USF Libraries
USF Digital Collections

Using in situ click chemistry to modulate protein-protein interactions

MISSING IMAGE

Material Information

Title:
Using in situ click chemistry to modulate protein-protein interactions Bcl-XL as a case study
Physical Description:
Book
Language:
English
Creator:
Malmgren, Lisa M
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla.
Publication Date:

Subjects

Subjects / Keywords:
Target-guided synthesis
Fragment-based synthesis
NMR
Triazole
Azide
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Protein-protein interactions are central to most biological processes. Although in the field of drug discovery there is a great interest in targeting protein-protein interactions, the discovery and development of small-molecules, which effect these interactions has been challenging. The purpose of this project is to determine if in situ click chemistry is a practical approach towards testing whether Bcl-XL is capable of assembling it's own inhibitory compounds. Abbott laboratories developed compound ABT-737, which binds with high affinity (Ki less than 1 nM) to the binding sites of Bcl-XL.36 Based on ABT-737, two acetylene anchor molecules AM3 and AM4 have been synthesized. These anchor molecules are distinguished by the reactivity of the their carbon-carbon triple bond. Compound AM3 contains an electron withdrawing carbonyl in the alpha-position to the acetylene resulting in an activating effect towards the 1,3-dipolar cycloaddition compared to compound AM4.To determine the reactivity of the activated system, 1H-NMR kinetic studies were performed to compare the relative rates of these two systems by reacting model alkynes 1,2,3, and 4 with azide AZ7. It was shown that the activated systems, 1 and 3, produce triazoles in an accelerated rate compared to the unactivated systems 2 and 3. To test for the self-assembly of inhibitory triazoles, the acetylenes AM3 and AM4 were incubated with Bcl-XL and 14 azide building blocks (AZ1-AZ12) for 12 hours at 37 degrees C. Subjecting these mixtures to LC/MS-SIM led to the discovery of two hit compounds, 35 and 36, of which 35 has been chemically synthesized confirming the hit. Future work includes the synthesis of all hit compounds. Since hit triazoles can be syn or anti, both need to be synthesized for each hit to investigate which regioisomer Bcl-XL generates. Tests to confirm if hit compounds are actually modulating Bcl-XL activity will be done using conventional bio-assays. This will validate that Bcl-XL is capable of assembling its own inhibitor via the in situ click chemistry approach to drug discovery.
Thesis:
Thesis (M.S.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
System Details:
System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Lisa M. Malmgren.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 111 pages.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001935516
oclc - 226251525
usfldc doi - E14-SFE0002233
usfldc handle - e14.2233
System ID:
SFS0026551:00001


This item is only available as the following downloads:


Full Text
xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.loc.govstandardsmarcxmlschemaMARC21slim.xsd
leader nam Ka
controlfield tag 001 001935516
003 fts
005 20080424163859.0
006 m||||e|||d||||||||
007 cr mnu|||uuuuu
008 080424s2007 flua sbm 000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0002233
035
(OCoLC)226251525
040
FHM
c FHM
049
FHMM
090
QD31.2 (ONLINE)
1 100
Malmgren, Lisa M.
0 245
Using in situ click chemistry to modulate protein-protein interactions :
b Bcl-XL as a case study
h [electronic resource] /
by Lisa M. Malmgren.
260
[Tampa, Fla.] :
University of South Florida,
2007.
520
ABSTRACT: Protein-protein interactions are central to most biological processes. Although in the field of drug discovery there is a great interest in targeting protein-protein interactions, the discovery and development of small-molecules, which effect these interactions has been challenging. The purpose of this project is to determine if in situ click chemistry is a practical approach towards testing whether Bcl-XL is capable of assembling it's own inhibitory compounds. Abbott laboratories developed compound ABT-737, which binds with high affinity (Ki less than 1 nM) to the binding sites of Bcl-XL.36 Based on ABT-737, two acetylene anchor molecules AM3 and AM4 have been synthesized. These anchor molecules are distinguished by the reactivity of the their carbon-carbon triple bond. Compound AM3 contains an electron withdrawing carbonyl in the alpha-position to the acetylene resulting in an activating effect towards the [1,3]-dipolar cycloaddition compared to compound AM4.To determine the reactivity of the activated system, 1H-NMR kinetic studies were performed to compare the relative rates of these two systems by reacting model alkynes 1,2,3, and 4 with azide AZ7. It was shown that the activated systems, 1 and 3, produce triazoles in an accelerated rate compared to the unactivated systems 2 and 3. To test for the self-assembly of inhibitory triazoles, the acetylenes AM3 and AM4 were incubated with Bcl-XL and 14 azide building blocks (AZ1-AZ12) for 12 hours at 37 degrees C. Subjecting these mixtures to LC/MS-SIM led to the discovery of two hit compounds, 35 and 36, of which 35 has been chemically synthesized confirming the hit. Future work includes the synthesis of all hit compounds. Since hit triazoles can be syn or anti, both need to be synthesized for each hit to investigate which regioisomer Bcl-XL generates. Tests to confirm if hit compounds are actually modulating Bcl-XL activity will be done using conventional bio-assays. This will validate that Bcl-XL is capable of assembling its own inhibitor via the in situ click chemistry approach to drug discovery.
502
Thesis (M.S.)--University of South Florida, 2007.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
538
System requirements: World Wide Web browser and PDF reader.
Mode of access: World Wide Web.
500
Title from PDF of title page.
Document formatted into pages; contains 111 pages.
590
Advisor: Roman Manetsch, Ph.D.
653
Target-guided synthesis.
Fragment-based synthesis.
NMR.
Triazole.
Azide.
690
Dissertations, Academic
z USF
x Chemistry
Masters.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.2233



PAGE 1

Using In Situ Click Chemistry to Modulate Protein-Protein Interactions: Bcl-X L as a Case Study by Lisa M. Malmgren A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Roman Manetsch, Ph.D. Edward Turos, Ph.D. Mark McLaughlin, Ph.D. Hong-Gang Wang, Ph.D. Date of Approval: November 9, 2007 Keywords: target guided synthesis, fragment based synthesis, NMR, triazole, azide Copyright 2007, Lisa M. Malmgren

PAGE 2

DEDICATION I would like to dedicate my thesis to my father, Mr. Ronald Malmgren, in appreciation for all of his hard work throughout life and yes dad, I am so excited. I also would like to mention my mother Mrs. Charlene Malmgren for the motivation she has given me. I would also like to thank my sister, Annie Voils, and my brother, Brandon Malmgren for making me laugh and to cheer me up in all of the rough times and always being there for me. Without the support of my family, I could of never reached this point in my life.

PAGE 3

ACKNOWLEDGEMENTS I would first like to acknowledge Roman Manetsch, Ph.D., my principle investigator for all of his support and guidance throughout my research. I would also like to thank my committee members Edward Turos, Ph.D., Mark McLaughlin, Ph.D., and Hong-Gang Wang, Ph.D. for all of their direction throughout this endeavor. It is my pleasure to acknowledge my lab mates Arun Babu Kumar, Shikha Mahajan, Richard Cross, Katya Nacheva, Sameer S. Kulkarni, and Kurt Van Horn for all of the thoughtful discussions. I could not have completed all of the work without the assistance of our one and only postdoctoral researcher, Dr. Xiangdong Hu. I would also like to mention the undergraduate researchers who helped me with the synthesis: Brandi Pennock, Matthew Kaplan, and Alexandra Athan. I would like to thank Ted Gauthier, Ph.D. for his assistance in the instrumentation lab as well as Edwin Rivera, Ph.D. and William Tay for their assistance and training with the NMR along with Ivan PerezSanchez, Ph.D. for the guidance with the interpretation of the NMRs. I appreciate the supply of protein from Sun Jiazhi (George), Ph.D. and the use of the microwave reactor from Nick Lawrence, Ph.D. and Harshani Lawrence, Ph.D.

PAGE 4

i TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi LIST OF SCHEMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii LIST OF ABBREVATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x Chapter One: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Introduction to Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Fragment-Based Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2.1 Non-Enzyme Templated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2.1.1 Functional Assay Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2.1.2 NMR-Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.2.1.3 X-ray Crystallography Screening . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.2.1.4 Mass Spectrometry Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.2.2 Enzyme Templated Fragment-Based Screening . . . . . . . . . . . . . . . 5 1.2.2.1 Introduction to Target-Guided Synthesis . . . . . . . . . . . . . . . . . . . 5 1.2.2.2 Dynamic Combinatorial Chemistry . . . . . . . . . . . . . . . . . . . . . . . 6 1.2.2.3 Catalyst-Supported Target-Accelerated Assembly . . . . . . . . . . . . 7 1.2.2.4. Kinetically Controlled Target-Guided Synthesis . . . . . . . . . . . . . 8 1.2.2.4.a In situ Click Chemistry Proof of Concept Acetylcholinesterase (AChE) . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 1.2.2.4.b Carbonic Anhydrase . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1.2.2.4.c HIV-1 Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.3. Protein-Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 1.4 B-Cell Lymphoma-2: Bcl-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 1.4.1 Bcl-X L Inhibitor ABT-737 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Chapter Two: 1 H-NMR Kinetic Study Comparing the Reaction Rates of Activated and Non-activated Acetylenes in the Cycloaddition with Azides to form Triazoles . . . . 21 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.2 Kinetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

PAGE 5

ii Chapter Three: Synthesis of Alkyne Anchor Molecules and Azide Building Blocks for the In Situ Bcl-X L Protein-Templated Cycloaddition of syn and anti Triazoles . . . 31 3.1 Design and Synthesis of Building Block Reagents: Sulfonylacetamide . . . . 31 3.2 Design and Synthesis of Building Block Reagents: Propynamide and Acetylenecarboxylate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.3 Design and Synthesis of Building Block Reagents: Azides . . . . . . . . . . . . . 40 3.5 Incubations With Bcl-X L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Chapter Four: Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 List of References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Appendix A: Spectra of Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

PAGE 6

iii LIST OF TABLES Table 2.1 Conditions and Yields for Triazoles in Scheme 2.3 . . . . . . . . . . . . . . . . . . . . 27 Table 2.2 Percent Triazole Conversion after 15 Days . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Table 3.1 General Procedure for the Synthesis of Aromatic Azides AZ1-AZ6 . . . . . . . 41 Table 3.2 General Procedure and Yields for Benzylic Azides AZ7-AZ10 . . . . . . . . . . . 43 Table 3.3 Azides AZ11-AZ14 Prepared Under TBAAz . . . . . . . . . . . . . . . . . . . . . . . . 44

PAGE 7

iv LIST OF FIGURES Figure 1.1 Dynamic Combinatorial Chemistry Using bCAII . . . . . . . . . . . . . . . . . . . . . . 7 Figure 1.2 Catalyst-Supported Target-Accelerated Assembly Using bCAII . . . . . . . . . . 8 Figure 1.3 Synthetic Routes for Syn and Anti -Triazoles . . . . . . . . . . . . . . . . . . . . . . . . 10 Figure 1.4 In situ Click Chemistry Using AChE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Figure 1.5 In situ Click Chemistry Using bCAII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Figure 1.6 In situ Click Chemistry Using HIV-1 Protease . . . . . . . . . . . . . . . . . . . . . . . 14 Figure 1.7 Protein-Protein Interactions Regulating Apoptosis . . . . . . . . . . . . . . . . . . . . 16 Figure 1.8 Development of ABT-737 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Figure 1.9 ABT-737 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Figure 2.1 Binding Site of Bcl-X L Occupied by ABT-737 or Azide and Alkyne Building Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Figure 2.2 NOE Comparison for Compound 6 and 7 . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Figure 2.3 Time-Product Profile for 2.0 M Ester 1 and Ether 2 with Azide AZ7 . . . . . . 29 Figure 3.1 Final Sulfonamide as Michael Type Acceptor . . . . . . . . . . . . . . . . . . . . . . . 34 Figure 3.2 Modification of Acetylene Anchor Molecules . . . . . . . . . . . . . . . . . . . . . . . 35 Figure 3.3 Binary In Situ Click Chemistry Incubations . . . . . . . . . . . . . . . . . . . . . . . . . 46 Figure 3.4 Triazole Hits from Bcl-XL Templated Reactions . . . . . . . . . . . . . . . . . . . . . 46 Figure 3.5 NOE for Compound 34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Figure 3.5 In Situ Hit Identification of AM3 and AZ1 by LC/MS-SIM . . . . . . . . . . . . . 48

PAGE 8

v Figure 3.6 In Situ Hit Identification of AM3 and AZ12 by LC/MS-SIM. . . . . . . . . . . . 49 Figure 3.7 NOE for Compound 34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Figure A1 1 H NMR and 13 C NMR of Compound 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Figure A2 1 H NMR of Compound 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Figure A3 1 H NMR and 13 C NMR of Compound 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Figure A4 1 H NMR and 13 C NMR of Compound 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Figure A5 1 H NMR and 13 C NMR of Compound 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Figure A6 1 H NMR of Compound 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Figure A7 1 H NMR of Compound 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Figure A8 1 H NMR of Compound 10 and 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Figure A9 1 H NMR of Compound 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Figure A10 1 H NMR of Compound 13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Figure A11 1 H NMR and 13 C NMR of Compound 17 . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Figure A12 1 H NMR and 13 C NMR of Compound 22 . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Figure A13 1 H NMR of Compound 24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Figure A14 1 H NMR of Compound 25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Figure A15 1 H NMR and 13 C NMR of Compound 26 . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Figure A16 1 H NMR and 13 C NMR of Compound 27 . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Figure A17 1 H NMR and 13 C NMR of Compound 28 . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Figure A18 1 H NMR and 13 C NMR of Compound AM3 . . . . . . . . . . . . . . . . . . . . . . . 96 Figure A19 1 H NMR and 13 C NMR of Compound AM4 . . . . . . . . . . . . . . . . . . . . . . . . 97 Figure A20 1 H NMR of Compound 34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

PAGE 9

vi Figure A21 1 H NMR of Compound 35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Figure A22 1 H NMR Compound AZ1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Figure A23 1 H NMR and 13 C NMR of Compound AZ2 . . . . . . . . . . . . . . . . . . . . . . . 100 Figure A24 1 H NMR and 13 C NMR of Compound AZ3 . . . . . . . . . . . . . . . . . . . . . . . 101 Figure A25 1 H NMR and 13 C NMR of Compound AZ4 . . . . . . . . . . . . . . . . . . . . . . . 102 Figure A26 1 H NMR Compound AZ5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Figure A27 1 H NMR Compound AZ6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Figure A28 1 H NMR Compound AZ7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Figure A29 1 H NMR Compound AZ8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Figure A30 1 H NMR Compound AZ9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Figure A31 1 H NMR of Compound AZ10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Figure A32 1 H NMR and 13 C NMR of Compound AZ11 . . . . . . . . . . . . . . . . . . . . . . 108 Figure A33 1 H NMR and 13 C NMR of Compound AZ12 . . . . . . . . . . . . . . . . . . . . . . 109 Figure A34 1 H NMR and 13 C NMR of Compound AZ13 . . . . . . . . . . . . . . . . . . . . . . 110 Figure A35 1 H NMR and 13 C NMR of Compound AZ14 . . . . . . . . . . . . . . . . . . . . . . 111

PAGE 10

vii LIST OF SCHEMES Scheme 2.1 Alkynes and Azide Used for 1 H NMR Kinetic Experiments . . . . . . . . . . . . 24 Scheme 2.2 Synthesis of Alkynes Used for 1 H NMR Kinetic Experiments . . . . . . . . . . 25 Scheme 2.3 Synthesis of Syn and Anti -Triazoles (Products of Kinetic Experiments) . . 26 Scheme 3.1 Initial Sulfonylacetamide Attempts Using DMTMM . . . . . . . . . . . . . . . . . 32 Scheme 3.2 Additional Sulfonylacetamide Coupling Attempts . . . . . . . . . . . . . . . . . . . 33 Scheme 3.3 Synthesis of Amine 25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Scheme 3.4 Attempted Synthesis of Azide 27 to be Reduced to Amine 21 . . . . . . . . . . 36 Scheme 3.5 Initial Benzylic Amine Synthesis Attempts . . . . . . . . . . . . . . . . . . . . . . . . 37 Scheme 3.6 Final Attempts Toward Synthesis of Amine 21 . . . . . . . . . . . . . . . . . . . . . 39 Scheme 3.7 Synthesis of Anchor Acetylenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Scheme 3.8 Coupling of Acid AZ3 to Secondary Amines . . . . . . . . . . . . . . . . . . . . . . . 42 Scheme 3.9 General Procedure for Preparation of Azido Amides . . . . . . . . . . . . . . . . . 43 Scheme 3.10 Synthesis of Hit Compound 35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

PAGE 11

viii LIST OF ABBREVIATIONS HTS high-throughput screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 MS mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 NMR nuclear magnetic resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 FBS target-guided synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 HSQC heteronuclear single quantum correlation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 ESI-MS electrospray ionization mass spectrometery . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 DCC dynamic combinatorial chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 BCAII bovine Carbonic Anhydrases II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 AChE acetylcholinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 LC/MS-SIM mass spectrometry in the select ion mode . . . . . . . . . . . . . . . . . . . . . . . . . 11 BSA bovine serum albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 PPIM protein-protein interaction modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Bcl-2 B-cell lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 BH Bcl-2 homology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 HAS human serum albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 DMTMM 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchlorine . . . . . 23 1 H NMR proton nuclear magnetic resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 HMBC heteronuclear multiple bond correlation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 NOE nuclear Overhauser effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

PAGE 12

ix CG gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 HPLC high pressure liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide . . . . . . . . . . . . . . . . . . . . . . 32 TLC thin layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 DCM dichloromethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 THF tetrahydrofuran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 DCC N,N'dicyclohexylcarbodiimide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 HOBT 1-hyroxybenzotriazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 TABAZz tetrabutylammonium azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 13 C NMR carbon nuclear magnetic resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 R f retention factor for chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 J coupling constant for NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

PAGE 13

x Using In Situ Click Chemistry to Modulate Protein-Protein Interactions: Bcl-X L as a Case Study Lisa M. Malmgren ABSTRACT Protein-protein interactions are central to most biological processes. Although in the field of drug discovery there is a great interest in targeting protein-protein interactions, the discovery and development of small-molecules, which effect these interactions has been challenging. The purpose of this project is to determine if in situ click chemistry is a practical approach towards testing whether Bcl-X L is capable of assembling its own inhibitory compounds. Abbott laboratories developed compound ABT-737, which binds with high affinity (K i 1 nM) to the binding sites of Bcl-X L 36 Based on ABT-737, two acetylene anchor molecules AM3 and AM4 have been synthesized. These anchor molecules are distinguished by the reactivity of the their carbon-carbon triple bond. Compound AM3 contains an electron withdrawing carbonyl in the -position to the acetylene resulting in an activating effect towards the [1,3]-dipolar cycloaddition compared to compound AM4 To determine the reactivity of the activated system, 1 H-NMR kinetic studies were performed to compare the relative rates of these two systems by reacting model alkynes 1 2 3 and 4 with azide AZ7 It was shown that the activated systems, 1 and 3 produce triazoles in an accelerated rate compared to the unactivated systems 2 and 3 To test for the self-assembly of inhibitory triazoles, the acetylenes AM3 and AM4 were incubated with Bcl-X L and 14 azide building blocks ( AZ1 AZ12 ) for 12 hours at

PAGE 14

xi 37 C. Subjecting these mixtures to LC/MS-SIM led to the discovery of two hit compounds, 35 and 36 of which 35 has been chemically synthesized confirming the hit. Future work includes the synthesis of all hit compounds. Since hit triazoles can be syn or anti both need to be synthesized for each hit to investigate which regioisomer BclX L generates. Tests to confirm if hit compounds are actually modulating Bcl-X L activity will be done using conventional bio-assays. This will validate that Bcl-X L is capable of assembling its own inhibitor via the in situ click chemistry approach to drug discovery.

