University of Southern California Dissertations and Theses

I. Microwave-assisted synthesis of phosphonic acids; II. Design and synthesis of polymerase β lyase domain inhibitors

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I. Microwave-assisted synthesis of phosphonic acids; II. Design and synthesis of polymerase β lyase domain inhibitors

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Content I. Microwave-assisted Synthesis of Phosphonic Acids
II. Design and Synthesis of Polymerase β Lyase Domain Inhibitors

By

Dana Mustafa

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

December 2013

Copyright 2013 Dana Mustafa

ii

DEDICATION

To my family, blood and otherwise, for their continued love and support.

iii

ACKNOWLEDGEMENTS

I joined the Ph.D. program at USC in the fall of 2008 with merely two
undergraduate courses in Chemistry under my belt. With a mix of trepidation and
excitement, I started this journey, uncertain of the challenges that lay ahead. Today,
five years later, I stand confident in knowing that I have grown not only as a chemist
and scientist, but also as a person. And for that, I have many people to thank.
I’d like to start by thanking Prof. Charles McKenna for his invaluable
mentorship and guidance throughout the years. His door remained open at all times,
and every time I stepped through it, he shared with me a wealth of Chemistry
knowledge, showed me the value of collaboration, and imparted countless encouraging
words that supported me in my quest for excellence. I am truly grateful to him for
taking me into his research group and for allowing me to realize my potential.
I would also like to extend my sincere gratitude to Dr. Boris Kashemirov, with
whom daily discussions have taught me almost everything I know about success in
synthetic Chemistry. His wisdom extends past solutions to overcoming difficulties in
the lab, and has helped me triumph over extraneous daily tribulations as well.
I am also very thankful to all of the faculty and staff in the Chemistry
department at USC who have helped me throughout the years. Thank you to Prof.
Travis Williams, Prof. Matthew Pratt, Prof. Surya Prakash, and Prof. Andrea Armani
for being on my qualifying committee. A special thank you to Travis for all of his help
with the NMR, and to Prof. Prakash for all of his helpful advice throughout the years.
Thank you to Marie de la Torre and Michele Dea for their help with administrative

iv

matters, Ross Lewis for always fixing our electronics with a smile, and Allan Kershaw
for maintaining the NMR facility that made my work possible. Thank you to Darrell
Karrfalt and Leo Bedoya for helping me acquire all the chemicals and glassware I
needed over the years, and to Phil Sliwoski for his glass-blowing expertise in
fashioning all of the special glassware that I required.
I owe a significant amount of gratitude to all of our collaborators who have
provided intellectual input, as well as biological data, over the course of my studies. I
would like to thank Prof. Myron Goodman at USC for input on the polymerase β
project, Dr. William Beard and Dr. Samuel Wilson at NIEHS for providing the
biological data for the pamoic acid derivatives, Dr. Mary Callanan at the Institut
Albert Bonnoit at Université Joseph Fourier in Grenoble, France, for the biological
testing of (+)-JQ1, and Dr. Hal Ebetino from Warner Chilcott for his input on the
bisphosphonate project. I would also like to thank Dr. Marcela Kreĉmerová from the
IOCB Research Centre, Academy of Sciences of the Czech Republic, for providing the
(S)-PMPDAP, and Melissa Williams for synthesizing the PMEDAP(OiPr)
2
and
PMEA(OiPr)
2
, that were used in the studies on microwave-assisted phosphonate ester
BTMS dealkylation.
I would also like to thank Procter & Gamble, the USC Chemistry Department,
the USC Graduate School (Grayson and Judith Manning Fellowship), the Partner
University Fund, and the Dana and David Dornsife College of Letters, Arts, and
Sciences for financial support.
I would like to extend a special thank you to the entire McKenna group,
especially those whose friendships made this journey so much more enjoyable. Dr.

v

Jorge Osuna, Elena Ferri, Melissa Williams, Kim Nguyen, Inah Kang and Candy
Hwang: I will miss all the colorful stories and endless laughter we shared over the
years in OCW 201. Dr. Ivan Krylov and Dr. Feng Ni, thank you for sharing countless
useful tricks and helping me in the lab when I needed it. Inah Kang, thank you so
much for all of your administrative help and for always knowing how to fix my
computer-related issues.
Finally, I would like to thank some of the most important people who have
played a pivotal role in making this all a reality. First and foremost, thank you to my
parents for raising me to be the person that I am, and for all of your love and support.
Rami and Samer, thank you for always being there for me when I needed you, for all
your support and advice, and for being the best brothers one could ever ask for. Sireen
Khan, Rasha Al-Safi, Siba Al-Adhami and Alejandra Beier, I cannot thank you enough
for you friendship, love, support, and words of encouragement throughout the years.
Janice Gaines, you have played a significant role in my success, and for that, I am
extremely thankful. Last, but certainly not least, thank you to Saif Al-Noami and his
family, to whom I am forever grateful for the kindness they have shown me.

vi

TABLE OF CONTENTS

Dedication ii
Acknowledgements iii
List of Tables ix
List of Figures x
List of Schemes xx
Abstract xxi
Chapter 1. Microwave-assisted Synthesis of Nitrogen-
containing Hydroxymethylenebisphosphonate
Drugs
1.1 Introduction 1
1.2 Results and Discussion 10
1.3 Conclusion 15
1.4 Experimental
1.4.1 Materials and Methods 15
1.4.2 Synthesis of Nitrogen-containing 1-
Hydroxymethylenebisphosphonates 16
1.5 Chapter References 21
Chapter 2. Microwave Studies on the BTMS Dealkylation of
Phosphonate Esters
2.1 Introduction 25
2.2 Results and Discussion 29
2.3 Conclusion 38
2.4 Experimental
2.4.1. Materials and Methods 39
2.4.2. Microwave BTMS Dealkylations of Dimethyl-,
Diethyl-, and Diisopropyl
methylphosphonate Esters 40
2.4.3. Microwave BTMS Dealkylations of
Trimethylphosphonoacetate,
Triethylphosphonoacetate, 2-
(Diethoxyphosphoryl)acetic Acid and
Diethyl (bromodifluoro)phosphonate 41
2.4.4. Microwave Coupling of (S)-PMPDAP to
Ethanol with PyBrOP and PyBOP 43

vii

2.4.5. Microwave BTMS Dealkylations of
PMEDAP(OiPr)
2
, (S)-PMPDAP(OEt)
2

and PMEA(OiPr)
2
44
2.5 Chapter References 46
Chapter 3. Pamoic Acid Derivatives as Inhibitors of the
Polymerase β Lyase Domain
3.1 Introduction 49
3.2 Results and Discussion
3.2.1. Molecular Docking Studies with Autodock
Vina 54
3.2.2. Fragment-Based Drug Design using the
MEDIT MED-SuMo Software 56
3.2.3. Molecular Docking Studies with PA
Derivatives 57
3.2.4. Synthesis of PA Derivatives 60
3.2.5. Preliminary Testing of Biological Activity
for the PA Derivatives 65
3.3 Conclusion 68
3.4 Experimental
3.4.1. Materials and Methods 69
3.4.2. Molecular Docking Studies with PA 70
3.4.3. Molecular Docking Studies with
Phosphonylated PA Derivatives 71
3.4.4. Synthesis of PA derivatives 71
3.4.5. Inhibitory Assays 77
3.5 Chapter References 79
Chapter 4. Synthesis of Enantiomerically Enriched (+)-JQ1
for Acute Myeloid Leukemia Studies
4.1 Introduction 81
4.2 Results and Discussion
4.2.1 Synthesis of Enantiomerically Enriched
(+)-JQ1 89
4.2.2. Biological Studies 91
4.3 Conclusion 91
4.4 Experimental
4.4.1. Materials and Methods 92
4.4.2. Synthesis of Enantiomerically Enriched
(+)-JQ1 93
4.5 Chapter References 97
Bibliography 101
Appendix A. Chapter 1 Supporting Data 110

viii

Appendix B. Chapter 2 Supporting Data 132
Appendix C. Chapter 3 Supporting Data 156
Appendix D. Chapter 4 Supporting Data 185

ix

LIST OF TABLES

Table 1.2.1 BPs synthesized via Scheme 1.2.1. For simplicity,
the compounds are depicted as acids.
13
Table 1.2.2 Results of the microwave-assisted synthesis of BPs
via Scheme 1.2.1 and comparison to conventional heating. All
MW programs for the synthesis of intermediate include a 3 min
ramp to 65 °C, while all hydrolysis programs include a 4 min
ramp to 150 °C. 14
Table 2.2.1. Results for the dealkylation reactions represented in
Scheme 2.2.1. 32
Table 2.2.2. Results for microwave BTMS dealkylation
reactions on mixed carboxylate-phosphonate esters, as well as on
2-(diethoxyphosphoryl)acetic acid and diethyl
(bromodifluoro)phosphonate. 34
Table 2.2.3. BTMS dealkylations of ANP esters using
microwave irradiation. 38

x

LIST OF FIGURES

Figure 1.1.1. The general structure for BPs, where R
1
and R
2

dictate the activity level of the drug and its affinity to bone. 1
Figure 1.1.2. Interaction of BPs with HAP in the bone is
mediated by the two phosphate groups, as well as the side chain
OH group and N atom.
10
2
Figure 1.1.3. Non-nitrogen containing BPs vs. nitrogen-
containing BPs. 3
Figure 1.1.4. Cellular mevalonate pathway and the intervention
of N-BPs.
2
4
Figure 1.1.5. X-ray structures illustrating the critical interaction
between the N atom in the side chains of the N-BPs Ris and Zol,
with a conserved Thr residue in FPPS.
10
5
Figure 1.1.6. The thermal effect in microwave irradation.
18
6
Figure 2.1.1. Suggested mechanism of the McKenna method for
phosphonate ester dealkylation by BTMS. The TMS esters are
hydrolyzed to phosphonic acids with water or methanol. 25
Figure 2.2.1. Possible intramolecular H-bonding that may
explain the decreased rate of BTMS dealkylation observed with
2-(diethoxyphosphoryl)acetic acid. 33
Figure 2.2.2. Structures for the ANPs PMEDAP(OiPr)
2
, (S)-
PMPDAP(OEt)
2
, and PMEA(OiPr)
2
. 36
Figure 3.1.1. The lyase domain of Pol β is involved in the
removal of the 5’dRP group during BER. 49
Figure. 3.1.2. The structure of pamoic acid, PA. 51
Figure 3.1.3. Overlay of the NMR solution structure of Pol β
(red) and the gapped DNA Pol β crystal structure (green).
Important residues involved in DNA binding and catalysis are
shown in magenta on the NMR solution structure and light green
on the crystal structure. Gapped DNA from the crystal structure
is shown in light blue.
5
52

xi

Figure 3.1.4. Some of the residues of the lyase domain of Pol ß
present in the binding pocket of PA.
2
52
Figure 3.1.5. Results of the docking experiments of PA with
1DK3 that were performed by Hazan et al.
4
53
Figure 3.2.1.1. The structures of BPDC, BQD and NCG, which
were utilized for docking studies with the lyase domain of Pol β,
to confirm the results obtained by Hu et al.
2
55
Figure 3.2.1.2. Results of docking studies performed with
Autodock Vina which utilized the lyase domain from PDB file
3JPT and PA as the ligand. Important residues include Lys35,
Tyr39, Gly64, Gly66, Lys68 and Lys72. 56
Figure 3.2.3.1. Overlap between the DNA substrate and the
mono-methylphosphonate PA analogue. 58
Figure 3.2.3.2. Overlap between the DNA substrate and the
mono-phosphonylated PA analogue. 59
Figure 3.2.3.3. Overlap between the DNA substrate and the di-
phosphonylated PA analogue. 59
Figure 3.2.5.1. Biological activities for (a) PA, (b) derivative 4,
and (c) derivative 3. Data provided by Dr. William Beard at the
NIEHS. 67
Figure 4.1.1. Phylogenetic tree of the human BRDs, based on
sequence alignments. The numbers in parentheses represent the
domain number, starting from the N-terminus.
3
82
Figure 4.1.2. Crystal structure of the first BRD (BD1) of the
BET family member BRD4 (PDB code: 3MUK). The protein
surface is in gray, and the acetyl-lysine binding site is located
between the ZA and BC loops.
11
83
Figure 4.1.3. The structures of the BET inhibitors (+)-JQ1 and I-
BET762, which are both triazolodiazepines, and I-BET151,
which contains a 3,5-dimethylisoxazole moiety. 85
Figure 4.1.4. The binding of (+)-JQ1 to the acetyl-lysine binding
pocket of BRD4(BD1), where (a) shows the contact residues
(labeled and depicted in stick form) and (b) shows the
interactions of (+)-JQ1 with the conserved asparagine and
tyrosine residues, as well as with structural water molecules,
based on the crystal structure with PDB code 3MXF.
17
87
Figure A1.
1
H NMR (500 MHz, D
2
O) of imidazol-1-yl acetic
acid. 110

xii

Figure A2.
1
H NMR spectrum (500 MHz, D
2
O, pH 8.9) of
risedronic acid ((1-hydroxy-1-phosphono-2-pyridin-3-yl-
ethyl)phosphonic acid) monohydrate, 1. 111
Figure A3.
31
P NMR spectrum (202 MHz, D
2
O, pH 8.9, 85%
H
3
PO
4
reference) of risedronic acid ((1-hydroxy-1-phosphono-2-
pyridin-3-yl-ethyl)phosphonic acid) monohydrate, 1. 112
Figure A4. Risedronate UV data. Using the average UV
absorption data from three experiments on an authentic sample of
risedronic acid disodium (found to contain 1.3 equivalents of
water by elemental analysis) supplied by Procter & Gamble, Inc.,
the extinction coefficient of risedronate was found to be 3687 M
-
1
cm
-1
in 0.1 M phosphate buffer pH 7.2. The absorption spectrum
for our microwave-synthesized sample 1 gave a purity of 99.6%
(monohydrate). 113
Figure A5. LC-MS data of risedronic acid, 1, using a mobile
phase of 60 µM ammonium acetate buffer with 2% acetonitrile,
pH 5.5.
114

Figure A6.
1
H NMR (500 MHz, D
2
O, pH 10.1) of zoledronic
acid ([1-hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-
diyl]bis(phosphonic acid)) monohydrate, 2. 115
Figure A7.
31
P NMR (202 MHz, D
2
O, pH 10.1, 85% H
3
PO
4

reference) of zoledronic acid ([1-hydroxy-2-(1H-imidazol-1-
yl)ethane-1,1-diyl]bis(phosphonic acid)) monohydrate, 2. 116
Figure A8. LC-MS data of zoledronic acid, 2, using a mobile
phase of 60 µM ammonium acetate buffer with 2% acetonitrile,
pH 5.5. 117
Figure A9.
1
H NMR (500 MHz, D
2
O, pH 8.8) of pamidronic
acid ((3-amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid))
monosodium monohydrate, 3. 118

Figure A10.
31
P NMR (202 MHz, D
2
O, pH 8.8, 85% H
3
PO
4

reference) of pamidronic acid ((3-amino-1-hydroxypropane-1,1-
diyl)bis(phosphonic acid)) monosodium monohydrate, 3.

119
Figure A11. Mass spectrum of pamidronate, 3. 120
Figure A12.
1
H NMR (500 MHz, D
2
O, pH 8.9) of alendronic
acid ([4-amino-1-hydroxy-1-(hydroxy-oxido-phosphoryl)-
butyl]phosphonic acid) monosodium dihydrate, 4.

121

Figure A13.
31
P NMR (202 MHz, D
2
O, pH 8.9, 85% H
3
PO
4

reference) of alendronic acid ([4-amino-1-hydroxy-1-(hydroxy-

xiii

oxido-phosphoryl)-butyl]phosphonic acid) monosodium
dihydrate, 4.
122

Figure A14. Mass spectrum of alendronate, 4. 123
Figure A15.
1
H NMR(500 MHz, D
2
O, pH 8.9) of neridronic acid
(6-amino-1-hydroxyhexane-1,1-diyl)bis(phosphonic acid))
monosodium monohydrate, 5. 124
Figure A16.
31
P NMR (202 MHz, D
2
O, pH 8.9, 85% H
3
PO
4

reference) of neridronic acid (6-amino-1-hydroxyhexane-1,1-
diyl)bis(phosphonic acid)) monosodium monohydrate, 5. 125
Figure A17. Mass spectrum of neridronate, 5.

126

Figure A18. Spiked
1
H NMR (500 MHz, D
2
O, pH 9.1) of
risedronic acid, 1. 5 mg of 1 was spiked with 8 mg of risedronate
disodium (98+% purity) supplied by Procter & Gamble, Inc. 127
Figure A19. Spiked
31
P NMR (202 MHz, D
2
O, pH 9.1, 85%
H
3
PO
4
reference) of risedronic acid, 1. 5 mg of 1 was spiked
with 8 mg of risedronate disodium (98+% purity) supplied by
Procter & Gamble, Inc. 127
Figure A20. Spiked
1
H NMR (500 MHz, D
2
O, pH 9.4) of Zol, 2.
5 mg of 2 was spiked with 5 mg zoledronic acid purchased from
Molekula, Inc. 128

Figure A21. Spiked
31
P NMR (202 MHz, D
2
O, pH 9.4, 85%
H
3
PO
4
reference) of Zol, 2. 5 mg of 2 was spiked with 5 mg
zolendronic acid purchased from Molekula, Inc. 128

Figure A22. Spiked
1
H NMR (500 MHz, D
2
O, pH 8.8) of
pamidronic acid monosodium, 3. 5 mg of 3 was spiked with 5
mg of pamidronate monosodium, supplied by Novartis, Inc. 129

Figure A23. Spiked
31
P NMR (202 MHz, D
2
O, pH 8.8, 85%
H
3
PO
4
reference) of pamidronic acid monosodium, 3. 5 mg of 3
was spiked with 5 mg of pamidronate monosodium, supplied by
Novartis, Inc. 129
Figure A24. Spiked
1
H NMR (500 MHz, D
2
O, pH 8.6) of
alendronic acid monosodium, 4. 6 mg of 4 was spiked with 6 mg
alendronate monosodium, supplied by Procter & Gamble, Inc. 130
Figure A25. Spiked
31
P NMR (202 MHz, D
2
O, pH 8.6, 85%
H
3
PO
4
reference) of alendronic acid monosodium, 4. 6 mg of 4
was spiked with 6 mg alendronate monosodium, supplied by
Procter & Gamble, Inc. 131

xiv

Figure B1.
1
H NMR (500 MHz, D
2
O) of the methylphosphonic
acid product from the microwave BTMS dealkylation of
dimethyl methylphosphonate in ACN at 40 °C for 10 min. 132
Figure B2.
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard
(0 ppm)) of the methylphosphonic acid product from the
microwave BTMS dealkylation of dimethyl methylphosphonate
in ACN at 40 °C for 10 min. 133
Figure B3.
1
H NMR (500 MHz, D
2
O) of the methylphosphonic
acid product from the microwave BTMS dealkylation of diethyl
methylphosphonate in ACN at 40 °C for 15 min. 134
Figure B4.
31
P NMR (202 MHz, D
2
O, adjusted with external
H
3
PO
4
standard (0 ppm)) of the methylphosphonic acid product
from the microwave BTMS dealkylation of diethyl
methylphosphonate in ACN at 40 °C for 15 min. 135
Figure B5.
31
P NMR (202 MHz, CDCl
3
) of the reaction mixture
containing the silyl ester of (2-methoxy-2-oxoethyl)phosphonic
acid after BTMS microwave reaction. The single peak is proof of
the complete selectivity of BTMS for phosphonate esters. 136
Figure B6.
1
H NMR (500 MHz, D
2
O, pH 7.6) of the (2-
methoxy-2-oxoethyl)phosphonic acid product from the
microwave BTMS dealkylation of trimethylphosphonoacetate. 137
Figure B7.
31
P NMR (202 MHz, D
2
O, pH 7.6, adjusted with
external H
3
PO
4
standard (0 ppm)) of the (2-methoxy-2-
oxoethyl)phosphonic acid product from the microwave BTMS
dealkylation of trimethylphosphonoacetate. 138
Figure B8.
31
P NMR (202 MHz, CDCl
3
) of the reaction mixture
containing the silyl ester of (2-ethoxy-2-oxoethyl)phosphonic
acid e after BTMS microwave reaction. The single peak is proof
of the complete selectivity of BTMS for phosphonate esters. 139
Figure B9.
1
H NMR (500 MHz, D
2
O, pH 8.2) of the (2-ethoxy-
2-oxoethyl)phosphonic acid product from the microwave BTMS
dealkylation of triethylphosphonoacetate. 140
Figure B10.
31
P NMR (202 MHz, D
2
O, pH 8.2, adjusted with
external H
3
PO
4
standard (0 ppm)) of the (2-ethoxy-2-
oxoethyl)phosphonic acid product from the microwave BTMS
dealkylation of triethylphosphonoacetate. 141
Figure B11.
31
P NMR (202 MHz, D
2
O) of
(bromodifluoromethyl)phosphonic acid. 142

xv

Figure B12.
1
H NMR (500 MHz, CDCl
3
) of 2-
(diethoxyphosphoryl)acetic acid. 143
Figure B13.
31
P NMR (202 MHz, CDCl
3
) of 2-
(diethoxyphosphoryl)acetic acid. 144
Figure B14.
1
H NMR (500 MHz, D
2
O, NaHCO
3
, pH 1.12) of
the 2-phosphonoacetic acid product from the microwave BTMS
dealkylation of 2-(diethoxyphosphoryl)acetic acid. 145
Figure B15.
31
P NMR (202 MHz, D
2
O, NaHCO
3
, pH 1.12) of
the 2-phosphonoacetic acid product from the microwave BTMS
dealkylation of 2-(diethoxyphosphoryl)acetic acid. 146
Figure B16.
31
P NMR (202 MHz, D
2
O capillary) of the product
mixture of MW PyBrOP-mediated coupling between (S)-
PMPDAP and ethanol. As the NMR shows, there is
approximately 80% monoethyl ester and 20% diethylester. The
peak at 14.20 ppm represents reacted PyBrOP. 147
Figure B17.
31
P NMR (202 MHz, D
2
O capillary) of the reaction
mixture of MW PyBOP-mediated coupling between (S)-
PMPDAP monoethyl ester and ethanol. The peak at 14.03 ppm
represents reacted PyBOP. 148
Figure B18.
31
P NMR (202 MHz, CD
3
OD) of (S)-
PMPDAP(OEt)
2
, after purification by silica column
chromatography. 149
Figure B19.
1
H NMR (500 MHz, D
2
O) of the 9-[2-
(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP)
product from the microwave BTMS dealkylation of
PMEDAP(OiPr)
2
. 150
Figure B20.
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard
(0 ppm)) of the 9-[2-(phosphonomethoxy)ethyl]-2,6-
diaminopurine (PMEDAP) product from the microwave BTMS
dealkylation of PMEDAP(OiPr)
2
. 151
Figure B21.
1
H NMR (500 MHz, D
2
O) of the 9-[(2-(S)-
(phosphonomethoxy)propyl-2,6-diaminopurine ((S)-PMPDAP)
product from the microwave BTMS dealkylation of (S)-
PMPDAP(OEt)
2
. 152
Figure B22.
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard
(0 ppm) of the 9-[(2-(S)-(phosphonomethoxy)propyl-2,6-
diaminopurine ((S)-PMPDAP) product from the microwave
BTMS dealkylation of (S)-PMPDAP(OEt)
2
. 153

xvi

Figure B23.
1
H NMR (500 MHz, D
2
O) of the 9-(2-
phosphonomethoxy)ethyl adenine (PMEA) product from the
microwave BTMS dealkylation of PMEA(OiPr)
2
. 154
Figure B24.
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard
(0 ppm)) of the 9-(2-phosphonomethoxy)ethyl adenine (PMEA)
product from the microwave BTMS dealkylation of
PMEA(OiPr)
2
. 155
Figure C1.
1
H NMR (500 MHz, CDCl
3
) of aryl dimethyl
phosphate (dimethyl naphthalen-2-yl phosphate), 1, with a
magnification of the aromatic section in the inset. 156
Figure C2.
31
P NMR (202 MHz, CDCl
3
) of aryl dimethyl
phosphate (dimethyl naphthalen-2-yl phosphate), 1. 157
Figure C3.
31
P NMR (202 MHz, CDCl
3
) of the product mixture
containing P-(3-hydroxy-2-naphthalenyl)-methyl phosponate, 2. 158

Figure C4. Mass spectrum of the product mixture containing P-
(3-hydroxy-2-naphthalenyl)-methyl phosponate, 2. 159
Figure C5.
1
H NMR (500 MHz, D
2
O) of the mono-
methylphosphonate PA derivative (3-hydroxy-4-((2-hydroxy-3-
(hydroxy(methoxy)phosphoryl)naphthalen-1-yl)methyl)-2-
naphthoic acid), 3. 160
Figure C6.
31
P NMR (202 MHz, D
2
O) of the mono-
methylphosphonate PA derivative (3-hydroxy-4-((2-hydroxy-3-
(hydroxy(methoxy)phosphoryl)naphthalen-1-yl)methyl)-2-
naphthoic acid), 3. 161

Figure C7. Mass spectrum of mono-methylphosphonate PA
derivative (3-hydroxy-4-((2-hydroxy-3-
(hydroxy(methoxy)phosphoryl)naphthalen-1-yl)methyl)-2-
naphthoic acid), 3. 162
Figure C8. HPLC chromatogram for the separation of mono-
methylphosphonate PA derivative (3-hydroxy-4-((2-hydroxy-3-
(hydroxy(methoxy)phosphoryl)naphthalen-1-yl)methyl)-2-
naphthoic acid), 3. 163
Figure C9.
1
H NMR (500 MHz, D
2
O) of deaklylated mono-
phosphonylated PA derivative (3-hydroxy-4-((2-hydroxy-3-
phosphononaphthalen-1-yl)methyl)-2-naphthoic acid), 4. 164

Figure C10.
31
P NMR (202 MHz, D
2
O) of deaklylated mono-
phosphonylated PA derivative (3-hydroxy-4-((2-hydroxy-3-
phosphononaphthalen-1-yl)methyl)-2-naphthoic acid), 4. 165

xvii

Figure C11. Mass spectrum of deaklylated mono-
phosphonylated PA derivative (3-hydroxy-4-((2-hydroxy-3-
phosphononaphthalen-1-yl)methyl)-2-naphthoic acid), 4. 166

