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Synthesis, structural analysis and in vitro antiviral activities of novel cyclic and acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs

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Synthesis, structural analysis and in vitro antiviral activities of novel cyclic and acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs

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SYNTHESIS, STRUCTURAL ANALYSIS AND IN VITRO ANTIVIRAL
ACTIVITIES OF NOVEL CYCLIC AND ACYCLIC (S)-HPMPA AND
(S)-HPMPC TYROSINAMIDE PRODRUGS

by

Ivan S. Krylov

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

December 2012

Copyright 2012 Ivan S. Krylov

ii
DEDICATION

To my parents, my wife and my son

for their endless love, tireless support and infinite encouragement

iii
ACKNOWLEDGEMENTS
An old proverb, stating that “it takes a whole village to raise a child” has acquired
an entirely new meaning for me after my son, Victor, was born on July 15, 2012. Now, as
I compose my Ph.D. thesis, I would like to paraphrase that proverb and say that “it takes
a whole university to grant a well-rounded Ph.D. degree.” Although it is impossible to
recognize everyone who helped me during my graduate studies, I would like to
acknowledge those who contributed and influenced me the most in the last five years on
my journey towards my Ph.D.
First, I would like to thank and express my most sincere appreciation to my Ph.D.
advisor, Professor Charles E. McKenna, a true mentor, for giving me the opportunity to
study Chemistry and perform research in his laboratory. I first met Professor McKenna as
a participant in the Research Experience for Undergraduates (REU) program at the USC
Chemistry Department in 2006. Throughout all these years, Professor McKenna provided
me with the guidance, support and encouragement necessary to facilitate my growth into
a positive young member of the scientific community. During this journey, I came a long
way from being in a bubble of idealism to realizing, that in order to be a successful
scientist, one needs to have more than just ideas. Thus, I am very thankful to Professor
McKenna for sharing not only his deep knowledge and passion for Chemistry, but also
for teaching me the values of collaboration, excellence in scientific writing and how to
give a quality presentation.
Secondly, I would like to thank Dr. Boris Kashemirov (an expert in phosphorus
Chemistry) for sharing ideas, providing invaluable training and working closely with me
iv
during all those years. I really appreciate that his door was always open for fruitful
discussions, valuable advice and interesting comments on a broad range of topics, from
science to politics and sports.
I would also like to acknowledge Professors G. K. Surya Prakash, Ian S. Haworth,
Travis Williams and Peter Z. Qin for serving on my guidance committee and for
providing helpful advice and suggestions throughout my Ph.D. career. Also, I am
thankful to Professors Thomas C. Flood, Nicos A. Petasis, Charles E. McKenna, Richard
W. Roberts, G. K. Surya Prakash, and Kyung W. Jung, whose classes I attended.
During my time at USC, I worked on a multidisciplinary project, which could not
move forward without a tremendous effort from our knowledgeable and passionate
collaborators. Thank you to Dr. John M. Hilfinger and other members of TSRL Inc. for
performing the transport studies on our prodrugs, Professor John C. Drach and his lab at
the University of Michigan for the antiviral evaluations of all our compounds, and
Professor Ralf Haiges at USC for the X-ray crystallographic analysis of our compounds.
Also, I would like to acknowledge the former members of the McKenna group who
worked on this project with me; Dr. Michaela Serpi for the synthesis and stability
evaluation of 4 prodrugs (2.23 – 2.25, 2.27) and Dr. Valeria Zakharova for the synthesis
of 2 diastereomers of the model phenyl cyclic (S)-HPMPC conjugates (R
p
)-4.5 and
(S
p
)-4.5.
Next, I would like to recognize and thank the past and present members of the
McKenna group who impacted my life not only by sharing knowledge, but also by setting
an example for how things should be done. I am especially grateful to Dr. Larryn
v
Peterson for all her help at the beginning of my Ph.D. career at USC, Dr. Brian
Chamberlain for the many fun and crazy times we had, Yue Wu for being an extremely
nice and helpful person, Dana Mustafa for her tireless proofreading and help with editing
my English throughout the years, and Candy Hwang for being the happiest person I
know.
I thank Allan Kershaw for helping me with the NMR experiments and for making
sure that all NMR instruments are working properly 24/7. Marie de la Torre, Michele
Dea, Inah Kang, Katie McKissick, and Heather Connor: thank you for taking good care
of the administrative side of my Ph.D.
I also thank my friends (too many to list here, but you know who you are) for
providing the support and friendship I needed during all these years.
Above all, I would like to thank my loving wife, Valentina Krylova, and my
parents, Irina Krylova and Sergey Krylov, for their endless support and encouragement.
There are no words to convey how much I love them and how much they mean to me.
Without them, none of this would have been possible.

vi
TABLE OF CONTENTS
DEDICATION .................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................... iii
LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
LIST OF SCHEMES......................................................................................................... xii
ABSTRACT ..................................................................................................................... xiv
CHAPTER 1 ...................................................................................................................... 1
Antiviral acyclic nucleoside phosphonates and their prodrugs .............................. 1
1.1 Acyclic nucleoside phosphonates ................................................................... 1
1.2 Mechanism of antiviral action of ANPs.......................................................... 3
1.3 Antiviral potential of the ANPs ...................................................................... 4
1.4 Limitations to the antiviral ANPs ................................................................... 5
1.5 Prodrug concept .............................................................................................. 6
1.6 Prodrug approaches ......................................................................................... 9
1.6.1 Acyloxyalkyl and alkyloxycarbonyloxymethyl approach .................... 9
1.6.2 Phosphoramidate approach ................................................................. 12
1.6.3 Lipidic prodrug approach .................................................................... 19
1.6.4 SATE approach ................................................................................... 26
1.6.5 CycloSal approach .............................................................................. 33
1.7 Perspectives................................................................................................... 40
1.8 References ..................................................................................................... 42
CHAPTER 2 .................................................................................................................... 61
Single amino acid prodrugs of (S)-HPMPA and (S)-HPMPC .............................. 61
2.1 Introduction ................................................................................................... 61
2.2 The concept of the amino acid phosphonate ester prodrug approach ........... 62
2.3 The Rationale: why amino acids? ................................................................. 63
2.4 Previously reported hydroxyl amino acid (S)-HPMPC prodrugs ................. 65
2.4.1 EG-linked amino acid (S)-HPMPC prodrugs ..................................... 65
2.4.2 Dipeptide (S)-HPMPC prodrugs ......................................................... 66
vii
2.5 Single amino acid prodrugs .......................................................................... 69
2.5.1 First round of SAR studies of single amino acid prodrugs ................. 71
2.5.2 Second round of SAR studies of single amino acid prodrugs ............ 78
2.5.3 Third round of SAR studies of single amino acid prodrugs ............... 84
2.6 Conclusions ................................................................................................... 90
2.7 Experimental section ..................................................................................... 92
2.8 References ..................................................................................................... 94
CHAPTER 3 .................................................................................................................... 99
Synthesis of the single amino acid (S)-HPMPA and (S)-HPMPC prodrugs ....... 99
3.1 Introduction ................................................................................................... 99
3.2 Synthesis of (S)-HPMPA and (S)-HPMPC ................................................ 100
3.3 Synthesis of the single amino acid prodrugs of (S)-HPMPA and
(S)-HPMPC ................................................................................................. 102
3.3.1 Synthesis of the cyclic (S)-HPMPA and (S)-HPMPC prodrugs…....102
3.3.2 Synthesis of the acyclic phosphonate ester prodrugs…………….....109
3.4 Synthesis of thio-(S)-HPMPA prodrugs ..................................................... 112
3.5 Conclusions ................................................................................................. 113
3.6 Experimental Section .................................................................................. 114
3.7 References ................................................................................................... 145
CHAPTER 4 .................................................................................................................. 147
The structure of cyclic nucleoside phosphonate ester prodrugs: an inquiry .... 147
4.1 Introduction ................................................................................................. 147
4.2 Synthesis and structural evaluation of individual diastereomers of
model phenyl (S)-HPMPA and (S)-HPMPC prodrugs ............................... 149
4.3 Conclusions ................................................................................................. 156
4.4 Experimental section ................................................................................... 157
4.5 References ................................................................................................... 163
BIBLIOGRAPHY ........................................................................................................... 168
APPENDIX A: Chapter 3 supporting data ..................................................................... 194
APPENDIX B: Chapter 4 supporting data...................................................................... 257

viii
LIST OF TABLES
Table
2.1
Half-lives (τ
1/2
, min) of single amino acid (S)-HPMPA prodrugs
2.12, 2.15 - 2.17 in PBS, pH 6.5 at 37 ºC.
74

2.2
In vitro antiviral activities against HCMV, Cowpox and Vaccinia
and cytotoxicities of the single amino acid prodrugs of (S)-
cHPMPA, 2.12, 2.15, 2.16.
77

2.3
Comparison of calculated log D values for the tyrosine-based (S)-
cHPMPA and (S)-cHPMPC derivatives with the values for parent
(S)-HPMPA and (S)-HPMPC.
79

2.4
Half-lives (τ
1/2
, min) of the tyrosine-based (S)-cHPMPA and (S)-
cHPMPC prodrugs 2.16, 2.23 - 2.27 in PBS and in rat intestinal
homogenate at pH 6.5 and 37 ºC.
80

2.5
In vitro antiviral activities against HCMV, Cowpox and Vaccinia
and cytotoxicities of the tyrosine-based prodrugs of (S)-cHPMPA
and (S)-cHPMPC 2.16, 2.23 - 2.28.
83

2.6
Comparison of the calculated logD values for the tyrosine N-alkyl
amide based (S)-cHPMPA, (S)-HPMPA, (S)-cHPMPC and (S)-
HPMPC prodrugs with the values of parent (S)-HPMPA, (S)-
cHPMPA, (S)-HPMPC and (S)-cHPMPC and the values of the
previously reported HDP- and ODE-prodrugs of (S)-HPMPC and
(S)-HPMPA.
87

2.7
In vitro antiviral activities against HCMV and cytotoxicities of
tyrosine N-alkyl amide prodrugs of (S)-HPMPA, (S)-cHPMPA, (S)-
HPMPC, (S)-cHPMPC 2.26, 2.28 - 2.41.
88

2.8
In vitro antiviral activities against cowpox and vaccinia virus and
cytotoxicities of the tyrosine N-alkyl amide prodrugs of (S)-HPMPA
and (S)-cHPMPA.
89

4.1
1
H NMR parameters for (R
p
)- and (S
p
)-diastereomers of 4.5 and 4.6
recorded at 25 ºC and 500 MHz.
152

4.2
Estimated population of conformation III for (S
p
)-4.5 and (S
p
)-4.6
diastereomers.
155

ix
LIST OF FIGURES
Figure
1.1
Selected examples of natural nucleosides (left) and acyclic nucleoside
analogues (right).
1

1.2 Structures of selected acyclic nucleoside phosphonates. 3

1.3
Mechanism of action of cidofovir (left) and adefovir (right). Reprinted by
permission from Macmillan Publishers Ltd: Nat. Rev. Drug Discovery,
2005, 4, 928-940, copyright 2012.
4

1.4
General representation of activation pathway of (A) bis(acyloxymethyl)
prodrug and (B) bis(alkyloxycarbonyloxymethyl) prodrug to the
corresponding nucleoside monophosphate analogue: a) enzymatic
hydrolysis; b) spontaneous chemical hydrolysis.
10

1.5 Structures of adefovir dipivoxyl and tenofovir disoproxil. 11

1.6
General representation of phosphoramidate activation pathway to the
corresponding nucleoside monophosphate inside the cell: a) ester
hydrolysis by carboxypeptidase or esterase type enzyme; b) spontaneous
cyclization; c) aqueous hydrolysis; d) enzymatic cleavage of P-N bond by
phosphoramidase type enzyme.
14

1.7 Phosphoramidate pronucleotides currently in clinical trials. 18

1.8
Activation pathway of cytarabine ocfosfate (YNK01) to cytarabine
monophosphate: a) ω-hydroxylation and subsequent oxidation to the
carboxylic acid; b) β-oxidation.
21

1.9 Activation pathway of HDP- and ODE-prodrugs of ANPs. 23

1.10 Structures of selected lipid ANP prodrugs. 24

1.11
General representation of bis(SATE) phosphotriester pronucleotides
activation pathway to the corresponding nucleoside monophosphate inside
the cell. a) esterase-mediated hydrolysis; b) spontaneous decomposition.
27

1.12 General representation of mixed SATE pronucleotide activation pathways. 30

1.13
General representation of the first, second and third generation cycloSal
pronucleotides activation pathways. a) hydrolysis of phenolate ester via
S
N
P mechanism; b) spontaneous cleavage of the benzyl ester group.
34
x
2.1
Amino acid phosphonate ester prodrugs: 1) cyclic phosphonate diesters; 2)
acyclic phosphonate monoesters.
62

2.2
General representation of the activation pathway of alcoholic amino acid
phosphonate ester prodrugs: a) enzymatic or pH-dependent chemical
hydrolysis; b) enzymatic hydrolysis.
63

2.3 Structures of acyclovir and its prodrug valacyclovir. 64

2.4
Selected structures of previously described amino acid (S)-HPMPC
prodrugs.
66

2.5 Modification sites of the single amino acid promoiety. 69

2.6
Structures of the serine-based (S)-HPMPA prodrugs evaluated for
hPEPT1-mediated transport in the electrophysiologic Xenopus laevis
oocytes assay.
70

2.7 Structures of single amino acid cyclic (S)-HPMPA prodrugs. 73

2.8
Chemical activation of the mono amino acid (S)-HPMPA prodrugs 2.12,
2.15 - 2.17 in phosphate buffer solution (PBS), pH 6.5 at 37 ºC.
74

2.9
Suggested mechanisms for the serine/threonine (left) and cysteine/tyrosine
(right) cyclic (S)-HPMPA prodrug hydrolysis in phosphate buffer solution
with physiological pH (6-8).
75

2.10
Enzymatic degradation of the mono amino acid cyclic (S)-HPMPA
prodrugs 2.12, 2.15, 2.16 in intestinal homogenate, pH 6.5 at 37 ºC.
76

2.11
Tyrosine (S)-HPMPA and (S)-HPMPC prodrugs 2.16, 2.23 - 2.27
synthesized and evaluated in the second round of SAR studies.
79

2.12
A) Chemical and enzymatic activation of (L)-TyrNH-i-Bu-cHPMPA 2.26
in phosphate buffer solution and intestinal homogenate at pH 6.5 and 37
ºC. B) HPLC analysis of (L)-TyrNHi-Bu-cHPMPA 2.28 metabolism at pH
6.5 and 37 ºC in PBS (left HPLC traces) and in intestinal homogenate
(right HPLC traces). Top HPLC traces correspond to the reaction mixtures
at t = 0 min; bottom HPLC traces correspond to the reaction mixtures after
incubation for 270 min or 300 min.
82

2.13
Structures of the previously reported hexadecyloxypropyl (HDP) or
octadecyloxyethyl (ODE) prodrugs of (S)-HPMPC and (S)-HPMPA.
85

xi
2.14
Tyrosine N-alkyl amide based (S)-cHPMPA, (S)-HPMPA, (S)-cHPMPC
and (S)-HPMPC prodrugs 2.26, 2.28, 2.29 - 2.40 synthesized and evaluated
in the third round of SAR studies.
86
3.1
Structures of (S)-HPMPC 2.1, (S)-HPMPC 2.2 and the single amino acid
prodrugs 2.12 - 2.17 and 2.23 - 2.40 synthesized for the SAR studies.
100

3.2 Gel
31
P NMR of (L)-SerOMe bound to the TCP-resin (3.23). 106

3.3
IR spectra of the free TCP-resin (left) and of the (L)-SerOMe bound resin
3.18 (right). The samples were analyzed in triplicate (KBr pellet).
108

4.1
Structures of (S)-HPMPC 4.1 and (S)-HPMPA 4.2 and corresponding
single amino acid and dipeptide prodrug forms 4.3 and 4.4.
147

4.2
Proposed cis/trans and axial/equatorial
31
P NMR correlations for different
cyclic ANP drug diastereomers.
148

4.3 X-ray crystal structure of (R
p
)-4.5. Ellipsoids enclose 50% probability. 151

4.4 X-ray crystal structure of (R
p
)-4.6. Ellipsoids enclose 50% probability. 151

4.5 X-ray crystal structure of (S
p
)-4.6. Ellipsoids enclose 50% probability. 153

4.6
Possible conformer equilibria of (R
p
)-4.5, -4.6 (I-II) (left) and (S
p
)-4.5, -
4.6 (III-V) (right) in solution (CDCl
3
stabilizes conformer III), based on
analysis of solvent effect on
1
H NMR coupling constant values. I is
favored by both steric (equatorial nucleobase) and electronic (anomeric
preference for axial P-OPh) effects.
154

xii
LIST OF SCHEMES
Schemes
3.1 Original synthesis of (S)-HPMPA by Holy et al. 100

3.2
Synthesis of (S)-HPMPA 2.2. Reagents and conditions: (a) Trityl
chloride, CH
2
Cl
2,
TEA, rt, 20 h; (b) K
2
CO
3
, DMF, 105
o
C, 22 h; (c)
3.4, NaH, DMF, 0-5
o
C, 2.5 h; r.t., 12 h; (d) 80% aq. AcOH, 80 °C
for 3h and r.t. for 12 h; (e) BTMS, DMF, r.t., overnight, H
2
O.
101

3.3
Conventional syntheses of mono amino acid cyclic (S)-HPMPA and
(S)-HPMPC prodrugs 3.7, 3.9 - 3.19. Reagents and conditions: i) and
ii) PyBOP, DIEA, DMF, 40 ºC; iii) TFA, CH
2
Cl
2
, r.t.
103

3.4
Synthesis of tBOC-tyrosine N-alkyl amides. Reagents and
conditions: i) RNH
2
, EDC, HOBt, CH
2
Cl
2
, 24 h, 25 ºC
104

3.5
Solid-phase synthesis of the single amino acid and dipeptide prodrugs
of cyclic (S)-HPMPA and (S)-HPMPC (2.12 – 2.16, 3.32, 3.33).
Reagents and conditions: i) PyBOP, DIEA, DMF, 40 ºC; ii) TFA,
CH
2
Cl
2
or HCl, dioxane, 25 ºC.
105

3.6
Immobilization of the mono amino acid esters 3.34 - 3.38 to TCP-
resin. Reagents and conditions: i) 2 eq. TBDMSCl, 3 eq. imidazole,
CH
2
Cl
2
, 25 ºC, 24 h; ii) TCP-resin, DIEA, CH
2
Cl
2
, 25 ºC, 18 h; iii)
TBAF, THF, 25 ºC, 5 h; iv) 2 eq. TBDMSCl, 3 eq. imidazole, DMF,
25 ºC, 24 h.
107

3.7
Formation of N-formyl derivatives 3.44 and 3.45 via a Vilsmeier-type
reagent. Reagents and conditions: i) imidazole, DMF, 18 h, 25 ºC; ii)
H
2
O.
12

107

3.8
Solid-phase synthesis of the dipeptides bound to the TCP-resin 3.28
and 3.29. Reagents and conditions: i) DIEA, CH
2
Cl
2
; ii) Pd(PPh
3
)
4
,
PhSiH
3
, CH
2
Cl
2
; iii) 3.34 or 3.36, PyBOP, DIEA, CH
2
Cl
2
, DMF; iv)
TBAF, THF.
109

3.9
Attempts to synthesize the tyrosine prodrug 2.28 from (S)-HPMPA.
Reagents and conditions: i) PyBOP, DIEA, DMF, 40 ºC; ii) 3.12,
PyBOP, DIEA, DMF, 40 ºC.
110

3.10
Synthesis of the single amino acid acyclic (S)-HPMPA and (S)-
HPMPC prodrugs 2.28, 2.35 - 2.40. Reagents and conditions: i) aq.
NH
4
OH, ACN, 45 ºC.
110
xiii
3.11
BTMS silylation-dealkylation of Ph-cHPMPA 3.48. Reagents and
conditions: i) BTMS, ACN, reflux, 18 h; ii) MeOH; iii) 50 mM aq.
K
2
CO
3
, 100 ºC.
111

3.12
Synthesis of (L)-TyrNH-i-Bu-SH-HPMPA prodrugs 3.52. Reagents
and conditions: i) BTMS, ACN, reflux, 18 h; ii) MeOH; iii) NaHS,
H
2
O, r.t.
112

3.13
Synthesis of thio cyclic (S)-HPMPA 3.54. Reagents and conditions:
i) PyBOP, DIEA, DMF, 40 ºC; ii) C
6
H
5
SH, Et
3
N/dioxane, r.t.
113

4.1
Preparation of individual cyclic (S)-HPMPC and (S)-HPMPA phenyl
ester diastereomers ((R
p
)-4.5, -4.6 and (S
p
)-4.5, -4.6). Reagents and
conditions: a) PhOH, PyBOP, N,N-diisopropylethylamine, DMF,
40 °C, 2 h; b) Cs
2
CO
3
, DMF, 0.1 eq PhOH; recrystallization from
CH
3
OH/acetone (for (R
p
)-4.5) or i-PrOH/EtOAc (for (R
p
)-4.6); c)
recrystallization from CH
3
OH/acetone/hexane (for (S
p
)-4.5) or
CH
3
CN (for (S
p
)-4.6).
149

xiv
ABSTRACT
Acyclic nucleoside phosphonates (ANPs), in particular (S)-HPMPC (Cidofovir,
Vistide®) and (S)-HPMPA are highly potent broad spectrum antiviral agents.
Unfortunately, unfavorable Absorption, Distribution, Metabolism and Excursion
(ADME) profiles limit the utilization of these therapeutics in the clinic, mainly due to
low cell membrane permeability, owing to the presence of a phosphonic acid group that
ionizes at physiological pH. To address this issue, an amino acid phosphonate ester
prodrug approach is being developed, which explores the use of benign single amino
acids as promoieties to mask the negative charges of the ANPs. The main objective of the
present work was to identify the promoiety, which, upon conjugation to the ANPs,
produces prodrugs able not only to withstand the rigors of metabolism, but also to
efficiently release the active parent drug at the target site in vivo.

In order to achieve this objective, three consecutive rounds of structure-activity
relationship (SAR) studies involving the synthesis, characterization and biological
evaluation of 24 single amino acid (S)-HPMPA and (S)-HPMPC prodrugs were
performed. The design of these prodrugs was aimed to “tune” the P-X-C linkage, the
amino acid stereochemistry and the C-terminal functional group, and to optimize the
length of the alkyl chain in the tyrosine N-alkyl amide moiety. Synthesis of the designed
prodrugs was accomplished using a solution-phase method, and a newly developed solid-
phase approach. Although the solid-phase approach produced the prodrugs in lower
yields (20-35%) as compared to the conventional method (40-65%), it required simpler
xv
purification, and can be easily scaled up and automated in the future. The tyrosine acyclic
phosphonate prodrugs were obtained by hydrolysis of their cyclic counterparts with
yields of 40-60%. The cyclic prodrugs were generated as pairs of diastereomers (S
p
or R
p
)
that in case of tyrosine-based prodrugs exhibit different pharmacokinetic properties. The
absolute configurations of both diastereomers were established based on X-ray
crystallographic and NMR analysis of the model compounds, phenyl esters of cyclic (S)-
HPMPA and (S)-HPMPC. Thus, the less stable diastereomer was identified to have (S
p
)-
configuration at the phosphorus atom and corresponded to the downfield
31
P NMR signal.

The first conclusion that followed from the SAR studies is that the tyrosine amino acid is
the most favorable promoiety among tested single amino acids, owing to its high
chemical stability and the efficient activation of the resulting tyrosine-based prodrugs.
Second, enzymatic stability of the tyrosine promoiety was significantly increased by
replacement of the carboxyl ester group with an N-alkyl amide moiety. Thus, the tyrosine
N-alkyl amide-based prodrugs undergo the same metabolism and exhibit identical
stabilities in enzymatic and non-enzymatic media. Third, the highest antiviral activities
(3-4-logs increase in activity compared to the parent ANP) of the tyrosine N-alkyl amide
(S)-HPMPA and (S)-HPMPC prodrugs against HCMV, cowpox and vaccinia viruses
were achieved by incorporation of a hexadecyl group (C
16
H
33
) into the tyrosine N-alkyl
amide moiety. As a result, the tyrosine N-hexadecyl amide was identified as a promising
single amino acid promoiety scaffold for further ANP prodrug development, surpassing
the previously reported ethylene glycol-linked amino acid and dipeptide promoieties.
1
CHAPTER 1
*

Antiviral acyclic nucleoside phosphonates and their prodrugs

1.1 Acyclic nucleoside phosphonates
Acyclic nucleoside phosphonates (ANPs) constitute an important class of antimetabolites
that are highly potent against various DNA and retroviruses, including clinical viruses
(HIV, hepatitis viruses, etc.) and viruses relevant to potential bioterrorism (variola,
vaccinia virus, etc.).
1, 2
Development of ANPs has begun after the discovery of acyclic
nucleoside analogues: 9-(2-hydroxyethoxy-methyl)guanine (Acyclovir),
3
a “gold
standard” for the treatment of herpes simplex virus (HSV) infections, and (S)-9-(2,3-
dihydroxypropyl)adenine ((S)-DHPA),
4
a broad-spectrum antiviral agent (Figure 1.1).

Figure 1.1 Selected examples of natural nucleosides (left) and acyclic nucleoside analogues
(right).

*
Chapter 1 is partially reproduced in Krylov, I. S.; McKenna, C. E. Enzymatically activated phosphate and
phosphonate prodrugs. In Enzyme Technology for Biotechnology and Pharmaceutical Applications. John
Wiley and Sons, 2012. In press.
2
Despite similar structures, these nucleoside analogues exhibit clearly distinct modes of
action. (S)-DHPA behaves as an inhibitor of S-adenosyl-L-homocysteine hydrolase, a
regulatory enzyme in S-adenosyl-L-methionine-mediated methylations, whereas
acyclovir is consecutively phosphorylated by a viral-specific thymidine kinase, GMP
kinase and NDP kinase yielding corresponding triphosphate metabolite, which then
inhibits viral DNA polymerase.
5
The first phosphorylation step, which affords
corresponding monophosphate derivatives, is often a bottleneck in this transformation
responsible for poor antiviral activity of the nucleoside analogues. In order to circumvent
this drawback, in the early 1980s Holy and De Clercq started to investigate acyclic
nucleoside phosphonates (ANPs), as catabolically stable, isopolar and possibly, isosteric
analogues of natural nucleoside monophosphates.
5

The prototype member of ANPs, (S)-9-[3-hydroxy-2-(phosphonomethoxy)-
propyl]adenine ((S)-HPMPA, Figure 1.2), which can be regarded as (S)-DHPA
derivative extended by a phosphonate moiety, was introduced in 1986 as a novel selective
broad-spectrum antiviral agent with activity against virtually all the DNA viruses that
were examined (polyoma, papiloma, adeno, herpes, and pox).
6
(S)-HPMPA was followed
by several generations of newer ANPs, that according to their structure can be classified
into two categories: (i) the “HPMP” derivatives that retain 3’-hydroxyl group in the
modified sugar moiety and are represented by (S)-HPMPA and its cytosine analogue, (S)-
HPMPC
7
((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, Cidofovir); and (ii)
the “PME” and “PMP” derivatives, represented by PMEA and PMPA respectively, that
3
lack the hydroxymethylene function in the modified sugar moiety (Figure 1.2). Selected
ANPs have been marketed as antiviral drugs.
8
For example, (S)-HPMPC has been
approved by the Food and Drug Administration (FDA) for the intravenous treatment of
cytomegalovirus (CMV) retinitis in AIDS patients.
9

Figure 1.2 Structures of selected acyclic nucleoside phosphonates.

1.2 Mechanism of antiviral action of ANPs
In order to exhibit antiviral activity, the ANPs mimic metabolism of natural nucleoside
monophosphates, but contain a non-hydrolyzable phosphonate group and modified sugar
moiety, in which the 2’-hydroxyl group is absent. Thus, after intracellular
phosphorylation to the corresponding triphosphate followed by incorporation into newly
synthesized DNA or RNA by a targeted viral nucleic acid polymerase, the acyclic
nucleoside triphosphate analogues terminate nucleic acid chain elongation.
10
The
incorporation of one molecule of PMEA (Adefovir) at the 3’-end of the growing DNA
chain suffices to terminate further chain elongation,
11
whereas (S)-HPMPC (Cidofovir)
requires two consecutive incorporations to efficiently terminate DNA elongation, as has
been shown for CMV inhibition (Figure 1.3).
12

4

Figure 1.3 Mechanism of action of cidofovir (left) and adefovir (right). Reprinted by permission
from Macmillan Publishers Ltd: Nat. Rev. Drug Discovery, 2005, 4, 928-940, copyright 2012.

Incorporation of enzymatically stable phosphonate group into the acyclic nucleoside
analogues to generate ANPs allowed not only to conveniently circumvent first
phosphorylation, the rate-limiting step in the nucleoside activation pathway, but also to
restore activity of the nucleoside analogues against thymidine kinase mutant viruses (e.g.
drug: cidofovir, gene UL-97, HCMV, drugs: ganciclovir, maribavir;
13
gene UL-23, HSV,
drug – acyclovir).
14, 15

1.3 Antiviral potential of the ANPs
Acyclic nucleoside phosphonates of HPMP-series, in particular (S)-HPMPC and (S)-
HPMPA were proven to be active against broad spectrum of DNA viruses, including
adenovirus (AV) serotypes, vaccinia virus (VV), variola (smallpox), cowpox (CPX),
herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), TK
-
mutants
of HSV-1, Varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus
(EBV), suine herpes virus type 1 (SHV-1), bovine herpesvirus type 1 (BHV-1, infectious
5
bovine rhinotracheitis virus), equine herpesvirus type 1 (EHV-1, equine abortion virus),
herpesvirus platyrrhinae (HPV), phocine herpesvirus type 1 (PV, seal herpesvirus), duck
hepatitis B virus (DHBV), human hepatitis virus B virus (HBV). Among this long list of
viruses, CMV and variola are of a particular interest. CMV is a herpes virus that infects
60-90% of the world’s population and is most dangerous for immunocompromised
persons, such as patients infected with human immunodeficiency virus (HIV). Variola is
a poxvirus that is relevant to potential bioterrorism
16
because a major part of the present
population has not been vaccinated against this deadly infection owing to its global
eradication in the 1977.
17

Cidofovir is 10 to 100-fold more potent against CMV compared to other antiviral agents,
including foscarnet and ganciclovir.
18
Furthermore, cidofovir exhibits long lasting
antiviral activity, thus enabling infrequent dosing,
19
and it does not lead to virus-drug
resistance, even after prolonged treatment.
20
In 1996, it was formally approved for the
intravenous treatment of cytomegalovirus (CMV) retinitis in AIDS patients.
9
Although,
cidofovir was reported to be the most active compound ever tested against poxviruses.
21

Thus, (S)-HPMPC and related nucleotide analogues are considered as the most promising
treatment of poxvirus infections in the event smallpox outbreak.

1.4 Limitations to the antiviral ANPs
Acyclic nucleoside phosphonates incorporate phosphonic acid group, which ionizes at
physiological pH and reduces cell membrane permeability of the nucleotide analogues.
22

6
Thus, ANPs in general suffer from low cellular uptake, low oral bioavailability and their
tendency to concentrate in the kidney proximal convoluted tubule (PCT) cells resulting in
nephrotoxicity.
23
For example, (S)-HPMPC exhibits oral bioavailability of less than 5%
24

and accumulates in kidney PCT cells, leading to severe nephrotoxic effects.
25

Recognition of an enormous therapeutic potential of the ANPs encouraged development
of various prodrug approaches modifying the phsophonate group with the goal to mask
its undesirable properties, to improve in vivo intracellular delivery and to reduce
cytotoxicity of the nucleotide analgues.
26-28

1.5 Prodrug concept
Prodrugs are derivatives of pharmacologically active agents that release the parent drug
upon enzymatic and/or chemical transformation in vivo.
29
The prodrug approach is often
used to improve various physicochemical properties of the parent drugs, such as water or
lipid membrane solubility, brain permeability, local delivery, pre-systemic metabolism,
toxicity or unacceptable taste. To achieve clinical objectives the designed prodrugs
should meet several basic criteria:
30
i) ready absorption from the site of administration; ii)
stability under delivery conditions and adequate transport to the target site; iii) conversion
to the active parent drug or metabolite in vivo by enzymatic or chemical reaction at a rate
consistent with pharmacological efficacy; iv) solubility in biological media. Additionally,
both the prodrug and the metabolized promoiety should be non-toxic. The release of the
parent drug via a purely chemical process has the advantage of being independent from
variability in levels of enzyme expression in various tissues and species, but examples of
7
successful prodrugs selectively activated via chemical reactions at the target site are
limited,
31-33
in part due to the difficulty in reaching an appropriate balance between
chemical stability and an efficient activation rate under physiological conditions
(e.g. pH).

On the other hand, enzymatic activation of chemically stable prodrugs allows not only
targeted and efficient release of the parent drug or metabolite, but also simplifies their
handling and development.
34
A drawback is that the expression pattern, selectivity and
structure-function dependence of the enzymes participating in prodrug activation is likely
to differ in various tissues, individuals and species. A potential prodrug activating
enzyme should meet the following criteria: i) be a member of a well-characterized
enzyme family that plays a known role in the target tissue or in the progression of
disease; and ii) be unique to the target tissue, or at least be present in significantly higher
concentrations compared to healthy tissues. Finally, the enzyme concentration and the
rate it selectively liberates the parent drug should be sufficient to generate the desired
pharmacological effect. Unfortunately, enzymes utilized for prodrug activation (e.g.
esterases) often demonstrate overlapping substrate selectivities and ubiquitous expression
throughout the body, thus diminishing the possibility for site-selective activation
(targeted delivery) and making assessment, which enzymes are responsible for
conversion of the prodrug, in vivo difficult.
35
As a result, these important parameters have
rarely been studied before a particular prodrug becomes a viable candidate for
preclinical/clinical development, except in cases where the prodrug is purpose-designed
8
for targeted drug delivery to tissues (e.g. tumor) or organs (e.g. liver). The fact that
relatively few prodrugs have reached the market despite hundreds of reports on the
design of prodrugs showing proof-of-concept emphasizes this missing or underdeveloped
mechanistic component in prodrug development.

Thus, ANPs and other nucleoside monophosphate analogues are prodrugs, since they
require additional intracellular transformations (phosphorylation) in order to exhibit
therapeutic activity. However, as was mentioned above, they exhibit poor cell penetration
owing to the anionic status of the phosph(on)ate group at physiological pH.
Consequently, various prodrug approaches modifying phosph(on)ate group have been
devised and investigated with the aim to generate prodrugs of the nucleotide analogues
with improved bioavailability and in case of the monophosphates, increased enzymatic
stability (nucleoside monophosphates undergo dephosphorylation by endogenous
phosphatases in most biological fluids).
27

Simple alkyl esters initially used as a promoiety to mask both negative charges of the
phosph(on)ate group failed to release the parent nucleotide analogue
36, 37
since chemical
hydrolysis of the phosphotriesters is particularly slow under physiological conditions and
specific enzymes (e.g. phosphotriesterases) that cleave them have not been identified in
mammalian cells.
38
In contrast to alkyl diesters, monoester prodrugs are potential
substrates for phosphodiesterases.
39, 40
In order to provide efficient intracellular delivery
of the nucleotides under physiological conditions, more sophisticated prodrug
9
approaches, that exploit intracellular enzymes to trigger the cascade of intramolecular
chemical reactions liberating the parent drug from the prodrug are been developed.
Several examples of the prodrug approaches are given below.

1.6 Prodrug approaches
1.6.1 Acyloxyalkyl and alkyloxycarbonyloxymethyl approach
In an effort to construct a hydrophobic promoiety exhibiting a good balance between
stability and efficient liberation of the parent drug, prodrugs have been proposed that
would utilize enzymatically labile acyloxy group connected to a phosph(on)ate group
through a linker, usually a hydroxymethyl group.
41-45
Promoiety stability was adjusted by
modification of the acyloxy structure. The proposed activation mechanism of the
bis(acyloxymethyl) phosph(on)ate prodrugs begins with enzymatic hydrolysis of the
acyloxy group catalyzed by esterases (including carboxyl easterases, paraoxonases and
cholinesterases) followed by spontaneous elimination of the linker as formaldehyde and
release of the mono acyloxyalkyl ester of the parent drug (Figure 1.4A).
46
Cleavage of the
second acyloxyalkyl promoiety may proceed via the same mechanism or may be
catalyzed by phosphodiesterases.
10

Figure 1.4 General representation of activation pathway of (A) bis(acyloxymethyl) prodrug and
(B) bis(alkyloxycarbonyloxymethyl) prodrug to the corresponding nucleoside monophosphate
analogue: a) enzymatic hydrolysis; b) spontaneous chemical hydrolysis.

Since 1984, when the acyloxyalkyl group was first studied as a promoiety for model
phenyl and benzyl phosphates,
42
this prodrug approach has been utilized to increase oral
bioavailability of numerous nucleotide analogues, including ddU monophosphate,
47

FUdR monophosphate,
41
AZT monophosphate,
44, 48
9-[2-(phosphonomethoxy)-
ethoxy]adenine (PMEA),
45
phosphonoformic acid (PFA, foscarnet),
43
and 9-[2-
(phosphonomethoxy)propyl]adenine (PMPA).
49
The bis(acyloxyalkyl) prodrug approach
has recently been applied to a non-nucleoside monophosphate, 4-phospho-D-
erythronohydroxamic acid (which is 6-phosphogluconate dehydrogenase inhibitor)
50
and
also to the novel nucleotide analogues 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-
2,6-diaminopurine (HPMPDAP)
51
and thymidine-but-2-enyl phosphonate.
52
An example
of successful application of this promoiety design is the bis(pivaloyloxymethyl) prodrug
11
of PMEA (adefovir dipivoxil, Hepsera®; Figure 1.5),
45
which has been approved by the
FDA and is currently being marketed for antiviral therapy against hepatitis B.
53
This
prodrug was selected from a range of acyloxylalkyl prodrugs of PMEA based on its high
oral bioavailability in rats.
36
Despite the success of adefovir dipivoxyl, application of the
pivaloyloxymethyl promoiety to high-dose drugs used for chronic treatment (e.g. (R)-
PMPA) could be limited due to concerns about the impact of pivalic acid on carnitine
homeostasis, which is released during metabolism of the promoiety.
54

O
N
N
N
N
NH
2
P
O
O
O
O
O
O
O O
N
N
N
N
NH
2
P
O O
O
O
O
O
O
O
O
H
3
C
Tenofovir Disoproxil Adefovirdipivoxil

Figure 1.5 Structures of adefovir dipivoxyl and tenofovir disoproxil.

In order to avoid liberation of pivalic acid, an alkyloxycarbonyloxymethyl promoiety
containing a carbonate diester group instead of a pivalate ester was utilized to improve
the pharmacokinetic properties of (R)-PMPA (tenofovir, TFV), a highly potent anti-HIV
and anti-HBV agent.
55
As with cleavage of the acyloxymethyl group, the activation
mechanism of alkyloxycarbonyloxymethyl promoiety is believed to proceed via
enzymatic hydrolysis of the carboxylic ester, but yields a carbonate monoester that
releases the nucleoside monophosphate analogue by spontaneous decarboxylation and
elimination of the hydroxymethyl linker as formaldehyde (Figure 1.4B). The generation
of the formaldehyde as a result of the hydrolysis of bis(acyloxymethyl) and
bis(alkyloxycarbonyloxymethyl) prodrugs could be a source of long-term cellular toxicity
12
arising from the interaction of formaldehyde with cellular nucleophiles, such as DNA,
RNA or proteins.
27
Alkyloxycarbonyloxymethyl prodrugs of tenofovir were reported to
be potent in vitro against HIV-1 and showed oral bioavailability ranging 16 – 30%, but
exhibited relatively short half-lives in intestinal homogenate (t
1/2
≤ 5 – 53 min).
56

Nonetheless, bis(isopropyl-oxycarbonyloxymethyl) prodrug of (R)-PMPA (Figure 1.5)
containing a more stable isopropyl carbonate ester group (t
1/2
= 53 min in intestinal
homogenate) was selected for further clinical development based on its antiviral potency,
water solubility and oral bioavailability.
56
It was approved by FDA as a fumarate salt
(tenofovir disoproxil fumarate, TDF, Viread®) for chronic treatment of HIV in 2001 and
for chronic treatment of hepatitis B infection in 2008.
57
As mentioned, the oral
bioavailability of acyloxymethyl and alkyloxycarbonyloxymethyl prodrugs can be limited
by premature hydrolysis of the promoiety in the GI tract where esterases, hydrolyzing
acyloxy and alkyloxycarbonyl groups are expressed in numerous tissues including small
intestine.
35

1.6.2 Phosphoramidate approach
The phosphoramidate pronucleotide (ProTide) approach introduced by McGuigan to
deliver monophosphorylated nucleosides into cells utilizes amino acid esters (connected
via amino group) to mask one negative charge and an alkyl or aryl ester group to mask
the remaining negative charge of the nucleotide phosphate group.
58
The mechanism of
intracellular release of the nucleoside monophosphate analogue from the aryloxy
phosphoramidate prodrug involves two enzyme-mediated and two spontaneous steps
13
(Figure 1.6), beginning with an enzymatic hydrolysis of the promoiety’s carboxylic ester
by carboxyesterases, lipases or proteases. Cleavage of the ester group was identified as a
necessary condition for antiviral activity, however the rate of enzymatic deesterification
did not always correlate with antiviral potency of the prodrug and was found to depend
on the structure of both the ester group and the amino acid side chain.
59
Thus, a tert-butyl
ester-derived phosphoramidate pronucleotide exhibited low potency because tert-butyl
esters are not good substrates for the cleaving enzymes.
60
Cathepsin A, a major enzyme
responsible for the cleavage of the ester group was unable to hydrolyze phosphoramidates
with heavily branched amino acids such as leucin, valine and isoleucine.
61
The second
step of the activation mechanism is proposed to be spontaneous nucleophilic substitution
of the aryl moiety by the carboxyl residue formed after ester hydrolysis yielding a five-
membered cyclic mixed anhydride. The rate of the cycle formation varies depending on
the structures of the nucleoside analogue and the amino acid, e.g. cyclization of AZT
phosphoramidates was more difficult than d4T, and phosphoramidates containing β-
alanine failed to cyclize.
62
The transient five-membered cyclic diester is unstable and
chemically hydrolyzes via nucleophilic attack of water at the phosphate group, to produce
an amino acyl metabolite (phosphoramidate), which finally undergoes phosphoramidase-
mediated P-N bond cleavage and releases corresponding nucleoside monophosphate
analogue.
14

Figure 1.6 General representation of phosphoramidate activation pathway to the corresponding
nucleoside monophosphate inside the cell: a) ester hydrolysis by carboxypeptidase or esterase
type enzyme; b) spontaneous cyclization; c) aqueous hydrolysis; d) enzymatic cleavage of P-N
bond by phosphoramidase type enzyme.

