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University of South Florida University of South Florida Scholar Commons Scholar Commons Graduate Theses and Dissertations Graduate School 7-8-2009 Design and Synthesis of Substituted 1,4-Hydrazine-linked Design and Synthesis of Substituted 1,4-Hydrazine-linked Piperazine-2,5- and 2,6-diones and 2,5-Terpyrimidinylenes as α- Piperazine-2,5- and 2,6-diones and 2,5-Terpyrimidinylenes as - Helical Mimetics Helical Mimetics Laura Anderson University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the American Studies Commons Scholar Commons Citation Scholar Commons Citation Anderson, Laura, "Design and Synthesis of Substituted 1,4-Hydrazine-linked Piperazine-2,5- and 2,6-diones and 2,5-Terpyrimidinylenes as α-Helical Mimetics" (2009). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/1830 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected].

Transcript of Design and Synthesis of Substituted 1,4-Hydrazine-linked ...

Page 1: Design and Synthesis of Substituted 1,4-Hydrazine-linked ...

University of South Florida University of South Florida

Scholar Commons Scholar Commons

Graduate Theses and Dissertations Graduate School

7-8-2009

Design and Synthesis of Substituted 1,4-Hydrazine-linked Design and Synthesis of Substituted 1,4-Hydrazine-linked

Piperazine-2,5- and 2,6-diones and 2,5-Terpyrimidinylenes as α-Piperazine-2,5- and 2,6-diones and 2,5-Terpyrimidinylenes as -

Helical Mimetics Helical Mimetics

Laura Anderson University of South Florida

Follow this and additional works at: https://scholarcommons.usf.edu/etd

Part of the American Studies Commons

Scholar Commons Citation Scholar Commons Citation Anderson, Laura, "Design and Synthesis of Substituted 1,4-Hydrazine-linked Piperazine-2,5- and 2,6-diones and 2,5-Terpyrimidinylenes as α-Helical Mimetics" (2009). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/1830

This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected].

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Design and Synthesis of Substituted 1,4-Hydrazine-linked Piperazine-2,5- and 2,6-diones

and 2,5-Terpyrimidinylenes as α-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, α-helix, PNA

© Copyright 2009, Laura Anderson

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DEDICATION

With my deepest gratitude,

To my dad (my angel) Luis Delio and my mom María Cenelia

To my love Patrick

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ACKNOWLEDGEMENTS

First, I want to thank my family, especially mom and dad, for allowing me the

possibility of a great education. 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 their 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 difficult to reach this goal. I

shall be grateful forever to him for this “our” achievement.

Very special thanks go to my advisor, 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, Professor Wayne C. Guida, Professor Roman

Manetsch, and Professor Hong-Gang Wang for taking the time to meet with me on

several occasions, for their guidance and insightful feedback. I thank Dr. Umut Oguz for

accepting to chair my defense and for all her 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 graduate school, but had trusted my academic

potential and welcomed me in his lab during my undergrad years. I also want to

recognize Jaisnover Villa, my first chemistry teacher in Colombia, whose enthusiasm and

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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, and 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 ocassions there was frustration 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 assisted by Dr. Vasudha Sharma and I am indebted

to her in many aspects, especially her “coaching” lessons and critical insight that helped

me to maintain my courage during the last steps 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 discussions, as well as good listeners of personal

experiences. I would like to acknowledge the Chemistry Department at USF and the H.

Lee Moffitt Cancer Center for providing the facilities to conduct my research. I

appreciate Dr. Ted Gauthier’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 all 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 friendship and Adriana for her positive and enviable attitude.

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TABLE OF CONTENTS TABLE OF CONTENTS ..................................................................................................... i LIST OF TABLES ............................................................................................................. iii 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: α-Helix ................................................................1 1.2 Protein-Protein Interactions (PPIs) ...................................................................3 1.3 Role of Bcl-2 Family and Hmd-2 Family Proteins in Apoptosis.......................5 1.4 Peptidic and Non-Peptidic α-Helical Mimetics .................................................8 1.5 References ........................................................................................................16 CHAPTER TWO: DESIGN AND SYNTHESIS OF 3-R-PIPERAZINE-2,5- AND 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-Piperazine-2,6-diones: DKPA ..............................25 2.2.2 Synthesis of Diacid Derivatives 2.6: Route 1 ...................................27 2.2.3 Cyclization and Coupling of Piperazine-2,6-diones .........................32 2.2.4 Synthesis of Piperazine-2,6-diones: Route 2 ....................................39 2.2.5 Synthesis of Piperazine-2,5-diones: Route 1 ....................................42

2.2.6 Synthesis of Piperazine-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-R- AND 4,6-R,R’-2,5-TERPYRIMIDINYLENES .......................................78

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3.1 Introduction ......................................................................................................78 3.1.1 Pyrimidines .......................................................................................78 3.1.2 2,5-Terpyrimidinylenes as Potential α-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,6-R-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 CRYSTALLOGRAPHIC DATA .............................................201 APPENDIX C: QIKPROP CALCULATIONS ...............................................................232 ABOUT THE AUTHOR ....................................................................................... End Page

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LIST OF TABLES Table 2.1 Model synthesis of hydrazine diester 2.5a .................................................30 Table 2.2 Failed attempts to isolate 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

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LIST OF FIGURES Figure 1.1 Hydrogen bonding pattern in an α-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 α-peptide and β-peptides ......................................................10 Figure 1.5 Depiction of a stapled peptide (Walensky, et al., 2006) ............................11 Figure 1.6 α-Helix mimics reported by Hamilton and co-workers .............................12 Figure 1.7 Polar α-helix mimics reported by Hamilton’s group .................................13 Figure 1.8 α-Helix mimics reported by Rebek’s and König’s groups ........................15 Figure 2.1 Structures of unsubstituted diketopiperazine rings ....................................22 Figure 2.2 DKPA and DKPB: Target α–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 hexameric 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

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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 terpyrimidinylene-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

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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-’,

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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

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LIST OF ABBREVIATIONS α Alpha Å Angstrom Ac2O Acetic anhydride AcOH Acetic acid Ala Alanine aq. Aqueous Ar aryl Asp Aspartic acid β 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 δ 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

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IC50 50% inhibitory concentration Ile Isoleucine J Coupling-constant(s) Ki 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 μL Microliter(s) μM Micromolar Val Valine

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Design and Synthesis of Substituted 1,4-Hydrazine-linked Piperazine-2,5- and

2,6-diones and 2,5-Terpyrimidinylenes as α-Helical Mimetics

Laura Anderson

ABSTRACT

The most common secondary structure of proteins is the α-helix. The α-helix can be

involved in various protein-protein interactions (PPIs) through the recognition of three or

more side chains along one face of the α-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 α-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 α-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 α-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.

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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 α-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.

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CHAPTER ONE

PROTEINS: GENERAL INTRODUCTION

1.1 Protein Secondary Structure: α-Helix

The biological function of a protein is greatly influenced 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 determined by the amino acid sequence. The

folding process also depends on the environment surrounding the protein, such as solvent,

temperature, pH, and the presence of chaperones (Cutler, et al., 2009; Williamson, 1994).

Proteins adopt well-defined 3-D structures to minimize the exposure of hydrophobic side

chain interactions to water counterbalancing overall entropic and enthalpic effects

(Branden and Tooze, 1992). Proteins organize at different levels including primary,

secondary, tertiary, and quaternary structures. The primary structure refers to the linear

sequence of the amino acids; secondary and tertiary structures are related to local and

global folding, respectively, of a single polypeptide chain; and the quaternary refers to

the stable organization of two or more polypeptide chains into an active sub-unit structure

(Garrett and Grisham, 1999).

The most common secondary structure of proteins is the α-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 interactions of the amino acid side residues. The hydrogen bonds are

almost parallel to the axis of the α-helix, whereas the side chain residues project almost

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orthogonally. Helices can be found in nature as right-handed and left-handed helices; the

most common in nature is the right-handed helix, which is promoted by L-amino acids

(Garrett and Grisham, 1999).

Figure 1.1. Hydrogen bonding pattern in an α-helix

The structural stability of an α-helix depends on the intramolecular hydrogen

bonding of the amino acids. The arrangement of the amino acids in an α-helix has 3.6

amino acid residues per turn. One turn places the ith and ith+4 residues 5.4 Å apart,

which is referred to as a pitch (5.4 Å = 3.6 residues x 1.5 Å, separation per residue).

Accordingly, hydrogen bonding occurs between C=O of residue ith and NH of residue

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ith+4, thus all NH and C=O groups in an α-helix are linked with hydrogen bonds except

for the first NH (the N-terminus) and the last CO group (the C-terminus) of the α-helix

(Figure 1.1) (Perez de Vega, et al., 2007).

1.2 Protein-Protein Interactions (PPIs)

The α-helix can be involved in many protein-protein interactions (PPIs) through

the recognition of three or more side chains along a single face of the α-helix (Wells and

McClendon, 2007). PPIs play significant roles in several biological systems including

signal transduction pathways, cellular processes (proliferation, growth, 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 Å2), as compared to protein-small molecule interaction

surfaces (~ 300 to 1000 Å2) or enzyme active sites, the relatively shallow surfaces, and

the noncontiguous binding regions involved in protein-protein interfacial domains (Wells

and McClendon, 2007). This implies that large molecular weight inhibitors that can

cover sufficient interactions between the inhibitor and the protein may be required to

displace the endogenous protein partner (Fletcher and Hamilton, 2006). Therapeutic

antibodies are good examples due to the high specificity binding to their molecular

targets and relative stability in human plasma. An example is adalimumab (Humira™,

MW = ~144,190 Da) with an absolute bioavailability of 64% (Reichert, 2008). However,

antibodies can also be problematic in terms of their high production costs, lack of cell-

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membrane permeability, and generation of undesirable side effects (Arkin and Wells,

2004; Saraogi and Hamilton, 2008; Verdine and Walensky, 2007).

Cl NN

HN

S

NH

O

O

SN

NO2

O

ABT-737

Cl NN

HN

S

NH

O

O

SN

SO2CF3

O

O

ABT-263

SP4206

Cl

N

NCl

N NHO

O

O

O

F FF

N N NO

O

Nutlin-3 SP304

SP4206 binds to IL-2, ABT-737 and ABT-263 bind to Bcl-xL, Nutlin-3 binds to Hdm-2,and SP304 binds to TNF (Wells and McClendon, 2007; Wendt, 2008; Domling, 2008)

NH

NH2H2N

HNO

O

N

N NCl Cl

O

OO

O

Figure 1.2. Examples of small molecular weight inhibitors of PPIs

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One of the myths about molecules that target protein-protein interfaces is that

these are too large to have “drug-like” properties 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 protein-protein interfaces with reasonable

oral bioavailability have also been reported (Toogood, 2002; Wells and McClendon,

2007; Zhao and Chmielewski, 2005). Some examples 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 (Nutlin-

3, MW=581 Da and JNJ-26854165, currently in Phase 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 these inhibitors (Wells and McClendon,

2007).

1.3 Role of Bcl-2 Family and Hmd-2 Family Proteins in Apoptosis

Many diseases, such as autoimmunity, inflammatory, neurodegenerative

disorders, diabetes, and cancer are the result of disregulation of apoptosis. Programmed

cell death, or apoptosis, is a highly regulated process that contributes to the elimination of

unnecessary or damaged cells; this occurs when a cell has fulfilled its biological function

(Afford and Randhawa, 2000). Apoptosis can occur via extrinsic and intrinsic pathways.

In both pathways, there is an activation of cysteinyl aspartate proteases (caspases) that

operate in proteolytic cascades. The extrinsic pathway starts outside the cell through an

activation signal caused by specific receptors called death receptors.

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Figure 1.3. Intrinsic pathway of apoptosis. Adapted from (Youle and Strasser, 2008)

The intrinsic pathway is initiated from inside the cell due to several stresses, such

as DNA damage, hypoxia, and defective cell cycle, among other cellular stresses (Youle

and Strasser, 2008). This pathway causes the release of pro-apoptotic proteins that

disrupt the mitochondrial membrane; the activation of specific caspases enzymes and the

release of cytochrome c and other proteins from the mitochondria ultimately 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 apoptotic process. Bcl-

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2 is a 239-amino acid integral membrane protein (Danial, 2007). The anti-apoptotic

proteins include B-cell lymphoma extra large (Bcl-xL) and Bcl-2, among others; the pro-

apoptotic proteins include Bcl-2-antagonist killer (Bak), Bcl-2-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 more specific conserved

regions known as Bcl-2 homology (BH) BH1, BH2, BH3, and BH4 domains. These

domains are α-helical regions that determine the function 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 form aggregates within the mitochondrial

outer membrane (MOM); this leads to the formation of pores in the membrane and

activates the apoptotic pathway by the cytochrome c releasing pathway. On the other

hand, the anti-apoptotic Bcl-2 and Bcl-xL proteins may heterodimerize with the death-

promoting region, BH3 domain, of Bak and Bax neutralizing their pro-apoptotic activity

(Walensky, et al., 2004; Wendt, et al., 2006). Accordingly, the overall balance of pro-

and anti-apoptotic protein interactions controls the susceptibility of a cell towards

programmed cell death.

