USF Libraries
USF Digital Collections

Design and synthesis of substituted 1,4-hydrazine-linked piperazine-2,5- and 2,6-diones and 2,5-terpyrimidinylenes as [a...

MISSING IMAGE

Material Information

Title:
Design and synthesis of substituted 1,4-hydrazine-linked piperazine-2,5- and 2,6-diones and 2,5-terpyrimidinylenes as alpha-helical mimetics
Physical Description:
Book
Language:
English
Creator:
Anderson, Laura
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Bcl-2
Mdm-2
Apoptosis
Alpha-helix
PNA
Dissertations, Academic -- Chemistry -- Doctoral -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: The most common secondary structure of proteins is the alpha-helix. The alpha-helix can be involved in various protein-protein interactions (PPIs) through the recognition of three or more side chains along one face of the alpha-helix (Wells and McClendon, 2007). In recent years, there has been an increasing interest in the development of peptidic and non-peptidic compounds that bind to PPI surfaces. We focused on the design and synthesis of compounds that mimic the orientation of side chain residues of an alpha-helical protein domain. Although our scaffolds could potentially inhibit various PPIs, we focused mainly on the disruption of interactions among the Bcl-2-family of proteins and the Mdm-2-family of proteins to favor apoptosis in cancer cells. A summary of Bcl-2 and Mdm-2 structure and function relationships that focuses on the possibility of using peptidic and non-peptidic alpha-helical mimics as PPI inhibitors is described in Chapter One.Chapter Two discusses the design and synthesis of 3-substituted-2,6- and 2,5-piperazinedione oligomers as more hydrophilic scaffolds compared to previously reported alpha-helical mimetics (Yin, et al., 2005). A key feature of this design is the linkage of the units by a hydrazine bond. While we were able to prepare several monomers containing the hydrazine linkage, synthesis of the dimers and trimers is very challenging. Due to the difficulty of synthesizing oligomeric piperazine-diones in practical yields, we next focused on the design and synthesis of novel 2,5-terpyrimidinylene scaffolds as an alternative to obtain alpha-helical mimetics; this is discussed in Chapter Three. The main outcome of this project was the efficient preparation of a "first-generation" non-peptidic compound library via a facile iterative synthesis enabled by the key conversion of 5-cyanopyrimidine to 5-carboxamidine.Chapter Three also discusses our progress towards the synthesis of structurally similar substituted-2,5-terpyrimidinylenes, but with more drug-like properties as determined by QikProp calculations. Chapter Four describes an independent study on the synthesis of a guanidine derivative as an alkylating agent for the synthesis of cysteine peptide nucleic acids, CPNA, which is another current project in our lab.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Laura Anderson.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 233 pages.
General Note:
Includes vita.

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 - 002317559
oclc - 660241077
usfldc doi - E14-SFE0003054
usfldc handle - e14.3054
System ID:
SFS0027371:00001


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 2200385Ka 4500
controlfield tag 001 002317559
005 20100902181951.0
007 cr cnu|||uuuuu
008 100902s2009 flua ob 000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0003054
035
(OCoLC)660241077
040
FHM
c FHM
049
FHMM
090
QD31.2 (Online)
1 100
Anderson, Laura.
0 245
Design and synthesis of substituted 1,4-hydrazine-linked piperazine-2,5- and 2,6-diones and 2,5-terpyrimidinylenes as [alpha]-helical mimetics
h [electronic resource] /
by Laura Anderson.
260
[Tampa, Fla] :
b University of South Florida,
2009.
500
Title from PDF of title page.
Document formatted into pages; contains 233 pages.
Includes vita.
502
Dissertation (Ph.D.)--University of South Florida, 2009.
504
Includes bibliographical references.
516
Text (Electronic dissertation) in PDF format.
520
ABSTRACT: The most common secondary structure of proteins is the alpha-helix. The alpha-helix can be involved in various protein-protein interactions (PPIs) through the recognition of three or more side chains along one face of the alpha-helix (Wells and McClendon, 2007). In recent years, there has been an increasing interest in the development of peptidic and non-peptidic compounds that bind to PPI surfaces. We focused on the design and synthesis of compounds that mimic the orientation of side chain residues of an alpha-helical protein domain. Although our scaffolds could potentially inhibit various PPIs, we focused mainly on the disruption of interactions among the Bcl-2-family of proteins and the Mdm-2-family of proteins to favor apoptosis in cancer cells. A summary of Bcl-2 and Mdm-2 structure and function relationships that focuses on the possibility of using peptidic and non-peptidic alpha-helical mimics as PPI inhibitors is described in Chapter One.Chapter Two discusses the design and synthesis of 3-substituted-2,6- and 2,5-piperazinedione oligomers as more hydrophilic scaffolds compared to previously reported alpha-helical mimetics (Yin, et al., 2005). A key feature of this design is the linkage of the units by a hydrazine bond. While we were able to prepare several monomers containing the hydrazine linkage, synthesis of the dimers and trimers is very challenging. Due to the difficulty of synthesizing oligomeric piperazine-diones in practical yields, we next focused on the design and synthesis of novel 2,5-terpyrimidinylene scaffolds as an alternative to obtain alpha-helical mimetics; this is discussed in Chapter Three. The main outcome of this project was the efficient preparation of a "first-generation" non-peptidic compound library via a facile iterative synthesis enabled by the key conversion of 5-cyanopyrimidine to 5-carboxamidine.Chapter Three also discusses our progress towards the synthesis of structurally similar substituted-2,5-terpyrimidinylenes, but with more drug-like properties as determined by QikProp calculations. Chapter Four describes an independent study on the synthesis of a guanidine derivative as an alkylating agent for the synthesis of cysteine peptide nucleic acids, CPNA, which is another current project in our lab.
538
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
590
Advisor: Mark L. McLaughlin, Ph.D.
653
Bcl-2
Mdm-2
Apoptosis
Alpha-helix
PNA
690
Dissertations, Academic
z USF
x Chemistry
Doctoral.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.3054



PAGE 1

Design and Synthesis of Substituted 1,4-Hydrazi ne-linked Piperazine-2,5and 2,6-diones and 2,5-Terpyrimidinylenes as -Helical Mimetics by Laura Anderson A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Mark L. McLaughlin, Ph.D. Wayne C. Guida, Ph.D. Roman Manetsch, Ph.D. Hong-Gang Wang, Ph.D. Date of Approval: July 8, 2009 Keywords: Bcl-2, Mdm-2, apoptosis, -helix, PNA Copyright 2009, Laura Anderson

PAGE 2

DEDICATION With my deepest gratitude, To my dad (my angel) Luis Delio and my mom Mara Cenelia To my love Patrick

PAGE 3

ACKNOWLEDGEMENTS First, I want to thank my family, esp ecially mom and dad, for allowing me the possibility of a great educati on. Although dad is not longer present, I could assure how happy and proud he would have felt to share this moment with me. My parents’ dedication and encouragement are invaluable and without their support I would not be writing this note. I also thank my brothers and sisters for thei r caring over the years. I want to reiterate my love and gratitude to my husband Patrick. Without his love, patience, and unconditional help it would have been very diffi cult to reach this goal. I shall be grateful forever to hi m for this “our” achievement. Very special thanks go to my adviso r, Professor Mark L. McLaughlin, for his determination, patience, and encouragement even through disappointing times. His knowledge and teaching passion were crucial in my progress as a scientist and I am grateful for the opportunity to work with a great person like him. I also want to acknowledge my committee members, Profe ssor Wayne C. Guida, Professor Roman Manetsch, and Professor Hong-Gang Wang fo r taking the time to meet with me on several occasions, for their guida nce and insightful feedback. I thank Dr. Umut Oguz for accepting to chair my defense and for all he r help in many educational and personal aspects. I want to convey my sincere thanks to Professor Dean F. Martin, who not only suggested and helped me to pursue gra duate school, but had trusted my academic potential and welcomed me in his lab durin g my undergrad years. I also want to recognize Jaisnover Villa, my first chemistry teacher in Colombia, whose enthusiasm and

PAGE 4

dedication influenced my interest in this field. I want to thank Drs. Turos, Baker, Bisht, and Zhang in the Chemistry Dept. at USF for general discussions and motivation to attend several “JACS” meetings, colloquia, an d seminars. I thank all the graduate and undergraduate students and post-docs in Dr. McLaughlin’s group that I met and worked with over the years. Although in many ocassi ons there was frustra tion and the research seemed to be unbearable, we were there to help each other and make this process more amenable. I was very fortunate to be assist ed by Dr. Vasudha Sharma and I am indebted to her in many aspects, especially her “coachin g” lessons and critical insight that helped me to maintain my courage during the last st eps of my graduate time. I thank HyunJoo and Mingzhou for carrying over my project. I thank everyone in Drs. Nick and Harshani Lawrence’s labs for their help and mainly for allowing me to share many of their research activities and discussions. Divya, Roberta, and Daniele were very helpful with instrumentation training and scientific discus sions, as well as good listeners of personal experiences. I would like to acknowledge th e Chemistry Department at USF and the H. Lee Moffitt Cancer Center for providing th e facilities to conduct my research. I appreciate Dr. Ted Gauthie r’s help with invaluable suggestions and mass spectra analysis. Dr. Edwin Rivera and Yunting Luo assisted me with NMR analysis. I thank Dr. Frank Fronczek, who very kindly solved al l the crystal structures reported in this work. Dr. Shen-Shu Sung and Daniel Santiago helped us with the molecular modeling studies. Dr. George Sung, Dr. Sharma, and Aleksandra Zajac performed the biological testing of our compounds. Last but not least, I want to thank my friends, especially Isabel Cristina, who has shared with me a truthful fr iendship and Adriana for her posi tive and enviable attitude.

PAGE 5

i TABLE OF CONTENTS TABLE OF C ONTENTS ..................................................................................................... i LIST OF TABLES ............................................................................................................. ii i LIST OF FIGURES ........................................................................................................... iv LIST OF SCHEMES .......................................................................................................... vi LIST OF ABBREVIATIONS .......................................................................................... viii ABSTRACT. ..................................................................................................................... ...x CHAPTER ONE: PROTEINS : GENERAL INTRODUCTION ........................................1 1.1 Protein Secondary Structure: -Helix ................................................................1 1.2 Protein-Protein Inte ractions (PPIs) ...................................................................3 1.3 Role of Bcl-2 Family and Hmd-2 Family Proteins in Apoptosis.......................5 1.4 Peptidic and Non-Peptidic -Helical Mimetics .................................................8 1.5 References ........................................................................................................16 CHAPTER TWO: DESIGN AND SYNTHE SIS OF 3-R-PIPERAZINE-2,5AND 2,6-DIONES ..............................................................................22 2.1 Introduction ......................................................................................................22 2.1.1 Piperazine-diones ..............................................................................22 2.2 Results and Discussion ....................................................................................25 2.2.1 Synthesis of 3-R-Pi perazine-2,6-diones: DKPA ..............................25 2.2.2 Synthesis of Diacid Derivatives 2.6: Route 1 ...................................27 2.2.3 Cyclization and Coupli ng of Piperazine-2,6-diones .........................32 2.2.4 Synthesis of Piper azine-2,6-diones: Route 2 ....................................39 2.2.5 Synthesis of Piper azine-2,5-diones: Route 1 ....................................42 2.2.6 Synthesis of Piperazi ne-2,5-diones: Route 2 ....................................45 2.3 Conclusion .......................................................................................................47 2.4 Experimental Section .......................................................................................48 2.4.1 Materials and Methods ......................................................................48 2.4.2 Experimental Procedures ..................................................................49 2.5 References ........................................................................................................72 CHAPTER THREE: DESIGN AND SYNTHESIS OF 4-RAND 4,6-R,R’-2,5-TERPYRIMIDINYLENES .......................................78

PAGE 6

ii 3.1 Introduction ......................................................................................................78 3.1.1 Pyrimidines .......................................................................................78 3.1.2 2,5-Terpyrimidinylenes as Potential -Helical Mimetics .................80 3.1.3 General Methods for the Synthesis of Pyrimidines ..........................81 3.2 Results and Discussion ....................................................................................83 3.2.1 Synthesis of a “First-generation” 4-R-2,5-Terpyrimidinylene Library ..............................................................................................83 3.2.2 In Vitro Biological Evaluation ........................................................102 3.2.3 Synthesis of a “Second-generation” 4-R-2,5-Terpyrimidinylene Library ................................................104 3.2.4 Synthesis of 4-R,6R-2,5-Terpyrimidinylenes ................................106 3.3 Conclusion .....................................................................................................109 3.4 Experimental Section .....................................................................................110 3.4.1 Experimental Procedures ................................................................110 3.5 References ......................................................................................................136 CHAPTER FOUR: SYNTHESIS OF A GUANIDINE DERIVATIVE FOR THE SYNTHESIS OF CPNA MONOMERS ................................141 4.1 Peptide Nucleic Acids (PNA): Introduction ..................................................141 4.1.1 Potential Applications of PNA .......................................................142 4.1.2 Cysteine-based PNA (CPNA) .........................................................143 4.2 Results and Discussion ..................................................................................145 4.2.1 Synthesis of Guanidine Derivative 4.4 ...........................................145 4.3 Conclusion .....................................................................................................152 4.4 Experimental Section .....................................................................................152 4.4.1 Experimental Procedures ................................................................152 4.5 References ......................................................................................................155 APPENDIX A: SELECTED 1H AND 13C NMR SPECTRA .........................................158 APPENDIX B: X-RAY CRY STALLOGRAPHIC DATA .............................................201 APPENDIX C: QIKPRO P CALCULATIONS ...............................................................232 ABOUT THE AUTHOR ....................................................................................... End Page

PAGE 7

iii LIST OF TABLES Table 2.1 Model synthesis of hydrazine diester 2.5a .................................................30 Table 2.2 Failed attempts to isol ate anhydride intermediates ....................................36 Table 3.1 Results of the synthesis of monomers 3.4a-j .............................................86 Table 3.2 Attempted routes to synthesize 5-carboxamidines ....................................90 Table 3.3 Reaction of compound 3.4a.3 with hydroxylamine HCl ...........................93 Table 3.4 Results of the synthesis of dimers 3.11 and trimers 3.12 ..........................97 Table 3.5 Conversion of 5-cyanopyrimidine to 5-carboxypyrimidine ....................101 Table 3.6 Results of the in vitro evaluation of monomeric and dimeric 2,5-pyrimidinylenes ................................................................................102 Table 3.7 Comparative in vitro evaluation of trimeric 2,5-pyrimidinylenes and Hamilton’s terphenylenes (Yin et al. 2005b) ..................................104 Table 4.1 Acylation conditions for the synthesis of guanidine 4.4 ..........................151

PAGE 8

iv LIST OF FIGURES Figure 1.1 Hydrogen bonding pattern in an -helix ......................................................2 Figure 1.2 Examples of small molecular weight inhibitors of PPIs ..............................4 Figure 1.3 Intrinsic pathway of apoptosis. Adapted from (Youle and Strasser, 2008) ...........................................................................6 Figure 1.4 Structures of -peptide and -peptides ......................................................10 Figure 1.5 Depiction of a stapled peptide (Walensky et al. 2006) ............................11 Figure 1.6 -Helix mimics reported by Hamilton and co-workers .............................12 Figure 1.7 Polar -helix mimics reported by Hamilton’s group .................................13 Figure 1.8 -Helix mimics reported by Rebek’s and Knig’s groups ........................15 Figure 2.1 Structures of unsubstitu ted diketopiperazine rings ....................................22 Figure 2.2 DKPA and DKPB: Target –helical peptidomimetics ..............................24 Figure 2.3 Retrosynthetic analysis of 3-R-piperazine-2,6-dione scaffold DKPA ...........................................................................................26 Figure 2.4 Docking studies of a hexa meric piperazine-2,6-dione analog (Pip) and Bcl-xL/Bak complex ...................................................................35 Figure 2.5 Retrosynthetic analysis of 3-R-piperazine-2,5-dione scaffold DKPB ...........................................................................................42 Figure 3.1 Structure of pyrimidine, bond angles and lengths (von Angerer, 2004) ..................................................................................78 Figure 3.2 Pyrimidine unit as component of biologically active compounds .............79 Figure 3.3 Structure of a trimeric 2,5-terpyrimidinylene scaffold ..............................80 Figure 3.4 Overlay of a 4,4’,4’’-trimethyl-2,5-terpyrimidylene and

PAGE 9

v an octa-alanine ..........................................................................................81 Figure 3.5 Retrosynthetic scheme of trimeric 2,5-pyrimidinylenes ............................84 Figure 3.6 Hypothetical model for H-bonding mediated synthesis of amidine ..........94 Figure 3.7 ORTEP diagram of carboxamide side product 3.10a.3 .............................95 Figure 3.8 ORTEP diagram of compound 3.12bac.3 ..................................................98 Figure 3.9 Typical 1H NMR spectrum of a trimeric 2,5-pyrimidinylene ....................99 Figure 3.10 QikProp calculations for terphenyl-based Bcl-xL-Bak inhibitor and a terpyrimidi nylene-based analog .....................................................100 Figure 4.1 Backbone structures of DNA and PNA ...................................................141 Figure 4.2 Schematic depiction of antisense and antigene inhibition .......................142 Figure 4.3 Cysteine-based PNA target scaffold (Yi Sung et al. 2009) ...................143 Figure 4.4 GPNA structure (Dragulescu-Andrasi et al. 2005) ................................144 Figure 4.5 CPNA building block target (Yi Sung et al. 2009) ................................145 Figure 4.6 1H NMR spectrum of compound 4.3 .......................................................150 Figure 4.7 S -alkylated CPNA monomers ..................................................................152

PAGE 10

vi LIST OF SCHEMES Scheme 2.1 Overall synthesis of diacid derivatives 2.6 ................................................27 Scheme 2.2 Attempted routes for the synthesis of hydrazine diester 2.5e ....................31 Scheme 2.3 General synthetic procedure of trimeric piperazine-2,6-dione 2.9bac via SPP ...........................................................................................33 Scheme 2.4 Synthesis of DKP1 monomer 2.13b ...........................................................38 Scheme 2.5 General synthesis of 2,6-DKP monomer, Route 2 .....................................39 Scheme 2.6 Cyclization of 2.19b with NaH ..................................................................40 Scheme 2.7 Failed attempt to synthesize monomer 2.13a .............................................41 Scheme 2.8 Attempted synthesis of 2,5-DKP monomer, Route 1 ................................43 Scheme 2.9 Failed attempt to synthesize 2.33f via N-H insertion ................................44 Scheme 2.10 General synthesis of 2,5-DKP monomer, Route 2 .....................................46 Scheme 3.1 Common method for the synthesis of substituted pyrimidines ..................82 Scheme 3.2 General synthesis of pyrimidinylene monomers........................................84 Scheme 3.3 Formation of side product 3.5a ..................................................................85 Scheme 3.4 Methods for the conversion of cyano group to amidine ............................89 Scheme 3.5 Hydroxylamine-mediated synthesis of amidine intermediates ..................91 Scheme 3.6 Attempted route to obtain amidine 3.8 ......................................................92 Scheme 3.7 Synthesis of compound 3.19a ..................................................................105 Scheme 3.8 Synthesis of 6-amino-4-substituted pyrimidinylene monomers ..............107 Scheme 3.9 General route for the synthesis of 6, 6’, 6”-triamino-4-R-, 4’-R-’,

PAGE 11

vii 4”-R”-substituted 2,5-pyrimidinylenes and Michael acceptors ...............108 Scheme 4.1 Synthesis of S -alkylating agent 4.4 ..........................................................146 Scheme 4.2 Failed attempt to synthesize guanidine derivative 4.3 .............................147 Scheme 4.3 Failed attempt to synthesize a triflylguanidine reagent ..........................148 Scheme 4.4 Synthesis of guanidine derivative 4.3 (Yong et al. 1997) .....................148

PAGE 12

viii LIST OF ABBREVIATIONS Alpha Angstrom Ac2O Acetic anhydride AcOH Acetic acid Ala Alanine aq. Aqueous Ar aryl Asp Aspartic acid Beta Bcl-2 B-Cell lymphoma 2 BH3 Bcl-2 homology region 3 Bn Benzyl Boc tert -Butoxycarbonyl br Broad (spectral) Bu Butyl Bz Benzoyl C Degree Celsius 13C NMR Carbon-13 Nuclear Magnetic Resonance Cbz Carboxybenzyl CPNA Cysteine-based peptide nucleic acid Delta or chemical shift DCM Dichloromethane DIEA Diisopropylethylamine DMF N,N -Dimethylformamide DMSO Dimethylsulfoxide Et Ethyl Et3N Triethylamine EtOAc Ethyl acetate EtOH Ethanol ESI Electrospray ionization equiv. Equivalent(s) g Gram(s) (g) Gas 1H NMR Proton Nuclear Magnetic Resonance h Hour(s) Hdm-2 Human double minute 2 HR High resolution Hz Hertz

PAGE 13

ix IC50 50% inhibitory concentration Ile Isoleucine J Coupling-constant(s) K i Inhibitor dissociation constant Leu Leucine LiHMDS Lithium hexamethyldisilazide LiOH Lithium hydroxide M Molar or moles per liter Mdm-2 Murine double minute 2 Me Methyl MeOH Methanol MeCN Acetonitrile mg Milligram (s) min. Minute (s) mL Milliliter(s) mmol Millimole(s) MOM Mitochondrial outer membrane m.p. Melting point MS Mass spectrum M.W Microwave MW Molecular weight NaOH Sodium hydroxide nM Nanomolar NMR Nuclear magnetic resonance spectrum ORTEP Oak Ridge thermal ellipsoid plot (crystallography) Pd/C Palladium on carbon Ph Phenyl Phe Phenylalanine PNA Peptide nucleic acid ppm Parts per million PPI(s) Protein-protein interaction (s) RMDS Root mean square deviation rt Room temperature SAR Structure activity relationship Sat’d Saturated SPPS Solid-phase peptide synthesis TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography Trp Tryptophan L Microliter(s) M Micromolar Val Valine

PAGE 14

x Design and Synthesis of Substituted 1,4Hydrazine-linked Piperazine-2,5and 2,6-diones and 2,5-Terpyrimidinylenes as -Helical Mimetics Laura Anderson ABSTRACT The most common secondary structure of proteins is the -helix. The -helix can be involved in various protein-protein interacti ons (PPIs) through the recognition of three or more side chains along one face of the -helix (Wells and McClendon, 2007). In recent years, there has been an increasing interest in the development of peptidic and non-peptidic compounds that bind to PPI surfaces. We focu sed on the design and synthesis of compounds that mimic the orientation of side chain residues of an -helical protein domain. Although our scaffolds could potentially inhibit various PPIs, we focused mainly on the disruption of interactions among the Bcl-2-family of proteins and the Mdm-2-family of proteins to favor apoptosis in cancer cells. A summary of Bcl-2 and Mdm-2 structure and function relationships that focuses on the possibility of using peptidic and non-peptidic -helical mimics as PPI inhibitors is described in Chapter One. Chapter Two disc usses the design and synthesis of 3-substituted2,6and 2,5-piperazinedione oligomers as more hydrophilic scaffolds compared to previously reported -helical mimetics (Yin et al. 2005). A key feature of this design is the linkage of the units by a hydrazine bond. While we were able to prepare several monomers containing the hydrazine linkage, synthesis of th e dimers and trimers is very challenging.

PAGE 15

xi Due to the difficulty of synthesizing oligomeric piperazine-diones in practical yields, we next focused on the design and synthesis of novel 2,5-terpyrimidinylene scaffolds as an alternative to obtain -helical mimetics; this is discussed in Chapter Three. The main outcome of this project was the efficient prep aration of a “first-generation” non-peptidic compound library via a facile iterative synt hesis enabled by the key conversion of 5cyanopyrimidine to 5-carboxamidine. Chapter Th ree also discusses our progress towards the synthesis of structurally similar substituted2,5-terpyrimidinylenes, but with more drug-like properties as determined by QikProp calculations Chapter Four describes an independent study on the synthesis of a guanidine derivative as an alkylating agent for the synthesis of cysteine peptide nucleic acids, CPNA, which is another current project in our lab.

PAGE 16

1 CHAPTER ONE PROTEINS: GENERAL INTRODUCTION 1.1 Protein Secondary Structure: -Helix The biological function of a protein is greatly influen ced by its three-dimensional (3-D) structure. Folding in proteins occurs due to the interactions of the amino acids mainly stabilized by hydrogen bonds and dete rmined by the amino acid sequence. The folding process also depends on the environmen t surrounding the protei n, such as solvent, temperature, pH, and the pres ence of chaperones (Cutler et al. 2009; Williamson, 1994). Proteins adopt well-defined 3-D structures to minimize the expos ure of hydrophobic side chain interactions to water counterbalanci ng overall entropic and enthalpic effects (Branden and Tooze, 1992). Prot eins organize at different levels including primary, secondary, tertiary, and quaternar y structures. The primary stru cture refers to the linear sequence of the amino acids; secondary and ter tiary structures are related to local and global folding, respectively, of a single polypep tide chain; and the quaternary refers to the stable organization of two or more polypep tide chains into an active sub-unit structure (Garrett and Grisham, 1999). The most common secondary st ructure of proteins is the -helix; this is stabilized by hydrogen bonds between the carbonyl oxygen and the amide hydrogen located four positions along the peptidic chain. Moreover, helices are stabilized by hydrophobic, ionic, and steric interacti ons of the amino acid side re sidues. The hydrogen bonds are almost parallel to the axis of the -helix, whereas the side chain residues project almost

PAGE 17

2 orthogonally. Helices can be f ound in nature as right-handed and left-handed helices; the most common in nature is th e right-handed helix, which is promoted by L-amino acids (Garrett and Grisham, 1999). Figure 1.1. Hydrogen bonding pattern in an -helix The structural stability of an -helix depends on the intramolecular hydrogen bonding of the amino acids. The arrangement of the amino acids in an -helix has 3.6 amino acid residues per turn. One turn places the i th and ith +4 residues 5.4 apart, which is referred to as a pi tch (5.4 = 3.6 residues x 1.5 , separation per residue). Accordingly, hydrogen bonding occu rs between C=O of residue ith and NH of residue

PAGE 18

3 ith +4, thus all NH and C=O groups in an -helix are linked with hydrogen bonds except for the first NH (the N -terminus) and the last CO group (the C -terminus) of the -helix (Figure 1.1 ) (Perez de Vega et al. 2007). 1.2 Protein-Protein Interactions (PPIs) The -helix can be involved in many proteinprotein interactions (PPIs) through the recognition of three or more side chains al ong a single face of the -helix (Wells and McClendon, 2007). PPIs play si gnificant roles in several biological systems including signal transduction pathways, cellular proce sses (proliferation, grow th, differentiation, and programmed cell death), and self-assembly of viruses (Fletcher and Hamilton, 2006; Gerrard et al. 2007; Toogood, 2002). As a result, the disruption of PPIs has become an attractive target for the development of novel biochemical tools and therapeutic agents. However, the disruption of PPIs is still a very challenging approach due to the large surface areas (~ 1500 to 3000 2), as compared to protein-small molecule interaction surfaces (~ 300 to 1000 2) or enzyme active sites, the relatively shallow surfaces, and the noncontiguous binding regions involved in protein-protein interfacial do mains (Wells and McClendon, 2007). This implies that larg e molecular weight inhibitors that can cover sufficient interactions between the i nhibitor and the protein may be required to displace the endogenous protein partner (Fle tcher and Hamilton, 2006). Therapeutic antibodies are good examples due to the high specificity binding to their molecular targets and relative stability in human plas ma. An example is adalimumab (Humira™, MW = ~144,190 Da) with an absolute bioavailab ility of 64% (Reicher t, 2008). However, antibodies can also be problem atic in terms of their high production costs, lack of cell-

PAGE 19

4 membrane permeability, and generation of undesirable side effects (Arkin and Wells, 2004; Saraogi and Hamilton, 2008; Verdine and Walensky, 2007). Cl N N H N S NH O O S N NO2 O ABT-737 Cl N N H N S NH O O S N SO2CF3 O O ABT-263 SP4206 Cl N N Cl N NH O O O O F F F N N N O O Nutlin-3 SP304SP4206bindstoIL-2,ABT-737andABT-263bindtoBcl-xL,Nutlin-3bindstoHdm-2, andSP304bindstoTNF(WellsandMcClendon,2007;Wendt,2008;Domling,2008) NH NH2 H2N H N O O N N N Cl Cl O O O O Figure 1.2. Examples of small mol ecular weight inhibitors of PPIs

PAGE 20

5 One of the myths about molecules that ta rget protein-protein interfaces is that these are too large to have “drug-like” propert ies based on the Lipinski’s rule of five (Lipinski, 2004; Lipinski and Hoffer, 2003). However, remarkable exceptions of small molecules (MW = 500 to 900 Da) that bind to pr otein-protein interfa ces with reasonable oral bioavailability have also been reported (Toogood, 2002; Wells and McClendon, 2007; Zhao and Chmielewski, 2005). Some ex amples are cytokine-interleukin-2 (IL-2) binders (Ro26-4550, MW = 560 Da and SP4206, MW = 663 Da), B-cell lymphoma 2 (Bcl-2) binders (ABT-737, MW = 813 Da and ABT-263, MW = 974 Da, in phase I ongoing trials) (Wendt, 2008), human protein double minute 2 (Hdm-2) binders (Nutlin3, MW=581 Da and JNJ-26854165, currently in Ph ase I human clinical trials for lung cancer) (Domling, 2008), human papilloma virus (HPV) E2 binder (compound 23, MW = 684 Da), and cytokine tumor-necrosis factor (TNF) disruptor (SP304, MW = 548 Da). Figure 1.2 shows the structures of some of th ese inhibitors (Wells and McClendon, 2007). 1.3 Role of Bcl-2 Family and Hmd-2 Family Proteins in Apoptosis Many diseases, such as autoimmunit y, inflammatory, neurodegenerative disorders, diabetes, and cancer are the result of disregulation of apoptosis. Programmed cell death, or apoptosis, is a high ly regulated process that contribu tes to the elimination of unnecessary or damaged cells; this occurs when a cell has fulfilled its biological function (Afford and Randhawa, 2000). Apoptosis can occu r via extrinsic and intrinsic pathways. In both pathways, there is an activation of cysteinyl aspartat e proteases (caspases) that operate in proteolytic cascades. The extrinsi c pathway starts outside the cell through an activation signal caused by specific r eceptors called death receptors.

PAGE 21

6 Figure 1.3. Intrinsic pathway of apoptosi s. Adapted from (Youle and Strasser, 2008) The intrinsic pathway is initiated from in side the cell due to several stresses, such as DNA damage, hypoxia, and defective cell cy cle, among other cellular stresses (Youle and Strasser, 2008). This pathway causes the release of pro-apoptot ic proteins that disrupt the mitochondrial membrane; the activa tion of specific caspases enzymes and the release of cytochrome c and other proteins fr om the mitochondria ul timately leading to cell death (Figure 1.3 ) (Afford and Randhawa, 2000). In the intrinsic pathway, proteins of the Bcl-2 family play a significant role in the regulation of the a poptotic process. Bcl-

PAGE 22

7 2 is a 239-amino acid integral membrane pr otein (Danial, 2007). The anti-apoptotic proteins include B-cell ly mphoma extra large (Bcl-xL) and Bcl-2, among others; the proapoptotic proteins include Bcl2-antagonist killer (Bak), Bcl2-associated x protein (Bax), and BH3 interacting domain death agonist (Bid), among others (Wendt et al. 2006). The Bcl-2 family proteins are characterized by sharing one or mo re specific conserved regions known as Bcl-2 homology (BH) BH 1, BH2, BH3, and BH4 domains. These domains are -helical regions that determine the f unction and structure of the proteins (Danial, 2007). Depending on the nature of the apoptotic stimuli, the multidomain Bax and Bak proteins may homo-oligomerize and fo rm aggregates within the mitochondrial outer membrane (MOM); this leads to the formation of pores in the membrane and activates the apoptotic pa thway by the cytochrome c releasing pathway. On the other hand, the anti-apoptotic Bcl-2 and Bcl-xL proteins may heterodimerize with the deathpromoting region, BH3 domain, of Bak and Bax neutralizing their pr o-apoptotic activity (Walensky et al. 2004; Wendt et al. 2006). Accordingly, the overall balance of proand anti-apoptotic protein interactions c ontrols the susceptibility of a cell towards programmed cell death. NMR studies have revealed that the BH3 domain of th e pro-apoptotic protein Bak is required for activity; this region takes an amphipathic -helical conformation when it binds to Bcl-xL. The Bak peptide interacts via hydr ophobic side chains (residues 72 to 87) projecting into the hydrophobic cleft of the Bcl-xL protein. This hydrophobic cleft (620 2) comprises four turns of the -helical BH3 domains of the pro-apoptotic protein partners (Wendt, 2008). Alanine scanning of the Bak peptide identified Val74, Leu78, Ile81, and Ile85 as key binding residues. Mo reover, electrostatic interactions between

PAGE 23

8 the charged side chains of Bak a nd oppositely charged residues of Bcl-xL stabilize the complex formation. Asp83 forms a salt bri dge with a lysine residue of the Bcl-xL protein (Sattler et al. 1997). Another important family of proteins i nvolved in the intrinsi c apoptotic pathway is the murine double minute 2 (Mdm-2) or Hd m-2 in humans. For clarification, Mdm-2 will be used to refer to both proteins. This family is a major regulator of the tumor suppressor protein 53 (p53). Blocking of p53 binding to a hydrophobic groove of the Mdm-2 protein inhibits the degradation of p53, thus leaving the wild type p53 free to be phosphorylated and activated for cell death stimulation. Similar to the Bcl-xL/Bak complex, a crystal structure of the Mdm-2/ p53 complex revealed that the p53 peptide (residues 16 to 28) forms an amphipathic -helix that binds proj ecting into a hydrophobic cleft on the globular Mdm-2 domain (residues 18 to 102) (Czarna et al. 2009; Chen et al. 2005; Popowicz et al. 2008; Yin et al. 2005a). Based on X-ray analysis, Phe19, Trp23, and Leu26 residues of the p53 peptide were re cognized as key binding residues of the Mdm-2/p53 interface (Kussie et al. 1996). Mdm-x is another protein homolog of Mdm-2 and it also binds the transactivati on domain of p53 (p53 AD) suppressing the activation of p53 target genes. Unlike Mdm-2, Mdm-x is not transcri ptionally induced by p53 and does not promote p53 degradation (Stad et al. 2001; Stad et al. 2000). Nevertheless, Mdm-x is able to heterodimerize with Mdm-2 stimulating the ubiquitination of Mdm-2 a nd degradation of p53 (Sharp et al. 1999). 1.4 Peptidic and Non-Peptidic -Helical Mimetics Previous studies have demonstrated that overexpression of Bcl-2, Bcl-xL, Mdm-2, and Mdm-x proteins is associated with tumo r progression and drug resistance (Strasser et

PAGE 24

9 al. 1997); therefore, the design and synthesis of compounds that can antagonize these proteins represents a great potential for medi cinal chemistry studies. More specifically, the synthesis of compounds designed to mimic the -helical region of the pro-apoptotic Bak BH3 domain and the NH2 terminus of p53 represen ts a potential in cancer therapeutics. Various strategies for the iden tification of small molecule inhibitors of protein-protein complexes have been describe d in the literature. One common method includes the design of inhibitors by scr eening through competitive binding, enzymatic, fluorometric, and phenotypic assays; also by virtual screening techniques. Another approach is the identification of inhibitors by structure-based desi gn; this relies on the understanding of the protein’ s 3-D structure usually ex plored by nuclear magnetic resonance (NMR), X-ray, and protein homology studies. From this, a template scaffold can be selected and side chains can be attached in a way that these project in the same spatial orientation as the known ligand’s key binding residues (Toogood, 2002). This rational design has been applied for the develo pment of small organi c molecules that can mimic secondary structures of proteins including -sheet and -helical conformations to inhibit PPIs. The synthesis of -peptides as potential mimi cs of protein secondary structures has also been reporte d (Chin and Schepartz, 2001; Kritzer et al. 2004). peptides have an additional carbon atom in the main backbone that increases metabolic resistance, as compared to corresponding -peptides (Figure 1.4 ). In general, -peptide oligomers can adopt an -helical conformation based on the substitu tion pattern of the comprising -amino acids; these are named based on the number of at oms in a ring closed by a hydrogen bonding network specific to the helix. These are classified as 8helix, 10-helix, 10/12-helix, 12-helix, and 14-helix.