Figure C12. HPLC chromatogram for the separation of
deaklylated mono-phosphonylated PA derivative (3-hydroxy-4-
((2-hydroxy-3-phosphononaphthalen-1-yl)methyl)-2-naphthoic
acid), 4. 167
Figure C13.
1
H (500 MHz, CDCl
3
) of aryl diethyl phosphate
(diethyl naphthalen-2-yl phosphate), 5. 168
Figure C14.
31
P (202 MHz, CDCl
3
) of aryl diethyl phosphate
(diethyl naphthalen-2-yl phosphate), 5. 169
Figure C15.
1
H NMR (500 MHz, CDCl
3
) of the isomer mixture
containing aryl diethyl phosphonate (P-(3-hydroxy-2-
naphthalenyl)-diethyl phosponate), 6, showing 12 aromatic
protons due to the presence of two isomers before separation. 170
Figure C16.
31
P NMR (CDCl
3
, 202 MHz) of aryl diethyl
phosphonate (P-(3-hydroxy-2-naphthalenyl)-diethyl phosponate),
6 with its isomer (the C-1 phosphonate) before separation. 171
Figure C17. Mass spectrum of the isomer mixture of aryl diethyl
phosphonate, 6. 172
Figure C18.
1
H NMR (500 MHz, CDCl
3
) of aryl diethyl
phosphonate (P-(3-hydroxy-2-naphthalenyl)-diethyl phosponate),
6, after separation from its C-1 phosphonate isomer. 173
Figure C19.
1
H NMR (500 MHz, CDCl
3
) of the aromatic region
of aryl diethyl phosphonate (P-(3-hydroxy-2-naphthalenyl)-
diethyl phosponate), 6, after separation from its C-1 phosphonate
isomer. The doublet at 8.00 ppm has a J
P-H
of 20 Hz, proving that
this is the C-3 phosphonate isomer. 174

Figure C20.
1
P NMR (202 MHz, CDCl
3
) of aryl diethyl
phosphonate (P-(3-hydroxy-2-naphthalenyl)-diethyl phosponate),
6, after separation from its C-1 phosphonate isomer. 175

Figure C21.
1
H NMR (500 MHz, CDCl
3
) of the C-1
phosphonate isomer of aryl diethyl phosphonate after separation
from the C-3 phosphonate. 176

Figure C22.
1
H NMR (500 MHz, CDCl
3
) of the aromatic region
of the C-1 phosphonate isomer of aryl diethyl phosphonate, after
separation from its mixture with the C-3 phosphonate. 177

xviii

Figure C23.
31
P NMR (202 MHz, CDCl
3
) of the C-1 phosphonate
isomer of aryl diethyl phosphonate after separation from the C-3
phosphonate. 178

Figure C24.
1
H NMR (500 MHz, CDCl
3
) of the tetraethyl di-
phosphonylated PA derivative (tetraethyl (methylenebis(3-
hydroxynaphthalene-4,2-diyl))bis(phosphonate)), 7. 179

Figure C25.
31
P NMR (202 MHz, CDCl
3
) of the tetraethyl
diphosphorylated PA derivative (tetraethyl (methylenebis(3-
hydroxynaphthalene-4,2-diyl))bis(phosphonate)), 7. 180

Figure C26. Mass spectrum of the tetraethyl di-phosphonylated
PA derivative (tetraethyl (methylenebis(3-hydroxynaphthalene-
4,2-diyl))bis(phosphonate)), 7. 181
Figure C27.
1
H NMR (500 MHz, D
2
O) of the dealkylated di-
phosphonylated PA derivative (methylenebis(3-
hydroxynaphthalene-4,2-diyl))diphosphonic acid, 8. 182
Figure C28.
31
P NMR (202 MHz, D
2
O) of the dealkylated di-
phosphonylated PA derivative (methylenebis(3-
hydroxynaphthalene-4,2-diyl))diphosphonic acid, 8. 183

Figure C29. Mass spectrum of the dealkylated di-
phosphonylated PA derivative (methylenebis(3-
hydroxynaphthalene-4,2-diyl))diphosphonic acid, 8. 184
Figure D1.
1
H NMR (500 MHz, CDCl
3
) of (2-amino-4,5-
dimethylthiophen-3-yl)(4-chlorophenyl)methanone, 2. 185
Figure D2.
1
H NMR (500 MHz, CDCl
3
) of (S)-tert-butyl-3-
({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-4-{[3-(4-
chlorobenzoyl)-4,5-dimethylthiophen-2-yl]amino}-4-
oxobutanoate, 3. 186
Figure D3.
1
H NMR (500 MHz, CDCl
3
) of (S)-tert-butyl 3-
amino-4-((3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-
yl)amino)-4-oxobutanoate, 4. 187
Figure D4.
1
H NMR (500 MHz, CDCl
3
) of (S)-tert-butyl 2-(5-
(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-
e][1,4]diazepin-3-yl)acetate, 5. 188

Figure D5.
1
H NMR (500 MHz, CDCl
3
) of (S)-tert-butyl 2-(4-
(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate, (+)-JQ1. 189

xix

Figure D6. Chiral analytical HPLC chromatogram of the
enantiomerically enriched sample of (+)-JQ1, showing the
relative ratios of (+)-JQ1 and (-)-JQ1 (70:30, respectively). 190
Figure D7. Mass spectrum of the enantiomerically enriched
sample of (+)-JQ1. 191

xx

LIST OF SCHEMES

Scheme 1.2.1. Modified method for the MAOS of nitrogen-
containing 1-hydroxymethylenebisphosphonic acids. The
preferred solvent in step 1 is sulfolane.
33
13
Scheme 2.2.1. General reaction scheme for the microwave-
assisted BTMS dealkylation reactions. 29
Scheme 2.2.2. Coupling of ethanol to (S)-PMPDAP to generate
the diethyl ester. 37
Scheme 3.2.4.1. Synthetic scheme for the synthesis of the novel
mono-methylphosphonate derivative of PA, 3. 61
Scheme 3.2.4.2. Dealkylation of 3 via reaction with BTMS. 62

Scheme 3.2.4.3. Synthetic scheme towards P-(3-hydroxy-2-
naphthalenyl)-diethyl phosphonate, (6). 64
Scheme 3.2.4.4. Synthetic scheme for the di-phosphonylated
derivative of PA, 8. 65
Scheme 4.2.1. Synthesis of enantiomerically enriched (+)-JQ1,
as described by Filippakopoulos et al.
17
90

xxi

ABSTRACT
Nitrogen-containing bisphosphonates (N-BPs) are a class of drugs that
accumulate in the bone and target osteoclasts, effectively preventing excessive bone
resorption, and providing treatment for diseases such as osteoporosis and Paget’s
disease. A library of novel N-BPs for biological testing is therefore of interest. One of
the methods to rapidly generate libraries of compounds is microwave-assisted organic
synthesis (MAOS), which relies on the direct heating of polar molecules to achieve
efficient heating. A rapid, simple, and efficient method for the small-scale synthesis of
N-BP drugs via microwave irradiation was established. Whereas the traditional
method for the preparation of these compounds typically requires heating for at least
9.5 h, this new microwave-assisted procedure is complete in less than 20 min, while
maintaining good yields of product.
Microwave irradiation was also utilized to devise a novel, more efficient
procedure for the McKenna method of selective phosphonate ester dealkylation using
bromotrimethylsilane (BTMS). Rates were vastly improved by microwave heating,
and the versatility of this reaction was demonstrated by varying the temperature, and
by utilizing a variety of solvents. Solvents with low polarities, such as dioxane, were
successfully utilized, and the reactions were also run in neat BTMS, suggesting that
the reactants themselves are polar enough to be directly heated via microwave
irradiation. BTMS dealkylation produced quantitative yields, and was applied towards
isopropyl, ethyl, and methyl esters. Furthermore, temperatures as low as 40 °C were
used, as well as equimolar amounts of BTMS, which, together with the short reaction
times, makes this approach mild and environmentally friendly. The use of pressure

xxii

was also obviated, and the reaction was compatible with carboxylate esters, and other
sensitive groups such as acyclic nucleoside phosphonate esters possessing diamino
purines.
Polymerase β (Pol β) is essential for maintaining the integrity of the genome by
participating in the base excision repair (BER) of damaged DNA bases via its lyase
domain. Pol β is overexpressed in several cancers, and is involved in the resistance of
tumors to DNA-damaging cancer therapeutics through the action of its lyase domain.
As a result, inhibiting the lyase domain of Pol β is of significant interest. The best
inhibitor known is pamoic acid (PA), which, due to its high K
d
and IC
50
values, cannot
be crystallized with the enzyme. Through the use of docking studies with Autodock
Vina, and fragment-based drug design with MEDIT MED-SuMo, mono- and di-
phosphonylated analogues of PA were designed and synthesized. Preliminary
biological data revealed that the inhibitory activity was increased 3-fold with the un-
methylated mono-phosphonylated derivative, while activity was increased 6-fold with
the mono-methylphosphonate derivative. Inhibitory activities for the di-
phosphonylated analogue have not yet been determined, and an X-ray crystal structure
of the lyase domain bound to any of the inhibitors is still pending.
Finally, enantiomerically enriched (+)-JQ1 was synthesized according to the
literature procedure by Filippakopoulos et al,,
1
yielding a 40% enantiomeric excess of
(+)-JQ1. This compound is a highly potent and specific inhibitor for the
bromodomains of the BET family of proteins, which recognize acetylated lysines on
the histone tails of chromatin, and participate in the epigenetic control of gene
expression. (+)-JQ1 was previously found to be effective in the treatment of several

xxiii

types of cancer, including many subtypes of acute myeloid leukemia (AML). (+)-JQ1
was tested against a novel aggressive subtype of AML for which current treatments are
ineffective. The in vitro treatment of AML cells with (+)-JQ1 resulted in the
downregulation of key onco-proteins that are linked to differentiation arrest and
resistance to senescence. (+)-JQ1 was also found to significantly constrain tumor
growth in a xenotransplant of this aggressive leukemia. As a result, BET inhibitors
represent a new class of molecules that may possess therapeutic potential for the
treatment of this aggressive AML subtype.

References:
(1) Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith, W. B.; Fedorov, O.;
Morse, E. M.; Keates, T.; Hickman, T. T.; Felletar, I.; et al. Selective Inhibition
of BET Bromodomains. Nature 2010, 468, 1067–1073.

1

Chapter 1. Microwave-assisted Synthesis of Nitrogen-containing
Hydroxymethylenebisphosphonate Drugs

1.1 Introduction
Bisphosphonates (BPs) are stable analogues of inorganic pyrophosphate, in which
the P-O-P bond is replaced by a non-hydrolyzable P-C-P bond.
1
Whereas the
phosphoanhydride bond is quite labile, the BP P-C bond is usually stable towards
degradation by chemicals, enzymes and high temperatures.
2
It was discovered in the
1960s that these compounds had a high affinity for bone and that they were effective for
the treatment of diseases associated with bone mineralization
3
such as osteoporosis,
Paget’s disease and hypercalcemia.
4
Additionally, these compounds have been found to
be active against the metastases of breast and prostate cancer to the bone.
2
Low bone
density and osteoporosis present a large threat to the health of millions of people
worldwide, and it has been predicted by the U.S. Surgeon General that by the year 2020,
50% of Americans over the age of 50 could be at risk for fractures due to low bone mass
and osteoporosis.
5
This decrease in bone mass is caused by an imbalance in the activities
of two types of bone cells; osteoclasts, which are responsible for bone resorption, and
osteoblasts, which are responsible for bone formation.
6

The general structure for BPs is shown in
Figure 1.1.1. There are two variable groups, R
1

and R
2
, attached to the geminal carbon. The
structure of these variable groups determines the
biological activity of the BP, as well as its affinity
Figure 1.1.1. The general structure for
BPs, where R
1
and R
2
dictate the activity
level of the drug and its affinity to bone.

2

towards bone.
2
This affinity to bone arises from the presence of two phosphonate groups
that are capable of bidentate chelation to divalent metal cations such as calcium, which is
present in copious amounts in the hydroxyapatite (HAP) of bone. In BPs where R
1

represents another OH group, the chelation becomes tridentate, as shown in Figure 1.1.2,
enhancing bone affinity.
2,3
Furthermore, the introduction of a nitrogen atom in the R
2

group may increase bone affinity (Figure 1.1.2)
3,7–9
, and can also significantly increase
the anti-resorptive potency of the BP. BPs accumulate at the surface of the bone,
particularly at areas of high bone turnover, thus being taken up primarily by osteoclasts,
or “bone digesting” cells, allowing them to limit the otherwise heightened activity level
of these cells, which is the underlying cause of the decrease in bone mass.
There are two classes of BPs, as shown
in Figure 1.1.3. The earlier generation BPs,
such as clodronate, etidronate and tiludronate,
do not contain a nitrogen atom in their side
chains, whereas the more potent ones, which
include alendronate, ibandronate, risedronate
(Ris), and zoledronate (Zol) all contain
nitrogen.
2
The difference in the potencies
arises from the different modes of action for
the two classes. Non-nitrogen-containing BPs are metabolized into non-hydrolyzable
ATP analogues that accumulate in osteoclasts, causing the inhibition of adenine
nucleotide translocase.
2
In turn, the inhibition of this enzyme leads to the downstream
Figure 1.1.2. Interaction of BPs with HAP in
the bone is mediated by the two phosphate
groups, as well as the side chain OH group
and N atom.
10

3

release of cytochrome C from the mitochondria, activating caspase-3 mediated apoptosis,
or cell death.
2

In contrast, as shown in Figure 1.1.4, the nitrogen-containing 1-
hydroxymethylene BPs (N-BPs), which are 10,000 times more potent, are not
metabolized in the cell, and instead inhibit the enzyme farnesyl diphosphate synthase
(FPPS) in osteoclasts at nM concentrations.
2
The presence of the nitrogen atom affects
binding substantially by forming a hydrogen bond to a critical and conserved threonine
residue in the active site of FPPS (Figure 1.1.5
10
).
2
This enzyme is involved in the
cellular mevalonate pathway, which is responsible for producing cholesterol and the
isoprenoid lipids necessary for the prenylation of signaling proteins such as GTPases.
2

Figure 1.1.3. Non-nitrogen-containing BPs vs. nitrogen-containing BPs.

4

Without these post-translational modifications, the proteins cannot perform their
functions, which include vital cellular processes such as cytoskeletal arrangement and
trafficking of intracellular vesicles, leading to the impairment of cell function and cell
death.
2
Because N-BPs selectively act upon osteoclasts, they effectively reduce excess
bone resorption.
2
As a result, these drugs are important in the fight against the rise in
osteoporosis and other bone mineralization diseases, and it is imperative for researchers
in this field to be able to generate libraries of novel BPs for continued studies.
Figure 1.1.4. Cellular mevalonate pathway and the intervention of N-BPs.
2

5

One of the methods that synthetic chemists have adapted over the last few
decades to increase the fecundity of their research relies on microwave irradiation. The
first published use of microwave ovens in organic synthesis was by Gedye et al. in 1986.
The authors utilized a domestic oven with 9 power settings, running their reactions at a
power setting of 7, which corresponded to approximately 558 Watts.
11
Four different
reactions were studied: the acid hydrolysis of benzamide to benzoic acid, the oxidation of
toluene to benzoic acid with permanganate, the esterification of benzoic acid using
methanol, propanol and butanol, and the reaction between sodium 4-cyanophenoxide and
benzyl chloride to give 4-cyanophenyl benzyl ether.
11
All reactions were carried out in
sealed Teflon vessels, and the reaction times and yields were compared to traditional
reflux conditions.
11
Due to the pressure and superheating (heating a solvent above its
boiling point)
12
effects associated with the use of sealed vessels, the largest enhancements
in reaction rates were seen with low boiling point solvents, with the largest increase being
Figure 1.1.5. X-ray structures illustrating the critical interaction between the N atom in the side
chains of the N-BPs Ris and Zol, with a conserved Thr residue in FPPS.
10

6

240 times over conventional heating.
11
However, due to the lack of control over pressure
with domestic ovens, violent explosions limited the scope of their studies.
11
Later that
year, Giguere et al. published the second study utilizing microwave irradiation in organic
synthesis.
13
Through their experiments, the authors were the first to demonstrate the
effect of solvent choice on reaction rate. Specifically, the authors determined that the
higher the dielectric constant of the solvent, the more rapid the heating in the microwave.
Since its introduction in 1986, there have been a substantial number of
publications illustrating the advantages of microwave-assisted organic synthesis, or
MAOS, over conventional heating. These advantages include the drastic reduction in
reaction times, the enhancement of product yields, as well as the elimination of unwanted
side products.
14,15
Furthermore, with the advent of research-grade commercial microwave
ovens, researchers can now precisely control the temperature and pressure profiles, as
well as the power outputs in
their reactions, ensuring the
facile reproduction of results.
12,15-16
Whereas domestic ovens
possess 3 to 6 different modes
– 3 dimensional wave patterns
created by microwaves
bouncing off the walls –
creating hot and cold spots,
commercial ovens are often
single-mode, effecting uniform
Figure 1.1.6. The thermal effect in microwave irradation.
18

7

heating.
17
Moreover, because it is possible to use closed vessels in MAOS, solvents with
low boiling points can be utilized, simplifying product workups
17
and allowing solvents
to be heated past their boiling points.
12
As such, almost all types of reactions have been
carried out by MAOS, including Diels-Alder, Heck, Suzuki, Mannich, hydrolysis,
dehydration, esterification, cycloaddition, epoxidation, reduction, condensation,
protection/deprotection, and cyclizations, to name a few.
12
In fact, it has been shown that
certain reactions, which are impossible under conventional heating, can be achieved in
the microwave.
12,18

The advantages that MAOS provides over conventional heating are believed to
stem from two mechanisms. The first is referred to as the thermal effect, which involves
the direct heating of polar molecules and ions present in the reaction mixture.
12,14,19
The
electric field generated by the microwaves alternates, usually at a frequency of 2.4
GHz,
14,20
causing the polar molecules and ions to rotate quickly as they align with the
external field,
12,14
as depicted in Figure 1.1.6. As a result of friction and dielectric loss,
heat is efficiently generated directly in the reaction mixture.
14
Consequently, the more
polar the solvent, the more efficient the heating, and the more pronounced the microwave
thermal effect,
19
explaining the finding by Giguere et al. that the higher the solvent’s
dielectric constant, the more rapid the heating.
13
In comparison, with conventional
heating methods, such as the use of an oil or sand bath, the heat comes from the outside
and is transferred by conduction, creating uneven heating zones and becoming less
effective towards the center of the reaction mixture.
19
Conventional heating may also
create hot zones on the surface of reaction vessels, causing decomposition, whereas the
direct heating of polar molecules creates a uniform mode of heating.
17

8

The second mechanism, known as the non-thermal effect, has been the subject of
much controversy in the literature. It is believed that the electric field generated within
the microwave interacts with polar molecules, such as starting materials and transition
states, stabilizing them.
21
This effect is also believed to allow for selectivity in chemical
reactions
12,21
– i.e., if two reaction pathways are possible, the one with the more polar
transition state(s) would predominate. The increase in reaction rates by the non-thermal
effect can be understood by examining the Arrhenius law, k = Ae
-Ea/RT
, which formulates
a relationship between temperature and time.
12
The value of A in this equation, the pre-
exponential factor, depends on the frequency of vibration of atoms at the interface of the
reaction.
12
It has been proposed that the values of A and the activation energy (E
a
) are
affected by orientation effects of polar molecules and intermediates during microwave
irradiation.
12,21
In fact, claims have been made that A can be increased by a factor of
3.3,
12
while E
a
can be decreased due to an increase in polarity in going from ground state
to transition state,
21
culminating in an increase in the overall reaction rate. As there is no
experimental proof for the non-thermal effect, it still remains a debatable topic. In an
attempt to debunk the theory of the non-thermal effect, Herrero et al. used an IR probe to
directly measure the temperatures of reaction mixtures undergoing microwave irradiation,
and found that there were large temperature fluctuations, particularly in the case of
viscous or heterogeneous mixtures.
21
However, the authors added that stirring usually
alleviates this problem.
21
As such, further studies are required in this area to ascertain the
validity of a non-thermal effect in MAOS.
Medicinal chemists are on an undying quest towards simplifying and improving
the efficiency of synthetic protocols, with the goal of rapidly generating compound

9

libraries that can be tested for biological activity.
15,17
The high rate of failure in clinical
trials is a substantial bottleneck, making it crucial to be able to quickly determine the
usefulness of a molecule as a possible drug, favoring a high throughput approach to drug
discovery.
15
MAOS is therefore an extremely valuable tool, as it allows medicinal
chemists and pharmaceutical companies to not only rapidly create libraries of compounds
for screening, but to also quickly and efficiently optimize reaction conditions.
17
As a
result, inactive molecules are eliminated early in the discovery process, and active ones
are placed into the market pipeline at a faster pace than would otherwise be possible.
14,17

This is particularly critical in the development of antivirals and antiobiotics, where it is
imperative to rapidly create new drugs to combat the ongoing development of
resistance.
18
MAOS is also considered to be a “greener” synthetic approach, resulting in
less power usage (shorter reaction times), which not only reduces pollution, but also
increases cost-effectiveness.
22

In an effort to improve the synthetic protocols for N-BPs, many studies have been
published
23–29
with variations in the solvent type and reagent ratios, but none of these
studies have employed MAOS. Other BPs, however, have been synthesized using
microwave irradiation. For example, unsubstituted tetraethyl BPs were synthesized from
dihaloalkanes and diethyl phosphite,
30
while α-hydroxymethylenebisphosphonate esters
were synthesized from α-oxophosphonates and dialkyl phosphites,
31
and α-
aminomethylenebisphosphonates were produced from amines, diethyl phosphite and
triethyl orthoformate.
32
This work represents the first example of the application of
MAOS in the synthesis of several well-known N-BP drugs currently on the market, in

10

good yields, and in much shorter reaction times than those required by conventional
synthetic methods.
33

1.2 Results and Discussion
One of the most commonly utilized protocols for the synthesis of N-BPs is
Kieczykowski’s method,
23
which involves a reaction between phosphorus trichloride,
phosphorus pentachloride, or phosphorus oxychloride with phosphorous acid and a
carboxylic acid. The mixture starts to thicken as the reaction proceeds, which impedes
stirring and can lead to sub-optimal yields. A hydrolysis step follows, furnishing the final
bisphosphonic acid products.
Many solvents have been used in the preparation of N-BPs, in attempts to mitigate
the problem of reaction mixture solidification. These include methanesulfonic acid
(MSA),
23
which is successful at solubilizing the reaction components, but is
disadvantageous due to its dangerous nature. MSA reacts with PCl
3
in an exothermic
manner that becomes uncontrollable
24
and problematic on a commercial scale
25
. It also
requires long reaction times (16-20 h),
23
furnishes low yields,
24
and requires a large
amount of alkali in the workup for neutralization.
25
Polar aprotic solvents such as
acetonitrile (ACN), dimethylol ethylene urea (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-
2(1H)-pyrimidinone (DMPU) and N-methylpyrrolidone (NMP) have also been employed
in the synthesis of N-BPs.
27
Although these solvents are safe to use, keep the reaction
mixture fluid, and are water miscible, they also require long reaction times.
Another solvent that was discovered to overcome the issues with solidification
and safety is sulfolane. This solvent proved to be advantageous for several additional

11

reasons. Firstly, the synthesis of N-BP intermediates was completed within 3.5 h via
conventional heating.
25
Also, it is water miscible and neutral, obviating the need for
removal prior to hydrolysis.
20
Additionally, sulfolane is non-toxic, and more
environmentally friendly than some of the other solvents commonly utilized.
19

In this study,
BP drugs with both aromatic (Ris and Zol), as well as aliphatic
(alendronate, pamidronate and neridronate) nitrogen-containing side chains (Table 1.2.1)
were successfully synthesized by MAOS.
33
The synthesis was carried out using a
modification of Kieczykowski’s method,
23
which conventionally uses a 1:1:2 ratio of
carboxylic acid: H
3
PO
3
: PCl
3
. Instead, a 1:3:3 ratio of carboxylic acid: H
3
PO
3
: PCl
3
was
used, and the reaction was run at 1/50
th
of the literature scale. Several reactions were
attempted with 3-pyridylacetic acid to synthesize Ris. The first combination of conditions
involved ACN as solvent, and included an 18 min microwave irradiation at 55 °C,
followed by an overnight hydrolysis via conventional heating, with the best yield
achieved being 46%. In order to improve the yield, reactions at 65 °C were attempted, but
the heating was uncontrollable in ACN. This is likely due to the exothermic nature of the
initial reaction between carboxylic acid and PCl
3
, whose effect seems to be magnified by
microwave irradiation. Conversely, reaction at 65 °C via conventional heating to
synthesize Ris required 5 h in ACN and resulted in a 72% yield of product.
In order to improve yield but avoid overheating, longer reaction times at 55 °C
were attempted in the microwave. A 1 h reaction yielded only 32% of pamidronate and
45% of alendronate, while a 1.5 h reaction yielded a mere 20% of Ris. In other trials,
PCl
3
was added drop-wise during microwave irradiation, but no improvements in yield
were realized. Also, a stepwise ramping up of temperature from room temperature to 35

12

°C, then to 55 °C was attempted, with the idea that completing the first exothermic step
of the reaction at the lower temperature could improve yield, but yields remained
unacceptably low. As a result, it became evident that a solvent with a higher boiling
point was required in order to control the heating. The solvent that was determined to be
most appropriate for our purposes was sulfolane. In addition to the reasons mentioned
above, sulfolane was chosen because, with a dielectric constant of 43, the high polarity of
this solvent improves the efficacy of microwave heating.
19
Furthermore, with its high
boiling point of 285 °C, this solvent allowed for the use of high reaction temperatures, if
necessary.
19

The synthetic method that was developed is shown in Scheme 1.2.1 and the N-
BPs synthesized are depicted in Table 1.2.1. The PCl
3
was added in one aliquot as
opposed to the conventional method, in which PCl
3
is added slowly.
23–29
Also, the scale
of the reaction was significantly reduced, from a gram or kilogram scale to a milligram
scale. One equiv of the appropriate carboxylic acid was added to three equiv of
phosphorous acid in a dry flask. 1.6 mL of dry sulfolane was added to the mixture that
was then warmed and stirred briefly to achieve dissolution. After cooling, three equiv of
phosphorus trichloride were added. The flask was then placed in a Milestone Ethos Synth
microwave reactor that was fitted with a condenser through which cold water was
passed.
33

13

Scheme 1.2.1. Modified method for the MAOS of nitrogen-containing 1-hydroxymethylenebisphosphonic
acids. The preferred solvent in step 1 is sulfolane.
33

Table 1.2.1 BPs synthesized via Scheme 1.2.1. For simplicity, the compounds are depicted as acids.

