The Pennsylvania State University

The Graduate School

The Integrative Biosciences Graduate Program

ADVANCES IN SYNTHESIS AND APPLICATIONS OF ARTIFICIAL CELL

SURFACE RECEPTORS AND METHODOLOGY FOR PREPARATION OF

NOVEL ANTIVIRAL AGENTS

A Dissertation in

Integrative Biosciences

by

Qi Sun

© 2008 Qi Sun

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

December 2008

The dissertation of Qi Sun was reviewed and approved* by the following:

Blake R. Peterson Regents Distinguished Professor of Medicinal Chemistry University of Kansas Dissertation Co-Advisor Co-chair of Committee Special Member

J. Martin Bollinger, Jr. Professor of Biochemistry and Molecular Biology Professor of Chemistry Dissertation Co-Advisor Co-Chair of Committee

Avery August Professor of Immunology

Gong Chen Assistant Professor of Chemistry

Peter Hudson Willaman Professor of Biology Director of Huck Institutes of Life Sciences

*Signatures are on file in the Graduate School

iii

ABSTRACT

In the past decade, our group has developed a novel technology that can

be used to dramatically enhance uptake of cell impermeable macromolecules by

mammalian cells. By mimicking small natural cell surface receptors, synthetic

receptors, composed of N-alkyl-3β-cholesterylamine membrane anchors linked to

protein-binding motifs, can be installed on cell surfaces. Mechanistic studies

have revealed that similar to certain natural cell surface receptors, the synthetic

receptors internalize cargo molecules through a clathrin-mediated endocytic

pathway. However, the lack of an efficient method for the synthesis of 3β-

cholesterylamine has limited the development of this technology. Additionally,

trapping of molecules delivered by synthetic receptors in confined intracellular

compartments, endosomes, restricts potential applications of this delivery system.

To address these issues, we developed novel and practical methodologies for

the large-scale preparation of 3β-cholesterylamine and related building blocks;

we also designed and synthesized novel 3β-cholesterylamine-capped PC4 lytic

peptides, which enable 3β-cholesterylamine-conjugated cargo bearing a disulfide

linkage to selectively escape from early/recycling endosomes of living

mammalian cells. Furthermore, study of the interactions of C-reactive protein with

phosphocholine-containing synthetic receptors led us to the discovery of a novel

method to promote apoptosis in Jurkat lymphocytes.

The other area of my research focused on the development of novel

methodologies for the preparation of antiviral agents and their phosphoylated

iv

metabolites. A novel one-pot approach for the synthesis of nucleoside 5’-

triphosphates from nucleoside 5’-H-phosphonate monoesters was explored. We

demonstrated that fully deprotected 5’-H-phosphonates of antiviral nucleosides

can be converted via pyridinium phosphoramidate intermediates to the

corresponding 5’-triphosphate products, which can be readily purified using a

two-step purification protocol. We also constructed a small 39-member library

based on a vinylbenzimidazolium Akt inhibitor through the discovery of a novel

and efficient Zr(IV)-catalyzed cyclization of substituted 1,2-arylenediamines and

α,β-unsaturated aldehydes.Biological screening of the new compound library

identifed four compounds with greater antiviral activity than the lead compound.

v

TABLE OF CONTENTS

LIST OF FIGURES .........................................................................................viii

LIST OF TABLES ...........................................................................................xiv

ACKNOWLEDGEMENTS...............................................................................xv

Chapter 1 Synthetic Mimics of Cell Surface Receptors .................................1

1.1 Synthetic Receptor Targeting as a Novel Delivery Strategy ..............1 1.2 Synthetic Cell Surface Receptors as Probes of Cellular Biology .......9 1.3 Current Limitations of Synthetic Receptor Targeting .........................11 1.4 Outline of This Dissertion...................................................................13 1.5 References ........................................................................................14

Chapter 2 Novel and Practical Synthesis of 3β-Amino-5-Cholestene (3β-Cholesterylamine), 3β-Amino-5α-Cholestane, and Related Anchor Building Blocks.........................................................................................19

2.1 Introduction........................................................................................19 2.2 A Novel Method for Conversion of Cholest-5-En-3β-Ol,

Methanesulfonate (21) to 3β-Azido-Cholest-5-Ene (18) ....................22 2.3 Effects of Leaving Groups and Solvents............................................25 2.4 A Novel and Efficient Route for Large-Scale Preparation of 3β-

Cholesterylamine (2)..........................................................................26 2.5 Study of the Reaction Mechanism by 1H NMR ..................................27 2.6 Novel Synthesis of Related 3β-Cholesterylhalides ............................30 2.7 A Modified Synthetic Route to 5α-Cholestane-3β-Amine (34) ...........31 2.8 Novel and Practical Synthesis of Boc-Protected Anchors Derived

from 5α-Cholestane-3β-Amine (34) and 3β-Cholesterylamine (2) .....33 2.9 Conclusions .......................................................................................35 2.10 Experimental Section .......................................................................36

2.10.1 General ......................................................................................................... 36 2.10.2 Synthetic Procedures and Compound Characterization

Data ..................................................................................................................... 37 2.10.3 1H NMR Spectra of the Conversion of 6β-Azido-3α,5-Cyclo-

5α-Cholestane (23) to 3β-Azido-5-Cholestene (18). ........................ 54 2.11 References .............................................................................................................. 57

Chapter 3 Selective Disruption of Early/Recycling Endosomes: Release of Disulfide-Linked Cargo Mediated by N-Alkyl-3β-Cholesterylamine-Capped Lytic Peptides .............................................................................64

vi

3.1 Introduction........................................................................................64 3.2 Design and Synthesis of N-Alkyl-3β-Cholesterylamine-Capped

Lytic Peptides (45, 46) and a Disulfide-Linked Fluorescent Probe (47) ....................................................................................................66

3.3 Confocal Microscopy of Early/Recycling Endosome Targeting of the Fluorescent N-Alkyl-3β-Cholesterylamine-Capped Lytic Peptide (46) and the Disulfide-Linked Probe (47) ..............................69

3.4 Confocal Microscopy of Endosome Disruption by the N-Alkyl-3β-Cholesterylamine-Capped Lytic Peptides (45, 46) and the Disulfide-Linked Fluorescent Probe (47)............................................71

3.5 Flow Cytometry of Endosome Disruption by the N-Alkyl-3β-Cholesterylamine-Capped Lytic Peptide (45) and the Disulfide-Linked Fluorescent Probe (47) ..........................................................74

3.6 Evaluation of Cytotoxicity of the N-Alkyl-3β-Cholesterylamine-Capped Lytic Peptide (45) .................................................................76

3.7 A Binary Drug Delivery System: Targeting a Disulfide-Linked Cytotoxin (52) to Endosomes and Activation by N-Alkyl-3β-Cholesterylamine-Capped Lytic Peptide (45) ....................................77

3.8 Conclusions .......................................................................................82 3.9 Experimental Section.........................................................................83

3.9.1 General............................................................................................................ 83 3.9.2 Synthetic Procedures and Compound Characterization Data ... 85 3.9.3 Biological Assays and Protocols ........................................................... 92

3.10 References .............................................................................................................. 95

Chapter 4 Mimicry of Exposed Phosphatidylcholine on Damaged Cells: Synthetic Cell Surface Receptors that Bind C-Reactive Protein Promote Apoptosis of Lymphocytes.........................................................103

4.1 Introduction........................................................................................103 4.2 Design and Synthesis of PC-containing Synthetic Receptors (70-

73) .....................................................................................................105 4.3 Cytotoxic effect of CRP to Jurkat Lymphocytes loaded with

Synthetic Receptors (73) ...................................................................108 4.4 Characterization of Cell Death Induced by 73 and CRP....................109 4.5 Dependence of Apoptosis and Viability on 73 and CRP....................115 4.6 Structure-Activity Relationships (SAR) of Synthetic Receptors (70-

73) .....................................................................................................117 4.7 Mechanisms of Apoptosis Induced by 73 and CRP...........................118 4.8 Conclusions .......................................................................................122 4.9 Experimental Section.........................................................................122

4.9.1 General............................................................................................................ 122 4.9.2 Synthetic Procedures and Compound Characterization Data ... 123 4.9.3 Biological Assays and Protocols ........................................................... 132

vii

4.10 References .............................................................................................................. 137

Chapter 5 A One-Pot Synthesis of Nucleoside 5’-Triphosphates from Nucleoside 5’-H-Phosphonates................................................................145

5.1 Introduction........................................................................................145 5.2 Synthesis of Nucleoside 5’-H-Phosphonates (83-88) ........................148 5.3 Synthesis and Purification of Nucleoside 5’-Triphosphates (96-101)

...........................................................................................................149 5.4 Mechanistic Studies of the Reaction by 31P NMR..............................150 5.5 Mass Spectrometric Study of Side-Products and Optimization of

Reaction Conditions...........................................................................152 5.6 Biological Activity of 6MePTP (101) Synthesized from 5’-H-

Phosphonate (88) ..............................................................................154 5.7 Conclusions .......................................................................................156 5.8 Experimental Section.........................................................................156

5.8.1 General............................................................................................................ 156 5.8.2 Synthetic Procedures and Compound Characterization Data ... 158 5.8.3 Biological Assays......................................................................172

5.9 References ........................................................................................173

Chapter 6 Synthesis of an Akt Inhibitor and Its Analogues via a Novel and Efficient Zr(IV)-Catalyzed Cyclization Reaction and Evaluation of Their Antiviral Activity ........................................................................................178

6.1 Introduction........................................................................................178 6.2 Synthesis of Lead Compound 111.....................................................183 6.3 A Novel Zr(IV)-Catalyzed Synthetic Method for Preparation of

Vinylbenzimidazoles ..........................................................................185 6.4 An Interesting Transamination Reaction on 111 ................................189 6.5 Design and Synthesis of a Benzimidazole-Based Compound

Library................................................................................................190 6.6 Biological Evaluation of Compound Library and Structure-Activity

Relationship (SAR) Analysis..............................................................196 6.7 Conclusions .......................................................................................200 6.8 Experimental Section.........................................................................201

6.8.1 General............................................................................................................ 201 6.8.2 Synthetic Procedures and Compound Characterization Data ... 202 6.8.3 Biological Assays......................................................................................... 251

6.9 References ........................................................................................251

viii

LIST OF FIGURES

Figure 1.1: Receptor-mediated endocytosis of the LDL. Panel A: LDL particle. Panel B: LDL receptor. Panel C: Receptor-mediated endocytotic pathway.................................................................................5

Figure 1.2: Structures of a natural cell surface receptor, ganglioside GM1 (1), 3β-cholesterylamine (2), and synthetic cell surface receptors (3-11).................................................................................................................7

Figure 1.3: A model of synthetic receptor-mediated endocytosis. ..................8

Figure 1.4: Structures of 3β-cholesterylamine-based fluorescent probes (12-14). ....................................................................................................9

Figure 1.5: Enhancement of membrane association by installation of negatively charged glutamic acid residues to the linker region. Panel A. Confocal microscopic analysis of membrane association of 13 and 14. Panel. B: Flow cytometric analysis of membrane association of 13 and 14. ............................................................................................................10

Figure 1.6: Previously reported synthesis of 3β-cholesterylamine (2) and its Nosyl-protected derivative (19)............................................................12

Figure 1.7: Confocal and DIC microscopy of entrapment of the delivered molecules in endosomes, illustrated by delivery of human IgG-AF488 by receptor 8 in Jurkat lymphocytes.........................................................13

Figure 2.1: Previoiusly reported routes to 3β-cholesterylamine (2) and proposed alternative synthetic route.. ......................................................20

Figure 2.2: A novel and stereoselective synthesis of 3β-azido-5-cholestene (18). .......................................................................................21

Figure 2.3: Optimized synthetic route for large-scale preparation of 3β-cholesterylamine ......................................................................................27

Figure 2.4: The mechanism of the transformation cholest-5-en-3β-ol, methanesulfonate (21) to 3β-azido-5-cholestene (18) monitored by 1H NMR Panel A: Stacked 1H NMR spectra of the reaction at different time points. Panel B: Proposed mechanism.............................................28

Figure 2.5: A modified route for the synthesis of 5α-cholestane-3β-amine (34)...........................................................................................................32

ix

Figure 2.6: A reductive amination method for the synthesis of a Boc-protected membrane anchor with an ethylvalerate linker (38)..................33

Figure 2.7: A new method for the synthesis of Boc-protected membrane anchors.. ..................................................................................................34

Figure 2.8: Compound 23 and TMSN3 at 0 min..............................................55

Figure 2.9: Compound 23 and TMSN3 plus BF3·Et2O at 20 min. ....................56

Figure 2.10: Compound 23 and TMSN3 plus BF3·Et2O at 2 h.. .......................57

Figure 3.1: Structures of N-alkyl-3β-cholesterylamine-capped lytic peptides (45, 46), a disulfide-Linked fluorescent probe (47), and its non-cleavable control (48)........................................................................67

Figure 3.2: Proposed molecular mechanism for the selective release of disulfide-linked fluorophore from early/recycling endosomes mediated by 45. Panel A: Products of cleavage of 47 by glutathione. Panel B: Mechanism of release of fluorophore 50 into the cytosol and nucleus of mammalian cells. .................................................................................69

Figure 3.3: Confocal laser scanning and differential interference contrast (DIC) micrographs of living CHO cells treated with green and red fluorescent compounds. Panel A: Cells treated with green fluorescent 47 (5 µM) for 12 h followed by red fluorescent Texas Red transferrin (500 nM) for 5 min at 37 °C. Panel B: Cells treated with green fluorescent 47 (5 µM) for 12 h followed by red fluorescent DiI-LDL (8 nM) for 5 min at 37 °C. Panel C: Cells treated with red fluorescent 46 (5 µM) for 12 h followed by green fluorescent Alexa Fluor 488 transferrin (610 nM) for 5 min at 37 °C. Panel D: Cells treated with red fluorescent 46 (2 µM) and green fluorescent 47 (5 µM) for 12 h at 37 °C.. ...........................................................................................................70

Figure 3.4: Confocal laser scanning and DIC micrographs of living Jurkat lymphocytes treated with N-alkyl-3β-cholesterylamine-capped lytic peptides and fluorescent probes. Panel A: Jurkat lymphocytes were treated with 47 only (2.5 µM) for 12 h at 37 °C. Panel B: Jurkat lymphocytes were treated with 45 (2 µM) and 47 (2.5 µM) for 12 h at 37 °C. Panel C: Jurkat lymphocytes were treated with 46 (2 µM) and 47 (2.5 µM) for 12 h at 37 °C. Panel D: Jurkat lymphocytes were treated with 45 (2 µM) and 48 (2.5 µM) for 12 h at 37 °C. Panel E: Jurkat lymphocytes were treated with 49 (2 µM) and 47 (2.5 µM) for 12 h at 37 °C.. ..........................................................................................72

x

Figure 3.5: Inhibition of endosomal escape with bafilomycin A1.....................73

Figure 3.6: Dose dependence and time dependence of endosome disruption quantified by flow cytometry. Panel A: Jurkat lymphocytes were treated with 47 (2.5 µM) and 45 or 49 for 12 h at 37 °C. Panel B: Jurkat lymphocytes were treated with 47 (2.5 µM) and 45 (2 µM) at 37 °C. ............................................................................................................75

Figure 3.7: Cytotoxicity of 45 to Jurkat lymphocytes.......................................76

Figure 3.8: Synthesis of colchicine-cholesterylamine conjugates (52, 53)......78

Figure 3.9: Cytotoxicity to Jurkat lymphocytes of 52 in the presence or absence of 45 (2 µM) and controls (61, 53) .............................................79

Figure 3.10: DIC micrographs of living Jurkat lymphocytes treated with colchicine-cholesterylamine conjugate 52 in the presence or absence of PC4 lipopeptide 45. Panel A: Jurkat lymphocytes were treated with 52 only (50 nM to 500 nM) for 48 h at 37 °C. Panel B: Jurkat lymphocytes were treated with 52 (50 nM to 500 nM) in the presence of 45 (2 µM) for 48 h at 37 °C...................................................................80

Figure 3.11: Proposed activation of prodrugs (52, 62) upon endosome disruption. Panel A: Cleavage of conjugate 52 enables release of cytotoxic colchicine derived headgroup 62. Panel B: Cleavage of designed conjugate 63 enables release of highly potent cytotoxin, colchifoline (65), via an intramolecular cyclization reaction......................81

Figure 3.12: Analytical HPLC profile of the peptide 49 after purification by preparative HPLC.....................................................................................89

Figure 3.13: Analytical HPLC profile of compound 45 after purification by preparative HPLC.....................................................................................90

Figure 3.14: Analytical HPLC profile of compound 46 after purification by preparative HPLC.....................................................................................92

Figure 4.1: Structure of CRP. Panel A. Side view of CRP and proposed interaction with C1q. Panel B. Bottom view of CRP and PC-binding sites..........................................................................................................104

Figure 4.2: Structures of synthetic receptors (70-73) that mimic phosphatidylcholine..................................................................................106

Figure 4.3: Synthesis of receptors (70-73) that mimic phosphatidylcholine....107

xi

Figure 4.4: Confocal micrographs of Jurkat lymphocytes treated with 73 and CRP-AF488.......................................................................................108

Figure 4.5: DIC micrographs of the cytotoxic effect of 73, CRP, and 73 and CRP on Jurkat lymphocytes. ....................................................................109

Figure 4.6: Morphological study of cell death induced by 73 and CRP. Panel A: Established morphological features of two distinctive types of cell death, necrosis and apoptosis. Panel B: Morphological features of Jurkat cells treated with 73 (1 µM) for 1 h followed by CRP (10 µg/mL) for 6 h at 37 °C.. .......................................................................................110

Figure 4.7: Confocal microscopy of phosphatidylserine (PS) exposure and pemeability of the membrane of dying cells induced by 73 and CRP. Panel A: Illustrations of the membrane features of live, apoptotic, and dead cells. Panel B: Confocal micrographs of Jurkat cells (treated with 73 (1 µM) for 1 h followed by CRP (10 µg/mL) for 6 h at 37 °C) stained with annexin V-AF488 and propidium iodide (PI). ....................................111

Figure 4.8: Flow cytometry of phosphatidylserine (PS) exposure and pemeability of the membrane of dying cells induced by 73 and CRP. Panel A: Dot plot of negative control Jurkat cells (treated with 1% DMSO for 1 h and media for 6 h at 37 °C) stained with annexin V-AF488 and PI. Panel B: Dot plot of Jurkat cells (treated with 73 (1 µM) for 1 h followed by CRP (10 µg/mL) for 6 h at 37 °C) stained with annexin V-AF488 and PI.. ........................................................................112

Figure 4.9: DNA Fragmentation assay. ..........................................................113

Figure 4.10: Annexin V-binding assays. Panel A: Dose-dependence of 73 (0-2 µM). Panel B: Dose-dependence of CRP (0-10 µg/mL)....................114

Figure 4.11: CellTiter-Glo luminescent cell viability assays. Panel A: Dose-dependence of 73 (0-2 µM). Panel B: Dose-dependence of CRP (0-10 µg/mL)......................................................................................................116

Figure 4.12: Structure-activity relationships (SAR) of receptors (70-73) in inducing Jurkat cell apoptosis.. ................................................................117

Figure 4.13: Effect of complement proteins in FBS on cell apoptosis induced by 73 and CRP. ..........................................................................118

Figure 4.14: Membrane association of CRP-AF488 and corresponding cell aggregation and apoptosis.......................................................................119

xii

Figure 4.15: Caspase inhibition assay by confocal microscopy......................120

Figure 4.16: Caspase inhibition assay by flow cytometry. ..............................121

Figure 5.1: Commonly used methods for the synthesis of NTPs. Panel A: The “one-pot, three-step” method. Panel B: Eckstein’s method...............146

Figure 5.2: The hypothesis of using nucleoside 5’-H-phosphonates to synthesize NTPs via a phosphoramdiate intermediate. Panel A: Borch’s new method via a pyrrolidinium phosphoramidate intermediate. Panel B: Our hypothesis involving use of a similar pyridinium phosphoramidate intermeidate to synthesize NTPs. ................................147

Figure 5.3: Synthesis of nucleoside 5’-H-phosphonates (89-94). ...................148

Figure 5.4: A one-pot method for conversion of nucleoside 5’-H-phosphonates (89-94) to nucleoside 5’-triphosphates (96-101). ..............149

Figure 5.5: Analysis of sequential conversion of H-phosphonate monoester 83 to triphosphate 105 by 31P NMR. Panel A: Synthetic route illustrating measured 31P chemical shifts of reagents, proposed intermediates, and product. Panel B: 31P NMR spectra of compounds prior to and after sequential addition of reagents shown in panel A. ........151

Figure 5.6: Mass spectral analysis of the reaction mixture obtained after conversion of H-phosphonate 13 to triphosphate 20, using 3 equiv of TMSCl, followed by partial purification by gel filtration (Sephadex LH-20). Panel A: Structures of compounds detected by mass spectrometry. Panel B: Mass spectrum obtained with a Waters ZQ-4000 single quadrupole instrument using electrospray ionization (negative-ion mode). ................................................................................153

Figure 5.7: Evaluation of 6-methylpurine ribonucleoside triphosphate (101) as a substrate of 3Dpol from poliovirus......................................................155

Figure 5.8: Analytical HPLC profile of 6MePTP (101) after purification by preparative HPLC.....................................................................................171

Figure 6.1: PI3K/Akt signaling pathway.. ........................................................179

Figure 6.2: A model for the involvement of Akt in viral RNA synthesis. ..........181

Figure 6.3: Structure of an Akt inhibitor, compound 111.................................182

Figure 6.4: Synthesis of lead compound 111.. ...............................................184

xiii

Figure 6.5: Scope of the Zr(IV)-catalyzed cyclization reaction........................189

Figure 6.6: Transamination reactions on vinylbenzimidazolium compound 111 and proposed mechanism.. ...............................................................190

Figure 6.7: Core benzimidazole and variations in the compound library. .......191

Figure 6.8: Structures of analogues of 111 in Group I. ...................................192

Figure 6.9: Synthesis and structures of analogues of 111 in Group II (158-163)..........................................................................................................193

Figure 6.10: Synthesis and structures of analogues of 111 in Group II (170-171) .................................................................................................194

Figure 6.11: Synthesis and structures of analogues of 111 in Group III (184-191).. ...............................................................................................195

Figure 6.12: Synthesis and structures of analogues of 111 in Group IV (192-194). ................................................................................................196

Figure 6.13: Preliminary screening of the compounds in Group I with a luciferase-based inhibition assay. ............................................................197

Figure 6.14: Preliminary screening of the compounds in Groups II-IV with a luciferase-based inhibition assay. .........................................................198

Figure 6.15: Secondary screening of active compounds. ...............................199

xiv

LIST OF TABLES

Table 2.1: Lewis acid-catalyzed formation of 3β-azido-5-cholestene (18).. ....23

Table 2.2: Effects of leaving groups. ..............................................................24

Table 2.3: Solvent effects.. .............................................................................25

Table 2.4: Application to the synthesis of cholesteryl 3β-halides (27, 29, and 30).. ...................................................................................................31

Table 6.1: Screening of metal catalyst to synthesize 120. ..............................186

Table 6.2: Solvent effects on Zr(IV)-catalyzed synthesis of 120. ....................187

Table 6.3: Comparison of microwave-assisted and thermal conditions for Zr(IV)-catalyzed synthesis of 120.............................................................188

xv

ACKNOWLEDGEMENTS

First of all, I need to thank my advisor, Dr. Blake R. Peterson for taking me

as his student and guiding me to achieve my academic goals. I would like to

show sincere gratitude to my thesis committee members, Dr. Juliette Lecomte,

Dr. J. Martin Bollinger, Dr. Avery August, and Dr. Gong Chen, for their precious

time and invaluable opinions.

I also would like to thank my dear colleagues, Dr. Siwarutt

Boonyarattanakalin, Dr. Sonalee Athavankar, Ms. Jocelyn Edathil, Mrs. Sutang

Cai, Ms. Runzhi Wu, and Mrs. Ewa Maddox, for the wonderful research

experience that we had together. Without their companion, my journey of

scientific adventure would have been a lot harder.

I dedicate this thesis to my family: my wife, Mrs. Qing Li, my father, Mr.

Mingxu Sun, my mother, Mrs. Shumin Gu, my sister, Mrs. Xin Sun, and my

brother-in-law, Mr. Ming Chen. I owe them too much as a husband, a son, and a

little brother. I want to let them know how important their support means to me. I

love you all, my dear family!

Chapter 1

Synthetic Mimics of Cell Surface Receptors

1.1 Synthetic Receptor Targeting as a Novel Delivery Strategy

Macromolecules, such as DNA, RNA, and proteins, and some drugs are

impermeable to cell membranes. Their entry into the interior of cells generally

requires specific active transport mechanisms.1 To circumvent this barrier,

impermeable molecules have been modified with polymers,2,3 liposomes,4,5 cell-

penetrating peptides (CPPs),6-9 or ligands targeting natural cell surface

receptors,10-14 to improve their cellular uptake properties. However, the

underlying mechanisms of these methods are not well understood, and the

delivery efficiencies of these methods vary substantially. Many applications of

these methods are limited by toxicity and cell-type specificity.

Receptor mediated endocytosis (RME) is an important process that

eukaryotic cells use to selectively internalize essential molecules in the

extracellular environment via natural cell surface receptors. RME is also

employed by cells to regulate transmembrane signaling in response to growth

factors and hormones, such as, epidermal growth factor (EGF) and insulin.

Certain pathogens, such as cholera toxin and adenovirus, have been found to

2

exploit RME to access the interior of the cells. RME involves many highly

concerted and complicated processes and is essential for many normal cellular

functions.15 RME can be generally divided into two major phases, the formation

of primary vesicles and the subsequent endocytic trafficking of internalized

ligands and their receptors (Figure 1.1).

The formation of clathrin-coated vesicle, the major type of primary

endocytic vesicles, starts with binding of the ligands to specific receptors on the

cell surface. Specialized sequences in cytoplasmic tails of transmembrane

receptors are then recognized by adaptor protein 2 (AP2), which recruits clathrin,

a complex of three molecules of heavy chain and three molecules of light chain in

a three-legged structure termed triskelion, to cluster ligand-receptor complexes in

dynamic regions called clathrin-coated pits. With the assistance of a variety of

accessory proteins, including epsins, endophilin, dynamin, and amphiphysin, the

coated pits actively invaginate and eventually pinch off to form the clathrin-coated

vesicles.

Once the coated vesicles are detached from the plasma membrane, they

are quickly uncoated in an ATP-dependent manner, which allows clathrin, AP2,

and accessory proteins, to dissociate and recycle back to the plasma membrane.

Mediated by SNAREs, early endosome antigen 1 (EEA1), and Rab5 protein, the

uncoated endocytic vesicles then fuse with sorting endosomes located near the

membrane. As the pivotal cellular compartments in RME trafficking pathway,

sorting endosomes, which possess tubular-vesicular structures with a lumenal

pH of ~6, are responsible for the determination of the destinations of internalized

3

molecules encapsulated in primary endocytic vesicles. In sorting endosomes,

due to increasing acidity, many ligands dissociate from their receptors and

remain in the vacuolar portion with other solute molecules, whereas the

membrane-associated protein components are transported with repeated

membrane budding of tubule-sharped vesicles to another endosomal organelle,

the endocytic recycling compartment (ERC), or directly to the plasma membrane.

Through this geometry-based sorting mechanism, most internalized membrane

proteins are rapidly and efficiently sorted from the soluble molecules for recycling.

The fusion with primary endosomes and the sorting process last only for 5-10

minutes. Subsequently, sorting endosomes begin to move along the

microtubules towards the center of cell, further decrease in pH to ~5, and acquire

acidic hydrolases, resulting in maturation to late endosomes and lysosomes,

where internalized substances are eventually digested.

As the major recipient of sorted membrane tubules released from sorting

endosomes, the ERC is composed of tubular organelles of narrow diameter that

attach to microtubules. While most of the membrane components in the ERC are

recycled back to the cell surface via transport vesicles or tubules with a t 1/2 = ~10

min, some molecules, such as trans-Golgi network protein 38 (TGN38) and

glycolipid-bound protein ligands, are transported to the trans-Golgi network,

indicating that ERC also functions as a sorting organelle.

Currently, the most well-understood examples of RME include the uptake

of cholesterol by LDL receptors,16 uptake of ion by transferrin receptors,17 and

cellular entry of cholera toxin bound to ganglioside GM1.15 Although these

4

processes exploit the general RME mechanism mentioned above, they also have

their own specific features.

Cholesterol is a key component of membranes that is essential for the

survival of vertebrate animals. This sterol is also a building block for the

biosynthesis of steroid hormones and bile acids. The endocytic uptake of

cholesterol-containing LDL particles is mainly mediated by the LDL receptor, a

single-pass transmembrane glycoprotein (~115 KDa). LDL ligands comprise

quasi-spherical particles (~22 nm in diameter and ~2,500 KDa). These particles

include a core of cholesterol esters that are encapsulated by a monolayer of

unesterified cholesterol, phospholipids, triglycerides, and a single large protein

termed apolipoprotein B-100 (apo-B, ~550 KDa). The LDL receptor (LDLR)

initially delivers LDL into sorting endosomes via clathrin-coated vesicles. Sorting

endosomes are acidified by the activation of proton pumps, which promotes the

dissociation of the LDLR from LDL. The geometry-based separation of receptor

from LDL particle allows cycling of the LDLR back to the cell surface via the ERC

and retention of LDL particles in the sorting endosome during its progression to

late endosomes and lysosomes. Finally, hydrolysis of the cholesteryl esters and

other components in LDL particles in lysosomes liberates free cholesterol and

other nutrients for use by the cell.

Another essential cell-impermeable nutrient and enzyme cofactor is iron.

In vertebrate animals, ferric iron is transported bound to the protein transferrin

(TF), a glycoprotein of ~80 KDa. Cellular uptake of TF is mediated by the

transferrin receptor (TFR), a homodimeric transmembrane protein (~85 KDa) that

5

binds two diferric transferrin molecules. Different from most other ligands (e.g.

LDL particles), internalized of TF releases iron ions in acidic sorting endosomes,

and remain continuously bound to TFR until it is recycled back to the cell surface

via the ERC.

Figure 1.1: A model of receptor-mediated endocytosis (RME). This model illustrates endocytic uptake of LDL mediated by the LDLR, uptake of transferrin(TF) by the transferrin receptor (TFR), and uptake of cholera toxin (CTX)mediated by ganglioside GM1. Figure courtesy of Professor Blake R. Peterson.

6

Unlike transmembrane receptor-mediated endocytosis, lipid-based RME,

such as the internalization of cholera toxin by ganglioside GM1, exploit multiple

endocytic pathways.15 In addition to or independent of the clathrin-dependent

mechanism, uncoated primary endocytic vesicles (e.g. caveolae) are employed

to deliver the binding complex through GPI-anchored protein-enriched early

endosomal compartments (GEECs) to the ERC, where the ligand and lipid-linked

receptor are sorted and tranported to the TGN and plasma membrane

respectively.

In the past decade, our group has created a novel delivery strategy

termed synthetic receptor targeting (SRT). This technology greatly enhances

uptake of cell impermeable macromolecules by decorating mammalian cell

surfaces with small artifical receptors. These receptors comprise the membrane

anchor N-alkyl-3β-cholesterylamine (2) linked to ligand-binding motifs, such as

fluorescein (3)18, biotin (4)19, protein-binding peptides (5-8)20,21, dinitrophenyl

(DNP, 9)22, nitrilotriacetic acid (NTA, 10)23 and drug-binding peptides (11)24.

Ligands of these synthetic receptors include anti-fluorescein IgG (receptor 3),

streptavidin (receptor 4 and 7), anti-hemagglutinin tag IgG (receptor 5), anti-flag

tag IgG (receptor 6), anti-DNP IgG (receptor 8), human IgG (receptor 9), (His)10

tagged AcGFP (receptor 10), and vancomycin (receptor 11). Quantitative flow

cytometry data established that these synthetic receptors (3-11) at a

concentration of 10 µM dramatically enhance the cellular uptake of poorly

permeable or impermeable molecules, ranging from 10 fold to 2000 fold, with

typically >99% cellular efficiency and low toxcity.

7

Figure 1.2: Structures of natural cell surface receptor, ganglioside GM1 (1), 3β-cholesterylamine (2), and synthetic receptors (3-11).

8

In synthetic receptor targeting, plasma membranes of mammalian cells

are loaded with synthetic receptors. When the ligands bind to the receptors on

the cell surface, the complex is internalized via endocytosis. In endosomes, the

receptors dissociate from their ligands and recycle back to the cell surface.

Studies by confocal microscopy, cholesterol depletion assays, sucrose density

gradient ultracentrifugation analysis, and clatherin-inhibition assays, have

established that these synthetic receptors likely associate with sphingolipid-

enriched lipid rafts, and the binding complex is internalized through a clathrin-

mediated pathway, a process analogous to the uptake of cholera toxin mediated

by the natural cell surface receptor, ganglioside GM1 (1).18

Figure 1.3: A model of synthetic receptor-mediated endocytosis. Courtesy of Professor Blake R. Peterson.

9

1.2 Synthetic Cell Surface Receptors as Probes of Cellular Biology

Another potential application of synthetic mimics of mammalian cell

surface receptors is as fluorescent probes of cellular biology (12-14). With the

help of Oregon green and 7-nitrobenz-2-oxa-1,3-diazole (NBD) probes (12, 13)22,

the kinetic and dynamic features of the recycling of synthetic receptors were

quantitatively determined. Probe 12 was measured to shuttle between the

plasma membrane and Endosomes making a round trip about every 10 min

using a methyl-β-cyclodextrin-mediated receptor depletion assay.22

Figure 1.4: Structures of 3β-cholesterylamine-based fluorescent probes (12-14).

10

Figure 1.5: Enhancement of membrane association by installation of negatively charged glutamic acid residues to the linker region. Panel A. Confocal microscopic analysis of membrane association of 13 and 14. Panel. B: Flow cytometric analysis of membrane association of 13 and 14. Jurkat lymphocytes were treated with 13 or 14 (10 µM) for 5 min or 1 h at 37 oC, and washed with fresh media to remove unincorporated receptors before analysis. (Unpublished results, Figure courtesy of Ms. Sutang Cai and Professor Blake R. Peterson)

11

Fluorenscence-quenching experiments with probe 13 and its analogues (with 1

or 2 β-alanine units in the linker region) revealed that insertion of β-alanine into

the linker region favors the distribution of receptors on the plasma membrane

rather than endosomes.22

More recently, confocal microscopy and flow cytometry data obtained by

Sutang Cai in our laboratorys established that negatively charged glutamic acid

residues in fluorescent probe 14 significantly enhance its membrane association

both in rate and magnitude. Comparison of the fluorescent confocal images of

cells treated with 13 and 14 showed dramatically increased cellular association

due to the presence of the negative charges (Figure 1.5, Panel A). In addiition,

analysis of fluorescence of the cells treated with 13 or 14 by quantitive flow

cytometry (Figure 1.5, Panel B) demonstrated that glutamic acid residues enable

14 to associate with plasma membrane over five times faster than 13 (at 5 min),

and with twice the magnitude of 13 (after 1 h). This important discovery has lead

to the use of glutamic residues in many second generation synthetic receptors to

improve their solubility and delivery efficacy.

1.3 Current Limitations of Synthetic Receptor Targeting

There are two major hurdles that restrain many applications of synthetic

receptors. One problem is the lack of efficient synthetic methods for preparation

of 3β-cholesterylamine and protected building blocks. Previously, our group has

12

reported a modified synthetic route to 2 (Figure 1.6), which involves the

transformation of cholesterol to epicholesterol by Swern oxidation, followed by

stereoselective reduction with expensive and unstable L-selectride, Mitsunobu

conversion of the 3α-alcohol to the 3β-azide, and LiAlH4 reduction to the amine.22

However, this method gives only moderate overall yields and is difficult to scale

up for multigram preparation. To further install linker units on 3β-cholesterylamine,

the primary amine was previously protected with a nosyl protecting group.

Although it enabled further alkylation with chlorinated alkyl linkers, removal of

nosyl protecting group at the end of the synthesis of receptors typically afforded

low and inconsistent yields.

Figure 1.6: Previously reported synthesis of 3β-cholesterylamine (2) and its Nosyl-protected derivative (19). Reagents and conditions: (a) oxalyl chloride,DMSO, CH2Cl2, TEA, -78 °C; (b) L-selectride, THF, -78 °C; (c) PPh3, HN3, DEAD, benzene; (d) LiAlH4, Et2O, 0 °C; (e) 2-nitrobenzenesulfonyl chloride, DIEA, THF.

13

A second problem is that impermeable ligands delivered by synthetic

receptors (3-12) typically become trapped in endosomes (Figure 1.7). These

intracellular compartments are acidic, tightly sealed, and contain hydrolases and

other digestive enzymes. Because many biologically active molecules have

targets either in the cytoplasm or nucleus, the entrapment of these molecules in

the endosomes limits the utility of the synthetic receptor-mediated delivery

system.

1.4 Outline of This Dissertation

Based on pioneering studies on synthetic mimics of mammalian cell

surface receptors by Dr. Hussey, Dr. Martin, and Dr. Boonyarattanakalin, my

research has been primarily focusing on addressing two major problems

Figure 1.7: Confocal and DIC microscopy of entrapment of delivered moleculesin endosomes, illustrated by delivery of human IgG-AF488 by receptor 8 in Jurkat lymphocytes. Figure Courtesy of Professor Blake R. Peterson.

14

mentioned above, and exploring novel biologicial applications of synthetic mimics

of cell surface receptors. Chapter 2 describes novel and highly efficient synthetic

methodologies that enable large-scale synthesis of 3β-cholesterylamine, 3β-

amino-5α-cholestane, and related anchor building blocks with Boc protection. A

novel endosomal escape strategy employing a 3β-cholesterylamine-capped lytic

peptide and disulfide-linked small molecular cargo, and studies on T-lymphocyte

apoptosis induced by the interactions of phosphocholine-containing synthetic

receptors and C-reactive proteins (CRP), are included in Chapters 3 and 4,

respectively. The last two chapters focus on the advances in research in the

development of antivirial agents and derivatives. While Chapter 5 introduces the

discovery of a novel method for the preparation of the 5’-triphosphates of antiviral

nucleosides, Chapter 6 reports the construction of a benzimidazole-based

compound library and biological evaluation of their inhibitory effect against

nonsegmented negative-stranded RNA viruses (NNSVs).

1.5 References

1. Smith, D. A.; van de Waterbeemd, H. Pharmacokinetics and metabolism

in early drug discovery. Curr. Opin. Chem. Biol. 1999, 3, 373-378.

2. Murphy, J. E.; Uno, T.; Hamer, J. D.; Cohen, F. E.; Dwarki, V.;

Zuckermann, R. N. A combinatorial approach to the discovery of efficient cationic

15

peptoid reagents for gene delivery. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1517-

1522.

3. Garnett, M. C. Gene-delivery systems using cationic polymers. Crit. Rev.

Ther. Drug Carrier Syst. 1999, 16, 147-207.

4. Bendas, G. Immunoliposomes: A promising approach to targeting cancer

therapy. Biodrugs 2001, 15, 215-224.

5. Zelphati, O.; Wang, Y.; Kitada, S.; Reed, J. C.; Felgner, P. L; Corbeil, J.

Intracellular delivery of proteins with a new lipid-mediated delivery system. J. Biol.

Chem. 2001, 276, 35103-35110.

6. Schwarze, S. R.; Ho, A.; Vocero-Akbani, A.; Dowdy, S. F. In vivo protein

transduction: delivery of a biologically active protein into the mouse. Science

1999, 285, 1569-1572.

7. Lindgren, M.; Hallbrink, M.; Prochiantz, A.; Langel, U. Cell-penetrating

peptides.Trends Pharmacol Sci. 2000, 21, 99-103.

8. El-Andaloussi, S.; Holm, T.; Langel, U. Cell-penetrating peptides:

Mechanisms and applications. Curr. Pharm. Des. 2005, 11, 3597-3611.

9. Snyder, E. L.; Dowdy, S. F. Cell-penetrating peptides in drug delivery

Pharm. Res. 2004, 21, 389-393.

10. Vyas, S. P.; Singh, A.; Sihorkar, V. Ligand-receptor-mediated drug

delivery: An emerging paradigm in cellular drug targeting. Crit. Rev. Ther. Drug

Carrier Syst. 2001, 18, 1-76.

16

11. Lu, Y.; Sega, E.; Leamon, C. P.; Low, P. S. Folate receptor-targeted

immunotherapy of cancer: mechanism and therapeutic potential. Adv. Drug

Delivery Rev. 2004, 56, 1161-1176.

12. Rui, Y. J.; Wang, S.; Low, P. S.; Thompson, D.H. Diplasmenylcholine-

folate liposomes: An efficient vehicle for intracellular drug delivery. J. Am. Chem.

Soc. 1998, 120, 11213-11218.

13. Qian, Z. M.; Li, H.; Sun, H.; Ho, K. Targeted drug delivery via the

transferrin receptor-mediated endocytosis pathway. Pharmacol. Rev. 2002, 54,

561-587.

14. Chung, N. S.; Wasan, K. M. Potential role of the low-density lipoprotein

receptor family as mediators of cellular drug uptake. Adv. Drug Delivery Rev.

2004, 56, 1315-1334.

15. (a) Conner, S. D.; Schmid, S. L. Regulated portals of entry into the cell.

Nature 2003, 422, 37-44. (b) Warren, G.; Smythe, E. The mechanism of

receptor-mediated endocytosis. Eur. J. Biochem. 1991, 202, 689-699. (c)

McGraw, T. E.; Maxfield, F. R. Endocytic recycling. Nat. Rev. Mol. Cell. Biol.

2004, 5, 121-132.

16. (a) Brown, M. S.; Goldstein, J. L. A receptor-mediated pathway for

cholesterol homeostasis. Science 1986, 232, 34-47. (b) Jeon, H.; Blacklow, S. C.;

Structure and physiological function of the low-density lipoprotein receptor. Annu.

Rev. Biochem. 2005, 74, 535-562.

17. Cheng, Y.; Zak, O.; Aisen, P.; Harrison, S. C.; Walz, T. Structure of the

human transferrin receptor-transferrin complex. Cell 2004, 116, 565-576.

17

18. Hussey S. L.; He, E.; Peterson, B. R. A synthetic membrane-anchored

antigen efficiently promotes uptake of antifluorescein antibodies and associated

Protein A by mammalian cells. J. Am. Chem. Soc. 2001, 123,12712-12713.

19. Hussey S. L.; Peterson, B. R. Efficient delivery of streptavidin to

mammalian cells: Clathrin-mediated endocytosis regulated by a synthetic ligand.

J. Am. Chem. Soc. 2002, 124, 6265-6273.

20. Martin, S. E.; Peterson, B. R. Non-natural cell surface receptors: Synthetic

peptides capped with N-cholesterylglycine efficiently deliver proteins into

mammalian cells. Bioconjugate Chem. 2003, 14, 67-74.

21. Boonyarattanakalin, S.; Martin, S. E.; Sun, Q.; Peterson, B. R. A synthetic

mimic of human Fc receptors: Defined chemical modification of cell surfaces

enables efficient endocytic uptake of human Immunoglobulin-G J. Am. Chem.

Soc. 2006, 128, 11463-11470.

22. Boonyarattanakalin, S.; Martin, S. E.; Dykstra, S. A.; Peterson, B. R.

Synthetic mimics of small mammalian cell surface receptors. J. Am. Chem. Soc.

2004, 126, 16379-16386.

23. Boonyarattanakalin, S.; Athavankar, S.; Sun, Q.; Peterson, B. R.

Synthesis of an artificial cell surface receptor that enables oligohistidine affinity

tags to function as metal-dependent cell-penetrating peptides. J. Am. Chem. Soc.

2006, 128, 386-387.

24. Boonyarattanakalin, S.; Hu, J.; Dykstra-Rummel, S.; August, A.; Peterson,

B. R. Endocytic delivery of vancomycin mediated by a synthetic cell surface

18

receptor: Rescue of bacterially infected mammalian cells and tissue targeting in

vivo. J. Am. Chem. Soc. 2007, 129, 268-269.

25. Peterson, B. R. Synthetic mimics of mammalian cell surface receptors:

Prosthetic molecules that augment living cells. Org. Biomol. Chem. 2005, 3,

3607-3612.

Chapter 2

Novel and Practical Synthesis of 3β-Amino-5-Cholestene (3β-

Cholesterylamine), 3β-Amino-5α-Chlestane, and Related Building Blocks

2.1 Introduction

Synthetic mimics of cholesterol represent important molecular tools in the

fields of bioorganic/medicinal chemistry and chemical biology. Cholesterol

derivatives have been used to facilitate the delivery of siRNA,1 enhance DNA

transfection, probe cellular membrane subdomains,2 and have been proposed for

tumor targeting applications.3 The cationic cholesterol mimic 3β-amino-5-

cholestene (3β-cholesterylamine, 2) is of particular interest because of its high

affinity for phospholipid membranes.4 Derivatives of 3β-amino-5-cholestene have

been used to construct photoaffinity and fluroscent probes,5,6 and some

members of this class of compounds exhibit antimicrobial acitivity.7 N-Alkyl

derivatives of 3β-amino-5-cholestene can insert into plasma membranes of living

mammalian cells and cycle between the cell surface and early/recycling

endosomes, mimicking the membrane trafficking of many cell surface receptors.8

These compounds are under investigation as tools for drug delivery,9 and when

20

linked to endosome disruptive peptides10 can deliver molecules into the cytosol

and nucleus of mammalian cells.

