Synthesis of the 5-phosphono-pent-2-en-1-yl nucleosides: A new class of antiviral acyclic nucleoside phosphonates

Article abstract of DOI:10.1016/j.bmc.2006.11.038

A new class of acyclic nucleoside phosphonates, the 5-phosphono-pent-2-en-1-yl nucleosides and their hexadecyloxypropyl esters, were synthesized from butyn-1-ol. Only the hexadecyloxypropyl esters showed antiviral activity against herpes simplex virus typ

Full text of DOI:10.1016/j.bmc.2006.11.038

Bioorganic & Medicinal Chemistry 15 (2007) 1771–1779
Synthesis of the 5-phosphono-pent-2-en-1-yl nucleosides:
A new class of antiviral acyclic nucleoside phosphonatesq
Hyunah Choo,a,b,c James R. Beadle,a,b Youhoon Chong,d
Julissa Trahana,b and Karl Y. Hostetlera,b,
*
aDepartment of Medicine, Division of Infectious Disease, University of California, San Diego, La Jolla, CA 92093-0676, USA
bVeterans Medical Research Foundation, San Diego, CA 92161, USA
cLife Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
dDivision of Biosciences and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
Received 18 September 2006; accepted 27 November 2006
Available online 30 November 2006
Abstract—A new class of acyclic nucleoside phosphonates, the 5-phosphono-pent-2-en-1-yl nucleosides and their hexadecyloxypro-
pyl esters, were synthesized from butyn-1-ol. Only the hexadecyloxypropyl esters showed antiviral activity against herpes simplex
virus type 1, in vitro. Hexadecyloxypropyl 1-(5-phosphono-pent-2-en-1-yl)-thymine was the most active and selective compound
among the synthesized nucleotides with an EC50 value of 0.90 lM.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
form of penciclovir, are also available now as effective
medications that can reduce the duration and severity
of HSV-1 infections.4 The general mode of action of
nucleoside analogs is through inhibition of viral DNA
polymerases by acting as competitive inhibitors and/or
DNA chain terminators. Nucleosides require intracellu-
lar conversion to their triphosphate form to be active as
antivirals, with the rate-determining step often being the
first phosphorylation which affords the corresponding
50-monophosphate. The effectiveness of nucleoside ana-
logs is often limited by either poor cellular penetration,
inefficient phosphorylation or weak interaction with the
viral DNA polymerase.
The herpes simplex viruses (HSV) belong to the family
of the Herpesviridae.1 There are eight known human
herpesviruses, including the two herpes simplex viruses
HSV-1 and HSV-2. HSV virions are 180–200 nm in
diameter and contain an icosahedral capsid surrounding
the linear double-stranded DNA genome. Cellular infec-
tion is initiated when the virus binds to heparin sulfate
chains on cell surface proteoglycans.2 HSV-1 is respon-
sible for the lesions at orofacial sites that are commonly
known as cold sores. Viral transmission is caused by
kissing, or by sharing the same utensils and towels.
HSV-2 is responsible for mucocutaneous genital infec-
tions and usually is spread by intimate sexual contact.
Resistance to selective HSV-1 agents such as acyclovir
and penciclovir5 is mostly mediated by deficiency of
HSV thymidine kinase, the enzyme normally responsible
for the initial phosphorylation in HSV-1-infected cells.6
The emergence of acyclovir-resistant variants, especially
in immunocompromised patients, has forced the devel-
opment of new drugs with activity against acyclovir-
resistant viruses.7
Nucleoside analogs became the mainstay for chemother-
apy of viral diseases in 1982 with the introduction of
acyclovir, the first selective antiviral agent.3 Valaciclovir,
an oral prodrug of acyclovir, and famciclovir, an oral
Keywords: Antivirals; Nucleosides; Nucleoside phosphonates;
Prodrugs.
q
Portions of this paper were presented in abstract form at the
In contrast to nucleoside analogs, acyclic nucleoside
phosphonates, such as cidofovir and adefovir, possess
a phosphonomethyl ether moiety instead of the nucleo-
tide phosphate ester and have advantages over the
nucleoside analogs because they bypass the limiting first
International Conference on Antiviral Research, April 11–14, 2005,
Barcelona, Spain.
Corresponding author. Tel.: +1 858 552 8585x2616; fax: +1 858 534
*
6133; e-mail: khostetl@ucsd.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.038

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H. Choo et al. / Bioorg. Med. Chem. 15 (2007) 1771–1779
phosphorylation step. As a result, acyclic nucleoside
phosphonates inhibit viruses that do not encode a thy-
midine kinase, as well as thymidine kinase deficient
strains of HSV-1. However, their double negative charge
at physiological pH reduces penetration into the cell,
reducing their antiviral activity. To overcome this prob-
lem, nucleotides are converted to phosphonoester forms
which are designed to penetrate into the cell by virtue of
their reduced polarity. The esters are hydrolyzed during
oral absorption by intestinal enzymes and the nucleo-
tides circulate as the antiviral nucleoside phosphonate.
After cellular uptake and anabolic phosphorylation to
corresponding diphosphate nucleotides, they can be
incorporated into the growing viral DNA strand causing
chain termination or otherwise inhibiting antiviral activ-
ity. One example is tenofovir disoproxil fumarate, which
is approved by the FDA for the treatment of HIV
(Fig. 1).8 Tenofovir disoproxil is a phosphonodiester
form of tenofovir. After hydrolysis and two anabolic
phosphorylations, tenofovir diphosphate shows antivi-
ral activity especially against wild type and drug-resis-
tant HIV.
sides (PPen-Ns) and their hexadecyloxypropyl deriva-
tives (HDP-PPen-Ns).
2. Results and discussion
2.1. Synthetic chemistry
Our synthetic strategy, shown in Scheme 1, was to intro-
duce the cis-double bond through partial hydrogenation
of a triple bond to give an intermediate (9) which could
be coupled to various nucleobases. Thus, 3-butyn-1-ol
(1) was treated with 3,4-dihydropran and PPTS to give
THP-protected butynol (2) in 86% yield. Reaction of
compound 2 with paraformaldehyde gave hydroxyme-
thylated compound 3 in 62% yield. Compound 3 was
protected (4, in 96% yield) and then treated with
PPTS/methanol to give alcohol 5 in 78% yield. Com-
pound 5 was converted to bromide 6 (99% yield), which
underwent Arbuzov reaction in P(OEt)3 to afford diethyl
phosphonate 7 in 98% yield. Partial hydrogenation of 7
over Lindlar’s catalyst13 gave 8 with a cis-double bond.
