5′-O-Aliphatic and amino acid ester prodrugs of (-)-β-d-(2R,4R)-dioxolane-thymine (DOT): Synthesis, anti-HIV activity, cytotoxicity and stability studies

Article abstract of DOI:10.1016/j.bmc.2008.10.078

A series of (-)-β-d-(2R,4R)-dioxolane-thymine-5′-O-aliphatic acid esters as well as amino acid esters were synthesized as prodrugs of (-)-β-d-(2R,4R)-dioxolane-thymine (DOT). The compounds were evaluated for anti-HIV activity against HIV-1_LAI i

Full text of DOI:10.1016/j.bmc.2008.10.078

Bioorganic & Medicinal Chemistry 17 (2009) 1404–1409
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
50-O-Aliphatic and amino acid ester prodrugs of (ꢀ)-b-D-(2R,4R)-dioxolane-thy-
mine (DOT): Synthesis, anti-HIV activity, cytotoxicity and stability studies
Yuzeng Liang a, Ashoke Sharon a, Jason P. Grier b, Kimberly L. Rapp b, Raymond F. Schinazi b, Chung K. Chu a,
*
a Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, Athens, GA 30602, USA
b Emory University School of Medicine/Veterans Affairs Medical Center, Decatur, GA 30033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2008
Revised 29 October 2008
Accepted 31 October 2008
Available online 5 November 2008
A series of (ꢀ)-b-
D
-(2R,4R)-dioxolane-thymine-50-O-aliphatic acid esters as well as amino acid esters
-(2R,4R)-dioxolane-thymine (DOT). The compounds were eval-
were synthesized as prodrugs of (ꢀ)-b-
D
uated for anti-HIV activity against HIV-1LAI in human peripheral blood mononuclear (PBM) cells as
well as for their cytotoxicity in PBM, CEM and Vero cells. Improved anti-HIV potency in vitro was
observed for the compound 2–4 (50-O-aliphatic acid esters) without increase in cytotoxicity in com-
parison to the parent drug. Chemical and enzymatic hydrolysis of the prodrugs was also studied, in
which the prodrugs exhibited good chemical stability with the half-lives from 3 h to 54 h at pH 2.0
and 7.4 phosphate buffer. However, the prodrugs were relatively labile to porcine esterase with the
half-lives from 12.3 to 48.0 min.
Keywords:
Anti-HIV activity
Nucleoside prodrugs
Dioxolane thymine (DOT)
50-O-aliphatic acid esters
50-O-amino acid esters
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
dioxolane-thymine (DOT).18,19 DOT is a pyrimidine 1,3-dioxo-
lane nucleoside, exhibiting interesting activity against drug
Nucleoside analogues continue to play important role as
antiviral agents,1 however, the therapeutic potential is often
hampered by their poor biopharmaceutical properties.2 These
properties may be due to their high polarity as well as low bio-
availability. Thus, various prodrugs of nucleosides have been
synthesized by chemical modification to improve their physico-
chemical and/or pharmacokinetic properties.3,4 Among the
nucleoside prodrugs, 50-O-esters5,6 and amino acid esters7,8 are
common to improve their physicochemical properties.3,9 The
rationale for this approach is to alter the lipophilicity, perme-
ability, stability and tissue specificity. Therefore, prodrugs could
potentially enhance the overall pharmaceutical properties over
the parent drugs which produce the active drugs to exert their
pharmacological action in vivo. Various esters, carbamates
and amide prodrugs can be hydrolyzed by esterases and
amidases, which are ubiquitous in vivo with a broad substrate
specificity.10
resistant mutants of HIV-1.18,19 Another dioxolane nucleoside,
amdoxovir is undergoing Phase II clinical studies as an anti-
HIV agent.20 The significant antiviral activity of the parent
nucleoside
of
DAPD,
(ꢀ)-b-
D-(2R,4R)–dioxolane-guanine
(DXG)21,22 along with its prodrug, amdoxovir (DAPD)17 and 2-
aminopurine dioxolane (APD)23 (another DXG prodrug)
prompted us to further enhance the antiviral potency of
DOT24,18 by a prodrug strategy, as it is a moderately potent
anti-HIV agent by itself, but with unique resistant profiles.18,19
Previously, we have reported the synthesis and anti-HIV activ-
ity of dendrimer, phosphoramidate and phosphate prodrugs of
DOT.25,26
Aliphatic and amino acids have been extensively used for
prodrugs due to their well-established chemistry, structural
diversities as well as lack of toxicity concerns.27,28 Lipophilic
aliphatic esters are known to preferentially be taken up by
mononuclear phagocyte system (MPS) after intravenous injec-
As a part of our continuing efforts to discover novel and
effective antiviral agents, we have reported the synthesis and
antiviral activity of various 1,3-oxathioalane and 1,3-dioxolane
nucleosides.11–16 From these studies, we discovered several
tion. Apart from CD4+ T-lymphocytes, cells of MPS play
a
decisive role in the pathogenesis of AIDS and are considered
as a major reservoir for HIV.29 The use of amino acid for a
prodrug strategy also provides advantages in the cellular
transport system.30 In view of these information, DOT-pro-
drugs with 50-O-aliphatic and amino acid esters were synthe-
sized and evaluated for their anti-HIV activity in vitro in
efforts to improve the pharmaceutical and pharmacological
properties of DOT.
clinical candidates, which include (ꢀ)-b-
D-(2R,4R)-2,6-diaminop-
urine-dioxolane (amdoxovir or DAPD)17 and (ꢀ)-b-
D-(2R,4R)-
* Corresponding author. Tel.: +1 706 542 5379; fax: +1 706 542 5381.
