Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Nucleoside phosphonate prodrugs: synthesis and antiviral activity
(USC Thesis Other)
Nucleoside phosphonate prodrugs: synthesis and antiviral activity
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
NUCLEOSIDE PHOSPHONATE PRODRUGS:
SYNTHESIS AND ANTIVIRAL ACTIVITY
by
Melissa Marie Williams
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
May 2015
Copyright 2015 Melissa Marie Williams
iii
DEDICATION
To my parents and siblings for their love and support
and to loved ones we have lost along the way.
Carpe Diem
iv
ACKNOWLEDGEMENTS
The chemistry Ph.D. experience has been an adventure filled with endless
amounts of learning experiences inside and outside of the research setting. And while
time and space will not allow me to thank every single human being that touched my life
during this humbling journey, I aim to mention those whose support and spirited nature I
will always cherish for inspiring me during this period.
First and foremost, I would like to give my sincerest gratitude and respect to my
Ph.D. advisor, Professor Charles E. McKenna, who welcomed me into his research
laboratory and has continuously provided opportunities for me to grow as a professional
scientist in the field of antiviral chemistry. I could not have asked for a better mentor to
provide instruction and advice in researching within the field of medicinal chemistry and
to teach me that scientific discovery comes from persistence, patience and hard work.
To Dr. Boris A. Kashemirov, the McKenna lab’s senior research scientist and
mentor, I have to say that I could not have accomplished or learned all I did without your
wisdom and guidance throughout my Ph.D. career. I wholeheartedly thank you our
research discussions and for teaching me that posing inquiries and discussing concepts
makes for a great learning experience.
I would like to extend my gratitude to my dissertation committee members, Prof.
Matthew Pratt, Prof. Wei-Chiang Shen, Prof. Susumu Takahashi and Prof. Kyung Jung,
and the USC professors of my graduate chemistry classes, Prof. Nicos Petasis, Prof.
Richard Brutchey, Prof. Mark Thompson, Prof. Travis Williams, Prof. Surya Prakash,
and Prof. Peter Qin. The excellent learning platform they provided challenged and
v
enriched my experience in the chemistry department at USC and I am very grateful for
their time and effort.
I would like to give a hearty thanks to all of the collaborators I had the pleasure of
working with and for whose efforts and support allowed the research to progress as far as
it did. I would like to warmly thank Dr. Marcela Krečmerová for inviting me to work in
her research group at Institute of Organic Chemistry and Biochemistry AS CR (IOCB)
and to her lab members, Dr. Tomáš Tichý (for synthesizing PMEO-DAPy compounds
3.19 and 3.20), Alice Pomeislová, and Dr. Karel Pomeisl, for being great colleagues and
friends. In remembrance of an amazing individual and scientist, I would like to express
my utmost appreciation and respect for the late Dr. John M. Hilfinger and his team at
TSRL, Inc. for all of the biological data and support they provided throughout our
collaboration. I would also like to give a huge thanks to Prof. Mark N. Prichard and his
lab in the Department of Pediatrics University of Alabama Birmingham, for providing the
in vitro antiviral data against DNA viruses, and Prof. Jae U. Jung and Prof. Zsolt Toth in
the USC Keck School of Medicine, Department of Molecular Biology and Immunology
for the means and instruction to perform the KSHV antiviral in vitro data on our
prodrugs.
It is with great pleasure that I thank past and present McKenna group members for
the pep talks, research advice as well as for making grad school a forum for learning and
fun-filled times. I would especially like to thank Dr. Dana A. Mustafa, Candy Hwang and
Kim Nguyen for their friendship and support throughout grad school. I would also like to
thank past group members Dr. Michaela Serpi, for assisting me in my first year of
vi
antiviral research and to Dr. Valeria Zakharova, for synthesizing PMEA and (R)-
PMPDAP compounds 2.33-2.35. A tremendous thank you to the ever-resourceful
energizer bunny of the group, Inah Kang, for being a lifesaver with all science and non-
science related events and materials. Thank you to the emotional support and ever-
vigilant OCW 201 guard dog and seat companion, Quinn.
Within the USC Department of Chemistry, I would first like to send a huge thank
you to an amazing woman and super hero, Michele Dea, for all of her help and advice
throughout the program. I would also like to thank Marie de la Torre and Magnolia
Benitez for all of their help in helping me maneuver through the administrative portion of
the Ph.D. program. To Darrell Karrafalt and Leo Bedoya, thank you for ensuring the
proper equipment and glassware were readily available. A warm thank you to Ross Lewis
for his kind demeanor and for taking care of all instrumental and technical issues our lab
threw at him. To Allan Kershaw, for sharing his expertise in NMR spectroscopy and to
Phil Sliwoski, for providing amazingly constructed glassware for the laboratory setting.
I cannot begin to express my feelings of gratitude for my friends and family. To
my dearest girlfriends, Jamie Hirami, Amber Eversole, Ana Ciric, Renee Sunseri, Felicia
Vo, and Ariel Thomke: thank you for keeping me grounded and for inspiring through
example. To Dr. Milan Dejmek, thank you for your constant faith in my abilities and for
being a huge support during the Ph.D. process. To Matthew Detrick, thank you for
reminding me to never settle for anything but the best in myself and in life. Last but not
least, I wish to thank my immediate family. To my parents, Donna Brown and Daniel
Williams, my stepmom, Mila Williams, my grandparents Angela Williams, Gene
vii
Williams and Belen Koyama, my aunt, Christine Williams, my siblings, Samantha
Williams, Veronica Williams, Matthew Williams, Danny Williams and my cousin,
Brittany Kilmer: Thank you for being your wonderful and caring selves and for your
endless love and support. I would especially like to thank my sister Samantha for being
my rock and for being a beacon of beautiful light and wisdom.
viii
TABLE OF CONTENTS
DEDICATION iii
ACKOWLEDGEMENTS iv
LIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF SCHEMES xv
ABSTRACT xvii
CHAPTER 1
Acyclic Nucleoside Phosphonates and Their Prodrugs 1
1.1 Brief history of acyclic nucleoside analogues as
antiviral agents 1
1.2 Mechanism of action 4
1.3 Limitations of acyclic nucleoside analogues 4
1.4 Acyclic nucleoside phosphonates 7
1.5 Mechanism of action 9
1.6 Drawbacks 10
1.7 Prodrug approaches 11
1.8 McKenna prodrug approach 15
1.8.1 Development of amino acid-based cyclic and
acyclic approach HPMP prodrugs 17
1.9 Chapter 1 references 22
CHAPTER 2
Synthesis of Tyrosine-Based 9-(2-Phosphonomethoxy)alkyl Purine
Analogues as Antiviral Agent 32
2.1 PME/PMP Background 32
2.2 Mechanism of action 34
2.3 Limitations and the clinical use of their prodrugs 34
2.4 Prodrug approaches to PME/PMP parent drugs 35
2.5 Synthesis of PME/PMP parent drugs 36
2.6 Rationale of prodrug design 37
2.7 PyBOP activating agent 37
2.8 Synthesis of PMEA monoester prodrug via
PyBOP coupling 39
2.8.1 Synthesis of long chain alkyl tyrosinamide
prodrugs 39
ix
2.8.2 Conjugation of amino acid promoiety with
PyBOP 40
2.8.3 Conjugation of alkyl groups with PyBOP 44
2.9 Coupling agent combinations: PyBOP and PyBrOP 46
2.10 Alternative synthetic approach to PME/PMP monoester
prodrugs 48
2.11 Antiviral activity studies 50
2.12 Conclusion 52
2.13 Experimental 53
2.14 Chapter 2 references 61
CHAPTER 3
Synthesis an Antiviral Activity Tyrosinamide Prodrugs of 2,4-Diamino
-Pyrimidine Acyclic Nucleoside Phosphonates 68
3.1 Introduction: Discovery of 2,4-diaminopyrimidines
(DAPys) 68
3.2 Antiviral activity of ANP-DAPy parent drugs 70
3.3 Mechanism of action 72
3.4 Limitations 72
3.5 Rationale for developing prodrugs of ANP-DAPys 73
3.6 Synthesis of PMEO-DAPy and (R)-HPMPO-DAPy 74
3.7 Synthesis of tyrosinamide and tyrosine-based prodrugs
of PMEO-DAPy 75
3.8 Synthesis of tyrosinamide cyclic and acyclic
(R)-HPMPO-DAPy prodrugs 78
3.9 Antiviral activity studies 80
3.10 Conclusion 82
3.11 Experimental section 82
3.12 Chapter 3 references 96
CHAPTER 4
Synthesis and Antiviral Activity of a Novel N-Alkyl Ser-Val
Dipeptide Analogue of Cyclic Cidofovir 100
4.1 Introduction: (S)-HPMPC 100
4.2 Mechanism of action 101
4.3 Limitations 102
4.4 Prodrugs of (S)-HPMPC 102
4.5 Rationale for prodrug design of long chain dipeptide
cHPMPC analogue 103
4.6 Synthesis of novel N-alkyl long chain Ser-Val dipeptide
promoiety 104
4.7 Synthesis of cyclic long chain N-alkyl Ser-Val dipeptide
prodrug of (S)-HPMPC 106
4.8 Antiviral activity against DNA viruses 107
x
4.9 Kaposi- sarcoma-associated herpesvirus 108
4.10 KSHV antiviral activity studies 109
4.11 Conclusion 113
4.12 Experimental 113
4.13 Chapter 4 references 118
CHAPTER 5
Scaled-up Procedure for Lipophilic N-Alkyl Tyrosinamide
Prodrugs of (S)-HPMPA and (S)-HPMPC 124
5.1 Introduction: (S)-HPMPA and (S)-HPMPC 124
5.2 Mechanism of action and limitations 124
5.3 Prodrugs of (S)-HPMPA and (S)-HPMPC 125
5.4 Large scale synthesis of cyclic and acyclic (S)-HPMPA
(S)-HPMPC lipophilic N-alkyl tyrosinamide prodrugs 127
5.4.1 Large scale synthesis of (L)-Tyr-NHC
16
H
33
promoiety 128
5.4.2 One-pot cyclization of parent drug and
conjugation of promoiety 128
5.4.3 Hydrolysis of intramolecular P-O bond 130
5.5 Conclusion 131
5.6 Experimental section 131
5.7 Chapter 5 references 138
CHAPTER 6
Synthesis of 5’-Phosphonate Analogues of Ribavirin as Antiviral
Agents Against Dengue Virus 140
6.1 Introduction: Dengue virus 140
6.2 Ribavirin and current uses 141
6.3 Mechanism of action 142
6.4 Limitations 143
6.5 Prodrugs of ribavirin 144
6.6 Ribavirin 5’-monophosphate mimics:
5’-monophosphonates 145
6.6.1.Synthesis of 5’-phosphonate analogue of
ribavirin 145
6.6.2. Synthesis of 5’-methylene phosphonate
analogue of ribavirin 148
6.7 Conclusion 150
6.8 Experimental section 151
6.9 Chapter 6 references 158
xi
CHAPTER 7
Insights and Perspectives 164
7.1 Introduction 164
7.2 Synthetic approach to monoester prodrugs
of PME/PMP parent drugs 164
7.3 PME/PMP monoester prodrugs and their antiviral
activity 167
7.4 Antiviral activity of the lipophilic N-alkyl Ser-Val
dipeptide cHPMPC prodrug 168
7.5 Synthetic approaches to 5’-phosphonate analogue of
ribavirin 169
BIBLIOGRAPHY 171
APPENDICES
APPENDIX A: Chapter 2 Supporting Data 196
APPENDIX B: Chapter 3 Supporting Data 209
APPENDIX C: Chapter 4 Supporting Data 226
APPENDIX D: Chapter 5 Supporting Data 229
APPENDIX E: Chapter 6 Supporting Data 234
xii
List of Tables
Table
2.1
Table 2.1 in vitro antiviral activities against HSV-2, VZV, CMV,
cytotoxicities and selectivity index values of the tyrosine prodrugs of
PMEA and (R)-PMPDAP.
a
51
2.2
Table 2.2 in vitro antiviral activities against VACV, CPXV, ADV,
cytotoxicities and selectivity index values of the tyrosine prodrugs of
PMEA and (R)-PMPDAP.
a
52
3.1
Table 3.1 in vitro antiviral activities against HCMV, cytotoxicities
and selectivity index values for 3.1, 3.2, 3.19-3.22, 3.29.
a
81
4.1
Table 4.1 in vitro antiviral activities against HSV-2, VZV, CMV,
cytotoxicities and selectivity index values of the cHPMPC prodrug
and the C
16
-dipeptide prodrug of cHPMPC.
a
107
4.2
Table 4.2 in vitro antiviral activities against VACV, CXPV, ADV,
cytotoxicities and selectivity index values of the cHPMPC prodrug
and the C
16
-dipeptide prodrug of cHPMPC.
a
108
xiii
List of Figures
Figure
1.1 Early acyclic nucleoside analogues 1
1.2 Examples of selected acyclic nucleoside analogues 2
1.3 Acyclic nucleoside analogues and some of their corresponding prodrugs 3
1.4 Phosphate vs. phosphonate linkage 6
1.5 Phosphonate analogues: PAA and PFA 7
1.6 Acyclic nucleoside phosphonates and their prodrugs 8
1.7 Prodrug approaches for nucleotide analogues 14
1.8 Peptidomimetic prodrugs of (S)-HPMPC and (S)-HPMPA 19
2.1 PME/PMP acyclic nucleoside phosphonates 33
2.2 Oral prodrugs of PMEA and (R)-PMPA 33
2.3 Common (benzotriazolyloxy)-phosphonium reagents in peptide coupling 38
2.4
31
P NMR (202.5 MHz) of reaction mixture (D
2
O capillary) of PMEA and
(L)-Ser-OMe (2.16)
42
2.5
31
P NMR (202.5 MHz, D
2
O capillary) of reaction mixture of 1) PMEA
+PyBOP (no nucleophile) 2) PMEA + PyBOP + MeOH and 3) PMEA +
PyBOP + (L)-Tyr-OMe at 40 °C. Observance of PMEA-OBt complex in
all cases.
45
2.6 Structure of PyBrOP 47
2.7 Tyrosine-based monoester prodrugs of PMEA and (R)-PMPDAP 50
3.1 FDA approved acyclic nucleoside phosphonates and their prodrugs 69
3.2 Structures of (R)-HPMPO-DAPy, PMEO-DAPy and (R)-PMPO-DAPy. 70
3.3 Tyrosine-based prodrugs of PMEO-DAPy 76
3.4 Cyclic and acyclic tyrosinamide prodrugs of (R)-HPMPO-DAPy 80
xiv
4.1 Cidofovir and cyclic cidofovir 100
4.2 Scaffolds for cyclic and acyclic prodrugs of (S)-HPMPC and (S)-HPMPA 103
4.3 (L)-Ser-NHC
16
H
33
-(L)-Val-cHPMPC 107
4.4 Nucleotide analogues screened against KSHV replication 111
4.5
A. KSHV infection and dosing cycle. B and C. Results from screening
nucleotide analogues that block KSHV replication.
112
5.1 ANPs of the HPMP Prototype: (S)-HPMPA and (S)-HPMPC 124
5.2 HDP and ODE monoester prodrugs of (S)-HPMPA and (S)-HPMPC 126
5.3 Cyclic and acyclic prodrugs of (S)-HPMPC and (S)-HPMPA 127
6.1 Structure of ribavirin 142
6.2 Prodrugs of ribavirin 144
6.3 Ribavirin 5-monophosphate mimics 145
6.4 Final compounds post HPLC purification 147
6.5
Left
1
H NMR spectra: Compound 6.13 post HWE olefination (
1
H NMR
(500 MHz, Methanol-d
4
). Right
1
H NMR spectra: Compound 6.14 post
hydrogenation (
1
H NMR (500 MHz, Methanol-d
4
)
149
xv
List of Schemes
Schemes
2.1
Synthesis of diisopropyl chloroethoxymethane phosphonate
synthon (2.9) a. Reflux, 120° C, N
2
, 4 h. Distill at reduced pressure,
heat to 110 °C – 115 °C.
36
2.2
Synthesis of PMEA a. K
2
CO
3
, DMF, 80 °C, 2h b. 2.9, 80 °C,
overnight c. SiMe
3
Br, MeCN, rt, overnight.
36
2.3 BOP coupling mechanism and formation of HMPA 39
2.4
Amidation of (L)-Boc-tyrosine; a. DCM, HOBt, EDC⋅HCl,
(NH
2
C
4
H
9
for 2.13; NH
2
C
8
H
17
for 2.14; NH
2
C
16
H
33
for 2.15), rt, 72
h.
39
2.5
General procedure for the synthesis of peptidomimetic prodrugs of
cyclic (S)-HPMPA and (S)-HPMPC
41
2.6
PyBOP condensation reaction with PMEA and promoiety yield
PMEA-OBt intermediate.
43
2.7 Successive PyBOP couping reactions 46
2.8 Consecutive PyBrOP reactions 47
2.9
a. DMF, DIEA, PyBrOP, EtOH or iPrOH, 40 °C, overnight b. DMF,
DIEA, PyBOP, 40 °C, promoiety (phenol, 2.13, 2.14 or 2.15), 2h c.
MeCN, BTMS, 75 °C, 4 h.
49
3.1
a. NaH, Dioxane, reflux; b. MeOH/ H
2
O, HCl, rt; c. DMTrCl,
pyridine, rt, 3 h; d. BrCH
2
P(O)(OiPr)
2
, NaH, DMF, -20 °C, 24 h; e.
HOAc, rt, 1 h; f. BTMS, MeCN, 80 °C, 24 h.
74
3.2
Synthesis of PMEO-DAPY and PMEO-DAPy(OiPr) a. Cs
2
CO
3
,
DMF, 100 °C, 16 h; b. Dioxane, KOH/H
2
O, reflux, 6 h; c. BTMS,
MeCN, 80 °C, 24 h.
75
3.3 a. PyBOP, DMF, DIEA, rt, 24 h; b. BTMS, MeCN, rt, 24 h. 77
3.4
a. DCC, DCMC, DMF, 100 °C, 6 h; b. PyBOP, DMF, DIEA, rt, 4 h;
c. TFA, CH
2
Cl
2
, 24 h; d. NH
4
OH, MeCN, 45 °C, 4 h.
79
4.1 Synthesis of the lipophilic N-alkyl (L)-Ser-C
16
-(L)-Val dipeptide 105
xvi
promoiety. a. EDC, HOBt, CH
2
Cl
2
, NH
2
C
16
H
33
, 0 °C, overnight; b.
TFA, CH
2
Cl
2
, rt, overnight; c. 0.2 M HCl/MeOH, MeOH, -20 °C, (x
3); d. EDC, HOBt, CH
2
Cl
2
, TEA, (L)-Boc-valine
4.2
Synthesis of cyclic dipeptide prodrug of cidofovir . a. DIEA, DMF,
4.7, PyBOP, 40 °C, overnight; b. TFA, CH
2
Cl
2
, rt, overnight
106
5.1
Synthesis of cyclic prodrugs of 5.1 and 4.1. a. DMF, DIEA, PyBOP,
40 °C, overnight b. CH
2
Cl
2
, TFA, rt, overnight
129
5.2
Synthesis of acyclic prodrugs from 5.2 and 5.3. a. NH
4
OH, MeCN,
45 °C, 6 h.
131
6.1
Synthesis of RBV-cP analogue. a. I
2
, PPh
3
, pyridine, 2 h, rt; b.
pyridine, BzCl, 1 h, rt; c. triethylphosphite, 100 °C, 15 h; d.
DMF:MeCN (1:1), BTMS, 6 h, rt; e. aq. NH
4
OH, 6 h, rt.
146
6.2
Synthesis of 2-iodoxybenzoic acid. a. H
2
O, 70 °C, 1 h; b. rt,
overnight
148
6.3
Synthesis of RBV-CCP analogue 6.3. a. TBSCl, imdazole, DMF, rt,
overnight; b. TCA, THF/H
2
O, rt, 6 h; c. 6.8, MeCN, 3 h, 80 °C; d.
THF, NaH, tetramethyl bisphosphonate, 3 h, rt; e. HCOOH: H
2
O
(1:1), 48 h, rt; f. Pd/C, H
2
, 3 h, rt; g. MeCN:DMF (1:1), BTMS, rt, 2
h.
150
xvii
Abstract
Acyclic nucleoside phosphonates, ANPs, are a drug class with broad-spectrum activity
against DNA and RNA viruses. However, limitations with ANP efficacy include: low cell
membrane permeability, low oral bioavailability (due to the presence of an ionizable
anionic group) and nephrotoxicity In an effort to curtail these effects, as well as to further
understand and demonstrate the full therapeutic potential of this drug class, a main focus
of the work presented in this dissertation is on the synthesis and antiviral activity of
various ANP parent drugs and their corresponding acyclic and cyclic nucleoside
phosphonate prodrugs.
Parent ANPs were masked with an amino acid or dipeptide promoiety, which contained a
lipophilic N-alkyl long chain on the C-terminal group as a carboxamide. PME and PMP
ANP monoester prodrugs required an alternative synthetic route involving PyBrOP
coupling with a small alkoxy nucleophile followed by PyBOP coupling with the desired
amino acid or dipeptide promoiety. A novel (L)-Ser N-alkyl dipeptide cyclic prodrug of
(S)-HPMPC was synthesized in order to evaluate and expand the ANP peptidomimetic
prodrug library. PME and HPMP prodrugs containing an N-alkyl C
16
long chain
demonstrated EC
50
values in the micromolar range against a variety of DNA viruses
herpes simplex virus type 1, varicella zoster virus, cytomegalovirus, cowpox virus,
adenovirus, and vaccinia virus. In addition, a selection of lipophilic N-alkyl amino acid-
and dipeptide-based ANP prodrugs were evaluated for in vitro antiviral activity against
xviii
kaposi-sarcoma associated herpesvirus. (L)-Tyr-NHC
18
H
37
and (L)-Ser-NHC
16
H
33
-(L)-
Val prodrugs of (S)-HPMPA and (S)-HPMPC demonstrated inhibition values requiring
further assessment of their application as KSHV antiviral agents. In addition, a large-
scale synthesis (1 g – 2 g) of (L)-Tyr-NHC
16
H
33
cyclic and acyclic HPMP prodrugs was
performed as a means to assess reaction conditions for optimal and efficient generation of
large quantities of lead candidates.
Ribavirin (RBV) is a guanosine ribonucleic analogue that acts as a nucleoside inhibitor. It
has been found to be active against various DNA and RNA viruses (including flaviviridae
viruses like yellow fever and hepatitis C). In an effort to expand the druggable
phosphonate library and potentially enhance its antiviral activity against dengue virus, an
emerging RNA pathogen to humans, two 5’-phosphonate analogues of RBV have been
synthesized. The structural modification lies in replacing the 5’-OH of the ribose ring
with an X-P(O)(OH)
2
linkage (where X is CH
2
or CH
2
-CH
2
). The 5’-phosphonate RBV
analogue was synthesized from a published procedure using the well-utilized Michaelis-
Arbuzov reaction and the Horner-Wadsworth-Emmons reaction was employed to extend
the alkyl length between the ribose ring and phosphonate to afford the 5’-methylene
phosphonate RBV analogue.
1
Chapter 1
Acyclic Nucleoside Phosphonates and Their Prodrugs
1.1 Brief history of acyclic nucleoside analogues as antiviral agents
The discovery and development of acyclic nucleoside phosphonates and their analogues
began more than 50 years ago when the era of antiviral therapy commenced. The age of a
new class of therapeutic drugs emerged in 1959 with the synthesis of idoxuridine (IDU,
Figure 1.1).
1
IDU started out as a potential anticancer agent but in 1961, after
demonstrating antiviral activity against herpes simplex virus (HSV) and vaccinia virus
(VV),
2
it became the first commercialized topical antiviral drug used to treat herpetic
keratitis.
3
Two years later, another uridine analogue, trifluridine (TFT, Figure 1.1), was
released by Kaufman and Heidelbrger as a topical remedy for herpetic keratitis.
4
Figure 1.1 Early acyclic nucleoside analogues
The 1960’s ushered in another antitumor agent to demonstrate antiviral activity known as
arabinosyladenine (Ara-A, Figure 1.1). Its gradual build up to being used in 1976
5
as a
systemic therapy for varicella zoster virus (VZV) began with Privat de Garilhe and de
NH
O
O N
O
OH
HO
I
Idoxuridine
NH
O
O N
O
OH
HO
F
3
C
Trifluridine
N
N
N
N
NH
2
O
HO
Arabinosyladenine
OH HO
2
Rudder reporting its antiviral potential against HSV and VV,
6
followed by Schabel
demonstrating its true status as an antiviral agent.
7
Its solubility and deamination issues,
as well as, the emergence of a new class of antiviral agents ended further usage as a
clinical therapeutic.
By the late 1970’s, the field of antiviral chemotherapy increasing the number of acyclic
nucleoside analogues to its successes. The first selective antiviral agents against the viral
DNA polymerases of HSV and VZV, acyclovir (ACV, Figure 1.2) and (E)-5-(2-
bromovinyl)-2’-deoxyuridine (BVDU, Figure 1.2) were discovered and studied.
8-11
Concurrently, as the result of a collaboration between Antonín Holý and Erik De Clercq,
in 1978 the first broad spectrum acyclic nucleoside analog, (S)-9-(2,3-
dihydroxypropyl)adenine (DHPA; Figure 1.2) was identified and demonstrated antiviral
activity against RNA and DNA viruses via inhibition of (S)-adenosylhomocysteine
hydrolase.
12,13
An interesting point, regardless of the structural similarities between ACV
and DHPA, their antiviral activities and modes of action are very different.
Figure 1.2 Examples of selected acyclic nucleoside analogues
NH
N
N
O
NH
2
N
O
HO
Acyclovir
ACV
HN
O
N
O
OH
HO
N
N
N
N
NH
2
HO
HO
Br
H
H
O
(E)-5-(2-bromovinyl)-2’-deoxyuridine
BVDU
(S)-9-(2,3-dihydroxypropyl)adenine
(S)-DHPA
3
Other antiviral acyclic nucleoside analogues, (penciclovir and ganciclovir; Figure 1.3),
and their prodrugs, (valacyclovir, famciclovir, and valganciclovir; Figure 1.3) have
followed the first set of antiviral agents in an effort to selectivity against certain DNA
viruses, as well as, overcome their therapeutic limitations (poor solubility, low oral
bioavailability). In terms of increasing oral bioavailability, the prodrug approach has been
applied to acyclovir and another nucleoside penciclovir and they were converted to their
oral prodrug forms, valaciclovir and famciclovir, respectively. Valacylovir (VACV,
Valitrex, Figure 1.3) replaced its predecessor in 1) becoming the oral treatment for HSV
and VZV infections, 2) the only antiviral drug approved for the once-daily dose for
treating genital herpes and 3) has been FDA approved for immunocompromised
individuals with HSV or VZV infections.
14,15
Figure 1.3 Acyclic nucleoside analogues and some of their corresponding prodrugs
NH
N
N
O
NH
2
N
O
O
O
H
2
N
Valacyclovir
NH
N
N
O
NH
2
N
O
HO
HO
Ganciclovir
NH
N
N
O
NH
2
N
O
O
HO
Valganciclovir
O
H
2
N
NH
N
N
O
NH
2
N
HO
HO
Penciclovir
NH
N
N
O
NH
2
N
O
O
Famciclovir
C H
3
C
O
C
O
H
3
C
4
1.2 Mechanism of action
The selectivity and potency of acyclic nucleoside analogues akin to ACV and BVDU
stems from their preferential initial phosphorylation by specific viral-encoded thymidine
kinase (TK). Once initial phosphorylation has occurred, nucleoside analogues require
conversion to their 5’-triphosphate metabolite form by cellular nucleoside and nucleotide
kinases in order to exert their therapeutic effect. Transformation of ACV and BVDU to
their triphosphate forms gives them the potential to be recognized by viral DNA
polymerase as natural nucleoside triphosphates. Incorporation into the viral DNA strand
and inhibition of viral DNA synthesis is considered to be the mode of antiviral action for
ACV, BVDU and related nucleoside analogues.
16
1.3 Limitations of acyclic nucleoside analogues
Three main limitations arise with antiviral nucleoside analogues: 1) The initial
phosphorylation, 2) nucleotide catabolism and 3) poor oral bioavailability.
17
The key to
the selectivity and antiviral potency of nucleoside drugs can also be a drawback. Initial
phosphorylation to the 5’-monophosphate analogue is typically a rate-limiting step that
can hinder their antiviral potential because it is performed by a virus-encoded TK (for
HSV and VZV) or a specific virus-encoded protein kinase (PK) (for cytomegalovirus,
CMV). Drawbacks of this nature are found in, but are not limited to, DNA viruses, such
as CMV and Epstein-Barr virus (EBV) lacking a specific viral TK
18
or are mutant strains
of HSV or VZV which display diminished TK activity.
19
As a result, acyclovir,
penciclovir and ganciclovir are ineffective against these types of DNA viruses that fail to
5
supply an enzyme for the phosphorylation to the monophosphate metabolite. Subsequent
phosphorylation to the triphosphate analogue is required for their conversion to become
alternative substrates that can compete with natural nucleoside triphosphates to be
incorporated into the viral DNA chain. On the other end of the spectrum, nucleoside and
nucleotide degradation by enzymes that can potentially cleave the nucleoside linkage, as
well as, phosphomonoester and phosphodiester linkages is another disadvantage.
17
Lastly,
solubility and oral bioavailability have been issues for nucleoside drugs such as ACV,
ganciclovir, penciclovir, and others.
6
As a result, development of modified nucleosides
and nucleotides involved attaining the following: 1) elimination of the initial
phosphorylation step, 2) resistance to enzymatic degradation, 3) maintain structural
isopolar properties and 4) increased oral absorption.
One possible solution included administering a nucleoside analogue in the form of its
monophosphate, thereby removing the necessary initial phosphorylation step.
20,21
However, a barrier for this approach is the presence of phosphohydrolases (phosphatases,
and 5’-nucleotidases) which can remove the phosphate group upon its entry into the
cell.
22
Due to this obstacle, the search and development of a mono-phosphorylated
nucleoside analogue was pursued.
As a part of the investigation of nucleoside analogues with enhanced potency, Erik De
Clercq and Antonín Holý began a collaboration in 1976 that resulted in the discovery of a
novel, broad-spectrum acyclic nucleoside analogue, DHPA (Figure 1.2), two years later
6
that lead to the investigation of phosphorus-containing derivatives of DHPA and related
analogues in search of metabolically stable, isopolar and isosteric nucleotide analogues.
From these studies, they elaborated the necessity of an oxygen atom near the phosphorus
moiety for enzyme recognition through their studies on phosphorus acid esters,
phosphothioates, alkylphosphonates and various substituted derivatives of the them.
23
The conclusion from their structure-activity studies indentified a phosphonomethyl ether
group (–O-CH2-P(O)(OH)2) in place of a phosphate to be optimal candidate for studying
catabolically stable acyclic nucleoside analogues with a phosphorus moiety.
17,24
Figure 1.4 Phosphate vs. phosphonate linkage
In the search for effective phosphorus-containing candidates for antiviral targets, the
therapeutic potentials of phosphonoacetic acid (PAA; Figure 1.5)
25
and phosphonoformic
acid (PFA; Figure 1.5)
26
were presented and evaluated. Surprisingly, conjugation of PAA
or PFA to the antiviral nucleoside agents BVDU, ACV and Ara-A did not result in higher
antiviral activity against HSV-1 and HSV-2 compared to the solo acyclic nucleoside
analogues.
27-29
However, a hybrid of PAA and DHPA produced a 2’-(S)-isomer with
potent against DNA viruses and cellular parasites
30
from a mixture of two regioisomers
and unleashed a whole new class of metabolites with enhanced antiviral potency and
capabilities: acyclic nucleoside phosphonates.
P
O
HO
OH
H
2
C O
Phosphate
linkage
P
O
HO
OH
O
H
2
C
Phosphonate
linkage
7
Figure 1.5 Phosphonate analogues: PAA and PFA
1.4 Acyclic nucleoside phosphonates
Since their discovery in the 1980’s, acyclic nucleoside phosphonates (ANPs) have
become an integral group of antiviral agents that demonstrate a wide range of biological
activities. Theses nucleotide analogues differ from their natural nucleotide counterparts
and nucleoside analogues in that they contain a nonhydrolyzable isopolar
phosphonomethyl ether moiety with a P-C linkage in place of the natural P-O nucleotide
phosphate ester group in order to prevent their enzymatic degradation
31
while
simultaneously eliminating initial intracellular phosphorylation necessary for nucleoside
activation. Presence of the phosphonate moiety allows for the rate-limiting initial
phosphorylation step to be bypassed and broadens their antiviral spectrum to include TK
deficient viruses. As a class, ANPs have demonstrated their wide range as a biological
therapeutic as antivirals, cytostatics, and immunomodulatories.
30,32-35
ANPs were first described in 1986 by Antonín Holý and Eric De Clercq starting with the
two of the main ANP prototypes: (S)-9-(3-hydroxyl-2-
phosphonylmethoxypropyl)adenine ((S)-HPMPA; 1, Figure 1.6)
30
and a non-chiral
adenine analogue 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; 2, Figure 1.6).
30
Upon
P
O
HO
OH
O
OH
Phosphonoacetic acid
PAA
P
O
HO
HO
O
OH
Phosphonoformic acid
Foscarnet
PFA
8
substituting the carboxylate group of PAA with (S)-DHPA, a novel ANP, (S)-HPMPA,
was developed and demonstrated a substantial change in antiviral activity that mimicked
PAA in inhibiting DNA viruses rather than the antiviral activity of (S)-DHPA. Included
in its antiviral spectrum was the activity observed against viruses lacking a viral TK, such
as TK
-
HCMV and TK
-
HSV, which had become resistant to the TK phosphorylation-
dependent nucleoside antiviral acyclovir.
30
Though the ANP prototype (S)-HPMPA has
not attained clinical approval, three antiviral ANP drugs, (S)-HPMPC (cidofovir,
Vistide
®
),
36
bis-POM PMEA (adefovir dipivoxil, Hepsera
®
) and bis-POC (R)-PMPA
(tenofovir disoproxil fumarate, Viread
®
) (Fig 1.6), are FDA approved to treat CMV
retinitis in AIDS patients, chronic hepatitis B infections and HIV infections
respectively.
23
Figure 1.6 Acyclic nucleoside phosphonates and their prodrugs
N
N
HO
O
P
O
OH HO
NH
2
O
HO
O
P
O
OH HO
N
N
N
N
NH
2
(S)-HPMPC
Cidofovir, Vistide
®
(S)-HPMPA
N
N
N
N
O P
O
OH
OH
NH
2
PMEA
Adefovir
N
N
N
N
O P
O
OH
OH
NH
2
(R)-PMPA
Tenofovir
(S)
(S)
(R)
N
N
N
N
O P
O
O
NH
2
N
N
N
N
O P
O
O
NH
2
O O
O
O
O
Adefovir Dipivoxil
Hepsera
®
Tenofovir disoproxil fumarate
Viread
®
O O
O
O
O O
O
CO
2
H
HO
2
C
9
ANPs are grouped into three classes (HPMP, PME and PMP derivatives), which contain
an overall structural framework similar to the prototypical ANPs (S)-HPMPC (cidofovir,
Vistide
®
), PMEA (adefovir) and (R)-PMPA (tenofovir) (Fig 1.6). ANPs within the
HPMP category display a broad antiviral activity mainly against DNA viruses (polyoma-,
papilloma-, adeno-, herpes- and poxviruses).
31,37
ANPs categorized in the PME class
demonstrate a slightly overlapping antiviral activity spectrum from the HPMP derivatives
in that it too was potent against some DNA viruses (HSV-1, HSV-2, EBV, VZV, and
HCMV) as well as hepadnaviruses (human and duck hepatitis B viruses) and retroviruses
(HIV-1, HIV-2, SIV, feline immunodeficiency virus (FIV), visna/maedi virus, feline
leukemia virus, LP-BMS (murine AIDS) virus and Moloney (murine) sarcoma virus).
31,37
Analogues of the PMP class demonstrate a narrower window of activity than the PME
class in that their antiviral activity against hepadna- (HBV) and retroviruses (HIV-1 and
HIV-2).
31,37
1.5 Mechanism of Action
In order to exhibit their antiviral potential, these compounds must be taken up into virally
infected cells and undergo two consecutive intracellular phosphorylation steps to achieve
their active 5’-triphosphate metabolite analogue form (ANPpp).
38
Unlike the original
acyclic nucleoside analogues, like ACV and ganciclovir, ANPs are initially
phosphorylated to their respective monophosphate analogue by a cellular nucleotide
kinase (GMP kinase or AMP kinase),
39
followed by additional phosphorylation by
cellular kinase to their active triphosphate analogue (ANPpp) which targets viral and
10
cellular
40,41
DNA polymerase. In addition, ANPpp analogues compete with natural
nucleotides as DNA polymerase substrates and upon incorporation into the viral DNA
chain can act as chain terminators or alternative substrates. As a result of ANPpp
conversion demonstrating a higher affinity for viral polymerases over cellular
polymerases α, β, ε, and γ,
42,43
and eliminating the TK-dependent initial phosphorylation
step, ANPs should demonstrate a wider activity spectrum than previous nucleoside
analogues. Therefore, incorporation of the phosphonate moiety serves to circumvent three
major limitations with acyclic nucleoside analogues: 1) the rate-limiting initial
phosphorylation step in acyclic nucleoside activation, as well as, 2) to provide a means of
treating thymidine kinase mutant viruses
44
and 3) infrequent dosing due to the long
intracellular half-life.
45,46
1.6 Drawbacks
The main limitations associated with the clinical applications of acyclic nucleoside
phosphonates is their reduced cell permeability and oral bioavailability due to the
ionizable phosphonic acid group.
47
In addition, ANPs generally demonstrate
nephrotoxicity due to their accumulation in the kidney proximal convoluted tubule (PCT)
cells.
48
In an effort to maximize the antiviral potential of this class of drugs, the prodrug
approach was applied to ANPs to mask undesirable properties and reduce adverse
biological effects.
11
1.7 Prodrug approaches
Of the approximate 60 antiviral drugs approved by the US Food and Drug Administration
(FDA), more than 25 are nucleoside and nucleotide analogues used against hepatitis, HIV
and HSV infections.
49
Nucleoside and nucleotide analogues must be phosphorylated to
their corresponding 5’-triphosphate form by intracellular or viral kinases in order to exert
their antiviral activity. Phosphorylation by endogenous kinases is often slow and a rate-
limiting step in the drug’s efficacious nature in human cells.
20
The option to administer
this class of drugs as their monophosphate analogues is dampened by the decreased oral
bioavailability due to the ionizable –O-P(O)(OH)
2
group nucleotide analogues
possess.
19,21
Phosphohydrolases, which remove the phosphate group, can pose an added
threat if the monophosphate analogues cross the cell membrane.
21
These two factors
render the isomeric ANP monophosphonate, which consists of a –O-CH
2
-P(O)(OH)
2
,
group, more favorable because dephosphorylation can be prevented. However, the
presence of an ionizable phosphonic acid moiety still displays decreased transport.
One of the most advantageous methods utilized to improve fundamental properties, such
as, pharmacokinetics, efficacy and adverse toxicological effects, of well known, active
drug molecules is to implement the prodrug approach and mask the physiologically
undesirable properties of the drug molecule, usually with a promoiety.
50-52
Ideally,
attachment of the promoiety serves to temporarily deactivate the drug’s therapeutic effect
until the biological target is reached and, through chemical or enzymatic alterations, the
active drug is released through cleavage of the promoiety.
50-52
Ideal qualifications of a
12
prodrug consist of 1) effectively releasing a non-toxic promoiety upon activation 2)
advantageous ADMET (absorption, distribution, metabolism, excretion and toxicity)
characteristics 3) demonstrates a favorable half-life in the body and 4) is chemically
stable in the form it is to be administered in. Prodrugs can be developed to address
various pharmacological barriers exhibited by active drug molecules, such as issues with
solubility, oral absorption/bioavailability, targeted drug delivery, chemical/enzymatic
stability, toxicity and other adverse effects. The main goal of this process is to introduce a
stable prodrug that is non-toxic and releases a non-toxic, metabolized promoiety along
with the active drug.
Activation of the parent drug and release of the promoiety has been reported to occur
through chemical or enzymatic processes. In the former transformation, the benefit of
parent drug liberation being independent of enzyme expression variability is outweighed
by the lack of success in target-specific activation of parent drugs due to difficulties in
fine-tuning chemical stability with pharmacologically sufficient activation rates.
53-55
In
order to effectively elicit the benefits of enzymatic activation, the activating enzyme
should possess the following: i) be associated with an extensively studied and
characterized enzyme class that takes part in the target (tissue or disease), ii) be
significantly expressed in the target tissue (so as to ensure selectivity over healthy cells
and tissue); and iii) be in substantial amount to generate a pharmacological effect upon
prodrug activation. Difficulties in this area of prodrug development include successfully
selecting a tissue/disease unique enzymatic target and in vivo identification of specific
13
activating enzyme.
56
Aside from the site specific target delivery prodrug approach (where
prodrugs are designed to enhance delivery to a specific tissue or organ), enzymatic
studies of prodrugs generally occurs after a lead compound (active drug) has been chosen
for clinical development.
Applications of the prodrug approach to nucleotide analogues can consist of various
modifications, however, formulation strategies
57-61
are not applicable to the work
presented in this document, therefore they will not be discussed. Focus will be placed on
attaching promoieties to the phosphonic/phosphoric acid moiety in hopes of enhancing
their in vivo efficacy and oral absorption. Delivery of the active drug can be increased by
attaching an appropriately designed promoiety that displays optimal biological and
physical properties, including lipophilicity, chemical and enzymatic stability, as well as
target specificity.
19,62,63
As a result of the limitations of nucleotide analogues, various
prodrug approaches have been applied in order to eliminate undesirable physiochemical
properties, improve in vivo efficacy, and minimize their cytotoxic effects.
64-66
Initial
attempts of utilizing simply dialkyl esters to mask the dual negative charge on PMEA
proved unsuccessful in that the parent drug due to insufficient chemical and enzymatic
release under physiological conditions.
67-69
As a result, monoester prodrugs as potential
substrates of phosphodiesterases,
70,71
and the exploitation of intracellular enzyme
activation of prodrugs has lead to the various prodrug approaches currently being
developed (Figure 1.7).
14
Figure 1.7 Prodrug approaches for nucleotide analogues
The most recent prodrug approaches that have been applied to known active drugs and
their analogues to increase their oral absorption include the acyloxyalkyl and
POM/POC Approach
N
OH
P
O
O(CH
2
)
3
O(CH
2
)
15
CH
3
O
OH
HDP-(S)-HPMPC
CMX001
Brincidofovir
Lipid Ester Approach
NH
O N
O
F OH
O
CH
3
P
O
NH
O O
O
O
Sofosbuvir
Solvadi
®
Phosphoramidate Protide Approach
N
O
NH
2
B
O
O
P
O
X
H
2
N
O
R
(S)
B
O
HO
P
O
X
H
2
N
O
R
HO
(S)
B = Cytosine ((S)-HPMPC)
B = Adenine ((S)-HPMPA)
Peptidomimetic Approach
P X
O
S
O
S
O
R
O
O
OR
1
OR
1
OR
1
R
1
O
R = t-Bu
R
1
= H; Ac
Mixed SATE pronucleotide
SATE Approach
O
P
O
O
X
R
CH
2 O
O
H
3
C
X = OCH
2
, CH
2
O
R = H; Me; t-Bu
5-(1-acetoxyvinyl)-cycloSal pronucleotides Amino acid and dipeptide prodrugs
CycloSal Approach
X = OCH
2
: OC
6
H
4
R = OAlk, NHAlk,
NH-Val, NH-Leu,
NH-Ala, NH-Phe
N
N
N
N
O P
O
O
NH
2
N
N
N
N
O P
O
O
NH
2
O O
O
O
O
Adefovir Dipivoxil
Hepsera
®
Tenofovir disoproxil fumarate
Viread
®
O O
O
O
O O
O
CO
2
H
HO
2
C
X = O, CH
2
NA = nucleoside
analogue
X = O, CH
2
NA = nucleoside
analogue
NA
NA
15
alkyloxycarbonyloxymethyl approach (POM/POC approach),
67,72-75
the lipid ester
approach,
76-79
the phosphoramidate approach,
80-81
the cycloSal approach,
55,82-84
the SATE
approach,
85,86
and the peptidomimetic approach.
87
Figure 1.7 depicts a prototype from
each of the prodrug approaches mentioned above. Recently, Gilead has announced the
release of Sofosbuvir (Solvaldi
®
, Figure 1.7),
88-90
a nucleotide analogue HCV NS5B
polymerase inhibitor, that, in combination with other antivirals, are interferon-free and
have demonstrated high rates of sustained virologic responses in patients with chronic
HCV infections.
91,92
In more recent news, Chimerix, Inc., has announced CMX001
(Figure 1.7) as an experimental antiviral agent for the emergency treatment of ebola virus
disease. The prodrugs approaches depicted in Figure 1.7 have been extensively
reviewed,
64-67
therefore they will not be discussed in length. However, the amino acid
phosphonate ester prodrug approach will be discussed in further detail below to describe
the evolution of this approach in the McKenna lab and explain the rationale for the novel
antiviral agents that have been described in the remaining chapters of this dissertation.
The rationale behind the structural design for each approach is based on addressing
known limitations for certain classes of drugs in order to enhance their pharmacokinetics
and efficacy by capitalizing on the in vivo activities of specific enzymes.
1.8 McKenna Prodrug Approach
The amino acid based prodrug approach developed in the McKenna laboratory has been
extensively review recently.
87
Depicted here will be a brief timeline of the evolution of
this approach in order to provide insight and research findings that lead to synthesizing
16
and investigating the novel compounds presented in this work originated from. The two
main ANP prototypes that have been published in previous studies by our group come
from the HPMP class: (S)-HPMPC and (S)-HPMPA. Their structure differs from the
PME and PMP class of ANPs in that they contain a 2’-hydroxymethylene function on the
modified sugar moiety (the phosphonomethoxy ether chain) that can undergo
intramolecular esterification with one of the phosphonic acid moieties to form a cyclic
ANP (cHPMPC and cHPMPA) with one free phosphonic acid moiety (cyclic prodrug).
The cyclic versions of the HPMP ANPs are prodrugs themselves, however, our group has
esterified the remaining free P-OH group with a natural and/or modified amino acid or
dipeptide (acyclic prodrug). Included in the studies described below are acyclic versions
of both HPMP prototypes where a single amino acid is esterified to the phosphonic acid
group leaving a free P-OH moiety.
Putting aside the type of promoiety, one of the main requirements for a prodrug to be
considered effective is proper and sufficient activation of the inactive prodrug such that
the promoiety is cleaved to allow for the bioactive parent drug to exert its therapeutic
activity. In terms of releasing HPMP parent drugs from amino acid- and dipeptide-based
promoieties, research findings indicate the enzymatic and pH-dependent hydrolysis of
simple aryl and alkyl esters of cyclic ANPs.
93
Of importance in developing this approach
has been investigating the process with which the promoieties are released and the
resulting metabolites. The activation pathway for cyclic ester prodrugs of HPMPC and
HPMPA releases cyclic drug (cHPMPC or cHPMPA) or the intramolecular P-O linkage
17
is cleaved and the acyclic monoester prodrug is released. Advantageously, both
metabolites would release the active parent HPMP drug upon further enzymatic
degradation.
87
1.8.1 Development of amino acid based cyclic and acyclic HPMP prodrugs
The reason behind implementing amino acids and dipeptides as promoieties stems from
their biologically native and non-toxic properties, formation of a stable,
pharmacologically efficacious P-X-C ester linkage via the side chain of the amino acid to
the parent drug and the ability to modify various functional groups (C(O)(OH), NH
2
) on
the amino acid for proper ADME (Absorption, Distribution, Metabolism, Excretion)
profiling.
87
Initially, targeting the human oligopeptide transporter 1 (hPEPT1) was also a
large factor in choosing amino acids and dipeptides based on the wide array of hPEPT1
substrates.
87
As a result, initial studies of HPMP prodrugs began with ethylene-glycol
(EG)-linked amino acid prodrugs of (S)-HPMPC designed to target hPEPT1.
Investigations were carried out in order to ascertain the structure-activity relationship
between the prodrugs and hPEPT1, as well as, determine the promoiety that elicits the
balance of metabolic stability and efficient target-specific release of the parent drug in
vivo.
94
After discovering the single amino acid derivatives were not hPEPT1 substrates,
studies were aimed at increasing physiochemical properties to better target the active
transporter. This was accomplished by conjugating a hydrophobic amino acid (Ala, Val,
Phe and Leu) to a serine molecule esterified to the P(O)(OH)
2
group of (S)-HPMPC
through its secondary OH side chain.
94-97
Although the dipeptide analogues of (S)-
18
HPMPC demonstrated increased antiviral activities and oral bioavailability in comparison
with the parent drug,
94,98,99
findings from a competitive binding assay for hPEPT1
demonstrated prodrug affinity for the transporter, but not increased cellular permeability
due to translocation.
96
In addition, inefficient release of the parent drug, as well as, the
multiple degradation pathways and metabolites decreased the efficacy of this generation
of prodrugs.
100
Serine-based derivatives of (S)-HPMPA were also evaluated for hPEPT1 transport and
from these studies it was determined that despite varying the nucleobase (adenine and
cytosine), the (L)/(D) configuration of serine and the alkyl ester groups on the C-terminus
(methyl and isopropyl), single amino acid prodrugs of cyclic (S)-HPMPC and (S)-
HPMPA were not transported via hPEPT1 but were transported into cells using passive
diffusion (endocytosis).
96
These findings prompted three rounds of SAR studies that
involved exploring the antiviral activity effects of varying promoiety linkages to the
phosphonic acid, changing the amino acid stereochemistry, modifying the C-terminal
functional group and investigating the effect of alkyl chain length.
87,101
The first round consisted of experimenting on the gastric stability and efficient parent
drug release at physiological pH of four single amino acid promoieties with side chains
that contained an oxygen or sulfur atom that could be esterified to the phosphonic acid
group of (S)-HPMPA. As shown in Figure 1.8, the investigation of single amino acid
19
prodrugs consisted of utilizing (L)-serine, (L)-tyrosine, (L)-threonine and (L)-cysteine
methyl esters as promoieties. Based on the parameters assessed in this SAR (chemical
Figure 1.8 Peptidomimetic prodrugs of (S)-HPMPC and (S)-HPMPA
N
O
O
P
O
O
(S)
N
NH
2
O
O R
R = Ala, Val
EG-linked amino acid prodrugs
N
O
O
P
O
O
N
NH
2
O
R
1
= Ala, Val, Phe or Leu
R
2
= Me or iPr
Dipeptide prodrugs
NHR
1
O
R
2
O
N
O
O
P
O
O
OR
3
O H
2
N
R
3
= Me or iPr
Serine-based prodrugs
N
N
N
NH
2
(S)
N
O
O
P
O
X
N
N
N
NH
2
Amino acid prodrugs
(S)
B
O
O
P
O
O
R
5
O H
2
N
R
5
= OMe, OiPr, or NH-i-Bu
Tyrosine-based prodrugs
B = Cytosine ((S)-HPMPC)
B = Adenine ((S)-HPMPA)
B
O
O
P
O
X
H
2
N
O
NHR
6
(S)
B
O
HO
P
O
X
H
2
N
O
NHR
6
HO
(S)
B = Cytosine ((S)-HPMPC)
B = Adenine ((S)-HPMPA)
Tyrosine N-alkyl amide prodrugs
cyclic prodrugs acyclic prodrugs
X = OC
6
H
4
OCH(CH
3
)
,
SHCH
2
(S) (S)
X = OC
6
H
4
R
6
= C
4
H
8,
C
8
H
17
,
C
12
H
25
,C
14
H
29
,
C
16
H
33
, or C
18
H
37
,
H
2
N
O
OMe
20
and enzymatic stability, activation metabolites, in vitro antiviral activities and
cytotoxicity) results of this study indicated tyrosine promoieties proved to be the best
candidate for further evaluation.
87,101
In summary, it demonstrated higher chemical
stability, facile prodrug activation (only cyclic parent drug and acyclic prodrug present)
and enhanced antiviral activity against HCMV, cowpox and vaccinia viruses.
In the second round, the functionality on the C-terminal group of the amino acid and the
(L)/(D) tyrosine configuration was modified with various functional groups to attempt to
enhance the enzymatic stability. Initially the methyl groups were replaced with isopropyl
groups but this was followed by substitution of the carboxylic moiety altogether with a
stable N-alkyl amido group (Figure 1.8) in order to further modify the lipophilicity of the
prodrug. Upon studying the chemical and enzymatic stabilities of the tyrosine-based
prodrugs in various media, the lead N-alkyl tyrosinamide prodrugs were found to
demonstrate enhanced enzymatic stability, efficient activation (cyclic parent drug and
acyclic tyrosine-based prodrug were the only metabolites) and enhanced oral
bioavailability.
87,101
These findings prompted the next round of studies to focus on the
correlation between hydrophobicity and antiviral activity.
In the third round, the chain length of the N-alkyl amide portion of the prodrug was
varied in order to study the effect of chain length on antiviral activity, cellular
permeability and oral bioavailability. A series of long chain N-alkyl tyrosinamide
21
prodrugs were synthesized and assessed for all properties named above. These findings
are reported in Dr. Ivan S. Krylov’s dissertation
93
and demonstrate certain lipophilic
tyrosinamide prodrugs were found to exhibit enhanced potencies against HCMV,
vaccinia and cowpox viruses.
87,101
Based on the progress and development of the amino acid based prodrug approach that is
abbreviated above, the work presented in this dissertation was performed in order to
make a contribution to this project by applying this approach to other ANP classes (2,4-
diaminopyrimidines (DAPys) and PME/PMP compounds), to new dipeptide promoieties
and to developing novel parent drugs for further investigation with this current prodrug
technology.
22
1.9 Chapter 1 References
1. Prusoff, W. H. Synthesis and biological activities of iododeoxyuridine, an analog
of thymidine. Biochem Biophys Acta. 1959, 32, 295-296.
