetate /

hexanes).

General Mitsunobu procedure: To a solution of the alcohol in anhydrous THF (5 mL) were

added PPh3, DIAD and the nucleophile. The mixture was stirred for 16 h. The mixture was

poured into brine and extracted with ethyl acetate (3 x 10 mL), dried over anhydrous Na2SO4 and

concentrated in vacuo.

General reductive amination procedure: To a solution of the aldehyde in dichloromethane (5

mL) was added the amine, and the mixture was stirred for 10 min. NaHB(OAc)3 was added and

the solution was stirred for 1 h.

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General procedure for benzyl group removal: To a solution of the compound in EtOH (5 mL)

was added Pd(OH)2 / C (~5 mg). The mixture was stirred under a hydrogen atmosphere at 50 °C for 48 h. The mixture was filtered through Celite and the solvent was removed in vacuo.

General procedure for Boc group removal: A solution of the compound in HCl in dioxanes (4

N, 3 mL) was stirred for 16 h. The solvent was removed under a stream of nitrogen and the

residue was dissolved in water (10 mL). The solution was washed with ethyl acetate and the

solvent was removed in vacuo. The resulting salt was dissolved in the minimum amount of

methanol, and anhydrous ether was added causing the salt to precipitate. The ether layer was

decanted to leave the pure salt as a white solid.

tert-Butyl 6-methylpyridin-2-ylcarbamate (III-7). To a solution of 2-amino-

BocHN N 6-methylpyridine (2.16 g, 20 mmol) in tert-butanol (50 mL) was added Boc2O

(4.36 g, 20 mmol). The solution was heated to 55 °C for 44 h. The solvent was removed in vacuo

and the residue was dissolved in ethyl acetate, washed with sat NaHCO3 (aq) and brine, dried

over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash

column chromatography (silica gel, ethyl acetate / hexanes, 1:19) to afford III-6 as a white solid

1 (2.2 g, 10.6 mmol, 53%). H NMR (500 MHz, CDCl3) δ 8.58 (s, 1H), 7.75 (d, J = 8Hz, 1H), 7.53

13 (t, J = 8 Hz, 1H), 6.79 (d, J = 7 Hz, 1H), 2.45 (s, 3H), 1.48 (s, 9H); C NMR (125 MHz, CDCl3)

δ 156.9, 153.0, 152.0, 138.6, 118.0, 109.6, 80.7, 28.4, 24.0; ESMS m/z = 209 (M + H)+.

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Boc tert-Butyl 3-((6-(tert-butoxycarbonylamino)pyridin-2-yl)-methyl)- N 4-hydroxypyrrolidine-1-carboxylate (III-8). A solution of III-7 N BocHN OH (572 mg, 2.75 mmol) in anhydrous THF (20 mL) was cooled to -

78 °C. n-BuLi (2.29 mL, 5.5 mmol, 2.4 M in hexanes) was added dropwise. The solution was

allowed to slowly warm to room temperature, stirred for 30 min, then cooled down to -78 °C. A

solution of II-10 (474 mg, 2.56 mmol) in THF (10 mL) was added dropwise. The solution was

allowed to warm over 2 h, then stirred at room temperature for an additional 4 h. The reaction

was quenched with brine (20 mL), and the product was extracted with ethyl acetate (3 x 20 mL).

The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo. The

crude product was purified using flash column chromatography (silica gel, ethyl acetate /

hexanes, 2:3) to afford III-8 as a white solid (707 mg, 1.8 mmol, 70%). 1H NMR (500 MHz,

CDCl3) δ 7.85-7.57 (m, 3H), 6.82 (m, 1H), 4.13 (m, 1H), 3.77-3.60 (m, 2H), 3.26-3.07 (m, 2H),

13 2.42 (m, 1H), 1.49 (s, 9H), 1.45 (s, 9H). C NMR (125 MHz, CDCl3) δ 157.9, 154.6, 152.4,

151.6, 139.2, 118.2, 110.3, 81.1, 79.5, (75.2 + 74.5), 64.5, 60.5, (52.7 + 52.3), (49.7 + 49.2),

(45.2 + 44.8), 39.2, 28.6, 28.3. ESMS: m/z = 394 (M + H) +.

NHBoc TsO 2-(tert-Butoxycarbonylamino)ethyl 4-methylbenzenesulfonate (III-10). To a

solution of tosyl chloride (1.05 g, 5.5 mmol) and TEA (1 mL, 7 mmol) in CH2Cl2 (10 mL) at

0 °C was added tert-butyl 2-hydroxyethylcarbamate (0.78 mL, 5 mmol) dropwise. DMAP (10

mg) was added and the solution was stirred for 18 h. The solution was washed with 1N HCl (3 x

20 mL), sat NaHCO3 (2 x 20 mL) and brine (20 mL), dried over Na2SO4 and concentrated in

vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl

177 acetate / hexanes, 2:3) to afford III-8 as a white solid (315 mg, 4.1 mmol, 82%). 1H NMR (500

MHz, CDCl3) δ 7.79 (d, J = 8 Hz, 2H), 7.36 (d, J = 8 Hz, 2H), 4.88 (br, 1H), 4.07 (t, J = 5 Hz,

13 2H), 3.39 (m, 2H), 2.46 (s, 3H), 1.41 (s, 9H); C NMR (125 MHz, CDCl3) δ 155.9, 145.3, 130.2,

128.2, 80.0, 69.7, 40.0, 28.5, 21.9.

Boc tert-Butyl 3-((6-(tert-butoxycarbonyl(cyanomethyl)amino)-pyridin- N 2-yl)methyl)-4-hydroxypyrrolidine-1-carboxylate (II-11). The N BocN OH CN general alkylation procedure was carried out on III-8 (393 mg, 1 mmol) using bromoacetonitrile (67 µL, 1.1 mmol) as the alkylating agent. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:1) to afford

1 III-11 as a white solid (260 mg, 0.6 mmol, 60%). H NMR (500 MHz, CDCl3) δ 7.63 (m, 2H),

6.93 (d, J = 7 Hz, 1H), 4.87 (s, 2H), 4.12 (m, 1H), 3.64 (m, 2H), 3.36 – 3.17 (m, 2H), 2.87 (m,

13 1H), 2.78 – 2.66 (m, 2H), 1.58 (s, 9H), 1.45 (s, 9H); C NMR (125 MHz, CDCl3) δ 157.7, 155.0,

152.6, 151.8, 138.5, 119.8, 117.3, 115.6, 84.0, 79.6, (74.6 + 74.0), (52.7 + 52.3), (49.6 + 49.0),

(45.8 + 45.2), 39.3, 34.8, 28.7, 28.4; ESMS m/z = 433 (M + H)+.

Boc tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)pyridin-2- N yl)methyl)-4-hydroxypyrrolidine-1-carboxylate (III-12). The N BocN OH Ph general alkylating procedure was carried out on III-8 (197 mg, 0.5 mmol) using benzyl bromide (71 µL, 0.6 mmol) as the alkylating agent. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:1) to afford

1 III-12 as a white solid (174 mg, 0.36 mmol, 72%). H NMR (500 MHz, CDCl3) δ 7.53 (m, 2H),

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7.25 (m, 5H), 6.85 (m, 1H), 7.16 (s, 2H), 4.02 (m, 1H), 3.69 – 3.52 (m, 2H), 3.17 (m, 1H), 3.08

13 (m, 1H), 2.85-2.69 (m, 2H), 2.44 (m, 1H), 1.43 (s, 18H). C NMR (125 MHz, CDCl3) δ 157.9,

154.8, 154.0, 139.5, 138.0, 128.5, 127.3, 127.0, 119.4, 117.7, 81.7, 79.5, (75.1 + 74.4), (52.8 +

52.4), 50.6, (49.8 + 49.4), (45.6 + 44.9), 39.5, 28.7, 28.4. ESMS m/z = 484 (M + H) +.

Boc tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-pyridin-2- N yl)methyl)-4-(cyanomethoxy)pyrrolidine-1-carboxylate (III- N CN BocN O 13). The general alkylating procedure was carried out on III-12 Ph (427 mg, 0.88 mmol) using bromoacetonitrile (66 µL, 1 mmol) as the alkylating agent. The crude

product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:3)

1 to afford III-13 as an oil (51 mg, 0.098 mmol, 11 %). H NMR (500 MHz, CDCl3) δ 7.66 (m,

1H), 7.56 (m, 1H), 7.27 (m, 5H), 6.82 (m, 1H), 5.20 (s, 2H), 4.00 – 3.83 (m, 2H), 3.57 (m, 1H),

3.40 (m, 2H), 3.20 – 3.11 (m, 2H), 2.74 – 2.53 (m, 3H), 1.44 (m, 18H); 13C NMR (125 MHz,

CDCl3) δ 157.8, 154.5, 154.2, 151.9, 140.0, 137.9, 128.5, 127.3, 126.9, 120.3, 119.0, 117.1, 82.3,

81.8, 79.9, 54.5, 50.1, 43.0, 42.1, 39.3, 28.7, 28.4; ESMS m/z = 523 (M + H)+, 446 (M + Na)+,

1067 (2M + Na)+.

Boc tert-Butyl 3-(2-aminoethoxy)-4-((6-(tert-butoxy-carbonyl- N amino)pyridin-2-yl)methyl)pyrrolidine-1-carboxylate N NH BocHN O 2 (III-14). To a solution of III-13 (51 mg, 0.098 mmol) in

MeOH (3 mL) and AcOH (1 mL) was added Pd(OH)2 / C (5 mg). The solution was heated to

60 °C and put under a hydrogen atmosphere for 14 h. The Pd(OH)2 / C was removed by filtration

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through Celite, the solvent was removed in vacuo, and the residue was dissolved in ethyl acetate, washed with NaHCO3 (aq), dried over anhydrous Na2SO4 and concentrated in vacuo to afford

III-14 as a white powder (14 mg, 0.022 mmol). The 1H NMR revealed that the benzyl group had

not been completely removed, so Boc2O (0.1 mmol) was added to protect the primary amine and

facilitate purification. See III-18 for 1H NMR.

H 6-((4-(2-Aminoethoxy)pyrrolidin-3-yl)methyl)-pyrid-in-2- N amine (III-1). The general procedure for the removal of Boc N NH H2N O 2 groups was carried out on III-18 (14 mg, 0.022 mmol) to give

the trihydrochloride salt of III-1 as a white powder (4.5 mg, 0.013 mmol, 59%). 1H NMR (500

MHz, D2O) δ 7.78 (br, 2H), 7.66 (t, J = 8 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 6.63 (d, J = 8 Hz, 1H),

4.00 (m, 1H), 3.65 (m, 2H), 3.55 – 3.37 (m, 5H), 2.98 (m, 2H), 2.83 – 2.65 (m, 2H). ESMS m/z =

237 (M + H)+.

Boc tert-Butyl 3-(allyloxy)-4-((6-(benzyl(tert-butoxycarb-onyl)- N amino)pyridin-2-yl)methyl)pyrrolidine-1-carboxy-late (III-15). BocN N O The general alkylating procedure was carried out on III-12 (334 Ph mg, 0.692 mmol) using allyl bromide (69 µL, 0.8 mmol). The crude product was purified using

flash column chromatography (silica gel, ethyl acetate / hexanes, 1:5) to afford III-15 as a

1 colorless oil (215 mg, 0.41 mmol, 59%). H NMR (500 MHz, CDCl3) δ 7.56 (m, 2H), 7.26 (m,

5H), 6.81 (d, J = 7.5 Hz, 1H), 5.79 (m, 1H), 5.19 (m, 4H), 3.84 (m, 2H), 3.70 (m, 1H), 3.62 –

3.41 (m, 2H), 3.34 – 3.09 (m, 2H), 2.78 (m, 1H), 2.63, (m, 2H), 1.43 (m, 18H); 13C NMR (125

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MHz, CDCl3) δ157.6, 155.0, 154.8, 154.1, 139.9, 137.7, 134.7, 128.4, 127.4, 126.8, 118.9, 117.2,

81.6, 80.7, 79.5, 70.4, 50.7, 50.0, 49.2, (43.7 + 42.7), 39.7, 28.8, 28.4; ESMS m/z = 524 (M +

H)+.

Boc tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)-amino)- N pyridin-2-yl)methyl)-4-(2-hydroxyethoxy)-pyrrol-idine-1- N OH BocN O carboxylate (III-16). A solution of III-15 (215 mg, 0.41 Ph mmol) in MeOH (10 mL) was cooled to -78 °C. Ozone was bubbled through the solution for 45 min. NaBH4 (57 mg, 1.5 mmol) was added and the mixture was allowed to warm to room temperature and stirred for 1 h. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:1) to afford III-16 as an oil (121 mg, 0.23

1 mmol, 57%). H NMR (500 MHz, CDCl3) δ 7.56 (m, 2H), 7.26 (m, 5H), 6.82 (m, 1H), 5.19 (s,

2H), 3.70 (m, 1H), 3.59 (m, 4H), 3.38 (m, 4H), 2.74 (m, 1H), 2.66 (m, 2H), 1.45 (m, 18H); 13C

NMR (125 MHz, CDCl3) δ 157.6, 155.0, 154.1, 147.7, 139.9, 137.8, 128.4, 127.3, 126.9, 118.9,

117.2, 81.7, 79.6, 70.5, 64.6, 62.0, 50.4, 49.2, (43.5 + 42.6), 39.7, 28.7, 28.4. ESMS m/z = 528

(M + H)+.

Boc tert-Butyl 3-((6-(tert-butoxycarbonylamino)-pyridin-2-yl)- N methyl)-4-(2-hydroxyethoxy)-pyrrol-idine-1-carboxylate N OH BocHN O (III-17). The general procedure for the removal of benzyl groups was carried out on III-16 (121 mg, 0.23 mmol). The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 2:1) to afford III-17 as an oil

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1 (74 mg, 0.17 mmol, 72 %). H NMR (500 MHz, CDCl3) δ 7.80 (m, 1H), 7.54 (m, 2H), 6.78 (m,

1H), 3.82 – 2.98 (m, 9H), 2.69 (m, 3H), 1.51 (s, 9H), 1.44 (s, 9H); ESMS m/z = 438 (M + H)+.

Boc tert-Butyl 3-(2-(tert-butoxycarbonylamino)-ethoxy)-4- N ((6-(tert-butoxycarbonylamino)-pyridin-2-yl)methyl)- N NHBoc BocHN O pyrrolidine-1-carboxylate (III-18). The general

Mitsunobu procedure was carried out on III-17 (74 mg, 0.17 mmol) using PPh3 (52 mg, 0.2 mmol), DIAD (38 µL, 0.2 mmol) and ethyl N-Boc oxamidate (57 mg, 0.3 mmol, Fluka) as the nucleophile. Aqueous NaOH (1N, 1 mL) was added to the mixture prior to extraction, and the mixture was stirred for 4 h. The crude product was purified using flash column chromatography

(silica gel, ethyl acetate / hexanes, 1:4) to afford III-18 as a colorless oil (75 mg, 0.14 mmol,

1 83%). H NMR (500 MHz, CDCl3) δ 7.69 (m, 1H), 7.50 (m, 1H), 7.18 (m, 1H), 6.70 (m, 1H),

4.84 (br, 1H), 3.70 – 3.02 (m, 9H), 2.57 (m, 3H), 1.46 (s, 9H), 1.38 (s, 18H); ESMS m/z = 537

(M + H)+.

Boc tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-pyridin-2- N yl)methyl)-4-(1,3-dioxoisoindolin-2-yl)pyrro-lidine-1-carboxylate BocN N NPhth (III-19). The general Mitsunobu procedure was carried out on III-12 Ph

(85 mg, 0.17 mmol) using PPh3 (52 mg, 0.2 mmol), DIAD (38 µL, 0.2 mmol) and phthalimide

(37 mg, 0.25 mmol) as the nucleophile. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:4) to afford III-19 as a white solid (84 mg,

1 0.13 mmol, 81%). Mp = 63 – 66 °C. H NMR (500 MHz, CDCl3) δ 7.81 (m, 2H), 7.73 (m, 2H),

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7.48 (m, 1H), 7.40 (m, 1H), 7.27 (m, 5H), 6.62 (d, J = 7.5 Hz, 1H), 5.15 (s, 2H), 4.83 (m, 1H),

3.80 (m, 2H), 3.57 – 3.48 (m, 1H), 3.39 (m, 1H), 3.02 (m, 1H), 2.72 (m, 1H), 2.58 (m, 1H), 1.48

13 (s, 9H), 1.42 (s, 9H); C NMR (125 MHz, CDCl3) δ 168.5, 156.9, 154.7, 154.6, 154.1, 139.9,

137.6, 134.4, 131.8, 128.3, 127.3, 127.2, 126.8, 123.6, 118.5, 116.9, 81.6, 79.6, (52.2 + 51.5),

(51.3 + 50.7), (50.1 + 49.5), (42.1 + 41.4), 36.4, 28.8, 28.4; ESMS m/z = 613 (M + H)+.

Boc tert-Butyl 3-azido-4-((6-(benzyl(tert-butoxycarbonyl)amino)- N pyridin-2-yl)methyl)pyrrolidine-1-carboxylate (III-20). The general BocN N N3 Mitsunobu procedure was carried out on III-12 (207 mg, 0.429 mmol) Ph

using PPh3 (131 mg, 0.5 mmol), DIAD (104 µL, 0.55 mmol) and DPPA (119 µL, 0.55 mmol) as

the nucleophile. The crude product was purified using flash column chromatography (silica gel,

ethyl acetate / hexanes, 1:4) to afford III-20 as an oil (187 mg, 0.368 mmol, 86%).1H NMR (500

MHz, CDCl3) δ 7.63 (m, 1H), 7.54 (m, 1H), 7.24 (m, 5H), 6.85 (d, J = 7 Hz, 1H), 5.18 (s, 2H),

3.76 (m, 1H), 3.59-3.29 (m, 3H), 3.00 (m, 1H), 2.90 (m, 1H), 2.77 (m, 1H), 2.61 (m, 1H), 1.45

13 (m, 18H); C NMR (125 MHz, CDCl3) δ 157.3, 154.6, 154.1, 139.9, 137.8, 128.4, 126.9, 118.8,

116.9, 81.7, 79.8, (63.4 + 62.7), (51.7 + 51.4), 50.1, (49.1 + 48.7), (42.7 + 42.1), 35.3, 28.7, 28.4.

ESMS m/z = 531 (M + Na)+.

Boc tert-Butyl 3-((6-(tert-butoxycarbonylamino)pyridin-2-yl)- N methyl)-4-(tert-butyldimethylsilyloxy)pyrrolidine-1-carboxylate BocHN N OTBS (III-21). To a solution of III-8 (162 mg, 0.42 mmol) in anhydrous

DMF (5 mL) were added imidazole (71 mg, 1.05 mmol) and TBSCl (79 mg, 0.525 mmol). The

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mixture was stirred at 40 °C for 16 h. The solvent was removed in vacuo and the residue was

dissolved in ethyl acetate, washed with brine, dried over anhydrous Na2SO4 and concentrated in

vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl

acetate / hexanes, 1:6) to afford III-21 as a white solid (154 mg, 0.31 mmol, 74%). 1H NMR

(500 MHz, CDCl3) δ 7.71 (m, 1H), 7.54 – 7.38 (m, 2H), 6.73 (d, J = 7.5 Hz, 1H), 3.94 (m, 1H),

3.62 – 3.38 (m, 2H), 3.14 – 2.98 (m, 2H), 2.78 (m, 1H), 2.42 (m, 2H), 1.47 (s, 9H), 1.40 (s, 9H),

13 0.81 (s, 9H), -0.03 (s, 6H); C NMR (125 MHz, CDCl3) δ 158.3, 154.9, 152.5, 151.6, 138.7,

117.9, 109.9, 81.0, 79.4, (75.3 + 74.5), (53.0 + 52.7), (49.1 + 48.8), (46.7 + 46.1), 39.3, 28.7,

28.4, 25.9, 18.2, -4.6; ESMS m/z = 508 (M + H)+.

Boc tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)pyridin-2-yl)- N methyl)-4-(tert-butyldimethylsilyloxy)pyrrolidine-1-carboxylate Boc N N 2 OTBS (III-22). To a solution of III-21 (154 mg, 0.31 mmol) in anhydrous

THF (5 mL) was added Boc2O (0.5 mmol) and DMAP (~10 mg). The solution was stirred at

room temperature for 16 h. The solvent was removed in vacuo and the crude product was

purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:6) to afford

1 III-22 as a white solid (172 mg, 0.28 mmol, 90%). H NMR (500 MHz, CDCl3) δ 7.61 (m, 1H),

7.10, m, 1H), 7.00 (m, 1H), 4.01 (m, 1H), 3.64 – 3.40 (m, 2H), 3.14 – 2.83 (m, 3H), 2.50 (m, 2H),

13 1.42 (m, 27H), 0.84 (s, 9H), -0.01 (s, 6H); C NMR (125 MHz, CDCl3) δ 159.2, 154.9, 152.0,

151.5, 138.3, 121.5, 118.9, 83.0, 79.4, (75.1 + 74.5), (53.2 + 52.6), (48.9 + 48.5), (46.7 + 46.1),

39.1, 28.7, 28.1, 25.9, 18.2, -4.5; ESMS m/z = 608 (M + H)+, 630 (M + Na)+.

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Boc tert-Butyl 3-((6-(bis(tert-butoxycarbonyl)amino)pyridin-2-yl)- N methyl)-4-hydroxypyrrolidine-1-carboxylate (III-23). To a solution Boc N N 2 OH of III-22 (172 mg, 0.28 mmol) anhydrous THF (5 mL) was added

TBAF (0.35 mL, 1M in THF, 0.35 mmol). The solution was stirred for 16 h. The mixture was poured into brine and extracted with ethyl acetate (3 x 10 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography

(silica gel, ethyl acetate / hexanes, 1:1) to afford III-22 as a white solid (125 mg, 0.254 mmol,

1 91%). H NMR (500 MHz, CDCl3) δ 7.70 (t, J = 7.5 Hz, 1H), 7.11 (m, 2H), 4.16 (m, 1H), 3.80-

3.61 (m, 2H), 3.19 (m, 1H), 3.09 (m, 1H), 2.91 (m, 2H), 2.47 (m, 1H), 1.46 (s, 27H). 13C NMR

(125 MHz, CDCl3) δ 159.4, 154.7, 151.8, 151.5, 139.1, (122.4 + 122.2), (120.0 + 119.8), 83.7,

79.5, (75.3 + 74.6), (53.0 + 52.5), (50.0 + 49.7), (45.7 + 44.9), (39.7 + 39.5), 28.7, 28.1. ESMS m/z = 494 (M + H)+, 516 (M + Na) +.

Boc tert-Butyl 3-azido-4-((6-(bis(tert-butoxycarbonyl)amino)-pyridin-2- N yl)methyl)pyrrolidine-1-carboxylate (III-24). The general N Boc2N N3 Mitsunobu procedure was carried out on III-23 (125 mg, 0.254 mmol) using PPh3 (79 mg, 0.3 mmol), DIAD (66 µL, 0.35 mmol) and DPPA (76 µL, 0.35 mmol) as the nucleophile. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:4) to afford III-24 as an oil (113 mg, 0.22 mmol, 85%). 1H NMR (500

MHz, CDCl3) δ 7.67 (m, 1H), 7.10 (m, 2H), 4.03 (m, 1H), 3.71-3.49 (m, 3H), 3.08 (m, 2H), 2.89

13 (m, 2H), 1.45 (s, 27H). C NMR (125 MHz, CDCl3) δ 158.7, 154.6, 154.3, 152.1, 151.5, 138.6,

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121.9, 119.4, 83.1, 79.9, (63.3 + 62.7), (51.8 + 51.5), (48.9 + 48.6), (43.2 + 42.5), 35.4, 28.7,

28.1. ESMS m/z = 519 (M + H) +, 541 (M + Na) +.

Boc tert-Butyl 3-((6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-2-yl)methyl)- N 4-hydroxypyrrolidine-1-carboxylate (III-25). The general procedure N N OH for the removal of benzyl groups was carried out on 1-benzyl-4-((6-

(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-2-yl)methyl)-pyrrolidin-3-ol (36 mg, 0.1 mmol). To the

crude product in methanol (5 mL) was added Boc2O (33 mg, 0.15 mmol) and TEA (15 µL, 0.1

mmol), and the mixture was stirred for 2 h. The solvent was removed in vacuo, and the crude

product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:1)

1 to afford III-25 as a yellow solid (33 mg, 0.089 mmol, 89%). H NMR (500 MHz, CDCl3) δ 7.78

(t, J = 7.5 Hz, 1H), 7.18 (m, 1H), 7.10 (d, J = 7.5 Hz, 1H), 5.89 (s, 2H), 4.11 (m, 1H), 3.78-3.62

(m, 2H), 3.21 (m, 1H), 3.09 (t, J = 8.5 Hz, 1H), 3.05-2.92 (m, 2H), 2.53 (m, 1H), 2.11 (s, 6H),

13 1.45 (s, 9H). C NMR (125 MHz, CDCl3) δ 159.9, 154.8, 151.7, 139.1, 128.7, 122.3, 120.2,

107.5, 79.7, (75.7 + 75.0), (53.2 + 52.7), (40.0 + 49.6), (45.5 + 44.6), 39.8, 28.7, 13.4. ESMS m/z

= 372 (M + H) +, 394 (M + Na) +.

Boc tert-Butyl 3-azido-4-((6-(2,5-dimethyl-1H-pyrrol-1-yl)-pyridin-2- N yl)methyl)pyrrolidine-1-carboxylate (III-26). The general Mitsunobu N N N3 procedure was carried out on III-25 (33 mg, 0.089 mmol) using PPh3

(26 mg, 0.1 mmol), DIAD (21 µL, 0.11 mmol) and DPPA (24 µL, 0.11 mmol) as the nucleophile.

The crude product was purified using flash column chromatography (silica gel, ethyl acetate /

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1 hexanes, 1:4) to afford III-26 as an oil (11 mg, 0.028 mmol, 31%). H NMR (500 MHz, CDCl3) δ

7.75 (q, J = 9 Hz, 1H), 7.19 (t, J = 8 Hz, 1H), 7.08 (t, J = 7 Hz, 1H), 5.90 ( s, 2H), 4.05 (m, 1H),

3.72-3.48 (m, 3H), 3.10 (m, 2H), 2.93 (m, 2H), 2.12 (s, 6H), 1.45 (s, 9H). 13C NMR (125 MHz,

CDCl3) δ 159.5, 154.4, 152.0, 138.6, 128.7, 122.2, 119.9, 107.2, 80.0, (63.6 + 62.8), (51.7 +

51.4), (49.1 + 48.7), (43.0 + 42.3), 35.8, 28.7, 13.5. ESMS m/z = 397 ([M + H]+), 419 ([M +

Na]+).

tert-Butyl 4,6-dimethylpyridin-2-ylcarbamate (II-9). To a solution of 2-

amino-4,6-dimethylpyridine (1.22 g, 10 mmol) in tert-butanol (20 mL) was BocHN N

added Boc2O (2.18 g, 10 mmol). The mixture was heated to 55 °C for 40 h. The solvent was

removed in vacuo. The residue was dissolved in ethyl acetate, washed with sat NaHCO3 (aq) and

brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified

using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:5) to afford II-9 as a

1 white solid (1.74 g, 7.8 mmol, 78%). Mp = 75 – 77 °C. H NMR (500 MHz, CDCl3) δ 8.20 (s,

1H), 7.60 (s, 1H), 6.64 (s, 1H), 2.39 (s, 3H), 2.29 (s, 3H), 1.49 (s, 9H); 13C NMR (125 MHz,

CDCl3) δ 156.5, 152.9, 151.6, 150.0, 119.4, 110.0, 80.8, 28.5, 24.0, 21.5; ESMS m/z = 223 (M +

H)+.