E-mail addresses: dchu@rx.uga.edu, david.dr.chu@gmail.com (C.K. Chu).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.078

Y. Liang et al. / Bioorg. Med. Chem. 17 (2009) 1404–1409
1405
group (6) showed enhanced cytotoxicity to Vero cells
(IC50 = 18.1 M). Compound 9 and compound 10 have shown
slightly reduced antiviral activity. Among the synthesized amino
acid esters, compounds 13–17 exhibited similar anti-HIV potency
as the parent drug without any cytotoxicity to PBM, CEM and Vero
cells.
2. Results and discussion
2.1. Chemistry
l
The chemical structure of synthesized aliphatic acid ester (1–
10) and amino acid ester prodrugs (11–22) of DOT are shown in
Figure 1. Eight aliphatic acids (acetic, valeric, octanoic, lauric, pal-
mitic, pivalic, cyclopropanecarboxylic and cyclohexanecarboxylic
acid) were chosen and were treated with DOT at room temperature
in the presence of DMAP and 1,3-diisopropylcarbodiimide (DIC) to
yield the prodrugs 1–8 (Scheme 1). Compound 9 was synthesized
by the reaction of chloromethyl pivalate with DOT in the presence
of NaH. The pivaloyl moiety is linked to DOT by an ether bond. The
DOT was reacted with Bis-dPEG8 acids in the presence of DMAP
and DIC to provide compound 10 as a dinucleoside. For the synthe-
sis of amino acid ester prodrugs of DOT (Scheme 1, compounds 11–
2.3. Hydrolysis of prodrugs
An ideal prodrug should exhibit a good chemical and enzymatic
stability before entering the cell, while requires enzymatic conver-
sion to the parent drug after being transported across the biological
membrane.31,32 In order to access the chemical and enzymatic sta-
bility, we have conducted chemical and enzymatic stability studies
of the synthesized prodrugs and the results are shown in Table 2.
The rate of chemical hydrolysis of the synthesized prodrug in phos-
phate buffer at pH 2.0 and pH 7.4 (Table 2) were obtained from the
linear regression of pseudo first order plot using concentration vs.
time. The most of the prodrugs showed a good chemical stability,
and it reveals that the structure of the prodrugs influences the rate
of hydrolysis (Table 2). In general, prodrugs with aliphatic esters
were hydrolyzed slower than the prodrugs with amino acid esters.
For aliphatic esters, the rates of chemical hydrolysis were reduced
with an increased length of aliphatic chains. Bis-PEG8 ester of DOT
(10) showed an increased rate of chemical hydrolysis in compari-
son to the prodrugs with aliphatic acids. Among amino acid pro-
drugs, the rate of chemical hydrolysis varied according to the
structural features. The stereochemistry of amino acids appeared
22), aromatic amino acids (
acids ( -Val, -Val, -Ala,
acid) and the polar amino acids (
L
-Phe,
D
-Phe and
-Ile, Gly and 2-aminoisobutyric
-Pro and -Gln) were selected.
L-Trp), aliphatic amino
L
D
L
L
-Leu,
L
L
L
In each case, Boc-protected amino acids were used to react with
DOT in the presence of DMAP and DIC to provide the Boc-protected
intermediates. The intermediates were purified by silica gel flash
chromatography. Further, deprotection of the Boc group was
achieved by treating with 50% TFA in DCM for 1 hour at 0 °C. After
removal of the excess TFA, the resulting residue was dissolved in
anhydrous methanol and treated with anhydrous HCl/ether to give
the HCl salt of amino acid esters. Subsequently, the pure product
was obtained by crystallization from methanol. The synthesized
DOT-prodrugs (1–22) were characterized by mass spectrometry
(ESI–MS), 1H NMR and elemental analysis.
to affect the chemical stability. The
higher stability than the -amino acid prodrugs (14 vs 15 and 16 vs
17). In general, -Gln, -Pro and -Trp prodrugs (20–22) were
hydrolyzed faster than the other amino acid prodrugs. The prodrug
with -Gln (20) showed the shortest half-life (190 min at pH 2.0
and 240 min at pH 7.4). The -Val prodrug (16) showed the longest
L-amino acid prodrugs showed
D
L
L
L
2.2. Anti-HIV activity and cytotoxicity
L
L
The anti-HIV activity of the prodrugs were evaluated against
HIV-1LAI in PBM cells and the cytotoxicity was evaluated in PBM,
CEM and Vero cells as shown in Table 1. Most of the synthesized
prodrugs exhibited potent anti-HIV activity against HIV-1LAI with-
out increased cytotoxicity in comparison to the parent drug, DOT.
Among the synthesized prodrugs, valeric, octanoic and lauric acid
ester prodrugs (2–4) exhibited improved antiviral activity with
half-life (960 min at pH 2.0 and 2,200 min at pH 7.4).
