Synthesis and anti-HIV activities of symmetrical dicarboxylate esters of dinucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.07.037

Three nucleoside analogues, 3′-fluoro-2′,3′- dideoxythymidine (FLT), 3′-azido-2′,3′-dideoxythymidine (AZT), and 2′,3′-dideoxy-3′-thiacytidine (3TC) were conjugated with three different dicarboxylic acids to afford the long chain dicarboxylate esters of nucleosides. In general, dinucleoside ester conjugates of FLT and 3TC with long chain dicarboxylic acids exhibited higher anti-HIV activity than their parent nucleosides. Dodecanoate and tetradecanoate dinucleoside ester derivatives of FLT were found to be the most potent compounds with EC ₅₀ values of 0.8-1.0 nM and 3-4 nM against HIV-1_US/92/727 and HIV-1_IIIB cells, respectively. The anti-HIV activity of the 3TC conjugates containing long chain dicarboxylate diester (EC₅₀ = 3-60 nM) was improved by 1.5-66 fold when compared to 3TC (EC₅₀ = 90-200 nM). This study reveals that the symmetrical ester conjugation of dicarboxylic acids with a number of nucleosides results in conjugates with improved anti-HIV profile.

Full text of DOI:10.1016/j.bmcl.2012.07.037

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5451–5454
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-HIV activities of symmetrical dicarboxylate esters
of dinucleoside reverse transcriptase inhibitors
Hitesh K. Agarwal a, Karen W. Buckheit b, Robert W. Buckheit Jr. b, Keykavous Parang a,
⇑
a Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA
b ImQuest BioSciences Inc, 7340 Executive Way, Suite R, Frederick, MD 21704, USA
a r t i c l e i n f o
a b s t r a c t
Three nucleoside analogues, 30-fluoro-20,30-dideoxythymidine (FLT), 30-azido-20,30-dideoxythymidine
(AZT), and 20,30-dideoxy-30-thiacytidine (3TC) were conjugated with three different dicarboxylic acids
to afford the long chain dicarboxylate esters of nucleosides. In general, dinucleoside ester conjugates
of FLT and 3TC with long chain dicarboxylic acids exhibited higher anti-HIV activity than their parent
nucleosides. Dodecanoate and tetradecanoate dinucleoside ester derivatives of FLT were found to be
the most potent compounds with EC50 values of 0.8–1.0 nM and 3–4 nM against HIV-1US/92/727 and
HIV-1IIIB cells, respectively. The anti-HIV activity of the 3TC conjugates containing long chain dicarboxyl-
ate diester (EC50 = 3–60 nM) was improved by 1.5–66 fold when compared to 3TC (EC50 = 90–200 nM).
This study reveals that the symmetrical ester conjugation of dicarboxylic acids with a number of nucleo-
sides results in conjugates with improved anti-HIV profile.
Article history:
Received 4 June 2012
Accepted 9 July 2012
Available online 17 July 2012
Keywords:
Anti-HIV
Dicarboxylic acid
Fatty acid
Nucleoside
Reverse transcriptase inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
Nucleoside reverse transcriptase inhibitors (NRTIs) are com-
monly used for the treatment of human immunodeficiency virus
(HIV) infection. NRTIs exert their pharmacological effects after
intracellular phosphorylation to monophosphates, diphosphates,
and triphosphates in the presence of the host cellular kinases.