PAGE 15

1 Chapter One: Introduction 1.1 Introduction to Drug Discovery Drug discovery today most often begins with the identification of a possible biological target, which is a protein that is known to be the major contributor to a disease. After a target has been discovered, it is then analyzed and validated to ensure that modulation of its activity by a drug enhances the therapeutic effects. Traditionally, the compounds that are likely to interact with the target protein are then synthesized, one by one, and tested for activity via high-throughput screening (HTS). When comparing the number of library members screened to the number of leads, the hit rates are usually low. 1,2 The few lead compounds are then structurally optimized to increase the biological activity. In the last decade, the traditional way of lead discovery approaches has seen improvements in methods for producing, handling, and screening large libraries of compounds. 3 Although improvements are in place there are still challenges of synthesis and purification, which are labor intensive and rather difficult. A recent study showed that the U.S. biomedical research funding rose from $48 to $94 billion over ten years starting in the early nineties. However, the number of drug candidates submitted to the FDA has decreased in comparison to the increase in investment. 3 Within a typical combinatorial library, over 99% of the compounds are inactive, therefore methods for producing only the compounds showing biological activity are highly desired. 4 Traditionally, lead-discovery has been aimed at preparing and screening large libraries of compounds in the molecular weight range of 250-600 Da and HTS hits commonly display affinities to the target protein in the low M to high nM range corresponding to 6.8-9.5 kcal/mol of binding energy. 2,5,6 Commonly, the large libraries of compounds fall short of probing the potential chemical diversity space. In the last years, various lead discovery approaches have been studied, in which libraries of small

PAGE 16

2 molecules (fragments) are screened for activity. Subsequently, the hit fragments are then converted into leads by merging, linking, and building additional fragments onto them. 5 These fragment based approaches allow for a more through sampling of the diversity space than traditional HTS by testing N 2 possibilities with N+N combinations. Theoretically, a collection of 10 4 fragments can probe a chemical diversity space of 10 8 molecules. 7 Fragment-based lead discovery entails the synthesis of libraries of lower molecular weight compounds compared to that of traditional combinational chemistry and parallel synthesis. These compound libraries contain molecules in the molecular weight of 120-250 Da, have less functionality, and are typically weak inhibitors ( MmM). Fragments are initially selected due to their ability to bind to the protein target or to inhibit the protein in a functional assay. Fragments with relevant binding properties are identified and expanded or combined to create new lead compounds for further drug discovery efforts. 7 Techniques for screening fragment-based libraries can be divided into two categories. The first entails detecting fragments and combining them by conventional ways. Several methods of detection include functional assay screening, nuclear magnetic resonance (NMR), X-ray crystallography, and mass spectrometry (MS). 5 The second category of fragment based library screening includes methods in which the protein target is directly involved in the synthesis of their potent inhibitor. Methods in this category include dynamic combinatorial chemistry and kinetically controlled target-guided synthesis (TGS).

PAGE 17

3 1.2 Fragment-Based Screening 1.2.1 Non-Enzyme Templated 1.2.1.1 Functional Assay Screening Functional assay screening differentiates the biological mediated reaction into several steps to obtain the hit compounds. First the libraries of building blocks are screened with the protein to determine which bind with a higher affinity. Next, those that bind with the highest affinity are then combined through conventional synthetic methods. Finally, the newly synthesized library of bidentant molecules is then screened with the protein using the same functional assay. Generally, fragments bind to the target with low affinity (mM range) and they are rather difficult to detect in traditional HTS assays. One way to overcome this issue is to increase the concentrations of the fragments for the assay. The Ellman group screened a library of fragments at 1mM concentration in a functional assay to detect inhibitors for the important oncogenic protein kinase c-Src. They were able to detect different fragments with inhibitory effects and synthetically linked them together to produce larger bidentant compounds. This newly synthesized library was then re-screened to identify inhibitors of c-Src in the nanomolar range. 8 There are several advantages of the functional screening method because the fragments are smaller. Also since fewer molecules are screened, there is less synthesis and characterization required compared to the traditional HTS. Prior knowledge of the protein structure, mechanism of action, or other inhibitory leads is not necessary required for this method.

PAGE 18

4 1.2.1.2 NMR-Screening One way to identify the binding properties of the fragments is by screening the libraries by NMR. This method is also called SAR-by-NMR, since quite often the structure-activity relationships can be determined. This method gives structuredetermination of the fragments that bind to the active site of the protein. SAR-by-NMR is done by screening the fragment library with 15 N-labeled biological target. Prior to any screening, the assignment of the amino acid backbone of the peptide must be determined, which takes a significant amount of time. The hit is detected by interpreting the spectra of the heteronuclear single quantum correlation (HSQC) experiment for changes in the amide chemical shifts at the binding site, once the compound binds to the labeled protein. 5 Therefore, SAR-by-NMR can detect fragments that bind with low affinity to the target, thus giving valuable information of where within the binding site the fragment has bound. This facilitates and accelerates structure-guided synthesis of inhibitory compounds consisting of various fragments. 2 This method is limited by compound solubility and the ability of the proteins to withstand being subjected to NMR techniques. 7 Other drawbacks include access to a high-field NMR spectrometer, significant amounts of 15 N-labeled protein, and the size of the protein (which can not be too large). 5 1.2.1.3 X-ray Crystallography Screening Like NMR, x-ray crystallography can detect fragments that bind with weak affinity and aid the optimization of hit compounds by providing a structural understanding about the binding site of the protein. Once an X-ray of the bound fragment

PAGE 19

5 with the protein is obtained, three-dimensional-structure guided optimization can begin. Solving crystal structures becomes a relatively high-throughput screening method due to advancements in robotics, X-ray technology, and computing power. 5 Although screening using X-ray has become high-throughput (500-1000 compounds per screen) it is significantly less high-throughput than other methods such as the standard high concentration functional bioassays (10-50K) and NMR (1-10K). 2 1.2.1.4 Mass Spectrometry Screening Ibis Therapeutics has developed a detection method to screen fragment libraries of low affinity compounds (millimolar range) which bind to the active site of bacterial 23S ribosomal RNA ( rRNA) using electrospray ionization mass spectrometery (ESIMS). 9 This technique allows for the identification of hit compounds due to the detection of the [M+H] + [M+NH 4 ] + [M-H] + and [ M+Na] + ions. This method is high-throughput by means of screening up to 1-10K compounds per screen. 1.2.2 Enzyme Templated Fragment-Based Screening 1.2.2.1 Introduction to Target-Guided Synthesis Although there has been a recent increase of fragment-based lead discovery methods, one, which is promising to deviate from the conventional lead identification process, is target-guided synthesis. This method of lead discovery deviates from traditional lead-discovery due to the fact that the preparation and screening of the ligand against a particular target is combined into one step, as the target itself chooses the lead compound. Thus, the biological target, a protein or DNA fragment, is actively involved in synthesizing its own inhibitory molecules. The biological target is incubated with a small library of low-molecular weight building blocks and when the target holds the two with the highest affinity in the correct alignment, the covalent bond formation is accelerated creating the inhibitory bidentant ligand. Hit identification can be as simple as

PAGE 20

6 screening the mixture to determine whether a given combination of building blocks have produced a product. This relatively new methodology of lead discovery has the potential to create a shortcut in the traditional drug discovery methods by combining synthesis and screening of libraries of low-molecular weight compounds in a single step. 10 Target-guided synthesis can be divided into three different approaches: (1) dynamic combinatorial chemistry; (2) catalyst-supported target-accelerated assembly; and (3) kinetically controlled target-guided synthesis, which are described below. 1.2.2.2 Dynamic Combinatorial Chemistry Dynamic combinatorial chemistry (DCC) uses the target proteins self-assembly processes to create libraries of chemical compounds. DCC takes advantage of the use of reversible reactions, covalent or non-covalent, by generating a random library of divalent molecules that are in dynamic equilibrium. 11 Addition of a target protein or receptor creates a driving force shifting the equilibrium towards the library members showing the highest affinity. 12 Thus, DCC allows for the target driven amplification of the active constituent(s) of the entire library, hence performing a self-screening process in which the high affinity products are expressed and retrieved from the library. 12 The processes of DCC approach include: (1) preparation of a variety of building blocks, which can reversibly react; (2) introducing a template which will result in an amplification of the best binder and (3) isolation or re-synthesis of the best inhibitor(s). 13 The selection of the building blocks must oblige to some requirements including the ability to cover as completely as possible the geometrical and functional space of the target sites, and reversibility of the linkage between the building blocks. 12 Common noncovalent exchanges between building blocks include hydrogen bonding and metal-ligand coordination. Although these interactions are usually fast and proceed under mild conditions, the isolation of the amplified product(s) is problematic due to the labile connections between the building blocks. 13 The use of covalent reactions can help reduce the associated problems. Common reactions used in DCC include trans-esterification, 14 olefin metathesis, 14 and disulfide exchange. 13

PAGE 21

7 Huc and Lehn showed that bCAII is able to synthesize its own inhibitory compounds by reversibly sampling a variety of amine and aldehyde building blocks which form imines through condensation reactions. This reversible reaction takes place in or near physiological conditions and allows the system to equilibrate in the presence of the target protein resulting in the best binding inhibitor. Once the imines are formed, they can be irreversibly reduced by hydride reduction using NaBH 3 CN which freezes the equilibrium generating stable amine compounds. This allows for the isolation and analysis of the final equilibrated mixture. 15 R O H S H 2 N O O + H 3 N R 1 ) b C A I I 2 ) N a B H 3 C N S H 2 N O O 4 a m i n e b u i l d i n g b l o c k s 3 a l d e h y d e b u i l d i n g b l o c k s N H 2 ( K d = 1 1 n M ) Figure 1.1 Dynamic Combinatorial Chemistry Using bCAII. Incubation of 3 aldehydes with 4 amines with bCAII resulted in 12 imines which were reduced with NaBH 3 CN to the corresponding amine. Treatment of the incubation samples with the reducing agent is necessary in order to freeze the equilibrium prior to HPLC analysis. 1.2.2.3 Catalyst-Supported Target-Accelerated Assembly Poulsen and Bornaghi showed that by using olefin cross metathesis (Grubbs catalyst), bovine Carbonic Anhydrases (bCAII) can selectively bind and assemble its own inhibitory compound from a mixture of ten olefin building blocks. (Figure 1.1) When the aromatic sulfonamide is used as an anchor molecule, it selectively chooses the terminal acetate to undergo cross metathesis resulting in an inhibitory olefin displaying a K i of 4.9

PAGE 22

8 nM. Thus, by reversibly sampling a variety of building blocks, bCAII was able to bind those with the highest affinity and create its own inhibitor. 16 Vancomycin is an antibiotic that has been used for the last 40 years to treat infections caused by Gram-positive bacteria including Staphylococcus aureus Nicolaou and co-workers were able to apply target-accelerated combinatorial synthesis to identify vancomycin ligands which form dimers when incubated with the target protein, Ac-DAla-D-Ala or Ac 2 -L-Lys-D-Ala-D-Ala. By utilizing olefin metathesis or disulfide bond formation reactions, the enzyme was able to accelerate the formation of covalent heterovancomycin dimers resulting in a vancomycin derivative with inhanced antibiotic properties. 17 S O O H 2 N O O + R S O O H 2 N O O O O K i = 4 9 n M G r u b b s C a t a l y s t b C A I I L i b r a r y o f 1 0 t e r m i n a l a l k e n e s Figure 1.2 Catalyst-Supported Target-Accelerated Assembly Using bCAII. Incubation of the sulfonamine anchor molecule with 10 terminal alkynes with bCAII and Grubbs catalyst, olefin metathesis resulted in the trans olefin with a K i = 4.9nM 1.2.2.4 Kinetically Controlled Target-Guided Synthesis Kinetically controlled TGS has been developed in the last 10 years and it slightly differs from DCC by allowing the protein to select its own building blocks that bind with the highest affinity to the active site. The protein samples the various pairs of building blocks reactants until it facilitates an irreversible reaction, which covalently connects the pair that binds with the highest affinity. 4 Although TGS is a novel methodology for the synthesis of lead compounds, it is limited by the use of highly reactive reagents such as strong electrophiles or nucleophiles and metal catalysts, which can sometimes harm the enzyme. A newly developed variant

PAGE 23

9 of kinetically controlled target-guided synthesis, termed in situ click chemistry, uses reactants that are bio-orthogonal and are thus tolerated by biological systems. As to date, the only example of in situ click chemistry utilitized in such applications is the Huisgen [1,3]-dipolar cycloaddition to irreversibly connect acetylene and azide building blocks all within the enzymes active site. This reaction produces fivemembered heterocycles, 1,2,3-triazoles, which are stable to acidic and basic hydrolysis as well as reductive/oxidative conditions. 18 Azides are perfect for click chemistry due to the resistance to H 2 O, O 2 and a majority of conditions used in organic chemistry. 19 The thermal triazole-forming reaction produces a mixture of 1,4and 1,5disubstituted regioisomers as shown in Figure 1.3 However, there are synthetic conditions available to obtain controlled regioselectivity of the disubstituted triazoles.The copper (I) catalyzed triazole formation produces only the 1,4-disubstituted 1,2,3-triazoles (anti-triazole) where as the magnesium acetylides reaction 20 and the ruthenium catalyzed reaction 21 result in the 1,5-disubsituted 1,2,3-triazoles (syn-triazole). The [1,3]-dipolar cycloaddition is an excellent candidate for TGS for a variety of reasons. Although there is a large thermodynamic driving force for the cycloaddition (>50 kcal/mol), the thermal reaction is slow at room temperature with a high activation barrier of approximately 25 kcal/mol. This is the feature that allows the enzyme to selectively build the best inhibitor by sampling the various pairs of building blocks until it binds the two with the highest affinity. These are the building blocks which potentially overcome the energy barrier to undergo the cycloaddition. Another reason that favors this cycloaddition is that it does not involve components that might disrupt the enzymatic binding sites (for example no catalysts or external reagents). The reaction does not produce any byproducts, which in theory may disrupt the protein-mediated reaction. Once formed, the triazole can actively participate in hydrogen bonding in addition to dipole-dipole and # # stacking. 18

PAGE 24

10 R N 3 + R N e a t N N N N N N R R R R + A n t i S y n C o p p e r c a t a l y s t N N N R R A n t i G r i g n a r d o r R u t h e n i u m c a t a l y s t N N N R R S y n Figure 1.3 Synthetic Routes for Syn and Anti -Triazoles. Triazole synthesis to obtain 1:1 equimolar synand antitriazoles (neat), syntriazoles (Gringard reagent or Ruthenium catalyst), and antitriazoles (Copper catalyst). 1.2.2.4.a In situ Click Chemistry Proof of Concept Acetylcholinesterase (AChE) The in situ click chemistry proof of concept experiments were done using acetylcholinesterase (AChE) as the first biological target to explore the use of the Huisgen cycloaddition. AChE is an important enzyme in the central and peripheral nervous system catalyzing the hydrolysis of the neurotransmitter acetylcholine. 22 AChE has two binding sites: the catalytic binding site is located at the end of a 20 narrow gorge whereas the second peripheral binding site is located at the top of the gorge, near the protein surface. 10 The experiments entailed using 8 tacrine reagents (active site ligand) of both azides and acetylenes to bind at the catalytic site as anchor molecules. An anchor molecule is a compound that once bound to the enzyme recruits other building

PAGE 25

11 blocks of the complementary functionality to undergo the irreversible cycloaddition to form a new, multivalent inhibitor. In addition to the 8 anchor molecules and 50 propidium mimics again both azides and acetylenes were synthesized to bind to the peripheral binding site. 23 For concept validation, the anchor molecules (20 M) were incubated as binary mixtures with each of the propidium mimics with eel or mouse AChE (1 M) at pH 7.4 for at least 6 hours. 23 Monitoring the reaction mixture using liquid chromatography combined with mass spectrometry in the select ion mode (LC/MS-SIM) allowed the identification of the mass peak of the resulting product. 10 LC/MS-SIM is a sensitive detection method that has been proven to be a reliable method for the detection of the trace amounts of hit compounds. The use of a highly sensitive instrument is essential due to the low amount of triazole formed in the enzyme-templated reaction. This technique is performed by comparing the SIM of the potential product from the proteintemplated reaction to that of the background reaction without the protein. When there is an increase of product in the in situ reaction compared to the background reaction, it can be considered as a potential hit. This method allows for the identification of the triazoles not only by molecular weight but also by retention times resulting in the regioisomer assignment of the triazole formed. Of the resulting mixtures, eight in situ hits were discovered with subpicomolar affinities and were validated by showing that no triazoles were formed when the building blocks were incubated without AChE or when AChE was replaced with bovine serum albumin ( BSA) 23 (Figure 1.4). The in situ hits were assigned to the correct regioisomer by comparing retention times of the incubation samples with the retention times of synthetically prepared compounds.

