An investigation into the anti-HIV activity of 2′,3′-didehydro- 2′,3′-dideoxyuridine (d4U) and 2′,3′-dideoxyuridine (ddU) phosphoramidate 'ProTide' derivatives

Article abstract of DOI:10.1039/b904276h

As part of our studies on the anti-HIV activities of 2′,3′- didehydro-2′,3′-dideoxyuridine (d4U), 2′,3′- dideoxyuridine (ddU) and their 'ProTides', we have prepared and evaluated the anti-HIV activity of a range of d4U and ddU aryl triester phosphoramidates. Besides elucidating SAR characteristics, we performed molecular modelling studies on both d4U and ddU in order to probe the first phosphorylation step required for the activation of these two nucleoside analogues. Overall, the application of the phosphoramidate approach turned the inactive ddU to a moderately active anti-HIV agent, while this was not the case with d4U. Enzymatic assays investigating the metabolism of d4U phosphoramidates revealed an efficient cleavage of the phosphoramidate motif to release the d4U monophosphate. Thus, a poor second and/or third phosphorylation step may be the most likely reason for the virtual lack of anti-HIV activity in this case. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b904276h

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www.rsc.org/obc | Organic & Biomolecular Chemistry
An investigation into the anti-HIV activity of 2¢,3¢-didehydro-2¢,3¢-
dideoxyuridine (d4U) and 2¢,3¢-dideoxyuridine (ddU) phosphoramidate
‘ProTide’ derivatives†
Youcef Mehellou,a Jan Balzarinib and Christopher McGuigan*a
Received 3rd March 2009, Accepted 9th April 2009
First published as an Advance Article on the web 30th April 2009
DOI: 10.1039/b904276h
As part of our studies on the anti-HIV activities of 2¢,3¢-didehydro-2¢,3¢-dideoxyuridine (d4U),
2¢,3¢-dideoxyuridine (ddU) and their ‘ProTides’, we have prepared and evaluated the anti-HIV activity
of a range of d4U and ddU aryl triester phosphoramidates. Besides elucidating SAR characteristics, we
performed molecular modelling studies on both d4U and ddU in order to probe the first
phosphorylation step required for the activation of these two nucleoside analogues. Overall, the
application of the phosphoramidate approach turned the inactive ddU to a moderately active anti-HIV
agent, while this was not the case with d4U. Enzymatic assays investigating the metabolism of d4U
phosphoramidates revealed an efficient cleavage of the phosphoramidate motif to release the d4U
monophosphate. Thus, a poor second and/or third phosphorylation step may be the most likely reason
for the virtual lack of anti-HIV activity in this case.
Introduction
Human immunodeficiency virus (HIV), the etiologic cause of
AIDS,1,2 is still one of the most fatal infections, and responsible for
killing millions of people worldwide. Currently dideoxynucleoside
analogues form a major class of drugs that are approved for com-
bating this infection.3 Such agents are structurally characterised
by lacking the 3¢-hydroxyl group, which gives them the ability
to be used as a substrate by HIV reverse transcriptase (HIV-
RT) resulting in DNA chain termination after being converted
to their corresponding 5¢-triphosphates by cellular enzymes.4,5
However, the emergence of resistance and toxicity as well as
dependence on host nucleoside kinase-mediated activation to
generate the bioactive phosphates, have limited the value of
these drugs. This highlights the need for new, potent anti-HIV
drugs with acceptable toxicity profiles. Encouraged by the success
of 2¢,3¢-didehydro-2¢,3¢-dideoxythymidine (d4T) as an anti-HIV
agent, we initiated a study aimed at investigating the anti-HIV
activity of 2¢,3¢-didehydro-2¢,3¢-dideoxyuridine (d4U), which is
structurally similar to d4T, as well as its 2¢,3¢-dideoxy analogue,
2¢,3¢-dideoxyuridine (ddU, Fig. 1).
