Synthesis and biological evaluation of fatty acyl ester derivatives of 2′,3′-didehydro-2′,3′-dideoxythymidine

Article abstract of DOI:10.1016/j.bmcl.2011.02.070

A number of 5′-O-fatty acyl derivatives of 2′,3′- didehydro-2′,3′-dideoxythymidine (stavudine, d4T) were synthesized and evaluated for anti-HIV activities against cell-free and cell-associated virus, cellular cytotoxicity, and cellular uptake studies. The conjugates were found to be more potent than d4T. Among these conjugates, 5′-O-12- azidododecanoyl derivative of d4T (2), displaying EC₅₀ = 3.1-22.4 μM, showed 4- to 9-fold higher activities than d4T against cell-free and cell-associated virus. Cellular uptake studies were conducted on CCRF-CEM cell line using 5(6)-carboxyfluorescein derivatives of d4T attached through β-alanine (9) or 12-aminododecanoic acid (10) as linkers. The fluorescein-substituted analog of d4T with long chain length (10) showed 12- to 15-fold higher cellular uptake profile than the corresponding analog with short chain length (9). These studies reveal that conjugation of fatty acids to d4T enhances the cellular uptake and anti-HIV activity of stavudine.

Full text of DOI:10.1016/j.bmcl.2011.02.070

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1917–1921
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of fatty acyl ester derivatives
of 20,30-didehydro-20,30-dideoxythymidine
Hitesh K. Agarwal a, Kelly Loethan a, Deendayal Mandal a, Gustavo F. Doncel b, , Keykavous Parang a,
⇑
⇑
a Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI 02881, USA
b CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23507, USA
a r t i c l e i n f o
a b s t r a c t
A number of 50-O-fatty acyl derivatives of 20,30-didehydro-20,30-dideoxythymidine (stavudine, d4T) were
synthesized and evaluated for anti-HIV activities against cell-free and cell-associated virus, cellular cyto-
toxicity, and cellular uptake studies. The conjugates were found to be more potent than d4T. Among these
Article history:
Received 7 January 2011
Revised 14 February 2011
Accepted 16 February 2011
Available online 18 February 2011
conjugates, 50-O-12-azidododecanoyl derivative of d4T (2), displaying EC50 = 3.1–22.4
lM, showed 4- to
9-fold higher activities than d4T against cell-free and cell-associated virus. Cellular uptake studies were
conducted on CCRF-CEM cell line using 5(6)-carboxyfluorescein derivatives of d4T attached through b-
alanine (9) or 12-aminododecanoic acid (10) as linkers. The fluorescein-substituted analog of d4T with
long chain length (10) showed 12- to 15-fold higher cellular uptake profile than the corresponding analog
with short chain length (9). These studies reveal that conjugation of fatty acids to d4T enhances the cel-
lular uptake and anti-HIV activity of stavudine.
Keywords:
Anti-HIV
Cellular uptake
20,30-Didehydro-20,30-dideoxythymidine
Fatty acids
Ó 2011 Elsevier Ltd. All rights reserved.
Stavudine
Stavudine (20,30-didehydro-20,30-dideoxythymidine, d4T) is a
nucleoside reverse transcriptase (RT) inhibitor that is used as an
anti-human immunodeficiency virus type 1 (HIV-1) drug. Stavu-
dine was initially synthesized for evaluation against cancer and
then identified to be active against HIV.1–3 The compound was
approved for clinical use in 1994. When compared with other
thymidine analogs, such as zidovudine (AZT), d4T demonstrates
comparable potency4 and is well absorbed orally. Once inside the
cells, d4T is converted to d4T 50-triphosphate. Application of d4T
as anti-HIV agent alone is limited due to the development of drug
resistance. Different point mutations in RT, such as V75T and K65R,
reduce d4T sensitivity against HIV-1.5,6 d4T shows synergistic
effect with other anti-HIV drugs and thus is generally used in
anti-HIV-1 combination therapy.
The replication of HIV-1 can be inhibited by heteroatom-con-
taining analogs of myristic acid without accompanying cellular
toxicity.7,8 It has been previously reported that several fatty acids,
such as 2-methoxydodecanoic acid, 4-oxatetradecanoic acid, and
12-thioethyldodecanoic acid, reduce HIV-1 replication in acutely
infected T-lymphocytes. For example, 12-thioethyldodecanoic acid
gag–pol, Pr55gag, p27nef).10 At N-terminal glycine, viral proteins
(gag and nef) are covalently attached to myristic acid in the pres-
ence of NMT. Myristic acid attachment makes the proteins more
hydrophobic, which improves protein–protein and protein–mem-
brane interactions.11 For example, after the N-myristoylation, p17
protein localizes itself towards the cell membrane, where new
virus is produced.12
In continuation of our efforts to design novel compounds target-
ing different steps in HIV life cycle13–19 and to improve the anti-
HIV activity and cellular uptake of nucleoside analogs, herein we
report the synthesis of fatty acyl derivatives of d4T, their anti-
HIV activities, and their cellular uptake profiles. It is hypothesized
that the attachment of nucleoside analogs to the long chain fatty
acid analogs enhances their lipophilicity and thus their cellular
uptake. Once the ester conjugate enters the cells; it gets
hydrolyzed by esterases; and generates two active molecules, a
nucleoside analog and a fatty acid, targeting RT and NMT enzymes,
respectively.
A number of 50-O-(fatty acyl) ester derivatives of d4T were syn-
thesized (Scheme 1) through conjugation with fatty acids. Myristic
acid, 12-azidododecanoic acid, 12-thioethyldodecanoic acid, and
12-bromododecanoic acid were selected based on the anti-HIV
activities of the fatty acids and the corresponding fatty acyl deriv-
atives of FLT and AZT.9,16,18 Compounds were synthesized from the
reaction of d4T with the commercially available myristoyl chloride
or freshly prepared fatty acyl chloride (i.e., 12-azidododecanoyl
chloride, 12-thioethyldodecanoyl chloride, and 12-bromododeca-
noyl chloride) in the presence of (dimethlyamino)pyridine (DMAP)
was moderately active (EC50 = 9.4 lM) against HIV-infected T4
lymphocytes.9 One potential mechanism for the anti-HIV activity
of fatty acids is the inhibition of N-myristoyl transferase (NMT)
enzyme, which is involved in catalyzing the myristoylation of
several proteins in HIV life cycle (e.g., capsid protein p17, Pr160
⇑
Corresponding author.
E-mail address: kparang@uri.edu (K. Parang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.070

