The presence of substituents on the aryl moiety of the aryl phosphoramidate derivative of d4T enhances anti-HIV efficacy in cell culture: A structure-activity relationship

Article abstract of DOI:10.1021/jm9803931

New substituted-aryl phosphoramidate derivatives of the anti-HIV drug d4T were synthesized as membrane-soluble intracellular prodrugs for the free bioactive phosphate to establish relationship(s) between compound structure and in vitro antiviral activity.

Full text of DOI:10.1021/jm9803931

J . Med. Chem. 1999, 42, 393-399
393
Th e P r esen ce of Su bstitu en ts on th e Ar yl Moiety of th e Ar yl P h osp h or a m id a te
Der iva tive of d 4T En h a n ces An ti-HIV Effica cy in Cell Cu ltu r e: A
Str u ctu r e-Activity Rela tion sh ip
Adam Q. Siddiqui, Carlo Ballatore, Christopher McGuigan,* Erik De Clercq,† and J an Balzarini†
Welsh School of Pharmacy, University of Wales Cardiff, King Edward VII Avenue, Cardiff CF1 3XF, U.K., and
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received J uly 2, 1998
New substituted-aryl phosphoramidate derivatives of the anti-HIV drug d4T were synthesized
as membrane-soluble intracellular prodrugs for the free bioactive phosphate to establish
relationship(s) between compound structure and in vitro antiviral activity. The majority of
compounds demonstrated an elevation of in vitro potency relative to that of the parent
nucleoside, and unlike d4T, all retained full activity in thymidine kinase-deficient cells. The
compound bearing a p-chloro aryl group (8e) expressed nanomolar activity in vitro, a 14-fold
increase in activity relative to that of the unsubstituted phosphoramidate (100-fold compared
to d4T). An assay using pig liver esterase was used to establish the stability of the compounds
to enzymatic degradation. While there was no apparent correlation between in vitro activity
and half-life of enzymatic degradation, there was a close correlation between compound
lipophilicity, determined by octanol/water partition coefficient, and in vitro potency. We suggest
that substitutions made to the aryl moiety of the aryl phosphoramidate of d4T that result in
enhancing lipophilicity may serve to increase the cellular uptake of the prodrug by passive
diffusion, leading to the expression of antiviral potency at reduced prodrug concentrations.
Ch a r t 1
In tr od u ction
Among the current diversity of nucleoside analogues
active against human immunodeficiency virus (HIV) in
cell culture, the 2′,3′-dideoxynucleosides (ddN’s) remain
among the most potent.1-6 In all cases the expression
of activity requires conversion to the corresponding 5′-
triphosphates. These may inhibit the progression of the
virus by competitive inhibition of the viral reverse
transcriptase and/or incorporation and subsequent chain
termination of the growing viral DNA strand. Anti-HIV
ddN derivatives rely on host cell nucleoside and nucleo-
tide kinases to deliver the corresponding 5′-triphos-
phates. However, many such compounds have been
shown to be poor substrates for nucleoside kinases.7-11
Furthermore their activation is restricted in cells where
nucleoside kinases are less prominent or deficient (e.g.,
monocyte/macrophages).12 Consequently, prodrug strat-
egies that deliver the dideoxynucleoside have been
sought to effectively bypass the initial nucleoside kinase-
mediated phosphorylation step (reviewed by J ones and
Bischofberger13 and recently by Meier14).
Previously we have reported the considerable success
of an approach using (aryloxy)phosphoramidate nucleo-
side prodrugs. In particular the (aryloxy)phosphorami-
dates 1 and 2 (Chart 1) (derivatives of d4A and d4T,
respectively) were found to exhibit greatly enhanced
activity against HIV-1 and HIV-2 in cell culture com-
pared to their parent ddN’s, with full retention of
activity in thymidine kinase-deficient cell lines (Chart
1).15-17 The masked-phosphate approach to d4T was
particularly appealing for several reasons. This nucleo-
side has been shown to be a potent inhibitor of HIV and
also to display reduced toxicity compared to, for ex-
ample, AZT in certain cell types (e.g., bone marrow
progenitor cells).4,18,19 Furthermore the kinetics of the
three phosphorylation steps leading to the nucleotide
triphosphate from the original nucleoside suggests the
initial monophosphorylation step to be rate-limiting in
this case. This is in contrast to AZT where the rate-
limiting step appears to be the phosphorylation of the
monophosphate to the diphosphate.20a,b
* Author for correspondence: tel/fax, +44 1222 874537; e-mail,
mcguigan@cardiff.ac.uk.
