Synthesis and anti-HIV activities of phosphate triester derivatives of 3′-fluoro-2′,3′-dideoxythymidine and 3′-azido-2′,3′-dideoxythymidine

Article abstract of DOI:10.1016/j.tetlet.2008.05.149

Fatty acyl-glycol phosphate triester conjugates of 3′-fluoro-2′,3′-dideoxythymidine (FLT) were prepared in three steps from the reaction of diisopropylphoramidous dichloride with fatty acyl-substituted glycols, followed by a coupling reaction with FLT and oxidation with tert-butyl hydroperoxide (t-BuOOH). Additionally, a number of fatty alcohols were reacted with diisopropylphoramidous dichloride to produce the phosphitylating intermediates, which underwent coupling reactions with 3′-azido-2′,3′-dideoxythymidine (AZT) and FLT followed by oxidation with t-BuOOH to yield fatty alcohol phosphate triester derivatives of AZT and FLT.

Full text of DOI:10.1016/j.tetlet.2008.05.149

Tetrahedron Letters 49 (2008) 4905–4907
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Tetrahedron Letters
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Synthesis and anti-HIV activities of phosphate triester derivatives of
30-fluoro-20,30-dideoxythymidine and 30-azido-20,30-dideoxythymidine
Hitesh K. Agarwal 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 Virgina 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
Fatty acyl-glycol phosphate triester conjugates of 30-fluoro-20,30-dideoxythymidine (FLT) were prepared
in three steps from the reaction of diisopropylphoramidous dichloride with fatty acyl-substituted glycols,
followed by a coupling reaction with FLT and oxidation with tert-butyl hydroperoxide (t-BuOOH). Addi-
tionally, a number of fatty alcohols were reacted with diisopropylphoramidous dichloride to produce the
phosphitylating intermediates, which underwent coupling reactions with 30-azido-20,30-dideoxythymi-
dine (AZT) and FLT followed by oxidation with t-BuOOH to yield fatty alcohol phosphate triester deriva-
tives of AZT and FLT.
Article history:
Received 5 May 2008
Revised 29 May 2008
Accepted 30 May 2008
Available online 5 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
20,30-Dideoxynucleoside analogs are used clinically against the
human immunodeficiency virus (HIV). There are numerous reasons
to utilize a nucleotide prodrug strategy in order to make anti-HIV
nucleosides more effective against the virus. Bypassing the first
rate-limiting phosphorylation step,1 increasing the lipophilicity,
and enhancing the cellular uptake and half-life in blood are some
of them.2
of a suitable phosphate-masking group. By judicious choice of the
alcohols used in triester formation, it may be possible to improve
cellular uptake and to direct intracellular hydrolysis to nucleoside
monophosphates. Thus, further research to identify prodrugs
containing both phosphotriester and lipophilic groups with
distinct advantages, relative to parent anti-HIV nucleosides, is
warranted.
On entering the cell, the majority of anti-HIV nucleoside ana-
logs, such as 30-fluoro-20,30-dideoxythymidine (FLT, alovudine)
and 30-azido-20,30-dideoxythymidine (AZT, zidovudine), are phos-
phorylated to monophosphates, diphosphates, and triphosphates
forms by host cellular kinases before they show antiviral activity.
Negatively-charged nucleoside monophosphates cannot be di-
rectly used because of their high polarity and poor cellular uptake.