The rates of enzymatic hydrolysis were also determined and are
also listed in the Table 2. As expected, the data show that the pro-
drugs were less stable (t1/2: from 13 to 48 min) in the presence of
porcine esterase than in phosphate buffer. For aliphatic acid pro-
drugs, the rates of hydrolysis were decreased with the increase
of the length of the aliphatic chain, in which the palmitic acid pro-
drug hydrolyzed with a t1/2 of 48 min while the prodrug with an
an EC50 < 0.6
lM
(EC90 < 4.3 lM) in comparison to DOT
(EC50 = 0.68 M, EC90 = 4.0
l
l
M). The prodrug with a t-butyl acid
O
O
H3C
O
NH
H3C
NH
O
N
O
O
O
O
O
N
N
O
O
6
O
R C O
O
O
HN
O
O
CH3
O
10
O
1-8
2. R = C4H9 3. R = C7H15
1. R = CH3
4. R = C11H23 5. R = C15H31 6. R = (CH3)3C
7. R =Cyclopropyl 8. R = Cyclohexyl
O
H3C
NH
NH2.HCl
N
O
COO
R
O
O
O
H3C
11-22
NH
O
11. Glycine
13. L-Alanine
15. D-Phenyl alanine 16. L-Valine
17. D-Valine
19. L-Isoleucine
21. L-Proline
12. 2-Amino isobutyric acid
14. L-Phenyl alanine
N
O
(H3C)3C C O H2C O
O
O
18. L-Leucine
20. L-Glutamine
22. L-Tryptophan
9
Figure 1. Structures of 50-O-aliphatic and amino acid esters of (ꢀ)-b-
D
-(2R,4R)-dioxolane-thymine (DOT).

1406
Y. Liang et al. / Bioorg. Med. Chem. 17 (2009) 1404–1409
O
O
N
CH3
CH3
NH2.HCl
OOC
NHBoc
DOT
NHBoc
COOH
HN
i) CF3COOH
ii) HCl, 0 °C
HN
O
O
N
R
OOC
O
O
R
R
DMAP,
DIPC,
6h, r.t.
O
O
Scheme 1. Synthesis of 50-O-amino acid esters of DOT.
Table 1
acetic acid has a t1/2 of 16.3 min. The prodrugs with amino acids
showed shorter half-lives (t1/2 12.4 to 19.4 min) than the aliphatic
acid prodrugs (t1/2 from 16.3 to 48 min). The Gly prodrug 11
Anti-HIV activity and cytotoxicity of 50-O-aliphatic and amino acid esters of
(ꢀ)-b-D-(2R,4R)-dioxolane-thymine (DOT).
Compounds
HIV-1LAI
EC50
(
l
M)a
Cytotoxicity (IC50
CEM
,
l
M)b
showed the shortest half-lives (12.4 min) while the D-Val prodrug
17 showed the longest half-lives (19.4 min). The results suggest
that the synthesized prodrugs are good substrates for the esterase
and can efficiently generate the parent drug. Although the struc-
tural features influenced the chemical and enzymatic stability of
two types of DOT-prodrugs, no clear correlation between the
anti-HIV activity and stability was observed in these series.
In conclusion, some of the synthesized aliphatic and amino acid
prodrugs of DOT have shown enhanced anti-HIV activity in vitro
without increase in cytotoxicity. In comparison to the previously
reported phosphoramidate, phosphate and dendrimer prodrugs,
the newly synthesized prodrugs have similar enhanced anti-HIV
activity, however, the overall cytotoxicity profile was more favor-
able. The synthesized prodrugs possess good chemical stability in
the buffer systems, and they are efficient substrates for porcine
esterase. The present study suggests that a certain class of DOT-
prodrugs may improve the overall biological profile. The detailed
pharmacokinetic and further biological evaluations may be war-
ranted to assess the full potential of these prodrugs.
EC90
PBM
Vero
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2.9
0.4
0.6
0.4
23.5
1.3
4.7
1.3
1.4
1.5
2.5
2.2
0.98
0.88
1.0
0.56
0.98
3.3
12.3
2.5
4.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
99.4
>100
49.0
>100
>100
92.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.2
>100
14.5
15.1
3.9
9.7
10.2
8.4
7.6
8.3
4.4
5.2
4.9
6.5
12.4
9.9
6.3
18.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.0
0.89
1.4
7.3
8.0
1.7
3. Materials and methods
3.1. Chemistry
DOT
AZT
0.68
0.007
4.0
0.04
>100
>100
>100
14.3
>100
50.6
a
Anti-HIV activity against HIV-1LAI in PBM cells.
Assay in PBM, CEM and Vero cells.
b
DOT was synthesized in our laboratory as described previ-
ously.11 Bis-dPEG8 acid was purchased from Quanta BioDesign,
Ltd. (Powell, Ohio). Porcine esterase and other chemicals were ob-
tained from Sigma Chemical Company (St. Louis, MO) and Ad-
vanced Chem. Tech. (Louisville, KY). Melting points were
determined on a MELl-TEMPII apparatus and were uncorrected.
TLC was performed on Uniplate (silica gel) purchased from Anal-
tech 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 column chromatography.
NMR spectra were recorded on Varian Inova 500 spectrometer at
500 MHz with software VNMRC6-1 with Me4Si as the internal
standard. Chemical 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 spectrophotometer. Optical rota-
tions were measured on a Jasco DIP-370 digital polarimeter. Mass
spectra were measured on a Micromass Autospec mass spectrom-
eter. Elemental analyses were performed by Atlantic Microlab, Inc.
(Norcross, GA). HPLC were performed using Waters 2996 instru-
ment manufactured by Waters Corporation with an analytical
symmetry C18 column (4.6 ꢁ 75 mm) and acetonitrile/water mix-
ture as eluent.
Table 2
Half-lives (t1/2, min) of the chemical and enzymatic hydrolysis of 50-O-aliphatic and
amino acid esters of (ꢀ)-b-D-(2R,4R)-dioxolane-thymine (DOT).