Significant evidence points toward an enzyme-acti-
vated degradation pathway leading from these and
† Katholieke Universiteit Leuven.
10.1021/jm9803931 CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/26/1999

394 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Siddiqui et al.
Sch em e 1
similar phosphate prodrugs to their corresponding free
nucleotides. Two possible degradation pathways have
been proposed (Scheme 1). Path A involves initial
esterase-catalyzed cleavage of the carboxy ester, fol-
lowed by the internal nucleophilic attack of the acid
residue on the phosphorus center, displacing the aryloxy
group.21 The putative transient, cyclic mixed-anhydride
is then rapidly hydrolyzed to the corresponding alaninyl
phosphomonoester 6. Path B involves hydrolysis of the
phosphodiester amidate to the phosphomonoester ami-
date followed by enzymatic cleavage of the carboxy ester
to give the alaninyl phosphomonoester. The alaninyl
residue of the phosphomonoester (formed by either
pathway) is then presumed to be cleaved by either
chemical or enzymatic means to give the nucleoside
monophosphate 7.
Previous metabolic studies on the (aryloxy)phospho-
ramidate of d4T (2)22 have revealed the presence of
significant amounts of alaninyl phosphomonoester 6 (in
addition to mono-, di-, and triphosphorylated nucleo-
sides) following incubation of 2 with cells. Similarly
studies by Imbach and co-workers on the (aryloxy)-
phosphoramidate (3) of isoddA21 revealed significant
amounts of alaninyl phosphomonoester on incubation
of 3 with cell extracts. Conversely when the correspond-
ing phosphomonoester amidate was incubated with cell
extracts, neither the alaninyl phosphomonoester nor the
nucleoside monophosphate were observed. These results
suggest that path A is the major route to the free
nucleoside monophosphate.
Exhaustive modifications to the amino acid moiety in
2 have established L-alanine as the moiety for optimal
antiviral activity.23 A small series of substituted (aryl-
oxy)phosphoramidate analogues of 2 was previously
synthesized to determine the effects on potency of modi-
fying the aryloxy moiety.17 The compounds were found
to be less active than the parent structure against HIV-1
and HIV-2 in cell culture. However, comparisons be-
tween the phenyl and p-nitrophenyl phosphoramidates
of isoddA 3 and 4 made by Imbach and co-workers
reveal interesting differences in the stability of the two
compounds.21 In culture medium and in human serum
(where esterase activity is less than in cell extracts),
the p-nitrophenyl phosphoramidate 4 is less stable than
the phenyl phosphoramidate 3 (likely due to the electron-
withdrawing characteristics of the nitro group). By
contrast, in cell extracts the p-nitrophenyl phosphor-
amidate 4 is twice as stable as the phenyl phosphora-
midate 3, indicating that the polar p-nitro substituent
may adversely affect the compound’s interaction with
cellular esterase(s). Furthermore, substitutions at the
aryloxy part of 2 may have the effect of altering not only
the rate and/or nature of prodrug degradation but also
the rate of membrane transport.
We present here a comprehensive structure-activity
relationship study on the effect of various substitutions
of the aryloxy moiety of 2 on anti-HIV activity in cell
culture. We will define some of the key chemical
characteristics required for lead optimization including
correlations between antiviral activity, compound parti-
tion coefficient, and compound stability to both enzy-
matic and chemical degradation.
