Synthesis and biological evaluation of fatty acyl ester derivatives of (-)-2′,3′-dideoxy-3′-thiacytidine

Article abstract of DOI:10.1021/jm300492q

A number of fatty acyl derivatives of (-)-2′,3′-dideoxy- 3′-thiacytidine (lamivudine, 3TC, 1) were synthesized and evaluated for their anti-HIV activity. The monosubstituted 5′-O-fatty acyl derivatives of 3TC (EC₅₀ = 0.2-2.3 μM) were more potent than the corresponding monosubstituted N₄-fatty acyl (EC₅₀ = 0.4-29.4 μM) and 5′-O-N₄-disubstituted (EC₅₀ = 72.6 to >154.0 μM) derivatives of the nucleoside. 5′-O-Myristoyl (16) and 5′-O-12-azidododecanoyl derivatives (17) were found to be the most potent compounds (EC₅₀ = 0.2-0.9 μM) exhibiting at least 16-36-fold higher anti-HIV activity against cell-free virus than 1 (EC₅₀ = 11.4-32.7 μM). The EC₉₀ values for 16 against B-subtype and C-subtype clinical isolates were several folds lower than those of 1. The cellular uptake studies confirmed that compound 16 accumulated intracellularly after 1 h of incubation with CCRF-CEM cells and underwent intracellular hydrolysis. 5′-O-Fatty acyl derivatives of 1 showed significantly higher anti-HIV activity than the corresponding physical mixtures against the B-subtype virus.

Full text of DOI:10.1021/jm300492q

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Fatty Acyl Ester Derivatives of
(−)-2′,3′-Dideoxy-3′-thiacytidine
Hitesh K. Agarwal,† Bhupender S. Chhikara,† Michael J. Hanley,† Guofeng Ye,† Gustavo F. Doncel,*,‡
and Keykavous Parang*,†
†Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, 41 Lower College Road,
Kingston, Rhode Island 02881, United States
‡CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia,
23507, United States
S
* Supporting Information
ABSTRACT: A number of fatty acyl derivatives of (−)-2′,3′-
dideoxy-3′-thiacytidine (lamivudine, 3TC, 1) were synthesized
and evaluated for their anti-HIV activity. The monosubstituted
5′-O-fatty acyl derivatives of 3TC (EC50 = 0.2−2.3 μM) were
more potent than the corresponding monosubstituted N4-fatty
acyl (EC50 = 0.4−29.4 μM) and 5′-O-N4-disubstituted (EC50
=
72.6 to >154.0 μM) derivatives of the nucleoside. 5′-O-
Myristoyl (16) and 5′-O-12-azidododecanoyl derivatives (17)
were found to be the most potent compounds (EC50 = 0.2−
0.9 μM) exhibiting at least 16−36-fold higher anti-HIV activity
against cell-free virus than 1 (EC50 = 11.4−32.7 μM). The EC90 values for 16 against B-subtype and C-subtype clinical isolates
were several folds lower than those of 1. The cellular uptake studies confirmed that compound 16 accumulated intracellularly
after 1 h of incubation with CCRF-CEM cells and underwent intracellular hydrolysis. 5′-O-Fatty acyl derivatives of 1 showed
significantly higher anti-HIV activity than the corresponding physical mixtures against the B-subtype virus.
Several fatty acid analogues of myristic acid have been shown
to inhibit NMT.10 HIV-1 replication can be inhibited by
heteroatom-containing analogues of myristic acid without
cellular toxicity.11,12 Several fatty acids, such as 2-methox-
ydodecanoic acid, 4-oxatetradecanoic acid, and 12-thioethyldo-
decanoic acid, reduce HIV-1 replication in acutely infected T-
lymphocytes. For example, 12-thioethyldodecanoic acid was
moderately active (EC50 = 9.4 μM) against HIV-infected CD4+
T lymphocytes.13
We previously reported the synthesis and evaluation of
several 5′-O-fatty acyl esters of AZT, FLT, and 2′,3′-didehydro-
2′,3′-dideoxythymidine (d4T) for inhibition of HIV-1.14−16
These conjugates were designed to achieve anti-HIV activity
through inhibition of reverse transcriptase and NMT by
nucleosides and fatty acids.
Herein, we report the synthesis of fatty acyl derivatives of 1,
their cellular uptake profiles, and the characterization of their
antiviral activity against lab-adapted HIV-1 and wild-type and
mutant clinical isolates. Three fatty acids, myristic acid, 12-
azidododecanoic acid, and 12-thioethyldodecanoic acid, were
conjugated at 5′-O- and N4-position of the nucleoside. Selection
of the fatty acids was based on the anti-HIV activity of the
previously reported corresponding fatty acyl derivatives of FLT
and d4T.14−16 We hypothesized that the attachment of the
INTRODUCTION
■
Lamivudine [(−)-2′,3′-dideoxy-3′-thiacytidine, 3TC, 1] is a
reverse transcriptase inhibitor known to block human
immunodeficiency virus-1 (HIV-1) and hepatitis B virus
replication (HBV).1 This anti-HIV drug is commonly used in
combination with two or more other anti-HIV drugs in Highly
Active Antiretroviral Therapy (HAART) programs for the
treatment of HIV infection.2 Compound 1 has been shown to
have a higher therapeutic index than 3′-azido-2′,3′-dideoxythy-
midine (AZT) and 3′-fluoro-2′,3′-dideoxythymidine (FLT) and
possesses anti-HIV activity against AZT-resistant virus.3
Although 1 has potent activity against wild-type HIV, over
time, mutations in HIV reverse transcriptase have generated
multiple strains of 3TC-resistant viruses.4−7 It is therefore
important to continue developing new and effective RT
inhibitors.
Myristoyl-CoA:protein N-myristoyltransferase (NMT) is a
crucial enzyme involved in catalyzing the myristoylation of
several proteins involved in the life cycle of HIV (e.g., capsid
protein p17, Pr160gag‑pol, Pr55gag, and p27nef). At N-terminal
glycine, viral proteins are covalently attached to myristic acid in
the presence of NMT, making proteins more hydrophobic. In
turn, this N-myristoylation improves protein−protein and
protein−membrane interactions.8 For example, after N-
myristoylation, p17 protein localizes itself toward the cell
membrane, where a new virus is produced.9
Received: April 7, 2012
Published: April 25, 2012
© 2012 American Chemical Society
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Scheme 1. Synthesis of Fatty Acyl Ester Derivatives of Compound 1
nucleoside to the long chain fatty acid analogues would
conjugates of 1 were envisioned and designed to be used as
enhance compound 1 lipophilicity and thus its cellular uptake.
Furthermore, once the ester conjugate entered the cells, it
would be hydrolyzed by esterases to yield two active species:
the nucleoside analogue and the fatty acid targeting RT and
NMT enzymes, respectively. The results of this study
demonstrate that conjugation of fatty acids with 1 yields anti-
HIV agents having enhanced lipophilicity, higher cellular
uptake, increased potency, and broader spectrum of antiviral
activity. Microbicides are topically applied agents that prevent
or reduce transmission of infectious disease, in particular HIV/
AIDS. Thus, a broad-based microbicide with an extended range
of activity against HIV would be highly advantageous. Fatty acyl
topical anti-HIV microbicides.
RESULTS AND DISCUSSION
■
Chemistry. Fatty Acyl Ester Derivatives of Compound 1.
5′-Hydroxyl and/or 4-amino positions were substituted with
the fatty acids to synthesize three classes of compounds: two
5′,N4-disubstituted (2 and 3), three N4-monosubstituted (8−
10), and three 5′-O-esters (16−18) of compound 1 (Scheme
1). Three fatty acids myristic acid, 12-azidododecanoic acid,
and 12-thioethyldodecanoic acid were used for the conjugation
with 1.
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5′,N4-Disubstituted derivatives (2 and 3) were synthesized by
reacting 1 with the appropriate fatty acyl chloride in the
presence of 4-dimethylaminopyridine (DMAP) as a base. N4-
Substituted derivatives (8−10) were synthesized by first
selectively protecting the 5′-hydroxyl group with tert-
butyldimethylsilyl chloride (TBDMS-Cl) in the presence of
imidazole as a base to afford 4. 5′-O-TBDMS-protected
nucleoside (4) was further reacted with the fatty acyl chloride
followed by deprotection of TBDMS to afford N4-substituted
derivatives (8−10).
5′-O-Fatty acyl derivatives of the nucleoside (16−18) were
synthesized by first protecting the 4-amino group of 4 with 4,4′-
dimethoxytrityl (DMTr) protecting group in the presence of
4,4′-dimethoxytrityl chloride (DMTr-Cl) and pyridine to afford
11. The TBDMS group was then removed from the 5′-O-
position by treatment of 11 with tetrabutylammonium fluoride
(TBAF) to yield the N4-DMTr derivative (12). Compound 12
was then reacted with the fatty acids in the presence of 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate (HBTU) and N,N-diisopropylethylamine (DIPEA)
followed by DMTr deprotection with acetic acid to afford 5′-
O-(fatty acyl) ester derivatives of the nucleoside (16−18).
The attachment of the long chain fatty acid analogues to 1
enhanced their lipophilicity as shown by calculated partition
coefficients (Log P) (Table 1). All compounds were initially
amino group in the presence of HBTU and DIPEA, followed by
DMTr deprotection to afford the 5(6)-carboxyfluorescein
derivatives of the nucleoside (23 and 24, Scheme 2). These
compounds were used to determine the cellular uptake profile
of the fatty acyl esters of 1. The nucleoside attached to FAM
through β-alanine (23) was used as a control compound 1
analogue. The nucleoside attached to FAM through 12-
aminododecanoyl (24) was used as an analogue of 5′-O-(12-
azidododecanoyl)-2′,3′-dideoxy-3′-thiacytidine (17) and other
5′-O-fatty acyl ester derivatives of compound 1.
