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Synthesis of protein-protein interaction inhibitors and development of new catalytic methods

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Synthesis of protein-protein interaction inhibitors and development of new catalytic methods

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SYNTHESIS OF PROTEIN-PROTEIN INTERACTION INHIBITORS AND

DEVELOPMENT OF NEW CATALYTIC METHODS

by

Jamie A. Jarusiewicz

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPY
(CHEMISTRY)

August 2012

Copyright 2012 Jamie A. Jarusiewicz
ii

Dedication

To Mom, Dad, and Holly.

iii

Acknowledgments
I would like to thank Professor Nicos Petasis, my advisor, for the guidance and
inspiration he shared with me. Thank you for giving me the opportunity to learn how to think
about science in new ways and for teaching me to appreciate the complexities of deceptively
straightforward situations.
I thank Professor Kyung Jung, who I am grateful to for having been able to start my
graduate career with and for emphasizing the importance of discipline and determination.
A special thank you to my qualifying exam and dissertation committee members,
Professors Daryl Davies; G.K. Surya Prakash; Richard Brutchey; and Nouri Neamati for their
time and helpful discussions. I also am greatly appreciative of the technical assistance and advice
Professors Matt Pratt and Travis Williams have provided.
Thank you to all past and present members of the Petasis group for making the lab a
pleasant environment for research: Kevin Gaffney; who pushed me (literally) when needed,
Jeremy Winkler, Min Zhu, Anne-Marie Finaldi, Charles Arden, Marcos Sainz, Dave Rosenberg,
Nikita Vlasenko, Steve Glynn, Dr. Kalyan Nagulapalli, Dr. Kenny Young, and Dr. Alex
Butkevich. Thanks also to the members of Prof. Lin Chen’s lab, Nimanthi and Xiao, who worked
with us. I also thank members of the Jung group with whom I was fortunate to work. Dr. Yoo,
you are greatly missed. I thank the friends I have made throughout the department for friendly
chats over coffee and never turning me away when I was searching for chemicals to “borrow.”
I thank the support staff of LHI and USC Department of Chemistry: Carole Phillips,
Jessy May, David Hunter, Dr. Robert Anizfeld, Michele Dea, and Katie McKissick.
Lastly, I thank my family for their continual support and encouragement. Without them,
this would not have been possible and I am forever grateful.

iv

Table of Contents
Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures vii
List of Schemes ix
Abstract xii
Chapter 1. Introduction. Protein-protein interactions and their therapeutic
potential. 1
1.1. The molecular structure of protein-protein interactions and the
challenges they present for drug development. 1
1.2. Techniques for characterizing protein-protein interactions and
development of their inhibitors. 4
1.3. Chapter 1. References 9

Chapter 2. Design and synthesis of inhibitors of the MEF2/HDAC
interaction. 11
2.1. Introduction. The role of myocyte enhancer factor (MEF2) as a
cellular regulator. 11
2.1.1. The role MEF2 family of transcription factors in signaling
pathways. 11
2.1.2. The association of MEF2 with activators and repressors. 13
2.1.3. MEF2-mediated cellular processes. 14
2.1.4. Inhibition of Histone Deacetylase (HDAC) activity. 15
2.1.5. Structure of MEF2 and implications for small molecule
inhibition. 16
2.2. Synthesis and evaluation of inhibitors of the MEF2/HDAC interaction. 17
2.2.1. Development of inhibitors. 17
2.2.2. Synthesis and investigation of capping regions. 21
2.2.3. Synthesis and evaluation of modified linker region. 29
2.3. Synthesis and evaluation of inhibitors containing reversed amide
bond orientation. 34
2.3.1. Overview of synthetic routes towards inhibitors. 34
2.3.2. Evaluation of linker length. 36
2.3.3. Synthesis and investigation of capping regions. 37
2.3.4. Synthesis and evaluation of aromatic linker. 45
v

2.3.5.
19
F NMR analysis of small molecule/MEF2 binding. 51
2.4. Synthesis of MEF2/HDAC interaction inhibitors related to the natural
product Trichostatin A. 53
2.4.1. Biological activity and prior syntheses of trichostatin A. 53
2.4.2. Synthesis of novel compounds related to trichostatin A. 56
2.5 Synthesis of helix-constrained peptides as MEF2 inhibitors. 61
2.5.1. Structure and biology of constrained peptides. 61
2.5.2. Copper assisted click chemistry cyclization method. 63
2.5.3. Hydrocarbon stapled method. 68
2.6. Experimental. 70
2.7. Chapter 2. References 191

Chapter 3. Synthesis and SAR study of benzimidazoles as inhibitors of
Bcl-2/Bcl-xL. 200
3.1. Introduction. The role of Bcl-2/Bcl-xL in apoptosis. 200
3.2. Design of benzimidazole core scaffold and analog synthesis. 202
3.3. Experimental. 207
3.4. Chapter 3. References 231

Chapter 4. Palladium-assisted reaction methodologies. 232
4.1. Introduction. Palladium catalyzed reactions. 232
4.1.1. N-heterocyclic carbene ligands in palladium catalysis. 232
4.1.2. Multicomponent Strecker synthesis of α-aminonitriles. 234
4.2. Regioselective Heck coupling of aryl halides and dihydropyran
using a NHC-Pd catalyst. 235
4.3. Strecker reaction using an NHC-amidate-Pd complex 243
4.4. Experimental. 248
4.4.1. Heck coupling methodology. 248
4.4.2. Strecker synthesis methodology. 256
4.5. Chapter 4. References. 267

Conclusions 274

Bibliography 275

Appendix: Selected spectra 292

vi

List of Tables
Table 4.1 Effect of catalyst and base on the cross-coupling reactions
of 4-iodoanisole and DHP 238

Table 4.2 Effect of solvent and temperature on the cross-coupling
reactions of 4-iodoanisole and DHP in the presence of
Pd-ligand complex 4.5 239

Table 4.3 Effect of arylhalide on the cross-coupling reactions with
DHP in the presence of Pd-ligand complex 4.5 240

Table 4.4 Effect of aryl halide on the cross-coupling reactions with
DHP in the presence of Pd-ligand complex 4.5 241

Table 4.5 Screening of Palladium Source and Catalyst Loading

245

Table 4.6 Strecker Reaction of Aldehydes and Amines in the
Presence of Palladium Catalyst 246

Table 4.7 Strecker Reaction of Ketones and Amines in the Presence
of Palladium Catalyst 247

vii

List of Figures

Figure 2.1 Amino acid sequence similarity of MEF2 proteins 11

Figure 2.2 Overview of the interaction of MEF2 with activators
and repressors 13

Figure 2.3 Represetative benzamides and hydroxamic acids previously
studied due to their ability to inhibit HDAC-related activites 16

Figure 2.4 Interactions between MEF2 and HDAC9 17

Figure 2.5 General structure of interest comprised of capping Region A, a
central linker, and capping Region B 18

Figure 2.6 Compounds with phenyl ring in Region A, a five carbon
aliphatic linker, and varied functional groups in Region B 22

Figure 2.7 Compounds containing a meta-bromo substituent in Region A,
a five carbon aliphatic linker, and varied functional groups in
Region B 23

Figure 2.8 Compounds containing pyridine functionality 23

Figure 2.9 Biphenyl-containing compounds 24

Figure 2.10 Compounds with varied meta-substitution in Region A 25

Figure 2.11 Fluorine-containing compounds 26

Figure 2.12 Structure of trichostatin A. 27

Figure 2.13 Evaluation of correlation between linker length and percent
Inhibition 30

Figure 2.14 Compounds containing a six-carbon aliphatic linker 30

Figure 2.15 Reversed orientation of the amide bond in the general scaffold
design 35

Figure 2.16 Investigation of aliphatic linker length 37

Figure 2.17 Compounds with varied meta- or para- substitution 37

Figure 2.18 Compounds containing N,N-dimethyl in para-position of
Region A 38

viii

Figure 2.19 Compounds including heterocyclic rings in Region A 40

Figure 2.20 Compounds with pyrrole or indole in Region A 43

Figure 2.21 Additional indole-containing compounds 43

Figure 2.22 Compound comprised of indole in Region A and extended linker 43

Figure 2.23 Invesigation of benzimidazole in Region A 44

Figure 2.24 Compounds with an aliphatic linker containing carbamate 44

Figure 2.25 Compounds containing a central aromatic ring 46

Figure 2.26 Variations in linker length between Region A indole and
aromatic linker 50

Figure 2.27 Shift in CF
3
resonance upon small molecule binding to MEF2 52

Figure 2.28 Structure of helix-constrained peptide synthesized by
olefin metathesis cyclization 70

Figure 3.1 Structure of ABT-737 201

Figure 3.2 Interaction of the hydrophobic BH3 binding groove of
Bcl-2 with an acylsulfonamide compound developed by a group
at Abbott Labs 201

Figure 3.3 Phenyl-benzimidazole core structures with variable
linkages/wing moieties 202

Figure 3.4 Compounds containing biphenyl right-wing substituents and
varied functionality on the left-wing 204

Figure 3.5 Compounds containing identical left and right-wing substituents 204

Figure 3.6 Compounds containing a 4-chloro benzoyl substituent fixed on
the right wing 205

Figure 3.7 Variations in the left-wing region 206

Figure 3.8 Additional variations in the left-wing region 206

Figure 4.1 Mechanism of a typical Heck cross-coupling reaction 233

Figure 4.2 New NHC-amidate palladium complexes 243

ix

List of Schemes

Scheme 2.1 General synthetic route starting with a dicarboyxlic acid 19

Scheme 2.2 Alternative synthetic route used to install ortho-anilide first 20

Scheme 2.3 Alternative synthetic route 21

Scheme 2.4 Synthesis of 2.34 and 2.35 employing Suzuki coupling
reactions 24

Scheme 2.5 Suzuki reaction to synthesize a hybrid of 2.39 and 2.41 26

Scheme 2.6 Inclusion of N,N-dimethylaniline functionality 27

Scheme 2.7 Dimethylation of 2.48 to provide 2.49 27

Scheme 2.8 Formylation of 2.50 to give 2.51 28

Scheme 2.9 Synthesis of carboxamide 2.54 28

Scheme 2.10 Synthesis of 2.56 29

Scheme 2.11 Synthesis of 2.66 31

Scheme 2.12 Unsuccessful route towards compound containing
piperazine core 31

Scheme 2.13 Unsuccessful synthetic route towards a piperazine-
containing compound 32

Scheme 2.14 Attempted route towards synthesis of piperazine-
containing compound 32

Scheme 2.15 Synthesis of 2.76 33

Scheme 2.16 Synthesis of 2.79 33

Scheme 2.17 Synthesis of 2.85 34

Scheme 2.18 General synthetic route for compounds with reversed amide
bond orientation 35

Scheme 2.19 Second strategy to synthesize compounds with reversed
orientation of the amide bond between Region A and the
aliphatic linker 36

x

Scheme 2.20 Synthesis of compound with reversed functional group
substitutions 39

Scheme 2.21 Initial attempt to synthesize 2.121 41

Scheme 2.22 Alternative route to obtain 2.123 41

Scheme 2.23 Synthesis of trifluoromethyl-functionalized carboxylic acid 42

Scheme 2.24 CDI-promoted coupling to synthesize intermediate 2.139 44

Scheme 2.25 Synthesis of 2.144 45

Scheme 2.26 Synthesis of trifluoromethylated compound 2.146 46

Scheme 2.27 Synthesis of 2.154 47

Scheme 2.28 Alternative strategy to install ortho-amine functional group 48

Scheme 2.29 Alternative plan used to install ortho-amine in last step 48

Scheme 2.30 Synthesis of 2.165 49

Scheme 2.31 Synthesis of 2.172 50

Scheme 2.32 First reported route for synthesis of trichostatin A (2.178)
in its racemic form 54

Scheme 2.33 Retrosynthesis use for first total synthesis of trichostatin A in
enantiopure form 55

Scheme 2.34 Retrosynthetic route implemented by Chatterjee et al 56

Scheme 2.35 Retrosynthetic route to obtain trichostatic acid based upon
work by Zhang et al 57

Scheme 2.36 Route which provided an unseparable mixture of isomers 58

Scheme 2.37 Wittig reactions to furnish diene 2.197 58

Scheme 2.38 Approach to 2.188 following Zhang et al. 59

Scheme 2.39 Synthesis of trichostatic acid (2.187) 59

Scheme 2.40 Synthesis of ortho-anilidine derivative of trichostatin A 60

Scheme 2.41 Synthesis of carboxamide of trichostatin A 61

xi

Scheme 2.42 Unsuccessful route towards N
α
-Fmoc-Abu(γ-N
3
)-OH 64

Scheme 2.43 Alternative synthesis of azide-functionalized amino
acid building block 64

Scheme 2.44 Modified diazotransfer reaction 65

Scheme 2.45 Synthesis of helix-constrained peptide using CuACC cyclization 66

Scheme 2.46 Use of Ala-Ni-BPB to synthesize α-methyl-α-amino acid 69

Scheme 3.1 Synthetic route for a variety of substituted phenyl-
benzimidazole analogues 203

Scheme 3.2 Synthetic route for analogues containing a sulfonamide linker 207

Scheme 4.1 Classical Strecker synthesis to form α-amino acids from
α-aminonitriles 234

Scheme 4.2 Synthetic route for preparation of NHC-ligand Pd-catalyst
complex 236

Scheme 4.3 Coupling reaction with hindered halides 242

Scheme 4.4 Coupling reaction with heterocyclic halide 242

Scheme 4.5 Synthesis of NHC-amidate palladium complex 4.21 for use in
Strecker synthesis 244

xii

Abstract
This dissertation comprises three projects, two related to the synthesis and evaluation of
small molecule inhibitors of protein-protein interactions, and one related to development of
palladium-assisted reaction methodologies.
The introduction (Chapter 1) briefly provides an overview of the challenges associated
with developing small molecule inhibitors of protein-protein interactions in relation to drug
discovery efforts.
Chapter 2 reviews the biological importance of the transcription factor MEF2 and details
its interaction with HDACs. The synthesis of a diverse set of over 100 molecules, including
compounds structurally related to the natural product trichostatin A, aimed at directly targeting
MEF2 is described. Evaluation of small molecule ligand/protein binding via
19
F NMR
spectroscopy is presented. The synthesis of helix-constrained peptides for investigation of their
interaction with MEF2 is discussed as well.
Chapter 3 describes the role of Bcl-2/Bcl-xL in apoptosis and cancer treatment. The
construction of a small molecule of benzimidazole-based inhibitors for investigation of their
structure-activity relationship is discussed.
Chapter 4 provides a brief description of the utility of Heck and Strecker reactions in
organic synthesis. Novel methodologies using palladium-promoted chemistry to form arylated
cyclic enol ethers and α-aminonitriles is described. The use of palladium-catalysis to achieve
selective oxidations is also briefly presented.

1

Chapter 1. Introduction. Protein-protein interactions and their
therapeutic potential.

1.1. The molecular structure of protein-protein interactions and the
challenges they present for drug development.
Biological processes often depend upon complex interactions between cellular
components. Functioning within these networks used for cellular communication and regulation
are protein-protein interactions, which allow for proper maintenance of living systems. In disease
states, however, cellular controls may not function properly. To ameliorate this, protein-protein
interactions present an interface which may be used as a target for development of thereapeutics
due to their ability to influence a diversity of signaling and metabolic pathways. This approach is
one that is challenging for many reasons, however, including the large size of the interface
involved in these interactions and the numerous cellular processes which a singular protein can
impact.
Despite this challenge, protein-protein interactions have attracted much interest because
of their central role in numerous biological processes and many structural, biophysical, and
chemical techniques have been developed to embark upon these studies. The characterization of
protein-protein interfaces can be used to elucidate key features in these interactions and the
evaluation of size, shape, and complementarity of protein surfaces can provide information into
intricate structures. Determining whether certain amino acids or structural motifs are important
for specific protein interactions and evaluating the strength of different binding contacts can
allow for insight towards the influence of molecular recognition on critical cellular functions.
Typically, the regions involved in protein-protein interactions are large, flat surfaces
comprised of many amino acid residues and, in general, these regions tend not to associate with
small molecule binding partners. This provides a challenge for structure-based drug design
2

efforts since a small molecule natural ligand to use for an initial scaffold design does not exist.
Most protein-protein interfaces are within the range of 1200-2000Å
2
, with short-lived and
unstable complexes having interfaces found on the lower end of this range, while interactions
involving some proteases and have been found to occupy even larger regions of nearly 5000Å
2
.
1

This is in stark contrast to the areas involved in the interactions between most small molecules
and proteins, which is usually within the range of 300-1,000Å
2
.
2

The size of protein interfaces can also be described in terms of their solvent accessible
surface area to more accurately measure regions able to form complexes. Evaluation of the
change in the solvent accessible surface area upon complexation can provide information about
how much of a protein structure becomes buried in this process. It has been found that in both
homodimerization and heterodimerization interactions the change in solvent accessible surface
area can be as great as several thousand Å
2
.
3
The shape of the portions of proteins involved in
binding interactions can also vary from two relatively flat surfaces to twisted interfaces.
Therefore, due to the large protein interfaces involved in protein-protein interactions and the
complexity of their conformations, targeting these interactions for the development of therapeutic
agents has been considered a formidable challenge.
Learning about the specific amino acid composition of protein binding interfaces can also
provide valuable information for drug design. The interfaces that make up protein-protein
interactions are often highly hydrophobic. Contacts between non-polar amino acid residues and
van der Waals interactions allow for these hydrophobic interactions to occur and tight packing
and burying of residues can occur upon binding, due to an energy profile favoring the movement
of these residues from an aqueous environment to one which is more non-polar.
4
Although
hydrophobic interactions provide a force which promotes protein-protein association, electrostatic
forces are important as well. Despite being rich in hydrophobic residues, many of the interfaces
3

involved in protein-protein interactions are similar to regions typically found on protein surfaces
rather than their interiors. Polar and charged amino acid residues can permit the formation of
stable complexes through ion pairing and the existence of salt bridges.
5
Additonally, large
amounts of hydrogen bonding contacts between the protein surfaces and the surrounding aqueous
environment promote protein-protein interactions.
1
In combination, these forces can impact the
rates for complex formation as well as the length of time the proteins associate. Evaluation of the
interactions between proteins has provided insight regarding the complementarity of the partner
surfaces in terms of their size and the packing density of the atoms involved.
6
This has allowed
for the development of filters useful in the creation of protein-protein docking algorithms to more
reliably investigate potential regions of interest in binding.
7
A combination of molecular
structure evaluation and computational strategies that consider structure complementarity can
therefore be useful tools in designing and optimizing small molecules for drug development.
Despite the large size of protein-protein interfaces it has been found that many protein-
protein interactions rely upon only small regions of these interfaces that form higher affinity
binding interactions, known as hot spots.
8
The development alanine scanning mutagenesis as a
technique to determine the contributions of individual amino acid residues in the overall energy
profile of a protein-protein complex has allowed for discernment of locations of important
binding sites within larger structures.
9
In this analysis, each amino acid residue in the region of
interest is systematically mutated to alanine and the change in binding energy upon complexation
is then compared to that of the wild-type complex. This allows for the determination of residues
that provide important interactions as well as those which do not significantly contribute to
binding energies. Although the alanine scanning mutagenesis technique can require a large
experimental effort, its utility in assessing the energetics at protein interfaces has prompted the
development of additional biophysical techniques, such as directed evolution, to reduce the
4

necessary experimental labor involved but still obtain valuable information.
10
Through the use of
these methods, it has been found that hot spot residues are often found in central part of the
binding site and are surrounded by less important residues; however, example of protein-protein
interfaces where crucial residues are located at the periphery, as well as those completely lacking
identifiable hot spots, have also been discovered.
8

When two proteins interact, many different amino acids can be important for binding,
even within hot spots themselves. Although certain residues may make more contacts than others
in a complex, there may be an uneven distribution of energy associated with these residues.
11

Additionally, although one type of amino acid may be discovered to provide a criticial interaction
in one protein-protein complex, this same interaction may be unimportant in another complex.
This highlights the importance of critically evaluating the interactions key in the particular
protein-protein complex of interest in the development of new therapeutics using structure-based
design. Interestingly, however, many proteins use the same amino acid residues within hot spots
to bind to multiple partners.
12
This indicates that small molecule inhibitors may be used to
effectively disrupt protein-protein interactions by focusing efforts upon specific regions for
structural design.
1.2. Techniques for characterizing protein-protein interactions and
development of their inhibitors.
Structural, biophysical, and chemical methods can be used in combination to understand
mechanisms of binding interactions. With the knowledge gained from these analyses,
development of small molecule therapeutics may not be as daunting a task as initially considered.
Within the 650,000 protein-protein interactions estimated to be found within humans, there are
bound to be a vast number of interactions amenable to inhibition by small molecules.
13

5

To find and develop small molecule inhibitors of protein-protein interactions,
qualititative biophysical assays can be used during compound screening. These techniques can be
used not only to identify binding but also to evaluate kinetics. Analytical ultracentrifugation,
often used to measure the ability of biomolecules to associate in solution, is a valuable method
that can also be employed to detect if a small molecule can bind to a particular protein.
12
This
method is used to determine the molecular weight of a species of interest after several hours of
high speed centrifugation through interpretation of its sedimentation profile as detected optically.
Binding stoichiometry can be estimated using analytical centrifugation, which is also useful for
evaluating whether aggregation occurs, a common mechanism of inhibition found to occur with
hydrophobic small molecules.
14
Surface plasmon resonance (SPR) is another technique used to
measure binding interactions between two molecules in which one is immobilized on a surface
and another is in solution.
15
This method uses refractivity of polarized light to verify ligand
association and can be used to gain a wealth of information about binding mechanisms, such as
equilibrium constants, rates of association and dissociation, and changes in free energy upon
binding. Such information about molecule binding can therefore be used to learn about relevant
interactions necessary for this to occur, which is useful in initial stages of therapeutic
development.
More detailed levels of characterization of protein-protein and protein-ligand interactions
can be obtained through application of x-ray crystallography and NMR spectroscopy. X-ray
crystallography is widely used for determining protein structures at the atomic level and allows
for analysis of features present at protein binding interfaces. This technique has been widely
implemented in pharmaceutical research due to the high resolution structural analyses x-
crystallography can permit.
16

6

A protein is flexible, however, and although x-ray crystallography can provide a detailed
structural model, it is important to consider that the structure obtained may in reality not be as
precisely ordered as it may seem. Although x-ray crystallography can help locate binding sites,
NMR spectroscopy can also be used for this type of structure elucidation. Additionally, NMR
can permit the investigation of dynamic perturbations that occur in the presence of ligands. One
particular NMR technique that has been demonstrated to provide detailed structural insight is the
1
H-
15
N heteronuclear single quantum correlation (HSQC) experiment.
12
Using isotopically
labeled protein, the NMR resonances associated with the protein backbone can be evaluated.
These peaks are sensitive to alterations in the local environment and can be used to detect effects
from ligand binding and conformational changes. Structure-activity-relationship (SAR) by NMR
has emerged as a powerful method for identifying and optimizing structures that bind to a site of
interest on a protein.
17
A combination of various experimental techniques can therefore be used to
learn about the interfaces involved in protein-protein interactions to shed light upon important
structural features that can guide the development of small molecule therapeutic agents.
In fact, this collection of biogical and chemical tools has been used to successfully
develop small molecule inhibitors of protein-protein interactions in the past. These small
molecules have been found to disrupt both extracellular and intracellular protein-protein
interactions.
18
Among the first small molecules that were identified as inhibitors of protein-
protein interactions were those that interacted with integrins. Located on the cell surface,
integrins act as adhesion receptors and are important in cell-cell communication. It was found
that the presence of an RGD motif in protein ligands was recognized for binding, and attempts to
mimic this scaffold lead to the successful design of compounds from a variety of chemical classes
that were abole to disrupt the interaction of the integrin receptor with its protein ligands.
19
This
7

consequently demonstrated the strategy of using a small portion of a relevant natural ligand to
initiate the design of a small molecule inhibitor.
Another example of a small molecule capable of inhibiting an extracellular protein-
protein interaction is Ro 26-4550, which was designed as a peptidomimetic of the cell signaling
cytokine IL-2.
20
Using both x-ray crystallography and HSQC NMR to evaluate protein structures
in the presence and absence of this compound, not only was the small molecule binding site
found, but drastic changes in protein conformation upon ligand binding were observed too.
21

Notably, this provided evidence of the binding site’s ability to adapt to its ligand and highlighted
the importance of being able to evaluate a dynamic structure versus considering a protein as a
static entity.
21

An added layer of difficulty emerges for the development of inhibitors of intracellular
protein-protein interactions. In this case, the small molecule must not only disrupt the intended
interaction, it must first be able to permeate the cell membrane to even reach its desired target.
Yet, some compounds in development are able to accomplish this task. Inhibition of the
interaction between the transcription factor p53 and associated proteins has been considered as a
target for cancer therapy. Using a combination of alanine scanning mutagenesis, high throughput
screening of molecules, and lead optimization, Nutlin-3 was identified as a p53-MDM2
antagonist.
22,23
Not only did this demonstrate the development of a small molecule inhibitor, it
also implemented high-throughput screening successfully, which had been considered to not
useful with protein-protein interactions.
Members of the B cell lymphoma 2 (Bcl-2) family are also of interest in drug
development efforts because they play an important role in apoptosis. Using virtual screening
methods, high-throughput screens, and ligand-based design several potent small molecule ligands
for Bcl-2 proteins have been identified.
12
Most notably, the compound navitoclax which is
8

currently undergoing clinical trials was developed using SAR by NMR techniques to improve the
oral bioavailability of an original lead.
24
Despite the challenges associated with developing small
molecule protein-protein inhibitors, it has even been able to bring such a compound to the market
as seen with the CCR5-receptor antagonist miraviroc, currently available for treatment against
HIV.
25

Although large protein-protein interfaces have often been considered ‘un-druggable’
targets, the investigation of small molecule inhibitors of protein-protein interactions has allowed
for progress in the identification, characterization, and optimization of such compounds.
Although it is not clear if existing databases of compounds for screening are covering the optimal
chemical space for the diversity of protein-protein interfaces, fragement-based methods may still
be used to identify synthetically viable candidates for starting points in drug development. The
use of a combination of biological, physical, and chemical methods has allowed for the
identification of binding hot spots and has also indicated that detailed analysis of protein-protein
interactions of interest can allow for meaningful structural data to emerge from large and complex
binding surfaces. By applying experimental methods capable of evaluating dynamic protein-
protein interactions, such as NMR, it has been possible to move beyond viewing proteins as
static structures and to account for binding interactions that are physically relevant. The
intriguing biology resulting from protein-protein interactions and the recent advances in using
small molecule inhibitors to disrupt these interfaces suggest that future efforts in this direction
may lead to the development of new therapeutic agents for a variety of diseases.

9

1.3. Chapter 1. References.
1
Moreira, I.S.; Fernandes, P.A.; Ramos, M.J. Hot spots – a review of the protein-protein interface
determinant amino acid residues. Proteins. 2007, 68, 803-812.
2
Cheng, A.C.; Coleman, R.G.; Smyth, K.T.; Cao, Q.; Soulard, P.; Caffrey, D.R.; Salzberg, A.C.;
Huang, E.S. Structure-based maximal affinity model predicts small-molecule druggability.
Nature Biotechnol. 2007, 25, 71-75.
3
Jones, S.; Thornton, J.M. Principles of protein-protein interfaces. Proc. Natl. Acad. Sci. USA.
1996, 93, 13-20.
4
Young, L.; Jernigan, R.L.; Covell, D.G. A role for surface hydrophobicity in protein-protein
recognition. Protein Sci. 1994, 3, 717-729.
5
Sheinerman, F.B.; Norel, R.; Honig, B. Electrostatic aspects of protein-protein interactions.
Curr. Opin. Struct. Biol. 2000, 10, 153-159.
6
Lawrence, M.C.; Colman, P.M. Shape complementarity at protein/protein interfaces. J. Mol.
Biol. 1993, 234, 946-950.
7
Kuntz, I.D.; Meng, E.C.; Shoichet, B.K. Structure-based molecular design. Acc. Chem. Res.
1994, 27, 117-123.
8
Bogan, A.A.; Thorn, K.S. Anatomy of hot spots in protein interfaces. J. Mol. Biol. 1998, 280, 1-
9.
9
Clackson, T.; Wells, J.A. A hot spot of binding energy in a hormone-receptor interface. Science.
1995, 267, 383-386.
10
Bonsor, D.A.; Sundberg, E.J. Dissecting protein-protein interactions using directed evolution.
Biochemistry. 2011, 50, 2394-2402.
11
DeLano, W.L. Unraveling hot spots in binding interfaces: progress and challenges. Curr. Opin.
Struct. Biol. 2002, 12, 14-20.
12
Arkin, M.R.; Wells, J.A. Small-molecule inhibitors of protein-protein interactions: progressing
towards the dream. Nat. Rev. Drug Discov. 2004, 3, 301-317.
13
Stumpf, M.P.; Thorne, T.; de Silva, E.; Stewart, R.; An, H.J.; Lappe, M; Wiuf, C. Estimating the
size of the human interactome. Proc. Natl. Acad. Sci. USA. 2008, 105, 6959-6964.
14
McGovern, S.L.; Caselli, E.; Grigorieff, N.; Shoichet, B.K. A common mechanism underlying
promiscuous inhibitors from virtual and high-throughput screening. J. Med. Chem. 2002, 45,
1712-1722.
10

15
Huber, W.; Mueller, F. Biomolecular interaction analysis in drug discovery using surface
plasmon resonance technology. Curr. Pharm. Design. 2006, 12, 3999-4021.
16
Blundell, T.L.; Jhoti, H.; Abell, C. High-throughput crystallography for lead discovery in drug
design. Nat. Rev. Drug Discov. 2001, 1, 45-54.
17
Hajduk, P.J.; Meadows, R.P.; Fesik, S.W. NMR-based screening in drug discovery. Q. Rev.
Biophys. 1999, 32, 211-240.
18
Buchwald, P. Small-molecule protein-protein interaction inhibitors: therapeutic potential in
light of molecular size, chemical space, and ligand binding efficiency considerations. IUBMB
Life, 2010, 62, 724-731.
19
Fry, D.C. Protein-protein interactions as targets for small molecule drug discovery. Biopolymers
(Pept. Sci.) 2006, 84, 535-552.
20
Tilley, J.W.; Chen, L.; Fry, D.C.; Emerson, D.; Powers, G.D.; Biodi, D.; Varnell, T.; Trilles, R.;
Guthrie, R.; Mennona, F.; Kaplan, G.; LeMahieu, R.A.; Carson, M.; Han, R.-J.; Liu, C.-M.;
Palermo, R.; Ju, G. Identification of a small molecule inhibitor of the IL-2/IL-2Rα receptor
interaction which binds to IL-2. J. Am. Chem. Soc. 1997, 119, 7589-7590.
21
Arkin, M.R.; Randal, M.; DeLano, W.L.; Hyde, J.; Luong, T.N.; Oslob, J.D.; Raphael, D.R.;
Taylor, L.; Wang, J.; McDowell, R.S.; Wells, J.A.; Braisted, A.C. Binding of small molecules to
an adaptive protein-protein interface. Proc. Natls. Acad. Sci. USA. 2003, 100, 1603-1608.
22
Picksley, S.M.; Vojtesek, B.; Sparks, A.; Lane, D.P. Immunochemical analysis of the
interaction of p53 with MDM2 – fine mapping of the MDM2 binding site on p53 using synthetic
peptides. Oncogene. 1994, 9, 2523-2529.
23
Kojima, K.; Burks, J.K.; Arts, J.; Andreeff, M. The novel tryptamine derivative JNL-26854165
induces wild-type p53- and E2F1-mediated apoptosis in acute myeloid and lymphoid leukemias.
Mol. Cancer Ther. 2010, 9, 2545-2557.
24
Park, C.M.; Bruncko, M.; Adickes, J.; Bauch, J.; Ding, H.; Kunzer, A.; Marsh, K.C.; Nimmer,
P.; Shoemaker, A.R.; Song, X.; Tahir, S.K.; Tse, C.; Wang, X.; Wendt, M.D.; Yang, X.; Zhang,
H.; Fesik, S.W.; Rosenberg, S.H.; Elmore, S.W. Discovery of an orally bioavailable small
molecule inhibitor of prosurvival B-cell lymphoma 2 proteins. J. Med. Chem. 2008, 51, 6902-
6915.
25
Dorr, P.; Westby, M.; Dobbs, S.; Griffin, P.; Irvine, B.; Macartney, M.; Mori, J.; Rickett, G.;
Smith-Burchnell, C.; Napier, C.; Webster, R.; Armour, D.; Price, D.; Stammen, B.; Wood, A.;
Perros, M. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule
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type 1 activity. Antimicrob. Agents. Chemother. 2005, 49, 4721-4732.
11

Chapter 2. Design and synthesis of inhibitors of the
MEF2/HDAC interaction.

2.1. Introduction. The role of myocyte enhancer factor (MEF2) as a
cellular regulator.

2.1.1. The role MEF2 family of transcription factors in signaling pathways.

The regulation of gene expression is influenced by the actions of a class of proteins
known as transcription factors. Transcription factors are able to bind to specific sequences of
DNA and control the transfer of genetic information from DNA to RNA. Capable of acting as
either activators or repressors they can control levels of gene transcription, and consequently,
gene expression.
One important transcription factor is the myocyte enhancer factor (MEF2) transcription
factor which plays a key role in the transmission of extracellular signals to the genome and
controls a wide range of cellular processes. Although a single MEF2 gene that expresses MEF2
protein has been identified in Saccharomyces cerevisia and Drosophila melanogaster, four genes
(MEF2A, -B, -C, and –D) have been found in vertebrates.
1
As shown in Figure 2.1., the MEF2
proteins, named for their ability to bind to DNA sequences found in genes specific to muscles, are
structurally related and have similar amino acid sequences.

Figure 2.1 - Amino acid sequence similarity of MEF2 proteins.
1

12

Each of the MEF2 proteins have similar features, including a MADS domain, a MEF2
domain, and a transcriptional activation domain. At the N-terminus is the MADS domain, which
is involved in binding to DNA.
2
Adjacent to the MADS domain is the MEF2 domain, which
controls DNA-binding affinity and interactions with other cofactors.
3
The transcriptional
activation domain is located at the C-terminus.
4
MEF2 transcription factors associate with
several different cofactors to control the expression of genes that are located downstream. The
specific genes that are activated or repressed vary, since MEF2 proteins can be expressed in
different cell types including cardiac, skeletal, and smooth muscle cells and neurons.
The MEF2 proteins are involved in several signaling pathways that control gene
expression. MEF2 has been demonstrated to play a role in cell-cycle regulation in response to
signals from many different receptors on the cell surface.
5
One signaling pathway that involves
MEF2 and consequently impacts cellular growth and differentiation can be activated by mitogen-
activated protein (MAP) kinases. These kinases can directly associate with MEF2 to allow gene
expression to occur after phosphorylating specific residues of the transcription factor.
6

Additionally, the extracellular signal-regulated protein kinase (ERK) can also associate with
MEF2 to enhance transcriptional activity by the phosphorylation of residues within the
MADS/MEF2 domain.
7

The activity of MEF2 is also controlled through calcium signaling pathways. Membrane
depolarization causes the flow of calcium into cells and elevated levels of calcium in the
cytoplasm activates calcium/calmodulin-dependent kinases (CaMKs), which can then in turn
activate MEF2 via phosphorylation.
8-10
It is also possible for CaMKs to transmit signals to MEF2
through the interaction of histone deacetylases (HDACs) with the MADS/MEF2 domain of
MEF2.
11

13

2.1.2. The association of MEF2 with activators and repressors.
HDACs and histone acetyltransferases (HATs) are two families of enzymes that act as
transcriptional repressors and activators. They provide a connection between signaling pathways
that are responsible for cellular regulation processes and the transcription factors that activate
genes. As depicted in Figure 2.2, MEF2 associates with HATs and HDACs which control the
acetylation state of histones.
12

Figure 2.2 - Overview of the interaction of MEF2 with activators and repressors.
12

Several transcription activators possess HAT activity and they can also permit
interactions with other transcription factors at the same time to enhance transcriptional
activity.
13,14
When acetylated, the lysine tails of histone proteins do not strongly interact with
negatively charged DNA. This allows chromatin to form a relaxed configuration and enables
transcription factors to associate with DNA, allowing for the expression of genes.
When deacetylated, however, the positively charged histone lysine residues can more
easily coordinate with negatively charged DNA. This leads to the formation of densely packed
structures and thus blocks transcription factors from associating with DNA. As a result, this
represses transcription and leads to gene silencing. In humans, there are at least 17 HDACs
capable of causing this type of activity. Among the four different classes of HDACs, class II
14

HDACs are catalytically inactive and mainly act as structural proteins or are involved in the
formation of complexes.
15
The class II HDACs (HDAC4, 5, 6, 7, 9, and 10) associate with the
MADS/MEF2 domains of MEF2 proteins through a specific amino acid sequence that is unique
to them in comparison with class I HDACs, which do not directly associate with MEF2.
12

Signaling by CaMK is capable of releasing MEF2 from the repressive activity of HDACs
by phosphorylation.
16
This phosphorylation creates a docking site for a chaperone protein that
disrupts the MEF2-HDAC interaction through its association with the phosphorylated HDAC.
17

Binding of the chaperone protein also leads to export of the HDACs to the cytoplasm.
18
MEF2 is
then released and able to associate with HATs to activate the expression of MEF2-dependent
genes.
2.1.3. MEF2-mediated cellular processes.
Through its central role in several signaling pathways, the activation and repression of
gene expression mediated by MEF2 has a large impact on developmental processes in a variety of
cell types. For example, MEF2 regulates skeletal muscle differentiation and controls gene
expression in cardiac muscle cells.
19,20
Additionally, MEF2 proteins are expressed in endothelial
and smooth muscle cells, which are required for vasculature development.
21
MEF2 proteins are
also found highly enriched in neurons, which allows for transcriptional regulation of neuron-
specific genes and can influence synapse development.
22,23
MEF2 also impacts immune and
inflammatory processes, due to its role in T-cell development.
24
After T-cell receptor activation,
dissociation of the HDAC from MEF2 permits activation of MEF2.
Therefore, as a mediator of signal-dependent transcription and cellular differentiation,
MEF2 serves as a central component in cell development processes. Due to its ability to
associate with many activators and repressors, MEF2 is a key intermediate in the transmission of
extracellular signals to the genome. By designing activators or inhibitors of the interactions of
15

MEF2 with its cofactors the functions of MEF2 may be modulated for disease treatment. One
such interaction that may be exploited for the creation of therapeutics is that between MEF2 and
HDACs.
2.1.4. Inhibition of Histone Deacetylase (HDAC) activity.
The development of HDAC inhibitors has received a large amount of attention, due to the
importance of HDACs in cancer, neurodegeneration, and inflammation. Previously, however,
many of these HDACs were designed with the intent of inhibiting the catalytic activity of
HDACs. In general, four chemical classes are represented by these inhibitors: hydroxamic
acids, benzamides, short chain fatty acids, and cyclic peptides. Most of these HDAC inhibitors
are comprised of a general three-part structure consisting of a region capable of binding to a zinc
atom in the catalytic active site of the HDAC, a linker region, and a surface recognition area.
25

The selectivity and potency of these classes varies, however. The hydroxamic acids tend to
exhibit low nanomolar potencies but lack class specificity, whereas benzamides and cyclic
peptides show some selectivity towards HDACs 1,2, and 3.
26
Despite this, investigation of
potential pharmacological use of these compounds is on-going. For example (Figure 2.3),
suberoylanilide hydroxamic acid Zolinza™ 2.1 (vorinostat, SAHA, Merck) has been approved for
treatment of advanced cutaneous T-cell lymphoma, while MS-275 2.2 (Syndax
Pharmaceuticals/Schering AG), CI-994 2.3 (Pfizer Inc.), MGCD0103 2.4 (MethylGene Inc.), and
LBH-589 2.5 (Novartis AG)

have undergone clinical trials.
27-34

16

Figure 2.3 - Represetative benzamides and hydroxamic acids previously studied due to their
ability to inhibit HDAC-related activites.

2.1.5. Structure of MEF2 and implications for small molecule inhibition.
In a series of structural studies, Prof. Lin Chen and his group have elucidated several
crystal structures of MEF2 and its protein complexes.
36,37
These structural studies of MEF2
protein complexes have shown a hydrophobic region exists on the surface of the MADS/MEF2
domain which can participate in protein-protein binding interactions.
35
Several hydrophobic
contacts and van der Waals interactions have been demonstrated to permit binding between
MEF2 and HDAC9 (Figure 2.4).
36
These interactions of MEF2 with HDACs suggested the
possibility to focus upon the direct inhibition of MEF2 and its activities. Rather than targeting
the active site of a HDAC, a more specific inhibition may be possible. This could be achieved by
targeting a particular protein-protein interaction between MEF2 and the classIIa subtype of
HDACs, which lack catalytic activity.
17

Figure 2.4 - Interactions between MEF2 and HDAC9.
36

Further studies which investigated the structure of the MADS-box/MEF2 domain of
MEF2 bound to DNA have indicated that the hydrophobic groove is present even in the absence
of a ligand.
37
The development of small molecules that could bind to this hydrophobic pocket of
MEF2 could potentially block the recruitment of class IIa HDACs and impact the expression of
MEF2-dependent genes.
2.2. Synthesis and evaluation of inhibitors of the MEF2/HDAC
interaction.
2.2.1. Development of inhibitors.
An initial high-throughput investigation by the Lin Chen group aimed at indentifying
small molecules that could disrupt the interaction between MEF2 and class IIa HDAC4 indicated
that compounds from the pimeloylanilide o-aminoanilide chemical class could serve as a lead. In
particular, N-(2-aminophenyl)-N’-phenyloctanediamide served as the basis for further structural
modification. These initial findings provided the basis for a collaboration between the Nicos
18

Petasis and Lin Chen groups aimed at the development and optimization of new MEF2/HDAC
inhibitors.
The design and synthesis efforts in the Petasis group were initiated by Kevin Gaffney and
continued as a joint effort with the work detailed herein.
99
Although prior syntheses of related
compounds have been reported, it was hoped to improve upon these methods to achieve our goal
of investigating structurally related compounds in their ability to alter the interaction of the
transcription factor MEF2 with associated HDACs by directly targeting MEF2.
38,39
These
compounds would be evaluated for their ability to disrupt this protein-protein interaction by our
collaborators in Lin Chen’s laboratory using a mammalian two-hybrid luciferase assay. The most
straight-forward strategy towards the synthesis of these analogs, comprised of an aliphatic linker
and two capping regions A and B, would involve amide bond formations. A general structure
depicted these regions of interest is shown in Figure 2.5.

Figure 2.5. – General structure of interest comprised of capping Region A, a central
linker, and capping Region B.

Typically, amide bonds are created from the condensation of a carboxylic acid and an
amine; however, increased temperatures of 200°C may be needed for the elimination of water to
occur.
40
To avoid use of high temperature reactions, which may cause unwanted side reactions or
decomposition of materials, another strategy is to activate the carboxylic acid coupling partner
and convert the hydroxyl of the acid into a better leaving group. Several methods have been used
to achieve this, such as those which can form acid chlorides, mixed anhydrides, or active esters
and the choice of coupling reagent can be a crucial factor in the success of the amide bond
19

forming reaction. Carbodiimides, such as dicyclohexylcarbodiimide (DCC) and water-soluble 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), activate the carboxylic acid by forming an
O-acylurea and can be used in combination with benzotriazole additives.
41,42
Many coupling
reagents have been based upon 1H-benzotriazoles as well, such us uranium and phosphonium
salts, which react with carboxylic acids to form active esters.
43
These reagents promote rapid
reactions and their efficiency has been demonstrated in their use in solid phase peptide synthesis
where many sequential reactions are conducted.
44
Consequently, it was decided to make use of
the 1H-benzotriazole-based reagents for our purposes.
Three different synthetic routes were used to provide compounds of the general scaffold
shown in Figure 2.5. The first of these routes is outlined in Scheme 2.1. Refluxing a selected
aniline under neat conditions in the presence of dicarboxylic acid 2.6 provided mono-acids 2.7.
Generally, modest yields were obtained in this amidation reaction due to incomplete consumption
of starting materials, as well as formation of the corresponding di-coupled product. Intermediate
mono-acid 2.7 was then submitted to another reaction with a second aniline using the peptide
coupling reagent O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate
(HBTU) to activate the carboxylic acid for nucleophilic attack by the aniline.
Reagents and conditions: (a) ArNH
2
, 110°C, 24 h, 18-75%; (b) ArNH
2
, HBTU, DIPEA, DMSO or DMF, rt,
3-24 h, 7-71%.
Scheme 2.1. – General synthetic route starting with a dicarboyxlic acid.
From ongoing structure-activity relationship studies it emerged that the presence of an
ortho-aniline substituent was beneficial for biological activity (to be discussed later in Chapter
2.2.2). Therefore, another synthetic route in which this functional group could be installed first
was undertaken in an attempt to more rapidly access a variety of analogs containing this moiety.
20

As shown in Scheme 2.2, two sequential HBTU-promoted amidations were conducted, the first
with Boc-protected 1,2-phenylenediamine and pimelic acid 2.9 followed by the reaction of
intermediate mono-acid 2.10 with a variety of anilines. Final deprotection of the Boc group using
trifluoroacetic acid then furnished analogs 2.12 to be studied in biological assays.

Reagents and conditions: (a) tert-butyl (2-aminophenyl)carbamate, HBTU, DIPEA, DMSO, rt, 24 h, 47%;
(b) ArNH
2
, HBTU, DIPEA, DMSO or DMF, rt, 3-24 h, 23-70%; (c) CF
3
COOH, CH
2
Cl
2
, 0° to rt, 3 h, 18-
79%.
Scheme 2.2. – Alternative synthetic route used to install ortho-anilide first.
Although it was originally envisioned that this route would be amenable to provide
several compounds possessing the ortho-amine functionality, it was found that yields of
intermediate 2.10 were variable and it was often difficult to purify this compound by
recrystallization or column chromatography. Additionally, this intermediate tended to degrade
easily, most likely due to cleavage of the Boc-protecting group. As a result, this scheme was not
universally adopted for synthesis of additional compounds, and in many cases the route shown in
Scheme 2.1 was found to be more reliable.
In a few instances a variation of the route shown in Scheme 2.2 was used which reversed
the order that the two different anilines were added to the dicarboxylic acid. After the first
HBTU-promoted coupling of an aniline with dicarboxylic acid 2.9, intermediate mono-acid 2.7
was coupled to Boc-protected phenylenediamine. This necessitated a final deprotection step to
provide compounds 2.12 (Scheme 2.3).
21

Reagents and conditions: (a) tert-buyl (2-aminophenyl)carbamate, HBTU, DIPEA, DMSO, rt, 3-24 h, 34-
70%; (b) CF
3
COOH, CH
2
Cl
2
, 0° to rt, 3 h, 18-79%.
Scheme 2.3. – Alternative synthetic route.
Using this route it was found that the polarity of intermediates 2.11 were similar to that of
the aniline substrates, which made chromatography at this intermediate stage tedious. Therefore,
due to trouble purifying intermediates 2.11 it was necessary for chromatography to be carried out
after the last step, whereas a recrystallization had originally been anticipated to be sufficient for
purification after deprotection. This route was consequently not used in preference to that shown
in Scheme 2.1, which was determined to be the most reliable of the three main synthetic routes
described.
2.2.2. Synthesis and investigation of capping regions.
Preliminary biological testing conducted by the Chen group using compounds
synthesized in the Petasis lab by Kevin Gaffney indicated that compounds comprised of a five-
carbon aliphatic central chain, a meta-bromophenyl or phenyl substituent in Region A and an
ortho-aniline in Region B provided good levels of activity. Analogs lacking the aniline functional
group were synthesized next (Figure 2.6) to gain insight regarding the structural relevance of the
amine group. Although the ortho-amine functionality had been investigated in prior studies of
HDAC inhibition, it had been thought that this group could serve as a zinc binding moiety at the
HDAC active site.
45
However, in contrast with that work, we were instead interested in the direct
binding of the synthesized ligands to MEF2, making the need for a metal binding interaction
irrelevant. Additionally, it had been shown through a DFT study that zinc binding of a
benzamide ligand is not favorable, with a ΔE of 0.6 kcal/mol and a ΔG of 3.3 kcal/mol relative to
22

water.
46
It was thus hoped to establish whether the ortho-aniline functionality was needed for an
interaction with a residue in MEF2.
To investigate this, a set of compounds which did not contain an ortho-aniline were
synthesized (Figure 2.6). Modest activity was observed (biological data from luciferase assay
not shown) in some compounds lacking the ortho-aniline, but at this stage it was not clear
whether removing the ortho-aniline in all future compounds would be desirable.

Figure 2.6. – Compounds with phenyl ring in Region A, a five carbon aliphatic linker, and varied
functional groups in Region B.
An additional series of compounds containing a meta-bromophenyl substituent in Region
A and varied functionality in Region B was also synthesized to probe the effect of removing the
ortho-anilide (Figure 2.7) However, based on resulting activity of these compounds, it appeared
that replacing the ortho-aniline with other functional groups did not improve the ability to disrupt
the MEF2/HDAC interaction.
23

Figure 2.7. – Compounds containing a meta-bromo substituent in Region A, a five carbon
aliphatic linker, and varied functional groups in Region B.
Next, in an attempt to improve the solubility of compounds, a series of compounds
containing pyridines were synthesized and tested (Figure 2.8). It was anticipated that the
inclusion of a polar nitrogen within the structure would potentially improve the characteristics of
designed analogs by lowering their lipophilicty.
47
These compounds were synthesized according
to the same general methods as outlined in 2.2.1, using two separate condensation reactions to
provide final compounds in modest yield.

Figure 2.8. – Compounds containing pyridine functionality.
The inclusion of more polar functionality within the analog design was not found to be
beneficial. Consequently, the synthesis of compounds containing larger hydrophobic moieties
was undertaken as an attempt to improve the ability of the designed compounds to disrupt the
MEF2/HDAC interaction. It was considered that inclusion of regions of extended hydrophobicity
would allow for a greater amount of interactions within the hydrophobic binding region of the
24

target protein. To achieve this, a series of biphenyl-functionalized compounds was synthesized
(Figure 2.9). At this point we also determined to fix the ortho-aniline in Region B.

Figure 2.9. – Biphenyl-containing compounds.
Analysis of the biphenyl-containing compounds indicated that a meta-biphenyl
substituent in Region A provided good activity; therefore, two additional analogs were
synthesized in which a heteroatom would be incorporated within this moiety. As indicated in
Scheme 2.4, Suzuki reactions were employed to install the pyridinyl functionality. A brief
screening of reaction conditions indicated that use of a small excess (1.2 equiv.) of boronic acid
and excess K
2
CO
3
in dioxane:water (1:1) solvent provided full conversion of the bromo-
compound to product. Final deprotection provided 2.34 and 2.35.

Reagents and conditions: (a) ArB(OH)
2
, K
2
CO
3
, Pd(Ph
3
)
4
, dioxane:water (1:1), 80°C, 18 h, 30-66%; (b)
CF
3
COOH, CH
2
Cl
2
, 0° to rt, 3 h, 73-79%.

Scheme 2.4. – Synthesis of 2.34 and 2.35 employing Suzuki coupling reactions.
25

It was determined that the location of the nitrogen within the biphenyl substituent
produced a large effect on bioactivity. A nitrogen atom in the meta position did not provide good
inhibition of the MEF2:HDAC interaction; however in the para position a high level of inhibition
was observed.
To further evaluate the effect meta-substitution, a set of compounds was synthesized in
which the ortho-aniline moiety in Region B was fixed and variable meta-substituted anilines were
included in Region A (Figure 2.10). In general, these compounds provided good inhibitory
activities, further indicating that substitution in the meta-position of this phenyl ring was well
tolerated.

Figure 2.10. – Compounds with varied meta-substitution in Region A.
To capitalize on the beneficial activity gained through the inclusion of a substituent in
Region A and for use as a probe to evaluate the binding interaction of the small molecule ligand
with MEF2 in solution (discussed in Chapter 2.3.5), a set of fluorinated compounds was
synthesized (Figure 2.11). Additionally, 2.39 was synthesized on large scale for evaluation in an
animal model.
26

Figure 2.11. – Fluorine-containing compounds.
It was found that compounds containing both meta-substitution and biphenyl
functionality in Region A and an ortho-aniline in Region B provided optimal biological activity.
Two compounds were therefore synthesized to potentially exploit a synergistic effect through
combination of these structural features. Compound 2.46 was synthesized from 2.44 and
phenylboronic acid via a Suzuki reaction as shown in Scheme 2.5. Initially, poor reactivity was
observed for this palladium-catalyzed coupling, presumably due to steric hinderance from the
neighboring trifluoromethyl group. A screening of alterative bases and catalysts/ligands was
carried out to determine that increasing catalyst loading lead to improved reactivity.

Reagents and conditions: (a) ArB(OH)
2
, K
2
CO
3
, Pd(Ph
3
)
4
, dioxane:water (1:1), 80°C, 18 h, 58%; (b)
CF
3
COOH, CH
2
Cl
2
, 0° to rt, 3 h, 67%.

Scheme 2.5. – Suzuki reaction to synthesize a hybrid of 2.39 and 2.41.
Surprisingly, the compounds intended to function as a hybrid of 2.39 and 2.41 and as a
hybrid of 2.38 and 2.41 exhibited lower levels of inhibitory activity than the parent compounds.
27

This may have been a result of steric congestion leading to the inability to form favorable binding
interactions.
To further investigate substitution in Region A, incorporation of the N,N-dimethyl
functionality present in the known HDAC inhibitor trichostatin A, was analyzed (Figure 2.12).
Inclusion of this functional group was tolerated in the para position of Region A (Scheme 2.6).

Figure 2.12. – Structure of trichostatin A.

Scheme 2.6. – Inclusion of N,N-dimethylaniline functionality.
The N,N-dimethyl functional group was also employed to evaluate the importance of the
ortho-amine hydrogen atoms in Region B. Alkylation of the amine using iodomethane was not
successful, but reductive amination of 2.48 following conditions adapted from the literature
provided 2.49 (Scheme 2.7).
48
It was found that replacement of the ortho-amine with N,N-
dimethyl in Region B lead to a reduction in inhibition of the MEF2:HDAC interaction.

Reagents and conditions: (a) paraformaldehyde, NaBH
4
, CF
3
COOH, THF, rt, 24 h, 38%.
Scheme 2.7. – Dimethylation of 2.48 to provide 2.49.
28

To further investigate the effect of altering Region B, a formylated compound (2.51) was
synthesized from 2.50, as shown in Scheme 2.8, using formic acid and sodium formate following
conditions from the literature.
49
Due to this compound’s lack of biological activity further
exploration of methods to improve reaction conditions for formylation of the primary amine were
not conducted.

Reagents and conditions: (a) HCOONa, HCOOH, rt, 18 h, 9%.
Scheme 2.8. – Formylation of 2.50 to give 2.51.
Previously it had been reported that one of the metabolites of the HDAC inhibitor
trichostatin A was a carboxamide.
50
It was thus considered that this structural motif could prove
to be useful for disrupting the interaction between MEF2 and HDACs. A survey of the literature
indicated that commonly used methods to install this functionality were not desirable, generally
requiring the reaction of the corresponding acid chloride with ammonia at high pressure, but a
method using EDC coupling of a carboxylic acid with ammonium hydroxide had been reported.
51

Therefore, an adaptation of our general HBTU coupling protocol was used, as outlined in Scheme
2.9, to provide 2.54 in 80% yield. Unfortunately, this modification was not helpful in improving
inhibitory activity.

Reagents and conditions: (a) NH
4
OH, HBTU, DIPEA, CH
3
CN, rt, 36 h, 80%.
Scheme 2.9. – Synthesis of carboxamide 2.54.
29

An additional modification to Region B through inclusion of a benzimidazole was
explored. This compound was obtained via an acid catalyzed cyclization was employed to form a
benzimidazole from the ortho-amine and amide bond linker of 2.55 (Scheme 2.10). The
importance of the ortho-amine substiuent in Region B was shown, as seen by a dramatic loss of
inhibitory activity.