Compound
Chemical hydrolysisa
Enzymatic hydrolysisb
pH 2.0
pH 7.4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1580 320c
1600 320
1800 380
NDd
2650 560
3180 620
3250 670
ND
16.3 1.1
18.5 0.4
29.0 0.3
41.0 0.4
48.0 1.6
17.0 0.7
17.3 0.3
19.3 0.3
17.6 0.5
18.3 0.5
12.4 0.4
13.4 0.5
13.9 0.4
14.6 0.6
15.6 0.6
18.9 0.7
19.4 0.8
17.9 0.9
17.5 1.2
13.4 0.5
13.6 0.4
17.8 0.3
ND
ND
1300 280
1200 270
1850 320
ND
2780 470
2800 480
3200 580
ND
380 40
520 90
920 90
870 58
550 65
430 50
960 80
730 48
870 55
800 60
190 40
230 40
320 35
520 65
1320 180
1870 280
1600 234
1070 28
940 25
2200 450
1800 270
2150 310
2040 290
240 60
380 50
420 40
3.2. General procedure for the synthesis of 50-O-aliphatic acid
ester of DOT (1–8)
A solution of DOT (1 mmol), aliphatic acid (2 mmol), DMAP
(2 mmol) and 1,3-diisopropylcarbodiimide (DIC) (2 mmol) in
dichloromethane (10 mL) was stirred at room temperature for 6
to 15 h. Dichloromethane (10 mL) was added and the reaction mix-
a
At pH 2.0 and pH at 7.4 phosphate buffer at 37 0.5 °C.
At pH 8.0 buffer solution of porcine esterase at 37 0.5 °C.
Half-lives expressed as mean SEM.
b
c
d
Not determined due to solubility.

Y. Liang et al. / Bioorg. Med. Chem. 17 (2009) 1404–1409
1407
ture was washed with aqueous sodium bicarbonate solution
(2 ꢁ 10 mL) followed by water (2 ꢁ 10 mL). The organic layer was
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The residue was purified using flash silica gel chromatog-
raphy to provide the desired product. The pure product was ob-
tained by crystallizing from the mixture of ethyl acetate and
hexane (1:1) solution.
H50), 4.34–4.45 (m, 2H, H20), 5.18 (s, 1H, H40), 6.22 (m, 1H, H10),
7.35 (s, 1H, H6), 8.95 (s, 1H, H3). MS: m/z 339 (M+1)+ Anal. calcd
for C16H22N2O6: C 56.80; H 6.55; N 8.28; Found: C 57.01; H 6.58;
N 8.26.
3.2.9. Dioxolane thymine-50-O-pivaloyloxymethyl ether (9)
A solution of DOT (1 mmol), chloromethyl pivalate (1.5 mmol),
NaH (1.5 mmol) in DMF (10 mL) was stirred at room temperature
for 5 h. Reaction mixture was cooled to 0 °C and methanol (2 mL)
was added. After usual work-up, the residue was purified using
flash silica gel chromatography to afford the desired product. The
compound was obtained by crystallizing with the mixture of ethyl
acetate and hexane (1:1) solution. Yield: 86%. Mp: 115.3 °C. 1H
NMR (CDCl3): d 0.98 (s, 9H, CH3), 1.92 (s, 3H, 5-Me), 4.12–4.22
(m, 2H, H50), 4.41 (s, 2H, OCH2O), 4.36–4.45 (m, 2H, H20), 5.17 (s,
1H, H40), 6.20 (m, 1H, H10), 7.35 (s, 1H, H6), 8.44 (s, 1H, H3). MS:
m/z 343 (M+1)+ Anal. calcd for C15H22N2O7: C 52.63; H 6.48; N
8.18; Found: C 52.44; H 6.41; N 8.10.
3.2.1. Dioxolane thymine-50-O-acetic ester (1)
Yield: 98% Mp: 190.5 °C. 1H NMR (CDCl3): d 1.91 (s, 3H, 5-Me),
2.05 (s, 3H, CH3COO–), 4.16–4.22 (m, 2H, H50), 4.34–4.43 (m, 2H,
H20), 5.17 (s, 1H, H40), 6.21 (m, 1H, H10), 7.31 (s, 1H, H6), 8.44 (s,
1H, H3). MS: m/z 271 (M+1)+ Anal. calcd for C11H14N2O6: C 48.89;
H 5.22; N 10.37; Found: C 48.80; H 5.26; N 10.17.
3.2.2. Dioxolane thymine-50-O-valeric ester (2)
Yield: 95% Mp: 162.9 °C. 1H NMR (CDCl3): d 0.92 (t, 3H, CH3–),
1.25–1.52 (m, 4H, –CH2–), 1.90 (s, 3H, 5-Me), 2.35 (m, 2H, CH2–
COO), 4.18–4.20 (m, 2H, H50), 4.30-4.40 (m, 2H, H20), 5.15 (s, 1H,
H40), 6.20 (m, 1H, H10), 7.28 (s, 1H, H6), 8.40 (s, 1H, H3). MS: m/z
313 (M+1)+ Anal. calcd for C14H20N2O6: C 53.84; H 6.45; N 8.97;
Found: C 53.86; H 6.52; N 8.89.
3.2.10. Dioxolane thymine-50-O-bis-PEG8 ester (10)
The same procedure given for the compounds 1–8 was followed
with bis-dPEG8 acid/DOT (1/2, mol/mol). Yield: 95% (for bis-dPEG8
acid). Mp: 85.2 °C. 1H NMR (CDCl3): d 1.92 (s, 3H, 5-Me), 2.24 (m,
4H, CH2), 3.89 (m, 28H, CH2O), 4.12–4.20 (m, 4H, H50), 4.31–4.40
(m, 4H, H20), 5.15 (s, 2H, H40), 6.31 (m, 2H, H10), 7.28 (s, 2H, H6),
8.38 (s, 2H, H3). MS: m/z 848 (M+1)+Anal. calcd for C36H54N4O19
0.3%H2O: C 50.74; H 6.46; N 6.57; Found: C 50.38; H 6.46; N 6.56.