Biological Activities. Anti-HIV Activities of Fatty Acyl-
Nucleoside Conjugates. The conjugates of compound 1 were
tested for cytotoxicity and anti-HIV activity against lab-adapted
and various strains of multidrug resistant viruses. Table 1
illustrates the anti-HIV-1 activity of the fatty acyl ester
derivatives of the nucleoside in comparison with parent
compound 1 against the cell-free (X4 and R5) virus. The
data provide structure−activity relationships for different fatty
acyl ester derivatives of 1 by comparing N4-substituted, 5′-O-
substituted, and 5′-O,N4-disubstituted compounds. The anti-
HIV activity of fatty acyl derivatives of the nucleoside was
dependent on the site of esterification. 5′-O,N4-Disubstituted
derivatives of the nucleoside (2 and 3) displayed significantly
less activity (EC50 = 72.6 to >154.0 μM) than the other
monosubstituted fatty acyl derivatives (EC50 = 0.2−29.4 μM)
and compound 1 alone (EC50 = 11.4−32.7 μM). In general,
minimal cytotoxicity was observed (EC50 > 148 μM) for
synthesized 5′-O,N4-, N4-, and 5′-O substituted fatty acyl esters
of 1.
Table 1. In Vitro Anti-HIV Activity of Fatty Acyl Derivatives
of Compound 1 against the Lab-Adapted Virus
anti-HIV activity
All the N4- or 5′-O-monosubstituted derivatives of the
nucleoside (8−10 and 16−18) exhibited a higher potency than
1 against cell-free HIV-1. 5′-O-Monosubstituted ester deriva-
tives (16−18) were the most potent compounds (EC50 = 0.2−
2.3 μM) among all the derivatives as determined by viral
inhibition single-round infection assays. 5′-O-Myristoyl ana-
logue (16) exhibited the highest anti-HIV activity (EC50 = 0.2−
0.5 μM). Compound 8 (EC50 = 0.7 μM) was the most potent
analogue against cell-associated HIV-1 showing approximately
115-fold higher antiviral activity than 1 (EC50 = 80.3 μM) (data
not shown).
Anti-HIV Activities of 5′-O-Myristoyl Analogue (16)
against Clinical Isolates and Multi-Drug Resistant HIV. On
the basis of the observed high antiviral activity against X4 and
R5 lab-adapted virus, 16 was further tested against clinical
isolates of HIV, and antiviral activity results were compared
with those of compound 1 (Table 2). The IC90 values for 16
against the B-subtype virus and C-subtype virus were
approximately 20- and 11-fold lower than those of 1,
respectively. When tested against drug resistant virus, the
IC90 values of 16 against NNRTI and NRTI-resistant viruses
were 9 and 38 times lower than those of 1. Improved IC50 and
IC90 values directly increased the therapeutic index of 16. Both
1 and 16 were poorly active against B-MDR strain of HIV
4755-5, containing multiple NRTI-resistant mutations. These
results indicate that conjugate 16 can be used against NNRTI
and NRTI-resistant viruses, significantly improving the anti-
HIV profile of 1.
a
c
d
cytotoxicity
X4
R5
b
e
compd EC(50) (μM) EC(50) (μM) EC(50) (μM) Log P (calcd)
1
>436.6
>154.0
>148.0
>227.5
>221.0
>212.0
>227.7
>221.1
>212.2
>100
32.7
>154.0
148.0
10.9
29.4
5.3
11.4
135.1
72.6
0.7
0.06
f
2
ND
ND
5.05
4.26
4.14
5.79
5.00
4.88
3
8
9
3.8
10
0.4
16
0.5
0.2
17
0.9
0.7
18
2.3
0.2
DMSO
>100
>100
0.4
g
C-2
>25
0.02
a
b
c
Cytotoxicity assay (MTS). 50% effective concentration. Single-
round infection assay (lymphocytotropic strain). Single-round
infection assay (monocytotropic strain). Calculated partition
coefficient using ChemBioDraw Ultra 12.0. Not determined. Assay
d
e
f
g
control (Dextran sulfate (50 KDa)).
synthesized at 100 mg scale and were tested to determine their
anti-HIV activities and cytotoxicity profiles. Compounds 8, 16,
and 17 were then synthesized in a larger scale (5 g) for further
biological evaluations. The compounds were first purified by
using silica gel column chromatography and then semi-
preparative HPLC. The purity of final products (>95%) was
confirmed by analytical HPLC.
5(6)-Carboxyfluorescein Derivatives of 1. Compound 1 was
attached to 5(6)-carboxyfluorescein (FAM) using β-alanine and
12-aminododecanoic acid as linkers. First, N4-DMTr protected
nucleoside (12) was reacted with the corresponding Fmoc-
amino acid in the presence of HBTU and DIPEA. Second, N-
Fmoc deprotection to the free amino group was achieved in the
presence of piperidine. Finally, FAM was attached to the free
Table 3 illustrates the anti-HIV-1 activity of 5′-O-ester
conjugates 16−18 and their corresponding physical mixtures
against the B-subtype virus. Physical mixtures were prepared by
mixing the fatty acid with 1 in similar equimolar ratios to that of
the corresponding conjugates. The physical mixtures of the
corresponding fatty acids and 1 showed significantly less anti-
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Scheme 2. Synthesis of 5′-Carboxyfluorescein Nucleoside Derivatives 23 and 24 through Different Linkers
hydrolysis and release of 1 and the fatty acid contribution to the
higher anti-HIV profile of the ester conjugates compared to the
physical mixtures.
Table 2. Anti-HIV Evaluation against Clinical Isolates and
MDR Viruses
a
clade/
resistance
IC50
(nM)
IC90
(nM)
CC50
(nM)
In summary, structure−function analysis revealed that the
anti-HIV activity of fatty acyl substituted derivatives of
nucleosides was clearly dependent on the nature of the
nucleoside and fatty acid analogue. The conjugation of RT
inhibiting nucleoside analogues with selected long chain fatty
acids (NMT inhibitors) exerted a synergistic anti-HIV effect.
Fatty acyl derivatives 8, 16, and 17 exhibited better anti-HIV
profiles than 1, with 16 being the most potent.
Cellular Uptake Studies. The presence of long chain fatty
acid at the 5′-position enhanced the lipophilicity of 1 as shown
by calculated Log P values (Table 1). The rate of cellular
uptake and the intracellular hydrolysis of lipophilic conjugates
to the parent nucleoside and fatty acids determine the overall
anti-HIV activities of the conjugates. An indirect evidence of
intracellular hydrolysis is that monosubstituted 5′-O-fatty acyl
ester derivatives of the nucleoside (16−18, EC50 = 0.2−2.3
μM) were more potent than the corresponding monosub-
stituted amide N4-fatty acyl (8−10, EC50 = 0.4−29.4 μM) and
5′-O-N4-disubstituted derivatives (2 and 3, EC50 = 72.6 to
>154.0 μM). Amide derivatives are known to be more stable
than the corresponding ester derivatives toward hydrolytic
enzymes. The lower anti-HIV activities of more stable amide
derivatives compared to the 5′-O-fatty acyl ester derivatives of 1
reflect that intracellular hydrolysis is critical for generating
higher potency.
compd
virus
1
94US3393IN
98USMSC5016
A-17
B
87.3
21.8
305.6
305.6
282.9
9,622
>13,097
15.0
>13,097
>13,097
>13,097
>13,097
>13,097
>6,830
>6,830
>6,830
>6,830
>6,830
C
B-NNRTI 92.1
71361-1
B-NRTI
812.0
>13,097
4.2
4755-5
B-MDR
16
94US3393IN
98USMSC5016
A-17
B
C
1.9
27.5
B-NNRTI 2.8
31.1
71361-1
B-NRTI
B-MDR
43.3
5,086.0
252.7
>6,830
4755-5
a
IC50, 50% of the maximal inhibitory concentration; IC90, 90% of the
maximal inhibitory concentration; CC50, 50% cytotoxic concentration.
Table 3. Anti-HIV Activity of 5′-O-Fatty Acyl Derivatives of 1
and the Corresponding Physical Mixtures against Clinical
a
Isolate (94US3393IN, Clade B) Virus
IC50
(nM)
IC90
(nM)
CC50
(nM)
composition
5′-O-(myristoyl)-3TC (16)
1 + myristic acid
4.2
15.0
>6,830
123.8
14.9
435.2
134.7
18,431.4
78.3
>21,869
>22,113
>21,266
>21,222
5′-O-(12-azidododecanoyl)-3TC (17)
1 + 12-azidododecanoic acid
9,871.8
14.7
5′-O-(12-thioethyldodecanoyl)-3TC
(18)
Cellular studies were performed to understand the uptake
profile of 5′-O-fatty acyl derivatives in comparison with 1 and to
confirm intracellular hydrolysis. Cellular uptake of compound
16 was monitored in human T lymphoblastoid cells (CCRF-
CEM, ATCC No. CCL-119). The growing cells (1 × 106) were
incubated with compound 16 (50 μM) for 1−72 h. The cellular
accumulation of compound 16 was monitored with HPLC
analysis and detection at 265 nm after cellular lysis. The cellular
analysis confirmed that compound 16 was accumulated
intracellularly after 1 h of incubation (analytical HPLC profile,
Supporting Information). Cellular hydrolysis was observed after
1 h of incubation of compound 16 (50 μM) with the main peak
1 + 12-thioethydodecanoic acid
257.5
609.1
>20,440
a
IC50, 50% of the maximal inhibitory concentration; IC90, 90% of the
maximal inhibitory concentration; CC50, 50% cytotoxic concentration.
HIV activity than the 5′-O-ester conjugates 16−18. For
example, 5′-O-myristoyl analogue (16) (IC50 = 4.2 nM)
exhibited 29-fold higher activity than the physical mixture of
1 and myristic acid (EC50 = 123.8 nM). In general, the physical
mixtures in equimolar ratio exhibited 8−137-fold lower anti-
HIV activity than the corresponding ester conjugates,
confirming the cellular uptake of the conjugate and intracellular
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Figure 1. Cellular uptake of 5(6)-carboxyfluorescein nucleoside derivatives 23 and 24 (10 μM) along with FAM and DMSO as controls at different
time intervals.
Figure 2. Cellular uptake of 5(6)-carboxyfluorescein nucleoside derivative 24 at different concentrations.
appearing at 15.3 min. Compound 1 and potential phosphory-
lated products peaks were detected at 1.7−2.7 min in the
HPLC profile and overlapped with cell extract peaks, making
this method challenging for comparative studies with the parent
analogue. Thus, 5(6)-carboxyfluorescein (FAM) derivatives of
the nucleoside were synthesized to quantify and visualize the
cellular uptake of the fatty acyl conjugates compared to parent
compound 1 more accurately.