NMR studies have revealed that the BH3 domain of the pro-apoptotic protein Bak

is required for activity; this region takes an amphipathic α-helical conformation when it

binds to Bcl-xL. The Bak peptide interacts via hydrophobic side chains (residues 72 to

87) projecting into the hydrophobic cleft of the Bcl-xL protein. This hydrophobic cleft

(620 Å2) comprises four turns of the α-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. Moreover, electrostatic interactions between

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the charged side chains of Bak and oppositely charged residues of Bcl-xL stabilize the

complex formation. Asp83 forms a salt bridge with a lysine residue of the Bcl-xL protein

(Sattler, et al., 1997).

Another important family of proteins involved in the intrinsic apoptotic pathway

is the murine double minute 2 (Mdm-2) or Hdm-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 α-helix that binds projecting 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 recognized 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 transactivation domain of p53 (p53AD) suppressing the

activation of p53 target genes. Unlike Mdm-2, Mdm-x is not transcriptionally 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 and degradation of p53 (Sharp, et al., 1999).

1.4 Peptidic and Non-Peptidic α-Helical Mimetics

Previous studies have demonstrated that overexpression of Bcl-2, Bcl-xL, Mdm-2,

and Mdm-x proteins is associated with tumor progression and drug resistance (Strasser, et

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al., 1997); therefore, the design and synthesis of compounds that can antagonize these

proteins represents a great potential for medicinal chemistry studies. More specifically,

the synthesis of compounds designed to mimic the α-helical region of the pro-apoptotic

Bak BH3 domain and the NH2 terminus of p53 represents a potential in cancer

therapeutics. Various strategies for the identification of small molecule inhibitors of

protein-protein complexes have been described in the literature. One common method

includes the design of inhibitors by screening through competitive binding, enzymatic,

fluorometric, and phenotypic assays; also by virtual screening techniques. Another

approach is the identification of inhibitors by structure-based design; this relies on the

understanding of the protein’s 3-D structure usually explored 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 development of small organic molecules that can

mimic secondary structures of proteins including β-sheet and α-helical conformations to

inhibit PPIs. The synthesis of β-peptides as potential mimics of protein secondary

structures has also been reported (Chin and Schepartz, 2001; Kritzer, et al., 2004). β-

peptides have an additional carbon atom in the main backbone that increases metabolic

resistance, as compared to corresponding α-peptides (Figure 1.4). In general, β-peptide

oligomers can adopt an α-helical conformation based on the substitution pattern of the

comprising β-amino acids; these are named based on the number of atoms in a ring

closed by a hydrogen bonding network specific to the helix. These are classified as 8-

helix, 10-helix, 10/12-helix, 12-helix, and 14-helix.

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Figure 1.4. Structures of α-peptide and β-peptides

Schepartz and co-workers synthesized a library of β3-peptide oligomers that

showed 14-helix character in aqueous media. One of these peptides labeled β53-1 was

reported to show significant nanomolar activity for the disruption of the Mdm-2/p53

complex (Kritzker, 2004). Gellman and co-workers also reported on β-peptides as α-

helical mimetics (Lee, et al., 2009; Raguse, et al., 2002; 2003).

The design of small compounds able to mimic α-helical structures has been

extensively investigated by several groups. Verdine and co-workers 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 peptidomimetics, known as stabilized α-helix of Blc-2

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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 synthesized 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-organized scaffolds (Yin and Hamilton,

2004), and benzoylurea oligomers (Rodriguez, et al., 2009b) as structural templates for

the design of functional α-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 the 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 α-helix (Fletcher and

Hamilton, 2006; Yin, et al., 2005b). Several PPIs were targeted with these terphenyl-

based compounds; an initial target was the interaction 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).

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Figure 1.6. α-Helix mimics reported by Hamilton and co-workers

Fluorescence polarization (FP) assays indicated that terphenylene derivative A,

shown in Figure 1.6, top panel-b, was their most potent antagonist of the Mdm-2 with a

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binding affinity in the nanomolar range (Ki = 182 nM) (Yin, et al., 2005a). Terphenyl-

based α-helical mimetic B, shown in Figure 1.6, top panel-c, was reported 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 affinity 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 α-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,

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et al., 2009a) were recently reported 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 α-helix and

the most potent inhibitor (Figure 1.7, b) for the Bcl-xL/Bak interaction have a Ki value of

1.8 µM by FP assay.

Rebek and co-workers have also published several analogs of the Hamilton’s

terphenylenes as α-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 α-helix mimics based on a piperazine scaffold (Figure 1.8, bottom panel-e)

(Restorp and Rebek, 2008). König’s group has recently published the synthesis of chiral

peptide mimetics based on a functionalized 1,4-dipiperazino benzene scaffold (Figure

1.8, bottom panel-f), which is another analog of Hamilton’s terphenylenes. These

compounds were described to adopt a staggered conformation with the substituents

resembling the side chain residues of an α-helix (Maity and Konig, 2008). Very recently,

Shaginian and co-workers reported a comprehensive compound library (8000

compounds) as α-helical mimetics for the disruption of the Mdm-2/p53 complex

(Shaginian, et al., 2009).

In summary, secondary structures show variations within proteins; however, it has

been demonstrated that conformationally restricted 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 based on protein-substructural

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components could hold great promise as valuable pharmacological tools and potential

new therapeutic agents.

Figure 1.8. α-Helix mimics reported by Rebek’s and König’s groups

While significant progress has been made in the rational design aimed at

mimicking peptide or protein structural conformations, as described in this chapter, this

field is still open to further investigation. Thus, 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

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synthesis of compounds that mimic the orientation of side chain residues of an α-helical

protein domain. Our scaffolds could potentially inhibit various PPIs; 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 design and our efforts towards the synthesis of

α-helical mimetics based on 3-R-1,4-hydrazine-linked piperazine-diones and Chapter

Three discusses our most recent results towards the synthesis of α-helical mimetics based

on substituted 2,5-terpyrimidinylenes. Our 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), 301-317. Biros, S. M.; Moisan, L.; Mann, 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 peptide-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.

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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 protein-protein 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 scaffold 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), 215-233. 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 oncoprotein 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.

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Lipinski, C. A. (2004) Lead- and 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 1,4-Dipiperazino Benzenes: Chiral Terphenyl-type Peptide Helix Mimetics. Organic Letters, 10 (7), 1473-1476. Moisan, L.; Dale, T. J.; Gombosuren, N.; Biros, 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.; Gombosuren, N.; Carella, A.; Rebek, J., Jr. (2008) Synthesis of an oxazole-pyrrole-piperazine scaffold 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 mimics 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 Chemistry (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 suppressor 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-chain 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: Striking a Balance between Shape Control and Functional Diversity. Journal of the American 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 mimetics with four side-chains. Bioorganic & Medicinal Chemistry Letters, 18 (22), 5909-5911.

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Rodriguez, J. M.; Nevola, L.; Ross, N. T.; Lee, G.-i.; Hamilton, A. D. (2009a) Synthetic inhibitors of extended helix-protein interactions 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-Derived alpha -Helix Mimetics Targeting the Bcl-xL/Bak Binding Interface. ChemMedChem, 4 (4), 649-656. Saraogi, I.; Hamilton, A. D. (2008) alpha -Helix mimetics as inhibitors of protein-protein interactions. Biochemical Society Transactions, 36 (6), 1414-1417. Sattler, M.; Liang, H.; Nettesheim, D.; Meadows, R. P.; Harlan, J. E.; Eberstadt, M.; Yoon, H. S.; Shuker, S. B.; Chang, B. S.; Minn, A. J.; Thompson, C. B.; Fesik, S. W. (1997) Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science (Washington, D. C.), 275 (5302), 983-986. Shaginian, A.; Whitby, L. R.; Hong, S.; Hwang, 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 Protein-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.; Frenk, 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.

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Volonterio, A.; Moisan, L.; Rebek, J., Jr. (2007) Synthesis of pyridazine-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 studies 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 target 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.; Park, 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-Molecular-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.

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

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CHAPTER TWO

DESIGN AND SYNTHESIS OF 3-R-PIPERAZINE-2,5- AND 2,6-DIONES

2.1 Introduction

2.1.1 Piperazine-diones

In recent years, there has been an increasing interest for the design and synthesis

of drug leads based on small heterocyclic library templates (Perrotta, et al., 2001). The

piperazine-dione or diketopiperazine unit (DKP), including 2,3-, 2,5-, and 2,6-DKPs

(Figure 2.1) are among the most attractive heterocyclic motifs due to their wide

representation in many biologically active compounds and their application as

structurally constrained active peptide analogs (Prasad, 1995). Many DKPs have been

reported to have antihistaminic, antibacterial (albonoursin, bicyclomycin), 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 unsubstituted 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-

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and 2,6-DKPs can be prepared from α-amino acids as starting materials by step-wise

intramolecular cyclization of the appropriate molecular fragments or tandem and

multicomponent reactions as used in combinatorial chemistry applications (Gellerman, et

al., 2008). DKPs can be efficiently prepared by solution phase or solid phase chemistry

and in many cases, 2,5-DKPs originate as unwanted byproducts during the process of

oligopeptide synthesis. DKP ring systems are the smallest cyclic peptides known and

their significance in the drug discovery field remains based on the intrinsic physical and

chemical properties of their rigid conformation and the presence of hydrogen bond

acceptor and donor groups for interactions with the biological targets. This feature makes

DKPs less susceptible to metabolic degradation of the amide bond compared to linear

peptides and it increases the drug-like properties of compounds containing the DKP unit

(Dinsmore and Beshore, 2002; Perrotta, et al., 2001).

In this project, we focused on the design and synthesis of peptidomimetics based

on semi-rigid scaffolds derived from 3-R-piperazine-2,5- and 2,6-dione 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 amino 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 α-helix.

More specifically, these compounds are designed to mimic the α-helical region of the

BH3 Bak peptide or the p53 peptide derived from the p53 N-terminus for their

interactions with anti-apoptotic 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 hydrophilic core that still conserves the

hydrophobic side chains required for activity (Yin, et al., 2005b; Yin, et al., 2005c).

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Figure 2.2. DKPA and DKPB: Target α–helical peptidomimetics

The R1, R1’, R2, R2

’, and R3, R3’, positions can be widely varied by selecting the

appropriate starting natural or unnatural amino 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 chiral or nonchiral α,α-substituted amino acid derivatives. The N-terminus-

like and C-terminus-like linker attachments to the ending 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

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compounds; some examples of drugs containing the hydrazine moiety include

antidepressants (isocarboxazid, phenelzine), antiparkinsonic agent (carbidopa), antiviral

(methiazone), antibacterial (sulfaphenazole), and alkylating agent (procarbazine) (Gilbert,

et al., 2000; Toth, 1996).

2.2 Results and Discussion

2.2.1 Synthesis of 3-R-Piperazine-2,6-diones: DKPA

3-R-piperazine-2,6-diones can be prepared by solution phase or solid phase

peptide (SPP) techniques. A general retrosynthetic analysis of scaffold DKPA via

solution phase is shown in Figure 2.3. Both routes require α-amino esters type A, which

are commercially available or can be readily prepared from corresponding α-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 cyclization after coupling of monoacid C or anhydride D

with a β-alanine linker derivative. In both 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 synthesis 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 phase peptide techniques will be

presented including the synthesis of a trimeric peptidomimetic type DKPA.

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

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2.2.2 Synthesis of Diacid Derivatives 2.6: Route 1

N-tert-butoxycarbonyl (Boc) protected 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 α-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 dimethylformamide (DMF); subsequent removal of the Boc

group by standard conditions with TFA/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 with thionyl chloride in

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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 Hünig’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 DMF, 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 reaction. 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 occur, it has been found that using the α-halo-alkyl acetate

is an efficient method for the preparation of diester 2.3 from good to excellent yields (60-

88%). The alternative reductive amination route 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 synthesis 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. Unsubstituted hydrazines can be prepared by reduction

of the hydrazone derived from an α-keto acid and reaction of an α-halo acid with

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hydrazine (Genari et al., 1996); optically active hydrazines 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 the 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 been 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-condensation of resulting free hydrazines

(Oguz, et al., 2001; Oguz, et al., 2002).