PAGE 26

12 N N H X N N H + R N R X 5 6 N N N N R R 5 6 ( 4 0 ) ( 2 0 ) A C h E ( m o u s e o r e e l ) 3 7 o C 1 2 h 8 t a c r i n e b u i l d i n g b l o c k s 5 0 p e r i p h e r a l b i n d i n g b u i l d i n g b l o c k s X = N 3 o r 8 h i t c o m p o u n d s ( K d = 3 3 f M t o 3 0 p M ) C H C Figure 1.4 In situ Click Chemistry Using AChE. Incubation of 8 tacrine with 50 propidium building blocks with AChE (mouse or eel) resulted in 8 hit compounds showing very strong inhibitory properties. 1.2.2.4.b Carbonic Anhydrase Carbonic Anhydrases (CA) are zinc-containing enzymes that catalyze the dehydration of bicarbonate and the hydration of carbon dioxide. They are central to many biological processes including the transport of CO 2 /HCO 3 respiration, bone resorption, calcification, acid secretion, and pH control. 24 S H 2 N O O N 3 R S H 2 N O O N N N R b o v i n e c a r b o n i c a n h y d r a s e I I a q b u f f e r p H 7 4 3 7 o C 4 0 h r s + ( K d = 3 7 n M ) 4 4 a z i d e b u i l d i n g b l o c k s 1 2 h i t c o m p o u n d s ( K d = 0 2 n M ) Figure 1.5 In situ Click Chemistry Using bCAII. Acetylene benzene sulfonamide and 44 azide building blocks were incubated with bCAII resulting in 12 hit compounds.

PAGE 27

13 Inhibitors of CA coordinate to the Zn 2+ ion within the active site. Para-substituted aromatic and heteroaromatic sulfonamides have been shown to be potent inhibitory compounds due to the ability of the sulfonamide to bind to the Zn 2+ center. 24,25, Mocharla and co-workers were able to show that CA is capable of assembling inhibitors by incubating the protein with acetylene benzene sulfonamide and a library totaling 44 azide building blocks (Figure 1.5). 27 LC/MS-SIM screening of the building blocks with bovine CA II (bCAII) led to a total of 12 antitriazole inhibitors (K d = 0.2-7.1nM). 28 1.2.2.4.c HIV-1 Protease HIV-1 protease (HIV-1-Pr) is an important target for the inhibition of viral replication thus, protease inhibitors that are effective against the wild-type and the emerging mutants is highly desirable. 18 In situ click chemistry was used to probe the enzyme-templated reaction to determine if this method could validate new in situ hits. Whiting and co-workers showed that incubation of an azide prepared from known inhibitors with a library of alkynes in the presence of HIV-1 protease (SF-2) resulted in the formation of the anti-substituted triazole (K i =1.7nM) (Figure 1.6). Binding studies showed that neither one of the building blocks used with HIV-1 protease showed high affinity to the active site such as the studies with AChE and bCAII (K i <37nM). However, it was found that the enzyme could selectively synthesize its own in situ inhibitor by saturating the active site through increasing the concentration of the reagents (200 M) while keeping the enzyme concentration low (15 M) in comparison to the reagents. The consequence of increasing the building block concentration was the increased rate of the background triazole reaction providing an approxiately 1:1 mixture of regioisomers. Still the enzyme templated reaction provided an enhanced regioselective ratio of 18:1 in favor of the anti-substituted triazole. 18

PAGE 28

14 O H N H O O O H N 3 N S O O M e O O H N N S O O M e O N N O O N H H O + S F 2 P r 0 1 M M E S 0 2 M N a C l ( a q ) 2 3 o C 2 4 h r s ( K i = 1 7 n M ) Figure 1.6 In situ Click Chemistry Using HIV-1 Protease. Neither azide and alkyne building blocks showed high affinity to the enzyme, resulting in incubations with building block concentrations at (200 M) and the enzyme at (15 M). However the inhibitory anti triazole was synthesized via the enzymetemplated reaction. 1.3 Protein-Protein Interactions Protein-protein interactions are central to most biological p rocesses including signal transduction, immune response, cell-cell recognition, cellular transcription and translation. Since protein-protein interactions are abundant in biological processes, they represent a large and important class of targets for human therapeutics. 29 Although in the field of drug discovery there is a great interest in targeting protein-protein interactions, the discovery and development of small-molecules, which effect these interactions, have been challenging. One challenge in particular is the size and shape of the protein-protein interface which is targeted by the small molecule. Approximately 750-1,500 2 of surface area is buried on either side of the interface 30 which exceeds the binding area required for small protein-protein interaction modulators (PPIM). X-ray structures of protein-protein pairs usually do not show the small, deep gorges resembling lowmolecular-weight binding sites. 29 Another challenge is that protein-protein interfaces are often flat lacking the potential binding sites such as small cavities or binding pockets for small molecules. Many protein-protein interfaces contain compact centralized regions of residues, also termed hot-spots, which are crucial for the affinity of the interactions. In crystal structures, hot-spots are found on both surfaces of the interacting proteins and seem to be

PAGE 29

15 complementary to each other. 29 The adaptive and flexible nature involved in hot spots interfaces creates difficulties in drug discovery since there might be binding-site conformations that can be occupied by a small molecule that is not visible in the crystal structure. 31,32,33 Thus, if protein-protein interface hot-spots are biologically designed for binding peptides and proteins, then these hot spots might be inherently well suited for the design of low-molecular-weight binding ligands to disrupt protein-protein interactions. 29 1.4 B-Cell Lymphoma-2: Bcl-2 Apoptosis is the natural programmed cellular death used to eliminate infected or damaged cells. The loss of control of apoptosis can lead to several abnormalities in cellular development, tissue homeostasis, and proper defense against pathogens. Once apoptosis becomes unregulated, it can promote serious conditions such as autoimmune and degenerative diseases as well as cancer. 34 The Bcl-2 (B-cell lymphoma) family of proteins composed of both pro-apoptotic and anti-apoptotic members play an important role in signaling the cellular death cascade. Members of this group contain at least one of four motifs known as Bcl-2 homology (BH) domains (BH1 to BH4). The proteins of the Bcl-2 family can be divided into two groups: anti-apoptotic (Bcl-2, Bcl-X L Bcl-w, Mcl-1, A1) and pro-apoptotic which itself is divided into two groups, Bax subfamily ( Bax, Bak, Bok) and the BH3-only proteins (Bad, Bid, Bim, Bik, and others) 35 Although most pro-apoptotic members contain BH1 and BH2 domains, those most similar to Bcl-2 contain all four BH domains. The Bax sub-family is very similar to Bcl-2 due to the presence of BH1, BH2, and BH3 domains where as the BH3-only proteins possess only the BH3 domain. Thus, the BH3 binding domain is essential for programmed cell death. 34

PAGE 30

16 Figure 1.7 Protein-Protein Interactions Regulating Apoptosis Bax and Bak (proapototic) directly mediate apoptosis and Bcl-2 family proteins (antiapoptotic) block the Bax/Bak activation. BH3-only proteins activate Bax/Bak mainly by binding to anti-apoptotic Bcl-2 proteins. This frees BH3-only proteins to activate Bak/Bax. The ratio of proto anti-apoptotic heterodimers is linked to cell survival. The top cartoon shows a healthy cell undergoing the natural cell death with the ratio of heterodimers to favor pro-apoptosis where as the bottom presents an over expression of Bcl-2 proteins resulting in favor of cell survival (anti-apoptosis).

PAGE 31

17 After the cellular death signal, pro-apoptotic proteins Bax (located in the cytosol) and Bak (mitochondria) form aggregates within the mitochondria outer membrane leading to the release of cytochrome c which triggers the mitochondrial apoptosis cascade. On the other hand, the anti-apoptotic Bcl-2 proteins (Bcl-2 and Bcl-X L ) inhibit the release of cytochrome c hence suppressing the apoptotic pathway by blocking Bak/Bax activation. These anti-apoptotic proteins can form heterodimers with the proapoptotic proteins of the Bcl-2 family and cell survival is correlated to the ratio of proto anti-apoptotic proteins. 36 (Figure 1.7) The three-dimensional structure of Bcl-X L along with mutagenesis experiments determined that the BH1, BH2, and BH3 domains have a large influence on the homoand hetero-dimerization regulating apoptosis. The BH1, BH2, and BH3 domains of BcLX L combine together to form an elongated hydrophobic groove in which the helical BH3 proteins can bind. 37a,b,38 Thus, the binding of a BH3 protein to the BcL-X L cleft may be responsible for all of the dimerization within the Bcl-2 protein family. The role of BcL-X L and Bcl-2 is to form heterodimers by sequestering the pro-apoptotic BH3-only family of proteins. 39 The BH3 proteins can act as either activators or sensitizers of apoptosis. The BH3 proteins can initiate apoptosis by activating pro-apoptotic Bcl-2 proteins or inhibiting the anti-apoptotic family proteins. The activating BH3 proteins including Bim, Bid, and Puma can directly activate Bax and Bak resulting in the induction of apoptosis. In contrast, other BH3 proteins such as Bad and Noxa, are called sensitizer proteins and are unable to bind to Bax and Bak, but rather they bind to the anti-apoptotic Bcl-2 family proteins. The result of this heterodimer interaction is that the activating proteins are then able to directly activate Bax and Bak. The design of a PPIM to imitate the role of the Bad-like protein-protein interaction with the BH3-binding grove of Bcl-X L is highly attractive 36 based on prior studies showing that Bcl-X L overexpression has a greater contribution to the progression of cancer compared to Bcl-2. 40

PAGE 32

18 1.4.1 Bcl-X L Inhibitor ABT-737 Researchers at Abbott laboratories screened a chemical library of low-molecular weight compounds using SAR-by-NMR and identified two molecules to bind to the BH3binding domain of Bcl-X L 4-Fluoro-biphenyl-4-carboxylic acid (A) (dissociation constant K d = 0.30 mM) and 5,6,7,8-tetrahydro-naphtalen-1-ol (B) (K d = 4.3 mM) were shown to bind to proximal binding sites within the BH3 binding groove (Figure 1.8). F O O H ( A ) K d = 0 3 0 m M S i t e 1 b i n d e r O H ( B ) K d = 4 3 m M S i t e 2 b i n d e r M o d i f i c a t i o n & L i n k a g e N O 2 H N S S H N O F O O ( C ) B c l X L b i n d e r ( K i = 3 6 n M ) S t r u c t u r e b a s e d s y n t h e s i s A B T 7 3 7 ( K i = < 1 n M ) Figure 1.8 Development of ABT-737. Compounds A and B bind to Bcl-X L with K d = 0.30 mM and 4.3 mM, respectively. Through synthetic modification and linkage, compound C was identified by structurebased design leading to ABT-737 with (K i 1 nM). Site 1 sits at the bottom of a deep, well-defined hydrophobic pocket formed by Tyr 101, Leu 108, Val 126, Phe 146 of the Bcl-X L binding cleft. This site is where two (Asp 83 and Leu 78) of the three most significant amino acid residues responsible for the high affinity of the BH3 domain of Bak dwell. It is this groove where the fluoric of compound A is shown to bind. Near the Arg 139 of Bcl-X L the carboxyl group of A was revealed to reside. The tetrahydronaphthalene derivative ( B ) binds to site 2, considered to be a hydrophobic region normally occupied by the third residue of Bak (Ile 85) when bound with Bcl-X L

PAGE 33

19 N N C l O H N S O O N O 2 N H S H N S i t e 1 S i t e 2 Figure 1.9 ABT-737. ABT-737 maintains high affinity (K i 1 nM) to the binding sites of Bcl-X L Bcl-2, and Bcl-w. Using SAR-by-NMR, the molecules were linked together by replacing the biphenyl carboxyl group with an acylsulfonamide while ensuring the correct position of the acidic proton. The tetrahydronaphthalene derivative was replaced with a 3-nitro-4 -(2phenylthioethyl)aminophenyl group which still occupies site 2 through intramolecular bent-back # # stacking. This newly designed compound (C) had an increased affinity (inhibitory constant K i =36 1.6nM). However binding studies with 1% human serum showed a decrease affinity by a factor of > 280 due to the tight binding to HAS-III. 41 The researchers then turned to structure-based synthesis to decrease the affinity to HSA while increasing that of Bcl-X L By adding polar groups to the molecule to occupy the aqueous, solvent exposed regions of Bcl-X L which correlate to the lipophilic regions of HSA, the molecule was able to maintain the affinity to the target protein while decreasing that to HSA. Two alterations were made including the addition of a basic 2-dimethylaminoethyl group attached to the 1-position of the thioethylamino group as well as replacement of the fluorophenyl group in site 1 with a piperazine derivative. The deep groove of site 1 in Bcl-X L contains additional space which is partially solvent exposed compared to HSA which is more buried by nonpolar residues. 36 This allowed for the attachment of a lipophilic 4-chlorobiphenyl group to the piperazine ring to yet increase the selective binding to Bcl-X L while rendering the binding to HAS impossible.

PAGE 34

20 It was shown that the newly designed compound, ABT-737, (Figure 1.9) binds with high affinity ( K i 1 nM) to the active sites of Bcl-X L Bcl-2, and Bcl-w. ABT-737 does not bind to the homologous proteins Bcl-B, Mcl-1, and A1 (K i = 0.46 0.11 M, > 1 M and > 1 M, respectively.) However, in the presence of 10% human serum, nanomolar activity was retained. Thus, ABT-737 was shown to show the same effect as the sensitizing Bad protein when tested in purified mitochondria.

PAGE 35

21 Chapter Two: 1 H-NMR Kinetic Study Comparing the Reaction Rates of Activated and Non-activated Acetylenes in the Cycloaddition with Azides to form Triazoles 2.1 Introduction The goal of this project is to investigate if Bcl-X L is capable of synthesizing its own multivalent inhibitory compound by in situ click chemistry. The development of protein-protein interaction modulators was based on low-molecular weight compounds that bind to the same binding sites as Asp 83, Leu 78, and Ile 85 residues of the proapoptotic Bak protein. Abbott laboratories developed compound ABT-737, which binds with high affinity ( K i 1 nM) to the binding sites of Bcl-X L 36 Thus, the project entailed synthesis of alkyne and azide building blocks that when incubated with Bcl-X L potentially form the triazole compounds through the [1,3]-dipolar cycloaddition. The building blocks were designed to position the triazole within the hydrophobic pocket (site 1) of the BH3 binding groove. In previous studies using AChE and bCAII to template the triazole formation the building blocks bind to the active site with relatively good affinities (K D = low M). For TGS, it is advantageous that the enzyme is capable of holding the building blocks in a defined geometry for sufficient time to allow the triazole formation to occur. Since the binding sites are not as well defined as the deep binding sites of AChE, it is anticipated that the building blocks will not bind to Bcl-X L with as high affinities and the proteinazide-acetylene complex will most likely display a shorter lifetime compared to the in situ click chemistry applications with AchE and bCAII. Therefore it is necessary to

PAGE 36

22 increase the reactivity of the building blocks to increase the rate of the [1,3]-dipolar cycloaddition (Figure 2.1). N N C l O N H S O O H N S H N H N S S N O 2 N O 2 N 3 0 3 R A B T 7 3 7 i n B c l X L b i n d i n g s i t e A l k y n e & a z i d e b u i l d i n g b l o c k s i n B c l X L b i n d i n g s i t e H N O O O Figure 2.1 Binding Site of Bcl-X L Occupied by ABT-737 or Azide and Alkyne Building Blocks. Binding pocket of Bcl-X L with inhibitory compound ABT-737 as well as the azide/acetylene building blocks occupying the same binding positions. In experiments done by Whiting and coworkers, it has been shown that neither one of the building blocks used with HIV-1 protease displayed good affinity to the active site. However, the enzyme selectively synthesize its own in situ inhibitor only by saturating the binding site by increasing the concentration of the building block reagents (200 M compared to 20 M for AChE and bCAII). The enzyme concentration for HIV

PAGE 37

23 1 was 15 M whereas for AChE and bCAII the concentration was 1 M. Consequently, the background reaction for HIV-1 significantly increases compared to the incubations with AChE and bCAII making the detection of hit compounds more difficult. Increasing the rate of the triazole formation would allow the building blocks to react even if they reside only for a short period of time within the active site of the protein. We hypothesize to enhance the building block reactivity by installation of an electron-withdrawing group in the -position to the triple bond. This suggests that the increased rate of reaction would allow for Bcl-X L to bind low affinity building blocks long enough to template the triazole formation. 2.2 Kinetic Studies In order to determine if the carbonyl functionality in the -position to the triple bond increases the rate of the [1,3]-dipolar cycloaddition reaction the rates of reactions for carbonyl and corresponding alkyl acetylenes were compared. Two sets of compounds were designed to test this hypothesis. The first set of compounds consists of ester 1 and ether 2 whereas the second set comprises amide 3 and amine 4 Our hypothesis is that the electron-poor alkynes 1 and 3 react faster compared to the unactivated alkynes 2 and 4 (Scheme 2.1). Compound 1 (ethyl propiolate) is commercially available and compound 2 was synthesized from butanol using sodium hydride and propargyl bromide (Scheme 2.2). 42 Compound 2 was purified by distillation. Compound 3 was prepared from N-methyl phenethylamine, 4-(4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chlorine (DMTMM) and propiolic acid. 43 DMTMM was synthesized as reported and its 1 H-NMR matched with literature values Compound 4 was prepared by simple alkylation of Nmethyl phenethylamine with propargyl bromide. 23

PAGE 38

24 Scheme 2.1 Alkynes and Azide Used for 1 H NMR Kinetic Experiments N O O O O N N 3 + + + + k 1 k 2 k 3 k 4 1 2 3 4 A Z 7 A Z 7 A Z 7 A Z 7 S y n a n d A n t i T r i a z o l e s H y p o t h e s i s : k 1 > k 2 & k 3 > k 4 Alkynes 1-4 were mixed with azide AZ7 in deuterated chloroform and the relative reaction rates k 1 -k 4 were by 1 H-NMR.

