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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
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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
DEDICATION
With my deepest gratitude,
To my dad (my angel) Luis Delio and my mom María Cenelia
To my love Patrick
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
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.
1
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
2
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
3
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-
4
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
5
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.
6
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
8
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.
10
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
11
domains (SAHB), were reported to have improved pharmacological properties as
compared to the native BH3 peptide (Figure 1.5) (Walensky, et al., 2004; Walensky, et
al., 2006).
Figure 1.5. Depiction of a stapled peptide (Walensky, et al., 2006)
Hamilton and co-workers designed and 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).
12
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
13
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,
14
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
15
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
16
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.
17
Chin, J. W.; Schepartz, A. (2001) Design and evolution of a miniature Bcl-2 binding protein. Angewandte Chemie, International Edition, 40 (20), 3806-3809. Danial, N. N. (2007) BCL-2 Family Proteins: Critical Checkpoints of Apoptotic Cell Death. Clinical Cancer Research, 13 (24), 7254-7263. Davis, J. M.; Truong, A.; Hamilton, A. D. (2005) Synthesis of a 2,3';6',3''-Terpyridine Scaffold as an alpha -Helix Mimetic. Organic Letters, 7 (24), 5405-5408. Domling, A. (2008) Small molecular weight 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.
22
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-
23
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).
24
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
25
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.
26
Figure 2.3. Retrosynthetic analysis of 3-R-piperazine-2,6-dione scaffold DKPA
27
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
28
methanol since both removal of the Boc group and formation of the ester occurred in a
one pot reaction. Methyl ester derivatives 2.2e-g were obtained as the HCl salts in
excellent yields (>90%).
In the next step, the methyl esters of the selected natural and unnatural amino
acids 2.2a-g were N-alkylated with ethyl bromoacetate (EBA) in the presence of 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
29
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
30
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
31
its decomposition by hydrolysis (Avancha, 2006). Oxaziridines are also common
reagents for oxygen transfer; in fact, the side product hydroxylamine 2.6a was isolated
and characterized (2.6a in Table 2.1). The hydrazine of 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
32
hydrolyzed to the diacid with 1N NaOH in MeOH. NaOH solutions were freshly
prepared to prevent prolonged contact with CO2 and consequent decrease of its basicity
by formation of carbonate species (Southgate, 2000). Treatment of more hindered amino
acid derivatives, such as valine (2.5c) and isoleucine (2.5d), with 1N NaOH resulted in a
partial hydrolysis to the monoacid. Although further attempts to hydrolyze with NaOH
were also successful without 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.
33
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
34
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.
35
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).
36
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
37
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.
38
Scheme 2.4. Synthesis of DKP1 monomer 2.13b
While it was reported that this acetic anhydride-mediated reaction afforded the
best results, a persistent problem was the concomitant acylation of the nitrogen of the
amide (Scheme 2.4, compound 2.14b’). Acylation of the amide, therefore, prevents
closure of the ring. In efforts to overcome this issue, a second route for the synthesis of
the target DKP units was developed.
39
2.2.4 Synthesis of Piperazine-2,6-diones: Route 2
As described in Section 2.2.1, the main difference between Routes 1 and 2 to
form the DKP monomer is the formation of an 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)
40
instead of EBA as in Route 1 (Scheme 2.1). Subsequent treatment of diesters 2.16 with
N-Boc oxaziridine 2.4 yielded Boc-protected hydrazines 2.17 in good yields (80-86%).
In the next step, removal of the benzyl group was achieved via hydrogenolysis with 5%
Pd/C in THF at 35 psi and at rt. It is 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
41
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.
42
2.2.5 Synthesis of Piperazine-2,5-diones: Route 1
Figure 2.5. Retrosynthetic analysis of 3-R-piperazine-2,5-dione scaffold DKPB
43
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
44
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
45
2.29 under basic conditions (Scheme 2.9). It should be noted that 2.31f does not contain
the N-Boc protected hydrazine, as we 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.