N-Boc-hydrazino derivatives 2.5 were then prepared by electrophilic 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 cleanly 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 nitrosation step, for example. Diester 2.3 was reacted

with N-Boc oxaziridine in MeOH, at -78 ºC to yield 2.5 derivatives. It has been reported

that best yields of the hydrazines are obtained using MeCN at -40 ºC to rt or MeOH at -

78 ºC 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

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different as various side products were observed by TLC and the percent yields were

lower than those reported (40- 65%). We observed that anhydrous methanol gives better

results than anhydrous acetonitrile 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 lowering the reaction temperature. The best

results were obtained when the reaction was performed at temperatures not exceeding 5

ºC 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

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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 α,α-disubstituted diester (2.5e)

was obtained in low yield as a result of the 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, such as phenylalanine (2.5a) and leucine (2.5b), were completely

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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 racemization, the use of LiOH formed the desired

compound with less harsh conditions that might lead to the racemization of the α-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 β-alanine-derivative linked to the

C-terminus and subsequent coupling with other DKP units can be pursued by both

solution and solid phase peptide (SPP) techniques. Dr. Umut Oguz, a former post-

doctoral researcher in our lab, pursued the cyclization and 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 piperazine-

2,6-diones by SPP were reported by Dr. Oguz. Trimeric scaffolds were assessed against

the Bcl-xL/Bak interaction by FP assay. Out of the 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 µM IC50 in this in vitro assay.

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Scheme 2.3. General synthetic procedure of trimeric piperazine-2,6-dione 2.9bac via SPP A computational docking model study performed at the Moffitt Cancer Center

suggested that longer α-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

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piperazine-2,6-dione analog Pip and Bcl-xL/Bax complex. As seen with Hamilton

terphenylenes (Yin, et al., 2005b) and terephthalamide derivatives (Yin and Hamilton,

2004), the computational docking modeling of our hexameric scaffold Pip and Bcl-xL

also indicates that our intended inhibitor binds in the same 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 α-helix (Rodriguez, et al., 2009a). Therefore, the synthesis of compounds that

can mimic the spatial projection of ith, ith+3/ith+4, and ith+7 sites on two turns of the α-

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 particularly, to improve the yield

of the coupling step to enable the synthesis of longer helical mimetics. Unfortunately,

our collaborative efforts to re-synthesize the desired trimer by SPP were unsuccessful;

cyclization of DKP units proved to be the most challenging step. Cleavage of the resin

after coupling of DKP1 with the β-alanine derivative was done to confirm if ring closure

had taken place, but only traces of the target monomer and uncyclized intermediates were

observed as determined by 1H NMR.

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a)

b)

Figure 2.4. Docking studies of a hexameric piperazine-2,6-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 shown 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).

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Table 2.2. Failed attempts to isolate anhydride intermediates

In order to advance this project, 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 corresponding cyclic acid anhydride.

Initially, we attempted the isolation of the anhydride to purify it from substituted urea

byproducts and uncyclized materials; several reaction conditions were done reacting

diacid hydrazines 2.6a and 2.6b with various condensing agents (Table 2.2). While the

desired anhydrides were observed by TLC and HPLC monitoring, isolation of these was

difficult since the compounds were unstable and in most cases these hydrolyzed back to

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starting diacids 2.6. Based on these results, we opted for generating the anhydride in situ

and coupling it to a β-alanine linker (2.10 or 2.11) in a one pot reaction to afford

derivatives 2.12 (Scheme 2.4). The N-Boc β-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/dioxane) for removal of the Boc protecting group gave salt

2.12. β-alanine methyl ester hydrochloride 2.11 was obtained from commercial sources.

As shown in Scheme 2.4, the coupling of anhydride intermediate 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 regioselectivity issue. The nitrogen

atom of the β-alanine moiety could attack the anhydride to either carbonyl site and two

possible products could be generated. However, the product 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 the 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 carboxylic acid of 2.12b with acetic

anhydride in presence of sodium acetate.

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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 efforts to overcome this issue, a second route for the synthesis of

the target DKP units was developed.

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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 orthogonally protected diester in step one

of Route 2. By doing this, we can generate a monoacid instead of a diacid to facilitate the

regioselective coupling of the C-terminal linker (β-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)

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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 worth mentioning that reactions 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 β-

alanine derivative 2.11 to obtain hydrazine diesters 2.19. Ring closure of compound 2.19

was obtained by a base-catalyzed reaction under 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 α-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 KOtBu 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 and consequently would facilitate the ring closure (Scheme

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2.7). While we attempted this, the approach did 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 synthesis and only reactions carried in small

quantities afforded the target compounds. Our 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 improve the synthesis of the piperazine-2,6-

diones, we decided to abandon this route and continue the project with a structurally

related scaffold.

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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

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A retrosynthetic analysis of a target scaffold based on 3-R-substituted piperazine-

2,5-dione (2,5-DKP) repeat units is shown in Figure 2.5. The synthesis of the 2,5-DKP

monomer has been also pursued by two routes from starting amino acids type A. By

introducing the 2,5-DKP ring instead of the 2,6-DKP, we would expect the cyclization

step of the 2,5-DKPs to be more accessible due 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 nucleophilic 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 synthetic approach for

Route 1 is described in Scheme 2.8.

Scheme 2.8. Attempted synthesis of 2,5-DKP monomer, Route 1

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The two-step reductive amination reaction of commercially available α-amino

acid ester HCl salts 2.2 with benzaldehyde was accomplished 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 α-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 β-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 literature procedures (Grange, et al., 1980; Ouihia, et al., 1993).

The advantage of preparing this reagent is its easier handling and high 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 insertion, we decided to treat compound 2.31f with reagent

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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 envisioned the free amine would facilitate the

diazoacetylation reaction. The use of diazoacetamides and diazoesters as carbene sources

to afford heterocyclic structures has been 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 this time we had also started working on the

synthesis of 2,5-terpyrimidinylene scaffolds (discussed in Chapter Three) and since this

new project had greater promise to give α-helical mimetics in an expedient manner, we

concentrated our efforts towards the synthesis of the new scaffold. During this course,

we revisited the synthesis of the 2,5-DKP scaffold 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 synthesis 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 coupling 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 readily available chiral and

nonchiral amino esters 2.2. This route also enables an easier installation of various linker

derivatives, R”, which are attached to the C-terminus-like position.

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Scheme 2.10. General synthesis of 2,5-DKP monomer, Route 2

In the first step, the HCl salts 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, β-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 installation of the hydrazine group, is under

investigation. A few options to achieve formation of the hydrazine includes the N-

nitrosation of the amide of the DKP monomer with nitrosonium 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

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al., 2009). It has been reported that acyl protected amines give very low to no yield in

the reduction step in reactions with stronger nitrosating agents and these were observed to

be unstable (Oguz, 2003). However, the advantage 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 hydrazine 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 these nitrosating agents, which require careful

handling since these are highly toxic compounds (d'Ischia, 2005; Mirvish, 1995). Future

work in this project will entail the continued efforts to prepare peptidomimetics based on

piperazine-2,5-dione repeat 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-2,6-

and 2,5-diones repeat units by several routes were described. 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 project has remained in the improvement of the

overall yield, especially during the cyclization step, to enable the synthesis of longer α-

helical mimetics. The synthesis of the 2,6-DKP 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 practical 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

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benzylamine or β-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 reagents (ACS grade), and solvents were

obtained from commercial sources and used as received unless otherwise noted.

Moisture- and air-sensitive reactions were carried out under an atmosphere of argon.

Thin layer chromatography (TLC) was performed 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 Å, #70-230

mesh (Selecto Scientific). Automated flash chromatography was performed in a

FlashMaster II system (Argonaut-Biotage) using Biotage silica cartridges. High

performance liquid chromatography was 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 chloroform-d (CDCl3), unless otherwise indicated.

Chemical shifts (δ) are reported in parts per million (ppm) relative to internal

tetramethylsilane (TMS) or chloroform (δ 7.26) for 1H NMR and chloroform (δ 77.0) for

13C NMR. Multiplicity is expressed as (s = singlet, br s = broad singlet, d = doublet, t =

triplet, q = quartet, p = pentet, or m = multiplet) 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 Series in the ESI-TOF mode. Microwave reactions were

performed in a Biotage Initiator I microwave reactor. Melting points (uncorrected) were

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determined using a Mel-Temp II®, Laboratory 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 α-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) was 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-methylpropanoate hydrochloride (2.2e):

Isolated yield: 93%, white solid, m.p. 179-182 ºC. 1H NMR (400 MHz, (CD3)2SO) δ

1.48 (s, 6H), 3.75 (s, 3H), 8.76 (br s, 2H). 13C NMR (100 MHz, CD3OD) δ 22.80, 52.81,

56.62, 171.97. HRMS (ESI) calcd. for C5H12NO2 [M+H]+ 118.0868, found 118.0859.

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(S)-methyl 2-amino-3-(naphthalene-2-yl)propanoate hydrochloride (2.2f):

Isolated yield: 97%, white solid. 1H NMR (400 MHz, (CD3)2SO) δ 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) δ

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) δ 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) δ 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 α-amino diester (2.3a):

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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-oxoethylamino)-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) δ 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) δ 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-oxoethylamino)-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) δ 1.25 (t, J = 7.0 Hz, 3H),

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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) δ 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:ethyl acetate, 3:2), yield: 80%, colorless

oil. 1H NMR (400 MHz, CDCl3) δ 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) δ 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-ethoxy-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) δ 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) δ 14.16, 38.56, 49.12, 51.83, 60.88, 62.23, 70.00, 114.91, 127.47, 127.92,

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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 α-amino hydrazine diester (2.5a):

A solution of diester 2.3a (1.88 mmol) in anhydrous methanol (7 mL) was cooled to -78

ºC and kept under an argon atmosphere. Tert-butyl 3- (trichloromethyl)-1,2-oxaziridine-

2-carboxylate 2.4 (5.64 mmol) was added slowly and the reaction was stirred at -78 ºC

for 4 h. Progress of the reaction was monitored by TLC. The solvent was evaporated

under reduced pressure to yield a yellow crude residue.

(S)-tert-butyl 2-(2-ethoxy-2-oxoethyl)-2-(1-methoxy-1-oxo-3-phenylpropan-2-

yl)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 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 14.35, 28.50, 36.83, 51.77, 61.11, 69.02, 80.53, 126.85,

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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 α-amino hydrazine diacid (2.6a):

Hydrazine compound 2.5a (2.10 mmol) was dissolved in MeOH (12 mL) and cooled to 0

ºC. Then freshly prepared 1N NaOH (6.3 mmol) was added and the mixture was stirred

at rt for 24 h. MeOH was removed 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-(tert- butoxycarbonyl)-1-(carboxymethyl)hydrazinyl)-3-phenylpropanoic

acid (2.6a):

NOH

O

HO

ONHBoc

Isolated yield: 75%, white fluffy solid, m.p. = 137-140 ºC. 1H NMR (400 MHz, CD3OD)

δ 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) δ 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.

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Experimental procedure for the synthesis of benzyl 3-aminopropanoate

hydrochloride (2.10’):

To a solution of N-Boc β-alanine (5.28 mmol) and benzyl bromide (6.34 mmol) in

acetone (15 mL) was added potassium carbonate (10.56 mmol). The reaction mixture

was stirred under refluxing for 5 h. The mixture was cooled to rt and the formed

precipitate was removed by filtration. The crude 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 pressure. Intermediate benzyl

3-(tert-butoxycarbonylamino)propanoate, 2.10’ was obtained after purification by

column chromatography on silica gel (hexanes:ethyl acetate, 4:1), yield: 85%, colorless

oil. 1H NMR (400 MHz, CDCl3) δ 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) δ 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-1-

phenyl-2,12-dioxa-6,9,10-triazatetradecan-9-yl)-4-methylpentanoic acid (2.12b):

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Hydrazine diacid 2.6b (0.82 mmol) was dissolved in anhydrous DCM (5 mL) and cooled

to 0 ºC. 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, β-alanine benzyl ester HCl, 2.10

(0.82 mmol) was added followed by triethylamine (1.64 mmol). The reaction mixture

was stirred under refluxing conditions for 18 h. The solvent was removed under reduced

pressure to obtain a yellow crude material. The crude was dissolved in water (5 mL) and

5% citric acid (5 mL) and extracted 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) δ 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 α-amino diester (2.16):

2.2a

HCl.H2N

R1 R1'

O

OBnBr

DIEA, MeCN,

2.16a

HN

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. Diisopropylethylamine (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

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acetate (3 x 15 mL). The combined organic layers 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-oxoethylamino)-3-phenylpropanoate (2.16a):

HN

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) δ 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) δ 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 α-amino hydrazine diester (2.17):

A solution of diester 2.16a (1.83mmol) in anhydrous methanol (7 mL) was cooled to -78

ºC and kept under argon atmosphere. Tert-butyl 3- (trichloromethyl)-1,2-oxaziridine-2-

carboxylate 2.4 (5.49 mmol) was added slowly and the reaction was stirred at -78 ºC for 4

h, then at rt for 12 h. Progress of the reaction was monitored by TLC. The solvent was

evaporated under reduced pressure to yield a crude oil.