Furthermore, they are readily dephosphorylated on cell surfaces
and in extracellular fluids by non-specific phosphohydrolases. In
order to bypass the first rate-limiting phosphorylation step in the
metabolic conversion of nucleoside analogs, numerous prodrugs
of 50-monophosphate types such as neutral species of phosphotri-
ester derivatives of nucleosides have been proposed2–9 with the
hope that these prodrugs would release the corresponding nucleo-
side-monophosphates intracellularly. The phosphotriesters must
have acceptable stability prior to cellular uptake and selective
intracellular biotransformation of the active species. Furthermore,
extensive efforts have been carried out to synthesize lipophilic pro-
drugs of anti-HIV nucleosides by an esterification approach.2,10–12
Both strategies have yet to provide an anti-HIV prodrug agent with
a clear-cut therapeutic advantage for clinical use. The major chal-
lenge of developing nucleotide prodrugs has been in the selection
Herein, we report the synthesis of uncharged fatty acyl and fatty
alcohol phosphotriester derivatives of AZT and FLT (Fig. 1). The
lipophilic moieties, fatty acyls or fatty alcohols, were incorporated
into the structure with the aim of improving interaction with
membrane bilayers and cellular uptake of anti-HIV nucleoside
phosphotriester derivatives and to release nucleoside monophos-
phates intracellularly, bypassing the first phosphorylation step.
In the first class of compounds, two identical fatty acids were
linked through a glycol linker to a phosphate group, which was
attached to 50-O-position of FLT to afford bis(fatty acyl-glycol)-
phosphate triester derivatives. The selection of fatty acids was
based on the previously reported moderate anti-HIV activities of
12-bromododecanoic acid, 12-azidododecanoic acid, and 12-thio-
ethydodecanoic acid.13 In the second class of compounds, fatty
O
N
O
N
O
O
R
R
NH
O
NH
O
O
O
O
O
R1O
R1O
O
O
P
O
P
O
O
O
R2
F
R
R
1 = CH3(CH2)9- or Br(CH2)11
-
R = CH3(CH2)11-, Br(CH2)11-,
N3(CH2)11-, or C2H5S(CH2)11-,
2 = -F or -N3
* Corresponding author.
E-mail address: kparang@uri.edu (K. Parang).
Figure 1. Fatty acyl and fatty alcohol phosphotriester derivatives of AZT and FLT.
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.05.149

4906
H. K. Agarwal et al. / Tetrahedron Letters 49 (2008) 4905–4907
alcohols and nucleosides, FLT and AZT, were directly attached to a
phosphotriester group.
R
R
O
O
Cl
Cl
R
O
O
O
P
N
DMAP, THF
-80 ºC
O
O
OH
The synthetic procedures used for the synthesis of phosphotri-
ester derivatives of nucleosides were based on P(III) chemistry
using phosphoramidite approach and consisted of three steps: (i)
derivatization of diisopropylphosphoramidous dichloride with
fatty-acyl-glycols or fatty alcohols to afford phosphoramidites,
(ii) reaction of resulting phophoramidates with FLT or AZT in the
presence of ethyl-1H-tetrazole, and (iii) oxidation of P(III) to P(V).
For the synthesis of compounds in the first class, we first
prepared fatty acid-glycol ester conjugates 3a–d (Scheme 1).
2-Hydroxyethyl tetradecanoate (3a) and 2-hydroxyethyl 12-bro-
mododecanoate (3b) were synthesized (70% yield) from the treat-
ment of the corresponding fatty acyl chloride (1a or 1b, 4 mmol)
and ethylene glycol (2, 18 mmol) in the presence of dimethyl-
aminopyridine (DMAP) (5 mmol) in benzene and DMF (Scheme 1).
2-Hydroxyethyl 12-bromododecanoate (3b) was used for the
synthesis of 2-hydroxyethyl 12-azidododecanoate (3c) and 2-
hydroxyethyl 13-thiapenatadecanoate (3d). Bromosubstituted es-
ter conjugate 3b (5.3 mmol) was treated first with sodium iodide
(10.7 mmol) in acetone to yield the corresponding iodosubstituted
glycol ester 3b0 (95%). Compound 3b0 (4.9 mmol) was reacted with
sodium azide (15.4 mmol) in the presence of 12-crown-4 ether
(15.9 mmol) in DMF to yield 3c in 55% yield. Similarly, nucleophilic
reaction of ethanethiol (7.6 mmol) with 3b (6.2 mmol) in the pres-
ence of sodium hydride (8.3 mmol) afforded 12-thioethyl substi-
tuted analog 3d in 60% yield (Scheme 1).