2. Hermann, E. C. Plaque inhibition test for detection of specific inhibitors of DNA
containing viruses. Proc Soc Exp Biol Med. 1961, 107, 142-145.
3. Kaufman, H. E. Clinical cure of herpes simplex keratitis by 5’-iodo-2’-
deoxyuridine. Proc Soc Exp Biol Med. 1962, 109, 251-253.
4. Kaufman, H. E.; Heidelberger, C. Therapeutic antiviral action of 5-
trifluoromethyl-2’-deoxyuridine in herpes simplex keratitis. Science. 1964, 145,
585-586.
5. Whitley, R. J.; Ch’ien, L. T.; Dolin, R., Galasso, G. J.; Alford, C. A. Jr. Adenine
arabinoside therapy of herpes zoster in the immuno-suppressed. NIAID
collaborative antiviral study. N Engl J Med. 1976, 294, 1193–9.
6. De Clercq, E. Milestones in the discovery of antiviral agents: nucleosides and
nucleotides. Acta Pharmaceut Sinica B. 2012, 2, 535-548.
7. Schabel, F. M. Jr. The antiviral activity of 9-b-D-arabinofuranosy- ladenine (Ara-
A). Chemotherapy. 1968, 13, 321–38.
8. Elion, G. B.; Furman, P. A.; Fyfe, J. A.; de Miranda, P.; Beauchamp, L.;
Schaeffer, H. J. Selectivity of action of an antiherpetic agent, 9-(2-
hydroxyethoxymethyl)guanine. Proc Natl Acad Sci USA. 1977, 74, 5716–20.
9. Schaeffer, H. J.; Beauchamp, L.; de Miranda, P.; Elion, G. B.; Bauer, D. J.;
Collins, P. 9-(2-Hydroxyethoxymethyl) guanine activity against viruses of the
herpes group. Nature 1978, 272, 583–5.
10. Dolin, R. Antiviral chemotherapy and chemoprophylaxis. Science. 1985, 227,
1296-1303.
11. De Clercq, E. 1985. Antiviral Agents. In: Greenwood, D.; O’Grady, F.; editors.
Symposium of the Society for General Microbiology. Scientific Basis of
Antimicrobial Chemotherapy. Cambridge: Cambridge University Press. p.155-
184.
12. De Clercq, E.; Descamps, J.; De Somer, P.; Holý, A. (S)-9-(2,3-
dihydroxypropyl)adenine: Aliphatic nucleoside analog with broad-spectrum anti-
viral activity. Science. 1978, 200, 563-565.
23
13. De Clercq, E.; Field, H. J. Antiviral prodrugs: the development of successful
prodrug strategies for antiviral chemotherapy. Brit J Pharmacol. 2006, 147, 1–11.
14. Brantley, J. S.; Hicks, L.; Sra, K.; Tyring, S. T. Valacyclovir for the treatment of
genital herpes. Expert Rev Anti Infect Ther. 2006, 4, 367–76.
15. De Clercq, E. The Discovery of Antiviral Agents: Ten Different Compounds, Ten
Different Stories. Med Res Rev. 2008, 28, 929-953.
16. De Clercq, E. Biochemical aspects of selective antiherpes activity of nucleoside
analogues. Biochem Pharmacol. 1984, 33, 2159-2169.
17. Larder, B. A.; Darby, G. Virus drug resistance: Mechanisms and consequences.
Antiviral Res. 1984, 4, 1-42.
18. Shipkowitz, N. L.; Bower, R. R.; Appell, R. N.; Nordeen, C. W.; Overby, L. R.;
Roderick, W. R.; Schleich, J. B.; Vonesch, A. M. Suppression of herpes simplex
virus infection by phosphonoacetic acid. Appl. Microbiol. 1973, 26, 264-267.
19. Field, A. K.; Biron, B. B. The end of innocence revisited: resistance of
herpesvirus to antiviral drugs. Clin Microbiol Rev. 1994, 7, 1-13.
20. Li, F.; Maag, H.; Alfredson, T., Prodrugs of nucleoside analogues for improved
oral absorption and tissue targeting. J Pharm Sci. 2008, 97, 1109-34.
21. Ariza, M. E. Current prodrug strategies for the delivery of nucleotides into cells.
Drug Des. Rev. 2005, 2, 373-387.
22. Wagner, C. R.; Iyer, V. V.; McIntee, E. J. Pronucleotides: Toward the in vivo
delivery of antiviral and anticancer nucleotides. Med Res Rev. 2000, 20, 417-451.
23. Holý, A. Antiviral acyclic nucleoside phosphonates structure activity studies.
Antiviral Res. 2006, 71, 248-253.
24. De Clercq, E.; Holý, A. Acyclic nucleoside phosphonates: A key class of antiviral
drugs. Nat. Rev. Drug Discov. 2005, 4, 928-940.
25. Sundquist, B.; Oberg, B., Phosphonoformate inhibits reverse transcriptase. J Gen
Virol. 1979, 45, 273-281.
26. Lambert, R. W.; Martin, J. A.; Thomas, G. J.; Duncan, I. B.; Hall, M. J.; Heimer,
E. P. Synthesis and antiviral activity of phosphonoacetic and phosphonoformic
acid esters of 5-bromo-2'-deoxyuridine and related pyrimidine nucleosides and
acyclonucleosides. J Med Chem. 1989, 32, 367-374.
24
27. Griengl, H.; Hayden, W.; Penn, G.; De Clercq, E.; Rosenwirth, B.
Phosphonoformate and phosphonoacetate derivatives of 5-substituted 2-
deoxyuridines: Synthesis and antiviral activity. J Med Chem. 1988, 31, 1831-
1839.
28. Vaghefi, M. M.; McKernan, P. A.; Robins, R. K., Synthesis and antiviral activity
of certain nucleoside 5'-phosphonoformate derivatives. J Med Chem. 1986, 29,
1389-1393.
29. De Clercq, E. Antivirals and antiviral strategies. Nature Rev Microbiol. 2004, 2,
704.
30. Srinivas, R.V.; Robbins, B.L.; Connelly, M.C.; Gong, Y.F.; Bischofberger, N.;
Fridland, A. Metabolism and in vitro antiretroviral activities of
bis(pivaloyloxymethyl) prodrugs of acyclic nucleoside phosphonates. Antimicrob
Agents Chemother. 1993, 37, 2247.
31. De Clercq, E.; Sakuma, T.; Baba, M.; Pauwels, R.; Balzarini, J.; Rosenberg, I.;
Holý, A. Antiviral Activity of Phosphonylmethoxyalkyl Derivatives of Purine and
Pyrimidines. Antiviral Res. 1987, 8, 261-272.
32. De Clercq, E. Acyclic nucleoside phosphonates: past, present, and future.
Bridging chemistry to HIV, HBV, HCV, HPV, adeno-, herpes-, and poxvirus
infections: The phosphonate bridge. Biochem Pharmacol. 2007, 73, 911-922.
33. De Clercq, E. Therapeutic of HPMPC as an antiviral drug. Rev Med Virol. 1993,
3, 85-96.
34. Holý, A.; Votruba, I.; Tloustova, E.; Masojídková, M. Synthesis and Cytostatic
Activity of N-[2-(Phosphonomethoxy)alkyl] Derivatives of N
6
-Substituted
Adenines, 2,6-Diaminopurines and Related Compounds. Collect Czech Chem
Comm. 2001, 66, 1545-1592.
35. Zídek, Z.; Potmesil, P.; Kmoníèková, E.; Holý, A. Immunobiological activity of
N-[2-(phosphonomethoxy)alkyl] derivatives of N6-substituted adenines, and 2,6-
diaminopurines. Eur J Pharmacol. 2003, 475, 149-159.
36. De Clercq, E., Holý, A., Rosenberg, I., Sakuma, T., Balzarini, J., Maudgal, P.C. A
Novel Selective Broad Spectrum Anti-DNA Virus Agent. Nature. 1986, 323, 464-
467.
37. De Clercq, E. Clinical potential of the acyclic nucleoside phosphonates cidofovir,
adefovir, and tenofovir in treatment of DNA virus and retrovirus infections. Clin
Microbiol Rev. 2003, 16, (4), 569-596.
25
38. De Clercq, E.; Neyts, J. Antiviral agents acting as DNA and RNA chain
terminators. Handb. Exp. Pharmacol. 2009, 189, 53-84.
39. Votruba, I.; Intracellular phosphorylation of broad-spectrum anti-DNA virus
agent (S)-9-(3-hydroxy-2-phosphorylmethoxypropyl) adenine and inhibition of
viral DNA synthesis. Mol Pharmacol. 1987, 32, 524-529.
40. Krejčová, R.; Horska, K.; Votruba, I.; Holý, A. Phosphorylation of Purine
(Phosphonomethoxy)alkyl Derivatives by Mitochondrial AMP Kinase (AK2
Type) from L1210 Cells. Collect Czech Chem Commun. 2000, 65, 1653-1668.
41. Krejčová, R.; Horska, K.; Votruba, I.; Holý, A. Interaction of Guanine
Phosphonomethoxyalkyl Derivatives with GMP Kinase Isoenzymes. Biochem
Pharmacol. 2000, 15, 1907-1913.
42. Kramata, P.; Votruba, I.; Otova, B.; Holý, A. Different Inhibitory Potencies of
Acyclic Phosphonomethoxyalkyl Nucleotide Analogs Toward DNA Polymerases
alpha, delta and epsilon. Mol Pharmacol. 1996, 49, 1005-1011.
43. Naesens, L.; Snoeck, R.; Graciela, A.; Balzarini, J.; Neyts, J.; De Clercq, E.
HPMPC (cidofovir), PMEA (adefovir) and related acyclic nucleoside
phosphonate analogues: a review of their pharmacology and clinical potential in
the treatment of viral infections. Antiviral Chem Chemother. 1997, 8, 1-23.
44. Chou, S. Cytomegalovirus UL-97 mutations in the era of ganciclovir and
maribavir. Rev Med Virol. 2008, 18, 233-246.
45. Neyts, J.; Snoeck, R.; Balzarini, J.; De Clercq, E. Particular characteristics of the
anti-human cytomegalovirus activity of (S)-1-(3-hydroxy-2-
phosphonylmethoxypropyl)-cytosine (HPMPC) in vitro. Antiviral Res. 1991, 16,
41–52.
46. Ho, H. T.; Woods, K. L.; Bronson, J. J; De Boeck, H.; Martin J. C.; Hitchcock, M.
J. Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-
(phosphonylmethoxy)propyl]cytosine. Mol Pharmacol. 1992, 41, 197–202 .
47. Holý, A. Phosphonomethoxyalkyl analogs of nucleotides. Curr. Pharm. Des.
2003, 9, 2567-2592.
48. Cundy, K. C. Clinical pharmacokinetics of the antiviral nucleotide analogs
cidofovir and adefovir. Clin Pharmacokinet. 1999. 36, 127-143.
26
49. Jordheim, L. P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the
development of nucleoside and nucleotide analogues for cancer and viral diseases.
Nat rev drug discov. 2013, 12, 447-64.
50. Huttunen, K. M.; Rautio, J. Prodrugs – An Efficient Way to Breach Delivery and
Targeting Barriers. Curr Top Med Chem. 2011, 11, 2265-2287.
51. Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Jarvinen, T.;
Savolainen, J. Prodrugs: Design and Clinical Applications. Nat Rev Drug Discov.
2008, 7, 255-270.
52. Ettmayer, P.; Amidon, G. L.; Clement, B.; Testa, B. Lessons learned from
marketed and investigational prodrugs. J Med Chem. 2004, 47, 2393-2404.
53. Denny, B. J.; Wheelhouse, R. T.; Stevens, M. F. G.; Tsang, L. L. H.; Slack, K. A.
NMR and Molecular modeling investigations of the mechanism of activation of
the anti-tumor drug Temozolomide and its interaction with DNA. Biochemistry.
1994, 33, 9045-51.
54. Krause, M.; Stark, H.; Schunack, W. Azomethine prodrugs of (R)-I±-
methylhistamine, a highly potent and selective histamine H3-receptor agonist.
Curr. Med. Chem. 2001, 8, 1329-1340.
55. Meier, C. Cyclosal phosphates as chemical Trojan horses for intracellular
nucleotide and glycosyl-monophosphate delivery – chemistry meets biology. Eur
J Org Chem. 2006, 1081-1102.
56. Liederer, B.M.; Borchardt, R.T. Enzymes involved in the bioconversion of ester-
based drugs. J. Pharm. Sci. 2006, 95, 1177-1195.
57. Hillaireau, H.; Le Doan, T.; Appel, M.; Couvreur, P. Hybrid polymer
nanocapsules enhance in vitro delivery of azidothymidine-triphosphate to
macrophages. J. Controlled Release 2006, 116, 346-352.
58. Vinogradov, S. V.; Zeman, A. D.; Batrakova, E. V.; Kabanov, A. V., Polyplex
Nanogel formulations for drug delivery of cytotoxic nucleoside analogs. J.
Controlled Release 2005, 107, 143-157.
59. Duzgunes, N.; Simoes, S.; Slepushkin, V.; Pretzer, E.; Flasher, D.; Salem, I. I.;
Steffan, G.; Konopka, K.; Pedroso de Lima, M. C., Delivery of antiviral agents in
liposomes. Methods Enzymol. 2005, 391, 351-373.
27
60. Rossi, L.; Serafini, S.; Pierige, F.; Antonelli, A.; Cerasi, A.; Fraternale, A.;
Chiarantini, L.; Magnani, M., Erythrocyte-based drug delivery. Expert Opin.
Drug Delivery 2005, 2, (2), 311-322.
61. Aguzzi, C.; Cerezo, P.; Viseras, C.; Caramella, C., Use of clays as drug delivery
systems: Possibilities and limitations. Appl Clay Sci. 2007, 36, 22-36.
62. Anastasi, C.; Quelever, G.; Burlet, S.; Garino, C.; Souard, F.; Kraus, J. -L., New
antiviral nucleoside prodrugs await application. Curr Med Chem. 2003, 10, 1825-
1843.
63. Van der Waterbeemdt, H. Drug Bioavailability: Estimation of solubility,
permeability, absorption and bioavailability. In Methods Princ. Med. Chem.
[online] Van de Waterbeemd, H.; Lennernas, H.; Artursson, P., Eds.; Wiley-VCH:
Weinheim, Germany, 2003. Vol 18.
http://onlinelibrary.wiley.com/book/10.1002/3527601473.
64. Peterson, L. W.; McKenna, C. E. Prodrug approaches to improving the oral
absorption of antiviral nucleotide analogues. Expert Opin Drug Delivery. 2009, 6,
405-420.
65. Freeman, S.; Ross, K. C. Prodrug design for phosphates and phosphonates. Prog
Med Chem. 1997, 34, 111-147.
66. Pertusati, F.; Serpi, M.; McGuigan, C. Medicinal chemistry of nucleoside
phosphonate prodrugs for antiviral therapy. Antiviral Chem Chemother. 2012, 22,
181-203.
67. Starrett Jr., J. E.; Tortolani, D. R.; Russell, J.; Hitchcock, M. J. M.; Whiterock, V.;
Martin, J. C.; Mansuri, M. M. Synthesis, oral bioavailability determination and in
vitro evaluation of prodrugs of the antiviral agent 9-[2-
(phosphonomethoxy)ethyl]adenine (PMEA). J Med Chem. 1994, 37, 1857-1864.
68. Serafinowska, H. T.; Ashton, R. J.; Bailey, S.; Handen, M. R.; Jackson, S. M.;
Sutton, D. Synthesis and in vivo evaluation of prodrugs of 9-[2-
(phosphonomethoxy)ethyl]adenine. J Med Chem. 1995, 38, 1372-1379.
69. Hecker, S. J.; Erion, M. D. Prodrugs of Phosphates and Phosphonates. J Med
Chem. 2008, 51, 2328-2345.
70. Kelly, S. J.; Dardinger, D. E.; Butler, L. G. Hydrolysis of phosphonate esters
catalyzed by 5’-nucleotide phosphodiesterase. Biochemistry. 1975, 14, 4983-
4988.
28
71. Kelly, S. J; Butler, L. G. Enzymatic hydrolysis of phosphonate esters. Reaction
mechanism of intestinal 5’-nucleotide phosphodiesterase. Biochemistry, 1977, 16,
1102-1104.
72. Farquar, D.; Kuttesch, N. J.; Wilkerson, M. G.; Winkler, T. Synthesis and
biological evaluation of neutral derivatives of 5-fluoro-2’-deoxyuridine 5’-
phosphate. J Med Chem. 1983, 26, 1153-1158.
73. Srivastva, D. N.; Farquar, D. Bioreversible phosphate protective groups.
Synthesis and stability of model acyloxymethyl phosphates. Bioorg Chem. 1984,
12, 118-129.
74. Iyer, R. P.; Phillips, L. R.; Biddle, J. A.; Thakker, D. R.; Egan, W.; Aoki, S.;
Mitsuya, H. Synthesis and acyloxyalkyl acylphosphonates as potential prodrugs
of the antiviral, trisodium phosponoformate (foscarnet sodium). Tetrahedron Lett.
1989, 30, 7141-7144.
75. McGuigan, C.; Nickson, C.; Petrik, J.; Karpas, A. Phosphate derivatives of AZT
display enhanced selectivity of action against HIV-1 by comparison to the parent
nucleoside. FEBS Lett. 1992, 310-171-174.
76. Warner, D. T.; Neil, G. L.; Taylor, A. J.; Wechler, W. J. Nucleic acids. 13. 3’-0-
and 2’-0-esters of 1.beta.-D-arabinofuranosylcytosine as antileukemic and
immunosuppressive agents. J Med Chem. 1972, 15, 790-792.
77. Wechter, W. J.; Johnson, M. A.; Hall, C. M.; Warner, D. T.; Berger, A. E.;
Wenzel, A. H.; Gish, D. T.; Neil, G. L. Nucleic acids. 14. Ara-Cytidine acylates.
Use of drug design predictors in structure-activity relation correlation. J Med
Chem. 1975, 18, 339-344.
78. Raetz, C. R. H; Chu, M. Y.; Srivastava, S. P.; Turcotte, J. G. A phospholipid
derivative of cytosine arabinoside and its conversion to phosphatidylinositol by
animal tissue. Science. 1977, 196, 303-305.
79. Hostetler, K. Y. Synthesis and antiviral evaluation of broad spectrum, orally
active analogs of cidofovir and other acyclic nucleoside phosphonates. Adv.
Antivir. Drug Des. 2007, 5, 167-184.
80. Cahard, D.; McGuigan, C.; Balzarini, J. Aryloxy phosphoramidate triesters as
protides. Mini-Rev Med Chem. 2004, 4, 371-381.
81. Mehellou, Y.; Balzarini, J.; McGuigan, C. Aryloxy phosphoramidate triesters: a
technology for delivering monophosphorylated nucleosides and sugars into cells.
Chem Med Chem, 2009, 4, 1779-1791.
29
82. Meier, C. cycloSal-pronucleotides, - design of chemical Trojan horses. Mini-Rev
Med Chem. 2002, 2, 219-234.
83. Meier, C.; Ducho, C.; Jessen, H.; Vukadinovic-Tenter, D.; Balzarini, J. Second-
generation cycloSal-d4TMP pro-nucleotides bearing esterase-cleavable sites – the
“trapping” concept. Eur J Org Chem. 2005, 197-206.
84. Girsch, N.; Pertenbreiter, F.; Balzarini, J.; Meier, C. 5-(1-acetoxyvinyl)-
cycloSaligenyl-2’3’-dideoxy-2’,3’-didehydrothymidine monophosphates, a
second type of new, enzymatically activated cycloSaligenyl pronucleotides. J
Med Chem. 2008, 51, 8115-8123.
85. Peyrottes, S.; Egron, D.; Lefebvre, I.; Gosselin, G.; Imach, J. L.; Periguad, C.
SATE pronucleotide approaches: an overview. Mini-Rev Med Chem. 2004, 4,
395-408.
86. Jochum, A.; Schlienger, N.; Egron, D.; Peyrottes, S.; Periguad, C. Biolabile
constructs for pronucleotide design. J Organomet Chem. 2005, 690, 2614-2625.
87. Krylov, I. S.; Kashemirov, B. A.; Hilfinger, J. M.; McKenna, C. E. Evolution of
amino acid based prodrug approach: stay tuned. Mol Pharmaceutics. 2013, 10,
445-458.
88. Sofia, M. J; Bao, D.; Chang, W.; Du, J; Nagarathnam, D.; Rachkonda, S.; Reddy,
P. G.; Ross, B. S.; Wang, P.; Zhang, H-R.; Bansal, S.; Espiritu, C.; Keilman, M.;
Lam, A. M.; Steuer, H. M. M.; Niu, C.; Otto, M. J.; Furman, P. A. Discovery of a
I-D-2’deoxy-2-I±fluoro-2-I2-C-methyluridine nucleotide prodrug (PSI-7977) for
the treatment of hepatitis C virus. J Med Chem. 2010, 53, 7202-7218.
89. Lam, A. M.; Murakami, E.; Espiritu, C.; Steuer, H.M.; Niu, C.; Keilman, M.; Bao,
H.; Zennou, V.; Bourne, N.; Julander, J. G.; Morrey, J. D.; Smee, D. F; Frick, D.
N.; Heck, J. A.; Wang, P.; Nagarathnam, D.; Ross, B. S.; Sofia, M. J.; Otto, M. J.;
Furman, P. A. PSI-7851, a pronucleotide of I2-D-2’-deoxy-2-fluoro-2’-
methyluridine monophosphate, is a potent and pan-genotype inhibitor of hepatitis
C virus replication. Antimicrob. Agents Chemother. 2010, 54, 3187-3196.
90. Murakami, E.; Tolstykh, T.; Bao, H.; Niu, C.; Steuer, H.M.; Bao, D.; Chang, W.;
Espiritu, C.; Bansal, S.; Lam, A. M.; Otto, M. J.; Sofia, M. J.; Furman, P. A.
Mechanism of activation of PSI-7851 and its diastereomer PSI-7977. J Biol
Chem. 2010, 285, 34337-34347.
91. Zeuzem, S.; Dusheiko, G. M.; Salupere, R.; Mangia, A.; Flisiak, R.; Hyland, R.
H.; Illeperuma, A.; Svarovskaia, E.; Brainard, D. M.; Symonds, W. T.;
30
Subramanian, G. M.; McHutchison, J. G.; Weiland, O.; Reesink, H. W.; Ferenci,
P.; Hezode, C.; Esteban, R.; VALENCE Investigators. Sofosbuvir and Ribavirin
in HCV Genotypes 2 and 3. N Engl J Med. 2014, 370, 1993-2001.
92. Sulkowski, M.S.; Gardiner, D. F.; Rodriquez-Torres, M.; Reddy, R.; Hassanein,
T.; Jacobson, I.; Lawitz, E.; Lok, A. S.; Hinestrosa, F.; Thuluvath, P. J.; Schwartz,
H.; Nelson, D. R.; Everson, G. T.; Eley, T.; Wind-Rotolo, M.; Hindes, R.;
Symonds, W.; Pasquinelli, C.; Grasela, D. M. Daclatasvir plus Sofosbuvir for
Previously Treated or Untreated Chronic HCV Infection. N Engl J Med. 2014,
370, 211-221.
93. Oliyai, R.; Shaw, J.-P.; Sueoka-Lennen, C. M.; Cundy, K. C.; Arimilli, M. N.;
Jones, R. J.; Lee, W. A. Aryl ester prodrugs of cyclic HPMPC. I: physicochemical
characterization and in vitro biological stability. Pharm Res. 1999, 16, 1687-1693.
94. 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 Pharm. 2008, 5, 598-609.
95. 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.
96. 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 Pharm. 2010,
7, 2349-2361.
97. 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.
98. 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.
99. Peterson, L. W.; Kim, J. S.; Kijek, P.; Mitchell, S.; Hilfinger, J. M.; Breitenbach,
J.; Borysko, K. Z.; Drach, J. C.; Kashemirov, B. A.; McKenna, C. E. Synthesis,
31
transport and antiviral activity of Ala-Ser and Val-Ser prodrugs of cidofovir.
Bioorg Med Chem Lett. 2011, 21, 4045-4049.
100. 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.
101. Krylov, I. S. Synthesis, structural analysis and in vitro antiviral activities
of novel cyclic and acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs.
Ph.D. Thesis, University of Southern California, Los Angeles, 2012.
32
CHAPTER 2
Synthesis of Tyrosine-Based Monoester 9-(2-
Phosphonomethoxy)alkyl Purine Analogues as Antiviral
Agents
2.1 PME/PMP Background
Among the subclasses of ANPs, 9-[2-(phosphonomethoxy)alkyl] purines, pyrimidines
and their analogues have attracted attention for their antiviral, cytostatic and
immunological activity.
1-8
9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir, 2.1),
(Fig. 2.1) is equipped with biological activity against DNA viruses (HSV-1, HSV-2,
VZV, EBV, and HCMV), hepadnaviruses (hepatitis B virus) and retroviruses (HIV-1,
HIV-2),
8-10
while 9-(R)-[(2-phosphonylmethoxy)propyl]adenine ((R)-PMPA, tenofovir,
2.3) (Fig. 2.1) demonstrates antiviral activity strictly against retroviruses (HIV-1, HIV-
2).
11
Their 2,6-diamino counterparts (2.2, 2.5 and 2.6 in Fig. 2.1) have also been
previously synthesized and evaluated against various viruses.
12-18
PMEDAP (2.2)
demonstrates potent in vivo activity against mammalian retroviruses,
12-15
while (R)-
PMPDAP (2.5) has been shown to exhibit up to 40-fold higher anti-HIV activity than it’s
adenine congener (2.3).
16-18
33
Figure 2.1 PME/PMP acyclic nucleoside phosphonates
Thus far, only two bis ester prodrugs are currently FDA approved. The
bis(pivaloyloxymethyl) ester of PMEA (adefovir dipivoxil, Hepsera
®
, Figure 2.2) and the
bis(isopropoxycarbonyloxymethyl) ester prodrug of (R)-PMPA (tenofovir disoproxil
fumarate, Viread
®
, Figure 2.2) are clinically approved for the treatment of hepatitis B
virus (HBV) and human immunodeficiency virus (HIV).
6
Figure 2.2 Oral prodrugs of PMEA and (R)-PMPA
N
N
N
N
O P
O
OH
OH
NH
2
R
R = H; R
1
= CH
3
; (R)-PMPA, 2.3
R = H; R
1
= CH
3
; (S)-PMPA, 2.4
R = NH
2
; R
1
= CH
3
; (R)-PMPDAP, 2.5
R = NH
2
; R
1
= CH
3
; (S)-PMPDAP, 2.6
N
N
N
N
O P
O
OH
OH
NH
2
R
R = H; PMEA, 2.1
R = NH
2
; PMEDAP, 2.2
R
1
N
N
N
N
O P
O
O
NH
2
N
N
N
N
O P
O
O
NH
2
O O
O
O
O
Adefovir Dipivoxil
Hepsera
®
Tenofovir disoproxil fumarate
Viread
®
O O
O
O
O O
O
CO
2
H
HO
2
C
34
2.2 Mechanism of action
The mechanism of action adefovir (2.1) begins with the drug successfully penetrating the
cell. Similar to the process of other ANPs and nucleoside antivirals, 2.1 must undergo
two subsequent phosphorylations via intracellular conversion by 5’adenosine
monophosphate activated protein kinase (AMP) or phosphoribosyl pyrophosphate
synthetase (PRPP) to achieve the diphosphate form where it can act as a competitive
inhibitor of hepatitis B (HBV) reverse transcriptase.
19,20
Adefovir acts as chain terminator
once incorporated into the viral DNA strand, halting viral DNA replication by preventing
DNA elongation.
20
2.3 Limitations and the clinical use of their prodrugs
A major limitation exhibited by ANPs, including PME and PMP prototypes, is the lack of
oral bioavailability and cell permeability at physiological pH due to the dual free
phosphonic acid motifs.
21
Thus, PMEA needs to be injected, which hinders its long term
clinical application.
22
Intravenous injection of [
3
H]PMEA demonstrates accumulation of
PMEA in the kidneys, which gives the parent drug a nephrotoxic drawback as well.
23
In
an attempt to overcome the low oral bioavailability of PMEA, attention has been given to
the prodrug approach. Several different cyclic and acyclic PME/PMP prodrugs have been
developed where the polar phosphonic group is masked by a labile moiety
24-31
in order to
facilitate penetration into the cell. Upon removal of the masking group, an active acyclic
nucleoside phosphonate is liberated to be transformed into a nucleoside triphosphate-like
substrate for viral polymerase. The bis(pivaloyloxymethyl) ester prodrug possesses
35
lipophilicity and greater intestinal permeability than the parent drug in vitro.
32,33
Greater
oral bioavailability is observed with Bis-POM PMEA allowing the prodrug to be orally
administered at a dose of 10 mg/day. However, adefovir dipivoxil doses from 30 mg/day
to 120 mg/day give rise to nephrotoxicity due to accumulation of PMEA and
phosphorylated PMEA metabolites found in the kidney. Renal tubular nephropathy has
been reported for adefovir dipivoxil by the US FDA, indicating a need for further
research in PMEA prodrugs.
2.4 Prodrug approaches to PME/PMP parent drugs
While an array of monoester PME and PMP prodrugs have been developed and their
antiviral activity assessed over the years,
15,18,24-31
an FDA approved monoester PME/PMP
prodrug has yet to be found. It would be of great interest to assess the antiviral activity,
toxicity and distribution of monoester prodrugs vs. already known diester prodrugs to
observe any possible changes. Previously, our lab has described the synthesis and
biological evaluation of tyrosine-based prodrugs of (S)-HPMPC and (S)-HPMPA.
34
The
tyrosine-based single amino acid and dipeptide promoieties in this study were modified at
one or more of its functional sites in order to assess potential antiviral properties.
Intramolecular cyclization of the hydroxymethylene group produces a cyclic ANP where
one of the negative charges is masked followed by conjugation via the phenolic side
chain of the tyrosine promoiety with the cyclic form of the ANP to mask the remaining
negatively charged phosphonic acid group. These compounds demonstrated more potent
antiviral activity and enhanced oral bioavailability compared to the parent drugs.
36
2.5 Synthesis of PME/PMP parent drugs
The chlorophosphonate synthon (2.9) was synthesized according to the procedure
described by Rejman and co-workers.
35
Upon the dropwise addition of 2.8 to 2.7, the
reaction was refluxed for 4 h and then distilled under reduced pressure to purify desired
compound (2.9) from by-products and impurities (Scheme 2.1).
Scheme 2.1 Synthesis of diisopropyl chloroethoxymethane phosphonate synthon (2.9) a.
Reflux, 120 °C, N
2
, 4 h. Distill (110 °C – 115 °C) under reduced pressure.
Conjugation of 2.9 with adenine was performed according to the procedures described by
Holý and co-workers
36
with one modification Petrov et al.
37
reported in using potassium
carbonate (K
2
CO
3
) in condensing the desired phosphonomethoxyalkyl ether synthon at
the N1-position of purines (Scheme 2.2).
Scheme 2.2 Synthesis of PMEA a. K
2
CO
3
, DMF, 80°C, 2 h b. 2.9, 80 °C, overnight c.
SiMe
3
Br, CH
3
CN, rt, overnight.
Cl
O Cl +
P
O
O O
a
Cl
O P
O
OiPr
OiPr
+
Cl
2.7 2.8 2.9 2.10
N
N
N
H
N
NH
2
a,b
N
N
N
N
NH
2
O P
O
OiPr
OiPr
N
N
N
N
NH
2
O P
O
OH
OH
c
2.11 2.12 2.1
37
Removal of the isopropyl groups was accomplished using McKenna reaction
38
conditions
followed by workup and recrystallization. The lab of Prof. Marcela Krečmerová (Institute
of Organic Chemistry, IOCB) synthesized and provided PMEA (2.1) and (R)-PMPDAP
(2.5) according to previously published procedures.
39-40
2.6 Rationale behind prodrug design
A detailed report of the development of the tyrosinamide prodrug design can be found in
Chapter 1.8. In this particular study, the aim was to extend and elaborate the tyrosine-
based prodrug approach to the PME/PMP class of ANPs. Our lab briefly described an
alternative approach for the synthesis of acyclic prodrugs of PME/PMP ANPs, where
masking one of the phosphonic acid groups is required before esterification with the
phenolic side chain of tyrosine can be successfully achieved.
41
The approach has been
elaborated to a three-step synthesis in making novel monoester peptidomimetic prodrugs
of PMEA (2.1, Fig. 2.1) and the 2-amino congener of tenofovir, (R)-PMPDAP (2.5, (Fig
2.1). To demonstrate the effect of the promoiety on the antiviral activity, a series of
PME/PMP monoester prodrugs were synthesized and their antiviral activity assessed
against the DNA viruses listed in Table 2.1 and Table 2.2 (Chapter 2.12).
2.7 PyBOP: activating agent
Direct coupling of the desired promoiety to the parent drug requires the appropriate
coupling agent. The right coupling agent should adequately activate the phosphonic acid
moiety of the parent drug to allow introduction of the amino acid or dipeptide promoiety.
38
It is possible to activate the phosphonic acid group under acidic conditions using the
Mitsunobu reaction,
42
however, the solubility issues of the parent drug in acidic
conditions prevented using this approach. Therefore, PyBOP,
43
(1H-benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate, Figure 2.3) was selected as the
efficient condensing agent for the synthesis of various ANP prodrugs in the McKenna
lab.
34,44-50
Formation of the diisopropylethylammonium salt of the parent drug under
basic conditions allows for solubilizing the compound for the coupling reaction in the
presence of PyBOP.
Figure 2.3 Common (benzotriazolyloxy)-phosphonium reagents in peptide coupling
BOP (Figure 2.3), the predecessor of PyBOP, formation of the potential human
carcinogenic, hexamethylphosphorotriamide (HMPA; Scheme 2.3) as the side product,
limits the application of this coupling reagent.
51
Also included in the advantages of using
PyBOP is being able to remove the by-product from the reaction mixture by washing
with diethyl ether. Another added feature is PyBOP’s ability to create a racemization-free
environment,
43
which is beneficial in conjugating amino acid and dipeptide promoieties.
N
N
N
O
P N
N
N
PF
6
N
N
N
O
P N
N
N
PF
6
BOP PyBOP
39
Scheme 2.3 BOP coupling mechanism and formation of HMPA
2.8 Synthesis of PMEA monoester prodrug via PyBOP coupling
2.8.1 Synthesis of long alkyl chain tyrosinamide promoieties
The Boc-protected single amino acid promoiety, (L)-Boc-tyrosine, was purchased from
Sigma Aldrich and amidated on the unprotected C-terminal carboxylic group using a
procedure developed by Grimm and co workers (Scheme 2.4).
52
Scheme 2.4 Amidation of (L)-Boc-tyrosine; a. DCM, HOBt, EDC⋅HCl, (NH
2
C
4
H
9
for
2.13; NH
2
C
8
H
17
for 2.14; NH
2
C
16
H
33
for 2.15), rt, 72 h.
N
N
N
O
P N
N
N
PF
6
+
R C
O
O
BOP
R C
O
O P(NMe
2
)
3
OBt
R C
O
OBt
+
O P(NMe
2
)
3
HMPA
Activated ester
N
N
N
O
= OBt
a
HO O OH
N
H
Boc
HO O R
N
H
Boc
R = NH-i-Bu; 2.13
R = NHC
8
H
17
; 2.14
R = NHC
16
H
33
; 2.15
(L)-Boc-Tyr-OH
40
Activation of the C-terminal carboxylic acid group was performed using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) and HOBt to allow for formation of the
desired tyrosinamide promoiety (2.13, 2.14 and 2.15; Scheme 2.4).
2.8.2 Conjugation of amino acid promoiety with PyBOP
In order to obtain acyclic monoester prodrugs of PMEA and PMPDAP, the direct
mediated PyBOP coupling, analogous to the method used for (S)-HPMPA and (S)-
HPMPC prodrug synthesis,
34,44-50
was initially attempted (Scheme 2.5). The prodrugs are
synthesized by conjugating a serine, threonine or tyrosine moiety through their side chain
hydroxyl group to a cyclic form of (S)-HPMPA or (S)-HPMPC (Scheme 2.5). As
described in a previous publication, the PMEA-DIEA salt was formed by performing
three additions and removals of DMF and DIEA to the parent drug in order to assist in
dissolving the parent drug. This was followed up by addition of DMF, DIEA, amino acid
((L)-Ser-OMe (2.16) or (L)-Tyr-OMe (2.17) and finally by PyBOP (Scheme 2.6). Prior
to conjugation of the desired promoiety, one of the negatively charged groups on the
phosphonic acid moiety of the parent drug ((S)-HPMPA or (S)-HPMPC) is masked via
intramolecular cyclization of the hydroxymethylene group on the phosphonomethoxy
ether chain. Once masked, the phosphonic acid moiety of the cyclic parent ANP
(cHPMPC or cHPMPA) is activated with a second equivalent of PyBOP to allow for
nucleophilic attack of the hydroxyl side chain of an amino acid promoiety (Ser or Tyr)
and formation of the cyclic ANP amino acid ester prodrug depicted in Scheme 2.5.
41
Scheme 2.5 General procedure for the synthesis of peptidomimetic prodrugs of cyclic
(S)-HPMPA and (S)-HPMPC
The presence of a phosphorus atom in the parent drug, the coupling agent and the desired
product permits monitoring this reaction via
31
P NMR (Figure 2.4). Unreacted PyBOP
and the tripyrrolidino oxide by-product of reacted PyBOP (O=P(NC
4
H
8
)
3
) demonstrate a
chemical shift at 32 ppm and 14.5 ppm respectively. The chemical shift of the PMEA
starting material resides around 10-9 ppm, which is not observed in Figure 2.4. The
presence of the tripyrrolidino oxide chemical shift at 14.5 ppm and lack of a peak at 32
ppm indicates PyBOP is entirely consumed. The peak at 19.5 ppm was originally
considered to be the desired (L)-Ser-OMe monoester prodrug of PMEA (2.16 in Scheme
2.6).
B
OH
P
O
OH
(S)
OH
B
O
O
P
O
X
H
2
N
O
R
*
(S)
PyBOP, DMF
DIEA, 40 °C
X
H
2
N
O
R
+
promoiety
B = Cytosine ((S)-HPMPC)
B = Adenine ((S)-HPMPA)
X = OCH
2
X = OCH
2
C
6
H
4
42
Figure 2.4
31
P NMR (202.5 MHz) of reaction mixture (D
2
O capillary) of PMEA and (L)-
Ser-OMe (2.16)
Despite following a previously successful coupling protocol, coupling the serine and
tyrosine O-Me derivatives under the reaction conditions described above did not result in
the desired monoester serine (2.16, Scheme 2.6) and tyrosine (2.14 Scheme 2.6) products
nor the diester analogues. This is due to the structural differences between PMEA and
ANPs of the HPMP class ((S)-HPMPC and (S)-HPMPA). The hydroxymethylene group
on the phosphonomethylether chain of HPMP derivatives allows for one of the anionic
charges on the free phosphonate to be masked by intramolecular conjugation. Being that
PMEA lacks this functional group, the large negative charge on the phosphonate group
P
O
N
N
N
tripyrrolidino phosphonium oxide
PyBOP by-product
43
under basic conditions prevents the negatively charged nucleophile from attacking the
phosphonate and displacing the leaving group (OBt). Therefore, the phosphorus peak
downfield of the tripyrrolidino oxide by-product peak in Figure 2.4 that was originally
thought to be the desired monoester PMEA prodrug 2.16 was confirmed to be a different
PMEA monoester. Instead, the acyclic OBt-intermediate monoester of PMEA (2.18) and
PMEA dimer (2.19, Scheme 2.6) were found to be more stable under reaction conditions.
The OBt monoester of PMEA (2.18) and PMEA dimer (2.19) were verified by
31
P-NMR
Scheme 2.6 PyBOP condensation reaction with PMEA and promoiety yield PMEA-OBt
intermediate.
N
N
N
N
O P
O
OH
OH
NH
2
N
N
N
N
O P
O
OH
O
NH
2
X
HN
O
OCH
3
O
O
DMF, DIEA
PyBOP,
amino acid
40°, 2hr
X = CH
2
; 2.16
X = CH
2
C
6
H
4
; 2.17
2.1
N
N
N
N
O P
O
O
OH
NH
2
N
N
N
PMEA-OBt
2.18
DMF, DIEA
PyBOP,
amino acid
40°, 2hr
+
N
N
N
N
NH
2
O P
O
O
OH
P O
N O
OH
N
N
N
H
2
N
PMEA dimer
2.19
44
(peak 19.51 ppm and the small doublet peaks upfield at 10 ppm and 6 ppm, Figure 2.4)
and mass spectrometry (MS-ESI (m/z) 389 and 526, respectively). Upon increasing the
temperature of the reaction to 80 °C, the
31
P-NMR PMEA-OBt intermediate peak did
decrease, however the PMEA dimer peak increased and still no sign of conjugation with
the amino acid promoiety. Thorough investigation of the reaction conditions to define
optimal coupling lead to using MeOH to determine if traditional coupling methods could
be applied to a smaller alkoxy nucleophile.
2.8.3 Conjugation of alkyl groups with PyBOP
To study the PyBOP coupling of PME and PMP parent drugs with alkoxy
nucleophiles, various conditions where the amounts and type of base, solvent,
nucleophiles, as well as, other reactions parameters (i.e. Mitsunobu,
42
converting PMEA
to dichloride) were modified and characterized by
31
P NMR (using a D
2
O capillary so as
not to alter the reaction conditions for analysis) in order to successfully synthesize the
corresponding monoesters and diesters. Initial reaction conditions consisted of using
equimolar amounts of PMEA and MeOH (nucleophile) in DMF under basic conditions
(excess of DIEA) in the presence of PyBOP. Generation of the PMEA-OBt monoester
under these reaction conditions (determined by MS) prompted further analysis. A
comparison of three different reaction mixtures is shown in Figure 2.5. The top
31
P NMR
spectrum is of a reaction mixture that contains PMEA, DMF as the solvent, DIEA,
PyBOP and no nucleophile. The two peaks observed (19 ppm and 14.5 ppm) are the
PMEA-OBt monoester and the tripyrrolidino by-product of reacted PyBOP (both
45
determined using TLC for separation and MS for identification). The middle and bottom
spectra contain the same reagents as the top, only the reaction mixtures contain MeOH
and (L)-Tyr-OMe as the nucleophiles, respectively. In all three spectra, only the PMEA-
OBt monoester peak and the PyBOP by-product peak are observed.
Figure 2.5
31
P NMR (202.5 MHz, D
2
O capillary) of reaction mixture of 1) PMEA
+PyBOP (no nucleophile), 2) PMEA + PyBOP + MeOH and 3) PMEA + PyBOP + (L)-
Tyr-OMe at 40 °C. Observance of PMEA-OBt complex in all cases.
46
These results lead to using a large excess of MeOH, elevated temperatures (80 °C) and
allowing the reaction mixture to stir overnight. Under these reaction conditions, the
monomethyl PMEA ester (2.20) was synthesized (Scheme 2.7).
Scheme 2.7 Successive PyBOP coupling reactions
A subsequent PyBOP coupling reaction of the PMEA monomethyl ester (2.20) with an
amino acid promoiety ((L)-Boc -Ser-OMe) did not afford the mixed PMEA diester (2.21;
Scheme 2.7). The
31
P-NMR of the reaction mixture indicated solely the presence of the
PMEA monomethyl ester (2.20). Analysis of the reaction mixture by
31
P-NMR and MS
indicated the presence of the stable PMEA-OBt monoester.
2.9 Coupling agent combinations: PyBrOP and PyBOP
After attempts to perform two consecutive PyBOP coupling reactions with conjugation of
MeOH first to mask one of the negative charges on the phosphonate followed by PyBOP
conjugation of the desired amino acid did not prove to be successful, the conjugation of
N
N
N
N
O P
O
OH
OH
NH
2
N
N
N
N
O P
O
OMe
OH
NH
2
DMF, DIEA
PyBOP, MeOH
80 °C
overnight
2.1
2.20
DMF, DIEA
PyBOP
(L)-Boc-Ser-OMe
40 °C
overnight
N
N
N
N
O P
O
OMe
O
NH
2
CH
2
HN
O
OMe
2.21
Boc
47
small alkyl alcohols (MeOH, EtOH and iPrOH) with PMEA were attempted with
PyBrOP
53
(Figure 2.6), the bromo derivative of PyBOP.
Figure 2.6 Structure of PyBrOP
The main reason for changing to the bromo derivative is the replacement of the OBt
moiety with a bromide ion in to eliminate formation of the stable PMEA-OBt monoester
observed in the previous PyBOP coupling attempts. Direct coupling of PMEA with the
desired amino acid promoiety in the presence of PyBrOP demonstrated low yields
(~17%), with a majority of the reaction going toward the PMEA dimer. Upon replacing
the amino acid with MeOH as the nucleophile, the reaction gave ~100% yield of the
monomethyl ester of PMEA (2.20; Scheme 2.8). Reaction mixture was extracted with
diethyl ether and a subsequent PyBrOP reaction with an amino acid promoiety was
performed (Scheme 2.8), which did not afford the desired mixed diester (2.21).
Scheme 2.8 Consecutive PyBrOP reactions
Br
P N
N
N
PF
6
PyBrOP
N
N
N
N
O P
O
OH
OH
NH
2
N
N
N
N
O P
O
OMe
OH
NH
2
DMF, DIEA
PyBrOP, MeOH
80 °C
overnight
2.1
2.20
DMF, DIEA
PyBrOP
(L)-Boc-Ser-OMe
40 °C
overnight
N
N
N
N
O P
O
OMe
O
NH
2
CH
2
HN
O
OMe
2.21
Boc
48
Based on these results, the presence of two deprotonated phosphonic acid groups under
these basic conditions (pKa = 2 and 6) may be preventing the anionic phenolic or
hydroxyl nucleophile on the bulkier amino acid promoiety (compared to MeOH) from
attacking the phosphorus atom. It was determined that acyclic nucleoside phosphonates
from the PME and PMP class require monoalkylation (with an ethyl or isopropyl group)
prior to conjugation with the promoiety in order to mask one of the negative charges on
the phosphonic acid group.
2.10 Alternative synthetic approach to PMEA monoester prodrugs
The target PME/PMP prodrugs were synthesized according to Scheme 2.9. Compounds
2.33-2.35 were synthesized by Dr. Valeria Zakharova, a postdoctoral scientist in
Professor McKenna’s research group. Due to lack of success with a PyBOP-mediated
coupling method developed in our lab with the free phosphonic acid of 2.1 or 2.5 and
production of the stable acyclic HOBt-intermediate (2.18, Scheme 2.6) under basic
reaction conditions,
41
monoalkyl (ethyl or isopropyl) esters were readily prepared using a
PyBrOP-mediated coupling approach in DMF and DIEA.
41
Completion of coupling
reaction was monitored using
31
P NMR. The reaction mixture of the synthesis of PMEA
and (R)-PMPDAP monoalkyl esters demonstrate three phosphorus peaks in their
31
P
NMR spectra upon completion: tripyrrolidino phosphonium oxide (14.5 ppm), the desired
monoalkyl ester (~12 ppm) and the dialkyl ester (~ 22 ppm). Monoethyl ANP esters
demonstrated yields ranging from 60-70% (as observed by
31
P NMR) while
49
monoisopropyl ANP esters demonstrated slightly higher yields (75-85%, as observed by
31
P NMR).
Scheme 2.9. a. DMF, DIEA, PyBrOP, EtOH or iPrOH, 40 °C, overnight b. DMF, DIEA,
PyBOP, 40 °C, promoiety (phenol, 2.13, 2.14 or 2.15), 2 h c. MeCN, BTMS, 75 °C, 4 h.
After solvent removal and washing with diethyl ether, the monoalkyl ester ANP was re-
dissolved in DMF, DIEA and the tyrosine-based promoiety of choice (phenol (HOC
6
H
5
),
2.13-2.15) along with PyBOP to give the mixed diester compounds (2.26-2.30; Scheme
2.9) in good yield (60-85% via
31
P-NMR). Following purification by silica gel
chromatography, the alkyl group (Et or iPr) was removed using the McKenna
silyldealkylation approach
54
by dissolving the mixed diester prodrug in MeCN and
adding equimolar amounts of BTMS overnight at rt. Pleasantly, the BOC protection on
the tyrosine amino group, which is removed under acidic conditions,
38,55,56
is removed by
the acidic environment produced by hydrolysis of the intermediate silyl ester. Following
methanolysis and recrystallization, compounds 2.31-2.35 (Figure 2.9) were afforded in
yields ranging from 39-70% (% active drug content, determined by UV).
N
N
N
N
O P
O
OH
OH
NH
2
R
R = H; R
1
= H; 2.1
R = NH
2
; R
1
= CH
3
; 2.5
R
1
b
N
N
N
N
O P
O
OH
OR
3
NH
2
R
R
1
R = H; R
1
= H; R
2
= iPr; R
3
= OC
6
H
5
; 2.26
R = H; R
1
= H; R
2
= Et; R
3
= (L)-Tyr-NH-i-Bu ; 2.27
R = H; R
1
= H; R
2
= Et; R
3
= (L)-Tyr-NH-C
8
H
17
; 2.28
R = H; R
1
= H; R
2
= Et; R
3
= (L)-Tyr-NH-C
16
H
33
; 2.29
R = NH
2
; R
1
= CH
3
; R
2
= Et; R
3
= (L)-Tyr-NH-C
8
H
17
; 2.30
a
N
N
N
N
O P
O
OR
2
OR
3
NH
2
R
R
1
c
R = H; R
1
= H; R
3
= OC
6
H
5
; 2.31
R = H; R
1
= H; R
3
= (L)-Tyr-NH-i-Bu ; 2.32
R = H; R
1
= H; R
3
= (L)-Tyr-NH-C
8
H
17
; 2.33
R = H; R
1
= H; R
3
= (L)-Tyr-NH-C
16
H
33
; 2.34
R = NH
2
; R
1
= CH
3
; R
3
= (L)-Tyr-NH-C
8
H
17
; 2.35
50
Figure 2.7 Tyrosine-based monoester prodrugs of PMEA and (R)-PMPDAP
2.11 Antiviral activity studies
Preliminary in vitro antiviral activities against DNA viruses (HSV-2, VZV, CMV,
VACV, CPXV and ADV) were evaluated for tyrosine-based prodrugs 2.31-2.35 (Table
2.1 and Table 2.2) and their corresponding parent drugs (PMEA, 2.1; (R)-PMPDAP, 2.5)
to observe any differences in antiviral activity between prodrugs with varying
promoieties from a simple phenolic group (2.31; Figure 2.7) to long chain N-alkyl
tyrosinamide (2.34, Figure 2.7). In vitro antiviral assays were performed by Professor
Mark N. Prichard and colleagues at the University of Alabama, Birmingham according to
previously reported procedures.
57-59
Compound 2.34 ((L)-Tyr-NHC
16
H
33
-PMEA)
demonstrated the highest antiviral activity in comparison with the other prodrugs and the
parent drug (2.1) for a majority of the DNA viruses listed below. Of particular interest for
PMEA prodrugs (2.31-2.34) is the antiviral activity against HSV-2, which is a one of the
DNA viruses the parent drug (PMEA, 2.1) has been found to be active against.
N
N
N
N
NH
2
O P
O
OH
O
PMEA-OPh
2.31
N
N
N
N
NH
2
O P
O
OH
O
H
2
N
O
NH
(L)-Tyr-NH-i-Bu-PMEA
2.32
N
N
N
N
NH
2
O P
O
OH
O
H
2
N
O
NHC
8
H
17
(L)-Tyr-NH-C
8
H
17
-PMEA
2.33
N
N
N
N
NH
2
O P
O
OH
O
H
2
N
O
NHC
16
H
33
(L)-Tyr-NH-C
16
H
33
-PMEA
2.34
N
N
N
N
NH
2
O P
O
OH
O
H
2
N
O
NHC
8
H
17
H
2
N
(L)-Tyr-NH-C
8
H
17
-(R)-PMPDAP
2.35
51
Table 2.1 in vitro antiviral activities against HSV-2, VZV, CMV, cytotoxicities and selectivity index
values of the tyrosine prodrugs of PMEA and (R)-PMPDAP.
a
HSV-2 VZV CMV
Compound
EC
50
(µM)
CC
50
(µM)
SI
EC
50
(µM)
CC
50
(µM)
SI
EC
50
(µM)
CC
50
(µM)
SI
2.1 24.8 60 2 30.05 60 2 0.02 60 3000
2.31 49.95 60 1 7.1 31.8 4 2.86 12 4
2.32 56.5 60 1 54.95 60 1 3.48 60 17
2.33 53.05 60 1 53.6 60 1 52 60 1
2.34 2.33 8.47 4 2.4 4.4 2 5.04 11.84 2
2.5 60 60 1 30.02 60 2 28.5 60 2
2.35 60 60 1 52 60 1 11.28 60 5
a
Data obtained by Prof. Mark N. Prichard et al. at the University of Alabama, Birmingham
For CMV, prodrugs 2.31 and 2.32 (PMEA-OPh and (L)-Tyr-NH-i-Bu-PMEA)
demonstrated the lowest EC
50
values with the isobutyl tyrosinamide PMEA monoester
(2.32) displaying the best selectivity index. All, except for 2.33 ((L)-Tyr-NHC
8
H
17
-
PMEA), demonstrated any activity against ADV. Due to previous research on PMP-
related compounds and their activity against mostly retroviruses, it is not surprising that
the parent drug 2.5 nor the prodrug 2.35 ((L)-Tyr-NHC
8
H
17
-(R)-PMPDAP) displayed any
significant activity against the DNA viruses in this preliminary studies (with CMV being
an exception, Table 2.1). It would be of great interest to have the prodrug 2.35 and its
parent drug (2.5) assessed for in vitro antiviral activity against suitable hepadnaviruses
(such as HBV) and retroviruses (such as HIV-1 and HIV-2) in future studies.