Boc tert-Butyl 3-((6-(tert-butoxycarbonylamino)-4-methyl-pyridin-2- N yl)methyl)-4-hydroxypyrrolidine-1-carboxylate (II-11). A solution BocHN N OH of II-9 (792 mg, 3.5 mmol) in anhydrous THF (20 mL) was cooled to

-78 °C. n-BuLi (4.4 mL, 7 mmol, 1.6 M in hexanes) was added dropwise. The solution was

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allowed to slowly warm to room temperature, stirred for 30 min, then cooled to -78 °C. A

solution of II-10 (648 mg, 3.5 mmol) in THF (10 mL) was added dropwise. The solution was allowed to warm over 2 h, then stirred at room temperature for an additional 4 h. The reaction was quenched with brine (20 mL), and the product was extracted with ethyl acetate (3 x 20 mL).

The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo. The

crude product was purified using flash column chromatography (silica gel, ethyl acetate /

hexanes, 2:3) to afford III-11 as a white solid (491 mg, 1.21 mmol, 35%). Mp = 70 – 72 °C. 1H

NMR (500 MHz, CDCl3) δ 7.66 (s, 1H), 7.28 (m, 1H), 6.67 (m, 1H), 5.56 (br, 1H), 4.13 (m, 1H),

3.85 – 3.60 (m, 2H), 2.80 (m, 2H), 2.43 – 2.31 (m, 4H), 1.50 (s, 9H), 1.46 (s, 9H); 13C NMR

(125 MHz, CDCl3) δ 157.6, 154.8, 154.6, 152.5, 151.3, 119.7, 110.9, 81.4, 79.6, (75.8 + 75.2),

(53.0 + 52.6), (50.1 + 49.6), (45.0 + 44.6), 39.5, 28.7, 28.5, 21.6; ESMS m/z = 408 (M + H)+.

The undesired regioisomer, formed by deprotonation of the 4-methyl group, eluted after the

1 desired regioisomer. H NMR (500 MHz, CDCl3) δ 8.10 (s, rotamers, 1H), 7.60 (s, rotamers, 1H),

6.65 (s, 1H), 4.06 (m, 1H), 3.66 (m, 1H), 3.52 (m, 1H), 3.27 (m, 1H), 3.10 (m, 1H), 2.40 (m, 5H),

13 1.49 (s, 9H), 1.45 (s, 9H); C NMR (125 MHz, CDCl3) δ 156.8, 154.9, 152.8, 151.9, 151.6,

118.7, 109.7, 80.9, 79.7, (74.3 + 73.7), (52.7 + 52.4), (49.1 + 48.9), (46.9 + 46.3), 37.1, 28.6,

28.4, 23.9; ESMS m/z = 408 (M + H)+.

Boc trans-tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-4- N methylpyridin-2-yl)methyl)-4-hydroxypyrrolidine-1-carboxylate BocN N OH Ph (III-27). The general alkylation procedure was carried out on II-11 (491 mg, 1.21 mmol) using benzyl bromide (148 µL, 1.25 mmol) as the alkylating agent. The

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crude product was purified using flash column chromatography (silica gel, ethyl acetate /

hexanes, 2:3) to afford III-27 as a white solid (402 mg, 0.81 mmol, 67%). 1H NMR (500 MHz,

CDCl3) δ 7.34 – 7.18 (m, 6H), 6.70 (m, 1H), 5.12 (s, 2H), 4.05 (m, 1H), 3.74 – 3.55 (m, 2H),

3.19 (m, 1H), 3.08 (m, 1H), 2.81 – 2.63 (m, 2H), 2.40 (m, 1H), 2.29 (s, 3H), 1.45 (s, 9H), 1.42 (s,

13 9H); C NMR (125 MHz, CDCl3) δ 157.5, 154.8, 154.5, 154.1, 149.4, 139.6, 128.4, 127.3,

127.0, 120.8, 118.4, 81.6, 79.5, (75.3 + 74.6), (52.9 + 52.5), 50.6, (49.9 + 49.5), (45.6 + 44.8),

39.4, 28.7, 28.4, 21.3; ESMS m/z = 498 (M + H)+.

Boc tert-Butyl 3-acetoxy-4-((6-(benzyl(tert-butoxycarbonyl)-amino)-4- N methylpyridin-2-yl)methyl)pyrrolidine-1-carboxylate (III-28). The BocN N OAc Ph general Mitsunobu procedure was carried out on III-27 (402 mg, 0.81

mmol) using PPh3 (262 mg, 1 mmol), DIAD (188 µL, 1 mmol) and acetic acid (86 µL, 1.5

mmol) as the nucleophile. The crude product was purified using flash column chromatography

(silica gel, ethyl acetate / hexanes, 1:3) to afford III-28 as a white solid (216 mg, 0.40 mmol,

1 49%). Unreacted III-27 was also recovered. H NMR (500 MHz, CDCl3) δ 7.44 (m, 1H), 7.29 –

7.18 (m, 5H), 6.62 (s, 1H), 5.16 (s, 2H), 4.11 (m, 1H), 3.43 (m, 3H), 3.10 (m, 1H), 2.85 (m, 1H),

13 2.66 (m, 2H), 2.29 (s, 3H), 2.05 (s, 3H), 1.45 (s, 9H), 1.41 (s, 9H); C NMR (125 MHz, CDCl3)

δ 170.7, 156.9, 154.5, 154.2, 148.9, 140.1, 128.3, 127.2, 126.8, 126.7, 119.9, 117.5, 81.4, 79.6,

(75.0 + 74.1), 60.6, (53.0 + 52.6), 50.2, (49.6 + 49.1), (42.0 + 41.4), 35.0, 28.7, 28.4, 21.3;

ESMS m/z = 540 (M + H)+, 562 (M + H)+.

189

Boc cis-tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-4- N methylpyridin-2-yl)methyl)-4-hydroxypyrrolidine-1-carb-oxylate BocN N OH Ph (III-29). To a solution of III-28 (216 mg, 0.40 mmol) in MeOH (3 mL) was added aqueous NaOH (1 N, 3 mL). The mixture was stirred at room temperature overnight.

The pH of the solution was adjusted to about 8, and the product was extracted with ethyl acetate

(3 x 15 mL). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated

in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl

acetate / hexanes, 2:3) to afford III-29 as a white solid (184 mg, 0.37 mmol, 93%). Mp = 51 –

1 53 °C. H NMR (500 MHz, CDCl3) δ 7.36 (d, 1H), 7.28 – 7.20 (m, 5H), 6.74 (d, 1H), 5.09 (s,

2H), 3.95 (m, 1H), 3.59 – 3.36 (m, 4H), 3.14 (m, 1H), 2.86 (m, 1H), 2.78 (m, 1H), 2.31 (s, 3H),

13 1.44 (s, 9H), 1.41 (s, 9H); C NMR (125 MHz, CDCl3) δ 157.8, 154.5, 154.2, 149.8, 139.2,

128.5, 127.0, 120.7, 118.7, 81.7, 79.3, (71.2 + 70.4), (53.9 + 53.6), 50.6, (49.6 + 49.2), (45.2 +

44.6), 35.3, 28.8, 28.4, 21.4; ESMS m/z = 498 (M + H)+, 520 (M + Na)+.

Boc tert-Butyl 3-(allyloxy)-4-((6-(benzyl(tert-butoxy-carbonyl)am- N ino)-4-methylpyridin-2-yl)methyl)pyrrol-idine-1-carboxylate BocN N O Ph (III-30). The general alkylating procedure was carried out on III- 29 (184 mg, 0.37 mmol) using allyl bromide (43 µL, 0.5 mmol) as the nucleophile. The crude

product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:4)

1 to afford III-30 as a colorless oil (154 mg, 0.287 mmol, 78%). H NMR (500 MHz, CDCl3) δ

7.40 (m, 1H), 7.27 – 7.17 (m, 5H), 6.69 (s, 1H), 5.80 (m, 1H), 5.22 – 5.11 (m, 4H), 3.96 (m, 1H),

3.72 – 3.64 (m, 2H), 3.55 – 3.37 (m, 2H), 3.21 – 3.09 (m, 2H), 2.93 (m, 1H), 2.72 (m, 1H), 2.59

190

13 (m, 1H), 2.29 (m, 3H), 1.45 – 1.41 (m, 18H); C NMR (125 MHz, CDCl3) δ 158.0, 154.9, 154.1,

148.7, 140.1, 134.9, 128.3, 127.2, 126.7, 120.4, 117.3, 116.8, 81.4, 79.3, 78.0, 70.4, (51.2 + 50.7),

50.1, (49.6 + 49.2), (43.1 + 42.4), 34.8, 28.8, 28.4, 21.4; ESMS m/z = 538 (M + H)+, 560 (M +

Na)+.

Boc tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-4- N methylpyridin-2-yl)methyl)-4-(2-oxoethoxy)-pyrrol-idine-1- BocN N O O carboxylate (III-31). A solution of III-30 (80 mg, 0.15 mmol) in Ph H

CH2Cl2 (5 mL) was cooled to -78 °C. Ozone was passed through the solution for 1 h. Zn powder

(29 mg, 0.45 mmol) and 50% aqueous acetic acid (5 mL) were added and the mixture was allowed to warm to room temperature. The mixture was stirred a further 1 h. The mixture was poured into NaHCO3 (aq) and the product was extracted with CH2Cl2 (3 x 20 ml). The organic

layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:2)

1 to afford III-31 as an oily white solid (41 mg, 0.076 mmol, 51%). H NMR (500 MHz, CDCl3) δ

9.56 (s, 1H), 7.44 (m, 1H), 7.28 – 7.16 (m, 5H), 6.69 (s, 1H), 5.17 (s, 2H), 4.06 – 3.94 (m, 1H),

3.81 – 3.63 (m, 2H), 3.54 – 3.41 (m, 2H), 3.22 – 3.11 (m, 2H), 2.98 (m, 1H), 2.77 (m, 1H), 2.71

13 – 2.53 (m, 1H), 2.30 (s, 3H), 1.46 (s, 9H), 1.42 (s, 9H); C NMR (125 MHz, CDCl3) δ 202.4,

157.6, 154.7, 154.1, 148.9, 140.0, 128.4, 127.1, 120.2, 117.2, 81.5, 80.7, 79.7, 74.7, (51.1 + 50.5),

50.1, (49.4 + 49.0), (42.9 + 42.3), 34.4, 28.7, 28.4, 21.3; ESMS m/z = 572 (M + MeOH)+.

191

Boc tert-Butyl 3-((6-(benzyl(tert-butoxy-carbon- N yl)amino)-4-methylpyridin-2-yl)methyl)-4-(2- Boc BocN N N F O (tert-butoxycarbonyl(3-fluorophenethyl)- Ph amino)ethoxy) pyrrol-idine-1-carboxylate (III-32). The general reductive amination procedure

was carried out using aldehyde III-31 (463 mg, 0.86 mmol), 3-fluorophenethylamine (224 µL,

1.72 mmol) and NaHB(OAc)3 (212 mg, 1 mmol). To the crude mixture DIEA (200 µL, 1.39

mmol) and Boc2O (218 mg, 1 mmol) were added and the solution was stirred for 2 h. The mixture was poured into brine (15 mL) and extracted with EtOAc (3 x 15 mL). The organic layers were combined, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:4) to afford III-32 as a white solid (249 mg, 0.33 mmol, 38%). 1H NMR (500 MHz,

CDCl3) δ 7.42 (m, 1H), 7.24 – 7.17 (m, 6H), 6.94 – 6.86 (m, 3H), 6.62 (m, 1H), 5.17 (s, 2H),

3.60 (m, 1H), 3.52 – 3.17 (m, 9H), 3.06 (q, J = 11 Hz, 1H), 2.88 – 2.79 (m, 3H), 2.68 (m, 1H),

13 2.58 (m, 1H), 2.28 (m, 3H), 1.43 (m, 27H); C NMR (125 MHz, CDCl3) δ 164.1, 162.1, 157.9,

155.5, 154.8, 154.6, 154.1, 148.8, 142.2, 140.1, 130.1, 128.3, 127.2, 126.7, 124.8, 120.2, 117.3,

115.9, 113.5, 81.4, 79.8, 79.3, 68.3, 64.5, 60.6, 50.3, 50.2, 49.5, 47.6, 43.0, 35.0, 34.5, 28.7, 28.4,

21.4; ESMS m/z = 763 (M + H)+.

Boc tert-Butyl 3-(2-(tert-butoxy-carbonyl-(3- N fluorophenethyl)amino)-ethoxy)-4-((6-(tert- Boc BocHN N N F O butoxycarbonyl-amino)-4-methylpyridin-2- yl)methyl)-pyrrol-idine-1-carboxylate (III-33). The general procedure for the removal of

192

benzyl groups was carried out on III-32 (249 mg, 0.33 mmol). The crude product was purified

using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:4) to afford III-33 as a

1 white solid (62 mg, 0.092 mmol, 28%). H NMR (500 MHz, CDCl3) δ 7.60 (m, 1H), 7.25 (m,

1H), 6.95 – 6.87 (m, 3H), 6.60 (m, 1H), 3.74 (m, 1H), 3.60 – 3.27 (m, 9H), 3.10 (m, 1H), 2.83 (m,

3H), 2.67 – 2.51 (m, 2H), 2.28 (m, 3H), 1.56 (s, 9H), 1.44 (m, 18H); 13C NMR (125 MHz,

CDCl3) δ 164.1, 162.2, 158.4, 155.6, 155.0, 152.7, 151.6, 150.2, 142.1, 130.1, 124.8, 119.4,

116.0, 113.2, 110.4, 81.0, 79.8, 78.9, 64.6, 54.7, 49.2, 47.7, 42.9, 35.2, 30.9, 28.6, 21.5; ESMS m/z = 673 (M + H)+.

H 6-((4-(2-(3-Fluorophenethylamino)-eth-oxy)- N pyrrolidin-3-yl)methyl)-4-methyl-pyridin-2- H H2N N N F O amine (III-1). The general procedure for the

removal of Boc groups was carried out on III-33 (62 mg, 0.092 mmol) to give III-2 as the white

1 trihydrochloride salt (41 mg, 0.085 mmol, 92%). H NMR (500 MHz, D2O) δ 7.24 (q, J = 7 Hz,

1H), 7.02 (d, J = 8 Hz, 1H), 6.97 (d, J = 10 Hz, 1H), 6.90 (t, J = 9 Hz), 6.57 (s, 1H), 6.45 (s, 1H),

4.10 (m, 1H), 3.75 (m, 1H), 3.70 (m, 1H), 3.63 – 3.53 (m, 4H), 3.38 (m, 1H), 3.29 – 3.20 (m, 4H),

13 3.04 (m, 1H), 2.96 (m, 1H), 2.84 (m, 1H), 2.68 (m, 1H), 2.21 (s, 3H); C NMR (125 MHz, D2O)

δ 163.8, 161.8, 158.4, 154.0, 145.9, 138.9, 130.8, 124.9, 115.6, 114.4, 110.6, 78.4, 64.0, 60.4,

49.5, 48.4, 47.2, 47.1, 41.7, 31.4, 29.3, 21.3; ESMS m/z = 373 (M + H)+.

193

Boc tert-Butyl 3-azido-4-((6-(benzyl(tert-butoxycarbonyl)-am-ino)-4- N methylpyridin-2-yl)methyl)pyrrolidine-1-carboxylate (III-34). The BocN N N3 Ph general Mitsunobu procedure was carried out on III-27 (519 mg, 1.04

mmol) using PPh3 (342 mg, 1.3 mmol), DIAD (255 µL, 1.35 mmol) and DPPA (281 µL, 1.30

mmol) as the nucleophile. The crude product was purified using flash column chromatography

(silica gel, ethyl acetate / hexanes, 1:4) to afford III-34 as a white solid (461 mg, 0.88 mmol,

1 85%) H NMR (500 MHz, CDCl3) δ 7.47 (m, 1H), 7.37 (m, 1H), 7.24 (m, 4H), 6.69 (s, 1H), 5.17

(s, 2H), 3.76 (m, 1H), 3.58 – 3.37 (m, 2H), 3.31 (m, 1H), 3.00 (q, J = 11 Hz, 1H), 2.85 (m, 1H),

2.72 (m, 1H), 2.62 (m, 1H), 2.30 (m, 3H), 1.45 (s, 9H), 1.42 (s, 9H); 13C NMR (125 MHz,

CDCl3) δ 156.8, 154.6, 154.2, 149.0, 140.0, 130.3, 128.3, 127.0, 126.8, 126.4, 120.5, 117.4, 81.5,

79.8, (63.5 + 62.7), 51.6, 50.2, 48.9, (42.8 + 42.2), 35.2, 28.7, 28.4, 21.4; ESMS m/z = 523 (M +

H)+, 545 (M + Na)+.

Boc tert-Butyl 3-amino-4-((6-(tert-butoxycarbonylamino)-4- N methylpyridin-2-yl)methyl)pyrrolidine-1-carboxylate (III-35). BocHN N NH 2 The general procedure for the removal of benzyl groups was carried

out on III-34 (461 mg, 0.88 mmol). The crude product was purified using flash column

chromatography (silica gel, ethyl acetate / methanol, 9:1) to afford III-35 as a white solid (170

1 mg, 0.42 mmol, 48%). H NMR (500 MHz, CDCl3) δ 7.62 (s, 1H), 6.64 (s, 1H), 3.56 – 3.14 (m,

4H), 2.78 – 2.61 (m, 3H), 2.46 (m, 1H), 2.30 (m, 3H), 1.52 (s, 9H), 1.44 (s, 9H); 13C NMR (125

MHz, CDCl3) δ 161.4, 158.1, 155.0, 152.6, 151.6, 150.4, 119.4, 110.5, 81.0, 79.4, (71.8 + 70.7),

194

(61.3 + 60.4), (50.0 + 49.6), (49.0 + 48.5), (35.3 + 34.7) , 28.7, 28.4, 21.5; ESMS m/z = 407 (M

+ H)+.

F O 1-(2-(Allyloxy)ethyl)-3-fluorobenzene (III-36). To a solution of 3-

fluorophenethanol (127 µL, 1 mmol) in anhydrous THF (5 mL) at

0 °C, was added NaH (45 mg, 60% in mineral oil, 1.1 mmol). On completion of effervescence,

allyl bromide (130 µL, 1.5 mmol) was added. The mixture was stirred for 16 h. The solvent was

removed in vacuo and the crude product was purified using flash column chromatography (silica

gel, ethyl acetate / hexanes, 1:6) to afford III-36 (143 mg, 0.79 mmol, 79%) as a colorless oil. 1H

NMR (500 MHz, CDCl3) δ 7.23 (m, 1H), 7.00 (d, J = 7.5 Hz, 1H), 6.91 (m, 2H), 5.89 (m, 1H),

5.25 (dd, J = 1, 17 Hz, 1H), 5.17 (d, J = 10 Hz, 1H), 3.98 (d, J = 5.5 Hz, 2H), 3.64 (t, J = 7 Hz,

13 2H), 2.89 (t, J = 7 Hz, 2H); C NMR (125 MHz, CDCl3) δ 162.1, 141.9, 134.9, 129.9, 124.8,

117.2, 116.0, 113.3, 72.2, 70.9, 36.3.

H 2-(3-Fluorophenethoxy)acetaldehyde (III-37). A solution of III-36 F O O (143 mg, 0.79 mmol) in CH2Cl2 (5 mL) was cooled to -78 °C. Ozone

was passed through the solution for 1 h. Zn powder (104 mg, 1.6 mmol) and 50% aqueous acetic

acid (5 mL) were added and the mixture was allowed to warm to room temperature. The mixture

was stirred a further 1 h. The mixture was poured into NaHCO3 (aq), and the product was

extracted with CH2Cl2 (3 x 20 mL). The organic layers were combined, dried over anhydrous

Na2SO4 and concentrated in vacuo. The crude product was purified using flash column

chromatography (silica gel, ethyl acetate / hexanes, 1:3) to afford III-37 as a white solid (110 mg,

195

1 0.61 mmol, 80%). H NMR (500 MHz, CDCl3) δ 9.69 (s, 1H), 7.25 (m, 1H), 6.94 (m, 3H), 4.07

13 – 3.43 (m, 4H), 2.90 (m, 2H); C NMR (125 MHz, CDCl3) δ 200.1, 163.0, 141.2, 130.1, 124.8,

116.1, 113.6, 73.3, 72.6, 36.1.

Boc tert-Butyl 3-(2-(tert-butoxycarbonyl-(3- N O Boc fluorophenethyl)amino)acetamido)-4-((6- BocHN N N F N H (tert-butoxycarbonylamino)-4-methylpyridin-

2-yl)methyl)pyrrol-idine-1-carboxylate (III-38). To a solution of II-51 (76 mg, 0.26 mmol)

and III-35 (106 mg, 0.25 mmol) in CH2Cl2 (10 mL) were added EDC (54 mg, 0.28 mmol),

HOBt (38 mg, 0.28 mmol) and DIEA (52 µL, 0.28 mmol). The mixture was stirred for 16 h. The

solution was diluted with CH2Cl2 (10 mL) and washed with 1N HCl (3 x 15 mL), sat NaHCO3

(aq. 2 x 15 mL) and brine (15 mL). The organic layer was dried over anhydrous Na2SO4 and

concentrated in vacuo. The crude product was purified using flash column chromatography

(silica gel, ethyl acetate / hexanes, 2:1) to afford III-38 (151 mg, 0.22 mmol, 88%) as a colorless

1 oil. H NMR (500 MHz, CDCl3) δ 7.62 (s, 1H), 7.24 (m, 1H), 6.91 (m, 3H), 6.60 (m, 1H), 4.57

(m, 1H), 3.81 (m, 2H), 3.56 – 3.39 (m, 4H), 3.25 – 3.06 (m, 2H), 2.86 – 2.60 (m, 5H), 2.27 (s,

13 3H), 1.52 (s, 9H), 1.42 (s, 9H), 1.38 (s, 9H); C NMR (125 MHz, CDCl3) δ 171.3, 164.1, 162.1,

157.2, 156.5, 154.4, 152.7, 151.6, 150.3, 141.2, 130.3, 124.8, 119.4, 116.0, 113.7, 110.7, 81.4,

80.9, 79.6, 52.4, 51.1, 50.5, 49.2, 49.0, 40.9, 36.3, 34.7, 28.6, 21.5; ESMS m/z = 686 (M + H)+,

708 (M + Na)+.

196

H N-(4-((6-Amino-4-methylpyridin-2-yl)-methyl)- N O pyrrolidin-3-yl)-2-(3-fluorophen-ethylamino)- H H2N N N F N H acetamide (III-3). The general procedure for the

removal of Boc groups was carried out on III-38 (151 mg, 0.22 mmol) to give III-3 (94 mg, 0.19

1 mmol, 86%) as a pale yellow trihydrochloride salt. H NMR (500 MHz, D2O) δ 7.24 (q, J = 7.5

Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H), 6.92 (m, 2H), 6.50 (s, 2H), 4.55 (m, 1H), 3.89 (s, 2H), 3.59 (m,

1H), 3.46 (m, 1H), 3.32 (m, 1H), 3.22 (m, 2H), 3.10 (m, 1H), 2.93 (t, J = 8 Hz, 2H), 2.83 (m, 1H),

13 2.73 (t, J = 7.5 Hz, 2H), 2.15 (s, 3H); C NMR (125 MHz, D2O) δ 166.3, 163.8, 161.9, 158.3,

154.0, 145.2, 138.8, 130.9, 124.9, 115.8, 114.4, 110.7, 50.5, 50.4, 48.6, 48.1, 48.0, 40.4, 31.5,

29.4, 21.4; ESMS m/z = 386 (M + H)+.

Boc tert-Butyl 3-(tert-butoxycarbonyl(2-(3- N fluorophenethoxy)ethyl)amino)-4-((6-(tert- BocHN N O F N Boc butoxycarbonylamino)-4-methylpyridin-2- yl)methyl)pyrrol-idine-1-carboxylate (III-39). The general reductive amination procedure was

carried out using amine III-35 (69 mg, 0.17 mmol) and aldehyde III-37 (27 mg, 0.15 mmol)

with NaHB(OAc)3 (42 mg, 0.2 mmol). DIEA (100 µL, 0.7 mmol) and Boc2O (50 mg, 0.23

mmol) were added, and the solution was stirred another 2 h. The mixture was poured into

NaHCO3 (aq) and the product was extracted with CH2Cl2 (3 x 20 mL). The organic layers were

combined, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was

purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:3) to afford

1 III-39 as a white solid (81 mg, 0.12 mmol, 80%). H NMR (500 MHz, CDCl3) δ 7.60 (m, 1H),

197

7.31 – 7.23 (m, 2H), 6.93 (m, 2H), 6.61 (m, 1H), 3.64 – 3.48 (m, 5H), 3.42 – 3.04 (m, 5H), 2.87

(m, 4H), 2.62 (m, 1H), 2.44 (m, 1H), 2.29 (m, 3H), 1.52 (s, 9H), 1.44 (s, 18H); ESMS m/z = 673

(M + H)+, 695 (M + Na)+.

H 6-((4-(2-(3-Fluorophenethoxy)ethyl-am- N ino)pyrrolidin-3-yl)methyl)-4-methyl-pyridin-2- H2N N O F N H amine (III-4). The general procedure for the

removal of Boc groups was carried out on III-39 (81 mg, 0.12 mmol) to give III-4 (54 mg, 0.11

1 mmol, 92%) as a white trihydrochloride salt. H NMR (500 MHz, D2O) δ 7.12 (m, 1H), 6.97 –

6.91 (m, 2H), 6.73 (m, 1H), 6.61 – 6.53 (m, 2H), 4.24 (m, 1H), 4.12 (m, 1H), 3.85 – 3.45 (m, 6H),

3.34 – 3.17 (m, 3H), 3.10 – 2.99 (m, 2H), 2.82 – 2.69 (m, 2H), 2.54 (m, 1H), 2.20 (s, 3H); ESMS

m/z = 373 (M + H)+.

Boc 2-(4-((6-(Benzyl(tert-butoxycarbonyl)amino)-4-methyl- N pyridin-2-yl)methyl)-1-(tert-butoxycarbonyl)-pyrrolidin-3- BocN N OH O yloxy)acetic acid (III-40). To a solution of III-31 (166 mg, Ph O 0.31 mmol) in anhydrous DMF (3 mL) was added oxone (190 mg, 0.31 mmol). The mixture was

stirred at room temperature for 3 h, and the solvent was removed in vacuo. The residue was dissolved in 1N HCl (aq) and extracted with ethyl acetate (5 x 15 mL). The organic layers were

combined, dried over anhydrous Na2SO4 and concentrated in vacuo to afford III-39 as a white

1 solid (143 mg, 0.258 mmol, 83%). H NMR (500 MHz, CDCl3) δ 7.25 (m, 6H), 6.92 – 6.70 (m,

1H), 5.18 (m, 2H), 4.12 (m, 1H), 3.89 (m, 2H), 3.48 (m, 2H), 3.17 (m, 2H), 2.92 (m, 2H), 2.59

198

13 (m, 1H), 2.32 (m, 3H), 1.44 (m, 18H); C NMR (125 MHz, CDCl3) δ 172.4, 158.4, 157.0, 155.1,

154.4, 151.5, 139.3, 128.5, 127.2, 120.7, 87.9, 82.1, 79.8, 74.8, 66.6, 51.1, 50.4, 49.4, 42.9, 34.8,

28.7, 28.4, 21.6; ESMS m/z = 556 (M + H)+, ESMS (negative ion mode) m/z = 554 (M – H)+.

Boc tert-Butyl 3-((6-(benzyl(tert-butoxy-carbonyl)- N

H amino)-4-methylpyridin-2-yl)methyl)-4-(2-(3- BocN N N F O fluorophenethyl-amino)-2-oxoethoxy)pyrrol- Ph O idine-1-carboxylate (III-41). To a solution of III-40 (143 mg, 0.258 mmol) in CH2Cl2 (3 mL)

were added 3-fluorophenethylamine (40 µL, 0.3 mmol), TEA (43 µL, 0.3 mmol, EDC (50 mg,

0.26 mmol), and HOBt (35 mg, 0.26 mmol). The mixture was stirred for 16 h. The solution was

diluted with CH2Cl2 (20 mL) and washed with 1N HCl (2 x 15 mL), sat NaHCO3 (15 mL) and

brine (15 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo.