Resu lts a n d Discu ssion
The synthesis of d4T from thymidine was conducted
essentially by the method of Horwitz24 noting the later
comments of Mansuri.25 (Aryloxy)phosphorochloridates,
and (aryloxy)aminophosphorochloridates were prepared
entirely as previously noted.26 Phosphoramidates
8a -8i (Table 1) and 2 were synthesized according to
the method described previously.17 The structure and
purity of compounds 8a -8i were determined by exten-
sive multinuclear NMR and analytical HPLC using two
distinct eluent gradients. The p-aryl substituents in
8a -8h were chosen to reflect a broad cross-section of
electron-donating/withdrawing effects (as measured by
Hammet σ values) with particular emphasis on the
latter (Table 1). Previous work with diaryl phosphates
of AZT demonstrated elevated antiviral activity for aryl
substituents containing electron-withdrawing substit-
uents.27 Although the mechanism(s) of degradation of
these AZT derivatives and the phosphoramidates syn-
thesized here may be different, it is possible that
electron-withdrawing substituents will increase the rate
at which the aryloxy group leaves after enzymatic
hydrolysis of the amino ester. The choice of substituents

Substituted-Aryl Phosphoramidate Derivatives of d4T
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 395
Ta ble 1a
pounds displayed significant antiviral activity against
Moloney murine sarcoma virus (MSV).
structure
X
σp
σm
π
Com p ou n d P a r tition Coefficien ts. The partition
coefficients of compounds 8a -8i and 2 were determined
in 1-octanol/aqueous buffer (pH 7.0). Each compound
was partitioned between 1-octanol and aqueous buffer
for 5 min. After separation of the two layers the relative
proportion of compound in each layer was determined
by measuring the UV absorption of the octanol layer
compared to a control solution. This method of deter-
mining the partition coefficients allows for a qualitative
comparison between the compounds in the series. The
results are shown in Table 3. The partition coefficients
correlated well with the Hansch constants of the respec-
tive substituents (Table 1) used initially to predict the
general order of lipophilicity. Compounds containing the
most polar substituents (8a , 8b, 8d , and 8i) gave rise
to the lowest Pa values, while those containing the least
polar substituents (8e and 8g) gave rise to the highest
values. Comparative data for d4T29 indicate significantly
increased lipophilicity for all the phosphoramidates
relative to their parent nucleoside. A plot of log Pa
versus-log EC50 (HIV-1) reveals a close correlation
(correlation coefficient, r ) 0.90) between antiviral
potency and compound hydrophobicity (Figure 1), with
the most lipophilic compound (8e) showing the greatest
biological activity. This may reflect an increased mem-
brane transport by passive diffusion for compounds
bearing lipophilic substituents. In addition it may also
reflect increased preference by cellular hydrolytic en-
zymes.
8a
8b
8c
8d
8e
8f
8g
8h
8i
p-NO2
p-CN
p-COOMe
p-COMe
p-Cl
p-F
p-Me
p-OMe
m-COMe
H
0.81
0.70
0.48
0.47
0.24
0.15
-0.14
-0.28
-0.28
-0.57
-0.01
-0.55
0.71
0.14
0.56
-0.02
0.35
0
2
0
0
Data for σ and π constants taken from Hansch and Leo.28
Quoted π values are for X-C6H5 and not for the specific structures
8a -8i.
a
was also influenced by the desire to examine the
influence of compound lipophilicity on antiviral activity.
Consequently, the substituents also reflect a broad
range of Hansch constants (π). Also, to compare the
possible effects of m-aryl versus p-aryl substituents, the
m-acyl-substituted analogue of 8d was synthesized (8i).
Given the enzymatic nature of the putative initial step
in phosphoramidate degradation, specific aryl substitu-
tion patterns may favor or disfavor interaction of the
phosphoramidate with the active site of the enzyme(s),
and substituent polarity and hydrogen-bonding char-
acteristics may also prove to be important in this
respect.
An tivir a l Activity. All of the synthesized phospho-
ramidates 8a -8i were evaluated for their ability to
inhibit the replication of HIV, as previously described.26
The results obtained using HIV-1- or HIV-2-infected
CEM cells are displayed in Table 2. The tests were also
conducted against HIV-2 in thymidine kinase-deficient
(TK-) CEM cells. For the purpose of reference, similar
data for d4T and 2 obtained previously17 are also
displayed.