3.2.3. Dioxolane thymine-50-O-octanotic ester (3)
Yield: 94% Mp: 158.0 °C. 1H NMR (CDCl3): d 0.92 (t, 3H, CH3),
1.30–1.52 (m, 10H, CH2), 1.91 (s, 3H, 5-Me), 2.33 (m, 2H, CH2COO),
4.16–4.24 (m, 2H, H50), 4.34–4.44 (m, 2H, H20), 5.16 (s, 1H, H40),
6.24 (m, 1H, H10), 7.34 (s, 1H, H6), 8.46 (s, 1H, H3). MS: m/z 355
(M+1)+ Anal. calcd for C17H26N2O6: C 57.61; H 7.39; N 7.90; Found:
C 57.63; H 7.47; N 7.84.
3.2.11. General procedure for the syntheses of 50-O-amino acid
ester of DOT (11–22)
A
solution of DOT (1 mmol), Boc-protected amino acid
3.2.4. Dioxolane thymine-50-O-dodecanoic ester (4)
(2.5 mmol), DMAP (2.5 mmol) and 1,3-diisopropylcarbodiimide
(DIC) (2.5 mmol) in dichloromethane (10 mL) was stirred at room
temperature from 6 to 16 h. Dichloromethane (10 mL) was added
and the mixture was washed with aqueous sodium bicarbonate
solution (2 ꢁ 10 mL) and water (2 ꢁ 10 mL). The organic layer
was dried over anhydrous Na2SO4, and concentrated under re-
duced pressure. The residue was purified by flash silica gel chro-
matography to provide dioxolane thymine 50-Boc-protected
amino acid esters. The 50-Boc-protected amino acid prodrug
(100 mg) was dissolved in dichloromethane (10 mL) and the solu-
tion was cooled to 0 °C. Trifluoroacetic acid (5 mL) was added to
the solution with vigorous stirring. The mixture was warmed to
room temperature and then stirred for 1 h. The excess trifluoroace-
tic acid was removed under reduced pressure. The solid was dis-
solved in methanol (10 mL) and the solution was cooled to 0 °C.
A mixture of 1.0 M HCl/ether (5 mL) was added with a vigorous
stirring. After 20 min, the excess HCl/ether was removed and the
solution was concentrated under reduced pressure. The pure
DOT-50-amino acid ester hydrochloride was obtained as an amor-
phous powder from methanol.
Yield: 94% Mp: 146.5 °C. 1H NMR (CDCl3): d 0.92 (t, 3H, CH3),
1.26 (m, 16H, CH2), 1.53 (m, 2H, CH2–CH2–COO), 1.91 (s, 3H, 5-
Me), 2.33 (m, 2H, CH2COO–), 4.14–4.21(m, 2H, H50), 4.37–4.45
(m, 2H, H20), 5.21 (s, 1H, H40), 6.23 (m, 1H, H10), 7.36 (s, 1H, H6),
8.78(s, 1H, H3). MS: m/z 412 (M+1)+ Anal. calcd for C20H34N2O6:
C 61.44; H 8.35; N 6.82; Found: C 61.17; H 8.36; N 6.58.
3.2.5. Dioxolane thymine-50-O-palmitic ester (5)
Yield: 93% Mp: 142.2 °C. 1H NMR (CDCl3): d 0.90 (t, 3H, CH3),
1.28–1.34 (m, 24H, CH2), 1.54 (m, 2H, CH2–CH2–COO), 1.91 (s,
3H, 5-Me), 2.29 (m, 2H, CH2COO), 4.18–4.25 (m, 2H, H50), 4.33–
4.46 (m, 2H, H20), 5.19 (s, 1H, H40), 6.20 (m, 1H, H10), 7.35 (s, 1H,
H6), 8.48 (s, 1H, H3). MS: m/z 468 (M+1)+ Anal. calcd for
C25H42N2O6: C 64.35; H 9.07; N 6.00; Found: C 64.44; H 9.20; N
6.05.
3.2.6. Dioxolane thymine-50-O-pivalic ester (6)
Yield: 91% Mp: 172.3 °C. 1H NMR (CDCl3): d 0.98 (s, 9H, (CH3)3–),
1.92 (s, 3H, 5-Me), 4.16–4.21 (m, 2H, H50), 4.33–4.41(m, 2H, H20),
5.17 (s, 1H, H40), 6.23 (m, 1H, H10), 7.32 (s, 1H, H6), 8.79 (s, 1H,
H3). MS: m/z 313 (M+1)+ Anal. calcd for C14H20N2O6: C 53.84; H
6.45; N 8.97; Found: C 53.94; H 6.47; N 8.87.
3.2.12. Dioxolane thymine-50-O-glycyl ester hydrochloride (11)
Yield: 91% 1H NMR (CDCl3): d 1.95 (s, 3H, 5-Me), 3.82–3.96 (m,
2H, CH2N), 4.21–4.42 (m, 2H, H50 and H20), 5.17 (s, 1H, H40), 6.38
(m, 1H, H10), 7.55 (s, 1H, H6). MS: m/z 286 (M+1)+ Anal. calcd for
C11H16ClN3O6 0.1% H2O: C 41.43; H 5.12; N 12.71; Found: C
41.30; H 5.11; N 12.52.