PAGE 25

10 Figure 1.4. Structures of -peptide and -peptides Schepartz and co-workers synthesized a library of 3-peptide oligomers that showed 14-helix character in aqueous me dia. One of these peptides labeled 53-1 was reported to show significant nanomolar act ivity for the disruption of the Mdm-2/p53 complex (Kritzker, 2004). Gellman and co-workers also reported on -peptides as helical mimetics (Lee et al. 2009; Raguse et al. 2002; 2003). The design of small compounds able to mimic -helical structures has been extensively investigated by se veral groups. Verdine and co-w orkers described the studies towards targeting the interaction between Bcl-2 and Bid. His approach was based on “hydrocarbon-stapling” the native BH3 peptide through a covalent cross-linking strategy. The resulting stapled BH3 peptid omimetics, known as stabilized -helix of Blc-2

PAGE 26

11 domains (SAHB), were reported to have improved pharmacological properties as compared to the native BH3 peptide (Figure 1.5 ) (Walensky et al. 2004; Walensky et al. 2006). Figure 1.5. Depiction of a stapled peptide (Walensky et al. 2006) Hamilton and co-workers designed and s ynthesized several scaffolds including oligoamide foldamers, such as trispyridylamides (Ernst et al. 2003), terphenylene (Fletcher and Hamilton, 2006; Kutzki et al. 2002; Orner et al. 2001; Yin et al. 2005a; Yin et al. 2005b) and terephthalamide pre-orga nized scaffolds (Yin and Hamilton, 2004), and benzoylurea oligomers (Rodriguez et al. 2009b) as structural templates for the design of functional -helical mimetics (Figure 1.6 a-e). The most successful compounds have been derived from the terphenylene template (Figure 1.6 top panel-b and c). The terphenylene was designed to adopt a staggered conformation in such a manner that appropriate ortho -substituents at th e 3, 2’, and 2” positions on the phenyl rings would project functionality in the same orientation of the ith ith +3 or ith +4, and ith +7 residues through two turns on a single face of a target -helix (Fletcher and Hamilton, 2006; Yin et al. 2005b). Several PPIs were targeted with these terphenylbased compounds; an initial target was the in teraction between the calmodulin (CaM) and smooth muscle myosin light-chain kinase (smMLCK) complex (Orner et al. 2001). More recently, the inhibition of the Mdm-2/p53 and the Bcl-xL/Bak interactions were described (Yin et al. 2005a; Yin et al. 2005b).

PAGE 27

12 Figure 1.6. -Helix mimics reported by Hamilton and co-workers Fluorescence polarization (FP) assays i ndicated that terphe nylene derivative A, shown in Figure 1.6 top panel-b, was their most potent antagonist of the Mdm-2 with a

PAGE 28

13 binding affinity in the nanomolar range ( Ki = 182 nM) (Yin et al. 2005a). Terphenylbased -helical mimetic B, shown in Figure 1.6 top panel-c, was re ported as a promising inhibitor of the Bcl-2/Bak complex ( Ki = 114nM) (Yin et al. 2005b). Although the versatility of the terphenylene scaffold has been demonstrated, there are some disadvantages based on reported challenging syntheses and relatively low hydrophilicity of the resulting compounds. The more polar terephthalamide scaffold (Figure 1.6 bottom panel-d) replaced the ending phenyl rings of the terphenylene by two functionalized carboxamide groups increasing the solubility and drug-like characteristics of the compound; but the binding affi nity of this derivative for Bcl-xL was found to decrease ( Ki = 780 nM) as compared to the earlier terphenylene analogs (Saraogi and Hamilton, 2008; Yin and Hamilton, 2004). Figure 1.7. Polar -helix mimics reported by Hamilton’s group In the same context, a series of more polar analogs of the terphenylene based on terpyridine (Davis et al. 2005) and biphenyl 4,4’-dicarboxamide scaffolds (Rodriguez

PAGE 29

14 et al. 2009a) were recently reporte d by Hamilton’s group (Figure 1.7 ). The latter was designed to mimic the ith ith +4, ith +7, and ith +11 residues of an extended -helix and the most potent inhibitor (Figure 1.7 b) for the Bcl-xL/Bak interaction have a Ki value of 1.8 M by FP assay. Rebek and co-workers have also publis hed several analogs of the Hamilton’s terphenylenes as -helical mimetics. Most of these trimeric heterocyclic scaffolds contain a pyridazine central ring and are described to be more drug-like than Hamilton’s terphenylene scaffolds (Figure 1.8 a-d) (Biros et al. 2007; Moisan et al. 2007; Moisan et al. 2008; Volonterio et al. 2007). Rebek’s group has also reported tetrameric heterocyclic -helix mimics based on a piperazine scaffold (Figure 1.8 bottom panel-e) (Restorp and Rebek, 2008). Knig’s group has recently published the synthesis of chiral peptide mimetics based on a functionalized 1 ,4-dipiperazino benzen e scaffold (Figure 1.8 bottom panel-f), which is another anal og of Hamilton’s terphenylenes. These compounds were described to adopt a stagge red conformation with the substituents resembling the side chain residues of an -helix (Maity and Koni g, 2008). Very recently, Shaginian and co-workers reported a comprehensive compound library (8000 compounds) as -helical mimetics for the disruption of the Mdm-2/p53 complex (Shaginian et al. 2009). In summary, secondary structures show va riations within proteins; however, it has been demonstrated that conformationally re stricted peptides can block PPIs and that advances in designing peptidomimetics from peptide sequences have proven successful. For this reason, it should be evident that mimetics base d on protein-substructural

PAGE 30

15 components could hold great pr omise as valuable pharmaco logical tools and potential new therapeutic agents. Figure 1.8. -Helix mimics reported by Rebek’s and Knig’s groups While significant progress has been made in the rational design aimed at mimicking peptide or protein structural confor mations, as described in this chapter, this field is still open to further investigation. T hus, encouraged by the recent interest in the development of small compounds that bind to PPI surfaces and inspired by the intense work and success of the groups previously mentioned, we focused on the design and

PAGE 31

16 synthesis of compounds that mimic the orie ntation of side chain residues of an -helical protein domain. Our scaffolds could potentia lly inhibit various PPI s; but we focused mainly in the disruption of interactions among the Bcl-2-family and the Mdm-2-family proteins since these protein families have been implicated in critical roles of cellular homeostasis. Chapter Two describes the desi gn and our efforts towards the synthesis of -helical mimetics based on 3-R-1,4-hydrazin e-linked piperazine-diones and Chapter Three discusses our most recent results towards the synthesis of -helical mimetics based on substituted 2,5-terpyrimidinylenes. Ou r approach is analogous to Hamilton’s approach with the terphenylenes but our semi-rigid scaffolds are further designed to have drug-like physical properties to increase the potential therapeutic applications of resulting PPI modulators. 1.5 References Afford, S.; Randhawa, S. ( 2000 ) Apoptosis. Molecular Pathology, 53 (2), 55-63. Arkin, M. R.; Wells, J. A. ( 2004 ) Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nature Reviews Drug Discovery, 3 (4), 301317. Biros, S. M.; Moisan, L.; Ma nn, E.; Carella, A.; Zhai, D.; Reed, J. C.; Rebek, J. ( 2007 ) Heterocyclic alpha -helix mimetics for targeting protein-protein interactions. Bioorganic & Medicinal Chemistry Letters, 17 (16), 4641-4645. Branden, C.; Tooze, J., Introduction to Protein Structure 1992; p 332 pp. Cutler, P.; Gemperline, P. J.; de Juan, A. ( 2009 ) Experimental monitoring and data analysis tools for protein folding. Analytica Chimica Acta, 632 (1), 52-62. Czarna, A.; Popowicz, G. M.; Pecak, A.; Wolf, S.; Dubin, G.; Holak, T. A. ( 2009 ) High affinity interaction of the p53 peptid e-analogue with human Mdm2 and Mdmx. Cell Cycle, 8 (8), 1176-1184. Chen, L.; Yin, H.; Farooqi, B.; Sebti, S.; Hamilton, A. D.; Chen, J. ( 2005 ) p53 alpha Helix mimetics antagonize p53/MDM2 interaction and activate p53. Molecular Cancer Therapeutics, 4 (6), 1019-1025.

PAGE 32

17 Chin, J. W.; Schepartz, A. ( 2001 ) Design and evolution of a miniature Bcl-2 binding protein. Angewandte Chemie, International Edition, 40 (20), 3806-3809. Danial, N. N. ( 2007 ) BCL-2 Family Proteins: Critical Checkpoints of Apoptotic Cell Death. Clinical Cancer Research, 13 (24), 7254-7263. Davis, J. M.; Truong, A.; Hamilton, A. D. ( 2005 ) Synthesis of a 2,3';6',3''-Terpyridine Scaffold as an alpha -Helix Mimetic. Organic Letters, 7 (24), 5405-5408. Domling, A. ( 2008 ) Small molecular weight proteinprotein interaction antagonists-an insurmountable challenge?, Current Opinion in Chemical Biology, 12 (3), 281-291. Ernst, J. T.; Becerril, J.; Park, H. S.; Yin, H.; Hamilton, A. D. ( 2003 ) Design and application of an alpha-helix-mimetic scaffo ld based on an oligoamide-foldamer strategy: antagonism of the Bak BH3/Bcl-xL complex. Angewandte Chemie (International ed. in English), 42 (5), 535-9. Fletcher, S.; Hamilton, A. D. ( 2006 ) Targeting protein-protein interactions by rational design: mimicry of protein surfaces. Journal of the Royal Society, Interface, 3 (7), 215233. Garrett, R. H. and Grisham, C. M. ( 1999 ) Biochemistry, 2nd Ed. ; Saunders College Publishing: Fort Worth. Gerrard, J. A.; Hutton, C. A.; Perugini, M. A. ( 2007 ) Inhibiting protein-protein interactions as an emerging paradigm for drug discovery. Mini-Reviews in Medicinal Chemistry, 7 (2), 151-157. Kritzer, J. A.; Stephens, O. M.; Guarracino, D. A.; Reznik, S. K.; Schepartz, A. ( 2004 ) beta -Peptides as inhibitors of protein-protein interactions. Bioorganic & Medicinal Chemistry, 13 (1), 11-16. Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.; Pavletich, N. P. ( 1996 ) Structure of the MDM2 onc oprotein bound to the p53 tumor suppressor transactivation domain. Science (New York, N.Y.), 274 (5289), 948-53. Kutzki, O.; Park Hyung, S.; Ernst Justin, T.; Orner Brendan, P.; Yin, H.; Hamilton Andrew, D. ( 2002 ) Development of a potent Bcl-x(L) antagonist based on alpha-helix mimicry. Journal of the American Chemical Society, 124 (40), 11838-9. Lee, E. F.; Sadowsky, J. D.; Smith, B. J.; Czabotar, P. E.; Peterson-Kaufman, K. J.; Colman, P. M.; Gellman, S. H.; Fairlie, W. D. ( 2009 ) High-Resolution Structural Characterization of a Helical alpha /beta -Peptide Foldamer Bound to the Anti-Apoptotic Protein Bcl-xL. Angewandte Chemie, International Edition, 48 (24), 4318-4322, S4318/1-S4318/6.

PAGE 33

18 Lipinski, C. A. ( 2004 ) Leadand drug-like compounds: the rule-of-five revolution. Drug Discovery Today Technologies, 1 (4), 337-341. Lipinski, C. A.; Hoffer, E. ( 2003 ) Compound properties and drug quality. Practice of Medicinal Chemistry (2nd Edition) 341-349. Maity, P.; Konig, B. ( 2008 ) Synthesis and Structure of 1,4Dipiperazino Benzenes: Chiral Terphenyl-type Peptide Helix Mimetics. Organic Letters 10 (7), 1473-1476. Moisan, L.; Dale, T. J.; Gombosuren, N.; Biro s, S. M.; Mann, E.; Hou, J.-L.; Crisostomo, F. P.; Rebek, J., Jr. ( 2007 ) Facile synthesis of pyridazine-based alpha -helix mimetics. Heterocycles, 73, 661-671. Moisan, L.; Odermatt, S.; Gombosure n, N.; Carella, A.; Rebek, J., Jr. ( 2008 ) Synthesis of an oxazole-pyrrole-piperazine scaffo ld as an alpha -helix mimetic. European Journal of Organic Chemistry, (10), 1673-1676. Orner, B. P.; Ernst, J. T.; Hamilton, A. D. ( 2001 ) Toward proteomimetics: terphenyl derivatives as structural and functional mimi cs of extended regions of an alpha-helix. Journal of the American Chemical Society, 123 (22), 5382-3. Perez de Vega, M. J.; Martin-Martinez, M.; Genzalez-Muniz, R. ( 2007 ) Modulation of protein-protein interactions by stabilizing/mimicking protein secondary structure elements. Current Topics in Medicinal Chemis try (Sharjah, United Arab Emirates), 7 (1), 33-62. Popowicz, G. M.; Czarna, A.; Holak, T. A. ( 2008 ) Structure of the human Mdmx protein bound to the p53 tumor suppresso r transactivation domain. Cell Cycle, 7 (15), 2441-2443. Raguse, T. L.; Lai, J. R.; Gellman, S. H. ( 2002 ) Evidence that the beta -peptide 14-helix is stabilized by beta 3-residues with side-c hain branching adjacent to the beta -carbon atom. Helvetica Chimica Acta, 85 (12), 4154-4164. Raguse, T. L.; Lai, J. R.; Gellman, S. H. ( 2003 ) Environment-Independent 14-Helix Formation in Short beta -Peptides: Stri king a Balance between Shape Control and Functional Diversity. Journal of the Americ an Chemical Society, 125 (19), 5592-5593. Reichert, J. M. ( 2008 ) Monoclonal antibodies as innovative therapeutics. Current Pharmaceutical Biotechnology, 9 (6), 423-430. Restorp, P.; Rebek, J. ( 2008 ) Synthesis of alpha -helix mi metics with four side-chains. Bioorganic & Medicinal Chemistry Letters, 18 (22), 5909-5911.

PAGE 34

19 Rodriguez, J. M.; Nevola, L.; Ross, N. T.; Lee, G.-i.; Hamilton, A. D. ( 2009a ) Synthetic inhibitors of extended helix-protein inter actions based on a biphenyl 4,4'-dicarboxamide scaffold. ChemBioChem, 10 (5), 829-833. Rodriguez, J. M.; Ross, N. T.; Katt, W. P.; Dhar, D.; Lee, G.-i.; Hamilton, A. D. ( 2009b ) Structure and Function of Benzoylurea-Derive d alpha -Helix Mimetics Targeting the BclxL/Bak Binding Interface. ChemMedChem, 4 (4), 649-656. Saraogi, I.; Hamilton, A. D. ( 2008 ) alpha -Helix mimetics as i nhibitors of protein-protein interactions. Biochemical Society Transactions, 36 (6), 1414-1417. Sattler, M.; Liang, H.; Nettesheim, D.; Meadow s, R. P.; Harlan, J. E.; Eberstadt, M.; Yoon, H. S.; Shuker, S. B.; Chang, B. S.; Mi nn, A. J.; Thompson, C. B.; Fesik, S. W. ( 1997 ) Structure of Bcl-xL-Bak peptide comp lex: recognition between regulators of apoptosis. Science (Washington, D. C.), 275 (5302), 983-986. Shaginian, A.; Whitby, L. R.; Hong, S.; Hwan g, I.; Farooqi, B.; Searcey, M.; Chen, J.; Vogt, P. K.; Boger, D. L. ( 2009 ) Design, Synthesis, and Evaluation of an alpha -Helix Mimetic Library Targeting Pr otein-Protein Interactions. Journal of the American Chemical Society, 131 (15), 5564-5572. Sharp, D. A.; Kratowicz, S. A.; Sank, M. J.; George, D. L. ( 1999 ) Stabilization of the MDM2 oncoprotein by interaction with the structurally related MDMX protein. The Journal of biological chemistry, 274 (53), 38189-96. Stad, R.; Little, N. A.; Xirodimas, D. P.; Fre nk, R.; van der Eb, A. J.; Lane, D. P.; Saville, M. K.; Jochemsen, A. G. ( 2001 ) Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms. EMBO reports, 2 (11), 1029-34. Stad, R.; Ramos, Y. F.; Little, N.; Grivell, S.; Attema, J.; van Der Eb, A. J.; Jochemsen, A. G. ( 2000 ) Hdmx stabilizes Mdm2 and p53. The Journal of biological chemistry, 275 (36), 28039-44. Strasser, A.; Huang, D. C. S.; Vaux, D. L. ( 1997 ) The role of the bcl-2/ced-9 gene family in cancer and general implications of defects in cell death control for tumorigenesis and resistance to chemotherapy. Biochimica et Biophysica Acta, Reviews on Cancer, 1333 (2), F151-F178. Toogood, P. L. ( 2002 ) Inhibition of Protein-Protein Association by Small Molecules: Approaches and Progress. Journal of Medicinal Chemistry, 45 (8), 1543-1558. Verdine, G. L.; Walensky, L. D. ( 2007 ) The Challenge of Drugging Undruggable Targets in Cancer: Lessons Learned from Targeting BCL-2 Family Members. Clinical Cancer Research, 13 (24), 7264-7270.

PAGE 35

20 Volonterio, A.; Moisan, L.; Rebek, J., Jr. ( 2007 ) Synthesis of pyridaz ine-based scaffolds as alpha -helix mimetics. Organic Letters, 9 (19), 3733-3736. Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.; Wagner, G.; Verdine, G. L.; Korsmeyer, S. J. ( 2004 ) Activation of Apoptosis in Vivo by a Hydrocarbon-Stapled BH3 Helix. Science (Washington, DC, United States), 305 (5689), 1466-1470. Walensky, L. D.; Pitter, K.; Morash, J.; Oh, K. J.; Barbuto, S.; Fisher, J.; Smith, E.; Verdine, G. L.; Korsmeyer, S. J. ( 2006 ) A stapled BID BH3 helix directly binds and activates BAX. Molecular Cell, 24 (2), 199-210. Wells, J. A.; McClendon, C. L. ( 2007 ) Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature (London, United Kingdom), 450 (7172), 1001-1009. Wendt, M. D. ( 2008 ) Discovery of ABT-263, a Bcl-family protein inhibitor: observations on targeting a large protein-protein interaction. Expert Opinion on Drug Discovery, 3 (9), 1123-1143. 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. ( 2006 ) Discovery and Structure-Activity Relationship of Antagonists of B-Cell Lymphoma 2 Family Proteins with Chemopotentiation Activity in Vitro and in Vivo. Journal of Medicinal Chemistry, 49 (3), 1165-1181. Williamson, M. P. ( 1994 ) Nuclear magnetic resonance st udies of peptides and their interactions with receptors. Biochemical Society Transactions, 22 (1), 140-4. Yin, H.; Hamilton, A. D. ( 2004 ) Terephthalamide derivatives as mimetics of the helical region of Bak peptide ta rget Bcl-xL protein. Bioorganic & Medicinal Chemistry Letters, 14 (6), 1375-1379. Yin, H.; Lee, G.-i.; Park, H. S.; Payne, G. A. ; Rodriguez, J. M.; Sebti, S. M.; Hamilton, A. D. ( 2005a ) Terphenyl-based helical mimetics that disrupt the p53/HDM2 interaction. Angewandte Chemie, International Edition, 44 (18), 2704-2707. Yin, H.; Lee, G.-i.; Sedey, K. A.; Kutzki, O.; Pa rk, H. S.; Orner, B. P.; Ernst, J. T.; Wang, H.-G.; Sebti, S. M.; Hamilton, A. D. ( 2005b ) Terphenyl-Based Bak BH3 alpha -Helical Proteomimetics as Low-Molecula r-Weight Antagonists of Bcl-xL. Journal of the American Chemical Society, 127 (29), 10191-10196. Youle, R. J.; Strasser, A. ( 2008 ) The BCL-2 protein family : opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology, 9 (1), 47-59.

PAGE 36

21 Zhao, L.; Chmielewski, J. ( 2005 ) Inhibiting protein-protein interactions using designed molecules. Current Opinion in Structural Biology, 15 (1), 31-34.

PAGE 37

22 CHAPTER TWO DESIGN AND SYNTHESIS OF 3R-PIPERAZINE-2,5AND 2,6-DIONES 2.1 Introduction 2.1.1 Piperazine-diones In recent years, there has been an increas ing interest for the design and synthesis of drug leads based on small heterocyclic library templates (Perrotta et al. 2001). The piperazine-dione or diket opiperazine unit (DKP), in cluding 2,3-, 2,5-, and 2,6-DKPs (Figure 2.1 ) are among the most attractive hetero cyclic motifs due to their wide representation in many biologically ac tive compounds and their application as structurally constrained active peptide an alogs (Prasad, 1995). Many DKPs have been reported to have antihistaminic, antibacter ial (albonoursin, bicyclom ycin), and antitumor properties (TAN-1496 A, C, and E), among others (Besada et al. 2005; Funabashi et al. 1994; Insaf and Witiak, 2000; Martins and Carvalho, 2007; Singh and Tomassini, 2001; Williams et al. 1985). DKPs are also found in processed foods and beverages (Gautschi et al. 1997). Figure 2.1. Structures of unsubsti tuted diketopiperazine rings The chemistry to synthesize DKPs is well known; an excellent review has been reported by Dinsmore and Beshore (Dinsmore and Beshore, 2002). In general, both 2,5

PAGE 38

23 and 2,6-DKPs can be prepared from -amino acids as starting materials by step-wise intramolecular cyclization of the appropr iate molecular frag ments or tandem and multicomponent reactions as used in combinatorial chemistry applications (Gellerman et al. 2008). DKPs can be efficientl y prepared by solution phas e or solid phase chemistry and in many cases, 2,5-DKPs originate as unw anted byproducts during the process of oligopeptide synthesis. DKP ring systems ar e the smallest cyclic peptides known and their significance in the drug di scovery field remains based on the intrinsic physical and chemical properties of their rigid conf ormation and the presence of hydrogen bond acceptor and donor groups for interactions with the biological targets. This feature makes DKPs less susceptible to metabolic degrada tion of the amide bond compared to linear peptides and it increases the drug-like prope rties of compounds c ontaining the DKP unit (Dinsmore and Beshore, 2002; Perrotta et al. 2001). In this project, we focused on the desi gn and synthesis of peptidomimetics based on semi-rigid scaffolds derived from 3-R-pi perazine-2,5and 2,6-di one repetitive units linked by hydrazine bonds. The proposed target molecule A is shown in Figure 2.2 This novel piperazine-2,6-dione scaffold holds ami no acid-like side chain residues in positions that structurally mimic the ith ith +3 or ith +4, and ith +7 sites of one face of an -helix. More specifically, these compounds are designed to mimic the -helical region of the BH3 Bak peptide or the p53 pe ptide derived from the p53 N -terminus for their interactions with anti-apopt otic Bcl-2 and Mdm-2 family proteins, respectively. This approach is analogous to that reported by Hamilton et al. (discussed in Chapter One), although our scaffold includes a more hydr ophilic core that still conserves the hydrophobic side chains re quired for activity (Yin et al. 2005b; Yin et al. 2005c).

PAGE 39

24 Figure 2.2. DKPA and DKPB: Target –helical peptidomimetics The R1, R1 ’, R2, R2 ’, and R3, R3 ’, positions can be widely varied by selecting the appropriate starting natural or unnatural ami no acids to originate the desired sequence DKP1-DKP2-DKP3 as shown in Figure 2.2 or any of the other possible sequences. In addition, the stereochemistry of the target scaffold can also be controlled by proper selection of chir al or nonchiral -substituted amino acid derivatives. The N -terminuslike and C -terminus-like linker attachments to the e nding DKP units can also be varied in length and charge to give various structural possibilities. A potential drawback for the design of this novel scaffold as a drug lead is the presence of the hydrazine bond linkage (nitrogen-nitrogen single bond) given that this could generate toxic metabolites derived from the hydrazine unit (Bollard Mary et al. 2005; Goodwin et al. 1996; Malca-Mor and Stark, 1982). However, the hydrazine group is also found in many bioactive

PAGE 40

25 compounds; some examples of drugs c ontaining the hydrazine moiety include antidepressants (isocar boxazid, phenelzine), antiparkins onic agent (carbidopa), antiviral (methiazone), antibacte rial (sulfaphenazole), and alkylat ing agent (procarbazine) (Gilbert et al. 2000; Toth, 1996). 2.2 Results and Discussion 2.2.1 Synthesis of 3-R-Pip erazine-2,6-diones: DKPA 3-R-piperazine-2,6-diones can be prep ared by solution phase or solid phase peptide (SPP) techniques. A general re trosynthetic analysis of scaffold DKPA via solution phase is shown in Figure 2.3 Both routes require -amino esters type A which are commercially available or can be readily prepared from corresponding -amino acids. The difference between Route 1 and Route 2 is the formation of monoacid derivatives C or anhydrides D which originate from respective diesters B A DKP1 monomer results from a two-step intramolecular cyc lization after coupling of monoacid C or anhydride D with a -alanine linker derivative. In bot h routes, coupling of the resulting DKP1 monomer with another monoacid C ’ or anhydride D’ can form the dimeric DKP1-DKP2 scaffold, which can successively form the target oligomeric DKPA over iteration of these steps. The intermediate anhydride derivatives D were prepared from corresponding diacids. In this study, the s ynthesis of diacids and monoacids and the formation of some individual building blocks DKP will be discussed. A more general description for the coupling of the DKP units via solution and solid pha se peptide techniques will be presented including the synthesis of a trimeric peptidomimetic type DKPA

PAGE 41

26 Figure 2.3. Retrosynthetic analysis of 3-R-piperazine-2,6-dione scaffold DKPA

PAGE 42

27 2.2.2 Synthesis of Diacid Derivatives 2.6: Route 1 N tert -butoxycarbonyl (Boc) prot ected hydrazine diacids 2.6 were synthesized in three or four steps (Scheme 2.1 ). The methyl esters of free amino acids 2.1e and 2.1g and the N -Boc protected amino ester 2.1f were prepared by a thionyl chloride-mediated reaction with methanol. Remaining -amino ester HCl salts 2.1a-d were obtained from commercial sources. Scheme 2.1. Overall synthesis of diacid derivatives 2.6 Initially, the N -Boc protected amino acid of 2.1f was treated with methyl iodide and potassium carbonate in dimethylformamid e (DMF); subsequent removal of the Boc group by standard conditions with TF A/DCM enabled the preparation of 2.2f Although this two-step process was successful in obtaining compound 2.2f in excellent yield, it was more convenient when compound 2.1f was treated directly wi th thionyl chloride in

PAGE 43

28 methanol since both removal of the Boc group and formation of the ester occurred in a one pot reaction. Methyl ester derivatives 2.2e-g were obtained as the HCl salts in excellent yields (>90%). In the next step, the methyl esters of the selected natural and unnatural amino acids 2.2a-g were N -alkylated with ethyl bromoacetate (EBA) in the presence of Hnig’s base, diisopropylethylamine (DIEA). Monoester derivatives 2.2a-d,f,g were not completely soluble in acetonitrile (MeCN), but homogenous solutions were obtained after the addition of DIEA and EBA. Conversely, compound 2.2e was poorly soluble in MeCN, thus the reaction was done using DM F, for which the product required further purification. During the N -alkylation reaction, we observed that temperature and reaction time were important to the outcome of the r eaction. When the reaction was stirred at rt for several hours, it was observed that side product formation decreased compared to those reactions where heating and shorter reaction times were involved. Although dialkylation side reactions can occu r, it has been found that using the -halo-alkyl acetate is an efficient method for the preparation of diester 2.3 from good to excellent yields (6088%). The alternative reductive amination rout e with glyoxalic acid or ethyl glyoxalate in the presence of sodium triacetoxyborohydride (Oguz et al. 2002) gave poorer yields, probably due to competition for reduction of the aldehyde or dialkylated side products (Abdel-Magid et al. 1996). A key step in this synt hesis is the formation of N -Boc protected hydrazino diester 2.5 Several procedures for the synthesis of unsubstituted and substituted hydrazines have been reported in literature. Unsubsti tuted hydrazines can be prepared by reduction of the hydrazone derived from an -keto acid and reaction of an -halo acid with

PAGE 44

29 hydrazine (Genari et al., 1996); optically active hydrazine s can be prepared via asymmetric electrophilic amination (Genari et al., 1996). Substituted hydrazines can be prepared by reaction of alkyl ureas with hypochlorite under basic conditions, direct substitution of hydrazine with triphenylbismuthane in th e presence of copper acetate (Ragnarsson, 2001), nitrosation of a secondary amine followed by its selective reduction and Boc protection (Oguz et al. 2002), and direct transfer of the Boc group by electrophilic amination (Vidal et al. 1993; Vidal et al. 1998). Although the formation of nitrosamines is widely used and it has b een previously explored in our lab, the latter method was adopted due to its efficiency. It was more convenient to obtain the hydrazines already substituted with the Boc protecting group to reduce the amount of steps of the entire synthesis and to prevent self-condensatio n of resulting free hydrazines (Oguz et al. 2001; Oguz et al. 2002). N -Boc-hydrazino derivatives 2.5 were then prepared by el ectrophilic amination with tert butyl 3(trichloromethyl)1,2-oxaziridine-2-carboxylate 2.4 ( N Boc oxaziridine). N -Boc oxaziridine 2.4 was prepared in our lab by adapting a literature procedure (Hannachi et al. 2004; Vidal et al. 1993; Vidal et al. 1998). One advantage of this method is the use of the N -alkoxycarbonyloxaziridine to cl eanly transfer the desired N -Boc protected group to the secondary amine. Also, this reagent is easier to handle compared to the t butyl nitrite reagent required for a ni trosation step, for example. Diester 2.3 was reacted with NBoc oxaziridine in MeOH, at -78 C to yield 2.5 derivatives. It has been reported that best yields of the hydr azines are obtained using MeCN at -40 C to rt or MeOH at 78 C to rt for 12 hours and a slight excess of oxaziridine (Avancha, 2006). When the same conditions were used with derivatives 2.3a and 2.3b the results were slightly

PAGE 45

30 different as various side products were obs erved by TLC and the percent yields were lower than those reported (4065%). We obs erved that anhydrous methanol gives better results than anhydrous acetonit rile as solvent for these reactions. To monitor the conditions for an efficient preparation of hydrazines 2.5 the temperature, reaction time, and the stoichiometric amount of oxaziridine were modified using 2.3a as a model substrate (Table 2.1 ). Table 2.1. Model synthesis of hydrazine diester 2.5a Side reactions were reduced by loweri ng the reaction temperature. The best results were obtained when the reaction was performed at temperatures not exceeding 5 C and using 3-fold excess of oxaziridine 2.4 (Table 2.1 entry 5). It should be noted that the oxaziridine reagent should be stored in an air-tight vessel at cold temperature to avoid

PAGE 46

31 its decomposition by hydrolysis (Avancha, 2006). Oxaziridines are also common reagents for oxygen transfer; in fact, the side product hydroxylamine 2.6a was isolated and characterized ( 2.6a in Table 2.1 ). The hydrazine of -disubstituted diester ( 2.5e ) was obtained in low yield as a result of th e more steric hindered 2-aminoisobutyric diester 2.3e (Scheme 2.2 top panel-a). In attempts to improve this yield, the hydrazine was prepared directly from amino ester 2.2e but subsequent N -alkylation of the resulting secondary amine 2.7e was unsuccessful (Scheme 2.2 bottom panel-b). Scheme 2.2. Attempted routes for the synthesis of hydrazine diester 2.5e The next step of the synthesis is the hydrolysis of the ester groups of 2.5 (Scheme 2.1 ), a required step for further intramolecular cyclization. In order to retain the Boc group, hydrolysis of compound 2.5 was performed under basic conditions. Less hindered amino acid derivatives, su ch as phenylalanine ( 2.5a ) and leucine ( 2.5b ), were completely

PAGE 47

32 hydrolyzed to the diacid with 1N NaOH in MeOH. NaOH solutions were freshly prepared to prevent prolonged contact with CO2 and consequent decrease of its basicity by formation of carbonate species (Southgate, 2000). Treatment of more hindered amino acid derivatives, such as valine ( 2.5c ) and isoleucine ( 2.5d ), with 1N NaOH resulted in a partial hydrolysis to the monoacid. Although further attempts to hydrolyze with NaOH were also successful without racemizati on, the use of LiOH formed the desired compound with less harsh condit ions that might lead to the racemization of the -carbon with some substrates (Anderson et al. 2009; Weiss, 2006). 2.2.3 Cyclization and Coupling of Piperazine-2,6-diones Cyclization of monomeric DKP1 containing a -alanine-derivative linked to the Cterminus and subsequent coupling with other DKP units can be pursued by both solution and solid phase peptide (SPP) tech niques. Dr. Umut Oguz, a former postdoctoral researcher in our lab, pursue d the cyclization a nd coupling of the DKP units via SPP. Details of the chemistry explored by Dr. Oguz are not described here, but only analogs prepared following her reported protocols or independently developed procedures are discussed. A general scheme is shown in Scheme 2.3 The synthesis of trimeric piperazine2,6-diones by SPP were reported by Dr. Oguz. Trimeric sca ffolds were assessed against the Bcl-xL/Bak interaction by FP assay. Out of th e possible sequences that can result from various starting amino acids, the trimer having Leu-Phe-Val amino acid-like side chain sequence ( 2.9bac) was the most promising lead of this series. Compound 2.9bac exhibited <5 M IC50 in this in vitro assay.