The nucleoside attached to FAM through β-alanine (23)
(Log P = 1.51) was used as the control compound 1 analogue.
The nucleoside attached to FAM through 12-aminododecanoic
acid (24) was used as a lipophilic analogue of 5′-O-(12-
azidododecanoyl)-2′,3′-dideoxy-3′-thiacytidine (16) (Log P =
5.42) and other fatty acid ester analogues of compound 1. Both
23 and 24 remained stable in culture medium after incubation
up to 24 h as shown by comparison of the analytical HPLC
profile with background cell culture medium without the
compound (Supporting Information), ruling out the extrac-
ellular hydrolysis of the compounds. The CCRF-CEM cells
were used for the study and were grown to 70% confluency in
the culture media. The cells were incubated with the
fluorescein-substituted conjugates (23 and 24) for different
time periods, at different concentrations, and in the presence or
absence of trypsin (Figures 1−3). DMSO and FAM were used
as controls. The cells were analyzed by flow cytometry
(FACSCalibur: Becton Dickinson) using FITC channel and
CellQuest software. The data presented are based on the mean
fluorescence signal for 10000 cells. All the assays were carried
out in triplicate.
In the time response study, the cells were first incubated with
10 μM of the compounds for varying amounts of time (0.5 h, 1
h, 2 h, 4 h, and 8 h; Figure 1). Compound 24 exhibited 3−6-
fold higher cellular uptake than 23 and FAM alone. The results
clearly indicate that the presence of a long chain enhances the
cellular uptake of compound 1, presumably by increasing
lipophilicity. The continuous incubation of cells with
compounds for up to 8 h did not show significant differences
in uptake, suggesting that cellular uptake was not time
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Figure 3. Cellular uptake studies of 23 and 24 (10 μM) with DMSO as controls with and without treatment with trypsin.
dependent and that most of the fatty acyl ester derivative is
absorbed into the cells within the first 30 min.
data indicate that the fatty acyl derivatives of nucleosides have
better cellular uptake than their parent nucleosides.
The cells were then incubated with different concentrations
(5, 10, 20, 40, and 100 μM) of the carboxyfluorescein derivative
24 for 1 h (Figure 2). The data suggest that cellular uptake was
concentration dependent.
CONCLUSIONS
■
Several 5′-O-substituted or N4-substituted fatty acyl derivatives
of 1 were synthesized, and their anti-HIV activity and cellular
uptake were evaluated. In general, the conjugation of selected
fatty acids with RT inhibiting nucleoside analogues resulted in
better anti-HIV profiles due to improved cellular uptake and
possibly intracellular hydrolysis of the conjugates yielding two
parent inhibitors targeting RT and NMT, two enzymes
involved in the life cycle of HIV-1. Although other fatty acyl
ester derivatives of other nucleosides were previously reported
by us,14−16 the 5′-O-ester conjugates of 1 exhibited significantly
higher anti-HIV activity than the parent analogue and the
corresponding physical mixtures, improved cellular uptake, and
demonstrated higher barrier to drug resistance.
Among all the ester derivatives, 8, 16, and 17 were found to
have better anti-HIV activity than 1 and the other fatty acyl
derivatives examined. For example, compound 16, was at least
57-fold more potent than 1. The data indicate that conjugation
of 1 with myristic acid analogues is effective in achieving higher
anti-HIV activity possibly by improving the cellular uptake of 1.
The presence of a long chain fatty acid at the 5′-position
enhanced the lipophilicity of 1 and its cellular uptake as shown
by calculated Log P values and cellular uptake studies of
compound 16 and 5′-carboxyfluoroscein derivatives of the
nucleoside containing short chain (23) and long chain (24)
alkyl ester groups, respectively. Cellular uptake and intracellular
hydrolysis of ester conjugate 16 was demonstrated in analytical
HPLC analysis. FACS experiments in CCRF-CEM cells
showed that 24 had significantly higher cellular uptake than
23. Fluorescence microscopy of cells incubated with these
compounds further confirmed these results. Increased inhib-
ition by fatty acyl ester derivatives of 1 may be due to their
increased rate of uptake and subsequent intracellular hydrolysis
yielding two antiviral agents with different targets, the parent
nucleoside and the fatty acid analogue. The potential dual
mechanism of action of the compounds increased the barrier to
resistance. The 5′-O-ester conjugates exhibited significantly
higher anti-HIV activity than the corresponding physical
mixtures. These data provide the basis for a new and rational
To confirm that the enhanced uptake of the 5(6)-
carboxyfluorescein derivative 24 was not due to compound
absorption onto the cell membrane surface, cells were
incubated with 10 μM of DMSO, 23, and 24 for 1 h, and
then half of the cells were treated with trypsin for 5 min to
wash the adsorbed molecules (if any) from the cell membrane.
The comparison of the data between trypsin-treated and
untreated cells indicates that only a small amount of the
fluorescence was due to the cell surface absorption. Cellular
uptake of the trypsin-treated cells with 24 was approximately 7
times higher than that of 23 (Figure 3). However, trypsin-
untreated cells incubated with 24 showed only 4-fold higher
cellular uptake than 23. These results suggest that the higher
cellular uptake of 24 is not due to artificial uptake and/or
absorption to the cell membrane.
Cell Viability Study. Cell viability studies were performed to
analyze the effect of FAM, 23, and 24 on live cells. CCRF-CEM
cells were incubated with 10 μM of compounds and mixed with
trypan blue (0.1%) to identify the dead cells. The percentage of
viability was calculated using Cellometer Auto T.4 (Nexcelom
Bioscience). It was observed that at least 80% of the cells were
viable in the presence of the compounds during a 24 h
incubation period (Figure S12, Supporting Information).
Differences in cell viability, therefore, did not account for the
observed differences in cellular uptake.
Real Time Fluorescence Microscopy in Live CCRF-CEM
Cells. CCRF-CEM cells were incubated with 10 μM DMSO,
FAM, 23, and 24 for 1 h and were imaged using a light
microscope (ZEISS Axioplan 2) equipped with transmitted
light microscopy, differential-interference contrast, and an
Achroplan 40× objective. Cells showed no significant
fluorescence when incubated with DMSO, FAM, and 23.
However, cells incubated with 24 showed fluorescence (Figure
S13, Supporting Information). The results further confirm
higher cellular uptake of 24, a fatty acyl derivative of compound
1, in comparison to 23 and FAM alone. Conceptually, these
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3.0 Hz, 1H, H-5′), 3.64 (dd, J = 5.1 and 12.6 Hz, 1H, H-2″), 3.29 (dd, J
= 3.2 and 12.6 Hz, 1H, H-2′), 2.47 (t, J = 7.6 Hz, 2H, CH2CONH),
2.40 (t, J = 7.6 Hz, 2H, CH2COO), 1.60−1.75 (m, 4H, CH2CH2CO),
1.20−1.40 (br m, 40H, methylene protons), 0.89 (t, J = 6.7 Hz, 6H,
CH3). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 175.06 (CONH),
173.39 (COO), 163.21 (C-4), 154.89 (C-2 CO), 145.87 (C-6),
95.59 (C-5), 88.70 (C-1′), 87.75 (C-4′), 62.49 (C-5′), 38.31 (C-2′),
37.00 (CH2CONH), 34.27, 32.00, 29.73, 29.63, 29.47, 29.43, 29.26,
29.16, 25.14, 22.77 (methylene carbons), 14.48 (CH3). HR-MS (ESI-
TOF) (m/z): C36H63N3O5S, calcd, 649.9675; found, 650.1885 [M +
H]+.
(−)-N4,5′-Di(12-azidododecanoyl)-2′,3′-dideoxy-3′-thiacytidine
(3). Yield (170 mg, 55%). 1H NMR (400 MHz, CDCl3, δ ppm): 8.90−
9.12 (br s, 1H, NH), 8.18 (d, J = 7.2 Hz, 1H, H-6), 7.49 (d, J = 7.2 Hz,
1H, H-5), 6.36 (m, 1H, H-1′), 5.43 (dd, J = 4.9 and 3.0 Hz, 1H, H-4′),
4.67 (dd, J = 12.5 and 4.9 Hz, 1H, H-5″), 4.47 (dd, J = 12.5 and 3.0
Hz, 1H, H-5′), 3.64 (dd, J = 12.6 and 5.4 Hz, 1H, H-2″), 3.20−3.34
(m, 5H, H-2′, CH2N3), 2.49 (t, J = 7.2 Hz, 2H, CH2CO), 2.42 (t, J =
7.2 Hz, 2H, CH2CO), 1.55−1.79 (m, 8H, −CH2CH2CO,
CH2CH2N3), 1.20−1.45 (br m, 28H, methylene protons). 13C NMR
(CDCl3, 100 MHz, δ ppm): 179.51 (CONH), 174.52 (COO), 163.20
(C-4), 146.10 (C-6), 96.50 (C-5), 88.60 (C-1′), 83.84 (C-4′), 64.82
(C-5′), 63.06, 51.87 (CH2N3), 39.43 (C-2′), 37.96 (CH2CONH),
34.80 (CH2COO), 34.47, 29.84, 29.78, 29.63, 29.53, 29.47, 29.23,
29.20, 27.10, 25.14 (methylene carbons). HR-MS (ESI-TOF) (m/z):
C32H53N9O5S, calcd, 675.8855; found, 676.6085 [M + H]+.
design of potent and safe anti-HIV agents as potential topical
anti-HIV microbicides using 1 as the parent nucleoside.
EXPERIMENTAL SECTION
■
Materials. Compound 1 was purchased from EuroAsia Trans
Continental (Bombay, India). 12-Bromododecanoic acid was
purchased from Sigma Aldrich Chemical Co. 5(6)-Carboxyfluorescein
(FAM) was purchased from Novabiochem. All the other reagents
including solvents were purchased from Fisher Scientific. The final
products were purified on a PhenomenexGemini 10 μm ODS
reversed-phase column (2.1 × 25 cm) with a Hitachi HPLC system
using a gradient system at a constant flow rate of 17 mL/min (Table
4).
