Synthesis and anti-HIV activities of bis-(cycloSaligenyl) pronucleotides derivatives of 3′-fluoro-3′-deoxythymidine and 3′-azido- 3′-deoxythymidine

Article abstract of DOI:10.1016/j.tetlet.2010.12.038

Anti-HIV nucleoside monophosphates have limited cellular uptake due to the presence of negatively-charged phosphate group. Bis-(cycloSaligenyl) derivatives containing two anti-HIV nucleosides, 3′-fluoro-3′-deoxythymidine (FLT) and 3′-azido-3′-deoxythymidi

Full text of DOI:10.1016/j.tetlet.2010.12.038

Tetrahedron Letters 52 (2011) 802–805
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and anti-HIV activities of bis-(cycloSaligenyl) pronucleotides
0
0
0
0
derivatives of 3 -fluoro-3 -deoxythymidine and 3 -azido-3 -deoxythymidine
a,b,
⇑
a
c
c
c
Yousef Ahmadibeni
, Rakesh Tiwari , Chelsie Swepson , Jui Pandhare , Chandravanu Dash ,
d
a,
⇑
Gustavo F. Doncel , Keykavous Parang
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island, Kingston, RI 02881, USA
Department of Chemistry, Columbus State University, Columbus, GA 31907, USA
Centre for AIDS Health Disparities Research, Meharry Medical College, Nashville, TN 37208, USA
CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23507, USA
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Anti-HIV nucleoside monophosphates have limited cellular uptake due to the presence of negatively-
Received 29 September 2010
Revised 7 December 2010
Accepted 8 December 2010
Available online 15 December 2010
charged phosphate group. Bis-(cycloSaligenyl) derivatives containing two anti-HIV nucleosides,
0
0
0
0
3
-fluoro-3 -deoxythymidine (FLT) and 3 -azido-3 -deoxythymidine (AZT) were synthesized to increase
intracellular delivery of nucleoside monophosphates. 2,5-Bis(hydroxymethylene)benzene-1,4-diol was
selected as monocyclic bidentate scaffold and synthesized by three different methods from
a
bis(hydroxymethylene)cyclohexan-1,4-diene-1,4-diol, or diethyl 2,5-dihydroxyterephthalate. The reac-
tion of the tetraol with diisopropylphosphoramidous dichloride in the presence of 2,6-lutidine, followed
by conjugation reactions with nucleosides (i.e., FLT and AZT) and oxidation afforded symmetrical and
unsymmetrical bis-(cycloSaligenyl) diphosphate triester products, AZT–AZT, FLT–FLT, and FLT–AZT con-
Keywords:
Nucleosides
Bis-(cycloSaligenyl)
AZT
jugates, in 63–74% overall yields and modest anti-HIV activities (IC50 = 2.8–69.6 lM).
FLT
Anti-HIV
Ó 2010 Elsevier Ltd. All rights reserved.
Nucleoside analogs are commonly used as antiviral or antican-
cer agents. Cellular nucleoside kinases mediate three phosphory-
tide delivery systems with higher ratios of masking unit, for
example, two to four for a single unit of drug (2:1–4:1). Ducho
1
8
0
lation steps, which convert the nucleosides into their active
