Aryl phosphoramidate derivatives of d4T have improved anti-HIV efficacy in tissue culture and may act by the generation of a novel intracellular metabolite

Article abstract of DOI:10.1021/jm950605j

New phosphate derivatives of the anti-HIV nucleoside analogue d4T were prepared as potential membrane-soluble prodrugs of the bioactive free nucleotide. The enhanced antiviral potency and/or reduced cytotoxicity of the derivatives leads to an increase in

Full text of DOI:10.1021/jm950605j

1748
J . Med. Chem. 1996, 39, 1748-1753
Ar yl P h osp h or a m id a te Der iva tives of d 4T Ha ve Im p r oved An ti-HIV Effica cy in
Tissu e Cu ltu r e a n d Ma y Act by th e Gen er a tion of a Novel In tr a cellu la r
Meta bolite
Christopher McGuigan,* Dominique Cahard, Hendrika M. Sheeka,† Erik De Clercq,‡ and J an Balzarini‡
Welsh School of Pharmacy, University of Wales Cardiff, King Edward VII Avenue, Cardiff, CF1 3XF, U.K.
Received August 14, 1995X
New phosphate derivatives of the anti-HIV nucleoside analogue d4T were prepared as potential
membrane-soluble prodrugs of the bioactive free nucleotide. The enhanced antiviral potency
and/or reduced cytotoxicity of the derivatives leads to an increase in selectivity relative to the
parent nucleoside analogue. Moreover, the derivatives appear to bypass the dependence of
the nucleoside on thymidine kinase-mediated activation, retaining full activity in thymidine
kinase-deficient cells. This strongly suggests the successful intracellular delivery of free
nucleotides by the masked phosphate triester prodrugs. This is further confirmed by studies
using radiolabeled compound which clearly demonstrate the generation of d4T mono-, di- and
triphosphates from the prodrug, even in thymidine kinase-deficient cells. Moreover, we herein
report the generation of a new metabolite, a partially hydrolyzed phosphate diester, alaninyl
d4T monophosphate. We suggest that at least part of the antiviral action of the prodrugs
derives from the intracellular generation of such novel diesters which may add considerable
weight to the suggested further preclinical development of the phosphate prodrugs.
In tr od u ction
phoramidates (2) derived from AZT (1).15 These phos-
phoramidates retained good activity in thymidine kinase-
deficient (TK-) cells, by comparison to thymidine kinase-
competent cells, indicating a (partial) independence of
thymidine kinase activation. We were particularly keen
to apply this technology to the 2′,3′-dideoxy-2′,3′-dide-
hydro analogue of thymidine (d4T) (3) for several
reasons. This nucleoside analogue has been noted to
be a potent inhibitor of HIV, but displays reduced
cytotoxicity in certain cells (e.g. bone marrow progenitor
cells) compared to AZT.3,16,17 Furthermore, it is known
that the kinetics of the three phosphorylation steps from
the nucleoside analogue to the (bioactive) triphosphate
differ in the case of d4T, by comparison to AZT and other
3′-modified nucleoside analogues. In particular, the
rate-limiting step for AZT appears to be the conversion
of mono- to diphosphate, whereas the conversion of
nucleoside to monophosphate may well be rate-limiting
for d4T.7 It could follow that the intracellular delivery
of preformed d4T monophosphate (d4TMP) may be more
useful than the delivery of AZT monophosphate. From
a series of blocked phosphate esters of d4T we have
noted significant and selective anti-HIV activity for a
phosphoramidate derivative So324 which we herein
describe. Particularly interesting is the observation
that this material may act by an additional mechanism
of action.
Several members of the 2′,3′-dideoxynucleoside (ddN)
series are potent inhibitors of human immunodeficiency
virus (HIV) in cell culture.1-5 The 5′-triphosphates of
these nucleoside analogues are potent inhibitors of HIV
reverse transcriptase.6-8 As a rule, the activation
(phosphorylation) of these nucleosides is in most cases
accomplished by cellular nucleoside and nucleotide
kinases. Thus, in contrast to other antiviral agents (e.g.
acyclovir) where (herpes simplex) virus-specific thymi-
dine kinase mediates the first step of the conversion of
the drug to the intracellular active species, the 2′,3′-
dideoxynucleoside analogues depend on cellular nucleo-
side kinases for their phosphorylation. However, in
many cases the ddN derivatives have a poor affinity for
nucleoside kinases (i.e. 2′,3′-dideoxycytidine for deoxy-
cytidine kinase, 2′,3′-didehydro-2′,3′-dideoxythymidine
and 2′,3′-dideoxyuridine for thymidine kinase, 2′,3′-
dideoxyadenosine for adenosine kinase and deoxycyti-
dine kinase, and 2′,3′-dideoxyinosine for 5′-nucleo-
tidase).9-13 Moreover, the dependence on phosphory-
lation for activation of the particular nucleoside ana-
logue may be a particular problem in cells where the
nucleoside kinase activity is known to be low or even
lacking (i.e. monocyte/macrophages).14 Therefore, we
have sought to overcome this dependence on nucleoside
kinase activation by the development of a suitable
nucleotide delivery strategy. The viability of such an
approach is entirely based on the ability to suitably
modify the phosphate structure of a membrane-soluble
masked nucleotide to enable intracellular delivery and
release of the free phosphate form. We have previously
noted the success of this strategy with (aryloxy)phos-
Resu lts a n d Discu ssion
Ch em istr y. D4T (3) was prepared from thymidine
essentially by the method of Horwitz et al.,18 noting the
later comments of Mansuri et al.19 Then, phenyl
methoxyalaninyl phosphorochloridate20 was allowed to
react with d4T using THF/N-methylimidazole to give
compound 4a in good yield (88). As anticipated, this
material displayed two closely spaced signals in the 31P
NMR (δP ca. 3.3 ppm),21 corresponding to the presence
of diastereoisomers, resulting from mixed stereochem-
istry at the phosphate center. Similar diastereomeric
splitting, and phosphorus coupling where appropriate,
† Department of Chemistry, University of Southampton, Southamp-
ton, SO9 5NH, U.K.
‡ Rega Institute for Medical Research, Katholieke Universiteit of
Leuven, B-3000, Leuven, Belgium.
* Author for correspondence. Tel. (+44) 1222 874537, FAX (+44)
1222 874180.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1996.
0022-2623/96/1839-1748$12.00/0 © 1996 American Chemical Society

Aryl Phosphoramidate Derivatives of d4T
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1749
Ta ble Aand Anti-HIV-2 Activity and Cytotoxicity of Test
Compounds 1, 3, 4a -f, 5a -e, and 6a ,b in vitroaand Anti-HIV-2
Activity and Cytotoxicity of Test Compounds 1, 3, 4a -f, 5a -e,
and 6a ,b in vitroa
against HIV-1 and HIV-2 in MT-4 and CEM cells.
Moreover, the >5-fold reduced cytotoxicity of 4a by
comparison to d4T 3 in MT-4 cells leads to a 50-fold
improved selectivity index for the phosphoramidate.