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(S)-tert-butyl 2-(2-(benzyloxy)-2-oxoethyl)-2-(1-methoxy-1-oxo-3-phenylpropan-2-

yl)hydrazinecarboxylate (2.17a):

Compound 2.17a was purified by column chromatography on silica gel (hexanes:ethyl

acetate, 4:1), yield: 86%, thick colorless oil. 1H NMR (400 MHz, CDCl3) δ 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-methyl-1-oxopentan-2-

yl)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) δ 1H NMR (400 MHz, CDCl3) δ 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 – 3.80 (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-((2S,3S)-1-methoxy-3-methyl-1-oxopentan-

2-yl)hydrazinecarboxylate (2.17d):

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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) δ 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 α-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 catalyst (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-2-

yl)hydrazinyl)acetic acid (2.18a):

Isolated yield: >90%, white solid. 1H NMR (400 MHz, CDCl3) δ 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) δ 28.49, 36.83,

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51.77, 52.01, 68.96, 80.57, 126.85, 128.59, 129.44, 137.26, 155.36, 170.63, 172.09. ESI-

MS 434.1 [M+H]+.

Representative procedure for the synthesis of α-amino hydrazine amide (2.19a):

A mixture of monoacid 2.18a (1.88 mmol) and the HCl salt of β-alanine methyl ester

2.11 in anhydrous DCM (5 mL) was cooled to 0 ºC under argon. Then 1-ethyl-3-(3-

dimethyllaminopropyl)carbodiimide hydrochloride (EDC.HCl) was added portionwise,

while the mixture stirred vigorously. The reaction was kept at 0 ºC 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 washed 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-phenylpropan-2-yl)-2-(2-(3-methoxy-3-

oxopropylamino)-2-oxoethyl)hydrazinecarboxylate (2.19a):

NO

O

NH

ONHBoc

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) δ 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) δ 14.40, 21.24, 28.43, 34.11, 34.94, 36.36, 51.86, 52.26, 60.59,

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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-oxopropylamino)-2-oxoethyl)-2-(1-methoxy-4-

methyl-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) δ 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) δ 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-dioxopiperazin-1-yl)propanoate (2.13a):

A solution of NaH (0.324 mmol) in anhydrous THF (1 mL) was cooled to 0 ºC under an

argon atmosphere. To this mixture, a solution of compound 2.19a (0.85 mmol) in

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62

anhydrous THF (5 mL) was added dropwise. The reaction was stirred at 0 ºC for 30 min,

then at rt for 22 h. The solvent was removed 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) δ 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) δ 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-2-

oxoethylamino)-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 methyl 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 with 5% citric acid (5 mL) and it was

extracted with ethyl acetate (3 x 10 mL). The combined organic 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) δ 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

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(100 MHz, CDCl3) δ 39.52, 48.98, 51.83, 51.87, 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-3-

phenylpropan-2-yl)-2-(2-methoxy-2-oxoethyl)hydrazinecarboxylate (2.23a):

DCM, 0 °C

O

NBocCl3C

2.4

2.23a

NO

O

O

ONHBoc

2.22a

HN

O

O

O

O

A solution of diester 2.22a (2.54 mmol) in anhydrous DCM (7 mL) was cooled to -78 ºC

and kept under argon atmosphere. Tert-butyl 3-(trichloromethyl)-1,2-oxaziridine-2-

carboxylate 2.4 (3.30 mmol) was added slowly and the reaction was stirred below 0 ºC

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% yield after purification by

column chromatography on silica gel (hexanes:ethyl acetate, 4:1) as a thick colorless oil.

1H NMR (400 MHz, CDCl3) δ 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) δ

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.

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Experimental procedure for the synthesis of (S)-2-(2-(tert-butoxycarbonyl)-1-(2-

methoxy-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) δ 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) δ 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-2-

yl)hydrazinecarboxylate (2.25a):

A mixture of monoacid 2.24a (0.85 mmol) and the HCl salt of β-alanine methyl ester

2.11 (0.94 mmol) in anhydrous DCM (10 mL) was cooled to 0 ºC under an argon

atmosphere. Then EDC.HCl (1.02 mmol) was added portionwise, while the mixture

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stirred vigorously. The mixture was stirred at 0 ºC 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 with 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 (hexanes:ethyl acetate, 1:1). 1H NMR (400 MHz, CD3OD)

δ 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) δ 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 α-amino ester (2.26):

Step 1: The HCl salt of amino ester 2.21a (2.32 mmol) was dissolved in anhydrous THF

(7 mL) and brought to 0 ºC. 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 stirred at rt under argon for 4 h. The crude

mixture was filtered and the solvent was removed 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 borohydride (4.64 mmol) was added slowly. The

reaction mixture was stirred at rt for 2 h under an argon atmosphere. The reaction

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mixture was quenched with 1N NaOH and extracted with diethyl 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) δ 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) δ 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):

HN

O

O

Compound 2.26c was prepared following the procedure described for 2.26a. Isolated

yield: 95% over two steps, colorless oil. 1H NMR (400 MHz, CDCl3) δ 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) δ 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]+.

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Experimental procedure for the synthesis of (S)-tert-butyl 2-benzyl-2-(1-methoxy-1-

oxo-3-phenylpropan-2-yl)hydrazinecarboxylate (2.27a):

DCM, -78 °C

O

NBocCl3C

2.4

2.27a

HN

O

O

2.26a

NO

ONHBoc

A solution of compound 2.26a (0.74 mmol) in anhydrous DCM (15 mL) was cooled to -

72 ºC and kept under an argon atmosphere. Tert-butyl 3- (trichloromethyl)-1,2-

oxaziridine-2-carboxylate 2.4 (0.96 mmol) was added slowly and the reaction was stirred

at -72 ºC for 2 h, then at rt for 18 h. Progress of the reaction was monitored by TLC. The

solvent was evaporated under reduced pressure and the crude residue was purified by

column chromatography on silica gel (hexanes:ethyl acetate, 4:1). Compound 2.27a was

obtained as a thick colorless oil in 46% yield. 1H NMR (400 MHz, CDCl3) δ 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 acid (12 mmol), and acetonitrile (50 mL)

was stirred at rt for 24 h. The solvent was removed under reduced pressure and the

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residue was triturated with water. The white crystalline product was removed by

filtration and air dried to yield glyoxylic acid tosylhydrazone intermediate 2.29’ in 87%

yield. m.p. = 148-152 ºC. 1H NMR (400 MHz, (CD3)2SO) δ 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) δ 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 glyoxylic acid tosylhydrazone 2.29’ (2.06 mmol) and N-hydroxy

succinimide (2.06 mmol) in ice-cold dioxane (10 mL) under an argon atmosphere, DCC

(2.03 mmol) in dioxane (4 mL) was added dropwise. 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 purification by flash column chromatography on

silica gel (dichloromethane), white solid, m.p. = 121-122 ºC. 1H NMR (400 MHz,

CDCl3) δ 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 solvent was removed under reduced pressure to

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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 ºC under an argon atmosphere. 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 mixture was stirred at 0 ºC for 40 min. then

at rt for 24 h. The solvent was removed under 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) δ 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 α-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 ºC. To this mixture, NaHCO3 (8.23 mmol) was added followed by the slow addition

of a solution of bromoacetyl bromide (3.58 mmol) in toluene (15 mL). The reaction was

mixture was brought to rt and stirred 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 combined organic layers were dried over Na2SO4 and

complete removal of the solvent gave a white solid.

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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. = 47- 48 ºC. 1H NMR (400 MHz,

CDCl3) δ 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) δ 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. = 64-

66 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 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.

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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) δ 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 synthesis of piperazine-2,5-dione DKP1 (2.33 and

2.35):

Compound 2.34a (0.85 mmol) was dissolved in methanol (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):

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Compound 2.35a was purified by column chromatography on silica gel (hexanes:ethyl

acetate, 1:4), yield: 62%, white solid, m.p. = 165- 166 ºC. 1H NMR (400 MHz, CDCl3) δ

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) δ

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-dioxopiperazin-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) δ 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).

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Besada, P.; Mamedova, L.; Thomas, C. J.; Costanzi, S.; Jacobson, K. A. (2005) Design and synthesis of new bicyclic diketopiperazines as scaffolds 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 metabonomics 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 chloride-sodium nitrite. A novel reagent for chemoselective N-nitrosation. Synthetic Communications, 39 (4), 604-612. d'Ischia, M. (2005) Nitrosation and nitration of bioactive 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-methyldiazoacetamides 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 Aminopurine Derivatives. Convenient Access to 6-Halo- and 2,6-Dihalopurine Nucleosides and 2'-Deoxynucleosides 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 Diazoacetate: An N-Heterocyclic Carbene-Cu System That Suppresses Diazo Coupling. Journal of the American Chemical Society, 126 (35), 10846-10847. Funabashi, Y.; Horiguchi, T.; Iinuma, 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.

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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 Keto- and 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 α-Amino and α-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 and therapeutic compositions. US Patent, 4218465. Hannachi, J.-C.; Vidal, J.; Mulatier, J.-C.; Collet, A. (2004) Electrophilic Amination of Amino Acids with N-Boc-oxaziridines: Efficient Preparation of N-Orthogonally Diprotected Hydrazino Acids and 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) diethyl ester and ethylenediaminetetraacetic acid tetraethyl ester scaffolds. Tetrahedron, 56 (16), 2359-2367. Kuang, R.; Ganguly, A. K.; Chan, T. M.; Pramanik, B. N.; Blythin, D. J.; McPhail, A. T.; Saksena, A. K. (2000) Enantioselective syntheses of carbocyclic 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 Ylide Generated through Cu(I) Carbene and Sulfides. Journal of the American Chemical Society, 127 (43), 15016-15017.

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Maity, P.; Konig, B. (2008) Synthesis and Structure of 1,4-Dipiperazino 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 National 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 and toxic potency in Salmonella typhimurium TA1538. Applied and Environmental Microbiology, 44 (4), 801-8. Martins, M. B.; Carvalho, I. (2007) Diketopiperazines: biological 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 bladder 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 dipeptide 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, United 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 from Amino Acids, from Solution-Phase to Solid-Phase Organic Synthesis. Journal of combinatorial chemistry, 3 (5), 453-460.

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Prasad, C. (1995) Bioactive cyclic dipeptides. Peptides (Tarrytown, New York), 16 (1), 151-64. Ragnarsson, U. (2001) Synthetic methodology for alkyl 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 interactions based on a biphenyl 4,4'-dicarboxamide scaffold. ChemBioChem, 10 (5), 829-833. Sattler, M.; Liang, H.; Nettesheim, D.; Meadows, R. P.; Harlan, J. E.; Eberstadt, M.; Yoon, H. S.; Shuker, S. B.; Chang, B. S.; Minn, A. J.; Thompson, C. B.; Fesik, S. W. (1997) Structure of Bcl-xL-Bak peptide complex: 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, Endonuclease 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 activity of carbapenem compounds: effect 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 N- and 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-3-Trichloromethyloxaziridine: a new, powerful 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-apoptotic 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.

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Yin, H.; Hamilton, A. D. (2004) Terephthalamide derivatives as mimetics of the helical region of Bak peptide target Bcl-xL protein. Bioorganic & Medicinal Chemistry Letters, 14 (6), 1375-1379. 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. (2005a) Terphenyl-Based Bak BH3 alpha -Helical Proteomimetics as Low-Molecular-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 American Chemical Society, 127 (15), 5463-5468.

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CHAPTER THREE

DESIGN AND SYNTHESIS OF 4-R- AND 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 lengths 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 properties of both molecules, benzene and

pyrimidine, are relatively modified depending 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 naturally occurring substances (Figure 3.2),

such as nucleobases (uracil, cytosine, and thymine), glycosides (vicine and convicine),

and thiamine or vitamin B1 (Zempleni, et al., 2007). It is also the key structure of several

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synthetic therapeutic agents including antibacterial (trimethoprim and sulfadiazine) and

anticancer agents (gemcitabine, tegafur, imatinib, nilotinib, and dasatinib) (Lagoja, 2005;

von Angerer, 2004).

Figure 3.2. Pyrimidine unit as component 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 general, pyrimidines are

thermally stable due to their aromaticity and amide-like structures (von Angerer, 2004).

While the characteristics mentioned above are more relevant to describe the chemistry of

pyrimidines, it is still significant to mention them since our objective is related to the

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synthesis of compounds that contain the pyrimidine core, but that can behave as

benzenoid structures with more drug-like characteristics.

3.1.2 2,5-Terpyrimidinylenes as Potential α-Helical Mimetics

Based on our efforts to synthesize the piperazine-dione oligomers (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 α-helix mimics.

Our 2,5-terpyrimidinylene scaffold target is shown in Figure 3.3. Specifically, this

template is structurally analogous to Hamilton’s terphenylene given that his work

provided a valuable reference point when designing the 2,5-terpyrimidinylenes 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 α-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 α-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

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the trimeric 2,5-pyrimidinylene scaffold are designed to mimic the ith, ith+4, and ith+7 of

the target α-helix. These groups can be varied broadly to optimize the bioactivity and

specificity of the resulting library compounds. Figure 3.4 shows a tube representation of

an idealized α-helical conformation of octa-alanine (gold with transparent ribbon) with its

ith, ith+4, and ith+7 methyl groups highlighted as gold spheres and a 4,4’,4”-trimethyl-

2,5-terpyrimidinylene with its methyl groups highlighted as green spheres. 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 α-

helix from the ith position.