The phosphoramidate concept has been applied to various antiviral and anticancer
nucleoside monophosphate/phosphonate analogues, including 4’-azidoadenosine
monophosphate,
63
4’-azidouridine monophosphate,
64
AZT monophosphate,
65
2’,3’-
dideoxyuridine (ddU) monophosphate,
66, 67
2’,3’-didehydro-2’,3’-dideoxyuridine (d4U)
monophosphate,
66, 67
2’,3’-didehydro-2’,3’-dideoxythymidine (d4T) monophosphate,
68

abacavir (ABC) monophosphate,
69
(E)-5-(2-bromovinyl)-2’-deoxyuridine (BVDU)
monophosphate,
70
FUdR monophosphate,
71
2’-C-methylguanosine monophosphate,
72

ribavirin monophosphate,
73
acyclovir monophosphate,
74
9-[2-(phosphonomethoxy)-
ethyl]adenine (PMEA)
75
and 9-[(R)-2-(phosphonomethoxy)propyl]-adenine ((R)-PMPA,
tenofovir).
75
Structure-activity relationship (SAR) studies have demonstrated that the
biological activity of the pronucleotide depends significantly on the structures of both
nucleoside and phosphoramidate promoiety, however (with some exceptions) the most
favorable combination of amino acid ester and aryloxy group was found to be L-Ala and
a phenyl (or naphthyl) group. In several cases, application of the phosphoramidate
15
approach to inactive nucleosides boosted their potency to a measurable level.
63, 66, 67
The
ProTide approach has been recently reviewed.
59
Below we discuss several
phosphoramidate prodrugs that have reached clinical trials.

The phosphoramidate approach has been applied to ameliorate the rate-limiting initial
phosphorylation of 2’-C-methylguanosine,
72
a nucleoside analogue, which undergoes
poor phosphorylation, but upon conversion into the triphosphate form serves as a potent
inhibitor of HCV RNA polymerase.
76, 77
In a search for the pronucleotide exhibiting the
most favorable antiviral potency and pharmacokinetic properties, various modifications
of aryl moiety, ester and amino acid groups of the phosphoramidate promoiety were
evaluated. Alanine ester pronucleotides assayed vs. HCV using a replicon assay, were
found to be 4 to 50-fold more potent (EC
50
= 0.045 - 0.21 μM) than the parent drug
(EC
50
= 3.5 μM), but exhibited inadequate stability to enzymatic hydrolysis in rodent
plasma. The most stable prodrug in the series, L-Ala benzyl ester, showed 40%
degradation after 30 min in rodent plasma at 2 – 4 ºC. Alteration of the amino acid
structure increased stability of the promoiety in plasma, but also resulted in decreased
pronucleotide antiviral activity.
72
Further development of the 2’-C-methylguanosine
pronucleotides included not only variation of the amino acid ester in the phosphoramidate
moiety to optimize enzymatic stability, but also modification of the purine base at the C-6
position to potentially increase antiviral potency.
78, 79
Combination of a naphthyl (L)-Ala
neopentyl ester phosphoramidate moiety with a C6-methoxy substituent at the purine
base resulted in a dual prodrug (INX-189; Figure 1.7) of 2’-C-methylguanosine
16
monophosphate, which was found to be 520-fold more active in the genotype 1 HCV
replicon assay (EC
50
= 0.01 μM) than the parent nucleoside analogue (EC
50
= 5.2 μM).
Nanomolar activity of INX-189 vs. HCV is consistent with intracellular levels of 2’-C-
methylguanosine triphosphate in primary human hepatocytes. INX-189 has successfully
completed phase 1b clinical trial in HCV-infected patients.
79

Another anti-HCV pronucleotide that has advanced to clinical trials is PSI-7977, a single
(S
p
)-diastereomer of the phosphoramidate prodrug of β-D-2’-deoxy-2’-fluoro-2’-C-
methyluridine-5’-monophosphate (Figure 1.7).
80
The triphosphate derivative of β-D-2’-
deoxy-2’-fluoro-2’-C-methyluridine is a potent inhibitor of HCV RNA polymerase
(NS5B RNA-directed), whereas the nucleoside analogue itself did not exhibit any
antiviral activity due to inability to be phosphorylated by cellular nucleoside kinases to its
monophosphate form.
81
Application of the phosphoramidate prodrug approach to bypass
inefficient first phosphorylation step yielded a number of pronucleotides exhibiting
potency in the HCV subgenomic replicon assay (EC
50
< 1 μM) and producing high levels
of β-D-2’-deoxy-2’-fluoro-2’-C-methyluridine-5’-triphosphate in primary hepatocytes
and in livers of rats, dogs, and monkeys when administered in vivo.
80
Based on its
antiviral, pharmacokinetic and toxicity profile, the phenyl (L)-alanine isopropyl ester
pronucleotide of β-D-2’-deoxy-2’-fluoro-2’-C-methyluridine (PSI-7851) was selected for
further development. PSI-7851 is a mixture of (S
p
)- and (R
p
)-diastereomers, with the (S
p
)-
diastereomer (PSI-7977) being the more potent (>10-fold) inhibitor of HCV RNA
polymerase in a clone A replicon-based assay. Studies of the activation mechanisms of
17
PSI-7851 and PSI-7977 in primary human hepatocytes indicate that (S
p
)- and (R
p
)-
diastereomers produce almost identical amounts of the corresponding triphosphate,
suggesting that both diastereomers are equally active in the liver cells, where infection of
HCV takes place.
82
Enzymes identified in activation process include human cathepsin A
(cat. A) and carboxylesterase 1 (CES1), catalyzing hydrolysis of the carboxyl ester, and
histidine triad nucleotide-binding protein 1 (Hint1) cleaving the amino acid moiety prior
to release of the free nucleoside monophosphate (Figure 1.6).
82
The (S
p
)-diastereomer
PSI-7977 is currently undergoing a phase II clinical trial for the treatment of HCV
infection.
80

Other phosphoramidate prodrugs undergoing clinical investigation include GS-7340 and
GS-9131, developed by Gilead Sciences to treat HIV/AIDS, and Thymectacin, developed
by NewBiotics to treat colon cancer (Figure 1.7). GS-7340 is the phenyl L-alanine
isopropyl ester of tenofovir. Use of the FDA-approved bis-isopropoxycarbonyloxymethyl
ester of (R)-PMPA (tenofovir disoproxil fumarate, TDF, Viread®) is limited due to
toxicity, development of resistance and rapid systemic degradation to the parent
nucleotide analogue.
83-85
In contrast, GS-7340 has shown 400-fold greater potency
against HIV in PBMCs and 200-fold greater stability in plasma compared to tenofovir
disoproxil.
86
Currently, GS-7340 is undergoing phase 1b clinical trial.
87

18

Figure 1.7 Phosphoramidate pronucleotides currently in clinical trials.

GS-9131 is a phenyl-(L)-alanine ethyl ester prodrug of the modified dAMP phosphonate
analogue 2’-α-fluoro-2’,3’-didehydro-2’,3’-dideoxyadenosine.
88
The prodrug moiety was
optimized for efficient delivery of GS-9148 together with its diphosphate, active
metabolite to lymphoid cells following oral administration. GS-9131 was chosen as a
candidate to be developed for treatment of drug-resistant HIV-1 infection based on its i)
anti-HIV potency against HIV-1-resistant virus strains; ii) favorable biological and
pharmacokinetic properties; and iii) ability to deliver the parent drug efficiently inside
cells.
88

Thymectacin, the phenyl L-alanine methoxy derivative of (E)-5-(2-bromovinyl)-2’-
deoxyuridine (BVDU), was reported to possess potent anticancer activity in tumor cells
with a high expression of thymidylate synthase (TS) and to be non-cross-resistant with
19
other TS-targeted agents.
89-91
Thymectacin was 10-fold more cytotoxic to 5-FU-resistant
TS over-expressing colorectal tumor cells than to normal cells
89
and was active in
fluoropyrimidine-resistant metastatic colorectal cancer (CRC) during monotherapy
trials,
92
and underwent combined phase I/II clinical trials for the treatment of colon
cancer.
93

In conclusion, the phosphoramidate prodrug approach can enhance intracellular delivery
of nucleotide analogues and has proven to increase activity of a number of antiviral or
anticancer nucleosides. Intracellular enzymatic activation of these pronucleotides to
liberate nucleoside monophosphates has been studied, but detailed information about all
the enzymes involved in the activation process is warranted, given successful progression
of a number of phosphoramide triester-based prodrugs (INX-189, PSI-7977, GS-7340,
GS-9131 and Thymectacin) to clinical trials.

1.6.3 Lipidic prodrug approach
Lipid ester prodrug approach aims to improve oral absorption and to reduce adverse side
effects of the parent drug by creating drug-lipid conjugates where the drug is covalently
bound to a long chain alkyl group, fatty acid, diglyceride or phosphoglyceride. Lipid ester
prodrugs can exhibit dramatically increased lipophilicity and can cross cell membranes
by passive diffusion, membrane flippase activity or phospholipid uptake. Once inside the
cell, they typically require enzymatic activation to release the parent drug. Development
of this concept began in the 1970s with lipid-like modification of cytarabine (ara-C), an
20
anticancer agent.
94-96
Incorporation of lipid and phospholipid moieties into ara-C’s
structure was intended: i) to prevent metabolic degradation of the drug by cytidine
deaminase; ii) to improve the intracellular drug concentration; and iii) to bypass the rate-
limiting first phosphorylation step in the ara-C activation pathway via release of ara-C
monophosphate from the phospholipid prodrug. This approach was then applied to a
variety of antiviral and antitumor agents including 5-fluorouridine monophosphate,
97

AZT monophosphate,
98, 99
3’-deoxy-3’-fluoro-thymidine (FLT) monophosphate,
100
ddC
monophosphate,
101
ddT monophosphate,
102
phosphonoformate (PFA),
103-105
(S)-HPMPC
(Cidofovir, CDV),
106
(S)-HPMPA,
107
PMEA,
108
PMEDAP.
108

An early example of orally administered phospholipid prodrug that reached clinical trials
and has been approved in Japan for treatment of leukemia is cytarabine ocfosfate
(YNK01, staracid), a stearyl ester of cytarabine monophosphate.
109
YNK01, synthesized
by Saneyoshi et al.
110
, was resistant to deoxycytidine deaminase and demonstrated high
antileukemic activity both in vitro and in vivo.
111
The mechanism of the parent drug
release was initially anticipated to proceed via enzymatic hydrolysis by
phosphodiesterases, however it was later proved to begin with hydroxylation of the side-
chain at the ω-end of the alkyl chain followed by oxidation to the carboxylic acid and
stepwise shortening of the alkyl chain by successive peroxisomal β-oxidation steps
(Figure 1.8).
112

21

Figure 1.8 Activation pathway of cytarabine ocfosfate (YNK01) to cytarabine monophosphate: a)
ω-hydroxylation and subsequent oxidation to the carboxylic acid; b) β-oxidation.

The oral bioavailability of cytarabine ocfosfate could not be measured in phase I/II
clinical studies due to intravascular hemolysis at the injection site after intravenous
administration. In toxicology studies in rodents, metabolic bioavailability of the drug was
defined by measuring the renal excretion of ara-C and ara-U taking into account that
>90% of YNK01 absorbed after oral administration was metabolized to ara-C and ara-U,
which were almost completely excreted. The metabolic bioavailability of YNK01 was
estimated to be 15.8% of the total dose.
113
Results of a phase II study of YNK01 in
combination with α–interferon used for the treatment of chronic myeloid leukemia have
recently been reported.
114
Other phospholipid prodrugs that have advanced to clinical
trials include fozivudine tidoxil
115
and fosalvudine tidoxil,
100
thioglycerolipid ester
prodrugs of AZT and 3’-deoxy 3’-fluoro-thymidine (FLT), respectively. The AZT
prodrug bypasses rate-limiting phosphorylation and increases concentration of active
drug in lymphoid tissues.
22
In addition to nucleoside analogues, Hostetler has applied a lipidic prodrug approach to
various acyclic nucleoside phosphonates
2
(ANPs), including (S)-HPMPC, (S)-HPMPA
and (R)-PMPA.
116
In this approach, a lipid ester promoiety consisting of glycerol linker
connected to a long alkyl chain was modeled after lysophosphatidylcholine, which, in
contrast to phospholipids, contains only one O-acyl chain and is known to diffuse across
cell membranes at a faster rate since their off-rate from phospholipid bilayer (higher
disturbance) membranes is higher than that of the diacylphospholipids.
117
Since only
about 40% of lysophosphatidylcholine is absorbed intact from the intestine,
118, 119
the
enzymatic and chemical stabilities of the promoiety were increased by replacement of the
acyl ester group with an ether linkage (to avoid hydrolysis by lysophospholipase) and by
substitution of the hydroxyl group at the sn-2 position of the glycerol linker with a
hydrogen atom (to prevent reacylation by lysophosphatidylcholine acyltransferases
present in small intestinal enterocytes and other tissues).
116
Liberation of the parent
phosphonate drug from the prodrug was proposed to occur by enzymatic cleavage of the
P-O-C bond by phospholipase C, an enzyme present in plasma and pancreatic
secretions.
116

Structural optimization studies of the promoiety examining variations in the linker and in
the alkyl chain length, suggested the combinations of a 16-C alkyl group with an
oxypropyl linker or 18-C alkyl group with an oxyethyl linker constituting the
hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) promoieties, respectively
(Figure 8).
120

23

Figure 1.9 Activation pathway of HDP- and ODE-prodrugs of ANPs.

Evaluation of the HDP- and ODE-derivatives of various ANPs for in vitro antiviral
activity vs. dsDNA viruses (cowpox, vaccinia, CMV, variola, HSV-1, VZV) and vs.
viruses relying on RNA polymerases (HBV and HIV-1) revealed that HDP- and ODE-
(S)-HPMPC conjugates were most potent against HCMV, exhibiting activity in the low
nanomolar range (EC
50
= 0.9 nM).
116
HDP- and ODE-prodrugs of (S)-HPMPA were
potent against HSV-1 and orthopoxviruses, but also were the most cytotoxic
compounds.
116, 121
HDP-PMPA prodrug demonstrated the highest activity against HIV-1
in MT-2 and PBM cells (EC
50
< 0.01 nM and 12 nM, respectively) than tenofovir
(EC
50
= 0.65 μM and 3.20 μM, respectively) (Figure 1.10).
122

The higher in vitro antiviral activities of HDP-(S)-HPMPC and HDP-(S)-HPMPA relative
to the parent drug were rationalized based on their 50 to 100-fold increased levels of
cellular uptake and conversion into the corresponding diphosphates, measured using
14
C -labeled HDP-(S)-HPMPC, HDP-(S)-HPMPA and corresponding parent drugs in
MRC-5 human lung fibroblast cells.
123, 124
The relative oral bioavailabilities of the
24
HDP-(S)-HPMPC and HDP-(S)-HPMPA were estimated to be 88% and 73%,
respectively (compared to the <5% and 24% for the parent ANPs), and the half-lives in
plasma were found to be 14.9 h and 11 h, respectively.
125, 126
Both prodrugs exhibited
reduced nephrotoxicities compared to the parent (S)-HPMPC and (S)-HPMPA.
116
Based
on these in vitro and in vivo results, HDP-prodrugs of (S)-HPMPC (CMX001) and (R)-
PMPA (CMX157) have advanced into clinical trials, CMX001 is developed as a
treatment for CMV, smallpox and BK viruses, whereas CMX157 is for HIV infection.

Figure 1.10 Structures of selected lipid ANP prodrugs.

In clinical trials, premature metabolism of HDP-(S)-HPMPC has not been reported to be
an issue, however, it was responsible for the failure of this prodrug to treat viral
infections in cynomolgus monkeys.
127
The proposed mechanism of rapid enzymatic
degradation of HDP-CDV includes hydroxylation of the ω or ω-1 carbon atom of the
alkyl chain with subsequent oxidation to a carboxylic group, followed by shortening of
the alkyl chain via β-oxidation, which is the same mechanism that leads to the release of
the nucleoside monophosphate from the phospholipid prodrug YNK01. Thus, to increase
enzymatic stability and prevent oxidation of the long alkyl chains present in HDP- and
ODE-prodrugs, Ruiz et al. synthesized and evaluated a variety of alkoxyalkyl (S)-
HPMPC and (S)-HPMPA prodrugs containing modified alkoxy or alkyl chains.
128
The
25
compounds were evaluated for in vitro antiviral potency vs. cowpox, vaccinia and
ectromelia viruses and their metabolic stabilities were estimated in guinea pig, monkey
and human liver S9 fractions. Modifications of the alkyl chain, e.g. by introduction of a
ω-1 methyl group, a terminal double bond, or a terminal cyclopropyl residue, had only a
moderate effect on the metabolic stabilities of the prodrugs, whereas alteration of the
alkoxy group by incorporation of a benzyloxy group at the sn-2 position increased the
metabolic stability of the (S)-HPMPC prodrug (88% of modified prodrug was intact after
90 min exposure to monkey liver S9 fractions vs. 16% in case of unmodified prodrug).
These structural modifications of the promoieties had little effect on antiviral activities of
the prodrugs and all the compounds retained antiviral potency in a submicromolar
range.
128

In summary, the lipidic prodrug approach – improvement of parent drugs hydrophobicity
via incorporation into its structure of a biolabile lipophilic alkyl chain – has been known
for decades and has been applied to a number of nucleotide analogues. To date, only one
phospholipid ester prodrug, cytarabine ocfosfate (YNK01) for treatment of leukemia, has
been approved for clinical use in Japan. An apparent reason for the relative lack of
success of lipid prodrugs in reaching the clinic may stem from elevated cytotoxicity,
decreased aqueous solubility and often insufficient enzymatic stability – properties that
they exhibit along with improved hydrophobicity and, in several cases, antiviral activity.
Structural modification of the alkyl group per se in order to ameliorate those drawbacks
by simple adjustment of the chain length may not suffice to achieve an ideal balance
26
between PK properties, cytotoxicity and antiviral activity of the prodrug. In this respect,
incorporation and structural alteration of an easily modifiable linker between the drug and
hydrophobic alkyl chain may be advantageous. In this connection, it is worth noting that
glycerol linker in two lipidic prodrugs of (S)-HPMPC and (R)-PMPA, CMX001 and
CMX157, did not show significant change in antiviral activity vs. HCMV when the ether
oxygen was replaced by a carbon atom.
115

1.6.4 SATE approach
The SATE prodrug approach was introduced for intracellular delivery of nucleoside
monophosphate/phosphonate analogues.
129, 130
It utilizes enzymatically cleavable (S)-
acyl-2-thioethyl (SATE) protecting groups as promoieties to mask the negative charges
of the phosphate/phosphonate group. According to metabolic studies of the radiolabeled
SATE pronucleotides in cell extracts and in intact cells,
48, 131
the mechanism of SATE
group cleavage from the pronucleotide and corresponding nucleoside monophosphate
release is initiated primarily via intracellular esterase-mediated hydrolysis of the acyl
group, yielding an unstable 2-mercaptoethyl ester (Figure 1.11). On the next step, the
newly formed thiol group intramolecularly attacks the α-carbon of the 2-mercaptoethyl
ester group, giving rise to the corresponding phosphate diester and ethylene sulfide. If the
second phosphate ester is also a SATE group, the above process is repeated, resulting in
release of the nucleoside monophosphate analogue.
27

Figure 1.11 General representation of bis(SATE) phosphotriester pronucleotides activation
pathway to the corresponding nucleoside monophosphate inside the cell. a) esterase-mediated
hydrolysis; b) spontaneous decomposition.

The SATE approach has been applied to a number of nucleoside
monophosphate/phosphonate analogues, including 2’,3’-dideoxyadenosine (ddA)
monophosphate,
132, 133
AZT monophosphate,
48, 134
d4T monophosphate,
135
cytarabine,
136

acyclovir (ACV) monophosphate,
137, 138
PMEA,
139
1-[1-(3,4-dimethylcyclopent-3-
enylmethoxymethyl)] cytosine phosphonic acid,
140
1-(4-amino-5H-pyrrolo[3,2-
d]pyrimidin-7-ylmethyl) cyclobutylmethyl phosphate,
141
3’,4’-dimethyl-5’-
norcarbocyclic adenosine phosphonic acid.
142
In all cases, the bis(SATE) prodrugs
exhibited increased in vitro antiviral activities compared to the parent nucleoside
analogues, e.g. bis(MeSATE)-ddA was 450-fold more potent against HIV-1 in peripheral
blood mononuclear (PBM) cells than the parent (ddA),
132, 133
and bis(MeSATE)-d4T
monophosphate was 10- to 17-fold more active than d4T vs. HIV-1 in wild-type CEM
and PBM cells and retained its potency in thymidine kinase-deficient cells.
135

28
The toxicity of the SATE promoiety and its metabolites, assessed assays in vitro (human
bone marrow progenitor cells) and in vivo (cynomolgus monkeys) of bis((S)-acetyl-2-
thioethyl) AZT (bis(MeSATE)-AZT) was comparable to that of the parent nucleoside
analogue (AZT),
129
indicating that neither the SATE promoiety nor its degradation
products (acetic acid and episulfide) induced additional toxicity. The acetate formed was
proposed to be metabolized by normal pH-homeostatic cellular mechanisms
143
and the
episulfide was hypothesized to conjugate with glutathione, the ubiquitous non-protein
thiol that serves as both a nucleophile and a reducing agent in playing a crucial role in
detoxification and repair of cell damage.
129
Elevated cytotoxicities observed for
hydrophobic SATE acyl prodrugs (e.g. R = tert-butyl) were attributed to increased
intracellular accumulation of corresponding phosphorylated nucleosides.
129, 144

The relative stability of the SATE promoieties in extracellular media (culture medium,
human serum) and rapid intracellular liberation of the corresponding nucleoside
monophosphate from the SATE prodrug was confirmed in all examined cell lines.
129
The
rate of the nucleoside monophosphate release was adjusted by varying the nature of the
SATE acyl moiety (R group; Figure 1.11). Phosphotriesters incorporating lipophilic
bis(tert-BuSATE) groups (R = tert-butyl) generally exhibited greater enzymatic
stabilities and antiviral activities than compounds with more labile MeSATE
promoieties.
145
In contrast to their in vitro stability, bis(tert-BuSATE) phosphotriester
derivatives were unable to survive presystemic metabolism and completely lost their
antiviral activity in vivo, presumably due to inadequate delivery of the corresponding
29
nucleoside analogues to infected cells and tissues.
146
No intact bis(tert-BuSATE)-AZT
monophosphate was observed in mouse plasma after oral administration, probably due to
extensive esterase-based metabolism in the GI tract or liver.
146
To address presystemic
degradation of the bis(tert-BuSATE) pronucleotides, two strategies were devised.
129, 130

One aimed to improve the enzymatic stability by modifying the SATE promoiety
147-149
on
the basis that carboxylesterases, the enzymes most likely involved in the degradation of
the SATE group, prefer lipophilic carboxyl esters over polar or charged esters.
150

Incorporation of a polar hydroxyl group into the acyl moiety of the tert-BuSATE group
(bis(HO-tert-BuSATE)) resulted in a decreased rate of enzymatic deacylation in human
serum.
149
However, efficient liberation of the active nucleoside monophosphate drug
precursor remained elusive because cleavage of the second SATE group in the
phosphodiesters proceeded at a much slower rate than hydrolysis of the first SATE
moiety in bis(SATE) phosphotriesters.
131
This difference arises because of the presence
in the SATE phosphodiesters of a phosphate negative charge, which greatly reduces the
rate of enzymatic deacylation of the second SATE promoiety and which also deters the
thiol group from attack at the α-carbon of the thioester group, retarding intramolecular
nucleophilic substitution and thus preventing release of the nucleoside
monophosphate.
129, 130

As a result, the prodrug design was revised with the goal of achieving not only effective
presystemic metabolism of the first SATE promoiety, but accelerated removal of the
second promoiety.
129, 130
A series of mixed SATE pronucleotides was synthesized
30
wherein one P-OH group was masked with a SATE group and other OH group by a
different, enzymatically labile promoiety, such as an aryl ester (removable by
phosphodiesterases),
39, 151
amino residues (amino acid, alkyl and aryl amine) to form a
phosphoramidate cleavable by phosphoramidase
62, 152
or a glucosyl residue containing a
2-thioethyl linkage connected to the phosphorus atom of the drug by a P-S bond
(isoSGTE group)
130
(Figure 1.12).

Figure 1.12 General representation of mixed SATE pronucleotide activation pathways.

A series of mixed SATE-AZT pronucleotides was prepared and evaluated for inhibition
of HIV-1 replication in a variety of cell lines, including thymidine-kinase (TK) deficient
cells where the parent drug (AZT) was inactive at concentrations up to 100 μM.
130
In
contrast to AZT, all the mixed SATE prodrugs examined had antiviral potencies in the
low micromolar range (EC
50
= 1-10 μM) both in wild type and TK-dificient cell lines.
130

Structure-activity relationship (SAR) studies involving modification of the second
31
promoiety (aryl ester group, amino residue or glucosyl group) and the SATE group has
been undertaken to further optimize pharmacological and pharmacokinetic (PK)
properties of these pronucleotides.

Modification of the aryl moiety in the mixed SATE pronucleotides was guided by the
hypothesis that they are cleaved by 5’-nucleotide phosphodiesterases, a family of
enzymes catalyzing hydrolysis of a wide range of naturally occurring, as well as synthetic
aryl phophodiesters to the corresponding phosphate monoesters (Figure 1.12).
39, 151
The
aqueous solubility of the mixed SATE aryl pronucleotides could be increased without
significant loss of antiviral activity by utilization of a (L)-tyrosinyl residue instead of a
phenyl ester group.
153
However, the (L)-tyrosinyl phosphodiesters released the parent
AZT monophosphate in cell extracts at a faster rate than the corresponding phenyl ester
derivatives, supposedly because they are better substrates for the phosphodiesterases.
129,
154
In another attempt to improve enzymatic stability of the mixed aryl SATE
pronucleotide, a hydroxyl group was incorporated into the pivaloyl moiety of the tert-
BuSATE group, which increased the half-life of the tert-BuSATE phenyl AZT
phophotriester in cell extracts 10-fold (t
1/2
increased from 1.5 to 15 h) with retention of
antiviral activity.
155
Replacement of this hydroxyl group with an amino residue
drastically decreased both the enzymatic stability (in culture medium and in cell extracts)
and the antiviral potency of the pronucleotide in a TK-deficient cell line, relative to the
mixed tert-BuSATE phenyl phosphotriester.
136, 155, 156
Introduction of a second hydroxyl
32
group into the pivaloyl residue increased the stability of the SATE group by an additional
12-fold, but also led to a total loss of antiviral potency in the TK-deficient cell line.
155

Another approach to enzymatically cleavable promoieties involving mixed SATE
pronucleotides has utilized amino residues connected to the phosphorus atom via a P-N
bond with the goal of achieving activation by a phosphoramidase to release the
nucleoside monophosphate drug (Figure 1.12).
129, 130
SAR studies of a library of mixed
tert-BuSATE phosphoramidates with varying amino group structures revealed that the
nitrogen atom basicity principally determines the rate of enzymatic P-N bond hydrolysis
to liberate the parent nucleotide analogue. Among mixed SATE phosphoramidate
pronucleotides of AZT, the isopropylamino derivative showed the greatest potency (EC
50

= 0.75 μM) against HIV-1 in a TK-deficient cell line, indicating that an amino acid is not
required for the pronucleotide to retain antiviral activity.
157

In summary, the SATE approach has been applied to a number of nucleoside
monophosphate/phosphonate analogues to create corresponding SATE pronucleotides
that exhibit in vitro antiviral activity both in wild type and TK-deficient cell lines.
However, rapid in vivo metabolism of bis(SATE) phosphotriesters yields chemically and
enzymatically stable SATE phosphodiesters, blocking ultimate liberation of the parent
nucleoside monophosphate drug. To circumvent this limitation, mixed SATE
pronucleotides were developed where one negative charge of the nucleoside
monophosphate was masked with a SATE group and the remaining P-OH was
33
derivatized with an enzymatically labile promoiety, such as an aryl ester or amide.
Unfortunately, further structural alterations of the SATE group and the second promoiety
to improve the pharmacokinetic (PK) properties of the mixed pronucleotides have led to
more cytotoxicity, without a compensating increase in antiviral activity, thus narrowing
their therapeutic windows. Currently, no information is available about SATE
pronucleotides undergoing clinical studies, most likely because additional studies need to
be conducted to balance their activity, PK properties and toxicity.

1.6.5 CycloSal approach
Originally developed by Meier the cycloSal (cyclo-saligenyl) concept was formulated as
a selective nucleotide delivery system that releases its cargo (nucleotide) exclusively via
a chemically induced cascade mechanism.
33, 158, 159
This approach utilizes salicyl alcohol
as a bifunctional masking unit that forms a phenyl and a benzyl ester with the targeted
phosphate/phosphonate group of the nucleotide analogue. Activation of the pronucleotide
is pH-dependent and achieved through a sequence of reactions which begins with
hydrolytic cleavage of phosphotriester and followed by substitution via S
N
P mechanism
of the best leaving group – the phenolate, thus releasing corresponding 2-hydroxy-benzyl
phosphate diester (Figure 1.13). In contrast to unsubstituted benzyl nucleotide diesters
that are chemically stable at physiological conditions, newly formed 2-hydroxy-benzyl
nucleoside monophosphate ester is activated by the strong electron-donating hydroxyl
group in the ortho position, and therefore is able to spontaneously release the nucleotide.
34

Figure 1.13 General representation of the first, second and third generation cycloSal
pronucleotides activation pathways. a) hydrolysis of phenolate ester via S
N
P mechanism; b)
spontaneous cleavage of the benzyl ester group.

The cycloSal prodrug approach has been applied to various nucleotide and nucleoside
monophosphate analogues, including 2’,3’dideoxyadenosine (ddA) monophosphate,
160

2’,3’-dideoxy-2’,3’-didehydroadenosine (d4A) monophosphate,
160
2’-F-ara-2’,3’-
dideoxyadenosine (F-ara-ddA) monophosphate,
161
2’-ribo-F-2’,3’-dideoxyadenosine (F-
ribo-ddA) monophosphate,
161
abacavir monophosphate,
162
3’-azidothymidine
monophosphate,
163
2’,3’-dideoxy-2’,3’-didehydrothymidine (d4T) monophosphate,
164-166

5-fluoro-2’-deoxyuridine (FUdR) monophosphate,
167, 168
5-[(E)-2-bromovinyl]-2’-
35
deoxyuridine (BVdU) monophosphate,
33, 169
acyclovir monophosphate
170, 171
and
PMEA.
33, 172

The first generation of cycloSal pronucleotides did not require enzymatic activity in order
to release corresponding nucleoside monophosphate under physiological conditions.
158

Half-lives of these pronucleotides in phosphate buffer at pH 7.3 ranged from 0.25 h to
96 h and could be adjusted by introduction of electron-donating or electron-withdrawing
substituents into the promoiety’s aromatic ring to increase or decrease hydrolytic
stability.
33
The predominance of chemical activation of the prodrugs over enzymatic
activation was demonstrated by comparing the half-lives of the pronucleotides in
phosphate buffer (pH 7.3) and in 10% human serum in phosphate buffer (pH 7.3), but
significant differences could not be found. The first generation cycloSal pronucleotides
exhibited similar or better antiviral activities compared to the parent nucleoside
analogues, e.g. 3-methyl-cycloSal-ddAMP was 100-fold more potent than ddA against
HIV-1 and HIV-2 in a wild-type T-lymphocytic cell-line (CEM/0)
173
and 3-methyl-
cycloSal-d4A proved to be 600-fold more potent than d4A.
160
CycloSal pronucleotides
exist as two diastereomers (S
p
and R
p
) that showed different pharmacokinetic properties
(metabolic stability and antiviral potency). The (R
p
)-diastereomers of the first generation
cycloSal pronucleotides released higher amounts of the parent nucleoside
monophosphates, resulting in more elevated intracellular levels of the corresponding
nucleotide triphosphates, resulting in higher antiviral potencies than the
(S
p
)-diastereomers.
174, 175

36
This first generation of cycloSal pronucleotides, which liberates the parent nucleoside
monophospate analogue via a non-enzymatic mechanism, does not depend on enzyme
expression differences in tissues, individuals and species and suffered from several other
drawbacks, namely extracellular release of the parent nucleotide and efflux of the
pronucleotide from the cell due to formation of a concentration equilibrium across the
cell membrane. To circumvent these limitations, an additional functional group was
added to the cycloSal promoiety to create a second generation of cycloSal pronucleotides
with increased chemical stability in extracellular media and also to promote enzymatic
conversion into a more polar group within the cell to retard efflux (“lock-in” concept).
159

Initially, the second generation of cycloSal pronucleotides included enzymatically
cleavable ester and ether groups connected to the aromatic ring of the cycloSal promoiety
through an ethylene linker,
159
to minimize the electronic effect of the ester group on the
hydrolysis of the pronucleotide triester. A carboxylic acid group was incorporated to trap
the pronucleotide inside the cell, since the free alcohol group released in case of ethers
was not polar enough to prevent efflux of the pronucleotides. Simple alkyl ester groups
connected to the aromatic ring through an alkyl linker were poor esterase substrates.
Acylal systems based on acetoxymethyl (AM) and pivaloyloxymethyl (POM)
modifications were explored as promoieties to release the carboxylic group
(Figure 13.1).
33
Introduction of the AM group resulted in low enzymatic stability (t
1/2
=
0.25 h) and decreased antiviral activity, whereas POM-modified pronucleotides were
only marginally more stable (t
1/2
= 0.9 h), while suffering from cytotoxic metabolites,
37
such as pivaloic acid.
33, 158
To avoid the cytotoxic metabolites and to expand structural
tunability, the pronucleotides were next modified with uncharged amino acid esters,
which upon rapid intracellular hydrolysis by esterases generate the carboxylate group.
176

Among the cycloSal amino acid ester derivatives of d4TMP studied, the L-alanine benzyl
ester derivative provided the most favorable balance between stability of the ester group
(t
1/2
= 0.5 h in CEM cell extracts, pH 6.9) and antiviral potency of the pronucleotide vs.
HIV-1 and HIV-2 in CEM/0 cells (EC
50
= 0.3 and 0.4 μM, respectively).
176

Although effective intracellular trapping was apparently achieved with these second
generation pronucleotides, liberation of the parent drug was slow due to the chemical
stability of the cycloSal moiety.
177, 178
To adjust the release rate of the parent drug, a third
generation of the cycloSal pronucleotides was developed in which the aromatic ring of
the cycloSal promoiety was decorated with a 5-diacetoxymethyl,
178
5-
diisobutyroxymethyl,
164
3-diacetoxymethyl
164
or 5-(1-acetoxyvinyl)
165
group
(Figure 1.13). Once these pronucleotides entered cell, the lipophilic, weakly electron-
withdrawing acylal substituent would be hydrolyzed by esterases to form a polar,
strongly electron-withdrawing aldehyde group, retaining the pronucleotide inside the cell
and also facilitating its chemical hydrolysis to a benzyl phosphodiester, which could
spontaneously release the parent nucleoside monophosphate. This concept was based on
the assumption that the enzymatic conversion occurs mainly inside the cell, since
intracellular concentration of esterases is higher than in the extracellular environment.
145,
179
5-Diacetoxymethyl and 5-(1-acetoxyvinyl) derivatives of cycloSal-d4TMP exhibited
38
decreased half-lives (referring to disappearance of the triesters) in CEM cell extracts at
pH 6.9 (t
1/2
= 0.08 h and 0.13 h, respectively) compared to half-lives in phosphate buffer
with pH 7.3 (t
1/2
= 1.2 h and 1.4 h, respectively),
165, 179
but showed up to 50-fold less in
antiviral activity vs. HIV-2 in CEM/TK
-
cells than vs. HIV-1 and HIV-2 in wild-type
CEM/0 cells.
165, 179

In an effort to maintain antiviral activity, the hydrolytic stability of the third generation
pronucleotides was increased by incorporation of 3-alkyl groups (methyl, tert-butyl) into
the cycloSal promoieties,
165
as such substitution had successfully increased the chemical
stability of the first generation cycloSal pronucleotides.
33
Introduction of 3-tert-butyl
group into 5-(1-acetoxyvinyl)-cycloSal-d4TMP increased its half-life in phosphate buffer
(pH 7.3) 10-fold (1.4 h to 13.5 h), but slightly decreased the stability of the pronucleotide
in CEM cell extracts (pH 6.9; 0.13 h to 0.04 h). Most importantly, however, the 3-alkyl
derivative of the 5-(1-acetoxyvinyl)-cycloSal pronucleotide retained their antiviral
potency in TK-deficient cells relative to the antiviral activities in wild-type CEM/0
cells.
164, 165
Of the two diastereomeric forms of these cycloSal pronucleotides, the (S
p
)-
diastereomers were significantly more active.
164, 165
This correlation between the
phosphorus stereochemistry and the pronucleotide antiviral activity is opposite to that
observed for the S
p
- and R
p
-diastereomers of the first generation cycloSal
pronucleotides.
174, 175

39
Recently, the cycloSal approach was applied to a new class of nucleoside cytostatics, 6-
heteroarly-7-deazapurine ribonucleosides, however the resulting prodrugs had only low
to moderate antiviral activity compared to the parent nucleosides.
180
In order to improve
the number of equivalents of released nucleotide per masking unit (mask-to-drug ratio to
1:2) bis-(cycloSal) pronucleotides of d4TMP were proposed in which two nucleotide
molecules are joined to form one “dimeric” cycloSal masking unit.
181-183
As with first
generation pronucleotides, the activation mechanism of the bis-(cycloSal) derivatives was
non-enzymatic. These pronucleotides showed not only reduced butyrylcholinesterase
(BChE) inhibition and marked antiviral activity against HIV-1 and HIV-2 in wild-type
CEM cells, but were considerably more toxic than the parent nucleoside, and often, partly
cost antiviral potency in TK-deficient CEM cells. The bis-(cycloSal) promoiety
(3,3’-bis(hydroxymethyl)-5,5’-di-tert-butlydiphenyl-4,4’-diol) itself exhibited an
independent antiviral activity against HIV-1 and HIV-2 in CEM/0 cells and against
HIV-2 in CEM/TK
-
cells only 10-fold inferior to the corresponding pronucleotide itself.
The cytotoxicities of this promoiety and the intact prodrug were comparable.
183

In summary, the cycloSal approach succeeded in improving intracellular delivery of
nucleotides with activation solely by a chemical mechanism. Salicyl alcohol was shown
to be a bifunctional phosphate/phosphonate masking unit that can also be removed non-
enzymatically in vitro. Antiviral evaluation showed that some cycloSal pronucleotides
have antiviral activity similar to, or better than, the parent nucleoside, particularly in
kinase-deficient cells. Although salicyl alcohol is non-toxic, its nucleotide prodrugs had
40
elevated levels of cytotoxicities. Recognized drawbacks of this approach, such as
extracellular release of the nucleotide and excessive efflux of the pronucleotide from the
cell, have been addressed in two subsequent generations of the pronucleotides by
introduction and further modification of the “lock-in” concept. However, lack of specific,
enzyme-dependent nucleotide release diminishes the benefit of the first generation
pronucleotides, an independence of activation mechanism from an enzyme activity.
Currently, there are no examples of cycloSal pronucleotides in the clinical trials.

1.7 Perspectives
Prodrug design continues to be one of the most important approaches in the medicinal
chemistry armamentarium in improving undesirable pharmacokinetic properties of drugs.
Drugs incorporating phosphoric or phosphonic acid group are a focus of modern prodrug
strategies. This chapter has surveyed several such strategies with an emphasis on the
utility of leveraging specific in vivo enzyme activities to render the activation process
(localize, selective and more rapid). At the same, in use of these promoieties, enzyme-
dependent activation can be a two-edged sword if insufficiently specific by
disadvantageously decreasing the prodrug half-life in plasma or elsewhere in vivo, this
necessitates careful tuning of the promoiety to optimize the balance between transport,
toxicity and activation. It should be noted that despite ongoing attempts to create
phosphate/phosphonate prodrugs liberating the parent drug solely via chemistry in vivo,
all phosphate/phosphonate prodrugs currently available on the market or undergoing
clinical trials partly or wholly depend on enzymatic activity during their activation
41
process. Further development of pronucleotides that can effectively achieve oral
absorption, bypass pre-systemic metabolism and deliver nucleotide analogues to exert
their therapeutic action is likely to depend on a more detailed knowledge of enzyme-
mediated pathways for prodrug activation.

42
1.8 References
1. De Clercq, E. The acyclic nucleoside phosphonates from inception to clinical use:
Historical perspective. Antiviral Res. 2007, 75, 1-13.

2. De Clercq, E.; Holy, A. Case history: Acyclic nucleoside phosphonates: a key
class of antiviral drugs. Nat. Rev. Drug Discovery 2005, 4, 928-940.

3. Schaeffer, H. J.; Beauchamp, L.; De, M. P.; Elion, G. B.; Bauer, D. J.; Collins, P.
9-(2-Hydroxyethoxymethyl)guanine activity against viruses of the herpes group. Nature
(London) 1978, 272, 583-5.

4. De Clercq, E.; Descamps, J.; De, S. P.; Holy, A. (S)-9-(2,3-
Dihydroxypropyl)adenine: an aliphatic nucleoside analog with broad-spectrum antiviral
activity. Science 1978, 200, 563-5.

5. De Clercq, E. Antiviral drug discovery: ten more compounds, and ten more
stories (part B). Med. Res. Rev. 2009, 29, 571-610.

6. De Clercq, E.; Holy, A.; Rosenberg, I.; Sakuma, T.; Balzarini, J.; Maudgal, P. C.
A novel selective broad-spectrum anti-DNA virus agent. Nature (London) 1986, 323,
464-7.

7. De Clercq, E.; Sakuma, T.; Baba, M.; Pauwels, R.; Balzarini, J.; Rosenberg, I.;
Holy, A. Antiviral activity of phosphonylmethoxyalkyl derivatives of purines and
pyrimidines. Antiviral Res. 1987, 8, 261-72.