The crude product was purified using flash column chromatography (silica gel, ethyl acetate /

hexanes, 2:1) to afford III-41 as a white solid (35 mg, 0.05 mmol, 20%). 1H NMR (500 MHz,

CDCl3) δ 7.44 (m, 1H), 7.23 (m, 6H), 6.95 (m, 1H), 6.89 (m, 2H), 6.52 (m, 1H), 5.14 (s, 2H),

3.87 (m, 1H), 3.67 – 3.36 (m, 6H), 3.13 (m, 1H), 2.99 (m, 1H), 2.79 (m, 2H), 2.69 (m, 1H), 2.54

13 (m, 2H), 2.30 (m, 3H), 1.45 (s, 9H), 1.40 (s, 9H); C NMR (125 MHz, CDCl3) δ 169.5, 164.2,

157.2, 154.3, 149.0, 140.0, 130.4, 128.4, 127.0, 126.9, 124.6, 120.0, 117.5, 115.9, 113.8, 83.7,

81.6, 80.4, 79.7, 68.9, (50.8 + 50.5), 50.2, (49.3 + 49.0), (42.7 + 42.2), 39.8, 35.6, 34.5, 28.7,

28.4, 21.4; ESMS m/z = 677 (M + H)+.

199

Boc tert-Butyl 3-((6-(tert-butoxycarbonyl-amino)- N

H 4-methylpyridin-2-yl)methyl)-4-(2-(3-fluoro- BocHN N N F O phenethylamino)-2-oxo-ethoxy)pyrrolidine-1- O carboxylate (III-42). The general procedure for the removal of benzyl groups was carried out on

III-41 (34 mg, 0.05 mmol). The crude product was purified using flash column chromatography

(silica gel, ethyl acetate / hexanes, 2:1) to afford III-42 as a yellow oil (21 mg, 0.036 mmol,

1 72%). H NMR (500 MHz, CDCl3) δ 7.61 (m, 1H), 7.05 (s, 1H), 6.98 (m, 1H), 6.89 (m, 2H),

6.71 (m, 1H), 6.53 (s, 1H), 4.01 (m, 1H), 3.84 (m, 2H), 3.65 – 3.46 (m, 4H), 3.29 (m, 1H), 3.07

(m, H), 2.84 (m, 2H), 2.71 (m, 1H), 2.57 (m, 2H), 2.30 (s, 3H), 1.52 (s, 9H), 1.46 (s, 9H); ESMS m/z = 587 (M + H)+.

H 2-(4-((6-Amino-4-methylpyridin-2-yl)-methyl)- N

H pyrrolidin-3-yloxy)-N-(3-fluoro-phenethyl)- H2N N N F O acetamide (III-5). The general procedure for the O removal of Boc groups was carried out on III-42 (0.036 mmol) to give III-5 as a greasy solid

1 (5.1 mg, 0.011 mmol, 31%). H NMR (500 MHz, D2O) δ 7.14 (q, J = 7 Hz, 1H), 6.95 (d, J = 7.5

Hz, 1H), 6.90 (d, J = 10 Hz, 1H), 6.75 (m, 1H), 6.55 (s, 1H), 6.26 (s, 1H), 4.00 (m, 1H), 3.86 (m,

1H), 3.70 (m, 1H), 3.42 (m, 6H), 3.13 (m, 1H), 3.01 (m, 1H), 2.75 – 2.58 (m, 3H), 2.16 (s, 3H);

ESMS m/z = 387 (M + H)+.

Boc tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)-amino)-4- N methylpyridin-2-yl)methyl)-4-(2-(benzyl-amino)ethoxy)- BocN N NHBn O Ph

200

pyrrolidine-1-carboxylate (III-43). The general reductive amination procedure was carried out

using aldehyde III-31 (59 mg, 0.11 mmol), benzylamine (36 µL, 0.33 mmol) and NaHB(OAc)3

(32 mg, 0.15 mmol). The crude product was purified using flash column chromatography (silica

gel, ethyl acetate) to afford III-43 as a white solid (63 mg, 0.1 mmol, 91%). 1H NMR (500 MHz,

CDCl3) δ 7.41 – 7.18 (m, 11H), 6.64 (s, 1H), 5.17 (s, 2H), 3.79 (s, 2H), 3.65 – 3.52 (m, 3H), 3.44

– 3.35 (m, 2H), 3.28 (m, 1H), 3.19 (m, 1H), 3.09 (m, 1H), 2.89 (m, 1H), 2.74 – 2.67 (m, 2H),

13 2.57 (m, 1H), 2.25 (s, 3H), 1.45 (s, 9H), 1.41 (s, 9H); C NMR (125 MHz, CDCl3) δ 157.9,

155.0, 154.7, 154.1, 148.8, 140.6, 140.1, 128.6, 128.3, 127.3, 127.2, 126.8, 126.7, 120.2, 117.3,

81.4, 79.7, 79.4, 78.8, (68.9 + 68.8), 54.0, (51.1 + 50.6), 50.2, 49.6, (49.0, (43.0 + 42.4), 34.7,

28.8, 28.5, 21.4; ESMS m/z = 631 (M + H)+.

Boc tert-Butyl 3-(2-aminoethoxy)-4-((6-(tert-butoxy-carbonyl- N amino)-4-methylpyridin-2-yl)methyl)-pyrrolidine-1- BocHN N NH2 O carboxylate (III-44). The general procedure for the removal

of benzyl groups was carried out on III-43 (63 mg, 0.10 mmol). The crude product was purified

using flash column chromatography (silica gel, EtOAc / MeOH, 9:1) to afford III-44 as a white

1 solid (13 mg, 0.03 mmol, 30%). H NMR (500 MHz, CDCl3) δ 7.55 (s, 1H), 7.20 (s, 1H), 6.58 (s,

1H), 3.73 (m, 1H), 3.58 – 3.20 (m, 6H), 3.08 (m, 1H), 2.82 (m, 2H), 2.61 (m, 1H), 2.46 (m, 1H),

2.22 (s, 3H), 1.45 (s, 9H), 1.39 (s, 9H); ESMS m/z = 451 (M + H)+.

201

Boc N-(2-(4-((6-Amino-4-methylpyridin-2-yl)- N

H methyl)pyrrolidin-3-yloxy)ethyl)-2-(3-fluoro- BocHN N N F O phenyl)acetamide (III-45). To a solution of III- O

44 (13 mg, 0.03 mmol) in anhydrous CH2Cl2 (3 mL) were added 3-fluorophenylacetic acid (5 mg,

0.03 mmol), EDC (6 mg, 0.03 mmol), HOBt (5 mg, 0.03 mmol) and TEA (3 µL, 0.03 mmol).

The mixture was stirred for 16 h, diluted with CH2Cl2 (10 mL), and washed with 1N HCl (2 x 10

mL), sat NaHCO3 (2 x 10 mL) and brine (10 mL), dried over anhydrous Na2SO4 and

concentrated in vacuo. The crude product was purified using flash column chromatography

(silica gel, ethyl acetate / hexanes, 3:1) to afford III-45 as a white solid (9 mg, 0.015 mmol,

1 51%). H NMR (500 MHz, CDCl3) δ 7.63 (m, 1H), 7.23 (m, 2H), 7.01 – 6.88 (m, 3H), 6.58 (s,

1H), 6.42 (s, 0.5H), 6.30 (s, 0.5H), 3.71 (m, 1H), 3.66 – 3.52 (m, 4H), 3.50 – 3.34 (m, 4H), 3.24

(m, 1H), 3.06 (m, 1H), 2.70 (m, 1H), 2.49 (m, 2H), 2.31 (s, 3H), 1.53 (s, 9H), 1.46 (s, 9H);

ESMS m/z = 587 (M + H)+, 609 (M + Na)+.

H N-(2-(4-((6-Amino-4-methylpyridin-2-yl)- N

H methyl)pyrrolidin-3-yloxy)ethyl)-2-(3-fluoro- H2N N N F O phenyl)acetamide (III-6). A solution of III-45 (9 O mg, 0.015 mmol) in 4N HCl in dioxanes (3 mL) was stirred overnight. The solvent was removed

under a stream of nitrogen and the salt was dissolved in water. The solution was washed with

ethyl acetate (2 x 15 mL) and concentrated to dryness to give III-6 as a greasy solid (3 mg,

1 0.0065 mmol, 44%). H NMR (500 MHz, D2O) δ 7.27 (s, 1H), 7.10 (q, J = 7.5 Hz, 1H), 6.97 (d,

J = 7.5, 1H), 6.92 (d, J = 8Hz, 1H), 6.72 (m, 1H), 6.52 (s, 1H), 6.16 (s, 1H), 3.86 (m, 1H), 3.67

202

(m, 1H), 3.59 (m, 1H), 3.52 (m, 1H), 3.50 – 3.39 (m, 2H), 3.29 (m, 3H), 3.08 (dd, J = 3, 13 Hz,

1H), 2.89 (t, J = 11.5 Hz, 1H), 2.50 – 2.37 (m, 3H), 2.13 (s, 3H); ESMS m/z = 387 (M + H)+.

203

Chapter 4

Increasing the Bioavailability of nNOS Inhibitors using a Prodrug Strategy 204

4.1 Introduction

The blood brain barrier (BBB) is a physical and enzymatic layer that protects the brain

parenchyma from blood-borne agents.1, 2 The endothelial cells of cerebral microvasculature have

tight junctions produced by the interaction of several transmembrane proteins that project into

and seal the paracellular pathway. They lack fenestrations or pores and engage in minimal

pinocytotic activity. The BBB also expresses various efflux transporters, including P-

glycoproteins (P-gp), breast cancer resistant protein (BCRP) and multi-drug resistant proteins

(MRPs), that actively pump unwanted molecules back into the blood.3, 4 Small hydrophilic

molecules that are necessary for brain function, such as glucose, electrolytes and amino acids,

are invariably taken up by transporter proteins, as they are unable to diffuse passively across the

BBB. Unfortunately, therapeutics intended to target the CNS are often prevented from accessing

the brain by the highly efficient barrier. In fact, 98 % of compounds intended for treatment of

neurological diseases never reach the market due to an inherent inability to cross the BBB.5-7

For a CNS drug to be effective it must be able to cross the blood brain barrier and reach a

reasonable concentration inside the brain. The ratio of brain concentration to plasma

8, 9 concentration at a steady state can be represented by the equilibrium constant Kp. For

marketed CNS drugs, Kp ranges from 0.1, indicating a low brain concentration relative to the

10 plasma, to 24. For Kp to be this high, the drug must be actively taken up into the brain.

However, Kp does not tell the whole story. The rate at which the drug crosses the BBB can also

be important, especially in the treatment of acute neurological diseases such as seizures. Also,

although a high Kp is favorable for passive diffusion across endothelial cell membranes, it does not necessarily equate to a high concentration of free drug in the cytoplasm of brain cells, as non-

205

specific binding to proteins can occur, reducing the effective concentration of drug in the brain.

A compound with a favorable Kp value may not necessarily demonstrate good efficacy if high

levels of non-specific binding to brain tissue constituents result in a low unbound drug

concentration available for binding to the target.8

The final concentration of free drug in the brain can be increased in several ways. The

best way is to increase active uptake, but this is usually difficult to do.11 The large neutral amino

acid transporter is used by the hydrophilic CNS therapeutics gabapentin, pregabalin and L-dopa,

leading to high concentrations in the brain.12-14 Attaching a compound that is known to be

actively taken up into the brain to a drug molecule can cause the drug to be actively

transported,15 but has no guarantee of success. Some examples of where this has been achieved will be given below. Another method of increasing Kp is to minimize efflux activity by designing

compounds that are not substrates for efflux pumps. To maintain high levels of drug in the brain,

metabolism of the drug by endothelial cells should also be kept to a minimum.

For a compound to diffuse passively across the BBB, the physicochemical requirements

on it are quite strict, more so than for general biomembrane permeability. Guidelines for

estimating whether a compound will be brain penetrant have been devised based upon analysis of

known neurological therapeutics. The potential CNS drug molecule must be small, typically less

than 400 Da. The average molecular weight for a data set of 48 CNS drugs was 313.16 The

number of hydrogen bond donors should be 3 or fewer, and the number of hydrogen bond

acceptors six or fewer.17, 18 The averages for the same data set were 0.85 and 3.56 respectively.

The polar surface area and logD are also important. Their averages were 40.5 Å2 and 2.08 in the

sample set studied. Although there are no listed requirements on charge, the number of positive

206

charges on compounds in the dataset typically ranged between zero and two, and there were

rarely any negative charges. The charge on the molecule affects the logD and therefore can be

included in that requirement. It is also believed that the number of rotatable bonds should be kept

to a minimum.6 While there are exceptions to each of these “rules”, especially when drugs are

transported actively, the more rules that are broken, the lower the likelihood of success. In

general, small drug-like molecules with greater lipophilicity have an increased rate of passive

diffusion across the BBB. However, increased lipophilicity often leads to increased non-specific

binding to plasma proteins and intracellular proteins,9, 19 increased uptake by non-target organs,

and increased metabolism.20 There is also an increased likelihood that the compound will be a

substrate for P-gp,21 further reducing free concentrations in the brain.

Predicting brain uptake prior to synthesis of a new class of drugs would significantly increase the chances of in vivo success. There are numerous in silico modeling programs that take into account H-bond donors and acceptors, polar surface area, charge, logD and size.

However, the datasets typically contain 300 molecules or fewer,22 and although these models

accurately predict the results for molecules similar to those used in the datasets, when

compounds that probe different chemical space are modeled, the predictions are usually less

accurate.

There are several in vitro and in vivo methods of measuring brain uptake, each with their

inherent advantages and disadvantages.23 The method used to obtain data for this chapter was an in vivo approach in which compounds were administered via intraperitoneal injection. The

animals were sacrificed at various time points and levels of the compound in blood and brain

samples were quantified to obtain a crude Kp. The advantage of this method is that the BBB is

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intact and all transporters and enzymes are present and functional. The disadvantages are that

distribution in and metabolism by other organs makes the actual kinetics difficult to determine accurately, and that the high initial plasma concentration do not correspond to an equilibrium state. However, it provides the most realistic evaluation of a compound’s brain uptake ability.

A prodrug is a compound that is metabolized into an active drug in the body. It typically shows lower potency against the target than the active drug but better pharmacokinetic characteristics. Prodrug moieties are often used to increase lipophilicity thereby increasing absorption through the intestinal wall.24 The moiety is then cleaved by esterases or another

common enzyme in plasma or the liver to release the active drug into the bloodstream. However,

few examples exist for which a prodrug moiety has been used to increase brain uptake

properties.25, 26 In principal, the same theory applies: increasing the lipophilicity of a polar compound should increase its ability to cross the BBB, and then once in the brain the prodrug is converted to the active drug. In an ideal situation, the prodrug should have a long half-life in the liver and in plasma, but a short half-life in the brain. In reality, this is difficult to achieve, as there are few enzymes specific to the brain that could be used to activate a prodrug.

There are a few successful examples of using a prodrug strategy to increase brain delivery. One of the oldest examples is the diacetylation of morphine to produce heroin, a molecule with increased ability to cross the BBB.27 Certain anti-HIV drugs have been modified

in numerous ways to increase brain uptake. AZT (Zidovudine) has been synthesized with a

complex monosaccharide prodrug moiety. While AZT itself cannot cross the BBB, when

(mannopyranosidyl)ethyl phospho diester analog IV-128 and hexadecyl-2-(α-D-

mannopyranosidyl)ethyl phospho diester analog IV-229 were administered, the active drug was

208

found at appreciable levels in the brain. In this case, the prodrugs are believed to be taken up actively by monosaccharide transporters.

HN HO O O N OH O O O P O O OH OR OH N3

IV-1,R=H IV-2,R=C16H34

Figure 4.1 Prodrugs of the anti-HIV therapeutic AZT, designed for increased brain uptake.

The anti-HIV drug 2’ F-ara-ddI (IV-4) suffers from poor uptake into the CNS. However,

the deoxygenated form (IV-3) is taken up more efficiently, and is converted to the active drug by

the enzyme xanthine oxidase (Fig. 4.2).30

O N HN N N N xanthine N N oxidase N O F O F HO HO

IV-3 IV-4

X O N HN N adenosine N X=Cl,Br,I N N N deaminase N O O HO HO

IV-5 IV-6

Figure 4.2 The conversion of brain penetrating prodrugs to the active drugs, 2’F-ara-ddI (IV-4)

and ddI (IV-6).

209

The brain levels of halogenated prodrugs (IV-5) of ddI (IV-6) were 20 times higher than that of

the parent drug. The prodrugs are converted to the drug by the enzyme adenosine deaminase.31

There have also been numerous attempts to increase BBB penetration with simple ester

prodrugs that have had little success.25, 32 Brain selective ester hydrolysis cannot be achieved, as the concentrations of esterases in plasma and the liver are much higher than those found in

neuronal cells. However, in certain cases, the approach may yield some benefit. If the parent

drug does not diffuse across the BBB, but a lipophilic ester prodrug version does, any prodrug

that crosses into the brain and is hydrolyzed will result in parent drug that is “locked in.” High

CNS concentrations will then rely on a basic equilibration of distribution, but this approach will

be foiled if the parent drug is a substrate for efflux transporters.

A chemical delivery system designed to target the brain uses a similar “lock-in” idea. It is

based on the in vivo oxidation of a dihydropyridine group to the pyridinium salt (trigonelline)

and has proved successful for a number of applications (Fig. 4.3). The dihydropyridine adds

significant lipophilicity to the parent drug and increases passive diffusion across the BBB. Once

in the brain, the heterocyclic ring is oxidized, resulting in a charged species that does not diffuse

back across the BBB into the blood. Enzymatic cleavage of the trigonelline group occurs to release the active drug over a prolonged period.

O O Enzymatic N N H oxidation H N N+

IV-7 IV-8

Figure 4.3 Dihydropyridine-based chemical delivery system.

210

For example, a dihydropyridine moiety was attached to phenethylamine via an amide

linkage to give IV-7.33 Significant levels of the oxidized form IV-8 were found in the brain

following intravenous dosing. In vitro metabolism studies showed that the oxidation occurred

rapidly in brain tissue (t1/2 ~ 30 min), blood (t1/2 ~ 14 min) and liver tissue (t1/2 ~ 14 min).

Hydrolysis of the amide bond occurred slowly (t1/2 ~ 3h), providing sustained release of

phenethylamine. The system has been used to deliver dopamine, peptides, hydrocortisone and

other steroids to the brain.34-37

There have been previous attempts in the Silverman group to increase a compound’s

brain uptake by masking amines with (acyloxy)alkyl carbamate drugs (Fig. 4.4). The potent and

selective nNOS inhibitor II-3 was found to show little activity in in vivo assays, presumably due to poor brain uptake. Several prodrug versions were made including IV-9.

HN NHNO2 O O HN NHNO2 NH HN O O NH NH2

H H N N H2N H2N

IV-9 II-3

Figure 4.4 Structure of IV-9, a pivaloyloxyethyl carbamate prodrug of the potent and selective

nNOS inhibitor II-3.

(Acyloxy)alkyl carbamate prodrug moieties have been used in multiple applications to protect amines.38, 39 The ester portion is hydrolyzed by esterases, and the unstable hemiacetal

211

decomposes to give an aldehyde and carbon dioxide, releasing the amine. The rate of ester

hydrolysis is affected by the size of the alkyl group. A tert-butyl group was chosen for IV-9 to

reduce the rate of hydrolysis and allow the compound sufficient time to reach the brain. Also, a

tert-butyl group is very lipophilic, and it minimizes the possibility of intramolecular acyl transfer

reactions, which are common when using this type of prodrug moiety to protect primary amines.

(Acyloxy)alkyl carbamate prodrugs have been successful in increasing the bioavailability

of a number of compounds.38 Gabapentin (IV-10) is taken across the BBB by the large neutral

amino acid transporter.14 However, its absorption across the intestinal wall is limited, particularly at higher doses, and varies between patients. An (acyloxy)alkyl carbamate prodrug of gabapentin

(IV-11, Fig. 4.5) was shown to be taken up by multiple intestinal transporters including a sodium-dependent multivitamin transporter.24 The prodrug moiety was cleaved in the blood,

intestinal epithelium and other tissues to release gabapentin, thus improving the oral

bioavailability of the drug.

O O OH H N OH 2 O O N O H O

IV-10 IV-11

Figure 4.5 Gabapentin (IV-10) and an (acyloxy)alkyl carbamate prodrug of gabapentin.

There are no examples of (acyloxy)alkyl carbamate prodrugs having been used to

improve the brain uptake of CNS drugs. The high concentration of esterases in plasma and the

liver will consume much of the prodrug before it reaches the brain.40 Also, the prodrug moiety

212 may not actually increase brain uptake. Although it removes a charge and increases the overall lipophilicity, it also adds numerous hydrogen bond acceptors, increases the number of rotatable bonds, and dramatically increases the molecular weight. It may also make the compound a better substrate for P-gp. The metabolic stability of IV-9 will be investigated in this chapter, both as a comparison to other types of prodrug and to determine the feasibility of using (acyloxy)alkyl carbamate prodrugs for increased brain uptake.

The nNOS inhibitor described in Chapter 3, compound III-2 (Fig. 4.6), has an ether bond in place of a secondary amine that is present in the lead compound, II-6, but has comparable potency and isoform selectivity. Whether the replacement results in an increase in bioavailability was investigated. Regardless, it was believed that the compound still carries too many charges at physiological pH and that masking one of those charges as a neutral moiety would substantially increase the compound’s ability to cross the blood brain barrier (BBB). For that reason, the pharmacokinetics of III-2 were investigated, along with several possible approaches to design a prodrug form of III-2 that could increase the compound’s concentration in the brain.

H H N N

N H N N H2N O 2 HN NH NH

F F

III-2 II-6

Figure 4.6 Structures of potent and selective nNOS inhibitors, III-2 and II-6.

213

4.2 Azides as prodrugs for primary amines

In general, aliphatic primary amines are charged at physiological pH, contribute to the polar surface area of the molecule and add hydrogen bond donors. All of these factors are believed to impede a molecule’s ability to passively diffuse across the BBB,18 and so it is

desirable to minimize the number of primary amines in potential CNS drugs. There is evidence

to suggest that in some cases an azide can undergo in vivo reduction to a primary amine.41-46 The

best example of this is the azide in the anti-HIV drug AZT (IV-12), a metabolite of which is the

amine form of the parent compound (IV-13).

O O

HN HN

O N O N O HO HO O

N3 NH2

IV-12 IV-13

Figure 4.7 Structures of AZT (IV-12) and its metabolite (IV-13).

As a prodrug moiety, azides have numerous beneficial characteristics. They are easily

synthesized, have low molecular weight, are non-toxic and are stable under physiological

conditions. For the purposes of BBB penetration, they are neutral at physiological pH, are not

hydrogen bond donors, and do not add rotatable bonds.

To be successful as an nNOS inhibitor prodrug, an azide containing compound must

fulfill three criteria. First, it must be able to cross the BBB more effectively than the

214 corresponding primary amine. Second, it must be metabolized to the active drug on a physiologically relevant timescale in the brain, or it will be eliminated before it is activated.

Third, the corresponding primary amine must be a potent and selective nNOS inhibitor.

As a means of testing the first two criteria, azide IV-14 (Fig. 4.8) and its corresponding amine (IV-15) were synthesized using chemistry similar to that described in previous chapters

(Scheme 4.1).

H H N N

H2N N N3 H2N N NH2 O O IV-14 IV-15

Figure 4.8 Structures of azide prodrug IV-14, and its primary amine analog IV-15.

Allyl ether III-30 underwent ozonolysis with reduction of the ozonide using sodium borohydride to give allyl alcohol IV-16. The benzyl group was removed under hydrogenation conditions, and the resulting alcohol IV-17 was submitted to Mitsunobu conditions using DPPA as a source of nucleophilic azide. The resulting compound IV-18 was split in two: one half was deprotected to give IV-14, the other half was reduced to the amine (III-46), and then deprotected to give IV-15.

215

Boc Boc N N Boc N i ii BocN N BocN N OH O O BocHN N OH O Ph Ph III-30 IV-16 IV-17

Boc Boc iii N iv N

BocHN N N3 BocHN N NH2 O O IV-18 III-44

v v

IV-14 IV-15

o o i) O3,MeOH,-78 C, 1h, then NaBH4,rt,3h;ii)H2,Pd(OH)2 /C,MeOH,60 C, 2d; iii) PPh3, DIAD, DPPA, THF, rt, 16h; iv) H2, Pd / C, MeOH, rt, 16h; v) 4N HCl, dioxanes, rt, 16h.

Scheme 4.1 Synthetic route to IV-14 and IV-15.

Both compounds were tested for inhibition of the three NOS isoforms using the standard hemoglobin capture assay. As one might predict, the azide was not a very potent nNOS inhibitor

(Table 4.1). IV-15 was not as potent as expected and is not potent enough to be a useful therapeutic. However, it was believed that these two compounds could be used for proof of principle. If the azide fulfilled the first two criteria for being a useful prodrug, further investigation into increasing the potency of primary amine containing analogs could be addressed at a later date.

216

Compound Ki vs nNOS (µM) Ki vs eNOS (µM) Ki vs iNOS (µM)

IV-14 3.8 >300 45

IV-15 0.3 250 17

Table 4.1 Inhibition constants for IV-14 and IV-15.

An assay was developed to determine the extent to which the azide penetrates the BBB.

Conditions were found for the extraction and partial purification of the azide from plasma and brain tissue. Biological samples cannot be loaded directly onto an HPLC without the removal of

macromolecules and lipids. The most convenient way to achieve this is through solid phase

extraction (SPE). The biological sample is loaded onto a cartridge containing a matrix (e.g. C18)

to which the compound binds. The column can be washed to remove polar impurities, and then

the compound of interest is eluted with organic solvent. Some form of SPE was used in the

pretreatment of all of the biological samples analyzed in this chapter.

The azide was quantified by HPLC with a UV detector, but the limits of quantification

were about 5 µM. A standard curve was established for each matrix. The azide was administered to mice via intraperitoneal injection at a dose of ~50 mg / kg (200 µL of 20 mM stock solution for a 20g mouse). This high dose was used to ensure that sufficient azide would be found in the brain to allow quantification. The animal exhibited signs of acute toxicity, such as labored breathing, impaired movement and visible discomfort. The animal was euthanized and the peritoneal cavity was examined. There were signs of vascular leak and damage to the internal

organs, particularly the liver. A second animal was given a dose of ~25 mg / kg (200 µL of 10

217 mM stock) and also showed similar signs of toxicity. Due to these obvious toxicity signs, the experiment was aborted.

The rate of metabolic conversion of the azide to the amine was also investigated. As a control to check for enzyme activity, and as a comparison to an established prodrug approach, the metabolism of IV-9 was also analyzed.

IV-9 and IV-14 (Fig. 4.9) were incubated in fresh mouse plasma at 37 °C in the presence of NADPH regenerating system, as azide reduction is believed to be NADPH dependent. At certain time intervals the reactions were quenched, and the amount of prodrug remaining was quantified by HPLC. Fig. 4.10 shows the normalized peak integrals for each compound.

HN NHNO 2 O O NH H HN O O N

H H2N N N3 N O H2N IV-14

IV-9

Figure 4.9 Structures of prodrugs IV-9 and IV-14.

218

Plasma Metabolism of IV-9 and IV-14

1.2

0.8 IV-9 A IV-9 B 0.6 IV-14

0.4

0.2 Normalized amount of compound

0 0 102030405060708090 Incubation time (min)

Figure 4.10 Incubation of IV-9 and IV-14 in fresh mouse plasma. The area under the peak was

integrated and the value was normalized based on the integration at 0 min. Two diastereoisomers

of IV-9 are shown separately (IV-9 A and IV-9 B).

As expected, no significant metabolism of azide IV-14 was observed over 80 minutes, as

the concentration of P450s in plasma is extremely low. In contrast, the conversion of IV-9 was

rapid due to the abundance of esterases in plasma. Due to the presence of a chiral center in the

prodrug moiety, IV-9 is a mixture of two diastereomers that are separable by HPLC. One diastereomer was metabolized with a half life of approximately 10 minutes, and was almost completely consumed by 80 minutes. The other was hydrolyzed more slowly with a half life of approximately 30 minutes, and could still be quantified at the 80 minute time point. The peak in

219 the HPLC corresponding to the parent drug, II-3, was also analyzed (Figure 4.11). Although the curve was not as smooth as was seen for the degradation of the parent compound, a clear increase in peak intensity over time was observed, indicating that the prodrug was indeed being converted to the active drug.