3.2.7. Dioxolane thymine-50-O-cyclopropanecarboxylic ester (7)
Yield: 87% Mp: 171.5 °C. 1H NMR (CDCl3): d 0.94 (m, 2H, CH2),
1.07 (m, 2H, CH2), 1.66 (m, 1H, –CHCOO), 1.91 (s, 3H, 5-Me),
4.17–4.24 (m, 2H, H50), 4.31–4.41 (m, 2H, H20), 5.19 (s, 1H, H40),
6.24 (m, 1H, H10), 7.33 (s, 1H, H6), 8.93(s, 1H, H3). MS: m/z 297
(M+1)+ Anal. calcd for C13H16N2O6 0.2%H2O: C 52.82; H 5.65; N
8.92; Found: C 53.21; H 5.54; N 9.28.
3.2.13. Dioxolane thymine-50-O-isobutyric ester hydrochloride
(12)
Yield: 94% 1H NMR (MeOH-d4): d 1.61 (s, 3H, CH3), 1.64 (s, 3H,
CH3), 1.91 (s, 3H, 5-Me), 4.21–4.57 (m, 4H, H20and H50), 5.26 (s, 1H,
H40), 6.39 (m, 1H, H10), 7.48 (s, 1H, H6). MS: m/z 314 (M+1)+ Anal.
calcd for C13H20ClN3O6 0.2 H2O%: C 44.19; H 5.82; N 11.89; Found:
C 43.90; H 5.91; N 11.75.
3.2.8. Dioxolane thymine-50-O-cyclohexanecarboxylic ester (8)
Yield: 90%. Mp: 176.5 °C. 1H NMR (CDCl3): d 1.20–1.88 (m, 10H,
CH2), 2.34 (m, 1H, CHCOO), 1.89 (s, 3H, 5-Me), 4.15–4.20 (m, 2H,

1408
Y. Liang et al. / Bioorg. Med. Chem. 17 (2009) 1404–1409
3.2.14. Dioxolane thymine-50-O-
(13)
L
-alanyl ester hydrochloride
3.2.22. Dioxolane thymine-50-O-
(21)
L-prolyl ester hydrochloride
Yield: 92% 1H NMR (MeOH-d4): d 1.58 (d, 3H, CH3), 1.92 (s, 3H,
5-Me), 4.12 (m, 1H, CHN), 4.21–4.61 (m, 4H, H20 and H50), 5.23 (s,
1H, H40), 6.39 (m, 1H, H10), 7.55 (s, 1H, H6). MS: m/z 300
(M+1)+Anal. calcd for C12H18ClN3O6 0.3H2O%: C 42.25; H 5.50; N
12.32; Found: C 42.50; H 5.26; N 11.97.
Yield: 90% 1H NMR (MeOH-d4): d 1.97 (s, 3H, 5-Me), 2.24 and
2.45 (m, 4H, CH2–CH2), 3.42 (m, 2H, CH2–N), 4.22 (m, 1H, CHN),
4.37–4.61 (m, 4H, H50 and H20), 5.27 (s, 1H, H40), 6.37 (m, 1H,
H10), 7.52 (s, 1H, H6). MS: m/z 355 (M+1)+ Anal. calcd for
C15H22ClN4O6 0.4H2O%: C 45.57; H 5.68; N 11.39; Found: C
45.28; H 5.95; N 10.99.
3.2.15. Dioxolane thymine-50-O-
L-phenylalanyl ester
hydrochloride (14)
3.2.23. Dioxolane thymine-50-O-
L-tryptophanyl ester hydro-
chloride (22)
Yield: 98% 1H NMR (CDCl3): d 1.92 (s, 3H, 5-Me), 3.01 (m, 2H,
PhCH2), 3.74 (m, 1H, CHN), 4.17–4.45 (m, 4H, H50 and H20), 5.17
(s, 1H, H40), 6.37 (m, 1H, H10), 7.18–7.32 (m, 5H, Ph), 7.51 (s, 1H,
H6). MS: m/z 376 (M+1)+ Anal. calcd for C18H22ClN3O6 1.1 H2O%:
C 50.02; H 5.65; N 9.73; Found: C 49.75; H 5.23; N 9.58.
Yield: 87% 1H NMR (CD3OD): d 1.98 (s, 3H, 5-Me), 3.42–3.50 (m,
2H, CH2), 4.17 (m, 1H, CHN), 4.27–4.54 (m, 4H, H50 and H20), 5.17
(s, 1H, H40), 6.37 (m, 1H, H10), 7.02–7.60 (m, 6H, Try H). MS: m/z
415 (M+1)+ Anal. calcd for C20H24Cl2N4O6: C 49.29; H 4.96; N
11.50; Found: C 49.67; H 5.33; N 11.42.
3.2.16. Dioxolane thymine-50-O-
D-phenylalanyl ester
hydrochloride (15)
3.3. Virology
Yield: 97% 1H NMR (CDCl3): d 1.91 (s, 3H, 5-Me), 3.08 (m, 2H,
PhCH2), 3.72 (m, 1H, CHN), 4.15–4.47 (m, 4H, H50 and H20), 5.16
(s, 1H, H40), 6.38 (m, 1H, H10), 7.17–7.34 (m, 5H, Ph), 7.52 (s, 1H,
H6). MS: m/z 376 (M+1)+ Anal. calcd for C18H22ClN3O6 1.0 H2O%:
C 50.09; H 5.65; N 9.73; Found: C 49.97; H 5.54; N 9.42.