8. De Clercq, E. The discovery of antiviral agents: ten different compounds, ten
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- 5,5'-Bis-(cycloSaligenyl-2',3'-dideoxy-2',3'-didehydrothymidine Monophosphates). J.
Med. Chem. 2009, 52, 3464-3473.

61
CHAPTER 2
*

Single amino acid prodrugs of (S)-HPMPA and (S)-HPMPC

2.1 Introduction
As pointed out in Chapter 1, acyclic nucleoside phosphonates (ANPs), in particular
(S)-HPMPC (Cidofovir) and (S)-HPMPA are highly potent broad spectrum antiviral
agents that exhibit low cell membrane permeability and minimal oral bioavailability due
to the presence of a phosphonic acid group, which is ionized at physiological pH.
Although several prodrug strategies aimed to ameliorate this undesirable property of
ANPs and other nucleotide analogues have been developed (see Chapter 1 for a review of
the prodrug approaches), currently there are no prodrugs for Cidofovir and (S)-HPMPA
approved by the FDA for use in the clinic. In order to address this issue, we are
developing the amino acid phosphonate ester prodrug approach that explores the use of
various single amino acids and dipeptides as promoieties to mask the negative charges of
the ANPs. The present chapter describes the concept of our prodrug approach and the
rationale behind the prodrug design, summarizes previously published results, and reports
the most recent findings about mono amino acid prodrugs.

*
Section 2.5.2 contains excerpts reproduced with permission from Zakharova, V. M.; Serpi, M.; Krylov, I.
S.; Peterson, L. W.; Breitenbach, J. M.; Borysko, K. Z.; Drach, J. C.; Collins, M.; Hilfinger, J. M.;
Kashemirov, B. A.; McKenna, C. E. J. Med. Chem. 2011, 54, 5680-5693. Copyright 2012 American
Chemical Society.
62
2.2 The concept of the amino acid phosphonate ester prodrug approach
Acyclic nucleoside phosphonates of the HPMP-series, such as (S)-HPMPC 2.1 and
(S)-HPMPA 2.2, incorporate in their structure a hydroxymethylene function, which
mimics the 2’-hydroxyl of natural nucleotides and is able to intramolecularly esterify the
ANP phosphonic acid group, producing cyclic forms of the ANPs. Thus, two classes of
(S)-HPMPA and (S)-HPMPC prodrugs have been investigated: 1) cyclic phosphonate
diesters and 2) acyclic phosphonate di- and monoesters (Figure 2.1). In the cyclic
phosphonate diester prodrugs, one phosphonic acid OH group of the ANPs is masked via
intramolecular cyclization with the hydroxyl group to afford the cyclic form of the drug.
1

The remaining P-OH group is esterified with the side chain group of a natural amino acid
(e.g. Ser, Thr, Tyr or Cys), serving as a single amino acid promoiety or incorporated into
a dipeptide promoiety (Figure 2.1, pathway 1). The second class of studied prodrugs,
acyclic phosphonate monoester prodrugs, incorporate a single amino acid promoiety to
mask one phosphonic acid P-OH, whereas the second P-OH group is unmasked (Figure
2.1, pathway 2).

Figure 2.1 Amino acid phosphonate ester prodrugs: 1) cyclic phosphonate diesters; 2) acyclic
phosphonate monoesters.
63
Although the mechanism of the parent drug release from the amino acid prodrugs varies
depending on the structure of the promoiety, it is hypothesized that in general, cyclic
phosphonate diester prodrugs undergo enzymatic or pH-dependent chemical hydrolysis,
releasing cyclic or acyclic phosphonate monoester prodrugs (Figure 2.2).
2
These
intermediates liberate the parent ANP drugs after enzymatic degradation given that
chemical hydrolysis of the phosphonate monoesters is particularly slow at physiological
conditions. Mendel et al. have shown that intracellular enzymatic conversion of
cHPMPC to the parent acyclic (S)-HPMPC is catalyzed by a cyclic CMP
phosphodiesterase.
3

Figure 2.2 General representation of the activation pathway of alcoholic amino acid phosphonate
ester prodrugs: a) enzymatic or pH-dependent chemical hydrolysis; b) enzymatic hydrolysis.

2.3 The Rationale: why amino acids?
The initial rationale behind the utilization of amino acids as the promoiety is based on:
1) the biologically benign properties of natural amino acids and their metabolites;
2) conjugation to the drug via naturally occurring P-X-C linkage (X = O, S), which is
stable at gastric acid pH (< 3.5) and can be activated at physiological pH (> 6.5); 3) the
presence of various functional groups, modification of which would allow “fine-tuning”
64
of the ADME (Absorption, Distribution, Metabolism and Excursion) profile of the
resulting prodrugs; and 4) the possibility to target human oligopeptide transporter 1
(hPEPT1). hPEPT1 is a proton-coupled di- and tripeptide transporter located in the brush
border membrane of enterocytes of the small intestine.
4
Broad substrate specificity of
hPEPT1 allows it to transport a wide array of chemically diverse compounds, such as
β-lactam antibiotics,
5-7
angiotensin-converting enzyme inhibitors
8-10
and non-peptidic
drugs,
11
thus making this transporter an attractive drug delivery target.
12
Prodrugs
successfully targeting the hPEPT1 transporter include valacyclovir (Valtrex®, Figure
2.3),
13
that is valine-containing conjugate of acyclovir (Zovirax®, Figure 2.3).

Figure 2.3 Structures of acyclovir and its prodrug valacyclovir.

Acyclovir does not contain a phosphonate group, but has a nucleoside base – guanine –
and a terminal hydroxyl group that is esterified by (L)-valine to form valacyclovir.
Valacyclovir has significantly higher oral bioavailability than the parent drug, associated
with transport of the prodrug by hPEPT1,
14-16
according to a study using radiolabeled
valacyclovir in Xenopus laevis oocytes expressing the transporter.
17
Among drugs
containing a phosphonate group, only two (fosinopril and pamidronic acid) have been
shown to be transported by hPEPT1.
18, 19
Efforts to define the structure-activity
relationship (SAR) and structure-transport relationship (STR) properties of hPEPT1 have
65
been made,
20-23
however reliable predictive rules for the affinity and transport have been
elusive.
24
In a search for the promoiety that, when conjugated to the phosphonate drug,
can be recognized and translocated by hPEPT1, and also can be efficiently cleaved from
the prodrug at the target site in vivo, various ethylene glycol (EG)-linked amino acid
prodrugs and dipeptide prodrugs of cyclic (S)-HPMPC (Figure 2.4, 2.5 - 2.11) have been
previously described and are reviewed below.

2.4 Previously reported hydroxyl amino acid (S)-HPMPC prodrugs
2.4.1 EG-linked amino acid (S)-HPMPC prodrugs
The ethylene glycol-linked amino acid conjugates of cHPMPC
25
closely resembled
valacyclovir’s structure (Figure 2.4, 2.5 - 2.6), but failed to demonstrate an increased oral
uptake and to efficiently release the parent drug at the target site in vivo, owing to the
insufficient stability of the promoiety under physiological conditions. For example, the
cHPMPC prodrug 2.5, incorporating the EG-linked (L)-valine, exhibited half-lives in
enzymatic media (liver and intestinal homogenates) ranging from 4–16 min and showed
submicromolar in vitro antiviral activity against human cytomegalovirus (HCMV).
However, this compound failed to demonstrate an improved oral uptake in a rat model
compared to the parent drug.
25

66

Figure 2.4 Selected structures of previously described amino acid (S)-HPMPC prodrugs.
25-27

2.4.2 Dipeptide (S)-HPMPC prodrugs
To increase the amino acid prodrug stability and to preserve the targeting of the hPEPT1
transporter, dipeptides composed of a hydrophobic amino acid, such as Ala, Val, Phe or
Leu, and a hydroxyl amino acid, such as Ser were employed as promoieties (Figure 2.4,
2.7 - 2.11).
27-30
The dipeptide promoiety was conjugated to the drug via an ester linkage
between the serine side chain OH and the P(O)OH group of ANP. Favorably, this design
allowed for the “fine-tuning” of the prodrugs’ pharmacokinetic properties by
modification of the multiple functional groups in the amino acids constructing the
promoiety. For example, utilization of the N-terminal amino acids with
D-stereochemistry to construct the dipeptide promoieties, generated additional enzymatic
stability of the prodrug, and thereby increased its level of transportation fraction
transported into the plasma in the intact form. Depending on the promoiety’s structure,
67
half-lives of the dipeptide prodrugs 2.7 - 2.11 ranged from 140 to 595 min in phosphate
buffer (pH 6.5 at 37 ºC) and from 43 to 216 min in intestinal homogenate (pH 6.5 at
37 ºC).
26
Furthermore, (S)-HPMPC prodrugs incorporating dipeptide promoieties
demonstrated 8-15-fold increase in oral bioavailability of total cidofovir species
compared to the parent drug in a murine model.
27, 31

In order to validate the idea that the observed enhancement of oral bioavailability is due
to active transport by hPEPT1, the prodrug, (L)-Val-(L)-Ser(OMe) cHPMPC 2.10a was
evaluated in a competitive binding assay in DC5 cells over-expressing hPEPT1 and
showed a significant affinity for the hPEPT1 transporter.
30
However, in a further
investigation of the dipeptide cHPMPC prodrugs 2.10a and 2.11 using radiotracer uptake
and electrophysiologic assays of Xenopus laevis oocytes overexpressing the transporter,
hPEPT1-mediated translocation of these compounds was not detected, despite the
confirmation that they bind to the transporter with low to high affinity.
29

Similar to the parent (S)-HPMPC 2.1, its dipeptide prodrugs 2.7 - 2.11 were found to be
up to 10-fold more potent in vitro against HCMV (IC
50
= 0.2-0.7 μM) compared to
ganciclovir, the positive control (IC
50
= 3 μM),
25, 27, 31
but in contrast, they failed to
exhibit any activity against orthopox viruses (vaccinia and cowpox virus,
IC
50
> 100 μM), despite the prodrugs’ improved metabolic stability and increased cell
membrane permeability. The difference in potency against various viruses was attributed
to the prodrugs’ inadequate activation in the viral assays caused by either lack of specific
68
viral-encoded enzymes that activate the prodrugs or different assay incubation times
(HCMV – 10 days; cowpox and vaccinia virus – 3 days).
27, 31
The shorter incubation
time in the poxvirus assays did not allow sufficient time for the prodrug to exert a
therapeutic effect via conversion to the parent drug. To test the latter hypothesis, the
dipeptide prodrug activation mechanism was evaluated in detail in phosphate buffer
(pH 6.5 and 7.4 at 37 ºC) and enzymatic media (intestinal and liver homogenates, and
plasma), mimicking physiological conditions.
26
The studies revealed not only the
existence of multiple prodrug activation mechanisms, including simple phosphonate ester
hydrolysis and β-elimination of the serine side-chain linked dipeptides, but also the
presence of several prodrug degradation pathways, such as deesterification of the
promoiety and cyclization of the promoiety to the corresponding diketopiperazine (DKP),
which reduce the released amount of the parent drug and significantly decrease the
efficiency of the dipeptide prodrug activation.
26

In summary, the dipeptide prodrugs of (S)-HPMPC, 2.7 - 2.11 exhibited sufficient
chemical and enzymatic stabilities and 8-15-fold enhanced oral bioavailability in a
murine model. However, these compounds lacked efficient activation, releasing only a
fraction of the parent drug along with various metabolically-stable and biologically-
inactive (S)-HPMPC-containing metabolites. Using the model system, it was found that
the dipeptide conjugates were tightly bound to hPEPT1, but were not translocated by it.
Finally, the dipeptide prodrugs were active against HCMV (IC
50
= 0.2-0.7 μM), but failed
to exhibit antiviral activity against orthopox viruses (cowpox and vaccinia).
69
2.5 Single amino acid prodrugs
In order to better define the structural effects on the interactions of the amino acid side
chain-linked peptidomimetic prodrugs with hPEPT1, with the goals of avoiding
inefficient dipeptide prodrug activation, and restoring the prodrug antiviral activity
against orthopox viruses, the effects of the removal of the non-linking, second amino acid
from the dipeptide moiety and of modification of the remaining single amino acid were
investigated by synthesizing and biologically evaluating mono amino acid prodrugs of
(S)-HPMPA and (S)-HPMPC. Similar to the dipeptides, the mono amino acid
promoieties contain several sites available for modification, including side chain,
stereochemistry, N-terminal and C-terminal groups (Figure 2.5). Structural alteration of
those sites allows for the “fine-tuning” of the physicochemical properties of the resulting
prodrugs.

Figure 2.5 Modification sites of the single amino acid promoiety.

Initially, several serine-based prodrugs of (S)-HPMPA (Figure 2.6, 2.12 - 2.14) were
synthesized and evaluated for hPEPT1-mediated transport in the electrophysiologic
Xenopus laevis oocytes assay.
29

70

Figure 2.6 Structures of the serine-based (S)-HPMPA prodrugs evaluated for hPEPT1-mediated
transport in the electrophysiologic Xenopus laevis oocytes assay.

In these experiments the serine prodrugs 2.12 – 2.14 showed inhibition of the Gly-Sar
uptake, but did not evoke an inward current. Variation of the serine stereochemistry
(L vs. D) and alkyl ester groups (Me vs. i-Pr) had no effect on the outcome of the
experiment.
29
Thus, it was concluded that, similar to the dipeptide prodrugs, the single
amino acid conjugates of the cyclic ANPs are not transported by hPEPT1, most likely
due to the presence of a phosphonic ester group. An analogous observation has been
recently reported for fosinopril (phosphorus-containing ACE inhibitor), which showed no
hPEPT1-mediated transport based on an electrogenic transport experiment.
32

Since active hPEPT1-mediated transport has not been confirmed, it was suggested that an
alternative transport mechanism, such as passive diffusion, operates for the single amino
acid and dipeptide (S)-HPMPC and (S)-HPMPA prodrugs.
29
The driving force for
diffusion of a compound from an aqueous phase (intestinal lumen) through GI cell
membranes is the concentration gradient of the drug across the membrane.
33
It is
essential for a molecule to have characteristics with low molecular weight and relatively
high lipophilicity in order to pass across the intestinal membrane by this pathway. The
lipophilicity of a drug is the most-used single physico-chemical property to predict
71
permeability across biological membranes. It is measured as the partition coefficient (for
non-ionized molecules) between water and n-octanol (log P) or as the distribution
coefficient (for ionized molecules) between buffer with fixed pH and n-octanol (log D).

Thus, in a further search for an adequately transported single amino acid promoiety,
calculated log D (Clog D) values at pH 6.5 and 7.4 were exploited to preliminarily
estimate cell membrane permeability. In order to identify prodrugs exhibiting suitable
balance between the prodrug’s stability and efficient conversion to the active parent drug
in vivo, a library of the mono amino acid prodrugs was synthesized and several rounds of
structure-activity relationship studies were performed.

2.5.1 First round of SAR studies of single amino acid prodrugs
The first round of SAR studies was aimed to identify a single amino acid side chain,
which, upon conjugation with the phosphonate drug, forms a pH-responsive P-X-C ester
linkage (X = O, S) sufficiently stable at gastric acid pH (< 3.5), but, at the same time,
efficiently hydrolyzable under physiological pH (6.5 - 7.4). Of all the natural amino
acids, only (L)-serine, (L)-threonine, (L)-tyrosine and (L)-cysteine contain side chains
that can be conjugated to the cyclic nucleoside phosphonates forming aliphatic (Ser, Thr),
aromatic (Tyr) and thio (Cys) phosphonate esters stable under acidic conditions (gastric
acid pH). Susceptibility of these phosphonate esters towards hydrolysis under neutral
conditions (physiological pH) was initially rationalized based on the literature data. For
example, hydrolysis of simple aliphatic phosphonate esters to the corresponding
72
phopshonic acid typically requires pH extremes and vigorous conditions.
34
However,
with the intramolecular assistance of a neighboring group, the hydrolysis of the aliphatic
phosphonate ester can be accomplished under mild physiological conditions. Several
examples of intramolecularly catalyzed hydrolyses of various phosphonate and phosphate
esters have been previously reported.
34-38
For example, it was demonstrated that
phosphorylethanolamine triesters can be rapidly hydrolyzed at neutral pH (6-8) via the
amino-group assisted C-O bond cleavage releasing the corresponding phosphodiester and
aziridine.
36

In contrast to aliphatic esters, aromatic phosphonate esters are more labile in alkaline
solution due to resonance stabilization of the leaving group, phenoxide anion. The
hydrolysis rate of these esters can also be increased by participation of a neighboring
group, for example at pH = 7.8, p-nitrophenyl phenacyl methylphosphonate was found to
be hydrolyzed ca. 9000 times faster (τ
1/2
= 41.0 s) compared to ethyl p-nitrophenyl
methylphosphonate (τ
1/2
= 3.63×10
5
s) due to assistance of the neighboring carbonyl
group.
39

Finally, when oxygen is substituted for sulfur in the P-O-C linkage in the case of
thiophosphonate esters, the rate of hydrolysis increases because, in contrast to oxygen,
the sulfur atom cannot conjugate effectively with phosphorus in order to reduce its
electron demand. In addition, this primary effect is enhanced by the fact that the
mercaptide anion is a better leaving group than the alkoxide anion.
40
Therefore,
73
hydrolysis of phosphonothioates can be accomplished under mild physiological
conditions without the need for catalysis from a neighboring group.

Thus, in the first round of SAR studies, four (S)-HPMPA prodrugs, incorporating
(L)-serine, (L)-threonine, (L)-tyrosine and (L)-cysteine methyl esters were synthesized
(Figure 2.7, 2.12, 2.15 - 2.17) and their chemical and enzymatic stabilities were
evaluated.

Figure 2.7 Structures of single amino acid cyclic (S)-HPMPA prodrugs.

All compounds were chemically robust under acidic conditions. Chemical stability was
determined by evaluating the hydrolysis rates of the prodrugs in 200 mM phosphate
buffer solution (PBS) at physiologically relevant pH (6.5 and 7.4) at 37 ºC. To assess the
enzymatic stability, the rate of disappearance of each prodrug was determined in rat
intestinal homogenate (pH 6.5) at 37 ºC. The chemical and enzymatic hydrolyses of each
prodrug followed pseudo-first-order kinetics over several half-lives (τ
1/2
). Half-lives of
the compounds 2.12, 2.15 - 2.17 in PBS, pH 6.5 at 37 ºC are reported in Table 2.1.

74
Table 2.1 Half-lives (τ
1/2
, min) of single amino acid (S)-HPMPA
prodrugs 2.12, 2.15 - 2.17 in PBS, pH 6.5 at 37 ºC
Prodrug
τ
1/2
, min
(average of two diastereomers)
(L)-SerOMe-cHPMPA, 2.12 19
(L)-ThrOMe-cHPMPA, 2.15 64
(L)-TyrOMe-cHPMPA, 2.16 147
(L)-CysOMe-cHPMPA, 2.17 complete hydrolysis after 45 min
Target prodrugs half-life 120 – 180

The prodrugs 2.12, 2.15 - 2.17 exhibited half-lives ranging from 19 min to 147 min
(average between the two diastereomers). The tyrosine-based prodrug 2.16 was found to
be 2–6-fold chemically more stable (τ
1/2
= 147 min) than the serine, threonine and
cysteine-based compounds 2.12, 2.15 and 2.17. Upon hydrolysis, the serine and
threonine (S)-HPMPA prodrugs 2.12, 2.15 mainly released the cyclic form of (S)-
HPMPA (cHPMPA, 2.4) with a yield ca. 90% (Figure 2.8). In contrast, the cysteine and
tyrosine cHPMPA prodrugs 2.16 and 2.17, when studied in the same conditions,
produced two products, cHPMPA 2.4 and corresponding acyclic prodrug 2.18 or 2.19
(Figure 2.8).

Figure 2.8 Chemical activation of the mono amino acid (S)-HPMPA prodrugs 2.12, 2.15 - 2.17 in
phosphate buffer solution (PBS), pH 6.5 at 37 ºC.
75
The difference in product distribution in case of serine/threonine and tyrosine/cysteine
containing prodrugs can be explained by the distinct mechanisms of hydrolysis involved.
The serine and threonine phosphonate ester prodrugs 2.12 and 2.15 contain the amino
group in the same position with respect to the P-O-C linkage as the previously studied
phosphorylethanolamine triesters.
36
Therefore, it is hypothesized that hydrolysis of the
prodrugs at physiological pH most likely proceeds through the same mechanism as in the
case of phosphorylethanolamine triesters – via C-O bond cleavage intramolecularly
assisted by the NH
2
-group (Figure 2.9, left).

Figure 2.9 Suggested mechanisms for the serine/threonine (left) and cysteine/tyrosine (right)
cyclic (S)-HPMPA prodrug hydrolysis in phosphate buffer solution with physiological pH (6-8).

In contrast, in case of the tyrosine/cysteine-containing prodrugs, no intramolecular
assistance of a neighboring amino group is possible and hydrolysis happens via
hydroxide attack at the phosphorus atom followed by P-X ester bond cleavage
(X = O, S). The OH
-
attack is non-selective and can happen either opposite to the exo-
cyclic P-X bond (Figure 2.9, right, pathway a), resulting in the placement of the exo-
cyclic ester in the axial position of the trigonal bipyramid intermediate and subsequent
76
breakdown to cyclic (S)-HPMPA 2.4, or opposite to the endocylclic P-O bond (Figure
2.9, right, pathway b), yielding the analogous placement for the endocyclic ester. Since
pseudorotation of the latter intermediate is energetically prohibited,
2
it is then
transformed to the acyclic monoesters 2.18 and 2.19.

When evaluated in intestinal homogenate, the single amino acid phosphonate ester
prodrugs 2.12, 2.15, and 2.16 exhibited significantly shorter half-lives (τ
1/2
< 30 min) than
in buffer solution, indicating enzymatic degradation of the prodrugs. All prodrugs tested
in the first round of SAR studies had a methyl carboxylate ester group in common, which
underwent rapid enzymatic deesterification by esterases in intestinal homogenate,
releasing the corresponding free carboxylic acid group (Figure 2.10, 2.12, 2.15, and
2.16).

Figure 2.10 Enzymatic degradation of the mono amino acid cyclic (S)-HPMPA prodrugs 2.12,
2.15, 2.16 in intestinal homogenate, pH 6.5 at 37 ºC.

In vitro antiviral activities of the single amino acid prodrugs 2.12, 2.15, 2.16 and of the
parent (S)-HPMPA 2.2 were evaluated against human cytomegalovirus (HCMV),
cowpox and vaccinia viruses and are summarized in Table 2.2. These prodrugs were also
tested for cytotoxicity in human nosapharyngeal carcinoma cells (KB cells). In these
77
experiments, the prodrugs 2.12, 2.15, 2.16 had antiviral potencies (IC
50
values) similar to
the activity of the parent (S)-HPMPA against all the viruses tested (Table 2.2). None of
the compounds showed any significant cytotoxicity towards growing KB cells up to a
concentration of at least 100 μM.

In conclusion, the first round of SAR studies revealed that the tyrosine (S)-HPMPA
prodrug 2.16 is 2-6-fold more chemically stable (τ
1/2
= 147 min) than the
serine/threonine/cystein-based conjugates. It was found that upon hydrolysis in PBS
(pH 6.5, 37 ºC), this prodrug released only two products: cHPMPA 2.4, and acyclic
tyrosine prodrug 2.18 (both compounds demonstrate in vitro antiviral activity against
HCMV, cowpox and vaccinia). Thus, the tyrosine-based prodrug 2.16 exhibited more
efficient activation compared to previously reported
26
dipeptide prodrugs. In enzymatic
media, the prodrugs 2.12, 2.15 and 2.16 underwent rapid enzymatic deesterification of
the amino acid ester group (τ
1/2
< 30 min in intestinal homogenate, pH 6.5, 37 ºC) and
released corresponding carboxylic acids, which are ionized at physiological pH and,
therefore, prevent the prodrug from efficient cell membrane penetration. When evaluated
Table 2.2 In vitro antiviral activities against HCMV, Cowpox and Vaccinia and
cytotoxicities of the single amino acid prodrugs of (S)-cHPMPA, 2.12, 2.15, 2.16.
a

Prodrug
Antiviral activity, IC
50
(μM) Toxicity, (μM)
HCMV Cowpox Vaccinia KB cells
(S)-HPMPA, 2.2 2.4 15 15 >100
(L)-SerOMe-cHPMPA, 2.12 0.5 20 25 >100
(L)-ThrOMe-cHPMPA, 2.15 0.62 20 30 >100
(L)-TyrOMe-cHPMPA, 2.16 4.7 10 2 >100
a
Data obtained by Prof. Drach et al. at the University of Michigan
78
in vitro against HCMV, Cowpox and Vaccinia viruses, these prodrugs showed activity
similar to or better than the parent (S)-HPMPA. Due to the high chemical stability and
efficient activation in PBS buffer of the tyrosine (S)-HPMPA prodrug 2.16, tyrosine was
identified as the most favorable amino acid promoiety for further prodrug development.

2.5.2 Second round of SAR studies of single amino acid prodrugs
In order to increase the enzymatic stability of the tyrosine methyl ester promoiety, a
second round of SAR studies involving alteration of the tyrosine stereochemistry (L vs.
D) and C-terminal group was designed. Utilization of (D)-tyrosine was rationalized
based on the findings that (D)-enantiomers of natural amino acids are not recognized by
most natural enzymes and therefore exhibit increased enzymatic stability.
41, 42

Replacement of the methyl ester with a sterically hindered isopropyl ester group was
justified taking into account the fact that the diisopropyl carbonate ester prodrug of
tenofovir, namely tenofovir disopropxil, was reported to exhibit sufficient chemical and
enzymatic stability and is currently approved by the FDA for the chronic treatment of
HIV and Hepatitis B infections.
43, 44
Finally, the tyrosine carboxyl ester group was
replaced by a non-toxic, enzymatically stable N-alkyl amido group (N-iso-butyl or N-tert-
butyl) in order to completely avoid enzymatic deesterification.
45-47
Thus, a library of six
(S)-HPMPA and (S)-HPMPC prodrugs, incorporating (L)-TyrOMe, (D)-TyrOMe, (L)-
TyrOi-Pr, (L)-TyrNHi-Bu and (L)-TyrNHt-Bu were synthesized and evaluated for
chemical and enzymatic stability, and antiviral potency (Figure 2.11, compounds 2.16,
2.24 - 2.28).
79

Figure 2.11 Tyrosine (S)-HPMPA and (S)-HPMPC prodrugs 2.16, 2.23 - 2.27 synthesized and
evaluated in the second round of SAR studies.

The hydrophobicities of the tyrosine prodrugs 2.16, 2.23 - 2.27 were preliminarily
estimated by calculating the log D (ClogD) values at pH 6.5 and 7.4 using Marvin Sketch
(version 5.2.0) software (Table 2.3).

Table 2.3 Comparison of calculated log D values for the tyrosine-based (S)-cHPMPA and
(S)-cHPMPC derivatives with the values for parent (S)-HPMPA and (S)-HPMPC.
Prodrug Clog D (pH 6.5) Clog D (pH 7.4)
(S)-HPMPA, 2.2 -4.51 -4.59
(S)-cHPMPA, 2.4 -3.43 -3.41
(L)-Tyr-OMe-cHPMPA, 2.16 -1.87 -1.62
(L)-Tyr-NHi-Bu-cHPMPA, 2.26 0.85 0.87

(S)-HPMPC, 2.1 -4.60 -4.70
(S)-cHPMPC, 2.3 -3.52 -3.52
(L)-Tyr-OMe-cHPMPC, 2.23 -0.41 0.04
(L)-Tyr-Oi-Pr-cHPMPC, 2.25 0.35 0.80
(L)-Tyr-NHt-Bu-cHPMPC, 2.27 -1.06 -0.25
80
A comparison of these values predicts the highest lipophilicity for the tyrosine iso-butyl
amide cHPMPA prodrug 2.26 (ClogD
(pH 6.5)
= 0.85) and for the tyrosine isopropyl ester
cHPMPC conjugate 2.25 (ClogD
(pH 6.5)
= 0.35). Interestingly, according to these
calculations, the Clog D value of the tyrosine tert-butyl amide conjugate of cHPMPC
2.27 (ClogD
(pH 6.5)
= -1.06) is more than 1 order of magnitude lower than that of the
corresponding N-alkyl amide (S)-HPMPA conjugate 2.26.

Table 2.4 Half-lives (τ
1/2
, min) of the tyrosine-based (S)-cHPMPA and (S)-cHPMPC
prodrugs 2.16, 2.23 - 2.27 in PBS and in rat intestinal homogenate at pH 6.5 and 37 ºC.
prodrug isomer τ
1/2
, min in PBS
τ
1/2
, min in rat intestinal
homogenate
2.16
R
p
330 <30
S
p
55 <30
2.23
R
p
256 <30
S
p
56 <30
2.24
R
p
385 210
S
p
91 72
2.25
R
p
770 <30
S
p
121 <30
2.26
R
p
771 771
S
p
122 114
2.27
R
p
1732 1732
S
p
210 231
Prodrugs 2.23 – 2.25, 2.27 were analyzed by Dr. Michaela Serpi.

The chemical and enzymatic stabilities of the tyrosine prodrugs were evaluated in PBS
and intestinal homogenate using the same procedure as in the first round of SAR studies.
Half-lives of the prodrugs 2.16, 2.23 - 2.27 are summarized in Table 2.4. Since a
significant difference in stability between the two diastereomers of the tyrosine prodrugs
was observed (about 3 to 10-fold), the half-life for each diastereomer is reported
separately.
28
Similar differences in stabilities of diastereomers were reported previously
81
for salicylate and aryl ester prodrugs of cHPMPC (5.4- and 9.4-fold) and were attributed
to the greater ground state free energy of one diastereomer relative to the other one.
2

As can be seen from table 2.4, the structural modifications described previously increase
the chemical stability of the tyrosine prodrugs by 1-6-fold when compared to the
(L)-tyrosine methyl ester conjugates. For example, incorporation of the iso-propyl ester,
iso-butyl amide or tert-butyl amide functionality increases 2-3-fold the chemical stability
of the tyrosine prodrugs 2.25 - 2.27 in PBS compared to the corresponding Me-ester
derivatives 2.16 and 2.23.

In intestinal homogenate, an enhanced enzymatic stability was observed when
(D)-tyrosine was used instead of the natural (L)-amino acid component of the promoiety
structure. However, the highest enzymatic stabilities among the compounds tested were
detected for the tyrosine N-alkyl amide prodrugs 2.26 and 2.27. These compounds
undergo the same activation pathway (release only two metabolites: the cyclic form of
the parent drug and the acyclic form of the prodrug) and exhibit nearly identical half-lives
in both enzymatic and non-enzymatic media at physiological conditions (Table 2.4). The
activation pathway of 2.26 in PBS and in intestinal homogenate, along with HPLC
analyses of the metabolites (cHPMPA 2.4 and (L)-TyrNH-i-Bu-HPMPA 2.28) at the
beginning of the reaction (t = 0 min) and after incubation of the reaction mixtures at
37 ºC for 4.5 h and 5 h, are depicted in Figure 2.12.
82

Figure 2.12 A) Chemical and enzymatic activation of (L)-TyrNH-i-Bu-cHPMPA 2.26 in
phosphate buffer solution and intestinal homogenate at pH 6.5 and 37 ºC. B) HPLC analysis of
(L)-TyrNHi-Bu-cHPMPA 2.28 metabolism at pH 6.5 and 37 ºC in PBS (left HPLC traces) and in
intestinal homogenate (right HPLC traces). Top HPLC traces correspond to the reaction mixtures
at t = 0 min; bottom HPLC traces correspond to the reaction mixtures after incubation for 270
min or 300 min.

Additional enzymatic stability studies of 2.26 were performed in rat plasma with pH 7.4
and revealed the same metabolic pathway as was observed in intestinal homogenate (the
same metabolites 2.4 and 2.28 were formed). The half-lives of the two diastereomers
under these conditions differed nearly 10 fold: 23 min for (S
p
)-diastereomer and 213 min
for (R
p
)-diastereomer.
28

As in the case of the tyrosine methyl ester (S)-HPMPA prodrug 2.16, the ratio of the
hydrolysis products for the tyrosine N-alkyl amide prodrugs 2.26 and 2.27 can be
83
explained by non-selective hydrolytic attack at the phosphorus opposite the exocyclic or
endocyclic P-O bonds to form a trigonal bipyramid intermediate that is transformed
subsequently to the respective products. The ratio of exocyclic and endocyclic P-O bond
cleavage for compound 2.26 reached 1:1 to 1:2.3, depending on the pH and the
diastereomeric content of the compound.
28

The antiviral potential of the tyrosine-based prodrugs 2.16, 2.23 - 2.27, the metabolites
2.4, 2.28 and the parent (S)-HPMPC 2.1 and (S)-HPMPA 2.2 was evaluated against
human cytomegalovirus (HCMV), cowpox and vaccinia viruses. The results obtained are
summarized in table 2.5.

All tyrosine conjugates were also tested for cytotoxicity in human carcinoma of the
nasopharynx (KB cells). Thus, the tyrosine conjugates 2.16, 2.23 - 2.27 and the
Table 2.5 In vitro antiviral activities against HCMV, Cowpox and Vaccinia and cytotoxicities
of the tyrosine-based prodrugs of (S)-cHPMPA and (S)-cHPMPC 2.16, 2.23 - 2.28.
a

Prodrug
Antiviral activity, IC
50
(μM) Toxicity, (μM)
HCMV Cowpox Vaccinia KB cells
(S)-HPMPA, 2.2 0.41 4 2 >100
(S)-cHPMPA, 2.4 1.3 0.6 1 >100
(L)-Tyr-OMe-cHPMPA, 2.16 0.45 7 3 >100
(L)-Tyr-NHi-Bu-cHPMPA, 2.26 0.3 7 3 >100
(L)-Tyr-NHi-Bu-HPMPA, 2.28 0.29 3 0.5 >100

(S)-HPMPC, 2.1 0.28 30 20 >100
(S)-cHPMPC, 2.3 0.25 30 40 >100
(L)-Tyr-OMe-cHPMPC, 2.23 0.23 50 4 >100
(D)-Tyr-OMe-cHPMPC, 2.24 0.2 35 25 >100
(L)-Tyr-Oi-Pr-cHPMPC, 2.25 0.12 40 30 >100
(L)-Tyr-NHt-Bu-cHPMPC, 2.27 0.1 20 10 >100
a
Data obtained by Prof. Drach et al. at the University of Michigan
84
metabolites 2.4, 2.28 exhibited antiviral potencies similar to, or better than the activity of
the parent ANPs 2.2 and 2.3 against all the viruses tested (Table 2.5). Antiviral activities
of these prodrugs were found to be independent of tyrosine stereochemistry and C-
terminal functional group. None of the tyrosine-based prodrugs showed any significant
cytotoxicity towards growing KB cells up to a concentration of at least 100 μM.

In summary, among all structural modifications of the tyrosine amino acid promoiety
performed in the second round of SAR studies, incorporation of an N-alkyl amide
functionality instead of a carboxyl ester group resulted in the most significant increase in
the enzymatic stability of the prodrugs. The tyrosine N-alkyl amide prodrugs 2.26 and
2.27 undergo the same activation pathway and demonstrate nearly identical half-lives in
both enzymatic (rat intestinal homogenate) and non-enzymatic (phosphate buffer
solution) media that mimic physiological conditions. Upon activation, these prodrugs
released only two metabolites, the cyclic form of the parent drug plus an acyclic form of
the respective prodrug. In addition, all the tyrosine-based prodrugs (2.16, 2.23 - 2.27) and
the metabolites (2.4 and 2.28) exhibited similar or better in vitro antiviral activities
against HCMV, cowpox and vaccinia virus compared to the parent ANP, (S)-HPMPC
and (S)-HPMPA.

2.5.3 Third round of SAR studies of single amino acid prodrugs
After discovering that the tyrosine N-alkyl amide prodrugs 2.26 and 2.27 were
sufficiently stable and efficiently activated in both enzymatic and non-enzymatic media, a
85
third round of SAR studies aimed at exploring the correlation between the hydrophobicity
and antiviral potency of the prodrugs was performed. Hydrophobic prodrugs exhibit
improved cell membrane permeability (by passive diffusion, membrane flippase activity
or phospholipid uptake), which might translate into enhanced potency. A well-known
strategy to increase a drug’s hydrophobicity is to conjugate it to a long alkyl chain. For
example, Hostetler et al. reported that esterification of the phopshonic acid moiety of (S)-
HPMPC and (S)-HPMPA with a hydrophobic alkyl group, particularly
hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE), resulted in prodrugs exhibiting
2.5 to 4-log increase in antiviral activity depending on the antiviral assay used (Figure
2.13).
48
The HDP-(S)-HPMPC prodrug (CMX-001) is currently in Phase II human
clinical studies.

Figure 2.13 Structures of the previously reported hexadecyloxypropyl (HDP) or
octadecyloxyethyl (ODE) prodrugs of (S)-HPMPC and (S)-HPMPA.
48

Thus, for the third round of SAR studies, a series of tyrosine N-alkyl amide cyclic (S)-
HPMPA and cyclic (S)-HPMPC prodrugs, incorporating alkyl chains varying in length
from C4 to C18 to “tune” the hydrophobicity of the prodrugs, were synthesized and
86
evaluated for in vitro antiviral activity (Figure 2.14, structures 2.26, 2.30 - 2.35). The
corresponding acyclic prodrugs 2.35 - 2.40 were also synthesized to assess their antiviral
potency, since the acyclic (L)-Tyr-NHi-Bu-cHPMPA prodrug 2.28 demonstrated antiviral
activity comparable to the parent drug.

Figure 2.14 Tyrosine N-alkyl amide based (S)-cHPMPA, (S)-HPMPA, (S)-cHPMPC and
(S)-HPMPC prodrugs 2.26, 2.28, 2.29 - 2.40 synthesized and evaluated in the third round of SAR
studies.

The hydrophobicities of the novel tyrosine prodrugs 2.27, 2.29 - 2.41 and of the
previously reported hexadecyloxypropyl (HDP) and octadecyloxyethyl (ODE) prodrugs
were predicted by calculating the logD values at pH 6.5 and 7.4 using Marvin Sketch
(version 5.2.0) software (Table 2.6). According to these calculations, the calculated logD
(ClogD) value increases by 0.5-0.7-log with every 2 carbon extension of the alkyl chain
of the prodrugs. The difference in ClogD values for cyclic and acyclic prodrugs
incorporating alkyl chains of the same length is 3-4-log (with the cyclic prodrug being
more hydrophobic). Finally, the comparison of ClogD values in table 2.6 suggested that
the (L)-TyrNH-C
16
H
33
acyclic (S)-HPMPA and (S)-HPMPC prodrugs 2.38, 2.40 were
nearly as hydrophobic as the HDP- and ODE-prodrugs of (S)-HPMPA and (S)-HPMPC.

87
Table 2.6 Comparison of the calculated logD values for the tyrosine N-alkyl amide based
(S)-cHPMPA, (S)-HPMPA, (S)-cHPMPC and (S)-HPMPC prodrugs with the values of
parent (S)-HPMPA, (S)-cHPMPA, (S)-HPMPC and (S)-cHPMPC and the values of the
previously reported HDP- and ODE-prodrugs of (S)-HPMPC and (S)-HPMPA.
Prodrug ClogD (pH 6.5) ClogD (pH 7.4)
(S)-HPMPA, 2.2 -4.51 -4.59
(L)-Tyr-NHi-Bu-(S)-HPMPA, 2.28 -2.84 -2.74
(L)-Tyr-NHC
8
H
17
-(S)-HPMPA, 2.35 -1.12 -1.02
(L)-Tyr-NHC
12
H
25
-(S)-HPMPA, 2.36 0.57 0.66
(L)-Tyr-NHC
14
H
29
-(S)-HPMPA, 2.37 1.41 1.50
(L)-Tyr-NHC
16
H
33
-(S)-HPMPA, 2.38 2.25 2.34
(L)-Tyr-NHC
18
H
33
-(S)-HPMPA, 2.39 3.09 3.18

(S)-cHPMPA, 2.4 -3.43 -3.41
(L)-Tyr-NHi-Bu-(S)-cHPMPA, 2.26 0.85 0.87
(L)-Tyr-NHC
8
H
17
-(S)-cHPMPA, 2.29 2.57 2.59
(L)-Tyr-NHC
12
H
25
-(S)-cHPMPA, 2.30 4.25 4.27
(L)-Tyr-NHC
14
H
29
-(S)-cHPMPA, 2.31 5.09 5.11
(L)-Tyr-NHC
16
H
33
-(S)-cHPMPA, 2.32 5.93 5.95
(L)-Tyr-NHC
18
H
33
-(S)-cHPMPA, 2.33 6.77 6.79

(S)-HPMPC, 2.1 -4.60 -4.70
(L)-Tyr-NHC
16
H
33
-(S)-HPMPC, 2.40 2.16 2.23

(S)-cHPMPC, 2.3 -3.52 -3.52
(L)-Tyr-NHC
16
H
33
-(S)-cHPMPC, 2.34 4.33 5.14

ODE-(S)-HPMPC 2.99 2.99
HDP-(S)-HPMPC 2.22 2.22
ODE-(S)-HPMPA 3.08 3.10
HDP-(S)-HPMPA 2.32 2.33

The activities of the prodrugs 2.26, 2.28 - 2.40 and the parent drugs 2.1 - 2.4 were
evaluated against human cytomegalovirus (HCMV) in an in vitro plaque reduction assays
and are summarized in table 2.7. The prodrugs 2.26, 2.28, 2.29, 2.32, 2.38 were also
evaluated against cowpox and vaccinia viruses (Table 2.8). Additionally, all the
88
compounds were tested for cytotoxicity in two human cell lines, KB cells and normal
human fibroblasts (HFF) (Tables 2.7 and 2.8).