Figure 3.4. Overlay of a 4,4’,4’’-trimethyl-2,5-terpyrimidinylene and an octa-alanine. The RMSD for the highlighted methyl groups is 0.68 Å. For simplicity, only polar hydrogens are shown. The details of these 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 Science of Synthesis series (von Angerer,

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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 including cross-coupling reactions (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 substituted pyrimidines involves the condensation of

1,3-dielectrophiles, such as β-dicarbonyls and synthetic equivalents, 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 synthesis of substituted pyrimidines

Pyrimidines have been well known for years and thousands of derivatives

containing unsubstituted and substituted monomeric pyrimidines have been reported in

the literature. However, dimeric and trimeric 2,5-pyrimidinylene scaffolds have not been

fully explored, especially an iterative synthetic approach and their potential application as

α-helical mimetics have not been reported previously. Only a few examples of

unsubstituted 2,5-terpyrimidinylenes have been described; these compounds have been

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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 α-helical

region of a protein as a PPI inhibitor, represents 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 some of the intrinsic disadvantages carried

when using peptides themselves as drug leads, such as metabolic degradation, poor

bioavailability, and high production costs (Ahmed and Kaur, 2009; Loffet, 2002).

3.2 Results and Discussion

3.2.1 Synthesis of a “First-generation” 4-R-2,5-Terpyrimidinylene Library

A retrosynthetic scheme for the targeted 4-R-2,5-terpyrimidinylene is shown in Figure

3.5. Due to its versatility, we adopted the most common method (Scheme 3.1) as the

general iterative approach for preparing the 4-R-2,5-terpyrimidinylenes. Accordingly,

the condensation of an α,β-unsaturated α-cyanoketone (shown in blue) with an amidine

derivative (shown in magenta) affords 5-cyano substituted pyrimidinylene monomer.

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Figure 3.5. Retrosynthetic scheme of trimeric 2,5-pyrimidinylenes

Iterative transformation of the 5-cyano group 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 synthesis of pyrimidinylene monomers

This synthesis began with the coupling of commercially available esters 3.1a-h

with acetonitrile in anhydrous THF in the presence of potassium t-amyloxide (KOt-amyl)

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to give β-ketonitriles 3.2a-h (Ji, et al., 2006). Reactions for step one were also carried

out in presence of other bases, such as sodium methoxide (NaOMe) (Sorger and Stohrer,

2007), lithium diisopropylamide (LDA), and potassium t-butoxide (KOt-Bu). While the

desired products were also afforded, the isolation and purification of the β-ketonitriles

were tedious resulting in fairly low yields (40-60%). The formation of side product 3.5a

was observed when KOt-Bu and LDA were used (Scheme 3.3).

Scheme 3.3. Formation of side product 3.5a

Alternatively, reactions with KOt-amyl in THF gave the products in good yields

(64-73%) and further purification 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 subsequent 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 was 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 β-ketonitriles 3.2 with N,N-dimethylformamide dimethyl

acetal (DMF-DMA) in THF gave 3.3 as an isomeric E>>Z mixture in excellent yields

(>90%) (Reuman, et al., 2008). The efficiency of this reaction allowed us to obtain

several α,β-unsaturated α-cyanoketones bearing hydrophobic alkyl, aryl, and

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heteroaromatic groups for introducing diversity in the 4-position of pyrimidinylenes 3.4.

The condensation of the α,β-unsaturated α-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 synthesis of the monomers are summarized in

Table 3.1.

Table 3.1. Results of the synthesis 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 was required because these compounds usually

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precipitated from the reaction mixtures; conversely, we observed that reactions to prepare

monomers having R’ = H, Me, and Cl typically afforded incomplete consumption of the

α,β-unsaturated α-cyanoketones and required additional purification to obtain the

products (Table 3.1, entries 1-3, 13). For instance, the observed low yield for the

preparation of 2-unsubstituted pyrimidinylene (Table 3.1, entry 1) could be attributed to

the ease of hydrolysis of formamidine under typical condensation 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 β-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 comparable yields and complete conversion of the

β-ketonitriles were not observed. Based on these 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 iterative synthesis of the dimeric and trimeric

2,5-pyrimidinylenes using mostly these derivatives via the condensation of the α,β-

unsaturated α-cyanoketones and amidine intermediates (Scheme 3.2).

The key step of this synthesis is the conversion of the 5-cyano group to a 5-

carboxamidine 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 cross-coupling-type reactions, which would require

more steps to synthesize the appropriate starting materials. Moreover, extensive research

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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 Nour-El-Din, 2008; Dunn, 2005; Gielen, et al., 2002; Lee, et al.,

1998), but we focused on the synthesis of 5-cyanopyrimidines due to the versatile access

to the β-ketonitriles. Many methods for the conversion 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 intermediate 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 incorporates 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-cyanopyrimidines (Table 3.2), none of these alternatives were better than the

two-step hydroxylamine route (Table 3.2, entry 10) (Judkins, et al., 1996; Nadrah and

Dolenc, 2007). We found excess hydroxylamine 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).

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

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Table 3.2. Attempted routes to synthesize 5-carboxamidines

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Scheme 3.5. Hydroxylamine-mediated synthesis of amidine intermediates

The N-hydroxylamine mediated transformation of ortho-substituted and di-ortho-

substituted arylnitriles to carboxamidines has been reported (Basso, et al., 2000; Judkins,

et al., 1996; Lukyanov, et al., 2008), but has not been used to synthesize oligo-

pyrimidinylenes. Based on our observations and literature reports on conversion of

nitriles to amidines, we could reasonably conclude 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 amidines through facile procedures (Lange, et al.,

1999; Lee, et al., 1998); however, most of the reported conditions have not been

described for an ortho-substituted aromatic ring or pyrimidine moiety. Moss

demonstrated the synthesis of amidines from 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 o-methylbenzonitrile with some of these conditions (as on entries 4, and 8 on

Table 3.2) gave indication that reaction of the nitrile group is inhibited by steric

hindrance. In most cases, the o-methylbenzonitrile 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

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to this substrate and it does not necessarily translate to any substrate containing a proton

on the ortho position. Further experimentation is required for validation and this is an

ongoing project in our lab. On the other hand, the direct addition of ammonia

(Beingessner, et al., 2008) to o-methylbenzonitrile in presence and absence of Lewis

acids, such as CuBr resulted in unreacted nitrile or traces 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 intermediate amidoximes were more efficiently

obtained by the hydroxylamine procedure (Scheme 3.5); however, this route 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 the 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 triethylamine afforded the amidoximes in higher yields compared

to reactions with weaker bases, such as NaHCO3 (Koryakova, et al., 2008), where the

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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

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It has been previously suggested that amide formation is due to an initial attack of

the oxygen atom of hydroxylamine to the cyano 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 indication of nitrile conversion to amidoxime.

Figure 3.6. Hypothetical model for H-bonding mediated synthesis of amidine

Based on this, we speculate that the attack of the nitrogen atom of hydroxylamine

to the cyanopyrimidine could be facilitated 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 reaction 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

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amidoximes in practical yields to continue the 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 formate 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 the hydrogen transfer reagent (Anbazhagan, et al.,

2003). Although the carboxamidine salts were not isolated and purified, our results were

consistent with the literature. Reactions with potassium formate in methanol after

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selective acylation of the amidoximes occurred faster. Also, complete conversion of the

amidoximes were observed as compared to reactions 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, separation of the polar carboxamidines from resulting

tin salts was laborious, which resulted in very low yields. Therefore, the method

described by Nadrah and Dolenc was the preferred procedure for the synthesis of 5-

carboxamidine intermediates (vide supra).

The intermediate amidine salts 3.7 were reacted with another desired α,β-

unsaturated α-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 dimers 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 isolated products as precipitates from the

reaction mixtures. The purity of all precipitated compounds was >90% as determined by

NMR spectroscopy.

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Table 3.4. Results of the synthesis of dimers 3.11 and trimers 3.12

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Figure 3.8. ORTEP diagram of compound 3.12bac.3

The ORTEP diagram of the 5”-cyano-4”-isopropyl-4’-phenylmethyl-4-isobutyl-2-

phenylterpyrimidine crystal structure 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 2,5-

pyrimidine 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 characteristic and useful indicator to

track the synthesis of these compounds.

An example of a typical 1H NMR spectrum of a trimer showing the protons at 6, 6’, and

6” (a, b, and c, respectively) positions is shown in Figure 3.9.

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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 chapter) could have been selected. We chose hydrophobic

substituents, such as isobutyl, benzyl, isopropyl, methyl, 1-naphthylmethyl, and 2-

naphthylmethyl (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 Hamilton and co-workers (Kutzki, et al., 2002; Yin, et al.,

2005b). The naphthalene group is often used to mimic the indole-containing side chain

of tryptophan (Moisan, et al., 2008), a key binding amino acid residue on one face of the

p53 α-helix (Shaginian, et al., 2009). Furthermore, these were selected with the idea that

these terpyrimidinylenes would mimic an α-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

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the Hamilton group (vide supra). Structure-activity studies indicated that terphenyl

derivatives with isobutyl, 1-naphthylmethyl, isobutyl (Figure 1.6, top panel-c, compound

14 from reference) (Yin, et al., 2005b) and isobutyl, 2-naphthylmethyl, isobutyl side

(Figure 1.6, top panel-b) (Yin, et al., 2005a) chain sequences were potent inhibitors of

Bcl-xL (Ki = 0.114 µM) and Mdm-2 (Ki = 0.180 µM), respectively. Also, it was

determined that the terminal carboxyl groups seemed to be necessary for the inhibitory

properties presumably due to the increased polarity, 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 calculations 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-terpyrimidinylene scaffold show the calculated log

Poctanol/water values ranged from quite hydrophobic for the Hamilton 1,4-terphenylene

scaffold (Yin, et al., 2005b) to within the desirable range for drug-like characteristics for

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the analogous 2,5-terpyrimidinylene scaffold. The full Qikprop output can be found in

Appendix C.

Modification of the R” group of dimeric and trimeric scaffolds to more polar

functionalities could be achieved via direct conversion of the nitrile to amide,

carboxylate, tetrazole, and methyl amine. Conversion of cyano to carboxylic acid was

efficiently obtained by following a procedure adapted by Dr. Vasudha Sharma, a post-

doctoral fellow in our group. The results and routes for the preparation of tetrazoles

explored by Dr. Sharma are not mentioned here; only the preparation of carboxylates is

discussed (Table 3.5).

Table 3.5. Conversion of 5-cyanopyrimidine to 5-carboxypyrimidine

The 5- and 5’-carboxylate pyrimidines were obtained by reacting 5- and 5’-

cyanopyrimidines with sodium hydroxide under microwave–assisted reaction conditions

(Table 3.5). Isolation and purification of monomeric and some dimeric 2,5-pyrimidine

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carboxylates (Table 3.5, entries 1-3) were easily performed, but the same was not true for

the trimeric compounds (Table 3.5, entries 4-5); rather, incomplete hydrolysis of the

cyano group was consistently observed. Both unreacted starting nitrile and intermediate

amides were observed by TLC and 1H NMR, even under prolonged 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 results to the reactions done under microwave-

assisted conditions.

3.2.2 In Vitro Biological Evaluation

Table 3.6. Results of the in vitro evaluation of monomeric and dimeric 2,5-

pyrimidinylenes

Several pyrimidinylene-based compounds were evaluated for their inhibitory

effect on the Bcl-xL/Bak and the Mdm-2, Mdm-x/p53 complexes in a competitive binding

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assay based on fluorescence polarization (FP). Based on the rationale behind the design

of α-helical mimetics (discussed in Chapter One), we would not anticipate monomeric

and dimeric 2,5-pyrimidines to exhibit any bioactivity 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 terpyrimidinylenes holding equal or similar hydrophobic R

groups as those of Hamilton’s best terphenylenes would inhibit these protein-protein

interactions. We used some of Professor Hamilton’s best terphenyl inhibitors (Yin, et al.,

2005b) as positive controls for the assay (Table 3.7, entries 5-7); unfortunately, 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-terphenylenes 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 replacement of the phenyl

ring by the pyrimidine ring on the analogous 1,4-terphenylene is not sufficient to achieve

water solubility. While none of our “first generation” library compounds were useful for

the inhibition of the Bcl-2/Bcl-xL or Mdm-2, Mdm-x/p53 protein-protein interactions, the

synthesis and biological evaluation of these provided us with significant tools for scaffold

optimization.