PAGE 39

25 Scheme 2.2 Synthesis of Alkynes Used for 1 H NMR Kinetic Experiments H N O H O D M T M M N O + N H B r + K 2 C O 3 N O O O H B r + N a H T H F O 1 2 3 4 A C N T H F r t 3 h r s 8 8 % r t 3 h r s 8 7 % r e f l u x o n 5 0 % Synthesis of alkynes 1-4 used for kinetic studies. All of the corresponding syn and anti triazoles were synthesized to allow for the identification of the products in the kinetic studies (Scheme 2.3). All of the reactions were done using benzyl azide, AZ7 which was synthesized from benzyl chloride with sodium azide. 44 Conditions and yields are presented in Table 2.1. Triazoles 6 and 7 were formed through a thermal reaction. The alkyne 1 was so reactive that the products were formed within ten minutes, resulting in a small explosion in the oven. The regioisomers 6 and 7 were assigned using NMR experiments (NOE and HMBC) to distinguish the protons on the triazole rings. The NOE experiment (Figure 2.2) showed the proton on the anti triazole 7 having a strong correlation when the benzylic protons were irradiated and vice versa. In comparison, the proton on the syn -triazole 6 did not show as strong of a correlation when the benzylic protons were irradiated.

PAGE 40

26 Scheme 2.3 Synthesis of Syn and Anti -Triazoles (Products of Kinetic Experiments) N O O O O N N 3 N 3 N 3 N 3 + + + + 1 2 3 4 O O N N N N N N O O + a O N N N N N N O + b o r c b b o r c N N N N N N N N + O O N N N N N N N N + 5 6 7 8 9 1 0 1 1 1 2 1 3 Conditions ( a ) neat, 80 C (equal molar syn and anti triazoles) ( b ) Copper (I) catalysis ( anti triazole) ( c ) ethyl magnesium chloride reaction ( syn -triazole ).

PAGE 41

27 Table 2.1 Conditions and Yields for Triazoles in Scheme 2.3 T r i a z o l e 6 7 8 9 1 0 + 1 1 1 2 1 3 C o n d i t i o n a a c b b c b 6 2 1 6 5 7 1 4 8 2 8 7 3 m i x t u r e Y i e l d % Conditions: (a) neat, 80 C (b) CuSO 4 H 2 O (5 mole %), LAscorbic Acid (30mole %), NaHCO 3 (30 mole %), tBuOH: H 2 O; rt, o.n. (c) EtMgCl,THF; reflux o.n. *mixture of syn and anti triazoles in 1:4 ratio. O O N N N N N N O O 8 1 3 5 9 2 7 9 6 5 5 7 W e a k N O E S t r o n g N O E Figure 2.2 NOE Comparison for Compound 6 and 7 For compound 6 NOE experiments show a strong correlation between the anti -triazole proton and the benzylic protons. In comparison, for the syntriazole 7 only a weak correlation between the syn -triazole proton and the benzylic protons were detected. Irradiation was performed at both the benzylic and triazole protons for both regioisomers. Values are reported in ppm.

PAGE 42

28 The syn -triazoles 8 and 12 were synthesized through the ethyl magnesium catalyzed reaction. 20 The copper (I) catalyst was used to prepare the anti triazoles 9 and 13 45 Both reactions are selective generating pure regioisomers as products. However, triazoles 10 and 11 were isolated as a mixture of 4:1 anti to syn and reported as such. In order to quantify to what extent the alkynes 1 4 differ in reactivity, attempts to determine the relative rates were undertaken. The progress of the reactions was monitored by GC-FID or HPLC-UV. Reactions at 2M alkyne and azide concentrations were incubated at 50 C for a period of several days. Aliquotes of the reaction mixtures were analyzed at intervals of 24 hours. As expected, the activated alkynes 1 and 3 displayed an increased reactivity compared to the unactivated compounds 2 and 4 Attempts to quantify the relative rates failed. Specifically, the detection and quantification of the small amounts of triazole products in the reactions of alkynes 2 and 4 were difficult due to the detection limits of the GC-FID and HPLC-UV. To overcome these limitations, it was decided to perform the reactions in NMR tubes and monitor the formation of triazoles and consumption of building blocks via 1 HNMR. Determination of the relative amounts was done by integrating the benzylic protons of the triazole and comparing it to the benzylic protons of the starting materials. Equal molar concentrations (2 M, 1 M, 0.5 M, and 0.25 M) of alkynes 1-4 and the prepared benzyl azide AZ7 were mixed in deuterated chloroform and the mixtures were kept at room temperature. The reactions were monitored over a period of five weeks. The 1 HNMR measurements were performed daily for the first ten days followed by 48 hour intervals for an additional ten days and then every 72 hours for the remaining two weeks. Due to the length of time of the experiments, the solvent line of the NMR tubes was noted and refilled prior to 1 H-NMR measurement. This maintained the initial concentrations for all of the reactions. For compound 3 there were problems associated with the solubility of triazoles 10 and 11 Especially for the reactions at the concentrations of 2 M, 1 M, and 0.5 M, which precipitated in the NMR tube. Hence it was difficult to obtain precise data with these mixtures. However, the products 10 and 11

PAGE 43

29 of the reaction at 0.25 M concentration remained in solution until the studies were complete. The time-product profile in Figure 2.3 sh ows the increase in concentration (%) of triazoles relative to time for the reaction using 2.0 M concentrations of ester 1 and ether 2 with azide AZ7 The half-life of ester 1 with AZ7 is shown to be 75 hours whereas the half-life of ether 2 and AZ7 can not be determined. It is shown that even after 350 hours, the triazole formation for the ether 2 and azide AZ7 is not more that 5 %. Table 2.2 shows a summary of the 1 H-NMR experiments after 15 days. For each experiment the percent of triazole is listed for the four concentrations (2 M, 1 M, 0.5 M, and 0.25 M). Amide 3 was not detectable for 2 M, 1 M, and 0.5 M due to the insolubility of the triazole products. The formation of triazoles for the reaction with ether 2 and amine 4 were not detectable for at concentrations of 1 M, 0.5 M, and 0.25 M. Figure 2.3 Time-Product Profile for 2.0 M Ester 1 and Ether 2 with Azide AZ7 The formation of triazoles for the reaction with ester 1 and azide AZ7 is depicted as solid circles whereas ether 2 and azide AZ7 is shown as white circles.

PAGE 44

30 Table 2.2 Percent Triazole Conversion after 15 Days 2 M 1 M 0 5 M 0 2 5 M E s t e r 1 E t h e r 2 A m i d e 3 A m i n e 4 3 3 4 8 8 0 6 5 1 2 4 2 n / d n / d n / d n / d n / d n / d n / d n / d n / d Not detectable (n/d) In conclusion it was shown that the activated alkynes 1 and 3 were faster in forming the triazoles than the nonactivated alkynes 2 and 4 When comparing the 0.25 M concentration for the two activated systems 1 and 3 the ester 1 is approximately three times faster (33 % conversion for 1 verses 12 % for 3 ).

PAGE 45

31 Chapter Three: Synthesis of Alkyne Anchor Molecules and Azide Building Blocks for the In Situ Bcl-X L Protein-Templated Cycloaddition of syn and anti -Triazoles 3.1 Design and Synthesis of Building Block Reagents: Sulfonylacetamide Our next experiments targeted the design and synthesis of building block reagents and to test these for Bcl-X L -templated triazole formation as shown in Figure 2.1. Based on the known inhibitor ABT-737 alkyne 18 was designed as an anchor molecule (Scheme 3.1) Our idea is that upon binding one of the alkynes to the protein, the protein-anchor molecule complex will recruit azide building blocks to undergo the [1,3]-dipolar cycloaddition resulting in triazole compounds. Based on previous experiments in synthesizing propynamides (Scheme 2.2), it was thought that installing a sulfonylacetamide in the same position would afford an anchor molecule 18 which is similar to one half of ABT-737. As shown earlier, similar to the synthesis of propynamide 3 compound 14 was reacted with commercially available propiolic acid using DMTMM, however compound 18 could not be isolated (Scheme 3.1) 43 Compound 14 was synthesized as reported and 1 H NMR matched with literature values. 36

PAGE 46

32 Scheme 3.1 Initial Sulfonylacetamide Attempts Using DMTMM H N O H O D M T M M T H F r t 3 h r 8 8 % N O + N N N N M e O M e O O M e C l D M T M M S N O 2 H N S N H 2 O O + O H O D M T M M T H F S N O 2 H N S H N O O O 3 1 4 1 8 Initial propynamides were synthesized using coupling conditions: DMTMM/THF, However, the sulfonylacetamide coupling attempts using DMTMM were unsuccessful. Owing to the failed results of the initial sulfonylacetamide coupling attempts using DMTMM, the attention was turned to using another coupling condition. However, attempts to couple sulfonamide 14 to propiolic acid (15) with 1-ethyl-3 -(3dimethylaminopropyl)carbodiimide (EDCI) and DMAP also failed. 36,43 The electron withdrawing nature of the sulfonylacetamide moiety most likely activates the acetylene for Michael additions. Alkynes 16 and 17 were designed to cause steric hindrance potentially decreasing the reactivity towards Michael additions. To prepare acid 17 ethynyldimethyl(phenyl)silane was added to a solution of ethyl magnesium chloride followed by passing CO 2 through the mixture to yield 17 in 58% yield. 46 The synthesis of 19 and 20 were attempted with both of the EDCI and DMTMM conditions, 36,43 however, no sulfonylacetamides 19 or 20 were obtained. The purification attempts for the sulfonylacetamide 18-20 were done using both silica and aluminum oxide (neutral) for the TLCs and columns.

PAGE 47

33 Scheme 3.2 Additional Sulfonylacetamide Coupling Attempts S N O 2 H N S N H 2 O O + O H O R S N O 2 H N S H N O O O R 1 5 R = H ; 1 6 R = C H 3 ; 1 7 R = S i ( C H 3 ) 2 P h R e a g e n t s a n d C o n d i t i o n s = 1 E D C I D M A P D C M r t o n 2 D M T M M T H F r t o n 1 4 1 8 R = H 1 9 R = C H 3 2 0 R = S i ( C H 3 ) 2 P h Further coupling conditions using bulkier acetylenes in attempts to decrease the susceptibility to Michael addition. Conditions included EDCI/DMAP and DMTMM. We decided to subject the mixture for high resolution mass (HRMS TOF) analysis specifically looking for the [M+H] + for the sulfonylacetamide 19 to which DMAP added in a Michael reaction. This was confirmed by HRMS (TOF) m/z 542.15319 of the product (Figure 3.1). The conclusion is that the reactivity of the sulfonylacetamides 18-20 goes beyond the synthetic challenge since such a reactive building block is not ideal for TGS. The key to TGS is using the biological target to template the triazole formation and if the acetylene is highly activated, such as in this case, introducing the acetylene to the target could result in other Michael type reactions before it even reaches the targeted proteins binding site.

PAGE 48

34 S N O 2 H N S H N O O O N N Figure 3.1 Final Sulfonylacetamide as Michael Acceptor. Final sulfonamide product with DMAP added in a Michael type addition. Product confirmed by HRMS (TOF) m/z 542.15319. 3.2 Design and Synthesis of Building Block Reagents: Propynamide and Acetylenecarboxylate Due to sulfonylacetamide activating the acetylene for Michael additions, the designs for the alkyne anchor molecules were changed. Attention was turned to the propynamide AM2 and ester AM3 The sulfonyl group in compound 14 would be replaced by the methylene to afford the corresponding amine 21 or alcohol 22 which could then be coupled to the series of acetylene acids to afford anchor molecules AM2

PAGE 49

35 and AM3 (Figure 3.2) Synthesis began with mesylating commercially available 4-fluoro3-nitrobenzyl alcohol with methanesulfonyl chloride to afford the corresponding 4fluoro-3-nitrobenzyl methanesulfonate 23 in 54% yield (Scheme 3.3). 47 Next, mesylate 23 was reacted with sodium azide to afford the benzylic azide 24 which was used without further purification. 47 In order to confirm that the azide moiety could be reduced to the amine in the presence of the nitro group, azide 24 was reduced to amine 25 using triphenyl phosphine. 48 S N O 2 H N S N H 2 O O 1 4 N O 2 H N S N H 2 N O 2 H N S O H 2 1 2 2 p r o p i o l i c a c i d S N O 2 H N S H N O O O N O 2 H N S H N O N O 2 H N S O O A M 2 A M 3 p r o p i o l i c a c i d p r o p i o l i c a c i d A M 1 Figure 3.2 Modification of Acetylene Anchor Molecules. Modification of 14 to 21 and 22 to be coupled to propiolic acid to obtain the acetylene anchor molecules AM1-AM3

PAGE 50

36 Scheme 3.3 Synthesis of Amine 25 F N O 2 O H M s C l T E A E t 2 O F N O 2 N 3 P P h 3 E t 2 O : E A ( 1 : 1 ) 5 % H C L 0 o C t o r t o n 3 7 % F N O 2 N H 2 2 4 2 5 0 o C t o r t o n 5 4 % F N O 2 O S O O N a N 3 H 2 O D M F r t o n ( u s e d w / o p u r i f i c a t i o n ) 2 3 Initial synthesis of amine 25 to be coupled to acids bearing acetylenes. Scheme 3.4 Attempted Synthesis of Azide 27 to be Reduced to Amine 21 F N O 2 N 3 S N H 2 + S H N N O 2 N 3 1 o r 2 2 4 2 6 2 7 N O 2 H N S N H 2 2 1 R e d u c t i o n Conditions for aromatic substitution: 1. DIPEA, DMSO; rt o.n .; 2. BuLi, THF; 0 o C to rt o.n. Once it was assured that the amine could be obtained, the focus shifted to the coupling of azide 24 with amine 26 to afford azide 27 which could then in turn be reduced to amine 21 (Scheme 3.4). 2-( Phenylthio) ethanamine) ( 26 ) was synthesized via a nucleophilic substitution reaction between thiophenol and 2-chloroethylamineHCL and was purified by distillation in 52% yield. 49 The initial coupling attempts of 24 and 26 were done with DIPEA as the base but no product was formed. Usage of the stronger base BuLi did not result in product formation and therefore the synthesis was redesigned. The idea was to use an aromatic carboxylic acid instead of the benzyl azide 24 for the aromatic substitution with 26 Compound 28 was obtained in 89% yield using compound 26 and the commercially available 4-fluoro-3-nitrobenzoic acid (Scheme 3.5) 50 This reaction was

PAGE 51

37 performed in a microwave reactor at 150 C for 15 min at 150W. Next the acid was reduced to obtain the alcohol 22 Initially the reaction was performed with sodium borohydride and borane trifluoride diethyl ether in diethylene glycol dimethyl ether (diglyme) 51 However, these conditions resulted in the reduction of the acid as well as the aromatic nitro group. Reduction using borane in THF afforded desired product 22 in 77% yield. 52 This reduction turned out to be very sensitive and best results have been obtained by keeping the reaction at room temperature with 12 equivalents of borane and the reaction was carefully monitored by TLC for the appearance of the side product (nitro reduced). Once the side product was observed, the reaction was immediately quenched and the unreacted acid 28 was recovered. Scheme 3.5 Initial Benzylic Amine Synthesis Attempts. S H N H C H 2 C H 2 C l H C l S K 2 C O 3 D C M 4 8 h r s r t 5 2 % F N O 2 O H O S K 2 C O 3 H 2 0 M i c r o w a v e 2 0 m i n 1 5 0 o C 2 5 W 8 9 % N H 2 B H 3 T H F T H F r t N H O 2 N O H O S N H O 2 N O H M s C l T E A D C M S N H O 2 N O M s N a N 3 H 2 O D M F r t o n 2 1 % S N H O 2 N N 3 P P h 3 E t 2 O : E A ( 1 : 1 ) 5 % H C L 0 o C t o r t o n S N H O 2 N N H 2 1 5 t o 4 h r s 7 7 % 2 6 2 8 2 2 2 9 2 7 2 1 Next, alcohol 22 was mesylated with methanesulfonyl chloride to afford the corresponding mesylate 29 which was used without further purification. 47 It was found that the mesylate was hydrolyzing back to alcohol 22 during column chromatography. Mesylate 29 was reacted with sodium azide to afford the benzylic azide 27 in 21%

PAGE 52

38 yield. 47 The benzylic azide 27 was then subjected to reduction conditions using triphenylphosphine and 5% solution of 1N HCL. However, amine 21 could not be obtained. 49 Then it was thought that the sodium azide could directly replace the alcohol functionality in compound 22 in a mixture of carbon tetrachloride in DMF (1:4) at 90 C followed by the addition of triphenylphosphine to afford the corresponding amine 21 ( Scheme 3.6). 53 However, these conditions did not afford benzylic amine 21 Next, attempts were made by dissolving mesylate 29 in a solution of 7M ammonia in methanol resulting in the addition of methanol obtaining compound 30 rather than the amine 21 Since there was a replacement of the alcohol functionality, although with the O-methoxy, it was thought to change the solvent from methanol to THF. Ammonia was bubbled into a solution of mesylate 29 in THF at -50 C. The mixture was kept at -50 C for 45 minutes then allowed to warm to room temperature. However, the reaction failed at forming compound 21 Finally we attempted to synthesize amine 21 from amide 31 ( Scheme 3.6). Acid 28 was mixed with DCC and HOBT followed by the addition of ammonium hydroxide to afford the corresponding amide 31 in 14% yield but when treated with borane in THF, a wide range of side products were generated. 54

PAGE 53

39 Scheme 3.6 Final Attempts Toward Synthesis of Amine 21 S N H O 2 N O H N a N 3 P P h 3 C C l 4 : D M F ( 1 : 4 ) 9 0 o C 1 8 h r s S N H O 2 N N H 2 N H 3 T H F 5 0 o C t o r t 4 8 h r s S N H O 2 N O M s K I M e O H 7 M N H 3 i n M e O H ( 1 2 e q ) r t 4 h r s 9 0 % S N H O 2 N O 1 D C C H O B T T H F 0 o C 3 5 h r s 2 N H 4 O H 1 4 % S N H O 2 N N H 2 O B H 3 T H F T H F 2 4 h r s 0 o C t o r t 2 2 2 9 2 9 2 8 2 1 2 1 2 1 3 0 3 1 The focus of the synthesis was then turned to ester AM3 (Figure 3.2) in place of the amide AM2 Alcohol 22 was reacted with DCC but did not yield the corresponding acetylene. 55 Alcohol 22 was coupled to 2-butyonic acid 16 using DMTMM and Nmethylmorpholine to obtain the corresponding acetylene AM3 in 68% yield. 43 In addition, the corresponding alkyl acetylene could be synthesized to incubate with the azide building blocks. The acetylene AM4 was prepared in 17% by alkylating alcohol 22 using propargyl bromide and sodium hydride. 42

