Synthesis and antiviral evaluation of alkoxyalkyl derivatives of 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)adenine against cytomegalovirus and orthopoxviruses

Article abstract of DOI:10.1021/jm050473m

9-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)adenine [(S)-HPMPA] was one of the first acyclic nucleoside phosphonates described and has been reported to have good antiviral activity against most double-stranded DNA viruses, including the herpes group viruses

Full text of DOI:10.1021/jm050473m

2010
J. Med. Chem. 2006, 49, 2010-2015
Synthesis and Antiviral Evaluation of Alkoxyalkyl Derivatives of
9-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)adenine against Cytomegalovirus and
Orthopoxviruses
James R. Beadle,† William B. Wan,† Stephanie L. Ciesla,† Kathy A. Keith,‡ Caroll Hartline,‡ Earl R. Kern,‡ and
Karl Y. Hostetler*,†
Department of Medicine, VA San Diego Healthcare System and the UniVersity of California, San Diego, La Jolla, California 92093-0676, and
Department of Pediatrics, UniVersity of Alabama School of Medicine, Birmingham, Alabama 35233
ReceiVed May 19, 2005
9-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)adenine [(S)-HPMPA] was one of the first acyclic nucleoside
phosphonates described and has been reported to have good antiviral activity against most double-stranded
DNA viruses, including the herpes group viruses and the orthopoxviruses. However, (S)-HPMPA is not
orally bioavailable and has not been developed for clinical use. We have prepared orally bioavailable lipid
esters of (S)-HPMPA and report their synthesis and antiviral evaluation against cytomegalovirus and
orthopoxviruses. These esters were evaluated in vitro in cells infected with human cytomegalovirus (HCMV),
murine cytomegalovirus (MCMV), vaccinia (VV), and cowpox viruses (CV). The most active compound,
oleyloxyethyl-(S)-HPMPA, was found to have EC50 value of 0.003 µM against HCMV vs 1.4 µM for
unmodified HPMPA. In cells infected with VV and CV, octadecyloxyethyl-(S)-HPMPA had EC50 values of
0.01-0.02 µM versus 2.7-4.0 µM for unmodified HPMPA. When compared with the alkoxyalkyl esters of
cidofovir, the corresponding alkoxyalkyl esters of (S)-HPMPA were equally active against HCMV and MCMV
but were 15-20-fold more active against VV and CV in vitro. The alkoxyalkyl esters of (S)-HPMPA are
promising new compounds worthy of further investigation for treatment of infections caused by herpes
viruses and orthopoxviruses.
Introduction
Of particular interest is the potent activity of CDV, (S)-
HPMPA, and other nucleoside phosphonates against orthopox-
viruses, including cowpox, vaccinia,13 ectromelia (monkeypox),
and smallpox (variola virus).14 If used as a weapon against an
unimmunized population, variola virus has the potential to infect
tens of thousands of individuals, kill 30% or more of those
infected, and trigger the vaccination of millions.15 However,
large numbers of people are not good candidates for vaccination
because of immunosuppression, skin diseases, and cardiovas-
cular disease. Another concern is suggested by the discovery
that recombinant poxviruses encoding interleukin-4 are lethal
despite prior vaccination.16 For these reasons, development of
therapeutics for orthopoxvirus infection is an important area of
research.17 Animal studies indicate that parenteral CDV may
be useful for prevention and/or treatment of vaccinia and variola
infections.14 Since intravenous agents would not be practical in
an emergency situation, safe orally active antiviral drugs for
orthopox infections are needed.
(S)-HPMPA [(S)-9-(3-hydroxy-2-phosphonomethoxypropyl)-
adenine] was the first acyclic nucleoside phosphonate to show
potent, broad-spectrum antiviral activity. Described by Holy´ and
De Clerq in 1986,1 (S)-HPMPA inhibits replication of many
double-stranded DNA viruses, including herpes simplex virus
(HSV) types 1 and 2, varicella zoster virus (VZV), thymidine
kinase-deficient (TK-) mutants of HSV and VZV, human
cytomegalovirus (HCMV), Epstein-Barr virus, hepatitis B
virus, adenoviruses, and various orthopoxviruses.2 (S)-HPMPA
also arrests the growth of Plasmodium falciparum (the causative
agent of malaria)3 and exhibits cytostatic activity against L1210
mouse leukemia cells in vitro.4
After the discovery of (S)-HPMPA, extensive studies of
acyclic nucleoside phosphonates led to the development of three
licensed antiviralsscidofovir (CDV; CMV retinitis), adefovir
(hepatitis B), and tenofovir (HIV/AIDS)5sand the identification
of many more compounds with potentially useful biological
activity.6 However, due to the presence of two negative charges,
free acyclic nucleoside phosphonates are not absorbed well
orally.7 After intravenous injection, drugs of this class ac-
cumulate in the kidney, the site of dose-limiting toxicity.8 CDV
(Vistide) is administered intravenously, while the oral formula-
tions of adefovir (Hepsera) and tenofovir (Viread) are the bis-
pivaloyloxymethyl and bis-(isopropoxycarbonyl)oxymethyl es-
ters, respectively.9,10 Several other phosphonate-masking prodrug
strategies, including aryl phosphonamidates11 and cycloSal
esters,12 have been applied to acyclic nucleoside phosphonates.