52
Table 2.2 in vitro antiviral activities against VACV, CPXV, ADV, cytotoxicities and selectivity index
values of the tyrosine prodrugs of PMEA and (R)-PMPDAP.
a
VACV CPXV ADV
Compound
EC
50
(µM)
CC
50
(µM)
SI
EC
50
(µM)
CC
50
(µM)
SI
EC
50
(µM)
CC
50
(µM)
SI
2.1 60 60 1 60 60 1 60 60 1
2.31 60 60 1 60 60 1 60 60 1
2.32 60 60 1 60 60 1 60 60 1
2.33 60 60 1 60 60 1 49 60 1
2.34 5.6 12.02 2 5.6 14.56 3 60 60 1
2.5 60 60 1 60 60 1 60 60 1
2.35 60 60 1 60 60 1 60 60 1
a
Data obtained by Prof. Mark N. Prichard et al. at the University of Alabama, Birmingham
Based on the data presented in the tables above, there would seem to be a correlation
between in vitro antiviral potency and lipophilicity for most of the viruses. Future
antiviral assessment will consist of testing these prodrugs and their parent drugs against
HBV and retroviruses of interest.
2.12 Conclusion
Five novel tyrosine-based monoester prodrugs of PMEA and (R)-PMPDAP (2.31-2.35)
were synthesized. Four of these prodrugs (2.32-2.35) contain a tyrosinamide promoiety
conjugated to PMEA or (R)-PMPDAP through the phenolic side chain moiety of the
tyrosine promoiety. The prodrug structure was varied at the C-terminal where the
carboxylic acid functional group was converted to an amide with varying alkyl length and
type. Due to 2’-OH hydroxyl group on the phosphonomethoxyether chain for PME and
PMP compounds, PyBOP mediated intramolecular cyclization to mask one of the free
phosphonic acid groups could not be accomplished and therefore prevented conjugating
53
the desired promoiety in the presence of PyBOP and the parent drug. An alternative
approach was applied such that one of the phosphonic acid groups was esterified with a
small alkoxy group (OEt or OiPr) followed by conjugation of the desired tyrosine
promoiety. Following the selective removal of the simple alkyl group and Boc protection
group, the antiviral activity of synthesized monoester prodrugs of PMEA and (R)-
PMPDAP were assessed against various DNA viruses (HSV-2, VZV, CMV, VACV,
CPXV and ADV). The antiviral studies indicated the C
16
length to demonstrate the best
potency.
2.13 Experimental section
General Experimental Methods
1
H and
31
P NMR spectra were obtained on 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); CHCl
3
(
1
H NMR δ = 7.26); H
2
O (
1
H NMR δ = 4.8).
31
P NMR spectra were proton-
decoupled, and
1
H NMR coupling constants (J values) were given in Hz. NMR
abbreviations used include s (singlet), d (doublet), m (unresolved multiplet), dd (doublet
of doublet), ddd (doublet of doublet of doublet), bs (broad signal). The UV spectra were
recorded using Beckman Coulter DU 800 spectrophotometer. MS analysis was performed
on a Thermo-Finnigan LCQ DECA Xp
max
Ion Trap LC/MS/MS eqipped with an ESI
Probe. Xcalibur software wa used to process the MS spectra. IUPAC names for
compounds 2.31-2.35 were obtained from MarvinSketch version 14.9.29.0. The >95%
content of active drug of the final compounds 2.31-2.35 were confirmed using UV
54
determination of the active compound content used the following extinction coefficients:
PMEA derivatives (ε = 14019 at 260 nm in EtOH, ε = 14191 at 260 nm at pH 7.0),
(R)/(S)-PMPDAP derivatives (ε = 10120 at 278 nm, pH 7.0), and tyrosine (ε = 612 at 260
nm and ε = 1300 at 274 nm, pH 7.0).
Synthesis of PMEA
Synthesis of Bis(propan-2-yl) [(2-chloroethoxy)methyl]phosphonate (2.9)
35
To a three neck round bottom flask, 2-chloroethyl chloromethyl ether (2.7; 4.86 mL, 48
mmol) was added and refluxed, under N
2
at 110 °C before dropwise addition of
triisopropyl phosphite (2.8; 14.3 mL, 58 mmol). Reaction mixture was refluxed under N
2
at 120 °C for 4 h followed by distillation under reduced pressure. The first fraction
(collected between 60-70 °C) consists of triisopropyl phosphite, while the last fraction
was collected at 115 °C.
31
P NMR indicated the desired diisopropyl chloroethoxymethane
phosphonate (2.9) in the last fraction as a colorless liquid. Yield 85%.
1
H NMR (400
MHz, CDCl
3
): δ 4.81-4.72 (m, 2H), 3.86-3.84 (t, J = 5.64 Hz, 2H), 3.81-3.79 (d, J = 8.36
Hz, 2H), 3.65-3.62 (t, J = 5.64 Hz, 2H), 1.35-1.33 (dd, J = 6.32 Hz, J = 1.16 Hz, 12H).
31
P NMR (202 MHz, CDCl
3
): δ 18.63.
Bis(propan-2-yl) ([2-(6-amino-9H-purin-9-yl)ethoxy]methyl)phosphonate
36,37
(2.12)
To three neck round bottom flask, adenine (950 mg, 7 mmol), K
2
CO
3
(1.94 g, 14 mmol)
and DMF (22 mL) were added and heated under N
2
for 30 min at 80 °C. Synthon 2.9
(1.79 mL, 7.7 mmol) was added under N
2
dropwise over a 5 min period. N
2
was removed
after 30 min of heating and reaction was stirred for 3 d at 80 °C. Reaction monitored by
55
31
P NMR. Solvent removed and purified silica gel column chromatography (product
eluted 5% MeOH/CH
2
Cl
2
). Yield 54%.
1
H NMR (400 MHz, CD
3
OD): δ 8.20 (s, 1H),
8.13 (s, 1H), 4.60-4.52 (m, 2H), 4.4-4.42 (m, 2H), 3.96-3.93 (m, 2H), 3.82-3.80 (d, J =
8.23 Hz, 2H), 1.23-1.17 (dd, J = 6.62 Hz, J = 18.79 Hz, 2H).
31
P NMR (202 MHz,
CD
3
OD): δ 19.30.
Synthesis of ([2-(6-Amino-9H-purin-9-yl)ethoxy]methyl)phosphonic acid (2.1)
Compound 2.12 (353 mg, 0.9 mmol) was dissolved in MeCN (3 mL) and stirred. BTMS
(0.28 mL, 2.1 mmol) was added dropwise and allowed to stir at rt for 72 h. Reaction was
monitored by thin layer chromatography and
31
P NMR. Solvent removed, co-distilled
with MeCN and H
2
O. The pH was adjusted to 7 by adding water (2 mL) and ammonia.
Solvent removed. Water added (2 mL) and pH adjusted to 2 using concentrated HBr.
Solvent removed and ethanol added. Filtration of precipitate afforded white crystals of
compound 2.1 in good yield (89% yield). Yield 89%.
1
H NMR (400 MHz, CD
3
OD): δ
8.30-8.29 (d, J = 2.47 Hz, 2H), 4.41-4.39 (m, 2H), 3.87-3.84 (m, 2H), 3.51-3.49 (d, J =
8.845 Hz, 2H).
31
P NMR (202 MHz, D
2
O): δ 15.41.
Conventional synthesis of monoester prodrugs of PMEA and (R)-PMPDAP
Amidation of Boc-protected (L)-Tyrosine. General procedure.
52
(L)-Boc-Tyrosine
(commercially available) (4.6 mmol, 1.30 g) was stirred in CH
2
Cl
2
(20 mL) at 0 °C (ice
bath) for 10 min prior to the addition of N-hydroxybenzotriazole (HOBt) hydrate (6.0
mmol, 0.81 g). After stirring this suspension for 15 min at 0 °C, hexadecylamine (5.1
56
mmol, 1.90 g) was added, followed by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC) hydrochloride (6.0 mmol, 1.15 g). The reaction mixture was removed from the ice
bath and allowed to stir at rt for 72 h. Reaction monitored by TLC (0.5:0.5:20,
MeOH:NH
4
OH:CH
2
Cl
2
, followed by ninhydrin test). An additional 30 mL of CH
2
Cl
2
is
added and the organic layer was washed consecutively with saturated citric acid (25 mL),
saturated NaHCO
3
(25 mL), and saturated NaCl (25 mL). The organic phase was dried
over Na
2
SO
4
and concentrated under reduced pressure. Product was purified by silica gel
column chromatography (eluted 2:100 MeOH:CH
2
Cl
2
). Fractions were combined and
solvent removed. Product was dried under reduced pressure.
tert-Butyl N-[(1S)-2-(4-hydroxyphenyl)-1-[(2-
methylpropyl)carbamoyl]ethyl]carbamate (2.13) Yield 89%.
1
H NMR (500 MHz,
CD
3
OD): δ 7.05-7.04 (m, 2H, aromatic), 6.72-6.70 (m, 2H, aromatic), 4.19-4.16 (t, J =
7.0 Hz, 1H), 3.20-3.15 (m, 1H), 3.11-3.05 (m, 1H), 2.95-2.91 (m, 1H), 2.78-2.74 (m, 1H),
1.41 (s, 9H), 1.31 (s, 6H), 0.93-0.91 (t, J = 6.5 Hz, 3H).
tert-Butyl N-[(1S)-2-(4-hydroxyphenyl)-1-(octylcarbamoyl)ethyl]carbamate (2.14)
Yield 88%.
1
H NMR (500 MHz, CD
3
OD): δ 7.05-7.04 (m, 2H, aromatic), 6.72-6.70 (m,
2H, aromatic), 4.19-4.16 (t, J = 7.0 Hz,1H), 3.20-3.15 (m, 1H), 3.11-3.05 (m, 1H), 2.95-
2.91 (m, 1H), 2.78-2.74 (m, 1H), 1.41 (s, 9H), 1.31 (s, 6H), 0.93-0.91 (t, J = 6.5 Hz, 3H).
57
tert-Butyl N-[(1S)-1-(hexadecylcarbamoyl)-2-(4-hydroxyphenyl)ethyl]carbamate
(2.15) Yield 84%.
1
H NMR (500 MHz, CD
3
OD): δ 7.06-7.04 (m, 2H, aromatic), 6.72-
6.71 (m, 2H, aromatic), 4.19-4.16 (t, J = 6.4 Hz,1H), 3.20-3.15 (m, 1H), 3.12-3.06 (m,
1H), 2.96-2.92 (m, 1H), 2.78-2.74 (m, 1H), 1.41 (s, 9H), 1.31 (s, 22 H), 0.93-0.91 (t, J =
6.5 Hz, 3H).
Synthesis of monoester PMEA and (R)-PMPDAP prodrugs (2.31-2.35). General
procedure. To a suspension of PMEA (2.1) or (R)-PMPDAP (2.5) (0.36 mmol) in
anhydrous DMF (3 mL), distilled and dry N,N-diisopropylethylamine (DIEA) (3.6 mmol,
0.647 mL), isopropyl or ethyl alcohol (7.2 mmol), and bromotripyrrolidino-phosphonium
hexafluorophosphate (PyBrOP) (0.59 mmol, 273 mg) were added. The reaction mixture
was stirred at 40 °C under N
2
overnight. The reaction was monitored by
31
P NMR. After
the starting material has disappeared and the reaction is complete, the solvent is removed
under reduced pressure. The residue was extracted with diethyl ether (50 mL) to remove
the PyBrOP by-product, tripyrrolidino phosphonium oxide, from the reaction mixture and
residual removed under reduced pressure. Without any further purification, fresh DMF
(2.2 mL), DIEA (0.425 mL), the desired promoiety 2.13-2.15 (1.1 mmol) and
(benzotriazol-1-yloxy)tripyrrolidino-phosphonium hexafluorophosphate (PyBOP) (0.55
mmol, 235 mg) were added sequentially. The reaction mixture is stirred at 40 °C under
N
2
for 2h. The reaction is monitored by
31
P NMR and additional portions of PyBOP were
added as necessary. Once the reaction is complete, the solvent is removed under reduced
pressure. The residue is extracted with diethyl ether (50 mL) and the reaction residue is
58
dissolved in CH
2
Cl
2
for purification using silica gel column chromatography (elutes at
10:30:60, MeOH:Acetone:CH
2
Cl
2
) or preparatory thin layer chromatography (chamber
solvent: 15:85, MeOH:CH
2
Cl
2
). For column purification, fractions were combined and
solvent removed. For thin-layer purification, product line is scratched and mixed with
15:85 MeOH:CH
2
Cl
2
to separate product from silica gel and solvents are removed.
Product is dried under reduced pressure and then MeCN (0.610 mL) is added, followed
by the drop-wise addition of bromotrimethylsilane (BTMS) (1.8 mmol, 0.160 mL). The
reaction mixture was stirred at 75 °C for 4 h. Reaction mixture was monitored using thin-
layer chromatography. Once the reaction has reached completion, solvent is removed,
followed by two rounds of MeOH additions and removal in order to neutralize unreacted
BTMS. Compounds were precipitated from ethanol: diethyl ether (0.5 mL: 2 mL),
filtered and dried under reduced pressure to give the HBr salts of the products.
([2-(6-Amino-9H-purin-9-yl)ethoxy]methyl)(phenoxy)phosphinic acid (2.31) Yield
50% (determined by UV). Obtained as an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ
8.38-8.36 (d, J = 8.46 Hz, 2H, aromatic), 7.33-7.30 (t, J = 8.72 Hz, 2H, aromatic), 7.18-
7.15 (t, J = 7.69 Hz,1H, aromatic), 7.09-7.07 (d, J = 8.46 Hz, 2H, aromatic), 4.56-4.54 (t,
J = 5.12 Hz, 2H), 4.03-4.01 (t, J = 6.15 Hz, 2H), 3.94-3.92 (d, J = 7.69 Hz, 2H).
31
P
NMR (202 MHz, CD
3
OD): δ 15.96. LRMS (MS-ESI): m/z [M + H]
+
calcd for
C
14
H
16
N
5
O
4
P: 350.1, found: 350.3 (M + H)
+
.
59
(4-[(2S)-2-Amino-2-[(2-methylpropyl)carbamoyl]ethyl]phenoxy)(([2-(6-amino-9H-
purin-9-yl)ethoxy]methyl))phosphinic acid (2.32) Yield 43% (determined by UV).
Obtained as an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ 8.45-8.39 (d, J = 8.42 Hz, 2H,
aromatic), 7.33-7.30 (t, J = 8.72 Hz, 2H, aromatic), 7.18-7.15 (d, J = 1H, aromatic), 4.57-
4.55 (t, J = 4.87 Hz, 2H), 4.03-4.00 (m, 3H), 3.83-3.81 (d, J = 7.76 Hz, 2H), 3.21-3.16
(m, 1H), 3.10-3.06 (m, 1H), 3.02-2.97 (m, 2H), 1.42-1.38 (m, 1H), 1.32 (s, 3H), 0.91-
0.88 (dd, J = 6.52 Hz, J = 1.23 Hz, 6H).
31
P NMR (202 MHz, CD
3
OD): δ 14.35. LRMS
(MS-ESI): m/z [M - H]
-
calcd for C
21
H
30
N
7
O
5
P: 490.19, found: 490.2 (M - H)
-
.
(4-[(2S)-2-Amino-2-(octylcarbamoyl)ethyl]phenoxy)(([2-(6-amino-9H-purin-9-
yl)ethoxy]methyl))phosphinic acid (2.33) Yield 39% (determined by UV). Obtained as
an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ 8.41-8.39 (d, J = 11.36 Hz, 2H, aromatic),
7.29-7.27 (d, J = 8.402 Hz, 2H, aromatic), 7.13-7.12 (d, J = 8.40 Hz, 2H, aromatic), 4.58-
4.56 (t, J = 2.966 Hz, 2H), 4.06-4.03 (m, 3H), 3.96-3.94 (d, J = 6.920 Hz, 2H), 3.23-3.15
(m, 4H), 3.09-3.06 (m, 1H), 1.47-1.44 (m, 3H), 1.31-1.30 (d, J = 6.920 Hz, 11H), 0.92-
0.89 (t, J = 6.425 Hz, 3H).
31
P NMR (202 MHz, CD
3
OD): δ 16.40. LRMS (MS-ESI): m/z
[M + H]
+
calcd for C
25
H
38
N
7
O
5
P: 548.3, found: 548.4 (M + H)
+
.
(4-[(2S)-2-Amino-2-(hexadecylcarbamoyl)ethyl]phenoxy)(([2-(6-amino-9H-purin-9-
yl)ethoxy]methyl))phosphinic acid (2.34) Yield 59% (determined by UV). Obtained as
an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ 8.41-8.40 (d, J = 3.05 Hz, 2H, aromatic),
7.26-7.25 (d, J = 8.69 Hz, 2H, aromatic), 7.14-7.13 (d, J = 7.39 Hz, 2H, aromatic), 4.58-
60
4.56 (t, J = 4.35 Hz, 2H), 4.05-4.03 (t, J = 4.35 Hz, 3H), 3.94-3.92 (d, J = 7.39 Hz, 2H),
3.22-3.13 (m, 4H), 3.07-3.03 (m, 1H), 1.47-1.45 (m, 3H), 1.30 (s, 27H), 0.93-0.90 (t, J =
7.395 Hz, 3H).
31
P NMR (202 MHz, CD
3
OD): δ 16.11. LRMS (MS-ESI): m/z [M + H]
+
calcd for C
33
H
54
N
7
O
5
P: 660.4, found: 660.7 (M + H)
+
.
(4-[(2S)-2-Amino-2-(octylcarbamoyl)ethyl]phenoxy)(([(2R)-1-(2,6-diamino-9H-
purin-9-yl)propan-2-yl]oxy)methyl)phosphinic acid (2.35) Yield 68% (determined by
UV). Obtained as an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ 7.99-7.98 (d, J = 3.87 Hz,
2H, aromatic), 7.28-7.26 (d, J = 7.263 Hz, 2H, aromatic), 7.14-7.12 (d, J = 7.26 Hz, 2H,
aromatic), 4.32-4.29 (dd, J = 4.11 Hz, J = 11.37 Hz, 1H), 4.18-4.14 (m, 1H), 4.04-4.00
(m, 3H), 3.89-3.84 (m, 1H), 3.21-3.14 (m, 4H), 3.08-3.04 (m, 1H), 1.46-1.43 (m, 3H),
1.31-1.30 (d, J = 5.81 Hz, 11H), 0.92-0.89 (t, J = 6.53 Hz, 3H).
31
P NMR (202 MHz,
CD
3
OD): δ 17.01. LRMS (MS-ESI): m/z [M + H]
+
calcd for C
26
H
41
N
8
O
5
P: 577.3, found:
577.6 (M + H)
+
.
Antiviral activity and cytotoxicity studies for tyrosine-based prodrugs of PMEA and
(R)-PMPDAP (2.31-2.35).
Antiviral and cytotoxic assays. All antiviral and cytotoxic assays were conducted by
Prof. Mark N. Prichard at the University of Alabama, Birmingham as part of a
collaboration.
57-59
61
2.14 Chapter 2 references
1. Holý, A.; Votruba, I.; Tloustova, E.; Masojídková, M. Synthesis and Cytostatic
Activity of N-[2-(Phosphonomethoxy)alkyl] Derivatives of N
6
-Substituted
Adenines, 2,6-Diaminopurines and Related Compounds. Collect Czech Chem
Comm. 2001, 66, 1545-1592.
2. Zídek, Z.; Potmesil, P.; Kmoníèková, E.; Holý, A. Immunobiological activity of
N-[2-(phosphonomethoxy)alkyl] derivatives of N6-substituted adenines, and 2,6-
diaminopurines. Eur J Pharmacol. 2003, 475, 149-159.
3. De Clercq, E. Broad-spectrum anti-DNA virus and anti-retrovirus activity of
phosphonylmethoxyalkylpurines and –pyrimidines. Biochem Pharmacol. 1991,
42, 963-972.
4. Holý, A. Synthesis and biological activity of isopolar acyclic nucleotide analogs.
[In: Recent Advances in Nucleosides: Chemistry and Chemotherapy.] Chu, C.K.,
Ed.; Elsevier. 2002, 167-238.
5. Naesens, L.; De Clercq, E. Therapeutic Potential of HPMPC (Cidofovir), PMEA
(Adefovir) and Related Acyclic Nucleoside Phosphonate Analogues as Broad-
Spectrum Antiviral Agents. Nucleos Nucleot. 1997, 16, 983-992.
6. De Clercq, E., Holý, A. Acyclic Nucleoside Phosphonates: A Key Class of
Antiviral Drugs. Nat Rev Drug Discov. 2005, 4, 928–940.
7. Zídek, Z.; Frankova, D.; Holý, A. Macrophage activation by antiviral acyclic
nucleoside phosphonates in dependence on priming immune stimuli. Int J
Immunopharmacol. 2000, 22, 1121-1129.
8. Holý, A. Phosphonomethoxyalkyl Analogs of Nucleotides. Curr Pharm Des.
2003, 9, 2567–2592.
9. De Clercq, E.; Holý, A.; Rosenberg, I.; Sakuma, T.; Balzarini, J.; Maudgal, P. C.
A Novel Broad-spectrum Anti-DNA virus agent. Nature. 1986, 323, 464.
10. Pauwels, R.; Balzarini, J.; Schols, D.; Baba, M.; Desmyter, P.; Rosenberg, I.;
Holý, A.; De Clercq, E. Phosphonylmethoxyethyl purine derivatives, a new class
of anti-human immunodeficiency virus agents. Antimicrob Agents Chemother.
1988, 32, 1025-1030.
11. Deeks, S. G.; Barditch,-Crovo, P.; Lietman, P. S.; Hwang, F.; Cundy, K. C.;
Rooney, J. F.; Hellmann, N. S.; Safrin, S.; Kahn, J. O. Safety, Pharmacokinetics,
and Antiretroviral Activity of Intravenous 9-[2-(R)-
62
(Phosphonomethoxy)propyl]adenine, a Novel Anti-Human Immunodeficiency
Virus (HIV) Therapy, in HIV-Infected Adults. Antimicrob Agents Chemother.
1998, 42, 2380.
12. Naesens, L.; Balzarini, J.; Rosenberg, I.; Holý, A.; De Clercq, E. 9-(2-
Phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP): a novel agent with
anti-human immunodeficiency virus activity in vitro and potent anti-Moloney
murine sarcoma virus activity in vivo. Eur J Clin Microbiol Infect Dis. 1989. 8,
1043.
13. Naesens., L.; Balzarini, J.; De Clercq, E. Single-dose administration of 9-(2-
phosphonylmethoxyethyl)adenine (PMEA) and 9-(2-phosphonylmethoxyethyl)-
2,6-diaminopurine (PMEDAP) in the prophylaxis of retrovirus infection in vivo.
Antiviral Res. 1991. 16, 53.
14. Naesens, L.; Neyts, J.; Balzarini, J.; Holý, A.; Rosenberg, I.; De Clercq, E.
Efficacy of oral 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) in
the treatment of retrovirus and cytomegalovirus infections in mice. J Med Virol.
1993, 39, 167.
15. Holý, A.; Gunter, J.; Dvořáková, H.; Masojídková, M.; Andrei, G.; Snoeck, R.;
Balzarini, J.; De Clercq, E. Structure-antiviral activity relationship in the series of
pyrimidine and purine N-[2-(2-phosphonomethoxy)ethyl] nucleotide analogues. 1.
Derivatives substituted at the carbon atoms of the base. J Med Chem. 1999. 42,
2064.
16. Balzarini, J.; Holý, A.; Jindrich, J.; Naesens, L.; Snoeck, R.; Schols, D.; De
Clercq. Differential antiherpesvirus and antiretrovirus effects of the (S) and (R)
enantiomers of acyclic nucleoside phosphonates: potent and selective in vitro and
in vivo antiretrovirus activities of (R)-9-(2-phosphonomethoxypropyl)-2,6-
diaminopurine. Antimicrob Agents Chemother. 1993. 37, 332.
17. Balzarini, J.; Aquaro, S.; Perno, C-F.; Witvrouw, M.; Holý, A.; De Clercq, E.
Activity of the (R)-enantiomers of 9-(2-phosphonylmethoxypropyl)-adenine and
9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine against human
immunodeficiency virus in different human cell systems. Biochem Biophys Res
Commun. 1996. 219, 337.
18. Krečmerová, M.; Jansa, P.; Dracinsky, M.; Sazelova, P.; Kasicka, V.; Neyts, J.;
Auwerx, J.; Kiss, E.; Goris, N.; Stephan, G.; Janeba, Z. 9-[2-(R)-
(Phosphonomethoxy)propyl]-2,6-diaminopurine (R)-PMPDAP and its prodrugs:
Optimized preparation, including identification of by-products formed, and
antiviral evaluation in vitro. Bioorg Med Chem. 2013. 21, 1199.
63
19. De Clercq, E. Antivirals and Antiviral Strategies. Nature Rev Microbiol. 2004, 2,
704.
20. De Clercq, E. Potential of acyclic nucleoside phosphonates in the treatment of
DNA virus and retrovirus infections. Expert Rev Antiinfect Ther. 2003, 1, 21–43.
21. Palu, G.; Stefanelli, S.; Rassu, M.; Parolin, C.; Balzarini, J.; De Clercq, E.
Cellular uptake of phosphonylmethoxyalkylpurine derivatives. Antiviral Res.
1991, 16, 115-119.
22. Naesens, L.; Balzarini, J.; Bischofberger, N.; De Clercq, E. Antiretroviral activity
and pharmacokinetics in mice of oral bis(pivaloyloxymethyl)-9-(2-
phosphonylmethoxyethyl)adenine, the bis)pivaloyloxymethyl) ester prodrug of 9-
(2-phosphonylmethoxyethyl) adenine. Antimicrob Agents Chemother 1991, 40,
22-28.
23. Naesens, L.; Balzarini, J.; De Clercq, E. Pharmacokinetics in mice of the anti-
retrovirus agent 9-(2-phosphonylmethoxyethyl)adenine. Drug Metab Dispos.
1992, 20, 747-752.
24. Benzaria, S.; Pelicano, H.; Johnson, R.; Maury, G.; Imbach, J.-L.; Aubertin, A.-
M.; Obert, G.; Gosselin, G. Synthesis, in vitro antiviral evaluation, and stability
studies of bis(S-acyl-2-thioethyl) ester derivatives of 9-[2-
(phosphonomethoxy)ethyl]adenine (PMEA) as potential PMEA prodrugs with
improved oral bioavailability. J Med Chem. 1996, 39, 4958.
25. Meris, C.; Gorbig, U.; Muller, C.; Balzarini, J. cycloSal-PMEA and cycloAmb-
PMEA: Potentially New Phosphonate Prodrugs Based on the cycloSal-
Pronucleotide Approach. J Med Chem. 2005, 48, 8079.
26. Erion, M. D.; Reddy, K. R.; Boyer, S. H.; Matelich, M. C.; Gomez-Galeno, J.;
Lemus, R. H.; Ugarkar, B. G.; Colby, T. J.; Schanzer, J.; van Poelje, P. D. Design,
synthesis, and characterization of a series of cytochrome P(450) 3A-activated
prodrugs (HepDirect prodrugs) useful for targeting phosph(on)ate-based drugs to
the liver. J Am Chem Soc. 2004, 126, 5154.
27. Ballatore, C.; McGuigan, C.; De Clercq, E.; Balzarini, J. Synthesis and evaluation
of novel amidate prodrugs of PMEA and PMPA. Bioorg Med Chem Lett. 2001.
11, 1053.
28. Reddy, K. R.; Matelich, M. C.; Ugarkar, B. G.; Gomez-Galeno, J. E.; DaRe, J.;
Ollis, K.; Sun, Z.; Craigo, W.; Colby, T. J; Fujitaki, J. M.; Boyer, S. H.; van
Poelje, P. D.; Erion, M. D. Pradefovir: a prodrug that targets adefovir to the liver
for the treatment of hepatitis B. J Med Chem. 2008. 51, 666.
64
29. Vrbkova, S.; Dracinsky, M.; Holý, A. Synthesis of phosphonomethoxyethyl or
1,3-bis(phosphonomethoxy)propan-2-yl lipophilic esters of acyclic nucleoside
phosphonates. Tetrahedron. 2007, 63, 11391.
30. Lin, C-C.; Yeh, L-T; Vitarella, D.; Hong, Z.; Erion, M. D. Remofovir mesylate: a
prodrug of PMEA with improved liver-targeting and safety in rats and monkeys.
Antiviral Chem Chemother. 2004, 15, 307-316.
31. Tichý, T.; Andrei, G.; Dracinsky, M.; Holý, A.; Balzarini, J.; Snoeck, R.;
Krečmerová, M. New Prodrugs of Adefovir and Cidofovir. Bioorg Med Chem.
2011, 19, 3527-3539.
32. Shaw, J.-P.; Cundy, K. C. abstract: Biological screens of PMEA prodrugs.
Pharmaceutical Research. 1993, 10, (Supplemental): S294.
33. Annaert, P.; Kinget, R.; Naesens, L.; De Clercq, E.; Augustijns, P. Transport,
uptake and metabolism of the bis(pivaloyloxymethyl)-ester prodrug of 9-(2-
phosphonylmethoxyethyl)adenine in an in-vitro cell culture system of the
intestinal mucosa (Caco-2). Pharm Res. 1997, 14, 492-496.
34. 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.
35. Rejman, D.; Masojikova, M.; De Clercq, E.; Rosenberg, I. 2’-C-alkoxy and 2’-C-
aryloxy derivatives of N-(2-phosphonomethoxyethyl)-purines and pyrimidines:
synthesis and biological activity. Nucleos Nucleot Nucleic. 2001, 20, 1497-1522.
36. Holý, A.; Rosenberg, I. Synthesis of 9-(2-phosphonylmethoxyethyl)adenine and
related compounds. Collect Czech Chem Commun. 1987, 52, 2792-2809.
37. Petrov, V.I.; Ozerov, A.A.; Novikov, M.S.; Pannecouque, C.; Balzarini, J.; De
Clercq, E. 9-(2-aryloxyethyl) derivatives of adenine – a new class of non-
nucleosidic antiviral agents. Chem Heterocycl Compd. 2003, 39, 1218-1226.
38. McKenna, C.E.; Schmidhauser, J. Functional Selectivity in Phosphonate Ester
Dealkylation with Bromotrimethylsilane. J Chem Soc Chem Commun. 1979, 739-
739.
65
39. Holý, A.; Rosenberg, I.; Dvořáková, H. Synthesis of N-(2-
phosphonylmethoxyethyl) derivatives of heterocyclic bases. Collect Czech Chem
Comm. 1989, 54, 2190-2207.
40. Holý, A.; Masojídková, M. Synthesis of enantiomeric N-(2-
phosphonylmethoxypropyl) derivatives of purine and pyrimidine bases I. The
stepwise approach. Collect Czech Chem Comm. 1995, 60, 1196-1212.
41. Williams, M.; Krylov, I. S.; Zakharova, V. M.; Serpi, M.; Peterson, L. W.;
Krečmerová, M.; Kashemirov, B. A.; McKenna, C. E. Cyclic and Acyclic
Phosphonate Tyrosine Ester Prodrugs of Acyclic Nucleoside Phosphonates. Czech
Coll Symp Ser. 2011, 12, 167-170.
42. Mitsunobu, O.; Eguchi, M. Preparation of carboxylic esters and phosphoric esters
by activation of alcohols. Bull Chem Soc Jpn. 1971, 44, 3427-3430.
43. Campagne, J-M.; Coste, J.; Jouin, P. (IH-benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate- and (1H-
benzotriazol-1-1yloxy)tripyrrolidinophosphonium hexafluorophosphate-mediated
activation of monophosphonate esters: synthesis of mixed phosphonate diesters,
the reactivity of the benzotriazolyl phosphonic esters vs. the reactivity of the
benzotriazolyl carboxylic esters. J Org Chem. 1995, 60, 5214-5223.
44. 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 Pharm. 2008, 5, 598.
45. Peterson, L. W.; Kashemirov, B. A.; Eriksson, U.; Kim, J. S.; Mitchell, S.; Kijek,
P.; Lee, K. D.; Hilfinger, J. M.; McKenna, C. E. Serine side-chain-linked
peptidomimetic prodrugs of cidofovir and cyclic cidofovir: C-ester effects on
transport and activation. Antiviral Res. 2008, 78, A46-A46.
46. Peterson, L. W.; Kashemirov, B. A.; Sala-Rabanal, M.; Kim, J. S.; Mitchell, S.;
Kijek, P.; Hilfinger, J. M.; McKenna, C. E. MEDI 183-Synthesis and transport
studies on serine side-chain-linked peptidomimetic prodrugs of cyclic cidofovir. J
Chem Soc Chem Commun 2008, 235.
47. Sala-Rabanal, M.; Peterson, L. W.; Serpi, M.; Krylov, I. S.; Kashemirov, B. A.;
Kim, J. S.; Mitchell, S.; Hilfinger, J. M.; McKenna, C. E. Interactions Between
the Human Oligopeptide Transporter, hPepT1 and Serine Side-chain-linked
Cidofovir Prodrugs. Antiviral Res. 2009, 82, A53.
66
48. 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 Pharmacol.
2010, 7, 2349.
49. McKenna C. E.; Peterson L. W.; Kashemirov B. A.; Serpi M.; Mitchell S.; Kim J.
S.; Hilfinger J. M.; Drach J. C. New peptidomimetic prodrugs of acyclic and
cyclic cidofovir: sat studies of chemical and enzymatic activation mechanisms.
Antiviral Res. 2009, 82, A75.
50. Peterson, L. W; Kim, J. S.; Kijek, P.; Mitchell, S.; Hilfinger, J.; Breitenbach, J.;
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.
51. Dykstra, R. R.; Paquette, L. A. (Ed.), Encyclopedia of Reagents for Organic
Synthesis. John Wiley and Sons Ltd., Chichester, UK. 1995, 4, 2668.
52. 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.
53. Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M-N.; Joun, P. PyBOP AND PyBrop:
Two reagents for the difficult coupling of the α,α-dialkyl amino acid, Aib.
Tetrahedron. 1991, 47, 259-270.
54. McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M-C. The facile
dealkylation of phosphonic acid dialkyl esters by bromotrimethylsilane.
Tetrahedron Lett. 1977, 18, 155.
55. McKenna, C. E.; Levy, J. N. α-Keto phosphonoacetates J Chem Soc Chem
Commun. 1989, 246.
56. Gross, H.; Keitel, I.; Coistella, B.; McKenna, C. E. Synthesis of Acid-labile
Geminal Bis- and Triphosphonic acids. Phosphorus Sulfur Silicon and Rel Elem.
1991, 61, 177.
57. Schormann, N.; Sommers, C. I.; Prichard, M. N.; Keith, K. A.; Noah, J. W.; Nuth,
M.; Ricciardi, R. P.; Chattopadhyay, D. Identification of Protein-Protein
Interaction Inhibitors Targeting Vaccinia Virus Processivity Factor for
Development of Antiviral Agents. Antimicrob Agents Chemother. 2011, 55, 5054-
5062.
67
58. James, S. H.; Price, N. B.; Hartline, C. B; Lanier, E. R.; Prichard, M. N. Selection
and Recombinant Phenotyping of Novel CMX001 and Cidofovir Resistance
Mutation in Human Cytomegalovirus. Antimicrob Agents. Chemother. 2013, 57,
3321-3325.
59. Prichard, M. N.; Williams, J. D.; Komazin-Meredith, G.; Khan, A. R.; Price, N.
B.; Jefferson, G. M.; Harden, E. A.; Hartline, C. B.; Peet, N. P.; Bowlin, T. L.
Synthesis and Antiviral Activities of Methylenecyclopropane Analogs with 6-
alkoxy and 6-alkylthio Substitutions that Exhibit Broad-Spectrum Antiviral
Activity Against Human Herpesviruses. Antimicrob Agents Chemother. 2013, 57,
3518-3527.
68
CHAPTER 3
Synthesis and Antiviral Activity of Tyrosinamide
Prodrugs of 2,4-Diaminopyrimidine Acyclic Nucleoside
Phosphonates
3.1 Introduction: discovery of 2,4-diaminopyrimidines (DAPys)
ANPs are a group of structurally homologous compounds that mimic natural nucleotides
and have demonstrated significant antiviral and cytostatic activity.
1,2
The key feature and
difference of these antivirally equipped entities from their biological cousins is the
replacement of the enzymatically degradable P-O bond with a P-C bond, which links to
the N-1 position of the pyrimidine base or the N-9 position of the purine base through an
aliphatic chain. This added feature eliminates problems associated with the initial, rate-
limiting intracellular phosphorylation necessary for nucleoside activation. In current
clinical use, there are three representatives of ANP drugs: cidofovir ((S)-HPMPC, i.e. (S)-
1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, Vistide
®
, Figure 3.1), approved for
the treatment of cytomegalovirus retinitis in AIDS patients, Adefovir Dipivoxil
(pivaloyloxymethyl prodrug of PMEA, i.e. 9-[2-(phosphonomethoxy)ethyl]adenine,
Hepsera
®
, Figure 3.1) and Tenofovir Disoproxil (isopropoxycarbonyloxymethyl prodrug
of (R)-PMPA, i.e. (R)-9-[2-(phosphonomethoxy)propyl]adenine, Viread
®
, Figure 3.1),
clinically approved for the treatment of HBV and HIV respectively.
3,4
69
Figure 3.1 FDA approved acyclic nucleoside phosphonates and their prodrugs
During more than twenty five years lasting history of development of ANPs,
dozens of other promising structures have been discovered.
5-8
Most biologically active
ANPs are predominantly purine-based compounds, derivatives of adenine, 2,6-
diaminopurine, a guanine and a marginal set containing 2-aminopurine as well.
9
Their
efficacy follows the order DAP~G>A>>AP.
1,10
Interesting antiviral activities vere found
also for their aza/deaza counterparts.
11,12
In the pyrimidine series, only cidofovir
powerfully and selectively inhibits all DNA viruses while substitution of cytosine with
other pyrimidine bases (thymine, uracil, 5-substituted cytosine) abolishes the antiviral
effect completely. The large scale of cidofovir activities has already been a topic of many
reviews.
13-15
Recently, a similar broad-spectrum of antiviral effects against DNA viruses
has been found also in 5-azacytosine analogue of cidofovir, (S)-HPMP-5-azaC.
16
N
N
HO
O
P
O
OH HO
NH
2
O
(S)-HPMPC
Cidofovir, Vistide
®
(S)
N
N
N
N
O P
O
O
NH
2
R
N
N
N
N
O P
O
O
NH
2
R
O O
O
O
O
Adefovir Dipivoxil
Hepsera
®
Tenofovir disoproxil fumarate
Viread
®
O O
O
O
O O
O
CO
2
H
HO
2
C
(R)
70
Figure 3.2 Structures of (R)-HPMPO-DAPy, PMEO-DAPy and (R)-PMPO-DAPy.
In 2002, Holý et al. reported the antiviral activity of a novel class of ANPs, 6-[2-
(phosphonomethoxy)alkoxy]pyrimidines, compounds bearing 2,4-diaminopyrimidine
(DAPy) as a nucleobase and the aliphatic phosphonate chain (PME-, PMP and/or HPMP
type) linked to the C-6 position of the pyrimidine ring via the oxygen atom instead of the
N-1 position (Figure 3.1).
17
Originally, the quaternary N-1 2,4-diaminopyrimidine ANP
isomers (Figure 3.3) were synthesized
18
and assessed against various viruses but were
found, along with the N-1 2-aminopyrimidine ANPs, to be devoid of antiviral and
cytostatic activity.
1,10,17
As a result, attention was given to their O-linked non-quaternary
analogues, which can be divided into two subclasses: 1) (R)-HPMPO-DAPy (3.1; Figure
3.2) derivatives and 2) PMEO-DAPy/(R)-PMPO-DAPy derivatives (3.2, 3.3; Figure 3.2).
3.2 Antiviral activity of ANP-DAPy parent drugs
This second generation of ANPs, also called “open-ring“ analogues are mimics of the
appropriate 2,6-diaminopurine derivatives with an open imidazole ring. Their antiviral
activity is essentially identical with the corresponding first generation ANP compounds
((S)-HPMPC, PMEA and (R)-PMPA), including the enantiomeric specificity: the (R)-
(R)-HPMPO-DAPy
3.1
PMEO-DAPy
3.2
N
N
NH
2
H
2
N O
O P
O
OH
OH
CH
3
N
N
NH
2
H
2
N O
O P
O
OH
OH
N
N
NH
2
H
2
N O
O P
O
OH
OH
(R)-PMPO-DAPy
3.3
OH
(R) (R)
71
PMP derivative is active, its (S)-enantiomer inactive. The (R)-HPMPO-DAPy, 2,4-
diamino-6-(R)-[-3-hydroxy-2-(phosphonomethoxy)propyl]pyrimidine (3.1), is analogous
in structure to (S)-HPMP derivatives and demonstrates an antiviral activity spectrum
similar to cidofovir, including the following: HSV-1, HSV-2, VZV, ADV, VV, CPXV,
orf virus (ORFV), and HPV.
19
In 2006, Stittelaar et al. reported cidofovir and (R)-HPMPO-DAPy as very promising
compounds in search for antipoxvirus agents with a goal to find effective treatment
against smallpox in a case of variola virus bioterrorist attack.
20
In experiments with the
monkeypox virus carried out in macaca monkeys, the antiviral treatment was thus proved
definitely to have an edge over vaccination.
20
Despite all above mentioned results, further
development of (R)-HPMPO-DAPy (3.1) as a drug candidate has never been continued,
including preparation and studies of appropriate prodrugs to improve its bioavailability
and the therapeutic potential in general.
The uncovering of PMEO-DAPy (3.2), a 2,4-diaminopyrimidine ANP which has been
shown to demonstrate antiviral activity against HSV-1, HSV-2, VZV, VV, CMV,
HBV, HIV-1, HIV-1 and MSV
9,18
has created an opportunity to develop a second sub
class of acyclic nucleoside phosphonates analogous to PME derivatives. The
pronounce antiretroviral activity was discovered at PMEO-DAPy derivatives bearing
substituents in C-5 position.
21
With the progressive increase of HBV strains harboring
mutations due to long term chronic hepatitis B treatment with lamivudine or adefovir
72
dipivoxil, the need for developing novel HBV inhibitors to overcome HBV drug
resistance as well as design new combination strategies to delay or prevent drug
resistance is of utmost importance.
22-25
It was reported by Brunelle et al. that most
drug mutants remained sensitive to PMEO-DAPy (3.2) indicating further evaluation of
this diaminopyrimidine and its analogues for the treatment of HBV infection.
22
3.3 Mechanism of action
The open, incomplete purine ring of the DAPy designates its class as mimics of the
2,6-diaminopurine ANP class. What makes PMEO-DAPy even more fascinating is
the report by Herman et al. in 2010 that demonstrated the efficient incorporation of the
2,4-diaminopyrimidine analogue of PMEA over (R)-PMPA by K65R HIV-1 reverse
transcriptase mutant.
26
On top of being recognized as a purine mimetic, the other
pleasantly surprising discovery was that it was not efficiently excised as (R)-PMPA by
the HIV-1 RT mutant.
26
From these studies and the various others that have been
performed over the years, further study and evaluation of this open ring ANP prodrugs
is of utmost importance to the field of ANP antiviral research.
3.4 Limitations
Similar to other acyclic nucleoside phosphonates, the drawback of DAPys is the presence
of the anionic charge on the phosphonic acid group at physiological pH. This prevents
them from penetrating the cell membrane and causes them to exhibit poor oral
73
bioavailability. That said, the prodrug approach has been implemented to overcome such
drawbacks.
3.5 Rationale for developing prodrugs of ANP-DAPys
A main focus in our lab has been the search for new acyclic and cyclic nucleoside
phosphonate prodrugs with enhanced, selective biological activity, emphasizing the
modification of the phosphonate bearing side chain and varying the nucleobase to
determine optimal biological effects. As was described in Chapter 1.9, peptidomimetic
serine, threonine and tyrosine prodrugs of (S)-HPMPC and (S)-HPMPA have been
thoroughly studied in our laboratories against various DNA viruses. As a result of a
collaboration formed between our lab and the research laboratory of Prof. Marcela
Krečmerová at the Institute of Organic Chemistry and Biochemistry (IOCB AS CR) in
Prague, Czech Republic, novel tyrosinamide prodrugs of PMEO-DAPy and (R)-HPMPO-
DAPy were synthesized. The synthesis of the (R)-HPMPO-DAPy parent drug (3.1,
Figure 3.2) was elaborated in this study as well. A series of tyrosine-based single amino
acid 3.19-3.22, 3.29-3.31 and dipeptide 3.23 P-O ester conjugates of 3.1 and 3.2 were
synthesized and their preliminary in vitro antiviral activity against HCMV was assessed
to examine their impact in antiviral drug discovery. In the current study we explore the
antiviral effect of the tyrosine promoiety and, in particular, its further modification at one
or more of its functional sites (e.g. NH
2
or CO
2
H) on the biological properties of 2,4-
diaminopyrimidines PMEO-DAPy and (R)-HPMPO-DAPy.
74
3.6 Synthesis of PMEO-DAPy and (R)-HPMPO-DAPy
The synthesis of 9-(R)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,4-diaminopyrimidine
((R)-HPMPO-DAPy, 3.1) described in Scheme 3.1 begins with the nucleophilic addition
of (S)-(+)-2,2-dimethyl-1-3-dioxolane-4-methanol (3.5) to 2,4-diamino-6-
choropyrimidine (3.4) in the presence of NaH, followed by the removal of the acetonide
group under acidic conditions to give 3.7. To prevent addition of the phosphonate moiety
to multiple positions on 3.7, the exocyclic amino groups on the nucleobase and the
primary hydroxyl group on the glycol ether chain are protected under basic conditions
using 4,4-dimethoxytrityl chloride (DMTrCl). Conjugation of the diisopropyl methyl
phosphonate moiety (BrCH
2
P(O)(OiPr)
2
) to compound 3.8 was performed in the
presence of NaH, followed by treatment with acetic acid is utilized to remove the DMTr
protecting groups. This is followed up with the McKenna approach to remove the
isopropyl groups from the phosphonate to give (R)-HPMPO-DAPy (3.1).
Scheme 3.1 a. NaH, dioxane, reflux; b. MeOH/ H
2
O, HCl, rt; c. DMTrCl, pyridine, rt, 3
h; d. BrCH
2
P(O)(OiPr)
2
, NaH, DMF, -20 °C, 24 h; e. HOAc, rt, 1 h; f. BTMS, MeCN, 80
°C, 24 h.
N
N
NH
2
H
2
N O
OH
OH
N
N
NHDMTr
DMTrHN O
OH
ODMTr
(R)-HPMPO-DAPy
3.1
N
N
NH
2
H
2
N Cl
O
O
HO
N
N
NH
2
H
2
N O
O
O
3.4 3.5
3.7
3.8 3.9
3.6
+
a b c
d,e f N
N
NH
2
H
2
N O
O
OH
P O OiPr
OiPr
N
N
NH
2
H
2
N O
O
OH
P O OH
OH
(R) (R)
75
A modified approach of the previously described synthesis of PMEO-DAPy (3.2)
17
is
shown in Scheme 3.2. The original synthesis describes the conjugation of the 2,4-
diamino-6-hydroxypyrimidine (3.10) with 2-(chloroethoxy)methylphosphonate (3.11) in
the presence of cesium carbonate to give 3.12 in 17% yield. From here, the parent drug
(3.2), can be made by removing both isopropyl groups in MeCN and BTMS or 3.13 can
be made by refluxing the diisopropyl ester of PMEO-DAPy for 6 h in a KOH/H
2
O
mixture. Being that the goal was to synthesize monoester tyrosinamide prodrugs, one
isopropyl group was removed and compound 3.13 was utilized in the next steps in
synthesizing prodrugs of PMEO-DAPy.
Scheme 3.2 Synthesis of PMEO-DAPY and PMEO-DAPy(OiPr) a. Cs
2
CO
3
, DMF, 100
°C, 16 h; b. Dioxane, KOH/H
2
O, reflux, 6 h; c. BTMS, MeCN, 80 °C, 24 h.
3.7 Synthesis of tyrosinamide and tyrosine-based prodrugs of PMEO-DAPy
Tyrosine-based prodrugs 3.19-3.22, 3.25 were synthesized according to a previously
described approach
27
outlined in Scheme 3.3. The Boc-protected tyrosine esters, amides
and dipeptides were synthesized according to literature procedures.
28,29
Synthesis of
PMEO-DAPy prodrugs consisted of utilizing the monoisopropyl ester 3.13 in
N
N
NH
2
H
2
N OH
+
Cl
O P
O
OiPr
OiPr
N
N
NH
2
H
2
N O
O P
O
OiPr
OiPr
N
N
NH
2
H
2
N O
O P
O
OiPr
OH
PMEO-DAPy-OiPr
3.13
3.12 3.11 3.10
N
N
NH
2
H
2
N O
O P
O
OH
OH
PMEO-DAPy
3.2
a
b
c
76
dimethylformamide (DMF), diisopropylethylamine (DIEA) as the base, the tyrosinamide
single amino acid or dipeptide moiety (3.14, 2.13-2.15) and benzotriazol-1-
yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) as the coupling reagent
overnight at rt.
Figure 3.3 Tyrosine-based prodrugs of PMEO-DAPy
Using
31
P NMR, the reaction was stopped upon the disappearance of starting material
3.13 (14.96 ppm) and the appearance of the mixed diester prodrug peak at 22 ppm for the
coupling reactions depicted in Scheme 3.3. After solvent removal and ether extractions to
remove the tripyrrolidinophosphonium reaction by-product, the residue is purified using
column chromatography to yield the Boc-protected mixed diester prodrugs (3.15-3.18,
3.24; Scheme 3.3) in moderate to good yields (65-75%).
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
O
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
HN
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
NHC
16
H
33
(L)-Tyr-OiPr-PMEO-DAPy
3.19
(L)-Tyr-NH-i-Bu-PMEO-DAPy
3.20
(L)-Tyr-NH-C
16
H
33
-PMEO-DAPy
3.21
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
NHC
8
H
17
N
N H
2
N
NH
2
O
O
P
O
HO
O O O
N
H
O
NH
2
(L)-Tyr-NH-C
8
H
17
-PMEO-DAPy
3.22
(L)-Tyr-OiPr-(L)-Val-PMEO-DAPy
3.25
77
Scheme 3.3 a. PyBOP, DMF, DIEA, rt, 24 h; b. BTMS, MeCN, rt, 24 h.
Following conjugation, the McKenna reaction
30,31
is utilized to simultaneously remove
the Boc group from the promoiety and the isopropyl group from the phosphonate ester.
The reaction mixture is allowed to stir at rt overnight and is monitored by thin later
chromatography (1:10; MeOH:CH
2
Cl
2
). After solvent removal, residue is coevaporated
with water and methanol, followed by purification with column chromatography using a
methanol-dichloromethane mobile phase. The prodrugs were dissolved in H
2
O where the
pH was increased to 9-10 using 14.8 M NH
4
OH and then decreased to a pH of 1-2 using
concentrated HCl to precipitate prodrugs 3.19-3.22, and 3.25. All compounds were dried
N
N H
2
N
NH
2
O
O P
O
OH
O
3.13
3.14 R
1
= OiPr
2.13 R
1
= NH-i-Bu
2.14 R
1
= NHC
8
H
17
2.15 R
1
= NHC
16
H
33
3.15-3.18 R
2
= Boc
R
3
= OiPr
a
HO O R
1
N
H
+
N
N H
2
N
NH
2
O
O P
O
OH
O
3.13
HO O O
N
H
+
O
NH
Boc
3.23
N
N H
2
N
NH
2
O
O
P
O
O
R
3
O
HN
O
R
1
3.19-3.22 R
2
= H
R
3
= H
3.24 R
2
= Boc
R
3
= OiPr
3.25 R
2
= H
R
3
= H
N
N H
2
N
NH
2
O
O
P
O
R
3
O
a
R
2
O O O
N
H
O
NH
R
2
b
b
Boc
78
under reduced pressure till solvent peaks were not present in the
1
H NMR. Compounds
3.19 and 3.20 were synthesized by Dr. Tomáš Tichý, a postdoctoral researcher at IOCB,
AS CR.
3.8 Synthesis of tyrosinamide cyclic and acyclic (R)-HPMPO-DAPy prodrugs
Cyclic and acyclic tyrosinamide prodrugs of (R)-HPMPO-DAPy (3.29-3.31; Figure 3.4)
were synthesized according to Scheme 3.4. Despite the structural similarity of 3.1 to (S)-
HPMPC and (S)-HPMPA, the traditional one-pot coupling procedure,
32,33
used previously
to synthesize cyclic HPMP prodrugs, was not successful in this case. Several trials with
modified reaction conditions (amounts of base, types of reagents, amount of promoiety)
gave yields no better than 25%. It was determined that masking the two negative charges
on the phosphonic acid would require two separate coupling steps for this parent drug.
Intramolecular cyclization to mask the first negative charge was accomplished with N,N’-
dicyclohexycarbodiimide (DCC) coupling
34,35
in the presence of N,N’-dicyclohexyl-4-
morpholinecarboxamidine (DCMC), followed by ion exchange on Dowex
®
(1 X 2, OAc
-
)
to give cyclic HPMPO-DAPy (3.26). Using this substrate to couple the desired tyrosine
promoiety (2.14-2.15; Scheme 3.4) with PyBOP gave the Boc-protected (R)-HPMPO
cyclic prodrugs (3.29, 3.30).
79
Scheme 3.4 a. DCC, DCMC, DMF, 100 °C, 6 h; b. PyBOP, DMF, DIEA, rt, 4 h; c. TFA,
CH
2
Cl
2
, 24 h; d. NH
4
OH, MeCN, 45 °C, 4 h.
Following the conjugation of the tyrosinamide promoiety and purification via column
chromatography, the Boc group was removed in an overnight reaction using TFA in
CH
2
Cl
2
. The products (3.29, 3.30) were purified with column chromatography, using a
methanol-dichloromethane mobile phase with a small amount (0.05% v/v) of TFA.