et al. recently reported synthesis of symmetrical 3,3 -bis-(cyclo-
2
0
0
0
0
nucleoside triphosphate analogs (Fig. 1). The first phosphorylation
Sal) derivatives of 2 ,3 -dideoxy-2 ,3 -didehydrothymidine (d4T)
using a bicyclic tetraol to deliver two similar nucleosides that
led to a significant improvement of anti-HIV activity when com-
3
to nucleoside monophosphate is often the rate-limiting step. Neg-
atively charged nucleoside monophosphates cannot be used as
therapeutic agents because they are unable to cross cell mem-
branes efficiently, and they are readily dephosphorylated on cell
surfaces and in extracellular fluids by non-specific enzymes. Thus,
several masked nucleoside phosphate mimics have been synthe-
9
pared to the parent nucleoside d4T. Gisch et al. synthesized
0
5,5 -bis-cycloSal-d4T to deliver intracellularly two molecules of
d4T-MP that showed improved anti-HIV activity when compared
0
to 3,3 -bis-cycloSal-d4T derivatives in thymidine kinase-deficient
sized as prodrugs with the aim of delivering the corresponding
cells.
0
5
-monophosphate derivative intracellularly and bypassing the ini-
The reported synthesis of the bis-(cycloSal) derivatives is chal-
lenging due to a number of synthetic steps for the synthesis of
bicyclic tetraol, side reactions between the reactants and reagents,
tedious purification steps, low yield, and instability of the interme-
diates. Therefore, alternative synthetic methodologies are still
required to improve the synthesis of bis-(cycloSal) derivatives
and their substituted pronucleotides. Furthermore, to the best of
our knowledge the synthesis of unsymmetrical bis-(cycloSal) deriv-
atives having two different nucleosides has not been previously re-
ported. Herein, we report the synthesis of novel symmetrical and
unsymmetrical derivatives of bis-(cycloSal) derivatives of AZT and
FLT (Fig. 2) from a new scaffold bis(hydroxymethylene)benzene-
4
tial phosphorylation step. cycloSaligenyl (cycloSal) pronucleotide
system has been introduced as a prodrug approach for cellular
delivery of several nucleoside monophosphates. The intracellular
cleavage of an optimal cycloSal pronucleotide to the monophos-
phate analog is based on intracellular pH-driven chemical
5
hydrolysis.
Two or more masking groups are required to generate neutral
phosphate triester prodrug of nucleosides. The cycloSal pronu-
cleotide system releases one molecule of drug per masking unit
mask to drug ratio, 1:1) as compared to some other pronucleo-
6,7
(
1
,4-diol (4). It was expected that the individual components of
⇑
Corresponding authors.
E-mail address: kparang@gmail.com (K. Parang).
multifunctional compounds (nucleoside monophosphates) would
exhibit biological activity, even enhanced efficacy, when released
0
040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.12.038