3.3.1. Antiviral assays
The procedures for the antiviral assays in human PBM cells have
been published previously.33,34 Briefly, PBM cells were isolated by
Ficoll–Hypaque discontinuous gradient centrifugation of whole
blood samples (obtained from Atlanta Red Cross) from healthy
seronegative donors. Cells were stimulated with phytohemaggluti-
3.2.17. Dioxolane thymine-50-O-
L-valyl ester hydrochloride (16)
Yield: 96% 1H NMR (CDCl3): d 1.10–1.12 (d, 6H, CH3), 1.91 (s, 3H,
5-Me), 2.61 (m, 1H, CH), 3.96 (m, 1H, CHN), 4.31–4.61 (m, 4H, H50
and H20), 5.32 (s, 1H, H40), 6.36(m, 1H, H10), 7.52 (s, 1H, H6). MS: m/
z 328 (M+1)+ Anal. calcd for C14H22ClN3O6: C 46.22; H 6.10; N
11.55; Found: C 46.37; H 6.18; N 11.35.
nin A (3 lg/mL; Sigma–Aldrich, St. Louis, MO) for 2–3 days prior to
use. HIV-1LAI obtained from the Centers for Disease Control and
Prevention (Atlanta, GA) was used as the standard reference virus
for the antiviral assays. Infections were done in bulk for 1 h, either
with 100 TCID50/1 ꢁ 107 cells for a flask (T25) assay or with 200
TCID50/2 ꢁ 106 cells/well for 24 well plate assay. Previous studies
indicated that this was the optimum virus concentration in order
to obtain good replication on day 5 when the virus is harvested
from the supernatant. Cells were added to a flask or plate contain-
ing a 10-fold serial dilution of the test compound. Assay medium
was RPMI-1640 supplemented with heat inactivated 16% fetal bo-
3.2.18. Dioxolane thymine-50-O-
D-valyl ester hydrochloride (17)
Yield: 93% 1H NMR (CDCl3): d 1.19 (d, 3H, CH3), 1.21 (d, 3H,
CH3), 1.91 (s, 3H, 5-Me), 2.33 (m, 1H, CH), 3.98 (m, 1H, CHN),
4.2–4.55 (m, 4H, H50and H20), 5.17 (s, 1H, H40), 6.40 (m, 1H, H10),
7.51 (s, 1H, H6). MS: m/z 328 (M+1)+ Anal. calcd for C14H22ClN3O6
0.7H2O%: C 44.46; H 6.29; N 11.11; Found: C 44.37; H 6.30; N
11.09.
vine serum, 1.6 mM L-glutamine, 80 IU/mL penicillin, 80 lg/mL
streptomycin, 0.0008% DEAE-Dextran, 0.045% sodium bicarbonate,
and 26 IU/mL recombinant interleukin-2 (Chiron Corp., Emeryville,
CA). AZT was used as the positive control for all the virologic as-
says. Uninfected PBM cells were grown in parallel at equivalent cell
concentrations as a control. The cell cultures were maintained in a
humidified 5% CO2-air at 37 °C for 5 day, and supernatants were
collected for reverse transcriptase activity. One mL of each super-
natant was centrifuged at 9740g for 2 h to pellet the virus. The pel-
3.2.19. Dioxolane thymine-50-O-
L-leucinyl ester hydrochloride
(18)
Yield: 96% 1H NMR (CDCl3): d 1.01–1.04 (dd, 6H, CH3), 1.73 (m,
1H, CH), 1.82 (m, 2H, CH2), 1.93 (s, 3H, 5-Me), 4.08 (m, 1H, CHN),
4.22–4.57 (m, 4H, H50 and H20), 5.27 (s, 1H, H40), 6.37 (m, 1H,
H10), 7.48 (s, 1H, H6). MS: m/z 342 (M+1)+ Anal. calcd for
C15H24ClN3O6 0.5H2O%: C 46.57; H 6.51; N 10.86; Found: C
46.49; H 6.19; N 10.52.
let was solubilized with vortexing in 100 lL of virus solubilization
buffer (0.5% Triton X-100, 0.8 M NaCl, 0.5 mM phenylmethylsul-
fonyl chloride, 20% glycerol, and 0.05 M Tris, pH 7.8). Each sample
3.2.20. Dioxolane thymine-50-O-
L-isoleucinyl ester
hydrochloride (19)
(10
7.8, 0.012 M MgCl2, 0.006 M dithiothreitol, 0.006 mg/mL poly (rA)n
oligo (dT)12–18, 96 g/mL dATP, and 1
M of 0.08 mCi/mL 3H-thy-
midine-50-triphosphate) (Perkin Elmer, Boston, MA) and incubated
at 37 °C for 2 h. The reaction was stopped by the addition of 100
lL) was added to 75 lL of RT reaction mixture (0.06 M Tris, pH
Yield: 97% 1H NMR (CDCl3): d 1.01 (t, 3H, CH3), 1.32 (d, 3H, CH3),
1.70 (m, 2H, CH2), 1.94 (s, 3H, 5-Me), 2.05 (m, 1H, CH), 4.08 (m, 1H,
CHN), 4.22–4.61 (m, 4H, H50 and H20), 5.27 (s, 1H, H40), 6.37 (m, 1H,
H10), 7.52 (s, 1H, H6). MS: m/z 342 (M+1)+ Anal. calcd for
C14H21ClN4O7: C 47.68; H 6.40; N 11.12; Found: C 47.69; H 6.46;
N 11.02.