Reagents and conditions: (a) acetic acid, 120°C, 24 h, 69%.
Scheme 2.10. – Synthesis of 2.56.
2.2.3. Synthesis and evaluation of modified linker region.
Modifications to the central region of the MEF2 inhibitors were also made. First, an
evaluation of optimal chain length for the aliphatic linker was conducted. For this series of
analogs the meta-trifluoroaniline in Region A and ortho-aniline in Region B were fixed while the
number of methylene groups in the central aliphatic region was altered. These compounds were
synthesized following the route outlined in Scheme 2.1. The use of a five-carbon linker (2.41)
provided excellent biological activity. Compounds with four, six, or seven carbon linkers (2.57,
2.58, and 2.59 respectively) exhibited moderate levels of activity, but a complete loss in activity
was observed when a compound with an eight-carbon linker (2.60) was analyzed in the two-
hybrid luciferase assay. This may not indicate that the compound is too large to fit in the
hydrophobic binding region of MEF2, but rather that the extended aliphatic chain could either
cause the compound to adopt a non-linear configuration or lead to poor solubility.
30

Figure 2.13. – Evaluation of correlation between linker length and percent inhibition.
Two additional compounds containing a six-carbon aliphatic linker were also evaluated
for biological activity (Figure 2.14). These compounds did not display good activity in the
luciferase assay.

Figure 2.14. – Compounds containing a six-carbon aliphatic linker.
Next, to provide more rigidity to the aliphatic linker, a compound with a central alkyne
was synthesized, as shown in Scheme 2.11. Base hydrolysis of the di-tert-butyl ester proceeded
sluggishly to provide carboxylic acid 2.64. Initially, formation of 2.65 was problematic. It was
attempted to condense 3-bromoaniline with 2.64 by refluxing neat or in various solvents, however
this lead to degradation of the alkyne. HBTU-promoted coupling provided minimal reactivity
based upon TLC analysis, but use of the more robust coupling agent HATU finally promoted
conversion to 2.65. Final coupling to furnish 2.66 was conducted using the standard HBTU
31

conditions. Based on results from our collaborator’s luciferase assay it was indicated that the
conformation of this compound was still amenable to binding with MEF-2.

Reagents and conditions: (a) LiOH, THF:H
2
O (1:1), 70°C, 48 h, 67%; (b) 3-bromoaniline, HATU, DIPEA,
CH
2
Cl
2
, rt, 18 h, 35%; (c) 1,2-phenylenediamine, HBTU, DIPEA, DMSO, rt, 18 h, 37%.

Scheme 2.11. – Synthesis of 2.66.
To determine whether constraining the aliphatic linker through inclusion of a cyclic core
would still allow for binding to MEF2, a compound featuring a central piperazine was targeted.
A route which began with the piperazine linker installed first, followed by nucleophilic
substitution to create a symmetric dicarboxylic acid was thought to be a straightforward method
to obtain this compound (Scheme 2.12). Two different amide bond forming reactions would then
lead to the desired final product. However, attempts to isolated mono-acid 2.69 were
unsuccessful.

Scheme 2.12. – Unsuccessful route towards compound containing piperazine core.
Next, instead of attempting to obtain mono-acid 2.69 from its di-acid precursor 2.68, a
linear route (Scheme 2.13) was implemented. Formation of chloroacetamide 2.71 proceeded well
32

and after nucleophilic substitution with Boc-piperazine intermediate 2.71 was obtained.
Following deprotection, substitution with bromoacetic acid was attempted, but due to the polarity
of 2.69, attempts to separate this material from unreacted bromoacetic acid through extraction or
via column chromatography proved unsuccessful. It was anticipated that separation of an ester
intermediate would be more amenable to chromatography, therefore 2.74 was synthesized
instead. Unfortunately, base hydrolysis of 2.74 did not produce the desired carboxylic acid
product.

Scheme 2.13 – Unsuccessful synthetic route towards a piperazine-containing compound.

Scheme 2.14 - Attempted route towards synthesis of piperazine-containing compound.
Knowing that the coupling of chloroacetamide 2.71 to Boc-piperazine was a successful
strategy, it was then decided to employ this coupling to install functionality on both sides of the
piperazine core. Substitution with tert-butyl (2-(2-chloroacetamido)phenyl)carbamate,
synthesized from Boc-protected 1,2-phenylenediamine, gave 2.75 albeit in low yield. Final
deprotection in the presence of trifluoroacetic acid then allowed for the successful formation of
2.76. This compound, however, did not display good activity in the standard assay.
33

Reagents and conditions: (a) tert-butyl (2-(2-chloroacetamido)phenyl)carbamate, K
2
CO
3
, DMF, 90°C, 24 h,
28%; (b) CF
3
COOH, CH
2
Cl
2
, rt, 3 h, 64%.

Scheme 2.15. – Synthesis of 2.76.
An alternative structure containing a benzene ring liner was also incorporated as part of
the linker region. As shown in Scheme 2.16, synthesis of this compound was straightforward.
Nucleophilic substitution with commercially available methyl 3-hydroxybenzoate while refluxing
in the presence of excess base gave intermediate 2.77 in good yield. Base hydrolysis and HBTU-
promoted coupling of carboxylic acid 2.78 with 1,2-phenylenediamine then gave 2.79. Biological
activity of this analog was modest. This indicated that structural modifications of the central
linker beyond the flexible, aliphatic chain could prove useful.

Reagents and conditions: (a) methyl 3-hydroxybenzoate, K
2
CO
3
, acetone, 60°C, 24 h, 80%; (b) LiOH,
THF, rt, 5 h, 34%; (c) 1,2-phenylenediamine, HBTU, DIPEA, DMSO, rt, 18 h, 61%.

Scheme 2.16. – Synthesis of 2.79.
To incorporate a five-membered heterocycle as part of the central linker, a furanyl analog
was then made. As outlined in Scheme 2.17, base hydrolysis of commercially available ethyl 3-
(2-furyl)propanoate provided carboxylic acid 2.81 in high yield, which was then coupled with 3-
34

bromoaniline under the general HBTU-promoted strategy. A radical reaction involving ethyl
iodoacetate, based upon a reported reaction, was then used to install the functionality necessary to
construct intermediate 2.83.
52
This then made synthesis of the remaining half of the final
compound straightforward and a second base hydrolysis followed by HBTU coupling of
intermediate 2.84 with 1,2-phenylenediamine thus provided 2.85.
Reagents and conditions: (a) KOH, MeOH:H
2
O (1:1), 80°C, 3 h, 86%; (b) 3-bromoaniline, HBTU, DIPEA,
DMSO, rt, 12 h, 59%; (c) ethyl iodoacetate, FeSO
4
6H
2
O, 30% H
2
O
2
aq., DMSO, 0°C to rt, 15 h, 15%; (d)
KOH, MeOH:H
2
O (1:1), 80°C, 8 h, 35%; (e) 1,2-phenylenediamine, HBTU, DIPEA, DMSO, rt, 12 h, 44%.

Scheme 2.17. – Synthesis of 2.85.
2.3. Synthesis and evaluation of inhibitors containing reversed amide bond
orientation.
2.3.1. Overview of synthetic routes towards inhibitors.
In relation to the analogs described in Chapter 2.2 it was considered that changing the
orientation of the amide bond used to link the central and capping regions could impact binding
affinity. To test this, another series of compounds was designed in which the reverse amide
linkage present between Region A and the aliphatic linker was switched to an amide bond, as
depicted in Figure 2.15.
35

Figure 2.15. – Reversed orientation of the amide bond in the general scaffold design.
Two synthetic strategies were devised to accomplish this, the first of which is depicted in
Scheme 2.18. The Cbz-protected intermediate 2.86 was submitted to an amide coupling reaction
with tert-butyl (2-aminophenyl)carbamate in the presence of HBTU and Hünig’s base to provide
intermediate 2.87. Deprotection under neutral conditions gave the free amine intermediate 2.88
which was used without purification. A second HBTU-promoted coupling of 2.88 with a variety
of commercially available carboxylic acids then gave intermediates 2.89. Lastly, deprotection of
the Boc-group using acidic conditions provided analogs 2.90 which were then analyzed for their
ability to disrupt the interaction between MEF2 and HDACs.

Reagents and conditions: (a) tert-butyl (2-aminophenyl)carbamate, HBTU, DIPEA, DMSO, rt, 5 h, 58%;
(b) H
2
, 5% Pd/C, MeOH, rt, 6 h, 65%; (c) ArCOOH or heteroarylCOOH, HBTU, DIPEA, DMSO or DMF,
3-24 h, 29-65%; (d) CF
3
COOH, CH
2
Cl
2
, 0°C to rt, 3-5 h, 24-96%.

Scheme 2.18. – General synthetic route for compounds with reversed amide bond orientation.
36

Alternatively, the route described in Scheme 2.19 was used, which was anticipated to
simplify purification steps. A number of different commercially available carboxylic acids were
reacted with compounds 2.91, synthesized by esterification of the corresponding carboxylic acids.
Coupling promoted by HBTU and an excess of base gave intermediates 2.92 of varying aliphatic
chain length. Base hydrolysis of the esters generally proceeded in high yield to provide the
carboxylic acids 2.93. Final coupling of 2.93 with 1,2-phenylenediamine using typical amide
coupling conditions gave analogs 2.94.

Reagents and conditions: (a) ArCOOH or heteroarylCOOH, HBTU, DIPEA, DMSO or DMF, rt, 3-24h,
23-88%; (b) LiOH, THF:H
2
O (1:1), rt, 2-5 h, 19-94%; (c) 1,2-phenylenediamine, HBTU, DIPEA, DMSO
or DMF, rt, 3-12 h, 24-81%.

Scheme 2.19. – Second strategy to synthesize compounds with reversed orientation of the amide
bond between Region A and the aliphatic linker.
2.3.2. Evaluation of linker length.
An initial investigation of the correlation between length of the aliphatic linker and
biological activity was first conducted. Based upon results obtained regarding activity of the
compounds summarized in Chapter 2.2, for this study all compounds contained an aromatic ring
with either a meta-trifluoromethyl or bis-trifluoromethyl substituent in Region A and an ortho-
aniline in Region B. Analogs were synthesized following the general procedure described in
2.3.1. with the chain length varied from four to six carbons (Figure 2.16). Although a four-carbon
linker (2.95 and 2.96) proved to be sub-optimal, both five and six carbon chains provided good
activity.
37

Figure 2.16. – Investigation of aliphatic linker length.

2.3.3. Synthesis and investigation of capping regions.
An evaluation of the structure activity relationship regarding substitution in Region A
was then made (Figure 2.17). A meta-bromo (2.101) and meta-phenyl (2.102) both provided
good inhibitory activity. Para-substitution was also well-tolerated (2.103).

Figure 2.17 – Compounds with varied meta- or para- substitution.
38

Although para-substitution was amenable to activity when included on a compound with
a five-carbon linker (2.105), a complete loss in activity was observed with a four-carbon chain
(2.104). This correlated with the reduction in activity seen from with shortest linkers studied in
Figure 2.16.

Figure 2.18 - Compounds containing N,N-dimethyl in para-position of Region A.
It was also investigated whether activity could be retained if the ortho-aniline moiety was
positioned in Region A. As shown in Scheme 2.20 a compound containing a meta-
trifluoromethyl group and the ortho-amine functionalities on opposite ends of the aliphatic linker
in comparison with 2.97 was synthesized. The Boc-protected ortho-aminobenzoic acid (2.106)
used to synthesize this analog was prepared according to a literature procedure.
53
Interestingly,
2.110 produced a loss in inhibitory activity in the two-hybrid luciferase assay. This implied that
altering the position of the carbonyl group near the ortho-amine removed a crucial binding
interaction.
39

Reagents and conditions: (a) 2.106, HBTU, DIPEA, DMSO, rt, 18 h, 53%; (b) LiOH, THF, rt, 8 h, 34%;
(c) 3-trifluoromethyl aniline, HBTU, DIPEA, DMSO, rt, 18 h, 46%; (d) CF
3
COOH, CH
2
Cl
2
, 0°C to rt, 3 h,
67%.

Scheme 2.20. – Synthesis of compound with reversed functional group substitutions.
A series of analogs which contained a heterocyclic system were also synthesized (Figure
2.19) In general, all compounds from this set displayed good levels of inhibition of the
MEF2:HDAC interaction. However, substitution in the 2-position of the heterocycle (2.111 and
2.113) provided better activity than substitution at the 3-position (2.112 and 2.114).
40

Figure 2.19 - Compounds including heterocyclic rings in Region A.
Prompted by the encouraging results obtained with heterocycle-containing compounds, a
set of compounds containing a pyrrole or indole were also synthesized and evaluated for their
ability to disrupt the MEF2/HDAC interaction. It was also considered that incorporating the
indole functional group along with an extended region of hydrophobicity could allow for
improved interactions with the hydrophobic groove of MEF2. To synthesize such a compound, it
was envisioned that a Suzuki coupling could be used to add this moiety to a bromo-indole.
Initially, this reaction was conducted utilizing 5-bromoindole-2-carboxylic acid and
phenylboronic acid, as shown in Scheme 2.21. Although this Suzuki coupling was successful,
the concomitant HBTU coupling between 2.120 and 2.88 was unsuccessful. It was thought that
this amidation reaction did not proceed due to excess acid remaining present from the workup
used to obtain 2.120, however addition of excess base still provided minimal conversion.
41

Reagents and conditions: (a) phenylboronic acid, Na
2
CO
3
, Pd(Ph
3
)
4
, toluene:ethanol (3:2), 80°C, 12 h,
95%.

Scheme 2.21. – Initial attempt to synthesize 2.121.
Therefore, a Suzuki reaction between 2.122 and phenylboronic acid was instead
attempted (Scheme 2.22). This reaction proceeded well, as did the final Boc-deprotection to
provide 2.123.

Reagents and conditions: (a) 2.88, HBTU, DIPEA, DMSO, rt, 16 h, 40%; (b) K
2
CO
3
, Pd(Ph
3
)
4
,
dioxane:water (1:1), 80°C, 18 h, 93%; (c) CF
3
COOH, CH
2
Cl
2
, 0°C to rt, 3 h, 71%.

Scheme 2.22 - Alternative route to obtain 2.123.

42

Based upon prior improvements in activity upon the inclusion of a trifluoromethyl moiety
as described in Chapter 2.2, an indole-containing analog containing this functional group was
also synthesized. Initially, a route involving a Vilsmeier-Haack reaction was explored to prepare
the necessary carboxylic acid. However, attempts at formylation of Cbz-protected 4-
(trifluoromethyl)aniline merely provided unreacted starting material. Alternatively, following a
previously developed method starting from commercially available 4-(trifluormethyl)aniline, the
desired carboxylic acid was prepared as shown in Scheme 2.23.
54

Reagents and conditions: (a) I
2
, AgSO
4
, ethanol, rt, dark, 18 h, 36%; (b) pyruvic acid, DABCO, Pd(OAc)
2
,
DMF, 110°C, 18 h, 78%.

Scheme 2.23. – Synthesis of trifluoromethyl-functionalized carboxylic acid.
Compounds in Figure 2.20, used to provide an initial evaluation of the impact on
biological activity resulting from the inclusion of a nitrogen-containing heterocycle in Region A,
were obtained via the general synthetic route described earlier in Scheme 2.19. Despite the
modest activity of 2.129, the added hydrophobicity provided by the phenyl substituent of 2.131
did not provide better activity than the un-brominated indole (2.128).
43

Figure 2.20. – Compounds with pyrrole or indole in Region A.
An additional set of select indole-containing compounds were synthesized to explore the
loss or gain of activity from the inclusion of additional methylene linkers, either within the central
linker or as part of the end capping group (Figure 2.21).

Figure 2.21 – Additional indole-containing compounds.
Extending the central aliphatic linker by one carbon (2.134) provided the most potent
compound synthesized in the course of the study.

Figure 2.22 - Compound comprised of indole in Region A and extended linker.
44

Based upon the high levels of activity given by the indole structure, the utility of a
benzimidazole was also briefly investigated (Figure 2.23). These analogs were synthesized
following the general method outlined in Scheme 2.18.

Figure 2.23. – Invesigation of benzimidazole in Region A.
To further evaluate modifications to the structural design, two compounds were
synthesized in which a carbamate replaced the amide bond between Region A and the aliphatic
linker (Figure 2.24).

Figure 2.24. – Compounds with an aliphatic linker containing carbamate.
These compounds were synthesized according to the general procedure described in
Scheme 2.18, with 2.136 furnished through deprotection of intermediate 2.86. Synthesis of 2.137
was carried out according to Scheme 2.14. However, the coupling of 3-pyridinemethanol with
2.88 was conducted using CDI, DBU, and triethylamine as described in Scheme 2.24 instead of
the typical HBTU reaction conditions.

Reagents and conditions: (a) 3-pyridinemethanol, CDI, DBU, NEt
3
, THF, 0°C to rt, 18 h, 29%.
Scheme 2.24. – CDI-promoted coupling to synthesize intermediate 2.139.
45

The alteration from the amide to carbamate linker resulted in modest activity. It was
found, however, that the inclusion of a pyridine (2.137) moiety lead to an improvement in the
response observed in the standard luciferase assay as compared to 2.136.
2.3.4. Synthesis and evaluation of aromatic linker.
It was next considered that a conformational change in the structural design resulting
from use of a carbmate in place of an amide bond and inclusion of an aromatic ring in the core
could be beneficial for binding to MEF2. Synthesis of 2.144 to test this is outlined in Scheme
2.25. Acylation of 2.142 provided carboxylic acid intermediate 2.143 and condensation of 2.143
with 1,2-phenylenediamine using HBTU and Hünig’s base furnished 2.144.

Reagents and conditions: (a) benzyl chloroformate, NaOH, dioxane, 0°C to rt, 10 h, 22%; (b) 1,2-
phenylenediamine, HBTU, DIPEA, CH
3
CN, rt, 12 h, 11%.

Scheme 2.25. – Synthesis of 2.144.
A similar fluorine-containing compound was also synthesized. Due to the low-yield
obtained through the route described in Scheme 2.25, an alternative strategy was used to
construct 2.146 (Scheme 2.26).
46

Reagents and conditions: (a) 3-(trifluoromethyl)benzyl alcohol, CDI, DBU, NEt
3
, THF, 0°C to rt, 16 h,
74%; (b) 1,2-phenylenediamine, HBTU, DIPEA, DMSO, rt, 8 h, 26%.

Scheme 2.26. – Synthesis of trifluoromethylated compound 2.146.
Using CDI-promoted coupling of commercially available 3-(trifluoromethyl)benzyl
alcohol and amine 2.142, intermediate 2.145 was obtained in good yield. Typical amino acid
coupling conditions were then used to furnish 2.146. This compound, however, lacked biological
activity and this prompted the investigation of other structural variations.
To achieve this, different synthetic routes were implemented to modify related structures
comprised of an aromatic core (Figure 2.25).

Figure 2.25 - Compounds containg a central aromatic ring.
In one synthetic route (Scheme 2.27) conversion of 2.150 to the corresponding acid
chloride followed by acylation of 2-nitroaniline gave intermediate 2.151. Even when it was
attempted to force the acylation by refluxing this step was plagued by low yield, due to the
presence of the strongly electron-withdrawing ortho-nitro group on the aniline. Fortunately, Boc-
47

deprotection of 2.151 under acidic conditions was not problematic and coupling of 2.152 to
pyridine acetic acid using HBTU provided the desired intermediate in moderate yield. Final
reduction of the nitro group did not proceed well. Low conversion to the amine was observed,
which may have been a result of coordination of the pyridine nitrogen to the palladium catalyst.
Therefore, the lack of reactivity necessitated the isolation of 2.154 via column chromatography.

Reagents and conditions: (a) (i) (COCl)
2
, pyridine, DMF, toluene, rt, 6 h; (ii) 2-nitroaniline, pyridine, rt, 12
h, 14%; (b) HCl/MeOH, rt, 12 h, 98%; (c) 3-pyridylacetic acid hydrochloride, HBTU, DIPEA, DMF, rt, 5
h, 65%; (d) H
2
, 5% Pd/C, MeOH, rt, 4 h, 22%.

Scheme 2.27. – Synthesis of 2.154.
The synthetic plan was then altered to avoid using the problematic nitro group. As
outlined in Scheme 2.28, the ortho-amine was alternatively installed using orthogonal protecting
groups. Phthalimide protection gave 2.155 in good yield. Coupling with Boc-protected
phenylenediamine and subsequent phthalimide deprotection with hydrazine hydrate then provided
the free amine 2.157 in high yield. Compound 2.148 was then readily furnished by the HBTU-
48

promoted coupling of 2.157 with commercially available nicotinic acid and Boc-deprotection (not
shown).

Reagents and conditions: (a) phthalic anhydride, acetic acid, 100°C, 4 h, 88%; (b) tert-butyl (2-
aminophenyl)carbamate, HBTU, DIPEA, DMF, rt, 16 h, 50%; (c) hydrazine hydrate, ethanol, 60°C, 12 h,
98%.

Scheme 2.28. – Alternative strategy to install ortho-amine functional group.
Another route which would leave installation of the ortho-aniline until the last step of the
synthesis was also explored (Scheme 2.29). Coupling of various carboxylic acids with amino
esters 2.158 using HBTU allowed for formation of intermediates 2.159. Subsequent hydrolysis of
the esters permitted another HBTU-coupling step to give final compounds 2.161 which were then
submitted to biological assay.

Reagents and conditions: (a) aryl or heteroaryl carboxylic acid, HBTU, DIPEA, DMF, r.t, 3-16 h, 52-74%;
(b) LiOH, THF, rt, 3-5 h, 43-83%; (c) 1,2-phenylenediamine, HBTU, DIPEA, DMF, rt, 3-16 h, 67-82%.

Scheme 2.29. – Alternative plan used to install ortho-amine in last step.
49

A variation of this synthetic plan was also used to obtain a compound which included
ortho-amine functionality on both sides of the central aromatic core. As depicted in Scheme
2.30, amino acid coupling, hydrolysis, and a second HBTU-promoted coupling were used to
obtain 2.164. Final deprotection of both Boc-protecting groups using an excess of trifluoroacetic
acid gave 2.165.

Reagents and conditions: (a) methyl 4-(aminomethyl) benzoate monohydrochloric acid, HBTU,
DIPEA, DMF, rt, 16 h, 59%; (b) LiOH, THF, rt, 12 h, 48%; (c) tert-butyl (2-aminophenyl)carbamate,
HBTU, DIPEA, DMF, rt, 16 h, 59%; (d) CF
3
COOH,CH
2
Cl
2
, 0°C to rt, 12 h, 26%.

Scheme 2.30. – Synthesis of 2.165.
Based upon our earlier studies (Chapter 2.2) which indicated that an indole was
beneficial for inhibition of the interaction between MEF2 and HDACs, a set of selected
compounds was also created to investigate the impact of alkyl spacers between the indole moiety,
amide bond, and aromatic core (Figure 2.26).
50

Figure 2.26 - Variations in linker length between Region A indole and aromatic linker.
The modest activity displayed by these compounds containing an indole and an aromatic
ring in the central linker region was encouraging.
To briefly investigate whether a smaller compound still containing the ortho-amine
functional group could still confer biological activity, 2.172 was synthesized. N-acetylation of
para-amino benzoic acid and HBTU-promoted coupling of intermediate 2.171 with 1,2-
phenylenediamine rapidly furnished this test compound (Scheme 2.31). Interestingly, this
compound displayed good activity in the standard luciferase assay.

Reagents and conditions: (a) NaOAc, acetic acid, 100°C, 18 h, 46%; (b) 1,2-phenylenediamine, HBTU,
DIPEA, DMF, rt, 12 h, 37%.

Scheme 2.31. – Synthesis of 2.172.

51

2.3.5.
19
F NMR analysis of small molecule/MEF2 binding.
The utility of NMR as a technique to identify ligand-binding interactions has become
apparent in recent years. To investigate the binding activity of a small molecule ligand with a
protein receptor, the most practical methods are those which can readily obtain useful information
without using large amounts of material. It is typically desirable to use procedures that are highly
sensitive, which is a benefit of NMR analysis, and includes the study of chemical shifts, NOEs,
and relaxation times.
55
NMR spectroscopy can be used to evaluate the signals arising from the
ligand of interest or to observe shift changes in the protein, making these techniques extremely
powerful.
Three fluorine atoms for biochemical screening (3-FABS) is a NMR-based method that
uses a trifluoromethylated probe molecule to evaluate substrate binding and can be used to
determine IC
50
values.
56
The advantages of 3-FABS arise from the sensitivity of the fluorine
nuclei, which are 0.83 times more sensitive than protons, and the signal strength of the
trifluoromethyl moiety. Due to the presence of three fluorines, the trifluoromethyl
19
F signal is
typically observed as an intense, sharp singlet. Without any other fluorine atoms present on the
probe molecule or protein, analysis of chemical shifts or signal broadening is unambiguous.
Also, in comparison with other biochemical assays, monitoring
19
F NMR does not require the use
of additional reagents or washes which can introduce additional variables to the experimental
design.
To examine the capability of small molecules to bind to MEF2 in solution, it was
therefore considered that use of
19
F-NMR would be a rapid method to detect this interaction. For
this purpose, an initial screen was conducted using 2.41 and 2.42 to determine optimal
concentration and acquisition parameters. Based upon observation of a more intense signal, due
to the presence of three fluorine atoms instead of one, 2.41 was selected for analysis.
52

Two separate samples were prepared, one which contained 5μM 2.41 and 5μM MEF2
protein and another which contained 5μM 2.41 and 10μM MEF2 protein. After 30 min. of
incubation of the CF
3
-labeled compound with protein the
19
F NMR spectra were acquired. As
shown in Figure 2.27, the CF
3
resonance of the free compound was observed at -62.86 ppm in
both the top and bottom spectra and that of the bound compound was at -63.23 ppm. This
demonstrated that in the presence of excess amounts of protein, the small molecule ligand was
able to bind to the protein in solution.

Figure 2.27 - Shift in CF
3
resonance upon small molecule binding to MEF2. Top spectrum: 5
μM 2.41 and 5 μM MEF2. Bottom spectrum: 5 μM 2.41 and 10 μM MEF2.

It was also possible to observe binding in solution by detecting a shift in CF
3
resonance
near the K
d
of the binding interaction in which the concentration of both 2.41 and MEF2 protein
in the sample solution was 500 nM. These experiments provided evidence of small molecule
binding in solution.

53

2.4. Synthesis of MEF2/HDAC interaction inhibitors related to the natural
product Trichostatin A.

2.4.1. Biological activity and prior syntheses of Trichostatin A.

The hydroxamic acid trichostatin A was first isolated from cultures of Streptomyces
hygroscopicus as an antifungal antibiotic against Trichophyton sp.
57
It was then found that
trichostatin A exhibited potent cytotoxicity and was capable of inducing differentiation of Friend
leukemia cells.
58
By conducting stereoselective syntheses of the natural R-(+)- and unnatural S-(-
)-trichosatin A molecules, it was determined that the natural enantiomer displayed 70 times
higher levels of biological activity.
59
Additionally, nanomolar concentrations of R-(+)-
trichostatin A were also found to cause an accumulation of acetylated histones and inhibit histone
deacetylase (HDAC) activity.
60

Due to its intriguing biological activity as a potent HDAC inhibitor, trichostatin A has been
used as a tool to study the enzymatic function of HDACs and as a lead compound for the
investigation of anti-cancer therapeutics.
61,62
Trichostatin A is capable of depleting mutant p53 in
cancer cells, indicating the relevance of non-cancer specific targets in cancer-related activities.
63

Also, a synergistic effect has been observed when human cancer cells were pretreated with
trichostatin A before being exposed to DNA-targeting anticancer drugs, which may result through
more ready access of the cytotoxic agents to the DNA in the presence of HDAC inhibitors.
64

A study of the metabolism of trichostatin A in mice determined that N-demethylation,
reduction of the hydroxamic acid to the amide, and oxidative deamination to provide the
carboxylic acid were the main metabolic pathways but the N-demethylated hydroxamic acid and
trichostatin A were the only metabolites which showed potent histone deacetylase activity.
65
It
was also shown that metabolism of trichostatin A occured within a rapid timeframe, which
indicated that this compound may not be amenable to drug development.
66

54

The hydrolysis product trichostatic acid was isolated from the culture fluid of Streptomyces
sioyaensis and similar to trichostatin A, the R-(+) enantiomer, but not the racemate, of trichostatic
acid displayed bioactivity.
58
Although the racemate did not induce differentiation of Friend
leukemia cells, it still exhibited cytotoxicity, however.
67

Despite the intriguing biological activity of trichostatin A, there are relatively few
reported syntheses of this natural product. Syntheses of the racemic and enantiopure forms of
trichostatin A have been carried out, the first of which was reported by Fleming and Iqbal in
1983.
68
They explored three different routes towards the racemate which involved the alkylation
of a silyl dienol ether and a silyl-protected cyanohydrin, two of which were successful. As shown
in Scheme 2.32, one method employed a Mukaiyama reaction, Wittig reaction, and DDQ
oxidation as key steps in the synthesis of trichostatin A in an overall yield of 22%. Fleming’s
second convergent synthesis using a bromo-dienoate and a silyl-protected cyanohydrin of p-
dimethylaminobenzaldehyde required a substantially longer route and provided the racemate of
trichostatin A (2.178) in 2.8% yield.

Scheme 2.32. – First reported route for synthesis of trichostatin A (2.178) in its racemic form.
68

55

In 1988 Mori and Koseki reported the total synthesis of both enantiomers of trichostatin
A and trichostatic acid.
69
To achieve high enantiopurity and avoid racemization at C-6 a strategy
was devised to install the C-7 carbonyl at a late stage, as shown in the retrosynthesis in Scheme
2.33. This route provided the natural R-(+)-trichostatin A in 6.1% overall yield in 18 steps.

Scheme 2.33. – Retrosynthesis use for first total synthesis of trichostatin A in enantiopure form.
69

More recently, there has been renewed interest in devising practical, efficient methods to
obtain this compound. A scalable route for the preparation of the racemate of trichostatin A in
61% overall yield was reported by Chatterjee et al. in 2009, as well as a method to provide
racemic trichostatic acid.
70
As shown in Scheme 2.34 the Suzuki-Miyaura coupling and
allenylmetal chemistry were used in key bond transformations. Despite the improvement in yield
and reduction in number of steps required, however, this route necessitated use of a toxic thallium
reagent. Therefore, this detracted from the practicality of this scheme.
56

Scheme 2.34. – Retrosynthetic route implemented by Chatterjee et al.
70

Another route, which made use of cheap and readily available starting materials to obtain
trichostatin A in an enantioselective manner, was reported by Zhang et al.
71
An L-proline
catalyzed aldol reaction was employed to set the stereochemistry in the first reaction of the
sequence and in 9 steps (+)-trichostatin A was obtained in 17.4% yield.
In addition to the synthesis of trichostatin A and trichostatic acid, the total synthesis of
trichostatin D, a glycosylated form of trichostatin A, has been reported.
72
Efforts have also been
made to synthesize rigid analogs of this bioactive natural product as well by including an
indanone in place of the benzene ring found in trichostatin.
73

2.4.2. Synthesis of novel compounds related to trichostatin A.
Despite the commercial availability of trichostatin A as a reference material for the
evaluation of HDAC activity, albeit at high cost, trichostatic acid is not available. To rapidly
obtain the carboxylic acid of this natural product to evaluate its biological activity and to employ
it as a building block for the synthesis of other trichostatin derivatives, it was determined that the
strategy used by Zhang et al. was the most efficient and practical method which could be adapted
for the synthesis of racemic trichostatic acid.
71
The retrosynthesis, which made use of Wittig and
aldol reactions as key steps, is shown in Scheme 2.35.
57

Scheme 2.35. – Retrosynthetic route to obtain trichostatic acid based upon work by Zhang et al.
71

Although it was necessary only to obtain the racemic form of trichostatic acid, due to the
low cost and ready availability of L-proline, the initial cross-aldol reaction between p-
nitrobenzaldehyde and propionaldehyde was carried out using this amino acid as a catalyst. This
reaction had been demonstrated to provide excellent enantioselectivity and an optimal anti/syn
ratio.
71
Due to instability of the aldol adduct 2.189, this crude compound was then converted to
ester 2.193 by a Wittig reaction which gave an unseparable mixture of 2.193 and 2.194. It was
attempted to proceed with the synthesis and separate the isomers at a later stage, however
additional elaboration of the molecule did not alter the chromatographic profile of the isomers
adequately to allow for their separation. As a result, a different route was implemented.
58

Reagents and conditions: (a) propionaldehyde, L-proline, DMF, 0°C, 6 h; (b) 2-
(triphenylphosphoranylidene)-propionaldehyde, toluene:CH
2
Cl
2
(5:1), 50°C, 6h.

Scheme 2.36. – Route which provided an unseparable mixture of isomers.
Alternatively, after the initial aldol reaction between 2.185 and 2.190 as shown in
Scheme 2.36, the Wittig reaction shown in Scheme 2.37 was carried out. DIBAL reduction of
ester 2.195 to alcohol 2.196 proceeded in high yield and subsequent oxidation of 2.196 gave
aldehyde 2.197 in 52% yield.

Reagents and conditions: (a) (1-methoxycarbonylethylidene)triphenylphosphorane, CH
2
Cl
2
, 50°C,
3.5 h, 45% over two steps from #.#; (b) DIBAL-H, THF, -78°C, 1 h, 95%; (c) MnO
2
, CH
2
Cl
2
, rt, 5 h, 52%.

Scheme 2.37. – Wittig reactions to furnish diene 2.197.
59

A Horner-Wadsworth-Emmons reaction of 2.197 with triethyl phosphonoacetate
provided ester 2.193 in 87% yield (Scheme 2.38). Following the method of Zhang et al.
reduction of the nitro group to the primary aromatic amine was carried out in the presence of
Lindlar’s catalyst and quinoline to avoid reduction of the diene.
71
Reductive amination of the
amine to form the dimethylamine occurred sluggishly to provide 2.188.

Reagents and conditions: (a) triethyl phosphonoacetate, NaH, THF, rt, 1 h, 87%; (b) H
2
, Lindlar's catalyst,
quinonline, MeOH, rt, 5.5 h, 84%;(c) HCHO, NaBH(OAc)
3
, THF, rt, 36 h, 50%.

Scheme 2.38. – Approach to 2.188 following Zhang et al.
71

At this point, our strategy diverged from that reported by Zhang et al. From intermediate
2.188, trichostatic acid could be furnished in two steps. Base hydrolysis of 2.188 using LiOH
provided 2.199, which was used without further purification. Finally, DDQ oxidation was
conducted rapidly to mitigate potential side reactions and trichostatic acid 2.187 was therefore
obtained in an overall yield of 6%.

Reagents and conditions: (a) LiOH, MeOH, 50°C, 12 h, 80%; (b) DDQ, CH
2
Cl
2
:H
2
O (2:1), 94%.
Scheme 2.39. – Synthesis of trichostatic acid (2.187).
60

Previous reports indicate that the hydroxamic acid functionality of trichostatin A permits
chelation to zinc in the active site of HDACs.
74
However, due to our interest in the alternative
target of MEF2 instead of the HDACs, it was considered that the hydroxamic acid would not be
necessary for ligand binding and potent activity. As a consequence, we sought to synthesize a
hybrid compound containing the p-dimethylaniline and diene of trichostatin and the ortho-
anilidine functionality of previously synthesized and analyzed compounds as discussed in
Chapter 2.2. Synthesis of this novel trichostatin derivative from trichostatic acid was
straightforward. As shown in Scheme 2.40, HBTU promoted coupling of trichostatic acid 2.187
with 1,2-phenylenediamine provided 2.200. The overall yield to obtain this compound was 5%.

Reagents and conditions: (a) 1,2-phenylenediamine, HBTU, DIPEA, CH
3
CN, rt, 18 h, 82%.
Scheme 2.40. – Synthesis of ortho-anilidine derivative of trichostatin A.
Knowing that the carboxamide of trichostatin A was reported to be one of its metabolites,
this compound was also of interest. Synthesis of 2.201 had not been reported, but the most
efficient route involved conversion of the carboxylic acid to the carboxamide. Typical methods
for this transformation did not appear to be ideal for this substrate because procedures for
carboxamide formation either involved condensation of an acid chloride with ammonium
hydroxide or necessitated the use of ammonia gas at high pressure. Formation of the acid chloride
of trichostatin was hoped to be avoided due to the presence of other acid sensitive functionality.
Therefore, a simple method was used in which HBTU promoted the coupling between trichostatic
61

acid and an excess of ammonium hydroxide, as shown in Scheme 2.41. Overall yield for this
synthetic route was 2%.

Reagents and conditions: (a) NH
4
OH, HBTU, DIPEA, CH
3
CN, rt, 18 h, 33%.

Scheme 2.41. – Synthesis of carboxamide of trichostatin A.
Biological analysis of 2.187, 2.188, 2.199, 2.200, and 2.201 conducted by the Lin Chen
lab indicated that the most potent inhibitory activity was demonstrated by the ortho-anilidine
functionalized trichostatin derivative 2.200.

2.5. Synthesis of helix-constrained peptides as MEF2 inhibitors.
2.5.1. Structure and biology of constrained peptides.
The α-helix, a common structural motif found in peptides, is capable of mediating
protein-protein interactions that impact physiological processes. However, despite their utility as
biological probes, peptides are often considered to be poor candidates for drug development due
to their loss of secondary structure in solution and lack of stability in the presence of proteases.
Recently, the use of chemical methods to enhance the structure of peptides and improve their
properties has garnered interest.
75
Commonly, α-helices have been stabilized through side chain
tethers via formation of disulfide bonds or cyclization to produce a lactam between residues
spaced two to eight positions apart.
76-82
The use of all hydrocarbon tethers has more recently
62

emerged as another powerful strategy to cyclize peptides while also imparting desirable
biological activity.
83-86

The incorporation of α,α-dialkylated amino acids within the peptide sequence has also
been demonstrated to provide enhancement of α-helical content and consequently improve
structural constraints in comparison to linear native peptide sequences.
85
Although ruthenium-
catalyzed olefin metathesis had been highlighted as a successful method for cross-linking peptide
side chains, this approach did not improve peptide helical content.
83
In a study screening several
configurations of all-hydrocarbon cross-linkers, while also making use of α,α-dialkyl amino
acids, it was determined that with the proper choice of configuration significant helix-
stabilization could be observed.
84

Triazolyl-amino acids can also help optimize secondary structure and triazoles have been
explored as structural mimics of amide bonds as well.
87,88
The 1,4-disubstituted [1,2,3]triazole
mimics a trans-amide bond because the distance between the 1 and 4 positions is approximately
one angstrom longer than that of two carbons separated by an amide bond. Additionally, the
dipole moment of the triazole allows for the N2 and N3 electron lone pairs to act as hydrogen
bond acceptors. Use of the copper(I)-mediated azide-alkyne cycloaddition (CuAAC) reaction
methodology to form a triazole is advantageous in solid-phase peptide synthesis because these
functional groups are stable to standard peptide synthesis and cleavage methods. The azide and
alkyne are slow to react without the addition of a catalyst; therefore until the CuAAC reaction is
conducted there is minimal risk for undesired by-product formation. Stepwise incorporation of
N
α
-Fmoc-protected amino acids with azidyl and propargylic amino acids at the i and i + 4
positions, followed by CuAAC cyclization, therefore serves as a method to synthesize helix-
constrained peptides.
63

To probe the interaction between MEF2 and HDACs the investigation of peptide ligands
would provide insight complementary to that gained from the series of small molecules studied,
as discussed in Chapters 2.2 and 2.3. Based on molecular modeling by Kevin Gaffney, a short
consensus sequence for MEF2 binding thus served as the basis for design of these synthetic
peptides and the use of structural constraints to promote helix-stabilization would improve the
stability of the peptides in solution.
99
Synthesis of linear peptides, followed by cyclization across
key side chains, was anticipated to permit analysis of novel modifications of the native peptide
ligands which bind to MEF2.
2.5.2. Copper assisted click chemistry cyclization method.
Before synthesis of the linear precursor could be undertaken, it was necessary to access
Fmoc-protected ε-azido norleucine, which would be used for triazole formation via CuACC
cyclization. Although it is possible to perform selective diazo transfer on-resin to provide the
azide functionality needed for cyclization, reported protocols for this require the use of triflyl
azide in dichloromethane.
87
Due to the potential formation of hazardous side-products, such as
azido-chloromethane and diazidomethane, and because of the desire to create a stock of the
amino acid building blocks, the preparation of azide-functionalized amino acids was carried out
prior to initiating solid phase peptide synthesis.
Initially, a multi-step strategy was attempted to convert L-hSer to N
α
-Fmoc-Abu(γ-N
3
)-
OH following a known protocol.
89
As shown in Scheme 2.42, L-hSer was first converted to α-
amino-γ-bromo-L-butyric acid·HBr (2.203) by refluxing in a solution of hydrogen bromide in
acetic acid. After conversion of 2.203 to the hydrochloride salt and Boc-protection, a Finkelstein
reaction provided iodo-compound 2.206 in good yield. Conversion to azide 2.207 also proceeded
well; however, the final Boc-deprotection, saponification, and Fmoc-protection sequence was
unsuccessful. Although these last steps were reported to have been carried out without isolation
64

or purification of intermediates, attempts to reproduce this strategy provided a mixture of several
compounds which were not separable.

Reagents and conditions: (a) HBr/AcOH, 115°C, 5 h, 82%; (b) SOCl
2
, MeOH, 0°C, 8 h, 99%;
(c) Boc
2
O, NaHCO
3
, dioxane/H
2
O, 0°C, 16 h, 73%; (d) NaI, acetone, 60°C, 2 h, 84%; (d) NaN
3
,
DMF, 90°C, 2 h, 82%; (e) (i) CF
3
COOH (ii) NaOH (iii) Fmoc-OSu, dioxane.

Scheme 2.42 - Unsuccessful route towards N
α
-Fmoc-Abu(γ-N
3
)-OH.
Alternatively, it was envisioned that use of an imidazole-1-sulfonate diazo transfer
reagent (Scheme 2.43) could provide the desired azidyl amino acid in one step.
90

Reagents and conditions: (a) imidazole-1-sulfonyl azide hydrochloride, K
2
CO
3
,Cu(SO
4
) 5H
2
O, MeOH, rt.

Scheme 2.43. – Alternative synthesis of azide-functionalized amino acid building block.
65

While revising the synthesis of the azidyl-amino acid, it was also decided to alter the
structure of the side chain tether of the helix-constrained peptide to make use of a commercially
available Fmoc-propargyl glycine as the alkyne precursor. Therefore, to compensate for the
shorter side chain of the alkynyl-amino acid building block, lysine was then to be used as a
precursor to the azidyl-amino acid rather than homoserine. The solid phase method to create the
linear peptide would follow an Fmoc strategy rather than a Boc method, and as a result it was
necessary to alter the conditions of the diazotransfer reaction from those reported.
90
As depicted
in Scheme 2.44, it was necessary to substitute sodium bicarbonate for potassium carbonate to
avoid deprotection of the Fmoc group. This procedure allowed for successful synthesis of Fmoc-
protected ε-azido norleucine in good yield on gram scale.

Reagents and conditions: (a) imidazole-1-sulfonyl azide hydrochloride, NaHCO
3
,Cu(SO
4
) 5H
2
O, MeOH,
rt, 16 h, 78%.

Scheme 2.44 - Modified diazotransfer reaction.
To investigate the interaction between HDACs and MEF2 a short amino acid sequence of
HDAC9 was selected for further study. Within this sequence, it had been demonstrated that
mutation of leucine to alanine in CABIN1, HDAC4, and HDAC9 abolished the ability of these
ligands to bind to the hydrophobic pocket formed by Leu66, Tyr69, and Thr70 of MEF2.
91
To
preserve this crucial binding interaction, the peptides to be used for analysis were designed in a
manner that would not only incorporate this leucine residue in the structure, but also not interfere
with this hydrophobic interaction after cyclization. To incorporate a crosslink over one turn,
66

unnatural amino acids would be placed at the i, i +4 positions. This location for modification was
chosen based upon a systematic study conducted in the Verdine laboratory which had shown that
placing two S-configured amino acids at the i and i +4 positions formed an effective crosslink.
84
Thus for our study (S)-amino acids would be incorporated at the crosslinking positions of the
designed peptides of interest.
To construct the linear peptide precursor a standard solid phase synthesis method using
sequential Fmoc-deprotection and amino acid couplings was employed. An overview of this
route is depicted in Scheme 2.45.

Scheme 2.45 - Synthesis of helix-constrained peptide using CuACC cyclization.
Rink amide resin was deprotected by treatment with 4-methylpiperidine (25% in DMF)
two times, first for five minutes and then for thirty minutes. The resin was then washed
67

sequentially with DMF and CH
2
Cl
2
. As illustrated in Scheme 2.45, the subsequent Fmoc-
protected amino acid was coupled for thirty minutes (or sixty minutes for cross-linkers) in the
presence of an excess of HATU and DIPEA in DMF while bubbling with nitrogen. After the
coupling step, the resin was again washed with DMF and then with CH
2
Cl
2
. A small portion of
resin was then removed to check for completion of the reaction using the colorimetric Kaiser test.
For incomplete reactions, the resin was re-subjected to coupling with the same Fmoc-amino acid.
If a positive Kaiser test was observed after three couplings, or if the initial coupling was
complete, the resin was capped with acetic anhydride in the presence of piperidine for fifteen
minutes. After again washing with DMF followed by CH
2
Cl
2
the Fmoc group was removed in
the presence of 4-methylpiperidine and the synthesis was continued using the next Fmoc-
protected amino acid in the same manner described. Global deprotection and cleavage of the
peptide from the resin was conducted using a solution of 5% H
2
O and 95% TFA. After
precipitation of the peptide in cold ether HPLC purification provided the pure linear precursor.
For the CuAAC reaction, a solution-phase intrachain reaction was carried out using and
excess of CuSO
4
·5H
2
0 and ascorbic acid in tBuOH/H
2
O (1:2 v/v). Conversion to the cyclized
product was monitored by a combination of HPLC and mass spectroscopic analysis.
Despite successfully attaining the desired constrained peptide 2.213, a substantial amount
of material was lost due to precipitation both in the reaction solvent and HPLC solvents.
Attempts to mitigate this by using MeOH/H
2
O or MeOH in place of the standard tBuOH/H
2
O
solvent system did not provide increased yield.
It was then attempted to carry out the click cross-linking cyclization with the synthesized
peptide still on the resin. Due to the inability of the Rink amide resin to swell properly in alcohol
or water solvents, alternative reaction conditions were investigated. A survey of the literature
indicated that the scarce examples of on-resin intramolecular CuAAC macrocyclizations were not
68

reliable, often giving inconsistent or undesired products.
89,92-95
Although use of CuI in DMF had
been demonstrated to promote macrocyclization, use of these conditions primarily gave a product
with a mass 127 molecular weight units higher than expected. This indicated formation of an
iodo-substituted triazole product rather than desired peptide 2.213. To reduce the potential for
peptide aggregation DMSO was used instead of DMF, but with no success. Unfortunately, no
reaction was observed in the presence of CuBr and sodium ascorbate either, which had been
demonstrated to promote cyclization of a helical peptide after the failure of a wide range of other
conditions.
96
Although in this instance it was not possible to successfully carry out the on-resin
cyclization, in the future it may be worthwhile to investigate the use of alternative resins, such as
Tenta-gel, which are compatible with aqueous solvent systems and therefore could be used under
the same reaction conditions used to promote the solution-phase cyclization.
2.5.3. Hydrocarbon stapled method.
To capitalize on the helix-stabilization imparted through the combination of an olefin
cross-linker and α,α-dimethyl amino acids, it was first necessary to access the requisite Fmoc-
protected amino acid which would be incorporated into the final peptide structure. Based on
reports of its utility in large scale asymmetric syntheses, an alanine-Ni(II)-benzophenone complex
was used to synthesize (S)-Fmoc-α-(2’-pentenyl)alanine.
97,98
As shown in Scheme 2.46, the Ni-
complex was synthesized in three steps and subsequent nucleophilic addition allowed for
installation of the aliphatic alkene. Acidic decomposition of the nickel complex provided the free
amino acid and final Fmoc-protection gave desired amino acid 2.219. The protection step was
found to be sensitive to pH; use of more than ten equivalents of base caused reformation of the
Ni-complex as a major side reaction.
69

Reagents and conditions: (a) (i)
i
PrOH, KOH, 40°C, 30 min.; (ii) benzyl chloride, 40°C, 16 h;
(iii) HCl, CH
3
Cl, rt, 18 h, 70%; (b) (i) SOCl
2
, CH
2
Cl
2
, 0°C to rt, 1 h; (ii) o-aminobenzophenone,
CH
2
Cl
2
, rt, 16 h; (iii) Na
2
CO
3
, H
2
O, rt,1 h, 50%; (c) alanine, Ni(NO
3
)
2
, KOH, MeOH, 65°C, 2 h,
90%; (d) 5-bromo-1-pentene, NaOH, DMF, rt, 1 h, 82%; (e) (i) HCl/MeOH, 80°C, 20 min.;
(ii) Fmoc-OSu, Na
2
CO
3
, water/acetone, rt, 30 min., 62%.

Scheme 2.46. – Use of Ala-Ni-BPB to synthesize α-mehyl-α-amino acid.
To synthesize the peptides of interest (Figure 2.28), a standard solid phase protocol was
used, as described earlier; however, olefin metathesis using Grubb’s generation I catalyst was
employed to form the ‘stapled’ hydrocarbon. Surprisingly, this transformation required a long
reaction time, although based on mass spectroscopic analysis it did proceed to completion upon
repeated addition of aliquots of catalyst. Deprotection of side chains and cleavage of the peptide
from the resin was finally conducted using a solution of 5% H
2
O and 95% TFA.
70

Figure 2.28 - Structure of helix-constrained peptide synthesized by olefin metathesis
cyclization.

2.6. Experimental.
All reactions, unless noted otherwise, were conducted using commercially available
solvents and reagents as received, without additional preparation or purification, in ordinary
glassware.
1
H,
13
C, and
19
F spectra were recorded on Mercury 400, Varian 400-MR (400 MHz),
Varian VNMRS-500 (500 MHz) 2-channel, or Varian VNMRS-600 (600 MHz) 3-channel NMR
spectrometers, using residual
1
H or
13
C signals of deuterated solvents as internal standards. Silica
gel (60 Å, 40-63 μm; Sorbent Technologies) was used as a sorbent for flash column
chromatography. Automated flash chromatography was performed on Isolera One flash
purification system (Biotage), default fraction volume – 12 mL. Peptide coupling reactions were
conducted in a fritted glass peptide synthesis vessel from Synthware.

71

GENERAL PROCEDURES
Method A: Monoacid Formation Using Neat Conditions
A mixture of dicarboxylic acid (1 equiv) and aniline (1 equiv) was heated to 140°C for 24 h. The
reaction mixture was cooled to room temperature, diluted with ethyl acetate (100 mL) and
extracted into 5% KOH solution (200 mL). The aqueous phase was cooled in an ice bath and
concentrated HCl was added dropwise to adjust the pH to 2. The aqueous phase was then
extracted into ethyl acetate (2 x 200 mL). The combined organic phases were dried over Na
2
SO
4
and concentrated under reduced pressure.
Method B: Amide Formation Using HBTU
To a mixture of carboxylic acid (1 equiv) in DMSO or DMF (1mL/mmol) was added
diisopropylethylamine (1-5 equiv) and the mixture was stirred for 5 min. Then HBTU (1 equiv)
and aniline (1 equiv), respectively, were added. After stirring at room temperature from 3-12 h
the reaction mixture was diluted with ethyl acetate (50 mL) and washed consecutively with brine
(50 mL) and saturated NaHCO
3
solution (3 x 50 mL). The organic layer was dried over Na
2
SO
4

and the solvent was evaporated under reduced pressure.
Method C: Boc-deprotection of Amine
To a solution of the Boc-protected amine in CH
2
Cl
2
(2mL/mmol) was added trifluoroacetic acid
(25 equiv) dropwise under nitrogen at 0°C and the mixture was stirred while warming to room
temperature for 3 h. The reaction mixture was diluted with CH
2
Cl
2
(25 mL) and washed with a
solution of saturated NaHCO
3
(3 x 50 mL). The organic layer was dried over Na
2
SO
4
and
concentrated under reduced pressure.
Method D: Suzuki coupling of Aryl Bromide
72

To a mixture of aryl bromide (1 equiv), boronic acid (1.2 equiv), and
tetrakis(triphenylphosphine)palladium (0) (3 mol%) in dioxane (10mL/mmol) was added a
solution of K
2
CO
3
(3 equiv) in water (0.5mL/mmol) at room temperature. After flushing with N
2

the reaction mixture was stirred at 80°C for 18 h. The reaction mixture was cooled to room
temperature, then poured into a saturated solution of NaHCO
3
(15 mL) and extracted with ethyl
acetate (3 x 15 mL). The combined organic layers were washed sequentially with water (25 mL)
and brine (25 mL), dried over Na
2
SO
4
and concentrated under reduced pressure.
Method E: Base Hydrolysis of Ester
To a solution of ester (1 equiv) in THF (2 mL/mmol) was added a 1M solution of aqueous LiOH
(2 equiv) at room temperature. After stirring at room temperature for 2-5 h a 0.5M NaOH
aqueous solution (10 mL) and ethyl acetate (50 mL) were added to the reaction mixture. The
phases were separated and the aqueous layer was acidified to pH 3 with 10% HCl solution. The
aqueous layer was extracted into ethyl acetate (3 x 25 mL) and the combined organic layers were
dried over Na
2
SO
4
and concentrated under reduced pressure.
Method F: Esterification of Aliphatic Amino Acid
To a suspension of amino acid (1 equiv) in methanol (10 equiv) at 0°C was added thionyl
chloride (2.5 equiv) dropwise. The reaction mixture was then refluxed for 16 h. After cooling to
room temperature, volatiles were evaporated under reduced pressure.
Characterization Data

73

2.10. 7-((2-((tert-butoxycarbonyl)amino)phenyl)amino)-7-oxoheptanoic acid (13). A
mixture of pimelic acid (0.500 g, 3.12 mmol), 12 (0.650 g, 3.12 mmol), DIPEA (0.543 mL, 3.12
mmol), and HBTU (1.18 g, 3.12 mmol) was dissolved in DMSO (5 mL) and stirred at room
temperature for 24 h. The reaction mixture was diluted with ethyl acetate (20 mL) and washed
with brine (20 mL). The organic layer was then extracted into 5% KOH aqueous solution. The
pH of the aqueous phase was adjusted to 3 using concentrated HCl and extracted into ethyl
acetate (50 mL). The organic layer was dried over Na
2
SO
4
and concentrated under reduced
pressure to give an orange oil. Recrystallization from acetonitrile/water gave 0.515 g as a white
solid (47%). TLC: 70% EtOAc/hexanes, R
f
≈ 0.1.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 7.88
(d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.48-7.58 (m, 3H), 7.36 (d, J = 7.0 Hz, 1H), 7.21 (t,
J = 7.5 Hz, 1H), 7.12 (t, J = 7.0 Hz, 1H), 2.44 (t, J = 7.5 Hz, 2H), 2.33 (t, J = 7.5 Hz, 2H), 1.75 (t,
J = 7.5 Hz, 2H), 1.68 (t, J = 7.5 Hz, 2H), 1.52 (s, 9H), 1.44-1.47 (m, 2H).
13
C NMR (125 MHz,
CD
3
OD) δC (ppm) 176.00, 173.74, 154.36, 131.74, 127.15, 125.99, 125.28, 124.46, 117.27,
110.06, 80.11, 35.87, 33.33, 28.29, 27.24, 25.16, 24.34.

2.13. N
1
-phenyl-N
7
-(pyridin-2-ylmethyl)heptanediamide. Synthesized according to general
procedures. Purification by automated flash chromatography using 40% EtOAc/hexanes as
eluent gave 0.047 g as a white solid (34%). TLC: 100% EtOAc, R
f
≈ 0.5.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 8.44 (d, J = 4.0 Hz, 1H), 7.75 (t, J = 6.0 Hz, 1H), 7.51 (d, J = 9.6 Hz, 2H),
7.24-7.34 (m, 4H), 7.06 (t, J = 7.2 Hz, 1H), 4.46 (s, 2H), 2.28-2.38 (m, 4H), 1.65-1.75 (m, 4H),
1.37-1.43 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 174.83, 172.98, 158.31, 148.28,
138.41, 137.42, 128.35, 123.69, 122.32, 121.43, 119.83, 46.95, 36.30, 35.36, 28.38, 25.17.
74

2.14. N
1
-(8-aminonaphthalen-1-yl)-N
7
-phenylheptanediamide. Synthesized according to
Method C. Purification by automated flash chromatography using ethyl acetate as eluent gave
0.031 g as a yellow solid (47%). TLC: 100% EtOAc, R
f
≈ 0.5.
1
H NMR (400 MHz, CD
3
OD)
δH (ppm) 7.49 (d, J = 4.0 Hz, 2H), 7.26 (t, J = 4.0 Hz, 2H), 7.00-7.08 (m, 5H), 6.44 (br s, 2H),
2.39 (t, J = 4.0 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.73-1.80 (m, 4H), 1.47-1.53 (m, 2H).
13
C NMR
(100 MHz, CD
3
OD) δC (ppm) 173.07, 159.03, 135.51, 130.38, 128.45, 127.82, 123.73, 123.66,
121.51, 120.02, 118.78, 36.27, 34.54, 28.29, 27.07, 25.13.