Windaus11 was the first to report that reaction of cholesteryl chloride with

ammonia, or reduction of cholestenone oxime, provides a mixture of 3-

substituted cholesterylamines in modest yields. More recently, stereoselective

syntheses of 3β-azido-5-cholestene (Figure 2.1), an important precursor to 3β-

amino-5-cholestene, have involved conversion of cholesterol to epicholesterol

followed by Mitsunobu reaction with hydrazoic acid4,12 or treatment of 6β-

methoxy-3α,5-cyclo-5α-cholestane and related compounds with hydrazoic

acid.5,13,14 However, because of the involvement of the homoallylic double bond

at the C5 position of the steroid, substitution reactions involving cholesterol and

derivatives can suffer from poor stereoselectivity, elimination, and

Figure 2.1: Previously reported synthesis of 3β-cholesterylamine (2) and proposed alternative synthetic route.

21

rearrangement.15-19 These complications limit existing synthetic methods to

small-scale preparation of 3β-azido-5-cholestene.

We report here a novel and practical three-step method (Figure 2.2), the

key step of which is the conversion of cholest-5-en-3β-ol, methanesulfonate (21)

into 3β-azido-cholest-5-ene (18) with complete retention of β configuration. This

unusal high regio- and stereoselectivity was elucidated to involve an i-steroid and

retro i-steroid rearrangement mechanism. Further application of this reaction with

trimethylsilyl halides provides a facile and efficient access to 3β-

cholesterylhalides.

A modified synthetic route for 3β-amino-5α-cholestane will be also

reported in this chapter. Development of an efficient method for the preparation

of diverse Boc-protected anchor building blocks is another highlight of this

chapter.

Figure 2.2: A novel and stereoselective synthesis of 3β-azido-5-cholestene (18).

22

2.2 A Novel Method for Conversion of Cholest-5-En-3β-Ol,

Methanesulfonate (21) to 3β-Azido-Cholest-5-Ene (18)

Our investigation of this approach was inspired in part by the use of

TMSN3 and lewis acids in neighboring group-assisted glycosylation reactions that

proceed with overall retention of configuration.20,21 Moreover, as elucidated by

Shoppee19 and Winstein,22 cholesterol and reactive derivatives have been shown

to undergo solvolysis with retention of 3β-configuration via the involvement of a

non-classical carbocation that is formed by neighboring participation of the

homoallylic alkene. This homoallylic carbocation reacts rapidly at the C6 position

of the steroid to afford 6β-substituted-3α,5-cyclosteroids through a process

termed the i-steroid rearrangement. Slower reaction of this cation at the C3

position yields cholesteryl 3β-derivatives.23,24 Based on these precedents, we

hypothesized that TMSN3 and lewis acids might efficiently convert 21 into 18 with

retention of configuration.

Table 2.1 lists our investigation of the effects of different lewis acids and

TMSN3 on the conversion of 21 to 18. Among the lewis acids investigated, the

addition of two equivalents of BF3·Et2O at ambient temperature (23 °C) for 2

hours proved optimal, providing a 93% yield of 21 from 18. In addition to azide 21,

SnCl4, TiCl4, and AlCl3 generated 3β-chloro-cholest-5-ene (27) as a major

byproduct. Reaction of fluoride derived from BF3·Et2O with TMSN3 is presumably

23

involved in the production of the nucleophilic azide, and high stability of TMSF

likely prevents formation of byproducts compared to the chlorinated lewis acids.

In control experiments, studies of the corresponding 3α-mesylate (24) and

the dihydro analogue (25) revealed that both the 3β-configuration of 21 and the

C5-alkene were required for reaction of 18 with BF3·Et2O/TMSN3. These results

Table 2.1: Lewis acid-catalyzed formation of 3β-azido-5-cholestene (18). aAll reactions (0.5 mmol scale) were conducted in anhydrous CH2Cl2 under N2 with 1.1 equiv. of TMSN3. bThe conditions of all reactions were optimized tomaximized the yield of 3β-azido-5-cholestene. cThe products were isolated by column chromatography (hexanes). dFor entry 1-3, yields of 3β-cholesteryl chloride as a major byproduct were shown in paratheses.

24

are consistent with previous studies of solvolytic rate enhancements of

cholesterol derivatives that compared the corresponding unsaturated and

saturated substrates and that examined the stereospecificity of related

reactions.19,25 These results indicate that the homoallylic double bond of 21 is a

key participant in the outcome of this reaction.

Table 2.2: Effects of leaving groups. aAll reactions (0.5 mmol scale) were conducted in anhydrous CH2Cl2 under N2 with 1.1 equiv. of TMSN3. bThe conditions of all reactions were optimized to maximized the yield of 3β-azido-5-cholestene. cThe products were isolated by column chromatography (hexanes).dFor entry 2, recovery yield of starting material was shown in parathesis.

25

2.3 Effects of Leaving Groups and Solvents

The effect of the leaving group on the reactivity of 21 was probed as listed

in Table 2.2 The less reactive tosylate (26) similarly furnished 3 in excellent yield

but required a larger excess of BF3·Et2O (5 eq.) and a longer reaction time than

the mesylate (21). Interestingly, whereas BF3·Et2O had little effect on conversion

of 3β-chloro-5-cholestene (27), SnCl4 converted 50% of this compound to the

azide (18). Substrates bearing poorer leaving groups, such as 28 and 15 were

unreactive.

Table 2.3: Solvent effects. aHexane,acetonitrile, and DMSO, were not tested dueto poor solubility of 21. bAll reactions (0.5 mmol scale) were conducted inanhydrous solvents under N2 with 1.1 equiv. of TMSN3. cThe conditions of all reactions were optimized to maximized the yield of 3β-azido-5-cholestene. dThe products were isolated by column chromatography (hexanes).

26

The nature of the solvent was also found to play a critical role in this

reaction (Table 2.3). Benzene could be substituted for dichloromethane or

chloroform without appreciably diminishing the yield of 18 from 21 (92%), but

required additional eqivalents of BF3·Et2O (5 eq.) and a longer reaction time (12 h)

for completion of the reaction. No reaction was observed in solvents bearing

heteroatoms that function as lewis bases, including tetrahydrofuran, acetone,

diethyl ether, or DMF, further emphasizing the role of the lewis acid in activating

the leaving group. Poor solubility of 21 precluded evaluation of reactivity in

hexane, acetonitrile, or DMSO.

2.4 A Novel and Efficient Route for Large-Scale Preparation of 3β-

Cholesterylamine (2)

Reduction of 3β-azido-5-cholestene (18) to 3β-cholesterylamine (2) was

achieved by treating the azide with LiAlH4.12 As illustrated in Figure 2.3, 3β-

cholesterylamine (2) was obtained in 88% overall yield over three steps on small

scale. The mild reaction conditions and high efficiency of each step allowed us to

scale up the synthesis to 100 grams with only simple modifications of purification

procedures (See experimental section).

27

2.5 Study of the Reaction Mechanism by 1H NMR

Because the conversion of 21 to 18 proceeded rapidly at ambient

temperature in the presence of BF3·Et2O and TMSN3, the course of the reaction

could be readily followed by 1H NMR. As shown in Figure 2.4, panel A, the

acquisition of 1H NMR spectra at different time points allowed clear observation

of the disappearance of signals of starting material and the emergence of

products. Importantly, these experiments detected the transient appearance of a

signal at 3.27 ppm (observed at 2 and 4 minutes) that did not correspond to the

starting material (21) or product (18), but rather could be assigned as the C6

proton of 6β-azido-3α,5-cyclo-5α-cholestane (23), a structure that was further

supported by distinctive cyclopropyl signals observed at 0.48 ppm (shown in the

Figure 2.3: Optimized synthetic route for large-scale preparation of 3β-cholesterylamine aYields at 0.5 mmol (~ 200 mg) scale. bYields at 100-gram scale. Reagents and conditions: (a) MsCl, DIEA, CH2Cl2, 4 °C to 22 °C, 6 h; (b) TMSN3, BF3·Et2O, CH2Cl2, 22 °C, 2 h; (c) LiAlH4, Et2O, 4 °C to 22 °C, 2 h.

28

Figure 2.4: The transformation cholest-5-en-3α-ol, methanesulfonate (21) to 3β-azido-5-cholestene (18) monitored 1H NMR. Panel A: Stacked 1H NMR spectra of the reaction at different time points. The arrows indicate the C-6 proton signal of intermediate product, 6β-azido-3α,5-cyclo-5α-cholestane (23). The reaction (0.5 mmol scale) was conducted in 0.5 ml of CDCl3 in a NMR tube. Panel B: Proposed mechanism.

29

experimental section).17 These data are consistent with generation of the i-

sterioid intermediate 23 derived from attack of azide on C6 of the homoallylic

cation (22) as shown in Figure 2.4, panel B.

The NMR data shown in Figure 1, panel A, in conjunction with the

necessity of both 3β-stereochemistry of the mesylate (21) and the presence of

the homoallylic double bond (Table 2.1) suggest that 21 is initially converted by

the lewis acid to a homoallylic carbocation that rapidly undergoes the i-steroid

rearrangement and retro-i-steroid rearrangement shown in Figure 2.4, panel B.

Winstein22 previously demonstrated that attack at the C6-carbon of cholesterol-

derived homoallylic carbocations is kinetically favored and that the structure of

the homoallylic cation engenders formation of the 6β-product over the 6α-product

by a factor of 10 to 100. Although the attack of nucleophiles at C3 of the

homoallylic carbocation is substantially slower than C6, this addition occurs to

form the thermodyamic product with exclusively β-stereochemistry. This

stereochemical outcome is a result of the non-classical carbocation forming a

partial bond between C5 and C3 only on the alpha face of the steroid.

To further support the idea that retro i-steroid rearrangement precedes the

generation of 18, we prepared 6β-azido-3α,5-cyclo-5α-cholestane (23) by

treatment of 21 with NaN3 in refluxing methanol, a modification of the method of

Freiberg.17 Importantly, treatment of 23 with BF3·Et2O (2 eq.) and TMSN3 (1 eq.)

resulted in quantitative conversion to 18 within 2 hours at ambient temperature

(data shown in the experimental section). In contrast, treatment of 23 with TMSN3

30

alone or TMSN3 and TBAF to generate the azide ion did not result in conversion

to 18, indicating that the homoallylic cation is also a required intermediate in the

retro i-steriod rearrangment. Addition of excess BF3·Et2O (2 eq.) alone resulted in

formation of 18 within 10 minutes. However, byproducts were associated with

treatment of 23 with BF3·Et2O in the absence of TMSN3. Correspondingly, the

addition of two equivalents of BF3·Et2O and 1.1 eq. of TMSN3 to 21 proved to be

optimal to both generate the homoallylic cation and convert 23 to 18 in high yield.

2.6 Novel Synthesis of Related 3β-Cholesterylhalides

We additionally examined the utility of other TMS derivatives for

preparation of 3β-substituted cholestenes. Correspondingly, as shown in Table

2.4, 3β-chloro-5-cholestene (27), 3β-bromo-5-cholestene (29), and 3β-iodo-5-

cholestene (30) were synthesized in high yield from lewis acids and cognate

TMS compounds. This approach is particularly useful for preparation of

compounds 29 and 30 due to their high susceptibility to elimination reactions.

31

2.7 A Modified Synthetic Route to 5α-Cholestane-3β-Amine (34)

Cholestanol (31), also called dihydrocholesterol, is a close analogue of

cholesterol, lacking the C-5 double bond. It is considered as a partial functional

substitute for cholesterol and can support proliferation of cholesterol-deficient

human cells in culture.26 Cholestanol is thought to lead to stronger sterol-

phospholipid association due to an increase in hydrophobicity and more effective

lipid contact with the B-ring of the sterol.27 Therefore, 5α-cholestane-3β-amine

(34) was proposed to have similar activities to 3β-cholesterylamine (2) and was

Table 2.4: Application to the synthesis of cholesteryl 3β-halides (17, 19, and 30).aAll reactions (1.0 mmol scale) were conducted in anhydrous CH2Cl2 under N2with 1.1 equiv. of TMS-halides. bThe conditions of all reactions were optimized tomaximized the yields of 3β-halo-5-cholestene. cThe products were isolated by column chromatography (hexanes).

32

synthesized as an surrogate of 3β-cholesterylamine. The preparation of 5α-

cholestane-3β-amine has been reported previously by Cushman et al.28 However,

their three-step method affords a moderate overall yield, which was significantly

improved from 41% to 83% in our research by modifications of the first and last

steps (Figure 2.5).

Instead of conversion into the corresponding iodide, cholestanol was

transformed into the reactive but more stable 3α-bromide (32) in higher yield.

The subsequent synthesis of 3β-azide (33) proceeded by the method of

Cushman28 without modification. The 3β-azide was efficiently reduced to the 3β-

amine (34) using a Staudinger reaction, a milder and safer method comopared

with the use of LiAiH4.

Figure 2.5: A modified route for the synthesis of 5α-cholestane-3β-amine (34). Reagents and conditions: (a) CBr4, PPh3, THF, 4 °C to 22 °C, 6 h; (b) NaN3, DMSO, 80 °C, 4 h; (c) PPh3, THF/H2O (10:1), 22 °C, 24 h.

33

2.8 Novel and Practical Synthesis of Boc-Protected Anchors Derived from

5α-Cholestane-3β-Amine (34) and 3β-Cholesterylamine (2)

To construct synthetic receptors, a primary linker (valeric acid or

propylamine) is typically added to 5α-cholestane-3β-amine and 3β-

cholesterylamine to allow coupling of other linker units and ligand-binding motifs.

Because the secondary amine product in alkylation reactions will react to form a

tertiary amine,29 our previous synthetic strategy employed Nosyl (2-nitrobenzene-

sulfonyl) as the protecting group, which enables alkylation of the sulfonamide for

installation of linker units.6 Two major problems are associated with use of the

Nosyl protecting group. First, it is not compatible with Fmoc chemistry, which is

one of the most efficient coupling methods. Second, the deprotection of the

Figure 2.6: A reductive amination method for the synthesis of a Boc-protected membrane anchor with an ethylvalerate linker (38). Reagents and conditions: (a) oxalyl chloride, DMSO, TEA, CH2Cl2, -78 °C, 2 h; (b) 5-aminoethylvalerate, 3Å molecular sieves, MeOH, 24 h; (c) NaBH4, MeOH, -78 °C, 30 min; (d) (Boc)2O, DIEA, CH2Cl2, 22 °C, 6 h. aThe acetate of β isomer was purified by recrystallation (CH2Cl2/Et2O).

34

Nosyl sulfonamide at the end of each synthesis gives only moderate and

inconsistent yields. Therefore, Boc-protected building blocks were chosen for a

more efficient synthesis of artifical receptors.

Our initial attempt to synthesize 5α-cholestane-3β-amine-based

membrane anchors bearing a ethylvalerate linker involved Swern oxidation of

cholestanol (31) to 3-cholestanone (35) and the subsequent generation of an

imine (36), which was directly reduced to the secondary amine by NaBH4.30 The

formation of the desired 3β isomer (37) was preferred (β:α, 9:1) at -78 °C. But the

3β isomer was inseparable from 3α isomer by column chromatography.

Alternatively, the β isomer was purified by recrystallizing its acetic acid salt from

Figure 2.7: A new method for the synthesis of Boc-protected membrane anchors. Reagents and conditions: (a) 5-bromoethylvalerate/3-bromopropylphthalimide, K2CO3, DMF, 60 °C, 24 h; (b) (Boc)2O, DIEA, CH2Cl2, 22 °C, 6 h.

35

CH2Cl2/Et2O. After the secondary amine was protected as a Boc carbamate, the

anchor moiety (38) was obtained in 46% overall yield at 200-mg scale. However,

this route was difficult to scale up to a multi-gram scale.

Although overalkylation is a typical problem in the synthesis of secondary

amines, Bhaduri et al. reported that if the alkyl bromide employed in the N-

alkylation reaction exceeds five carbons in length, monoalkylation is favored from

the resulting steric hindrance.31 By treating 5α-cholestane-3β-amine (34) with 3-

bromopropylphthalimide according to conditions previously described,31 the

desired product (44) was obtained through protection of the secondary amine

with Boc in situ. However, the yield (35%) was much lower than reported in the

literature (>80%).31 Additional research revealed that DMF was a better solvent

than DMSO for this reaction and a lower temperature (60 °C) gave a much higher

yield (71%). When this new method was applied to the synthesis of other related

anchor modules, similar yields were obtained in reactions up to a 20-gram scale

(Figure 2.8). Further deprotection of either phthalimide or ethyl ester afforded the

amine or carboxylic acid for subsequent coupling reactions in very high yields

with only simple purification procedures.32-34

2.9 Conclusions

In summary, we developed a novel and highly efficient route for the

synthesis of 3β-cholesterylamine and related 3β-halides. The advantages of this

36

synthetic method include its practicability in large-scale synthesis and regio- and

stereoselective conversion of cholest-5-en-3β-ol, methanesulfonate to 3β-azido-

and 3β-halo-cholest-5-ene, the mechanism of which was elucidated to involve an

i-steroid and retro i-steroid rearrangement pathway. This method may potentially

facilitate the synthesis of other aminosteroids bearing similar or even more

conjugated systems, such as conessine and ergosterol.

We also developed a modified three-step route for the synthesis of 5α-

cholestane-3β-amine with significantly improved overall yield. In addition, a novel

method for the large-scale preparation of diverse Boc-protected anchors was

established, utilizing the steric hindrince of the steroid moiety to favor the

formation of the secondary amine.

2.10 Experimental Section

2.10.1 General

Chemical reagents were obtained from Acros, Aldrich, or Alfa Aesar.

Solvents were from EM Science. Commercial grade reagents were used without

further purification unless otherwise noted. Epicholesterol were synthesized

according to literature procedures.4 Anhydrous solvents were obtained after

passage through a drying column of a solvent purification system from

GlassContour (Laguna Beach, CA). All reactions were performed under an

37

atmosphere of dry argon or nitrogen. Reactions were monitored by analytical

thin-layer chromatography on plates coated with 0.25 mm silica gel 60 F254 (EM

Science). TLC plates were visualized by UV irradiation (254 nm) or stained with a

solution of phosphomolybdic acid. Flash column chromatography employed ICN

SiliTech Silica Gel (32-63 mm). Melting points were measured with a Thomas

Hoover capillary melting point apparatus and were uncorrected. Infrared spectra

were obtained with a Perkin Elmer 1600 Series FTIR. NMR spectra were

obtained with Bruker CDPX-300 and AV-400 instruments with chemical shifts

reported in parts per million (ppm) referenced to either CDCl3 (1H 7.27 ppm; 13C

77.2 ppm) or TMS (0 ppm). High-resolution mass spectra were obtained from the

Pennsylvania State University Mass Spectrometry Facilities. Peaks are reported

as m/z.

2.10.2 Synthetic Procedures and Compound Characterization Data

Cholest-5-en-3β-ol, 3-(4-methylbenzenesulfonate) (26). To a solution of

cholesterol (1.93 g, 5.0 mmol) in anhydrous CH2Cl2 (30 mL) at 4 °C was added

pyridine (2 mL, 25.0 mmol) and p-toluenesulfonyl chloride (1.14 g, 6.0 mmol).

The reaction was stirred at 22 °C for 16 h. When cholesterol completely

38

disappeared on TLC (hexanes/ethyl acetate, 5:1), the reaction solution was

diluted with CH2Cl2 (150 mL). The organic phase was washed by aqueous HCl

solution (1.0 M, 100 mL), saturated aqueous NaHCO3 solution (100 mL), and

saturated aqueous NaCl solution (100 mL). The organic phase was dried over

anhydrous Na2SO4, and concentrated in vacuo. Flash column chromatography

afforded the product (2.46 g, 91%) as a white solid, mp 130.5-131.5 °C (Lit.35 mp

130-132 °C); 1H NMR (300 MHz, CDCl3) δ 7.79 (d, J = 8.2 Hz, 2H), 7.32 (d, J =

8.2 Hz, 2H), 5.30 (d, 1H), 4.32 (m, 1H), 2.44 (s, 3H), 2.41 (m, 1H), 2.27 (m, 1H),

2.01-0.85 (m, 38H), 0.65 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 144.4, 138.8, 13.7,

129.7 (× 2), 127.6 (× 2), 123.5, 82.3, 56.6, 56.0, 49.9, 42.3, 39.6, 39.5, 38.9, 36.9,

36.3, 36.1, 35.7, 31.8, 31.7, 28.6, 28.2, 28.0, 24.3, 23.8, 22.8, 22.6, 21.6, 21.0,

19.1, 18.7, 11.8; IR (film) ν max 3033, 2950, 2901, 2868, 1599, 1467, 1440, 1380,

1365, 1334,1189, 1175, 1098, 965, 941, 889, 867, 815.5, 733, 690, 668 cm-1.

Cholest-5-en-3β-ol, acetate (28). To a solution of cholesterol (3.86 g, 10.0 mmol)

in anhydrous CH2Cl2 (50 mL) at 4 °C was added pyridine (4 mL, 50.0 mmol) and

acetic anhydride (1.42 mL, 15.0 mmol). The reaction was stirred at 22 °C for 4 h.

When cholesterol completely disappeared on TLC (hexanes/ethyl acetate, 5:1),

the reaction solution was diluted with CH2Cl2 (150 mL). The organic phase was

39

washed by aqueous HCl solution (1.0 M, 100 mL), saturated aqueous NaHCO3

solution (100 mL), and saturated aqueous NaCl solution (100 mL). The organic

phase was dried over anhydrous Na2SO4, and concentrated in vacuo. Flash

column chromatography afforded the product (4.07 g, 95%) as white a solid, mp

113-114 °C (Lit.36 mp 113-114 °C); 1H NMR (300 MHz, CDCl3) δ 5.37 (d, 1H),

4.60 (m, 1H), 2.31 (m, 2H), 2.03 (s, 3H), 2.00-0.85 (m, 38H), 0.68 (s, 3H); 13C

NMR (75 MHz, CDCl3) δ 170.5, 139.6, 122.6, 74.0, 56.7, 56.1, 50.0, 42.3, 39.7,

39.5, 38.1, 38.1, 37.0, 36.6, 36.2, 31.9, 31.8, 28.2, 28.0, 27.8, 24.3, 23.8, 22.9,

22.6, 21.4, 21.0, 19.3, 18.7, 11.9; IR (film) ν max 3016, 2938, 2901, 2868, 1731,

1467, 1440, 1375, 1366, 1251, 1216, 1034, 759 cm-1.

Cholest-5-en-3β-ol, methanesulfonate (21). To a solution of cholesterol (100.0

g, 0.259 mol) in anhydrous CH2Cl2 (1 L) at 4 °C was added freshly distilled

triethylamine (54 mL, 0.388 mol) and a solution of methanesulfonyl chloride (21.3

mL, 0.272 mol) in anhydrous CH2Cl2 (100 mL) dropwise. The reaction was

maintained at 4 °C for 30 min, warmed to 22 °C, and stirred for 6 h. When

cholesterol completely disappeared on TLC (hexanes/ethyl acetate, 5:1), the

reaction was concentrated in vacuo. The resulting residue was redissolved in

CH2Cl2 (80 mL), and the product was recrystallized by adding MeOH (500 mL)

40

and collected by vacuum filtration. The filrate was concentrated in vacuo.

Recrystallization of the resulting residue with methanol afforded the second crop

of the product. The combined solid was dried under vacuum to afford the product

(117.1 g, 98%) as a white solid, mp 119-120 °C; 1H NMR (300 MHz, CDCl3) δ

5.41 (s, 1H), 4.56-4.45 (m, 1H), 3.00 (s, 3H), 2.57-2.43 (m, 2H), 2.04-0.81 (m,

38H), 0.67 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 138.6, 123.7, 81.9, 56.5, 56.0,

49.9, 42.2, 39.6, 39.4, 39.1, 38.7, 36.8, 36.3, 36.1, 35.7, 31.8, 31.7, 28.9, 28.1,

27.9, 24.2, 23.8, 22.8, 22.5, 20.9, 19.1, 18.6, 11.8; IR (film) ν max 3029, 2944,

2908, 2861, 1468, 1440, 1415, 1353, 1330, 1173 cm-1; HRMS (ESI+) m/z

487.3237 (M+Na+, C28H48O3SNa requires 487.3222).

Cholest-5-en-3α-ol, methanesulfonate (24). To a solution of epicholesterol

(386 mg, 1.0 mmol) in anhydrous CH2Cl2 (10 mL) at 4 °C was added freshly

distilled triethylamine (280 µL, 2.0 mmol) and methanesulfonyl chloride (85 uL,

1.1 mmol). The reaction was maintained at 4 °C for 30 min, warmed to 22 °C,

and stirred for 6 h. When epicholesterol completely disappeared on TLC

(hexanes/ethyl acetate, 5:1), the reaction was diluted with CH2Cl2 (40 mL). The

organic phase was washed with saturated NaHCO3 solution (50 mL), dried over

anhydrous Na2SO4, and concentrated in vacuo. Flash column chromatography

41

afforded the product (440 mg, 95%) as a white solid; mp 122-123.5 °C (Decomp.);

1H NMR (400 MHz, CDCl3) δ 5.36 (m, 1H), 4.99 (s, 1H), 2.99 (s, 3 H), 2.58 (m,

1H), 2.37 (m, 1H), 2.05-0.86 (m, 40H), 0.68 (s, 3H); 13C NMR (100 MHz, CDCl3)

δ 136.7, 123.6, 79.6, 56.7, 56.1, 49.7, 42.3, 39.7, 39.5, 38.8, 37.3, 36.8, 36.2,

35.8, 32.8, 31.8, 31.7, 28.2, 28.0, 27.3, 24.2, 23.8, 22.8, 22.6, 20,7, 18.9, 18.7,

11.8; IR (film) ν max 3412, 3016, 2961, 2932, 2868, 1457, 1432, 1369, 1344,

1328, 1202, 1174, 1165, 1147, 988, 963, 921, 912, 897, 867, 760 cm-1.

5α-Cholestan-3β-ol, methanesulfonate (25). To a solution of dihydrocholesterol

(3.88 g, 10.0 mmol) in anhydrous CH2Cl2 (50 mL) at 4 °C was added freshly

distilled triethylamine (2.8 mL, 20.0 mmol) and methanesulfonyl chloride (0.85

mL, 11.0 mmol). The reaction was maintained at 4 °C for 30 min, warmed to 22

°C, and stirred for 6 h. When dihydrocholesterol completely disappeared on TLC

(hexanes/ethyl acetate, 5:1), the reaction was concentrated in vacuo. The

resulting residue was redissolved in CH2Cl2 (5 mL). The product was

recrystallized by adding MeOH (50 mL) and collected by vacuum filtration. The

filrate was concentrated in vacuo. Recrystallization of the resulting residue

afforded the second crop of the product. The combined solid was dried under

vacuum to afford product (4.38 g, 94%) as a white solid, mp 112-113 °C (Lit.37

42

mp 116.5-118.5 °C) ; 1H NMR (400 MHz, CDCl3) δ 4.61 (m, 1H), 2.99 (s, 3H),

1.96 (m, 2H), 1.82-0.82 (m, 41H), 0.65 (s, 3H); 13C NMR (100 MHz, CDCl3) δ

82.2, 56.3, 56.2, 54.1, 44.8, 42.6, 39.9, 39.5, 38.8, 36.8, 36.2, 35.8, 35.4, 35.2,

35.1, 31.9, 28.7, 28.5, 28.2, 28.0, 24.2, 23.8, 22.8, 22.6, 21.2, 18.7, 12.2, 12.1;

IR (film) ν max 3027, 2950, 2933, 2863, 1470, 1449, 1416, 1355,1333, 1215,

1173, 1132, 968, 934, 867, 845, 798, 758 cm-1.

3β-Azido-5-cholestene (18). To a solution of 3β-cholest-5-en-3-ol,

methanesulfonate (117.1 g, 0.252 mol) in anhydrous CHCl3 (800 mL), TMSN3

(37.1 mL, 0.277 mol) was added, followed by the addition of BF3·Et2O (63 mL,

0.504 mol). The reaction was stirred at 22 °C for 3 h. When starting material

disappeared on TLC (hexanes), the reaction was slowly poured into aqueous

NaOH solution (1.0 M, 500 mL) and stirred for 5 min. The organic phase was

separated and the aqueous layer was extracted with CHCl3 (200 mL). The

combined organic phase was washed with saturated NaCl solution (500 mL),

dried over anhydrous Na2SO4, and concentrated in vacuo to give the crude

product as light yellow solid. The crude product was dissolved in minimum

amount of hexanes, and loaded to a short (10 cm) and tightly packed pad of

silica gel in a frit funnel (diameter = 15 cm). The azide was then flushed out with

43

hexanes under vacuum. The fractions containing the product were identified by

TLC, combined, and concentrated to afforded the product (88.5 g, 85%) as a

white solid, mp 84-86 °C (Lit.4,17 mp 84-85 °C); 1H NMR (300 MHz, CDCl3) δ 5.38

(s, 1H), 3.22 (m, 1H), 2.28 (d, J = 7.9 Hz, 2H), 2.05-0.85 (m, 38H), 0.68 (s, 3H);

13C NMR (75 MHz, CDCl3) δ 139.8, 122.5, 61.1, 56.7, 56.1, 50.1, 42.3, 39.7, 39.5,

38.1, 37.6, 36.6, 36.2, 35.8, 31.8,31.7, 28.2, 28.0, 27.9, 24.2, 23.8, 22.8, 22.5,

21.0, 19.2, 18.7, 11.8; IR (film) ν max 2934, 2896, 2861, 2097, 1464, 1443, 1377,

1367,1243, 1231 cm-1; HRMS (APCI+) m/z 411.3607 (M+, C27H45N3 requires

411.3613).

3β-Amino-5-cholestene (2). To a solution of 3β-azido-5-cholestene (88.5 g,

0.215 mol) in anhydrous diethyl ether (800 mL) in a 2 L round bottom flask at 4

°C was added LiAlH4 powder (10.0 g, 0.263 mol) in five equal portions. The

reaction was maintained at 4 °C for 30 min, warmed to 22 °C, stirred for 2 h.

When the azide disappeared completely on TLC (hexanes), the reaction was

cooled back to 4 °C and VERY carefully quenched by slow addition of ice-cold

water dropwise. When the bubbling of H2 gas ceased, the resulting solution was

poured into water (500 mL). The organic phase was separated, and the aqueous

phase was extracted with ethyl acetate (300 mL × 2). The combined organic

44

phase was washed with saturated NaCl solution (500 mL), dried over anhydrous

Na2SO4, and concentrated in vacuo. The resulting solid was redissolved in 200

mL of CHCl3 and residual inorganic salts were removed by filtration.

Concentration of the filtrate afforded the product (71.2 g, 86%) as a white solid,

mp 92-94 °C (Lit.4,38 mp 90-91 °C); 1H NMR (300 MHz, CDCl3) δ 5.27 (s, 1H),

2.56 (m, 1H), 2.10-0.81 (m, 40H), 0.64 (s, 3H); 13C NMR (75 MHz, CDCl3) δ

141.7, 120.5, 56.7, 56.1, 51.9, 50.2, 43.3, 42.2, 39.7, 39.4, 38.1, 36.5, 36.1, 35.7,

32.6, 31.8 (× 2), 28.2, 27.9, 24.2, 23.8, 22.7, 22.5, 20.9, 19.4, 18.6, 11.8; IR (film)

ν max 3354, 3260, 3154, 2936, 2896, 2849, 1464, 1437, 1381 cm-1; HRMS (ESI+)

m/z 386.37 (M+H+, C27H48N requires 386.3787).

6β-Azido-3α,5-cyclo-5α-cholestane (23). To a solution of cholest-5-en-3β-ol,

methanesulfonate (5.0 g, 10.8 mmol) in MeOH (100 mL) was added NaN3 (13.0 g,

200.0 mmol). The reaction was refluxed for 8 h. When the mesylate completely

disappeared on TLC (hexanes/ethyl acetate, 5:1), the reaction solution was

concentrated in vacuo. The residue was dissolved in CH2Cl2 (100 mL), washed

with saturated NaHCO3 aqueous solution (100 mL). The organic phase was dried

over Na2SO4 and concentrated in vacuo. Column chromatography (hexanes)

afforded the product (790 mg, 18%) as a white solid, mp 66.5-67.5 °C (Lit.17 mp

45

67-68 °C); 1H NMR (400 MHz, CDCl3) δ 3.27 (t, J = 2.8 Hz, 1H), 2.00 (m, 1H),

1.97-0.78 (m, 40H), 0.72 (s, 1H), 0.70 (dd, J1 = 4.2 Hz, J2 = 4.9 Hz, 1H), 0.53 (dd,

J1 = 5.5 Hz, J2 = 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 64.4, 56.3, 56.1, 47.6,

43.0, 42.7, 40.1, 39.5, 36.2, 36.2, 35.8, 35.3, 33.2, 30.8, 28.3, 28.0, 24.8, 24.2,

23.9, 22.8, 22.6, 22.5, 22.2, 19.0, 18.7, 12.4, 12.2; IR (film) ν max 3319, 3060,

2950, 2869, 2093, 1467, 1382, 1337, 1292, 1268, 1249, 1220, 1019, 969 cm-1.

3β-Chloro-cholest-5-ene (27). To a solution of 3β-cholest-5-en-3-ol,

methanesulfonate (465 mg, 1.0 mmol) in anhydrous CH2Cl2 (10 mL) was added

TMSCl (160 µL, .1.1 mmol). The reaction was cooled to -20 °C. TiCl4 (1.0 M, 0.5

mL, 0.5 mmol) was added dropwise. After 5 min, the reaction was quenched by

adding CH2Cl2 (25 mL) and saturated NaHCO3 aqueous solution (20 mL). The

organic phase was separated and the aqueous phase was extracted with CH2Cl2

(20 mL). The combined organic phase was washed with saturated NaCl aqueous

solution, dried over anhydrous Na2SO4, and concentrated in vacuo. Column

chromatography (hexanes) afforded the product (384 mg, 95%) as a white solid,

mp 93-94 °C (Lit.39 mp 95-95.5 °C); 1H NMR (400 MHz, CDCl3) δ 5.36 (m, 1H),

3.75 (m, 1H), 2.59-2.45 (m, 2H), 2.09-1.78 (m, 6H), 1.61-0.85 (m, 34H), 0.67 (s,

3H); 13C NMR (100 MHz, CDCl3) δ 140.8, 122.4, 60.2, 56.7, 56.1, 50.1, 43.4,

46

42.3, 39.7, 39.5, 39.1, 36.3, 36.2, 35.8, 33.4, 31.8, 31,7, 28.2, 28.0, 24.3, 23.9,

22.8, 22.6, 21.0, 19.2, 18.7, 11.8; IR (film) ν max 3033, 2950, 2935, 2868, 2848,

1668, 1468, 1445, 1432, 1378, 1334, 1216, 1158, 1133, 1026, 1005, 990, 960,

881, 870, 827, 799, 760 cm-1.

3β-Bromo-cholest-5-ene (29). To a solution of 3β-cholest-5-en-3-ol,

methanesulfonate (466 mg, 1.0 mmol) in anhydrous CH2Cl2 (10 mL) was added

TMSBr (145 µL, 1.1 mmol), followed by the addition of BF3·Et2O (250 µL, 2.0

mmol). The reaction was stirred at 22 °C for 1 h. When the mesylate disappeared

on TLC (hexanes), the reaction was quenched by adding CH2Cl2 (25 mL) and

saturated NaHCO3 aqueous solution (20 ml). The organic phase was separated

and the aqueous phase was extracted with CH2Cl2 (20 mL). The combined

organic phase was dried over anhydrous Na2SO4, and concentrated in vacuo.

Column chromatography (hexanes) afforded the product (412 mg, 92%) as a

white solid, mp 97-98 °C (Lit.39 mp 100 °C); 1H NMR (400 MHz, CDCl3) δ 5.36 (m,

1H), 3.92 (m, 1H), 2.75 (m, 1H), 2.58 (m, 1H), 2.19-0.85 (m, 38H), 0.67 (s, 3H);

13C NMR (100 MHz, CDCl3) δ 141.6, 122.3, 56.7, 56.1, 52.6, 50.2, 44.3, 42.3,

40.3, 39.7, 39.5, 36.4, 36.2, 35.8, 34.4, 31.8, 31.7, 28.2, 28.0, 24.3, 23.8, 22.8,

47

22.6, 20.9, 19.3, 18.7, 11.9; IR (film) ν max 3033, 2950, 2902, 2867, 2850, 1665,

1466, 1432, 1376, 1330, 1187, 1151, 1025, 1001, 988, 960, 863, 817cm-1.

3β-Iodo-cholest-5-ene (30). To a solution of 3β-cholest-5-en-3-ol,

methanesulfonate (465 mg, 1.0 mmol) in anhydrous CH2Cl2 (10 mL) at -20 °C

was added TMSI (160 µL, 1.1 mmol), immediately followed by BF3·Et2O (250 µL,

2.0 mmol).The reaction was stirred for 10 min, and quenched by adding CH2Cl2

(20 mL) and saturated NaHCO3 aqueous solution (10 ml). The organic phase

was separated, and the aqueous phase was washed with CH2Cl2 (20 mL). The

combined organic phases was dried over anhydrous Na2SO4 and concentrated in

vacuo. Column chromatography (hexanes) afforded the product (407 mg, 82%)

as a white solid, mp 105-106 °C (Lit.40 mp 106-107 °C); 1H NMR (400 MHz,

CDCl3) δ 5.33 (d, 1H), 4.04 (m, 1H), 2.93 (m, 1H), 2.67 (m,1H), 2.41-0.85 (m,

38H), 0.67 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 142.7, 121.7, 56.7, 56.1, 50.3,

46.4, 42.3, 41.9, 39.6, 39.5, 36.6, 36.4, 36.2, 35.8, 31.7, 31.6, 30.5, 28.2, 28.0,

24.2, 23.8, 22.8, 22.6, 20.8, 19.2, 18.7, 11.8; IR (film) ν max 3033, 2948, 2907,

2850, 1663, 1460, 1429, 1374, 1328, 1179, 1136, 1117, 1020, 996, 988, 957,

818, 798 cm-1.

48

3α-Bromo-5α-cholestane (32). A solution of cholestanol (1.17 g, 3.0 mmol) in

anhydrous THF (70 mL) was cooled to 4 °C by ice-water bath. PPh3 (1.18 g, 4.5

mmol) was added, the reaction was stirred for 5 min, and a solution of CBr4 (1.50

g, 4.5 mmol) in anhydrous THF (30 mL) was added dropwise. The reaction was

slowly warmed to 22 °C and stirred for 16 h. The solvent was removed in vacuo.

The resulting residue was resuspended in hexanes (100 mL) and filtered under

vacuum to remove insoluble material. The filtrate was concentrated in vacuo to

give the crude product as yellow solid. Column chromatography (hexanes)

afforded the product (1.29 g, 95%) as a white solid, mp 102-104 °C; 1H NMR

(400 MHz, CDCl3) δ 4.73 (s, 1H), 1.99-0.81 (m, 40H), 0.79 (s, 3H), 0.65 (s, 3H);

13 C NMR (100 MHz, CDCl3) δ 56.4, 56.2, 55.9, 53.9, 42.6, 40.1, 40.0, 39.5, 37.3,

36.2, 36.2, 35.8, 35.4, 32.9, 31.8, 31.0, 28.2, 28.0, 27.9, 24.2, 23.9, 22.8, 22.6,

20.8, 18.7, 12.3, 12.1; IR (film) v max 2942, 2906, 2861, 1466, 1444, 1383, 1368,

1247, 1208, 760 cm-1; HRMS (CI+) m/z 451.2934 (M+H+ ,C27H48Br requires

451.2939).

49

3β-Azido-5α-cholestane (33). To a solution of 3α-bromo-5α-cholestane (903 mg,

2.0 mmol) in DMSO (20 mL) was added NaN3 (1.3 g, 20.0 mmol). The reaction

was heated to 80 °C and stirred for 6 h. The reaction was cooled to 22 °C and

poured into deionized H2O containing ice (100 mL). The product was extracted

with diethyl ether (40 mL × 4). The combined organic phase was washed with

deionized H2O (100 mL), dried over anhydrous Na2SO4, and concentrated in

vacuo. Flash column chromatography (hexanes) afforded the product (745 mg,

90%) as a white solid, mp 65-67 °C (Lit.28 mp 65-66 °C); 1H NMR (400 MHz,

CDCl3) δ 3.25 (m, 1H), 1.99-0.85 (m, 40H), 0.80 (s, 3H), 0.65 (s, 3H); 13 C NMR

(100 MHz, CDCl3) δ 60.6, 56.4, 56.3, 54.2, 45.2, 42.5, 40.0, 39.5, 37.1, 36.2,

35.8, 35.5 (× 2), 34.0, 31.9, 28.6, 28.2, 28.0, 27.6, 24.2, 23.9, 22.8, 22.5, 21.1,

18.6, 12.2, 12.0; IR (film) v max 2933, 2866, 2091, 1466, 1446, 1382, 1253 cm-1;

HRMS (CI+) m/z 414.3843 (M+H+,C27H48N3 requires 414.3848).

50

3α-Amino-5α-cholestane (34). To a solution of 3α-azido-5α-cholestane (620 mg,

1.5 mmol) in THF (30 mL) and deionized H2O (3 mL) was added PPh3 (1.18 g,

4.5 mmol). The reaction was stirred for 24 h at 22 °C and concentrated in vacuo.

Flash column chromatography (CH2Cl2/MeOH, 10:1 followed by MeOH/Et3N, 99:

1) afforded the product (552 mg, 95%) as a white solid, mp 118-120 °C (Lit.28 mp

118 °C); 1H NMR (400 MHz, CDCl3) δ 2.67 (m, 1H), 1.99-0.85 (m, 40H), 0.77 (s,

3H), 0.64 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 56.4, 56.2, 54.4, 51.1, 45.5, 42.5,

40.0, 39.4, 39.2, 37.6, 36.1, 35.7, 35.4 (× 2), 32.4, 32.0, 28.7, 28.2, 27.9, 24.1,

23.8, 22.7, 22.5, 21.1, 18.6, 12.3, 12.0; IR (film) v max 3354, 3272, 2933, 2911,

2849, 1466, 1449, 1382 cm-1; HRMS (CI+) m/z 388.3939 (M+H+ ,C27H50N

requires 388.3943).

Ethyl 5-[(tert-butoxycarbonyl)-3β-cholestan-3-yl-amino]pentanoate (38). To

DMF (5 mL) was added 3β-amino-5α-cholestane (388 mg, 1.0 mmol), ethyl 5-

bromovalerate (174 µL, 1.1 mmol) and K2CO3 (276 mg, 2.0 mmol). The solution

was heated to 60 °C and stirred for 24 h. The reaction was cooled to 22 °C and

DMF was removed in vacuo. To the resulting residue was added CH2Cl2 (10 mL),

insoluble salts were removed by filtration. The solid was washed with CH2Cl2 (5

mL). To this solution containing the crude secondary amine product was added

51

(Boc)2O (327 mg, 1.5 mmol) and DIEA (0.5 mL, 3.0 mmol). The reaction was

stirred for 4 h at 22 °C and concentrated in vacuo. Flash column chromatography

(hexanes/ethyl acetate, 10:1) afforded the product (431 mg, 67%) as a white

solid, mp 85-87 °C; 1H NMR (400 MHz, CDCl3) δ 4.12 (q, J = 7.1 Hz, 2H), 3.73

(br, 1H), 3.09 (br, 2H), 2.30 (t, J = 7.1 Hz, 2H), 2.01-0.83 (m, 57H), 0.66 (s, 3H);

13C NMR (100 MHz, CDCl3) δ 173.6, 155.5, 79.3, 60.4, 56.9, 56.3 (× 2), 50.3,

42.6 (× 2), 41.6, 39.9, 39.7, 38.6, 37.1, 36.8, 36.3, 35.9, 34.2, 32.0 (× 2), 29.3,

28.7 (Boc, × 3), 28.4, 28.1 (× 2), 26.9, 24.4, 24.0, 23.0, 22.7, 22.6, 21.2, 19.6,

18.9, 14.4, 12.0; IR (film) ν max 2936, 2865, 1736, 1692, 1462, 1409, 1355, 1246,

1165 cm-1; HRMS (CI+) m/z 616.5344 (M+H+, C39H70NO4 requires 616.5305).

tert-Butyl-3β-cholest-5-en-3-yl[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)pro-

pyl]carbamate (40) . To DMF (10 mL) was added 3β-amino-5-cholestene (386

mg, 1.0 mmol), N-(3-bromopropyl)phthalimide (295 mg, 1.1 mmol) and K2CO3

(276 mg, 2.0 mmol). The solution was heated to 60 °C and stirred for 24 h. The

reaction was cooled to 22 °C, and DMF was removed in vacuo. To the resulting

residue was added CH2Cl2 (10 mL), and the insoluble salt was removed by

filtration. The insoluble material was washed with CH2Cl2 (5 mL × 2). To the

combined filtrate containing the crude secondary amine product was added

(Boc)2O (327 mg, 1.5 mmol) and DIEA (0.5 mL, 3.0 mmol). The reaction was

52

stirred for 4 h at 22 °C and concentrated in vacuo. Flash column chromatography

(hexanes/ethyl acetate, 8:1) afforded the product (465 mg, 69%) as a white foam,

mp 59-61 °C; 1H NMR (400 MHz, CDCl3) δ 7.83 (m, 2H), 7.69, (m, 2H), 5.30 (d,

1H), 3.68 (t, 2H), 3.13 (br, 3H), 2.01-0.83 (m, 51H), 0.65 (s, 3H); 13C NMR (100

MHz, CDCl3) δ 168.2 (× 2), 155.2, 141.3, 133.8 (× 2), 132.0 (× 2), 123.2 (× 2),

79.3, 56.6, 56.1 (× 2), 50.1, 42.6 (× 2), 39.8, 39.4 (× 2), 38.3, 36.8, 36.5, 36.1,

35.9, 35.7, 31.2 (× 2), 28.4 (Boc, × 3), 28.2, 27.9, 26.7, 24.2, 23.8 (× 2), 22.8,

22.5, 20.9, 19.3, 18.7, 11.8; IR (film) ν max 2935, 2867, 1772, 1715, 1689, 1467,

1395, 1365, 1238, 1172, 1146, 1031, 888, 756, 720 cm-1; HRMS (CI+) m/z

673.4946 (M+H+, C43H65N2O4, requires 673.4944).