PAGE 48

33 Scheme 2.3. General synthetic procedure of trimeric piperazine-2,6-dione 2.9bac via SPP A computational docking model study perf ormed at the Moffitt Cancer Center suggested that longer -helical mimetics (four or six DKP units) could bind to Bcl-xL with higher binding affinity. Figure 2.4 shows the results of the docking of a hexameric

PAGE 49

34 piperazine-2,6-dione analog Pip and Bcl-xL/Bax complex. As seen with Hamilton terphenylenes (Yin et al. 2005b) and terephthalamide deri vatives (Yin and Hamilton, 2004), the computational docking mode ling of our hexameric scaffold Pip and Bcl-xL also indicates that our inte nded inhibitor binds in the sa me hydrophobic region as the Bak peptide (Figure 2.4 top panel-a). This also supports the idea that many PPIs take place through the contact of three or more side residues, usually along a single face of an extended -helix (Rodriguez et al. 2009a). Therefore, the s ynthesis of compounds that can mimic the spatial projection of ith ith +3/ ith +4, and ith +7 sites on two turns of the helix (our trimeric peptidomimetics) or five turns (hexameric proteomimetics) by the ith ith +3/ ith +4, ith +7, and ith +11 sites remains the main interest in our group. Encouraged by these observations and the results of the in vitro evaluation, we envisioned to reproduce the synthesis of compound 2.9bac and most particularl y, to improve the yield of the coupling step to enable the synthesis of longer helic al mimetics. Unfortunately, our collaborative efforts to re-synthesize th e desired trimer by SPP were unsuccessful; cyclization of DKP units proved to be the most challe nging step. Cleavage of the resin after coupling of DKP1 with the -alanine derivative was done to confirm if ring closure had taken place, but only traces of the targ et monomer and uncyclized intermediates were observed as determined by 1H NMR.

PAGE 50

35 a) b) Figure 2.4. Docking studies of a hexameric piperazine2,6-dione analog (Pip) and Bcl-xL/Bak complex. ( a ) Full view. ( b ) Close up of overlay. Bak peptide is shown in green and its key binding side chains are s hown in red as stick representations. Bcl-xL protein is shown in pink (adapted from a PDB file created by Dr. Shen-Shu Sung). Original PDB file 1BXL of Bcl-xL / Bak complex was reported by Fesik’s group (Sattler et al. 1997).

PAGE 51

36 Table 2.2. Failed attempts to is olate anhydride intermediates In order to advance this pr oject, we then focused our attention on the cyclization and coupling of the DKP monomers by solution phase chemistry. Accordingly, hydrazine diacid 2.6a was treated with a condensing agent, usually diisopropylcarbodiimide (DIC), to generate the corre sponding cyclic acid anhydride. Initially, we attempted the isolation of the an hydride to purify it from substituted urea byproducts and uncyclized materials; severa l reaction conditions were done reacting diacid hydrazines 2.6a and 2.6b with various condens ing agents (Table 2.2 ). While the desired anhydrides were obs erved by TLC and HPLC monito ring, isolation of these was difficult since the compounds were unstable and in most cases these hydrolyzed back to

PAGE 52

37 starting diacids 2.6 Based on these results, we opted for generating the anhydride in situ and coupling it to a -alanine linker ( 2.10 or 2.11 ) in a one pot reaction to afford derivatives 2.12 (Scheme 2.4 ). The N -Boc -alanine benzyl ester 2.10 was prepared in our lab by adapting a procedure described in the literature (Sunagawa et al. 1995); standard conditions (HCl/di oxane) for removal of the Boc protecting group gave salt 2.12 -alanine methyl ester hydrochloride 2.11 was obtained from comm ercial sources. As shown in Scheme 2.4 the coupling of anhydride in termediate with compounds 2.10 and 2.11 afforded hydrazine monoacids 2.12b and 2.13b in modest yields (30-36%). A possible explanation for this can be related to a regiosel ectivity issue. The nitrogen atom of the -alanine moiety could attack the anhydr ide to either carbonyl site and two possible products could be ge nerated. However, the produc t from a reaction at carbon b seemed to be favored as determined by 1H NMR (Scheme 2.4 bottom panel-b). Formation of 2.12b can also be rationalized by the steric hindrance of carbon b over carbon site a due to the presence of th e alpha side chain of carbon a Generation of either product can still afford monomer 2.13b but this leads to more tedious isolation and purification procedures. The cyclization step in solution phase to afford target DKP1 unit 2.14b was performed by Dr. Oguz, who tried to optimize this step with several coupling reagents (optimization data is not discussed here). The best method to obtain compound 2.14b occurred via activation of the carboxy lic acid of 2.12b with acetic anhydride in presence of sodium acetate.

PAGE 53

38 Scheme 2.4. Synthesis of DKP1 monomer 2.13b While it was reported that this acetic anhydride-mediated reaction afforded the best results, a persistent problem was the concomitant acylation of the nitrogen of the amide (Scheme 2.4 compound 2.14b’ ). Acylation of the amide, therefore, prevents closure of the ring. In effort s to overcome this issue, a se cond route for the synthesis of the target DKP units was developed.

PAGE 54

39 2.2.4 Synthesis of Piperazine-2,6-diones: Route 2 As described in Section 2.2.1 the main difference between Routes 1 and 2 to form the DKP monomer is the formation of an ort hogonally protected diester in step one of Route 2 By doing this, we can generate a monoaci d instead of a diacid to facilitate the regioselective coupling of the C -terminal linker ( -alanine derivative 2.10 ). Scheme 2.5. General synthesis of DKP monomers 2.15, Route 2 The forward synthetic route is shown in Scheme 2.5 Synthesis of diester 2.16 was achieved by the N -alkylation of amino ester 2.2 with benzyl bromoacetate (BBA)

PAGE 55

40 instead of EBA as in Route 1 (Scheme 2.1 ). Subsequent treatment of diesters 2.16 with N -Boc oxaziridine 2.4 yielded Boc-protected hydrazines 2.17 in good yields (80-86%). In the next step, removal of the benzyl group was achieved via hydrogenolysis with 5% Pd/C in THF at 35 psi and at rt. It is wo rth mentioning that react ions performed with 10% Pd/C gave products with cleavage of the hydrazine N-N bond as determined by NMR spectroscopy. Formation of monoacids 2.18 allowed regioselective coupling of alanine derivative 2.11 to obtain hydrazine diesters 2.19 Ring closure of compound 2.19 was obtained by a base-catal yzed reaction unde r thermal conditions. The monomer having the Phe-like side chain ( 2.15a ) was obtained in good yield (71%) by using a catalytic amount of NaH in anhydrous THF under thermal conditions; no racemization of the -carbon was observed in this case. However, treatment of compound 2.19b with NaH gave traces of the target product 2.15b in addition to side product 2.20b (Scheme 2.6 ). Compounds 2.15b and 2.15d were obtained when KO t Bu was used, but the yields were rather low (Scheme 2.5 ). Scheme 2.6. Cyclization of 2.19b with NaH We also attempted to optimize the cyclization step by inverting the groups of diester 2.16a in step one. We envisioned that by doing this, the steric hindrance of the reaction site would decrease a nd consequently would facilita te the ring closure (Scheme

PAGE 56

41 2.7 ). While we attempted this, the approach di d not give us better results than the one previously explored as shown in Scheme 2.5 Scheme 2.7. Failed attempt to synthesize monomer 2.13a Based on these results, we could conclude that synthesis of the 2,6-DPK monomer was better achieved by following Route 2 (Scheme 2.5 ); however, the cyclization step remained a challenge throughout the entire synt hesis and only reactions carried in small quantities afforded the target compounds. Ou r attempts to scale up the reactions were unsuccessful. Therefore, yields obtained in general were not practical to continue building the scaffolds to have them readily available for hit to lead focused library design. After several failed attempts to im prove the synthesis of the piperazine-2,6diones, we decided to abandon this route and continue the project with a structurally related scaffold.

PAGE 57

42 2.2.5 Synthesis of Piperazine-2,5-diones: Route 1 Figure 2.5. Retrosynthetic analysis of 3-R-piperazine-2,5-dione scaffold DKPB

PAGE 58

43 A retrosynthetic analysis of a target scaffold based on 3-R-substituted piperazine2,5-dione (2,5-DKP) repeat un its is shown in Figure 2.5 The synthesis of the 2,5-DKP monomer has been also pursued by two r outes from starting amino acids type A By introducing the 2,5-DKP ring instead of the 2, 6-DKP, we would expe ct the cyclization step of the 2,5-DKPs to be more accessible du e the increased nucleophilicity of the amino group in either route. For example, in Route 1 the cyclization is facilitated by a metal catalyzed N-H insertion of compound type C In Route 2 the cyclization is favored by a bimolecular nuc leophilic substitution (SN2) reaction displacing the X halide of compound type D Whereas cyclization of the 2,6-DKP has to occur at less reactive ester sites of compounds type C and D in Figure 2.2 The general synt hetic approach for Route 1 is described in Scheme 2.8 Scheme 2.8. Attempted synthesi s of 2,5-DKP monomer, Route 1

PAGE 59

44 The two-step reductive amination reaction of commercially available -amino acid ester HCl salts 2.2 with benzaldehyde was accomplis hed adapting a procedure from literature (Oguz et al. 2002). Benzyl protected amino esters 2.26a and 2.26b were obtained in excellent yields. Compound 2.26a was then reacted with N -Boc oxaziridine 2.4 to form pure -hydrazino ester 2.27a in modest yield. In the next step, 2.27 was submitted to hydrogenolysis in presence of 5% Pd/C; this enabled the removal of the benzyl groups and the resulting acid intermediate was coupled with -alanine methyl ester HCl 2.11 to afford the corresponding ester 2.28 The next step would be the reaction of 2.28 with diazoacetate derivative 2.29 Succinimidyl diazoacetate 2.29 was synthesized following liter ature procedures (Grange et al. 1980; Ouihia et al. 1993). The advantage of preparing this reagent is its easier handling and hi gh stability compared to the reported moisture-labile analog acid chloride (Blankley et al. 1969; House and Blankley, 1968). Scheme 2.9. Failed attempt to synthesize 2.33f via N-H insertion To examine the diazoacetylation reaction and subsequent intramolecular cyclization reaction via N-H insert ion, we decided to treat compound 2.31f with reagent

PAGE 60

45 2.29 under basic conditions (Scheme 2.9 ). It should be noted that 2.31f does not contain the N -Boc protected hydrazine, as we envisi oned the free amine would facilitate the diazoacetylation reaction. The use of diazoace tamides and diazoesters as carbene sources to afford heterocyclic structures has b een reported (Doyle and Kalinin, 1996; Fructos et al. 2004). As shown in Scheme 2.9 diazoacetylation of compound 2.31f afforded the diazoacetamide derivative 2.32f in only 12% yield after two steps. An initial effort to cyclize the 2,5-DKP unit by treating 2.32f with the Cu(I) catalysts (Ma et al. 2005) was unsuccessful. Other catalysts based on Rh and Cu (Doyle and Kalinin, 1996; Morilla et al. 2002) were also considered; however, at th is time we had also started working on the synthesis of 2,5-terpyrimidinylene scaffolds (d iscussed in Chapter Th ree) and since this new project had greate r promise to give -helical mimetics in an expedient manner, we concentrated our efforts towards the synthesi s of the new scaffold. During this course, we revisited the synthesis of the 2,5-DKP sca ffold and developed a different strategy for the synthesis of the 3-substituted-2,5-DKPs by adapting a literature procedure (Scheme 2.10 ) (Maity and Konig, 2008). 2.2.6 Synthesis of Piperazine-2,5-diones: Route 2 The second route to pursue the synthesi s of the 2,5-DKPs is shown in Scheme 2.10 In this synthesis we intend to form the monomeric DKP first and install the hydrazine moiety before c oupling it with a second DKP unit (Scheme 2.10 bottom panel-b). The main advantage of following Route 2 over Route 1 (Scheme 2.8 ) is the facile two-step preparation of the 2,5-DKP units from r eadily available chiral and nonchiral amino esters 2.2 This route also enables an ea sier installation of various linker derivatives, R”, which are attached to the C -terminus-like position.

PAGE 61

46 Scheme 2.10. General synthesi s of 2,5-DKP monomer, Route 2 In the first step, the HCl salt s of corresponding amino esters 2.2 were N -acylated with bromoacetyl bromide in a toluene and aqueous sodium bicarbonate biphasic system to afford compounds 2.34 in good yields. Subsequent intramolecular cyclocondensation of a primary amine derivative, -alanine derivative 2.10 or benzylamine, with 2.34 in methanol gave the corresponding 2,5-DKP units 2.33 and 2.35 in modest to good yields. The next step, which includes the inst allation of the hydrazi ne group, is under investigation. A few options to achiev e formation of the hydrazine includes the N nitrosation of the amide of the DKP monomer with nitroson ium tetrafluoroborate (NOBF4) and subsequent reduction (Kuang et al. 2000) or the in situ generation of nitrosyl chloride (NOCl) or nitrosyl bromide (NOBr) (Francom and Robins, 2003). Another alternative is the use of bismuth (III) chloride and sodium nitrite, which has been recently reported as a mild, chemoselective, and efficient nitrosating agent (Chaskar et

PAGE 62

47 al. 2009). It has been reported that acyl protec ted amines give very low to no yield in the reduction step in reactions with stronger nitrosating agen ts and these were observed to be unstable (Oguz, 2003). However, the adva ntage of these methods is the direct formation of the nitroso intermediates in the acylated amine of DKP monomers. This way, it is not required to form the hydrazi ne bond previous to the ring closure as discussed in Section 2.2.2 and this may facilitate access to target monomers. A concerning aspect remains in the use of thes e nitrosating agents, which require careful handling since these are highl y toxic compounds (d'Ischia, 2005; Mirvish, 1995). Future work in this project will entail the continue d efforts to prepare peptidomimetics based on piperazine-2,5-dione re peat units and the potential use of the DKP monomers in other scaffolds applications. 2.3 Conclusion Our efforts towards the synthesis of peptidomimetics based on piperazine-2,6and 2,5-diones repeat units by several routes were descri bed. While various monomers, some dimers and trimers were synthesized in our lab and the trimeric 2,6-DKP derivative holding Leu-Phe-Val-like side chains 2.9bac (Scheme 2.3 ) had shown promising bioactivity, the main challenge of this proj ect has remained in the improvement of the overall yield, especially during the cycliza tion step, to enable the synthesis of longer helical mimetics. The synthesis of the 2,6DKP units was slightly improved by following Route 2 (Scheme 2.5 ), but this approach was still unsatisfactory due to limitation to the reaction scale to afford practic al quantities. Preparation of the 2,5-DKPs may be a more promising approach, although formation of the hydrazine bond and subsequent coupling of the units are still to be investigated. Synthesis of various 2 ,5-DKP monomers having a

PAGE 63

48 benzylamine or -alanine-derived linker at the C -terminus-like site was accomplished and this can be seen as preliminary results for the synthesis of the target trimeric peptidomimetics. 2.4 Experimental Section 2.4.1 Materials and Methods Starting materials, organic and inorganic r eagents (ACS grade), and solvents were obtained from commercial sources and used as received unless otherwise noted. Moistureand air-sensitive reactions were car ried out under an atmo sphere of argon. Thin layer chromatography (TLC) was perfor med on glass plates precoated with 0.25 mm thickness of silica gel (60 F-254) with fluorescent indicator (EMD or Whatman). Column chromatographic purification was performed using silica gel 60 , #70-230 mesh (Selecto Scientific). Automated flash chromatography was performed in a FlashMaster II system (Argonaut-Biotage ) using Biotage silic a cartridges. High performance liquid chromatography wa s performed on a Jasco LC-NetII/ADC instrument. 1H NMR and 13C NMR spectra were obtained using a 400 MHz Varian Mercury plus instrument at 25 C in chloroformd (CDCl3), unless otherwise indicated. Chemical shifts ( ) are reported in parts per million (ppm) relative to internal tetramethylsilane (TMS) or chloroform ( 7.26) for 1H NMR and chloroform ( 77.0) for 13C NMR. Multiplicity is expressed as (s = si nglet, br s = broad singlet, d = doublet, t = triplet, q = quartet, p = pentet, or m = multip let) and the values of coupling constants ( J ) are given in Hertz (Hz). High Resolution Mass Spectrometry (HRMS) spectra were carried out on an Agilent 1100 Se ries in the ESI-TOF mode. Microwave reactions were performed in a Biotage Initiator I microwave reactor. Melting point s (uncorrected) were

PAGE 64

49 determined using a Mel-Temp II, Laborator y Devices, MA, USA. Hydrogenation was performed in a Parr hydrogenator in a closed-vessel system at room temperature or in a H-Cube™ continuous-flow hydrogenation reactor, Thales Technology. 2.4.2 Experimental Procedures Representative procedure for the synthesis of -amino ester (2.2f): N -Boc protected amino acid 2.1f (1.59 mmol) was dissolved in MeOH (5 mL) and cooled to 0 C. Thionyl chloride (3.17 mmol) wa s added slowly and the reaction mixture was stirred at rt for 6 h; then under refluxing conditions for 12 h. The solvent was removed under reduced pressure to afford compound 2.2f in quantitative yield. Compounds 2.2e,g were obtained following the procedure described for 2.2f from commercially available free amino acids 2.1e,g All compounds were obtained in >95% purity as determined by NMR spectroscopy. Methyl 2-amino-2-methylpropan oate hydrochloride (2.2e): Isolated yield: 93%, wh ite solid, m.p. 179-182 C. 1H NMR (400 MHz, (CD3)2SO) 1.48 (s, 6H), 3.75 (s, 3H), 8.76 (br s, 2H). 13C NMR (100 MHz, CD3OD) 22.80, 52.81, 56.62, 171.97. HRMS (ESI) calcd. for C5H12NO2 [M+H]+ 118.0868, found 118.0859.

PAGE 65

50 ( S )-methyl 2-amino-3-(naphthalene-2-y l)propanoate hydrochloride (2.2f): Isolated yield: 97%, white solid. 1H NMR (400 MHz, (CD3)2SO) 3.28 (m, 1H), 3.37(m, 1H), 3.69 (s, 3H), 4.41 (t, J = 6.6 Hz, 1H), 7.49-7.54 (m, 2H), 7.38 (d, J = 8.4 Hz, 1H), 7.76 (s, 1H), 7.86-7.92 (m, 3H), 8.58 (br s, 2H). 13C NMR (100 MHz, (CD3)2SO) 35.93, 52.60, 54.81, 125.88, 126.16, 127.26, 127.43, 127.49, 128.11, 128.15, 132.04, 132.13, 132.87, 169.32. ESI-MS calcd. for C14H16NO2 [M+H]+ 230.11, found 230.11. ( R )-methyl 2-amino-3-( tert -butyldisulfanyl)propanoate hydrochloride (2.2g): Isolated yield: 96%, white solid. 1H NMR (400 MHz, CD3OD) 1.37 (s, 9H), 3.18-3.28 (m, 2H), 3.86 (s, 3H), 4.37-4.40 (m, 1H). 13C NMR (100 MHz, CD3OD) 23.02, 28.91, 39.32, 51.86, 52.74, 171.48. ESI-MS calcd. for C8H18NO2S2 [M+H]+ 224.08, found 224.1. Representative procedure for the synthesis of -amino diester (2.3a):

PAGE 66

51 The HCl salt of amino ester 2.2a (4.63mmol) was dissolved in anhydrous acetonitrile (20 mL). Then diisopropylethylamine (9.26 mmol) and ethyl bromoacetate (6.94 mmol) were added, while the reaction mixture was kept under an argon atmosphere; then it was stirred at rt for 24 h. The reaction mixture was quenched with 5% citric acid (5 mL) and it was extracted with ethyl acetate (3 x 15 mL ). The combined organic layers were washed with brine (2 x 15 mL), dried over Na2SO4, and evaporated in vacuo to yield compound 2.3a as a colorless crude oil. ( S )-methyl 2-(2-ethoxy-2-oxoethylam ino)-3-phenylpropanoate (2.3a): 2.3a was purified by column chromatography on silica gel (hexanes:ethyl acetate, 7:3), yield: 88%, colorless oil. 1H NMR (400 MHz, CDCl3) 1.23 (t, J = 6.8 Hz, 3H), 2.11 (br s, 1H), 2.95-2.98 (m, 1H), 3.04 (dd, J = 13.4 and 6 Hz, 1H), 3.33 and 3.41 (2d, J = 17.2 Hz, 2H), 3.66 (s, 3H), 4.14 (q, J = 7.2 Hz, 2H), 7.18–7.30 (m, 5H). 13C NMR (100 MHz, CDCl3) 14.38, 39.74, 49.39, 52.04, 61.06, 62.37, 127.08, 128.73, 129.40, 137.12, 171.80, 174.29. ESI-MS calcd. for C14H20NO4 [M+H]+ 266.13, found 266.13. Methyl 2-(2-ethoxy-2-oxoethylamin o)-2-methylpropanoate (2.3e): 2.3e was prepared following the procedure described for 2.3a using DMF as solvent. 2.3e was purified by column chromatography on silica gel (hexanes:ethyl acetate, 7:3), yield: 60%, colorless thick oil. 1H NMR (400 MHz, CDCl3) 1.25 (t, J = 7.0 Hz, 3H),

PAGE 67

52 1.34 (s, 6H), 3.35 (s, 2H), 3.68 (s, 3H), 4.17 (q, J = 7.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) 14.36, 25.34, 46.25, 52.34, 58.93, 61.25, 171.83, 176.62. ESI-MS calcd. for C9H18NO4 [M+H]+ 204.12, found 204.12. ( R )-methyl 3-( tert -butyldisulfanyl)-2-(2 -ethoxy-2-oxoethylamino)propanoate (2.3g): 2.3g was prepared following the procedure described for 2.3a and it was purified by column chromatography on silica gel (hexanes:e thyl acetate, 3:2), yield: 80%, colorless oil. 1H NMR (400 MHz, CDCl3) 1.26 (t, J = 7.2 Hz, 3H), 1.32 (s, 9H), 3.48 (d, J = 7.2 Hz, 2H), 3.68 (t, J = 6.2 Hz, 1H), 3.75 (s, 3H), 4.18 (q, J = 7.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) 14.40, 30.08, 43.41, 48.35, 49.28, 52.48, 60.29, 61.27, 171.53, 172.97. ESI-MS calcd. for C12H24NO4S2 [M+H]+ 310.11, found 310.1. ( S )-methyl 3-(4-(benzyloxy)phenyl)-2-(2-eth oxy-2-oxoethylamino)propanoate (2.3h): 2.3h was prepared following the procedure described for 2.3a Isolated yield: 90%, light yellow oil. 1H NMR (400 MHz, CDCl3) 1.24 (m, 3H), 2.38 (br s, 1H), 2.89 – 3.02 (m, 2H), 3.31 – 3.44 (m, 2H), 3.58 (t, J = 6.7, 1H), 3.65 – 3.67 (m, 3H ), 4.14 (m, 2H), 5.03 (s, 2H), 6.88 – 6.93 (m, 2H), 7.10 – 7.14 (m, 2H), 7.29 – 7.44 (m, 5H). 13C NMR (100 MHz, CDCl3) 14.16, 38.56, 49.12, 51.83, 60.88, 62.23, 70.00, 114.91, 127.47, 127.92,

PAGE 68

53 128.55, 129.06, 130.21, 137.05, 157.77, 171.46, 173.99. ESI-MS calcd. for C21H26NO5 [M+H]+ 372.18, found 372.18. Representative procedure for the synthesis of -amino hydrazine diester (2.5a): A solution of diester 2.3a (1.88 mmol) in anhydrous methanol (7 mL) was cooled to -78 C and kept under an argon atmosphere. Tert -butyl 3(trichloro methyl)-1,2-oxaziridine2-carboxylate 2.4 (5.64 mmol) was added slowly and th e reaction was stir red at -78 C for 4 h. Progress of the reaction was mon itored by TLC. The solvent was evaporated under reduced pressure to yi eld a yellow crude residue. ( S )tert -butyl 2-(2-ethoxy-2-oxoe thyl)-2-(1-methoxy-1-oxo-3-phenylpropan-2yl)hydrazine carboxylate (2.5a): 2.5a was purified by column chromatography on silica gel (hexanes:ethyl acetate, 3:2), yield: 90%, white solid, m.p. = 63-68 C. 1H NMR (400 MHz, CDCl3) 1.23 (t, J = 7.2 Hz, 3H), 1.44 (s, 9H), 2.98 – 3.01 (m, 1H), 3.14 (dd, J = 13.2 and 5.2 Hz, 1H), 3.55 (s, 3H), 3.64-3.85 (m, 3H), 4.12 (q, J = 6.8 Hz, 2H), 6.95 (br s, 1H), 7.17–7.28 (m, 5H). 13C NMR (100 MHz, CDCl3) 14.35, 28.50, 36.83, 51.77, 6 1.11, 69.02, 80.53, 126.85,

PAGE 69

54 128.60, 129.46, 137.31, 172.06. ESI-MS calcd. for C19H28N2NaO6 [M+Na]+ 403.18, found 403.18. Representative procedure for the synthesis of -amino hydrazine diacid (2.6a): Hydrazine compound 2.5a (2.10 mmol) was dissolved in MeOH (12 mL) and cooled to 0 C. Then freshly prepared 1N NaOH (6.3 mmol) was added and th e mixture was stirred at rt for 24 h. MeOH was re moved under reduced pressure and the residue was diluted with water. The aqueous solution was acidified to pH=1-2 with 1.2 N HCl and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo to give pure 2.6a as a white solid. ( S )-2-(2-( tertbutoxycarbonyl)-1-(carboxymethyl )hydrazinyl)-3-phenylpropanoic acid (2.6a): N OH O HO O NHBoc Isolated yield: 75%, white fl uffy solid, m.p. = 137-140 C. 1H NMR (400 MHz, CD3OD) 1.46 (s, 9H), 3.02 (d, J = 7.2 Hz, 2H), 3.71 (s, 2H), 3.90 (t, J = 7.2 Hz, 1H), 7.17–7.26 (m, 5H). 13C NMR (100 MHz, CD3OD) 27.36, 35.96, 57.20, 69.26, 80.83, 126.41, 128.21, 129.04, 137.60, 157.20, 172.70, 173.41. ESI-MS calcd. for C16H21N2O6 [M H]+ 337.14, found 337.1.

PAGE 70

55 Experimental procedure for the synt hesis of benzyl 3-aminopropanoate hydrochloride (2.10’): To a solution of N -Boc -alanine (5.28 mmol) and benz yl bromide (6.34 mmol) in acetone (15 mL) was added potassium carbona te (10.56 mmol). The reaction mixture was stirred under refluxing for 5 h. The mi xture was cooled to rt and the formed precipitate was removed by filtration. The cr ude filtrate was concentrated under reduced pressure and the resulting crude oil was dissolved in ethyl acetate, washed with brine (2 x 20 mL), dried over CaCl2, and concentrated under reduced pr essure. Intermediate benzyl 3-( tert -butoxycarbonylamino)propanoate, 2.10’ was obtained after purification by column chromatography on silica gel (hexanes:e thyl acetate, 4:1), yield: 85%, colorless oil. 1H NMR (400 MHz, CDCl3) 1.43 (s, 9H), 2.58 (t, J = 6.0, 2H), 3.37 – 3.45 (m, 2H), 5.02 (br s, 1H), 5.14 (s, 2H), 7.31 – 7.40 (m, 5H). 13C NMR (100 MHz, CDCl3) 28.37, 64.63, 36.07, 66.48, 79.44, 128.20, 128.35, 128.61, 135.65, 155.75, 172.34. ESI-MS 179.1 [M-Boc]+. Experimental procedure for the synthesis of ( S )-2-(13,13-dimethyl-3,7,11-trioxo-1phenyl-2,12-dioxa-6,9,10-triazatetradecan-9yl)-4-methylpentanoic acid (2.12b):

PAGE 71

56 Hydrazine diacid 2.6b (0.82 mmol) was dissolved in anhydrous DCM (5 mL) and cooled to 0 C. Then diisopropylcarbodiimide ( 0.90 mmol) was added; the mixture was then stirred under refluxing conditions for 24 h. The formed precipitate was filtered and washed with DCM (2 x 5 mL). To the crude filtrate, -alanine benzyl ester HCl, 2.10 (0.82 mmol) was added followed by triethyl amine (1.64 mmol). The reaction mixture was stirred under refluxing conditions for 18 h. The solvent was removed under reduced pressure to obtain a ye llow crude material. The crude wa s dissolved in water (5 mL) and 5% citric acid (5 mL) and extr acted with ethyl acetate (2 x 8 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo to give 2.12b as a yellow crude oil. Compound 2.12b was purified by column chromatography on silica gel (hexanes:ethyl acetate, 3:2), yield: 36%, off white solid. 1H NMR (400 MHz, CD3OD) 0.95 – 0.86 (m, 6H), 1.07 – 1.13 (m, 3H), 1.46 (s, 9H), 2.53 – 2.66 (m, 2H), 3.37 – 3.49 (m, 2H), 3.49 – 3.59 (m, 2H), 3.71 – 3.83 (m, 1H), 5.12 (s, 2H), 7.39 – 7.27 (m, 5H). Representative procedure for the synthesis of -amino diester (2.16): 2.2a HCl.H2N R1 R1' O OBn Br DIEA,MeCN,2.16a H N R1 R1' O O O O O O The HCl salt of amino ester 2.2a (2.32 mmol) was dissolved in anhydrous acetonitrile (10 mL) under an argon atmosphere. Diisopro pylethylamine (4.64 mmol) and benzyl bromoacetate (3.47 mmol) were added and the mixture was stirred at rt for 24 h. The reaction mixture was quenched with 5% citric acid (5 mL) and it was extracted with ethyl

PAGE 72

57 acetate (3 x 15 mL). The combined organic laye rs were washed with water and brine (2 x 15 mL), dried over Na2SO4, and evaporated under reduced pressure to yield a crude yellow oil. ( S )-methyl 2-(2-(benzyloxy)-2-oxoethyl amino)-3-phenylpropanoate (2.16a): H N O O O O 2.16a was purified by column chromatography on silica gel (hexanes:ethyl acetate, 7:3), yield: 86%, thick colorless oil. 1H NMR (400 MHz, CDCl3) 2.30 (br s, 1H), 2.94 – 3.08 (m, 2H), 3.37 – 3.51 (m, 2H), 3.60 – 3.64 (m, 1H), 3.65 (s, 3H), 5.13 (s, 1H), 5.22 (s, 1H), 7.17 – 7.39 (m, 10H). 13C NMR (100 MHz, CDCl3) 39.65, 49.33, 52.07, 62.30, 66.88, 127.11, 128.57, 128.62, 128.70, 128.74, 128.78, 128.81, 128.89, 128.90, 129.41, 135.68, 137.01, 171.55, 174.10. ESI-MS 434.1 [M+H]+. Representative procedure for the synthesis of -amino hydrazine diester (2.17): A solution of diester 2.16a (1.83mmol) in anhydrous metha nol (7 mL) was cooled to -78 C and kept under argon atmosphere. Tert -butyl 3(trichloromethyl)-1,2-oxaziridine-2carboxylate 2.4 (5.49 mmol) was added slowly and the reaction was stirred at -78 C for 4 h, then at rt for 12 h. Progr ess of the reaction was monito red by TLC. The solvent was evaporated under reduced pre ssure to yield a crude oil.