Prodrugs 3.29 and 3.30 were precipitated from diethyl ether as TFA salts. The acyclic
prodrug of HPMPO-DAPy 3.31 was obtained by hydrolysis with ammonia. Upon
completion of the reaction, the solvent is removed and the cyclic by-product of the
reaction is removed by dissolving the residue in H
2
O, sonicating to get the by-product to
3.1
N
N O
P O
O
HN
O
R
1
NH
2
H
2
N
OH
OH
O
3.26
3.27 R
1
= NHC
16
H
33
; R
2
= Boc
3.28 R
1
= NHC
8
H
17
; R
2
= Boc
3.29 R
1
= NHC
16
H
33
; R
2
= H
3.30 R
1
= NHC
8
H
17
; R
2
= H
3.31 R
1
= NHC
16
H
33
; R
2
= H
R
2
2.14 R
1
= NHC
8
H
17
2.15 R
1
= NHC
16
H
33
HO O R
1
N
H
Boc +
c
a b
d
N
N
NH
2
H
2
N O
O P
O
OH
OH
OH
N
N O
P
O
O
O
OH
NH
2
H
2
N
N
N O
P
O
O
O
O
HN
O
R
NH
2
H
2
N
R
2
80
precipitate and separating the solid via filtration. The filtrate is collected, solvent
removed and purified using silica gel chromatography (45% MeOH/CH
2
Cl
2
).
Recrystallization from diethyl ether afforded compound 3.31.
Figure 3.4 Cyclic and acyclic tyrosinamide prodrugs of (R)-HPMPO-DAPy
3.9 Antiviral activity studies
Preliminary in vitro antiviral activity studies of the ANP-DAPy parent drugs (3.1 and 3.2)
and corresponding prodrugs (3.19-3.22, and 3.29) were evaluated against HCMV. In
vitro antiviral assays were performed by Professor Mark N. Prichard and colleagues at
UAB according to previously reported procedures.
36,37
The inhibitory values, cytotoxicity
levels and selectivity values are summarized in Table 3.1.
N
N O
P
O
O
O
H
2
N
O
NHC
16
H
33
NH
2
H
2
N
(L)-Tyr-NH-C
16
H
33
-cHPMPO-DAPy
3.29
N
N O
P O
O
H
2
N
O
NHC
16
H
33
NH
2
H
2
N
OH
OH
O
N
N O
P
O
O
O
O
H
2
N
O
NHC
8
H
17
NH
2
H
2
N
(L)-Tyr-NH-C
8
H
17
-cHPMPO-DAPy
3.30
(L)-Tyr-NH-C
16
H
33
-HPMPO-DAPy
3.31
O
81
Table 3.1 In vitro antiviral activities against HCMV, cytotoxicities and selectivity
index values for 3.1, 3.2, 3.19-3.22, 3.25, 3.29.
a
Compound EC
50
(µM) CC
50
(µM) SI
Ganciclovir 3.17 >100 >31.5
PMEO-DAPy (3.2) >10 >10 >1
(L)-Tyr-(OiPr) (3.19) >10 >10 >1
(L)-Tyr-NH-i-Bu-PMEO-DAPy (3.20) >10 >10 >1
(L)-Tyr-(OiPr)-(L)-Val-PMEO-DAPy (3.25) >10 >10 >1
(L)-Tyr-NHC
16
H
33
-PMEO-DAPy (3.21) 1.08 10.0 9.2
(L)-Tyr-NHC
8
H
17
-PMEO-DAPy (3.22) >10 >10 >11
(R)-HPMPO-DAPY (3.1) >10 >10 >1
(L)-Tyr-NHC
16
H
33
-cHPMPO-DAPy (3.29) 0.61 >10 >16.4
a
Data obtained by Prof. Mark N. Prichard et al. at the University of Alabama, Birmingham
The PMEO-DAPy and (R)-HPMPO-DAPy tyrosine-based prodrugs did not exhibit
potencies different from the parent drug 3.2, with the exception of the (L)-Tyr-NHC
16
H
33
-
PMEO-DAPy analogue (3.21) and (L)-Tyr-NHC
16
H
33
-cHPMPO-DAPy analogue (3.29).
Both Tyr-C
16
DAPy derivatives (3.21 and 3.29) demonstrated better antiviral activity
than the gold standard for HCMV, ganciclovir (Table 3.1). The selectivity index for
ganciclovir did, however, demonstrate values 2-3 fold higher than the C
16
analogues
(3.21 and 3.29). As can be seen from the data table above, the C
16
derivatives were the
only ones to demonstrate significant antiviral activity against HCMV in this preliminary
in vitro assessment. This may be due to enhanced permeability as a result of the lipophilic
C
16
alkyl chain on the tyrosine promoiety, which is allowing the drug to be more
accessible to inhibit HMCV viral replication.
82
3.10 Conclusion
Eight novel long chain alkyl tyrosinamide prodrugs of PMEO-DAPy and (R)-HPMPO-
DAPy (3.19-3.22, 3.25, 3.29-3.31) were synthesized. The five PMEO-DAPy monoester
prodrugs were synthesized by masking one of the phosphonic acid charges with an alkyl
group (ethyl or isopropyl) followed by coupling the desired tyrosine-based promoiety
through the phenolic side chain. An alternate route in the synthesis had to be applied in
the first step due to low yields during initial attempts to simultaneously cyclize and
conjugate the promoiety. Synthesis of the four (R)-HPMPO-DAPy cyclic and acyclic
tyrosinamide prodrugs required first cyclizing the parent drug with DCC, followed by
PyBOP-mediated coupling of the desired lipophilic Tyr-C
16
promoiety. The prodrugs
were evaluated for antiviral activity against HCMV. The tyrosinamide C
16
ester prodrugs
of PMEO-DAPy and cHPMPO-DAPy demonstrated the best activity against HCMV and
will be further evaluated against HIV-1 to determine their antiviral potency.
3.11 Experimental Section
General Experiment Procedures.
1
H and
31
P NMR spectra were obtained on 400 MHz 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).
31
P NMR spectra were
proton-decoupled, and
1
H NMR coupling constants (J values) were given in Hz. NMR
abbreviations used include s (singlet), d (doublet), p (pentet), m (unresolved multiplet),
dd (doublet of doublet), ddd (doublet of doublet of doublet), bs (broad signal). The UV
83
spectra were recorded using Beckman Coulter DU 800 spectrophotometer. MS analysis
was performed on a Thermo-Finnigan LCQ DECA Xp
max
Ion Trap LC/MS/MS eqipped
with an ESI Probe. Xcalibur software wa used to process the MS spectra. IUPAC names
for compounds 3.19-3.22, 3.25, and 3.29-3.31 were obtained from MarvinSketch version
14.9.29.0. The >95% content of active drug of the final compounds 3.19-3.22, 3.25, and
3.29-3.31 were confirmed using UV determination of the active compound content used
the following extinction coefficients: PMEO-DAPy derivatives (ε = 7500 at 276 nm at
pH 7.0),
17
(R)-HPMPO-DAPy derivatives (ε = 7500 at 276 nm at pH 7.0),
17
and tyrosine
(ε = 612 at 260 nm and ε = 1300 at 274 nm, pH 7.0).
Synthesis of (2R)-3-[(2,6-Diaminopyrimidin-4-yl)oxy]propane-1,2-diol (3.7)
To a suspension of NaH (60% suspension in oil,) in freshly distilled dioxane (350 mL)
(S)-(+)-2,2-dimethyl-1-3-dioxalane-4-methanol (17.1 mL) was slowly added and H
2
bubbled through an oil chamber. Mixture was stirred until H
2
bubbling ceased (2 h). Then
4-chloro-2,6-diaminopyrimidine (10 g, 69.2 mmol) was added and the suspension was
refluxed for 4 h with exclusion of moisture. Upon completion, reaction mixture was
neutralized with acetic acid (1.97 mL, 5 mmol) and evaporated. Residue was partitioned
between chloroform (100 mL) and aqueous NaCl (100 mL). The aqueous layer was
extracted with chloroform (3 X 50 mL). Combined organic extracts were dried with
MgSO
4
and evaporated. Residue was purified using silica gel column chromatography
(mobile phase was 7% MeOH/EtOAc). Residue from the previous step was dissolved in
MeOH:H
2
O (1:4; 40 mL). The mixture was acidified with concentrated HCl (to pH 2)
84
and stirred at rt for 4 h. Complete conversion was monitored by TLC (10%
MeOH/EtOAc). The mixture was applied to onto a column of Dowex 50 X 8 (H+ form),
washed with H
2
O until neutral pH was achieved. Product was eluted with 2.5% aqueous
ammonia. Fractions were combined and evaporated. Product was crystallized from
MeOH-ether as a yellow-white solid. Yield 70%. Precipitated as an NH
4
+
salt.
1
H NMR
(500 MHz, CD
3
OD): δ 5.25 (s, 1H), 4.20-4.09 (m, 2H), 3.90-3.85 (m 1H), 3.75-3.67 (m,
2H), 3.62-3.54 (m, 2H), 3.30-3.28 (p, J = 1.629 Hz, 2H)
31
P NMR (500 MHz, D
2
O): δ
13.33.
Synthesis of (([(2R)-1-[(2,6-diaminopyrimidin-4-yl)oxy]-3-hydroxypropan-2-
yl]oxy)methyl)phosphonic acid (3.1)
3.7 (10.82 g, 54 mmol) in pyridine (600 mL), 4,4-dimethoxy tritylchloride (56.7 g, 167.4
mmol) was added and the mixture was stirred at rt overnight. Complete conversion was
checked by TLC with Hubert 1 (18:3:1:1; EtOAc:(CH
3
)
2
CO:EtOH:H
2
O). The reaction
was quenched with EtOH (3 M eq. of 100% ethanol) and the solvent removed under
reduced pressure. The syrupy residue was extracted with chloroform (500 mL) and
washed with saturated aqueous NaHCO
3
(1 L). The aqueous portion was extracted with
chloroform (500 mL, X 2) and the organic layers were combined and dried with MgSO
4
.
Solution was filtered and concentrated under reduced pressure. Residue was co-distilled
with toluene (X 3) to get remove residual H
2
O. The residue in anhydrous
dimethylformamide (500 mL) was stirred at 0 °C for 30 min (ice bath). Sodium hydride
(60% suspension in min oil, 6.48 g, 162 mmol) was added and stirred at 0 °C for 20 min
85
(ice bath). Diisoproxyphosphorylmethyl bromide (25.28 g, 81 mmol, 83% solution) was
added and the mixture was stirred at rt overnight. Solvent was removed under reduced
pressure and the residue was dissolved in 80% acetic acid (360 mL). Mixture was stirred
at rt for 1 h, acetic acid evaporated followed by co-distillation with H
2
O once and EtOH
twice. Completion of reaction was checked by TLC 10% MeOH/CHCl
3
. Sample was
dissolved in CHCl
3
and applied on a silica gel column (product eluted 0-15%
MeOH/CHCl
3
). Fractions containing product were collected, evaporated and dried under
reduced pressure. The remaining 5.1 g was dissolved in 160 mL of MeCN followed by 16
mL of BTMS. The reaction was stirred overnight at rt and checked the following day by
TLC. An additional equivalent of BTMS was added to the mixture and allowed to stir at
rt overnight. Solvent was removed under reduced pressure and co-distilled with 200 mL
of H
2
O. Compound was re-dissolved in H
2
O and alkalized to a pH of 9-10 with 25%
ammonia. The pH was then brought back down through the addition of HCl and left to
crystallize in the fridge over the weekend. Solvent was removed by vacuum filtration,
solid washed with small portions of H
2
O, followed by (CH
3
)
2
CO to remove traces of
H
2
O. Solid was dried under reduced pressure and weighed to afford 3.1. Filtrate is saved,
solvent removed and residue is re-dissolved in H
2
O to repeat recrystallization process and
recover as much product as possible. Yield 70% (determined by UV). Precipitated as an
HCl salt.
1
H NMR (500 MHz, D
2
O, 1 drop NH
4
OH): δ 5.49 (s, 1H), 4.26 (s, 2H), 3.85-
3.83 (m 2H), 3.75-3.67 (m, 2H), 3.60-3.56 (m, 1H).
31
P NMR (202 MHz, D
2
O): δ 13.33.
LRMS (MS-ESI): m/z [M - H]
-
calcd for C
8
H
15
N
4
O
6
P: 293.06, found: 293.2 (M - H)
-
.
86
Synthesis of PMEO-DAPy-OiPr (3.13) and PMEO-DAPy (3.1).
General Procedure.
7
A mixture of 2,4-diamino-6-hydroxypyrimidine (10 g, 79.3 mol), cesium carbonate (12.9
g, 39.65 mol) and 158 mL of DMF were stirred at 80 °C for 30 min and diisopropyl 2-
chloroethoxymethylphosphonate (14.3 mL, 92.78 mol) was added. The mixture was
stirred at 110 °C for 16 h and filtered from salts. The filtrate was taken down under
reduced pressure and the residue was purified using silica gel column chromatography.
Residue was dissolved in ethyl acetate and applied to ethyl acetate silica gel column.
Product was eluted with 10% MeOH/EtOAc. All fractions combined and solvent
removed. The diisopropyl ester of PMEO-DAPy (4.00 g, 11.48 mmol) was dissolved in
dioxane (20 mL). An alkaline solution of KOH/H
2
O (1.290 g, 22.96 mmol in 15mL of
H
2
O) was added and the pH adjusted for alkalinity. The mixture was refluxed for 24 h.
Solvent was removed and dissolved in H
2
O. The residue was deionized on a Dowex
®
50
X 8 (H+ form), washed with H
2
O and the UV-absorbing fraction of the ammonia elute
(2.5% ammonia used to elute product) was collected. Solvent was removed, residue
redissolved in H
2
O, and acidified to a pH of 2 with aq. HCl. The product crystallized, so
solvent was removed by filtration and the compound was dried under reduced pressure in
an oil bath at 50°C.
((2-[(2,6-Diaminopyrimidin-4-yl)oxy]ethoxy)methyl)(propan-2-yloxy)phosphinic
acid (3.13) Yield 11%. Precipitated as an HBr salt.
1
H NMR (500 MHz, D
2
O, 1 drop
NH
4
OH): δ 5.44 (s, 1H), 4.71-4.64 (m, 2H), 4.46-4.45 (m 2H), 4.00-3.98 (m, 2H), 3.82-
87
3.81 (d, J = 9.033 Hz, 2H), 1.41-1.40 (d, J = 6.486 Hz, 6H),
31
P NMR (202 MHz, D
2
O): δ
14.96. LRMS (MS-ESI): m/z [M + H]
+
calcd for C
10
H
19
N
4
O
5
P: 307.1, found: 307.3 (M +
H)
+
.
General Procedure for dealkylation of diisopropyl-2,4-diamino pyrimidine.
From the diisopropyl derivative of PMEO-DAPy (3.13), the free phosphonic acid was
achieved by dealkylation with BTMS as performed according to the literature
procedure.
30,31
((2-[(2,6-Diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphonic acid) (3.2) Yield
15% (determined UV). Precipitated as an HBr salt.
1
H NMR (500 MHz, D
2
O, 1 drop
NH
4
OH): δ 5.31 (s, 1H), 4.16-4.14 (m, 2H), 3.77-3.75 (m 2H), 3.44-3.42 (d, J = 8.682
Hz, 2H),
31
P NMR (202 MHz, D
2
O): δ 13.54. LRMS (MS-ESI): m/z [M - H]
-
calcd for
C
7
H
13
N
4
O
5
P: 263.05, found: 263.2 (M - H)
-
.
Synthesis of dipeptide (L)-Tyr(OiPr)-(L)-Val (3.23). General Procedure. The
dipeptide promoiety was synthesized according to a procedure described by Miyazawa et
al.
29
(L)-Tyr-OiPr (3.14) was purchased from Sigma Aldrich.
Synthesis of (L)-Tyr-NH-i-Bu, (L)-Tyr-NHC
16
H
33
, and (L)-Tyr-NHC
8
H
17
synthons
(2.13, 2.14 and 2.15). General procedure. The isobutyl, C
16
and C
8
tyrosinamide
88
promoieties were synthesized according to the procedure previously described in the
literature
28
and in the experimental section of Chapter 2.
Synthesis of monoester PMEO-DAPy amino acid and dipeptide monoester prodrugs
(numbers). General procedure.
27
To a suspension of 3.13 (1.5 mmol, 460 mg) in
anhydrous DMF (35 mL), distilled and dry N,N-diisopropylethylamine (DIEA) (15
mmol, 2.658 mL), (benzotriazol-1-yloxy)tripyrrolidino-phosphonium
hexafluorophosphate (PyBOP) (3 mmol, 1.56 mg) and the desired promoiety (3.14, 3.15,
2.13-2.15) (2.25 mmol) were added. The reaction mixture was stirred at 40 °C under N
2
overnight. The reaction was monitored by
31
P NMR and additional portions of PyBOP
are added as necessary. Once the reaction is complete, the solvent is removed under
reduced pressure. The residue is extracted with diethyl ether and washed with H
2
O (100
mL each). Ether fractions collected and solvent removed to be dissolved in CH
2
Cl
2
for
purification using silica gel column chromatography (elutes at 10:30:60;
MeOH:(CH
3
)
2
CO:CH
2
Cl
2
). For column purification, fractions were combined and
solvent removed. Product is dried under reduced pressure and then MeCN (10 mL) is
added, followed by the drop-wise addition of BTMS (15 mmol, 10 mL). The reaction
mixture was stirred at rt overnight. Reaction mixture was monitored using thin-layer
chromatography. Once the reaction has reached completion, solvent is removed, followed
by co-distillation with H
2
O. Residue washed with EtOH and removed under reduced
pressure to remove traces of H
2
O. Residue was dissolved in 20% MeOH/CH
2
Cl
2
and
purified via silica gel column chromatography (mobile phase 20% MeOH/CH
2
Cl
2
).
89
Fractions combined and solvent removed. Compounds were dried under reduced pressure
to give the HBr salts of the PMEO-monoester prodrugs.
(4-[(2S)-2-Amino-3-oxo-3-(propan-2-yloxy)propyl]phenoxy)((2-[(2,6-
diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic acid (3.19) Yield 47.7%
(determined by UV). Obtained as an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ 7.20-7.13
(d, J = 9.071 Hz, 2H, aromatic), 7.15-7.13 (d, J = 9.071 Hz, 2H, aromatic), 5.29 (s, 1H),
5.11-5.06 (m, 1H), 4.33-4.31 (t, J = 5.400 Hz, 2H), 4.16-4.13 (t, J = 7.668 Hz, 1H), 3.87-
3.85 (t, J = 5.076 Hz, 2H), 3.78-3.76 (d, J = 9.071 Hz, 2H), 3.22-3.21 (dd, J = 8.747 Hz,
J = 6.156 Hz, 1H), 3.05-3.00 (dd, J = 6.480 Hz, J = 7.668 Hz, 1H), 1.30-1.26 (dd, J =
10.467 Hz, J = 6.280 Hz, 6H).
31
P NMR (202 MHz, CD
3
OD): δ 13.81. LRMS (MS-ESI):
m/z [M - H]
-
calcd for C
19
H
28
N
5
O
7
P: 468.2, found: 468.3 (M - H)
-
.
(4-[(2S)-2-Amino-2-[(2-methylpropyl)carbamoyl]ethyl]phenoxy)(2-[(2,6-
diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic acid (3.20) Yield 46.5%
(determined by UV). Obtained as an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ 7.18-7.16
(d, J = 9.735 Hz, 2H, aromatic), 7.13-7.11 (d, J = 7.66 Hz, 2H, aromatic), 5.28 (s, 1H),
4.29 (s, 2H), 4.14-4.09 (q, J = 7.664 Hz, 1H), 3.84 (s, 2H), 3.75-3.73 (d, J = 9.735 Hz,
2H), 3.59-3.56 (t, J = 6.835 Hz, 1H), 3.03-2.91 (m, 3H), 2.80-2.76 (m, 1H), 1.74-1.68 (m,
1H), 1.29-1.24 (m, 1H), 0.86-0.84 (t, J = 6.214 Hz, 6H).
31
P NMR (202 MHz, CD
3
OD): δ
13.30. LRMS (MS-ESI): m/z [M - H]
-
calcd for C
20
H
31
N
6
O
6
P: 481.2, found: 481.2 (M -
H)
-
.
90
(4-[(2S)-2-Amino-2-(hexadecylcarbamoyl)ethyl]phenoxy)((2-[(2,6-
diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic acid (3.21) Yield 48%
(determined by UV). Obtained as an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ 7.24-7.20
(m, 4H, aromatic), 5.40 (s, 1H), 4.47 (s, 2H), 4.02-3.99 (q, J = 8.411 Hz, 1H), 3.93 (s,
2H), 3.84-3.82 (d, J = 9.093 Hz, 2H), 3.25-3.16 (m, 3H), 3.03-2.99 (m, 1H), 1.51-1.49
(m, 2H), 1.33-1.32 (d, J = 7.501 Hz, 27H), 0.95-0.92 (t, J = 6.820 Hz, 3H).
31
P NMR
(500 MHz, CD
3
OD): δ 13.94. HRMS (MS-ESI): m/z [M - H]
-
calcd for C
32
H
55
N
6
O
6
P:
649.3, found: 649.3 (M - H)
-
.
(4-[(2S)-2-Amino-2-(octylcarbamoyl)ethyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.22) Yield 36% (determined by UV). Obtained
as an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ 7.21-7.19 (m, 4H, aromatic), 5.38 (s,
1H), 4.45-4.43 (m, 2H), 3.98-3.96 (m, 1H), 3.89 (m, 2H), 3.81-3.79 (d, J = 8.951 Hz,
2H), 3.21-3.13 (m, 3H), 3.00-2.96 (m, 1H), 1.49-1.46 (m, 2H), 1.31 (s, 11H), 0.92-0.89
(t, J = 5.520 Hz, 3H).
31
P NMR (202 MHz, CD
3
OD): δ 13.87. LRMS (MS-ESI): m/z [M
+ H]
+
calcd for C
24
H
39
N
6
O
6
P: 539.3, found: 539.4. 2119 (M + H)
+
.
(4-[(2S)-2-[(2S)-2-Amino-3-methylbutanamido]-3-oxo-3-(propan-2-
yloxy)propyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.25) Yield 47% (determined by UV).
Obtained as an HBr salt.
1
H NMR (500 MHz, CD
3
OD): δ 7.12 (s, 4H), 5.29 (s, 1H), 5.02-
91
4.97 (p, J = 10.516 Hz, J = 6.510 Hz, 1H), 4.71-4.68 (m, 1H), 4.29-4.27 (m, 2H), 3.84-
3.82 (m, 2H), 3.75-3.73 (d, J = 9.765 Hz, 2H), 3.17-3.13 (dd, J = 5.508 Hz, J = 14.522
Hz, 1H), 2.93-2.89 (m, 1H), 2.15-2.09 (m, 1H), 1.26-1.17 (m, 6H), 1.04-0.97 (dd, J =
6.510 Hz, J = 26.541 Hz, 6H).
31
P NMR (202 MHz, CD
3
OD): δ 13.44. HRMS (MS-ESI):
m/z [M + H]
+
calcd for C
24
H
37
N
6
O
8
P: 569.4, found: 569.2 (M + H)
+
.
Synthesis of monoester cyclic HPMPO-DAPy prodrugs (3.29, 3.30). General
procedure.
32,34,35
(R)-HPMPO-DAPy (1.56 mmol, 460 mg), DCC (1.99 mmol, 413 mg), and DCMC (1.89
mmol, 555 mg) were added, followed by 20 mL of DMF. Reaction flask was placed in an
oil bath (100 °C) for 3 h. TLC (2:1; IPAV:H
2
O) indicated completion of reaction.
Reaction was allowed to cool, added 20 mL of H
2
O, filtered and washed with H
2
O. All
filtrates combined and concentrated under reduced pressure to 10 mL. MeOH was added
and applied to Dowex 1 X 2 (AcO
-
form) column. MeOH (250 mL), H
2
O (150 mL) and
acetic acid dilutions (0.1 M, 0.3 M and 0.5 M) were added. Fractions 8-13 were
combined and solvent removed followed by co-distillation with H
2
O (50 mL, X 3). Clear
sludge was dried under reduced pressure was 2 h then 20 mL of H
2
O was added and set
in fridge overnight. DMF (15 mL) was added and removed under reduced pressure (X 2).
Then dried under reduced pressure for 1.5 h to yield 3.26 as a white sludge.
DMF (20 mL) and DIEA (2.8 mL) were added to clear sludge and sonicated for 25 min.
2.14 or 2.15 (945 mg, 1.1 eq.) was added and suspension was sonicated for 10 min.
PyBOP (905 mg, 1.73 mmol) was added and mixture was sonicated and swirled till red
92
color ceased getting darker. Mixture was left to stir at rt under argon. Additional portions
of PyBOP were added until reaction demonstrated completion. Solvent was removed and
toluene was added and removed (20 mL, x 3) to remove traces of DMF. Residue was
washed with 125 mL H
2
O and extracted with 150 mL diethyl ether and add fresh ether
aliquots until ether layer is clear. Ether fractions were combined and solvent was
removed. Residue was redissolved in MeOH and the solvent was removed under reduced
pressure. Residue was dissolved in CHCl
3
and applied to a CHCl
3
silica gel column.
Gradual addition of MeOH to stationary phase (product elutes 7%-9% MeOH/CHCl
3
)
elutes cyclic prodrug. Fractions were combined, solvent removed and dried under
reduced pressure to afford 3.27 and 3.28.
To fractions of 3.27 and 3.28 obtained from purification, dry CHCl
3
(8 mL) was added
and mixture was sonicated. Residue did not dissolve until TFA was added (1.5 mL).
After 4h stirring at rt, reaction was complete (monitored by TLC). Solvent was removed,
fresh CHCl
3
added and removed (X 2). Residue was re-dissolved in 1% TFA/CHCl
3
and
applied to 1% TFA/CHCl
3
silica gel column. Product eluted at 18% MeOH/CHCl
3
(constant 1% TFA throughout column chromatography), fractions combined and solvent
removed. Residue was co-distilled in CHCl
3
, dried for 6 h under reduced pressure
(heating at 50 °C) until glass-like. Diethyl ether added, filtered and dried to yield 3.29
and 3.30.
93
(2S)-2-Amino-3-(4-([(5R)-5-([(2,6-diaminopyrimidin-4-yl)oxy]methyl)-2-oxo-1,4,2λ⁵-
dioxaphosphinan-2-yl]oxy)phenyl)-N-hexadecylpropanamide (3.29) Yield 18.3%
(determined by UV). Obtained as a TFA salt. Mixture of diastereomers.
1
H NMR (500
MHz, CD
3
OD): δ 7.35-7.28 (m, 4H, aromatic), 5.44 (s, 1H), 4.74-4.56 (m, 3H), 4.43-4.35
(m, 3H), 4.29-4.27 (m, 2H), 4.20-4.17 (d, J = 15.318 Hz, 2H), 4.01-3.98 (m, 1H), 3.24-
3.07 (m, 5H), 1.46-1.40 (m, 2H), 1.31 (s, 11H), 0.93-0.90 (t, J = 5.617 Hz, 3H).
31
P NMR
(202 MHz, CD
3
OD): δ 10.46, 9.22 (74:26). LRMS (MS-ESI): m/z [M + H]
+
calcd for
C
33
H
55
N
6
O
6
P: 663.4, found: 663.5 (M + H)
+
.
(2S)-2-Amino-3-(4-([(5R)-5-([(2,6-diaminopyrimidin-4-yl)oxy]methyl)-2-oxo-1,4,2λ⁵-
dioxaphosphinan-2-yl]oxy)phenyl)-N-octylpropanamide (3.30) Yield 56.2%
(determined by UV). Obtained as a TFA salt. Mixture of diastereomers.
1
H NMR (500
MHz, CD
3
OD): δ 7.34-7.23 (m, 4H, aromatic), 5.43 (s, 1H), 4.74-4.68 (m, 2H), 4.64-4.58
(m, 2H), 4.50-4.47 (m, 1H), 4.43-4.39 (m, 2H), 4.28-4.24 (m, 2H), 4.02-3.98 (m, 1H),
3.24-3.06 (m, 5H), 1.43-1.40 (m, 2H), 1.30 (s, 27H), 0.92-0.90 (t, J = 6.159 Hz, 3H).
31
P
NMR (202 MHz, CD
3
OD): δ 10.61, 9.20 (28:82). LRMS (MS-ESI): m/z [M + H]
+
calcd
for C
25
H
39
N
6
O
6
P: 552.3, found: 552.5 (M + H)
+
.
Synthesis of monoester acyclic HPMPO-DAPy prodrug (3.31). General procedure
3.29 (0.47 mmol, 310.8 mg) was added to a round bottom flask, followed by 76 mL of a
MeCN/NH
4
OH solution (60 mL of MeCN in 16 mL of 3.7 M NH
4
OH). This suspension
94
was sonicated for 10 min in which the remainder of the MeCN/NH
4
OH solution was
added. The reaction mixture was refluxed at 45 °C overnight and monitored by
31
P NMR.
Upon completion, additional MeCN (100 mL) was added and solvent removed. Added
MeOH (125 mL) to dissolve compound and remove solvent. To precipitate product,
HPLC grade H
2
O (50 mL) was added and suspension sonicated for 15 min. Filtered till
and decanted H
2
O layer, solid was dissolved in MeOH, combined and solvent removed
several times (to remove all traces of H
2
O). Dissolve sample in 5% MeOH/CH
2
Cl
2
and
purify via silica gel chromatography (5-40% MeOH/CH
2
Cl
2
in increments of 5%).
Fractions combined and solvent removed under reduced pressure. Residue was
recrystallized from diethyl ether to afford 3.31.
(4-[(2S)-2-Amino-2-(hexadecylcarbamoyl)ethyl]phenoxy)(([(2R)-1-[(2,6-
diaminopyrimidin-4-yl)oxy]-3-hydroxypropan-2-yl]oxy)methyl)phosphinic acid
(3.31) Yield 71.4% (determined by UV). Obtained as an NH
4
+
salt.
1
H NMR (500 MHz,
CD
3
OD): δ 7.18-7.16 (d, J = 7.852 Hz, 2H, aromatic), 7.12-7.10 (d, J = 7.852 Hz, 2H,
aromatic), 5.27 (s, 1H), 4.25-4.24 (d, J = 4.625 Hz, 2H), 3.99-3.95 (m, 1H), 3.80-3.73 (m,
3H), 3.65-3.64 (m, 1H), 3.55-3.52 (t, J = 7.206 Hz, 2H), 3.15-3.12 (m, 2H), 2.99-2.95 (m,
1H), 2.79-2.75 (m, 1H), 1.46-1.43 (m, 2H), 1.30 (s, 27H), 0.93-0.90 (t, J = 6.131 Hz,
3H).
31
P NMR (202 MHz, CD
3
OD): δ 13.69. LRMS (MS-ESI): m/z [M - H]
-
calcd for
C
33
H
57
N
6
O
7
P: 679.4, found: 679.6 (M - H)
-
.
95
Antiviral activity and cytotoxicity studies for tyrosine-based prodrugs of (R)-
HPMPO-DAPy and PMEO-DAPy (3.19-3.22, 3.25 3.29).
Antiviral and cytotoxic assays. All antiviral and cytotoxic assays were conducted by
Prof. Mark N. Prichard at the University of Alabama, Birmingham as part of a
collaboration.
36,37
96
3.12 Chapter 3 references
1. De Clercq, E.; Holý, A. Acyclic Nucleoside Phosphonates: A Key Class of Antiviral
Drugs. Nat Rev Drug Discov. 2005, 4, 928-940.
2. Holý, A. Synthesis and Biological Activity of Isopolar Acyclic Nucleotide Analogues. [In
Recent Advances in Nucleosides: Chemistry and Chemotherapy.] Chu, C.K., Ed.;
Elsevier Science B.V. 2002, 167-238.
3. De Clercq, E. Clinical Potential of the acyclic nucleoside phosphonates cidofovir,
adefovir, and tenofovir in treatment of DNA virus and retrovirus infections. Clin
Microbiol Rev. 2003, 16, 569-596.
4. De Clercq, E. Acyclic nucleoside phosphonates: Past, present and future Bridging
chemistry to HIV, HBV, HCV, HPV, adeno-, herpes-, and poxvirus infections: The
phosphonate bridge. Biochem Pharmacol. 2007, 73, 911-922.
5. De Clercq, E. Broad-spectrum anti-DNA virus and antiretrovirus activity of
phosphonomethoxyalkylpurine and –pyrimidines. Biochem Pharmacol. 1991. 42, 963-
972.
6. De Clercq, E. In search of a selective antiviral chemotherapy. Clin Microbiol Rev. 1997,
10, 674-693.
7. Holý, A.; Gunter, J.; Dvořáková, H.; Masojídková, M.; Andrei, G.; Snoeck, R.; Balzarini,
J.; De Clercq, E. Structure-antiviral activity relationship in the series of pyrimidine and
purine N-[2-(2-phosphonomethoxy)ethyl] nucleotide analogues. 1. Derivatives
substituted at the carbon atoms of the base. J Med Chem. 1999, 42, 2064-2086.
8. Naesens, L.; Balzarini, J.; Rosenberg, I.; Holý, A.; De Clercq, E. 9-(2-
phosphonomethoxyethyl)-2,6-diaminopurine (PMEDAP): a novel agent with anti-human
immunodeficiency virus activity in vitro and potent anti-Moloney murine sarcoma virus
activity. Eur J Clin Microbiol Infect. 1989, 8, 1043-1047.
9. Balzarini, J.; Pannecouque, C.; De Clercq, E.; Aquaro, S.; Perno, C-F.; Egberink, H.;
Holý, A. Antiretrovirus Activity of a Novel Class of Acyclic Pyrimidine Nucleoside
Phosphonates. Antimicrob Agents Chemother. 2002, 46, 2185-2193.
10. Holý, A. Phosphonomethoxyalkyl analogs of nucleotides. Current Pharm Design, 2003,
9, 2567-2692.
11. Dvořáková, H.; Holý, A.; Alexander, P. Synthesis and biological effects of 9-(3-hydroxy-
2-phosphonomethoxypropyl) derivatives of deazapurine bases. Collect Czech Chem
Commun. 1993, 58, 1403-1418.
97
12. Holý, A.; Dvořáková, H.; Jindřich, J.; Masojídková, M.; Buděšínský, M.; Balzarini, J.;
Andrei, G. & De Clercq, E. Acyclic nucleotide analogs derived from 8-azapurines:
synthesis and antiviral activity. J Med Chem. 1996, 39, 4073-4088.
13. Naesens, L.; De Clercq, E. Therapeutic potential of HPMPC (cidofovir), PMEA
(adefovir) and related acyclic nucleoside phosphonate analogues as broad-spectrum
antiviral agents. Nucleosides Nucleotides 1997, 16, 983 – 992.
14. Naesens, L.; Snoeck, R.; Andrei, G; Balzarini, J.; Neyts, J.; De Clercq, E. HPMPC
(cidofovir), PMEA (adefovir) and related acyclic nucleoside phosphonate analogues: A
review of their pharmacology and clinical potential in the treatment of viral infections.
Antivir Chem Chemother. 1997, 8, 1–23.
15. De Clercq E. Towards an effective chemotherapy of virus infections: Therapeutic
potential of cidofovir [(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine,
HPMPC] for the treatment of DNA virus infections. Collect Czech Chem Commun. 1998,
63, 480-506.
16. Krečmerová, M.; Holý, A.; Pískala, A.; Masojídková, M.; Andrei, G.; Naesens, L.; Neyts,
J.; Balzarini, J.; De Clercq, E.; Snoeck, R. Antiviral activity of triazine analogues of 1-
(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (cidofovir) and related
compounds. J Med Chem. 2007, 50, 1069-1077.
17. Holý, A.; Votruba, I.; Masojídková, M.; Andrei, G.; Snoeck, R.; Naesens, L.; De Clercq,
E.; Balzarini, J. 6-[2-(Phosphonomethoxy)alkoxy]pyrimidines with Antiviral Activity. J
Med Chem. 2002, 45, 1918-1929.
18. Holý, A.; Buděšínský, M.; Podlaha, J.; Císařová, I. Synthesis of N1-[2-
(phosphonomethoxy)ethyl] derivatives of 2,4-diaminopyrimidine and related acyclic
nucleotide analogues. Collect Czech Chem Comm. 1999. 64, 242-256.
19. De Clercq, E.; Andrei, G.; Balzarini, J.; Leyssen, P.; Naesens, L.; Neyts, J.;
Pannecouque, C.; Snoeck, R.; Ying, C.; Hocková, D.; and Holý, A. Antiviral Potential of
a New Generation of Acyclic Nucleoside Phosphonates, the 6-[2-
(phosphonomethoxy)alkoxy]-diaminopyrimidines. Nucleosides Nucleotides Nucleic
Acids. 2005, 24, 331-341.
20. Stittelar, K. J.; Neyts, J.; Naesens, L.; van Amerongen, G.; van Lavieren, R. F.; Holý, A.;
De Clercq, E.; Niesters, H. G. M.; Fries, E.; Maas, C.; Mudler, P. G. H.; van der Zeijst, B.
A. M.; Osterhaus, A.D.M. Antiviral Treatment is more effective than smallpox
vaccination upon lethal monkeypox viral infection. Nature 2006, 439, 745-748.
98
21. Hocková, D.; Holý, A.; Masojídková, M.; Andrei, G.; Snoeck, R.; De Clercq, E.;
Balzarini, J. 5-Substituted-2,4-Diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidines –
Acyclic Nucleoside Phosphonate Analogues with Antiviral Activity. J Med Chem. 2003,
46, 5064-5073.
22. Brunelle, M. N.; Lucifora, J.; Neyts, J.; Villet, S.; Holý, A.; Trepo, C.; Zoulim, F. In
Vitro Activity of 2,4-Diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine against
Multi-Drug-Resistant Hepatitis B Virus Mutants. Antimicrob Agents Chemother. 2007,
51, 2240-2243.
23. Hadziyannis, S. J.; Tassopoulos, N. C.; Heathcote, E. J.; Chang, T. T.; Kitis, G.; Rizzetto,
M.; Marcellin, P.; Lim, S. G; Goodman, Z.; Ma, J.; Arterburn, S.; Xiong, S; Currie, G.;
Brosgart, C. L. Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic
hepatitis B. N Engl J Med. 2005, 352, 2673-2681.
24. Lai, C. L.; Dienstag, J.; Schiff, E.; Leung, N. W.; Atkins, M.; Hunt, C.; Brown, N.;
Woessner, M.; Boehme, R.; Condreay, L. Prevalence and clinical correlates of YMDD
variants during lamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis.
2003, 36, 687-696.
25. Zoulin, F. Antiviral therapy of chronic hepatitis B. Antiviral Res. 2006, 71, 206-215.
26. Herman, B. D.; Votruba, I.; Holý, A.; Sluis-Cremer, N.; Balzarini, J. The Acyclic 2,4-
Diaminopyrimidine Nucleoside Phosphonate Acts as a Purine Mimetic in HIV-1 Reverse
Transcriptase DNA Polymerization. J Biol Chem. 2010, 285, 12101-12108.
27. Williams, M.; Krylov, I. S.; Zakharova, V. M.; Serpi, M.; Peterson, L. W.; Krečmerová,
M.; Kashemirov, B. A.; McKenna, C. E. Cyclic and Acyclic Phosphonate Tyrosine Ester
Prodrugs of Acyclic Nucleoside Phosphonates. Czech Coll Symp Ser. 2011, 12, 167-170.
28. 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.
29. Miyazawa, T.; Hiramatsu, S.; Tsuboi, Y.; Yamada, T.; Kuwata, S. Studies of unusual
amino acids and their peptides. XVII. The synthesis of peptides containing N-
carboxymethyl amino acids. II. Bull Chem Soc Jpn. 1985, 58, 1976–1982.
30. McKenna, C.E.; Schmidhauser, J. Functional Selectivity in Phosphonate Ester
Dealkylation with Bromotrimethylsilane. J Chem Soc Chem Commun. 1979, 739-739.
31. McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M-C. The facile dealkylation of
phosphonic acid dialkyl esters by bromotrimethylsilane. Tetrahedron Lett. 1977, 18, 155.
99
32. 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 Pharmacol. 2010, 7,
2349.
33. 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.
34. Krečmerová, M.; Holý, A.; Pohl, R.; Masojídková, M.; Andrei, G.; Naesens, L.; Neyts,
J.; Balzarini, J.; De Clercq, E.; Snoeck, R. Ester Prodrugs of Cyclic 1-(S)-[3-Hydroxy-2-
(phosphonomethoxy)propyl]-5-azacytosine: Synthesis and Antiviral Activity. J Med
Chem. 2007, 50, 5765-5772.
35. Krečmerová, M.; Holý, A.; Andrei, G.; Pomeisl, K.; Tichý, T.; Břehová, P.;
Masojídková, M.; Dracinsky, M.; Pohl, R.; Laflamme, G.; Naesens, L.; Hui, H.; Cihlar,
T.; Neyts, J,; De Clercq, E.; Balzarini, J.; Snoeck, R. Synthesis of ester Prodrugs of 9-(S)-
[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) as Anti-
Poxvirus Agents. J Med Chem. 2010, 53, 6825-6837.
36. Schormann, N.; Sommers, C. I.; Prichard, M. N.; Keith, K. A.; Noah, J. W.; Nuth, M.;
Ricciardi, R. P.; Chattopadhyay, D. Identification of Protein-Protein Interaction Inhibitors
Targeting Vaccinia Virus Processivity Factor for Development of Antiviral Agents.
Antimicrob Agents Chemother. 2011, 55, 5054-5062.
37. Prichard, M. N.; Williams, J. D.; Komazin-Meredith, G.; Khan, A. R.; Price, N. B.;
Jefferson, G. M.; Harden, E. A.; Hartline, C. B.; Peet, N. P.; Bowlin, T. L. Synthesis and
Antiviral Activities of Methylenecyclopropane Analogs with 6-alkoxy and 6-alkylthio
Substitutions that Exhibit Broad-Spectrum Antiviral Activity Against Human
Herpesviruses. Antimicrob Agents Chemother. 2013, 57, 3518-3527.
100
CHAPTER 4
Synthesis and Antiviral Activity of a Novel N-Alkyl Ser-
Val Dipeptide Analogue of Cyclic Cidofovir
4.1 Introduction: (S)-HPMPC
Of the main prototypes of acyclic nucleoside phosphonates, the HPMP class of
derivatives demonstrate a wide range of antiviral activity against DNA viruses. Shortly
following the emergence of (S)-HPMPA, the cytosine equivalent, (S)-HPMPC,
1
(cidofovir, Vistide
®
; Figure 4.1), was outlined to demonstrate an extraordinary spectrum
of antiviral activity against DNA viruses.
1
Included in it’s viral adversaries are HPV,
polyomavirus, HSV-1, HSV-2, Epstein-Barr virus (EBV), HCMV, VZV, human
herpesviruses (HHV-6, HHV-7, HHV-8), ADV, poxvirus, VV, CPXV, molluscum
contagiosum, and orf infections.
2,3
Figure 4.1 Cidofovir and cyclic cidofovir
N
N
NH
2
O
O
OH
P
O
OH
OH
N
N
NH
2
O
O
P
O
OH
(S)-HPMPC
Cidofovir
Vistide
!
4.1
cyclic Cidofovir
4.2
O
101
Compound 4.1 was determined to be particularly active against HCMV
3
and in 1996
became the first FDA approved ANP for the systemic treatment of CMV retinitis among
AIDS patients. Due to its success in eradicating CMV infection in AIDS patients, 4.1 is
still currently used off-label (topical and systemic) in treating polyoma, papilloma, adeno
and poxvirus infections.
4.2 Mechanism of action
Similar to the mechanism of action of PME and PMP derivatives, cidofovir requires
cellular uptake in order to exert its antiviral activity. Once inside the cell, initial
phosphorylation of cidofovir to its diphosphate analogue (HPMPCp) is performed by
pyrimidine nucleoside monophosphate kinase.
4
It is then phosphorylated to its active
metabolite triphosphate form (HPMPCpp) by nucleoside diphosphate kinase, pyruvate
kinase or creatine kinase.
4
The triphosphate analogue of cidofovir exerts its antiviral
potential by being recognized by viral DNA polymerase and being incorporated into the
viral DNA strand.
5
HPMPCpp competes with its natural nucleoside counterpart
deoxycytidine triphosphate (dCTP) and acts as a chain terminator and alternative
substrate for viral DNA polymerase.
5
Halting viral DNA replication and terminating
DNA chain elongation requires two consecutive cidofovir metabolites to be incorporated
into the viral DNA strand.
5
Therapeutic advantages of administering cidofovir includes
infrequent dosing due to the observed long half life of HPMPCpp. As with other ANPs,
conversion of cidofovir to its active metabolite via cellular enzymes bypasses the rate-
limiting initial phosphorylation step by viral-encoded enzymes that acyclic nucleoside
102
analogues (e.g. acyclovir and ganciclovir) require.
6
The higher affinity for the cellular
DNA polymerases α, β, γ, and ε that diphosphorylated ANP metabolites demonstrate is
the basis for their antiviral potential.
7,8
4.3 Limitations
Despite the remarkable therapeutic advantages of cidofovir, one of the major limitations
of its therapeutic use include nephrotoxicity.
9
Another issue with cidofovir is the
presence of an ionizable –P(O)(OH)
2
group which hinders cell permeability and oral
bioavilability.
10
The 5% oral bioavailability demonstrated by cidofovir
11
limits its
therapeutic potential.
4.4 Prodrugs of (S)-HPMPC
The prodrug approach has been applied to cidofovir to address the limitations described
in Chapter 4.3 above. Of the prodrug approaches mentioned in Chapter 1.8, the lipid ester
approach developed by Hostetler and co workers has been applied to various antivirals in
need of pharmacokinetic enhancement. In particular, the HDP analogue of (S)-HPMPC
(CMX001) demonstrated higher in vitro antiviral activities,
12-14
oral bioavailability
15
and
reduced nephrotoxicity
14
compared to the parent drug. As a result, CMX001 has
advanced to clinical trials as a treatment of CMV, smallpox and BK viruses. More
recently, Chimerix, Inc. has announced the use of CMX001 as an experimental antiviral
in emergency uses against ebola virus disease. Other prodrug approaches that have been
applied to (S)-HPMPC include salicylate and aryl esters,
16,17
and amphiphilic prodrugs,
18
103
have been developed. There are currently no clinically approved prodrugs of (S)-HPMPC
and (S)-HPMPA.
4.5 Rationale for prodrug design of long chain dipeptide cHPMPC analogue
As mentioned in Chapter 1.8, the first peptidomimetic prodrugs of (S)-HPMPC to be
designed and evaluated using the McKenna prodrug approach were ethylene glycol
linked amino acid prodrugs of cyclic (S)-HPMPC followed by dipeptide prodrugs of
cyclic (S)-HPMPC. When the EG-linked amino acid ((L)-alanine or (L)-valine) cHPMPC
analogues demonstrated short half lives against HCMV,
19
variations in the prodrug
scaffold were implemented (linkage between X and phosphonic acid, amino acid vs.
dipeptide promoiety, converting functional groups on the promoiety (R), Figure 4.2) in
order to study and assess the in vitro and in vivo efficacy of this class of prodrugs.
Figure 4.2 Scaffolds for cyclic and acyclic prodrugs of (S)-HPMPC and (S)-HPMPA
B
OH
P
O
OH
B = Cytosine ((S)-HPMPC)
B = Adenine ((S)-HPMPA)
B
(S)
O
O
P
O
X
H
2
N
O
R
(R / S)
(L / D)
OH
acyclic phosphonate
prodrugs
*
*
B
O
HO
P
O
X
H
2
N
O
R
(R / S)
(L / D)
*
*
HO
(S)
(S)
cyclic phosphonate
prodrugs
104
After assessing amino acid and dipeptide based serine promoieties on 4.1,
20-23
studies
shifted to modifying position X (Figure 4.2) in order to enhance prodrug stability. This
included replacing (L)-serine methyl ester with (L)-tyrosine, (L)-threonine or (L)-cysteine
methyl esters to study amino acid prodrugs of (S)-HPMPA in order to determine optimal
efficacy for esterified amino acid promoieties.
24
In this studies, it was found that tyrosine-
based derivatives (varying at position R, Figure 4.2) demonstrated the favorable half-
lives and antiviral in vitro activities against various viruses
24,25
and so serine, along with
the other two amino acids, were not included in the following stages of development.
Prodrugs in which the C-terminal on tyrosine had been converted to an amide with a long
alkyl chain had been found to demonstrate remarkable in vitro antiviral activity and
stability,
25
which prompted further investigation into developing lipophilic N-alkyl
tyrosine prodrugs of (S)-HPMPA and (S)-HPMPC. Evolution of this prodrug approach to
include a hydrophobic moiety on the modified carboxylic group of the tyrosine amino
acid is what prompted synthesizing a dipeptide promoiety with a similar alkyl long chain.
It is of interest to investigate the in vitro and, in the future, the stability effect of the
lipophilic N-alkyl long chain group on a serine-based prodrug of HPMPC and compare
the findings with previously studied serine prodrugs and lipophilic tyrosine prodrugs of
the same ANP class.
4.6 Synthesis of novel N-alkyl long chain Ser-Val dipeptide promoiety
The synthesis of the N-alkyl long chain dipeptide promoiety consisted of using the
amidation procedure
26
described in Chapters 2 to synthesize the tyrosinamide promoieties
105
(2.13-2.15) depicted in Scheme 4.1. (L)-Boc-Serine (4.3) is amidated using standard EDC
coupling procedures in the presence of HOBt and hexadecylamine (NH
2
C
16
H
33
).
Following purification by silica gel column chromatography, the Boc group is removed
with TFA and purified by silica gel column chromatography to afford the TFA salt (4.5).
In order to conjugate the (L)-Boc-Valine, the (L)-Ser-NHC
16
H
33
molecule (4.5) was
dissolved in MeOH and converted to the HCl salt using a 0.2 M HCl/MeOH solution.
Once 4.5 was in its HCl salt form 4.6, it was conjugated with (L)-Boc-Valine using the
EDC coupling procedure used to make 4.4 in Scheme 4.1. However, 4.6 was not readily
soluble in CH
2
Cl
2
, so triethylamine (TEA) had to be incorporated in order to successfully
afford compound 4.7 in 87% yield.
Scheme 4.1 Synthesis of the lipophilic N-alkyl (L)-Ser-C
16
-(L)-Val dipeptide promoiety.
a. EDC, HOBt, CH
2
Cl
2
, NH
2
C
16
H
33
, 0 °C, overnight; b. TFA, CH
2
Cl
2
, rt, overnight; c.
0.2 M HCl/MeOH, -20 °C (X 3); d. EDC, HOBt, CH
2
Cl
2
, TEA, (L)-Boc-valine
a
b
c
HO
O
NH
3
NHC
16
H
33
Cl
HO
O
NH
3
NHC
16
H
33
O
O
CF
3
d
HO
O
NH
OH
Boc
4.3
HO
O
NH
NHC
16
H
33
4.4
Boc
4.5
HO
O
HN
NHC
16
H
33
O
NH
4.7
Boc
4.6
106
4.7 Synthesis of cyclic long chain N-alkyl Ser-Val dipeptide prodrug of (S)-HPMPC
Conjugation of 4.1 to the (L)-Ser-NHC
16
H
33
-(L)-Boc-Val dipeptide promoiety (4.7) was
performed according to the previous one-pot PyBOP coupling procedure used to make
cyclic (S)-HPMPC and (S)-HPMPA peptidomimetic prodrugs in the McKenna lab.
20-23
4.1 is dissolved in DMF and DIEA due to the formation of the DIEA-salt of 4.1. Upon
addition of 4.7 and PyBOP, the reaction was stirred at 40 °C for 4 h. The reaction was
monitored by
31
P NMR and additional amounts of PyBOP were added accordingly.
Disappearance of the cyclic cidofovir (3 ppm) and the presence of the two peaks
belonging to the diastereoisomers of the Boc-protected 4.8 (Scheme 4.2) indicated the
reaction was complete. After removing solvent and extracting the residue with diethyl
ether, the remaining tripyrrolidino PyBOP by-product is separated from the desired
prodrug via silica gel column chromatography. Boc-deprotection was performed using
TFA, followed by column chromatography and recrystallization from diethyl ether to
yield 4.9.
Scheme 4.2 Synthesis of cyclic dipeptide prodrug of cidofovir . a. DIEA, DMF, 4.7,
PyBOP, 40 °C, overnight; b. TFA, CH
2
Cl
2
, rt, overnight.
4.9 4.8
b
N
N
NH
2
O
O
OH
P
O
OH
OH
(S)
4.1
a
N
N
NH
2
O
O
P
O
O
O C
16
H
33
HN
H
N
O
N
H
(S)
O
Boc
N
N
NH
2
O
O
P
O
O
O C
16
H
33
HN
H
N
O
NH
2
(S)
O
107
4.8 Antiviral activity against DNA viruses
Figure 4.3 (L)-Ser-NHC
16
H
33
-(L)-Val-cHPMPC
Compound 4.9 was screened against various DNA viruses HSV-2, VZV, CMV, VACV,
CPXV and ADV in vitro via plaque reduction assay (PRA) using previously described
procedures.
27,28
The data is shown in Table 4.1 and Table 4.2 below. Also submitted for
in vitro analysis against the same viruses for comparison were the parent drug (HPMPC,
4.1) and cyclic parent drug (cHPMPC, 4.2), which are listed in Table 4.1 and Table 4.2.
Table 4.1 in vitro antiviral activities against HSV-2, VZV, CMV, cytotoxicities and selectivity index
values of 4.1, 4.2 and 4.9.
a
HSV-2 VZV CMV
Compound
EC
50
(µM)
CC
50
(µM)
SI
EC
50
(µM)
CC
50
(µM)
SI
EC
50
(µM)
CC
50
(µM)
SI
4.1 26.8 60 2 60 60 1 0.4 60 150
4.2 5.5 60 11 20 68.9 3 1.7 139 82
4.9 0.13 5.68 44 0.02 1.43 72 0.02 1.42 71
a
Data obtained by Prof. Mark N. Prichard et al. at the University of Alabama, Birmingham
(L)-Ser-(NH-C
16
H
33
)-(L)-Val-cHPMPC
4.9
N
N
NH
2
O
O
P
O
O
O C
16
H
33
HN
H
N
O
NH
2
(S)
O
108
Aside from the low micromolar values observed across the board, another interesting
point about this data is the decent selectivity values for all viruses in comparison to the
parent and cyclic parent drug (which demonstrate fairly weak selectivity values, except
for the value observed for compound 4.1 against CMV; Table 4.1).
Table 4.2 In vitro antiviral activities against VACV, CXPV, ADV, cytotoxicities and selectivity index
values of 4.1, 4.2 and 4.9.
a
VACV CPXV ADV
Compound
EC
50
(µM)
CC
50
(µM)
SI
EC
50
(µM)
CC
50
(µM)
SI
EC
50
(µM)
CC
50
(µM)
SI
4.1 18.6 60 3 23.8 60 3 5.5 60 11
4.2 19.1 60 3 44.5 60 1 - - -
4.9 0.3 12.3 41 0.95 17.83 19 0.06 2.1 35
a
Data obtained by Prof. Mark N. Prichard et al. at the University of Alabama, Birmingham
In comparison with the parent drug (4.1), cyclic HPMPC (4.2), and the Ser-C
16
-Val
cyclic prodrug of HPMPC (4.9) demonstrates enhanced antiviral activity and superior
selectivity indices in regard to all viruses on the table. Discrepancies of the selectivity
values obtained for prodrug 4.9 and the parent drug 4.1 (or the cyclic prodrug 4.2) may
be attributed to an instrumentation reading error. Based on the preliminary in vitro
findings for 4.9, which is a cHPMPC prodrug, it would be of great interest to synthesize
and evaluate more lipophilic serine-based prodrugs in the future.
4.9 Kaposi sarcoma-associated herpesvirus
Kaposi-Sarcoma associated Herpesvirus (KSHV) is the etiological agent of Kaposi-
Sarcoma (KS), an endothelial cell derived vascular malignancy.
29-31
KSHV, also known
as human herpesvirus-8 (HHV-8) and is one of seven currently known oncoviruses, is
109
also linked to at least two rare B-cell lymphoproliferative disorders: primary effusion
lymphoma (PEL) and multicentric Castleman’s disease (MCD).
29,32,33
As is the case for
other herpesviruses, KSHV is comprised of latent and lytic replication cycles.
34
The
pathologic process of KSHV to KS tumor development is expedited by a debilitated
immune system and is therefore a common neoplasm found in HIV and atrogenic
patients.
35
In the course of latent infection, the viral genome of KSHV is characterized as
a double stranded DNA encapsulated in an episome in the nucleus with only a few viral
latent genes being expressed in the latency locus and no virion production. It is due to the
latent state of KSHV that allows elusive detection by the immune system and establishes
it as a lifelong persistent infection. Studies have indicated most KS tumors are infected
with latent KSHV, indicating the strong implications in the development of KS
tumorgenesis.
36
While there are currently no FDA approved antiviral treatments for
KSHV, antiherpetic agents that target the viral DNA polymerase such as, acyclovir,
ganciclovir, foscarnet and (S)-HPMPC, have been shown to demonstrate in vitro antiviral
activity against KSHV and another gammaherpesvirus, Epstein-Barr virus (EBV).
37-41
4.10 KSHV antiviral activity studies
Extensive research has been dedicated toward the development of both cell and animal
models to study KSHV.
42-47
In relation to the preliminary KSHV antiviral activity
analysis presented here, a recent study by Coen et al.,
48
illustrated the antiviral in vitro
potencies of various acyclic nucleoside phosphonates against gammaherpesviruses
(including KSHV and EBV) through lytic cycle reactivation by phorbol esters of latently
110
infected B cells. In a different approach to fully characterize the KSHV gene expression
in an in vitro model, herpesvirus mutagenesis has resulted in utilizing recombinant virus
strategies consisting of bacterial artificial chromosome (BAC) and recombineering
technologies.
49
The Jae U. Jung research lab at USC has recently developed a full length
KSHV viral genome clone called BAC16 that is stable in E. Coli and can produce
infectious viruses when restored in mammalian cells.
50,51
Initial KSHV viral replication
inhibition screening of (L)-Ser-NHC
16
H
33
-(L)-Val-cHPMPC (4.9) and the nucleotide
analogues shown in Figure 4.3 was carried out according to previously reported
methods
50,51
elaborated and verified in the Jung lab (Figure 4.4A). Included in the list of
analogues tested in this model were the following: DAPy compounds from Chapter 3 of
this dissertation ((R)-HPMPO-DAPy (3.1) PMEO-DAPy (3.2), (L)-Tyr-NHC
16
H
33
-
PMEO-DAPy (MMW-3-101, 3.21), and (L)-Tyr-NHC
16
H
33
-HPMPO-DAPy (MMW-3-
179, 3.31)), cyclic and acyclic (L)-Tyr-NHC
18
H
37
prodrugs of (S)-HPMPA synthesized
by Dr. Ivan S. Krylov (IK-2-110 and IK-2-119), and (S)-HPMPA (5.1).
In vitro ANP prodrug inhibition of KSHV infection was monitored in iSLKBAC16
infected cells, which are obtained by introducing recombineered KSHV BAC clone
(BAC16) to an engineered endothelial cell line with a doxycycline-inducible RTA gene
(iSLK). The iSLKBAC16 model contains a green fluorescent protein (GFP) tag, which
allows for monitoring of the inhibition of KSHV viral replication of the prodrugs and
their parent drugs (Figure 4.3) by measuring the percentage of GFP-positive cells using a
fluorescence activated cell sorter (FACS). A sample with an infected (no drug treatment)
111
iSLKBAC16 was used as the standard for this investigation. Plot B in Figure 4.4
demonstrates the amount of infected GFP-positive cells present after treatment with the
corresponding drugs listed in the bottom columns. A concentration-dependent study was
performed for each of the eight drugs listed in Plot B (10 µM, 2.5 µM, 0.6 µM and 0.15
µM) with each column indicating the percent GFP-positive cells counted for the different
concentrations for each drug. Upon analyzing Plot B, the first three columns, which
belong to parent drugs (no promoiety), indicate significantly higher levels of infected
cells (lower inhibition) than their corresponding prodrugs in the last five columns (higher
inhibition of viral replication). In particular, prodrug 4.9, IK-2-110 and
Figure 4.4 Nucleotide analogues screened against KSHV replication
HO
O
P
O
OH HO
N
N
N
N
NH
2
(S)-HPMPA
5.1
(S)
MMW-3-79
(R)-HPMPO-DAPy
3.1
MMW-3-237
PMEO-DAPy
3.2
MMW-3-101
(L)-Tyr-NHC
16
H
33
-PMEO-DAPy
3.21
N
N
NH
2
H
2
N O
O P
O
OH
OH
OH
(R)
N
N
NH
2
H
2
N O
O P
O
OH
OH
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
NHC
16
H
33
N
O
HO
P
O
H
2
N
O
NHC
18
H
37
O
N
N
N
NH
2
HO
(L)-Tyr-NHC
18
H
37
-HPMPA
IK-2-119
(S)
N
O
O
P
O
H
2
N
O
NHC
18
H
37
O
N
N
N
NH
2
(L)-Tyr-NHC
18
H
37
-cHPMPA
IK-2-110
(S)
MMW-3-179
(L)-Tyr-NHC
16
H
33
-HPMPO-DAPy
3.31
N
N O
P
O
O
O
H
2
N
O
NHC
16
H
33
NH
2
H
2
N
O
N
N
NH
2
O
O
O
P
O
O
O C
16
H
33
HN
H
N
O
NH
2
(S)
MMW-3-231
(L)-Ser-NHC
16
H
33
-(L)-Val-cHPMPC
4.9
112
IK-2-119 demonstrated the lowest number of GFP-positive cells, which is indicative of
higher inhibition of KSHV infected cells across the four concentrations when compared
to the other prodrugs and parent drugs for this round. From this round of studies, the
HPMPA parent drug and corresponding cyclic and acyclic prodrugs (IK-2-110 and IK-2-
119) were further assessed in the same KSHV in vitro model at lower concentrations
(Plot C, Figure 4.4). When screened at 150 nM (0.15µM), 38 nM, 9 nM and 2.3 nM, the
same trend in inhibition of viral replication was observed with the lowest concentration
giving the highest number of GFP-positive cells. Based on this preliminary KSHV data,
the HPMP-prodrugs (amino acid and dipeptide) afford the best viral replication
inhibition. Further assays (westernblot, qPCR, RT-PCR, DNA and RNA preparation) will
Figure 4.5 A. KSHV infection and dosing cycle. B and C. Results from screening
nucleotide analogues that block KSHV replication.
113
have to be administered to HPMP prodrugs from this preliminary analysis to gain a better
understanding of the mechanism of inhibition of KSHV viral replication by lipophilic
prodrugs of (S)-HPMPA and (S)-HPMPC.
4.11 Conclusion
A novel long chain N-alkyl amide dipeptide prodrug of cyclic (S)-HPMPC (4.9) was
synthesized and its antiviral activity against various DNA viruses was assessed. Synthesis
of the (L)-Ser-NHC
16
H
33
-(L)-Val promoiety consisted of utilizing standard peptide
coupling and deprotection procedures, as well as, the amidation procedure utilized
extensively in our lab. The long chain N-alkyl dipeptide cHPMPC prodrug was
synthesized according to previously reported procedures and evaluated against HSV-2,
VZV, CMV, VACV, CPXV, and ADV in cell culture. In addition, 4.9, along with a
variety of other ANP prodrugs synthesized in our lab, was also assessed for antiviral
activity against KSHV in cell culture. Compound 4.9 demonstrated excellent in vitro
activity against all DNA viruses in this work and is a candidate for further development.
4.12 Experimental Section
General Experimental Procedures.
1
H and
31
P NMR spectra were obtained on a 500
MHz spectrometer. 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).
31
P
NMR spectra were proton-decoupled, and
1
H NMR coupling constants (J values) were
given in Hz. NMR abbreviations used include s (singlet), d (doublet), m (unresolved
114
multiplet), dd (doublet of doublet), ddd (doublet of doublet of doublet), bs (broad signal).
4.9 was obtained as diastereomeric mixtures. The ratio of diastereomers was determined
by
31
P NMR. The UV spectra were recorded using Beckman Coulter DU 800
spectrophotometer. MS analysis was performed on a Thermo-Finnigan LCQ DECA
Xp
max
Ion Trap LC/MS/MS eqipped with an ESI Probe. Xcalibur software wa used to
process the MS spectra. IUPAC names for compounds 4.9 were obtained from
MarvinSketch version 14.9.29.0. The >95% content of active drug of the final
compounds 4.9 were confirmed using UV determination of the active compound content
used the following extinction coefficients: (S)-HPMPC derivatives (ε = 8362 at 274 nm
in EtOH; ε = 9000 at 274 nm at pH 7.0).
Amidation of (L)-Boc-serine. General Procedure.
21,26
(L)-Boc-Ser-NHC
16
H
33
(4.7) was
synthesized using the EDC coupling procedure previously described in Chapter 2.9.
26
4.3
(4.6 mmol, 1.30 g) was stirred in CH
2
Cl
2
(20 mL) at 0 °C for 10 min prior to the addition
of HOBt hydrate (6.0 mmol, 0.81 g). After stirring this suspension for 15 min at 0 °C,
hexadecylamine (5.1 mmol, 1.90 g) was added, followed by EDC•Cl (6.0 mmol, 1.15 g).
The reaction mixture was removed from the ice bath and allowed to stir at rt for 72 h.
Reaction monitored by TLC (0.5:0.5:20, MeOH:NH
4
OH:CH
2
Cl
2
, followed by ninhydrin
test). An additional 30 mL of CH
2
Cl
2
is added and the organic layer was washed
consecutively with saturated citric acid (25 mL), saturated NaHCO
3
(25 mL), and
saturated NaCl (25 mL). The organic phase was dried over Na
2
SO
4
and concentrated
under reduced pressure. Product was purified by silica gel column chromatography
115
(eluted 2:100 MeOH:CH
2
Cl
2
). Fractions were combined and solvent removed. 4.4 was
dried under reduced pressure. 4.4 (7.6 mmol, 3.255 g) was dissolved in CH
2
Cl
2
(30 mL)
and TFA added (1.52 mol, 11.63 mL) to remove Boc protection group. Reaction mixture
was allowed to stir at rt overnight. Solvent removed and 4.5 was converted to the HCl salt
(4.6) in order to facilitate the coupling with (L)-valine in the next step. The solid was
washed with 0.2 M HCl/MeOH at -20 °C according to a previously reported procedure.
21
4.6 was dried and dissolved in CH
2
Cl
2
(120 mL) with (L)-Boc-valine (9.9 mmol, 2.15 g).
This mixture was stirred at 0 °C for 20 min prior to the addition of HOBt (14.8 mmol,
2.00 g) and TEA (49.4 mmol, 6.9 mL) which was stirred at 0 °C for an additional 15 min.
After addition of EDC•Cl (12.4 mmol, 2.374 g), the reaction mixture was allowed to
reach rt and stirred for 2 d. Workup of product resembled that used for 2.14 and 2.15 in
Chapter 2.
26
After purification via column chromatography (2% MeOH/CH
2
Cl
2
),
fractions combined, solvent removed under reduced pressure and product co-distilled
with DMF to prepare the synthon for coupling reaction with 4.1.
tert-Butyl N-(1-([(1S)-1-(hexadecylcarbamoyl)-2-hydroxyethyl]carbamoyl)-2-
methylpropyl)carbamate (4.7) Yield 87% (determined by weight).
1
H NMR (500 MHz,
CD
3
OD): δ 7.06-7.04 (m, 2H, aromatic), 6.72-6.71 (m, 2H, aromatic), 4.19-4.16 (t, J =
6.4 Hz,1H), 3.20-3.15 (m, 1H), 3.12-3.06 (m, 1H), 2.96-2.92 (m, 1H), 2.78-2.74 (m, 1H),
1.41 (s, 9H), 1.31 (s, 22 H), 0.93-0.91 (t, J = 6.5 Hz, 3H). LRMS (MS-ESI): m/z [M +
H]
+
calcd for C
29
H
57
N
3
O
5
: 528.4, found: 528.9 (M + H)
+
.
116
Synthesis of cyclic (S)-HPMPC dipeptide prodrug (4.9).
General Procedure.
20-23
In dry DMF (35 mL), 4.1 (0.73 mmol, 203.6 mg), DIEA (7.3
mmol, 1.29 mL), PyBOP (1.6 mmol, 835 mg) and 4.7 (1.0 mmol, 539 mg) were added
sequentially. Reaction mixture was stirred at 40 °C for 3 h and monitored by
31
P NMR.
Solvent removed under reduced pressure and residue extracted with diethyl ether (150
mL). Reaction residue was dissolved in CH
2
Cl
2
:(CH
3
)
2
CO (60:30) and purified with
silica gel chromatography (column packed in CH
2
Cl
2
; started with CH
2
Cl
2
:(CH
3
)
2
CO;
60:30 followed addition of 1.5% increments of MeOH; product eluted 5-8% MeOH.
Product eluted at ~7-9% MeOH/CH
2
Cl
2
:(CH
3
)
2
CO). Fractions containing product were
combined, solvent removed and dried under reduced pressure for 1 h. To this flask,
CH
2
Cl
2
(10 mL) and TFA (0.139 mL) were added and stirred at rt overnight. Reaction
was monitored by TLC (20% MeOH/CH
2
Cl
2
). Solvent was removed under reduced
pressure and residue dissolved in 0.75% TFA/ CH
2
Cl
2
for purification by silica gel
chromatography (1%-18% MeOH/CH
2
Cl
2
, increased increments of 2%). Fractions
containing UV active compound were collected, solvent removed and dried for 3 h to
give 39 mg of 5.2.
(2S)-2-Amino-N-[(1S)-2-({5-[(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)methyl]-2-
oxo-1,4,2λ⁵-dioxaphosphinan-2-yl}oxy)-1-(hexadecylcarbamoyl)ethyl]-3-
methylbutanamide (4.9). Yield 8% (determined by UV). Obtained as a TFA salt;
mixture of diastereomers.
1
H NMR (500 MHz, CD
3
OD) δ 7.73-7.65 (dd, J = 8.023 Hz, J
= 7.178 Hz, 1H), 5.98-5.94 (m, 1H), 5.51 (s, 1H), 4.76-4.68 (dt, J = 4.383 Hz, J = 5.341
117
Hz, 1H), 4.55-4.4.43 (m, 2H), 4.38-4.25 (m, 4H), 4.17-4.11 (m, 1H), 4.10-4.00 (m, 2H),
3.81-3.76 (m, 2H), 3.29-3.17 (m, 2H), 2.28-2.19 (m, 1H), 1.56-1.53 (m, 2H), 1.31 (s,
28H), 1.11-1.05 (m, 6H), 0.93-0.90 (t, J = 6.163 Hz, 3H).
31
P NMR (202 MHz, CD
3
OD)
δ 14.01, 12.96 (42:58). LRMS (MS-ESI): m/z [M + H]
+
calcd for C
32
H
59
N
6
O
7
P: 671.4,
found: 671.5 (M + H)
+
.
Antiviral activity and cytotoxicity studies for (S)-HPMPC, cHPMPC and (L)-Ser-
NHC
16
H
33
-(L)-Val-cHPMPC (4.1, 4.2, 4.9).
Antiviral and cytotoxic assays. All antiviral and cytotoxic assays were conducted by
Prof. Mark N. Prichard at the University of Alabama, Birmingham as part of a
collaboration.
27,28
KSHV Antiviral activity studies for (L)-Ser-NHC
16
H
33
-(L)-Val-cHPMPC (4.9), N-
alkyl tyrosine-based 2,4-diaminopyrmidine prodrugs (Chapter 3) and N-alkyl
tyrosine-based prodrugs of (S)-HPMPA.
Antiviral assays. The in vitro cell culture method used to analyze the inhibition of
KSHV viral replication was conducted by Dr. Zsolt Toth and myself at the University of
Southern California, Keck School of Medicine under the direction of Prof. Jae U.
Jung.
50,51
118
4.13 Chapter 4 references
1. De Clercq, E.; Sakuma, T.; Baba, M.; Pauwels, R.; Balzarini, J.; Rosenberg, I.;
Holy, A., Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and
pyrimidines. Antiviral Res. 1987, 8, 261-272.
2. De Clercq, E. Clinical potential of the acyclic nucleoside phosphonates cidofovir,
adefovir, and tenofovir in treatment of DNA virus and retrovirus infections. Clin
Microbiol Rev. 2003, 16, 569-596.
3. Snoeck, R.; Sakuma, T.; De Clercq, E.; Rosenberg, I.; Holy, A. (S)-1-(3-hydroxy-
2-phosphonomethoxypropyl)cytosine, a potent and selective inhibitor of human
cytomegalovirus replication. Antimicrob Agents Chemother. 1988, 32, 1839-1844.
4. Cihlar, T.; Chen, M. S. Identification of enzymes catalyzing two-step
phosphorylation of cidofovir and the effect of cytomegalovirus infection on their
activities in host cells. Mol Pharmacol. 1996, 50, 1502-1510.
5. De Clercq, E. Cidofovir in the therapy and short-term prophylaxis of poxvirus
infections. Trends Pharmacol Sci. 2002, 23, 456-458.
6. Mendel, D. B.; Cihlar, T.; Moon, K.; Chen, M. S. Conversion of 1-[((S)-2-
hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosine to cidofovir by an
intracellular cyclic CMP phosphodiesterase. Antimicrob Agents Chemother. 1997,
41, 641-646.
7. Naesens, L.; Snoeck, R.; Andrei, G.; Balzarini, J.; Neyts, J.; De Clercq, E.
HPMPC (cidofovir), PMEA (adefovir) and related acyclic nucleoside
phosphonate analogues: A review of their pharmacology and clinical potential in
the treatment of viral infections. Antiviral Chem Chemother. 1997, 8, 1-23.
8. Kramata, P.; Votruba, I.; Otova, B.; Holy, A. Different inhibitory potencies of
acyclic phosphonomethoxyalkyl nucleotide analogs toward DNA polymerases
alpha, delta, and epsilon. Mol Pharmacol. 1996, 49, 1005-1011.
9. Cundy, K. C. Clinical Pharmacokinetics of the antiviral nucleotide analogs
cidofovir and adefovir. Clin Pharmacokinet. 1999, 36, 127-143.
10. Holy, A. Phosphonomethoxyalkyl analogs of nucleotides. Curr Pharm Des. 2003,
9, 2567-2592.
11. Wachsman, M.; Petty, B. G.; Cundy, K. C.; Jaffe, H. S.; Fisher, P. E.; Pastelak,
A.; Lietman, P. S. Pharmacokinetics, safety and bioavailability of HPMPC
119
(cidofovir) in human immunodeficiency virus-infected subjects. Antiviral Res.
1996, 29, 153-161.
12. Aldern, K. A.; Ciesla, S. L.; Winegarden, K. L.; Hostetler, K. Y. Increased
antiviral activity of 1-O-hexadexyloxypropyl-[2-14C]cidofovir in MRC-5 human
lung fibroblasts is explained by unique cellular uptake and metabolism. Mol
Pharm. 2003, 63, 678-681.
13. Magee, W. C.; Aldern, K. A.; Hostetler, K. Y.; Evans, D. H. Cidofovir and (S)-9-
[3-hydroxy-(2-phosphonomethoxy)propyl]adenine are highly effective inhibitors
of vaccinia virus DNA polymerase when incorporated into the template strand.
Antimicrob Agents Chemother. 2008, 52, 586-597.
14. Hostetler, K. Y. Alkoxyalkyl prodrugs of acyclic nucleoside phosphonates
enhance oral antiviral activity and reduce toxicity: current state of the art.
Antiviral Res. 2009, 82, A84-A98.
15. Ciesla, S. L.; Trahan, J.; Wan, W. B.; Beadle, J. R.; Aldern, K. A.; Painter, G. R.;
Hostetler, K. Y. Esterification of cidofovir and alkylalkanols increases oral
bioavailability and diminishes drug accumulation in kidney. Antiviral Res. 2003,
59, 163-171.
16. Oliyai, R.; Arimilli, M. N.; Jones, R. J.; Lee, W. A. Pharmacokinetics of salicylate
ester prodrugs of cyclic HPMPC in dogs. Nucleosides Nucleotides Nucleic Acids.
2001, 20, 1411-1414.
17. Oliyai, R.; Shaw, J. P.; Sueoka-Lenne, C. M.; Cundy, K. C.; Arimilli, M. N.;
Jones, R. J.; Lee, W. A. Aryl ester prodrugs of cyclic HPMPC. I: Physicochemical
characterization and in vitro biological stability. Pharm Res. 1999, 16, 1687-1693.
18. Tichý, T.; Andrei, G.; Dracinsky, M,; Holy, A.; Balzarini, J.; Snoeck, R.;
Krečmerová, M. New prodrugs of Adefovir and Cidofovir. Bioorg Med Chem.
2011, 19, 3527-3539.
19. 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.
20. 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 Pharm. 2008, 5, 598.
120
21. 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.
22. 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 Pharmacol.
2010, 7, 2349-2361.
23. 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.
24. Krylov, I. S.; Kashemirov, B. A.; Hilfinger, J. M.; McKenna, C. E. Evolution of
amino acid based prodrug approach: stay tuned. Mol Pharmaceutics. 2013, 10,
445-458.
25. Krylov, I. S. Synthesis, structural analysis and in vitro antiviral activities of novel
cyclic and acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs. Ph.D.
Thesis, University of Southern California, Los Angeles, 2012.
26. 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.
27. Schormann, N.; Sommers, C. I.; Prichard, M. N.; Keith, K. A.; Noah, J. W.; Nuth,
M.; Ricciardi, R. P.; Chattopadhyay, D. Identification of Protein-Protein
Interaction Inhibitors Targeting Vaccinia Virus Processivity Factor for
Development of Antiviral Agents. Antimicrob Agents Chemother. 2011, 55, 5054-
5062.
28. Prichard, M. N.; Williams, J. D.; Komazin-Meredith, G.; Khan, A. R.; Price, N.
B.; Jefferson, G. M.; Harden, E. A.; Hartline, C. B.; Peet, N. P.; Bowlin, T. L.
Synthesis and Antiviral Activities of Methylenecyclopropane Analogs with 6-
alkoxy and 6-alkylthio Substitutions that Exhibit Broad-Spectrum Antiviral
Activity Against Human Herpesviruses. Antimicrob Agents Chemother. 2013, 57,
3518-3527.
29. Boshoff, C.; Schultz, T. F.; Kennedy, M. M.; Graham, A. K.; Fisher, C.; Thomas,
A.; McGee, J. O.; Weiss, R. A.; O’Leary, J. J. Kaposi’s sarcoma-associated
herpesvirus infects endothelial and spindle cells. Nat Med. 1995, 1, 1274-1278.
121
30. Ganem, D. KSHV and the pathogenesis of Kaposi Sarcoma: listening to human
biology and medicine. J Clin Invest. 2010, 120, 939-949.
31. Mesri, E. A.; Cesarman, E.; Boshoff, C. Kaposi’s sarcoma and its associated
herpesvirus. Nat Rev Cancer. 2010, 10, 707-719.
32. Du, M. Q.; Bacon, C. M.; Isaacson, P. G. Kaposi sarcoma-associated
herpesvirus/human herpesvirus 8 and lymphoproliferative disorders. J Clin
Pathol. 2007, 60, 1350-1357.
33. Wen. K. W.; Damania, B. Kaposi sarcoma-associated herpesvirus (KSHV):
molecular biology and ontogenesis. Cancer Lett. 2010, 289, 140-150.
34. Boshoff, C.; Chang, Y. Kaposi’s sarcoma-associated herpesvirus: a new DNA
tumor virus. Annu Rev Med. 2001, 52, 453–470.
35. Dittmer, D. P; Damania, B. KSHV-Associated Disease in AIDS Patient. In Aids-
Associated Viral Oncogenesis. C. Meyers., Eds.; Cancer Treatment and Research.
2007, 133, 129-139.
36. Sturzl, M.; Blasig, C.; Schreier, A.; Neipel, F.; Hohenadl, C.; Cornali, E.; Ascherl,
G.; Esser, S.; Brockmeyer. N. H.; Ekman, M.; Kaaya, E. E.; Tschachler, E.;
Biberfeld, P. Expression of HHV-8 latency–associated T0.7 RNA in spindle cells
and endothelial cells of AIDS-associated classical and African Kaposi’s sarcoma.
Int J Cancer. 1997, 72, 68-71.
37. Friedrichs, C.; Neyts, J.; Gaspar, G.; De Clercq, E.; Wutzler, P. Evaluation of
antiviral activity against human herpesvirus 8 (HHV-8) and Epstein-Barr virus
(EBV) by a quantitative real-time PCR assay. Antiviral Res. 2004, 62, 121–123.
38. Lin, J. C.; De Clercq, E.; Pagano, J. S. Novel acyclic adenosine analogs inhibit
Epstein-Barr virus replication. Antimicrob Agents Chemother. 1987, 31, 1431–
1433.
39. Lin, J. C.; De Clercq, E.; Pagano, J. S. Inhibitory effects of acyclic nucleoside
phosphonate analogs, including (S)-1-(3-hydroxy-2-
phosphonylmethoxypropyl)cytosine, on Epstein-Barr virus replication.
Antimicrob Agents Chemother. 1991, 35, 2440 –2443.
40. Meerbach, A.; Holy, A.; Wutzler, P.; De Clercq, E.; Neyts, J. Inhibitory effects of
novel nucleoside and nucleotide analogues on Epstein-Barr virus replication.
Antivir Chem Chemother. 1998, 9, 275–282.
122
41. Neyts, J.; De Clercq, E. Antiviral drug susceptibility of human herpesvirus 8.
Antimicrob Agents Chemother. 1997, 41, 2754 –2756.
42. Chang H.; Wachtman, L. M; Pearson, C. B.; Lee, J-S.; Lee, H-R.; Lee, S. H.;
Vieira, J.; Mansfield, K.G.; Jung, J. U. Non-human primate model of Kaposi’s
sarcoma- associated herpesvirus infection. PLoS Pathog. 2009, 5(10), e1000606,
1-10.
43. Jones T.; Ye, F.; Bedolla, R.; Huang, Y.; Meng, J.; Qian, L.; Pan, H.; Zhou, F.;
Moody, R.; Wagner, B.; Arar, M.; Gao, S. J. Direct and efficient cellular
transformation of primary rat mesenchymal precursor cells by KSHV. J Clin
Invest. 2012, 122, 1076 -1081.
44. Matthews, N. C.; Goodier, M. R.; Robey, R. C.; Bower, M.; Gotch, F. M. Killing
of Kaposi’s sarcoma-associated herpesvirus-infected fibroblasts during latent
infection by activated natural killer cells. Eur J Immunol. 2001, 41, 1958 –1968.
45. Mutlu, A. D.; Cavallin, L. E.; Vincent, L.; Chiozzini, C.; Eroles, P.; Duran, E. M.;
Asgari, Z.; Hooper, A. T.; La Perle, K. M. D.; Hilsher, C.; Gao, S-J.; Dittmer, D.
P.; Rafii, S.; Mesri, E. A. In vivo-restricted and reversible malignancy induced by
human herpesvirus-8 KSHV: a cell and animal model of virally induced Kaposi’s
sarcoma. Cancer Cell. 2007, 11, 245–258.
46. Parsons, C. H.; Adang, L. A.; Overdevest, J.; O’Connor, C. M.; Taylor, J. R. Jr.;
Camerini, D.; Kedes, D. H. KSHV targets multiple leukocyte lineages during
long-term productive infection in NOD/SCID mice. J Clin Invest. 2006, 116,
1963–1973.
47. Wu, W.; Vieira, J.; Fiore, N.; Banerjee, P.; Sieburg, M.; Rochford, R.; Harrington,
W. Jr.; Feuer, G. KSHV/HHV-8 infection of human hematopoietic progenitor
(CD34ჼ) cells: persistence of infection during hematopoiesis in vitro and in vivo.
Blood 2006, 108, 141–151.
48. Coen, N.; Duraffour, S.; Naesens, L.; Krečmerová, M.; Van den Oord, J.; Snoeck,
R.; Andrei, G. Evaluation of Novel Acyclic Nucleoside Phosphonates against
Human and Animal Gammaherpesviruses Revealed an Altered Metabolism of
Cyclic Prodrugs upon Epstein-Barr Virus Reactivation in P3HR-1 Cells. J Virol.
2013, 87, 12422-12432.
49. Warden, C.; Tang, Q. Y.; Zhu, H. Herpesvirus BACs: past, present, and future. J
Biomed Biotechnol. 2011, 27, 124595.
50. Brulois, K. F.; Chang, H.; Le, A. S-Y.; Ensser, A.; Wong, L-Y.; Toth, Z.; Lee, S.
H.; Lee, H-R.; Myoung, J.; Ganem, D.; Oh, T-K.; Kim, J. F.; Gao, S-H.; Jung, J.
123
U. Construction and Manipulation of New Kaposi’s Sarcoma-Associated
Herpesvirus Bacterial Artificial Chromosome Clone. J Virol. 2012, 86, 9708-
9720.
51. Brulois, K.; Toth, Z.; Wong, L-Y.; Feng, P.; Gao, S-J; Ensser, A.; Jung, J. U.
Kaposi’s Sarcoma-Associated Herpesvirus K3 and K5 Ubiquitin E3 Ligases Have
Stage-Specific Immune Evasion Roles during Lytic Replication. J Virol. 2014, 88,
9335-9349.
124
CHAPTER 5
Scaled-up Procedure for Lipophilic N-Alkyl
Tyrosinamide Prodrugs of (S)-HPMPA and (S)-
HPMPC
5.1 Introduction: (S)-HPMPA and (S)-HPMPC
The first ANPs to prelude the advent of a novel class of nucleotide analogues as antiviral
therapeutics are (S)-HPMPA and (S)-HPMPC (5.1 and 4.1; Figure 5.1).
1,2
The antiviral
activities demonstrated by these two ANPs spans a wide variety of DNA viruses
1
which
has attributed to 5.1 being labeled as the prototype for ANPs and 4.1 being the first of the
ANPs to be licensed for clinical use.
Figure 5.1 ANPs of the HPMP Prototype: (S)-HPMPA and (S)-HPMPC
5.2 Mechanism of action and limitations
In order for 5.1 and 4.1 to exert their antiviral potential, they must first be taken up into
cells through a process called endocytosis. Once inside the virally infected cell, they must
HO
O
P
O
OH HO
N
N
N
N
NH
2
(S)-HPMPA
5.1
(S)
N
N
HO
O
P
O
OH HO
NH
2
O
(S)-HPMPC
Cidofovir, Vistide
®
4.1
(S)
125
be phosphorylated by cellular enzymes to their monophosphorylphosphonate derivatives
(HPMPAp and HPMPCp) followed by phosphorylation to their diphosphorylphosphonate
analogues (HPMPApp and HPMPCpp). After intracellular phosphorylation to their
triphosphate analogue form, they are recognized by viral DNA polymerase as natural
nucleotide analogues and incorporated in the viral DNA chain to inhibit viral replication
by terminating chain elongation.
3
While the presence of the nonhydrolyzable
phosphonate moiety in ANPs enhances the antiviral potency by increasing the half-life
and the presence of the anionic phosphonate structure at physiological pH limits cellular
permeability.
4
In addition, ANPs exhibit nephrotoxicity due to concentration in the
kidney proximal tubule.
5
In order exploit the therapeutic potential of ANPs and increase
their oral bioavailability, the prodrug approach of masking the phosphonic acid groups
has been developed and studied.
5.3 Prodrugs of (S)-HPMPA and (S)-HPMPC
As mentioned in Chapter 4, there are currently no FDA approved prodrugs of 5.1 and 4.1.
The extensive efforts into examining various prodrug approaches for ANPs have been
mentioned in Chapter 1.7 and Chapter 1.8 so they will not be discussed in length here.
More recently there has been mention of the Hostetler approach, which has been applied
to both 5.1 and 4.1 (Figure 5.2) to generate hexadecyloxypropyl (HDP) and
octadecyloxyethyl (ODE) monoester prodrugs to enhance antiviral activity
6,7
by
increasing cellular permeation
8,9
and simultaneously augment oral bioavailability.
10,11
Despite improving the hydrophobic character of these nucleotide analogues, some of their
126
drawbacks may include elevated cytotoxicity, poor enzymatic stability and decreased
aqueous solubility.
Figure 5.2 HDP and ODE monoester prodrugs of (S)-HPMPA and (S)-HPMPC
In our group, a prodrug approach has been developed that focuses on utilizing an amino
acid ester-based scaffold where the hydroxyl side chain of the amino acid is esterified to
the phosphonic acid group to mask its negative charge. The aims of this approach are
focused on enhancing cellular permeability, antiviral activity and oral bioavailability of
ANPs. The design and development of this amino acid based prodrug approach is
described in Chapter 1.8 and has been reviewed recently.
12
As a result of our recent
findings,
12,13
the lipophilic N-alkyl tyrosinamide prodrugs of (S)-HPMPC and (S)-
HPMPA (Figure 5.3) are candidates for further evaluation (toxicity, PK studies, in vivo
models) to determine their clinical application. As opposed to in vitro antiviral analysis,
which traditionally requires small amounts of compound for testing (10 mg - 20 mg),
preclinical analysis will require larger quantities (100 mg – 2 g) in order to run these
prodrugs through the necessary preclinical assessments. This necessity for providing
larger amount of compound has resulted in the need to analyze the potential of our
previously described small scale synthesis of these prodrugs in order to develop a large-
scale synthetic method for the prodrugs shown in Figure 5.3 below.
B
O
OH
P
O
O
OH
(S)
O(CH
2
)
15
CH
3
B = adenine; HDP-(S)-HPMPA
B = cytosine; HDP-(S)-HPMPC
B
O
OH
P
O
O
OH
(S)
O(CH
2
)
17
CH
3
B = adenine; ODE-(S)-HPMPA
B = cytosine; ODE-(S)-HPMPC
127
5.4 Large scale synthesis of cyclic and acyclic (S)-HPMPA and (S)-HPMPC
lipophilic N-alkyl tyrosinamide prodrugs
The scale-up procedure for the HPMP prodrugs depicted in Figure 5.3 were performed
for the purpose of re-synthesizing the prodrugs in larger amounts (from 100 mg – 200 mg
scale to 1 g – 2 g scale) for pharmacokinetic and in vivo studies. With this in mind, the
standard prodrug synthetic approach in the McKenna lab was used to investigate its
applicability in a large-scale synthesis.
Figure 5.3 Cyclic and acyclic prodrugs of (S)-HPMPC and (S)-HPMPA
N
O
O
P
O
H
2
N
O
NHC
16
H
33
(S)
O
N
NH
2
O N
O
O
P
O
H
2
N
O
NHC
16
H
33
O
N
N
N
NH
2
(L)-Tyr-NHC
16
H
33
-cHPMPA; 5.2 (L)-Tyr-NHC
16
H
33
-cHPMPC; 5.3
N
O
HO
P
O
H
2
N
O
NHC
16
H
33
O
N
N
N
NH
2
HO
(L)-Tyr-NHC
16
H
33
-HPMPA; 5.4
N
O
HO
P
O
H
2
N
O
NHC
16
H
33
O
N
NH
2
O
(L)-Tyr-NHC
16
H
33
- (S )HPMPC; 5.5
HO
(S)
(S)
(S)
128
5.4.1 Large-scale synthesis of (L)-Tyr-NHC
16
H
33
promoiety
The synthesis of the (L)-Tyr-NHC
16
H
33
synthon was performed using 5 g of the (L)-Boc-
tyrosine starting material. The synthesis was carried out according to a previously
reported procedure,
15
which consists of allowing the amidation reaction to stir at rt for 3
d, a basic and acidic extractions and quick purification by column chromatography
(elutes at 2% MeOH/CH
2
Cl
2
) to afford the Tyr-C
16
synthon in good yield (87%).
5.4.2 One-pot cyclization of parent drug and conjugation of promoiety
The procedure used for the synthesis of the cyclized prodrugs of 5.1 and 4.1 was
previously reported by Zakharova et al.
40
In the scale-up procedure, the amount of
starting material used ranged from 1.00 g to 2.00 g of 5.1 or 4.1. In the one-pot coupling
reaction, one equivalent of PyBOP is required to cyclize the parent drug and another
equivalent to couple the tyrosinamide promoiety. Despite only requiring two equivalents
of PyBOP for the reactions described above, extra PyBOP is usually required. Because
the starting material, cyclic parent drug, desired prodrug, coupling reagent and the
tripyrrolidino PyBOP by-product all contain a phosphorus atom, the reaction can be
monitored by
31
P NMR. Upon only adding 2 eq. of PyBOP, the cyclic parent drug peak
around 6 ppm can still be seen while unreacted PyBOP peak around 32 ppm is not
observed. The reaction usually requires anywhere from 3-5 eq. of PyBOP in order for the
reaction to go to completion. In order to efficiently remove the tripyrrolidino by-product
of PyBOP from the reaction mixture prior to purification by column chromatography, the
residue from the reaction is extracted with diethyl ether. The desired cyclic prodrugs 5.2
129
and 5.3 are not very soluble in diethyl ether while the TP by-product is readily soluble.
Column chromatography is used to separate the desired prodrugs from small by-products
detected in
31
P NMR and the large amount of HOBt that accumulates during the PyBOP
coupling reaction. Because the amount of PYBOP used in these coupling reactions is in
large excess (and therefore a large amount of the tripyrrolidino and HOBt by-products),
effectively removing these impurities is necessary before proceeding to the next step.
Once purified by column chromatography, the N-Boc protection group is removed in the
presence of TFA and CH
2
Cl
2
. Once the reaction has reached completion, column
chromatography purification is applied followed by recrystallization with diethyl ether to
afford 5.2 and 5.3 (Scheme 5.1). While column chromatography worked for this gram
scale (1 g to 2 g), another means of purification may have to be explored if the amount of
starting material were to increase. Overloading a column and not achieving good
separation becomes much easier as the amount of starting materials increase, with
PyBOP impurities being of the biggest concern.
Scheme 5.1 Synthesis of cyclic prodrugs of 5.1 and 4.1. a. DMF, DIEA, PyBOP, 40 °C,
overnight b. CH
2
Cl
2
, TFA, rt, overnight.
(L)-Boc-Tyr-NH-C
16
H
33
; 2.15
HO O NHC
16
H
33
N
H
Boc
+
B
O
OH
P
O
OH
OH
B = adenine; (S)-HPMPA; 5.1
B = cytosine; (S)-HPMPC; 4.1
a
B = adenine; (S)-HPMPA; 5.2
B = cytosine; (S)-HPMPC; 5.3
(S)
b
B
O
O
P
O
(S)
O
NHC
16
H
33
O
H
2
N
130
5.4.3. Hydrolysis of intramolecular P-O bond
Converting to the acyclic prodrug requires hydrolysis in a MeCN/NH
4
OH solution. In
this reaction, hydrolysis of the intramolecular phosphonate bond yields two products: the
desired acyclic prodrug (5.4 and 5.5; Scheme 5.2) and the cyclic parent drug of 5.1 or 4.1
(cHPMPA or cHPMPC) with the promoiety cleaved. This reaction is easily monitored via
31
P NMR because the diastereomeric mixture of cyclic prodrug, cyclic parent drug and
acyclic prodrug demonstrate distinct chemical shifts (10/8 ppm, 5 ppm and 14 ppm,
respectively). Despite playing with the concentration of ammonia used, both products are
observed (with the best ratio amounting to 60:40 acyclic prodrug: cyclic parent drug,
verified by
31
P NMR). Once the reaction is complete, separating the cyclic parent drug
from the desired acyclic prodrug (5.4 or 5.5) is crucial. Due to the solubility differences
between the two products, removal of the cyclic parent drug is achieved by dissolving the
reaction residue in H
2
O to precipitate 5.4 and 5.5. Proper filtration or centrifugation is
required to separate the precipitate from the H
2
O layer. Column chromatography is
performed in order to remove the remaining cyclic parent drug and other impurities. The
fractions are collected, solvent removed and recrystallized from diethyl ether to afford the
product as a solid in good purity. The low yields observed for both 5.4 and 5.5 can be
attributed to the hydrolysis reaction and difficulty in successfully separating the cyclic
parent drug from the acyclic prodrug. The best yields are attained when the cyclic parent
drug (cHPMPA or cHPMPC) is removed prior to column chromatography during the
H
2
O extraction process.
131
Scheme 5.2 Synthesis of acyclic prodrugs from 5.2 and 5.3. a. NH
4
OH, MeCN, 45 °C, 6
h.
5.5 Conclusion
A scaled-up synthetic procedure for lipophilic N-alkyl tyrosinamide cyclic and acyclic
prodrugs of (S)-HPMPC and (S)-HPMPA was performed according to synthetic
procedures previously developed in the McKenna lab and reported in the Ph.D.
dissertation of Dr. Ivan S. Krylov.
13
Being able to monitor the coupling and hydrolysis
reaction using
31
P NMR has proven to be advantageous in this synthesis. Performing the
coupling and hydrolysis reaction on a scale larger than 2 g may require further
investigation into using column chromatography and conditions for the hydrolysis
reaction to optimize yields and purity on a much larger scale.
5.6 Experimental section
General Experimental Procedures.
1
H and
31
P NMR spectra were obtained on 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).
31
P
B = adenine; (S)-HPMPA; 5.2
B = cytosine; (S)-HPMPC; 5.3
a
B = adenine; (S)-HPMPA; 5.4
B = cytosine; (S)-HPMPC; 5.5
B
O
O
P
O
O
H
2
N NHC
16
H
33
O
(S)
B
O
HO
P
O
(S)
O
HO
H
2
N
NHC
16
H
33
O
B
O
O
P
O
(S)
OH
+
cHPMPA
or
cHPMPC
132
NMR spectra were proton-decoupled, and
1
H NMR coupling constants (J values) were
given in Hz. NMR abbreviations used include s (singlet), d (doublet), m (unresolved
multiplet), dd (doublet of doublet), ddd (doublet of doublet of doublet), bs (broad signal).
Compounds 5.2-5.5 were obtained as diastereomeric mixtures. The ratio of diastereomers
was determined by
31
P NMR. The UV spectra were recorded using Beckman Coulter DU
800 spectrophotometer. MS analysis was performed on a Thermo-Finnigan LCQ DECA
Xp
max
Ion Trap LC/MS/MS eqipped with an ESI Probe. Xcalibur software wa used to
process the MS spectra. IUPAC names for compounds 5.2-5.5 were obtained from
MarvinSketch version 14.9.29.0. The >95% content of active drug of the final
compounds 5.2-5.5 were confirmed using UV determination of the active compound
content used the following extinction coefficients: (S)-HPMPA derivatives (ε = 14109 at
260 nm in EtOH, ε = 14019 at 260 nm at pH 7.0), (S)-HPMPC derivatives (ε = 8362 at
274 nm in EtOH; ε = 9000 at 274 nm at pH 7.0), and tyrosine (ε = 612 at 260 nm and ε =
1300 at 274 nm, pH 7.0).
Amidation of Boc-Protected (L)-Tyrosine. General Procedure. (L)-Boc-Tyr-
NHC
16
H
33
promoiety 2.15 was synthesized as previously described in Chapter 2.9.
15
Large Scale Synthesis of (L)-Tyr-NHC
16
H
33
-cHPMPA (5.2).
General Procedure.
In dry DMF (70 mL), 5.1 (3.3 mmol, 1.0g), DIEA (33 mmol, 4.0 mL), PyBOP (9.9
mmol, 5.19 g) and 2.15 (1.2 eq.) were added sequentially. Reaction mixture was stirred at
133
40 °C for 3 h and monitored by
31
P NMR. Solvent removed under reduced pressure and
residue extracted with diethyl ether (150 mL). Residue was dissolved in
CH
2
Cl
2
:(CH
3
)
2
CO (60:30) and purified with silica gel chromatography (column packed
in CH
2
Cl
2
; started with CH
2
Cl
2
:(CH
3
)
2
CO (60:30) followed by 1.5% increments of
MeOH added; product eluted 5-8% MeOH). Fractions containing product were
combined, solvent removed under reduced pressure and dried for 1 h. To this flask,
CH
2
Cl
2
(30 mL) and TFA (4.5 mL) were added and stirred at rt overnight. Reaction
monitored by thin layer chromatography. Solvent was removed under reduced pressure
and residue dissolved in 0.5% TFA/CH
2
Cl
2
for purification by silica gel chromatography
(0%-12% MeOH/CH
2
Cl
2
, increased increments of 2.5%; 0.5% TFA kept constant in
mobile phase). Fractions containing UV active compound were collected, solvent
removed under reduced pressure and dried for 4 h to give 1.6 g of 5.2.
(2S)-2-Amino-3-(4-([(5S)-5-[(6-amino-9H-purin-9-yl)methyl]-2-oxo-1,4,2λ⁵-
dioxaphosphinan-2-yl]oxy)phenyl)-N-hexadecylpropanamide (5.2). Yield 72.4%
(determined by UV). Obtained as a TFA salt; mixture of diastereomers.
1
H NMR (500
MHz, CD
3
OD) δ 8.34 (s, 1H), 8.29 (s, 1H), 7.30-7.29 (d, J = 8.736 Hz, 2H), 7.21-7.20
(d, J = 8.212 Hz, 2H), 4.78-4.72 (m, 1H), 4.66-4.63 (m, 1H), 4.56-4.36 (m, 5H), 4.21-
4.17 (dd, J = 3.888 Hz, J = 14.095 Hz, 1H) 4.11-4.08 (d, J = 15.715 Hz, 1H) 4.00-3.97
(m, 1H), 3.22-3.04 (m, 4H), 1.42-1.39 (m, 2H), 1.28 (s, 27H), 0.92-0.89 (t, J = 7.121 Hz,
3H).
31
P NMR (202 MHz, CD
3
OD) δ 10.26, 8.74 (61:39). MS-ESI (m/z) 670.6 (M+H)
+
,
692.3 (M+Na)
+
.
134
Large Scale Synthesis of cyclic (L)-Tyr-NHC
16
H
33
-cHPMPC (5.3).
General Procedure.
4.1 (7.16 mmol, 2.00 g), anhydrous DMF (60 mL), and DIEA (71.6 mmol, 13 mL) were
added to a 250 mL RBF followed by the addition of 2.15 (78.8 mmol, 3.6 g) and PyBOP
(15.8 mmol, 8.23 g). Mixture was stirred at 40 °C for 2 hr followed overnight at rt.
Reaction was monitored by
31
P NMR and additional amounts of PyBOP were
administered accordingly. Solvent removed under reduced pressure, extracted with
diethyl ether (125 mL) and ether removed. Residue was dissolved in 2% MeOH/CH
2
Cl
2
and purified by silica gel chromatography (product eluted 7-9% MeOH/CH
2
Cl
2
). 12.84
mL TFA (26 eq.) and 35 mL anhydrous CH
2
Cl
2
. Stirred at rt overnight. Solvent removed
under reduced pressure and residue dissolved in 0.5% TFA/CH
2
Cl
2
for purification by
silica gel chromatography (0%-12% MeOH/CH
2
Cl
2
, increased increments of 2.5%; 0.5%
TFA kept constant in mobile phase). Product eluted 9-13% MeOH/CH
2
Cl
2
/0.5%TFA.
Fractions containing product collected and combined, solvent removed and compound
dried for 4 h. The solid was recrystallized from ether and dried under reduced pressure to
afford 0.9988 g of 5.3.
(2S)-2-Amino-3-(4-([(5S)-5-[(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)methyl]-2-
oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-hexadecylpropanamide (5.3). Yield
21.6% (determined by UV). Obtained as a TFA salt; mixture of diastereomers.
1
H NMR
(500 MHz, CD
3
OD) δ 7.82-7.78 (m, 1H), 7.33-7.20 (m, 4H), 4.78-4.72 (m, 1H), 6.03-
6.02 (d, J = 7.726 Hz, 1H), 4.66-4.61, 4.40-4.36 (m, 1H), 4.53-4.36 (m, 2H), 4.25-4.22
135
(m, 2H) 4.15-3.97 (m, 3H) 3.23-3.04 (m, 4H), 1.43-1.41 (m, 2H), 1.29 (s, 27H), 0.92-
0.89 (t, J = 6.570 Hz, J = 6.570 Hz, 3H).
31
P NMR (202, MHz, CD
3
OD) δ 10.42, 8.87
(65:35). MS-ESI (m/z) 646.8 (M+H)
+
, 668.5 (M+Na)
+
.
Large Scale Synthesis of (L)-Tyr-NHC
16
H
33
-HPMPA (5.4).
General procedure.
5.2 (2.4 mmol, 1.6 g) was added to a round bottom flask, followed by 35 mL of a
MeCN/NH
4
OH solution (70 mL of MeCN in 12 mL of 7.9M NH
4
OH). This suspension
was sonicated for 10 min in which the remainder of the MeCN/NH
4
OH solution was
added. The reaction mixture was refluxed at 45 °C overnight and monitored by
31
P NMR.
Upon completion, additional MeCN (100 mL) was added and solvent removed under
reduced pressure. Added MeOH to dissolve compound and remove solvent. To
precipitate product, HPLC grade H
2
O (55 mL) was added and suspension sonicated for
15 min. Decanted H
2
O until ~10 mL remaining then centrifuged. The H
2
O layer was
extracted, solid was dissolved in MeOH, combined and solvent removed several times (to
remove all traces of H
2
O). Dissolve sample in 5% MeOH/CH
2
Cl
2
and purify via silica gel
chromatography (5-40% MeOH/CH
2
Cl
2
in increments of 5%). Due to presence of 3%
cHPMPA (indicative at ~6ppm in
31
P NMR), 10 mL of HPLC H
2
O added to flask and
centrifuged once more. Decanted H
2
O layer, dissolved solid in MeOH, removed solvent
under reduced pressure. Recrystallized from diethyl ether to afford 320 mg of 5.4.
136
(4-[(2S)-2-Amino-2-(hexadecylcarbamoyl)ethyl]phenoxy)(([(2S)-1-(6-amino-9H-
purin-9-yl)-3-hydroxypropan-2-yl]oxy)methyl)phosphinic acid (5.4). Yield 14 %
(determined by UV). Obtained as an NH
4
+
salt. ! = 14190 (260 nm, EtOH), 94%.
1
H
NMR (500 MHz, CD
3
OD) δ 8.19 (s, 1H), 8.17 (s, 1H), 7.13-7.11 (d, J = 7.698 Hz, 2H),
7.05-7.03 (d, J = 8.340 Hz, 2H), 4.48-4.37 (m, 2H), 3.91-3.88 (m, 1H), 3.86-3.84 (m,
1H), 3.81-3.77 (m, 1H) 3.73-3.67 (m, 2H) 3.55-3.51 (dd, J = 4.816 Hz, J = 12.806 Hz,
1H), 3.22-3.19 (m, 3H), 3.15-3.11 (m, 1H), 2.93-2.88 (m, 1H), 1.50-1.48 (m, 2H), 1.28
(s, 28H), 0.92-0.89 (t, J = 7.498 Hz, J = 5.563 Hz, 3H).
31
P NMR (202 MHz, CD
3
OD) δ
13.94. MS-ESI (m/z) 688.9 (M+H)
+
, 710.4 (M+Na)
+
.
Large Scale Synthesis of (L)-Tyr-NHC
16
H
33
-HPMPC (5.5).
5.5 was synthesized according to the procedure described for compound 5.4 above.
Amount of starting material for hydrolysis reaction was 0.998 g (1.54 mmol).
(4-[(2S)-2-Amino-2-(hexadecylcarbamoyl)ethyl]phenoxy)(([(2S)-1-(4-amino-2-oxo-
1,2-dihydropyrimidin-1-yl)-3-hydroxypropan-2-yl]oxy)methyl)phosphinic acid (5.5).
Yield 5% (determined by UV). Obtained as an NH
4
+
salt.
1
H NMR (500 MHz, CD
3
OD) δ
7.62 (s, 1H), 7.60 (s, 1H), 7.15-7.13 (d, J = 8.426 Hz, 2H), 7.10-7.09 (d, J = 8.102 Hz,
2H), 5.81-5.79 (d, J = 7.256 Hz, 1H), 4.08-4.04 (m, 1H), 3.85-3.78 (m, 2H), 3.73-3.65
(m, 3H), 3.63-3.61 (m, 1H) 3.53-3.50 (m, 1H) 3.17-3.15 (t, J = 7.911 Hz, J = 7.655 Hz,
2H) 3.03-2.99 (dd, J = 5.869 Hz, J = 13.014 Hz, 1H), 2.82-2.78 (dd, J = 7.655 Hz, J =
12.759 Hz, 1H), 1.48-1.46 (m, 2H), 1.31 (s, 27H), 0.93-0.90 (t, J = 7.320 Hz, J = 7.320
137
Hz, 3H).
31
P NMR (202 MHz, CD
3
OD) δ 13.07. MS-ESI (m/z) 664.5 (M+H)
+
, 686.3
(M+Na)
+
.
138
5.7 Chapter 5 References
1. De Clercq, E.; Sakuma, T.; Baba, M.; Pauwels, R.; Balzarini, J.; Rosengberg, I.;
Holy, A. Antiviral activity of phosphonomethoxyalkyl derivatives of purine and
pyrimidines. Antiviral Res. 1987, 8 261-272.
2. De Clercq, E.; Holy, A. Acyclic nucleoside phosphonates: a key class of antiviral
drugs. Nat Rev Drug Discov. 2005, 4, 928-940.
3. De Clercq, E.; Neyts, J. Antiviral agents acting as DNA or RNA chain
terminators. Handb Exp Pharmacol. 2009, 189, 53-84.
4. Holy, A.; Phosphonomethoxyalkyl analogs of nucleotides. Curr Pharm Des.
2003, 9, 2567-2592.
5. Cundy, K. C.; Clinical Pharmacokinetics of the Antiviral Nucleotide Analogs
Cidofovir and Adefovir. Clin Pharmacokinet. 1999, 36, 127-143.
6. Hostetler, K.Y. Alkoxyalkyl prodrugs of acyclic nucleoside phosphonates
enhance oral bioavailability and reduce toxicity: current state of the art. Antiviral
Res. 2009, 82, A84-AA98.
7. Valiaeva, N.; Prichard, M. N.; Buller, R. M.; Beadle, J. R.; Hartline, C. B.; Keith,
K. A. Schriewer, J., Trahan, J., Hostetler, K.Y. Antiviral evaluation of
octadecyloxyethyl esters of (S)-3-hydroxy-2-(phosphonomethoxy)propyl
nucleosides against herpesviruses and orthopoxviruses. Antiviral Res. 2009, 84,
254-259.
8. Aldern, K. A.; Ciesla, S. L.; Winegarden, K. L.; Hostetler, K.Y. Increased
antiviral activity of 1-O-hexadexyloxypropyl-[2-14C]cidofovir in MRC-5 human
lung fibroblasts is explained by unique cellular uptake and metabolism. Mol
Pharm. 2003, 63, 678-681.
9. Magee, W. C.; Aldern, K. A.; Hostetler, K. Y.; Evans, D. H. Cidofovir and (S)-9-
[3-hydroxy-(2-phosphonomethoxy)propyl]adenine are highly effective inhibitors
of vaccinia virus DNA polymerase when incorporated into the template strand.
Antimicrob Agents Chemother. 2008, 52, 586-597.
10. Ciesla, S. L.; Trahan, J.; Wan, W. B.; Beadle, J. R.; Aldern, K. A.; Painter, G. R.;
Hostetler, K.Y. Esterification of cidofovir and alkylalkanols increases oral
bioavailability and diminishes drug accumulation in kidney. Antiviral Res. 2003,
59, 163-171.
139
11. Quenelle, D. C.; Collins, D. J.; Herrod, B. P.; Keith, K. A.; Trahan, J.; Beadle, J.
R.; Hostetler, K. Y.; Kern, E. R. Effect of oral treatment with
hexadecyloxypropyl-[(S)-9-(hydroxy-2-phosphonomethoxypropyl)adenine]
[HDP-(S)-HPMPA)] or octadecyloxyethyl-(S)-HPMPA on cowpox or vaccinia
virus infections in mice. Antimicrob Agents Chemother. 2007, 51, 3940-3947.
12. Krylov, I. S.; Kashemirov, B. A.; Hilfinger, J. M.; McKenna, C. E. Evolution of
amino acid based prodrug approach: stay tuned. Mol Pharmaceutics. 2013, 10,
445-458.
13. Krylov, I. S. Synthesis, structural analysis and in vitro antiviral activities of novel
cyclic and acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs. Ph.D.
Thesis, University of Southern California, Los Angeles, 2012.
14. 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.
15. 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.
140
CHAPTER 6
Synthesis of 5’-Phosphonate Analogues of Ribavirin as
Antiviral Drugs Against Dengue Virus
6.1 Introduction: dengue virus
With over 2.5 billion of the world’s population at risk for infection, dengue virus has re-
emerged as a major public health concern. Dengue virus is a mosquito-borne illness
encountered mainly in tropical and sub-tropical regions of the world with extensive
documentation in Southeast Asia and Latin America.
1
Transmitted by Aedes aegypti and
Aedes albopictus mosquito vectors,
2
dengue virus is caused by four distinct serotypes
(DENV1-4) that can manifest into the acutely febrile dengue fever (DF) and progress into
potentially life-threatening forms, such as dengue hemorrhagic fever (DHF) and dengue
shock syndrome (DSS).
3
A recent report on the number of annual dengue-related
incidents indicate nearly 100 million dengue cases and around 400 million dengue
infections worldwide,
1
which has lead to 25,000 deaths annually – most of which are
children.
4
Dengue virus is a positive, single strand RNA virus
5
belonging to the Flaviviridae family,
which also contains flaviviruses such as West Nile virus (WNV), Japanese encephalitis
virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV).
6
While
human vaccines are available for JEV, YFV and TBEV, there are no commercially
available antiviral treatments for flaviviruses nor vaccines for dengue virus, prompting
141
the necessity for research efforts to be directed toward their discovery and development.
While both viral and host proteins are potential targets for treating dengue infections,
7,8
the focus of this work will be from the viral target-based approach. Within the dengue
viral genome, which consists of three structural proteins and seven non-structural (NS)
proteins, of great interest are NS viral proteins that play a significant role in viral
replication. With the occurrence of four distinct DENV serotypes (DENV 1-4) that can
result in an infection, the antiviral approach concerning inhibition of the DENV NS5
RNA-dependent RNA polymerases would be advantageous to study in that all DENV
serotypes should be equally susceptible. Of the antiviral drugs available, nucleoside
inhibitors make up the largest group of them. Inhibitors of this class act as chain
terminators within the viral DNA, retro and RNA virus replication cycle
9
after they have
been converted to their 5’-triphosphate metabolite form.
10
Various nucleoside analogues
containing a ribose ring with an intact 3’-OH have been found to inhibit HCV
infections,
11,12
a close relative to flaviviruses, and DENV infections.
13-15
Combination
therapies involving the broad spectrum ribonucleoside analogue, ribavirin, paired with
other classes of viral target-based inhibitors have also demonstrated the therapeutic
potential of ribavirin in studying and treating DENV infections.
17,18
6.2 Ribavirin and current uses
Since its introduction in 1972, ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-
carboxamide, RBV, Virazole®, 6.1; Figure 6.1) still remains the only nucleotide
analogue to be approved as a broad-spectrum antiviral drug.
19
Ribavirin is a guanosine
142
ribonucleoside analogue that acts as a nucleoside inhibitor by halting viral RNA or DNA
synthesis. With demonstrated in vitro and in vivo antiviral activity against a variety of
RNA and DNA viruses,
19
RBV is clinically approved to treat respiratory syncytial virus
20
Lassa fever infections
21
and as a treatment for hepatitis C when paired with pegylated
interferon.
22
In the past, ribavirin has been utilized in combination with other
therapeutics (6-mercapto-9-tetrahydro-2-furylpurine (6-MPTF),
23
mycophenolic acid,
18
and cellular α-glucosidase I and II enzymes
17
) to attempt to treat dengue infections.
Figure 6.1 Structure of ribavirin
6.3 Mechanism of action
In order for ribavirin to exert its antiviral activity, three consecutive phosphorylations to
the triphosphate form (RTP) by cellular enzymes are required.
24
The mode of action of
ribavirin and its phosphorylated derivatives is still a mystery and various hypotheses have
been proposed depending on the virus at hand. One proposed mechanism includes the 5-
monophosphate of ribavirin (RMP) reduces guanosine triphosphate (GTP) pools by
inhibiting the cellular enzyme inosine monophosphate dehydrogenase (IMPDH)
25,26
and
N
N
N
O
HO
OH
NH
2
O
HO
Ribavirin
Virazole
®
6.1
143
may therefore allow more frequent incorporation of ribavirin into viral RNA strand.
27
In
terms of the influenza virus, RTP has demonstrated viral RNA inhibition by inhibiting
viral RNA polymerase.
28
A report put out by Benarroch et al.
29
describes the prevention
of 5’capping of viral mRNAs via inhibition of the NS5 dengue virus mRNA 2’-O-
methyltransferase domain or viral guanylyl transferase by ribavirin-5’-triphosphate (RTP)
and ribavirin-5’-monophosphate (RMP), respectively. In addition, impaired function can
occur when ribavirin is incorporated into the 5’-cap as well as a build up of mutations
when integrated into viral RNA that can block viral replication.
30
Not to mention,
ribavirin has also been linked to various immune-system related activities.
31,32
6.4 Limitations
Initial phosphorylation by adenosine kinase is considered to be one of the drawbacks to
the clinical potential of ribavirin.
33
In addition, the occurrence of dose-limiting induced
hemolytic anemia due to ribavirin’s active transport and toxic accumulation in red blood
cells
34-36
and differences in patient responses to ribavirin as a result of poor cellular
uptake with the equilibrative nucleoside transporter 1 (ENT1) and all possible isoforms,
37
have been reported to limit the therapeutic application of ribavirin. The focal point of
these issues point toward developing an approach that eliminates the initial
phosphorylation step in order to determine if the efficacy, safety and dosing can be
enhanced.
144
6.5 Prodrugs of ribavirin
In order to ameliorate the clinical limitations of ribavirin associated with the initial
phosphorylations step, applying the prodrug approach to this compound has been
attempted by several groups in the past. The main goal has been to implement a
phosphorus moiety and in order to bypass the rate-limiting step in the mechanism of
action of ribavirin. Previously developed prodrug approaches applied to ribavirin include
the ProTide approach,
38
alkyloxyalkyl phosphodiester approach,
39
macromolecular
prodrugs,
40
and a self-assembling galactosylated micelle approach.
41
To date, the only
ribavirin derivative proven to be as successful as 6.1 is its 3-carboxamidine counterpart,
taribavirin (viramidine; Figure 6.2). It was first reported in 1973 by Witkowski et al.
42
to
demonstrate similar antiviral activity to ribavirin and is currently in phase III of clinical
trials.
Figure 6.2 Prodrugs of ribavirin
N
N
N
O
HO
OH
NH
2
NH
HO
Taribavirin
Viramidine
N
N
N
O
HO
OH
NH
2
O
O
O OH O
m n
macromolecular prodrug
N
N
N
O
HO
OH
NH
2
O
O
P
O
H
3
C(H
2
C)
17
OH
2
CH
2
CO
HO
alkyloxyalkyl phosphoryl prodrug
N
N
N
O
HO
OH
NH
2
O
P
NH
O
O
R
O
OBn
R = CH
3
, CH
2
Ph or H
Ribavirin protide
O
145
6.6 Ribavirin 5’-monophosphate mimics: 5’-monophosphonates
Application of the prodrug approach serves to enhance ribavirin’s efficacy by reducing
the side effects associated with the initial phosphorylation step by introducing a
phosphonic acid moiety. Compound 6.2 has been synthesized previously
43
and its
inhibitory activity against Inosine Monophosphate (IMP) Dehydrogenase assessed. Since
it has not been tested against dengue virus, it was included in this study to compare to the
monophosphate mimic 6.3, which has also been previously synthesized by Fuertes et al.
44
Compounds 6.2 and 6.3 depicted in Figure 6.3 were synthesized and will undergo in vitro
assessment as antiviral parent drugs against dengue virus RNA-dependent RNA
polymerase (RdRp). If a lead candidate is identified, an additional prodrug approach will
be administered in order to mask the negative charges (at physiological pH) on the
phosphonic acid group with efficacious promoieties.
Figure 6.3 Ribavirin 5’-monophosphate mimics
6.6.1 Synthesis of 5’-phosphonate of analogues ribavirin
Chemical transformations involving the incorporation of a phosphoryl or phosphonate
moiety have been developed and applied to nucleoside analogue scaffolds in order to
enhance their cellular permeability and antiviral activity bypassing the standard initial
N
N
N
O
HO
OH
NH
2
O
5'-phosphonate ribavirin analogue
6.2
5'-methylene phosphonate ribavirin analogue
6.3
N
N
N
O
HO
OH
NH
2
O
P
O
HO
HO
P
O
HO
HO
146
rate-limiting phosphorylation step. One possible synthetic pathway used in making
nucleoside 5’-phosphonate analogues involves a method utilized by Koh et al,
45
and
originally described by Raju et al.
46
However, that particular method involves
glycosylation of various nucleobases with a furanose diethylphosphonate. Therefore, the
synthesis of 6.2 was carried out according to a procedure developed by Wang and co-
workers.
43
Selective iodination of the 5’-OH group was performed under basic conditions
followed by basic benzoylation of the 2’- and 3’-OH groups on the ribose ring to afford
6.5 in good yield. In accordance with Michaelis-Arbuzov reaction conditions, 6.5 was
refluxed at 100 °C for 15 h in the presence of triethylphosphite (P(OEt)
3
) to afford low
yields.
Scheme 6.1 Synthesis of RBV-cP analogue. a. I
2
, PPh
3
, pyridine, 2 h, rt; b. pyridine,
BzCl, 1 h, rt; c. triethylphosphite, 100 °C, 15 h; d. DMF:MeCN (1:1), BTMS, 6 h, rt; e.
aq. NH
4
OH, 6 h, rt.
N
N
N
O
HO
OH
NH
2
O
HO
N
N
N
O
HO
OH
NH
2
O
I
N
N
N
O
BzO
OBz
NH
2
O
I
N
N
N
O
BzO
OBz
NH
2
O
P
O
EtO
EtO
N
N
N
O
HO
OH
NH
2
O
P
O
HO
HO
d,e
c
b
a
6.1
6.2
6.4
6.5
6.6
147
A possible explanation for the low yield for this reaction is accumulation of the side
product ethyl iodide, which is expelled from the molecule after nucleophilic attack by the
phosphorus atom occurs. In most reported Arbuzov reactions, the halogen of choice as
the leaving group is either a bromide ion (Br
-
) or chloride ion (Cl
-
), which would produce
a more volatile alkyl halogen (Cl or Br) by-product that would be removed during the
heated reaction and not have a chance to compete with 6.5 for triethylphosphite to
produce an unwanted side product. After purification by column chromatography, the
ethyl groups are removed using the McKenna reaction followed by removal of the
benzoyl groups in aqueous ammonia. HPLC purification gives 6.2 as a triethylammonium
salt. Also retrieved in the HPLC sample, as determined by mass spectrometry, was the
monophosphate analogue of this derivative (6.7, Figure 6.4). The contamination, thought
to be a by-product of the Arbuzov reaction, was not able to be separated from the desired
compound, so the commercially available monophosphate RBV analogue (6.7) was
purchased and sent along with 6.2 to be tested against dengue virus.
Figure 6.4 Final compounds post HPLC purification
N
N
N
O
HO
OH
NH
2
O
N
N
N
O
HO
OH
NH
2
O
P
O
HO
HO
6.2
O
P
O
HO
OH
6.7
148
6.6.2 Synthesis of 5’-methylene phosphonate analogue of ribavirin
With the original synthesis of 6.3 utilizing the Wittig reaction,
44
Wittig-like conditions,
including those reported by Montgomery and co workers
47
and those adapted by Pradere
et al.,
48
were of interest in the synthesis of phosphonate derivatives of nucleosides.
Introduction of a phosphonate moiety to prepare 5’-methylene alkyl phosphonates can be
prepared using a variety of synthetic pathways (Wittig reaction,
44,49-51
Michaelis-Arbuzov
condensation,
52-54
alkylphosphonate anions,
55-57
and via the Barton reaction
58-60
). With
respect to time and synthetic efficiency, previously reported Horner-Wadsworth-Emmons
(HWE) olefination reaction conditions
48
were employed to introduce the ethylene
phosphonate moiety. Initial protection of the 2’-, 3’- and 5’-OH groups with tert-
butyldimethylsilyl (TBDMS) groups in the presence of imidazole was performed,
followed by selective deprotection of the 5’-OH group using trichloroacetic acid (TCA)
and aqueous conditions was utilized to yield compound 6.11 (Scheme 6.3). The 5’-OH
was first oxidized to an aldehyde by refluxing with 2-idoxybenzoic acid (IBX, 6.8,
Scheme 6.2) in MeCN for 3 h. Commercially available IBX is stored as a 45% wt. IBX
mixture with a stabilizer and has previously demonstrated low yields in IBX oxidation
reactions performed in our laboratory. Therefore, IBX was prepared according to a
procedure reported by Frigerio et al. (Scheme 6.2)
61
in good yield (70%).
Scheme 6.2. Synthesis of 2-iodoxybenzoic acid. a. H
2
O, 70 °C, 1 h b. rt, overnight.
I
CO
2
H
+
2KHSO
5
· KHSO
4
· K
2
SO
4
a
O
I
O
OH
O
6.8 6.9 6.10
b
149
Figure 6.5 Left
1
H NMR spectra: Compound 6.13 post Horner-Wadsworth-Emmons
olefination (
1
H NMR (500 MHz, Methanol-d
4
). Right
1
H NMR spectra: Compound 6.14
post hydrogenation (
1
H NMR (500 MHz, Methanol-d
4
)
Using the freshly prepared IBX (6.8), the 5’-OH on 6.11 was oxidized to an aldehyde,
which is depicted in Scheme 6.3. Due to the sensitivity of the 5’-aldehydic ribavirin
analogue (6.12), immediately following the workup and drying of 6.12, the residue was
dissolved in THF (and a small amount of dimethylformamide (DMF) to assist with
solubility issues) in order to perform the olefination reaction. HWE olefination began
with first preparing a THF solution of the tetramethyl bisphosphonate carbanion
(tetramethyl bisphosphonate in the presence of sodium hydride (NaH)). After stirring at 0
°C for 20 min, the solution is added to the THF/DMF solution of 6.12 to form the
dimethyl phosphonate olefin ribavirin analogue 6.13. The presence of a set of triplets at
6.83 ppm and 6.25 ppm in the
1
H NMR indicated formation of the double bond between
the phosphorus atom and the ribose ring. The TBDMS protecting groups were removed
under acidic conditions and the double bond was reduced using Pd/C in the presence of
H
2
to afford 6.14 (Scheme 6.3). Disappearance of the olefinic protons between 6-7 ppm
150
and the appearance of four protons around 2 ppm in
1
H NMR indicated the double bond
had successfully been reduced. The methyl groups on the phosphonate moiety were
removed using the McKenna reaction followed by two separate reverse phase HPLC
purifications to afford 6.3. Taking into account the use of different solvents, the
1
H NMR
data obtained in this study (with additional triethylamine proton peaks from purification)
is comparable to the literature values previously reported.
44
Scheme 6.3 Synthesis of RBV-CCP analogue 6.3. a. TBSCl, imdazole, DMF, rt,
overnight; b. TCA, THF/H
2
O, rt, 6 h; c. 6.8, MeCN, 3 h, 80 °C; d. THF, NaH,
tetramethyl bisphosphonate, 3 h, rt; e. HCOOH: H
2
O (1:1), 48 h, rt; f. Pd/C, H
2
, 3 h, rt; g.
MeCN:DMF (1:1), BTMS, rt, 2 h.
6.7 Conclusion
A ribavirin 5’-methylene phosphonate RBV analogue (6.3) and a ribavirin 5’-
phosphonate RBV analogue (6.2) were synthesized to be assessed as potential antiviral
N
N
O
NH
2
6.1 6.11 6.12
6.13 6.3 6.14
O
OTBS
OTBS
N
N
N
O
HO
OH
NH
2
O
HO
N
N
N
O
TBSO
OTBS
NH
2
O
HO
N
N
N
O
TBSO
OTBS
NH
2
O P
O
MeO
MeO
N
N
N
O
HO
OH
NH
2
O P
O
MeO
MeO
N
N
N
O
HO
OH
NH
2
O P
O
HO
HO
O
H
a
b
c
d e
f
g
151
agents for dengue virus. For the one carbon linker between the phosphonate and the
ribose ring, the Arbuzov reaction was used to attach the phosphonic acid moiety while a
modified Horner-Wadsworth-Emmons (HWE) olefination was utilized to synthesize the
5’methylene RBV phosphonate (6.3).
6.8 Experimental section
General Experimental Procedures.
1
H and
31
P NMR spectra were obtained on 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);
DMSO (
1
H NMR δ = 2.5).
31
P NMR spectra were proton-decoupled, and
1
H NMR
coupling constants (J values) were given in Hz. NMR abbreviations used include s
(singlet), d (doublet), m (unresolved multiplet), dd (doublet of doublet), ddd (doublet of
doublet of doublet), bs (broad signal). MS analysis was performed on a Thermo-Finnigan
LCQ DECA Xp
max
Ion Trap LC/MS/MS eqipped with an ESI Probe. Xcalibur software
wa used to process the MS spectra. HPLC separations for 6.2 and 6.3 were performed on
a C18 HPLC column (5 µm, 250 mm x 4.6 mm) with a 5% MeCN gradient or 5%
isocratic in 50 mM triethylammonium bicarbonate, pH 7.3 or 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 220 or 235 nm for 6.2 and 6.3.
152
Synthesis of 1-[(2R,3R,4S)-3,4-Dihydroxy-5-(iodomethyl)oxolan-2-yl]-1H-1,2,4-
triazole-3-carboxamide (6.4). General Procedure.
24
A solution of triphenylphosphine
(1.75 g, 6.7 mmol) and iodine (1.56 g, 6.2 mmol) in pyridine (10 mL) was stirred at rt for
20 min and then 6.1 (1.0 g, 4.1 mmol) was added. The reaction mixture was stirred at rt
for 2 h, concentrated to dryness, and co-evaporated with toluene (X 2). Precipitation by
EtOH (50 mL), filtered and washed with fresh EtOH. Solvent removed and dried under
reduced pressure to yield 1.45 g of 6.4. Yield ~100 %.
1
H NMR (500 MHz, DMSO-d
6
) δ
8.84 (d, J = 11.8 Hz, 1H), 5.88 (s, 1H), 4.48 (s, 1H), 4.18 (d, J = 5.4 Hz, 3H), 4.03 (d, J =
8.0 Hz, 2H), 3.56 (s, 2H).
Synthesis of (2S,3S,4R,5R)-4-(Benzoyloxy)-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)-2-
(iodomethyl)oxolan-3-yl benzoate (6.5). A solution of 6.4 was dissolved in anhydrous
pyridine (8 mL) and stirred at 0 °C prior to the addition of benzoyl chloride (1.05 mL, 9.0
mmol). The reaction was allowed to stir at 0 °C for 1 h then at rt overnight (covered in
foil). The reaction mixture was poured into saturated sodium bicarbonate (100 mL) and
extracted with ethyl acetate (300 mL). The organic phases were combined, dried over
sodium sulfate and solvent removed. The residue was purified by silica gel
chromatography (CH
2
Cl
2
:MeOH, 7:3) to afford 6.5. Yield 40%.
1
H NMR (500 MHz,
CDCl
3
) δ 8.49 (s, 1H), 7.95 – 7.85 (m, 4H), 7.58 – 7.48 (m, 2H), 7.35 (t, J = 7.8 Hz, 6H),
6.54 (s, 1H), 6.27 (s, 1H), 6.09 (d, J = 8.3 Hz, 1H), 5.85 (t, J = 5.8 Hz, 1H), 4.61 (d, J =
6.0 Hz, 1H).
153
Synthesis of ([(3S,4R,5R)-5-(3-Carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-
dihydroxyoxolan-2-yl]methyl)phosphonic acid (6.2). 6.5 was dissolved in
triethylphosphite and stirred at 100 °C for 15 h. The reaction was monitored by
31
P NMR.
Solvent was removed and purified via silica gel chromatography using CH
2
Cl
2
:MeOH
(10:7). Fractions collected, solvent removed and dried to afford 6.6. Compound 6.6 was
dissolved in DMF:ACN (1:1) and BTMS (0.180 mL, 1.36 mmol) added. The reaction
mixture was stirred at rt for 6 h followed by drying under reduced pressure. Residue was
co-distilled with methanol and toluene three times. The residue was then dissolved in aq
NH
4
OH (28%, 3 mL) and stirred at rt for 6 h. After solvent removal, the compound was
purified by reverse-phase HPLC to give 40 mg of the desired product and a small amount
(15% by
31
P NMR) of the monophosphate analogue 6.2. Yield 10%.
1
H NMR (500 MHz,
D
2
O) δ 8.66 (d, J = 34.8 Hz, 1H), 5.89 (s, 1H), 4.62 – 4.58 (m, 1H), 4.31 – 4.25 (m, 2H),
3.09 (q, J = 7.3 Hz, 28H), 1.99 (d, J = 17.9 Hz, 2H), 1.16 (t, J = 7.3 Hz, 42H).
31
P NMR
(202 MHz, D
2
O) δ 18.99. LRMS (MS-ESI): m/z [M - H]
-
calcd for C
8
H
13
N
4
O
7
P: 307.0,
found: 307.4 (M - H)
-
.
Synthesis of 5’-methylene phosphonate ribavirin analogue (6.3).
26
Synthesis of 1-[(3R,4R,5R)-3,4-Bis[(tert-butyldimethylsilyl)oxy]-5-
(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide (6.11).
General Procedure.
6.1 (1.0 g, 4.1 mmol) was dissolved in DMF (8 mL), followed by
the addition of imidazole (2.09 g, 30.7 mmol) and tert-butyldimethylsilyl chloride (4.63
g, 30.7 mmol) under N
2
. The reaction mixture was stirred overnight at rt and then stirred
154
with saturated aqueous sodium bicarbonate (40 mL) for 30 min. The mixture was
extracted with CH
2
Cl
2
(3 x 75 mL), washed with water (200 mL) and brine (125 mL) and
dried over sodium sulfate. Solvent was removed and purified by silica gel
chromatography (3% MeOH/CH
2
Cl
2
). Fractions containing product were collected (~ 2.8
g, 4.8 mmol), solvent removed and dissolved in THF (35 mL). The mixture was stirred at
0 °C when a freshly prepared solution of trichloroacetic acid (TCA, 16 g, 97.7 mmol) in
8.8 mL of water was added. The reaction mixture was stirred at 0 °C for 3 h. Upon
completion, solid sodium bicarbonate was added until the evolution of gas ceased, then
the mixture was extracted with EtOAc (3 X 150 mL). The organic layers were combined
and dried over Na
2
SO
4
followed by removal of solvent and purification using silica gel
chromatography (7% MeOH/CH
2
Cl
2
) to afford 6.11 in good yield (1.84 g). Yield 95%.
1
H NMR (500 MHz, CD
3
OD) δ 8.83 (s, 1H), 5.89 (s, 1H), 4.75 – 4.67 (m, 1H), 4.41 –
4.30 (m, 1H), 4.11 (q, J = 4.0 Hz, 1H), 3.76 (ddd, J = 55.3, 12.3, 4.1 Hz, 2H), 3.36 (s,
0H), 3.32 (p, J = 1.7 Hz, 18H), 0.91 (d, J = 55.0 Hz, 21H), 0.25 – -0.03 (m, 10H), -0.14
(s, 3H). m/z (APCI): 471, m/z(+) 472.
Synthesis of 2-iodoxybenzoic acid.
60
In 70 mL of HPLC water, 2-iodobenzoic acid (2 g, 8 mmol) and oxone (14.8 g, 24 mmol)
were added and stirred at 70 °C for 2 h followed by stirring at rt overnight. The reaction
mixture was stirred at 0 °C for 30 min, filtered, washed with 40 mL of water and 16 mL
of acetone. The residue was left out to dry for 24 h to afford 1.56 g as a white powder.
155
Yield 70%. (500 MHz, DMSO-d6):
1
H NMR δ 8.15-8.13 (d, J = 8.353 Hz, 1H), 8.04-
7.98 (m, 2H), 7.85-7.82 (t, J = 7.913 Hz, J = 7.327 Hz, 1H).
Synthesis of Dimethyl [(E)-2-[(3R,4R,5R)-3,4-bis[(tert-butyldimethylsilyl)oxy]-5-(3-
carbamoyl-1H-1,2,4-triazol-1-yl)oxolan-2-yl]ethenyl]phosphonate (6.13).
General Procedure. A suspension of 6.11 (1.02 g, 2.2 mmol), IBX (665 mg, 2.4 mmol)
and MeCN (125 mL) was stirred at 80 °C for 3 h. Solvent was removed and residue was
re-dissolved in EtOAc:THF (1:1) and filtered through a short silica pad using 500 mL of
EtOAc:THF (1:1). The solvent was removed and dried for 1 h. Anhydrous THF (100 mL)
was added to the flask and the mixture stirred at 0 °C for 10 min prior to the drop-wise
addition of freshly prepared tetramethylbisphosphonate sodium salt (prepared by
addition, at 0 °C, of tetramethylmethylene bisphosphonate (0.8 mL, 4.3 mmol) to a
suspension of NaH (96 mg, 4.2 mmol) in THF and stirring for 10 min). The reaction
mixture was stirred at rt for 3 h followed by the addition of saturated aqueous NH
4
Cl.
The solution was extracted with EtOAc (350 mL), organic layers combined and washed
with water (2 x 100mL) and brine (100 mL). Solvent was removed to afford a mixture of
E/Z isomers of 6.13 and excess tetramethylmethylene bisphosphonate.
1
H NMR (500
MHz, CD
3
OD) δ 8.86 (s, 1H), 7.05 – 6.91 (m, 2H), 6.32 (d, J = 17.1 Hz, 1H), 6.08 (s,
1H), 4.76 (d, J = 2.2 Hz, 2H), 4.53 (s, 1H), 3.38 (d, J = 3.2 Hz, 1H), 2.28 (s, 1H), 2.07 (s,
13H), 1.46 (s, 9H), 1.30 (s, 18H), 0.97 (t, J = 32.6 Hz, 27H), 0.18 (d, J = 81.2 Hz, 12H), -
0.07 (s, 3H).
31
P NMR (202 MHz, CD
3
OD) δ 30.74, 21.10 (1:4).
156
Synthesis of Dimethyl (2-[(3S,4R,5R)-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-
dihydroxyoxolan-2-yl]ethyl)phosphonate (6.14). The mixture of 6.13 and excess
tetramethylmethylene bisphosphonate was dissolved in HCOOH:H
2
O (1:1) and stirred at
rt for 72 h. Following solvent removal, the residue was re-dissolved in EtOAc:MeOH
(1:1; 50 mL) and Pd/C was added (2.1 g, 10% Pd on activated carbon). After flushing the
system with N
2
several times, H
2
gas was introduced and reaction was stirred for 4 h. The
mixture was then filtered over a short celite pad and washed with MeOH (200 mL) to
afford crude compound 6.14 with tetramethylene bisphosphonate impurity still present.
Yield 54%.
1
H NMR (500 MHz, CD
3
OD) δ 8.71 (s, 1H), 5.95 (s, 1H), 4.56 (d, J = 4.4
Hz, 1H), 4.31 (t, J = 5.2 Hz, 1H), 4.09 (s, 1H), 2.07 – 1.79 (m, 4H), 1.40 (s, 1H).
31
P
NMR (202 MHz, CD
3
OD) δ 35.72, 31.08.
Synthesis of (2-[(3S,4R,5R)-5-(3-Carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-
dihydroxyoxolan-2-yl]ethyl)phosphonic acid (6.3). DMF was added to residue 6.14 and
removed under reduced pressure (3x) to remove any residual MeOH. To 6.14, DMF (2
mL) and MeCN (8 mL) were added. BTMS (1.9 mL) was added dropwise to the solution
and stirred for 2 h at rt. Solvent was removed followed by MeOH added and removed
(3x). H
2
O (2 mL) and NH
4
OH (5 drops) were added to dissolved residue and Chelex
(Na+ form) was added to remove traces of palladium (Pd) still present from
hydrogenation. Liquid was extracted from Chelex, solvent removed and process Chelex
process repeated. Reverse phase C18 HPLC purification using a gradient between 0.05 N
triethylammonium bicarbonate (pH 7.3) and 0.05 N triethylammonium bicarbonate/ 5%
157
acetonitrile (pH 7.5) was applied to afford compound 6.3 with a small impurity (3%)
detected in the
31
P NMR. An additional reverse phase C18 HPLC purification under
isocratic conditions with 60 mM ammonium acetate/ 5% acetonitrile (pH 5.5) was
utilized to afford 6.3 in high purity (98%). Yield 10%. Obtained as an NH
4
+
salt. ! =
14190 (220 nm, EtOH), 94%.
1
H NMR (500 MHz, D
2
O) δ 8.63 (s, 1H), 5.87 (s, 1H), 4.60
– 4.54 (m, 1H), 4.22 (t, J = 4.9 Hz, 1H), 4.11 – 4.00 (m, 1H), 2.91 (q, J = 7.3, 5.5 Hz,
20H), 1.85 – 1.74 (m, 2H), 1.47 – 1.24 (m, 2H), 1.09 (td, J = 7.3, 1.6 Hz, 30H).
31
P NMR
(202 MHz, D
2
O) δ 20.83. LRMS (MS-ESI): m/z [M - H]
-
calcd for C
9
H
15
N
4
O
7
P: 321.0,
found: 321.0 (M - H)
-
.
158
6.9 Chapter 6 references
1. Bhatt, S.; Gething, P. W.; Brady, O. J.; Messina, J. P.; Farlow, A. W.; Moyes, C.
L.; Drake, J. M.; Brownstein, J. S.; Hoen, A. G.; Sankoh, O.; Myers, M. F.;
George, D. B.; Jaenisch, G. R.; Wint, G. R. W.; Simmons, C. P.; Scott, T. W.;
Farrar, J. J.; Hay, S. I. The global distribution and burden of dengue. Nature.
2013, 496, 504–7.
2. Kyle, J. L.; Harris, E. Global spread and persistence of dengue. Annu Rev
Microbiol. 2008, 62, 71–92.
3. World Health Organization. Dengue haemorrhagic fever: diagnosis, treatment,
prevention, and control. 2nd ed. Geneva: World Health Organization; 1997.
4. Guzman, M. G.; Halstead, S. B.; Artsob, H.; Buchy, P.; Farrar, J.; Gubler, D. J.;
Hunsperger, E.; Kroeger, A.; Margolis, H. S.; Martinez, E.; Nathan, M. B.;
Pelegrino, J. L; Simmons, C.; Yoksan, S.; Peeling, R. W. Dengue: a continuing
global threat. Nat Rev Microbiol. 2010, 8, S7–S16.
5. Clyde, K.; Kyle, J. L.; Harris, E. Recent advances in deciphering viral and host
determinants of dengue virus replication and pathogenesis. J Virol. 2006, 80,
11418–11431.
6. Schmaljohn, A. L.; McClain, D. Alphaviruses (Togaviridae) and Flaviviruses
(Flaviviridae) In: Baron S, editor. Medical Microbiology. 4th edition. Galveston
(TX): University of Texas Medical Branch at Galveston; 1996. Chapter 54.
7. Krishnan, M. N.; Garcia-Blanco, M. A. Targeting Host Factors to Treat West Nile
and Dengue Viral Infections. Viruses. 2014, 6, 683-708.
8. Noble, C. G.; Chen, Y-L.; Dong, H.; Gu, F.; Lim, S. P.; Schul, W.; Wang, Q-Y.;
Shi, P-Y. Strategies for Development of Dengue Virus Inhibitors. Antiviral Res.
2010, 85, 450-462.
9. De Clercq, E.; Neyts, J. Antiviral agents acting as DNA or RNA chain
terminators. Handb Exp Pharmacol. 2009, 189, 53–84.
10. Carroll, S. S.; Olsen, D. B. Nucleoside analog inhibitors of hepatitis C virus
replication. Infect Disord Drug Targets. 2006, 6, 17–29.
11. Carroll, S. S.; Ludmerer, S.; Handt, L.; Koeplinger, K.; Zhang, N. R.; Graham,
D.; Davies, M. E.; MacCoss, M.; Hazuda, D.; Olsen, D. B. Robust antiviral
159
efficacy upon administration of a nucleoside analog to hepatitis C virus-infected
chimpanzees. Antimicrob Agents Chemother. 2009, 53, 926–934.
12. Carroll, S. S.; Tomassini, J. E.; Bosserman, M.; Getty, K.; Stahlhut, M. W.;
Eldrup, A. B.; Bhat, B.; Hall, D.; Simcoe, A. L.; LaFemina, R.; Rutkowski, C. A.;
Wolanski, B.; Yang, Z.; Migliaccio, G.; De Francesco, R.; Kuo, L. C.; MacCoss,
M.; Olsen, D. B. Inhibition of hepatitis C virus RNA replication by 2-modified
nucleoside analogs. J Biol Chem. 2003, 278, 11979–11984.
13. Klumpp, K.; Kalayanov, G.; Ma, H.; Le Pogam, S.; Leveque, V.; Jiang, W. R.;
Inocencio, N.; De Witte, A.; Rajyaguru, S.; Tai, E.; Chanda, S.; Irwin, M. R.;
Sund, C.; Winqist, A.; Maltseva, T.; Eriksson, S.; Usova, E.; Smith, M.; Alker,
A.; Najera, I.; Cammack, N.; Martin, J. A.; Johansson, N. G.; Smith, D. B. 2ჼ-
Deoxy-4-azido nucleoside analogs are highly potent inhibitors of hepatitis C virus
replication despite the lack of 2-alpha-hydroxyl groups. J Biol Chem. 2008, 283,
2167–2175.
14. Klumpp, K.; Leveque, V.; Le Pogam, S.; Ma, H.; Jiang, W. R.; Kang, H.;
Granycome, C.; Singer, M.; Laxton, C.; Hang, J. Q.; Sarma, K.; Smith, D. B.;
Heindl, D.; Hobbs, C. J.; Merrett, J. H.; Symons, J.; Cammack, N.; Martin, J. A.;
Devos, R.; Najera, I. The novel nucleoside analog R1479 (4-azido-cytidine) is a
potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus
replication in cell culture. J Biol Chem. 2006, 281, 3793–3799.
15. Chen, Y-L.; Yin, Z.; Duraiswamy, J.; Schul, W.; Lim, C. C.; Liu, B.; Xu, H. Y.;
Qing, M.; Yip, A.; Wang, G.; Chan, W. L.; Tan, H. P.; Lo, M.; Liung, S.;
Kondreddi, R. R.; Rao, R.; Gu, H.; He, H.; Keller, T. H.; Shi, P-Y. Inhibition of
Dengue Virus RNA Synthesis by an Adenosine Nucleoside. Antimicrob Agents
Chemother. 2010, 54, 2932-2939.
16. Chen, Y-L.; Yin, Z.; Lakshminarayana, S. B.; Qing, M.; Schul, W.; Duraiswamy,
J.; Kondreddi, R. R.; Goh, A.; Xu, H. Y.; Yip, A.; Liu, B.; Weaver, M.; Dartois,
V.; Keller, T. H.; Shi, P-Y. Inhibition of Dengue Virus by an Ester Prodrug of an
Adenosine Analog. Antimicrob Agents Chemother. 2010, 54, 3255-3261.
17. Chang, J.; Schul, W.; Butters, T. D.; Yip, A.; Liu, B.; Goh, A.; Lakshminarayana,
S. B.; Alonzi, D.; Reinkensmeier, G.; Pan, X.; Qu, X.; Weidner, J. M.; Wang, L.;
Yu, W.; Borune, N.; Kinch, M. A.; Rayahin, J. E.; Moriarity, R.; Xu, X.; Shi, P-
Y.; Guo, J-T.; Block, T. M. Combination of α-Glucosidase Inhibitor and
Ribavirin for the Treatment of Dengue Virus Infection In Vitro and In Vivo.
Antiviral Res. 2011, 89, 26-34.
18. Takhampunya, R.; Ubol, S.; Huong, H-S.; Cameron, C. E.; Padmanabhan, R.
Inhibition of Dengue Virus Replication by Mycophenolic acid and Ribavirin. J
Gen Virol. 2006, 87, 1947-1952.
160
19. Sidwell, R. W.; Huffman, J. H.; Khare, G. P.; Allen, L. B.; Witkowski, J. T.;
Robins, R. K. Broad-spectrum antiviral activity of Virazole: 1-beta-D-
ribofuranosyl- 1,2,4-triazole-3-carboxamide. Science. 1972, 177, 705–706.
20. Snell, N. J. Ribavirin—Current State of a Broad Spectrum Antiviral Agent. Exp
Opin Pharmacother. 2001, 2, 1317.
21. Crotty, S.; Cameron, C.; Andino, R. Ribavirin’s antiviral mechanism of action:
lethal mutagenesis? J Mol Med. 2002, 80, 86–95.
22. Saracco, G.; Ciancio, A.; Olivero, A.; Smedile, A.; Roffi, L.; Croce, G.; Colletta,
C.; Cariti, G.; Andreoni, M.; Biglino, A.; Calleri, G.; Maggi, G.; Tappero, G. F.;
Orsi, P. G.; Terreni, N.; Macor, A.; Di Napoli, A.; Rinaldi, E.; Ciccone, G.;
Rizzetto, M. A randomized 4-arm multicenter study of interferon alfa-2b plus
ribavirin in the treatment of patients with chronic hepatitis C not responding to
interferon alone. Hepatology. 2001, 34, 133.
23. Koff, W. C.; Elm Jr., J. L.; Halstead, S. B. Antiviral effects of ribavirin and 6-
mercapto-9-tetrahydro-2-furylpurine against dengue virus in vitro. Antiviral Res.
1982, 2, 69-79.
24. Graci, J. D.; Cameron, C. E. Quasispecies, Error Catastrophe, and the Antiviral
Activity of Ribavirin. Virology. 2002, 298, 175-180.
25. Leyssen, P.; Balzarini, J.; De Clercq, E.; Neyts, J. J. The Predominant Mechanism
by Which Ribavirin Exerts Its Antiviral Activity in Vitro against Flaviviruses and
Paramyxoiruses is Mediated by Inhibition of IMP Dehydrogenase. Virology.
2005, 79, 1943.
26. Leyssen, P.; De Clercq, E.; Neyts, J. The Anti-Yellow Fever Virus Activity of
Ribavirin Is Independent of Error-Prone Replication. Mol Pharmacol. 2006, 69,
1461.
27. Crotty, S.; Maag, D.; Arnold, J. J.; Zhong, W.; Lau, J. Y. N.; Hong, Z.; Andino,
R.; Cameron, C. E. The Broad Spectrum Antiviral Ribonucleoside Ribavirin is an
RNA Virus Mutagen. Nat Med. 2000, 6, 1375.
28. Eriksson, B.; Helgstrand, E.; Johansson, N. G.; Larsson, A.; Misiorny, A.; Noren,
J. O.; Philipson, L.; Stenberg, K.; Stening, G.; Stridh, S.; Oberg, B. Inhibition of
Influenza Virus Ribonucleic Acid Polymerase by Ribavirin Triphosphate.
Antimicrob Agents Chemother. 1977, 11, 946.
29. Benarroch, D.; Egloff, M. P.; Mulard, L.; Guerreiro, C.; Romette, J. L.; Canard,
B. J. A structural basis for the inhibition of the NS5 dengue virus mRNA 2'-O-
161
methyltransferase domain by ribavirin 5'-triphosphate. Biol Chem. 2004, 279,
35638.
30. Crotty, S.; Cameron, C. E.; Andino, R. RNA virus error catastrophe: Direct
molecular test by using ribavirin. Proc Natl Acad Sci U.S.A. 2001, 98, 6895.
31. Bergamini, A.; Bolacchi, F.; Cepparulo, M.; Demin, F.; Uccella, I.; Bongiovanni,
B.; Ombres, D.; Angelico, F.; Liuti, A.; Hurtova, M.; Francioso, S.; Carvelli, C.;
Cerasari, G.; Angelico, M.; Rocchi, G. Treatment with ribavirin and interferon-α
reduces interferon-γ expression in patients with chronic hepatitis C. Clin Exp
Immunol. 2001, 123, 459.
32. Hultgren, C.; Milich, D. R.; Weiland, O.; Sallberg, M. The antiviral compound
ribavirin modulates the T helper (Th) 1/Th2 subset balance in hepatitis B and C
virus-specific immune responses. J Gen Virol. 1998, 79, 2381.
33. Willis, R. C.; Carson, D. A.; Seegmiller, J. E. Adenosine kinase initiates the major
route of ribavirin activation in a cultured human cell line. Proc Natl Acad Sci
U.S.A. 1978, 75, 3042.
34. Russmann, S.; Grattagliano, I.; Portincasa, P.; Palmieri, V. O.; Palasciano, G.
Ribavirin-induced anemia: mechanisms, risk factors and related targets for future
research. Curr Med Chem. 2006, 13, 3351–3357.
35. Page, T.; Connor, J. D. The metabolism of ribavirin in erythrocytes and nucleated
cells. Int. J Biochem. 1990, 22, 379–383.
36. Inoue, Y.; Homma, M.; Matsuzaki, Y.; Shibata, M.; Matsumura, T.; Ito, T.;
Kohda, Y. Erythrocyte ribavirin concentration for assessing hemoglobin reduction
in interferon and ribavirin combination therapy. Hepatol Res. 2006, 34, 23–27.
37. Fukuchi, Y.; Furihata, T.; Hashizume, M.; Iikura, M.; Chiba, K. Characterization
of ribavirin uptake systems in human hepatocytes. J Hepatol. 2010, 52, 486–492.
38. Derudas, M.; Brancale, A.; Naesens, L.; Neyts, J.; Balzarini, J.; McGuigan, C.
Application of the phosphoramidate ProTide approach to the antiviral drug
ribavirin. Bioorg Med Chem. 2010, 18, 2748-2755.
39. Dong, S. D.; Lin, C-C.; Schroeder, M. Synthesis and evaluation of a new
phosphorylated ribavirin prodrug. Antiviral Res. 2013, 99, 18-26.
40. Kryger, M. B. L..; Wohl, B. M.; Smith, A. A. A.; Zelikin, A. N. Macromolecular
prodrugs of ribavirin combat side effects and toxicity with no loss of activity of
the drug. Chem Comm. 2013, 49, 2643-2649.
162
41. Craparo, E. F.; Triolo, D.; Pitarresi, G.; Giammona, G.; Cavallaro, G.
Galactosylated Micelles for a Ribavirin Prodrug Targeting to Hepatocytes.
Biomacromolecules. 2013, 14, 1838-1849.
42. Witkowski, J. T.; Robins, R. K.; Khare, G. P.; Sidwell, R. W. Synthesis and
antiviral activity of 1,2,4-triazole-3-thiocarboxamide and 1,2,4-triazole-3-
carboxamidine ribonucleosides. J Med Chem. 1973, 16, 935–7.
43. Wang, G.; Sakthivel, K.; Rajappan, V.; Bruice, T. W.; Tucker, K.; Fagan, P.;
Brooks, J. L.; Hurd, T.; Leeds, J. M.; Cook, P. D. Synthesis of Azole nucleoside
5’-phosphate mimics (P1Ms) and their inhibitory properties of IMP
dehydrogenases. Nucleosides Nucleotides Nucleic Acids. 2004. 23, 317-337.
44. Fuertes, M.; Witkowski, J. T.; Streeter, D. G.; Robins, R. K. Synthesis and
Enzymatic Activity of 1,2,4-Triazole-3-carboxamide 6’-
Deoxyhomoribonucleoside-6’-phosphonic acid and related compounds. J Med
Chem. 1974, 17, 642-645.
45. Koh, Y-H.;
Shim, J. H.;
Wu, J. Z.; Zhong, W.; Hong, Z.; Girardet, J-L. Design,
Synthesis, and Antiviral Activity of Adenosine 5'′-Phosphonate Analogues as
Chain Terminators against Hepatitis C Virus. J Med Chem. 2005, 48, 2867-2875.
46. Raju, N.; Smee, D. F.; Robins, R. K.; Vaghefi, M. M. Synthesis and biological
properties of purine and pyrimidine 5′-deoxy-5′- (dihydroxyphosphinyl)-beta-D-
ribofuranosyl analogues of AMP, GMP, IMP, and CMP. J Med Chem. 1989, 32,
1307-1313.
47. Montgomery, J. A.; Hewson, K. The synthesis of phosphonic acid analogues of
purine ribonucleotides: an exception to the trans rule. Chem Commun. 1969, 15-
16.
48. Pradere, U.; Amblard, F.; Coats, S. J.; Schinazi, R. F. Synthesis of 5’-methylene
phosphonate furanonucleoside prodrugs: application to D-2’-deoxy-α-fluoro-2’-
β-C-methyl nucleosides. Org Lett. 2012, 14, 4426-4429.
49. Gallier, F.; Alexandre, J. A. C.; El Amri, C.; Deville-Bonne, D.; Peyrottes, S.;
Perigaud, C. 5',6'-nucleoside phosphonate analogues architecture: synthesis and
comparative evaluation towards metabolic enzymes. Chem Med Chem. 2011, 6,
1094.
50. Cosyn, L.; Van Calenbergh, S.; Joshi, B. V.; Ko, H.; Carter, R. L.; Harden, T. K.;
Jacobson, K. A. Synthesis and P2Y receptor activity of nucleoside 5'-phosphonate
derivatives. Bioorg Med Chem Lett. 2009, 19, 3002.
163
51. Jones, G. H.; Moffatt, J. G. The synthesis of 6'-deoxyhomonucleoside 6'-
phosphonic acids. J Am Chem Soc. 1968, 90, 5337.
52. Padyukova, N. S.; Karpeisky, M. Y.; Kolobushkina, L. I.; Mikhailov, S. N.
Oxidation of alkynes using PdCl
2
/CuCl
2
in PEG as a recyclable catalytic system:
one-pot synthesis of quinoxalines. Tetrahedron Lett. 1987, 28, 3623.
53. Mikhailov, S. N.; Paoyukova, N. S.; Karpeiskii, M. Y.; Kolobushkina, L. I.;
Beigelman, L. N. Use of 5-deoxy-ribo-hexofuranose derivatives for the
preparation of 5'-nucleotide phosphonates and homoribonucleosides. Collect
Czech Chem Commun. 1989, 54, 1055.
54. Ioannidis, P.; Classon, B.; Samuelsson, B.; Kvarnstrom, I. Synthesis of some 3',5'-
dideoxy-5'-C-phosphonomethyl nucleosides. Acta Chem Scand. 1991, 45, 746.
55. Tanaka, H.; Fukui, M.; Haraguchi, K.; Masaki, M.; Miyasaka, T. Cleavage of a
nucleosidic oxetane with carbanions: synthesis of a highly promising candidate
for anti-HIV agents — a phosphonate isostere of AZT 5′-phosphate. Tetrahedron
Lett. 1989, 30, 2567.
56. Hutter, D.; Blaettler, M. O.; Benner, S. A. From Phosphate to Bis(methylene)
Sulfone: Non-Ionic Backbone Linkers in DNA. Helv Chim Acta. 2002, 85, 2777.
57. Barral, K.; Priet, S.; De Michelis, C.; Sire, J.; Neyts, J.; Balzarini, J.; Canard, B.;
Alvarez, K. Synthesis and antiviral activity of boranophosphonate isosteres of
AZT and d4T monophosphates. Eur J Med Chem. 2010, 45, 849-856.
58. Barton, D. H. R.; Gero, S. D.; Quiclet-Sire, B.; Samadi, M. J. Radical addition to
vinyl phosphonates. A new synthesis of isosteric phosphonates and phosphonate
analogues of α-amino acids. Chem Soc Chem Commun. 1989, 1000-1001.
59. Barton, D. H. R.; Gero, S. D.; Quiclet- Sire, B.; Samadi, M. Stereoselectivity in
radical reactions of 2′-deoxynucleosides. A synthesis of an isostere of 3′-azido-3′-
deoxythymidine-5′-monophosphate (AZT-5′ monophosphate). Tetrahedron Lett.
1989, 30, 4969-4972.
60. Barton, D. H. R.; Gero, S. D.; Quiclet-Sire, B.; Samadi, M. New synthesis of
sugar, nucleoside and α-amino acid phosphonates. Tetrahedron. 1992, 48, 1627.
61. Frigerio, M.; Santagostino, M.; Sputore, S. A User-Friendly Entry to 2-
Iodoxybenzoic Acid (IBX). J Org Chem. 1999, 64, 4537-4538.
164
Chapter 7
Insights and Perspectives
7.1 Introduction
The work presented in this dissertation is comprised of the synthesis and antiviral activity
of various amino acid- and dipeptide-based prodrugs of acyclic nucleoside phosphonates,
as well as, the synthesis of phosphonate analogues of RBV. The overall goal within each
research project described above was to contribute to the field of antiviral research by
extending the prodrug approach developed in our lab to other acyclic nucleoside
analogues, as well as, to begin developing new active phosphonate drugs. This section
will serve to discuss the research findings presented in earlier chapters.