Ethyl 5-[(tert-butoxycarbonyl)-3β-cholest-5-en-3-yl-amino]pentanoate (42).

To DMF (5 mL) was added 3β-amino-5-cholestene (386 mg, 1.0 mmol), ethyl 5-

bromovalerate (174 µL, 1.1 mmol) and K2CO3 (276 mg, 2.0 mmol). The solution

was heated to 60 °C and stirred for 24 h. The reaction was cooled to 22 °C and

DMF was removed in vacuo. To the resulting residue was added CH2Cl2 (10 mL),

insoluble salts were removed by filtration. The solid was washed with CH2Cl2 (5

mL). To this solution containing the crude secondary amine product was added

(Boc)2O (327 mg, 1.5 mmol) and DIEA (0.5 mL, 3.0 mmol). The reaction was

53

stirred for 4 h at 22 °C and concentrated in vacuo. Flash column chromatography

(hexanes/ethyl acetate, 10:1) afforded the product (417 mg, 68%) as a white

solid, mp 78-79°C; 1H NMR (400 MHz, CDCl3) δ 5.30 (d, 1H), 4.10 (q, J = 7.1 Hz,

2H), 3.73 (br s, 1H), 3.09 (br s, 2H), 2.30 (t, J = 7.1 Hz, 2H), 2.01-0.83 (m, 56H),

0.66 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 173.6, 155.5, 141.3, 121.4, 79.3, 60.4,

56.9, 56.3 (× 2), 50.3, 42.6 (× 2), 39.9, 39.7, 38.6, 37.1, 36.8, 36.3, 35.9, 34.2,

32.0 ( 2), 28.7 (Boc, × 3), 28.4, 28.1 (× 2), 26.9, 24.4, 24.0, 23.0, 22.7, 22.6, 21.2,

19.6, 18.9, 14.4, 12.0; IR (film) ν max 2935, 2867, 1737, 1692, 1466, 1409, 1365,

1247, 1173 cm-1; HRMS (CI+) m/z 614.5146 (M+H+, C39H68NO4 requires

614.5148).

tert-Butyl-3β-cholestan-3-yl[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)pro-

pyl]carbamate (44). To a solution of 3β-amino-5α-cholestane (388 mg, 1.0 mmol)

in DMF (5 mL) was added N-(3-bromopropyl)phthalimide (295 mg, 1.1 mmol) and

K2CO3 (276 mg, 2.0 mmol). The reaction was heated to 60 °C and stirred for 24 h.

The solvent was removed in vacuo, and the resulting residue was resuspended

in CH2Cl2 (10 mL). Insoluble salts were removed by vacuum filtration and washed

with CH2Cl2 (5 mL). To the combined filtrate was added (Boc)2O (327 mg, 1.5

mmol) and DIEA (0.5 mL, 3.0 mmol). The reaction was stirred for 4 h at 22 °C

54

and concentrated in vacuo. Flash column chromatography (hexanes/ethyl

acetate, 10:1) afforded the product (479 mg, 71%) as a white solid, mp 65-68°C;

1H NMR (300 MHz, CDCl3) δ 7.74 (m, 2H), 7.63 (m, 2H), 3.81 (br s, 1H), 3.60 (t,

J = 6.8 Hz, 2H), 2.79 (br, 2H), 1.91-0.76 (m, 51H), 0.62 (s, 3H), 0.54 (s, 3H); 13C

NMR (75 MHz, CDCl3) δ 168.2, 155.2, 133.9, 132.1, 123.2, 79.2, 56.4, 56.2, 55.2,

54.2, 45.9, 42.5, 40.8, 40.0, 39.5, 37.8, 36.2, 35.9, 35.8, 35.5, 35.4, 32.9, 32.0,

29.9, 28.7, 28.4 (Boc, × 3), 28.3, 28.0, 26.2, 24.2, 23.8, 22.9, 22.6, 21.1, 18.7,

12.3, 12.1; IR (film) v max 2931, 2866, 1772, 1716, 1689, 1468, 1395, 1366,

1306, 1242, 1172 cm-1; HRMS (CI+) m/z 675.5107 (M+H+, C43H67N2O4 requires

675.5101).

2.10.3 1H NMR Spectra of the Conversion of 6β-Azido-3α,5-Cyclo-5α-

Chlestane (23) to 3β-Azido-5-Chlestene (18).

Compound 23 (103 mg, 0.25 mmol) and TMSN3 (40 µL, 0.28 mmol) were

dissolved in 0.5 mL of CDCl3 in a NMR tube and 1H NMR was taken as 0 min.

(Figure 2.8) Then, BF3·Et2O (63 µL, 0.5 mmol) was added into the tube. 1H NMR

experiments were conducted every 20 min until 120 min. Only the spectra at 20

min (Figure 2.9) and 2 h (Figure 2.10) were shown.

55

Figure 2.8: Compound 23 and TMSN3 at 0 min. 23 (103 mg, 0.25 mmol) and TMSN3 (40 µL, 0.28 mmol) were dissolved in 0.5 mL of CDCl3 in a NMR tube and the 1H NMR spectrum was acquired (0 min).

56

Figure 2.9: Compound 23 and TMSN3 plus BF3·Et2O at 20 min. To the previous mixture in the NMR tube was added BF3·Et2O (63 uL, 0.5 mmol). A 1H NMR spetrum was acquired after 20 min. Over 70% conversion of starting material tothe product could be observed at this time point.

57

2.11 References

1. Wolfrum, C.; Shi, S.; Jayaprakash, K. N.; Jayaraman, M.; Wang, G.;

Pandey, R. K.; Rajeev, K. G.; Nakayama, T.; Charrise, K.; Ndungo, E. M.;

Zimmermann, T.; Koteliansky, V.; Manoharan, M.; Stoffel, M. Mechanisms and

Figure 2.10: Compound 23 and TMSN3 plus BF3·Et2O at 2 h. To the previous mixture in the NMR tube was added BF3·Et2O (63 uL, 0.5 mmol). A 1H NMR was acquired after 2 h. Total disappearance of the starting material indicated that thereaction was complete after 2 h.

58

optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotechnol. 2007, 25,

1149-1157.

2. Sato, S. B.; Ishii, K.; Makino, A.; Iwabuchi, K.; Yamaji-Hasegawa, A.;

Senoh, Y.; Nagaoka, I.; Sakuraba, H.; Kobayashi, T. Distribution and Transport

of Cholesterol-rich Membrane Domains Monitored by a Membrane-impermeant

Fluorescent Polyethylene Glycol-derivatized Cholesterol. J. Biol. Chem. 2004,

279, 23790-23796.

3. Firestone, R. A. Low-density lipoproteins as a vehicle for targeting

antitumor compounds to cancer cells. Bioconjug. Chem. 1994, 5, 105-113.

4. Kan, C. C.; Yan, J.; Bittman, R. Rates of spontaneous exchange of

synthetic radiolabeled sterols between lipid vesicles. Biochemistry 1992, 31,

1866-1874.

5. Spencer, T. A.; Wang, P.; Li, D.; Russel, J. S.; Blank, D. H.; Huuskonen, J.;

Fielding, P. E.; Fielding, C. J. Benzophenone-containing cholesterol surrogates:

synthesis and biological evaluation. J. Lipid Res. 2004, 45, 1510-1518.

6. Mottram L. F.; Boonyarattanakalin, S.; Kovel, R. E.; Peterson, B. R. The

Pennsylvania Green fluorophore: A hybrid of oregon green and Tokyo Green for

the construction of hydrophobic and pH-insensitive molecular probes. Org. Lett.

2006, 8, 581-584.

7. Salmi, C.; Loncle, C.; Vidal, N.; Laget, M.; Letourneux, Y.; Brunel, J. M.

New 3-Aminosteroid Derivatives as a New Family of Topical Antibacterial Agents

Active against Methicillin-Resistant Staphylococcus aureus (MRSA). Lett. Drug

Design Discov. 2008, 5, 169-172.

59

8. Peterson, B. R. Synthetic mimics of mammalian cell surface receptors:

prosthetic molecules that augment living cells. Org. Biomol. Chem. 2005, 3,

3607-3612.

9. Boonyarattanakalin, S.; Hu, J.; Dykstra-Rummel, S.; August, A.; Peterson,

B. R. Endocytic delivery of vancomycin mediated by a synthetic cell surface

receptor: Rescue of bacterially infected mammalian cells and tissue targeting in

vivo. J. Am. Chem. Soc. 2007, 129, 268-269.

10. Sun, Q.; Cai, S.; Peterson, B. R. Selective disruption of early/recycling

endosomes: Release of disulfide-linked cargo mediated by an N-alkyl-3β-

cholesterylamine-capped peptide. J. Am. Chem. Soc. 2008, 130, 10064-10065.

11. Windaus, A.; Adamla, J. Cholesterol. XIII Cholesterylamine. Berichte der

Deutschen Chemischen Gesellschaft 1912, 44, 3051-3058.

12. Boonyarattanakalin, S.; Martin, S. E.; Dykstra, S. A.; Peterson, B. R.

Synthetic mimics of small mammalian cell surface receptors. J. Am. Chem. Soc.

2004, 126, 16379-16386.

13. Patel, M. S.; Peal, W. J. A modifed procedure for the preparation of 3,5-

cyclosteroids. J. Chem. Soc. 1963, 1544-1546.

14. Jarreau, F. X.; Khuonghuu, Q.; Goutarel, R. Steroid alkaloids. XIX. New

method of synthesis of 3-beta-amino-5-steroids. Bull. Soc. Chim. France 1963,

1861-1865.

15. Corey, E. J.; Nicolaou, K. C.; Shibmasaki, M.; Machida, Y.; Shiner, C. S.

Superoxide ion as a synthetically useful oxygen nucleophile. Tetrahedron Lett.

1975, 37, 3183-3186.

60

16. Aneja, R.; Davis, A. P.; Knaggs, J. A. Formation of a 3,5-cyclocholestan-

6α-yl derivative in a nucleophilic substitution reaction of cholesterol. Tetrahedron

Lett. 1975, 12, 1033-1036.

17. Freiberg, L. 6α-Azido-3α, 5α-cyclocholestane. J. Org. Chem. 1965, 30,

2476-2479.

18. Haworth, R. D.; Lunts, L. H. C.; McKenna, J. The constitution of conessine.

Part VIII. Reaction of cholesteryl toluene-p-sulphonate with liquid ammonia. J.

Chem. Soc. 1955, 986-991.

19. Shoppee, C. W.; Summers, G. H. R. Steroids and walden inversion. Part

VII. The stereochemistry and the mechanism of the i-steroid rearrangement. J.

Chem. Soc. 1952, 3361-3374.

20. Stimac, A.; Kobe, Studies on the origin of stereoselectivity in the synthesis

of 1,2-trans glycofuranosyl azides. J. Carbohydr. Res. 2000, 324, 149-160.

21. El Akri, K.; Bougrin, K.; Balzarini, J.; Faraj, A.; Benhida, R. Efficient

synthesis and in vitro cytostatic activity of 4-substituted triazolyl-nucleosides.

Bioorg. Med. Chem. Lett. 2007, 17, 6656-6659.

22. Winstein, S.; Kosower, E. M. Neighboring carbon and hydrogen. XXXIII.

Reactivities of 3,5-cyclocholestan-6-yl derivatives. Strain and reactivity in

homoallylic systems. J. Am. Chem. Soc. 1959, 81, 4399-4408.

23. Galynker, I.; Still, W. C. A simple method for tosylation with inversion.

Tetrahedron Lett. 1982, 23, 4461-4464.

24. Koen, M. J.; Guvader, F. L.; Motherwall, W. B. Observations on the

reaction of xanthate esters with 4-methyl(difluoroiodo)benzene: A new method

61

for the conversion of alcohols to alkyl fluorides. J. Chem. Soc., Chem. Commun.

1995, 1241-1242.

25. Story, P. R.; Clark, B. C. In Carbonium Ions, Volume III; Olah, G. A.,

Schleyer, P. v. R., Eds.; Wiley and Sons: 1972, p 1007-1016.

26. Suarez, Y.; Fernandez, C.; Ledo, B.; Martin, M.; Gomez-Coronado, D.;

Lasuncion, M. A. Sterol stringency of proliferation and cell cycle progression in

human cells. Biochim. Biophys. Acta 2005, 1734, 203-213.

27. Cao, H.; Tokutake, N.; Regen, S. L. Unraveling the mystery surrounding

cholesterol's condensing effect. J. Am. Chem. Soc. 2003, 125, 16182-16183.

28. Casimiro-Garcia, A.; De Clercq, E.; Pannecouque, C.; Witvrouw, M.; Stup,

T. L.; Turpin, J. A.; Buckheit, R. W. J.; Cushman, M. Synthesis and anti-HIV

activity of cosalane analogues incorporating nitrogen in the linker chain. Bioorg.

Med. Chem. 2000, 8, 191-200.

29. Salvatore, R. N.; Yoon, C. H.; Jung, K. W. Synthesis of secondary amines.

Tetrahedron 2001, 57, 7785-7811.

30. Zhou, X-D.; Cai, F.; Zhou, W-S. A stereoselective synthesis of squalamine.

Tetrahedron, 2002, 58, 10293-10299.

31. Srivastava, S. K.; Chauhan, P. M. S.; Bhaduri, A. P. A novel strategy for

N-alkylation of primary amines. Synth. Commun. 1999, 29, 2085-2091.

32. Kung P-P.; Bharadwaj, R.; Allister, S. F.; Cook, D. R.; Kawasaki, A. M.;

Cook, P. D. Solution-phase synthesis of novel liner oxyamine combinatorial

libaries with antibacterial activity. J. Org. Chem. 1998, 63, 1846-1852.

62

33. Boonyarattanakalin, S.; Athavankar, S.; Sun, Q.; Peterson, B. R.

Synthesis of an artificial cell surface receptor that enables oligohistidine affinity

tags to function as metal-dependent cell-penetrating peptides. J. Am. Chem. Soc.

2006, 128, 386-387.

34. Boonyarattanakalin, S.; Martin, S. E.; Sun, Q.; Peterson, B. R. A synthetic

mimic of human Fc receptors: Defined chemical modification of cell surfaces

enables efficient endocytic uptake of human Immunoglobulin-G. J. Am. Chem.

Soc. 2006, 128, 11463-11470.

35. Barragan-Montero, V.; Winum J-V.; Molès, J-P.; Juan E.; Clavel C.;

Montero, J-L. Synthesis and properties of isocannabinoid and cholesterol

derivatized rhamnosurfactants: application to liposomal targeting of keratinocytes

and skin. Eur. J. Med. Chem. 2005, 40, 1022–1029.

36. Eshghi, H.; Shafieyoon, P. P2O5 on SiO2 as a mild and efficient reagent for

acylation of alcohols, phenols and amines under solvent-free conditions J. Chem.

Res. 2004, 802-805.

37. Pinkus, J. L. Pinkus, G.; Cohen, T. A convenient stereospecific synthesis

of axial amines in some steroidal, decalyl, and cyclohexyl systems. J. Org. Chem.

1962, 27, 4356-4360.

38. Haworth, R. D.; McKenna, J.; Powell, R. G. The constitution of conessine.

Part V. Synthesis of some basic steroids. J. Chem. Soc. 1953, 1110-1115.

39. Wagner, A. F.; Wolff, N. E.; Wallis, E. S. Molecular rearrangements in the

sterols. VII. The chemistry of the epi-i-sterols and their rearrangement product. J.

Org. Chem. 1952, 17, 529-541.

63

40. Powell, D. A.; Maki, T.; Fu, G. Stille cross-couplings of unactivated

secondary alkyl halides using monoorganotin reagents. J. Am. Chem. Soc. 2005,

127, 510-511.

Chapter 3

Selective Disruption of Early/Recycling Endosomes: Release of Disulfide-

Linked Cargo Mediated by N-Alkyl-3β-Cholesterylamine-Capped Lytic

Peptides

3.1 Introduction

As mentioned in Chapter 1, the synthetic receptor targeting strategy has

achieved significant success in the delivery of impermeable bioactive molecules

into mammalian cells. However, the entrapment of transported cargo in

endosomes often blocks the access of these molecules to targets in cytosol and

nucleus.1-6 Other carrier delivery systems involving endocytosis mechanisms,

such as liposomes, polymers, and cell-penetrating peptides (CPPs), generally

suffer reduced activity from this issue as well. To attempt to solve this problem,

our laboratory has explored several approaches known to facilitate endosome

disruption in other systems. We have preliminarily explored the use of

endosome-disruptive polyamines,7-9 endoporter,10 proton-pump inhibitors,11-13

synthetic receptors containing pH-responsive imidazoles,14-17 and generation of

reactive radicals by irradiation of a malachite green-containing synthetic

65

receptor.18-21 However, none of these experiments were effective for non-toxic

release of cell impermeable molecules delivered by synthetic receptors.

Many viruses enter host cells via endocytotic mechanisms and use

specific molecules on viral caspids, called fusion proteins (FPs), to disrupt

endosomes and release genetic material into the cytoplasm. For example,

Semliki Forest virus disrupts early endosomes and influenza virus disrupts late

endosomes during the course of infection.22 Therefore, the structures and

mechanism of viral fusion proteins have been intensively studied.23-25 One of the

best studied viral fusion protein is influenza hemagglutinin (HA). It has been

elucidated that after proteolytic cleavage at Arg329 of HA0 (the precursor of HA),

the resulting HA1 subunit is released, whereas the HA2 subunit is activated. In

the lowered pH of endosomes, the HA2 subunit undergoes a conformational

change, relocates the N-terminal domain (20 amino acids, fusion peptide) to the

same side of the transmembrane domain, and inserts deeply into endosomal

membrane to induce viral and host cell membrane mixing.26-27 To mimic this

endosomal disruption strategy, various fusion peptides have been incorporated

into liposomes,28-31 combined with polymers,32-33 or linked to CPPs34 to facilitate

the release of cargo molecules, delivered though a variety of mechanisms. Most

previously reported fusion peptides comprise 20~30 residues.25 Although these

peptides can be prepared on solid phase, their length makes the synthesis costly

and low-yielding, and these peptides can be toxic to cells, which limits their

applications. More recently, Thomas Weber et al. identified a pH-dependent lytic

peptide, PC4, by phage display technology.35 PC4 promotes liposomal leakage

66

comparable to the HA2 fusion peptide, INF7 (24 amino acids), but PC4 is

significantly smaller (12 amino acids), which allowed us to easily tether it to 3β-

cholesterylamine to target this lytic peptide to endosomes.

We describe in this chapter the synthesis of novel 3β-cholesterylamine-

capped PC4 lytic peptides designed to enable membrane-bound disulfide-linked

cargo to selectively escape from early/recycling endosomes of living mammalian

cells.36 Because these endosomes are less acidic and less hydrolytically active

than late endosomes/lysosomes, this approach may be advantageous when

compared to delivery methods that penetrate deeper into the endosomal system.

3.2 Design and Synthesis of N-Alkyl-3β-Cholesterylamine-Capped Lytic

Peptides (45, 46) and a Disulfide-Linked Fluorescent Probe (47)

To selectively deliver compounds into early/recycling endosomes, we

designed and synthesized four derivatives of the dynamic membrane anchor N-

alkyl-3β-cholesterylamine4,5 (45-48, Figure 3.1). Two of these compounds (45,

and red fluorescent 46) incorporate PC4, a pH-dependent membrane-lytic

dodecapeptide previously reported6 by Weber to disrupt membranes of

67

liposomes. Two others comprise the green fluorophore 5-carboxyfluorescein

linked through disulfide (47) and amide (48) bonds. The unmodified PC4 peptide

Figure 3.1: Structures of N-alkyl-3β-cholesterylamine-capped lytic peptides (45,46), a disulfide-Linked fluorescent probe (47), and its non-cleavable control (48).

68

with the amino acid sequence AcNH(SSAWWSYWPPVA)CONH2 (49) was

additionally prepared as a control peptide.

When added to mammalian cells, derivatives of N-alkyl-3β-

cholesterylamine become avidly incorporated in cellular plasma membranes and

engage a membrane trafficking pathway that involves rapid cycling between the

cell surface and intracellular endosomes, similar to many natural cell surface

receptors.37 The partitioning of these compounds between the plasma membrane

and endosomes is affected by the structure of the linker region proximal to the

membrane anchor. In 45-48, the two glutamic acid residues in this region were

installed to enhance the localization of these compounds in endosomes

compared to the plasma membrane. Because early/recycling endosomes are

thought to be oxidizing,38 we hypothesized that the disulfide of 47 should be

relatively stable in these compartments. However, if 47 were exposed to reduced

glutathione (GSH), a thiol present at mM concentrations in the cytosol, this

functional group would be cleaved (Figure 3.2, Panel A).39 Correspondingly,

disruption of early/recycling endosomes loaded with 47 by compounds 45 or 46

was proposed as a mechanism to enable GSH to access these compartments,

reduce the disulfide of 47, and release the soluble fluorophore 50 into the

cytoplasm and nucleus of cells (Figure 3.2, Panel B).

69

3.3 Confocal Microscopy of Early/Recycling Endosome Targeting of the

Fluorescent N-Alkyl-3β-Cholesterylamine-Capped Lytic Peptide (46) and

Disulfide-Linked Probe (47)

To examine the subcellular localization of fluorescent compounds (46, 47),

confocal laser scanning microscopy was employed to analyze living mammalian

cells after treatment with 46 or 47. In Chinese hamster ovary (CHO) cells,

compound 47 was found to become localized in defined intracellular

compartments that reside outside of the cell nucleus. These compartments were

Figure 3.2: Proposed molecular mechanism for the selective release of disulfide-linked fluorophore from early/recycling endosomes mediated by 45. Panel A: Products of cleavage of 47 by glutathione. Panel B: Mechanism of release offluorophore 50 into the cytosol and nucleus of mammalian cells.

70

Figure 3.3: Confocal laser scanning and differential interference contrast (DIC) micrographs of living CHO cells treated with green and red fluorescentcompounds. Panel A: Cell treated with green fluorescent 47 (5 µM) for 12 h followed by red fluorescent Texas Red transferrin (500 nM) for 5 min at 37 °C.Panel B: Cell treated with green fluorescent 47 (5 µM) for 12 h followed by red fluorescent DiI-LDL (8 nM) for 5 min at 37 °C. Panel C: Cell treated with redfluorescent 46 (5 µM) for 12 h followed by green fluorescent Alexa Fluor 488transferrin (610 nM) for 5 min at 37 °C. Panel D: Cell treated with red fluorescent46 (2 µM) and green fluorescent 47 (5 µM) for 12 h at 37 °C. Colocalization of red and green fluorescence is shown as yellow pixels in the DIC overlay images.Arrows point to distinct red fluorescence. Scale bar = 10 microns.

71

identified as early/recycling endosomes by essentially complete intracellular

colocalization with a highly selective early/recycling endosome marker, red

fluorescent transferrin protein (Figure 3.3, Panel A).40 As a control, cells were

similarly treated with red fluorescent DiI-labeled low density lipoprotein (LDL), a

selective marker of late endosomes and lysosomes (Figure 3.3, Panel B).41

Treatment with DiI-LDL showed distinct red fluorescence, indicating that in CHO

cells the 3β-cholesterylamine membrane anchor targets the fluorophore of 47

specifically to early/recycling endosomes rather than late endosomes/lysosomes.

When CHO cells were incubated with red fluorescent 46, followed by green

fluorescent Transferrin-AlexaFluor488, essentially complete colocalization of red

and green fluorescence was observed, indicating that 46 also becomes

selectively localized in early/recycling endosomes (Figure 3.3, Panel C). This

result was further confirmed by the observation of complete overlap of 46 and 47

at concentrations that minimize the disruption of endosomes (Figure 3.3, Panel

D).

3.4 Confocal Microscopy of Endosome Disruption by the N-Alkyl-3β-

Cholesterylamine-Capped Lytic Peptides (45, 46) and the Disulfide-

Linked Fluorescent Probe (47)

Compared to 47 alone, living Jurkat cells treated with 47 and 45 (or 47

and 46) showed a strikingly different pattern of intracellular fluorescence (Figure

72

3.4, Panel A). When combined with 45 or 46, the green fluorescence of 47 was

released from entrapment in early/recycling endosomes and fluorescence was

Figure 3.4: Confocal laser scanning and DIC micrographs of living Jurkatlymphocytes treated with N-alkyl-3β-cholesterylamine-capped lytic peptides and fluorescent probes. Panel A: Jurkat lymphocytes were treated with 47 only (2.5 µM) for 12 h at 37 °C. Panel B: Jurkat lymphocytes were treated with 45 (2 µM) and 47 (2.5 µM) for 12 h at 37 °C. Panel C: Jurkat lymphocytes were treated with 46 (2 µM) and 47 (2.5 µM) for 12 h at 37 °C. Panel D: Jurkat lymphocytes were treated with 45 (2 µM) and 48 (2.5 µM) for 12 h at 37 °C. Panel E: Jurkatlymphocytes were treated with 49 (2 µM) and 47 (2.5 µM) for 12 h at 37 °C. Scale bars = 10 microns.

73

observed in the cytosol and nucleus (Figure 3.3, Panel B and C). Consistent with

the model shown in Figure 3.2, replacement of the disulfide of 47 with the amide

bond of 48 blocked release of the fluorophore (Figure 3.4, Panel D). The red

fluorescence of 46 allowed simultaneous visualization of the linked PC4 peptide

in early/recycling endosomes (Figure 3.4, Panels C). Colocalization of 45 or 46

with 47 in these compartments was required to promote efficient cargo release.

Little effect was observed with the unmodified PC4 peptide (49, Figure 3.4,

Panels E).

Figure 3.5: Inhibition of endosomal escape with bafilomycin A1. Jurkatlymphocytes were treated with green fluorescent 47 (2.5 µM) and red fluorescent 46 (2 µM) for 12 h at 37 °C. In Panel B, bafilomycin A1 (1 µM), a vacuolar H+

ATPase inhibitor that blocks acidification of endosomes, was added to the mediaand was present during the 12 h incubation period. Cells were washed withmedia and imaged by DIC and confocal laser scanning microscopy. Scale bar = 10 microns.

74

To investigate the importance of endosomal acidity on the function of the

PC4 peptide,35 we increased the pH of acidic endosomes with bafilomycin A1.42

This compound blocked release of the fluorophore (Figure 3.5, compare panels A

and B), indicating that 45 or 46 disrupted the endosomal membranes in a pH

dependent manner, and low pH (~ 6) is required to activate the PC4 peptide.

3.5 Flow Cytometry of Endosome Disruption by the N-Alkyl-3β-

Cholesterylamine-Capped Lytic Peptide (45) and the Disulfide-Linked

Fluorescent Probe (47)

To quantitatively evaluate endosome disruption by 45 and 47, flow

cytometry was employed to measure the increase in cellular fluorescence

resulting from release of the fluorophore from the endosomes. To analyze the

dose dependence of endosome disruption by 45 and 47, Jurkat lymphocytes

were treated with 47 (2.5 µM) and 45 (0 to 8 µM) or 49 (2 µM) for 12 h at 37 °C.

The acidity of early/recycling endosomes partially quenches the fluorescence of

47, and treatment with increasing concentration of 45 was observed to enhance

cellular fluorescence as a consequence of disruption of these acidic

compartments (Figure 3.6, Panel A). Through these experiments, 2 µM was

determined to be the most effective concentration of 45. In a time-dependence

experiment, Jurkat lymphocytes were treated with 47 (2.5 µM) and 45 (2 µM) at

37 °C, and cellular fluorescence was measured at different time points (Figure

75

Figure 3.6: Dose dependence and time dependence of endosome disruptionquantified by flow cytometry. Panel A: Jurkat lymphocytes were treated with 47(2.5 µM) and 45 or 49 for 12 h at 37 °C. Panel B: Jurkat lymphocytes weretreated with 47 (2.5 µM) and 45 (2 µM) at 37 °C.

76

3.6, Panel B). Maximum cellular fluorescence was reached at 12 h. The

decrease of flurorescence at longer times is possibly due to leakage or

degradation of intracellular flurophores.

3.6 Evaluation of Cytotoxicity of the N-Alkyl-3β-Cholesterylamine-Capped

Lytic Peptide (45)

The pH-dependence likely reduces the toxicity of endosome disruption by

45 or 46 by limiting effects on the plasma membrane under physiological

conditions (pH ~ 7.4). Consistent with this idea, a CellTiter-Glo luminecence cell

viability assay (Figure 3.7) revealed that 45 is non-toxic under conditions that

disrupt early/recycling endosomes in Jurkat lymphocytes (2 µM).

Figure 3.7: Cytotoxicity of 45 to Jurkat lymphocytes. The viability of the cells wasdetermined by Celltiter-Glo luminecent assay after incubation of 48 h at 37 °C.

77

3.7 A Binary Drug Delivery System: Targeting a Disulfide-Linked Cytotoxin

(52) to Endosomes and Activation by N-Alkyl-3β-Cholesterylamine-

Capped Lytic Peptide (45)

A major challenge associated with cancer chemotherapy is the low

therapeutic index of many cytotoxic agents. Colchicine (61, Figure 3.8) is one

such highly toxic alkaloid that has been isolated from plants for centuries.

Colchicine exerts its toxicity by binding tubulin in the cytosol, and consequently

inhibiting the polymerization of microtubules during mitosis. Unfortunately, poor

cell-type specificity greatly limits applications of colchicine as an anticancer agent

and results in severe side effects.43

To explore a potential application of our novel early/recycling endosome-

targeting system for drug delivery, a colchicine-derived and disulfide-linked

prodrug (52) and a non-cleavable control (53) were designed and synthesized via

the scheme illustrated in Figure 3.8. The disulfide-linked prodrug was designed to

target the tubulin-binding colchicine moiety into the early/recycling endosomes to

minimize its cytotoxic effect. Only upon the activation by a PC4 lipopeptide would

the colchicine moiety be released into the cytosol and kill the cells. This binary

drug delivery strategy was expected to modulate the cytotoxic effect of a

nonselective cytotoxin by entrapping the prodrug in early/recycling endosomes.

The PC4 lytic lipopeptide (45) would be used to release the toxic headgroup in a

controlled manner.

78

To investigate this concept, Jurkat lymphocytes were treated with 52 in the

presence or absence of the lytic PC4 lipopeptide (45). Both confocal microscopy

Figure 3.8: Synthesis of colchicine-cholesterylamine conjugates (52, 53). Reagents and conditions: (a) NH2NH2, EtOH, 50 °C, 4 h; (b) EDC, HOBt, Fmoc-Glu(OtBu)-OH, 4 °C to 22 °C, 12 h; (c) 20 % piperidine, DMF, 22 °C, 30 min; (d)EDC, HOBt, 56/57, 4 °C to 22 °C, 12 h; (e) EDC, HOBt, 60, 22 °C, 12 h; (f) 15 % TFA, CH2Cl2, 22 °C, 12 h.

79

and a CellTiter-Glo luminecent cell viability assay were employed to study this

drug delivery system. As hypothesized, the cell viability assay showed that the

colchicine-cholesterylamine conjugate 52 (IC50 ~380 nM) significantly reduced

the toxicity

of parent compound colchicine (IC50 ~10 nM) by trapping the cytotoxin in

endosomes. However, the estimated IC50 of 52 indicated that this compound still

possesses some toxicity, which is likely due to slight reduction of the disulfide in

endosomes during the 48 hour incubation. This interpretation is supported by the

fact that non-cleavable control 53 was completely nontoxic even at 500 nM.

These results established that the cytotoxicity of an anticancer agent can be

Figure 3.9: Cytotoxicity to Jurkat lymphocytes of 52 the presence or absence of 45 (2 µM) and controls (61, 53). The viability of the cells was determined byCellTiter-Glo luminecent assay after incubation with compounds (0 to 500 nM) for48 h at 37 °C.

80

controlled by linking these molecules to the endosome-targeting 3β-

cholesterylamine membrane anchor. On the other hand, the combination of 52

and 45 (IC50 ~140 nM), was nearly 3 fold as potent as 52 alone, indicating that

activation by 45 induced the release of the colchicine-derived cytotoxic

headgroup (62, Figure 3.11, Panel A).43

The DIC micrographs shown in Figure 3.10 confirmed the results of the

cell viability assay. It was observed that 52 alone didn’t exhibit a significant

cytotoxic effect until the concentration reached 300 nM. In contrast, in the

presence of 2 µM of 45, significant cell death could be observed at 100 nM.

Figure 3.10: DIC micrographs of living Jurkat lymphocytes treated withcolchicine-cholesterylamine conjugate 52 in the presence or absence of PC4 lipopeptide 45. Panel A: Jurkat lymphocytes were treated with 52 only (50 nM to 500 nM) for 48 h at 37 °C. Panel B: Jurkat lymphocytes were treated with 52 (50 nM to 500 nM) in the presence of 45 (2 µM) for 48 h at 37 °C.

81

Inspired by the results from this research, the construction of a more

sophisticated prodrug system (63), designed to release highly toxic colchifoline

(66, IC50 ~10 nM)44 upon cleavage of the disulfide bond through an

intramolecular cyclization reaction (Figure 3.11, Panel B),45 is currently underway.

Figure 3.11: Proposed activation of prodrugs (52, 62) upon endosome disruption. Panel A: Cleavage of conjugate (52) enables release of cytotoxic colchicine derived headgroup (62). Panel B: Cleavage of designed conjugate (63) enables release of highly potent cytotoxin, colchifoline (65), via an intramolecular cyclization reaction upon activation by 45.

82

This system is designed to achieve a more dramatic cytotoxic effect upon

activation by 45.

3.8 Conclusions

In summary, the use of endocytic uptake pathways to deliver poorly

permeable molecules into mammalian cells is often plagued by entrapment and

degradation in late endosomes and lysosomes. As a strategy to prevent the

exposure of delivered molecules to these highly hydrolytic membrane-sealed

compartments, we synthesized derivatives of the membrane anchor N-alkyl-3β-

cholesterylamine that selectively target linked compounds to less hydrolytic

early/recycling endosomes. By targeting a pH-dependent membrane-lytic

dodecapeptide and a disulfide-linked fluorophore to these compartments in

Chinese hamster ovary cells or Jurkat lymphocytes, membranes of

early/recycling endosomes were selectively disrupted, resulting in cleavage of

the disulfide and escape of the fluorophore into the cytosol and nucleus with low

toxicity. Although other pH-dependent peptides, polymers, and liposomes that

disrupt endosomes have been reported,30,31,46 the ability of appropriately

designed N-alkyl-3β-cholesterylamines to deliver cargo into and release disulfide-

linked cargo from relatively nonhydrolytic early/recycling endosomes may be

useful for the delivery of a variety of sensitive molecules into living mammalian

cells.

83

We also demonstrated that the cytotoxic effect of a cell permeable

cytotoxin, colchicine, could be diminished by trapping the molecule in sealed

endocytic vesicles and reactivated by disrupting the endosomes with the PC4

lipopeptide to release its thiol derivative. Future studies will examine a potentially

more potent prodrug that is capable of release of unmodified colchifoline.

3.9 Experimental Section

3.9.1 General

Chemical reagents and solvents were obtained from Acros, Aldrich and

EMD Biosciences. Media and antibiotics were purchased from Mediatech. The

CellTiter Glo reagent was from Promega. Commercial grade reagents were used

without further purification unless otherwise noted. Detailed synthetic procedures

and characterization data for compounds 54-59 are described in the 2008

Masters’ thesis of Ms. Sutang Cai (Peterson Lab, Department of Chemistry,

Penn State University) or reference 36. Compound 60 was synthesized

according to precedures reported in literature.41 Anhydrous solvents were

obtained after passage through a drying column of a solvent purification system

from GlassContour (Laguna Beach, CA). All reactions were performed under an

atmosphere of dry nitrogen. Reactions were monitored by analytical thin-layer

chromatography on plates coated with 0.25 mm silica gel 60 F254 (EMD

84

Chemicals). TLC plates were visualized by UV irradiation (254 nm) or stained

with a solution of phosphomolybdic acid in ethanol (20%). Flash column

chromatography employed ICN SiliTech Silica Gel (32-63 µm). Purification by

preparative reverse phase HPLC employed an Agilent 1100 preparative

pump/gradient extension instrument equipped with a Hamilton PRP-1

(polystyrene-divinylbenzene) reverse phase column (7 µm particle size, 21.5 mm

x 25 cm). The HPLC flow rate was maintained at 25 mL/min for the entire run

unless otherwise noted. Melting points were measured with a Thomas Hoover

capillary melting point apparatus and are uncorrected. Infrared spectra were

obtained with a Perkin Elmer 1600 Series FTIR. NMR spectra were obtained with

Bruker CDPX-300, DPX-300, AMX-360, or DRX-400 instruments with chemical

shifts reported in parts per million (ppm, δ ) referenced to either CDCl3 (1H 7.27

ppm; 13C 77.2 ppm), MeOH-d4 (1H 4.80 ppm; 13C 49.2 ppm), DMSO-d6 (1H 2.50

ppm; 13C 39.5 ppm), or (CH3)4Si (0 ppm). High-resolution mass spectra were

obtained from the Penn State University Mass Spectrometry Facility (ESI and CI).

Peaks are reported as m/z.

85

3.9.2 Synthetic Procedures and Compound Characterization Data

(4S,7S)-7-(2-Carboxyethyl)-4-[({3-(3β-cholest-5-en-3-ylamino)propyl}amino)

carbonyl]-6,9,17,21-tetraoxo-21-{[(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-

tetrahydrobenzo[a]heptalen-7-yl]amino}-12,13-dithia-5,8,16-triazahenicos-

an-1-oic acid (52). Compound 58 (45 mg, 0.034 mmol) was dissolved in DMF (2

mL) containing piperidine (20%) and stirred for 30 min at 22 °C. The solvent was

removed in vacuo, and the primary amine derived from 58 was dissolved in

anhydrous DMF (2 mL). To a solution of N,N’-deacetyl-colchicine-hemiglutaric

acid 60 (19 mg, 0.040 mmol) were added EDC (10 mg, 0.05 mmol) and HOBt (7

mg, 0.05 mmol). After 30 min, the DMF solution of the primary amine derived

from 58 was added. The reaction was stirred for 12 h at 22 °C, the reaction

solution was concentrated in vacuo. The residue was dissolved in CH2Cl2 (10 mL)

containing TFA (15%) and stirred for 12 h at 22 °C. The reaction was

concentrated in vacuo, and the crude product was purified by preparative

reverse-phase HPLC (gradient: 9.95% MeCN, 89.95% H2O, and 0.1% TFA to

99.9% MeCN, 0% H2O, and 0.1% TFA over 25 min; retention time = 18.0 min

(215 nm)), which afforded 52 (42 mg, 95%) as a light yellow solid, mp 153-155

86

°C; 1H NMR (400 MHz, MeOH-d4) δ 7.29 (m, 2H), 7.08 (d, J = 11.1 Hz, 1H), 6.60

(s, 1H), 5.34 (d, J = 4.5 Hz, 1H), 4.35 (m, 1H), 4.11(m, 2H), 3.87 (s, 3H), 3.76 (s,

3H), 3.74 (s, 3H), 3.47 (s, 3H), 3.35 (m, 2H), 3.19 (m, 2H), 2.92(m, 2H), 2.84 (m,

3H), 2.69 (t, J = 6.6 Hz, 2H), 2.57 (m, 2H), 2.49 (m, 1H), 2.30-0.73 (m, 59H), 0.57

(s, 3H); 13C NMR (100 MHz, MeOH-d4) δ 180.7, 176.5, 176.3, 175.3, 174.8,

174,7, 174.6, 174.3, 165.5, 161.0, 155.2, 154.5, 152.2, 142.7, 139.5, 138.8,

137.9, 136.0, 131.3, 126.7, 124.8, 115.3, 108.8, 61.9, 61.7, 59.4, 58.0, 57.5, 57.1,

56.6, 55.1, 54.9, 53.8, 51.3, 43.5, 43.2, 41.0, 40.7, 39.6, 38.6, 38.2, 37.8, 37.3,

37.2, 37.1, 36.9, 36.3, 36.0, 35.7, 35.3, 33.1, 32.9, 31.2 (× 2), 30.6, 29.3, 29.1,

27.6 (× 2), 27.5, 26.2, 25.3, 24.9, 23.2, 22.9, 22.8, 22.1, 19.6, 19.2, 12.3; IR (film)

ν max 3700-2500 (br), 3284, 2942, 2861, 1713, 1651, 1588, 1538, 1488, 1463,

1434, 1254, 1199, 1139, 1018, 840, 793, 755 cm-1; HRMS (ESI-) m/z 1315.6908

(M-H-, C70H103N6O14S2 requires 1315.6974).

(4S,7S)-7-(2-carboxyethyl)-4-[({3-(3β-cholest-5-en-3-ylamino)propyl}amino)

carbonyl]-6,9,13,17,21-pentaoxo-21-{[(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,

7,9-tetrahydrobenzo[a]heptalen-7-yl]amino}-5,8,12,16-tetraazahenicosan-1-

oic acid (53). Compound 59 (45 mg, 0.035 mmol) was dissolved in DMF (2 mL)

87

containing piperidine (20%) and stirred for 30 min at 22 °C. The solvent was

removed in vacuo, and the primary amine derived from 59 was dissolved in

anhydrous DMF (2 mL). To a solution of 57 (19 mg, 0.040 mmol) were added

EDC (10 mg, 0.050 mmol) and HOBt (7 mg, 0.050 mmol). After 30 min, the DMF

solution of the primary amine derived from 59 was added. The reaction was

stirred for 12 h at 22 °C, the reaction solution was concentrated in vacuo. The

residue was dissolved in CH2Cl2 (10 mL) containing TFA (15%) and stirred for 12

h at 22 °C. The reaction was concentrated in vacuo, and the crude product was

purified by preparative reverse-phase HPLC (gradient: 9.95% MeCN, 89.95%

H2O, and 0.1% TFA to 99.9% MeCN, 0% H2O, and 0.1% TFA over 25 min;

retention time = 16.2 min (215 nm)), which afforded 53 (41 mg, 91%) as a yellow

solid, mp 135-137 °C; 1H NMR (300 MHz, MeOH-d4) δ 7.38 (s, 1H), 7.36 (d, J =

11.1 Hz, 1H), 7.16 (d, J = 11.1 Hz, 1H), 6.67 (s, 1H), 5.40 (d, J = 4.4 Hz, 1H),

4.41 (m, 1H), 4.16 (m, 2H), 3.94 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.53 (s, 3H),

3.35 (m, 5H), 2.98-2.88 (m, 3H), 2.57 (m, 1H), 2.41-0.79 (m, 62H), 0.64 (s, 3H);

13C NMR (75 MHz, MeOH-d4) δ 180.8, 176.5, 176.3, 175.1, 174.9, 174.8 (× 2),

174.4, 174.0, 165.5, 155.3, 154.5, 152.2, 142.7, 139.5, 138.8,138.0, 136.0, 131.3,

126.7, 124.8, 115.4, 108.8, 61.9, 61.7, 59.4, 58.0, 57.5, 57.1, 56.6, 55.1, 54.9,

53.9, 51.4, 43.5, 43.2, 41.0, 40.7, 38.2, 37.8, 37.4, 37.3, 37.2 (× 2), 37.1, 37.0,

36.9, 36.8, 36.3, 35.9, 35.6, 33.7, 32.9, 31.3 (× 2), 30.6, 29.3, 29.1, 27.1, 27.5,

26.3, 25.3, 24.9, 23.2, 22.9, 22.8, 22.1, 19.6, 19.2, 12.3; IR (film) ν max 3700-

2500 (br), 3284, 3060, 2943, 2861, 1713, 1652, 1590, 1545, 1489, 1444, 1255,

88

1202, 1179, 1140, 1096, 1019, 840, 722 cm-1; HRMS (ESI-) m/z 1294.7600 (M-

H-, C71H104N7O15 requires 1294.7590).