PAGE 73

58 ( S )tert -butyl 2-(2-(benzyloxy)-2-oxoethyl)-2 -(1-methoxy-1-oxo-3-phenylpropan-2yl)hydrazinecarboxylate (2.17a): Compound 2.17a was purified by column chromatogr aphy on silica gel (hexanes:ethyl acetate, 4:1), yield: 86%, thick colorless oil. 1H NMR (400 MHz, CDCl3) 1.45 (s, 9H), 2.99 – 3.16 (m, 2H), 3.52 (s, 3H), 3.71 – 3.83 (m, 2H), 3.83 – 3.93 (m, 1H), 5.07 – 5.17 (m, 2H), 6.97 (br s, 1H), 7.18 – 7.25 (m, 5H), 7.32 – 7.38 (m, 5H). ( S )tert -butyl 2-(2-(benzyloxy)-2-oxoethyl)2-(1-methoxy-4-meth yl-1-oxopentan-2yl)hydrazinecarboxylate (2.17b): 2.17b was prepared following the procedure described for 2.17a and it was purified by column chromatography on silica gel (hexanes :ethyl acetate, 4:1) yield: 86%, thick colorless oil. 1H NMR (400 MHz, CDCl3) 1H NMR (400 MHz, CDCl3) 0.88 – 0.93 (m, 6H), 1.43 (s, 9H), 1.48 (s, 3H), 1 .61 – 1.69 (m, 1H), 1.80 – 1.94 (m, 1H), 3.63 (d, J = 8.2, 1H), 3.65 (s, 3H), 3.71 (s, 1H), 3.75 – 3.8 0 (m, 1H), 5.13 (s, 2H), 6.89 (br s, 1H), 7.30 – 7.41 (m, 5H). tert -butyl 2-(2-(benzyloxy)-2-oxoethyl)-2-((2 S ,3 S )-1-methoxy-3-methyl-1-oxopentan2-yl)hydrazinecarboxylate (2.17d):

PAGE 74

59 2.17d was prepared following the procedure described for 2.17a and it was purified by column chromatography on silica gel (hexanes :ethyl acetate, 7:3) yield: 62%, thick colorless oil. 1H NMR (400 MHz, CDCl3) 0.83 (d, J = 6.7, 3H), 1.17 (m, 1H), 1.43 (s, 9H), 1.47 – 1.51 (m, 3H), 1.70 – 1.82 (m, 1H), 1.91 – 2.02 (m, 1H), 3.19 (d, J = 9.9, 1H), 3.65 (s, 3H), 3.78 – 3.90 (m, 1H), 5.12 (s, 2H), 7.00 (br s, 1H), 7.40 – 7.30 (m, 5H). Representative procedure for the synthesis of -amino hydrazine monoacid (2.18a): A solution of hydrazine 2.17a (1.87 mmol) in THF (5 mL) was placed in a hydrogenation vessel along with the Pd/C catalys t (5% mol). Hydrogenolysis of 2.17a took place at 35 psi for 30 min. at rt. Upon complete consumption of starting material 2.17a as determined by TLC, the catalyst was filtered through Celite™ (1 g) and washed with THF (3 x 10 mL). The filtrate was concentrated in vacuo to yield monoacid 2.18a ( S )-2-(2-( tert -butoxycarbonyl)-1-(1-methoxy-1-oxo-3-phenylpropan-2yl)hydrazinyl)acetic acid (2.18a): Isolated yield: >90%, white solid. 1H NMR (400 MHz, CDCl3) 1.46 (s, 9H), 1.68 (br s, 1H), 2.98 – 3.06 (m, 1H), 3.11 – 3.18 (m, 1H), 3.58 (s, 2H), 3.68 (s, 3H), 3.77 – 3.86 (m, 1H), 6.94 (br s, 1H), 7.17 – 7.30 (m, 5H). 13C NMR (100 MHz, CDCl3) 28.49, 36.83,

PAGE 75

60 51.77, 52.01, 68.96, 80.57, 126.85, 128.59, 129.44, 137.26, 155.36, 170.63, 172.09. ESIMS 434.1 [M+H]+. Representative procedure for the synthesis of -amino hydrazine amide (2.19a): A mixture of monoacid 2.18a (1.88 mmol) and the HCl salt of -alanine methyl ester 2.11 in anhydrous DCM (5 mL) was cooled to 0 C under argon. Then 1-ethyl-3-(3dimethyllaminopropyl)carbodiimide hydrochloride (EDC.HCl) was added portionwise, while the mixture stirred vigorousl y. The reaction was kept at 0 C for 30 min., then at rt for 16 h. The mixture was quenched with sat’d. NaHCO3 and extracted with CHCl3 (3 x 5 mL). The combined organic layers were wa shed with 1N HCl, water, and brine; then dried over MgSO4. The solvent was removed in vacuo to obtain a yellow crude oil. ( S )tert -butyl 2-(1-methoxy-1-oxo-3-pheny lpropan-2-yl)-2-(2-(3-methoxy-3oxopropylamino)-2-oxoethyl)hy drazinecarboxylate (2.19a): N O O N H O NHBoc O O Compound 2.19a was purified by column chromatography on silica gel (hexanes:ethyl acetate, 1:1), yield: 79%, white solid. 1H NMR (400 MHz, CDCl3) 1.45 (s, 9H), 1.91 (br s, 1H), 2.40 (t, J = 7.2, 2H), 2.92 – 3.09 (m, 2H), 3.29 – 3.41 (m, 2H), 3.41 – 3.53 (m, 2H), 3.69 (s, 3H), 3.70 (s, 3H), 6.88 (br s, 1H ), 7.22 – 7.35 (m, 5H), 8.09 (br s, 1H). 13C NMR (100 MHz, CDCl3) 14.40, 21.24, 28.43, 34.11, 34.94, 36.36, 51.86, 52.26, 60.59,

PAGE 76

61 69.88, 81.11, 127.08, 128.71, 129.35, 137.38, 156.14, 169.36, 171.34, 172.10, 172.97. ESI-MS 438.22[M+H]+. ( S )-tert-butyl 2-(2-(3-methoxy-3-oxopro pylamino)-2-oxoethyl)-2-(1-methoxy-4methyl-1-oxopentan-2-yl)hydrazinecarboxylate (2.19b): Compound 2.19b was prepared following the procedure described for 2.19a and it was purified by column chromatography on silica gel (hexanes:ethyl acetate, 7:3), yield: 72%, off white solid. 1H NMR (400 MHz, CDCl3) 0.98 – 0.88 (m, 6H), 1.43 (s, 9H), 1.48 – 1.66 (m, 2H), 1.78 (s, 1H), 1.99 (br s, 1H), 2.55 (t, J = 7.1, 2H), 3.35 – 3.42 (m, 1H), 3.50 – 3.62 (m, 3H), 3.69 (s, 3H), 3.74 (s, 3H), 6.83 (br s, 1H), 8.54 (br s, 1H). 13C NMR (100 MHz, CDCl3) 21.42, 23.33, 24.64, 28.39, 34.32, 35.02, 39.16, 39.31, 51.85, 52.18, 66.60, 80.91, 156.26, 169.74, 172.20, 174.39. HRMS (ESI) calcd. for C18H34N3O7 [M + H]+ 404.2397, found 404.2386. Experimental procedure for the synthesis of ( S )-methyl 3-(3-benzyl-4-( tert butoxycarbonylamino)-2,6-dioxopipera zin-1-yl)propanoate (2.13a): A solution of NaH (0.324 mmol) in anhydrous TH F (1 mL) was cooled to 0 C under an argon atmosphere. To this mixture, a solution of compound 2.19a (0.85 mmol) in

PAGE 77

62 anhydrous THF (5 mL) was added dropwise. Th e reaction was stirred at 0 C for 30 min, then at rt for 22 h. The solvent was rem oved under reduced pressure to obtain a yellow crude. Monomer 2.13a was obtained in 71% yield as a white solid after purification by column chromatography on silica gel (hexanes:ethyl acetate, 4:1). 1H NMR (400 MHz, CDCl3) 1.44 (s, 9H), 2.58 (t, J = 6.9, 2H), 3.12 – 3.31 (m, 2H), 3.67 (s, 3H), 3.90 – 3.96 (m, 1H), 4.01 – 4.16 (m, 4H), 6.61 (br s, 1H), 7.21 – 7.31 (m, 5H). 13C NMR (100 MHz, CDCl3) 28.46, 31.93, 34.86, 35.39, 52.22, 57.42, 67.02, 126.96, 128.69, 129.48, 137.74, 154.88, 169.01, 170.99, 172.27. ESI-MS 428.17[M+Na]+. Experimental procedure for the synthesis of ( S )-benzyl 2-(2-methoxy-2oxoethylamino)-3-phenylpropanoate (2.22a): The HCl salt of amino ester 2.21a (3.43 mmol) was suspended in anhydrous acetonitrile (25 mL). Then DIEA (5.14 mmol) and met hyl bromoacetate (4.11 mmol) were added, while the reaction mixture was kept under an argon atmosphere; then it was stirred at rt for 24 h. The crude mixture was quenched w ith 5% citric acid (5 mL) and it was extracted with ethyl acetate (3 x 10 mL). The combined orga nic layers were washed with water and brine (2 x 15 mL), dried over Na2SO4, and evaporated in vacuo to yield pure compound 2.22a Isolated yield: 80%, colorless oil. 1H NMR (400 MHz, CDCl3) 1.74 (br s, 1H), 2.97 – 3.06 (m, 2H), 3.32 – 3.45 (m, 2H), 3.64 (t, J = 6.7, 1H), 3.67 (s, 3H), 5.08 (s, 2H), 7.13 – 7.17 (m, 2H), 7.20 – 7.29 (m, 5H), 7.31 – 7.36 (m, 3H). 13C NMR

PAGE 78

63 (100 MHz, CDCl3) 39.52, 48.98, 51.83, 51.8 7, 62.17, 62.22, 66.68, 126.84, 128.35, 128.38, 128.50, 128.55, 129.24, 135.41, 136.73, 171.96, 173.50. HRMS (ESI) calcd. for C19H22NO4 [M + H]+ 328.1549, found 328.1551. Experimental procedure for the synthesis of ( S )tert -butyl 2-(1-(benzyloxy)-1-oxo-3phenylpropan-2-yl)-2-(2-methoxy-2oxoethyl)hydrazinecarboxylate (2.23a): DCM,0C O NBoc Cl3C 2.42.23a N O O O O NHBoc 2.22a H N O O O O A solution of diester 2.22a (2.54 mmol) in anhydrous DCM (7 mL) was cooled to -78 C and kept under argon atmosphere. Tert -butyl 3-(trichloromethyl)-1,2-oxaziridine-2carboxylate 2.4 (3.30 mmol) was added slowly and the reaction was stirred below 0 C for 6 h, then at rt for 16 h. The solvent was evaporated under reduced pressure to obtain a colorless crude oil. Compound 2.23a was isolated in 70% yi eld after purification by column chromatography on silica gel (hexanes:ethyl acetate, 4:1) as a thick colorl ess oil. 1H NMR (400 MHz, CDCl3) 1.45 (s, 9H), 2.98 – 3.07 (m, 1H), 3.14 – 3.22 (m, 1H), 3.63– 3.66 (m, 3H), 3.66 – 3.73 (m, 1H), 3.75 – 3.88 (m, 2H), 4.99 (q, J = 12.3, 2H), 6.97 (br s, 1H), 7.05 – 7.13 (m, 2H), 7.16 – 7.31 (m, 8H). 13C NMR (100 MHz, CDCl3) 28.87, 36.79, 51.79, 56.65, 66.46, 68.72, 80.31, 126.63, 128.12, 128.22, 128.40, 128.46, 128.86, 129.30, 135.20, 136.83, 155.13, 170.35, 171.40. ESI-MS 364.11[M+Na]+-Boc.

PAGE 79

64 Experimental procedure for the synthesis of ( S )-2-(2-(tert-butoxycarbonyl)-1-(2methoxy-2-oxoethyl)hydrazinyl)-3-phenylpropanoic acid (2.24a): Hydrazine 2.23a (0.27 mmol) was dissolved in THF (5 mL) and this solution was passed through 10% Pd/C catalyst in a flow reactor (H -Cube, Thales Technology) at a flow rate of 1 mL/min for 1 h at 10 bar. The solvent was evaporated in vacuo to yield 2.24a in 88 % yield. 1H NMR (400 MHz, CDCl3) 1.47 (s, 9H), 3.00 – 3.23 (m, 2H), 3.64 (s, 3H), 3.65 – 3.72 (m, 2H), 3.93 – 4.03 (m, 1H), 6.80 (br s, 1H), 7.07 – 7.38 (m, 5H). 13C NMR (100 MHz, CDCl3) 28.19, 35.88, 52.09, 56.39, 69.59, 82.46, 126.96, 128.71, 128.97, 130.15, 133.66, 137.05, 173.38. Experimental procedure for the synthesis of ( S )tert -butyl 2-(2-methoxy-2-oxoethyl)2-(1-(3-methoxy-3-oxopropylamino)-1-oxo-3-phenylpropan-2yl)hydrazinecarboxylate (2.25a): A mixture of monoacid 2.24a (0.85 mmol) and the HCl salt of -alanine methyl ester 2.11 (0.94 mmol) in anhydrous DCM (10 mL) was cooled to 0 C under an argon atmosphere. Then EDC.HCl (1.02 mmol) was added portionwise, while the mixture

PAGE 80

65 stirred vigorously. The mixture was stirred at 0 C for 30 min, then at rt for 24 h. The reaction crude was quenched with sat’d. NaHCO3 and extracted with CHCl3 (3 x 5 mL). The combined organic layers were washed wi th 1N HCl, water, and brine; then dried over MgSO4. The solvent was removed under reduced pressure to yield a yellow crude oil. 2.25a was obtained in 57% yield as a thick colorless oil upon purification by column chromatography on silica gel (hex anes:ethyl acetate, 1:1). 1H NMR (400 MHz, CD3OD) 1.47 (s, 9H), 2.30 – 2.47 (m, 2H), 2.87 – 3.06 (m, 2H), 3.31 – 3.35 (m, 2H), 3.59 – 3.63 (m, 2H), 3.64 (s, 3H), 3.67 (s, 3H), 3.69 – 3.77 (m, 1H), 7.14 – 7.28 (m, 5H). 13C NMR (400 MHz, CD3OD) 27.43, 33.20, 34.76, 36.30, 50.97, 51.10, 55.94, 70.10, 80.34, 126.42, 128.22, 129.16, 137.44, 156.69, 170.40, 172.36, 172.42. Representative procedure for the synthesis of benzyl protected -amino ester (2.26): Step 1 : The HCl salt of amino ester 2.21a (2.32 mmol) was dissolv ed in anhydrous THF (7 mL) and brought to 0 C. Then MgSO4 (0.5 g), benzaldehyde (4.64 mmol), and triethylamine (2.32 mmol) were added, while the reaction mixture was under an argon atmosphere. The reaction mixture was stir red at rt under argon for 4 h. The crude mixture was filtered and the solvent was rem oved under reduced pressure to yield a crude yellow oil, which was used for step 2 without further purification. Step 2 : Schiff base obtained from step 1 (2.32 mmol) was dissolved in anhydrous methanol (20 mL); then sodium borohydr ide (4.64 mmol) was added slowly. The reaction mixture was stirred at rt for 2 h under an argon atmosphere. The reaction

PAGE 81

66 mixture was quenched with 1N NaOH and extracted with diet hyl ether (3 x 5 mL). Ether layers were washed with brine and dried over Na2SO4. Compound 2.26a was obtained upon removal of the solvent under reduced pressure. ( S )-methyl 2-(benzylamino)-3 -phenylpropanoate (2.26a): Isolated yield: 96% over two steps, colorless oil. 1H NMR (400 MHz, CDCl3) 1.82 (br s, 1H), 2.94 – 2.98 (m, 2H), 3.52 – 3.57 (m, 1H), 3.60 – 3.67 (m, 4H), 3.76 – 3.84 (m, 1H), 7.14 – 7.38 (m, 10H). 13C NMR (100 MHz, CDCl3) 39.73, 51.64, 51.99, 62.06, 126.67, 126.97, 127.00, 127.65, 128.11, 128.32, 128.37, 128.46, 128.56, 128.58, 129.20, 137.30, 175.03. ESI-MS 270.14[M+H]+. ( S )-methyl 2-(benzylamino )-3-methylbutanoate (2.26c): H N O O Compound 2.26c was prepared following th e procedure described for 2.26a Isolated yield: 95% over two steps, colorless oil. 1H NMR (400 MHz, CDCl3) 0.91 – 0.98 (m, 6H), 1.68 (br s, 1H), 1.86 – 1.97 (m, 1H), 3.02 (d, J = 6.1, 1H), 3.55 – 3.60 (m, 1H), 3.72 (s, 3H), 3.80 – 3.85 (m, 1H), 7.27 – 7.40 (m, 5H). 13C NMR (100 MHz, CDCl3) 18.88, 19.52, 31.93, 51.62, 52.77, 66.59, 126.96, 127.67, 128.23, 128.27, 128.57, 140.09, 175.77. ESI-MS 222.17[M+H]+.

PAGE 82

67 Experimental procedure for the synthesis of ( S )tert -butyl 2-benzyl-2-(1-methoxy-1oxo-3-phenylpropan-2-yl)hydrazinecarboxylate (2.27a): DCM,-78C O NBoc Cl3C 2.42.27a H N O O 2.26a N O O NHBoc A solution of compound 2.26a (0.74 mmol) in anhydrous DC M (15 mL) was cooled to 72 C and kept under an argon atmosphere. Tert -butyl 3(trichloromethyl)-1,2oxaziridine-2-carboxylate 2.4 (0.96 mmol) was added slowly and the reaction was stirred at -72 C for 2 h, then at rt for 18 h. Progr ess of the reaction was monitored by TLC. The solvent was evaporated under reduced pressu re and the crude resi due was purified by column chromatography on silica gel (h exanes:ethyl acetate, 4:1). Compound 2.27a was obtained as a thick colorl ess oil in 46% yield. 1H NMR (400 MHz, CDCl3) 1.47 (s, 9H), 2.92 – 3.17 (m, 2H), 3.49 – 3.59 (m, 1H), 3.65 (s, 3H), 3.78 – 4.02 (m, 2H), 6.66 (br s, 1H), 7.14 – 7.36 (m, 10H). Experimental procedure for the synthesis of 2,5-dioxopyrrolidin-1-yl 2-diazoacetate (2.29): Step 1 : A mixture of p -toluene sulfonyl hydrazide (20 mmol), glyoxylic acid monohydrate (26 mmol), conc. hydrochloric ac id (12 mmol), and acetonitrile (50 mL) was stirred at rt for 24 h. The solvent was removed under redu ced pressure and the

PAGE 83

68 residue was triturated with water. The white crysta lline product was removed by filtration and air dried to yield glyoxy lic acid tosylhydrazone intermediate 2.29’ in 87% yield. m.p. = 148-152 C. 1H NMR (400 MHz, (CD3)2SO) 2.39 (s, 3H), 7.18 (s, 1H), 7.44 (d, J = 8.0, 2H), 7.71 (d, J = 8.3, 1H), 12.30 (s, 1H). 13C NMR (100 MHz, (CD3)2SO) 20.93, 117.95, 124.36, 125.37, 126.99, 127.91, 129.32, 129.80, 135.59, 137.32, 143.92, 163.43. Step 2 : To a mixture of glyoxyl ic acid tosylhydrazone 2.29’ (2.06 mmol) and N -hydroxy succinimide (2.06 mmol) in ice-cold dioxa ne (10 mL) under an argon atmosphere, DCC (2.03 mmol) in dioxane (4 mL) was added dr opwise. The reaction mixture was allowed to warm up to rt and stirred for 4 h. The resulting urea precipitate was removed by filtration and the filtrate was evaporated to dryness under reduced pressure. Compound 2.29 was obtained in 51% yield after purific ation by flash column chromatography on silica gel (dichloromethane), white solid, m.p. = 121-122 C. 1H NMR (400 MHz, CDCl3) 2.86 (s, 4H), 5.11 (br s, 1H). Experimental procedure for the synthesis of ( S )-methyl 3-(2-(2-diazoacetamido)-3(naphthalen-2-yl)propanamido)propanoate (2.32f): Step 1 : Compound 2.31f (0.99 mmol) was dissolved in a solution of TFA : DCM (1:1, 2 mL) and stirred at rt for 45 min. The solv ent was removed under reduced pressure to

PAGE 84

69 obtain a light yellow solid crude, which was used in the next step without further purification. Step 2 : The TFA salt obtained in step 1 ( 0.96 mmol) was suspended in anhydrous DCM (2 mL) and cooled to 0 C under an argon at mosphere. Then triethylamine (1.15 mmol) was added followed by the slow addition of a solution of succinimidyl diazoacetate 2.29 (1.05 mmol) in anhydrous DCM (3 mL). The mi xture was stirred at 0 C for 40 min. then at rt for 24 h. The solvent was removed unde r reduced pressure to obtain a pale yellow oil crude. Compound 2.32f was obtained in 12% over two steps after purification by column chromatography on silica gel (hexanes:ethyl acetate, 1:1). 1H NMR (400 MHz, CDCl3) 2.17 – 2.41 (m, 2H), 3.07 – 3.15 (m, 1H ), 3.25 – 3.35 (m, 2H), 3.43 (s, 3H), 3.44 – 3.51 (m, 1H), 4.67 – 4.74 (m, 1H), 4.76 (s, 1H), 5.77 (br s, 1H), 6.11 (br s, 1H), 7.32 – 7.37 (m, 1H), 7.43 – 7.51 (m, 2H), 7.64 (s, 1H), 7.75 – 7.84 (m, 3H). Representative procedure for the synthesis of -amino ester bromide (2.34): The HCl salt of amino ester 2.2c (3.58 mmol) was dissolved in water (20 mL) and cooled to 0 C. To this mixture, NaHCO3 (8.23 mmol) was added follo wed by the slow addition of a solution of bromoacetyl bromide (3.58 mmo l) in toluene (15 mL). The reaction was mixture was brought to rt and st irred until consumption of ester 2.2c was observed as determined by TLC (6 h). The layers were separated and the aqueous layer was extracted with toluene (2 x 20 mL). The combin ed organic layers were dried over Na2SO4 and complete removal of the solvent gave a white solid.

PAGE 85

70 Note : The aqueous layer was also extracted with DCM and results were comparable based on product yield and purity. ( S )-methyl 2-(2-bromoacetamido) -3-methylbutanoate (2.34c): Isolated yield: 83 %, crystalline white solid, m.p. = 4748 C. 1H NMR (400 MHz, CDCl3) 0.92 – 0.98 (m, 6H), 2.16 – 2.28 (m, 1H), 3.77 (s, 3H), 3.92 (s, 2H), 4.50 – 4.56 (m, 1H), 6.90 (br s, 1H). 13C NMR (100 MHz, CDCl3) 17.91, 19.10, 29.18, 31.54, 52.55, 57.91, 165.58, 171.99. HRMS (ESI) calcd. for C8H15BrNO3 [M + H]+ 252.0235, found 252.0222 and 254.0202. ( S )-benzyl 2-(2-bromoacetamido)-3 -phenylpropanoate (2.34a’): Compound 2.34a’ was prepared following the procedure described for 2.34a from the HCl salt of phenylalanine benzyl ester 2.21a Isolated yield: 70%, white solid, m.p. = 6466 C. 1H NMR (400 MHz, CDCl3) 3.09 – 3.19 (m, 2H), 3.85 (s, 2H), 4.86 – 4.92 (m, 1H), 5.11 – 5.22 (m, 2H), 6.85 (br s, 1H), 6.99 – 7.04 (m, 2H), 7.22 – 7.25 (m, 3H), 7.30 – 7.40 (m, 5H). 13C NMR (100 MHz, CDCl3) 28.89, 37.87, 53.97, 67.71, 127.52, 128.88, 129.54, 135.10, 135.33, 165.30, 170.87. HRMS (ESI) calcd. for C18H19BrNO3 [M + H]+ 376.0548, found 376.0508 and 378.0490.

PAGE 86

71 methyl 2-(2-bromoacetamido )-2-methylpropanoate (2.34d): 2.34d was prepared following the procedure described for 2.34a Isolated yield: 52%, white solid. 1H NMR (400 MHz, CDCl3) 1.63 (s, 6H), 3.75 (s, 3H), 4.19 (s, 2H). HRMS (ESI) calcd. for C7H13BrNO3 [M + H]+ 238.0079, found 238.0088 and 240.0065. Representative procedure for the synthesi s of piperazine-2,5-dione DKP1 (2.33 and 2.35): Compound 2.34a (0.85 mmol) was dissolved in metha nol (2 mL); then triethylamine (2.55 mmol) was added. To this mixture, a solution of benzylamine (1.02 mmol) in methanol (2 mL) was added slowly. The reaction mixture was stirred under refluxing conditions until consumption of ester bromide 2.34a was observed by TLC monitoring (8 h). The crude solution was cooled to rt and concentrated under reduced pressure to yield a colorless oil crude. This crude was dissolved in DCM, washed with 5% aqueous citric acid (10 mL), NaHCO3 (10 mL), brine (10 mL), and dried over Na2SO4. Removal of the solvent under reduced pressure gave a white solid crude. ( S )-1,3-dibenzylpiperazine -2,5-dione (2.35a):

PAGE 87

72 Compound 2.35a was purified by column chromatography on silica gel (hexanes:ethyl acetate, 1:4), yield: 62%, white solid, m.p. = 165166 C. 1H NMR (400 MHz, CDCl3) 3.00 – 3.06 (m, 1H), 3.11 – 3.24 (m, 2H), 3.54 (d, J = 17.6, 1H), 4.32 – 4.37 (m, 1H), 4.44 – 4.55 (m, 2H), 6.16 (s, 1H), 7.13 – 7.35 (m, 10H). 13C NMR (100 MHz, CDCl3) 40.96, 48.70, 49.97, 56.82, 127.78, 128.38, 128.86, 129.09, 130.07, 135.00, 135.03, 164.41, 165.82. HRMS (ESI) calcd. for C18H19N2O2 [M + H]+ 295.1447, found 295.1434. ( S )-methyl 3-(3-isopropyl-2,5-dioxop iperazin-1-yl)propanoate (2.33c): 2.33c was prepared following the procedure described for 2.35a Isolated yield: 75%, thick colorless oil. 1H NMR (400 MHz, CDCl3) 0.89 – 0.96 (m, 6H), 2.14 – 2.25 (m, 1H), 2.52 – 2.59 (m, 1H), 2.81 – 3.09 (m, 1H), 3.14 – 3.35 (m, 2H), 3.68 (s, 3H), 3.69 – 3.71 (m, 2H), 4.47 – 4.54 (m, 1H), 7.50 (d, J = 8.9, 1H). 2.5 References Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. ( 1996 ) Reductive Amination of Aldehydes and Ke tones with Sodium Triacetoxyborohydride. Studies on Direct and Indirect Reductive Amination Procedures. Journal of Organic Chemistry, 61 (11), 3849-3862. Anderson, L.; Topper, M.; Jain, P.; Mc Intosh, E.; McLaughlin Mark, L. ( 2009 ) Synthesis of N -Boc protected hydrazine diacids as key stru ctural units for the formation of alphahelix mimics. Advances in experimental medicine and biology, 611, 211-2. Avancha, K. K. V. R. ( 2006 ) Design and synthesis of core structural intermediates for novel HIV-1 protease inhibitors and synthe sis, biological activity and molecular modeling of novel 20S proteasome inhibitors. Di ssertation, University of South Florida.

PAGE 88

73 Besada, P.; Mamedova, L.; Thomas, C. J.; Costanzi, S.; Jacobson, K. A. ( 2005 ) Design and synthesis of new bicyclic diketopiperazines as scaffo lds for receptor probes of structurally diverse functionality. Abstracts of Papers, 229th ACS National Meeting, San Diego, CA, United States, March 13-17, 2005 MEDI-553. Blankley, C. J.; Sauter, F. J.; House, H. O. ( 1969 ) Crotyl diazoacetate. Organic Syntheses, 49, No pp 99. Bollard Mary, E.; Keun Hector, C.; Beckonert, O. ; Ebbels Tim, M. D.; Antti, H.; Nicholls Andrew, W.; Shockcor John, P.; Cantor Glenn, H.; Stevens, G.; Lindon John, C.; Holmes, E.; Nicholson Jeremy, K. ( 2005 ) Comparative metabonomic s of differential hydrazine toxicity in the rat and mouse. Toxicology and applied pharmacology, 204 (2), 135-51. Chaskar, A. C.; Langi, B. P.; Deorukhkar, A.; Deokar, H. ( 2009 ) Bismuth chloridesodium nitrite. A novel reagent for chemoselective N-nitrosation. Synthetic Communications, 39 (4), 604-612. d'Ischia, M. ( 2005 ) Nitrosation and nitration of bioact ive molecules: toward the basis of disease and its prevention. Comptes Rendus Chimie, 8 (5), 797-806. Dinsmore, C. J.; Beshore, D. C. ( 2002 ) Recent advances in the synthesis of diketopiperazines. Tetrahedron, 58 (17), 3297-3312. Doyle, M. P.; Kalinin, A. V. ( 1996 ) Highly Enantioselective Intramolecular Cyclopropanation Reactions of N-Allylic-N-me thyldiazoacetamides Catalyzed by Chiral Dirhodium(II) Carboxamidates. Journal of Organic Chemistry, 61 (6), 2179-84. Francom, P.; Robins, M. J. ( 2003 ) Nucleic Acid Related Compounds. 118. Nonaqueous Diazotization of Aminopurin e Derivatives. Convenient Access to 6-Haloand 2,6Dihalopurine Nucleosides and 2'-Deoxynucle osides with Acyl or Silyl Halides. Journal of Organic Chemistry, 68 (2), 666-669. Fructos, M. R.; Belderrain, T. R.; Nicasio, M. C.; Nolan, S. P.; Kaur, H.; Diaz-Requejo, M. M.; Perez, P. J. ( 2004 ) Complete Control of the Chemoselectivity in Catalytic Carbene Transfer Reactions from Ethyl Di azoacetate: An N-Heterocyclic Carbene-Cu System That Suppresses Diazo Coupling. Journal of the American Chemical Society, 126 (35), 10846-10847. Funabashi, Y.; Horiguchi, T.; Iinu ma, S.; Tanida, S.; Harada, S. ( 1994 ) TAN-1496 A, C and E, diketopiperazine antibiotics with inhibitory activity against mammalian DNA topoisomerase I. The Journal of antibiotics, 47 (11), 1202-18.

PAGE 89

74 Gautschi, M.; Schmid, J. P.; Peppard, T. L. ; Ryan, T. P.; Tuorto, R. M.; Yang, X. ( 1997 ) Chemical Characterization of Diketopiperazines in Beer. Journal of Agricultural and Food Chemistry, 45 (8), 3183-3189. Gellerman, G.; Hazan, E.; Brider, T.; Traube, T.; Albeck, A.; Shatzmiler, S. ( 2008 ) Facile Synthesis of Orthogonally Protected Optically Pure Ketoand Diketopiperazine Building Blocks for Combinatorial Chemistry. International Journal of Peptide Research and Therapeutics, 14 (2), 183-192. Gilbert, J. A.; Frederick, L. M.; Ames, M. M. ( 2000 ) The aromatic-L-amino acid decarboxylase inhibitor carbidopa is selectively cytotoxic to human pulmonary carcinoid and small cell lung carcinoma cells. Clinical cancer research an official journal of the American Association for Cancer Research, 6 (11), 4365-72. Gennari, C., Colombo, L., Bertolini, G. ( 1986 ) Asymmetric Electrophilic Amination: Synthesis of -Amino and -Hydrazino Acids with High Optical Purity, Journal of the American Chemical Society 108, 6394-6395. Goodwin, D. C.; Aust, S. D.; Grover, T. A. ( 1996 ) Free radicals produced during the oxidation of hydrazines by hypochlorous acid. Chemical Research in Toxicology, 9 (8), 1333-1339. Grange, E. W.; Henry, D. W.; Lee, W. W. ( 1980 ) Glyoxylic acid hydrocarbylsulfonylhydrazones a nd therapeutic compositions. US Patent, 4218465. Hannachi, J.-C.; Vidal, J.; Mu latier, J.-C.; Collet, A. ( 2004 ) Electrophilic Amination of Amino Acids with N-Boc-oxaziridines: Efficient Preparati on of N-Orthogonally Diprotected Hydrazino Acids an d Piperazic Acid Derivatives. Journal of Organic Chemistry, 69 (7), 2367-2373. House, H. O.; Blankley, C. J. ( 1968 ) Preparation and decomposition of unsaturated esters of diazoacetic acid. Journal of Organic Chemistry, 33 (1), 53-60. Insaf, S. S.; Witiak, D. T. ( 2000 ) Synthesis of all distinct alpha -methyl-substituted isomers of amino bis(2,2'-ethanoic acid) diethy l ester and ethylenedi aminetetraacetic acid tetraethyl ester scaffolds. Tetrahedron, 56 (16), 2359-2367. Kuang, R.; Ganguly, A. K.; Chan, T. M.; Pramani k, B. N.; Blythin, D. J.; McPhail, A. T.; Saksena, A. K. ( 2000 ) Enantioselective syntheses of carboc yclic ribavirin and its analogs: linear versus convergent approaches. Tetrahedron Letters, 41 (49), 9575-9579. Ma, M.; Peng, L.; Li, C.; Zhang, X.; Wang, J. ( 2005 ) Highly Stereoselective [2,3]Sigmatropic Rearrangement of Sulfur Y lide Generated through Cu(I) Carbene and Sulfides. Journal of the American Chemical Society, 127 (43), 15016-15017.