1918
H. K. Agarwal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1917–1921
RCOOH
O
O
N
Oxalyl chloride
NH
NH
Benzene
N
O
O
R
HO
O
RCOCl, DMAP, Benzene
O
O
O
d4T
R
1
2
3
4
CH3(CH2)12
N3(CH2)11
CH3CH2S(CH2)11
Br(CH2)11
-
-
-
-
Scheme 1. Synthesis of 50-O-(fatty acyl) ester derivatives of d4T (1–4).
as a base. Fatty acyl chloride derivatives were synthesized from the
reaction of fatty acids with oxalyl chloride in benzene in situ.
d4T was attached to 5(6)-carboxyfluorescein (FAM) through
b-alanine (9) and 12-aminododecanoyl (10) as linkers. Compounds
9 and 12 were used as representative fluorescein-conjugate of d4T
and 50-O-fatty acyl derivative of d4T, respectively, for cellular up-
take studies. d4T was reacted with the corresponding Fmoc-amino
acid in the presence of 1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HBTU), 1-hydroxybenzotriazole (HOBt), N,N0-diisopro-
pylcarbodiimide (DIC), and N,N-diisopropylethylamine (DIPEA)
followed by N-Fmoc deprotection to free amino group in the pres-
ence of piperidine. Finally, FAM was attached to free amino group
in the presence of 5(6)-carboxyfluorescein diisobutyrate (CFDI),
HBTU and DIPEA, followed by isobutyrate deprotection using 20%
piperidine to afford 5(6)-carboxyfluorescein derivatives of d4T 9
and 10 (Scheme 2).
higher anti-HIV activity (EC50 = 2.3–14.9
lM) against cell-free
virus than d4T (EC50 = 26.8–28.1 M) in a single-round infection
l
assay. The anti-HIV activity of 50-substituted derivatives of d4T
was dependent on the nature of the 50-substituent. Among all fatty
acyl ester derivatives of d4T, 12-azidododecanoyl derivative 2 was
the most potent compound and showed 4- to 9-fold higher anti-
HIV activity against cell-free and cell-associated virus than d4T
(Table 1).
The differences in anti-HIV potency of conjugates 1–4 compared
to each other and to d4T may be due to their increased rate of
cellular uptake and intracellular hydrolysis, rendering higher
amounts of parent nucleoside and fatty acids. Thus, cellular uptake
profiles of 50-O-fatty acyl derivatives were investigated in compar-
ison with d4T using fluorescein-labeled compounds 9 and 10. d4T
attached to FAM through b-alanine (9) was used as a control d4T
analog. d4T attached to FAM through 12-aminododecanoic acid
(10) was used as an analog of 50-O-fatty acid ester analogues of
d4T.
The anti-HIV activity of the compounds was evaluated accord-
ing to the previously reported procedure.13,19,20 Table 1 illustrates
cytotoxicity and anti-HIV-1 activity against cell-free and cell-asso-
ciated virus. 50-O-(Fatty acyl) ester derivatives d4T 2–4 showed
Human T lymphoblastoid cells (CCRF-CEM, ATCC No. CCL-119)
were grown to 70% confluency in culture medium. The cells were
O
O
NH
NH
N
O
N
O
Fmoc-NH-(CH2)n-OH, HBTU,
HOBt, DIC, DIPEA, DMF
HO
O
O
FmocHN
O
n
O
5
n = 2
O
d4T
6 n = 11
NH
1. CFDI, HBTU, HOBt,
DIC, DIPEA, DMF
2. 20% Piperidine
N
O
O
Piperidine (20%)
H2N
O
n
O
7
n = 2
8 n = 11
O
NH
HOOC
HO
O
N
O
5
O
N
H
6
O
n
O
O
9
n = 2
10 n = 11
O
Scheme 2. Synthesis of 50-O-carboxyfluorescein derivatives of d4T (9 and 10) containing short and long linkers.