Fig. 1 Chemical structure of d4T (an FDA-approved anti-HIV drug),
d4U and ddU.
of several clinically approved anti-HIV drugs, and compared their
docking to that of thymidine. We also prepared a series of d4U and
ddU ‘ProTides’ in order to bypass the first phosphorylation step,
which out of the three phosphorylations required for the activation
of nucleoside analogues, is often considered to be the bottleneck
for activation of such compounds. In this ‘ProTide’ approach,
the phosphate group is masked to improve the poor membrane
permeability seen with the free (charged) nucleotides and the
inherent susceptibility to dephosphorylation. Upon entering the
cell, the group masking the phosphate moiety may undergo
enzymatic metabolism to release the corresponding nucleoside
monophosphate, which may be subsequently phosphorylated by
human kinases into the di- and triphosphates.6
In this work, we will report both the results of the molecular
modelling studies as well as the effect of the ‘ProTide’ approach on
the anti-HIV activity of d4U and ddU. In addition, we will report
on the results of the enzymatic metabolic assays carried out to
investigate the efficiency of the d4U monophosphate release from
the ProTide.
Both of these nucleoside analogues, d4U and ddU, have been
found to possess poor anti-HIV activity.6,7 In this study, we inves-
tigated whether or not the first phosphorylation step is behind the
poor anti-HIV activity seen with these agents. We accomplished
this via two different strategies. Firstly, we performed the docking
of both d4U and ddU into the human thymidine kinase (TK-1),
which has been shown to be responsible for the phosphorylation
aWelsh School of Pharmacy, Cardiff University, King Edward VII Av-
enue, Cardiff, UK CF10 3NB. E-mail: mcguigan@cf.ac.uk; Fax: +44
2920874537; Tel: +44 2920874537
Results and discussion
bRega Institute for Medical Research, Katholieke Universiteit Leuven,
B-3000, Leuven, Belgium
1. Docking
† Electronic supplementary information (ESI) available: Experimental
details for the preparation and characterisation of d4U, ddU and their
phosphoramidate derivatives. See DOI: 10.1039/b904276h
The fact that d4U and ddU exerted poor anti-HIV activities has led
us to hypothesise that this could be due to the failure of the host cell
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thymidine kinase to efficiently phosphorylate these two nucleoside
analogues to their 5¢-mono phosphate derivatives. This hypothesis
was more of a conceivable explanation after the observations by
Hao et al.,7 that 2¢,3¢-dideoxyuridine (ddU) was a poor inhibitor of
HIV, while its triphosphate derivative was found to possess potent
activity versus HIV reverse transcriptase (Ki, 0.05 mM). Working
upon our hypothesis, we carried out some molecular modelling
studies investigating the first phosphorylation step required for
the activation of d4U and ddU. Thus, both 2¢,3¢-didehydro-2¢,3¢-
dideoxyuridine (d4U) and 2¢,3¢-dideoxyuridine (ddU) were docked
(see experimental) separately into the active site of thymidine
kinase (human TK-1) and compared with the natural substrate
thymidine. The resulting structures for the two nucleoside ana-
logues were ranked according to the relative position of the
uracil base with the corresponding position of the thymine base
in the crystal structure of thymidine, and only the results with
were prepared bearing different ester and aryl moieties, while the
amino acid was kept as L-alanine. Phosphoramidates bearing
L-alanine were found to be effective in improving the biological
activity of different nucleoside analogues, particularly d4T,11 as
were phosphoramidates with either phenyl or naphthyl as their aryl
moieties.12 Thus, we chose to make phosphoramidate derivatives
of both d4U and ddU with different amino acids, mainly L-alanine,
and two aryl moieties of phenyl and 1-naphthyl.
D4U can be synthesised via different routes.13,14 In this study, it
was prepared via the same procedure adopted by McGuigan et al.15
(see ESI†), which was originally described by Horwitz et al.13 Thus,
2¢-deoxyuridine was firstly 3¢,5¢-dimesylated and then reacted with
aqueous sodium hydroxide in 52% yield, which was followed by
treating the product with sodium hydride in DMF to give the
desired product (d4U) in a moderate yield, 43%. Hydrogenation
of d4U in the presence of palladium on activated carbon gave ddU
in a 69% yield.