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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 bioactivity of the “first generation” library, our next

objective was to synthesize terpyrimidinylenes holding polar moieties at each end of the

scaffold. The use of the chemistry already explored 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 replacing the phenyl group derived from

benzamidine, with a carboxylate derived from 4-cyanobenzoic acid. Treatment of 4-

cyanobenzoic acid 3.16 with excess hydroxylamine afforded intermediate amidoxime in

good yield (Scheme 3.7). In this case, formation of amide side product 3.18 was

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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. After isolation and purification,

monomer 3.19a was afforded in modest yield (35%). Condensation with more polar α,β-

unsaturated α-cyanoketone, such as 3.3b, gave much lower yield of the monomer as a

result of a tedious isolation of the highly polar pyrimidines. A potential alternative to

overcome the isolation issue of the terpyrimidinylenes carboxylates may be the use of 4-

cyanobenzoic 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 improvised an alternative route to

introduce the polar functionality at the N-terminus-like site as part of a “second-

generation” library. Several monomeric 2-aminopyrimidinylenes and a dimer were

efficiently prepared. To our delight, these compounds were as easy to isolate as the

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monomers having R’ = Ph and the majority of the 2-aminopyrimidinylenes precipitated

from the reaction mixtures and were obtained 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 advantages of pursuing the 2-

aminoterpyrimidinylene 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 nucleophilic displacement of

the amino group via diazotization reaction could lead to the placement of the carboxylate;

for instance, β-alanine or glycine ester can be placed in that position to yield carboxylates

similar to the 1,4-terphenylenes (Table 3.7, entries 5-7). The direct N-alkylation of the 2-

aminopyrimidinylenes was also considered, but our efforts were unsuccessful. This most

likely was due to a decrease of the nucleophilicity of the amino group exerted by the

electron-withdrawing effect of the pyrimidine ring (Hurst, 1980). The synthesis,

optimization, and biological evaluation of this ‘second-generation’ compound library are

under investigation.

3.2.4 Synthesis of 4-R,6-R-2,5-Terpyrimidinylenes

Previously, we showed our efforts to synthesize the semi-rigid terpyrimidinylene

scaffolds and only the early steps toward the synthesis of this new structurally related, but

more polar di-ortho-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 2,5-

pyrimidinylene monomers compared to the scaffold previously described (Figure 3.3).

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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 α-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 Michael acceptor used in this case is

derived directly from malonitrile and not from the ketonitrile; 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-4-substituted pyrimidinylene monomers

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We have successfully synthesized a few 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 synthesis of 6, 6’, 6”-triamino-4-R-, 4’-R-’, 4”-R”-2,5-pyrimidinylenes and Michael acceptors

A general proposed route for the synthesis 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

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cyano group to amidine as previously 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 phenyl at the 4-position of the

pyrimidine unit, the ethylidene malonates (Michael acceptor in Scheme 3.9) can be

prepared by a Knoevenagel-type condensation with procedures previously 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 surrogates 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 dimethyl sulfate. It is expected that iteration of the steps

described in Scheme 3.9 will furnish di-ortho-substituted dimeric and trimeric 2,5-

terpyrimidinylenes. The potential bioactivity of these 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 through a facile iterative condensation between

amidines and α,β-unsaturated α-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 4,6-

R,R’-2,5-terpyrimidinylenes were presented. Although none of the compounds

synthesized so far have shown significant bioactivity for disrupting various protein-

protein interactions, the value of these syntheses lies in the assessment of several

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synthetic routes to obtain the desired compounds. Also, we learned 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 β-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 t-pentylate (~1.7 M in

toluene, 106 mmol) and anhydrous THF (20 mL) was cooled at 0 ºC 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 stirred 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 consumption of the ester (3.1a). The mixture was filtered and the

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filtered cake was washed thoroughly with hexanes (80 mL). Then, the filtered residue

was transferred to a separatory funnel and it was 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 washed with brine (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) δ 3.46 (s, 2H), 3.86 (s, 2H), 7.21–7.40

(m, 5H). 13C NMR (100 MHz, CDCl3) δ 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) δ 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 ) δ 22.53, 24.66, 32.56, 51.08,

113.96, 197.25.

2-acetyl-3-oxo-4-phenylbutanenitrile (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 t-pentylate. 3.5a was purified

by column chromatography on silica gel (hexanes:ethyl acetate, 3:2), yield: 39%,

colorless thick oil. 1H NMR (400 MHz, CDCl3) δ 2.37 (s, 3H), 3.70 (s, 2H), 4.32 (s, 1H),

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7.28 – 7.46 (m, 5H). 13C NMR (100 MHz, CDCl3 ) δ 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 α,β-unsaturated α-cyanoketones (3.3):

β-ketonitrile 3.2a (0.75 mmol) was dissolved in dry THF (3 mL) under an argon

atmosphere. Then N,N-dimethylformamide dimethyl acetal (0.98 mmol) was added and

the mixture was stirred at rt. Progress 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 reaction, the mixture was cooled to 0 ºC and

then it was filtered and rinsed with cold THF to isolate the desired compound.

(E)-2-((dimethylamino)methylene)-3-oxo-4-phenylbutanenitrile (3.3a):

Isolated yield: >90 %, colorless needle-like crystal, m.p. = 134-138 ºC. 1H NMR (400

MHz, CDCl3) δ 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) δ 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 structure is reported in Appendix B.

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(E)-2-((dimethylamino)methylene)-5-methyl-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 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 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-methyl-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 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ

19.10, 37.05, 38.99, 48.11, 79.35, 120.48, 157.98, 199.77. HRMS (ESI) calcd. for

C9H15N2O [M + H]+ 167.1184, found 167.1184.

(E)-2-((dimethylamino)methylene)-4,4-dimethyl-3-oxopentanenitrile (3.3i):

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3.3i was prepared by the same method described for 3.3a. Yield: >90%, white solid. 1H

NMR (400 MHz, CDCl3) δ 1.32 (s, 9H), 3.23 (s, 3H), 3.42 (s, 3H), 7.92 (s, 1H). 13C

NMR (100 MHz, CDCl3) δ 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):

OCN

N

3.3j was prepared by the same method described for 3.3a. Isolated yield: >90%, white

solid. 1H NMR (400 MHz, CDCl3) δ 1.32 (s, 9H), 3.23 (s, 2H), 3.42 (s, 2H), 7.92 (s, 1H).

13C NMR (100 MHz, CDCl3) δ 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 synthesis of pyrimidinylenes (3.4):

3.4a.4

OCN

3.3a

N NPh

CNN

NH2

Method A: A mixture of compound 3.3a (15.17 mmol) and guanidine hydrochloride

(30.34 mmol) in ethanol (absolute, 200 proof, 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 crystals in 83% yield, m.p. = 129-132 ºC.

1H NMR (400 MHz, CDCl3) δ 4.10 (s, 2H), 5.58 (br s, 2H), 7-24-7.38 (m, 5H), 8.45 (s,

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1H). 13C NMR (100 MHz, CDCl3) δ 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 structure 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 pyrimidinylenes. For example, to a mixture of α,β-

unsaturated α-cyanoketone 3.3b (0.933 mmol) and guanidine hydrochloride (1.87 mmol)

in ethanol (5 mL) was added sodium ethoxide (0.933 mmol) suspended in ethanol (1

mL). The reaction mixture was placed on a microwave reactor for 40 min at 120 ºC. A

colorless crystal-like precipitate formed in the solution upon cooling to rt. The

precipitate was filtered and rinsed 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 using 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) δ 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) δ 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.

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4-benzyl-2-methylpyrimidine-5-carbonitrile (3.4a.2):

3.4a.2 was prepared following method A using 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) δ 2.79 (s, 3H),

4.26 (s, 2H), 7.24 – 7.41 (m, 5H), 8.78 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 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-phenylpyrimidine-5-carbonitrile (3.4a.3):

N N

CN

3.4a.3 was prepared following method A using commercially available benzamidine

hydrochloride. Isolated yield: 91%, white solid, m.p. = 138-141 ºC. 1H NMR (400 MHz,

CDCl3) δ 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) δ 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.

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4-isobutyl-2-phenylpyrimidine-5-carbonitrile (3.4b.3):

3.4b.3 was prepared following method A. Isolated yield: 70%, colorless crystals, m.p. =

68-69 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 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. Isolated yield: 75%, white solid, m.p. = 175-

178 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 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.

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4-tert-butyl-2-phenylpyrimidine-5-carbonitrile (3.4i.3):

3.4i.3 was prepared following method A. Isolated yield: 83%, white solid, m.p. = 101-

103 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 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-4-tert-butylpyrimidine-5-carbonitrile (3.4i.4):

NH2

N N

CN 3.4i.4 was prepared following method B. Isolated yield: 75%, colorless crystals. 1H

NMR (400 MHz, CDCl3) δ 1.43 (s, 9H), 5.56 (br s, 2H), 8.44 (s, 1H). 13C NMR (100

MHz, CDCl3) δ 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):

N N

Cl

CN

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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) δ 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):

N N

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) δ 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) δ 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 synthesis of 4-(4-benzyl-5-cyanopyrimidin-2-

yl)benzoic acid (3.19a):

CN

3.16

N NH2HO

3.17

O OH OH.Et3NO1) Ac2O, HOAc, MeOH

HCOOK, 10% Pd/C

OHO

N N

CN

3.19a

Et3N, MeOH

1) NH2OH.HCl

2) 3.3a, NaOEt, EtOH

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Step 1: To a mixture of commercially available 4-cyanobenzoic acid (2.72 mmol) and

hydroxylamine hydrochloride (6.8 mmol) in methanol (7 mL) was added triethylamine

(6.8 mmol). The reaction mixture was stirred at rt for 18 h. The solvent was removed

under reduced pressure to obtain 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 monitored 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 filtrate 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

OHO

Isolated yield: >80%, white solid, m.p. = >243 ºC. 1H NMR (400 MHz, CD3OD) δ 7.89-

7.91(m, 2H), 8.21-8.23 (m, 2H). 13C NMR (100 MHz, CD3OD) δ 128.04, 130.20,

132.31, 135.82, 166.84. HRMS (ESI) calcd. for C8H9N2O2 [M + H]+ 165.0664, found

165.0666.

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Step 3: Amidine 3.17’ from step 2 was dissolved in ethanol (2 mL) and heated to 70 ºC.

Then, excess sodium ethoxide was added and 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 chromatography in silica gel (hexanes: 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 3-o-tolylisoquinolin-1-amine (3.9):

A solution of o-methylbenzonitrile (16.88 mmol) in anhydrous THF was cooled to 0 ºC

and kept under an argon atmosphere. Then 1M NaHMDS in THF (20.26 mmol) was

added and the mixture was stirred for 1 h at rt. The mixture was filtered and the filtrate

was concentrated under reduced pressure to yield 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). The 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,

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(CD3)2SO)) δ 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 synthesis 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 added triethylamine (10.53 mmol). The reaction

mixture was stirred under refluxing 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-phenylpyrimidine-5-carboximidamide (3.6b.3):

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3.6b.3 was purified by column chromatography in silica gel (hexanes: ethyl acetate 3:2),

70% yield, white solid, m.p. = 125-127 ºC. 1H NMR (400 MHz, (CD3)2SO) δ 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)) δ

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-phenylpyrimidine-5-carboxamide (3.10b.3):

N N

NH2O

3.10b.3 was purified by column chromatography in silica gel (hexanes: ethyl acetate 3:2),

20%, white solid, m.p. = 124-127 ºC. 1H NMR (400 MHz, (CD3)2SO) δ 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)) δ 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 dissolved in glacial acetic acid (1

mL) and acetic anhydride (1.01 mmol). After 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 addition 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 pressure to obtain a yellow crude residue.

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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 dissolved 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 conditions 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 ºC. 13C NMR

(100 MHz, CDCl3) δ 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'-bipyrimidine-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) δ 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) δ 22.64, 22.80, 28.57, 29.10, 44.97, 45.85, 107.10, 115.28, 127.69, 128.85,

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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'-bipyrimidine-5-carbonitrile (3.11af.3):

N N

N N

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 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 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'-bipyrimidine-5-carbonitrile (3.11aa.3):

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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) δ 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'-bipyrimidine-5-carbonitrile (3.11ag.3):

N N

N N

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 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 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'-bipyrimidine-5-carbonitrile (3.11ba.4):

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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 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 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'-bipyrimidine-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 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ

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.

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Representative procedure for the synthesis of trimeric 2,5-pyrimidinylenes (3.12):

N N

N N

3.12bac.3

steps 1,2,3

N N

N N

CN

3.11ba.3

N N

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 refluxing 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 was 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 anhydride (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 until the TLC indicated

completion of the reaction. The crude was filtered through CeliteTM (1 g) and rinsed with

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129

methanol (40 mL). The filtrate was concentrated 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 conditions 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% yield after three steps.