Compound

R
1

R
2

Risedronic acid (1)

OH

Zoledronic acid (2)

OH

Pamidronic acid (3)

OH

Alendronic acid (4)

OH

Neridronic acid (5)

OH

The first step of the reaction, which yielded an intermediate product mixture,
required 3 min and 15 s for the shortest reaction, where the maximum power reached was

14

300-400 W, and 7 min for the longest reaction, where the maximum power reached was
200-300 W. All reactions were carried out at 65 °C. The intermediate mixtures were then
quenched with 6 mL of water, transferred to a sealed 50 mL quartz vessel, and
hydrolyzed in the microwave for 10 min at 150 °C. The maximum power reading for this
step was 450-500 W. A yellow-orange side product precipitates after the first step and
can be removed by centrifugation following the addition of water, either before or after
the hydrolysis step.
33

Following hydrolysis, the pH was adjusted to yield the BPs as either free acids or
monosodium salts. As a control, compounds 2, 3 and 4 were synthesized via conventional
heating, which required 3.5 h at 65 °C for the first step, followed by a 6+ h reflux in
water for hydrolysis to the final BP products. As Table 1.2.2 shows, the yields obtained
via microwave irradiation were comparable to the yields via conventional heating, but
required less than 20 min in total, as opposed to at least 9.5 h.
33

Table 1.2.2 Results of the microwave-assisted synthesis of BPs via Scheme 1.2.1 and comparison to
conventional heating. All MW programs for the synthesis of intermediate include a 3 min ramp to 65 °C,
while all hydrolysis programs include a 4 min ramp to 150 °C.

Compound

Time for MW synthesis
of intermediate (65 °C)

Time for MW
hydrolysis
(150 °C)

Isolated
yield %,
MW

Isolated yield %,
conventional heating
(3.5 h at 65 °C followed
by reflux for 6+ h)

1

3 min 15 s

10 min

74

-

2

3 min 45 s

10 min

70

67

3

3 min 15 s

10 min

64

72

4

7 min

10 min

41

38

5

7 min

10 min

53

-

15

1.3 Conclusion
A rapid, simple, and efficient method for the small-scale synthesis of nitrogen-
containing 1-hydroxymethylenebisphosphonate drugs via microwave irradiation was
established. Whereas the traditional method for the preparation of these compounds
typically requires heating from 3.5 h to overnight for the first step, followed by a 6+ h
hydrolysis step, this new microwave-assisted procedure is complete in less than 20 min.
Thus, the total reaction time was effectively reduced from at least 9.5 h to minutes, while
maintaining good yields of product. These results illustrate the powerful potential of
MAOS in the quick, facile and cost-effective generation of libraries of N-BPs and other
drugs for biological testing.
1.4 Experimental
1.4.1 Materials and Methods
3-Pyridylacetic acid hydrochloride was purchased from Alfa Aesar, imidazol-1-
yl-acetic acid and 6-aminohexanoic acid from AK Scientific, β-alanine from Aldrich and
γ-aminobutyric acid from Sigma-Aldrich. Imidazol-1-yl-acetic acid was also synthesized
following a published procedure
34
from imidazole, purchased from Aldrich, ethyl
bromoacetate purchased from Lancaster Synthesis, and benzyltriethylammonium
bromide, purchased from Alfa Aesar. Sulfolane was purchased from Sigma-Aldrich and
was distilled under reduced pressure prior to use.
The NMR operating frequencies were 500 MHz for
1
H and 202 MHz for
31
P.
1
H
NMR spectra were referenced to residual HDO (δ 4.79) in D
2
O
35
, while
31
P NMR spectra
were referenced against an external 85% H
3
PO
4
standard (δ 0.00). All chemical shift

16

values (δ) are given in ppm, the concentrations of all NMR samples was approximately 1-
3 mg/mL, and NMR sample pH values were measured in 99.9% D
2
O without deuterium
isotope correction. A Thermo-Finnigan Deca XP Max mass spectrometer with an ESI
probe was utilized for all low-resolution mass spectra.
1
H and
31
P NMR spectra, as well
as mass spectra for 1-5 are presented in Appendix A. LC-MS (1 and 2) and UV-vis data
(1) are also presented in Appendix A. Additionally, Appendix A includes
1
H and
31
P
NMRs of microwave synthesized Ris, Zol, pamidronate and alendronate products, each
spiked with commercially acquired samples. These samples were 98+% pure Ris
disodium sample supplied by Procter & Gamble, Inc., Zol purchased from Molekula,
Inc., pamidronate monosodium supplied by Novartis, Inc., and alendronate monosodium
supplied by Procter & Gamble, Inc., respectively. All NMR data agreed with the
literature values.
1.4.2 Synthesis of Nitrogen-containing 1-Hydroxymethylenebisphosphonates
Imidazol-1-yl acetic acid. The starting carboxylic acid for the synthesis of Zol,
imidazol-1-yl acetic acid, was synthesized according to a published procedure.
34
To a dry
500 mL flask, 73.6 mmol (2 equiv) of KOH, 73.6 mmol (2 equiv) of K
2
CO
3
and 150 mL
of dry CH
2
Cl
2
(distilled over P
2
O
5
) were added. The contents were stirred at room
temperature, and 73.6 mmol (2 equiv) of imidazole and 0.37 mmol (0.01 equiv) of
benzyltriethylammonium bromide were added. The mixture was stirred vigorously for 10
min, after which 36.7 mmol (1 equiv) of ethyl bromoacetate was added. The reaction
mixture was stirred under nitrogen for 8 h at room temperature. The mixture was then
filtered and the residue was washed with 20 mL of CH
2
Cl
2
three times. The filtrate was
then washed with 50 mL of chilled water, and the organic layer was collected and dried

17

over sodium sulfate overnight. After filtration, the CH
2
Cl
2
was removed in vacuo,
yielding approximately 5 mL of an oily ester, which was transferred to a flask and
hydrolyzed with 25 mL of water in an overnight reflux. The water was then removed
under reduced pressure, and the product was refluxed for an additional 3 h with 25 mL of
200 proof ethanol. The product was then filtered and dried under vacuum until constant
weight.
1
H NMR (500 MHz, D
2
O) δ: 4.87 (s, 2H), 7.48 (s, 2H), 8.72 (s, 1H).
General procedure for the synthesis of bisphosphonates 1-5. 11.4 mmol (3
equiv) of H
3
PO
3
were added to a dry flask. 3.8 mmol (1 equiv) of the appropriate
carboxylic acid was then added. This corresponded to 658 mg of 3-pyridylacetic acid
hydrochloride for 1, 478 mg of imidazol-1-yl-acetic acid for 2, 339 mg of β-alanine for 3,
392 mg of γ-aminobutyric acid for 4, and 499 mg of 6-aminohexanoic acid for 5. 1.6 mL
of dry (distilled) sulfolane was then added and the contents of the flask were briefly
heated and stirred to dissolve the solids. The solution was cooled down to approximately
25-35 °C, and then 11.4 mmol (3 equiv) of PCl
3
were added immediately. The flask was
placed in a Milestone Ethos Synth Microwave Synthesis Labstation and was fitted with a
condenser through which cold water was passed.
The microwave programs for the synthesis of 1-5 included an initial 3 min ramp
to 65 °C. For the synthesis of 1 and 3, the microwave program was set to 15 s at 65 °C
after the initial ramp, while for 2, irradiation at 65 °C for 45 s was required to achieve
completion. As for 4 and 5, the microwave temperature was set to 65 °C for 4 min
following the 3 min ramp to that temperature. The power was set to a maximum of 1200
W, and was allowed to adjust automatically to reach and maintain the temperature
designated by the program. The temperature was measured accurately by an IR sensor

18

that is built in to the microwave reactor. For the synthesis of the intermediates of 1 and 2,
the power fluctuated between 0 and a maximum of 300-400 W, while for 3, 4, and 5, the
maximum power reached was typically 200-300 W.
After microwave irradiation, a solid mixture forms which consists of intermediate
phosphorus compound together with an undesired yellow-orange side product. This
mixture was quenched with 6 mL of water, and the side product was easily removed by
centrifugation after dissolution in water, either before or after the hydrolysis step. The
intermediate solution was transferred to a 50 mL sealed quartz reaction vessel, in which
the hydrolysis to bisphosphonic acid was carried out. The program for the microwave
was set to a 6 min ramp to 150 °C, followed by 4 min at that temperature. The power was
again allowed to adjust automatically, and generally fluctuated between 0 and a
maximum of 450-500 W.
Following hydrolysis, the pH of the mixtures for acids 1, 3, 4, and 5 was
adjusted
23
with NaOH and the mixture was aged at 0-5 °C until crystallization was
complete. 4 and 5 were precipitated as monosodium salts by stirring with 2-5 mL of
ethanol for 1-2 h at room temperature. 2 was precipitated as an acid by the addition of 9
mL of acetone to the acidic hydrolysis mixture and stirring for 3-4 h at room temperature.
The white crystalline products were then filtered and washed with cold water and acetone
or ethanol, and dried under vacuum at 45 °C to a constant weight. 1-5 were characterized
by
31
P NMR,
1
H NMR, and mass spectrometry. 1 and 2 were additionally characterized
by LC-MS, and 1 by UV-vis analysis. All data are presented in Appendix A. NMR data
are reported for solutions whose pH was adjusted with sodium bicarbonate.

19

Control experiments were carried out for the synthesis of 2, 3, and 4 by following
the same one-pot protocol, with the exception of using conventional heating in a sand
bath as opposed to microwave irradiation. The first step, towards the synthesis of the
intermediate, required heating for 3.5 h at 65 °C in sulfolane, while the hydrolysis step
required a reflux for 6-15 h.
Risedronic acid ((1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic
acid) monohydrate, 1.
1
H NMR (500 MHz, D
2
O, pH 8.9) δ: 3.26-3.31 (t, 2H), 7.37-7.39
(q, 1H), 7.94-7.96 (d, 1H), 8.36-8.37 (d, 1H), 8.53 (s, 1H).
31
P NMR (202 MHz, D
2
O, pH
8.9, 85% H
3
PO
4
reference) δ: 18.22. MS (ESI) C
7
H
12
NO
7
P
2
(M+H)
+
m/z calcd: 284.12.
Found: 283.99. Content of risedronic acid monohydrate calculated by UV (Appendix A):
99.6%.
1
H and
31
P NMRs (Appendix A) of synthesized Ris spiked with a Ris disodium
sample provided by Procter & Gamble, Inc. (98+% purity) proved that the two
compounds were identical.
Zoledronic acid ([1-hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-
diyl]bis(phosphonic acid)) monohydrate, 2.
1
H NMR (500 MHz, D
2
O, pH 10.1) δ:
4.54-4.58 (m, 2H), 7.13 (s, 1H), 7.39 (s, 1H), 8.17 (s, 1H).
31
P NMR (202 MHz, D
2
O, pH
10.1, 85% H
3
PO
4
reference) δ: 15.69. MS (ESI) C
5
H
11
N
2
O
7
P
2
(M+H)
+
m/z calcd: 273.10.
Found: 272.96.
1
H and
31
P NMRs (Appendix A) of synthesized Zol spiked with a Zol
sample purchased from Molekula, Inc. showed that the two compounds were identical.
Pamidronic acid ((3-amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid))
monosodium monohydrate, 3.
1
H NMR (500 MHz, D
2
O, pH 8.8) δ: 2.22-2.30 (m, 2H),
3.31-3.34 (t, 2H).
31
P NMR (202 MHz, D
2
O, pH 8.8, 85% H
3
PO
4
reference) δ: 18.32. MS

20

(ESI) C
3
H
10
NO
7
P
2
(M-Na)
-
m/z calcd: 234.06. Found: 234.2. Anal calcd. for
C
3
H
9
NO
7
P
2
Na.H
2
O: C, 13.15%; H, 4.05%; N, 5.11%. Found: C, 12.94%; H, 4.23%; N,
4.92%.
1
H and
31
P NMRs (Appendix A) of synthesized pamidronate spiked with a
pamidronate monosodium sample provided by Novartis, Inc. showed that the two
compounds were identical.
Alendronic acid ([4-amino-1-hydroxy-1-(hydroxy-oxido-phosphoryl)-
butyl]phosphonic acid) monosodium dihydrate, 4.
1
H NMR (500 MHz, D
2
O, pH 8.9)
δ: 1.96-2.0 (m, 4H), 3.04-3.06 (t, 2H).
31
P NMR (202 MHz, D
2
O pH 8.9, 85% H
3
PO
4

reference) δ: 18.90. MS (ESI) C
4
H
12
NO
7
P
2
(M-Na)
-
m/z calcd.: 248.09. Found: 248.30.
Anal calcd. for C
4
H
11
NO
7
P
2
Na.2H
2
O: C, 15.70%; H, 4.94%; N, 4.58%. Found: C,
15.47%; H, 5.08%; N, 4.40%.
1
H and
31
P NMRs (Appendix A) of synthesized
alendronate spiked with an alendronate monosodium sample provided by Procter &
Gamble, Inc. showed that the two compounds were identical.
Neridronic acid (6-amino-1-hydroxyhexane-1,1-diyl)bis(phosphonic acid))
monosodium monohydrate, 5.
1
H NMR (500 MHz, D
2
O, pH 8.9) δ: 1.38-1.44 (m, 2H),
1.57-1.63 (m, 2H), 1.68-1.74 (m, 2H), 1.88-1.94 (m, 2H), 3.01-3.04 (t, 2H).
31
P NMR
(202 MHz, D
2
O, pH 8.9, 85% H
3
PO
4
reference) δ: 19.46 MS (ESI) C
6
H
16
NO
7
P
2
(M-Na)
-

m/z calcd.: 276.14. Found: 276.1. Anal calcd. for C
6
H
15
NO
7
P
2
Na.H
2
O: C, 22.80%; H,
5.42%; N, 4.43%. Found: C, 22.83%; H, 5.61%; N, 4.33%.

21

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Houben-Weyl Methods of Molecular Transformation. In; Trost, B. M., Ed.; Georg
Thieme: Stuttgart, 2009; Vol. 42, p. 804.

(2) Thompson, K.; Rogers, M. J. The Molecular Mechanisms of Action of
Bisphosphonates. Clin. Rev. Bone Miner. Metab. 2007, 5, 130–144.

(3) Widler, L.; Jaeggi, K. A.; Glatt, M.; Müller, K.; Bachmann, R.; Bisping, M.; Born,
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Bisphosphonates. From Pamidronate Disodium (Aredia) to Zoledronic Acid
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(4) Russell, R. G. G.; Watts, N. B.; Ebetino, F. H.; Rogers, M. J. Mechanisms of
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Treatment of Low Bone Density and Osteoporosis. J. Med. Chem. 2006, 49, 3060–
3063.

(6) Xie, Y.; Ding, H.; Qian, L.; Yan, X.; Yang, C.; Xie, Y. Synthesis and Biological
Evaluation of Novel Bisphosphonates with Dual Activities on Bone in Vitro.
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(7) Kavanagh, K. L.; Guo, K.; Dunford, J. E.; Wu, X.; Knapp, S.; Ebetino, F. H.;
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Nitrogen-containing Bisphosphonates as Antiosteoporosis Drugs. Proc. Natl.
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(8) Rondeau, J.-M.; Bitsch, F.; Bourgier, E.; Geiser, M.; Hemmig, R.; Kroemer, M.;
Lehmann, S.; Ramage, P.; Rieffel, S.; Strauss, A.; et al. Structural Basis for the
Exceptional in Vivo Efficacy of Bisphosphonate Drugs. ChemMedChem 2006, 1,
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(11) Gedye, R.; Smith, F.; Westaway, K.; Ali, H.; Baldisera, L.; Laberge, L.; Rousell, J.
The Use of Microwave Ovens for Rapid Organic Synthesis. Tetrahedron Lett.
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(12) Man, A. K.; Shahidan, R. Microwave-assisted Chemical Reactions. J. Macromol.
Sci. Part 2007, 44, 651–657.

(13) Giguere, R. J.; Bray, T. L.; Duncan, S. M.; Majetich, G. Application of
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(14) Kappe, C. O.; Dallinger, D. The Impact of Microwave Synthesis on Drug
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(15) Colombo, M.; Peretto, I. Chemistry Strategies in Early Drug Discovery: An
Overview of Recent Trends. Drug Discov. Today 2008, 13, 677–684.

(16) Zhang, S.; Arvidsson, P. I. Facile Synthesis of N-protected Amino Acid Esters
Assisted by Microwave Irradiation. Int. J. Pept. Res. Ther. 2008, 14, 219–222.

(17) Mavandadi, F.; Pilotti, Å. The Impact of Microwave-assisted Organic Synthesis in
Drug Discovery. Drug Discov. Today 2006, 11, 165–174.

(18) Leonetti, F.; Capaldi, C.; Carotti, A. Microwave-assisted Solid Phase Synthesis of
Imatinib, a Blockbuster Anticancer Drug. Tetrahedron Lett. 2007, 48, 3455–3458.

(19) Yin, J.; Zhang, A.; Liew, K. Y.; Wu, L. Synthesis of Poly(ether Ether Ketone)
Assisted by Microwave Irradiation and Its Characterization. Polym. Bull. 2008, 61,
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Common Solvents and Application in a Diels–Alder Organic Synthesis. Org.
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(21) Herrero, M. A.; Kremsner, J. M.; Kappe, C. O. Nonthermal Microwave Effects
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(23) Kieczykowski, G. R.; Jobson, R. B.; Melillo, D. G.; Reinhold, D. F.; Grenda, V. J.;
Shinkai, I. Preparation of (4-Amino-1-Hydroxybutylidene)bisphosphonic Acid
Sodium Salt, MK-217 (Alendronate Sodium). An Improved Procedure for the
Preparation of 1-Hydroxy-1,1-bisphosphonic Acids. J. Org. Chem. 1995, 60,
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(24) Singh, G. P.; Jadhav, H. S.; Maddireddy, N. V.; Srivastava, D. Process for the
Production of 4-Amino-1-hydroxybutylidene-1,1-bisphosphonic Acid or Salts
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(25) Patel, V. M.; Chitturi, T. R.; Thennati, R. A Process for Preparation of
Bisphosphonic Acid Compounds.
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(26) Gore, V. G.; Shukla, V. K.; Ghadge, M. M.; Avadhut, R. M. Novel Process for the
Preparation of Bisphosphonic Acids. WO 2008/004000 A1.

(27) Baptista, J.; Mendes, Z. Process for the Preparation of Bisphosphonic Acids and
Salts Thereof. WO 2008/056129 A1.

(28) Danda, S. R.; Garimella, K. A. S. S. N.; Divvela, V. N. S. R.; Dandala, R.;
Meenakshisunderam, S. An Improved Process for the Preparation of
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WO 2007/083240 A2.

(29) Dembrowski, L.; Krzyzanowski, M.; Rynkiewicz, R.; Szramka, R.; Roznerski, Z.;
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24

(30) Meziane, D.; Hardouin, J.; Elias, A.; Guénin, E.; Lecouvey, M. Microwave
Michaelis-Becker Synthesis of Diethyl Phosphonates, Tetraethyl Diphosphonates,
and Their Total or Partial Dealkylation. Heteroat. Chem. 2009, 20, 369–377.

(31) Grün, A.; Molnár, I. G.; Bertók, B.; Greiner, I.; Keglevich, G. Synthesis of Α-
hydroxy-methylenebisphos-phonates by the Microwave-assisted Reaction of Α-
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(32) Kaboudin, B.; Alipour, S. A Microwave-assisted Solvent- and Catalyst-free
Synthesis of Aminomethylene Bisphosphonates. Tetrahedron Lett. 2009, 50,
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25

Chapter 2. Microwave Studies on the BTMS Dealkylation of
Phosphonate Esters
2.1 Introduction
Phosphonic acids and their salts have elicited a lot of synthetic interest due to the
breadth of utility they display.
1
For example, phosphonates have been used as antibiotic
and antiviral agents, as enzyme inhibitors, as chelating agents in imaging, catalysis and
ion exchange, and as components of the polymers used in paints and adhesives.
1

When phosphonates are present as part of a complex molecule, they are usually
maintained in their ester form to prevent side reactions during multi-step synthetic
pathways, and are cleaved back to the acid form in the final step towards the product of
interest.
2
Though there are several ways to achieve this, the most commonly used method
for phosphonate ester dealkylation is the McKenna reaction (Figure 2.1.1)
3–5
which
involves the use of
bromotrimethylsilane
(BTMS) to convert the
esters into their
corresponding
trimethylsilyl
phosphonates, which are
then facilely converted into
phosphonic acids by
hydrolysis with water or methanol.
4
This reaction is not only simple, but is mild and
efficient, usually achieving quantitative yields. The McKenna phosphonate ester
Figure 2.1.1. Suggested mechanism of the McKenna method
for phosphonate ester dealkylation by BTMS. The TMS esters
are hydrolyzed to phosphonic acids with water or methanol.

26

dealkylation is also compatible with a wide range of functional groups including alkenyl,
alkoxyalkyl, benzyl, benzoyl, and diazomethyl groups, making it very useful in
phosphorus chemistry.
5
A further advantage to the use of BTMS over other dealkylation
methods is its complete selectivity towards phosphonate esters when a molecule contains
both phosphonate and carboxylate esters.
4–7

As discussed in depth in Chapter 1, MAOS has rapidly gained popularity in the
last two to three decades. This is because, in comparison to conventional heating
methods, the use of microwave irradiation results in improved efficiencies, due to the
drastic reduction in reaction times (hours or days to minutes),
2,8
cleaner reaction profiles,
9

increased reproducibility,
10
and higher product yields.
2,9
Briefly, this is because
microwave irradiation results in the direct heating of polar molecules in the reaction
mixture, whereas conventional heating by sand or oil bath relies on convection, which is
less uniform and efficient.
8,11–13
For example, in the work described in Chapter 1, we
showed that the synthesis of nitrogen-containing 1-hydroxymethylenebisphosphonate
drugs can be transformed from a 1-2 day process, to one complete in minutes.
14

Very few attempts to improve the efficiency of phosphonate ester dealkylation via
microwave dielectric heating can be found in the literature today. The first study of the
kind, published by Kumar et al. in 2006, utilized BTMS to dealkylate the ethyl (Et) esters
of phosphonates, as well as those of phosphoramides and phosphates.
2
With respect to
phosphonate Et ester cleavage, it was found that over 95% yield could be achieved with 2
equiv of BTMS at 40 °C, in 10 min when the reactions were carried out in the
microwave. The dealkylations were performed in a septum-sealed vessel using
acetonitrile (ACN) as a solvent, and cleavage of the trimethylsilyl phosphonates was

27

achieved by methanolysis. In comparison, conventional heating required 400 min, and
achieved slightly lower yields. Through their work, the authors showed that not only was
BTMS dealkylation possible under microwave irradiation, but it increased the rate of the
reaction by 40 times. Furthermore, the authors demonstrated that the microwave BTMS
reactions were tolerant of labile functional groups such as bromoalkyl, piperazine and
nitrile moieties.
2

In another study published in 2009, Meziane et al.
1
reproduced the procedure
developed by Kumar et al.
2
successfully. In their hands, the authors were able to
quantitatively convert simple phosphonate Et esters into phosphonic acids in 2 min using
BTMS in the microwave. All reactions were performed in an open reaction vessel with a
drying tube, as opposed to a septum-sealed vessel, indicating both that pressure is not
required, and that the reaction can be exposed to air if a drying tube is installed,
increasing the simplicity of the set-up. The authors also used a mixture of
trimethylsilylchloride (TMSCl) and sodium iodide to achieve the dealkylations
successfully, but commented on the need for tedious purification, which led them to
lower yields than those achieved with BTMS. Additionally, 6 equiv each of TMSCl and
NaI were required, compared to only 2 of BTMS, highlighting the greater efficiency and
convenience of the BTMS method for the dealkylation of phosphonate esters.
An alternate method to dealkylate phosphonate esters is to reflux them with HCl.
Recently, Jansa et al., studied the acid-catalyzed hydrolysis of acyclic nucleoside
phosphonate (ANP) esters under microwave irradiation.
12
ANPs are a group of nucleotide
analogues that demonstrate a broad spectrum of biological activity.
16,17
Most notably,
these compounds possess antiviral activity against both DNA and RNA viruses, and have

28

been successfully applied against cytomegalovirus (CMV), human immunodeficiency
virus (HIV), hepatitis B and C viruses (HBV and HBC), herpes simplex virus (HSV), and
poxvirus (such as vaccinia virus, VACV), among many others.
16,18
While various
synthetic routes towards the free phosphonic acids of ANPs have been reported, a widely
used approach involves the condensation of a natural or derivatized heterocyclic base and
an organophosphorus synthon bearing alkyl groups (Et or isopropyl (iPr)) on the
phosphonate to prevent the formation of side products.
18–20
After purification by silica gel
chromatography, the alkyl groups on the phosphonate are typically removed via the
McKenna approach, overnight at room temperature, with equimolar amounts of BTMS.