H. K. Agarwal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1917–1921
1919
Table 1
and 9 (Fig. 2). Cellular uptake for the trypsin-treated cells with 10
was approximately seven times higher than that of 9.
Anti-HIV activity of fatty acyl ester derivatives of d4T
Compd Cytotoxicitya
Anti-viral activity
R5 virusd
CCRF-CEM cells were incubated with 10 lM DMSO, FAM, 9, and
b
EC50
(lM)
10 for 1 h, and were imaged using fluorescent light microscopy
(ZEISS Axioplan 2). Cells showed no significant fluorescence when
incubated with DMSO, FAM, and 9 (Fig. 3). On the other hand, cells
incubated with 10 showed clearly evident fluorescence. The results
further confirm the higher cellular uptake of 10, a fatty acyl deriv-
ative of d4T, in comparison to 9 and FAM alone. In general, these
data indicate that the fatty acyl derivatives of d4T have better cel-
lular uptake than the parent nucleoside.
These fatty acyl derivatives of anti-HIV nucleosides represent
potential candidates for active ingredients in topical microbicidal
formulations being developed for the prevention of HIV sexual
transmission.21 Since fatty acids intercalate into the sperm mem-
brane altering its functionality22, and vaginal microbicides are
likely to come in contact with sperm, we investigated the effects
of our conjugates on sperm motility and viability.23 In addition, a
compound that displays anti-HIV and sperm-immobilizing prop-
erties would be an ideal candidate for a dual-protection, micro-
bicidal and contraceptive, technology.24 The spermicidal activity
of d4T and their fatty acyl ester derivatives 1–3 was compared
(Fig. 4) with that of nonoxynol-9 (N-9), a commercially available
spermicidal product. In a dose–response study, unlike N-9, com-
pounds 1–3 did not show any significant sperm immobilizing or
spermicidal activity, even at their maximum concentrations
(1 mg/mL).
In general, the conjugation of selected fatty acids with d4T to
generate bifunctional 50-O-substituted fatty acyl derivatives of
d4T resulted in better anti-HIV profile than the parent nucleoside,
possibly through improved cellular uptake of the conjugates. It is
also possible that the fatty acyl moiety might have had a direct
inhibitory effect, either on the myristoylation of critical HIV pro-
teins or the entry of the virus.9,25 Among all the ester derivatives,
50-O-12-azidododecanoyl derivative of d4T (2) was found to have
better anti-HIV activity profile than d4T and other fatty acyl deriv-
atives. The presence of long chain fatty acid at 50-position
enhanced the lipophilicity of d4T and the cellular uptake. These
results showed increased inhibition of HIV-1 infection by fatty acyl
ester derivatives of d4T and suggest that the increased potency
may be due to their higher rate of uptake and intracellular
X4 virusc
CA virusꢀe
EC50 M)
EC50
(lM)
EC50
(
lM)
(l
d4T
1
2
3
4
>200
>200
>200
>200
>200
26.8
78.3
6.7
14.4
14.9
28.1
12.4
3.1
5.8
2.3
136.1
>230.3
22.4
46.5
>206.5
>1000
DMSOf >1000
>1000
>1000
a
b
c
d
e
f
Cytotoxicity assay (MTS).
50% effective concentration.
Single-round infection assay (lymphocytotropic strain, IIIB).
Single-round infection assay (monocytotropic strain, BaL).
Cell-associated transmission assay (SupT1-IIIB).
Solvent control.
incubated with fluorescein-substituted conjugates (9 and 10) at
different time periods and in the presence or absence of trypsin
(Figs. 1 and 2). DMSO and FAM were used as controls for the study.
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
10,000 cells. All the assays were carried out in triplicate.
First, cells were incubated with 10 lM of the compounds for 1,
2, 4, and 8 h (Fig. 1). Compound 10 exhibited 10- to 16-fold higher
cellular uptake than that of 9 and FAM alone. The results clearly
indicate that presence of long chain enhances the cellular uptake
of d4T, by increasing lipophilicity. The continuous incubation of
cells with compounds for up to 8 h did not show significant differ-
ence in the cellular uptake, suggesting that most of the fatty acyl
ester derivative is absorbed into cells within the first hour and that
cellular uptake was not time dependent.
To confirm that the enhanced uptake of 5(6)-carboxyfluorescein
derivative of d4T, 10, is not due to the absorption of the compound
to the cell membrane surface, cells were incubated with 10 lM
DMSO, FAM, 9, or 10 for 1 h, and then half of the cells were treated
with trypsin for 5 min to wash off the adsorbed molecules (if any)
from the cell membrane. These studies showed that cellular uptake
of 10 was still much higher than those of control compounds, FAM
450
400
350
300
250
200
150
100
50
0
Figure 1. Cellular uptake studies for 5(6)-carboxyfluorescein derivatives of d4T (9 and 10) along with FAM and DMSO as controls at different time intervals.