PAGE 54

40 Scheme 3.7 Synthesis of Anchor Acetylenes. S N H O 2 N O H + O H O T H F 0 o C D C C r t o n S N H O 2 N O O + O H O D M T M M N M e t h y l m o r p h o l i n e S N H O 2 N O O T H F r t o n 6 8 % B r N a H T H F o n r t S N H O 2 N O + 1 7 % 2 2 2 2 2 2 1 6 1 6 A M 3 A M 4 3.3 Design and Synthesis of Building Block Reagents: Azides With the anchor molecules AM3 and AM4 prepared, the focus was turned to synthesize the azide building blocks. The target azides AZ1-AZ6 were synthesized via a diazotization followed by substitution with sodium azide (Table 3.1) 56 The amide linkage of AZ4-AZ6 were synthesized by coupling acid AZ3 to the various secondary amines using EDCI (Scheme 3.8). 36

PAGE 55

41 Table 3.1 General Procedure for the Synthesis of Aromatic Azides AZ1-AZ6 N H 2 R N 3 R 1 N a N O 2 H C l ( 2 M ) 2 N a N 3 N a O A c U r e a H 2 O e t h e r 5 t o 0 o C C o m p o u n d R Y i e l d ( % ) O N O O A Z 2 A Z 4 O H O A Z 3 F A Z 1 8 6 4 3 8 5 N O N O N 6 2 5 8 A Z 5 A Z 6 4 8

PAGE 56

42 Scheme 3.8 Coupling of Acid AZ3 to Secondary Amines N 3 H O O D M A P E D C I D C M r t A r g o n N 3 N O R 1 R 2 + H N R 2 R 1 General coupling conditions for EDCI coupling of acid AZ3 to the various secondary amines AZ4-AZ6 Benzylic azides AZ7-AZ10 were prepared from the corresponding benzyl chlorides via a S N 2 reaction in the presence of sodium azide 44 (Table 3.2). AZ11 was prepared by reacting N-(3-bromopropyl)phthalimide with tetrabutylammonium azide (TBAAz) conditions. 57 AZ12-AZ14 were prepared from the corresponding anilines using chloroacetyl chloride followed by treatment with TBAAz to afford the desired azides (Scheme 3.9). 57

PAGE 57

43 Table 3.2 General Procedure and Yields for Benzylic Azides AZ7-AZ10 C o m p o u n d R Y i e l d ( % ) C l R N 3 R N a N 3 D M F H 2 O r t H F O O H A Z 7 A Z 8 A Z 9 7 1 8 3 5 7 N 3 A Z 1 0 9 1 Scheme 3.9 General Procedure for Preparation of Azido Amides R N H 2 K O H C h l o r o a c e t y l c h l o r i d e H 2 O : E t h y l A c e t a t e R H N O C l n T B A A Z T H F R H N O N 3 n 0 o C 3 0 m i n r t o n

PAGE 58

44 Table 3.3 Azides AZ11-AZ14 Prepared With TBAAz C o m p o u n d R Y i e l d ( % ) N O O N 3 H N O N 3 H N O N 3 H N O N 3 A Z 1 1 A Z 1 2 A Z 1 3 A Z 1 4 5 0 6 5 7 1 5 5

PAGE 59

45 3.5 Incubations With Bcl-X L The in situ click chemistry approach allows the enzyme to selectively build the best inhibitor by sampling the various pairs of building blocks until it facilitates an irreversible reaction which covalently connects the pair that binds with the highest affinity. 4 In order to determine if Bcl-X L is capable of synthesizing its own inhibitory triazole, incubations of Bcl-X L with the building blocks were done followed by LC/MSSIM analysis of the incubation mixtures. This method is ideal because it allows for identification of the triazoles by both molecular weight and retention time. Incubations with Bcl-X L were performed using anchor alkynes AM3 and AM4 The alkynes were incubated as binary mixtures with building block azides AZ1-AZ14 resulting in a total of 28 incubations. This, in theory, produces 56 triazole products since each incubation can possibly form 2 products ( syn or anti triazole). Incubation mixtures of acetylene and azide building blocks without Bcl-X L were also done as a control. The incubations were done in a sealed 96-well plated which was heated to 38.5 C for 12 hours. Subsequently each sample was subjected to LC/MS-SIM analysis to determine if the corresponding triazole products were formed in the presence of Bcl-X L

PAGE 60

46 S N H O 2 N O O S N H O 2 N O O R + N 3 R L i b r a r y o f 1 4 a z i d e b u i l d i n g b l o c k s ( 2 0 M ) ( 2 0 M ) B c l X L ( 2 0 M ) 3 8 5 o C 1 2 h r s N N N R R A M 3 A M 4 Figure 3.3 Binary In Situ Click Chemistry Incubations. Binary mixtures of alkyne AM3 or AM4 with 14 azide building blocks. Reagents were kept at 20 M whereas the protein, Bcl-X L was at 2.0 M. After heating the mixtures for 12 hours, the samples were analyzed by LC/MS-SIM for triazole formation. O O N O 2 N H S N N N F O O N O 2 N H S N N N O N H 3 5 3 6 Figure 3.4 Triazole hits from Bcl-X L Templated Reactions.

PAGE 61

47 Scheme 3.10 Synthesis of Hit Compound 35 N 3 F + O H O N e a t 9 0 o C o n 9 9 % N F N N O H O D M T M M N M e t h y l m o r p h o l i n e T H F r t o n 4 0 % O O N O 2 N H S N N N F 3 5 O H N O 2 N H S A Z 1 1 6 3 4 The incubations resulted in two potential hits, 35 and 36 which are shown in Figure 3.4. The incubations show that Bcl-X L was in fact able to synthesize its own inhibitory compounds as seen in Figure 3.5 and Figure 3.6. The top HP/LC-SIM trace in Figure 3.5 shows the incubation of AM3 and AZ1 without Bcl-X L resulting in a triazole peak ( 35 ) at 5.13 minutes with an integration area of 236 whereas the bottom trace shows the protein-templated reaction displaying the same triazole peak at 5.13 minutes with an integration area of 3,602. As for hit triazole 36 Figure 3.6 shows in the top trace the incubation of AM3 and AZ12 without Bcl-X L missing any triazole peaks. However, the bottom trace in Figure 3.6 shows the incubation of AM3 and AZ12 with Bcl-X L with a triazole peak ( 36 ) at 5.08 minutes with the integration area of 684. The increase in peak areas indicates that Bcl-X L is capable of synthesizing its own in situ inhibitory molecule. Compound 34 was synthesized through a thermal reaction of AZ1 and 2-butyonic acid ( 16) which afforded the anti triazole 34 in 99% yield. Triazole 34 was then coupled to alcohol 22 using DMTMM to afford the desired anti triazole 35 43 NOESY was used to confirm the regioisomer by irradiating the methyl hydrogen at 2.6 ppm and observing a correlation at 7.6ppm. (Figure 3.7)

PAGE 62

48 Figure 3.5 In Situ Hit Identification of AM3 and AZ1 by LC/MS-SIM Top trace: AM3 (20 M) and AZ1 (20 M) after incubation without Bcl-X L showing a small amount triazole 35 formation with the correct peak at 5.13 minutes ([M+H] + 480.1). Bottom trace: AM3 (20 M) and AZ1 (20 M) after incubation with Bcl-X L (2.0 M) clearly showing the triazole 35 peak at 5.13 minutes.

PAGE 63

49 Figure 3.6 In Situ Hit Identification of AM3 and AZ12 by LC/MS-SIM Top trace: AM3 (20 M) and AZ12 (20 M) after incubation without Bcl-X L showing no distinct triazole 36 peak at 5.08 minutes. Bottom trace: AM3 (20 M) and AZ12 (20 M) after incubation with Bcl-X L (2.0 M) showing the triazole 36 peak at 5.08 minutes ([M+H] + 582.6).

PAGE 64

50 N F N N C H 3 O H O 2 6 7 6 H S t r o n g N O E Figure 3.7 NOE for Compound 34. By irradiating the hydrogen on the methyl, it was confirmed that the regioisomer was the anti-triazole 3.6 Conclusion Protein-protein interactions are central to most biological processes. Although in the field of drug discovery there is a great interest in targeting protein-protein interactions. The discovery and development of smallmolecules which effect these interactions has been challenging. The purpose of this project is to determine if in situ click chemistry is a practical approach towards testing whether Bcl-X L is capable of assembling its own inhibitory compounds. Abbott laboratories developed compound ABT-737 which binds with high affinity (K i 1 nM) to the binding sites of Bcl-X L 36 Based on ABT-737, two acetylene anchor molecules AM3 and AM4 have been synthesized. These anchor molecules are distinguished by the reactivity of their carbon-carbon triple bond. Compound AM3 contains an electron withdrawing carbonyl in the -position to the acetylene resulting in an activating effect towards the [1,3]-dipolar cycloaddition compared to compound AM4 To determine the reactivity of the activated system, 1 H-NMR kinetic studies were performed to compare the relative rates of these two systems by reacting model alkynes 1 2 3 and 4 with azide AZ7 It was shown that the activated systems, 1 and 3 produces triazoles in an accelerated rate compared to the unactivated systems 2 and 3

PAGE 65

51 To test for the self-assembly of inhibitory triazoles, the acetylenes AM3 and AM4 were incubated with Bcl-X L and 14 azide building blocks ( AZ1 AZ12 ) for 12 hours at 37 C. Subjecting these mixtures to LC/MS-SIM lead to the discovery of two hit compounds, 35 and 36 of which one ( 35 ) has been chemically synthesized confirming the hit. Future work includes the synthesis of all hit compounds. Since hit triazoles can be syn or anti both need to be synthesized for each hit to investigate which regioisomer BclX L generates. Tests to confirm if hit compounds are actually modulating Bcl-X L activity will be done using conventional bio-assays. This will validate that Bcl-X L is capable of assembling its own inhibitor via the in situ click chemistry approach to drug discovery.

PAGE 66

52 Chapter Four: Experimental General: All reactions requiring an inert atmosphere were conducted under dry argon, and the glassware was flame dried. All common reagents and solvents were obtained from commercial suppliers and used without any further purification unless otherwise indicated. Solvents used for the reactions requiring an inert atmosphere were dried before their use by passing the degassed solvent through a column of activated alumina prior to use in reactions. Analytical TLC was performed on glass-backed plates coated with silica gel 60 with F 254 indicator or EMD 100DC-Pattern aluminum-backed aluminum oxide with 60 with F 254 indicator; the chromatograms were visualized under ultraviolet light and/or by staining with a Ce/Mo or ninhydrin (amines) bromo creol green (acids) reagent and subsequent heating. R f values are reported on silica gel. Flash column chromatography was carried out on EMD flash chromatography silica gel (40 m) or on ACROS aluminium oxide, activated, neutral (50-200 micron). Routine NMR measurements were recorded on Bruker DPX-250 or Varian INOVA-400 spectrometers. Chemical shifts are given in ppm relative to tetramethylsilane, which is used as an internal standard, and coupling constants ( J ) are reported in Hz. 1 H-NMR: splitting pattern abbreviations are: s, singlet; br s, broad singlet; d, doublet; br d, broad doublet; dd, double doublet; dt, double triplet; dq, double quartet; t, triplet; td, triple doublet; tt, triple triplet; q, quartet; quint., quintet; m, multiplet. 13 C-NMR: multiplicities were determined by DEPT, HMQC, HMBC NOE and NOESY experiments. Standard pulse sequences were employed for the DEPT experiments. Mass spectra was determined with a Agilent Technologies for low resolution mass spectra (LCMSD), and high resolution mass spectra (LC-MS-TOF), respectively; the intensities are reported as a percentage relative to the base peak after the corresponding m / z value. ESI Electron spray ionization technique was employed. Infrared spectra were obtained with

PAGE 67

53 Thermo Electron CorporationNicolet IR 100. Microwave reactions were done in a Biotage Initiator microwave reactor. In Situ Click Chemistry General Procedure for Binary Reagent Mixtures (Incubation) : There are two incubations f or each pair of anchor molecules AM3 and AM4 with 14 azide building blocks ( AZ1-AZ14 ), the first set as a control (without protein) and the second as the incubation with the protein. Acetylene AM3 (20 mM, 1 L) was added to phosphate buffer solution (98 L ; 58 mM Na 2 HPO 4 17 mM NaH 2 PO 4 68 mM NaCl, 1 mM NaN 3 pH=7.40) immediately followed by AZ1 (20 mM, 1 L) and were mixed and kept at 3 8.5 o C for 12 hours. The final concentrations were as followed: Acetylene anchor molecules and azide building blocks: 20 M. This set of binary mixtures was used as the control. The incubation was done same as the control with the exception of the addition of Bcl-X L (2 M). The final concentrations were as followed: Bcl-X L : (2 M), acetylene anchor molecules and azide building blocks: 20 M. The measurements were performed on Agilent 1100 series LC-MSD instrument to perform the LC/MS-SIM analysis. 1 ( p r o p 2 y n y l o x y ) b u t a n e ( 2 ) O The sodium hydride (60% in mineral oil) (4.37 g, 109.3 mmol, 1 eq) was washed with hexanes (2x20mL) to remove the mineral oil. Dry THF (50 mL) was added and 1-butanol (10.0 mL, 109.3 mmol, 1 eq) was added slowly and stirred for 10 min. The mixture was then heated to 50 C for 15 min. and then cooled back down to room temperature. This

PAGE 68

54 mixture was then slowly added to a second flask containing propargyl bromide (80% in toluene) (19.50 g, 131.1 mmol, 1.2 eq) dissolved in dry THF (60 mL). The resulting mixture was refluxed overnight and reaction complete by TLC. The mixture was diluted with Na 2 CO 3 (50 mL) and extracted with ether (3x100 mL) The combined organic layers were dried with Na 2 SO 4 The organic layer was removed under reduced vacuum. The crude was purified by distillation (61 C) to afford the corresponding alkyne as a clear oil in good yield (6.13 g, 54.6 mmol, 50%); 1 H NMR (250 MHz, CDCl 3 ): $ 0.93 (t, J = 7.3, 3H), 1.31-1.64 (m, 4H), 2.41 (t, J = 2.4, 1H), 3.52 (t, J = 6.5, 2H), 4.13 (d, 2H); 13 C NMR (63 MHz, CDCl 3 ): $ 13.6 (CH 3 ), 19.0 (CH 2 ), 31.3 (CH 2 ), 57.7 (CH 2 ), 69.6 (CH 2 ), 73.8 (CH), 79.7(C) ppm. N m e t h y l N p h e n e t h y l p r o p 2 y n a m i d e ( 3 ) N O To a solution of N-methyl phenethylamine (298 mg, 2.2 mmol, 1.1 eq) in dry THF (10 mL) propiolic acid (300 mg, 2.0 mmol, 1.0 eq) was added dropwise. After 10 min, DMTMM (610 mg, 2.2 mmol, 1.1 eq) was added and the solution was stirred for 4.5 hrs at rt. THF was removed under reduced vacuum and the resulting solution was dissolved in H 2 O ( 50 mL) and extracted with ethyl acetate (4x50 mL) The combined organic layers were dried with Na 2 SO 4 The organic layer was removed under reduced vacuum. The crude was purified by column chromatography on silica gel ( Hx:EtOAc, 1:1) to afford the corresponding alkyne as a clear oil with a brown tint in (330 mg, 1.76 mmol, 88%). Rf = 0.42 (Hx:EtOAc, 1:1); 1 H NMR (250 MHz, CDCl 3 ): $ 3.09 (s, 1H), 3.10 (s, 3H), 3.63 (t, J = 7.5, 2H), 3.80 (t, J = 7.6, 2H), 7.18-7.35 (m with s at 7.260, 13H); HRMS (TOF) m/z calcd (C 12 H 13 NO) 187.0997, found 188.1066 [M+H] +

PAGE 69

55 N m e t h y l N p h e n e t h y l p r o p 2 y n 1 a m i n e ( 4 ) N To a solution of N-methyl phenethylamine (269 mg, 1.99 mmol, 3 eq) in ACN (20 mL) a solution of K 2 CO 3 (275 mg, 1.99 mmol, 3 eq) dissolved in H 2 O (2 mL) was added followed by propargyl bromide (79.0 mg, 0.637 mmol, 1 eq). The mixture was left to stir at rt overnight. After 18hrs an additional 1eq of propargyl bromide was added and left to stir 3 hours. ACN was removed under reduced vacuum and redissolved in brine and extracted with EA (3x50 mL). The organic layers were combined and washed with H 2 O and dried with Na 2 SO 4 Solvent was removed under reduced pressure. The crude was purified by column chromatography on silica gel (Hx:EtOAc, 3:1) to afford the corresponding alkyne as a colorless oil in (280 mg, 1.61 mmol, 87%); Rf = 0.37 (Hx:EtOAc, 3:1); 1 H NMR (250 MHz, CDCl 3 ): $ 2.23 (t, J = 2.4, 1H), 2.38 (s, 3H), 2.652.72 (m, 2H), 2.75-2.82 (m, 2H), 3.41 (d, J = 2.4, 2H), 7.20-7.29 ppm (m, 5H); 13 C NMR (63 MHz, CDCl 3 ): $ 34.0 (CH 2 ), 41.6 (CH 3 ), 45.4 (CH 2 ), 57.2 (CH 2 ), 73.1 (C), 78.2 (CH), 125.9 (CH), 128.2 (CH), 128.5 (CH), 139.9 ppm (C); HRMS (TOF) m/z calcd (C 12 H 15 N) 173.1205, found 174.1289 [M+H] + e t h y l 1 b e n z y l 1 H 1 2 3 t r i a z o l e 5 c a r b o x y l a t e ( 6 ) a n d e t h y l 1 b e n z y l 1 H 1 2 3 t r i a z o l e 4 c a r b o x y l a t e ( 7 ) O O N N N N N N O O 6 7 Benzyl azide (563 mg, 4.23 mmol) and ethyl propiolate (415 mg, 4.23 mmol) were added neat to a tube and sealed. The mixture was placed in the oven at 60 C. After five minutes,