Furthermore, it is particularly striking that, whereas
d4T is virtually inactive in thymidine kinase-deficient
CEM cells, the phosphoramidate 4a retains full activity,
being ca. 400-fold more potent than d4T in these cells.
Similarly, while AZT is inherently more potent than
either d4T or the d4T phosphate in thymidine kinase-
competent CEM cells, the phosphoramidate derivative
4a is >1300 times more active than AZT in the kinase-
deficient CEM cell line.
EC50 (µM)
CC50 (µM)
MT-4 MT-4 CEM/0 CEM/0 CEM/TK-
compd HIV-1 HIV-2 HIV-1 HIV-2
HIV-1
MT4 CEM/0
1
3
0.002 0.003 0.003 0.004
0.651 0.770 0.800 0.775
0.066 0.067 0.182 0.200
>100
33.3
0.075
7
4
0.4
0.33
0.34
0.025
0.11
8.5
0.11
0.06
>250
48
6
19
>500
174
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
6a
6b
>100 100
g250 g250
>250 >250
61.5
34.1
22
1.34
3.58
0.2
0.19
0.23
ND
0.046 0.11
1.72 4.07
0.037 0.12
6.71
11.2
0.47
0.38
0.38
ND
6
12.5
1.1
0.8
0.6
0.04
0.15
2.5
6
12.5
2.23
1.35
0.8
0.055
0.15
3.7
0.15
0.16
50
g250
216
g250
16
29.8
115
26.9
In terms of structure-activity relationships in opera-
tion, it is apparent that relatively small changes in the
amino acid region lead to significant changes in activity.
Thus, of the series 4a -f, the alanine compound 4a is
the most potent, with leucine 4d , phenylalanine 4e, and
methionine 4f analogues being approximately 10-fold
less active, depending on the virus and cell line in
question. The glycine compound 4b is less active still,
and the valine analogue 4c is the least active of the
series, being approximately 100-fold less potent than the
lead alanine compound 4a . However, throughout this
series, it is notable that full activity is always retained
in TK- cells by comparison to the activity shown in TK+
cells.
ND
9.75
82.2
10.5
>100 60
>250 >250
g250 g250
0.11
0.057 0.063 0.07
25.4
28.5
50.9
62.5
20
48
240
a
EC50 is the 50% effective concentration or compound concen-
tration required to protect MT-4 or CEM cells against the
cytopathogenicity of HIV by 50%. Data are the mean of two to
four independent experiments. CC50 is the 50% cytotoxic concen-
tration or compound concentration required to reduce MT-4 or
CEM cell viability by 50%.
were also noted in the H-decoupled 13C spectrum. The
1
presence of diastereoisomers was also apparent from H
Similarly, there is some variation in activity with
varying substitution in the aryloxy moiety, studying the
series 4a , and 5a -e. The dibromophenyl analogue 5a
is perhaps slightly more active than the parent phenyl
compound 4a , although this is at the cost of some
cytotoxicity in CEM cells. The (trifluoromethyl)phenyl
(5b), dichlorophenyl (5d ), and the ethylphenyl (5e)
compounds are approximately equipotent with the par-
ent phenyl system, while the pentafluorophenyl ana-
logue 5c is approximately 50-fold less potent. Again,
unlike the parent phenyl compound, some of the sub-
stituted aryl analogues appear to exhibit significant
cytotoxicity. As a result, the highest selectivity index
is displayed by the parent phenyl compound 4a .
NMR spectroscopy, and analytical HPLC studies on 4a .
Similarly prepared were the analogues of 4a with
other amino acids: glycine 4b, valine 4c, leucine 4d ,
phenylalanine 4e, and methionine 4f. Each of the
analogues was isolated as a mixture of [phosphate]
diastereoisomers, as evidenced from spectroscopic and
chromatographic data.
We were also interested in studying the structure-
activity relationships operating in the aryl region of the
phosphoramidates. In particular, we had previously
noted that, for diaryl phosphates of AZT, the introduc-
tion of electron-withdrawing groups within the aryl
moieties leads to a substantial increase in antiviral
activity.22 This was taken as corresponding to the
increased ability of such systems to undergo [intracel-
lular] liberation of the aryl moiety and release of the
phosphate diester en route to the free nucleotides. Thus,
we prepared the 2,4-dibromo- (5a ), 3-trifluoromethyl
(5b), pentafluoro (5c), and 3,5-dichloro (5d ) analogues,
each with some degree of electron withdrawal. For
purposes of comparison, we also prepared the 4-ethyl
compound 5e which lacks any electron-withdrawing
nature.
Finally, we prepared the analogues lacking the aryl
moiety entirely, and instead carrying a methyl group
(6a ) or an ethyl group (6b ). These were designed to
simply probe the necessity for antiviral action of an
aryloxy group.
An tivir a l Activity. The parent nucleoside d4T (3)
and phosphoramidates (4a -f, 5a -e, and 6a ,b) were
tested for their ability to inhibit the replication of HIV,
as previously described,23 and the results obtained using
HIV-1- or HIV-2-infected CEM and MT4 cells are
displayed in Table 1. The clinically used nucleoside
analogue AZT (1) was included as reference material,
and tests were also conducted in thymidine kinase-
deficient CEM cells.
Finally, the importance of an aryl moiety for antiviral
activity is most evident noting the data for the methyl
(6a ) and ethyl (6b) phosphates. These show very little
antiviral activity, being approximately 1000-fold less
potent than the lead compound 4a .
Data in TK- versus TK+ cells clearly demonstrate
that the antiviral activity of the phosphoramidates 4a -f
and 5a -e is independent of thymidine kinase-mediated
phosphorylation. The most obvious mechanism of action
consistent with this observation is of intracellular
delivery of the free nucleotide d4TMP from the phos-
phoramidates, and further phosphorylation to generate
the active metabolite d4T triphosphate (d4TTP). In
order to test this hypothesis, we incubated CEM cells
3
with H-labeled compound 4a and subsequently studied
the formation of radiolabeled intracellular metabolites
by HPLC.24 Markers of authentic d4T mono-, di-, and
triphosphate were included, along with d4T (3) and
blocked phosphoramidate 4a . The study was compared
with labeled d4T (3), and both experiments were also
performed using thymidine kinase-deficient CEM cells.
Measurable levels of d4T mono-, di-, and triphosphate
were formed from either d4T incubation or incubation
with 4a using kinase-competent cells. However, with
kinase-deficient cells there was no detectable phospho-
It is notable that the phosphoramidate derivative 4a
is approximately 4-10-fold more potent than d4T itself

1750 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
McGuigan et al.
F igu r e 1.
rylation of d4T. On the other hand, levels of d4T
triphosphate generated by 4a incubation were entirely
maintained in the thymidine kinase-deficient cells.