1920
H. K. Agarwal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1917–1921
450
400
350
300
250
200
150
100
50
0
DMSO DMSO-Trypsin FAM FAM-Trypsin
9
9-Trypsin
10
10-Trypsin
Figure 2. Cellular uptake studies for 9 and 10 with DMSO as controls with and without treatment with trypsin.
Figure 3. Real time fluorescence microscopy of 9 and 10 in live CCRF-CEM cell line. Control = DMSO, FAM = 5(6)-carboxyfluorescein.
120
Motility @ 10 min
Viability
100
80
60
40
20
0
d4T
1
2
3
N9
Compounds (mg/ml)
Figure 4. In vitro assays for spermicidal activity of d4T, 1, 2, 3 and N-9 (control).

H. K. Agarwal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1917–1921
1921
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Support for this subproject (MSA-03-367) was provided by
CONRAD, Eastern Virginia Medical School under a Cooperative
Agreement (HRN-A-00-98-00020-00) with the United States
Agency for International Development (USAID) and National Sci-
ence Foundation, Grant Number CHE 0748555. The views ex-
pressed by the authors do not necessarily reflect the views of
USAID or CONRAD. We also acknowledge National Center for
Research Resources, NIH, and Grant Number 1 P20 RR16457 for
sponsoring the core facility.
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