PAGE 70

56 a small blast was noticed and smoke was emitted from the oven. The tube was removed and dark crystals were noticed. The solution was dissolved in brine (100 mL) and extracted with ethyl acetate (3x75 mL). The organic layers were combined and dried with Na 2 SO 4 and condensed under reduced vacuum. The crude was purified by column chromatography on silica gel (Hx:EtOAc, 4:1 to 2:1) to afford the corresponding synand anti-triazoles in 6% and 21%, respectively. Syn-substituted triazole 6 : Light brown oil in (60 mg, 0.259 mmol, 6%) Rf = 0.44 (Hx:EtOAc, 2:1); 1 H NMR (250 MHz, CDCl 3 ): $ 1.35 (t, J = 7.1, 3H), 4.35 (q, J = 7.1, 2H), 5.92 (s, 2H), 7.24-7.35 (m, 5H), 8.13 ppm (s, 1H); 13 C NMR (63 MHz, CDCl 3 ): $ 14.0 (CH 3 ), 53.2 (CH 2 ), 61.6 (CH 2 ), 127.6 (CH), 127.9 (CH), 128.2 (CH), 128.6 (CH), 134.8 (C), 138.0 (CH), 158.2 ppm (C=O). HRMS (TOF) m/z calcd (C 12 H 13 N 3 O 2 ) 213.1008, found 232.1081 [M+H] + Antisubstituted triazole 7 : Light orange crystal solid in (202 mg, 0.873 mmol, 21%) Rf = 0.31 ( Hx:EtOAc, 2:1); 1 H NMR (250 MHz, CDCl 3 ): $ 1.38 (t, J = 7.1, 3H), 4.4 (q, J = 7.1, 2H), 5.57 (s, 2H), 7.29-7.41 (m, 5H), 7.96 ppm (s, 1H); 13 C NMR (63 MHz, CDCl 3 ): $ 14.0 (CH 3 ), 54.1 (CH 2 ), 61.1 (CH 2 ), 126.7 (CH), 128.0 (CH), 128.8 (CH), 129.0 (CH), 133.6 (CH), 140.3 (C), 160.4 ppm (C=0). HRMS (TOF) m/z calcd (C 12 H 13 N 3 O 2 ) 213.1008, found 232.1081 [M+H] +

PAGE 71

57 N ( ( 1 b e n z y l 1 H 1 2 3 t r i a z o l 4 y l ) m e t h y l ) N m e t h y l 2 p h e n y l e t h a n a m i n e ( 1 3 ) N N N N General Procedure for anti triazole: To a solution of benzyl azide (154 mg, 1.15 mmol, 1 eq) and N -methylN -phenethylprop-2-yn-1-amine (200 mg, 1.15 mmol, 1 eq) in a 1:1 mixture of tBuOH: H 2 O (2.3 mL each), CuSO 4 H 2 O (14.4 mg, 0.058 mmol ., 5 mol %) as a solution in 100 L was added via a micro pipette. The copper solution was made by adding 0.58 mM to 1 mL and using 100 L. To a solution of L-ascorbic acid (57.8 mg, 0.35 mmole, 30 mol %) in 0.75 mL H 2 O sodium bicarbonate (29.0 mg, 0.35 mmol, 30 mol %) was added. Upon addition, the solution fizzed. This mixture was added to the reaction flask and left to stir over night at room temperature. The reaction was complete by TLC so the reaction mixture was diluted with sat. sodium bicarbonate (100 mL) and rinsed with dichloromethane (3x50 mL). The organic layers were combined and dried with Na 2 SO 4 and condensed under reduced vacuum. The crude was purified by column chromatography on silica gel (DCM:MeOH, 95:1) to afford the corresponding antitriazole as a light brown solid in (352 mg, 1.15 mmol, 65%); Rf = 0.25 (DCM:MeOH, 95:5); 1 H NMR (250 MHz, CDCl 3 ): $ 2.33 (s, 3H), 2.59-2.65 (m, 2H), 2.75-2.81 (m, 2H), 3.72 (s, 2H), 5.48 (s, 2H),

PAGE 72

58 7.12-7.34 ppm (m, 11H); HRMS (TOF) m/z calcd (C 19 H 22 N 4 ) 306.1845, found 307.1921 [M+H] + 1 b e n z y l 4 ( b u t o x y m e t h y l ) 1 H 1 2 3 t r i a z o l e ( 9 ) N N N O Triazole 9 was prepared from azide AZ7 (163 mg, 1.22 mmol) and acetylene 2 (137 mg, 1.22 mmol) according to the procedure for the synthesis of triazole 13 Obtained as a brown oil in (212 mg, 0.865 mmol, 71%); Rf = 0.30 (Hx:EtOAc, 2:1); 1 H NMR (250 MHz, CDCl 3 ): $ 0.89 (t, J = 7.3, 3H), 1.30-1.41 (m, 2H), 1.50-1.61 (m, 2H), 3.50 (t, J = 6.6, 2H), 4.59 (s, 2H), 5.52 (s, 2H), 7.25-7.43 ppm (m, 6H). ESI calculated for (C 14 H 19 ON 3 ) 245.193, found 246.1 (M+H] + 1 b e n z y l N m e t h y l N p h e n e t h y l 1 H 1 2 3 t r i a z o l e 5 c a r b o x a m i d e ( 1 0 ) 1 b e n z y l N m e t h y l N p h e n e t h y l 1 H 1 2 3 t r i a z o l e 4 c a r b o x a m i d e ( 1 1 ) N N N N N N N N O O 1 0 1 1 Triazoles 10 and 11 were prepared from azide AZ7 (142 mg, 1.07 mmol) and acetylene 3 (200 mg, 1.07 mmol) according to the procedure for the synthesis of triazole 13 Obtained

PAGE 73

59 as a white solid in (250 mg, 0.781 mmol, 73%) Rf = 0.33 (Hx:EtOAc, 1:1); 1 H NMR (250 MHz, CDCl 3 ): $ 2.90-3.03 (m, 4H), 3.10 (s, 3H), 3.48 (s, 3H), 3.72 (t, J = 7.7, 2H), 4.20 (t, J = 7.5, 2H), 5.50 (s, 2H), 5.54 (s, 2H), 7.12-7.42 (m, 20H), 7.76, (s, 1H), 7.98ppm, (s, 1H); HRMS (TOF) m/z calcd (C 19 H 20 N 4 O) 320.1637, found 321.1722 [M+H] + N ( ( 1 b e n z y l 1 H 1 2 3 t r i a z o l 5 y l ) m e t h y l ) N m e t h y l 2 p h e n y l e t h a n a m i n e 1 2 N N N N General Procedure for syn triazole: To a flame dried flask, N -methylN -phenethylprop2-yn-1-amine (150 mg, 0.87 mmol, 1 eq) was dissolved in dry THF (0.6 mL). ethylmagnesium chloride (0.32 mL, 0.87 mmol, 1 eq) (in 25% THF) was added dropwise. The solution was heated to 50 C for 30 min. and cooled back down to room temperature. Benzyl azide (115 mg, 0.87 mmol, 1 eq) was added dropwise and the mixture was refluxed overnight. The reaction was complete by TLC and was diluted with 15mL sat. NH 4 CL and extracted with ethyl acetate (3x30 mL). The organic layers were combined and dried with Na 2 SO 4 and condensed under reduced vacuum. The crude was purified by column chromatography on silica gel ( Hx: EtOAc, 2:1) to afford the corresponding syn triazole in (140 mg, 0.239 mmol, 28%); Rf = 0.28 ( Hx:EtOAc, 2:1); 1 H NMR (250 MHz, CDCl 3 ): $ 2.30 (s, 3H), 2.51-2.58 (m, 2H), 2.70-2.75 (m, 2H), 3.32 (s, 2H), 5.36 (s, 2H), 6.99-7.51 ppm (m, 11H). ESI m/z calculated for (C 19 H 22 N 4 ) 306.1845, found 307.2 (M+H] +

PAGE 74

60 1 B e n z y l 5 ( b u t o x y m e t h y l ) 1 H 1 2 3 t r i a z o l e ( 8 ) O N N N Triazole 8 was prepared from azide AZ7 (238 mg, 1.78 mmol) and acetylene 2 (200 mg, 1.78 mmol) according to the procedure for the synthesis of triazole 12 Obtained as a light brown oil in (212 mg, 0.86 mmol, 48%); Rf = 0.33 (Hx:EtOAc, 2:1); 1 H NMR (250 MHz, CDCl 3 ): $ 0.89 (t, J = 7.2, 3H), 1.23-1.4 (m, 2H), 1.45-1.57 (m, 2H), 3.24 (t, J = 6.5, 2H), 4.37 (s, 2H), 5.62 (s, 2H), 7.17-7.38 (m, 5H), 7.62 ppm (s, 1). ESI calculated for (C 14 H 19 ON 3 ) 245.193, found 246.1 (M+H] + 3 ( d i m e t h y l ( p h e n y l ) s i l y l ) p r o p 2 y n o i c a c i d ( 1 7 ) S i O H O To a solution of ethyl magnesium chloride (4.23 g, 48.2 mmol) in 16 mL dry THF at 0 C, a solution of ethynyldimethyl(phenyl)silane (4.00 g, 25.0 mmol), in 8 mL dry THF was added and stirred for 2 hr at room temperature. The mixture was cooled to C and CO 2 was passed through for 30 min and left to warm to rt for 2 hours. HCL (1N, 40 mL) was added and extracted with hexanes, dried with Na 2 SO 4 and hexanes removed under reduced pressure to yield bright yellow oil in (2.95 g, 14.5 mmol, 58%); Rf = 0.23 (Hx:EtOAc, 1:1); 1 H NMR (250 MHz, CDCl 3 ): $ 0.51 (s, 6H), 7.39-7.44 (m, 2H), 7.59-7.63 (m, 2H); 13 C NMR (63 MHz, CDCl 3 ): $ -1.9 (CH 3 ), 95.0 (C), 95.1 (C), 128.1 (CH), 130.1 (CH), 133.6 (CH), 134.0 (C), 157.2 ppm (C); HRMS (TOF) m/z calcd (C 11 H 12 O 2 Si) 204.0607, found 227.0517 [M+Na] +

PAGE 75

61 ( 3 N i t r o 4 ( 2 ( p h e n y l t h i o ) e t h y l a m i n o ) p h e n y l ) m e t h a n o l ( 2 2 ) S N H O 2 N O H To a dry flask containing 3-nitro-4-(2-(phenylthio)ethylamino)benzoic acid (1.00 g, 3.14 mmol, 1 eq) 20mL of dry THF was added. Borane (80% in THF) (3.7 mL, 37.73 mmol, 12 eq) was added dropwise. After 4hr, THF was removed under reduced pressure and the residue was redissolved in H 2 O. K 2 CO 3 was added until neutral to pH paper. The mixture was extracted with DCM (3x50 mL)and combined organic layers were dried with Na 2 SO 4 and DCM was removed under reduced pressure. The crude was purified by column chromatography on silica gel (Hx:EtOAc, 1:1) to afford the corresponding alcohol as to yield a bright orange solid in (740 mg, 2.43 mmol, 77%); Rf = 0.43 (Hx:EtOAc, 1:1); 1 H NMR (250 MHz, CDCl 3 ): $ 2.31(br s, 1H), 3.18 (t, J = 6.7, 2H), 3.52 (apparent q, J = 6.4, 2H), 4.55 (s, 2H), 6.72 (d, J = 8.9, 1H), 7.23-7.43 (m, 6H), 8.08 (d, J =1.6, 1H), 8.26 (t, J =5.1, 1H); 13 C NMR (63 MHz, CDCl 3 ): $ 33.1 (CH 2 ), 41.8 (CH 2 ), 63.8 (CH 2 ), 113.8 (CH), 125.1 (CH), 126.9 (CH), 128.3(C), 129.0 (CH), 130.6 (CH), 131.3(C), 134.1(CH), 135.6(CH), 144.3(C). HRMS (TOF) m/z calcd (C 15 H 14 N 2 O 4 S) 318.0674, found 336.1083 [M+NH 4 ] + 4 f l u o r o 3 n i t r o b e n z y l m e t h a n e s u l f o n a t e ( 2 3 ) F N O 2 O S O O A solution of 4-fluoro-3-nitrobenzyl alcohol (1.00 g, 5.84 mmol, 1 eq) and triethylamine (2.46 mL, 17.53 mmol, 3 eq) dissolved in ethyl ether (40 mL) was cooled to 0 C.

PAGE 76

62 Methanesulfonyl chloride (1.00 g, 8.77 mmol, 1.5 eq) was added dropwise and the mixture was left to warm to room temperature overnight. The reaction was complete by TLC and was diluted with brine solution (100 mL) and extracted with ethyl ether (3x100 mL). The organic layers were combined and dried with MgSO 4 then condensed under reduced vacuum. The crude was used without further purification to afford the corresponding mesylate in (783 mg, 3.14 mmol, 53%); Rf = 0.45 (Hx:EtOAc, 1:1) 4 ( a z i d o m e t h y l ) 1 f l u o r o 2 n i t r o b e n z e n e ( 2 4 ) F N O 2 N 3 To a solution of 4-fluoro-3-nitrobenzyl methanesulfonate (750 mg, 2.81 mmol, 1eq) dissolved in DMF (20 mL) a solution of sodium azide (650 mg, 9.99 mmol, 3.56 eq) dissolved in H 2 O (4 mL) was added dropwise. The mixture was left to stir at room temperature over night. Ethyl acetate (100 mL) was added and extracted with ice water (3x25 mL). The organic layers were combined and dried with MgSO 4 then reduced under vacuum to yield a yellow oil in (539 mg, 2.75 mmol, 98%); Rf = 0.40 (Hx:EtOAc, 1:1); 1 H NMR (250 MHz, CDCl 3 ): $ 4.44 (s, 2H), 7.35 (d, J = 8.3, 1H), IR: broad stretch 2100 (N 3 ).

PAGE 77

63 ( 4 f l u o r o 3 n i t r o p h e n y l ) m e t h a n a m i n e ( 2 5 ) F N O 2 N H 2 A solution of 4-(azidomethyl)-1-fluoro-2-nitrobenzene (94 mg, 0.479 mmol, 1 eq) dissolved in Et 2 O: ethyl acetate (1:1, 1.0 mL each) was cooled to 0 C. HCL (5% 1N, 0.5 mL) was slowly added followed by addition of triphenyl phosphine (126 mg, 0.479 mmol) in portions over 15 min. The ice bath was removed and the reaction was left to stir at room temperature overnight. The reaction was complete by TLC and the organic layer was discarded. The aqueous layer was washed with dichloromethane (2x25 mL). The aqueous layer was then basified with 1M NaOH to pH=8 then washed with dichloromethane (3x50 mL). The organic layers were combined and dried with Na 2 SO 4 and condensed under reduced vacuum. The crude was purified by column chromatography on silica gel (Hx :EtOAc, 4:1) to afford the corresponding amine as a bright yellow solid in (30 mg, 0.176 mmol, 37%); Rf = 0.20 (Hx:EtOAc, 4:1); 1 H NMR (250 MHz, CDCl 3 ): Note: these values are approximates due to loss of file. $ 4.32 (s, 2H), 6.15 (br s, 2H), 6.88 (d, 1H) 7.40 (dd, 1H), 8.10 (d ,1H). 2 ( p h e n y l t h i o ) e t h a n a m i n e ( 2 6 ) S N H 2 To a flame dried flask, dry dichloromethane (40 mL) was added. Thiophenol (1.03 g, 10 mmol, 1 eq), 2-chloroethylamineHCL (1.51 g, 13 mmol, 1.3 eq), and K 2 CO 3 (4.15 g, 30

PAGE 78

64 mmol, 3 eq) were added. The mixture was stirred for 2 days and was complete by TLC. The mixture was diluted with 50 mL H 2 O and extracted with dichloromethane (4x50mL) The combined organic layers were dried with Na 2 SO 4 The organic layer was removed under reduced vacuum. The crude was purified by vacuum distillation (53 C) to afford the corresponding amine as a clear oil in (804 mg, 5.25 mmol, 52%); Rf = 0.30 (DCM:MeOH, 9:1); 1 H NMR (250 MHz, CDCl 3 ): $ 1.30 (s, 2H), 2.89-3.07 (m, 4H), 7.18-7.41 (m, 5H)ppm ; 13 C NMR (63 MHz, CDCl 3 ): $ 38.0 (CH 2 ), 40.8 (CH 2 ), 126.1 (C), 128.8 (CH), 129.6 (CH), 135.6 (C) ppm; HRMS (TOF) m/z calcd (C 8 H 11 NS) 153.0612, found 154.0692 [M+H] + 4 ( a z i d o m e t h y l ) 2 n i t r o N ( 2 ( p h e n y l t h i o ) e t h y l ) a n i l i n e ( 2 7 ) S N H O 2 N N 3 To a dried flask containing 3-nitro-4-(2-(phenylthio)ethylamino)benzyl methanesulfonate (1.04 g, 5.70 mmol, 1 eq) dissolved in DMF (16 mL) a solution of sodium azide (740 mg, 11.4 mmol, 2 eq) dissolved in 3 mL H 2 O was added dropwise. To facilitate the sodium azide to dissolve in the water, sonication was used. The reaction was stirred at room temperature overnight. The reaction mixture was diluted with H 2 O and was extracted with ethyl acetate (3x100 mL). Ice was added to maintain that the DMF remained in the organic layer. The combined organic layers were dried with Na 2 SO 4 and the organic layer was removed under reduced pressure. The crude was purified by column chromatography on silica gel (Hx:EtOAc, 5:1) to afford the corresponding azide as a n orange oil in (394 mg, 1.20 mmol, 21%); Rf = 0.37 (Hx:EtOAc, 5:1); 1 H NMR (250 MHz, CDCl 3 ): $ 3.15 (t, J = 6.8, 2H), 3.50 (apparent q, J = 6.4, 2H), 4.20 (s, 2H), 6.72 (d, J = 8.8, 2H), 7.20-7.39 (m, 6H), 8.07 (d, J = 2.0, 1H), 8.28 (br s, 1H); 13 C NMR (63 MHz, CDCl 3 ): $ 33.3 (CH 2 ), 42.0 (CH 2 ), 53.6 (CH 2 ), 114.4 (CH), 122.7 (C), 126.9 (CH), 127.1 (CH), 129.2 (CH), 130.9