˚
RMSD values below 1.0 A were further analysed. The best results
(Fig. 2) showed that both d4U and ddU had their 5¢-hydroxyl
group placed significantly far away from the optimal (ideal) site
of phosphorylation where thymidine placed its 5¢-hydroxyl group,
D4U and ddU phosphoramidates were synthesised according to
the previously reported synthetic routes developed by McGuigan
et al.15–18 (Scheme 1). Aryl phosphochloridates were prepared
by the reaction of phenyl/naphthyl dichlorophosphates with
the appropriate amino acid ester hydrochloride. The obtained
phosphochloridates were allowed to react with d4U or ddU in
THF and tBuMgCl to give target phosphoramidates in moderate
yields. 31P NMR investigations of the phosphoramidates displayed
two closely spaced signals, corresponding to two diastereoisomers
resulting from mixed phosphate stereochemistry. All the phospho-
ramidate samples presented in this work (Table 1) were further
investigated as a 1 : 1 mixture of phosphate diastereoisomers,
unless stated otherwise (see experimental and ESI†).
The parent nucleoside d4U and its ‘ProTide’ derivatives were
evaluated for their activity against HIV-1 and HIV-2. The bio-
logical data revealed that neither the parent compound nor any
of its phosphoramidates possessed significant anti-HIV activity
(Table 2). Generally, all the tested compounds were shown to have
low, if any, toxicity.
˚
˚
1 A for ddU and 2 A for d4U. Thus, this would most likely have
a negative effect on the efficiency of the first phosphorylation step
and as a result decreases the chances of d4U and ddU to be further
phosphorylated to their di- and (potentially active) triphosphate
derivatives. In addition, the conformations of the sugars of ddU
and thymidine were different. Indeed, ddU adopted a North
conformation while the natural substrate, thymidine, adopted
a South conformation. The sugar conformation of nucleoside
analogues is well documented to be crucial for the efficiency of
phosphorylation,8–10 as the non-preferred sugar conformation for
kinases may result in reduced efficiency of the phosphorylation
process or even prevent phosphorylation at all. Overall, the reasons
presented discussed above led us to hypothesize that bypassing the
first phosphorylation step, using the phosphoramidate ProTide
approach, might improve the biological activity of d4U and ddU.
The failure of d4U phosphoramidates to exert significant anti-
HIV activity could be attributed to three different reasons. Firstly,
the inability of d4U ‘ProTides’ to deliver d4U monophosphate.
Secondly, the failure of d4U-monophosphate to be further phos-
phorylated to its di- and triphosphate forms and/or thirdly, the
triphosphate of d4U being inactive as an inhibitor of HIV-RT.
Recently, researchers from Gilead sciences reported the inhibitory
2. D4U and ddU phosphoramidates
Since the docking results (Fig. 2) suggested the first phosphoryla-
tion might be responsible for the poor anti-HIV activity of both
d4U and ddU, we decided to synthesise d4U and ddU and then
apply to them the ‘ProTide’ approach in order to bypass the first
phosphorylation step. A series of d4U and ddU phosphoramidates
Fig. 2 Docking results of d4U (purple) and thymidine (left) as well as ddU (green) and thymidine (right).
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Table 1 Structures of d4U and ddU phosphoramidates
Compound
R1
R2
Compound
R1
R2
1a
1b
1c
1d
1e
1f
Phenyl
1-Naphthyl
Phenyl
1-Naphthyl
Phenyl
1-Naphthyl
Phenyl
1-Naphthyl
Phenyl
1-Naphthyl
Methyl
Methyl
Ethyl
2a
2b
2c
2d
2e
2f
2g
—
—
—
Phenyl
1-Naphthyl
Phenyl
Phenyl
Phenyl
1-Naphthyl
4-Methoxy-1-naphthyl
—
—
—
Methyl
Methyl
Ethyl
tButyl
Benzyl
Benzyl
Benzyl
—
Ethyl
iPropyl
iPropyl
tButyl
tButyl
Benzyl
Benzyl
1g
1h
1i
—
—
1j
Scheme 1 Reagents and conditions: (i) Et3N, -78 ◦C (ii) THF, d4U, tBuMgC1, N2, rt (iii) THF, d4U, tBuMgC1,N2, rt.