Scheme 2 outlines the synthesis of bis(fatty acyl-glycol)phos-
photriester derivatives of FLT (7a–d) using P(III) chemistry. In
general, fatty acyl-glycol ester conjugates 3a–d (3 mmol) were
treated in THF with diisopropylphosphoramidous dichloride
(1.5 mmol) in the presence of DMAP (3 mmol) at ꢀ80 °C to afford
intermediate bis(fatty acyl-glycol) diisopropyl phosphoramidite
conjugates (5a–d). Low temperature proved to be important for
the success of this coupling reaction as shown by the failure of
the reaction of 3a with diisopropylphosphoramidous dichloride
in the presence of pyridine at room temperature. The intermedi-
ates 5a–d should be used immediately in the next reaction without
purification because of the activity of the phosphorous in trivalent
form in these compounds. Subsequent conversion of phosphorami-
dite intermediates to phosphotriesters was accomplished by treat-
ment with FLT (1.5 mmol) in presence of 5-ethyl-1H-tetrazole
(4.5 mmol) followed by in situ oxidation with tert-butyl hydroper-
oxide (t-BuOOH, 4.5 mmol) to obtain bis(fatty acyl-glycol)phos-
photriester derivatives of FLT (7a–d). The chemical structures of
7a–d were determined by 1H NMR, 13C NMR, 31P NMR, and high-
resolution ESI mass spectrometry (Table 1).
P N
O
3a-d
5a-d
O
N
NH
R
R
O
O
O
O
O
O
FLT
5-ethyl-1H-tetrazole
P
O
t-BuOOH
O
O
F
6a-d
O
N
NH
R
O
R
O
O
O
O
a
CH3(CH2)12
Br(CH2)11
N3(CH2)11
C2H5SCH2)11
-
O
O
b
c
d
P
-
O
R
-
O
-
O
F
7a-d
Scheme 2. Synthesis of fatty acyl-glycol ester conjugates 7a–d.
amino)chlorophosphine in the presence of pyridine. However, the
intermediate was not stable during purification by silica gel col-
umn chromatography. Alternatively, the synthesis of fatty alcohol
phosphotriester derivatives of AZT and FLT (11a–c) was accom-
plished by the reaction of dialkoxy substituted phosphitylating re-
agents, diisopropylamino dialkoxyphosphine, with AZT and FLT in
the presence of 5-ethyl-1H-tetrazole in THF (Scheme 3). First dialk-
oxy substituted phosphitylating reagents were synthesized using
diisopropylphosphoramidous dichloride and different fatty alco-
hols (i.e., decanol and 11-bromoundecanol). Solution of alcohols
8a–c (3 mmol) in THF (20 mL) was added to a mixture of dii-
sopropylphosphoramidous dichloride (1.5 mmol) and DMAP
(3 mmol) in THF (100 mL) at ꢀ80 °C to afford 9a–c. FLT or AZT
(1.5 mmol) and 5-ethyl-1H-tetrazole (4.5 mmol) were added to
the reaction mixture to yield 10a–c. Oxidation of phosphite tries-
ters 10a–c to phosphate triesters was accomplished with t-BuOOH
(4.5 mmol) to afford 11a–c. The chemical structures of 11a–c were
R1 (CH2)10
Cl
Cl
O
DMAP, THF
-80 ºC
P
N
P
N
R1 (CH2)10 OH
O
R1 (CH2)10
8a-c
9a-c
O
For the synthesis of fatty alcohol phosphotriester derivatives of
the nucleosides, AZT was first attached to bis(diisopropyl-
NH
O
N
O
NH
HO
R1 (CH2)10
R1 (CH2)10
O
R
Cl
R
N
O
O
HO
O
OH
OH
P
O
Benzene, DMF
DMAP
t-BuOOH
O
R2
5-ethyl-1
O
O
O
1a or 1b
3a
R = CH3(CH2)12
2
-
H
-tetrazole
3b R = Br(CH2)11
-
R2
10a-c
NaI, acetone
I (CH2)11
O
3b
O
N
OH
3b'
O
NH
C2H5SH, NaH
THF
NaN3,12-crown-4 ether
DMF
R1 (CH2)10
R1 (CH2)10
O
R1
-H
R2
O
O
O
P
O
O
a
b
c
F
F
-CH2Br
-H
C2H5
S
(CH2)11
N3 (CH2)11
O
O
N3
OH
OH
O
R2
O
11a-c
3d
3c
Scheme 3. Synthesis of fatty alcohol phosphotriester derivatives of AZT and FLT
(11a–c).