PAGE 79

65 (CH), 131.7 (CH), 134.3 (C), 136.2 (C), 144.7 (C); HRMS (TOF) m/z calcd (C 15 H 15 N 5 O 2 S) 329.09465, found 330.10198 [M+H] + 3 N i t r o 4 ( 2 ( p h e n y l t h i o ) e t h y l a m i n o ) b e n z o i c a c i d ( 2 8 ) S N H O 2 N O H O To a microwave reaction vessel, 4-Fluoro-3-nitro-benzoic acid (350 mg, 1.89 mmol, 1 eq), 2-(phenylthio)ethanamine (319 mg, 2.08 mmol, 1.1 eq) were added to H 2 O (3 mL). Sodium bicarbonate (548 mg, 3.97 mmol, 2.1 eq) was added and the solution fizzed and became bright yellow. The tube was sealed and subjected to microwave at 150 C for 15 min at 150W. The solution was cooled and diluted with 1N HCL (50 mL) and extracted with ethyl acetate (3x50 mL). The combined organic layers were dried with Na 2 SO 4 The organic layer was removed under reduced vacuum. The crude was purified by column chromatography on silica gel (Hx:EtOAc, 9:1) to afford the corresponding acid as a bright yellow crystal (1.61 mg, 5.07 mmol, 89%); Rf = 0.23 ( Hx:EtOAc 9:1); 1 H NMR (250 MHz, CDCl 3 ): $ 3.13(t, J = 6.7 2H), 3.50 (apparent q, J = 6.3, 2H), 6.66 (d, J = 9.0, 1H), 7.13-7.34 (m, 6H), 7.94 ( dd, J = 9.0, 1.9, 1H), 8.49 (t, J = 5.3, 1H), 8.78 (d, J =1.9, 1H); 13 C NMR (63 MHz, CDCl 3 ): $ 32.9 (CH 2 ), 41.7 (CH 2 ), 112.9 (CH), 118.0 (C), 126.9 (CH), 128.9 (CH), 129.4 (CH), 130.6 (CH), 131.2 (C), 133.7 (C), 136.4 (C), 146.8 (C), 166.8 (C=O); HRMS (TOF) m/z calcd (C 15 H 14 N 2 O 4 S) 318.0674, found 318.0747 [M+H] + 3 N i t r o 4 ( 2 ( p h e n y l t h i o ) e t h y l a m i n o ) b e n z y l m e t h a n e s u l f o n a t e ( 2 9 ) S N H O 2 N O M s A solution of (3-nitro-4-(2-(phenylthio)ethylamino)phenyl)methanol (158 mg, 0.520 mmol, 1 eq) and triethylamine (0.219 mL, 1.56 mmol, 3 eq) dissolved in ethyl ether (4

PAGE 80

66 mL) was cooled to 0 C. Methanesulfonyl chloride (0.06 mL, 0.78 mmol, 1.5 eq) was added dropwise and the mixture was left to warm to room temperature overnight. The reaction was complete by TLC and was diluted with brine solution (50 mL) and extracted with ethyl ether (3x50 mL). The organic layers were combined and dried with MgSO 4 then condensed under reduced vaccum to afford the corresponding mesylate which was used without further purification. 3 N i t r o 4 ( 2 ( p h e n y l t h i o ) e t h y l a m i n o ) b e n z y l b u t 2 y n o a t e ( A M 3 ) S N H O 2 N O O To a dried flask under argon, (3-nitro-4-(2-( phenylthio)ethylamino)phenyl)methanol (75.0 mg, 0.25 mmol, 1 eq.), 2-butynoic acid (20.7 mg, 0.25 mmol, 1 eq.), and DMTMM (203 mg, 0.73 mmol, 3 eq) were dissolved in dry THF (5 mL). N-Methylmorpholine (0.081 mL, 0.73 mmol, 3 eq) was then added dropwise. The reaction was left to stir overnight at room temperature. The reaction was complete after 16hrs and poured into H 2 O (75 mL) and extracted into ethyl acetate (4x50 mL). The combined organic layers were dried with Na 2 SO 4 and condensed under reduced pressure. The crude was purified by column chromatography on silica gel ( Hx:EtOAc, 4:1) to afford the corresponding alkyne as an orange solid (68 mg, 0.212 mmol, 68%); Rf = 0.26 (Hx:EtOAc, 4:1); 1 H NMR (250 MHz, CDCl 3 ): $ 1.98 (s, 3H), 3.18 (t, J = 6.8, 2H), 3.54 (apparent q, J =6.8, 2H), 5.06 (s, 2H), 6.73 (d, J = 8.8, 1H), 7.28-7.43 (m, 6H), 8.19 (s, 1H), 8.30 ppm ( br s, 1H); 13 C NMR (63 MHz, CDCl 3 ): $ 3.8 (CH 3 ), 33.2 (CH 2 ), 41.9 (CH 2 ), 66.3 (CH 2 ), 72.1 (C), 86.3 (C), 113.9

PAGE 81

67 (CH), 122.4 (C), 127.1 (CH), 127.7 (CH), 129.1 (CH), 130.9 (CH), 131.7 (C), 134.1 (C), 136.8 (CH), 144.8 (C), 153.4 ppm (C). HRMS (TOF) m/z calcd (C 19 H 18 N 2 O 4 S) 370.0987, found 371.1062 [M+H] + 2 N i t r o N ( 2 ( p h e n y l t h i o ) e t h y l ) 4 ( ( p r o p 2 y n y l o x y ) m e t h y l ) a n i l i n e ( A M 4 ) S N H O 2 N O The sodium hydride (11.0 mg, 0.27 mmol, 1.1 eq) was washed with hexanes (2x20 mL) to remove the mineral oil. THF (3 mL) was added followed by a solution of alcohol (75 mg, 0.25 mmol, 1 eq) in THF. After 30 min, propargyl bromide (40.3 mg, 0.27 mmol, 30 L, 1.1 eq) was added dropwise. The reaction was left at room temperature overnight. The reaction was neutralized with sat. NH 4 Cl and THF was removed under reduced vacuum. The residue was redissolved H 2 O and washed with ethyl acetate (3x75 mL) and dried with Na 2 SO 4 The organic layer was removed under vacuum. The crude was purified by column chromatography on silica gel ( Hx:EtOAc, 4:1) to afford the corresponding alkyne as an orange oil in (14.0 mg, 0.043 mmol, 17%); Rf = 0.45 (Hx:EtOAc, 4:1); 1 H NMR (250 MHz, CDCl 3 ): $ 2.49 (t, J = 2.4, 1H), 2.98-3.25 (m, 2H), 3.63-3.97 (m, 2H), 4.16 (d, J = 2.4, 2H), 4.49 (s, 2H), 6.84 (d, J = 8.8, 1H), 7.50-7.64 (m, 6H), 8.11-8.14 ppm (m, 2H); 13 C NMR (63 MHz, CDCl 3 ): $ 35.8 (CH 2 ), 54.8 (CH 2 ), 57.2 (CH 2 ), 70.3 (CH 2 ), 75.0 (CH), 79.3 (C), 113.7 (CH), 123.8 (CH), 125.3 (C), 126.8 (CH), 129.5 (CH), 131.3 (CH), 132.0

PAGE 82

68 (C), 136.6 (CH), 142.8 (C), 144.1 ppm (C); HRMS (TOF) m/z calcd (C 18 H 18 N 2 O 3 S) 342.1038, found 360.1483 [M+NH 4 ] + 1 a z i d o 4 m e t h o x y b e n z e n e ( A Z 2 ) N 3 O General Procedure for Azide Synthesis from an Amine. Preparation of 1-azido-4methoxybenzene AZ2: To a solution of p-anisidine (777 mg, 6.31 mmol) in 2M hydrochloric acid (10 mL ) at 0 C, a solution of sodium nitrite (518 mg, 7.51 mmol 1.19 eq) dissolved in distilled water (2 mL ) at 0 C was added dropwise. Urea (62.9 mg, 1.01 mmol, 0.16 eq) was added, followed by the drop wise addition of a chilled solution of sodium acetate (1.48 g, 18.1 mmol, 2.87 eq) and sodium azide (816 mg, 12.6 mmol, 1.99 eq) in distilled water (10 mL). Ether (6 mL) was added, and the solution stirred over night at room temperature. The solution was diluted with ether (75 mL) and washed with 1M NaOH (2x50 mL) followed by H 2 O (1x75 mL). The organic layer was dried with Na 2 SO 4 and solvent removed under reduced vacuum. The crude was purified by column chromatography on silica gel (Hx) to afford the corresponding azide as a light brown crystal in 43% yield (402 mg, 2.70 mmol); Rf = 0.18 ( Hx); 1 H NMR (250 Hz, CDCl 3 ) $ 3.80 (s, 3H), 6.93 (q, J = 9.3, 4H); 13 C NMR (63 mHz) 55.5 (CH 3 ), 115.1 (CH), 119.9 (CH) ppm. 1 a z i d o 4 f l u o r o b e n z e n e ( A Z 1 ) N 3 F The preparation of AZ1 was accomplished via the same procedure used to prepare AZ2 (3.38 g, 30.4 mmol, 58%); Rf = 0.43 (Hx); 1 H NMR (250 Hz, CDCl 3 ) $ 6.95-7.09 (m,

PAGE 83

69 4H); 13 C NMR (63 mHz) 116.3 (CH), 116.7 (CH), 120.2 (CH), 120.3 (CH), 135.7 (C), 135.8 (C), 157.9 (C), 161.8 (C) ppm. 3 ( 4 a z i d o p h e n y l ) p r o p a n o i c a c i d ( A Z 3 ) N 3 O H O The preparation of AZ3 was accomplished via the same procedure used to prepare AZ2 with the exception of purification by column chromatography. Purification AZ3 was accomplished by diluting the reaction mixture with distilled water, and adding 1M NaOH (75 mL). The aqueous layer was extracted with ether (3x75 mL), and the ether layer was removed to obtain crude AZ3 in the form of pale yellow crystals (3.98 g, 20.8 mmol, 86%); Rf = 0.41 ( Hx:EtOAc, 1:1); 1 H NMR (250 Hz, CDCl 3 ) $ 2.67 (t, J = 7.6 Hz, 2H), 2.94 (t, J = 7.6 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H). 3 ( 4 a z i d o p h e n y l ) 1 m o r p h o l i n o p r o p a n 1 o n e ( A Z 4 ) N 3 N O O General coupling procedure for azide acid and amines. (AZ4): Under Argon, AZ3 (500 mg, 2.62 mmol) and morpholine (456 mg, 5.23 mmol, 2 eq), in the presence of EDCI (2.0 g, 10.5 mmol, 4 eq) and DMAP (320 mg, 2.62 mmol, 1 eq), were dissolved in DCM (50

PAGE 84

70 mL) at room temperature and stirred over night. Solution was extracted by washing with DCM and distilled water. The crude was purified by column chromatography on silica gel (Hx:EtOAc, 5:1) to afford the corresponding azide as orange crystals in (580 mg, 2.23 mmol, 85%); Rf = 0.47 (EtOAc); 1 H NMR (250 Hz, CDCl 3 ) $ 2.51 (t, J = 7.7, 2H), 2.87 (t, J = 7.6, 2H), 3.29 (t, J = 4.6, 2H), 3.47 (t, J = 4.8, 2H), 3.52 (s, 4H), 6.85 (d, J = 8.4, 2H), 7.12 (d, J = 8.4, 2H); 13 C NMR (63 mHz) 30.2 (CH 2 ), 34.2 (CH 2 ), 41.5 (CH 2 ), 45.5 (CH 2 ), 66.1 (CH 2 ), 66.4 (CH 2 ), 118.7 (CH), 129.4 (CH), 137.5 (C), 170.1 (C=O) ppm. ESI : Calculated (C 13 H 16 O 2 N 4 ) 260.29 found [M+H] + 261.1 3 ( 4 a z i d o p h e n y l ) N N d i m e t h y l p r o p a n a m i d e ( A Z 5 ) N 3 N O The preparation of AZ5 was accomplished via the same procedure used to prepare AZ4 The reaction yielded the corresponding amide as light orange crystals in (352 mg, 1.61 mmol, 62%); Rf = 0.38 (Hx:EtOAc, 5:1); 1 H NMR (250 Hz, CDCl 3 ) $ 2.57 (t, J = 7.7 Hz, 2H), 2.93 (t with s at 2.92 ppm, J = 7.7 Hz, 8H), 6.93 (d, J = 8.5 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H); 13 C NMR (63 mHz) 30.5 (CH 2 ), 35.0 (CH 2 ),118.9 (CH), 129.7 (CH), 137.7 (C), 138.2 (C), 171.8 (C=O) ppm. ESI : Calculated (C 11 H 14 ON 4 ) 218.23 found [M+H] + 219.1

PAGE 85

71 3 ( 4 a z i d o p h e n y l ) 1 ( 4 p h e n y l p i p e r a z i n 1 y l ) p r o p a n 1 o n e ( A Z 6 ) N 3 N O N The preparation of AZ6 was accomplished via the same procedure used to prepare AZ4 The reaction yielded thick, light orange crystals (848 mg, 2.53 mmol, 48%). Rf = 0.40 (Hx:EtOAc, 1:1; 1 H NMR (250 Hz, CDCl 3 ) $ 2.97 (t, J = 7.7 Hz, 2H), 3.28-3.43 (m, 6H), 3.87 (t, J = 2.6 Hz, 2H), 4.10 (t, J = 2.6 Hz, 2H), 7.21-7.29 (m, 5H), 7.53-7.64 (m, 4H); 13 C NMR (63 mHz) 30.6 (CH 2 ), 34.7 (CH 2 ), 41.4 (CH 2 ), 49.5 (CH 2 ), 116.6 (CH), 119.0 (CH), 120.6 (CH), 129.1 (CH), 129.7 (CH), 137.9 (C), 150.6 (C), 170.3 (C=O). ESI : Calculated (C 18 H 21 ON 5 ) 335.18 found [M+H] + 336.1. B e n z y l a z i d e ( 5 ) / ( A Z 7 ) N 3 General Procedure for Azides from Chlorides: Benzyl chloride (5.0 g, 39.5 mmol) and sodium azide (7.70 g, 119 mmol, 3 eq) in a solution of DMF (50 mL) and distilled water (30 mL) were stirred over night at room temperature. The solution was extracted with ether (2x100 mL) followed by H 2 O (1x100 mL). The organic layers were combined and dried with Na 2 SO 4 and solvent removed under reduced vacuum. The crude was purified by column chromatography on silica gel (Hx) to afford the corresponding azide as a clear, colorless oil in (3.4 g, 28.1 mmol, 71%); Rf = 0.4 (Hx); 1 H NMR (250 Hz, CDCl 3 ) $ 4.35 (s, 2H), 7.32-7.43 (m, 5H).

PAGE 86

72 1 ( a z i d o m e t h y l ) 4 f l u o r o b e n z e n e ( A Z 8 ) F N 3 To a solution of 4-fluorobenzyl chloride (1.00 g, 6.92 mmol., 1 eq) dissolved in DMSO (0.5M, 19.83 mL) a solution of sodium azide (674 mg, 10.4 mmol., 1.5 eq) dissolved in H 2 O (15 mL) was added dropwise. To facilitate the sodium azide to dissolve in the water, sonication was used. The reaction was stirred at 70 C overnight. The reaction mixture was diluted with H 2 O (75 mL) was extracted with ethyl acetate (3x75 mL). The combined organic layers were dried with Na 2 SO 4 and the organic layer was removed under reduced pressure. The crude was purified by column chromatography on silica gel ( Hx) to afford the corresponding azide as a clear oil (864 mg, 5.71 mmol, 83%); Rf = 0.30 (Hx); 1 H NMR (250 MHz, CDCl 3 ): $ 4.32 (s, 2H), 7.08 (t, J = 8.7, 2H), 7.27-7.33 (m, 2H). GC/MS: Calculated (C 7 H 6 N 4 F) 151.2 found [M] + 151. 4 ( a z i d o m e t h y l ) b e n z o i c a c i d ( A Z 9 ) N 3 H O O The preparation of AZ9 was accomplished via the same procedure used to prepare AZ7 (939 mg, 5.30 mmol, 57%); Rf = 0.47 (Hx:EtOAc, 3:2); 1 H NMR (250 MHz, CDCl 3 ): $ 4.45 (s, 2H), 7.44 (d, J = 8.1, 2H), 8.14 (d, J = 8.3, 2H); HRMS (TOF) m/z calcd (C 8 H 7 N 3 O 2 ) 177.05383, found 178.06214 [M+H] +

PAGE 87

73 1 ( a z i d o m e t h y l ) n a p h t h a l e n e ( A Z 1 0 ) N 3 The preparation of AZ10 was accomplished via the same procedure used to prepare AZ7 (2.87 g, 15.7 mmol, 91%); Rf = 0.22 (Hx:EtOAc, 3:1); 1 H NMR (250 MHz, CDCl 3 ): $ 4.78 (s, 2H), 7.42-8.06 (m, 7H). 2 ( 2 a z i d o e t h y l ) i s o i n d o l i n e 1 3 d i o n e ( A Z 1 1 ) N O O N 3 The preparation of AZ11 was accomplished via the same procedure used to prepare AZ7 (323 mg, 1.49 mmol, 95%); 1 H NMR (250 MHz, CDCl 3 ): $ 3.58 (t, J = 5.9, 2H), 3.90 (t, J = 5.9, 2H), 7.71-7.88 (m, 4H); 13 C NMR (63 MHz, CDCl 3 ): $ 36.8 (CH 2 ), 48.9 (CH 2 ), 123.4 (2xCH), 131.8 (2xC), 134.1 (2xCH), 167.9 (2xC); HRMS (TOF) m/z calcd (C 10 H 8 N 4 O 2 ) 216.06473, found 217.07174 [M+H] + 2 a z i d o N p h e n y l e t h a n a m i d e ( A Z 1 3 ) H N O N 3 General Procedure for Preparation of Azido Amides (AZ13): To a solution of analine (500 mg, 5.37 mmol, 1.2 eq) in H 2 O:EA (11 mL each, 1:1) KOH (904 mg, 16.11 mmol, 3.3 eq) was added and stirred vigorously. Chloroacetyl chloride (619 mg, 4.88 mmol, 1.0