In their paper, Jansa et al. used 2-3 equiv of HCl, and temperatures of 130-140
°C, to hydrolyze Et and iPr phosphonate esters of ANPs in 20-30 min in a sealed reaction
vessel.
15
However, in the case of the diaminopurine (DAP) ANP derivatives, partial
hydrolysis of the C6 amino groups was observed, leading to the formation of 10%
guanine derivatives.
15
This side reaction was reduced to 5% by lowering the temperature
to 130 °C and using a lower concentration of HCl.
15
As such, yields of products varied
between 77-93%.
15
Though this method was presented as a possible alternative to BTMS
dealkylation, it presents many drawbacks, including the need for high temperatures and
pressures, and the incompatibility of HCl with sensitive groups such as the DAP class of
ANPs. Also, while BTMS is 100% selective towards P-O ester dealkylation, HCl would
also dealkylate C-O esters if present in the molecule.
A clear and in-depth investigation of the scope of microwave-assisted BTMS
dealkylation of phosphonate esters has not been performed as of yet. This study aims to
tackle this issue, by not only comparing the effects of Me, Et and iPr ester groups on

29

reaction rate, but by also examining the role of the solvent and the effects of electron
withdrawing groups, and by demonstrating the selectivity and sensitivity of BTMS
dealkylation in the cases of phosphonoacetates and phosphonates possessing sensitive
groups, respectively. In the latter case, we demonstrate the stability of the acyclic
nucleosides 9-(2-phosphonomethoxy)ethyl adenine (PMEA), 9-[2-
(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) and 9-[(2-(S)-
(phosphonomethoxy)propyl-2,6-diaminopurine ((S)-PMPDAP) when their iPr or Et esters
undergo BTMS-mediated microwave dealkylation.
2.2 Results and Discussion
Simple dimethyl-,
diethyl-, and
diisopropylmethylphosphonate
esters were dealkylated using
BTMS in the microwave.
Solvent type and temperatures
were varied to study the effect on reaction times and yields. Scheme 2.2.1 shows the
general reaction for the phosphonate ester microwave-assisted BTMS dealkylation. The
Me, Et or iPr esters of methylphosphonate were reacted with BTMS in one of five
different solvents: ACN, dioxane, neat BTMS, N,N-dimethylformamide (DMF) or
sulfolane. The reaction temperature was varied between 40 °C and 110 °C, but never
exceeded the boiling point of the solvent, as ambient pressure was used. The open
reaction system included a reflux condenser fitted with a drierite drying tube. The results
are shown in Table 2.2.1. Results for representative control reactions are also included,
Scheme 2.2.1. General reaction scheme for the microwave-
assisted BTMS dealkylation reactions.

30

which were performed by holding all reaction conditions constant, aside from the use of
conventional heating with a sand bath.
As illustrated by Figure 2.1.1, BTMS dealkylation likely proceeds via an SN
2

mechanism. Thus, as expected, the results in Table 2.2.1 demonstrate a decrease in rate
when going from Me to Et to iPr esters. Whereas most of the reactions (with the
exception of entry (5) utilized equimolar amounts of BTMS (2 equiv), the neat reactions
were carried out in 6 equiv of BTMS (3x excess). As such, the reactions in neat BTMS
proceeded with the fastest rates. Since MAOS garners its efficiency from the direct
heating of polar molecules, the neat reactions were possible due to the polar nature of the
reactants themselves. In the cases where solvent was utilized, the more polar the solvent,
the more efficient the microwave heating should be, and the faster the reaction. Although
this was generally found to be true, it was not as clearly evident in all of the results. The
solvents that were used varied in polarity, with sulfolane (ε = 43)
12
being the most polar,
ACN (ε = 37.5) and DMF (ε = 36.7) being similar and intermediate in polarity, and
dioxane (ε = 2.25) being the least polar.
Although the reactions were generally faster in sulfolane than in ACN, which is
less polar, they were also faster in DMF, which is similar in polarity to ACN. This is
most evident in the reactions involving Et and iPr esters. On the other hand, the reactions
in dioxane, which has been previously used in the BTMS dealkylation of phosphonate
esters,
21
were only slightly slower overall, despite its much lower polarity. As for the Me
esters, the reactions were fastest in sulfolane, followed by DMF, dioxane and finally,
ACN. It is likely that due to the polar nature of the reactants themselves, the polarity of
the solvent is not as crucial in determining reaction rates as it is in other microwave

31

irradiated reactions, particularly in the case of Me esters, which are the easiest to remove
via BTMS dealkylation.
Overall, the results revealed that reactions proceeded fastest in neat BTMS,
followed by sulfolane, DMF, ACN and dioxane. Additionally, reactions were 6-120 times
faster in the microwave than they were by conventional heating, and almost always
resulted in better yields. Of particular notability is the ease with which Et and iPr esters
can be removed under microwave irradiation with BTMS, as compared to HCl. As Table
2.2.1 demonstrates, Et esters are completely cleaved in as little as 5 min at 60 °C (in
ACN), while iPr esters are cleaved in as little as 2 min at 80 °C (in DMF), with equimolar
amounts of BTMS.
All reactions were carried out in an open system with a drying tube, thereby
obviating the need for elevated pressure. Furthermore, it was not necessary to run the
reactions under an inert atmosphere, as the drying tube was sufficient to protect against
moisture with such short reaction times. When compared to HCl, which requires 20-30
min under pressure at 130-140 °C, our method is milder and much greener in terms of
energy consumption. Work-up is also very simple, requiring stirring with methanol for
10 min, followed by drying under reduced pressure. A scale of approximately 100-300
mg was utilized, and yields were quantitative by
31
P and
1
H NMR, remaining above 93%
following isolation.

32

Table 2.2.1. Results for the dealkylation reactions represented in Scheme 2.2.1.

Entry

R

Solvent

BTMS
Equiv.

Temp.
(°C)

Time:
MW
(min)

%
Yield

Time:
Conventional
heating
(min)

% Yield
(
31
P NMR)
1. Me ACN 2 40 10 93
a
60 91
2. Me ACN 2 60 4 97
a
- -
3. Et ACN 2 40 15 93
a
420 80
4. Et ACN 2 60 5 97
a
- -
5. iPr ACN 4 40 90 97
a
- -
6. iPr ACN 2 60 30 98
a
360 85
7. Me Dioxane 2 40 5 >99
a
60 95
8. Me Dioxane 2 60 2 >99
a
- -
9. Me Dioxane 2 80 2 >99
a
- -
10. Et Dioxane 2 40 15 95
a
420 74
11. Et Dioxane 2 60 6 96
a
- -
12. Et Dioxane 2 80 3 96
a
120 48
13. iPr Dioxane 2 80 30 >99
a
240 89
14. Me Neat 6 40 2 >99
a
- -
15. Et Neat 6 40 2 98
a
- -
16. iPr Neat 6 40 15 >99
a
- -
17. Me DMF 2 40 4 97
a
- -
18. Me DMF 2 60 2 94
a
- -
19. Et DMF 2 40 6 98
a
- -
20. Et DMF 2 60 2 98
a
240 62
21. Et DMF 2 80 2 95
a
240 98
22. iPr DMF 2 80 20 96
a
- -
23. iPr DMF 2 100 5 97
a
60 73
24. iPr DMF 2 110 2 95
a
- -
25. Me Sulfolane 2 40 4 >99
b
- -
26. Me Sulfolane 2 60 2 >99
b
- -
27. Et Sulfolane 2 40 15 100
b
- -
28. Et Sulfolane 2 60 2 100
b
240 40
29. iPr Sulfolane 2 60 10 100
b
- -
30. iPr Sulfolane 2 80 2 min 100
b
4 hr 92
a
Isolated yield,
b
yield by
31
P NMR.

33

Another advantage to the use of BTMS is its complete selectivity towards P-O
silyldealkylation in carboxylate-phosphonate mixed esters.
4,6,7
To demonstrate this effect
in the microwave, two simple mixed esters, trimethyl- and triethylphosphonoacetate,
were reacted with BTMS under microwave irradiation in ACN at 60 °C. The results,
displayed in Table 2.2.2, show that the dealkylation of these compounds requires slightly
longer times than it does for their simple methylphosphonate ester counterparts. As
expected, the Et ester required a longer reaction time than the Me ester. The slower
dealkylation rates observed for these compounds are likely due to the fact that the first
step of the McKenna reaction (Figure 2.1.1) involves the silylation of the oxygen on the
phosphonate group, which is slowed down in the presence of an adjacent electron-
withdrawing group (EWG). Therefore, the presence of the carboxylate group in the
phosphonoacetate compounds is likely responsible for the slight decrease in rates when
compared to the methylphosphonates.

Interestingly, the BTMS dealkylation of 2-(diethoxyphosphoryl)acetic acid (Table
2.2.2, entry 3, prepared from the dealkylation of the carboxyl ester of
triethylphosphonoacetate) required not only longer reaction times than
triethylphosphonoacetate, but also required
three times as much BTMS. This may be due
to the formation of an intramolecular
hydrogen bond between the free carboxyl
group and the P=O, which can decrease the
nucleophilicity of the P=O oxygen (Figure
2.2.1). Furthermore, the dealkylation of
Figure 2.2.1. Possible intramolecular H-
bonding that may explain the decreased rate
of BTMS dealkylation observed with 2-
(diethoxyphosphoryl)acetic acid.

34

diethyl (bromodifluoro)phosphonate, with a very strong EWG, required 6 equiv of BTMS
and microwave irradiation at 60 °C for 1 h, as shown in Table 2.2.2. Conversion to
phosphonic acids was again quantitative by
31
P and
1
H NMR, and isolated yields
remained excellent.
Table 2.2.2. Results for microwave BTMS dealkylation reactions on mixed carboxylate-phosphonate
esters, as well as on 2-(diethoxyphosphoryl)acetic acid and diethyl (bromodifluoro)phosphonate.

Slight modifications to the work-up procedures for the carboxylate-phosphonate
dealkylation products were necessary. For the silyl ester intermediate of 2-
phosphonoacetic acid (Table 2.2.2, Entry 3), hydrolysis to final product was performed
with water because the use of methanol produces a methyl ester at the free carboxyl end.
Importantly, for the compounds in which the carboxylate is alkylated (Table 2.2.2 Entries
1 and 2), it should be noted that, as evidenced by a single peak in the
31
P NMR spectra
(Figures B5 and 8, Appendix B), the silylation by BTMS is 100% selective towards the
P-O esters. However, the presence of trace amounts of unreacted BTMS will produce

Entry

Structure

Solvent

BTMS
Equiv

Temp.
(°C)

Time:
MW

% Yield
(NMR/isolated)
1.

ACN 2 60 10 min 100/98
2.

ACN

2

60

15 min

100/96
3.

ACN 6 60 30 min 100/99
4.

ACN 6 60 1 h 100/85

35

HBr upon exposure to moisture during work-up, which can result in slight (5-10%)
dealkylation at the carboxylate ester.
6,7,22
In order to avoid this, any excess BTMS must
be removed under reduced pressure before hydrolysis or methanolysis of the silyl esters.
Also, in addition to the drying tube, the BTMS reactions with the phosphonoacetates
were carried out under nitrogen as an extra precaution against moisture. As mentioned
previously, this complete selectivity towards phosphonate esters is not possible with the
use of HCl as a dealkylating agent.
As mentioned previously, ANP esters, particularly those containing DAP groups,
can be sensitive towards dealkylation, as evidenced by the degradation which
accompanied HCl dealkylation in the work of Jansa et al.
15
As such, we devised a method
for the microwave BTMS dealkylation of the ANP esters PMEDAP(OiPr)
2
, (S)-
PMPDAP(OEt)
2
and PMEA(OiPr)
2
(Figure 2.2.2). The PMEDAP(OiPr)
2
and
PMEA(OiPr)
2
starting materials were synthesized by Melissa Williams, while the free
phosphonic acid of (S)-PMPDAP was supplied by Dr. Marcela Kreĉmerová from the
IOCB Research Centre, Academy of Sciences of the Czech Republic. In addition to the
microwave’s utility in the BTMS dealkylation of ANP esters, we decided to investigate
the coupling reactions between free phosphonic acid ANPs and alcohols in the
microwave, using (S)-PMPDAP and ethanol as an example, to yield the (S)-
PMPDAP(OEt)
2
starting material used for the BTMS reactions.

36

Figure 2.2.2. Structures for the ANPs PMEDAP(OiPr)
2
, (S)-PMPDAP(OEt)
2
, and PMEA(OiPr)
2
.

While the coupling of cyclic nucleoside phosphonic acids (such as (S)-HPMPA
and (S)-HPMPC) with ethanol or isopropanol is possible in a one-pot synthesis using
PyBOP as a coupling reagent, this is not the case with acyclic derivatives.
23
This was
determined in our group to be due to the formation of a stable acyclic HOBt-intermediate
that does not react with the alcohols.
23
Therefore, an alternative method was developed in
which the first P-OH group is coupled to an ethyl group using PyBrOP, generating
approximately 80% mono-ethyl ester and 20% di-ethyl ester. Conversion of the
monoester to diester is then achieved using a second coupling with PyBOP as the
coupling reagent,
23
as shown in Scheme 2.2.2. An attempt to synthesize the diester using
only PyBrOP was unsuccessful, as it becomes significantly less active once one ester is
synthesized.
As depicted by the results in Table 2.2.3, the dealkylation reactions of ANP esters
with BTMS in ACN reached completion in as little as 15 min with 6 equiv of BTMS, or
30 min with 4 equiv of BTMS, at 60 °C for iPr esters, and 15 min with 4 equiv of BTMS
for the Et ester. The use of BTMS allows for lower temperatures and ambient pressure to
be used, which is not only green, but is mild enough to preserve the integrity of the DAP
groups. In addition, small scales were successful (30-50 mg) and yields were quantitative

37

by
31
P and
1
H NMR. On the contrary, HCl requires 20-30 min under pressure at 130-140
°C to achieve dealkylation of ANPs, and results in 5-10% degradation of the DAP
groups.
15

Scheme 2.2.2. Coupling of ethanol to (S)-PMPDAP to generate the diethyl ester.

As such, we have demonstrated that microwave-assisted BTMS dealkylation
reactions for ANPs are fast, efficient, facile, mild and high yielding, making them highly
convenient. Control reactions were carried out in which all the reaction conditions except
for the heating method (sand bath in lieu of microwave) were held constant. As the
results in Table 2.2.3 demonstrate, BTMS dealkylation is 16-20 times faster in the
microwave.

38

Table 2.2.3. BTMS dealkylations of ANP esters using microwave irradiation.
a 31
P and
1
H NMR yields, which were quantitative.
b
Isolated yields after crystallization of the product.

2.3 Conclusion
In conclusion, we have shown that the rates of phosphonate ester dealkylation by
BTMS are vastly improved by microwave dielectric heating, and have demonstrated the
versatility of this reaction by varying temperatures and utilizing different solvents with a
range of polarities. Solvents with low polarities, such as dioxane, can be successfully
utilized, and the reactions can also be run in neat BTMS, since the reactants themselves
are polar enough to be directly heated via microwave irradiation. Additionally, not only is
BTMS dealkylation highly efficient and facile, producing quantitative yields, but it can
be applied towards not only iPr and Et esters, but also towards Me esters. Furthermore,
temperatures as low as 40 °C can be used, as well as equimolar amounts of BTMS,
which, together with the short reaction times, makes this approach mild and
environmentally friendly. The use of pressure is also obviated, and the reaction is

Entry

Name

Solvent

BTMS
Equiv.

Temp.
(°C)

Time:
MW
(min)

% Yield
MW

Time
conventional
heating
(min)

ACN

4

60

30

>99
a

-

1.

PMEDAP(OiPr)
2

ACN

6

60

15

>99
a
/91
b

240

2.

(S)-PMPDAP(OEt)
2
ACN 4

60

15

> 99
a
/82
b

240

ACN

4

60

30

>99
a

-

3.

PMEA(OiPr)
2

ACN

6

60

15
>99
a
/85
b

300

39

compatible with carboxylate esters, and other sensitive groups such as acyclic nucleoside
phosphonate esters possessing diamino purines. As a result, the McKenna reaction, which
clearly still remains the most convenient and effective method for the selective and mild
P-O ester dealkylation, can be vastly accelerated with the use of microwave irradiation.
2.4 Experimental
2.4.1. Materials and Methods
BTMS was purchased from Sigma-Aldrich and was distilled under nitrogen prior
to its use. Dimethyl methylphosphonate, trimethylphosphonoacetate and
triethylphosphonoacetate were purchased from Aldrich. Diethyl methylphosphonate was
purchased from Alfa Aesar, while diisopropyl methylphosphonate was purchased from
Lancaster Synthesis. Diethyl (bromodifluoro)phosphonate was purchased from Oakwood
Products. Acetonitrile was purchased from EMD Chemicals and distilled prior to use,
Drisolv N,N-dimethylformamide was purchased from EMD Chemicals, dioxane was
purchased from Macron Chemicals and distilled over sodium, and sulfolane was
purchased from Sigma-Aldrich and distilled under reduced pressure. The PMEDAP- and
PMEA diisopropyl ester starting materials were synthesized by Melissa Williams, while
the free phosphonic acid of (S)-PMPDAP was supplied by Dr. Marcela Kreĉmerová from
the IOCB Research Centre, Academy of Sciences of the Czech Republic. (Benzotriazol-
1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) was purchased from
Novabiochem, while bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP)
was purchased from Creosalus Advanced Chemtech.

40

The NMR operating frequencies were 500 MHz for
1
H and 202 MHz for
31
P.
1
H
NMR spectra were referenced to residual HDO (δ 4.79) in D
2
O or residual CHCl
3
(δ
7.26) in CDCl
3,
24
while
31
P NMR spectra were referenced against an external 85% H
3
PO
4

standard (δ 0.00). All chemical shift values (δ) are given in ppm and NMR sample pH
values were measured in 99.9% D
2
O without deuterium isotope correction. The
approximate concentration of the NMR samples was 1-3 mg/mL for isolated compounds.
1
H and
31
P NMR spectra are presented in Appendix B, and agree with literature values.
2.4.2. Microwave BTMS Dealkylations of Dimethyl-, Diethyl-, and Diisopropyl
methylphosphonate Esters.
General procedure for the microwave-assisted dealkylation of
methylphosphonate esters. 1.8 mmol of the appropriate methylphosphonate ester was
dissolved in 1 mL of solvent (6 equiv of BTMS were added instead of solvent in neat
reactions) in a dry 10 mL flask. The appropriate equiv of BTMS were quickly added via a
1 mL glass pipette, and the reaction mixture was placed in a Milestone Ethos Synth
Microwave Synthesis Labstation. All reactions were carried out in an open system, fitted
with a reflux condenser and drying tube filled with drierite. All microwave reaction times
include a one min ramp to the listed temperature. After the microwave reactions were
complete, the reaction mixtures were allowed to cool for 3-10 min, after which excess
methanol was added and the mixture was left to stir at room temperature for 10 min. The
products, which were brown oils, were isolated by drying in vacuo until constant weight.
In the case of reactions in sulfolane, the products were not isolated. All experiments were
repeated in triplicate to ensure reproducibility. Control reactions were also performed in
which all reaction conditions were held constant except for the use of a sand bath for

41

heating. Since all starting materials were dealkylated to methylphosphonic acid,
regardless of the starting ester,
1
H and
31
P NMRs of all final products were very similar,
and NMRs of the products from only two representative reactions are included in
Appendix B. For the methylphosphonic acid product from the microwave BTMS
dealkylation of dimethyl methylphosphonate in ACN at 40 °C for 10 min (Table 2.2.1,
Entry 1):
1
H NMR (500 MHz, D
2
O) δ: 1.20-1.24 (d).
31
P NMR (202 MHz, D
2
O, external
H
3
PO
4
standard) δ: 31.05.

For the methylphosphonic acid product from the microwave
BTMS dealkylation of diethyl methylphosphonate in ACN at 40 °C for 15 min (Table
2.2.1, Entry 3):
1
H NMR (500 MHz, D
2
O) δ: 1.33-1.37 (d).
31
P NMR (202 MHz, D
2
O,
external H
3
PO
4
standard) δ: 31.21.
2.4.3. Microwave BTMS Dealkylations of Trimethylphosphonoacetate.
Triethylphosphonoacetate, 2-(Diethoxyphosphoryl)acetic Acid and Diethyl
(bromodifluoro)phosphonate .
General procedure for the microwave dealkylation of trimethyl- and triethyl
phosphonoacetate, and diethyl (bromodifluoro)phosphonate. The microwave BTMS
dealkylation procedure was the same as the procedure utilized for the simple
methylphosphonates. However, in the case of methyl- and ethyl phosphonoacetate, the
solvent and any excess BTMS were removed under reduced pressure before
methanolysis, as opposed to after, and the reactions were carried out under nitrogen as an
extra precaution against moisture. This is necessary due to the sensitivity of the
carboxylate esters to the HBr produced from the exposure of BTMS to water.
31
P NMRs
of product mixture in CDCl
3
were taken for the silyl esters of (2-methoxy-2-

42

oxoethyl)phosphonic acid and (2-ethoxy-2-oxoethyl)phosphonic acid, in addition to the
1
H and
31
P NMR spectra of the final products.
Silyl ester of (2-methoxy-2-oxoethyl)phosphonic acid.
31
P NMR (202 MHz,
CDCl
3
) δ: 0.06.
(2-Methoxy-2-oxoethyl)phosphonic acid.
1
H NMR (500 MHz, D
2
O, pH 7.6) δ:
2.66-2.70 (d, 2H), 3.69 (s, 3H).
31
P NMR (202 MHz, D
2
O, H
3
PO
4
external standard, pH
7.6) δ: 10.99.
Silyl ester of (2-ethoxy-2-oxoethyl)phosphonic acid.
31
P NMR (202 MHz,
CDCl
3
) δ: 1.76.
(2-Ethoxy-2-oxoethyl)phosphonic acid.
1
H NMR (500 MHz, D
2
O, pH 8.2) δ:
1.25-1.28 (t, 3H), 2.65-2.69 (d, 2H), 4.12-4.16 (q, 2H).
31
P NMR (202 MHz, D
2
O, H
3
PO
4

external standard, pH 8.2) δ: 10.97.
(Bromodifluoromethyl)phosphonic acid.
31
P NMR (202 MHz, D
2
O) δ: (-1.62)-
(-0.79) (t).
Preparation of 2-(diethoxyphosphoryl)acetic acid and its microwave BTMS
dealkylation to 2-phosphonoacetic acid. 50 mmol (1 equiv) of
triethylphosphonoacetate was dissolved in 50 mL of 75% ethanol solution, and 50 mmol
(1 equiv) of KOH was added. The reaction was stirred for 2 d at room temperature, after
which the solvent was removed under reduced pressure. The product was washed with 10
mL of chloroform to remove any starting triester, and then it was acidified with HCl. The
product was then extracted with 30 mL of chloroform, followed by 10 mL and 5 mL. The

43

chloroform was then removed under reduced pressure to furnish 6.3 g (65% yield) of 2-
(diethoxyphosphoryl)acetic acid (97% purity). For BTMS dealkylation, the same
procedure utilized for the methylphosphonate esters was applied, but the silyl ester was
hydrolyzed with water instead of methanol to avoid the formation of a methyl ester at the
free carboxyl group of the product.
2-(diethoxyphosphoryl)acetic acid.
1
H NMR (500 MHz, CDCl
3
) δ: 1.21-1.24 (t,
6H), 2.88-2.92 (d, 2H), 4.05-4.11 (m, 4H), 10.47 (s, br, 1H).
31
P NMR (202 MHz,
CDCl
3
) δ: 21.15.
2-phosphonoacetic acid.
1
H NMR (500 MHz, D
2
O, NaHCO
3
, pH 1.12) δ: 2.77-
2.81 (d).
31
P NMR (202 MHz, D
2
O, NaHCO
3
, pH 1.12) δ: 15.88.
2.4.4. Microwave Coupling of (S)-PMPDAP to Ethanol with PyBrOP and PyBOP.
Procedure for the PyBrOP mediated coupling of (S)-PMPDAP to ethanol.
446 mg (1.47 mmol, 1 equiv) of (S)-PMPDAP, 25 mL of dry DMF, 2.59 mL (10 equiv)
of distilled DIEA and 2.58 mL (30 equiv) of ethanol were added to a dry flask and the
contents were briefly sonicated. 1.24 g (1.8 equiv) of PyBrOP was then added, and the
reaction mixture was placed in a Milestone Ethos Synth Microwave Synthesis Labstation.
The reaction was carried out in an open system, where it was fitted with a reflux
condenser and drying tube filled with drierite. The drying tube was connected to a
nitrogen supply. The microwave program was set to heat at 60 °C for 45 min (including a
1 min ramp to 60 °C), after which the reaction mixture was allowed to cool for 10 min. A
31
P NMR spectrum was then taken of the product mixture with a D
2
O capillary.
31
P NMR

44

(202 MHz, D
2
O capillary) δ: 10.80 (80%, monoethyl ester) and 21.28 (20%, diethyl
ester). The peak at 14.20 ppm represents reacted PyBrOP.
Procedure for the PyBOP mediated coupling of the monoethyl ester of (S)-
PMPDAP to ethanol. The contents of the flask containing the product mixture from the
PyBrOP coupling of (S)-PMPDAP to ethanol were dried under vacuum. To the 1.18
mmol (1 equiv) of monoethyl ester, 25 mL of DMF, 2.06 mL (30 equiv) of ethanol, and
2.08 mL of DIEA (10 equiv) were added, and the contents were briefly sonicated. 921 mg
(1.5 equiv) of PyBOP was then added and the flask was placed in the microwave and the
reaction heated using the same program as before. After 45 min at 60 °C, a
31
P NMR of
the reaction mixture with a D
2
O capillary was taken.
31
P NMR (202 MHz, D
2
O capillary)
δ: 21.23. The peak at 14.03 represents reacted PyBOP. After verifying that the reaction
was complete, the diethyl ester product was purified by silica column chromatography
using a gradient of methanol in DCM (0 to 10%). The product fractions were then
collected and dried under vacuum to give 414 mg (86% yield) of the diethyl ester of (S)-
PMPDAP.
31
P NMR (202 MHz, CD
3
OD) δ: 21.85.
2.4.5. Microwave BTMS Dealkylations of PMEDAP(OiPr)
2
, (S)-PMPDAP(OEt)
2
and
PMEA(OiPr)
2
.