7.2 Synthetic approach to monoester prodrugs of PME/PMP parent drugs
In this chapter, a series of monoester prodrugs of acyclic nucleoside phosphonates PMEA
and (R)-PMPDAP were synthesized according to a three-step synthetic scheme designed
within the McKenna group. In regards to developing the synthetic strategy, a tactic
similar to the design of experiment (DOE) approach was utilized to optimize reaction
conditions toward synthesizing novel and attractive tyrosine-based monoester prodrugs of
PMEA and (R)-PMPDAP in high yields. As has been demonstrated in synthetic reactions
involving the conjugation of peptidomimetic promoieties with HPMP acyclic nucleoside
phosphonates ((S)-HPMPC and (S)-HPMPA), masking one of the negatively charged
phosphonic acid groups on PME and PMP is required in order to achieve conjugation of a
desired amino acid or dipeptide promoiety in high yields. Initially, extensive screening of
165
solvents, stoichiometric ratios, temperature and coupling reagents with regards to a
coupling reaction between a desired ANP and promoiety to produce a monoester prodrug
resulted in yields of 25% at best (according to
31
P NMR) at higher temperatures. This low
yield was observed with (L)-Ser-OMe and (L)-Tyr-OMe as promoieties and PMEA as the
parent drug. With the main hindrance in obtaining high yields for this reaction revolving
around formation of the stable PMEA-OBt monoester, higher temperatures only provided
a slightly increased reaction yield (according to
31
P NMR, 10% yield for rt reaction
mixtures and 25% yield for reactions run at 80 °C). Formation of the stable PMEA-OBt
monoester under all reaction conditions indicated the necessity for developing a different
pathway to activating the phosphonic acid group toward nucleophilic attack by the
desired promoiety. Utilizing the bromo derivative (PyBrOP) eliminates the presence of
the hydroxybenzotriazole ion (
-
OBt) in the reaction mixture and prevents formation of its
stable corresponding PMEA-OBt monoester.
Despite successful conjuation of a small alkoxy molecule (MeOH, EtOH or iPrOH) with
PMEA in the presence of PyBrOP, replacement of the nucleophile with an amino acid
promoiety did not result in the same outcome. However, nucleophilic attack of a
deprotonated amino acid promoiety with the monomethyl (ethyl or isopropyl) ester of
PMEA occurred to form the mixed PMEA diester. Selective dealkylation of the alkyl
group using the McKenna reaction affords the desired PMEA-promoiety monoester. It
was determined from these studies that successful esterification of OH-side chain
containing promoieties with HPMP ANPs requires basic reaction conditions in order to
166
allow for nucleophilic attack of the deprotonated OH-side chain of the promoiety on the
phosphonic acid group of the desired ANP. Attempts to utilize the acidic Mitsunobu
reaction conditions for phosphorus-containing parent compounds, which requires the
phosphonic acid group (P(O)(OH)
2
) on ANPs to be converted to the corresponding
dichloride (P(O)(Cl)
2
), does not deprotonate the desired nucleophile and therefore does
not allow for successful nucleophilic attack by OH-side chain containing promoieties.
These results indicate that despite structural differences between HPMP and PME/PMP
acyclic nucleoside phosphonates (PME/PMP parent drugs lack a hydroxymethylene
functional group on the phosphonomethoxy ether chain), both classes of ANPs require
masking one of the phosphonic acid groups through either intramolecular cyclization
(HPMP parent drugs) or a simple alkyl group (Et or iPr) in order to achieve successful
conjugation of the desired functional group (hydroxyl side chain) on the promoiety.
Based on this observation, future application of the McKenna prodrug approach to other
phosphonic acid drugs and their isoteres, may require an initial masking step if
introduction of the promoiety requires its nucleophilic attack on the phosphorus-
containing parent drug.
The alternative synthetic approach used to synthesize monoester PME/PMP prodrugs in
Chapter 2 was successfully employed to synthesize PMEO-DAPy monoester prodrugs
3.19-3.22, 3.25. The novel cyclic and acyclic HPMPO-DAPy prodrugs 3.29-3.31 were
synthesized according to a previously published synthetic procedure established in the
McKenna laboratory. The main alteration that was applied due to difficulties with the
167
one-pot coupling reaction was to use DCC coupling to mask one of the phosphonic acid
groups followed by conjugation of the desired promoiety using PyBOP.
7.3 PME/PMP monoester prodrugs and their antiviral activity
Based on Tables 2.1, 2.2 and 3.1, the common trend that is observed between structure
and antiviral activity indicates the (L)-Tyr-NHC
16
H
33
monoester prodrugs of PMEA
(Chapter 2) and PMEO-DAPy (Chapter 3) demonstrates the best antiviral activity.
Compounds 2.31-2.34 are all PMEA monoester prodrugs with varying promoieties (2.31
= HO-C
6
H
5
, 2.32-2.34 = (L)-Tyr-NH-Alk). PMEA itself demonstrates antiviral activity
against DNA viruses (mostly herpesviruses) and retroviruses (HBV and HIV). Based on
this, the enhanced observed antiviral activity against various DNA viruses (HSV-2, VZV,
CMV, VACV, CPXV and ADV) for the PMEA monoester tyrosine-based prodrugs
compared to PMEA is not surprising. The enhanced antiviral activity observed for 2.34
((L)-Tyr-NHC
16
H
33
-PMEA) across all viruses indicated in tables 2.1 and 2.2 (except for
ADV) and the lack of consistent enhanced antiviral activity for the other PMEA
monoester prodrugs indicates further studies to determine the reason for these
observations. A similar trend is seen among the monoester tyrosine-based prodrugs of
PMEO-DAPy (3.19-3.22). While their antiviral assessment was limited to the DNA virus
HCMV, according to the studies presented in Table 3.1, compound 3.21 ((L)-Tyr-
NHC
16
H
33
-PMEO-DAPy) demonstrated the best antiviral activity and selectivity in
comparison to other PMEO-DAPy tyrosine-based prodrugs. Included in this list is a Tyr-
Val dipeptide monoester PMEO-DAPy prodrug and the shorter N-alkyl chain length
168
monoester PMEO-DAPy prodrug ((L)-Tyr-NHC
8
H
17
PMEO-DAPy), which did not
demonstrate comparable antiviral activity to the C
16
counterpart. As was seen for PMEA
monoester prodrugs in Chapter 2, the (L)-Tyr-NHC
16
H
33
prodrug of PMEO-DAPy
outperformed the other tyrosine-based derivatives for this parent drug in this preliminary
in vitro study. 2.33 ((L)-Tyr-NHC
8
H
17
-PMEA) and 2.35 ((L)-Tyr-NHC
8
H
17
-(R)-
PMPDAP)) did not demonstrate antiviral activity against various DNA viruses (Tables
2.1 and 2.2). This would indicate that C
8
Tyr derivatives of PME/PMP compounds do not
show appreciable in vitro antiviral activity against DNA viruses.
7.4 Antiviral activity of the lipophilic N-alkyl Ser-Val dipeptide cHPMPC prodrug
Initial stability and antiviral studies of cyclic HPMP prodrugs containing (L)-serine, (L)-
tyrosine, (L)-cysteine or (L)-threonine amino acid promoieties indicated the tyrosine
promoiety to be the lead candidate to continue further development on. Continued
assessments were focused on the antiviral activity and stability of modifications on the C-
terminal of the tyrosine promoiety. This lead to studying a lipophilic N-alkyl chain of
varying length on the C-terminal, which demonstrated the (L)-Tyr-NHC
16
H
33
promoiety
to demonstrate enhanced antiviral activity. In the interest of studying the effect of the
lipophilic N-alkyl modification, another suitable promoiety, an (L)-Ser-NHC
16
H
33
-(L)-
Val dipeptide promoiety was synthesized and conjugated to (S)-HPMPC to produce a
novel lipophilic N-alkyl Ser-Val dipeptide cHPMPC prodrug. The antiviral activities of
(S)-HPMPC, cHPMPC and 4.9 ((L)-Ser-NHC
16
H
33
-(L)-Val-cHPMPC) against various
DNA viruses (HSV-2, VZV, CMV, VACV, CPXV and ADV) depict the N-alkyl
169
dipeptide to demonstrate significant antiviral activity against all DNA viruses. In
addition, 4.9 also demonstrated an elevated selectivity index against all DNA viruses
compared to the parent drug and cyclic parent drug. These preliminary in vitro results
indicate that the lipophilic N-alkyl approach may in fact be applicable for increasing the
antiviral activity and possibly even the stability of other amino acid and/or dipeptide
promoieties in addition to tyrosine.
7.5 Synthetic approaches to 5’-phosphonate analogue of ribavirin
In the synthesis of the 5’-phosphonate RBV analogue 6.2, introduction of the
phosphonate moiety consisted of performing the Michaelis-Arbuzov reaction. The
previously reported synthesis for this compound was used and carried out the Arbuzov
reaction with an iodine-alkylated 5’-OH position on ribavirin and triethyl phosphite. One
issue that prevented higher yields from being obtained for this step of the synthesis was
the use of an iodide ion (I
-
) as a leaving group for the 5’-OH position on the ribavirin
substrate. In typical Arbuzov reactions, chloride ion (Cl
-
) or bromide ion (Br
-
) are more
commonly used as a leaving group. Based on the
31
P NMR of the synthesis of 5’-I-
ribavirin, the presence of additional phosphorus peaks than the expected triethyl
phosphite starting material, desired product, and the by-product of this reaction indicated
the issue with the low yield for the reaction. One hypothesis is based on the formation of
the ethyl iodide after the nucleophilic phosphorus atom on triethyl phosphite kicks off
iodide from the ribavirin molecule and subsequently picks of an ethyl group from the
phosphonium-ribavirin intermediate to give the desired diester ribavirin phosphonate.
170
The issue with the formation of ethyl iodide is that in comparison to its Br and Cl
counterparts, this ethyl iodide by-product is heavier, which may prevent this by-product
from being volatile and light enough to leave the reaction vessel. Being heavier, ethyl
iodide will reside within the reaction mixture and react with triethyl phosphite to produce
an unwanted diethyl ethyl phosphonate. The reaction mixture was partitioned at 10 h with
one half being worked up and the other half being heated for an additional 10 h with
31
P
NMR being used to monitor reaction. The yield of the reaction did not increase the longer
the mixture was heated. It is possible the ethyl iodide by-product is competing with the
5’-I-ribavirin substrate. It would be interesting to substitute other leaving groups at the
5’-OH position of ribavirin to optimize the yields for this compound. In comparison, the
synthesis of the 5’-methylene phosphonate prodrug of ribavirin involved utilizing a
different synthetic method for introducing the phosphonate moiety due to the extension of
the carbon chain between the phosphorus moiety and the sugar ring. The Horner-
Wadsworth-Emmons (HWE) reaction was utilized, which consisted of first oxidizing the
5’-OH group on the ribose ring to an aldehyde using 2-iodoxybenzoic acid (IBX),
followed by introduction of the phosphonate with tetramethyl bisphosphonate in the
presence of NaH to afford the dimethyl olefinic phosphonate derivative of ribavirin
(6.13). Using this method required only filtration in between oxidation and introduction
of the phosphonate moiety and the distinct chemical shift of the olefinic protons in this
chemical transformation allowed for ease in monitoring the success of the reaction (by
1
H
NMR). Therefore, utilization of the HWE reaction allowed the straightforward, step-wise
synthesis of the 5’-methylene phosphonate derivative of ribavirin (6.3).
171
Bibliography
Aguzzi, C.; Cerezo, P.; Viseras, C.; Caramella, C., Use of clays as drug delivery systems:
Possibilities and limitations. Appl Clay Sci. 2007, 36, 22-36.
Aldern, K. A.; Ciesla, S. L.; Winegarden, K. L.; Hostetler, K. Y. Increased antiviral activity
of 1-O-hexadexyloxypropyl-[2-14C]cidofovir in MRC-5 human lung fibroblasts is
explained by unique cellular uptake and metabolism. Mol Pharm. 2003, 63, 678-681.
Anastasi, C.; Quelever, G.; Burlet, S.; Garino, C.; Souard, F.; Kraus, J. -L., New antiviral
nucleoside prodrugs await application. Curr Med Chem. 2003, 10, 1825-1843.
Annaert, P.; Kinget, R.; Naesens, L.; De Clercq, E.; Augustijns, P. Transport, uptake and
metabolism of the bis(pivaloyloxymethyl)-ester prodrug of 9-(2-
phosphonylmethoxyethyl)adenine in an in-vitro cell culture system of the intestinal
mucosa (Caco-2). Pharmaceutical Research. 1997, 14, 492-496.
Ariza, M. E. Current prodrug strategies for the delivery of nucleotides into cells. Drug Des.
Rev. 2005, 2, 373-387.
Ballatore, C.; McGuigan, C.; De Clercq, E.; Balzarini, J. Synthesis and evaluation of novel
amidate prodrugs of PMEA and PMPA. Bioorg Med Chem Lett. 2001. 11, 1053.
Balzarini, J.; Aquaro, S.; Perno, C-F.; Witvrouw, M.; Holý, A.; De Clercq, E. Activity of the
(R)-enantiomers of 9-(2-phosphonylmethoxypropyl)-adenine and 9-(2-
phosphonylmethoxypropyl)-2,6-diaminopurine against human immunodeficiency
virus in different human cell systems. Biochem Biophys Res Commun. 1996. 219,
337.
Balzarini, J.; Holý, A.; Jindrich, J.; Naesens, L.; Snoeck, R.; Schols, D.; De Clercq.
Differential antiherpesvirus and antiretrovirus effects of the (S) and (R) enantiomers
of acyclic nucleoside phosphonates: potent and selective in vitro and in vivo
antiretrovirus activities of (R)-9-(2-phosphonomethoxypropyl)-2,6-diaminopurine.
Antimicrob Agents Chemother. 1993. 37, 332.
Balzarini, J.; Pannecouque, C.; De Clercq, E.; Aquaro, S.; Perno, C-F.; Egberink, H.; Holý,
A. Antiretrovirus Activity of a Novel Class of Acyclic Pyrimidine Nucleoside
Phosphonates. Antimicrob Agents Chemother. 2002, 46, 2185-2193.
Barral, K.; Priet, S.; De Michelis, C.; Sire, J.; Neyts, J.; Balzarini, J.; Canard, B.; Alvarez, K.
Synthesis and antiviral activity of boranophosphonate isosteres of AZT and d4T
monophosphates. Eur J Med Chem. 2010, 45, 849-856.
172
Barton, D. H. R.; Gero, S. D.; Quiclet-Sire, B.; Samadi, M. J. Radical addition to vinyl
phosphonates. A new synthesis of isosteric phosphonates and phosphonate analogues
of α-amino acids. Chem Soc Chem Commun. 1989, 1000-1001.
Barton, D. H. R.; Gero, S. D.; Quiclet- Sire, B.; Samadi, M. Stereoselectivity in radical
reactions of 2′-deoxynucleosides. A synthesis of an isostere of 3′-azido-3′-
deoxythymidine-5′-monophosphate (AZT-5′ monophosphate). Tetrahedron Lett.
1989, 30, 4969-4972.
Barton, D. H. R.; Gero, S. D.; Quiclet-Sire, B.; Samadi, M. New synthesis of sugar,
nucleoside and α-amino acid phosphonates. Tetrahedron. 1992, 48, 1627.
Benarroch, D.; Egloff, M. P.; Mulard, L.; Guerreiro, C.; Romette, J. L.; Canard, B. J. A
structural basis for the inhibition of the NS5 dengue virus mRNA 2'-O-
methyltransferase domain by ribavirin 5'-triphosphate. Biol Chem. 2004, 279, 35638.
Benzaria, S.; Pelicano, H.; Johnson, R.; Maury, G.; Imbach, J.-L.; Aubertin, A.-M.; Obert,
G.; Gosselin, G. Synthesis, in vitro antiviral evaluation, and stability studies of bis(S-
acyl-2-thioethyl) ester derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine
(PMEA) as potential PMEA prodrugs with improved oral bioavailability. J Med
Chem. 1996, 39, 4958.
Bergamini, A.; Bolacchi, F.; Cepparulo, M.; Demin, F.; Uccella, I.; Bongiovanni, B.;
Ombres, D.; Angelico, F.; Liuti, A.; Hurtova, M.; Francioso, S.; Carvelli, C.;
Cerasari, G.; Angelico, M.; Rocchi, G. Treatment with ribavirin and interferon-α
reduces interferon-γ expression in patients with chronic hepatitis C. Clin Exp
Immunol. 2001, 123, 459.
Bhatt, S.; Gething, P. W.; Brady, O. J.; Messina, J. P.; Farlow, A. W.; Moyes, C. L.; Drake,
J. M.; Brownstein, J. S.; Hoen, A. G.; Sankoh, O.; Myers, M. F.; George, D. B.;
Jaenisch, G. R.; Wint, G. R. W.; Simmons, C. P.; Scott, T. W.; Farrar, J. J.; Hay, S. I.
The global distribution and burden of dengue. Nature. 2013, 496, 504–7.
Boshoff, C.; Chang, Y. Kaposi’s sarcoma-associated herpesvirus: a new DNA tumor virus.
Annu Rev Med. 2001, 52, 453–470.
Boshoff, C.; Schultz, T. F.; Kennedy, M. M.; Graham, A. K.; Fisher, C.; Thomas, A.;
McGee, J. O.; Weiss, R. A.; O’Leary, J. J. Kaposi’s sarcoma-associated herpesvirus
infects endothelial and spindle cells. Nat Med. 1995, 1, 1274-1278.
Brantley, J. S.; Hicks, L.; Sra, K.; Tyring, S. T. Valacyclovir for the treatment of genital
herpes. Expert Rev Anti Infect Ther. 2006, 4, 367–76.
173
Brulois, K. F.; Chang, H.; Le, A. S-Y.; Ensser, A.; Wong, L-Y.; Toth, Z.; Lee, S. H.; Lee, H-
R.; Myoung, J.; Ganem, D.; Oh, T-K.; Kim, J. F.; Gao, S-H.; Jung, J. U. Construction
and Manipulation of New Kaposi’s Sarcoma-Associated Herpesvirus Bacterial
Artificial Chromosome Clone. J Virol. 2012, 86, 9708-9720.
Brulois, K.; Toth, Z.; Wong, L-Y.; Feng, P.; Gao, S-J; Ensser, A.; Jung, J. U. Kaposi’s
Sarcoma-Associated Herpesvirus K3 and K5 Ubiquitin E3 Ligases Have Stage-
Specific Immune Evasion Roles during Lytic Replication. J Virol. 2014, 88, 9335-
9349.
Brunelle, M. N.; Lucifora, J.; Neyts, J.; Villet, S.; Holý, A.; Trepo, C.; Zoulim, F. In Vitro
Activity of 2,4-Diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine against Multi-
Drug-Resistant Hepatitis B Virus Mutants. Antimicrob Agents Chemother. 2007, 51,
2240-2243.
Cahard, D.; McGuigan, C.; Balzarini, J. Aryloxy phosphoramidate triesters as protides. Mini-
Rev Med Chem. 2004, 4, 371-381.
Campagne, J-M.; Coste, J.; Jouin, P. (IH-benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate- and (1H-benzotriazol-
1-1yloxy)tripyrrolidinophosphonium hexafluorophosphate-mediated activation of
monophosphonate esters: synthesis of mixed phosphonate diesters, the reactivity of
the benzotriazolyl phosphonic esters vs. the reactivity of the benzotriazolyl carboxylic
esters. J Org Chem. 1995, 60, 5214-5223.
Carroll, S. S.; Ludmerer, S.; Handt, L.; Koeplinger, K.; Zhang, N. R.; Graham, D.; Davies,
M. E.; MacCoss, M.; Hazuda, D.; Olsen, D. B. Robust antiviral efficacy upon
administration of a nucleoside analog to hepatitis C virus-infected chimpanzees.
Antimicrob Agents Chemother. 2009, 53, 926–934.
Carroll, S. S.; Olsen, D. B. Nucleoside analog inhibitors of hepatitis C virus replication.
Infect Disord Drug Targets. 2006, 6, 17–29.
Carroll, S. S.; Tomassini, J. E.; Bosserman, M.; Getty, K.; Stahlhut, M. W.; Eldrup, A. B.;
Bhat, B.; Hall, D.; Simcoe, A. L.; LaFemina, R.; Rutkowski, C. A.; Wolanski, B.;
Yang, Z.; Migliaccio, G.; De Francesco, R.; Kuo, L. C.; MacCoss, M.; Olsen, D. B.
Inhibition of hepatitis C virus RNA rep- lication by 2ჼ-modified nucleoside analogs.
J Biol Chem. 2003, 278, 11979–11984.
Chang H.; Wachtman, L. M; Pearson, C. B.; Lee, J-S.; Lee, H-R.; Lee, S. H.; Vieira, J.;
Mansfield, K.G.; Jung, J. U. Non-human primate model of Kaposi’s sarcoma-
associated herpesvirus infection. PLoS Pathog. 2009, 5(10), e1000606, 1-10.
174
Chang, J.; Schul, W.; Butters, T. D.; Yip, A.; Liu, B.; Goh, A.; Lakshminarayana, S. B.;
Alonzi, D.; Reinkensmeier, G.; Pan, X.; Qu, X.; Weidner, J. M.; Wang, L.; Yu, W.;
Borune, N.; Kinch, M. A.; Rayahin, J. E.; Moriarity, R.; Xu, X.; Shi, P-Y.; Guo, J-T.;
Block, T. M. Combination of α-Glucosidase Inhibitor and Ribavirin for the
Treatment of Dengue Virus Infection In Vitro and In Vivo. Antiviral Res. 2011, 89,
26-34.
Chen, Y-L.; Yin, Z.; Duraiswamy, J.; Schul, W.; Lim, C. C.; Liu, B.; Xu, H. Y.; Qing, M.;
Yip, A.; Wang, G.; Chan, W. L.; Tan, H. P.; Lo, M.; Liung, S.; Kondreddi, R. R.;
Rao, R.; Gu, H.; He, H.; Keller, T. H.; Shi, P-Y. Inhibition of Dengue Virus RNA
Synthesis by an Adenosine Nucleoside. Antimicrob Agents Chemother. 2010, 54,
2932-2939.
Chen, Y-L.; Yin, Z.; Lakshminarayana, S. B.; Qing, M.; Schul, W.; Duraiswamy, J.;
Kondreddi, R. R.; Goh, A.; Xu, H. Y.; Yip, A.; Liu, B.; Weaver, M.; Dartois, V.;
Keller, T. H.; Shi, P-Y. Inhibition of Dengue Virus by an Ester Prodrug of an
Adenosine Analog. Antimicrob Agents Chemother. 2010, 54, 3255-3261.
Chou, S. Cytomegalovirus UL-97 mutations in the era of ganciclovir and maribavir. Rev Med
Virol. 2008, 18, 233-246.
Ciesla, S. L.; Trahan, J.; Wan, W. B.; Beadle, J. R.; Aldern, K. A.; Painter, G. R.; Hostetler,
K. Y. Esterification of cidofovir and alkylalkanols increases oral bioavailability and
diminishes drug accumulation in kidney. Antiviral Res. 2003, 59, 163-171.
Cihlar, T.; Chen, M. S. Identification of enzymes catalyzing two-step phosphorylation of
cidofovir and the effect of cytomegalovirus infection on their activities in host cells.
Mol Pharmacol. 1996, 50, 1502-1510.
Clyde, K.; Kyle, J. L.; Harris, E. Recent advances in deciphering viral and host determinants
of dengue virus replication and pathogenesis. J Virol. 2006, 80, 11418–11431.
Coen, N.; Duraffour, S.; Naesens, L.; Krečmerová, M.; Van den Oord, J.; Snoeck, R.;
Andrei, G. Evaluation of Novel Acyclic Nucleoside Phosphonates against Human and
Animal Gammaherpesviruses Revealed an Altered Metabolism of Cyclic Prodrugs
upon Epstein-Barr Virus Reactivation in P3HR-1 Cells. J Virol. 2013, 87, 12422-
12432.
Cosyn, L.; Van Calenbergh, S.; Joshi, B. V.; Ko, H.; Carter, R. L.; Harden, T. K.; Jacobson,
K. A. Synthesis and P2Y receptor activity of nucleoside 5'-phosphonate derivatives.
Bioorg Med Chem Lett. 2009, 19, 3002.
175
Craparo, E. F.; Triolo, D.; Pitarresi, G.; Giammona, G.; Cavallaro, G. Galactosylated
Micelles for a Ribavirin Prodrug Targeting to Hepatocytes. Bio Macromol. 2013, 14,
1838-1849.
Crotty, S.; Cameron, C. E.; Andino, R. RNA virus error catastrophe: Direct molecular test by
using ribavirin. Proc Natl Acad Sci U.S.A. 2001, 98, 6895.
Crotty, S.; Cameron, C.; Andino, R. Ribavirin’s antiviral mechanism of action: lethal
mutagenesis? J Mol Med. 2002, 80, 86–95.
Crotty, S.; Maag, D.; Arnold, J. J.; Zhong, W.; Lau, J. Y. N.; Hong, Z.; Andino, R.;
Cameron, C. E. The Broad Spectrum Antiviral Ribonucleoside Ribavirin is an RNA
Virus Mutagen. Nat Med. 2000, 6, 1375.
Cundy, K. C. Clinical pharmacokinetics of the antiviral nucleotide analogs cidofovir and
adefovir. Clin Pharmacokinet. 1999. 36, 127-143.
De Clercq, E. Biochemical aspects of selective antiherpes activity of nucleoside analogues.
Biochem Pharmacol. 1984, 33, 2159-2169.
De Clercq, E. Antiviral Agents. In Scientific Basis of Antimicrobial Chemotherapy.
Greenwood, D.; O’Grady, F., Eds.; Symposium of the Society for General
Microbiology. Cambridge University Press. Cambridge, 1995. pp 155-184.
De Clercq, E. Broad-spectrum anti-DNA virus and anti-retrovirus activity of
phosphonylmethoxyalkylpurines and pyrimidines. Biochem Pharmacol. 1991, 42,
963-972.
De Clercq, E. Therapeutic of HPMPC as an antiviral drug. Rev Med Virol. 1993, 3, 85-96.
De Clercq, E. In search of a selective antiviral chemotherapy. Clin Microbiol Rev. 1997, 10,
674-693.
De Clercq, E. Towards an effective chemotherapy of virus infections: Therapeutic potential
of cidofovir [(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, HPMPC] for
the treatment of DNA virus infections. Collect Czech Chem Commun. 1998, 63, 480-
506.
De Clercq, E. Cidofovir in the therapy and short-term prophylaxis of poxvirus infections.
Trends Pharmacol Sci. 2002, 23, 456-458.
De Clercq, E. Potential of acyclic nucleoside phosphonates in the treatment of DNA virus
and retrovirus infections. Expert Rev Antiinfect Ther. 2003, 1, 21–43.
176
De Clercq, E. Clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir,
and tenofovir in treatment of DNA virus and retrovirus infections. Clin Microbiol
Rev. 2003, 16, 4, 569-596.
De Clercq, E. Antivirals and antiviral strategies. Nature Rev Microbiol. 2004, 2, 704.
De Clercq, E. Acyclic nucleoside phosphonates: past, present, and future. Bridging chemistry
to HIV, HBV, HCV, HPV, adeno-, herpes-, and poxvirus infections: The phosphonate
bridge. Biochem Pharmacol. 2007, 73, 911-922.
De Clercq, E. The Discovery of Antiviral Agents: Ten Different Compounds, Ten Different
Stories. Med Res Rev. 2008, 28, 929-953.
De Clercq, E.; Andrei, G.; Balzarini, J.; Leyssen, P.; Naesens, L.; Neyts, J.; Pannecouque,
C.; Snoeck, R.; Ying, C.; Hocková, D.; and Holý, A. Antiviral Potential of a New
Generation of Acyclic Nucleoside Phosphonates, the 6-[2-
(phosphonomethoxy)alkoxy]-diaminopyrimidines. Nucleosides Nucleotides Nucleic
Acids. 2005, 24, 331-341.
De Clercq, E.; Descamps, J.; De Somer, P.; Holý, A. (S)-9-(2,3-dihydroxypropyl)adenine:
Aliphatic nucleoside analog with broad-spectrum anti- viral activity. Science. 1978,
200, 563-565.
De Clercq, E.; Field, H. J. Antiviral prodrugs: the development of successful prodrug
strategies for antiviral chemotherapy. Brit J Pharmacol. 2006, 147, 1–11.
De Clercq, E. Milestones in the discovery of antiviral agents: nucleosides and nucleotides.
Acta Pharmaceut Sinica B. 2012, 2, 535-548.
De Clercq, E., Holý, A., Rosenberg, I., Sakuma, T., Balzarini, J., Maudgal, P.C. A Novel
Selective Broad Spectrum Anti-DNA Virus Agent. Nature. 1986, 323, 464-467.
De Clercq, E.; Holý, A., Acyclic nucleoside phosphonates: A key class of antiviral drugs. Nat
Rev Drug Disco. 2005, 4, 928-940.
De Clercq, E.; Neyts, J. Antiviral agents acting as DNA and RNA chain terminators. Handb
Exp Pharmacol. 2009, 189, 53-84.
De Clercq, E.; Sakuma, T.; Baba, M.; Pauwels, R.; Balzarini, J.; Rosenberg, I.; Holý, A.
Antiviral Activity of Phosphonylmethoxyalkyl Derivatives of Purine and
Pyrimidines. Antiviral Res. 1987, 8, 261-272.
Deeks, S. G.; Barditch-Crovo, P.; Lietman, P. S.; Hwang, F.; Cundy, K. C.; Rooney, J. F.;
Hellmann, N. S.; Safrin, S.; Kahn, J. O. Safety, Pharmacokinetics, and Antiretroviral
177
Activity of Intravenous 9-[2-(R)-(Phosphonomethoxy)propyl]adenine, a Novel Anti-
Human Immunodeficiency Virus (HIV) Therapy, in HIV-Infected Adults. Antimicrob
Agents Chemother. 1998, 42, 2380.
Denny, B. J.; Wheelhouse, R. T.; Stevens, M. F. G.; Tsang, L. L. H.; Slack, K. A. NMR and
Molecular modeling investigations of the mechanism of activation of the anti-tumor
drug Temozolomide and its interaction with DNA. Biochemistry. 1994, 33, 9045-51.
Derudas, M.; Brancale, A.; Naesens, L.; Neyts, J.; Balzarini, J.; McGuigan, C. Application of
the phosphoramidate ProTide approach to the antiviral drug ribavirin. Bioorg Med
Chem. 2010, 18, 2748-2755.
Dittmer, D. P; Damania, B. Dittmer, D. P; Damania, B. KSHV-Associated Disease in AIDS
Patient. In Aids-Associated Viral Oncogenesis. C. Meyers., Eds.; Cancer Treatment
and Research. 2007, 133, 129-139.
Dolin, R. Antiviral chemotherapy and chemoprophylaxis. Science. 1985, 227, 1296-1303.
Dong, S. D.; Lin, C-C.; Schroeder, M. Synthesis and evaluation of a new phosphorylated
ribavirin prodrug. Antiviral Res. 2013, 99, 18-26.
Du, M. Q.; Bacon, C. M.; Isaacson, P. G. Kaposi sarcoma-associated herpesvirus/human
herpesvirus 8 and lymphoproliferative disorders. J Clin Pathol. 2007, 60, 1350-1357.
Duzgunes, N.; Simoes, S.; Slepushkin, V.; Pretzer, E.; Flasher, D.; Salem, I. I.; Steffan, G.;
Konopka, K.; Pedroso de Lima, M. C., Delivery of antiviral agents in liposomes.
Methods Enzymol. 2005, 391, 351-373.
Dvořáková, H.; Holý, A.; Alexander, P. Synthesis and biological effects of 9-(3-hydroxy-2-
phosphonomethoxypropyl) derivatives of deazapurine bases. Collect Czech Chem
Commun. 1993, 58, 1403-1418.
Dykstra, R. R.; In Encyclopedia of Reagents for Organic Synthesis. Paquette, L. A., Ed.;
John Wiley and Sons Ltd., Chichester, UK. 1995, 4, 2668.
Elion, G. B.; Furman, P. A.; Fyfe, J. A.; de Miranda, P.; Beauchamp, L.; Schaeffer, H. J.
Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl)guanine.
Proc Natl Acad Sci USA. 1977, 74, 5716–20.
Eriksson, B.; Helgstrand, E.; Johansson, N. G.; Larsson, A.; Misiorny, A.; Noren, J. O.;
Philipson, L.; Stenberg, K.; Stening, G.; Stridh, S.; Oberg, B. Inhibition of Influenza
Virus Ribonucleic Acid Polymerase by Ribavirin Triphosphate. Antimicrob Agents
Chemother. 1977, 11, 946.
178
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.
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 Pharm. 2008, 5, 598-609.
Erion, M. D.; Reddy, K. R.; Boyer, S. H.; Matelich, M. C.; Gomez-Galeno, J.; Lemus, R. H.;
Ugarkar, B. G.; Colby, T. J.; Schanzer, J.; van Poelje, P. D. Design, synthesis, and
characterization of a series of cytochrome P(450) 3A-activated prodrugs (HepDirect
prodrugs) useful for targeting phosph(on)ate-based drugs to the liver. J Am Chem Soc.
2004, 126, 5154.
Ettmayer, P.; Amidon, G. L.; Clement, B.; Testa, B. Lessons learned from marketed and
investigational prodrugs. J Med Chem. 2004, 47, 2393-2404.
Farquar, D.; Kuttesch, N. J.; Wilkerson, M. G.; Winkler, T. Synthesis and biological
evaluation of neutral derivatives of 5-fluoro-2’-deoxyuridine 5’-phosphate. J Med
Chem. 1983, 26, 1153-1158.
Field, A. K.; Biron, B. B. The end of innocence revisited: resistance of herpesvirus to
antiviral drugs. Clin Microbiol Rev. 1994, 7, 1-13.
Freeman, S.; Ross, K. C. Prodrug design for phosphates and phosphonates. Prog Med Chem.
1997, 34, 111-147.
Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M-N.; Joun, P. PyBOP AND PyBrop: Two
reagents for the difficult coupling of the α,α-dialkyl amino acid, Aib. Tetrahedron.
1991, 47, 259-270.
Friedrichs, C.; Neyts, J.; Gaspar, G.; De Clercq, E.; Wutzler, P. Evaluation of antiviral
activity against human herpesvirus 8 (HHV-8) and Epstein-Barr virus (EBV) by a
quantitative real-time PCR assay. Antiviral Res. 2004, 62, 121–123.
Frigerio, M.; Santagostino, M.; Sputore, S. A User-Friendly Entry to 2-Iodoxybenzoic Acid
(IBX). J Org Chem. 1999, 64, 4537-4538.
Fuertes, M.; Witkowski, J. T.; Streeter, D. G.; Robins, R. K. Synthesis and Enzymatic
Activity of 1,2,4-Triazole-3-carboxamide 6’-Deoxyhomoribonucleoside-6’-
phosphonic acid and related compounds. J Med Chem. 1974, 17, 642-645.
179
Fukuchi, Y.; Furihata, T.; Hashizume, M.; Iikura, M.; Chiba, K. Characterization of ribavirin
uptake systems in human hepatocytes. J Hepatol. 2010, 52, 486–492.
Gallier, F.; Alexandre, J. A. C.; El Amri, C.; Deville-Bonne, D.; Peyrottes, S.; Perigaud, C.
5',6'-nucleoside phosphonate analogues architecture: synthesis and comparative
evaluation towards metabolic enzymes. Chem Med Chem. 2011, 6, 1094.
Ganem, D. KSHV and the pathogenesis of Kaposi Sarcoma: listening to human biology and
medicine. J Clin Invest. 2010, 120, 939-949.
Girsch, N.; Pertenbreiter, F.; Balzarini, J.; Meier, C. 5-(1-acetoxyvinyl)-cycloSaligenyl-2’3’-
dideoxy-2’,3’-didehydrothymidine monophosphates, a second type of new,
enzymatically activated cycloSaligenyl pronucleotides. J Med Chem. 2008, 51, 8115-
8123.
Graci, J. D.; Cameron, C. E. Quasispecies, Error Catastrophe, and the Antiviral Activity of
Ribavirin. Virology. 2002, 298, 175-180.
Griengl, H.; Hayden, W.; Penn, G.; De Clercq, E.; Rosenwirth, B. Phosphonoformate and
phosphonoacetate derivatives of 5-substituted 2- deoxyuridines: Synthesis and
antiviral activity. J Med Chem. 1988, 31, 1831-1839.
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.
Gross, H.; Keitel, I.; Coistella, B.; McKenna, C. E. Synthesis of Acid-labile Geminal Bis-
and Triphosphonic acids. Phosphorus Sulfur Silicon and Rel Elem. 1991, 61, 177.
Guzman, M. G.; Halstead, S. B.; Artsob, H.; Buchy, P.; Farrar, J.; Gubler, D. J.; Hunsperger,
E.; Kroeger, A.; Margolis, H. S.; Martinez, E.; Nathan, M. B.; Pelegrino, J. L;
Simmons, C.; Yoksan, S.; Peeling, R. W. Dengue: a continuing global threat. Nat Rev
Microbiol. 2010, 8, S7–S16.
Hadziyannis, S. J.; Tassopoulos, N. C.; Heathcote, E. J.; Chang, T. T.; Kitis, G.; Rizzetto,
M.; Marcellin, P.; Lim, S. G; Goodman, Z.; Ma, J.; Arterburn, S.; Xiong, S; Currie,
G.; Brosgart, C. L. Long-term therapy with adefovir dipivoxil for HBeAg-negative
chronic hepatitis B. N Engl J Med. 2005, 352, 2673-2681.
Hecker, S. J.; Erion, M. D. Prodrugs of Phosphates and Phosphonates. J Med Chem. 2008,
51, 2328-2345.
Herman, B. D.; Votruba, I.; Holý, A.; Sluis-Cremer, N.; Balzarini, J. The Acyclic 2,4-
Diaminopyrimidine Nucleoside Phosphonate Acts as a Purine Mimetic in HIV-1
Reverse Transcriptase DNA Polymerization. J Biol Chem. 2010, 285, 12101-12108.
180
Hermann, E. C. Plaque inhibition test for detection of specific inhibitors of DNA containing
viruses. Proc Soc Exp Biol Med. 1961, 107, 142-145.
Hillaireau, H.; Le Doan, T.; Appel, M.; Couvreur, P. Hybrid polymer nanocapsules enhance
in vitro delivery of azidothymidine-triphosphate to macrophages. J Controlled
Release 2006, 116, 346-352.
Ho, H. T.; Woods, K. L.; Bronson, J. J; De Boeck, H.; Martin J. C.; Hitchcock, M. J.
Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-
(phosphonylmethoxy)propyl]cytosine. Mol Pharmacol. 1992, 41, 197–202 .
Hocková, D.; Holý, A.; Masojídková, M.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J.
5-Substituted-2,4-Diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidines – Acyclic
Nucleoside Phosphonate Analogues with Antiviral Activity. J Med Chem. 2003, 46,
5064-5073.
Holý, A. Synthesis and biological activity of isopolar acyclic nucleotide analogs. [In: Recent
Advances in Nucleosides: Chemistry and Chemotherapy.] Chu, C.K., Ed.; Elsevier.
2002, 167-238.
Holý, A. Phosphonomethoxyalkyl analogs of nucleotides. Curr Pharm Des. 2003, 9, 2567-
2592.
Holý, A. Antiviral acyclic nucleoside phosphonates structure activity studies. Antiviral Res.
2006, 71, 248-253.
Holý, A.; Buděšínský, M.; Podlaha, J.; Císařová, I. Synthesis of N1-[2-
(phosphonomethoxy)ethyl] derivatives of 2,4-diaminopyrimidine and related acyclic
nucleotide analogues. Collect Czech Chem Comm. 1999. 64, 242-256.
Holý, A.; Dvořáková, H.; Jindřich, J.; Masojídková, M.; Buděšínský, M.; Balzarini, J.;
Andrei, G. & De Clercq, E. Acyclic nucleotide analogs derived from 8-azapurines:
synthesis and antiviral activity. J Med Chem. 1996, 39, 4073-4088.
Holý, A.; Gunter, J.; Dvořáková, H.; Masojídková, M.; Andrei, G.; Snoeck, R.; Balzarini, J.;
De Clercq, E. Structure-antiviral activity relationship in the series of pyrimidine and
purine N-[2-(2-phosphonomethoxy)ethyl] nucleotide analogues. 1. Derivatives
substituted at the carbon atoms of the base. J Med Chem. 1999. 42, 2064-2086.
Holý, A.; Masojídková, M. Synthesis of enantiomeric N-(2-phosphonylmethoxypropyl)
derivatives of purine and pyrimidine bases I. The stepwise approach. Collect Czech
Chem Comm. 1995, 60, 1196-1212.
181
Holý, A.; Rosenberg, I. Synthesis of 9-(2-phosphonylmethoxyethyl)adenine and related
compounds. Collect Czech Chem Commun. 1987, 52, 2792-2809.
Holý, A.; Rosenberg, I.; Dvořáková, H. Synthesis of N-(2-phosphonylmethoxyethyl)
derivatives of heterocyclic bases. Collect Czech Chem Comm. 1989, 54, 2190-2207.
Holý, A.; Votruba, I.; Tloustova, E.; Masojídková, M. Synthesis and Cytostatic Activity of
N-[2-(Phosphonomethoxy)alkyl] Derivatives of N
6
-Substituted Adenines, 2,6-
Diaminopurines and Related Compounds. Collect Czech Chem Comm. 2001, 66,
1545-1592.
Holý, A.; Votruba, I.; Masojídková, M.; Andrei, G.; Snoeck, R.; Naesens, L.; De Clercq, E.;
Balzarini, J. 6-[2-(Phosphonomethoxy)alkoxy]pyrimidines with Antiviral Activity. J
Med Chem. 2002, 45, 1918-1929.
Hostetler, K. Y. Synthesis and antiviral evaluation of broad spectrum, orally active analogs
of cidofovir and other acyclic nucleoside phosphonates. Adv. Antivir. Drug Des.
2007, 5, 167-184.
Hostetler, K. Y. Alkoxyalkyl prodrugs of acyclic nucleoside phosphonates enhance oral
antiviral activity and reduce toxicity: current state of the art. Antiviral Res. 2009, 82,
A84-A98.
Hultgren, C.; Milich, D. R.; Weiland, O.; Sallberg, M. The antiviral compound ribavirin
modulates the T helper (Th) 1/Th2 subset balance in hepatitis B and C virus-specific
immune responses. J Gen Virol. 1998, 79, 2381.
Hutter, D.; Blaettler, M. O.; Benner, S. A. From Phosphate to Bis(methylene) Sulfone: Non-
Ionic Backbone Linkers in DNA. Helv Chim Acta. 2002, 85, 2777.
Huttunen, K. M.; Rautio, J. Prodrugs – An Efficient Way to Breach Delivery and Targeting
Barriers. Curr Top Med Chem. 2011, 11, 2265-2287.
Iyer, R. P.; Phillips, L. R.; Biddle, J. A.; Thakker, D. R.; Egan, W.; Aoki, S.; Mitsuya, H.
Synthesis and acyloxyalkyl acylphosphonates as potential prodrugs of the antiviral,
trisodium phosphonoformate (foscarnet sodium). Tetrahedron Lett. 1989, 30, 7141-
7144.
Inoue, Y.; Homma, M.; Matsuzaki, Y.; Shibata, M.; Matsumura, T.; Ito, T.; Kohda, Y.
Erythrocyte ribavirin concentration for assessing hemoglobin reduction in interferon
and ribavirin combination therapy. Hepatol Res. 2006, 34, 23–27.
Ioannidis, P.; Classon, B.; Samuelsson, B.; Kvarnstrom, I. Synthesis of some 3',5'-dideoxy-
5'-C-phosphonomethyl nucleosides. Acta Chem Scand. 1991, 45, 746.
182
James, S. H.; Price, N. B.; Hartline, C. B; Lanier, E. R.; Prichard, M. N. Selection and
Recombinant Phenotyping of Novel CMX001 and Cidofovir Resistance Mutation in
Human Cytomegalovirus. Antimicrob Agents. Chemother. 2013, 57, 3321-3325.
Jochum, A.; Schlienger, N.; Egron, D.; Peyrottes, S.; Periguad, C. Biolabile constructs for
pronucleotide design. J Organomet Chem. 2005, 690, 2614-2625.
Jones, G. H.; Moffatt, J. G. The synthesis of 6'-deoxyhomonucleoside 6'-phosphonic acids. J
Am Chem Soc. 1968, 90, 5337.
Jones T.; Ye, F.; Bedolla, R.; Huang, Y.; Meng, J.; Qian, L.; Pan, H.; Zhou, F.; Moody, R.;
Wagner, B.; Arar, M.; Gao, S. J. Direct and efficient cellular transformation of
primary rat mesenchymal precursor cells by KSHV. J Clin Invest. 2012, 122, 1076 -
1081.
Jordheim, L. P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the development of
nucleoside and nucleotide analogues for cancer and viral diseases. Nat Rev Drug
Discov. 2013, 12, 447-64.
Kaufman, H. E. Clinical cure of herpes simplex keratitis by 5’-iodo-2’-deoxyuridine. Proc
Soc Exp Biol Med. 1962, 109, 251-253.
Kaufman, H. E.; Heidelberger, C. Therapeutic antiviral action of 5-trifluoromethyl-2’-
deoxyuridine in herpes simplex keratitis. Science. 1964, 145, 585-586.
Kelly, S. J.; Dardinger, D. E.; Butler, L. G. Hydrolysis of phosphonate esters catalyzed by 5’-
nucleotide phosphodiesterase. Biochemistry. 1975, 14, 4983-4988.
Kelly, S. J; Butler, L. G. Enzymatic hydrolysis of phosphonate esters. Reaction mechanism
of intestinal 5’-nucleotide phosphodiesterase. Biochemistry, 1977, 16, 1102-1104.
Klumpp, K.; Leveque, V.; Le Pogam, S.; Ma, H.; Jiang, W. R.; Kang, H.; Granycome, C.;
Singer, M.; Laxton, C.; Hang, J. Q.; Sarma, K.; Smith, D. B.; Heindl, D.; Hobbs, C.
J.; Merrett, J. H.; Symons, J.; Cammack, N.; Martin, J. A.; Devos, R.; Najera, I. The
novel nucleoside analog R1479 (4-azido-cytidine) is a potent inhibitor of NS5B-
dependent RNA synthesis and hepatitis C virus replication in cell culture. J Biol
Chem. 2006, 281, 3793–3799.
Klumpp, K.; Kalayanov, G.; Ma, H.; Le Pogam, S.; Leveque, V.; Jiang, W. R.; Inocencio, N.;
De Witte, A.; Rajyaguru, S.; Tai, E.; Chanda, S.; Irwin, M. R.; Sund, C.; Winqist, A.;
Maltseva, T.; Eriksson, S.; Usova, E.; Smith, M.; Alker, A.; Najera, I.; Cammack, N.;
Martin, J. A.; Johansson, N. G.; Smith, D. B. 2-Deoxy-4-azido nucleoside analogs are
highly potent inhibitors of hepatitis C virus replication despite the lack of 2-alpha-
hydroxyl groups. J Biol Chem. 2008, 283, 2167–2175.
183
Koff, W. C.; Elm Jr., J. L.; Halstead, S. B. Antiviral effects of ribavirin and 6-mercapto-9-
tetrahydro-2-furylpurine against dengue virus in vitro. Antiviral Res. 1982, 2, 69-79.
Koh, Y-H.;
Shim, J. H.;
Wu, J. Z.; Zhong, W.; Hong, Z.; Girardet, J-L. Design, Synthesis,
and Antiviral Activity of Adenosine 5'′-Phosphonate Analogues as Chain Terminators
against Hepatitis C Virus. J Med Chem. 2005, 48, 2867-2875.
Kramata, P.; Votruba, I.; Otova, B.; Holý, A. Different Inhibitory Potencies of Acyclic
Phosphonomethoxyalkyl Nucleotide Analogs Toward DNA Polymerases alpha, delta
and epsilon. Mol Pharmacol. 1996, 49, 1005-1011.
Krause, M.; Stark, H.; Schunack, W. Azomethine prodrugs of (R)-I±-methylhistamine, a
highly potent and selective histamine H3-receptor agonist. Curr Med Chem. 2001, 8,
1329-1340.
Krečmerová, M.; Holý, A.; Andrei, G.; Pomeisl, K.; Tichý, T.; Břehová, P.; Masojídková,
M.; Dracinsky, M.; Pohl, R.; Laflamme, G.; Naesens, L.; Hui, H.; Cihlar, T.; Neyts,
J,; De Clercq, E.; Balzarini, J.; Snoeck, R. Synthesis of ester Prodrugs of 9-(S)-[3-
Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) as Anti-
Poxvirus Agents. J Med Chem. 2010, 53, 6825-6837.
Krečmerová, M.; Jansa, P.; Dracinsky, M.; Sazelova, P.; Kasicka, V.; Neyts, J.; Auwerx, J.;
Kiss, E.; Goris, N.; Stephan, G.; Janeba, Z. 9-[2-(R)-(Phosphonomethoxy)propyl]-
2,6-diaminopurine (R)-PMPDAP and its prodrugs: Optimized preparation, including
identification of by-products formed, and antiviral evaluation in vitro. Bioorg Med
Chem. 2013. 21, 1199.
Krečmerová, M.; Holý, A.; Pískala, A.; Masojídková, M.; Andrei, G.; Naesens, L.; Neyts, J.;
Balzarini, J.; De Clercq, E.; Snoeck, R. Antiviral activity of triazine analogues of 1-
(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (cidofovir) and related
compounds. J Med Chem. 2007, 50, 1069-1077.
Krečmerová, M.; Holý, A.; Pohl, R.; Masojídková, M.; Andrei, G.; Naesens, L.; Neyts, J.;
Balzarini, J.; De Clercq, E.; Snoeck, R. Ester Prodrugs of Cyclic 1-(S)-[3-Hydroxy-2-
(phosphonomethoxy)propyl]-5-azacytosine: Synthesis and Antiviral Activity. J Med
Chem. 2007, 50, 5765-5772.
Krejčová, R.; Horska, K.; Votruba, I.; Holý, A. Phosphorylation of Purine
(Phosphonomethoxy)alkyl Derivatives by Mitochondrial AMP Kinase (AK2 Type)
from L1210 Cells. Collect Czech Chem Commun. 2000, 65, 1653-1668.
184
Krejčová, R.; Horska, K.; Votruba, I.; Holý, A. Interaction of Guanine
Phosphonomethoxyalkyl Derivatives with GMP Kinase Isoenzymes. Biochem
Pharmacol. 2000, 15, 1907-1913.
Krishnan, M. N.; Garcia-Blanco, M. A. Targeting Host Factors to Treat West Nile and
Dengue Viral Infections. Viruses. 2014, 6, 683-708.
Kryger, M. B. L..; Wohl, B. M.; Smith, A. A. A.; Zelikin, A. N. Macromolecular prodrugs of
ribavirin combat side effects and toxicity with no loss of activity of the drug. Chem
Commun. 2013, 49, 2643-2649.
Krylov, I. S. Synthesis, structural analysis and in vitro antiviral activities of novel cyclic and
acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs. Ph.D. Thesis,
University of Southern California, Los Angeles, 2012.
Krylov, I. S.; Kashemirov, B. A.; Hilfinger, J. M.; McKenna, C. E. Evolution of amino acid
based prodrug approach: stay tuned. Mol Pharm. 2013, 10, 445-458.
Kyle, J. L.; Harris, E. Global spread and persistence of dengue. Annu Rev Microbiol. 2008,
62, 71–92.
Lai, C. L.; Dienstag, J.; Schiff, E.; Leung, N. W.; Atkins, M.; Hunt, C.; Brown, N.;
Woessner, M.; Boehme, R.; Condreay, L. Prevalence and clinical correlates of
YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin
Infect Dis. 2003, 36, 687-696.
Lam, A. M.; Murakami, E.; Espiritu, C.; Steuer, H.M.; Niu, C.; Keilman, M.; Bao, H.;
Zennou, V.; Bourne, N.; Julander, J. G.; Morrey, J. D.; Smee, D. F; Frick, D. N.;
Heck, J. A.; Wang, P.; Nagarathnam, D.; Ross, B. S.; Sofia, M. J.; Otto, M. J.;
Furman, P. A. PSI-7851, a pronucleotide of I2-D-2’-deoxy-2-fluoro-2’-methyluridine
monophosphate, is a potent and pan-genotype inhibitor of hepatitis C virus
replication. Antimicrob Agents Chemother. 2010, 54, 3187-3196.
Lambert, R. W.; Martin, J. A.; Thomas, G. J.; Duncan, I. B.; Hall, M. J.; Heimer, E. P.
Synthesis and antiviral activity of phosphonoacetic and phosphonoformic acid esters
of 5-bromo-2'-deoxyuridine and related pyrimidine nucleosides and
acyclonucleosides. J Med Chem. 1989, 32, 367-374.
Larder, B. A.; Darby, G. Virus drug resistance: Mechanisms and consequences. Antiviral
Res. 1984, 4, 1-42.
Leyssen, P.; Balzarini, J.; De Clercq, E.; Neyts, J. J. The Predominant Mechanism by Which
Ribavirin Exerts Its Antiviral Activity in Vitro against Flaviviruses and
185
Paramyxoiruses is Mediated by Inhibition of IMP Dehydrogenase. Virology. 2005,
79, 1943.
Leyssen, P.; De Clercq, E.; Neyts, J. The Anti-Yellow Fever Virus Activity of Ribavirin Is
Independent of Error-Prone Replication. Mol Pharmacol. 2006, 69, 1461.
Li, F.; Maag, H.; Alfredson, T., Prodrugs of nucleoside analogues for improved oral
absorption and tissue targeting. J Pharm Sci. 2008, 97, 1109-34.
Liederer, B.M.; Borchardt, R.T. Enzymes involved in the bioconversion of ester-based drugs.
J Pharm Sci. 2006, 95, 1177-1195.
Lin, C-C.; Yeh, L-T; Vitarella, D.; Hong, Z.; Erion, M. D. Remofovir mesylate: a prodrug of
PMEA with improved liver-targeting and safety in rats and monkeys. Antiviral Chem
Chemother. 2004, 15, 307-316.
Lin, J. C.; De Clercq, E.; Pagano, J. S. Novel acyclic adenosine analogs inhibit Epstein-Barr
virus replication. Antimicrob Agents Chemother. 1987, 31, 1431–1433.
Lin, J. C.; De Clercq, E.; Pagano, J. S. Inhibitory effects of acyclic nucleoside phosphonate
analogs, including (S)-1-(3-hydroxy-2- phosphonylmethoxypropyl)cytosine, on
Epstein-Barr virus replication. Antimicrob Agents Chemother. 1991, 35, 2440 –2443.
Magee, W. C.; Aldern, K. A.; Hostetler, K. Y.; Evans, D. H. Cidofovir and (S)-9-[3-hydroxy-
(2-phosphonomethoxy)propyl]adenine are highly effective inhibitors of vaccinia virus
DNA polymerase when incorporated into the template strand. Antimicrob Agents
Chemother. 2008, 52, 586-597.