Finally, key intermediate 9 was obtained in 99% yield
after deprotection of 8 with TBAF.
Our laboratory recently reported a new type of phos-
phonoester, alkoxyethyl and alkoxypropyl phosphono-
ester prodrugs which greatly increase the penetration
of nucleotide analogs into cells.9 This strategy increases
antiviral activity dramatically and makes acyclic nucleo-
side phosphonates orally active.10 Cidofovir (CDV),
which is used intravenously for the treatment of
cytomegalovirus (CMV) retinitis, was converted to
hexadecyloxypropyl-CDV (HDP-CDV) or octadecyl-
oxyethyl-CDV (ODE-CDV) (Fig. 1), resulting in a
>100-fold increase of antiviral activity against CMV.11
HDP-CDV and ODE-CDV were orally active against
lethal poxvirus infections and in murine CMV infec-
tion.12 Acyclic nucleoside phosphonates are very prom-
ising antiviral agents, especially when their high polarity
is minimized by esterification with alkoxyalkyl groups.
Scheme 2 illustrates Mitsunobu reaction of compound 9
with various nucleobases to give the PPen nucleosides.
Adenine and 2-amino-6-chloropurine were coupled with
9 to give compounds 10 and 11 in 42% and 50% yield,
respectively. Compound 10 was then dealkylated using
bromotrimethylsilane (TMSBr) to give 9-(5-phospho-
nopent-2-en-1-yl)-adenine (13, PPen-A). Each 5-phos-
phonopent-2-en-1-yl nucleoside was purified by
DOWEX-1X2 ion-exchange column chromatography
with gradient elution (0–0.25 M HCO2H). Compound
11 was hydrolyzed with 88% HCO2H at 100 ꢁC for 8 h
to give guanine derivative 12 which was dealkylated
to give 9-(5-phosphono-pent-2-en-1-yl)-guanine (15,
PPen-G). Hexadecyloxypropyl 9-(5-phosphono-pent-2-
en-1-yl)-adenine (16, HDP-PPen-A) was obtained by
DCC coupling of PPen-A (13) and 3-(hexadecyloxy)-1-
propanol. Similarly, compound 11 was hydrolyzed by
TMSBr, esterified with 3-hexadecyl-1-propanol, and
hydrolyzed with HCO2H to afford HDP-PPen-G 18.
Herein we describe for the first time the synthesis and
anti-HSV activity of a new type of acyclic nucleoside
phosphonates, the 5-phosphono-pent-2-en-1-yl nucleo-
O
O
N
N
N
N
NH
NH2
NH
NH2
Intermediate 9 was also coupled with 3-benzoyluracil
and 3-benzoylthymine under Mitsunobu conditions, fol-
lowed by treatment with methanolic ammonia, to give
compounds 19 and 21 in 81% and 78% yield, respective-
ly. PPen-U (22) and PPen-T (24) were obtained by treat-
ment with TMSBr in acetonitrile in 98% and 85% yield,
respectively. Compound 19 was treated with 2,4,6-tri-
isopropylbenzenesulfonyl chloride, TEA, and DMAP
followed by NH4OH to give the cytosine derivative 20
in 75% yield. Compound 20 was converted to PPen-C
(23) in 71% yield. Finally, hexadecyloxypropyl deriva-
tives 25, 26, and 27 were obtained by DCC coupling in
44%, 38%, and 15% yield, respectively.
HO
HO
N
N
O
O
HO
acyclovir
penciclovir
NH2
N
NH2
N
N
O
O
O
O
N
O
N
O
N
O
P
O
O
R
Na+ -
O
P
O
O
O
CH3
O
O
OH
tenofovir disoproxil
hexadecyloxypropyl cidofovir,
R = -(CH2)3O(CH2)15CH3
octadecyloxyethyl cidofovir,
R = -(CH2)2O(CH2)17CH3
2.2. Antiviral evaluation
The antiviral activity of the synthesized phosphonopen-
tenyl nucleosides (PPen-Ns) and their hexadecyloxypro-
Figure 1. Structures of antiviral nucleosides and acyclic nucleoside
phosphonates.

H. Choo et al. / Bioorg. Med. Chem. 15 (2007) 1771–1779
1773
THPO
HO
THPO
THPO
c
a
b
OTBDPS
OH
2
3
1
4
d
Br
HO
(EtO)2(O)P
f
e
OTBDPS
OTBDPS
OTBDPS
OTBDPS
7
8
5
6
g
OH
h
(EtO)2(O)P
(EtO)2(O)P
9
Scheme 1. Synthesis of the key intermediate 9. Reagents and conditions: (a) DHP, PPTS, CH2Cl2, rt; (b) n-BuLi, (CH2O)n, 0 ꢁC; (c) TBDPSCl,
imidazole, CH2Cl2, rt; (d) MeOH, PPTS, rt; (e) CBr4, PPh3, CH2Cl2, ꢀ78 ꢁC; (f) P(OEt)3, reflux; (g) H2, Lindlar’s catalyst, MeOH, rt; (h) TBAF,
acetonitrile, 0 ꢁC.