Table 4. HPLC Method Used for the Purification of the
Final Compounds
time
(min)
water concentration
A (%)
acetonitrile
concentration B (%)
flow rate
(mL/min)
0.00
1.0
100.0
100.0
0.0
0.0
1.0
0.0
17.0
17.0
17.0
17.0
1.0
45.0
55.0
59.0
60.0
100.0
100.0
0.0
0.0
100.0
100.0
0.0
(−)-5′-O-(t-Butyldimethylsilyl)-2′,3′-dideoxy-3′-thiacytidine (4).
Compound 1 (1.09 mmol, 250 mg), tert-butyldimethylsilyl chloride
(500 mg, 3.27 mmol), and imidazole (230 mg, 3.27 mmol) were
dissolved in dry DMF (10 mL), and the reaction mixture was stirred
for 18 h at room temperature. The solvent was concentrated at
reduced pressure, and the residue was purified with silica gel column
chromatography using dichloromethane and methanol (0−5%) as
eluents to yield 4 (350 mg, 95%).
The purity of the compounds was confirmed by using a Hitachi
analytical HPLC system on a C18 column (Grace Allsphere ODS 2−3
μm, 150 × 4.6 mm) using a gradient system (water/acetonitrile 30:70
v/v) at constant flow rate of 1 mL/min with UV detection at 265 nm.
The purity of final products (>95%) was confirmed by analytical
HPLC. The chemical structures of final products were characterized by
nuclear magnetic resonance spectrometry (1H NMR and 13C NMR)
determined on a Bruker NMR spectrometer (400 MHz) and
confirmed by a high-resolution PE Biosystems Mariner API time-of-
flight electrospray mass spectrometer. Chemical shifts are reported in
parts per millions (ppm). For cellular uptake studies, cells were
analyzed by flow cytometry (FACSCalibur: Becton Dickinson) using
FITC channel and CellQuest software. Cell-viability studies were
conducted using Cellometer Auto T.4 (Nexcelom Biosciences). The
real time microscopy in live CCRF-CEM cell line with or without
compounds were imaged using a ZEISS Axioplan 2 light microscope
equipped with transmitted light microscopy with a differential-
interference contrast method and an Achroplan 40× objective.
Chemistry. (−)-N4,5′-(Ditetradecanoyl)-2′,3′-dideoxy-3′-thiacyti-
dine (2) and (−)-N4,5′-di(12-azidododecanoyl)-2′,3′-dideoxy-3′-
thiacytidine (3). In general, a reaction mixture consisting of the
appropriate fatty acid (1.0 mmol), oxalyl chloride (100 μL, 1.2 mmol),
and anhydrous benzene (18 mL) was stirred at room temperature (25
°C) for 1 h. The obtained yellow solution was evaporated to dryness
under reduced pressure to prepare the acid chloride. Lamivudine (1,
100 mg, 0.44 mmol) and 4-dimethylaminopyridine (DMAP, 160 mg,
1.3 mmol) were dissolved in dry benzene (20 mL). The freshly
prepared acid chloride (1.1 mmol) from the reaction of the fatty acid
with oxalyl chloride was added to the mixture. The reaction mixture
was refluxed at 100 °C for 4 h. After the completion of reaction, the
reaction mixture was cooled down to room temperature and
neutralized with 5% sodium bicarbonate solution. The benzene layer
was separated, and the aqueous layer was extracted with dichloro-
methane (3× 100 mL). The organic layer was separated and mixed
with the benzene layer and concentrated at reduced pressure. The
residue was purified with silica gel column chromatography using
dichloromethane and methanol (0−1%) as eluents.
1H NMR (400 MHz, CDCl3, δ ppm): 8.21 (d, J = 7.6 Hz, 1H, H-6),
6.32 (dd, J = 5.2 and 2.8 Hz, 1H, H-1′), 6.02 (d, J = 7.6 Hz, 1H, H-5),
5.26 (t, J = 3.0 Hz, 1H, H-4′), 4.18 (dd, J = 11.8 and 3.0 Hz, 1H, H-5″),
3.96 (dd, J = 11.8 and 3.0 Hz, 1H, H-5′), 3.54 (dd, J = 12.5 and 5.2 Hz,
1H, H-2″), 3.19 (dd, J = 12.5 and 2.8 Hz, 1H, H-2′), 0.95 (s, 9H,
(CH3)3C), 0.15 (s, 6H, CH3Si). HR-MS (ESI-TOF) (m/z):
C14H25N3O3SSi, calcd, 343.1386; found, 344.3933 [M + H]+,
686.4590 [2M + H]+.
(−)-5′-O-(t-Butyldimethylsilyl)-N4(tetradecanoyl)-2′,3′-dideoxy-
3′-thiacytidine (5), (−)-5′-O-(t-butyldimethylsilyl)-N4(12-azidodode-
canoyl)-2′,3′-dideoxy-3′-thiacytidine (6), and (−)-5′-O-(t-butyldi-
methylsilyl)-N4(12-thioethyldodecanoyl)-2′,3′-dideoxy-3′-thiacyti-
dine (7). Compound 4 (140 mg, 0.4 mmol) and DMAP (70 mg, 0.6
mmol) were dissolved in dry benzene (10 mL). The corresponding
acid chloride (0.48 mmol) (prepared as described above) was added
dropwise, and the reaction mixture was refluxed for 4 h at 100 °C. The
reaction mixture was cooled down to room temperature and
neutralized with saturated sodium bicarbonate solution (100 mL).
The benzene layer was separated, and the aqueous layer was extracted
with dichloromethane (3 × 100 mL). The organic layer was separated
and mixed with the benzene layer and concentrated at reduced
pressure. The residue was purified with silica gel column
chromatography using dichloromethane and methanol (0−1%) as
eluents to afford 5−7.
(−)-5′-O-(t-Butyldimethylsilyl)-N4(tetradecanoyl)-2′,3′-dideoxy-
3′-thiacytidine (5). Yield (110 mg, 50%). 1H NMR (400 MHz,
CD3OD, δ ppm): 8.12 (d, J = 7.5 Hz, 1H, H-6), 6.24 (dd, J = 5.3, 3.5
Hz, 1H, H-1′), 5.81 (d, J = 7.5 Hz, 1H, H-5), 5.26 (t, J = 3.3 Hz, 1H,
H-4′), 4.09 (dd, J = 11.7 and 3.3 Hz, 1H, H-5″), 3.96 (dd, J = 11.7 and
3.3 Hz, 1H, H-5′), 3.49 (dd, J = 12.2 and 5.3 Hz, 1H, H-2″), 3.11 (dd, J
= 12.2 and 3.5 Hz, 1H, H-2′), 2.24 (t, J = 7.4 Hz, 2H, CH2CO), 1.56
(t, J = 7.1 Hz, 2H, CH2CH2CO), 1.20−1.35 (br m, 20H, methylene
protons), 0.91 (s, 9H, (CH3)3C), 0.86 (t, J = 6.8 Hz, 3H, CH3), 0.11
(s, 6H, CH3Si). HR-MS (ESI-TOF) (m/z): C28H51N3O4SSi, calcd,
553.8727; found, 554.1020 [M + H]+.
(−)-N4,5′-(Ditetradecanoyl)-2′,3′-dideoxy-3′-thiacytidine (2).
1
Yield (155 mg, 53%). H NMR (400 MHz, CDCl3, δ ppm): 8.90−
9.40 (br s, 1H, NH), 8.16 (d, J = 7.5 Hz, 1H, H-6), 7.48 (d, J = 7.5 Hz,
1H, H-5), 6.34 (dd, J = 3.2 and 5.1 Hz 1H, H-1′), 5.39−5.43 (m, 1H,
H-4′), 4.65 (dd, J = 12.5 and 4.8 Hz, 1H, H-5″), 4.45 (dd, J = 12.5 and
4867
dx.doi.org/10.1021/jm300492q | J. Med. Chem. 2012, 55, 4861−4871

Journal of Medicinal Chemistry
Article
(−)-5′-O-(t-Butyldimethylsilyl)-N4(12-azidododecanoyl)-2′,3′-di-
deoxy-3′-thiacytidine (6). Yield (110 mg, 50%). 1H NMR (400 MHz,
CDCl3, δ ppm): 8.40−8.69 (br s, 2H, NH and H-6), 7.44 (d, J = 7.3
Hz, 1H, H-5), 6.37−6.41 (m, 1H, H-1′), 5.33 (t, J = 2.5 Hz, 1H, H-4′),
4.27 (dd, J = 11.9 and 2.5 Hz, 1H, H-5″), 4.01 (dd, J = 11.9 and 2.5
Hz, 1H, H-5′), 3.64 (dd, J = 12.7 and 5.2 Hz, 1H, H-2″), 3.27−3.30
(m, 3H, H-2′, CH2N3), 2.47 (s, 2H, CH2CO), 1.60−1.82 (m, 4H,
CH2CH2CO, CH2CH2N3), 1.28−1.45 (br m, 14H, methylene
protons), 0.99 (s, 9H, (CH3)3C), 0.18 (s, 6H, CH3Si). HR-MS
(ESI-TOF) (m/z): C26H46N6O4SSi, calcd, 556.8317; found, 567.0023
[M + H]+.
(−)-5′-O-(t-Butyldimethylsilyl)-N4(12-thioethyldodecanoyl)-2′,3′-
dideoxy-3′-thiacytidine (7). Yield (110 mg, 50%). 1H NMR (400
MHz, CDCl3, δ ppm) 9.29−9.41 (br s, 1H, NH), 8.60 (d, J = 7.5, 1H,
H-6), 7.46 (d, J = 7.5 Hz, 1H, H-5), 6.37 (dd, J = 5.2 and 2.3 Hz, 1H,
H-1′), 5.32 (t, J = 2.2 Hz, 1H, H-4′), 4.68 (dd, J = 11.9 and 2.2 Hz, 1H,
H-5″), 4.00 (dd, J = 11.9 and 2.2 Hz, 1H, H-5′), 3.63 (dd, J = 12.7, 5.2
Hz, 1H, H-2″), 3.26−3.33 (m, 1H, H-2′), 2.53−2.62 (m, 4H,
CH2SCH2), 2.39 (t, J = 7.5 Hz, 2H, CH2COO), 1.52−1.83 (m, 4H,
CH2CH2COO and SCH2CH2), 1.21−1.50 (br m, 17H, methylene
protons, CH3CH2S), 0.99 (s, 9H, (CH3)3C), 0.18 (s, 6H, CH3Si). HR-
MS (ESI-TOF) (m/z): C28H51N3O4S2Si, calcd, 585.3077; found,
585.8926 [M + H]+, 607.8040 [M + Na]+.