PAGE 90

75 Maity, P.; Konig, B. ( 2008 ) Synthesis and Structure of 1,4Dipiperazino Benzenes: Chiral Terphenyl-type Peptide Helix Mimetics. Organic Letters, 10 (7), 1473-1476. Maity, P.; Konig, B. ( 2008 ) Synthesis of 1,4-dipiperazino benzene scaffolds as alpha helix mimetic. Abstracts of Papers, 235th ACS Na tional Meeting, New Orleans, LA, United States, April 6-10, 2008 ORGN-370. Malca-Mor, L.; Stark, A. A. ( 1982 ) Mutagenicity and toxicity of carcinogenic and other hydrazine derivatives: correlation between toxic potency in animals an d toxic potency in Salmonella typhimurium TA1538. Applied and Environmental Microbiology, 44 (4), 801-8. Martins, M. B.; Carvalho, I. ( 2007 ) Diketopiperazines: biologi cal activity and synthesis. Tetrahedron, 63 (40), 9923-9932. Mirvish, S. S. ( 1995 ) Role of N-nitroso compounds (NOC ) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bl adder cancer and contribution to cancer of known exposures to NOC. Cancer Letters (Shannon, Ireland), 93 (1), 17-48. Morilla, M. E.; Morfes, G.; Nicasio, M. C. ; Belderrain, T. R.; Diaz-Requejo, M. M.; Graiff, C.; Tiripicchio, A.; Sanchez-Delgado, R.; Perez, P. J. ( 2002 ) Intramolecular dealkylation of chelating diamines with Ru(ii) complexes. Chemical Communications (Cambridge, United Kingdom), (17), 1848-1849. Oguz, U. ( 2003) Design and synthesis of constrained di peptide units for use as beta-sheet promoters. Dissertation, Louisiana State University. Oguz, U.; Gauthier, T. J.; McLaughlin, M. L. ( 2001 ) Facile synthesis of a constrained dipeptide unit (DPU) for use as a beta -sheet promoter. Peptides The Wave of the Future, Proceedings of the Second International and the Seventeenth American Peptide Symposium, San Diego, CA, Un ited States, June 9-14, 2001 46-47. Oguz, U.; Guilbeau, G. G.; McLaughlin, M. L. ( 2002 ) A facile stereospecific synthesis of alpha -hydrazino esters. Tetrahedron Letters, 43 (15), 2873-2875. Ouihia, A.; Rene, L.; Guilhem, J.; Pascard, C.; Badet, B. ( 1993 ) A new diazoacylating reagent: preparation, structure, and use of succinimidyl diazoacetate. Journal of Organic Chemistry, 58 (7), 1641-1642. Perrotta, E.; Altamura, M.; Barani, T.; Bindi, S.; Giannotti, D.; Harmat, N. J. S.; Nannicini, R.; Maggi, C. A. ( 2001 ) 2,6-Diketopiperazines fr om Amino Acids, from Solution-Phase to Solid-Pha se Organic Synthesis. Journal of combinatorial chemistry, 3 (5), 453-460.

PAGE 91

76 Prasad, C. ( 1995 ) Bioactive cyclic dipeptides. Peptides (Tarrytown, New York), 16 (1), 151-64. Ragnarsson, U. ( 2001 ) Synthetic methodology for al kyl substituted hydrazines. Chemical Society Reviews, 30 (4), 205-213. Rodriguez, J. M.; Nevola, L.; Ross, N. T.; Lee, G.-i.; Hamilton, A. D. ( 2009 ) Synthetic inhibitors of extended helix-protein inter actions based on a biphenyl 4,4'-dicarboxamide scaffold. ChemBioChem, 10 (5), 829-833. Sattler, M.; Liang, H.; Nettesheim, D.; Meadow s, R. P.; Harlan, J. E.; Eberstadt, M.; Yoon, H. S.; Shuker, S. B.; Chang, B. S.; Mi nn, A. J.; Thompson, C. B.; Fesik, S. W. ( 1997 ) Structure of Bcl-xL-Bak peptide comp lex: recognition between regulators of apoptosis. Science (Washington, D. C.), 275 (5302), 983-986. Singh, S. B.; Tomassini, J. E. ( 2001 ) Synthesis of Natural Flutimide and Analogous Fully Substituted Pyrazine-2,6-diones, Endonucl ease Inhibitors of Influenza Virus. Journal of Organic Chemistry, 66 (16), 5504-5516. Southgate, D. A. T., Food Analysis, 2nd ed Edited by S. Suzanne Nielsen ( 2000) ; Vol. 35, p 449-451. Sunagawa, M.; Matsumura, H.; Sumita, Y.; Nouda, H. ( 1995 ) Structural features resulting in convulsive activ ity of carbapenem compounds: e ffect of C-2 side chain. Journal of Antibiotics, 48 (5), 408-16. Toth, B. ( 1996 ) A review of the antineoplastic action of certain hydrazines and hydrazine-containing natural products. In Vivo, 10 (1), 65-96. Vidal, J.; Guy, L.; Sterin, S.; Collet, A. ( 1993 ) Electrophilic amination: preparation and use of N-Boc-3-(4-cyanophenyl) oxaziridine, a new reagent that transfers a N-Boc group to Nand C-nucleophiles. Journal of Organic Chemistry, 58 (18), 4791-3. Vidal, J.; Hannachi, J.-C.; Hourdin, G.; Mulatier, J.-C.; Collet, A. ( 1998 ) N-Boc-3Trichloromethyloxaziridine: a new, power ful reagent for electrophilic amination. Tetrahedron Letters, 39 (48), 8845-8848. Weiss, S. T. The theoretical modeling, design, and synthesis of key structural units for novel molecular clamps and pro-apopto tic alpha helix peptidomimetics. ( 2006 ). Williams, R. M.; Armstrong, R. W.; Dung, J. S. ( 1985 ) Synthesis and antimicrobial evaluation of bicyclomycin analogues. Journal of Medicinal Chemistry, 28 (6), 733-40.

PAGE 92

77 Yin, H.; Hamilton, A. D. ( 2004 ) Terephthalamide derivatives as mimetics of the helical region of Bak peptide ta rget Bcl-xL protein. Bioorganic & Medicinal Chemistry Letters, 14 (6), 1375-1379. Yin, H.; Lee, G.-i.; Sedey, K. A.; Kutzki, O.; Pa rk, H. S.; Orner, B. P.; Ernst, J. T.; Wang, H.-G.; Sebti, S. M.; Hamilton, A. D. ( 2005a ) Terphenyl-Based Bak BH3 alpha -Helical Proteomimetics as Low-Molecula r-Weight Antagonists of Bcl-xL. Journal of the American Chemical Society, 127 (29), 10191-10196. Yin, H.; Lee, G.-i.; Sedey, K. A.; Rodriguez, J. M.; Wang, H.-G.; Sebti, S. M.; Hamilton, A. D. ( 2005b ) Terephthalamide Derivatives as Mimetics of Helical Peptides: Disruption of the Bcl-xL/Bak Interaction. Journal of the Americ an Chemical Society, 127 (15), 5463-5468.

PAGE 93

78 CHAPTER THREE DESIGN AND SYNTHESIS OF 4-RAND 4,6-R,R’-2,5-TERPYRIMIDINYLENES 3.1 Introduction 3.1.1 Pyrimidines Pyrimidines are compounds containing two nitrogen atoms in positions 1 and 3. Pyrimidines are considered among the most important class of heteroaromatic compounds due to their unique chemical and biological properties (Hurst, 1980). Compared to the benzene ring, the pyrimidine is a planar structure, but it contains four different bond angles and six different bond le ngths forming an irregular hexagonal shape (Figure 3.1 ) (von Angerer, 2004). Unlike benzene, pyrimidine is a water-soluble compound and a weak base, but the proper ties of both molecules, benzene and pyrimidine, are relatively modified dependi ng on the absence or presence of ring substituents (Hurst, 1980). Figure 3.1. Structure of pyrimidine, bond angles and lengths (von Angerer, 2004) The pyrimidine core is found in many na turally occurring substances (Figure 3.2 ), such as nucleobases (uracil, cytosine, and t hymine), glycosides (vicine and convicine), and thiamine or vitamin B1 (Zempleni et al. 2007). It is also the key structure of several

PAGE 94

79 synthetic therapeutic agents including antibacterial (trimethoprim and sulfadiazine) and anticancer agents (gemcitabine, tegafur, ima tinib, nilotinib, and dasa tinib) (Lagoja, 2005; von Angerer, 2004). Figure 3.2. Pyrimidine unit as componen t of biologically active compounds The electron-deficient nature of pyrimidines is exerted by the two nitrogen atoms, which is comparable to having two nitro groups in positions 1 and 3 of benzene; whereas substitution on the pyrimidine ring with electron-donating groups counteracts this deficiency by making it behave more like a benzene ring. In gene ral, pyrimidines are thermally stable due to their aromaticity a nd amide-like structures (von Angerer, 2004). While the characteristics menti oned above are more relevant to describe the chemistry of pyrimidines, it is still significant to mention them since our objective is related to the

PAGE 95

80 synthesis of compounds that contain the pyr imidine core, but that can behave as benzenoid structures with mo re drug-like characteristics. 3.1.2 2,5-Terpyrimidinylenes as Potential -Helical Mimetics Based on our efforts to synthesize the pi perazine-dione olig omers (discussed in Chapter Two), we revisited the idea of using the scaffolds reported by Hamilton and Rebek (Section 1.3 in Chapter One) as a guide for designing potential -helix mimics. Our 2,5-terpyrimidinylene scaffold target is shown in Figure 3.3 Specifically, this template is structurally analogous to Ha milton’s terphenylene given that his work provided a valuable reference point when designing the 2,5-te rpyrimidinylenes described here. Figure 3.3. Structure of a trimeric 2,5-terpyrimidinylene scaffold. R1, R2, and R3 = selected alkyl or aryl groups specific to mimic the ith ith +4, and ith +7 residues of an -helix; R' = H, alkyl, or aryl; and R'' = CN, CO2H, CONH2, C(NH2)-NH, C(NH2)-NOH. Our novel scaffold replaces the phenyl rings of Hamilton’s 1,4-terphenylenes by the pyrimidine units to make the synthesis much easier, highly iterative, and afford more hydrophilic compounds. The N -terminus-like and the C -terminus-like can be modified to improve the drug-like physical properties of the scaffold. Moreover, the monomeric and dimeric pyrimidinylenes are structural mimics of the biphenyl core, a fairly well known structure in drug discovery (Horton et al. 2003). Independent of the intended -helical mimic design of trimeric 2,5-pyrimidinylenes, these could be used as probes for biological targets that are still undefined. The 4, 4’, and 4” positions (R1, R2, and R3) of

PAGE 96

81 the trimeric 2,5-pyrimidinylene scaffold are designed to mimic the ith ith +4, and ith +7 of the target -helix. These groups can be varied br oadly to optimize the bioactivity and specificity of the resulting library compounds. Figure 3.4 shows a tube representation of an idealized -helical conformation of oc ta-alanine (gold with transparent ribbon) with its ith ith +4, and ith +7 methyl groups highlighted as gol d spheres and a 4,4’,4”-trimethyl2,5-terpyrimidinylene with its methyl groups high lighted as green sphere s. As seen with the Hamilton designed 1,4-terphenylene scaffolds (Yin et al. 2005a; Yin et al. 2005b), there is good overlap of these positions both in orientation and distance. Residues at the ith +4 position are one turn plus 40 and ith +7 residues are 20 less than two turns of an helix from the ith position. Figure 3.4. Overlay of a 4,4’,4’’-trimethy l-2,5-terpyrimidinylene and an octaalanine. The RMSD for the highlighted methyl groups is 0.68 . For simplicity, only polar hydrogens are shown. The details of th ese calculations can be found in Appendix C (calculations were done by Daniel N. Santiago). 3.1.3 General Methods for the Synthesis of Pyrimidines Several methods for the synthesis of pyrimidines have been reported; a comprehensive review is described in the Sc ience of Synthesis se ries (von Angerer,

PAGE 97

82 2004). In general, the pyrimidine ring can be prepared directly from cyclization of different aliphatic fragments, rearrangements, ring contraction or expansion of other ring systems, or modification of substituents in cluding cross-coupling r eactions (Hurst, 1980; Kang et al. 2005; Schomaker and Delia, 2001; von Angerer, 2004). Substituted pyrimidines can be formed by the Biginelli method where aromatization of the resulting dihydropyrimidinone gives access to the pyrimidine ring (Kang et al. 2005). The most common method for the synthesis of substitute d pyrimidines involves the condensation of 1,3-dielectrophiles, such as -dicarbonyls and synthetic equi valents, with unsubstituted amidine derivatives, such as amidine salts, guanidine, ureas, and thioureas, usually in the presence of a base (Scheme 3.1 ) (Brown, 1994; Hill and Movassaghi, 2008; von Angerer, 2004). Scheme 3.1. Common method for the sy nthesis of substituted pyrimidines Pyrimidines have been well known for years and thousands of derivatives containing unsubstituted and substituted mono meric pyrimidines have been reported in the literature. However, dimeric and trimer ic 2,5-pyrimidinylene scaffolds have not been fully explored, especially an iterative syntheti c approach and their potential application as -helical mimetics have not been reported previously. Only a few examples of unsubstituted 2,5-terpyrimidinylenes have b een described; these compounds have been

PAGE 98

83 reported for their electrochemical properties and a few for highly functionalized dimeric pyrimidines, which have been prepared via Stille and Suzuki couplings (Brandl et al. 1996; Gompper et al. 1996; Gompper et al. 1997; Saygili et al. 2004). Thus, our novel non-peptidic scaffold (Figure 3.3 ), which is designed to mimic a specific -helical region of a protein as a PPI inhibitor, re presents a great opportunity for targeted therapeutics. The scaffold based on repeated pyrimidine units should improve the hydrophilicity of the resulting compounds as compared to previously described peptidomimetics (discussed in Chapter One, Section 1.3 ). Also, the non-peptidic nature of our compounds is expected to overcome so me of the intrinsic disadvantages carried when using peptides themselves as drug l eads, such as metabolic degradation, poor bioavailability, and high pr oduction costs (Ahmed and Kaur, 2009; Loffet, 2002). 3.2 Results and Discussion 3.2.1 Synthesis of a “First-generation” 4-R2,5-Terpyrimidinylene Library A retrosynthetic scheme for the targeted 4R-2,5-terpyrimidinylen e is shown in Figure 3.5 Due to its versatility, we adopt ed the most common method (Scheme 3.1 ) as the general iterative approach fo r preparing the 4-R-2,5-terpyr imidinylenes. Accordingly, the condensation of an -unsaturated -cyanoketone (shown in blue) with an amidine derivative (shown in magenta) affords 5-cy ano substituted pyrimidinylene monomer.

PAGE 99

84 Figure 3.5 Retrosynthetic scheme of trimeric 2,5-pyrimidinylenes Iterative transformation of the 5-cyano gr oup of that unit into an amidine allows the modular synthesis of 2,5-terpyrimidinylene trimers with variable groups at the 4, 4’, 4”, and 5” positions. A representative general procedure for the synthesis of pyrimidinylene monomers ( 3.4a-d ) is shown in scheme 3.2 Scheme 3.2. General synthesi s of pyrimidinylene monomers This synthesis began with the coupli ng of commercially available esters 3.1a-h with acetonitrile in anhydrous TH F in the presence of potassium t -amyloxide (KO t -amyl)

PAGE 100

85 to give -ketonitriles 3.2a-h (Ji et al. 2006). Reactions for step one were also carried out in presence of other bases, such as s odium methoxide (NaOMe) (Sorger and Stohrer, 2007), lithium diisopropylamide (LDA), and potassium t -butoxide (KO t -Bu). While the desired products were also afforded, the isolation and pur ification of the -ketonitriles were tedious resulting in fairly low yields (40-60%). The formation of side product 3.5a was observed when KO t -Bu and LDA were used (Scheme 3.3 ). Scheme 3.3. Formation of side product 3.5a Alternatively, reactions with KO t -amyl in THF gave the products in good yields (64-73%) and further purifica tion was not required as unpurified compounds showed clean TLC and NMR spectra with complete conversion of esters 3.1a-h The precipitated ketonitrile salts were easily isolated by filtration and subs equent hydrolysis with aqueous acid allowed us to obtain the products 3.2a-h A slight variation of the protocol described by Ji and co-workers was the use of the carboxylic ester as the limiting reagent since in our case the required acetonitrile wa s significantly cheaper and readily available compared to esters 3.1 (Ji et al. 2006). Both enolizable and nonenolizable esters reacted with acetonitrile at room temperature in similar reaction rates and yields. Subsequent treatment of -ketonitriles 3.2 with N,N -dimethylformamide dimethyl acetal (DMF-DMA) in THF gave 3.3 as an isomeric E>>Z mi xture in excel lent yields (>90%) (Reuman et al. 2008). The efficiency of this reaction allowed us to obtain several -unsaturated -cyanoketones bearing hyd rophobic alkyl, aryl, and

PAGE 101

86 heteroaromatic groups for introducing diversity in the 4-position of pyrimidinylenes 3.4 The condensation of the -unsaturated -cyanoketones 3.2a-j with several amidines in presence of a base gave monomeric pyrimidinylenes 3.4a-j (Hill and Movassaghi, 2008; von Angerer, 2004). The results for the synthe sis of the monomers are summarized in Table 3.1 Table 3.1. Results of the sy nthesis of monomers 3.4a-j Monomers having R’ = Ph (Table 3.1 entries 4-12), were generally isolated in higher yields and no further purification wa s required because these compounds usually

PAGE 102

87 precipitated from the reaction mixtures; convers ely, we observed that reactions to prepare monomers having R’ = H, Me, and Cl typically afforded incomplete consumption of the -unsaturated -cyanoketones and required additi onal purification to obtain the products (Table 3.1 entries 1-3, 13). For instance, the obser ved low yield for the preparation of 2-unsubstitu ted pyrimidinylene (Table 3.1 entry 1) could be attributed to the ease of hydrolysis of formamidine unde r typical condensati on conditions (Hurst, 1980) and the low yield of the 2-chloropyrimidine (Table 3.1 entry 3) was possibly due to the poor solubility of 2-chloroamidine in the required aprotic solvent. Monomers having R’ = NH2 were also isolated in good yields (Table 3.1 entries 14-18). Some pyrimidinylenes ( 3.4i.3 and 3.4j.3 ) were also obtained by the direct condensation of the -ketonitriles ( 3.2i and 3.2j ) and amidines (Baran et al. 2006) in presence of the orthoformate (DMF-DMA) without a base; however, these required much longer reaction times (>48 h) to obtain comparab le yields and complete conversion of the -ketonitriles were not observed. Based on th ese experimental results and encouraged by the excellent yields and purities (>90% as determined by NMR spectroscopy) obtained when R’ = Ph, we decided to focus on the ite rative synthesis of the dimeric and trimeric 2,5-pyrimidinylenes using mostly these de rivatives via the condensation of the unsaturated -cyanoketones and amidine intermediates (Scheme 3.2 ). The key step of this synthesis is the conversion of the 5-cyano group to a 5carboxamidine intermediate to further synthesize dimeric 2,5-pyrimidinylenes. The advantage of this transformation is that it allows obtaining the amidine intermediates in fewer steps compared to alternative crosscoupling-type reactions, which would require more steps to synthesize the appropriate star ting materials. Moreove r, extensive research

PAGE 103

88 based on this key transformation is available in the literature since it is also the most common method to prepare unsubstituted amidines (Aly and Nour-El-Din, 2008). Methods for the synthesis of amidines from amides, thioamides, and esters have also been reported (Aly and Nou r-El-Din, 2008; Dunn, 2005; Gielen et al. 2002; Lee et al. 1998), but we focused on the synthesis of 5-cy anopyrimidines due to the versatile access to the -ketonitriles. Many methods for the conver sion of nitriles to amidines have been reported (Dunn, 2005) and some are depicted in Scheme 3.4 These include the >150 year old Pinner reaction, where a nitrile is activated to an intermed iate salt in the presence of HCl and ethanol. The resulting imino ester, known as the Pinner salt, is treated with ammonia or amines to afford the desired amidine (Pinner’s method) (Balo et al. 2007; Han et al. 2000). A variation of this method inco rporates the addition of amines to thioimidates (Thio-Pinner method) (Lange et al. 1999). Amidines have been also prepared by the direct addition of amines to nitriles in presence or absence of Lewis acids, such as ZnCl2 (Lee et al. 2007), Cu(I) salts (Rousselet et al. 1999), and ytterbium amides (Wang et al. 2008). Treatment of nitriles with Na and LiHMDS (Bruning, 1997; Hu et al. 2008) and with alkylchloroaluminum amides (Garigipati’s method) (Garigipati, 1990) followed by aqueous hydrolysis allows the synthesis of amidines as well. While several of these procedures were performed with some 5-cyanopyr imidines (Table 3.2 ), none of these alternativ es were better than the two-step hydroxylamine route (Table 3.2 entry 10) (Judkins et al. 1996; Nadrah and Dolenc, 2007). We found excess hydr oxylamine hydrochloride followed by an in situ reduction of the resulting amidoxime to be the best route to obtain the intermediate carboxamidine salts 3.6 (Scheme 3.5 ).

PAGE 104

89 Scheme 3.4. Methods for the conversion of cyano group to amidine

PAGE 105

90 Table 3.2. Attempted routes to synthesize 5-carboxamidines

PAGE 106

91 Scheme 3.5. Hydroxylamine-mediated synthesis of amidine intermediates The N -hydroxylamine mediated transformation of ortho -substituted and diortho substituted arylnitriles to carboxa midines has been reported (Basso et al. 2000; Judkins et al. 1996; Lukyanov et al. 2008), but has not been used to synthesize oligopyrimidinylenes. Based on our observations and literature report s on conversion of nitriles to amidines, we could reasonably concl ude that the inefficiency of most of these reactions is essentially due to an steric effect exerted by the ortho -substituents of the pyrimidine ring (Dunn, 2005; von Angerer, 2004; Watanabe et al. 2009). Many of these publications claim the formation of amid ines through facile procedures (Lange et al. 1999; Lee et al. 1998); however, most of the repo rted conditions have not been described for an ortho -substituted aromatic ring or pyrimidine moiety. Moss demonstrated the synthesis of amidines fr om sterically hindered alkyl nitriles via Garigipati’s method (Moss et al. 1995), but in our hands, the procedure was not reproducible when using ortho -substituted pyrimidinylenes. An initial study where we treated omethylbenzonitrile with some of these conditions (as on entries 4, and 8 on Table 3.2 ) gave indication that reaction of th e nitrile group is inhibited by steric hindrance. In most cases, the omethylbenzonitrile was recovered unreacted. It was particularly noted that in presence of strong bases, such as Na and LiHMDS, the predominant product was isoquinazoline 3 9 (Scheme 3.6 ). This observation was specific

PAGE 107

92 to this substrate and it does not necessarily translate to any substrate containing a proton on the ortho position. Further experimentation is re quired for validation and this is an ongoing project in our lab. On the othe r hand, the direct addition of ammonia (Beingessner et al. 2008) to omethylbenzonitrile in pres ence and absence of Lewis acids, such as CuBr resulted in unreact ed nitrile or tr aces of amidine 3.8 in addition to decomposition products as evidenced by TLC and 1H NMR. Scheme 3.6. Attempted route to obtain amidine 3.8 As previously mentioned, the intermed iate amidoximes were more efficiently obtained by the hydroxylamine procedure (Scheme 3.5 ); however, this ro ute continued to show the formation of carboxamide side product 3.10a.3 (Table 3.3 ). The desired amidoximes were separated from the amide side products by column chromatography. We also attempted to improve the yields of the corresponding amidoximes by changing the reaction conditions; we could observe from these reactions that the ratio of amidoxime : amide was mostly affected by th e type of base used and possibly by the steric hindrance of the ortho substituent in the cyanopyrimidine as this has been previously suggested (Judkins et al. 1996; Lukyanov et al. 2008). It was observed that reactions in presence of trie thylamine afforded the amidoximes in higher yields compared to reactions with weaker bases, such as NaHCO3 (Koryakova et al. 2008), where the

PAGE 108

93 formation of the amide was predominant (Table 3.3 entry 7). Variations of reaction time, temperature, or solvent, including anhydrous conditions did not improve the yields of amidoximes 3.6a.3 but it was noted that excess of hydroxylamine (>2 eq.) gave the best results. Table 3.3. Reaction of compound 3.4a.3 with hydroxylamine HCl

PAGE 109

94 It has been previously suggested that amide formation is due to an initial attack of the oxygen atom of hydroxylamine to the cy ano group and not to hydrolysis of the amidoxime or nitrile (Lukyanov et al. 2008). To follow up with this observation, we then treated cyanopyrimidine 3.4b.3 with O-benzyl hydroxylamine, but this only resulted in unreacted starting materials with no indi cation of nitrile conversion to amidoxime. Figure 3.6. Hypothetical model for H-bo nding mediated synthesis of amidine Based on this, we speculate that the a ttack of the nitrogen atom of hydroxylamine to the cyanopyrimidine could be facilita ted by simultaneous hydrogen bonding from the OH of hydroxylamine as depicted in Figure 3.6 which could explain why the O-benzyl hydroxylamine reaction did not occur; we conclude the use of unsubstituted hydroxylamine is therefore essential for the re action to take place. Formation of the amide side product has been inevitable. For instance, amide 3.10a.3 was purified and recrystallized (Figure 3.7 ); nevertheless, we have been able to obtain the desired

PAGE 110

95 amidoximes in practical yields to continue th e iterative synthesis of dimeric and trimeric scaffolds. Figure 3.7. ORTEP diagram of carboxamide side product 3.10a.3 In the next step, the resulting amidoximes 3.6 were reduced by a mild procedure via transfer hydrogenation using potassium fo rmate in the presence of 10% Pd/C to afford the carboxamidine intermediates (Step two in Scheme 3.5 ) (Nadrah and Dolenc, 2007). The advantage of this methodology is the easier handling of reactants and the reduced need for hydrogen gas and high pressure reactions. Some amidoximes were also reduced with ammonium formate as th e hydrogen transfer reagent (Anbazhagan et al. 2003). Although the carboxamidine sa lts were not isolated and purified, our results were consistent with the literature. Reactions with potassium formate in methanol after

PAGE 111

96 selective acylation of the amidoximes occurred faster. Also, complete conversion of the amidoximes were observed as compared to re actions with ammonium formate in acetic acid as solvent (Nadrah and Dolenc, 2007). Treatment of amidoximes with stannous chloride (1M SnCl2.2H2O in EtOH) (Cesar et al. 2004) or Zn in HOAc (Lepore et al. 2002) provided incomplete reductions; most of the starting material remained by TLC monitoring, even when extended reaction times or microwave-assisted reduction conditions were used. In addition, separatio n of the polar carboxamidines from resulting tin salts was laborious, which resulted in ve ry low yields. Therefore, the method described by Nadrah and Dolenc was the pr eferred procedure for the synthesis of 5carboxamidine intermediates ( vide supra ). The intermediate amidine salts 3.7 were reacted with another desired unsaturated -cyanoketone 3.3 to yield dimeric 2,5-pyrimidines 3.11 Several of these compounds were synthesized in our group in modest yields (Table 3.4 entries 1-9). Repetition of the steps described for the dime rs afforded target 2,5-terpyrimidinylenes 3.12 in lower yields compared to dimers (Table 3.4 entries 10-14). The yields reported for both dimers and trimers are indicated for is olated products as precipitates from the reaction mixtures. The purity of all precip itated compounds was >90% as determined by NMR spectroscopy.

PAGE 112

97 Table 3.4. Results of the synthesi s of dimers 3.11 and trimers 3.12

PAGE 113

98 Figure 3.8. ORTEP diagram of compound 3.12bac.3 The ORTEP diagram of the 5”-cyano-4 ”-isopropyl-4’-phenylmethyl-4-isobutyl-2phenylterpyrimidine crystal st ructure is shown in Figure 3.8 The dynamic nature of the crystal structure infers the low barrier to rotation about the single bonds between the 2,5pyrimidine rings. In addition, the simple 1H NMR spectra of these compounds is also consistent with this low barrier to rotation. The chemical shift of the proton in position 6 of the pyrimidine ring (Figure 3.1 ) is also a very characterist ic and useful indicator to track the synthesis of these compounds. An example of a typical 1H NMR spectrum of a trimer show ing the protons at 6, 6’, and 6” (a, b, and c, respectively) positions is shown in Figure 3.9

PAGE 114

99 Figure 3.9. Typical 1H NMR spectrum of a trimeric 2,5-pyrimidinylene For the synthesis of trimeric 2,5-pyrimidinylenes, any R1, R2, and R3 substituent that would be unreactive to the conversion of cyano to amidine or carboxylic acid (details described later in this ch apter) could have been sel ected. We chose hydrophobic substituents, such as isobutyl, benzyl, isopropyl, methyl, 1-naphthylmethyl, and 2naphthylmethyl (Table 3.3 ), as surrogates of common side chains of DNA-encoded amino acids. These R1, R2, and R3 groups play important roles in binding interactions, a subject investigated by Ham ilton and co-workers (Kutzki et al. 2002; Yin et al. 2005b). The naphthalene group is often used to mimic the i ndole-containing side chain of tryptophan (Moisan et al. 2008), a key binding amino acid residue on one face of the p53 -helix (Shaginian et al. 2009). Furthermore, these were selected with the idea that these terpyrimidinylenes would mimic an -helical conformation. Structurally analogous terphenylenes (Figure 1.6 in Chapter One) that contain polar substituents at the N -terminus-like and C -terminus-like sites have been reported by

PAGE 115

100 the Hamilton group ( vide supra ). Structure-activity studies indicated th at terphenyl derivatives with isobutyl, 1-na phthylmethyl, isobutyl (Figure 1.6 top panel-c, compound 14 from reference) (Yin et al. 2005b) and isobutyl, 2-napht hylmethyl, isobutyl side (Figure 1.6 top panel-b) (Yin et al. 2005a) chain sequences were potent inhibitors of Bcl-xL ( K i = 0.114 M) and Mdm-2 ( K i = 0.180 M), respectively. Also, it was determined that the terminal carboxyl groups seemed to be necessary for the inhibitory properties presumably due to the increased pol arity, solubility in aqueous solutions, and potential interactions with a Bcl-xL lysine residue possibly by mimicking aspartic acid residue 83 of the Bak peptide (Yin et al. 2005b). Figure 3.10. QikProp calculatio ns for terphenyl-based Bcl-xL-Bak inhibitor and a terpyrimidinylene-based analog. Calculations were done by Daniel N. Santiago According to the QikProp calculations summarized in Figure 3.10 structurally analogous 1,4-terphenylene and 2,5-terpyrimidi nylene scaffold show the calculated log Poctanol/water values ranged from quite hydroph obic for the Hamilton 1,4-terphenylene scaffold (Yin et al. 2005b) to within the desirable ra nge for drug-like characteristics for

PAGE 116

101 the analogous 2,5-terpyrimidinylene scaffol d. The full Qikprop out put can be found in Appendix C Modification of the R” group of dimeric and trimeric scaffolds to more polar functionalities could be achieved via dir ect conversion of the nitrile to amide, carboxylate, tetrazole, and methyl amine. Conversion of cyano to carboxylic acid was efficiently obtained by following a proce dure adapted by Dr. Vasudha Sharma, a postdoctoral fellow in our group. The results and routes for the preparation of tetrazoles explored by Dr. Sharma are not mentioned he re; only the preparation of carboxylates is discussed (Table 3.5 ). Table 3.5. Conversion of 5-cyanopyr imidine to 5-carboxypyrimidine The 5and 5’-carboxylate pyrimidines were obtained by reacting 5and 5’cyanopyrimidines with sodium hydroxide unde r microwave–assisted reaction conditions (Table 3.5 ). Isolation and purifi cation of monomeric and some dimeric 2,5-pyrimidine

PAGE 117

102 carboxylates (Table 3.5 entries 1-3) were easily performe d, but the same was not true for the trimeric compounds (Table 3.5 entries 4-5); rather, inco mplete hydrolysis of the cyano group was consistently observed. Both unreacted starting nitrile and intermediate amides were observed by TLC and 1H NMR, even under prolonge d heating and reaction times with the trimers. This problem could possibly be due to the lower solubility of some dimers and all trimers because this was not observed for monomers. Reactions done by conventional heating gave similar re sults to the reactions done under microwaveassisted conditions. 3.2.2 In Vitro Biological Evaluation T able 3.6. Results of the in vitro evaluation of monomeric and dimeric 2,5pyrimidinylenes Several pyrimidinylene-based compounds were evaluated for their inhibitory effect on the Bcl-xL/Bak and the Mdm-2, Mdm-x/p53 comp lexes in a competitive binding

PAGE 118

103 assay based on fluorescence polarization (FP). Based on the rationale behind the design of -helical mimetics (discussed in Chapter On e), we would not anticipate monomeric and dimeric 2,5-pyrimidines to exhibit any bio activity at least for these target assays; nevertheless, these were tested as SAR probes for the potential activity of the trimeric compounds (Table 3.6 ). We envisioned that terpyrimidinylen es holding equal or similar hydrophobic R groups as those of Hamilton’s best terpheny lenes would inhibit these protein-protein interactions. We used some of Professo r Hamilton’s best terphenyl inhibitors (Yin et al. 2005b) as positive controls for the assay (Table 3.7 entries 5-7); unfor tunately, none of our trimeric derivatives tested so far have shown promising bioactivity (Table 3.7 entries 1-4). It is worth noting that preparation of the solutions in DMSO at equal concentrations to those of the 1,4-t erphenylenes for the in vitro test demonstrated that most of our compounds, especially the trimeric scaffolds having R’ = Ph and R’’ = CN, precipitated from the solutions by forming aggregates. This suggests the repl acement of the phenyl ring by the pyrimidine ring on the analogous 1,4terphenylene is not sufficient to achieve water solubility. While none of our “first ge neration” library compounds were useful for the inhibition of the Bcl-2/Bcl-xL or Mdm-2, Mdm-x/p53 proteinprotein interactions, the synthesis and biological evaluati on of these provided us with significant tools for scaffold optimization.