Formation of II-3 during incubation of IV-9 in plasma

4 II-3

3 Integral of II-3 peak of Integral 2

0 0 102030405060708090 Incubation Time (min)

Figure 4.11 Formation of active drug II-3 from prodrug IV-9 during incubation in fresh mouse plasma.

Next, the two compounds were incubated in brain homogenate to investigate the extent of metabolism in brain tissue. The compounds were incubated with fresh mouse brain homogenate in phosphate buffered saline (pH 7.4) at 37 °C with NADPH added in excess. At various time

220 points the reactions were stopped by heating to 95 °C for 1 minute. The mixtures were partially purified by solid phase extraction (SPE), and the amount of compound was quantified by HPLC

(Fig. 4.12). The amount of each diastereomer of IV-9 decreased over the course of the experiment. As in the case of the plasma metabolism, one of the diastereomers was metabolized slightly faster than the other. However, the rate of metabolism was not nearly as rapid as it was in plasma owing to the decreased abundance of esterases. The concentration of IV-14 did not decrease over the course of the experiment, indicating that no significant metabolism occurred.

Metabolism of IV-9 and IV-14 in Brain Homogenate

1.2

0.8 IV-14 IV-9 A 0.6 IV-9 B

0.4 Relative concentration

0.2

0 0 5 10 15 20 25 30 35 40 45 Incubation time (min)

Figure 4.12 Metabolism of IV-9 and IV-14 in brain homogenate.

The brain homogenate system was complex and prone to error, so a simpler system was used to check for azide reduction. Microsomes, a concentrated mixture of liver enzymes rich in

221

cytochrome P450s, were added to the compounds at several low concentrations, and the mixtures were incubated in the presence of excess NADPH. At certain time points, the reactions were stopped by the addition of acetonitrile, and the samples were processed and analyzed by HPLC.

Typically, microsomes tend to metabolize compounds very rapidly under these conditions, and it was expected that the azide would be degraded over time. It would then be necessary to prove whether the degradation was due to reduction of the azide to the amine, or due to some other type of metabolism. The experiment was performed with a control to determine whether the microsomes contained active P450s. Minaprine, a prescription drug that is known to be rapidly metabolized under similar assay conditions (t½ = 4 min in rodent liver microsome assays), was

tested along with the compounds.

As expected, the IV-9 diastereomers were metabolized over time at different rates (Fig.

4.13). A peak corresponding to the active drug did grow in, but was difficult to quantify as it was

small and overlapped with other peaks. The concentration of esterases in the mixture is not very

high, and the microsomes become denatured fairly rapidly at 37 °C, and so IV-9 was not fully

degraded, and the concentration of prodrug remaining leveled off. The minaprine was rapidly

degraded, confirming the P450 activity of the microsome mixture. However, no statistically

significant loss of the azide was seen. This was surprising, as it was expected that even if the

azide was not converted to the amine it would be metabolized in other ways.

222

Microsome metabolism of IV-9 and IV-14

1.2

0.8 IV-9 A IV-9 B 0.6 Minaprine IV-14 0.4

0.2 Normalized amount of compound

0 0 1020304050 Incubation time (min)

Figure 4.13 Metabolism of IV-9, IV-14 and the prescription CNS drug minaprine by liver microsomes at 37 °C.

In vitro azide reduction by P450s is reported to be inhibited by oxygen.44 The incubation was repeated with various increasingly stringent anaerobic conditions, ultimately using vials with rubber septa under a balloon of nitrogen with the reaction run in deoxygenated buffer. However, although the minaprine was metabolized as expected, no statistically significant loss in azide was seen. Mass spectral analysis of the reaction mixtures showed no peak corresponding to the amine.

Closer analysis of the literature shows that when azide reduction does occur, it is slow.

For example, the prodrug FAAddP (IV-19) is reported to be converted to the active drug, 2’-F- araddA (IV-20) by cytochrome P450s (Fig 4.14).43 However, the half-life of the prodrug in brain

223 homogenate was 6 h, and only 5% of the active drug was seen after 6 h of incubation. Clearly, alternative mechanisms of degradation occurred faster than azide reduction, and the conclusion of the authors was that azide reduction was too slow for FAAddP to be effective for brain delivery.

NH N3 2 N N

N N N N F F O HO O HO

IV-19 IV-20

Figure 4.14 Structures of 2’-F-araddA (IV-20) and a potential azide prodrug (IV-19).

The Km for AZT as a substrate of P450s is ~3 mM, suggesting that AZT is a poor substrate for the enzyme responsible for azide reduction.44 Although reduction may occur to produce detectable levels of metabolite IV-13 after administration of AZT, the actual rate of amine formation is quite low. In addition, if the reduction of azides is sensitive to oxygen, the rate would be slow in brain tissue as the brain is well supplied with oxygenated blood. In our hands, there was no convincing evidence to suggest that azide reduction occurred at a rate that would be necessary for prodrug purposes. Given the problems with the in vivo experiments, the lack of convincing evidence of azide reduction in vitro, and the fact that the corresponding primary amine is only a weak nNOS inhibitor, the azide prodrug approach was set aside to investigate other ideas.

224

4.3 Microsomal stability of ether III-2

Although the use of azides as prodrugs had not looked promising, it was still possible that another type of prodrug could be used to increase the brain uptake of the most active nNOS inhibitors. It was envisioned that one of the secondary amines of ether III-2 (Fig. 4.15) could be capped with a carbamate prodrug moiety, similar to that used in IV-9, or with another kind of prodrug moiety that would neutralize the amine. Before this was attempted, more information about III-2 was needed. For example, how metabolically stable is the compound? If III-2 is metabolized relatively quickly, it will make the study of the conversion of prodrug to drug more complicated. Also, how much III-2 penetrates the BBB? There needed to be a baseline for comparison if we are to determine whether a prodrug moiety offers any advantage.

H N

N H2N O NH

III-2

Figure 4.15 Structure of potent and selective nNOS inhibitor, III-2.

To address the first point, III-2 was subjected to the microsome assay described above.

The assay was performed both in the presence and absence of NADPH, to determine whether any metabolism observed was NADPH dependent. The results are shown in Fig. 4.16.

225

Metabolism of III-2 by microsomes

1.2

0.8

III-2 ox 0.6 III-2 red min

0.4

0.2 Normalized amount of compound

0 0 5 10 15 20 25 30 35 40 45 Incubation time (min)

Figure 4.16 Metabolic stability of III-2 with microsomes at 37 °C. The experiment was performed in the presence (III-2 red) and absence (III-2 ox) of additional NADPH. Minaprine concentration at 20 min was >0 but was not quantifiable.

Clearly, there is some degree of metabolism under both sets of conditions, suggesting that the metabolism is not NADPH dependent. However, the amount of compound lost is relatively small: approximately 20% is lost over 30 minutes. Given that the microsomes are concentrated enzymes, this equates to reasonable metabolic stability. The background metabolism of III-2 should not interfere with possible prodrug conversion studies.

226

4.4 Brain uptake study on III-2

As with azide IV-14, it was believed that a high concentration of III-2 would need to be administered to a mouse to see sufficient compound in the brain for reliable quantification by

HPLC. III-2 was administered at a dose of 24 mg / kg producing mild toxic effects. Although the effect was not nearly as severe as that seen when IV-14 was administered, the dose would need to be lowered in a full brain uptake study. The blood and brains were harvested from the animals used in this test, and SPE was performed followed by HPLC analysis. The peaks corresponding to III-2 were small and overlapped with other peaks in the spectrum, making them difficult to quantify. It became clear that since the dose would need to be lowered, and the levels of III-2 were already difficult to quantify, a more sensitive method of detection was needed.

LCMS/MS has a sensitivity for these compounds approximately 1000 times that of HPLC with UV detection. The spectra are also very clean, as the mass spectrometer first separates ions based on the molecular weight of the parent compound and then measures the quantity of daughter ions. The likelihood of two unrelated compounds having identical mass of both parent and daughter ions is very low, resulting in the spectrum showing only one peak corresponding to the compound of interest, even when the compound was extracted from a complex matrix such as brain tissue. Because of this, quantification is very reliable and consistent.

The LCMS/MS measures the amount of compound in a given sample. However, as the extraction and SPE processes are prone to error, quantification is most reliable when an internal standard is used. The internal standard should have physical properties similar to those of the compound of interest, such that both compounds will be lost or retained during the extraction process to similar degrees. Compound III-4 was found to be the ideal internal standard, as it is a

227 structural isomer of III-2, yet elutes differently by LC. A known amount of the internal standard was added prior to all extractions, and the final quantification was performed as a ratio of the compound of interest to the internal standard. This ratio should be maintained throughout the extraction process, such that mechanical loss does not affect the results.

Quantification of real samples required a standard curve for comparison. To plasma samples were added a fixed quantity of III-4 and varying known amounts of III-2. Five concentrations were used with samples prepared in triplicate. The samples were diluted and subjected to SPE, and the ratio of the two compounds was measured by LCMS/MS. An example of this curve is shown in Fig. 4.17. This served as the standard curve for future plasma samples.

Biological samples that were believed to contain III-2 were first given the same fixed amount of

III-4, and then processed in the same way as the standards. The ratio of III-2 to III-4 was measured by LCMS/MS, and this ratio was compared to the standard curve. From this, the original concentration of III-2 in the samples could be calculated.

228

Standard Curve for III-2 in plasma

1.2

R2 = 0.9999 0.8

0.6

0.4 Ratio of III-2 to III-4 to III-2 of Ratio

0.2

0 0 0.2 0.4 0.6 0.8 1 1.2 [III-2] (ug / mL)

Figure 4.17 Standard curve for the quantification of III-2 in plasma.

A similar standard curve was needed for quantification of III-2 in brain tissue. Initially, mice brains were used to make the standards. However, these were scarce, so rat pup brains and pieces of pig brain cut to the same mass as mice brains were also used. There was no difference found between the resulting ratios regardless of the type of brain used. The brains were homogenized in acidic extraction solution containing a fixed amount of III-4 and varying known amounts of III-2. The samples were centrifuged and the supernatant was subjected to SPE. The ratios of III-2 to III-4 in the samples were measured by LCMS/MS, and a brain standard curve was created (Figure 4.18).

229

Standard curve for III-2 in brain

0.8

0.7

R2 = 0.9998 0.6

0.5

0.4

0.3 Ratio of III-2 to III-4 to III-2 of Ratio 0.2

0.1

0 0 0.1 0.2 0.3 0.4 0.5 0.6 [III-2] (ug / mL)

Figure 4.18 Standard curve for the quantification of III-2 in brain tissue.

A brain uptake study of III-2 was performed using mice. Six mice were given III-2 by intraperitoneal injection at 3.7 mg / kg, a concentration at which no toxicity was observed. Three mice were sacrificed 5 minutes after administration; the other three were sacrificed 10 minutes after administration. Blood and brain samples were taken from each mouse, and the extraction protocol described above was carried out. At a later date, three more mice were given III-2 and sacrificed 20 minutes after administration. The data are shown in Fig. 4.19 and 4.20. The amount of III-2 was calculated in µg / mL of plasma, and µg / g of brain. This value was converted to

µM for display to allow a straightforward comparison between compounds in this chapter. The conversion for calculating the concentration in the brain assumed that the brain density is 1.0 g /

230 mL. In actual fact the density of brain may be greater than this, which would mean that the real concentrations may be higher than shown.

Plasma concentration of III-2

15 [III-2] (uM)

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.19 Concentration of III-2 in plasma after an i.p. dose of 3.7 mg / kg. Error bars show standard error mean.

231

Brain concentration of III-2

0.09

0.08

0.07

0.06

0.05

0.04 [III-2] (uM) 0.03

0.02

0.01

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.20 Concentration of III-2 in the brain after an i.p. dose of 3.7 mg / kg. Error bars show standard error mean.

Although more data would be needed to fully understand the kinetic profile of this compound, the data are consistent with what would be expected and provide solid numbers for future comparisons. In all of the brain uptake experiments shown in this chapter, the 5 min time point shows the most variance. At this point the plasma levels are changing at a great rate and have not yet stabilized. It is very difficult to sacrifice the animal at exactly 5 min, and even a variation as little as 30 seconds either way could produce a wide disparity in results. Also, the animals may process the compounds differently depending on heart rate etc. In most cases, the levels are consistent at 10 and 20 min and the data are reproducible with small error bars. It

232

should also be noted that the experiments are only continued to the 20 min time point, and so all interpretations are only valid up until this time. For a full understanding of the pharmacokinetics, including rate of elimination, longer time points are needed in each case. However, the data is sufficiently reliable to allow comparisons between compounds and for conclusions about brain

uptake to be drawn.

The amount of III-2 in the blood is very high at 5 min, but drops and then remains

constant from 10 to 20 minutes at about 1.6 µM. This indicates that III-2 shows good

bioavailability; it is absorbed rapidly into the blood from the peritoneal cavity, and then appears

to be distributed throughout tissues. The brain levels rapidly reach their maximum level and are

maintained throughout the course of the 20 min experiment. This indicates that III-2 crosses the

BBB to some extent and is not actively pumped out of the brain by efflux pumps. The ratio of

brain concentration to blood concentration is approximately 5% (Kp ~0.05), which is low

compared to other CNS penetrating drugs. However, it should be noted that the brain

concentration is greater than 60 nM for much of the experiment and does not appear to be

dropping at any great rate. This value is four times the Ki for this compound. Given that the Km

for arginine is 1.3 µM for nNOS, even concentrations of inhibitor as low as the Ki would be

expected to have an effect. A relatively low dose was administered to the mice, yet there is

clearly sufficient compound in the brain to potentially cause a therapeutic response. In addition,

the plasma levels peak at about 25 µM and quickly drop to about 1.6 µM. This is well below the

Ki for III-2 against eNOS (31 µM), and so there should be no inhibition of eNOS, and therefore

no hypertensive effect, if the compound is administered at this dose.

233

At this stage it was of interest to determine the extent to which II-6 crosses the BBB, to determine whether any improvement was made in the modification described in Chapter 3 to give III-2. II-6 was synthesized using the route shown in Scheme 4.2.

Boc O N Boc N F i + H BocHN N NH2 III-35 II-49

Boc H N N ii Boc H BocHN N N F H2N N N F N N H H IV-21 II-6

i) CH2Cl2,15min,thenNaHB(OAc)3, rt, 1h; ii) 4N HCl, dioxanes, rt, 16h.

Scheme 4.2 Synthesis of II-6.

Plasma and brain standard curves were obtained for II-6 using similar conditions to those used for III-2, and using III-4 as the internal standard. II-6 was administered to mice at 3.7 mg / kg, the dose that had been used for III-2. Animals were sacrificed at 5, 10 and 20 minutes post dose, and the blood and brains were harvested. The samples were processed and analyzed by

LCMS. The data are shown in Fig 4.21 and 4.22.

234

Plasma Concentration of II-6

2.5

1.5 [II-6] (uM) [II-6] 1

0.5

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.21 Concentration of II-6 in plasma after a dose of 3.7 mg / kg in mice. Error bars show standard error mean.

The plasma level at 5 minutes post dose is not as high as it is in the case of III-2, and the level is dropping much more slowly. This suggests that II-6 is not as bioavailable as III-2; it does not pass as quickly into the blood, and once in the blood it does not pass as quickly into tissues. The fact that the plasma concentration does not level out means that the system never really reaches equilibrium, so any calculated Kp values will be approximations.

235

Brain concentration of II-6

0.07

0.06

0.05

0.04

0.03 [II-6] (uM) [II-6]

0.02

0.01

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.22 Concentration of II-6 in the brain after a dose of 3.7 mg / kg in mice. Error bars

show standard error mean.

The level of II-6 in the brain also appears to be fairly constant, just as was seen with III-

2. The levels are around 0.015 µg / g (35 nM). As is the case with III-2, the brain concentration

peaks rapidly but maintains a constant level throughout the experiment, suggesting that II-6

passes across the BBB and is not a good substrate for efflux pumps. The ratio of brain to blood

concentrations equates to a Kp of about 0.02 at 5 to 10 minutes, rising to 0.08 at 20 minutes.

However, as the system is not in equilibrium, it is difficult to interpret what these values mean.

Comparing III-2 and II-6, it appears that the two have a very similar brain uptake profile

over the first 20 min. The average brain concentration for III-2 is higher than that of II-6, at

236 about 0.025 µg / g (60 nM) versus 0.015 µg / g (35 nM) (Fig. 4.23). The 5 min values overlap, but this is because there is a wide degree of variation at this time point. However, by 10 min there is a clear difference in the brain levels of the two compounds.

Brain concentrations of III-2 and II-6

0.09

0.08

0.07

0.06

0.05 II-6 III-2 0.04

0.03 Concentration (uM) 0.02

0.01

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.23 Comparison of the averaged brain concentrations of III-2 and II-6 when both compounds were administered at 3.7 mg / kg. Error bars show standard error mean.

The results indicate that substituting the secondary amine of II-6 with an ether to give

III-2 does result in a small increase in the compound’s ability to cross the blood brain barrier.

Pharmacologists often refer to the area under the curve (AUC) when viewing pharmacokinetic graphs. As the AUC is a function of both the maximum concentration reached and the time that this level is maintained, it provides a value as to how much of the dose is delivered to the site of

237 action. The AUC for III-2 in the brain is significantly greater than that of II-6 over the course of the 20 min experiment, indicating an improvement in bioavailability. This was anticipated, as one hydrogen bond donor has been removed and the overall logD of the molecule is slightly higher. However, the increase is modest, and III-2 is still poor at crossing the BBB relative to marketed CNS penetrating drugs. Additional modifications were made to try to further improve its pharmacokinetic properties.

4.5 The effect of removing a charge on brain uptake

The purpose of a carbamate prodrug moiety, such as that used in IV-9, is to first convert a positively charged amine to a neutral group, and second to add increased hydrophobicity to the molecule. We wanted to determine whether these two factors resulted in a significant increase in

BBB penetration. The examples of carbamate prodrug moieties shown in Table 4.2 are cleaved by esterases found in the plasma and liver. Also shown are amide prodrug moieties that are cleaved via a different mechanism. The table shows the structure of the prodrug moiety and its molecular weight, and summarizes the pros and cons of each moiety.

238

Mol. Entry Structure Advantages Disadvantages Weight

O O Chiral center, toxicity I 174 Highly lipophilic of pivalic acid O O unknown

O O Low mw, ease of Formaldehyde toxic? II 148 O O O synthesis, achiral Unknown stability

O O By-products non- Unknown in literature, III 162 O O O toxic chiral center

By-products non- O toxic, achiral, well Unstable at high pH, IV O O 158 O known, half-life 11- storage concerns. O 26 min (lit.)

O “Lock-in”design, V 123 Amide bond stability, low mw, achiral N

O Unknown toxicity of VI 220 Achiral, lipophilic by-product, high mw, unknown stability O Table 4.2 A comparison of various carbamate and amide based prodrug moieties.

239

Entries I through IV are all variations of (acyloxy)alkyl carbamates. Entry I is the prodrug moiety used in IV-9. Its disadvantages include the unknown toxicity of pivalic acid, one of the by-products of activation. This problem is solved by using carbonate based moieties such as that found in entry II, which, when hydrolyzed, produce a molecule of ethanol as a by-

product.47 Unfortunately, when this moiety is hydrolyzed, a molecule of formaldehyde is also

produced, the toxicity of which is controversial.48 Entry III is a combination of I and II that

produces no toxic byproducts, but is unknown in the literature. Both I and III have a chiral center

in the pro-moiety, and this can cause differences in the rate of hydrolysis, as seen with IV-9 (Fig.

4.10). Moiety I is hydrolyzed relatively quickly in plasma (t1/2 = 10 min), but II is hydrolyzed

even faster (t1/2 ~ 2-4 min). Much of the prodrug would be metabolized before it had a chance to

reach the brain and cross the BBB. All the moieties I-III add significant molecular weight,

hydrogen bond acceptors and rotatable bonds to the molecule, which may limit their usefulness

for increasing brain uptake. Moiety IV may be a better choice in this class, as it is small, rigid

and achiral, and does not produce toxic byproducts.48-50

The dihydropyridine “lock-in” moiety, entry V, has been discussed earlier in this chapter.

One major disadvantage of this moiety is that the dihydropyridine would need to be connected to

III-2 via an amide linkage forming a tertiary amide, which may be resistant to enzymatic

hydrolysis.

The quinone group of entry VI is reduced by P450s and the resulting hydroxyl group

cleaves the amide bond. The methyl groups are necessary for forcing a conformation that

promotes intramolecular attack. The advantages of this system are that the moiety is rigid and

lipophilic, which may help with brain uptake. It is achiral so there is no issue with diastereomers

240 that are metabolized at different rates. It will be stable in plasma, and will only be activated in liver and brain tissue. However, the group is extremely large, and the toxicity of the metabolite is unknown, as there are no reports of it having been used in animals.

These prodrug moieties will be considered, and one or more may be chosen for attachment to III-2 to increase its concentration in the brain. First, the effect on brain uptake of masking one of the secondary amines as a neutral moiety was investigated. At this stage, one of the prodrug moieties shown in Table 4.2 could have been used. However, as was shown in Fig

4.9, esterases in the plasma can rapidly cleave even hindered esters to release the drug. This means that once an (acyloxy)alkyl carbamate prodrug is administered, it begins to be converted to the drug form. Measuring the levels of prodrug in the brain then becomes complex, as some prodrug is metabolized in plasma, some is metabolized in the brain, and some of the drug formed in the plasma also crosses into the brain. To simplify the experiment, a group that neutralizes the secondary amine, but which does not add significant hydrophobicity and will not be rapidly metabolized was chosen. An acetyl group was used to cap the secondary amine in the chain of

III-2 to give amide IV-22.

H N O

H2N N N F O

IV-22

Figure 4.24 Structure of IV-22, an acetylated version of III-2.

241

The synthesis of IV-22 was carried out using late-stage intermediates from Chapter 3

(Scheme 4.3). Aldehyde III-31 underwent a reductive amination reaction with 3-

fluorophenethylamine to give IV-23. The secondary amine was acetylated with acetic anhydride

to give IV-24. The benzyl group was removed under hydrogenation conditions, and the Boc groups were removed with acid to give IV-22.

Boc N i ii III-31 H BocN N N F O Bn

IV-23

Boc N O iv IV-22 BocN N N F O R

IV-24, R=Bn iii IV-25, R=H

i) 3-fluorophenethylamine, CH2Cl2,10min,thenNaHB(OAc)3,1h;ii)Ac2O, MeOH, rt, 1h; o iii) H2,Pd(OH)2 /C,MeOH,60 C,1-2d;iv)4NHCl,dioxanes,rt16h

Scheme 4.3 Synthesis of IV-22.

Amide IV-22 was tested in the standard microsome assay and was found to be quite stable (Fig. 4.37, Experimental Procedures section). Conditions for extraction from blood and brain were developed, and standard curves were established in the same way as had been done for III-2 and II-6, again using III-4 as the internal standard. A brain uptake study of IV-22 was

carried out in mice. The data are shown in Figures 4.21 and 4.22.

242

The blood profile of IV-22 does not match that of III-2 in that the plasma concentration appears to increase continually up to at least 20 minutes. This could mean that IV-22 is taking

longer to diffuse across the lining of the peritoneal cavity into the blood stream.

Plasma Concentration of IV-22

4 [IV-22] (uM) [IV-22]

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.25 Concentration of IV-22 in plasma after a dose of 4.1 mg / kg. Error bars show

standard error mean.

The brain concentration of IV-22 (Fig. 4.26) peaks at 5 min and then drops, suggesting that the compound is a substrate for efflux pumps that are actively removing the compound from the brain. Analysis of the brain samples showed no presence of III-2, indicating that the acetyl

group was not cleaved during the 20 min experiment.

243

Brain Concentration of IV-22

0.14

0.12

0.1

0.08

0.06 [IV-22] (uM) [IV-22]

0.04

0.02

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.26 Concentration of IV-22 in brain after a dose of 4.1 mg / kg. Error bars show

standard error mean.

Comparing the brain curves of III-2 and IV-22 (Fig. 4.27) gives a surprising result. The

concentration of IV-22 at 5 minutes is slightly higher than that of III-2, but drops to a level that is below that of III-2 by the 10 min time point, and so the AUC for each compound over the 20

min experiment is roughly the same. As the two compounds were administered at the same dose,

this indicates that the acetylation of III-2 to give IV-22 results in no positive increase in brain uptake. It was predicted that the elimination of one positive charge would have a significant positive effect on brain uptake, but this is not the case with IV-22.

244

Brain Concentrations of III-2 and IV-22

0.14

0.12

0.1 III-2 IV-22 0.08

0.06

Concentration (uM) 0.04

0.02

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.27 Comparison of the brain concentrations of III-2 and IV-22 after administration of

3.7 mg / kg and 4.1 mg / kg respectively. Error bars show standard error mean.

Several of the prodrug moieties in Table 4.2 are attached to the drug molecule by a

carbamate linkage, which is significantly less polarized than an amide bond. It is possible that

capping the amine with a carbamate has more of an impact on brain uptake as carbamates are

less polarized and so the passive diffusion rate may be much greater. To determine whether this

is the case, methyl carbamate IV-26 was synthesized (Fig 4.28).

H H N N O OMe O OBn

H2N N N F H2N N N F O O

IV-26 IV-27

Figure 4.28 Structures of carbamates IV-26 and IV-27.

245

Also, prodrug moieties can add hydrophobicity to the molecule. To determine whether adding

non-polar groups to the molecule increases brain uptake, benzyl carbamate IV-27 was synthesized. Intermediate IV-23 was used to create both of the carbamates (Scheme 4.4). The

benzyl group was removed under hydrogenation conditions to give IV-28. Also, due to the high temperatures, some IV-29 was produced as a result of loss of the Boc group. The IV-28 that was

formed was mixed with methylchloroformate to give IV-30. The Boc groups were removed to give IV-26. The IV-29 portion was combined with benzylchloroformate to give IV-31. The Boc groups were removed to give IV-27.

Boc N i IV-23 H HN N N F R O

IV-28, R=Boc IV-29, R=H

Boc N ii O OMe iii IV-28 IV-26 BocHN N N F O

IV-30

Boc N ivO OBn iii IV-29 IV-27 H2N N N F O

IV-31

o i) H2,Pd(OH)2 /C,MeOH,60 C, 2 d; ii) ClCOOMe, MeOH, rt, 4h; iii) 4N HCl, dioxanes, rt 16h; iv) ClCOOBn, MeOH, rt 4h.

Scheme 4.4 Synthesis of IV-26 and IV-27.

246

Methyl carbamate IV-26 was subjected to the standard microsome assay and was found

to be quite stable (Fig. 4.37). Standard curves in plasma and brain were obtained, again using

III-4 as the internal standard. Six mice were given IV-26 at 4.3 mg / kg. Two mice were sacrificed at 5, 10 and 20 minutes, and their blood and brains were harvested. The plasma levels were calculated by LCMS/MS and are shown in Fig. 4.29.

Plasma Concentration of IV-26

1.4

1.2

0.8

0.6 [IV-26] (uM)[IV-26]

0.4

0.2

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.29 Plasma concentration of IV-26 after a dose of 4.3 mg / kg. Error bars show standard error mean.

The plasma levels of IV-26 have peaked and are declining slowly over the 20 minutes.

The scatter in the data is extremely small suggesting that the results are very reliable.

247

Brain Concentration of IV-26

0.04

0.035

0.03

0.025

0.02

[IV-26] (uM) [IV-26] 0.015

0.01

0.005

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.30 Concentration of IV-26 in the brain after a dose of 4.3 mg / kg. Error bars show standard error mean.

The brain level of IV-26 is very low at 5 – 10 minutes. However, the level is continuing to rise at 20 minutes, and may peak at a higher level. This data seems to suggest that the rate at which IV-26 crosses the BBB is slow. Although the brain levels have not peaked, it is unlikely that they will reach a level that is significantly higher than that of III-2 (Fig. 4.31).