46
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
47
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
48
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
49
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.
50
(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):
51
The HCl salt of amino ester 2.2a (4.63mmol) was dissolved in anhydrous acetonitrile (20
mL). Then diisopropylethylamine (9.26 mmol) and ethyl bromoacetate (6.94 mmol)
were added, while the reaction mixture was kept under an argon atmosphere; then it was
stirred at rt for 24 h. The reaction mixture was quenched with 5% citric acid (5 mL) and
it was extracted with ethyl acetate (3 x 15 mL). The combined organic layers were
washed with brine (2 x 15 mL), dried over Na2SO4, and evaporated in vacuo to yield
compound 2.3a as a colorless crude oil.
(S)-methyl 2-(2-ethoxy-2-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),
52
1.34 (s, 6H), 3.35 (s, 2H), 3.68 (s, 3H), 4.17 (q, J = 7.2 Hz, 2H). 13C NMR (100 MHz,
CDCl3) 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,
53
128.55, 129.06, 130.21, 137.05, 157.77, 171.46, 173.99. ESI-MS calcd. for C21H26NO5
[M+H]+ 372.18, found 372.18.
Representative procedure for the synthesis of 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,
54
128.60, 129.46, 137.31, 172.06. ESI-MS calcd. for C19H28N2NaO6 [M+Na]+ 403.18,
found 403.18.
Representative procedure for the synthesis of 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.
55
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):
56
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
57
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.
58
(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):
59
2.17d was prepared following the procedure described for 2.17a and it was purified by
column chromatography on silica gel (hexanes:ethyl acetate, 7:3), yield: 62%, thick
colorless oil. 1H NMR (400 MHz, CDCl3) 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,
60
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,
61
69.88, 81.11, 127.08, 128.71, 129.35, 137.38, 156.14, 169.36, 171.34, 172.10, 172.97.
ESI-MS 438.22[M+H]+.
(S)-tert-butyl 2-(2-(3-methoxy-3-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
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
63
(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.
64
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
65
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
66
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]+.
67
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
68
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
69
obtain a light yellow solid crude, which was used in the next step without further
purification.
Step 2: The TFA salt obtained in step 1 (0.96 mmol) was suspended in anhydrous DCM
(2 mL) and cooled to 0 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.
70
Note: The aqueous layer was also extracted with DCM and results were comparable
based on product yield and purity.
(S)-methyl 2-(2-bromoacetamido)-3-methylbutanoate (2.34c):
Isolated yield: 83 %, crystalline white solid, m.p. = 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.
71
methyl 2-(2-bromoacetamido)-2-methylpropanoate (2.34d):
2.34d was prepared following the procedure described for 2.34a. Isolated yield: 52%,
white solid. 1H NMR (400 MHz, CDCl3) 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):
72
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).
2.5 References Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. (1996) Reductive Amination of Aldehydes and Ketones with Sodium Triacetoxyborohydride. Studies on Direct and Indirect Reductive Amination Procedures. Journal of Organic Chemistry, 61 (11), 3849-3862. Anderson, L.; Topper, M.; Jain, P.; McIntosh, E.; McLaughlin Mark, L. (2009) Synthesis of N-Boc protected hydrazine diacids as key structural units for the formation of alpha-helix mimics. Advances in experimental medicine and biology, 611, 211-2. Avancha, K. K. V. R. (2006) Design and synthesis of core structural intermediates for novel HIV-1 protease inhibitors and synthesis, biological activity and molecular modeling of novel 20S proteasome inhibitors. Dissertation, University of South Florida.
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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
80
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,
82
2004). In general, the pyrimidine ring can be prepared directly from cyclization of
different aliphatic fragments, rearrangements, ring contraction or expansion of other ring
systems, or modification of substituents 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
83
reported for their electrochemical properties and a few for highly functionalized dimeric
pyrimidines, which have been prepared via Stille and Suzuki couplings (Brandl, et al.,
1996; Gompper, et al., 1996; Gompper, et al., 1997; Saygili, et al., 2004). Thus, our
novel non-peptidic scaffold (Figure 3.3), which is designed to mimic a specific 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
86
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
87
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).