Nucleoside triphosphates inhibit the reverse transcriptase, a cru-
cial enzyme for HIV life cycle, through substrate competition with
natural deoxynucleosides and incorporation into the viral DNA that
leads to the chain termination.1,2 Some examples of commer-
cially available NRTIs are 20,30-dideoxy-30-thiacytidine (3TC),
20,30-didehydro-20,30-dideoxythymidine (d4T), 20,30-dideoxy-5-flu-
oro-30-thiacytidine (FTC), 20,30-dideoxycytidine (ddC), 30-azido-30-
deoxythymidine (AZT), and (R)-9-(2-phosphonomethoxypropyl)
adenine (TFV). In spite of the potent anti-HIV activities, NRTIs face
major challenges such as high levels of clinical toxicity including
bone marrow suppression and neuropathy, drug resistance, low
therapeutic index, and/or mitochondrial toxicity.3–6 Combination
therapy in Highly Active Antiretroviral Therapy (HAART) programs
using different classes of anti-HIV drugs provides several potential
advantages, such as reducing the drug resistance and controling
HIV-1 infection.7
of lipophilic fatty acyl esters of anti-HIV nucleosides; AZT, 30-flu-
oro-20,30-dideoxythymidine (FLT), 3TC, and d4T.9–13 Lipophilic fatty
acyl esters of anti-HIV nucleosides demonstrated higher anti-HIV
activity compared to the parent nucleosides.10,14 Cellular uptake
studies indicated that the long chain fatty acyl esters of 3TC had
improved cellular uptake due to improved lipophilicity and were
able to hydrolyze to parent nucleoside intracellularly.14
Herein, we report the synthesis of long chain dicarboxylic acid
esters of FLT, AZT and 3TC to determine whether the presence of
two nucleosides on fatty acyl chains will affect the overall anti-
HIV activity. Three long dicarboxylic acids with chain lengths of
C10 (sebacic acid), C12 (dodecandioic acid), and C14 (tetra-
decandioic acid) were selected for the conjugation. The conjugates
were expected to have high lipophilic nature because of the pres-
ence of long chain dicarboxylate esters and thereby to have im-
proved cellular uptake properties. We have previously shown the
lipophilic nature and/or higher cellular uptake of the fatty acyl es-
ters of the nucleosides compared to the their parent nucleo-
sides.9,10,14 Furthermore, the conjugation of anti-HIV nucleosides
with three long chain dicarboxylic acids will provide insights about
the structure–activity relationships of these conjugates. Once the
ester enters the cells, it is expected to release two parent nucleo-
sides after intracellular ester hydrolysis as shown previously with
fatty acyl ester conjugates of 3TC.14 The results of this study dem-
onstrate that conjugation of two similar nucleosides with dicar-
boxylic fatty acids yields anti-HIV agents having enhanced
lipophilicity and increased potency. These conjugates were envi-
sioned and designed to be used as topical anti-HIV microbicides
Despite the success of HAART, the hydrophilic nature of NRTIs
leads to limited cellular uptake and bioavailability. Various nucle-
oside prodrugs have been synthesized in the past to overcome
these short comings.3,8 We have previously reported the synthesis
⇑
Corresponding author.
E-mail address: kparang@uri.edu (K. Parang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.037

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H. K. Agarwal et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5451–5454
which are topically applied agents that prevent or reduce transmis-
sion of HIV/AIDS.
3–66 times enhanced anti-HIV activity in comparison to 3TC.
Dodecandicarboxylate ester of 3TC (23, EC50 = 9–20 nM) exhibited
4.5–22-fold higher anti-HIV activity than 3TC. The activity of
decanoate ester of 3TC (22) was 1.5–6.6 times higher than 3TC.
However, succinate ester of 3TC (7, EC50 = 130–150 nM) had com-
parable anti-HIV activity with 3TC (EC50 = 90–200 nM). These data
indicate that the anti-HIV activity of dicarboxylate ester conjugates
of dinucleosides depends on the chain length of the spacer be-
tween two nucleosides. The optimal antiviral activity for 3TC con-
jugates was obtained with dodecandioic and tetradecandioic ester
conjugates.
In general, dinucleoside ester conjugates of FLT (6–8) and 3TC
(22–24) with long chain dicarboxylic acids exhibited higher anti-
HIV activity than their parent nucleosides. On the other hand, the
AZT conjugates (9–11, EC50 = 2–5 nM) showed comparable activity
with AZT (EC50 = 2–8 nM) against HIV in both PBMC and CEM-SS
assays.
The dicarboxylic esters of FLT (6–8, EC50 = 0.76–1 nM) exhib-
ited slightly higher anti-HIV activity than their parent nucleoside
FLT in PBMC assay against HIV-1US/92/727. Among all the conju-
gates, FLT esters 7 and 8 were the most potent conjugates against
HIV in the series with EC50 values of 0.97 and 0.76 nM, respec-
tively, that was approximately 2 times higher than that of FLT
(EC50 = 2 nM). The anti–HIV activity of FLT conjugates (6–8,
EC50 = 3–4 nM) in CEM–SS’s against HIV-1III–B was found to be
5–6.6 times higher than FLT (EC50 = 20 nM). The attachment of
the long chain dicarboxylic acid analogues to 1 and 12 enhanced
their lipophilicity as shown by calculated partition coefficients
(Log P) (Table 1). There was a correlation between lipophilicity
and anti-HIV activity of the compounds since more lipophilic
compounds exhibited higher anti-HIV activity compared to polar
parent nucleoside analogues. As shown previously with other
fatty acyl derivatives of nucleosides,14 the highly lipophilic conju-
gates could have higher cellular uptake that contributed to their
improved anti-HIV activity.