Scheme 1. Synthesis of fatty acid-glycol ester conjugates 3a–d.

H. K. Agarwal et al. / Tetrahedron Letters 49 (2008) 4905–4907
4907
Table 1
extracellularly. The utility of phosphotriester derivatives of nucleo-
sides will be enhanced by a clearer understanding of the mecha-
nisms pertaining to their bioconversion, uptake, and cellular
incorporation.
The physicochemical characteristics and anti-HIV activities of compounds 7a–d and
11a–c
Compd.
no.
31P NMRa
(d, ppm)
HR-MS (ESI-TOF)
Anti-HIV
Overall
yield (%)
b
IC50
(
lM)
7a
7b
4.79
4.78
855.4673 [M+Na]+
955.4436 [M+Na]+,
976.3518, [M+2Na]+
859.3552 [M+H]+,
881.3245 [M+Na]+,
897.2941 [M+K]+
897.3566 [M+H]+
605.0470 [M+H]+,
642.9381 [M+K]+
789.9278 [M+H]+
628.3108 [M+H]+
>100
63
6.3
7.7
Acknowledgments
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). The views ex-
pressed by the authors do not necessarily reflect the views of
USAID or CONRAD.
7c
2.81
76
6.5
7d
11a
2.82
5.09
87
>100
12.5
14.1
11b
11c
5.03
5.08
33
>100
7.0
19.8
Supplementary data
a
The spectra were measured on a 400 MHz spectrometer using CDCl3 as the
solvent (H3PO4 85% in water as external standard).
Detailed synthetic procedure, 1H NMR, 13C NMR, and/or 31P
NMR spectra of compounds. Supplementary data associated with
this article can be found, in the online version, at doi:10.1016/
j.tetlet.2008.05.149.
b
IC50: 50% inhibitory concentration.
determined by 1H NMR, 13C NMR, 31P NMR, and high-resolution ESI
mass spectrometry (Table 1).
Using a single-round infection assay14 with HIV-1 IIIB and
transformed HeLa cells expressing HIV receptors (CD4) and core-
ceptors (CXCR4 and CCR5), the newly synthesized triester deriva-
tives showed only modest antiviral activity, significantly lower
than that of their parent nucleosides, AZT and FLT (IC50 = 10 and
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sis of different classes of lipophilic phosphate triesters of FLT and
AZT can be successfully accomplished by using P(III) chemistry.
The extension of this methodology should prove to be useful for
the development of lipophilic phosphotriester prodrugs of other
nucleosides. The premature hydrolysis of the phosphate-masking
group bond in the extracellular medium, however, may have
yielded a negatively-charged diester with low cellular uptake and
reduced antiviral potency. The phosphotriesters must have accept-
able stability in cell culture prior to cellular uptake and selective
intracellular transformation of the active species. We were not able
to determine the stability of compounds because of their extre-
mely low water solubility. The extracellular hydrolysis of phospho-
triester derivatives of nucleosides has been previously reported.
For example, McGuigan et al.15,16 reported that some of dialkyl
and diaryl phosphotriester derivatives of AZT were inactive be-
cause of the rapid in vitro hydrolysis to release the nucleotide
16. McGuigan, C.; Pathirana, R. N.; Davis, M. P. H.; Balzarini, J.; De Clercq, E. Bioorg.
Med. Chem. Lett. 1994, 4, 427–430.