248

Brain Concentrations of III-2 and IV-26

0.09

0.08

0.07

0.06

0.05 III-2 IV-26 0.04

0.03 Concentration (uM) 0.02

0.01

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.31 Comparison of the brain concentrations of III-2 and IV-26 after a dose of 3.7 and

4.3 mg / kg, respectively. Error bars show the standard error mean.

The AUC up to 20 min for III-2 is clearly much greater than that of IV-26. This implies

that for overall brain uptake the methyl carbamate modification is even more detrimental than

acetylation, and certainly is not beneficial.

Conditions were found for the extraction of IV-27 from brain, and standard curves in

plasma and brain were created. In this case, IV-26 was used as the internal standard as the two compounds were more similar than IV-27 and III-4, and the chromatography was therefore more reliable. IV-27 was administered to mice at a dose of 5.1 mg / kg. Two mice were sacrificed at

249

each time point, and the plasma and brain samples were analyzed by LCMS. The data are shown

in Fig. 4.32 and 4.33.

Plasma Concentration of IV-27

1.2

0.8

0.6 [IV-27] (uM)[IV-27] 0.4

0.2

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.32 Plasma concentration of IV-27 after a dose of 5.1 mg / kg. Error bars show standard error mean.

The plasma concentration curve for IV-27 is similar to that of IV-26, peaking at around 1

µM and declining slowly over the course of the experiment.

250

Brain Concentration of IV-27

0.018

0.016

0.014

0.012

0.01

0.008 [IV-27] (uM) [IV-27] 0.006

0.004

0.002

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.33 Brain concentration of IV-27 after a dose of 5.1 mg / kg. Error bars show standard

error mean.

The curve for the brain concentration of IV-27 also resembles that of IV-26. The levels at

5 min are extremely low, but are steadily increasing throughout the course of the experiment.

However, even by the 20 min time point, the concentration of IV-27 is still far below that of III-

2 (Fig 4.34), and the AUC over the 20 min experiment is much lower.

251

Brain concentrations of III-2 and IV-27

0.09

0.08

0.07

0.06

0.05 IV-27 III-2 0.04

0.03 Concentration (uM) 0.02

0.01

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.34 Comparison of the brain concentrations of III-2 and IV-27 after a dose of 3.7 and

5.1 mg / kg, respectively. Error bars show the standard error mean.

In the case of both carbamates, the modification is detrimental to brain uptake over the

first 20 min. The brain levels of each compound are increasing, whereas those of III-2 are

beginning to decrease, and so over the course of a longer uptake study the concentration of

carbamate may eventually exceed that of III-2. However, this is not encouraging for the use of

(acyloxy)alkyl carbamate prodrugs, as these would be hydrolyzed long before they could offer

any advantage for brain uptake. A prodrug moiety may help with oral bioavailability as in the

case of gabapentin, but the results seen with the two carbamates indicate that an (acyloxy)alkyl

carbamate prodrug moiety is unlikely to provide any positive effect on brain uptake.

A comparison of the in vivo concentrations of all of the compounds tested in this chapter allows some conclusions to be drawn. Fig. 4.35 shows the plasma levels of all of the compounds

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used in this study. For clarity, the error bars are not shown. Also, the 5 min point for III-2 was

excluded as it is so much higher than the others.

Comparison of plasma levels

6 III-2 IV-22 5 IV-26 4 IV-27 II-6 3 Concentration (uM) 2

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.35 Comparison of the plasma levels after i.p. injection.

The two carbamates behave very similarly, in that their levels are fairly constant over the

20 min but are lower than those of III-2. This may be caused by a low rate of diffusion from the

peritoneal cavity into the blood, but the more likely scenario is that these compounds bind to

more proteins and cellular components than III-2 such that their free plasma concentrations are

lower. The levels of II-6 are declining indicating that it is being eliminated from the body.

Although a longer experiment would be needed to establish true clearance rates and half lives, this is not encouraging. The compound on this chart that is displaying an obviously different profile is IV-22, the acetylated compound. In the case of this compound, the plasma levels are

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higher than the other compounds and are actually increasing with time. It could be that although this compound is diffusing slowly into the blood, it is ultimately more bioavailable than the other compounds.

A comparison of the brain levels of each compound is shown in Fig. 4.36. It is apparent that over the course of the 20 min experiment there was not a great deal of difference between the overall brain concentrations of the compounds tested. III-2 and IV-22 have a similar AUC,

indicating similar overall brain penetration. The AUC for II-6 is slightly less, but not dramatically so. Surprisingly, the brain concentrations of the two carbamates are less than the other compounds. Even though their plasma levels were lower, it was expected that the brain levels would be higher, as the compounds are more lipophilic and so should diffuse across the

BBB more readily. Some hypotheses on why this turns out not to be the case will be discussed in the next section.

Comparison of Brain Levels

0.1

0.09

0.08

0.07 III-2 0.06 IV-22 IV-26 0.05 IV-27 0.04 II-6 0.03 Concentration (uM) Concentration

0.02

0.01

0 0 5 10 15 20 25 Time after administration (min)

Figure 4.36 Comparison of brain concentrations after i.p. injection.

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4.6 Summary, conclusions and future directions

An investigation into whether an azide can be used as a prodrug for a primary amine was carried out. Although there is literature evidence of the metabolism of azides into primary amines, in our hands the data proved unconvincing, and the approach was ultimately abandoned.

Several studies of the brain uptake properties of nNOS inhibitors and their analogs were carried out and gave some interesting results. The data suggested that the modification of potent inhibitor II-6 to replace an amine with an ether linkage to give III-2 did indeed have a positive impact on brain uptake. This is likely the result of an increase in logP, a decrease in total polar surface area and the removal of one hydrogen bond donor, all factors that must be considered for

CNS drugs. However, the simple removal of a charge by acetylating a secondary amine had no positive impact on BBB penetration. This may have been because the changes made to the molecule did not affect the molecular properties enough to improve the rate of passive diffusion, or it may be because the acetylated compound is a better substrate for efflux pumps. Basic CNS drugs are usually substrates for efflux pumps to some degree, and the more lipophilic the compound is, the better a substrate it is.8 The use of a methyl carbamate to neutralize a secondary amine did not give favorable brain penetration either. In this case, a greater increase in logP is expected, but the brain levels appear to be lower. It could be that the addition of another H-bond acceptor, even more molecular weight and more rotatable bonds is unfavorable for BBB penetration, but it is more likely that the more lipophilic compound is binding to proteins and is an even better substrate for efflux pumps. The benzyl carbamate, which has an even higher logP, was found at even lower levels in the brain, confirming this hypothesis. The brain curves for the two carbamates show a slow increase in brain concentration over the course of the 20 min

255

experiment. This is further evidence of efflux pump activity, as compounds that are substrates for

efflux pumps tend to reach equilibrium more slowly.9

The overall conclusion must be that charge is not the only factor affecting brain uptake.

We had believed that removing a charge would result in a dramatic increase in BBB penetration,

but this was simply not the case. Nor does a modest increase in logP automatically guarantee

greater brain penetration. There are many factors that must be considered, including molecular

weight, number of H-bond donors, number of H-bond acceptors, number of rotatable bonds, and

total polar surface area. Selecting one of these factors for modification in isolation may not lead

to more potent CNS drugs. How a compound will behave in vivo is a complex problem that is difficult to predict. While the removal of a charge and subsequent raising of logD and reduction of H-bond donors is expected to cause an increase in BBB penetration based on the literature evidence discussed in the introduction, clearly in our case it did not have the desired effect. This may have been because other properties of the molecule were adversely affected by our modifications. Alternatively, increasing the logD may have caused a significant increase in binding to plasma proteins and cellular components, which decreases brain penetration.8 In addition, we may have produced molecules that are better substrates for efflux pumps. This

explanation seems to fit the data for the two carbamates.

All of these findings must be considered when designing ways to improve the BBB

penetration of our potent nNOS inhibitors. Using the molecular properties of III-2 predicted by

Molinspiration it is apparent that III-2 is a viable CNS drug.51 The molecular weight is in the correct range, at just less than 400. The polar surface area is acceptable at 84.1 Å2. Upper limits

on this value vary between 70 and 90 Å2,52, 53 so III-2 either passes or fails, but it can be

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assumed to be at the threshold. The logD of the compound is low at 0.11. Typical CNS drugs have a logD between 1 and 3. The number of H-bond donors is much too high at 6, and is a number that should be reduced to increase brain uptake. As mentioned in the introduction, this number should be around 3. The number of oxygens and nitrogens should be less than 5.6, 54 III-

2 has a total of 5. Finally, the compound should have as few rotatable bonds as possible. No upper limit is quoted, but CNS drugs typically have less than 10. III-2 has 9.

The molecular properties of III-2 are close to the limits for it to cross the BBB by passive

diffusion, which is reflected in its low Kp value. Increasing the logD by one unit and reducing the number of H-bond donors by 2 or more should have a positive impact on the brain uptake of the molecule. This points to capping one of the secondary amines with a lipophilic prodrug moiety that can be cleaved inside the brain. However, addition of any of the prodrug moieties listed in

Table 4.2 would change other molecular properties so that they become undesirable. In some cases the molecular weight would be dramatically increased; in others the number of oxygens and number of rotatable bonds would be outside the limits for a CNS drug. In addition to this, several of the prodrug moieties have other drawbacks, such as toxic byproducts or extensive cleavage in plasma. The increase in logD may also have negative effects in terms of pharmacokinetics. It will reduce the aqueous solubility, could increase binding to plasma proteins, and could increase efflux pump activity.

If the prodrug approach is used to increase the brain concentration of III-2, the moiety

must be chosen with care, taking into account all of the factors listed above. However, a return to

a SAR type of approach may yield more promising results. Although most of the modifications

listed in Chapter 3 failed, other types of modification may have more success. These could

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include lowering the pKa of the chain amine with electron withdrawing groups, rigidifying the molecule with rings, and adding functionality to the molecule to cause a modest increase in its lipophilicity.

The results presented in this chapter indicate that III-2 already has properties that make it a viable drug candidate. The Kp value for III-2 was low, at about 0.05. However, even a compound with a Kp as low as 0.1, such as sulpride, can still be a successful CNS drug,

8 suggesting that it is difficult to assess efficacy based on Kp alone. When III-2 was administered at about 5 mg / kg, a fairly low dose, a sufficient amount was taken up into the brain to produce levels that are four times the Ki against nNOS. Importantly, the blood levels are sufficiently low that eNOS should not be inhibited. The enantiomerically pure compound should show pharmacokinetic properties similar to the racemic compound used in this study, but has an even greater affinity for nNOS and so could be administered at half the dose. III-2 has other properties that make it a good drug candidate. It has excellent aqueous solubility. It does not appear to be metabolized at a significant rate (Fig. 4.16). Its high polarity should limit plasma protein binding. This, along with other properties such as oral bioavailability, elimination rate and toxicity, needs to be investigated. However, the properties exhibited already, combined with the therapeutic success of II-6 in animal studies, indicate that the next step for III-2 is an animal study in an nNOS disease model.

The design of highly selective, potent nNOS inhibitors that have good pharmacological properties is extremely challenging. However, a successful candidate could have an impact on several debilitating neurological diseases that affect millions of people, so it is a goal well worth pursuing.

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4.7 Experimental Details

See Chapter 2 for general chemistry procedures. Purity of compounds and bioanalytical

analyses (e.g. for microsome assay) were determined on a Dionex HPLC system (Dionex,

Sunnyvale, CA) using a Phenomenex (Torrance, CA) Luna C18 column (250 x 2.0 mm; 5 µM) and guard column with a flow rate of 0.2 mL / min. All water used was Milli-Q water obtained using a Biocel A10 water purification system from Millipore Corporation (Bedford, MA). Solid phase extraction was carried out using Waters Oasis HLB or MCX 1cc cartridges or MCX micro-elution plates (Waters Chromatography, Milford, MA). Analysis of biological samples was carried out using an API 3000 liquid chromatography-tandem mass spectrometry system

(Applied Biosystems, Foster City, CA) equipped with an Agilent 1100 series HPLC system

(Agilent Technologies, Wilmington, DE). Sample concentration was performed in a Genevac

EZ-2plus (Genevac Inc., Valley Cottage, NY). NADPH regeneration solutions and rat liver

microsomes were purchased from BD.

Boc tert-Butyl 3-((6-(benzyl(tert-butoxycarbonyl)amino)-4- N methylpyridin-2-yl)methyl)-4-(2-hydroxyethoxy)-pyrrol- BocN N OH O Ph idine-1-carboxylate (IV-16). A solution of III-30 (220 mg, 0.41 mmol) in methanol (5 mL) was cooled to -78 °C. Ozone was bubbled through the solution for 1 h. NaBH4 (57 mg, 1.5 mmol) was added and the mixture was allowed to warm to room

temperature. The solution was poured into sat NH4Cl and extracted with ethyl acetate (3 x 15

mL). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in

vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl

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acetate / hexanes, 1:1) to afford IV-16 as an oil (133 mg, 0.246 mmol, 60%). 1H NMR (500 MHz,

CDCl3) δ 7.34 (m, 1H), 7.24 (m, 5H), 6.72 (s, 1H), 5.14 (s, 2H), 3.69 – 3.43 (m, 5H), 3.26 – 3.11

(m, 3H), 2.97 (m, 1H), 2.72 (m, 1H), 2.56 (m, 2H), 2.30 (s, 3H), 1.46 (s, 9H), 1.42 (s, 9H); 13C

NMR (125 MHz, CDCl3) δ 158.2, 155.0, 154.7, 154.2, 149.1, 139.9, 128.3, 127.3, 127.2, 126.8,

120.5, 118.0, 81.4, 79.5, 78.5, 70.6, 61.9, (51.1 + 50.7), 50.5, (49.6 + 49.1), (43.4 + 42.8), 34.6,

28.8, 28.4, 21.3; ESMS m/z = 542 (M + H)+.

Boc tert-Butyl 3-((6-(tert-butoxycarbonylamino)-4-methyl- N pyridin-2-yl)methyl)-4-(2-hydroxyethoxy)-pyrrolidine-1- BocHN N OH O carboxylate (IV-17). To a solution of IV-12 (133 mg, 0.246

mmol) in ethanol (5 mL) was added Pd(OH)2 / C (~10 mg). The mixture was stirred under a

hydrogen atmosphere at 60 °C for 2 days. The mixture was filtered through Celite, and the

solvent was removed in vacuo. The crude product was purified using flash column

chromatography (silica gel, ethyl acetate / hexanes, 3:2) to afford IV-13 as an oil (41 mg, 0.091

1 mmol, 37%). H NMR (500 MHz, CDCl3) δ 8.07 (m, 1H), 7.72 (s, 1H), 6.66 (m, 1H), 5.59 (br,

1H), 3.84 – 3.51 (m, 6H), 3.33 (m, 1H), 3.19 (m, 2H), 2.64 (m, 1H), 2.31 (m, 4H), 1.52 (s, 9H),

13 1.46 (m, 9H); C NMR (125 MHz, CDCl3) δ 158.2, 155.0, 154.8, 153.0, 152.2, 150.9, 119.0,

110.9, 81.0, 79.6, 78.2, 70.4, 61.0, (51.0 + 50.6), (49.7 + 49.3), (45.7 + 45.0), 34.2, 28.8, 28.5,

21.6; ESMS m/z = 452 (M + H)+.

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Boc tert-Butyl 3-(2-azidoethoxy)-4-((6-(tert-butoxy-carb- N onylamino)-4-methylpyridin-2-yl)methyl)-pyrrol-idine-1- BocHN N N3 O carboxylate (IV-18). To a solution of IV-17 (41 mg, 0.091 mmol) in anhydrous THF (5 mL) were added PPh3 (26 mg, 0.1 mmol), DPPA (26 µL, 0.12 mmol) and DIAD (21 µL, 0.11 mmol). The mixture was stirred for 16 h. The solvent was removed in vacuo and the crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:3) to afford IV-14 as an oil (34 mg, 0.072 mmol, 79%). 1H NMR

(500 MHz, CDCl3) δ 7.60 (m, 1H), 7.17 (s, 1H), 6.69 (m, 1H), 3.99 – 3.63 (m, 3H), 3.53 – 3.28

(m, 5H), 3.15 (m, 1H), 2.95 (m, 1H), 2.76 – 2.60 (m, 2H), 2.31 (m, 3H), 1.52 (s, 9H), 1.45 (s,

13 9H); C NMR (125 MHz, CDCl3) δ 158.4, 155.1, 152.7, 151.6, 150.2, 119.6, 110.4, (81.1 +

80.3), (79.6 + 79.2), 70.3, (68.6 + 68.4), 51.3, (50.9 + 50.4), (43.5 + 42.8), (34.9 + 34.7), (28.8 +

28.5), 22.2, 21.6; ESMS m/z = 477 (M + H)+.

Boc tert-Butyl 3-(2-aminoethoxy)-4-((6-(tert-butoxy-carbonyl- N amino)-4-methylpyridin-2-yl)methyl)-pyrrolidine-1- BocHN N NH2 O carboxylate (III-44). To a solution of IV-18 (14 mg, 0.03

mmol) in ethanol (5 mL) was added Pd / C (~10 mg). The mixture was stirred under a hydrogen

atmosphere for 16 h. The mixture was filtered through Celite and the solvent was removed in

vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl

acetate / methanol, 9:1) to afford III-44 as a colorless oil (11 mg, 0.025 mmol, 83%). See

Chapter 3 for characterization data.

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H 6-((4-(2-Azidoethoxy)pyrrolidin-3-yl)methyl)-4-methyl- N pyridin-2-amine (IV-14). A solution of IV-18 (19 mg, 0.04 H2N N N3 O mmol) in 4N HCl in dioxanes (3 mL) was stirred for 16 h. The solvent was removed under a stream of nitrogen and the residue was dissolved in water, washed with ethyl acetate and concentrated to give IV-14 as a white dihydrochloride salt (7.3 mg, 0.02

1 mmol, 50%). H NMR (500 MHz, CDCl3) δ 6.55 (s, 2H), 4.00 (m, 1H), 3.62 (m, 2H), 3.50 (m,

1H), 3.43 (m, 1H), 3.34 (m, 2H), 3.17 (m, 1H), 3.05 (m, 1H), 2.84 (m, 2H), 2.68 (m, 1H), 2.20 (s,

3H); ESMS m/z = 277 (M + H)+.

H 6-((4-(2-Aminoethoxy)pyrrolidin-3-yl)methyl)-4-methyl- N pyridin-2-amine (IV-15). A solution of III-46 (11 mg, 0.025 H2N N NH2 O mmol) in 4N HCl in dioxanes (3 mL) was stirred for 16 h. The solvent was removed under a stream of nitrogen and the residue was dissolved in water, washed with ethyl acetate and concentrated to give IV-15 as a white trihydrochloride salt (5.5 mg, 0.015

1 mmol, 60%). H NMR (500 MHz, CDCl3) δ 6.55 (s, 1H), 6.50 (s, 1H), 4.06 (m, 1H), 3.67 (m,

1H), 3.51 (m, 2H), 3.40 (m, 1H), 3.21 – 3.04 (m, 4H), 2.80 (m, 1H), 2.68 (m, 1H), 2.19 (s, 3H);

ESMS m/z = 251 (M + H)+.

Boc tert-Butyl 3-(2-(tert-butoxycarbonyl-(3- N

Boc fluorophenethyl)amino)ethyl-amino)-4-((6- BocHN N N F N H (tert-butoxycarbonyl-amino)-4-methyl-

pyridin-2-yl)methyl)-pyrrolidine-1-carboxylate (IV-21). To a solution of III-35 (138 mg, 0.28

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mmol) in CH2Cl2 (3 mL) was added II-49 (85 mg, 0.3 mmol) in CH2Cl2 (2 mL). The solution

was stirred for 10 min. before NaHB(OAc)3 (70 mg, 0.33 mmol) was added. The mixture was

stirred for 1 h. The solution was poured into sat NaHCO3 and extracted with CH2Cl2 (3 x 20 mL).

The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate /

1 hexanes, 3:1) to afford IV-21 as an oil (127 mg, 0.19 mmol, 68%). H NMR (500 MHz, CDCl3)

δ 7.58 (m, 2H), 7.23 (m, 1H), 6.90 (m, 3H), 6.62 (s, 1H), 3.41 – 3.10 (m, 9H), 2.78 (m, 4H), 2.56

(m, 3H), 2.28 (s, rotamers, 3H), 1.50 (s, 9H), 1.43 (s, 18H); ESMS m/z = 672 (M + H)+.

1 H N -(4-((6-Amino-4-methylpyridin-2-yl)-methyl)- N pyrrolidin-3-yl)-N2-(3-fluoro-phen-ethyl)- H H2N N N F N H ethane-1,2-diamine (II-6). A solution of IV-21

(127 mg, 0.19 mmol) in 4N HCl in dioxanes (3 mL) was stirred for 16 h. The solvent was removed under a stream of nitrogen and the residue was dissolved in water, washed with ethyl acetate and concentrated to give II-6 as a white tetrahydrochloride salt (37 mg, 0.10 mmol, 53%).

1 H NMR (500 MHz, D2O) δ 7.260 (q, J = 6.5 Hz, 1H), 7.02 – 6.91 (m, 3H), 6.59 (s, 1H), 6.56 (s,

1H), 4.02 (q, J = 5.5 Hz, 1H), 3.73 – 3.51 (m, 4H), 3.41 – 3.26 (m, 6H), 3.18 (m, 1H), 3.00 (m,

3H), 2.66 (m, 1H), 2.21 (s, 3H); ESMS m/z = 372 (M + H)+.

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Boc tert-Butyl 3-((6-(benzyl(tert-butoxy-carbonyl)- N amino)-4-methylpyridin-2-yl)methyl)-4-(2-(3- H BocN N N F O fluorophenethyl-amino)ethoxy)pyrrolidine-1- Ph

carboxylate (IV-23). To a solution of III-31 (318 mg, 0.59 mmol) in CH2Cl2 (5 mL) was added

3-fluorophenethylamine (78 µL, 0.6 mmol). The mixture was stirred for 10 min before

NaHB(OAc)3 (127 mg, 0.6 mmol) and MeOH (1 mL) were added. The mixture was stirred for 1

h. The solution was poured into sat NaHCO3 and extracted with dichloromethane (3 x 15 mL).

The organic layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate /

1 methanol, 9:1) to afford IV-23 as an oil (274 mg, 0.41 mmol, 70%). H NMR (500 MHz, CDCl3)

δ 7.42 (m, 1H), 7.23 (m, 6H), 6.98 – 6.83 (m, 3H), 6.61 (s, 1H), 5.17 (s, 2H), 3.63 (m, 1H), 3.53

(m, 1H), 3.68 (m, 2H), 3.27 (m, 1H), 3.18 (m, 1H), 3.04 (m, 1H), 2.87 (m, 2H), 2.80 – 2.73 (m,

13 5H), 2.57 (m, 2H), 2.29 (m, 3H), 1.45 (m, 9H), 1.41 (s, 9H); C NMR (125 MHz, CDCl3) δ

164.1, 162.1, 157.9, 155.0, 154.6, 154.1, 148.7, 142.8, 140.0, 130.1, 128.7, 128.3, 127.5, 127.2,

127.1, 126.8, 124.6, 120.2, 117.2, 115.8, 115.7, 113.4, 113.2, 81.4, 79.7, 79.4, 78.9, 68.6, 50.9,

50.8, 50.5, 50.2, 49.4, 49.3, 43.0, 42.3, 36.2, 34.7, 28.8, 28.4, 21.4; ESMS m/z = 663 (M + H)+.

Boc tert-Butyl 3-((6-(benzyl(tert-butoxy-carbonyl)- N O amino)-4-methylpyridin-2-yl)methyl)-4-(2-(N- BocN N N F O (3-fluorophenethyl)-acetamido)ethoxy)- Ph pyrrolidine-1-carboxylate (IV-24). To a solution of IV-23 (79 mg, 0.12 mmol) in methanol (5

mmol) was added acetic anhydride (13 µL, 0.14 mmol) and triethylamine (20 µL, 0.14 mmol).

264

The mixture was stirred for 4 h. The solvent was removed in vacuo and the residue was dissolved in sat NaHCO3 and extracted with ethyl acetate (3 x 20 mL). The organic layers were combined,

dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using

flash column chromatography (silica gel, ethyl acetate / hexanes, 4:1) to afford IV-24 as a

1 colorless oil (84 mg, 0.12 mmol, quant). H NMR (500 MHz, CDCl3) δ 7.41 (m, 1H), 7.23 (m,

6H), 6.98 – 6.86 (m, 3H), 6.60 (m, 1H), 5.15 (s, 2H), 3.57 (m, 4H), 3.39 (m, 4H), 3.15 (m, 2H),

3.03 (m, 1H), 2.83 (m, 3H), 2.69 – 2.50 (m, 2H), 2.28 (m, 3H), (2.11 + 1.94 + 1.91) (s, rotamers,

13 3H), 1.44 (m, 9H), 1.41 (s, 9H); C NMR (125 MHz, CDCl3) δ 171.1, 164.1, 162.2, 157.8, 155.0,

154.6, 154.2, 148.9, 142.1, 140.1, 130.6, 130.2, 128.3, 127.2, 127.1, 127.0, 126.8, 124.7, 120.1,

117.4, 115.9, 113.4, 81.5, 79.9, 79.6, 79.1, 61.7, 52.0, 50.7, 50.2, 49.4, 48.1, 46.5, 42.8, 35.2,

33.9, 28.8, 28.4, 22.1, 21.5, 21.3; ESMS m/z = 705 (M + H)+.

Boc tert-Butyl 3-((6-(tert-butoxycarbonyl-amino)- N O 4-methylpyridin-2-yl)methyl)-4-(2-(N-(3- BocHN N N F O fluorophenethyl)acet-amido)ethoxy)- pyrrolidine-1-carboxylate (IV-25). To a solution of IV-24 (84 mg, 0.12 mmol) in ethanol (5 mL) was added Pd(OH)2 / C (~10 mg). The mixture was stirred under a hydrogen atmosphere at

60 °C for 2 days. The mixture was filtered through Celite, and the solvent was removed in vacuo.

The crude product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 5:1) to afford IV-25 as a colorless oil (55 mg, 0.09 mmol, 75%). 1H NMR (500 MHz,

CDCl3) δ 7.59 (m, 1H), 7.43 (m, 1H), 7.25 (m, 1H), 7.00 – 6.88 (m, 3H), 6.58 (m, 1H), 3.76 –

265

3.24 (m, 10H), 3.08 (m, 1H), 2.85 (m, 3H), 2.67 – 2.48 (m, 2H), 2.27 (m, 3H), (2.16 + 1.96 +

1.93) (s, rotamers, 3H), 1.52 (s, 9H), 1.43 (m, 9H); ESMS m/z = 615 (M + H)+.

H N-(2-(4-((6-Amino-4-methylpyridin-2-yl)- N O methyl)pyrrolidin-3-yloxy)ethyl)-N-(3-fluoro- H2N N N F O phenethyl)acetamide (IV-22). A solution of IV-

25 (55 mg, 0.09 mmol) in 4N HCl in dioxanes (3 mL) was stirred for 16 h. The solvent was

removed under a stream of nitrogen and the residue was dissolved in water, washed with ethyl

acetate and concentrated to give IV-17 as a white dihydrochloride salt (29 mg, 0.06 mmol, 67%).

1 Mp = 59 – 61 °C. H NMR (500 MHz, D2O) δ 7.21 (m, 1H), 6.89 (m, 3H), (6.53 + 6.47) (s, rotamers, 1H), (6.41 + 6.34) (s, rotamers, 1H), 3.97 (m, 1H), 3.69 (m, 1H), 3,62 – 3,42 (m, 5H),

3.39 – 3.15 (m, 4H), 3.01 (m, 1H), 2.79 – 2.60 (m, 4H), (2.17 + 2.13) (s, rotamers, 3H), (2.02 +

1.71) (s, rotamers, 3H); ESMS m/z = 415 (M + H)+.