All of the (substituted-aryloxy)phosphoramidates apart
from 8a , 8b, and 8d showed a notable elevation in
antiviral potency against HIV-1 and HIV-2 compared
to the free nucleoside, d4T. Furthermore, whereas d4T
showed a pronounced decrease in activity in CEM/TK-
cells, all of the synthesized phosphoramidates appeared
to retain full antiviral activity, in common with 2. The
activity displayed by most of the compounds was
comparable to that of the lead structure 2. The notable
exception to this is the p-Cl derivative 8e, which
displayed an approximately 14-fold greater activity than
2 (100 times greater than d4T). This increased potency
was also reflected in CEM/TK- cells. Significantly
enhanced activity was also shown by the p-COOMe
derivative 8c.
Compounds containing the most electron-withdrawing
substituents (highest σ value) appear to be among the
least potent in the series. Also, the compounds showing
greatest antiviral activity have σ values in the range
0.24-0.48. The exceptions to this are the p- and m-acyl-
substituted compounds (8d and 8i) which have σ values
within this range but do not display a substantially
elevated potency compared to 2. The cytotoxicity data
(CC50) show all of the synthesized phosphoramidates to
exhibit greater toxicity than 2. Furthermore, a strong
positive correlation exists between toxicity and potency,
indicating that toxicity most likely arises from the same
pharmacophore in each case. None of the tested com-
En zym a tic Degr a d a tion Stu d ies. In an attempt to
establish the dependence of drug potency on enzymatic
degradation, compounds 8a -8i and 2 were incubated
with pig liver esterase (PLE) in pH 7.6 buffer. PLE is a
mixture of five isozymes; the two major isozymes, I and
V, hydrolyze aromatic/long-chain esters and short-chain
esters, respectively. An addition of acetone was made
in each case to solubilize the test compound, and it
should be noted that acetone has been found to inhibit
isozyme V.30 Evidence suggests that most mammalian
esterases are similar but not identical. For example,
PLE has a virtually identical subunit weight and similar
amino acid sequence as human esterase. They differ in
that human esterase contains fewer isozymes.31 The
progress of degradation was followed by 31P NMR
spectroscopy, by monitoring the loss of starting material.
In all cases the degradation of the substrate gave rise
to a signal in the phosphorus NMR spectra previously
determined32 to correspond to compound 6 (Scheme 1)
(δP(D2O) 8.1 ppm). The results (summarized in Table
3) show the overall half-lives of degradation to be
remarkably similar for the majority of compounds,
varying between 100 and 150 h, with some notable
exceptions. The lead structure 2 and the p-fluoro
derivative 8f exhibited the greatest stabilities, both
having half-lives of 300 h. The p-nitro (8a ) and p-
methoxy (8h ) derivatives were the least stable with half-
lives of 60 and 11 h, respectively. Control experiments
were conducted to establish the independent rates of
chemical hydrolysis, by incubating the compounds in the
same buffer in the absence of enzyme (results shown in
Table 3). The degradation half-lives in the presence of
enzyme are labeled with an asterisk for those com-

396 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Siddiqui et al.
Ta ble 2. Anti-HIV-1, Anti-HIV-2, and Cytotoxicity of Compounds 8a -8i, 2, and d4T in Vitro
a
b
EC50 (µM)
CC50 (µM)
EC50 (µM)
MCCc (µM)
compd
CEM/0 (HIV-1)
CEM/0 (HIV-2)
CEM/TK- (HIV-1)
CEM/0 (HIV-2)
C3H/3T3 (MSV)
C3H/3T3
8a
8b
8c
8d
8e
8f
8g
8h
8i
0.130
0.150
0.025
0.110
0.005
0.053
0.040
0.057
0.080
0.075
0.651
0.190
0.130
0.022
0.100
0.007
0.060
0.050
0.053
0.090
0.075
0.770
0.09
0.09
73
60
31
40
11
26
34
35
50
9.3
30
5.6
13
2.2
7.4
10
16
26
15
d
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
d
0.025
0.050
0.006
0.022
0.025
0.047
0.045
0.075
33
2
d4T
>100
174
a
EC50 is the 50% effective compound concentration required to protect CEM cells against the cytopathogenicity of HIV by 50% or to
b
inhibit MSV-induced transformation of C3H/3T3 cells by 50%. Data are the means of 2-4 independent experiments. CC50 is the 50%
cytotoxic compound concentration required to inhibit CEM cell proliferation. c MCC is the minimum cytotoxic concentration that causes
a microscopically detectable alteration of cell morphology. EC50 and CC50 data for d4T are taken from McGuigan et al.17 Not determined.
d
Ta ble 3. Partition Coefficient Data and Degradation
Half-Lives at pH 7.6 and 37 °C in the Presence and Absence of
Pig Liver Esterase (Principle Product: 6, Scheme 1)
may have a diminished rate of membrane transport by
passive diffusion compared to the phosphate diester.