PAGE 119

104 Table 3.7. Comparative in vitro evaluation of trimeric 2,5-pyrimidinylenes and Hamilton’s terphenylenes (Yin et al. 2005b) 3.2.3 Synthesis of a “Second-generation” 4-R-2,5-Terpyrimidinylene Library Based on the results of the bi oactivity of the “first ge neration” library, our next objective was to synthesize terpyrimidinylenes holding polar moieties at each end of the scaffold. The use of the chemistry already e xplored for the “first-generation” compounds seemed to be the most practical approach. Thus, to introduce the desired carboxylic acid at the N -terminus-like site, we looked for re placing the phenyl gr oup derived from benzamidine, with a carboxylate derived from 4-cyanobenzoic acid. Treatment of 4cyanobenzoic acid 3.16 with excess hydroxylamine afforded intermediate amidoxime in good yield (Scheme 3.7 ). In this case, forma tion of amide side product 3.18 was

PAGE 120

105 significantly decreased, most likely due to the absence of substituents flanking the cyano group of compound 3.16 Scheme 3.7. Synthesis of compound 3.19a The intermediate amidoxime 3.17 was obtained as a triethylamine salt and further reduction gave intermediate amidine carboxylate salt, which was also used in the condensation step with 3.3a without further purification. Af ter isolation and purification, monomer 3.19a was afforded in modest yield (35 %). Condensation with more polar unsaturated -cyanoketone, such as 3.3b gave much lower yield of the monomer as a result of a tedious isolation of the highly pol ar pyrimidines. A potential alternative to overcome the isolation issue of the terpyrimid inylenes carboxylates may be the use of 4cyanobenzoic ester as starting material for this library, although formation of N hydroxyamides could be also problematic if the hydroxylamine route is used (Massaro et al. 2007). Encouraged by the fact that monomers having R’ = NH2 were also obtained in excellent yields (Table 3.1 entries 14-18), we improvise d an alternative route to introduce the polar functionality at the N -terminus-like site as part of a “secondgeneration” library. Several monomeric 2-aminopyrimidinylenes and a dimer were efficiently prepared. To our delight, thes e compounds were as easy to isolate as the

PAGE 121

106 monomers having R’ = Ph and the majority of the 2-ami nopyrimidinylenes precipitated from the reaction mixtures and were obtaine d in excellent yields and purity. Most importantly, the dimeric 2-amino-2,5-pyrimidine 3.11ba.4 showed some bioactivity when evaluated in the FP assay (Table 3.6 entry 4 ). The advant ages of pursuing the 2aminoterpyrimidinylene synthesis are: first, the guanidine reagent is readily available making this reaction cost effective; second, the 2-aminopyrimidines are already more polar than the 2-phenylpyrimidinylenes; and third, potential nucleophili c displacement of the amino group via diazotization reaction coul d lead to the placement of the carboxylate; for instance, -alanine or glycine ester can be placed in that position to yield carboxylates similar to the 1,4-te rphenylenes (Table 3.7 entries 5-7). The direct N -alkylation of the 2aminopyrimidinylenes was also considered, but our efforts were unsuccessful. This most likely was due to a decrease of the nucleophili city of the amino group exerted by the electron-withdrawing effect of the pyrimidine ring (Hur st, 1980). The synthesis, optimization, and biological evaluation of this ‘second-ge neration’ compound library are under investigation. 3.2.4 Synthesis of 4-R,6-R2,5-Terpyrimidinylenes Previously, we showed our efforts to s ynthesize the semi-rigid terpyrimidinylene scaffolds and only the early steps toward the synt hesis of this new stru cturally related, but more polar diortho -substituted structure will be discussed. The target trimeric scaffold is shown in Figure 3.10 Substitution at the 4, 4’, and 4” and 6, 6’, and 6” positions of these novel library reduces the rotational freedom along the single bond between the 2,5pyrimidinylene monomers compared to the scaffold previously described (Figure 3.3 ).

PAGE 122

107 Figure 3.10. Target 4-R,6-R-2,5-terpyrimidinylene. R1, R2, and R3 = selected alkyl or aryl groups specific to mimic the ith ith +4, and ith +7 residues of an -helix; R' = H, alkyl, or aryl; and R'' = CN, CO2H. The synthesis of 6-amino-4-substituted building block is shown in Scheme 3.8 This synthetic route is very similar to the one followed for the “first-generation” library (Scheme 3.2 ). The only difference is that the Mi chael acceptor used in this case is derived directly from malonitr ile and not from the ketonitril e; by doing this, one of the cyano groups of the malonitrile derivative acts as an acceptor to originate the pyrimidine with the amino group in position 6 and the R group in position 4. Scheme 3.8. Synthesis of 6-amino-4substituted pyrimidinylene monomers

PAGE 123

108 We have successfully synthesized a fe w monomers from some commercially available Michael acceptors. The synthesis of the monomers is shown in Scheme 3.8 It should be noted that monomer 3.22a.3 (Scheme 3.8 ) was obtained without further treatment with an oxidizing agent (Peters et al. 2004). We believe the intermediate dihydropyrimidinylene aromatized by simple air oxidation during the course of the reaction. Scheme 3.9. General route for the synthesi s of 6, 6’, 6”-triamino -4-R-, 4’-R-’, 4”R”-2,5-pyrimidinylenes and Michael acceptors A general proposed route for the synthesi s of the 6, 6’, 6”-triamino-4-R-, 4’-R-’, 4”-R”-2,5-pyrimidinylenes is summarized in Scheme 3.9 top panel-a. The intermediate amidine can also be obtained by the two-step hydroxylamine-mediated conversion of the

PAGE 124

109 cyano group to amidine as pr eviously described (Scheme 3.5 ). Subsequent condensation with a Michael acceptor holding the desired R group can generate dimeric scaffolds. To have other alkyl or aryl groups different than methyl and ph enyl at the 4-position of the pyrimidine unit, the ethylidene malonates (Michael acceptor in Scheme 3.9 ) can be prepared by a Knoevenagel-type condensatio n with procedures pr eviously described (Sylla et al. 2006). A disadvantage of this method is that it is efficient only with aromatic, highly branched, and non enolizable aldehydes. An alternative to get the R groups we desire as amino acid residue surr ogates is by adapting the procedure outlined in Scheme 3.9 bottom panel-b (Kraybill et al. 2002). Any synthesized acid chloride most likely would be converted to the methyl ether after appropriate O-alkylation of the resulting dicyanoenol with dimet hyl sulfate. It is expected that iteration of the steps described in Scheme 3.9 will furnish diortho -substituted dimeri c and trimeric 2,5terpyrimidinylenes. The pot ential bioactivity of thes e compounds may increase or decrease, but the water solubility is expected to be much improved. 3.3 Conclusion A “first-generation” focused library based on a novel 4-R-2,5-terpyrimidinylene scaffold was successfully synthesized thr ough a facile iterative condensation between amidines and -unsaturated -cyanoketones in the presence of a base. In a similar fashion, the initial steps for the synthesis of related but more polar scaffolds based on 4,6R,R’-2,5-terpyrimidinylenes were pres ented. Although none of the compounds synthesized so far have shown significant bioactivity for disrupting various proteinprotein interactions, th e value of these syntheses lies in the assessment of several

PAGE 125

110 synthetic routes to obtain the desired compounds. Also, we l earned that the presence of polar functionalities, such as the carboxylate at the N -terminus-like and C -terminus-like sites play an important role in improving the drug-like characteristics and potentially the bioactivity of these libraries. 3.4 Experimental Section 3.4.1 Experimental Procedures Representative procedure for the synthesis of -ketonitriles (3.2): All compounds described in Section 3.2.1 were labeled based on the substituents as follows: 3-oxo-4-phenylbutanenitrile (3.2a) : A mixture of potassium tpentylate (~1.7 M in toluene, 106 mmol) and anhydrous THF (20 mL) was cooled at 0 C in an ice-bath and under an argon atmosphere. Then anhydrous acetonitrile (106 mmol) and the methyl ester 3.1a (71 mmol) were added simultaneously to the cold solution. The reaction mixture was allowed to warm to rt and s tirred under an argon atmosphere for 22 h. A precipitate was formed within a few minutes (3-5 min.) of reaction and it remained cloudy until completion. The reaction was monitored by TLC and it was stopped when the TLC indicated the cons umption of the ester ( 3.1a ). The mixture was filtered and the

PAGE 126

111 filtered cake was washed thoroughly with hexa nes (80 mL). Then, the filtered residue was transferred to a se paratory funnel and it wa s acidified to pH= ~2-3 with saturated aq. KHSO4 solution (400 mL). An equal volume amount of DCM (400 mL) was used to extract the desired compound ( 3.2a ). The organic layer was wa shed with brin e (100 mL), dried over Na2SO4, and concentrated under reduced pressure to yield 3.2a in 68% yield, as a pale yellow oil. 1H NMR (400 MHz, CDCl3) 3.46 (s, 2H), 3.86 (s, 2H), 7.21–7.40 (m, 5H). 13C NMR (100 MHz, CDCl3) 31.10, 49.20, 113.51, 127.99, 129.27, 129.40, 131.88, 195.11. 5-methyl-3-oxohexanenitrile (3.2b): 3.2b was prepared by the same method described for 3.2a Isolated yield: 73%, yellow oil. 1H NMR (400 MHz, CDCl3) 0.95-0.97 (m, 6H), 2.11-2.25 (m, 1H), 2.49 (d, J = 6.88 Hz, 2H), 3.43 (s, 2H). 13C NMR (100 MHz, CDCl3 ) 22.53, 24.66, 32.56, 51.08, 113.96, 197.25. 2-acetyl-3-oxo-4-phenyl butanenitrile (3.5a): 3.5a was isolated as a side product from the reaction of 3.1a and anhydrous acetonitrile in the presence of potassium t -butoxide instead of potassium tpentylate. 3.5a was purified by column chromatography on silica gel (hex anes:ethyl acetate, 3:2), yield: 39%, colorless thick oil. 1H NMR (400 MHz, CDCl3) 2.37 (s, 3H), 3.70 (s, 2H), 4.32 (s, 1H),

PAGE 127

112 7.28 – 7.46 (m, 5H). 13C NMR (100 MHz, CDCl3 ) 21.01, 45.01, 104.89, 115.37, 128.17, 129.42, 129.55, 133.05, 156.26, 168.98. Representative procedure for the synthesis of -unsaturated -cyanoketones (3.3): -ketonitrile 3.2a (0.75 mmol) was dissolved in dry THF (3 mL) under an argon atmosphere. Then N,N -dimethylformamide dimethyl a cetal (0.98 mmol) was added and the mixture was stirred at rt. Progre ss of the reaction was monitored by TLC until complete consumption of starting material 3.2a was observed (16 h). The solvent was evaporated under reduced pressure and the desired compound was obtained as an E>>Z isomeric mixture in nearly quantitative yield (> 90%). Note: If a precipitate was formed during the r eaction, the mixture was cooled to 0 C and then it was filtered and rinsed with co ld THF to isolate the desired compound. ( E )-2-((dimethylamino)methylene)-3oxo-4-phenylbutanenitrile (3.3a) : Isolated yield: >90 %, colorless needle-like crystal, m.p. = 134-138 C. 1H NMR (400 MHz, CDCl3) 3.20 (s, 3H), 3.38 (s, 3H), 3.95 (s, 2H), 7.21–7.34 (m, 5H), 7.80 (s, 1H). 13C NMR (100 MHz, CDCl3) 9.05, 46.51, 48.19, 120.54, 126.99, 128.70, 129.80, 129.85, 129.86, 135.07, 158.02, 192.88. HRMS (ESI) calcd. for C13H15N2O [M + H]+ 215.1184, found 215.1194. A crystal structur e is reported in Appendix B.

PAGE 128

113 ( E )-2-((dimethylamino)methylene)-5-me thyl-3-oxohexanenitrile (3.3b): 3.3b was prepared by the same method described for 3.3a Isolated yield: >90%, yellow solid, m.p. = 43-45 C. 1H NMR (400 MHz, CDCl3) 0.95 (d, J = 1.48 Hz, 3H), 0.96 (d, J = 1.47 Hz, 3H), 2.25-2.13 (m, 1H), 2.54 (m, 2H), 3.24 (s, 3H), 3.40 (s, 3H), 7.82 (s, 1H). 13C NMR (100 MHz, CDCl3) 22.77, 25.78, 38.99, 48.05, 48.80, 80.79, 120.59, 157.57, 195.37. HRMS (ESI) calcd. for C10H17N2O [M + H]+ 181.1341, found 181.1333. ( E )-2-((dimethylamino)methylene)-4-me thyl-3-oxopentanenitrile (3.3c): 3.3c was prepared by the same method described for 3.3a Yield: >90%, pale yellow solid, m.p. = 40-42 C. 1H NMR (400 MHz, CDCl3) 1.12 (d, J = 2.26 Hz, 3H), 1.14 (d, J = 2.26 Hz, 3H), 3.24 (s, 3H), 3.41 (s, 3H), 7.84 (s, 1H). 13C NMR (101 MHz, CDCl3) 19.10, 37.05, 38.99, 48.11, 79.35, 120.48, 157.98, 199.77. HR MS (ESI) calcd. for C9H15N2O [M + H]+ 167.1184, found 167.1184. ( E )-2-((dimethylamino)methylene)-4,4-di methyl-3-oxopentanenitrile (3.3i):

PAGE 129

114 3.3i was prepared by the sa me method described for 3.3a Yield: >90%, white solid. 1H NMR (400 MHz, CDCl3) 1.32 (s, 9H), 3.23 (s, 3H), 3.42 (s, 3H), 7.92 (s, 1H). 13C NMR (100 MHz, CDCl3) 27.01, 39.06, 43.84, 48.47, 121.54, 160.54, 200.42. HRMS (ESI) calcd. for C10H17N2O [M + H]+ 181.1341, found 181.1340. ( E )-2-benzoyl-3-(dimethylamino)acrylonitrile (3.3j): O CN N 3.3j was prepared by the same method described for 3.3a Isolated yield: >90%, white solid. 1H NMR (400 MHz, CDCl3) 1.32 (s, 9H), 3.23 (s, 2H), 3.42 (s, 2H), 7.92 (s, 1H). 13C NMR (100 MHz, CDCl3) 27.0, 39.1, 43.8, 48.5, 121.5, 160.5, 200.4. HRMS (ESI) calcd. for C10H17N2O [M + H]+ 181.1341, found 181.1340. Representative procedure for the sy nthesis of pyrimidinylenes (3.4): 3.4a.4 O CN 3.3a NN Ph CN N NH2 Method A : A mixture of compound 3.3a (15.17 mmol) and guani dine hydrochloride (30.34 mmol) in ethanol (absolute, 200 pr oof, 10mL) was stirred under refluxing conditions until the TLC indicated completion of the reaction. The mixture was brought to rt and the precipitate was filtered by vacuum and rinsed with ice cold ethanol (5 mL x 3). Compound 3.4a.4 was isolated as colorless crys tals in 83% yield, m.p. = 129-132 C. 1H NMR (400 MHz, CDCl3) 4.10 (s, 2H), 5.58 (br s, 2H), 7-24-7.38 (m, 5H), 8.45 (s,

PAGE 130

115 1H). 13C NMR (100 MHz, CDCl3) 42.89, 97.76, 116.44, 127.51, 129.00, 129.46, 136.04, 162.36, 162.80, 173.45. HRMS (ESI) calcd. for C12H11N4 [M + H]+ 211.0984, found 211.0972. A crystal structur e is reported in Appendix B. Compounds 3.4a.1 and 3.4a.2 required additional purification since these did not precipitate from the mixtures. Method B: Microwave-assisted reactions were also done in the presence of base (e.g. Et3N or NaOEt) to obtain the desired pyrimid inylenes. For example, to a mixture of unsaturated -cyanoketone 3.3b (0.933 mmol) and guanidine hydrochloride (1.87 mmol) in ethanol (5 mL) was adde d sodium ethoxide (0.933 mmol) suspended in ethanol (1 mL). The reaction mixture was placed on a microwave reactor for 40 min at 120 C. A colorless crystal-like precipitate formed in the solution upon cooling to rt. The precipitate was filtered and rins ed with ice cold ethanol (3 mL x 2) to isolate compound 3.4b.4 as needle-like crystals. 4-benzylpyrimidine-5-carbonitrile (3.4a.1): 3.4a.1 was prepared following method A usin g commercially available formamidine hydrochloride. It was purified by column chromatography on silica gel (hexanes:ethyl acetate, 4:1), yield: 39%, pale yellow thick oil. 1H NMR (400 MHz, CDCl3) 4.25 (s, 2H), 7.17 – 7.26 (m, 2H), 7.26 – 7.33 (m, 3H), 8.83 (s, 1H), 9.20 (s, 1H). 13C NMR (100 MHz, CDCl3) 43.1, 109.2, 114.8, 127.8, 129.2, 129.5, 135.7, 160.4, 160.5, 172.0. HRMS (ESI) calcd. for C12H10N3 [M + H]+ 196.0875, found 196.0868.

PAGE 131

116 4-benzyl-2-methylpyrimidin e-5-carbonitrile (3.4a.2): 3.4a.2 was prepared following method A usi ng commercially available acetamidine hydrochloride. It was purified by column chromatography on silica gel (hexanes:ethyl acetate, 4:1), yield: 63%, colorless thick oil. 1H NMR (400 MHz, CDCl3) 2.79 (s, 3H), 4.26 (s, 2H), 7.24 – 7.41 (m, 5H), 8.78 (s, 1H). 13C NMR (100 MHz, CDCl3) 26.90, 43.19, 105.84, 115.40, 127.61, 129.08, 129.42, 136.03, 160.44, 171.26, 171.65. HRMS (ESI) calcd. for C13H12N3 [M + H]+ 210.1031, found 210.1022. 4-benzyl-2-phenylpyrimidin e-5-carbonitrile (3.4a.3): NN CN 3.4a.3 was prepared following method A using commercially available benzamidine hydrochloride. Isolated yiel d: 91%, white solid, m.p. = 138-141 C. 1H NMR (400 MHz, CDCl3) 4.37 (s, 2H), 7.27 – 7.30 (m, 1H), 7.35 (dd, J = 10.1, 4.6, 2H), 7.45 – 7.59 (m, 5H), 8.49 – 8.54 (m, 2H), 8.92 (s, 1H). 13C NMR (100 MHz, CDCl3) 43.34, 105.94, 115.69, 127.59, 129.03, 129.08, 129.46, 129.53, 132.61, 136.12, 136.17, 160.82, 166.02, 171.85. HRMS (ESI) calcd. for C18H14N3 [M + H]+ 272.1188, found 272.1196.

PAGE 132

117 4-isobutyl-2-phenylpyrimidin e-5-carbonitrile (3.4b.3): 3.4b.3 was prepared following method A. Isolat ed yield: 70%, colorless crystals, m.p. = 68-69 C. 1H NMR (400 MHz, CDCl3) 1.05 (d, J = 6.7, 6H), 2.39 (m, 1H), 2.93 (d, J = 7.2, 2H), 7.49 – 7.59 (m, 3H), 8.50 – 8.53 (m, 2H), 8.93 (s, 1H). 13C NMR (100 MHz, CDCl3) 22.57, 28.88, 45.71, 106.71, 115.80, 129.00, 129.38, 132.44, 136.36, 160.37, 165.72, 173.29. HRMS (ESI) calcd. for C15H16N3 [M + H]+ 238.1344, found 238.1337. A crystal structure is reported in Appendix B 2-amino-4-isobutylpyrimidine -5-carbonitrile (3.4b.4): 3.4b.4 was prepared following method B. Isol ated yield: 75%, white solid, m.p. = 175178 C. 1H NMR (400 MHz, CDCl3) 0.98 (d, J = 6.7, 6H), 2.18 (m, 1H), 2.66 (d, J = 7.3, 2H), 5.72 (br s, 2H), 8.45 (s, 1H). 13C NMR (100 MHz, CDCl3) 22.56, 28.86, 45.51, 98.44, 116.62, 162.06, 162.81, 174.96. HRMS (ESI) calcd. for C9H13N4 [M + H]+ 177.1140, found 177.1136.

PAGE 133

118 4tert -butyl-2-phenylpyrimidine5-carbonitrile (3.4i.3): 3.4i.3 was prepared following method A. Isol ated yield: 83%, white solid, m.p. = 101103 C. 1H NMR (400 MHz, CDCl3) 1.60 (s, 9H), 7.49 – 7.58 (m, 3H), 8.51 – 8.55 (m, 2H), 8.94 (s, 1H). 13C NMR (100 MHz, CDCl3) 28.8, 40.2, 104.0, 117.2, 128.9, 129.4, 132.4, 136.5, 162.5, 164.8, 179.3. HRMS (ESI) calcd. for C15H16N3 [M + H]+ 238.1344, found 238.1347. 2-amino-4tert -butylpyrimidine-5-car bonitrile (3.4i.4): NH2 NN CN 3.4i.4 was prepared following method B. Isol ated yield: 75%, colorless crystals. 1H NMR (400 MHz, CDCl3) 1.43 (s, 9H), 5.56 (br s, 2H), 8.44 (s, 1H). 13C NMR (100 MHz, CDCl3) 28.5, 39.5, 95.6, 118.3, 162.4, 164.0, 181.2. HRMS (ESI) calcd. for C9H13N4 [M + H]+ 177.1140, found 177.1148. 4-benzyl-2-chloropyrimidine-5-carbonitrile (3.4a.5): NN Cl CN

PAGE 134

119 3.4a.5 was prepared following method A using 2-chloroamidine hydrochloride and anhydrous dioxane instead of ethanol. It was purified by column chromatography on silica gel (hexanes:ethyl acetate, 4:1), yield: 18%, white solid. 1H NMR (400 MHz, CDCl3) 4.29 (s, 2H), 7.42 – 7.28 (m, 5H), 8.77 (s, 1H). HRMS (ESI) calcd. for C12H9ClN3 [M + H]+ 230.0485, found 230.0488. A crystal structure is reported in Appendix B 4-benzyl-2-isopropoxypyrimidine-5-carbonitrile (3.4a.6): NN O CN 3.4a.6 was prepared following method A using 2-chloroamidine hydrochloride and isopropanol instead of ethanol It was purified by column chromatography on silica gel (hexanes:ethyl acetate, 7:3), yield: 68%, white solid. 1H NMR (400 MHz, CDCl3) 1.39 (d, J = 6.1, 6H), 4.19 (s, 2H), 5.29 – 5.38 (m, 1H ), 7.24 – 7.36 (m, 5H), 8.65 (s, 1H). 13C NMR (100 MHz, CDCl3) 21.87, 40.68, 72.71, 101.31, 115.71, 127.18, 128.73, 129.51, 163.36, 165.38, 174.89. Experimental procedure for the synthesi s of 4-(4-benzyl-5-cyanopyrimidin-2yl)benzoic acid (3.19a): CN 3.16 N NH2 HO 3.17 O OH OH.Et3N O 1)Ac2O,HOAc,MeOH HCOOK,10%Pd/C OH O NN CN 3.19aEt3N,MeOH 1)NH2OH.HCl 2) 3.3a ,NaOEt,EtOH

PAGE 135

120 Step 1 : To a mixture of commercially availa ble 4-cyanobenzoic acid (2.72 mmol) and hydroxylamine hydrochloride (6.8 mmol) in meth anol (7 mL) was added triethylamine (6.8 mmol). The reaction mixture was stirre d at rt for 18 h. The solvent was removed under reduced pressure to obt ain intermediate amidoxime as a triethylamine salt ( 3.17 ). Compound 3.17 was isolated as a white solid in >85% yield and it was used in the next step without further purification. Step 2 : Intermediate from step 1 (2.72mmol) was dissolved in glacial acetic acid (2 mL). To this mixture was added ammonium formate (13.6 mmol) followed by the addition of 10% Pd/C (0.4 g). The reaction mixture was heated to refluxing under an argon atmosphere. The reaction was monitore d by TLC until consumption of amidoxime 3.17 was observed. The crude was cooled to rt and filtered through CeliteTM (1 g) and rinsed with glacial acetic acid (3 mL x 2). The f iltrate was concentrated under reduced pressure to obtain intermediate amidine 3.17’ which was used in step 3 without further purification. 3.17’ was >90% pure as determined by NMR spectroscopy. 5-carbamimidoylpicolinic acid (3.17’): HN NH2 OH O Isolated yield: >80%, white solid, m.p. = >243 C. 1H NMR (400 MHz, CD3OD) 7.897.91(m, 2H), 8.21-8.23 (m, 2H). 13C NMR (100 MHz, CD3OD) 128.04, 130.20, 132.31, 135.82, 166.84. HRMS (ESI) calcd. for C8H9N2O2 [M + H]+ 165.0664, found 165.0666.

PAGE 136

121 Step 3 : Amidine 3.17’ from step 2 was dissolved in et hanol (2 mL) and heated to 70 C. Then, excess sodium ethoxide was added a nd the mixture was stirred under refluxing conditions for 20 min. before compound 3 3a was added. After addition of 3.3a (0.23 mmol) the mixture was refluxed until the TLC indicated complete consumption of 3.3a The crude mixture was cooled to rt and concentrated under reduced pressure. Compound 3.19a was purified by flash column chromatogr aphy in silica gel (h exanes: ethyl acetate 1:9), yield: 35%, off-white solid. HRMS (ESI) calcd. for C19H14N3O2 [M + H]+ 316.1086, found 316.1093. Experimental procedure for the synthesis of 3o -tolylisoquinolin-1-amine (3.9): A solution of omethylbenzonitrile (16.88 mmol) in anhydrous THF was cooled to 0 C and kept under an argon atmosphere. Then 1M NaHMDS in THF (20.26 mmol) was added and the mixture was stirre d for 1 h at rt. The mixture was filtered and the filtrate was concentrated under reduced pressure to yi eld a yellow crude solid. The crude residue was dissolved in water (5 mL) and acidified to pH= ~5-6 with 5% citric acid; then it was extracted with ethyl acetate (3 x 5 mL). Th e combined organic layers were washed with aqueous Na2CO3 solution (10 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain a yellow solid crude. 3.9 was purified by column chromatography in silica gel (hexanes: ethyl acetate 3:2), 58% yield, white solid. 13C NMR (100 MHz,

PAGE 137

122 (CD3)2SO)) 21.03, 110.03, 116.62, 124.57, 125.94, 126.13, 127.33, 128.01, 130.05, 130.92, 136.05, 138.14, 141.84, 152.54, 157.29. Experimental procedure for the synthesi s of 4-benzyl-4'-isobutyl-2'-phenyl-2,5'bipyrimidine-5-carbonitrile (3.11ba.3): Step 1 : To a mixture of compound 3.4b.3 (4.21 mmol) and hydroxylamine hydrochloride (10.53 mmol) in methanol (12 mL) was adde d triethylamine (10.53 mmol). The reaction mixture was stirred under refl uxing conditions until the TLC indicated the consumption of starting material 3.4b.3 The solvent was removed under reduced pressure to obtain a white crude solid. The crude was dissolved in DCM (50 mL), washed with water (40 mL), dried over Na2SO4, and concentrated under vacuum to obtain a white solid. Further purification by column chromatography gave amidoxime 3.6b.3 and side product 3.10b.3 ( E )-N'-hydroxy-4-isobutyl-2-phenylpyrim idine-5-carboximidamide (3.6b.3):

PAGE 138

123 3.6b.3 was purified by column chromatography in silica gel (hexanes: ethyl acetate 3:2), 70% yield, white solid, m.p. = 125-127 C. 1H NMR (400 MHz, (CD3)2SO) 0.97 (d, J = 6.7, 6H), 2.32 – 2.43 (m, 1H), 2.92 (d, J = 7.1, 2H), 6.10 (br s, 2H), 7.57 – 7.62 (m, 3H), 8.45 – 8.50 (m, 2H), 8.77 (s, 1H), 9.78 (s, 1H). 13C NMR (100 MHz, (CD3)2SO)) 23.15, 27.94, 44.15, 126.63, 128.42, 129.42, 131.57, 137.70, 149.29, 157.50, 162.91, 168.64. HRMS (ESI) calcd. for C15H18N4O [M + H]+ 270.1481, found 270.1480. 4-isobutyl-2-phenylpyrimid ine-5-carboxamide (3.10b.3): NN NH2 O 3.10b.3 was purified by column chromatography in silica gel (hexanes: ethyl acetate 3:2), 20%, white solid, m.p. = 124-127 C 1H NMR (400 MHz, (CD3)2SO) 0.90 (d, J = 6.7, 6H), 2.16 – 2.30 (m, 1H), 2.86 (d, J = 7.1, 2H), 7.51 – 7.55 (m, 3H ), 7.72 (br s, 1H), 8.11 (br s, 1H), 8.38 – 8.42 (m, 2H), 8.80 (s, 1H). 13C NMR (100 MHz, (CD3)2SO)) 23.06, 28.45, 43.99, 128.55, 128.72, 129.46, 131.78, 137.49, 156.38, 163.42, 168.21, 168.32. Step 2 : Intermediate 3.6b from step 1 (0.92 mmol) was dissol ved in glacial acetic acid (1 mL) and acetic anhydride (1.01 mmol). Af ter 5 min. of stirring, potassium formate prepared in situ from K2CO3 (5 mmol), formic acid (10 mmol) in methanol (2.5 mL) was added to the mixture followed by the additi on of 10% Pd/C (10 mol %). The reaction mixture was stirred at rt until the TLC indicated the consumption of starting material. The crude was filtered through CeliteTM (1.5 g) and rinsed with methanol (3 mL x 3). The filtrate was concentrated under reduced pr essure to obtain a yellow crude residue.

PAGE 139

124 To this residue, DCM (6 mL) was added and the white precipitate that was formed was removed by vacuum filtration. The filtrate was concentrated under reduced pressure to obtain the crude acetate salt of carboxamidine as a yellow solid, which was used without further purification in the next step. Step 3 : To the crude carboxamidine salt dissol ved in ethanol (0.8 mL) was added compound 3.3a (0.69 mmol) and triethylamine (1.38 mmol). The reaction mixture was stirred under refluxing conditi ons for 2 h and then it was stirred at rt for 18 h. The precipitate that formed was filtered and rinsed with cold ethanol (5 mL) to yield dimer 3.11ba.3 as a white solid in 28% yield after three steps, m.p. = 144-147 C. 13C NMR (100 MHz, CDCl3) 22.57, 28.88, 45.71, 106.71, 115.80, 129.00, 129.38, 132.44, 136.36, 160.37, 165.72, 173.29. HRMS (ESI) calcd. for C26H24N5 [M + H]+ 406.2032, found 406.2049. 4,4'-diisobutyl-2'-phenyl-2,5'-bipyrim idine-5-carbonitrile (3.11bb.3): 3.11bb.3 was prepared following the procedure described for 3.11ba.3 Isolated yield: 38% after 3 steps, white fluffy solid. 1H NMR (400 MHz, CDCl3) 0.96 (d, J = 6.7, 6H), 1.06 (d, J = 6.7, 6H), 2.24 – 2.44 (m, 2H), 2.97 (d, J = 7.2, 2H), 3.24 (d, J = 7.1, 2H), 7.50 – 7.55 (m, 3H), 8.54 – 8.59 (m, 2H), 9.02 (s, 1H), 9.41 (s, 1H). 13C NMR (100 MHz, CDCl3) 22.64, 22.80, 28.57, 29.10, 44.97, 45.85, 107.10, 115.28, 127.69, 128.85,

PAGE 140

125 128.96, 131.46, 137.44, 159.92, 160.22, 164.66, 170.29, 173.45. HRMS (ESI) calcd. for C23H26N5 [M + H]+ 372.2188, found 372.2208. 4'-benzyl-4-(naphthalen-2-ylmethyl)-2'phenyl-2,5'-bipyrimidin e-5-carbonitrile (3.11af.3): NN NN CN 3.11af.3 was prepared following the procedure described for 3.11ba.3 It was purified by column chromatography on silica gel (hexanes:ethyl acetate, 4:1), yield: 22% after 3 steps, off-white solid, m.p. = 136-138 C. 1H NMR (400 MHz, CDCl3) 4.51 (s, 2H), 4.70 (s, 2H), 7.08 – 7.18 (m, 5H), 7.42 – 7.54 (m, 6H), 7.76 – 7.83 (m, 4H), 8.49 – 8.55 (m, 2H), 9.00 (s, 1H), 9.47 (s, 1H). 13C NMR (100 MHz, CDCl3) 42.4, 43.4, 106.5, 115.2, 126.4, 126.5, 126.7, 127.1, 127.2, 127.9, 128.0, 128.4, 128.9, 129.3, 131.6, 132.8, 133.1, 133.7, 137.1, 138.5, 160.4, 160.6, 165.1, 165.2, 169.0, 172.0. HRMS (ESI) calcd. for C33H24N5 [M + H]+ 490.2032, found 490.2038. 4,4'-dibenzyl-2'-phenyl-2,5'-bipyr imidine-5-carbonitrile (3.11aa.3):

PAGE 141

126 3.11aa.3 was prepared following the procedure described for 3.11ba.3 It was purified by column chromatography on silica gel (hexanes :ethyl acetate, 4:1), yield: 40% after 3 steps, white solid. 1H NMR (400 MHz, CDCl3) 4.35 (s, 2H), 4.72 (s, 2H), 7.11 – 7.23 (m, 5H), 7.27 – 7.39 (m, 5H), 7.48 – 7.54 (m, 3H ), 8.51 – 8.55 (m, 2H), 8.98 (s, 1H), 9.46 (s, 1H). HRMS (ESI) calcd. for C29H22N5 [M + H]+ 440.1875, found 440.1887. 4'-benzyl-4-methyl-2'-phenyl-2,5'-bip yrimidine-5-carbonitrile (3.11ag.3): NN NN CN 3.11ag.3 was prepared following the procedure described for 3.11ba.3 Isolated yield: 44% after 3 steps, tan solid, m.p. = 169 C. 1H NMR (400 MHz, CDCl3) 2.82 (s, 3H), 4.78 (s, 2H), 7.14 – 7.25 (m, 5H), 7.48 – 7.56 (m, 3H), 8.53 – 8.60 (m, 2H), 8.96 (s, 1H), 9.49 (s, 1H). 13C NMR (100 MHz, CDCl3) 23.83, 42.63, 106.93, 115.02, 126.57, 127.13, 128.48, 128.87, 129.05, 129.36, 131.63, 137.17, 138.59, 159.93, 160.28, 164.94, 168.97, 170.49. HRMS (ESI) calcd. for C23H18N5 [M + H]+ 364.1562, found 364.1579. 2'-amino-4-benzyl-4'-isobutyl-2,5'-bip yrimidine-5-carbonitrile (3.11ba.4):

PAGE 142

127 3.11ba.4 was prepared following the procedure described for 3.11ba.3 Isolated yield: 46% after 3 steps, buff solid, m.p. = 184-186 C. 1H NMR (400 MHz, CDCl3) 0.85 (d, J = 6.7, 6H), 1.98– 2.09 (m, 1H), 3.05 (d, J = 7.1, 2H), 4.33 (s, 2H ), 5.43 (s, 2H), 7.26– 7.43 (m, 5H), 8.90 (s, 1H), 9.07 (s, 1H). 13C NMR (100 MHz, (CD3CN) 21.9, 28.3, 42.8, 44.6, 105.1, 115.8, 127.4, 129.0, 129.6, 136.8, 161.0, 161.9, 163.6, 165.9, 171.5, 172.2, 174.1. HRMS (ESI) calcd. for C20H21N6 [M + H]+ 345.1828, found 345.1820. 4'-benzyl-4-isobutyl-2'-phenyl-2,5'-bip yrimidine-5-carbonitrile (3.11ab.3): 3.11ab.3 was prepared following the procedure described for 3.11ba.3 It was purified by column chromatography on silica gel (hexanes :ethyl acetate, 9:1), yield: 27% after 3 steps, white solid, m.p. = >245 C. 1H NMR (400 MHz, CDCl3) 1.00 (d, J = 6.7, 6H), 2.27 (m, 1H), 2.91 (d, J = 7.2, 2H), 4.77 (s, 2H), 7.24 – 7.13 (m, 5H), 7.48 – 7.55 (m, 3H), 8.51 – 8.58 (m, 2H), 8.98 (s, 1H), 9.47 (s, 1H). 13C NMR (100 MHz, CDCl3) 22.57, 29.09, 42.52, 45.75, 107.22, 115.22, 126.55, 127.31, 128.48, 128.86, 129.03, 131.61, 137.18, 138.60, 160.14, 160.32, 164.89, 168.87, 173.52. HRMS (ESI) calcd. for C26H24N5 [M + H]+ 406.2032, found 406.2028.

PAGE 143

128 Representative procedure for the synthesi s of trimeric 2,5-pyri midinylenes (3.12): NN NN 3.12bac.3 steps1,2,3 NN NN CN 3.11ba.3 NN CN Step 1 : To a mixture of compound 3.11ba.3 (0.6 mmol) and hydroxylamine hydrochloride (1.5 mmol) in methanol (8 mL) was added triethylamine (1.5 mmol). The reaction mixture was stirred under refluxi ng conditions until the TLC indicated the consumption of the starting material 3.11ba.3 The solvent was removed under reduced pressure to obtain a yellow crude solid. The crude was dissolved in DCM (10 mL), washed with water (8 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain an off-white, fluffy solid, which wa s used in the next step without further purification. Step 2 : Intermediate amidoxime obtained from step 1 (0.50 mmol) was dissolved in glacial acetic acid (2 mL) and acetic anhydr ide (0.55 mmol). After 5 min. of stirring, potassium formate prepared in situ from K2CO3 (5 mmol, 0.69 g), formic acid (10 mmol, 0.37 mL) in methanol (2.5 mL) was added to the mixture followed by the addition of 10% Pd/C (10 mol %). The reaction mixture was stirred at rt unt il the TLC indicated completion of the reaction. The crude was filtered through CeliteTM (1 g) and rinsed with

PAGE 144

129 methanol (40 mL). The filtrate was concen trated under reduced pressure to obtain a yellow crude residue. To this residue, DCM (6 mL) was added and the white precipitated that formed was removed by vacuum filtration. The filtrate was concentrated under reduced pressure to obtain the crude carboxamidine acetate salt as a yellow solid. The crude was used without further purification in the next step. Step 3 : The crude salt from step 2 was dissolved in ethanol (3 mL); to this mixture was added compound 3.3c (0.41 mmol). Triethylamine was not used in this step. The reaction mixture was stirred under refluxing co nditions for 28 h. The precipitate that formed was filtered and rinsed with cold ethanol (3 mL x 3) to yield trimer 3.12bac.3 as a tan solid in 41% yiel d after three steps. 5”-cyano-4”-isopropyl-4’-phenylmethyl-4-is obutyl-2-phenylterpyrimidine (3.12bac.3): Isolated yield: 41% after 3 steps, white solid, m.p. = 166-168 C. 1H NMR (400 MHz, CDCl3) 0.89 (d, J = 6.7, 6H), 1.39 (d, J = 6.8, 6H), 2.29 (m, 1H), 3.18 (d, J = 7.1, 2H), 3.53 (m, 1H), 4.80 (s, 2H), 7.16 – 7.25 (m, 5H ), 7.48 – 7.54 (m, 3H), 8.52 – 8.58 (m, 2H), 9.03 (s, 1H), 9.36 (s, 1H), 9.56 (s, 1H). 13C NMR (100 MHz, CDCl3) 21.34, 22.79, 28.45, 35.34, 42.23, 44.67, 106.24, 114.82, 126.80, 128.69, 128.79, 128.83, 129.40,

PAGE 145

130 131.14, 137.77, 138.25, 159.55, 160.16, 160.53, 164.76, 169.02, 169.99, 178.80. HRMS (ESI) calcd. for C33H32N7 [M + H]+ 526.2719, found 526.2707. 5”-cyano-4,4”-diisobutyl-4’-(2-naphthylm ethyl)-2-phenylterpyr imidine (3.12bfb.3): NN NN NN CN 3.12bfb.3 was prepared following the procedure described for 3.12bac.3 Isolated yield: 48% after 3 steps, off-wh ite solid, m.p. = 164-167 C. 1H NMR (400 MHz, CDCl3) 0.83 (d, J = 6.7, 6H), 0.97 (d, J = 6.7, 6H), 2.20 – 2.32 (m, 2H), 2.93 (d, J = 7.2, 2H), 3.17 (d, J = 7.1, 2H), 4.93 (s, 2H), 7.36 – 7.46 (m, 3H), 7.49 – 7.54 (m, 3H), 7.58 (br s, 1H), 7.65 – 7.80 (m, 3H), 8.55 (m, 2H), 9.03 (s 1H), 9.40 (s, 1H), 9.55 (s, 1H). 13C NMR (100 MHz, CDCl3) 22.52., 22.68, 28.52, 29.18, 42.52, 44.94, 45.78, 107.79, 114.94, 125.92, 126.39, 127.67, 127.72, 127.75, 127.83, 127.86, 128.34, 128.78, 129.05, 129.22, 132.06, 132.40, 133.63, 135.68, 153.93, 157.76, 160.23, 160.28, 164.42, 169.12, 173.88. HRMS (ESI) calcd. for C38H36N7 [M + H]+ 590.3032, found 590.3054. 5”-cyano-4,4”-diisobutyl-4’-(2-phenylmet hyl)-2-phenylterpyrimidine (3.12bab.3):

PAGE 146

131 NN NN NN CN 3.12bab.3 was prepared following the procedure described for 3.12bac.3 Isolated yield 37% after 3 steps, white solid. 1H NMR (400 MHz, CDCl3) 0.90 (d, J = 6.7, 6H), 1.02 (d, J = 6.7, 6H), 2.22 – 2.36 (m, 2H), 2.95 (d, J = 7.2, 2H), 3.20 (d, J = 7.1, 2H), 4.77 (s, 2H), 7.17 – 7.25 (m, 5H), 7.49 – 7.54 (m, 3H), 8.52 – 8.58 (m, 2H), 9.03 (s, 1H), 9.38 (s, 1H), 9.52 (s, 1H). 13C NMR (100 MHz, CDCl3) 22.59, 22.80, 28.47, 29.20, 42.30, 44.71, 45.80, 107.64, 115.04, 126.79, 127.43, 128.57, 128.67, 128.79, 128.83, 129.34, 131.14, 137.76, 138.27, 159.56, 160.12, 160.26, 164.18, 164.62, 164.75, 168.98, 169.98, 173.80. HRMS (ESI) calcd. for C34H34N7 [M + H]+ 540.2876, found 540.2869. Experimental procedure for the synthesi s of 4-isobutyl-2-phenylpyrimidine-5carboxylic acid (3.13b.3): 3.4b.3 (0.25 mmol) was dissolved in a mixtur e of 20% aqueous solution NaOH and methanol (3 mL : 2 mL). The reaction mixt ure was placed on a microwave reactor for 40

PAGE 147

132 min. at 160 C. TLC monitoring indicated complete conversion of starting material 3.4b.3 The crude mixture was transferred to a separatory funnel and hexanes were added (30 mL); the layers were separated and the aqueous la yer was acidified to pH= ~23 with 1M HCl (22 mL). The precipitate that formed was filtered and rinsed with cold methanol (3 mL) to isolate pure compound 3.13b.3 in 75% yield as a white powder, m.p. = 175-177 C. 1H NMR (400 MHz, CD3OD) 0.99 (s, 3H), 1.00 (s, 3H), 2.28 (m, 1H), 3.17 (d, J = 7.1, 2H), 7.51 -8.48 (m, 5H), 9.19 (s, 1H). 13C NMR (100 MHz, CD3OD) 21.70, 28.71, 44.45, 128.45, 128.57, 131.35, 137.00, 159.44, 161.90, 165.17. 4-benzyl-4'-isobutyl-2'-phenyl-2,5'-bipyr imidine-5-carboxylic acid (3.14ba.3): NN NN OH O 3.14ba.3 was prepared following the procedure described for 3.13ba.3 Isolated yield: 93% yield, white solid, m.p. = 167-169 C. 1H NMR (400 MHz, (CD3)2SO) 0.78 (s, 3H), 0.79 (s, 3H), 2.12 (m, 1H), 3.06 (d, J = 7.1, 2H), 4.60 (s, 2H), 7.23 – 7.31 (m, 5 H), 7.57 8.47 (m, 5 H), 9.26 (s, 1H), 9.30 (s, 1H). 13C NMR (100 MHz, (CD3)2SO) 22.89, 28.41, 41.68, 44.24, 122.77, 127.13, 128.79, 129.03, 129.10, 129.49, 129.88, 132.00, 137.35, 138.70, 159.72, 160.24, 163.60, 164.80, 166.67, 169.49, 170.02.

PAGE 148

133 4'-isobutyl-4-(naphthalen-2-ylmethyl)-2'-phe nyl-2,5'-bipyrimidine-5-carboxylic acid (3.14bf.3): NN NN OH O 3.14bf.3 was prepared following the procedure described for 3.13ba.3 Isolated yield: 68% yield, off-white solid. 1H NMR (400 MHz, CDCl3) 0.84 (d, J = 6.6, 6H), 2.18 – 2.31 (m, 1H), 3.16 (d, J = 7.0, 2H), 4.85 (s, 2H), 7.39 – 7.56 (m, 5H), 7.72 – 7.81 (m, 4H), 8.50 – 8.56 (m, 3H), 9.41 (s, 1H), 9.43 (s, 1H). 13C NMR (100 MHz, CD3OD) 21.52, 28.27, 41.51, 44.05, 104.98, 104.99, 125.42, 125.86, 127.41, 127.47, 127.68, 127.81, 127.84, 128.38, 128.40, 131.01, 131.33, 132.59, 133.85, 135.87, 137.20, 158.96, 159.79, 169.90, 170.46. Experimental procedure for the synthesi s of 4-amino-2,6-diph enylpyrimidine-5carbonitrile (3.22a.3): To a mixture of commercially available 3.20a (5.18 mmol) and benzamidine hydrochloride (5.70 mmol) in ethanol (absol ute, 200 proof, 8 mL) was added sodium ethoxide (5.70 mmol). The mixture was stirred under refl uxing conditions until the TLC

PAGE 149

134 indicated complete consumpti on of the starting material 3.20a The mixture was cooled to rt and the precipitate that formed was isolated by vacuum filtration and rinsed with ice cold ethanol (5 mL x 3). Compound 3.22a.3 was isolated in 84% yield, tan solid, m.p. = 208-210 C. 1H NMR (400 MHz, CDCl3) 5.77 (br s, 2H), 7.53 (m, 6H), 8.13 (m, 2H), 8.51 (m, 2H). 13C NMR (100 MHz, CDCl3) 85.41, 116.67, 128.74, 128.93, 129.03, 129.25, 131.67, 132.10, 136.47, 136.58, 164.82, 165.38, 168.37. Experimental procedure for the sy nthesis of pyrimidinylene (3.22): NC CN O 3.21b R=H 3.21c R=Me R' NH2.HCl HN K2CO3,EtOH reflux,18hNN R' CN R 3.22b.2 R=H,R'=Me 3.22b.3 R=H,R'=Ph 3.22c.3 R=Me,R'=Ph H2N R To a mixture of compound 3.21b (6.55 mmol) and benzamidine hydrochloride (4.36 mmol) in ethanol (absolute, 200 proof, 12 mL) was added potassium carbonate (10.9 mmol). The mixture was stirred under refl uxing conditions until the TLC indicated the complete consumption of starting material 3.21b The crude mixture was concentrated under reduced pressure and dissolved in ethyl acetate (20 mL) and washed with water (15 mL x 2). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to yield a yellow crude solid. 4-amino-2-phenylpyrimidine5-carbonitrile (3.22b.3): NN CN H2N

PAGE 150

135 Compound 3.22b.3 was purified by column chromatography on silica gel (hexanes:ethyl acetate, 7:3), yield: 74%, white solid. 1H NMR (400 MHz, CDCl3) 5.58 (br s, 2H), 7.44 – 8.33 (m, 5H), 8.59 (s, 1H). Experimental procedure for the synthesis of 4'-amino-4tert -butyl-2',6'-diphenyl2,5'-bipyrimidine-5-ca rbonitrile (3.23a.3): Step 1 : To a mixture of compound 3.22a.3 (1.47 mmol) and hydroxylamine hydrochloride (3.67 mmol) in methanol (10 mL) was added triethylamine (3.67 mmol). The reaction mixture was stirred under refluxi ng conditions until the TLC indicated the consumption of the starting material 3.22a.3 (48 h). The precipit ate that formed was removed by vacuum filtration and washed with methanol (2 x 10 mL). The filtrate was concentrated under reduced pressu re to yield a yellow crude solid. The crude was used in the next step without further purification. Step 2 : Intermediate amidoxime obtained from step 1 (1.47 mmol) was dissolved in glacial acetic acid (3 mL) and acetic anhydr ide (1.61 mmol). After 5 min. of stirring, potassium formate prepared in situ from K2CO3 (5 mmol, 0.69 g), formic acid (10 mmol, 0.37 mL) in methanol (2.5 mL) was added to the mixture followed by the addition of 10% Pd/C (10 mol %). The reaction mixture was stirred at rt unt il the TLC indicated

PAGE 151

136 completion of the reaction. The cr ude was filtered through CeliteTM (0.5 g) and rinsed with methanol (30 mL). The filtrate was c oncentrated under reduced pressure to obtain a yellow crude residue, which was used without further purification in the next step. Step 3 : The crude salt from step 2 was suspended in ethanol (8 mL); to this mixture was added compound 3.3i (1.03 mmol) and sodium ethoxide (1.61 mmol). The reaction mixture was stirred under re fluxing conditions for 18 h. The crude was concentrated under reduced pressure to obtai n a crude yellow solid. Compound 3.23a.3 was purified by column chromatography on silica gel (hexan es:ethyl acetate, 9:1), yield: 42%, white solid, m.p. = 166-169 C. 1H NMR (400 MHz, CDCl3) 1.12 (s, 9H), 5.71 (br s, 2H), 7.27 – 7.59 (m, 5H), 8.10 – 8.15 (m, 2H), 8.48 – 8.54 (m, 3H), 8.86 (s, 1H). 13C NMR (100 MHz, CDCl3) 28.16, 28.16, 39.75, 103.06, 109.41, 116.70, 116.76, 128.37, 128.93, 129.69, 136.52, 141.00, 161.00, 162.82, 164.02, 164.94, 166.09, 168.34, 168.45, 178.53. HRMS (ESI) calcd. for C27H25N4 [M + H]+ 405.2079, found 405.1980. 3.5 References Ahmed, S.; Kaur, K. ( 2009 ) The proteolytic stability and cytotoxicity studies of Laspartic acid and L-diaminopr opionic acid derived beta -peptides and a mixed alpha /beta -peptide. Chemical Biology & Drug Design, 73 (5), 545-552. Aly, A. A.; Nour-El-Din, A. M. ( 2008 ) Functionality of amid ines and amidrazones. ARKIVOC (Gainesville, FL, United States), (1), 153-194. Anbazhagan, M.; Boykin, D. W.; Stephens, C. E. ( 2003 ) Direct conversion of amidoximes to amidines via transfer hydrogenation. Synthesis, (16), 2467-2469. Balo, C.; Lopez, C.; Brea, J. M.; Fernandez, F.; Caamano, O. ( 2007 ) Synthesis and evaluation of adenosine antagonist activ ity of a series of [1,2,4]triazolo[1,5c]quinazolines. Chemical & Pharmaceutical Bulletin, 55 (3), 372-375. Baran, P. S.; Shenvi, R. A.; Nguyen, S. A. ( 2006 ) One-step synthesis of 4,5-disubstituted pyrimidines using commercially available and inexpensive reagents. Heterocycles, 70, 581-586.

PAGE 152

137 Basso, A.; Pegg, N.; Evans, B.; Bradley, M. ( 2000 ) Solid-phase synthesis of amidinebased GP IIb-IIIa antagonist s on dendrimer resin beads. European Journal of Organic Chemistry, (23), 3887-3891. Beingessner, R. L.; Deng, B.-L.; Fanwick, P. E.; Fenniri, H. ( 2008 ) A Regioselective Approach to Trisubstituted 2(or 6)-Ary laminopyrimidine-5-carbaldehydes and Their Application in the Synthesis of Structur ally and Electronically Unique G-C Base Precursors. Journal of Organic Chemistry, 73 (3), 931-939. Brandl, S.; Gompper, R.; Polborn, K. ( 1996 ) An efficient new pyrimidine synthesis. A pathway to octupoles. Journal fuer Praktische Chemie/Chemiker-Zeitung, 338 (5), 451459. Brown, D. J. ( 1994 ) The Pyrimidines [In: Chem. Heterocycl. Compd., 28] ; p 1059 pp. Bruning, J. ( 1997 ) Lithium and potassium bis(tr imethylsilyl)amide: utilizing nonnucleophile bases as nitrogen sources. Tetrahedron Letters, 38 (18), 3187-3188. Cesar, J.; Nadrah, K.; Sollner Dolenc, M. ( 2004 ) Solid-phase synthesis of amidines by the reduction of amidoximes. Tetrahedron Letters, 45 (40), 7445-7449. Dunn, P. J. ( 2005 ) Amidines and N-substituted amidines. Comprehensive Organic Functional Group Transformations II, 5, 655-699. Garigipati, R. S. ( 1990 ) An efficient conversion of nitriles to amidines. Tetrahedron Letters, 31 (14), 1969-72. Gielen, H.; Alonso-Alija, C.; Hendrix, M.; Niewohner, U.; Schauss, D. ( 2002 ) A novel approach to amidines from esters. Tetrahedron Letters, 43 (3), 419-421. Gompper, R.; Brandl, S.; Mair, H.-J. Use of pyrimidine group-c ontaining conjugated compounds as electroluminescent materials. 95-109928690052, 19950626., 1996 Gompper, R.; Mair, H. J.; Polborn, K. ( 1997 ) Synthesis of oligo(diazaphenyls). Tailormade fluorescent heteroaromatics a nd pathways to nanostructures. Synthesis, (6), 696718. Han, Q.; Dominguez, C.; Stouten, P. F. W.; Pa rk, J. M.; Duffy, D. E.; Galemmo, R. A., Jr.; Rossi, K. A.; Alexander, R. S.; Sm allwood, A. M.; Wong, P. C.; Wright, M. M.; Leuttgen, J. M.; Knabb, R. M.; Wexler, R. R. ( 2000 ) Design, Synthesis, and Biological Evaluation of Potent and Selective Amid ino Bicyclic Factor Xa Inhibitors. Journal of Medicinal Chemistry, 43 (23), 4398-4415. Hill, M. D.; Movassaghi, M. ( 2008 ) New strategies for the synthesis of pyrimidine derivatives. Chemistry--A European Journal, 14 (23), 6836-6844.

PAGE 153

138 Horton, D. A.; Bourne, G. T.; Smythe, M. L. ( 2003 ) The combinatorial synthesis of bicyclic privileged structures or privileged substructures. Chemical Reviews (Washington, DC, United States), 103 (3), 893-930. Hu, L.; Arafa, R. K.; Ismail, M. A.; Wenz ler, T.; Brun, R.; Munde, M.; Wilson, W. D.; Nzimiro, S.; Samyesudhas, S.; Werbovetz, K. A.; Boykin, D. W. ( 2008 ) Azaterphenyl diamidines as antileishmanial agents. Bioorganic & Medicinal Chemistry Letters, 18 (1), 247-251. Hurst, D. T. ( 1980 ) An Introduction to the Chemistry and Biochemistry of Pyrimidines, Purines, and Pteridines ; p 266 pp. Ji, Y.; Trenkle, W. C.; Vowles, J. V. ( 2006 ) A high-yielding prepar ation of beta -keto nitriles. Organic Letters, 8 (6), 1161-1163. Judkins, B. D.; Allen, D. G.; Cook, T. A.; Evans, B.; Sardharwala, T. E. ( 1996 ) A versatile synthesis of amidines from nitriles via amidoximes. Synthetic Communications, 26 (23), 4351-4367. Kang, F.-A.; Kodah, J.; Guan, Q.; Li, X.; Murray, W. V. ( 2005 ) Efficient conversion of Biginelli 3,4-dihydropyrimidin2(1H)-one to pyrimidines vi a PyBroP-mediated coupling. Journal of Organic Chemistry, 70 (5), 1957-1960. Koryakova, A. G.; Ivanenkov, Y. A.; Ryzhova, E. A.; Bulanova, E. A.; Karapetian, R. N.; Mikitas, O. V.; Katrukha, E. A.; Kazey, V. I.; Okun, I.; Kravchenko, D. V.; Lavrovsky, Y. V.; Korzinov, O. M.; Ivachtchenko, A. V. ( 2008 ) Novel aryl and heteroaryl substituted N-[3-(4-phenylpi perazin-1-yl)propyl]-1,2,4-oxadiazole-5carboxamides as selective GSK-3 inhibitors. Bioorganic & Medicinal Chemistry Letters, 18 (12), 3661-3666. Kraybill, B. C.; Elkin, L. L.; Blethrow, J. D.; Morgan, D. O.; Shokat, K. M. ( 2002 ) Inhibitor scaffolds as new alle le specific kinase substrates. Journal of the American Chemical Society, 124 (41), 12118-12128. Kutzki, O.; Park Hyung, S.; Ernst Justin, T.; Orner Brendan, P.; Yin, H.; Hamilton Andrew, D. ( 2002 ) Development of a potent Bcl-x(L) antagonist based on alpha-helix mimicry. Journal of the American Chemical Society, 124 (40), 11838-9. Lagoja, I. M. ( 2005 ) Pyrimidine as constituent of natural biologically active compounds. Chemistry & Biodiversity, 2 (1), 1-50. Lange, U. E. W.; Schafer, B.; Baucke, D.; Buschmann, E.; Mack, H. ( 1999 ) A new mild method for the synthesis of amidines. Tetrahedron Letters, 40 (39), 7067-7070.

PAGE 154

139 Lee, H. K.; Ten, L. N.; Pak, C. S. ( 1998 ) Facile synthesis of amidines from thioamides. Bulletin of the Korean Chemical Society, 19 (11), 1148-1149. Lee, J. H.; Choi, B. S.; Chang, J. H.; Lee, H. B.; Yoon, J.-Y.; Lee, J.; Shin, H. ( 2007 ) The Decarboxylative Blaise Reaction. Journal of Organic Chemistry, 72 (26), 10261-10263. Lepore, S. D.; Schacht, A. L.; Wiley, M. R. ( 2002 ) Preparation of 2hydroxybenzamidines from 3-aminobenzisoxazoles. Tetrahedron Letters, 43 (48), 87778779. Loffet, A. ( 2002 ) Peptides as drugs: Is there a market?, Journal of Peptide Science, 8 (1), 1-7. Lukyanov, S. M.; Bliznets, I. V.; Shorshnev, S. V. ( 2008 ) Synthesis of sterically hindered 3-(azolyl)pyridines. ARKIVOC (Gainesville, FL, United States), (4), 21-45. Massaro, A.; Mordini, A.; Reginato, G.; Russo, F.; Taddei, M. ( 2007 ) Microwaveassisted transformation of esters into hydroxamic acids. Synthesis, (20), 3201-3204. Moisan, L.; Odermatt, S.; Gombosure n, N.; Carella, A.; Rebek, J., Jr. ( 2008 ) Synthesis of an oxazole-pyrrole-piperazine scaffo ld as an alpha -helix mimetic. European Journal of Organic Chemistry, (10), 1673-1676. Moss, R. A.; Ma, W.; Merrer, D. C.; Xue, S. ( 1995 ) Conversion of 'obstinate' nitriles to amidines by Garigipati's reaction. Tetrahedron Letters, 36 (48), 8761-4. Nadrah, K.; Dolenc, M. S. ( 2007 ) Preparation of amidines by amidoxime reduction with potassium formate. Synlett, (8), 1257-1258. Peters, J.-U.; Weber, S.; Kritter, S.; Weiss, P.; Wallier, A.; Boehringer, M.; Hennig, M.; Kuhn, B.; Loeffler, B.-M. ( 2004 ) Aminomethylpyrimidines as novel DPP-IV inhibitors: A 100 000-fold activity increase by optim ization of aromatic substituents. Bioorganic & Medicinal Chemistry Letters, 14 (6), 1491-1493. Reuman, M.; Beish, S.; Davis, J.; Batchelor, M. J.; Hutchings, M. C.; Moffat, D. F. C.; Connolly, P. J.; Russell, R. K. ( 2008 ) Scalable Synthesis of the VEGF-R2 Kinase Inhibitor JNJ-17029259 Using Ultrasound-Mediat ed Addition of MeLi-CeCl3 to a Nitrile. Journal of Organic Chemistry, 73 (3), 1121-1123. Rousselet, G.; Capdevielle, P.; Maumy, M. ( 1999 ) Conversion of nitriles to tertiary amines: N,N-dimethylhomoveratrylamin e (Benzeneethanamine, 3,4-dimethoxy-N,Ndimethyl-). Organic Syntheses, 76, 133-141.

PAGE 155

140 Saygili, N.; Batsanov, A. S.; Bryce, M. R. ( 2004 ) 5-Pyrimidylboronic acid and 2methoxy-5-pyrimidylboronic acid: new hetero arylpyrimidine derivatives via Suzuki cross-coupling reactions. Organic & Biomolecular Chemistry, 2 (6), 852-857. Schaefer, F. C.; Krapcho, A. P. ( 1962 ) Preparation of amidine salts by reaction of nitriles with ammonium salts in the presence of ammonia. Journal of Organic Chemistry, 27, 1255-8. Schomaker, J. M.; Delia, T. J. ( 2001 ) Arylation of Halogenated Pyrimidines via a Suzuki Coupling Reaction. Journal of Organic Chemistry, 66 (21), 7125-7128. Shaginian, A.; Whitby, L. R.; Hong, S.; Hwan g, I.; Farooqi, B.; Searcey, M.; Chen, J.; Vogt, P. K.; Boger, D. L. ( 2009 ) Design, Synthesis, and Evaluation of an alpha -Helix Mimetic Library Targeting Pr otein-Protein Interactions. Journal of the American Chemical Society, 131 (15), 5564-5572. Sorger, K.; Stohrer, J. ( 2007 ) Procedure for the production of beta -ketonitriles and their Group IA or IIA salts by the acy lation of acetonitriles with carboxylate esters in the presence of Group IA or IIA alkoxides with azeotropic distillative removal of byproduct alcohols. 2005-102005057461102005057461, 20051201. Sylla, M.; Joseph, D.; Chevallier, E.; Camara, C.; Dumas, F. ( 2006 ) A simple and direct access to ethylidene malonates. Synthesis, (6), 1045-1049. von Angerer, S. ( 2004 ) Product class 12: pyrimidines. Science of Synthesis, 16, 379-572. Wang, J.; Xu, F.; Cai, T.; Shen, Q. ( 2008 ) Addition of Amines to Nitriles Catalyzed by Ytterbium Amides: An Efficient OneStep Synthesis of Monosubstituted NArylamidines. Organic Letters, 10 (3), 445-448. Watanabe, K.; Kogoshi, N.; Miki, H.; Torisawa, Y. ( 2009 ) Improved pinner reaction with CPME as a solvent. Synthetic Communications, 39 (11), 2008-2013. Yin, H.; Lee, G.-i.; Park, H. S.; Payne, G. A. ; Rodriguez, J. M.; Sebti, S. M.; Hamilton, A. D. ( 2005a ) Terphenyl-based helical mimetics that disrupt the p53/HDM2 interaction. Angewandte Chemie, International Edition, 44 (18), 2704-2707. Yin, H.; Lee, G.-i.; Sedey, K. A.; Kutzki, O.; Pa rk, H. S.; Orner, B. P.; Ernst, J. T.; Wang, H.-G.; Sebti, S. M.; Hamilton, A. D. ( 2005b ) Terphenyl-Based Bak BH3 alpha -Helical Proteomimetics as Low-Molecula r-Weight Antagonists of Bcl-xL. Journal of the American Chemical Society, 127 (29), 10191-10196. Zempleni, J. s.; Rucker, R. B.; McCormick, D. B.; Suttie, J. W.; Editors, Handbook of Vitamins 2007 ; p 593 pp.

PAGE 156

141 CHAPTER FOUR SYNTHESIS OF AN ALKYLATING GUANIDINE DERIVATIVE FOR THE SYNTHESIS OF CPNA MONOMERS 4.1 Peptide Nucleic Acids (PNA): Introduction Peptide Nucleic Acids (PNA) were introdu ced by Nielsen and co-workers in 1991 (Nielsen et al. 1991). PNA are mimic structures of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in which the phosphate backbone has been substituted for a 2aminoethylglycine scaffold, a pe ptide-like backbone, and the four natural nucleobases are still attached (Figure 4.1 )(Wang and Xu, 2004). Figure 4.1. Backbone structures of DNA and PNA According to the Watson-Crick base pair ing rules, PNA can selectively bind to complementary DNA and RNA to form PN A-DNA and PNA-RNA duplexes and even (PNA)2-DNA triplexes, whose bindings are st ronger than that of DNA-DNA or RNARNA bindings. In addition, the duplexes formed exhibit high stability towards enzymatic degradation by nucleases and prot eases (Hyrup and Nielsen, 1996).

PAGE 157

142 4.1.1 Potential Applications of PNA Potential uses of PNA include their app lication as antisense and antigene drugs. Antisense inhibition involves the use of short oligonucleo tide sequences of DNA, RNA, or chemical analogs (15 to 20 bases) synt hesized to be complementary to a specific messenger RNA (mRNA) sequence, which is responsible for the coding of a target protein. Once in the cell, the antisense agent forms a heteroduplex with the corresponding mRNA, thus inhibi ting the translation of the protein coded by that mRNA. This can occur because mRNA needs to be single stranded in order to be translated. In antigene inhibition, the oligonucleotides or poten tial analogs, such PN A, are designed to identify and bind to complementary sequences in a specific gene interfering with the transcription of that gene (Dias and Stei n, 2002; Maher, 1996; Ray and Norden, 2000). Figure 4.2 depicts the basics of antise nse and antigene inhibition. Figure 4.2. Schematic depiction of an tisense and antigene inhibition The applications of antisense and antigene agents have been investigated for several diseases including cancer, diabetes, HIV, cardiovascular diseases, and nervous system disorders, among others (Nielsen, 2004; Weiss et al. 1999; Zaffaroni et al. 2004). Out of the different types of these therapeutic agents, the PNA are of great interest due to their good st ability, high affinity hybridizat ion properties with DNA and

PAGE 158

143 RNA, and lack of toxicity (Dias and Stei n, 2002; Nielsen, 1999; Ray and Norden, 2000). Previous in vitro studies have indicated that PNA coul d in fact inhibit both translation and transcription of genes; however, unm odified PNA (naked PNA) cannot easily penetrate the cell membrane due to the high molecular ma ss (Nielsen, 2004), poor water solubility (Capasso et al. 2001), and their uncharged nature (Wang and Xu, 2004). 4.1.2 Cysteine-based PNA (CPNA) The PNA original structure has been submitted to modifications in many ways with the aim of improving the intracellular delivery and uptake in eukaryotic cells (Capasso et al. 2001; Ganesh and Ni elsen, 2000; Yi Sung et al. 2009; Zhou et al. 2003). One of the strategies involves m odification of the backbone by introducing a cysteine and an aminoethyl group giving ri se to cysteine-based PNA (CPNA, Figure 4.3 ) (Jain et al. 2008; Yi Sung et al. 2009). Figure 4.3. Cysteine-based PNA target scaffold (Yi Sung et al. 2009) This has been done in our lab and several monomeric CPNA have been successfully synthesized using this strategy. The details of th e synthesis of CPNA monomers are not described in this docum ent; some data has been already published (refer to Sung Wook Yi, 2008) and the remaining work is under curren t investigation.

PAGE 159

144 A method that has been reported for deliver y of PNA is a covalent attachment of PNA to peptide carriers. Studies based on bacterial agents suggested that when permeability is increased, the modified PNA can conjugate with cell-permeating peptides, allowing PNA to readily penetrate the memb rane. Although the mechanistic basis for this delivery process has not been yet establis hed, it has been suggest ed that the anionic segments of the lipopolysaccharide layer (LPS) attracts the cationic residues of the modified PNA, while the neutral portions of the PNA enables the penetration across the hydrophobic region of the cell membrane (Eriksson et al. 2002). Another approach is based on the introduction of positively char ged residues into the backbone which has been explored by other groups to improve cellular uptake. Figure 4.4. GPNA structure (Dragulescu-Andrasi et al. 2005) Ly’s group reported guanidine-based PNA (GPNA), which incorporated an arginine residue into the PNA backbone (Figure 4.4 ); this modified PNA was found to be more soluble in water compared to the unmodified version of PNA. Their studies revealed that GPNA can bind to DNA, while having increased cellular uptake; however, the GPNA’s capacity to bind to RNA has not been yet esta blished (Dragulescu-Andrasi et al. 2005).

PAGE 160

145 The proposed target scaffold for th is project is sh own in Figure 4.3 Unlike Ly’s GPNA, our structure does not contain the side chain at the -L-position and it is not synthesized from an arginine derivative. Instead, the positively charged residue is attached at the ethylene un it between the two nitrogen at oms of the backbone and its synthesis derives from cysteine. By in troducing the cationic guanidine residue, the solubility of the PNA in water should increa se; thus, efficient PNA delivery and antisense effects should be enhanced. This approach is expected to be functional not only for prokaryotic, but also for eukaryotic cells allowing inhibition of translation or transcription of a targeted gene. The details about the synthesis of this alkylating guanidine derivative ( R group in the CPNA target scaffold in Figure 4.3 ) will be discussed. 4.2 Results and Discussion 4.2.1 Synthesis of Guanid ine Derivative 4.4 Figure 4.5. CPNA building block target (Yi Sung et al. 2009)

PAGE 161

146 The CPNA building block target is shown in Figure 4.5 This structure contains a C N and S termini and it is composed of a base and a modified backbone derived from cysteine, which includes a di-benzyloxycar bonyl (Cbz) protected guanidine group, R1, attached to the cysteine -like side chain (Yi Sung et al. 2009). The overall synthesis of this S -alkylating agent is described in Scheme 4.1 Compound 4.4 was obtained in four steps from 1,3-diaminopropane 4.1 The first step of this synthesis is the monoprotection of commerci ally available diamine 4.1 with t butoxycarbonyl group (Boc) (Montero et al. 2002). Compound 4.1 was dissolved in chloroform and a solution of Boc anhydride in chloroform was added dropwise for 30 min. at 0 C. Upon removal of the solvent, a pale oil was obtained, which solidified to a white solid after standing under high vacuum Pure compound 4.2 was obtained in 93% yield. Scheme 4.1. Synthesis of S -alkylating agent 4.4

PAGE 162

147 Compound 4.2 was guanidinylated in the next st ep. Several methods have been reported for the preparation of guanidines; including reactions with triflylguanidines (Feichtinger et al. 1998a; Feichtinger et al. 1998b); derivatives of pyrazole-1carboxamidine (Bernatowicz et al. 1993); treatment with electr ophilic agents, such as S methyl(aryl)-sulfanylisothioureas (Kent et al. 1996); protected thioureas and Mukaiyama’s reagent (Yong et al. 1997); protected isothiour eas in the presence of mercury chloride (HgCl2) (Guo et al. 2000); and benzotriazole-based guanylating reagents (Katritzky et al. 2005). Two of these methods were explored for the synthesis of guanidine 4.3 The first approach invo lves the reaction of amine 4.2 with a triflylguanidine derivative in the pr esence of triethylamine in DCM (Scheme 4.2 ). Scheme 4.2. Failed attempt to synt hesize guanidine derivative 4.3 Preparation of the triflic reagent wa s required since this compound was not readily available. As shown in Scheme 4.3 this reagent could be prepared in two steps from commercially available gua nidine hydrochloride (Feichtinger et al. 1998b). The first step consists of protecting the guani dine with benzyloxycar bonyl chloride in the presence of NaOH in DCM. This step was achieved and pure di-Cbz guanidine 4.5 was obtained in good yield (79%). The next st ep is to introduce a triflyl group on the unprotected nitrogen of the guanidine. Unfort unately, the results re ported by Feichtinger

PAGE 163

148 were not reproducible; the desi red pure compound was not isol ated since purification of the crude by column chromatography even w ith different eluents was not efficient. Scheme 4.3. Failed attempt to synt hesize a triflylguanidine reagent In order to move forward with the synthesis, a sec ond route was adopted (Scheme 4.4 ). The use of a diprotected isothiourea as the guanidinylating agent has been widely used as these reagents usually react very efficiently with primary and secondary amines. It has been suggested that this reaction occurs easily due to the formation of a carbodiimide intermediate, which is a highly electrophilic species (Yong et al. 1997). Also, the presence of both Cbz protecting gr oups, which are in conjugation with the reaction center, may increase not only the electr ophilicity of the reagent, but its solubility in organic solvents (Orner and Hamilton, 2001). Scheme 4.4. Synthesis of guanidine derivative 4.3 (Yong et al. 1997) Treatment of amine 4.2 with 1,3-bis(benzyloxycarbonyl)-2-methyl-2pseudothiourea in presence of triethylamine and HgCl2 provided a good method for the synthesis of guanidine 4.3 in excellent yield (90%). However, the use of toxic mercury salts was a disadvantage along with the increased difficulty in the purification of the

PAGE 164

149 guanidine. Purification of the target guani dine by column chromatography was always required. The possibility of replacing the HgCl2 by using other reagents, such as Mukaiyama’s reagent or nickel catalyst s (Bhat and Georg, 2000) was considered. Instead, a protocol reported by Gers and co -workers was followed since better results were obtained based on the need for synthe sizing pure compounds in practical scale and under mild conditions (Gers et al. 2004). Amine derivative 4.2 was treated with the di-Cbz-p rotected pseudothiourea in the absence of the thiophilic agent HgCl2. Accordingly, guanidine derivative 4.3 was obtained in 92 % yiel d by the reaction of 4.2 with 1,3-bis(benzyloxycarbonyl)-2-methyl2-pseudothiourea at rt. The adaptation of this procedure was very useful because it not only avoided the use of heavy metal reagen ts, but it also did not require additional reagents or purification techni ques to isolate the desired pr oduct in excellent yield. The reaction time depended on how fast the pse udothiourea reagent was consumed; this was monitored by TLC and it varied between 6 an d 24 h, independent of the reaction scale. As expected, temperatures over rt helped reducing the reaction times, but not in significant proportion. 1H NMR of 4.3 showed the appearance of two singlets for the methylenes of the Cbz groups (Figure 4 6 ). The first peak appeared around 5.0 ppm and the second around 5.19 ppm, the latter indicatin g that the major product isolated had the double bond located in conjugation with the carbonyl of the protecting group and not between the guanidine carbon and the attached amine, for which one single peak would be observed.

PAGE 165

150 Figure 4.6. 1H NMR spectrum of compound 4.3 The next step in the synthesis is the removal of the Boc protecting group. The standard methods include treatment with a mi xture of trifluoroace tic acid (TFA, 40%) and (DCM, 60%) or HCl in dioxane (Green e and Wuts, 1991). Both methods were followed and the best results were obtained with HCl in dioxane. The acylation of HCl or TFA salts of compound 4.3 with bromoacetyl bromide was performed under different conditions to afford the target alkylating guanidine derivative 4.4 (Table 4.1 ). It was observed that presence of TFA from the TFA salt of compound 4.3 became a major issue since it seemed to activate the bromoacetyl bromide to form side product 4.5a It was observed that ratio of formation of desired product 4.4 and side product 4.5a was approximately of 50 : 40, respect ively. Both compounds were is olated and characterized. Another major issue observed in this step was the formation of side product 4.5b via an undesired intramolecula r cyclization.

PAGE 166

151 Table 4.1 Acylation conditions for the synthesis of guanidine 4.4 The formation of the stable six-membered ring of 4.5b was observed to increase when stronger basic conditions and temperatures above 0 C were used, whether the reaction was performed in an organic or aque ous phase according to the protocol (Table 4.1 entries 2-4). The best method to obtain compound 4.4 was the formation of the HCl salt of 4.3 and subsequent treatment with bromo acetyl bromide in a two-phase system (Table 4.1 entry 1). The desired product was isol ated in 68% yield after purification by column chromatography on silica gel. The alkylating guanidine derivative 4.4 has been installed in several CPNA building blocks ( R1 in target CPNA af ter alkylation step, Figure 4.3 ).

PAGE 167

152 Figure 4.7. S -alkylated CPNA monomers These compounds (Figure 4.7 compounds a h ) were prepared by other members of our lab (Dr. Sung Wook Yi and Priyesh Jain ) and subsequent research will disclose if the presence of this positively charged group R1, as part of the CPNA backbone, assists on the delivery and uptake of CPNA to cellular targets. 4.3 Conclusion The synthesis of a guanidine-derived alkyl bromide for the alkylation of the S terminus of novel CPNA monomers was su ccessfully synthesized using mild and efficient conditions (absence of heavy metals). In addition, the overall yield to obtain compound 4.4 was improved by using a biphasic reac tion system to allow the preparation of practical quantities for further advancement with oligomeric CPNA synthesis. 4.4 Experimental Section 4.4.1 Experimental Procedures Experimental procedure for the synthesis of tert -butyl 3-aminopropylcarbamate (4.2):

PAGE 168

153 Compound 4.1 (1.64 mol) was dissolved in CHCl3 (250 mL) and cooled to 0 C. Then solid t -Boc2O (0.03 mol) dissolved in CHCl3 (36 mL) was added dropwise from an addition funnel during 30 min. The reaction mixt ure was brought to rt and stirred for 2 h. The mixture was filtered and the filtrate wa s concentrated under reduced pressure; the resulting crude oil was dissolved in ethyl aceta te (125 mL), washed with brine, and dried over MgSO4. Solvent was removed under high vacuum to give compound 4.2 as a white solid in 93 % yield. 1H NMR (400 MHz, CDCl3) 1.44 (s, 9H), 1.67 (q, J = 6.6, 2H), 2.39 (br s, 2H), 2.81 (t, J = 6.4 Hz, 2H), 3.20-3.23 (m, 2H), 4.93 (br s, 1H). 13C NMR (100 MHz, CDCl3) 28.63, 32.89, 38.43, 39.44, 79.29, 156.41. HRMS (ESI) calcd. for C8H19N2O2 [M + H]+ 175.1447, found 175.1439. Experimental procedure for the synthesis of tert -butyl 3-(2,3dibenzyloxycarbonylguanidino)propylcarbamate (4.3): 1,3-bis(benzyloxycarbonyl)-2-met hyl-2-pseudothiourea (8.9 mmol) was dissolved in DCM. Then compound 4.2 (14.83 mmol) was added and the mixture was stirred at rt for 20 h. Progress of the reaction was monitored by TLC until consumption of the pseudothiourea compound was observed. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (50 mL), washed with 10% citric acid (3 x 30 mL), sat’d. NaHCO3 (3 x 30 mL), water (3 x 30 mL), and dried over MgSO4. The solvent was removed under reduced pressure to yield 4.3 as a colorless oil in 92% yield. 1H NMR (400 MHz, CDCl3) 1.41 (s, 9H), 1.71 (p, J = 6.4, 2H), 3.11 – 3.19 (m,

PAGE 169

154 2H), 3.46 – 3.54 (m, 2H), 3.55 (br s, 1H), 5.13 (s, 2H), 5.18 (s, 2H), 7.26 – 7.41 (m, 10H), 8.45 (br s, 1H), 11.70 (br s, 1H). 13C NMR (100 MHz, CDCl3) 28.62, 30.15, 37.37, 38.29, 67.33, 68.49, 76.23, 128.12, 128.17, 128.64, 128.72, 128.92, 129.03, 134.78, 136.95, 153.97, 156.57. HRMS (ESI) calcd. for C25H33N4O6 [M + H]+ 485.2400, found 485.2399. Experimental procedure for the synthesis of 2-bromoN -(3-(2,3dibenzyloxycarbonylguanidino)propyl)acetamide (4.4): Compound 4.3 (4.13 mmol) was treated with HCl-di oxane and stirred for 10 min. The solvent was removed under reduced pressure and the resulting HCl salt was dried under high vacuum overnight. The resulting HCl salt intermediate (4.12 mmol) was dissolved in a mixture of DCM: sat. Na2CO3 (15 mL : 15 mL) and cooled to -10 C. Bromoacetyl bromide (4.13 mmol) was added and stirring wa s continued for 30 min. at 0 C; then a second portion of bromoacetyl bromide ( 2.15 mmol) was added. The solution was brought to rt and stirred for 4 h. The reaction mixture was transferred to a separatory funnel containing a mixture of ethyl acetate (50 mL) and water (40 mL). The organic layer was washed with 5% NaHCO3 (40 mL), 1M HCl (40 mL), brine (2 x 40 mL), and dried over Na2SO4. The solvent was removed under reduced pressure and the crude residue was purified by column chromatography on silica gel (hexanes:ethyl acetate, 1:1) to afford 4.4 in 68% yield as a white solid, m.p. = 95-98 C. 1H NMR (400 MHz, CDCl3)

PAGE 170

155 1.68 – 1.76 (m, 2H), 3.26 – 3.33 (m, 2H), 3.48 – 3.53 (m, 2H), 3.64 (s, 2H), 5.11 (s, 2H), 5.20 (s, 2H), 7.31–7.41 (m, 10H), 7.70 (br s, 1H), 8.52 (br s, 1H), 11.67 (s, 1H). 13C NMR (100 MHz, CDCl3) 29.21, 29.67, 36.37, 37.80, 67.59, 68.63, 128.47, 128.59, 128.76, 128.79, 128.96, 129.11, 134.68, 136.49, 153.96, 157.01, 163.49. HRMS (ESI) calcd. for C22H26BrN4O5 [M+H]+ 505.1087, found 505.1095 and 507.1075. 4.5 References Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. ( 1993 ) Urethane protecte d derivatives of 1guanylpyrazole for the mild and effi cient preparation of guanidines. Tetrahedron Letters, 34 (21), 3389-92. Bhat, L.; Georg, G. I. Nickel-promoted guanyl ation of amines with (iso)thioureas. 994128446100428, 19991006., 2000. Capasso, D.; De Napoli, L.; Di Fabio, G.; Messere, A.; Montesarchio, D.; Pedone, C.; Piccialli, G.; Saviano, M. ( 2001 ) Solid phase synthesis of DNA-3'-PNA chimeras by using Bhoc/Fmoc PNA monomers. Tetrahedron, 57 (46), 9481-9486. Dias, N.; Stein, C. A. ( 2002 ) Antisense oligonucleotides: basic concepts and mechanisms. Molecular Cancer Therapeutics, 1 (5), 347-355. Dragulescu-Andrasi, A.; Zhou, P.; He, G.; Ly, D. H. ( 2005 ) Cell-permeable GPNA with appropriate backbone stereochemistry and spac ing binds sequence-spec ifically to RNA. Chemical Communications (Cambridge, United Kingdom), (2), 244-246. Eriksson, M.; Nielsen, P. E.; Good, L. ( 2002 ) Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli. Journal of Biological Chemistry, 277 (9), 7144-7147. Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman, M. ( 1998a ) Triurethane-protected guanidines and trif lyldiurethane-protected guanidines: new reagents for guanidinylation reactions. Journal of Organic Chemistry, 63 (23), 84328439. Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. ( 1998b ) Diprotected Triflylguanidines: A New Class of Guanidinylation Reagents. Journal of Organic Chemistry, 63 (12), 3804-3805. Ganesh, K. N.; Nielsen, P. E. ( 2000 ) Peptide nucleic acids: analogs and derivatives. Current Organic Chemistry, 4 (9), 931-943.

PAGE 171

156 Gers, T.; Kunce, D.; Markowski, P.; Izdebski, J. ( 2004 ) Reagents for efficient conversion of amines to protected guanidines. Synthesis, (1), 37-42. Greene, T. W.; Wuts, P. G. M., Protective Groups in Or ganic Synthesis. 2nd Ed 1991; p 473 pp. Guo, Z. X.; Cammidge, A. N.; Horwell, D. C. ( 2000 ) Dendroid peptide structural mimetics of w-Conotoxin M VIIA ba sed on a 2(1H)-quinolinone core. Tetrahedron, 56 (29), 5169-5175. Hyrup, B.; Nielsen, P. E. ( 1996 ) Peptide nucleic acids (PNA) : synthesis, properties and potential applications. Bioorganic & Medicinal Chemistry, 4 (1), 5-23. Jain, P.; Yi, S. W.; Kaulagari, S. R.; Ajme ra, M.; Anderson, L.; Topper, M.; McLaughlin, M. L. ( 2008 ) Design and synthesis of cy steine based PNA monomers. Abstracts of Papers, 236th ACS National Meeting, Philade lphia, PA, United States, August 17-21, 2008 MEDI-419. Katritzky, A. R.; Khashab, N. M.; Bobrov, S. ( 2005 ) The preparation of 1,2,3trisubstituted guanidines. Helvetica Chimica Acta, 88 (7), 1664-1675. Kent, D. R.; Cody, W. L.; Doherty, A. M. ( 1996 ) Two new reagents for the guanidylation of primary, secondary and aryl amines. Book of Abstracts, 212th ACS National Meeting, Orlando, FL, August 25-29 MEDI-146. Maher, L. J., III. ( 1996 ) Prospects for the therapeutic use of antigene oligonucleotides. Cancer Investigation, 14 (1), 66-82. Nielsen, P. E. ( 1999 ) Peptide nucleic acids as therapeutic agents. Current Opinion in Structural Biology, 9 (3), 353-7. Nielsen, P. E. ( 2004 ) The many faces of PNA. Letters in Peptide Science, 10 (3-4), 135147. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. ( 1991 ) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science (Washington, DC, United States), 254 (5037), 1497-500. Orner, B. P.; Hamilton, A. D. ( 2001 ) The guanidinium group in molecular recognition: design and synthetic approaches. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 41 (1-4), 141-147. Ray, A.; Norden, B. ( 2000 ) Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future. FASEB Journal, 14 (9), 1041-1060.

PAGE 172

157 Wang, G.; Xu, X. S. ( 2004 ) Peptide nucleic acid (PNA) binding-mediated gene regulation. Cell Research, 14 (2), 111-116. Weiss, B.; Davidkova, G.; Zhou, L. W. ( 1999 ) Antisense RNA gene therapy for studying and modulating biolog ical processes. Cellular and Molecular Life Sciences, 55 (3), 334358. Yi Sung, W.; Jain, P.; Ajmera, M.; Kaulagar i Sridhar, R.; Topper, M.; Anderson, L.; McLaughlin Mark, L. ( 2009 ) Cysteine based PNA (CPNA) : design and synthesis of novel CPNA monomers. Advances in experimental medicine and biology, 611, 553-4. Yi Sung, W.; Jain, P.; Ajmera, M.; Kaulagar i Sridhar, R.; Topper, M.; Anderson, L.; McLaughlin Mark, L. ( 2008 ) Cysteine Based PNA (CPNA) : Design, Synthesis, and Applications. Dissertation, Un iversity of South Florida. Yong, Y. F.; Kowalski, J. A.; Lipton, M. A. ( 1997 ) Facile and Efficient Guanylation of Amines Using Thioureas and Mukaiyama's Reagent. Journal of Organic Chemistry, 62 (5), 1540-1542. Zaffaroni, N.; Villa, R.; Folini, M. ( 2004 ) Therapeutic uses of peptide nucleic acids (PNA) in oncology. Letters in Peptide Science, 10 (3-4), 287-296. Zhou, P.; Wang, M.; Du, L.; Fisher, G. W.; Waggoner, A.; Ly, D. H. ( 2003 ) Novel Binding and Efficient Cellular Uptake of Guanidine-Based Peptide Nucleic Acids (GPNA). Journal of the American Chemical Society, 125 (23), 6878-6879.

PAGE 173

158 APPENDIX A: SELECTED 1H AND 13C NMR SPECTRA

PAGE 174

159 Methyl 2-amino-2-methylprop anoate hydrochloride (2.2e) 6.00 2.74 H2N O O H2N O O

PAGE 175

160( S )-methyl 3-(4-(benzyloxy)phenyl)-2-(2-ethoxy-2-oxoethylamino)propanoate (2.3h) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 f1(ppm) 3.0 3.2 3.4 3.6 f1(ppm) H N O O O O O H N O O O O O

PAGE 176

161 benzyl 3-( tert -butoxycarbonylamino)propanoate (2.10’) -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) O O NHBoc O O NHBoc

PAGE 177

162 ( S )-methyl 3-(3-benzyl-4-( tert -butoxycarbonylamino)-2,6-dioxopiperazin-1yl)propanoate (2.13a) -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 2.6 2.8 3.0 3.2 f1(ppm) O O NN O O NHBoc O O NN O O NHBoc

PAGE 178

163 tert -butyl 2-(2-(benzyloxy)-2-oxoethyl)-2-((2 S ,3 S )-1-methoxy-3-methyl-1-oxopentan2-yl)hydrazinecarboxylate (2.17d) -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) N O O O O NHBoc N O O O O NHBoc

PAGE 179

164 ( S )-tert-butyl 2-(1-meth oxy-1-oxo-3-phenylpropan2-yl)-2-(2-(3-methoxy-3oxopropylamino)-2-oxoethyl) hydrazinecarboxylate (2.19a) -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) N O O N H O NHBoc O O N O O N H O NHBoc O O

PAGE 180

165 tert -butyl 2-(2-(3-methoxy-3-oxopropylamino )-2-oxoethyl)-2-(1-methoxy-4-methyl-1oxopentan-2-yl)hydrazinecarboxylate (2.19b) 6.00 8.69 1.71 0.66 0.72 1.94 0.87 3.85 2.98 2.66 0.67 0.82 N O O N H O NHBoc O O N O O N H O NHBoc O O

PAGE 181

166 ( S )-benzyl 2-(2-methoxy-2-oxoethylamin o)-3-phenylpropanoate (2.22a) H N O O O O H N O O O O

PAGE 182

167 ( S )-tert-butyl 2-(1-(benzy loxy)-1-oxo-3-phenylpropan-2-yl)-2-(2-methoxy-2oxoethyl)hydrazinecarboxylate (2.23a) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) N O O O O NHBoc N O O O O NHBoc

PAGE 183

168 ( S) -2-(2-(tert-butoxycarbonyl)-1-(2methoxy-2-oxoethyl)hydrazinyl)-3phenylpropanoic acid (2.24a) -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) N OH O O O NHBoc N OH O O O NHBoc

PAGE 184

169 ( S )tert -butyl 2-(2-methoxy-2-oxo ethyl)-2-(1-(3-methoxy-3 -oxopropylamino)-1-oxo3-phenylpropan-2-yl)hydrazinecarboxylate (2.25a) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 2.4 2.6 2.8 3.0 f1(ppm) N N H O O O NHBoc O O N N H O O O NHBoc O O

PAGE 185

170 ( S )-methyl 2-(benzylamino)-3 -phenylpropanoate (2.26a) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 f1(ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) H N O O H N O O

PAGE 186

171 ( S )-methyl 2-(benzylamino )-3-methylbutanoate (2.26c) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 1.92 2.00 f1(ppm) 3.6 3.7 3.8 f1(ppm) 3.00 3.03 f1(ppm) H N O O H N O O

PAGE 187

172 ( E )-2-(2-tosylhydrazono)acetic acid (2.29’) -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) S N H O N O O OH S N H O N O O OH

PAGE 188

173 2,5-dioxopyrrolidin-1-yl 2-diazoacetate (2.29) -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) ( S )-methyl 3-(2-(2-diazoacetamido)-3-(na phthalen-2-yl)propanamido)propanoate (2.32f) -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 2.2 2.3 2.4 f1(ppm) 7.3 7.4 7.5 7.6 7.7 7.8 7.9 f1(ppm) O O NO O N2 H N N H O O O O N2

PAGE 189

174 ( S )-benzyl 2-(2-bromoa cetamido)-3-phenylpropanoate (2.34a’) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 5.13 5.22 f1(ppm) 4.85 4.90 f1(ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) H N O O O Br H N O O O Br

PAGE 190

175 ( S )-1,3-dibenzylpiperazine-2,5-dione (2.35a) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 3.10 3.15 3.20 3.25 f1(ppm) NH N O O NH N O O

PAGE 191

176 ( S )-methyl 3-(3-isopropyl-2,5-dioxop iperazin-1-yl)propanoate (2.33c) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 f1(ppm) 2.8 2.9 3.0 3.1 3.2 3.3 f1(ppm) ( S )-2-(2-(tert-butoxycarbonyl)-1-( 1-methoxy-1-oxo-3-phenylpropan-2yl)hydrazinyl)acetic acid (2.18a) NH N O O O O N O O HO O NHBoc

PAGE 192

177 3-oxo-4-phenylbutanenitrile (3.2a) O CN O CN

PAGE 193

178 2-acetyl-3-oxo-4-phenyl butanenitrile (3.5a) 3.31 2.39 1.00 5.29 0.83 O O CN O O CN

PAGE 194

179 ( E / Z )-2-((dimethylamino)methylene)-3oxo-4-phenylbutanenitrile (3.3a) O CN N O CN N

PAGE 195

180 ( E / Z )-2-((dimethylamino)methylene)-4,4-di methyl-3-oxopentanenitrile (3.3i) 8.52 3.08 3.00 0.88 O CN N O CN N

PAGE 196

181 4-benzylpyrimidine-5-carbonitrile (3.4a.1) NN CN NN CN

PAGE 197

182 4-benzyl-2-methylpyrimidin e-5-carbonitrile (3.4a.2) 2.99 2.24 5.18 0.82 NN CN NN CN

PAGE 198

183 4-benzyl-2-phenylpyrimidin e-5-carbonitrile (3.4a.3) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 2.21 0.77 1.94 5.00 1.94 0.817.5 8.0 8.5 f1(ppm) 0.77 1.94 5.00 1.94 NN CN NN CN

PAGE 199

184 2-amino-4-benzylpyrimidi ne-5-carbonitrile (3.4a.4) 2.01 1.75 5.12 0.81 5.12 NH2 NN CN NH2 NN CN

PAGE 200

185 2-amino-4tert -butylpyrimidine-5-c arbonitrile (3.4i.4) 9.00 2.08 0.78 NH2 NN CN NH2 NN CN

PAGE 201

186 4tert -butyl-2-phenylpyrimidin e-5-carbonitrile (3.4i.3) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 7.5 7.7 7.9 8.1 8.3 8.5 f1(ppm) NN CN NN CN

PAGE 202

187 ( E )N '-hydroxy-4-isobutyl-2-phenylpyrim idine-5-carboximidamide (3.6b.3) 6.00 1.03 2.12 1.92 3.15 2.09 0.92 0.87 1.03 NN NH2 N HO NN NH2 N HO

PAGE 203

188 4-isobutyl-2-phenylpyrimid ine-5-carboxamide (3.10b.3) NN NH2 O NN NH2 O

PAGE 204

189 4-isobutyl-2-phenylpyrimidin e-5-carboxylic acid (3.13b.3) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 3.22 3.30 1.12 2.28 3.04 2.00 0.89 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) NN O HO NN O HO

PAGE 205

190 4,4'-diisobutyl-2'-phenyl-2,5'-bipyrim idine-5-carbonitrile (3.11bb.3) 5.96 6.05 2.12 2.05 2.04 2.95 1.92 0.80 0.81 2.12 NN NN CN NN NN CN

PAGE 206

191 4'-benzyl-4-(naphthalen-2-ylmethyl)-2'phenyl-2,5'-bipyrimidin e-5-carbonitrile (3.11af.3) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) NN NN CN NN NN CN

PAGE 207

192 4'-benzyl-4-methyl-2'-phenyl-2,5'-bip yrimidine-5-carbonitrile (3.11ag.3) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) 122 124 126 128 130 132 134 f1(ppm) NN NN CN NN NN CN

PAGE 208

193 2'-amino-4-benzyl-4'-isobutyl-2,5'-bip yrimidine-5-carbonitrile (3.11ba.4) 5.60 1.07 2.01 2.00 1.83 5.57 0.77 0.78 1.07 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) NH2 NN NN CN NH2 NN NN CN

PAGE 209

194 5”-cyano-4”-isopropyl-4’-phenylmethy l-4-isobutyl-2-phenylterpyrimidine (3.12bac.3) 6.08 6.16 1.12 2.13 1.09 2.00 4.44 3.07 2.01 0.78 0.80 0.81 1.12 2.13 1.09 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) NN NN NN CN NN NN NN CN

PAGE 210

195 5”-cyano-4,4”-diisobutyl-4’-(2-phenylmet hyl)-2-phenylterpyrimidine (3.12bab.3) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 2.20 2.25 2.30 2.35 f1(ppm) 0.85 0.90 0.95 1.00 1.05 f1(ppm) NN NN NN CN NN NN NN CN

PAGE 211

196 5”-cyano-4,4”-diisobutyl-4’-(2-napht hylmethyl)-2-phenylterpyrimidine (3.12bfb.3)-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) 5.94 6.14 2.18 2.12 2.10 2.00 2.94 3.28 1.08 3.27 2.14 0.85 0.89 0.887.4 7.5 7.6 7.7 7.8 f1(ppm) 2.94 3.28 1.08 3.272.2 2.3 f1(ppm) 2.18 NN NN NN CN NN NN NN CN

PAGE 212

197 4'-amino-4tert -butyl-2',6'-diphenyl-2,5'-bipyr imidine-5-carbonitrile (3.23a.3) -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 f1(ppm) NN CN H2N NN CN H2N

PAGE 213

198 tert -butyl 3-aminopropylcarbamate (4.2) 9.00 1.99 2.32 1.78 2.13 0.96 1.78 1.99 NHBoc H2N NHBoc H2N

PAGE 214

199 tert -butyl 3-(2,3-dibenzyloxycarbonylg uanidino)propylcarbamate (4.3) 8.75 2.16 2.01 2.08 2.15 2.08 9.41 0.85 0.86 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 f1(ppm) NHCbz NCbz N H BocHN NHCbz NCbz N H BocHN

PAGE 215

200 2-bromoN -(3-(2,3-dibenzyloxycarbonylgua nidino)propyl)acetamide (4.4) NHCbz NCbz N H N H O Br NHCbz NCbz N H N H O Br

PAGE 216

201 APPENDIX B: X-RAY CRYSTALLOGRAPHIC DATA

PAGE 217

202 ORTEP diagram for compound 3.2a

PAGE 218

203

PAGE 219

204 ORTEP diagram for compound 3.4b.3

PAGE 220

205

PAGE 221

206

PAGE 222

207

PAGE 223

208 ORTEP diagram for compound 3.4i.4

PAGE 224

209 ORTEP diagram for compound 3.4a.4

PAGE 225

210

PAGE 226

211

PAGE 227

212 ORTEP diagram for compound 3.4a.5

PAGE 228

213

PAGE 229

214

PAGE 230

215

PAGE 231

216

PAGE 232

217 ORTEP diagram for compound 3.10a.3

PAGE 233

218

PAGE 234

219

PAGE 235

220

PAGE 236

221

PAGE 237

222 ORTEP diagram for compound 3.9

PAGE 238

223

PAGE 239

224

PAGE 240

225 ORTEP diagram for compound 3.12bac.3

PAGE 241

226

PAGE 242

227

PAGE 243

228

PAGE 244

229

PAGE 245

230

PAGE 246

231 APPENDIX C: QIKPROP CALCULATIONS

PAGE 247

232 QikProp Calculation Parameters The following parameters correspond to the overlay of a 4,4’,4”-trimethyl-2,5terpyrimidinylene and an octa-alanine and th e QikProp calculations for a terphenyl-based Bcl-xL-Bak inhibitor and a terpyrimidin e-based analog shown in Figures 3.4 and 3.10 respectively (Chapter Three). These calculations were done by Daniel N. Santiago. Maestro (2) was used to view and build molecular models of 4,4’,4”-trimethyl2,5-terpyrimidinylene and an ideal -helix composed of 8 alanine residues. MacroModel (3) performed a conformational search of TMOP using OPLS 2005 force fields and GB/SA solvation (4). Maestro was used to superimpose the ith ith + 4, and ith + 7 methyl carbons of the octa-alanine with the me thyl carbons of the terp yrimidinylene. Out of 100,000 conformations gene rated, approximately 5,000 (5%) of the conformations aligned well with the -helix. Most of the aligned c onformations exhibited the fourth lowest potential energy calcul ated by MacroModel with an RMSD of 0.68 . PyMol (5) was used to create the image of TMOP superimposed on octa-alanine. ClogP determination using QikProp was performed on two compounds: compound 14 reported by Hang Y. et al (Yin et al. 2005) and its terpyrimidinylene scaffold analog. The full output is copied below: 1. QikProp, 3.1; Schrdi nger, LLC: New York, NY, 2008 2. Maestro, 8.5; Schrd inger, LLC: New York, NY, 2008 3. MacroModel, 9.6; Schrdinger, LLC: New York, NY, 2008 4. Qiu, D.; Shenkin, P. S.; Hollinger, F. P.; Still, W. C. ( 1997 ) The GB/SA Continuum Model for Solvation. A Fast Analy tical Method for the Calculation of Approximate Born Radii. Journal of Physical Chemistry 101, (16), 3005-3014.

PAGE 248

233 5. Delano, W. L. The PyMol Molecular Graphics System, Delano Scientific: Palo Alto, CA, 2002. 6. Yin, H.; Lee, G.-i.; Sedey, K. A.; Kutzki, O.; Park, H. S.; Orner, B. P.; Ernst, J. T.; Wang, H.-G.; Sebti, S. M.; Hamilton, A. D. ( 2005 ) Terphenyl-Based Bak BH3 alpha -Helical Proteomimetics as LowMolecular-Weight Antagonists of Bcl-xL. Journal of the American Chemical Society, 127 (29), 10191-10196.

PAGE 249

ABOUT THE AUTHOR Laura Anderson (Laura Len Daz) was born in Montenegro, Colombia. After graduating from the University of Quindo in 1996, where she received her first degree in chemistry with emphasis in natural products, she moved to the United States in 1999. She earned her bachelors degree majoring in chemistry from the University of South Florida in May 2004 and continued her graduate studies in August 200 4. She joined the laboratory of Professor Mark L. McLaughlin at the University of South Florida in the Moffitt Cancer Center. Laura will receive her doctoral degree in chemistry with emphasis in organic and medicinal chemistry in July 2009.