Table 2.7. In vitro antiviral activities against HCMV and cytotoxicities of tyrosine N-alkyl
amide prodrugs of (S)-HPMPA, (S)-cHPMPA, (S)-HPMPC, (S)-cHPMPC 2.26, 2.28 - 2.41.
a

Prodrug
Antiviral
activity,
IC
50
(μM)
Toxicity, (μM)
Selectivity
Index, (μM)
HCMV
(Towne strain)
KB cells HFF cells
(S)-HPMPA, 2.2 0.4 >100 >100 > 5 x 10
2

(L)-Tyr-NHi-Bu-(S)-HPMPA, 2.28 0.3 >100 nd > 2 x 10
2

(L)-Tyr-NHC
8
H
17
-(S)-HPMPA, 2.35 <0.1 >100 nd > 1 x 10
3

(L)-Tyr-NHC
12
H
25
-(S)-HPMPA, 2.36 4 x 10
-3
40 0.3 1 x 10
4
(L)-Tyr-NHC
14
H
29
-(S)-HPMPA, 2.37 1 x 10
-5
20 0.3 2 x 10
6

(L)-Tyr-NHC
16
H
33
-(S)-HPMPA, 2.38 2 x 10
-5
20 0.3 1 x 10
6

(L)-Tyr-NHC
18
H
37
-(S)-HPMPA, 2.39 1 x 10
-5
10 0.3 1 x 10
6

(S)-cHPMPA, 2.4 1.3 >100 >100 > 1 x 10
2

(L)-Tyr-NHi-Bu-(S)-cHPMPA, 2.26 0.6 >100 >100 > 1.7 x 10
2

(L)-Tyr-NHC
8
H
17
-(S)-cHPMPA, 2.29 0.3 100 nd > 1 x 10
3

(L)-Tyr-NHC
12
H
25
-(S)-cHPMPA, 2.30 2 x 10
-2
15 0.3 7.5 x 10
2

(L)-Tyr-NHC
14
H
29
-(S)-cHPMPA, 2.31 1 x 10
-4
15 0.3 1.5 x 10
5

(L)-Tyr-NHC
16
H
33
-(S)-cHPMPA, 2.32 6 x 10
-5
6 0.3 1 x 10
5

(L)-Tyr-NHC
18
H
37
-(S)-cHPMPA, 2.33 1 x 10
-5
10 0.3 1 x 10
6

(S)-HPMPC, 2.1 0.05 >100 >100 2 x 10
3

(L)-Tyr-NHC
16
H
33
-(S)-HPMPA, 2.40 1 x 10
-5
90 0.3 9 x 10
6

(S)-cHPMPC, 2.3 0.12 >100 >100 > 8.3 x 10
2

(L)-Tyr-NHC
16
H
33
-(S)-cHPMPC, 2.34 1 x 10
-4
15 0.3 1 x 10
5

HDP-(S)-HPMPC 2 x 10
-5
10 nd 5 x 10
5

a
Data obtained by Prof. Drach et al. at the University of Michigan

In these experiments, the Tyr amide ester prodrugs of (S)-HPMPA, (S)-cHPMPA,
(S)-HPMPC and (S)-cHPMPC exhibited antiviral potencies (IC
50
values) against HCMV
similar to or several logs better compared to the parent ANP drugs (Table 2.7). The
prodrugs having shorter alkyl chains of 4 to 8 atoms were not highly active, with IC
50
s of
89
0.6-0.1 μM. The most potent prodrugs contained longer N-alkyl amide chains, in
particular the C14, C16 and C18-alkyl groups, and exhibited reductions in infectious
virus (HCMV) with up to a 10,000-fold improvement at non-cytotoxic concentrations.
When compared to HDP-(S)-HPMPC, the tyrosine N-alkyl amide prodrugs 2.32, 2.33,
2.37 - 2.40 were found to be at least as potent (Table 2.7). Furthermore, (S)-HPMPA and
its Tyr N-alkyl amide prodrugs were active against strains of virus that are resistant to
Ganciclovir (GCV), an antiviral agent currently approved for the treatment of HCMV
(data not shown). Although the most active prodrugs were found to be the most cytotoxic
in both KB and HFF cells, they also possessed very high selectivity indices ranging from
10
2
to 10
6
μM, due to the significantly increased antiviral activity of these analogs (Table
2.7).

Table 2.8. In vitro antiviral activities against cowpox and vaccinia virus and cytotoxicities
of the tyrosine N-alkyl amide prodrugs of (S)-HPMPA and (S)-cHPMPA.
a

Prodrug
Antiviral activity,
IC
50
(μM)
Toxicity, (μM)
Cowpox Vaccinia KB cells HFF cells
(S)-HPMPA, 2.2 4 2 >100 >100
(L)-Tyr-NHi-Bu-(S)-HPMPA, 2.28 3 0.5 >100 Nd
(L)-Tyr-NHC
16
H
33
-(S)-HPMPA, 2.38 3 x 10
-3
<0.1 20 0.3

(S)-cHPMPA, 2.4 0.6 1 >100 >100
(L)-Tyr-NHi-Bu-(S)-cHPMPA, 2.26 7 4.5 >100 >100
(L)-Tyr-NHC
8
H
17
-(S)-cHPMPA, 2.29 3.0 1.5 >100 >100
(L)-Tyr-NHC
16
H
33
-(S)-cHPMPA, 2.32 6 x 10
-3
<0.1 6 0.3
a
Data obtained by Prof. Drach et al. at the University of Michigan

When evaluated against orthopox viruses, the prodrugs 2.26, 2.28, 2.29, 2.32, 2.38 were
active against cowpox and vaccinia viruses with IC
50
values varying over a range of
90
7-0.003 μM (Table 2.8), with the N-hexadecyl tyrosine amide prodrugs 2.32 and 2.38
showing up to 3-logs better activity than the parent (S)-HPMPA. Although, the long
chain conjugates 2.32 and 2.38 showed increased potency against orthopox viruses, the
magnitude of the enhancement over the activity of the parent ANPs 2.2 and 2.4 was
generally less than that seen with the herpesviruses, particularly HCMV.

In summary, the third round of structure-activity relationship studies clearly showed that
the antiviral activities of the tyrosine N-alkyl amide (S)-HPMPA and (S)-HPMPC
prodrugs against HCMV, cowpox and vaccinia viruses strongly depend on the length of
the alkyl chain attached to the tyrosine amide promoiety with optimal activity obtained at
16-18 atoms. Surprisingly, the tyrosine N-alkyl amide prodrugs of the cyclic and acyclic
ANPs were equally active when evaluated in vitro against cytomegalovirus and orthopox
viruses (Tables 2.7 and 2.8) despite the 3-4-logs difference in their hydrophobicities
(ClogD values, Table 2.6). The significantly increased potency against HCMV resulting
from a single variation in the tyrosine N-alkyl amide promoiety is hypothesized to reflect
a virus-dependent, highly efficient mechanism of prodrug activation within the cell in
addition to enhanced cell membrane permeability due to the presence of a lipophilic alkyl
group in the tyrosine N-alkyl amide promoiety.

2.6 Conclusions
Initially, 24 mono amino acid ANP prodrugs were designed and investigated with the
aims: 1) to define the structural effects on the interactions of the peptidomimetic
91
phosphonate prodrugs with hPEPT1, and 2) to identify the promoiety, which upon
conjugation to the ANPs, produces prodrugs able not only to withstand the rigors of
metabolism, but also to efficiently release the active parent drug at the target site in vivo.
Thus, after evaluation of the serine-based (S)-HPMPA prodrugs 2.12 - 2.14 for hPEPT1-
mediated transport in the electrophysiologic Xenopus laevis oocytes assay, it was
concluded that the single amino acid prodrugs were not transported by hPEPT1, most
likely due to the presence of a phosphonate ester group. In order to satisfy the second
objective of the present project, three consecutive rounds of SAR studies involving the
“tuning” of the P-X-C linkage, the amino acid stereochemistry and the C-terminal
functional group, and optimizing the length of alkyl chain in the tyrosine N-alkyl amide
moiety were performed. The first conclusion that followed from these studies is that the
tyrosine amino acid was the most favorable single amino acid promoiety, owing to the
high chemical stability and the efficient activation of the resulting tyrosine-based
prodrugs. Second, the enzymatic stability of the tyrosine promoiety was significantly
increased by replacement of the carboxyl ester group with an N-alkyl amide moiety.
Thus, the tyrosine N-alkyl amide-based prodrugs 2.26, 2.27 undergo the same
metabolism and exhibit identical stabilities in enzymatic and non-enzymatic media.
Third, the highest antiviral activities (3-4-log increase in activity compared to the parent
ANP) of the tyrosine N-alkyl amide (S)-HPMPA and (S)-HPMPC prodrugs against
HCMV, cowpox and vaccinia virus were achieved by incorporation of the hexadecyl
group (C
16
H
33
) into the tyrosine N-alkyl moiety (prodrugs 2.32, 2.34, 2.38, 2.40). Thus,
the tyrosine N-hexadecyl amide was identified as a promising single amino acid
92
promoiety scaffold for further ANP prodrug development, surpassing previously reported
ethylene glycol-linked amino acid and dipeptide promoieties.

2.7 Experimental section

Chemical and enzymatic stability of the single amino acid prodrugs 2.12, 2.15 - 2.17,
2.23 – 2.27
pH-Dependent phosphate buffer stability. Compounds 2.12, 2.15 - 2.17, 2.23 – 2.27
were incubated in 200 mM phosphate buffer at pH 6.5 and 7.4 at 37 ºC in a water bath.
The incubation mixture was prepared by dissolving 1 mg/mL stock solution of
compounds 2.12, 2.15 - 2.17, 2.23 – 2.27 in H
2
O in preheated buffer solutions (1 mL).
Aliquots of 25 μL were withdrawn at appropriate time intervals and analyzed by LC-MS.
LC-MS analysis of compounds 2.12, 2.15 - 2.17, 2.23 – 2.27 was performed on a
C18 HPLC column (5 μm, 250 mm × 4.6 mm) with a 0-30% CH
3
CN gradient in 60 mM
ammonium acetate buffer, pH 5.5, at a flow rate of 1.0 mL/min. MS parameters were
optimized as follows: sheath gas (N
2
) flow rate 20 arb, I spray voltage 5 kV, capillary
temperature 275 °C, capillary voltage 35 V, tube lens offset 55 V. Full scan mass spectra
were recorded over a range of m/z 200-1400. The UV detector was operated at 274 or
260 nm for (S)-HPMPC or (S)-HPMPA derivatives, respectively. Stabilities of the
prodrugs 2.23 – 2.25 and 2.27 were evaluated by Dr. Michaela Serpi.

93
Stability of prodrugs in rat tissue intestinal homogenates at pH 6.5. The enzymatic
stability of compounds 2.12, 2.15, 2.16, 2.23 – 2.27 was investigated by exposing the
compound to rat tissue intestinal homogenates, provided by TSRL Inc. The incubation
mixture for enzymatic stability study of compounds 2.12, 2.15, 2.16, 2.23 – 2.27 was
prepared by dissolving 400 μL of 1 mg/mL stock solution in 260 μL of phosphate buffer
at pH 6.5 and 140 μL of intestinal homogenate. The mixture was incubated at 37 ºC in a
water bath. Samples (25 μL) were drawn at appropriate intervals mixed with methanol
(25 μL) and centrifuged (2 min at 6000 rpm). Aliquots of 25 μL of the supernatant liquid
were withdrawn at appropriate time intervals and analyzed by LC-MS using conditions
described in the previous section.

Antiviral activity and cytotoxicity evaluation of the single amino acid prodrugs 2.12,
2.15 - 2.17, 2.23 – 2.40
Antiviral and cytotoxicity assays. All antiviral activity and cytotoxicity assays were
conducted by Julie Breitenbach and Kathy Borysko under the supervision of Prof. John
Drach and the University of Michigan as part of a collaborative effort funded by the
National Institutes of Health. The data was obtained using previously described
experimental procedure.
28

94
2.8 References
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4. Charrier, L.; Merlin, D. The oligopeptide transporter hPepT1: gateway to the
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5. Ganapathy, M. E.; Brandsch, M.; Prasad, P. D.; Ganapathy, V.; Leibach, F. H.
Differential recognition of Î²-lactam antibiotics by intestinal and renal peptide
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6. Wenzel, U.; Gebert, I.; Weintraut, H.; Weber, W.-M.; Clauss, W.; Daniel, H.
Transport characteristics of differently charged cephalosporin antibiotics in oocytes
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7. Bretschneider, B.; Brandsch, M.; Neubert, R. Intestinal transport of Î²-lactam
antibiotics: analysis of the affinity at the H+/peptide symporter (PEPT1), the uptake into
Caco-2 cell monolayers and the transepithelial flux. Pharm. Res. 1999, 16, 55-61.

8. Boll, M.; Markovich, D.; Weber, W. M.; Korte, H.; Daniel, H.; Murer, H.
Expression cloning of a cDNA from rabbit small intestine related to proton-coupled
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9. Temple, C. S.; Boyd, C. A. R. Proton-coupled oligopeptide transport by rat renal
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10. Zhu, T.; Chen, X.-Z.; Steel, A.; Hediger, M. A.; Smith, D. E. Differential
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11. Doring, F.; Walter, J.; Will, J.; Focking, M.; Boll, M.; Amasheh, S.; Clauss, W.;
Daniel, H. Delta-aminolevulinic acid transport by intestinal and renal peptide transporters
and its physiological and clinical implications. J. Clin. Invest. 1998, 101, 2761-2767.

12. Nielsen, C. U.; Brodin, B. Di/tri-peptide transporters as drug delivery targets:
Regulation of transport under physiological and patho-physiological conditions. Curr.
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13. Antman, M. D.; Gudmundsson, O. S. Valacyclovir: a prodrug of acyclovir.
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Valacyclovir: a substrate for the intestinal and renal peptide transporters PEPT1 and
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15. Sinko, P. J.; Balimane, P. V. Carrier-mediated intestinal absorption of
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anions and organic cations in rats. Biopharm. Drug Dispos. 1998, 19, 209-217.

16. Sawada, K.; Terada, T.; Saito, H.; Hashimoto, Y.; Inui, K.-I. Recognition of L-
amino acid ester compounds by rat peptide transporters PEPT1 and PEPT2. J.
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17. Balimane, P. V.; Tamai, I.; Guo, A.; Nakanishi, T.; Kitada, H.; Leibach, F. H.;
Tsuji, A.; Sinko, P. J. Direct evidence for peptide transporter (PepT1)-mediated uptake of
a nonpeptide prodrug, valacyclovir. Biochem Biophys Res Commun 1998, 250, 246-51.

18. Ezra, A.; Hoffman, A.; Breuer, E.; Alferiev, I. S.; Moenkkoenen, J.; El Hanany-
Rozen, N.; Weiss, G.; Stepensky, D.; Gati, I.; Cohen, H.; Toermaelehto, S.; Amidon, G.
L.; Golomb, G. A Peptide Prodrug Approach for Improving Bisphosphonate Oral
Absorption. J. Med. Chem. 2000, 43, 3641-3652.

19. Shu, C.; Shen, H.; Hopfer, U.; Smith, D. E. Mechanism of intestinal absorption
and renal reabsorption of an orally active ACE inhibitor: uptake and transport of
fosinopril in cell cultures. Drug Metab. Dispos. 2001, 29, 1307-1315.

20. Larsen, S. B.; Jorgensen, F. S.; Olsen, L. QSAR Models for the Human
H+/Peptide Symporter, hPEPT1: Affinity Prediction Using Alignment-Independent
Descriptors. J. Chem. Inf. Model. 2008, 48, 233-241.
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21. Pedretti, A.; De, L. L.; Marconi, C.; Negrisoli, G.; Aldini, G.; Vistoli, G.
Modeling of the intestinal peptide transporter hPepT1 and analysis of its transport
capacities by docking and pharmacophore mapping. ChemMedChem 2008, 3, 1913-1921.

22. Winiwarter, S.; Hilgendorf, C. Modeling of drug-transporter interactions using
structural information. Curr. Opin. Drug Discovery Dev. 2008, 11, 95-103.

23. Kottra, G.; Frey, I.; Daniel, H. Inhibition of intracellular dipeptide hydrolysis
uncovers large outward transport currents of the peptide transporter PEPT1 in Xenopus
oocytes. Pfluegers Arch. 2009, 457, 809-820.

24. Foley, D.; Pieri, M.; Pettecrew, R.; Price, R.; Miles, S.; Lam, H. K.; Bailey, P.;
Meredith, D. The in vitro transport of model thiodipeptide prodrugs designed to target the
intestinal oligopeptide transporter, PepT1. Org. Biomol. Chem. 2009, 7, 3652-3656.

25. Eriksson, U.; Hilfinger, J. M.; Kim, J.-S.; Mitchell, S.; Kijek, P.; Borysko, K. Z.;
Breitenbach, J. M.; Drach, J. C.; Kashemirov, B. A.; McKenna, C. E. Synthesis and
biological activation of an ethylene glycol-linked amino acid conjugate of cyclic
cidofovir. Bioorg. Med. Chem. Lett. 2007, 17, 583-586.

26. Peterson, L. W. Peptidomimetic prodrugs of cidofovir: Design, synthesis,
transport, mechanism of activation, and antiviral activity. Ph. D. Thesis, University of
Southern California, Los Angeles, 2009.

27. Eriksson, U.; Peterson, L. W.; Kashemirov, B. A.; Hilfinger, J. M.; Drach, J. C.;
Borysko, K. Z.; Breitenbach, J. M.; Kim, J. S.; Mitchell, S.; Kijek, P.; McKenna, C. E.
Serine Peptide Phosphoester Prodrugs of Cyclic Cidofovir: Synthesis, Transport, and
Antiviral Activity. Mol. Pharmaceutics 2008, 5, 598-609.

28. Zakharova, V. M.; Serpi, M.; Krylov, I. S.; Peterson, L. W.; Breitenbach, J. M.;
Borysko, K. Z.; Drach, J. C.; Collins, M.; Hilfinger, J. M.; Kashemirov, B. A.; McKenna,
C. E. Tyrosine-based 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine and -
adenine ((S)-HPMPC and (S)-HPMPA) prodrugs: Synthesis, stability, antiviral activity,
and in vivo transport studies. J. Med. Chem. 2011, 54, 5680-5693.

29. Peterson, L. W.; Sala-Rabanal, M.; Krylov, I. S.; Serpi, M.; Kashemirov, B. A.;
McKenna, C. E. Serine Side Chain-Linked Peptidomimetic Conjugates of Cyclic
HPMPC and HPMPA: Synthesis and Interaction with hPEPT1. Mol. Pharmaceutics
2010, 7, 2349-2361.

30. McKenna, C. E.; Kashemirov, B. A.; Eriksson, U.; Amidon, G. L.; Kish, P. E.;
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Organomet. Chem. 2005, 690, 2673-2678.

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Borysko, K.; Drach, J. C.; Kashemirov, B. A.; McKenna, C. E. Synthesis, transport and
antiviral activity of Ala-Ser and Val-Ser prodrugs of cidofovir. Bioorg. Med. Chem. Lett.
2011, 21, 4045-4049.

32. Knuetter, I.; Wollesky, C.; Kottra, G.; Hahn, M. G.; Fischer, W.; Zebisch, K.;
Neubert, R. H. H.; Daniel, H.; Brandsch, M. Transport of angiotensin-converting enzyme
inhibitors by H+/peptide transporters revisited. J. Pharmacol. Exp. Ther. 2008, 327, 432-
441.

33. Brandl, M.; Flaten, G. E.; Bauer-Brandl, A. Passive diffusion across membranes.
In Wiley Encyclopedia of Chemical Biology, John Wiley & Sons, Inc.: 2009; Vol. 3, pp
541-550.

34. Gordon, M.; Notaro, V. A.; Griffin, C. E. Phosphonic acids and esters. VII.
Intramolecular electrophilic catalysis of phosphonate ester hydrolysis by neighboring
carboxyl. J. Am. Chem. Soc. 1964, 86, 1898-9.

35. Lazarus, R. A.; Benkovic, P. A.; Benkovic, S. J. Mechanism of hydrolysis of
phosphorylethanolamine diesters. Intramolecular nucleophilic amine participation. J.
Chem. Soc., Perkin Trans. 2 1980, 373-9.

36. Lazarus, R. A.; Benkovic, S. J. Mechanism of hydrolysis of
phosphorylethanolamine triesters. Multiple catalytic effects of an intramolecular amino
group. J. Am. Chem. Soc. 1979, 101, 4300-12.

37. Kluger, R.; Davis, P. P.; Adawadkar, P. D. Mechanism of urea participation in
phosphonate ester hydrolysis. Mechanistic and stereochemical criteria for enzymic
formation and reaction of phosphorylated biotin. J. Am. Chem. Soc. 1979, 101, 5995-
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38. Lieske, C. N.; Miller, E. G., Jr.; Zeger, J. J.; Steinberg, G. M. Participation of a
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1966, 88, 188-9.

39. Hudson, R. F.; Keay, L. Hydrolysis of phosphonate esters. J. Chem. Soc. 1956,
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40. Cox, J. R., Jr.; Ramsay, O. B. Mechanisms of nucleophilic substitution in
phosphate esters. Chem. Rev. 1964, 64, 317-52.

41. Guichard, G.; Benkirane, N.; Zeder-Lutz, G.; Van, R. M. H. V.; Briand, J.-P.;
Muller, S. Antigenic mimicry of natural L-peptides with retro-inversopeptidomimetics.
Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 9765-9.
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42. Benkirane, N.; Friede, M.; Guichard, G.; Briand, J. P.; Van, R. M. H. V.; Muller,
S. Antigenicity and immunogenicity of modified synthetic peptides containing D-amino
acid residues. Antibodies to a D-enantiomer do recognize the parent L-hexapeptide and
reciprocally. J. Biol. Chem. 1993, 268, 26279-85.

43. Jenh, A. M.; Thio, C. L.; Pham, P. A. Tenofovir for the treatment of hepatitis B
virus. Pharmacotherapy 2009, 29, 1212-1227.

44. Shaw, J.-P.; Sueoka, C. M.; Oliyai, R.; Lee, W. A.; Arimilli, M. N.; Kim, C. U.;
Cundy, K. C. Metabolism and pharmacokinetics of novel oral prodrugs of 9-[(R)-2-
(phosphonomethoxy)propyl]adenine (PMPA) in dogs. Pharm. Res. 1997, 14, 1824-1829.
45. Chicca, A.; Raduner, S.; Pellati, F.; Strompen, T.; Altmann, K.-H.; Schoop, R.;
Gertsch, J. Synergistic immunomopharmacological effects of N-alkylamides in
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46. Matthias, A.; Penman, K. G.; Matovic, N. J.; Bone, K. M.; De, V. J. J.; Lehmann,
R. P. Bioavailability of Echinacea constituents: Caco-2 monolayers and pharmacokinetics
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47. Matthias, A.; Gillam, E. M. J.; Penman, K. G.; Matovic, N. J.; Bone, K. M.; De,
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48. Wan, W. B.; Beadle, J. R.; Hartline, C.; Kern, E. R.; Ciesla, S. L.; Valiaeva, N.;
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Agents Chemother. 2005, 49, 656-662.

99
CHAPTER 3
*

Synthesis of the single amino acid (S)-HPMPA and (S)-HPMPC
prodrugs

3.1 Introduction
As part of the prodrug approach development process (see Chapter 2), it was necessary to
identify a leading single amino acid promoiety scaffold, which, upon conjugation to the
ANPs, produces prodrugs able not only to withstand the rigors of metabolism, but also to
efficiently release the active parent drug at the target site in vivo. In order to accomplish
this task, we performed three consecutive rounds of structure-activity relationship (SAR)
studies that involved the evaluation of 24 (S)-HPMPA and (S)-HPMPC prodrugs,
incorporating various single amino acids with different stereochemistry (L vs. D) and C-
terminal functional groups (Figure 3.1, prodrugs 2.12 - 2.17 and 2.23 - 2.40). The
rationale behind this prodrug design for the SAR studies, along with a discussion of the
obtained results (prodrug stabilities, in vitro antiviral activities and oral bioavailabilities)
are reported in Chapter 2, whereas syntheses and structural characterization of the
evaluated single amino acid prodrugs and of the parent (S)-HPMPA and (S)-HPMPC are
described in the present chapter. Elucidation of stereochemistry of the cyclic
phosphonate prodrugs is reported in Chapter 4.

*
Sections 3.3 and 3.6 contain excerpts reproduced with permission from Peterson, L. W.; Sala-Rabanal,
M.; Krylov, I. S.; Serpi, M.; Kashemirov, B. A.; McKenna, C. E. Mol. Pharmaceutics 2010, 7, 2349-2361;
and from Zakharova, V. M.; Serpi, M.; Krylov, I. S.; Peterson, L. W.; Breitenbach, J. M.; Borysko, K. Z.;
Drach, J. C.; Collins, M.; Hilfinger, J. M.; Kashemirov, B. A.; McKenna, C. E. J. Med. Chem. 2011, 54,
5680-5693. Copyright 2012 American Chemical Society.
100

Figure 3.1 Structures of (S)-HPMPC 2.1, (S)-HPMPC 2.2 and the single amino acid prodrugs
2.12 - 2.17 and 2.23 - 2.40 synthesized for the SAR studies.

3.2 Synthesis of (S)-HPMPA and (S)-HPMPC
(S)-HPMPA 2.2 was originally synthesized by Holy et al. from the 9-(2,3-
dihydroxypropyl)adenine ((S)-DHPA) as depicted in general Scheme 3.1.
1
This method
required separation of isomers and ion-exchange chromatography/desalting, procedures
deemed impractical on a large scale.
2

Scheme 3.1 Original synthesis of (S)-HPMPA by Holy et al.
1

101
In order to obtain (S)-HPMPA 2.2 in our laboratory, the procedure for the synthesis of
(S)-HPMPA from bis-tritylated (S)-DHPA
3
was combined with the reaction sequence for
the synthesis of its cytosine analogue, (S)-HPMPC from (R)-gycidol.
4
As a result, our
synthesis of (S)-HPMPA included 5 steps (Scheme 3.2). The first step was tritylation of
(R)-glycidol 3.1 using trityl chloride in CH
2
Cl
2
/Et
3
N. Tritylated (S)-glycidol 3.2 (note:
change of R,S-nomenclature) was then reacted with adenine in the presence of anhydrous
K
2
CO
3
in dimethylformamide (DMF) at 105 ºC, selectively generating the 3’-O-tritylated
(S)-DHPA derivative 3.3 with 72% yield.

Scheme 3.2 Synthesis of (S)-HPMPA 2.2. Reagents and conditions: (a) Trityl chloride, CH
2
Cl
2,
TEA, rt, 20 h; (b) K
2
CO
3
, DMF, 105
o
C, 22 h; (c) 3.4, NaH, DMF, 0-5
o
C, 2.5 h; r.t., 12 h; (d)
80% aq. AcOH, 80 °C for 3h and r.t. for 12 h; (e) BTMS, DMF, r.t., overnight, H
2
O.

102
Intermediate 3.3 was alkylated with (toluenesulfonyloxy)methyl-diethylphosphonate 3.4
in the presence of NaH in DMF, giving crude phosphonate 3.5, which was subsequently
detritylated using 80% aqueous AcOH at 80 ºC. After purification by column
chromatography on silica gel, phosphonate 3.6 was de-esterified using the McKenna
reaction,
5
yielding pure (S)-HPMPA 2.2. This method produces (S)-HPMPA in 19%
overall yield from adenine, does not require isomer separations, ion-exchange
chromatography or desalting isolation procedures, and provides ready access to
(S)-HPMPA on a large scale. (S)-HPMPC 2.1 and (toluenesulfonyloxy)methyl-
diethylphosphonate 3.4 were synthesized using previously described methods.
4

3.3 Synthesis of the single amino acid prodrugs of (S)-HPMPA and (S)-HPMPC
The single amino acid (S)-HPMPA and (S)-HPMPC prodrugs can be classified according
to their structure into two categories: 1) cyclic phosphonate diesters 2.12 – 2.17, 2.23 –
2.27 and 2.29 – 2.34, and 2) acyclic phosphonate monoesters 2.28, 2.35 – 2.40 (Figure
3.1). The cyclic phosphonate diester and acyclic phosphonate monoester prodrugs were
synthesized using different routes as described below.

3.3.1 Synthesis of the cyclic (S)-HPMPA and (S)-HPMPC prodrugs
The cyclic phosphonate diester prodrugs have been synthesized using a solution-phase
method (Scheme 3.3, prodrugs 2.17, 2.23 - 2.27 and 2.29 – 2.34) and a solid-phase
approach (Scheme 3.5, prodrugs 2.12 - 2.16). The solution-phase procedure was
originally developed for the synthesis of the ethylene glycol-linked amino acid and
103
dipeptide (S)-HPMPC prodrugs.
6, 7
When adapted to the synthesis of single amino acid
prodrugs, this method included three steps (Scheme 3.3) and was used to obtain cysteine
methyl ester cHPMPA 2.17 and tyrosine (S)-cHPMPA and (S)-HPMPC prodrugs
2.23 - 2.27 and 2.29 – 2.34. The first two steps – intramolecular cyclization of the parent
ANP drug and subsequent esterification of the remaining P-OH group with the side chain
hydroxyl group of a tBoc-protected amino acid (3.7 – 3.17) – were accomplished in one
pot using a PyBOP-mediated coupling procedure (Scheme 3.3, steps i) and ii)). This
reaction was monitored by
31
P NMR. The last step was tBoc-deprotection of the amino
acid conjugated to the drug using trifluoroacetic acid (TFA) in dichloromethane (DCM).

Scheme 3.3 Conventional syntheses of mono amino acid cyclic (S)-HPMPA and (S)-HPMPC
prodrugs 3.7, 3.9 - 3.19. Reagents and conditions: i) and ii) PyBOP, DIEA, DMF, 40 ºC;
iii) TFA, CH
2
Cl
2
, r.t.

104
The final prodrugs were obtained in overall moderate to good yields (40-65%). Starting
tBOC-protected cysteine and tyrosine methyl esters 3.7 - 3.9 were commercially
available, the t-BOC-protected tyrosine N-alkyl amides 3.11 - 3.17 were synthesized from
the corresponding t-BOC protected carboxylic acid 3.44 using previously reported
procedure (Scheme 3.4).
8

Scheme 3.4 Synthesis of tBOC-tyrosine N-alkyl amides. Reagents and conditions: i) RNH
2
,
EDC, HOBt, CH
2
Cl
2
, 24 h, 25 ºC

Although the solution-phase approach is time-efficient, it requires tedious purification
(column chromatography at each step), making it less practical for large scale synthesis if
needed in the future. In order to address the scalability issue, a new solid-phase approach
to the mono amino acid and dipeptide cyclic phosphonate prodrugs was developed and is
outlined in Scheme 3.5.
6, 9
In contrast to the solution phase method, this strategy involved
the replacement of the tBoc-protecting group at the amino acid with a tritylchloride
polystyrene (TCP) resin. The cyclization and conjugation of the (S)-HPMPC 2.1 or
(S)-HPMPA 2.2 to the amino acid or dipeptides bound to the resin (3.18 - 3.22,
3.28, 3.29) were accomplished using the same conditions as in the solution-phase
approach (PyBOP, DIEA, DMF, 40 ºC).
105

Scheme 3.5 Solid-phase synthesis of the single amino acid and dipeptide prodrugs of cyclic
(S)-HPMPA and (S)-HPMPC (2.12 – 2.16, 3.32, 3.33). Reagents and conditions: i) PyBOP,
DIEA, DMF, 40 ºC; ii) TFA, CH
2
Cl
2
or HCl, dioxane, 25 ºC.

After completion of the coupling reaction, all the byproducts were washed, leaving the
product on the resin. This can be visualized by
31
P gel NMR (Figure 3.2). The two peaks
in the NMR spectrum correspond to the two different diastereomers. Finally, the
products 2.12 - 2.16, 3.32, 3.33 were cleaved from the resin using either TFA in DCM or
HCl in dioxane, and were subsequently crystallized from a saturated methanolic solution
by precipitation with diethyl ether.

106

Figure 3.2 Gel
31
P NMR of (L)-SerOMe bound to the TCP-resin (3.23).

The solid-phase strategy produced the single amino acid prodrugs 3.3 - 3.6, 3.8 and the
dipeptide conjugates 3.32, 3.33 in 20-35% overall yield from (S)-HPMPA or
(S)-HPMPC, and provided the following advantages over the solution-phase prodrug
synthesis: easy product purification (column chromatography is not required), scalability
and potential for automation, if needed in the future.

For the solid-phase approach, it was necessary to immobilize the amino acids and
dipeptides to the TCP-resin exclusively via the amino group. Therefore, the side chain
hydroxyl group of the mono amino acids 3.18 - 3.22 was first protected with a tert-butyl
dimethyl silyl (TBDMS) group by stirring 1 eq. of the amino acid with 2 eq. of
TBDMSCl and 3 eq. of imidazole in CH
2
Cl
2
at 25 ºC overnight (Scheme 3.6, step i).
10

Interestingly, when DMF
11
was used as a solvent in this reaction instead of CH
2
Cl
2
, the
corresponding N-formyl derivatives of TBDMS-protected amino acids were formed
(Scheme 3.6, step vi, compounds 3.44 and 3.45). Structures of 3.44 and 3.45 were
confirmed by
1
H and
13
C NMR spectroscopy.
107

Scheme 3.6 Immobilization of the mono amino acid esters 3.34 - 3.38 to TCP-resin. Reagents
and conditions: i) 2 eq. TBDMSCl, 3 eq. imidazole, CH
2
Cl
2
, 25 ºC, 24 h; ii) TCP-resin, DIEA,
CH
2
Cl
2
, 25 ºC, 18 h; iii) TBAF, THF, 25 ºC, 5 h; iv) 2 eq. TBDMSCl, 3 eq. imidazole, DMF,
25 ºC, 24 h.

According to the literature, the N-formylation under such conditions proceeds through a
Vilsmeier-type reagent formed by reaction of TBDMSCl and DMF (Scheme 3.7).
12

Scheme 3.7 Formation of N-formyl derivatives 3.44 and 3.45 via a Vilsmeier-type reagent.
Reagents and conditions: i) imidazole, DMF, 18 h, 25 ºC; ii) H
2
O.
12

The resulting TBDMS-protected amino acid 3.39 - 3.43 were reacted with the TCP-resin
in the presence of N,N-diisopropylethylamine (DIEA).
13
Finally, the TBDMS-protecting
group was removed using tetra-n-butylammonium fluoride (TBAF) in THF at 25 ºC,
yielding the amino acids connected to the resin via their NH
2
-group (Scheme 3.6). The
(L)-Serine methyl ester bound to the resin 3.18 was visualized using IR spectroscopy.
108
The characteristic peak at 1735 cm
-1
corresponding to the carboxyl ester group was
observed (Figure 3.3, right).

Figure 3.3 IR spectra of the free TCP-resin (left) and of the (L)-SerOMe bound resin 3.18 (right).
The samples were analyzed in triplicate (KBr pellet).

The dipeptides used as promoieties in the prodrug syntheses contained a modified C-
terminal group and a free amino group, therefore, they were assembled on a solid support
in the N → C direction, which is reverse compared to typical solid-phase peptide
synthesis (SPPS).
14
The solid-phase syntheses of target dipeptides 3.28 and 3.29 bound
to the TCP-resin was accomplished in 4 steps as outlined in Scheme 3.8.
15
First, (L)-
Valine allyl ester was anchored to the TCP-resin via amino group. Then the allyl-
protecting group was orthogonally removed under neutral conditions using phenylsilane
(PhSiH
3
) in the presence of palladium-catalyst, Pd(PPh
3
)
4
.
16

1735 cm
-1

1735 cm
-1

No peaks at 1700-1800 cm
-1

109

Scheme 3.8 Solid-phase synthesis of the dipeptides bound to the TCP-resin 3.28 and 3.29.
Reagents and conditions: i) DIEA, CH
2
Cl
2
; ii) Pd(PPh
3
)
4
, PhSiH
3
, CH
2
Cl
2
; iii) 3.34 or 3.36,
PyBOP, DIEA, CH
2
Cl
2
, DMF; iv) TBAF, THF.

In the next step, the (L)-Valine bound to the resin was coupled to TBDMS-protected (L)-
serine esters 3.34, 3.36 using PyBOP as a coupling reagents. Finally, the TBDMS-
protecting group was removed using TBAF in THF at room temperature, giving the target
dipeptides 3.28 and 3.29 connected to the resin via their NH
2
-groups.

3.3.2 Synthesis of the acyclic phosphonate ester prodrugs 2.28, 2.35 - 2.40
Synthesis of the acyclic phosphonate monoester prodrugs 2.28, 2.35 - 3.40 directly from
(S)-HPMPC 2.1 and (S)-HPMPA 2.2 using the PyBOP-coupling procedure is challenging
due to the rapid intramolecular esterification of the phosphonic acid group by the 3’-OH
group, resulting in the formation of the cyclic phosphonates, cHPMPC 2.3 or cHPMPA
2.4 (Scheme 3.9, R = H). Protection of the ANP’s 3’-hydroxyl group with a trityl group
and subsequently attempting to selectively esterify one of the two P-OH groups with the
amino acid side-chain hydroxyl group using the PyBOP-mediated coupling procedure
also failed to produce the desired product, due to the formation of a stable benzotriazolyl
phosphonate monoester intermediate 3.47, that was clearly observed by
31
P NMR (δ ≈ 19
ppm) and confirmed by mass-spectrometry (Scheme 3.9, R = CPh
3
). Most likely, the
110
negatively charged second P-OH group protects phosphorus from the incoming
nucleophile, thus preventing further re-esterification of the intermediate.
17

Scheme 3.9 Attempts to synthesize tyrosine-based acyclic phosphonate monoester prodrugs from
(S)-HPMPA. Reagents and conditions: i) PyBOP, DIEA, DMF, 40 ºC; ii) 3.12, PyBOP, DIEA,
DMF, 40 ºC.

Since initial attempts to synthesize acyclic phosphonate monoester prodrugs directly from
(S)-HPMPA or (S)-HPMPC failed, another approach, based on the hydrolysis of the
cyclic phosphonate diester prodrugs in basic media, was utilized. Thus, the tyrosine
acyclic phosphonate monoester prodrugs 2.28, 2.35 - 2.40 were synthesized via
hydrolysis of their cyclic counterparts 2.26, 2.29 - 2.34 in an aqueous NH
4
OH-ACN
mixture, at 45 ºC with 40-60% yields (Scheme 3.10). Completion of the prodrug
hydrolysis was monitored by
31
P NMR.

Scheme 3.10 Synthesis of the single amino acid acyclic (S)-HPMPA and (S)-HPMPC prodrugs
2.28, 2.35 - 2.40. Reagents and conditions: i) aq. NH
4
OH, ACN, 45 ºC.
111
In an effort to optimize selective cleavage of the endocyclic P-O-ester bond of the
tyrosine cyclic ANP prodrugs, we initially utilized a model compound, phenyl ester of
cyclic (S)-HPMPA 3.48, and studied its hydrolysis under various conditions (aq. NH
4
OH,
40 °C, 2 M aq. NaOH, r.t.; 1 M t-BuOK/DMSO, r.t.; H
2
O, 100 ºC). In these conditions
the phenyl ester of cyclic (S)-HPMPA 3.48 produced either a mixture of cHPMPA 2.4
and of the phenyl ester of acyclic (S)-HPMPA 3.49 or exclusively cHPMPA (Scheme
3.11). To this end, it was determined that bromotrimethylsilane (BTMS) silylation-
dealkylation
5
in acetonitrile regioselectively removes endocyclic alkyl ester group with
nearly quantitative yield (according to
31
P NMR analysis), but generates 3’-bromo-
derivative 3.50 (Scheme 3.11).

Scheme 3.11 BTMS silylation-dealkylation of Ph-cHPMPA 3.48. Reagents and conditions: i) aq.
NH
4
OH, 40 °C; ii) 2 M aq. NaOH, r.t.; iii) 1 M t-BuOK/DMSO, r.t.; iv) H
2
O, 100 ºC; v) BTMS,
ACN, reflux, 18 h; vi) MeOH; vii) 50 mM aq. K
2
CO
3
, 100 ºC.

Subsequent attempts to hydrolyze the bromine under basic conditions using 50 mM
aqueous K
2
CO
3
at 100 ºC (at 25 ºC no reaction was observed) and to selectively obtain
the phenyl ester of (S)-HPMPA 3.49 failed due to rapid intramolecular nucleophilic
substitution of the bromine by the free P-OH group resulting in formation of the starting
112
cyclic phosphonate ester 3.48. This product immediately hydrolyzed under those
conditions and released cHPMPA and phenyl ester of acyclic (S)-HPMPA. Although the
BTMS dealkylation-silylation could not be utilized for the synthesis of the tyrosine
acyclic prodrugs from their cyclic counterparts, it found an application in preparation of
the tyrosine prodrugs of PMEA and PMPA.
18
Also, it was used in the reaction sequence
to obtain the tyrosine prodrug of 3’-thio (S)-HPMPA 3.52 (Scheme 3.12).

3.4 Synthesis of thio-(S)-HPMPA prodrugs
(L)-TyrNH-i-Bu prodrug of acyclic 3’-thio-(S)-HPMPA 3.52 was obtained as outlined in
scheme 3.12. First, the corresponding tyrosine cyclic (S)-HPMPA prodrug 2.26 was
treated with BTMS in acetonitrile to generate the bromo-derivative 3.51. On the next
step, the bromine was substituted with thio-group using NaSH hydrate.
19
The reaction
was monitored by LC-MS and once completed the product 3.52 was purified using
preparative RP-HPLC.

Scheme 3.12 Synthesis of (L)-TyrNH-i-Bu-SH-HPMPA prodrugs 3.52. Reagents and conditions:
i) BTMS, ACN, reflux, 18 h; ii) MeOH; iii) NaHS, H
2
O, r.t.

Synthesis of another thio-(S)-HPMPA prodrug, particularly thio cyclic (S)-HPMPA 3.54,
was accomplished as outlined in scheme 3.13. First, (S)-HPMPA was conjugated to
113
benzyl mercaptan using the PyBOP-mediated coupling procedure with yield. The
reaction was monitored by
31
P NMR. On the next step, obtained thio ester 3.53 was
dealkylated by treatment with thiophenoxide anion generated in situ (C
6
H
5
SH in
Et
3
N/dioxane)
20
giving thio cyclic (S)-HPMPA 3.54. The final product 3.54 was purified
using preparative RP-HPLC.