General procedure for the microwave BTMS dealkylation of ANPs. 0.134
mmol of PMEDAP(OiPr)
2
, 0.140 mmol of (S)-PMPDAP(OEt)
2
, or 0.140 mmol of
PMEA(OiPr)
2
were dissolved in 1.5 mL of ACN in a dry 10 mL flask. 4 or 6 equiv of
BTMS were then quickly added with a 1 mL glass pipette, and the reaction mixture was
placed in a Milestone Ethos Synth Microwave Synthesis Labstation. All reactions were

45

carried out in an open system, fitted with a reflux condenser and drying tube filled with
drierite that was connected to a nitrogen supply. All microwave reaction times include a
one min ramp to 60 °C (Table 2.2.3). After microwave irradiation for 15 or 30 min, the
reaction mixtures were allowed to cool for 10 min, after which excess methanol was
added and the mixture was left to stir at room temperature for 10 min. The products were
then dried under reduced pressure and precipitated by dissolution in ammonia and water,
and then adjusting the pH to 2-2.5 with HBr. The crystals were filtered and dried to
constant weight.
9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) (Table 2.2.3,
Entry 1, bottom).
1
H NMR (500 MHz, D
2
O) δ: 3.42-3.44 (d, 2H), 3.84-3.86 (t, 2H),
4.19-4.21 (t, 2H), 7.89 (s, 1H).
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard) δ:
13.55.
9-[(2-(R)-(phosphonomethoxy)propyl-2,6-diaminopurine ((S)-PMPDAP)
(Table 2.2.3, Entry 2).
1
H NMR (500 MHz, D
2
O) δ: 1.91 (s, 3H), 2.59 (s, 1H), 3.52-3.57
(t, 1H), 3.76-3.81 (m, 1H), 3.88-3.90 (m, 1H), 4.08-4.13 (m, 1H), 4.30-4.33 (d, 1H), 8.68
(s, 1H).
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard) δ: 32.94.
9-(2-phosphonomethoxy)ethyl adenine (PMEA) (Table 2.2.3, Entry 3,
bottom).
1
H NMR (500 MHz, D
2
O) δ: 3.71-3.72 (d, 2H), 3.92-3.94 (t, 2H), 4.45-4.47 (t,
2H), 8.34-8.35 (d, 2H).
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard) δ: 19.63.

46

2.5 Chapter References
(1) Meziane, D.; Hardouin, J.; Elias, A.; Guénin, E.; Lecouvey, M. Microwave
Michaelis-Becker Synthesis of Diethyl Phosphonates, Tetraethyl Diphosphonates,
and Their Total or Partial Dealkylation. Heteroat. Chem. 2009, 20, 369–377.

(2) Kishore Kumar, G. D.; Saenz, D.; Lokesh, G. L.; Natarajan, A. Microwave-
assisted Cleavage of Phosphate, Phosphonate and Phosphoramide Esters.
Tetrahedron Lett. 2006, 47, 6281–6284.

(3) McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M.-C. The Facile
Dealkylation of Phosphonic Acid Dialkyl Esters by Bromotrimethylsilane.
Tetrahedron Lett. 1977, 18, 155–158.

(4) McKenna, C. E.; Schmidhuser, J. Functional Selectivity in Phosphonate Ester
Dealkylation with Bromotrimethylsilane. J. Chem. Soc. Chem. Commun. 1979,
739.

(5) Thottathil, J. Chapter 2: Developments in the Preparation and Use of Silicon-
containing Organophosphorus Compounds. In Handbook of Organophosphorus
Chemistry.; Engel, R., Ed.; Marcel Dekker, Inc., 1992; p. 61.

(6) Marma, M. S.; Khawli, L. A.; Harutunian, V.; Kashemirov, B. A.; McKenna, C. E.
Synthesis of Α-fluorinated Phosphonoacetate Derivatives Using Electrophilic
Fluorine Reagents: Perchloryl Fluoride Versus 1-chloromethyl-4-fluoro-1,4-
diazoniabicyclo[2.2.2]octane Bis(tetrafluoroborate) (Selectfluor®). J. Fluor.
Chem. 2005, 126, 1467–1475.

(7) Chougrani, K.; Niel, G.; Boutevin, B.; David, G. Regioselective Ester Cleavage
During the Preparation of Bisphosphonate Methacrylate Monomers. Beilstein J.
Org. Chem. 2011, 7, 364–368.

(8) Kappe, C. O.; Dallinger, D. The Impact of Microwave Synthesis on Drug
Discovery. Nat. Rev. Drug Discov. 2005, 5, 51–63.

(9) Colombo, M.; Peretto, I. Chemistry Strategies in Early Drug Discovery: An
Overview of Recent Trends. Drug Discov. Today 2008, 13, 677–684.

(10) Zhang, S.; Arvidsson, P. I. Facile Synthesis of N-protected Amino Acid Esters
Assisted by Microwave Irradiation. Int. J. Pept. Res. Ther. 2008, 14, 219–222.

47

(11) Mavandadi, F.; Pilotti, Å. The Impact of Microwave-assisted Organic Synthesis in
Drug Discovery. Drug Discov. Today 2006, 11, 165–174.

(12) Yin, J.; Zhang, A.; Liew, K. Y.; Wu, L. Synthesis of Poly(ether Ether Ketone)
Assisted by Microwave Irradiation and Its Characterization. Polym. Bull. 2008, 61,
157–163.

(13) Man, A. K.; Shahidan, R. Microwave-assisted Chemical Reactions. J. Macromol.
Sci. Part 2007, 44, 651–657.

(14) Mustafa, D. A.; Kashemirov, B. A.; McKenna, C. E. Microwave-assisted Synthesis
of Nitrogen-containing 1-hydroxymethylenebisphosphonate Drugs. Tetrahedron
Lett. 2011, 52, 2285–2287.

(15) Jansa, P.; Baszczyňski, O.; Procházková, E.; Dračínský, M.; Janeba, Z.
Microwave-assisted Hydrolysis of Phosphonate Diesters: An Efficient Protocol for
the Preparation of Phosphonic Acids. Green Chem. 2012, 14, 2282.

(16) Clercq, E. D.; Holý, A. Case History: Acyclic Nucleoside Phosphonates: a Key
Class of Antiviral Drugs. Nat. Rev. Drug Discov. 2005, 4, 928–940.

(17) Holý, A. Phosphonomethoxyalkyl Analogs of Nucleotides. Curr. Pharm. Des.
2003, 9, 2567–2592.

(18) Krečmerová, M.; Holý, A.; Andrei, G.; Pomeisl, K.; Tichý, T.; Břehová, P.;
Masojídková, M.; Dračínský, M.; Pohl, R.; Laflamme, G.; et al. Synthesis of Ester
Prodrugs of 9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine
(HPMPDAP) as Anti-Poxvirus Agents. J. Med. Chem. 2010, 53, 6825–6837.

(19) Holý, A.; Rosenberg, I.; Dvořáková, H. Synthesis of N-(2-
phosphonylmethoxyethyl) Derivatives of Heterocyclic Bases. Collect. Czechoslov.
Chem. Commun. 1989, 54, 2190–2210.

(20) Holý, A.; Masojídková, M. Synthesis of Enantiomeric N-(2-
Phosphonomethoxypropyl) Derivatives of Purine and Pyrimidine Bases. I. The
Stepwise Approach. Collect. Czechoslov. Chem. Commun. 1995, 60, 1196–1212.

48

(21) Salomon, C. Efficient and Selective Dealkylation of Phosphonate Diisopropyl
Esters Using Me
3
SiBr. Tetrahedron Lett. 1995, 36, 6759–6760.

(22) Blackburn, G. M.; Ingleson, D. Specific Dealkylation of Phosphonate Esters Using
Iodotrimethylsilane. J. Chem. Soc. Chem. Commun. 1978, 870.

(23) Williams, M.; Krylos, I. S.; Zakharova, V. M.; Serpi, M.; Peterson, L., W.;
Krečmerová, M.; Kashemirov, B. A.; McKenna, C. E. Cyclic and Acyclic
Phosphonate Tyrosine Ester Prodrugs of Acyclic Nucleoside Phosphonates.
Collection Symposium Series (Chemistry of Nucleic Acid Components) 2011, 12,
167–170.

(24) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. NMR Chemical Shifts of Common
Laboratory Solvents as Trace Impurities. J. Org. Chem. 1997, 62, 7512–7515.

49

Chapter 3. Pamoic Acid Derivatives as Inhibitors of the Polymerase β
Lyase Domain
3.1 Introduction
DNA polymerases are responsible for maintaining an organism’s genome through
DNA replication and repair.
1
Errors in these processes can lead to mutations, including
those with serious consequences such as disease or death.
1
Therefore, understanding the
mechanism of action for these enzymes is crucial. One of the mutations that occur in
DNA, and that may be propagated through errors in repair, are single nucleotide base
lesions. There are several methods by which base lesions can occur in DNA, including
spontaneous base loss, base oxidation and base deamination.
2
It can also occur upon the
exposure of DNA to alkylating agents, such as
chemotherapeutic drugs.
2

Single base lesions can be very harmful,
and one of the enzymes responsible for protecting
the cell against these errors is polymerase β (Pol
β), a member of the X family of polymerases, and
one of 17 polymerases that are encoded for by the
human genome.
3
This polymerase is characterized
by having a very low degree of processivity – the
ability to incorporate multiple nucleotides into the
DNA strand before dissociating from the
template-primer – implicating it in the synthesis
Figure 3.1.1. The lyase domain of Pol β
is involved in the removal of the 5’dRP
group during BER.

50

of short patches for DNA repair.
3
In fact, Pol β is the major enzyme in mammals that is
responsible for base excision repair (BER)
2-4
and the repair of single-stranded breaks.
4
As
illustrated by Figure 3.1.1, the BER of single nucleotides involves five steps: the excision
of the damaged base by a glycosylase, cleavage of the strand by an apurinic/apyrimidinic
(AP) endonuclease, removal of the remaining deoxyribose phosphate (dRP) and synthesis
of DNA by Pol β, and finally, the ligation of the nick in the strand by DNA ligase.
2,5

DNA Pol β is the smallest of the human DNA polymerases and is comprised of
two domains, the 31-kDa domain, which is at the C-terminal and is responsible for
double-stranded DNA binding and DNA polymerase activity, and the 8-kDa domain,
which is at the N-terminal, binds single-stranded DNA, and is responsible for dRP lyase
activity during BER.
2,4,5
The importance of Pol β for BER has been demonstrated by
studies in which the Pol β gene was deleted from the genome of mice, resulting in
hypersensitivity to alkylating agents such as methylmethanesulfonate (MMS),
2,4,6,
which
are often used as indicators of BER activity in the cell.
2
Pol β is also able to bypass
lesions in the DNA which normally obstruct replicative DNA polymerases, such that
adducts like those resulting from the administration of cisplatin may be replicated,
promoting errors and reducing the efficacy of these chemotherapeutic drugs.
4
As a result,
this enzyme plays a significant role in the resistance of tumors to anticancer therapeutics.
4

In fact, the down-regulation of Pol β elicited a better response of cells to cisplatin.
2

Furthermore, studies have shown a direct correlation between the overexpression of Pol β
and a decreased response in breast, colon and prostate cancers to anticancer therapies
which utilize cisplatin.
4

51

Due to the genetic instability and resistance to cancer
therapies induced by Pol β, there is much interest in the
development of inhibitors for this enzyme.
4
One of the
known inhibitors of Pol β is a natural product known as
koetjapic acid (KJA).
2
This molecule was found to bind to
the lyase (8-kDa) domain of the enzyme via NMR chemical
shift mapping studies.
2
However, KJA does not bind very
strongly, and there are currently no potent inhibitors of Pol β, with most known inhibitors
having a micromolar binding affinity. At present, the most active and specific known
inhibitor of Pol β is pamoic acid (PA, Figure 3.1.2),
2,4
with a K
d
value of 13 ± 5 µM
4
and
an IC
50
of 100 µM.
2
PA is capable of inhibiting the dRP lyase activity of Pol β, and
increasing cellular sensitivity to MMS,
4
indicating a decrease in BER activity. Therefore,
modifying PA to increase its binding affinity may give rise to a potential drug candidate
that can be used in conjugation with cisplatin and other chemotherapeutic agents, with the
goal of increasing their therapeutic indices.
Fragment based drug design, or FBDD, is one of the most powerful tools for
inhibitor design. It relies on the premise that fragments with relatively weak binding
affinities individually can be covalently linked to create a molecule with a much higher
affinity to the original target.
4
In order to utilize this approach, a lot of information is
needed about the nature of the target’s binding site. One of the methods used to elucidate
the structure of the binding site and its specific interactions with inhibitors or ligands is
NMR chemical shift mapping.
4
NMR chemical shift analysis of the interactions between
the lyase domain of Pol β and DNA have given rise to the published refined solution
Figure. 3.1.2. The structure
of pamoic acid, PA.

52

structure of the lyase domain with
PDB code 1DK3.
5
The structure of
the lyase domain consists of 4
alpha helices, present as two
antiparallel pairs, with the binding
pocket of the DNA substrate sitting
between helices 2 and 4.
4
Figure
3.1.3
5
illustrates some of the
important residues involved in the
DNA-enzyme interaction, namely
His34, Lys35, Tyr39, Lys68 and
Lys72.
5

Figure 3.1.4
2
illustrates the result of a
later study that utilized 1DK3 to examine the
interactions between PA and the Pol β lyase
domain.
2
The results highlighted several
amino acids whose chemical shifts appeared
to be most affected by the binding of PA,
including Lys35, Tyr39, Gly64 and Ile69.
2

Further studies
4
utilizing 1DK3 in docking
experiments have corroborated that the binding pocket sits between helices 2 and 4 of the
lyase domain and involves amino acids His34, Lys35, Asn37, Ala38, Lys41, Gly64,
Gly66, Lys68 and Ile69, as illustrated in Figure 3.1.5.
4

Figure 3.1.3. Overlay of the NMR solution structure of
Pol β (red) and the gapped DNA Pol β crystal structure
(green). Important residues involved in DNA binding and
catalysis are shown in magenta on the NMR solution
structure and light green on the crystal structure. Gapped
DNA from the crystal structure is shown in light blue.
5
Figure 3.1.4. Some of the residues of the
lyase domain of Pol β present in the binding
pocket of PA.
2

53

Understanding
the interactions between
PA and the amino acids
in the binding pocket of
the lyase domain will
allow us to construct a
better inhibitor by
employing an FBDD
approach. Due to the
low binding affinity of
PA, there is currently
no x-ray crystal
structure, and therefore, definitive interactions between the lyase domain and PA have
not yet been elucidated. However, the interactions suggested in previous studies via NMR
chemical shift mapping and computational docking provide a good starting point for our
analysis. Since the inhibition of the lyase domain of Pol ß has the potential to improve the
therapeutic index for several DNA-damaging anti-cancer agents, such as Pt-containing
drugs and DNA-alkylating agents, by preventing the survival of cancer cells propagated
via DNA repair, and because, as of yet, no potent inhibitors of the lyase domain have
been identified, this remains an area of great interest. As mentioned previously, PA, with
an IC
50
of 100 µM
2
and a K
d
of approximately 13 ± 5 µM,
4
is currently the best known
inhibitor of the lyase domain. The aim of this study was to analyze the binding pocket of
PA with computational docking, to compare it to the DNA binding site, and to design a
Figure 3.1.5. Results of the docking experiments of PA with 1DK3 that
were performed by Hazan et al.
4

54

more potent inhibitor for synthesis by utilizing Autodock Vina for modeling, and the
MEDIT MED-SuMo software for FBDD. Furthermore, we were interested in
synthesizing an inhibitor potent enough to allow for the generation of an x-ray crystal
structure with the Pol β lyase domain. This will provide the necessary information
regarding specific interactions that is required for the design of even more potent
inhibitors in the future.
3.2 Results and Discussion
3.2.1 Molecular Docking Studies with Autodock Vina
Docking studies were performed with the Autodock Vina
7
software, and two
different crystal structures for the 8-kDa lyase domain of Pol β were utilized: the NMR-
based structure with PDB ID 1DK3, and the x-ray based structure with PDB ID 3LK9.
The inhibitors used were PA, as well as three other structurally related molecules, which
also contain aromatic groups and two carboxylate groups: 4,4’-biphenyl dicarboxylic acid
(BPDC), biquinoline-dicarboxylic acid (BQD), and naphthochrome green (NCG) (Figure
3.2.1.1). These examples were drawn from the study by Hu et al. that confirmed, via
NMR chemical shift mapping, that all of these compounds bind to the same, or a very
similar region, on the surface of Pol β.
2
Docking was performed with both the original,
rigid protein structures and by varying the flexibility at different amino acid residues,
particularly at Lys 35, 68 and 72, which have been implicated in the binding of inhibitors.
When docking PA, BPDC, BQD, and NCG with the rigid, unaltered 8-kDa
domains of Pol β, the results were very similar for both lyase structures in terms of
energies and amino acid involvement. The energies ranged from -5.9 kcal/mol for BQD

55

docked with 3LK9, to -6.8
kcal/mol for PA with 3LK9.
For the most part, the amino
acids found within, and
proximal to, the binding sites
for these 4 ligands included
His34, Lys35, Asn37, Ala38,
Tyr39, Lys68, Lys72, Lys84,
Gly64, Gly66, Thr67 and Ile69,
suggesting that the binding pocket described independently by Hazan,
4
Hu
2
and
Maciejewski
5
is correct.
Next, docking studies were performed in which the flexibilities of some of the
residues in the binding pocket were varied, particularly the lysines, since the long side
chain inherently creates flexibility. Specifically, Lys35, 68, 72 and 84, Tyr39 and Asn37
were made flexible in many different combinations. In almost all cases, the energy levels
were lowered significantly by making Lys35, 68, and 72 flexible. Docking with PA
resulted in energy levels as low as -8.6 kcal/mol and binding to the same pocket
mentioned above. Furthermore, with 3LK9, all inhibitors bind in the same site as with the
rigid, unaltered protein, but with 1DK3, BQD, BPDC and NCG appear to dock in a
nearby hydrophobic pocket, involving the amino acids Phe76, Met18, Leu19, Leu22 and
Leu82. However, docking of 1DK3 with PA yielded the expected results.
To further verify the suggested binding site of PA, docking studies were
performed with Autodock Vina
7
on the X-ray defined crystal structure of the lyase
Figure 3.2.1.1. The structures of BPDC, BQD and NCG,
which were utilized for docking studies with the lyase domain
of Pol β, to confirm the results obtained by Hu et al.
2

56

domain from the PDB file 3JPT.
As summarized in Figure 3.2.1.2,
our results indicate the
involvement of the same amino
acids identified previously, most
importantly Glu26, Lys35, Tyr39,
Gly64, Gly66, Lys68 and Lys72.
Of particular interest is the fact that
one of the COO
–
groups of PA
mimics the phosphoryl group on
the DNA substrate and H bonds to
the lysines in the vicinity, as well
as to Tyr39.
3.2.2 Fragment-Based Drug Design using the MEDIT MED-SuMo Software
Studying the 3-dimensional structure of proteins gives researchers insight into the
functions of these macromolecules, and the easy access to protein structure databases
now allows them to employ computational modeling for rational drug design.
8
The
publicly accessible protein data bank (PDB) currently contains information on the
structures of over 91,000 proteins, with the number continually rising, as approximately
600
9
new entries are released each month. MEDIT MED-SuMo is a target-based in silico
drug design tool that compares proteins within the PDB and finds small regions within
the proteins that exhibit similar structures.
8
The software then compares known protein-
Figure 3.2.1.2. Results of the docking studies performed
with Autodock Vina which utilized the lyase domain from
PDB file 3JPT and PA as the ligand. Important residues
include Lys35, Tyr39, Gly64, Gly66, Lys68 and Lys72.

57

ligand interactions with a library of small molecules that can be uploaded to the program,
outputting a list of ligand fragments in order of binding affinity to the target of interest.
9

After performing a search with MEDIT MED-SuMo for PA and the lyase domain,
the software returned a number of suggested ligands with expected superior binding to
PA, one of which included PA appended with a phosphoryl group in lieu of a carboxyl
group at one side of the molecule. Some of the other hits incorporated different groups
including sugars such as β-glucose and/or nucleosides onto the PA framework. After
factoring in synthetic simplicity and accessibility, the inhibitors chosen for this study all
involved modification with a phosphonyl group. Also, as mentioned previously, docking
studies revealed that one of the COO
–
groups of PA mimics the phosphoryl group on the
DNA substrate and H bonds to the lysines and Tyr39 in the vicinity. As such, replacing
the carboxyl group on PA with a phosphonyl group may result in stronger binding of the
ligand to the lyase active site.
3.2.3 Molecular Docking Studies with PA derivatives
The mono- and di-phosphonylated analogs of pamoic acid were chosen for this
study in an attempt to improve the interactions between the inhibitors and the lyase
domain binding site. One of the mono-phosphonylated derivatives contained a single
phosphonate methyl ester. Prior to the synthesis of the derivatives, docking studies with
Autodock Vina utilizing the PDB file 3JPT, which is the X-ray crystallography structure
of the Pol β ternary complex, were performed. These studies revealed that the phosphonyl
groups on the inhibitors overlap quite well with the phosphoryls from the DNA substrate,
as shown below. Furthermore, the presence of a methyl on the phosphonyl group did not

58

decrease binding affinity significantly, but may instead improve the efficacy of the
inhibitor by slightly improving its lipophilicity (by masking the negative charge on the
phosphonate), thereby enhancing entry into the cell. All three inhibitors showed a binding
affinity similar to, or slightly better than that for pamoic acid (approximately -7
kcal/mol). Figures 3.2.3.1-3.2.3.3 illustrate the overlap between the natural DNA
substrate and the PA analogues in the binding site of the lyase domain.

Figure 3.2.3.1. Overlap between the DNA substrate and the mono-methylphosphonate PA analogue.

59

Figure 3.2.3.2. Overlap between the DNA substrate and the mono-phosphonylated PA analogue.
Figure 3.2.3.3. Overlap between the DNA substrate and the di-phosphonylated PA analogue.

60

3.2.4 Synthesis of PA Derivatives
The synthesis of the novel mono-methylphosphonate derivative (3) of PA was
achieved in three steps as depicted by Scheme 3.2.4.1. First, triethyl amine was added to
an ice-cold carbon tetrachloride solution of 2-naphthol and dimethyl phosphite.
10
The
reaction mixture was stirred overnight at room temperature. The resulting aryl dimethyl
phosphate (1) was produced in a 58% yield, and then rearranged into a phosphonate via
reaction with LDA, generated in situ from n-butyllithium and diisopropylamine, as
described previously.
11,12
During the rearrangement step, one of the methyl groups is
removed and two isomers are produced; one with the phosphonate group at the 1 position
of the naphthol ring (not shown) and one with the phosphonate at the 3 position, in an
approximate ratio of 1:1.8 (by
31
P NMR, Appendix C) respectively, and with a combined
yield of 48%. The assignment of the isomer peaks in the
31
P NMR was made according to
the observation by Dhawan and Redmore that the peak corresponding to the C-3
phosphonate appeared more upfield than the C-1 isomer peak, in the case of the
analogous aryl diethyl phosphonate mixture.
11
The assignments were later verified by
isolating the C-1 and C-3 diethyl analogues via column chromatography and examining
their
1
H and
31
P NMRs (vide infra). Dhawan and Redmore also reported that the major
isomer produced in their hands was the C-3 phosphonate, as the signal intensities in the
31
P NMR showed a 1:2 ratio of the C-1: C-3 phosphonates,
11
which is very similar to our
results. Other studies reported the successful synthesis of the aryl dimethyl phosphonate
via a slightly modified procedure, where reaction with LDA was limited to 2 h,
13
as
opposed to the 4 h in Dhawan and Redmore’s procedure, which we utilized.
11
The shorter
reaction time may be key for avoiding the removal of one of the methyl ester groups.

61

Since the isomer at the 1 position is unreactive towards coupling, the mixture was
not separated. The product P-(3-hydroxy-2-naphthalenyl)-methyl phosphonate (2) was
then coupled with 3-hydroxy-2-naphthoic acid (commercially available) by utilizing a
published procedure.
14,15
Briefly, the two compounds were reacted with excess
formaldehyde in glacial acetic acid, while utilizing a sulfuric acid catalyst to yield the
final product 3. The naphthoic acid was added in excess (2x) to ensure complete reaction
of the phosphonylated derivative, and the reaction mixture was refluxed for 6 h. As
expected, coupling of naphthoic acid with itself yielded an excess of PA as side product.

Scheme 3.2.4.1. Synthetic scheme for the synthesis of the novel mono-methylphosphonate derivative of
PA, 3.

62

After the reaction was complete (as determined by mass spec), product 3 was
separated from the rest of the reaction mixture via reverse phase HPLC. The optimal
mobile phase was determined to be 0.1 N triethyl ammonium carbonate buffer, pH 7.2-
7.5, with 28% acetonitrile. The buffer was removed under reduced pressure, and
1
H NMR
showed that 3 was isolated as a pure triethyl ammonium salt. The product was then
converted to a sodium salt via dowex exchange, and then lyophilized to yield
approximately 4 mg of 3. The overall yield of 3 from the coupling reaction was estimated
to be approximately 25%, based on HPLC yields.
To synthesize the fully acidic version of 3, the second methyl group on the
phosphonate group was removed. To achieve this, 591 mg of the crude product mixture
from the coupling reaction (containing 3) was dissolved in DMF and reacted with excess
BTMS for 1 h at room temperature, resulting in a quantitative yield, as depicted by
Scheme 3.2.4.2. The solvent and excess BTMS were removed under reduced pressure
and the product mixture was re-dissolved in methanol and ammonia. The HPLC
procedure used to purify 3 was followed to separate 4 from the product mixture as a clean
triethyl ammonium salt. Dowex exchange yielded a sodium salt that was then lyophilized
to produce the final product, 4, as a light pink-brown solid (~13 mg).
Scheme 3.2.4.2. Dealkylation of 3 via reaction with BTMS.