Table 2 Anti-HIV activity and cytostatic properties of d4U and its
being inactive against HIV (Table 3). In particular the 1-naphthyl
derivatives were more active than their phenyl counterparts. This
indicates that a successful bypass of the first phosphorylation step
can turn ddU from an inactive compound to a moderately active
anti-HIV agent. Interestingly, only compound 2d, which has a
tbutyl as an ester moiety was inert against HIV. This strongly sup-
ports phosphate delivery via ester cleavage as the mode of action
of the other ddU ProTides.20 Notably, ddU phosphoramidates
retained their anti-HIV activity in cells deficient of thymidine
kinase, thus confirming the ability of the phosphoramidates to
deliver ddU monophosphate into cells. As for d4U, the tested
compounds showed low, if any, cytostatic activity.
In terms of SAR, we have found the naphthyl derivatives
to be more active than the phenyl counterparts. This could be
attributed to the higher logP of the naphthyl ‘ProTides’ compared
to those of the phenyl compounds. These higher logP values could
translate in vitro to a better membrane permeability and eventu-
ally better delivery of the nucleoside analogue monophosphate.
Generally, the tbutyl ester phosphoramidates are poor substrates
for ester hydrolysis as suggested by Perrone et al.20 Therefore,
phosphoramidates bearing tbutyl esters are unlikely to undergo
the first hydrolysis activation step required for the activation of
phosphoramidates.
phosphoramidates in CEM cell cultures
EC50 / mMa
Compound
HIV-1
HIV-2
CC50 / mMb
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
d4U
d4T
>50
>10
>50
>50
>250
200 70
>250
>50
>10
>10
>50
>50
>250
185 92
>250
>50
97 13
17 13
212 54
214 62
>250
>250
>250
102
0
>50
>50
106 7.1
NDc
>50
NDc
>50
NDc
207 61
174
0.65
0.77
a EC50: the effective concentration (mM) required to protect CEM cells
against the cytopathogenicity of HIV by 50%. b CC50: the cytostatic
concentration (mM) required to inhibit CEM cell proliferation by 50%.
c ND: no data.
effect of d4U triphosphate on HIV reverse transcriptase, and
noted potent inhibiton of this enzyme, IC50 = 0.55 mM.19 Thus,
in this study we decided to investigate the first possibility, which
is the failure of d4U phosphoramidates to be metabolised to d4U
monophosphate, as will be discussed later.
3. Metabolic enzymatic assay
By comparison, the ‘ProTide’ derivatives of ddU were found to
exert modest anti-HIV activity despite the parent nucleoside, ddU,
The metabolism of aryl triester phosphoramidates is thought to
proceed in four steps to eventually release the nucleoside analogue
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Table 3 Anti-HIV activity and cytostatic properties of ddU and its
To reveal whether CEM cells contain the enzymes required for
hydrolysis (activation) of the prodrugs, the naphthyl L-alanine
benzyl ester phosphoramidates of d4U and ddU were exposed
to CEM cell extracts for 30, 60 and 120 minutes at 37 ◦C. The
prodrugs were hydrolysed for more than 90% (d4U prodrug) or
95% (ddU prodrug) within 30 min. After 60 to 120 min, no intact
parent compound remained. Since marked differences in cellular
uptake of both prodrugs would be rather unlikely, and given the
fact that d4U-TP has been reported to be a potent HIV-1 RT
inhibitor,7 our data suggest that the bottleneck for the eventual
antiviral activity of the prodrug of d4U-MP may reside in the
phosphorylation to the di- and/or triphosphate derivative.