We previously reported that esterification of cidofovir with
certain alkanols or alkoxyalkanols enhances the antiviral activity
and selectivity of CDV in vitro.18-20 Optimal in vitro activity
was found with 20-atom alkyl, alkenyl, alkylglyceryl, or
alkoxyalkyl groups esterified to CDV.21 Oral dosing of several
CDV esters showed good bioavailability, greatly reduced
accumulation of drug in kidney,22 and anti-orthopoxvirus
efficacy in three lethal animal models of orthopoxvirus disease,
including ectromelia.23-25 On the basis of these results, hexa-
decyloxypropyl cidofovir (HDP-CDV) is currently under de-
velopment for possible use as an oral treatment in case of a
smallpox bioterror attack.26 CDV-resistant strains of CMV have
been isolated from treated patients27 and laboratory-induced
CDV resistance has been observed in camelpox, cowpox,
monkeypox, and vaccinia viruses.28 Additional agents against
* Corresponding author. Telephone: (858) 552-8585, ext 2616. Fax:
(858) 534-6133. E-mail: khostetl@ucsd.edu.
† VA San Diego Healthcare System and the University of California,
San Diego.
‡ University of Alabama School of Medicine.
10.1021/jm050473m CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/18/2006

Alkoxyalkyl DeriVatiVes of HPMPA
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2011
Scheme 1. Synthesis of Alkoxyalkyl Esters of (S)-HPMPAa
a (a) TMSBr, CH2Cl2; (b) oxalyl chloride, cat. N,N-DMF, CH2Cl2; (c) 2a-d, ethyl ether, pyridine; (d) NaH, Et3N (solvent), 50 °C; (e) 80% aq CH3COOH,
60 °C.
poxvirus disease would be useful. Since (S)-HPMPA was
Solutions of 1 in triethylamine were treated with sodium
hydride and then phosphonoesters 3a-d and the mixture was
heated to 50 °C to obtain fully protected (S)-HPMPA esters
4a-d. Interestingly, the use of triethylamine as the solvent
facilitated the alkylation reaction. Lower yields resulted when
alkylation was attempted in either THF or N,N-DMF solvent.
Reaction of 3a and 3b with 1 provided satisfactory yields of
fully protected (S)-HPMPA analogues. However, unsaturated
esters 3c and 3d gave lower yields of alkylated product and
complete separation of products 4c and 4d from residual 3c-d
by flash chromatography was difficult. However, impure 4c and
4d, could be deprotected (80% aq acetic acid) and then purified
easily by flash chromatography on silica gel to give 5c and 5d.
In contrast to the unsaturated esters, saturated phosphonates 3a
and 3b reacted with (S)-9-(3-trityloxy-2-hydroxypropyl)-N6-
trityladenine in triethylamine to give fully protected intermedi-
ates 4a and 4b, and they were isolable in reasonable yields and
high purity. Compounds 4a and 4b were deprotected (80% acetic
acid) to provide (S)-HPMPA derivatives 5a and 5b.
Biological Activity. In Vitro Antiviral Activity. The (S)-
HPMPA alkoxylalkyl esters 5a-d were evaluated for their
inhibitory activity in cells infected with HCMV, MCMV, VV,
and CV. The unmodified phosphonates (S)-HPMPA and CDV
were used as comparators. All four alkoxyalkyl esters were more
active than (S)-HPMPA against HCMV and MCMV (Table 1).
Against HCMV, EC50 values of 3 nM were obtained for all
four analogues, which is about 270-fold more potent than
HPMPA itself. The most selective compound was OLP-(S)-
HPMPA (5d), which had a selectivity index of 25 000 against
HCMV due to its lower cytotoxicity. Against MCMV, the most
active compound was HDP-(S)-HPMPA (5a) and the most
selective was OLP-(S)-HPMPA (5d).
reported earlier to be more active against poxviruses than
CDV,13,14,29 we began investigating (S)-HPMPA derivatives to
address the need for more active and selective antiviral drugs.
In this report, we describe the synthesis and biological evaluation
of several new alkoxyalkyl analogues of (S)-HPMPA.
Results
Chemistry. We previously synthesized alkoxyalkyl CDV
esters by either esterifying cyclic CDV (cCDV) with alkoxyalkyl
bromides or, more efficiently, using the Mitsunobo reaction to
couple alkoxyalkanols to cCDV.21 Since we did not have access
to premade (S)-HPMPA or cyclic (S)-HPMPA, we envisioned
the route outlined in Scheme 1 as a more direct approach to
the target compounds. Our strategy was based on previous (S)-
HPMPA syntheses reported by Webb30 and Holy´.31 Both groups
prepared (S)-9-(2,3-dihydroxypropyl)adenine derivatives with
the 3-hydroxyl and 6-amino groups protected and then etherified
the 2-hydroxyl using a dialkyl p-toluenesulfonyloxymethylphos-
phonate in the presence of a strong base. Deprotection, including
dealkylation of the phosphonodiesters with bromotrimethyl-
silane, gave (S)-HPMPA.
We prepared (S)-9-(3-trityloxy-2-hydroxypropyl)-N6-trityl-
adenine according to Webb’s method, expecting that it would
be more stable than the N6-benzoyl derivative under the basic
conditions of the impending alkylation step. Adenine, heated
with (S)-trityl glycidyl ether in N,N-DMF/DBU, gave (S)-9-(3-
trityloxy-2-hydroxypropyl)adenine. Further reaction of (S)-9-
(3-trityloxy-2-hydroxypropyl)adenine with trityl chloride in
refluxing pyridine afforded (S)-9-(3-trityloxy-2-hydroxypropyl)-
N6-trityladenine (1) in 53% yield.
Rather than alkylate 1 with a dialkyl toluenesulfonyloxy-
methylphosphonate, the standard method for HPMP compounds,
we prepared alkoxyalkyl toluenesulfonyloxymethylphosphonates
3a-d, with the lipophilic groups preattached. Initial attempts
to couple toluenesulfonyloxymethylphosphonic acid to alkoxy-
alkanols 2a-d using DCC/pyridine resulted in low yields of
the monoesters due to loss of the tosyl group during the
condensation. A more efficient procedure was to treat the diethyl
p-tolueneoxymethylphosphonate with TMSBr and then oxalyl
chloride. Reaction of the resulting phosphonodichloridate with
2a-d, followed by hydrolysis, gave phosphonoesters 3a-d in
good yield.
When the compounds were evaluated against VV and CV
they were also more active than (S)-HPMPA (Table 2). The
most active compound was OLE-(S)-HPMPA (5c), with an EC50
value of 3 nM. Against VV and CV, the alkoxyalkyl esters were
150-900 and 117-561-fold more active, respectively, than (S)-
HPMPA.
Discussion
Esterification of (S)-HPMPA to alkoxyalkyl moieties having
20 or 21 atoms clearly increases antiviral activity and selectivity,
as shown in Tables 1 and 2, extending our findings with CDV

2012 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Beadle et al.
Table 1. Inhibition of Human and Murine Cytomegalovirus (HCMV and MCMV) Replication by Alkoxyalkyl Esters of (S)-HPMPA
HCMV
MCMV
cytotoxicity
compd
(CC50, µM)a,b
EC50 (µM)b
selectivityc
EC50 (µM)b
selectivityc
CDV
(S)-HPMPA
HDP-(S)-HPMPA (5a)
ODE-(S)-HPMPA (5b)
OLE-(S)-HPMPA (5c)
OLP-(S)-HPMPA (5d)
>317 ( 0
>289 ( 38
9.7 ( 6.2
1.5 ( 0.04
6.3 ( 1.6
74.8 ( 6.3
1.2 ( 0.4
>264
>352
3233
500
2100
25000
0.04 ( 0.03
0.16 ( 0.06
0.002 ( 0
0.003 ( 0.002
0.04 ( 0.007
0.1 ( 0.03
>7925
>181
485
500
158
0.82 ( 0.23
0.003 ( 0.0007
0.003 ( 0.001
0.003 ( 0.0007
0.003 ( 0
750
a Cytotoxicity by neutral red uptake in HFF cells. b Values are the mean of two or more assays ( standard deviation. c Selectivity index (SI) ) CC50/
EC50.
Table 2. Inhibition of Vaccinia and Cowpox Virus Replication by Alkoxyalkyl Esters of (S)-HPMPA
vaccinia Copenhagen
cytotoxicity
cowpox Brighton
compd
(CC50, µM)a,b
EC50 (µM)b
selectivityc
EC50 (µM)b
selectivityc
CDV
(S)-HPMPA
HDP-(S)-HPMPA (5a)
ODE-(S)-HPMPA (5b)
OLE-(S)-HPMPA (5c)
OLP-(S)-HPMPA (5d)
>317 ( 0
>289 ( 38
9.7 ( 6.2
1.5 ( 0.4
6.3 ( 1.6
74.8 ( 6.3
25.3 ( 6.1
2.7 ( 2.4
0.01 ( 0.004
0.01 ( 0.003
0.003 ( 0.001
0.018 ( 0.005
>12.5
>107
970
150
2100
4156
30.7 ( 6.3
4.0 ( 3.8
>10.3
>72
485
75
900
0.02 ( 0.006
0.02 ( 0.017
0.007 ( 0
0.034 ( 0.004
2200
a Cytotoxicity by neutral red uptake. b Values are the mean of two or more assays ( standard deviation. c Selectivity index (SI) ) CC50/EC50.
than that reported for the CMV DNA polymerase.34 With the
VV DNA polymerase, CDV can be incorporated into the
Table 3. Inhibition of Viral Replication by Alkoxyalkyl Esters of CDV
and HPMPAa
EC50 (µM)
growing DNA strand opposite template G’s; however, the
enzyme exhibits a lower catalytic efficiency compared with
dCTP. CDV-terminated primers are good substrates for the next
dNMP addition, but these CDV + 1 base reaction products are
not good substrates for further DNA synthesis. Although CDV
can be excised from the primer 3′-terminus by the 3′-to-5′
proofreading exonuclease activity of the VV DNA polymerase,
DNAs bearing CDV as the penultimate 3′-residue are completely
resistant to exonuclease attack.33 It will be interesting to
determine the mechanism of (S)-HPMPA diphosphate inhibition
of the vaccinia DNA polymerase in view of the multiple log
increase in antiviral activity noted with HPMPA and its
alkoxyalkyl esters as compared with CDV.
In conclusion, the alkoxyalkyl esters of HPMPA exhibit
multiple log increases in antiviral activity versus HCMV,
MCMV, VV, and CV versus unmodified HPMPA. HDP-(S)-
HPMPA and ODE-(S)-HPMPA are particularly active against
the poxviruses with antiviral activity 15-30-fold greater than
the corresponding CDV analogue against VV and CV, respec-
tively. Compounds of this class are potentially useful against
infections caused by the orthopoxviruses and are worthy of
further development.
compd
HCMV
MCMV
vaccinia
cowpox
cidofovir (HPMPC)
HDP-CDV
ODE-CDV
(S)-HPMPA
HDP-(S)-HPMPA (5a)
ODE-(S)-HPMPA (5b)
1.20
0.04
46.2
0.80
0.20
2.70
0.01
0.01
44.7
0.60
0.30
4.00
0.02
0.02
0.0009
0.0009
0.82
0.003
0.003
0.0009
0.0010
0.16
0.002
0.003
a Data abstracted from Tables 1 and 2 and from refs 18, 19, and 21.
(HPMPC) to (S)-HPMPA, a related acyclic nucleoside phos-
phonate.18-21 HDP-CDV gains its increased antiviral activity
because of its greatly enhanced cell uptake and conversion to
CDV by cellular phospholipases C and/or phosphodiesterases.32
The same mechanisms are presumed to increase the antiviral
activity of HDP-(S)-HPMPA compared with (S)-HPMPA. In
vitro studies with 14C-labeled HDP-(S)-HPMPA are currently
in progress.
We compared the antiviral activity of HDP and ODE esters
of CDV and HPMPA against HCMV and MCMV and with VV
and CV using previously published data for the CDV analogues
(Table 3).18,19,21 Against HCMV and MCMV the HDP and ODE
esters of CDV and HPMPA differed little in activity, the CDV
esters being slightly more active (2-3-fold) than the HPMPA
analogues. Unmodified CDV and (S)-HPMPA had similar
antiviral activity against the cytomegaloviruses. However,
against both poxviruses, (S)-HPMPA was much more active
than CDV (11-16-fold). Strikingly, the HDP and ODE esters
of HPMPA were substantially more active than the correspond-
ing esters of CDV: 80- and 20-fold for VV and 30- and 15-
fold for CV. The increased activity of HDP- and ODE-(S)-
HPMPA versus HDP- and ODE-CDV against poxviruses but
no difference in activity versus CMV is not likely to be due to
cellular uptake and metabolism of the alkoxyalkyl esters of CDV
and HPMPA to their respective diphosphates, because both the
CMV and poxvirus studies were done in the same HFF cell
line. Therefore, the marked increase in (S)-HPMPA analogue
activity against the poxviruses may be due to differences in their
effect on the vaccinia E9L DNA polymerase. Evans and co-
workers33 found that the mechanism of inhibition of the VV
DNA polymerase by CDV-diphosphate was slightly different
Experimental Section
Chemistry Methods. Elemental microanalyses were performed
1
by Atlantic Microlabs of Norcross, GA. H and 31P NMR spectra
were recorded on a Varian HG-400 NMR spectrophotometer with
tetramethylsilane (internal) and 85% D3PO4 in D2O (external) as
1
references for H and 31P (0.00 ppm), respectively. Electospray
ionization mass spectra (MS ES) were obtained by HT Laboratories
(San Diego, CA). Analytical thin-layer chromatography (TLC) was
performed on Analtech 250 µm silica gel GF Uniplates visualized
under UV light, with Phospray (Supelco, Bellafonte, PA) and
charring at 400 °C. Chromatographic purification was done by the
flash method using Merck silica gel (240-400 mesh). All com-
mercially obtained reagents were used as received unless otherwise
noted.
Diethyl toluenesulfonyloxymethylphosphonate was synthesized
from diethyl hydroxymethylphosphonate.35 (S)-9-[3-Trityloxy-2-
hydroxypropyl]-N6-trityladenine was prepared from adenine and (S)-
trityl glycidyl ether (Daiso Co., Ltd.) following the method of
Webb.30 Lipophilic alkoxyalkanols (2a-d, Scheme 1) were syn-
thesized according to procedures we previously described.36

Alkoxyalkyl DeriVatiVes of HPMPA
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2013
General Procedure for the Synthesis of Alkoxyalkyl Tolu-
enesulfonyloxymethylphosphonates (3a-d). Bromotrimethylsilane
(27 g, 175 mmol) was added to a solution of diethyl toluenesul-
fonyloxymethylphosphonate (9.5 g, 29.5 mmol) in dichloromethane
(anhydrous, 150 mL). The mixture was stirred at room temperature
under a N2 atmosphere for 18 h. The mixture was then concentrated
under vacuum to remove solvent and excess TMSBr and then
redissolved in dichloromethane (150 mL) and cooled to 0 °C with
an ice bath. N,N-DMF (0.5 mL) was added, a solution of oxalyl
chloride (22 g, 175 mmol) in CH2Cl2 (50 mL) was added dropwise
over 30 min, and then the mixture was stirred for an additional 5
h. The mixture was evaporated to an oil which was then redissolved
in Et2O (100 mL). A solution of the alkoxyalkanol (2a-d, 21.5
mmol) and pyridine (10 mL) in Et2O (50 mL) was added, and
stirring was continued for about 3 h or until TLC analysis (1:1
hexanes/ethyl acetate) indicated complete phosphonylation of the
alcohol. The reaction mixture was then added to cold, saturated
NaHCO3 and vigorously stirred for 1 h. After hydrolysis was
complete, the organic layer was separated, dried over MgSO4, and
evaporated under vacuum to give the crude esters, which were
purified by flash chromatography (elution solvent: 15% EtOH/
CH2Cl2).
3-(Hexadecyloxy)propyl toluenesulfonyloxymethylphosphonate,
sodium salt (3a), was prepared from 3-hexadecyloxy-1-propanol
(2a) in 67% yield: 1H NMR (CDCl3) δ 0.88 (t, 3H, -CH3), 1.26
(br s, 26H, OCH2CH2(CH2)13CH3), 1.54 (m, 2H, OCH2CH2(CH2)13-
CH3), 1.83 (qt, 2H, -OCH2CH2CH2O-), 2.46 (s, 3H, toluyl CH3),
3.38 (t, 2H, -CH2-), 3.47 (t, 2H, -CH2-), 3.91 (dt, 2H, -P(O)-
OCH2-), 4.02 (d, 2H, -CH2P(O)O-), 7.37 (d, 2H, aromatic), 7.80
(d, 2H, aromatic); MS (ES) m/z 571.32 [M + Na]+, 593.27 [M -
H + 2Na]+, 547.02 [M - H]-. Anal. (C27H48O7NaPS‚0.5H2O) C,
H, N.
7.2-7.5 (br m, 30H), 7.82 (s, 1H), 8.20 (s, 1H); MS (ES) m/z 1071
[M + H]+, 1069 [M - H]-. Anal. (C66H79NaN5O6P‚0.5H2O) C,
H, N.
2-(Octadecyloxy)ethyl (S)-9-[3-trityloxy-2-(phosphonometh-
oxy)propyl]-N6-trityladenine (4b) was prepared from 3b in 56%
yield: 1H NMR (CDCl3) δ 0.90 (t, 3H), 1.26 (br s, 30H), 1.41 (m,
2H), 3.3-3,7 (m, 7H), 3.8 (m, 2H), 4.21 (m, 2H), 7.2-7.6 (m,
30H), 7.93 (s, 1H), 8.10 (s, 1H); MS (ES) m/z 1085 [M + H]+,
1083 [M - H]-. Anal. (C67H81NaN5O6P‚H2O) C, H, N.
2-(Oleyloxy)ethyl (S)-9-[3-trityloxy-2-(phosphonomethoxy)-
propyl]-N6-trityladenine (4c) was prepared from 3c in 12% yield.
After flash chromatography, TLC analysis indicated that the product
was contaminated with 3c (<5%). The crude material was depro-
tected without further purification. Analysis by mass spectroscopy
indicated that 4c was the major product: MS (ES) m/z 1083 [M +
H]+, 1081 [M - H]-.
3-(Oleyloxy)propyl (S)-9-[3-trityloxy-2-(phosphonomethoxy)-
propyl]-N6-trityladenine (4d) was prepared from 3d in 28% yield.
After flash chromatography, TLC analysis indicated that the product
was contaminated with 3d (<5%). The crude material was
deprotected without further purification. Analysis by mass spec-
troscopy indicated that 4d was the major product: MS (ES) m/z
1097 [M + H]+, 1095 [M - H]-.
General Procedure for Deprotection and Isolation of (S)-
HPMPA Alkoxyalkyl Esters (5a-d). Derivatives 4a-d were
suspended in 80% aqueous acetic acid (20 mL/mmol) and heated
to 60 °C for 1 h or until detritylation was complete as determined
by TLC analysis. After cooling, the solvent was evaporated, and
the products were purified by flash chromatography. Elution with
30% MeOH/CH2Cl2 provided compounds 5a-d as white powdery
solids.
3-(Hexadecyloxy)propyl (S)-9-[3-hydroxy-2-(phosphonometh-
oxy)propyl]adenine, sodium salt (HDP-HPMPA, 5a), was pre-
1
2-(Octadecyloxy)ethyl toluenesulfonyloxymethylphosphonate,
sodium salt (3b), was prepared from 2-octadecyloxy-1-ethanol (2b)
in 71% yield: 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.26 (br s, 30H),
1.54 (m, 2H), 2.60 (s, 3H), 3.51 (t, 2H), 3.65 (t, 2H), 4.03 (dt,
2H), 4.16 (d, 2H), 7.35 (d, 2H), 7.78 (d, 2H); MS (ES) m/z 563 [M
+ H]+, 585 [M + Na]+, 561 [M - H]-. Anal. (C28H50NaO7PS‚
1.5H2O) C, H, N.
pared from 4a in 81% yield: H NMR (CD3OD) δ 0.84 (t, 3H),
1.20 (br s, 18H), 1.23 (m, 2H), 1.63 (qt, 2H), 3.67 (m, 2H), 3.20-
3.55 (m, 11H), 4.05-4.34 (pair dd, 2H), 8.13 (s, 1H), 8.17 (s, 1H);
31P NMR δ 15.18; MS (ES) m/z 586 [M + H]+, 608 [M + Na]+,
585 [M - H]-. Anal. (C28H52NaN5O6P‚0.5H2O) C, H, N.
2-(Octadecyloxy)ethyl (S)-9-[3-Hydroxy-2-(phosphonomethoxy)-
propyl]adenine, sodium salt (ODE-HPMPA, 5b). Deprotection
of 4b gave ODE-HPMPA in 73% yield: 1H NMR (CD3OD) δ 0.85
(t, 3H), 1.22 (br s, 30H), 1.43 (m, 2H), 1.89 (t, 2H), 3.35-3.80
(m, 11H), 4.00-4.15 (m, 2H), 8.11 (s, 1H), 8.15 (s, 1H); 31P NMR
δ 15.06; MS (ES) m/z 600 [M + H]+, 598 [M - H]-. Anal. (C29H53-
NaN5O6P‚1.5H2O) C, H, N.