Y. Ahmadibeni et al. / Tetrahedron Letters 52 (2011) 802–805
803
O
P
O
X
ONu
O
OH
OH
pH-driven hydrolysis
X
after interacellular delivery
O
P
O
P
O
P
O
O
O
Cellular Kinase
rate-limiting step
Cellular Kinase
Cellular Kinase
Nu
NuO
P
O
NuO
P
O
P
O
NuO
O
O
O
O
O
O
O
O
O
Nu = Nucleoside
X = substituent
Figure 1. Nucleoside phosphorylation and cycloSal delivery system of nucleoside monophosphate.5
O
time-of-flight electrospray mass spectrometry (ESI-TOF), and was
used as a scaffold for the synthesis of bis-(cycloSal) derivatives.
O
O
P
P
O
O
O
OR
2
The reaction of tetraol 4 (0.5 mmol) with phosphorous
R
1
O
trichloride (1 mmol) and 2,6-lutidine (2 mmol) in anhydrous
tetrahydrofuran afforded the bis-(cycloSal chlorophosphite) 5
under controlled temperature (ꢁ78 °C to ꢁ15 °C) (Scheme 2). The
addition of 2,6-lutidine (1 mmol) followed by the dry nucleoside
R OH and R OH = nucleosides (AZT, FLT)
1
2
Figure 2. Symmetrical and unsymmetrical derivatives of bis-(cycloSal) derivatives
of AZT and FLT.
(
AZT or FLT) (1 mmol), to the same reaction mixture under the con-
trolled conditions of the temperature (ꢁ55 °C to ꢁ10 °C) afforded
the corresponding symmetrical phosphite triester derivatives 6a
and 6b. The oxidation of 6a and 6b by TBHP and purification by
preparative HPLC afforded the symmetrical bis-(cycloSal) phos-
phate triester derivatives FLT–FLT (7a, 74%) and AZT–AZT (7b,
71%) as a diastereomeric mixture. The separation of diastereomers
was not possible for 7a and 7b.
intracellularly by degradative metabolism of the conjugate. Devel-
opment of resistant virus is a serious problem for any antiviral
drug. This combination of nucleosides using unsymmetrical bis-
(cycloSal) may enhance the activity of conjugates against resistant
strains. The development of resistance to nucleosides would occur
at a slower rate than to either compound alone.
Bis(hydroxymethylene)benzene-1,4-diol (4) was introduced as
a novel monocyclic tetraol skeleton for the synthesis of bis-cycloSal
derivatives of dideoxynucleosides. The scaffold was synthesized by
using three different methods and was reacted with phosphorous
trichloride to afford the cycloSal chlorophosphite. The intermediate
was reacted with nucleosides under the controlled conditions of
stoichiometry and temperature to afford the trivalent phosphite
derivatives of nucleosides. Subsequent oxidation using tert-butyl
hydroperoxide (TBHP) and purification afforded bis-(cycloSal)
derivatives of AZT and FLT in moderate yields.
In a separate reaction, tetraol 4 was reacted with phosphorus
trichloride in the presence of 2,6-lutidine at ꢁ78 °C in anhydrous
tetrahydrofuran to afford intermediate 5 as described above
(Scheme 2). Then the reaction temperature was kept at ꢁ55 °C
using dry ice and acetone. To the reaction mixture was added
2,6-lutidine (1 mmol) followed by dry nucleoside AZT (0.5 mmol)
(Scheme 3). The reaction was continued for 85 min with steady in-
crease of the temperature to ꢁ50 °C. The second nucleoside FLT
(0.5 mmol) was added and the temperature was raised to ꢁ10 °C
over a period of 95 min. Oxidation with TBHB and HPLC purifica-
tion afforded the unsymmetrical bis-(cycloSal) phosphate triester
of AZT–FLT 10 (63%) as a diastereomeric mixture (Scheme 3).
Scheme 1 illustrates the synthesis of 2,5-bis(hydroxymethyl-
ene)benzene-1,4-diol 4. 2,5-Bis(methoxycarbonyl)cyclohexa-1,4-
diene-1,4-diol 1 was oxidized by refluxing in the presence of
excess of activated manganese dioxide in toluene to afford the
Compound 7b showed only modest antiviral activity (IC50
69.6 M), significantly lower than that of the parent nucleoside,
FLT (IC = 0.8 M), when tested using a single-round infection as-
=
l
2
,5-bis(methoxycarbonyl)benzene-1,4-diol 2. Reduction of com-
l
5
0
1
0
pound 2 using lithium borohydride in methanol and anhydrous
diethyl ether or lithium aluminium hydride in THF afforded the
corresponding tetraol 4 after purification in 44% and 52% overall
yields, respectively. An alternative method was used to synthesize
say using HIV-1 X4 tropic strains and transformed HeLa cells
expressing HIV receptors (CD4) and coreceptors (CXCR4). Com-
pounds 7a and 10 exhibited IC50 values of 12.3 and 2.8
lM, respec-
tively, while control scaffold 4 had an IC50 value of 86.5
lM. These
4
(60% overall yield) by reducing the corresponding diethyl-2,5-
data suggest that the presence of both AZT and FLT in the same
conjugate generates higher anti-HIV activity as was shown in 10
when compared with 7a and 7b. There are several factors which
determine in vitro anti-HIV activity of each analog that may
dihydroxyterephthalate 3 with BH
tions. In all cases the crude product 4 was purified by column
chromatography and characterized by NMR and high-resolution
3
ꢀTHF under anhydrous condi-
O
O
OH
OH
LiBH , MeOH/ether
MnO2
MeO
4
MeO
HO
OMe
Toluene
OMe
or LiAlH , THF
4
HO
1
O
2
O
OH
OH
EtOOC
HO
OH
HO
BH
3
HO
COOEt
THF
4
3
Scheme 1. Synthesis of 2,5-bis(hydroxymethylene)benzene-1,4-diol 4.