Indeed, CEM (wild type) and CEM-TK- cells generate
0.25 and 0.48 nmol/109 cells of d4TTP respectively after
20 h of incubation with 0.2 µM 4a . There was thus firm
evidence that 4a could give rise to significant levels of
the bioactive metabolite d4T triphosphate, by a mech-
anism which was entirely independent of thymidine
kinase, unlike d4T which could give similar levels of the
triphosphate, but by an entirely thymidine kinase-
dependent process. This is consistent with the sug-
gested intracellular hydrolysis of phosphoramidates
such as 4a to give d4T monophosphate and the thymidy-
late kinase-mediated phosphorylation of this material
to the higher phosphates. The alternative hydrolysis
of 4a to give d4T could not play a major role in the
activation pathway, otherwise the generation of d4T
triphosphate (and the antiviral activity) would have
been significantly reduced in thymidine kinase-deficient
cells.24
However, we noted in the HPLC chromatogram
arising from incubation with radiolabeled 4a , using
either kinase-competent or kinase-deficient cells, the
formation of high levels of a new metabolite, of apparent
polarity intermediary between d4T mono- and diphos-
phate. The concentration of this material, as estimated
from the radiolabel, was approximately 10-200 times
the levels of d4T triphosphate generated, depending on
the initial concentration of the phosphoramidate 4a . At
its EC50 (ca. 0.2 µM) 4a generates levels of the new
metabolite 10-15-fold higher than the levels of d4T
triphosphate (d4TTP) generated from d4T administra-
tion to the cell cultures over a comparable incubation
time (24 h). At longer incubation times (72 h) the new
metabolite was still present at ca. 10-fold higher levels
than those of d4TTP. Eventually we were able to
prepare a synthetic material identical to this metabolite,
on the basis of HPLC retention time in two different
systems, by the regioselective base-catalyzed hydrolysis
of compound 4a . This product was then identified as
the novel phosphate diester 7 arising from cleavage of
both the phenyl and methyl ester groups. We noted that
this material could also be generated from 4a using hog
liver esterase, incubated for 24 h at 37 °C. Further
evidence for the structure of the metabolite, identified
as 7, came from the observation that compounds 4c, 4e,
and 4f gave products with hog liver esterase with
similar, but nonidentical HPLC retention times. This
is entirely consistent with the suggestion that the amino
acid moiety is retained in the metabolites. Further-
more, the metabolite and the synthetic material 7 were
both stable to alkaline phosphatase, as anticipated for
the proposed structure. We wondered whether the
metabolite 7 could exert antiviral action via release of
d4T (at least in TK+ cells) or d4T monophosphate, and
subsequent phosphorylation to the triphosphate act in
an entirely novel way. Indeed, we suggest that the
much greater concentrations of 7 generated, by com-
parison to the intracellular levels of d4T triphosphate,

Aryl Phosphoramidate Derivatives of d4T
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1751
to 20% water at 30 min, with a flow rate of 1 mL/min and
detection by UV at 265 nm. Final products showed purities
exceeding 99% with undetectable levels (<0.02) of parent
nucleosides in every case. (Aryloxy)phosphorodichloridates,
and (aryloxy)aminophosphorochloridates were prepared en-
tirely as previously noted.15
indicates that 7 could contribute to the antiviral efficacy
of phosphoramidates such as 4a via intracellular release
of d4TMP. However, it is likely that the contribution
of each mechanism to the overall antiviral effect will
vary with the exact structure of the phosphoramidate,
the initial drug concentration, and the cell type studied.
It is quite feasible that some of the structure-activity
relationships we noted above, particularly involving
variations in the aryl moiety, may relate to the efficiency
of the intracellular generation of d4T monophosphate
and/or the formation of the phosphate diester metabolite
7. Alternatively, and particularly in the case of the
amino acid variations, some of the evident preference
for certain amino acids may also relate either to the
relative efficiency of formation of the analogues of (7)
or to the efficiency of their subsequent activation to
d4TTP. This is currently under active investigation in
our laboratory.
Gen er a l P r oced u r e. Phenyl methoxyalaninyl phospho-
rochoridate (250 mg, 0.9 mmol, 2.0 equiv) was added to a
stirred solution of d4T (3) (100 mg, 0.45 mmol) and N-
methylimidazole (143.5 µL, 1.8 mmol, 4 equiv) in tetrahydro-
furan (THF) (2 mL) at ambient temperature. After 4 h, the
solvent was removed under reduced pressure. The gum was
dissolved in chloroform (10 mL) and washed with 1 M HCl (8
mL), sodium bicarbonate solution (10 mL), and water (15 mL).
The organic phase was dried (MgSO4), and the solvent was
removed in vacuo. The residue was purified by column
chromatography on silica with elution by chloroform-metha-
nol (97:3). Pooling and evaporation of appropriate fractions
gave the product as a white foam.
2′,3′-Dideoxy-2′,3′-dideh ydr oth ym idin e 5′-(ph en yl m eth -
oxya la n in yl p h osp h a te) (4a ): yield 88%; δP 3.20, 3.86; δH
1.32, 1.34 (d, 3H, J ) 6.8Hz, Ala-Me), 1.81, 1.84 (d, 3H, 5-Me),
3.69, 3.70 (s, 3H, OMe), 3.84-4.00 (m, 2H, Ala-CH, Ala-NH),
4.32 (m, 2H, H5′), 5.02 (m, 1H, H4′), 5.88 (m, 1H, H2′), 6.33
(m, 1H, H3′), 7.03 (m, 1H, H1′), 7.15-7.35 (m, 6H, Ph, H6),
9.22, 9.26 (bs, 1H, NH); δC 12.52 (5-Me), 21.02 (Ala-Me), 50.22-
50.35 (Ala-CH), 52.74 (OMe), 66.62-67.29 (C5′), 84.80-84.88
(C4′), 89.69-89.93 (C1′), 111.44-111.57 (C5), 120.13-120.31
(Ph ortho), 125.30 (Ph para), 127.49-127.65 (C2′), 129.87-
129.93 (Ph meta), 133.19-133.50 (C3′), 135.77-136.06 (C6),
150.51 (Ph ipso), 151.16 (C2), 164.14 (C4), 174.12 (Ala-CO);
MS m/e FAB 466 (MH+, 7), 340 (MH+ - base), 200 (17), 136
(47), 89 (25), 81 (C5H5O, 100), HPLC tR 22.48, 22.87 min (see
experimental methods for HPLC conditions).
In conclusion, the phosphoramidate derivatives of d4T
herein described show advantage over d4T itself, par-
ticularly in thymidine kinase-deficient cells. This leads
to enhanced antiviral selectivity relative to the parent
drug. Metabolic studies indicate that the phosphora-
midates lead to the intracellular generation of bioactive
d4T triphosphate by a mechanism which is entirely
independent of thymidine kinase, with full retention of
d4TTP levels in TK- cells. We also note the novel
discovery of a new metabolite, a phosphate diester
retaining the amino acid. We thus report on a new
mechanism of inhibition of HIV; the d4T phosphorami-
dates can act via a d4T or d4TMP depot form, which
may be entirely independent of thymidine kinase. We
suggest that this new discovery offers great promise for
the development of new improved therapies for HIV
infection and AIDS.