Scheme 3.13 Synthesis of thio cyclic (S)-HPMPA 3.54. Reagents and conditions: i) PyBOP,
DIEA, DMF, 40 ºC; ii) C
6
H
5
SH, Et
3
N/dioxane, r.t.
20

3.5 Conclusions
Twenty-four novel (S)-HPMPA and (S)-HPMPC prodrugs incorporating Serine,
Threonine, Tyrosine or Cysteine amino acids were synthesized. The cyclic phosphonate
prodrugs 3.3 - 3.19 were obtained using a solution-phase method and a newly developed
solid-phase approach, involving the replacement of the tBoc-protecting group at the
amino acid with TCP-resin. Although the solid-phase approach produced the prodrugs in
a lower yield (26-35%) compared to the conventional method (40-65%), it required
simpler purification (only washing as opposed to column chromatography), and can be
easily scaled up and automated in the future. The tyrosine acyclic phosphonate prodrugs
3.20 - 3.26 were obtained by hydrolysis of their cyclic counterparts 3.13 - 3.19 in an
114
aqueous NH
4
OH-ACN mixture at 45 ºC with yields 40-60%. Additionally, two
thio-(S)-HPMPA prodrugs 3.52 and 3.54 were synthesized to study their antiviral
potencies. The structures of all compounds were confirmed by
1
H and
31
P NMRs and by
HR-MS and purities of all compounds were verified by LC-MS analysis.

3.6 Experimental Section
General Experimental Methods.
1
H,
13
C and
31
P NMR spectra were recorded on 250,
400 and 500 MHz spectrometers. Chemical shifts (δ) are reported in parts per million
(ppm) relative to internal CH
3
OH (
1
H NMR, δ = 3.34;
13
C NMR, δ = 49.86); CHCl
3
(
1
H
NMR, δ = 7.26) or external 85% H
3
PO
4
(
31
P NMR, δ = 0.00).
31
P NMR spectra were
proton-decoupled, and
1
H and
13
C coupling constants (J values) are given in Hz. The
following NMR abbreviations are used: s (singlet), d (doublet), m (unresolved multiplet),
dd (doublet of doublets), ddd (doublet of doublet of doublet), br (broad signal). LC-MS
analysis of compounds 3.3 - 3.26 was performed on a mass spectrometer in positive ion
mode (ESI), equipped with PDA detector and HPLC solvent delivery system. HPLC
separations were performed on a C18 HPLC column (5 μm, 250 mm × 4.6 mm) with a
0-30% CH
3
CN gradient or 35% or 85% CH
3
CN isocratic in 60 mM ammonium acetate
buffer, pH 5.5, at a flow rate of 1.0 mL/min. MS parameters were optimized as follows:
sheath gas (N
2
) flow rate 20 arb, I spray voltage 5 kV, capillary temperature 275 °C,
capillary voltage 35 V, tube lens offset 55 V. Full scan mass spectra were recorded over
a range of m/z 200-1400. The UV detector was operated at 274 or 260 nm for (S)-
HPMPC or (S)-HPMPA derivatives, respectively. Compounds 2.12 - 2.17, 2.23 - 2.27
115
and 2.29 - 2.34 were obtained as diastereomeric mixtures. The ratio of diastereomers was
determined based on
31
P NMR. Where possible, the characteristic parameters of the
signals of S
p
and R
p
isomers are described separately. The assignment of the signals in
1
H and
13
C NMR was done based on the analysis of coupling constants and additional
two-dimensional experiments (COSY, HSQC). The >95% purity of the final compounds
2.12 - 2.17 and 2.23 – 2.40 was confirmed using HPLC analysis and UV determinations
of the active compound content used the following extinction coefficients: (S)-HPMPA
derivatives (ε = 14019 at 260 nm in EtOH, ε = 14191 at 260 nm, pH 7.0), (S)-HPMPC
prodrugs (ε = 8362 at 274 nm in EtOH, ε = 9000 at 274 nm, pH 7.0), and tyrosine
(ε = 612 at 260 nm and ε = 667 at 274 nm, pH 7.0).

Synthesis of (S)-HPMPA
Synthesis of (S)-2-Trityloxymethyl-oxirane (3.2). Trityl chloride (36.96 g, 0.133 mol,
1.1 equiv) was dissolved in 200 mL CH
2
Cl
2
and cooled to 0 °C under N
2
. TEA (18.5 mL,
0.133 mol, 1.1 equiv) was added dropwise to the solution followed by the addition of
8.85 g R-(+)-glycidol (97% ee) 3.1 (0.119 mol, 1.0 equiv) after 10 min. The reaction was
stirred at room temperature for 20 h before being quenched by addition of 100 mL sat.
NH
4
Cl solution and 30 mL of H
2
O. The organic layer was concentrated under vacuum,
and 100 mL EtOH was added to initiate precipitation of the product, (S)-tritylglycidol 3.2
as a white crystalline solid (33.68 g, yield 89%).
1
H NMR (400 MHz, CDCl
3
): δ 7.47-
7.45 (m, 6H, aromatic), 7.32-7.22 (m, 9H, aromatic), 3.32 (dd, 1H, J = 9.8, 2.2 Hz), 3.17-
3.11 (m, 2H), 2.79-2.76 (m, 1H), 2.62 (dd, J = 5.3, 2.4 Hz, 1H).
116
Synthesis of 1-(6-Amino-purin-9-yl)-3-trityloxy-propan-2-ol (3.3). Adenine (5.36 g,
0.0396 mol, 1.0 equiv), anhydrous K
2
CO
3
(7.12 g, 0.0515 mol, 1.3 equiv), and anhydrous
DMF (100 mL) were stirred for 2 h at 105 ºC under N
2
. A solution of 3.2 (16.30 g, 51.5
mmol, 1.3 equiv) and 100 mL anhydrous DMF was added under N
2
, and the reaction was
complete after 22 h at 105 ºC. Solvent was removed under reduced pressure. The residue
was dissolved in 300 mL of CHCl
3
, and obtained solution was washed two times using 30
mL of H
2
O. The organic phase was dried over Na
2
SO
4
, and evaporated in vacuum to give
brown residue. The residue was recrystallized from chloroform to give 12.87 g (72%) of
of pure product 3.3.
1
H NMR (400 MHz, CDCl
3
): δ 8.28 (s, 1H, 2-H), 7.74 (s, 1H, 8-H),
7.44-7.24 (m, 15H, aromatic), 5.73 (brs, 2H), 4.91 brs (1H), 4.44 (dd, J = 14.2, 2.4 Hz,
1H), 4.34 (dd, J = 14.3, 6.6 Hz, 1H), 4.26-4.20 (m, 1H), 3.31 (dd, J = 9.6, 5.5 Hz, 1H),
3.06 (dd, 1H).

Synthesis of diethyl p-tolylsulfonyloxymethylphosphonate (3.4). Diethyl
hydroxymethylphosphonate (17.10 g, 0.102 mol, 1.1 equiv), DMAP (1.82 g), and TEA
(9.29 g, 13 mL, 1.0 equiv) were diluted with 40 mL anhydrous CH
2
Cl
2
, which was added
via an addition funnel. The reaction mixture was cooled to 0 ºC. A solution of p-
toluenesulfonyl chloride (17.60 g, 0.092 mol, 1.0 equiv) in 100 mL anhydrous CH
2
Cl
2

was added dropwise to the reaction mixture. The solution was kept at 5 ºC during the
addition and the reaction then continued for 6 h at that temperature. The organic phase
was washed with 2 x 50 mL H
2
O, 50 mL sat. citric acid, 50 mL sat. NaHCO
3
, and 50 mL
H
2
O. The organic layer was dried over Na
2
SO
4
and concentrated in vacuum to afford
117
25.20 g of 3.4 (77%).
1
H NMR (400 MHz, CDCl
3
): δ 7.78-7.76 (m, 2H, aromatic), 7.36-
7.34 (m, 2H, aromatic), 4.16 (d, J = 9.9 Hz, 2H), 4.16-4.10 (m, 4H), 2.43 (s, 3H), 1.29 (t,
J = 7.1 Hz, 6H).
31
P NMR (162 MHz, CDCl
3
): δ 15.1 (s).

Synthesis of [2-(6-Amino-purin-9-yl)-1-trityloxymethyl-ethoxymethyl]-phosphonic
acid diethyl ester (3.5). A mixture of NaH (60% in oil, 4.39 g) and 50 mL of DMF
(anh.)

was cooled to 0-5
o
C under N
2
. A solution of 3.3 (11.00 g, 0.0244 mol, 1.0 equiv) in 100
mL anhydrous DMF was added dropwise to the reaction mixture then it was stirred for
1.5 h at 0-5
o
C. A solution of 11.77 g of 3.4 (36.5 mmol, 1.5 equiv) in 120 mL anhydrous
DMF was added dropwise at 0-5 ºC under N
2
to the reaction mixture. The reaction was
stirred under N
2
for 2 h at 0-5 ºC followed by stirring at 25 ºC overnight. The reaction
mixture was added dropwise to a stirred solution of 300 mL H
2
O-saturated ethyl acetate.
Solvents were removed under vacuum and the residue was redissolved in 300 mL of
EtOAc and washed using 40 ml of sat. NaCl solution. The organic layer was dried over
Na
2
SO
4
and concentrated under reduced pressure. The crude brown residue 3.5 was used
directly for the subsequent deprotection step.

Synthesis of [2-(6-Amino-purin-9-yl)-1-hydroxymethyl-ethoxymethyl]-phosphonic
acid diethyl ester (3.6). Acid catalyzed detritylation of 3.5 was achieved by stirring the
residue in 300 mL of 80% aq. AcOH at 80
o
C for 3 h and additional 12 h at rt. Acetic acid
and water were removed under vacuum. 100 mL of water was added to the residue and
the precipitate (TrOH) was filtered away. The solution was concentrated and dried under
118
vacuum. Purification of 7 was accomplished by silica gel column chromatography using
a mobile phase consisting of CH
2
Cl
2
followed by EtOAc containing 1.5%, 2%, and 5%
MeOH to give 3.50 g of 3.6 as yellow-orange crystals. Product after column
chromatography was recrystallized from mixture of CHCl
3
/hexanes (2:1) yielding 2.92 g
of pure 3.6.
1
H NMR (400 MHz, CDCl
3
): δ 8.37 (s, 1H, 2-H), 7.95 (s, 1H, 8-H), 5.78
(brs, 2H), 4.82 brs (1H), 4.51 (dd, J = 14.7, 4.9 Hz, 1H), 4.45 (dd, J = 14.7, 4.0 Hz, 1H),
4.22-4.15 (m, 4H), 3.91-3.88 (m, 3H), 3.66-3.60 (m, 1H), 3.49-3.44 (m, 1H), 1.39 (t,
J = 5.3 Hz, 3H), 1.35 (t, J = 5.3 Hz, 3H).
31
P NMR (162 MHz, CDCl
3
): δ 21.1 (s).

Synthesis of [2-(6-Amino-purin-9-yl)-1-hydroxymethyl-ethoxymethyl]-phosphonic
acid (2.2, (S)-HPMPA). BTMS (1.5 mL) was added to a solution of 3.6 (0.5 g,
0.0014 mol) in 10 mL DMF
(anh.)
to initiate the desired dealkylation reaction. The reaction
was carried out at room temperature for 15 h under N
2
. The residue was concentrated in
vacuum, and 5 mL of H
2
O was added, and the procedure was repeated two subsequent
times. Finally, the residue was dissolved in 3 mL of H
2
O and the product was precipitated
by addition of 50 mL of acetone. Precipitate was filtered, washed using 50 mL of
acetone, and dried under vacuum. Yield of 2.2 is 0.33 g (79%).
1
H NMR (400 MHz,
D
2
O): δ 8.28 (s, 1H, 2-H), 8.28 (s, 1H, 8-H), 4.43 (dd, J = 14.8, 3.7 Hz, 1H), 4.32 (dd,
J = 14.8, 7.3 Hz, 1H), 3.78-3.73 (m, 1H), 3.66 (dd, J = 12.5, 4.1 Hz, 1H), 3.58 (dd,
J = 13.0, 9.4 Hz, 1H), 3.46 (dd, J = 12.5, 4.6 Hz, 1H), 3.39 (dd, J = 13.0, 9.7 Hz, 1H).
31
P
NMR (162 MHz, CDCl
3
): δ 15.3 (s).

119

Conventional synthesis of single amino acid prodrugs
Amidation of Boc-Protected (L)-Tyrosine. General procedure. To a suspension of
Boc-(L)-tyrosine (commercially available) (9.4 mmol, 2.64 g) in CH
2
Cl
2
(60 ml) were
added N-hydroxybenzotriazole (HOBt) hydrate (14.1 mmol, 1.91 g) and corresponding
amine (10 mmol). The reaction mixture was cooled to 0 - 5 ºC before 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide (EDC) hydrochloride (11.8 mmol, 2.25 g) was
added. The reaction mixture was stirred at room temperature for 24 h. An additional
70 ml of CH
2
Cl
2
was added, and the organic layer was washed consecutively with 1.6 M
citric acid (25 ml), saturated NaHCO
3
(25 ml), and saturated NaCl (40 ml). The organic
phase was dried over Na
2
SO
4
and concentrated under vacuum. Obtained Boc-protected
tyrosine N-alkyl amides (3.11 – 3.17) were used in the next step without further
purification.

N
α
-(tert-butoxycarbonyl)-N-(2-methylpropyl)-(L)-tyrosinamide (3.12). Yield 89 %.
1
H NMR (400 MHz, CDCl
3
): δ 6.98-6.96 (m, 2H, aromatic), 6.70-6.68 (m, 2H, aromatic),
5.82 (br, 1H, NH), 5.03 (br, 1H, NH), 4.15 (dd, 1H, J = 14.4, 7.2 Hz, CHNH (Tyr)), 2.95-
2.88 (m, 4H, CH
a
H
b
, CH
a
H
b
(Tyr), CH
2
(iBu)), 1.61-1.51 (m, 1H, CH(CH
3
)
2
), 1.35 (s,
9H, C(CH
3
)
3
), 0.73 (dd, J = 5.8 Hz, 6H, CH(CH
3
)
2
).

N
α
-(tert-butoxycarbonyl)-N-dodecyl-(L)-tyrosinamide (3.14). Yield 83%.
1
H NMR
(500 MHz, CDCl
3
): δ 7.07-7.05 (m, 2H, aromatic), 6.77-6.74 (m, 2H, aromatic), 4.22-
120
4.17 (m, 1H, CHNH
2
), 3.15 (dd, J = 12.9, 6.9 Hz, 2H, CH
2
CH
2
NH), 3.01 (dd, J = 13.8,
6.1 Hz, 1H, CH
a
H
b
(Tyr)), 2.93 (dd, J = 13.8, 6.1 Hz, 1H, CH
a
H
b
(Tyr)), 1.42 (s, 9H,
C(CH
3
)
3
), 1.38-1.35 (m, 2H, CH
2
CH
2
NH
2
), 1.28-1.25 (m, 18H, 9CH
2
), 0.88 (t, J = 7.0
Hz, 3H, CH
3
CH
2
).

N
α
-(tert-butoxycarbonyl)-N-tetradecyl-(L)-tyrosinamide (3.15). Yield 94%.
1
H NMR
(500 MHz, CDCl
3
): δ 7.07-7.05 (m, 2H, aromatic), 6.77-6.74 (m, 2H, aromatic), 4.21-
4.17 (m, 1H, CHNH
2
), 3.15 (dd, J = 13.0, 6.6 Hz, 2H, CH
2
CH
2
NH), 3.01 (dd, J = 13.8,
6.1 Hz, 1H, CH
a
H
b
(Tyr)), 2.93 (dd, J = 13.7, 7.7 Hz, 1H, CH
a
H
b
(Tyr)), 1.42 (s, 9H,
C(CH
3
)
3
), 1.38-1.35 (m, 2H, CH
2
CH
2
NH
2
), 1.30-1.25 (m, 22H, 11CH
2
), 0.88 (t, J = 7.0
Hz, 3H, CH
3
CH
2
).

N
α
-(tert-butoxycarbonyl)-N-hexadecyl-(L)-tyrosinamide (3.16). Yield 89%.
1
H NMR
(400 MHz, CDCl
3
): δ 7.07-7.05 (m, 2H, aromatic), 6.77-6.74 (m, 2H, aromatic), 4.20 (dd,
J = 13.9, 7.3 Hz, 1H, CHNH
2
), 3.15 (dd, J = 13.2, 6.9 Hz, 2H, CH
2
CH
2
NH), 3.00 (dd, J
= 13.8, 6.3 Hz, 1H, CH
a
H
b
(Tyr)), 2.93 (dd, J = 13.8, 7.7 Hz, 1H, CH
a
H
b
(Tyr)), 1.42 (s,
9H, C(CH
3
)
3
), 1.38-1.35 (m, 2H, CH
2
CH
2
NH
2
), 1.30-1.26 (m, 26H, 13CH
2
), 0.88 (t, J =
6.9 Hz, 3H, CH
3
CH
2
).

N
α
-(tert-butoxycarbonyl)-N-octadecyl-(L)-tyrosinamide (3.17). Yield 84%.
1
H NMR
(500 MHz, CDCl
3
): δ 7.08-7.06 (m, 2H, aromatic), 6.77-6.75 (m, 2H, aromatic), 4.20-
4.17 (m, 1H, CHNH
2
), 3.15 (dd, J = 13.3, 7.1 Hz, 2H, CH
2
CH
2
NH), 3.01 (dd, J = 13.7,
121
5.7 Hz, 1H, CH
a
H
b
(Tyr)), 2.93 (dd, J = 13.7, 7.8 Hz, 1H, CH
a
H
b
(Tyr)), 1.42 (s, 9H,
C(CH
3
)
3
), 1.38-1.35 (m, 2H, CH
2
CH
2
NH
2
), 1.30-1.25 (m, 30H, 15CH
2
), 0.88 (t, J = 7.0
Hz, 3H, CH
3
CH
2
).

Synthesis of cyclic (S)-HPMPA and (S)-HPMPC prodrugs 2.17, 2.23 - 2.27,
2.29 - 2.34. General procedure. To a suspension of (S)-HPMPC 2.1 or (S)-HPMPA 2.2
(0.6 mmol) in dry DMF (10 mL), dry N,N-diisopropylethylamine (DIEA) (11.5 mmol,
2.0 mL), 3.7 - 3.17 (0.9 mmol) and (benzotriazol-1-yloxy)tripyrrolidino-phosphonium
hexafluorophosphate (PyBOP) (1.2 mmol, 0.62 g) were added. The reaction mixture was
stirred under N
2
at 40 °C for 2 h. The reaction was monitored by
31
P NMR, and additional
portions of PyBOP were added as necessary. After reaction completion, DMF and DIEA
were removed under vacuum. The residue was washed with diethyl ether and purified
using silica gel column chromatography [CH
2
Cl
2
, then CH
2
Cl
2
:acetone (2:1), then
CH
2
Cl
2
:acetone:CH
3
OH (6:3:1)]. Solvents were removed under vacuum to furnish the
Boc-protected intermediates that were used in the next step without further purification.
TFA (3 ml) was added to a suspension of the Boc-protected intermediates in CH
2
Cl
2
(10
ml). After the mixture was stirred for overnight at room temperature, volatiles were
removed under vacuum and the residue was purified by silica gel column
chromatography [CH2Cl2/MeOH (10 : 0.2-1.5), with addition of 0.5% v/v TFA]. After
evaporation of the solvents, compounds 2.17, 2.23 - 2.27, 2.29 - 2.34 were precipitated
with diethyl ether, filtered, and dried in vacuum to give TFA salts of the product obtained
122
as a mixture of (S
p
)- and (R
p
)-diastereomers (ratio assignment based on integration of the
corresponding signals in
31
P NMR).

Methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-
2-yl}-(L)-cysteinate (2.17). Yield 46%.Obtained as a TFA salt; mixture of two
diastereomers (S
P
/R
P
).
1
H NMR (400 MHz, CD
3
OD): δ 8.41 (s, 0.17H), 8.38 (s, 0.83H),
8.28 (s, 0.17H), 8.25 (s, 0.83H), 4.69-4.61 (m, 1H), 4.57-4.34 (m, 8H), 4.26-4.15 (m,
1H), 3.90 and 3.89 (2s, 3H).
31
P NMR (202 MHz, CD
3
OD): δ 43.3 (s, 0.12P), 42.6 (s,
0.88P).

O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-
(2-methylpropyl)-(L)-tyrosinamide (2.26). Yield 62%. Obtained as a TFA salt; mixture
of diastereomers (S
p
/R
p
, 5:1).
1
H NMR (400 MHz, CD
3
OD): δ 8.39 and 8.38 (s, 1H, 2-H,
(S
p
) and (R
p
)), 8.32 (s, 0.85H, 8-H, (S
p
)), 8.28 (s, 0.15H, 8-H, (R
p
)), 7.34-7.32 (m, 2H,
aromatic), 7.25-7.23 (m, 2H, aromatic), 4.78 (ddd, J = 12.0, 12.0, 2.8 Hz, 0.85H,
CH
a
H
b
O, (S
p
)), 4.67 (dd, J = 14.9, 8.4 Hz, 0.85H, CH
a
H
b
N, (S
p
)), 4.59-4.34 (m, 4.3H,
CH
a
H
b
O, (R
p
), CH
a
H
b
N (R
p
), CH
a
H
b
O, CH
a
H
b
N, CH
a
H
b
P and CHO), 4.22 (dd, J = 14.8,
4.3 Hz, 0.85H, CH
a
H
b
P, (S
p
)), 4.12 (dd, J = 15.2, 1.1 Hz, 0.15H, CH
a
H
b
P, (R
p
)), 4.05-
4.03 (t, J = 7.4 Hz, 1H, CHNH
2
, (S
p
and R
p
)), 3.19 (dd, J = 13.9, 7.4 Hz, 1H, CH
a
H
b

(Tyr)), 3.11-3.05 (m, 2H, CH
a
H
b
(Tyr) and CH
a
H
b
(iBu)), 2.93 (dd, J = 13.2, 7.1 Hz, 1H,
CH
a
H
b
(iBu)), 1.75-1.65 (m, 1H, CH(CH
3
)
2
), 0.86 (d, J = 6.7 Hz, 3H, CH
3
), 0.83 (d, J =
6.7 Hz, 3H, CH
3
).
31
P NMR (162 MHz, CD
3
OD): δ 11.7 (0.85P (S
P
)), 10.2 (0.15P (R
P
)).
123
HRMS: m/z calcd 504. 2119 (M + H)
+
, found 504.2128 (M + H)
+
. HPLC: t
R
17.66 min
(R
P
), 18.32 min (S
P
).

O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-
octyl-(L)-tyrosinamide (2.29). Yield 48%. Obtained as a TFA salt; mixture of
diastereomers (S
p
/R
p
, 4.2:1).
1
H NMR (400 MHz, CD
3
OD): δ 8.36 (s, 0.8H, 2-H, (S
p
)),
8.35 (s, 0.2H, 2-H, (R
p
)), 8.29 (s, 0.8H, 8-H, (S
p
)), 8.25 (s, 0.2H, 8-H, (R
p
)), 7.33-7.30 (m,
2H, aromatic), 7.24-7.21 (m, 2H, aromatic), 4.77 (ddd, J = 12.1, 12.1, 2.8 Hz, 0.8H,
CH
a
H
b
O, (S
p
)), 4.67 (dd, J = 14.9, 8.4 Hz, 0.8H, CH
a
H
b
N, (S
p
)), 4.59-4.32 (m, 4.4H,
CH
a
H
b
O, (R
p
), CH
a
H
b
N (R
p
), CH
a
H
b
O, CH
a
H
b
N, CH
a
H
b
P and CHO), 4.21 (dd, J = 14.8,
4.3 Hz, 0.8H, CH
a
H
b
P, (S
p
)), 4.12 (dd, J = 15.3, 1.3 Hz, 0.2H, CH
a
H
b
P, (R
p
)), 4.00-3.97
(m, 1H, CHNH
2
, (S
p
and R
p
)), 3.23-3.05 (m, 4H, CH
2
(Tyr), CH
2
NHCO), 1.46-1.41 (m,
2H, NHCH
2
CH
2
), 1.35-1.24 (m, 10H, 5CH
2
), 0.92 (t, J = 6.9 Hz, 3H, CH
3
CH
2
).
31
P
NMR (162 MHz, CD
3
OD): δ 11.4 (0.81P (S
p
)), 9.9 (0.19P, (R
p
)). MS-ESI (m/z) 560.47
(M+H)
+
, 582.19 (M+Na)
+
. HPLC: t
R
, 8.16 min (S
p
and R
p
).

O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-
dodecyl-(L)-tyrosinamide (2.30).Yield 60%. Obtained as a TFA salt; mixture of
diastereomers (S
p
/R
p
, 3.8:1).
1
H NMR (400 MHz, CD
3
OD): δ 8.37 (s, 1H, 2-H, (S
p
and
R
p
)), 8.30 (s, 0.8H, 8-H, (S
p
)), 8.25 (s, 0.2H, 8-H, (R
p
)), 7.33-7.30 (m, 2H, aromatic),
7.24-7.21 (m, 2H, aromatic), 4.77 (ddd, J = 12.1, 12.1, 2.8 Hz, 0.8H, CH
a
H
b
O, (S
p
)), 4.67
(dd, J = 15.0, 8.4 Hz, 0.8H, CH
a
H
b
N, (S
p
)), 4.59-4.33 (m, 4.4H, CH
a
H
b
O, (R
p
), CH
a
H
b
N
124
(R
p
), CH
a
H
b
O, CH
a
H
b
N, CH
a
H
b
P and CHO), 4.21 (dd, J = 14.8, 4.3 Hz, 0.8H, CH
a
H
b
P,
(S
p
)), 4.12 (dd, J = 15.3, 1.3 Hz, 0.2H, CH
a
H
b
P, (R
p
)), 4.01-3.97 (m, 1H, CHNH
2
, (S
p
and
R
p
)), 3.25-3.05 (m, 4H, CH
2
(Tyr), CH
2
NHCO), 1.46-1.39 (m, 2H, NHCH
2
CH
2
), 1.36-
1.26 (m, 18H, 9CH
2
), 0.92 (t, J = 6.9 Hz, 3H, CH
3
CH
2
).
31
P NMR (162 MHz, CD
3
OD): δ
11.4 (0.79P (S
p
)), 9.9 (0.19P, (R
p
)). MS-ESI (m/z) 616.63 (M+H)
+
, 638.34 (M+Na)
+
.
HPLC: t
R
, 3.73 min (S
p
and R
p
).

O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-
tetradecyl-(L)-tyrosinamide (2.31). Yield 49%. Obtained as a TFA salt; mixture of
diastereomers (S
p
/R
p
, 4.1:1).
1
H NMR (500 MHz, CD
3
OD): δ 8.35 (s, 0.8H, 2-H, (S
p
)),
8.34 (s, 0.2H, 2-H, (R
p
)), 8.28 (s, 0.8H, 8-H, (S
p
)), 8.23 (s, 0.2H, 8-H, (R
p
)), 7.33-7.31 (m,
2H, aromatic), 7.24-7.21 (m, 2H, aromatic), 4.77 (ddd, J = 12.1, 12.1, 3.0 Hz, 0.8H,
CH
a
H
b
O, (S
p
)), 4.67 (dd, J = 14.9, 8.4 Hz, 0.8H, CH
a
H
b
N, (S
p
)), 4.59-4.33 (m, 4.4H,
CH
a
H
b
O, (R
p
), CH
a
H
b
N (R
p
), CH
a
H
b
O, CH
a
H
b
N, CH
a
H
b
P and CHO), 4.21 (dd, J = 14.8,
4.3 Hz, 0.8H, CH
a
H
b
P, (S
p
)), 4.12 (dd, J = 15.1, 1.3 Hz, 0.2H, CH
a
H
b
P, (R
p
)), 4.00-3.97
(m, 1H, CHNH
2
, (S
p
and R
p
)), 3.25-3.05 (m, 4H, CH
2
(Tyr), CH
2
NHCO), 1.46-1.40 (m,
2H, NHCH
2
CH
2
), 1.35-1.25 (m, 22H, 11CH
2
), 0.93 (t, J = 7.0 Hz, 3H, CH
3
CH
2
).
31
P
NMR (202 MHz, CD
3
OD): δ 11.7 (0.81P (S
p
)), 10.1 (0.19P, (R
p
)). MS-ESI (m/z) 644.87
(M+H)
+
, 666.47 (M+Na)
+
. HPLC: t
R
, 5.14 min (S
p
and R
p
).

O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-
hexadecyl-(L)-tyrosinamide (2.32). Yield 33%. Obtained as a TFA salt; mixture of
125
diastereomers (S
p
/R
p
, 3.8:1).
1
H NMR (500 MHz, CD
3
OD): δ 8.35 (s, 1H, 2-H, (S
p
and
R
p
)), 8.28 (s, 0.8H, 8-H, (S
p
)), 8.23 (s, 0.2H, 8-H, (R
p
)), 7.33-7.31 (m, 2H, aromatic),
7.24-7.21 (m, 2H, aromatic), 4.77 (ddd, J = 12.0, 12.0, 2.8 Hz, 0.8H, CH
a
H
b
O, (S
p
)), 4.66
(dd, J = 15.0, 8.4 Hz, 0.8H, CH
a
H
b
N, (S
p
)), 4.59-4.33 (m, 4.4H, CH
a
H
b
O, (R
p
), CH
a
H
b
N
(R
p
), CH
a
H
b
O, CH
a
H
b
N, CH
a
H
b
P and CHO), 4.21 (dd, J = 14.8, 4.3 Hz, 0.8H, CH
a
H
b
P,
(S
p
)), 4.12 (dd, J = 15.2, 1.4 Hz, 0.2H, CH
a
H
b
P (R
p
)), 4.02-3.97 (m, 1H, CHNH
2
, (S
p
and
R
p
)), 3.23-3.06 (m, 4H, CH
2
(Tyr), CH
2
NHCO), 1.44-1.40 (m, 2H, NHCH
2
CH
2
), 1.35-
1.27 (m, 26H, 13CH
2
), 0.93 (t, J = 6.9 Hz, 3H, CH
3
CH
2
).
31
P NMR (202 MHz, CD
3
OD):
δ 11.7 (0.80P (S
p
)), 10.1 (0.20P, (R
p
)). MS-ESI (m/z) 672.89 (M+H)
+
, 694.58 (M+Na)
+
.
HPLC: t
R
, 8.12 min (S
p
and R
p
).

O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-
octadecyl-(L)-tyrosinamide (2.33). Yield 53%. Obtained as a TFA salt; mixture of
diastereomers (S
p
/R
p
, 3.8:1).
1
H NMR (500 MHz, CD
3
OD): δ 8.36 (s, 1H, 2-H), 8.28 (s,
0.8H, 8-H (S
p
)), 8.24 (s, 0.2H, 8-H (R
p
)), 7.33-7.31 (m, 2H, aromatic), 7.24-7.21 (m, 2H,
aromatic), 4.77 (ddd, J = 12.0, 12.0, 2.8 Hz, 0.8H, CH
a
H
b
O (S
p
)), 4.67 (dd, J = 15.0, 8.5
Hz, 0.8H, CH
a
H
b
N (S
p
)), 4.59-4.33 (m, 4.4H, CH
a
H
b
O (R
p
), CH
a
H
b
N (R
p
), CH
a
H
b
O,
CH
a
H
b
N, CH
a
H
b
P and CHO), 4.21 (dd, J = 14.8, 4.3 Hz, 0.8H, CH
a
H
b
P (S
p
)), 4.12 (dd, J
= 15.4, 1.2 Hz, 0.2H, CH
a
H
b
P (R
p
)), 4.02-3.98 (m, 1H, CHNH
2
, (S
p
and R
p
)), 3.25-3.06
(m, 4H, CH
2
(Tyr), CH
2
NHCO), 1.46-1.40 (m, 2H, NHCH
2
CH
2
), 1.35-1.25 (m, 30H,
15CH
2
), 0.93 (t, J = 6.9 Hz, 3H, CH
3
CH
2
).
31
P NMR (202 MHz, CD
3
OD): δ 11.7 (0.77P
126
(S
p
)), 10.1 (0.23P, (R
p
)). MS-ESI (m/z) 700.93 (M+H)
+
, 722.48 (M+Na)
+
. HPLC: t
R
,
13.54 min (S
p
and R
p
).

O-[(5S)-5-[(4-amino-2-oxopyrimidin-1(2H)-yl)methyl]-2-oxido-1,4,2-dioxa-
phosphinan-2-yl]-N-hexadecyl-(L)-tyrosinamide (2.34). Yield 36%. Obtained as a
TFA salt; mixture of diastereomers (S
p
/R
p
, 3.0:1).
1
H NMR (400 MHz, CD
3
OD): δ 7.77
(d, J = 7.5 Hz, 0.75H, 6-H, (S
p
)), 7.73 (d, J = 7.5 Hz, 0.25H, 6-H, (R
p
)), 7.34-7.22 (m,
4H, aromatic), 6.01-5.98 (m, 1H, 5-H), 4.69-4.62 (m, 0.75H, CH
a
H
b
O, (S
p
)), 4.55-4.36
(m, 2.25H, CH
a
H
b
O (R
p
), CH
a
H
b
O, CH
a
H
b
P), 4.27-4.23 (m, 1.75H, CH
a
H
b
P (S
p
), CHO),
4.17-3.98 (m, 3H, CH
a
H
b
P (R
p
), CH
a
H
b
N, CH
a
H
b
N (S
p
), CHNH
2
), 3.83 (dd, J = 14.4, 7.5
Hz, 0.25 H, CH
a
H
b
N (R
p
)), 3.26-3.05 (m, 4H, CH
2
(Tyr), CH
2
NHCO), 1.46-1.43 (m, 2H,
NHCH
2
CH
2
), 1.35-1.27 (m, 26H, 13CH
2
), 0.93 (t, J = 6.9 Hz, 3H, CH
3
CH
2
).
31
P NMR
(162 MHz, CD
3
OD): δ 11.5 (0.75P (S
p
)), 10.0 (0.25P, (R
p
)). MS-ESI (m/z) 648.80
(M+H)
+
, 670.53 (M+Na)
+
. HPLC: t
R
, 7.22 min (S
p
and R
p
).

Solid-phase synthesis of the single amino acid and dipeptide prodrugs

TBDMS-protection of serine, threonine and tyrosine esters. General procedure. The
corresponding amino acid ester 3.34 - 3.38 (2.3 mmol, 1 equiv) and tert-
butylchlorodimethylsilane (TBDMSCl) (4.8 mmol, 2.1 equiv) were mixed in 20 mL of
dry CH
2
Cl
2
. The reaction mixture was cooled to 0
o
C before addition of imidazole (7.1
mmol, 3.1 equiv). The reaction mixture was stirred overnight at RT. An additional 100
mL of CH
2
Cl
2
was added to the reaction mixture, and the organic layer was washed with
127
a sat. citric acid solution (25 mL) and sat. NaHCO
3
solution (25 mL). Combined water
phases were extracted twice with 30 mL of CH
2
Cl
2
(to increase the yield, product as a
salt is soluble in water). All organic phases were combined and dried over Na
2
SO
4
and
concentrated under reduced pressure yielding a white solid. The solid was washed on a
filter with 20 mL of hexanes to remove the TBDMSOH, giving a clean product
3.39 - 3.43.

Methyl O-[tert-butyl(dimethyl)silyl]-(L)-serinate (3.39). Yield 69%.
1
H NMR (400
MHz, CDCl
3
): δ 8.72 (brs, 2H), 4.33-4.31 (m, 1H), 4.26 (dd, J = 10.9, 2.6 Hz, 1H), 4.15
(dd, J = 10.9, 2.7 Hz, 1H), 3.83 (s, 3H), 0.88 (s, 9H), 0.11 (s, 3H), 0.08 (s, 3H).

Methyl O-[tert-butyl(dimethyl)silyl]-(D)-serinate (3.40). Yield 72%.
1
H NMR (400
MHz, CDCl
3
): δ 8.73 (brs, 2H), 4.33 (t, J = 3.0 Hz, 1H), 4.25 (dd, J = 10.7, 3.0 Hz, 1H),
4.14 (dd, J = 10.7, 3.0 Hz, 1H), 3.81 (s, 3H), 0.87 (s, 9H), 0.10 (s, 3H), 0.07 (s, 3H).

Propan-2-yl O-[tert-butyl(dimethyl)silyl]-(L)-serinate (3.41). Yield 76%.
1
H NMR
(400 MHz, CDCl
3
): δ 8.72 (brs, 2H), 5.11 (sept, J = 6.7 Hz, 1H), 4.25 (dd, J = 10.7, 2.6
Hz, 1H), 4.20 (t, J = 2.6 Hz, 1H), 4.13 (dd, J = 10.7, 2.6 Hz, 1H), 1.29 (d, J = 6.4 Hz,
6H), 0.88 (s, 9H), 0.12 (s, 3H), 0.08 (s, 3H).

128
Methyl O-[tert-butyl(dimethyl)silyl]-(L)-threoninate (3.42). Yield 85%.
1
H NMR (400
MHz, CDCl
3
): δ 8.78 (brs, 2H), 4.51 (qd, J = 6.5, 1.6 Hz, 1H), 4.11 (d, J = 1.7 1H), 3.81
(s, 3H), 1.49 (d, J = 6.6 Hz, 3H), 0.86 (s, 9H), 0.08 (s, 3H), 0.02 (s, 3H).

Methyl O-[tert-butyl(dimethyl)silyl]-(L)-tyrosinate (3.43). Yield 68%.
1
H NMR (400
MHz, CD
3
OD): δ 6.95-6.92 (m, 2H, aromatic), 6.66-6.63 (m, 2H, aromatic), 4.71 (brs,
2H), 4.07 (t, J = 6.7, 1H), 3.59 (s, 3H), 2.99 (dd, J = 14.4, 6.2 Hz, 1H), 2.92 (dd, J = 14.4,
7.2 Hz, 1H), 0.79 (s, 9H), 0.00 (s, 6H).

Synthesis of N-formyl TBDMS-protected serine esters 3.44 and 3.45. General
procedure. (L)-Serine ester 3.34 or 3.36 (10 mmol, 1 equiv) and tert-
butylchlorodimethylsilane (20 mmol, 2.0 equiv) were mixed in 25 mL of dry DMF. The
reaction mixture was cooled to 0 ºC before addition of imidazole (30 mmol, 3.0 equiv).
The reaction mixture was stirred overnight at RT. An additional 100 mL of CH
2
Cl
2
was
added to the reaction mixture, and the organic layer was washed with a sat. citric acid
solution (25 mL) and sat. NaHCO
3
solution (25 mL). Combined water phases were
extracted twice with 30 mL of CH
2
Cl
2
(to increase the yield, product as a salt is soluble in
water). All organic phases were combined and dried over Na
2
SO
4
and concentrated
under reduced pressure yielding an oily residue. The residue was purified using SiO
2

column chromatography.

129
Methyl O-[tert-butyl(dimethyl)silyl]-N-formyl-(L)-serinate (3.44). Yield 67%.
1
H
NMR (400 MHz, CDCl
3
): δ 8.24 (s, 1H), 6.46 (brs, 1H), 4.74 (dt, J = 8.5, 2.7 Hz, 1H),
4.06 (dd, J = 10.1, 2.6 Hz, 1H), 3.82 (dd, J = 10.1, 3.0 Hz, 1H), 3.74 (s, 3H), 0.83 (s, 9H),
0.01 (s, 3H), 0.00 (s, 3H).

Propan-2-yl O-[tert-butyl(dimethyl)silyl]-N-formyl-(L)-serinate (3.45). Yield 56%.
1
H
NMR (400 MHz, CD
3
OD): δ 8.07 (s, 1H), 4.97 (hept, J = 6.3 Hz, 1H), 4.52-4.51 (m, 1H),
3.99 (dd, J = 10.5, 3.6 Hz, 1H), 3.79 (dd, J = 10.2, 3.6 Hz, 1H), 1.22-1.19 (m, J = 6.4 Hz,
6H), 0.83 (s, 9H), 0.01 (s, 3H), 0.00 (s, 3H).
13
C NMR (100 MHz, CD
3
OD): δ 169.2
(CO), 162.2 (CO), 69.1, 63.0, 53.4, 24.9, 20.8, 20.7, 17.7, -6.7, -6.8.

Addition of the TBDMS-protected amino acid to the TCP-resin. General procedure.
The TBDMS-protected serine alkyl ester 3.39 - 3.43 (4 equiv) was added to the trityl
chloride polystyrene (TCP) resin (1 equiv) in CH
2
Cl
2
in the presence of DIEA (15 equiv).
Obtained reaction mixture was shaken at 25 ºC for 12 h. Then the resin was filtered,
washed using 150 mL of CH
2
Cl
2
, and shaken with a mixture of CH
2
Cl
2
/MeOH (10:1, 20
ml) for 30 min to cap any unreacted sites. The resin was washed with MeOH, CH
2
Cl
2
and
dried. Removal of TBDMS-protecting group was accomplished with TBAF (4 equiv) in
THF for 5 h at 25 ºC. After that the resin was filtered and washed using 50 mL of THF
followed by 100 mL of CH
2
Cl
2
. Finally, the amino acid bound to TCP-resin 3.18 - 3.22
was dried under vacuum in a desiccator before being used for the coupling reaction.

130
Solid-phase synthesis of the TBDMS-protected dipeptides 3.28, 3.29. General
procedure. Incorporation of the first (L)-valine allyl ester (0.5 equiv) to the TCP-resin (1
equiv) was performed in CH
2
Cl
2
in the presence of DIEA (10 equiv) for overnight. The
resin was filtered and washed with 100 mL of CH
2
Cl
2
. Removal of the allyl group was
accomplished under an Ar atmosphere with Pd(PPh
3
)
4
(20 mol %, based on estimated
substitution of the resin) and PhSiH
3
(24 equiv)in CH
2
Cl
2
(2 × 15 min).
15
After filtering
the Pd solution, the resin was washed consecutively with 150 mL of CH
2
Cl
2
, 40 mL of
H
2
O-dioxane (1:9) and 120 mL of DMF. The following coupling reaction was conducted
by adding the TBDMS-protected (L)-serine methyl or isopropyl ester (4 equiv) 3.34 or
3.36 dissolved in DCM-DMF (9:1), DIEA (20 equiv) and then PyBOP (5 equiv) as a
solid. Obtained reaction mixture was shaken for overnight at r. t. The resin was then
filtered and washed with 100 ml of DMF followed by 150 ml of CH
2
Cl
2
. Removal of
TBDMS-protecting group was accomplished with TBAF (4 equiv) in THF for 5 h at
25 ºC. Finally, the resin was filtered and washed using 80 mL of THF followed by
100 mL of CH
2
Cl
2
. The dipeptides bound to the TCP-resin 3.28 and 3.29 were dried in
the desiccator before being used for the coupling reaction.