Boc tert-Butyl 3-((6-(tert-butoxycarbonyl-amino)- N 4-methylpyridin-2-yl)methyl)-4-(2-(3-fluoro- H BocHN N N F O phenethylamino)-ethoxy)pyrrolidine-1- carboxylate (IV-28). To a solution of IV-23 (199 mg, 0.3 mmol) in ethanol (5 mL) was added

Pd(OH)2 / C (~10 mg). The mixture was stirred under a hydrogen atmosphere at 60 °C for 2 days.

The mixture was filtered through Celite, and the solvent was removed in vacuo. A mixture of IV-

28 and IV-29 were formed. The crude products were purified using flash column chromatography (silica gel, ethyl acetate / methanol, 1:9) to afford IV-28 (61 mg, 0.11 mmol,

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1 37 %) and IV-29 (42 mg, 0.09 mmol, 30 %) as white solids. IV-28 H NMR (500 MHz, CDCl3)

δ 7.60 (d, J = 10 Hz, 1H), 7.28 – 7.18 (m, 2H), 6.90 (m, 3H), 6.58 (s, 1H), 3.67 (m, 2H), 3.46 (m,

2H), 3.34 (m, 1H), 3.24 (dd, J = 4, 12 Hz, 1H), 3.08 (m, 1H), 2.95 – 2.73 (m, 6H), 2.57 (m, 2H),

13 2.47 (m, 1H), 2.30 (s, rotamers, 3H), 1.52 (s, 9H), 1.45 (s, 9H); C NMR (125 MHz, CDCl3) δ

164.1, 162.1, 158.4, 154.9, 152.9, 151.9, 150.0, 142.7, 130.1, 124.6, 119.0, 115.7, 113.2, 110.3,

80.8, 79.5, 78.6, 68.8, 50.9, 50.5, (49.5 + 49.2), (44.1 + 43.3), 36.3, 29.0, 28.8, 28.3, 21.5; ESMS

m/z = 573 (M + H)+.

Boc tert-Butyl 3-((6-(tert-butoxy-carbonyl-amino)- N O OMe 4-methylpyridin-2-yl)methyl)-4-(2-((3- BocHN N N F O fluorophenethyl)-(methoxy- carbonyl)amino)ethoxy)pyrrolidine-1-carboxylate (IV-30). To a solution of IV-28 (61 mg,

0.11 mmol) in CH2Cl2 (5 mL) were added methyl chloroformate (10 µL, 0.12 mmol) and TEA

(16 µL, 0.11 mmol). The mixture was stirred for 4 h. The solvent was removed in vacuo, and the residue was dissolved in sat NaHCO3 and extracted with ethyl acetate (3 x 20 mL). The organic

layers were combined, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude

product was purified using flash column chromatography (silica gel, ethyl acetate / hexanes, 1:1)

1 to afford IV-30 as a colorless oil (55 mg, 0.087 mmol, 91%). H NMR (500 MHz, CDCl3) δ 7.60

(m, 1H), 7.28 (m, 2H), 6.91 (m, 3H), 6.57 (s, 1H), 3.70 (m, 4H), 3.59 – 3.25 (m, 8H), 3.10 (m,

1H), 2.84 (m, 3H), 2.60 (m, 3H), 2.28 (m, 3H), 1.52 (s, 9H), 1.44 (m, 9H); 13C NMR (125 MHz,

CDCl3) δ 164.1, 162.2, 158.4, (157.0 + 156.8), (155.0 + 154.8), (152.7 + 151.7), 150.1, 141.8,

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130.1, 124.7, 119.3, 115.9, 113.5, 110.5, 81.0, 79.9, 79.5, 68.3, 60.6, 52.8, 50.8, 50.4, 49.5, 49.1,

48.4, 47.9, 43.7, 35.2, 28.9, 28.4, 21.5; ESMS m/z = 631 (M + H)+.

Boc tert-Butyl 3-((6-amino-4-methylpyridin-2-yl)- N O OBn methyl)-4-(2-((benzyloxycarbonyl)-(3-fluoro- H2N N N F O phenethyl)amino)ethoxy)-pyrrolidine-1-

carboxylate (IV-31). To a solution of IV-29 (42 mg, 0.09 mmol) in methanol (3 mL) was added

benzyl chloroformate (20 µL, 0.1 mmol). The mixture was stirred for 4 h. The solvent was

removed in vacuo, and the residue was dissolved in sat NaHCO3 and extracted with ethyl acetate

(3 x 20 mL). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated

in vacuo. The crude product was purified using flash column chromatography (silica gel, ethyl acetate / methanol, 9:1) to afford IV-31 (45 mg, 0.075 mmol, 83%) as a white solid. 1H NMR

(500 MHz, CDCl3) δ 7.34 (m, 5H), 7.21 (m, 1H), 6.88 (m, 3H), 6.25 (m, 1H), 6.14 (m, 1H), 5.13

(m, 2H), 4.33 (m, 2H), 3.78 – 3.25 (m, 10H), 3.07 (m, 1H), 2.90 – 2.75 (m, 3H), 2.57 (m, 2H),

2.15 (m, 3H), 1.43 (m, 9H); ESMS m/z = 507 (M + H)+.

H Methyl 2-(4-((6-amino-4-methylpyridin-2-yl)- N O OMe methyl)pyrrolidin-3-yloxy)ethyl(3-fluoro- H2N N N F O phenethyl)carbamate (IV-26). A solution of IV-

30 (55 mg, 0.087 mmol) in 4N HCl in dioxanes (3 mL) was stirred for 16 h. The solvent was

removed under a stream of nitrogen and the residue was dissolved in water, washed with ethyl

acetate and concentrated to give IV-26 as a greasy colorless solid (8.4 mg, 0.02 mmol, 23%). 1H

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NMR (500 MHz, D2O) δ 7.16 (q, J = 6 Hz, 1H), 6.83 (m, 3H), 6.48 (s, 1H), 6.33 (s, 1H), 3.91 (m,

1H), 3.66 (m, 1H), 3.58 (s, 1H), 3.52 – 3.40 (m, 4H), 3.33 – 3.13 (m, 7H), 2.99 (m, 1H), 2.70 –

2.63 (m, 4H), 2.12 (s, 3H); ESMS m/z = 431 (M + H)+.

H Benzyl 2-(4-((6-amino-4-methylpyridin-2-yl)- N O OBn methyl)pyrrolidin-3-yloxy)ethyl(3-fluoro- H2N N N F O phenethyl)carbamate (IV-27). A solution of IV-

31 (45 mg, 0.075 mmol) in 4N HCl in dioxanes (3 mL) was stirred for 16 h. The solvent was

removed under a stream of nitrogen, and the residue was dissolved in methanol, which was then

removed in vacuo. Anhydrous diethyl ether was added, causing a white precipitate to form. The

ether was decanted, and the precipitate was dried to give IV-27 as a greasy colorless solid (18

mg, 0.031 mmol, 42%). HPLC and LCMS revealed that IV-27 was contaminated with III-2

(~10%, See Appendix 1). It is unclear whether this was caused by loss of the CBZ group during deprotection, or whether it is from an intermediate being carried through. 1H NMR (500 MHz,

CD3OD) δ 7.35 (m, 6H), 6.97 (m, 3H), 6.67 (s, 1H), 6.50 (m, 1H), 5.08 (m, 2H), 4.21 (m, 1H),

3.99 (m, 1H), 3.75 – 3.23 (m, 9H), 3.23 (m, 1H), 2.88 (m, 2H), 2.78 (m, 2H), 2.32 (s, 3H); ESMS

m/z = 507 (M + H)+.

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HPLC analysis

The mobile phase consisted of 0.1% (v/v) formic acid (Fluka) in water as solvent A and

80% acetonitrile, 0.08% formic acid in water as solvent B.

Method A: Isocratic 5 min, 0% B, gradient 0 – 50% B, 25 min, gradient 50 – 100% B, 5 min,

gradient 100 – 0% B, 5 min, isocratic 0% B, 2 min. Monitored at 260, 280, 300 and 310 nm.

Column equilibrated in 0% B for 5 min prior to injection. All samples injected in aqueous

solutions (typically 2% formic acid), 20 µL injection volume. See Appendix 1 for actual

chromatograms.

Compound Wavelength (nm) Retention time (min)

III-2 310 14.4

II-6 310 14.0

IV-26 310 22.0

IV-27 310 26.5

IV-9 260 27.8, 28.3

IV-14 260 14.5

II-3 260 14.0

Minaprine 310 17.7

Table 4.3 Retention times of various compounds using HPLC conditions described above,

Method A.

270

Determination of metabolism of IV-9 and IV-14 in plasma

Three mice were sacrificed under anesthesia by exsanguination and decapitation. The blood was collected in a pre-heparinized tube. The tubes were centrifuged to precipitate the blood cells, and the plasma supernatant was transferred to separate vials. To an aliquot of plasma

(84 µL) was added NADPH regeneration solution A (5 µL), NADPH regeneration solution B (1

µL), and either IV-9 or IV-14 (10 µL of 200 µM stock). Samples were incubated at 37 °C and

removed at various time points (t = 0, 10, 20, 40 min). To quench the sample, acetonitrile (400

µL) and III-4 (10 µL of 200 µM stock) were added. The samples were centrifuged, and the supernatant was transferred to borosilicate tubes and concentrated to dryness using a Genevac.

The residues were reconstituted in 2% formic acid / water (50 µL) and the mixtures were analyzed by HPLC monitoring at 310 nm for IV-14 and III-4, and at 260 nm for IV-9. For each sample, the area under the peak corresponding to the compound of interest (IV-9, diastereomer 1,

IV-9, diastereomer 2, or IV-14) and to the internal standard (III-4) was integrated. The ratio of

compound of interest to internal standard was measured and normalized by dividing by the ratio

at t = 0 min.

Determination of metabolism of IV-9 and IV-14 in brain homogenate

Three fresh mice brains were homogenized, each in PBS (Phosphate buffered saline, pH

7.4, 1 mL per brain) with NADPH regeneration buffer A (50 µL) and NADPH regeneration

buffer B (10 µL). The homogenates were combined to form one stock. To an aliquot of

homogenate (190 µL) was added either IV-9 or IV-14 (10 µL of 200 µM stock). The samples

were incubated at 37 °C for varying lengths of time (0, 10, 20 and 40 min.) At each time point, a

271

sample was removed from the water bath and heated to 95 °C for 1 min. A mixture of 2% formic

acid in water (500 µL) was added to each sample, followed by III-4 (20 µL of 100 µM stock).

The IV-14 samples were loaded onto 1cc Oasis MCX SPE columns. The columns were washed

with 2% formic acid in water (1 mL), and with 80% acetonitrile, 2% formic acid in water (1 mL),

and the compounds were eluted with 5% NH4OH in methanol (2 mL). The eluents were

concentrated to dryness and reconstituted in 2% formic acid (50 µL). The IV-9 samples were

loaded onto 1cc Oasis HLB SPE columns. The columns were washed with 2% formic acid in

water (1 mL), and the compounds were eluted with 80% acetonitrile, 2% formic acid in water (2

mL). The eluents were concentrated to dryness and reconstituted in 2% formic acid (50 µL). The

mixtures were analyzed by HPLC monitoring at 310 nm for IV-14 and III-4, and at 260 nm for

IV-9. For each sample, the area under the peak corresponding to the compound of interest (IV-9, diastereomer 1, IV-9, diastereomer 2, or IV-14) and to the internal standard (III-4) was integrated. The ratio of compound of interest to internal standard was measured and normalized by dividing by the ratio at t = 0 min.

Standard microsome assay

To PBS (257 µL) was added NADPH regeneration buffer A (15 µL) and NADPH

regeneration buffer B (3 µL), and the compound of interest, e.g. minaprine (10 µL of 300 µM

stock). The tubes were vortexed and equilibrated at 37 °C. Rat liver microsomes (15 µL) were

added, and the samples were incubated at 37 °C for varying lengths of time (0, 10, 20, 40 min.).

To quench the reaction, acetonitrile (500 µL) was added, and the tubes were chilled to 0 °C to

precipitate out the proteins. The samples were centrifuged (14 krpm, 10 min), and the

272

supernatants were transferred to separate tubes. The samples were concentrated to dryness and reconstituted in 2% formic acid (50 µL). The mixtures were analyzed by HPLC. Peak integrals were normalized by dividing by the area for the peak at t = 0 min.

Rat Liver Microsome Assay

1.2

0.8 Min III-2 IV-22 0.6 II-6 IV-26 IV-27 0.4 Normalized [inhibor]

0.2

0 0 5 10 15 20 25 30 35 40 45 Incubation Time (min)

Figure 4.37 Results of standard rat liver microsome assay performed on compounds used in

brain uptake studies. The prescription CNS drug minaprine was used as a control.

Anaerobic microsome assay for azide metabolism

Nitrogen gas was bubbled through PBS (2.35 mL) for 15 min to deoxygenate it. To the

buffer was added NADPH regeneration buffer A (125 µL) and NADPH regeneration buffer B

(25 µL). To 2 mL vials with PTFE faced rubber septa were added the deoxygenated PBS mixture

(240 µL) and the azide (30 µL of 100 µM stock). The vials were sealed, and the volume above

273

the solution in the vial was purged with nitrogen. A balloon was used to maintain a nitrogen atmosphere inside the vial. The vials were equilibrated in a water bath at 37 °C. Microsomes (30

µL) were added through the rubber septum, and the mixtures were incubated at 37 °C for various lengths of time (0, 10, 20 and 40 minutes). To quench the reaction, acetonitrile (600 µL) was added. The mixtures were transferred to centrifuge tubes and cooled to 0 °C to precipitate the proteins. The samples were centrifuged at 14 krpm for 10 minutes. The supernatants were concentrated to dryness and the residues were reconstituted in 2% formic acid (50 µL) and analyzed by HPLC. Peak integrals were normalized by dividing by the area for the peak at t = 0 min.

Preparation of plasma standard curve

Samples were prepared to obtain final concentrations in µg / mL. A typical standard

curve would contain the concentrations 0.01, 0.05, 0.1, 0.5 and 1.0 µg / mL, but additional points were prepared as needed.

Example: to prepare a sample of 0.01 µg / mL of III-2 in plasma, III-2 (13.45 µL of 1 µM stock) was added to blank plasma (487 µL). Molecular weight of III-2 = 372. 1µg = 0.00269 µmol, therefore 1 µg / mL = 2.69 µM. 13.45 µL in 500 µL of 1 µM stock = 0.0269 µM = 0.01 µg / mL.

To an aliquot of the sample (100 µL) was added III-4 (10 µL of 10 µM stock). The

mixture was diluted with 2% acetic acid (500 µL) and loaded onto a preconditioned Oasis MCX

micro-elution plate. The well was washed with 2% formic acid (500 µL), and methanol (500 µL),

274

Standards were run in triplicate.

Preparation of brain standard curve

To a homogenizer was added 2% formic acid in water / acetonitrile (2:1, 700 µL) and III-

4 (10 µL of 100 µM stock). The compound of interest was added to obtain a final concentration

in µg / mL based on a total volume of 1 mL. A typical standard curve would contain the

concentrations 0.001, 0.005, 0.01, 0.05 and 0.1 µg / mL, but additional points were prepared as needed. For example, to obtain a concentration of 0.005 µg / mL of IV-22, 12.08 µL of 1 µM stock were added. To the solution was added a mouse brain, a rat pup brain or a piece of pig brain weighing between 300 and 500 mg. The brain was homogenized and transferred to a centrifuge tube. The homogenizer was washed with 2% formic acid in water / acetonitrile (2:1,

300 µL), which was also added to the centrifuge tube. The samples were centrifuged (10 krpm,

12 min) and the supernatant was transferred to a separate tube. An aliquot (400 µL) was loaded onto an Oasis MCX micro-elution plate. The well was washed with 2% formic acid (500 µL), and methanol (500 µL), and the compound was eluted with 5% NH4OH in methanol (2 x 400

µL). The solvent was removed under a stream of nitrogen and the residue was reconstituted in

LCMS/MS mobile phase (200 µL). An aliquot (20 µL) was analyzed by LCMS. Standards were

run in triplicate.

275

Administration of compounds to mice

Compounds were diluted in PBS to form a 1 mM solution. Mice were weighed and then

given the compound (100 µL per 10 g) via intraperitoneal injection. Three min before the desired

time of sacrifice, the mouse was injected with pentobarbital (100 µL of a 50 mg / mL solution in

PBS). Once the animal was no longer responsive, its thoracic cavity was opened and blood was

removed from the right ventricle of the heart using a syringe that had been treated with heparin

and transferred to Microtainer PST pre-heparinized tubes. The tubes were centrifuged (5 min at 6

krpm), and the plasma supernatants were transferred to labeled tubes. The brain was perfused

with PBS by inserting a needle through the left ventricle and allowing PBS under pressure to

flow into it. The right atrium was cut to relieve the pressure. The mouse was decapitated, and the

brain was removed. All samples were flash-frozen in liquid nitrogen and stored at -20 °C until

analyzed.

Preparation of plasma samples for quantification

To an aliquot of the sample (100 µL) was added III-4 (10 µL of 10 µM stock). The

mixture was diluted with 2% acetic acid (500 µL) and loaded onto a preconditioned Oasis MCX

micro-elution plate. The well was washed with 2% formic acid (500 µL) and methanol (500 µL),

and the compound was eluted with 5% NH4OH in methanol (2 x 400 µL). The solvent was removed under a stream of nitrogen and the residue was reconstituted in LCMS/MS mobile phase (200 µL). An aliquot (20 µL) was analyzed by LCMS. Quantification of compound was carried out by calculating the ratio of the mass spectral peak intensity of the compound to the mass spectral peak intensity of the internal standard and comparing it to the standard curve.

276

The data were originally obtained in µg / mL, but were converted to µM for ease of

comparison. The error bars on the charts represent the standard error mean. This was calculated

by dividing the standard deviation of the data for each time point by the square root of the

number of animals used for that point.

Preparation of brain samples for quantification

Brains were thawed and weighed. The brain was added to a homogenizer containing 2%

formic acid / acetonitrile (2:1, 700 µL) and III-4 (10 µL of 100 µM). The brain was

homogenized and transferred to a centrifuge tube. The homogenizer was washed with 2% formic

acid in water / acetonitrile (2:1, 300 µL), which was also added to the centrifuge tube. The

samples were centrifuged (10 krpm, 12 min), and the supernatant was transferred to a separate

tube. An aliquot (400 µL) was loaded onto an Oasis MCX micro-elution plate. The well was

washed with 2% formic acid (500 µL), and methanol (500 µL), and the compound was eluted

with 5% NH4OH in methanol (2 x 400 µL). The solvent was removed under a stream of nitrogen, and the residue was reconstituted in LCMS/MS mobile phase (200 µL). An aliquot (20 µL) was analyzed by LCMS. Quantification of compound was carried out by calculating the ratio of the peak intensity of the compound to the peak intensity of the internal standard and comparing it to the standard curve. The final value in µg / mL was divided by the mass of the brain to obtain a concentration in µg of compound / g of brain. For display on the charts, this was converted to a concentration in µM by assuming that the density of the brain tissue is approximately 1 g / mL.

277

LCMS/MS Conditions

Samples containing III-2, IV-22 and III-4 were eluted isocratically from a Phenomenex

MAX column (50 x 2.0 mm, Phenomenex, Torrance, CA) with a mobile phase consisting of

water and methanol (80:20) containing 0.1 % TFA at a flow rate of 250 µL / min. The tandem

mass spectrometer was operated with its electrospray source in the positive ionization mode. The

mass to charge ratios of the precursor-to-product ion reactions monitored were 373.3→123.1 for

III-2 and III-4 and 415.3→208.2 for IV-22. The retention time of III-2 was 2.38 min, that of

III-4 was 4.74 min, and that of IV-22 was 6.12 min.

Samples containing II-6, III-4 and IV-26 were eluted isocratically from a Phenomenex

MAX column (50 x 2.0 mm, Phenomenex, Torrance, CA) with a mobile phase consisting of

water and methanol (75:25) containing 0.1 % TFA at a flow rate of 125 µL / min. The tandem

mass spectrometer was operated with its electrospray source in the positive ionization mode. The

mass to charge ratios of the precursor-to-product ion reactions monitored were 372.3→123.2 for

II-6, 415.3→208.2 for IV-26. The retention time of II-6 was 2.46 min, that of III-4 was 4.74 min, and that of IV-26 was 9.27 min.

Samples containing IV-27 and IV-26 were eluted isocratically from a Phenomenex MAX column (50 x 2.0 mm, Phenomenex, Torrance, CA) with a mobile phase consisting of water and methanol (70:30) containing 0.1 % TFA at a flow rate of 125 µL / min. The tandem mass spectrometer was operated with its electrospray source in the positive ionization mode. The mass to charge ratios of the precursor-to-product ion reactions monitored were 507.3→91.1 for IV-27.

The retention time of IV-26 was 3.58 min and that of IV-27 was 15.7 min.

278

References for Chapter 1

1. Dermitzakis, E. T.; Reymond, A.; Lyle, R.; Scamuffa, N.; Ucla, C.; Deutsch, S.; Stevenson, B. J.; Flegel, V.; Bucher, P.; Jongeneel, C. V.; Antonarakis, S. E., Numerous potentially functional but non-genic conserved sequences on human chromosome 21. Nature 2002, 420, 578-582.

2. Wassarman, K. M., Small RNAs in bacteria: Diverse regulators of gene expression in response to environmental changes. Cell 2002, 109, 141-144.

3. Bashan, A.; Zarivach, R.; Schluenzen, F.; Agmon, I.; Harms, J.; Auerbach, T.; Baram, D.; Berisio, R.; Bartels, H.; Hansen, H. A. S.; Fucini, P.; Wilson, D.; Peretz, M.; Kessler, M.; Yonath, A., Ribosomal crystallography: Peptide bond formation and its inhibition. Biopolymers 2003, 70, 19-41.

4. Winkler, W.; Nahvi, A.; Breaker, R. R., Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 2002, 419, 952-956.

5. Mironov, A. S.; Gusarov, I.; Rafikov, R.; Lopez, L. E.; Shatalin, K.; Kreneva, R. A.; Perumov, D. A.; Nudler, E., Sensing small molecules by nascent RNA: A mechanism to control transcription in bacteria. Cell 2002, 111, 747-756.

6. Kruger, K.; Grabowski, P. J.; Zaug, A. J.; Sands, J.; Gottschling, D. E.; Cech, T. R., Self- Splicing RNA - Auto-Excision and Auto-Cyclization of the Ribosomal-RNA Intervening Sequence of Tetrahymena. Cell 1982, 31, 147-157.

7. Doudna, J. A.; Cech, T. R., Self-Assembly of a Group-I Intron Active-Site from Its Component Tertiary Structural Domains. RNA 1995, 1, 36-45.

8. Gestland, R. F.; Cech, T. R.; Atkin, J. F., Eds.; The RNA World; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1999.

9. Fields, B. N.; Knipe, D. M.; Howley, P. M., Eds.; Fundamental Virology; Lipincott- Raven: Philadelphia, PA, 1996.

10. DeJong, E. S.; Luy, B.; Marino, J. P. Curr. Top. Med. Chem. 2002, 2, 289.

11. Moazed, D.; Noller, H. F., Interaction of Antibiotics with Functional Sites in 16S Ribosomal-RNA. Nature 1987, 327, 389-394.

12. Pearson, N. D.; Prescott, C. D., RNA as a drug target. Chem. Biol. 1997, 4, 409-414.

13. Hermann, T., Strategies for the design of drugs targeting RNA and RNA - Protein complexes. Angew. Chem. Int. Edit. 2000, 39, 1891-1905. 279

14. Chow, C. S.; Bogdan, F. M., A structural basis for RNA-ligand interactions. Chem. Rev. 1997, 97, 1489-1513.

15. Frankel, A. D.; Young, J. A. T., HIV-1: Fifteen proteins and an RNA. Annu. Rev. Biochem. 1998, 67, 1-25.

16. Jones, K. A.; Peterlin, B. M., Control of RNA Initiation and Elongation at the Hiv-1 Promoter. Annu. Rev. Biochem. 1994, 63, 717-743.

17. Gait, M. J.; Karn, J., RNA Recognition by the Human-Immunodeficiency-Virus Tat- Protein and Rev-Protein. Trends Biochem. Sci. 1993, 18, 255-259.

18. Pollard, V. W.; Malim, M. H., The HIV-1 Rev protein. Annu. Rev. Microbiol. 1998, 52, 491-532.

19. Litovchick, A.; Evdokimov, A. G.; Lapidot, A., Aminoglycoside-arginine conjugates that bind TAR RNA: Synthesis, characterization, and antiviral activity. Biochemistry 2000, 39, 2838-2852.

20. Hwang, S.; Tamilarasu, N.; Kibler, K.; Cao, H.; Ali, A.; Ping, Y. H.; Jeang, K. T.; Rana, T. M., Discovery of a small molecule Tat-trans-activation-responsive RNA antagonist that potently inhibits human immunodeficiency virus-1 replication. J. Biol. Chem. 2003, 278, 39092-39103.

21. Luedtke, N. W.; Tor, Y., Fluorescence-based methods for evaluating the RNA affinity and specificity of HIV-1 Rev-RRE inhibitors. Biopolymers 2003, 70, 103-119.

22. Cundcliffe, E. The Molecular Basis of Antibiotic Action, Wiley: NY, 1981.

23. Carter, A. P.; Clemons, W. M.; Brodersen, D. E.; Morgan-Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V., Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 2000, 407, 340-348.

24. Schlunzen, F.; Zarivach, R.; Harms, J.; Bashan, A.; Tocilj, A.; Albrecht, R.; Yonath, A.; Franceschi, F., Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature 2001, 413, 814-821.

25. Hansen, J. L.; Ippolito, J. A.; Ban, N.; Nissen, P.; Moore, P. B.; Steitz, T. A., The structures of four macrolide antibiotics bound to the large ribosomal subunit. Mol. Cell 2002, 10, 117-128.

26. Spahn, C. M. T.; Prescott, C. D., Throwing a spanner in the works: Antibiotics and the translation apparatus. J. Mol. Med. 1996, 74, 423-439.

280

27. Ogle, J. M.; Brodersen, D. E.; Clemons, W. M.; Tarry, M. J.; Carter, A. P.; Ramakrishnan, V., Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 2001, 292, 897-902.

28. Slee, A. M.; Wuonola, M. A.; McRipley, R. J.; Zajac, I.; Zawada, M. J.; Bartholomew, P. T.; Gregory, W. A.; Forbes, M., Oxazolidinones, a New Class of Synthetic Antibacterial Agents - Invitro and Invivo Activities of Dup-105 and Dup-721. Antimicrob. Agents Chemother. 1987, 31, 1791-1797.

29. Barbachyn, M. R.; Ford, C. W., Oxazolidinone structure-activity relationships leading to linezolid. Angew. Chem. Int. Edit. 2003, 42, 2010-2023.

30. Zapp, M. L.; Stern, S.; Green, M. R., Small Molecules That Selectively Block Rna- Binding of Hiv-1 Rev Protein Inhibit Rev Function and Viral Production. Cell 1993, 74, 969-978.

31. Schroeder, R.; Waldsich, C.; Wank, H., Modulation of RNA function by aminoglycoside antibiotics. Embo. J. 2000, 19, 1-9.

32. Hermann, T.; Westhof, E., Aminoglycoside binding to the hammerhead ribozyme: A general model for the interaction of cationic antibiotics with RNA. J. Mol. Biol. 1998, 276, 903-912.

33. Tor, Y.; Hermann, T.; Westhof, E., Deciphering RNA recognition: aminoglycoside binding to the hammerhead ribozyme. Chem. Biol. 1998, 5, R277-R283.

34. Wang, S. H.; Huber, P. W.; Cui, M.; Czarnik, A. W.; Mei, H. Y., Binding of neomycin to the TAR element of HIV-1 RNA induces dissociation of Tat protein by an allosteric mechanism. Biochemistry 1998, 37, 5549-5557.

35. Hermann, T.; Westhof, E., Docking of cationic antibiotics to negatively charged pockets in RNA folds. J. Med. Chem. 1999, 42, 1250-1261.