2-(Oleyloxy)ethyl toluenesulfonyloxymethylphosphonate,
sodium salt (3c), was prepared from 2-oleyloxy-1-ethanol (2c) in
1
55% yield: H NMR (CDCl3) δ 0.88 (t, 3H), 1.27 (br d, 18H),
1.58 (m, 2H), 2.15 (m, 4H), 2.44 (s, 3H), 3.37 (t, 2H), 3.49 (t,
2H), 4.03 + 4.13 (dt, 2H), 4.23 (d, 2H), 5.30 (t, 2H), 7.41 (d, 2H),
7.77 (d, 2H); MS (ES) m/z 583 [M + Na]+, 559 [M - H]-. Anal.
(C28H48NaO7PS) C, H, N.
2-(Oleyloxy)ethyl (S)-9-[3-hydroxy-2-(phosphonomethoxy)-
propyl]adenine, sodium salt (OLE-HPMPA, 5c), was prepared
from 4c in 85% yield: 1H NMR (CD3OD) δ 0.88 (t, 3H), 1.29 (br
s, 18H), 1.47 (m, 2H), 2.00 (d, 4H), 3.35 (t, (2H), 3.45 (t, 2H),
3.52-3.85 (m, 4H), 3.78 (m, 1H), 3.90 (dd, 2H), 4.45-4.34 (pair
dd, 2H), 5.38 (t, 2H), 8.22 (s, 1H), 8.23 (s, 1H); 31P NMR δ 15.12;
MS (ES) m/z 598 [M + H]+, 596 [M - H]-. Anal. (C29H51-
NaN5O6P‚2.0H2O) C, H; N: calcd, 10.68; found, 9.96.
3-(Oleyloxy)propyl
toluenesulfonylmethylphosphonate,
sodium salt (3d), was prepared from 3-oleyloxy-1-propanol (2d)
in 54% yield: 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.27 (br d, 18H),
1.56 (m, 2H), 1.81 (qt, 2H), 2.02 (m, 4H), 2.45 (s, 3H), 3.41 (t,
2H), 3.49 (t, 2H), 3.93 + 3.91 (dt, 2H), 4.00 (d, 2H), 5.33 (t, 2H),
7.42 (d, 2H), 7.81 (d, 2H); MS (ES) m/z 597 [M + Na]+, 573 [M
- H]-. Anal. (C29H50NaO7PS‚0.5H2O) C, H, N.
General Procedure for Alkylation of (S)-9-[3-Trityloxy-2-
hydroxypropyl]-N6-trityladenine (1) with 3a-d. Sodium hydride
(24 mg, 1.0 mmol) was added to a stirred solution of (S)-9-[3-
trityloxy-2-hydroxypropyl]-N6-trityladenine (640 mg, 0.62 mmol)
in dry triethylamine (10 mL). After 15 min, the appropriate
alkoxyalkyl toluenesulfonyloxymethylphosphonate (3a-d, 0.65
mmol) was added, and the reaction mixture was heated to 50 °C
and kept there overnight. After cooling, the mixture was quenched
with brine and extracted with ethyl acetate (3 × 15 mL). The
organic extracts were dried over MgSO4 and then concentrated in
vacuo. The residue was purified by flash chromatography, where
the products were eluted with 10% EtOH/CH2Cl2.
3-(Oleyloxy)propyl (S)-9-[3-Hydroxy-2-(phosphonomethoxy)-
propyl]adenine, sodium salt (OLP-HPMPA, 5d). Deprotection
1
of 4d yielded OLP-HPMPA in 77% yield: H NMR (CD3OD) δ
0.88 (t, 3H), 1.27 (br s, 18H), 1.50 (m, 2H), 1.79 (qt, 2H), 2.00 (d,
4H), 3.35 (t, 2H), 3.45 (t, 2H), 3.48-3.73 (m, 4H), 3.78 (m, 1H),
3.88 (dd, 2H), 4.45-4.34 (pair dd, 2H), 5.32 (t, 2H), 8.21 (s, 1H),
8.25 (s, 1H); 31P NMR δ 15.20; MS (ES) m/z 612 [M + H]+, 634
[M + Na]+, 611 [M - H]-. Anal. (C30H53NaN5O6P‚1.25H2O) C,
H, N.