Coupling of (S)-HPMPA to the amino acid or dipeptide promoiety bound to the
TCP-resin. General procedure. PyBOP (1.2 equiv) was added to a solution of (S)-
HPMPA (1 equiv) and DIEA (15 equiv) in DMF (10 mL). The cyclization reaction
proceeded at 25 ºC. After 1 h an additional portion of PyBOP (3-4 equiv) and the TCP-
resin containing corresponding amino acid alkyl ester 3.18 - 3.22 or dipeptide 3.28, 3.29
131
were added to the reaction mixture. The reaction was shaken for overnight at 38 ºC. The
resin was then filtered and washed with 60 mL of DMF followed by 200 mL of CH
2
Cl
2
.
The product was cleaved from the resin using TFA (2 mL) in CH
2
Cl
2
(10 mL) in the case
of the single amino acid and dipeptide conjugates (2.12, 2.13, 2.15, 2.16, 3.32, 3.33), and
with 1.4 M HCl in dioxane/CH
2
Cl
2
(10 mL/5 mL) in case of serine isopropyl ester
conjugate 2.14. The products were precipitated as trifluoroacetic acid or hydrochloric
acid salts from methanol by addition of diethyl ether and collected by filtration.

Methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-
2-yl}-(L)-serinate (2.12) Yield 29%. Obtained as a TFA salt; mixture of two
diastereomers (S
P
/R
P
).
1
H NMR (400 MHz, CD
3
OD): δ 8.36 (s, 1H), 8.27 (s, 0.7H), 8.20
(s, 0.3H), 4.76-4.46 (m, 7H), 4.39-4.31 (m, 2H), 4.12-4.05 (m, 1H), 3.90 and 3.89 (2s,
3H).
31
P NMR (202 MHz, CD
3
OD): δ 14.2 (s, 0.68P), 13.1 (s, 0.32P); HRMS-FAB m/z
[M+H]
+
calcd for C
13
H
19
N
6
O
6
P: 387.1176, found: 387.1181.

Methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-
2-yl}-(D)-serinate (2.13) Yield 26%. Obtained as a TFA salt; mixture of two
diastereomers (S
P
/R
P
).
1
H NMR (400 MHz, CD
3
OD): δ 8.38 and 8.37 (2s, 1H), 8.28 (s,
0.7H), 8.23 (s, 0.3H), 4.74-4.68 (m, 1H), 4.66-4.58 (m, 1H), 4.57-4.30 (m, 7H), 4.13-4.02
(m, 1H), 3.92 and 3.91 (2s, 3H).
31
P NMR (202 MHz, CD
3
OD): δ 14.3 (s, 0.64P), 12.9 (s,
0.36P); HRMS-FAB m/z [M+H]
+
calcd for C
13
H
19
N
6
O
6
P: 387.1176, found: 387.1181.

132
Propan-2-yl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxa-
phosphinan-2-yl}-(L)-serinate (2.14). Yield 30%. Obtained as a HCl salt; mixture of
two diastereomers (S
P
/R
P
).
1
H NMR (400 MHz, CD
3
OD): δ 8.47 (s, 1H), 8.41 (s, 0.6H),
8.36 (s, 0.4H), 5.19-5.13 (m, 1H), 4.73-4.32 (m, 9H), 4.20-4.05 (m, 1H), 1.39-1.33 (m,
6H).
31
P NMR (202 MHz, CD
3
OD): δ 13.8 (0.5P), 12.8 (0.5P). HRMS-FAB m/z [M+H]
+

calcd for C
15
H
23
N
6
O
6
P:

415.1489, found: 415.1495.

Methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-
2-yl}-(L)-threoninate (2.15). Yield 28%. Obtained as a TFA salt; mixture of two
diastereomers (S
P
/R
P
).
1
H NMR (400 MHz, CD
3
OD): δ 8.37 (s, 1H), 8.27 (s, 0.75H), 8.22
(s, 0.25H), 5.31-5.26 (m, 1H), 4.68-4.27 (m, 7H), 4.07-4.00 (m, 1H), 3.91 and 3.88 (2s,
3H), 1.57 (d, J = 6.7 Hz, 3H).
31
P NMR (202 MHz, CD
3
OD): δ 15.3 (s, 0.76P), 13.1 (s,
0.24P). HRMS-FAB m/z [M+H]
+
calcd for C
14
H
22
N
6
O
6
P:

401.1333, found: 401.1344.

Methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-
2-yl}-(L)-tyrosinate (2.16). Yield 34%. Obtained as a TFA salt; mixture of two
diastereomers (S
P
/R
P
, 1.9:1).
1
H NMR (400 MHz, CD
3
OD): δ 8.31 (s, 0.65H, N=CH-N
(S
P
)), 8.29 (s, 0.35H, N=CH-N (R
P
)), 8.23 (s, 0.65H, N=CH-N (S
P
)), 8.19 (s, 0.35H,
N=CH-N (R
P
)), 7.29-7.27 (m, 2H, aromatic), 7.22-7.19 (m, 2H, aromatic), 4.73 (ddd, J =
12.1, 12.1, 2.9 Hz, 0.65H, CH
a
H
b
O (S
P
)), 4.64 (m, 6.4H, CH
a
H
b
N (S
P
), CH
a
H
b
O (R
P
),
CH
a
H
b
N (R
P
), CH
a
H
b
N, CH
a
H
b
O, CH
a
H
b
P, CHO and CHNH
2
), 4.19 (dd, J = 14.8, 4.2
Hz, 0.65H, CH
a
H
b
P (S
P
)), 4.08 (dd, J = 15.3, 1.4 Hz, 0.35H, CH
a
H
b
P (R
P
)), 3.81 (s, 1H,
133
OCH
3
(R
P
)), 3.80 (s, 2H, OCH
3
(S
P
)), 3.31-3.23 (m, 1H, CH
a
H
b
(Tyr)), 3.17-3.11 (m, 1H,
CH
a
H
b
(Tyr)).
31
P NMR (202 MHz, CD
3
OD): δ 11.7 (0.65P (S
P
)), 10.1 (0.35P (R
P
)).
HRMS: m/z calcd 463.1489 (M + H)
+
, found 463.1499 (M + H)
+
. HPLC: t
R
17.34 min
(R
P
), 18.24 min (S
P
).

Methyl (L)-valyl-O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxa-
phosphinan-2-yl}-(L)-serinate (3.32) Yield 15%. Obtained as a TFA salt; mixture of
two diastereomers (S
P
/R
P
).
1
H NMR (400 MHz, CD
3
OD): δ 8.35 (s, 1H), 8.25 (s, 0.4H),
8.19 (s, 0.6H), 4.63-4.28 (m, 9H), 4.12-4.03 (m, 1H), 3.80 and 3.79 (2s, 3H), 2.29-2.19
(m, 1H), 1.15-1.07 (m, 6H).
31
P NMR (202 MHz, CD
3
OD): δ 14.1 (s, 0.4P), 12.9 (s,
0.6P).

Propan-2-yl (L)-valyl-O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-
dioxaphosphinan-2-yl}-(L)-serinate (3.33). Yield 19%. Obtained as a TFA salt; mixture
of two diastereomers (S
P
/R
P
).
1
H NMR (400 MHz, CD
3
OD): δ 8.34 (s, 1H), 8.24 (s,
0.5H), 8.17 (s, 0.5H), 5.10-5.01 (m, 1H), 4.89-4.83 (m, 1H), 4.67-4.29 (m, 8H), 4.08-4.04
(m, 1H), 3.79-3.78 (m, 1H), 2.29-2.23 (m, 1H), 1.32-1.28 (m, 6H), 1.15-1.09 (m, 6H).
31
P
NMR (202 MHz, CD
3
OD): δ 13.9 (0.4P), 12.8 (0.6P).

Synthesis of acyclic (S)-HPMPA and (S)-HPMPC prodrugs 2.28, 2.35 - 2.40. General
procedure. The suspension of cyclic (S)-HPMPA or (S)-HPMPC phenyl or tyrosine N-
alkyl amide prodrug 2.26, 2.29-2.34, 3.48 (0.3 mmol) in 8 mL of 14.8M NH
4
OH and 60
134
mL of acetonitrile was stirred at 45
o
C for 72 h (reaction was monitored by
31
P NMR).
After reaction completion volatiles were evaporated under vacuum and the residue was
washed with H
2
O to remove cHPMPA or cHPMPC. Remaining acyclic prodrugs
2.36-2.40 that are insoluble in H
2
O were further purified from corresponding tyrosine N-
alkyl amide using silica gel column chromatography [eluent: CH
2
Cl
2
:CH
3
OH (10-35%)].
Due to increased aqueous solubility, compounds 2.28, 2.35 and 3.49 were purified on a
Varian Dynamax Microsorb 100-8 C
18
HPLC column (5μm, 41.4 mm × 250 mm) using
isocratic 30% ACN in ammonium acetate buffer (pH 5.5), with a flow rate of 80 mL/min.
The product was detected at 260 nm and collected. After evaporation of the solvents the
compounds 2.28, 2.35 – 2.40 and 3.49 were crystallized with diethyl ether (Et
2
O),
filtered, and dried in vacuum yielding final products as white crystals.

O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)-
phosphoryl]-N-(2-methylpropyl)-(L)-tyrosinamide (2.28). Yield 56%. Obtained as
ammonium acetate salt.
1
H NMR (400 MHz, CD
3
OD): δ 8.17 (s, 1H, 2-H), 8.14 (s, 1H,
8-H), 7.11-7.09 (m, 2H, aromatic), 7.02-7.00 (m, 2H, aromatic), 4.44 (dd, J = 14.7, 3.9
Hz, 1H, CH
a
H
b
N), 4.37 (dd, J = 14.5, 6.8 Hz, 1H, CH
a
H
b
N), 3.86-3.62 (m, 5H, CHNH
2
,
CH
a
H
b
O, CHO, CH
a
H
b
P), 3.51 (dd, J = 12.5, 4.2 Hz, 1H, CH
a
H
b
P), 3.12-2.83 (m, 4H,
CH
2
(Tyr), CH
2
(iBu)), 1.76-1.69 (m, 1H, CH(CH
3
)
2
), 0.87 (t, J = 6.1 Hz, 6H, 2CH
3
).
13
C
NMR (126 MHz, CD
3
OD): δ 171.0 (CO), 155.9 (C
6
, adenine), 152.2 (C
2
, adenine), 151.8
(d,
2
J
CP
= 7.6 Hz, COP, aromatic), 149.5 (C
4
, adenine), 142.5 (C
8
, adenine), 130.4 (2CH,
aromatic), 129.8 (ipso-C, aromatic), 120.9 (d,
3
J
CP
= 3.8 Hz, 2CH, aromatic), 118.4 (C
5
,
135
adenine), 80.4 (d,
3
J
CP
= 11.8 Hz, CHO), 65.0 (d,
1
J
CP
= 162.3 Hz, CH
2
P), 60.2 (CH
2
OH),
55.3 (CHNH
2
), 46.7 (CH
2
N), 44.0 (CH
2
NH), 38.3 (CH
2
C
6
H
4
), 28.1 (CH), 19.1 (2CH
3
).
31
P NMR (202 MHz, CD
3
OD): δ 14.8 (s). HRMS: m/z calcd 522.2224 (M + H)
+
, found
522.2224 (M + H)
+
.

O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)-
phosphoryl]-N-octyl-(L)-tyrosinamide (2.35). Yield 44%. Obtained as ammonium
acetate salt.
1
H NMR (500 MHz, CD
3
OD): δ 8.20 (s, 1H, 2-H), 8.18 (s, 1H, 8-H), 7.14-
7.13 (m, 2H, aromatic), 7.06-7.04 (m, 2H, aromatic), 4.48 (dd, J = 14.5, 3.7 Hz, 1H,
CH
a
H
b
N), 4.41 (dd, J = 14.5, 7.0 Hz, 1H, CH
a
H
b
N), 3.92-3.68 (m, 5H, CHNH
2
,
CH
a
H
b
O, CH
a
H
b
P, CH
a
H
b
P, CHO), 3.56 (dd, J = 12.3, 4.5 Hz, 1H, CH
a
H
b
O), 3.23-3.18
(m, 2H, CH
2
NHCO), 3.13 (dd, J = 13.6, 5.5 Hz, 1H, CH
a
H
b
(Tyr)), 2.94 (dd, J = 13.6, 8.1
Hz, 1H, CH
a
H
b
(Tyr)), 1.51-1.48 (m, 2H, NHCH
2
CH
2
), 1.35-1.32 (m, 10H, 5CH
2
), 0.92
(t, J = 6.9 Hz, 3H, CH
3
CH
2
).
13
C NMR (126 MHz, CD
3
OD): δ 171.6 (CO), 158.0 (C
6
,
adenine), 154.5 (C
2
, adenine), 154.3 (d,
2
J
CP
= 7.5 Hz, COP, aromatic), 151.8 (C
4
,
adenine), 144.8 (C
8
, adenine), 132.1 (2CH, aromatic), 131.4 (ipso-C, aromatic), 123.3 (d,
3
J
CP
= 3.8 Hz, 2CH, aromatic), 120.6 (C
5
, adenine), 82.7 (d,
3
J
CP
= 11.8 Hz, CHO), 67.4
(d,
1
J
CP
= 162.6 Hz, CH
2
P), 62.5 (CH
2
OH), 57.2 (CHNH
2
), 46.3 (CH
2
N), 41.5 (CH
2
NH),
39.7 (CH
2
C
6
H
4
), 33.9 (CH
2
), 31.2 (2CH
2
), 31.1 (CH
2
), 28.9 (CH
2
), 24.6 (CH
2
), 15.3
(CH
3
).
31
P NMR (202 MHz, CD
3
OD): δ 14.7 (s). MS-ESI (m/z) 578.39 (M+H)
+
, 600.18
(M+Na)
+
. HPLC: t
R
, 4.55 min.

136
O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)-
phosphoryl]-N-dodecyl-(L)-tyrosinamide (2.36). Yield 25%.
1
H NMR (500 MHz,
CD
3
OD): δ 8.20 (s, 1H, 2-H), 8.19 (s, 1H, 8-H), 7.15-7.13 (m, 2H, aromatic), 7.06-7.04
(m, 2H, aromatic), 4.48 (dd, J = 14.5, 3.7 Hz, 1H, CH
a
H
b
N), 4.41 (dd, J = 14.6, 7.0 Hz,
1H, CH
a
H
b
N), 3.99 (dd, J = 8.3, 6.3 Hz, 1H, CHNH
2
), 3.90-3.86 (m, 1H, CHO), 3.82
(dd, J = 13.0, 9.3 Hz, 1H, CH
a
H
b
P), 3.76-3.69 (m, 2H, CH
a
H
b
O, CH
a
H
b
P,), 3.56 (dd, J =
12.4, 4.5 Hz, 1H, CH
a
H
b
O), 3.27-3.19 (m, 2H, CH
2
NHCO), 3.15 (dd, J = 14.1, 6.1 Hz,
1H, CH
a
H
b
(Tyr)), 2.95 (dd, J = 14.1, 8.5 Hz, 1H, CH
a
H
b
(Tyr)), 1.52-1.49 (m, 2H,
NHCH
2
CH
2
), 1.35-1.30 (m, 18H, 9CH
2
), 0.92 (t, J = 6.9 Hz, 3H, CH
3
CH
2
).
13
C NMR
(126 MHz, CD
3
OD): δ 170.6 (CO), 158.0 (C
6
, adenine), 154.5 (C
2
, adenine), 154.4 (d,
2
J
CP
= 7.6 Hz, COP, aromatic), 151.8 (C
4
, adenine), 144.8 (C
8
, adenine), 132.1 (2CH,
aromatic), 131.4 (ipso-C, aromatic), 123.5 (d,
3
J
CP
= 3.9 Hz, 2CH, aromatic), 120.5 (C
5
,
adenine), 82.7 (d,
3
J
CP
= 11.8 Hz, CHO), 67.5 (d,
1
J
CP
= 162.7 Hz, CH
2
P), 62.5 (CH
2
OH),
56.9 (CHNH
2
), 46.3 (CH
2
N), 41.5 (CH
2
NH), 39.1 (CH
2
C
6
H
4
), 33.9 (CH
2
), 31.64 (CH
2
),
31.62 (CH
2
), 31.60 (CH
2
), 31.5 (CH
2
), 31.3 (CH
2
), 31.2 (CH
2
), 31.1 (CH
2
), 28.9 (CH
2
),
24.6 (CH
2
), 15.3 (CH
3
).
31
P NMR (202 MHz, CD
3
OD): δ 14.9 (s). MS-ESI (m/z) 634.72
(M+H)
+
, 656.59 (M+Na)
+
. HPLC: t
R
, 3.32 min.

O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)-
phosphoryl]-N-tetradecyl-(L)-tyrosinamide (2.37). Yield 38%.
1
H NMR (500 MHz,
CD
3
OD): δ 8.20 (s, 1H, 2-H), 8.19 (s, 1H, 8-H), 7.15-7.13 (m, 2H, aromatic), 7.06-7.05
(m, 2H, aromatic), 4.48 (dd, J = 14.6, 3.8 Hz, 1H, CH
a
H
b
N), 4.42 (dd, J = 14.6, 7.0 Hz,
137
1H, CH
a
H
b
N), 3.99 (dd, J = 8.5, 6.2 Hz, 1H, CHNH
2
), 3.90-3.86 (m, 1H, CHO), 3.82
(dd, J = 13.1, 9.3 Hz, 1H, CH
a
H
b
P), 3.76-3.69 (m, 2H, CH
a
H
b
O, CH
a
H
b
P,), 3.56 (dd, J =
12.4, 4.6 Hz, 1H, CH
a
H
b
O), 3.26-3.19 (m, 2H, CH
2
NHCO), 3.16 (dd, J = 14.0, 6.0 Hz,
1H, CH
a
H
b
(Tyr)), 2.95 (dd, J = 14.1, 8.6 Hz, 1H, CH
a
H
b
(Tyr)), 1.52-1.49 (m, 2H,
NHCH
2
CH
2
), 1.36-1.30 (m, 22H, 11CH
2
), 0.92 (t, J = 7.0 Hz, 3H, CH
3
CH
2
).
13
C NMR
(126 MHz, CD
3
OD): δ 170.5 (CO), 158.0 (C
6
, adenine), 154.5 (C
2
, adenine), 154.4 (d,
2
J
CP
= 7.6 Hz, COP, aromatic), 151.8 (C
4
, adenine), 144.8 (C
8
, adenine), 132.1 (2CH,
aromatic), 131.4 (ipso-C, aromatic), 123.5 (d,
3
J
CP
= 3.9 Hz, 2CH, aromatic), 120.5 (C
5
,
adenine), 82.7 (d,
3
J
CP
= 11.8 Hz, CHO), 67.5 (d,
1
J
CP
= 162.6 Hz, CH
2
P), 62.5 (CH
2
OH),
56.9 (CHNH
2
), 46.3 (CH
2
N), 41.5 (CH
2
NH), 39.0 (CH
2
C
6
H
4
), 33.9 (CH
2
), 31.66 (2CH
2
),
31.64 (CH
2
), 31.62 (CH
2
), 31.60 (CH
2
), 31.54 (CH
2
), 31.33 (CH
2
), 31.26 (CH
2
), 31.1
(CH
2
), 28.9 (CH
2
), 24.6 (CH
2
), 15.3 (CH
3
).
31
P NMR (202 MHz, CD
3
OD): δ 14.9 (s).
MS-ESI (m/z) 662.74 (M+H)
+
, 684.58 (M+Na)
+
. HPLC: t
R
, 4.34 min.

O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)-
phosphoryl]-N-hexadecyl-(L)-tyrosinamide (2.38). Yield 46%.
1
H NMR (500 MHz,
CD
3
OD): δ 8.21 (s, 1H, 2-H), 8.19 (s, 1H, 8-H), 7.15-7.13 (m, 2H, aromatic), 7.07-7.05
(m, 2H, aromatic), 4.48 (dd, J = 14.6, 3.8 Hz, 1H, CH
a
H
b
N), 4.41 (dd, J = 14.6, 7.0 Hz,
1H, CH
a
H
b
N), 3.92 (dd, J = 8.6, 6.0 Hz, 1H, CHNH
2
), 3.89-3.85 (m, 1H, CHO), 3.81
(dd, J = 13.1, 9.3 Hz, 1H, CH
a
H
b
P), 3.76-3.69 (m, 2H, CH
a
H
b
O, CH
a
H
b
P,), 3.55 (dd, J =
12.4, 4.6 Hz, 1H, CH
a
H
b
O), 3.26-3.19 (m, 2H, CH
2
NHCO), 3.15 (dd, J = 14.1, 6.0 Hz,
1H, CH
a
H
b
(Tyr)), 2.93 (dd, J = 14.0, 8.5 Hz, 1H, CH
a
H
b
(Tyr)), 1.52-1.49 (m, 2H,
138
NHCH
2
CH
2
), 1.36-1.31 (m, 26H, 13CH
2
), 0.93 (t, J = 7.0 Hz, 3H, CH
3
CH
2
).
13
C NMR
(126 MHz, CD
3
OD): δ 171.3 (CO), 158.0 (C
6
, adenine), 154.5 (C
2
, adenine), 154.3 (d,
2
J
CP
= 7.7 Hz, COP, aromatic), 151.8 (C
4
, adenine), 144.8 (C
8
, adenine), 132.0 (2CH,
aromatic), 131.7 (ipso-C, aromatic), 123.4 (d,
3
J
CP
= 3.9 Hz, 2CH, aromatic), 120.5 (C
5
,
adenine), 82.7 (d,
3
J
CP
= 11.8 Hz, CHO), 67.4 (d,
1
J
CP
= 162.7 Hz, CH
2
P), 62.5 (CH
2
OH),
57.2 (CHNH
2
), 46.3 (CH
2
N), 41.5 (CH
2
NH), 39.5 (CH
2
C
6
H
4
), 33.9 (CH
2
), 31.65 (4CH
2
),
31.63 (CH
2
), 31.62 (CH
2
), 31.60 (CH
2
), 31.5 (CH
2
), 31.33 (CH
2
), 31.26 (CH
2
), 31.15
(CH
2
), 28.9 (CH
2
), 24.6 (CH
2
), 15.3 (CH
3
).
31
P NMR (202 MHz, CD
3
OD): δ 14.9 (s).
MS-ESI (m/z) 690.89 (M+H)
+
, 712.68 (M+Na)
+
. HPLC: t
R
, 6.45 min.

O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)-
phosphoryl]-N-octadecyl-(L)-tyrosinamide (2.39). Yield 47%.
1
H NMR (500 MHz,
CD
3
OD): δ 8.20 (s, 1H, 2-H), 8.19 (s, 1H, 8-H), 7.15-7.13 (m, 2H, aromatic), 7.06-7.05
(m, 2H, aromatic), 4.48 (dd, J = 14.6, 3.8 Hz, 1H, CH
a
H
b
N), 4.42 (dd, J = 14.6, 7.0 Hz,
1H, CH
a
H
b
N), 3.99 (dd, J = 8.5, 6.2 Hz, 1H, CHNH
2
), 3.90-3.86 (m, 1H, CHO), 3.82
(dd, J = 13.1, 9.3 Hz, 1H, CH
a
H
b
P), 3.76-3.69 (m, 2H, CH
a
H
b
O, CH
a
H
b
P,), 3.56 (dd, J =
12.4, 4.6 Hz, 1H, CH
a
H
b
O), 3.27-3.19 (m, 2H, CH
2
NHCO), 3.16 (dd, J = 14.1, 6.0 Hz,
1H, CH
a
H
b
(Tyr)), 2.95 (dd, J = 14.1, 8.6 Hz, 1H, CH
a
H
b
(Tyr)), 1.52-1.49 (m, 2H,
NHCH
2
CH
2
), 1.36-1.30 (m, 30H, 15CH
2
), 0.93 (t, J = 6.9 Hz, 3H, CH
3
CH
2
).
13
C NMR
(126 MHz, CD
3
OD): δ 170.5 (CO), 158.0 (C
6
, adenine), 154.5 (C
2
, adenine), 154.4 (d,
2
J
CP
= 7.7 Hz, COP, aromatic), 151.8 (C
4
, adenine), 144.8 (C
8
, adenine), 132.1 (2CH,
aromatic), 131.4 (ipso-C, aromatic), 123.5 (d,
3
J
CP
= 3.9 Hz, 2CH, aromatic), 120.5 (C
5
,
139
adenine), 82.7 (d,
3
J
CP
= 11.8 Hz, CHO), 67.5 (d,
1
J
CP
= 162.6 Hz, CH
2
P), 62.5 (CH
2
OH),
56.9 (CHNH
2
), 46.3 (CH
2
N), 41.6 (CH
2
NH), 39.1 (CH
2
C
6
H
4
), 33.9 (CH
2
), 31.65 (7CH
2
),
31.62 (CH
2
), 31.61 (CH
2
), 31.55 (CH
2
), 31.34 (CH
2
), 31.26 (CH
2
), 31.15 (CH
2
), 28.9
(CH
2
), 24.6 (CH
2
), 15.3 (CH
3
).
31
P NMR (202 MHz, CD
3
OD): δ 14.9 (s). MS-ESI (m/z)
718.69 (M+H)
+
, 740.52 (M+Na)
+
. HPLC: t
R
, 10.68 min.

O-[([[(2S)-1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yl]oxy]methyl)-
(hydroxy)phosphoryl]-N-hexadecyl-(L)-tyrosinamide (2.40). Yield 45%.
1
H NMR
(500 MHz, CD
3
OD): δ 7.63 (s, 1H, 6-H), 7.19-7.17 (m, 2H, aromatic), 7.13-7.11 (m, 2H,
aromatic), 5.82 (s, 1H, 5-H), 4.06 (dd, J = 13.9, 3.4 Hz, 1H, CH
a
H
b
N), 3.90-3.87 (m, 1H,
CHNH
2
), 3.85-3.80 (m, 2H, CH
a
H
b
N, CH
a
H
b
P), 3.76-3.68 (m, 3H, CH
a
H
b
O, CH
a
H
b
P,
CHO), 3.53 (dd, J = 12.0, 3.8 Hz, 1H, CH
a
H
b
O), 3.21 (t, 2H, CH
2
CH
2
NH), 3.13 (dd, J =
13.9, 6.0 Hz, 1H, CH
a
H
b
(Tyr)), 2.91 (dd, J = 14.0, 8.5 Hz, 1H, CH
a
H
b
(Tyr)), 1.53-1.50
(m, 2H, CH
2
CH
2
NH), 1.34-1.32 (m, 26H, 13CH
2
), 0.93 (t, J = 6.9 Hz, 3H, CH
3
CH
2
).
13
C
NMR (126 MHz, CD
3
OD): δ 172.0 (CONH), 168.8 (NCON), 160.2 (CNH
2
), 154.3 (d,
2
J
CP
= 7.3 Hz, COP, aromatic), 149.8 (C
6
, cytosine), 132.2 (2CH, aromatic), 123.3 (d,
3
J
CP
= 4.0 Hz, 2CH, aromatic), 96.5 (C
5
, cytosine), 82.3 (d,
3
J
CP
= 12.2 Hz, CHO), 67.2
(d,
1
J
CP
= 161.6 Hz, CH
2
P), 62.5 (CH
2
OH), 57.3 (CHNH
2
), 52.4 (CH
2
N), 41.5 (CH
2
NH),
39.9 (CH
2
C
6
H
4
), 33.9 (CH
2
), 31.66 (5CH
2
), 31.62 (CH
2
), 31.61 (CH
2
), 31.56 (CH
2
),
31.34 (CH
2
), 31.28 (CH
2
), 31.18 (CH
2
), 28.9 (CH
2
), 24.6 (CH
2
), 15.3 (CH
3
).
31
P NMR
(202 MHz, CD
3
OD): δ 14.7 (s). MS-ESI (m/z) 666.49 (M+H)
+
, 688.35 (M+Na)
+
. HPLC:
t
R
, 6.09 min.
140
Phenyl hydrogen ({[(2S)-1-(6-amino-9H-purin-9-yl)-3-hydroxypropan-2-
yl]oxy}methyl)-phosphonate (3.49). Yield 49%. Obtained as ammonium acetate salt.
1
H
NMR (500 MHz, CD
3
OD): δ 7.85 (s, 1H, 2-H), 7.82 (s, 1H, 8-H), 6.87-6.83 (m, 2H,
aromatic), 6.71-6.65 (m, 3H, aromatic), 4.09 (dd, J = 14.5, 3.8 Hz, 1H, CH
a
H
b
N), 4.03
(dd, J = 14.6, 7.1 Hz, 1H, CH
a
H
b
N), 3.48-3.44 (m, 1H, CHO), 3.41 (dd, J = 13.1, 9.4 Hz,
1H, CH
a
H
b
O), 3.36 (dd, J = 12.4, 4.2 Hz, 1H, CH
a
H
b
P), 3.30 (dd, J = 13.0, 9.1 Hz, 1H,
CH
a
H
b
O), 3.17 (dd, J = 12.4, 4.5 Hz, 1H, CH
a
H
b
P).
13
C NMR (126 MHz, CD
3
OD): δ
155.8 (C
6
, adenine), 152.5 (d,
2
J
CP
= 7.5 Hz, COP, aromatic), 152.2 (C
2
, adenine), 149.5
(C
4
, adenine), 142.5 (C
8
, adenine), 128.7 (2CH, aromatic), 123.0 (CH, aromatic), 120.6
(d,
3
J
CP
= 4.0 Hz, 2CH, aromatic), 118.3 (C
5
, adenine), 80.4 (d,
3
J
CP
= 12.0 Hz, CHO),
64.8 (d,
1
J
CP
= 162.1 Hz, CH
2
P), 60.1 (CH
2
OH), 46.7 (CH
2
N), 44.1 (CH
2
NH).
31
P NMR
(202 MHz, CD
3
OD): δ 14.5 (s). MS-ESI (m/z) 378.18 (M-H)
-
, 378.09 (M-H)
-
.

Synthesis of acyclic Br-(S)-HPMPA prodrugs 3.50 and 3.51. General procedure. The
suspension of cyclic (S)-HPMPA phenyl or tyrosine N-iso-butyl amide prodrug 2.26,
3.48 (0.08 mmol) in 1.2 mL of bromotrimethylsilane and 40 mL of acetonitrile was
refluxed
o
C for 16 h. After completion, the volatiles were removed under vacuum and 15
mL of MeOH were added to the residue. Sovents were removed under vacuum and a new
portion of 15 mL of MeOH was added to the residue. Solvent was removed under
vacuum and remaining acyclic prodrugs 3.50 and 3.51 were dried in vacuum. Compound
3.51 was further purified on a Varian Dynamax Microsorb 100-8 C
18
HPLC column
(5μm, 41.4 mm × 250 mm) using gradient 0-30% ACN in ammonium acetate buffer (pH
141
5.5), with a flow rate of 75 mL/min. The product was detected at 260 nm and collected.
After evaporation of the solvents the compound 3.51 was lyophilized yielding final
product as white crystals.

Phenyl hydrogen ({[(2S)-1-(6-amino-9H-purin-9-yl)-3-bromopropan-2-yl]oxy}-
methyl)-phosphonate (3.50).
1
H NMR (500 MHz, CD
3
OD): δ 8.24 and 8.23 (2s, 2H, 2-
H and 8-H), 7.24-7.21 (m, 2H, aromatic), 7.09-7.06 (m, 1H, aromatic), 6.97-6.96 (m, 2H,
aromatic), 4.57 (dd, J = 14.6, 3.2 Hz, 1H, CH
a
H
b
N), 4.44 (dd, J = 14.6, 8.0 Hz, 1H,
CH
a
H
b
N), 4.13-4.08 (m, 1H, CHO), 4.03-3.98 (m, 1H, CH
a
H
b
P), 3.65 (dd, J = 14.0, 9.3
Hz, 1H, CH
a
H
b
P), 3.56-3.55 (m, 2H, CH
2
Br).
31
P NMR (202 MHz, CD
3
OD): δ 15.8 (s).
MS-ESI m/z calcd 442.03 (M+H)
+
, found 442.10 (M+H)
+
.

O-[({[(2S)-1-(6-amino-9H-purin-9-yl)-3-bromopropan-2-yl]oxy}methyl)(hydroxy)-
phosphoryl]-N-(2-methylpropyl)-(L)-tyrosinamide (3.51). Obtained as ammonium
acetate salt. Yield 32%.
1
H NMR (500 MHz, CD
3
OD): δ 8.24 (s, 1H, 2-H), 8.19 (s, 1H,
8-H), 7.13-7.11 (m, 2H, aromatic), 7.05-7.04 (m, 2H, aromatic), 4.58 (dd, J = 14.6, 3.4
Hz, 1H, CH
a
H
b
N), 4.44 (dd, J = 14.6, 6.9 Hz, 1H, CH
a
H
b
N), 4.15-4.10 (m, 1H, CHO),
3.92-3.85 (m, 2H, CHNH
2
,CH
a
H
b
P), 3.65 (dd, J = 13.2, 9.2 Hz, 1H, CH
a
H
b
P), 3.61-3.54
(m, 2H, CH
2
Br), 3.14 (dd, J = 14.0, 5.9 Hz, 1H, CH
a
H
b
(Tyr)), 3.07 (dd, J = 13.2, 6.9 Hz,
1H, CH
a
H
b
NHCO), 3.00 (dd, J = 13.2, 7.0 Hz, 1H, CH
a
H
b
NCO), 2.89 (dd, J = 13.9, 8.8
Hz, 1H, CH
a
H
b
(Tyr)), 1.79-1.73 (m, 1H, CH(CH
3
)
2
), 0.89 (t, J = 6.6 Hz, 6H, 2CH
3
).
31
P
142
NMR (202 MHz, CD
3
OD): δ 13.9 (s). MS-ESI m/z calcd 584.14 (M+H)
+
, found 584.21
(M+H)
+
.

Synthesis of the tyrosine N-iso-butyl amide thio (S)-HPMPA prodrug 3.52. The
suspension of 74 mg (0.13 mmol) of (L)-TyrNHi-Bu-Br-(S)-HPMPA 3.51 in 4.0 mL of
water was mixed with 340 mg (1.4 mmol) of sodium sulfide nonahydrate. Obtained
reaction mixture was stirred overnight at r. t under N
2
atmosphere. The reaction was
monitored by HPLC. After reaction completion, the mixture was separated on a Varian
Dynamax Microsorb 100-8 C
18
HPLC column (5μm, 41.4 mm × 250 mm) using gradient
0-30% ACN in ammonium acetate buffer (pH 5.5), with a flow rate of 75 mL/min. The
product 3.52 was detected at 260 nm and collected. After evaporation of the solvents the
compound 3.52 was lyophilized yielding final product as white crystals.

O-[({[(2S)-1-(6-amino-9H-purin-9-yl)-3-sulfanylpropan-2-yl]oxy}methyl)(hydroxy)-
phosphoryl]-N-(2-methylpropyl)-(L)-tyrosinamide (3.52). Yield 25%. Obtained as
ammonium acetate salt.
1
H NMR (500 MHz, CD
3
OD): 8.26 (s, 1H, 2-H), 8.17 (s, 1H,
8-H), 7.12-7.10 (m, 2H, aromatic), 7.05-7.03 (m, 2H, aromatic), 4.59 (dd, J = 14.6, 3.2
Hz, 1H, CH
a
H
b
N), 4.43 (dd, J = 14.6, 6.2 Hz, 1H, CH
a
H
b
N), 4.09-4.05 (m, 1H, CHO),
3.97 (dd, J = 8.2, 6.2 Hz, 1H, CHNH
2
), 3.87 (dd, J = 13.1, 9.0 Hz, 1H, CH
a
H
b
P), 3.67
(dd, J = 13.0, 9.7 Hz, 1H, CH
a
H
b
P), 3.11 (dd, J = 14.1, 6.1 Hz, 1H, CH
a
H
b
NCO), 3.06
(dd, J = 13.2, 6.9 Hz, 1H, CH
a
H
b
(Tyr)), 2.98 (dd, J = 13.2, 7.0 Hz, 1H, CH
a
H
b
(Tyr)),
2.95-2.86 (m, 3H, CH
a
H
b
NCO and CH
2
SH), 1.78-1.70 (m, 1H, CH(CH
3
)
2
), 0.87 (t,
143
J = 6.6 Hz, 6H, 2CH
3
).
13
C NMR (126 MHz, CD
3
OD): δ 169.4 (CO), 155.6 (C
6
,
adenine), 152.3 (C
2
, adenine), 152.0 (d,
2
J
CP
= 7.5 Hz, COP, aromatic), 149.6 (C
4
,
adenine), 142.7 (C
8
, adenine), 129.9 (2CH, aromatic), 129.6 (ipso-C, aromatic), 121.2 (d,
3
J
CP
= 3.5 Hz, 2CH, aromatic), 118.1 (C
5
, adenine), 78.8 (d,
3
J
CP
= 12.6 Hz, CHO), 65.1
(d,
1
J
CP
= 161.7 Hz, CH
2
P), 54.8 (CHNH
2
), 46.3 (CH
2
N), 45.1 (CH
2
NH), 39.8
(CH
2
C
6
H
4
), 37.2 (CH
2
SH), 28.1 (CH), 19.1 (2CH
3
).
31
P NMR (202 MHz, CD
3
OD):
δ 13.8 (s). MS-ESI m/z calcd 537.19 (M)
+
, found 537.39 (M)
+
. HPLC: t
R
, 17.41 min.

Synthesis of cyclic thio (S)-HPMPA prodrug 3.54. To a suspension of (S)-HPMPA 2.2
(0.6 mmol, 0.182 g) in dry DMF (10 mL), dry DIEA (11.5 mmol, 2.0 mL), benzyl
mercaptan (0.9 mmol) and PyBOP (1.2 mmol, 0.62 g) were added. The reaction mixture
was stirred under N
2
at 40 °C for 2 h. The reaction was monitored by
31
P NMR, and
additional portions of PyBOP were added as necessary. After reaction completion, DMF
and DIEA were removed under vacuum. The residue was washed with diethyl ether and
purified using silica gel column chromatography [CH
2
Cl
2
, then CH
2
Cl
2
:acetone (2:1),
then CH
2
Cl
2
:acetone:CH
3
OH (6:3:1)]. Solvents were removed under vacuum and
obtained phosphonate thio-ester intermediate was used in the next step without further
purification. A solution of thiophenol:Et
3
N:dioxane
20
(2:2:1) (6 ml) was added to the
intermediate and obtained reaction mixture was stirred at r. t. overnight. The volatiles
were removed under vacuum the residue was crystallized using Et
2
O. The final product
was purified on a Varian Dynamax Microsorb 100-8 C
18
HPLC column (5μm, 41.4 mm ×
250 mm) using gradient 0-30% ACN in ammonium acetate buffer (pH 5.5), with a flow
144
rate of 75 mL/min. The product 3.54 was detected at 260 nm and collected. After
evaporation of the solvents the compound 3.54 was lyophilized yielding final product as
white crystals.

9-[((5S)-2-oxido-2-sulfanyl-1,4,2-dioxaphosphinan-5-yl)methyl]-9H-purine-6-amine
(3.54) Obtained as ammonium acetate salt, mixture of two diastereomers (S
P
/R
P
).
1
H
NMR (400 MHz, D
2
O): δ 8.24 (s, 1H, 2-H), 8.17 (s, 1H, 8-H), 4.40 (m, 5H), 3.99-3.85
(m, 1H), 3.76-3.69 (m, 1H).
31
P NMR (202 MHz, CD
3
OD): δ 60.8 (s, 0.36P), 58.5 (s,
0.64P).

145
3.7 References
1. Holy, A.; Rosenberg, I. Acyclic nucleotide analogs. Part I. Synthesis of isomeric
and enantiomeric O-phosphonylmethyl derivatives of 9-(2,3-dihydroxypropyl)adenine.
Collect. Czech. Chem. Commun. 1987, 52, 2775-91.

2. Webb, R. R., II; Martin, J. C. A convenient synthesis of S-HPMPA [(S)-9-(3-
hydroxy-2-phosphonylmethoxypropyl)adenine]. Tetrahedron Lett. 1987, 28, 4963-4.
3. Webb, R. R., II. The bis-trityl route to (S)-HPMPA. Nucleosides Nucleotides
1989, 8, 619-24.

4. Peterson, L. W. Peptidomimetic prodrugs of cidofovir: Design, synthesis,
transport, mechanism of activation, and antiviral activity. Ph. D. Thesis, University of
Southern California, Los Angeles, 2009.

5. 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, 155-8.

6. Peterson, L. W.; Sala-Rabanal, M.; Krylov, I. S.; Serpi, M.; Kashemirov, B. A.;
McKenna, C. E. Serine Side Chain-Linked Peptidomimetic Conjugates of Cyclic
HPMPC and HPMPA: Synthesis and Interaction with hPEPT1. Mol. Pharmaceutics
2010, 7, 2349-2361.
7. McKenna, C. E.; Kashemirov, B. A.; Eriksson, U.; Amidon, G. L.; Kish, P. E.;
Mitchell, S.; Kim, J.-S.; Hilfinger, J. M. Cidofovir peptide conjugates as prodrugs. J.
Organomet. Chem. 2005, 690, 2673-2678.

8. Grimm, J. B.; Wilson, K. J.; Witter, D. J. Suppression of racemization in the
carbonylation of amino acid-derived aryl triflates. Tetrahedron Lett. 2007, 48, 4509-
4513.

9. Krylov, I. S.; Kashemirov, B. A.; McKenna, C. E. In Solid-phase synthesis of
peptidomimetic antiviral nucleotide prodrugs, 2009; American Chemical Society: 2009;
pp MEDI-338.

10. Novachek, K. A.; Myers, A. I. A convenient procedure for the reduction of S-
(+)silyl serine methyl ester to chiral serinol derivatives. Tetrahedron Lett. 1996, 37,
1743-6.

11. Hakimelahi, G. H.; Jarrahpour, A. A. Synthesis of ethyl cis-2-
[(diethoxyphosphoryl)methyl]-7-oxo-3-phenyl-6-phthalimido-1-azabicyclo[3.2.0]hept-3-
ene-2-carboxylate and methyl cis-2-bromo-3-methyl-8-oxo-7-phthalimido-4-oxa-1-
azabicyclo[4.2.0]octane-2-carboxylate. Helv. Chim. Acta 1989, 72, 1501-5.

146
12. Djuric, S. W. A mild and convenient procedure for the N-formylation of
secondary amines using organosilicon chemistry. J. Org. Chem. 1984, 49, 1311-12.

13. Abbenante, G.; Leung, D.; Bond, T.; Fairlie, D. P. An efficient Fmoc strategy for
the rapid synthesis of peptide para-nitroanilides. Lett. Pept. Sci. 2001, 7, 347-351.

14. Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to the
Synthesis of Peptides and Proteins. CRC: 1997; p 297.