2′,3′-Dideoxy-2′,3′-dideh ydr oth ym idin e 5′-(ph en yl m eth -
oxyglycin yl p h osp h a te) (4b): yield 90%; δP 4.89, 5.52; δH
1.79, 1.83 (s, 3H, 5-Me), 3.69 (s, 3H, OMe), 3.70-4.05 (m, 3H,
Gly-CH2, Gly-NH), 4.32 (m, 2H, H5′), 4.99 (m, 1H, H4′), 5.92
(m, 1H, H2′), 6.38 (m, 1H, H3′), 6.98 (m, 1H, H1′), 7.05-7.38
(m, Ph, H6), 9.44, 9.46 (s, 1H, NH); δC 12.75 (5-Me), 43.15 (Gly-
CH2), 52.94 (OMe), 66.78-67.52 (C5′), 84.98-85.10 (C4′),
89.68-90.16 (C1′), 111.69-111.80 (C5), 120.46-120.59 (Ph
ortho), 125.66 (Ph para), 127.66-127.91 (C2′), 130.22 (Ph
meta), 133.48-133.87 (C3′), 136.11-136.40 (C6), 150.65 (Ph
ipso), 151.45 (C2), 164.46 (C4), 171.41-171.51 (Gly-CO); MS
m/e FAB 452 (MH+, 74), 474 (M + Na, 46), HPLC tR 27.21
min.
Exp er im en ta l Section
All experiments involving water-sensitive compounds were
conducted under scrupulously dry conditions. Triethylamine
and dichloromethane were dried by heating under reflux over
calcium hydride for several hours followed by distillation.
Dichloromethane was further dried over activated 4 Å molec-
ular sieves. Tetrahydrofuran was dried by heating under
reflux over sodium and benzophenone followed by distillation.
N-Methylimidazole was purified by distillation. Nucleosides
were dried by storage at elevated temperature in vacuo over
P2O5. Proton, carbon, and phosphorus Nuclear Magnetic
Resonance (1H, 13C, 31P NMR) spectra were recorded on a
Bruker Avance DPX spectrometer operating at 300, 75.5, and
121.5 MHz, respectively, with 13C and 31P spectra being
recorded proton-decoupled. All NMR spectra were recorded
in CDCl3 at room temperature (20 °C ( 3 °C). 1H and 13C
chemical shifts are quoted in parts per million downfield from
tetramethylsilane. J values refer to coupling constants, and
signal splitting patterns are described as singlet (s), broad
singlet (bs), doublet (d), triplet (t), quartet (q), multiplet (m),
or combinations thereof. 31P chemical shifts are quoted in
parts per million relative to an external phosphoric acid
standard. Many proton and carbon NMR signals were split
due to the presence of (phosphate) diastereoisomers in the
samples. The mode of ionization for mass spectroscopy unless
stated was fast atom bombardment (FAB) with MNOBA as
matrix. Chromatography refers to flash column chromatog-
raphy and was carried out using Merck silica gel 60 (40-60
µM) as stationary phase. Thin layer chromatography was
performed using Alugram SIL G/UV254 aluminum-backed
silica gel plates. HPLC was conducted on an ACS quaternary
system, using an ODS5 column and an eluant of water/
acetonitrile, with 82% water 0-10 min, then a linear gradient
2′,3′-Dideoxy-2′,3′-dideh ydr oth ym idin e 5′-(ph en yl m eth -
oxyva lin yl p h osp h a te) (4c): yield 86%; δP 4.85, 5.40; δH 0.92
(m, 6H, Val-Me), 1.82 (m, 1H, Val-iPrCH), 1.89, 1.91 (s, 3H,
5-Me), 3.76 (s, 3H, OMe), 3.82 (m, 2H, Val-CH, Val-NH), 4.30-
4.48 (m, 2H, H5′), 5.07 (m, 1H, H4′), 5.96 (m, 1H, H2′), 6.38
(m, 1H, H3′), 7.10 (m, 1H, H1′), 7.18-7.35 (m, 6H, Ph, H6),
9.31 (s, 1H, NH); δC 12.80 (5-Me), 17.77-19.24 (Val-Me),
32.43-32.62 (Val-iPrCH), 52.67 (OMe), 60.32-60.38 (Val-CH),
66.92-67.65 (C5′), 85.04 (C4′), 89.98-90.24 (C1′), 111.76-
111.87 (C5), 120.45-120.56 (Ph ortho), 125.54-125.59 (Ph
para), 127.81-127.86 (C2′), 130.13-130.17 (Ph meta), 133.51-
133.72 (C3′), 136.01-136.28 (C6), 150.83 (Ph ipso), 150.87-
151.34 (C2), 164.30-164.37 (C4), 173.56-173.65 (Val-CO); MS
m/e FAB 493.6 (MH+, 100), HPLC tR 28.50 min.
2′,3′-Dideoxy-2′,3′-dideh ydr oth ym idin e 5′-(ph en yl m eth -
oxyleu cin yl p h osp h a te) (4d ): yield 87%; δP 4.18, 4.83; δH
0.91 (m, 6H, Leu-Me), 1.42-1.70 (m, 3H, Leu-CH2CH), 1.91,
1.93 (s, 3H, 5-Me), 3.73 (s, 3H, OMe), 3.76-3.98 (m, 2H, Leu-
CHm Leu-NH), 4.28-4.46 (m, 2H, H5′), 5.08 (m, 1H, H4′), 5.96
(m, 1H, H2′), 6.36 (m, 1H, H3′), 7.09 (m, 1H, H1′), 7.18-7.35
(m, 6H, Ph, H6), 9.35 (s, 1H, NH); δC 12.76 (5-Me), 22.23-
23.01 (Leu-Me), 24.75 (CH(CH3)2), 43.86-44.11 (CH2CH),
52.75 (OMe), 53.42-53.60 (Leu-CH), 66.92-67.55 (C5′), 85.62
(C4′), 89.92-90.19 (C1′), 111.69-111.83 (C5), 120.37-120.62
(Ph ortho), 125.55-125.58 (Ph para), 127.79 (C2′), 130.12 (Ph
meta), 133.51-133.70 (C3′), 136.00-136.36 (C6), 151.05 (Ph
ipso), 151.38 (C2), 164.39-164.50 (C4), 174.55-174.88 (Leu-
CO); MS m/e FAB 508 (MH+, 62), 530 (M + Na, 59); HPLC tR
30.17 min.

1752 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
McGuigan et al.