Matthews, N. C.; Goodier, M. R.; Robey, R. C.; Bower, M.; Gotch, F. M. Killing of Kaposi’s
sarcoma-associated herpesvirus-infected fibroblasts during latent infection by
activated natural killer cells. Eur J Immunol. 2001, 41, 1958 –1968.
McGuigan, C.; Nickson, C.; Petrik, J.; Karpas, A. Phosphate derivatives of AZT display
enhanced selectivity of action against HIV-1 by comparison to the parent nucleoside.
FEBS Lett. 1992, 310-171-174.
McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M-C. The facile dealkylation of
phosphonic acid dialkyl esters by bromotrimethylsilane. Tetrahedron Lett. 1977, 18,
155.
McKenna, C. E.; Levy, J. N. α-Keto phosphonoacetates J Chem Soc Chem Commun. 1989,
246.
186
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.
McKenna C. E.; Peterson L. W.; Kashemirov B. A.; Serpi M.; Mitchell S.; Kim J. S.;
Hilfinger J. M.; Drach J. C. New peptidomimetic prodrugs of acyclic and cyclic
cidofovir: sat studies of chemical and enzymatic activation mechanisms. Antiviral
Res. 2009, 82, A75.
McKenna, C.E.; Schmidhauser, J. Functional Selectivity in Phosphonate Ester Dealkylation
with Bromotrimethylsilane. J Chem Soc Chem Commun. 1979, 739-739.
Meerbach, A.; Holý, A.; Wutzler, P.; De Clercq, E.; Neyts, J. Inhibitory effects of novel
nucleoside and nucleotide analogues on Epstein-Barr virus replication. Antivir Chem
Chemother. 1998, 9, 275–282.
Mehellou, Y.; Balzarini, J.; McGuigan, C. Aryloxy phosphoramidate triesters: a technology
for delivering monophosphorylated nucleosides and sugars into cells. Chem Med
Chem, 2009, 4, 1779-1791.
Meier, C. cycloSal-pronucleotides, - design of chemical Trojan horses. Mini-Rev Med Chem.
2002, 2, 219-234.
Meier, C. Cyclosal phosphates as chemical Trojan horses for intracellular nucleotide and
glycosyl-monophosphate delivery – chemistry meets biology. Eur J Org Chem. 2006,
1081-1102.
Meier, C.; Ducho, C.; Jessen, H.; Vukadinovic-Tenter, D.; Balzarini, J. Second-generation
cycloSal-d4TMP pro-nucleotides bearing esterase-cleavable sites – the “trapping”
concept. Eur J Org Chem. 2005, 197-206.
Mendel, D. B.; Cihlar, T.; Moon, K.; Chen, M. S. Conversion of 1-[((S)-2- hydroxy-2-oxo-
1,4,2-dioxaphosphorinan-5-yl)methyl]cytosine to cidofovir by an intracellular cyclic
CMP phosphodiesterase. Antimicrob Agents Chemother. 1997, 41, 641-646.
Meris, C.; Gorbig, U.; Muller, C.; Balzarini, J. cycloSal-PMEA and cycloAmb-PMEA:
Potentially New Phosphonate Prodrugs Based on the cycloSal-Pronucleotide
Approach. J Med Chem. 2005, 48, 8079.
Mesri, E. A.; Cesarman, E.; Boshoff, C. Kaposi’s sarcoma and its associated herpesvirus. Nat
Rev Cancer. 2010, 10, 707-719.
187
Mikhailov, S. N.; Paoyukova, N. S.; Karpeiskii, M. Y.; Kolobushkina, L. I.; Beigelman, L.
N. Use of 5-deoxy-ribo-hexofuranose derivatives for the preparation of 5'-nucleotide
phosphonates and homoribonucleosides. Collect Czech Chem Commun. 1989, 54,
1055.
Mitsunobu, O.; Eguchi, M. Preparation of carboxylic esters and phosphoric esters by
activation of alcohols. Bull Chem Soc Jpn. 1971, 44, 3427-3430.
Miyazawa, T.; Hiramatsu, S.; Tsuboi, Y.; Yamada, T.; Kuwata, S. Studies of unusual amino
acids and their peptides. XVII. The synthesis of peptides containing N-carboxymethyl
amino acids. II. Bull Chem Soc Jpn. 1985, 58, 1976–1982.
Montgomery, J. A.; Hewson, K. The synthesis of phosphonic acid analogues of purine
ribonucleotides: an exception to the trans rule. Chem Commun. 1969, 15-16.
Mutlu, A. D.; Cavallin, L. E.; Vincent, L.; Chiozzini, C.; Eroles, P.; Duran, E. M.; Asgari, Z.;
Hooper, A. T.; La Perle, K. M. D.; Hilsher, C.; Gao, S-J.; Dittmer, D. P.; Rafii, S.;
Mesri, E. A. In vivo-restricted and reversible malignancy induced by human
herpesvirus-8 KSHV: a cell and animal model of virally induced Kaposi’s sarcoma.
Cancer Cell. 2007, 11, 245–258.
Murakami, E.; Tolstykh, T.; Bao, H.; Niu, C.; Steuer, H.M.; Bao, D.; Chang, W.; Espiritu,
C.; Bansal, S.; Lam, A. M.; Otto, M. J.; Sofia, M. J.; Furman, P. A. Mechanism of
activation of PSI-7851 and its diastereomer PSI-7977. J Biol Chem. 2010, 285,
34337-34347.
Naesens., L.; Balzarini, J.; De Clercq, E. Single-dose administration of 9-(2-
phosphonylmethoxyethyl)adenine (PMEA) and 9-(2-phosphonylmethoxyethyl)-2,6-
diaminopurine (PMEDAP) in the prophylaxis of retrovirus infection in vivo. Antiviral
Res. 1991. 16, 53.
Naesens, L.; Balzarini, J.; De Clercq, E. Pharmacokinetics in mice of the anti-retrovirus
agent 9-(2-phosphonylmethoxyethyl)adenine. Drug Metab Dispos. 1992, 20, 747-
752.
Naesens, L.; Balzarini, J.; Rosenberg, I.; Holý, A.; De Clercq, E. 9-(2-
Phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP): a novel agent with anti-
human immunodeficiency virus activity in vitro and potent anti-Moloney murine
sarcoma virus activity in vivo. Eur J Clin Microbiol Infect Dis. 1989. 8, 1043-1047.
Naesens, L.; De Clercq, E. Therapeutic Potential of HPMPC (Cidofovir), PMEA (Adefovir)
and Related Acyclic Nucleoside Phosphonate Analogues as Broad-Spectrum
Antiviral Agents. Nucleosides Nucleotides. 1997, 16, 983-992.
188
Naesens, L.; Neyts, J.; Balzarini, J.; Holý, A.; Rosenberg, I.; De Clercq, E. Efficacy of oral 9-
(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) in the treatment of
retrovirus and cytomegalovirus infections in mice. J Med Virol. 1993, 39, 167.
Naesens, L.; Snoeck, R.; Graciela, A.; Balzarini, J.; Neyts, J.; De Clercq, E. HPMPC
(cidofovir), PMEA (adefovir) and related acyclic nucleoside phosphonate analogues:
a review of their pharmacology and clinical potential in the treatment of viral
infections. Antiviral Chem Chemother. 1997, 8, 1-23.
Neyts, J.; De Clercq, E. Antiviral drug susceptibility of human herpesvirus 8. Antimicrob
Agents Chemother. 1997, 41, 2754 –2756.
Neyts, J.; Snoeck, R.; Balzarini, J.; De Clercq, E. Particular characteristics of the anti-human
cytomegalovirus activity of (S)-1-(3-hydroxy-2- phosphonylmethoxypropyl)-cytosine
(HPMPC) in vitro. Antiviral Res. 1991, 16, 41–52.
Noble, C. G.; Chen, Y-L.; Dong, H.; Gu, F.; Lim, S. P.; Schul, W.; Wang, Q-Y.; Shi, P-Y.
Strategies for Development of Dengue Virus Inhibitors. Antiviral Res. 2010, 85, 450-
462.
Oliyai, R.; Arimilli, M. N.; Jones, R. J.; Lee, W. A. Pharmacokinetics of salicylate ester
prodrugs of cyclic HPMPC in dogs. Nucleosides Nucleotides Nucleic Acids. 2001, 20,
1411-1414.
Oliyai, R.; Shaw, J.-P.; Sueoka-Lennen, C. M.; Cundy, K. C.; Arimilli, M. N.; Jones, R. J.;
Lee, W. A. Aryl ester prodrugs of cyclic HPMPC. I: physicochemical
characterization and in vitro biological stability. Pharm Res. 1999, 16, 1687-1693.
Padyukova, N. S.; Karpeisky, M. Y.; Kolobushkina, L. I.; Mikhailov, S. N. Oxidation of
alkynes using PdCl
2
/CuCl
2
in PEG as a recyclable catalytic system: one-pot synthesis
of quinoxalines. Tetrahedron Lett. 1987, 28, 3623.
Page, T.; Connor, J. D. The metabolism of ribavirin in erythrocytes and nucleated cells. Int J
Biochem. 1990, 22, 379–383.
Palu, G.; Stefanelli, S.; Rassu, M.; Parolin, C.; Balzarini, J.; De Clercq, E. Cellular uptake of
phosphonylmethoxyalkylpurine derivatives. Antiviral Res. 1991, 16, 115-119.
Parsons, C. H.; Adang, L. A.; Overdevest, J.; O’Connor, C. M.; Taylor, J. R. Jr.; Camerini,
D.; Kedes, D. H. KSHV targets multiple leukocyte lineages during long-term
productive infection in NOD/SCID mice. J Clin Invest. 2006, 116, 1963–1973.
189
Pauwels, R.; Balzarini, J.; Schols, D.; Baba, M.; Desmyter, P.; Rosenberg, I.; Holý, A.; De
Clercq, E. Phosphonylmethoxyethyl purine derivatives, a new class of anti-human
immunodeficiency virus agents. Antimicrob Agents Chemother. 1988, 32, 1025-1030.
Pertusati, F.; Serpi, M.; McGuigan, C. Medicinal chemistry of nucleoside phosphonate
prodrugs for antiviral therapy. Antiviral Chem Chemother. 2012, 22, 181-203.
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.
Peterson, L. W.; Kashemirov, B. A.; Eriksson, U.; Kim, J. S.; Mitchell, S.; Kijek, P.; Lee, K.
D.; Hilfinger, J. M.; McKenna, C. E. Serine side-chain-linked peptidomimetic
prodrugs of cidofovir and cyclic cidofovir: C-ester effects on transport and activation.
Antiviral Res. 2008, 78, A46-A46.
Peterson, L. W.; Kashemirov, B. A.; Sala-Rabanal, M.; Kim, J. S.; Mitchell, S.; Kijek, P.;
Hilfinger, J. M.; McKenna, C. E. MEDI 183-Synthesis and transport studies on serine
side-chain-linked peptidomimetic prodrugs of cyclic cidofovir. J Chem Soc Chem
Commun 2008, 235.
Peterson, L. W.; Kim, J. S.; Kijek, P.; Mitchell, S.; Hilfinger, J. M.; Breitenbach, J.; Borysko,
K. Z.; 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.
Peterson, L. W.; McKenna, C. E. Prodrug approaches to improving the oral absorption of
antiviral nucleotide analogues. Expert Opin Drug Delivery. 2009, 6, 405-420.
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 Pharm. 2010, 7, 2349-2361.
Petrov, V.I.; Ozerov, A.A.; Novikov, M.S.; Pannecouque, C.; Balzarini, J.; De Clercq, E. 9-
(2-aryloxyethyl) derivatives of adenine – a new class of non-nucleosidic antiviral
agents. Chem Heterocycl Compd. 2003, 39, 1218-1226.
Peyrottes, S.; Egron, D.; Lefebvre, I.; Gosselin, G.; Imach, J. L.; Periguad, C. SATE
pronucleotide approaches an overview. Mini-Rev Med Chem. 2004, 4, 395-408.
Pradere, U.; Amblard, F.; Coats, S. J.; Schinazi, R. F. Synthesis of 5’-methylene phosphonate
furanonucleoside prodrugs: application to D-2’-deoxy-α-fluoro-2’-β-C-methyl
nucleosides. Org Lett. 2012, 14, 4426-4429.
190
Prichard, M. N.; Williams, J. D.; Komazin-Meredith, G.; Khan, A. R.; Price, N. B.; Jefferson,
G. M.; Harden, E. A.; Hartline, C. B.; Peet, N. P.; Bowlin, T. L. Synthesis and
Antiviral Activities of Methylenecyclopropane Analogs with 6-alkoxy and 6-alkylthio
Substitutions that Exhibit Broad-Spectrum Antiviral Activity Against Human
Herpesviruses. Antimicrob Agents Chemother. 2013, 57, 3518-3527.
Prusoff, W. H. Synthesis and biological activities of iododeoxyuridine, an analog of
thymidine. Biochem Biophys Acta. 1959, 32, 295-296.
Quenelle, D. C.; Collins, D. J.; Herrod, B. P.; Keith, K. A.; Trahan, J.; Beadle, J. R.;
Hostetler, K. Y.; Kern, E. R. Effect of oral treatment with hexadecyloxypropyl-[(S)-
9-(hydroxy-2-phosphonomethoxypropyl)adenine] [HDP-(S)-HPMPA)] or
octadecyloxyethyl-(S)-HPMPA on cowpox or vaccinia virus infections in mice.
Antimicrob Agents Chemother. 2007, 51, 3940-3947.
Raetz, C. R. H; Chu, M. Y.; Srivastava, S. P.; Turcotte, J. G. A phospholipid derivative of
cytosine arabinoside and its conversion to phosphatidylinositol by animal tissue.
Science. 1977, 196, 303-305.
Raju, N.; Smee, D. F.; Robins, R. K.; Vaghefi, M. M. Synthesis and biological properties of
purine and pyrimidine 5′-deoxy-5′- (dihydroxyphosphinyl)-beta-D-ribofuranosyl
analogues of AMP, GMP, IMP, and CMP. J Med Chem. 1989, 32, 1307-1313.
Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Jarvinen, T.; Savolainen, J.
Prodrugs: Design and Clinical Applications. Nat Rev Drug Discov. 2008, 7, 255-270.
Reddy, K. R.; Matelich, M. C.; Ugarkar, B. G.; Gomez-Galeno, J. E.; DaRe, J.; Ollis, K.;
Sun, Z.; Craigo, W.; Colby, T. J; Fujitaki, J. M.; Boyer, S. H.; van Poelje, P. D.;
Erion, M. D. Pradefovir: a prodrug that targets adefovir to the liver for the treatment
of hepatitis B. J Med Chem. 2008. 51, 666.
Rejman, D.; Masojídková, M.; De Clercq, E.; Rosenberg, I. 2’-C-alkoxy and 2’-C-aryloxy
derivatives of N-(2-phosphonomethoxyethyl)-purines and pyrimidines: synthesis and
biological activity. Nucleosides Nucleotides Nucleic Acids. 2001, 20, 1497-1522.
Rossi, L.; Serafini, S.; Pierige, F.; Antonelli, A.; Cerasi, A.; Fraternale, A.; Chiarantini, L.;
Magnani, M., Erythrocyte-based drug delivery. Expert Opin Drug Delivery 2005, 2,
(2), 311-322.
Russmann, S.; Grattagliano, I.; Portincasa, P.; Palmieri, V. O.; Palasciano, G. Ribavirin-
induced anemia: mechanisms, risk factors and related targets for future research. Curr
Med Chem. 2006, 13, 3351–3357.
191
Sala-Rabanal, M.; Peterson, L. W.; Serpi, M.; Krylov, I. S.; Kashemirov, B. A.; Kim, J. S.;
Mitchell, S.; Hilfinger, J. M.; McKenna, C. E. Interactions Between the Human
Oligopeptide Transporter, hPepT1 and Serine Side-chain-linked Cidofovir Prodrugs.
Antiviral Res. 2009, 82, A53.
Saracco, G.; Ciancio, A.; Olivero, A.; Smedile, A.; Roffi, L.; Croce, G.; Colletta, C.; Cariti,
G.; Andreoni, M.; Biglino, A.; Calleri, G.; Maggi, G.; Tappero, G. F.; Orsi, P. G.;
Terreni, N.; Macor, A.; Di Napoli, A.; Rinaldi, E.; Ciccone, G.; Rizzetto, M. A
randomized 4-arm multicenter study of interferon alfa-2b plus ribavirin in the
treatment of patients with chronic hepatitis C not responding to interferon alone.
Hepatology. 2001, 34, 133.
Schabel, F. M. Jr. The antiviral activity of 9-b-D-arabinofuranosyladenine (Ara-A).
Chemotherapy. 1968, 13, 321–38.
Schaeffer, H. J.; Beauchamp, L.; de Miranda, P.; Elion, G. B.; Bauer, D. J.; Collins, P. 9-(2-
Hydroxyethoxymethyl) guanine activity against viruses of the herpes group. Nature.
1978, 272, 583–5.
Schmaljohn, A. L.; McClain, D. Alphaviruses (Togaviridae) and Flaviviruses (Flaviviridae).
In Medical Microbiology. 4th ed. Baron, S., Ed.; University of Texas Medical
Branch at Galveston: Galveston, Texas, 1996; Ch 54.
Schormann, N.; Sommers, C. I.; Prichard, M. N.; Keith, K. A.; Noah, J. W.; Nuth, M.;
Ricciardi, R. P.; Chattopadhyay, D. Identification of Protein-Protein Interaction
Inhibitors Targeting Vaccinia Virus Processivity Factor for Development of Antiviral
Agents. Antimicrob Agents Chemother. 2011, 55, 5054-5062.
Serafinowska, H. T.; Ashton, R. J.; Bailey, S.; Handen, M. R.; Jackson, S. M.; Sutton, D.
Synthesis and in vivo evaluation of prodrugs of 9-[2-
(phosphonomethoxy)ethyl]adenine. J Med Chem. 1995, 38, 1372-1379.
Shaw, J.-P.; Cundy, K. C. abstract: Biological screens of PMEA prodrugs. Pharmaceutical
Research. 1993, 10, (Supplemental): S294.
Shipkowitz, N. L.; Bower, R. R.; Appell, R. N.; Nordeen, C. W.; Overby, L. R.; Roderick,
W. R.; Schleich, J. B.; Vonesch, A. M. Suppression of herpes simplex virus infection
by phosphonoacetic acid. Appl Microbiol. 1973, 26, 264-267.
Sidwell, R. W.; Huffman, J. H.; Khare, G. P.; Allen, L. B.; Witkowski, J. T.; Robins, R. K.
Broad-spectrum antiviral activity of Virazole: 1-beta-D-ribofuranosyl- 1,2,4-triazole-
3-carboxamide. Science. 1972, 177, 705–706.
192
Snell, N. J. Ribavirin—Current State of a Broad Spectrum Antiviral Agent. Exp Opin
Pharmacother. 2001, 2, 1317.
Snoeck, R.; Sakuma, T.; De Clercq, E.; Rosenberg, I.; Holý, A. (S)-1-(3-hydroxy-2-
phosphonomethoxypropyl)cytosine, a potent and selective inhibitor of human
cytomegalovirus replication. Antimicrob Agents Chemother. 1988, 32, 1839-1844.
Sofia, M. J; Bao, D.; Chang, W.; Du, J; Nagarathnam, D.; Rachkonda, S.; Reddy, P. G.;
Ross, B. S.; Wang, P.; Zhang, H-R.; Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A.
M.; Steuer, H. M. M.; Niu, C.; Otto, M. J.; Furman, P. A. Discovery of a I-D-
2’deoxy-2-I±fluoro-2-I2-C-methyluridine nucleotide prodrug (PSI-7977) for the
treatment of hepatitis C virus. J Med Chem. 2010, 53, 7202-7218.
Srinivas, R.V.; Robbins, B.L.; Connelly, M.C.; Gong, Y.F.; Bischofberger, N.; Fridland, A.
Metabolism and in vitro antiretroviral activities of bis(pivaloyloxymethyl) prodrugs
of acyclic nucleoside phosphonates. Antimicrob Agents Chemother. 1993, 37, 2247.
Srivastva, D. N.; Farquar, D. Bioreversible phosphate protective groups. Synthesis and
stability of model acyloxymethyl phosphates. Bioorg Chem. 1984, 12, 118-129.
Starrett Jr., J. E.; Tortolani, D. R.; Russell, J.; Hitchcock, M. J. M.; Whiterock, V.; Martin, J.
C.; Mansuri, M. M. Synthesis, oral bioavailability determination and in vitro
evaluation of prodrugs of the antiviral agent 9-[2-(phosphonomethoxy)ethyl]adenine
(PMEA). J Med Chem. 1994, 37, 1857-1864.
Stittelar, K. J.; Neyts, J.; Naesens, L.; van Amerongen, G.; van Lavieren, R. F.; Holý, A.; De
Clercq, E.; Niesters, H. G. M.; Fries, E.; Maas, C.; Mudler, P. G. H.; van der Zeijst,
B. A. M.; Osterhaus, A.D.M. Antiviral Treatment is more effective than smallpox
vaccination upon lethal monkeypox viral infection. Nature. 2006, 439, 745-748.
Sturzl, M.; Blasig, C.; Schreier, A.; Neipel, F.; Hohenadl, C.; Cornali, E.; Ascherl, G.; Esser,
S.; Brockmeyer. N. H.; Ekman, M.; Kaaya, E. E.; Tschachler, E.; Biberfeld, P.
Expression of HHV-8 latency–associated T0.7 RNA in spindle cells and endothelial
cells of AIDS-associated classical and African Kaposi’s sarcoma. Int J Cancer. 1997,
72, 68-71.
Sulkowski, M.S.; Gardiner, D. F.; Rodriquez-Torres, M.; Reddy, R.; Hassanein, T.;
Jacobson, I.; Lawitz, E.; Lok, A. S.; Hinestrosa, F.; Thuluvath, P. J.; Schwartz, H.;
Nelson, D. R.; Everson, G. T.; Eley, T.; Wind-Rotolo, M.; Hindes, R.; Symonds, W.;
Pasquinelli, C.; Grasela, D. M. Daclatasvir plus Sofosbuvir for Previously Treated or
Untreated Chronic HCV Infection. N Engl J Med. 2014, 370, 211-221.
Sundquist, B.; Oberg, B., Phosphonoformate inhibits reverse transcriptase. J Gen Virol.
1979, 45, 273-281.
193
Takhampunya, R.; Ubol, S.; Huong, H-S.; Cameron, C. E.; Padmanabhan, R. Inhibition of
Dengue Virus Replication by Mycophenolic acid and Ribavirin. J Gen Virol. 2006,
87, 1947-1952.
Tanaka, H.; Fukui, M.; Haraguchi, K.; Masaki, M.; Miyasaka, T. Cleavage of a nucleosidic
oxetane with carbanions: synthesis of a highly promising candidate for anti-HIV
agents — a phosphonate isostere of AZT 5′-phosphate. Tetrahedron Lett. 1989, 30,
2567.
Tichý, T.; Andrei, G.; Dracinsky, M.; Holý, A.; Balzarini, J.; Snoeck, R.; Krečmerová, M.
New Prodrugs of Adefovir and Cidofovir. Bioorg Med Chem. 2011, 19, 3527-3539.
Vaghefi, M. M.; McKernan, P. A.; Robins, R. K., Synthesis and antiviral activity of certain
nucleoside 5'-phosphonoformate derivatives. J Med Chem. 1986, 29, 1389-1393.
Valiaeva, N.; Prichard, M. N.; Buller, R. M.; Beadle, J. R.; Hartline, C. B.; Keith, K. A.
Schriewer, J., Trahan, J., Hostetler, K.Y. Antiviral evaluation of octadecyloxyethyl
esters of (S)-3-hydroxy-2-(phosphonomethoxy)propyl nucleosides against
herpesviruses and orthopoxviruses. Antiviral Res. 2009, 84, 254-259.
Van der Waterbeemdt, H. Drug Bioavailability: Estimation of solubility, permeability,
absorption and bioavailability. In Methods Princ. Med. Chem. [online] Van de
Waterbeemd, H.; Lennernas, H.; Artursson, P., Eds.; Wiley-VCH: Weinheim,
Germany, 2003. Vol 18. http://onlinelibrary.wiley.com/book/10.1002/3527601473.
Vinogradov, S. V.; Zeman, A. D.; Batrakova, E. V.; Kabanov, A. V., Polyplex Nanogel
formulations for drug delivery of cytotoxic nucleoside analogs. J Controlled Release
2005, 107, 143-157.
Votruba, I.; Intracellular phosphorylation of broad-spectrum anti-DNA virus agent (S)-9-(3-
hydroxy-2-phosphorylmethoxypropyl) adenine and inhibition of viral DNA synthesis.
Mol Pharmacol. 1987, 32, 524-529.
Vrbkova, S.; Dracinsky, M.; Holý, A. Synthesis of phosphonomethoxyethyl or 1,3-
bis(phosphonomethoxy)propan-2-yl lipophilic esters of acyclic nucleoside
phosphonates. Tetrahedron. 2007, 63, 11391.
Wachsman, M.; Petty, B. G.; Cundy, K. C.; Jaffe, H. S.; Fisher, P. E.; Pastelak, A.; Lietman,
P. S. Pharmacokinetics, safety and bioavailability of HPMPC (cidofovir) in human
immunodeficiency virus-infected subjects. Antiviral Res. 1996, 29, 153-161.
Wagner, C. R.; Iyer, V. V.; McIntee, E. J. Pronucleotides: Toward the in vivo delivery of
antiviral and anticancer nucleotides. Med Res Rev. 2000, 20, 417-451.
194
Wang, G.; Sakthivel, K.; Rajappan, V.; Bruice, T. W.; Tucker, K.; Fagan, P.; Brooks, J. L.;
Hurd, T.; Leeds, J. M.; Cook, P. D. Synthesis of Azole nucleoside 5’-phosphate
mimics (P1Ms) and their inhibitory properties of IMP dehydrogenases. Nucleosides
Nucleotides Nucleic Acids. 2004. 23, 317-337.
Warden, C.; Tang, Q. Y.; Zhu, H. Herpesvirus BACs: past, present, and future. J Biomed
Biotechnol. 2011, 27, 124595.
Warner, D. T.; Neil, G. L.; Taylor, A. J.; Wechler, W. J. Nucleic acids. 13. 3’-0- and 2’-0-
esters of 1.beta.-D-arabinofuranosylcytosine as antileukemic and immunosuppressive
agents. J Med Chem. 1972, 15, 790-792.
Wechter, W. J.; Johnson, M. A.; Hall, C. M.; Warner, D. T.; Berger, A. E.; Wenzel, A. H.;
Gish, D. T.; Neil, G. L. Nucleic acids. 14. Ara-Cytidine acylates. Use of drug design
predictors in structure-activity relation correlation. J Med Chem. 1975, 18, 339-344.
Wen. K. W.; Damania, B. Kaposi sarcoma-associated herpesvirus (KSHV): molecular
biology and ontogenesis. Cancer Lett. 2010, 289, 140-150.
Whitley, R. J.; Ch’ien, L. T.; Dolin, R., Galasso, G. J.; Alford, C. A. Jr. Adenine arabinoside
therapy of herpes zoster in the immuno-suppressed. NIAID collaborative antiviral
study. N Engl J Med. 1976, 294, 1193–9.
Williams, M.; Krylov, I. S.; Zakharova, V. M.; Serpi, M.; Peterson, L. W.; Krečmerová, M.;
Kashemirov, B. A.; McKenna, C. E. Cyclic and Acyclic Phosphonate Tyrosine Ester
Prodrugs of Acyclic Nucleoside Phosphonates. Czech Coll Symp Ser. 2011, 12, 167-
170.
Willis, R. C.; Carson, D. A.; Seegmiller, J. E. Adenosine kinase initiates the major route of
ribavirin activation in a cultured human cell line. Proc Natl Acad Sci U.S.A. 1978, 75,
3042.
Witkowski, J. T.; Robins, R. K.; Khare, G. P.; Sidwell, R. W. Synthesis and antiviral activity
of 1,2,4-triazole-3-thiocarboxamide and 1,2,4-triazole-3-carboxamidine
ribonucleosides. J Med Chem. 1973, 16, 935–7.
WHO. Dengue haemorrhagic fever: diagnosis, treatment, prevention, and control. 2nd ed.
Geneva: World Health Organization; 1997.
Wu, W.; Vieira, J.; Fiore, N.; Banerjee, P.; Sieburg, M.; Rochford, R.; Harrington, W. Jr.;
Feuer, G. KSHV/HHV-8 infection of human hematopoietic progenitor (CD34ჼ) cells:
persistence of infection during hematopoiesis in vitro and in vivo. Blood. 2006, 108,
141–151.
195
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.
Zeuzem, S.; Dusheiko, G. M.; Salupere, R.; Mangia, A.; Flisiak, R.; Hyland, R. H.;
Illeperuma, A.; Svarovskaia, E.; Brainard, D. M.; Symonds, W. T.; Subramanian, G.
M.; McHutchison, J. G.; Weiland, O.; Reesink, H. W.; Ferenci, P.; Hezode, C.;
Esteban, R.; Sofosbuvir and Ribavirin in HCV Genotypes 2 and 3. N Engl J Med.
2014, 370, 1993-2001.
Zídek, Z.; Frankova, D.; Holý, A. Macrophage activation by antiviral acyclic nucleoside
phosphonates in dependence on priming immune stimuli. Int J Immunopharmacol.
2000, 22, 1121-1129.
Zídek, Z.; Potmesil, P.; Kmoníèková, E.; Holý, A. Immunobiological activity of N-[2-
(phosphonomethoxy)alkyl] derivatives of N6-substituted adenines, and 2,6-
diaminopurines. Eur J Pharmacol. 2003, 475, 149-159.
Zoulin, F. Antiviral therapy of chronic hepatitis B. Antiviral Res. 2006, 71, 206-215.
196
APPENDIX A: Chapter 2 Supporting Data
197
A.1
31
P NMR (202 MHz, CDCl
3
) Bis(propan-2-yl)[(2-chloroethoxy)methyl]phosphonate
(2.9)
A.2
1
H NMR (500 MHz, CDCl
3
) Bis(propan-2-yl) [(2-chloroethoxy)methyl]phosphonate
(2.9)
198
A.3
31
P NMR (202 MHz, CD
3
OD) Bis(propan-2-yl)([2-(6-amino-9H-purin-9-
yl)ethoxy]methyl)phosphonate (2.12)
A.4
1
H NMR (500 MHz, CD
3
OD) Bis(propan-2-yl)([2-(6-amino-9H-purin-9-
yl)ethoxy]methyl)phosphonate (2.12)
199
A.5
31
P NMR (202 MHz, D
2
O, NH
4
OH; pH 9-10) ([2-(6-Amino-9H-purin-9-
yl)ethoxy]methyl)phosphonic acid (2.1)
A.6
1
H NMR (500 MHz, D
2
O, NH
4
OH; pH 9-10) ([2-(6-Amino-9H-purin-9-
yl)ethoxy]methyl)phosphonic acid (2.1)
200
A.7
1
H NMR (500 MHz, CD
3
OD) tert-Butyl N-[(1S)-2-(4-hydroxyphenyl)-1-
(octylcarbamoyl)ethyl]carbamate (2.14)
A.8
1
H NMR (500 MHz, CD
3
OD) tert-Butyl N-[(1S)-1-(hexadecylcarbamoyl)-2-(4-
hydroxyphenyl)ethyl]carbamate (2.15)
201
A.9
31
P NMR (202 MHz, CD
3
OD) ([2-(6-Amino-9H-purin-9-
yl)ethoxy]methyl)(phenoxy)phosphinic acid (2.31)
A.10
1
H NMR (500 MHz, CD
3
OD) ([2-(6-Amino-9H-purin-9-
yl)ethoxy]methyl)(phenoxy)phosphinic acid (2.31)
202
A.11 LRMS of ([2-(6-Amino-9H-purin-9-yl)ethoxy]methyl)(phenoxy)phosphinic acid
(2.31)
A.12
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-[(2-
methylpropyl)carbamoyl]ethyl]phenoxy)(([2-(6-amino-9H-purin-9-
yl)ethoxy]methyl))phosphinic acid (2.32)
203
A.13
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-[(2-
methylpropyl)carbamoyl]ethyl]phenoxy)(([2-(6-amino-9H-purin-9-
yl)ethoxy]methyl))phosphinic acid (2.32)
A.14 LRMS of (4-[(2S)-2-Amino-2-[(2-methylpropyl)carbamoyl]ethyl]phenoxy)(([2-(6-
amino-9H-purin-9-yl)ethoxy]methyl))phosphinic acid (2.32)
204
A.15
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(octylcarbamoyl)ethyl]phenoxy)(([2-(6-amino-9H-purin-9-yl)ethoxy]methyl))phosphinic
acid (2.33)
A.16
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(octylcarbamoyl)ethyl]phenoxy)(([2-(6-amino-9H-purin-9-yl)ethoxy]methyl))phosphinic
acid (2.33)
205
A.17 LRMS of (4-[(2S)-2-Amino-2-(octylcarbamoyl)ethyl]phenoxy)(([2-(6-amino-9H-
purin-9-yl)ethoxy]methyl))phosphinic acid (2.33)
A.18
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)(([2-(6-amino-9H-purin-9-
yl)ethoxy]methyl))phosphinic acid (2.34)
206
A.19
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)(([2-(6-amino-9H-purin-9-
yl)ethoxy]methyl))phosphinic acid (2.34)
A.20 LRMS of (4-[(2S)-2-Amino-2-(hexadecylcarbamoyl)ethyl]phenoxy)(([2-(6-amino-
9H-purin-9-yl)ethoxy]methyl))phosphinic acid (2.34)
207
A.21
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(octylcarbamoyl)ethyl]phenoxy)(([(2R)-1-(2,6-diamino-9H-purin-9-yl)propan-2-
yl]oxy)methyl)phosphinic acid (2.35)
A.22
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(octylcarbamoyl)ethyl]phenoxy)(([(2R)-1-(2,6-diamino-9H-purin-9-yl)propan-2-
yl]oxy)methyl)phosphinic acid (2.35)
N
N
N
N
NH
2
O P
O
OH
O
H
2
N
O
NHC
8
H
17
H
2
N
N
N
N
N
NH
2
O P
O
OH
O
H
2
N
O
NHC
8
H
17
H
2
N
208
A.23 LRMS of (4-[(2S)-2-Amino-2-(octylcarbamoyl)ethyl]phenoxy)(([(2R)-1-(2,6-
diamino-9H-purin-9-yl)propan-2-yl]oxy)methyl)phosphinic acid (2.35)
N
N
N
N
NH
2
O P
O
OH
O
H
2
N
O
NHC
8
H
17
H
2
N
209
APPENDIX B: Chapter 3 Supporting Data
210
B.1
31
P NMR (202 MHz, D
2
O, NH
4
OH; pH 9-10) (([(2R)-1-[(2,6-Diaminopyrimidin-4-
yl)oxy]-3-hydroxypropan-2-yl]oxy)methyl)phosphonic acid (3.1)
B.2
1
H NMR (500 MHz, D
2
O, NH
4
OH; pH 9-10) (([(2R)-1-[(2,6-Diaminopyrimidin-4-
yl)oxy]-3-hydroxypropan-2-yl]oxy)methyl)phosphonic acid (3.1)
211
B.3 LRMS of (([(2R)-1-[(2,6-Diaminopyrimidin-4-yl)oxy]-3-hydroxypropan-2-
yl]oxy)methyl)phosphonic acid (3.1)
B.4
31
P NMR (202 MHz, D
2
O, NH
4
OH; pH 9-10) ((2-[(2,6-Diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphonic acid) (3.2)
212
B.5
1
H NMR (500 MHz, D
2
O, NH
4
OH; pH 9-10) ((2-[(2,6-Diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphonic acid) (3.2)
B.6
1
H NMR (500 MHz, D
2
O) ((2-[(2,6-Diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphonic acid) (3.2)
213
B.7
31
P NMR (202 MHz, D
2
O) ((2-[(2,6-Diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)(propan-2-yloxy)phosphinic acid (3.13)
B.8
1
H NMR (500 MHz, D
2
O) ((2-[(2,6-Diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)(propan-2-yloxy)phosphinic acid (3.13)
214
B.9
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-3-oxo-3-(propan-2-
yloxy)propyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic
acid (3.19)
B.10
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-3-oxo-3-(propan-2-
yloxy)propyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic
acid (3.19)
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
O
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
O
215
B.11 LCMS of (4-[(2S)-2-Amino-3-oxo-3-(propan-2-yloxy)propyl]phenoxy)((2-[(2,6-
diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic acid (3.19)
B.12
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-[(2-
methylpropyl)carbamoyl]ethyl]phenoxy)(2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.20)
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
HN
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
O
216
B.13
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-[(2-
methylpropyl)carbamoyl]ethyl]phenoxy)(2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.20)
B.14 LRMS of (4-[(2S)-2-Amino-2-[(2-methylpropyl)carbamoyl]ethyl]phenoxy)(2-[(2,6-
diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic acid (3.20)
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
HN
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
HN
217
B.15
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.21)
B.16
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.21)
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
NHC
16
H
33
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
NHC
16
H
33
218
B.17 HRMS of (4-[(2S)-2-Amino-2-(hexadecylcarbamoyl)ethyl]phenoxy)((2-[(2,6-
diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic acid (3.21)
B.18
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(octylcarbamoyl)ethyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.22)
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
NHC
8
H
17
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
NHC
16
H
33
219
B.19
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(octylcarbamoyl)ethyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.22)
B.20 LRMS of (4-[(2S)-2-Amino-2-(octylcarbamoyl)ethyl]phenoxy)((2-[(2,6-
diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic acid (3.22)
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
NHC
8
H
17
N
N H
2
N
NH
2
O
O
P
O
O
HO
H
2
N
O
NHC
8
H
17
220
B.21
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-[(2S)-2-Amino-3-methylbutanamido]-3-
oxo-3-(propan-2-yloxy)propyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.25)
B.22
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-[(2S)-2-Amino-3-methylbutanamido]-3-
oxo-3-(propan-2-yloxy)propyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-
yl)oxy]ethoxy)methyl)phosphinic acid (3.25)
221
B.23 HRMS of (4-[(2S)-2-[(2S)-2-Amino-3-methylbutanamido]-3-oxo-3-(propan-2-
yloxy)propyl]phenoxy)((2-[(2,6-diaminopyrimidin-4-yl)oxy]ethoxy)methyl)phosphinic
acid (3.25)
B.24
31
P NMR (202 MHz, CD
3
OD) (2S)-2-Amino-3-(4-([(5R)-5-([(2,6-
diaminopyrimidin-4-yl)oxy]methyl)-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-
hexadecylpropanamide (3.29)
N
N O
P
O
O
O
H
2
N
O
NHC
16
H
33
NH
2
H
2
N
O
222
B.25
1
H NMR (500 MHz, CD
3
OD) (2S)-2-Amino-3-(4-([(5R)-5-([(2,6-
diaminopyrimidin-4-yl)oxy]methyl)-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-
hexadecylpropanamide (3.29)
B.26 LRMS of (2S)-2-Amino-3-(4-([(5R)-5-([(2,6-diaminopyrimidin-4-yl)oxy]methyl)-2-
oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-hexadecylpropanamide (3.29)
N
N O
P
O
O
O
H
2
N
O
NHC
16
H
33
NH
2
H
2
N
O
N
N O
P
O
O
O
H
2
N
O
NHC
16
H
33
NH
2
H
2
N
O
223
B.27
31
P NMR (202 MHz, CD
3
OD) (2S)-2-Amino-3-(4-([(5R)-5-([(2,6-
diaminopyrimidin-4-yl)oxy]methyl)-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-
octylpropanamide (3.30)
B.28
1
H NMR (500 MHz, CD
3
OD) (2S)-2-Amino-3-(4-([(5R)-5-([(2,6-
diaminopyrimidin-4-yl)oxy]methyl)-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-
octylpropanamide (3.30)
N
N O
P
O
O
O
O
H
2
N
O
NHC
8
H
17
NH
2
H
2
N
N
N O
P
O
O
O
O
H
2
N
O
NHC
8
H
17
NH
2
H
2
N
224
B.29 LRMS of (2S)-2-Amino-3-(4-([(5R)-5-([(2,6-diaminopyrimidin-4-yl)oxy]methyl)-2-
oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-octylpropanamide (3.30)
B.30
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)(([(2R)-1-[(2,6-diaminopyrimidin-4-yl)oxy]-3-
hydroxypropan-2-yl]oxy)methyl)phosphinic acid (3.31)
N
N O
P O
O
H
2
N
O
NHC
16
H
33
NH
2
H
2
N
OH
OH
O
N
N O
P
O
O
O
O
H
2
N
O
NHC
8
H
17
NH
2
H
2
N
225
B.31
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)(([(2R)-1-[(2,6-diaminopyrimidin-4-yl)oxy]-3-
hydroxypropan-2-yl]oxy)methyl)phosphinic acid (3.31)
B.32 LRMS of (4-[(2S)-2-Amino-2-(hexadecylcarbamoyl)ethyl]phenoxy)(([(2R)-1-[(2,6-
diaminopyrimidin-4-yl)oxy]-3-hydroxypropan-2-yl]oxy)methyl)phosphinic acid (3.31)
N
N O
P O
O
H
2
N
O
NHC
16
H
33
NH
2
H
2
N
OH
OH
O
N
N O
P O
O
H2N
O
NHC16H33
NH2
H2N
OH
OH
O
226
APPENDIX C: Chapter 4 Supporting Data
227
C.1
1
H NMR (500 MHz, CD
3
OD) tert-Butyl N-(1-([(1S)-1-(hexadecylcarbamoyl)-2-
hydroxyethyl]carbamoyl)-2-methylpropyl)carbamate (4.7)
C.2
31
P NMR (202 MHz, CD
3
OD) (2S)-2-Amino-N-[(1S)-2-((5-[(4-amino-2-oxo-1,2-
dihydropyrimidin-1-yl)methyl]-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl)oxy)-1-
(hexadecylcarbamoyl)ethyl]-3-methylbutanamide (4.9)
HO
O
HN
NHC
16
H
33
O
NH
Boc
228
C.3
1
H NMR (500 MHz, CD
3
OD) (2S)-2-Amino-N-[(1S)-2-((5-[(4-amino-2-oxo-1,2-
dihydropyrimidin-1-yl)methyl]-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl)oxy)-1-
(hexadecylcarbamoyl)ethyl]-3-methylbutanamide (4.9)
C.4 LRMS of (2S)-2-Amino-N-[(1S)-2-((5-[(4-amino-2-oxo-1,2-dihydropyrimidin-1-
yl)methyl]-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl)oxy)-1-(hexadecylcarbamoyl)ethyl]-3-
methylbutanamide (4.9)
229
APPENDIX D: Chapter 5 Supporting Data
230
D.1
31
P NMR (202 MHz, CD
3
OD) (2S)-2-Amino-3-(4-([(5S)-5-[(6-amino-9H-purin-9-
yl)methyl]-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-hexadecylpropanamide
(5.2).
D.2
1
H NMR (500 MHz, CD
3
OD) (2S)-2-Amino-3-(4-([(5S)-5-[(6-amino-9H-purin-9-
yl)methyl]-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-hexadecylpropanamide
(5.2).
N
O
O
P
O
H
2
N
O
NHC
16
H
33
O
N
N
N
NH
2
(S)
N
O
O
P
O
H
2
N
O
NHC
16
H
33
O
N
N
N
NH
2
(S)
231
D.3
31
P NMR (202 MHz, CD
3
OD) (2S)-2-Amino-3-(4-([(5S)-5-[(4-amino-2-oxo-1,2-
dihydropyrimidin-1-yl)methyl]-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-
hexadecylpropanamide (5.3).
D.4
1
H NMR (500 MHz, CD
3
OD) (2S)-2-Amino-3-(4-([(5S)-5-[(4-amino-2-oxo-1,2-
dihydropyrimidin-1-yl)methyl]-2-oxo-1,4,2λ⁵-dioxaphosphinan-2-yl]oxy)phenyl)-N-
hexadecylpropanamide (5.3).
N
O
O
P
O
H
2
N
O
NHC
16
H
33
(S)
O
N
NH
2
O
N
O
O
P
O
H
2
N
O
NHC
16
H
33
(S)
O
N
NH
2
O
232
D.5
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)(([(2S)-1-(6-amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy)methyl)phosphinic acid (5.4).
D.6
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)(([(2S)-1-(6-amino-9H-purin-9-yl)-3-
hydroxypropan-2-yl]oxy)methyl)phosphinic acid (5.4).
N
O
HO
P
O
H
2
N
O
NHC
16
H
33
O
N
N
N
NH
2
HO
(S)
N
O
HO
P
O
H
2
N
O
NHC
16
H
33
O
N
N
N
NH
2
HO
(S)
233
D.7
31
P NMR (202 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)(([(2S)-1-(4-amino-2-oxo-1,2-dihydropyrimidin-1-
yl)-3-hydroxypropan-2-yl]oxy)methyl)phosphinic acid (5.5).
D.8
1
H NMR (500 MHz, CD
3
OD) (4-[(2S)-2-Amino-2-
(hexadecylcarbamoyl)ethyl]phenoxy)(([(2S)-1-(4-amino-2-oxo-1,2-dihydropyrimidin-1-
yl)-3-hydroxypropan-2-yl]oxy)methyl)phosphinic acid (5.5).
N
O
HO
P
O
H
2
N
O
NHC
16
H
33
O
N
NH
2
O
HO
(S)
N
O
HO
P
O
H
2
N
O
NHC
16
H
33
O
N
NH
2
O
HO
(S)
234
APPENDIX E: Chapter 6 Supporting Data
235
E.1
1
H NMR (500 MHz, DMSO-d
6
) 1-[(2R,3R,4S)-3,4-Dihydroxy-5-(iodomethyl)oxolan-
2-yl]-1H-1,2,4-triazole-3-carboxamide (6.4)
E.2
1
H NMR (500 MHz, CDCl
3
) (2S,3S,4R,5R)-4-(Benzoyloxy)-5-(3-carbamoyl-1H-
1,2,4-triazol-1-yl)-2-(iodomethyl)oxolan-3-yl benzoate (6.5)
236
E.3
31
P NMR (202 MHz, D
2
O) ([(3S,4R,5R)-5-(3-Carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-
dihydroxyoxolan-2-yl]methyl)phosphonic acid (6.2)
E.4
1
H NMR (500 MHz, D
2
O) ([(3S,4R,5R)-5-(3-Carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-
dihydroxyoxolan-2-yl]methyl)phosphonic acid (6.2)
237
E.5 LRMS of ([(3S,4R,5R)-5-(3-Carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-dihydroxyoxolan-
2-yl]methyl)phosphonic acid (6.2)
E.6
1
H NMR (500 MHz, CD
3
OD) 1-[(2R,3R,4R,5R)-3,4-Bis[(tert-
butyldimethylsilyl)oxy]-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide
(6.11)
N
N
N
O
TBSO
OTBS
NH
2
O
HO
238
E.7
1
H NMR (500 MHz, DMSO-d
6
) 2-Iodoxybenzoic acid (6.8)
E.8
1
H NMR (500 MHz, CD
3
OD) Dimethyl [(E)-2-[(3R,4R,5R)-3,4-bis[(tert-
butyldimethylsilyl)oxy]-5-(3-carbamoyl-1H-1,2,4-triazol-1-yl)oxolan-2-
yl]ethenyl]phosphonate (6.13)
N
N
N
O
TBSO
OTBS
NH
2
O P
O
MeO
MeO
239
E.9
1
H NMR (500 MHz, CD
3
OD) Dimethyl (2-[(3S,4R,5R)-5-(3-carbamoyl-1H-1,2,4-
triazol-1-yl)-3,4-dihydroxyoxolan-2-yl]ethyl)phosphonate (6.14)
E.10
31
P NMR (202 MHz, D
2
O) (2-[(3S,4R,5R)-5-(3-Carbamoyl-1H-1,2,4-triazol-1-yl)-
3,4-dihydroxyoxolan-2-yl]ethyl)phosphonic acid (6.3)
N
N
N
O
HO
OH
NH
2
O P
O
MeO
MeO
N
N
N
O
HO
OH
NH
2
O P
O
HO
HO
240
E.11
1
H NMR (500 MHz, D
2
O) (2-[(3S,4R,5R)-5-(3-Carbamoyl-1H-1,2,4-triazol-1-yl)-
3,4-dihydroxyoxolan-2-yl]ethyl)phosphonic acid (6.3)
E.12 LRMS of
(2-[(3S,4R,5R)-5-(3-Carbamoyl-1H-1,2,4-triazol-1-yl)-3,4-
dihydroxyoxolan-2-yl]ethyl)phosphonic acid (6.3)
N
N
N
O
HO
OH
NH
2
O P
O
HO
HO
N
N
N
O
HO
OH
NH
2
O P
O
HO
HO
Abstract (if available)
Abstract
Acyclic nucleoside phosphonates, ANPs, are a drug class with broad-spectrum activity against DNA and RNA viruses. However, limitations with ANP efficacy include: low cell membrane permeability, low oral bioavailability (due to the presence of an ionizable anionic group) and nephrotoxicity In an effort to curtail these effects, as well as to further understand and demonstrate the full therapeutic potential of this drug class, a main focus of the work presented in this dissertation is on the synthesis and antiviral activity of various ANP parent drugs and their corresponding acyclic and cyclic nucleoside phosphonate prodrugs. ❧ Parent ANPs were masked with an amino acid or dipeptide promoiety, which contained a lipophilic N-alkyl long chain on the C-terminal group as a carboxamide. PME and PMP ANP monoester prodrugs required an alternative synthetic route involving PyBrOP coupling with a small alkoxy nucleophile followed by PyBOP coupling with the desired amino acid or dipeptide promoiety. A novel (L)-Ser N-alkyl dipeptide cyclic prodrug of (S)-HPMPC was synthesized in order to evaluate and expand the ANP peptidomimetic prodrug library. PME and HPMP prodrugs containing an N-alkyl C₁₆ long chain demonstrated EC₅₀ values in the micromolar range against a variety of DNA viruses herpes simplex virus type 1, varicella zoster virus, cytomegalovirus, cowpox virus, adenovirus, and vaccinia virus. In addition, a selection of lipophilic N-alkyl amino acid- and dipeptide-based ANP prodrugs were evaluated for in vitro antiviral activity against Kaposi-sarcoma associated herpesvirus. (L)-Tyr-NHC₁₈H₃₇ and (L)-Ser-NHC₁₆H₃₃-(L)-Val prodrugs of (S)-HPMPA and (S)-HPMPC demonstrated inhibition values requiring further assessment of their application as KSHV antiviral agents. In addition, a large-scale synthesis (1 g – 2 g) of (L)-Tyr-NHC₁₆H₃₃ cyclic and acyclic HPMP prodrugs was performed as a means to assess reaction conditions for optimal and efficient generation of large quantities of lead candidates. ❧ Ribavirin (RBV) is a guanosine ribonucleic analogue that acts as a nucleoside inhibitor. It has been found to be active against various DNA and RNA viruses (including flaviviridae viruses like yellow fever and hepatitis C). In an effort to expand the druggable phosphonate library and potentially enhance its antiviral activity against dengue virus, an emerging RNA pathogen to humans, two 5’-phosphonate analogues of RBV have been synthesized. The structural modification lies in replacing the 5’-OH of the ribose ring with an X-P(O)(OH)₂ linkage (where X is CH₂ or CH₂-CH₂). The 5’-phosphonate RBV analogue was synthesized from a published procedure using the well-utilized Michaelis-Arbuzov reaction and the Horner-Wadsworth-Emmons reaction was employed to extend the alkyl length between the ribose ring and phosphonate to afford a novel 5’-methylene phosphonate RBV analogue.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Synthesis, structural analysis and in vitro antiviral activities of novel cyclic and acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs
PDF
Design, synthesis, and antiviral activities of novel tyrosinamide prodrugs
PDF
Peptidomimetic prodrugs of cidofovir: design, synthesis, transport, mechanism of activation, and antiviral activity
PDF
Syntheses of P-O-C linked foscarnet-peptide conjugates
PDF
I. Microwave-assisted synthesis of phosphonic acids; II. Design and synthesis of polymerase β lyase domain inhibitors
PDF
Catalytic applications of palladium-NHC complexes towards hydroamination and hydrogen-deuterium exchange and development of acid-catalyzed hydrogen-deuterium exchange methods for preparative deut...
PDF
Fluorescent imaging probes of nitrogen-containing bone active drugs: design, synthesis and applications
PDF
Design and synthesis of a series of methylenebisphosphonates: a nucleotide analogue toolkit to probe nucleic acid polymerase structure and function
PDF
Carbon-hydrogen bond activation: radical methane functionalization; unactivated alkene coupling; saccharide degradation; and carbon dioxide hydrogenation
PDF
Post-translational processing and activation of the human myeloid alpha-defensin HNP1 precursor is mediated by neutrophil serine proteases
Asset Metadata
Creator
Williams, Melissa Marie (author)
Core Title
Nucleoside phosphonate prodrugs: synthesis and antiviral activity
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Chemistry
Publication Date
01/26/2016
Defense Date
10/14/2014
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
acyclic nucleoside phosphonates,lipophilic promoiety,OAI-PMH Harvest,prodrugs,tyrosinamide
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
McKenna, Charles E. (
committee chair
), Jung, Kyung Woon (
committee member
), Pratt, Matthew R. (
committee member
), Shen, Wei-Chiang (
committee member
), Takahashi, Susumu (
committee member
)
Creator Email
melissamariewilliams9@gmail.com,mmw@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-529681
Unique identifier
UC11297648
Identifier
etd-WilliamsMe-3137_rev.pdf (filename),usctheses-c3-529681 (legacy record id)
Legacy Identifier
etd-WilliamsMe-3137_rev.pdf
Dmrecord
529681
Document Type
Dissertation
Format
application/pdf (imt)
Rights
Williams, Melissa Marie
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
Tags
acyclic nucleoside phosphonates
lipophilic promoiety
prodrugs
tyrosinamide