X
X
N
X
N
N
N
N
N
N
N
N
N
N
N
Y
O
P
HO
OH
Y
Y
a
c
d
HDPO
(EtO)2(O)P
(EtO)2(O)P
(HO)2(O)P
9
10 X = NH2, Y = H
13 X = NH2, Y = H
16 X = NH2, Y = H
17 X = Cl, Y = NH2
18 X = OH, Y = NH2
11 X = Cl, Y = NH2
12 X = OH, Y = NH2
14 X = Cl, Y = NH2
15 X = OH, Y = NH2
b
b
X
X
X
Y
Y
Y
N
N
N
N
O
N
O
N
O
O
P
a
c
d
(EtO)2(O)P
(HO)2(O)P
HDPO
HO
19 X = OH, Y = H
20 X = NH2, Y = H
21 X = OH, Y = CH3
22 X = OH, Y = H
23 X = NH2, Y = H
24 X = OH, Y = CH3
25 X = OH, Y = H
26 X = NH2, Y = H
e
27 X = OH, Y = CH3
Scheme 2. Synthesis of 5-phosphono-pent-2-en-1-yl nucleosides and their phosphonoesters. Reagents and conditions: (a) DIAD, PPh3, nucleobases,
DMF, 0 ꢁC or rt; (b) HCOOH, reflux; (c) TMSBr, acetonitrile, rt; (d) HDP-OH, DCC, DMAP, DMF, 60 ꢁC; (e) i—2,4,6-triisopropylbenzenesulfonyl
chloride, TEA, DMAP, acetonitrile, rt; ii—NH4OH.
pyl derivatives (HDP-PPen-Ns) was tested against HSV-
1 in vitro using a DNA reduction assay method as
reported previously11 and the results are summarized
in Table 1. The unmodified PPen nucleosides, such as
compounds 13, 15, 22, 23, and 25, show no inhibitory
activity against HSV-1 and no cytotoxicity up to
100 lM. Nucleoside phosphonates sometimes fail to
exhibit biological activity in vitro because they carry a
double negative charge at physiological pH conditions
and do not readily enter cells. Previously, it was reported
that lipid-modified cidofovir, HDP-CDV, enters cells
rapidly and is metabolized to the active form, cidofovir
diphosphate, resulting in increased antiviral activity ver-
sus CDV.9,11 Therefore, we prepared hexadecyloxypro-
pyl esters of the PPen compounds. Some of the
esterified PPen nucleosides showed potent inhibitory
activity against HSV-1. Of these, HDP-PPen-T (27) is
the most active compound against HSV-1 with an

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H. Choo et al. / Bioorg. Med. Chem. 15 (2007) 1771–1779
Table 1. Antiviral activity and selectivity of the PPen nucleosidesa
Compounds
Anti-HSV-1 activityb EC50, lM
Cytotoxicityc CC50, lM
Selective index
PPen-A 13
PPen-G 15
>30
>30
>100
>100
—
—
PPen-U 22
PPen-C 23
PPen-T 24
>30
>30
>100
>100
>100
—
—
>30
—
HDP-PPen-A 16
HDP-PPen-G 18
HDP-PPen-U 25
HDP-PPen-C 26
HDP-PPen-T 27
18 4.5
5.8 2.2
>30
17.3 4.2
12.2 6.2
51.0 11.8
11.2 4.2
7.4 2.4
0.96
2.10
—
4.7 5.4
0.90 0.5
2.38
8.22
a The values are the means SD of 3 experiments.
b Anti-HSV-1 activity was determined by DNA reduction assay in MRC-5 cells.
c Antiproliferative activity and cytotoxicity was determined by neutral red uptake assay method in MRC-5 cells.
EC50 value of 0.90 lM. The lack of antiviral activity of
HDP-PPen-U (25) may be explained by the fact that uri-
dine triphosphate (RNA) is not a HSV-1 DNA polymer-
ase substrate. The other HDP-PPen nucleosides such as
compounds 16, 18, and 26 show anti-HSV-1 activity
with EC50 values of 18.0, 5.8, and 4.7 lM. Selective
indexes were low for HDP-PPen-A and HDP-PPen-U,
intermediate with HDP-PPen-C and HDP-PPen-G,
and high with HDP-PPen-T, the most active compound.
diphosphate to the target enzyme that is similar to that
of acyclovir. In addition, a possible hydrophobic p–p
interaction between the cis-double bond of PPen-T
diphosphate and the aromatic side chain of the steric
gate (Tyr722), which lies beneath the incoming nucleo-
tide binding site, might increase the binding interaction
of PPen-T diphosphate with the target enzyme.15
3. Conclusion
Based on the observed HSV-1 antiviral activity, it is rea-
sonable to suggest that the active HDP-PPen nucleo-
sides enter cells and are metabolized to the
corresponding PPen nucleoside diphosphates and bind
tightly to the HSV-1 DNA polymerase to inhibit HSV-
1 replication. The compounds may also be incorporated
resulting in termination of the growing DNA chain. Fig-
ure 2 shows a model of the replication complex of HSV-
1 DNA polymerase complexed with PPen-T diphos-
phate constructed by following the protocol of Liu
et al.14 The model suggests a binding mode of PPen-T
In summary, hexadecyloxypropyl 5-phosphono-pent-2-
en-1-yl nucleosides (HDP-PPen-Ns) were synthesized
and identified as novel antivirals with activity against
HSV-1, in vitro. HDP-PPen-T (27) was the most active
compound among the synthesized nucleotides with an
EC50 value of 0.90 lM. A model of PPen-T bound at
the active site of HSV-1 polymerase suggests that its
binding mode is similar to that of acyclovir and the
cis-double bond of HDP-PPen-Ns might serve to in-
crease the binding interaction to the target enzyme. Fur-
ther modification of the synthesized nucleotides is in
progress and their antiviral activity against other viruses
is being explored.
4. Experimental
4.1. General chemistry methods
1H and 31P nuclear magnetic resonance (NMR) spectra
were recorded on a Varian HG-300 spectrometer at
300 MHz for 1H NMR. Chemical shifts (d) are reported
as s (singlet), d (doublet), t (triplet), q (quartet), m (mul-
tiplet), or br s (broad singlet). Mass spectra were record-
ed on a Finnigan LCQDECA mass spectrometer and a
ThermoFinnigan MAT900XL high-resolution mass
spectrometer at the small molecule facility in Depart-
ment of Chemistry at University of California, San Die-
go. Thin-layer chromatography (TLC) was performed
on silica gel-GF Uniplates (250 lm) purchased from
Analtech Inc. (Newark, DE) and visualized by UV light,
phospray (Supelco, Bellefonte, PA), and charring at
400 ꢁC. Flash column chromatography was performed
using silica gel-60 (240–400 mesh).
Figure 2. A model of PPen-T diphosphate Æ HSV-1 polymerase com-
plex. HSV-1 polymerase is shown in ribbons and the key enzyme
residues are represented as sticks. Hydrogen bonds involving PPen-T
diphosphate are shown in black dashes.