15. Thieriet, N.; Guibe, F.; Albericio, F. Solid-Phase Peptide Synthesis in the Reverse
(Nâ†’C) Direction. Org. Lett. 2000, 2, 1815-1817.

16. Thieriet, N.; Alsina, J.; Giralt, E.; Guibe, F.; Albericio, F. Use of Alloc-amino
acids in solid-phase peptide synthesis. Tandem deprotection-coupling reactions using
neutral conditions. Tetrahedron Lett. 1997, 38, 7275-7278.

17. Campbell, D. A. The synthesis of phosphonate esters; an extension of the
Mitsunobu reaction. J. Org. Chem. 1992, 57, 6331-5.

18. Williams, M.; Krylov, I. S.; Zakharova, V. M.; Serpi, M.; Peterson, L. W.;
Krecmerova, 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.

19. Fevig, T. L.; Phillips, W. G.; Lau, P. H. A Novel and Expeditious Approach to
Thiophene-3-carboxylates. J. Org. Chem. 2001, 66, 2493-2497.

20. Lesnikowski, Z. J.; Jaworska, M. M. Studies on stereospecific formation of P-
chiral internucleotide linkage. Synthesis of (Rp,Rp)- and (Sp,Sp)-
thymidylyl(3',5')thymidylyl(3',5')thymidine di(O,O-phosphorothioate) using 2-
nitrobenzyl group as a new S-protection. Tetrahedron Lett. 1989, 30, 3821-4.

147
CHAPTER 4
*

The structure of cyclic nucleoside phosphonate ester prodrugs: an
inquiry

4.1 Introduction
Six-membered phosphonate rings constitute an essential structural feature of cyclic
prodrug diesters described in Chapters 2 and 3. More generally, six-membered ring
phosphates and related compounds have been a durable topic of interest in organic
chemistry.
1-9
HPMP-based ANPs, such as (S)-HPMPC 4.1 and (S)-HPMPA 4.2 are
chiral compounds, exerting their maximal antiviral effect as a single (S)-enantiomer.
12

Intramolecular cyclization followed by esterification of the remaining P-OH group in the
cyclic form of 4.1 and 4.2 by the promoiety leads to the formation of a new stereocenter
at the phosphorus atom, resulting in generation of (S
p
)- and (R
p
)-diastereomers (4.3, 4.4;
Figure 4.1).

Figure 4.1 Structures of (S)-HPMPC 4.1 and (S)-HPMPA 4.2 and corresponding single amino
acid and dipeptide prodrug forms 4.3 and 4.4.

*
Adapted with permission from Krylov, I. S.; Zakharova, V. M.; Serpi, M.; Haiges, R.; Kashemirov, B. A.;
McKenna, C. E. J. Org. Chem. 2012, 77, 684-689. Copyright 2012 American Chemical Society.
Information on author contributions can be found in the Acknowledgements section.
148
The diastereomers of cyclic 4.1 aryl esters
22
and also amino acid and dipeptide
prodrugs
17, 19, 21
4.3 and 4.4 have significantly different pharmacokinetic properties (see
Chapter 2). It was clearly important to define the phosphorus configuration and
phostonate
23
ring conformations of these prodrugs in order to understand their structure-
activity relationships.
1
H,
13
C,
31
P and 2D NMRs
22, 24, 25
and dipole moment calculations
21

have been previously applied to predict their absolute configurations (AC). The prodrug
diastereomers of 4.5 (Scheme 4.1) were distinguished as axial/equatorial
22
depending on
the stipulated position of the exocyclic aryl phosphonate ester group, with the axial
isomer assigned to the upfield
31
P NMR peak (Figure 4.2). Conversely, phosphonate
ester prodrugs of cyclic 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-
diaminopurine (HPMPDAP) and cyclic 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-
5-azacytosine were distinguished as cis/trans
24, 25
with the cis isomer assigned to the
downfield
31
P chemical shift (Figure 4.2).

Figure 4.2 Proposed cis/trans
24, 25
and axial/equatorial
22

31
P NMR correlations for different
cyclic ANP drug diastereomers.

A key assumption underlying the axial/equatorial descriptives frequently used
22, 26-28
to
distinguish cyclic (S)-HPMPC and (S)-HPMPA diastereomeric diesters is that the
nucleobase is invariably equatorial. However, trans-2-phenyloxy-2-oxo-5-phenyl-1,3,2-
149
dioxaphosphorinane
29
and trans-2-methoxy-2-oxo-5-tert-butyl-1,3,2-
dioxaphosphorinane
30
(a, a) conformers have been observed in both the solution and
solid states, suggesting that in six-membered cyclic phosphates the anomeric axial
preference of the PhO- or CH
3
O-ester groups can compensate for steric repulsions when
a 5-phenyl or 5-tert-butyl group is axial.

4.2 Synthesis and structural evaluation of individual diastereomers of model
phenyl (S)-HPMPA and (S)-HPMPC prodrugs
In an attempt to elucidate this seeming contradiction, we have synthesized and isolated
and analyzed by X-ray crystallography the individual diastereomers of the phenyl ester
prodrugs 4.5 and 4.6 (Scheme 4.1, (R
p
)-4.5, (R
p
)-4.6 and (S
p
)-4.6),
31
which may be
regarded as model compounds for 4.3 or 4.4 where the promoiety is tyrosine.
32

Scheme 4.1 Preparation of individual cyclic (S)-HPMPC and (S)-HPMPA phenyl ester
diastereomers ((R
p
)-4.5, -4.6 and (S
p
)-4.5, -4.6). Reagents and conditions: a) PhOH, PyBOP,
N,N-diisopropylethylamine, DMF, 40 °C, 2 h; b) Cs
2
CO
3
, DMF, 0.1 eq PhOH; recrystallization
from CH
3
OH/acetone (for (R
p
)-4.5) or i-PrOH/EtOAc (for (R
p
)-4.6); c) recrystallization from
CH
3
OH/acetone/hexane (for (S
p
)-4.5) or CH
3
CN (for (S
p
)-4.6). Compounds (R
p
)-4.5 and (S
p
)-4.5
were synthesized by Dr. Valeria Zakharova.
150
The solution phase conformations of the 4.5 and 4.6 diastereomers were studied by
correlating the
31
P NMR  values with the X-ray structures and by examining solvent
polarity effects on the
3
J
HCOP
and
3
J
HH
coupling constant values. Esters 4.5 and 4.6 were
prepared by our previously described procedure for the synthesis of tyrosine side chain
ester conjugates of 4.1 and 4.2.
17
After PyBOP-mediated coupling, 4.5 and 4.6 were
obtained as diastereomeric mixtures enriched with the diastereomer having the more
downfield
31
P NMR resonance (11.2-11.3 ppm) (Scheme 4.1). These mixtures could be
substantially enriched in the initially minor diastereomer (
31
P NMR resonance 9.8-10.1
ppm) by treating with Cs
2
CO
3
and 0.1 eq of phenol in DMF at r.t. The pure (R
p
)-4.5, -4.6
and (S
p
)-4.5, -4.6 diastereomers were obtained by recrystallization and characterized by
1
H,
13
C,
31
P and 2D HSQC NMR, LC-MS and by X-ray crystallography.

(R
p
)-4.5 crystallizes with one molecule of methanol in the monoclinic space group P2(1)
with a = 6.9573(7) Å, b = 6. 9976(7) Å, c = 34.145(4) Å, and  = 90.050(3)° (Z = 4)
(Figure 4.3). (R
p
)-4.6 and (S
p
)-4.6 crystallize in the monoclinic space group P2(1) with
the unit cell parameters a = 8.2341(9) Å, b = 8.3291(9) Å, c = 12.9089(14) Å,
 = 104.660(2)° (Z = 2) (Figure 4.4) and a = 5.7894(15) Å, b = 43.207(11) Å,
c = 6.8100(18) Å,  = 112.184(4)° (Z = 4) (Figure 4.5), respectively.

151

Figure 4.3. X-ray crystal structure of (R
p
)-4.5. Ellipsoids enclose 50% probability.

In the (R
p
)-diastereomers of 4.5 and 4.6, the PhO-ester group is axial and the nucleobase
is equatorial (Figures 4.3 and 4.4). However, in the (S
p
)-4.6 diastereomer (Figure 4.5) the
nucleobase is axial not equatorial as was previously postulated for its (S)-HPMPC
analogue (S
p
)-4.5.
22

Figure 4.4. X-ray crystal structure of (R
p
)-4.6. Ellipsoids enclose 50% probability.

152
With the solid phase structures of the (R
p
)-diastereomer of 4.5 and the (R
p
)- and
(S
p
)-distereomers of 4.6 in hand, we then turned to analysis
33-36
of their behavior in
solution. Assignment of the signals in their
1
H and
13
C NMR spectra was based in part on
2D HSQC NMR experiments. Karplus relationships between the HCOP dihedral angle
and the
3
J
HCOP
coupling constants for a variety of six-membered phosphorus heterocycles,
e.g. 1,3,2-dioxaphosphorinanes have been previously defined experimentally.
29, 35, 37
The
3
J
HH
,
3
J
HCOP
and
2
J
HP
values for the individual 4.5 and 4.6 diastereomers were measured
in two solvents of differing polarities, CDCl
3
(4.81) and CD
3
CN (37.5). The NMR data
are summarized in Table 4.1.

Table 4.1
1
H NMR parameters for (R
p
)- and (S
p
)-diastereomers of 4.5 and 4.6 recorded at
25 ºC and 500 MHz.
Compd. Solvent δ (A) δ (B) δ (C)

δ (D)

δ (X) δ (
31
P) J
AP
J
BP
J
CP
J
DP
J
AX
J
BX

(R
p
)-4.5
CD
3
CN 4.47 4.43 4.24 4.02 4.15 9.1 16.6 2.3 10.8 1.3 2.7 10.3
CDCl
3
4.44 4.32 4.17 3.92 4.08 8.0 17.6 1.4 11.0 1.1 2.3 10.5
(S
p
)-4.5
CD
3
CN 4.62 4.41 4.36 4.12 4.16 10.5 11.2 8.6 5.7 5.2 2.9 6.7
CDCl
3
4.46 4.40 4.21 3.96 4.16 9.1 14.3 5.6 8.8 2.2 2.9 8.5
(R
p
)-4.6
CD
3
CN 4.52 4.41 4.26 4.02 4.29 8.9 17.2 1.6 10.9 1.4 2.1 9.3
CDCl
3
4.50 4.33 4.28 4.00 4.20 7.6 17.4 1.6 10.8 1.2 2.1 10.5
(S
p
)-4.6
CD
3
CN 4.71 4.47 4.43 4.09 4.34 10.3 10.0 9.9 4.6 6.1 2.9 5.9
CDCl
3
4.54 4.43 4.31 3.97 4.19 9.0 12.7 7.1 7.2 3.8 2.7 7.7

The
1
H NMR coupling constant values for (R
p
)-diastereomers of 4.5 and 4.6 were
insensitive to the change in solvent polarity, suggesting that their dominant conformation
in solution is I, the solid state structure found by X-ray crystallography, which is favored
by both steric and electronic factors (Figure 4.6 (left), Table 4.1). The vicinal P-O-C-H
153
coupling constant values (J
AP
= 16.0-17.4 Hz, J
BP
= 1.4-2.3 Hz) are consistent with an
antiperiplanar orientation of the H
A
and P atoms and a synclinal relation of the H
B
and P
atoms confirming a chair conformation.
34
The H
B
-C-C-H
X
dihedral angle is close to 180º
(J
BX
= 9.3-10.5 Hz, Table 4.1) corresponding to a sterically favorable equatorial position
of the nucleobase.
38
The P-OPh group is axial in this structure, as predicted
4
by the
anomeric effect.

Figure 4.5. X-ray crystal structure of (S
p
)-4.6. Ellipsoids enclose 50% probability.

Antithetically, the
3
J
HCOP
and
3
J
HH
values of 4.5 and 4.6 and their solvent polarity
dependence in the (S
p
)-diastereomers are consistent with a system of equilibrating
conformers as shown in Figure 4.6 (right).
39
In these stereoisomers, the equatorial
preference of the nucleobase (due to a steric effect
38
) and the axial preference of the OPh
group (due to the anomeric effect) are opposed, thus stabilizing both chair conformations
154
III and IV (Figure 4.6 (right)). Alternatively, these effects could mutually reinforce
stabilization of twist conformations V, where the OPh remains axial and the nucleobase is
pseudoequatorial.

Figure 4.6 Possible conformer equilibria of (R
p
)-4.5, -4.6 (I-II) (left) and (S
p
)-4.5, -4.6 (III-V)
(right) in solution (CDCl
3
stabilizes conformer III), based on analysis of solvent effect on
1
H
NMR coupling constant values. I is favored by both steric (equatorial nucleobase) and electronic
(anomeric preference for axial P-OPh) effects.

The mole fractions (N) of chair conformations III and IV in CDCl
3
or CD
3
CN were
estimated by measuring the time averaged coupling constants at 25 C.
5
Equillibrium of
III and IV interchanges the (S
p
)-4.5 and (S
p
)-4.6 H
A
and H
B
protons, decreasing J
AP
and
increasing J
BP
, while the sum J
AP
+ J
BP
remains nearly equal to the sum of the
corresponding constants observed for the 4.5 and 4.6 (R
p
)-diastereomers (Figure 4.6). For
(S
p
)-6 in CDCl
3
, J
AP
(12.7 Hz) > J
BP
(7.1 Hz). This difference becomes smaller in the
more polar solvent, CD
3
CN, indicating that the conformer ratio approaches 1. The same
trend is observed for (S
p
)-4.5. In contrast, the (R
p
)-diastereomers of 4.5 and 4.6 show no
solvent-dependent differences in their coupling constants. On the reasonable assumption
155
that the (R
p
)-diastereomers essentially retain one conformation, namely that shown in the
X-ray structure, we can make the following approximations: J
AP
(I) = J
AP
(III); J
BP
(I) =
J
AP
(IV). Using these values and equations (1)-(4),
35, 40
the ratio of the conformers III and
IV can be estimated (Table 4.2).
Table 4.2 Estimated population of conformation III for (S
p
)-4.5 and (S
p
)-4.6 diastereomers.
Assumed J, Hz Percentage (%)
of III based on
J
AP
(obsd)
Percentage (%)
of III based on
J
BP
(obsd)
Average
% of III
Compd. Solvent J
AP
(III) J
BP
(III) J
AP
(IV) J
BP
(IV)
(S
p
)-4.5
CD
3
CN
17.6 1.4 1.4 17.6
60 56 58
CDCl
3
80 74 77
(S
p
)-4.6
CD
3
CN
17.6 1.4 1.4 17.6
53 47 50
CDCl
3
70 65 68

The calculations reveal that for (S
p
)-4.5 and (S
p
)-4.6 in CD
3
CN

50-58% of III is present,
whereas in CDCl
3
, the proportion of III increases to 68-77%. The assignment of structure
III rather than IV to the major conformer is based upon the relatively large values (5.9
Hz or 6.7 Hz) observed for J
BX
, with respect to the 2-4 Hz value expected for IV.
N(III) J
AP
(III) + N(IV) J
AP
(IV) = J
AP
(obsd) (1)
N(IV) = 1 – N(III) (2)
therefore
N(III) = [J
AP
(obsd) – J
AP
(IV)] / [J
AP
(III) – J
AP
(IV)] (3)
Similarly, for J
BP

N(III) = [J
BP
(obsd) – J
BP
(IV)] / [J
BP
(III) – J
BP
(IV)] (4)

156
Taking into consideration earlier work describing the conformations of 2-oxo-1,3,2-
oxazaphosphorinane,
33, 34
we conclude that the contribution of twist conformations such
as V (with the nucleobase equatorial to minimize steric repulsion and the OPh
pseudoaxial to maximize the anomeric effect) to the equilibrium is negligible, given that
the sums of the corresponding spin-spin couplings (J
AP
+ J
BP
) are nearly equal throughout
the series of diastereomers. If twist conformations were substantially present, the sums of
these J values should decrease by 2-3 Hz because protons H
A
and H
B
are not interchanged
by interconverting the twist and chair conformations.
40

4.3 Conclusions
In summary, the structures of three of the four individual diastereomers of cyclic (S)-
HPMPC and (S)-HPMPA phenyl esters 4.5 and 4.6 have been defined by X-ray
crystallography. The (S
p
)-4.6 stereoisomer crystallized with an (a, a) arrangement of the
C-5 nucleobase and phenyl phosphonate ester groups, contradicting the assumption that
the nucleobase is exclusively equatorial and indicating that the anomeric effect of the
axial OPh group can outweigh steric repulsion of the axial nucleobase. The dependence
of the
3
J
HCOP
and
3
J
HH
values of (S
p
)-4.5 and (S
p
)-4.6 on solvent polarity suggests that
they significantly exist as two conformers at equilibrium in solution, thus the
axial/equatorial terminology often used to differentiate cyclic (S)-HPMPC and
(S)-HPMPA prodrug diastereomers, and similar compounds, could be misleading.
Assignment of the more upfield
31
P NMR chemical shifts
22
to axial P-OPh is supported
157
by our work, thus for the cyclic (S)-HPMPC and (S)-HPMPA phenyl ester prodrugs, the
more downfield
31
P NMR signal corresponds to the (S
p
) configuration at phosphorus.

4.4 Experimental section
General Experimental Methods.
1
H,
13
C and
31
P NMR spectra were recorded on 400,
500, 600 MHz spectrometers. Chemical shifts (δ) are reported in parts per million (ppm)
relative to internal CH
3
OH (
1
H NMR, δ=3.34;
13
C NMR, δ=49.86); CHCl
3
(
1
H NMR,
δ=7.26); CH
3
CN (
1
H NMR, δ=1.96); or external 85% H
3
PO
4
(
31
P NMR, δ=0.00).
31
P
NMR spectra were proton-decoupled, and
1
H and
13
C coupling constants (J values) are
given in Hz. The following NMR abbreviations are used: s (singlet), d (doublet), m
(unresolved multiplet), dd (doublet of doublets), ddd (doublet of doublet of doublet), br
(broad signal). LC-MS analysis of compounds (S
p
)-4.5, (R
p
)-4.5, (S
p
)-4.6 and (R
p
)-4.6
was performed on a mass spectrometer in positive ion mode (ESI), equipped with PDA
and UV detectors and HPLC solvent delivery system. HPLC separations were performed
on a C18 HPLC column (5 μm, 250 mm × 4.6 mm) with a 0 to 30% CH
3
CN gradient in
60 mM ammonium acetate buffer, pH 5.5, at a flow rate of 1.0 mL/min. MS parameters
were optimized as follows: sheath gas (N
2
) flow rate 20 arb, I spray voltage 5 kV,
capillary temperature 275 °C, capillary voltage 35 V, tube lens offset 55 V. Full scan
mass spectra were recorded over a range of m/z 200-600. The UV detector was operated
at 274 or 260 nm for (S)-HPMPC or (S)-HPMPA derivatives, respectively.

158
General procedure for synthesis of cyclic 4.1 and 4.2 phenyl esters. To a suspension
of (S)-HPMPC (4.1) or (S)-HPMPA (4.2) (0.42 mmol) in dry DMF (5 mL), dry N,N-
diisopropylethylamine (DIEA) (10 mmol, 1.8 mL), PhOH (60 mg, 0.63 mmol) and
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) (1.05
mmol, 0.546 g) were added. The reaction mixture was stirred under N
2
at 40 °C for 2 h.
The reaction was monitored by
31
P NMR, and additional portions of PyBOP were added
as necessary. After reaction completion, DMF and DIEA were removed under vacuum.
The residue was washed with diethyl ether and purified using silica gel column
chromatography [CH
2
Cl
2
, then CH
2
Cl
2
:acetone (2:1), then CH
2
Cl
2
:acetone:CH
3
OH
(6:3:1)]. Solvents were removed under vacuum yielding, the product as a mixture of (S
p
)-
and (R
p
)-diastereomers in a ratio 3:1 (assignment based on integration of the
corresponding signals in
31
P NMR and the X-ray crystallographic structures) for
cHPMPC-Ph (4.5) (83% yield) and 4:1 for cHPMPA-Ph (4.6) (71% yield).
Diastereomeric mixtures of 4.5 or 4.6 enriched in the (S
p
)-diastereomers were
recrystallized as described below to furnish the pure (S
p
)-diastereomers used for X-ray
crystallography and NMR experiments.

General procedure for isomerization. Molecular sieves (0.4 nm) were added to a
solution of PhOH (0.02 mmol) and a mixture of cHPMPC-Ph or cHPMPA-Ph
diastereomers (0.2 mmol) enriched with the (S
p
)-diastereomer in absolute DMF (5 mL).
Cs
2
CO
3
(0.4 mmol, 130 mg) was added after 30 min in N
2
atmosphere. The reaction
mixture was stirred for 24 h at room temperature and monitored by
31
P NMR until the
159
ratio of (S
p
):(R
p
) diastereomers was ~ 1:9. In case of cHPMPC-Ph, the reaction mixture
was additionally heated for 2 h at 40 °C, affording a ~ 1:13 ratio of (S
p
):(R
p
)
diastereomers. Molecular sieves and Cs
2
CO
3
were removed by filtration, DMF was
evaporated under vacuum and the residue was purified utilizing the same procedure as
described above, yielding enriched products (cHPMPC-Ph (4.5), 74% yield; cHPMPA-Ph
(4.6), 84% yield), which were recrystallized as described below to furnish the pure (R
p
)-
diastereomers used for X-ray crystallography and NMR experiments.

4-Amino-1-{[(5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-
2(1H)-one (4.5)
The (R
p
)-4.5 diastereomer was obtained by recrystallization from CH
3
OH/acetone.
1
H
NMR (400 MHz, CD
3
OD): δ = 7.50 (d, J = 7.4 Hz, 1H, 6-H), 7.41-7.37 (m, 2H, 2 arom.
m-CH), 7.26-7.21 (m, 3H, 3 arom. CH), 5.83 (d, J = 7.6 Hz, 1H, 5-H), 4.52 (ddd,
3
J
PH
=
16.9 Hz, J
gem
= 12.0 Hz, J
vic
= 3.0 Hz, 1H, CH
A
H
B
O), 4.44 (ddd, J
gem
= 12.1 Hz, J
vic
=
10.1 Hz,
3
J
PH
= 1.8 Hz, 1H, CH
A
H
B
O), 4.32 (dd, J
gem
= 14.9 Hz,
2
J
HP
= 10.6 Hz, 1H,
CH
C
H
D
P), 4.20-4.15 (m, 1H, CH
X
O), 4.07 (dd, J
gem
= 14.9 Hz,
2
J
PH
= 1.2 Hz, 1H,
CH
C
H
D
P), 4.01 (dd, J
gem
= 14.5 Hz, J
vic
= 3.9 Hz, 1H, CHHN), 3.77 ppm (dd, J
gem
= 14.3
Hz, J
vic
= 6.8 Hz, 1H, CHHN);
13
C NMR (126 MHz, CD
3
OD): δ 166.7 (CNH
2
), 157.6
(CO), 149.6 (d,
2
J
CP
= 8.4 Hz, arom. ipso-C), 147.0 (C-6), 129.8 (2 arom. m-CH), 125.4
(arom. p-CH), 120.0 (d,
3
J
CP
= 4.3 Hz, 2 arom. o-CH), 94.1 (C-5), 74.3 (d,
3
J
CP
= 5.5 Hz,
CHO), 73.2 (d,
2
J
CP
= 8.6 Hz, CH
2
OP), 62.1 (d,
1
J
CP
= 144.0 Hz, CH
2
P), 48.5 ppm
(CH
2
N);
31
P NMR (162 MHz, CD
3
OD): δ = 10.1 ppm.
160

The (S
p
)-4.5 diastereomer was obtained by recrystallization from
CH
3
OH/acetone/hexane.
1
H NMR (500 MHz, CD
3
OD): δ = 7.53 (d, J = 7.4 Hz, 1H, 6-
H), 7.40-7.36 (m, 2H, 2 arom. m-CH), 7.25-7.18 (m, 3H, 3 arom. CH), 5.82 (d, J = 7.6
Hz, 1H, 5-H), 4.52 (ddd,
3
J
PH
= 12.1 Hz, J
gem
= 12.0 Hz, J
vic
= 2.7 Hz, 1H, CH
A
H
B
O),
4.48-4.42 (m, 1H, CH
A
H
B
O), 4.32 (dd, J
gem
= 14.4 Hz,
2
J
HP
= 7.4 Hz, 1H, CH
C
H
D
P),
4.19-4.15 (m, 1H, CH
X
O), 4.17 (dd, J
gem
= 15.0 Hz,
2
J
PH
= 3.0 Hz, 1H, CH
C
H
D
P), 4.01
(dd, J
gem
= 14.2 Hz, J
vic
= 3.3 Hz, 1H, CHHN), 3.91 ppm (dd, J
gem
= 14.2 Hz, J
vic
= 8.0
Hz, 1H, CHHN);
13
C NMR (126 MHz, CD
3
OD): δ = 166.7 (CNH
2
), 157.5 (CO), 149.5
(d,
2
J
CP
= 8.7 Hz, arom. ipso-C), 146.7 (C-6), 129.7 (d,
4
J
CP
= 1.0 Hz, 2 arom. m-CH),
125.5 (arom. p-CH), 120.1 (d,
3
J
CP
= 4.1 Hz, 2 arom. o-CH), 94.3 (C-5), 73.3 (d,
3
J
CP
=
5.2 Hz, CHO), 71.6 (d,
2
J
CP
= 7.5 Hz, CH
2
OP), 62.1 (d,
1
J
CP
= 146.2 Hz, CH
2
P), 48.2 ppm
(CH
2
N);
31
P NMR (162 MHz, CD
3
OD): δ = 11.3 ppm.

9-{[(5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine
(4.6)
The (R
p
)-4.6 diastereomer was obtained by recrystallization from i-PrOH/EtOAc.
1
H
NMR (600 MHz, CD
3
OD): δ = 8.24 (s, 1H, 2-H), 8.13 (s, 1H, 8-H), δ 7.41-7.38 (m, 2H,
2 arom. m-CH), 7.26-7.24 (m, 1H, arom. p-CH), 7.19-7.17 (m, 2H, 2 arom. o-CH), 4.61
(ddd,
3
J
HP
= 17.6 Hz, J
gem
= 11.6 Hz, J = 2.0 Hz, 1H, CH
A
H
B
O), 4.46 (dd, J = 14.4, 2.9
Hz, 1H, CHHN), 4.42 (ddd, J
gem
= 11.6,
3
J
HP
= 1.2 Hz, CH
A
H
B
O), 4.39-4.33 (m, 3H,
CHHN, CH
C
H
D
P, CH
X
O), 4.10 ppm (dd, J
gem
= 15.3 Hz,
2
J
HP
= 1.4 Hz, 1H, CH
C
H
D
P);
161
13
C NMR (126 MHz, CD
3
OD): 156.05 (C-NH
2
), 152.51 (C-2), 149.56 (d,
2
J
CP
= 8.3 Hz,
arom. ipso-C), 149.42 (NCC=CNN), 142.05 (C-8), 129.73 (d,
4
J
CP
= 0.7 Hz, 2 arom. m-
CH), 125.34 (arom. p-CH), 119.90 (d,
3
J
CP
= 4.3 Hz, 2 arom. o-CH), 118.33
(NCC=CNN), 73.90 (d,
3
J
CP
= 5.5 Hz, CHO), 72.96 (d,
2
J
CP
= 9.1 Hz, CH
2
OP), 62.08 (d,
1
J
CP
= 144.1 Hz, CH
2
P), 42.55 ppm (CH
2
N);
31
P NMR (162 MHz, CD
3
OD): δ = 9.8 ppm.

The (S
p
)-4.6 diastereomer was obtained by recrystallization from CH
3
CN.
1
H NMR
(600 MHz, CD
3
OD): δ = 8.20 (s, 1H, 2-H), 8.11 (s, 1H, 8-H), δ 7.38-7.35 (m, 2H, 2
arom. m-CH), 7.23-7.21 (m, 1H, arom. p-CH), 7.19-7.17 (m, 2H, 2 arom. o-CH), 4.71
(ddd, J = 12.1 Hz, J = 2.8 Hz, 1H, CH
A
H
B
O), 4.54 (dd, J = 15.1, 8.8 Hz, 1H, CH
a
H
b
N),
4.50 (m, 1H, CH
A
H
B
O), 4.46 (dd, J = 15.0, 7.0, 1H, CH
C
H
D
P), 4.44 (dd, J = 14.7, 3.5
Hz, 1H, CHHN), 4.32 (m, CH
X
O), 4.15 ppm (dd, J = 14.8, 4.0 Hz, CH
C
H
D
P);
13
C NMR
(126 MHz, CD
3
OD): δ = 156.01 (C-NH
2
), 152.53 (C-2), 149.51 (d,
2
J
CP
= 8.6 Hz, arom.
ipso-C), 149.41 (NCC=CNN) 141.74 (C-8), 129.70 (2 arom. m-CH), 125.48 (arom. p-
CH), 120.11 (d,
3
J
CP
= 4.0 Hz, 2 arom. o-CH), 118.37 (NCC=CNN), 72.97 (d,
3
J
CP
= 5.3
Hz, CHO), 71.42 (d,
2
J
CP
= 7.4 Hz, CH
2
OP), 61.47 (d,
1
J
CP
= 145.8 Hz, CH
2
P), 42.16 ppm
(CH
2
N);
31
P NMR (202 MHz, CD
3
OD): δ = 11.2 ppm.

X-ray crystallography
Crystals were grown from CH
3
OH/acetone ((R
p
)-4.5), i-PrOH/EtOAc ((R
p
)-4.6) or neat
acetonitrile ((S
p
)-4.6). The single crystal x-ray diffraction data were collected on a 3-
circle platform diffractometer equipped with a CCD detector with the -axis fixed at
162
54.74  and using MoK
α
radiation (  = 0.71073 Å) from a fine-focus tube. This
diffractometer was equipped with an apparatus for low temperature data collection using
controlled liquid nitrogen boil off. A complete hemisphere of data was scanned on omega
(0.3°) with a run time of 10-second per frame at a detector resolution of 512 x 512 pixels.
The structure was solved by the direct method using the SHELX-90 program and refined
by the least squares method on F
2
using SHELXL-97.
41
All non-hydrogen atoms were
refined anisotropically. Crystallographic data have been deposited with the Cambridge
Crystallographic Center, CCDC No. 795825, 795826, 795827. Copies of the data can be
obtained, free of charge, from CCDC, 12 Union Road, Cambridge, CB21EZ, UK (fax:
+44-1233-336033; e-mail: deposit@ccdc.cam.ac.uk; internet:
http://www.ccdc.cam.ac.uk).

163
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4
which was
our own experience.

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31
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194

APPENDIX A: Chapter 3 supporting data

195

Figure A.1
1
H NMR spectrum (400 MHz, CDCl
3
) of (S)-2-Trityloxymethyl-oxirane
(3.2).

Figure A.2
1
H NMR spectrum (400 MHz, CDCl
3
) of 1-(6-Amino-purin-9-yl)-3-trityloxy-
propan-2-ol (3.3).
196

Figure A.3
1
H NMR spectrum (400 MHz, CDCl
3
) of diethyl p-tolylsulfonyloxy-
methylphosphonate (3.4).

Figure A.4
31
P NMR spectrum (162 MHz, CDCl
3
) of diethyl p-tolylsulfonyloxy-
methylphosphonate (3.4).
197

Figure A.5
1
H NMR spectrum (400 MHz, CDCl
3
) of [2-(6-Amino-purin-9-yl)-1-
hydroxymethyl-ethoxymethyl]-phosphonic acid diethyl ester (3.6).

Figure A.6
31
P NMR spectrum (162 MHz, CDCl
3
) of [2-(6-Amino-purin-9-yl)-1-
hydroxymethyl-ethoxymethyl]-phosphonic acid diethyl ester (3.6).
198

Figure A.7
1
H NMR spectrum (400 MHz, D
2
O) of [2-(6-Amino-purin-9-yl)-1-
hydroxymethyl-ethoxymethyl]-phosphonic acid (2.2, (S)-HPMPA).

Figure A.8
31
P NMR spectrum (162 MHz, D
2
O) of [2-(6-Amino-purin-9-yl)-1-
hydroxymethyl-ethoxymethyl]-phosphonic acid (2.2, (S)-HPMPA).
199

Figure A.9
1
H NMR (400 MHz, CDCl
3
) of N
α
-(tert-butoxycarbonyl)-N-(2-
methylpropyl)-(L)-tyrosinamide (3.12).

Figure A.10
1
H NMR (500 MHz, CDCl
3
) of N
α
-(tert-butoxycarbonyl)-N-dodecyl-(L)-
tyrosinamide (3.14).
200

Figure A.11
1
H NMR (500 MHz, CDCl
3
) of N
α
-(tert-butoxycarbonyl)-N-tetradecyl-(L)-
tyrosinamide (3.15).

Figure A.12
1
H NMR (400 MHz, CDCl
3
) of N
α
-(tert-butoxycarbonyl)-N-hexadecyl-(L)-
tyrosinamide (3.16).
201

Figure A.13
1
H NMR (500 MHz, CDCl
3
) of N
α
-(tert-butoxycarbonyl)-N-octadecyl-(L)-
tyrosinamide (3.17).

Figure A.14
1
H NMR spectrum (400 MHz, CDCl
3
) of methyl O-[tert-butyl(dimethyl)-
silyl]-(L)-serinate (3.39).
202

Figure A.15
1
H NMR spectrum (400 MHz, CDCl
3
) of methyl O-[tert-
butyl(dimethyl)silyl]-(D)-serinate (3.41).

Figure A.16
1
H NMR spectrum (400 MHz, CDCl
3
) of propan-2-yl O-[tert-
butyl(dimethyl)silyl]-(L)-serinate (3.41).
203

Figure A.17
1
H NMR spectrum (400 MHz, CDCl
3
) of methyl O-[tert-butyl(dimethyl)-
silyl]-(L)-threoninate (3.42).

Figure A.18
1
H NMR spectrum (400 MHz, CDCl
3
) of methyl O-[tert-butyl(dimethyl)-
silyl]-(L)-tyrosinate (3.43).

204

Figure A.19
1
H NMR spectrum (400 MHz, CDCl
3
) of methyl O-[tert-butyl(dimethyl)-
silyl]-N-formyl-(L)-serinate (3.44).

Figure A.20
1
H NMR spectrum (400 MHz, CD
3
OD) of propan-2-yl O-[tert-butyl-
(dimethyl)silyl]-N-formyl-(L)-serinate (3.45).

205

Figure A.21
13
C NMR spectrum (100 MHz, CD
3
OD) of propan-2-yl O-[tert-butyl-
(dimethyl)silyl]-N-formyl-(L)-serinate (3.45).

Figure A.22
1
H NMR spectrum (400 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-serinate (2.12).
206

Figure A.23
31
P NMR spectrum (202 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-serinate (2.12).

Figure A.24 LC-MS trace of methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-
oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-serinate (2.12).
C:\Xcalibur\...\SercHPMPAonly_2 5/30/2008 4:15:39 PM
RT: 0.00 - 14.98 SM: 11B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min)
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
uAU
14.25
9.00
10.92 9.51 7.08 4.00 6.74 7.37 6.08 5.62 4.86 2.91 2.25 1.77 1.34 0.05
NL:
6.26E5
Channel A
UV
SercHPMP
Aonly_2
SercHPMPAonly_2 #1921 RT: 14.27 AV: 1 NL: 1.30E9
T: + c ESI Full ms [250.00-520.00]
260 280 300 320 340 360 380 400 420 440 460 480 500 520
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
386.99
409.04
286.18
391.04 459.97 490.62 312.83 418.80 503.12 330.33 267.94 341.37 366.98 476.71 444.86
207

Figure A.25
1
H NMR spectrum (400 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(D)-serinate (2.13).

Figure A.26
31
P NMR spectrum (202 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(D)-serinate (2.13).

208

Figure A.27 LC-MS trace of methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-
oxido-1,4,2-dioxaphosphinan-2-yl}-(D)-serinate (2.13).

Figure A.28
1
H NMR spectrum (400 MHz, CD
3
OD) of propan-2-yl O-{(5S)-5-[(6-
amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-serinate (2.14).
C:\Xcalibur\...\(D)-SerOMe-cHPMPA_061909 6/19/2009 12:49:41 PM
RT: 0.00 - 8.75 SM: 11B
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
Time (min)
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
uAU
7.61
5.08
5.99
4.82 5.75 6.89 2.84
NL:
1.62E6
Channel A
UV
(D)-
SerOMe-
cHPMPA_0
61909
(D)-SerOMe-cHPMPA_061909 #897 RT: 7.62 AV: 1 NL: 2.70E8
T: + c ESI Full ms [250.00-600.00]
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
m/z
10
20
30
40
50
60
70
80
90
100
Relative Abundance
387.37
409.34
460.22
441.36 286.54
419.32 503.99 485.07 391.47 544.98 260.27 567.13 313.68 591.20 531.35 329.80 344.03 360.54
cHPMPA 2.4
209

Figure A.29
31
P NMR spectrum (202 MHz, CD
3
OD) of propan-2-yl O-{(5S)-5-[(6-
amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-serinate (2.14).

Figure A.30 LC-MS trace of propan-2-yl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-
oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-serinate (2.14).
C:\Xcalibur\...\prodrug#1 10/22/2008 5:48:35 PM
RT: 0.00 - 12.01 SM: 11B
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (min)
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
uAU
10.30
10.19
3.66
6.47
NL:
1.26E6
Channel A
UV
prodrug#1
prodrug#1 #1414 RT: 10.30 AV: 1 NL: 4.02E8
T: + c ESI Full ms [250.00-450.00]
260 280 300 320 340 360 380 400 420 440
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
415.16
437.13 286.24
417.21 438.13
287.16 379.25 268.13 373.08 395.62 318.67 302.21 345.95 327.98 404.08 278.80 256.85 362.95
210

Figure A.31
1
H NMR spectrum (400 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-threoninate (2.15).

Figure A.32
31
P NMR spectrum (202 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-threoninate (2.15).

211

Figure A.33 LC-MS trace of methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-
oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-threoninate (2.15).

Figure A.34
1
H NMR spectrum (400 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-tyrosinate (2.16).
C:\Xcalibur\...\prodrug_4_070909 7/8/2009 3:52:41 PM
RT: 0.00 - 14.98 SM: 11B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min)
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
uAU
10.38
10.13
5.77
3.04 11.56 12.28 7.46 13.61 0.03
NL:
1.12E6
Channel A
UV
prodrug_4
_070909
prodrug_4_070909 #1382 RT: 10.34 AV: 1 NL: 5.38E8
T: + c ESI Full ms [250.00-530.00]
260 280 300 320 340 360 380 400 420 440 460 480 500 520
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
401.30
286.54
423.32 519.61
474.23 260.24 330.09 320.21 439.30 499.12 300.89 338.62 462.14 360.17 376.71 392.10
212

Figure A.35
31
P NMR spectrum (202 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-tyrosinate (2.16).

Figure A.36 LC-MS trace of methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-
oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-tyrosinate (2.16).
C:\Xcalibur\...\prodrugt5 6/10/2009 5:48:21 PM
RT: 0.00 - 21.39 SM: 11B
0 2 4 6 8 10 12 14 16 18 20
Time (min)
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
uAU
18.24
17.34
6.93 4.60 3.62 13.69 5.89 11.11 1.63 2.56 1.23
NL:
9.91E5
Channel A
UV
prodrugt5
prodrugt5 #2017 RT: 18.30 AV: 1 NL: 2.02E8
T: + c ESI Full ms [250.00-600.00]
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
463.69
485.55 501.41
561.10 536.44 268.29 279.10 584.08 359.44 314.52 403.21 375.47 449.59 419.72 326.62
213

Figure A.37
1
H NMR spectrum (400 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-cysteinate (2.17).

Figure A.38
31
P NMR spectrum (202 MHz, CD
3
OD) of methyl O-{(5S)-5-[(6-amino-9H-
purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-cysteinate (2.17).

214

Figure A.39 LC-MS trace of methyl O-{(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-
oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-cysteinate (2.17).

Figure A.40
1
H NMR spectrum (400 MHz, CD
3
OD) of methyl (L)-valyl-O-{(5S)-5-[(6-
amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxa-phosphinan-2-yl}-(L)-serinate (3.32)
C:\Xcalibur\data\Ivan\CysteinHPMPA 2/5/2009 4:31:33 PM
RT: 0.00 - 8.82 SM: 11B
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
Time (min)
0
50
100
0
50
100
0
50
100
0
500000
uAU
4.19 5.89
3.59
5.31
6.05
0.03 7.90 6.73 7.59 7.09 2.71 0.31 8.25 2.89 8.55 0.97 1.52 1.74 2.48 4.35 3.77 5.57 3.58 4.52 5.10
4.33
4.70 5.05 0.07 6.05 5.52 8.19 2.75 0.37 6.34 0.74 6.59 8.00 2.42 1.26 7.07 8.64 1.66 2.19 7.42 2.95 3.95 3.74
4.34
6.05
3.73
4.74 5.06 5.66 0.32 6.60 0.50 7.90 0.86 6.91 2.69 1.41 1.88 7.58 2.19 2.95 8.11 3.41 8.52
NL: 6.74E5
Channel A UV
CysteinHPMPA
NL: 9.54E7
Base Peak m/z=
402.50-403.50 MS
CysteinHPMPA
NL: 7.66E7
Base Peak m/z=
420.50-421.50 MS
CysteinHPMPA
NL: 2.72E7
Base Peak m/z=
285.50-286.50 MS
CysteinHPMPA
CysteinHPMPA #784 RT: 6.05 AV: 1 NL: 1.05E8
T: + c ESI Full ms [270.00-570.00]
280 300 320 340 360 380 400 420 440 460 480 500 520 540 560
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
403.32
425.23
286.30
441.17
300.39
421.38 475.67 328.25 560.83 545.01 500.73 339.04 360.18 465.04 386.58 518.01
215

Figure A.41
31
P NMR spectrum (202 MHz, CD
3
OD) of methyl (L)-valyl-O-{(5S)-5-[(6-
amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxa-phosphinan-2-yl}-(L)-serinate (3.32)

Figure A.42
1
H NMR spectrum (400 MHz, CD
3
OD) of propan-2-yl (L)-valyl-O-{(5S)-5-
[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-serinate
(3.33).
216

Figure A.43
31
P NMR spectrum (202 MHz, CD
3
OD) of propan-2-yl (L)-valyl-O-{(5S)-5-
[(6-amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl}-(L)-serinate
(3.33).