2.15. N
1
-phenyl-N
7
-(quinolin-8-yl)heptanediamide. Synthesized according to general
procedures. Purification by automated flash chromatography using 40% EtOAc/hexanes as
eluent gave 0.045 g as a white solid (27%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.6.
1
H NMR (400
MHz, CD
3
OD) δH (ppm) 8.84 (d, J = 4.0 Hz, 1H), 8.60 (d, J = 8.8 Hz, 1H), 8.28 (d, J = 8.4 Hz,
1H), 7.60 (d, J = 9.6 Hz, 1H), 7.48-7.54 (m, 4H), 7.24 (t, J = 10 Hz, 2H), 7.05 (t, J = 7.2 Hz, 1H),
2.61 (t, J = 7.6 Hz, 2H), 2.39 (t, J = 7.6 Hz, 2H), 1.74-1.87 (m, 4H), 1.48-1.54 (m, 2H).
13
C
NMR (100 MHz, CD
3
OD) δC (ppm) 173.07, 172.98, 148.53, 138.44, 136.23, 134.17, 128.29,
128.21, 126.54, 123.66, 122.04, 121.59, 119.85, 116.97, 36.86, 36.35, 28.37, 25.19, 25.13.
75

2.16. N
1
-(2-methoxyphenyl)-N
7
-phenylheptanediamide. Synthesized according to the general
procedures. Purification by automated flash chromatography using 60% EtOAc/hexanes as eluent
gave 0.065 g as a white solid (45%). TLC: 60% EtOAc/hexanes, R
f
≈ 0.8.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 7.92 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 7.6 Hz, 2H), 7.32 (t, J = 7.2 Hz, 2H),
7.08-7.15 (m, 2H), 7.02 (d, J = 8.0 Hz, 1H), 6.93 (t, J = 8.0 Hz, 1H) 3.88 (s, 3H), 2.40-2.50 (m,
4H), 1.75-1.82 (m, 4H), 1.47-1.53 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.24,
173.14, 150.33, 138.56, 128.37, 125.96, 123.71, 122.35, 120.02, 119.87, 110.42, 54.80, 36.37,
36.19, 28.36, 25.23.

2.17. 4-methoxy-N-(6-oxo-6-(phenylamino)hexyl)benzamide. Sythesized according to general
procedures. Purification by automated flash chromatography using 60% EtOAc/hexanes as
eluent gave 0.033 g as a white solid (23%). TLC: 60% EtOAc/hexanes, R
f
≈ 0.3.
1
H NMR (600
MHz, CD
3
OD) δH (ppm) 7.51 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.27 (t, J = 7.8 Hz,
2H), 7.05 (t, J = 7.8 Hz, 1H), 6.83 (d, J = 12.0 Hz, 2H), 3.75 (s, 3H), 2.34-2.38 (m, 4H), 1.71-
1.76 (m, 4H), 1.42-1.47 (m, 2H).
13
C NMR (151 MHz, CD
3
OD) δC (ppm) 173.07, 172.82,
156.45, 138.43, 131.36, 128.30, 123.67, 121.69, 119.85, 113.46, 54.40, 36.29, 36.14, 28.33,
25.21, 25.14.
76

2.18. N
1
-(3-bromophenyl)-N
7
-(pyridin-2-ylmethyl)heptanediamide. Synthesized according to
general procedures. Purification by automated flash chromatography using 40% EtOAc/hexanes
as eluent gave 0.105 g as a white solid (71%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.2.
1
H NMR
(400 MHz, CD
3
OD) δH (ppm) 8.45 (d, J = 4.8 Hz, 1H), 7.88 (s, 1H), 7.76 (t, J = 9.6 Hz, 1H),
7.42 (d, J = 8.8 Hz, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.24-7.28 (m, 1H), 7.19-7.25 (m, 1H), 4.46 (s,
2H), 2.36 (t, J = 7.2 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H), 1.67-1.73 (m, 4H), 1.37-1.45 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 174.83, 173.12, 157.89, 148.28, 140.08, 137.42, 129.88,
126.32, 122.34, 122.32, 121.82, 121.37, 117.99, 44.10, 37.45, 36.26, 35.33, 28.33, 25.13.

2.19. N
1
-(3-bromophenyl)-N
7
-(quinolin-8-yl)heptanediamide. Synthesized according to
general procedures. Purification by automated flash chromatography using 60% EtOAc/hexanes
as eluent gave 0.015 g as a white solid (11%). TLC: 40%EtOAc/hexanes, R
f
≈ 0.3.
1
H NMR
(600 MHz, CD
3
OD) δH (ppm) 8.84 (s, 1H), 8.60 (d, J = 7.2 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H),
7.85 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.50-7.53 (m, 2H), 7.41 (d, J = 7.8 Hz, 1H), 7.15-7.19 (m,
2H), 2.61 (t, J = 7.2 Hz, 2H), 2.39 (t, J = 7.2 Hz, 2H), 1.82-1.85 (m, 2H), 1.76-1.79 (m, 2H),
1.51-1.53 (m, 2H).
13
C NMR (151 MHz, CD
3
OD) δC (ppm) 173.19, 172.97, 148.53, 136.37,
134.11, 129.83, 128.30, 126.34, 122.37, 122.05, 121.81, 121.57, 118.09, 117.04, 99.21, 36.87,
36.34, 28.33, 25.04.
77

2.21. N
1
-(3-bromophenyl)-N
7
-(2-methoxyphenyl)heptanediamide. Synthesized according to
general procedures. Purification by automated flash chromatography using 60% EtOAc/hexanes
as eluent gave 0.065 g as a white solid (48%). TLC: 60% EtOAc/hexanes, R
f
≈ 0.8.
1
H NMR
(400 MHz, CD
3
OD) δH (ppm) 7.89 (s, 2H), 7.41 (d, J = 5.2 Hz, 1H), 7.15-7.20 (m, 2H), 7.06-
7.09 (m, 1H), 6.96 (d, J = 5.2 Hz, 1H), 6.87 (t, J = 4.0 Hz, 1H), 3.83 (s, 3H), 2.43 (t, J = 5.2 Hz,
2H), 2.38 (t, J = 4.8 Hz, 2H), 1.70-1.75 (m, 4H), 1.42-1.47 (m, 2H).
13
C NMR (100 MHz,
CD
3
OD) δC (ppm) 173.25, 173.20, 150.20, 140.13, 129.92, 126.71, 126.38, 124.86, 122.41,
122.22, 121.83, 119.99, 118.13, 110.44, 36.35, 36.21, 28.29, 25.18, 25.03.

2.20. N-(7-((2-aminophenyl)amino)-7-oxoheptyl)-1H-indole-2-carboxamide. Synthesized
according to general procedures. Purification by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.068 g as a yellow solid (59%). TLC: 90% EtOAc/hexanes, R
f
≈
0.35.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.53 (s, 1H), 9.10 (s, 1H), 8.42 (s, 1H), 7.60 (d,
J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.16-7.18 (m, 2H), 7.11 (s, 1H), 7.05 (t, J = 7.2 Hz,
1H), 6.90 (t, J = 7.2 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 6.54 (t, J = 7.2 Hz, 1H), 4.81 (br s, 1H),
3.28-3.34 (m, 3H), 2.33 (t, J = 7.2 Hz, 2H), 1.56-1.62 (m, 4H), 1.35-1.38 (m, 3H).
13
C NMR (100
MHz, DMSO-d
6
) δC (ppm)171.40, 161.41, 142.24, 136.61, 132.24, 127.49, 126.04, 125.60,
78

123.89, 123.46, 121.85, 119.99, 116.66, 116.40, 112.69, 102.55, 39.05, 36.10, 29.60, 28.89,
26.72, 25.67.

2.21. N
1
-(3-bromophenyl)-N
7
-(4-methoxyphenyl)heptanediamide. Synthesized according to
general procedures. Purification by automated flash chromatography using 60% EtOAc/hexanes
as eluent gave 0.078 g as a white solid (46%). TLC: 60%EtOAc/hexanes, R
f
≈ 0.75.
1
H NMR
(400 MHz, CD
3
OD) δH (ppm) 7.93 (s, 1H), 7.43-7.48 (m, 3H), 7.21-7.27 (m, 2H), 6.88 (d, J =
7.2 Hz, 2H), 3.80 (s, 3H), 2.38-2.44 (m, 4H), 1.74-1.81 (m, 4H), 1.45-1.51 (m, 2H).
13
C NMR
(100 MHz, CD
3
OD) δC (ppm) 173.21, 172.86, 156.47, 140.09, 131.35, 129.86, 126.34, 122.36,
121.82, 121.66, 118.06, 113.37, 54.40, 36.33, 36.12, 28.25, 25.16, 24.95.

2.22. N
1
-(2-aminopyridin-3-yl)-N
7
-phenylheptanediamide. Synthesized according to general
procedures. Purification by automated flash chromatography using 10% MeOH/CH
2
Cl
2
as eluent
gave 0.043 g as an orange solid (14%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈ 0.5.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 7.80 (d, J = 4.8 Hz, 1H), 7.52 (d, J = 8.0 Hz, 3H), 7.28 (t, J = 8.0 Hz, 2H),
7.06 (t, J = 8.0 Hz, 1H), 6.65 (t, J = 8.0 Hz, 1H), 2.37-2.46 (m, 4H), 1.72-1.79 (m, 4H), 1.44-1.52
(m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.90, 173.05, 144.19, 138.43, 133.93,
128.32, 123.68, 119.82, 118.83, 113.00, 36.26, 34.41, 28.33, 25.08, 24.99.
79

2.23. N
1
-(2-aminopyridin-3-yl)-N
7
-(3-bromophenyl)heptanediamide. Synthesized according
to general procedures. Purification by automated flash chromatography using 10%
MeOH/CH
2
Cl
2
as eluent gave 0.033 g as a tan solid (26%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈ 0.5.
1
H NMR (600 MHz, CD
3
OD) δH (ppm) 7.88 (s, 1H), 7.80 (d, J = 5.4 Hz, 1H), 7.52 (d, J = 7.8
Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.17-7.22 (m, 2H), 6.65-6.67 (m, 1H), 2.44 (t, J = 7.2 Hz, 2H),
2.39 (t, J = 7.2 Hz, 2H), 1.72-1.79 (m, 4H), 1.44-1.48 (m, 2H).
13
C NMR (151 MHz, CD
3
OD) δC
(ppm) 158.08, 144.09, 133.97, 129.89, 126.34, 122.34, 121.82, 120.33, 120.19, 118.87, 118.03,
115.20, 113.01, 104.99, 36.24, 35.54, 28.29, 24.96, 24.92.

2.24. N
1
-(4-aminopyridin-3-yl)-N
7
-phenylheptanediamide. Synthesized according to general
procedures. Purification by automated flash chromatograph using 10% MeOH/CH
2
Cl
2
as eluent
gave 0.020 g as a white solid (14%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈ 0.5.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 8.03 (s, 1H), 7.77 (d, J = 5.2 Hz, 1H), 7.47-7.52 (m, 3H), 7.27 (t, J = 7.6 Hz,
2H), 7.06 (t, J = 6.8 Hz, 1H), 2.47 (t, J = 7.6 Hz, 2H), 2.38 (t, J = 7.6 Hz, 2H), 1.71-1.79 (m, 4H),
1.43-1.48 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.44, 173.07, 138.43, 138.05,
137.94, 136.76, 131.50, 128.33, 123.71, 119.85, 117.59, 36.26, 35.84, 28.30, 25.09, 24.93.
80

2.25. N
1
-(2-aminophenyl)-N
7
-(pyridin-3-yl)heptanediamide. Synthesized according to
Method C. Purification by automated flash chromatography using 10% MeOH/CH
2
Cl
2
as eluent
gave 0.008 g as a white solid (21%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈ 0.4.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 7.80 (d, J = 4.8 Hz, 1H), 7.52 (d, J = 8.0 Hz, 3H), 7.28 (t, J = 8.0 Hz, 2H),
7.06 (t, J = 8.0 Hz, 1H), 6.65 (t, J = 8.0 Hz, 1H), 2.37-2.46 (m, 4H), 1.72-1.79 (m, 4H), 1.44-1.52
(m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.52, 173.49, 143.61, 141.89, 140.39,
136.11, 127.52, 126.83, 125.71, 123.86, 123.69, 123.38, 118.06, 117.09, 36.14, 35.55, 28.34,
25.22, 24.89.

2.26. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-[1,1'-biphenyl]-2-carboxamide. Synthesized
according to Method C. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.010 g as a white solid (43%). TLC: 80% EtOAc/hexanes, R
f
≈
0.5.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.46 (d, J = 7.2 Hz, 1H), 7.31-7.40 (m, 7H), 7.06 (d,
J = 8.0 Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.67 (t, J = 7.6 Hz, 1H), 2.38
(t, J = 8.0 Hz, 2H), 2.23 (t, J = 7.6 Hz, 2H), 1.58-1.72 (m, 4H), 1.32-1.38 (m, 2H).
13
C NMR
(100 MHz, CD
3
OD) δC (ppm) 173.79, 173.49, 141.84, 139.18, 137.89, 134.06, 130.09, 129.38,
128.73, 128.04, 127.55, 127.09, 126.87, 126.79, 126.31, 125.68, 123.30, 118.07, 117.11, 35.59,
35.56, 28.35, 25.23, 24.99.
81

2.27. N
1
-([1,1'-biphenyl]-3-yl)-N
7
-(2-aminophenyl)heptanediamide. Synthesized according to
Method C. Purified by automated flash chromatography using ethyl acetate as eluent gave 0.067
g as a white solid (50%). TLC: 100% EtOAc, R
f
≈ 0.4.
1
H NMR (600 MHz, CD
3
OD) δH (ppm)
7.84 (s, 1H), 7.58 (d, J = 7.2 Hz, 2H), 7.51 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.2 Hz, 2H), 7.31-7.38
(m, 3H), 7.05 (d, J = 6.6 Hz, 1H), 6.99 (t, J = 3.6 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.66 (d, J =
7.8 Hz, 1H), 2.42-3.30 (m, 4H), 1.76-1.81 (m, 4H), 1.48-1.54 (m, 2H).
13
C NMR (151 MHz,
CD
3
OD) δC (ppm) 173.53, 173.20, 141.76, 138.97, 128.81, 128.40, 127.05, 126.79, 126.56,
122.28, 118.60, 118.30, 118.08, 117.09, 109.99, 36.35, 35.57, 28.38, 25.27, 25.13.

2.28. N
1
-([1,1'-biphenyl]-4-yl)-N
7
-(2-aminophenyl)heptanediamide. Synthesized according to
Method C. Purification by automated flash chromatography using ethyl acetate as eluent gave
0.025 g as a white solid (4%). TLC: 100% EtOAc, R
f
≈ 0.3.
1
H NMR (400 MHz, CD
3
OD) δH
(ppm) 7.54-7.64 (m, 6H), 7.40 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 8.8 Hz,
1H), 7.00 (t, J = 7.2 Hz), 6.82 (d, J = 7.6 Hz, 1H), 6.68 (t, J = 7.6 Hz, 1H), 2.40-2.46 (m, 4H),
1.74-1.82 (m, 4H), 1.47-1.54 (m, 2H).
13
C NMR (151 MHz, CD
3
OD) δC (ppm) 173.19, 173.17,
150.28, 140.06, 129.88, 126.66, 126.32, 124.83, 122.34, 122.29, 121.81, 119.90, 118.04, 110.39,
36.31, 36.14, 28.27, 25.27, 25.01.
82

2.29. N
1
-([1,1'-biphenyl]-4-ylmethyl)-N
7
-(2-aminophenyl)heptanediamide. Synthesized
according to Method C. Purification by automated flash chromatography using 5%
MeOH/EtOAc as eluent gave 0.012 g as a white solid (31%). TLC: 100% EtOAc, R
f
≈ 0.5.
1
H
NMR (400 MHz, CD
3
OD) δH (ppm) 7.56 (t, J = 8.4 Hz, 4H), 7.28-7.41 (m, 5H), 7.06 (d, J = 8.0
Hz, 1H), 7.00 (t, J = 8.4 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.67 (t, J = 6.8 Hz, 1H), 4.39 (s, 2H),
2.41 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 8.0 Hz, 2H), 1.67-1.78 (m, 4H), 1.40-1.48 (m, 2H).
13
C
NMR (100 MHz, CD
3
OD) δC (ppm) 175.00, 173.00, 128.42, 127.68, 126.88, 126.79, 126.67,
126.49, 125.67, 118.09, 117.11, 42.35, 35.60, 35.44, 28.30, 27.21, 25.19.

2.31. tert-butyl (2-(7-oxo-7-((3-(pyridin-3-yl)phenyl)amino)heptanamido)phenyl)carbamate.
Synthesized according to Method D. Purification by automated flash chromatography using ethyl
acetate as eluent gave 0.098 g as a white solid (66%). TLC: 100% EtOAc, R
f
≈ 0.7.
1
H NMR
(400 MHz, CD
3
OD) δH (ppm) 8.77 (s, 1H), 8.51 (d, J = 4.8 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H),
7.92 (s, 1H), 7.33-7.65 (m, 7H), 7.17 (t, J = 7.6 Hz, 1H), 7.07 (t, J – 8.0 Hz, 1H), 2.41-2.46 (m,
4H), 1.74-1.82 (m, 4H), 1.50-1.54 (m, 11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.65,
173.18, 154.33, 147.47, 146.91, 139.40, 137.79, 127.03, 135.11, 131.67, 129.31, 128.59, 128.47,
83

125.84, 125.13, 124.40, 124.04, 122.26, 119.54, 118.24, 80.01, 36.30, 35.81, 28.27, 27.21, 25.10,
25.00.

2.32. tert-butyl (2-(7-oxo-7-((3-(pyridin-4-yl)phenyl)amino)heptanamido)phenyl)carbamate.
Synthesized according to Method D. Purification by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.045 g as a white foam (30%). TLC: 90% EtOAc/hexanes, R
f
≈
0.1.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.55 (d, J = 6.4 Hz, 1H), 8.02 (s, 1H), 7.66 (d, J =
6.4 Hz, 2H), 7.60-7.62 (m, 2H), 7.56-7.58 (m, 1H), 7.53-7.54 (m, 1H), 7.42-7.44 (m, 1H), 7.16 (t,
J = 6.4 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 2.41-2.45 (m, 4H), 1.74-1.81 (m, 4H), 1.43-1.51 (m,
10H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.66, 173.20, 149.21, 139.54, 138.12, 132.36,
131.70, 131.60, 129.62, 129.36, 128.64, 128.49, 125.86, 125.13, 124.45, 122.18, 121.75, 120.55,
118.08, 80.01, 36.32, 35.81, 28.30, 27.22, 25.09, 24.97.

2.33. N
1
-(2-aminophenyl)-N
7
-(3-(pyridin-3-yl)phenyl)heptanediamide. Synthesized
according to Method C. Purification by automated flash chromatography using 5%
MeOH/CH
2
Cl
2
as eluent gave 0.055 g as a white solid (79%). TLC: 5% MeOH/CH
2
Cl
2
, R
f
≈ 0.1.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.77 (s, 1H), 8.51 (d, J = 3.6 Hz, 1H), 8.06 (d, J = 8.0
84

Hz, 1H), 7.92 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 8.8 Hz, 1H), 7.39-7.45 (m, 2H), 7.04
(d, J = 7.6 Hz, 1H), 6.99 (t, J = 7.2 Hz, 1H0, 6.80 (d, J = 10.4 Hz, 1H), 6.65 (t, J = 11.6 Hz, 1H),
2.42-2.45 (m, 4H), 1.75-1.82 (m, 4H), 1.47-1.55 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC
(ppm) 173.52, 173.26, 147.47, 146.91, 141.85, 139.40, 137.79, 137.02, 135.12, 129.31, 126.79,
125.67, 124.04, 123.60, 122.26, 119.56, 118.25, 118.05, 117.08, 36.35, 35.56, 28.27, 25.25,
25.07.

2.34. N
1
-(2-aminophenyl)-N
7
-(3-(pyridin-4-yl)phenyl)heptanediamide. Synthesized
according to Method C. Purification by automated flash chromatography using 5%
MeOH/EtOAc as eluent gave 0.024 g as a white solid (73%). TLC: 100% EtOAc, R
f
≈ 0.15.
1
H
NMR (500 MHz, CD
3
OD) δH (ppm) 8.58 (d, J = 4.5 Hz, 2H), 8.04 (s, 1H), 7.70 (d, J = 4.0 Hz,
2H), 7.62 (d, J = 7.5 Hz, 1H), 7.45-7.49 (m, 2H), 7.07 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 8.0 Hz,
1H), 6.83 (d, J = 8.0 Hz, 1H), 6.68 (t, J = 7.5 Hz, 1H), 2.45-2.48 (m, 4H), 1.79-1.82 (m, 4H),
1.52-1.57 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.51, 173.27, 149.13, 149.04,
141.85, 139.47, 138.08, 129.35, 126.79, 125.67, 122.18, 121.74, 121.64, 121.05, 120.57, 118.17,
118.05, 117.08, 109.99, 36.34, 35.36, 28.36, 25.25, 25.06.

2.35. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-1H-benzo[d]imidazole-5-carboxamide.
Synthesized according to Method B. Purification by automated flash chromatography using 10%
85

MeOH/CH
2
Cl
2
as eluent gave 0.043 g as a yellow solid (68%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈
0.1.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.29 (s, 1H), 8.15 (s, 1H), 7.78 (d, J = 6.8 Hz, 1H),
7.65 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 7.02 (t, J = 6.4 Hz, 1H), 6.84 (d, J = 8.4 Hz,
1H), 6.68 (t, J = 7.2 Hz, 1H), 3.46 (t, J = 6.8 Hz, 2H), 2.46 (t, J = 7.2 Hz, 2H), 1.70-1.85 (m, 4H),
1.49-1.59 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.64, 169.15, 143.17, 141.81,
126.73, 125.74, 123.69, 121.57, 117.95, 117.04, 39.55, 35.71, 28.85, 26.21, 25.24.

2.35. N
1
-(2-aminophenyl)-N
7
-(3-methoxyphenyl)heptanediamide. Synthesized according to
general procedures. Purification by automated flash chromatography using 80% EtOAc/hexanes
as eluent gave 0.073 g as a yellow solid (27%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.20.
1
H NMR
(400 MHz, CD
3
OD) δH (ppm) 7.27 (s, 1H), 7.19 (t, J = 8.4Hz, 1H), 6.98-7.04 (m, 3H), 6.82 (d, J
= 7.6 Hz, 1H), 6.63-6.71 (m, 2H), 3.76 (s, 3H), 2.36-2.45 (m, 4H) 1.48-1.80 (m, 4H), 1.44-1.52
(m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.53, 173.09, 160.06, 141.76, 139.61,
129.03, 126.79, 125.69, 123.74, 118.13, 117.14, 111.93, 109.99, 109.17, 106.52, 105.60, 54.20,
36.36, 35.57, 28.37, 25.26, 25.11.

2.36. N
1
-(2-aminophenyl)-N
7
-(3-ethylphenyl)heptanediamide. Synthesized according to
Method B. Purification by automated flash chromatography using 70% EtOAc/hexanes as eluent
gave 0.111 g as a yellow solid (55%). TLC: 70% EtOAc/hexanes, R
f
≈ 0.25.
1
H NMR (400 MHz,
86

CD
3
OD) δH (ppm) 7.40 (s, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 9.2
Hz, 1H), 7.01 (t, J = 6.0 Hz, 1H), 6.92 (d, J = 7.2 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.67 (t, J =
7.6 Hz, 1H), 2.60 (q, J = 7.6 Hz, 2H), 2.37-2.57 (m, 4H), 1.72-1.81 (m, 4H), 1.45-1.53 (m, 2H),
1.21 (t, J = 8.0 Hz, 3H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.56, 173.07, 144.81, 141.86,
138.43, 128.27, 126.82, 125.71, 123.73, 123.28, 119.32, 118.09, 117.28, 117.11, 36.34, 35.58,
28.46, 28.39, 25.27, 25.17, 14.66.

2.37. N
1
-(2-aminophenyl)-N
7
-(m-tolyl)heptanediamide. Synthesized according to Method B.
Purification by automated flash chromatography using 80% EtOAc/hexanes as eluent gave 0.088
g as a yellow solid (43%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.2.
1
H NMR (400 MHz, CD
3
OD) δH
(ppm) 7.39 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.01-7.07 (m, 2H), 6.90 (d,
J = 8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.67 (t, J = 6.0 Hz, 1H), 2.43 (t, J = 7.6 Hz, 2H), 2.40
(t, J = 7.6 Hz, 2H), 1.72-1.81 (m, 4H), 1.45-1.53 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC
(ppm) 173.55, 173.07, 141.83, 138.27, 128.23, 126.85, 125.71, 124.42, 123.69, 120.45, 118.09,
117.11, 117.02, 36.33, 35.03, 28.38, 25.16, 20.12.

2.38. N
1
-(2-aminophenyl)-N
7
-(3-isopropylphenyl)heptanediamide. Synthesized according to
Method B. Gave 0.064 g as a yellow solid (32%).
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.42
(s, 1H) 7.34 (8.0 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.95-7.06 (m, 3H), 6.82 (d, J = 7.6 Hz, 1H),
87

6.68 (t, J = 7.6 Hz, 1H), 2.81-2.90 (m, 1H), 2.37-2.45 (m, 4H), 1.72-1.80 (m, 4H), 1.45-1.53 (m,
2H), 1.22 (d, J = 8.0 Hz, 6H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.55, 173.06, 149.39,
141.84, 138.43, 128.29, 126.74, 125.69, 123.70, 121.83, 118.14, 117.87, 117.40, 117.13, 36.33,
35.57, 33.99, 28.37, 25.27, 25.14, 22.93.

2.39. N
1
-(2-aminophenyl)-N
7
-(3-bromophenyl)heptanediamide. Synthesized by method C
from 2.229. Purification by flash chromatography using 70% EtOAc/hexanes as eluent gave
0.117 g as a white solid (58%). TLC: 60% EtOAc/hexanes, R
f
≈ 0.3.
1
H-NMR (400 MHz,
CD
3
OD): δ 7.94 (s, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.21-7.25 (m, 2H), 7.05-7.11 (m, 2H), 6.87 (d,
J = 8.0 Hz, 1H), 6.73 (t, J = 8.0 Hz, 1H), 2.41-2.49 (m, 4H), 1.76-1.83 (m, 4H), 1.49-1.56 (m,
2H).
13
C-NMR (100 MHz, CD
3
OD): δ 173.6, 173.2, 141.9, 140.2, 130.0, 126.9, 126.4, 125.7,
123.8, 122.3, 121.8, 118.2, 118.1, 117.2, 36.3, 35.6, 28.4, 25.3, 25.0. HRMS (ESI): m/z 404.0961,
[M+H]
+
, calc. 404.0974).

2.40. N
1
-(2-aminophenyl)-N
7
-(4-fluorophenyl)heptanediamide. Synthesized according to
general procedures. Purification by automated flash chromatography using 80% EtOAc/hexanes
as eluent gave 0.095 g as a yellow solid (47%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.2.
1
H NMR
(400 MHz, CD
3
OD) δH (ppm) 7.51-7.54 (m, 2H), 6.99-7.06 (m, 4H), 6.82 (d, J = 9.6 Hz, 1H),
6.67 (t, J = 8.0 Hz, 1H), 2.36-2.45 (m, 4H), 1.71-1.80 (m, 4H), 1.44-1.52 (m, 2H).
13
C NMR (100
88

MHz, CD
3
OD) δC (ppm) 174.03, 172.97, 126.80, 125.68, 121.70, 121.65, 118.07, 117.10, 114.
67, 114.35, 36.27, 35.56, 29.23, 28.36, 25.31, 25.10.

2.41. N
1
-(2-aminophenyl)-N
7
-(3-(trifluoromethyl)phenyl)heptanediamide. Synthesized by
method B from 2.234. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.266 g as a yellow solid (68%). TLC: 80% EtOAc/hexanes, R
f
≈
0.2.
1
H-NMR (400 MHz, CD
3
OD): δ 8.01 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 7.2 Hz,
1H), 7.34 (d, J = 7.6 Hz, 1H), 6.98-7.06 (m, 2H), 6.82 (d, J = 7.6 Hz, 1H), 6.68 (t, J = 7.2 Hz,
1H), 2.41-2.44 (m, 4H), 1.74-1.78 (m, 4H), 1.47-1.50 (m, 2H).
13
C-NMR (100 MHz, CD
3
OD): δ
173.5, 173.3, 141.9, 139.4, 129.2, 126.9, 125.7, 123.7, 122.7, 119.8, 119.7, 118.1, 117.1, 115.9,
36.3, 35.6, 28.4, 25.2, 24.9.
19
F-NMR (376 MHz, CD
3
OD): δ -64.3. HRMS (ESI): m/z 394.1738,
[M+H]
+
, calc. 394.1737).

2.42. N
1
-(2-aminophenyl)-N
7
-(3-fluorophenyl)heptanediamide. Synthesized by Method B.
Purification by automated flash chromatography using 80% EtOAc/hexanes as eluent gave 0.033
g as a yellow solid (6%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.2.
1
H NMR (600 MHz, CD
3
OD) δH
(ppm) 7.52 (d, J = 10.8 Hz, 1H), 7.21-7.29 (m, 2H), 7.05 (d, J = 7.8 Hz, 1H), 7.01 (t, J = 7.8 Hz,
1H), 6.82 (d, J = 8.4 Hz, 1H), 6.77 (t, J = 7.8 Hz, 1H), 6.68 (t, J = 7.8 Hz, 1H), 2.38-2.44 (m,
4H), 1.72-1.79 (m, 4H), 1.45-1.51 (m, 2H).
13
C NMR (151 MHz, CD
3
OD) δC (ppm) 173.52,
89

173.18, 141.84, 129.70, 129.63, 126.80, 125.68, 118.07, 117.10, 114.92, 109.76, 106.49, 36.32,
35.56, 28.36, 25.24, 25.00.

2.43. N
1
-(2-aminophenyl)-N
7
-(3,5-bis(trifluoromethyl)phenyl)heptanediamide. Synthesized
according to Method B. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.040 g as a yellow solid (51%). TLC: 80% EtOAc/hexanes, R
f
≈
0.5.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.22 (s, 2H), 7.63 (s, 1H), 7.01-7.08 (m, 2), 6.85 (d,
J = 8.0 Hz, 1H), 6.69 (t, J = 7.6 Hz, 1H), 2.44-2.48 (m, 4H), 1.78-1.80 (m, 4H), 1.51-1.53 (m,
2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.55, 141.87, 140.84, 132.19, 131.93, 131.67,
131.40, 126.83, 125.68, 124.37, 123.72, 122.29, , 118.89, 118.86, 118.09, 117.13, 116.01, 36.31,
35.55, 28.35, 25.24, 24.76.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.65.

2.44.tert-butyl(2-(7-((4-bromo-3-(trifluoromethyl)phenyl)amino)-7-oxoheptanamido)
phenyl)carbamate. Synthesized according to general procedures. Purification by automated
flash chromatography using 60% EtOAc/hexanes as eluent gave 0.069 g as a white foam (69%).
TLC: 60% EtOAc/hexanes, R
f
≈ 0.5.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.10 (s, 1H), 7.67
(s, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.18 (t, J = 7.2 Hz, 1H), 7.10 (t, J = 7.6
90

Hz, 1H), 2.39-2.45 (m, 4H), 1.72-1.79 (m, 4H), 1.48-1.49 (m, 11H).
13
C NMR (100 MHz,
CD
3
OD) δC (ppm) 173.62, 173.21, 152.04, 138.56, 135.08, 131.64, 125.84, 125.08, 124.40,
123.66, 118.42, 111.38, 109.99, 108.23, 80.05, 36.22, 35.78, 28.20, 27.20, 25.05, 24.77.

2.46. N
1
-(2-aminophenyl)-N
7
-(2-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)heptanediamide.
Synthesized according to Method C. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.055 g as a yellow solid (67%). TLC: 80% EtOAc/hexanes, R
f
≈
0.25.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 7.99 (s, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.26-7.28
(m, 3H), 7.16-7.18 (m, 3H), 6.96 (d, J = 8.0 Hz, 1H), 6.91 (t, J = 8.0 Hz, 1H), 6.74 (d, J = 6.4 Hz,
1H), 6.59 (t, J = 7.5 Hz, 1H), 2.33-2.36 (m, 4H), 1.65-1.68 (m, 4H), 1.39-1.42 (m, 2H).
13
C NMR
(125 MHz, CD
3
OD) δC (ppm) 173.55, 173.38, 141.87, 139.53, 138.24, 136.46, 132.40, 128.80,
128.79, 128.29, 127.49, 127.20, 126.83, 125.71, 125.10, 123.72, 122.11, 118.10, 117.12, 116.81,
116.72, 36.34, 35.58, 28.39, 25.27, 25.00.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -58.31.

2.47. N
1
-(2-aminophenyl)-N
7
-(4-(dimethylamino)phenyl)heptanediamide. Synthesized
according to general procedures. Purification by automated flash chromatography using ethyl
acetate as eluent gave 0.031 g as a white solid. TLC: 100% EtOAc, R
f
≈ 0.1.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 7.36 (d, J = 9.5 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H),
91

6.84 (d, J = 7.0 Hz, 1H), 6.75 (d, J = 10 Hz, 2 H), 6.70 (t, J = 10 Hz, 1H), 2.89 (s, 6H), 2.45 (t, J
= 7.5 Hz, 2H), 2.37 (t, J = 7.0 Hz, 2H), 1.73-1.81 (m, 4H), 1.47-1.53 (m, 2H).
13
C NMR (125
MHz, CD
3
OD) δC (ppm) 173.52, 172.87, 148.14, 141.87, 128.63, 126.82, 125.70, 123.74,
121.64, 118.11, 117.09, 113.09, 39.99, 36.16, 35.60, 28.39, 25.29.

2.49. N
1
-(2-(dimethylamino)phenyl)-N
7
-(3-(trifluoromethyl)phenyl)heptanediamide. To a
mixture of amine # (0.100g, 0.254 mmol), paraformaldehyde (0.076 g, 2.54 mmol), and sodium
borohydride (0.048 g, 1.27 mmol) in THF (1 mL) was added trifluoroacetic acid (2 mL, 26 mmol)
dropwise. The reaction mixture was stirred at room temperature for 24 h then poured into a cold
10% NaOH aqueous solution (10 mL) which was then diluted with brine (10 mL) and extracted
into CH
2
Cl
2
(3 x 25 mL).The combined organic layers were dried over Na
2
SO
4
and concentrated
onto celite. Purification by automated flash chromatography using 50% EtOAc/hexanes as eluent
gave 0.041 g as a colorless oil (38%). TLC: 50% EtOAc/hexanes, R
f
≈ 0.3.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 8.03 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.0
Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 7.02 (t, J =
7.5 Hz, 1H), 2.64 (s, 6H), 2.48 (t, J = 7.5 Hz, 2H), 2.42 (t, J = 7.0 Hz, 2H), 1.75-1.78 (m, 4H),
1.46-1.52 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.29, 172.88, 144.79, 139.50,
132.10, 130.90 (q, J = 33 Hz), 130.37, 129.25, 125.20, 124.56, 123.41, 123.00, 122.72, 121.77,
119.75, 119.27, 115.90, 43.27, 36.48, 36.32, 28.38, 25.19, 24.99.
19
F NMR (470 MHz, CD
3
OD)
δF (ppm) -64.26.
92

2.51. N
1
-(2-formamidophenyl)-N
7
-(3-(trifluoromethyl)phenyl)heptanediamide. To a mixture
of amine 2.41 (0.200 g, 0.508 mmol) in formic acid (0.076 mL, 2.03 mmol) was added sodium
formate (0.007 g, 0.102 mmol) at room temperature. After stirring at room temperature for 18 h
water (10 mL) and ethyl acetate (10 mL) were added to reaction mixture and the material was
sonicated for 10 min. The phases were separated and the aqueous layer was extracted into ethyl
acetate (3 x 10 mL). The combined organic layers were dried over Na
2
SO
4
and concentrated onto
celite. Purification by automated flash chromatography using 90% EtOAc/hexanes as eluent
provided 0.020 g as a colorless oil (9%). TLC: 90% EtOAc/hexanes, R
f
≈ 0.6.
1
H NMR (500
MHz, CDCl
3
) δH (ppm) 8.77 (s, 1H), 7.94 (s, 1H), 7.66 (d, J = 7.5 Hz, 1H), 7.49-7.50 (m, 2H),
7.33-7.38 (m, 2H), 7.18-7.20 (m, 2H), 2.92 (t, J = 7.0 Hz, 2H), 2.33 (t, J = 7.5 Hz, 2H), 1.78-1.84
(m, 2H), 1.67-1.73 (m, 2H), 1.35-1.40 (m, 2H).
13
C NMR (126 MHz, CDCl
3
) δC (ppm) 172.38,
154.92, 139.01, 131.41, 129.43, 124.92, 122.98, 122.68, 122.29, 120.75, 116.75, 36.70, 28.41,
28.09, 27.50, 24.33.
19
F NMR (470 MHz, CDCl
3
) δF (ppm) -62.74.

2.54. N
1
-(3-(trifluoromethyl)phenyl)heptanediamide. To a suspension of carboxylic acid
2.234 (0.100 g, 0.346 mmol) in acetonitrile (4 mL) at room temperature was added
diisopropylethylamine (0.060 mL, 0.346 mmol) followed by HBTU (0.131 g, 0.346 mmol).
After stirring for 5 min. ammonium hydroxide (0.117 mL, 1.73 mmol) was added and stirring
93

was continued at room temperature for 3 days. The reaction mixture was then diluted with ethyl
acetate (20 mL) and washed sequentially with brine (20 mL) and saturated NaHCO
3
solution (2 x
20 mL). The organic layer was dried over Na
2
SO
4
and concentrated onto celite. Purification by
automated flash chromatography using 10% MeOH/CH
2
Cl
2
as eluent gave 0.084 g as a white
solid (80%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈ 0.45.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm)
10.21 (s, 1H), 8.11 (s, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz,
1H), 7.22 (br s, 1H), 6.68 (br s, 1H), 2.33 (t, J = 6.8 Hz, 2H), 2.05 (t, J = 6.8 Hz, 2H), 1.47-1.62
(m, 4H), 1.29-1.33 (m, 2H). ).
13
C NMR (150 MHz, DMSO-d
6
) δC (ppm) 174.64, 172.25,
140.49, 130.34, 129.95, 129.67, 125.91, 123.22, 122.90, 119.69, 115.39, 39.33, 36.74, 35.39,
28.74, 25.30, 25.18.
19
F NMR (150 MHz, CD
3
OD) δF (ppm) -61.39.

2.56. 6-(1H-benzo[d]imidazol-2-yl)-N-phenylhexanamide. A mixture of N
1
-(2-aminophenyl)-
N
7
-phenylheptanediamide (0.053 g, 0.16 mmol) was refluxed in acetic acid for 24 h. The
reaction mixture was cooled to room temperature then solvent was removed under nitrogen. To
the crude material was added saturated NaHCO
3
solution (10 mL) and this was extracted with
ethyl acetate (2 x 15 mL). The combined organic layers were washed with water (20 mL), dried
over Na
2
SO
4
and concentrated onto celite. Purification by flash chromatography using 5%
MeOH/CH
2
Cl
2
as eluent gave 0.034 g as a white solid (69%). TLC: 5% MeOH/CH
2
Cl
2
, R
f
≈
0.5.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 12.15 (br s, 1H), 9.81 (s, 1H), 7.54 (d, J = 8.4
Hz, 2H), 7.42-7.44 (m, 2H), 7.24 (t, J = 7.6 Hz, 2H), 7.06-7.09 (m, 2H), 6.98 (t, J = 6.8 Hz, 1H),
2.79 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 1.78 (t, J = 7.2 Hz, 2H), 1.62 (t, J = 7.6 Hz, 2H),
94

1.35 (t, J = 7.2 Hz).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 171.60, 155.45, 139.75, 129.05,
123.34, 121.53, 119.46, 36.73, 28.77, 28.75, 27.78, 25.31.

2.57. N
1
-(2-aminophenyl)-N
6
-(3-(trifluoromethyl)phenyl)adipamide. Synthesized according
to Method B. Purification by automated flash chromatography using 80% EtOAc/hexanes as
eluent gave 0.094 g as a yellow solid (57%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.15.
1
H NMR (400
MHz, DMSO-d6) δH (ppm) 10.21 (s, 1H), 9.07 (s, 1H), 8.09 (s, 1H), 7.74 (d, J = 7.6 Hz, 1H),
7.51 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 6.86 (t, J = 7.2 Hz,
1H), 6.68 (d, J = 6.8 Hz, 1H), 6.48-6.52 (m, 1H), 4.84 (br s, 2H), 2.32-2.48 (m, 4H), 1.60-1.63
(m, 4H).
13
C NMR (100 MHz, DMSO-d6) δC (ppm) 172.15, 171.38, 142.28, 140.46, 130.36,
129.98, 129.66, 126.14, 125.92, 125.75, 125.72, 123.96, 119.69, 116.32, 115.46, 36.73, 36.04,
25.43, 25.13.
19
F NMR (376 MHz, DMSO-d6) δF (ppm) -61.37.

2.58. N
1
-(2-aminophenyl)-N
8
-(3-(trifluoromethyl)phenyl)octanediamide. Synthesized
according to Method B. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.067 g as a white solid (52%). TLC: 80% EtOAc/hexanes, R
f
≈
0.25.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.02 (s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.49 (t, J =
8.0 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.07 (d, J = 9.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.84 (d, J
= 9.0 Hz, 1H), 6.72 (t, J = 8.0 Hz, 1H), 2.40-2.45 (m, 4H), 1.74-1.77 (m, 4H), 1.46-1.48 (m, 4H).
95

13
C NMR (126 MHz, CD
3
OD) δC (ppm) 173.66, 173.48, 141.94, 139.50, 129.25, 126.79, 125.67,
125.23, 123.73, 122.63, 119.87, 118.12, 117.05, 115.97, 103.40, 36.44, 35.79, 28.56, 25.46,
25.14.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.33.

2.59. N
1
-(2-aminophenyl)-N
9
-(3-(trifluoromethyl)phenyl)nonanediamide. Synthesized
according to Method B. Purification by automated flash chromatography using 70%
EtOAc/hexanes as eluent gave 0.065 g as a white solid (51%). TLC: R
f
≈ 0.4.
1
H NMR (500
MHz, DMSO- d
6
) δH (ppm) 10.19 (s, 1H), 9.07 (s, 1H), 8.10 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H),
7.52 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.87 (t, J = 8.0 Hz,
1H), 6.69 (d, J = 7.0 Hz, 1H), 6.52 (t, J = 8.0 Hz, 1H), 4.79 (br s, 2H), 2.28-2.34 (m, 4H), 1.59-
1.61 (m, 4H), 1.30-1.32 (m, 6H).
13
C NMR (126 MHz, DMSO-d
6
) δC (ppm) 172.31, 171.58,
142.32, 140.50, 130.36, 129.96, 129.71, 126.11, 125.70, 125.67, 124.03, 123.51, 122.92, 119.70,
119.67, 116.61, 116.33, 115.45, 36.88, 36.22, 29.07, 25.74, 25.40.
19
F NMR (470 MHz, DMSO-
d
6
) δF (ppm) -61.35.

2.60. N
1
-(2-aminophenyl)-N
10
-(3-(trifluoromethyl)phenyl)decanediamide. Synthesized
according to Method B. Purification by automated flash chromatography using 70%
96

EtOAc/hexanes as eluent gave 0.031 g as a pale yellow solid (25%). TLC: 70% EtOAc/hexanes,
R
f
≈ 0.5.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.16 (s, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.62 (t, J
= 8.0 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.97 (d,
J = 8.5 Hz, 1H), 6.84 (t, J = 7.5 Hz, 1H), 2.53 (m, 4H), 1.85-1.88 (m, 4H), 1.50-1.55 (m, 8H).
13
C
NMR (126 MHz, CD
3
OD) δC (ppm) 173.76, 73.56, 141.90, 139.47, 130.83, 130.57, 129.25,
126.86, 125.67, 123.78, 122.62, 119.80, 118.12, 117.14, 115.96, 36.56, 35.83, 28.85, 28.83,
25.60, 25.29.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.33.

2.61. N
1
-(3-aminophenyl)-N
8
-phenyloctanediamide. Synthesized according to Method C.
Purification by automated flash chromatography using 5% MeOH/CH
2
Cl
2
as eluent gave 0.088 g
as a white, solid (61%). TLC: 5% MeOH/CH
2
Cl
2
, R
f
≈ 0.3.
1
H NMR (400 MHz, CD
3
OD) δH
(ppm) 7.51 (d, J = 8.4 Hz, 2H), 7.28 (t, J = 7.2 Hz, 2H), 7.06 (t, J = 7.6 Hz, 1H), 6.98-7.01 (m,
2H), 6.77 (d, J = 8.0 Hz, 1H), 6.44 (d, J = 8.0 Hz, 1H), 2.31-2.38 (m, 4H), 1.68-1.73 (m, 4H),
1.41-1.44 (m, 4H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.19, 173.08, 147.84, 128.82,
128.31, 123.66, 119.82, 111.13, 109.94, 107.05, 36.48, 36.43, 28.57, 25.37, 25.32.

2.64. oct-4-ynedioic acid. A mixture of di-tert-butyl-oct-4-ynedioate 2.63 (0.481 g, 1.7 mmol) in
THF (4 mL) and water (2 mL) was treated with LiOH (0.083 g, 3.5 mmol) in water (2 mL). The
97

reaction mixture was stirred at 70°C. Additional LiOH (5 equiv) was added after 5 h and again
after another 24 h. After a total of 48 h the reaction mixture was cooled to room temperature and
volatiles were evaporated. The crude material was diluted with water (5 mL) and acidified to pH
2 using 10% HCl solution. The aqueous layer was extracted into ethyl acetate (3 x 5 mL), dried
over Na
2
SO
4
and concentrated under reduced pressure to provide 0.193 g as an orange solid
(67%).
1
H NMR (400 MHz, DMSO-d6) δH (ppm) 12.17 (br s, 2H), 2.29-2.33 (m, 8H).
13
C NMR
(100 MHz, DMSO-d6) δC (ppm) 173.38, 79.76, 33.80, 14.53.

2.65. 8-((3-bromophenyl)amino)-8-oxooct-4-ynoic acid. To a mixture of 2.64 (0.046 g, 0.27
mmol) in CH
2
Cl
2
(3 mL) was added diisopropylethylamine (0.047 mL, 0.27 mmol) and the
mixture was stirred for 5 min. HATU (0.103 g, 0.27 mmol) and 3-bromoaniline (0.029 mL, 0.27
mmol), respectively, were then added. After stirring at room temperature for 18 h the reaction
mixture was poured into a solution of 10% HCl (10 mL). The aqueous phase was extracted with
ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na
2
SO
4
and the solvent
was evaporated under reduced pressure. Purification by automated flash chromatography using
80% EtOAc/hexanes as eluent gave 0.031 g as a yellow solid (35%). TLC: 80% EtOAc/hexanes,
R
f
≈ 0.3.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.88 (s, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.17-
7.23 (m, 2H), 2.49-2.51 (m, 4H), 2.39-2.41 (m, 4H).
13
C NMR (151 MHz, CD
3
OD) δC (ppm)
174.39, 171.45, 139.94, 129.92, 126.50, 122.42, 121.76, 118.14, 79.03, 78.39, 35.94, 33.26,
14.44, 13.96.
98

2.66. N
1
-(2-aminophenyl)-N
8
-(3-bromophenyl)oct-4-ynediamide. Synthesized according to
method B from 2.65 and 1,2-phenylenediamine. Purification by automated flash chromatography
using 60% EtOAc/hexanes as eluent gave 0.014 g orange solid (37%). TLC: 80%
EtOAc/hexanes, R
f
≈ 0.5.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.07 (s, 1H), 7.62 (d, J = 7.6
Hz, 1H), 7.36-7.40 (m, 2H), 7.19-7.25 (m, 2H), 7.01 (d, J = 7.2 Hz, 1H), 6.87 (t, J = 9.2 Hz, 1H),
2.27 (s, 8H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 171.89, 171.40, 142.23, 140.00, 129.91,
127.03, 126.38, 125.99, 123.28, 122.37, 121.84, 118.08, 117.93, 116.80, 79.07, 78.99, 35.85,
35.19, 14.85, 14.44.

2.71. 2-chloro-N-(3-(trifluoromethyl)phenyl)acetamide. To a mixture of 3-
(trifluoromethyl)aniline (0.500 mL, 4.00 mmol) and potassium carbonate (0.829 g, 6.00 mmol) in
THF (40 mL) at room temperature was added chloroacetyl chloride (0.478 mL, 6.00 mmol)
dropwise. The reaction mixture was refluxed for 2 h, cooled to room temperature, and the solvent
was evaporated. The crude material was diluted with water (50 mL) and filtered to provide 0.922
g as a white solid (97%) which was used without further purification. TLC: 20% EtOAc/hexanes,
R
f
≈ 0.25.
1
H NMR (500 MHz, CDCl
3
) δH (ppm) 8.35 (br s, 1H), 7.85 (s, 1H), 7.76 (d, J = 8.0
Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 4.21 (s, 2H).
13
C NMR (125 MHz,
CDCl
3
) δC (ppm) 164.05, 137.20, 131.60 (q, J = 40 Hz), 129.74, 124.79, 123.14, 122.61, 121.88,
121.85, 121.79, 120.43, 116.89, 42.77.
19
F NMR (470 MHz, CDCl
3
) δF (ppm) -62.81.
99

2.72. tert-butyl 4-(2-oxo-2-((3-(trifluoromethyl)phenyl)amino)ethyl)piperazine-1-
carboxylate. A mixture of 2.71 (0.300 g, 1.26 mmol), Boc-piperazine (0.235 g, 1.26 mmol), and
potassium carbonate (0.174 g, 1.26 mmol) in DMF (5 mL) was stirred at 90°C for 20 h. The
reaction mixture was then cooled to room temperature, diluted with ethyl acetate (25 mL) and
washed sequentially with saturated NaHCO
3
solution (25 mL) and brine (25 mL). The organic
layer was dried over Na
2
SO
4
and concentrated onto celite. Purification by automated flash
chromatography using 40% EtOAc/hexanes as eluent provided 0.390 g as a yellow solid (80%).
TLC: 40% EtOAc/hexanes, R
f
≈ 0.25.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.06 (s, 1H), 7.80
(d, J = 8.5 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H).
13
C NMR (125 MHz,
CD
3
OD) δC (ppm) 169.73, 154.98, 138.72, 130.70 (q, J = 40 Hz), 129.29, 125.15, 123.04,
122.99, 120.23, 116.28, 79.89, 52.72, 47.58, 27.23.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -
64.29.

2.73. 2-(piperazin-1-yl)-N-(3-(trifluoromethyl)phenyl)acetamide. Synthesized by method C
from 2.72. Gave 0.144 g as a yellow solid (65%).
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.03
(s, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 3.20 (s, 2H),
2.93-2.95 (m, 4H), 2.60 (br s, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 169.86, 138.67,
129.31, 122.94, 120.13, 116.21, 62.04, 53.44, 44.76.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -
64.32.
100

2.75. tert-butyl (2-(2-(4-(2-oxo-2-((3-(trifluoromethyl)phenyl)amino)ethyl)piperazin-1-
yl)acetamido)phenyl)carbamate. A mixture of 2.73 (0.318 g, 1.11 mmol), 2.244 (0.315 g, 1.11
mmol), and potassium carbonate (0.153 g, 1.11 mmol) was stirred in DMF (5 mL) at 90°C for 24
h. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (50 mL) and
washed sequentially with saturated NaHCO
3
solution (50 mL) and brine (50 mL). The organic
phase was dried over Na
2
SO
4
and concentrated onto celite. Purification by automated flash
chromatography using 80% EtOAc/hexanes as eluent provided 0.268 g as a yellow solid (28%).
TLC: 80% EtOAc/hexanes, R
f
≈ 0.35.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.06 (s, 1H), 7.83
(d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H),
7.33 (d, J = 7.2 Hz, 1H), 7.13-7.18 (m, 2H), 3.26 (s, 2H), 3.23 (s, 2H), 2.75 (s, 8H), 1.53 (s, 9H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 169.77, 155.11, 138.98, 130.96, 130.79, 129.47, 125.94,
125.03, 122.96, 120.38, 116.28, 80.06, 61.44, 61.36, 52.99, 52.78, 27.41.
19
F NMR (376 MHz,
CD
3
OD) δF (ppm) -64.30.

2.76. N-(2-aminophenyl)-2-(4-(2-oxo-2-((3-(trifluoromethyl)phenyl)amino)ethyl)piperazin-
1-yl)acetamide. Synthesized from 2.75 by method C. Purification by automated flash
chromatography using ethyl acetate as eluent gave 0.080 g as a yellow solid (64%). TLC: 100%
EtOAc, R
f
≈ 0.1.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.06 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H),
101

7.51 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 9.2 Hz, 1H), 7.02 (t, J = 9.2 Hz,
1H), 6.86 (d, J = 6.8 Hz, 1H), 6.72 (t, J = 6.4 Hz, 1H), 3.25 (s, 2H), 3.24 (s, 2H), 2.72-2.76 (m,
8H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 170.07, 169.85, 141.28, 138.74, 130.61, 129.29,
126.65, 124.97, 123.70, 122.90, 120.17, 118.39, 117.31, 116.17, 61.36, 61.00, 52.85, 52.77.
19
F
NMR (376 MHz, CD
3
OD) δF (ppm) -64.32.

2.77. methyl 3-(2-oxo-2-((3-(trifluoromethyl)phenyl)amino)ethoxy)benzoate. To a mixture
of methyl 3-hydroxybenzoate (0.200 g, 1.31 mmol) in acetone (20 mL) were added potassium
carbonate (0.908 g, 6.57 mmol) followed by 2.71 at room temperature then the temperature was
increased to 60°C. After 24 h the reaction mixture was cooled to room temperature, filtered to
remove base, and rinsed with acetone. The filtrate was concentrated and purified by flash
chromatography using 20% EtOAc/hexanes as eluent to provide 0.372 g as a white solid (80%).
TLC: 20% EtOAc/hexanes, R
f
≈ 0.25.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.08 (s, 1H), 7.85
(d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.43 (t, J = 7.0 Hz, 2H),
7.32 (d, J = 8.0 Hz, 1H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 167.87, 166.69, 157.85,
138.49, 131.46, 129.48, 129.32, 123.48, 122.52, 120.55, 120.53, 119.52, 116.73, 115.09, 671.7,
51.33.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.31.

2.78. 3-(2-oxo-2-((3-(trifluoromethyl)phenyl)amino)ethoxy)benzoic acid. Synthesized from
2.77 by method E. Gave 0.099 g as a white solid (34%).
1
H NMR (500 MHz, CD
3
OD) δH (ppm)
102

8.08 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.70 (t, J = 6.5 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.45 (t, J
= 8.0 Hz, 1H), 7.42-7.44 (m, 2H), 7.30 (d, J = 5.0 Hz, 1H), 4.77 (s, 2H).
13
C NMR (125 MHz,
CD
3
OD) δC (ppm) 167.92, 157.90, 138.57, 132.64, 130.96, 129.37, 123.53, 122.80, 120.54,
119.43, 116.83, 115.28, 67.18.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.32.