phosphoramidates
EC50 / mMa
CC50 / mMb
CEM/0
CEM/TK-
CEM/0
Compound
HIV-1
HIV-2
≥50
HIV-2
40.0 014.1
>250
175 106
>250
50.8 41.6
22.5 10.6
21.0 8.5
>250
2a
36.7 11.5
≥50
121 1.4
101.9 4.7
≥250
2b
≥20
2c
43.3 20.8
>250
93.3 32.1
>250
33.2 5.1
20.0 7.1
30.0 7.1
>250
2d
>250
2e
40.8 31.7
15.0 0.0
20.0 7.1
>250
96.4 82
96.9 0.85
90.9 10.0
>250
2f
2g
ddU
d4T
0.65
0.77
—
174
Conclusion
a EC50: the effective concentration (mM) required to protect CEM cells
against the cytopathogenicity of HIV by 50%. b CC50: the cytostatic
concentration (mM) required to inhibit CEM cell proliferation by 50%.
In conclusion, d4U and ddU are two nucleoside analogues
with poor anti-HIV activity. We have shown in this study,
using molecular modelling, that these two nucleoside analogues
occupy a position at the active site of thymidine kinase, which
is not favourable for phosphorylation. Applying the ‘ProTide’
approach to bypass the first phosphorylation step turned ddU
from an inactive compound into a moderately active anti-HIV
compound. However, this was not the case for d4U as none
of its ‘ProTides’ showed any appreciable anti-HIV activity. Our
enzymatic metabolic assays revealed that d4U phosphoramidates
were metabolised to release d4U monophosphate, and since d4U
triphosphate is a potent inhibitor of HIV reverse transcriptase, we
concluded that d4U phosphoramidates lack potent anti-HIV ac-
tivity because of poor, if any second and/or third phosphorylation
steps.
monophosphate (Scheme 2). The first step in this metabolic
process is mediated by esterase-type activity, which cleaves the
ester motif of the aryl triester phosphoramidate molecule. One
enzyme responsible for this intracellular cleavage has been recently
described as cathepsin A.21
Since we found d4U phosphoramidates to be devoid of any
anti-HIV activity, we hypothesised that one of the reasons for the
absence of activity was that d4U phosphoramidates did not un-
dergo intracellular metabolism to release the nucleoside analogue
monophosphate. To investigate this possibility, we conducted the
enzymatic metabolism assays of d4U phosphoramidates using
cathepsin A. In addition, we synthesised d4U-monophosphate to
be used as a reference in this enzymatic assay.22
Experimental
The naphthyl L-alanine benzyl ester phosphoramidate of d4U
(1j) was incubated with cathepsin A for 12 h at room temperature,
and the progress of the reaction was monitored by 31P NMR. The
results (Fig. 3) show that d4U phosphoramidates were metabolised
primarily to the aminoacyl intermediate iii (dp ca. 6.7, Scheme 2)23
and to some extent to the corresponding d4U-monophosphate.
Standard procedure for phosphoramidate synthesis
tBuMgCl (1.3 equivalents) was added to a stirring solution of the
nucleoside analogue, d4U or ddU (1 equivalent) in THF (10 mL)
under nitrogen. After 15 minutes and at room temperature, the
appropriate phosphochloridate (2 equivalents) was added and
Scheme 2 Postulated mechanism of action of aryl triester phosphoramidates.
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Fig. 3 The metabolism of naphthyl L-alanine benzyl ester d4U phosphoramidate in the presence of the esterase enzyme cathepsin A. (Spectra were
recorded at 1 hour intervals except when stated otherwise.)
the progress of the reaction was monitored by TLC using
MeOH–DCM (1 : 9) as an eluant. At the end of the reaction,
the solvent was evaporated and the crude product was dissolved in
DCM and purified by flash column chromatography with eluant
MeOH–DCM (3 : 97). Pooling and evaporation of the appropriate
fractions gave the desired phosphoramidate as a solid.
3.4.16.1) that had already been dissolved in 200 mL of TRIZMA
buffer. 31P NMR of the reaction mixture was then carried out every
30 minutes for 12 h at room temperature.