H. Choo et al. / Bioorg. Med. Chem. 15 (2007) 1771–1779
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4.2. Synthesis of 2-but-3-ynyloxy-tetrahydro-pyran (2)
4.6. Synthesis of (5-bromo-pent-2-ynyloxy)-tert-butyl-di-
phenyl-silane (6)
A solution of 3-butyn-1-ol (1, 1.00 g, 14.3 mmol) and
PPTS (0.72 g, 2.9 mmol) in CH2Cl2 (20 ml) was treated
dropwise with 3,4-dihydropyran (1.7 ml, 19 mmol) and
the resulting mixture was stirred overnight. The reaction
mixture was diluted with CH2Cl2 (80 ml) and washed
with 0.02 N NaOH (50 ml) and brine (100 ml). The
organic layer was dried over MgSO4 and concentrated,
and the residue was purified by flash chromatography
(3–5% EtOAc in hexanes) to give 1.90 g of compound
A solution of compound 5 (1.09 g, 3.22 mmol) and CBr4
(1.28 g, 3.86 mmol) in CH2Cl2 (70 ml) was treated with
a solution of Ph3P (1.27 g, 4.84 mmol) in CH2Cl2
(30 ml) dropwise at ꢀ78 ꢁC. After 30 min, the reaction
mixture was slowly warmed up to room temperature for
2 h and then stirred for overnight. The reaction mixture
was filtered through a silica gel pad. The filtrate was con-
centrated to dryness. After concentration, the residue was
purified with 0–2% EtOAc in hexanes by flash chromatog-
raphy to give 1.28 g of product 6 (3.19 mmol, 99% yield);
1H NMR (CDCl3) d 7.80–7.65 (m, 4H), 7.50–7.32 (m,
6H), 4.34 (t, J = 1.9 Hz, 1H), 3.35 (t, J = 5.8 Hz, 2H),
2.73 (tt, J = 2.1, 6.0 Hz, 2H) 1.08 (s, 9H).
1
2 (12.3 mmol, 86% yield); H NMR (CDCl3) d 4.63 (t,
J = 3.4 Hz, 1H), 3.92–3.77 (m, 2H), 3.59–3.45 (m, 2H),
2.47 (td, J = 7.1, 2.8 Hz, 2H), 1.96 (t, J = 2.5 Hz, 1H),
1.90–1.44 (m, 6H).
4.3. Synthesis of 5-(tetrahydro-pyran-2-yloxy)-pent-2-yn-
1-ol (3)
4.7. Synthesis of [5-(tert-butyl-diphenyl-silanyloxy)-pent-
3-ynyl]-phosphonic acid diethyl ester (7)
A solution of compound 2 (11.0 g, 71.3 mmol) in THF
(80 ml) was treated with a 1.6 M solution of n-BuLi in
hexane (58 ml, 92.8 mmol) dropwise at 0 ꢁC for
20 min. After 30 min, paraformaldehyde (6.4 g) was
added to the reaction mixture at 0 ꢁC. After 5 h, the
reaction mixture was quenched with aqueous NH4Cl
at 0 ꢁC, diluted with EtOAc (200 ml), and washed
with aq NH4Cl and brine. The organic layer was dried
over MgSO4 and concentrated. The residue was puri-
fied with 20% EtOAc in hexanes by flash chromatog-
raphy to give 8.15 g of compound 3 (44.2 mmol, 62%
yield); 1H NMR (CDCl3) d 4.63 (t, J = 3.4 Hz, 1H),
4.22 (t, J = 2.1 Hz, 2H), 3.91–3.76 (m, 2H), 3.59–3.46
(m, 2H), 2.51 (tt, J = 7.1, 2.2 Hz, 2H), 1.86–1.46 (m,
6H).
A mixture of compound 6 (3.24 g, 8.07 mmol) and triethyl
phosphite (40 ml) was refluxed under nitrogen atmo-
sphere overnight. After evaporation, the residue was puri-
fied with 50% EtOAc in hexanes by flash chromatography
1
to give 3.26 g of product 7 (7.90 mmol, 98% yield); H
NMR (CDCl3) d 7.77–7.64 (m, 4H), 7.46–7.32 (m, 6H),
4.29 (t, J = 1.9 Hz, 2H), 4.16–4.02 (m, 4H), 2.50–2.34
(m, 2H), 1.96–1.82 (m, 2H), 1.31 (t, J = 7.1 Hz, 6H),
1.04 (s, 9H); 31P NMR (CDCl3) d 30.43.
4.8. Synthesis of [5-(tert-butyl-diphenyl-silanyloxy)-pent-
3-enyl]-phosphonic acid diethyl ester (8)
A mixture of compound 7 (5.00 g, 10.9 mmol) and Lind-
lar’s catalyst (5% palladium on calcium carbonate poi-
soned with lead) in MeOH was treated with H2 using a
balloon. After overnight, the reaction mixture was filtered
and concentrated to dryness to give 2.50 g of product 8
4.4. Synthesis of tert-butyl-diphenyl-[5-(tetrahydro-pyran-
2-yloxy)-pent-2-ynyloxy]-silane (4)
1
A solution of compound 3 and imidazole in CH2Cl2 was
treated with TBDPSCl dropwise at 0 ꢁC for 2 h. The
reaction mixture was diluted with CH2Cl2 (300 ml) and
washed with water (200 ml). The organic layer was dried
over MgSO4 and concentrated. The residue was purified
with 10% EtOAc in hexanes by flash chromatography to
(5.5 mmol, 50% yield); H NMR (MeOH-d4) 7.73–7.56
(m, 4H), 7.44–7.32 (m, 6H), 5.68–5.57 (m, 1H), 5.45–5.34
(m, 1H), 4.25 (d, J = 6.0 Hz, 2H), 4.08–3.97 (m, 4H),
2.24–2.10 (m, 2H), 1.74–1.61 (m, 2H), 1.26 (t, J = 7.1 Hz,
6H), 1.03 (s, 9H); 31P NMR (MeOH-d4) d 32.17.