89
Scheme 3.4. Methods for the conversion of cyano group to amidine
90
Table 3.2. Attempted routes to synthesize 5-carboxamidines
91
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
92
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
96
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
98
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
103
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
105
significantly decreased, most likely due to the absence of substituents flanking the cyano
group of compound 3.16.
Scheme 3.7. Synthesis of compound 3.19a
The intermediate amidoxime 3.17 was obtained as a triethylamine salt and further
reduction gave intermediate amidine carboxylate salt, which was also used in the
condensation step with 3.3a without further purification. 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).
107
Figure 3.10. Target 4-R,6-R-2,5-terpyrimidinylene. R1, R2, and R3 = selected alkyl or aryl groups specific to mimic the ith, ith+4, and ith+7 residues of an 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
108
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
110
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),
112
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.
113
(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):
114
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,
115
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.
116
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.
117
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.
118
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
119
3.4a.5 was prepared following method A using 2-chloroamidine hydrochloride and
anhydrous dioxane instead of ethanol. It was purified by column chromatography on
silica gel (hexanes:ethyl acetate, 4:1), yield: 18%, white solid. 1H NMR (400 MHz,
CDCl3) 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
120
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.
121
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,
122
(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):
123
3.6b.3 was purified by column chromatography in silica gel (hexanes: ethyl acetate 3:2),
70% yield, white solid, m.p. = 125-127 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.
124
To this residue, DCM (6 mL) was added and the white precipitate that was formed was
removed by vacuum filtration. The filtrate was concentrated under reduced pressure to
obtain the crude acetate salt of carboxamidine as a yellow solid, which was used without
further purification in the next step.
Step 3: To the crude carboxamidine salt 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,
125
128.96, 131.46, 137.44, 159.92, 160.22, 164.66, 170.29, 173.45. HRMS (ESI) calcd. for
C23H26N5 [M + H]+ 372.2188, found 372.2208.
4'-benzyl-4-(naphthalen-2-ylmethyl)-2'-phenyl-2,5'-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):
126
3.11aa.3 was prepared following the procedure described for 3.11ba.3. It was purified
by column chromatography on silica gel (hexanes:ethyl acetate, 4:1), yield: 40% after 3
steps, white solid. 1H NMR (400 MHz, CDCl3) 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):
127
3.11ba.4 was prepared following the procedure described for 3.11ba.3. Isolated yield:
46% after 3 steps, buff solid, m.p. = 184-186 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.
128
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
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,
130
131.14, 137.77, 138.25, 159.55, 160.16, 160.53, 164.76, 169.02, 169.99, 178.80. HRMS
(ESI) calcd. for C33H32N7 [M + H]+ 526.2719, found 526.2707.
5”-cyano-4,4”-diisobutyl-4’-(2-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):
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
132
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.
133
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
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
135
Compound 3.22b.3 was purified by column chromatography on silica gel (hexanes:ethyl
acetate, 7:3), yield: 74%, white solid. 1H NMR (400 MHz, CDCl3) 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
136
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.
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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.
144
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).
145
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
148
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
149
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.
151
Table 4.1. Acylation conditions for the synthesis of guanidine 4.4
The formation of the stable six-membered ring of 4.5b was observed to increase
when stronger basic conditions and temperatures above 0 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).
152
Figure 4.7. S-alkylated CPNA monomers
These compounds (Figure 4.7, compounds a-h) were prepared by other members
of our lab (Dr. Sung Wook Yi and Priyesh Jain) and subsequent research will disclose if
the presence of this positively charged group R1, as part of the CPNA backbone, assists
on the delivery and uptake of CPNA to cellular targets.
4.3 Conclusion
The synthesis of a guanidine-derived alkyl bromide for the alkylation of the S-
terminus of novel CPNA monomers was 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):
153
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,
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)
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.
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.
158
APPENDIX A: SELECTED 1H AND 13C NMR SPECTRA
159
Methyl 2-amino-2-methylpropanoate hydrochloride (2.2e)
6.00
2.74
H2NO
O
H2NO
O
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
161
benzyl 3-(tert-butoxycarbonylamino)propanoate (2.10’)
-101234567891011121314f1 (ppm)
-100102030405060708090100110120130140150160170180190200210220f1 (ppm)
O
O
NHBoc
O
O
NHBoc
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
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
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
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
166
(S)-benzyl 2-(2-methoxy-2-oxoethylamino)-3-phenylpropanoate (2.22a)
HN
O
O
O
O
HN
O
O
O
O
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
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
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
170
(S)-methyl 2-(benzylamino)-3-phenylpropanoate (2.26a)
-2-1012345678910111213f1 (ppm)
-100102030405060708090100110120130140150160170180190200210220f1 (ppm)
HN
O
O
HN
O
O
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
172
(E)-2-(2-tosylhydrazono)acetic acid (2.29’)
-101234567891011121314f1 (ppm)
-100102030405060708090100110120130140150160170180190200210220f1 (ppm)
SNH
ON
O O
OH
SNH
ON
O O
OH
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
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
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
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
177
3-oxo-4-phenylbutanenitrile (3.2a)
OCN
OCN
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
179
(E/Z)-2-((dimethylamino)methylene)-3-oxo-4-phenylbutanenitrile (3.3a)
OCN
N
OCN
N
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
181
4-benzylpyrimidine-5-carbonitrile (3.4a.1)
N N
CN
N N
CN
182
4-benzyl-2-methylpyrimidine-5-carbonitrile (3.4a.2)
2.99
2.24
5.18
0.82
N N
CN
N N
CN
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
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
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
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
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
188
4-isobutyl-2-phenylpyrimidine-5-carboxamide (3.10b.3)
N N
NH2O
N N
NH2O
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
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
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
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
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
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
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
196
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
197
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
198
tert-butyl 3-aminopropylcarbamate (4.2)
9.00
1.99
2.32
1.78
2.13
0.96
1.78 1.99
NHBocH2N
NHBocH2N
199
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
200
2-bromo-N-(3-(2,3-dibenzyloxycarbonylguanidino)propyl)acetamide (4.4)
NHCbz
NCbz
NH
NH
OBr
NHCbz
NCbz
NH
NH
OBr
201
APPENDIX B: X-RAY CRYSTALLOGRAPHIC DATA
202
ORTEP diagram for compound 3.2a
203
204
ORTEP diagram for compound 3.4b.3
205
206
207
208
ORTEP diagram for compound 3.4i.4
209
ORTEP diagram for compound 3.4a.4
210
211
212
ORTEP diagram for compound 3.4a.5
213
214
215
216
217
ORTEP diagram for compound 3.10a.3
218
219
220
221
222
ORTEP diagram for compound 3.9
223
224
225
ORTEP diagram for compound 3.12bac.3
226
227
228
229
230
231
APPENDIX C: QIKPROP CALCULATIONS
232
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.
233
5. Delano, W. L. The PyMol Molecular Graphics System, Delano Scientific: Palo Alto, CA, 2002.
6. Yin, H.; Lee, G.-i.; Sedey, K. A.; Kutzki, O.; Park, H. S.; Orner, B. P.; Ernst, J. T.; Wang, H.-G.; Sebti, S. M.; Hamilton, A. D. (2005) Terphenyl-Based Bak BH3 alpha -Helical Proteomimetics as Low-Molecular-Weight Antagonists of Bcl-xL. Journal of the American Chemical Society, 127 (29), 10191-10196.
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.