2 CO), 145.85 (C-6), 96.73 (C-5), 88.61 (C-1′), 88.48 (C-4′), 63.14
(C-5′), 39.38 (C-2′), 38.10 (CH2CONH), 34.83, 32.06, 26.31−30.10,
25.32 (methylene carbons), 15.23 (CH3). HR-MS (ESI-TOF) (m/z):
C22H37N3O4S2, calcd, 471.6769; found, 472.1656 [M + H]+, 941.7961
[2M + H]+.
(−)-5′-O-(t-Butyldimethylsilyl)-N4-(4,4′-dimethoxytrityl)-2′,3′-di-
deoxy-3′-thiacytidine (11). Compound 4 (600 mg, 1.75 mmol) was
dissolved in dry pyridine (10 mL). A solution of 4,4′-dimethoxytrityl
chloride (DMTr-Cl, 1.4 mg, 4.4 mmol) in 10 mL of pyridine was
added to the reaction mixture dropwise at 0 °C. The reaction mixture
was stirred for 30 min. The temperature was raised to room
temperature, and stirring was continued overnight. The reaction
mixture was neutralized with saturated sodium bicarbonate solution
(500 mL) and was extracted with dichloromethane (3 × 200 mL). The
organic layer was separated and concentrated in vacuo. The residue was
purified with silica gel column chromatography using dichloromethane
and methanol (0−1%) as eluents to yield 11 (1.05 g, 90%).
1H NMR (400 MHz, CDCl3, δ ppm): 7.81 (d, J = 7.7 Hz, 1H, H-6),
7.74 (dd, J = 5.7 and 3.3 Hz, 1H, DMTr proton), 7.55 (dd, J = 5.7 and
3.2 Hz, 1H, DMTr proton), 7.12−7.33 (m, 7H, DMTr protons),
6.82−6.88 (m, 4H, DMTr protons), 6.33 (dd, J = 5.2 and 3.0 Hz, 1H,
H-1′), 5.17−5.22 (m, 1H, H-4′), 5.03 (d, J = 7.7 Hz, 1H, H-5), 4.36−
4.40 (m, 1H, H-5″), 4.06 (dd, J = 11.8 and 3.1 Hz, 1H, H-5′), 3.81 (s,
6H, DMTr-OCH3), 3.52 (dd, J = 12.2 and 5.2 Hz, 1H, H-2″), 3.17
(dd, J = 12.2 and 3.0 Hz, 1H, H-2′), 0.80 (s, 9H, (CH3)3C), 0.06 (s,
6H, CH3Si). HR-MS (ESI-TOF) (m/z): C35H43N3O5SSi, calcd,
645.8835; found, 686.4624 (M + K)+, 1289.3671 (2M + H)+.
(−)-N4-(4,4′-Dimethoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (12).
Compound 11 (1 g, 1.55 mmol) was dissolved in tetrabutylammo-
nium fluoride (4.65 mL, 1 M, 4.65 mmol,) and stirred for 3 h. The
reaction mixture was concentrated at reduced pressure, and the residue
was purified with silica gel column chromatography using dichloro-
methane (2% triethylamine) and methanol (0−1%) as eluents to yield
12 (820 mg, 90%).
(−)-N4(Tetradecanoyl)-2′,3′-dideoxy-3′-thiacytidine (8),
(−)-N4(12-azidododecanoyl)-2′,3′-dideoxy-3′-thiacytidine (9), and
(−)-N4(12-thioethyldodecanoyl)-2′,3′-dideoxy-3′-thiacytidine (10).
Tetrabutylammonium fluoride (1.5 mL, 1M) was added to 5−7
(150 mg), and the reaction mixture was stirred at room temperature
for 3 h. The solvent was concentrated at reduced pressure, and the
residue was purified with silica gel column chromatography using
dichloromethane and methanol (0−1%) as eluents to afford 8−10.
20
(−)-N4(Tetradecanoyl)-2′,3′-dideoxy-3′-thiacytidine (8). [α]D
−26.7 (c 0.15 g/100 mL, MeOH/AcCN (1:1 v/v)). Yield (55 mg,
65%). H NMR (400 MHz, DMSO-d6, δ ppm): 10.85 (s, 1H, NH),
1
1H NMR (400 MHz, CD3OD, δ ppm): 8.53 (d, J = 7.8 Hz, 1H, H-
6), 7.32−7.34 (m, 2H, DMTr protons), 7.17−7.23 (m, 6H, DMTr
protons), 7.11−7.13 (m, 1H, DMTr protons), 6.75−7.78 (m, 4H,
DMTr protons), 6.22 (dd, J = 5.3 and 2.5 Hz, 1H, H-1′), 6.01 (d, J =
7.8 Hz, 1H, H-5), 5.25 (t, J = 3.2, 1H, H-4′), 3.99 (dd, J = 12.8 and 3.2
Hz, 1H, H-5″), 3.84 (dd, J = 12.8 and 3.2 Hz, 1H, H-5′), 3.69 (s, 6H,
DMTr-OCH3), 3.51 (dd, J = 12.6 and 5.3 Hz, 1H, H-2″), 3.27 (dd, J =
12.6 and 2.5 Hz, 1H, H-2′). HR-MS (ESI-TOF) (m/z): C29H29N3O5S,
calcd, 531.1828; found, 531.92 [M + H]+, 632.6916 [M + TEA)]+,
1061.1780 [2M + 1]+.
(−)-N4-(4,4′-Dimethoxytrityl)-5′-O-(tetradecanoyl)-2′,3′-dideoxy-
3′-thiacytidine (13), (−)-5′-O-(12-azidododecanoyl)-N4-(4,4′-dime-
thoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (14), and (−)-N4-(4,4′-
dimethoxytrityl)-5′-O-(12-thioethyldodecanoyl)-2′,3′-dideoxy-3′-
thiacytidine (15). Compound 12 (150 mg, 0.30 mmol), the
corresponding fatty acid (0.60 mmol), and HBTU (250 mg, 0.65
mmol) were dissolved in dry DMF (10 mL). Diisopropylethylamine
(DIPEA, 2 mL, 15 mmol) was added to the reaction mixture, and
stirring was continued overnight at room temperature. The reaction
mixture was concentrated at reduced pressure, and the residue was
purified with silica gel column chromatography using dichloromethane
(2% triethylamine) as eluents to afford 13−15. These compounds
were used directly for subsequent deprotection reactions.
(−)-N4-(4,4′-Dimethoxytrityl)-5′-O-(tetradecanoyl)-2′,3′-dideoxy-
3′-thiacytidine (13). Yield (100 mg, 50%). 1H NMR (400 MHz,
CD3OD, δ ppm) 8.62 (d, J = 7.8 Hz, 1H, H-6), 7.40−7.43 (m, 2H,
DMTr protons), 7.26−7.30 (m, 6H, DMTr protons), 7.18−7.22 (m,
1H, DMTr proton), 6.85 (d, J = 8.9 Hz, 4H, DMTr protons), 6.28−
6.32 (m, 1H, H-1′), 6.08 (d, J = 7.8 Hz, 1H, H-5), 5.34 (t, J = 3.1 Hz,
1H, H-4′), 4.07 (dd, J = 12.8 and 3.1 Hz, 1H, H-5″), 3.92 (dd, J = 12.8
and 3.1 Hz, 1H, H-5′), 3.78 (s, 6H, DMTr-OCH3), 3.60 (dd, J = 5.3
and 12.3 Hz, 1H, H-2″), 3.36 (d, J = 12.3 Hz, 1H, H-2′), 2.29 (t, J = 7.3
Hz, 2H, CH2CO), 1.60 (t, J = 6.8 Hz, 2H, CH2CH2CO), 1.20−1.42
(br m, 20H, methylene protons), 0.91 (s, J = 6.6 Hz, 3H, CH3). HR-
MS (ESI-TOF) (m/z): C43H55N3O6S, calcd, 741.3812; found, 742.35
[M + H]+, 843.4629 [M + TEA]+, 1483.7079 [2M + H]+.
8.38 (d, J = 7.5 Hz, 1H, H-6), 7.23 (d, J = 7.5 Hz, 1H, H-5), 6.21 (dd, J
= 5.2 and 3.2 Hz, 1H, H-1′), 5.26 (t, J = 4.1 Hz, 1H, H-4′), 3.79−3.89
(m, 2H, H-5″ and H-5′), 3.56 (dd, J = 12.3 and 5.2 Hz, 1H, H-2″), 3.20
(dd, J = 12.3 and 3.2 Hz, 1H, H-2′), 2.38 (t, J = 7.3 Hz, 2H, CH2CO),
1.53 (t, J = 6.5 Hz, 2H, CH2CH2CO), 1.15−1.35 (s, 20H, methylene
protons), 0.89 (t, J = 6.7 Hz, 3H, CH3). 13C NMR (DMSO-d6, 100
MHz, δ ppm): 174.80 (COO), 163.41 (C-4), 155.11 (C-2 CO),
146.17 (C-6), 95.8 (C-5), 88.90 (C-1′), 87.94 (C-4′), 62.70 (C-5′),
38.51 (C-2′), 37.16 (CH2CONH), 32.16, 29.92, 29.88, 29.73, 29.57,
29.30, 25.28, 22.96 (methylene carbons), 14.77 (CH3). HR-MS (ESI-
TOF) (m/z): C22H37N3O4S, calcd, 439.6119; found, 440.3352 [M +
H]+, 462.2543 [M + Na]+, 878.1789 [2M + H]+, 900.0877 [2M +
Na]+.
(−)-N4(12-Azidododecanoyl)-2′,3′-dideoxy-3′-thiacytidine (9).
1
Yield (50 mg, 60%). H NMR (400 MHz, CDCl3, δ ppm): 8.45−
8.47 (br s, 2H, NH, H-6), 7.49 (d, J = 7.5 Hz, 1H, H-5), 6.38 (dd, J =
5.3 and 3.4 Hz, 1H, H-1′), 5.41 (t, J = 3.2 Hz, 1H, H-4′), 4.21 (dd, J =
12.7 and 3.2 Hz, 1H, H-5″), 4.01 (dd, J = 12.7 and 3.2 Hz, 1H, H-5′),
3.68 (dd, J = 12.5 and 5.3 Hz, 1H, H-2″), 3.27−3.31 (m, 3H, H-2′,
CH2N3), 2.48 (t, J = 6.8 Hz, 2H, CH2CO), 1.58−1.68 (m, 4H,
CH2CH2CO, CH2CH2N3), 1.25−1.45 (br m, 14H, methylene
protons). 13C NMR (CDCl3, 100 MHz, δ ppm): 173.65 (COO),
162.40 (C-4), 155.04 (C-2 CO), 145.47 (C-6), 96.28 (C-5), 88.32
(C-1′), 88.23 (C-4′), 62.99 (C-5′), 51.67 (CH2N3), 39.18 (C-2′), 38.05
(CH2CONH), 29.61, 29.54, 29.45, 29.31, 29.19, 29.02, 26.89, 25.03
(methylene carbons). HR-MS (ESI-TOF) (m/z): C20H32N6O4S,
calcd, 452.5709; found, 453.2421 [M + H]+, 903.9628 [2M + H]+.
(−)-N4(12-Thioethyldodecanoyl)-2′,3′-dideoxy-3′-thiacytidine
1
(10). Yield (50 mg, 50%). H NMR (400 MHz, CDCl3, δ ppm): 8.58
(s, 1H, NH), 8.02−8.07 (br s, 1H, H-6), 6.26−6.30 (br s, 1H, H-5),
6.13−6.17 (m, 1H, H-1′), 5.35 (d, J = 2.8 Hz, 1H, H-4′), 4.59−4.72
(m, 1H, H-5″), 4.35−4.45 (m, 1H, H-5′), 3.53−3.65 (m, 1H, H-2″),
3.20−3.32 (m, 1H, H-2′), 2.45−2.75 (m, 4H, CH2SCH2), 2.33−2.43
(m, 2H, CH2COO), 1.53−1.70 (m, 4H, SCH2CH2, CH2CH2CO),
1.20−1.40 (br m, 17H, methylene protons, CH3CH2S). 13C NMR
(CDCl3, 100 MHz, δ ppm): 174.08 (COO), 163.02 (C-4), 155.44 (C-
4868
dx.doi.org/10.1021/jm300492q | J. Med. Chem. 2012, 55, 4861−4871

Journal of Medicinal Chemistry
Article
(−)-5′-O-(Tetradecanoyl)-2′,3′-dideoxy-3′-thiacytidine (16),
(−)-5′-O-(12-azidododecanoyl)-2′,3′-dideoxy-3′-thiacytidine (17),
and (−)-5′-O-(12-thioethyldodecanoyl)-2′,3′-dideoxy-3′-thiacyti-
dine (18). Acetic acid (AcOH, 80%, 10 mL) was added to compounds
13−15 (0.25 mmol). The reaction mixture was heated at 80 °C for 30
min. The reaction mixture was concentrated at reduced pressure, and
the residue was purified with silica gel column chromatography using
dichloromethane as the eluent to afford 16−18.
(−)-5′-O-(3-Aminopropanoyl)-N4-(4,4′-dimethoxytrityl)-2′,3′-di-
deoxy-3′-thiacytidine (21) and (−)-5′-O-(12-aminododecanoyl)-N4-
(4,4′-dimethoxytrityl)-2′,3′-dideoxy-3′-thiacytidine (22). The crude
products were dissolved in THF (10 mL). To the reaction mixture was
added piperidine (6 μL, 0.06 mmol) and 1-octanethiol (10 mmol
solution in THF, 0.6 mL, 6 mmol). The reaction mixture was allowed
to stir for 1 h at room temperature. The reaction solution was
concentrated at reduced pressure. The residue was purified with
reversed phase HPLC using C18 column and water/acetonitrile as
solvents as described above to yield 21 and 22.
(−)-5′-O-(3-Aminopropanoyl)-N4-(4,4′-dimethoxytrityl)-2′,3′-di-
deoxy-3′-thiacytidine (21). Overall yield (200 mg, 55%). HR-MS
(ESI-TOF) (m/z): C32H34N4O6S, calcd, 602.2199; found, 603.1806
[M + H]+, 1205.0313 [2M + H]+.
(−)-5′-O-(12-aminododecanoyl)-N4-(4,4′-dimethoxytrityl)-2′,3′-
dideoxy-3′-thiacytidine (22). Overall yield = 210 mg, 52%). HR-MS
(ESI-TOF) (m/z): C41H52N4O6S, calcd, 728.3608; found, 729.2265
[M + H]+, 1458.1201 [2M + H]+.
(−)-5′-O-(Tetradecanoyl)-2′,3′-dideoxy-3′-thiacytidine (16).
20
[α]D −34.6 (c 0.61 g/100 mL, MeOH/AcCN (3:1 v/v)). Yield
1
(50 mg, 65%). H NMR (400 MHz, CDCl3, δ ppm): 7.72 (d, J = 7.5
Hz, 1H, H-6), 6.30 (t, J = 4.7 Hz, 1H, H-1′), 5.97 (d, J = 7.5 Hz, 1H,
H-5), 5.32 (dd, J = 3.5 and 5.5 Hz, 1H, H-4′), 4.51 (dd, J = 12.1 and
5.5 Hz, 1H, H-5″), 4.35 (dd, J = 12.1 and 3.5 Hz, 1H, H-5′), 3.52 (dd, J
= 12.0 and 5.3 Hz, 1H, H-2″), 3.09 (dd, J = 12.0 and 4.2 Hz, 1H, H-2′),
2.34 (t, J = 7.6 Hz, 2H, CH2CO), 1.49−1.72 (m, 2H, CH2CH2CO),
1.18−1.42 (br m, 20H, methylene protons), 0.87 (s, J = 6.7 Hz, 3H,
CH3). 13C NMR (CDCl3, 100 MHz, δ ppm): 173.39 (COO), 165.98
(C-4), 155.64 (C-2 CO), 140.73 (C-6), 94.96 (C-5), 87.79 (C-1′),
83.21 (C-4′), 64.48 (C-5′), 38.22 (C-2′), 34.24 (CH2COO), 32.10,
29.87, 29.84, 29.79, 29.65, 29.54, 29.45, 29.31, 25.03, 22.88
(methylene carbons), 14.33 (CH3). HR-MS (ESI-TOF) (m/z):
C22H37N3O4S, calcd, 439.2505; found, 440.2835 [M + H]+.
General Procedure for the Synthesis of 5′-O-(5(6)-Carboxyfluor-
escein) Derivatives 23 and 24. A mixture of 5(6)-carboxyfluoroscein
(430 mg, 1.15 mmol), the corresponding N4-DMTr-5′-O-aminoacyl
derivative of lamivudine (21 or 22, 0.29 mmoL), and HBTU (440 mg,
1.15 mmol) was dissolved in a mixture of dry DMF (10 mL) and
DIPEA (2 mL, 15 mmol) and stirred overnight at room temperature.
The reaction mixture was concentrated and dried under vacuum.
Acetic acid (80%, 10 mL) was added to the reaction mixture and was
heated at 80 °C for 30 min. The final compounds were purified with
reversed phase HPLC using C18 column and using water/acetonitrile
as solvents as described above.
(−)-5′-O-(3-(N(5(6)-Carboxyfluorescein)aminopropanoyl)-2′,3′-
dideoxy-3′-thiacytidine (23). Yield (40 mg, 20%). 1H NMR (400
MHz, CD3CN + D2O, δ ppm) 8.41 (s, 0.5H, FAM−Ar-H, 5 or 6
isomer), 8.11 and 8.12 (two d, J = 8.0 Hz, 1H, H-6), 8.00−8.05 (m,
1H, FAM−Ar-H, 5 or 6 isomer), 7.93 (d, J = 8.0 Hz, 0.5H, FAM−Ar-
H, 5 or 6 isomer), 7.53 (s, 0.5H, FAM−Ar-H, 5 or 6 isomer), 7.29 (d, J
= 8.0 Hz, 0.5H, FAM−Ar-H, 5 or 6 isomer), 6.78−6.90 (m, 4H,
FAM−Ar-H), 6.71 (dd, J = 2.4 and 8.9 Hz, 2H, FAM−Ar-H, 5 or 6
isomer), 6.10 and 6.17 (two d, J = 8.0, 2H, H-1′, H-5), 5.36−5.42 and
5.25−5.31 (two m, 1H, H-4′), 4.55 (dd, J = 12.6 and 4.4 Hz, 1H, H-
5″), 4.32 (dd, J = 12.6 and 2.9 Hz, 1H, H-5′), 3.45−3.69 (m, 3H, H-2″
and CH2NH), 3.13−3.21 (m, 1H, H-2′), 2.72 and 2.61 (two t, J = 6.5
Hz, 2H, CH2CO). 13C NMR (CD3CN + D2O, 100 MHz, δ ppm):
172.49, 172.32 (COO), 168.80 (CONH), 167.24, 167.11 (COO-
FAM), 160.71 (Ar-C-FAM), 159.78, 159.72 (C-4), 154.66 (C-2 C
O), 147.62, 147.55 (Ar-C-FAM), 144.50, 144.39 (C-6), 136.44,
133.91, 130.70, 129.60, 128.39, 115.06, 111.93, 102.87 (FAM-C),
94.32, 94.26 (C-5), 87.38, 87.21 (C-1′), 85.17, 84.98 (C-4′), 63.93,
63.81 (C-5′), 49.09 (CH2NH2), 37.98, 37.87 (C-2′), 36.15, 36.06
(CH2COO). HR-MS (ESI-TOF) (m/z): C32H26N4O10S, calcd,
658.137; found, 330.2546 [M + 2H]2+, 659.2739 [M + H]+,
1317.2294 [2M + H]+.