N-Acetyl-L-seryl-L-seryl-L-alanyl-L-tryptophyl-L-tryptophyl-L-seryl-L-tyrosyl

-L-tryp-tophyl-L-prolyl-L-prolyl-L-valyl-L-alaninamide (49). Peptide synthesis

employed a Burrell Wrist-Action TM Laboratory shaker and standard N-Fmoc

methodology. The peptide was contructed with Rink amide Novagel resin (0.62

mmol/g, 50 mg, 0.032 mmol) using the following Fmoc-protected amino acids:

Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Trp-OH, Fmoc-Tyr(t-Bu)-OH,

Fmoc-Ser(t-Bu)-OH, Fmoc-Glu(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-β-Ala-OH.

Amino acids were consecutively coupled to the resin by addition of DMF solution

(2 mL) of amino acid (4.0 eq.), HOBT (3.8 eq.), HBTU (3.8 eq.) and DIEA (8.0 eq.)

with shaking at 22 °C for 2 h. Deprotection of Fmoc carbamates on the resin was

carried out by addition of piperidine (20%) in DMF (2 mL for 5 min followed by 2

mL for 15 min followed by 2 mL for 5 min (× 2)). After removal of the final N-Fmoc

group of the N-terminal amino acid of 49, the free amine was capped by shaking

the resin for 5 min with acetic anhydride (0.5 M) and 2, 6-lutidine (0.5 M) in DMF

(2 mL). The capped resin was subsequently washed with DMF (2 mL × 5). The

89

peptide was cleaved from the resin with concurrent removal of t-Bu side chain

protecting groups by treatment with TFA/TIPS/H2O (90:8:2) with shaking for 2 h.

The resin was removed by filtration and washed with CH2Cl2 (1 mL × 3). The

filtrates were combined and concentrated in vacuo. The crude product was

dissolved in MeOH (1 mL) and purified by preparative reverse-phase HPLC

(gradient: 90% H2O, 9.9% MeCN, and 0.1 % TFA to 99.9% MeCN and 0.1% TFA

over 20 min; retention time = 10.8 min) to afford 49 as a white solid (8.3 mg,

18.0%). LRMS (ESI+) m/z 1478.0 (M+H+, C74H93N16O17 requires 1478.6).

N-[6-({6-[(N-{5-(3β-Cholest-5-en-3-ylammonio)pentanoyl}-5-oxidanidyl-5-oxi-

danylidene-L-norvalyl-5-oxidanidyl-5-oxidanylidene-L-norvalyl)amino]hex-

anoyl}amino)hexanoyl]-L-seryl-L-seryl-L-alanyl-L-tryptophyl-L-tryptophyl-L-

seryl-L-tyrosyl-L-tryptophyl-L-prolyl-L-prolyl-L-valyl-L-alaninamide (45). The

Figure 3.12: Analytical HPLC profile of the peptide 49 after purification by preparative HPLC. Retention time = 10.8 min. Purity by HPLC > 99%.

90

side-chain protected peptide sequence (EE-ε-Ahx-ε-Ahx-SSAWWSYWPPVA)

was constructed on Rink amide Novagel resin (0.62 mmol/g, 120 mg, 0.076

mmol) using the method described for preparation of peptide 49. After removal of

the N-Fmoc group of the N-terminal amino acid, the free amine was acylated with

5-{(tert-butoxycarbonyl)[(3β)-cholest-5-en-3-yl]amino}pentanoic acid5 using the

standard amino acid coupling protocol. The product was cleaved from the resin

with TFA/TIPS/H2O (90:8:2) by shaking for 2 h and purified by preparative

reverse phase-HPLC (gradient: 90% H2O, 9.9% MeCN, and 0.1% TFA to 99.9%

MeCN and 0.1% TFA over 20 min; retention time = 16.7 min) to afford 45 as a

white solid (27 mg, 15.0%). LRMS (ESI+) m/z 2389.1 (M+H+, C126H180N21O25

requires 2388.9).

Figure 3.13: Analytical HPLC profile of compound 45 after purification by preparative HPLC. Retention time = 16.7 min. Purity by HPLC > 99%.

91

N-(6-{[6-({N-{5-(3β-Cholest-5-en-3-ylammonio)pentanoyl}-5-oxidanidyl-5-oxi-

danyl-idene-L-norvalyl-N6-[4-carboxy-2,5-dichloro-3-(2,4,5,7-tetrachloro-6-

hydroxy-3-oxo-3H-xanthen-9-yl)benzoyl]-L-lysyl}amino)hexanoyl]amino}he-

xanoyl)-L-seryl-L-seryl-L-alanyl-L-tryptophyl-L-tryptophyl-L-seryl-L-tyrosyl-

L-tryptophyl-L-prolyl-L-prolyl-L-valyl-L-alaninamide (46). The side-chain

protected peptide sequence (EK-ε-Ahx-ε-Ahx-SSAWWSYWPPVA) was

constructed on Rink amide Novagel resin (0.62 mmol/g, 60 mg, 0.038 mmol)

using the method described for preparation of 45. After removal of the N-Fmoc

group of the N-terminal amino acid, the free amine was acylated with 5-{(tert-

butoxycarbonyl) [(3β)-cholest-5-en-3-yl]amino}pentanoic acid.5 The product was

cleaved from resin with TFA/TIPS/H2O (90:8:2) by shaking for 2 h and purified by

preparative reverse-phase HPLC (gradient: 90% H2O, 9.9% MeCN, and 0.1 %

TFA to 99.9% MeCN and 0.1% TFA over 30 min; retention time = 18.4 min) to

afford white solid (12 mg, 13.6%), the fully deprotected intermediate bearing an

unmodified Lys residue. This material was dissolved in DMSO (2 mL) and treated

with 6-carboxy-2', 4, 4', 5', 7, 7'-hexachlorofluorescein, succinimidyl ester (5 mg,

92

1.5 eq.) and DIEA (9 mL, 10 eq.). The reaction was stirred overnight at 22 °C.

The DIEA was removed in vacuo, and the product was purified by preparative

reverse-phase HPLC (gradient: 90% H2O, 9.9% MeCN, and 0.1% TFA to 99.9%

MeCN and 0.1% TFA over 20 min; retention time = 18.5 min) to afford 46 as a

purple solid (7.2 mg, 6.6%). LRMS (ESI+) m/z 2952.3 (M+H+, C148H189Cl6N22O29

requires 2952.9).

3.9.3 Biological Assays and Protocols

Cell culture: Jurkat lymphocytes (human acute leukemia, ATCC #TIB-152) were

cultivated in Roswell Park Memorial Institute (RPMI) 1640 media supplemented

with Fetal Bovine Serum (FBS, 10%), penicillin (100 units /mL), and streptomycin

(100 µg/mL). CHO-K1 cells (Chinese hamster ovary cells, ATCC# CCL-61) were

cultivated in F-12K medium supplemented with Fetal Bovine Serum (FBS, 10%),

penicillin (100 units /mL), and streptomycin (100 µg/mL). Both cell lines were

Figure 3.14: Analytical HPLC profile of compound 46 after purification by preparative HPLC. Retention time = 18.5 min. Purity by HPLC > 99%.

93

propagated in a humidified 5% CO2 incubator at 37 °C. Media used for cell

culture and wash steps contained antibiotics and FBS unless otherwise noted.

Microscopy: A Zeiss LSM 5 Pascal confocal laser-scanning microscope fitted

with a Plan Apochromat objective (63×) was employed. Fluorescein and Alexa

Fluor-488 were excited with a 488 nm Argon ion laser (25 mW, 1% laser power)

and emitted photons were collected through 505 nm LP filter. Excitation of 6-Hex,

Texas Red, and DiI employed a 543 nm HeNe laser and emitted photons were

collected through a 560 nm LP filter.

Flow cytometry: Analyses were performed with a Beckman-Coulter XL-MCL

bench-top flow cytometer. Forward-scatter (FS) and side-scatter (SSC) dot plots

afforded cellular physical properties of size and granularity that allowed gating of

live cells. After gating, 10,000 cells were counted. In studies of endosomal

release of fluorescein derivative 50 mediated by 45, the fluorophore was excited

at 488 nm with a 15 mW air-cooled argon-ion laser, the emission was split with a

550 nm dichroic and filtered through a 510 nm long pass filter and 530/30-nm

band pass filter using the XL-MCL cytometer. The PMT voltage for this

instrument was set to 501 for Jurkat cells and 524 for CHO cells.

Colocalization assays by confocal microscopy: CHO cells (1.2 × 105) in

media (2 mL) were cultivated on round collagen-coated coverslips (22 mm, BD

BioCoatTM) in a 6-well plate. After incubation at 37 °C for 24 h, media was

94

discarded and replaced with fresh media (2 mL). The cells were treated with 46

(final concentration = 2 µM) and/or 47 (final concentration = 5 µM) in DMSO (final

[DMSO] = 1%) at 37 °C for 12 h followed by washing with fresh media (2 mL).

Cells treated with 46 and 47 were immediately analyzed by confocal microscopy

after the wash step. Cells treated with 48 alone were incubated with media

containing Texas Red Transferrin (Invitrogen, final concentration = 500 nM) or

DiI-LDL (final concentration = 8 nM). Cells treated with 46 were incubated with

media containing Transferrin-Alexa Fluor488 (final concentration = 610 nM).

Cells treated with these fluorescent protein markers of early/recycling or late

endosomal/lysosomal compartments were incubated at 37 °C for 5 min, washed

with fresh media (2 mL), and imaged by confocal microscopy.

Cytotoxicity assay of 45: Jurkat lymphocytes (7 × 104) in media (100 µL) were

loaded on a 96-well plate. To these cells, increasing concentrations of 45 in

DMSO (final [DMSO] = 1%) were added. After 48 h of incubation, cells from each

well (10 µL) were transferred to an opaque 96-well plate and treated with

CellTiter-Glo reagent (20 µL, Promega) according to the Promega protocol

provided with the reagent. After incubation at 22 °C for 10 min, the luminescence

of the samples was measured with a Packard Fusion microplate reader.

Cell viability assay and microscopic analysis of colchicine-

cholesterylamine conjugate (52): Jurkat lymphocytes (7 × 104) in media (100

µL) were loaded on a 96-well plate. To these cells, increasing concentrations of

95

61, 53, 52, and 52 with 45 (2 µM) in DMSO (final [DMSO] = 1%) were added.

After 48 h of incubation, cells from each well (10 µL) were transferred to an

opaque 96-well plate and treated with CellTiter-Glo reagent (20 µL, Promega)

according to the manufacturer’s instruction. After incubation at 22 °C for 10 min,

the luminescence of the samples was measured with a Packard Fusion

microplate reader. For microscopic study, the cells were spinned down and

resuspended in 30 uL of fresh media, and loaded to microplates for imaging.

　

Endosome inhibition assay with bafilomycin A1: Jurkat lymphocytes (5 × 105)

in media (500 µL) were pretreated with bafilomycin A1 (final concentration = 1

µM) in DMSO (final [DMSO] = 2%) for 1 h. To these cells was added 47 (final

concentration = 2.5 µM) and 46 (final concentration = 2 µM) in DMSO (final

[DMSO] = 1%). After incubation at 37 °C for 12 h, the cells were washed with

fresh media (500 µL) and resuspended in fresh media (125 µL) for analysis by

confocal microscopy.

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96

2. Hussey S. L.; Peterson, B. R. Efficient delivery of streptavidin to

mammalian cells: Clathrin-mediated endocytosis regulated by a synthetic ligand.

J. Am. Chem. Soc. 2002, 124, 6265-6273.

3. Martin, S.; Peterson, B. R. Non-natural cell surface receptors: Synthetic

peptides capped with N-cholesterylglycine efficiently deliver proteins into

mammalian cells. Bioconjugate Chem. 2003, 14, 67-74.

4. Boonyarattanakalin, S.; Martin, S. E.; Dykstra, S. A.; Peterson, B. R.

Synthetic mimics of small mammalian cell surface receptors. J. Am. Chem. Soc.

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5. Boonyarattanakalin, S.; Athavankar, S.; Sun, Q.; Peterson, B. R.

Synthesis of an artificial cell surface receptor that enables oligohistidine affinity

tags to function as metal-dependent cell-penetrating peptides. J. Am. Chem. Soc.

2006, 128, 386-387.

6. Boonyarattanakalin, S.; Martin, S. E.; Sun, Q.; Peterson, B. R. A synthetic

mimic of human Fc receptors: Defined chemical modification of cell surfaces

enables efficient endocytic uptake of human Immunoglobulin-G. J. Am. Chem.

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7. Lemkine, G. F.; Demeneix, B. A. Polyethylenimines for in vivo gene

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U.S.A. 1990, 87, 3655-3659. (b) Cotton, M.; Langle-Rousault, F.; Kirlappos, H.;

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Wagner, E.; Zatloukal, K.; Phillips, S.; Curiel, D. T.; Birnstiel, M. L. High-efficiency

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14. Verma, S. K.; Mani, P.; Sharma, N. R.; Krishnan, A.; Kumar, V. V.; Reddy,

B. S.; Chaudhuri, A.; Roy, R. P.; Sarkar, D. P. Histidylated lipid-modified sendai

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15. Kumar, V. V.; Pichon, C.; Refregiers, M.; Guerin, B.; Midoux, P.;

Chaudhuri, A. Single histidine residue in head-group region is sufficient to impart

remarkable gene transfection properties to cationic lipids: evidence for histidine-

mediated membrane fusion at acidic pH. Gene Therapy, 2003, 10, 1206-1215.

16. Ihm, J-E.; Han, K-O.; Han, I-K.; Ahn, K-D.; Han, D-K.; Cho, C-S. High

transfection efficiency of poly(4-vinylimidazole) as a new gene carrier.

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17. Park, J. S.; Han, T. H.; Lee, K. Y. ; Han, S. S.; Hwang, J. J.; Moon, D. H.;

Kim, S. Y.; Cho, Y. W. N-acetyl histidine-conjugated glycol chitosan self-

assembled nanoparticles for intracytoplasmic delivery of drugs: Endocytosis,

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18. Maiolo, J. R.; Ottinger, E. A.; Ferrer, M. Specific redistribution of cell-

penetrating peptides from endosomes to the cytoplasm and nucleus upon laser

illumination. J. Am. Chem. Soc. 2008, 126, 15376-15377.

19. Prasmickaite, L.; Høgset, A.; Selbo P. K.; Engesaeter, B. Ø.; Hellum, M.;

Berg K. Photochemical disruption of endocytic vesicles before delivery of drugs:

a new strategy for cancer therapy. Br. J. Cancer. 2002, 86, 652-657.

20. Shiraishi, T.; Nielsen, P. E. Photochemically enhanced cellular delivery of

cell penetrating peptide-PNA conjugates. FEBS Lett. 2006, 580,1451-1456.

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21. Liao, J. C.; Roider, J.; Jay, D. G. Chromophore-assisted laser inactivation

of proteins is mediated by the photogeneration of free radicals. Proc. Natl. Acad.

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22. Lakadamyali, M.; Rust, M. J.; Zhuang, X. Endocytosis of influenza viruses.

Microbes Infect. 2004, 6, 929-836.

23. Tamm, L. K.; Crane, J.; Kiessling, V. Membrane fusion: a structural

perspective on the interplay of lipids and proteins. Curr. Opin. Struc. Biol. 2003,

13, 453-466.

24. Dimitrov D. S. Virus entry: molecular mechanisms and biomedical

applications. Nat. Rev. Microbiol. 2004, 2, 109-122.

25. Martin, I.; Ruysschaert, J-M. Common properties of fusion peptides from

diverse systems. Bioscience Rep. 2000, 20, 483-500.

26. Han, X.; Bushweller, J. H.; Cafiso, D. S.; Tamm, L. K. Membrane structure

and fusion-triggering conformational change of the fusion domain from influenza

hemagglutinin. Nat. Struct. Biol. 2001, 8, 715-720.

27. Skehel, J. J.; Cross, K.; Steinhauer, D.; Wiley, D. C. Influenza fusion

peptides. Biochem. Soc. T. 2001, 29, 623-626.

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mediated release of folate-targeted liposome contents from endosomal

compartments. J. Am. Chem. Soc. 1996, 118, 1581-1586.

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Storm W. Functional characterization of an endosome-disruptive peptide and its

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application in cytosolic delivery of immunoliposome-entrapped proteins. J. Biol.

Chem. 2002, 277, 27135-27143.

30. Turk, M. J.; Reddy, J. A.; Chmielewski, J. A.; Low, P. S. Characterization

of a novel pH-sensitive peptide that enhances drug release from folate-targeted

liposomes at endosomal pHs. Biochim. Biophys. Acta 2002, 1559, 56-68

31. Kakudo, T.; Chaki, S.; Futaki, S.; Nakase, I.; Akaji, K.; Kawakami, T.;

Maruyama, K.; Kamiya, H.; Harashima, H. Transferrin-modified liposome

equipped with a pH-sensitive fusogenic peptide: an artificial viral-like delivery

system. Biochemistry 2004, 43, 5618-5628.

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peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.

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endosomes: Release of disulfide-linked cargo mediated by an N-alkyl-3β-

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of disulfide-based antibody-drug conjugates. Proc. Natl. Acad. Sci. U.S.A. 2005,

102, 17987-17992.

39. Saito, G.; Swanson, J. A.; Lee, K. D. Drug delivery strategy utilizing

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Transferrin receptor recycling in the absence of perinuclear recycling endosomes.

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Chapter 4

Mimicry of Exposed Phosphatidylcholine on Damaged Cells: Synthetic Cell

Surface Receptors that Bind C-Reactive Protein Promote Apoptosis of

Lymphocytes

4.1 Introduction

C-Reactive protein (CRP), a member of the pentraxin protein family, is an

ancient component of the innate immune system.1,2 Homologous proteins are

found in species as evolutionarily diverse as humans and horseshoe crabs.3

Dramatically upregulated (up to 1,000 fold) in blood during inflammation, this

pentameric protein binds the abundant lipid phosphocholine exposed on the

surface of foreign pathogens4, apoptotic, or dead host cells.5,6 Ordinarily,

phosphatidylcholine (67) is tightly packed in cellular plasma membranes and is

inaccessible to CRP. However, on diseased or damaged cells, the

phosphocholine (PC) headgroup of phosphatidylcholine or other oxidized

phospholipids aberrantly protrudes from the lipid bilayer, enabling binding of CRP

to cell surfaces in a Ca2+ dependent manner.7 This molecular recognition event

can activate the classic complement system8-11 to lyse foreign pathogens or

opsonize damaged cells for elimination by phagocytic cells.12-17 It has been

104

elucidated that the plate-shaped CRP is composed of five noncovalently

associated subunits.18 Each identical subunit has a PC-binding site on the

recognition face, whereas the other face of CRP, known as the effector face,

interacts with complement C1q8-11 and Fcγ receptors12-17 on phagocytes (Figure

4.1).

Other than important roles in acute phase immune response, a

relationship of minor eleviated CRP level (2 µg/mL-10 µg/mL) and various

chronic diseases, such as atherosclerosis19-24, diabetes25-27, and colon

cancer,28,29 has recently emerged. Intensive investigations have been devoted to

examine potential biological effects of CRP on various types of cells at normal

levels (<2 µg/ mL) or minor elevated plasma levels.

Figure 4.1: Structure of CRP. Panel A: Side view of CRP and proposed interaction with C1q. Panel B: Bottom view of CRP and PC-binding sites.1 This figure is modified from Figure 2. in C-reactive Protein [Black S. et al. J. Biol. Chem., 2004] under the copyright permission policy of J. Biol. Chem.

105

We report here an efficient synthesis of a series of PC-containing artificial

cell surface receptors (68-71) that are designed to mimic the phosphatidylcholine

(67) exposed on damaged cells. Our studies of the binding of CRP to the

synthetic receptors on the surface of Jurkat lymphocytes lead to the discovery

that apoptosis is induced by the interactions of both components. This type of cell

apoptosis appears to be a consequence of intensive cell aggregation that results

from the multivalent binding of CRP to the receptors on the surface of adjacent

cells, and this process is induced through a caspase-dependent pathway.

4.2 Design and Synthesis of PC-containing Synthetic Receptors (70-73)

3β-Cholesterylamine derived synthetic receptors can be efficiently loaded

onto mammalian cell plasma membranes.30 Derivatives of this membrane anchor

rapidly cycle between the cell surface and intracellular endosomes, similar to

many natural cell surface receptors. Hence, we hypothesized that synthetic

receptors containing PC headgroups (70-73, Figure 4.2) might mimic the

exposure of phosphocholine on damaged or apoptotic cells and promote the

binding of CRP to plasma membranes. β-Alanine subunits were installed in the

linker region of these compounds to confer metabolic stability, favor localization

of compounds on the cell surface, and maximize accessibility of the attached

headgroup.31 The recruitment of CRP to the cell surface loaded with the PC-

106

containing synthetic receptors could be a novel platform for the study of the

interactions of CRP with plasma membranes of living cells.

To test this hypothesis, we synthesized derivatives of N-alkyl-3β-

cholesterylamine linked to phosphocholine with β-alanine and hexanoic acid

subunits (70-73, Figure 4.3). Starting from the membrane anchor building block

Figure 4.2: Structures of synthetic receptors (70-73) that mimic phosphatidylcholine.

107

(40), the phthalimide was deprotected with hydrazine in excellent yield to provide

the free amine. The β-alanine linker units were installed by standard Fmoc

deprotection and EDC coupling reactions to generate the precursors with

different linker lengths (74-76). The PC headgroup was installed by coupling

phosphocholine hydroxyhexanoic acid (69, see experimental section), which was

prepared from phosphocholine calcium salt (68), to the corresponding amine

precursors (40, 74-76) in high yields. Finally, after the Boc protecting group was

removed with TFA, the final products (70-73) were purified by preparative

reverse phase HPLC.

Figure 4.3: Synthesis of synthetic receptors (70-73) that mimic phosphatidylcholine. Reagents and conditions: (a) NH2NH2, EtOH, 50 °C, 4 h; (b) EDC, HOBt, 69, CH2Cl2/iPrOH, 22 °C, 12 h; (c) 15 % TFA, CH2Cl2, 22 °C, 12 h.(d) EDC, HOBt, Fmoc-β-alanine, 4 °C to 22 °C, 12 h; (e) 20% piperidine, DMF,22 °C, 30 min.

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4.3 Cytotoxic Effect of CRP to Jurkat Lymphocytes Loaded with Synthetic

Receptors (73)

To test the binding of CRP to the synthetic receptor on the cell surface,

commercially available CRP was labeled with green fluorescent Alexa Fluor 488

(AF488).32 In a cellular binding assay, Jurkat lymphocytes were treated with 73 (1

µM) for 1 hour and washed to remove unincorporated receptors. The cells were

subsequently incubated with CRP-AF488 for 6 h. After washing with media, the

cells were subjected to confocal microscopy. The confocal micrographs (Figure

4.4) showed that CRP-AF488 bound cells loaded with 73. However, massive cell

death was observed.

Figure 4.4: Confocal micrographs of Jurkat lymphocytes treated with 73 and CRP-AF488. Jurkat lymphocytes were treated were treated with 73 (1 µM) for 1 h at 37 °C, then washed with fresh media, and incubated with CRP-AF488 (10 µg/mL) for 6 h at 37 °C.

109

To examine the cause of the cell death, a qualitative cellular viability assay

was conducted. In this assay, Jurkat cells were treated with 1% DMSO or 73 at 1

µM for 1 hour. After washing with media, cells were incubated in media only or

media containing 10 µg/mL of CRP at 37 °C for 12 h before microscopic anaylsis.

The DIC images (Figure 4.5) revealed that 73 or CRP alone are non-toxic to the

cells, treatment with 73 and CRP engendered significant cytotoxicity.

4.4 Characterization of Cell Death Induced by 73 and CRP

To identify the type of cell death observed, we characterized this cellular

event with both cellular and biochemical assays. First, the morphological

changes associated with cell death were studied by microscopy. The DIC images

Figure 4.5: DIC micrographs of the cytotoxic effect of 73, CRP, and 73 and CRP. Jurkat lymphocytes were treated were treated with 1% DMSO or 73 (1 µM) for 1 h at 37 °C, then washed with fresh media, and incubated in the presence orabsence of CRP (10 µg/mL) for 12 h at 37 °C.

110

showed that the dying cells had some distinctive features, such as cell shrinkage,

nuclear condensation, budding, and formation of small apoptotic bodies, all of

which match the morphological features of apoptosis (Figure 4.6).33,34

Compared to necrosis, also known as accidental cell death, apoptosis is a

programmed, physiological mode of cell death. During early apoptosis,

phosphatidylserine (PS), normally located on the inner leaflet of the cell

membrane, is translocated to the outer leaflet without imparing membrane

integrity.35-38 Annexin V, a human vascular anticoagulant, has high affinity for PS.

Green fluoresent annexin V has been widely used with a red membrane

Figure 4.6: Morphological study of cell death induced by 73 and CRP. Panel A: Established morphological features of two distinctive types of cell death, necrosis and apoptosis.33 Panel B: Morphological features of Jurkat cells treated with 73(1 µM) for 1 h followed by CRP (10 µg/mL) for 6 h at 37 °C. The representitive cells were pointed with arrows.

111

impermeable DNA stain, such as propidium iodide (PI) and 7-amino-actinomycin

D (7-AAD), to distinguish normal, apoptotic, and dead cells.39-40 Staining of

treated Jurkat cells with annexin V-AF488 and PI revealed that most of the dying

cell exposed PS on their surface but maintained their membrane integrity,

indicating that this type of cell death was apoptosis.

Figure 4.7: Confocal microscopy of phosphatidylserine (PS) exposure andpermeability of the membrane of dying cells induced by 73 and CRP. Panel A: Illustrations of the membrane features of live, apoptotic, and dead cells. Panel B: Confocal micrographs of the Jurkat lymphocytes (treated with 73 (1 µM) for 1 h followed by CRP (10 µg/mL) for 6 h at 37 °C) stained with annexin V-AF488 and propidium iodide (PI). The live cells were double negative, the apoptotic cellswere green, and the dead cells were both red and green.

112

To confirm the observations from confocal microscopy, flow cytometry was

employed to quantitatively measure the percentage of the apoptotic and dead

cells in a population after treatment with 73 and CRP (Figure 4.8). It was

observed that most cells in the control group were living cells. However, in the

group treated with receptor 73 (1 µM) and CRP (10 µg/mL), 35.1% of the cells

were apoptotic, and 4.3% of the cells were dead.

Figure 4.8: Flow cytometry of phosphatidylserine (PS) exposure and permeabilityof the membrane of dying cells induced by 73 and CRP. Panel A: Dot plot of negative control Jurkat lymphocytes (treated with 1% DMSO for 1 h and mediafor 6 h at 37 °C) stained with annexin V-AF488 and PI. Panel B: Dot plot of Jurkat lymphocytes (treated with 73 (1 µM) for 1 h followed by CRP (10 µg/mL) for 6 h at 37 °C) stained with annexin V-AF488 and PI. The live cells were gated as doubleFITC-/PI-, the apoptotic cells were gated as FITC+/PI-, and the dead cells were gated as both FITC+/PI+.

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Other than morphological changes and PS externalization, extensive DNA

degradation from the activation of endonucleases is another hallmark of

apoptosis.41-43 Since the products of DNA degradation are nucleosomal and

oligonucleosomal DNA sections, apoptotic DNA samples typically exhibit a

characteristic “Ladder” pattern on an agarose gel. A DNA fragmentation assay

was performed to further confirm cell death via apoptosis (Figure 4.9). The gel

indicated that the DNA sample of the cells treated with receptor 73 and CRP

(lane 3) showed a typical DNA ladder comparable to that of the cells treated with

Figure 4.9: DNA Fragmentation assay. The DNA sample of the Jurkat cellstreated with 73 and CRP (73 (1 µM) for 1 h followed by CRP (10 µg/mL) for 6 h at 37 °C) exhibited a distinctive DNA ladder pattern on gel.

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Figure 4.10: Annexin V-binding assays. Upper panel: Dose-dependence on 73 (0 to 2 µM). Lower Panel: Dose-dependence on CRP (0 to 10 µg/mL). Jurkat lymphocytes were treated with 73 for 1 h at 37 °C, washed with media, and treated with CRP for 6 h at 37 °C. The cells were stained with annexin V-AF488 and PI before flow cytometric analysis.

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the apoptosis-inducing agent camptothecin (lane 7). When the broad-spectrum

caspase inhibitor zVAD-fmk was added to the cells treated with 73 and CRP

(lane 1), the DNA ladder completely disappeared, indicating that DNA

degradation is downstream of caspase activation in this type of apoptosis.

4.5 Dependence of Apoptosis and Viability on 73 and CRP

To quantify the dose dependence of cell apoptosis on receptor 73, the

concentration of CRP was fixed at 10 µg/mL and the concentration of 73 was

increased from 0 to 2 µM. The percentages of apoptotic and dead cells were

determined by annexin V-binding assay using flow cytometry (Figure 4.10, Panel

A). The data showed clear dose dependence on the concentration of 73, and

apoptosis dramatically increased when the concentration of 73 reached 1 µM. In

another annexin V-binding assay, the concentration of 73 was fixed at 2 µM and

concentrations of CRP were increased from 0 to 10 µg/mL (Figure 4.10, Panel B).

CRP similarly induced dose-dependent binding of annexin V, indicating that the

cell apoptosis is induced by the interaction of 73 and CRP.

The cytotoxicity of this system was evaluated using commercial CellTiter-

Glo luminescent cell viability assays. Cell death was dependent on the

concentrations of both receptor 73 and CRP (Figure 4.11)., Nearly 90% of the

cells were dead after 24 h, using 2 µM of 73 and 10 µg/mL of CRP.

116

Figure 4.11: CellTiter-Glo luminescent cell viability assays. Upper panel: Dose-dependence on 73 (0 to 2 µM). Lower panel: Dose-dependence on CRP (0 to 10 µg/mL). Jurkat lymphocytes were treated with 73 for 1 h at 37 °C, washed with media, and treated with CRP for 24 h at 37 °C. The cell samples were treatedwith CellTiter-Glo reagent (Promega) for 30 min at 22 °C, before theluminescence of the samples were measured.

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4.6 Structure-Activity Relationships (SAR) of Synthetic Receptors (70-73)

Binding of CRP to the cell surface depends on the extent of exposure of

phosphocholine on the plasma membrane. To investigate the relationship

between linker length of synthetic receptors and potency in inducing cell

apoptosis, Jurkat lymphocytes were treated with 70-73 (2 µM) and CRP (10

µg/mL). Apoptosis and death were evaluted by flow cytometry with an annexin V-

binding assay.

Whereas the receptor with shortest linker (70) showed the least activity,

the longest compound (73) was the most active in inducing cell apoptosis.

Meanwhile, it was also observed that there was no significant difference between

Figure 4.12: Structure-activity relationships (SAR) of receptors (70-73) in inducing Jurkat cell apoptosis. Jurkat cells were treated with 70-73 for 1 h at 37 °C, washed with media, and treated with CRP (2 µg/mL) for 6 h at 37 °C. The cells were stained with annexin V-AF488 and PI before flow cytometric analysis.

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the potency of the receptors with one (71), two (72), and three β-alanine units

(73).

4.7 Mechanisms of Apoptosis Induced by 73 and CRP

Due to the fact that CRP is a highly conserved protein among different

species, it is possible that the human CRP used in these experiments cross

reacted with bovine complement proteins in the culture media to cause cell

apoptosis.44

Figure 4.13: Effect of complement proteins in FBS on cell apoptosis induced by73 and CRP. Jurkat lymphocytes were treated with 73 (2 µM) for 1 h at 37 °C, washed with media, and treated with CRP (10 µg/mL) for 6 h at 37 °C. Panel A: Experiment with media containing 10% FBS. Panel B: Experiment with mediacontaining 10% heat-inactivated FBS. The cells were stained with annexin V-AF488 and PI before confocal microscopic analysis.

119

However, this hypothesis was not supported by confocal microscopic

analysis of Jurkat cells treated with 73 and CRP in heat-inactivated FBS, which

had no effect on cell apoptosis (Figure 4.13).

Through the use of confocal microscopy, a distinctive feature of Jurkat

cells treated with 73 and CRP, intensive cell aggregation, was found to be

correlated with the cell apoptosis. By using green fluorescent CRP-AF488, CRP

was found to bind synthetic receptors on cell surfaces within 5 min. CRP

Figure 4.14: Membrane association of CRP-AF488 and corresponding cell aggregation and apoptosis. Jurkat lymphocytes were treated with 73 for 1 h at 37 °C, washed with media, and treated with CRP-AF488 for 6 h at 37 °C. The cells were washed with fresh media before confocal microscopic analysis.

120

subsequently aggregated on the cell surface. At low receptor and CRP

concentrations (Figure 4.14, Row 1), the binding complexes of 73 and CRP-

AF488 were mostly internalized by the cells with little cytotoxic effect. However,

at higher concentrations of 73 and CRP (Figure 4.14, Row 2 and 3), the

aggregation of binding complexes on the cell surface caused adjacent cells to

cluster tightly after 30 min and cell apoptosis was observed after 6 h. The

confocal micrographs in Figure 4.14 revealed a clear relationship between the

intensity of cell aggregation and the extent of cell apoptosis.

Figure 4.15: Caspase inhibition assay by confocal microscopy. Panel A: Jurkatlymphocytes were treated with 73 (2 µM) for 1 h at 37 °C with zVAD-fmk (50 µM), washed with media, and treated with CRP-AF488 for 6 h at 37 °C with zVAD-fmk (50 µM). Panel B: Jurkat lymphocytes were treated with 73 (2 µM) for 1 h at 37 °C, washed with media, and treated with CRP-AF488 for 6 h at 37 °C.The cells were stained with annexin V-AF488 and PI before confocal microscopic analysis.

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Caspases are cysteine-aspartic acid proteases with specificity for an

aspartic acid in the P1 position of the substrate and play an important role in

many types of cell apoptosis.45-48 Our previous DNA fragmentation assay showed

that caspases were involved in the DNA degradation event. Therefore, we

performed a caspase inhibtion assay to further investigate the involvement of

caspases in this type of cell apoptosis. The confocal microscopic analysis

showed that addition of pan-caspase inhibitior zVAD-fmk significantly alleviated

cell apoptosis induced by 73 and CRP, which was further quantitatively

confirmed by the flow cytometry, establishing that this type of apoptosis is

induced through a caspase-dependent pathway.

Figure 4.16: Caspase inhibition assay by flow cytometry. Jurkat lymphocyteswere treated with 73 (2 µM) for 1 h at 37 °C w/wo zVAD-fmk (50 µM), washed with media, and treated with CRP-AF488 for 6 h at 37 °C w/wo zVAD-fmk (50 µM). The cells were stained with annexin V-AF488 and PI before flow cytometric analysis.

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4.8 Conclusions

In conclusion, we synthesized a series of PC-containing receptors (70-73)

that recruit CRP to the surface of Jurkat lymphocytes. The binding of CRP to

synthetic receptor 73 on Jurkat cells induced significant cell death, which was

identified as apoptosis by morphological and biochemical assays. The

investigation of the mechanism of this type of apoptosis suggested that intensive

cell aggregation induced by 73 and CRP induced cell apoptosis through a

caspase-dependent pathway. The synthetic receptor 73 could be a useful tool for

the study of CRP-PC interactions and functions of CRP on cellular

membranes.49-51

4.9 Experimental Section

4.9.1 General

Chemical reagents were obtained from Acros, Aldrich or Alfa Aesar. Solvents

were from EM Science. Commercial grade reagents were used without further

purification unless otherwise noted. Anhydrous solvents were obtained after

passage through a drying column of a solvent purification system from

GlassContour (Laguna Beach, CA). All reactions were performed under an

atmosphere of dry argon or nitrogen. Reactions were monitored by analytical

123

thin-layer chromatography on plates coated with 0.25 mm silica gel 60 F254 (EM

Science). TLC plates were visualized by UV irradiation (254 nm) or stained with a

solution of phosphomolybdic acid. Flash column chromatography employed ICN

SiliTech Silica Gel (32-63 mm). Melting points were measured with a Thomas

Hoover capillary melting point apparatus and are uncorrected. Infrared spectra

were obtained with a Perkin Elmer 1600 Series FTIR. NMR spectra were

obtained with Bruker CDPX-300 and AMX-360 instruments with chemical shifts

reported in parts per million (ppm, δ ) referenced to either CDCl3 (1H 7.27 ppm;

13C 77.2 ppm), MeOH-d4 (1H 4.80 ppm; 13C 49.2 ppm), or TMS (0 ppm). 31 P

NMR analyses performed in MeOH-d4 were indirectly referenced to 85% H3PO4

(0 ppm). High-resolution mass spectra were obtained from the Pennsylvania

State University Mass Spectrometry Facilities (ESI). Peaks are reported as m/z.

4.9.2 Synthetic Procedures and Compound Characterization Data

tert-Butyl-3β-cholest-5-en-3-yl[3-({3-[(9H-fluoren-9-ylcarbonyl)amino]propa-

noyl}amino)propyl]carbamate (74). To A solution of phthalimide protected

anchor (40, 1.344 g, 2.0 mmol) in ethanol (50 mL) was added anhydrous

124

hydrazine (0.25 mL, 8.0 mmol). The reaction was stirred at 50 °C for 4 h. The

white precipitate was removed by filtration. The filtrate was concentrated in vacuo.

The resulting residue was redissolved in CHCl3 (5 mL) and filtered again. The

filtrate was concentrated to afford the product (1.05 g, 97%) as a white solid.

Without further purification, the amine was directly applied to the next step. To a

solution of Fmoc-β-alanine (281 mg, 1.0 mmol) and HOBt (162 mg, 1.2 mmol) in

CH2Cl2 (25 mL) at 4 °C was added EDC (211 mg, 1.2 mmol). The reaction was

stirred for 30 min, followed by the addition of phthalimide deprotected anchor

(542 mg, 1.0 mmol). The reaction was warmed to 22 °C and stirred for 12 h. The

reaction was diluted with CH2Cl2 (75 mL) and washed with NaOH aqueous

solution (1.0 M, 50 mL). The organic phase was dried over anhydrous Na2SO4,

and concentrated in vacuo. Flash column chromatography (hexane/ethyl acetate,

3:1) afforded the product (769 mg, 92%) as a white foam; mp 80-82 °C; 1H NMR

(400 MHz, CDCl3) δ 7.74 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.38 (dd,

J1 = J2 = 7.5 Hz, 2H), 7.29 (dd, J1 = J2 = 7.5 Hz, 2H), 7.03 (br, 1H), 5.76 (br, 1H),

5.32 (d, J = 4.6 Hz, 1H), 4.35 (d, J = 7.1 Hz, 2H), 4.19 (t, J = 7.1 Hz, 1H), 3.64 (br,

2H), 3.25 (m, 4H), 2.44 (m, 3H), 2.03-0.85 (m, 51H), 0.67 (s, 3H); 13C NMR (100

MHz, CDCl3) δ 171.5, 156.5, 144.0 (× 3), 141.2 (× 2), 127.6 (× 2), 127.0 (× 2),

125.1 (× 2), 121.4, 119.9 (x 2), 79.9, 66.7, 56.7, 56.1 (× 2), 50.0, 47.2, 42.2 (× 2),

41.3, 39.7, 39.5, 38.4, 37.2, 36.7, 36.1, 35.9, 35.7, 31.8 (× 2), 28.5 (× 6), 28.2,

28.0, 26.7, 24.2, 23.8, 22.7, 22.5, 20.9, 19.4, 18.7, 11.8; IR (film) ν max 3321,

3067, 2936, 2867, 1688, 1667, 1542, 1450, 1412, 1366, 1248, 1168, 1141, 1009,

125

757, 741 cm-1; HRMS (ESI+) m/z 836.5961, (M+H+, C53H78N3O5 requires

836.5941).

tert-Butyl-3β-cholest-5-en-3-yl(3-{[3-({3-[(9H-fluoren-9-ylcarbonyl)amino]

propanoyl}amino)propanoyl]amino}propyl)carbamate (75). To 74 (420 mg,

0.5 mmol) was added 20% piperidine in DMF (5 mL). The reaction was stirred at

22 °C for 30 min and concentrated in vacuo. The resulting residue was dissolved

in anhydrous CH2Cl2 (5 mL). To a solution of Fmoc-β-alanine (141 mg, 0.5 mmol)

and HOBt (81 mg, 0.6 mmol) in CH2Cl2 (15 mL) at 4 °C was added EDC (106 mg,

0.6 mmol). The reaction was stirred for 30 min, followed by the addition of the

solution of primary amine derived from 74. The reaction was warmed to 22 °C

and stirred for 12 h. The reaction solution was diluted with CH2Cl2 (50 mL) and

washed with NaOH aqueous solution (1.0 M, 30 mL). The organic phase was

dried over anhydrous Na2SO4, and concentrated in vacuo. Flash column

chromatography (hexane/ethyl acetate, 2:1) afforded the product (417 mg, 92%)

as a white foam; mp 151-153 °C; 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 7.5

Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.37 (dd, J1 = J2 = 7.5 Hz, 2H), 7.29 (dd, J1 = J2

= 7.5 Hz, 2H), 7.20 (br, 1H), 7.04 (br, 1H), 5.79 (br, 1H), 5.31 (d, J = 4.6 Hz, 1H),

4.34 (d, J = 7.1 Hz, 2H), 4.18 (t, J = 7.1 Hz, 1H), 3.50 (m, 4H), 3.22 (m, 4H), 2.41

126

(m, 5H), 2.03-0.85 (m, 51H), 0.67 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 171.5 (×

2), 156.5, 144.0 (× 3), 141.2 (× 2), 127.6 (× 2), 127.0 (× 2), 125.1 (× 2), 121.4,

119.9 (× 2), 79.9, 66.7, 56.7, 56.1 (x 2), 50.0, 47.1, 42.2 (× 2), 41.3, 39.7, 39.5 (×

2), 38.4, 37.2, 36.6, 36.1, 35.9, 35.7, 31.8 (× 2), 28.5 (× 6), 28.2, 27.9, 26.8, 24.2,

23.8, 22.7, 22.5, 20.9, 19.4, 18.7, 11.8; IR (film) ν max 3307, 3067, 2937, 2867,

1652, 1540, 1450, 1450, 1412, 1366, 1251, 1168, 1141, 758 cm-1; HRMS (ESI+)

m/z 907.6310 (M+H+, C56H83N4O6 requires 907.6313).

tert-Butyl-3β-cholest-5-en-3-yl[17-(9H-fluoren-9-yl)-5,9,13,17-tetraoxo-4,8,12,

16-tetraazaheptadec-1-yl]carbamate (76). To 75 (272 mg, 0.3 mmol) was

added 20% piperidine in DMF (5 mL). The reaction was stirred at 22 °C for 30

min and concentrated in vacuo. The resulting residue was dissolved in

anhydrous CH2Cl2 (5 mL). To a solution of Fmoc-β-alanine (85 mg, 0.3 mmol)

and HOBt (47 mg, 0.35 mmol) in CH2Cl2 (15 mL) at 4 °C was added EDC (67 mg,

0.35 mmol). The reaction was stirred for 30 min, followed by the addition of the

solution of primary amine derived from 75. The reaction was warmed to 22 °C

and stirred for 12 h. The reaction solution was diluted with CH2Cl2 (50 mL) and

washed with NaOH aqueous solution (1.0 M, 30 mL). The organic phase was

dried over anhydrous Na2SO4, and concentrated in vacuo. Flash column

127

chromatography (hexane/ethyl acetate, 1:1) afforded the product (264 mg, 90%)

as a white solid; mp 143-144 °C; 1H NMR (300 MHz, CDCl3/MeOH-d4) δ 7.71 (d,

J = 7.5 Hz, 2H), 7.56 (d, J = 7.5 Hz, 2H), 7.34 (dd, J1 = J2 = 7.5 Hz, 2H), 7.26 (dd,

J1 = J2 = 7.5 Hz, 2H), 7.20 (br, 1H), 7.04 (br, 1H), 5.27 (d, J = 4.5 Hz, 1H), 4.32

(d, J = 7.1 Hz, 2H), 4.15 (t, J = 7.1 Hz, 1H), 3.40 (m, 6H), 3.14 (m, 4H), 2.33 (m,

7H), 1.98-0.80 (m, 51H), 0.60 (s, 3H); 13C NMR (100 MHz, CDCl3 / MeOH-d4) δ

172.4 (× 3), 157.1, 143.8 (× 3), 141.2 (× 2), 127.8 (× 2), 127.1 (× 2), 125.1 (× 2),

121.6, 120.0 (× 2), 80.1, 66.8, 56.8, 56.2 (× 2), 50.2, 47.3, 42.4 (× 2), 42.1, 39.8,

39.6 (× 2), 38.4, 37.3, 37.2, 36.8, 36.2, 36.1, 36.0, 35.9 (× 2), 35.7, 31.9 (× 2),

28.5 (× 6), 28.3, 28.1 (× 2), 26.8, 24.3, 23.9, 22.8, 22.5, 21.0, 19.4, 18.7, 11.9; IR

(film) ν max 3293, 3072, 2934, 2849, 1684, 1644, 1539, 1449, 1366, 1253, 1167,

1138, 1029, 756, 747 cm-1; HRMS (ESI+) m/z 978.6707 (M+H+, C56H83N4O6

requires 978.6684).

6-(O-Phosphorylcholine)-N-{3-[3β-cholest-5-en-3-ylamino]propyl}hexan-

amide (70). To a solution of 40 (67 mg, 0.1 mmol) in ethanol (5 mL) was added

anhydrous hydrazine (30 µL, 1 mmol). The reaction was stirred at 50 °C for 4 h.