2′,3′-Dideoxy-2′,3′-dideh ydr oth ym idin e 5′-(ph en yl m eth -
oxyp h en yla la n in yl p h osp h a te) (4e): yield 89%; δP 3.96,
4.35; δH 1.89 (s, 3H, 5-Me), 3.00 (m, 2H, CH2Ph), 3.74 (s, 3H,
OMe), 3.80-4.28 (m, 4H, Phe-CH, Phe-NH, H5′), 4.94 (m, 1H,
H4′), 5.91 (m, 1H, H2′), 6.21-6.30 (m, 1H, H3′), 7.04-7.32 (m,
12H, Ph, H1′, H6), 9.35 (s, 1H, NH); δC 12.54 (5-Me), 40.55
(CH2Ph), 52.63 (OMe), 55.72-56.01 (Phe-CH) 66.50-67.10
(C5′), 84.78 (C4′), 89.71-89.95 (C1′), 111.53-111.64 (C5),
120.28 (OPh ortho), 125.40 (OPh para), 127.52 (C2′), 128.86,
129.65, 129.98 (CH2Ph), 129.86-129.92 (OPh meta), 133.18-
133.50 (C3′), 135.72 (CH2Ph ipso), 135.79-136.06 (C6), 150.46
(OPh ipso), 151.13-151.17 (C2), 164.12-164.18 (C4), 173.00
(Phe-CO); MS m/e FAB 542 (MH+, 77), 564 (M + Na, 29);
HPLC tR 29.88 min.
2′,3′-Dideoxy-2′,3′-dideh ydr oth ym idin e 5′-(ph en yl m eth -
oxym eth ion in yl p h osp h a te) (4f): yield 81%; δP 4.09, 4.86;
δH 1.74, 1.79 (s, 3H, MeS), 1.94, 1.97 (s, 3H, 5-Me), 1.80-2.40
(m, 5H, CHCH2CH2S), 3.72, 3.74 (s, 3H, OMe), 3.98-4.32 (m,
4H, H5′, Met-CH, Met-NH), 4.96 (m, 1H, H4′), 5.84 (m, 1H,
H2′), 6.26 (m, 1H, H3′), 6.96 (m, 1H, H1′), 7.05-7.25 (m, 6H,
Ph, H6), 9.58 (bs, 1H, NH); δC 12.80 (5-Me), 15.68 (CH3S), 29.95
(CH2SCH3), 33.73-33.85 (CH2CH2S), 53.06 (OMe), 53.81-
54.07 (Met-CH), 67.05-67.70 (C5′), 84.90-85.03 (C4′), 89.98-
90.23 (C1′), 111.66-111.86 (C5), 120.39-120.66 (Ph ortho),
125.63 (Ph para), 127.81-127.91 (C2′), 130.18 (Ph meta),
133.44-133.69 (C3′), 136.00-136.38 (C6), 150.72-150.80 (Ph
ipso), 151.41 (C2), 164.52 (C4), 173.61-173.94 (Met-CO); MS
m/e FAB 526 (MH+, 46), 548 (M + Na, 21); HPLC tR 29.92
min.
5.14 (1H, m, H-4′), 6.04 (1H, m, H-2′), 6.44 (1H, m, H-3′), 7.14
(1H, m, H-1′), 7.29 (3H, m, Ph), 7.40 (1H, s, H-6), 9.74 (1H, s,
NH); δC 12.51 (5-Me), 20.93 (Ala-Me), 50.26 (Ala-CH), 52.85
(OMe), 66.98, 67.68 (C-5′), 84.60 (C-4′), 89.74, 90.03 (C-1′),
111.40, 111.54 (C-5), 119.40 (Ph), 125.69 (Ph), 127.83 (C-2′),
132.89, 133.14 (C-3′), 135.60 (C-6), 136.01 (Ph), 151.06 (C-2)
151.27 (Ph), 164.09 (C-4), 173.93 (Ala-CO); MS m/e FAB
534.0589 (MH+, C20H23N3O8PCl2 requires 534.0600, 8), 408
(MH+ - thymine, 12), 391 (10), 149 (12), 127 (thymine H+),
12), 81 (C5H5O, 100); HPLC tR 32.19 min.
2′,3′-Dideh ydr o-2′,3′-dideoxyth ym idin e 5′-(4-eth ylph en -
yl m eth oxya la n in yl p h osp h a te) (5e): yield 79%; δP 3.43 ;
δH 1.19 (3H, m, Ala-Me), 1.31 (3H, m, CH2CH3), 1.80, 1.84 (3H,
d, 5-Me, J ) 1.2 Hz), 2.60 (2H, q, CH2CH3, J ) 7.5 Hz), 3.67,
3.70 (3H, s, OMe), 3.93 (2H, m, Ala-NH, Ala-CH), 4.38-4.25
(2H, m, H-5′), 5.00 (1H, m, H-4′), 5.88 (1H, m, H-2′), 6.28 (1H,
m, H-3′), 7.00 - 7.14 (5H, m, Ph, H-1′), 7.33, 7.34 (1H, s, H-6),
9.23, 9.25 (1H, s, NH); δC 12.41, 12.45 (5-Me), 15.69 (CH2CH3),
20.90, 20.97 (d, Ala-Me, J ) 4.9 Hz), 28.19 (Ph-CH2), 50.13,
50.26 (Ala-CH), 52.65 (OMe), 66.48, 67.11 (d, C-5′, J ) 4.9 Hz),
84.70, 84.88 (d, C-4′,), 111.40, 111.51 (C-5), 119.90, 120.08 (d,
Ph, J ) 3.9, 4.9 Hz), 127.36, 127.54 (C-2′), 129.05, 129.11 (Ph),
133.15, 133.50 (C-3′), 135.76, 136.06 (C-6), 141.19, 141.24 (Ph),
148.16, 148.29 (Ph), 151.12, 151.15 (C-2), 164.17, 164.22 (C-
4), 174.12, 174.25 (Ala-CO); MS m/e FAB 494.1693 (MH+,
C22H29N3O8P requires 494.1692, 5), 368 (MH+ - thymine, 25),
228 (15), 81 (C5H5O, 100); HPLC tR 27.23, 27.48 min.
2′,3′-Dideoxy-2′,3′-dideh ydr oth ym idin e 5′-(m eth yl m eth -
oxya la n in yl p h osp h a te) (6a ): yield 86%; δP 9.36, 9.70; δH
1.29, 1.31 (d, 3H, Ala-Me, J ) 6.7Hz), 1.81, 1.83 (s, 3H, 5-Me)
3.61-3.68 (m, 6H, OMe), 3.84 (m, 2H, Ala-CH, Ala-NH), 4.16
(m, 2H, H5′), 4.95 (bs, 1H, H4′), 5.83 (bs, 1H, H2′), 6.26 (m,
1H, H3′), 6.97 (m, 1H, H1′), 7.26 (d, 1H, H6), 9.30 (bs, 1H,
NH); δC 12.26-12.48 (5-Me), 21.11 (Ala-Me), 49.93-50.06 (Ala-
CH), 52.74 (OMe), 53.19-53.54 (MeOP), 65.93-66.75 (C5′),
84.83-84.94 (C4′), 89.61-89.87 (C1′), 111.37-11.43 (C5),
127.41-127.63 (C2′), 133.22-133.64 (C3′), 135.90-136.21 (C6),
151.07 (C2), 164.10 (C4), 174.39 (Ala-CO); MS m/e FAB
404.1223 (MH+, C15H22O8N3P requires 404.1248, 18), 278 (MH+
- base, 39); 81 (C5H5O, 100); HPLC tR ) 18.72, 22.19 min.