Symmetrical long chain fatty acyl dicarboxylate esters of FLT–
FLT, AZT–AZT, and 3TC–3TC were synthesized. Scheme 1 depicts
the general synthesis of dinucleoside dicarboxylate ester deriva-
tives of FLT–FLT (6–8) and AZT–AZT (9–11). The compounds were
synthesized by reaction of commercially available nucleosides
with three different dicarboxylic acids (sebacic acid, dodecandioic
acid, and tetradecandioic acid) in the presence of 1,1,3,3-tetram-
ethyluronium hexafluorophosphate (HBTU) as coupling reagent
and N,N-diisopropylethylamine (DIPEA) as the base (Scheme 1) in
DMF at room temperature. The final products were purified by
using a reversed phase HPLC.
The Synthesis of fatty acyl dicarboxylate esters of 3TC is shown
in Scheme 2. N4-amino protection of 3TC was required before con-
jugation with dicarboxylic acids. First, tert-butyldimethylsilyl chlo-
ride (TBDMS–Cl) was reacted with 3TC in the presence of imidazole
to afford 50-O-TBDMS-3TC. Then N4-amino group of 50-O-TBDMS-
3TC was further protected with 4,40-dimethoxytrityl (DMTr) pro-
tecting group by reaction with DMTr-Cl in the presence of pyridine.
Finally, TBDMS was removed by using tetrabutylammonium fluo-
ride (TBAF) to yield N4-DMTr-3TC according to the previously re-
ported procedure by us.15 The esterification was carried out with
four different dicarboxylic acids (e.g., succinic acid, sebacic acid,
dodecandioic acid, and tetradecandioic acid) in the presence of
HBTU and DIPEA to afford N4-DMTr protected dinucleoside dicar-
boxylate esters (17–20) as crude products that were isolated by
using extraction. The DMTr group was deprotected by heating
the reaction mixture in 80% acetic acid in water (v/v) at 80 °C to
give di-3TC-dicarboxylate esters (21–24) (Scheme 2).
The chemical structures of the final products were character-
ized by nuclear magnetic resonance spectrometry (1H NMR and
13C NMR), and were confirmed by a high–resolution PE Biosystems
Mariner API time-of-flight electrospray mass spectrometer. The
purity of the final products (>95%) was confirmed by using a
Hitachi analytical HPLC system on a C18 column using a gradient
system (water:acetonitrile 30:70 v/v) at constant flow rate of
1 mL/min with UV detection at 265 nm.
Synthesized dinucleoside ester conjugates were evaluated for
their ability to inhibit HIV-1 (subtype B, US/92/727) and (subtype
IIIB) replication in human PBMC and CEM-SS cells, respec-
tively.16,17 Table 1 illustrates the anti-HIV-1 activity (EC50) and
cytotoxicity (TC50) of the synthesized compounds compared with
their corresponding parent nucleosides. No cytotoxicity was ob-
served up to the highest tested concentration (TC50 >1000 nM)
for the synthesized conjugates.
The anti-HIV activity of 1 (FLT, EC50 = 2 nM) in PBMC’s against
HIV-1US/92/727 was found to be 4 and 45 times higher than 2
(AZT, EC50 = 8 nM) and 12 (3TC, EC50 = 45 nM), respectively. Simi-
larly, the FLT esters of long chain dicarboxylic acids (6–8,
EC50 = 0.76–1.0 nM) were more potent than the corresponding
AZT conjugates (9–11, EC50 = 2.0–4.0 nM) and 3TC conjugates
(22–24, EC50 = 20–60 nM).
A number of symmetrical 50-O-substituted dicarboxylate ester
derivatives of NRTIs were synthesized, and their anti–HIV activity
was evaluated. In general, the conjugation of selected long chain
dicarboxylic acids NRTIs resulted in better anti-HIV profiles than
the corresponding parent nucleosides.
Among all the dinucleoside diester derivatives, FLT–FLT conju-
gates 7 and 8 were found to have better anti-HIV activity than 1
and the other dicarboxylate dinucleoside derivatives. The data
The anti-HIV activity of the 3TC conjugates containing long
chain dicarboxylate (22–24, EC50 = 3–60 nM) was improved by
1.5–66 fold when compared to 3TC (EC50 = 90–200 nM). The 3TC
ester of tetradecandicarboxylic acid (24, EC50 = 3–30 nM) showed
O
O
R1
NH
NH
O
N
O
N
O
OH
OH
HBTU, DIPEA
DMF, rt
O
O
HO
O
n
O
O
n
N
O
O
O
O
O
R1
HN
R1
3
4
5
Sebacic acid, n = 8
Dodecandioic acid, n = 10
Tetradecandioic acid, n = 12
1 (FLT) R1 = F
2 (AZT) R1 = N3
6 R1 = F, n = 8 (48%)
7 R1 = F, n = 10 (42%) 10 R1 = N3, n = 10 (30%)
11
9 R1 = N3, n = 8 (30%)
8
R1 = F, n = 12 (30%)
R1 = N3, n = 12 (30%)
Scheme 1. Synthesis of 50,50-dinucleoside dicarboxylate ester derivatives of FLT (6–8) and AZT (9–11).