2.79. N-(2-aminophenyl)-3-(2-oxo-2-((3-(trifluoromethyl)phenyl)amino)ethoxy)benzamide.
Synthesized from 2.78 and 1,2-phenylenediamine according to method B. Purification by
automated flash chromatography using 60% EtOAc/hexanes as eluent gave 0.070 g as a white
solid (61%). TLC: 60% EtOAc/hexanes, R
f
≈ 0.4.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.09
(s, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.65-7.68 (m, 2H), 7.51-7.57 (m, 2H), 7.47 (d, J = 7.5 Hz, 1H),
7.31 (d, J = 8.5 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.09 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 6.5 Hz,
1H), 6.78 (t, J = 7.5 Hz, 1H), 4.81 (s, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 167.95,
167.01, 157.94, 142.37, 138.50, 135.79, 129.54, 129.33, 127.20, 126.23, 123.78, 123.48, 120.69,
120.52, 118.18, 117.31, 116.73, 113.82, 67.18.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.31.

2.81. 3-(furan-2-yl)propanoic acid. To a solution of ethyl 3-(2-furyl)propanoate (2.00 mL, 12.5
mmol) in methanol (20 mL) was added a solution of KOH (1.40 g, 25 mmol) in water (20 mL)
dropwise at room temperature. The reaction mixture was then stirred at 80°C for 3 h. After
cooling to room temperature the methanol was evaporated under reduced pressure and the
aqueous phase was acidified using 10% HCl solution. The aqueous phase was extracted into
ethyl acetate (2 x 25 mL) and the combined organic layers were dried over Na
2
SO
4
and
103

concentrated to provide 1.59 g as orange crystals (86%).
1
H NMR (400 MHz, DMSO-d6) δH
(ppm) 12.18 (br s, 1H), 7.47 (d, J = 1.6 Hz, 1H), 6.31 (d, J = 3.2 Hz, 1H), 6.06 (d, J = 3.6 Hz,
1H), 2.80 (t, J = 7.6 Hz, 2H), 2.48 (t, J = 3.6 Hz).
13
C NMR (100 MHz, DMSO-d6) δC (ppm)
173.82, 154.75, 141.80, 110.78, 105.56, 32.42, 23.38.

2.82. N-(3-bromophenyl)-3-(furan-2-yl)propanamide. Synthesized according to Method B.
Purification by automated flash chromatography using 10% EtOAc/hexanes as eluent gave 0.621
g as a yellow oil (59%). TLC: 10% EtOAc/hexanes, R
f
≈ 0.1.
1
H NMR (400 MHz, CD
3
OD) δH
(ppm) 7.91 (s, 1H), 7.46 (d, J = 7.2 Hz, 1H), 7.38 (s, 1H), 7.23-7.24 (m, 2H), 6.32 (t, J = 2.8 Hz,
1H), 6.11 (t, J = 3.2 Hz, 1H), 3.04 (t, J = 7.6 Hz, 2H), 2.72 (t, J = 7.6 Hz, 2H).
13
C NMR (100
MHz, CD
3
OD) δC (ppm) 171.99, 154.58, 141.09, 140.04, 129.96, 126.45, 122.40, 121.87,
118.10, 109.83, 104.97, 34.91, 23.36.

2.83. ethyl 2-(5-(3-((3-bromophenyl)amino)-3-oxopropyl)furan-2-yl)acetate. To a mixture of
N-(3-bromophenyl)-3-(furan-2-yl)-propanamide 2.82 (0.400 g, 1.36 mmol), ethyl iodoacetate
(0.161 mL, 1.36 mmol), and iron sulfate heptahydrate (0.189 g, 0.68 mmol) in DMSO (5 mL) at
0°C was added a 30% aqueous solution of H
2
O
2
(0.154 mL, 1.36 mmol). The reaction mixture
was stirred for 15 hours while warming to room temperature then diluted with brine (10 mL) and
extracted into ethyl ether (3 x 15 mL). The combined organic layers were dried over Na
2
SO
4
and
concentrated onto celite. The crude material was purified by automated flash chromatography
104

using 30% EtOAc/hexanes as eluent to provide 0.080 g as a yellow oil (15%).
1
H NMR (400
MHz, CD
3
OD) δH (ppm) 9.93 (br s, 1H), 7.89 (s, 1H), 7.46 (d, J = 7.2 Hz, 1H), 7.19-7.26 (m,
2H), 6.13 (s, 1H), 6.05 (s, 1H), 4.15 (q, 2H), 3.66 (s, 2H), 3.00 (t, J = 7.6 Hz, 2H), 2.70 (t, J = 7.6
Hz, 2H), 1.26 (t, J = 7.6 Hz, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 171.92, 170.13,
153.76, 146.51, 139.88, 129.91, 126.43, 122.63, 121.85, 118.14, 108.31, 105.98, 60.78, 34.95,
33.32, 23.46, 13.04.

2.84. 2-(5-(3-((3-bromophenyl)amino)-3-oxopropyl)furan-2-yl)acetic acid. To a solution of
ethyl 2-(5-(3-(3-bromophenylamino)-3-oxopropyl)furan-2-yl)acetate (0.080 g, 0.21 mmol) in
methanol (1 mL) was added a solution of KOH (0.024 g, 0.42 mmol) in water (1 mL) dropwise at
room temperature. The reaction mixture was then stirred at 80°C for 8 h. After cooling to room
temperature the methanol was evaporated under reduced pressure and the aqueous phase was
acidified to pH 2 using 10% HCl solution. The aqueous phase was extracted into ethyl acetate (2
x 10 mL) and the combined organic layers were dried over Na
2
SO
4
and concentrated.
Purification by flash chromatography using 5% MeOH/CH
2
Cl
2
as eluent gave 0.026 g as a yellow
solid (35%). TLC: 5% MeOH/CH
2
Cl
2
, R
f
≈ 0.1.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.82
(s, 1H), 7.38 (d, J = 9.2 Hz, 1H), 7.12-7.16 (m, 2H), 5.96 (s, 1H), 5.91 (s, 1H), 3.42 (s, 2H), 2.91
(t, J = 7.2 Hz, 2H), 2.63 (t, J = 7.2 Hz, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 171.11,
152.20, 149.14, 148.89, 139.15, 129.16, 125.86, 121.75, 120.99, 117.41, 106.18, 104.87, 35.55,
34.00, 22.77.
105

2.85. 3-(5-(2-((2-aminophenyl)amino)-2-oxoethyl)furan-2-yl)-N-(3-
bromophenyl)propanamide. Synthesized according to Method B. Gave 0.055 g as a yellow
solid (44%).
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.83 (s, 1H), 7.39 (d, J = 9.6 Hz, 1H), 7.17-
7.38 (m, 2H), 7.07 (d, J = 9.6 Hz, 1H), 7.02 (t, J = 8.4 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.69 (t, J
= 8.0 Hz, 1H), 6.17 (d, J = 3.2Hz, 1H), 6.03 (d, J = 4.0 Hz, 1H).
13
C NMR (100 MHz, CD
3
OD)
δC (ppm) 171.76, 169.16, 153.87, 147.59, 141.91, 139.93, 129.87, 127.01, 126.39, 125.79,
123.38, 122.40, 121.79, 118.12, 118.04, 116.97, 108.30, 106.11, 35.40, 34.91, 23.48.

2.86. 6-(((benzyloxy)carbonyl)amino)hexanoic acid. A mixture of 6-aminocaproic acid (4.00
g, 30.5 mmol) and aqueous NaOH solution (2 M, 22 mL) was cooled to 0°C. Benzyl
chloroformate (4.49 mL, 30.5 mmol) and aqueous NaOH solution (4 M, 7.6 mL) were then added
slowly to the reaction mixture simultaneously. Stirring was continued over 20 h while warming
to room temperature. The reaction mixture was then washed with ethyl ether (3 x 10 mL). The
aqueous phase was cooled on an ice bath and acidified to pH 3 using concentrated HCl. The
white precipitate was filtered, washed with water (100 mL), and dried to obtain 7.36 g as a white
solid (91%).
1
H NMR (500 MHz, DMSO-d
6
) δH (ppm) 11.98 (br s, 1H), 7.22-7.37 (m, 5H), 5.00
(s, 2H), 2.95-2.99 (m, 2H), 2.16-2.19 (m, 2H), 1.46-1.49 (m, 2H), 1.36-1.40 (m, 2H), 1.23-1.27
(m, 2H).
13
C NMR (125 MHz, DMSO- d
6
) δC (ppm) 174.89, 156.54, 137.78, 128.79, 128.16,
66.53, 34.07, 29.59, 26.25, 24.66.
106

2.87. Tert-butyl-2-amino-N-benzyloxycarbonyl-6-aminocaproate. Synthesized according to
Method B. Purification by automated flash chromatography using 50% EtOAc/hexanes as eluent
gave 0.835 g as a colorless oil (58%). TLC: 50% EtOAc/hexanes, R
f
≈ 0.35.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 7.52 (d, J = 8.0 Hz, 1H), 7.28-7.38 (m, 6H), 7.20 (t, J = 6.5 Hz, 2H), 7.12 (t, J
= 7.0 Hz, 1H), 5.06 (s, 2H), 3.15 (t, J = 7.0 Hz, 2H), 2.41 (t, J = 7.5 Hz, 2H), 1.70-1.76 (m, 2H),
1.55-1.56 (m, 2H), 1.51 (s, 9H), 1.40-1.45 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm)
173.85, 157.61, 154.35, 137.05, 131.75, 129.64, 128.06, 127.54, 127.36, 125.91, 124.58, 80.11,
65.91, 40.23, 36.00, 29.21, 27.30, 25.95, 25.14.

2.88. tert-butyl (2-(6-aminohexanamido)phenyl)carbamate. To Cbz-protected compound
2.87 (2.50 g, 5.49 mmol) in methanol (50 mL) was added palladium on charcoal (5% wet) (1.00
g). The reaction vessel was degassed then placed under a balloon of H
2
for 6 h. The reaction
mixture was filtered through celite then concentrated under reduced pressure to obtain 0.381 g as
a colorless oil (65%).
1
H NMR (500 MHz, CDCl
3
) δH (ppm) 8.32 (br s, 1H), 7.40 (d, J = 5.0 Hz,
1H), 7.32 (d, J = 7.5 Hz, 1H), 7.05-7.12 (m, 2H), 2.62 (t, J = 5.5 Hz, 2H), 2.30 (t, J = 7.0 Hz,
107

2H), 1.83 (br s, 2H), 1.63-1.69 (m, 2H), 1.48 (s, 9H), 1.34-1.48 (m, 2H).
13
C NMR (125 MHz,
CDCl
3
) δC (ppm) 172.48, 154.24, 130.90, 129.82, 126.09, 125.25, 124.52, 124.43, 80.67, 41.20,
36.70, 31.84, 28.32, 26.10, 25.26.

2.95. N-(5-((2-aminophenyl)amino)-5-oxopentyl)-3-(trifluoromethyl)benzamide.
Synthesized according to Method B. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.042 g as an orange solid (32%). TLC: 80% EtOAc/hexanes, R
f
≈
0.2.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.16 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.83 (d, J =
7.5 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1), 6.86 (d, J =
8.0 Hz, 1H), 6.72 (t, J = 8.0 Hz, 1H), 3.47 (t, J = 6.5 Hz, 2H), 2.50 (t, J = 7.5 Hz, 2H), 1.72-1.85
(m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.50, 167.15, 141.89, 135.35, 130.59,
129.32, 127.57, 126.93, 125.66, 123.74, 118.20, 117.11, 39.23, 35.24, 28.53, 22.85.
19
F NMR
(470 MHz, CD
3
OD) δF (ppm) -64.24.

2.96. N-(5-((2-aminophenyl)amino)-5-oxopentyl)-3,5-bis(trifluoromethyl)benzamide.
Synthesized according to Method B. Purification by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.089 g as an orange solid (36%). TLC: 90% EtOAc/hexanes, R
f
≈
0.35.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.34 (s, 2H), 8.05 (s, 1H), 6.98 (d, J = 8.0 Hz, 1H),
108

6.92 (t, J = 8.0 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 7.5 Hz, 1H), 3.38 (t, J = 5.6 Hz,
2H), 2.39 (t, J = 7.0 Hz, 2H), 1.63-1.74 (m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.38,
165.20, 141.78, 136.81, 132.06, 127.58, 126.84, 125.67, 124.48, 124.26, 123.77, 122.11, 118.18,
117.20, 110.04, 39.53, 35.26, 28.41, 22.91.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.40.

2.97. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-3-(trifluoromethyl)benzamide. Synthesized
according to Method B. Recrystallization from acetonitrile/water gave 0.154 g as an orange solid
(48%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.1.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.14 (s,
1H), 8.07 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.02-7.07 (m,
2H), 6.85 (d, J = 8.0 Hz, 1H), 6.69 (t, J = 7.5 Hz, 1H), 3.45 (t, J = 7.0 Hz, 2H), 2.45 (t, J = 7.5
Hz, 2H), 1.70-1.81 (m, 4H), 1.51-1.53 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.57,
167.01, 141.86, 135.44, 130.51, 129.15, 127.67, 126.83, 125.71, 123.78, 118.04, 117.13, 39.54,
35.68, 28.72, 26.24, 25.27.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.22.

2.98. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-3,5-bis(trifluoromethyl)benzamide.
Synthesized according to Method B. Purification by automated flash chromatography using 70%
EtOAc/hexanes as eluent gave 0.061 g as a yellow solid (24%). TLC: 70% EtOAc/hexanes, R
f
≈
0.4.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.43 (s, 2H), 8.16 (s, 1H), 7.01-7.05 (m, 2H), 6.84
109

(d, J = 8.5 Hz, 1H), 6.69 (t, J = 8.0 Hz, 1H), 3.46 (t, J = 7.5 Hz, 2H), 2.45 (t, J = 7.5 Hz, 2H),
1.73-1.80 (m, 4H), 1.49-1.52 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.54,165.12,
141.84, 136.78, 131.87, 131.57, 127.58, 126.68, 125.64, 124.48, 124.11, 123.74, 122.17, 118.09,
117.15, 39.73, 35.66, 26.24, 25.21.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.39.

2.99. N-(7-((2-aminophenyl)amino)-7-oxoheptyl)-3-(trifluoromethyl)benzamide.
Synthesized according to Method B. Purification by automated flash chromatography using 80%
EtOAc/hexanes gave 0.092 g as a white solid (72%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.2.
1
H
NMR (500 MHz, CD
3
OD) δH (ppm) 8.14 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz,
1H), 7.66 (t, J = 7.5 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 7.0 Hz, 1H), 6.84 (d, J = 8.5
Hz, 1H), 6,71 (t, J = 7.5 Hz, 1H), 3.42 (t, J = 7.5 Hz, 2H), 2.43 (t, J = 7.5 Hz, 2H), 1.67-1.76 (m,
4H), 1.47-1.49 (m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.64, 166.99, 141.87, 135.46,
130.50, 129.18, 127.64, 126.79, 125.68, 123.75, 118.13, 117.17, 39.66, 35.73, 28.84, 28.59,
26.37, 25.53.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.22.

2.100. N-(7-((2-aminophenyl)amino)-7-oxoheptyl)-3,5-bis(trifluoromethyl)benzamide.
Synthesized according to Method B. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.025 g as a white solid (76%). TLC: 80% EtOAc/hexanes, R
f
≈
110

0.5.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.32 (s, 2H), 8.05 (s, 1H), 6.96 (d, J = 7.5 Hz, 1H),
6.91 (t, J = 7.5 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 7.5 Hz, 1H), 3.33 (t, J = 7.0 Hz,
2H), 2.31 (t, J = 6.5 Hz, 2H), 1.58-1.65 (m, 4H), 1.35-1.37 (m, 4H).
13
C NMR (125 MHz,
CD
3
OD) δC (ppm) 173.66, 165.12, 141.87, 132.00, 131.6 (q, J = 41 Hz), 127.55, 126.86, 125.67,
124.50, 124.26, 123.74, 122.13, 118.10, 117.13, 39.84, 35.73, 28.73, 28.57, 26.38, 25.51.
19
F
NMR (470 MHz, CD
3
OD) δF (ppm) -64.40.

2.102.N-(6-((2-aminophenyl)amino)-6-oxohexyl)-[1,1'-biphenyl]-3-carboxamide.
Synthesized according to Method B. Purification by automated flash chromatography using ethyl
acetate as eluent gave 0.250 g as a pale yellow solid (81%). TLC: 100% EtOAc, R
f
≈ 0.3.
1
H
NMR (400 MHz, CD
3
OD) δH (ppm) 8.07 (s, 1H), 7.78 (d, J = 9.6 Hz, 2H), 7.66 (d, J = 8.0 Hz,
2H), 7.52 (t, J = 8.0 Hz, 2H), 7.46 (t, J = 7.2 Hz, 2H), 7.37 (t, J = 7.2 Hz, 2H), 7.05 (d, J = 8.8
Hz, 1H), 7.00 (t, J = 9.2 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.67 (t, J = 8.0 Hz, 1H), 3.44 (t, J =
7.2 Hz, 2H), 2.45 (t, J = 7.2 Hz, 2H), 1.69-1.83 (m, 4H), 1.48-1.55 (m, 2H).
13
C NMR (100 MHz,
CD
3
OD) δC (ppm) 173.69, 168.74, 141.88, 141.49, 140.27, 134.96, 129.60, 128.67, 128.55,
127.35, 126.79, 126.67, 125.66, 125.42, 123.71, 118.08, 117.10, 39.44, 35.68, 28.80, 26.24,
25.28.
111

2.103. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-[1,1'-biphenyl]-4-carboxamide.
Synthesized according to Method C. Purification by automated flash chromatography using 5%
MeOH/EtOAc as eluent gave 0.047 g as a pale yellow solid (59%). TLC: 100% EtOAc, R
f
≈ 0.2.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 9.30 (s, 1H), 8.56 (t, J = 5.6 Hz, 1H), 7.99 (d, J = 8.4
Hz, 2H), 7.80 (t, J = 8.4 Hz, 3H), 7.56 (t, J = 7.2 Hz, 2H), 7.47 (t, J = 7.2 Hz, 1H), 7.20 (d, J =
7.6 Hz, 1H), 6.94 (t, J = 8.0 Hz, H), 6.58 (t, J = 8.8 Hz, 1H), 4.87 (s, 2H), 3.33-3.36 (m, 2H), 2.39
(t, J = 7.2 Hz, 2H), 1.62-1.72 (m, 4H), 1.50-1.63 (m, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC
(ppm) 171.60, 166.16, 142.89, 142.24, 139.61, 133.96, 129.45, 128.48, 128.25, 127.28, 126.88,
126.10, 125.73, 123.98, 116.52, 116.28, 36.18, 29.41, 29.31, 28.91, 27.71, 26.64, 25.54.

2.104. N-(5-((2-aminophenyl)amino)-5-oxopentyl)-4-(dimethylamino)benzamide.
Synthesized according to Method B. Purification by automated flash chromatography using ethyl
acetate as eluent gave 0.086 g as a yellow solid (64%). TLC: 100% EtOAc, R
f
≈ 0.1.
1
H NMR
(500 MHz, CD
3
OD) δH (ppm) 7.71 (d, J = 9.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 1H), 7.02 (t, J = 8.0
Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.69-6.74 (m, 3H), 3.40-3.43 (m, 2H), 3.01 (s, 6H), 2.48 (t, J =
7.5 Hz, 2H), 1.77-1.83 (m, 2H), 168-1.73 (m, 2H).
13
C NMR (126 MHz, CD
3
OD) δC (ppm)
112

173.46, 169.14, 152.81, 141.82, 128.29, 126.83, 125.72, 123.74, 120.72, 118.05, 117.12, 110.74,
39.09, 38.97, 38.83, 35.38, 28.86, 23.00.

2.105. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-4-(dimethylamino)benzamide.
Synthesized according to Method B. Purification by automated flash chromatography using ethyl
acetate as eluent gave 0.078 g as a yellow foam (59%). TLC: 10% MeOH/EtOAc, R
f
≈ 0.5.
1
H
NMR (400 MHz, CD
3
OD) δH (ppm) 7.69 (d, J = 9.2 Hz, 2H), 6.99-7.06 (m, 2H), 6.82 (d, J = 9.2
Hz, 1H), 6.66-6.72 (m, 3H), 3.35-3.40 (m, 2H), 2.95 (s, 6H), 2.43 (t, J = 7.6 Hz, 2H), 1.73-1.81
(m, 2H), 1.63-1.70 (m, 2H), 1.44-1.52 (m, 2H).
13
C NMR (126 MHz, CD
3
OD) δC (ppm) 173.68,
169.06, 152.81, 141.80, 128.27, 126.72, 125.72, 123.63, 120.71, 118.19, 117.17, 110.79, 39.20,
38.84, 35.71, 28.98, 26.22, 25.33.

2.107. methyl 6-(2-((tert-butoxycarbonyl)amino)benzamido)hexanoate. Synthesized
according to Method B. Purification by automated flash chromatography using 30%
EtOAc/hexanes as eluent gave 0.536 g as a clear oil (54%). TLC: 30% EtOAc/hexanes, R
f
≈ 0.5.
1
H NMR (600 MHz, CD
3
OD) δH (ppm) 8.14 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 9.6 Hz, 1H), 7.42
(t, J = 7.2 Hz, 1H), 7.04 (t, J = 6.0 Hz, 1H), 3.63 (s, 3H), 3.33 (t, J = 7.2 Hz, 2H), 2.34 (t, J = 7.2
Hz. 2H), 1.59-1.68 (m, 4H), 1.50 (s, 9H), 1.39-1.42 (m, 2H).
13
C NMR (150 MHz, CD
3
OD) δC
113

(ppm) 174.43, 169.45, 153.17, 138.98, 131.61, 127.26, 121.53, 119.13, 79.67, 50.53, 47.99,
39.20, 33.13, 28.55, 27.16, 25.98, 24.27.

2.108. 6-(2-((tert-butoxycarbonyl)amino)benzamido)hexanoic acid. Synthesized according to
Method E. Gave 0.167g as a clear oil (34%).
1
H NMR (600 MHz, CD
3
OD) δH (ppm) 8.14 (d, J
= 8.4 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 8.4 Hz, 1H), 7.02 (t, J = 7.8 Hz, 1H), 3.34 (t,
J = 4.8 Hz, 2H), 2.30 (t, J = 7.8 Hz, 2H), 1.59-1.67 (m, 4H), 1.49 (s, 9H), 1.39-1.43 (m, 2H).
13
C
NMR (150 MHz, CD
3
OD) δC (ppm) 176.21, 169.61, 153.06, 139.09, 131.56, 127.41, 120.82,
119.15, 79.94, 39.21, 33.43, 28.63, 27.21, 26.16, 24.32.

2.109.tert-butyl(2-((6-oxo-6-((3-(trifluoromethyl)phenyl)amino)
hexyl)carbamoyl)phenyl)carbamate. Synthesized according to Method B. Purification by
automated flash chromatography using 40% EtOAc/hexanes as eluent gave 0.100 g as a white
foam (46%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.4.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.52
(d, J = 8.5 Hz, 1H), 8.37 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.75-7.84 (m,
2H), 7.70 (d, J = 7.5 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 3.73 (t, J = 7.0 Hz, 2H), 2.78 (t, J = 7.5
Hz, 2H), 2.14 (t, J = 7.5 Hz, 2H), 2.04 (t, J = 7.0 Hz, 2H), 1.86 (s, 9H), 1.81-1.83 (m, 2H).
13
C
NMR (126 MHz, CD
3
OD) δC (ppm) 173.33, 169.62, 153.22, 139.43, 139.15, 131.59, 131.07,
114

130.60 (q, J = 27.64), 129.24, 127.41, 125.19, 123.03, 122.65, 121.52, 120.82, 119.80, 119.19,
115.97, 79.94, 39.18, 36.40, 28.64, 27.19, 26.18, 24.93.
19
F NMR (470 MHz, CD
3
OD) δF (ppm)
-64.25.

2.110. N
1
-(2-aminophenyl)-N
8
-(3-(trifluoromethyl)phenyl)octanediamide. Synthesized
according to Method C. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.067 g as a white solid (52%). TLC: 80% EtOAc/hexanes, R
f
≈
0.25.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.02 (s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.49 (t, J =
8.0 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.07 (d, J = 9.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.84 (d, J
= 9.0 Hz, 1H), 6.72 (t, J = 8.0 Hz, 1H), 2.40-2.45 (m, 4H), 1.74-1.77 (m, 4H), 1.46-1.48 (m, 4H).
13
C NMR (126 MHz, CD
3
OD) δC (ppm) 173.66, 173.48, 141.94, 139.50, 129.25, 126.79, 125.67,
125.23, 123.73, 122.63, 119.87, 118.12, 117.05, 115.97, 103.40, 36.44, 35.79, 28.56, 25.46,
25.14.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.33.

2.111. N-(6-((2-aminophenyl)amino)-6-oxohexyl)furan-2-carboxamide. Synthesized
according to Method C. Purification by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.068 g as a white solid (89%). TLC: 90% EtOAc/hexanes, R
f
≈
0.25.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.53 (s, 1H), 6.92-6.98 (m, 3H), 6.75 (d, J = 7.2
Hz, 1H), 6.60 (t, J = 7.0 Hz, 1H), 6.45 (s, 1H), 3.27 (t, J = 6.8 Hz, 2H), 2.33 (t, J = 7.6 Hz, 2H),
115

1.63-1.70 (m, 2H), 1.53-1.60 (m, 2H), 1.34-1.41 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC
(ppm)173.62, 159.47, 147.74, 144.71, 141.86, 126.81, 125.70, 123.69, 118.09, 117.11, 113.56,
111.46, 38.56, 35.68, 28.91, 26.15, 25.26.

2.112. N-(6-((2-aminophenyl)amino)-6-oxohexyl)furan-3-carboxamide. Synthesized
according to Method C. Purification by automated flash chromatography using ethyl acetate as
eluent gave 0.059 g as a white solid (61%). TLC: 100% EtOAc, R
f
≈ 0.25.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 8.02 (s, 1H), 7.54 (s, 1H), 7.00-7.06 (m, 2H), 6.84 (d, J = 8.0 Hz, 1H), 6.77 (s,
1H), 6.65 (t, J = 8.0 Hz, 1H), 3.34 (t, J = 7.2 Hz, 2H), 2.43 (t, J = 7.6 Hz, 2H), 1.70-1.76 (m,
2H), 1.60-1.68 (m, 2H), 1.40-1.48 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.55,
163.82, 144.96, 143.78, 141.87, 126.77, 125.69, 123.67, 122.37, 118.08, 117.11, 108.10, 38.83,
35.67, 28.82, 26.19, 25.25.

2.113. N-(6-((2-aminophenyl)amino)-6-oxohexyl)thiophene-2-carboxamide. Synthesized
according to Method C. Purificaiton by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.078 g as a white solid (87%). TLC: 90% EtOAc/hexanes, R
f
≈
0.35.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 7.66 (d, J = 4.0 Hz, 1H), 7.62 (d, J = 5.0 Hz, 1H),
7.11 (t, J = 4.0 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 7.02 (t, J = 7.0 Hz, 1H), 6.84 (d, J = 8.0 Hz,
1H), 6.70 (t, J = 7.0 Hz, 1H), 3.38 (t, J = 6.5 Hz, 2H), 2.44 (t, J = 7.5 Hz, 2H), 1.69-1.78 (m, 2H),
116

1.62-1.67 (m, 2H), 1.39-1.49 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.66, 163.11,
141.88, 139.03, 130.02, 127.96, 127.32, 126.80, 125.73, 123.74, 118.11, 117.13, 39.26, 35.70,
28.88, 26.20, 25.27.

2.114. N-(6-((2-aminophenyl)amino)-6-oxohexyl)thiophene-3-carboxamide. Synthesized
according to Method C. Purification by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.032 g as a white solid (71%). TLC: 90% EtOAc/hexanes, R
f
≈
0.25.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.00 (s, 1H), 7.43-7.48 (m, 2H), 6.99-7.06 (m, 2H),
6.82 (d, J = 8.0 Hz, 1H), 6.67 (t, J = 7.6 Hz, 1H), 3.36 (t, J = 7.2 Hz, 2H), 2.43 (t, J = 7.2 Hz,
2H), 1.72-1.80 (m, 2H), 1.62-1.70 (m, 2H), 1.43-1.51 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC
(ppm) 173.50, 164.25, 141.87, 137.91, 128.16, 126.83, 126.05, 125.99, 125.72, 123.66, 118.08,
117.11, 39.07, 35.68, 28.83, 26.20, 25.26.

2.115. N-(6-((2-aminophenyl)amino)-6-oxohexyl)benzofuran-2-carboxamide. Synthesized
according to Method C. Purification by automated flash chromatography using ethyl acetate as
eluent gave 0.011 g as a yellow oil (31%). TLC: 100% EtOAc, R
f
≈ 0.2.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 7.61 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.35-7.37 (m, 2H), 7.22 (t,
J = 7.5 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.90 (t, J = 7.5 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 6.56
(t, J = 7.5 Hz, 1H), 3.35 (t, J = 7.5 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.60-1.69 (m, 4H), 1.19-1.42
117

(m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.54, 159.80, 154.99, 148.73, 141.86,
127.43, 126.80, 126.68, 125.68, 123.43, 122.29, 118.03, 117.10, 111.3, 109.64, 38.80, 35.68,
28.86, 26.17, 26.27.

2.116. N-(6-((2-aminophenyl)amino)-6-oxohexyl)pyrimidine-5-carboxamide. Synthesized
according to Method B. Purification by automated flash chromatography using ethyl acetate as
eluent gave 0.060 g as a yellow solid (47%). TLC: 100% EtOAc, R
f
≈ 0.1.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 9.24 (s, 1H), 8.99 (d, J = 5.0 Hz, 1H), 8.07 (d, J = 5.5 Hz, 1H), 7.04-7.06 (m,
2H), 6.84 (d, J = 8.0 Hz, 1H), 6.69 (t, J = 7.5 Hz, 1H), 3.47 (t, J = 7.0 Hz, 2H), 2.44 (t, J = 7.5
Hz, 2H), 1.70-1.79 (m, 4H), 1.49-1.51 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.48,
163.46, 158.99, 157.57, 156.73, 141.87, 126.80, 125.66, 123.72, 118.71, 118.04, 117.10, 19.03,
35.65, 28.72, 26.13, 25.24.

2.118. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-5-phenylfuran-2-carboxamide.
Synthesized according to Method C. Purificaiton by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.026 g as a white solid (67%). TLC: 90% EtOAc/hexanes, R
f
≈
0.3.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.85 (d, J = 7.6 Hz, 2H), 7.42 (t, J = 8.0 Hz, 2H),
7.38 (t, J = 8.0 Hz, 1H), 7.16 (d, J = 3.2 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 7.00 (t, J = 7.5 Hz,
118

1H), 6.89 (d, J = 3.6 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.64 (t, J = 7.5 Hz, 1H), 3.42 (t, J = 7.2
Hz, 2H), 7.42 (t, J = 7.2 Hz, 2H), 1.65-1.79 (m, 3H), 1.42-1.58 (m, 2H).
13
C NMR (100 MHz,
CD
3
OD) δC (ppm) 173.72, 159.49, 156.02, 146.64, 141.86, 129.65, 128.49, 128.28, 126.80,
125.69, 124.20, 123.67, 118.10, 117.11, 115.73, 106.61, 38.68, 35.71, 29.04, 26.20, 25.30.

2.125. 2-iodo-4-(trifluoromethyl)aniline.
54
In a foil-covered flask iodine (1.27g, 5.00 mmol)
was added to ethanol (100 mL), followed by silver sulfate (1.56 g, 5.00 mmol) and 4-
(trifluoromethyl)aniline (0.628 mL, 5.00 mmol). After stirring at room temperature for 16 h the
reaction mixture was filtered through celite. The filtrate was concentrated under reduced pressure
then dissolved in CH
2
Cl
2
(100 mL) and washed sequentially with 5% NaOH aqueous solution (50
mL) and water (50 mL). The organic phase was dried over Na
2
SO
4
and concentrated.
Purification by column chromatography using 20% EtOAc/hexanes as eluent gave 0.517 g as an
orange oil (36%). TLC: 20% EtOAc/hexanes, R
f
≈ 0.5.
1
H NMR (400 MHz, CDCl
3
) δH (ppm)
7.87 (s, 1H), 7.38 (d, J = 8.8 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 4.41 (br s, 2H).
13
C NMR (100
MHz, CDCl
3
) δC (ppm) 149.52, 136.14, 126.49, 124.82, 122.13, 121.59, 121.20, 113.46, 82.11.
19
F NMR (376 MHz, CDCl
3
) δF (ppm) -61.39.

2.126. 5-(trifluoromethyl)-1H-indole-2-carboxylic acid.
54
To a flame-dried flask was added
Pd(OAc)
2
(0.019 g, 5 mol%), DABCO (0.576 g, 5.13 mmol), and 2-iodo-4-
(trifluoromethyl)aniline (0.491 g, 1.71 mmol) in DMF (15 mL) at room temperature. Pyruvic
119

acid (0.355 mL, 5.13 mmol) was then added at room temperature and the reaction vessel was
degassed for 5 min. The reaction mixture was stirred at 110°C for 12 h. After cooling to room
temperature ethyl acetate (25 mL) and 5 M HCl (25 mL) were added. The phases were separated
and the aqueous layer was extracted with ethyl acetate (3 x 20 mL). The combined organic
phases were dried over Na
2
SO
4
and concentrated. Purification by flash chromatography using
57%EtOAc/hexanes and 3%AcOH as eluent followed by recrystallization from hexanes gave
0.307 g as an orange solid (78%) TLC: 60% EtOAc/hexanes, R
f
≈ 0.3.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 8.00 (s, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.28 (s, 1H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 162.91, 138.60, 130.25, 126.55, 123.96, 122.34, 121.98,
120.32, 119.52, 112.51, 108.34.
19
F NMR (376 MHz, CD
3
OD) δF (ppm) -62.22.

2.127. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-1H-pyrrole-2-carboxamide. Synthesized
according to Method C. Purification by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.026 g as a yellow solid (58%). TLC: 90% EtOAc/hexanes, R
f
≈
0.3.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 6.99-7.07 (m, 2H), 6.88 (s, 1H), 6.83 (d, J = 8.4 Hz,
1H), 6.76 (d, J = 2.4 Hz, 1H), 6.70 (t, J = 6.8 Hz, 1H), 6.15 (t, J = 4.0 Hz, 1H), 3.34 (t, J = 7.2
Hz, 2H), 2.43 (t, J = 7.6 Hz, 2H), 1.73-1.80 (m, 2H), 1.61-1.67 (m, 2H), 1.44-1.51 (m, 2H).
13
C
NMR (100 MHz, CD
3
OD) δC (ppm) 173.65, 162.42, 141.88, 126.82, 125.74, 123.87, 121.68,
121.26, 118.16, 117.28, 117.11, 113.86, 110.10, 108.71, 38.64, 35.71, 29.08, 26.20, 25.30.

120

2.128. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-1H-indole-2-carboxamide. Synthesized
according to Method C. Purification by automated flash chromatography using ethyl acetate as
eluent gave 0.017 g as a yellow solid (24%). TLC: 100% EtOAc, R
f
≈ 0.2.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 7.59 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.21 (t, J = 8.5 Hz, 2H),
7.01-7.08 (m, 4H), 6.82 (d, J = 7.0 Hz, 1H), 6.66 (t, J = 6.5 Hz, 1H), 3.44 (t, J = 7.0 Hz, 2H), 2.45
(t, J = 7.5 Hz, 2H), 1.77-1.83 (m, 2H), 1.70-1.74 (m, 2H), 1.49-1.54 (m, 2H).
13
C NMR (125
MHz, CD
3
OD) δC (ppm) 173.60, 162.78, 141.87, 136.81, 130.91, 127.65, 126.80, 125.73,
123.74, 123.49, 121.35, 119.70, 118.08, 117.11, 111.61, 102.79, 38.92, 35.69, 28.96, 26.20,
25.28.

2.129. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-5-bromo-1H-indole-2-carboxamide.
Synthesized according to Method C. Purification by automated flash chromatography using 5%
MeOH/CH
2
Cl
2
as eluent gave 0.045 g as a pale yellow solid (96%). TLC: 5% MeOH/CH
2
Cl
2
, R
f

≈ 0.2.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.77 (s, 1H), 9.10 (s, 1H), 8.56 (t, J = 6.0 Hz,
1H), 7.83 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 6.4 Hz, 1H), 7.10
(s, 1H), 6.88 (t, J = 8.0 Hz, 1H), 6.71 (d, J = 8.8 Hz, 1H), 6.52 (t, J = 6.4 Hz, 1H), 4.82 (br s, 2H),
3.27-3.30 (m, 2H), 2.33 (t, J = 7.2 Hz, 2H), 1.57-1.68 (m, 4H), 1.36-1.42 (m, 2H).
13
C NMR (100
121

MHz, DMSO-d
6
) δC (ppm) 171.60, 161.04, 142.31, 135.30, 133.66, 129.23, 126.12, 125.59,
123.96, 116.66, 116.28, 114.65, 112.44, 102.07, 36.16, 29.43, 26.60, 25.51.

2.130. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-5-(trifluoromethyl)-1H-indole-2-
carboxamide. Synthesized according to Method B. Purification by automated flash
chromatography using ethyl acetate as eluent gave 0.033 g as a yellow solid (46%). TLC: 100%
EtOAc, R
f
≈ 0.25.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.94 (s, 1H), 7.58 (d, J = 8.8 Hz, 1H),
7.45 (d, J = 8.8 Hz, 1H), 7.17 (s, 1H), 6.98-7.06 (m, 2H), 6.83 (d, J = 8.0 Hz, 1H), 6.65 (t, J = 8.8
Hz, 1H), 3.45 (t, J = 6.8 Hz, 2H), 2.46 (t, J = 7.2 Hz, 2H), 1.68-1.84 (m, 4H), 1.49-1.57 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm)173.53, 162.04, 141.83, 137.93, 13.18, 126.78, 125.67,
123.70, 122.15, 119.65, 118.07, 117.09, 112.30, 109.99, 103.29, 38.94, 35.62, 28.80, 26.25,
25.25.
19
F NMR (376 MHz, CD
3
OD) δF (ppm) -62.11

2.131. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-5-phenyl-1H-indole-2-carboxamide.
Synthesized according to Method C. Purification by automated flash chromatography using ethyl
acetate as eluent gave 0.019 g as a white solid (70%). TLC: 100% EtOAc, R
f
≈ 0.2.
1
H NMR (400
MHz, DMSO-d
6
) δH (ppm) 11.61 (s, 1H), 9.09 (s, 1H), 8.51 (t, J = 5.6 Hz, 1H), 7.88 (s, 1H), 7.68
122

(d, J = 7.6 Hz, 2H), 7.51 (s, 2H), 7.45 (t, J = 8.0 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 7.15-7.17 (m,
2H), 6.89 (t, J = 6.8 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.53 (t, J = 8.0 Hz, 1H), 4.81 (br s, 2H),
2.34 (t, J = 7.2 Hz, 2H), 1.59-1.71 (m, 4H), 1.39-1.42 (m, 2H). ).
13
C NMR (100 MHz, DMSO-d
6
)
δC (ppm) 171.52, 161.30, 142.29, 141.88, 136.35, 133.09, 132.58, 129.21, 128.16, 127.13,
126.81, 125.71, 123.90, 123.11, 119.78, 116.58, 116.28, 113.12, 103.08, 36.18, 29.52, 26.63,
25.53.

2.132. 6-(2-(1H-indol-2-yl)acetamido)-N-(2-aminophenyl)hexanamide. Synthesized
according to Method B. Purification by automated flash chromatography sing 10%
MeOH/CH
2
Cl
2
as eluent gave 0.041 g as a white foam (32%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈
0.4.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 10.84 (s, 1H), 9.07 (s, 1H), 7.86 (s, 1H), 7.54 (d, J
= 8.0 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.15-7.17 (m, 2H), 7.07 (t, J = 6.4 Hz, 1H), 6.98 (t, J =
6.8 Hz, 1H), 6.89 (t, J = 7.6 Hz, 1H), 6.72 (d, J = 6.8 Hz, 1H), 6.54 (t, J = 6.4 Hz, 1H), 4.81 (s,
2H), 3.49 (s, 2H), 3.03-3.08 (m, 2H), 2.29 (t, J =7.2 Hz, 2H), 1.54-1.63 (m, 2H), 1.40-1.47 (m,
2H), 1.27-1.34 (m, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 171.50, 170.88, 142.31,
136.53, 127.69, 126.10, 125.71, 124.12, 123.97, 121.32, 119.09, 118.66, 116.58, 116.32, 111.71,
109.43, 39.00, 36.14, 33.17, 29.42, 26.57, 25.48.

123

2.133. 6-(3-(1H-indol-3-yl)propanamido)-N-(2-aminophenyl)hexanamide. Synthesized
according to Method B. Purification by automated flash chromatography using 5%
MeOH/EtOAc as eluent gave 0.025 g as a white solid (71%). TLC: 5% MeOH/EtOAc, R
f
≈ 0.35.
1
H NMR (500 MHz, DMSO-d
6
) δH (ppm) 10.73 (br s, 1H), 9.07 (s, 1H), 7.81 (t, J = 5.5 Hz, 1H),
7.51 (d, J = 7.5 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 7.0 Hz, 1H), 7.04-7.08 (m, 2H),
6.96 (t, J = 7.5 Hz, 1H), 6.88 (t, J = 8.5 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.53 (t, J = 8.0 Hz,
1H), 4.81 (br s, 2H), 3.03-3.08 (m, 2H), 2.91 (t, J = 8.0 Hz, 2H), 2.42 (t, J = 8.0 Hz, 2H), 2.30 (t,
J = 7.5 Hz, 2H), 1.55-1.61 (m, 2H), 1.38-1.44 (m, 2H), 1.26-1.29 (m, 2H).
13
C NMR (125 MHz,
DMSO-d
6
) δC (ppm) 1721.10, 171.55, 142.32, 136.66, 127.49, 126.12, 125.73, 124.01, 122.50,
121.30, 118.79, 118.54, 116.65, 116.32, 114.36, 111.72, 38.85, 36.81, 36.18, 29.45, 26.61, 25.53,
21.54.

2.135. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-1H-benzo[d]imidazole-5-carboxamide.
Synthesized according to Method B. Purification by automated flash chromatography using 10%
MeOH/CH
2
Cl
2
as eluent gave 0.043 g as a yellow solid (68%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈
0.1.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.29 (s, 1H), 8.15 (s, 1H), 7.78 (d, J = 6.8 Hz, 1H),
7.65 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 7.02 (t, J = 6.4 Hz, 1H), 6.84 (d, J = 8.4 Hz,
1H), 6.68 (t, J = 7.2 Hz, 1H), 3.46 (t, J = 6.8 Hz, 2H), 2.46 (t, J = 7.2 Hz, 2H), 1.70-1.85 (m, 4H),
1.49-1.59 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.64, 169.15, 143.17, 141.81,
126.73, 125.74, 123.69, 121.57, 117.95, 117.04, 39.55, 35.71, 28.85, 26.21, 25.24.
124

2.136. benzyl (6-((2-aminophenyl)amino)-6-oxohexyl)carbamate. Synthesized according to
Method E. Recrystallization from acetonitrile/water gave 0.073 g as a white solid (78%).
1
H
NMR (400 MHz, CD
3
OD) δH (ppm) 9.07 (s, 1H), 7.33-7.37 (m, 6H), 7.15 (d, J = 7.0 Hz, 1H),
6.89 (t, J = 7.0 Hz, 1H), 6.71 (d, J = 6.8 Hz, 1H), 6.53 (t, J = 7.0 Hz, 1H), 5.01 (s, 1H), 4.80 (s,
2H), 2.97-3.01 (m, 2H), 2.28 (t, J = 7.2 Hz, H), 1.49-1.58 (m, 2H), 1.40-1.47 (m, 2H), 1.25-1.35
(m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 171.60, 156.63, 142.27, 137.75, 128.78,
128.23, 126.04, 125.57, 123.93, 116.63, 116.18, 65.51, 36.15, 29.66, 26.40, 25.47.

2.137. pyridin-3-ylmethyl (6-((2-aminophenyl)amino)-6-oxohexyl)carbamate. Synthesized
according to Method B. Purification by automated flash chromatography using 5%
MeOH/CH
2
Cl
2
as eluent gave 0.070 g as a yellow solid (55%). TLC: 5% MeOH/CH
2
Cl
2
, R
f
≈
0.35.
1
H NMR (400 MHz, CDCl
3
) δH (ppm) 8.62 (s, 1H), 8.56 (d, J = 2.8 Hz, 1H), 7.67 (d, J =
6.0 Hz, 1H), 7.38 (br s, 1H), 7.28-7.30 (m, 1H), 7.19 (d, J = 6.0 Hz, H), 7.06 (t, J = 6.0 Hz, 1H),
6.78-6.81 (m, 2H), 5.09 (s, 2H), 4.89 (br s, 1H), 3.21-3.25 (m, 2H), 2.40 (t, J = 6.0 Hz, 2H), 1.75-
1.79 (m, 2H), 1.55-1.58 (m, 2H), 1.42-1.46 (m, 2H).
13
C NMR (100 MHz, CDCl
3
) δC (ppm)
171.45, 156.16, 149.45, 149.42, 140.72, 135.86, 132.36, 127.11, 125.10, 123.41, 119.56, 118.29,
63.99, 40.67, 36.76, 29.56, 26.06, 25.12.

125

2.143. 4-((((benzyloxy)carbonyl)amino)methyl)benzoic acid. To 4-(aminomethyl)benzoic acid
(1.50 g, 9.92 mmol) in a mixture of dioxane (20 mL) and 1 M NaOH aqueous solution (19.84
mL, 19.48 mmol) at 0°C was added benzyl chloroformate (1.42 mL, 9.92 mmol) dropwise.
Additional 1 M NaOH solution (10 mL) was added dropwise and the reaction mixture was stirred
at room temperature for 16 h. An aqueous solution of 1 M HCl (10 mL) was then added. The
reaction mixture was diluted with CH
2
Cl
2
and filtered to obtain 0.615 g as a white solid (22%).
1
H
NMR (400 MHz, DMSO-d
6
) δH (ppm) 12.81 (br s, 1H), 7.90 (d, J = 8.0 Hz, 3H), 7.34-7.38 (m,
6H), 5.06 (s, 2H), 4.28 (d, J = 6.4 Hz, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 167.61,
156.87, 145.26, 137.54, 129.81, 128.81, 128.20, 127.41, 65.94, 44.10.

2.144. benzyl 4-((2-aminophenyl)carbamoyl)benzylcarbamate. Synthesized according to
Method B. Purification by automated flash chromatography using 70% EtOAc/hexanes as eluent
gave 0.015 g as a white solid (11%). TLC: 70% EtOAc/hexanes, R
f
≈ 0.5.
1
H NMR (400 MHz,
DMSO-d
6
) δH (ppm) 9.58 (br s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.36-7.38 (m, 6H), 7.31 (s, 1H),
7.16 (t, J = 7.6 Hz, 1H), 6.78 (d, J = 6.8 Hz, 1H), 6.59 (t, J = 7.2 Hz, 1H), 5.05 (s, 2H), 4.86 (d, J
= 6.0 Hz, 1H), 4.27 (d, J = 6.4 Hz, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 165.52,
126

156.84, 143.68, 143.54, 137.56, 133.62, 128.80, 128.25, 128.19, 128.12, 127.99, 127.16, 127.09,
127.05, 126.84, 123.76, 116.67, 116.54, 65.89, 44.03.

2.145. 4-(((((3-(trifluoromethyl)benzyl)oxy)carbonyl)amino)methyl)benzoic acid. To a
suspension of CDI (0.268 g, 1.65 mmol) in THF (1 mL) at 0°C was added 3-
(trifluoromethyl)benzyl alcohol (0.225 mL, 1.65 mmol) and stirred while warming to room
temperature for 1 h. This solution was then added to a suspension of 4-(aminomethyl)benzoic
acid (0.250 g, 1.65 mmol), DBU (0.247 mL, 1.65 mmol), and triethylamine (0.230 mL, 1.65
mmol) in THF (5 mL). After stirring at room temperature for 18 h the solvent was evaporated
under reduced pressure. The crude material was dissolved in water (5 mL), acidified to pH 2
using 10% HCl solution and filtered. The white precipitate was rinsed with methanol then diluted
in ethyl acetate and concentrated onto celite. Purification by automated flash chromatography
using 60% EtOAc/hexanes as eluent provided 0.071 g as a white solid (13%). TLC: 60%
EtOAc/hexanes, R
f
≈ 0.3.
1
H NMR (600 MHz, CD
3
OD) δH (ppm) 12.84 (br s, 1H), 7.99 (s, 1H),
7.87 (d, J = 7.8 Hz, 2H), 7.70 (s, 1H), 7.64-7.66 (m, 2H), 7.60-7.61 (m, 1H), 7.33 (d, J = 8.4 Hz,
2H), 5.14 (s, 2H), 4.26 (d, J = 6.0 Hz, 2H).
13
C NMR (150 MHz, CD
3
OD) δC (ppm) 167.63,
156.68, 145.20, 139.15, 132.05, 129.96, 129.82, 127.39, 124.93, 124.32, 65.07, 44.06.
19
F NMR
(564 MHz, CD
3
OD) δF (ppm) -61.15.
127

2.146. 3-(trifluoromethyl)benzyl 4-((2-aminophenyl)carbamoyl)benzylcarbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 60%
EtOAc/hexanes as eluent gave 0.020 g as a white solid (26%). TLC: 60% EtOAc/hexanes, R
f
≈
0.45.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 7.94 (d, J = 7.5 Hz, 2H), 7.70 (s, 1H), 7.61-7.63
(m, 2H), 7.42-7.44 (m, 2H), 7.18 (d, J = 7.5 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.90 (d, J = 8.0 Hz,
1H), 6.78 (t, J = 7.0 Hz, 1H), 5.20 (s, 2H), 4.40 (s, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm)
167.23, 157.32, 143.39, 142.33, 138.48, 132.94, 130.90, 128.93, 127.64, 127.43, 127.12, 127.01,
126.86, 126.78, 126.22, 124.29, 123.91, 123.83, 118.28, 117.33, 109.99, 65.23, 43.77.
19
F NMR
(470 MHz, CD
3
OD) δF (ppm) -64.15.

2.148. N-(4-((2-aminophenyl)carbamoyl)benzyl)nicotinamide. Synthesized according to
Method C. Purification by automated flash chromatography using 10% MeOH/CH
2
Cl
2
as eluent
gave 0.040 g as a white solid (63%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈ 0.1.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 9.05 (s, 1H), 8.71 (d, J = 6.4 Hz, 1H), 8.31 (d, J = 8.0 Hz, 1H), 7.98 (d, J =
8.4 Hz, 2H), 7.52-7.59 (m, 3H), 7.20 (d, J = 8.0 Hz, H), 7.09 (t, J = 7.6 Hz, 1H), 6.91 (d, J = 7.6
Hz, 1H), 6.79 (t, J = 7.6 Hz, 1H), 4.70 (s, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 166.34,
128

151.37, 147.75, 142.61, 142.34, 135.64, 133.04, 130.46, 127.66, 127.21, 127.11, 126.21, 123.87,
123.62, 118.21, 117.31, 42.73.

2.149. N-(2-aminophenyl)-4-((4-(dimethylamino)benzamido)methyl)benzamide.
Synthesized according to Method B. Gave 0.037 g as a yellow solid (71%).
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 7.94 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 9.2 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H),
7.17 (d, J = 8.8 Hz, 1H), 7.06 (t, J = 8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.73-6.77 (m, 3H),
4.62 (s, 2H), 3.01 (s, 6H).
13
C NMR (150 MHz, CD
3
OD) δC (ppm) 168.98, 167.27, 152.95,
143.66, 142.31, 132.77, 128.40, 127.57, 126.19, 123.90, 120.26, 118.23, 117.28, 110.73, 42.59,
38.79.

2.150. 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid. To 4-(aminomethyl)benzoic acid
(5.00 g, 33.1 mmol) in a mixture of 10% NaOH aqueous solution (45 mL) and ethanol (125 mL)
at 0°C was slowly added di-tert-butyl-dicarbonate (8.36 mL, 36.4 mmol). After stirring at room
temperature for 20 h the reaction mixture was concentrated under reduced pressure and water
(250 mL) was added to the crude material. While cooled in an ice bath saturated citric acid
solution (100 mL) was added. The precipitate was filtered and washed with water to obtain 3.09
g as a white solid (37%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 12.78 (br s, 1H), 7.89 (d, J =
129

8.0 Hz, 2H), 7.46 (t, J = 6.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 4.18 (d, J = 6.4 Hz, 2H), 1.40 (s,
9H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 167.65, 156.25, 145.69, 129.78, 129.60, 127.31,
78.37, 43.73, 28.65.

2.151. tert-butyl 4-((2-nitrophenyl)carbamoyl)benzylcarbamate. To a solution of acid
chloride of 2.150 (3.98 mmol) in pyridine (12 mL) was added 2-nitroaniline (0.605 g, 4.38
mmol). After stirring at room temperature for 8 h the reaction mixture was concentrated under
reduced pressure then dissolved in CH
2
Cl
2
(50 mL). The crude material was washed with 10%
HCL aqueous solution (3 x 50 mL), followed by saturated NaHCO
3
solution (3 x 50 mL), and
brine (3 x 50 mL). The organic layer was dried over Na
2
SO
4
and concentrated. Purification by
automated flash chromatography using 20% EtOAc/hexanes as eluent provided 0.111 g as a
yellow solid (14%). TLC: 20% EtOAc/hexanes, R
f
≈ 0.25.
1
H NMR (400 MHz, CD
3
OD) δH
(ppm) 8.43 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.8 Hz, 2H), 7.76 (t, J = 7.6
Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.37 (t, J = 7.2 Hz, 1H), 4.33 (s, 2H), 1.47 (s, 9H).
13
C NMR
(100 MHz, CD
3
OD) δC (ppm) 166.28, 134.58, 133.19, 132.48, 127.32, 127.08, 125.20, 125.16,
124.36, 123.82, 78.94, 43.25, 27.31.

2.152. 4-(aminomethyl)-N-(2-nitrophenyl)benzamide hydrochloride. To a suspension of tert-
butyl-4-((2-nitrophenyl)carbamoyl)benzylcarbonate # (0.084 g, 0.226 mmol) in methanol (8 mL)
130

was added concentrated HCl (0.500 mL). After stirring at room temperature for 24 h the reaction
mixture was concentrated under reduced pressure to provide 0.060 g as a yellow solid (98%).
1
H
NMR (400 MHz, CD
3
OD) δH (ppm) 8.36 (d, J = 6.8 Hz, 1H), 8.20 (d, J = 6.4 Hz, 1H), 8.07 (d, J
= 6.8 Hz, 2H), 7.79 (t, J = 7.2 Hz, 1H), 7.67 (d, J = 6.8 Hz, 2H), 7.40 (t, J = 7.2 Hz, 1H), 4.25 (s,
2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 165.94, 140.38, 137.50, 134.61, 132.79, 129.08,
127.93, 125.25, 124.80, 124.22, 42.42.

2.153. N-(2-nitrophenyl)-4-((2-(pyridin-3-yl)acetamido)methyl)benzamide. Synthesized
according to Method B. Purification by automated flash chromatography using 5%
MeOH/EtOAc as eluent gave 0.051 g as a yellow solid (65%). TLC: 5% MeOH/EtOAc, R
f
≈
0.25.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 10.68 (s, 1H), 8.73 (t, J = 6.0 Hz, 1H), 8.45-8.49
(m, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.71-7.82 (m, 3H), 7.40-7.42 (m, 3H),
7.33-7.35 (m, 1H), 4.36 (d, J = 6.0 Hz, 2H), 3.56 (s, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC
(ppm) 170.19, 165.51, 150.47, 148.14, 144.40, 143.19, 137.06, 134.48, 132.39, 131.96, 128.23,
127.72, 126.38, 125.92, 125.45, 123.80, 42.44.

131

2.154. N-(2-aminophenyl)-4-((2-(pyridin-3-yl)acetamido)methyl)benzamide. To a solution of
2.153 (0.050 g, 0.128 mmol) in methanol (10 mL) was added palladium on charcoal (5% wet)
(0.027 g). The reaction vessel was degassed and then stirred under a balloon of H
2
for 4 h. The
reaction mixture was filtered through celite and concentrated under reduced pressure.
Purification by automated flash chromatography using 10% MeOH/CH
2
Cl
2
as eluent gave 0.010 g
as a yellow solid (22%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈ 0.4.
1
H NMR (400 MHz, CD
3
OD) δH
(ppm) 8.49 (s, 1H), 8.44 (d, J = 6.0 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 7.6 Hz, 1H),
7.39-7.41 (m, 3H), 7.18 (d, J = 8.8 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H),
6.77 (t, J = 7.6 Hz, 1H), 4.46 (s, 2H), 3.64 (s, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm)
171.40, 149.05, 147.12, 142.64, 137.50, 133.01, 127.65, 127.11, 126.21, 123.77, 118.21, 117.31,
42.51, 39.18.

2.155. 4-((1,3-dioxoisoindolin-2-yl)methyl)benzoic acid. A mixture of phthalic anhydride
(0.980 g, 6.62 mmol) and 4-(aminomethyl)benzoic acid (1.00 g, 6.62 mmol) was refluxed in
acetic acid (15 mL) for 4 h. The reaction mixture was then cooled to room temperature and
concentrated under reduced pressure to obtain a white solid. Trituration with water and filtration
provided 1.63 g as a white solid (88%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 12.19 (s, 1H),
7.85-7.92 (m, 6H), 7.41 (d, J = 5.6 Hz, 2H), 4.84 (s, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC
(ppm) 168.12, 167.53, 141.98, 135.05, 132.01, 130.06, 127.80, 123.73, 40.92.
132

2.156. tert-butyl (2-(4-((1,3-dioxoisoindolin-2-yl)methyl)benzamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 40%
EtOAc/hexanes as eluent gave 0.417 g as a yellow solid (50%). TLC: 40% EtOAc/hexanes, R
f
≈
0.3.
1
H NMR (500 MHz, DMSO-d
6
) δH (ppm) 9.81 (s, 1H), 8.62 (s, 1H), 7.87-7.93 (m, 6H),
7.54-7.56 (m, 2H), 7.48 (d, J = 7.5 Hz, 2H), 7.20 (t, J = 8.5 Hz, 1H), 7.13 (t, J = 5.5 Hz, 1H), 4.87
(s, 2H), 1.46 (s, 9H).
13
C NMR (125 MHz, DMSO-d
6
) δC (ppm) 168.16, 165.53, 153.82, 140.94,
136.55, 135.08, 133.96, 132.28, 132.10, 130.01, 128.41, 127.82, 126.52, 126.02, 124.51, 124.14,
123.75, 80.04, 41.10, 28.47.

2.157. tert-butyl (2-(4-(aminomethyl)benzamido)phenyl)carbamate. A mixture of 2.156
(0.300 g, 0.636 mmol) and hydrazine hydrate (0.061 mL, 1.27 mmol) was stirred in ethanol at
60°C for 16 h. After cooling to room temperature the white precipitate was filtered and washed
with ethanol (25 mL). The filtrate was concentrated under reduced pressure and purification by
automated flash chromatography using 20% MeOH/CH
2
Cl
2
as eluent gave 0.212 g as a white
solid (98%). TLC: 20% MeOH/CH
2
Cl
2
, R
f
≈ 0.3.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 9.81
(s, H), 8.70 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 9.2 Hz, 1H), 7.48-7.53 (m, 3H), 7.14-
7.22 (m, 2H), 3.81 (s, 2H), 1.47 (s, 9H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 165.64,
133

153.95, 148.82, 132.53, 131.98, 130.35, 127.85, 127.41, 126.38, 125.92, 125.59, 124.60, 124.38,
80.09, 45.71, 28.47.
2.162. methyl 4-((2-((tert-butoxycarbonyl)amino)benzamido)methyl)benzoate. Synthesized
according to Method B. Purification by automated flash chromatography using 30%
EtOAc/hexanes as eluent gave 0.192 g as a white solid (59%). TLC: 30% EtOAc/hexanes, R
f
≈
0.4.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.19 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 6.8 Hz, 2H),
7.69 (d, J = 8.4 Hz, 1H), 7.44-7.48 (m, 3H), 7.07 (t, J = 6.8 Hz, 1H), 4.62 (s, 2H), 3.90 (s, 3H),
1.51 (s, 9H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 169.57, 166.92, 153.04, 144.29, 139.34,
129.38, 128.77, 127.06, 127.01, 121.58, 119.33, 79.95, 42.55, 27.13.

2.163. 4-((2-((tert-butoxycarbonyl)amino)benzamido)methyl)benzoic acid. Synthesized
according to Method E. Purification by automated flash chromatography using 10%
MeOH/CH
2
Cl
2
as eluent gave 0.084 g as a white solid (48%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈
0.5.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 12.95 (br s, 1H), 10.69 (s, 1H), 9.37 (t, J = 6.0 Hz,
1H), 8.22 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 7.6 Hz, 1H), 7.52 (t, J = 7.6
Hz, 1H), 7.43 (d, J = 11.2 Hz, 2H), 7.10 (t, J = 7.2 Hz, 1H), 4.54 (d, J = 5.2 Hz, 2H), 1.46 (s, 9H).
134

13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 168.91, 167.01, 152.57, 144.72, 140.26, 130.07,
127.48, 121.96, 119.51, 118.93, 80.22, 42.79, 28.37.

2.164. tert-butyl (2-(4-((2-(4-tertbutylbenzamido)methylbenzamido)phenyl)carbamate.
Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent gave 0.068
g as a white foam (59%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.4.
1
H NMR (400 MHz, CD
3
OD) δH
(ppm) 8.20 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 7.6
Hz, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.42-7.48 (m, 2H), 7.18-7.25 (m, 2H), 7.08 (t, J = 7.6 Hz, 1H),
4.63 (s, 2H), 1.51 (s, 9H), 1.48 (s, 9H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 169.57, 166.72,
154.83, 153.02, 143.25, 139.43, 132.80, 131.87, 131.61, 130.12, 127.49, 127.27, 126.00, 125.69,
124.80, 124.15, 120.30, 119.28, 80.28, 79.27, 42.53, 27.20, 27.19.

2.165. 2-amino-N-(4-((2-aminophenyl)carbamoyl)benzyl)benzamide. Purification by
automated flash chromatography using 80% EtOAc/hexanes as eluent gave 0.011 g as a white
solid (26%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.4.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 9.60
(br s, 1H), 8.85 (t, J = 9.2 Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 7.2 Hz, 1H), 7.43 (d, J =
135

8.0 Hz, 2H), 7.14-7.18 (m, 2H), 6.97 (t, J = 8.0 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.71 (d, J = 8.4
Hz, 1H), 6.60 (t, J = 7.6 Hz, 1H), 6.54 (t, J = 7.2 Hz, 2H), 6.43 (br s, 2H), 4.88 (br s, 2H), 4.49 (d,
J = 5.6 Hz, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 169.33, 165.57, 150.23, 143.97,
143.51, 133.47, 143.51, 133.47, 132.25, 128.47, 128.20, 127.29, 127.06, 126.84, 123.77, 116.84,
116.65, 116.53, 115.00, 114.66, 42.47.

2.166. N-(4-((2-aminophenyl)carbamoyl)benzyl)-1H-indole-2-carboxamide. Synthesized
according to general procedures. Purification by automated flash chromatography using
20%MeOH/CH
2
Cl
2
as eluent gave 0.071 g as a yellow solid (64%). TLC: 20% MeOH/CH
2
Cl
2
, R
f

≈ 0.25.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.62 (s, 1H), 9.63 (s, 1H), 9.12 (t, J = 6.0 Hz,
1H), 7.96 (d, J = 8.4 Hz, 7.63 (d, J = 8.0 Hz, 1H), 7.44-7.48 (m, 3H), 7.17-7.21 (m, 3H), 7.05 (t, J
= 7.2 Hz, 1H), 6.98 (t, J = 10 Hz, 6.78 (d, J = 7.6 Hz), 6.61 (t, J = 7.6 Hz, 1H), 4.89 (s, 2H), 4.59
(d, J = 6.0 Hz, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 165.52, 161.65, 143.62, 143.53,
136.92, 133.60, 131.92, 128.29, 127.51, 127.34, 127.08, 126.87, 123.78, 123.75, 121.95, 120.17,
116.7, 116.54, 112.74, 103.09, 42.40.

2.167. 4-((2-(1H-indol-2-yl)acetamido)methyl)-N-(2-aminophenyl)benzamide. Synthesized
according to Method B. Purification by automated flash chromatographyusing ethyl acetate as
136

eluent gave 0.106 g as a yellow solid (82%). TLC: 100% EtOAc, R
f
≈ 0.3.
1
H NMR (400 MHz,
DMSO-d
6
) δH (ppm) 10.88 (s, 1H), 9.60 (s, 1H), 8.47 (t, J = 6.4 Hz, 1H), 7.89 (d, J = 7.6 Hz,
2H), 7.56 (d, J = 7.6 Hz, 1H), 7.32-7.36 (m, 3H), 7.21 (s, 1H), 7.15 (d, J = 7.2 Hz, 1H), 7.08 (t, J
= 8.0 Hz, H), 6.95-6.98 (m, 2H), 6.77 (d, J = 8.4 Hz, 1H), 6.60 (t, J = 8.8 Hz, 1H), 4.87 (s, 2H),
4.34 (d, J = 6.4 Hz, 2H), 3.60 (s, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 171.42, 143.69,
143.55, 136.56, 133.45, 128.16, 127.65, 127.28, 127.07, 126.88, 124.25, 123.69, 121.40, 119.10,
118.61, 116.55, 111.76, 109.12, 42.38, 33.13.

2.168. 4-((3-(1H-indol-3-yl)propanamido)methyl)-N-(2-aminophenyl)benzamide.
Synthesized according to Method C. Purification by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.018 g as a white solid (41%). TLC: 90% EtOAc/hexanes, R
f
≈
0.3.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 10.11 (br s, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.57 (d, J
= 8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.14-7.20 (m, 3H), 7.07-7.10 (m, 2H), 6.89-7.02 (m, 2H),
6.78 (d, J = 4.8 Hz, 1H), 6.77 (t, J = 7.2 Hz, 1H), 4.37 (s, 2H), 3.11 (t, J = 7.6 Hz, 2H), 2.65 (t, J
= 7.6 Hz, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 174.53, 142.15, 136.72, 132.71, 127.49,
127.06, 126.86, 126.17, 123.77, 121.93, 120.87, 118.38, 117.91, 117.28, 113.40, 110.66, 42.15,
36.78, 21.20.

137

2.169. N-(4-((2-aminophenyl)carbamoyl)phenethyl)-1H-indole-2-carboxamide. Synthesized
according to Method B. Purification by automated flash chromatography using 90%
EtOAc/hexanes as eluent gave 0.043 g as a pale pink solid (67%). TLC: 90% EtOAc/hexanes, R
f

≈ 0.4.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.54 (s, 1H), 9.60 (s, 1H), 8.58 (t, J = 5.2 Hz,
1H), 7.92 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.39-7.43 (m, 3H), 7.15-7.19 (m, 2H),
7.10 (s, 1H), 7.03 (t, J = 5.6 Hz, 1H), 6.95 (t, J = 8.8 Hz, 1H), 6.77 (d, J = 6.8 Hz, 1H), 6.60 (t, J
= 7.2 Hz, 1H), 4.87 (s, 2H), 3.56-3.61 (m, 2H), 2.97 (t, J = 6.8 Hz, 2H).
13
C NMR (100 MHz,
DMSO-d
6
) δC (ppm) 161.48, 143.56, 136.71, 132.94, 132.19, 128.99, 128.28, 127.47, 127.07,
126.95, 123.80, 123.63, 121.88, 120.11, 116.56, 116.52, 112.36, 102.72, 35.46.

2.171. 4-acetamidobenzoic acid. A mixture of 4-aminobenzoic acid (1.00 g, 7.29 mmol) and
sodium acetate (0.718 g, 8.75 mmol) was refluxed in acetic acid (3 mL) for 16 h. The reaction
mixture was cooled to room temperature then poured into cold water (10 mL). The precipitate
was filtered and washed with water (50 mL). Recrystallization from 2:1 water/ethanol provided
0.599 g as a white solid (46%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈ 0.2.
1
H NMR (400 MHz,
DMSO-d
6
) δH (ppm) 12.67 (br s, 1H), 10.24 (s, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 9.2 Hz,
2H), 2.09 (s, 3H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 169.27, 167.40, 143.75, 130.76,
125.25, 118.58, 24.58.

138

2.172. 4-acetamido-N-(2-aminophenyl)benzamide. Synthesized according to Method B.
Purification by automated flash chromatography using 90% EtOAc/hexanes as eluent gave 0.028
g as a white solid (37%). TLC: 90% EtOAc/hexanes, R
f
≈ 0.2.
1
H NMR (400 MHz, DMSO-d
6
)
δH (ppm) 10.19 (s, 1H), 9.55 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H), 7.16 (d, J
= 7.6 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.60 (t, J = 7.5 Hz, 1H), 4.88 (s,
2H), 2.09 (s, 3H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 169.16, 165.16, 143.55, 142.48,
129.26, 129.09, 127.07, 126.78, 123.93, 118.45, 116.70, 116.56, 24.57.

2.187. (2E,4E)-7-(4-(dimethylamino)phenyl)-4,6-dimethyl-7-oxohepta-2,4-dienoic acid.
71
To
a solution of 2.199 (0.018 g, 0.062 mmol) in CH
2
Cl
2
/H
2
O (2:1) (1.3 mL) at 0°C was added DDQ
(0.017 g, 0.075 mmol) in two portions. After stirring for 15 min. the reaction mixture was diluted
with water (5 mL) and extracted into CH
2
Cl
2
(3 x 15 mL). The combined organic layers were
washed successively with water (5 x 15 mL) and brine (15 mL), dried over Na
2
SO
4
, then
concentrated to obtain 0.017 g as an orange solid (94%).
1
H NMR (500 MHz, CDCl
3
) δH (ppm)
7.85 (d, J = 7.5 Hz, 2H), 7.36 (d, J = 15.5 Hz, 1H), 6.65 (d, J = 9.5 Hz, 2H), 6.09 (d, J = 8.5 Hz,
1H), 5.83 (d, J = 16 Hz, 1H), 4.37-4.43 (m, 1H), 3.06 (s, 6H), 1.93 (s, 3H), 1.32 (d, J = 6.5 Hz,
3H).

139

2.188. (2E,4E)-ethyl 7-(4-(dimethylamino)phenyl)-7-hydroxy-4,6-dimethylhepta-2,4-
dienoate.
71
To a solution of 2.198 (0.073 g, 0.241 mmol) in THF (4 mL) was added 37.4%
formaldehyde solution (0.058 mL, 0.722 mmol) at room temperature. After stirring for 10 min.
NaBH(OAc)
3
(0.153 g, 0.722 mmol) was added and stirring was continued at room temperature
for 36 h. The reaction mixture was then diluted with ethyl acetate (5 mL) and filtered. The
filtrate was concentrated onto celite under reduced pressure and purified by flash chromatography
using 40% EtOAc/hexanes as eluent to obtain 0.038 g as a colorless oil (50%). TLC: 40%
EtOAc/hexanes R
f
≈ 0.5.
1
H NMR (400 MHz, CDCl
3
) δH (ppm) 7.36 (d, J = 15.6 Hz, 1H), 7.19
(d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H), 5.86 (d, J = 9.2 Hz, 1H), 5.81 (d, J = 15.6 Hz, 1H),
4.37 (d, J = 7.6 Hz, 1H), 4.18-4.24 (m, 2H), 2.95 (s, 6H), 2.83-2.87 (m, 1H), 1.78 (s, 3H), 1.25-
1.32 (m, 3H), 0.86 (d, J = 6.4 Hz, 3H).

2.189. (2S,3R)-3-hydroxy-2-methyl-3-(4-nitrophenyl)propanal.
71
To a mixture of 4-
nitrobenzaldehyde (0.378 g, 2.50 mmol) and L-proline (0.058 g, 0.50 mmol) in DMF (8 mL) at
0°C was added propionaldehyde (0.361 mL, 5.00 mmol) in one portion, then stirring was
continued for 6 h while warming to room temperature. Water (10 mL) was then added to the
reaction mixture and the crude mixture was extracted with ethyl acetate (3 x 20 mL). The
combined organic layers were washed with brine (50 mL), dried over Na
2
SO
4
and concentrated
under reduced pressure to obtain 0.653 g as a yellow oil which was used without further
purification.
1
H NMR (500 MHz, CDCl
3
) δH (ppm) 9.81 (s, 1H), 8.24 (d, J = 8.5 Hz, 2H), 7.55
140

(d, J = 8.5 Hz, 2H), 4.98 (d, J = 8.5 Hz, 1H), 3.16 (br s, 1H), 2.78 (t, J = 7.5 Hz, 1H), 1.01 (d, J =
7.0 Hz, 3H).

2.193 (2E,4E)-ethyl 7-hydroxy-4,6-dimethyl-7-(4-nitrophenyl)hepta-2,4-dienoate.
71
To a
suspension of NaH (60% in mineral oil) (0.062 g, 1.56 mmol) in anhydrous THF (2 mL) was
added dropwise a solution of triethyl phosphonoacetate (0.418 mL, 2.09 mmol) in THF (2 mL)
under argon over 10 min. at room temperature. The mixture was stirred at room temperature for
20 min. then was added 2.197 (0.130 g, 0.52 mmol) in THF (2 mL) dropwise. After stirring at
room temperature for 30 min. the reaction mixture was quenched with saturated sodium
bicarbonate solution (5 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic
phases were washed with brine (25 mL), dried over Na
2
SO
4
, then concentrated under reduced
pressure. Purification by flash chromatography using 30% EtOAc/hexanes as eluent gave 0.150 g
as a colorless oil (87%). TLC: 30% EtOAc/hexanes R
f
≈ 0.35.
1
H NMR (500 MHz, CDCl
3
) δH
(ppm) 8.20 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 10.4 Hz, 2H), 7.28 (d, J = 15.2 Hz, 1H), 5.82 (d, J =
15.2 Hz, 1H), 5.79 (d, J = 10.0 Hz, 1H), 4.65 (d, J = 6.0 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.83-
2.92 (m, 1H), 2.07 (d, J = 3.2 Hz, 1H), 1.69 (d, J = 1.2 Hz, 3H), 1.31 (t, J = 6.8 Hz, 3H), 0.98 (d,
J = 6.4 Hz, 3H).

141

2.195. (E)-methyl 5-hydroxy-2,4-dimethyl-5-(4-nitrophenyl)pent-2-enoate.
71
To a solution of
aldehyde 2.189 (0.523 g, 2.50 mmol) in dry CH
2
Cl
2
(5 mL) was added methyl 2-
(triphenylphosphoranylidene)propanoate (1.05 g, 3.00 mmol). The reaction mixture was refluxed
for 3.5 h, cooled to room temperature, then concentrated onto celite under reduced pressure.
Purification by flash chromatography using 30% EtOAc/hexanes as eluent gave 0.315 g yellow
oil (45%). TLC: 30% EtOAc/hexanes R
f
≈ 0.4.
1
H NMR (400 MHz, CDCl
3
) δH (ppm) 8.20 (d, J
= 8.0 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 6.69 (d, J = 10.4 Hz, 1H), 4.67 (d, J = 6.4 Hz, 1H), 3.75
(s, 3H), 2.79-2.88 (m, 1H), 2.17 (s, 1H), 1.77 (d, J = 1.6 Hz, 3H), 0.96 (d, J = 6.4 Hz, 3H).

2.196. (E)-2,4-dimethyl-5-(4-nitrophenyl)pent-2-ene-1,5-diol.
71
To a solution of ester 2.195
(0.319 g, 1.14 mmol) in anhydrous THF (3 mL) at -78°C under argon was added dropwise a
solution of DIBAL-H in CH
2
Cl
2
(3.94 mL, 3.94 mmol) over 10 min. The reaction mixture was
stirred at -78°C for 1 h and was then slowly quenched with a saturated solution of sodium tartrate
and allowed to warm to room temperature. The crude mixture was then extracted with ethyl
acetate (3 x 10 mL). The combined organic phases were washed with brine (25 mL), dried over
Na
2
SO
4
and concentrated under reduced pressure to obtain 0.271 g (95%) which was used
without further purification. TLC: 50% EtOAc/hexanes R
f
≈ 0.2.
1
H NMR (500 MHz, CDCl
3
)
δH (ppm) 8.19 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), 5.35 (d, J = 9.5 Hz, 1H), 4.48 (d, J =
7.0 Hz, 1H), 4.05 (d, J = 6.0 Hz, 2H), 2.68-2.75 (m, 1H), 2.26 (s, 1H), 1.64 (s, 3H), 0.87 (d, J =
6.0 Hz, 3H).
142

2.197. (E)-5-hydroxy-2,4-dimethyl-5-(4-nitrophenyl)pent-2-enal.
71
To a solution of alcohol
2.196 (0.259 g, 1.03 mmol) in dry CH
2
Cl
2
(13 mL) was added activated manganese (IV) oxide
(0.896 g, 10.3 mmol) at room temperature. After stirring for 5 h the reaction mixture was diluted
with ethyl acetate (50 mL) and filtered through celite. The filtrate was concentrated under
reduced pressure and purification by flash chromatography using 50% EtOAc/hexanes as eluent
provided 0.134 g as yellow crystals (52%). TLC: 50% EtOAc/hexanes R
f
≈ 0.5.
1
H NMR (500
MHz, CDCl
3
) δH (ppm) 9.44 (s, 1H), 8.23 (d, J = 9.0 Hz, 2H), 7.53 (d, J = 9.0 Hz, 2H), 6.46 (d, J
= 9.5 Hz, 1H), 4.82 (d, J = 5.5 Hz, 1H), 3.01-3.09 (m, 1H), 2.13 (d, J = 3.0 Hz, 1H), 1.63 (s, 3H),
1.10 (d, J = 6.5 Hz, 3H).

2.198. (2E,4E)-ethyl 7-(4-aminophenyl)-7-hydroxy-4,6-dimethylhepta-2,4-dienoate.
71
To a
solution of 2.197 (0.105 g, 0.329 mmol) in methanol (3.3 mL) at room temperature was added
quinolone (0.031 mL, 0.262 mmol) and Lindlar’s catalyst (0.073 g). The reaction mixture was
stirred under a balloon of H2 for 5.5 h then diluted with methanol and filtered through celite. The
filtrate was concentrated under reduced pressure and purified by flash chromatography using 50%
EtOAc/hexanes as eluent to obtain 0.084 g as a white solid (84%). TLC: 50% EtOAc/hexanes R
f
≈ 0.4.
1
H NMR (400 MHz, CDCl
3
) δH (ppm) 7.34 (d, J = 18.8 Hz, 1H), 7.10 (d, J = 8.0 Hz, 2H),
143

6.65 (d, J = 8.0 Hz, 2H), 5.75-5.85 (m, 2H), 4.35 (d, J = 9.6 Hz, 1H), 4.17-4.23 (m, 2H), 3.65 (br
s, 2H), 2.81-2.87 (m, 1H), 1.77 (d, J = 1.2 Hz, 3H), 1.28-1.31 (m, 3H), 0.85 (d, J = 8.8 Hz, 1H).

2.199. (2E,4E)-7-(4-(dimethylamino)phenyl)-7-hydroxy-4,6-dimethylhepta-2,4-dienoic
acid.
71
To a solution of 2.188 (0.084 g, 0.264 mmol) in methanol was added an aqueous solution
of 0.5 M LiOH (0.741 mL, 0.370 mmol) at room temperature. The reaction mixture was stirred at
50°C for 12 h, then cooled to room temperature. The pH was adjusted to 7 using phosphate
buffer, then concentrated under reduced pressure. The crude material was redissolved in water (5
mL) and the pH was adjusted to 2 using 10% aqueous HCl solution. The aqueous phase was
extracted with chloroform/methanol (95:5) (5 x 20 mL). The combined organic layers were
washed with brine (75 mL), dried over Na
2
SO
4
, and concentrated under reduced pressure to
obtain 0.061 g as a yellow solid (80%).
1
H NMR (400 MHz, 10% CD
3
OD + 90% CDCl
3
) δH
(ppm) 7.28 (d, J = 14.4 Hz, 1H), 7.11 (d, J = 6.4 Hz, 2H), 6.65 (d, J = 6.8 Hz, 2H), 5.80 (d, J =
7.6 Hz, 1H), 5.70 (d, J = 13.6 Hz, 1H), 4.30 (d, J = 6.0 Hz, 1H), 2.86 (s, 6H), 2.85-2.87 (m, 3H),
1.71 (s, 3H), 0.78 (d, J = 5.6 Hz, 3H).

2.200. (2E,4E)-N-(2-aminophenyl)-7-(4-(dimethylamino)phenyl)-4,6-dimethyl-7-oxohepta-
2,4-dienamide. A mixture of trichostatic acid (0.017 g, 0.059 mmol), 1,2-phenylenediamine
144

(0.006 g, 0.059 mmol), HBTU (0.022 g, 0.059 mmol), and DIPEA (0.010 mL, 0.059 mmol) was
stirred at room temperature in acetonitrile (1 mL) for 18 h. The reaction mixture was diluted with
ethyl acetate (10 mL) then washed sequentially with brine (10 mL) and saturated sodium
bicarbonate solution (2 x 10 mL). The organic phase was dried over Na
2
SO
4
, concentrated under
reduced pressure, and purified by flash chromatography using 60% EtOAc/hexanes as eluent to
obtain 0.018 g as a yellow film (82%). . TLC: 60% EtOAc, R
f
≈ 0.4.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 7.88 (d, J = 4.5 Hz, 2H), 7.28 (d, J = 15.5 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H),
7.03 (t, J = 7.5 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.72-6.76 (m, 3H), 6.26 (d, J = 16 Hz, 1H), 5.97
(d, J = 9.5 Hz, 1H), 4.56-4.60 (m, 1H), 3.08 (s, 6H), 2.01 (s, 3H), 1.29 (d, J = 7.0 Hz, 3H).
13
C
NMR (125 MHz, CD
3
OD) δC (ppm) 199.99, 180.48, 165.94, 154.06, 145.64, 141.62, 140.39,
133.09, 130.48, 126.71, 125.22, 123.90, 123.29, 119.03, 118.15, 117.24, 110.49, 40.35, 38.67,
16.87, 11.45.

2.201. (2E,4E)-7-(4-(dimethylamino)phenyl)-4,6-dimethyl-7-oxohepta-2,4-dienamide. To a
mixture of trichostatic acid (0.012 g, 0.042 mmol), HBTU (0.016 g, 0.042 mmol), and DIPEA
(0.015 mL, 0.084 mmol) in acetonitrile (1 mL) at room temperature was added ammonium
hydroxide (0.014 mL, 0.21 mmol). After stirring for 36 h the reaction mixture was diluted with
ethyl acetate (10 mL), washed successively with brine (10 mL) and saturated sodium bicarbonate
solution (2 x 10 mL), dried over Na
2
SO
4
, the concentrated under reduced pressure onto celite.
Purification by flash chromatography using 5% MeOH/CH
2
Cl
2
as eluent gave 0.004 g as a yellow
oil (33%). . TLC: 5% MeOH/EtOAC, R
f
≈ 0.45.
1
H NMR (500 MHz, CDCl
3
) δH (ppm) 7.84 (d,
145

J = 9.0 Hz, 2H), 7.18 (d, J = 15.5 Hz, 1H), 6.64 (d, J = 8.5 Hz, 2H), 6.02 (d, J = 10.0 Hz, 1H),
5.84 (d, J = 16.0 Hz, 1H), 5.34 (br s, 2), 4.33-4.40 (m, 1H), 3.06 (s, 6H), 1.91 (s, 3H), 1.30 (d, J =
7.0 Hz, 1H).
13
C NMR (125 MHz, CDCl
3
) δC (ppm) 198.47, 168.04, 153.43, 146.50, 141.09,
132.29, 130.61, 123.92, 118.13, 110.71, 109.98, 40.88, 40.00, 17.71, 12.66.

2.212. To a mixture of Fmoc-Lys-OH HCl (2.00 g, 4.94 mmol) and copper sulfate pentahydrate
(0.024 g, 2 mol%) in methanol/water (8:2) (40 mL) at room temperature was added sodium
bicarbonate (1.45 g, 17.29 mmol), followed by imidazole-1-sulfonyl-azide HCl (1.03 g, 5.93
mmol). The pH of the reaction mixture was adjusted to 8 using solid sodium bicarbonate and
stirring was continued for 16 h. After concentrating to remove methanol the reaction mixture was
acidified to pH 2 using 10% aqueous HCl solution then extracted with ethyl acetate (4 x 50 mL).
The combined organics were washed with brine (100 mL), then dried over sodium sulfate and
concentrated onto celite. Purification by column chromatography using 10% MeOH/DCM
provided 1.53 g as a yellow oil (78%).
1
H NMR (400 MHz, CDCl
3
) δH (ppm) 7.74 (d, J = 7.6
Hz, 2H), 7.57 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.6 Hz, 2 H), 7.30 (t, J = 7.6 Hz, 2H), 5.29 (d, J =
8.4 Hz, 1H), 4.41 (d, J = 7.2 Hz, 2H), 4.21 (t, J = 7.2 Hz, 1H), 3.27 (t, J = 6.8 Hz, 2H), 1.82-1.99
(m, 1H), 1.59-1.64 (m, 1H), 1.45-1.47 (m, 2H), 1.23-1.25 (m, 2H).
13
C NMR (100 MHz, CDCl
3
)
δC (ppm) 176.80, 155.99, 143.67, 141.31, 127.75, 127.06, 124.95, 120.00, 67.12, 53.52, 51.02,
47.12, 31.80, 28.32, 22.42.
146

2.215. (S)-1-benzylpyrrolidine-2-carboxylic acid. A solution of (S)-proline (5.00 g, 43.0 mmol)
and KOH (9.26 g, 165 mmol) in isopropanol was stirred at 40°C until the mixture became
transparent. To the reaction mixture was added benzyl chloride (7.42 mL, 64.5 mmol) dropwise
and stirring was continued for 16 h. The reaction mixture was cooled to room temperature, then
the pH was adjusted to 5 with concentrated HCl (10 mL). To this was added chloroform (25 mL)
and stirring was continued for 18 h at room temperature. The reaction mixture was then filtered
and rinsed with chloroform. The filtrate was concentrated to obtain a yellow oil. To this oil was
added acetone and after filtration and drying on hi-vac was obtained 6.2 g as a white solid (70%).
1
H NMR (400 MHz, CDCl
3
) δH (ppm) 9.51 (br s), 7.41-7.43 (m, 2H), 7.33-7.36 (m, 3H), 4.24 (q,
J = 12.8 Hz, 2H), 3.79 (t, J = 6 Hz, 1H), 3.64-3.70 (m, 1H), 2.84 (q, J = 1.6 Hz, 1H), 2.23-2.29
(m, 2H), 1.72-2.01 (m, 2H).
13
C NMR (100 MHz, CDCl
3
) δC (ppm) 171.10, 131.10, 130.44,
129.39, 129.11, 67.59, 57.89, 53.38, 28.97, 22.97.

2.216. (S)-N-(3-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide. A mixture of 2.215 (4.1
g, 20 mmol) in dry CH
2
Cl
2
(10 mL) was cooled to 0°C and thionyl chloride (1.8 mL, 25 mmol)
147

was added dropwise. The reaction mixture was stirred on ice until it became transparent, then o-
aminobenzophenone (2.27 g, 12.5 mmol) in dry CH
2
Cl
2
(10 mL) was added. The reaction
mixture was stirred while warming to room temperature for 16 h. The reaction mixture was
quenched by addition of a solution of sodium carbonate (2 eq.) in water at 0°C while stirring.
The layers were then separated and the aqueous was extracted with CH
2
Cl
2
(3 x 25 mL). The
combined organics were dried over sodium sulfate and concentrated to obtain a yellow solid
which was recrystallized from ethanol to obtain 3.9 g as a yellow solid (50%).
1
H NMR (400
MHz, DMSO-d
6
) δH (ppm) 11.03 (s, 1H), 8.28 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 6.8 Hz, 2H),
7.68-7.71 (m, 1H), 7.59-7.61 (m, 3H), 7.46-7.49 (m, 1H), 7.31 (d, J = 9.2 Hz, 2H), 7.21-7.23 (m,
1H), 7.11-7.12 (m, 3H), 3.75 (d, J = 12.8 Hz, 1H), 3.54 (d, J = 12.8 Hz, 1H), 2.99-3.20 (m, 1H),
2.99 (t, J = 8.8 Hz, 1H), 2.34-2.40 (m, 1H), 2.10-2.16 (m, 1H), 1.51-1.65 (m, 3H).

2.217. Ala – Ni (II) – BPB. A mixture of 2.126 (0.283 g, 0.74 mmol), nickel nitrate hexahydrate
(0.430 g, 5.90 mmol), and alanine (0.131 g, 1.47 mmol) was stirred at 55°C in methanol (2.6 mL)
under argon. To this green reaction mixture was added KOH (0.331 g, 5.90 mmol) in methanol
(1.1 mL) dropwise, which formed a white precipitate that then became red. After stirring for an
additional 2 h at 55°C reaction mixture was cooled on ice. While stirring at 0°C acetic acid
(0.337 mL) was added, then the reaction mixture was diluted with water (8 mL) and allowed to
stand for 16 h. After cooling on ice and scratching to initiate crystallization of the red oil, the
148

material was filtered and rinsed with water to obtain 0.340 g of a red solid (90%). %).
1
H NMR
(400 MHz, CDCl
3
) δH (ppm) 8.05-8.09 (m, 3H), 7.44-7.51 (m, 3H), 7.36 (t, J = 7.6 Hz, 2H), 7.18
(t, J = 7.6 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 6.94 (d, J = 6.94 Hz, 1H), 6.61-6.67 (m, 2H), 4.40 (d,
J = 12.8 Hz, 1H), 3.90 (q, J = 6.0 Hz, 1H), 3.61-3.72 (m, 1H), 3.46-3.57 (m, 3H), 2.65-2.73 (m,
1H), 2.49-2.55 (m, 1H), 2.16-2.22 (m, 2H), 2.05-2.10 (m, 1H), 1.58 (d, J = 6.8 Hz, 3H).
13
C
NMR (100 MHz, CDCl
3
) δC (ppm) 180.49, 180.38, 170.27, 142.06, 133.47, 133.28, 133.10,
13.06, 131.52, 129.67, 128.93, 128.89, 128.87, 127.48, 127.81, 126.45, 123.87, 120.76, 66.55,
63.00, 57.23, 30.74, 24.08, 21.81.

2.218. Ala-Ni-BPB 2.217 (0.10 g, 0.20 mmol) and powdered NaOH (0.080 g, 2.0 mmol) were
added to a round bottom flask which was purged with argon. Dry DMF (1 mL) was added and
then the mixture was stirred at room temperature for 5 min. At room temperature was added 5-
bromo-1-pentene (0.024 mL, 0.20 mmol) and stirring was continued for 45 min. The reaction
mixture was then slowly poured into a cold solution of 5% acetic acid in water (10 mL). After
extraction with CH
2
Cl
2
(3 x 10 mL) the organic phase was dried over sodium sulfate and
concentrated onto celite. Purification by column chromatography using 5%MeOH/DCM
provided 0.065 g (62%) of a red solid.
1
H NMR (400 MHz, CDCl
3
) δH (ppm) 8.05 (d, J = 7.2
Hz, 1H), 7.99 (d, J = 10.4 Hz, 1H), 7.45-7.47 (m, 2 H), 7.42-7.45 (m, 2H), 7.27-7.32 (m, 2H),
7.10-7.14 (m, H), 6.96 (d, J = 7.2 Hz, H), 6.59-6.65 (m, 2H), 5.81-5.91 (m, 1H), 5.07 (d, J = 6.8
149

Hz, 1H), 5.02 (d, J = 9.6 Hz, 1H), 4.49 (d, J = 12.8 Hz, 1H), 3.69 (d, J = 12.8 Hz, 1H), 3.62-3.68
(m, 1H), 3.42-3.44 (m, 1H), 3.21-3.31 (m, 1H), 2.88-2.95 (m, 1H), 2.35-2.51 (m, 2H), 1.65-1.71
(m, 2H), 1.30 (s, 3H).
13
C NMR (100 MHz, CDCl
3
) δC (ppm) 182.37, 180.40, 172.36, 141.45,
137.81, 136.64, 133.31, 131.68, 131.51, 130.26, 129.26, 128.88, 128.63, 128.27, 127.90, 127.37,
127.00, 123.85, 120.78, 115.36, 78.04, 63.36, 56.93, 39.83, 33.68, 30.53, 29.45, 25.27, 23.17.

2.219. To a solution of 3N HCl in MeOH (1:1) (7.75 mL) at 70°C was added 2.218 (0.600 g, 1.03
mmol) in MeOH (2 mL) dropwise. After stirring at 70°C for 15 min. the reaction mixture was
cooled to room temperature and concentrated to obtain a green/yellow solid. To this crude
material was added a solution of 10% aqueous Na
2
CO
3
(10.3 mL), then dioxane (10.3 mL) at
0°C. Fmoc-OSu (0.417 g, 1.24 mmol) in dioxane (1 mL) was then added and the reaction
mixture was stirred while warming to room temperature for 12 h. The mixture was then diluted
with ethyl acetate (50 mL) and 10% aqueous HCl (50 mL), then washed with additional 10%
aqueous HCl (3 x 50 mL). The organic phase was dried over sodium sulfate, concentrated, and
purified by column chromatography using 5%MeOH/DCM with 1% AcOH to obtain 0.089 g of a
yellow oil (62%).
1
H NMR (400 MHz, MeOD) δH (ppm) 7.78 (d, J = 7.2 Hz, 2H), 7.63 (d, J =
7.6 Hz, 2H), 7.38 (t, J = 7.6 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 5.73-5.83 (m, H), 4.96 (d, J = 17.2
Hz, 1H), 4.90 (d, J = 10 Hz, 1H), 4.27-4.34 (m, 2H), 4.18-4.21 (m, 1H), 1.98-2.01 (3H), 1.77-
1.84 (m, 1H), 1.46 (s, 3H).
13
C NMR (100 MHz, MeOD) δC (ppm) 186.28, 155.24, 143.95,
150

143.89, 141.15, 127.28, 126.67, 126.55, 126.47, 124.70, 119.46, 113.50, 65.76, 35.92, 33.48,
23.59, 23.46, 22.70.

2.221. tert-butyl (8-aminonaphthalen-1-yl)carbamate. To a solution of 1,8-
diaminonaphthalene (1.00 g, 6.32 mmol) in dry CH
2
Cl
2
(10 mL) under nitrogen was added di-tert-
buyl-dicarbonate (1.60 mL, 6.95 mmol). After stirring at room temperature for 2 h the reaction
mixture was washed with brine (3 x 25 mL). The organic layer was dried over Na
2
SO
4
and
concentrated onto celite under reduced pressure. The crude material was purified by automated
flash chromatography using 20% EtOAc/hexanes as eluent to provide 0.515 g as a red oil (32%).
TLC: 20% EtOAc/hexanes, R
f
≈ 0.6.
1
H NMR (600 MHz, CD
3
OD) δH (ppm) 7.59 (br s, 1H),
7.50 (d, J = 8.4 Hz, 1H), 7.25-7.31 (m, 1H), 7.19 (d, J = 6.0 Hz, 1H), 6.82 (d, J = 7.8 Hz, 1H),
1.51 (s, 9H).
13
C NMR (151 MHz, CD
3
OD) δC (ppm) 136.56, 134.56, 125.71, 125.16, 124.99,
124.89, 119.93, 119.78, 79.67, 27.32.

2.222. tert-butyl (8-(7-oxo-7-(phenylamino)heptanamido)naphthalen-1-yl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 40%
EtOAc/hexanes as eluent gave 0.086 g as a white solid (23%). TLC: 40% EtOAc/hexanes, R
f
≈
151

0.2.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.74-7.79 (m, 2H), 7.51 (d, J = 7.6 Hz, 4H), 7.38-
7.44 (m, 2H), 7.27 (t, J = 7.2 Hz, 2H), 7.17-7.21 (m, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.06 (t, J =
7.2 Hz, 1H), 6.51 (d, J = 6.4 Hz, 1H), 2.54 (t, J = 7.6 Hz, 2H), 2.41 (t, J = 7.2 Hz, 2H), 1.75-1.86
(m, 4H), 1.47-1.56 (m, 11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.92, 173.01, 154.92,
138.46, 136.90, 136.08, 132.48, 130.94, 130.49, 128.31, 127.54, 126.53, 124.78, 123.66, 119.80,
109.99, 104.11, 79.98, 36.29, 28.50, 27.34, 25.16, 24.68.

2.223. tert-butyl 2-aminophenylcarbamate. To a mixture of 1,2-phenylenediamine (1.00 g,
9.25 mmol) in CH
2
Cl
2
(10 mL) at 0 ⁰C was added di-tert-butyl-dicarbonate (2.50 mL, 11.1 mmol).
The reaction mixture was stirred while warming to room temperature for 8 h. The reaction
mixture was diluted with CH
2
Cl
2
(50 mL) then washed with saturated NaCl solution (3 x 50 mL).
The organic phase was dried over sodium sulfate and concentrated to obtain a pale yellow solid.
Recrystallization from chloroform/hexanes gave 1.71 g as a white solid (61%). TLC: 50%
EtOAc/hexanes, R
f
≈ 0.75.
1
H-NMR (600 MHz, CDCl
3
): 7.26 (s, 1H), 6.99 (d, J = 7.8 Hz, 1H),
6.75-6.69 (m, 2H), 6.24 (br s, 1H), 3.70 (br s, 2H), 1.51 (s, 9H).
13
C-NMR (150 MHz, CDCl
3
):
153.8, 139.9, 126.1, 124.8, 124.6, 119.6, 117.6, 80.4, 28.3.

152

2.224. tert-butyl (2-(7-oxo-7-(pyridin-3-ylamino)heptanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.048 g as a white solid (27%). TLC: 80% EtOAc/hexanes, R
f
≈
0.2.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.71 (s, 1H), 8.22 (d, J = 5.2 Hz, 1H), 8.09 (d, J =
11.2 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.34-7.37 (m, 2H), 7.17 (t, J = 12.0 Hz, 1H), 7.10 (t, J =
7.6 Hz, 1H), 2.45 (t, J = 7.6 Hz, 4H), 1.78 (t, J = 7.2, 4H), 1.45-1.49 (m, 11H).
13
C NMR (100
MHz, CD
3
OD) δC (ppm) 173.64, 173.40, 154.39, 143.61, 140.36, 136.12, 131.65, 129.57,
127.50, 125.86, 125.11, 124.42, 123.82, 80.02, 36.09, 35.81, 28.26, 27.23, 25.07, 24.83.

2.225. tert-butyl (2-(7-([1,1'-biphenyl]-2-ylamino)-7-oxoheptanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 50%
EtOAc/hexanes as eluent gave 0.029 g as a colorless oil (20%). TLC: 50% EtOAc/hexanes, R
f
≈
0.4.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.21 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H),
8.00-8.12 (m, 8H), 7.88 (t, J = 7.2 Hz, 1H), 7.79 (t, J = 8.0Hz, 1H), 3.07 (t, J = 7.6 Hz, 2H), 2.92
(t, J = 7.2 Hz, 2H), 2.27-2.41 (m, 4H), 2.27 (s, 9H), 1.99-2.06 (m, 2H).
13
C NMR (100 MHz,
CD
3
OD) δC (ppm) 173.68, 173.62, 154.33, 139.23, 137.84, 134.00, 131.59, 130.12, 129.57,
128.75, 128.06, 127.57, 127.11, 126.83, 126.31, 125.87, 125.11, 124.44, 124.02, 79.95, 35.84,
35.60, 28.27, 27.30, 25.09, 24.98.
153

2.226. tert-butyl (2-(7-([1,1'-biphenyl]-3-ylamino)-7-oxoheptanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 60%
EtOAc/hexanes as eluent gave 0.151 g as a clear oil (70%). TLC: 60% EtOAc/hexanes, R
f
≈ 0.4.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.84 (s, 1H), 7.56 (d, J = 6.8 Hz, 2H), 7.49-7.52 (m, 2H),
7.28-7.41 (m, 6H), 7.15 (t, J = 6.4 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 2.37-2.43 (m, 4H), 1.17-1.79
(m, 4H), 1.46-1.51 (m, 11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.68, 173.11, 154.31,
141.74, 140.76, 138.99, 131.66, 129.59, 128.87, 127.10, 126.60, 125.88, 125.15, 124.43, 124.01,
122.31, 118.64, 118.37, 79.93, 36.34, 35.85, 28.29, 27.26, 25.13.

2.227. tert-butyl (2-(7-([1,1'-biphenyl]-4-ylamino)-7-oxoheptanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 50%
EtOAc/hexanes as eluent gave 0.060 g as a white solid (28%). TLC: 50% EtOAc/hexanes, R
f
≈
0.3.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.49-7.63 (m, 6H), 7.40 (d, J = 5.6 Hz, 1H), 7.38 (t,
J = 8.0 Hz, 2H), 7.30-7.36 (m, 1H), 7.28 (t, J= 6.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.10 (t, J =
7.6 Hz, 1H), 2.40-2.46 (m, 4H), 1.74-1.81 (m, 4H), 1.48-1.54 (m, 11H).
13
C NMR (100 MHz,
CD
3
OD) δC (ppm) 173.69, 173.03, 154.37, 140.45, 138.43, 137.81, 136.72, 135.26, 128.40,
154

126.79, 126.65, 126.23, 125.86, 125.10, 124.43, 120.00, 79.62, 36.30, 35.84, 28.29, 27.22, 25.12,
25.07.

2.228.tert-butyl(2-(7-(([1,1'-biphenyl]-4-ylmethyl)amino)-7-oxoheptanamido)
phenyl)carbamate. Synthesized according to Method B. Purification by automated flash
chromatograpy using 60% EtOAc/hexanes as eluent gave 0.048 g as a colorless oil (7%). TLC:
60% EtOAc/hexanes, R
f
≈ 0.3.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.49-7.57 (m, 5H), 7.28-
7.49 (m, 6H), 7.15 (t, J = 8.0 Hz, 1H), 7.09 (t, J = 7.6 Hz, 1H), 4.37 (s, 2H), 2.40 (t, J = 8.0 Hz,
2H), 2.27 (t, J = 6.8 Hz, 2H), 1.66-1.77 (m, 4H), 1.41-1.47 (m, 11H).
13
C NMR (100 MHz,
CD
3
OD) δC (ppm) 174.61, 173.67, 154.25, 140.61, 139.97, 137.78, 131.62, 129.67, 128.42,
127.66, 126.88, 126.68, 126.48, 125.86, 125.11, 124.43, 80.34, 42.52, 35.84, 35.48, 28.30, 27.24,
25.23, 25.07.

2.229. tert-butyl 2-(7-(3-bromophenylamino)-7-oxoheptanamido)phenylcarbamate.
Synthesized according to Method B. Purification by flash chromatography using 60%
EtOAc/hexanes as eluent gave 0.421 g as a white solid (52%). TLC: 60% EtOAc/hexanes, R
f
≈
0.4.
1
H-NMR (400 MHz, CD
3
OD): δ 7.93 (s, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.2 Hz,
155

1H), 7.39 (d, J = 8.0 Hz, 1H) 7.23 (m, 3H), 7.15 (t, J = 7.2 Hz, 1H), 2.42-2.49 (m, 4H), 1.75-1.84
(m, 4H), 1.53-1.54 (m, 11H).
13
C-NMR (100 MHz, CD
3
OD): δ 173.7, 173.1, 154.5, 140.1, 131.7,
129.9, 129.6, 126.3, 125.8, 125.1, 124.4, 122.4, 121.8, 118.0. 80.0, 36.3, 35.8, 28.2, 27.2, 25.1,
24.9. HRMS (ESI): m/z 526.1303, [M+Na]
+
, calc. 526.1317).

2.230. 7-((3-ethylphenyl)amino)-7-oxoheptanoic acid. Synthesized according to Method A.
Recrystallization from acetonitrile/water gave 0.470 g as a white solid (29%).
1
H NMR (400
MHz, CD
3
OD) δH (ppm) 7.39 (s, 1H), 7.33 (d, J = 7.5 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 6.92 (d,
J = 7.2 Hz, 1H, 1.58-1.63 (m, 2H), 2.28-2.38 (m, 4H), 1.63-1.74 (m, 4H), 1.33-1.43 (m, 2H), 1.21
(t, J = 6.8 Hz, 3H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 176.09, 173.05, 144.79, 138.42,
128.27, 123.27, 119.31, 117.27, 36.37, 33.35, 18.46, 28.37, 25.19, 24.37, 14.64.

2.231. 7-oxo-7-(m-tolylamino)heptanoic acid. Synthesized according to Method A. Gave
0.530 g as a white solid (34%). TLC: 50% EtOAc/hexanes, R
f
≈ 0.1.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 7.61 (s, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.41 (t, J = 7.5Hz, 1H), 7.14 (d, J = 7.5
Hz, 1H), 2.61 (t, J = 7.5 Hz, 2H), 2.54 (s, 3H), 1.88-1.97 (m, 4H), 1.67-1.68 (m, 2H).
13
C NMR
(125 MHz, CD
3
OD) δC (ppm) 176.07, 173.04, 156.87, 138.33, 128.17, 124.42, 120.45, 117.02,
36.37, 33.35, 28.38, 25.19, 24.37, 20.12.

156

2.232. 7-((3-isopropylphenyl)amino)-7-oxoheptanoic acid. Synthesized according to Method
A. Gave 0.587 g as a gray solid (54%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.98 (br s,
1H), 9.78 (s, 1H), 7.41-7.45 (m, 2H), 7.18 (t, J = 8.0 Hz, 1H), 6.89 (d, J = 9.2 Hz, 1H), 2.50-2.87
(m, 1H), 2.26-2.29 (m, 2H), 2.19-2.23 (m, 1H), 1.56-1.65 (m, 4H), 1.48-1.52 (m, 2H), 1.32 (d, J =
7.6 Hz, 6H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 174.88, 171.50, 149.27, 139.77, 128.93,
121.43, 117.36, 117.07, 36.70, 33.99, 33.91, 28.65, 25.30, 24.72, 24.28.

2.233. 7-((4-fluorophenyl)amino)-7-oxoheptanoic acid. Synthesized according to Method A.
Recrystallized from CH
2
Cl
2
to provide 0.523 g as a pale blue solid (34%). TLC: 10%
MeOH/CH
2
Cl
2
, R
f
≈ 0.6.
1
H NMR (600 MHz, CD
3
OD) δH (ppm) 7.52 (d, J = 9.0 Hz, 2H), 7.02
(d, J = 8.4 Hz, 2H), 2.35 (t, J = 7.2 Hz, 2H), 2.29 (t, J = 7.2 Hz, 2H), 1.62-1.69 (m, 4H), 1.40-
1.43 (m, 2H).
13
C NMR (151 MHz, CD
3
OD) δC (ppm) 176.18, 172.96, 159.98, 134.67, 121.69,
121.63, 114.80, 114.65, 36.20, 33.43, 28.35, 25.12, 24.38.

2.234. 7-oxo-7-(3-(trifluoromethyl)phenylamino)heptanoic acid. Synthesized by method A
from 3-(trifluoromethyl)aniline and pimelic acid. Recrystallization from acetonitrile/water gave
0.490 g a pale orange solid (26%). TLC: 60%EtOAc/hexanes, R
f
≈ 0.1.
1
H-NMR (500 MHz,
CD
3
OD): δ 8.01 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 9.0 Hz,
1H), 2.42 (t, 1.5 Hz, 2H), 2.33 (t, 1.5 Hz, 2H), 1.64-1.76 (m, 4H), 1.42 (m, 2H).
13
C-NMR (125
MHz, CD
3
OD): δ 176.1, 173.3, 139.4, 130.7 (q, J = 34 Hz), 129.2, 125.2, 123.0, 122.7, 119.8,
157

116.0, 36.3, 33.3, 28.3, 25.0, 24.3.
19
F-NMR (470 MHz, CD
3
OD): δ -64.3. HRMS (ESI): m/z
304.1146, [M+H]
+
, calc. 304.1161).

2.235. 7-((3-fluorophenyl)amino)-7-oxoheptanoic acid. Synthesized according to Method A.
Purification by automated flash chromatography using 10% MeOH/CH
2
Cl
2
as eluent gave 0.044
g as a white solid (27%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈ 0.6.
1
H NMR (400 MHz, CD
3
OD) δH
(ppm) 7.58 (d, J = 12.0 Hz, 1H), 7.25-7.32 (m, 2H), 6.81 (t, J = 9.6 Hz, 1H), 2.41 (t, J = 7.2 Hz,
2H), 2.35 (t, J = 7.6 Hz, 2H), 1.64-1.78 (m, 4H), 1.42-1.48 (m, 2H).
13
C NMR (100 MHz,
CD
3
OD) δC (ppm) 176.32, 173.19, 164.06, 161.65, 140.38, 140.28, 129.72, 129.63, 114.94,
109.96, 106.72, 36.36, 33.46, 28.35, 25.01, 24.38.

2.236. 7-((3,5-bis(trifluoromethyl)phenyl)amino)-7-oxoheptanoic acid. Synthesized by
Method A. Recrystallization from acetonitrile/water gave 0.069 g as a white solid (25%).
1
H
NMR (400 MHz, CD
3
OD) δH (ppm) 8.20 (s, 2H), 7.60 (s, 1H), 2.42 (t, J = 7.6 hz, 2H), 2.30 (t, J
= 7.2 Hz, 2H), 1.63-1.74 (m, 4H), 1.40-1.44 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm)
176.09, 173.54, 140.75, 131.62, 118.94, 118.80, 116.08, 115.92, 36.36, 33.31, 28.33, 24.79,
24.34.
19
F NMR (376 MHz, CD
3
OD) δF (ppm) -64.69.
158

2.237. N
1
-(2-aminophenyl)-N
7
-(4-bromo-3-(trifluoromethyl)phenyl)heptanediamide.
Synthesized according to Method C. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.035 g as a yellow solid (18%). TLC: 80% EtOAc/hexanes, R
f
≈
0.1.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.09 (s, 1H), 7.67 (s, 2H), 6.98-7.05 (m, 2H), 6.82
(d, J = 7.6 Hz, 1H), 6.67 (t, J = 8.4 Hz, 1H), 2.39-2.44 (m, 4H), 1.71-1.79 (m, 4H), 1.44-1.49 (m,
2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.52, 173.33, 141.88, 138.57, 135.10, 129.88,
129.57, 126.80, 125.68, 123.70, 121.54, 118.51, 118.08, 117.11, 112.40, 36.28, 35.53, 28.31,
25.20, 24.84.

2.238.tert-butyl(2-(7-((4-(dimethylamino)phenyl)amino)-7-oxoheptanamido)
phenyl)carbamate. Synthesized according to general procedures. Purification by automated
flash chromatography using 70% EtOAc/hexanes as eluent gave 0.131 g as a yellow solid (39%).
TLC: 70% EtOAc/hexanes, R
f
≈ 0.25.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 7.50 (d, J = 8.0
Hz, 1H), 7.32-7.36 (m, 3H), 7.18 (t, J = 9.0 Hz, 1H), 7.10 (t, J = 9.0 Hz, 1H), 6.72 (d, J = 9.5 Hz,
2H), 2.87 (s, 6H), 2.43 (t, J = 7.5 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.71-1.79 (m, 4H), 1.49-1.71
(m, 11H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.83, 172.70, 157.73, 154.42, 148.14,
159

131.78, 128.47, 125.96, 125.15, 124.33, 121.61, 113.08, 80.04, 40.02, 36.19, 35.86, 28.31, 27.24,
25.23, 25.14.

2.239. 6-oxo-6-((3-(trifluoromethyl)phenyl)amino)hexanoic acid. Synthesized by method A
from 3-(trifluoromethyl)aniline and adipic acid. Recrystallization from acetonitrile/water gave
0.182 g as a white solid (18%).
1
H NMR (400 MHz, DMSO-d6) δH (ppm) 11.99 (s, 1H), 10.19
(s, 1H), 8.07 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H)), 7.51 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H),
2.32 (t, J = 6.8 Hz, 2H), 2.22 (t, J = 7.6 Hz, 2H), 1.51-1.58 (m, 4H).
13
C NMR (100 MHz,
DMSO-d6) δC (ppm) 174.75, 172.07, 140.44, 130.35, 129.97, 129.66, 122.91, 119.69, 115.49,
115.40, 36.55, 33.83, 24.91, 24.52.
19
F NMR (376 MHz, DMSO-d
6
) δF (ppm) -61.37.

2.240. 8-oxo-8-((3-(trifluoromethyl)phenyl)amino)octanoic acid. Synthesized by method A
from 3-(trifluoromethyl)aniline and suberic acid. Recrystallization from acetonitrile/water gave
0.407 g as a white solid (22%).
1
H NMR (500 MHz, DMSO- d
6
) δH (ppm) 11.96 (br s, 1H), 10.19
(s, 1H), 8.09 (s, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 7.5 Hz, 1H),
2.50 (s, 1H), 2.32 (t, J = 7.0 Hz, 2H), 2.18 (t, J = 7.0 Hz, 2H), 1.57-1.58 (m, 2H, 1.49-1.51 (m,
2H), 1.44-1.47 (m, 4H). ).
13
C NMR (126 MHz, DMSO-d
6
) δC (ppm) 173.31, 170.69, 138.90,
128.74, 128.37, 124.07, 121.90, 121.32, 118.10, 118.07, 116.89, 113.84, 35.22, 32.47, 27.21,
23.66, 23.22.
19
F NMR (470 MHz, DMSO-d
6
) δF (ppm) -61.39.
160

2.241. 9-oxo-9-((3-(trifluoromethyl)phenyl)amino)nonanoic acid. Synthesized by method A
from 3-(trifluoromethyl)aniline and azelaic acid. Recrystallization from acetonitrile/water gave
0.807 g as a white solid (75%).
1
H NMR (500 MHz, DMSO- d
6
) δH (ppm) 11.95, (br s, 1H),
10.18 (s, 1H), 8.09 (s, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz,
1H), 2.49 (t, J = 2.0 Hz, 2H), 2.32 (t, J = 7.5 Hz, 2 H), 1.57-1.60 (m, 2H), 1.47-1.49 (m, 3H),
1.22-1.27 (m, 7H).
13
C NMR (126 MHz, DMSO-d
6
) δC (ppm) 173.34, 170.72, 138.92, 128.50 (q,
J = 17.50), 124.08, 121.93, 121.33, 118.13, 113.84.
19
F NMR (470 MHz, DMSO-d
6
) δF (ppm) -
61.39.

2.242. 10-oxo-10-((3-(trifluoromethyl)phenyl)amino)decanoic acid. To a solution of sebacic
acid (0.500 g, 2.47 mmol) in CH
2
Cl
2
(45 mL) at room temperature was added EDC (0.472 g,
2.47 mmol) followed by a solution of 3-(trifluoromethyl)aniline (0.309 mL, 2.47 mmol) in DMF
(1.5 mL). The reaction mixture was stirred at room temperature for 36 h then was washed
sequentially with 10% HCl solution (2 x 50 mL) and brine (50 mL). The organic layer was dried
over Na
2
SO
4
and concentrated. Recrystallization from acetonitrile/water provided 0.183 g as a
white solid (21%). TLC: 50% EtOAc/hexanes, R
f
≈ 0.25.
1
H NMR (600 MHz, DMSO- d
6
) δH
(ppm) 11.93 (s, 1H), 10.16 (s, 1H), 8.08 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 7.0 Hz,
1H), 7.35 (d, J = 7.8 Hz, 1H), 2.16 (t, J = 7.2 Hz, 2H), 2.16 (t, J = 7.2 Hz, 1H), 1.54-1.57 (m,
161

2H), 1.45-1.47 (m, 1H), 1.22-1.28 (m, 6H).
13
C NMR (150 MHz, DMSO-d
6
) δC (ppm) 174.96,
172.30, 140.56, 130.34, 122.79, 119.69, 115.42, 36.87, 34.11, 29.11, 29.05, 28.93, 25.35, 24.91.
19
F NMR (470 MHz, DMSO-d
6
) δF (ppm) -61.37.

2.243. tert-butyl (3-(8-oxo-8-(phenylamino)octanamido)phenyl)carbamate. Synthesized
according to general procedures. Purification by automated flash chromatography using 60%
EtOAc/hexanes as eluent gave 0.187 g as a white solid (53%). TLC: 60% EtOAc/hexanes, R
f
≈
0.5.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.68 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.27 (t, J =
7.6 Hz, 2H), 7.09-7.17 (m, 2H), 7.03-7.08 (m, 2H), 2.34-2.36 (m, 4H), 1.50-1.81 (m, 4H), 1.49 (s,
9H), 1.35-1.43 (m, 4H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.17, 153.72, 139.61, 138.83,
128.48, 128.26, 123.63, 119.84, 114.26, 36.42, 28.59, 27.26, 25.30.

2.244. tert-butyl (2-(2-chloroacetamido)phenyl)carbamate. To a suspension of 2.222 (0.500
g, 2.40 mmol) and NaHCO
3
(0.403 g, 4.80 mmol) in CH
2
Cl
2
(10 mL) at 0°C was added dropwise
chloroacetyl chloride (0.191 mL, 2.40 mmol) under N
2
. The reaction mixture was stirred while
warming to room temperature for 2 h then diluted with ethyl acetate (25 mL) and washed
sequentially with water (25 mL) and brine (25 mL). The organic layer was dried over Na
2
SO
4

162

and concentrated onto celite. Purification by automated flash chromatography using 30%
EtOAc/hexanes as eluent provided 0.602 g as a white solid (88%). TLC: 30% EtOAc/hexanes,
R
f
≈ 0.4.
1
H NMR (500 MHz, CDCl
3
) δH (ppm) 9.08 (br s, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.18-
7.28 (m, 3H), 6.64 (br s, 1H), 4.20 (s, 2H), 1.52 (s, 9H).
13
C NMR (100 MHz, CDCl
3
) δC (ppm)
164.95, 154.18, 130.16, 126.60, 126.12, 125.39, 124.80, 81.45, 42.89, 28.28.

2.245. Methyl 5-aminopentanoate, monohydrochloric acid. Synthesized according to Method
F. Gave 2.78 g as ayellow solid (98%).
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 4.84 (br s, 3H),
3.66 (s, 3H), 2.92-2.95 (m, 2H), 4.20 (t, J = 6.8 Hz, 2H), 1.69-1.71 (m, 4H).
13
C NMR (100 MHz,
CD
3
OD) δC (ppm) 173.82, 50.81, 39.05, 32.57, 26.50, 21.34.

2.246. Methyl 7-aminoheptanoate monohydrochloric acid. Synthesized according to Method
F. Gave 0.310 g as a yellow solid (71%).
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 4.75 (br s
(3H), 3.56 (s, 3H), 2.84 (t, J = 7.0 Hz, 2H), 2.26 (t, J = 7.0 Hz, 2H), 1.54-1.59 (m, 4H), 1.28-1.31
(m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 174.10, 50.66, 39.37, 33.20, 28.17, 26.96,
25.71, 24.30.

163

2.247. methyl 5-(3-(trifluoromethyl)benzamido)pentanoate. Synthesized according to
Method B. Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent
gave 0.480 g as an amber oil (60%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.25.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 8.14 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.67 (t, J = 7.5
Hz, 1H), 4.86 (s, 3H), 3.42 (t, J = 7.0 Hz, 2H), 2.40 (t, J = 6.5 Hz, 2H), 1.66-1.71 (m, 4H).
13
C
NMR (125 MHz, CD
3
OD) δC (ppm) 174.29, 167.02, 135.41, 130.51, 129.15, 127.63, 125.01,
123.68, 122.88, 50.59, 39.23, 32.90, 28.36, 21.95.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -
64.24.

2.248. 5-(3-(trifluoromethyl)benzamido)pentanoic acid. Synthesized according to Method E.
Gave 0.303 g as a white solid (71%).
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.14 (s, 1H), 8.08
(d, J = 8.0 Hz, 1H), 7.83 (d, J = 7.0 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 3.41 (t, J = 6.0 Hz, 2H),
2.36 (t, J = 6.0 Hz, 2H), 1.67-1.69 (m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 175.95,
167.04, 135.43, 130.51, 129.14, 127.62, 123.79, 123.76, 123.70, 39.31, 33.05, 28.42, 22.02.
19
F
NMR (470 MHz, CD
3
OD) δF (ppm) -64.25.

2.249. methyl 5-(3,5-bis(trifluoromethyl)benzamido)pentanoate. Synthesized according to
Method B. Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent
gave 0.526 g as an amber oil (73%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.6.
1
H NMR (500 MHz,
164

CD
3
OD) δH (ppm) 8.43 (s, 2H), 8.16 (s, 1H), 3.66 (s, 3H), 3.44 (t, J = 7.0 Hz, 2H), 2.41 (t, J =
7.0 Hz, 2H), 1.68-1.71 (m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 174.28, 165.18, 136.81,
131.7 (q, J = 41 Hz) 127.56, 126.46, 124.48, 124.23, 122.08, 50.59, 39.41, 32.87, 28.24, 21.93.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.42.

2.250. 5-(3,5-bis(trifluoromethyl)benzamido)pentanoic acid. Synthesized according to
Method E. Gave 0.301 g as a white solid (65%). TLC: 60% EtOAc/hexanes, R
f
≈ 0.3.
1
H NMR
(500 MHz, CD
3
OD) δH (ppm) 8.32 (s, 2H), 8.04 (s, 1H), 3.34 (t, J = 6.0 Hz, 2H), 2.26 (t, J = 6.5
Hz, 2H), 1.59 (m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 175.96, 165.15, 136.82, 131.60
(q, J = 33 Hz), 127.56, 124.40, 124.24, 122.08, 119.90, 39.48, 32.99, 28.30, 22.00.
19
F NMR (470
MHz, CD
3
OD) δF (ppm) -64.41.

2.251. methyl 6-(3-(trifluoromethyl)benzamido)hexanoate. Synthesized according to Method
B. Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent gave
0.674 g as a colorless oil (88%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.35.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 7.78 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.31 (t, J =
7.5 Hz, 1H), 3.28 (s, 3H), 3.04 (t, J = 7.0 Hz, 2H), 1.99 (t, J = 7.0 Hz, 2H), 1.28-1.31 (m, 4H),
1.06-1.08 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 174.41, 166.96, 135.44, 130.49,
165

129.13, 127.60, 127.57, 123.73, 123.70, 50.54, 39.51, 33.23, 28.61, 26.09, 24.25.
19
F NMR (470
MHz, CD
3
OD) δF (ppm) -64.22.

2.252. 6-(3-(trifluoromethyl)benzamido)hexanoic acid. Synthesized according to Method E.
Gave 0.488 g as a white solid (83%).
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.70 (br s, 1H),
8.14 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 3.41 (t, J
= 7.0 Hz, 2H), 2.33 (t, J = 7.0 Hz, 2H), 1.64-1.70 (m, 4H), 1.43-1.48 (m, 2H).
13
C NMR (125
MHz, CD
3
OD) δC (ppm) 176.10, 167.25, 135.45, 130.51, 129.11, 127.60, 127.54, 123.71,
123.68, 39.56, 33.37, 28.66, 26.16, 24.33.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.26.

2.253. methyl 6-(3,5-bis(trifluoromethyl)benzamido)hexanoate. Synthesized according to
Method B. Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent
gave 0.666 g as a colorless oil (63%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.7.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 8.43 (s, 2H), 8.15 (s, 1H), 3.64 (s, 3H), 3.43 (t, J = 7.0 Hz, 2H), 2.36 (t, J =
7.5 Hz, 2H), 1.66-1.69 (m, 4H), 1.42-1.47 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm)
174.42, 165.14, 136.85, 132.06, 131.79, 131.50, 131.23, 127.55, 124.49, 124.43, 124.39, 124.20,
122.07, 50.53, 39.66, 33.19, 28.48, 26.06, 24.21.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.41.
166

2.254. 6-(3,5-bis(trifluoromethyl)benzamido)hexanoic acid. Synthesized according to Method
E. Gave 0.381 g as a white solid (61%).
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.43 (s, 2H),
8.15 (s, 1H), 3.43 (t, J = 6.5 Hz, 2H0, 2.32 (t, J = 7.0 Hz, 2H), 1.48-1.71 (m, 4H), 1.43-1.48 (m,
2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 176.07, 165.21, 136.83, 132.06, 131.78, 131.52,
131.27, 127.53, 124.42, 124.25, 122.08, 119.92, 39.74, 33.34, 28.56, 26.16, 24.31.
19
F NMR (470
MHz, CD
3
OD) δF (ppm) -64.41.

2.255. methyl 7-(3-(trifluoromethyl)benzamido)heptanoate. Synthesized according to
Method B. Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent
gave 0.310 g as a yellow oil (71%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.45.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 8.14 (s, 1H), 8.07 (d, J = 7.5 Hz, 1H), 7.84 (d, J = 7.0 Hz, 1H), 7.68 (t, J =
7.5 Hz, 1H), 2.74 (s, 3H), 3.40 (t, J = 7.0 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.63-1.64 (m, 4H),
1.39-1.41 (m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 174.54, 167.01, 135.54, 130.50,
129.14, 127.57, 123.74, 50.53, 39.64, 33.27, 28.78, 28.42, 26.31, 24.48.
19
F NMR (470 MHz,
CD
3
OD) δF (ppm) -64.25.
167

2.256. 7-(3-(trifluoromethyl)benzamido)heptanoic acid. Synthesized according to Method E.
Gave 0.161 g as a white solid (56%). TLC: 100% EtOAc, R
f
≈ 0.5.
1
H NMR (500 MHz, CD
3
OD)
δH (ppm) 8.14 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.67 (t, J = 8.0 Hz,
1H), 3.40 (t, J = 7.5 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H), 1.62-1.67 (m, 4H), 1.41-1.43 (m, 4H).
13
C
NMR (125 MHz, CD
3
OD) δC (ppm) 176.21, 167.03, 135.47, 130.44, 129.14, 127.57, 125.04,
123.74, 122.88, 39.68, 33.41, 28.81, 28.47, 26.34, 24.56.
19
F NMR (470 MHz, CD
3
OD) δF (ppm)
-64.25.

2.257. methyl 7-(3,5-bis(trifluoromethyl)benzamido)heptanoate. Synthesized according to
Method B. Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent
gave 0.093 g as a colorless oil (23%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.6.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 8.43 (s, 2H), 8.16 (s, 1H), 3.65 (s, 3H), 3.43 (t, J = 7.5 Hz, 2H), 2.32-2.35 (m,
2H), 1.64-1.66 (m, 4H), 1.40-1.41 (m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 174.53,
165.16, 136.86, 131.60 (q, J = 33 Hz), 127.52, 124.40, 124.24, 122.09, 50.53, 39.81, 33.26,
28.66, 28.39, 26.27, 24.47.
19
F NMR (470 MHz, CD
3
OD) δF (ppm) -64.42.
168

2.258. 7-(3,5-bis(trifluoromethyl)benzamido)heptanoic acid. Synthesized according to
Method E. Gave 0.017 g as a white solid (19%).
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 8.32 (s,
2H), 8.05 (s, 1H), 3.33 (t, J = 5.5 Hz, 2H), 2.20 (t, J = 7.0 Hz, 2H), 1.52-1.57 (m, 4H), 1.31-1.33
(m, 4H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 176.22, 165.18, 136.84, 131.60 (q, J = 31 Hz),
127.84, 124.40, 124.24, 122.09, 39.87, 33.42, 28.71, 28.45, 26.33, 24.55.
19
F NMR (470 MHz,
CD
3
OD) δF (ppm) -64.42.

2.259. methyl 6-(3-bromobenzamido)hexanoate. Synthesized according to Method B.
Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent gave 0.344
g as a colorless oil (39%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.4.
1
H NMR (500 MHz, CD
3
OD) δH
(ppm) 7.97 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 174.42, 167.03, 136.58, 133.99, 129.97, 129.94, 125.62,
122.05, 50.56, 39.46, 33.24, 28.61, 26.08, 24.25.

2.260. 6-(3-bromobenzamido)hexanoic acid. Synthesized according to Method E. Gave 0.210
g as a white solid (72%).
1
H NMR (500 MHz, DMSO-d
6
) δH (ppm) 11.97 (br s, 1H), 8.55 (s,
169

1H), 8.00 (s, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.41-7.44 (m, 2H), 3.21-3.25
(m, 2H), 2.20 (t, J = 7.0 Hz, 2H), 1.58-1.53 (m, 4H), 1.28-1.31 (m, 2H).
13
C NMR (125 MHz,
DMSO-d
6
) δC (ppm)174.88, 164.95, 137.28, 134.19, 130.98, 130.24, 126.74, 122.07, 34.05,
29.17, 26.46, 24.70.

2.261. N-(6-((2-aminophenyl)amino)-6-oxohexyl)-3-bromobenzamide. Synthesized according
to Method B. Purification by automated flash chromatography using 80% EtOAc/hexanes as
eluent gave 0.119 g as a yellow solid (62%). TLC: 80% EtOAc/hexanes, , R
f
≈ 0.0.1.
1
H NMR
(500 MHz, CD
3
OD) δH (ppm) 7.88 (s, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H),
7.27 (t, J = 7.5 Hz, H), 6.90-6.96 (m, 2H), 6.73 (d, J = 8.0 Hz, 1H), 6.59 (t, J = 9.0 Hz, 1H), 6.59
(t, J = 7.5 Hz, 1H), 3.30 (t, J = 7.0 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.65-1.71 (m, 2H), 1.55-1.58
(m, 2H), 1.38-1.42 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.55, 141.85, 136.57,
133.99, 129.97, 129.95, 126.81, 125.68, 125.62, 123.73, 122.00, 118.11, 117.13, 39.48, 35.68,
28.72, 26.22, 25.27.

2.262. methyl 6-([1,1'-biphenyl]-3-ylcarboxamido)hexanoate. Synthesized according to
Method B. Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent
170

gave 0.283 g as an orange oil (63%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.4.
1
H NMR (400 MHz,
CDCl
3
) δH (ppm) 7.99 (s, 1H), 7.70-7.73 (m, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.43-7.51 (m, 3H),
7.36 (t, J = 8.0 Hz, 1H), 6.25 (br s, 1H), 3.64 (s, 3H), 3.48 (q, J = 8.0 Hz, 2H), 2.34 (t, J = 9.0 Hz,
2H), 1.64-1.72 (m, 4H), 1.39-1.47 (m, 2H).
13
C NMR (100 MHz, CDCl
3
) δC (ppm) 174.12,
167.44, 141.62, 140.28, 135.32, 130.01, 128.96, 128.85, 127.70, 127.18, 125.74, 125.52, 51.46,
39.72, 33.78, 29.21, 26.32, 24.31.

2.263. 6-([1,1'-biphenyl]-3-ylcarboxamido)hexanoic acid. Synthesized according to Method
E. Gave 0.240 g as a white solid (94 %).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.94 (s,
1H), 8.53 (t, J = 5.6 Hz, 1H), 8.08 (s, 1H), 7.79 (t, J = 8.0 Hz, 2H), 7.69 (d, J = 7.2 Hz, 2H), 7.45-
7.53 (m, 2H), 7.35-7.39 (m, 1H), 3.21-3.29 (m, 2H), 2.18 (t, J = 7.2 Hz, 2H), 1.46-1.52 (m, 4H),
1.27-1.32 (m, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 174.89, 166.34, 140.59, 140.07,
135.77, 129.63, 129.41, 129.39, 128.18, 127.29, 126.79, 125.70, 34.04, 29.31, 26.50, 24.71.

2.264. methyl 6-(4-(dimethylamino)benzamido)hexanoate. Synthesized according to Method
B. Purification by automated flash chromatography using 50% EtOAc/hexanes as eluent gave
0.551 g as a colorless oil (69%). TLC: 50% EtOAc/hexanes, R
f
≈ 0.4.
1
H NMR (500 MHz,
171

CDCl
3
) δH (ppm) 7.66 (d, J = 9.0 Hz, 2H), 6.66 (d, J = 9.0 Hz, 2H), 3.66 (s, 3H), 3.53 (q, 2H),
2.33 (t, J = 5.5 Hz, 2H), 1.67-1.70 (m, 2H), 1.60-1.63 (m, 2H), 1.38-1.44 (m, 2H). ).
13
C NMR
(126 MHz, CDCl
3
) δC (ppm) 174.23, 167.63, 152.37, 128.27, 121.52, 111.09, 51.50, 40.14,
39.55, 33.89, 29.49, 26.44, 24.52.

2.265. 6-(4-(dimethylamino)benzamido)hexanoic acid. Synthesized according to Method E.
Gave 0.272 g as a white solid (58%).
1
H NMR (500 MHz, DMSO-d
6
) δH (ppm) 11.97 (s, 1H),
8.06 (s, 1H), 7.69 (d, J = 6.5 Hz, 2H), 6.67 (d, J = 9.0 Hz, 2H), 3.17-3.21 (m, 2H), 2.95 (s, 6H),
2.20 (t, J = 7.0 Hz, 2H), 1.45-1.54 (m, 4H), 1.25-1.32 (m, 2H).
13
C NMR (126 MHz, DMSO-d
6
)
δC (ppm) 174.92, 166.56, 152.43, 128.87, 121.91, 111.21, 34.10, 29.58, 26.55, 24.76.

2.266. tert-butyl (2-(6-([1,1'-biphenyl]-4-ylcarboxamido)hexanamido)phenyl)carbamate.
Synthesized according to general procedures. Purification by automated flash chromatography
using 60% EtOAc/hexanes as eluent gave 0.124g as a white solid (53%). TLC: 60%
EtOAc/hexanes, R
f
≈ 0.35.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.89 (d, J =8.4 Hz, 2H), 7.69
(d, J = 8.0 Hz, 2H), 7.65 (d, J = 7.6 Hz, 2H), 7.45-7.53 (m, 3H), 7.37 (t, J = 8.0 H, 2H), 7.19 (t, J
= 8.8 Hz, 1H), 7.11 (t, J = 6.8 Hz, 1H), 3.44 (t, J = 7.2 Hz, 2H), 2.46 (t, J = 7.6 Hz, 2H), 1.70-
172

1.84 (m, 4H), 1.50-1.56 (m, 11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.77, 168.45,
154.28, 144.14, 139.77, 133.02, 131.63, 128.58, 127.62, 127.43, 126.61, 125.85, 125.11, 124.42,
79.98, 39.37, 35.93, 28.77, 27.22, 26.11, 25.09.

2.267. methyl 5-(4-(dimethylamino)benzamido)pentanoate. Synthesized according to Method
B. Purification by automated flash chromatography using 50% EtOAc/hexanes as eluent gave
0.554 g as a yellow solid (66%). TLC: 50% EtOAc/hexanes, R
f
≈ 0.5.
1
H NMR (500 MHz,
CD
3
OD) δH (ppm) 7.77 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 9.5 Hz, 2H), 3.73 (s,3H), 3.43-3.45 (m,
2H), 2.46 (t, J = 6.5 Hz, 2H), 1.67-1.78 (m, 4H).
13
C NMR (126 MHz, CD
3
OD) δC (ppm) 174.41,
169.11, 152.85, 128.26, 120.66, 110.73, 50.58, 39.05, 38.83, 32.98, 28.71, 21.99.

2.268. 5-(4-(dimethylamino)benzamido)pentanoic acid. Synthesized according to Method B.
Gave 0.210 g as a white solid (40%).
1
H NMR (500 MHz, DMSO-d
6
) δH (ppm) 11.98 (br s, 1H),
8.07 (t, J = 6.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 6.68 (d, J = 9.0 Hz, 2H), 3.20-3.22 (m, 2H),
2.95 (s, 6H), 2.20-2.23 (m, 2H), 1.49-1.51 (4H).
13
C NMR (126 MHz, DMSO-d
6
) δC (ppm)
174.91, 166.44, 152.44, 128.88, 121.86, 111.22, 39.06, 33.84, 29.36, 22.52.
173

2.269. tert-butyl (2-(6-(furan-2-carboxamido)hexanamido)phenyl)carbamate. Synthesized
according to Method B. Purification by automated flash chromatography using 70%
EtOAc/hexanes as eluent gave 0.113 g as a white solid (62%). TLC: 70% EtOAc/hexanes, R
f
≈
0.3.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.61 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.35 (d, J =
8.4Hz, 1H), 7.20 (t, J = 7.2 Hz, 1H), 7.12 (d, J = 6.4 Hz, 1H), 7.07 (d, J = 9.2 Hz, 1H), 6.55 (d, J
= 4.8 Hz, 1H), 3.39 (t, J = 7.2 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 1.73-1.82 (m, 2H), 1.62-1.70 (m,
2H), 1.49-1.51 (m, 11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.78, 159.49, 154.39,
147.77, 144.71, 131.70, 129.78, 125.84, 125.21, 124.43, 124.04, 113.60, 111.30, 79.94, 38.55,
35.94, 28.88, 27.22, 26.06, 25.10.

2.270. tert-butyl (2-(6-(furan-3-carboxamido)hexanamido)phenyl)carbamate. Synthesized
according to Method B. Purification by automated flash chromatography using 70%
EtOAc/hexanes as eluent gave 0.141 g as a white solid (65%). TLC: 70% EtOAc/hexanes, R
f
≈
0.4.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.02 (s, 1H), 7.50-7.54 (m, 2H), 7.35 (d, J = 8.0 Hz,
1H), 7.19 (t, J = 8.4 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 6.78 (s, 1H), 3.34 (t, J = 7.2 Hz, 2H), 2.44
(t, J = 7.6 Hz, 2H), 1.73-1.80 (m, 2H), 1.61-1.69 (m, 2H), 1.46-1.51 (m, 11H).
13
C NMR (100
174

MHz, CD
3
OD) δC (ppm) 173.78, 163.87, 154.40, 144.98, 143.76, 131.88, 129.60, 125.91,
124.43, 124.04, 122.47, 108.15, 80.06, 38.86, 35.97, 28.81, 27.29, 26.12, 25.12.

2.271. tert-butyl (2-(6-(thiophene-2-carboxamido)hexanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 70%
EtOAc/hexanes as eluent gave 0.050 g as a colorless oil (42%). TLC: 70% EtOAc/hexanes, R
f
≈
0.5.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 7.66 (s, 1H), 7.62 (d, J = 5.0 Hz, 1H), 7.52 (d, J =
7.5 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.10-7.13 (m, 2H), 3.38 (t, J = 6.0
Hz, 2H), 2.44 (d, J = 6.0 Hz, 2H), 1.76-1.79 (m, 2H), 1.66-1.69 (m, 2H), 1.48-1.50 (m, 11H).
13
C
NMR (125 MHz, CD
3
OD) δC (ppm) 173.73, 163.11, 162.09, 154.44, 138.95, 131.74, 130.05,
129.69, 127.99, 127.35, 125.91, 125.18, 124.46, 80.07, 39.26, 35.97, 28.85, 27.26, 26.11, 25.12.

2.272. tert-butyl (2-(6-(thiophene-3-carboxamido)hexanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 70%
EtOAc/hexanes as eluent gave 0.059 g as a white foam (38%). TLC: 70% EtOAc/hexanes, R
f
≈
0.4.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.00 (d, J = 3.6 Hz, 1H), 7.43-7.52 (m, 3H), 7.35 (d,
J = 8.4 Hz, 1H), 7.19 (d, J = 7.2 Hz, 1H), 7.11 (d, J = 6.8 Hz, 1H), 3.37 (t, J = 7.2 Hz, 2H), 2.44
175

(t, J = 7.2 Hz, 2H), 1.73-1.81 (m, 2H), 1.65-1.71 (m, 2H), 1.48-1.52 (m, 11H).
13
C NMR (100
MHz, CD
3
OD) δC (ppm) 173.76, 164.91, 154.39, 146.95, 137.12, 131.73, 128.15, 126.04,
125.97, 125.88, 125.13, 124.45, 124.03, 79.94, 39.05, 35.94, 28.80, 27.23, 26.10, 25.10.

2.273. tert-butyl (2-(6-(benzofuran-2-carboxamido)hexanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 60%
EtOAc/hexanes as eluent gave 0.046 g as a yellow oil (29%). TLC: 60% EtOAc/hexanes, R
f
≈
0.3.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.71 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 9.2 Hz, 1H),
7.50 (d, J = 7.6 Hz, 1H), 7.45 (t, J = 6.0 Hz, 2H), 7.30-7.36 (m, 2H), 7.18 (t, J = 6.4 Hz, 1H), 7.08
(t, J = 8.8 Hz, 1H), 3.45 (t, J = 6.8 Hz, 2H), 2.45 (t, J = 7.6 Hz, 2H), 1.68-1.83 (m, 4H), 1.48-1.52
(m, 11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.65, 159.76, 154.96, 154.32, 148.68,
131.66, 127.40, 126.66, 125.83, 125.09, 124.39, 123.41, 122.27, 111.33, 109.63, 79.99, 48.19,
48.11, 38.75, 35.91, 28.05, 27.07, 25.10, 23.88.

2.274. methyl 6-(pyrimidine-5-carboxamido)hexanoate. Synthesized according to Method B.
Gave 0.238 g as a yellow solid (24%).
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 9.26 (s, 1H), 9.01
(d, J = 10 Hz, 1H), 8.07 (d, J = 5.0 Hz, 1H), 3.64 (s, 3H), 3.44 (t, J = 6.5 Hz, 2H), 2.35 (t, J = 7.5
176

Hz, 2H), 1.65-1.68 (m, 4H), 1.38-1.41 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 174.38,
163.44, 159.03, 157.60, 156.75, 118.29, 50.54, 39.01, 33.22, 28.65, 26.03, 24.23.

2.275. 6-(pyrimidine-5-carboxamido)hexanoic acid. Synthesized according to Method E.
Gave 0.092 g as a white solid (49%).
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 9.26 (s, 1H), 9.00
(s, 5.0 Hz, 1H), 8.07 (d, J = 5.5 Hz, 1H), 3.44 (t, J = 7.5 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.64-
1.70 (m, 4H), 1.41-1.47 (m, 2H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 176.01, 163.43,
159.00, 157.58, 156.75, 118.27, 39.05, 33.35, 28.70, 26.10, 24.31.

2.276. tert-butyl (2-(6-(5-phenylfuran-2-carboxamido)hexanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 80%
EtOAc/hexanes as eluent gave 0.063 g as a white solid (31%). TLC: 80% EtOAc/hexanes, R
f
≈
0.6.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.85 (d, J = 7.2 Hz, 2H), 7.49 (d, J = 8.4 Hz, 1H),
7.42 (t, J = 6.8 Hz, 1H), 7.36 (d, J = 10 Hz, 2H), 7.17=7.19 (m, 2H), 7.09 (t, J = 7.6 Hz, 1H), 6.89
(d, J = 3.6 Hz, 1H), 3.40-3.43 (m, 2H), 2.45 (t, J = 7.6 Hz, 2H), 1.77-1.82 (m, 2H), 1.67-1.72 (m,
2H), 1.48-1.52 (m, 11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.76, 159.42, 156.03,
177

154.36, 146.63, 129.69, 128.49, 128.28, 125.75, 125.16, 124.44, 124.20, 115.74, 106.59, 80.00,
38.59, 35.95, 28.81, 27.22, 25.93, 24.98.

2.277. tert-butyl (2-(6-(1H-pyrrole-2-carboxamido)hexanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 70%
EtOAc/hexanes as eluent gave 0.071 g as awhite solid (40%). TLC: 70% EtOAc/hexanes, R
f
≈
0.3.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 7.52 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H),
7.20 (t, J = 7.5 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.89 (s, 1H), 6.76 (d, J = 4.0 Hz, 1H), 6.15 (d, J
= 4.0 Hz, 1H), 3.35 (t, J = 6.5 Hz, 2H), 2.44 (t, J = 6.5 Hz, 2H), 1.77 (t, J = 7.0 Hz, 2H), 1.65 (t, J
= 7.5 Hz, 2H), 1.48-1.51 (m, 11H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 173.78, 162.54,
154.33, 131.76, 125.88, 125.56, 125.16, 124.51, 121.26, 110.08, 108.70, 79.83, 38.67, 35.98,
29.07, 27.26, 26.13, 25.16.

2.278. tert-butyl (2-(6-(1H-indole-2-carboxamido)hexanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 50%
EtOAc/hexanes as eluent gave 0.089 g as a white solid (57%). TLC: 50% EtOAc/hexanes, R
f
≈
178

0.25.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.59 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H),
7.42 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 9.2 Hz, 1H), 7.15-7.22 (m, 2H), 7.03-7.08 (m, 3H), 3.42 (t,
J = 6.8 Hz, 2H), 2.45 (t, J = 7.6 Hz, 2H), 1.77-1.82 (m, 2H), 1.67-1.72 (m, 2H), 1.48-1.53 (m,
11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.66, 162.83, 154.40, 136.85, 131.78, 130.88,
127.62, 125.89, 125.16, 124.43, 123.56, 121.31, 119.73, 111.64, 102.84, 80.11, 38.94, 35.97,
28.94, 27.25, 26.13, 25.13.

2.279. tert-butyl (2-(6-(5-bromo-1H-indole-2-carboxamido)hexanamido)phenyl)carbamate.
Synthesized according to Method B. Purification by automated flash chromatography using 70%
EtOAc/hexanes as eluent gave 0.064 g as a white solid (40%). TLC: 70% EtOAc/hexanes, R
f
≈
0.4.
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.75 (s, 1H), 9.43 (s, 1H), 8.54 (t, J = 5.2 Hz,
1H), 8.31 (s, 1H), 7.82 (s, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.36-7.41 (m, 2H), 7.27 (d, J = 8.8 Hz,
1H), 7.03-7.13 (m, 3H), 3.30-3.31 (m, 2H), 2.36 (t, J = 7.2 Hz, 2H), 1.55-1.69 (m, 4H), 1.55 (s,
9H), 1.37-1.44 (m, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 172.25, 161.05, 153.57,
147.62, 135.34, 133.63, 131.51, 130.24, 129.49, 126.17, 125.53, 124.36, 123.95, 114.66, 112.51,
102.07, 79.82, 36.35, 29.41, 28.48, 26.44, 25.39.
179

2.280. tert-butyl (2-(6-(5-phenyl-1H-indole-2-carboxamido)hexanamido)phenyl)carbamate.
Synthesized according to Method D. Purification by automated flash chromatography using 60%
EtOAc/hexanes as eluent gave 0.088 g as a pale yellow solid (93%). TLC: 60% EtOAc/hexanes,
R
f
≈ 0.2.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.80 (s, 1H), 7.61 (d, J = 7.6 Hz, 2h, 7.49-7.51
(m, 3H), 7.40 (t, J = 7.2 Hz, 2H), 7.33 (d, J = 8.0 Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.17 (t, J =
7.6 Hz, 1H), 7.10 (s, 1H), 7.06 (t, J = 8.0 Hz, 1H), 3.43 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 7.2 Hz,
2H), 1.69-1.83 (m, 4H), 1.49-1.54 (m, 11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.63,
162.60, 154.35, 142.07, 136.44, 133.48, 129.58, 128.29, 128.14, 126.67, 125.99, 125.80, 125.13,
124.44, 123.50, 119.43, 111.96, 103.16, 79.98, 38.91, 35.93, 28.89, 27.21, 26.09, 25.01.

2.281. methyl 6-(5-(trifluoromethyl)-1H-indole-2-carboxamido)hexanoate. Synthesized
according to Method B. Purification by automated flash chromatography using 40%
EtOAc/hexanes as eluent gave 0.105 g as a pale orange solid (68%). TLC: 40% EtOAc/hexanes,
R
f
≈ 0.25.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.94 (s, 1H), 7.57 (d, J = 8.8 H, 1H), 7.43 (d, J
= 10 Hz, 1H), 7.16 (s, 1H), 3.63 (s, 3H), 3.39 (t, J = 7.2 Hz, 2H), 2.34 (t, J = 7.2 Hz, 2H), 1.61-
180

1.71 (m, 4H), 1.38-1.46 (m, 2H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 174.40, 162.00,
137.93, 133.18, 126.80, 119.65, 119.62, 119.03, 112.30, 103.20, 53.00, 38.97, 33.22, 28.81,
26.06, 24.25.
19
F NMR (376 MHz, CD
3
OD) δF (ppm) -62.11, -74.08, -75.96.

2.282. 6-(5-(trifluoromethyl)-1H-indole-2-carboxamido)hexanoic acid. Synthesized
according to Method E. Gave 0.056 g as a white solid (60%). TLC: 10% MeOH/CH
2
Cl
2
, R
f
≈
0.35
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 12.10 (br s, 1H), 8.67 (t, J = 5.2 Hz, 1H), 8.05 (s,
1H), 7.59 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.26 (s, 1H), 3.26-3.31 (m, 2H), 2.17 (t, J
= 8.0 Hz, 2H), 1.53-1.57 (m, 4H), 1.31-1.37 (m, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm)
174.86, 162.89, 160.99, 138.14, 134.62, 126.67, 124.65, 121.36, 120.54, 119.89, 113.47, 103.48,
34.04, 29.33, 26.46, 24.68.
19
F NMR (376 MHz, DMSO-d
6
) δF (ppm) -58.70.

2.283. methyl 6-(2-(1H-indol-2-yl)acetamido)hexanoate. Synthesized according to Method B.
Purification by automated flash chromatography using 80% EtOAc/hexanes as eluent gave 0.379
g as an orange solid (44%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.25.
1
H NMR (400 MHz, CDCl
3
)
δH (ppm) 8.38 (br s, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.24 (d, J = 7.6 Hz,
1H), 7.13-7.15 (m, 2H), 5.70 (br s, 1H), 3.73 (s, 2H), 3.64 (s, 3H), 3.16 (q, J = 6.8 H, 2H), 2.21 (t,
J = 8.8 Hz, 2H), 1.49-1.57 (m, 2H), 1.33-1.40 (m, 2H), 1.14-1.21 (m, 2H).
13
C NMR (100 MHz,
181

CDCl
3
) δC (ppm) 174.00, 171.41, 136.42, 126.98, 123.75, 122.60, 120.04, 118.69, 111.41,
109.07, 51.40, 39.23, 33.80, 33.43, 29.12, 26.21, 24.42.

2.284. 6-(2-(1H-indol-2-yl)acetamido)hexanoic acid. Synthesized according to Method E.
Gave 0.259 g as an orange solid (89%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.97 (s, 1H),
10.84 (s, 1H), 7.84 (t, J = 6.0 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.16 (s,
1H), 7.07 (t, J = 5.6 Hz, 1), 6.95 (t, J = 6.0 Hz, 1H), 3.48 (s, 2H), 3.02 (q, J = 7.2 Hz, 2H), 2.16 (t,
J = 7.6 Hz, 2H), 1.45-1.49 (m, 2H), 1.37-1.40 (m, 2H), 1.21-1.26 (m, 2H).
13
C NMR (100 MHz,
DMSO-d
6
) δC (ppm) 174.91, 170.90, 136.50, 127.65, 121.32, 119.16, 118.64, 111.65, 109.45,
38.88, 34.02, 33.16, 29.31, 26.38, 24.63.

2.285. methyl 7-(1H-indole-2-carboxamido)heptanoate. Synthesized according to Method B.
Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent gave 0.125
g as a gray solid (54%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.4.
1
H NMR (400 MHz, CD
3
OD δH
(ppm) 7.59 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.21 (t, J = 6.4 Hz, 1H), 7.05 (t, J = 8.8
Hz, 2H), 3.64 (s, 3H), 3.39 (t, J = 7.2 Hz, 2H), 2.33 (t, J = 7.6 Hz, 2H), 1.60-1.68 (m, 4H), 1.39-
1.41 (m, 4H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 174.53, 162.71, 136.81, 130.96, 127.59,
123.48, 121.25, 119.66, 111.51, 102.70, 50.55, 39.05, 33.27, 29.05, 28.43, 26.27, 24.43.
182

2.286. 7-(1H-indole-2-carboxamido)heptanoic acid. Synthesized according to Method E.
Gave 0.101 g as a yellow solid (81%). TLC: 100% EtOAc, R
f
≈ 0.3.
1
H NMR (400 MHz, DMSO-
d
6
) δH (ppm) 11.97 (s, 1H), 11.51 (s, 1H), 8.41 (t, J = 5.6 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.41
(d, J = 8.4 Hz, 1H), 7.17 (t, J = 6.4 Hz, H), 7.09 (s, 1H), 7.02 (t, J = 8.4 Hz, 1H), 3.24-3.29 (m,
2H), 2.20 (t, J = 7.2 Hz, 2H), 1.49-1.55 (m, 4H), 1.30-1.32 (m, 4H).
13
C NMR (100 MHz,
DMSO-d
6
) δC (ppm) 174.92, 161.40, 136.76, 132.37, 127.54, 123.47, 121.77, 102.60, 34.06,
29.55, 28.74, 26.64, 24.90.

2.287.tert-butyl(2-(6-(1H-benzo[d]imidazole-5-carboxamido)hexanamido)
phenyl)carbamate. Synthesized according to Method B. Purification by automated flash
chromatography using 10% MeOH/EtOAc as eluent gave 0.091 g as a yellow solid (31%). TLC:
10% MeOH/CH
2
Cl
2
, R
f
≈ 0.4.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 8.29 (s, 1H), 8.15 (br s,
1H), 7.77 (d, J = 7.6 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 8.4
Hz, 1H), 7.19 (t, J = 7.2 Hz, 1H), 7.09 (t, J = 7.6 Hz, 1H), 3.45 (t, J = 6.8 Hz, 2H), 2.46 (t, J = 7.6
Hz, 2H), 1.71-1.85 (m, 4H), 1.50-1.57 (m, 11H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm)
173.71, 169.13, 154.34, 143.25, 131.65, 129.64, 125.83, 125.11, 124.36, 80.00, 60.08, 39.47,
35.85, 28.88, 27.20, 26.14, 25.19.
183

2.288. tert-butyl (2-(6-(3-(1H-benzo[d]imidazol-2-yl)propanamido)hexanamido)
phenyl)carbamate. Synthesized according to Method B. Purification by automated flash
chromatography using 5% MeOH/EtOAc as eluent gave 0.121 g as a white foam. TLC: 5%
MeOH/EtOAc, R
f
≈ 0.6.
1
H NMR (400 MHz, CD
3
OD) δH (ppm) 7.50-7.53 (m, 3H), 7.37 (d, J =
8.0 Hz, H), 7.15-7.21 (m, 3H), 7.10-7.13 (m, 1H), 3.15-3.21 (m, 4H), 2.73 (t, J = 7.6 Hz, 2H),
2.35 (t, J = 7.6 Hz, 2H), 1.65-1.71 (m, 2H), 1.48-1.51 (m, 11H), 1.30-1.38 (m, 2H).
13
C NMR
(100 MHz, CD
3
OD) δC (ppm) 173.59, 172.50, 154.06, 131.63, 125.83, 125.07, 124.42, 121.84,
121.82, 80.02, 38.78, 35.87, 33.38, 28.63, 27.21, 25.98, 25.01, 24.35.

2.289. tert-butyl (2-(4-(nicotinamidomethyl)benzamido)phenyl)carbamate. Synthesized
according to Method B. Purification by automated flash chromatography using ethyl acetate as
eluent gave 0.091 g as a yellow foam (69%). TLC: 100% EtOAc, R
f
≈ 0.2.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 9.06 (s, 1H), 8.73 (d, J = 3.2 Hz, 1H), 8.30 (d, J = 10 Hz, 1H), 7.98 (d, J = 8.4
Hz, 2H), 7.54-7.64 (m, 4H), 7.45 (d, J = 7.6 Hz, 1H), 7.24-7.26 (m, 2H), 4.71 (s, 2H), 2.84 (s,
1H), 1.50 (s, 9H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 166.52, 154.84, 151.44, 147.75,
143.03, 135.68, 132.94, 131.64, 130.40, 130.16, 127.48, 127.36, 125.93, 125.69, 124.81, 123.74,
80.27, 42.81, 27.17.
184

2.290.tert-butyl(2-(4-((3-(1H-indol-3-yl)propanamido)methyl)benzamido)
phenyl)carbamate. Synthesized according to general procedures. Purification by automated
flash chromatography using 70% EtOAc/hexanes as eluent gave 0.065 g as a white solid (78%).
TLC: 70% EtOAc/hexanes, R
f
≈ 0.3.
1
H NMR (500 MHz, CD
3
OD) δH (ppm) 7.83 (d, J = 7.5 Hz,
2H), 7.58-7.62 (m, 2H), 7.44 (d, J = 9.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.21-7.26 (m, 2H),
7.16 (d, J = 8.5 Hz, 2H), 7.08 (t, J = 8.0 Hz, 1H), 6.99-7.02 (m, 2H), 4.39 (s, 2H), 3.12 (t, J = 7.5
Hz, 2H), 2.66 (t, J = 7.5 Hz, 2H), 1.48 (s, 9H).
13
C NMR (125 MHz, CD
3
OD) δC (ppm) 174.54,
154.86, 143.17, 136.73, 132.59, 131.63, 127.27, 126.93, 126.02, 125.70, 124.86, 121.87, 120.93,
118.15, 117.98, 113.35, 110.81, 81.69, 80.33, 42.15, 36.89, 27.21, 21.24.

2.291. methyl 4-((1H-indole-2-carboxamido)methyl)benzoate. Synthesized according to
Method B. Purification by automated flash chromatography using 40% EtOAc/hexanes as eluent
gave 0.074 g as a pale yellow solid (48%). TLC: 40% EtOAc/hexanes, R
f
≈ 0.3.
1
H NMR (400
MHz, CD
3
OD) δH (ppm) 7.98 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 8.4Hz,
2H), 7.45 (d, J = 7.6 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.07 (t, J = 8.0 Hz, 1H), 4.66
(s, 2H), 3.88 (s, 3H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 166.98, 162.81, 144.65, 137.25,
130.76, 129.28, 128.91, 127.41, 126.97, 123.63, 121.32, 119.75, 111.59, 103.08, 51.10, 42.30.
185

2.292. 4-((1H-indole-2-carboxamido)methyl)benzoic acid. Synthesized according to Method
E. Gave 0.036 g as a yellow solid (60%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 12.85 (br s,
1H), 11.60 (s, 1H), 9.10 (t, J = 6.0 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 7.6 Hz, 1H),
7.42-7.45 (m, 3H), 7.18 (t, J = 7.2 Hz, 2H), 7.03 (t, J = 8.0 Hz, 1H), 4.58 (d, J = 6.0 Hz, 2H).
13
C
NMR (100 MHz, DMSO-d
6
) δC (ppm) 168.30, 162.81, 144.23, 136.92, 130.50, 129.55, 129.31,
127.53, 126.92, 123.67, 121.25, 119.80, 111.63, 103.10, 42.33.

2.293. methyl 4-((2-(1H-indol-2-yl)acetamido)methyl)benzoate. Synthesized according to
Method B. Purification by automated flash chromatography using 80% EtOAc/hexanes as eluent
gave 0.340 g as a white solid (74%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.6.
1
H NMR (400 MHz,
CD
3
OD) δH (ppm) 7.89 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 7.2 Hz, 1H), 7.36 (d, J = 7.6 Hz, H),
7.28 (d, J = 8.0 Hz, 2H), 7.20 (s, 1H), 7.12 (t, J = 7.2 Hz, 1H), 7.01 (t, J = 8.0 Hz, 1H), 4.42 (s,
2H), 3.88 (s, 3H), 3.37 (s, 2H), 2.81 (s, 1H).
13
C NMR (100 MHz, CD
3
OD) δC (ppm) 173.55,
166.93, 144.27, 136.75, 129.20, 128.51, 126.84, 123.65, 121.13, 118.45, 118.00, 110.86, 107.91,
51.05, 42.36, 32.60.
186

2.294. 4-((2-(1H-indol-2-yl)acetamido)methyl)benzoic acid. Synthesized according to Method
E. Gave 0.149 g as a white solid (78%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 12.81 (s, 1H),
10.85 (s, 1H), 8.42 (t, J = 6.0 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 7.6 Hz, 1H), 7.28-
7.32 (m, 3H), 7.17 (s, 1H), 7.04 (t, J = 7.2 Hz, 1H), 6.94 (t, J = 7.2 Hz, 1H), 4.29 (d, J = 5.6 Hz,
2H), 3.56 (s, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm)171.40, 167.59, 145.35, 136.56,
129.61, 127.58, 124.32, 121.39, 119.10, 118.69, 111.72, 109.09, 42.41. 33.13.

2.295. (4-(methoxycarbonyl)phenyl)methanaminium chloride. To a mixture of 4-
(aminomethyl)benzoic acid (2.00 g, 13.23 mmol) in methanol (60 mL) was added concentrated
HCl (10 mL) at room temperature. After refluxing for 16 h the reaction mixture was cooled to
room temperature and concentrated under reduced pressure. Ether was added to the crude
material and filtered to provide 1.98 g as a pale yellow solid (75%).
1
H NMR (400 MHz,
DMSO-d
6
) δH (ppm) 8.63 (br s, 3H), 7.97 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 4.08 (d, J
= 5.2 Hz, 2H), 3.85 (s, 3H).

187

2.296. methyl 4-(2-aminoethyl)benzoate hydrochloride. To a suspension of 4-(2-
aminomethyl)benzoic acid hydrochloride (0.375 g, 2.53 mmol) in methanol (20 mL) was added
concentrated HCl (1 mL). After refluxing for 48 h the reaction mixture was cooled to room
temperature and concentrated under reduced pressure. Trituration with ether and filtration
provided 0.390 g as a white solid (71%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 8.19 (br s,
3H), 7.91 (d, J = 7.6 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 3.84 (s, H), 2.98-3.04 (m, 4H).
13
C NMR
(100 MHz, DMSO-d
6
) δC (ppm) 166.58, 143.71, 130.03, 129.87, 129.55, 129.35, 128.54, 52.53,
33.24.

2.297. methyl 4-(2-(1H-indole-2-carboxamido)ethyl)benzoate. Synthesized according to
Method B. Purification by automated flash chromatography using 60% EtOAc/hexanes as eluent
gave 0.169 g as a pale yellow solid (52%). TLC: 60% EtOAc/hexanes, R
f
≈ 0.6.
1
H NMR (400
MHz, DMSO-d
6
) δH (ppm) 11.53 (s, 1H), 8.56 (t, J = 5.6 Hz, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.59
(d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 3H), 7.17 (t, J = 8.4 Hz, 1H), 7.07 (s, 1H), 7.02 (t, J = 8.0
Hz, 1H), 3.83 (s, 3H), 3.56 (q, J = 6.0 Hz, 2H), 2.96 (t, J = 7.2 Hz, 2H).
13
C NMR (100 MHz,
DMSO-d
6
) δC (ppm) 166.62, 161.49, 145.83, 136.80, 132.15, 129.65, 129.57, 128.01, 127.49,
123.71, 121.88, 120.10, 112.70, 102.72, 52.43, 35.60.
188

2.298. 4-(2-(1H-indole-2-carboxamido)ethyl)benzoic acid. Synthesized according to Method
E. Gave 0.080 g as a pink solid (83%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.58 (s, 1H),
8.61 (t, J = 5.6 Hz, 1H), 7.87 (d, J = 8.4 H, 2H), 7.60 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.0 Hz,
1H), 7.37 (d, J = 8.0 Hz, 2H), 7.17 (t, J = 6.8 Hz, 1H), 7.08 (s, 1H), 7.03 (t, J = 7.6 Hz, 1H), 3.53-
3.59 (m, 2H), 2.95 (t, J = 7.2 Hz, 2H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 167.95, 161.49,
144.79, 136.82, 132.20, 129.80, 129.25, 127.49, 123.65, 121.90, 119.92, 112.71, 102.76, 35.60.

2.299. methyl 4-((4-(dimethylamino)benzamido)methyl)benzoate. Synthesized according to
Method B. Purification by automated flash chromatography using 80% EtOAc/hexanes as eluent
gave 0.274 g as a white solid (71%). TLC: 80% EtOAc/hexanes, R
f
≈ 0.7.
1
H NMR (400 MHz,
CDCl
3
) δH (ppm) 7.99 (d, J = 7.2 Hz, 2H), 7.71 (d, J = 9.2 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H),
6.66 (d, J = 9.2 Hz, 2H), 6.39 (br s, 1H), 4.68 (d, J = 6.0 Hz, 2H), 3.91 (s, 3H), 3.02 (s, 6H).
13
C
NMR (100 MHz, CDCl
3
) δC (ppm) 167.51, 166.79, 152.57, 144.20, 129.94, 129.25, 128.47,
127.51, 120.79, 111.06, 52.05, 43.46, 40.09.

189

2.300. 4-((4-(dimethylamino)benzamido)methyl)benzoic acid. Synthesized according to
Method E. Gave 0.061 g as a white solid (43%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 7.90
(d, J = 8.0 Hz, 2H), 7.78 (d, J = 9.2 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 4.51 (d, J = 6.4 Hz, 2H),
2.98 (s, 6H).
13
C NMR (100 MHz, DMSO-d
6
) δC (ppm) 167.66, 166.62, 152.63, 145.93, 129.78,
129.54, 129.05, 127.55, 121.16, 111.26, 42.69.

2.301. methyl 6-(((pyridin-3-ylmethoxy)carbonyl)amino)hexanoate. To a suspension of CDI
(0.309 g, 1.91 mmol) in THF (2 mL) at 0°C was added 3-pyridinemethanol (0.185 mL, 1.91
mmol). After stirring for 1 h this mixture was added to a suspension of 6-aminocaproic acid
(0.250 g, 1.9 mmol), DBU (0.286 mL, 1.91 mmol), and triethylamine (0.266 mL, 1.91 mmol) in
THF (3 mL) at room temperature. After 18 h the reaction mixture was concentrated and diluted
with water (10 mL). The pH was adjusted to 4 using 40% HCl aqueous solution and methanol (5
mL) was added. The mixture was refluxed for 24 h. After cooling to room temperature the
reaction mixture was evaporated under reduced pressure. The crude material was diluted with
water (10 mL) and extracted into ethyl acetate (3 x 10 mL). The combined organic layers were
dried over over Na
2
SO
4
and concentrated. Purification by automated flash chromatography using
ethyl acetate as eluent gave 0.155 g as a yellow oil (29%). TLC: 100% EtOAc, R
f
≈ 0.35.
1
H
NMR (400 MHz, CDCl
3
) δH (ppm) 8.53-8.59 (m, 2H), 7.66 (d, J = 7.6 Hz, 7.25-7.28 (m, 2H),
5.08 (s, 2H), 4.94 (br s, 1H), 3.64 (s, 3H), 3.15-3.20 (m, 2H), 2.28 (t, J = 7.2 Hz, 2H), 1.58-1.65
(m, 2H), 1.46-1.54 (m, 2H), 1.30-1.36 (m, 2H).
13
C NMR (100 MHz, CDCl
3
) δC (ppm) 173.94,
156.05, 149.48, 149.40, 135.82, 132.19, 123.36, 63.94, 51.48, 40.84, 33.80, 29.53, 26.14, 24.42.
190

2.302. 6-(((pyridin-3-ylmethoxy)carbonyl)amino)hexanoic acid. Synthesized according to
Method E. Gave 0.109 g as a white solid (74%).
1
H NMR (400 MHz, DMSO-d
6
) δH (ppm) 11.98
(s, 1H), 8.51-8.56 (m, 2H), 7.76 (d, J = 9.6 Hz, 1H), 7.37-7.41 (m, 1H), 7.27 (s, 1H), 5.03 (s, 2H),
2.94-2.99 (m, 2H), 2.17 (t, J = 7.6 Hz, 2H), 1.26-1.49 (m, 4H), 1.21-1.25 (m, 2H).
13
C NMR (100
MHz, DMSO-d
6
) δC (ppm) 174.86, 156.33, 149.54, 149.46, 136.21, 133.27, 123.94, 63.30, 34.02,
29.51, 26.20, 24.61.

2.303. methyl 2-(triphenylphosphoranylidene)propanoate.

Methyl(triphenylphosphoranylidene)acetate (5.00 g, 14.95 mmol) was dissolved in dry CH
2
Cl
2

(25 mL) and cooled to 0°C in a foil-covered round bottom flask. Iodomethane (1.40 mL, 22.4
mmol) was then added and the reaction mixture was stirred while warming to room temperature
for 16 h. The reaction mixture was concentrated under reduced pressure then redissolved in
CH
2
Cl
2
(50 mL) and a solution of NaOH (1.64 g, 41 mmol) in water (33 mL). After stirring at
room temperature for 2 h the phases were separated. The aqueous phase was extracted with
CH
2
Cl
2
(2 x 50 mL). The combined organic layers were dried over Na
2
SO
4
and concentrated
under reduced pressure to obtain 4.44 g as a yellow solid (85%) which was obtained as two
rotamers and was used without further purification.
1
H NMR (500 MHz, CDCl
3
) δH (ppm) 7.45-
7.70 (m, 36H), 3.62 (t, J = 15 Hz, 3H), 3.14 (s, 3H), 1.60-1.64 (m, 5H).
31
P NMR (202 MHz,
CDCl
3
) δP (ppm) 34.60, 29.00, 22.69, 22.48, 18.00.
191

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200

Chapter 3. Synthesis and SAR study of benzimidazoles as
inhibitors of Bcl-2/Bxl-xL.

3.1. Introduction. The role of Bcl-2/Bcl-xL in apoptosis.

Apoptosis, or programmed cell death, is a highly regulated natural process which
removes defective cells, such as those containing mutations. Disruption of this process can alter
the balance between cell growth and cell death, often leading to cancer.
1
The intrinsic pathway of
apoptosis involves release of cytochrome c from the mitochondria which leads to the activation of
downstream caspases and causes a death response.
2
Members of the B-cell lymphoma 2 (Bcl-2)
protein family are localized to the mitochondria and can either prevent or promote the release of
cytochrome c, consequently halting or promoting apoptosis.
3
Therefore, the Bcl-2 proteins can
serve as a potential target for cancer therapy because they function within the apoptotic pathway.
4
Proteins in the Bcl-2 family are divided into pro-apoptotic proteins (BAK, BAD, BID,
and BAX) and anti-apoptotic proteins (Bcl-2 and Bcl-xL).
5
The pro-apoptotic proteins function
by binding to the BH3 binding domain of the anti-apoptotic proteins, which then inhibits the
activity of the anti-apoptotic proteins and promotes cell growth.
6
The anti-apoptotic proteins
function by inhibiting the release of cytochrome c, thus allowing cell proliferation to continue.
Therefore, a molecule which mimics the protein-protein interaction between the pro-apoptotic
and anti-apoptotic proteins may inhibit the activity of the anti-apoptotic proteins. This could
consequently restore apoptosis and lead to the death of cancer cells. One anti-apoptotic protein of
interest for the development of such inhibitors is Bcl-2, a 26 kDa protein localized to the
mitochondria and endoplasmic reticulum.
7
Inhibition of Bcl-2 activity is an attractive target for
the treatment of cancer because it has been found that expression of Bcl-2 is essential for the
growth of certain tumor cell lines and it has been found to be upregulated in many tumor types.
8

201

Much research has been carried out to exploit the interaction of the BH3 binding domain
to inhibit Bcl-2 activity. Both peptidic and non-peptidic inhibitors have been investigated, but
currently, non-peptidic small-molecule inhibitors of signaling pathways are the developmental
drugs of choice, mainly due to their low antigenicity.
7
Additionally, these types of inhibitors are
easily modified to improve bioavailability and affinity for the desired target. Although the area of
non-peptidic small-molecule inhibitors is one which is relatively new for Bcl-2 inhibitors, a
compound developed by Abbott Labs (Compund 3.1, ABT-737), shown in Figure 3.1, has
demonstrated low nanomolar affinity for Bcl-2.
9

Figure 3.1 – Structure of ABT-737.
ABT-737 was rationally designed by investigation of the elongated hydrophobic groove
of the BH3 binding domain of Bcl-2, as shown in Figure 3.2.
10

Figure 3.2 – Interaction of the hydrophobic BH3 binding groove of Bcl-2 with an
acylsulfonamide compound developed by a group at Abbott Labs (PDB code 202F).
10

202

3.2. Design of benzimidazole core scaffold and synthesis.
By evaluating the critical interactions of ABT-737 with Bcl-2, another series of potential
BH3-binding domain mimics, shown in Figure 3.3, were designed by the Neamati laboratory at
the USC School of Pharmacy using a pharmacophore model. Compounds developed for
investigation of their structure-activity relationship were comprised of a phenyl-benzimidazole
core. These compounds could be varied in the left and right wing portions, as well as in the
linkage used between the core and each wing, to facilitate development of a small library of test
compounds. Therefore, using the designed scaffold, the main objective of this study was to
synthesize a series of selected compounds which would include variable functional groups in the
side chains to provide compounds capable of demonstrating excellent potency against a panel of
cancer cell lines.

Figure 3.3 - Phenyl-benzimidazole core structures with variable linkages/wing moieties.
Synthesis of the first set of test molecules was carried out following the route illustrated
in Scheme 3.1.
11
To construct the benzimidazole backbone, acid catalyzed dehydrative
cyclization in the presence of polyphosphoric acid provided compound 3.5 in modest yield
(52%). Subsequent coupling of the free amine with different acid chlorides using pyridine as
both base and solvent allowed for access to a number of amides 3.6, generally in good yield.
Reduction of the nitro group on the benzimidazole backbone was followed by a second acylation
reaction between the newly formed amine with a number of acid chlorides. This provided final
203

compounds 3.8, which then were evaluated for their biological activity in several cancer cell lines
using a standard MTT assay in our collaborators’ laboratory (biological data not shown).

Reagents and conditions: (a) 4-aminobenzoic acid, polyphosphoric acid, xylenes, 150°C, 12 h, 52%;
(b) ArCOCl, pyridine, 0°C to rt, 12 h, 19-98%; (c) H
2
, 5% Pd/C, MeOH, 16 h, 2-98%; (d) ArCOCl,
pyridine, 0°C to rt, 12 h, 10-91%.

Scheme 3.1 - Synthetic route for a variety of substituted phenyl-benzimidazole
analogues.
11

Variation in the acid chlorides used in the synthetic route allowed for development of an
initial set of compounds which were intended to possess a long, planar structure to optimize
binding of the molecules in the hydrophobic groove of the Bcl-2 BH-3 binding domain. As
shown in Figure 3.4, these preliminary compounds possessed a biphenyl substituent fixed on the
right wing and a variety of substituents on the left wing. However, poor to modest biological
activity was observed in breast cancer cell lines. It was therefore determined that to improve
activity, the biphenyl substituent on the right wing would need to be altered.
204

Figure 3.4 – Compounds containing biphenyl right-wing substituents and varied functionality on
the left-wing.
Next, a set of compounds containing the same functionality on the left and right wing
substituents was synthesized (Figure 3.5). This set of analogues was evaluated because an initial
lead compound tested for biological activity had the same substituent on both sides of the phenyl-
benzimidazole core.

Figure 3.5 - Compounds containing identical left and right-wing substituents.
Compounds containing identical substituents at either wing provided an improvement in
biological activity when screened against several cancer cell lines. Although 2-naphthyl
205

substitution was not well tolerated (3.19), a 1-naphthyl substituent on either wing provided
modest activity (3.20). Although inclusion of a 4-methoxy benzoyl substituent in the scaffold
structure did not display desirable activity (3.16), the compound containing two 4-chloro benzoyl
substituents provided the best activity (3.17) out of compounds evaluated.
Using this information, an additional set of test compounds with the 4-chloro benzoyl
substituent fixed on the right wing was then synthesized and evaluated for bioactivity. These
compounds, shown in Figure 3.6, did not provide a drastic improvement in activity compared
with compound 3.17.

Figure 3.6 - Compounds containing a 4-chloro benzoyl substituent fixed on the right wing.
To further evaluate the role of the 4-chloro benzoyl substituent on the right wing,
compounds with either benzoyl or 4-methoxy benzoyl fixed on the right were evaluated for their
activity (Figures 3.7 and 3.8).

206

Figure 3.7 – Variations in the left-wing region.
Interestingly, compounds with 4-methoxy benzoyl subsituents on the right wing
displayed modest activity against several cancer cell lines when either benzoyl (3.30) or 4-chloro
benzoyl (3.31) substituents were present on the left wing. However, the 4-methoxy benzoyl
substituent was not well-tolerated when included on both wings (3.32).

Figure 3.8 - Additional variations in the left-wing region.
To probe the effect of a different bond linkage, compounds using a sulfonamide to link the
phenyl-benzimidazole core to the right wing were also synthesized as well, as shown in Scheme
3.2. The core was synthesized in a two-step procedure involving mono-acylation of 3.4 followed
by an acid-promoted cyclization.
12
Base hydrolysis readily converted the nitrile 3.33 to the
carboxylic acid 3.34 in excellent yield.
12
However, EDCI coupling of the carboxylic acid to a
variety of sulfonamides was more problematic, due to the weak nucleophilicity of the
207

sulfonamide coupling partners and poor solubility of the carboxylic acid.
13
Reduction of the nitro
group of the benzimidazole provided the amine in modest yield as well. Unfortunately, out of
small number of compounds tested from this synthetic route, none demonstrated promising
biological activity in the cancer cells against which they were screened.

Reagents and conditions: (a) (i) 4-cyanobenzoyl chloride, NEt
3
, THF, 0°C to rt, 12 h (ii) acetic acid, 120°C,
24 h, 58%; (b) NaOH, ethanol:THF (1:1), 80°C, 24 h, 78%; (c) ArSO
2
NH
2
, EDC, DMAP, CH
2
Cl
2
, 0°C to
rt, 24 h, 2-36%; (d) H
2
, Pd/C, MeOH, 16 h, 45%.

Scheme 3.2 - Synthetic route for analogues containing a sulfonamide linker.
3.3 Experimental.

All commercially available reagents and solvents were used as received by Aldrich and
Acros chemical without further purification.
1
H and
13
C spectra were recorded on a 250 and
63MHz Bruker or 400 and 100MHz Varian instrument. Thin-layer chromatography (TLC) was
performed using commercially prepared 60 mesh silica gel plates visualized with short-
wavelength UV light (254 nm). Silica gel 60 (9385, 230-400 mesh) was used for column
chromatography.

208

3.5. 4-(5-Nitro-1H-benzoimidazol-2-yl)-phenylamine: To a mixture of 4-nitro-1,2-phenylene
diamine 3.4 (5.60 g, 36.6 mmol) and 4-aminobenzoic acid (5.12 g, 37.3 mmol) in a 1 L two-neck
round bottom flask was added polyphosphoric acid (60 g) and xylenes (400 mL).
11
The reaction
mixture was stirred mechanically at 150 ⁰C for 12 h, then cooled to room temperature and xylenes
solvent was decanted. Water was then added to the crude solid and the reaction mixture was
neutralized with solid sodium bicarbonate. The dark green solid was then filtered and washed
with hot water (5 x 500 mL). The crude solid was stirred in hot THF (500 mL) for 3 hrs.
Decolorizing charcoal (3 g) was added to the mixture, which was then filtered. The filtrate was
concentrated in vacuo and purification of the resulting dark brown solid product by silica gel
chromatography with ethyl acetate provided 4-(5-nitro-1H-benzimidazole-2-yl)-phenylamine 3 as
a red solid (4.80 g, 52%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 13.13 (br s, 1H), 8.41 (br s, 1H),
8.26 (dd, J = 3.2 Hz, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.65 (br s, 1H), 6.70 (d, J = 9.2 Hz, 2H), 5.85
(br s, 2H).

3.6. (R=4-Cl): To a solution of 4-(5-nitro-1H-benzimidazole-2-yl)-phenylamine 3.5 (1.00 g, 3.93
mmol) in anhydrous pyridine (10 mL) at 0 ⁰C was added acid chloride (0.520 mL, 4.05 mmol).
11

The reaction mixture was stirred for 12 h, while warming to room temperature, then quenched
with water and filtered. The gummy solid was stirred in 10% NaOH aqueous solution (1 mL) at
90⁰C for 30 min., then water was added. The solid 3.6 was filtered, washed with water, and dried
209

to obtain an orange solid (1.31 g, 85%).
1
H NMR (DMSO-d
6
, 400 MHz) δ
1
H NMR (DMSO-d
6
,
400 MHz) δ 12.22 (br s, 1H), 10.47 (br s, 1H), 8.33 (s, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.03 (d, J =
8.4 Hz, 2H), 7.89 (d, J = 8.8 Hz, 3H), 7.64 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 1H).

3.7. (R = 4-Cl): A solution of 3.6 (1.00 g, 2.55 mmol) was sonicated in MeOH (50 mL) for 5 min
and then was added 5% Pd/C.
11
The reaction mixture was shaken under H
2
(50 psi) at room
temperature for 16 h, filtered through a pad of celite, and then concentrated in vacuo. Purification
by silica gel chromatography, eluting with 10% MeOH in ethyl acetate, provided 3.7 as a yellow
solid (500 mg, 54 %).
1
H NMR (DMSO-d
6
, 400 MHz) δ
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.22
(br s, 1H), 10.50 (br s, 1H), 7.99-8.06 (m, 5H), 7.90-7.94 (m, 3H), 7.65 (d, J = 8.8 Hz, 2H), 7.58
(d, J = 8.0 Hz, 1H), 7.29 (d, J = 11.6 Hz, 1H), 6.66 (s, 1H), 6.51 (dd, J = 8.8 Hz, 1H), 4.99 (br s,
2H).

3.8. (R = 4-Cl): To a solution of 3.7 (100 mg, 0.28 mmol) in anhydrous pyridine (10 mL) at 0 ⁰C
was added acid chloride (0.036 mL, 0.28 mmol).
11
The reaction mixture was stirred for 12 h,
while warming to room temperature, then quenched with water and filtered. The gummy solid
was stirred in 10% NaOH aqueous solution (1 mL) at 90 ⁰C for 30 min., then water was added.
The solid 6was filtered, washed with water, and dried to obtain a solid which was triturated with
methanol to provide a white solid (122 mg, 87%).
1
H NMR (DMSO-d
6
, 400 MHz) δ
1
H NMR
210

(DMSO-d
6
, 400 MHz) δ 12.84 (br s, 1H), 10.58 (br s, 1H), 10.39 (br s, 1H), 8.15-8.17 (d, J = 7.6
Hz, 3H), 8.03 (d, J = 8.40, 4H), 7.97 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.0 Hz, 4H), 7.57 (br s,
1H), 7.52 (br s, 1H).
13
C NMR (DMSO-d
6
, 100 MHz) δ 165.09, 164.73, 140.73, 136.98, 136.62,
134.34, 133.93, 130.15, 130.15, 130.04, 128.97, 128.87, 127.26, 120.78, 116.45. ESI-MS (m/z)
501.0879 (M + H).

3.11. N-(4-(5-benzamido-1H-benzo[d]imidazol-2-yl)phenyl)-[1,1'-biphenyl]-4-carboxamide:
Synthesized according to general procedure for 3.7 to obtain a yellow solid (0.003 g, 49%).
1
H
NMR (MeOD-d
3
, 400 MHz) δ 8.57 (br s, 1H), 8.22 (s, 1H), 8.15 (d, J = 8.8 Hz, 1H), 8.10 (d, J =
8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H), 7.96 (dd, J = 4.8 Hz, 1H), 7.84 (d, J = 10.4 Hz, 2H), 7.75
(d, J = 7.2 Hz, 2H), 7.62-7.65 (m, 1H), 7.58 (d, J = 10.8 Hz, 1H), 7.52 (t, J = 8.0 Hz, 3H), 7.54-
7.57 (m, 1H), 7.35-7.39 (m, 1H), 6.83 (d, J = 10.8 Hz, 1H).

3.12. N-(4-(5-(4-methoxybenzamido)-1H-benzo[d]imidazol-2-yl)phenyl)-[1,1'-biphenyl]-4-
carboxamide: Synthesized according to general procedure for 3.8 to obtain a yellow solid (0.196
g, 76%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.81 (br s, 1H), 10.57 (br s, 1H), 10.18 (br s, 1H),
8.23 (s, 1H), 8.11-8.20 (m, 4H), 8.01 (d, J = 8.8 Hz, 4H), 7.89 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 8.0
Hz, 2H), 7.53-7.62 (m, 3H), 7.45-7.48 (m, 2H), 7.09 (d, J = 8.8 Hz, 2H), 3.87 (s, 3H).
211

3.13. N-(4-(5-(4-cyanobenzamido)-1H-benzo[d]imidazol-2-yl)phenyl)-[1,1'-biphenyl]-4-
carboxamide: Synthesized according to general procedure for 3.8 to obtain a yellow solid (0.010
g, 73%).
1
H NMR (MeOD-d
3
, 400 MHz) δ 8.21 (s, 1H), 8.09 (d, J = 8.0 Hz, 3H), 7.82-7.88 (m,
5H), 7.74 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 10.8 Hz, 1H), 7.53 (t, J = 7.2 Hz, 3H), 7.41-7.47 (m,
2H), 6.84 (d, J = 10.0 Hz, 2H).

3.14. N-(4-(5-(4-chlorobenzamido)-1H-benzo[d]imidazol-2-yl)phenyl)-[1,1'-biphenyl]-4-
carboxamide: Synthesized according to general procedure for 3.8 to obtain a yellow solid
(0.003g , 46%).
1
H NMR (MeOD-d
3
, 400 MHz) δ 8.58 (s, 1H), 8.15 (d, J = 8.8 Hz, 2H), 8.08-
8.12 (m, 2H), 8.0 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.82-7.87 (m, 2H), 7.74 (d, J =
8.0 Hz, 1H), 7.53 (t, J = 6.4 Hz, 2H), 7.36-7.41 (m, 4H), 7.24 (d, J = 7.6 Hz, 1H), 6.83 (d, J = 8.4
Hz, 1H).

212

3.15. N-(4-(5-benzamido-1H-benzo[d]imidazol-2-yl)phenyl)benzamide: Synthesized
according to general procedure for 3.8 using 3.48 (0.200 g, 0.892 mmol) and 2.06 eq. acid
chloride to obtain a brown solid (0.040 g, 10%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.73 (br s,
1H), 10.52 (br s, 1H), 10.29 (br s, 1H), 8.16 (d, J = 8.4, 2H), 7.97-8.01 (m, 5H), 7.49-7.65 (m,
6H). ESI-MS (m/z) 433.3 (M + H)

3.16. 4-methoxy-N-(4-(5-(4-methoxybenzamido)-1H-benzo[d]imidazol-2-
yl)phenyl)benzamide: Synthesized according to general procedure for 3.8 using 3.48 (0.060 g,
0.268 mmol) and 2.06 eq. acid chloride to obtain a tan solid (0.061 g, 52%).
1
H NMR (DMSO-d
6
,
400 MHz) δ 12.75 (br s, 1H), 10.33 (br s, 1H), 10.17 (br s, 1H), 8.14 (d, J = 9.6 Hz, 3H), 7.96-
8.03 (m, 6H), 7.50 (br s, 2H), 7.08-7.12 (m, 4H). ESI-MS (m/z) 493.3 (M + H)

3.17. 4-chloro-N-(4-(5-(4-chlorobenzamido)-1H-benzo[d]imidazol-2-yl)phenyl)benzamide:
Synthesized according to general procedure for 3.8 to obtain a white solid (122 mg, 87%).
1
H
NMR (DMSO-d
6
, 400 MHz) δ 12.84 (br s, 1H), 10.58 (br s, 1H), 10.39 (br s, 1H), 8.15-8.17 (d, J
= 7.6 Hz, 3H), 8.03 (d, J = 8.40, 4H), 7.97 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.0 Hz, 4H), 7.57 (br
s, 1H), 7.52 (br s, 1H).
13
C NMR (DMSO-d
6
, 100 MHz) δ 165.09, 164.73, 140.73, 136.98,
213

136.62, 134.34, 133.93, 130.15, 130.15, 130.04, 128.97, 128.87, 127.26, 120.78, 116.45. ESI-
MS (m/z) 501.0879 (M + H)

3.18. N-(4-(5-([1,1'-biphenyl]-4-ylcarboxamido)-1H-benzo[d]imidazol-2-yl)phenyl)-[1,1'-
biphenyl]-4-carboxamide: Synthesized according to general procedure for 3.8 using 3.48 (0.100
g, 0.446 mmol) and 2.06 eq. acid chloride to obtain a tan solid (0.138 g, 53%).
1
H NMR (DMSO-
d
6
, 400 MHz) δ 12.82 (br s, 1H), 10.56 (br s, 1H), 10.39 (br s, 1H), 8.28 (s, 1H), 8.17 (d, J = 8.4
Hz, 2H), 8.12 (d, J = 7.6 Hz, 4H), 8.02 (d, J = 8.8 Hz, 2H), 7.89 (t, J = 8.0 Hz, 4H), 7.80 (d, J =
7.2 Hz, 4H), 7.63 (d, J = 8.4 Hz, 1H), 7.51-7.56 (m, 5H), 7.37-7.47 (m, 2H). ESI-MS (m/z) 585.3
(M + H)

3.19. N-(4-(5-(2-naphthamido)-1H-benzo[d]imidazol-2-yl)phenyl)-2-naphthamide:
Synthesized according to general procedure for 3.8 from 3.48 (0.200 g, 0.892 mmol) and acid
chloride (2.06 eq., 1.84 mmol) to obtain a tan solid (0.225 g, 47%).
1
H NMR (DMSO-d
6
, 400
MHz) δ 12.85 (br s, 1H), 10.78 (br s, 1H), 10.54 (br s, 1H), 8.65 (s, 2H), 8.32 (s, 1H), 8.20-8.32
(m, 1H), 8.15-8.18 (m, 2H), 8.10-8.13 (m, 2H), 8.04-8.09 (m, 7 H), 7.64-7.71 (m, 5H), 7.53-7.56
(m, 1H). ESI-MS (m/z) 533.5 (M + H)
214

3.20. N-(4-(5-(1-naphthamido)-1H-benzo[d]imidazol-2-yl)phenyl)-1-naphthamide:
Synthesized according to general procedure for 3.8 using 3.48 (0.200 g, 0.892 mmol) and 2.06 eq.
acid chloride to obtain a tan solid (0.273 g, 58%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.89 (br s,
1H), 10.87 (br s, 1H), 10.65 (br s, 1H), 8.75 (d, J = 9.6 Hz, 1H), 8.17-8.24 (m, 4H), 8.12 (t, J =
7.6 Hz, 2H), 8.08-8.10 (m, 2H), 8.01 (d, J = 8.8 Hz, 2H), 7.82 (t, J = 7.2 Hz, 2H), 7.62-7.67 (m,
8H).

3.22. 4-chloro-N-(4-(5-(4-fluorobenzamido)-1H-benzo[d]imidazol-2-yl)phenyl)benzamide:
Synthesized according to general procedure for 3.8 to obtain a yellow solid (0.042 g, 62%).
1
H
NMR (DMSO-d
6
, 400 MHz) δ 12.80 (br s, 1H), 10.51 (br s, 1H), 10.36 (br s, 1H), 8.22 (s, 1H),
8.14-8.18 (m, 2H), 8.07-8.11 (m, 2H), 7.96-8.04 (m, 4H), 7.63-7.67 (m, 1H), 7.56-7.60 (m, 2H),
7.47-7.51 (m, 1H), 7.37-7.42 (m, 2H). ESI-MS (m/z) 485.3 (M + H)

215

3.23. N-(4-(5-benzamido-1H-benzo[d]imidazol-2-yl)phenyl)-4-chlorobenzamide: Synthesized
according to general procedure for 3.8 to obtain a tan solid (0.148 g, 46%).
1
H NMR (DMSO-d
6
,
400 MHz) δ 12.86 (br s, 1H), 10.57 (br s, 1H), 10.32 (br s, 1H), 8.25 (br s, 1H), 8.16 (d, J = 8.0
Hz, 2H), 7.97-8.05 (m, 6H), 7.52-7.68 (m, 7H). ESI-MS (m/z) 467.5 (M + H)

3.25. N-(2-(4-(4-chlorobenzamido)phenyl)-1H-benzo[d]imidazol-5-yl)-1-naphthamide:
Synthesized according to general procedure for 3.8 to obtain an orange solid (0.157 g, 55%).
1
H
NMR (DMSO-d
6
, 400 MHz) δ 12.83 (br s, 1H), 10.65 (br s, 1H), 10.51 (br s, 1H), 8.24 (d, J = 8.8
Hz, 1H), 8.17 (d, J = 7.6 Hz, 2H), 8.10 (d, J = 8.0 Hz, 1H), 7.97-8.07 (m, 5H), 7.80 (d, J = 6.4
Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 7.61-7.65 (m, 4H), 7.56-7.58 (m, 2H). ESI-MS (m/z) 517.12
(M + H)

3.26. 4-chloro-N-(4-(5-(4-methoxybenzamido)-1H-benzo[d]imidazol-2-yl)phenyl)benzamide:
Synthesized according to general procedure for 3.8 to obtain a tan solid (0.173 g, 73%).
1
H NMR
(DMSO-d
6
, 400 MHz) δ 12.76 (br s, 1H), 10.50 (br s, 1H), 10.20 (br s, 1H), 8.23 (s, 1H), 8.11-
8.18 (m, 2H), 7.97-8.05 (m, 6H), 7.56-7.68 (m, 3H), 7.47 (d, J = 10.4 Hz, 1H), 7.09 (d, J = 8.8
Hz, 2H), 3.87 (s, 3H).
216

3.28. N-(2-(4-benzamidophenyl)-1H-benzo[d]imidazol-5-yl)-4-chlorobenzamide: Synthesized
according to general procedure for 3.8 to obtain a tan solid (0.096g, 34%).
1
H NMR (DMSO-d
6
,
400 MHz) δ 12.82 (br s, 1H), 10.53 (br s, 1H), 10.42 (br s, 1H), 8.23 (s, 1H), 8.15 (d, J = 8.8 Hz,
2H), 7.98-8.05 (m, 6H), 7.64-7.66 (m, 3H), 7.51-7.60 (m, 3H), 7.49 (t, J = 6.8 Hz, 1H). ESI-MS
(m/z) 467.5 (M + H)

3.29. N-(2-(4-benzamidophenyl)-1H-benzo[d]imidazol-5-yl)-[1,1'-biphenyl]-4-carboxamide:
Synthesized according to general procedure for 3.8 to obtain a tan solid (0.074 g, 24%).
1
H NMR
(DMSO-d
6
, 400 MHz) δ 12.82 (br s, 1H), 10.51 (br s, 1H), 10.39 (br s, 1H), 8.28 (s, 1H), 8.15-
8.17 (m, 3H), 8.11 (d, J = 8.0 Hz, 2H), 7.99-8.01 (m, 4H), 7.87 (d, J = 8.0 Hz, 2H), 7.79 (d, J =
8.8 Hz, 2H), 7.52-7.66 (m, 6H), 7.46 (d, J = 6.8 Hz, 1H). ESI-MS (m/z) 509.5 (M + H)

3.30. N-(4-(5-benzamido-1H-benzo[d]imidazol-2-yl)phenyl)-4-methoxybenzamide:
Synthesized according to general procedure for 3.8 to obtain a tan solid (0.031 g, 12%).
1
H NMR
217

(DMSO-d
6
, 400 MHz) δ 12.79 (br s, 1H), 10.32 (br s, 1H), 8.24 (s, 1H), 8.13 (d, J = 8.4 Hz, 2H),
7.97-8.03 (m, 5H), 7.57-7.62 (m, 4H), 7.48 (d, J = 11.2 Hz, 1H), 7.10 (d, J = 10.8 Hz, 2H), 3.87
(s, 3H).

3.31. 4-chloro-N-(2-(4-(4-methoxybenzamido)phenyl)-1H-benzo[d]imidazol-5-yl)benzamide:
Synthesized according to general procedure for 3.8 to obtain a tan solid (0.118 g, 72%).
1
H NMR
(DMSO-d
6
, 400 MHz) δ 12.80 (br s, 1H), 10.40 (br s, 1H), 10.34 (br s, 1H), 8.22 (s, 1H), 8.12-
8.17 (m, 3H), 7.97-8.05 (m, 7H), 7.64 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 3.87 (s, 3H).

3.33. 4-(5-nitro-1H-benzo[d]imidazol-2-yl)benzonitrile: To a solution of acetic acid (125 mL)
was added 3.63 (5.00 g, 17.7 mmol). The reaction mixture was stirred under reflux for 24 h, then
cooled to room temperature and solvent was evaporated under nitrogen.
12
To the crude solid was
added methanol (50 mL) and the compound was filtered to obtain a white solid (2.85 g, 61%).
1
H
NMR (DMSO-d
6
, 400 MHz) δ 13.92 (br s, 1H), 8.57 (br s, 1H), 8.49 (d, J = 8.8 Hz, 2H), 8.20
(dd, J = 9.6 Hz, 1H), 8.11 (d, J = 8.0 Hz, 2H), 7.85 (br s, 1H).
13
C NMR (DMSO-d
6
, 63 MHz) δ
154.64, 143.78, 133.86, 133.81, 133.36, 129.54, 129.41, 128.27, 119.15, 119.12, 115.85, 113.65,
113.43.
218

3.34. 4-(5-nitro-1H-benzo[d]imidazol-2-yl)benzoic acid: To a (1:1:1) solution of 1M aqueous
NaOH:ethanol:THF (120 mL) was added 3.33 (1.00 g, 3.78 mmol). The reaction mixture was
stirred under reflux for 24 h, then cooled to room temperature and concentrated in vacuo to half
volume. The reaction mixture was diluted with water (50 mL) and washed with chloroform (80
mL).
12
While on ice, the aqueous layer was then acidified with 5M HCl to pH 2 and filtered to
obtain a pale yellow solid (830 mg, 78%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 11.99 (br s, 1H),
8.54 (d, J = 2.4 Hz, 1H), 8.40 (d, J = 6.4 Hz), 8.18 (dd, J = 6.4 Hz, 1H), 8.15 (d, J = 8.8 Hz, 2H),
7.84 (d, J = 9.6 Hz, 2H).
13
C NMR (DMSO-d
6
, 100 MHz) δ 167.10, 154.50, 143.75, 133.36,
132.15, 130.46, 128.77, 127.96, 119.21, 115.50, 112.34.

3.35. 4-(5-nitro-1H-benzo[d]imidazol-2-yl)-N-(phenylsulfonyl)benzamide: Synthesized
according to general procedure for 3.64 to obtain a yellow solid (0.035 g, 23%).
1
H NMR
(DMSO-d
6
, 400 MHz) δ 13.78 (br s, 1H), 8.51 (br s, 1H), 8.20 (d, J = 8.4 Hz, 2H), 8.15 (d, J =
8.8 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.80 (br s, 1H), 7.41-7.43 (m, 3H),
6.70 (d, J = 6.0 Hz, 1H).
13
C NMR (DMSO-d
6
, 100 MHz) δ 169.67, 156.26, 156.04, 146.45,
143.20, 143.13, 143.08, 141.78, 130.71, 130.40, 130.29, 129.37, 129.02, 128.68, 128.65, 128.15,
127.44, 127.15, 126.84, 118.39, 107.28.
219

3.36 4-(5-amino-1H-benzo[d]imidazol-2-yl)-N-((4-chlorophenyl)sulfonyl)benzamide:
Synthesized according to general procedure for 3.7 to obtain a brown solid (0.067 g, 45%).
1
H
NMR (DMSO-d
6
, 400 MHz) δ 8.23 (d, J = 6.4 Hz, 1H), 8.06-8.07 (m, 2H), 7.99-8.02 (m, 1H),
7.88 (d, J = 10.4 Hz, 1H), 7.45-7.50 (m, 2H), 7.31 (d, J = 11.2 Hz, 1H), 6.98 (d, J = 6.4 Hz, 1H),
6.75 (br s, 1H), 6.60 (d, J = 8.0 Hz, 1H), 4.15 (br s, 2H).
13
C NMR (DMSO-d
6
, 100 MHz) δ
169.63, 145.14, 135.14, 131.00, 129.33, 129.29, 129.25, 128.37, 128.23, 127.29, 125.83, 125.78,
124.59, 122.05.

3.37. N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)-[1,1'-biphenyl]-4-carboxamide:
Synthesized according to general procedure for 3.6 (0.900 g, 3.5 mmol) to obtain an orange solid
(1.504 g, 98%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.84 (br s, 1H), 10.61 (br s, 1H), 8.47 (s, 1H),
8.25 (d, J = 9.2 Hz, 2H), 8.13 (d, J = 6.0 Hz, 3H), 8.06 (d, J = 8.8 Hz, 2H), 7.89 (d, J = 8.0 Hz,
2H), 7.79 (d, J = 8.0 Hz, 2H), 7.75 (d, J = 9.6 Hz, 1H), 7.54 (t, J = 7.6 Hz, 2H), 7.46 (d, J = 6.8
Hz, 1H).

220

3.38. N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)-1-naphthamide: Synthesized according
to general procedure from 3.6 (0.500 g, 1.97 mmol) to obtain a tan solid (0.569 g, 70%).
1
H NMR
(DMSO-d
6
, 400 MHz) δ 13.40 (br s, 1H), 10.89 (br s, 1H), 8.59 (d, J = 6.4 Hz, 1H), 8.44 (s, 1H),
8.27 (d, J = 9.2 Hz, 1H), 8.23 (d, J = 10.4 Hz, 1H), 8.12 (d, J = 7.6 Hz, 1H), 8.06 (d, J = 9.6 Hz,
2H), 8.01 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 6.0 Hz, 1H), 7.62-7.70 (m, 4H).
13
C NMR (DMSO-d
6
,
100 MHz) δ 167.84, 150.06, 140.82, 136.57, 135.11, 133.62, 130.64, 130.11, 128.79, 128.00,
127.51, 126.79, 126.00, 124.33, 120.08, 116.29, 114.98, 112.28.

3.39. 3,5-dimethoxy-N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)benzamide: Synthesized
according to general procedure from 3.6 (0.634 g, 2.49 mmol) to obtain a tan solid (0.195 g,
19%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 10.25 (br s, 1H), 8.25-8.30 (m, 2H), 7.97 (d, J = 8.8 Hz,
2H), 7.72-7.83 (m, 2H), 7.15 (s, 1H), 6.60 (d, J = 9.6 Hz, 2H), 3.86 (s, 3H), 3.72 (s, 3H).

3.40. 4-ethoxy-N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)benzamide: Synthesized
according to general procedure from 3.6 (0.668 g, 2.63 mmol) to obtain an orange solid (0.550 g,
53%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.00 (br s, 1H), 10.17 (br s, 1H), 8.26 (br s, 2H), 7.98
221

(d, J = 9.2 Hz, 2H), 7.93 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 7.46-7.53 (m, 1H), 7.07 (d,
J = 8.4, 2H), 6.64 (d, J = 8.8 Hz, 2H), 4.41 (q, 2H), 1.38 (t, J = 7.2 Hz, 3H).

3.41. N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)benzamide: Synthesized according to
general procedure from 3.6 (0.900 g, 3.5 mmol) to obtain an orange solid (1.130 g, 90%).
1
H
NMR (DMSO-d
6
, 400 MHz) δ 12.23 (br s, 1H), 10.56 (br s, 1H), 8.46 (s, 1H), 8.24 (d, J = 8.8 Hz,
2H), 8.11 (dd, J = 8.0 Hz, 1H), 8.00 (t, J = 8.8 Hz, 4H), 7.73 (d, J = 8.4 Hz, 1H), 7.58-7.66 (m,
3H).

3.42. N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)-2-naphthamide: Synthesized according
to general procedure from 3.6 (0.900 g, 3.5 mmol) to obtain a tan solid (1.1 g, 77%).
1
H NMR
(DMSO-d
6
, 400 MHz) δ 12.90 (br s, 1H), 10.67 (br s, 1H), 8.64 (s, 1H), 8.40 (s, 1H), 8.28 (d, J =
8.8 Hz, 2H), 8.12-8.15 (m, 1H), 8.02-9.08 (m, 3H), 7.99 (d, J = 6 Hz, 2H), 7.66-7.71 (m, 2H),
7.62 (d, J = 8.8 Hz).

3.43. 4-fluoro-N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)benzamide: Synthesized
according to general procedure from 3 (0.900 g, 3.5 mmol) to obtain a brown solid (1.175 g,
222

88%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.60 (br s, 1H), 10.53 (br s, 1H), 8.41 (s, 1H), 8.26 (d, J
= 8.4 Hz, 2H), 8.07-8.11 (m, 4H), 8.01 (dd, J = 7.2 Hz, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.64 (d, J =
9.2 Hz, 1H), 7.42 (t, J = 10.8 Hz, 2H).

3.44. 4-methoxy-N-(4-(5-nitro-1H-benzo[d]imidazol-2-yl)phenyl)benzamide: Synthesized
according to general procedure from 3.6 (0.900 g, 3.5 mmol) to obtain a brown solid (0.737 g,
54%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 10.33 (br s, 1H), 8.40 (s, 1H), 8.24 (d, J = 8.8 Hz, 2H),
8.01 (d, J = 9.6 Hz, 3H), 7.96 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 8.0 Hz,
2H), 3.87 (s, 3H).

3.45. N-(4-(5-amino-1H-benzo[d]imidazol-2-yl)phenyl)-1-naphthamide: Synthesized
according to general procedure for 3.7 to obtain a yellow solid (0.032 g, 98%).
1
H NMR (DMSO-
d
6
, 400 MHz) δ 12.31 (br s, 1H), 10.80 (br s, 1H), 8.22 (m, 1H), 8.05-8.13 (m, 5H), 7.95 (d, J =
6.4 Hz, 2H), 7.80 (d, J = 6.4 Hz, 2H), 7.62-7.67 (m, 4H), 7.28 (d, J = 7.6 Hz, 1H), 6.70 (s, 1H),
6.53 (d, J = 8.4 Hz, 1H), 4.94 (br s, 2H).

223

3.46. N-(4-(5-amino-1H-benzo[d]imidazol-2-yl)phenyl)-4-fluorobenzamide: Synthesized
according to general procedure for 3.7 to obtain a brown solid (0.382 g, 41%).
1
H NMR (DMSO-
d
6
, 400 MHz) δ 12.30 (br s, 1H), 10.47 (br s, 1H), 8.05-8.10 (m, 5H), 7.91 (d, J = 8.4 Hz, 2H),
7.42 (t, J = 6.8 Hz, 3H), 7.28 (br s, 1H), 6.68 (s, 1H), 6.53 (d, J = 7.6 Hz, 1H), 4.99 (br s, 2H).

3.47. N-(4-(5-amino-1H-benzo[d]imidazol-2-yl)phenyl)-4-methoxybenzamide: Synthesized
according to general procedure for 3.7 to obtain a red solid (0.489 g, 51%).
1
H NMR (DMSO-d
6
,
400 MHz) δ 12.23 (br s, 1H), 10.29 (br s, 1H), 7.99-8.05 (m, 5H), 7.91 (d, J = 8.4 Hz, 2H), 7.27
(br s, 1H), 7.10 (d, J = 8.4 Hz, 2H), 6.67 (br s, 1H), 4.97 (br s, 1H), 4.14 (br s, 1H), 3.87 (s, 3H).

3.48. 2-(4-aminophenyl)-1H-benzo[d]imidazol-5-amine: Synthesized according to general
procedure for 3.7 to obtain a red solid (0.273 g, 51%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 11.87
(br s, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 7.6 Hz, 1H), 6.63 (d, J = 8.4 Hz, 2H), 6.60 (s,
1H), 6.44 (d, J = 8.4 Hz, 1H), 5.48 (br s, 2H), 4.85 (br s, 2H).

224

3.49. N-(4-(5-(4-chlorobenzamido)-1H-benzo[d]imidazol-2-yl)phenyl)-1-naphthamide:
Synthesized according to general procedure for 3.8 to obtain a yellow solid (0.005 g, 91%).
1
H
NMR (DMSO-d
6
, 400 MHz) δ 12.84 (br s, 1H), 10.86 (br s, 1H), 10.41 (br s, 1H), 8.24 (m, 1H),
8.17 (d, J = 9.2 Hz, 1H), 8.12 (m, 1H), 8.02 (m, 4H), 7.96 (m, 1H), 7.82 (t, J = 6.8 Hz, 1H), 7.65
(m, 5H), 7.50 (t, J = 9.2 Hz, 1H), 7.41 (m, 1H), 7.30 (d, J = 8.8 Hz, 1H).

3.50. N-(4-(5-amino-1H-benzo[d]imidazol-2-yl)phenyl)-2-naphthamide: Synthesized
according to general procedure for 3.7 to obtain a red solid (0.176, 16%).
1
H NMR (DMSO-d
6
,
400 MHz) δ 12.26 (br s, 1H), 10.65 (br s, 1H), 8.64 (s, 1H), 8.05-8.14 (m, 6H), 7.98 (d, J = 8.0
Hz, 2H), 7.63-7.70 (m, 3H), 7.29 (s, 1H), 6.67 (s, 1H), 6.53 (d, J = 8.0 Hz, 1H), 4.98 (br s, 1H),
4.14 (br s, 1H).

3.51. N-(4-(5-([1,1'-biphenyl]-4-ylcarboxamido)-1H-benzo[d]imidazol-2-yl)phenyl)-1-
naphthamide: Synthesized according to general procedure for 3.8 to obtain a yellow solid (0.002
g, 68%).
1
H NMR (MeOD-d
3
, 400 MHz) δ 8.59 (s, 1H), 7.86 (d, J = 5.2 Hz, 1H), 7.31-7.37 (m,
5H), 7.15-7.26 (m, 9H), 7.01 (d, J = 10.0 Hz, 1H), 6.88-6.90 (m, 1H), 6.64 (s, 2H).
225

3.52. N-(4-(5-amino-1H-benzo[d]imidazol-2-yl)phenyl)-[1,1'-biphenyl]-4-carboxamide:
Synthesized according to general procedure for 3.7 to obtain a yellow solid (0.217 g, 23%).
1
H
NMR (DMSO-d
6
, 400 MHz) δ 12.24 (br s, 1H), 10.50 (br s, 1H), 8.10 (d, J = 8.8 Hz, 2H), 8.06
(d, J = 8.8 Hz, 1H), 7.95-7.97 (m, 2H), 7.88 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 7.2 Hz, 2H), 7.79 (d,
J = 12.4 Hz, 1H), 7.54 (t, J = 7.6 Hz, 2H), 7.46 (d, J = 6.8 Hz, 1H), 7.30 (d, J = 8.8 Hz, 1H),
6.66 (s, 1H), 6.51 (d, J = 8.8 Hz, 1H), 4.99 (br s, 1H).

3.53. N-(4-(5-amino-1H-benzo[d]imidazol-2-yl)phenyl)-3,5-dimethoxybenzamide:
Synthesized according to general procedure for 3.7 to obtain a yellow solid (0.005 g, 2%).
1
H
NMR (MeOD-d
3
, 400 MHz) δ 8.17 (s, 1H), 8.13 (s, 2H), 7.81-7.87 (m, 1H), 7.67 (dd, J = 4.8 Hz,
2H), 7.16 (br s, 2H), 6.88 (d, J = 6.8 Hz, 2H), 6.75-6.76 (m, 2H), 3.84 (s, 3H), 3.34 (s, 3H).

3.54. N-(4-(5-amino-1H-benzo[d]imidazol-2-yl)phenyl)-4-ethoxybenzamide: Synthesized
according to general procedure for 3.7 to obtain a brown solid (0.257 g, 35%).
1
H NMR (DMSO-
d
6
, 400 MHz) δ 12.28 (br s, 1H), 10.29 (br s, 1H), 8.04 (d, J = 7.6 Hz, 2H), 7.98 (d, J = 8.4 Hz,
226

2H), 7.91 (d, J = 8.4 Hz, 2H), 7.27 (br s, 1H), 7.08 (d, J = 9.6 Hz, 2H), 6.68 (br s, 1H), 6.52 (d, J
= 7.60 Hz, 1H), 4.96 (br s, 1H), 4.14 (q, J = 6.8 Hz, 2H), 1.38 (t, J = 6.8 Hz).

3.55. N-(4-(5-amino-1H-benzo[d]imidazol-2-yl)phenyl)-4-chlorobenzamide: Synthesized
according to general procedure for 3.7 to obtain a yellow solid (0.500 g, 54%).
1
H NMR (DMSO-
d
6
, 400 MHz) δ 12.22 (br s, 1H), 10.50 (br s, 1H), 7.99-8.06 (m, 5H), 7.90-7.94 (m, 3H), 7.65 (d,
J = 8.8 Hz, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 11.6 Hz, 1H), 6.66 (s, 1H), 6.51 (dd, J =
8.8 Hz, 1H), 4.99 (br s, 2H).

3.56. N-(4-(5-amino-1H-benzo[d]imidazol-2-yl)phenyl)benzamide: Synthesized according to
general procedure for 3.7 to obtain a red solid (0.474 g, 66%).
1
H NMR (DMSO-d
6
, 400 MHz) δ
12.25 (br s, 1H), 10.45 (br s, 1H), 7.92-8.06 (m, 5H), 7.55-7.63 (m, 6H), 6.66 (s, 1H), 4.98 (br s,
2H).

3.57. 4-fluoro-N-(4-(5-(4-fluorobenzamido)-1H-benzo[d]imidazol-2-yl)phenyl)benzamide:
Synthesized according to general procedure for 3.8 using 3.48 (0.200 g, 0.892 mmol) and 2.06 eq.
227

acid chloride to obtain a pink solid (0.083 g, 20%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.89 (br s,
1H), 10.55 (br s, 1H), 10.31 (br s, 1H), 8.16 (d, J = 8.4 Hz, 3H), 8.08-8.11 (m, 4H), 7.96 (d, J =
8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 10 Hz, 1H), 7.38-7.44 (m, 4H).

3.58. 4-chloro-N-(4-(5-(4-(trifluoromethyl)benzamido)-1H-benzo[d]imidazol-2-
yl)phenyl)benzamide: Synthesized according to general procedure for 3.8 to obtain a dark purple
solid (0.034 g, 58%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.84 (br s, 1H), 10.50 (br s, 2H), 8.12-
8.21 (m, 4H), 8.00 (t, J = 7.2 Hz, 4H), 7.96 (d, J = 8.4 Hz, 2H), 7.56-7.68 (m, 4H).

3.59. 3-chloro-N-(4-(5-(3-chlorobenzamido)-1H-benzo[d]imidazol-2-yl)phenyl)benzamide:
Synthesized according to general procedure for 3.8 using 3.48 (0.100 g, 0.446 mmol) and 2.06 eq.
acid chloride to obtain a tan solid (0.069 g, 31%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.78 (br s,
1H), 10.66 (br s, 1H), 10.41 (br s, 1H), 8.17 (d, J = 8.4 Hz, 3H), 8.06 (s, 2H), 7.97 (d, J = 9.6 Hz,
4H) 7.68-7.73 (m, 2H), 7.57-7.64 (m, 3H), 7.51 (d, J = 8.4 Hz, 1H).

228

3.60. 4-chlorophenyl (2-(4-(4-chlorobenzamido)phenyl)-1H-benzo[d]imidazol-5-
yl)carbamate: Synthesized according to general procedure for 3.8 to obtain a tan solid (0.024 g,
42%).
1
H NMR (MeOD-d
3
, 400 MHz) δ 8.12 (d, J = 8.4 Hz, 2H), 7.95-8.01 (m, 5H), 7.58-7.60
(m, 3H), 7.45 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 9.6 Hz, 2H).

3.61. N-(2-(4-(4-chlorobenzamido)phenyl)-1H-benzo[d]imidazol-5-yl)-[1,1'-biphenyl]-4-
carboxamide: Synthesized according to general procedure for 3.8 to obtain a yellow solid (0.023
g, 31%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 12.82 (br s, 1H), 10.47 (br s, 1H), 10.31 (br s, 1H),
8.18 (s, 1H), 8.14 (d, J = 9.6 Hz, 2H), 8.09 (d, J = 8.4 Hz, 2H), 7.95-7.99 (m, 4H), 7.84 (d, J = 8.0
Hz, 2H), 7.76 (d, J = 6.8 Hz, 2H), 7.60 (d, J = 6.8 Hz, 1H), 7.50-7.57 (m, 6), 7.41-7.44 (m, 1H).

3.62. 4-bromo-N-(2-(4-(4-chlorobenzamido)phenyl)-1H-benzo[d]imidazol-5-yl)benzamide:
Synthesized according to general procedure for 3.8 to obtain a brown solid (0.041 g, 39%).
1
H
NMR (DMSO-d
6
, 400 MHz) δ 12.82 (br s, 1H), 10.50 (br s, 1H), 10.37 (br s, 1H), 8.16 (d, J = 8.4
Hz, 3H), 7.96-8.02 (m, 6H), 7.78 (d, J = 8.4 Hz, 2H), 7.56-7.60 (m, 3H) 7.52 (d, J = 8.4 Hz, 1H).
229

3.63 N-(2-amino-5-nitrophenyl)-4-cyanobenzamide: To a solution of 4-nitro-1,2-
phenylenediamine 3.4 in (5.00 g, 32.6 mmol) and triethylamine (5.90 mL, 42.4 mmol) in
anhydrous THF (250 mL) at 0 ⁰C was added 4-cyanobenzoyl chloride 7 (5.57g, 30.2 mmol) in
THF (10 mL).
12
The reaction mixture was stirred while warming to room temperature for 12 h,
then concentrated in vacuo to obtain an orange solid (8.74 g, 95%).
1
H NMR (DMSO-d
6
, 400
MHz) δ 10.02 (br s, 1H), 8.18 (d, J = 8.0 Hz, 2H), 8.12 (d, J = 2.8 Hz, 1H), 8.04 (d, J = 8.4 Hz,
2H), 7.95 (dd, J = 6.0 Hz, 1H), 6.81 (d, J = 9.6 Hz, 1H), 6.71 (br s, 2H).
13
C NMR (DMSO-d
6
,
100 MHz) δ 165.31, 151.34, 138.81, 135.63, 132.69, 129.37, 124.32, 121.03, 118.92, 114.23.

3.64. N-((4-chlorophenyl)sulfonyl)-4-(5-nitro-1H-benzo[d]imidazol-2-yl)benzamide: A
mixture of carboxylic acid 3.34 (200 mg, 0.71 mmol), 4-chlorobenzenesulfonamide (135 mg,
0.71 mmol), and DMAP (86 mg, 0.71 mmol) was stirred 0 ⁰C for 30 min and then was added
EDCI (135 mg, 0.71 mmol).
13
The reaction mixture was stirred while warming to room
temperature for 24 h then concentrated in vacuo onto silica gel. Purification by silca gel
chromatography eluting with 10% MeOH in ethyl acetate provided a yellow solid (116 mg, 36%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 13.92 (br s, 1H), 8.53 (br s, 1H), 8.38 (br s, 1H), 8.21 (d, J =
7.6 Hz, 2H), 8.15 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 6.8 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.48 (dd,
230

J = 6.4 Hz, 2H).
13
C NMR (DMSO-d
6
, 100 MHz) δ 169.71, 145.32, 143.27, 143.17, 141.54,
134.96, 130.79, 129.39, 129.28, 128.16, 126.84.

3.65. N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(5-nitro-1H-benzo[d]imidazol-2-yl)benzamide:
Synthesized according to general procedure for 3.64 to obtain a yellow solid (0.021 g, 2%).
1
H
NMR (MeOD-d
3
, 400 MHz) δ 8.56 (d, J = 1.6 Hz, 1H), 8.51 (br s, 1H), 8.22 (dd, J = 6.4 Hz, 2H),
8.19 (d, J = 8.8 Hz, 3H), 8.11 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 7.6 Hz,
1H).

3.66. 4-(5-nitro-1H-benzo[d]imidazol-2-yl)-N-((3-nitro-4-((2-
(phenylthio)ethyl)amino)phenyl)sulfonyl)benzamide: Synthesized according to general
procedure for 3.64 using a sulfonamide coupling partner which was prepared following known
procedures
10,14
to obtain an orange solid (0.069 g, 4%).
1
H NMR (DMSO-d
6
, 400 MHz) δ 13.72
(br s, 1H), 8.54-8.57 (m, 2H), 8.17-8.20 (m, 2H), 8.08 (d, J = 7.6 Hz, 2H), 7.97 (s, 1H), 7.91 (d, J
= 9.2 Hz, 1H), 7.73 (d, J = 9.2 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.32 (t, J = 8.4 Hz, 1H), 7.22 (d,
J = 8.0 Hz, 1H), 7.01 (d, J = 10.4 Hz, 1H), 3.61 (q, 2H), 3.28 (t, J = 7.2 Hz).
13
C NMR (DMSO-
d
6
, 100 MHz) δ 193.15, 169.29, 149.96, 148.81, 146.75, 145.79, 144.40, 142.98, 141.57, 135.49,
135.40, 133.45, 130.01, 129.55, 129.37, 129.23, 126.69, 126.62, 126.24, 124.35, 118.26, 113.96.

231

3.4. Chapter 3. References.
1
McDonald, E.R., III; El-Deiry, W.S. Mammalian cell death pathways: intrinsic and extrinsic.
Death Recept. Cancer Ther. 2005, 1-41.

2
Budihardjo, I.; Oliver, H.; Lutter, M.; Luo, X.; Wang, X. Biochemical pathways of caspase
activation during apoptosis. Annu. Rev. Cell Dev. Biol. 1999, 15, 269-290.

3
Yoshino, T.; Shiina, H.; Urakami, S.; et. al. Bcl-2 expression as a predictive marker of hormone-
refactory prostate cancer treated with taxane-based chemotherapy. Clin. Cancer Res. 2006, 12,
6116-6124.

4
Minn, A.J.; Rudin, C.M.; Boise, L.H.; Thompson, C.B. Expression of Bcl-xL can confer a
multidrug resistance phenotype. Blood. 1995, 86, 1903-1910.

5
Fesik, S.W. Promoting apoptosis as a strategy for cancer drug discovery. Nature Rev. Cancer.
2005, 5, 876-885.

6
Boatright, K.M.; Salvesen, G.S. Mechanisms of caspase activation. Curr. Opin. Cell Biol. 2003,
15, 725-731.

7
Zeitlin, B.D.; Zeitlin, I.J.; Nor, J.E. Expanding Circle of Inhibition: Small-Molecule Inhibitors of
Bcl-2 as Anticancer Cell and Antiangiogenic Agents. J. Clinic. Oncol. 2008, 26, 4180-4188.

232

Chapter 4. Palladium-assisted reaction methodologies.
4.1. Introduction. Palladium catalyzed reactions.

4.1.1. N-heterocyclic carbene ligands in palladium catalysis.

After the synthesis of free carbenes was first investigated by Arduengo, a later series of
work conducted by Herrmann demonstrated that N-heterocyclic carbenes (NHCs) exhibit similar
properties to electron-rich organophosphanes, particularly in their coordination to metals.
1,2
Due
to these important findings, N-heterocyclic carbenes (NHCs) have since become a popular ligand
class for transition metals, and in addition to the abundance of research regarding the
coordination chemistry of these ligands, much experimental work has been conducted regarding
practical applications of NHCs.
3-11
For example, these ligands have shown promise to be utilized
as catalysts in Heck coupling reactions.
12-20
It should therefore be possible to use an NHC ligand
to create a robust catalyst which could withstand high temperature reaction conditions. Thus,
such a ligand could be comprised of a benzimidazolium carbene tethered to pyridine by a short
alkyl group. In particular, this type of ligand should have application in a Heck-type coupling
reaction.
The Heck reaction, which follows the generally accepted mechanism shown in Figure
4.1, is the palladium catalyzed cross-coupling of an aryl halide and an olefin.
12
The catalytic cycle
initiates with oxidative addition of an organic halide to a Pd(0) complex. This is followed by
olefin insertion to form a Pd(II) alkyl complex, which subsequently undergoes β-hydride
elimination to form the new C-C bond. Use of a base then regenerates the Pd(0) complex and
allows for continuation of the catalytic cycle.
233

Figure 4.1 – Mechanism of a typical Heck cross-coupling reaction.
12

This reaction is a versatile and powerful carbon-carbon bond forming reaction and has
been widely used by chemists in a variety of syntheses.
21-27
For example, many successful Heck
reactions have been used in natural products syntheses by Overman and Shibasaki.
28-31
Although
most applications of the Heck reaction have focused on a substrate scope which encompassed
olefins containing electron-withdrawing groups as a coupling partner, the utility of this reaction
for olefins possessing an electron-donating group have been under-developed.
32-37
The cyclic enol
ether substrate inverts the polarization of the double bond with respect to the traditional Heck
reaction, and therefore is an interesting substrate to further expand the scope of this reaction.
32,33

The use of olefins containing an electron-donating group has potential synthetic utility as well.
For example, the Heck reaction employing cyclic enol ethers as a coupling partner could serve as
a route to form biologically important glycosides and the cyclic enol ether motif is one found in
numerous toxins and other natural products which display bioactivity.
38,39
However, despite this
potential utility, the use of the Heck reaction involving cyclic enol ethers has been relatively
234

under-explored. Most prior work involving cyclic enol ethers focused on the use of 2,3-
dihydrofuran.
40-43
Reactions employing 3,4-dihydro-2H-pyran (DHP) as a coupling partner
provided only modest yields and mixtures of regioisomers, with only one enantioselective
example reported.
32-37
Therefore, a more versatile catalyst system for the Heck reaction using an
NHC-ligand complexed to palladium would be useful to develop because NHCs are known to be
good σ-donors, and could thereby enhance the rate of oxidative addition.
44
In addition, the steric
bulk of a benzimidazolium-pyridine ligand could assist with reductive elimination, which would
also be a beneficial property for a catalyst of Heck-type reactions.
4.1.2. Multicomponent Strecker synthesis of α-aminonitriles.
The Strecker reaction, the hydrocyanation of imines, has been a widely studied reaction
since its first report in 1850 and is shown in its classical form in Scheme 4.1.
45
This efficient,
multicomponent reaction is useful as a method to synthesize α-amino acids from α-aminonitriles.
The α-aminonitrile is typically formed by the condensation of an aldehyde or ketone with an
amine and a cyanide source. Subsequent hydrolysis of this α-aminonitrile product provides ready
access to a variety of amino acids. Since it is possible to create a versatile amount of
intermediates with the multicomponent Strecker reaction, it can be utilized as an efficient method
for the preparation of a library of biologically relevant molecules.
46-52

Scheme 4.1 – Classical Strecker synthesis to form α-amino acids from α-aminonitriles.
45

This simple and economical reaction has been the focus of a wide body of research in the
years following its discovery and many improvements to the original protocol have been made.
235

Successful examples of the Strecker reaction have been reported involving a wide variety of
catalysts, including tranisiton metal catalysis using such metals as iron, titanium, or zirconium.
53-
58
Lewis bases and Schiff bases have been demonstrated to promote reactivity and Lewis acids
such as gallium triflate have proven to be of great utility too.
59-62
Recently, Vachal and Jacobsen
have employed chiral organocatalysts for a metal-free asymmetric Strecker reaction.
63-68

However, despite the considerable amount of interest in this reaction, there is still a need
to develop additional efficient and practical methods to further advance the utility of the
multicomponent Strecker reaction. While many one-pot multicomponent procedures of this
reaction involving aldehydes and amines have been developed, the Strecker synthesis applied to
ketones and aliphatic amines still remains a more difficult reaction. Typically, with these more
difficult substrates it is necessary to either use a stepwise synthetic sequence which requires prior
formation of the imine or high-pressure reaction conditions.
69-71
Although there have been reports
of multicomponent Strecker reactions promoted by Lewis acids; such as scandium triflate,
vanadyl triflate, ytterbium triflate, lithium perchlorate, zinc halides, and montmorillonite; the use
of these expensive reagents with extended reaction times, harsh conditions, fast hydrolysis, and
tedious workup procedures can lead to the generation of large amounts of waste and limits the
application of these methodologies.
72-77

4.2. Regioselective Heck coupling of aryl halides and dihydropyran using
a NHC-Pd catalyst.

The NHC-ligand precursor 4.5 was prepared as shown in Scheme 4.2. First, pyridinyl
benzimidazole compound 4.3 was prepared by N-alkylation of benzimidazole 4.1 with 2-
bromomethyl pyridine 4.2 in the presence of KOH in THF to provide 4.3 in high yield.
Subsequent N-methylation of 4.3 with iodomethane while refluxing in THF for 12 h then
236

provided 4.4, also in high yield. Synthesis of ligand 4.4 was previously reported; however, this
newly developed synthetic scheme was shown to be a more efficient route.
78

Reagents and conditions: (a) KOH, THF, 100°C, 48 h, 81%; (b) iodomethane, THF, 100°C, 16 h,
80%;(c) (i) Ag
2
O, CH
2
Cl
2
, rt, dark, 4 h; (ii) Pd(OAc)
2
, CH
3
CN, rt, 24 h, 38%.

Scheme 4.2 - Synthetic route for preparation of NHC-ligand Pd-catalyst complex.
Synthesis of the NHC-ligand was then followed by metal-complexation to form the
catalyst, as illustrated in Scheme 4.2. Compound 4.4 was reacted with silver (I) oxide in
anhydrous dichloromethane at room temperature while excluding light to afford the silver NHC
complex. Metal exchange with Pd(OAc)
2
in acetonitrile then provided the palladium(II)/NHC
ligand complex 4.5 as a pale orange solid in 38% yield over two steps. The structure was
confirmed to have coordinated to palladium by the complete disappearance of the imidazole
proton (9.14 ppm) of 4.4, in addition to a downfield shift (δ = 8.48 ppm to 8.98 ppm) of the
pyridine ortho-proton
1
H-NMR resonance. Broadening of the methylene at δ = 5.96, which was
observed in complex 4.5, also indicated successful coordination of the NHC-ligand to the metal
center.
After preparing palladium complex 4.5, we then turned our attention to the Heck reaction
of a cyclic enol ether to investigate the cross-coupling of 4-iodoanisole with DHP. Our initial
237

goal was to determine if yields and regioselectivity of this particular reaction could be improved
in comparison to earlier work.
32-37
As shown in Table 4.1, the possibility of using a catalytic
system comprised of Pd(OAc)
2
and commercially available amine ligands, such as
phenanthroline, was evaluated but under these conditions a mixture of regioisomers 4.8 and 4.9
was obtained with poor to modest yields (entries 1 - 3). When using a combination of Pd(OAc)
2

and 1,10-phenanthroline or 2,9-dimethylphenanthroline the catalyst and ligand were pre-mixed in
DMF at room temperature for 30 min. before the addition of substrates. However, when 4.5 was
used as the catalyst, 4.8 was obtained as the exclusive product (entries 4-8), and as seen
previously in reactions of cyclic enol ether substrates, arylation was observed only at the α-
carbon.
32,33
The use of K
2
CO
3
as base in this initial screening of reaction conditions provided the
best isolated yield (entry 5).
Previous reports had demonstrated that an oxygen atom in the olefin substrate could
electronically assist with favorable coordination of the Pd
II
center to the olefin coupling partner
and therefore promote reactivity.
44
In earlier reports, the double bond isomerization of the Heck
product had been attributed to high temperature reaction conditions; however, it was determined
by Jeffery and David that the selectivity of this reaction could be directed through the use of an
appropriate catalytic system.
36
With the use of NHC-ligand Pd-catalyst complex 4.5 isolation of
the arylated vinylic ether as the sole product was possible. These results thus indicated that this
particular combination of catalytic system and reaction conditions were able to impart
regioselectivity. This selectivity may in part be due to the unsymmetric structure of the designed
NHC-ligand palladium complex 4.5 or its ability to act as an internal base.

238

Table 4.1. Effect of catalyst and base on the cross-coupling reactions of 4-iodoanisole and DHP
a

a) All reactions were carried out with 4-iodoanisole (0.5 mmol), DHP (6 mmol), base (0.75
mmol), and Pd-L (5 mol%) in solvent (1 mL) under Argon atmosphere. b) Isolated yield. c) phen:
1,10-phenanthroline, dmphen: 2,9-dimethylphenanthroline.

After this initial screening of conditions demonstrated that 4.5 and K
2
CO
3
were the best
combination of catalytic complex and base for reaction selectivity, the role of solvent and
temperature was then evaluated, as presented in Table 4.2. Reactions were incomplete and low-
yielding (entries 2 - 4) when toluene, benzene, or THF were used as solvent. However, use of a
polar aprotic solvent such as DMF, provided the desired product 4.8 in 80 % yield (entry 1).
Formation of the isomeric side product 4.9 was not detected under any of these reaction
conditions. Regardless of the base used, the cross-coupling reaction was not efficient at
temperatures lower than 100 ⁰C (entries 5 and 6). Using this information, it was determined that
the use of a polar aprotic solvent, such as DMF, in the presence of K
2
CO
3
base at a reaction
temperature of 100 ⁰C would provide optimal reaction conditions. This set of reaction conditions
239

was then used to further evaluate the substrate scope of the cross-coupling of aryl halides with
DHP.
Table 4.2. Effect of solvent and temperature on the cross-coupling reactions of 4-iodoanisole and
DHP in the presence of Pd-ligand complex 4.5
a

a) All reactions were carried out with 4-iodoanisole (0.5 mmol), DHP (6 mmol), K
2
CO
3
(0.75
mmol), and 4.5 (5 mol%) in solvent (1 mL) under Argon atmosphere. b) Isolated yield.

The use of less active aryl halides in the cross-coupling reaction was investigated as well
to diversify the scope of the reaction and broaden the utility of this methodology. However, as
shown in Table 4.3, when either aryl bromides (entries 2 and 4) or aryl chlorides (entry 5) were
used as coupling partners, a reduction in yields was observed, in comparison to reactions
employing aryl iodides as coupling partners. This result is consistent with the fact that the
oxidative addition of aryl bromides and aryl chlorides to Pd
0
is more difficult due to the greater
bond strengths of the Ph-X bond in aryl bromides and aryl chlorides, respectively, compared to
that of aryl iodides.
79
Therefore, these developed reaction conditions are best-suited for the
coupling of aryl iodides to DHP.
240

Table 4.3. Effect of arylhalide on the cross-coupling reactions with DHP in the presence of Pd-
ligand complex 4.5
a

a) All reactions were carried out with aryl halide (0.5 mmol), DHP (6 mmol), K
2
CO
3
(0.75
mmol), and 4.5 (5 mol%) in solvent (1 mL) under Argon atmosphere. b) Isolated yield

The functionality of the aryl iodide coupling partner was then varied to investigate the
range of halide substrates amenable to the optimized reaction conditions.

As shown in Table 4.4,
the cross-coupling reaction proceeded well with electron donating and neutral substrates (entries
1 and 2). Reactions of ortho-, meta-, and para-methyl or methoxy phenyl iodide with DHP took
place to provide the desired products 4.11 (62 - 75%) and 4.12 (58 - 62%), respectively, in
modest to good yields. However, halide coupling partners substituted with an electron
withdrawing group were not well-tolerated, as seen in the reactions with trifluoromethyl, cyano,
and acetyl phenyl iodides (entries 3 - 5), which showed little reactivity. Removal of unreacted
starting materials proved laborious and further contributed to low isolated yields (< 16%).
241

Therefore, aryl iodides including an electron donating or neutral substituent proved to be best
coupling partners for the Heck reaction with DHP under these conditions.
Table 4.4 Effect of aryl halide on the cross-coupling reactions with DHP in the presence of Pd-
ligand complex 4.5
a

a) All reactions were carried out with aryl iodide (0.5 mmol), DHP (6 mmol), K
2
CO
3
(0.75
mmol), and 4.5 (5 mol%) in solvent (1 mL) under Argon atmosphere. b) Isolated yield

Additionally, sterically hindered aryl iodide substrates reacted smoothly, but for the most
hindered aryl iodide coupling partner an increase in temperature was necessary to drive the
242

reaction to completion. The coupling reaction of 2,6-dimethyl iodobenzene with DHP at 150
o
C
for 48 hours, shown in Scheme 4.3, proceeded to give the corresponding cross-coupling
compound 4.17 in 55 % yield.

Scheme 4.3 - Coupling reaction with hindered halides.
This methodology can also be extended for application to nitrogen-containing
heterocycles as coupling partners as well, demonstrated by the successful coupling of 3-
iodopyridine with DHP to afford 4.19 in 61% yield (Scheme 4.4). The utility of this particular
example is important due to the use of heterocyclic compounds as building blocks for natural
products and biologically active molecules.

Scheme 4.4 - Coupling reaction with heterocyclic halide.
NHC-pyridinyl ligand-palladium complex 4.5 was therefore synthesized successfully and
it was possible to demonstrate its utility in regioselective Heck reactions with DHP. Synthesis of
the catalyst is straight-forward and efficient, which indicates the potential utility of this complex
in additional palladium-catalyzed reactions. By developing a methodology for a previously
243

under-explored cross coupling reaction, which provides a substituted cyclic enol ether product in
good yield and in a highly regioselective manner, the utility of this methodology has been
demonstrated. Further application of this methodology in broader synthetic schemes may be of
future value.
4.3. Strecker reaction using an NHC-amidate-Pd complex.
It was considered that a palladium source could also act as a Lewis acid to catalyze a
multicomponent reaction to synthesize α-aminonitriles. Previously, it had been found that an N-
heterocyclic carbene (NHC) amidate palladium (II) complex 4.20, shown in Figure 4.2, was able
to act as an effective catalyst of asymmetric boron-Heck type carbon-carbon coupling reactions
under mild conditions.
80
This prior work also indicated that catalysis was not inhibited by
coordination of water to the metal center because the presence of strongly electron donating
groups such as the amidate nitrogen and oxygen, as well as the NHC, could increase the electron
density of palladium and allow for only a weak interaction between the electrophilic metal and
water. Additionally, an ester moiety on the ligand could function as a portable chelating group to
easily form an open site on palladium. With this in mind, a new NHC-amidate palladium
complex 4.21, shown in Figure 4.2, was prepared to evaluate its potential utility in the one-pot
multicomponent Strecker reaction to synthesize α-aminonitriles due to the potential of this
complex to act as a Lewis acid that could tolerate water released in the Strecker reaction.

Figure 4.2 - New NHC-amidate palladium complexes.
244

The NHC-amidate palladium (II) complex was prepared by Dr. Chan Pil Park in Prof.
Kyung Jung’s laboratory, as illustrated in Scheme 4.5. Compound 4.22, prepared by amidation of
valine methyl ester and bromoacetyl bromide, was treated with benzimidazole in the presence of
base to provide 4.23. N-alkylation efficiently gave 4.24, and the palladium catalytic complex
4.21 was then obtained in a two-step sequence involving formation of the carbene with silver(I)
oxide and subsequent metal exchange.

Reagents and conditions: (a) benzimidazole, KOH, DMF, rt, 81%; (b) iodomethane, THF, 80°C, 89%; (c)
(i) Ag
2
O, CH
2
Cl
2
, rt, dark; (ii) PdCl
2
(CH
3
CN)
2
, CH
3
CN, rt, 81%.

Scheme 4.5 – Synthesis of NHC-amidate palladium complex 4.21 for use in Strecker synthesis.
An initial screening of palladium catalysts was first conducted, as shown in Table 4.5.
All reactions were conducted in dichloromethane solvent at room temperature with TMSCN as
the cyanide source and in the presence of sodium sulfate dessicant. In the absence of a palladium
catalyst, reactivity was poor and low conversion was observed for the reaction of acetophenone
with benzophenone (entry 1). The use of PdCl
2
gave low conversion for reactions involving
either a ketone or aldehyde (entries 2 and 5); however, an improvement in reaction conversion
(>95%) was observed when reactions were conducted in the presence of 3 mol % loading of
245

NHC-palladium complex 4.21 using either the ketone or aldehyde as the carbonyl source (entries
4 and 6).
Table 4.5 - Screening of Palladium Source and Catalyst Loading
a

a
To a mixture of palladium catalyst, sodium sulfate (100 mg, 0.7 mmol), 4.25 (0.2 mmol), and
4.26 (0.2 mmol) in 1 mL of CH
2
Cl
2
in a Schlenk tube was added dropwise 4.27 (0.4 mmol). The
mixture was stirred for 24 h at room temperature.
b
Conversion.
For reactions of aldehydes, both PdCl
2
and 4.21 were found to be suitable catalysts.
However, due to its stability to water released during the course of the reaction, 4.21 appeared to
promote better reactivity than PdCl
2
for reactions employing ketones as the carbonyl source.
Therefore, palladium complex 4.21 was evaluated for application to a wider substrate scope for
the multicomponent Strecker reaction, as indicated in Table 4.6. First, reactions were carried out
using aliphatic and aromatic aldehydes and amines. In all cases, regardless of differences in the
electronic structure of the aldehyde substrates, high yields were observed. For example, an
electron-withdrawing group present on the aldehyde (entries 2 and 9) was compatible with these
reaction conditions, as were heteroatom-containing aldehydes (entries 3-5 and 10-12). Aliphatic
aldehydes (entries 7,14, and 15) were also suitable substrates. Similar to prior reports of the
246

Strecker synthesis catalyzed by other Lewis acids, both aromatic (entries 1-7) and aliphatic
(entries 8-15) amines were compatible with these reaction conditions and gave the desired
product in good to excellent yields.
72-77
However, it was found that reactions conducted without
the use of a drying agent proceeded slowly and low conversion to the α-aminonitrile product was
observed (not shown).
Table 4.6 – Strecker Reaction of Aldehydes and Amines in the Presence of Palladium Catalyst
a

a
To a mixture of palladium catalyst, sodium sulfate (100 mg, 0.7 mmol), 4.29 (0.2 mmol), and
4.30 (0.2 mmol) in 1 mL of CH
2
Cl
2
in a Schlenk tube was added dropwise 4.27 (0.4 mmol). The
mixture was stirred for 24 h at room temperature.
b
Isolated yields.
Due to the encouraging reactivity of palladium complex 4.21 as a Lewis acid catalyst to
promote the Strecker reaction of a variety of aldehydes, its ability to be utilized in reactions
employing ketones was then investigated, as shown in Table 4.7. Good reactivity was observed
for reactions of an aromatic amine and a ketone containing an electron-donating substitutent
(entry 2) in the presence of complex 4.21. Although an electron-withdrawing bromine substituent
was tolerated (entry 3), a nitro group was found to not be a feasible substrate (entry 4). The
247

particularly low yield with this substrate may be attributed to low conversion to the imine
intermediate, and consequently, little formation of the desired product. Generally, when aniline
was used as the amine, the desired α-aminonitrile products were obtained in higher conversion
than reactions conducted using benzylamine. In this case again, electron-withdrawing
substituents on the ketone substrate were not well-tolerated. However, electron-donating (entry
8) and heteroatom (entry 10) functionality allowed for modest reactivity to occur.
Table 4.7 – Strecker Reaction of Ketones and Amines in the Presence of Palladium Catalyst
a

a
To a mixture of palladium catalyst, sodium sulfate (100 mg, 0.7 mmol), 4.47 (0.2 mmol), and 4.0
(0.2 mmol) in 1 mL of CH
2
Cl
2
in a Schlenk tube was added dropwise 4.27 (0.4 mmol). The
mixture was stirred for 24 h at room temperature. If necessary, compounds were purified by silica
gel column chromatography eluting with a gradient system of hexanes/ethyl acetate.
b
Isolated
yields.
248

It has been therefore been demonstrated that NHC-amidate ester palladium (II) complex
4.21 can act as a Lewis acid catalyst to promote one-pot multicomponent Strecker reactions for
the synthesis of α-aminonitriles. Of particular note is that the application of 4.21 in reactions
using ketone substrates allowed for the formation of α-aminonitrile products containing a
quaternary carbon. This methodology is useful due to the simplicity of the procedure involved
and its efficiency.
4.4. Experimental.
4.4.1. Heck coupling methodology.
All commercially available reagents and solvents were used as received by Aldrich and
Acros chemical without further purification.
1
H and
13
C spectra were recorded on a 250 and
63MHz Bruker or 400 and 100MHz Varian instrument. Thin-layer chromatography (TLC) was
performed using commercially prepared 60 mesh silica gel plates visualized with short-
wavelength UV light (254 nm). Silica gel 60 (9385, 230-400 mesh) was used for column
chromatography. The isolated yields are the average of two runs. MS analysis was performed
using a Thermo Scientific DSQ II GS-MS (ESI) instrument (He gas, 25 minute run time, the first
5 minutes at 40 degrees Celsius, an increase of 15 degrees/min for the next 15 minutes, and the
final 5 minutes at 250 degrees Celsius, with a constant flow of 1.5 mL/min.).

249

General Procedure for the Synthesis of Palladium (II) Complex

4.3: To a mixture of 2-(Bromomethyl)pyridine HBr (20 mmol) and benzimidazole (20 mmol) was
added THF (100 mL). Then, potassium hydroxide (80 mmol) was added and the solution was
heated to 100 ⁰C for 2 days. The reaction mixture was cooled to room temperature and solvent
was removed in vacuo. The crude product was diluted with DCM (50 mL) and washed with
water (2 x 50 mL). The organic layer was dried over sodium sulfate and filtered. Solvent was
removed in vacuo to obtain 4.3 (3.39 g, 81%) as a brown solid.
1
H NMR (250 MHz, CDCl
3
) δ:
8.61 (d, J = 4.8 Hz, 1H), 8.07 (s, 1H), 7.83-7.86 (m, 1H), 7.56-7.64 (m, 2H), 7.20 – 7.36 (m, 3H),
6.93 (d, J = 12.5 Hz), 5.50 (s, 2H).
13
C NMR (62.5 MHz, CDCl
3
) δ: 155.7, 150.0, 144.0, 143.6,
137.4, 134.2, 123.4, 123.2, 122.5, 121.2, 120.7, 110.1, 50.74. GC-MS m/z calc’d: 207.9, found:
209.2.

4.4: To a solution of 4.3 (5 mmol) in THF (100 mL) was added iodomethane (15 mmol). The
reaction mixture was heated to 100 ⁰C for 16 h. The reaction mixture was then allowed to cool to
room temperature and then gravity filtered. Solvent was evaporated under nitrogen to obtain 4.4
(1.40 g, 80%) as a white solid.
1
H NMR (250 MHz, CDCl
3
) δ: 9.14 (s, 1H), 8.48-8.49 (m, 1H),
7.87-7.91 (m, 1H), 7.73-7.81 (m, 2H), 7.57-7.71 (m, 3H), 7.27-7.31 (m, 1H), 5.98 (s, 2H), 4.26 (s,
250

3H).
13
C NMR (62.5 MHz, CDCl
3
) δ: 151.9, 149.7, 142.6, 137.7, 131.8, 131.5, 127.3, 127.2,
124.0, 123.8, 114.3, 112.6, 52.30, 34.10.

4.5: To a solution of 4.4 (0.6 mmol) in dry DCM (20 mL) was added silver (I) oxide (0.3 mmol)
and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was then
gravity filtered and dried under nitrogen to obtain a silver complex as a white solid. The silver
complex (0.4 mmol) was then suspended in a solution of acetonitrile (20 mL) in a foil-covered
RB flask. To the reaction mixture was then added palladium (II) acetate (0.4 mmol) and the
reaction mixture was stirred at room temperature for 24 h. The reaction mixture was then gravity
filtered and the filtrate was concentrated in vacuo to obtain 5 (100 mg, 38% over two steps) as an
orange solid.
1
H NMR (250 MHz, CDCl
3
) : 8.98-9.02 (m, 1H), 7.82-7.90 (m, 1H), 7.59 (d, J =
10.0 Hz, 1H), 7.49-7.55 (m, 1H), 7.32-7.45 (m, 4H), 5.96 (br s, 2H), 4.06 (s, 3H), 2.02 (s, 6H).
13
C NMR (62.5 MHz, CDCl
3
)  178.1, 153.5, 153.4, 139.8, 134.5, 132.7, 125.0, 124.9, 124.4,
124.1, 110.9, 110.4, 51.40, 33.40, 22.60.
GENERAL PROCEDURE FOR SYNTHESIS OF SUBSTITUTED DI-HYDRO PYRANS
An oven-dried resealable Schlenk flask was evacuated and filled with argon, then were
added 4-iodoanisole (117 mg, 0.5 mmol), 3,4-dihydro-2H-pyran (0.55 mL, 6 mmol), potassium
carbonate (104 mg, 0.75 mmol), dimethylformamide (1 mL), palladium complex 4.5 (22 mg, 0.05
mmol). The reaction mixture was stirred at 100 ⁰C. After 48 h the solution was then allowed to
cool to room temperature. Ethyl acetate (20 mL) was added to the reaction mixture and then the
reaction mixture was washed with water (3 x 10 mL). The organic layer was dried over sodium
sulfate. After filtration, solvent was evaporated and purified by column chromatography
251

(hexanes/ethyl acetate, 19:1), to afford 2-(4-methoxy-phenyl)-3,4-dihydro-2H-pyran (76 mg,
80%) as a light orange oil. NMR yields were obtained using toluene as an internal standard.

4.8. 2-(4-methoxyphenyl)-3,4-dihydro-2H-pyran: Synthesized and purified according to the
general procedure to afford a light orange oil (76 mg, 80%).
1
H NMR (250 MHz, CDCl
3
) δ: 7.28
(d, J = 10. 0 Hz, 2H), 6.98 (d, J = 10.0 Hz, 2H), 6.50 (d, J = 5.0 Hz, 1H), 4.79 (d, J = 7.5 Hz, 1H),
4.75 (m, 1H), 3.80 (s, 3H), 2.15-2.30 (m, 2H), 1.85-2.10 (m, 2H).
13
C NMR (62.5 MHz, CDCl
3
)
δ: 159.0, 144.2, 128.6, 127.2, 113.7, 100.5, 76.73, 55.22, 30.09, 20.38. GC-MS m/z calc’d:
190.10, found: 189.97.

4.9. 2-(4-methoxyphenyl)-3,6-dihydro-2H-pyran:
1
H NMR (250 MHz, CDCl
3
) δ: 7.30 (d, J =
10.0 Hz, 2H), 6.88 (d, J = 10.0 Hz, 2H), 5.76-5.96 (m, 2H), 4.51 (dd, J = 10.0 Hz, 2H), 4.35 (m,
1H), 3.80 (s, 3H), 2.16-2.46 (m, 2H).

4.10 2-phenyl-3,4-dihydro-2H-pyran: Synthesized and purified according to the general
procedure to afford a yellow oil (41 mg, 51 %).
1
H NMR (250 MHz, CDCl
3
) δ: 7.16-7.51 (m,
252

5H), 6.45 (d, J = 7.5 Hz, 1H), 4.75-4.77 (m, 1H), 4.65-4.76 (m, 1H), 2.05-2.24 (m, 2H), 1.75-2.03
(m, 2H).
13
C NMR (62.5 MHz, CDCl
3
) δ: 144.3, 141.4, 128.5, 127.7, 127.4, 127.3, 126.0, 100.8,
77.19, 30.42, 20.44. GC-MS m/z calc’d: 160.1, found: 160.0.

4.11a. 2-(o-tolyl)-3,4-dihydro-2H-pyran: Synthesized according to general procedure to provide
a yellow oil (62 mg, 71%).
1
H NMR (250 MHz, CDCl
3
) : 7.43 (m, 1H), 7.17-7.26 (m, 3H), 6.56
(d, J = 7.5 Hz, 1H), 5.00 (dd, J = 10.0 Hz, 1H), 4.80 (m, 1H), 2.35 (s, 3H), 2.20-2.32 (m, 2H),
1.84-2.12 (m, 2H).
13
C NMR (62.5 MHz, CDCl
3
)  144.7, 140.0, 134.6, 130.4, 127.5, 126.3,
125.6, 100.6, 74.37, 29.29, 20.93,18.95. GC-MS m/z calc’d: 174.1, found: 173.9.

4.11b. 2-(m-tolyl)-3,4-dihydro-2H-pyran: Synthesized according to general procedure to afford
a yellow oil (65 mg, 75%).
1
H NMR (250 MHz, CDCl
3
) : 7.45-7.48 (m, 1H), 7.38-7.42 (m, 1H),
7.31-7.35 (m, 1H), 7.17-7.27 (m, 1H), 6.61 (d, J = 7.5 Hz, 1H), 4.87-4.89 (m, 1H), 4.83-4.87 (m,
1H), 2.44 (s, 3H), 2.26-2.36 (m, 2H), 1.96-2.16 (m, 2H).
13
C NMR (62.5 MHz, CDCl
3
)  144.1,
137.9, 128.5, 127.9, 126.5, 124.2, 122.9, 100.5, 77.06, 30.22, 21.35, 20.29. GC-MS m/z calc’d:
174.1, found: 174.0

253

4.11c. 2-(p-tolyl)-3,4-dihydro-2H-pyran: Synthesized according to general procedure to afford a
yellow oil (54 mg, 62%).
1
H NMR (250 MHz, CDCl
3
) : 7.48 (d, J = 10.0 Hz, 2H), 7.22 (d, J =
10.0 Hz, 2H), 6.53 (d, J = 7.5 Hz, 1H), 4.82 (m, 1H), 4.77 (m, 1H), 2.35 (s, 3H), 2.13-2.25 (m,
2H), 1.90-2.08 (m, 2H). ).
13
C NMR (62.5 MHz, CDCl
3
)  140.1, 137.0, 129.0, 128.9, 126.8,
125.9, 92.47, 77.50, 32.67, 22.49, 21.10. GC-MS m/z calc’d: 174.1, found: 174.0.

4.12a. 2-(2-methoxyphenyl)-3,4-dihydro-2H-pyran: Synthesized according to general
procedure to afford a yellow oil (59 mg, 62 %).
1
H NMR (250 MHz, CDCl
3
) : 7.43 (d, J = 10
Hz, 1H), 7.22-7.35 (m, 1H), 6.98 (t, J = 7.5 Hz, 1H), 6.88 (d, J = 7.5 Hz, 1H), 6.66 (d, J = 7.5 Hz,
1H), 5.21 (d, J = 7.5 Hz, 1H), 4.74-4.80 (m, 1H), 3.84 (s, 3H), 1.94-2.30 (m, 2H), 1.72-1.88 (m,
2H).
13
C NMR (62.5 MHz, CDCl
3
)  158.1, 144.5, 128.1, 128.0, 126.4, 122.5, 111.0, 100.6,
72.47, 56.26, 29.58, 20.50. GC-MS m/z calc’d: 190.1, found: 190.0.

4.12b. 2-(3-methoxyphenyl)-3,4-dihydro-2H-pyran: Synthesized according to the general
procedure to afford an orange oil (71 mg, 75%).
1
H NMR (250 MHz, CDCl
3
) : 7.24-7.31 (m,
1H), 6.92-6.96 (m, 1H), 6.82-6.86 (m, 2H), 6.53 (d, J = 7.5 Hz, 1H), 4.82-4.85 (m, 1H), 4.75-4.81
(m, 1H), 3.02 (s, 3H), 2.16-2.32 (m, 2H), 1.84-2.10 (m, 2H).
13
C NMR (62.5 MHz, CDCl
3
) 
254

159.8, 144.2, 129.5, 118.3, 113.2, 111.5, 100.7, 77.05, 55.29, 30.38, 20.30. GC-MS m/z calc’d:
190.1, found: 190.0.

4.13a. 2-(2-(trifluoromethyl)phenyl)-3,4-dihydro-2H-pyran: Synthesized according to the
general procedure to afford a yellow oil (17 mg, 15%).
1
H NMR (250 MHz, CDCl
3
) : 7.62 (d, J
= 10.0 Hz, 2H), 7.47 (d, J = 10.0 Hz, 2H), 6.54 (d, J = 7.5 Hz, 1H), 4.90 (d, J = 7.5 Hz, 1H),
4.75-4.84 (m, 1H), 1.65-2.15 (m, 2H), 1.55-1.64 (m, 2H). GC-MS m/z calc’d: 230.1, found:
229.7.

4.14a. 3-(3,4-dihydro-2H-pyran-2-yl)benzonitrile: Synthesized according to the general
procedure to afford a yellow oil (17 mg, 15%).
1
H NMR (250 MHz, CDCl
3
) : 7.45-7.80 (m, 4H),
6.51 (d, J = 7.5 Hz, 1H), 4.70-4.80 (m, 2H), 2.25-2.43 (m, 2H), 1.60-1.98 (m, 2H). GC-MS m/z
calc’d: 187.10, found: 184.95.

255

4.15b. 3-(3,4-dihydro-2H-pyran-2-yl)phenyl acetate: Synthesized according to the general
procedure to afford a yellow oil (17 mg, 16%).
1
H NMR (250 MHz, CDCl
3
) : 7.96 (d, J = 10.0
Hz, 2H), 7.45 (d, J = 10.0 Hz, 2H), 6.53 (d, J = 7.5 Hz, 1H), 4.90 (d, J = 7.5 Hz, 1H), 4.76-4.84
(m, 1H), 2.08-2.18 (m, 2H), 1.82-1.99 (m, 2H). GC-MS m/z calc’d: 204.26, found: 201.94.

4.17. 2-(2,6-dimethylphenyl)-3,4-dihydro-2H-pyran: Synthesized according to the general
procedure at 150 ⁰C to afford an orange oil (52 mg, 55%).
1
H NMR (250 MHz, CDCl
3
) : 6.98-
7.11 (m, 3H), 6.51 (d, J = 7.5 Hz, 1H), 5.20 (d, J = 10.0 Hz, 1H), 4.76-4.82 (m, 1H), 2.39 (s, 6H),
2.02-2.30 (m, 2H), 1.80-1.94 (m, 2H).
13
C NMR (62.5 MHz, CDCl
3
)  143.9, 137.1, 136.0, 129.2,
127.2, 100.2, 75.11, 26.03, 20.80, 20.51. GC-MS m/z calc’d: 188.1, found: 188.0.

4.19. 2-(3,4-dihydro-2H-pyran-2-yl)pyridine: Synthesized according to the general procedure
to afford a yellow oil (49 mg, 61%).
1
H NMR (250 MHz, CDCl
3
) : 8.61 (s, 1H), 8.53-8.57 (m,
1H), 7.67-7.73 (m, 1H), 7.27-7.33 (m, 1H), 6.53 (d, J = 7.5 Hz, 1H), 4.88 (d, J = 10.0 Hz, 1H),
4.78-4.84 (m, 1H), 2.18-2.30 (m, 2H), 1.88-2.14 (m, 2H).
13
C NMR (62.5 MHz, CDCl
3
)  149.1,
147.8, 143.9, 133.6, 123.4, 101.0, 74.82, 30.14, 20.02. GC-MS m/z calc’d: 161.1, found: 161.0.

256

4.4.2. Strecker synthesis methodology.
All commercially available reagents and solvents were used as received by Aldrich and
Acros chemical without further purification. 1H and 13C NMR spectra were recorded on a 250
and 63MHz Bruker or 400 and 100MHz Varian instrument. Thin-layer chromatography (TLC)
was performed using commercially prepared 60 mesh silica gel plates visualized with short-
wavelength UV light (254 nm). Silica gel 60 (9385, 230-400 mesh) was used for column
chromatography. The reported conversions are based upon consumption of amine substrate and
yields are isolated yields and are the average of two runs. MS analysis was performed using a
Thermo Scientific DSQ II GC-MS (ESI) instrument (He gas, 25 minute run time, the first 5
minutes at 40 degrees Celsius, an increase of 15 degrees/min for the next 15 minutes, and the
final 5 minutes at 250 degrees Celsius, with a constant flow of 1.5 mL per minute).

General procedure of synthesis of α-aminonitrile compounds

4.32. 2-phenyl-2-(phenylamino)acetonitrile: To a mixture of palladium complex (3 mol %),
sodium sulfate (100 mg, 0.7 mmol), benzaldehyde (0.020 mL, 0.2 mmol), and aniline (0.018 mL,
0.2 mmol) in 1 mL CH
2
Cl
2
in a pressure tube was added dropwise TMSCN (0.053 mL, 0.4
mmol). The pressure tube was closed and stirred for 24 h at 23 C and reaction progress was
monitored by TLC. The mixture was then filtered and the residue was washed with CH
2
Cl
2
(10
mL). The filtrate was collected and the solvent was removed under reduced pressure. If
necessary, column chromatography on silica gel with an ethyl acetate/hexanes gradient elution
257

was performed to obtain 2-phenyl-2-(phenylamino)acetonitrile (33 mg, 79%) as a light yellow
solid m.p. 76-79 C.
1
H NMR (250 MHz) δ 4.67 (br s, 1H), 5.36 (s, 1H), 6.72 (d, J = 7.8Hz, 2H),
6.84 (t, J = 7.4 Hz, 1H), 7.22 (t, J = 8.0 Hz, 2 H), 7.37-7.44 (m, 3H), 7.52-7.55 (m, 2H);
13
C
NMR (63 MHz) δ 50.1, 114.1, 118.1, 120.2, 127.2, 128.4, 129.2, 129.4, 134.0, 144.7. This
compound is known.
82

4.33. 2-(4-chlorophenyl)-2-(phenylamino)acetonitrile: Synthesized according to the general
procedure to obtain 2-(4-chlorophenyl)-2-(phenylamino)acetonitrile as a white solid (20 mg,
42%). m.p. 95-97 C.
1
H NMR (CDCl
3
, 250 MHz) δ 4.04 (br s, 1H), 5.43 (s, 1H), 6.77 (d, J = 7.5
Hz, 2H), 6.92 (t, J = 7.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.44 (d, J = 5 Hz), 7.55 (d J = 7.5 Hz).
This compound is known.
82

4.34. 2-(furan-2-yl)-2-(phenylamino)acetonitrile: Synthesized according to the general
procedure to obtain 2-(furan-2-yl)-2-(phenylamino)acetonitrile as a light yellow solid (31 mg,
79%).
1
H NMR (CDCl
3
, 250 MHz) δ 4.19 (br s, 1H), 5.51 (s, 1H), 6.44-6.46 (m, 1H), 6.61 (d, J
= 2.5 Hz, 1H), 6.80 (d, J = 10.0 Hz, 2H), 6.94 (t, J = 7.5 Hz, 1H), 7.30 (t, J = 7.5 Hz, 2H), 7.50-
7.51 (m, 1 H). This compound is known.
82

258

4.35. 2-(phenylamino)-2-(thiophen-2-yl)acetonitrile: Synthesized according to the general
procedure to obtain 2-(phenylamino)-2-(thiophen-2-yl)acetonitrile as a white solid (39 mg, 91%).
1
H NMR (CDCl
3
, 400 MHz) δ 5.65 (s, 1H), 6.81 (d, J = 8.0 Hz, 2H), 6.94 (t, J = 8.0 Hz, 1H),
7.05-7.07 (m, 1H), 7.26-7.32 (m, 3H), 7.39-7.40 (m, 2H);
13
C NMR (CDCl
3
, 63 MHz) δ 46.2,
114.7, 117.6, 120.8, 125.5, 127.1, 127.2, 127.3, 130.0, 144.2. This compound is known.
82

4.36. 2-(phenylamino)-2-(pyridin-3-yl)acetonitrile: Synthesized according to the general
procedure to obtain 2-(phenylamino)-2-(pyridin-3-yl)acetonitrile as a light yellow solid (40 mg,
95%). m.p. 74-83 C.
1
H NMR (CDCl
3
, 400 MHz) δ 4.16 (br s, 1H), 5.53 (d, J = 8.0 Hz, 1H),
6.81, (d, J = 8.0 Hz, 2H), 6.96, (t, J = 8.0 Hz, 1H), 7.28-7.34 (m, 3H), 7.42-7.45 (m, 1H), 7.98 (d,
J = 8.0 Hz, 1H), 8.71 (d, J = 4.0 Hz, 1H), 8.88 (s, 1H);
13
C NMR (CDCl
3
, 63 MHz) δ 48.3, 114.5,
117.3, 120.9, 123.9, 129.7, 134.9, 144.3, 148.7, 150.8. This compound is known.
83

4.37. (E)-4-phenyl-2-(phenylamino)but-3-enenitrile: Synthesized according to the general
procedure to obtain (E)-4-phenyl-2-(phenylamino)but-3-enenitrile as a yellow solid (39 mg,
259

83%). m.p. 55-61 C.
1
H NMR (CDCl
3
, 400 MHz) δ 3.50 (br s, 1H), 5.08 (dd, J = 8.0 Hz, 1H),
6.30 (dd, J = 16.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 2H), 6.93 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 16.0 Hz,
1H), 7.27-7.32 (m, 2H), 7.34-7.40 (m, 3H), 7.44-7.46 (m, 2H). This compound is known.
82

4.38. 2-cyclohexyl-2-(phenylamino)acetonitrile: Synthesized according to the general
procedure to obtain 2-cyclohexyl-2-(phenylamino)acetonitrile as a white solid (38 mg, 88%).
1
H
NMR (CDCl
3
, 250 MHz) δ 1.17-1.35 (m, 5H), 1.74 (d, J = 5.0 Hz, 1H), 1.82-1.88 (m, 3H), 1.96-
2.01 (m, 2H), 3.79 (br s, 1H), 4.04-4.08 (m, 1H), 6.72 (d, J = 5.0 Hz, 2H), 6.86 (t, J = 5.0 Hz,
1H), 7.23-7.27 (t, J = 5.0 Hz, 2H);
13
C NMR (CDCl
3
, 63 MHz) δ 25.7, 29.7, 30.4, 40.8, 51.8,
114.1, 118.9. 119.9, 129.5, 145.3. This compound is known.
54

4.39. 2-(benzylamino)-2-phenylacetonitrile: Synthesized according to the general procedure to
obtain 2-(benzylamino)-2-phenylacetonitrile as a white solid (42 mg, 95%).
1
H NMR (CDCl
3
,
400 MHz) δ 3.99 (AB, q, J = 11.0 Hz, 2H), 4.77 (s, 1H), 7.31-7.45 (m, 8H), 7.55 (d, J = 4.0 Hz,
2H);
13
C NMR (CDCl
3
, 63 MHz) δ 51.1, 53.3, 118.5, 127.2, 127.5, 128.3, 128.5, 128.8, 128.9,
134.6, 137.9. This compound is known.
82

260

4.40. 2-(benzylamino)-2-(4-chlorophenyl)acetonitrile: Synthesized according to the general
procedure to obtain 2-(benzylamino)-2-(4-chlorophenyl)acetonitrile as a white solid (46 mg,
90%).
1
H NMR (CDCl
3
, 250 MHz) δ 1.89(br s, 1H), 4.00 (AB, q, J= 12.5 Hz, 2H), 4.74 (s, 1H),
7.32-7.51 (m, 9H). This compound is known.
84

4.41. 2-(benzylamino)-2-(furan-2-yl)acetonitrile: Synthesized according to the general
procedure to obtain 2-(benzylamino)-2-(furan-2-yl)acetonitrile as a light brown oil (36 mg, 86%).
1
H NMR (CDCl
3
, 250 MHz) δ 2.03 (br s, 1H), 3.99 (AB, q, J = 15.0 Hz, 2H), 4.80 (s, 1H), 6.38-
6.40 (m, 1H), 6.48-6.50 (m, 1H), 7.29-7.41 (m, 5H), 7.44-7.45 (m, 1H). This compound is
known.
75

4.42. 2-(benzylamino)-2-(thiophen-2-yl)acetonitrile: Synthesized according to the general
procedure to obtain 2-(benzylamino)-2-(thiophen-2-yl)acetonitrile as a white solid (42 mg, 91%).
1
H NMR (CDCl
3
, 250 MHz) δ 2.47 (br s, 1H), 4.03 (AB, q, J = 17.5 Hz, 2H), 4.97, (s, 1H), 6.99-
261

7.02 (m, 1H), 7.25-7.43 (m, 6H);
13
C NMR (CDCl
3
, 63 MHz) δ 49.3, 50.9, 118.1, 126.5, 126.9,
127.8, 128.4, 128.8, 137.9, 138.2; MS Anal. Calcd [-CN]: 228.07 Found: 228.10. This compound
is known.
75

4.43. 2-(benzylamino)-2-(pyridin-3-yl)acetonitrile: Synthesized according to the general
procedure to obtain 2-(benzylamino)-2-(pyridin-3-yl)acetonitrile as a yellow solid (39 mg, 87%).
m.p. 103-105 C.
1
H NMR (CDCl
3
, 250 MHz) δ 2.47 (br s, 1H), 4.03 (AB, q, J = 15.0 Hz, 2H),
4.81 (s, 1H), 7.27-7.44 (m, 6H), 7.88-7.93 (m, 1H), 8.63-8.65 (m, 1H), 8.80 (d, J = 5.0 Hz, 1H);
13
C NMR (CDCl
3
, 63 MHz) δ 51.0, 117.5, 123.4, 127.6, 128.1, 128.5, 130.5, 134.8, 137.4, 148.5,
150.1. This compound is known.
85

4.44. (E)-2-(benzylamino)-4-phenylbut-3-enenitrile: Synthesized according to the general
procedure to obtain (E)-2-(benzylamino)-4-phenylbut-3-enenitrile as a yellow solid (49 mg,
98%). m.p. 109-115 C.
1
H NMR (CDCl
3
, 400 MHz) δ 1.62 (br s, 1H), 4.01 (AB, q, J = 14.0 Hz,
2H), 4.40 (dd, J = 8.0 Hz, 1H), 6.19 (dd, J = 12.0 Hz, 1H), 6.93 (d, J = 20.0 Hz), 7.28-7.42 (m,
10H). This compound is known.
74

262

4.45. 2-(benzylamino)pentanenitrile: Synthesized according to the general procedure to obtain
2-(benzylamino)pentanenitrile as a white oil (27 mg, 70%).
1
H NMR (CDCl
3
, 250 MHz) δ 0.94 (t,
J = 7.5 Hz, 3H), 1.46-1.61 (m, 2H), 1.71-1.80 (m, 2H), 3.50 (t, J = 7.5 Hz, 1H), 3.95 (AB, q, J =
12.5 Hz, 2H), 7.27-7.36 (m, 5H). This compound is known.
86

4.46. 2-(benzylamino)-2-cyclohexylacetonitrile: Synthesized according to the general procedure
to obtain 2-(benzylamino)-2-cyclohexylacetonitrile as a white solid (28 mg, 62%).
1
H NMR
(CDCl
3
, 250 MHz) δ 1.09-1.30 (m, 5H), 1.67-1.92 (m, 6H), 3.31 (d, J = 5.0 Hz, 1H), 3.96 (AB, q,
J = 12.5 Hz, 2H), 7.27-7.38 (m, 5H);
13
C NMR (CDCl
3
, 63 MHz) δ 25.9, 26.3, 29.2, 30.0, 51.8,
55.8, 119.9, 128.6, 128.8, 138.8.

4.48. 2-(naphthalen-2-yl)-2-(phenylamino)propanenitrile: Synthesized according to the
general procedure to obtain 2-(naphthalen-2-yl)-2-(phenylamino)propanenitrile as an orange solid
(50 mg, 92%).
1
H NMR (CDCl
3
, 250 MHz) δ 2.02 (s, 3H), 4.37 (br s, 1H), 6.57 (d, J = 7.5 Hz,
263

2H), 6.76 (t, J = 7.5 Hz, 1H), 7.09 (t, J = 7.5 Hz, 2H), 7.51-7.56 (m, 2H), 7.68 (d, J = 7.5 Hz,
1H), 7.86-7.90 (m, 3H), 8.15 (s, 1H);
13
C NMR (CDCl
3
, 63 MHz) δ 33.2, 57.3, 113.2, 115.8,
120.0, 122.0, 124.3, 126.6, 127.6, 128.2, 129.0, 129.3, 133.1, 143.6. This compound is known.
82

4.49. 2-(4-methoxyphenyl)-2-(phenylamino)propanenitrile: Synthesized according to the
general procedure to obtain 2-(4-methoxyphenyl)-2-(phenylamino)propanenitrile as a white solid
(22 mg, 44%).
1
H NMR (CDCl
3
, 250 MHz) δ 1.92 (s, 3H), 3.82 (s, 3H), 4.26 (br s, 1H), 6.56 (d, J
= 7.5 Hz, 2 H), 6.80 (t, J = 10.0 Hz, 1H), 6.92 (d, J = 10.0 Hz, 2H), 7.13 (t, J = 7.5 Hz, 2H), 7.52
(d, J = 7.5 Hz, 2H);
13
C NMR (CDCl
3
, 63 MHz) δ 33.4, 55.3, 56.7, 114.5, 115.9, 119.9, 126.2,
129.0, 143.6, 159.6; MS Anal. Calcd [-CN]: 225.12 Found: 225.00.

4.50. 2-(4-bromophenyl)-2-(phenylamino)propanenitrile: Synthesized according to the
general procedure to obtain 2-(4-bromophenyl)-2-(phenylamino)propanenitrile as a white solid
(55 mg, 88%).
1
H NMR (CDCl
3
, 250 MHz) δ 1.92 (s, 3H), 4.30 (br s, 1H), 6.53 (d, J = 7.5 Hz,
2H), 6.77-6.86 (m, 1H), 7.14 (t, J = 5.0 Hz, 2H), 7.52-7.63 (m, 3H), 7.84 (t, J = 7.5 Hz, 1H);
13
C
NMR (CDCl
3
, 63 MHz) δ 33.4, 56.8, 115.8, 120.4, 126.8, 129.2, 132.5, 143.2. This compound is
known.
62
264

4.51. 2-(4-nitrophenyl)-2-(phenylamino)propanenitrile: Synthesized according to the general
procedure to obtain 2-(4-nitrophenyl)-2-(phenylamino)propanenitrile as a yellow solid (8 mg,
15%).
1
H NMR (CDCl
3
, 400 MHz) δ 1.98 (s, 3H), 4.39 (br s, 1H), 6.49 (d, J = 8.0 Hz, 2H), 6.85
(t, J = 8.0 Hz, 1H), 7.14 (t, J = 8.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H), 8.27 (d, J = 8.0 Hz, 2H);
13
C
NMR (CDCl
3
, 100 MHz) δ 32.7, 56.4, 115.3, 119.3, 120.4, 123.5, 124.3, 125.8, 128.8, 142.3,
147.7. This compound is known.
82

4.52. 2-(phenylamino)-2-(pyridin-3-yl)propanenitrile: Synthesized according to the general
procedure to obtain 2-(phenylamino)-2-(pyridin-3-yl)propanenitrile as an orange solid (18 mg,
40%).
1
H NMR (CDCl
3
, 400 MHz) δ 2.00 (s, 3H), 4.33 (br s, 1H), 6.55 (d, J = 8.0 Hz, 2H), 6.85
(t, J = 8.0 Hz), 1H), 7.15 (t, J = 8.0 Hz, 2H), 7.35-7.38 (m, 1H), 7.93 (d, J = 8 Hz, 1H), 8.65 (d, J
= 4.0 Hz, 1H), 8.93 (s, 1H);
13
C NMR (CDCl
3
, 63 MHz) δ 33.0, 55.4, 116.1, 120.5, 124.0, 129.3,
133.2, 135.6, 143.1, 147.2, 150.1, 153.5. This compound is known.
87

265

4.53. 2-(furan-2-yl)-2-(phenylamino)propanenitrile: Synthesized according to the general
procedure to obtain 2-(furan-2-yl)-2-(phenylamino)propanenitrile as a light yellow solid (35 mg,
83%). m.p. 83-85 C.
1
H NMR (CDCl
3
, 400 MHz) δ 2.02 (s, 3H), 4.11 (br s, 1H), 6.37-6.38 (m,
1H), 6.48 (d, J = 4.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 2H), 6.90 (t, J = 8.0 Hz, 1 H), 7.20 (t, J = 4.0
Hz, 2H), 7.43 (s, 1H);
13
C NMR (CDCl
3
, 63 MHz) δ 28.8, 52.8, 108.4, 110.9, 117.2, 119.3, 121.3,
129.4, 143.4, 150.8, 152.2, 156.6. This compound is known.
87

4.54. 2-(benzylamino)-2-(naphthalen-2-yl)propanenitrile: Synthesized according to the general
procedure to obtain 2-(benzylamino)-2-(naphthalen-2-yl)propanenitrile as a white solid (19 mg,
33%).
1
H NMR (CDCl
3
, 400 MHz) δ 1.87 (s, 3H), 3.76 (AB, q, J = 12.0 Hz, 2H), 7.25-7.38 (m,
6H), 7.51-7.56 (m, 2H), 7.76-7.79 (m, 1H), 7.85-7.93 (m, 3H), 8.19 (s, 1H);
13
C NMR (CDCl
3
, 63
MHz) δ 31.0, 49.6, 60.6, 122.6, 125.2, 126.5, 126.6, 127.3, 127.5, 128.1, 128.2, 128.4, 129.0,
132.9, 133.2, 136.9; MS Anal. Calcd [-CN]: 259.14 Found: 258.11.

4.55. 2-(benzylamino)-2-(4-methoxyphenyl)propanenitrile: Synthesized according to the
general procedure to obtain 2-(benzylamino)-2-(4-methoxyphenyl)propanenitrile as a light brown
solid (11 mg, 35%).
1
H NMR (CDCl
3
, 250 MHz) δ 1.80 (s, 3H), 3.76 (AB, q, J = 7.5 Hz, 2H),
266

3.85 (s, 3H), 6.98 (d, J = 7.5Hz, 2H), 7.27-7.41 (m, 5H), 7.65 (d, J = 7.5 Hz, 2H);
13
C NMR
(CDCl
3
, 63 MHz) δ 31.2, 49.4, 55.3, 59.9, 114.1, 126.7, 127.3, 128.2, 128.4, 139.0, 159.7; MS
Anal. Calcd [-CN]: 239.13 Found: 238.08. This compound is known.
88

4.56. 2-(benzylamino)-2-(4-bromophenyl)propanenitrile: Synthesized according to the general
procedure to obtain 2-(benzylamino)-2-(4-bromophenyl)propanenitrile as a white solid (35 mg,
55%).
1
H NMR (CDCl
3
, 400 MHz) δ 1.61 (br s, 1H), 1.75 (s, 3H), 3.71 (AB, q, J = 12.0 Hz, 2H),
7.27-7.36 (m, 5H), 7.53-7.63 (m, 3H), 8.39-8.42 (m, 1H);
13
C NMR (CDCl
3
, 63 MHz) δ 31.3,
49.6, 60.1, 122.7, 127.4, 127.5, 128.2, 128.6, 132.1. This compound is known.
88

4.57. 2-(benzylamino)-2-(pyridin-3-yl)propanenitrile: Synthesized according to the general
procedure to obtain 2-(benzylamino)-2-(pyridin-3-yl)propanenitrile as a white solid (28 mg,
60%).
1
H NMR (CDCl
3
, 250 MHz) δ 1.84 (s, 3H), 2.86 (br s, 1H), 3.75 (AB, q, J = 7.5 Hz, 2H),
7.28-7.47 (m, 6H), 7.99-8.04 (m, 1H), 8.62-8.65 (m, 1H), 8.98 (d, J = 2.5 Hz, 1H);
13
C NMR
(CDCl
3
, 63 MHz) δ 31.2, 49.7, 59.0, 123.6, 127.7, 128.3, 133.6, 135.6, 147.6, 150.2.

267

4.5. Chapter 4. References.
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274

Conclusions

1. Novel small molecules, natural product derivatives, and helix-constrained
peptides have been prepared and characterized to investigate their utility in
disrupting the interaction between the transcription factor MEF2 and HDACs.
Evidence for small molecule ligand binding to MEF2 in solution was obtained
using NMR spectroscopy.

2. Small molecules comprised of a benzimidazole core scaffold were synthesized to
determine structural features important to provide inhibitory activity towards Bcl-
2/Bcl-xL proteins.

3. New catalytic methodologies were developed using novel NHC-ligand palladium
complexes. These catalytic complexes were able to promote Heck and Strecker
reactions to obtain substituted cyclic enol ethers and α-aminonitriles, respectively.
The benefit of these methodologies was their ability to obtain good
regioselectivity in Heck-type reactions and use mild reaction conditions to
promote Strecker syntheses.

275

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Abstract (if available)

Abstract This dissertation comprises three projects, two related to the synthesis and evaluation of small molecule inhibitors of protein-protein interactions, and one related to development of palladium-assisted reaction methodologies. ❧ The introduction (Chapter 1) briefly provides an overview of the challenges associated with developing small molecule inhibitors of protein-protein interactions in relation to drug discovery efforts. ❧ Chapter 2 reviews the biological importance of the transcription factor MEF2 and details its interaction with HDACs. The synthesis of a diverse set of over 100 molecules, including compounds structurally related to the natural product trichostatin A, aimed at directly targeting MEF2 is described. Evaluation of small molecule ligand/protein binding via 19F NMR spectroscopy is presented. The synthesis of helix-constrained peptides for investigation of their interaction with MEF2 is discussed as well. ❧ Chapter 3 describes the role of Bcl-2/Bcl-xL in apoptosis and cancer treatment. The construction of a small molecule of benzimidazole-based inhibitors for investigation of their structure-activity relationship is discussed. ❧ Chapter 4 provides a brief description of the utility of Heck and Strecker reactions in organic synthesis. Novel methodologies using palladium-promoted chemistry to form arylated cyclic enol ethers and α-aminonitriles is described. The use of palladium-catalysis to achieve selective oxidations is also briefly presented.

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Asset Metadata

Creator Jarusiewicz, Jamie A. (author)

Core Title Synthesis of protein-protein interaction inhibitors and development of new catalytic methods

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 07/31/2012

Defense Date 07/31/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag BCL-2 inhibitors,Heck reaction,MEF2,N-heterocyclic carbenes,OAI-PMH Harvest,organic synthesis,Strecker reaction

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Petasis, Nicos A. (committee chair), Davies, Daryl L. (committee member), Jung, Kyung Woon (committee member)

Creator Email jarusiej@yahoo.com,jarusiew@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-80293

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