2′,3′-Did eh yd r o2′,3′-d id eoxyth ym id in e 5′-(eth yl m eth -
oxya la n in yl p h osp h a te) (6b): yield 74%; δP 7.66, 7.69 ; δH
1.26 (6H, m, Ala-Me, CH2CH3), 1.83, 1.85 (3H, d, 5-Me, J )
1.2 Hz), 3.67, 3.68 (3H, s, OMe), 3.82-4.16 (6H, m, Ala-NH,
Ala-CH, CH2OP, H-5′), 4.94 (1H, m, H-4′), 5.88 (1H, m, H-2′),
6.28 (1H, m, H-3′), 6.95 (1H, m, H-1′), 7.22, 7.31 (1H, d, H-6,
J ) 1.2, 1.3 Hz) 10.01 (1H, s, NH); δC 12.28, 12.41(5-Me), 16.18
(d, CH2CH3, J ) 6.8 Hz), 20.84, 20.92 (d, Ala-Me, J ) 5.2 Hz),
49.83, 49.90 (Ala-CH), 52.51 (OMe), 62.90, 63.07 (d, CH2OP,
J ) 4.9 Hz), 66.63, 66.93 (C-5′), 84.85 (d, C-4′, J ) 8.8 Hz),
89.48, 89.75 (C-1′), 111.25, 111.31 (C-5), 127.28, 127.39 (C-2′),
133.20, 133.51 (C-3), 135.80, 136.03 (C-6), 151.15, 151.19 (C-
2), 164.30 (C-4), 174.26, 174.32 (d, Ala-CO, J ) 6.9 Hz); HPLC
tR ) 38.90, 40.82 min.
2′,3′-Did eh yd r o-2′,3′-d id eoxyt h ym id in e 5′-(Ala n in yl
p h osp h a te) (7). Compound 4a (0.116 g, 0.25 mmol) was
dissolved in a 1:1 mixture of triethylamine and water (8 mL).
After 3 h at room temperature the triethylamine phase was
removed and the aqueous phase evaporated under high
vacuum at ambient temperature. The resulting crude product
was purified on silica using the chromatotron with the mixture
CHCl3/MeOH/H2O/NH4OH: 120/70/10/1 as eluent. Pooling
and freeze-drying of appropriate fractions gave the pure
compound: yield 0.051 g, 54); δP (D2O) 7.63; δH 1.12 (d, 3H, J
) 6.9Hz, Ala-Me), 1.73 (s, 3H, 5-Me), 3.42 (m, 1H, Ala-CH),
3.83 (m, 2H, H5′), 4.93 (m, 1H, H4′), 5.80 (m, 1H, H2′), 6.34
(m, 1H, H3′), 6.78 (m, 1H, H1′), 7.45 (s, 1H, H6); δC (D2O) 11.80
(5-Me), 19.65 (Ala-Me), 50.21 (Ala-CH), 65.32 (C5′), 86.26 (C4′),
90.38 (C1′), 111.57 (C5), 125.19 (C2′), 134.86 (C3′), 138.67 (C6),
152.57 (C2), 166.92 (C4), 179.30 (Ala-CO2H); MS m/e FAB 398
(MNa+, 17), 376 (MH+, 6); HPLC tR 3.15 min.
2′,3′-Did eh yd r o-2′,3′-d id eoxyt h ym id in e 5′-(2,4-d ib r o-
m op h en yl m eth oxy a la n in yl p h osp h a te) (5a ): yield 88%;
δP 3.07, 3.62; δH 1.26, 1.28 (d, 3H, J ) 6.8 Hz, Ala-Me), 1.75,
1.80 (s, 3H, 5-Me), 2.11 (s, 1H, NH), 3.64 (s, 3H, OMe), 3.92-
4.30 (m, 3H, Ala-CH, H5′), 4.98 (m, 1H, H4′), 5.87 (m, 1H, H2′),
6.26 (m, 1H, H3′), 6.96 (m, 1H, H1′), 7.30-7.60 (m, 4H, Ph,
H6), 9.41(bs, 1H, NH); δC 12.51 (5-Me), 21.00 (Ala-Me), 50.24
(Ala-CH), 52.80 (OMe), 67.37-67.83 (C5′), 84.49-84.61 (C4′),
89.80-89.92 (C1′), 111.60 (C5), 115.49 (Ph), 118.26 (Ph),
122.61-122.89 (Ph), 127.70 (C2′), 131.86 (Ph), 133.06-133.21
(C3′), 135.64 (Ph), 135.75-135.88 (C6), 147.01 (Ph), 151.07
(C2), 164.03 (C4), 173.71-173.82 (Ala-CO); MS m/e FAB 626
(MH+, 2 × 81Br, 3), 624 (MH+, 81Br, 6), 621.9507 (MH+,
C20H22O8N3PBr2 requires 621.9516, 3), 500, 498, 496 (MH+
base, 5,9,5), 81 (100); HPLC tR 41.17, 41.30 min.
-
2′,3′-Did eh yd r o-2′,3′-d id eoxyt h ym id in e 5′-(3-t r iflu o-
r om eth ylp h en yl m eth oxya la n in yl p h osp h a te) (5b): yield
80%; δP 2.49, 3.16; δH 1.36 (3H, m, Ala-Me), 1.80, 1.86 (3H, d,
5-Me), 3.70, 3.71 (3H, s, OMe) 3.97 (2H, m, Ala-NH, Ala-CH),
4.32 (2H, m, H-5′), 5.03 (1H, m, H-4′), 5.92 (1H, m, H-2′), 6.31
(1H, m, H-3′), 7.03 (1H, m, H-1′), 7.45 (5H, m, Ph, H-6), 9.06
(1H, s, NH); δC 12.55, 12.47 (5-Me,) 21.11, 20.99 (d, Ala-Me, J
) 4.9 Hz), 50.32, 50.26 (d, Ala-CH, J ) 4.8 Hz), 52.87 (OMe),
67.60, 66.89 (d, C-5′, J ) 4.9 Hz), 84.61 (d, C-4′, J ) 7.8 Hz),
90.04, 89.77 (C-1′), 111.61, 111.44 (C-5), 117.54 (d, Ph, J )
3.9 Hz), 122.14 (Ph), 123.98, 123.79 (Ph), 123.84 (q, CF3, J )
272.0 Hz), 127.84, 127.74 (C-2′), 130.66 (Ph), 132.00 (q, Ph, J
) 32.0 Hz), 133.30, 133.02 (C-3′), 135.86, 135.66 (C-6), 150.71
(d, Ph, J ) 5.9 Hz), 150.96 (C-2), 163.91, 163.86 (C-4), 174.06,
173.89 (d, Ala-CO, J ) 6.8 Hz); MS m/e FAB 534.1201(MH+,
C21H24N3O8PF3 requires 534.1253, 6), 408 (MH+ - thymine,
8), 268 (10), 149 (10), 81 (C5H5O, 100); HPLC tR 30.56 min.