For the preparation of human T-lymphocyte extracts and
stability measurements of test compounds, human T-lymphocyte
CEM cells were grown in RPMI-1640 culture medium in a 1 litre
culture bottle to a density of 2 ¥ 106 cells per mL. Cells were
◦
Molecular modelling
centrifuged at 1200 rpm at 4 C for 10 min, washed three times
with cold PBS, and resuspended in 20 ml of PBS at 100 ¥ 106 cells
per mL. After 2 rounds of 6 ¥ 20 sec sonication on ice, the cell
extract was centrifuged at 13 000 rpm for 30 min at 4 ◦C and the
supernatant divided in aliquots of 1 mL and frozen at -80 ◦C until
use. Stability of the test compounds was performed as follows:
200 mL of cell extract were mixed with 200 mL of PBS and 200 mL
of test compound (300 mM) to obtain a total volume of 600 mL
(100 mM test compound). The reaction mixture was incubated for
0, 30, 60 and 120 minutes at 37 ◦C. At each time point, 100 mL were
withdrawn, added to 200 mL cold methanol, centrifuged and the
supernatant analysed for compound stability by HPLC (Reverse
Phase RP-8 (Lichrocard 125–4), Merck, Darmstadt, Germany).
The following gradient was used: 2 min buffer A (acetonitrile)
2% + buffer B (50 mM NaH2PO4 + 5 mM heptane sulfonic acid
pH 3.2); 6 min linear gradient to 20% buffer A + 80% buffer B;
2 min linear gradient to 25% buffer A + 75% buffer B; 2 min
linear gradient to 35% buffer A + 65% buffer B; 8 min linear
gradient to 50% buffer A + 50% buffer B; 10 min same buffer
mixture; 5 min linear gradient to 2% buffer A + 98% buffer B;
5 min equilibration at 2% buffer A + 98% buffer B. The naphthyl
L-alanine benzyl ester phosphoramidate enantiomers of d4U and
ddU had retention times of 29.2 and 29.6 min (d4U prodrug) and
29.9 and 30.2 min (ddU prodrug).
All the molecular modelling studies were performed on a RM
Innovator with Pentium IV 2.8 GHz processor, running Linux
Fedora Core 3 using Molecular Operating Environment (MOE)
2006.08 (Tripos Inc., 1699 South Hanley Rd, St Louis, MO 63144,
USA. http://www.tripos.com) and FlexX module in SYBYL
7.2.24 The thymidine kinase structure was downloaded from the
PDB data bank (PDB code: 1kim).25 All the minimisations were
performed with MOE until an RMSD gradient of 0.05 kcal
-1
˚
A
mol-1
was reached with the MMFF94x force field. Docking
experiments were carried out using the FlexX docking program
of SYBYL 7.2 using the default settings. The output of FlexX
docking was visualised with MOE, and the scoring.svl26 was used
to identify interaction types between ligand and protein.
Enzymatic assays
5 mg of the appropriate phosphoramidate derivative was first
dissolved in 200 mL of d6-acetone and then 400 mL of TRIZMA
(tris(hydroxymethyl)aminomethane) buffer solution (pH = 7.4)
was added. A 31P NMR was conducted at this stage to see the
peaks of the phosphoramidate in d6-acetone and TRIZMA, so
that it acts as a reference (t = 0 h). To this mixture was added 0.3 mg
of cathepsin A (carboxypeptidase Y, >50 units mg-1, EC number
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Antiretroviral evaluation
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Human immunodeficiency virus type 1 (HIV-1) was originally
obtained from a persistently HIV-infected H9 cell line, as described
previously and was kindly provided by Dr R. C. Gallo (then at the
National Institutes of Health, Bethesda, MD, USA). Virus stocks
were prepared from the supernatants of HIV-1-infected MT-4 cells.
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required to inhibit virus-induced cytopathicity by 50%. The CC50
was defined as the compound concentration required to inhibit
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Acknowledgements
The authors would like to thank Dr Andrea Brancale for his
assistance with the molecular modelling and Mr Marco Deruds
for his help with some NMR studies.
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