63

The acidic nature of the mono-methyl phosphonate derivatives 2 and 3 prevents
the use of silica gel column chromatography for isolation, and instead requires HPLC for
separation from the side products and excess starting materials. In order to obviate the
need for HPLC purification, which is tedious, time consuming, and inefficient, the di-
phosphonylated derivative of PA (8) was synthesized by way of the diethyl phosphonate,
6, as illustrated by Scheme 3.2.4.3. The same procedure
11
to synthesize 1 was followed to
generate the aryl diethylphosphate 5, except that diethyl phosphite was used instead of
dimethyl phosphite. The yield for this reaction was 52%. Phosphate 5 was then
rearranged to phosphonate 6 via reaction with LDA, as described above, with the
exception that both ethyl groups remained intact, as they are not as labile as methyl
groups. Similar to its methyl counterpart, however, two isomers were produced; one with
the phosphonate group at the 1 position of the naphthol ring and one with the
phosphonate at the 3 position, in an approximate ratio of 1:1 by
31
P NMR respectively,
and with a combined yield of 100%. Therefore, we obtained less of the isomer of interest
when compared to the published results (1:2),
11
and the results we obtained with the
methyl derivative (1:1.8). The
31
P NMR chemical shifts we obtained for the isomer
mixture matched very closely to the published
11
values, but in order to confirm the
correct assignment of the isomer peaks, the two products were isolated by silica gel
column chromatography, using a gradient of acetone in chloroform from 0-10%. The
isomer believed to be the C-3 phosphonate showed an aromatic proton doublet with
chemical shift 8.00-8.04 ppm, and J
P-H
= 20 Hz. This coupling constant indicates that this
proton is attached to the carbon atom adjacent to the phosphonate group. The
31
P NMR
chemical shift value for this isomer corresponded to the more upfield value, confirming

64

that the literature assignments were correct and that they agreed with our findings. The
literature value for this J
P-H
was measured as 16.5 Hz.
11
We also measured the J

value for
the same proton doublet on the other isomer, and determined that it was 10 Hz, indicating
that this was the C-1 phosphonate, in which there is no proton on the carbons directly
next to the phosphonate group.

Scheme 3.2.4.3. Synthetic scheme towards P-(3-hydroxy-2-naphthalenyl)-diethyl phosphonate, (6).

After synthesizing P-(3-hydroxy-2-naphthalenyl)-diethyl phosphonate (6), the
product was coupled with itself. However, another procedure was utilized, in which water
was used as the solvent instead of glacial acetic acid, and no acidic catalyst was included,
since PA was previously synthesized under these conditions.
15
6 was refluxed with excess
formaldehyde for 4.5 h in water to yield the coupled product, 7 (Scheme 3.2.4.4). The
tetraethyl di-phosphonylated product 7 was then purified via column chromatography
with a gradient of acetone in chloroform as the mobile phase, providing pure 7 in an 18%

65

yield. The purified product was then subjected to dealkylation by BTMS for 13 h at room
temperature to yield the final product 8, which was then recrystallized from methanol and
lyophilized to yield a pale pink-brown solid, at a 21% yield, and with > 99% purity.

Scheme 3.2.4.4. Synthetic scheme for the di-phosphonylated derivative of PA, 8.

3.2.5 Preliminary Testing of Biological Activity for the PA Derivatives
Biological testing of our compounds was performed by our collaborators Dr.
William Beard and Dr. Samuel Wilson at the National Institute of Environmental Health
Sciences (NIEHS). PA (Sigma), 3, and 4 were dissolved in 25 mM phosphate buffer, pH

66

7.5. 8 was dissolved in 20 mM Tris buffer, pH 8.4, resulting in a cloudy solution.
Addition of small quantities of KOH or DMSO did not improve its appearance; therefore,
testing was only performed on PA and compounds 3 and 4. HPLC-purified
oligonucleotides were purchased from IDT (Integrated DNA Technologies), dissolved in
10 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and their concentrations were determined by
UV absorbance at 260 nm. A single-nucleotide gap was produced by annealing three
oligonucleotides: a primer, downstream oligos, and template oligos, whose sequences
were (5´–3´): CTG CAG CTG ATG CGC (15-mer), GTA CGG ATC CCC GGG TAC
(18-mer), and GTA CCC GGG GAT CCG TAC GGC GCA TCA GCT GCA G (34-mer;
coding base in the gap is underlined), respectively. The upstream primer was 5´-labeled
with [γ-
32
P] ATP and free radioactive ATP was removed.
DNA synthesis was assayed on the prepared single-nucleotide gapped DNA
substrates (where the templating base in the gap was guanine).
16
Enzyme activities were
determined at various concentrations of inhibitor, and the radiolabeled 15-mer primer
substrate and 16-mer product were resolved by denaturing 16% polyacrylamide gel
electrophoresis and quantified in the dried gels by phosphorimagery. The concentration
of inhibitor (I) that produces 50% inhibition (IC
50
) was determined by fitting fractional
activity (activity with inhibitor/activity without inhibitor) to equation (1):
Fractional Activity = 1/[1 + (I/IC
50
)]
h
Eq. (1),
where h is the Hill coefficient.
The results of the enzyme inhibition assay, which are displayed in Figure 3.2.5.1,
revealed that, in comparison to PA, with an IC
50
of 142 µM, the mono-phosphonylated

67

derivatives, 4 and 3, are 3- and 6-folds more potent, respectively. Furthermore, the mono-
methylphosphonate derivative (3) was twice as active as the un-methylated version (4).
Since this assay was not carried out in cells, this increased activity cannot be attributed to
enhanced cellular permeability. It would be of interest to test the ethyl ester version of
these PA derivatives to see the effect on inhibitory activity. Although a crystal structure
with bound inhibitor is not yet available, the increased activity of our inhibitors, and
therefore, the inferred increased binding affinity, may allow for the generation of an x-ray
crystal structure that will confirm the predicted binding sites within the lyase domain, and
provide the necessary information for the design of even more potent inhibitors in the
future.

Figure 3.2.5.1. Biological activities for (a) PA, (b) derivative 4, and (c) derivative 3. Data provided by Dr.
William Beard at the NIEHS.

a
.
[3] (µM)
c.
b.
[4] (µM)

68

3.3 Conclusion
Docking studies were performed on PA and three other molecules that are known
to bind to the lyase domain of Pol β to corroborate the binding site that has been
suggested in the literature via NMR chemical shift mapping and other docking studies.
Once the enzyme-ligand interactions were established, a search was performed utilizing
the fragment-based drug design software MEDIT MED-SuMo, which returned a number
of hits, including a modification of PA to include a phosphoryl group in lieu of the
carboxyl group. Docking studies with mono- and di-phosphonylated analogues of PA
showed that the derivatives would most likely bind to the same pocket within the Pol β
lyase domain, and that the phosphonyl groups from the analogues overlap well with the
phosphoryl groups on the natural DNA substrate. As such, synthetic procedures were
devised to generate the mono-methylphosphonate and un-methylated mono-phosphonate
analogues of PA, as well as a free acid version incorporating two phosphonyl groups.
Preliminary biological data revealed that the inhibitory activity was increased 3-
fold with the un-methylated mono-phosphonylated derivative 4, while activity was
increased 6-fold with the mono-methylphosphonate derivative 3. Inhibitory activities of
the di-phosphonylated analogue 8 have not yet been determined, and an X-ray crystal
structure of the lyase domain bound to any of the inhibitors has not yet been produced.
Future studies will aim to improve upon the activities of these derivatives by optimizing
the structures. To achieve this, it is imperative to produce an x-ray crystal structure that
will provide further, more concrete data regarding the nature of the binding between the
PA derivatives and the lyase domain active site, allowing us to more intelligently design
an inhibitor with increased potency.

69

3.4 Experimental
3.4.1 Materials and Methods
2-Naphthol was purchased from Alfa Aesar, dimethyl phosphite from Aldrich,
and diethyl phosphite from Sigma-Aldrich. N-butyllithium for the generation of LDA was
purchased as a 1.6 M solution in hexane from Alfa Aesar, while diisopropylamine was
purchased from Sigma-Aldrich and distilled prior to use. 3-Hydroxy-2-naphthoic acid
was purchased from Acros Organics, while 37% formaldehyde solution and glacial acetic
acid were purchased from Mallinckrodt. BTMS, which was distilled under nitrogen
before use, was purchased from Aldrich, and the dowex used in this study was a 50WX8-
200 ion exchange resin purchased from Sigma-Aldrich.
For HPLC separation, the solid product mixtures were dissolved in a 1:1
H
2
O/methanol mixture with 1 drop of ammonia and separated using a Varian Prostar
HPLC system with an SPD-10A VP Shimadzu UV detector (set to 254 nm), and a
Phenomenex Luna 5u C18(2) preparative column (250 x 21.2 mm). The mobile phase
used was a 0.1 N triethylammonium carbonate buffer, pH 7.2-7.5, with 28% acetonitrile,
and the flow rate used was 8 mL/min over 30-40 min. The NMR operating frequencies
were 500 MHz for
1
H and 202 MHz for
31
P.
1
H NMR spectra were referenced to residual
HDO (δ 4.79) in D
2
O or residual CHCl
3
(δ 7.26) in CDCl
3
.
17
All chemical shift values (δ)
are given in ppm, and the concentration of all NMR samples was approximately 1-3
mg/mL. Our NMR data matched literature data
11
where available. A Thermo-Finnigan
Deca XP Max mass spectrometer with an ESI probe was utilized for all low-resolution
mass spectra.
1
H spectra for compounds 1 and 3-8, and
31
P NMR spectra for compounds

70

1-8, as well as mass spectra for compounds 2-4 and 6-8, are presented in Appendix C.
HPLC traces for compounds 3 and 4 and
1
H and
31
P NMRs of the separated aryl diethyl
phosphonate isomers are also included in Appendix C.
3.4.2. Molecular Docking Studies with PA
PA was docked into the lyase domain active sites of the PDB files 1DK3 (NMR-
based), 3LK9 (X-ray based), and 3JPT (X-ray based), using the Autodock Vina 1.0.2
software.
7
The exhaustiveness parameter was set to 32, and all other parameters were left
as their default values. The cell dimensions used were 24 x 24 x 24 Å, centered on the
active site with coordinates x = 17.329, y = 30.702, z = -5.615 for 1DK3, x = 22.763, y =
-3.107, z = 10.288 for 3LK9, and x = 26.97, y = -4.102, z = 10.275 for 3JPT.
The receptor files were prepared with the Autodock Tools 1.5.4. software by
extracting the lyase domain from the entire polymerase β PDB files. The receptor was
kept rigid in some docking studies, while Lys 35, 68, 72 and 89, Tyr39, and Asn37 were
made flexible in different combinations for the flexible residue docking studies. AD4
type assignment was applied to all atoms before the receptor files were converted to
pdbqt format. The PA ligand was prepared in the Spartan ’08 software, where the total
charge was set to -2 and the optimum geometry was calculated by the Hartee Fock
method, and the atomic charges were estimated by Mulliken population analysis. The
docking results were analyzed in MacPyMOL, which was also used to prepare the
images.

71

3.4.3 Molecular Docking Studies with Phosphonylated PA Derivatives
The lyase domain was extracted from the X-ray crystal structure of the ternary
complex of DNA polymerase β with a dideoxy terminated primer and 2'-deoxyguanosine
5'-beta, gamma-fluoro, chloro, methylene triphosphate (PDB ID 3JPT). The receptor file
was prepared using the Autodock Tools 1.5.4. software. The receptor was kept rigid and
AD4 type assignment was applied to all atoms before the receptor file was converted to
pdbqt format. The phosphonylated PA ligands were prepared in the Spartan ’08 software,
where the optimum geometry was calculated by the Hartee Fock method, and the atomic
charges were estimated by Mulliken population analysis.
Docking studies were performed using the Autodock Vina 1.0.2 software.
7
The
exhaustiveness parameter was set to 32, and all other parameters were left as their default
values. The cell dimensions used were 24 x 24 x 24 Å, centered on the active site with
coordinates x = 26.97, y = -4.102, z = 10.275. MacPyMOL was used to analyze the
docking results and to prepare the picture files.
3.4.4 Synthesis of PA Derivatives
Aryl dimethyl phosphate (dimethyl naphthalen-2-yl phosphate), 1. 0.02 mol
of 2-naphthol (1 equiv) was added to 6 mL of CCl
4
on ice, after which 0.0208 mol (1.04
equiv) of dimethyl phosphite were added. 0.0208 mol (1.04 equiv) of Et
3
N were then
added slowly, while stirring on ice. After addition was complete, the reaction mixture
was left to stir overnight at room temperature. The mixture was then diluted and washed
with 5 mL of water. The organic layer was collected and washed with 1 N HCl (1 x 5
mL) followed by 1 N NaOH (4 x 5 mL) and H
2
O (1 x 5 mL). After drying over potassium

72

carbonate, the organic layer was filtered and dried in vacuo to give a brown oil in a 58%
yield.
1
H NMR (500 MHz, CDCl
3
) δ: 3.88-3.91 (d, 6H), 7.35-7.37 (dd, 1H), 7.43-7.51
(m, 2H), 7.68 (s, 1H), 7.79-7.84 (m, 3H).
31
P NMR (202 MHz, CDCl
3
) δ: -4.00.
P-(3-hydroxy-2-naphthalenyl)-methyl phosphonate, 2. 3.2 mL (2 equiv) of
distilled diisopropylamine was added to a flask that was then flushed with nitrogen gas.
9.25 mL of dry THF were then added and the flask was placed under nitrogen, at -78 °C.
14.4 mL of 1.6 M n-BuLi in hexane (2 equiv) were then added and the mixture was
stirred at -78 °C for 30 min. 2.9 g (1 equiv) of phosphate 1, dissolved in 9.25 mL dry
THF, was added to the flask with the (in situ formed) LDA, and the mixture was left to
stir at -78 °C for 1 h, after which the flask was warmed up to room temperature slowly
and left to stir for 3 h, giving a deep red solution. To extract the product, the reaction
mixture was poured over a mixture of 58 mL saturated NH
4
Cl solution and 71 mL of
CH
2
Cl
2
. The organic layer was separated and washed with 32 mL of H
2
O, dried over
Na
2
SO
4
, and then the dichloromethane was removed under reduced pressure. A deep red
sticky oil was produced, at a yield of 48%.
31
P NMR (202 MHz, CDCl
3
) δ: -4.00 (starting
material), 14.38 (correct isomer, with phosphonate at 3 position, 59%), 17.01 (other
isomer, with phosphonate at 1 position, 32%). MS (ESI) C
11
H
10
O
4
P (M-H)
-
m/z calcd:
237.17. Found: 237.1.
Mono-methyphosphonate PA derivative (3-hydroxy-4-((2-hydroxy-3-
(hydroxy(methoxy)phosphoryl)naphthalen-1-yl)methyl)-2-naphthoic acid), 3. 0.934
mmol of 2 (1 equiv) and 1.87 mmol (2 equiv) of 3-hydroxy-2-naphthoic acid were added
to a flask, together with 6 mL of 37% formaldehyde solution and 8 mL of glacial acetic
acid. 10 drops of sulfuric acid were then added, and the reaction mixture was refluxed for

73

6 h. Mass spectrometry revealed the mono-methylphosphonate product of interest, as
well as an excess of PA, as expected from the coupling of 3-hydroxy-2-naphthoic acid
with itself. The products, which precipitated together out of the reaction mixture as a
yellow solid, were filtered and collected for HPLC separation. The solid mixture was
dissolved in a 1:1 H
2
O/methanol mixture with 1 drop of ammonia, and separated on a
Phenomenex Luna 5u C18(2) preparative column (250 x 21.2 mm) using a mobile phase
of 0.1 N triethylammonium carbonate buffer with 28% acetonitrile and pH 7.3, over 30-
40 min with an 8 mL/min flow rate. Products were detected at 254 nm and the retention
time for 3 was 16.4 min, while the retention time for the PA side product was 17.9 min,
giving good separation. After collecting the peak of interest, the buffer was removed
under vacuum and the purity of the product was confirmed via
1
H and
31
P NMRs. The
product was then converted from a triethyl ammonium salt into a sodium salt via
exchange with dowex resin (50WX8-200), dissolved in water, and lyophilized to yield a
pale brown solid. The estimated yield for the coupling reaction was 25%, based on HPLC
yields.
1
H NMR (500 MHz, D
2
O) δ: 3.52-3.54 (d, 3H), 7.20-7.25 (q, 2H), 7.31-7.34 (t,
2H), 7.79-7.81 (dd, 2H), 8.04-8.09 (m, 3H), 8.34 (s, 1H).
31
P NMR (202 MHz, D
2
O) δ:
16.84. MS (ESI) C
11
H
10
O
4
P (M-H)
-
m/z calcd: 437.08. Found: 437.1.
Dealkylated mono-phosphonylated PA derivative (3-hydroxy-4-((2-hydroxy-
3-phosphononaphthalen-1-yl)methyl)-2-naphthoic acid), 4. Approximately 100 mg of
product mixture from the reaction to produce 3 was dissolved in 2 mL dry DMF and an
excess (approximately 2.75 mL) of BTMS was quickly added. The reaction was capped
and stirred at room temperature for 1 h. The product mixture was dried under reduced
pressure, dissolved in a 1:1 H
2
O/methanol mixture with 1 drop of ammonia, and

74

subjected to the same HPLC procedure as for 3. Retention time was 8.2 min for product 4
and 15.3 min for the PA side product, providing excellent separation. After HPLC
separation, the buffer was removed under reduced pressure to yield the product as a pure
triethyl ammonium salt, which was exchanged to a sodium salt via dowex exchange
(50WX8-200 resin) and lyophilized to furnish 13 mg of a pink-brown solid.
1
H NMR
(500 MHz, D
2
O) δ: 7.16-7.27 (m, 3H), 7.35-7.38 (t, 1H), 7.77-7.80 (t, 2H), 8.01-8.04 (d,
1H), 8.10-8.12 (d, 2H), 8.33 (s, 1H).
31
P NMR (202 MHz, D
2
O) δ: 11.74. MS (ESI)
C
22
H
16
O
7
P (M-H)
-
m/z calcd: 423.06. Found: 423.1.
Aryl diethyl phosphate (diethyl naphthalen-2-yl phosphate), 5. 0.02 mol of 2-
naphthol (1 equiv) was added to 6 mL of CCl
4
on ice, after which 0.0208 mol (1.04
equiv) of diethyl phosphite were added. 0.0208 mol (1.04 equiv) of Et
3
N were then added
slowly while stirring on ice. The reaction mixture was then left to stir overnight at room
temperature. The mixture was then diluted and washed with 5 mL of water. The organic
layer was collected and washed with 1 N HCl (1 x 5 mL) followed by 1 N NaOH (4 x 5
mL) and H
2
O (1 x 5 mL). After drying over potassium carbonate, the organic layer was
filtered and dried in vacuo to give a brown oil in a 52% yield.
1
H NMR (500 MHz,
CDCl
3
) δ: 1.35-1.37 (t, 6H), 4.20-4.31 (m, 4H), 7.37-7.39 (dd, 1H), 7.43-7.51 (m, 2H),
7.70 (s, 1H), 7.80-7.84 (t, 3H).
31
P NMR (202 MHz, CDCl
3
) δ: -6.21.
P-(3-hydroxy-2-naphthalenyl)-diethyl phosphonate, 6. 2.4 mL (2 equiv) of
distilled diisopropylamine was added to a flask that was then flushed with nitrogen gas.
6.9 mL of dry THF were then added, and the flask was placed under nitrogen at -78 °C.
10.7 mL of 1.6 M n-BuLi in hexane (2 equiv) were then added and the mixture was
stirred at -78 °C for 30 min. 2.4 g (1 equiv) of phosphate 5, dissolved in 6.9 mL dry THF,

75

was added to the flask with the (in situ formed) LDA, and the mixture was left to stir at -
78 °C for 1 h, after which the flask was warmed up to room temperature gradually, and
left to stir for 3 h to give a deep red solution. To extract the product, the reaction mixture
was poured over a mixture of 30 mL saturated NH
4
Cl solution and 55 mL of CH
2
Cl
2
. The
organic layer was separated and washed with 20 mL of H
2
O, dried over Na
2
SO
4
, and
dried under reduced pressure to remove the dichloromethane. A deep red sticky oil was
produced, at a quantitative yield. The product consisted of a 1:1 mixture of the two
phosphonate isomers (with the phosphonate group at the 1 or 3 position), as shown by
31
P
NMR (Appendix C). The aromatic region of the proton NMR of this mixture (Appendix
C), which is complicated, shows 12 protons, as expected for two isomers.
31
P NMR (202
MHz, CDCl
3
) δ: 21.23 (C-3 phosphonate), 25.23 (C-1 phosphonate). MS (ESI)
C
14
H
16
O
4
P (M-H)
-
m/z calcd: 279.25. Found: 279.4. In order to separate the two isomers,
silica gel column chromatography with a gradient of acetone in chloroform from 0-10%
was utilized.
C-3 phosphonate isomer:
1
H NMR (500 MHz, CDCl
3
) δ: 1.34-1.37 (t, 6H), 4.06-
4.26 (m, 4H), 7.33-7.35 (m, 2H), 7.48-7.51 (t, 1H), 7.69-7.71 (d, 1H), 7.77-7.79 (d, 1H),
8.00-8.04 (d, 20 Hz, 1H), 9.97 (s, 1H).
31
P NMR (202 MHz, CDCl
3
) δ: 21.23.
C-1 phosphonate isomer:
1
H NMR (500 MHz, CDCl
3
) δ: 1.28-1.31 (t, 6H),
3.95-4.24 (m, 4H), 7.12-7.15 (m, 1H), 7.35-7.38 (t, 1H), 7.50-7.53 (t, 1H), 7.75-7.76 (d,
1H), 7.90-7.91 (d, 1H), 8.08-8.10 (d, 10 Hz, 1H), 11.82 (s, 1H).
31
P NMR (202 MHz,
CDCl
3
) δ: 25.23.

76

Tetraethyl di-phosphonylated PA derivative (tetraethyl (methylenebis(3-
hydroxynaphthalene-4,2-diyl))bis(phosphonate)), 7. 472 mg of phosphonate mixture
6, together with 7 mL of water and 4 mL of 37% formaldehyde solution were added to a
flask and refluxed for 4.5 h. The product mixture was dried under reduced pressure, dry
loaded onto a silica gel column (25 x 4 cm) and washed with 250 mL of CHCl
3
. The
product was separated by an increasing gradient of acetone (3% - 8%) in chloroform. The
fractions containing product were dried under vacuum to yield 89 mg (18% yield) of
product 7.
1
H NMR (500 MHz, CDCl
3
) δ: 1.34-13.7 (t, 12H), 4.08-4.29 (m, 8H), 4.95 (s,
2H), 7.19-7.22 (t, 2H), 7.30-7.33 (t, 2H), 7.66-7.68 (d, 2H), 7.89-7.93 (d, 2H), 8.33-.8.35
(d, 2H).
31
P NMR (202 MHz, CDCl
3
) δ: 22.17. MS (ESI) C
22
H
16
O
7
P (M-H)
-
m/z calcd:
571.17. Found: 571.1.
Dealkylated diphosphonylated PA derivative (methylenebis(3-
hydroxynaphthalene-4,2-diyl))diphosphonic acid, 8. 59 mg of 7 was added to a dry
flask and an excess (2 mL) of distilled BTMS was added. The reaction mixture was
stirred at room temperature overnight (13 h). The product mixture was then dried under
reduced pressure, recrystallized from methanol, and then lyophilized to furnish
approximately 10.5 mg of a light brown solid (21% yield).
1
H NMR (500 MHz, D
2
O) δ:
7.19-7.22 (t, 2H), 7.30-7.33 (t, 2H), 7.77-7.79 (d, 2H), 8.00-8.03 (d, 2H), 8.06-8.08 (d,
2H).
31
P NMR (202 MHz, D
2
O) δ: 11.80. MS (ESI) C
11
H
10
O
4
P (M-H)
-
m/z calcd: 459.04.
Found: 459.0.

77

3.4.5. Inhibitory Assays
Inhibitor preparation. PA (Sigma), 3, and 4 were dissolved in 25 mM phosphate
buffer (pH 7.5). 8 was dissolved in 20 mM Tris buffer (pH 8.4) that resulted in a cloudy
solution. Addition of small quantities of KOH or DMSO did not improve its appearance.
Since the concentration could not be verified, the observed 50% inhibition at 250 µM
compound is probably a maximum estimate, but significantly greater than those observed
with the other compounds.
DNA preparation. High-pressure liquid chromatography-purified
oligonucleotides were purchased from IDT (Integrated DNA Technologies), dissolved in
10 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and their concentrations were determined by
UV absorbance at 260 nm. A single-nucleotide gap was produced by annealing three
oligonucleotides (1/1.2/1.2, 15-mer primer/18-mer downstream oligonucleotide/34-mer
template). The annealing reactions were carried out by incubating a solution of 10 µM
primer with 12 µM of downstream and template oligonucleotides at 90–100 ˚C for 3 min,
followed by 30 min at 65 ˚C and then slow cooling to room temperature. The downstream
oligonucleotide was synthesized with a 5´-phosphate. The sequence of the primer,
downstream, and template oligonucleotides were (5´–3´): CTG CAG CTG ATG CGC
(15-mer), GTA CGG ATC CCC GGG TAC (18-mer), GTA CCC GGG GAT CCG TAC
GGC GCA TCA GCT GCA G (34-mer; coding base in the gap is underlined),
respectively. The upstream primer was 5´-labeled with [γ-
32
P] ATP using Optikinase
(United States Biochemical Corp.) and free radioactive ATP was removed using a Bio-
Spin 6 column (Bio-Rad).

78

DNA synthesis assays. DNA synthesis was assayed on single-nucleotide
gapped DNA substrates where the templating base in the gap was guanine.
16
Enzyme
activities were determined using a reaction mixture containing 50 mM Tris-HCl, pH 7.4,
10 mM MgCl
2
, 200 nM single-nucleotide gapped DNA, 20 µM dCTP concentrations, and
various concentrations of inhibitor. Reactions were initiated with 1 nM enzyme at 37 ºC
and stopped with EDTA (150 mM final concentration) mixed with formamide dye after 1
min. The radiolabeled 15-mer primer substrate and 16-mer product were resolved by
denaturing 16% polyacrylamide gel electrophoresis [31 × 38.5 × 0.04 cm, 16% (v/v)
polyacrylamide, 8 M urea gels] and quantified in the dried gels by phosphorimagery. The
concentration of inhibitor (I) that produces 50% inhibition (IC
50
) was determined by
fitting fractional activity (activity with inhibitor/activity without inhibitor) to equation
(1):
Fractional Activity = 1/[1 + (I/IC
50
)]
h
Eq. (1),
where h is the Hill coefficient.