2′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-(p en ta flu o-
r op h en yl m eth oxya la n in yl p h osp h a te) (5c): yield 76%;
δP 4.74, 5.66; δH 1.34, 1.36 (d, 3H, Ala-Me, J ) 6.7Hz), 1.75,
1.81 (s, 3H, 5-Me), 3.69 (s, 3H, OMe), 3.92-4.40 (m, 4H, Ala-
CH, Ala-NH, H5′), 4.97 (m, 1H, H4′), 5.85 (m, 1H, H2′), 6.29
(m, 1H, H3′), 6.93 (m, 1H, H1′), 7.19 (m, 1H, H6), 9.38 (bs,
1H, NH); δC 12.23-12.43 (5-Me), 20.83 (Ala-Me), 50.22-50.34
(Ala-CH), 52.99 (OMe), 67.75-68.37 (C5′), 84.42-84.52 (C4′),
89.87-90.17 (C1′), 111.75 (C5), 127.69-127.93 (C2′), 132.86-
133.13 (C3′), 132-143 (m, Ph), 135.74-135.96 (C6), 151.11
(C2), 164.15 (C4), 173.64-173.76 (Ala-CO); MS m/e FAB 556
(MH+, 31), 578 (M + Na, 100); HPLC tR 35.90 min.
An tir etr ovir a l Eva lu a tion . HIV-1 (HTLV-IIIB) was ob-
tained from persistently HIV-infected H9 cells as described
previously.25 Virus stocks were prepared from the superna-
tants of HIV-1 (IIIB)-infected MT4 cells. HIV-2 (ROD) was
provided by Dr. L. Montagnier (Pasteur Institute, Paris,
France). MT-4 cells were provided by Dr. N. Yamamoto (Tokyo
2′,3′-Did eh yd r o-2′,3′-d id eoxyth ym id in e 5′-(3, 5-d ich lo-
r op h en yl m eth oxya la n in yl p h osp h a te) (5d ): yield 70%;
δP 2.83, 3.42; δH 1.48 (3H, m, Ala-Me), 1.92, 1.97 (3H, s, 5-Me),
3.84 (3H, s, OMe), 4.07 - 4.48 (4H, m, Ala-NH, Ala-CH, H-5′),

Aryl Phosphoramidate Derivatives of d4T
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1753
(7) Balzarini, J .; Herdewijn, P.; De Clercq, E. Differential patterns
of intracellular metabolism of 2′,3′-didehydro-2′,3′-dideoxythy-
midine and 3′-azido-2′,3′-dideoxythymidine, two potent anti-
human immunodeficiency virus compounds. J . Biol. Chem. 1989,
264, 6127-6133.
(8) Hao, Z.; Cooney, D. A.; Hartman, N. R.; Perno, C. F.; Fridland,
A.; De Vico, A. L.; Sarngadharan, M. G.; Broder, S.; J ohns, D.
G. Factors determining the activity of 2′,3′-dideoxynucleosides
in suppressing human immunodeficiency virus in vitro. Mol.
Pharmacol. 1988, 34, 431-435.
(9) Balzarini, J .; Cooney, D. A.; Dalal, M.; Kang, G.-J .; Cupp, J . E.;
De Clercq, E.; Broder, S.; J ohns, D. G. 2′,3′-Dideoxycytidine:
regulation of its metabolism and anti-retroviral potency by
natural pyrimidine nucleosides and by inhibitors of pyrimidine
nucleotide synthesis. Mol. Pharmacol. 1987, 32, 798-806.
(10) Starnes, M. C.; Cheng, Y.-C. Cellular metabolism of 2′,3′-
dideoxycytidine, a compound active against human immunode-
ficiency virus in vitro. J . Biol. Chem. 1987, 262, 988-991.
(11) Hao, Z.; Cooney, D. A.; Farquhar, D.; Perno, C. F.; Zhang, K.;
Masood, R.; Wilson, Y.; Hartman, N. R.; Balzarini, J .; J ohns, D.
G. Potent chain termination activity and selective inhibition of
human immunodeficiency virus reverse transcriptase by 2′,3′-
dideoxyuridine-5′-triphosphate. Mol. Pharmacol. 1990, 37, 157-
163.
Medical and Dental University School of Medicine, Tokyo,
J apan). CEM/0 cells were obtained from the American Tissue
Culture Collection (Rockville, MD), and CEM/TK- cells were
a gift from Prof. S. Eriksson and Dr. A. Karlsson (Karolinska
Institute, Stockholm, Sweden).
MT-4 and CEM cells were infected with HIV-1 as previously
described.23 Briefly, 5 × 105 MT-4 or CEM cells/mL were
infected with HIV-1 or HIV-2 at approximately 100 CCID50
(50% cell culture infective dose)/mL of cell suspension. Then,
100 µL of the infected cell suspension were transferred to
microtiter plate wells and mixed with 100 µL of the appropri-
ate dilutions of the test compounds. After 4 days, giant cell
formation was recorded microscopically in the HIV-infected
CEM cell cultures, and after 5 days, the number of viable cells
was determined by trypan blue staining of the HIV-infected
MT-4 cell cultures. The 50% effective concentration (EC50) and
50% cytotoxic concentration (CC50) were defined as the com-
pound concentrations required to reduce by 50% the number
of viable cells (MT-4) or giant cells (CEM) in the virus-infected
and mock-infected cell cultures, respectively.
C3H/3T3 cells were seeded into Costar Tissue Culture
Cluster plates (Costar Broadway, Cambridge, MA) at 20 000
cells/mL into 1 cm2 wells and grown to confluency. Cell
cultures were then infected by 75 foci-forming units of Moloney
murine sarcoma virus (MSV) during 90 min, whereafter
medium was replaced by 1 mL of fresh culture medium
containing different concentrations of test compound. After
6 days the transformation of the test cultures was examined
microscopically. The EC50 was defined as the compound
concentration that is required to inhibit MSV-induced trans-
formation by 50%.
(12) J ohnson, M. A.; Ahluwalia, G.; Connelly, M. C.; Cooney, D. A.;
Broder, S.; J ohns, D. G.; Fridland, A. Metabolic pathways for
the activation of the antiretroviral agent 2′,3′-dideoxyadenosine
in human lymphoid cells. J . Biol. Chem. 1988, 263, 15354-
15357.
(13) J ohnson, M. A.; Fridland, A. Phosphorylation of 2′,3′-dideoxy-
inosine by cytosolic 5′-nucleotidase of human lymphoid cells. Mol.
Pharmacol. 1989, 36, 291-295.
(14) Perno, C. F.; Yarchoan, R.; Cooney, D. A.; Hartman, N. R.; Webb,
D. S. A.; Hao, Z.; Mitsuya, H.; J ohns, D. G.; Broder, S.
Replication of human immunodeficiency virus in monocytes. J .
Exp. Med. 1989, 169, 933-951.
(15) McGuigan, C.; Pathirana, R. N.; Balzarini, J .; De Clercq, E.
Intracellular delivery of bioactive AZT nucleotides by aryl
phosphate derivatives of AZT. J . Med. Chem. 1993, 36, 1048-
1052.
(16) Baba, M.; Pauwels, R.; Herdewijn, P.; De Clercq, E.; Desmyter,
J .; Vandeputte, M. Both 2′,3′-dideoxythmidine and its 2′,3′-
unsaturated derivative (2′,3′-dideoxythymidinene) are potent
and selective inhibitors of human immunodeficiency virus
replication in vitro. Biochem. Biophys. Res. Commun. 1987, 142,
128-134.
(17) Mansuri, M. M.; Hitchcock, M. J . M.; Buroker, R. A.; Bregman,
C. L.; Ghazzouli, I; Desiderio, J . V.; Starrett J r, J . E.; Sterzycki,
R. Z.; Martin, J . C. Comparison of in vitro biological properties
and mouse toxicities of three thymidine analogs active against
human immunodeficiency virus. Antimicrob. Agents Chemother.
1990, 34, 637-641.
Ester a se Hyd r olysis. Compound 4a was incubated with
hog liver esterase (20 units/µL) at pH 7.6 in 25 mM Tris‚HCl
containing 10 mM magnesium dichloride at 37 °C and samples
were analyzed by HPLC at appropriate time points.
Ack n ow led gm en t. We thank the AIDS Directed
Programme of the MRC, the Biomedical Research
Programme and the Human Capital and Mobility Pro-
gramme of the European Commission, the Belgian
Geconcerteerde Onderzoeksacties, and the Belgian Na-
tionaal Fonds voor Wetenschappelijk Onderzoek for
financial support and Mrs. A. Absillis for excellent
technical assistance.
(18) Horwitz, J . P.; Chua, J .; DaRooge, M. A.; Noel, M.; Klundt, I. L.
Nucleosides. IX. The formation of 2′,3′-unsaturated pyrimidine
nucleosides via a novel â-elimination reaction. J . Org. Chem.
1966, 31, 205-211.
Refer en ces
(1) Mitsuya, H.; Weinhold, K. J .; Furman, P. A.; St. Clair, M. H.;
Nusinoff-Lehrman, S.; Gallo, R. C.; Bolognesi, D.; Barry, D. W.;
Broder, S. (1985) 3′-Azido-3′-deoxythymidine (BWA509U): An
agent that inhibits the infectivity and cytopathic effect of human
T-cell lymphotropic virus type III/lymphadenopathy-associated
virus in vitro. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 7096-7100.
(2) Mitsuya, H.; Broder, S. Inhibition of the in vitro infectivity and
cytopathic effect of human T-lymphotropic virus, type III/
lymphadenopathy-associated virus (HTLV III/LAV) by 2′,3′-
dideoxynucleosides. Proc. Natl. Acad. Sci. U.S.A. 1986, 83,
1911-1915.
(3) Balzarini, J .; Kang, G.-J .; Dalal, M.; Herdewijn, P.; De Clercq,
E.; Broder, S.; J ohns, D. G. The anti-HTLV-III (anti-HIV) and
cytotoxic activity of 2′,3′-didehydro-2′,3′-dideoxyribonucleosides.
A comparison with their parental 2′,3′-dideoxyribonucleosides.
Mol. Pharmacol. 1987, 32, 162-167.
(4) Herdewijn, P.; Balzarini, J .; De Clercq, E.; Pauwels, R.; Baba,
M.; Broder, S.; Vanderhaeghe, H. 3′-Substituted 2′,3′-dideoxy-
nucleoside analogues as potential anti-HIV (HTLV-III/LAV)
agents. J . Med. Chem. 1987, 30, 1270-1278.
(5) Balzarini, J .; Baba, M.; Pauwels, R.; Herdewijn, P.; De Clercq,
E. Anti-retrovirus activity of 3′-fluoro- and 3′-azido-substituted
pyrimidine 2′,3′-dideoxynucleoside analogues. Biochem. Phar-
macol. 1988, 37, 2847-2856.
(19) Mansuri, M. M.; Starrett J r, J . E.; Ghazzouli, I.; Hitchcock, M.
J . M.; Sterzycki, R. Z.; Brankovan, V; Lin, T.-S.; August, E. M.;
Prusoff, W. H.; Sommadossi, J .-P.; Martin, J . C. 1-(2,3-dideoxy-
â-D-glycero-pent-2-enfuranosyl)thymine. A Highly potent and
selective anti-HIV agent. J . Med. Chem. 1989, 32, 461-466.
(20) McGuigan, C.; Pathirana, R. N.; Mahmood, N.; Devine, K. G.;
Hay, A. J . Aryl phosphate derivatives of AZT retain activity
against HIV1 in cell lines which are resistant to the action of
AZT. Antiviral Res. 1992, 17, 311-321.
(21) Mark, V.; Dungan, C. H.; Crutchfield, M. M.; Van Wazer, J . R.
In Compilation of 31P NMR data; Grayson, M., Griffith, E. J .,
Ed,.; Topics in Phosphorus Chemistry, vol. 5; Wiley: New York,
1969.
(22) McGuigan, C.; Davies, M.; Pathirana, R. N.; Mahmood, N.; Hay,
A. J . Synthesis and anti-HIV activity of some novel diaryl
phosphate derivatives of AZT. Antiviral Res. 1994, 24, 69-77.
(23) Balzarini, J .; Karlsson, A.; Perez-Perez, M.-J .; Camarasa, M.-
J .; De Clercq, E. Knocking-out concentrations of HIV-1-specific
inhibitors completely suppress HIV-1 infection and prevent the
emergence of drug-resistant virus. Virology 1993, 196, 576-585.
(24) Balzarini, J .; Karlsson, A; Perno, C.-F.; Cahard, D.; Naesens,
L.; De Clercq, E.; McGuigan, C. Dual mechanism of action of
masked alaninyl d4TMP derivatives. Manuscript submitted for
publication.
(6) Furman, P. A.; Fyfe, J . A.; St. Clair, M. H.; Weinhold, K.;
Rideout, J . L.; Freeman, G. A.; Nusinoff Lehrman, S.; Bolognesi,
D. P.; Broder, S.; Mitsuya, H.; Barry, D. W. Phosphorylation of
3′-azido-3′-deoxythymidine and selective interaction of the 5′-
triphosphate with human immunodeficiency virus reverse tran-
scriptase. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8333-8337.
(25) Popovic, M.; Sarngadharan M. G.; Read, E.; Gallo, R. C.
Detection, isolation, and continuous production of cytopathic
retroviruses (HTLV-III) from patients with AIDS and pre-AIDS.
Science 1984, 224, 497-500.
J M950605J