PAGE 88

74 eq) was added dropwise over a period of 5 minutes. The reaction was left to stir at room temperature for 30 minutes. The mixture was bored into brine and extracted into ethyl acetate (3x50 mL). The organic layers were combined and dried with Na 2 SO 4 and condensed under reduced vacuum. The crude was purified by column chromatography on silica gel to afford the corresponding chloroacetanilide in (847 mg, 5.0 mmol, 94%). To a solution of chloroacetanilide (300 mg, 1.77 mmol) in THF (1 mL) TBAAz (2M solution in THF) (756 mg, 2.66 mmol) was added and left to stir overnight at room temperature. The crude was purified by column chromatography on silica gel (Hx: EtOAc, 1:1) to afford the corresponding azide in (220 mg, 1.25 mmol, 71%); 1 H NMR (250 MHz, CDCl 3 ): $ 4.16 (s, 2H), 7.13-7.56 (m, 6H), 7.98 ( br s, 1H); 13 C NMR (63 MHz, CDCl 3 ): $ 52.8 (CH 2 ), 120.0 (CH), 124.9 (CH), 129.0 (CH), 136.6 (C), 164.5 (C); HRMS (TOF) m/z calcd (C 8 H 8 N 4 O) 176.06981, found 177.07683 [M+H] + 2 a z i d o N ( n a p h t h a l e n 1 y l ) e t h a n a m i d e ( A Z 1 2 ) H N O N 3 The preparation of AZ12 was accomplished via the same procedure used to prepare AZ13 Obtained in 55% yield. 1 H NMR (250 MHz, CDCl 3 ): $ 4.32 (s, 2H), 7.45-8.03 (m, 7H), 8.54 ( br s, 1H); 13 C NMR (63 MHz, CDCl 3 ): $ 53.2 (CH 2 ), 120.1 (CH), 120.4 (CH), 125.6 (CH), 126.1 (CH), 126.2 (CH), 126.5 (CH), 128.8 (CH), 131.0 (C), 134.0 (C), 164.9 (C) ppm; HRMS (TOF) m/z calcd (C 12 H 10 N 4 O) 226.08546, found 227.09193 [M+H] +

PAGE 89

75 2 a z i d o N ( 4 i s o p r o p y l p h e n y l ) e t h a n a m i d e ( A Z 1 4 ) H N O N 3 The preparation of AZ14 was accomplished via the same procedure used to prepare AZ13 (292 mg, 1.34 mmol, 94%); 1 H NMR (250 MHz, CDCl 3 ): $ 1.27 (d, J = 6.9, 6H), 2.88-2.99 (m, 1H), 4.17 (s, 2H) 7.23-7.50 (m, 4H), 8.02 (br s, 1H); 13 C NMR (63 MHz, CDCl 3 ): $ 23.9 (2xCH 3 ), 33.6 (CH), 52.9 (CH 2 ), 120.2 (CH), 126.9 (CH), 134.3 (C), 145.7 (C), 164.3 (C); HRMS (TOF) m/z calcd (C 11 H 14 N 4 O) 218.11676, found 219.12373 [M+H] + 1 ( 4 f l u o r o p h e n y l ) 5 m e t h y l 1 H 1 2 3 t r i a z o l e 4 c a r b o x y l i c a c i d ( 3 4 ) N F N N O H O Azide AZ1 (400 mg, 2.92 mmol) and butynoic acid 16 (245 mg, 3.39 mmol) were heated as a neat solution at 80 C overnight. The reaction was then diluted with ethyl acetate and extracted with brine solution. The corresponding triazole was obtained as a brown solid without any further purification in (639 mg, 2.89 mmol, 99%). Rf = 0.35 (DCM:MeOH, 9:1); 1 H NMR (250 Hz, CDCl 3 ) 2.56 (s, 3H), 7.36-7.41 (m, 2H), 7.60-7.63 (m, 2H).

PAGE 90

76 3 n i t r o 4 ( 2 ( p h e n y l t h i o ) e t h y l a m i n o ) b e n z y l 1 ( 4 f l u o r o p h e n y l ) 5 m e t h y l 1 H 1 2 3 t r i a z o l e 4 c a r b o x y l a t e ( 3 5 ) O O N O 2 N H S N N N F To a dried flask under argon, (3-nitro-4-(2-(phenylthio) ethylamino)phenyl)methanol (0.25 mmol, 1 eq), 34 (40 mg, 0.18 mmol, 1 eq), and DMTMM (0.73 mmol, 3 eq) were dissolved in dry THF (5 mL). N-Methylmorpholine (0.73 mmol, 3eq) was then added dropwise. The reaction was left to stir overnight at room temperature. The reaction was complete after 16hrs and poured into H 2 O (75 mL) and extracted into ethyl acetate (4x50 mL). The combined organic layers were dried with Na 2 SO 4 and condensed under reduced pressure. The crude was purified by column chromatography on silica gel (Hx:EtOAc, 1:1) to afford the corresponding triazole in 40% yield. Rf = 0.40 ( Hx:EtOAc, 1:1); 1 H NMR (250 MHz, CDCl 3 ): $ 1.86 (s, 3H), 3.21 (t, J = 6.8, 2H), 3.54 (apparent q, J =6.4, 2H), 5.11 (s, 2H), 6.72 (d, J = 8.8, 1H), 7.11-7.44 (m, 9H), 8.08 (d, J =1.9, 1H), 8.34 (t, J =4.9, 1H).

PAGE 91

77 List of References 1. Mestres, J.; Veeneman, G.H. J. Med Chem 2003, 46 3441-3444. 2. Gribbon, P.; Andreas, S. Drug Discov. Today 2005, 10 17-22. 3. Borman, S. Improving Efficiency. C&EN, June 19, 2006, p 56-78. 4. Manetsch, R.; Krasiski, A.; Radi, Z.; Raushel, J.; Taylor, P.; Sharpless, K.B.; Kolb, H.C. J. Am. Chem. Soc. 2004, 126, 12809-12818. 5. Erlanson, D.A.; McDowell, R.S.; O'Brien, T. J. Med Chem. 2004, 47 (14) 34633482. 6. Rees, D.C.; Congreve, M.; Murray, C.W.; Carr, R. Nature Reviews Drug Discovery 3 660 672. 7. Erlanson, D.A.; Hansen, S.K. Current Opinion in Chemical Biology, 2004, 8 399406. 8. Dustin J. Maly; Ingrid C. Choong ; Jonathan A. Ellman Proc. Natl. Acad. Sci. U.S.A Vol. 97, No. 6 (Mar., 2000), pp. 2419-2424. 9. Hofstadler, S.A.; Griffey, R.H. Chem. Rev. 2001, 101 377-390. 10. Sharpless, K.B.; Manetsch, R. Expert Opin. Drug Discov. 2006 1 (6), 525-538. 11. De Bruin, B.; Hauwert, P.; Reek, J.N.H. Angew. Chem. Int. Ed. 2006 45 2660-2663. 12. Ramstrm, O.; Lehn, J.M. Nature Reviews Drug Discovery 2002, 1, 26 36. (01 Jan 2002) Review. 13. Otto, S., Furlan, R.L.E., Sanders, J.K.M. Drug Discov. Today 2002 7 (2), 117-125. 14. Rowan, S.J.; Sanders, J.K.M. J. Org. Chem 1998, 63 1536-1546. 15. Huc I., Luhn J.M. Proc. Natl. Acad. Sci. USA 1997, 94 (6) 2106-2110. 16. Poulsen ,S.A.; Bornaghi L.F. Bioorg. Med. Chem. 2006, 14 (10), 3275-3284.

PAGE 92

78 17. Nicolaou, K.C.; Hughes, R.; Cho, S.Y.; Winssinger, N.; Smethurst, C.; Labischinski, H.; Endermann, R. Angew. Chem., Int. Ed. 2000 39 3823-3828. 18. Whiting, M.; Muldoon, J.; Lin, Y.C.; Silverman, S.M.; Lindstrom, W.; Olson, A.J.; Kolb, H.C.; Finn, M.G.; Sharpless, B.K.; Elder, J.H.; Fokin, V.V. Angew. Chem. Int. Ed. 2006, 45 (9),1435-1439. 19. Moore, D.R.; Cheng, M.; Lobkovsky, E.B.; Coates, G.W. Angew. Chem. Int. Ed. 2002 41 (14)2599-2602. 20. Krasiski, A.; Fokin, V.V.; Sharpless, B.K. Org. Lett. 2004, 6 (8) ,1237-1240. 21. Zhang, L.; Chen, X.; Xue, P.; Sun, H.H.Y.; Williams, I.D.; Sharpless, B.K.; Fokin, V.V; Jia, G. J. Am. Chem. Soc. 2005, 127 (46),15998-15999. 22. Taylor, P., Radic, Z. Ann. Rev. Pharmacol. Toxicol. 1994 34 281-320. 23. Krasiski, A.; Radi, Z.; Manetsch, R.; Raushel, J.; Taylor, P.; Sharpless, B.K.; Kolb, H.C.; J. Am. Chem. Soc. 2005 127 6686-6692. 24. Casomo, A.; Mincione, F.; Ilies, M.A.; Menabuoni, L.; Scozzafa, A.; Supuran, C.T.; J Enzym Inhib. 2001, 16 (2),113-123. 25. Gao, J.; Cheng, X.; Chen, R.; Sigal, G.B.; Bruce, G.E.; Schwartz, B.L.; Hofstadler, S.A.; Anderson, G.A.; Smith, R.D.; Whitesides, G.M. J. Med. Chem 1996, 39 (10),1949-1955. 26. Grybowski, B.A.; Ishchenko, A.V.; Kim, C.Y.; Topalov, G.; Chapman, R.; Christianson, D.W.; Whitesides, G.M.; Shakhnovich, E.I. Proc. Natl. Acad. Sci. USA 2002, 99 (3),1270-1273. 27. Mocharla, V.P.; Colasson, B.; Lee, L.V.; Rper, S.; Sharpless, B.K.; Wong, C.H.; Kolb, H.C. Angew. Chem. Int. Ed. 2004, 44 (1), 116-120. 28. Wang, J.; Sui, G.; Mocharla, V.P.; Lin, R.J.; Phelps, M.E.; Kolb, H.C.; Tseng, H.R. Angew. Chem. Int. Ed. 2006 45 5276-5281. 29. Arkin, M.R.; Wells ,J.A. Nature Rev. Drug Discov 2004 3 301-317. 30. Conte, L.L.; Chothia, C.; Janin,J. J. Mol. Biol. 1999, 285 2177-2198. 31. Teague, S.J. Nature Reviews Drug Discovery 2003, 2 527 541.

PAGE 93

79 32. Proteins S4, 63-71 (2000) 33. Ma, B.; Shatsky, M.; Wolfson, H.J.; Nussinov, R. Protein Sci. 2002, 11 184-197. 34. Adams J.; Cory S. Science 1998 281 1322-1326. 35. Cory, S.; Adams, J.M. Nature Reviews Cancer 2002 2 647 656. 36. Wendt, M. D.; Shen, W.; Kunzer, A.; McClellan, W. J.; Bruncko, M.; Oost, T. K.; Ding, H.; Joseph, M. K.; Zhang, H.; Nimmer, P. M.; Ng, S.-C.; Shoemaker, A. R.; Petros, A. M.; Oleksijew, A.; Marsh, K.; Bauch, J.; Oltersdorf, T.; Belli, B. A.; Martineau, D.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. J. Med. Chem ( Article) 2006 49 (3), 1165-1181. 37. a)Chittenden, T.; Flemington, C.; Houghton, A.B.; Ebb, R.G.; Gallo, G.J.; Elangovan, B.; Chinnadurai, G.; Lutz, R.J. EMBO J. 1995 14 (22), 5589-5596. b) Kelekar, A.; Thompson, C.G. Trends Cell Biol. 1998, 8 324. 38. Yin, X.M.; Oltvai, Z.N.; Korsmeyer, S.J. Nature 1994, 369 321 323. 39. Cheng E. H.-Y.A.; Wei A.C.;Weiler S.; Flavell, R.A.; Mak, T.W.; Lindsten,T. Korsmeyer, S.J. Mol. Cell 2001 8 705-711. 40. Amundson, S.A.; Myers, T.G.; Scudiero, D.; Kitada, S.; Reed, J.C.; Fornace, A.J. Jr. Cancer Res. 2000 60 6101-6110. 41. Oltersdorf, T.; Elmore, S.W.; Shoemaker, A.R.; Armstrong, R.C.; Augeri, D.J.; Belli, B.A.; Bruncko, M.; Deckwerth, T.L.; Dinges, J.; Hajduk, P.J.; Joseph, M.K.; Kitada, S.; Korsmeyer, S.J.; Kunzer, A.R.; Letai, A.; Li, C.; Mitten, M.J.; Nettesheim, D.J.; Ng, S.C.; Nimmer, P.M.; O'Connor, J.M.; Oleksijew, A.; Petros, A.M.; Reed, J.C.; Shen, W.; Tahir, S.K.; Thompson, K. B.; Tomaselli, K.J.; Wang, B.; Wendt, M.D.; Zhang, H.; Fesik, S.W.; Rosenberg S.H. Nature 2005 435 677 681. 42. DeBoef, B.; Counts, R.W.; Gilbertson, S. R. J. Org. Chem. 2007 72 (3), 799-804. 43. .Kunishima, M.; Kawachi, C.; Monta, J.; Terao, K.; Iwasaki, F.; Tani, S. Tetrahedron 1999, 55 (46), 13159-13170. 44. Manetsch, R.; Krasiski, A.; Radi, Z.; Raushel, J.; Taylor, P.; Sharpless, K.B.; Kolb, H.C. J. Am. Chem. Soc. 2004, 126, 12809-12818. 45. Rostovtsev, V.V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. Angew. Chem. Int. Ed. 2002, 41 (14), 2596-2599.

PAGE 94

80 46. Fleming, I.; Lawrence, N.J. J. Che. Soc., Perkin Trans. 1998 1 2679-2686. 47. Dullin A. ; Dufrasne, F.; Gelbcke, M.; Gust, R. Arch. Pharm. Med. Chem 2004 337 (12), 654-667. 48. Lee, J.W.; Jun S.I.; Kim K. Tetrahedron Lett. 2001, 42 (14), 2709-2711. 49. Katritzky, A.R.; Xu, Y.J.; He, H.Y.; Mehta, S. J. Org. Chem. 2001, 66, 5590 5594. 50. Semple, G.; Skinner, P.J.; Cherrier, M.C.; Webb, P.J.; Sage, C.R.; Tamura, S.Y.; Chen, R.; Richman, J.G.; Connolly, D.T. J. Med Chem 2006 49, 1227-1230. 51. Feely, W.; Lehn, W.L.; Boekelheide, V. Org. Chem. 1957, 22. 1135 1135. 52. Yoon, N.M.; Pak, C.S.; Brown, H.C.; Krishnamurthy, S.; Stocky, T.P. Org. Chem. 1973, 38 (16), 2786 2792. 53. Reddy, G.V.S.; Rao, G.V.; Subramanyam, R.V.K.; Iyenger, D.S. Synthetic Communications 2000, 30 (12), 2233-2237. 54. Roy, A.D.; Sharma, S.; Grover, R.K.; Kundu, B.; Roy, R. Org. Letters 2004, 6 (25), 4763 4766. 55. Kubo I., Fujita, K.I; and Nihei, K.I. J. Agric. Food. Chem. 2002, 50, 6692-6696. 56. Kawada, K.; Dolence, E.K.; Morita, H.; Kometani, T.; Watt, D.S.; Balapure, A.; Fitz, T.A.; Orlicky, D.J.; Gerschenson, L.E. J. Med Chem 1989 32 (1), 257-264. 57. Kaplan, M.; Manetsch, M. Unpublished Results.

PAGE 95

81 Appendices Appendix A: Spectra of Compounds

PAGE 96

82 Appendix A: (Continued) Figure A1 1 H NMR and 13 C NMR of Compound 2

PAGE 97

83 Appendix A: (Continued) Figure A2 1 H NMR of Compound 3

PAGE 98

84 Appendix A: (Continued) Figure A3 1 H NMR and 13 C NMR of Compound 4

PAGE 99

85 Appendix A: (Continued) Figure A4 1 H NMR and 13 C NMR of Compound 6

PAGE 100

86 Appendix A: (Continued) Figure A5 1 H NMR and 13 C NMR of Compound 7

PAGE 101

87 Appendix A: (Continued) Figure A6 1 H NMR of Compound 8 Figure A7 1 H NMR of Compound 9

PAGE 102

88 Appendix A: (Continued) Figure A8 1 H NMR of Compound 10 and 11 Figure A9 1 H NMR of Compound 12

PAGE 103

89 Appendix A: (Continued) Figure A10 1 H NMR of Compound 13

PAGE 104

90 Appendix A: (Continued) Figure A11 1 H NMR and 13 C NMR of Compound 17

PAGE 105

91 Appendix A: (Continued) Figure A12 1 H NMR and 13 C NMR of Compound 22

PAGE 106

92 Appendix A: (Continued) Figure A13 1 H NMR of Compound 24 Figure A14 1 H NMR of Compound 25

PAGE 107

93 Appendix A: (Continued) Figure A15 1 H NMR and 13 C NMR of Compound 26

PAGE 108

94 Appendix A: (Continued) Figure A16 1 H NMR and 13 C NMR of Compound 27

PAGE 109

95 Appendix A: (Continued) Figure A17 1 H NMR and 13 C NMR of Compound 28

PAGE 110

96 Appendix A: (Continued) Figure A18 1 H NMR and 13 C NMR of Compound AM3

PAGE 111

97 Appendix A: (Continued) Figure A19 1 H NMR and 13 C NMR of Compound AM4

PAGE 112

98 Appendix A: (Continued) Figure A20 1 H NMR of Compound 34 Figure A21 1 H NMR of Compound 35

PAGE 113

99 Appendix A: (Continued) Figure A22 1 H NMR and 13 C NMR of Compound AZ1

PAGE 114

100 Appendix A: (Continued) Figure A23 1 H NMR and 13 C NMR of Compound AZ2

PAGE 115

101 Appendix A: (Continued) Figure A24 1 H NMR Compound AZ3

PAGE 116

102 Appendix A: (Continued) Figure A25 1 H NMR and 13 C NMR of Compound AZ4

PAGE 117

103 Appendix A: (Continued) Figure A26 1 H NMR and 13 C NMR of Compound AZ5

PAGE 118

104 Appendix A: (Continued) Figure A27 1 H NMR and 13 C NMR of Compound AZ6

PAGE 119

105 Appendix A: (Continued) Figure A28 1 H NMR Compound AZ7

PAGE 120

106 Appendix A: (Continued) Figure A29 1 H NMR Compound AZ8 Figure A30 1 H NMR Compound AZ9

PAGE 121

107 Appendix A: (Continued) Figure A31 1 H NMR Compound AZ10

PAGE 122

108 Appendix A: (Continued) Figure A32 1 H NMR and 13 C NMR of Compound AZ11

PAGE 123

109 Appendix A: (Continued) Figure A33 1 H NMR and 13 C NMR of Compound AZ12

PAGE 124

110 Appendix A: (Continued) Figure A34 1 H NMR and 13 C NMR of Compound AZ13

PAGE 125

111 Appendix A: (Continued) Figure A35 1 H NMR and 13 C NMR of Compound AZ14