5”-cyano-4”-isopropyl-4’-phenylmethyl-4-isobutyl-2-phenylterpyrimidine (3.12bac.3):

Isolated yield: 41% after 3 steps, white solid, m.p. = 166-168 ºC. 1H NMR (400 MHz,

CDCl3) δ. 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) δ 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,

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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-naphthylmethyl)-2-phenylterpyrimidine (3.12bfb.3):

N N

N N

N N

CN

3.12bfb.3 was prepared following the procedure described for 3.12bac.3. Isolated yield:

48% after 3 steps, off-white solid, m.p. = 164-167 ºC. 1H NMR (400 MHz, CDCl3) δ

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) δ 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-phenylmethyl)-2-phenylterpyrimidine (3.12bab.3):

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131

N N

N N

N N

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) δ 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 synthesis of 4-isobutyl-2-phenylpyrimidine-5-

carboxylic acid (3.13b.3):

3.4b.3 (0.25 mmol) was dissolved in a mixture of 20% aqueous solution NaOH and

methanol (3 mL : 2 mL). The reaction mixture was placed on a microwave reactor for 40

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min. at 160 ºC. 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 layer was acidified to pH= ~2-

3 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 ºC. 1H NMR (400 MHz, CD3OD) δ 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) δ

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'-bipyrimidine-5-carboxylic acid (3.14ba.3):

N N

N N

OHO

3.14ba.3 was prepared following the procedure described for 3.13ba.3. Isolated yield:

93% yield, white solid, m.p. = 167-169 ºC. 1H NMR (400 MHz, (CD3)2SO) δ 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) δ 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.

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4'-isobutyl-4-(naphthalen-2-ylmethyl)-2'-phenyl-2,5'-bipyrimidine-5-carboxylic acid

(3.14bf.3):

N N

N N

OHO

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) δ 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) δ

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 synthesis of 4-amino-2,6-diphenylpyrimidine-5-

carbonitrile (3.22a.3):

To a mixture of commercially available 3.20a (5.18 mmol) and benzamidine

hydrochloride (5.70 mmol) in ethanol (absolute, 200 proof, 8 mL) was added sodium

ethoxide (5.70 mmol). The mixture was stirred under refluxing conditions until the TLC

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134

indicated complete consumption 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 ºC. 1H NMR (400 MHz, CDCl3) δ 5.77 (br s, 2H), 7.53 (m, 6H), 8.13 (m, 2H),

8.51 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 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 synthesis of pyrimidinylene (3.22):

NC CN

O

3.21b R = H3.21c R = Me

R'

NH2.HClHN

K2CO3, EtOHreflux, 18 h

N N

R'

CNR

3.22b.2 R = H, R' = Me3.22b.3 R = H, R' = Ph3.22c.3 R = Me, R' = Ph

H2NR

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 refluxing 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-phenylpyrimidine-5-carbonitrile (3.22b.3):

N N

CNH2N

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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) δ 5.58 (br s, 2H), 7.44

– 8.33 (m, 5H), 8.59 (s, 1H).

Experimental procedure for the synthesis of 4'-amino-4-tert-butyl-2',6'-diphenyl-

2,5'-bipyrimidine-5-carbonitrile (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 refluxing conditions until the TLC indicated the

consumption of the starting material 3.22a.3 (48 h). The precipitate that formed was

removed by vacuum filtration and washed with methanol (2 x 10 mL). The filtrate was

concentrated under reduced pressure 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 anhydride (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 until the TLC indicated

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completion of the reaction. The crude was filtered through CeliteTM (0.5 g) and rinsed

with methanol (30 mL). The filtrate was concentrated 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 refluxing conditions for 18 h. The crude was concentrated

under reduced pressure to obtain a crude yellow solid. Compound 3.23a.3 was purified

by column chromatography on silica gel (hexanes:ethyl acetate, 9:1), yield: 42%, white

solid, m.p. = 166-169 ºC. 1H NMR (400 MHz, CDCl3) δ 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) δ 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

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Basso, A.; Pegg, N.; Evans, B.; Bradley, M. (2000) Solid-phase synthesis of amidine-based GP IIb-IIIa antagonists 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)-Arylaminopyrimidine-5-carbaldehydes and Their Application in the Synthesis of Structurally 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), 451-459. Brown, D. J. (1994) The Pyrimidines. [In: Chem. Heterocycl. Compd., 28]; p 1059 pp. Bruning, J. (1997) Lithium and potassium bis(trimethylsilyl)amide: utilizing non-nucleophile 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-containing conjugated compounds as electroluminescent materials. 95-109928690052, 19950626., 1996. Gompper, R.; Mair, H. J.; Polborn, K. (1997) Synthesis of oligo(diazaphenyls). Tailor-made fluorescent heteroaromatics and pathways to nanostructures. Synthesis, (6), 696-718. Han, Q.; Dominguez, C.; Stouten, P. F. W.; Park, J. M.; Duffy, D. E.; Galemmo, R. A., Jr.; Rossi, K. A.; Alexander, R. S.; Smallwood, 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 Amidino 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.

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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.; Wenzler, 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 preparation 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-dihydropyrimidin-2(1H)-one to pyrimidines via 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-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamides 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 allele 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.

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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 2-hydroxybenzamidines from 3-aminobenzisoxazoles. Tetrahedron Letters, 43 (48), 8777-8779. 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) Microwave-assisted transformation of esters into hydroxamic acids. Synthesis, (20), 3201-3204. Moisan, L.; Odermatt, S.; Gombosuren, N.; Carella, A.; Rebek, J., Jr. (2008) Synthesis of an oxazole-pyrrole-piperazine scaffold 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 optimization 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-Mediated 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-dimethylhomoveratrylamine (Benzeneethanamine, 3,4-dimethoxy-N,N-dimethyl-). Organic Syntheses, 76, 133-141.

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Saygili, N.; Batsanov, A. S.; Bryce, M. R. (2004) 5-Pyrimidylboronic acid and 2-methoxy-5-pyrimidylboronic acid: new heteroarylpyrimidine 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.; Hwang, 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 Protein-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 acylation 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 One-Step Synthesis of Monosubstituted N-Arylamidines. 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.; Park, 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-Molecular-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.

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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 introduced 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 2-

aminoethylglycine scaffold, a peptide-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 pairing rules, PNA can selectively bind to

complementary DNA and RNA to form PNA-DNA and PNA-RNA duplexes and even

(PNA)2-DNA triplexes, whose bindings are stronger than that of DNA-DNA or RNA-

RNA bindings. In addition, the duplexes formed exhibit high stability towards enzymatic

degradation by nucleases and proteases (Hyrup and Nielsen, 1996).

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4.1.1 Potential Applications of PNA

Potential uses of PNA include their application as antisense and antigene drugs.

Antisense inhibition involves the use of short oligonucleotide sequences of DNA, RNA,

or chemical analogs (15 to 20 bases) synthesized 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 inhibiting 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 potential analogs, such PNA, are designed to

identify and bind to complementary sequences in a specific gene interfering with the

transcription of that gene (Dias and Stein, 2002; Maher, 1996; Ray and Norden, 2000).

Figure 4.2 depicts the basics of antisense and antigene inhibition.

Figure 4.2. Schematic depiction of antisense 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 stability, high affinity hybridization properties with DNA and

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RNA, and lack of toxicity (Dias and Stein, 2002; Nielsen, 1999; Ray and Norden, 2000).

Previous in vitro studies have indicated that PNA could in fact inhibit both translation

and transcription of genes; however, unmodified PNA (naked PNA) cannot easily

penetrate the cell membrane due to the high molecular mass (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 Nielsen, 2000; Yi Sung, et al., 2009; Zhou, et al.,

2003). One of the strategies involves modification of the backbone by introducing a

cysteine and an aminoethyl group giving rise 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 the synthesis of CPNA

monomers are not described in this document; some data has been already published

(refer to Sung Wook Yi, 2008) and the remaining work is under current investigation.

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A method that has been reported for delivery 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 membrane. Although the mechanistic basis for

this delivery process has not been yet established, it has been suggested 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 charged 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 established (Dragulescu-Andrasi,

et al., 2005).

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The proposed target scaffold for this project is shown in Figure 4.3. Unlike Ly’s

GPNA, our structure does not contain the side chain at the α-L-position and it is not

synthesized from an arginine derivative. Instead, the positively charged residue is

attached at the ethylene unit between the two nitrogen atoms of the backbone and its

synthesis derives from cysteine. By introducing the cationic guanidine residue, the

solubility of the PNA in water should increase; 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 Guanidine Derivative 4.4

Figure 4.5. CPNA building block target (Yi Sung, et al., 2009)

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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-benzyloxycarbonyl (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 commercially 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 ºC. 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

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Compound 4.2 was guanidinylated in the next step. 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-1-

carboxamidine (Bernatowicz, et al., 1993); treatment with electrophilic agents, such as S-

methyl(aryl)-sulfanylisothioureas (Kent, et al., 1996); protected thioureas and

Mukaiyama’s reagent (Yong, et al., 1997); protected isothioureas 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 involves the reaction of amine 4.2 with a

triflylguanidine derivative in the presence of triethylamine in DCM (Scheme 4.2).

Scheme 4.2. Failed attempt to synthesize guanidine derivative 4.3

Preparation of the triflic reagent was 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 guanidine hydrochloride (Feichtinger, et al., 1998b). The

first step consists of protecting the guanidine with benzyloxycarbonyl 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 step is to introduce a triflyl group on the

unprotected nitrogen of the guanidine. Unfortunately, the results reported by Feichtinger

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were not reproducible; the desired pure compound was not isolated since purification of

the crude by column chromatography even with different eluents was not efficient.

Scheme 4.3. Failed attempt to synthesize a triflylguanidine reagent

In order to move forward with the synthesis, a second 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 groups, which are in conjugation with the

reaction center, may increase not only the electrophilicity 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-2-

pseudothiourea 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

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guanidine. Purification of the target guanidine by column chromatography was always

required. The possibility of replacing the HgCl2 by using other reagents, such as

Mukaiyama’s reagent or nickel catalysts (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 synthesizing pure compounds in practical scale and

under mild conditions (Gers, et al., 2004).

Amine derivative 4.2 was treated with the di-Cbz-protected pseudothiourea in the

absence of the thiophilic agent HgCl2. Accordingly, guanidine derivative 4.3 was

obtained in 92 % yield by the reaction of 4.2 with 1,3-bis(benzyloxycarbonyl)-2-methyl-

2-pseudothiourea at rt. The adaptation of this procedure was very useful because it not

only avoided the use of heavy metal reagents, but it also did not require additional

reagents or purification techniques to isolate the desired product in excellent yield. The

reaction time depended on how fast the pseudothiourea reagent was consumed; this was

monitored by TLC and it varied between 6 and 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 indicating 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.

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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 mixture of trifluoroacetic acid (TFA, 40%)

and (DCM, 60%) or HCl in dioxane (Greene 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, respectively. Both compounds were isolated and characterized.

Another major issue observed in this step was the formation of side product 4.5b via an

undesired intramolecular cyclization.

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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 ºC were used, whether the

reaction was performed in an organic or aqueous 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 bromoacetyl bromide in a two-phase system

(Table 4.1, entry 1). The desired product was isolated 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 after alkylation step,

Figure 4.3).

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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 successfully 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 reaction 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):

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Compound 4.1 (1.64 mol) was dissolved in CHCl3 (250 mL) and cooled to 0 ºC. Then

solid t-Boc2O (0.03 mol) dissolved in CHCl3 (36 mL) was added dropwise from an

addition funnel during 30 min. The reaction mixture was brought to rt and stirred for 2 h.

The mixture was filtered and the filtrate was concentrated under reduced pressure; the

resulting crude oil was dissolved in ethyl acetate (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) δ 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) δ 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-(2,3-

dibenzyloxycarbonylguanidino)propylcarbamate (4.3):

1,3-bis(benzyloxycarbonyl)-2-methyl-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) δ 1.41 (s, 9H), 1.71 (p, J = 6.4, 2H), 3.11 – 3.19 (m,

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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) δ 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-bromo-N-(3-(2,3-

dibenzyloxycarbonylguanidino)propyl)acetamide (4.4):

Compound 4.3 (4.13 mmol) was treated with HCl-dioxane 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 ºC. Bromoacetyl

bromide (4.13 mmol) was added and stirring was continued for 30 min. at 0 ºC; 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 ºC. 1H NMR (400 MHz, CDCl3)

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155

δ 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) δ 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 protected derivatives of 1-guanylpyrazole for the mild and efficient preparation of guanidines. Tetrahedron Letters, 34 (21), 3389-92. Bhat, L.; Georg, G. I. Nickel-promoted guanylation of amines with (iso)thioureas. 99-4128446100428, 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 spacing binds sequence-specifically 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 triflyldiurethane-protected guanidines: new reagents for guanidinylation reactions. Journal of Organic Chemistry, 63 (23), 8432-8439. 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.

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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 Organic 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 based 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.; Ajmera, M.; Anderson, L.; Topper, M.; McLaughlin, M. L. (2008) Design and synthesis of cysteine based PNA monomers. Abstracts of Papers, 236th ACS National Meeting, Philadelphia, PA, United States, August 17-21, 2008, MEDI-419. Katritzky, A. R.; Khashab, N. M.; Bobrov, S. (2005) The preparation of 1,2,3-trisubstituted 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), 135-147. 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.

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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 biological processes. Cellular and Molecular Life Sciences, 55 (3), 334-358. Yi Sung, W.; Jain, P.; Ajmera, M.; Kaulagari 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.; Kaulagari Sridhar, R.; Topper, M.; Anderson, L.; McLaughlin Mark, L. (2008) Cysteine Based PNA (CPNA): Design, Synthesis, and Applications. Dissertation, University 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.

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158

APPENDIX A: SELECTED 1H AND 13C NMR SPECTRA

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159

Methyl 2-amino-2-methylpropanoate hydrochloride (2.2e)

6.00

2.74

H2NO

O

H2NO

O

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160

(S)-methyl 3-(4-(benzyloxy)phenyl)-2-(2-ethoxy-2-oxoethylamino)propanoate (2.3h)

-2-1012345678910111213f1 (ppm)

3.03.23.43.6f1 (ppm)

HN

O

O

O

O

O

HN

O

O

O

O

O

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161

benzyl 3-(tert-butoxycarbonylamino)propanoate (2.10’)

-101234567891011121314f1 (ppm)

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

O

O

NHBoc

O

O

NHBoc

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162

(S)-methyl 3-(3-benzyl-4-(tert-butoxycarbonylamino)-2,6-dioxopiperazin-1-yl)propanoate (2.13a)

-101234567891011121314f1 (ppm)

2.62.83.03.2f1 (ppm)

O

O

N N

O

O

NHBoc

O

O

N N

O

O

NHBoc

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163

tert-butyl 2-(2-(benzyloxy)-2-oxoethyl)-2-((2S,3S)-1-methoxy-3-methyl-1-oxopentan-2-yl)hydrazinecarboxylate (2.17d)

-101234567891011121314f1 (ppm)

NO

O

O

ONHBoc

NO

O

O

ONHBoc

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164

(S)-tert-butyl 2-(1-methoxy-1-oxo-3-phenylpropan-2-yl)-2-(2-(3-methoxy-3-oxopropylamino)-2-oxoethyl)hydrazinecarboxylate (2.19a)

-101234567891011121314f1 (ppm)

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

NO

O

NH

ONHBoc

O

O

NO

O

NH

ONHBoc

O

O

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165

tert-butyl 2-(2-(3-methoxy-3-oxopropylamino)-2-oxoethyl)-2-(1-methoxy-4-methyl-1-oxopentan-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

NO

O

NH

ONHBoc

O

O

NO

O

NH

ONHBoc

O

O

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166

(S)-benzyl 2-(2-methoxy-2-oxoethylamino)-3-phenylpropanoate (2.22a)

HN

O

O

O

O

HN

O

O

O

O

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167

(S)-tert-butyl 2-(1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl)-2-(2-methoxy-2-oxoethyl)hydrazinecarboxylate (2.23a)

-2-101234567891011121314f1 (ppm)

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

NO

O

O

ONHBoc

NO

O

O

ONHBoc

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168

(S)-2-(2-(tert-butoxycarbonyl)-1-(2-methoxy-2-oxoethyl)hydrazinyl)-3-phenylpropanoic acid (2.24a)

-101234567891011121314f1 (ppm)

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

NOH

O

O

ONHBoc

NOH

O

O

ONHBoc

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169

(S)-tert-butyl 2-(2-methoxy-2-oxoethyl)-2-(1-(3-methoxy-3-oxopropylamino)-1-oxo-3-phenylpropan-2-yl)hydrazinecarboxylate (2.25a)

-2-101234567891011121314f1 (ppm)

2.42.62.83.0f1 (ppm)

NNH

O

O

ONHBoc

O

O

NNH

O

O

ONHBoc

O

O

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170

(S)-methyl 2-(benzylamino)-3-phenylpropanoate (2.26a)

-2-1012345678910111213f1 (ppm)

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

HN

O

O

HN

O

O

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171

(S)-methyl 2-(benzylamino)-3-methylbutanoate (2.26c)

-2-101234567891011121314f1 (ppm)

1.922.00f1 (ppm)3.63.73.8

f1 (ppm)3.003.03

f1 (ppm)

HN

O

O

HN

O

O

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172

(E)-2-(2-tosylhydrazono)acetic acid (2.29’)

-101234567891011121314f1 (ppm)

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

SNH

ON

O O

OH

SNH

ON

O O

OH

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173

2,5-dioxopyrrolidin-1-yl 2-diazoacetate (2.29)

-101234567891011121314f1 (ppm)

(S)-methyl 3-(2-(2-diazoacetamido)-3-(naphthalen-2-yl)propanamido)propanoate (2.32f)

-101234567891011121314f1 (ppm)

2.22.32.4f1 (ppm)7.37.47.57.67.77.87.9

f1 (ppm)

O

O

N O

O N2

HN

NH

O

OO

ON2

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174

(S)-benzyl 2-(2-bromoacetamido)-3-phenylpropanoate (2.34a’)

-2-101234567891011121314f1 (ppm)

5.135.22f1 (ppm)

4.854.90f1 (ppm)

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

HN

O

O

OBr

HN

O

O

OBr

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175

(S)-1,3-dibenzylpiperazine-2,5-dione (2.35a)

-2-101234567891011121314f1 (ppm)

3.103.153.203.25f1 (ppm)

NHN

O

O

NHN

O

O

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176

(S)-methyl 3-(3-isopropyl-2,5-dioxopiperazin-1-yl)propanoate (2.33c)

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)

2.82.93.03.13.23.3f1 (ppm)

(S)-2-(2-(tert-butoxycarbonyl)-1-(1-methoxy-1-oxo-3-phenylpropan-2-

yl)hydrazinyl)acetic acid (2.18a)

NHN

O

O

O

O

NO

O

HO

ONHBoc

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177

3-oxo-4-phenylbutanenitrile (3.2a)

OCN

OCN

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178

2-acetyl-3-oxo-4-phenylbutanenitrile (3.5a)

3.31

2.39

1.00

5.29

0.83

O O

CN

O O

CN

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179

(E/Z)-2-((dimethylamino)methylene)-3-oxo-4-phenylbutanenitrile (3.3a)

OCN

N

OCN

N

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180

(E/Z)-2-((dimethylamino)methylene)-4,4-dimethyl-3-oxopentanenitrile (3.3i)

8.52

3.08

3.00

0.88

OCN

N

OCN

N

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181

4-benzylpyrimidine-5-carbonitrile (3.4a.1)

N N

CN

N N

CN

Page 198: Design and Synthesis of Substituted 1,4-Hydrazine-linked ...

182

4-benzyl-2-methylpyrimidine-5-carbonitrile (3.4a.2)

2.99

2.24

5.18

0.82

N N

CN

N N

CN

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183

4-benzyl-2-phenylpyrimidine-5-carbonitrile (3.4a.3)

-2-101234567891011121314f1 (ppm)

2.21

0.77

1.94

5.00

1.94

0.81

7.58.08.5f1 (ppm)

0.77

1.94

5.00

1.94

N N

CN

N N

CN

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184

2-amino-4-benzylpyrimidine-5-carbonitrile (3.4a.4)

2.01

1.75

5.12

0.81

5.12

NH2

N N

CN

NH2

N N

CN

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185

2-amino-4-tert-butylpyrimidine-5-carbonitrile (3.4i.4)

9.00

2.08

0.78

NH2

N N

CN

NH2

N N

CN

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186

4-tert-butyl-2-phenylpyrimidine-5-carbonitrile (3.4i.3)

-2-101234567891011121314f1 (ppm)

7.57.77.98.18.38.5f1 (ppm)

N N

CN

N N

CN

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187

(E)-N'-hydroxy-4-isobutyl-2-phenylpyrimidine-5-carboximidamide (3.6b.3)

6.00

1.03

2.12

1.92

3.15

2.09

0.92

0.87

1.03

N N

NH2NHO

N N

NH2NHO

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188

4-isobutyl-2-phenylpyrimidine-5-carboxamide (3.10b.3)

N N

NH2O

N N

NH2O

Page 205: Design and Synthesis of Substituted 1,4-Hydrazine-linked ...

189

4-isobutyl-2-phenylpyrimidine-5-carboxylic acid (3.13b.3)

-2-101234567891011121314f1 (ppm)

3.22

3.30

1.12

2.28

3.04

2.00

0.89

0102030405060708090100110120130140150160170180190200210220f1 (ppm)

N N

OHO

N N

OHO

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190

4,4'-diisobutyl-2'-phenyl-2,5'-bipyrimidine-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

N N

N N

CN

N N

N N

CN

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191

4'-benzyl-4-(naphthalen-2-ylmethyl)-2'-phenyl-2,5'-bipyrimidine-5-carbonitrile (3.11af.3)

-2-101234567891011121314f1 (ppm)

N N

N N

CN

N N

N N

CN

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192

4'-benzyl-4-methyl-2'-phenyl-2,5'-bipyrimidine-5-carbonitrile (3.11ag.3)

-2-101234567891011121314f1 (ppm)

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

122124126128130132134f1 (ppm)

N N

N N

CN

N N

N N

CN

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193

2'-amino-4-benzyl-4'-isobutyl-2,5'-bipyrimidine-5-carbonitrile (3.11ba.4)

5.60

1.07

2.01

2.00

1.83

5.57

0.77

0.78

1.07

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

NH2

N N

N N

CN

NH2

N N

N N

CN

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194

5”-cyano-4”-isopropyl-4’-phenylmethyl-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

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

N N

N N

N N

CN

N N

N N

N N

CN

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195

5”-cyano-4,4”-diisobutyl-4’-(2-phenylmethyl)-2-phenylterpyrimidine (3.12bab.3)

-2-101234567891011121314f1 (ppm)

2.202.252.302.35f1 (ppm)

0.850.900.951.001.05f1 (ppm)

N N

N N

N N

CN

N N

N N

N N

CN

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5”-cyano-4,4”-diisobutyl-4’-(2-naphthylmethyl)-2-phenylterpyrimidine (3.12bfb.3)

-2-101234567891011121314f1 (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

0.88

7.47.57.67.77.8f1 (ppm)

2.94

3.28

1.08

3.27

2.22.3f1 (ppm)

2.18

N N

N N

N N

CN

N N

N N

N N

CN

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4'-amino-4-tert-butyl-2',6'-diphenyl-2,5'-bipyrimidine-5-carbonitrile (3.23a.3)

-2-101234567891011121314f1 (ppm)

N N

CNH2N

N N

CNH2N

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tert-butyl 3-aminopropylcarbamate (4.2)

9.00

1.99

2.32

1.78

2.13

0.96

1.78 1.99

NHBocH2N

NHBocH2N

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tert-butyl 3-(2,3-dibenzyloxycarbonylguanidino)propylcarbamate (4.3)

8.75

2.16

2.01

2.08

2.15

2.08

9.41

0.85

0.86

-100102030405060708090100110120130140150160170180190200210220f1 (ppm)

NHCbz

NCbz

NH

BocHN

NHCbz

NCbz

NH

BocHN

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2-bromo-N-(3-(2,3-dibenzyloxycarbonylguanidino)propyl)acetamide (4.4)

NHCbz

NCbz

NH

NH

OBr

NHCbz

NCbz

NH

NH

OBr

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APPENDIX B: X-RAY CRYSTALLOGRAPHIC DATA

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ORTEP diagram for compound 3.2a

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ORTEP diagram for compound 3.4b.3

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ORTEP diagram for compound 3.4i.4

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ORTEP diagram for compound 3.4a.4

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ORTEP diagram for compound 3.4a.5

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ORTEP diagram for compound 3.10a.3

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ORTEP diagram for compound 3.9

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ORTEP diagram for compound 3.12bac.3

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APPENDIX C: QIKPROP CALCULATIONS

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QikProp Calculation Parameters

The following parameters correspond to the overlay of a 4,4’,4”-trimethyl-2,5-

terpyrimidinylene and an octa-alanine and the QikProp calculations for a terphenyl-based

Bcl-xL-Bak inhibitor and a terpyrimidine-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”-trimethyl-

2,5-terpyrimidinylene and an ideal α-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 methyl carbons of the terpyrimidinylene. Out

of 100,000 conformations generated, approximately 5,000 (5%) of the conformations

aligned well with the α-helix. Most of the aligned conformations exhibited the fourth

lowest potential energy calculated by MacroModel with an RMSD of 0.68 Å. 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; Schrödinger, LLC: New York, NY, 2008. 2. Maestro, 8.5; Schrödinger, LLC: New York, NY, 2008. 3. MacroModel, 9.6; Schrödinger, 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 Analytical Method for the Calculation of Approximate Born Radii. Journal of Physical Chemistry, 101, (16), 3005-3014.

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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 Low-Molecular-Weight Antagonists of Bcl-xL. Journal of the American Chemical Society, 127 (29), 10191-10196.

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ABOUT THE AUTHOR

Laura Anderson (Laura León Díaz) was born in Montenegro, Colombia. After

graduating from the University of Quindío 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 bachelor’s degree majoring in chemistry from the University of South

Florida in May 2004 and continued her graduate studies in August 2004. 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.