l
l
lL
of 10% trichloroacetic acid containing 0.05% sodium pyrophos-
phate. The acid-insoluble product was harvested onto filter paper
using a Packard Harvester (Meriden, CT), and RT activity was read
on a Packard Direct Beta Counter (Meriden, CT). RT results were ex-
pressed in counts per minute (CPM) per milliliter. The antiviral 50%
effective concentration (EC50) and 90% effective concentration
(EC90) was determined from the concentration–response curve
using the median effect method.35
3.2.21. Dioxolane thymine-50-O-
L-Glutminyl ester
hydrochloride (20)
Yield: 91% 1H NMR (MeOH-d4): d 1.93 (s, 3H, 5-Me), 2.17 (m 2H,
CH2), 2.34 (m, 2H, CH2), 4.18 (m, 1H, CHN), 4.22–4.55 (m, 4H, H50
and H20), 5.19 (s, 1H, H40), 6.38 (m, 1H, H10), 7.48 (s, 1H, H6),
9.80 (s, 1H, 3-NH). MS: m/z 357 (M+1)+ Anal. calcd for
C14H21ClN4O7 0.2H2O%: C 42.42; H 5.44; N 14.13; Found: C
42.15; H 5.34; N 13.94.
3.3.2. Cytotoxicity assays
The compounds were evaluated for their potential toxic effects
on uninfected PHA-stimulated human PBM cell, CEM (T-lympho-

Y. Liang et al. / Bioorg. Med. Chem. 17 (2009) 1404–1409
1409
2. Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Jarvinen, T.;
Savolainen, J. Nat. Rev. Drug Discov. 2008, 7, 255.
blastoid cell line obtained from American Type Culture Collection,
Rockville, MD) and Vero (African green monkey kidney) cells. The
log phase Vero, CEM and PHA-stimulated human PBM cells were
seeded at a density of 5 ꢁ 103, 2.5 ꢁ 103, and 5 ꢁ 104 cells/well,
respectively. All of the cells were plated in 96-well cell culture
plates containing 10-fold serial dilutions of the test drug. The cul-
tures 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 tetrazolium dye solution (cell titer 96, Promega,
Madison, WI) was added to each well and incubated overnight. The
reaction was stopped with stop solubilization solution (Promega,
Madison, WI). The plates were incubated for 5 h to ensure that
the formazan crystals were dissolved. The plates were read at
570 nm using an ELISA plate reader (Bio-Tek Instruments, Inc.,
Winooski, VT, Model EL 312e). The 50% inhibition concentration
(IC50) was determined from the concentration–response curve
using the median effect method.35,36
3. Li, F.; Maag, H.; Alfredson, T. J. Pharm. Sci. 2008, 97, 1109.
4. Heimbach, T.; Fleisher, D.; Kaddoumi, A. In Prodrugs, 2007; pp. 157–215.
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Roey, P. V.; Schinazi, R. F. Tetrahedron Lett. 1991, 32, 3791.
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Shanmuganathan, K.; Alves, A. J.; McMillan, A.; Chu, C. K.; Mathis, R. J. Med.
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13. Jeong, L. S.; Schinazi, R. F.; Beach, J. W.; Kim, H. O.; Shanmuganathan, K.;
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3.4. Hydrolysis of prodrugs
15. Kim, H. O.; Schinazi, R. F.; Shanmuganathan, K.; Jeong, L. S.; Beach, J. W.;
Nampalli, S.; Cannon, D. L.; Chu, C. K. J. Med. Chem. 1993, 36, 519.
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3.4.1. Kinetics in aqueous buffer
Hydrolysis of prodrugs was studied at pH 2.0 and pH 7.4. An ali-
quot (1 mL) of 1 mM solution of prodrugs in DMSO was diluted to
10 mL using phosphate buffer (10 mM). A constant ionic strength
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(l) of 0.1 was maintained by the addition of an appropriate quan-
tity of NaCl to the solutions. The double dilution with the buffer
solutions yielded to ester prodrugs solution, which contain 5%
DMSO (v/v) and were maintained at 37 0.5 °C in screw-capped
vials in a water bath. The samples were withdrawn at appropriate
time intervals, filtered and the filtrate was analyzed by HPLC.
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3.4.2. Enzyme study
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The prodrug stock solution (2.8 mM) was prepared by dissolv-
ing into 1:0.1 mixture of Tris–HCl buffer (pH = 8.0) and DMSO. Pro-
cine esterase stock solution (40 lg/mL) was made using Tris–HCl
buffer (pH = 8.0). The mixing of the equal amount of nucleoside
prodrug solution and procine esterase solution yielded to nucleo-
side prodrugs solution (1.4 mM) in Tris–HCl buffer (pH = 8.0) con-
taining 5% DMSO (v/v) along with 20
l
g/mL porcine esterase. The
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quots were removed at intervals, quenched with ice-cold metha-
nol/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
33. Schinazi, R. F.; Sommadossi, J. P.; Saalmann, V.; Cannon, D. L.; Xie, M. Y.;
Hart, G. C.; Smith, G. A.; Hahn, E. F. Antimicrob. Agents Chemother. 1990, 34,
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34. Schinazi, R. F.; Cannon, D. L.; Arnold, B. H.; Martino-Saltzman, D. Antimicrob.
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This research was supported by the US Public Health Service
Grant 4R37-AI25899 and by the Department of Veterans Affairs.
35. Belen’kii, M. S.; Schinazi, R. F. Antiviral Res. 1994, 25, 1.
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M.; Choi, Y.; Chong, Y.; Choo, H.; Chu, C. K.; Otto, M. J.; Schinazi, R. F. Antimicrob.
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References and notes
1. De Clercq, E. J. Clin. Virol. 2001, 22, 73.