H. K. Agarwal et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5451–5454
5453
NH2
NH2
N
N
N
O
N
O
OH
O
TBDMS
DMTr-Cl, Pyridine, rt
TBDMS-Cl,
Imidazole, DMF, rt
O
O
S
S
12 (3TC)
13
DMTr
DMTr
HN
N
HN
O
O
N
O
N
OH
OH
N
O
n
OH
TBDMS
O
TBAF, rt
O
O
HBTU, HOAt, DIC, DIPEA, DMF, rt
S
S
15 (3TC-DMTr)
16 (Succinic acid) n = 2
14
3 (Sebacic acid) n = 8
4 (Dodecandioic acid) n = 10
5 (Tetradecandioic acid) n = 12
NHDMTr
N
NH2
N
S
S
N
O
N
O
O
O
O
O
Acetic acid, 80%,
80 °C
O
O
O
O
n
n
N
O
N
O
O
O
S
O
S
O
N
N
17 n = 2
18
21 n = 2 (45%)
n = 8
19 n = 10
20
22
23
24
n = 8 (51%)
n = 10 (46%)
n = 12 (56%)
NHDMTr
NH2
n = 12
Scheme 2. Synthesis of symmetrical 50,50-dinucleoside dicarboxylate ester derivatives of 3TC (21–24).
Table 1
Anti-HIV activity of dicarboxylic acid esters of dinucleoside conjugates
Compd chemical name PBMC/HIV-1US/92/727
CEM-SS/HIV-1IIIB
Log Pd (Calcd)
EC50 (nM)
TC50 (nM)
TIc
>250
>125
>5.6
>1000
>1031
>1316
>250
>500
>333
>7
EC50 (nM)
TC50 (nM)
TIc
a
b
a
b
1 (FLT)
2 (AZT)
12 (3TC)
6
7
8
9
30-Fluoro-20,30-deoxythymidine
2.0
8.0
90.0
1.0
0.97
0.76
4.0
2.0
3.0
150.0
60.0
20.0
30.0
>500
>1000
>500
20.0
2.0
200.0
4.0
4.0
3.0
5.0
4.0
3.0
>500
>500
>500
>25
>250
>2.5
>250
>250
>333
>200
>250
>333
ꢀ0.41
NDe
0.06
2.12
2.96
3.79
ND
30-Azido-20,30-dideoxythymidine
(ꢀ)-20,30-Dideoxy-30-thiacytidine
1,10-Di-FLT-decanoate
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
1,12-Di-FLT-dodecanoate
1,14-Di-FLT-tetradecanoate
1,10-Di-AZT-decanoate
10
11
21
22
23
24
1,12-Di- AZT-dodecanoate
1,14-Di-AZT-tetradecanoate
1,4-Di-3TC-succinate
1,10-Di-3TC-decanoate
1,12-Di-3TC-dodecanoate
1,14-Di-3TC-tetradecanoate
ND
ND
0.56
3.06
3.90
4.73
130.0
30.0
9.0
>7.7
>17
>50
>33
>33
>333
>333
3.0
a
b
c
EC50 (50% effective concentration), All the assays were carried out in triplicate (n = 3).
TC50 (50% toxic concentration), All the assays were carried out in triplicate (n = 3).
Therapeutic index (TC50/EC50).
Calculated partition coefficient using ChemBioDraw Ultra 12.0.
Not determined.
d
e
indicate that conjugation of the NRTIs with dicarboxylic acids is an
effective strategy in achieving higher anti-HIV activity possibly by
improving the lipophilicity and potentially cellular uptake of the
parent nucleosides. These data provide the basis for rational opti-
mization of NRTIs through conjugation of two nucleosides with
long chain dicarboxylic acids.
Acknowledgements
We acknowledge the National Science Foundation, Grant Num-
ber CHE 0748555 for the financial support and National Center for
Research Resources, NIH, and Grant Number 1 P20 RR16457 for
sponsoring the core facility. The authors wish to acknowledge

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Supplementary data
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037.
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