36. Greenberg, W. A.; Priestley, E. S.; Sears, P. S.; Alper, P. B.; Rosenbohm, C.; Hendrix, M.; Hung, S. C.; Wong, C. H., Design and synthesis of new aminoglycoside antibiotics containing neamine as an optimal core structure: Correlation of antibiotic activity with in vitro inhibition of translation. J. Am. Chem. Soc. 1999, 121, 6527-6541.

37. Luedtke, N. W.; Baker, T. J.; Goodman, M.; Tor, Y., Guanidinoglycosides: A novel family of RNA ligands. J. Am. Chem. Soc. 2000, 122, 12035-12036.

38. Edwards, T. E.; Okonogi, T. M.; Sigurdsson, S. T., Investigation of RNA-protein and RNA-metal ion interactions by electron paramagnetic resonance spectroscopy: The HIV TAR-Tat motif. Chem. Biol. 2002, 9, 699-706.

281

39. Alper, P. B.; Hendrix, M.; Sears, P.; Wong, C. H., Probing the specificity of aminoglycoside ribosomal RNA interactions with designed synthetic analogs. J. Am. Chem. Soc. 1998, 120, 1965-1978.

40. Kirk, S. R.; Luedtke, N. W.; Tor, Y., Neomycin-acridine conjugate: A potent inhibitor of Rev-RRE binding. J. Am. Chem. Soc. 2000, 122, 980-981.

41. Hamy, F.; Brondani, V.; Florsheimer, A.; Stark, W.; Blommers, M. J. J.; Klimkait, T., A new class of HIV-1 Tat antagonist acting through Tat-TAR inhibition. Biochemistry 1998, 37, 5086-5095.

42. Peytou, V.; Condom, R.; Patino, N.; Guedj, R.; Aubertin, A. M.; Gelus, N.; Bailly, C.; Terreux, R.; Cabrol-Bass, D., Synthesis and antiviral activity of ethidium-arginine conjugates directed against the TAR RNA of HIV-1. J. Med. Chem. 1999, 42, 4042-4053.

43. Ratmeyer, L.; Zapp, M. L.; Green, M. R.; Vinayak, R.; Kumar, A.; Boykin, D. W.; Wilson, W. D., Inhibition of HIV-1 Rev-RRE interaction by diphenylfuran derivatives. Biochemistry 1996, 35, 13689-13696.

44. Gelus, N.; Bailly, C.; Hamy, F.; Klimkait, T.; Wilson, W. D.; Boykin, D. W., Inhibition of HIV-1 Tat-TAR interaction by diphenylfuran derivatives: Effects of the terminal basic side chains. Bioorgan. Med. Chem. 1999, 7, 1089-1096.

45. Du, Z. H.; Lind, K. E.; James, T. L., Structure of TAR RNA complexed with a Tat-TAR interaction nanomolar inhibitor that was identified by computational screening. Chem. Biol. 2002, 9, 707-712.

46. Mei, H. Y.; Cui, M.; Heldsinger, A.; Lemrow, S. M.; Loo, J. A.; Sannes-Lowery, K. A.; Sharmeen, L.; Czarnik, A. W., Inhibitors of protein-RNA complexation that target the RNA: Specific recognition of human immunodeficiency virus type 1 TAR RNA by small organic molecules. Biochemistry 1998, 37, 14204-14212.

47. Zhao, M.; Janda, L.; Nguyen, J.; Strekowski, L.; Wilson, W. D., The interaction of substituted 2-phenylquinoline intercalators with Poly(A).Poly(U) - classical and threading intercalation modes with Rna. Biopolymers 1994, 34, 61-73.

48. Tan, R. Y.; Chen, L.; Buettner, J. A.; Hudson, D.; Frankel, A. D., RNA Recognition by an Isolated Alpha-Helix. Cell 1993, 73, 1031-1040.

49. Garcia, J. A.; Harrich, D.; Soultanakis, E.; Wu, F.; Mitsuyasu, R.; Gaynor, R. B., Human Immunodeficiency Virus Type-1 Ltr Tata and Tar Region Sequences Required for Transcriptional Regulation. Embo. J. 1989, 8, 765-778.

282

50. Jakobovits, A.; Smith, D. H.; Jakobovits, E. B.; Capon, D. J., A Discrete Element 3' of Human Immunodeficiency Virus-1 (Hiv-1) and Hiv-2 Messenger-Rna Initiation Sites Mediates Transcriptional Activation by an Hiv Trans Activator. Mol. Cell. Biol. 1988, 8, 2555-2561.

51. Selby, M. J.; Bain, E. S.; Luciw, P. A.; Peterlin, B. M., Structure, Sequence, and Position of the Stem Loop in Tar Determine Transcriptional Elongation by Tat through the Hiv-1 Long Terminal Repeat. Gene Dev. 1989, 3, 547-558.

52. Kesavan, V.; Tamilarasu, N.; Cao, H.; Rana, T. M., A new class of RNA-binding oligomers: Peptoid amide and ester analogues. Bioconjugate Chem. 2002, 13, 1171-1175.

53. Litovchick, A.; Rando, R. R., Stereospecificity of short Rev-derived peptide interactions with RRE IIB RNA. RNA 2003, 9, 937-948.

54. Gelman, M. A.; Richter, S.; Cao, H.; Umezawa, N.; Gellman, S. H.; Rana, T. M., Selective binding of TAR RNA by a tat-derived beta-peptide. Org. Lett. 2003, 5, 3563- 3565.

55. Runyon, S. T.; Puglisi, J. D., Design of a cyclic peptide that targets a viral RNA. J. Am. Chem. Soc. 2003, 125, 15704-15705.

56. Tuerk, C.; Gold, L., Systematic Evolution of Ligands by Exponential Enrichment - RNA Ligands to Bacteriophage-T4 DNA-Polymerase. Science 1990, 249, 505-510.

57. Ellington, A. D.; Szostak, J. W., In vitro Selection of RNA Molecules That Bind Specific Ligands. Nature 1990, 346, 818-822.

58. Hermann, T.; Patel, D. J., Biochemistry - Adaptive recognition by nucleic acid aptamers. Science 2000, 287, 820-825.

59. Gold, L.; Polisky, B.; Uhlenbeck, O.; Yarus, M., Diversity of Oligonucleotide Functions. Annu. Rev. Biochem. 1995, 64, 763-797.

60. Baumann, M.; Bischoff, H.; Schmidt, D.; Griesinger, C., Combinatorial synthesis of cholesterol ester transfer protein-mRNA ligands and screening by nondenaturating gel- electrophoresis. J. Med. Chem. 2001, 44, 2172-2177.

61. Tamilarasu, N.; Huq, I.; Rana, T. M., Design, synthesis, and biological activity of a cyclic peptide: An inhibitor of HIV-1 Tat-TAR interactions in human cells. Bioorg. Med. Chem. Lett. 2000, 10, 971-974.

62. Carlson, C. B.; Stephens, O. M.; Beal, P. A., Recognition of double-stranded RNA by proteins and small molecules. Biopolymers 2003, 70, 86-102.

283

63. Krishnamurthy, M.; Gooch, B. D.; Beal, P. A., Peptide quinoline conjugates: A new class of RNA-binding molecules. Org Lett 2004, 6, 63-66.

64. Ecker, D. J.; Griffey, R. H., RNA as a small-molecule drug target: doubling the value of genomics. Drug Discov. Today 1999, 4, 420-429.

65. Lacourciere, K. A.; Stivers, J. T.; Marino, J. P., Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy. Biochemistry 2000, 39, 5630-5641.

66. DeJong, E. S.; Chang, C. E.; Gilson, M. K.; Marino, J. P., Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1. Biochemistry 2003, 42, 8035-8046.

67. Peng, J. W.; Lepre, C. A.; Fejzo, J.; Abdul-Manan, N.; Moore, J. M., Nuclear magnetic resonance-based approaches for lead generation in drug discovery. Method Enzymol. 2001, 338, 202-230.

68. Olejniczak, M.; Gdaniec, Z.; Fischer, A.; Grabarkiewicz, T.; Bielecki, L.; Adamiak, R. W., The bulge region of HIV-1 TAR RNA binds metal ions in solution. Nucleic Acids Res. 2002, 30, 4241-4249.

69. Luy, B.; Werner, M.; Marino, J. P., F-19NMR as a method for monitoring ligand binding to RNA and RNA-protein complexes. Biophys. J. 2001, 80, 567a-567a.

70. Hofstadler, S. A.; Griffey, R. H., Analysis of noncovalent complexes of DNA and RNA by mass spectrometry. Chem Rev. 2001, 101, 377-390.

71. SannesLowery, K. A.; Hu, P. F.; Mack, D. P.; Mei, H. Y.; Loo, J. A., HIV 1 Tat peptide binding do to TAR RNA by electrospray ionization mass spectrometry. Anal. Chem. 1997, 69, 5130-5135.

72. Hendrix, M.; Priestley, E. S.; Joyce, G. F.; Wong, C. H., Direct observation of aminoglycoside-RNA interactions by surface plasmon resonance. J. Am. Chem. Soc. 1997, 119, 3641-3648.

73. Geiger, A.; Burgstaller, P.; vonderEltz, H.; Roeder, A.; Famulok, M., RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity. Nucleic Acids Res. 1996, 24, 1029-1036.

74. Austin, R. J.; Xia, T. B.; Ren, J. S.; Takahashi, T. T.; Roberts, R. W., Designed arginine- rich RNA-binding peptides with picomolar affinity. J. Am. Chem. Soc. 2002, 124, 10966- 10967.

284

75. Kaul, M.; Barbieri, C. M.; Pilch, D. S., Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: Comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences. J. Am. Chem. Soc. 2004, 126, 3447-3453.

76. Tamilarasu, N.; Zhang, J.; Hwang, S.; Rana, T. M., A new strategy for site-specific protein modification: Analysis of a Tat peptide - TAR RNA interaction. Bioconjugate Chem. 2001, 12, 135-138.

77. Murchie, A. I. H.; Davis, B.; Isel, C.; Afshar, M.; Drysdale, M. J.; Bower, J.; Potter, A. J.; Starkey, I. D.; Swarbrick, T. M.; Mirza, S.; Prescott, C. D.; Vaglio, P.; Aboul-ela, F.; Karn, J., Structure-based drug design targeting an inactive RNA conformation: Exploiting the flexibility of HIV-1 TAR RNA. J. Mol. Biol. 2004, 336, 625-638.

78. Lee, J. K.; Kwon, M. Y.; Lee, K. H.; Jeong, S. J.; Hyun, S.; Shin, K. J.; Yu, J. H., An approach to enhance specificity against RNA targets using heteroconjugates of aminoglycosides and chloramphenicol (or linezolid). J. Am. Chem. Soc. 2004, 126, 1956- 1957.

79. Tok, J. B. H.; Rando, R. R., Simple aminols as aminoglycoside surrogates. J. Am. Chem. Soc. 1998, 120, 8279-8280.

80. Hall, D. G.; Laplante, C.; Manku, S.; Nagendran, J., Mild oxidative cleavage of borane- amine adducts from amide reductions: Efficient solution- and solid-phase synthesis of N- alkylamino acids and chiral oligoamines. J. Org. Chem. 1999, 64, 698-699.

81. Manku, S.; Hall, D. G., Combinatorial approach to selective multivalent ion pairing in mixed aqueous-organic media using bead-supported libraries of unnatural polyamines. Org. Lett. 2002, 4, 31-34.

82. Manku, S.; Wang, F.; Hall, D. G., Synthesis and high performance liquid chromatography/electrospray mass spectrometry single-bead decoding of split-pool structural libraries of polyamines supported on polystyrene and polystyrene/ethylene glycol resins. J. Comb. Chem. 2003, 5, 379-391.

83. Coy, D. H.; Hocart, S. J.; Sasaki, Y., Solid-Phase Reductive Alkylation Techniques in Analog Peptide-Bond and Side-Chain Modification. Tetrahedron 1988, 44, 835-841.

84. Matthews, J.; Rivero, R. A., Base-promoted solid-phase synthesis of substituted hydantoins and thiohydantoins. J. Org. Chem. 1997, 62, 6090-6092.

85. Sasaki, Y.; Murphy, W. A.; Heiman, M. L.; Lance, V. A.; Coy, D. H., Solid-Phase Synthesis and Biological Properties of Psi-[CH2NH] Pseudopeptide Analogs of a Highly Potent Somatostatin Octapeptide. J. Med. Chem. 1987, 30, 1162-1166.

285

86. Fehrentz, J. A.; Castro, B., An Efficient Synthesis of Optically-Active Alpha-(tert- Butoxycarbonylamino)-Aldehydes from Alpha-Amino-Acids. Synthesis-Stuttgart 1983, 676-678.

87. AbdelMagid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D., Reductive amination of aldehydes and ketones with sodium triacetoxyborohydride. Studies on direct and indirect reductive amination procedures. J. Org. Chem. 1996, 61, 3849-3862.

88. Wen, J. J.; Crews, C. M., Synthesis of 9-fluorenylmethoxycarbonyl-protected amino aldehydes. Tetrahedron: Asymmetry 1998, 9, 1855-1858.

89. Lee, N.; Gorelick, R. J.; Musier-Forsyth, K., Zinc finger-dependent HIV-1 nucleocapsid protein-TAR RNA interactions. Nucleic Acids Res. 2003, 31, 4847-4855.

90. Jeong, S.; Sefcikova, J.; Tinsley, R. A.; Rueda, D.; Walter, N. G., Trans-acting hepatitis delta virus ribozyme: Catalytic core and global structure are dependent on the 5' substrate sequence. Biochemistry 2003, 42, 7727-7740.

91. Walter, N. G.; Yang, N.; Burke, J. M., Probing non-selective cation binding in the hairpin ribozyme with Tb(III). J. Mol. Biol. 2000, 298, 539-555.

92. Hargittai, M. R. S.; Musier-Forsyth, K., Use of terbium as a probe of tRNA tertiary structure and folding. RNA 2000, 6, 1672-1680.

93. Hargittai, M. R. S.; Mangla, A. T.; Gorelick, R. J.; Musier-Forsyth, K., HIV-1 nucleocapsid protein zinc finger structures induce tRNA(Lys,3) structural changes but are not critical for primer/template annealing. J. Mol. Biol. 2001, 312, 985-997.

94. Soukup, G. A.; Breaker, R. R., Relationship between internucleotide linkage geometry and the stability of RNA. RNA 1999, 5, 1308-1325.

95. Winkler, W. C.; Cohen-Chalamish, S.; Breaker, R. R., An mRNA structure that controls gene expression by binding FMN. Proc. Natl. Acad. Sci. USA 2002, 99, 15908-15913.

96. Mandal, M.; Boese, B.; Barrick, J. E.; Winkler, W. C.; Breaker, R. R., Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 2003, 113, 577-586.

97. Milligan, J. F.; Groebe, D. R.; Witherell, G. W.; Uhlenbeck, O. C., Oligoribonucleotide Synthesis Using T7 Rna-Polymerase and Synthetic DNA Templates. Nucleic Acids Res. 1987, 15, 8783-8798.

286

98. Ho, P. T.; Chang, D.; Zhong, J. W. X.; Musso, G. F., An Improved Low Racemization Solid-Phase Method for the Synthesis of Reduced Dipeptide (Psi-CH2NH) Bond Isosteres. Peptide Res. 1993, 6, 10-12.

99. Loukas, V.; Noula, C.; Kokotos, G., Efficient protocols for the synthesis of enantiopure gamma-amino acids with proteinogenic side chains. J. Pept. Sci. 2003, 9, 312-319.

100. Salituro, F. G.; Agarwal, N.; Hofmann, T.; Rich, D. H., Inhibition of Aspartic Proteinases by Peptides Containing Lysine and Ornithine Side-Chain Analogs of Statine. J. Med. Chem. 1987, 30, 286-295.

101. Reddy, D. R.; Iqbal, M. A.; Hudkins, R. L.; Messina-McLaughlin, P. A.; Mallamo, J. P., A simple synthetic protocol for the protection of amides, lactams, ureas, and carbamates. Tetrahedron Lett. 2002, 43, 8063-8066.

102. Guichard, G.; Briand, J. P.; Friede, M., Synthesis of Arginine Aldehydes for the Preparation of Pseudopeptides. Peptide Res. 1993, 6, 121-124.

103. Kaiser, E.; Colescot.Rl; Bossinge.Cd; Cook, P. I., Color Test for Detection of Free Terminal Amino Groups in Solid-Phase Synthesis of Peptides. Anal. Biochem. 1970, 34, 595-6.

104. Lambert, J. B.; Shurvell, H. F.; Lightner, D. A.; Cooks, R. G. Organic Structural Spectroscopy; Prentice Hall, Inc.: Upper Saddle River, NJ, 1998, p 294.

105. Blount, K. F.; Tor, Y., Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding. Nucleic Acids Res. 2003, 31, 5490-5500.

References for Chapter 2

1. Nathan, C. F.; Hibbs, J. B., Role of Nitric-Oxide Synthesis in Macrophage Antimicrobial Activity. Curr. Opin. Immunol. 1991, 3, 65-70.

2. Knowles, R. G.; Moncada, S., Nitric-Oxide Synthases in Mammals. Biochem. J. 1994, 298, 249-258.

3. Palmer, R. M. J.; Ferrige, A. G.; Moncada, S., Nitric-Oxide Release Accounts for the Biological-Activity of Endothelium-Derived Relaxing Factor. Nature 1987, 327, 524-526.

4. Denninger, J. W.; Marletta, M. A., Guanylate cyclase and the (NO)-N-./cGMP signaling pathway. Biochim. Biophys. Acta- Bioenergetics 1999, 1411, 334-350.

287

5. Garthwaite, J., Glutamate, Nitric-Oxide and Cell Cell Signaling in the Nervous-System. Trends Neurosci. 1991, 14, 60-67.

6. Moncada, S.; Palmer, R. M. J.; Higgs, E. A., Biosynthesis of Nitric-Oxide from L- Arginine - a Pathway for the Regulation of Cell-Function and Communication. Biochem. Pharmacol. 1989, 38, 1709-1715.

7. Schuman, E. M.; Madison, D. V., A Requirement for the Intercellular Messenger Nitric- Oxide in Long-Term Potentiation. Science 1991, 254, 1503-1506.

8. MacMicking, J.; Xie, Q. W.; Nathan, C., Nitric oxide and macrophage function. Annu. Rev. Immunol. 1997, 15, 323-350.

9. Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A., Peroxynitrite-Induced Membrane Lipid-Peroxidation - the Cytotoxic Potential of Superoxide and Nitric-Oxide. Arch. Biochem. Biophys. 1991, 288, 481-487.

10. Schmidt, H. H. H. W.; Walter, U., No at Work. Cell 1994, 78, 919-925.

11. Forstermann, U.; Pollock, J. S.; Schmidt, H. H. H. W.; Heller, M.; Murad, F., Calmodulin-Dependent Endothelium-Derived Relaxing Factor Nitric-Oxide Synthase Activity Is Present in the Particulate and Cytosolic Fractions of Bovine Aortic Endothelial-Cells. Proc. Natl. Acad. Sci. USA 1991, 88, 1788-1792.

12. Fischmann, T. O.; Hruza, A.; Niu, X. D.; Fossetta, J. D.; Lunn, C. A.; Dolphin, E.; Prongay, A. J.; Reichert, P.; Lundell, D. J.; Narula, S. K.; Weber, P. C., Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation. Nat. Struct. Biol. 1999, 6, 233-242.

13. Siddhanta, U.; Presta, A.; Fan, B. C.; Wolan, D.; Rousseau, D. L.; Stuehr, D. J., Domain swapping in inducible nitric-oxide synthase - Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer. J. Biol. Chem. 1998, 273, 18950- 18958.

14. Griffith, O. W.; Stuehr, D. J., Nitric Oxides Synthases - Properties and Catalytic Mechanism. Annu. Rev. Physiol. 1995, 57, 707-736.

15. Titheradge, M. A., Nitric oxide in septic shock. Biochim. Biophys. Acta-Bioenergetics 1999, 1411, 437-455.

16. Hobbs, A. J.; Higgs, A.; Moncada, S., Inhibition of nitric oxide synthase as a potential therapeutic target. Annu. Rev. Pharmacol. 1999, 39, 191-220.

288

17. Kato, Y.; Miura, Y.; Yamamoto, N.; Ozaki, N.; Oiso, Y., Suppressive effects of a selective inducible nitric oxide synthase (iNOS) inhibitor on pancreatic beta-cell dysfunction. Diabetologia 2003, 46, 1228-1233.

18. van't Hof, R. J.; Hocking, L.; Wright, P. K.; Ralston, S. H., Nitric oxide is a mediator of apoptosis in the rheumatoid joint. Rheumatology 2000, 39, 1004-1008.

19. van't Hof, R. J.; Ralston, S. H., Nitric oxide and bone. Immunology 2001, 103, 255-261.

20. Ross, C. A.; Bredt, D.; Snyder, S. H., Messenger Molecules in the Cerebellum. Trends Neurosci. 1990, 13, 216-222.

21. Iravani, M. M.; Kashefi, K.; Mander, P.; Rose, S.; Jenner, P., Involvement of inducible nitric oxide synthase in inflammation-induced dopaminergic neurodegeneration. Neuroscience 2002, 110, 49-58.

22. Meininger, C. J.; Kelly, K. A.; Li, H.; Haynes, T. E.; Wu, G. Y., Glucosamine inhibits inducible nitric oxide synthesis. Biochem. Bioph. Res. Commun. 2000, 279, 234-239.

23. Moncada, S.; Higgs, E. A., Molecular Mechanisms and Therapeutic Strategies Related to Nitric-Oxide. Faseb J. 1995, 9, 1319-1330.

24. Huang, P. L.; Huang, Z. H.; Mashimo, H.; Bloch, K. D.; Moskowitz, M. A.; Bevan, J. A.; Fishman, M. C., Hypertension in Mice Lacking the Gene for Endothelial Nitric-Oxide Synthase. Nature 1995, 377, 239-242.

25. Sims, N. R.; Anderson, M. F., Mitochondrial contributions to tissue damage in stroke. Neurochem. Int. 2002, 40, 511-526.

26. Elibol, B.; Soylemezoglu, F.; Unal, I.; Fujii, M.; Hirt, L.; Huang, P. L.; Moskowitz, M. A.; Dalkara, T., Nitric oxide is involved in ischemia-induced apoptosis in brain: A study in neuronal nitric oxide synthase null mice. Neuroscience 2001, 105, 79-86.

27. Huang, Z. H.; Huang, P. L.; Panahian, N.; Dalkara, T.; Fishman, M. C.; Moskowitz, M. A., Effects of Cerebral-Ischemia in Mice Deficient in Neuronal Nitric-Oxide Synthase. Science 1994, 265, 1883-1885.

28. Zhang, L.; Dawson, V. L.; Dawson, T. M., Role of nitric oxide in Parkinson's disease. Pharmacol. Therapeut. 2006, 109, 33-41.

29. Uehara, T.; Nakamura, T.; Yao, D. D.; Shi, Z. Q.; Gu, Z. Z.; Ma, Y. L.; Masliah, E.; Nomura, Y.; Lipton, S. A., S-Nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. Nature 2006, 441, 513-517.

289

30. Dorheim, M. A.; Tracey, W. R.; Pollock, J. S.; Grammas, P., Nitric-Oxide Synthase Activity Is Elevated in Brain Microvessels in Alzheimers-Disease. Biochem. Bioph. Res. Commun. 1994, 205, 659-665.

31. Dawson, V. L.; Dawson, T. M., Nitric oxide in neurodegeneration. Prog. Brain Res. 1998, 118, 215-229.

32. Iversen, H. K., Human migraine models. Cephalalgia 2001, 21, 781-785.

33. Baba, H.; Suzuki, T.; Arai, H.; Emson, P. C., Expression of nNOS and soluble guanylate cyclase in schizophrenic brain. Neuroreport 2004, 15, 677-680.

34. Catania, M. V.; Aronica, E.; Yankaya, B.; Troost, D., Increased expression of neuronal nitric oxide synthase spliced variants in reactive astrocytes of amyotrophic lateral sclerosis human spinal cord. J. Neurosci. 2001, 21.

35. Rajasekaran, K.; Jayakumar, R.; Venkatachalam, K., Increased neuronal nitric oxide synthase (nNOS) activity triggers picrotoxin-induced seizures in rats and evidence for participation of nNOS mechanism in the action of antiepileptic drugs. Brain Res. 2003, 979, 85-97.

36. Ferriero, D. M.; Holtzman, D. M.; Black, S. M.; Sheldon, R. A., Neonatal mice lacking neuronal nitric oxide synthase are less vulnerable to hypoxic-ischemic injury. Neurobiol. Dis. 1996, 3, 64-71.

37. Jenner, P.; Olanow, C. W., Oxidative stress and the pathogenesis of Parkinson's disease. Neurology 1996, 47, S161-S170.

38. Brouillet, E.; Beal, M. F., Nmda Antagonists Partially Protect against Mptp Induced Neurotoxicity in Mice. Neuroreport 1993, 4, 387-390.

39. Hantraye, P.; Brouillet, E.; Ferrante, R.; Palfi, S.; Dolan, R.; Matthews, R. T.; Beal, M. F., Inhibition of neuronal nitric oxide synthase prevents MPTP-induced parkinsonism in baboons. Nat. Med. 1996, 2, 1017-1021.

40. van den Tweel, E. R. W.; van Bel, F.; Kavelaars, A.; Peeters-Scholte, C. M. P. C. D.; Haumann, J.; Nijboer, C. H. A.; Heijnen, C. J.; Groenendaal, F., Long-term neuroprotection with 2-iminobiotin, an inhibitor of neuronal and inducible nitric oxide synthase, after cerebral hypoxia-ischemia in neonatal rats. J. Cerebr. Blood F. Met. 2005, 25, 67-74.

41. Ishida, A.; Trescher, W. H.; Lange, M. S.; Johnston, M. V., Prolonged suppression of brain nitric oxide synthase activity by 7-nitroindazole protects against cerebral hypoxic- ischemic injury in neonatal rat. Brain Dev.-Jpn. 2001, 23, 349-354.

290

42. Tsuji, M.; Higuchi, Y.; Shiraishi, K.; Kume, T.; Akaike, A.; Hattori, H., Protective effect of aminoguanidine on hypoxic-ischemic brain damage and temporal profile of brain nitric oxide in neonatal rat. Pediatr. Res. 2000, 47, 79-83.

43. Yoshida, T.; Limmroth, V.; Irikura, K.; Moskowitz, M. A., The NOS Inhibitor, 7- Nitroindazole, Decreases Focal Infarct Volume but Not the Response to Topical Acetylcholine in Pial Vessels. J. Cerebr. Blood F. Met. 1994, 14, 924-929.

44. Zicha, J.; Pechanova, O.; Dobesova, Z.; Kunes, J., Hypertensive response to chronic N- G-nitro-L-arginine methyl ester (L-NAME) treatment is similar in immature and adult Wistar rats. Clin. Sci. 2003, 105, 483-489.

45. Buchan, A. M.; Gertler, S. Z.; Huang, Z. G.; Li, H.; Chaundy, K. E.; Xue, D., Failure to Prevent Selective Ca1 Neuronal Death and Reduce Cortical Infarction Following Cerebral-Ischemia with Inhibition of Nitric-Oxide Synthase. Neuroscience 1994, 61, 1-11.

46. Alderton, W. K.; Cooper, C. E.; Knowles, R. G., Nitric oxide synthases: structure, function and inhibition. Biochem. J. 2001, 357, 593-615.

47. Tinker, A. C.; Wallace, A. V., Selective inhibitors of inducible nitric oxide synthase: Potential agents for the treatment of inflammatory diseases? Curr. Top. Med. Chem. 2006, 6, 77-92.

48. Erdal, E. P.; Litzinger, E. A.; Seo, J. W.; Zhu, Y. Q.; Ji, H. T.; Silverman, R. B., Selective neuronal nitric oxide synthase inhibitors. Curr. Top. Med. Chem. 2005, 5, 603-624.

49. Furfine, E. S.; Carbine, K.; Bunker, S.; Tanoury, G.; Harmon, M.; Laubach, V.; Sherman, P., Potent inhibition of human neuronal nitric oxide synthase by N-ω-nitro-L-arginine methyl ester results from contaminating N-G-nitro-L-arginine. Life Sci. 1997, 60, 1803- 1809.

50. Narayanan, K.; Spack, L.; Mcmillan, K.; Kilbourn, R. G.; Hayward, M. A.; Masters, B. S. S.; Griffith, O. W., S-Alkyl-L-Thiocitrullines - Potent Stereoselective Inhibitors of Nitric- Oxide Synthase with Strong Pressor Activity in vivo. J. Biol. Chem. 1995, 270, 11103- 11110.

51. Collins, J. L.; Shearer, B. G.; Oplinger, J. A.; Lee, S. L.; Garvey, E. P.; Salter, M.; Duffy, C.; Burnette, T. C.; Furfine, E. S., N-phenylamidines as selective inhibitors of human neuronal nitric oxide synthase: Structure-activity studies and demonstration of in vivo activity. J. Med. Chem. 1998, 41, 2858-2871.

52. Wolff, D. J.; Lubeskie, A., Aminoguanidine Is an Isoform-Selective, Mechanism-Based Inactivator of Nitric-Oxide Synthase. Arch. Biochem. Biophys. 1995, 316, 290-301.

291

53. Kengatharan, M.; Szabo, C.; Dekimpe, S.; Southan, G. J.; Thiemermann, C., S-Methyl- Isothiourea, a Potent Inhibitor of the Inducible Isoform of Nitric-Oxide Synthase, Has Beneficial Hemodynamic-Effects in Gram-Positive and Gram-Negative Forms of Circulatory Shock. Faseb J. 1995, 9, A28-A28.

54. Ijuin, R.; Umezawa, N.; Higuchi, T., Design, synthesis, and evaluation of new type of L- amino acids containing pyridine moiety as nitric oxide synthase inhibitor. Bioorg. Med. Chem. 2006, 14, 3563-3570.

55. Hagmann, W. H.; Caldwell, C. G.; Chen, P.; Durette, P. L.; Esser, C. K.; Lanza, T. J.; Kopka, I. E.; Guthikonda, R.; Shah, S. K.; MacCoss, M.; Chabin, R. M.; Fletcher, D.; Grant, S. K.; Green, B. G.; Humes, J. L.; Kelly, T. M.; Luell, S.; Meurer, R.; Moore, V.; Pacholok, S. G.; Pavia, T.; Williams, H. R.; Wong, K. K., Substituted 2-aminopyridines as inhibitors of nitric oxide synthases. Bioorg. Med. Chem. Lett. 2000, 10, 1975-1978.

56. Strub, A.; Ulrich, W. R.; Hesslinger, C.; Eltze, M.; Fuchss, T.; Strassner, J.; Strand, S.; Lehner, M. D.; Boer, R., The novel imidazopyridine 2-[2-(4-methoxy-pyridin-2-yl)- ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) is a highly selective inhibitor of the inducible nitric-oxide synthase. Mol. Pharmacol. 2006, 69, 328-337.

57. Mete, A.; Connolly, S., Inhibitors of the NOS enzymes: A patent review. Idrugs 2003, 6, 57-65.

58. Garvey, E. P.; Oplinger, J. A.; Furfine, E. S.; Kiff, R. J.; Laszlo, F.; Whittle, B. J. R.; Knowles, R. G., 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J. Biol. Chem. 1997, 272, 4959-4963.

59. Alderton, W. K.; Angell, A. D. R.; Craig, C.; Dawson, J.; Garvey, E.; Moncada, S.; Monkhouse, J.; Rees, D.; Russell, L. J.; Russell, R. J.; Schwartz, S.; Waslidge, N.; Knowles, R. G., GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo. Brit. J. Pharmacol. 2005, 145, 301- 312.

60. Silverman, R. B.; Huang, H.; Marletta, M. A.; Martasek, P., Selective inhibition of neuronal nitric oxide synthase by N-ω-nitroarginine- and phenylalanine-containing dipeptides and dipeptide esters. J. Med. Chem. 1997, 40, 2813-2817.

61. Huang, H.; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B., N-ω- nitroarginine-containing dipeptide amides. Potent and highly selective inhibitors of neuronal nitric oxide synthase. J. Med. Chem. 1999, 42, 3147-3153.

62. Gomez-Vidal, J. A.; Martasek, P.; Roman, L. J.; Silverman, R. B., Potent and selective conformationally restricted neuronal nitric oxide synthase inhibitors. J. Med. Chem. 2004, 47, 703-710.

292

63. Huang, H.; Martasek, P.; Roman, L. J.; Silverman, R. B., Synthesis and evaluation of peptidomimetics as selective inhibitors and active site probes of nitric oxide synthases. J. Med. Chem. 2000, 43, 2938-2945.

64. Hah, J. M.; Martasek, P.; Roman, L. J.; Silverman, R. B., Aromatic reduced amide bond peptidomimetics as selective inhibitors of neuronal nitric oxide synthase. J. Med. Chem. 2003, 46, 1661-1669.

65. Hah, J. M.; Roman, L. J.; Martasek, P.; Silverman, R. B., Reduced amide bond peptidomimetics. (4S)-N-(4-amino-5-[aminoalkyl]aminopentyl)-N '-nitroguanidines, potent and highly selective inhibitors of neuronal nitric oxide synthase. J. Med. Chem. 2001, 44, 2667-2670.

66. Flinspach, M. L.; Li, H. Y.; Jamal, J.; Yang, W. P.; Huang, H.; Hah, J. M.; Gomez-Vidal, J. A.; Litzinger, E. A.; Silverman, R. B.; Poulos, T. L., Structural basis for dipeptide amide isoform-selective inhibition of neuronal nitric oxide synthase. Nat. Struct. Mol. Biol. 2004, 11, 54-59.

67. Bohm, H. J., The Computer-Program Ludi - a New Method for the de novo Design of Enzyme-Inhibitors. J. Comput. Aid. Mol. Des. 1992, 6, 61-78.

68. Ertl, P.; Rohde, B.; Selzer, P., Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties. J. Med. Chem. 2000, 43, 3714-3717.

69. Bohm, H. J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Muller, K.; Obst-Sander, U.; Stahl, M., Fluorine in medicinal chemistry. Chembiochem 2004, 5, 637-643.

70. Ji, H.; Igarashi, J.; Li, H.; Martásek, P.; Roman, L. J.; Vásquez Vivar, L.; Derrick, M.; Poulos, T. L.; Tan, S.; Silverman, R. B., Selective Neuronal Nitric Oxide Synthase Inhibitors for Prevention of Cerebral Palsy. In preparation 2007.

71. Martin, Y. C., A bioavailability score. J. Med. Chem. 2005, 48, 3164-3170.

72. Ajay; Bemis, G. W.; Murcko, M. A., Designing libraries with CNS activity. J. Med. Chem. 1999, 42, 4942-4951.

73. Schmidt am Busch, M.; Knapp, E. W., Accurate pKa determination for a heterogeneous group of organic molecules. Chemphyschem 2004, 5, 1513-1522.

74. Bagley, M. C.; Dale, J. W.; Xiong, X.; Bower, J., Synthesis of dimethyl sulfomycinamate. Org. Lett. 2003, 5, 4421-4424.

293

75. Barton, A.; Breukelman, S. P.; Kaye, P. T.; Meakins, G. D.; Morgan, D. J., The Preparation of Thiazole-4-Carboxylates and Thiazole-5-Carboxylates, and an Infrared Study of Their Rotational Isomers. J. Chem. Soc. Perk. T. 1 1982, 159-164.

76. van Well, R. M.; Overkleeft, H. S.; van Boom, J. H.; Coop, A.; Wang, J. B.; Wang, H. Y.; van der Marel, G. A.; Overhand, M., Synthesis of novel sugar amino acids by Curtius rearrangement. Eur. J. Org. Chem. 2003, 1704-1710.

77. Bagley, M. C.; Chapaneri, K.; Glover, C.; Merritt, E. A., Simple microwave-assisted method for the synthesis of primary thioamides from nitriles. Synlett 2004, 2615-2617.

78. Dudin, L.; Pattenden, G.; Viljoen, M. S.; Wilson, C., Synthesis of novel N-methylated thiazole-based cyclic octa and dodecapeptides. Tetrahedron 2005, 61, 1257-1267.

79. Taylor, E. C.; Wolinsky, J.; Lee, H.-H., The Reaction of a-Cyanobenzyl Benzenesulfonate with Thioureas. J. Am. Chem. Soc. 1954, 76, 1866-1870.

80. Mancuso, A. J.; Huang, S. L.; Swern, D., Oxidation of Long-Chain Alcohols to Carbonyls by Activated DMSO. Abstr. Pap. Am. Chem. S. 1977, 174, 164-164.

81. Mancuso, A. J.; Huang, S. L.; Swern, D., Oxidation of Long-Chain and Related Alcohols to Carbonyls by Dimethyl-Sulfoxide Activated by Oxalyl Chloride. J. Org. Chem. 1978, 43, 2480-2482.

82. Mancuso, A. J.; Swern, D., Activated Dimethylsulfoxide - Useful Reagents for Synthesis. Synthesis-Stuttgart 1981, 165-185.

83. Allevi, P.; Galligani, M.; Anastasia, M., A simple and convenient transformation of L- lysine into pyridinoline and deoxypyridinoline, two collagen cross-links of biochemical interest. Tetrahedron-Asymmetry 2002, 13, 1901-1910.

84. Roman, L. J.; Sheta, E. A.; Martasek, P.; Gross, S. S.; Liu, Q.; Masters, B. S. S., High- Level Expression of Functional-Rat Neuronal Nitric-Oxide Synthase in Escherichia coli. Proc. Natl. Acad. Sci. USA 1995, 92, 8428-8432.

85. Hevel, J. M.; White, K. A.; Marletta, M. A., Purification of the Inducible Murine Macrophage Nitric-Oxide Synthase - Identification as a Flavoprotein. J. Biol. Chem. 1991, 266, 22789-22791.

86. Martasek, P.; Liu, Q.; Liu, J. W.; Roman, L. J.; Gross, S. S.; Sessa, W. C.; Masters, B. S. S., Characterization of bovine endothelial nitric oxide synthase expressed in E. coli. Biochem. Bioph. Res. Commun. 1996, 219, 359-365.

294

87. Hevel, J. M.; Marletta, M. A., Nitric-Oxide Synthase Assays. Method Enzymol. 1994, 233, 250-258.

88. Little, T. L.; Webber, S. E., A Simple and Practical Synthesis of 2-Aminoimidazoles. J. Org. Chem. 1994, 59, 7299-7305.

89. Birman, V. B.; Jiang, X. T., Synthesis of sceptrin alkaloids. Org. Lett. 2004, 6, 2369- 2371.

90. Abou-Jneid, R.; Ghoulami, S.; Martin, M. T.; Dau, E. T. H.; Travert, N.; Al-Mourabit, A., Biogenetically inspired synthesis of marine C6N4 2-aminoimidazole alkaloids: Ab initio calculations, tautomerism, and reactivity. Org. Lett. 2004, 6, 3933-3936.

References for Chapter 3

1. Berree, F.; Michelot, G.; Le Corre, M., N-Boc ethyl oxamate: a new nitrogen nucleophile for use in Mitsunobu reactions. Tetrahedron Lett. 1998, 39, 8275-8276.

2. Denmark, S. E.; Schnute, M. E., Nitroalkene [4+2]-Cycloadditions with 2- (Acyloxy)Vinyl Ethers - Stereoselective Synthesis of 3-Hydroxy-4-Substituted- Pyrrolidines. J. Org. Chem. 1994, 59, 4576-4595.

3. Travis, B. R.; Sivakumar, M.; Hollist, G. O.; Borhan, B., Facile oxidation of aldehydes to acids and esters with oxone. Org. Lett. 2003, 5, 1031-1034.

4. Dagnall, S. P.; Hague, D. N.; McAdam, M. E., C-13 Nuclear Magnetic-Resonance Study of the Protonation Sequence of Some Linear Aliphatic Polyamines. J. Chem. Soc. Perk. T. 2 1984, 1111-1114.

References for Chapter 4

1. Rubin, L. L.; Staddon, J. M., The cell biology of the blood-brain barrier. Annu. Rev. Neurosci. 1999, 22, 11-28.

2. Ballabh, P.; Braun, A.; Nedergaard, M., The blood-brain barrier: an overview - Structure, regulation, and clinical implications. Neurobiol. Dis. 2004, 16, 1-13.

3. Demeule, M.; Regina, A.; Jodoin, J.; Laplante, A.; Dagenais, C.; Berthelet, F.; Moghrabi, A.; Beliveau, R., Drug transport to the brain: Key roles for the efflux pump P- glycoprotein in the blood-brain barrier. Vasc. Pharmacol. 2002, 38, 339-348.

4. Schinkel, A. H.; Jonker, J. W., Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv. Drug Deliv. Rev. 2003, 55, 3-29.

295

5. Clark, R. D.; Wolohan, P. R. N., Molecular design and bioavailability. Curr. Top. Med. Chem. 2003, 3, 1269-1288.

6. Clark, D. E., In silico prediction of blood-brain barrier permeation. Drug Discov. Today 2003, 8, 927-933.

7. Pardridge, W. M., Crossing the blood-brain barrier: are we getting it right? Drug Discov. Today 2001, 6, 1-2.

8. Liu, X. R.; Chen, C. P., Strategies to optimize brain penetration in drug discovery. Curr. Opin. Drug Disc. 2005, 8, 505-512.

9. Liu, X. R.; Smith, B. J.; Chen, C. P.; Callegari, E.; Becker, S. L.; Chen, X.; Cianfrogna, J.; Doran, A. C.; Doran, S. D.; Gibbs, J. P.; Hosea, N.; Liu, J. H.; Nelson, F. R.; Szewc, M. A.; Van Deusen, J., Use of a physiologically based pharmacokinetic model to study the time to reach brain equilibrium: An experimental analysis of the role of blood-brain barrier permeability, plasma protein binding, and brain tissue binding. J. Pharmacol. Exp. Ther. 2005, 313, 1254-1262.

10. Doran, A.; Obach, R. S.; Smith, B. J.; Hosea, N. A.; Becker, S.; Callegari, E.; Chen, C. P.; Chen, X.; Choo, E.; Cianfrogna, J.; Cox, L. M.; Gibbs, J. P.; Gibbs, M. A.; Hatch, H.; Hop, C. E. C. A.; Kasman, I. N.; LaPerle, J.; Liu, J. H.; Liu, X. R.; Logman, M.; Maclin, D.; Nedza, F. M.; Nelson, F.; Olson, E.; Rahematpura, S.; Raunig, D.; Rogers, S.; Schmidt, K.; Spracklin, D. K.; Szewc, M.; Troutman, M.; Tseng, E.; Tu, M. H.; Van Deusen, J. W.; Venkatakrishnan, K.; Walens, G.; Wang, E. Q.; Wong, D.; Yasgar, A. S.; Zhang, C. H., The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: Evaluation using the MDR1A/1B knockout mouse model. Drug Metab. Dispos. 2005, 33, 165-174.

11. Pardridge, W. M., Blood-brain barrier genomics and the use of endogenous transporters to cause drug penetration into the brain. Curr. Opin. Drug Disc. 2003, 6, 683-691.

12. Matsuo, H.; Tsukada, S.; Nakata, T.; Chairoungdua, A.; Kim, D. K.; Cha, S. H.; Inatomi, J.; Yorifuji, H.; Fukuda, J.; Endou, H.; Kanai, Y., Expression of a system L neutral amino acid transporter at the blood-brain barrier. Neuroreport 2000, 11, 3507-3511.

13. Uchino, H.; Kanai, Y.; Kim, D. K.; Wempe, M. F.; Chairoungdua, A.; Morimoto, E.; Anders, M. W.; Endou, H., Transport of amino acid-related compounds mediated by L- type amino acid transporter 1 (LAT1): Insights into the mechanisms of substrate recognition. Mol. Pharmacol. 2002, 61, 729-737.

14. Tamai, I.; Tsuji, A., Transporter-mediated permeation of drugs across the blood-brain barrier. J. Pharm. Sci. 2000, 89, 1371-1388.

296

15. Markovitz, D. C.; Fernstrom, J. D., Diet and Uptake of Aldomet by Brain - Competition with Natural Large Neutral Amino-Acids. Science 1977, 197, 1014-1015.

16. Doan, K. M. M.; Humphreys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L. J.; Serabjit-Singh, C. J.; Adkison, K. K.; Polli, J. W., Passive permeability and P- glycoprotein-mediated efflux differentiate central nervous system (CNS) and non-CNS marketed drugs. J. Pharmacol. Exp. Ther. 2002, 303, 1029-1037.

17. Lipinski, C., Drug Discovery Development and Delivery for Neurodegenerative Diseases. 2007.

18. Zhao, Y. H.; Abraham, M. H.; Ibrahim, A.; Fish, P. V.; Cole, S.; Lewis, M. L.; de Groot, M. J.; Reynolds, D. P., Predicting penetration across the blood-brain barrier from simple descriptors and fragmentation schemes. J. Chem. Inf. Model 2007, 47, 170-175.

19. Lewis, D. F. V.; Lake, B. G.; Ito, Y.; Dickins, M., Lipophilicity relationships in inhibitors of CYP2C9 and CYP2C19 enzymes. J. Enzym. Inhib. Med. Ch. 2006, 21, 385-389.

20. Yabuki, M.; Mine, T.; Iba, K.; Nakatsuka, I.; Yoshitake, A., Pharmacokinetics of Sm- 10888 and Its Metabolites Depending on Their Physicochemical Properties. Drug Metab. Dispos. 1994, 22, 294-297.

21. Hu, L. Q., Prodrugs: Effective Solutions for Solubility Permeability and Targeting Challenges - 28-29 June 2004, Philadelphia, PA, USA. Idrugs 2004, 7, 736-742.

22. Abraham, M. H.; Ibrahim, A.; Zhao, Y.; Acree, W. E., A data base for partition of volatile organic compounds and drugs from blood/plasma/serum to brain, and an LFER analysis of the data. J. Pharm. Sci. 2006, 95, 2091-2100.

23. Nicolazzo, J. A.; Charman, S. A.; Charman, W. N., Methods to assess drug permeability across the blood-brain barrier. J. Pharm. Pharmacol. 2006, 58, 281-293.

24. Cundy, K. C.; Branch, R.; Chernov-Rogan, T.; Dias, T.; Estrada, T.; Hold, K.; Koller, K.; Liu, X. L.; Mann, A.; Panuwat, M.; Raillard, S. P.; Upadhyay, S.; Wu, Q. Q.; Xiang, J. N.; Yan, H.; Zerangue, N.; Zhou, C. X.; Barrett, R. W.; Gallop, M. A., XP13512 [(+/-)-1- ([(alpha-isobutanoyloxyethoxy)carbonyl] aminomethyl)-1-cyclohexane acetic acid], a novel gabapentin prodrug: I. Design, synthesis, enzymatic conversion to gabapentin, and transport by intestinal solute transporters. J. Pharmacol. Exp. Ther. 2004, 311, 315-323.

25. Anderson, B. D., Prodrugs for improved CNS delivery. Adv. Drug Deliver. Rev. 1996, 19, 171-202.

26. Jong, A. S.-H. H., Blood-brain barrier drug discovery for central nervous system infections. Current drug targets- infectious disorders 2005, 5, 65-72.

297

27. May, E. L.; Jacobson, A. E., Chemistry and Pharmacology of Homologs of 6-Acetyl- Morphine and 3,6-Diacetylmorphine. J. Pharm. Sci. 1977, 66, 285-286.

28. Henin, Y.; Gouyette, C.; Schwartz, O.; Debouzy, J. C.; Neumann, J. M.; Huynhdinh, T., Lipophilic Glycosyl Phosphotriester Derivatives of Azt - Synthesis, Nmr Transmembrane Transport Study, and Antiviral Activity. J. Med. Chem. 1991, 34, 1830-1837.

29. Namane, A.; Gouyette, C.; Fillion, M. P.; Fillion, G.; Huynhdinh, T., Improved Brain Delivery of AZT Using a Glycosyl Phosphotriester Prodrug. J. Med. Chem. 1992, 35, 3039-3044.

30. Jones, D. B.; Rustgi, V. K.; Kornhauser, D. M.; Woods, A.; Quinn, R.; Hoffnagle, J. H.; Jones, E. A., The Disposition of 6-Deoxyacyclovir, a Xanthine Oxidase-Activated Prodrug of Acyclovir, in the Isolated Perfused-Rat-Liver. Hepatology 1987, 7, 345-348.

31. Morgan, M. E.; Chi, S. C.; Murakami, K.; Mitsuya, H.; Anderson, B. D., Central- Nervous-System Targeting of 2',3'-Dideoxyinosine Via Adenosine Deaminase-Activated 6-Halo-Dideoxypurine Prodrugs. Antimicrob. Agents Ch. 1992, 36, 2156-2165.

32. Anderson, B. D.; Hoesterey, B. L.; Baker, D. C.; Galinsky, R. E., Kinetics of ddI in Plasma, Brain, and CSF of Rats after Administration of ddI and an Ester Prodrug of ddI. Ann. NY Acad. Sci. 1990, 616, 472-474.

33. Bodor, N.; Farag, H. H., Improved Delivery through Biological-Membranes .11. A Redox Chemical Drug-Delivery System and Its Use for Brain-Specific Delivery of Phenylethylamine. J. Med. Chem. 1983, 26, 313-318.

34. Perioli, L.; Ambrogi, V.; Bernardini, C.; Grandolini, G.; Ricci, M.; Giovagnoli, S.; Rossi, C., Potential prodrugs of non-steroidal anti-inflammatory agents for targeted drug delivery to the CNS. Eur. J. Med. Chem. 2004, 39, 715-727.

35. Bodor, N.; Prokai, L.; Wu, W. M.; Farag, H.; Jonalagadda, S.; Kawamura, M.; Simpkins, J., A Strategy for Delivering Peptides into the Central-Nervous-System by Sequential Metabolism. Science 1992, 257, 1698-1700.

36. Simpkins, J. W.; Mccornack, J.; Estes, K. S.; Brewster, M. E.; Shek, E.; Bodor, N., Sustained Brain-Specific Delivery of Estradiol Causes Long-Term Suppression of Luteinizing-Hormone Secretion. J. Med. Chem. 1986, 29, 1809-1812.

37. Bodor, N.; Farag, H. H., Improved Delivery through Biological-Membranes .13. Brain- Specific Delivery of Dopamine with a Dihydropyridine-Reversible-Pyridinium Salt Type Redox Delivery System. J. Med. Chem. 1983, 26, 528-534.

298

38. Alexander, J.; Cargill, R.; Michelson, S. R.; Schwam, H., (Acyloxy)Alkyl Carbamates as Novel Bioreversible Prodrugs for Amines - Increased Permeation through Biological- Membranes. J. Med. Chem. 1988, 31, 318-322.

39. Rahmathullah, S. M.; Hall, J. E.; Bender, B. C.; McCurdy, D. R.; Tidwell, R. R.; Boykin, D. W., Prodrugs for amidines: Synthesis and anti-Pneumocystis carinii activity of carbamates of 2,5-bis(4-amidinophenyl)furan. J. Med. Chem. 1999, 42, 3994-4000.

40. Davidsen, S. K.; Summers, J. B.; Albert, D. H.; Holms, J. H.; Heyman, H. R.; Magoc, T. J.; Conway, R. G.; Rhein, D. A.; Carter, G. W., N-(Acyloxyalkyl)Pyridinium Salts as Soluble Prodrugs of a Potent Platelet-Activating-Factor Antagonist. J. Med. Chem. 1994, 37, 4423-4429.

41. Kotra, L. P.; Manouilov, K. K.; CrettonScott, E.; Sommadossi, J. P.; Boudinot, F. D.; Schinazi, R. F.; Chu, C. K., Synthesis, biotransformation, and pharmacokinetic studies of 9-(beta-D-arabinofuranosyl)-6-azidopurine: A prodrug for ara-A designed to utilize the azide reduction pathway. J. Med. Chem. 1996, 39, 5202-5207.

42. Kotra, L. P.; Manouilov, K. K.; Sommadossi, J. P.; Boudinot, D. F.; Schinazi, R. F.; Chu, C. K., A novel approach in the design of prodrugs: 6-azido-ara-purine (6-AAP) as a prodrug of ara-A with enhanced brain delivery. Abstr. Pap. Am. Chem. S. 1996, 211, 16- Medi.

43. Koudriakova, T.; Manouilov, K. K.; Shanmuganathan, K.; Kotra, L. P.; Boudinot, F. D.; CrettonScott, E.; Sommadossi, J. P.; Schinazi, R. F.; Chu, C. K., In vitro and in vivo evaluation of 6-azido-2',3'-dideoxy-2'-fluoro-beta-D-arabinofuranosylpurine and N-6- methyl-2',3'-dideoxy-2'-fluoro-beta-D-arabinofuranosyladenine as prodrugs of the anti- HIV nucleosides 2'-F-ara-ddA and 2'-F-ara-ddI. J. Med. Chem. 1996, 39, 4676-4681.

44. Fayz, S.; Inaba, T., Zidovudine azido-reductase in human liver microsomes: Activation by ethacrynic acid, dipyridamole, and indomethacin and inhibition by human immunodeficiency virus protease inhibitors. Antimicrob. Agents Ch. 1998, 42, 1654-1658.

45. Inaba, T.; Fayz, S., AZT (zidovudine) metabolism by human liver: Inhibition and activation of toxic metabolite formation in vitro. Clin. Pharmacol. Ther. 1997, 61, 58-58.

46. Damen, E. W. P.; Nevalainen, T. J.; van den Bergh, T. J. M.; de Groot, F. M. H.; Scheeren, H. W., Synthesis of novel paclitaxel prodrugs designed for bioreductive activation in hypoxic tumour tissue. Bioorgan. Med. Chem. 2002, 10, 71-77.

47. Li, Z.; Bitha, P.; Lang, S. A.; Lin, Y. I., Synthesis of (alkoxycarbonyloxy)methyl, (acyloxy)methyl and (oxodioxolenyl)methyl carbamates as bioreversible prodrug moieties for amines. Bioorg. Med. Chem. Lett. 1997, 7, 2909-2912.

299

48. Alexander, J.; Bindra, D. S.; Glass, J. D.; Holahan, M. A.; Renyer, M. L.; Rork, G. S.; Sitko, G. R.; Stranieri, M. T.; Stupienski, R. F.; Veerapanane, H.; Cook, J. J., Investigation of (oxodioxolenyl)methyl carbamates as nonchiral bioreversible prodrug moieties for chiral amines. J. Med. Chem. 1996, 39, 480-486.

49. Zhang, Q.; Guan, J.; Sacci, J.; Ager, A.; Ellis, W.; Milhous, W.; Kyle, D.; Lin, A. J., Unambiguous synthesis and prophylactic antimalarial activities of imidazolidinedione derivatives. J. Med. Chem. 2005, 48, 6472-6481.

50. Sun, X. C.; Rodriguez, M.; Zeckner, D.; Sachs, B.; Current, W.; Boyer, R.; Paschal, J.; McMillian, C.; Chen, S. H., Synthesis and evaluation of oxodioxolenylmethyl carbamate prodrugs of pseudomycins. J. Med. Chem. 2001, 44, 2671-2674.

51. http://www.molinspiration.com/cgi-bin/properties

52. van de Waterbeemd, H.; Camenisch, G.; Folkers, G.; Chretien, J. R.; Raevsky, O. A., Estimation of blood-brain barrier crossing of drugs using molecular size and shape, and H-bonding descriptors. J. Drug Target 1998, 6, 151-165.

53. Kelder, J.; Grootenhuis, P. D. J.; Bayada, D. M.; Delbressine, L. P. C.; Ploemen, J. P., Polar molecular surface as a dominating determinant for oral absorption and brain penetration of drugs. Pharmaceut. Res. 1999, 16, 1514-1519.

54. Norinder, U.; Haeberlein, M., Computational approaches to the prediction of the blood- brain distribution. Adv. Drug Deliver. Rev. 2002, 54, 291-313.

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Appendix 1: HPLC spectra for final compounds HPLC spectra were obtained using the instrument and methods described in Chapter 4, Experimental Procedures.

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III-2

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III-6

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IV-22

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