Figure A.44.
1
H NMR (400 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-(2-methylpropyl)-(L)-tyrosinamide
(2.26).
217

Figure A.45
31
P NMR (162 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-(2-methylpropyl)-(L)-tyrosinamide
(2.26).

Figure A.46 LC-MS trace of O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-
dioxaphosphinan-2-yl]-N-(2-methylpropyl)-(L)-tyrosinamide (2.26).
C:\Xcalibur\...\(L)TyrNHisoBucHPMPAH20 6/9/2009 12:23:37 PM
RT: 0.00 - 24.64 SM: 11B
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
200000
400000
600000
800000
1000000
1200000
1400000
uAU
21.19
20.45
15.59 4.31 3.62 0.05 2.63
NL:
1.50E6
Channel A
UV
(L)TyrNHis
oBucHPMP
AH20
(L)TyrNHisoBucHPMPAH20 #2256 RT: 20.52 AV: 1 NL: 2.93E8
T: + c ESI Full ms [250.00-600.00]
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
504.56
526.44
542.35
577.17 403.34 312.76 279.08 375.39 488.72 332.21 259.93 592.53 360.55 447.84 462.09 520.41 416.50
218

Figure A.47
1
H NMR (400 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-octyl-(L)-tyrosinamide (2.29).

Figure A.48
31
P NMR (162 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-octyl-(L)-tyrosinamide (2.29).

219

Figure A.49 LC-MS trace of O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-
dioxaphosphinan-2-yl]-N-octyl-(L)-tyrosinamide (2.29).

Figure A.50
1
H NMR (400 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-dodecyl-(L)-tyrosinamide (2.30).
C:\Xcalibur\...\IK-2-73_long_3_C18_col 1/13/2012 12:25:54 PM
RT: 0.00 - 14.50 SM: 11B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min)
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
uAU
8.16
8.48 7.25 14.10 13.75 13.05 9.60 12.38 11.68 9.94 6.89 6.36 5.73 5.22 4.56 4.13 3.14 2.52 2.22 1.70 1.11
NL:
1.88E5
Channel A
UV
IK-2-
73_long_3
_C18_col
IK-2-73_long_3_C18_col #453 RT: 8.19 AV: 1 NL: 3.02E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
560.47
582.19
1140.83
1118.94 403.42
268.23 658.04 740.04 1216.92 821.57 344.97 973.82 1382.72 863.97 1304.69 503.33
220

Figure A.51
31
P NMR (162 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-dodecyl-(L)-tyrosinamide (2.30).

Figure A.52 LC-MS trace of O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-
dioxaphosphinan-2-yl]-N-dodecyl-(L)-tyrosinamide (2.30).
C:\Xcalibur\...\IK-2-113_long_C18_col 1/12/2012 1:37:30 PM
RT: 0.00 - 10.99 SM: 11B
0 1 2 3 4 5 6 7 8 9 10
Time (min)
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
uAU
3.73
4.11 5.11 5.46 5.81 10.98 10.39 9.77 8.46 9.46 8.21 6.30 6.67 3.10 7.53 2.86 2.41 1.91 1.54 1.13 0.64
NL:
1.10E6
Channel A
UV
IK-2-
113_long_
C18_col
IK-2-113_long_C18_col #212 RT: 3.80 AV: 1 NL: 9.32E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
616.63
638.34
1231.12
1253.04
268.26 403.70 654.18 943.29 714.29 1329.89 887.70 1153.43 831.15 333.33 1056.35 564.15 457.10
221

Figure A.53
1
H NMR (500 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-tetradecyl-(L)-tyrosinamide (2.31).

Figure A.54
31
P NMR (202 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-tetradecyl-(L)-tyrosinamide (2.31).
222

Figure A.55 LC-MS trace of O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-
dioxaphosphinan-2-yl]-N-tetradecyl-(L)-tyrosinamide (2.31).

Figure A.56
1
H NMR (500 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-hexadecyl-(L)-tyrosinamide (2.32).

C:\Xcalibur\...\IK-2-103_long_C18_col 1/12/2012 3:46:11 PM
RT: 0.00 - 10.99 SM: 11B
0 1 2 3 4 5 6 7 8 9 10
Time (min)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
uAU
5.14
5.33
6.40 6.64 3.34 7.15 3.73 8.17 7.49 8.40 10.87 9.26 4.41 2.42 9.60 10.13 2.67 2.22 0.77 1.28 0.56
NL:
6.01E4
Channel A
UV
IK-2-
103_long_
C18_col
IK-2-103_long_C18_col #282 RT: 5.15 AV: 1 NL: 1.86E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
644.87
666.47
1309.30
1287.34
742.32 268.24 403.36 824.22 1326.18 906.00 584.54 328.05 1190.35 985.13 1044.22 1141.53 536.73 460.95
223

Figure A.57
31
P NMR (202 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-hexadecyl-(L)-tyrosinamide (2.32).

Figure A.58 LC-MS trace of O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-
dioxaphosphinan-2-yl]-N-hexadecyl-(L)-tyrosinamide (2.32).

C:\Xcalibur\...\IK-2-97_3_long_C18_col 1/12/2012 1:19:20 PM
RT: 0.00 - 10.99 SM: 11B
0 1 2 3 4 5 6 7 8 9 10
Time (min)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
uAU
8.12
8.45 7.94
10.18 10.57 7.58 7.04 6.62 6.20 5.41 5.16 4.64 4.28 3.79 3.45 2.89 2.51 1.99 1.75 1.30 0.70 0.31
NL:
5.58E4
Channel A
UV
IK-2-
97_3_long
_C18_col
IK-2-97_3_long_C18_col #457 RT: 8.18 AV: 1 NL: 3.15E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
672.89
694.58
1343.43
710.44 403.38 268.40 1027.22 770.24 934.06 1077.45 1142.33 519.87 1241.06 332.63 606.66 821.92 461.47
224

Figure A.59
1
H NMR (500 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-octadecyl-(L)-tyrosinamide (2.33).

Figure A.60
31
P NMR (202 MHz, CD
3
OD) of O-[(5S)-5-[(6-Amino-9H-purin-9-
yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-octadecyl-(L)-tyrosinamide (2.33).

225

Figure A.61 LC-MS trace of O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-
dioxaphosphinan-2-yl]-N-octadecyl-(L)-tyrosinamide (2.33).

Figure A.62 COSY 2D NMR (CD
3
OD, 500 MHz for
1
H NMR) of O-[(5S)-5-[(6-Amino-
9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-octadecyl-(L)-
tyrosinamide (2.33).
C:\Xcalibur\...\IK-2-110_2_long_C18_col 1/12/2012 4:25:49 PM
RT: 0.00 - 19.99 SM: 11B
0 2 4 6 8 10 12 14 16 18
Time (min)
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
uAU
13.54
14.28
17.34 17.85 8.22 2.43 18.80 10.78 8.80 11.75 2.99 9.43 5.11 4.37 6.62 5.57 7.17 0.72
NL:
1.35E5
Channel A
UV
IK-2-
110_2_lon
g_C18_col
IK-2-110_2_long_C18_col #748 RT: 13.62 AV: 1 NL: 4.81E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
700.93
722.48
1399.31
403.47 798.51 268.29 1069.68 836.61 357.32 977.76 554.69 502.08 1186.49 690.35 1237.71 930.42 1378.54
226

Figure A.63 HSQC 2D NMR (CD
3
OD, 500 MHz for
1
H NMR and 126 MHz for
13
C
NMR) of O-[(5S)-5-[(6-Amino-9H-purin-9-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-
yl]-N-octadecyl-(L)-tyrosinamide (2.33).

Figure A.64
1
H NMR (400 MHz, CD
3
OD) of O-[(5S)-5-[(4-amino-2-oxopyrimidin-
1(2H)-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-hexadecyl-(L)-tyrosinamide
(2.34).
227

Figure A.65
31
P NMR (162 MHz, CD
3
OD) of O-[(5S)-5-[(4-amino-2-oxopyrimidin-
1(2H)-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-hexadecyl-(L)-tyrosinamide
(2.34).

Figure A.66 LC-MS trace of O-[(5S)-5-[(4-amino-2-oxopyrimidin-1(2H)-yl)methyl]-2-
oxido-1,4,2-dioxaphosphinan-2-yl]-N-hexadecyl-(L)-tyrosinamide (2.34).
C:\Xcalibur\...\IK-2-127_long_C18_col 1/12/2012 5:07:13 PM
RT: 0.00 - 17.99 SM: 11B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time (min)
0
20000
40000
60000
80000
100000
120000
140000
160000
uAU
7.22
12.48 2.34 2.75 4.78 11.49 3.51 15.45 13.97 5.94 15.90 16.74
NL:
1.70E5
Channel A
UV
IK-2-
127_long_
C18_col
IK-2-127_long_C18_col #395 RT: 7.23 AV: 1 NL: 2.81E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
648.80
1317.23
670.53
1295.41
746.28 991.15 1333.29 379.33 828.38 955.59 1205.14 469.77 303.80 543.11 1062.74 1134.28 622.73
228

Figure A.67 COSY 2D NMR (CD
3
OD, 500 MHz for
1
H NMR) of O-[(5S)-5-[(4-amino-
2-oxopyrimidin-1(2H)-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-hexadecyl-
(L)-tyrosinamide (2.34).

Figure A.68 COSY 2D NMR (CD
3
OD, 500 MHz for
1
H NMR) of O-[(5S)-5-[(4-amino-
2-oxopyrimidin-1(2H)-yl)methyl]-2-oxido-1,4,2-dioxaphosphinan-2-yl]-N-hexadecyl-
(L)-tyrosinamide (2.34).
229

Figure A.69
1
H NMR (400 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-
tyrosinamide (2.28).

Figure A.70
13
C NMR (126 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-
tyrosinamide (2.28).
230

Figure A.71
31
P NMR (202 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-
tyrosinamide (2.28).

Figure A.72 LC-MS trace of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-
yl]oxy]methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-tyrosinamide (2.28).
L-TyrNHi-Bu-HPMPA_clean_2 8/11/2009 1:02:33 PM
RT: 0.00 - 18.80 SM: 11B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Time (min)
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
uAU
14.20
6.88
NL:
1.66E6
Channel A
UV
L-TyrNHi-
Bu-
HPMPA_cl
ean_2
L-TyrNHi-Bu-HPMPA_clean_2 #1579 RT: 14.28 AV: 1 NL: 1.32E9
T: + c ESI Full ms [250.00-600.00]
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
522.49
567.07 595.21 421.47 286.19 542.21 452.08 381.84 497.04 267.97 316.09
231

Figure A.73
1
H NMR (500 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-octyl-(L)-tyrosinamide (2.35).

Figure A.74
13
C NMR (126 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-octyl-(L)-tyrosinamide (2.35).
232

Figure A.75
31
P NMR (202 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-octyl-(L)-tyrosinamide (2.35).

Figure A.76 LC-MS trace of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-
yl]oxy]methyl)(hydroxy)phosphoryl]-N-octyl-(L)-tyrosinamide (2.35).
C:\Xcalibur\...\IK-2-75_long_C18_col 1/13/2012 12:57:41 PM
RT: 0.00 - 14.98 SM: 11B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min)
0
50000
100000
150000
200000
250000
300000
350000
400000
uAU
4.55
14.78 14.32 13.58 12.78 12.12 8.44 11.32 10.59 9.79 9.22 7.42 5.70 7.12 5.99 2.22 2.52 4.19 3.73 1.93 1.30 0.73
NL:
4.37E5
Channel A
UV
IK-2-
75_long_C
18_col
IK-2-75_long_C18_col #257 RT: 4.64 AV: 1 NL: 4.82E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
578.39
1155.05 600.18
1177.04 286.24 622.25 421.34 1141.90 329.12 1296.40 1354.33 1044.99 946.01 697.85 802.17 860.30 561.36 459.21
233

Figure A.77 COSY 2D NMR (CD
3
OD, 500 MHz for
1
H NMR) of O-[([[(2S)-1-(6-
Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-
octyl-(L)-tyrosinamide (2.35).

Figure A.78 HSQC 2D NMR (CD
3
OD, 500 MHz for
1
H NMR and 126 MHz for
13
C
NMR) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)-
(hydroxy)phosphoryl]-N-octyl-(L)-tyrosinamide (2.35).
234

Figure A.79
1
H NMR (500 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-dodecyl-(L)-tyrosinamide
(2.36).

Figure A.80
13
C NMR (126 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-dodecyl-(L)-tyrosinamide
(2.36).
235

Figure A.81
31
P NMR (202 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-dodecyl-(L)-tyrosinamide
(2.36).

Figure A.82 LC-MS trace of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-
yl]oxy]methyl)-(hydroxy)phosphoryl]-N-dodecyl-(L)-tyrosinamide (2.36).
C:\Xcalibur\...\IK-2-122_long_C18_col 1/12/2012 3:29:55 PM
RT: 0.00 - 10.99 SM: 11B
0 1 2 3 4 5 6 7 8 9 10
Time (min)
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
700000
uAU
3.32
3.63 2.30 4.31 4.72 5.06 5.39 10.87 6.60 3.03 10.48 5.85 6.87 8.42 7.39 9.67 8.12 8.65 2.05 1.58 0.06 0.55
NL:
7.11E5
Channel A
UV
IK-2-
122_long_
C18_col
IK-2-122_long_C18_col #182 RT: 3.30 AV: 1 NL: 9.58E7
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
634.72
656.59
1267.46
678.64 286.31 1311.48 754.40 421.50 998.04 620.80 1185.27 797.81 332.37 1093.49 959.87 864.73 461.31 521.06
236

Figure A.83 COSY 2D NMR (CD
3
OD, 500 MHz for
1
H NMR) of O-[([[(2S)-1-(6-
Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-
dodecyl-(L)-tyrosinamide (2.36).

Figure A.84 HSQC 2D NMR (CD
3
OD, 500 MHz for
1
H NMR and 126 MHz for
13
C
NMR) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)-
(hydroxy)phosphoryl]-N-dodecyl-(L)-tyrosinamide (2.36).
237

Figure A.85
1
H NMR (500 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-tetradecyl-(L)-tyrosinamide
(2.37).

Figure A.86
13
C NMR (126 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-tetradecyl-(L)-tyrosinamide
(2.37).

238

Figure A.87
31
P NMR (202 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-tetradecyl-(L)-tyrosinamide
(2.37).

Figure A.88 LC-MS trace of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-
yl]oxy]methyl)-(hydroxy)phosphoryl]-N-tetradecyl-(L)-tyrosinamide (2.37).
C:\Xcalibur\...\IK-2-116_long_C18_col 1/12/2012 2:24:57 PM
RT: 0.00 - 10.99 SM: 11B
0 1 2 3 4 5 6 7 8 9 10
Time (min)
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
uAU
4.34
4.76 6.57 6.05 3.34 7.15 7.44 10.84 2.23 8.01 8.47 9.71 10.01 3.77 9.30 8.93 2.51 2.00 1.40 1.12 0.70
NL:
9.36E5
Channel A
UV
IK-2-
116_long_
C18_col
IK-2-116_long_C18_col #244 RT: 4.33 AV: 1 NL: 5.32E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
662.74
1323.36
684.58 294.54 1345.41 744.71 404.47 892.99 594.60 828.34 1295.51 966.87 450.54 1174.76 1045.44 543.43
239

Figure A.89 COSY 2D NMR (CD
3
OD, 500 MHz for
1
H NMR) of O-[([[(2S)-1-(6-
Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-
tetradecyl-(L)-tyrosinamide (2.37).

Figure A.90 HSQC 2D NMR (CD
3
OD, 500 MHz for
1
H NMR and 126 MHz for
13
C
NMR) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)-
(hydroxy)phosphoryl]-N-tetradecyl-(L)-tyrosinamide (2.37).
240

Figure A.91
1
H NMR (500 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)-phosphoryl]-N-hexadecyl-(L)-tyrosinamide
(2.38).

Figure A.92
13
C NMR (126 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)-phosphoryl]-N-hexadecyl-(L)-tyrosinamide
(2.38).
241

Figure A.93
31
P NMR (202 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)-phosphoryl]-N-hexadecyl-(L)-tyrosinamide
(2.38).

Figure A.94 LC-MS trace of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-
yl]oxy]methyl)-(hydroxy)phosphoryl]-N-hexadecyl-(L)-tyrosinamide (2.38).
C:\Xcalibur\...\IK-2-107_long_C18_col 1/12/2012 1:59:21 PM
RT: 0.00 - 10.99 SM: 11B
0 1 2 3 4 5 6 7 8 9 10
Time (min)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
uAU
6.45
6.71
7.90 8.13 10.95 8.58 10.18 9.90 9.00 3.79 5.98 5.42 4.54 5.18 2.27 3.31 2.83 2.02 1.64 1.15 0.80
NL:
5.94E4
Channel A
UV
IK-2-
107_long_
C18_col
IK-2-107_long_C18_col #364 RT: 6.50 AV: 1 NL: 1.42E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
690.89
1379.47
712.68
735.00 286.58 1075.23 421.59 791.41 944.97 987.79 632.04 1188.41 573.92 473.45 858.38 376.36 1361.02 1253.11
242

Figure A.95 COSY 2D NMR (CD
3
OD, 500 MHz for
1
H NMR) of O-[([[(2S)-1-(6-
Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)phosphoryl]-N-
hexadecyl-(L)-tyrosinamide (2.38).

Figure A.96 HSQC 2D NMR (CD
3
OD, 500 MHz for
1
H NMR and 126 MHz for
13
C
NMR) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)-
(hydroxy)phosphoryl]-N-hexadecyl-(L)-tyrosinamide (2.38).

243

Figure A.97
1
H NMR (500 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)-phosphoryl]-N-octadecyl-(L)-tyrosinamide
(2.39).

Figure A.98
13
C NMR (126 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)-phosphoryl]-N-octadecyl-(L)-tyrosinamide
(2.39).

244

Figure A.99
31
P NMR (202 MHz, CD
3
OD) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy]methyl)(hydroxy)-phosphoryl]-N-octadecyl-(L)-tyrosinamide
(2.39).

Figure A.100 LC-MS trace of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-
2-yl]oxy]methyl)-(hydroxy)phosphoryl]-N-octadecyl-(L)-tyrosinamide (2.39).
C:\Xcalibur\...\IK-2-119_2_long_C18_col 1/12/2012 3:00:01 PM
RT: 0.00 - 14.88 SM: 11B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (min)
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
uAU
10.68
11.19 6.56 13.58 14.03 4.46 8.42 7.11 8.70 8.08 9.78 2.32 5.63 2.70 4.82 4.08 2.01 0.19 1.16
NL:
4.73E5
Channel A
UV
IK-2-
119_2_lon
g_C18_col
IK-2-119_2_long_C18_col #597 RT: 10.69 AV: 1 NL: 5.48E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
718.69
740.52
286.23 421.46 373.36 1145.24 796.31 690.51 1242.11 1081.68 497.73 556.32 1014.22 894.98 1374.29 612.90
245

Figure A.101 COSY 2D NMR (CD
3
OD, 500 MHz for
1
H NMR) of O-[([[(2S)-1-(6-
Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)-(hydroxy)phosphoryl]-N-
octadecyl-(L)-tyrosinamide (2.39).

Figure A.102 HSQC 2D NMR (CD
3
OD, 500 MHz for
1
H NMR and 126 MHz for
13
C
NMR) of O-[([[(2S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl]oxy]methyl)-
(hydroxy)phosphoryl]-N-octadecyl-(L)-tyrosinamide (2.39).
246

Figure A.103
1
H NMR (500 MHz, CD
3
OD) of O-[([[(2S)-1-(4-amino-2-oxopyrimidin-
1(2H)-yl)-3-hydroxypropan-2-yl]oxy]methyl)-(hydroxy)phosphoryl]-N-hexadecyl-(L)-
tyrosinamide (2.40).

Figure A.104
13
C NMR (126 MHz, CD
3
OD) of O-[([[(2S)-1-(4-amino-2-oxopyrimidin-
1(2H)-yl)-3-hydroxypropan-2-yl]oxy]methyl)-(hydroxy)phosphoryl]-N-hexadecyl-(L)-
tyrosinamide (2.40).
247

Figure A.105
31
P NMR (202 MHz, CD
3
OD) of O-[([[(2S)-1-(4-amino-2-oxopyrimidin-
1(2H)-yl)-3-hydroxypropan-2-yl]oxy]methyl)-(hydroxy)phosphoryl]-N-hexadecyl-(L)-
tyrosinamide (2.40).

Figure A.106 LC-MS trace of O-[([[(2S)-1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-
hydroxypropan-2-yl]oxy]-methyl)(hydroxy)phosphoryl]-N-hexadecyl-(L)-tyrosinamide
(2.40).
C:\Xcalibur\...\IK-2-143_2_long_C18_col 1/12/2012 5:51:35 PM
RT: 0.00 - 17.99 SM: 11B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Time (min)
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
uAU
6.09
4.61 7.66 2.26 9.76 4.16 2.91 11.96 1.83
NL:
5.03E5
Channel A
UV
IK-2-
143_2_lon
g_C18_col
IK-2-143_2_long_C18_col #339 RT: 6.10 AV: 1 NL: 4.71E8
T: + c ESI sid=35.00 Full ms [250.00-1400.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
666.49
1331.46
1353.29 688.35
262.06 397.95 830.58 763.94 1022.60 648.82 356.17 865.22 963.12 1136.21 1265.51 577.93 448.12
248

Figure A.107 COSY 2D NMR (CD
3
OD, 500 MHz for
1
H NMR) of O-[([[(2S)-1-(4-
amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yl]oxy]methyl)(hydroxy)-
phosphoryl]-N-hexadecyl-(L)-tyrosinamide (2.40).

Figure A.108 HSQC 2D NMR (CD
3
OD, 500 MHz for
1
H NMR and 126 MHz for
13
C
NMR) of O-[([[(2S)-1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yl]oxy]-
methyl)(hydroxy)-phosphoryl]-N-hexadecyl-(L)-tyrosinamide (2.40).
249

Figure A.109
1
H NMR (500 MHz, CD
3
OD) of phenyl hydrogen ({[(2S)-1-(6-amino-9H-
purin-9-yl)-3-hydroxypropan-2-yl]oxy}methyl)phosphonate (3.49).

Figure A.110
13
C NMR (126 MHz, CD
3
OD) of phenyl hydrogen ({[(2S)-1-(6-amino-9H-
purin-9-yl)-3-hydroxypropan-2-yl]oxy}methyl)phosphonate (3.49).
250

Figure A.111
31
P NMR (202 MHz, CD
3
OD) of phenyl hydrogen ({[(2S)-1-(6-amino-9H-
purin-9-yl)-3-hydroxypropan-2-yl]oxy}methyl)phosphonate (3.49).

Figure A.112 Mass-spectrum of phenyl hydrogen ({[(2S)-1-(6-amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy}methyl)phosphonate (3.49).

C:\Xcalibur\...\Ph-HPMPA_101123170649 11/23/2010 5:06:49 PM
Ph-HPMPA_101123170649 #2 RT: 0.04 AV: 1 NL: 2.74E6
T: - c ESI Full ms [150.00-2000.00]
150 200 250 300 350 400 450 500 550 600 650 700
m/z
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
378.18
460.03
178.67
541.93
623.98 222.91
705.94
193.90
305.03
255.96 386.89 557.92 535.92 436.10 468.94 618.07 639.99
315.12 663.68
362.81
251

Figure A.113
1
H NMR (500 MHz, CD
3
OD) of phenyl hydrogen ({[(2S)-1-(6-amino-9H-
purin-9-yl)-3-bromopropan-2-yl]oxy}methyl)phosphonate (3.50).

Figure A.114
31
P NMR (202 MHz, CD
3
OD) of phenyl hydrogen ({[(2S)-1-(6-amino-9H-
purin-9-yl)-3-bromopropan-2-yl]oxy}methyl)phosphonate (3.50).

252

Figure A.115
1
H NMR (500 MHz, CD
3
OD) of O-[({[(2S)-1-(6-amino-9H-purin-9-yl)-3-
bromopropan-2-yl]oxy}methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-
tyrosinamide (3.51).

Figure A.116
31
P NMR (202 MHz, CD
3
OD) of O-[({[(2S)-1-(6-amino-9H-purin-9-yl)-3-
bromopropan-2-yl]oxy}methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-
tyrosinamide (3.51).

253

Figure A.117 Mass-spectrum of O-[({[(2S)-1-(6-amino-9H-purin-9-yl)-3-bromopropan-
2-yl]oxy}methyl)-(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-tyrosinamide (3.51).

Figure A.118
1
H NMR (500 MHz, CD
3
OD) of O-[({[(2S)-1-(6-amino-9H-purin-9-yl)-3-
sulfanylpropan-2-yl]oxy}methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-
tyrosinamide (3.52).
C:\Xcalibur\...\L-TyrNHi-Bu-Br-HPMPA 10/11/2010 1:23:49 PM
L-TyrNHi-Bu-Br-HPMPA #4 RT: 0.09 AV: 1 NL: 4.61E7
T: + c ESI Full ms [150.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
584.21
294.31
606.10
399.94
279.17 221.89
538.32
628.06
264.95
485.17 1041.36
204.94 683.85 1168.89
303.98 739.91 998.86 462.02 1101.14 839.77 909.65
254

Figure A.119
13
C NMR (126 MHz, CD
3
OD) of O-[({[(2S)-1-(6-amino-9H-purin-9-yl)-3-
sulfanylpropan-2-yl]oxy}methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-
tyrosinamide (3.52).

Figure A.120
31
P NMR (202 MHz, CD
3
OD) of O-[({[(2S)-1-(6-amino-9H-purin-9-yl)-3-
sulfanylpropan-2-yl]oxy}methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-
tyrosinamide (3.52).
255

Figure A.121 LC-MS trace of O-[({[(2S)-1-(6-amino-9H-purin-9-yl)-3-sulfanylpropan-
2-yl]oxy}methyl)(hydroxy)phosphoryl]-N-(2-methylpropyl)-(L)-tyrosinamide (3.52).

Figure A.122
1
H NMR (400 MHz, D
2
O) of 9-[((5S)-2-oxido-2-sulfanyl-1,4,2-dioxa-
phosphinan-5-yl)methyl]-9H-purine-6-amine (3.54).
C:\Xcalibur\...\L-TyrNHi-Bu-SH-HPMPA 10/27/2010 5:47:33 PM
RT: 0.00 - 30.00 SM: 11B
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (min)
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
1100000
1200000
uAU
17.41
5.30
NL:
1.23E6
Channel A
UV
L-TyrNHi-
Bu-SH-
HPMPA
L-TyrNHi-Bu-SH-HPMPA #1921 RT: 17.47 AV: 1 NL: 2.87E8
T: + c ESI Full ms [250.00-650.00]
250 300 350 400 450 500 550 600 650
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
537.39
548.49
580.67
436.48
556.58 478.42 492.22 349.91 526.60 279.03 378.33 611.02 335.97 302.82 399.62 449.92
256

Figure A.123
31
P NMR (162 MHz, D
2
O) of 9-[((5S)-2-oxido-2-sulfanyl-1,4,2-dioxa-
phosphinan-5-yl)methyl]-9H-purine-6-amine (3.54).

257

APPENDIX B: Chapter 4 supporting data

258

Figure B.1
1
H NMR spectrum (600 MHz, CD
3
OD) of 4-amino-1-{[(2R,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((R
p
)-4.5).

Figure B.2
13
C NMR spectrum (126 MHz, CD
3
OD) of 4-amino-1-{[(2R,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((R
p
)-4.5).
259

Figure B.3 HSQC 2D NMR spectrum (CD
3
OD, 500 MHz for
1
H-NMR and 126 MHz for
13
C) of 4-amino-1-{[(2R,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-
pyrimidin-2(1H)-one ((R
p
)-4.5).

Figure B.4
31
P NMR spectrum (162 MHz, CD
3
OD) of 4-amino-1-{[(2R,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((R
p
)-4.5).
260

Figure B.5 LC-MS trace of 4-amino-1-{[(2R,5S)-2-oxido-2-phenoxy-1,4,2-
dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((R
p
)-4.5).

Figure B.6
1
H NMR spectrum (500 MHz, CD
3
CN) of 4-amino-1-{[(2R,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((R
p
)-4.5).
261

Figure B.7 Enlarged region (3.60-4.55 ppm) of
1
H NMR spectrum (500 MHz, CD
3
CN)
of 4-amino-1-{[(2R,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-
pyrimidin-2(1H)-one ((R
p
)-4.5).

Figure B.8
1
H NMR spectrum (500 MHz, CDCl
3
) of 4-amino-1-{[(2R,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((R
p
)-4.5).
262

Figure B.9 Enlarged region (3.45-4.60 ppm) of
1
H NMR spectrum (500 MHz, CDCl
3
) of
4-amino-1-{[(2R,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-
2(1H)-one ((R
p
)-4.5).

Figure B.10
1
H NMR spectrum (600 MHz, CD
3
OD) of 4-amino-1-{[(2S,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((S
p
)-4.5).
263

Figure B.11
13
C NMR spectrum (126 MHz, CD
3
OD) of 4-amino-1-{[(2S,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((S
p
)-4.5).

Figure B.12 HSQC 2D NMR spectrum (CD
3
OD, 500 MHz for
1
H-NMR and 126 MHz
for
13
C) of 4-amino-1-{[(2S,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-
yl]methyl}pyrimidin-2(1H)-one ((S
p
)-4.5).
264

Figure B.13
31
P NMR spectrum (162 MHz, CD
3
OD) of 4-amino-1-{[(2S,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((S
p
)-4.5).

Figure B.14 LC-MS trace of 4-amino-1-{[(2S,5S)-2-oxido-2-phenoxy-1,4,2-
dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((S
p
)-4.5).
265

Figure B.15
1
H NMR spectrum (500 MHz, CDCl
3
) of 4-amino-1-{[(2S,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((S
p
)-4.5).

Figure B.16 Enlarged region (4.05-4.60 ppm) of
1
H NMR spectrum (500 MHz, CDCl
3
)
of 4-amino-1-{[(2S,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-
pyrimidin-2(1H)-one ((S
p
)-4.5).
266

Figure B.17
1
H NMR spectrum (500 MHz, CD
3
CN) of 4-amino-1-{[(2S,5S)-2-oxido-2-
phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}pyrimidin-2(1H)-one ((S
p
)-4.5).

Figure B.18 Enlarged region (4.05-4.70 ppm) of
1
H NMR spectrum (500 MHz, CD
3
CN)
of 4-amino-1-{[(2S,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-
pyrimidin-2(1H)-one ((S
p
)-4.5).
267

Figure B.19
1
H NMR spectrum (600 MHz, CD
3
OD) of 9-{[(2R,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((R
p
)-4.6).

Figure B.20
13
C NMR spectrum (126 MHz, CD
3
OD) of 9-{[(2R,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((R
p
)-4.6).
268

Figure B.21 HSQC 2D NMR spectrum (CD
3
OD, 500 MHz for
1
H-NMR and 126 MHz
for
13
C) of 9-{[(2R,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-9H-
purin-6-amine ((R
p
)-4.6).

Figure B.22
31
P NMR spectrum (162 MHz, CD
3
OD) of 9-{[(2R,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((R
p
)-4.6).
269

Figure B.23 LC-MS trace of 9-{[(2R,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-
yl]methyl}-9H-purin-6-amine ((R
p
)-4.6).

Figure B.24
1
H NMR spectrum (500 MHz, CDCl
3
) of 9-{[(2R,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((R
p
)-4.6).
270

Figure B.25 Enlarged region (4.23-4.57 ppm) of
1
H NMR spectrum (500 MHz, CDCl
3
)
of 9-{[(2R,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-
amine ((R
p
)-4.6).

Figure B.26
1
H NMR spectrum (500 MHz, CD
3
CN) of 9-{[(2R,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((R
p
)-4.6).
271

Figure B.27 Enlarged region (4.18-4.60 ppm) of
1
H NMR spectrum (500 MHz, CD
3
CN)
of 9-{[(2R,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-
amine ((R
p
)-4.6).

Figure B.28
1
H NMR spectrum (600 MHz, CD
3
OD) of 9-{[(2S,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((S
p
)-4.6).
272

Figure B.29
13
C NMR spectrum (126 MHz, CD
3
OD) of 9-{[(2S,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((S
p
)-4.6).

Figure B.30 HSQC 2D NMR spectrum (CD
3
OD, 500 MHz for
1
H-NMR and 126 MHz
for
13
C) of 9-{[(2S,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-9H-
purin-6-amine ((S
p
)-4.6).
273

Figure B.31
31
P NMR spectrum (202 MHz, CD
3
OD) of 9-{[(2S,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((S
p
)-4.6).

Figure B.32 LC-MS trace of 9-{[(2S,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-
yl]methyl}-9H-purin-6-amine ((S
p
)-4.6).
274

Figure B.33
1
H NMR spectrum (500 MHz, CDCl
3
) of 9-{[(2S,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((S
p
)-4.6).

Figure B.34 Enlarged region (4.23-4.60 ppm) of
1
H NMR spectrum (500 MHz, CDCl
3
)
of 9-{[(2S,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-
amine ((S
p
)-4.6).
275

Figure B.35
1
H NMR spectrum (500 MHz, CD
3
CN) of 9-{[(2S,5S)-2-oxido-2-phenoxy-
1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-amine ((S
p
)-4.6).

Figure B.36 Enlarged region (4.35-4.77 ppm) of
1
H NMR spectrum (500 MHz, CD
3
CN)
of 9-{[(2S,5S)-2-oxido-2-phenoxy-1,4,2-dioxaphosphinan-5-yl]methyl}-9H-purin-6-
amine ((S
p
)-4.6).
276
Crystallographic Experimental Details for (R
p
)-4.5, (S
p
)-4.6, and (R
p
)-4.6

Figure B.37 The (R
p
)-4.5 molecule in the crystal structure.

Figure B.38 The asymmetric unit in the crystal structure of (R
p
)-4.5·CH
3
OH.

Figure B.39 The unit cell of the crystal structure of (R
p
)-4.5·CH
3
OH.
277
Table 1. Crystal data and structure refinement for (R
p
)-4.5.
Identification code (R
p
)-4.5
Empirical formula C15 H20 N3 O6 P
Formula weight 369.31
Temperature 133(2) K
Wavelength 0.71073 Å
Crystal system Monoclinic
Space group P2(1)
Unit cell dimensions a = 6.9573(7) Å α= 90°.
b = 6.9976(7) Å β= 90.050(3)°.
c = 34.145(4) Å γ = 90°.
Volume 1662.3(3) Å
3

Z 4
Density (calculated) 1.476 Mg/m
3

Absorption coefficient 0.204 mm
-1

F(000) 776S
Crystal size 0.65 x 0.07 x 0.04 mm
3

Theta range for data collection 1.79 to 27.67°.
Index ranges -9<=h<=9, -9<=k<=9, -39<=l<=43
Reflections collected 14574
Independent reflections 7330 [R(int) = 0.0398]
Completeness to theta = 27.67° 96.8 %
Absorption correction multiscan
Transmission factor min/max: 0.898
Refinement method Full-matrix least-squares on F
2

Data / restraints / parameters 7330 / 1 / 477
Goodness-of-fit on F
2
1.010
Final R indices [I>2sigma(I)] R1 = 0.0491, wR2 = 0.0913
R indices (all data) R1 = 0.0719, wR2 = 0.1021
Absolute structure parameter -0.13(9)
Largest diff. peak and hole 0.252 and -0.297 e.Å
-3

278

Figure B.40 The (S
p
)-4.6 molecule in the crystal structure.

Figure B.41 The asymmetric unit in the crystal structure of (S
p
)-4.6.

Figure B.42 The unit cell of the crystal structure of (S
p
)-4.6.

279
Table 1. Crystal data and structure refinement for (S
p
)-4.6
Identification code (S
p
)-4.6
Empirical formula C15 H16 N5 O4 P
Formula weight 361.30
Temperature 163(2) K
Wavelength 0.71073 Å

Crystal system Monoclinic
Space group P2(1)
Unit cell dimensions a = 5.7894(15) Å α= 90°.
b = 43.207(11) Å β= 112.184(4)°.
c = 6.8100(18) Å γ = 90°.
Volume 1577.4(7) Å
3

Z 4
Density (calculated) 1.521 Mg/m
3

Absorption coefficient 0.208 mm
-1

F(000) 752
Crystal size 0.22 x 0.05 x 0.04 mm
3

Theta range for data collection 1.89 to 27.54°.
Index ranges -7<=h<=7, -55<=k<=48, -8<=l<=8
Reflections collected 13406
Independent reflections 6226 [R(int) = 0.1073]
Completeness to theta = 27.54° 98.2 %
Absorption correction None
Refinement method Full-matrix least-squares on F
2

Data / restraints / parameters 6226 / 1 / 451
Goodness-of-fit on F
2
0.733
Final R indices [I>2sigma(I)] R1 = 0.0498, wR2 = 0.0554
R indices (all data) R1 = 0.1105, wR2 = 0.0657
Absolute structure parameter -0.05(10)
Largest diff. peak and hole 0.237 and -0.333 e.Å
-3

280

Figure B.43 The (R
p
)-4.6 molecule in the crystal structure.

Figure B.44 The unit cell of the crystal structure of (R
p
)-4.6

281
Table 1. Crystal data and structure refinement for (R
p
)-4.6.
Identification code (R
p
)-4.6
Empirical formula C15 H16 N5 O4 P
Formula weight 361.30
Temperature 133(2) K
Wavelength 0.71073 Å
Crystal system Monoclinic
Space group P2(1)
Unit cell dimensions a = 8.2341(9) Å α= 90°.
b = 8.3291(9) Å β= 104.660(2)°.
c = 12.9089(14) Å γ = 90°.
Volume 856.50(16) Å
3

Z 2
Density (calculated) 1.401 Mg/m
3

Absorption coefficient 0.192 mm
-1

F(000) 376
Crystal size 0.33 x 0.11 x 0.03 mm
3

Theta range for data collection 1.63 to 27.49°.
Index ranges -10<=h<=10, -10<=k<=10, -16<=l<=16
Reflections collected 9644
Independent reflections 3733 [R(int) = 0.0327]
Completeness to theta = 27.49° 98.0 %
Absorption correction multiscan
Transmission factors min/max: 0.850
Refinement method Full-matrix least-squares on F
2

Data / restraints / parameters 3733 / 1 / 226
Goodness-of-fit on F
2
1.036
Final R indices [I>2sigma(I)] R1 = 0.0514, wR2 = 0.1054
R indices (all data) R1 = 0.0781, wR2 = 0.1182
Absolute structure parameter 0.09(12)
Largest diff. peak and hole 0.411 and -0.223 e.Å
-3

Abstract (if available)

Abstract Acyclic nucleoside phosphonates (ANPs), in particular (S) HPMPC (Cidofovir, Vistide®) and (S) HPMPA are highly potent broad spectrum antiviral agents. Unfortunately, unfavorable Absorption, Distribution, Metabolism and Excursion (ADME) profiles limit the utilization of these therapeutics in the clinic, mainly due to low cell membrane permeability, owing to the presence of a phosphonic acid group that ionizes at physiological pH. To address this issue, an amino acid phosphonate ester prodrug approach is being developed, which explores the use of benign single amino acids as promoieties to mask the negative charges of the ANPs. The main objective of the present work was to identify the promoiety, which, upon conjugation to the ANPs, produces prodrugs able not only to withstand the rigors of metabolism, but also to efficiently release the active parent drug at the target site in vivo. ❧ In order to achieve this objective, three consecutive rounds of structure-activity relationship (SAR) studies involving the synthesis, characterization and biological evaluation of 24 single amino acid (S)-HPMPA and (S)-HPMPC prodrugs were performed. The design of these prodrugs was aimed to “tune” the P-X-C linkage, the amino acid stereochemistry and the C-terminal functional group, and to optimize the length of the alkyl chain in the tyrosine N-alkyl amide moiety. Synthesis of the designed prodrugs was accomplished using a solution-phase method, and a newly developed solid-phase approach. Although the solid-phase approach produced the prodrugs in lower yields (20-35%) as compared to the conventional method (40-65%), it required simpler purification, and can be easily scaled up and automated in the future. The tyrosine acyclic phosphonate prodrugs were obtained by hydrolysis of their cyclic counterparts with yields of 40-60%. The cyclic prodrugs were generated as pairs of diastereomers (Sp or Rp) that in case of tyrosine-based prodrugs exhibit different pharmacokinetic properties. The absolute configurations of both diastereomers were established based on X-ray crystallographic and NMR analysis of the model compounds, phenyl esters of cyclic (S)-HPMPA and (S)-HPMPC. Thus, the less stable diastereomer was identified to have (Sp)-configuration at the phosphorus atom and corresponded to the downfield ³¹P NMR signal. ❧ The first conclusion that followed from the SAR studies is that the tyrosine amino acid is the most favorable promoiety among tested single amino acids, owing to its high chemical stability and the efficient activation of the resulting tyrosine-based prodrugs. Second, enzymatic stability of the tyrosine promoiety was significantly increased by replacement of the carboxyl ester group with an N-alkyl amide moiety. Thus, the tyrosine N-alkyl amide-based prodrugs undergo the same metabolism and exhibit identical stabilities in enzymatic and non-enzymatic media. Third, the highest antiviral activities (3-4-logs increase in activity compared to the parent ANP) of the tyrosine N-alkyl amide (S)-HPMPA and (S)-HPMPC prodrugs against HCMV, cowpox and vaccinia viruses were achieved by incorporation of a hexadecyl group (C16H33) into the tyrosine N-alkyl amide moiety. As a result, the tyrosine N-hexadecyl amide was identified as a promising single amino acid promoiety scaffold for further ANP prodrug development, surpassing the previously reported ethylene glycol-linked amino acid and dipeptide promoieties.

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Asset Metadata

Creator Krylov, Ivan S. (author)

Core Title Synthesis, structural analysis and in vitro antiviral activities of novel cyclic and acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 05/09/2013

Defense Date 10/17/2012

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

Tag (S)-HPMPA,acyclic nucleoside phosphonates,amino acid,antiviral,cidofovir,OAI-PMH Harvest,prodrugs,tyrosinamide

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles E. (committee chair), Haworth, Ian S. (committee member), Prakash, G. K. Surya (committee member)

Creator Email ikrylov@usc.edu,ivkryl@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-109157

Unique identifier UC11290215

Identifier usctheses-c3-109157 (legacy record id)

Legacy Identifier etd-KrylovIvan-1277.pdf

Dmrecord 109157

Document Type Dissertation

Rights Krylov, Ivan S.

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