The white precipitate was filtered off. The filtrate was concentrated in vacuo. The

resulting residue was redissolved in CHCl3 (2 mL) and filtered again. To a

128

solution of 6-(O-phosphorylcholine)hydroxyhenxanoic acid (69, 30 mg, 0.1

mmol)52 and HOBt (16 mg, 0.12 mmol) in iPrOH/CH2Cl2 (1 mL/2 mL) at 4 °C was

added EDC (23 mg, 0.12 mmol). The reaction was stirred for 10 min, followed by

the addition of the solution of primary amine derived from 40. The reaction was

warmed to 22 °C and stirred for 12 h. The reaction solution concentrated in

vacuo. Flash column chromatography (iPrOH/ CH2Cl2/H2O, 3:1:1, with 0.5% Et3N)

afforded the crude product as a glassy solid. The crude product was purified by

preparative reverse-phase HPLC (gradient: 9.95% MeCN, 89.95% H2O, and

0.1% TFA to 99.9% MeCN, 0% H2O, and 0.1% TFA over 10 min; retention time =

9.43 min (215 nm)), which afforded the product (71 mg, 85%) as a white solid;

mp 142-144 °C; 1H NMR (400 MHz, MeOH-d4) δ 5.30 (d, J = 4.6 Hz, 1H), 4.12

(m, 2H), 3.74 (q, J = 6.2 Hz, 2H), 3.47 (m, 2H), 3.11 (m, 2H), 3.04 (s, 9H), 2.80

(m, 3H), 2.23-0.67 (m, 54H), 0.53 (s, 3H); 13C NMR (100 MHz, MeOH-d4) δ 176.8,

139.6, 124,7, 67.3, 67.2, 60.7, 59.1, 58.1, 57.5, 54.7 (× 3), 51.4, 43.5 (× 2), 43.3,

41.0, 40.7, 38.3, 37.8, 37.4, 37.1, 37.0, 36.7, 36.2, 33.1, 32.9, 31.2, 29.3, 29.1,

27.9, 26.4, 26.2, 25.3, 24.9, 23.2, 23.0, 22.1, 19.6, 19.3, 12.3; 31P NMR (146

MHz, MeOH-d4) δ 0.46; IR (film) ν max 3393, 2940, 2868, 2509, 2361, 1766,

1676, 1637, 1560,1469, 1437, 1380, 1203, 1179, 1140, 1047, 975 cm-1; HRMS

(ESI+) m/z 722.5594 (M+H+, C41H77N3O5P requires 722.5601).

129

6-(O-Phosphorylcholine)-N-[7-[-(3β-cholest-5-en-3-ylamino)-3-oxo-4-aza]

hexanamide (71). To 74 (42 mg, 0.05 mmol) was added 20% piperidine in DMF

(2 mL). The reaction was stirred at room temperature for 30 min and

concentrated in vacuo. The resulting residue was dissolved in anhydrous CH2Cl2

(3 mL). To a solution of 69 (15 mg, 0.05 mmol) and HOBt (8 mg, 0.06 mmol) in

iPrOH/CH2Cl2 (1 mL/2 mL) at 4 °C was added EDC (12 mg, 0.06 mmol). The

reaction was stirred for 10 min, followed by the addition of the solution of primary

amine derived from 74. The reaction was warmed to 22 °C and stirred for 12 h.

The reaction solution concentrated in vacuo. Flash column chromatography

(iPrOH/CH2Cl2/H2O, 3:1:1, with 0.5% Et3N) afforded the crude product as a

glassy solid. The crude product was purified by preparative reverse-phase HPLC

(gradient: 9.95% MeCN, 89.95% H2O, and 0.1% TFA to 99.9% MeCN, 0% H2O,

and 0.1% TFA over 10 min; retention time = 9.20 min (215 nm)), which afforded

the product (39 mg, 86%) as a white solid; mp 163-165 °C; 1H NMR (300 MHz,

MeOH-d4) δ 5.41 (d, J = 4.6 Hz, 1H), 4.23 (m, 2H), 3.84 (q, J = 6.3 Hz, 2H), 3.60

(m, 2H), 3.36 (m, 2H), 3.22, (m, 2H), 3.16 (s, 9H), 2.95 (m, 3H), 2.23-0.79 (m,

55H), 0.65 (s, 3H); 13C NMR (100 MHz, MeOH-d4) δ 176.2, 174.9, 139.7, 124.8,

67.5, 67.3, 60.8, 59.4, 58.2, 57.7, 54.8 (× 3), 51.6, 43.6 (× 2), 43.4, 41.2, 40.8,

38.4, 38.0, 37.5, 37.3 (× 2), 37.1, 37.0, 36.9, 36.4, 33.2, 33.1, 31.5, 29.5, 29.3,

27.9, 26.6, 26.4, 26.4, 25.4, 25.1, 23.4, 23.1, 22.2, 19.8, 19.4,12.5; 31P NMR (146

MHz, MeOH-d4) δ 0.37; IR (film) ν max 3425, 3299, 3039, 2940, 2860, 2510,

1772, 1673, 1643, 1556, 1469, 1443, 1378, 1202, 1179, 1138, 1044, 974 cm-1;

HRMS (ESI+) m/z 793.5967, (M+H+, C44H82N4O7P requires 793.5972).

130

6-(O-Phosphorylcholine)-N-[11-[-(3β-cholest-5-en-3-ylamino)-3,7-dioxo-4,8-

diaza]hexanamide (72). To 75 (45 mg, 0.05 mmol) was added 20% piperidine in

DMF (2 mL). The reaction was stirred at 22 °C for 30 min and concentrated in

vacuo. The resulting residue was dissolved in anhydrous CH2Cl2 (3 mL). To a

solution of 69 (15 mg, 0.05 mmol) and HOBt (8 mg, 0.06 mmol) in iPrOH/CH2Cl2

(1 mL/2 mL) at 4 °C was added EDC (12 mg, 0.06 mmol). The reaction was

stirred for 10 min, followed by the addition of the solution of primary amine

derived from 75. The reaction was warmed to 22 °C and stirred for 12 h. The

reaction solution concentrated in vacuo. Flash column chromatography (iPrOH /

CH2Cl2 / H2O, 3:1:1, with 0.5% Et3N) afforded the crude product as a glassy solid.

The crude product was purified by preparative reverse-phase HPLC (gradient:

9.95% MeCN, 89.95% H2O, and 0.1% TFA to 99.9% MeCN, 0% H2O, and 0.1%

TFA over 10 min; retention time = 9.14 min (215 nm)), which afforded the product

(40 mg, 82%) as a white solid; mp 184-186 °C; 1H NMR (400 MHz, MeOH-d4) δ

5.31 (d, J = 4.6 Hz, 1H), 4.15 (m, 2H), 3.75 (q, J = 6.3 Hz, 2H), 3.50 (m, 2H),

3.25 (m, 4H), 3.13 (m, 2H), 3.06 (s, 9H), 2.84 (m, 3H), 2.25-0.69 (m, 58H), 0.55

(s, 3H); 13C NMR (100 MHz, MeOH-d4) δ 176.1, 174.8, 173.7, 139.4, 124.7, 67.3

(x 2), 60.8, 59.2, 58.1, 57.5, 54.7 (× 3), 51.5, 43.5 (× 2), 43.3, 41.0, 40.7, 38.2,

131

37.8, 37.4, 37.1, 37.0 (× 2), 36.9, 36.8 (× 2), 36.3, 33.0, 31.3, 29.3, 29.1, 27.8,

26.5, 26.3, 26.2, 25.3, 24.9, 23.2, 22.9, 22.1, 19.6, 19.2, 12.3; 31P NMR (146

MHz, MeOH-d4) δ 0.43; IR (film) ν max 3425, 3300, 3039, 2939, 2849, 2520,

1770, 1672, 1634, 1557, 1470, 1443, 1381, 1203, 1138, 1046, 974 cm-1; HRMS

(ESI+) m/z 864.6343 (M+H+, C47H87N5O7P requires 864.6343).

6-(O-Phosphorylcholine)-N-[15-[-(3β-cholest-5-en-3-ylamino)-3,7,11-trioxo-

4,8,12-triaza]hexanamide (73). To 76 (98 mg, 0.1 mmol) was added 20%

piperidine in DMF (3 mL). The reaction was stirred at 22 °C for 30 min and

concentrated in vacuo. The resulting residue was dissolved in anhydrous CH2Cl2

(5 mL). To a solution of 69 (30 mg, 0.1 mmol) and HOBt (16 mg, 0.12 mmol) in

iPrOH/CH2Cl2 (1 mL/2 mL) at 4 °C was added EDC (23 mg, 0.12 mmol). The

reaction was stirred for 10 min, followed by the addition of the solution of primary

amine derived from 76. The reaction was warmed to 22 °C and stirred for 12 h.

The reaction solution concentrated in vacuo. Flash column chromatography

(iPrOH/CH2Cl2/H2O, 3:1:1, with 0.5% Et3N) afforded the crude product as a

glassy solid. The crude product was purified by preparative reverse-phase HPLC

(gradient: 9.95% MeCN, 89.95% H2O, and 0.1% TFA to 99.9% MeCN, 0% H2O,

and 0.1% TFA over 10 min; retention time = 8.65 min (215 nm)), which afforded

132

the product (84 mg, 80%) as a white solid; mp 206-208 °C; 1H NMR (300 MHz,

MeOH-d4) δ 5.40 (d, J = 4.6 Hz, 1H), 4.20 (m, 2H), 3.80 (q, J = 6.2 Hz, 2H), 3.56

(m, 2H), 3.34 (m, 6H), 3.21 (m, 2H), 3.14 (s, 9H), 2.96 (m, 3H), 2.35-0.78 (m,

61H), 0.64 (s, 3H); 13C NMR (75 MHz, MeOH-d4) δ 176.1, 174.9, 173.8, 173.7,

139.6, 124.8, 67.4, 66.9, 60.5, 59.3, 58.1, 57.5, 54.7 (× 3), 51.5, 43.5 (× 2), 43.3,

41.0, 40.7, 38.2, 37.8, 37.4, 37.2, 37.1 (× 2), 37.0 (× 2), 36.9, 36.9 (× 2), 36.8,

36.3, 33.1, 33.0, 31.4, 29.3, 29.2,27.8, 26.5, 26.4, 26.3, 25.3, 24.9, 23.2, 22.9,

22.1, 19.6, 19.2, 12.3; 31P NMR (146 MHz, MeOH-d4) δ 0.22; IR (film) ν max

3396, 3292, 3086, 2936, 2880, 1675, 1633, 1543, 1466, 1374, 1204, 1135, 1048,

973 cm-1; HRMS (ESI+) m/z 935.6747 (M+H+, C50H92N6O8P requires 935.6714).

4.9.3 Biological Assays and Protocols

Cell culture: Jurkat lymphocytes (human acute leukemia, ATCC #TIB-152) were

cultivated in Roswell Park Memorial Institute (RPMI) 1640 media supplemented

with Fetal Bovine Serum (FBS, 10%), penicillin (100 units /mL), and streptomycin

(100 µg/mL). The Jurkat cell line was propagated in a humidified 5% CO2

incubator at 37 °C. Media used for cell culture and wash steps contained

antibiotics and FBS unless otherwise noted.

Microscopy: A Zeiss LSM 5 Pascal confocal laser-scanning microscope fitted

with a Plan Apochromat objective (63×) was employed. Alexa Fluor-488 was

excited with a 488 nm Argon ion laser (25 mW, 1% laser power) and emitted

133

photons were collected through 505 nm LP filter. Excitation of PI employed a 543

nm HeNe laser and emitted photons were collected through a 560 nm LP filter.

Flow cytometry: Analyses were performed with a Beckman-Coulter XL-MCL

bench-top flow cytometer. Forward-scatter (FS) and side-scatter (SSC) dot plots

afforded cellular physical properties of size and granularity that allowed gating of

live cells. After gating, 10,000 cells were counted. The AF-488 and PI were

excited with an Ar ion laser with an excitation wavelength of 488 nm. Two

wavelengths of fluorescence, 525 nm (for the detection of annexin V-AF 488

emission) and 610 nm (for PI emission) were measured. The PMT voltage for

this instrument was set to 501 for Jurkat cells.

CRP labeling: Human C-reactive protein (300 µL, 1 mg/mL, Calbiochem,

#23660) in Tris buffer (pH 7.5) was used for protein labeling. The Tris buffer was

replaced with PBS buffer (pH 7.4, 500 mL) by dialysis at 4 °C for 1 h. The protein

solution was transferred to a vial of Alexa Fluor 488 labeling dye with a magnetic

stir bar (Molecular Probes, #A10235), followed by the addition of 30 µL of 1.0 M

NaHCO3 aqueous solution. The solution was stirred for 30 min and the labeled

protein was purified through a size exclusion column provided with the labeling

reagent, and collected as the second fluorescent band. The CRP-AF488 was

immediately redialyzed back to the Tris buffer (NaCl 150 mM, Tris 20 mM, CaCl2

2 mM, 0.1% NaN3, pH 7.5). The protein concentration and degree of labeling

(DOL) were determined to be 0.56 mg/mL and 3.1 fluorophores per pentamer

134

respectively by measuring the absorbances at 280 nm and 494 nm. The labelled

protein was maitained at 4 °C and protected from light.

Annexin V-binding assays: Jurkat lymphocytes (2 × 105) in media (300 µL)

were treated with synthetic receptor in DMSO (final [DMSO] = 1%) at 37 °C for 1

h. The cells were washed with fresh media, resuspended in the media (200 µL)

containing CRP or CRP-AF488, which was predialyzed in Tris buffer without

NaN3 (300 mL) at 4 °C for 1 h, and incubated at 37 °C for 6h. The treated cells

were washed with cold PBS, resuspended in annexin V-binding buffer (40 µL, pH

7.4) with annexin V-AF488 (4 µL, Molecular Probes, #A13201) at 22 °C for 10

min. For confocal microscopy, propidium iodide solution (5 µL, 30 µM) was

added to the cells in annexin-binding buffer and incubated for 2 min. The cells

were centrifuged, and washed with PBS (50 µL) before confocal microscopy. For

flow cytometry, propidium iodide solution (5 µL, 30 µM) was added to the cells in

annexin-binding buffer. After 1 min, 450 µL of PBS was added, and the sample

was immediately analyzed by flow cytometry.

DNA fragmentation assay: Jurkat lymphocytes (1 × 106) in media (1 mL) were

treated with synthetic receptor 73 in DMSO (2 µM , final [DMSO] = 1%) at 37 °C

for 1 h. The cells were washed with fresh media, and resuspended in fresh media

(1 mL) containing CRP (10 µg/mL) and incubated at 37 °C for 8 h to induce

apoptosis. To confirm the specificity of DNA fragmentation, appropriate omission

135

controls consisting of (1) cells treated with 1% DMSO alone for 9 h at 37 °C, or (2)

receptor 73 (10 µM) for 1 h followed by incubation with media only for 8 h at 37

°C, and (3) cells treated with 1% DMSO for 1h followed by incubation with media

containing CRP (10 µg/mL) for 8 h at 37 °C, were also evaluated for DNA

degradation. As a positive control for DNA laddering, cells were treated with

camptothecin in DMSO (50 µM, final [DMSO]= 1%). In addition to the omission

controls, cells were treated with 73 (10 µM) for 1 h followed by CRP (10 µg/mL)

for 8 h at 37 °C, in the presence of the broad spectrum caspase inhibitor zVAD-

fmk in DMSO (50 µM, Calbiochem, final [DMSO] = 1%). The cells were analyzed

for DNA degradation using the Suicide Track DNA ladder isolation kit

(Calbiochem, #AM41). Extraction of genomic DNA, separation of high molecular

weight chromatin and DNA precipitation was carried out according to the

manufacturer’s instructions. The precipitated lower molecular weight DNA was

reconstituted in 10 mM Tris buffer (pH 7.5, 1 mM EDTA) supplied by the

manufacturer (40 µL) and analyzed by agarose gel (1.5%) electrophoresis. The

gel was stained using ethidium bromide (5 µg/mL in 100 mL Tris Acetate EDTA

(TAE) buffer (1×)) for 2 h. The gel was destained in Tris acetate EDTA (TAE)

buffer for 1 h prior to visualization using UV irradiation.

Cytotoxicity assays: Jurkat lymphocytes (7 × 104) in media (100 µL) were

treated with synthetic receptor 73 in DMSO (final [DMSO] = 1%) at 37 °C for 1 h.

The cells were washed with fresh media, resuspended in the media (100 µL)

136

containing CRP, which was predialyzed in Tris buffer without NaN3 (300 mL), and

transferred onto a 96-well plate. After 24 h of incubation at 37 °C, cells from each

well (10 µL) were transferred to an opaque 96-well plate and treated with

CellTiter-Glo reagent (20 µL, Promega) according to the manufacturer’s

instructions. After incubation at 22 °C for 10 min, the luminescence of the

samples was measured with a Packard Fusion microplate reader.

Heat inactivation of FBS: The frozen FBS (10 mL) in a Falcon tube (15 mL)

was thawed to 22 °C and then placed in a preequilibrated water bath at 56 °C for

30 min. The FBS was swirled every 5 min during incubation, cooled on ice

immediately after 30 min, and maintained at -20 °C. 　

Caspase Inhibition assay: Jurkat lymphocytes (2 × 105) in media (300 µL) were

treated with synthetic receptor 73 in DMSO (2 µM, final [DMSO] = 1%) with or

without pan-caspase inhibitor, zVAD-fmk (50 µM, final [DMSO] = 1%) at 37 °C for

1 h. The cells were washed with fresh media (200 µL), resuspended in the media

(200 µL) containing CRP (10 µg/mL) with or without zVAD-fmk (50 µM, final

[DMSO] = 1%), and incubated at 37 °C for 6 h. The treated cells were then

subjected to confocal microscopy and flow cytometry according to the

procedures described for annexin V-binding assays.

137

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22. Torzewski, J.; Torzewski, M.; Bowyer, D. E.; Fröhlich, M.; Koenig, W.;

Waltenberger, J.; Fitzsimmons, C.; Hombach, V. C-reactive protein frequently

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23. Paul, A.; Ko, K. W. S.; Li, L.; Yechoor, V.; McCrory, M. A.; Szalai, A. J.;

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Zinman, B. C-reactive protein and estational diabetes: The central role of

maternal obesity J. Clin. Endocr. Metab. 2003, 88, 3507-3512.

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Greenberg, E. R.; Mandel, J. S.; Marcon, N.; Mott, L. A.; Pearson, L.; Saibil, F.;

van Stolk, R. U. A randomized trial of aspirin to prevent colorectal adenomas.

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Ultrasensitive confocal fluorescence microscopy of C-reactive protein interacting

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F. Cytometry in cell necrobiology: Analysis of apoptosis and accidental cell death

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Chapter 5

A One-Pot Synthesis of Nucleoside 5’-Triphosphates from Nucleoside 5’-H-

Phosphonates

5.1 Introduction

Nucleoside 5’-triphosphates (NTPs) are critical mediators of myriad

biological processes including DNA replication, transcription, and translation.

Correspondingly, synthetic mimics of NTPs have been widely used as molecular

probes, biological assay components, and represent active metabolites of certain

drugs such as the antiviral agent ribavirin. Despite their importance in biology

and medicine, the diversity of commercially available NTPs is limited because

these compounds are often difficult to prepare and isolate in pure form.1 Most

commonly used methods for the synthesis of NTPs include the “one-pot, three-

step” method and Eckstein’s method (Figure 5.1).2,3 Although these strategies

work well for some substrates, others are plagued by low yields and difficulties in

purification.4

More recently, Ahmadibeni reported a solid-phase route to NTPs,5 and

Borch reported a method for the preparation of activated phosphoramidates that

can be converted to NTPs by reaction with tris(tetra-n-butylammonium) hydrogen

146

pyrophosphate.6 The final coupling step employed by Borch was found to

proceed in remarkably high yield, but the required four-step synthesis of the

phosphoramidate precursor increased the complexity of the synthesis compared

with other approaches. We hypothesized that nucleoside 5’-H-phosphonates

might provide novel and more readily synthesized precursors to NTPs. This

hypothesis was based on previously reported syntheses of phosphates,

phosphoramidates, and other phosphate derivatives from these precursors.7-10

After conversion to silyl phosphites with TMSCl, H-phosphonate monoesters can

be readily oxidized by elemental iodine and other reagents to generate

electrophilic pyridinium phosphoramidate intermediates, similar but even more

reactive to the ones used in Borch’s approach (Figure 5.2).11,12 These

Figure 5.1: Commonly used methods for the synthesis of NTPs. Panel A: The“one-pot, three-step” method. Panel B: Eckstein’s method.

147

intermediates are known to react with a variety of nucleophiles to afford addition

products, but surprisingly this strategy has not been previously investigated for

the synthesis of NTPs.

We report here a one-pot approach for the synthesis of NTPs from

nucleoside 5’-H-phosphonate monoesters, compounds that can be readily

prepared from 2’,3’-O-isopropylidene-protected nucleosides.13 We demonstrate

that fully deprotected ribonucleoside 5’-H-phosphonate monoesters can be

converted in situ to pyridinium phosphoramidate intermediates. Upon addition of

nucleophilic tris(tetra-n-butylammonium) hydrogen pyrophosphate, NTPs can be

readily isolated by use of a two-step purification protocol.

Figure 5.2: The hypothesis of using nucleoside 5’-H-phosphonates to synthesize NTPs via a phosphoramidate intermediate. Panel A: Borch’s new method via apyrrolidinium phosphoramidate intermediate. Panel B: Our hypothesis involvinguse of a similar pyridinium phosphoramidate intermediate to synthesize NTPs.

148

5.2 Synthesis of Nucleoside 5’-H-Phosphonates (83-88)

As shown in Figure 5.3, starting with the known 2’,3’-O-isopropylidene-

protected nucleosides (77-82),13-15 phosphitylation16 with salicyl

phosphorochlorodite or PCl3 provided 2’,3’-O-isopropylidene-protected

nucleoside 5’-H-phosphonate monoesters (83-88). Treatment of these

compounds with aqueous TFA yielded 5’-H-phosphonates derived from the four

natural nucleosides, uridine (89), cytidine (90), guanosine (91), and adenosine

(92) as well as 5’-H-phosphonates derived from the antiviral agents ribavirin (93)

and 6-methylpurine ribonucleoside (94).

Figure 5.3: Synthesis of nucleoside 5’-H-phosphonates (89-94).

149

5.3 Synthesis and Purification of Nucleoside 5’-Triphosphates (96-101)

Based on previous literature precedents,7-11,17 we hypothesized that

addition of excess TMSCl and pyridine to the unprotected nucleoside 5’-H-

phosphonate monoesters (89-94) would accomplish two objectives: 1)

conversion of the H-phosphonate to a silyl-H-phosphonate or more reactive bis-

silyl phosphite, and 2) transient modification of spurious water and/or other

reactive nucleophiles. Subsequent addition of elemental iodine would rapidly

oxidize the phosphite and generate a reactive pyridinium phosphoramidate in situ.

We further proposed that nucleophilic attack by tris(tetra-n-butylammonium)

Figure 5.4: A one-pot method for conversion of nucleoside 5’-H-phosphonates (89-94) to nucleoside 5’-triphosphates (96-101).

150

hydrogen pyrophosphate (95)18 would yield the NTP product. As shown in Figure

5.4, applying this sequence to 89-94 indeed afforded triphosphates 96-101.

Athough the yields of the pure triphosphates were modest, ranging from 26% to

41%, the ability to easily control the production of the NTP in this one-pot

process proved to be of significant benefit for the sythesis of certain substrates

such as ribavirin triphosphate (100), which can be difficult to synthesize by non-

enzymatic methods.

To purify triphosphates prepared with this method, a simple prepurification

step involving passage through a column of Sephadex LH-20 gel filtration media

with aqueous triethylammonium bicarbonate (TEAB) buffer as the eluent was

found to remove the majority of non-phosphate byproducts. Crude products were

subsequently lyophilized and further purified by preparative reverse phase HPLC

to afford 10-30 mg quantities of triphosphates (96-101) as

tetra(triethylammonium) salts in >90% purity, which was determined by reverse-

phase analytic HPLC.

5.4 Mechanistic Studies of the Reaction by 31P NMR

To examine the mechanism proposed for formation of NTPs, a related

reaction involving a more soluble, protected substrate was analyzed by 31P NMR.

In this experiments, 2’,3’-O-isopropylidene uridine 5’-H-phosphonate (83)19 and

tris(tetra-n-butylammonium) hydrogen pyrophosphate (95)18 were prepared

according to literature procedures. The 5’-H-phosphonate was treated with 3

151

equiv. of TMSCl in anhydrous pyridine-d5, followed by rapid oxidation with iodine

to generate the pyridinium phosphoramidate in situ. As shown in Figure 5.5,

chemical shifts observed by 31P NMR were consistent with the clean generation

of two silylated intermediates (102, 103) that upon oxidation are efficiently

Figure 5.5: Analysis of sequential conversion of H-phosphonate monoester 83 to triphosphate 105 by 31P NMR. Panel A: Synthetic route illustrating measured 31P chemical shifts of reagents, proposed intermediates, and product. Panel B: 31P NMR spectra of compounds prior to and after sequential addition of reagents shown in panel A. From the bottom of the spectral stack: the first two spectra areof purified starting materials (83, 95), the third spectrum was obtained after addition of TMSCl, the fourth spectrum was obtained after addition of I2, and the fifth spectrum was obtained after addition of 95. Inset: expansion of resonances of 105 showing assignments of phosphorus atoms.

152

converted to the activated pyridinium phosphoramidate (104). Addition of

tris(tetra-n-butylammonium) hydrogen pyrophosphate afforded the triphosphate

(105) as the major product with 60% conversion (calculated by integration). In

contrast, if the less nucleophilic tris(tri-n-butylammonium) pyrophosphate was

employed as the nucleophile, only a trace of triphosphate was produced (data

not shown), demonstrating the importance of the more nucleophilic6,18 tris(tetra-n-

butylammonium) hydrogen pyrophosphate in the reaction.

5.5 Mass Spectrometric Study of Side-Products and Optimization of

Reaction Conditions

To optimize the reaction and probe the mechanism of conversion of

nucleoside 5’-H-phosphonates to triphosphates by mass spectrometry, reaction

of deprotected uridine 5’-H-phosphonate monoester (89) was investigated. Initial

attempts to synthesize UTP (96) explored the addition of only 3 equiv. of TMSCl

in the first step. As shown in Figure 5.6, analysis of the crude products by mass

spectrometry revealed the formation of the monophosphate (107), the

diphosphate (109), and dimeric derivatives (108 and 110). Since tris(tetra-n-

butylammonium) hydrogen pyrophosphate typically contains more than 3 equiv.

of water,18 we reasoned that the phosphoramidate intermediate might be partially

hydrolyzed and form these byproducts through the routes shown in Figure 5.6.

To suppress these side reactions, the addition of 5 to 8 equiv. of TMSCl,

depending on the nucleoside, was found to minimize the formation of undesired

153

side-products without adversely affecting the nucleophilicity of the pyrophosphate.

For example, during the synthesis of UTP (96) from 89, a 31% yield was

Figure 5.6: Mass spectral analysis of the reaction mixture obtained afterconversion of H-phosphonate 13 to triphosphate 20, using 3 equiv of TMSCl, followed by partial purification by gel filtration (Sephadex LH-20). Panel A: Structures of compounds detected by mass spectrometry. Panel B: Massspectrum obtained with a Waters ZQ-4000 single quadrupole instrument using electrospray ionization (negative-ion mode).

154

obtained using 3 equiv. of TMSCl, whereas the yield was increased to 41% with

5 equiv. of TMSCl. We also explored the utility of other nucleophilic bases known

to yield stable phosphoramidate intermediates20-22 including N-methyl morpholine,

DMAP, N-methyl imidazole and N-methyl pyrrolidine, but pyridine proved to be

superior, likely because these alternatives are presumably too basic or

nucleophilic for compatability with TMSCl. Correspondingly, anhydrous DMF

containing 25 equiv. of anhydrous pyridine proved to be the optimal solvent/base

combination for both solubilization and preparation of NTPs from deprotected

nucleoside 5’-H-phosphonates such as 89-94.

5.6 Biological Activity of 6MePTP (101) Synthesized from 5’-H-Phosphonate

(88)

To examine the biological activity of a triphosphate prepared by this

method, we investigated the incorporation of 6-methylpurine ribonucleoside

triphosphate (101) into RNA by RNA-dependent RNA polymerase from poliovirus

(3Dpol).The cognate ribonucleoside, an antiviral and anticancer agent with activity

against RNA viruses, lacks a clearly defined mechanism of action.23 As shown in

Figure 5.7, in an assay that was previously employed to investigate other

NTPs,24,25 101 was found to be efficiently incorporated by 3Dpol into a

symmetrical RNA substrate, with activity comparable to natural ATP. These

results establish that a high level of biological activity of NTPs can be obtained

following synthesis from a 5’-H-phosphonate precursor. Additionally, the ability of

155

101 to efficiently mimic ATP during synthesis of RNA by 3Dpol suggests that if 6-

methylpurine ribonucleoside were converted into 101 in mammalian cells, it could

become incorporated into viral RNA, potentially altering RNA structure, function,

or coding. Although further study is needed to determine the mechanism of

action of 101, related purine analogues such as ribavirin are known to function by

a mechanism that is dependent on incorporation of the analogue into the viral

genome.26-28

Figure 5.7: Evaluation of 6-methylpurine ribonucleoside triphosphate (101) as a substrate of 3Dpol from poliovirus. Bars illustrate the amount of incorporation of101 into RNA relative to correct natural NTPs templated by U, C, A, or G(efficiency of correct incorporation of natural NTPs 100%). Data was acquired by first assembling 3Dpol on a symmetrical RNA primer template duplex (end-labeled with 32P ATP) for 2 min at 30 °C. Incorporation of nucleotides into RNA wasinitiated, and the reaction was quenched after 1, 5, or 10 min. Extension productswere separated from unmodified RNA by denaturing PAGE, bands werevisualized with a phosphorimager, and radioactivity was quantified usingImageQuant software.

156

5.7 Conclusions

In summary, we developed a one-pot method for the synthesis of

biologically active nucleoside 5’-triphosphates from nucleoside 5’-H-

phosphonates. These precursors can be readily synthesized in high yield from

2’,3’-O-isopropylidene-protected nucleosides. Nucleoside 5’-H-phosphonates

silylated with excess TMSCl (5-8 eq.) are efficiently activated under mild

conditions with I2/pyridine, enabling conversion to triphosphates upon addition of

nucleophilic tris(tetra-n-butylammonium) hydrogen pyrophosphate. This oxidative

coupling method does not require protection of ribose or nucleobases and has

the potential to be applied to a variety of nucleosides and analogues.

5.8 Experimental Section

5.8.1 General

Chemical reagents and solvents were obtained from Acros, Aldrich, and EMD

Biosciences. Commercial grade reagents were used without further purification

unless otherwise noted. Ribavirin and 6-methylpurine ribonucleoside were

synthesized by previously reported methods.14,29 Natural ribonucleosides (A, C,

G, U) protected with the 2’,3’-O-isopropylidene group were prepared as

previously reported.13 Ribavirin and 6-methylpurine ribonucleoside protected with

157

the 2’,3’-O-isopropylidene group have been previously described.30,15 Tris(tetra-

n-butylammonium) hydrogen pyrophosphate (95) was prepared as previously

reported,18 dried over P2O5, and stored at -20 °C. Anhydrous solvents were

obtained after passage through a drying column of a solvent purification system

from GlassContour (Laguna Beach, CA). All reactions were performed under an

atmosphere of dry nitrogen. Reactions were monitored by analytical thin-layer

chromatography on plates coated with 0.25 mm silica gel 60 F254 (EMD

Chemicals). TLC plates were visualized by UV irradiation (254 nm) or stained

with 20% sulfuric acid in ethanol. Flash column chromatography employed silica

gel (ICN SiliTech, 32-63 µm) or Sephadex LH-20 (GE Healthcare, column

dimensions: 2.5 cm × 40 cm). Purification by preparative HPLC employed an

Agilent 1100 series instrument equipped with an Atlantis C18 preparative column

(19 × 150 mm, 5 µm; Waters Corporation). The HPLC flow rate was maintained

at 25 mL/min for the duration of the run unless otherwise noted. NMR spectra

were obtained with Bruker CDPX-300 or AMX-360 instruments with chemical

shifts reported in parts per million (ppm, δ) referenced to D2O (1H, 4.80 ppm).

Chemical shifts for 13C NMR and 31P NMR analyses performed in D2O were

indirectly referenced to 10% acetone in D2O (CH3, 30.89 ppm) and 85% H3PO4

(0 ppm) respectively. Low-resolution mass spectra were obtained with a Waters

ZQ-4000 mass spectrometer. High-resolution mass spectra were obtained from

the proteomics and mass spectrometry core facility at Penn State University,

University Park, PA. Peaks are reported as m/z.

158

5.8.2 Synthetic Procedures and Compound Characterization Data

2’,3’-O-Isopropylidene uridine 5’-H-phosphonate (83). To a solution of

phosphorus trichloride19 (350 µL, 3.9 mmol) in anhydrous CH2Cl2 (5 mL) at -20

°C was added 2’,3’-O-isopropylidene uridine13 (77, 100 mg, 0.35 mmol) and the

reaction mixture stirred at -20 °C for 1 h. The reaction was warmed over ~30 min

to 22 °C and stirred for 4 h. The solution was concentrated in vacuo and the

residue was treated with aqueous TEA (10%, 1 mL) for 5 min. The solution was

concentrated in vacuo, the residue was dissolved in CH2Cl2, and the product was

purified by flash column chromatography on silica gel eluting with a step gradient

of CH2Cl2/MeOH (10:1 to 1:1) containing triethylamine (0.5%) to afford 83 as the

triethylammonium salt, a glassy solid (141 mg, 90%). 1H NMR (300 MHz, D2O) δ

7.68 (d, J = 8.0 Hz, 1H), 6.66 (d, JP,H = 626 Hz, 1H), 5.82 (d, 1H), 5.77 (d, 1H),

4.94 (m, 1H), 4.90 (m, 1H), 4.40 (m, 1H), 3.99 (m, 2H), 3.10 (m, 6H, N(CH2CH3)3),

1.52 (s, 3H), 1.32 (s, 3H), 1.20 (t, 9H, N(CH2CH3)3); 13C NMR (75 MHz, D2O) δ

168.7, 153.1, 142.7, 114.9, 102.3, 92.9, 85.2 (JC,P = 33 Hz), 84.5, 81.0, 63.7 (JC,P

= 15 Hz), 47.0 (3, N(CH2CH3)3), 26.6, 24.7, 8.6 (3, N(CH2CH3)3); 31P NMR (146

159

MHz, D2O) δ 6.5 (m); LRMS (ESI-) m/z 347.2 (M-H-, C12H116N2O8P, requires

347.2).

Uridine 5’-H-phosphonate (89). Method A: To a solution of phosphorus

trichloride19 (450 µL, 5.0 mmol) in anhydrous CH2Cl2 (5 mL) at -20 °C, 2’,3’-O-

isopropylidene uridine13 (77, 142 mg, 0.5 mmol) was added and stirred for 1 h at

-20 °C. The reaction was slowly warmed over ~ 30 min to 22 °C and stirred for 4

h. The solution was concentrated in vacuo and the residue was treated with

aqueous TFA (2 mL, 50%) for 45 min. The solvent was removed in vacuo, the

crude product was dissolved in triethylammonium bicarbonate (TEAB) buffer (1

mL, 10 mM, pH = 8), and the product was loaded on a Sephadex LH-20 column

(2.5 cm × 40 cm) pre-equilibrated with TEAB buffer. Elution with TEAB buffer (10

mM) and lyophilization afforded the product as the triethylammonium salt, a

colorless glassy solid (174 mg, 85%). 1H NMR (300 MHz, D2O) δ 7.75 (d, J = 8.1

Hz, 1H), 6.62 (d, JP,H = 637 Hz, 1H), 5.80 (d, J = 4.7 Hz, 1H), 5.77 (d, J = 8.1 Hz,

1H), 4.17 (m, 2H), 4.10 (m, 1H), 3.97 (m, 2H), 3.04 (m, 6H, N(CH2CH3)3), 1.12 (t,

9H, N(CH2CH3)3); 13C NMR (75 MHz, D2O) δ 166.4, 152.0, 142.0, 102.8, 89.0,

83.4 (d, JC,P= 33 Hz), 74.0, 70.0, 63.0 (d, JC,P=16 Hz), 47.0 (3, N(CH2CH3)3), 8.6

160

(3, N(CH2CH3)3); 31P NMR (146 MHz, D2O) δ 13.4 (m); LRMS (ESI-) m/z 307.3

(M-H-, C9H12N2O8P, requires 307.2).

Cytidine 5’-H-phosphonate (90). Method B: To a solution of salicyl

phosphorochloridate20 (76 mg, 0.375 mmol) in anhydrous pyridine (2 mL) and

dioxane (2 mL), 2’,3’-O-isopropylidene cytidine13 (78, 71 mg, 0.25 mmol) was

added and stirred at 22 °C for 1 h. The reaction was quenched by addition of

deionized water (1 mL) and stirred for 10 min before concentration in vacuo. The

residue was dissolved in CH2Cl2 and purified by flash column chromatography on

silica gel eluting with a step gradient of CH2Cl2/MeOH (10:1 to 1:1) containing

triethylamine (0.5%) to afford 2’,3’-O-isopropylidene cytidine 5’-H-phosphonate

(84) as the triethylammonium salt, a glassy solid. This solid was dissolved in

aqueous TFA (2 mL, 50%) and stirred for 45 min at 22 °C. After the solvent was

removed in vacuo, the residue was dissolved in TEAB buffer (1 mL, 10 mM, pH =

8) and loaded onto a Sephadex LH-20 column (2.5 cm × 40 cm) pre-equilibrated

with TEAB buffer. Elution with TEAB buffer (10 mM) followed by lyophilization

afforded the product as the triethylammonium salt, a colorless glassy solid (72

mg, 71%). 1H NMR (360 MHz, D2O) δ 7.71 (d, J = 7.6 Hz, 1H), 6.60 (d, JP,H= 637

161

Hz, 1H), 5.90 (d, J = 7.6 Hz, 1H), 5.79 (d, J = 2.9 Hz, 1H), 4.11 (m, 2H), 4.06 (br,

1H), 3.99 (m, 1H), 3.90 (m, 1H), 3.00 (m, 6H, N(CH2CH3)3), 1.10 (t, 9H,

N(CH2CH3)3); 13C NMR (90 MHz, D2O) δ 166.0, 157.5, 141.3, 96.3, 89.4, 82.4 (d,

JC,P = 32 Hz), 74.0, 69.3, 62.4 (d, JC,P = 15 Hz), 46.5 (3, N(CH2CH3)3), 8.2 (3,

N(CH2CH3)3); 31P NMR (145.8 MHz, D2O) δ 13.5 (m); LRMS (ESI-) m/z 306.4 (M-

H-, C9H13N3O7P requires 306.2).

Guanosine 5’-H-phosphonate (91). To a solution of salicyl

phosphorochloridate20 (91 mg, 0.45 mmol) in anhydrous pyridine (2 mL) and

dioxane (2 mL) was added 2’,3’-O-isopropylidene guanosine13 (79, 97 mg, 0.30

mmol). Compound 91 was synthesized by Method B. The product was purified by

chromatography on silica gel and Sephadex LH-20, and lyophilization afforded

the product as the triethylammonium salt, a colorless glassy solid (102 mg, 76%).

1H NMR (300 MHz, D2O) δ 7.80 (s, 1H), 6.54 (d, JP,H= 637 Hz, 1H), 5.68 (d, J =

5.7 Hz, 1H), 4.53 (dd, J1= 5.7 Hz, J2= 5.1 Hz, 1H), 4.27 (dd, J1 = 4.0 Hz, J2 = 5.1

Hz, 2H), 4.10 (m, 1H), 3.90 (m, 2H), 2.98 (dd, 6H, N(CH2CH3)3), 1.04 (t, 9H,

N(CH2CH3)3); 13C NMR (75 MHz, D2O) δ 160.0, 155.0, 151.8, 137.3, 116.4, 87.2,

83.8 (d, JC,P = 32 Hz), 73.9, 70.6, 63.3 (d, JC,P = 16 Hz), 46.9 (3, N(CH2CH3)3),

162

8.6 (3, N(CH2CH3)3); 31P NMR (145.8 MHz, D2O) δ 7.2 (m); LRMS (ESI-) m/z

346.1 (M-H-, C10H13N5O7P requires 346.2).

Adenosine 5’-H-phosphonate (92). To a solution of salicyl

phosphorochloridate20 (121 mg, 0.60 mmol) in anhydrous pyridine (2 mL) and

dioxane (2 mL) was added 2’,3’-O-isopropylidene adenosine13 (80, 123 mg, 0.40

mmol). Compound 92 was synthesized by Method B. The product was purified by

chromatography on silica gel and Sephadex LH-20, and lyophilization afforded

the product as the triethylammonium salt, a colorless glassy solid (135 mg, 78%).

1H NMR (300 MHz, D2O) δ 8.18 (s, 1H), 7.91 (s, 1H), 6.58 (d, JP,H= 639 Hz, 1H),

5.88 (d, J = 5.5 Hz, 1H), 4.57 (dd, J = 5.5 Hz, J = 5.1 Hz, 1H), 4.32 (dd, J = 4.0

Hz, J = 5.1 Hz, 1H), 4.20 (m, 1H), 3.96 (m, 2H), 2.96 (dd, 6H, N(CH2CH3)3), 1.09

(t, 9H, N(CH2CH3)3); 13C NMR (75 MHz, D2O) δ 155.1, 152.5, 148.6, 139.4, 118.1,

86.9, 83.5 (d, JC,P = 32 Hz), 74.1, 70.2, 62.9 (d, JC,P = 16 Hz), 46.4 (3,

N(CH2CH3)3), 8.3 (3, N(CH2CH3)3); 31P NMR (145.8 MHz, D2O) δ 7.3 (m); LRMS

(ESI-) m/z 330.1 (M-H-, C10H13N5O6P requires 330.2).

163

Ribavirin 5’-H-phosphonate (93). To a solution of salicyl phosphorochloridate20

(71 mg, 0.35 mmol) in anhydrous pyridine (2 mL) and dioxane (2 mL) was added

2’,3’-O-isopropylidene ribavirin30 (81, 65 mg, 0.23 mmol). Compound 93 was

synthesized by Method B. The product was purified by chromatography on silica

gel and Sephadex LH-20, and lyophilization afforded the product as the

triethylammonium salt, a colorless glassy solid (70 mg, 75%). 1H NMR (300 MHz,

D2O) δ 8.62 (s, 1H), 6.53 (d, JP,H = 640 Hz, 1H), 5.90 (d, J = 3.9 Hz, 1H), 4.56 (dd,

J1= 5.0 Hz, J2 = 3.9 Hz, 1H), 4.39 (dd, J1 = 5.0 Hz, J2 = 5.1 Hz, 1H), 4.19 (m, 1H),

3.93 (m, 2H), 2.84 (dd, 6H, N(CH2CH3)3), 1.02 (t, 9H, N(CH2CH3)3); 13C NMR (75

MHz, D2O) δ 163.1, 156.7, 146.4, 92.1, 84.2 (d, JC,P = 29 Hz), 74.8, 70.5, 63.3 (d,

JC,P = 17 Hz), 46.9 (3, N(CH2CH3)3), 8.6 (3, N(CH2CH3)3); 31P NMR (145.8 MHz,

D2O) δ 13.8 (m); LRMS (ESI-) m/z 306.9 (M-H-, C8H12N4O7P requires 307.2).

164

6-Methylpurine ribonucleoside 5’-H-phosphonate (94). To a solution of

phosphorus trichloride19 (200 µL, 2.2 mmol) in anhydrous CH2Cl2 (5 mL) at -20

°C was added 2’,3’-O-isopropylidene 6-methylpurine ribonucleoside15 (82, 58 mg,

0.19 mmol). Compound 94 was synthesized by Method A. The product was

purified by chromatography on Sephadex LH-20, and lyophilization afforded the

product as the triethylammonium salt, a colorless glassy solid (67 mg, 82%). 1H

NMR (300 MHz, D2O) δ 8.96 (s, 1H), 8.83 (s, 1H), 6.59 (d, JP,H= 640 Hz, 1H),

6.17 (d, J = 4.8 Hz, 1H), 4.56 (dd, J1 = 4.8 Hz, J2 = 4.4 Hz, 1H), 4.40 (dd, J1 = 4.4

Hz, J2 = 4.5 Hz, 1H), 4.27 (m, 1H), 4.02 (m, 2H), 3.05 (dd, 6H, N(CH2CH3)3), 2.87

(s, 3H), 1.13 (t, 9H, N(CH2CH3)3); 13C NMR (75 MHz, D2O) δ 156.0, 152.2, 148.0,

147.6, 132.6, 88.9, 84.4 (d, JC,P = 31 Hz), 74.7, 70.5, 63.0 (d, JC,P = 15 Hz), 47.0

(3, N(CH2CH3)3), 16.1, 8.6 (3, N(CH2CH3)3); 31P NMR (145.8 MHz, D2O) δ 7.5

(m); IR (film) ν max 3245 (br), 2990, 2944, 2681, 2496, 2360, 2342, 1655, 1601,

1495, 1460, 1401, 1336, 1213, 1058, 985, 900, 816, 751 cm-1; HRMS (ESI-) m/z

329.0639 (M-H-, C11H14N4O6P requires 329.0651).