79

3.5 Chapter References
(1) Cisneros, G. A.; Perera, L.; García-Díaz, M.; Bebenek, K.; Kunkel, T. A.;
Pedersen, L. G. Catalytic Mechanism of Human DNA Polymerase λ with Mg2+
and Mn2+ from Ab Initio Quantum Mechanical/molecular Mechanical Studies.
Dna Repair 2008, 7, 1824–1834.

(2) Hu, H.-Y. Identification of Small Molecule Synthetic Inhibitors of DNA
Polymerase by NMR Chemical Shift Mapping. J. Biol. Chem. 2004, 279, 39736–
39744.

(3) Garcia-Diaz, M.; Bebenek, K.; Gao, G.; Pedersen, L. C.; London, R. E.; Kunkel, T.
A. Structure–function Studies of DNA Polymerase Lambda. Dna Repair 2005, 4,
1358–1367.

(4) Hazan, C.; Boudsocq, F.; Gervais, V.; Saurel, O.; Ciais, M.; Cazaux, C.; Czaplicki,
J.; Milon, A. Structural Insights on the Pamoic Acid and the 8 kDa Domain of
DNA Polymerase Beta Complex: Towards the Design of Higher-affinity
Inhibitors. Bmc Struct. Biol. 2008, 8, 22.

(5) Maciejewski, M. W.; Liu, D.; Prasad, R.; Wilson, S. H.; Mullen, G. P. Backbone
Dynamics and Refined Solution Structure of the N-terminal Domain of DNA
Polymerase Β. Correlation with DNA Binding and dRP Lyase Activity. J. Mol.
Biol. 2000, 296, 229–253.

(6) Barakat, K.; Tuszynski, J. Relaxed Complex Scheme Suggests Novel Inhibitors for
the Lyase Activity of DNA Polymerase Beta. J. Mol. Graph. Model. 2011, 29,
702–716.

(7) Trott, O.; Olson, A. J. AutoDock Vina: Improving the Speed and Accuracy of
Docking with a New Scoring Function, Efficient Optimization, and
Multithreading. J. Comput. Chem. 2009, 455–461.

(8) Doppelt-Azeroual, O.; Moriaud, F.; Adcock, S. A.; Delfaud, F. A Review of MED-
SuMo Applications. Infect. Disord. Drug Targets 2009, 9, 344–357.

(9) Moriaud, F.; Doppelt-Azeroual, O.; Martin, L.; Oguievetskaia, K.; Koch, K.;
Vorotyntsev, A.; Adcock, S. A.; Delfaud, F. Computational Fragment-Based
Approach at PDB Scale by Protein Local Similarity. J. Chem. Inf. Model. 2009, 49,
280–294.

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(10) Kenner, G. W.; Williams, N. R. A Method of Reducing Phenols to Aromatic
Hydrocarbons. J. Chem. Soc. Resumed 1955, 522.

(11) Dhawan, B.; Redmore, D. Lithiation-induced 1,3-migrations of phosphorus(IV)
Groups from Heteroatom to the Naphthalene Ring. J. Org. Chem. 1991, 56, 833–
835.

(12) Hatano, M.; Miyamoto, T.; Ishihara, K. Enantioselective Dialkylzinc Addition to
Aldehydes Catalyzed by Chiral Zn(II)-BINOLates Bearing Phosphonates and
Phosphoramides in the 3,3′-Positions. Synlett 2006, 2006, 1762–1764.

(13) Li, X.; Hewgley, J. B.; Mulrooney, C. A.; Yang, J.; Kozlowski, M. C.
Enantioselective Oxidative Biaryl Coupling Reactions Catalyzed by 1,5-
Diazadecalin Metal Complexes: Efficient Formation of Chiral Functionalized
BINOL Derivatives. J. Org. Chem. 2003, 68, 5500–5511.

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Synthesis, Characterization and First Crystal Structure of a Dimeric Ti(IV)
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Chapter 4. Synthesis of Enantiomerically Enriched (+)-JQ1 for Acute
Myeloid Leukemia Studies
4.1 Introduction
Histones, which are the core proteins involved in chromatin structural
organization, undergo several types of post-translational modifications (PTMs). These
covalent modifications are referred to collectively as the “histone code”, and play an
important role in epigenetics: the heritable patterns of gene expression and phenotype that
are not encoded for by the underlying DNA sequence.
1
One of these PTMs is the N-
acetylation of lysine residues, which has been shown to either promote or silence gene
expression.
1,2
As a result, the improper degree of histone acetylation can cause the
expression of disease-causing genes, such as those that result in cancer and
inflammation.
3

There are three classes of proteins involved in the acetylation of lysines. The
histone acetyltransferases (HATs) function as “writers”, transferring acetyl groups from
acetyl CoA to the lysines, while the histone deacetylases (HDACs) are “erasers”, which
remove the acetyl groups from the histone tails.
1,3,4
The third group is the “readers”,
which are proteins containing bromodomains (BRDs) that recognize the acetylated
lysines.
1,3,4
BRDs are α-helical protein domains consisting of approximately 110 residues,
that recognize the acetylated lysines on histone 3 and 4 proteins in chromatin.
5-7
BRDs
play an important role in regulating gene expression and chromatin organization by
recruiting nuclear proteins to the acetylated sites on the chromatin.
7
The phylogenetic tree
of the human BRDs is shown in Figure 4.1.1.
3
In humans, there are 61 known BRDs that

82

have been found in 46 different proteins, and studies have uncovered clear links between
certain BRDs and several diseases.
4,5
Thus, selectively targeting and inhibiting these
protein domains has
elicited much interest
recently as a novel
therapeutic approach.
One class of
proteins that contain
BRDs is the
bromodomains and
extraterminal (BET)
family of proteins. These
proteins are
transcriptional regulators
that each consist of two
BRDs, BD1 and BD2,
followed by an
extraterminal (ET)
domain.
2,6
In mammals, there are four members in the BET family; BRD2, BRD3, BRD4
and BRDT.
7,8
The BET family controls cellular processes that may result in cancer,
inflammation or viral infection, making the bromodomains within this family a popular
target for drug discovery.
9
Although most transcriptional regulators dissociate from
chromosomes during mitosis, BETs do the opposite, allowing growth-associated genes to
Figure 4.1.1. Phylogenetic tree of the human BRDs based on sequence
alignments. The numbers in parentheses represent the domain number,
starting from the N-terminus.
3

83

be transcribed after cell division.
3,7,8,10
This imparts an epigenetic “memory” that is
transferred to daughter cells after cell division, such that they inherit a certain pattern of
gene expression.
3,10

Crystal and NMR solution
structures have both revealed that
BRDs, although diverse in
sequence, all contain 4 α helices,
Z, A, B and C, with a ZA-loop
connecting helices Z and A, and a
BC-loop connecting helices B and
C.
3,5,8
These features can be seen in
the crystal structure of
BRD4(BD1),
11
shown in Figure
4.1.2. The acetyl-lysine binding
site within the BRDs lies between
the two loops and is hydrophobic in nature, with several aromatic residues.
3,5,11
This
hydrophobicity is necessary because the acetylation masks the charge on the lysines,
making them less polar.
3
In all BRDs, the BC-loop contains a conserved asparagine
residue, whose side chain forms a hydrogen bond to the carbonyl of the acetylated lysine
side chain, while both the BC and ZA loops contain a conserved tyrosine residue
each.
3,11,12
These conserved residues are required for the recognition of acetyl-lysine
residues.
11
On the other hand, the hydrophobic, variable (less conserved) amino acid
sequences on the BC and ZA loops of BRDs mainly allow for specific interactions
Figure 4.1.2. Crystal structure of the first BRD (BD1)
of the BET family member BRD4 (PDB code:
3MUK). The protein surface is in gray, and the acetyl-
lysine binding site is located between the ZA and BC
loops.
11

84

between each BRD and its target protein, but also allow for the recognition of the acetyl-
lysine.
11,13
Additionally, crystal structures have revealed a series of structured water
molecules that mediate hydrogen-bonding interactions between the BRD and acetyl-
lysine, enhancing the binding.
5,11

As mentioned previously, BET proteins are responsible for many cellular
processes. In particular, they play a critical role in modulating the proliferation of cells
and the progression of the cell cycle.
7
For example, BRD4 plays a role in the regulation
of transcription in cells by recruiting the positive transcription elongation factor, P-
TEFb.
3,8,14,15
P-TEFb contains a kinase (a Cdk9-cyclin T1 heterodimer) that
phosphorylates a serine on RNA polymerase II, initiating transcriptional
elongation.
3,8,10,14,16
When BRD4 fails to function properly in cells, it can lead to several
types of cancer, including acute myeloid leukemia and breast cancer,
15
as it supports
tumor progression by activating the transcription of genes on mitotic chromosomes that
promote growth and prevent apoptosis.
7,16
As an example, BRD4 recruits P-TEFb to
mitotic chromosomes, causing the expression of c-Myc, a well-known growth-promoting
gene.
16-18
Also, a genetic mutation that produces a chimeric protein consisting of the N-
terminal BRD of BRD4 fused with the nuclear protein in testis (NUT), leads to the
development of the incurable and fatal subtype of squamous carcinoma known as NUT
midline carcinoma (NMC).
1,3,7,11
Importantly, the RNA silencing of BRD4-NUT has been
successful in halting proliferation and leading to terminal squamous differentiation,
indicating the significant role that BRDs play in disease progression, and the potential of
targeting them for therapeutic intervention.
17

85

In addition to its role in cancer, BRD4 is also involved in the regulation of
transcription in viruses such as the human immunodeficiency virus (HIV) and Epstein-
Barr Virus (EBV).
2
Furthermore, BRD4 is involved in mediating the viral transcription of
the human papillomavirus (HPV), an oncovirus that can cause cervical cancer, via its
interaction with the viral E2 protein factor.
8,14,15,19,20
BRD4 is also known to activate the
transcription of pro-inflammatory genes.
2
Due to the obvious involvement of BETs in
several diseases, specific and potent BET inhibitors are highly sought after in current
research.

Two of the BET-specific BRD inhibitors that were recently developed are (+)-
JQ1 and I-BET762 (Figure 4.1.3), which are based on triazolodiazepine scaffolds, and
exhibit nM potencies against BRD4.
4,9
Compounds containing a 3,5-dimethylisoxazole
moiety have also been designed as acetyl lysine mimetics for the inhibition of BET
bromodomains, such as I-BET151 (Figure 4.1.3).
4,21
Overall, these inhibitors have shown
a lot of promise. I-BET151 and (+)-JQ1 have both displayed significant activity against
Figure 4.1.3. The structures of the BET inhibitors (+)-JQ1 and I-BET762, which are both
triazolodiazepines, and I-BET151, which contains a 3,5-dimethylisoxazole moiety.

86

NMC, multiple myeloma, acute myeloid leukemia and mixed lineage leukemia in murine
models,
17,21–23
while I-BET762 has recently entered clinical trials for the treatment of
NMC.
24
Intriguingly, (+)-JQ1 was also found to work against another member of the
BET family, the testis-specific BRDT, which is necessary for spermatogenesis,
effectively acting as a non-hormonal male contraceptive.
25

The studies leading to the development of (+)-JQ1 arose from the observation that
simple thienodiazepines can bind to BRD4.
17
This discovery was made by Mitsubishi
Pharmaceuticals during their application of anti-inflammatory phenotypic studies in the
search for molecules that can be utilized in the treatment of ailments such as autoimmune
diseases.
26
Molecular modeling studies performed by Bradner et al. then examined the
binding of different potential ligands within the binding pocket in the crystal structure of
the first BRD of BRD4 (BRD4(BD1), giving rise to the structure-activity relationships
that were used to design the novel thieno-triazolo-1,4,-diazepine (+)-JQ1.
17
This novel
molecule was found to exhibit potent and selective inhibition of BRD4(BD1), with a K
d

value of 50 nM, and an IC
50
of 77 nM.
4,17
The authors designed the molecule to contain a
bulky tert-butyl ester functional group at the C6 position to both mediate binding to the
central benzodiazepine receptor,
27
and to allow for future diversification of the pendant
group, if necessary.
17

Crystal structures revealed that the (+)-JQ1 enantiomer occupied the whole
acetyl-lysine pocket of BRD4(BD1), displaying excellent shape complementarity, as
shown in Figure 4.1.4 (a). Similar to the interactions between BRD4 and its natural
acetyl-lysine substrate, the (+)-JQ1 triazole ring on the molecule forms a hydrogen bond
to the conserved Asn140 residue in BRD4(BD1) (Figure 4.1.4 (b)), and binding is

87

stabilized by hydrophobic interactions with the residues in the ZA- and BC- loop
regions.
17
Furthermore, studies showed that (+)-JQ1 acts as a potent and specific
competitive inhibitor of acetyl-lysine for the BET BRDs, and that it is capable of
displacing BRD4 from nuclear chromatin in cells.
17
Since (+)-JQ1 has already exhibited
impressive activity levels against several forms of cancer, it is of interest to test its
efficacy against new cancer types that have yet to be explored and that are in dire need of
novel therapeutic strategies.

One of these important cancer targets is acute myeloid leukemia, or AML. AML
is an aggressive, life-threatening stem cell malignancy in which the myeloblast cells in
the blood, bone marrow, and organs of patients proliferate and accumulate
uncontrollably.
28
While some patients respond to chemotherapy and/or hematopoietic
a. b.
Figure 4.1.4. The binding of (+)-JQ1 to the acetyl-lysine binding pocket of BRD4(BD1), where (a)
shows the contact residues (labeled and depicted in stick form) and (b) shows the interactions of (+)-
JQ1 with the conserved asparagine and tyrosine residues, as well as with structural water molecules,
based on the crystal structure with PDB code 3MXF.
17

88

stem cell transplantation (SCT), others exhibit a poor response to the currently available
drugs or cannot undergo SCT.
28
One of the ways by which AML is propagated is via the
generation of self-renewing leukemia stem cells, which arise due to the failure of cell-fate
programs.
29
This process has been linked to alterations in the epigenetic pathways which
are created by chromatin modifications,
30
suggesting a possible alterative therapy by
targeting those pathways. In fact, by utilizing an RNA interference (RNAi) screening
study in an AML mouse model, it was recently discovered that BRD4 was a potential
therapeutic target for this disease.
28

Consequently, studies on the links between BRD4 and several different AML
subtypes have been carried out. For example, BRD4 knockdown with small hairpin
RNAs (shRNAs) was shown to result in cell-cycle arrest in leukemia cells, including two
MLL-AF9
+
human AML cell lines.
23
Furthermore, JQ1, which targets BRD4, was found
to inhibit growth in 13 out of 14 AML cell lines, as well as in 12 out of 15 primary
human AMLs with a diverse set of subtypes at submicromolar concentrations, by
inducing cell-cycle arrest and apoptosis.
23
Overall, the suppression of BRD4 using
shRNAs or JQ1 resulted in both in vitro and in vivo anti-leukemic effects, and in the
terminal differentiation of self-renewing leukemia stem cells.
23
In other studies, JQ1
exhibited significant anti-leukemic effects in several AML subtypes including those in
relapsed and refractory patients, and CD34
+
/CD38
-
and CD34
+
/CD38
+
AML cells.
28
(+)-
JQ1 was also found to suppress the expression of the myc gene in several AML subtypes
from human and mouse samples,
23
and exhibited significant anti-proliferative effects on
mouse xenograft models of MV4-11 AML cells.
31

89

Thus, it is clearly evident that (+)-JQ1 has been effective in the suppression of
several subtypes of AML to date, and that it shows promise for the treatment of other
subtypes. In this study, enantiomerically enriched (+)-JQ1 was synthesized in order to
evaluate it for its therapeutic efficacy in an aggressive subtype of AML for which current
therapies are ineffective.
4.2 Results and Discussion
4.2.1 Synthesis of Enantiomerically Enriched (+)-JQ1
The synthesis of enantiomerically enriched (+)-JQ1 was carried out by following
the five-step synthetic procedure published by Filippakopoulos et al,
17
with minimal
changes, as illustrated in Scheme 4.2.1. The authors also published a method to produce a
racemic mixture of JQ1, but since only the (+)-JQ1 isomer is biologically active against
BRD4, we chose to synthesize it via the pathway that would yield more of the active
isomer. The scale of our synthesis was three times the published scale. The first step
involved a Gewald reaction, with a Knoevenagel-type condensation between 2-butanone
and 4-chlorobenzoyl acetonitrile, forming an intermediate that then underwent a sulfur-
mediated cyclization, and finally tautomerized to form the 2-amino-thiophene product, 2.
The amino group on 2 was then coupled to an aspartic acid residue with an Fmoc
protected amine and a tert-butyl protected side chain acid via PyBOP-mediated coupling
to form 3. Amine Fmoc deprotection was then achieved by reaction with a 20%
piperidine in DMF solution, forming 4. An acid-catalyzed cyclization reaction between
the free amine and the keto group using silica in toluene was then carried out to convert 4
to 5. In the final step, a Pellizzari reaction between the amide group in 5 and acetic

90

hydrazide was used to produce a 1,2,4-triazole. Briefly, the amide was converted via
reaction with potassium tert-butoxide and diethylchlorophosphate to a reactive enol-
phosphate, that then underwent a reaction with acetic hydrazide, and two sequential
dehydrations to furnish (+)-JQ1, in a 40% enantiomeric excess, as determined by
analytical chiral HPLC. Each product was purified by flash silica column
chromatography, and analyzed by
1
H NMR before proceeding to the next step. While we
obtained a 70:30 ratio of (+)- : (-)-JQ1, the authors reported a 90% enantiomeric purity of
(+)-JQ1, and further purified the product by preparative chiral HPLC.
17

Scheme 4.2.1. Synthesis of enantiomerically enriched (+)-JQ1, as described by Filippakopoulos et al.
17

91

4.2.2 Biological Studies
Biological studies were performed by Dr. Mary Callanan at the Institut Albert
Bonnoit at Université Joseph Fourier in Grenoble, France. (+)-JQ1 was evaluated for its
therapeutic efficacy in an aggressive subtype of AML for which current therapies are
ineffective. Treatment of leukemic cells with (+)-JQ1 in vitro was found to
downregulate the expression of key onco-proteins linked to differentiation arrest and
resistance to senescence. Furthermore, in a xenotransplant of this aggressive leukemia,
(+)-JQ1 treatment was found to significantly constrain tumor growth (8 animals tested
per control and (+)-JQ1 treatment groups). This raises the possibility of applying BET
inhibitors for the successful treatment of this aggressive AML subtype.
4.3 Conclusion
In conclusion, enantiomerically enriched (+)-JQ1 was synthesized according to
the literature procedure by Filippakopoulos et al,
17
yielding a 40% enantiomeric excess of
the isomer of interest. The in vitro treatment of AML cells by our collaborators with the
(+)-JQ1 sample we provided resulted in the downregulation of key onco-proteins that are
linked to differentiation arrest and resistance to senescence. (+)-JQ1 was also found to
significantly constrain tumor growth in a xenotransplant of this aggressive leukemia. As a
result, BET inhibitors represent a new class of molecules that may possess therapeutic
potential for the treatment of this aggressive AML subtype.

92

4.4 Experimental
4.4.1 Materials and Methods
4-Chlorobenzoyl acetonitrile and acetic hydrazide were purchased from Sigma-
Aldrich. Morpholine, piperidine and diethyl chlorophosphate were purchased from
Aldrich and were distilled prior to use. PyBOP was purchased from Novabiochem, Fmoc-
Asp(Ot-Bu)-OH was purchased from Aldrich, silica powder was purchased from Macron
Fine Chemicals, sulfur was purchased from Malinckrodt, and potassium tert-butoxide
was purchased from Acros Organics. 2-Butanone was purchased from Malinckrodt, while
n-butanol was purchased from Fischer Scientific, and both were distilled prior to use.
The NMR operating frequency for
1
H was 500 MHz.
1
H NMR spectra were
referenced to residual CHCl
3
(δ 7.26) in CDCl
3
.
32
All chemical shift values (δ) are given
in ppm. A Thermo-Finnigan Deca XP Max mass spectrometer with an ESI probe was
utilized for low-resolution mass spectra.
1
H NMR spectra for compounds 2-5 and for the
JQ1 product matched literature values
17
and are presented in Appendix D. All NMR
sample concentrations were approximately 1-3 mg/mL. Analytical HPLC to determine
the relative ratios of the (+)- and (-)-JQ1 isomers was performed on a Varian Prostar
HPLC system with an SPD-10A VP Shimadzu UV detector (set to 254 nm), using an
analytical ChiralCel OD-H chiral column (250 mL x 4.6 mm x 5 µm, Diacel Chemical
Ind. Ltd.) and a mobile phase of 15% isopropanol in hexane, with a flow rate of 1
mL/min over 16 min. The HPLC trace and mass spectrum of the enantiomerically
enriched (+)-JQ1 sample are included in Appendix D.

93

4.4.2 Synthesis of Enantiomerically Enriched (+)-JQ1
(2-Amino-4,5-dimethylthiophen-3-yl)(4-chlorophenyl)methanone, 2. 21.9
mmol (1 equiv) of 4-chlorobenzoyl acetonitrile (1) was added to a dry flask, to which 60
mL of ethanol, 21.9 mmol (1 equiv) of distilled morpholine and 21.9 mmol (1 equiv) of
distilled 2-butanone were added. The mixture was stirred at room temperature to dissolve
the solids, after which 21.9 mmol (1 equiv) of sulfur was added. The reaction mixture
was then heated to 70 °C and the reaction was stirred for 12 h under nitrogen. The
reaction mixture was allowed to cool down and then poured into approximately 200 mL
of brine, and extracted with 150 mL of ethyl acetate three times. The organic layers were
combined and washed with 50 mL of brine, dried over sodium sulfate, and then filtered
and dried under vacuum to yield crude product 2. A smaller amount of brine was used in
comparison to the published procedure in order to avoid loss of product to the aqueous
layer. 2 was then purified by flash silica column chromatography using a gradient of
ethyl acetate in hexanes from 0-50%. The fractions determined to contain 2 by TLC were
then collected and dried under vacuum to yield 2.98 g (52% yield) of bright yellow solid
product 2.
1
H NMR (500 MHz, CDCl
3
) δ: 1.56 (s, 3H), 2.14 (s, 3H), 7.38-7.39 (d, 2H),
7.46-7.48 (d, 2H).
(S)-tert-Butyl-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-4-{[3-(4-
chlorobenzoyl)-4,5-dimethylthiophen-2-yl]amino}-4-oxobutanoate, 3. In a dry flask,
3.5 mmol (1 equiv) of 9-fluorenylmethoxycarbonyl-aspartic acid β-tert-butyl ester
(Fmoc-Asp(Ot-Bu)-OH) was dissolved in 3.5 mL dry DMF. 3.3 mmol (0.95 equiv) of
(benzotriazol-1-yloxyl)tripyrrolidinophosphonium (PyBOP) was then added, followed by
9.63 mmol (2.75 equiv) of distilled DIEA. 3.5 mmol (1 equiv) of 2 was added to the

94

mixture, which was then stirred at room temperature for 5.5 h. The published procedure
called for a 4 h reaction, but it was determined by TLC that 5.5 h were required to reach
completion in our hands. After the reaction, 30 mL of brine were added to the reaction
mixture, and the aqueous layer was extracted with 70 mL of ethyl acetate three times.
Less brine was utilized than the published procedure, to avoid loss of product. The
organic layer was washed with brine, dried over sodium sulfate, filtered, and then
concentrated under vacuum. The crude product 3 was then purified by flash silica column
chromatography utilizing a gradient of ethyl acetate in hexanes from 0-10%, to give 1.1 g
(49% yield) of 3 as a sticky yellow solid.
1
H NMR (500 MHz, CDCl
3
) δ: 1.43 (s, 9H),
1.68 (s, 3H), 2.26 (s, 3H), 2.71-2.75 (d, 1H), 3.11-3.15 (d, 1H), 4.24-4.32 (m, 2H), 4.63
(s, br, 1H), 4.78 (s, br, 1H), 6.09-6.11 (d, 1H), 7.21-7.26 (m, 2H), 7.31-7.33 (d, 2H), 7.36-
7.39 (t, 2H), 7.43-7.45 (d, 2H), 7.61-7.65 (dd, 2H), 7.75-7.76 (d, 2H), 11.73 (s, br, 1H).
(S)-tert-Butyl 3-amino-4-((3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-
yl)amino)-4-oxobutanoate, 4. 1.7 mmol (1 equiv) of 3 was dissolved in 8.1 mL of a 20%
piperidine in DMF solution (0.22 M), and the mixture was stirred at room temperature for
30 min. 80 mL of ethyl acetate and 45 mL of brine were then added to the reaction
mixture. The aqueous layer was extracted with 2 x 80 mL of ethyl acetate and the organic
layers were combined, washed with 50 mL of brine three times, dried over sodium
sulfate, filtered, and then dried under vacuum to yield crude product 4. Again, less brine
was used than the published procedure to minimize loss of product. The crude product
was then purified by flash silica column chromatography, using a gradient of ethyl
acetate in hexanes from 0-20%. The solvent was removed in vacuo to give a quantitative
yield of 4 as a sticky yellow solid.
1
H NMR (500 MHz, CDCl
3
) δ: 1.42 (s, 9H), 1.71 (s,

95

3H), 1.85 (s, br, 1H), 2.26 (s, 3H), 2.66-2.75 (m, 1H), 2.85-2.90 (m, 1H), 3.85 (s, br, 1H),
7.40-7.43 (d, 2H), 7.57-7.59 (d, 2H), 12.10 (s, br, 2H).
(S)-tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-
thieno[2,3-e][1,4]diazepin-3-yl)acetate, 5. 1.6 mmol (1 equiv) of 4 was dissolved in 54
mL of toluene, and 1.6 g of silica powder was added. The reaction mixture was heated for
3 h at 90 °C, after which it was cooled down to room temperature. The silica was not
filtered off as described in the published procedure. Instead, the solvent was removed
under vacuum and the product-infused silica was dry loaded onto a silica gel column. The
crude product 4 was purified by flash silica column chromatography using an ethyl
acetate in hexanes mobile phase with a gradient from 0-20%, furnishing 401 mg (60%
yield) of compound 5.
1
H NMR (500 MHz, CDCl
3
) δ: 1.47 (s, 9H), 1.61 (s, 3H), 2.29 (s,
3H), 3.07-3.11 (m, 1H), 3.32-3.37 (m, 1H), 4.22-4.24 (t, 1H), 7.34-7.36 (d, 2H), 7.43-
7.45 (d, 2H).
(S)-tert-Butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate, (+)-JQ1. A 0.15 M solution of 5
(0.96 mmol, 1 equiv) in dry THF (6.4 mL) was added to a dry 3-necked flask and cooled
to -78 °C, after which 1.1 mL of a 1 M solution of potassium tert-butoxide in THF (1.05
mmol, 1.1 equiv) was added drop-wise. The reaction mixture was then gradually warmed
to -10 °C and then to room temperature, and stirred for 30 min. Next, the mixture was
cooled back down to -78 °C and 1.15 mmol (1.2 equiv) of diethyl chlorophosphate was
added. Next, the mixture was warmed to -10 °C slowly over 45 min, after which 1.44
mmol (1.5 equiv) of acetic hydrazide was added. The reaction mixture was then stirred
for 1 h at room temperature, followed by the addition of 8 mL of n-butanol. The mixture

96

was then heated at 90 °C for 2 h, as opposed to the published procedure which involved
heating for only 1 h. The solvents were then removed under vacuum and the crude
product was purified twice by flash silica column chromatography utilizing a gradient of
ethyl acetate in hexanes from 0-100%, to give 101 mg of enantiomerically enriched (+)-
JQ1. Chiral HPLC using an analytical ChiralCel OD-H chiral column (250 mL x 4.6 mm
x 5 µm, Diacel Chemical Ind. Ltd.) and a mobile phase of 15% isopropanol in hexane,
with a flow rate of 1 mL/min over 16 min was used to determine the ratio of the (+)- and
(-)- JQ1 isomers, which was found to be 70:30, respectively. On the other hand, the work
by Filippakopoulos et al.
17
yielded (+)-JQ1 with a 90% enantiomeric purity. The authors
also further purified the product via chiral preparative HPLC using an OD-H column,
generating the (+)-JQ1 isomer in greater than 99% ee.
1
H NMR (500 MHz, CDCl
3
) δ:
1.50 (s, 9H), 1.71 (s, 3H), 2.43 (s, 3H), 2.80 (s, 3H), 3.52-3.59 (m, 2H), 4.59-4.62 (t, 1H),
7.34-7.36 (d, 2H), 7.41-7.43 (d, 2H). MS (ESI) C
23
H
26
ClN
4
O
2
S (M+H)
+
m/z calcd.:
457.15. Found: 457.