Biological Methods. Cells and Viruses. Human foreskin
fibroblast (HFF) cells were prepared as primary cultures from
freshly obtained newborn human foreskins (UAB or Brookwood
Hospital, Birmingham, AL) as soon as possible after circumcision.
Vero cells were obtained from the American Type Culture
Collection (Manassas, VA). Mouse embryo fibroblast (MEF) cells
were also prepared as primary cultures. Cells were propagated in
minimal essential medium (MEM) containing 10% fetal bovine
serum (FBS), L-glutamine (2 mM), penicillin (100 U/mL), and
3-(Hexadecyloxy)propyl (S)-9-[3-trityloxy-2-(phosphonometh-
oxy)propyl]-N6-trityladenine (4a) was prepared from 3a in 66%
yield: 1H NMR (CDCl3) δ 0.88 (t, 3H), 1.33 (br s, 26H), 1.46 (m,
2H), 1.77 (qt, 2H), 3.37-3.8 (m, 9H), 3.86 (m, 2H), 4.03 (m, 2H),

2014 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Beadle et al.
gentamicin (25 µg/mL) in T-175 cm2 tissue culture flasks (BD
interests and serve as consultants to Chimerix Inc. The terms
of these arrangements have been reviewed and approved by the
University of California, San Diego and the University of
Alabama at Birmingham, respectively, in accordance with their
conflict of interest policies.
Falcon, Bedford, MA).
HCMV strain AD-169 and MCMV strain Smith were propagated
in HFF or MEF cells using standard virological techniques. Vaccinia
virus (VV) strain Copenhagen and cowpox virus (CV) strain
Brighton stock pools were obtained from Dr. John Huggins of the
U.S. Army Medical Research Institute for Infectious Diseases,
Frederick, MD. These pools had been prepared in Vero cells and
were diluted 1:50 in our laboratory to provide working stocks.
Supporting Information Available: Elemental analysis data
for compounds 3a-d, 4a-d, and 5a-d. This material is available
free of charge via the Internet at http://pubs.acs.org.
Activity against HCMV and MCMV. The antiviral assays were
performed as described previously.37 HFF or MEF cells were grown
in 6- or 12-well plates for 2 days (HFF) or 1 day (MEF) before
virus was added (diluted in MEM containing 10% FBS to 20-30
plaque forming units per well). The medium was aspirated, and
0.2 mL of virus was added to each well in triplicate with 0.2 mL
of medium added to drug toxicity wells. Ganciclovir and CDV were
used as positive controls. The plates were incubated for 1 h with
shaking every 15 min. The infected cells were incubated with MEM
containing varying concentrations of the test compounds. Com-
pounds were serially diluted 1:5 in MEM with 2% FBS starting at
100 µM. Following incubation at 37 °C (7 days for MCMV or 8
days for HCMV), the cells were stained for 6 h with 2 mL of 0.02%
neutral red in phosphate-buffered saline (PBS). After the stain was
aspirated, viral plaques were enumerated with the aid of a 10×
stereomicroscope. The antiviral activity was expressed as EC50,
which represents the compound concentration required to reduce
virus plaque formation by 50%.
Activity against VV and CV. Two days prior to use, HFF cells
were plated in six-well plates and incubated at 37 °C with 5% CO2
and 90% humidity. On the day of assay, the drugs were made up
at twice the desired concentration in 2× MEM containing 10%
FBS and antibiotics and diluted serially 1:5 in 2× MEM to provide
six concentrations of test compound. The initial concentration was
usually 200 µM and ranged down to 0.06 µM. The virus to be
used was diluted in MEM containing 10% FBS to the desired
concentration that would give 20-30 plaques per well. The medium
was then aspirated from the wells, and 0.2 mL of virus was added
to each well in triplicate, with 0.2 mL of medium being added to
drug toxicity wells. The plates were incubated for 1 h with shaking
every 15 min. After the incubation period, an equal amount of 1%
agarose was added to an equal volume of each drug dilution. This
gave final drug concentrations beginning with 100 µM and ending
with 0.03 µM and a final agarose overlay concentration of 0.5%.
The drug agarose mixture was added to each well in 2 mL volumes,
and the plates were incubated for 3 days, after which the cells were
stained with a 0.02% solution of neutral red in PBS. After a 5-6
h incubation period, the stain was aspirated, and the plaques were
counted using a stereomicroscope at 10× magnification. The Mac-
Synergy II, version 1 computer program was used to calculate the
50% effective concentration (EC50).
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