Uridine 5’-triphosphate (UTP, 96) and the general method for the synthesis

and purification of NTPs. Prior to the reaction, tris(tetra-n-butylammonium)

165

hydrogen pyrophosphate18 (95, 45 mg, 0.05 mmol) and uridine 5’-H-phosphonate

(89, 20 mg, 0.05 mmol) were dried overnight under vacuum at 22 °C in two

separate round bottom flasks (25 mL). To the round bottom flask containing 89,

anhydrous DMF (1.5 mL) and pyridine (100 µL, 1.24 mmol) were added. After the

solid was dissolved, TMSCl (32 µL, 0.25 mmol) was added by microsyringe. After

5 min, a solution of I2 in DMF (0.2 M, 350 µL) was added dropwise by

microsyringe. Upon mixing, the brown color of iodine initially disappeared, but

addition of the I2 solution was continued until the color of iodine was maintained.

The brown solution was stirred for 2 min at 22 °C. Tris(tetra-n-butylammonium)

hydrogen pyrophosphate in anhydrous DMF (0.5 mL) was quickly injected into

the reaction mixture. The reaction was stirred for 30 min at 22 °C and

concentrated in vacuo. The resulting residue of 96 was dissolved in cold

deionized water (1 mL) by sonication for ~ 1 min. The precipitated iodine was

removed by filtration of the solution through a small plug of cotton inserted in the

bottom of a 12 cm glass Pasteur pipet. The flask was washed once with cold

deionized water (1 mL), and this solution was also passed through the pipet

microfilter. The combined filtrate was loaded onto a Sephadex LH-20 column (2.5

cm × 40 cm) pre-equilibrated with TEAB buffer, and the crude triphosphate was

eluted with ice-cold TEAB buffer (10 mM, pH = 7.5) at the rate of 2 mL/min under

moderate pressure. The fractions containing the triphosphate (the typical volume

required for elution of the triphosphate is 30 to 45 mL) were identified by mass

spectrometry, combined, and lyophilized to afford crude UTP (96). Further

purification was achieved by preparative reverse-phase HPLC with a linear

166

gradient of 0% to 10% CH3CN in TEAB buffer (10 mM, pH = 7.5) over 30 min;

retention time = 6.8-8.2 min (detected by absorbance at 215 nm). Repeated

lyophilization and resuspension in ddH2O (5 mL × 3) afforded the product (18 mg,

41%) as the tetra(triethylammonium) salt, a colorless glassy solid. 1H NMR (300

MHz, D2O) δ 7.85 (d, J = 8.1 Hz, 1H), 5.86 (d, J = 5.6 Hz, 1H), 5.84 (d, J = 8.1 Hz,

1H), 4.29 (m, 2H), 4,15 (m, 1H), 4.11 (m, 2H), 3.07 (dd, 24H), 1.12 (t, 36H); 31P

NMR (146 MHz, D2O) δ -8.9 (d, JP,P = 19 Hz), -10.7 (d, JP,P = 19.0 Hz), -22.3 (t,

JP,P = 19.0 Hz); LRMS (ESI-) m/z 483.3 (M-H-, C9H14N2O15P3 requires 483.1).

Cytidine 5’-triphosphate (CTP, 97). Prior to reaction, tris(tetra-n-

butylammonium) hydrogen pyrophosphate18 (95, 40 mg, 0.045 mmol) and

cytidine 5’-H-phosphonate (90, 18 mg, 0.045 mmol) were dried overnight under

vacuum at 22 °C in two separate round bottom flasks (25 mL). As described in

detail in the general method for the synthesis and purification of NTPs, to the

round bottom flask containing 90 was added pyridine (100 µL, 1.24 mmol) and

anhydrous DMF (1.5 mL). TMSCl (45 µL, 0.35 mmol) was added, followed by I2

(0.2 M in DMF, 350 µL), and pyrophosphate. Further purification on Sephadex,

by preparative reverse-phase HPLC (linear gradient of 0% to 10% CH3CN in

167

TEAB buffer (10 mM, pH = 7.5) over 30 min; retention time = 6.8-8.7 min (215

nm)), and thorough lyophilization afforded the product (10 mg, 26%) as the

tetra(triethylammonium) salt, a colorless glassy solid. 1H NMR (360 MHz, D2O) δ

7.96 (d, J = 7.6 Hz, 1H), 6.12 (d, J = 7.6 Hz, 1H), 5.98 (d, J = 4.3 Hz, 1H), 4.38

(dd, J1 = 4.3 Hz, J2 = 4.5 Hz,1H), 4.30 (dd, J1 = 4.5 Hz, J2 = 5.1 Hz, 1H), 4.27 (m,

1H), 4.24 (m, 2H), 3.17 (dd, 24H, N(CH2CH3)3 × 4), 1.25 (t, 36H, N(CH2CH3)3 ×

4); 31P NMR (145.8 MHz, D2O) δ -9.5 (d, JP,P = 19.8 Hz), -10.7 (d, JP,P = 19.8 Hz),

-22.4 (t, JP,P = 19.8 Hz); LRMS (ESI-) m/z 482.3 (M-H-, C9H15N3O14P3 requires

482.1).

Guanosine 5’-triphosphate (GTP, 98). Prior to reaction, tris(tetra-n-

butylammonium) hydrogen pyrophosphate18 (95, 40 mg, 0.045 mmol) and

guanosine 5’-H-phosphonate (91, 20 mg, 0.045 mmol) were dried overnight

under vacuum at 22 °C in two separate round bottom flasks (25 mL). As

described in detail in the general method for the synthesis and purification of

NTPs, to the round bottom flask containing 91 was added pyridine (100 µL, 1.24

mmol) and anhydrous DMF (1.5 mL). TMSCl (45 µL, 0.35 mmol) was added,

followed by I2 (0.2 M in DMF, 350 µL), and pyrophosphate. Further purification on

Sephadex, by preparative reverse-phase HPLC (linear gradient of 0% to 10%

168

CH3CN in TEAB buffer (10 mM, pH = 7.5) over 30 min; retention time = 10.0-

11.9 min (215 nm)), and thorough lyophilization afforded the product (14 mg,

33%) as the tetra(triethylammonium) salt, a colorless glassy solid. 1H NMR (360

MHz, D2O) δ 8.1 (s, 1H), 5.89 (d, J = 5.6 Hz, 1H), 4.55 (dd, 1H), 4.32 (dd, 1H),

4.20 (m, 3H), 3.15 (dd, 24H, N(CH2CH3)3 × 4), 1.23 (t, 36H, N(CH2CH3)3 × 4); 31P

NMR (145.8 MHz, D2O) δ -8.9 (d, JP,P = 19.6 Hz), -10.6 (d, JP,P = 19.6 Hz), -22.2

(t, JP,P = 19.6 Hz); LRMS (ESI-) m/z 522.3 (M-H-, C10H15N5O14P3 requires 522.2).

Adenosine 5’-triphosphate (ATP, 99). Prior to reaction, tris(tetra-n-

butylammonium) hydrogen pyrophosphate18 (95, 42 mg, 0.046 mmol) and

adenosine 5’-H-phosphonate (92, 20 mg, 0.046 mmol) were dried overnight

under vacuum at 22 °C in two separate round bottom flasks (25 mL). As

described in detail in the general method for the synthesis and purification of

NTPs, to the round bottom flask containing 92 was added pyridine (100 µL, 1.24

mmol) and anhydrous DMF (1.5 mL). TMSCl (47 µL, 0.37 mmol) was added,

followed by I2 (0.2 M in DMF, 350 µL), and pyrophosphate. Further purification on

Sephadex, by preparative reverse-phase HPLC (linear gradient of 0% to 10%

CH3CN in TEAB buffer (10 mM, pH = 7.5) over 30 min; retention time = 12.1-14.6

169

min (215 nm)), and thorough lyophilization afforded the product (13 mg, 31%) as

the tetra(triethylammonium) salt, a colorless glassy solid. 1H NMR (360 MHz,

D2O) δ 8.40 (s, 1H), 8.09 (s, 1H), 5.98 (d, J = 6.0 Hz, 1H), 4.72 (dd, 1H), 4.63 (dd,

1H), 4.25 (m, 1H), 4.05 (m, 2H), 3.03 (dd, 24H, N(CH2CH3)3 × 4), 1.11 (t, 36H,

N(CH2CH3)3 × 4); 31P NMR (145.8 MHz, D2O) δ -8.7 (d, JP,P = 19.3 Hz), -10.6 (d,

JP,P = 19.3 Hz), -22.4 (t, JP,P = 19.3 Hz); LRMS (ESI-) m/z 506.2 (M-H-,

C10H15N5O13P3 requires 506.2).

Ribivarin 5’-triphosphate (RTP, 100). Prior to reaction, tris(tetra-n-

butylammonium) hydrogen pyrophosphate18 (95, 35 mg, 0.039 mmol) and

ribivarin 5’-H-phosphonate (93, 16 mg, 0.039 mmol) were dried overnight under

vacuum at 22 °C in two separate round bottom flasks (25 mL). As described in

detail in the general method for the synthesis and purification of NTPs, to the

round bottom flask containing 93 was added pyridine (100 µL, 1.24 mmol) and

anhydrous DMF (1.5 mL). TMSCl (40 µL, 0.31 mmol) was added, followed by I2

(0.2 M in DMF, 300 µL), and pyrophosphate. Further purification on Sephadex,

by preparative reverse-phase HPLC (linear gradient of 0% to 10% CH3CN in

TEAB buffer (10 mM, pH = 7.5) over 30 min; retention time = 7.0-8.4 min (215

nm)), and thorough lyophilization afforded the product (14 mg, 40%) as the

170

tetra(triethylammonium) salt, a colorless glassy solid. 1H NMR (360 MHz, D2O) δ

8.70 (s, 1H), 5.90 (d, J = 4.4 Hz, 1H), 4.61 (dd, 1H), 4.47 (dd, 1H), 4.26 (m, 1H),

4.11 (m, 2H), 3.06 (dd, 24H, N(CH2CH3)3 × 4), 1.14 (t, 36H, N(CH2CH3)3 × 4); 31P

NMR (145.8 MHz, D2O) δ -9.1 (d, JP,P = 19.9 Hz), -10.6 (d, JP,P = 19.9 Hz), -22.3

(t, JP,P = 19.9 Hz); LRMS (ESI-) m/z 483.2 (M-H-, C8H14N4O14P3 requires 483.1).

6-Methylpurine ribonucleoside 5’-triphosphate (6MePTP, 101). Prior to

reaction, tris(tetra-n-butylammonium) hydrogen pyrophosphate18 (95, 90 mg,

0.10 mmol) and 6-methylpurine ribonucleoside 5’-H-phosphonate (94, 43 mg,

0.10 mmol) were dried overnight under vacuum at 22 °C in two separate round

bottom flasks (25 mL). As described in detail in the general method for the

synthesis and purification of NTPs, to the round bottom flask containing 94 was

added pyridine (100 µL, 1.24 mmol) and anhydrous DMF (1.5 mL). TMSCl (64 µL,

0.50 mmol) was added, followed by I2 (0.2 M in DMF, 300 µL), and

pyrophosphate. Further purification on Sephadex, by preparative reverse-phase

HPLC (linear gradient of 0% to 10% CH3CN in TEAB buffer (10 mM, pH = 7.5)

over 30 min; retention time = 11.1-14.8 min (215 nm)), and thorough

lyophilization afforded the product (24 mg, 27%) as the tetra(triethylammonium)

171

salt, a colorless glassy solid. 1H NMR (360 MHz, D2O) δ 8.70 (s, 1H), 8.66 (s,

1H), 6.12 (d, J = 6.0 Hz, 1H), 4.73 (dd, 1H), 4.51 (dd, 1H), 4.28 (m, 1H), 4.13 (m,

2H), 3.05 (dd, 24H, N(CH2CH3)3 × 4), 2.68 (s, 3H), 1.13 (t, 36H, N(CH2CH3)3 × 4);

31 P NMR (145.8 MHz, D2O) δ -9.7 (d, JP,P =19.8 Hz), -10.6 (d, JP,P = 19.8 Hz), -

22.3 (t, JP,P = 19.8 Hz); HRMS (ESI-) m/z 504.9900 (M-H-, C11H16N4O13P3

requires 504.9927). The purity of 101 was further examined by analytic HPLC

with UV detection (flow rate = 1 mL/min; linear gradient of 1% to 80% CH3CN in

KH2PO4 buffer (100 mM, pH = 6.0) over 30 min). As shown in Figure 5.8, 101

was eluted at 6.3 min in 92% purity.

Figure 5.8: Analytical HPLC profile of 6MePTP (101) after purification by preparative HPLC.

172

5.8.3 Biological Assays

Analysis of incorporation of 6MePTP (101) into RNA mediated by poliovirus

RNA-dependent RNA polymerase (3Dpol): Incorporation of 6MePTP (101)

opposite each of the four templating bases (A, C, G and U) was examined using

pre-assembled 3Dpol-primer/template complexes. Incorporation of the correct

nucleotide across each templating base served as a comparative control. As

previously reported,24,25 symmetrical substrates (sym/subs) served as the primer

and template RNA for 3Dpol nucleotide incorporation assays. Annealing of 32P-

end-labeled and unlabeled sym/sub oligos to form the primer/template duplex

was preformed as previously described.24,25 3Dpol was allowed to preassemble

with sym/sub duplex for 2 min at 30°C. Incorporation assays with A, C and U as

templating bases were performed at 30°C in HEPES buffer (pH 7.5, 50 mM)

containing 2-mercaptoethanol (10 mM), MgCl2 (5 mM), 3Dpol (1 µM), sym/sub

duplex RNA (0.5 µM) and nucleotide (500 µM). The incorporation assay with G

as the templating base was accomplished using a sym/sub duplex initially having

C in the first templating position and G in the second. Assembly of 3Dpol with the

sym/sub duplex was performed in the presence of GTP (5 µM) to create a

sym/sub duplex having G in the first templating position. The subsequent

incorporation assay was identical to those described for A, C and U as templating

bases. All incorporation assays were initiated by addition of nucleotide, reaction

products were separated by denaturing PAGE, gels were visualized using a

173

phosphorimager, and radioactivity was quantified using ImageQuant software

(Molecular Dynamics).

5.9 References

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2000, 100, 2047-2060.

2. Ludwig, J. A new route to nucleoside 5'-triphosphates. Acta Biochim.

Biophys. Hung. 1981, 16, 131-133.

3. Ludwig, J.; Eckstein, F. Rapid and efficient synthesis of nucleoside 5'-O-

(1-thiotriphosphates), 5'-triphosphates and 2',3'-cyclophosphorothioates using 2-

chloro-4H-1,3,2-benzodioxaphosphorin-4-one. J. Org. Chem. 1989, 54, 631-635.

4. Wu, W. D.; Bergstrom, D. E.; Davisson, V. J. A combination chemical and

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6. Wu, W. D.; Meyers, C. L. F.; Borch, R. F. A novel method for the

preparation of nucleoside triphosphates from activated nucleoside

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phosphorofluoridate, nucleoside phosphorofluoridothioate, and nucleoside

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9. Kers, I.; Stawinski, J.; Kraszewski, A. A new synthetic method for the

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1219-1222.

10. Wada, T.; Mochizuki, A.; Sato, Y.; Sekine, M. A convenient method for

phosphorylation involving a facile oxidation of H-phosphonate monoesters via

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11. Garegg, P. J.; Regberg, T.; Stawinski, J.; Stromberg, R. Nucleoside

phosphonates: Part 7. Studies on the oxidation of nucleoside phosphonate esters.

J. Chem. Soc., Perkin Trans. 1 1987, 6, 1269-1273.

12. Cullis, P. M.; Lee, M. The mechanism of iodine-water oxidation of H-

phosphonate diesters. J. Chem. Soc., Chem. Commun. 1992, 17, 1207–1208.

13. Hampton, A. Nucleotides. Il. A new procedure for the conversion of

ribonucleosides to 2‘,3’-O-isopropylidene derivatives. J. Am. Chem. Soc. 1961,

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antiviral activity of ribavirin and its derivatives. Chin. J. Org. Chem. 2004, 24,

1432-1435.

15. Silamkoti, A. V.; Allan, P. W.; Hassan, A. E. A.; Fowler, A. T.; Sorscher, E.

J.; Parker, W. B.; Secrist, J. A. Synthesis and biological activity of 2-fluoro

adenine and 6-methyl purine nucleoside analogs as prodrugs for suicide gene

therapy of cancer. Nucleos. Nucleot. Nucl. 2005, 24, 881-885.

16. Marugg, J. E.; Tromp, M.; Kuylyeheskiely, E.; Vandermarel, G. A.;

Vanboom, J. H. A convenient and general approach to the synthesis of property

protected d-nucleoside-3'-hydrogenphosphonates via phosphite intermediates.

Tetrahedron Lett. 1986, 27, 2661-2664.

17. Nilsson, J.; Kraszewski, A.; Stawinski, J. Chemical and stereochemical

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diesters. Reactions with N,N-, N,O-and O,O-binucleophiles. Lett. Org. Chem.

2005, 2, 188-197.

18. Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremler, K. E.;

Muehlbacher, M.; Poulter, C. D. Phosphorylation of isoprenoid alcohols. J. Org.

Chem. 1986, 51, 4768-4779.

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phosphonate derivatives of d4T and AZT. Chem. Commun. 2002, 20, 2414-2415.

20. van Boom, J. H.; Crea, R.; Luyten, W. C.; Vink, A. B. 2,2,2-Tribromoethyl

phosphoromorphalinochlorioate: A convenient reagent for the synthesis of

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2782.

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22. Bogachev, V. S.; Ulanov, P. A., The interaction of trihalogenoacetic

anhydrides and trihalogenoacetyl chlorides with thymidine 5'-phosphate as an

approach to new activating agents in the phosphorylation reactions for

nucleotides. Russ. J. Bioorg. Chem. 2003, 29, 56-65.

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Herdewijn, P. A. Antiviral activity of C-alkylated purine nucleosides obtained by

cross-coupling with tetraalkyltin reagents. J. Med. Chem. 1993, 36, 2938-2942.

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Peterson, B. R. Synthesis of a universal 5-nitroindole ribonucleotide and

incorporation into RNA by a viral RNA-dependent RNA polymerase.

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25. Harki, D. A.; Graci, J. D.; Korneeva, V. S.; Ghosh, S. K.; Hong, Z.;

Cameron, C. E.; Peterson, B. R. Synthesis and antiviral evaluation of a

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ribofuranosyl-3-nitropyrrole. Biochemistry 2002, 41, 9026-9033.

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A.; Galarraga, J. E.; Bollinger, J. M., J.; Peterson, B. R.; Loakes, D.; Brown, D.

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27. Crotty, S.; Maag, D.; Arnold, J. J.; Zhong, W.; Lau, J. Y. N.; Hong, Z.;

Andino, R.; Cameron, C. E. The broad-spectrum antiviral ribonucleoside ribavirin

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Chapter 6

Synthesis of an Akt Inhibitor and Its Analogues via a Novel and Efficient

Zr(IV)-Catalyzed Cyclization Reaction and Evaluation of Antiviral Activity

6.1 Introduction

Akt, also called protein kinase B, is an important serine/theronine kinase

homologous to protein kinase A and C in humans. The Akt gene was first

discovered as the human homologue of the oncogene, v-akt, in the transforming

retrovirus AKT8.1-4 The three isoforms of Akt, which are derived from distinct

genes (Akt1/PKB-α, Akt2/PKB-β, and Akt3/PKB-γ), contain a Pleckstrin

Homology (PH) domain, which binds 3’-phosphoinositides to recruit Akt to

membranes with the assistance of other Akt interaction partners. Upon anchoring

to the plasma membrane, the two phosphorylation sites of Akt (Thr 308 and Ser

473 of Akt1) are phosphorylated by phosphoinositide-dependent kinase 1 and 2

(PDK1 and 2) respectively to fully activate its kinase activity.5 As the major

downsteam mediator of the effect of phosphatidylinositol-3-kinase (PI3K), Akt

plays a key role in the PI3K signaling pathway, which is critical in the regulation

179

of cell growth and metabolism.6,7 Among the numerous downstream targets of

Akt, the most important ones include the mammalian target of rapamycin

(mTOR), Bcl-2-associated death promoter (BAD), procaspase-9, Forkhead

(FKHR) transcription factors, cyclic-AMP response element-binding protein

(CREB), IκB kinase (IKK), glycogen synthase kinase 3 (GSK-3), and mdm2

Figure 6.1: The PI3K/Akt signaling pathway. Akt is activated downstream of PI3Kand has multiple targets.

180

protein.5,8-13 It has been revealed that the PI3K/Akt signaling pathway is

aberrantly activated in many tumors, which results in promoted proliferation and

inhibited apoptosis of cancer cells, metastasis, and therapeutic resistance.14-20

Therefore, Akt has been recognized as a novel therapeutic target for anticancer

drug discovery.6,7,21-24

Recently, Biao He et al. from Penn State University discovered that Akt

plays a critical role in replication of nonsegmented negative-stranded RNA

viruses (NNSVs).25 In their earlier research, it was discovered that V protein of

Parainfluenza virus 5 (PIV5), also known as simian virus 5, plays an important

role in regulating viral RNA synthesis.26 Though the mechanism of action was still

not clear, it was speculated that a host protein was involved. In subsequent

research, He and his coworkers examined the interactions between V protein

and Akt1, MAPK1, and PIP3E, on the basis of bioinformatics screening using

Scanite (http://scanite.mit.edu). They found that V interacts with Akt by using

both yeast two-hybrid screening and affinity copurification methods. Further

experiments with both chemical inhibitors and small interfering RNA against Akt1

reduced PIV5 replication, indicating that Akt is indeed involved in PIV5 replication.

Since Akt is a protein kinase, and phosphorylation of viral P protein is essential

for its activation for viral RNA synthesis, interactions between Akt and P protein

were studied.27 It was found that inhibition of Akt reduced the phosphorylation of

P protein, and Akt1 can directly phosphorylate recombinant P protein in an in

vitro kinase assay. On the basis of these results, a model of the role of Akt in

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viral replication was proposed (Figure 6.2). Akt possibly promotes viral RNA

synthesis by phosphorylating P protein to form the P/L complex.25

Among the small molecule Akt inhibitors tested, compound 11128 (EMD

biosciences, Figure 6.3), which targets all three isoforms of Akt, exhibited the

most potent inhibition of PIV5 replication. It was determined that 111 was also

effective against a number of other paramyxoviruses, such as mumps virus

(MuV), measles virus (MeV), sendai virus (SeV), respiratory syncytial virus (RSV),

Figure 6.2: A model for the involvement of Akt in viral RNA synthesis. Thereplication of viral RNA and synthesis of viral mRNA require P and L complex. The phosphorylation status of the P protein plays a critical in viral RNA synthesis.Akt phosphorylates P, thus leading to the activation of P. For viruses encoding aV protein, the V protein can regulate viral RNA synthesis via its interaction with Akt through an unknown mechanism. The V-Akt1 interaction may also contribute to regulation of innate immune responses by the V protein. Activation of Akt1 isoften through the PI3K pathway. However, it is not clear what activates Akt1 invirus-infected cells, since PI3K inhibitors had no effect on virus replication. Figurecourtesy of Professor Biao He.

182

and a prototypical rhabdovirus, vesicular stomatitis virus (VSV). These

experiments demonstrated the universal role of Akt in NNSV replication and

implied for the first time the potential of Akt as a novel therauputic target in the

treatment of numerous human infectious diseases caused by NNSVs.25

Another very interesting question from this research is how Akt is

activated in the infected host cells. Since Akt is primarily activated through the

PI3K pathway, two PI3K inhibitors, wortmannin and LY294002, were tested on

PIV5-infected cells in He’s research.25 However, these two compounds failed to

inihibit viral replication, indicating that Akt was activated by a kinase downstream

of PI3K, or an unknown kinase, in infected cells. It has been reported that unlike

many other small molecule Akt inhibitors targeting ATP-binding site, allosteric

sites, or PH domain, 111 targets the ATP-binding site of an unknown kinase

downstream of PI3K.28 Therefore, 111 exhibits its antiviral activity possibly by

inhibiting this specific unknown kinase, resulting in the activation of Akt after viral

infection. Consequently, 111 may intercept viral replication with minimum

interruption of the normal of PI3K/Akt signaling pathway in host cells, which could

be a major advantage over other direct Akt inhibitors. Due to its excellent antiviral

Figure 6.3: Structure of an Akt inhibitor, compound 111.

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activity and unique mechanism of action, we selected compound 111 as the lead

for further development of more potent small-molecule agents against NNSVs.

We report in this chapter the synthesis of 111 and its analogues via a

novel and efficient Zr(IV)-catalyzed cyclization of substituted 1,2-arylenediamines

and α,β-unsaturated aldehydes. Preliminary biological screening of the new

compound library has led to the identification of four compounds with higher

potency than 111 and provided valuble information for the analysis of structure-

activity relationships (SAR).

6.2 Synthesis of Lead Compound 111

Based on previously reported syntheses of structurally similar

benzimidazole compounds,29,30 our synthetic route to lead compound 111

employed the construction of substituted 1,2-arylenediamine 116 and

subsequent condensation with α,β-unsaturated aldehyde 117. Alkylation of the

benzimidazole 120 afforded benzimidazolium salt 111. The preparation of 116

started with 2-aminothiophenol 112 and 3-chloro-4-nitro benzaldehyde 113. After

48 h of refluxing in ethanol,31 benzothiazole 114 precipitated as a white solid.

After filtration and evaporation under vacuum, 114 was treated with freshly

distilled aniline and heated at 80 °C for 10 h.32,33 After acidic workup, column

chromatography afforded 115 as an orange solid, which was refluxed with

hydrazine in the presence of Pd (II) catalyst for 15 min to reduce the nitro group

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to amine.34 Recrystallization of 116 from methylene chloride/hexanes provided a

light yellow solid. Condensation of un- or simply- substituted 1,2-arylenediamines

with unconjugated aldehydes at alleviated temperatures is known to afford

benzimidazoles.32,33,35 Mild oxidants, such as Oxone,32 MnO2,36 and DDQ,37 have

been reported to facilitate the formation of benzimidazoles by oxidizing

benzimidazoline intermediates. When 116 and α,β-unsaturated aldehyde 117,

which was prepared as previously described,38 were refluxed in ethanol, the

reaction was extremely slow. Addition of the oxidants mentioned above resulted

in decomposition of 116 within 5 min, and complex reaction mixtures were

Figure 6.4: Synthesis of lead compound 111. Reagents and conditions: (a) EtOH, reflux, 48 h; (b) aniline, DMSO, 70°C, 12 h; (c) NH2NH2, EtOH, reflux, 15 min; (d) 117, 3Å molecular seives, EtOH, reflux, 48 h; (e) MnO2, EtOH, reflux, 5 min; (f) EtI, reflux, 12 h.

185

obtained. These results were in agreement with the fact that α,β-unsaturated

aldehydes are known to be poor substrates for the preparation of

benzimidazoles.32,36,37 Therefore, the cyclization and oxidation steps were

conducted separately. It was found that in the presence of 3Å molecular sieves,

imine intermediate 118 was generated along with 2H-benzimidazole 119 as a

major byproduct in refluxing ethanol, and only MnO2 was mild enough to

generate 120 without decomposing the intermediate. However, this reaction was

low-yielding, time-consuming, and poorly reproducible at larger scale. Alkylation

of 120 with a large excess of ethyl iodide afforded the target compound 111.

6.3 A Novel Zr(IV)-Catalyzed Synthetic Method for the Preparation of

Vinylbenzimidazoles

Some metals, such as ZrOCl2, CuSO4, and FeCl3, are capable of

promoting the cyclization of structurally similar benzimidazoles, benzothiazoles,

and purines.39-41 Based on this precedent, we explored the possibility of

synthesizing benzimidazole 120 with metal catalysts. Towards this end, the metal

lewis acids listed in Table 6.1, were screened for their activities to promote

cyclization of 116 and 117 in refluxing ethanol. It was found that while CuSO4,

Cu(OAc)2, CeCl3, InCl3, PdCl2 and ZnCl2 failed to give desired product, AlCl3 and

FeCl3 promoted low to moderate conversion of reactants to desired product. In

contrast, 0.5 equiv. of ZrOCl2 and ZrCl4 afforded product 120 in 68% and 75%

186

yields, respectively, after in situ oxidation with MnO2. Therefore, ZrCl4·was used

as the best metal catalyst for further studies of solvent effects on this reaction. As

shown in Table 6.2, reactions were performed in different organic solvents, and

Table 6.1: Metal lewis acid screening for synthesis of 120. aThe reaction conditions were optimized to maximize the yield of 120. bAll reactions (0.1 mmol scale) were performed in EtOH (10 mL). CThe product was purified by column chromatography after in situ oxidation of the imine intermdiate with MnO2. dIn case of NR, no further oxidation with MnO2 was performed. NR: No reaction observed.

187

the yields revealed that this reaction was quite solvent-specific; ethanol was the

best solvent for a smooth reaction.

Since it is also well known that microwave irradiation often enhances

organic reactions, especially condensation and cyclization reactions, in rate and

yield,42,43 microwave-assisted conditions were tested and compared with thermal

conditions. The advantage of microwave irradiation is that it greatly accelerated

the reaction, shortening the reaction time from 30 min to only 2 min. However, it

also slightly promoted the formation of 2H-benzimidazole (119) and the over-

Table 6.2: Solvent effects on Zr(IV)-catalyzed synthesis of 120. aAll reactions (0.1 mmol scale) were performed in solvent (10 mL). bThe product was purified by column chromatography after in situ oxidation of the imine intermdiate with MnO2. cIn case of NR, no further oxidation with MnO2 was performed. NR: No reaction observed.

188

alkylated benzimidazolium byproducts, resulting in a lower yield of desired

product.

To explore the scope and generality of this novel Zr(IV)-catalyzed

synthetic method of vinylbenzimidazoles, other α,β-unsaturated and

unconjugated aldehyde substrates were tested. Compound 116 was reacted with

the four α,β-unsaturated aldehyde substrates 121-124. As summarized in Figure

6.5., all four benzimidazole products 127-130 were obtained in good to high

yields directly without MnO2 oxidation, indicating that oxidation of these imine

intermediates preceeded by spontaneous disproportionation.32,33 This reaction

also works for unconjugated alkyl and aryl aldehydes 125 and 126. However, the

yields for the corresponding products 131 and 132 were significantly lower than

Table 6.3: Comparison of microwave-assisted and thermally heated conditions for Zr(IV)-catalyzed synthesis of 120. aAll reactions (0.1 mmol scale) were performed in EtOH (10 mL). bThe product was purified by column chromatography after in situ oxidation of the imine intermdiate with MnO2.

189

those of α,β-unsaturated aldehydes, indicating that this Zr(IV)-catalyzed

cyclization specifically favors conjugate aldehyde substrates.

6.4 An Interesting Transamination Reaction on 111

Interestingly, when benzimidazolium iodide 111 was treated with large

excess of amines, such as N-methylcyclohexylamine, N-methylbenzylamine, and

1-phenylpiperazine in DMF, unexpected amine-exchange reactions of the N-

methylaniline moiety were observed and three new vinylbenzimidazolium

analogs 140-142 were obtained in high yields. However, when the unalkylated

Figure 6.5: Scope of the Zr(IV)-catalyzed cyclization reaction. aAll reactions (0.2 mmol scale) were performed in EtOH (10 mL).

190

compound 120 was treated with excess amines, no reaction happened,

indicating that the positive charge in the vinylbenzimidazolium salt 111 is crucial

for the transamination reaction to proceed (Figure 6.6).

6.5 Design and Synthesis of a Benzimidazole-Based Compound Library

To explore structure-activity relationships (SAR) of AKT inhibitor 111 as an

antiviral agent, a vinylbenzimidazole-based compound library was designed and

synthesized via the novel Zr(IV)-catalyzed methodology. In the structure of lead

compound 111, the benzimidazole moiety was considered as the core of the

molecule because benzimidazole is a key heterocyclic scaffold for a great variety

Figure 6.6: Transamination reactions on vinylbenzimidazolium compound 111 and proposed mechanism. aAll reactions (0.025 mmol scale) were performed in DMF (1 mL).

191

of biologically active molecules30-38 and defines in the center of compound 111.

The four functional groups at different positions of 111 were either removed or

replaced with other functionalities, leading to the generation of four groups of

analogues (Groups I-IV).

Group I is focused on the analogues with different substitutions at the R1

position. Since the fixed charge in lead compound 111 may limit intestinal

absorption, charge-free precursors without ethylation at R4 were also examined

to determine whether the positive charge could be removed without affecting the

acitivity of these compounds. The R1 was eliminated in 119 and 144. The

conjugated vinyl system was maintained with variations at the side chain

terminus in 127-130, 140-142, and 145-148, whereas the vinyl substituent was

eliminated in 131, 132, 149, and 150 (Figure 6.8). The synthesis of these

analogues utilized the schemes described in previous sections (6.2, 6.3, and 6.4).

Group II involves the analogues that have variations at R2 position. The

phenyl group was replaced with different alkyl and aryl substitutions in 158-160

Figure 6.7: Core benzimidazole and variations (R1-R4) in the compound library.

192

and 161-163 as shown in Figure 6.9. Because regioselective alkylation of the

benzimidazole precursor via the scheme employed for the synthesis of 161-163

was a potential problem, the R2-eliminated analogues were synthesized via the

scheme shown in Figure 6.10. However, due to the poor yield and solubility

issues with benzothiazole-substituted substrate, the similar benzoxazole-

Figure 6.8: Structures of analogues of 111 in Group I.

193

substituted precusor 165 was used to generate the R2-eliminated benzimidazole

derivatives (170-171).

Figure 6.9: Synthesis and structures of analogues of 111 in Group II (158-163). Reagents and conditions: (a) 1-hexylamine/ cyclohenxylamine, DMSO, 45 °C, 24h, or p-anisidine, 80 °C, 12 h; (b) NH2NH2, Pd/C, EtOH, reflux, 15 min; (c) 117, ZrCl4, EtOH, reflux, 30 min; (d) MnO2, EtOH, reflux, 5 min; (e) EtI, reflux, 24 h.

194

The analogues of Group III and IV have varitations at R3 and R4 position

respectively. The original benzothiazole group at R3 was either removed or

replaced to test the structural tolerance at this positon in compounds 184-187

and 188-191 (Figure 6.11). Compound 120 was also alkylated with either longer

alkyl chains or an ethylphenyl group to increase the lipophilicity of these

compounds 192-194 (Figure 6.12).

Figure 6.10: Synthesis and structures of analogues of 111 in Group II (170-171). Reagents and conditions: (a) 2-aminophenol, HATU, DMF, 2 h; (b) Xylene, p-TsOH, relux, 1 h; (c) ethylamine/hexylamine, DMSO/THF (1:1), 22 °C, 48 h; (d)NH2NH2, Pd/C, EtOH, reflux, 15 min; (e) 117, ZrOCl2, EtOH, reflux, 30 min; (f) MnO2, EtOH, reflux, 5 min

195

Figure 6.11: Synthesis and structures of analogues of 111 in Group III (184-191). Reagents and conditions: (a) aniline, DMSO, 80 °C, 24 h; (b) NH2NH2, Pd/C, EtOH, reflux, 15 min; (c) 117, ZrCl4, EtOH, reflux, 30 min; (d) MnO2, EtOH, reflux, 5 min; (e) EtI, reflux, 24 h.

196

6.6 Biological Evaluation of Compound Library and Structure-Activity

Relationship (SAR) Analysis

To evaluate the inhibitory effects of novel benzimidazole-based

compounds on the replication of NNSVs, a preliminary screening, employing the

luciferase-based (r-PIV5-R-Luc) infection assay,25 was conducted by Professor

Biao He. In these experiments, Hela cells were infected with r-PIV5-R-Luc at a

MOI of 1 and treated with the compounds at 1 µM. DMSO and lead compound

111 were used as negative and positive controls. The cells were collected at 24

hours postinfection and assayed for luciferase activity. The quantitative results of

Figure 6.12: Synthesis and structures of analogues of 111 in Group IV (192-194).

197

the inhibition of viral replication by these compounds are summarized in Figures

6.13 and 6.14.

The trans-vinyl group at R1 position proved to be of high importance for

antiviral activity. Compounds with the trans-vinyl group (145-148) all resulted in

over 50% inhibition of viral replication at 1 µM, whereas compounds without it

(149-151) were much less effective, and the R1-deletion compound 144 was

essentially inactive. When the N-methylaniline moiety was substituted by other

secondary amines, compounds 140-142 showed better activity, possibly due to

stronger H-bonding interactions. Interestingly, all of the benzimidazole

Figure 6.13: Preliminary screening of the compounds in Group I with a luciferase-based inhibition assay.

198

precursors (119-120, 127-132, and 143) were all inactive, indicating that the

positive charge is crucial for antiviral activity.

All the variations on R2 (161-163) showed remarkable antiviral activity

comparing to lead 111, indicating a relatively high structural tolerance at this

position. However, their benzimidazole precursors (158-160) and R2-deletion

analogues (170 and 171) again lost activity, demonstrating the important role of

the positive charge in blocking viral replication.

In contrast, among the analogues with R3 variations, only the structurally

similar benzoxozole moiety maintained antiviral activity (190). Elimination of the

benzothiazole in the molecule (188) resulted in lost of about 50% of the inhibitory

Figure 6.14: Preliminary screening of the compounds in Groups II-IV with a luciferase-based inhibition assay.

199

activity, whereas a methyl ester and benzamide at the R3 position (189 and 191)

were inactive. Neutral benzimidazole precursors were again confirmed to be

inactive.

When the ethyl group at R4 position was replaced with longer alkyl chains

or an ethylphenyl group (compounds 192-194), the activity of these compounds

was enhanced. This result suggested that either R4 may have strong

hydrophobic interactions at the binding site or cell permeability may be enhanced

by the increased lipophilicity.

In the preliminary screening, 11 compounds exhibited similar (145, 147,

and 190) or higher (140-142, 161, 163, 192-194) potency than the lead

Figure 6.15: Secondary screening of active compounds.

200

compound 111. Currently, a second round of screening of these 11 hits at a

concentration of 500 nM is in progress. At least 4 compounds (140, 141, 192,

and 193) have been shown to maintain high antiviral activity (~ 90% inhibition) at

500 nM (Figure 6.15).

6.7 Conclusions

In summary, we developed a practical synthetic route for the preparation

of the putative Akt inhibitor 111 and 39 new analogues. A highlight of this

research is the development of a novel and efficient Zr(IV)-catalyzed cyclization

reaction for the construction of vinylbenzimidazoles, which are difficult to

synthesize using known methods. An unexpected but interesting transamination

reaction was observed, when 111 was treated with excess secondary amines,

leading to esay access to novel vinylbenzimidazolium analogues. Preliminary

biological screening of the new compound library identifed 11 active compounds

with comparable or better antiviral activity than the lead compound 111 at 1 µM.

Currently, the ongoing second screening for more active compounds at 500 nM

has revealed that at least 4 compounds have higher potency than lead

compound 111.

201

6.8 Experimental Section

6.8.1 General

Chemical reagents and solvents were obtained from Acros, Aldrich, and EMD

Biosciences. Commercial grade reagents were used without further purification

unless otherwise noted. Anhydrous solvents were obtained after passage

through a drying column of a solvent purification system from GlassContour

(Laguna Beach, CA). All reactions were performed under an atmosphere of dry

nitrogen. Reactions were monitored by analytical thin-layer chromatography on

plates coated with 0.25 mm silica gel 60 F254 (EMD Chemicals). TLC plates

were visualized by UV irradiation (254 nm) or stained with 20% sulfuric acid in

ethanol. Flash column chromatography employed silica gel (ICN SiliTech, 32-63

µ m). NMR spectra were obtained with Bruker CDPX-300 or AMX-360

instruments with chemical shifts reported in parts per million (ppm, δ ) referenced

to. Low-resolution mass spectra were obtained with a Waters ZQ-4000 mass

spectrometer. High-resolution mass spectra were obtained from the proteomics

and mass spectrometry core facility at Penn State University, University Park, PA.

Peaks are reported as m/z.

202

6.8.2 Synthetic Procedures and Compound Characterization Data

(4-Benzothiazol-2-yl-2-nitrophenyl)phenylamine (115). To a solution of

compound 114, 2.9 g, 10 mmol) in DMSO (50 mL) was added freshly distilled

aniline (4.6 mL, 50 mmol). The reaction was heated up to 70 °C and stirred for 8

h. The reaction was cooled to 22 °C and poured into water (200 mL). The

aqueous phase was extracted with diethyl ether (100 mL × 4). The combined

organic phase was washed with saturated aqueous NaCl solution (200 mL), dried

over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by

column chromatography on silica gel with CH2Cl2 to afford the product (2.46 g,

71%) as an orange solid; mp 160-162 °C; Rf 0.56 (hexane/ethyl acetate, 3:1); 1H

NMR (300 MHz, CDCl3) δ 9.75 (s, 1H), 8.86 (d, 1H, J = 2.1 Hz), 8.07 (dd, 1H, J1

= 2.1 Hz, J2 = 9.0 Hz), 8.00 (d, 1H, J = 8.1 Hz), 7.86 (d, 1H, J = 9 Hz), 7.49-7.44

(m, 3H), 7.36-7.25 (m, 5H); 13C NMR (75 MHz, CDCl3) δ 165.6, 156.6, 144.5,

137.8, 134.7, 134.0, 132.7, 129.9 (× 2), 126.5, 126.4, 126.0, 125.2, 124.8 (× 2),

123.3, 123.0, 121,6, 116.4; IR (film) ν max 3333, 1624, 1594, 1567, 1531, 1496,

1353, 1266, 1210, 753 cm-1; HRMS (ESI+) m/z 348.0833 (M+H+, C19H14N3O2S

requires 348.0807).

203

4-Benzothiazol-2-yl-N1-phenylbenzene-1,2-diamine (116). To a slurry of

compound 115 (1.74 g, 5 mmol) in ethanol (50 mL) were added 10% palladium

on carbon (530 mg, 0.5 mmol) and anhydrous hydrazine (0.5 mL, 16 mmol). The

reaction was heated to 80 °C, and refluxed for 30 min. The reaction was cooled

to 22 °C and filtered through a frit funnel. The palladium was washed with ethanol

(10 mL). The combined filtrate was concentrated in vacuo to afford the crude

product. Recrystallization from CH2Cl2/Hexanes afforded the product (1.52 g,

95%) as a light yellow solid; mp 154-156 °C; Rf 0.13 (hexane / ethyl acetate, 3:1);

1H NMR (300 MHz, DMSO-d6) δ 8.06 (d, 1H, J = 7.5 Hz), 7.97 (d, 1H, J = 7.7 Hz),

7.55 (d, 1H, J = 2.0 Hz), 7.52-7.46 (m, 1H) 7.44 (s, 1H) 7.41-7.36 (m, 1H) 7.29-

7.19 (m, 4H), 7.01 (d, 2H, J = 8 Hz ) 6.83 (t, 1H, J = 7.3 Hz), 5.17 (s, 2H); 13C

NMR (75 MHz, DMSO-d6) δ 168.0, 153.8, 143.7, 140.2, 134.1, 132.3, 129.1 (× 2),

126.4, 126.3, 124.8, 122.2, 122.1, 119.7, 118.4, 117.0 (× 2), 116.4, 113.2; IR

(film) ν max 3360, 3037, 1595, 1496, 1434, 1313, 1194, 1000, 810, 749, 728,

693 cm-1; HRMS (ESI+) m/z 318.1066 (M+H+, C19H16N3S requires 318.1065).

204

2-(1-Phenyl-1H-benzoimidazol-5-yl)benzothiazole (119). To a solution of 116

(130 mg, 0.4 mmol) and 117 (65 mg, 0.4 mmol) in ethanol (15 mL) was added

activated 3Å molecular seives (0.5 g). The reaction was heated to 80 °C and

refluxed for 48 h. The reaction was cooled and filtered through a frit funnel. The

molecular seives were washed with ethanol (10 mL). The combined filtrate was

concentrated in vacuo. Flash column chromatography (hexanes/ethyl acetate,

2:1) afforded the product (20 mg, 31%) as a white solid; mp 138-140 °C; Rf 0.17

(hexane/ethyl acetate, 1:1); 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 1.1 Hz, 1H),

8.15 (s, 1H), 8.14 (dd, J1 = 1.4 Hz, J2 = 9.9 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.87

(d, J = 7.9 Hz, 1H), 7.58-7.54 (m, 3H), 7.49-7.44 (m, 4H), 7.34 (t, J = 7.3 Hz, 1H);

13C NMR (75 MHz, CDCl3) δ 168.4, 154.2, 144.3, 143.7, 135.8, 135.5, 135.0,

130.2 (× 2), 128.8, 128.4, 126.9, 126.2, 124.9, 124.0 (× 2), 123.2, 123.0, 121.5,

120.3, 111.0; IR (film) ν max 3382, 3064, 2954, 2360, 1617, 1599, 1509, 1461,

1435, 1314, 1284, 1240, 1216, 1167, 750, 719, 692 cm-1; HRMS (ESI+) m/z

328.0897 (M+H+, C20H14N3S requires 328.0908).

205

[(E)-2-(5-Benzothiazol-2-yl-1-phenyl-1H-benzoimidazol-2-yl)ethenyl]methyl

phenylamine (120). To a solution of compound 116 (64 mg, 0.2 mmol) and 117

(32 mg, 0.2 mmol) in ethanol (15 mL) was added ZrCl4 (24 mg, 0.1 mmol). The

reaction was heated to 80 °C and refluxed for 30 min. When the starting material

dissapeared on TLC, MnO2 (70mg, 0.8 mmol) was added and stirred for 5 min.

The reaction was cooled and filtered through a frit funnel. The MnO2 was washed

with ethanol (10 mL). The combined filtrate was concentrated in vacuo. Flash

column chromatography (hexanes/ethyl acetate, 4:1) afforded the product (68 mg,

75%) as a yellow solid; mp 215-217 °C; Rf 0.13 (hexane/ethyl acetate, 3:1); 1H

NMR (300 MHz, CDCl3) δ 8.34 (s, 1H), 8.31 (d, 1H, J = 0.9 Hz), 8.05 (d, 1H, J =

6.0 Hz), 7.99 (dd, 1H, J1 = 1.2 Hz, J2 = 6.3 Hz), 7.89 (d, 1H, J = 5.8 Hz), 7.64-

7.44 (m, 5H), 7.34 (dd, 3H, J1 = 5.6 Hz, J2 = 6.0 Hz), 7.18 (d, 2H, J = 5.8 Hz),

7.13 (d, 1H, J = 6.3 Hz), 7.08 (t, 1H, J = 5.5 Hz), 5.22 (d, 1H, J = 9.9 Hz), 3.19 (s,

3H); 13C NMR (75 MHz, CDCl3) δ 169.8, 155.9, 154.8, 147.0, 143.2 (× 2), 139.1,

136.2, 135.5, 130.4 (× 2), 129.8 (× 2), 129.2, 128.7, 128.0 (× 2), 126.4, 125.1,

123.9, 121.9, 121.3, 119.8, 117.7, 114.9, 111.7, 109.9 87.5, 36.5; IR (film) ν max

3059, 2944, 2901, 1627, 1594, 1492, 1464, 1435, 1347, 1298, 1270, 1127, 978,

206

910, 758, 730, 696 cm-1; HRMS (ESI+) m/z 459.1663 (M+H+, C29H23N4S requires

459.1643).

2-(1-Phenyl-2(E)-styryl-1H-benzoimidazol-5-yl)benzothiazole (127). To a

solution of Compound 116 (64 mg, 0.2 mmol) and trans-cinnamaldehyde (26 µL,

0.2 mmol) in ethanol (15 mL) was added ZrCl4 (24 mg, 0.1 mmol). The reaction

was heated to 80 °C and refluxed for 30 min. The reaction was cooled and

poured into 5% ammonium hydroxide aqueous solution (50 mL). The product

was extracted with CH2Cl2 (50 mL × 2). The combined organic phase was dried

over anhydrous Na2SO4, and concentrated in vacuo. Flash column

chromatography (hexanes/ethyl acetate, 5:1) afforded the product (73 mg, 85%)

as a yellow solid; mp 218-220 °C; Rf 0.20 (hexane/ethyl acetate, 3:1); 1H NMR

(300 MHz, CDCl3) δ 8.48 (d, 1H, J = 1.4 Hz), 8.11 (dd, 1H, J1 = 1.6 Hz, J2 = 8.5

Hz), 8.07 (d, 1H, J = 8.1 Hz), 8.01 (d, 1H, J = 16.0 Hz), 7.90 (d, 1H, J = 7.6 Hz),

7.66-7.55 (m, 3H), 7.50-7.45 (m, 5H), 7.37-7.32 (m, 5H), 6.85 (d, 1H, J = 16.0

Hz); 13C NMR (75 MHz, CDCl3) δ 168.8, 154.3, 152.4, 143.4, 138.5, 138.1, 135.8,

130.14 (× 2), 129.3 (× 2), 129.2 (× 2), 128.8 (× 2), 127.4, 126.6 (× 2), 126.2,

123.0 (× 2), 122.7, 121.5, 119.2 (× 2), 113.5, 110.7 (× 2); IR (film) ν max 3059,

207

2217, 1633, 1596, 1499, 1433, 1386, 1340,1274, 1216, 909, 756, 729 cm-1;

HRMS (ESI+) m/z 430.1393 (M+H+, C28H20N3S requires 430.1378).

2-[2-((E)-2-Furan-2-yl-ethenyl)-1-phenyl-1H-benzoimidazol-5-yl]benzo-

thiazole (128). Compound 116 (64 mg, 0.2 mmol) and trans-3-(2-furyl)acrolein

117 (24 mg, 0.2 mmol) were used to synthesize 128 according the procedures

described for 127. Flash column chromatography (hexanes/ethyl acetate, 5:1)

afforded the product (69 mg, 82%) as a yellow solid; mp 97-99 °C; Rf 0.18

(hexane/ethyl acetate, 3:1); 1H NMR (400 MHz, CDCl3) δ 8.47 (d, 1H, J = 1.1 Hz),

8.11 (dd, 1H, J1 = 1.5 Hz, J2 = 8.5 Hz), 8.07 (d, 1H, J = 7.9 Hz), 7.91 (d, 1H, J =

7.8 Hz), 7.83 (d, 1H, J = 15.7 Hz), 7.68-7.58 (m, 3H), 7.50-7.46 (m, 3H), 7.40-

7.37 (m, 2H), 7.26 (d, 1H, J = 8.5 Hz), 6.71 (d, 1H, J = 15.7 Hz), 6.57 (d, 1H, J =

3.3 Hz), 6.45 (dd, 1H, J1 = 1.8 Hz, J2 = 3.3 Hz); 13C NMR (100 MHz, CDCl3) δ

168.7, 154.3, 152.3, 152.0, 144.2, 138.4, 135.2, 134.9, 130.3 (× 2), 130.1, 129.5,

127.7 (× 2), 127.4, 125.7, 123.1, 123.0, 122.9 (× 2), 118.8, 118.7, 113.5, 112.5,

112.3, 110.9, 110.7; IR (film) ν max 3284, 3060, 2931, 2860, 1652, 1608, 1595,

1498, 1434, 1380, 1314, 1273, 1224, 1016, 757, 695 cm-1; HRMS (ESI+) m/z

420.1166 (M+H+, C26H18N3OS requires 420.1171).

208

2-{2-[(E)-2-(4-Methoxyphenyl)ethenyl]-1-phenyl-1H-benzoimidazol-5-yl}

benzothiazole (129). Compound 116 (64 mg, 0.2 mmol) and trans-4-

methoxycinnamaldehyde (33 mg, 0.2 mmol) were used to synthesize 129

according the procedures described for 127. Flash column chromatography

(hexanes/ethyl acetate, 5:1) afforded the product (79 mg, 86%) as a yellow solid;

mp 177-179 °C; Rf 0.17 (hexane/ethyl acetate, 3:1); 1H NMR (300 MHz, CDCl3) δ

8.46 (d, 1H, J = 1.4 Hz), 8.10 (dd, 1H, J1 = 1.6 Hz, J2 = 8.6 Hz), 8.07 (d, 1H, J =

7.2 Hz), 7.98 (d, 1H, J = 16.0 Hz), 7.91 (d, 1H, J = 7.9 Hz), 7.66-7.59 (m, 3H),

7.49-7.34 (m, 6H), 7.25 (d, 1H, J = 8.5 Hz), 6.88 (d, 2H, J = 8.7 Hz), 6.69 (d, 1H,

J = 16.0 Hz), 3.81 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 168.9, 160.6, 154.3,

152.9, 143.3, 138.5, 137.9, 135.2, 135.1, 130.2 (× 2), 129.3, 129.1 (× 2), 129.0,

128.6 (× 2), 127.5, 126.2, 124.9, 123.0, 122.5, 121.5, 118.9, 114.3 (× 2), 111.0,

110.5, 55.3; IR (film) ν max 3061, 2954, 2929, 2837, 1603, 1514, 1500, 1434,

1304, 1254, 1173, 759 cm-1; HRMS (ESI+) m/z 460.1473 (M+H+, C29H22N3OS

requires 460.1484).

209

2-(2-Nona-1(E),3(E)-dienyl-1-phenyl-1H-benzoimidazol-5-yl)benzothiazole

(130). Compound 116 (64 mg, 0.2 mmol) and trans,trans-2,4-decadienal (35 µL,

0.2 mmol) were used to synthesize 130 according the procedures described for

127. Flash column chromatography (hexanes/ethyl acetate, 8:1) afforded the

product (63 mg, 67%) as a yellow glassy solid; Rf 0.40 (hexane/ethyl acetate,

3:1); 1H NMR (300 MHz, CDCl3) δ 8.43 (d, J = 1.5 Hz, 1H), 8.07 (m, 2H), 7.89 (d,

J = 7.9 Hz, 1H), 7.64-7.56 (m, 4H), 7.50-7.33 (m, 4H), 7.23 (d, J = 19.0 Hz, 1H),

6.24-6.04 (m, 3H), 2.18-2.11 (m, 2H), 1.47-1.23 (m, 6H), 0.88 (t, J = 6.9 Hz, 3H);

13C NMR (75 MHz, CDCl3) δ 168.9, 154.4, 152.9, 143.5, 142.0, 139.1, 138.5,

135.3, 135.1, 130.0 (× 2), 129.5, 129.2, 129.0 (× 2), 127.5, 126.2, 124.8, 123.0,

122.4, 121.5, 119.0, 114.4, 110.4, 33.0, 31.4, 28.6, 22.5, 14.0; IR (film) ν max

3048, 2925, 2855, 1638, 1614, 1596, 1498, 1455, 1434, 1388, 1314, 1291, 993,

757 cm-1; HRMS (ESI+) m/z 450.2025 (M+H+, C29H28N3S requires 450.2004).

210

2-(2-Phenethyl-1-phenyl-1H-benzoimidazol-5-yl)benzothiazole (131). 116 (64

mg, 0.2 mmol) and 3-phenylpropionaldehyde (26 µL, 0.2 mmol) were used to

synthesize 131 according the procedures described for 127. Flash column

chromatography (hexanes/ethyl acetate, 3:1) afforded the product (41 mg, 48%)

as a light yellow solid; mp 56-58 °C; Rf 0.10 (hexane/ethyl acetate, 3:1); 1H NMR

(300 MHz, CDCl3) δ 8.48 (s, 1H), 8.07 (dd, 2H, J1 = 3.2 Hz, J2 = 4.8 Hz), 7.91 (d,

1H, J = 7.9 Hz), 7.55-7.31 (m, 5H), 7.25-7.14 (m, 6H), 7.08 (d, 2H, J = 7.0 Hz),

3.21 (t, 4H, J = 6.5 Hz); 13C NMR (75 MHz, CDCl3) δ 168.9, 156.0, 154.3, 142.8,

140.6, 138.4, 135.3, 135.1, 130.0 (× 2), 129.3, 128.5 (× 2), 128.4 (× 2), 127.3 (×

2), 126.3, 126.2, 124.9, 123.0 (× 2), 122.4, 121.5, 119.1, 110.6, 34.0, 29.8; IR

(film) ν max 3052, 3028, 2921, 2862, 1596, 1514, 1498, 1462, 1434, 1393, 1314,

1274, 758, 728, 699 cm-1; HRMS (ESI+) m/z 432.1542 (M+H+, C28H22N3S

requires 432.1534).

2-(1,2-Diphenyl-1H-benzoimidazol-5-yl)benzothiazole (132). 116 (64 mg, 0.2

mmol) and benzaldehyde (20 µL, 0.2 mmol) were used to synthesize 132

according the procedures described for 127. Flash column chromatography

(hexanes/ethyl acetate, 6:1) afforded the product (42 mg, 52%) as a light yellow

solid solid; mp 184-186 °C; Rf 0.28 (hexane/ethyl acetate, 3:1); 1H NMR (300

211

MHz, CDCl3) δ 8.54 (d, J = 1.3 Hz, 1H), 8.14 (dd, J1 = 1.7 Hz, J2 = 8.6 Hz, 1H),

8.07 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.60-7.43 (m, 6H), 7.38-7.29 (m,

7H); 13C NMR (75 MHz, CDCl3) δ168.8, 154.3, 153.9, 143.2, 139.2, 136.6, 135.1,

130.0 (× 2), 129.8, 129.5 (× 2), 129.0, 128.9, 128.4 (× 2), 127.3 (× 2), 126.6,

126.2, 123.0, 122.9 (× 2), 121.5, 119.7, 111.0 cm-1; HRMS (ESI+) m/z 404.1249

(M+H+, C26H18N3S requires 404.1221).

3-(5-Benzothiazol-2-yl-1-phenyl-1H-benzoimidazol-2-yl)chromen-2-one (143).

To coumarin-3-carboxylic acid (40 mg, 0.2 mmol) in a round bottom flask (25 mL)

with a condensor was added thionyl chloride (2 mL, 10 mmol). The reaction was

heated to 80 °C and refluxed for 2 h. After excess thionyl chloride was distilled off,

the residue was further dried under high vacuum from 30 min. The residue was

then dissolved in anhydrous toluene (5 mL). 116 (64 mg, 0.2 mmol) was added

and the reaction was stirred at 22 °C for 30 min. Then, the reaction was heated

to 110 °C and refluxed for 12 h. The reaction was cooled and concentrated in

vacuo. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the

product (36 mg, 38%) as an off-white solid; mp 164-166 °C; Rf 0.20 (hexane/ethyl

acetate, 3:1); 1H NMR (300 MHz, CDCl3) δ 8.57 (d, J = 1.4 Hz, 1H), 8.40 (s, 1H),

8.21 (dd, J1 = 1.6 Hz, J2 = 8.5 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.94 (d, J = 7.9

212

Hz, 1H), 7.64-7.35 (m, 12H); 13C NMR (75 MHz, CDCl3) δ 168.5, 157.8, 154.5,

154.3, 149.0, 146.4, 142.9, 138.6, 136.1, 135.1, 133.1, 129.8 (× 2), 129.2, 128.9,

128.8, 126.2 (× 2), 126.1,125.0, 124.9, 123.7, 124.9, 123.7, 123.0, 121.6, 119.9,

119.3, 118.5, 116.8, 111.2; IR (film) ν max 3060, 2966, 1735, 1608, 1499, 1456,

1434, 1386, 1325, 1282, 1242, 1215, 756 cm-1; HRMS (ESI+) m/z 472.1113

(M+H+, C29H18N3O2S requires 472.1120).

5-Benzothiazol-2-yl-3-ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl -

3H-benzoimidazol-1-ium, iodide (111). A slurry of 120 (35 mg, 0.076 mmol) in

ethyl iodide (3 mL) was heated to 75 °C and refluxed for 24 h. The solvent was

removed under vacuum. The crude product was purified by flash column

chromatography (CH2Cl2/MeOH, 20:1) to afford the product (41 mg, 88%) as a

yellow solid; mp 165-167 °C; Rf 0.18 (CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz,

MeOH-d4) δ 8.43 (s, 1H), 8.10-8.00 (m, 3H), 7.83-7.69 (m, 5H), 7.53-7.14 (m, 7H),

6.74 (d, J = 6.5 Hz, 2H), 5.52 (d, J = 12.3 Hz, 1H), 4.61 (q, J = 7.1 Hz, 2H), 3.41

(s, 3H), 1.61 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, MeOH-d4) δ 167.2, 154.1,

152.1, 150.7 (× 2), 135.9, 135.3, 135.0, 132.1, 131.9 (× 2), 131.5, 131.2, 129.7 (×

2), 128.1 (× 2), 127.0, 126.3, 126.0, 125.5, 123.0, 122.1, 120.4 (× 2), 112.0 (× 2),

109.6, 78.2, 40.3, 12.9; IR (film) ν max 3290, 3058, 2924, 1688, 1618, 1507,

213

1537, 1494, 1465, 1371, 1308, 1203, 1132, 1029, 800, 763, 697 cm-1; LRMS

(ESI+) m/z 487.8, (M+, C31H27N4S requires 487.2).

5-Benzothiazol-2-yl-3-ethyl-1-phenyl-3H-benzoimidazol-1-ium, iodide (144).

Compound 119 (35 mg, 0.11 mmol) and ethyl iodide (3 mL) were used to

synthesize 144 according to the procedures described for 111. Flash column

chromatography (CH2Cl2/MeOH, 20:1) afforded the product (49 mg, 93%) as a

white solid; mp 122-124 °C; Rf 0.30 (CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz,

MeOH-d4) δ 9.95 (s, 1H), 8.71 (d, J = 1.0 Hz, 1H), 8.34 (dd, J1 = 1.5 Hz, J2 = 8.8

Hz, 1H), 7.98 (t, J = 7.1 Hz, 2H), 7.87 (d, J = 8.8 Hz, 1H), 7.76-7.68 (m, 5H),

7.49-7.32 (m, 2H), 4.70 (q, J = 7.3 Hz, 2H), 1.72 (t, J = 7.3 Hz, 3H); 13C NMR (75

MHz, MeOH-d4) δ 167.4, 155.1, 144.4, 136.6, 134.7, 134.5, 134.2, 133.5, 132.2,

131.8 (× 2), 128.3, 128.1, 127.3, 126.3 (× 2), 124.3, 123.1, 115.7, 113.4, 44.4,

14.5; IR (film) ν max 3434, 3131, 3064, 1780, 1736, 1686, 1561, 1499, 1478,

1446, 1416, 1314, 1200, 1143, 758, 706 cm-1; HRMS (ESI+) m/z 356.1200 (M+,

C22H18N3S requires 356.1221).

214

5-Benzothiazol-2-yl-3-ethyl-1-phenyl-2(E)-styryl-3H-benzoimidazol-1-ium,

iodide (145). Compound 127 (35 mg, 0.08 mmol) and ethyl iodide (3 mL) were

used to synthesize 145 according to the procedures described for 111. Flash

column chromatography (CH2Cl2/MeOH, 20:1) afforded the product (43 mg, 92%)

as a light yellow solid; mp 199-202 °C; Rf 0.33 (CH2Cl2/MeOH, 10:1); 1H NMR

(300 MHz, MeOH-d4) δ 8.76 (d, J = 0.9 Hz, 1H), 8.34 (dd, J1 = 1.5 Hz, J2 = 8.7 Hz,

1H), 8.06 (t, J = 7.7 Hz, 2H), 7.82-7.76 (m, 6H), 7.57-7.42 (m, 9H), 7.21 (q, J =

7.2 Hz, 2H), 1.73 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, MeOH-d4) δ 166.6,

154.1, 149.5, 148.9, 135.6, 135.4, 134.1, 133.6, 133.0, 132.1, 132.0, 131.8,

131.2 (× 2), 129.4 (× 2), 128.5 (× 2), 127.8 (× 2), 127.2, 127.1, 126.3, 123.3,

122.2, 114.0, 111.6, 106.6, 42.0, 13.9; IR (film) ν max 3416, 3061, 2988, 1688,

1632, 1503, 1470, 1441, 1199, 1135, 760 cm-1; HRMS (ESI+) m/z 458.1684 (M+,

C30H24N3S requires 458.1691).

215

5-Benzothiazol-2-yl-3-ethyl-2-((E)-2-furan-2-yl-ethenyl)-1-phenyl-3H-benzo

imidazol-1-ium, iodide (146). Compound 128 (30 mg, 0.07 mmol) and ethyl

iodide (3 mL) were used to synthesize 146 according to the procedures

described for 111. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded

the product (35 mg, 88%) as a yellow solid; mp 83-85 °C; Rf 0.30 (CH2Cl2/MeOH,

10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.60 (d, 1H, J = 1.0 Hz), 8.19 (dd, J1 = 1.5

Hz, J2 = 8.7 Hz, 1H), 7.95 (t, J = 7.3 Hz, 2H), 7.72-7.62 (m, 6H), 7.47-7.35 (m,

3H), 6.85 (d, J = 1.4 Hz, 2H), 6.73 (d, J = 3.5 Hz, 1H), 6.51 (q, J = 1.8 Hz, 1H),

4.73 (q, J = 7.3 Hz, 2H), 1.61 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, MeOH-d4) δ

168.0, 155.5, 152.0, 150.4, 149.0, 136.9, 136.8, 134.8, 134.7, 134.3, 133.6,

133.4, 132.7, 131.8, 129.2 (× 2), 128.5 (× 2), 127.6, 120.2, 115.1, 114.9, 112.7,

104.4, 43.2, 15.2; IR (film) ν max 3429, 3063, 2342, 1688, 1626, 1447, 1199,

1128, 762 cm-1; HRMS (ESI+) m/z 448.1454 (M+, C28H22N3OS requires

448.1484).

5-Benzothiazol-2-yl-3-ethyl-2-[(E)-2-(4-methoxyphenyl)ethenyl]-1-phenyl-3H-

benzoimidazol-1-ium, iodide (147). Compound 129 (35 mg, 0.076 mmol) and

ethyl iodide (3 mL) were used to synthesize 147 according to the procedures

described for 111. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded

216

the product (42 mg, 90%) as a yellow solid; mp 188-192 °C; Rf 0.33

(CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.51 (d, J = 1.0 Hz, 1H),

8.09 (d, J = 1.4 Hz, 1H), 7.88 (t, J = 7.5 Hz, 1H), 7.68-7.66 (m, 5H), 7.60-7.33 (m,

5H), 6.90 (d, J = 3.1 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 4.67 (q, J = 7.2 Hz, 2H),

3.68 (s, 3H), 1.55(t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, MeOH-d4) δ 170.4, 167.2,

157.8, 153.4, 152.3, 139.2, 139.0, 137.4, 136.4, 135.7, 135.5, 135.0 (× 2), 134.4

(× 2), 131.6 (× 2), 130.9, 130.8, 130.7, 130.0 126.9, 125.9, 118.5 (× 2), 117.4,

115.0, 107.0, 58.8, 46.2, 17.6; IR (film) ν max 3404, 3064, 1688, 1628, 1599,

1573, 1520, 1470, 1256, 1199, 1176, 1116, 763 cm-1; HRMS (ESI+) m/z

488.1770 (M+, C31H26N3OS requires 488.1797).

5-Benzothiazol-2-yl-3-ethyl-2-nona-1(E),3(E)-dienyl-1-phenyl-3H-benzo

imidazol-1-ium, iodide (148). Compound 130 (30 mg, 0.067 mmol) and ethyl

iodide (3 mL) were used to synthesize 148 according to the procedures

described for 111. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded

the product (36 mg, 89%) as a yellow solid; mp 66-68 °C; Rf 0.33 (CH2Cl2/MeOH,

10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.68 (d, J = 1.0 Hz, 1H), 8.26 (dd, J1 = 1.5

Hz, J2 = 8.7 Hz, 1H), 8.04 (t, J = 6.4 Hz, 2H), 7.81-7.30 (m, 9H), 6.73-6.62 (m,

1H), 6.42-6.34 (m, 1H), 6.11-6.01 (m, 1H), 4.73 (q, J = 7.2 Hz, 2H), 2.22-2.16 (m,

217

2H), 1.65 (t, J = 7.2 Hz, 3H), 1.44-1.29 (m, 6H), 0.89 (t, J = 7.1 Hz, 3H); 13C NMR

(75 MHz, MeOH-d4) δ 166.7, 154.1, 149.6, 149.4, 135.6, 135.4, 134.0, 133.5,

132.8, 132.0, 131.9, 131.2 (× 2), 129.6, 127.8 (× 2), 127.1, 127.0, 126.2, 123.2,

122.2, 113.7, 111.3, 107.4, 41.7, 33.2, 31.5, 28.3, 22.5, 13.8, 13.6; IR (film) ν

max 3416, 3051, 2950, 2923, 2862, 1687, 1632, 1613, 1501, 1469, 1198, 1166,

1126, 762 cm-1; HRMS (ESI+) m/z 478.2298 (M+, C31H32N3S requires 478.2317).

5-Benzothiazol-2-yl-3-ethyl-2-phenethyl-1-phenyl-3H-benzoimidazol-1-ium,

iodide (149). Compound 131 (35 mg, 0.08 mmol) and ethyl iodide (3 mL) were

used to synthesize 149 according to the procedures described for 111. Flash

column chromatography (CH2Cl2/MeOH, 20:1) afforded the product (44 mg, 93%)

as a white solid; mp 131-133 °C; Rf 0.30 (CH2Cl2/MeOH, 10:1); 1H NMR (300

MHz, MeOH-d4) δ 8.73 (s, 1H), 8.30 (dd, J1 = 1.4 Hz, J2 = 8.6 Hz, 1H), 8.04 (t, J

= 8.1 Hz, 2H), 7.83-7.72 (m, 3H), 7.58-7.44 (m, 5H), 7.24-7.21 (m, 3H), 6.99-6.96

(m, 2H), 4.70 (q, J = 7.2 Hz, 2H), 3.52 (t, J = 7.6 Hz, 2H), 2.98 (t, J = 7.6 Hz, 2H),

1.65 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, MeOH-d4) δ 167.5, 155.8, 155.0,

139.4, 136.6, 135.8, 133.9, 133.2, 132.9, 132.5, 132.0 (× 2), 130.2 (× 2), 129.4 (×

2), 128.5 (× 2), 128.4, 128.1, 128.0, 127.2, 124.2, 123.1, 115.1, 112.8, 42.9, 33.8,

27.8, 14.9; IR (film) ν max 3416, 3064, 1733, 1688, 1506, 1471, 1455, 1199,

218

1135, 762, 701 cm-1; HRMS (ESI+) m/z 460.1833 (M+, C30H26N3S requires

460.1847).

5-Benzothiazol-2-yl-3-ethyl-1,2-diphenyl-3H-benzoimidazol-1-ium, iodide

(150). Compound 132 (30 mg, 0.074 mmol) and ethyl iodide (3 mL) were used to

synthesize 150 according to the procedures described for 111. Flash column

chromatography (CH2Cl2/MeOH, 20:1) afforded the product (37 mg, 90%) as a

white solid; mp 72-74 °C; Rf 0.33 (CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz,

MeOH-d4) δ 8.88 (s, 1H), 8.43 (d, J = 8.6 Hz, 1H), 8.09 (t, J = 8.2 Hz, 2H), 7.73-

7.47 (m, 13H), 4.65 (q, J = 7.1 Hz, 2H), 1.64 (t, J = 7.1 Hz, 3H); 13C NMR (75

MHz, MeOH-d4) δ 166.6, 154.1, 152.5, 135.6, 135.0, 133.4, 133.3, 132.9, 132.0,

131.2, 130.7 (× 2), 130.5 (× 2), 129.7 (× 2), 127.7 (× 2), 127.4, 127.1, 126.3,

123.3, 122.2, 121.4, 114.6, 112.2, 42.6, 14.0; IR (film) ν max 3425, 3060, 2990,

1778, 1737, 1688, 1502, 1454, 1434, 1198, 1151, 1138, 760, 703 cm-1; HRMS

(ESI+) m/z 432.1518 (M+, C28H22N3S requires 432.1534).

219

5-Benzothiazol-2-yl-3-ethyl-2-(2-oxo-2H-chromen-3-yl)-1-phenyl-3H-

benzoimidazol-1-ium, iodide (151). Compound 143 (20 mg, 0.042 mmol) and

ethyl iodide (3 mL) were used to synthesize 151 according to the procedures

described for 111. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded

the product (21 mg, 81%) as a light yellow solid; mp 206-208 °C; Rf 0.25

(CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, DMSO-d6) δ 8.88 (d, J = 1.0 Hz, 1H),

8.63 (s, 1H), 8.43 (dd, J1 = 1.5 Hz, J2 = 8.8 Hz, 1H), 8.06 (dd, J1 = 1.0 Hz, J2 =

8.1 Hz, 2H), 7.78-7.38 (m, 12H), 4.76 (q, J = 7.2 Hz, 2H), 1.60 (t, J = 7.2 Hz, 3H);

13C NMR (75 MHz, DMSO-d6) δ 166.7, 158.2, 155.8, 154.7, 153.5, 147.2, 136.7,

136.2, 135.5, 134.1, 133.0, 132.5, 132.4, 131.4 (× 2), 131.3, 128.4, 128.0 (× 2),

127.9, 127.0, 123.1, 118.3, 117.8, 115.6, 113.3, 110.8, 43.7, 15.0; IR (film) ν max

3412, 3060, 1725, 1687, 1609, 1576, 1503, 1437, 1255, 1201, 1174, 1128, 761

cm-1; HRMS (ESI+) m/z 500.1440 (M+, C31H22N3O2S requires 500.1433).

220

5-Benzothiazol-2-yl-2-[(E)-2-(cyclohexylmethylamino)ethenyl]-3-ethyl-1-

phenyl-3H-benzoimidazol-1-ium, iodide (140). To a solution of 111 (15 mg,

0.024 mmol) in DMF (2 mL) was added N-methylcyclohexylamine (0.66 mL, 5.0

mmol). The reaction was stirred for 12 h. The solvent and excess reagent was

removed under vacuum. Flash column chromatography (CH2Cl2/MeOH, 20:1)

afforded 140 (14 mg, 93%) as a yellow solid; mp 55-57 °C; Rf 0.11

(CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.28 (s, 1H), 7.98-7.93 (m,

3H), 7.78-7.72 (m, 3H), 7.62-7.60 (m, 2H), 7.48-7.32 (m, 2H), 7.04 (d, J = 8.5 Hz,

1H), 6.90 (d, J = 12.9 Hz, 1H), 5.05 (d, J = 12.9 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H),

2.92 (s, 3H), 2.88-2.78 (m, 1H), 1.78-1.46 (m, 7H), 1.25-1.09 (m, 3H), 0.96-0.72

(m, 3H); 13C NMR (75 MHz, MeOH-d4) δ 168.5, 155.1, 153.7, 151.7, 137.0, 136.6,

136.3, 133.2, 132.9 (× 2), 132.3, 131.7, 129.4 (× 2), 128.0, 126.9, 125.9, 123.9,

123.1, 112.4, 110.0, 74.3, 67.6, 40.8, 35.4, 32.6 (× 2), 26.3 (× 2), 26.2, 13.6; IR

(film) ν max 3356, 2936, 2869, 1688, 1620, 1548, 1471, 1454, 1415, 1302, 1202,

1176, 1131 cm-1; HRMS (ESI+) m/z 493.2444 (M+, C31H33N4S requires 493.2426).

5-Benzothiazol-2-yl-2-[(E)-2-(benzylmethylamino)ethenyl]-3-ethyl-1-phenyl-

3H-benzoimidazol-1-ium, iodide (141). To a solution of 111 (15 mg, 0.024

mmol) in DMF (2 mL) was added N-methylbenzylamine (0.5 mL, 3.9 mmol). The

221

reaction was stirred for 2 h. The solvent and excess reagent was removed under

vacuum. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded the

product (14 mg, 92%) as a yellow solid; mp 65-67 °C; Rf 0.11 (CH2Cl2/MeOH,

10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.45 (s, 1H), 8.11-8.05 (m, 3H), 7.85-7.39

(m, 10H), 7.21 (d, J = 8.5 Hz, 1H), 6.83-6.76 (m, 3H), 5.21 (d, J = 13.1 Hz, 1H),

4.57 (q, J = 7.2 Hz, 2H), 4.25 (s, 2H), 2.97 (s, 3H), 1.60 (t, J = 7.2 Hz, 3H); 13C

NMR (75 MHz, MeOH-d4) δ 168.9, 155.5, 154.9, 154.1, 137.4, 136.9, 136.7 (× 2),

136.5, 133.6, 133.1 (× 2), 132.9, 132.4, 130.6 (× 2), 130.0, 129.6 (× 2), 129.4,

128.4, 127.4, 126.5, 124.4, 123.5, 113.1, 110.7, 75.5, 63.4, 41.5, 36.2, 14.1; IR

(film) ν max 3332, 3059, 2928, 1688, 1620, 1547, 1477, 1444, 1409, 1292, 1253,

1204, 1134 cm-1; HRMS (ESI+) m/z 501.2099 (M+, C32H29N4S requires 501.2113).

5-Benzothiazol-2-yl-3-ethyl-1-phenyl-2-[(E)-2-(4-phenylpiperazin-1-yl)ethenyl]

-3H-benzoimidazol-1-ium, iodide (142). To a solution of 111 (15 mg, 0.024

mmol) in DMF (2 mL) was added 1-phenylpiperazine (0.6 mL, 3.9 mmol). The

reaction was stirred for 4 h. The solvent was removed under vacuum. Flash

column chromatography (CH2Cl2/MeOH, 20:1) of the crude product afforded the

product (14 mg, 89%) as a yellow solid; mp 129-131 °C; Rf 0.11 (CH2Cl2/MeOH,

10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.41 (d, J = 1.2 Hz, 1H), 8.05 (dd, J1 = 1.5

222

Hz, J2 = 8.5 Hz, 1H), 7.99 (t, J = 6.0 Hz, 2H), 7.76-7.73 (m, 3H), 7.64-7.61 (m,

2H), 7.49 (t, J = 7.5 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.19 (t, J = 8.1 Hz, 3H),

6.90 (d, J = 7.9 Hz, 2H), 6.84-6.77 (m, 3H), 5.30 (d, J = 14.2 Hz, 1H), 4.53 (q, J =

7.2 Hz, 2H), 3.65-3.47 (m, 4H), 3.17-3.02 (m, 4H), 1.54 (t, J = 7.2 Hz, 3H); 13C

NMR (75 MHz, MeOH-d4) δ 168.9, 155.5, 154.4, 153.8, 152.4, 137.4, 136.7,

136.4, 135.3, 133.7, 133.0 (× 2), 132.8, 130.6 (× 2), 129.6 (× 2), 128.4, 127.4,

126.6, 124.3, 123.5, 122.3, 118.4 (× 2), 113.1, 110.8, 75.3, 41.5, 14.2; IR (film) ν

max 3389, 3060, 2926, 1690, 1616, 1544, 1496, 1438, 1286, 1226, 760 cm-1;

HRMS (ESI+) m/z 542.2373 (M+, C34H32N5S requires 542.2378).

(4-Benzothiazol-2-yl-2-nitrophenyl)hexylamine (152). To a solution of 114

(580 mg, 2.0 mmol) in DMSO (5 mL) was added hexylamine (1.3 mL, 10.0 mmol).

The reaction was stirred at 22 °C for 48 h. The reaction was poured into 1.0 M

HCl slution (50 mL), and the product was extracted with diethyl ether (50 mL × 3).

The combined organic phase was washed with saturated aqueous NaCl solution

(100 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue

was purified by column chromatography on silica gel with CH2Cl2 to afford the

product (625 mg, 88%) as an orange solid, mp 98-100 °C; Rf = 0.50

223

(hexane/ethyl acetate, 3:1); 1H NMR (300 MHz, CDCl3) δ 8.78 (t, J = 2.0 Hz, 1H),

8.28 (s, br, 1H), 8.15 (d, J = 6.9 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.84 (d, J = 8.0

Hz, 1H), 7.45 (dd, J1 = 7.2 Hz, J2 = 8.1 Hz, 1H), 7.34 (dd, J1 = 7.3 Hz, J2 = 7.8 Hz,

1H), 6.89 (dd, J1 = 2.4 Hz, J2 = 9.1 Hz, 1H), 3.31 (m, 2H), 1.73 (m, 2H), 1.45 (m,

2H), 1.35 (m, 4H), 0.91 (t, J = 6.8 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 166.0,

154.0, 146.7, 134.6, 134.4, 131.3, 126.3, 126.2, 125.0, 122.8, 121.5, 121.1,

114.3, 43.3, 31.4, 28.8, 26.7, 22.5, 14.0; IR (film) ν max 3372, 2955, 2931, 2858,

1626, 1566, 1488, 1360, 1269, 1214, 1159, 758 cm-1; HRMS (ESI+) m/z

356.1413 (M+H+, C19H22N3O2S, requires 356.1433).

(4-Benzothiazol-2-yl-2-nitrophenyl)cyclohexylamine (153). Compound 114

(580 mg, 2.0 mmol) and cyclohexylamine (1.16 mL, 10.0 mmol) was used to

synthesize 153 according to the procedures described for the synthesis of 152.

Column chromatography (CH2Cl2) afforded the product (609 mg, 87%) as an

orange solid, mp 138-140 °C; Rf = 0.47 (hexane/ethyl acetate, 3:1); 1H NMR (300

MHz, CDCl3) δ 8.78 (s, 1H), 8.28 (d, J = 7.2 Hz,, 1H), 8.13 (d, J = 9.0 Hz, 1H),

7.98 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.45 (dd, J1 = 7.4 Hz, J2 = 7.9

Hz, 1H), 7.36 (dd, J1 = 7.6 Hz, J2 = 7.6 Hz, 1H), 6.92 (d, J = 9.1 Hz, 1H), 3.56 (m,

1H), 2.05 (m, 2H), 1.81 (m, 2H), 1.69-1.21 (m, 6H); 13C NMR (75 MHz, CDCl3) δ

224

166.1, 154.0, 145.8, 135.1, 134.6, 134.3, 131.2, 126.5, 126.3, 124.9, 122.7,

121.5, 120.8, 114.7, 51.3, 32.6 (× 2), 25.5, 24.5 (× 2); IR (film) ν max 3556, 2931,

2854, 1624, 1567, 1534, 1488, 1436, 1361, 1266, 1217, 1155, 757 cm-1; HRMS

(ESI+) m/z 354.1280 (M+H+, C19H20N3O2S requires 354.1276).

(4-Benzothiazol-2-yl-2-nitrophenyl)-(4-methoxyphenyl)amine (154).

Compound 114 (580 mg, 2.0 mmol) and p-anisidine (1.23 g, 10.0 mmol) was

used to synthesize 154 according to the procedures described for the synthesis

of 152. Column chromatography (CH2Cl2) afforded the product (560 mg, 74%) as

an orange solid, mp 188-190 °C; Rf = 0.40 (hexane/ethyl acetate, 3:1); 1H NMR

(300 MHz, CDCl3) δ 9.65 (s, 1H), 8.84 (s, 1H), 8.02 (m, 2H), 7.85 (d, J = 9.0 Hz,

1H), 7.48-6.96 (m, 7H), 3.85 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 165.7, 158.4,

154.0, 145.7, 134.7, 134.0, 132.1, 130.3, 127.2 (× 2), 126.4, 126.0, 125.1, 122.9,

122.7, 121.6, 116.3, 115.1 (× 2); IR (film) ν max 3337, 1626, 1593, 1568, 1510,

1486, 1352, 1247, 1208, 1147, 753 cm-1; HRMS (ESI+) m/z 378.0919 (M+H+,

C20H16N3O3S requires 378.0912).

225

4-Benzothiazol-2-yl-N1-hexylbenzene-1,2-diamine (155). Compound 152 (356

mg, 1.0 mmol) was treated with anhydrous hydrazine (0.2 mL, 6.4 mmol) with

Pd/C (106mg, 0.1 mmol) as catalyst according to the precedures described for

116. Flash column chromatography (hexane/ethyl acetate, 8:1) afforded the

product (282 mg, 87%) as a yellow solid, mp 151-153 °C; Rf = 0.40 (hexane/ethyl

acetate, 3:1); 1H NMR (300 MHz, CDCl3) δ 7.88 (d, J = 8.1 Hz, 1H), 7.81 (d, J =

7.8 Hz, 1H), 7.53 (m, 1H), 7.42 (dd, J1 = 7.3 Hz, J2 = 7.8 Hz, 1H), 7.29 (dd, J1 =

7.3 Hz, J2 = 7.8 Hz, 1H), 6.64 (d, J = 8.8 Hz, 1H), 3.77 (br, 1H) 3.38 (br, 2H), 3.14

(t, J =7.1 Hz, 2H), 1.65 (m, 2H), 1.39 (m, 2H), 1.32 (m, 4H), 0.90 (t, J = 6.6 Hz,

3H); 13C NMR (75 MHz, CDCl3) δ 169.0, 154.3, 141.7, 134.6, 133.2, 126.0, 124.3,

122.9, 122.3, 121.8, 121.4, 115.4, 110.2, 43.9, 31.6, 29.4, 26.9, 22.6, 14.0; IR

(film) ν max 3379.8, 3212.9, 3018.9, 2952.1, 2916.7, 2848.5, 1647.9, 1594.3,

1459.8, 1438.6, 1364.1, 1307.4, 1215.5, 1160.7 769.8, 753.6, 726.3 cm-1; HRMS

(ESI+) m/z 326.1666 (M+H+, C19H24N3S, requires 326.1691).

226

4-Benzothiazol-2-yl-N1-cyclohexylbenzene-1,2-diamine (156). Compound 153

(354 mg, 1.0 mmol) was treated with anhydrous hydrazine (0.2 mL, 6.4 mmol)

with Pd/C (106mg, 0.1 mmol) as catalyst according to the precedures described

for 116. Flash column chromatography (hexane/ethyl acetate, 8:1) afforded the

product (291 mg, 90%) as a yellow solid, mp 168-170 °C; Rf = 0.37 (hexane/ethyl

acetate, 1:1); 1H NMR (300 MHz, CDCl3) δ 8.08 (d, J = 7.9 Hz,, 1H), 7.97 (d, J =

8.1 Hz, 1H), 7.51 (m, 2H), 7.42 (dd, J1 = 7.7 Hz, J2 = 7.7 Hz, 1H), 7.29 (dd, J1 =

7.6 Hz, J2 = 7.6 Hz, 1H), 6.65 (d, J = 8.7 Hz, 1H), 3.48 (br, 3H), 3.32 (m, 1H),

2.08 (m, 2H), 1.78 (m, 2H), 1.66 (m, 1H), 1.29 (m, 5H); 13C NMR (75 MHz, CDCl3)

δ 169.1, 154.3, 140.6, 134.6, 133.1, 126.0, 124.2, 122.5, 121.9, 121.3, 115.9,

110.6, 51.4, 33.3 (× 2), 25.9, 24.9 (× 2); IR (film) ν max 3369, 3215, 3050, 2930,

2862, 1604, 1462, 1431, 1300, 1256, 1147, 756 cm-1; HRMS (ESI+) m/z

324.1506 (M+H+, C19H22N3S requires 324.1534).

227

4-Benzothiazol-2-yl-N1-cyclohexylbenzene-1,2-diamine (157). Compound 154

(378 mg, 1.0 mmol) was treated with anhydrous hydrazine (0.2 mL, 6.4 mmol)

with Pd/C (106 mg, 0.1 mmol) as catalyst according to the precedures described

for 116. Flash column chromatography (hexane/ethyl acetate, 6:1) afforded the

product (295 mg, 85%) as a yellow solid, mp 155-157 °C; Rf = 0.33 (hexane/ethyl

acetate, 1:1); 1H NMR (300 MHz, CDCl3) δ 8.01 (d, J = 8.0 Hz, 1H), 7.85 (d, J =

7.7 Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.47-7.30 (m, 3H), 7.04 (d, J = 8.2 Hz, 1H),

6.95 (d, J = 8.9 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H), 5.40 (s, 1H), 3.79 (s, 3H), 3.68

(br, 2H); 13C NMR (75 MHz, CDCl3) δ 168.4, 155.1, 154.2, 137.3, 136.2, 135.7,

134.8, 127.1, 126.1, 124.6, 122.6, 121.5, 121.0 (× 2), 120.2, 117.3, 115.3, 114.8

(× 2), 55.6; IR (film) ν max 3361, 3260, 3058, 3003, 2953, 2833, 1600, 1509,

1477, 1437, 1311, 1243, 1034, 822, 758 cm-1; HRMS (ESI+) m/z 348.1156

(M+H+, C20H18N3OS requires 348.1156).

[(E)-2-(5-Benzothiazol-2-yl-1-hexyl-1H-benzoimidazol-2-yl)ethenyl]methyl

phenylamine (158). Compound 155 (65 mg, 0.2 mmol), 117 (32 mg, 0.2 mmol),

and ZrCl4 (24 mg, 0.1 mmol) were used to synthesize 158 according to the

procedures described for 120. Flash column chromatography (hexane/ethyl

228

acetate, 6:1) afforded the product (66 mg, 71%) as a yellow solid; mp 60-62 °C;

Rf = 0.53 (hexane/ethyl acetate, 1:1); 1H NMR (300 MHz, CDCl3) δ 8.34 (d, J =

13.1 Hz, 1H), 8.24 (d, J = 1.5 Hz, 1H), 8.03 (m, 2H) 7.87 (d, J = 7.9 Hz, 1H), 7.43

(t, J = 8.0 Hz, 1H), 7.38-7.23 (m, 6H), 7.09 (t, J = 8.2 Hz, 1H), 5.36 (d, J = 13.1

Hz, 1H), 4.10 (t, J = 7.1 Hz, 2H) 3.37 (s, 3H), 1.82 (m, 2H), 1.33 (m, 6H), 0.90 (t,

J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 169.7, 155.2, 154.4, 146.7, 143.7,

142.9, 137.8, 135.1, 129.4 (× 2), 127.6, 126.0, 124.6, 123.5, 122.8, 121.5, 120.2,

119.5 (× 2), 117.4, 108.7, 86.3, 43.3, 36.4, 31.4, 29.8, 22.5, 14.0; IR (film) ν max

3059, 2953, 2928, 2856, 1628, 1593, 1491, 1467, 1437, 1325, 1302, 1265, 1127,

756 cm-1; HRMS (ESI+) m/z 467.2257 (M+H+, C29H31N4S requires 467.2269).