97

4.5 Chapter References
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110

Appendix A. Chapter 1 Supporting Data

Figure A1.
1
H NMR (500 MHz, D
2
O) of imidazol-1-yl acetic acid.

111

Figure A2.
1
H NMR spectrum (500 MHz, D
2
O, pH 8.9) of risedronic acid ((1-hydroxy-
1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acid) monohydrate, 1.

112

Figure A3.
31
P NMR spectrum (202 MHz, D
2
O, pH 8.9, 85% H
3
PO
4
reference) of
risedronic acid ((1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acid)
monohydrate, 1.

113

Figure A4. Risedronate UV data. Using the average UV absorption data from three
experiments on an authentic sample of risedronic acid disodium (found to contain 1.3
equivalents of water by elemental analysis) supplied by Procter & Gamble, Inc., the
extinction coefficient of risedronate was found to be 3687 M
-1
cm
-1
in 0.1 M phosphate
buffer, pH 7.2. The absorption spectrum for our microwave-synthesized sample 1 gave a
purity of 99.6% (monohydrate).

114

Figure A5. LC-MS data of risedronic acid, 1, using a mobile phase of 60 µM ammonium
acetate buffer with 2% acetonitrile, pH 5.5.

115

Figure A6.
1
H NMR (500 MHz, D
2
O, pH 10.1) of zoledronic acid ([1-hydroxy-2-(1H-
imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid)) monohydrate, 2.

116

Figure A7.
31
P NMR (202 MHz, D
2
O, pH 10.1, 85% H
3
PO
4
reference) of zoledronic acid
([1-hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid)) monohydrate, 2.

117

Figure A8. LC-MS data of zoledronic Acid, 2, using a mobile phase of 60 µM
ammonium acetate buffer with 2% acetonitrile, pH 5.5.

118

Figure A9.
1
H NMR (500 MHz, D
2
O, pH 8.8) of pamidronic acid ((3-amino-1-
hydroxypropane-1,1-diyl)bis(phosphonic acid)) monosodium monohydrate, 3.

119

Figure A10.
31
P NMR (202 MHz, D
2
O, pH 8.8, 85% H
3
PO
4
reference) of pamidronic
acid ((3-amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid)) monosodium
monohydrate, 3

120

Figure A11. Mass spectrum of pamidronate, 3.

121

Figure A12.
1
H NMR (500 MHz, D
2
O, pH 8.9) of alendronic acid ([4-amino-1-hydroxy-
1-(hydroxy-oxido-phosphoryl)-butyl]phosphonic acid) monosodium dihydrate, 4.

122

Figure A13.
31
P NMR (202 MHz, D
2
O, pH 8.9, 85% H
3
PO
4
reference) of alendronic acid
([4-amino-1-hydroxy-1-(hydroxy-oxido-phosphoryl)-butyl]phosphonic acid)
monosodium dihydrate, 4.

123

Figure A14. Mass spectrum of alendronate, 4.

124

Figure A15.
1
H NMR (500 MHz, D
2
O, pH 8.9) of neridronic acid (6-amino-1-
hydroxyhexane-1,1-diyl)bis(phosphonic acid)) monosodium monohydrate, 5.

125

Figure A16.
31
P NMR (202 MHz, D
2
O, pH 8.9, 85% H
3
PO
4
reference) of neridronic acid
(6-amino-1-hydroxyhexane-1,1-diyl)bis(phosphonic acid)) monosodium monohydrate, 5.

126

Figure A17. Mass spectrum of neridronate, 5.

127

Figure A18. Spiked
1
H NMR (500 MHz, D
2
O, pH 9.1) of risedronic acid, 1. 5 mg of 1
was spiked with 8 mg of risedronate disodium (98+% purity) supplied by Procter &
Gamble, Inc.

Figure A19. Spiked
31
P NMR (202 MHz, D
2
O, pH 9.1, 85% H
3
PO
4
reference) of
risedronic acid, 1. 5 mg of 1 was spiked with 8 mg of risedronate disodium (98+% purity)
supplied by Procter & Gamble, Inc.

128

Figure A20. Spiked
1
H NMR (500 MHz, D
2
O, pH 9.4) of Zol, 2. 5 mg of 2 was
spiked with 5 mg zoledronic acid purchased from Molekula, Inc.

Figure A21. Spiked
31
P NMR (202 MHz, D
2
O, pH 9.4, 85% H
3
PO
4
reference) of
Zol, 2. 5 mg of 2 was spiked with 5 mg zolendronic acid purchased from
Molekula, Inc.

129

Figure A22. Spiked
1
H NMR (500 MHz, D
2
O, pH 8.8) of pamidronic acid
monosodium, 3. 5 mg of 3 was spiked with 5 mg of pamidronate monosodium,
supplied by Novartis, Inc.

Figure A23. Spiked
31
P NMR (202 MHz, D
2
O, pH 8.8, 85% H
3
PO
4
reference) of
pamidronic acid monosodium, 3. 5 mg of 3 was spiked with 5 mg of pamidronate
monosodium, supplied by Novartis, Inc.

130

Figure A24. Spiked
1
H NMR (500 MHz, D
2
O, pH 8.6) of alendronic acid monosodium,
4. 6 mg of 4 was spiked with 6 mg alendronate monosodium, supplied by Procter &
Gamble, Inc.

131

Figure A25. Spiked
31
P NMR (202 MHz, D
2
O, pH 8.6, 85% H
3
PO
4
reference) of
alendronic acid monosodium, 4. 6 mg of 4 was spiked with 6 mg alendronate
monosodium, supplied by Procter & Gamble, Inc.

132

Appendix B. Chapter 2 Supporting Data

Figure B1.
1
H NMR (500 MHz, D
2
O) of the methylphosphonic acid product from the
microwave BTMS dealkylation of dimethyl methylphosphonate in ACN at 40 °C for 10
min.

133

Figure B2.
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard (0 ppm)) of the
methylphosphonic acid product from the microwave BTMS dealkylation of dimethyl
methylphosphonate in ACN at 40 °C for 10 min.

134

Figure B3.
1
H NMR (500 MHz, D
2
O) of the methylphosphonic acid product from the
microwave BTMS dealkylation of diethyl methylphosphonate in ACN at 40 °C for 15
min.

135

Figure B4.
31
P NMR (202 MHz, D
2
O, adjusted with external H
3
PO
4
standard (0 ppm)) of
the methylphosphonic acid product from the microwave BTMS dealkylation of diethyl
methylphosphonate in ACN at 40 °C for 15 min.

136

Figure B5.
31
P NMR (202 MHz, CDCl
3
) of the reaction mixture containing the silyl ester
of (2-methoxy-2-oxoethyl)phosphonic acid after BTMS microwave reaction. The single
peak is proof of the complete selectivity of BTMS for phosphonate esters.

137

Figure B6.
1
H NMR (500 MHz, D
2
O, pH 7.6) of the (2-methoxy-2-oxoethyl)phosphonic
acid product from the microwave BTMS dealkylation of trimethylphosphonoacetate.

138

Figure B7.
31
P NMR (202 MHz, D
2
O, pH 7.6, adjusted with external H
3
PO
4
standard (0
ppm)) of the 2-methoxy-2-oxoethyl)phosphonic acid product from the microwave BTMS
dealkylation of trimethylphosphonoacetate.

139

Figure B8.
31
P NMR (202 MHz, CDCl
3
) of the reaction mixture containing the silyl ester
of (2-ethoxy-2-oxoethyl)phosphonic acid after BTMS microwave reaction. The single
peak is proof for the complete selectivity of BTMS for phosphonate esters.

140

Figure B9.
1
H NMR (500 MHz, D
2
O, pH 8.2) of the (2-ethoxy-2-oxoethyl)phosphonic
acid product from the microwave BTMS dealkylation of triethylphosphonoacetate.

141

Figure B10.
31
P NMR (202 MHz, D
2
O, pH 8.2, adjusted with external H
3
PO
4
standard (0
ppm)) of the (2-ethoxy-2-oxoethyl)phosphonic acid product from the microwave BTMS
dealkylation of triethylphosphonoacetate.

142

Figure B11.
31
P NMR (202 MHz, D
2
O) of (bromodifluoromethyl)phosphonic acid.

143

Figure B12.
1
H NMR (500 MHz, CDCl
3
) of 2-(diethoxyphosphoryl)acetic acid.

144

Figure B13.
31
P NMR (202 MHz, CDCl
3
) of 2-(diethoxyphosphoryl)acetic acid.

145

Figure B14.
1
H NMR (500 MHz, D
2
O, NaHCO
3
, pH 1.12) of the 2-phosphonoacetic
acid product from the microwave BTMS dealkylation of 2-(diethoxyphosphoryl)acetic
acid.

146

Figure B15.
31
P NMR (202 MHz, D
2
O, NaHCO
3
, pH 1.12) of the 2-phosphonoacetic
acid product from the microwave BTMS dealkylation of 2-(diethoxyphosphoryl)acetic
acid.

147

Figure B16.
31
P NMR (202 MHz, D
2
O capillary) of the product mixture of MW
PyBrOP-mediated coupling between (S)-PMPDAP and ethanol. As the NMR shows,
there is approximately 80% monoethyl ester and 20% diethylester. The peak at 14.20
ppm represents reacted PyBrOP.

148

Figure B17.
31
P NMR (202 MHz, D
2
O capillary) of the reaction mixture of MW PyBOP-
mediated coupling between (S)-PMPDAP monoethyl ester and ethanol. The peak at 14.03
ppm represents reacted PyBOP.

149

Figure B18.
31
P NMR (202 MHz, CD
3
OD) of (S)-PMPDAP(OEt)
2
, after purification by
silica gel column chromatography.

150

Figure B19.
1
H NMR (500 MHz, D
2
O) of the 9-[2-(phosphonomethoxy)ethyl]-2,6-
diaminopurine (PMEDAP) product from the microwave BTMS dealkylation of
PMEDAP(OiPr)
2
.

151

Figure B20.
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard (0 ppm)) of the 9-[2-
(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) product from the microwave
BTMS dealkylation of PMEDAP(OiPr)
2
.

152

Figure B21.
1
H NMR (500 MHz, D
2
O) of the 9-[(2-(S)-(phosphonomethoxy)propyl-2,6-
diaminopurine ((S)-PMPDAP) product from the microwave BTMS dealkylation of (S)-
PMPDAP(OEt)
2
.

153

Figure B22.
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard (0 ppm)) of the 9-[(2-
(S)-(phosphonomethoxy)propyl-2,6-diaminopurine ((S)-PMPDAP) product from the
microwave BTMS dealkylation of (S)-PMPDAP(OEt)
2
.

154

Figure B23.
1
H NMR (500 MHz, D
2
O) of the 9-(2-phosphonomethoxy)ethyl adenine
(PMEA) product from the microwave BTMS dealkylation of PMEA(OiPr)
2
.

155

Figure B24.
31
P NMR (202 MHz, D
2
O, external H
3
PO
4
standard (0 ppm)) of the 9-(2-
phosphonomethoxy)ethyl adenine (PMEA) product from the microwave BTMS
dealkylation of PMEA(OiPr)
2
.

156

Appendix C. Chapter 3 Supporting Data

Figure C1.
1
H NMR (500 MHz, CDCl
3
) of aryl dimethyl phosphate (dimethyl
naphthalen-2-yl phosphate), 1, with a magnification of the aromatic section in the inset.

157

Figure C2.
31
P NMR (202 MHz, CDCl
3
) of aryl dimethyl phosphate (dimethyl
naphthalen-2-yl phosphate), 1.

158

Figure C3.
31
P NMR (202 MHz, CDCl
3
) of the product mixture containing P-(3-
hydroxy-2-naphthalenyl)-methyl phosponate, 2.

159

Figure C4. Mass spectrum of the product mixture containing P-(3-hydroxy-2-
naphthalenyl)-methyl phosponate, 2.

160

Figure C5.
1
H NMR (500 MHz, D
2
O) of the mono-methylphosphonate PA derivative (3-
hydroxy-4-((2-hydroxy-3-(hydroxy(methoxy)phosphoryl)naphthalen-1-yl)methyl)-2-
naphthoic acid), 3.

161

Figure C6.
31
P NMR (202 MHz, D
2
O) of the mono-methylphosphonate PA derivative (3-
hydroxy-4-((2-hydroxy-3-(hydroxy(methoxy)phosphoryl)naphthalen-1-yl)methyl)-2-
naphthoic acid), 3.

162

Figure C7. Mass spectrum of mono-methylphosphonate PA derivative (3-hydroxy-4-((2-
hydroxy-3-(hydroxy(methoxy)phosphoryl)naphthalen-1-yl)methyl)-2-naphthoic acid), 3.

163

Figure C8. HPLC chromatogram for the separation of the mono-methylphosphonate PA
derivative (3-hydroxy-4-((2-hydroxy-3-(hydroxy(methoxy)phosphoryl)naphthalen-1-
yl)methyl)-2-naphthoic acid), 3.

164

Figure C9.
1
H NMR (500 MHz, D
2
O) of deaklylated mono-phosphonylated PA
derivative (3-hydroxy-4-((2-hydroxy-3-phosphononaphthalen-1-yl)methyl)-2-naphthoic
acid), 4.

165

Figure C10.
31
P NMR (202 MHz, D
2
O) of deaklylated mono-phosphonylated PA
derivative (3-hydroxy-4-((2-hydroxy-3-phosphononaphthalen-1-yl)methyl)-2-naphthoic
acid), 4.

166

Figure C11. Mass spectrum of deaklylated mono-phosphonylated PA derivative (3-
hydroxy-4-((2-hydroxy-3-phosphononaphthalen-1-yl)methyl)-2-naphthoic acid), 4.

167

Figure C12. HPLC chromatogram for the separation of deaklylated mono-
phosphonylated PA derivative (3-hydroxy-4-((2-hydroxy-3-phosphononaphthalen-1-
yl)methyl)-2-naphthoic acid), 4.

168

Figure C13.
1
H (500 MHz, CDCl
3
) of aryl diethyl phosphate (diethyl naphthalen-2-yl
phosphate), 5.

169

Figure C14.
31
P (202 MHz, CDCl
3
) of aryl diethyl phosphate (diethyl naphthalen-2-yl
phosphate), 5.

170

Figure C15.
1
H NMR (500 MHz, CDCl
3
) of the isomer mixture containing aryl diethyl
phosphonate (P-(3-hydroxy-2-naphthalenyl)-diethyl phosponate), 6, showing 12 aromatic
protons due to the presence of two isomers before separation.

171

Figure C16.
31
P NMR (CDCl
3
, 202 MHz) of aryl diethyl phosphonate (P-(3-hydroxy-2-
naphthalenyl)-diethyl phosponate), 6 with its isomer (the C-1 phosphonate) before
separation.

172

Figure C17. Mass spectrum of the isomer mixture of aryl diethyl phosphonate, 6.

173

Figure C18.
1
H NMR (500 MHz, CDCl
3
) of aryl diethyl phosphonate (P-(3-hydroxy-2-
naphthalenyl)-diethyl phosponate), 6, after separation from its C-1 phosphonate isomer.

174

Figure C19.
1
H NMR (500 MHz, CDCl
3
) of the aromatic region of aryl diethyl
phosphonate (P-(3-hydroxy-2-naphthalenyl)-diethyl phosponate), 6, after separation from
its C-1 phosphonate isomer. The doublet at 8.00 ppm has a J
P-H
of 20 Hz, proving that
this is the C-3 phosphonate isomer.

175

Figure C20.
1
P NMR (202 MHz, CDCl
3
) of aryl diethyl phosphonate (P-(3-hydroxy-2-
naphthalenyl)-diethyl phosponate), 6, after separation from its C-1 phosphonate isomer.

176

Figure C21.
1
H NMR (500 MHz, CDCl
3
) of the C-1 phosphonate isomer of aryl diethyl
phosphonate after separation from the C-3 phosphonate.

177

Figure C22.
1
H NMR (500 MHz, CDCl
3
) of the aromatic region of the C-1 phosphonate
isomer of aryl diethyl phosphonate, after separation from its mixture with the C-3
phosphonate.

178

Figure C23.
31
P NMR (202 MHz, CDCl
3
) of the C-1 phosphonate isomer of aryl diethyl
phosphonate after separation from the C-3 phosphonate.

179

Figure C24.
1
H NMR (500 MHz, CDCl
3
) of the tetraethyl di-phosphonylated PA
derivative (tetraethyl (methylenebis(3-hydroxynaphthalene-4,2-diyl))bis(phosphonate)),
7.

180

Figure C25.
31
P NMR (202 MHz, CDCl
3
) of the tetraethyl di-phosphonylated PA
derivative (tetraethyl (methylenebis(3-hydroxynaphthalene-4,2-diyl))bis(phosphonate)),
7.

181

Figure C26. Mass spectrum of the tetraethyl di-phosphonylated PA derivative (tetraethyl
(methylenebis(3-hydroxynaphthalene-4,2-diyl))bis(phosphonate)), 7.

182

Figure C27.
1
H NMR (500 MHz, D
2
O) of the dealkylated di-phosphonylated PA
derivative (methylenebis(3-hydroxynaphthalene-4,2-diyl))diphosphonic acid, 8.

183

Figure C28.
31
P NMR (202 MHz, D
2
O) of the dealkylated di-phosphonylated PA
derivative (methylenebis(3-hydroxynaphthalene-4,2-diyl))diphosphonic acid, 8.

184

Figure C29. Mass spectrum of the dealkylated di-phosphonylated PA derivative
(methylenebis(3-hydroxynaphthalene-4,2-diyl))diphosphonic acid, 8.

185

Appendix D Chapter 4 Supporting Data

Figure D1.
1
H NMR (500 MHz, CDCl
3
) of (2-amino-4,5-dimethylthiophen-3-yl)(4-
chlorophenyl)methanone, 2.

186

Figure D2.
1
H NMR (500 MHz, CDCl
3
) of (S)-tert-butyl-3-({[(9H-fluoren-9-
yl)methoxy]carbonyl}amino)-4-{[3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-
yl]amino}-4-oxobutanoate, 3.

187

Figure D3.
1
H NMR (500 MHz, CDCl
3
) of (S)-tert-butyl 3-amino-4-((3-(4-
chlorobenzoyl)-4,5-dimethylthiophen-2-yl)amino)-4-oxobutanoate, 4.

188

Figure D4.
1
H NMR (500 MHz, CDCl
3
) of (S)-tert-butyl 2-(5-(4-chlorophenyl)-6,7-
dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate, 5.

189

Figure D5.
1
H NMR (500 MHz, CDCl
3
) of (S)-tert-butyl 2-(4-(4-chlorophenyl)-2,3,9-
trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate, (+)-JQ1.

190

Figure D6. Chiral analytical HPLC chromatogram of the enantiomerically enriched
sample of (+)-JQ1, showing the relative ratios of (+)-JQ1 and (-)-JQ1 (70:30,
respectively).

191

Figure D7. Mass spectrum of the enantiomerically enriched sample of (+)-JQ1.

Abstract (if available)

Abstract Nitrogen-containing bisphosphonates (N-BPs) are a class of drugs that accumulate in the bone and target osteoclasts, effectively preventing excessive bone resorption, and providing treatment for diseases such as osteoporosis and Paget’s disease. A library of novel N-BPs for biological testing is therefore of interest. One of the methods to rapidly generate libraries of compounds is microwave-assisted organic synthesis (MAOS), which relies on the direct heating of polar molecules to achieve efficient heating. A rapid, simple, and efficient method for the small-scale synthesis of N-BP drugs via microwave irradiation was established. Whereas the traditional method for the preparation of these compounds typically requires heating for at least 9.5 h, this new microwave-assisted procedure is complete in less than 20 min, while maintaining good yields of product. ❧ Microwave irradiation was also utilized to devise a novel, more efficient procedure for the McKenna method of selective phosphonate ester dealkylation using bromotrimethylsilane (BTMS). Rates were vastly improved by microwave heating, and the versatility of this reaction was demonstrated by varying the temperature, and by utilizing a variety of solvents. Solvents with low polarities, such as dioxane, were successfully utilized, and the reactions were also run in neat BTMS, suggesting that the reactants themselves are polar enough to be directly heated via microwave irradiation. BTMS dealkylation produced quantitative yields, and was applied towards isopropyl, ethyl, and methyl esters. Furthermore, temperatures as low as 40 °C were used, as well as equimolar amounts of BTMS, which, together with the short reaction times, makes this approach mild and environmentally friendly. The use of pressure was also obviated, and the reaction was compatible with carboxylate esters, and other sensitive groups such as acyclic nucleoside phosphonate esters possessing diamino purines. ❧ Polymerase β (Pol β) is essential for maintaining the integrity of the genome by participating in the base excision repair (BER) of damaged DNA bases via its lyase domain. Pol β is overexpressed in several cancers, and is involved in the resistance of tumors to DNA-damaging cancer therapeutics through the action of its lyase domain. As a result, inhibiting the lyase domain of Pol β is of significant interest. The best inhibitor known is pamoic acid (PA), which, due to its high Kd and IC₅₀ values, cannot be crystallized with the enzyme. Through the use of docking studies with Autodock Vina, and fragment-based drug design with MEDIT MED-SuMo, mono- and di-phosphonylated analogues of PA were designed and synthesized. Preliminary biological data revealed that the inhibitory activity was increased 3-fold with the un-methylated mono-phosphonylated derivative, while activity was increased 6-fold with the mono-methylphosphonate derivative. Inhibitory activities for the di-phosphonylated analogue have not yet been determined, and an X-ray crystal structure of the lyase domain bound to any of the inhibitors is still pending. ❧ Finally, enantiomerically enriched (+)-JQ1 was synthesized according to the literature procedure by Filippakopoulos et al.,¹ yielding a 40% enantiomeric excess of (+)-JQ1. This compound is a highly potent and specific inhibitor for the bromodomains of the BET family of proteins, which recognize acetylated lysines on the histone tails of chromatin, and participate in the epigenetic control of gene expression. (+)-JQ1 was previously found to be effective in the treatment of several types of cancer, including many subtypes of acute myeloid leukemia (AML). (+)-JQ1 was tested against a novel aggressive subtype of AML for which current treatments are ineffective. The in vitro treatment of AML cells with (+)-JQ1 resulted in the downregulation of key onco-proteins that are linked to differentiation arrest and resistance to senescence. (+)-JQ1 was also found to significantly constrain tumor growth in a xenotransplant of this aggressive leukemia. As a result, BET inhibitors represent a new class of molecules that may possess therapeutic potential for the treatment of this aggressive AML subtype. ❧ References: ❧ (1) Filippakopoulos, P.

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Creator Mustafa, Dana (author)

Core Title I. Microwave-assisted synthesis of phosphonic acids; II. Design and synthesis of polymerase β lyase domain inhibitors

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 11/12/2013

Defense Date 10/24/2013

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

Tag bisphosphonates,BTMS,microwave-assisted organic synthesis,myase domain,OAI-PMH Harvest,pamoic acid,polymerase β,TMSBr

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles E. (committee chair), Armani, Andrea M. (committee member), Williams, Travis J. (committee member)

Creator Email dana.mustafa@gmail.com,dmustafa@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-347124

Unique identifier UC11296067

Identifier etd-MustafaDan-2149.pdf (filename),usctheses-c3-347124 (legacy record id)

Legacy Identifier etd-MustafaDan-2149.pdf

Dmrecord 347124

Document Type Dissertation

Format application/pdf (imt)

Rights Mustafa, Dana

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA