Phosphoramidate and phosphate prodrugs of (-)-β-D-(2R,4R)-dioxolane- thymine: Synthesis, anti-HIV activity and stability studies

Article abstract of DOI:10.1016/j.bmc.2005.11.008

A series of phosphoramidate and phosphate prodrugs of DOT were synthesized via dichlorophosphate or H-phosphonate chemistry and evaluated for their anti-HIV activity against LAI M184V mutants in PBM cells as well as for their cytotoxicity. The antiviral and cytotoxic profiles of the prodrugs were compared with that of the parent compound (DOT), and it was found that four aryl phosphoramidates 5, 18, 20, and 26 showed a significant enhancement (8- to 12-fold) in anti-HIV activity without cytotoxicity. Chemical stability of these prodrugs was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 2.0 and 7.4). Enzymatic hydrolysis was also studied in esterase or lipase in buffer solution. Chemical stability studies indicate that the phosphoramidates have good chemical stability at pH 2.0 and at pH 7.4 phosphate buffer. Phosphoramidate prodrugs were hydrolyzed in vitro by esterase or lipase and found to be better substrates for lipases than for esterases. 1,3-Diol cyclic phosphates showed potent anti-HIV activity without increasing the cytotoxicity compared with that of DOT and have good chemical and enzymatic stability. Long-chain lipid phosphates, although showed potent anti-HIV activity, exhibited increased cytotoxicity.

Full text of DOI:10.1016/j.bmc.2005.11.008

Bioorganic & Medicinal Chemistry 14 (2006) 2178–2189
Phosphoramidate and phosphate prodrugs
of (ꢀ)-b-D-(2R,4R)-dioxolane-thymine: Synthesis,
anti-HIV activity and stability studies
Yuzeng Liang,a Janarthanan Narayanasamy,a Raymond F. Schinazib and Chung K. Chua,*
aThe University of Georgia, College of Pharmacy, Athens, GA 30602, USA
bEmory University School of Medicine, Veterans Affairs Medical Center, Atlanta, GA 30033, USA
Received 29 September 2005; revised 31 October 2005; accepted 1 November 2005
Available online 28 November 2005
Abstract—A series of phosphoramidate and phosphate prodrugs of DOT were synthesized via dichlorophosphate or H-phospho-
nate chemistry and evaluated for their anti-HIV activity against LAI M184V mutants in PBM cells as well as for their cytotoxicity.
The antiviral and cytotoxic profiles of the prodrugs were compared with that of the parent compound (DOT), and it was found that
four aryl phosphoramidates 5, 18, 20, and 26 showed a significant enhancement (8- to 12-fold) in anti-HIV activity without cyto-
toxicity. Chemical stability of these prodrugs was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 2.0 and
7.4). Enzymatic hydrolysis was also studied in esterase or lipase in buffer solution. Chemical stability studies indicate that the phos-
phoramidates have good chemical stability at pH 2.0 and at pH 7.4 phosphate buffer. Phosphoramidate prodrugs were hydrolyzed
in vitro by esterase or lipase and found to be better substrates for lipases than for esterases. 1,3-Diol cyclic phosphates showed
potent anti-HIV activity without increasing the cytotoxicity compared with that of DOT and have good chemical and enzymatic
stability. Long-chain lipid phosphates, although showed potent anti-HIV activity, exhibited increased cytotoxicity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
strategy, the phosphate groups are masked with
lipophilic groups in order to obtain lipophilic mem-
brane-permeable derivatives and to be able to access
intracellular target sites, wherein enzymatic or chemical
hydrolysis yields the nucleoside monophosphates.4b 1,3-
Diol cyclic phosphate prodrugs, known as HepDirect
prodrugs, represent a new class of prodrugs for improv-
ing nucleoside-based drug therapies.5
Nucleoside reverse transcriptase inhibitors (NRTIs)
have been widely used in the treatment of HIV-1 infec-
tion. Their antiviral activity depends on the cellular up-
take and conversion to the corresponding nucleoside
triphosphates (NTPs) by cellular kinases.1,2 NTPs act
either as inhibitors of viral DNA polymerase or as chain
terminators following incorporation into a growing
DNA of HIV. Unfortunately, nucleoside kinases often
have narrow substrate specificity and are unable to cat-
alyze the initial phosphorylation, that is, conversion of
the nucleoside to the nucleoside 50-monophosphates
(NMPs), while the subsequent phosphorylations are
not usually rate-limiting. Phosphoramidate triester of
nucleoside is a prodrug methodology increasingly being
used to bypass the required initial phosphorylation and
to improve the pharmacological activity of existing
nucleosides.3,4a In another potential phosphate prodrug
(ꢀ)-b-D-(2R,4R)-Dioxolane-thymine (DOT) was report-
ed from our laboratory and shows anti-HIV activity
against most of the nucleoside resistant HIV-1
mutants.6a We carried out molecular modeling studies
for DOT and found that dioxolane moiety plays a signif-
icant role in stabilizing the binding between the mutant
HIV-1 RT and nucleoside triphosphate.6b Due to DOTÕs
pronounced activity, we were interested in synthesizing
prodrugs of DOT. Accordingly, herein we report aryl
and long-chain lipid phosphoramidates, 1,3-diol cyclic
phosphates, and long-chain lipid phosphates of DOT
with the objective of further enhancing the anti-HIV
activity of DOT. We also studied chemical and enzymat-
ic hydrolysis of aryl phosphoramidates and 1,3-diol
cyclic phosphates of DOT.
Keywords: Nucleoside; Prodrug; Dioxolane-thymine; Anti-HIV
activity.
*
Corresponding author. Tel.: +1 706 542 5379; fax: +1 706 542 5381;
e-mail: dchu@rx.uga.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.008

Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
2179
2. Results and discussion
(ODE), and oleyl alcohol. In order to obtain the phos-
phoramidates (31–35), one equivalent of DOT was add-
ed to two equivalents of diphenyl phosphite (29) in
pyridine at 0 ꢁC over a period of 1 h (by this method,
the formation of by-product, di-DOT H-phosphonates,
could be greatly reduced). Long-chain lipid alcohol
was then added to yield the corresponding 50-O-phos-
pholipids of DOT (30). The 50-O-phospholipids of
DOT (30) were readily oxidized and produced the corre-
sponding phosphoramidates (31–35) by reacting with
amino acid methyl ester in a mixture of Et3N/CCl4/
H2O/MeCN (1/1/1/10, v/v).11
2.1. Chemistry
Aryl phosphoramidates of DOT (3–28) were synthesized
according to the method described by McGuigan et al.9
as shown in Scheme 1. Thus, phenol or naphthol-deriv-
atives were first reacted with phosphoryl chloride in
diethyl ether to give aryl dichlorophosphates (1), which
were reacted with amino acid alkyl ester hydrochloride
in dichloromethane in the presence of triethylamine at
ꢀ78 ꢁC to yield arylalkoxy-amino acid phosphorochlo-
ridates (2). These arylalkoxy-amino acid phosphorochl-
oridates (2), without further purification, were reacted
with DOT in THF in the presence of N-methylimidazole
to provide the target compounds, aryl phosphorami-
dates of DOT, in various yields. The chirality at the
phosphorus center results in the formation of two dia-
stereomeric phosphoramidates in an approximately 1:1
ratio, which was inseparable by silica gel chromatogra-
phy. They displayed two closely spaced signals in the
31P NMR and two isomers were also apparent from
1H NMR spectroscopy as well as by HPLC. Variation
in the amino acid side chains was studied by the prepa-
ration of the glycine (3), 2-aminoisobutyric acid (4),
L- and D-alanine (5 and 6), L- and D-valine (7 and 8),
and L- and D-phenylalanine (9 and 10). Variation of
the ester group in the amino acids was also studied by
synthesizing L-alanine phosphoramidates of tert-butyl
(11) and benzyl esters (12, 21, 23, and 28). The structural
modification with different substituents of phenyl group
was also conducted (13–25). The long-chain lipid phos-
phoramidates of DOT (31–35) were synthesized using
the H-phosphonate approach as shown in Scheme 2.10
The long-chain lipid alcohols used were 3-hexadecyl-
For the synthesis of aryl-1,3-propanediol-cyclic phos-
phates of DOT (Scheme 3),12 the corresponding diol
enantiomers were first prepared. The benzoyl chloride
(36) was reacted with potassium ethyl malonate using
MgCl2ꢀEt3N base system and then treated with HCl
to give a b-keto ester, 3-phenyl-2-oxo-propionate (37).
The b-keto ester was reduced by NaBH4 to provide race-
mic diols. In order to separate the mixture, the racemic
diols first reacted with (ꢀ)-menthone to form diastereo-
meric (ꢀ)-menthone ketals. The ketals were separated
by column chromatography on silica gel and the remov-
al of (ꢀ)-menthone yielded the enantiomeric 1,3-pro-
panediol with the high optical purity.13 Reaction of
1,3-propanediol with p-nitrophenyl dichlorophosphate
in the presence of 4 equiv of p-nitrophenol and Et3N
gave the cyclic phosphate intermediate (40). The cyclic
phosphate intermediate (40) was then reacted with
DOT in the presence of tert-BuMgCl to give cyclic phos-
phoramidates with high optical purity (41–44). Coupling
of the phosphate intermediate (40) with nucleosides
proved to be the most challenging reaction, resulting
in poor yields in various reaction conditions. The mag-
nesium salt of the 50-hydroxyl DOT reacted with the
oxy-1-propanol
(HDP),
3-octadecyloxy-1-ethanol
Scheme 1. Synthesis of aryl phosphoramidates. Reagents and conditions: (a) amino acid ester hydrochloride, Et3N, ꢀ78 ꢁC; (b) DOT,
N-methylimidazole, THF, rt.
Scheme 2. Synthesis of long-chain lipid phosphoramidates. Reagents and conditions: (a) DOT, Py, 0 ꢁC; (b) R1OH, Py, rt; (c) amino acid methyl
ester, Et3N/CCl4/H2O/MeCN(1/1/1/10, v/v).

2180
Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
Scheme 3. Synthesis of cyclic phosphates. Reagents and conditions: (a) (i) potassium ethyl malonate, MgCl2, Et3N; (ii) HC1; (b) NaBH4, MeOH,
THF, reflux; (c) (i) HMDS, TMSOTf; (ii) (ꢀ)-menthone, TMSOTf; (iii) HC1, MeOH; (d) 4-nitrophenyloxy dichlorophosphate, Et3N, p-nitrophenol;
(e) DOT, tert-buty MgCl.
Scheme 4. Synthesis of long-chain lipid phosphates. Reagents and conditions: (a) DOT, 1,2,4-triazole; Et3N, rt; (b) ROH, N-methylimidazole, THF,
rt; (c) 0.5 N NaOH, THF/H2O.
phosphate intermediate with inversion of configuration
and afforded the prodrugs of DOT in ca. 50% yield.
2.2. Anti-HIV activity (SAR studies)
The anti-HIV activity of all prodrugs and the parent
nucleoside DOT was evaluated against LAI M184V
strain in PBM cells. Cytotoxicity was evaluated in
PBM, CEM, and Vero cells. AZT was used as the posi-
tive control and the results are summarized in Table 1
and Table 2. The results of aryl phosphoramidates of
DOT (3–12) with the different amino acid methyl esters
indicate that changes in the amino acid lead to signifi-
cant changes in activity (Table 1). Among the series, 2-
amino-3-methyl-butyric acid 4 and alanine prodrugs 5
were the most potent, while the other prodrugs being
less potent in comparison to DOT. Prodrug 5 was found
to be approximately 7.5-fold more potent than DOT.
The results are similar to the SAR studies of the phos-
phoramidates of D4T by McGuigan.15 In general, the
phoshoramidates with L-amino acid methyl ester were
more active than those with D-amino acid methyl ester;
the prodrug 5, 7, and 9 were more active than 6, 8, and
10. The D-valine derivative 10 was the least active of the
series, being approximately 12-fold less potent than that
of DOT. There was some variation in activity with the
The long-chain lipid phosphates of DOT (47 and 48)
were synthesized using the phosphotriester approach
as shown in Scheme 4.14 DOT was reacted with
2-chlorophenyl dichlorophosphate (45) in the presence
of 1,2,4-triazole and triethylamine to provide the inter-
mediate 46. Without further purification, the intermedi-
ate 46 was treated with long-chain lipid alcohol
(3-hexadecyloxy-1-propanol or 2-octadecyloxy-1-etha-
nol) to give the fully protected phosphotriester, which
was isolated by flash chromatography in good yield.
The phosphotriester displayed two closely spaced signals
in the 31P NMR corresponding to the two diastereoiso-
mers. The presence of diastereoisomers was also appar-
ent from 1H NMR spectroscopy. For the removal of the
2-chlorophenyl groups, the phosphotriester was dis-
solved in THF and treated with 0.5 N NaOH at 50 ꢁC
for 1.5 h to give the target prodrugs. The identity of
all the prodrugs was confirmed by electron-spray ioniza-
1
tion-mass spectrometry (ESI-MS), H NMR, and ele-
mental analysis.

Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
2181
Table 1. Anti-HIV activity and cytotoxicity of phosphoramidates and long-chain lipid phosphoramidates
Compound
R1
R2
R3
M184V(lM)a
EC50 EC90
14.2
Cytotoxicity (lM)
PBM
CEM
Vero
3
4
Phenyl
Phenyl
H
(CH3)2
CH3
CH3
2.2
0.62
0.2
4.5
1.9
>100
>100
>100
>100
>100
>100
>100
39.2
>100
>100
>100
>100
>100
3.3
1.2
5
Phenyl
Phenyl
(L)CH3
(D)CH3
(L)PhCH2
(D)PhCH2
(L)(CH3)2CH
(D)(CH3)2CH
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(L)CH3
(CH3)2
CH3
CH3
6
38.6
8.4
>100
>100
49.2
7
Phenyl
Phenyl
CH3
CH3
8
13.7
49.9
37.4
82.8
40.1
59.3
9
Phenyl
Phenyl
CH3
CH3
9.6
18.0
24.8
>100
>100
>100
>100
>100
>100
>100
>100
63.8
>100
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
31
32
33
34
35
DOT
Phenyl
Phenyl
C(CH3)3
CH2Ph
CH3
>100
58.2
1.08
4.6
3.4
1.6
4.7
2.6
1.6
0.62
7.3
1.4
8.9
2.6
4.7
>100
>100
4-Methylphenyl
4-Propylphenyl
4-Neopentylphenyl
4-Methoxyphenyl
4-Cyanophenyl
4-Chlorophenyl
4-Fluorophenyl
4-Bromophenyl
4-Bromophenyl
2-Chlorophenyl
2-Chlorophenyl
2-Allylphenyl
2,4-Dichlorophenyl
Naphthyl
0.61
0.2
74.3
CH3
CH3
25.6
15.4
21.1
13.5
43.5
36.6
91.5
>100
0.99
0.56
0.54
0.18
1.7
CH3
CH3
>100
>100
>100
>100
>100
15.7
>100
78.4
>100
32.0
>100
>100
>100
>100
>100
CH3
CH3
CH3
CH2Ph
CH3
0.15
1.9
84.7
0.53
0.57
3.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
77.1
24.3
>100
>100
>100
>100
>100
>100
>100
CH2Ph
CH3
CH3
12.9
2.8
0.85
0.14
0.38
2.2
>100
>100
>100
22.2
CH3
CH3
0.64
2.6
Naphthyl
Naphthyl
C16H33O(CH2)3
(L)CH3
H
CH2Ph
CH3
CH3
11.6
11.9
80.5
15.0
13.6
35.1
23.7
8.8
9.31
4.8
13.71
8.1
4.96
C
16H33O(CH2)3
C16H33O(CH2)3
18H37O(CH2)2
Oleyl
(L)CH3
(L)(CH3)2CH
(L)CH3
(L)CH3
2.7
2.8
4.4
4.9
1.5
19.7
CH3
CH3
10.7
61.4
31.3
2.42
C
15.7
3.7
33.6
11.4
>100
CH3
>100
>100
a LAI M184V mutants in PBM cells unless otherwise indicated.
sized and their antiviral activity and cytotoxicity were
evaluated. Similarly, there were some variations in activ-
ity and toxicity. Except for the 4-fluorophenyl-19 and 2-
allylphenyl-prodrug 24, which were 2-fold less potent
than DOT, all other prodrugs showed increased antivi-
ral activity with an IC50 value ranging from 0.15 to
0.99 lM. 4-Bromophenyl prodrug 20 was found to be
very active, approximately 10-fold more potent than
DOT. 4-Chlorophenyl prodrug 18 was also found to
be highly potent and showed an 8-fold increase in the
EC50 and the 14-fold enhancement in the EC90 value
in comparison to DOT. Introduction of the lipophilic
substituent in the aryloxy moiety (13–15), although led
to increased activity, resulted in enhanced cytotoxicity.
However, introduction of the electron-withdrawing sub-
stituents in the aryloxy moiety (16–25) did not result in
increased cytotoxicity.
Table 2. Anti-HIV activity and cytotoxicity of cyclic phosphates and
long-chain lipid phosphates
Compound
M184V
(lM)a
Cytotoxicity (lM)
EC50
EC90
PBM
CEM
Vero
41
42
1.8
2.0
0.48
3.2
2.0
0.3
1.5
55.9
40
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
43
44
10.1
22.7
15.4
3.6
47
48
66.19
15.04
>100
9.83
3.91
>100
23.63
10.08
>100
DOT
8.8
a LAI M184V mutants in PBM cells unless otherwise indicated.
benzyl or tert-butyl group instead of the methyl group
for alanine prodrugs 11 and 12. The alanyl-benzyl ester
prodrug 12 exhibited increased activity with an IC50 val-
ue of 1.08 lM, while alanyl-tert-butyl ester prodrug 11
showed decreased activity with an IC50 value of
24.8 lM in comparison to DOT. These prodrugs, except
for D-phenylalanine prodrug 8, did not show any
increased cytotoxicity. After having completed the
alanyl-methyl- and alanyl-benzyl- and 2-amino-3-meth-
ylbutryic methyl ester of the phosphoramidates of
DOT, the phosphoramidate series from 13 to 25 with
varying substitutions in the aryloxy moiety were synthe-
It is reported that, naphthyl phosphoramidates of
nucleosides can increase the anticancer activity. 1-Naph-
thyloxy L-alanine benzyl ester phosphoramidate of
BVdU showed a 100-fold increase in activity against
prostate cancer when compared to the parent drug
BvdU.16 Thus, three naphthyl phosphoramidates were
synthesized (26, 27, and 28), and their anti-HIV activity
and cytotoxicity were investigated (Table 1). All three
prodrugs showed increased antiviral activity and did

2182
Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
Table 3. Chemical and enzymatic hydrolysis recovery (%) of phos-
phoramidates and 1,3-diol cyclic phosphates
not show any enhanced cytotoxicity in comparison to
DOT. Prodrugs 26 and 28 showed 11-fold and 27 exhib-
ited 4-fold enhanced activity when compared with DOT.
Long-chain lipid phosphoramidates 31–35 exhibited the
decreased activity and the increased cytotoxicity. Pro-
drug 31 showed the most decreased activity, being 8-fold
less potent than DOT. Thus, the long-chain lipid phos-
phoramidates of DOT should not be considered as the
prodrugs of DOT. Among the aryl phosphoramidates
of DOT, compounds 5, 18, 20, and 26 emerged as the
most potent inhibitors of HIV in PBM cells. The struc-
ture–activity relationship studies suggest that the
decreasing order of activity is phenyl = substituted
phenyl = naphthyl > alkyl. For amino acids, L-alanine
methyl ester seems to be the most active. The results
are in consistent with the results of the phosphorami-
dates of AZT by McGuigan.17
Compound
Chemical
hydrolysisa
Enzymatic
hydrolysisb
pH 2.0
pH 7.4
Esterase
Lipase
5
7
95.1
96.5
96.3
93.9
97.4
98.7
97.3
96.3
95.7
92.1
94.3
96.5
98.6
99.2
98.8
98.3
99.4
98.4
99.7
99.4
99.8
98.8
98.4
97.2
98.1
98.6
98.3
98.5
87.7
75.3
86.2
94.1
94.2
77.5
79.1
86.4
86.3
87.4
88.7
77.3
96.5
97.6
77.8
29.6
84.5
32.4
45.6
37.8
40.5
36.7
36.1
68.4
47.8
66.7
95.4
96.4
11
13
16
18
20
21
22
26
27
28
41
42
Cyclic phosphate prodrugs are known to be highly sta-
ble in plasma and tissue.18a These prodrugs undergo
an oxidative cleavage catalyzed reaction by a cyto-
chrome P450 (CYP) expressed in the liver. They are capa-
ble of bypassing the rate-limiting step of nucleoside
kinase in vivo and generating high levels of liver
NTP.18b All synthesized cyclic phosphates of DOT
exhibited potent anti-HIV activity (Table 2). Prodrug
43 showed the most increased activity, being 3-fold more
potent than DOT, while others showed a slightly de-
creased activity. The anti-HIV activity and cytotoxicity
of long-chain lipid phosphates of DOT (47 and 48) are
shown in Table 2. According to the study by Hostetler
and co-workers,19 the addition of an alkoxyalkyl ester
group to the phosphonate of cidofovir or cyclic cidofo-
vir resulted in a remarkable increase in antiviral activity
against CMV and HSV-1 in vitro. In the case of DOT,
the cytotoxicity of the analogs was also increased. The
long-chain lipid phosphates of DOT, however, were
not so effective. Prodrug 47 showed decreased activity
compared with that of DOT. Although the prodrug 48
showed increased antiviral activity as well as cytotoxic-
ity thus, decreasing the selectivity. It was found that
the introduction of the long-chain lipids led DOT to
be more cytotoxic in vitro in its phosphoramidate or
phosphate form.
a pH 2.0 and pH 7.4 phosphate buffer at 37 ꢁC.
b pH 8.0 buffer solution of esterase or lipase at 37 ꢁC.
pH 7.4 at 37 ꢁC. Enzymatic hydrolysis was studied at
pH 8.0, phosphate buffer at 37 ꢁC (Table 3). The studies
indicated that they are stable both in acidic and neutral
media. The decomposition by hydrolysis was less than
8% over 24 h. These phosphoramidates were also stable
to esterase. McGuigan et al.21 demonstrated that phos-
phoramidates of AZT with electron-withdrawing
substituents on the phenyl ring could increase the anti-
HIV activity, being associated with the possibility that
electron-withdrawing substituents can increase the rate
of hydrolysis. For the phosphoramidates of DOT, how-
ever, no clear correlation was found between electron-
donating (p-Me) or electron-withdrawing (p-Cl, p-Br)
substituents on the phenyl ring. Uckun et al.22 found
that halogen-substituted stavudine phosphoramidates
could be activated by lipase-mediated hydrolysis. In
our studies, we noticed that the phosphoramidates of
DOT were less stable in the presence of lipases than in
esterases, suggesting that lipase promotes the activation
of the parent drug DOT. The phosphoramidates of
DOT were better substrates for lipases than for esteras-
es. The hydrolysis by lipases was 15–70% over 24 h, and
hydrolysis by esterases was 6–25% over 24 h. It was also
observed that the anti-HIV activity of phosphorama-
dates of DOT does not correlate with the hydrolytic rate
of lipase hydrolysis. Two cyclic phosphates (41 and 42)
showed good chemical stability at pH 2.0 and at pH
7.4, and they also exhibited good enzymatic stability
to esterase and lipase (Table 3). The decomposition
was 0.8–4.6% over 24 h in chemical or enzymatic media.
2.3. Hydrolysis
Documented work strongly suggests that the carboxyl
ester cleavage is a necessary prerequisite for the activa-
tion of the phosphoramidate prodrugs to parent nucleo-
sides for antiviral activity.20 The role of esterase in the
activation step of the prodrugs was confirmed in this
study. However, in vivo the prodrugs can reach the tar-
get cells only if they are resistant to hydrolysis by extra-
cellular and intracellular carboxylesterases during the
absorption and distribution process. Partial conversion
of the prodrugs to the intermediates or free nucleosides
would almost certainly result in a lower cell penetration
and reduce antiviral potency. Thus, in vitro chemical
and enzymatic hydrolysis of representative phospho-
ramidates and cyclic phosphates was investigated.
Chemical hydrolysis was studied under experimental
conditions of biological relevance, i.e., at pH 2.0 and
In conclusion, the synthesized aryl phosphoramidate
and cyclic phosphate prodrugs of DOT appear to have
advantages in antiviral therapy over the parent nucleo-
side. In some cases, newly synthesized prodrugs exhibit-
ed a significantly increased anti-HIV activity with
increased selectivity. They also exhibited good chemical
and enzymatic stability. Thus, suggesting that both
phosphoramidates and cyclic phosphates of DOT may
have improved pharmacological activity in vivo.

Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
2183
3. Materials and methods
(m, 2H, H20), 5.18 (s, 1H, H40), 6.41 (m, 1H, H10),
7.14–7.35 (m, 5H, Ph), 7.46 (s, 1H, H6), 9.80 (s, 1H,
H3). MS: m/z 469 (M + 1)+. Anal. Calcd for
C19H24N3O9P: C, 47.48; H, 4.87; N, 9.23. Found: C,
47.44; H, 5.10; N, 9.11.
3.1. Chemistry
DOT was synthesized in our laboratory as described be-
fore.8 Bis-dPEG8 acid was purchased from Quanta Bio-
Design, Ltd (Powell, Ohio). Porcine esterases and
lipases (from Candida rugosa) enzyme and other chem-
icals were obtained from Sigma Chemical Company (St.
Louis, MO) and Advanced Chem. Tech. (Louisville,
KY). Melting points were determined with a Mel-temp
II apparatus and are uncorrected. TLC was performed
on a Uniplate (silica gel) purchased from Analtech Co.
Column chromatography was performed using either
silica gel-60 (220–240 mesh) for flash chromatography
or silica gel G (TLC grade, >440 mesh) for vacuum flash
chromatography. NMR spectra were recorded on a Var-
ian Inova 500 spectrometer at 500 MHz with software
VNMRC6-1 with Me4Si as the internal standard. Chem-
ical shifts (d) are reported as s (single), d (doublet), t
(triplet), q (quartet), m (multiplet) or b (broad). UV
spectra were recorded on a Beckman DU-650 spectro-
photometer. Optical rotations were measured on a Jasco
DIP-370 digital polarimeter. Mass spectra were mea-
sured on a Micromass Autospec mass spectrometer. Ele-
mental analyses were performed by Atlantic Micro lab,
Inc. (Norcross, GA). HPLCs were performed using
Waters 2996 instrument manufactured by Waters Cor-
poration with an analytical Symmetry C18 column
(4.6 · 75 mm) and acetonitrile/water mixture as eluent.
3.2. Dioxolane-thymine 50-(phenyl methoxy-2-aminoiso-
butyric phosphate) (4)
Yield 83%. Mp: 146.3 ꢁC. 31P NMR (CDCl3): d 3.0, 2.8
(1:0.6); 1H NMR (CDCl3): d 1.51 and 1.57 (s, 6H,
(CH3)2C), 1.91 (s, 3H, 5-Me), 3.70 (s, 3H, OCH3), 4.08
(m, 1H, NH), 4.20 (m, 2H, H50), 4.38 (m, 2H, H20), 5.18
(s, 1H, H40), 6.39 (m, 1H, H10), 7.12–7.35 (m, 5H, Ph),
7.43 (s, 1H, H6), 8.24 (s, 1H, H3). MS: m/z 483
(M + 1)+. Anal. Calcd for C20H26N3O9P 0.3%H2O: C,
49.14; H, 5.48; N, 8.60. Found: C, 48.77; H, 5.38; N, 8.33.
3.3. Dioxolane-thymine 50-(phenyl methoxy-L-alanyl phos-
phate) (5)
Yield 81%. Mp: 145.2 ꢁC. 31P NMR (CDCl3): d 3.3, 3.0
(1:0.3); 1H NMR (CDCl3): d 1.30–1.39 (d, 3H, Ala-
CH3), 1.91 (s, 3H, 5-Me), 3.66 (m, 1H, Ala-CH), 3.74
(s, 3H, OCH3), 4.02 (m, 1H, NH), 4.20 (m, 2H, H50),
4.39 (m, 2H, H20), 5.18 (s, 1H, H40), 6.39 (m, 1H,
H10), 7.12–7.35 (m, 5H, Ph), 7.43 (s, 1H, H6), 8.24 (s,
1H, H3). MS: m/z 469 (M + 1)+. Anal. Calcd for
C19H24N3O9P: C, 48.62; H, 5.15; N, 8.95. Found: C,
48.67; H, 5.24; N, 8.86.
3.1.1. General procedure for the synthesis of aryl phospho-
ramidates. A solution of triethylamine (9.48 mmol) in
anhydrous dichloromethane(25 mL) was added dropwise
with vigorous stirring to a solution of amino acid ester
hydrochloride (4.74 mmol) and phenyl phosphorodichlo-
ride (4.74 mmol) in dichloromethane (20 mL) at ꢀ78 ꢁC
over a period of 2 h. The reaction temperature was slowly
raised to room temperature and stirred for another 6 h.
The solvent was removed under reduced pressure and sol-
id residue was dissolved in diethyl ether (20 mL) and fil-
tered. The filtrate was evaporated in vacuum at room
temperature to yield the phosphorochloromidates as a
colorless oil. Without further purification, the colorless
oil was dissolved in 2 mL of anhydrous THF and added
to the reaction mixture of DOT (4.74 mmol) in THF
(2 mL) and N-methylimidazole (7.35 mmol) with vigor-
ous stirring. The mixture was stirred at room temperature
for 8 h. The solvent was removed under reduced pressure.
The residue was dissolved in dichloromethane (15 mL)
and washed with 1 N HCl solution (2 · 15 mL), saturated
NaHCO3 aqueous solution (2 · 10 mL), water
(3 · 10 mL), and brine (2 · 20 mL), dried over Na2SO4,
and evaporated under reduced pressure. The residue
was purified by flash chromatography on silica gel by elu-
tion with 2–12% methanol in dichloromethane to yield
aryl phosphoramidates (3–28) as white solids.
3.4. Dioxolane-thymine 50-(phenyl methoxy-D-alanyl phos-
phate) (6)
Yield 78%. Mp:145.3 ꢁC. 31P NMR (CDCl3): d 3.1, 3.0
(1:0.8); 1H NMR (CDCl3): d 1.30–1.39 (d, 3H, Ala-
CH3), 1.91 (s, 3H, 5-Me), 3.66 ( m, 1H, Ala-CH), 3.74
(s, 3H, OCH3), 4.02 (m, 1H, NH), 4.20 (m, 2H, H50),
4.39 (m, 2H, H20), 5.18 (s, 1H, H40), 6.39 (m, 1H,
H10), 7.12–7.35 (m, 5H, Ph), 7.43 (s, 1H, H6), 8.24 (s,
1H, H3). MS: m/z 469 (M + 1)+. Anal. Calcd for
C19H24N3O9P: C, 48.62; H, 5.15; N, 8.95. Found: C,
48.73; H, 5.18; N, 8.81.
3.5. Dioxolane-thymine 50-(phenyl methoxy-L-phenylalanyl
phosphate) (7)
Yield 84%. Mp: 147.1 ꢁC. 31P NMR (CDCl3): d 3.1, 3.0
(1:0.7); 1H NMR (CDCl3): d 1.91 (s, 3H, 5-Me), 3.02 (m,
2H, Ph-CH2), 3.46 (m, 1H, Ph-CH), 3.64 (s, 3H, OCH3),
4.05 (m, 1H, NH), 4.37 (m, 4H, H50 and H20), 5.07 (s,
1H, H40), 6.39 (m, 1H, H10), 7.12–7.35 (m, 10H, Ph),
7.43 (s, 1H, H6), 8.24 (s, 1H, H3). MS: m/z 546
(M + 1)+. Anal. Calcd for C25H28N3O9P: C, 55.05; H,
5.17; N, 7.70. Found: C, 55.27; H, 5.31; N, 7.82.
3.6. Dioxolane-thymine 50-(phenyl methoxy-D-phenylalanyl
phosphate) (8)
3.1.2. Dioxolane-thymine 50-(phenyl methoxy-glycinyl
phosphate) (3). Yield 85%. Mp: 147.5 ꢁC. 31P
Yield 88%. Mp: 147.3 ꢁC. 31P NMR (CDCl3): d 3.4, 2.9
(1:0.9); 1H NMR (CDCl3): d 1.91 (s, 3H, 5-Me), 3.02 (m,
2H, Ph-CH2), 3.46 (m, 1H, Ph-CH), 3.64 (s, 3H, OCH3),
4.05 (m, 1H, NH), 4.37 (m, 4H, H50 and H20), 5.07 (s,
1
NMR (CDCl3): d 2.1, 1.8 (1:1); H NMR (CDCl3): d
1.91 (s, 3H, 5-Me), 3.72 (s, 3H, OCH3), 3.77 (m, 2H,
Gly-CH2), 4.03 (m, 1H, NH), 4.22 (m, 2H, H50), 4.40

2184
Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
1H, H40), 6.39 (m, 1H, H10), 7.12–7.35 (m, 10H, Ph),
7.41 (s, 1H, H6), 8.04 (s, 1H, H3). MS: m/z 546
(M + 1)+. Anal. Calcd for C25H28N3O9P: C, 55.05; H,
5.17; N, 7.70. Found: C, 54.69; H, 5.29; N, 8.40.
NH), 4.20 (m, 2H, H50), 4.39 (m, 2H, H20), 5.18 (s,
1H, H40), 6.39 (m, 1H, H10), 7.08–7.31 (m, 4H, Ph),
7.43 (s, 1H, H6), 8.84 (s, 1H, H3). MS: m/z 483
(M + 1)+. Anal. Calcd for C20H26N3O9P: C, 49.69; H,
5.42; N, 8.69. Found: C, 49.59; H, 5.47; N, 8.64.
3.7. Dioxolane-thymine 50-(phenyl methoxy-L-valyl
phosphate) (9)
3.12. Dioxolane-thymine 50-(4-propylphenyl methoxy-L-
alanyl phosphate) (14)
Yield 87%. Mp: 141.2 ꢁC. 31P NMR (CDCl3): d 3.4, 3.1
(1:0.5); 1H NMR (CDCl3): d 1.20–1.28 (d, 6H, Val-
CH3), 1.91 (s, 3H, 5-Me), 2.81 (m, 1H, Val-CH), 3.66
(m, 1H, Val-CH), 3.74 (s, 3H, OCH3), 4.02 (m, 1H,
NH), 4.20 (m, 2H, H50), 4.39 (m, 2H, H20), 5.18 (s,
1H, H40), 6.39 (m, 1H, H10), 7.12–7.35 (m, 5H, Ph),
7.43 (s, 1H, H6), 8.24 (s, 1H, H3). MS: m/z 497
(M + 1)+. Anal. Calcd for C21H28N3O9P: C, 50.70; H,
5.67; N, 8.45. Found: C, 50.60; H, 5.85; N, 8.23.
Yield 61%. Mp: 135.5 ꢁC. 31P NMR (CDCl3): d 3.2, 2.9
1
(1:0.5); H NMR (CDCl3): d 0.91 (m, 3H, CH3), 1.30–
1.42 (d, 3H, Ala-CH3), 1.61 (m, 2H, CH2), 1.91 (s, 3H,
5-Me), 3.69 (s, 3H, OCH3), 3.78 (m, 1H, Ala-CH), 4.05
(m, 1H, NH), 4.20 (m, 2H, H50), 4.39 (m, 2H, H20), 5.18
(s, 1H, H40), 6.39 (m, 1H, H10), 7.08–7.31 (m, 4H, Ph),
7.43 (s, 1H, H6), 8.81 (s, 1H, H3). MS: m/z 511
(M + 1)+. Anal. Calcd for C22H30N3O9P: C, 51.66; H,
5.91; N, 8.22. Found: C, 51.36; H, 5.99; N, 7.98.
3.8. Dioxolane-thymine 50-(phenyl methoxy-D-valyl
phosphate) (10)
3.13. Dioxolane-thymine 50-(4-neopentylphenyl methoxy-
L-alanyl phosphate) (15)
Yield 85%. Mp: 141.5 ꢁC. 31P NMR (CDCl3): d 3.3, 2.8
(1:0.6); 1H NMR (CDCl3): d 1.20–1.28 (d, 6H, Val-
CH3), 1.91 (s, 3H, 5-Me), 2.81 (m, 1H, Val-CH), 3.66
(m, 1H, Val-CH), 3.74 (s, 3H, OCH3), 4.02 (m, 1H,
NH), 4.20 (m, 2H, H50), 4.39 (m, 2H, H20), 5.18 (s,
1H, H40), 6.39 (m, 1H, H10), 7.12–7.35 (m, 5H, Ph),
7.43 (s, 1H, H6), 8.24 (s, 1H, H3). MS: m/z 497
(M + 1)+. Anal. Calcd for C21H28N3O9P: C, 50.70; H,
5.67; N, 8.45. Found: C, 50.74; H, 5.75; N, 8.20.
Yield 55%. Mp: 134.1 ꢁC. 31P NMR (CDCl3): d 3.2, 2.9
(1:0.5); 1H NMR (CDCl3): d 0.86 and 1.45–1.64 (m,
11H, neopentyl-CH2 and CH3), 1.30–1.42 (d, 3H, Ala-
CH3), 1.91 (s, 3H, 5-Me), 3.69 (s, 3H, OCH3), 3.78 (m,
1H, Ala-CH), 4.05 (m, 1H, NH), 4.20 (m, 2H, H50), 4.39
(m, 2H, H20), 5.18 (s, 1H, H40), 6.39 (m, 1H, H10), 7.08–
7.28 (m, 4H, Ph), 7.43 (s, 1H, H6), 8.85 (s, 1H, H3). MS:
m/z 539 (M + 1)+. Anal. Calcd for C24H34N3O9P: C,
53.43; H, 6.35; N, 7.79. Found: C, 53.53; H, 6.30; N, 7.62.
3.9. Dioxolane-thymine 50-(phenyl isobutyloxy-L-alayl
phosphate) (11)
3.14. Dioxolane-thymine 50-(4-methyoxyphenyl methoxy-
L-alanyl phosphate) (16)
Yield 86%. Mp: 141. 3 ꢁC. 31P NMR (CDCl3): d 3.1, 2.9
1
(1:1); H NMR (CDCl3): d 1.30–1.39 (d, 3H, Ala-CH3),
Yield 62%. Mp: 142.5 ꢁC. 31P NMR (CDCl3): d 3.4, 13.2
(1:0.7); 1H NMR (CDCl3): d 1.28–1.40 (d, 3H, Ala-
CH3), 1.91 (s, 3H, 5-Me), 3.64 (s, 3H, OCH3), 3.71 (s,
3H, CH3OPh), 3.78 (m, 1H, Ala-CH), 4.04 (m, 1H,
NH), 4.20 (m, 2H, H50), 4.39 (m, 2H, H20), 5.08 (s,
1H, H40), 6.41 (m, 1H, H10), 6.83 and 7.15 (m, 4H,
Ph), 7.45 (s, 1H, H6), 8.96 (s, 1H, H3). MS: m/z 499
(M + 1)+. Anal. Calcd for C20H26N3O10P: C, 48.10; H,
5.25; N, 8.41. Found: C, 47.93; H, 5.34; N, 8.34.
1.41 (s, 9H, C(CH3)3), 1.91 (s, 3H, 5-Me), 3.66 (m, 1H,
Ala-CH), 4.02 (m, 1H, NH), 4.20 (m, 2H, H50), 4.39 (m,
2H, H20), 5.18 (s, 1H, H40), 6.39 (m, 1H, H10), 7.12–7.35
(m, 5H, Ph), 7.43 (s, 1H, H6), 8.24 (s, 1H, H3). MS: m/z
511 (M + 1)+. Anal. Calcd for C22H30N3O9P: C, 51.66;
H, 5.91; N, 8.22. Found: C, 51.38; H, 5.75; N, 7.99.
3.10. Dioxolane-thymine 50-(phenyl benzyloxy-L-alayl
phosphate) (12)
3.15. Dioxolane-thymine 50-(4-cyanophenyl methoxy-L-
alanyl phosphate) (17)
Yield 84%. Mp: 128.2 ꢁC. 31P NMR (CDCl3): d 3.1, 2.7
(1:0.5); 1H NMR (CDCl3): d 1.30–1.39 (d, 3H, Ala-
CH3), 1.91 (s, 3H, 5-Me), 3.66 (m, 1H, Ala-CH), 4.02
(m, 1H, NH), 4.20 (m, 2H, H50), 4.39 (m, 2H, H20),
5.12 (s, 1H, H40), 5.17 (m, 2H, PhCH2O), 6.39 (m, 1H,
H10), 7.12–7.35 (m, 10H, Ph), 7.43 (s, 1H, H6), 8.34 (s,
1H, H3). MS: m/z 545 (M + 1)+. Anal. Calcd for
C25H28N3O9P: C, 55.05; H, 5.17; N, 7.70. Found: C,
54.93; H, 5.21; N, 7.30.
Yield 56%. Mp: 178.9 ꢁC. 31P NMR (CDCl3): d 3.3, 3.0
(1:1.1); 1H NMR (CDCl3): d 1.33–1.40 (d, 3H, Ala-
CH3), 1.90 (s, 3H, 5-Me), 3.61 (m, 1H, Ala-CH), 3.72 (s,
3H, OCH3), 4.05 (m, 1H, NH), 4.20 (m, 2H, H50), 4.39
(m, 2H, H20), 5.18 (s, 1H, H40), 6.39 (m, 1H, H10), 7.01–
7.19 (m, 4H, Ph), 7.43 (s, 1H, H6), 8.15 (s, 1H, H3). MS:
m/z 494 (M + 1)+. Anal. Calcd for C20H23N4O9P: C,
48.59; H, 4.69; N, 11.33. Found: C, 48.86; H, 4.52; N,
11.25.
3.11. Dioxolane-thymine 50-(4-methylphenyl methoxy-L-
alanyl phosphate) (13)
3.16. Dioxolane-thymine 50-(4-chlorophenyl methoxy-L-
alanyl phosphate) (18)
Yield 68%. Mp: 138.0 ꢁC. 31P NMR (CDCl3): d 3.2, 2.9
(1:0.6); 1H NMR (CDCl3): d 1.30–1.42 (d, 3H, Ala-
CH3), 1.91 (s, 3H, 5-Me), 2.33 (m, 3H, CH3Ph), 3.64
(s, 3H, OCH3), 3.78 (m, 1H, Ala-CH), 4.04 (m, 1H,
Yield 58%. Mp: 153.5 ꢁC. 31P NMR (CDCl3): d 3.1, 2.8
(1:0.5); 1H NMR (CDCl3): d 1.36–1.38 (d, 3H, Ala-

Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
2185
CH3), 1.86 (s, 3H, 5-Me), 3.61 (m, 1H, Ala-CH), 3.73 (s,
3H, OCH3), 4.05 (m, 1H, NH), 4.21 (m, 2H, H50), 4.40
(m, 2H, H20), 5.19 (s, 1H, H40), 6.42 (m, 1H, H10), 7.18–
7.29 (m, 4H, Ph), 7.43 (s, 1H, H6), 8.35 (s, 1H, H3). MS:
m/z 503 (M + 1)+. Anal. Calcd for C19H23ClN3O9P: C,
45.29; H, 4.60; N, 8.34. Found: C, 45.55; H, 4.67; N, 8.30.
(m, 1H, NH), 4.21 (m, 2H, H50), 4.40 (m, 2H, H20), 5.15
(s, 1H, H40), 5.19 (m, 2H, PhCH2), 6.42 (m, 1H, H10),
7.08–7.42 (m, 9H, Ph), 7.43 (s, 1H, H6), 7.96 (s, 1H,
H3). MS: m/z 580 (M + 1)+. Anal. Calcd for
C25H27ClN3O9P: C, 51.78; H, 4.69; N, 7.25. Found: C,
52.09; H, 4.85 ; N, 7.05.
3.17. Dioxolane-thymine 50-(4-fluorophenyl methoxy-L-
alanyl phosphate) (19)
3.22. Dioxolane-thymine 50-(2-allylphenyl methoxy-L-alanyl
phosphate) (24)
Yield 63%. Mp: 141.3 ꢁC. 31P NMR (CDCl3): d 2.9, 2.5
Yield 45%. Mp: 127.0 ꢁC. 31P NMR (CDCl3): d 2.8, 2.7
1
1
(1:1); H NMR (CDCl3): d 1.36–1.38 (d, 3H, Ala-CH3),
(1:1); H NMR (CDCl3): d 1.32–1.41 (d, 3H, Ala-CH3),
1.86 (s, 3H, 5-Me), 3.73 (s, 3H, OCH3), 3.97 (m, 1H,
Ala-CH), 4.05 (m, 1H, NH), 4.21 (m, 2H, H50), 4.40 (m,
2H, H20), 5.19 (s, 1H, H40), 6.42 (m, 1H, H10), 7.25–7.42
(m, 4H, Ph), 7.63 (s, 1H, H6), 9.08 (s, 1H, H3). MS: m/z
487 (M + 1)+. Anal. Calcd for C19H23FN3O9P: C, 46.82;
H, 4.76; N, 8.62. Found: C, 47.10; H, 4.52; N, 8.89.
1.85 (s, 3H, 5-Me), 3.40 (m, 2H, All-CH2), 3.45 (m, 1H,
Ala-CH), 3.77 (s, 3H, OCH3), 4.07 (m, 1H, NH), 4.21
(m, 2H, H50), 4.43 (m, 2H, H20), 5.17 (s, 1H, H40),
5.19 and 5.98 (m, 3H, CH@CH2), 6.41 (m, 1H, H10),
7.12–7.42 (m, 4H, Ph), 7.47 (s, 1H, H6), 8.76 (s, 1H,
H3). MS: m/z 509 (M + 1)+. Anal. Calcd for
C22H28N3O9P 0.4%H2O: C, 51.14; H, 5.62; N, 8.13.
Found: C, 50.56; H 5.32; N, 7.91.
3.18. Dioxolane-thymine 50-(4-bromophenyl methoxy-L-
alanyl phosphate) (20)
3.23. Dioxolane-thymine 50-(2,4-dichlorophenyl methoxy-
L-alanyl phosphate) (25)
Yield 58%. Mp: 176.5 ꢁC. 31P NMR (CDCl3): d 3.0, 2.7
(1:0.5); 1H NMR (CDCl3): d 1.36–1.38 (d, 3H, Ala-
CH3), 1.86 (s, 3H, 5-Me), 3.63 (m, 1H, Ala-CH), 3.73 (s,
3H, OCH3), 4.05 (m, 1H, NH), 4.21 (m, 2H, H50), 4.40
(m, 2H, H20), 5.19 (s, 1H, H40), 6.42 (m, 1H, H10), 7.08–
7.42 (m, 4H, Ph), 7.43 (s, 1H, H6), 7.96 (s, 1H, H3). MS:
m/z 547 (M + 1)+. Anal. Calcd for C19H23BrN3O9P: C,
41.62; H, 4.23; N, 7.66. Found: C, 41.86; H, 4.29; N, 7.59.
Yield 52%. Mp: 171.5 ꢁC. 31P NMR (CDCl3): d 2.9, 2.8
1
(1:1); H NMR (CDCl3): d 1.34–1.42 (d, 3H, Ala-CH3),
1.86 (s, 3H, 5-Me), 3.68 (m, 1H, Ala-CH), 3.73 (s, 3H,
OCH3), 4.09 (m, 1H, NH), 4.21 (m, 2H, H50), 4.43 (m,
2H, H20), 5.19 (s, 1H, H40), 6.41 (m, 1H, H10), 7.22–7.42
(m, 3H, Ph), 7.54 (s, 1H, H6), 8.89 (s, 1H, H3). MS: m/z
537 (M + 1)+. Anal. Calcd for C19H22N3O9P: C, 42.40;
H, 4.12; N, 7.81. Found: C, 42.69; H, 4.19; N, 7.58.
3.19. Dioxolane-thymine 50-(4-bromophenyl benzyloxy-L-
alanyl phosphate) (21)
3.24. Dioxolane-thymine 50-(naphthyl methoxy-L-alanyl
phosphate) (26)
Yield 58%. Mp: 142.1 ꢁC. 31P NMR (CDCl3): d 3.4, 2.9
(1:0.8); 1H NMR (CDCl3): d 1.36–1.38 (d, 3H, Ala-
CH3), 1.88 (s, 3H, 5-Me), 3.68 (m, 1H, Ala-CH), 4.05
(m, 1H, NH), 4.21 (m, 2H, H50), 4.40 (m, 2H, H20),
5.15 (s, 1H, H40), 5.19 (m, 2H, PhCH2), 6.42 (m, 1H,
H10), 7.08–7.42 (m, 9H, Ph), 7.43 (s, 1H, H6), 7.96 (s,
1H, H3). MS: m/z 624 (M + 1)+. Anal. Calcd for
C25H27BrN3O9P: C, 48.09; H, 4.36; N, 6.73. Found: C,
48.33; H, 4.41; N, 6.55.
Yield 45%. Mp: 142.7 ꢁC. 31P NMR (CDCl3): d 3.3, 3.1
(1:0.7); 1H NMR (CDCl3): d 1.34–1.42 (d, 3H, Ala-
CH3), 1.86 (s, 3H, 5-Me), 3.68 (m, 1H, Ala-CH), 3.73
(s, 3H, OCH3), 4.09 (m, 1H, NH), 4.21 (m, 2H, H50),
4.43 (m, 2H, H20), 5.19 (s, 1H, H40), 6.41 (m, 1H,
H10), 7.23–7.82 (m, 7H, naphthyl), 7.96 (s, 1H, H6),
8.87 (s, 1H, H3). MS: m/z 2519 (M + 1)+. Anal. Calcd
for C23H26N3O9P: C, 53.18; H, 5.05; N, 8.09. Found:
C, 52.97; H, 5.17; N, 8.06.
3.20. Dioxolane-thymine 50-(2-chlorophenyl methoxy-L-
alanyl phosphate) (22)
3.25. Dioxolane-thymine 50-(naphthyl methoxy-2-amino
isobutyric phosphate) (27)
Yield 78%. Mp: 149.5 ꢁC. 31P NMR (CDCl3): d 3.4, 3.2
(1:0.9); 1H NMR (CDCl3): d 1.32–1.41 (d, 3H, Ala-
CH3), 1.85 (s, 3H, 5-Me), 3.68 (m, 1H, Ala-CH), 3.73
(s, 3H, OCH3), 4.09 (m, 1H, NH), 4.21 (m, 2H, H50),
4.43 (m, 2H, H20), 5.19 (s, 1H, H40), 6.41 (m, 1H,
H10), 7.12–7.42 (m, 4H, Ph), 7.44 (s, 1H, H6), 7.86 (s,
1H, H3). MS: m/z 503 (M + 1)+. Anal. Calcd for
C19H23ClN3O9P: C, 45.29; H, 4.60; N, 8.34. Found: C,
45.28; H, 4.71; N, 8.816.
Yield 43%. Mp: 140.5 ꢁC. 31P NMR (CDCl3): d 3.4, 3.1
1
(1:0.7); H NMR (CDCl3): d 1.44–1.53 (d, 6H, Isobut-
CH3), 1.86 (s, 3H, 5-Me), 3.75 (s, 3H, OCH3), 4.08 (m,
1H, NH), 4.21 (m, 2H, H50), 4.43 (m, 2H, H20), 5.19
(s, 1H, H40), 6.41 (m, 1H, H10), 7.23–7.82 (m, 7H, naph-
thyl), 8.06 (s, 1H, H6), 8.57 (s, 1H, H3). MS: m/z 533
(M + 1)+. Anal. Calcd for C24H28N3O9P: C, 54.03; H,
5.29; N, 7.88. Found: C, 52.89; H, 5.16; N, 7.68.
3.21. Dioxolane-thymine 50-(2-chlorophenyl benzyloxy-L-
alanyl phosphate) (23)
3.26. Dioxolane-thymine 50-(naphthyl benzyloxy-L-alanyl
phosphate) (28)
Yield 77%. Mp: 137.1 ꢁC. 31P NMR (CDCl3): d 3.3, 3.1
(1:0.8); 1H NMR (CDCl3): d 1.33–1.42 (d, 3H, Ala-
CH3), 1.88 (s, 3H, 5-Me), 3.68 (m, 1H, Ala-CH), 4.05
Yield 48%. Mp: 132.1 ꢁC. 31P NMR (CDCl3): d 3.3, 3.2
(1:0.8); 1H NMR (CDCl3): d 1.34–1.47 (d, 6H, Ala-

2186
Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
CH3), 1.88 (s, 3H, 5-Me), 3.68 (m, 1H, Ala-CH), 4.08
(m, 1H, NH), 4.21 (m, 2H, H50), 4.43 (m, 2H, H20),
5.16 (s, 1H, H40), 5.20 (m, 2H, PhCH2), 6.41 (m, 1H,
H10), 7.23–7.74 (m, 12H, Ph and naphthyl), 8.01 (s,
1H, H6), 8.77 (s, 1H, H3). MS: m/z 596 (M + 1)+. Anal.
Calcd for C29H30N3O9P: C, 58.49; H, 5.08; N, 7.06.
Found: C, 58.45; H, 5.27; N, 6.77.
1.95 (m, 33H, HDP-H), 1.96 (s, 3H, 5-Me), 2.05 (m,
1H, Val-CH), 3.34 and 3.75 (m, 2H, Val-CHNH),
3.38, 3.48, and 4.21 (m, 6H, OCH2), 3.78 (s, 3H,
OCH3), 4.18 (m, 2H, H50), 4.39 (m, 2H, H20), 5.15 (s,
1H, H40), 6.39 (m, 1H, H10), 7.48 (s, 1H, H6), 8.17 (s,
1H, H3). MS: m/z 704 (M + 1)+. Anal. Calcd for
C34H62N3O10P 0.1%H2O: C, 57.87; H, 8.88; N, 5.95.
Found: C, 57.99; H, 8.95; N, 5.94.
3.27. Dioxolane-thymine 50-(3-hexadecyloxypropyl
methoxy-L-alanyl phosphate) (32)
3.30. Dioxolane-thymine 50-(2-octadecyloxyethyl
methoxy-L-alanyl phosphate) (34)
To a solution of diphenyl phosphite (234 mg, 1.0 mmol)
in pyridine (10 mL) was added DOT (114 mg, 0.5 mmol)
in pyridine (10 mL) over a period of 1 h under nitrogen
atmosphere at 0 ꢁC, and the reaction mixture was stirred
for 30 min. Anhydrous long-chain alcohol 3-hexadecyl-
oxypropyl-1-ol (450 mg, 1.5 mmol) was added to the
above mixture and stirred for an additional 2 h at room
temperature. The solvent was removed under reduced
pressure and the residue was purified by flash chromatog-
raphy on silica gel (2% MeOH in CH2Cl2) to provide
dioxolane thymine 50-(3-hexadecyloxypropyl phosphite)
in good yield. (130 mg, 70%). 31P NMR (CDCl3): d 4.5;
1H NMR (CDCl3): d 0.89–1.95 (m, 33H, HDP-H), 1.96
(s, 3H, 5-Me), 3.38, 3.48 and 4.21 (m, 6H, OCH2), 4.18
(m, 2H, H50), 4.39 (m, 2H, H20), 5.15 (s, 1H, H40), 6.39
(m, 1H, H10), 6.18 and 7.62 (d, 1H, P-H), 7.48 (s, 1H,
H6), 8.20 (s, 1H, H3). The above H-phosphonate
(1 mmol) in CH2Cl2 (2 mL) was added dropwise to a solu-
tion of the corresponding L-amino acid methyl ester
hydrochloride (1.2 mmol) in Et3N (0.5 mL)-CCl4
(0.5 mL)-H2O (0.5 mL)-MeCN (5 mL) at 0 ꢁC. The mix-
ture was stirred at room temperature for 20 min. The sol-
vent was removed under reduced pressure and the residue
was purified by flash chromatography on silica gel (5%
MeOH in CH2Cl2) to afford 32. Yield 98%. 31P NMR
(CDCl3): d 3.7, 3.4 (1:1); 1H NMR (CDCl3): d 0.89–1.95
(m, 33H, HDP-H), 1.35 (d, 3H, Ala-CH3), 1.96 (s, 3H,
5-Me), 3.34 (m, 1H, Ala-CH), 3.38, 3.48, and 4.21 (m,
6H, OCH2), 3.74 (m, 1H, NH), 3.78 (s, 3H, OCH3), 4.18
(m, 2H, H50), 4.39 (m, 2H, H20), 5.15 (s, 1H, H40), 6.39
(m, 1H, H10), 7.48 (s, 1H, H6), 8.17 (s, 1H, H3). MS:
m/z 675 (M + 1)+. Anal. Calcd for C32H58N3O10P: C,
56.87; H, 8.65; N, 6.22. Found: C, 57.04; H, 8.86; N, 6.18.
Overall yield 68%. 31P NMR (CDCl3): d 3.7, 3.6 (1:1);
1H NMR (CDCl3): d 0.93–1.90 (m, 35H, ODE-H),
1.33 (d, 3H, Ala-CH3), 1.95 (s, 3H, 5-Me), 3.34 (m,
1H, Ala-CH), 3.38, 3.48, and 4.21 (m, 6H, OCH2),
3.74 (m, 1H, NH), 3.78 (s, 3H, OCH3), 4.18 (m, 2H,
H50), 4.39 (m, 2H, H20), 5.15 (s, 1H, H40), 6.39 (m,
1H, H10), 7.48 (s, 1H, H6), 8.17 (s, 1H, H3); MS: m/z
689 (M + 1)+. Anal. Calcd for C33H60N3O9P: C, 57.46;
H, 8.77; N, 6.09. Found: C, 57.58; H, 9.05; N, 5.95.
3.31. Dioxolane-thymine 50-(oleyl methoxy-L-alanyl
phosphate) (35)
Overall yield 67%. 31P NMR (CDCl3): d 3.7, 3.6 (1:1);
1H NMR (CDCl3): d 0.90–1.80 (m, 27H, Oleyl-H),
1.35 (d, 3H, Ala-CH3), 1.95 (s, 3H, 5-Me), 2.03 (m,
4H, CH2C@), 3.34 (m, 1H, Ala-CH), 3.66 (m, 2H,
OCH2), 3.74 (m, 1H, NH), 3.78 (s, 3H, OCH3), 4.18
(m, 2H, H50), 4.39 (m, 2H, H20), 5.15 (s, 1H, H40),
5.38 (m, 2H, CH@CH), 6.39 (m, 1H, H10), 7.48 (s,
1H, H6), 8.17 (s, 1H, H3). MS: m/z 643 (M + 1)+. Anal.
Calcd for C32H58N3O9P: C, 57.84; H, 8.45; N, 6.53.
Found: C, 57.80; H, 8.39; N, 6.38.
3.32. (+)-cis-50-O-[4-(S)-(3-Chlorophenyl)-2-oxo-1,3,2-
dioxaphosphorinan-2-yl] dioxolane-thymine (41)
A solution of diol (120 mg, 0.79 mmol) and triethyl-
amine (0.44 mL, 3.17 mmol) in THF (10 mL) was added
dropwise to a solution of 4-nitrophenoxyphosphorod-
ichloridate (405 mg, 1.58 mmol) in THF (2 mL) at
0 ꢁC. After 2h, additional triethylamine (0.44 mL,
3.17 mmol) and 4-nitrophenol (1.76 g, 14.68 mmol) were
added and the reaction mixture was stirred overnight.
The solvent was evaporated, and the residue was parti-
tioned between EtOAc and water. The organic phase
was washed with 0.2 M NaOH (1 · 10 mL), water
(2 · 10 mL), brine (2 · 10 mL), and dried over MgSO4.
The mixture was concentrated under reduced pressure
and purified by flash chromatography (50% EtOAc in
hexanes) to yield 50-O-[4-(S)-(3-chlorophenyl)-2-oxo-
1,3,2-dioxaphosphorinan-2-yl]-4-nitrobenzene (143 mg,
88%). 31P NMR (CDCl3): d ꢀ4.8, ꢀ4.2 (1:0.1); 1H
NMR (CDCl3): d 2.09–2.48 (m, 2H, CH2), 4.61 (m,
2H, CH2O), 5.60 (m, 1H, PhCH), 7.37–8.22 (m, 9H, Ph).
3.28. Dioxolane-thymine 50-(3-hexadecyloxypropyl
methoxy-glycinyl phosphate) (31)
Overall yield 67%. 31P NMR (CDCl3): d 4.1, 3.8 (1:1);
1H NMR (CDCl3): d 0.89–1.95 (m, 33H, HDP-H),1.96
(s, 3H, 5-Me), 3.36 (m, 2H, Gly-CH2), 3.38, 3.48, and
4.21 (m, 6H, OCH2), 3.76 (m, 1H, NH), 3.79 (s, 3H,
OCH3), 4.18 (m, 2H, H50), 4.39 (m, 2H, H20), 5.15 (s,
1H, H40), 6.39 (m, 1H, H10), 7.49 (s, 1H, H6), 8.18 (s,
1H, H3). MS: m/z 662 (M + 1)+. Anal. Calcd for
C31H56N3O10P 0.1%H2O: C, 56.11; H, 8.54; N, 6.33.
Found: C 56.16; H, 8.59; N, 6.17.
3.29. Dioxolane-thymine 50-(3-hexadecyloxypropyl
methoxy-L-valyl phosphate) (33)
A solution of dioxolane-thymine (45 mg, 0.18 mmol) in
DMF (5 mL) was treated with a THF solution of 1 M
tert-BuMgCl (20 mg, 0.23 mmol) and stirred at room
temperature. After 30 min., 50-O-[4-(S)-(3-chlorophe-
nyl)-2-oxo-1,3,2-dioxaphosphorinan-2-yl]-4-nitrobenzene
Overall yield 68%. 31P NMR (CDCl3): d 3.7, 3.6 (1:1);
1H NMR (CDCl3): d 0.85 (dd, 6H, Val-CH3), 0.97-

Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
2187
(100 mg, 0.31 mmol) was added. The reaction mixture
was stirred for 20 h at room temperature and then
quenched with saturated NH4Cl (10 mL) and extracted
with EtOAc. The organic extract was washed with 1 N
phate (1.96 g, 8 mmol) in THF (10 mL). The mixture
was stirred for 30 min. and then filtered. To the filtrate
were added sequentially, additional 30 mL of THF,
dioxolane thymine (1.37 g, 6 mmol), and 1-methylimidaz-
ole (0.66 g, 8 mmol). After 1 h, 3-hexadecyl-1-propanol
(HDP, 1.8 g, 6 mmol) was added to the mixture and stir-
red overnight at room temperature. The solvent was evap-
orated and the residue was purified by flash
chromatography on silica gel (4% MeOH in CH2Cl2) to
afford dioxolane thymine 50-[(2-chlorophenyl 3-hexade-
cyloxypropyl) phosphate] (1.88 g, 60%). 31P NMR
NaOH
(2 · 20 mL),
water
(2 · 20 mL),
brine
(2 · 20 mL), and dried (Na2SO4), concentrated to a resi-
due, and purified by flash chromatography (5% MeOH
22
D
in CH2Cl2) to give 41. Yield 52%. Mp: 128.7 ꢁC. ½aꢁ
+25.13 (c 0.99, CHCl3). 31P NMR (CDCl3): d ꢀ4.2,
1
ꢀ4.1 (1:0.1); H NMR (CDCl3): d 1.90 (s, 3H, 5-Me),
2.05–2.35 (m, 2H, CH2), 4.19 (m, 2H, H50), 4.43 (m,
2H, H20), 4.41 and 4.71 (m, 2H, CH2O), 5.16 (s, 1H,
H40), 5.68 (m, 2H, PhCH), 6.41 (m, 1H, H10), 7.29–7.44
(m, 4H, Ph), 7.50 (s, 1H, H6), 8.21 (s, 1H, H3). MS: m/
z 458 (M + 1)+. Anal. Calcd for C18H20ClN2O8P
0.1%H2O: C, 46.94; H, 4.42; N, 6.08. Found: C46.70;
H, 4.45; N, 5.96.
1
(CDCl3): d 6.7, 6.5 (1:0.9); H NMR (CDCl3): d 0.88–
1.87 (m, 33H, HDP-H), 1.90 (s, 3H, 5-Me), 3.35, 3.47,
and 4.35 (m, 6H, OCH2), 4.20 (m, 2H, H50), 4.45 (m,
2H, H20), 5.18 (s, 1H, H40), 6.40 (m, 1H, H10), 7.11–7.40
(m, 4H, Ph), 7.46 (s, 1H, H6), 8.65 (s, 1H, H3). Then it
was dissolved in THF and 0.5N NaOH was added at
0 ꢁC. The mixture was stirred at 50
3 ꢁC for 1.5 h
3.33. (ꢀ)-cis-50-O-[4-(R)-(3-Chlorophenyl)-2-oxo-1,3,2-
and neutralized with HCl at 0 ꢁC. The solvent was evapo-
rated and the residue was purified by flash chromatogra-
phy on silica gel (10% MeOH in CH2Cl2) to provide 47.
Yield 93%. 31P NMR (CDCl3): d 5.4; 1H NMR (CDCl3):
d 0.88–1.88 (m, 33H, HDP-H), 1.96 (s, 3H, 5-Me), 3.33,
3.45, and 4.21 (m, 6H, OCH2), 3.87 (m, 2H, H50), 4.15
(m, 2H, H20), 5.16 (s, 1H, H40), 6.32 (m, 1H, H10), 7.78
(s, 1H, H6). MS: m/z 591 (M + 1)+. Anal. Calcd for
C28H51N2O9P 1.2% H2O: C, 54.92; H, 8.69; N, 4.58.
Found: C, 54.52; H, 8.32; N, 4.45.
dioxaphosphorinan-2- yl]dioxolane-thymine (42)
22
Yield 61%. Mp: 126.8 ꢁC. ½aꢁ ꢀ26.75 (c 1.01, CHCl3).
D
31P NMR (CDCl3): d ꢀ4.4, ꢀ4.2(1:0.1); 1H NMR
(CDCl3): d 1.90 (s, 3H, 5-Me), 2.07–2.40 (m, 2H,
CH2), 4.20 (m, 2H, H50), 4.43 (m, 2H, H20), 4.46 and
4.71 (m, 2H, CH2O), 5.18 (s, 1H, H40), 5.65 (m, 2H,
PhCH), 6.41 (m, 1H, H10), 7.29–7.43 (m, 4H, Ph), 7.50
(s, 1H, H6), 8.20 (s, 1H, H3). MS: m/z 458 (M + 1)+.
Anal. Calcd for C18H20ClN2O8P: C, 47.12; H, 4.39; N,
6.11. Found: C, 47.06; H, 4.54; N, 5.96.
3.37. Dioxolane-thymine 50-[(2-octadecyloxyethyl)
phosphate] (48)
3.34. (+)-cis-50-O-[4-(S)-(3-Phenyl)-2-oxo-1,3,2-dioxa-
phosphorinan-2-yl]dioxolane -thymine (43)
Yield 94%. 31P NMR (CDCl3): d 5.3; 1H NMR (CDCl3):
d 0.88–1.92 (m, 35H, HDP-H), 1.96 (s, 3H, 5-Me), 3.43,
3.60, and 4.01 (m, 6H, OCH2), 4.17 (m, 2H, H50), 4.19
(m, 2H, H20), 5.15 (s, 1H, H40), 6.34 (m, 1H, H10),
7.68 (s, 1H, H6). MS: m/z 619 (M + 1)+. Anal. Calcd
for C30H55N2O9P 0.7% H2O: C, 57.07; H, 9.00; N,
4.44. Found: C, 56.69; H, 8.81; N, 4.44.
22
Yield 57%. Mp: 121.5 ꢁC. ½aꢁ +32.70 (c 0.99, CHCl3).
D
31P NMR (CDCl3): d ꢀ3.8, ꢀ3.7(1:0.1); 1H NMR
(CDCl3): d 1.90 (s, 3H, 5-Me), 2.07-2.40 (m, 2H,
CH2), 4.20 (m, 2H, H50), 4.41 (m, 2H, H20), 4.44 and
4.71 (m, 2H, CH2O), 5.16 (s, 1H, H40), 5.67 (m, 2H,
PhCH), 6.41 (m, 1H, H10), 7.29–7.43 (m, 5H, Ph), 7.50
(s, 1H, H6), 8.20 (s, 1H, H3). MS: m/z 424 (M + 1)+.
Anal. Calcd for C18H21N2O8P: C, 50.95; H, 4.99; N,
6.60. Found: C, 50.68; H, 5.34; N, 6.32.
3.38. Virology
3.38.1. Antiviral assays. The procedures for the antiviral
assays in human peripheral blood mononuclear (PBM)
cells have been published previously.7a,7b PBM cells (ob-
tained from Atlanta Red Cross) were isolated by Ficoll–
Hypaque discontinuous gradient centrifugation from
healthy seronegative donors. Cells were stimulated with
phytohemagglutinin A (Difco, Sparks, MD) for 2–3
days prior to use. HIV-1 LAI obtained from the Center
for Disease Control and Prevention (Atlanta, GA) was
used as the standard reference virus for the antiviral as-
says. The molecular infectious clones HIV-1 M184 V
pitt were obtained from Dr. John Mellors (University
of Pittsburgh). Infections were done in bulk for 1 h,
either with 100 TCID50/1 · 107 cells for a flask (T25) as-
say or with 200 TCID50/6 · 107 cells/well for the 24-well
plate assay. Cells were added to a plate or a flask con-
taining a 10-fold serial dilution of the test compound.
Assay medium was RPMI-1640 supplemented with
heat-inactivated 16% fetal bovine serum. 1.6 mM L-glu-
tamine, 80 IU/mL penicillin, 80 lg /mL streptomycin,
0.0008% DEAE–Dextran, 0.045% sodium bicarbonate,
3.35. (ꢀ)-cis-50-O-[4-(R)-(3-Phenyl)-2-oxo-1,3,2-dioxa-
phosphorinan-2-yl]dioxolane-thymine (44)
22
Yield 56%. Mp: 121.3 ꢁC. ½aꢁ ꢀ28.52 (c 0.98, CHCl3).
D
31P NMR (CDCl3): d ꢀ3.9, ꢀ3.8 (1:0.1); 1H NMR
(CDCl3): d 1.90 (s, 3H, 5-Me), 2.07–2.40 (m, 2H,
CH2), 4.20 (m, 2H, H50), 4.41 (m, 2H, H20), 4.44 and
4.71 (m, 2H, CH2O), 5.18 (s, 1H, H40), 5.67 (m, 2H,
PhCH), 6.40 (m, 1H, H10), 7.29–7.45 (m, 5H, Ph), 7.50
(s, 1H, H6), 8.20 (s, 1H, H3). MS: m/z 424 (M + 1)+.
Anal. Calcd for C18H22N2O8P: C, 50.95; H, 4.99; N,
6.60. Found: C, 50.45; H, 5.27; N, 6.77.
3.36. Dioxolane-thymine 50-[(3-hexadecyloxypropyl)
phosphate] (47)
To a solution of 1,2,4-trizole (1.1 g, 16 mmol) and trieth-
ylamine (91.62 g, 16 mmol) in anhydrous THF (10 mL)
was added a solution of 2-chlorophenyl dichlorophos-