8
04
Y. Ahmadibeni et al. / Tetrahedron Letters 52 (2011) 802–805
OH
OH
PCl (1.0 mmol)
O
Cl
R-OH (1 mmol)
HO
HO
3
O
P
P
O
2
-
,6-Lutidine (2 mmol)
78ºC to -15ºC, THF
2,6-Lutidine, (1 mmol)
-55ºC to -10ºC, THF
Cl
O
4
(0.5 mmol)
5
OR
O
O
OR
7a, ROH=AZT
O
P
P
O
O
P
P
O
TBHP (4 mmol)
3h, -10ºC to RT
O
O
7
b, ROH=FLT
O
RO
O
RO
6
6
a, ROH=AZT
b, ROH=FLT
H
O
N
O
O
N
O
O
O
O
P
O
O
N
3
P
7a
N
3
O
O
H
N
N
O
O
O
O
N
H
O
N
O
O
O
O
O
P
O
F
P
O
7b
F
O
O
N
O
O
N
O
H
Scheme 2. Synthesis of symmetrical bis-(cycloSal) derivatives of AZT–AZT 7a and FLT–FLT 7b.
R -OH, (0.5 mmol)
O
Cl
R -OH, (0.5 mmol)
2
1
5
O
P
P
O
(
0.5 mmol)
THF, -55 ºC to -10 ºC
2,6-Lutidine, (1 mmol)
R
1
O
O
-
55 ºC to- 50 ºC, THF
8
OR2
O
OR2
O
TBHP (4 mmol)
h, -10 ºC RT
O
P
P
O
O
P
P
O
R OH=FLT
1
2
O
O
O
N
R
OH=AZT
3
O
R
1
O
O
R
1
1
0
9
H
O
N
O
O
O
O
O
P
O
O
F
P
O
10
N
3
O
O
N
O
O
N
H
O
Scheme 3. Synthesis of unsymmetrical derivative of bis-(cycloSal) analog of FLT and AZT (10).
include stability, rate of cellular uptake, nature of nucleoside, and
extracellular and intracellular rate of hydrolysis. Structure–func-
tion analysis revealed that the anti-HIV activity of bis-(cycloSalige-
nyl) diphosphate triester products, AZT–AZT, FLT–FLT, and
FLT–AZT was clearly dependent on the nature of the nucleoside
in the conjugate. These results suggest that the increased inhibi-
tion by 10 may be due to the higher cellular uptake, enhanced
intracellular release of FLT and AZT, and/or stability of the conju-
gate when compared with 7a and 7b. The extracellular hydrolysis
of phosphate triester derivatives of FLT and AZT has been previ-
uptake and decreased anti-HIV activity. The rate of extracellular
hydrolysis remains to be determined to compare the stability of
the conjugates. Cytotoxicity assays revealed that compounds were
not cytotoxic at the highest concentration tested (CC50 >100 lM).
To the best of our knowledge, this is first report on the synthesis
of symmetrical and unsymmetrical bis-(cycloSal) derivatives of FLT
and AZT from a monocyclic scaffold, bis(hydroxymethylene)ben-
zene-1,4-diol. This one-pot strategy from a tetraol to the target
compounds minimizes side reactions and eliminates the need for
the purification of intermediates. The synthesis of these com-
pounds provides insights for design of more optimal scaffolds of
bis-(cycloSal) nucleoside derivatives with higher stability, cellular
uptake, and anti-HIV activity.
1
1
ously reported. The partial or complete premature extracellular
hydrolysis of the bis-(cycloSal) group in all analogs may produce
negatively-charged phosphate derivatives with limited cellular

Y. Ahmadibeni et al. / Tetrahedron Letters 52 (2011) 802–805
805
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.
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Acknowledgments
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Balzarini, J.; De Clercq, E. 2 ,3 -Dideoxynucleoside Analogues as Anti-HIV
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Y. Ahmadibeni and R. Tiwari contributed equally. We acknowl-
edge the financial support from National Science Foundation, Grant
Number CHE 0748555. Partial Support for this subproject (MSA-
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under a Cooperative Agreement (GPO-8-00-08-00005-00) with the
United States Agency for International Development (USAID). The
views expressed 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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Supplementary data
Supplementary data (experimental procedures and character-
ization of final compounds with 1H NMR, C NMR, P NMR, and
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