[(E)-2-(5-Benzothiazol-2-yl-1-cyclohexyl-1H-benzoimidazol-2-yl)ethenyl]

methylphenylamine (159). Compound 156 (65 mg, 0.2 mmol), 117 (32 mg, 0.2

mmol), and ZrCl4 (24 mg, 0.1 mmol) were used to synthesize 156 according to

the procedures described for 120. Flash column chromatography (hexane/ethyl

acetate, 6:1) afforded the product (64 mg, 69%) as a yellow solid; mp 114-116 °C;

Rf = 0.53 (hexane/ethyl acetate, 1:1); 1H NMR (300 MHz, CDCl3) δ 8.30 (d, J =

13.1 Hz,, 1H), 8.25 (d, J = 1.5 Hz,, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.95 (dd, J1 =

8.1 Hz, J2 = 1.5 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.45

229

(dd, J1 = J2 = 7.7 Hz, 1H), 7.36-7.20 (m, 5H), 7.06 (t, J = 7.3 Hz, 1H), 5.46 (d, J =

13.2 Hz, 1H), 4.26 (m, 1H), 3.39 (s, 3H), 2.25 (m, 2H), 2.02 (m, 4H), 1.84 (m, 1H),

1.45 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 169.6, 155.1, 154.4, 146.8, 144.2,

143.1, 136.5, 135.1, 129.4 (× 2), 127.2, 126.0, 124.6, 123.4, 122.8, 121.5, 119.8,

119.5 (× 2), 117.6, 111.2, 87.0, 56.0, 36.4, 30.1 (× 2), 26.2 (× 2), 25.4; IR (film) ν

max 3060, 2931, 2849, 1625, 1593, 1490, 1466, 1436, 1301, 1127, 755 cm-1;

HRMS (ESI+) m/z 465.2129 (M+H+, C29H29N4S requires 465.2113).

{(E)-2-[5-Benzothiazol-2-yl-1-(4-methoxyphenyl)-1H-benzoimidazol-2-yl]ethe

nyl}methylphenylamine (160). Compound 157 (70 mg, 0.2 mmol), 117 (32 mg,

0.2 mmol), and ZrCl4 (24 mg, 0.1 mmol) were used to synthesize 160 according

to the procedures described for 120. Flash column chromatography

(hexane/ethyl acetate, 5:1) afforded the product (70 mg, 72%) as a yellow solid;

mp 182-184 °C; Rf = 0.53 (hexane/ethyl acetate, 1:1); 1H NMR (300 MHz, CDCl3)

δ 8.31-8.27 (m, 2H), 8.04 (d, J = 8.2 Hz,, 1H), 7.96 (dd, J1 = 8.3 Hz, J2 = 1.6 Hz,

1H), 7.87 (d, J = 7.9 Hz, 1H), 7.45-7.30 (m, 6H), 7.17-6.92 (m, 6H), 5.18 (d, J =

13.3 Hz, 1H), 3.90 (s, 3H), 3.16 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 169.5,

159.7, 155.9, 154.4, 146.6, 143.8, 142.4, 139.2, 135.1, 129.3 (× 2), 128.8 (× 2),

230

128.3, 128.1, 126.0, 124.6, 123.4, 122.8, 121.5, 120.7, 119.2 (× 2), 117.7, 115.1

(× 2), 109.5, 87.3, 55.6, 36.0; IR (film) ν max 3060, 2007, 2934, 2836, 1627,

1592, 1514, 1491, 1465, 1435, 1296, 1251, 1127, 755 cm-1; HRMS (ESI+) m/z

489.1754 (M+H+, C30H25N4OS requires 489.1749).

5-Benzothiazol-2-yl-3-ethyl-1-hexyl-2-[(E)-2-(methylphenylamino)ethenyl]-

3H-benzoimidazol-1-ium, iodide (161). Compound 158 (30 mg, 0.064 mmol)

and ethyl iodide (3 mL) were used to synthesize 161 according to the procedures

described for 111. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded

the product (36 mg, 91%) as a yellow solid; mp 128-131 °C; Rf = 0.25

(CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.33 (d, J = 1.1 Hz, 1H),

8.13 (m, 1H), 7.98 (m, 2H), 7.80 (m, 2H), 7.49-7.17 (m, 7H), 5.45 (d, J = 13.2 Hz,

1H), 4.50 (q, J = 7.3 Hz, 2H) 4.32 (t, J = 7.7 Hz, 2H), 3.53 (s, 3H), 1.87 (m, 2H),

1.53 (t, J = 7.2 Hz, 1H) 1.33 (m, 2H), 1.27 (m, 4H), 0.83 (t, J = 7.0 Hz, 3H); 13C

NMR (75 MHz, MeOH-d4) δ 168.7, 155.5, 153.5, 151.4, 136.8, 135.6, 133.8,

132.5, 131.5 (× 3), 128.4, 127.9, 127.4, 126.7, 124.4, 123.5, 122.9, 122.8, 113.9,

111.4, 79.8, 47.5, 42.7, 32.9 (× 2), 30.2, 27.8, 24.0, 14.7, 14.6; IR (film) ν max

3412.8, 3060.3, 2942.7, 2919.2, 2860.5, 1689.5, 1620.5, 1587.7, 1535.6, 1493.0,

231

1357.9, 1199.8, 1130.2, 760.2 cm-1; HRMS (ESI+) m/z 495.2572 (M+, C31H35N4S

requires 495.2582).

5-Benzothiazol-2-yl-1-cyclohexyl-3-ethyl-2-[(E)-2-(methylphenylamino)

ethenyl]-3H-benzoimidazol-1-ium, iodide (162). Compound 159 (30 mg, 0.065

mmol) and ethyl iodide (3 mL) were used to synthesize 162 according to the

procedures described for 111. Flash column chromatography (CH2Cl2/MeOH,

20:1) afforded the product (36 mg, 90%) as a yellow solid; mp 112-114 °C; Rf =

0.28 (CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.40 (s, 1H), 8.11 (m,

2H), 7.98 (m, 2H), 7.66 (dd, J1 = 7.3 Hz, J2 = 6.3 Hz, 1H), 7.52-7.31 (m, 6H), 7.23

(dd, J1 = J2 = 7.3 Hz, 1H), 5.50 (d, J = 13.5 Hz, 1H), 4.63 (m, 1H), 4.49 (q, J = 7.2

Hz, 2H), 3.53 (s, 3H), 2.35 (m, 2H), 2.02 (m, 4H), 1.78 (m 1H), 1.54 (t, J = 7.2 Hz,

3H), 1.56-1.37 (m, 3H); 13C NMR (75 MHz, MeOH-d4) δ 168.6, 155.5, 154.4,

154.3 (x 2), 151.3 (× 2), 148.0, 136.8, 134.5, 134.0, 132.3, 131.4 (× 2), 128.4,

127.5, 126.3, 124.4, 123.5, 122.4, 116.8, 111.9, 79.4, 61.6, 43.1, 38.3, 32.0 (× 2),

27.3 (× 2), 26.3, 14.8; IR (film) ν max 3342 (br), 3060, 2931, 2948, 1688, 1616,

1588, 1528, 1494, 1461, 1363, 1305, 1199, 1172, 1130, 761 cm-1; HRMS (ESI+)

m/z 493.2415 (M+, C31H33N4S requires 493.2426).

232

5-Benzothiazol-2-yl-3-ethyl-1-(4-methoxyphenyl)-2-[(E)-2-(methylphenyl

amino)ethenyl]-3H-benzoimidazol-1-ium, iodide (163). Compound 160 (35 mg,

0.072 mmol) and ethyl iodide (3 mL) were used to synthesize 163 according to

the procedures described for 111. Flash column chromatography (CH2Cl2/MeOH,

20:1) afforded the product (42 mg, 92%) as a yellow solid; mp 174-176 °C; Rf =

0.22 (CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.34 (s, 1H), 7.92 (m,

2H), 7.48 (d, J = 8.8 Hz, 2H), 7.41-7.03 (m, 9H), 6.70 (d, J = 7.5 Hz, 2H), 5.36 (d,

J = 14.0 Hz, 1H), 4.47 (q, J = 6.9 Hz, 2H), 3.85 (s, 3H), 3.30 (s, 3H), 1.49 (t, J =

7.1 Hz, 3H); 13C NMR (75 MHz, MeOH-d4) δ 168.6, 163.7, 155.5, 153.5, 151.9,

147.6, 137.6, 136.7, 133.3, 132.4, 131.1 (× 2), 130.8 (× 2), 128.4, 127.8, 127.4,

124.4, 123.5, 121.8, 118.3 (× 2), 113.4, 110.9, 79.8, 57.0, 41.7, 37.3, 14.4; IR

(film) ν max 3412 (br), 3050, 2932, 2840, 1682, 1613, 1588, 1504, 1410, 1369,

1256, 1201, 1126, 762 cm-1; HRMS (ESI+) m/z 517.2045 (M+, C32H29N4OS

requires 517.2062).

233

2-(3-Chloro-4-nitrophenyl)benzooxazole (165). To a slurry of 3-chloro-4-nitro-

benzoic acid (1.0 g, 5.0 mmol) in DMF (15 mL) were added HATU (1.9 g, 5.0

mmol) and DIEA (1.7 mL, 10 mmol). The reaction was stirred for 5 min before 2-

aminophenol (550 mg, 5.0 mmol) was added. The reaction was stirred for 4 h

and then poured into saturated NaCl aqueous solution (150 mL). The aqueous

phase was extracted with diethyl ether (100 mL × 3). The combined organic

phase was washed with saturated NaCl aqueous solution (100 mL), dried over

anhydrous Na2SO4, and concentrated in vacuo. To the slurry of resulting residue

in xylene (30 mL) was added p-toluenesulfonic acid (3.8 g, 20 mmol). The

reaction was refluxed for 1 h, then cooled down, and poured into NaOH

acqueous solution (1.0 M, 100 mL). The aqueous phase was extracted with

diethyl ether (100 mL × 3). The combined organic phase was washed with

saturated NaCl aqueous solution (100 mL), dried over anhydrous Na2SO4, and

concentrated in vacuo. Flash column chromatography (hexanes/ethyl acetate,

10:1) offorded the product (1.02 g, 71%) as an off-white solid; mp 162-164 °C; Rf

= 0.58 (hexanes/ethyl acetate, 3:1); 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 1.7

Hz, 1H), 8.23 (dd, J1 = 1.7 Hz, J2 = 8.5 Hz, 2H), 7.99 (d, J = 8.5 Hz, 1H), 7.79 (m,

1H), 7.61 (m, 1H), 7.41 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 159.4, 151.0,

149.0, 141.7, 131.8, 130.6, 128.0, 126.6, 126.2 (× 2), 125.4, 120.7, 111.0; IR

234

(film) ν max 3093, 1610, 1580, 1547, 1523, 1468, 1446, 1331, 1067, 762, 750

cm-1; HRMS (ESI+) m/z 275.0238 (M+H+, C13H8N2O3Cl requires 275.0224).

(5-Benzooxazol-2-yl-2-nitrophenyl)ethylamine (166). To a slurry of 165 (200

mg, 0.73 mmol) of DMSO (2 mL) was added ethylamine (2.0 M in THF, 4 mL).

The reaction was stirred at 22 °C for 48 h. The reaction solution was poured into

HCl slution, (1.0 M, 30 mL). The aqueous phase was extracted with diethyl ether

(50 mL × 3). The combined organic phase was washed with saturated aqueous

NaCl solution (50 mL), dried over anhydrous Na2SO4, and concentrated in vacuo.

Recrystallization of the crude product from CH2Cl2/MeOH afforded the product

(190 mg, 92%) as an orange solid; mp 168-170 °C; Rf = 0.55 (hexanes/ethyl

acetate, 3:1); 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 8.9 Hz, 1H), 8.01 (br, 1H),

7.80 (m, 1H), 7.70 (d, J = 1.6 Hz, 1H), 7.59 (m, 1H), 7.46 (d, J1 = 1.6 Hz, J2 = 8.9

Hz, 1H), 7.39 (m, 2H), 3.48 (m, 2H), 1.43 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz,

CDCl3) δ 161.2, 150.9, 145,3, 141,9, 133.6, 132.8, 127.7, 126.1, 125.1, 120.1,

113.6,112.8, 110.8, 38.0, 14.4; IR (film) ν max 3379, 2969, 2912, 2850, 1627,

1621, 1580, 1553, 1523, 1490, 1402, 1339, 1320, 1281, 1215, 1188, 1048, 743

cm-1; HRMS (ESI+) m/z 284.1036 (M+H+, C15H14N3O3 requires 284.1035).

235

(5-Benzooxazol-2-yl-2-nitrophenyl)hexylamine (167). Compound 165 (200 mg,

0.73 mmol) and hexylamine (1.0 mL, 7.7 mmol) were reacted in DMSO/THF (2

mL/2 mL) for 48 h. The reaction was worked up according to the procedures

described for 166. Recrystallization of the crude product from CH2Cl2/MeOH

afforded the product (233 mg, 94%) as an orange solid; mp 134-135 °C; Rf =

0.68 (hexanes/ethyl acetate, 3:1); 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 8.9

Hz, 1H), 8.09 (br, 1H), 7.81 (m, 1H), 7.72 (d, J = 0.9 Hz, 1H), 7.60 (m, 1H), 7.46

(d, J1 = 0.9 Hz, J2 = 8.9 Hz, 1H), 7.40 (m, 2H), 3.42 (q, J = 5.2 Hz, 2H), 1.80 (m,

2H), 1.49 (m, 2H), 1.38 (m, 4H), 0.93 (t, J = 5.2 Hz, 3H); 13C NMR (100 MHz,

CDCl3) δ 161.3, 150.9, 145.4, 141.9, 133.6, 132.8, 127.7, 126.1, 125.1, 120.5,

113.5, 112.9, 110.8, 43.3, 31.5, 28.9, 26.7, 22.6, 14.0; IR (film) ν max 3368, 3077,

2950, 2924, 2857, 1630, 1622, 1582, 1556, 1523, 1491, 1470, 1451, 1407, 1318,

1284, 1260, 1244, 1211, 1191, 1180,1050, 760, 744 cm-1; HRMS (ESI+) m/z

340.1646 (M+H+, C19H22N3O3 requires 340.1661).

236

4-Benzooxazol-2-yl-N2-ethylbenzene-1,2-diamine (168). Compound 166 (160

mg, 0.56 mmol) was treated with anhydrous hydrazine (0.1 mL, 3.2 mmol) with

Pd/C (64 mg, 0.06 mmol) as catalyst according to the precedures described for

116. Recrystallization from CH2Cl2/hexanes afforded the product (132 mg, 93%)

as a light yellow solid; mp 172-173 °C; Rf = 0.30 (hexanes/ethyl acetate, 1:1); 1H

NMR (400 MHz, DMSO-d6) δ 7.65 (br, 2H), 7.36-7.18 (m, 4H), 6.66 (d, J = 7.6 Hz,

1H), 5.44 (s, 2H), 4.70 (br, 1H), 3.15 (m, 2H), 1.27 (m, 3H); 13C NMR (100 MHz,

DMSO-d6) δ 164.7, 150.4, 142.7, 140.4, 135.8, 124.7, 124.4, 119.1, 118.3, 114.7,

113.3, 110.7, 108.0, 38.4, 14.8; IR (film) ν max 3429, 3379, 3335, 3225, 2973,

2956, 2923, 2852, 1643, 1607, 1586, 1495, 1451, 1036, 1278, 1240, 1150, 1053,

856, 793, 759, 746 cm-1; HRMS (ESI+) m/z 254.1287 (M+H+, C15H16N3O requires

254.1293).

4-Benzooxazol-2-yl-N2-hexylbenzene-1,2-diamine (169). Compound 167 (160

mg, 0.47 mmol) was treated with anhydrous hydrazine (0.1 mL, 3.2 mmol) with

Pd/C (53 mg, 0.05 mmol) as catalyst according to the precedures described for

116. Recrystallization from CH2Cl2/hexanes afforded the product (136 mg, 94%)

as a light yellow solid; mp 126-127 °C; Rf = 0.24 (hexanes/ethyl acetate, 3:1); 1H

NMR (400 MHz, CDCl3) δ 7.72 (m, 2H), 7.62 (dd, J1 = 1.8 Hz, J2 = 8.0 Hz, 1H),

7.53 (m, 2H), 7.28 (m, 2H), 6.77 (d, J = 8.0 Hz, 1H), 3.6 (br, 3H), 3.20 (t, J = 7.2

237

Hz, 2H), 1.69 (m, 2H), 1.44 (m, 2H), 1.34 (m, 4H), 0.91 (t, J = 7.0 Hz, 3H); 13C

NMR (100 MHz, CDCl3) δ 164.3,150.6, 142.4, 138.5, 137.5,124.2 (× 2), 119.3 (×

2), 118.7, 115.4, 110.8, 110.2, 44.4, 31.7, 29.7, 27.0, 22.6, 14.1; IR (film) ν max

3410, 3360, 3220, 2950, 2927, 2846, 1646, 1591, 1582, 1557, 1505, 1484, 1455,

1443, 1318, 1292, 1158, 955, 851, 797, 758, 743 cm-1; HRMS (ESI+) m/z

310.1899 (M+H+, C19H24N3O requires 310.1919).

[(E)-2-(6-Benzooxazol-2-yl-1-ethyl-1H-benzoimidazol-2-yl)ethenyl]methyl

phenylamine (170). To a solution of 168 (51 mg, 0.2 mmol) and 117 (32 mg, 0.2

mmol) in ethanol (10 mL), ZrOCl2·8H2O (32 mg, 0.1 mmol) was added. The

reaction was stirred at 22 °C for 30 min. Then, the reaction was heated to 80 °C

and refluxed for 5 min before the addition of MnO2 (86 mg, 1.0 mmol). After 10

min, the reaction solution was cooled to 22 °C and filter through a frit funnel. The

MnO2 was washed with ethanol (10 mL). The combined filtrate was concentrated

in vacuo. Flash column chromatography (hexanes/ethyl acetate, 3:1) afforded the

product (53 mg, 68%) as a yellow solid; mp 171-173 °C; Rf 0.15 (hexane/ethyl

acetate, 1:1); 1H NMR (300 MHz, CDCl3) δ 8.37 (d, J = 13.0 Hz, 1H), 8.12 (m,

2H),7.72 (m, 2H), 7.58 (m, 1H), 7.39-7.22 (m, 6H), 7.12 (t, J = 6.7 Hz, 1H), 5.38

(d, J = 13.0 Hz, 1H), 4.23 (q, J = 6.7 Hz, 2H), 3.38 (s, 3H), 1.47 (t, J = 6.7 Hz,

238

3H); 13C NMR (75 MHz, CDCl3) δ 164.5, 156.0, 150.8, 146.8, 146.6, 143.5 (x 2),

142.5, 135.3, 129.4(x 2), 124.4, 124.3, 123.7, 121.9, 119.7 (x 2), 119.4, 119.0,

117.6, 110.3, 107.8, 85.9, 38.0, 36.5, 15.0; IR (film) ν max 3379, 3060, 2978,

2934, 1628, 1594, 1558, 1491, 1453, 1410, 1347, 1326, 1291, 1242, 1128, 809,

747 cm-1; HRMS (ESI+) m/z 395.1849 (M+H+, C25H23N4O requires 395.1872).

[(E)-2-(6-Benzooxazol-2-yl-1-hexyl-1H-benzoimidazol-2-yl)ethenyl]methyl

phenylamine (171). Compound 169 (62 mg, 0.2 mmol) and 117 (32 mg, 0.2

mmol) in ethanol (10 mL), ZrOCl2·8H2O (32 mg, 0.1 mmol) was used to

synthesize 171 according to the precedures described for 170. Flash column

chromatography (hexanes/ethyl acetate, 5:1) afforded 171 (58 mg, 64%) as a

yellow solid; mp 88-89 °C; Rf 0.40 (hexane/ethyl acetate, 1:1); 1H NMR (300 MHz,

CDCl3) δ 8.35 (d, J = 13.1 Hz, 1H), 8.11-8.09 (m, 2H), 7.76 (m, 1H), 7.67 (dd, J1

= 0.9 Hz, J2 = 8.0 Hz, 1H), 7.56 (m, 1H), 7.38-7.30 (m, 4H), 7.23-7.21 (m, 2H),

7.10 (m, 1H), 5.37 (d, J = 13.1 Hz, 1H), 4.15 (t, J = 7.4 Hz, 2H), 3.37 (s, 3H), 1.85

(m, 2H), 1.35 (m, 6H), 0.89 (t, J = 7.4 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 164.5,

156.3, 150.8, 146.6, 143.4, 142.5, 135.8, 129.4 (× 2), 124.4, 124.3, 123.7, 121.9,

119.7 (× 3), 119.4, 119.0, 117.6, 110.3, 108.0, 86.2, 43.3, 36.5, 31.5, 29.8, 26.6,

22.6, 14.0; IR (film) ν max 3357, 3060, 2950, 2929, 2853, 1628, 1594, 1559,

239

1491, 1452, 1409, 1326, 1294, 1268, 1243, 1128, 1001, 823, 746, 695 cm-1;

HRMS (ESI+) m/z 451.2472 (M+H+, C29H31N4O requires 451.2498).

(4-Benzooxazol-2-yl-2-nitrophenyl)phenylamine (178). 2-(4-Chloro-3-nitro-

phenyl)benzooxazole (1.2 g, 4.5 mmol) and freshly distilled aniline (1.8 mL, 20

mmol) were used to synthesize 178 according to the procedures described for

115. Column chromatography (CH2Cl2) afforded the product (2.24 g, 84%) as an

orange solid, mp 164-166 °C; Rf 0.15 (hexanes/ethyl acetate, 3:1); 1H NMR (300

MHz, CDCl3) δ 9.82 (s, 1H), 9.09 (s, 1H), 8.20 (d, J = 9.0 Hz, 1H), 7.73 (m, 1H),

7.57 (m, 1H), 7.52-7.47 (m, 2H), 7.36-7.29 (m, 5H); 13C NMR (75 MHz, CDCl3) δ

161.3, 150.6, 144.9, 142.0, 137.6, 134.0, 132.6, 130.0 (× 2), 126.7, 126.4, 125.1,

125.0 (× 2), 124.7, 119.7, 116.4 (× 2), 110.5; IR (film) ν max 3339, 1628, 1596,

1507, 1452, 1352, 1264, 1242, 1152, 1077 cm-1. LRMS (ESI+) m/z 331.9 (M+H+,

C19H14N3O3 requires 332.1).

240

3-Nitro-N-phenyl-4-phenylamino-benzamide (179). 4-Chloro-3-nitro-N-phenyl-

benzamide (2.2 g, 8 mmol) and freshly distilled aniline (3.7 mL, 40 mmol) were

used to synthesize 179 according to the procedures described for 115. Flash

column chromatography (hexanes/ethyl acetate, 8:1) afforded the product (2.24 g,

84%) as an orange solid, mp 212-215 °C; 1H NMR (300 MHz, CDCl3) δ 9.81 (s,

1H), 8.74 (d, J = 1.5 Hz, 1H), 7.98 (dd, J1 = 1.3 Hz, J2 = 6.7 Hz, 1H), 7.79 (s, 1H),

7.66 (d, J = 5.9 Hz, 2H), 7.50 (t, J = 5.9 Hz, 2H), 7.43 (t, J = 5.6 Hz, 2H), 7.34-

7.27 (m, 3H), 7.19 (t, J = 5.6 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 165.8, 144.1,

139.8, 139.6, 131.9, 129.0 (× 2), 128.8, 128.5 (× 2), 123.1, 120.1 (× 2), 119.3,

118.3, 116.5 (× 2), 116.3, 114.7; IR (film) ν max 3331, 1648, 1623, 1596, 1440,

1319, 1267, 1214, 1152, 753, 690 cm-1; HRMS (ESI+) m/z 334.1181 (M+H+,

C19H16N3O3 requires 334.1192).

4-Benzooxazol-2-yl-N1-phenylbenzene-1,2-diamine (182). Compound 178

(900 mg, 2.7 mmol) was treated with anhydrous hydrazine (0.5 mL, 16 mmol)

with Pd/C (318 mg, 0.3 mmol) as catalyst according to the precedures described

for 116. Flash column chromatography (hexane/ethyl acetate, 8:1) afforded the

product (758 mg, 93%) as a yellow solid; Rf 0.53 (hexanes/ethyl acetate, 1:1); mp

142-143 °C; 1H NMR (300 MHz, CDCl3) δ 7,79-7.69 (m, 3H), 7.60-7.57 (m, 1 H),

241

7.37-7,29 (m, 5H), 7.01-6.98 (m, 3H), 5,60 (s, 1H), 3.65 (s, 2H); 13C NMR (75

MHz, CDCl3) δ 163.4, 150.6, 142.9, 142.2, 138.7, 134.2, 129.4 (× 2), 124.6,

124.4, 121.6, 121.1, 119.7, 119.7, 119.5, 117.6 (× 2) ,115.6, 110.5; IR (film) ν

max 3364, 3048, 1614, 1594, 1557, 1497, 1454, 1313, 1244, 744 cm-1; HRMS

(ESI+) m/z 302.1302 (M+H+, C19H16N3O requires 302.1293).

3-Amino-N-phenyl-4-phenylamino-benzamide (183). 179 (1.0 g, 3.0 mmol)

was treated with anhydrous hydrazine (0.5 mL, 16 mmol) with Pd/C (318 mg, 0.3

mmol) as catalyst according to the precedures described for 116. Flash column

chromatography (hexane/ethyl acetate, 8:1) afforded the product (875 mg, 96%)

as a white solid; Rf 0.53 (hexanes/ethyl acetate, 1:1); mp 176-178 °C; 1H NMR

(300 MHz, DMSO-d6) δ 7.76 (d, J = 7.6 Hz, 2H), 7.34-7.29 (m, 4H), 7.23-7.10 (m,

4H), 7.05 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 7.6 Hz, 2H), 6.79 (t, J = 7.2 Hz, 1H), 5.0

(s, 2H), 7.19 (t, J = 5.6 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ 165.8, 144.1,

139.8, 139.6, 131.9, 129.1 (× 2), 128.8, 128.5 (× 2), 123.1, 120.1 (× 2), 119.3,

118.3, 116.5 (× 2), 116.4, 114.7; IR (film) ν max 3324, 1643, 1594, 1497, 1434,

1316, 1241, 886, 774, 691 cm-1; HRMS (ESI+) m/z 304.1457 (M+H+, C19H18N3O

requires 304.1450).

242

Methyl-phenyl-[(E)-2-(1-phenyl-1H-benzoimidazol-2-yl)ethenyl]amine (184).

N-Phenylbenzene-1,2-diamine (37 mg, 0.2 mmol), 117 (32 mg, 0.2 mmol), and

ZrCl4 (24 mg, 0.1 mmol) were used to synthesize 184 according to the

procedures described for 120. Flash column chromatography (hexane/ethyl

acetate, 5:1) afforded the product (39 mg, 61%) as a yellow solid; mp 118-121 °C;

Rf 0.54 (hexanes/ethyl acetate, 1:1); 1H NMR (300 MHz, CDCl3) δ 8.30 (d, J =

13.3 Hz, 1H), 7.71(d, J = 7.9 Hz, 1H), 7.64-7.46 (m, 5H), 7.34 (t, J = 7.9 Hz, 2H),

7.25-7.05 (m, 6H), 5.26 (d, J = 13.3 Hz, 1H), 3.20 (s, 3H); 13C NMR (75 MHz,

CDCl3) δ 153.8, 146.7, 142.0, 136.5, 136.3, 129.8 (× 2), 129.3 (× 2), 128.5, 127.6

(× 2), 123.1, 122.4, 121.1, 119.2 (× 2), 117.6, 109.2 (× 2), 87.6, 35.9; IR (film) ν

max 3060, 1629, 1594, 1492, 1454, 1347, 1301, 1286, 1267, 1127, 757, 695

cm-1; HRMS (ESI+) m/z 326.1651 (M+H+, C22H20N3 requires 326.1657).

243

2-[(E)-2-(Methylphenylamino)ethenyl]-1-phenyl-1H-benzoimidazole-5-carbo

xylic acid methyl ester (185). 3-Amino-4-phenylamino-benzoic acid methyl

ester (48 mg, 0.2 mmol), 117 (32 mg, 0.2 mmol), and ZrCl4 (24 mg, 0.1 mmol)

were used to synthesize 185 according to the procedures described for 120.

Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the product

(50 mg, 65%) as a light yellow solid; mp 169-170 °C; Rf 0.64 (hexanes/ethyl

acetate, 1:1); 1H NMR (300 MHz, CDCl3) δ 8.37 (d, J = 1.3 Hz, 1H), 8.28 (d, J =

13.3 Hz, 1H), 7.82 (dd, J1 = 1.5 Hz, J2 = 8.4 Hz, 1H), 7.63-7.53 (m, 3H), 7.45-

7.43 (m, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.15 (d, J = 7.7 Hz, 2H), 7.07 (m, 2H),

5.20 (d, J = 13.3 Hz, 1H), 3.93 (s, 3H), 3.17 (s, 3H); 13C NMR (75 MHz, CDCl3) δ

167.9, 155.7, 146.6, 143.3, 142.7, 139.9, 135.8, 130.0 (× 2), 129.3 (× 2), 128.8,

127.5 (× 2), 124.4, 123.5, 123.0, 119.6, 119.3 (× 2), 108.6, 87.1, 52.0, 36.1; IR

(film) ν max 1712, 1628, 1492, 1438, 1348, 1297, 1224, 1127, 1085, 751, 696

cm-1; HRMS (ESI+) m/z 384.1731 (M+H+, C24H22N3O2 requires 384.1712).

[(E)-2-(5-Benzooxazol-2-yl-1-phenyl-1H-benzoimidazol-2-yl)ethenyl]methyl

phenylamine (186). Compound 182 (60 mg, 0.2 mmol), 117 (32 mg, 0.2 mmol),

and ZrCl4 (24 mg, 0.1 mmol) were used to synthesize 182 according to the

procedures described for 120. Flash column chromatography (hexane/ethyl

244

acetate, 5:1) afforded the product (48 mg, 54%) as a light yellow solid; mp 194-

196 °C; Rf 0.54 (hexanes/ethyl acetate, 1:1); 1H NMR (300 MHz, CDCl3) δ 8.55

(d, J = 1.1 Hz, 1H), 8.35 (d, J = 13.2 Hz, 1H), 8.09 (dd, J1 = 1.5 Hz, J2 = 8.4 Hz,

1H), 7.78 (m, 1H), 7.68-7.54 (m, 4 H), 7.51-7.49 (m, 2H), 7.39-7.31 (m, 4H),

7.21-7.02 (m, 4H), 5.36 (d, J = 13.2 Hz, 1H), 3.15 (s, 3H); 13C NMR (75 MHz,

CDCl3) δ 164.3, 155.6, 150.8, 146.6, 142.8, 142.4, 140.0, 135.7, 130.0 (× 2),

129.3 (× 3), 128.9, 127.6 (× 2), 124.4, 124.3, 123.5, 121.4, 121.4, 119.6, 119.4 (×

2), 117.1, 110.4, 109.5, 87.0, 36.1; IR (film) ν max 3060, 1627, 1594, 1582, 1492,

1453, 1347, 1297, 1244, 1127, 746, 696 cm-1; HRMS (ESI+) m/z 443.1875

(M+H+, C29H23N4O requires 443.1872).

2-[(E)-2-(Methylphenylamino)ethenyl]-1-phenyl-1H-benzoimidazole-5-carbo

xylic acid phenylamide (187). Compound 183 (61 mg, 0.2 mmol), 117 (32 mg,

0.2 mmol), and ZrCl4 (24 mg, 0.1 mmol) were used to synthesize 187 according

to the procedures described for 120. Flash column chromatography

(hexane/ethyl acetate, 5:1) afforded the product (50 mg, 56%) as a yellow solid;

mp 121-123 °C; Rf 0.50 (hexanes/ethyl acetate, 1:1); 1H NMR (300 MHz, MeOH-

d4) δ 8.28 (d, J = 17.3 Hz, 1H), 8.12(s, 1H), 7.72-7.07 (m, 18H), 5.21 (d, J = 13.4

Hz, 1H), 3.16 (s, 3H); 13C NMR (75 MHz, MeOH-d4) δ 169.3, 158.3, 157.5, 148.0,

245

144.5, 143.7, 140.1, 140.0, 136.8, 131.4 (× 2), 130.8, 130.6 (× 2), 130.5, 129.8 (×

2), 128.7, 125.4, 124.8, 122.6, 122.4 (× 2), 120.3 (× 2), 117.4, 110.2, 87.1, 36.3;

IR (film) ν max 3290, 3059, 1625, 1595, 1540, 1498, 1433, 1316, 1297, 1127,

751 cm-1; HRMS (ESI+) m/z 445.2021 (M+H+, C29H25N4O requires 445.2028).

1-Ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-3-phenyl-3H-benzoimidazol-1-

ium, iodide (188). Compound 184 (30 mg, 0.092 mmol) and ethyl iodide (3 mL)

were used to synthesize 188 according to the procedures described for 111.

Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded the product (37 mg,

85%) as a yellow solid; mp 43-45 °C; Rf = 0.28 (CH2Cl2/MeOH, 10:1); 1H NMR

(300 MHz, MeOH-d4) δ 7.83-7.77 (m, 4H), 7.69-7.66(m, 2H), 7.52 (t, J = 7.2, 1H),

7.44 (t, J = 7.3 Hz, 1H), 7.31-7.08 (m, 5H), 6.74 (d, J = 7.9 Hz, 2H), 5.56 (d, J =

11.3 Hz, 1H), 4.58 (d, J = 7.3 Hz, 2H), 3.41 (s, 3H), 1.58 (t, J = 7.3 Hz, 3H); 13C

NMR (75 MHz, MeOH-d4) δ 152.1, 151.6, 151.5, 147.6, 136.8, 135.5, 133.2 (× 2),

132.9, 132.7, 131.1 (× 2), 129.7 (× 2), 127.4, 127.3, 127.2, 121.8, 112.9, 112.8,

79.5, 41.6, 37.5, 14.4; IR (film) ν max 3360, 1688, 1623, 1585, 1533, 1494, 1361,

1310, 1201, 1177, 1129, 759, 697 cm-1; HRMS (ESI+) m/z 354.1949 (M+,

C24H24N3 requires 354.1970).

246

1-Ethyl-6-methoxycarbonyl-2-[(E)-2-(methylphenylamino)ethenyl]-3-phenyl-

3H-benzoimidazol-1-ium, iodide (189). Compound 185 (30 mg, 0.078 mmol)

and ethyl iodide (3 mL) were used to synthesize 189 according to the procedures

described for 111. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded

the product (39 mg, 93%) as a yellow solid, mp 43-46 °C; Rf = 0.25

(CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, DMSO-d6) δ 8.47 (s, 1H), 8.02 (dd, J1

= 8.6 Hz, J2 = 1.2 Hz, 1H), 7.84-8.63 (m, 5H), 7.29 (t, J = 8.1 Hz, 2H), 7.21-7.14

(m, 3H), 6.70 (d, J = 7.9 Hz, 2H), 5.76 (d, J = 13.5 Hz, 1H), 4.70 (q, J = 7.1 Hz,

2H), 3.92 (s, 3H), 3.41 (s, 3H), 1.46 (t, J = 7.0 Hz, 3H); 13C NMR (75 MHz,

DMSO-d6) δ 165.6, 152.0, 149.5, 145.2, 136.6, 134.5, 131.5 (× 2), 131.1, 130.9,

129.5 (× 3), 128.0 (× 2), 126.5, 126.4, 125.6, 119.6, 112.7, 111.3, 79.1, 52.5,

39.4, 36.4, 13.7; IR (film) ν max 3412, 1716, 1692, 1622, 1588, 1538, 1621, 1588,

1538, 1494, 1463, 1373, 1312, 1290, 1266, 1198, 1134, 764, 698 cm-1; HRMS

(ESI+) m/z 412.2000 (M+, C26H26N3O2 requires 412.2028).

247

5-Benzooxazol-2-yl-3-ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl-

3H-benzoimidazol-1-ium, iodide (190). Compound 186 (35 mg, 0.079 mmol)

and ethyl iodide (3 mL) were used to synthesize 190 according to the procedures

described for 111. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded

the product (40 mg, 86%) as a yellow solid, mp 181-184 °C; Rf = 0.22

(CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H), 8.44 (d, J =

12.2 Hz, 1H), 7.71-7.56 (m, 7H), 7.31-7.03 (m, 7H), 6.52 (m, 2H), 5.41 (d, J =

11.6 Hz, 1H), 4.50 (q, J = 6.7 Hz, 2H), 3.17 (s, 3H), 1.50 (t, J = 6.5 Hz, 3H); 13C

NMR (75 MHz, CDCl3) δ 163.4, 153.3, 152.2, 151.8, 142.9, 137.3, 136.0, 132.9

(× 3), 132.6, 130.7 (× 3), 129.2 (× 3), 127.3, 127.0, 126.3, 126.2, 125.4, 120.8,

113.1, 111.9, 111.3, 79.1, 41.1, 36.9, 13.9; IR (film) ν max 3425, 1681, 1617,

1586, 1537, 1494, 1453, 1365, 1298, 1197, 1136, 761, 697 cm-1; HRMS (ESI+)

m/z 471.2168 (M+, C31H27N4O requires 471.2185).

248

3-Ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl-5-phenylcarbamoyl-

3H-benzoimidazol-1-ium, iodide (191). Compound 187 (35 mg, 0.079 mmol)

and ethyl iodide (3 mL) were used to synthesize 191 according to the procedures

described for 111. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded

the product (40 mg, 84%) as a yellow solid, mp 285-287 °C (decomp.); Rf = 0.44

(CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, DMSO-d6) δ 10.4 (s, 1H), 8.52(s, 1H),

8.06 (d, J = 8.4 Hz, 1H), 7.82 (m, 7H), 7.40 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.2 Hz,

2H), 7.24-7.15 (m, 4H), 6.73 (d, J = 7.6 Hz, 2H), 5.77 (d, J = 12.8 Hz), 4.69 (q, J

= 6.5 Hz, 2H), 3.43 (s, 3H), 1.53 (t, J = 6.2 Hz, 3H); 13C NMR (75 MHz, DMSO-d6)

δ 164.8, 152.0, 149.7, 145.6, 139.3, 135.8, 134.9, 132.2, 131.9 (× 2), 131.4,

131.1, 129.9 (× 2), 129.1 (× 2), 128.4 (× 2), 125.9, 125.7, 124.4, 121.0 (× 2),

119.9 (× 2), 111.6, 111.3, 79.6, 40.9, 36.8, 14.2; IR (film) ν max 3260, 1662,

1620, 1587, 1525, 1493, 1463, 1440, 1369, 1306, 1251, 1128, 1209, 758 cm-1;

HRMS (ESI+) m/z 473.2335 (M+, C31H29N4O requires 473.2341).

5-Benzothiazol-2-yl-3-hexyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl-

3H-benzoimidazol-1-ium, iodide (192). Compound 120 (35 mg, 0.076 mmol)

and 1-iodo-hexane (3 mL) were used to synthesize 192 according to the

procedures described for 111. Flash column chromatography (CH2Cl2/MeOH,

249

30:1) afforded 192 (47 mg, 92%) as a yellow solid, mp 86-88 °C; Rf = 0.37

(CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.40 (d, J = 1.1 Hz, 1H),

8.06-7.95 (m, 3H), 7.78-7.74 (m, 3H), 7.67-7.64 (m, 2H), 7.48-7.38 (m, 2H), 7.26

(m, 2H), 7.23-7.12 (m, 3H), 6.72 (br, 2H), 5.47 (d, J = 11.9 Hz, 1H), 4.50 (m, 2H),

3.36 (s, 3H), 1.95 (m, 2H), 1.51 (m, 2H), 1.36 (m, 4H), 0.88 (t, J = 7.0 Hz, 3H);

13C NMR (75 MHz, MeOH-d4) δ 168.6, 155.5, 153.8, 152.1, 137.2, 136.7, 136.4,

134.0, 133.3 (× 2), 133.0, 132.6, 131.1 (× 2), 129.6 (× 2), 128.4, 127.4, 126.9,

124.4, 123.5, 121.9, 113.5, 111.2, 79.9, 46.5, 36.8, 33.0, 30.0, 27.9, 24.1, 14.8;

IR (film) ν max 3331, 3060, 2943, 2931, 2849, 1688, 1617, 1586, 1537, 1494,

1464, 1369, 1310, 1120, 1167, 1127, 799, 761, 697 cm-1; HRMS (ESI+) m/z

543.2548 (M+, C35H35N4S requires 543.2582).

5-Benzothiazol-2-yl-3-dodecyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-

phenyl-3H-benzoimidazol-1-ium, iodide (193). Compound 120 (30 mg, 0.066

mmol) and 1-iodo-dodecane (3 mL) were used to synthesize 193 according to

the procedures described for 111. Flash column chromatography (CH2Cl2/MeOH,

30:1) afforded the product (43 mg, 87%) as a yellow glassy solid; Rf = 0.44

(CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.42 (s, 1H), 8.07-7.95 (m,

250

3H), 7.79-7.64 (m, 5H), 7.51-7.36 (m, 2H), 7.27-7.15 (m, 5H), 6.69 (br, 2H), 5.53

(d, J = 12.3 Hz, 1H), 4.52 (m, 2H), 3.36 (s, 3H), 1.97 (m, 2H), 1.50-1.00 (m, 20H),

0.78 (t, J = 6.1 Hz, 3H); 13C NMR (75 MHz, MeOH-d4) δ 168.3, 155.1, 153.4,

151.7, 136.8, 136.4, 136.1, 133.6, 132.9 (× 2), 132.6, 132.3, 130.1 (× 2), 129.2 (×

2), 128.0, 127.1, 126.6, 124.0, 123.1, 121.4, 113.1, 110.9, 79.5, 46.1, 37.1, 33.0,

30.8, 30.7, 30.6, 30.5, 30.3, 29.5, 27.7, 23.7, 14.4; IR (film) ν max 3425, 3060,

2919, 2849, 1689, 1620, 1586, 1537, 1494, 1465, 1370, 1310, 1200, 1131, 799,

762, 697 cm-1; HRMS (ESI+) m/z 627.3523 (M+, C41H47N4S requires 627.3521).

5-Benzothiazol-2-yl-2-[(E)-2-(methylphenylamino)ethenyl]-3-phenethyl-1-

phenyl-3H-benzoimidazol-1-ium, iodide (194). Compound 120 (30 mg, 0.066

mmol) and (2-iodoethyl)benzene (3 mL) were used to synthesize 194 according

to the procedures described for 194. Flash column chromatography

(CH2Cl2/MeOH, 30:1) afford the product (43 mg, 87%) as a yellow solid; mp 83-

85 °C; Rf = 0.41 (CH2Cl2/MeOH, 10:1); 1H NMR (300 MHz, MeOH-d4) δ 8.31 (d, J

= 1.3 Hz, 1H), 8.18-8.03 (m, 3H), 7.78-7.75 (m, 3H), 7.62-7.42 (m, 4H), 7.31-7.02

(m, 9H), 6.83 (d, J = 13.5 Hz, 1H), 6.65 (br, 2H), 5.03-4.84 (m, 5H), 4.52 (m, 2H),

3.23 (s, 3H); 13C NMR (75 MHz, MeOH-d4) δ 168.5, 155.5, 154.1, 151.4, 139.2,

136.9, 136.8, 136.3, 133.5, 133.3 (× 2), 132.9, 132.7, 131.1(× 2), 130.9 (× 2),

251

130.4 (× 2), 129.4 (× 2), 128.9, 128.4, 127.7, 127. 4, 126.9, 124.4, 123.5, 121.6,

113.4, 111.6, 79.8, 48.0, 37.0, 35.9; IR (film) ν max 3428, 3061, 2943, 2860,

1689, 1616, 1586, 1535, 1494, 1464, 1370, 1307, 1200, 1167, 1127, 799, 760,

697 cm-1; HRMS (ESI+) m/z 563.2261 (M+, C37H31N4S requires 563.2269).

6.8.3 Biological Assays

Inhibition of luciferase expression from r-PIV5-R-Luc by compounds of

Group I-IV: Hela cells were infected with r-PIV5-R-Luc at a MOI of 1 and treated

with DMSO (0.1%), lead compound 111, and compounds of Groups I-IV at 1 µM.

The cells were collected at 24 hours postinfection and assayed for luciferase

activity by using a Renilla luciferase assay system (Promega), following the

manufacturer’s instructions.25 In the second round screening, active hits, which

exhibited inhibitory effect on viral replication (comparable to lead compound 111

at 1 µM), were tested both at 1 µM and 500 nM.

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VITA

Qi Sun

Qi Sun was born in Beijing, China. Upon graduation from Donzhimen high

school, he was admitted to the Department of Chemistry at Tsinghua University,

the top university in China. He received his B.S. degree in Chemistry in July,

2000 and his M.S. degree in Organic Chemistry in July, 2003. A month later, he

joined the Chemical Biology Option of the Integrative Biosciences Graduate

Program at the Pennsylviania State University, where he pursued his Ph.D.

degree under the supervision of Professor Blake R. Peterson. Upon completion

of his doctoral study, Qi Sun is looking forward to pursuing a career in chemical

industry.