2188
Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
and 26 IU/mL recombinant interleukin-2 (Chiron
Corp., Emeryville, CA). AZT was used as a positive
control for the assay. Uninfected PBM cells were grown
in parallel at equivalent cell concentrations as control.
The cell cultures were maintained in a humidified 5%
CO2-air at 37 ꢁC for 5 days, and supernatants were col-
lected for reverse transcriptase activity. Supernatants
were centrifuged at 12,000 rpm for 2 h to pellet the virus.
The pellet was solubilized with vortexing in 100 lL of
virus solubilization buffer (0.5% Triton X-100, 0.8 M
NaCl, 0.5 mM phenylmethylsulfonyl chloride, 20% glyc-
erol, and 0.05 M Tris, pH 7.8). 10 lL of each sample was
added to 70 lL RT reaction mixture [0.06 M Tris, pH
7.8, 0.012 M MgCl2, 0.006 M dithiothreitol, 0.006 mg/
mL poly(rA)n oligo(dT)12–18, 96 lg/mL dATP, and
1 lM of 0.08 mCi/mL [3H]thymidine triphosphate
(Moravek Biochemicals, Brea, CA) and incubated at
37 ꢁC for 2 h. The reaction was stopped by the addition
of 100 lL of 10% trichloroacetic acid containing 0.05%
sodium perophosphate. The acid-insoluble product
was harvested onto filter paper using a Packard Harvest-
er (Meriden, CT), and RT activity was read on a Pack-
ard Direct Beta Counter (Meriden, CT). RT results were
expressed in counts per minute (CPM) per milliliter. The
antiviral 50% effective concentration (EC50) and 90%
effective concentration (EC90) were determined from
the concentration–response curve using the median ef-
fect method.
37
0.5 ꢁC in screw-capped vials in a water bath. The
samples were withdrawn at appropriate time intervals
and filtered. The filtration was analyzed by HPLC.
3.39.2. Enzyme study. Prodrugs (1.4 mM) were incubat-
ed at 37 ꢁC in pH 8.0 phosphate buffer containing 20 lg/
mL porcine esterase. Aliquots were removed at inter-
vals, quenched with ice-cold methanol/acetonitrile 1/1
(v/v), filtered, and analyzed by HPLC. The enzyme
activity was determined using ethyl butyrate as substrate
with HPLC to monitor substrate disappearance.
Acknowledgments
This research was supported by the U.S. Public Health
Service Grant AI-25899 and Veterans Affairs.
References and notes
1. Shirasaka, J.; Chokekijchal, S.; Yamada, A.; Gosselin, G.;
Imbach, J. L.; Mitsuya, H. Antimicrob. Agents Chemother.
1995, 39, 2555.
2. Tan, X.; Chu, C. K.; Boudinot, F. D. Adv. Drug Deliv.
Rev. 1999, 39, 117.
3. McGuigan, C.; Pathirana, R. N.; Balzarini, J.; De Clercq,
E. J. Med. Chem. 1993, 36, 1048.
4. (a) Chang, S.-L.; Griesgraber, G. W.; Southern, P. J.;
Wagner, C. R. J. Med. Chem. 2001, 44, 223; (b) Siccardi,
D.; Gumbleton, M.; Omidi, Y.; Mcguigan, C. Eur. J.
Pharm. Sci. 2004, 22, 25.
5. Erion, M. D.; Reddy, K. R.; Boyer, S. H.; Matelich, M.
C.; Gomez-Geleno, J.; Lemus, R. H.; Ugarkar, B. G.;
Colby, T. J.; Schanzer, J.; Van Poelje, P. D. J. Am. Chem.
Soc. 2004, 126, 5154.
6. (a) Chong, Y.; Chu, C. K. Antiviral Res. 2004, 63, 7; (b)
Chu, C. K.; Yadav, V.; Chong, Y.; Schinazi, R. F. J. Med.
Chem. 2005, 48, 3949.
7. (a) Schinazi, R. F.; Cannon, D. L.; Arnold, B. H.;
Martinosaltzman, D. Antimicrob. Agents Chemother.
1988, 32, 1784; (b) Schinazi, R. F.; Sommadossi, J. P.;
Saalmann, V.; Cannon, D. L.; Xie, M.-W.; Hart, G. C.;
Smith, G. A.; Hahn, E. F. Antimicrob. Agents Chemother.
1990, 34, 1061; (c) Stuyver, L. J.; Lostia, S.; Admas, M.;
Mathew, J.; Pai, B. S.; Grier, J.; Tharnish, P.; Choi, Y.;
Chong, Y.; Choo, H.; Chu, C. K.; Otto, M. J.; Schinazi,
R. F. Antimicrob. Agents Chemother. 2002, 46, 3854.
8. Chu, C. K.; Ahn, S. K.; Kim, H. O.; Beach, J. W.; Alves,
A. J.; Jeong, L. S.; Islam, Q.; Roey, P. V.; Schinazi, R. F.
Tetrahedron Lett. 1991, 32, 3791.
9. (a) McGuigan, C.; Pathirana, R. N.; Mahmood, N.;
Devine, K. G.; Hay, A. G. Antiviral Res. 1992, 17, 311; (b)
Van Boom, J. H.; Burgers, P. M. J.; Crea, R.; Luyten, V.
M.; Reese, C. B. Tetrahedron 1975, 31, 2953.
10. Stawinski, J. In Handbook of Organophosphorus Chemis-
try; Engel, E., Ed.; Marcel Dekker: New York, 1992; p
377.
11. Xiao, Q.; Sun, J.; Sun, Q.; Ju, Y.; Zhao, Y.-F.; Cui, Y.-X.
Synthesis 2003, 107.
12. Takamura, M.; Yabu, K.; Nishi, T.; Yanagisawa, H.;
Kanai, M.; Shibasaki, M. Synlett 2003, 353.
13. Harada, T.; Kurokawa, H.; Oku, A. Tetrahedron Lett.
1987, 28, 4843.
14. Beadle, J.; Kini, G.; Aldern, K.; Gaedner, M.; Wright, K.;
Rybak, R.; Kern, E.; Hostetler, K. Nucleosides, Nucleo-
tides & Nucleic Acids 2000, 19, 471.
3.38.2. Cytotoxicity assays. The compounds were evalu-
ated for their potential toxic effects on uninfected PHA-
stimulated human PBM cell, in CEM (T-lymphoblas-
toid cell line obtained from American Type Culture
Collection, Rockville, MD) and Vero (African green
monkey kidney) cells. Log-phase Vero, CEM, and
PHA-stimulated human PBM cells were seeded at densi-
ty at 5 · 103, 2.5 · 103, and 5 · 104 cells/well, respective-
ly. All cells were planted in 96-well cell culture plates
containing 10-fold serial dilutions of the test drug. The
cultures were incubated for 2, 3, and 4 days for Vero,
CEM, and PBM cells, respectively, in a humidified 5%
CO2-air at 37 ꢁC. At the end of incubation, MTT tetra-
zolium dye solution (cell titer 96, Promega, Madison,
WI) was added to each well and incubated overnight.
The reaction was stopped with stop solubilization solu-
tion (Promega, Madison, WI). The plates were incubat-
ed for 5 h to ensure that the formazan crystals were
dissolved. The plates were read at a wavelength of
570 nm using an ELISA plate reader (Bio-Tek Instru-
ments, Inc., Winooski, VT, Model EL 312 e). The 50%
inhibition concentration (IC50) was determined from
the concentration–response curve using the median ef-
fect method.7c
3.39. Hydrolysis of prodrugs
3.39.1. Aqueous buffer kinetics. Hydrolysis of prodrugs
was studied in pH 2.0 and 7.4. A 3 mL aliquot of a
1 mM solution of prodrugs in DMSO was diluted to
10 mL using phosphate buffer (10 mM). A constant ion-
ic strength (l) of 0.1 was maintained by the addition of
an appropriate quantity of NaCl to the solutions. Buffer
solutions containing ester prodrugs were maintained at

Y. Liang et al. / Bioorg. Med. Chem. 14 (2006) 2178–2189
2189
15. Mcguigan, C.; Cahard, D.; Ballatore, C.; Siddiqui, A.; De
Clercq, E.; Balzarini, J. Bioorg. Med. Chem. Lett. 1998, 8,
2949.
19. Beadle, J. R.; Hartline, C.; Aldern, K. A.; Rodriguez, N.;
Harden, E.; Kern, E. R.; Hostetler, K. Y. Antimicrob.
Agents Chemother. 2002, 46, 2381.
16. McGuigan, C.; Harris, S. A.; Daluge, S. M.; Gudm-
undsson, K. S.; McLean, E. W.; Burnette, T. C.;
Marr, H.; Hazen, R.; Condreary, L. D.; Johnson, L.;
Clercq, E. D.; Balzarini, J. J. Med. Chem. 2005, 48,
3504.
20. (a) Siccardi, D.; Gumbleton, M.; Omodi, Y.; McGuigan,
C. Eur. J. Pharm. Sci. 2004, 22, 25; (b) Saboulard, D.;
Naesens, L.; Cahard, D.; Salgado, A.; Pathirana, R.;
Velazquez, S.; McGuigan, C.; De Clercq, E.; Balzarini, J.
Mol. Pharmacol. 1999, 56, 693.
17. McGuigan, C.; Pathirana, R. N.; Mahmood, N.; Devine,
K. G.; Hay, A. J. Antiviral Res. 1992, 17, 311.
18. (a) Ludeman, S. M.; Boyd, V. L.; Regan, J. B.; Gallo, K.
A.; Zon, G.; Ishii, K. J. Med. Chem. 1986, 29, 716; (b)
Zon, G.; Ludeman, S. M.; Brandt, J. A.; Boyd, V. L.;
Ozkan, G.; Egan, W.; Shao, K.-L. J. Med. Chem. 1984, 27,
466.
21. McGuigan, C.; Davies, M.; Pathirana, R.; Mahmood, N.;
Hay, A. J. Antiviral Res. 1994, 24, 69.
22. (a) Uckun, F. M.; Pendergrass, S.; Qazi, S.; Samuel, P.;
Venkatachalam, T. K. Eur. J. Med. Chem. 2004, 39, 225; (b)
Venkatachalam, T. K.; Samuel, P.; Li, G.; Qazi, S.;
Pendergrass, S.; Uckun, F. M. Bioorg. Med. Chem. 2004,
12, 3371.