(−)-5′-O-(12-(N(5(6)-Carboxyfluorescein)aminododecanoyl)-
2′,3′-dideoxy-3′-thiacytidine (24). Yield (30 mg, 16%). 1H NMR
(400 MHz, CD3CN + D2O, δ ppm) 8.28−8.35 (m, 0.5H, FAM−Ar-H,
5 or 6 isomer), 8.11 and 8.12 (two d, J = 8.0 Hz, 1H, H-6), 7.96−8.04
(m, 1.5H, FAM−Ar-H), 7.52 (s, 0.5H, FAM−Ar-H, 5 or 6 isomer),
7.25 (d, J = 8.0 Hz, 0.5H, FAM−Ar-H, 5 or 6 isomer), 6.68−6.74 (m,
2H, FAM−Ar-H), 6.59−6.67 (m, 2H, FAM−Ar-H), 6.56 (dd, J = 2.3
and 8.8 Hz, 2H, FAM−Ar-H), 6.13−6.22 (m, 1H, H-1′), 6.08 and 6.09
(two d, J = 8.0, 2H, H-5), 5.33−5.40 (m, 1H, H-4′), 4.50 (dd, J = 12.5
and 4.8 Hz, 1H, H-5″), 4.38 (dd, J = 12.5 and 3.1 Hz, 1H, H-5′), 3.54
(dd, J = 5.5 and 12.6 Hz, 1H, H-2″), 3.34 (m, 3H, H-2′ and CH2NH),
2.25−2.34 (m, 2H, CH2COO), 1.40−1.60 (m, 4H, CH2CH2CO,
CH2CH2NH), 1.11−1.25 (br m, 14H, methylene protons). 13C NMR
(CD3CN + D2O, 100 MHz, δ ppm): 174.02 (COO), 170.02
(CONH), 167.21 (COO-FAM), 160.84 (C-4), 153.67 (C-2 CO),
148.19, 145.23 (Ar-C-FAM), 142.19 (C-6), 137.63, 135.24, 130.52,
130.39, 130.10, 128.11, 126.38, 125.50, 124.85, 123.85, 113.80, 110.95,
103.48 (FAM-C), 94.73 (C-5), 88.12 (C-1′), 85.72 (C-4′), 64.39 (C-
5′), 49.69 (CH2NH2), 38.55 (C-2′), 34.54 (CH2COO), 30.17, 30.13,
(−)-5′-O-(12-Azidododecanoyl)-2′,3′-dideoxy-3′-thiacytidine
(17). [α]D20 −55.0 (c −0.42 g/100 mL, MeOH). Yield (50 mg, 65%).
1H NMR (400 MHz, CD3OD, δ ppm): 7.71 (d, J = 7.5 Hz, 1H, H-6),
6.29 (dd, J = 5.2 and 3.8 Hz, 1H, H-1′), 6.01 (d, J = 7.5 Hz, 1H, H-5),
5.33 (dd, J = 3.2 and 5.4 Hz, 1H, H-4′), 4.51 (dd, J = 12.2 and 5.4 Hz,
1H, H-5″), 4.35 (dd, J = 12.2 and 3.2 Hz, 1H, H-5′), 3.52 (dd, J = 12.0
and 5.2 Hz, 1H, H-2″), 3.21−3.28 (m, 2H, CH2N3), 3.08 (dd, J = 12.0
and 3.8 Hz, 1H, H-2′), 2.34 (t, J = 7.5 Hz, 2H, CH2CO), 1.54−1.64
(m, 4H, CH2CH2CO, CH2CH2N3), 1.22−1.38 (br m, 14H, methylene
protons). 13C NMR (CD3OD, 100 MHz, δ ppm): 173.40 (COO),
164.96 (C-4), 154.96 (C-2 CO), 140.98 (C-6), 95.31 (C-5), 87.64
(C-1′), 83.51 (C-4′), 64.32 (C-5′), 51.64 (CH2N3), 38.20 (C-2′), 34.19
(CH2CO), 29.88, 29.71, 29.60, 29.54, 29.39, 29.29, 29.24, 28.98, 26.86,
24.98, (methylene carbons). HR-MS (ESI-TOF) (m/z):
C20H32N6O4S, calcd, 452.2206; found, 453.1729 [M + H]+.
(−)-5′-O-(12-Thioethyldodecanoyl)-2′,3′-dideoxy-3′-thiacytidine
(18). Yield (50 mg, 65%). 1H NMR (400 MHz, CD3OD, δ ppm): 7.64
(d, J = 7.4 Hz, 1H, H-6), 6.29−6.35 (m, 1H, H-1′), 5.91 (d, J = 7.4 Hz,
1H, H-5), 5.30−5.34 (m, 1H, H-4′), 4.49 (dd, J = 12.0 and 5.7 Hz, 1H,
H-5″), 4.35 (dd, J = 12.0 and 3.2 Hz, 1H, H-5′), 3.50 (dd, J = 12.0 and
5.3 Hz, 1H, H-2″), 3.04 (dd, J = 12.0 and 4.6 Hz, 1H, H-2′), 2.44−2.63
(m, 4H, CH2SCH2), 2.34 (t, J = 7.4 Hz, 2H, CH2CO), 1.50−1.79 (m,
4H, SCH2CH2, CH2CH2CO), 1.18−1.48 (br m, 17H, methylene
protons). 13C NMR (CD3OD, 100 MHz, δ ppm): 173.36 (COO),
166.01 (C-4), 155.60 (C-2 CO), 140.49 (C-6), 95.35 (C-5), 87.73
(C-1′), 83.03 (C-4′), 64.55 (C-5′), 38.04 (C-2′), 34.20 (CH2COO),
31.82, 29.80, 29.67, 29.58, 29.41, 29.62, 29.11, 28.90, 25.00, 22.88
(methylene carbons), 14.33 (CH3). HR-MS (ESI-TOF) (m/z):
C22H37N3O4S2, calcd, 471.2225; found, 472.2418 [M + H]+,
941.9318 [2M + H]+.
(−)-5′-O-(12(N-Fmoc-aminododecanoyl)-N4-(4,4′-dimethoxytri-
tyl)-2′,3′-dideoxy-3′-thiacytidine (19) and (−)-5′-O-(3(N-Fmoc-
aminopropanoyl)-N4-(4,4′-dimethoxytrityl)-2′,3′-dideoxy-3′-thiacy-
tidine (20). Compound 12 (320 mg, 0.60 mmol), the corresponding
Fmoc-amino acid (1.2 mmoL), and HBTU (500 mg, 1.3 mmol) were
dissolved in a mixture of dry DMF (10 mL) and DIPEA (2 mL, 15
mmol). The reaction mixture was stirred overnight at room
temperature. The reaction mixture was concentrated and dried
under reduced pressure to afford crude 5′-O-Fmoc-amino acid
derivatives of N4-DMTr-2′,3′-dideoxy-3′-thiacytidine, 19 and 20.
(−)-5′-O-(12(N-Fmoc-aminododecanoyl)-N4-(4,4′-dimethoxytri-
tyl)-2′,3′-dideoxy-3′-thiacytidine (19). HR-MS (ESI-TOF) (m/z):
C47H44N4O8S, calcd, 824.288; found, 825.2218 [M + H]+, 1650.0664
[M + H]+.
(−)-5′-O-(12(N-Fmoc-aminododecanoyl)-N4-(4,4′-dimethoxytri-
tyl)-2′,3′-dideoxy-3′-thiacytidine (19). HR-MS (ESI-TOF) (m/z):
C56H62N4O8S, calcd, 950.4288; found, 951.8527 [M + H]+.
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Journal of Medicinal Chemistry
Article
30.07, 30.01, 29.94, 29.87, 29.80, 29.71, 29.59 (methylene carbons),
27.58 (CH2CH2NH2), 25.54 (CH2CH2COO). HR-MS (ESI-TOF)
(m/z): C41H44N4O10S, calcd, 784.2778; found, 393.0862 [M + 2H]2+,
784.9019 [M]+, 1569.4510 [2M + H]+.
serum. Upon reaching about 70% confluency, the cells were treated as
described below and incubated for 1 h or longer at 37 °C.
Cellular Uptake of FAM, 23 and 24 at Different Time Points.
When the cells reached about 70% confluency, FAM, 23, or 24 (1 mL,
20 μM) in RMPI-1640 medium was added to 1 mL of cells to make
the final concentration 10 μM. The cells were incubated for 0.5, 1, 2, 4,
and 8 h at 37 °C. Then, the flow cytometry assays were performed as
described below.
Cellular Uptake of 24 at Different Concentrations. When the cells
reached about 70% confluency, 1 mL of graded concentrations (0, 10,
20, 40, 80, and 200 μM in RMPI-1640) of 24 was added to 1 mL of
cells to make the final concentration 0, 5, 10, 20, 40, and 100 μM. The
cells were incubated for 1 h at 37 °C. Then, the flow cytometry assays
were performed as described in the General Information.
Cellular Uptake of 23 and 24 with Trypsin Treatment. When the
cells reached about 70% confluency, FAM, 23, or 24 (1 mL, 20 μM) in
RMPI-1640 medium was added to 1 mL of cells to make the final
concentration 10 μM. The cells were incubated for 0.5, 1, 2, 4, and 8 h
at 37 °C. The cells used were incubated with 0.25% trypsin/0.53 mM
EDTA for 5 min before washing with PBS (pH 7.4).
Flow Cytometry. The cells were washed twice with PBS (pH 7.4) at
2000 rpm for 5 min. Then the cells were analyzed by flow cytometry
(FACS Calibur: Becton Dickinson) using FITC channel and
CellQuest software. The data presented are based on the mean
fluorescence signal for 10000 cells collected. All the assays were done
in triplicate.
Cell Viability Assay. When the cells reached about 70% confluency,
the cells were incubated with a solution of CCRF-CEM cell alone or
10 μM FAM, 23, or 24 for 24 h at 37 °C. Then, 20 μL of the cells from
each flask was treated with 2 μL of trypan blue (0.1%) for 1 min. The
cells were then transferred to a Cellometer counting slide and analyzed
using Cellometer Auto T.4 (Nexcelom Bioscience). All the assays were
performed in triplicate.
Stability of Compounds 23 and 24 in Culture Media. Compounds
23 and 24 were dissolved in cell culture media to make a final
concentration of 10 μM. The incubation was continued in culture
media for 24 h at 37 °C. The stability of compounds was determined
with a Hitachi analytical HPLC system using a C18 Shimadzu Premier
3 μm column (150 cm ×4.6 mm) using Method B at a flow rate of 1
mL/min with detection at 265 nm. Dimethyl sulfoxide and FAM were
used as controls.
Real Time Fluorescence Microscopy in the Live CCRF-CEM Cell
Line. The cellular uptake studies and intracellular localization of
CCRE-CEM cell alone, or incubated with 23 and 24 were imaged
using a ZEISS Axioplan 2 light microscope equipped with transmitted
light microscopy with a differential-interference contrast method and
an Achroplan 40× objective. The human T lymphoblastoid cells
CCRF-CEM (ATCC No. CCL-119) were grown on 60 mm Petri
Dishes with RPMI-1640 medium containing 10% fetal bovine serum.
Upon reaching about 70% confluency, the cells were incubated with a
solution of 10 μM of 23 and 24 for 1 h at 37 °C. They were then
observed under the fluorescent microscope under bright field and
FITC channels (480/520 nm).
Anti-HIV Assays. The anti-HIV activity of the compounds was
evaluated according to the previously reported procedure.14,17−19
Compound anti-HIV activity was evaluated in single-round (MAGI)
infection assays using X4 (IIIB) and R5 (BaL) HIV-1 and P4R5 cells
expressing CD4 and coreceptors. In summary, P4R5MAGI cells were
cultured at a density of 1.2 × 104 cells/well in a 96 well plate
approximately 18 h prior to infection. Cells were incubated for 2 h at
37 °C with purified, cell-free HIV-1 laboratory strains IIIB or BaL
(Advanced Biotechnologies, Inc., Columbia, MD) in the absence or
presence of each agent. After 2 h, cells were washed, cultured for an
additional 46 h, and subsequently assayed for HIV-1 infection using
the Galacto-Star β-Galactosidase Reporter Gene Assay System for
Mammalian Cells (Applied Biosystems, Bedford, MA). Reductions in
infection were calculated as a percentage relative to the level of
infection in the absence of agents, and 50% inhibitory concentrations
(EC50) were derived from regression analysis. Each compound
concentration was tested in triplicate wells. Cell toxicity was evaluated
using the same experimental design but without the addition of virus.
The impact of compounds on cell viability was assessed using an MTT
(reduction of tetrazolium salts) assay (Invitrogen, Carlsbad, CA).
For the assessment of compounds against wild-type (WT; R5;
clones = 94US3393IN [B subtype] and 98USMSC5016 [C subtype])
and drug resistant (clones = 4755-5, 71361-1, and A17) HIV-1 clinical
isolates, PHA-P stimulated cells from at least two normal donors were
pooled, diluted in fresh media, and plated in the interior wells of a 96
well round-bottom microplate. Each plate contains virus/cell control
wells (cells + virus), experimental wells (drug + cells + virus), and
compound control wells (drug + media, no cells, necessary for MTS
monitoring of cytotoxicity). Test drug dilutions were prepared in
microtiter tubes, and each concentration was placed in appropriate
wells. Following addition of the drug dilutions to the PBMCs, a
predetermined dilution of virus stock was then placed in each test well
(final MOI ≅ 0.1). Since HIV-1 is not cytopathic to PBMCs, the same
assay plate can be used for both antiviral efficacy and cytotoxicity
measurements. Compounds were incubated with the virus and cells in
a 96 well format for 6 h. The cells were then washed by removing 75%
of the medium (150 μL) and replacing with 150 μL of fresh (no drug)
medium. The plates were then centrifuged (∼200g) for 10 min, after
which 150 μL of medium was removed, and an additional 150 μL of
fresh medium was added to each well and further incubated for 6 days
or until peak reverse transcriptase (RT) activity was detected. A
microtiter plate-based RT reaction was utilized.20 Incorporated
radioactivity (counts per minute, CPM) was quantified using standard
liquid scintillation techniques. Compound IC50 (50%, inhibition of
virus replication) was calculated using statistical software and
regression analysis.
Cellular Uptake of Compound 16. Cellular uptake and
accumulation of compound 16 was evaluated in CCRF-CEM cells
by analytical HPLC studies. CCRF-CEM cells were grown in 75 cm2
culture flasks with serum free RPMI medium to ∼70−80% confluency
(1 × 106 cells/mL). The medium was replaced with RPMI medium
containing compound 16 (50 μM). The cells were incubated at 37 °C
for 1−72 h. After incubation for the indicated time, the cells were
collected by centrifugation. The medium was removed carefully by
decantation, and the cell pallets were washed with ice-cold PBS to
remove any medium. The cell pallets were thoroughly extracted with
equal volume of methanol, chloroform, and isopropanol mixture
(4:3:1 v/v/v) and filtered through 0.2 μm filters. Compound 16 in cell
lysates was detected by analytical HPLC analysis (20 μL injection) at
265 nm using a gradient of water (0.1% TFA)/acetonitrile (0.1%
TFA).
ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical HPLC methods; cellular uptake analysis of
compound 16 by analytical HPLC; stability of compound 24
in cell culture medium; cell viability study of 23 and 24 in
CCRF-CEM cells; real time fluorescence microscopy of 23 and
24 in live CCRF-CEM cell line; and 1H NMR and 13C NMR of
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Cellular Uptake Study of Fluorescence-Labeled Nucleoside
Analogues. All of the stock solutions for compounds FAM, 23, and
24 were prepared in DMSO. The human T lymphoblastoid cells
CCRF-CEM (ATCC No. CCL-119) were grown on 25 cm2 cell
culture flasks with RMPI-1640 medium containing 10% fetal bovine
4870
dx.doi.org/10.1021/jm300492q | J. Med. Chem. 2012, 55, 4861−4871

Journal of Medicinal Chemistry
Article
Gordon, J. I. 4-Oxatetradecanoic acid is fungicidal for Cryptococcus
neoformans and inhibits replication of human immunodeficiency virus
I. J. Biol. Chem. 1992, 267, 17159−17169.
AUTHOR INFORMATION
Corresponding Author
*(G.F.D.) Tel: +1-757-446-8908.; Fax: +1-757-446-8998. E-
mail: DoncelGF@evms.edu. (K.P.) Tel.: +1-401-874-4471. Fax:
+1-401-874-5787. E-mail: kparang@uri.edu.
■
(11) Bryant, M. L.; McWherter, C. A.; Kishore, N. S.; Gokel, G. W.;
Gordon, J. I. MyristoylCoA:protein N-myristoyltransferase as a
therapeutic target for inhibiting replication of human immunodefi-
ciency virus-1. Perspect. Drug Dis. Des. 1993, 1, 193−209.
(12) Takamune, N.; Hamada, H.; Misumi, S.; Shoji, S. Novel strategy
for anti-HIV-1 action: selective cytotoxic effect of N-myristoyltransfer-
ase inhibitor on HIV-1 infected cells. FEBS Lett. 2002, 527, 138−142.
(13) Parang, K.; Wiebe, L. I.; Knaus, E. E.; Huang, J.-S.; Tyrrell, D.
L.; Csizmadia, F. In vitro antiviral activities of myristic acid analogs
against human immunodeficiency and hepatitis B viruses. Antiviral Res.
1997, 34, 75−90.
Notes
The views expressed by the authors do not necessarily reflect
the views of USAID or CONRAD.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support for this subproject (MSA-03-367) was provided by
CONRAD, Eastern Virginia Medical School under a Cooper-
ative Agreement (GPO-A-8-00-08-00005-00) with the United
States Agency for International Development (USAID).
(14) Agarwal, H. K.; Loethan, K.; Mandal, D.; Gustavo, D. F.; Parang,
K. Synthesis and biological evaluation of fatty acyl ester derivatives of
2′, 3′-didehydro-2′,3′-dideoxythymidine. Bioorg. Med. Chem. Lett. 2011,
7, 1917−1921.
(15) Parang, K.; Knaus, E. E.; Wiebe, L. I. Synthesis, in vitro anti-HIV
activity, and biological stability of 5′-O-myristoyl analogue derivatives
of 3′-fluoro-2′,3′-dideoxythymidine (FLT) as potential prodrugs of
FLT. Nucleosides Nucleotides 1998, 17, 987−1008.
(16) Parang, K.; Knaus, E. E.; Wiebe, L. I. Synthesis, in vitro anti-HIV
structure-activity relationships and stability of 5′-O-myristoyl analogue
derivatives of 3′-azido-2′,3′- dideoxythymidine as potential prodrugs of
3′-azido-2′,3′-dideoxythymidine (AZT). Antiviral Chem. Chemother.
1998, 9, 311−323.
(17) Agarwal, H. K.; Kumar, A.; Doncel, G. F.; Parang, K. Synthesis,
antiviral and contraceptive activities of nucleoside-sodium cellulose
sulfate acetate and succinate conjugates. Bioorg. Med. Chem. Lett. 2010,
20, 6993−6997.
(18) Ahmadibeni, Y.; Tiwari, R.; Swepson, C.; Pandhare, J.; Dash, C.;
Doncel, G. F.; Parang, K. Synthesis and anti-HIV activities of bis-
(cycloSaligenyl) pronucleotides derivatives of 3′-fluoro-3′-deoxythymi-
dine and 3′-azido-3′-deoxythymidine. Tetrahedron Lett. 2011, 52, 802−
805.
ABBREVIATIONS USED
■
AZT, 3′-azido-2′,3′-dideoxythymidine; d4T, 2′,3′-didehydro-
2′,3′-dideoxythymidine; FLT, 3′-fluoro-2′,3′-dideoxythymidine;
DIPEA, N,N-diisopropylethylamine; DMAP, 4-dimethylamino-
pyridine; DMF, N,N-dimethylformamide; DMTr, 4,4′-dime-
thoxytrityl; FTC, (−)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine;
HAART, highly active antiretroviral therapy; HBTU, 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate; HBV, hepatitis B virus; NMM, N-methylmorpholine;
NRTI, nucleoside reverse transcriptase inhibitors; TBAF,
tetrabutylammonium fluoride; TBDMS-Cl, tert-butyldimethyl-
silyl chloride; 3TC, (−)-2′,3′-dideoxy-3′-thiacytidine; TFA,
trifluoroacetic acid
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