1
give 18.0 g of compound 4 (42.6 mmol, 96% yield); H
4.9. Synthesis of (5-hydroxy-pent-3-enyl)-phosphonic acid
diethyl ester (9)
NMR (CDCl3) d 7.80–7.64 (m, 4H), 7.49–7.34 (m,
6H), 4.63 (t, J = 3.6 Hz, 1H), 4.31 (t, J = 1.9 Hz, 2H),
3.93–3.83 (m, 1H), 3.77 (dt, J = 9.7, 7.1 Hz, 1H), 3.55–
3.44 (m, 2H), 2.48 (tt, J = 7.1, 2.1 Hz, 2H), 1.89–1.46
(m, 6H), 1.06 (s, 9H).
A solution of compound 8 (0.67 g, 1.46 mmol) in acetoni-
trile (20 ml) was treated with a 1.0 M solution of TBAF
in THF (1.7 ml) at 0 ꢁC. After 1 h, the reaction mixture
was concentrated and purified with 5% MeOH in CH2Cl2
by flash chromatography to give 0.32 g of product 9
(1.44 mmol, 99% yield); 1H NMR (CDCl3) 5.66–5.47 (m,
2H), 4.16–4.02 (m, 6H), 2.43–2.28 (m, 2H), 1.94–1.79 (m,
2H), 1.32 (t, J = 7.1 Hz, 6H); 31P NMR (CDCl3) d 33.47.
4.5. Synthesis of 5-(tert-butyl-diphenyl-silanyloxy)-pent-
3-yn-1-ol (5)
A solution of compound 4 (6.12 g, 14.5 mmol) in MeOH
(100 ml) was treated with PPTS (0.36 g, 1.43 mmol) at
room temperature overnight. After concentration, the
residue was purified by flash chromatography with
20% EtOAc in hexanes to give 3.84 g of product 5
4.10. Synthesis of 9-(5-phosphono-pent-2-en-1-yl)-adenine
diethyl phosphonoester (10)
1
(11.3 mmol, 78%); H NMR (CDCl3) d 7.80–7.66 (m,
A solution of adenine (0.49 g, 3.6 mmol) in DMF was
added to a flask containing compound 9 (0.32 g,
1.4 mmol). The resulting mixture was treated with
Ph3P (0.94 g, 3.6 mmol) and DIAD (0.70 ml, 3.6 mmol)
4H), 7.49–7.35 (m, 6H), 4.33 (t, J = 1.9 Hz, 1H), 3.60
(t, J = 6.0 Hz, 2H), 2.40 (tt, J = 2.2, 6.1 Hz, 2H) 1.05
(s, 9H).

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H. Choo et al. / Bioorg. Med. Chem. 15 (2007) 1771–1779
successively at 0 ꢁC. After overnight, the mixture was
concentrated and the residue was purified with 5–10%
MeOH in CH2Cl2 by flash chromatography to give
0.20 g of product 10 (0.589 mmol, 42% yield); 1H
NMR (CDCl3) d 8.36 (s, 1H), 7.86 (s, 1H), 5.83 (br s,
2H), 5.82–5.61 (m, 2H), 4.86 (d, J = 6.6 Hz, 2H), 4.18–
4.02 (m, 4H), 2.63–2.49 (m, 2H), 1.88 (dt, J = 17.9,
7.4 Hz, 2H), 1.32 (t, J = 6.9 Hz, 6H); 31P NMR (CDCl3)
d 31.71; MS (ESI) m/z 340 (M+H)+.
out further purification, compound 14 was used for the
next reaction; 1H NMR (MeOH-d4) d 9.09 (s, 1H), 5.93–
5.81 (m, 1H), 5.78–5.63 (m, 1H), 4.91 (d, J = 7.4 Hz,
1H), 2.72–2.54 (m, 2H), 1.96–1.81 (m, 2H); 31P NMR
(MeOH-d4) d 30.23.
4.15. Synthesis of 9-(5-phosphono-pent-2-en-1-yl)-guanine
(15, PPen-G)
See the procedure for the preparation of compound 13.
Compound 15 was obtained on 0.563 mmol-scale in 95%
yield; 1H NMR (MeOH-d4) d 8.71 (s, 1H), 5.84–5.73 (m,
1H), 5.54–5.48 (m, 1H), 4.74 (d, J = 6.9 Hz, 1H), 2.44–
2.29 (m, 2H), 1.79–1.65 (m, 2H); 31P NMR (MeOH-
d4) d 29.97; MS (ESI) m/z 300 (M+H)+, 298 (MꢀH)ꢀ.
4.11. Synthesis of 2-amino-6-chloro-9-(5-phosphono-pent-
2-en-1-yl)-purine diethyl phosphonoester (11)
A solution of 2-amino-6-chloropurine (0.61 g, 3.6 mmol)
in DMF was added to a flask containing compound 9
(0.32 g, 1.4 mmol). The resulting mixture was treated with
Ph3P (0.94 g, 3.6 mmol) and DIAD (0.70 ml, 3.6 mmol)
successively at 0 ꢁC. After overnight, the mixture was con-
centrated and the residue was purified with 5–10% MeOH
in CH2Cl2 by flash chromatography to give 0.33 g of
product 11 (0.92 mmol, 66 % yield) 1H NMR (CDCl3) d
7.76 (s, 1H), 5.75 (br s, 2H), 5.72–5.48 (m, 2H), 4.67 (d,
J = 6.1 Hz, 2H), 4.17–4.01 (m, 4H), 2.74–2.56 (m, 2H),
1.94–1.78 (m, 2H), 1.30 (t, J = 7.1 Hz, 6H); 31P NMR
(CDCl3) d 32.37; MS (ESI) m/z 374 (M+H)+, 372
(MꢀH)ꢀ.
4.16. Synthesis of 9-(5-phosphono-pent-2-en-1-yl)-adenine
mono-(3-hexadecyloxy-1-propyl) phosphonoester (16)
A solution of compound 13 (0.120 g, 0.424 mmol), 3-
hexadecyloxy-propan-1-ol
(HDP-OH)
(0.191 g,
0.64 mmol), and DMAP (0.078 g, 0.64 mmol) in DMF
(10 ml) was treated with DCC (0.262 g, 1.26 mmol) at
room temperature. The reaction mixture was warmed
up to 80 ꢁC and stirred overnight. After concentration,
the residue was purified with a gradient mixture of chlo-
roform, methanol, con NH4OH, and water (100:40:3:3
to 80:20:1:1) by flash chromatography to give 0.065 g
of product 16 (0.115 mmol, 27% yield); 1H NMR
(MeOH-d4) d 8.21 (s, 1H), 8.19 (s, 1H) 5.84–5.74 (m,
1H), 5.65–5.57 (m, 1H), 4.92 (d, J = 7.0 Hz, 2H), 3.94
(q, J = 6.2 Hz, 2H), 3.52 (t, J = 6.2 Hz, 2H), 3.37 (t,
J = 6.6 Hz, 2H), 2.58–2.44 (m, 2H), 1.90–1.78 (m, 2H),
1.74–1.62 (m, 2H), 1.54–1.43 (m, 2H), 1.36–1.14 (m,
12H), 0.89 (t, J = 7.0 Hz, 3H); 31P NMR (MeOH-d4) d
25.89; MS (ESI) m/z 566 (M+H)+, 564 (MꢀH)ꢀ.
4.12. Synthesis of 9-(5-phosphono-pent-2-en-1-yl)-guanine
diethyl phosphonoester (12)
A solution of compound 11 (0.200 g, 0.53 mmol) in 30 ml
of 88% HCO2H was stirred at 100 ꢁC for 8 h. After concen-
tration, the residue was purified with 10% MeOH in
CH2Cl2 byflashchromatographytogive0.170 gofproduct
12(0.478 mmol, 89% yield);1H NMR (MeOH-d4) d 8.97(s,
1H), 5.90–5.77 (m, 1H), 5.75–5.63 (m, 1H), 4.89 (d,
J = 7.1 Hz, 1H), 4.17–4.04 (m, 4H), 2.70–2.50 (m, 2H),
2.06–1.87 (m, 2H), 1.33 (t, J = 6.9 Hz, 6H); 31P NMR
(CDCl3) d 33.48;MS(ESI)m/z 356 (M+H)+, 354 (MꢀH)ꢀ.
4.17. Synthesis of 2-amino-6-chloro-9-(5-phosphono-pent-
2-en-1-yl)-purine mono-(3-hexadecyloxy-1-propyl) phos-
phonoester (17)
4.13. Synthesis of 9-(5-phosphono-pent-2-en-1-yl)-adenine
(13, PPen-A)
See the procedure for the preparation of compound 16.
Compound 17 was obtained on 0.598 mmol-scale in
56% yield; H NMR (MeOH-d4) d 7.79 (s, 1H) 5.76–
5.68 (m, 1H), 5.57–5.49 (m, 1H), 4.72 (d, J = 7.3 Hz,
2H), 3.95 (q, J = 6.6 Hz, 2H), 3.53 (t, J = 6.2 Hz, 2H),
3.39 (t, J = 6.6 Hz, 2H), 2.62–2.52 (m, 2H), 1.90–1.82
(m, 2H), 1.74–1.64 (m, 2H), 1.56–1.46 (m, 2H), 1.36–
1.12 (m, 12H), 0.89 (t, J = 6.6 Hz, 3H); 31P NMR
(MeOH-d4) d 26.46.
1
A solution of compound 10 (0.300 g, 0.884 mmol) in ace-
tonitrile (10 ml) was treated with TMSBr (5 ml) at room
temperature overnight. After concentration, the residue
was dissolved in water (20 ml) and the resulting mixture
was stirred for 1 h. The reaction mixture was concentrated
to dryness. The residue was dissolved in water (4 ml) and
adjusted to ca. pH 8. The resulting mixture was loaded to
a column containing DOWEX-1X2 resin and purified
with gradient eluent (0–0.25 M HCO2H) to give 0.180 g
of product 13 (0.636 mmol, 72% yield); 1H NMR
(MeOH-d4) d 8.20 (s, 1H), 8.13 (s, 1H), 5.74–5.62 (m,
1H), 5.53–5.41 (m, 1H), 4.78 (d, J = 6.9 Hz, 1H), 2.33–
2.19 (m, 2H), 1.57–1.43 (m, 2H); 31P NMR (DMSO-d6)
d 26.31; MS (ESI) m/z 284 (M+H)+, 282 (MꢀH)ꢀ.
4.18. Synthesis of 9-(5-phosphono-pent-2-en-1-yl)-guanine
mono-(3-hexadecyloxy-1-propyl) phosphonoester (18,
HDP-PPen-G)
A solution of compound 17 (0.200 g, 0.333 mmol) in 88%
HCO2H (40 ml) was stirred at 100 ꢁC overnight. After con-
centration, the residue was purified with a mixture of chlo-
roform, methanol, con NH4OH, and water (80:20:1:1 to
70:58:8:8) to give 0.120 g of product 18 (0.206 mmol,
62% yield); 1H NMR (MeOH-d4) d 7.75 (s, 1H) 5.76–5.66
(m, 1H), 5.56–5.48 (m, 1H), 4.71 (d, J = 7.3 Hz, 2H), 3.94
(q, J = 5.9 Hz, 2H), 3.53 (t, J = 6.6 Hz, 2H), 3.39 (t,
4.14. Synthesis of 2-amino-6-chloro-9-(5-phosphono-pent-
2-en-1-yl)-purine (14)
See the procedure for the preparation of compound 13.
Compound 14 was obtained on 0.576 mmol-scale. With-

H. Choo et al. / Bioorg. Med. Chem. 15 (2007) 1771–1779
1777
J = 6.6 Hz, 2H), 2.62–2.52 (m, 2H), 1.90–1.82 (m, 2H),
1.72–1.62 (m, 2H), 1.54–1.47 (m, 2H), 1.34–1.22 (m,
12H), 0.89 (t, J = 7.0 Hz, 3H); 31P NMR (MeOH-d4) d
26.14; MS (ESI) m/z 582 (M+H)+, 580 (MꢀH)ꢀ.
J = 7.6 Hz, 1H), 5.68–5.57 (m, 1H), 5.52 (d, J = 6.7 Hz,
1H), 5.39–5.18 (m, 1H), 4.28 (d, J = 6.6 Hz, 2H), 2.36–
2.22 (m, 2H), 1.64–1.51 (m, 2H); 31P NMR (DMSO-d6)
d 26.38; MS (ESI) m/z 261 (M+H)+, 259 (MꢀH)ꢀ.
4.19. Synthesis of 1-(5-phosphono-pent-2-en-1-yl)-uracil
diethyl phosphonoester (19)
4.23. Synthesis of 1-(5-phosphono-pent-2-en-1-yl)-cyto-
sine (23, PPen-C)
A solution of compound 9 (0.20 g, 0.90 mmol), 3-benzo-
yl-uracil (0.24 g, 1.1 mmol), and Ph3P (0.29 g, 1.1 mmol)
in DMF was treated with DIAD (0.21 ml, 1.1 mmol)
dropwise at 0 ꢁC. After 2 h, the reaction mixture was
concentrated and purified with 2% MeOH in CH2Cl2
by flash chromatography. The benzoyl-protected inter-
mediate was dissolved in methanolic ammonia (50 ml)
and stirred overnight. The resulting mixture was concen-
trated and purified with 5% MeOH in CH2Cl2 by silica
gel column chromatography to give 0.23 g of product
See the procedure for the preparation of compound 13.
Compound 23 was obtained on 1.30 mmol-scale in 71%
yield; H NMR (DMSO-d6) d 7.52 (d, J = 7.1 Hz, 1H),
7.04 (br s, 1H), 6.98 (br s, 1H), 5.62 (d, J = 7.7 Hz,
1H), 5.66–5.53 (m, 1H), 5.37–5.26 (m, 1H), 4.26 (d,
J = 6.9 Hz, 2H), 2.37–2.23 (m, 2H), 1.63–1.50 (m, 2H);
31P NMR (DMSO-d6) d 26.14; MS (ESI) m/z 260
(M+H)+, 258 (MꢀH)ꢀ.
1
4.24. Synthesis of 1-(5-phosphono-pent-2-en-1-yl)-thy-
mine (24, PPen-T)
1
19 (0.73 mmol, 81% yield); H NMR (CDCl3) d 8.66
(br s, 1H), 7.31 (d, J = 8.1 Hz, 1H), 5.76–5.66 (m, 1H),
5.69 (dd, J = 7.7, 2.0 Hz, 1H), 4.41 (d, J = 7.0 Hz, 2H),
4.16–4.02 (m, 4H), 2.53–2.42 (m, 2H), 1.89–1.80 (m,
2H), 1.32 (t, J = 7.0 Hz, 3H); 31P NMR (CDCl3) d
31.80; MS (ESI) m/z 317 (M+H)+, 315 (MꢀH)ꢀ.
See the procedure for the preparation of compound 13.
Compound 24 was obtained on 1.12 mmol-scale in 85%
yield; 1H NMR (MeOH-d4) d 7.41 (s 1H), 5.80–5.65 (m,
1H), 5.53–5.41 (m, 1H), 4.39 (d, J = 5.5 Hz, 2H), 2.56–
2.40 (m, 2H), 1.85 (s, 3H), 1.85–1.74 (m, 2H); 31P
NMR (MeOH-d4) d 30.40; HRMS (ESI) obsd, m/z
274.0727, calcd for C10H15N2O5P, m/z 274.0713 M+.
4.20. Synthesis of 1-(5-phosphono-pent-2-en-1-yl)-cyto-
sine diethyl phosphonoester (20)
A solution of compound 19 (0.73 g, 2.3 mmol), TEA
(0.97 ml, 7.0 mmol), and DMAP (0.28 g, 2.3 mmol) in
acetonitrile was treated with 2,4,6-triisopropylbenzene-
sulfonyl chloride (2.1 g, 6.9 mmol) at room temperature
for 3 h. Concentrated NH4OH (5 ml) was added to the
reaction mixture. The resulting mixture was stirred for
1 h. After concentration, the residue was purified with
a mixture of chloroform, methanol, con NH4OH, and
water (240:20:1:1) by flash chromatography to give
4.25. Synthesis of 1-(5-phosphono-pent-2-en-1-yl)-uracil
mono-(3-hexadecyloxy-1-propyl) phosphonoester (25,
HDP-PPen-U)
See the procedure for the preparation of compound 16.
Compound 25 was obtained on 0.50 mmol-scale in 44%
1
yield; H NMR (MeOH-d4) d 7.62 (d, J = 8.0 Hz, 1H),
5.82–5.71 (m, 1H), 5.65 (d, J = 8.0 Hz, 1H), 5.50–5.37 (m,
1H), 4.44 (d, J = 5.8 Hz, 2H), 3.93 (q, J = 6.3 Hz, 2H),
3.53 (t, J = 6.3 Hz, 2H), 3.41 (t, J = 6.6 Hz, 2H), 2.50–2.36
(m, 2H), 1.90–1.81 (m, 2H), 1.70–1.48 (m, 4H), 1.40–1.20
(m, 12H), 0.89 (t, J = 6.2 Hz, 3H); 31P NMR (MeOH-d4)
d 25.90; MS (ESI) m/z 543 (M+H)+, 541 (MꢀH)ꢀ.
1
0.55 g of product 20 (1.74 mmol, 75% yield); H NMR
(CDCl3) d 7.34 (d, J = 7.1 Hz, 1H), 5.80 (d, J = 7.4 Hz,
1H), 5.72–5.61 (m, 1H), 5.56–5.46 (m, 1H), 4.44 (d,
J = 6.9 Hz, 2H), 4.16–4.02 (m, 4H), 2.54–2.39 (m, 2H),
2.17 (br s, 2H), 1.91–1.78 (m, 2H), 1.32 (t, J = 7.1 Hz,
6H); 31P NMR (CDCl3) d 32.06; MS (ESI) m/z 316
(M+H)+, 314 (MꢀH)ꢀ.
4.26. Synthesis of 1-(5-phosphono-pent-2-en-1-yl)-cyto-
sine mono-(3-hexadecyloxy-1-propyl) phosphonoester (26,
HDP-PPen-C)
4.21. Synthesis of 1-(5-phosphono-pent-2-en-1-yl)-thy-
mine diethyl phosphonoester (21)
See the procedure for the preparation of compound 16.
Compound 17 was obtained on 0.58 mmol-scale in 38%
yield; H NMR (MeOH-d4) d 7.86 (d, J = 7.4 Hz, 1H),
5.96 (d, J = 7.6 Hz, 1H), 5.84–5.72 (m, 1H), 5.52–5.51
(m, 1H), 4.50 (d, J = 6.6 Hz, 2H), 3.92 (q, J = 6.3 Hz,
2H), 3.53 (t, J = 6.0 Hz, 2H), 3.41 (t, J = 6.6 Hz, 2H),
2.49–2.35 (m, 2H), 1.91–1.80 (m, 2H), 1.70–1.45 (m,
4H), 1.36–1.22 (m, 12H), 0.89 (t, J = 6.6 Hz, 3H);
31P NMR (MeOH-d4) d 25.93; HRMS (ESI) obsd, m/z
541.3645, calcd for C28H52N3O5P, m/z 541.3639 M+.
1
See the procedure for the preparation of compound 19.
Compound 21 was obtained on 1.44 mmol-scale in 78%
1
yield; H NMR (CDCl3) d 8.46 (br s, 1H), 7.11 (s, 1H),
5.78–5.66 (m, 1H), 5.52–5.41 (m, 1H), 4.38 (d,
J = 6.6 Hz, 2H), 4.20–4.02 (m, 4H), 2.55–2.41 (m, 2H),
1.91 (s, 3H), 1.91–1.78 (m, 2H), 1.33 (t, J = 7.1 Hz,
6H); 31P NMR (CDCl3) d 31.84; HRMS (ESI) obsd,
m/z 330.1352, calcd for C14H23N2O5P, m/z 330.1339 M+.
4.22. Synthesis of 1-(5-phosphono-pent-2-en-1-yl)-uracil
(22, PPen-U)
4.27. Synthesis of 1-(5-phosphono-pent-2-en-1-yl)-thy-
mine mono-(3-hexadecyloxy-1-propyl) phosphonoester
(27, HDP-PPen-T)
See the procedure for the preparation of compound 13.
Compound 22 was obtained on 1.26 mmol-scale in 98%
yield; H NMR (DMSO-d6) d 11.23 (s, 1H), 7.69 (d,
See the procedure for the preparation of compound 16.
Compound 17 was obtained on 0.58 mmol-scale in 15%
1

1778
H. Choo et al. / Bioorg. Med. Chem. 15 (2007) 1771–1779
1
yield; H NMR (MeOH-d4) d 7.44 (d, J = 1.1 Hz, 1H),
5.80–5.69 (m, 1H), 5.47–5.36 (m, 1H), 4.42 (d,
J = 6.9 Hz, 2H), 3.92 (q, J = 6.3 Hz, 2H), 3.53 (t,
J = 6.3 Hz, 2H), 3.41 (t, J = 6.6 Hz, 2H), 2.50–2.36 (m,
2H), 1.91–1.81 (m, 2H), 1.87 (d, J = 1.1 Hz, 3H), 1.70–
1.48 (m, 4H), 1.35–1.24 (m, 12H), 0.89 (t, J = 6.6 Hz,
3H); 31P NMR (MeOH-d4) d 25.62; HRMS (ESI) obsd,
m/z 556.3643, calcd for C29H53N2O6P, m/z 556.3636 M+.
4.30. Neutral red uptake assay for cytotoxicity
MRC-5 cells were seeded into 96-well tissue culture
plates at 2.5 · 104 cells/well. After 24-h incubation, med-
ia were replaced with MEM containing 2% FBS, drug
was added to the first row, and then diluted serially five-
fold from 100 lM to 0.03 lM. The plates were incubat-
ed for 7 days and cells stained with neutral red and
incubated for 1 h. Plates were shaken on a plate shaker
for 15 min. and neutral red was solubilized with 1% gla-
cial acetic acid/50% ethanol. The optical density was
read at 540 nm. The concentration of drug that reduced
cell viability by 50% (CC50) was calculated using com-
puter software. Since the cells were plated sparse and
are dividing during the 7 day period, the results reflect
both the antiproliferative and cytotoxicity of the
compounds.
4.28. Molecular modeling
All molecular modeling of the enzyme-ligand com-
plexes was carried out on a Linux enterprise opera-
tion system using SYBYL 7.2 software packages
(Tripos Inc. St Louis, Mo.). The crystal structure of
herpes polymerase was downloaded from the protein
data bank (PDB ID: 2GV9), and a model of the rep-
lication complex of HSV POL which includes tem-
plate, primer, and inhibitor was constructed by
following the protocol reported by Liu et al.14 Thus,
individual domains of HSV POL were superimposed
onto those of the replication complex of RB69 POL
(PDB ID: 1IG9).16 The palm domain of the replicat-
ing RB69 POL complex was chosen as the reference,
and the HSV POL domains were individually super-
imposed onto the RB69 POL structure. The key ac-
tive site residues of RB69 POL (Asp411, Leu412,
Leu415, Tyr416, Arg482, Lys486, Lys560, Asn564,
Tyr567, Asp623, and Ser624) were in good match
with the corresponding residues in HSV POL
(Asp717, Phe718, Leu721, Tyr722, Arg785, Arg789,
Asn815, Tyr818, Asp888, and Ser889, respectively).
Template, primer, active site calcium ions, and thymi-
dine-50-triphosphate (TTP) of the RB69 POL were
merged into the HSV POL to provide a model of
catalytically active HSV POL structure. Crystallo-
graphic structure of TTP bound at the active site
was modified to construct PPen-T, which was ener-
gy-minimized to give the stable conformation inside
the active site of HSV POL. The resulting HSV
POL:PPen-T complex was used for investigation of
the possible binding mode of PPen-T to the active
site of HSV POL.
Acknowledgments
The studies were supported in part by NIH Grants
AI-66499, and AI-64615 from National Institute of
Allergy and Infectious Disease and EY-11832 and
EY-07366 from the National Eye Institute.
References and notes
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HSV-1 in vitro
Antiviral activities of the compounds were determined
against HSV-1 by DNA reduction with MRC-5 human
lung fibroblast cells using HSV-1 DNA probes as de-
scribed previously.17 Briefly, subconfluent MRC-5 cells
in 24-well culture plates were inoculated by removing
the medium and adding HSV-1 at a dilution that caus-
es 3–4+ cytopathic effect in a nondrug well in 24 h. The
virus was absorbed for 1 h at 37 ꢁC, aspirated, and re-
placed with various concentrations of drugs as indicat-
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hybridization using an HSV antiviral susceptibility kit
from Diagnostic Hybrids (Athens, Ohio) according to
the manufacturer’s instructions. Results are expressed
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