Showing no independent chemical hydrolysis over the
time scale of the enzyme experiments, the rapid rate of
t1/2 (h)
compd
log Pa
enzymatic
chemical
degradation shown by the p-methoxy derivative 8h in
the presence of enzyme must reflect the rate of its
enzymatic activation. p-Methoxyphenol is an inferior
chemical leaving group compared to any of the phenols
having electron-withdrawing substituents. Further-
more, the lipophilicity of 8h is similar to that of 2 (log
Pa 8h ) 1.09, log Pa 2 ) 1.04); therefore, specific
structural characteristics of 8h most likely serve to
enhance its recognition as a substrate compared to 2.
8a
8b
8c
8d
8e
8f
8g
8h
8i
0.90
0.91
1.01
0.90
1.43
1.10
1.19
1.09
0.92
1.04
*60 ( 6
*100 ( 10
*100 ( 10
*150 ( 15
151 ( 15
301 ( 30
151 ( 15
11 ( 1
151 ( 15
151 ( 15
301 ( 30
301 ( 30
a
a
a
a
*151 ( 15
301 ( 30
301 ( 30
2
a
No apparent correlations were noted between the
degradation half-lives in the presence of enzyme and
the antiviral activity of the different compounds. An
obvious trend is also absent when the half-lives of those
compounds shown not to undergo independent chemical
hydrolysis (over the time scale of the enzyme experi-
ments) are compared to biological activity. In these cases
(compounds 8e-8h and 2), there would be a direct
correlation between the biological activity and the rate
of enzymatic degradation. These data show that quan-
titative kinetic comparisons between the enzymatic
assay and the in vitro antiviral potency are not evident
for this series of compounds. However, they demonstrate
the formation of 6 from all compounds in the series 8a -
8i, via esterase-mediated degradation. These results are
consistent with earlier observations that 6 is the major
metabolite of 2 in six discrete cell lines and is presumed
to degrade by chemical or enzymatic means to the
corresponding nucleoside monophosphate.22
a
*Chemical hydrolysis observed. Stable to chemical hydrolysis.
A simple correlation between substituent σ values and
overall degradation half-life is not apparent, since the
least stable compounds (8a and 8h ) have the highest
and lowest σ values, respectively. The relatively rapid
degradation of the p-nitro derivative 8a cannot be
entirely explained in terms of additional chemical
hydrolysis during the course of the enzyme experiment,
since the p-cyano derivative 8b, although chemically
hydrolyzed at the same rate as 8a , was almost twice as
stable in the presence of enzyme. Also, the lipophilicities
of compounds 8a and 8b are similar (log Pa 8a ) 0.90,
log Pa 8b ) 0.91). Therefore, improved molecular
recognition of the p-nitro derivative by the enzyme is
the probable explanation. This is in contrast to the
conclusion drawn by Imbach and co-workers21 regarding
F igu r e 1. Antiviral potency in CEM/0 cells (-log EC50 HIV-1
in µM) plotted against partition coefficient (log Pa). Correlation
coefficient (r) ) 0.90.
pounds where independent chemical hydrolysis was
observed. Of the compounds containing electron-with-
drawing substituents, all exhibited independent chemi-
cal hydrolysis apart from the p-fluoro (8f) and p-chloro
(8g) derivatives. Such chemical hydrolysis may be
problematic in terms of drug delivery, as substantial
hydrolysis in the extracellular environment may lead
to the phosphate monoester, which, carrying a charge,

Substituted-Aryl Phosphoramidate Derivatives of d4T
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 397
Partition coefficients were determined using 1-octanol and
water (Fisher, HPLC grade); UV absorptions were determined
using a Kontron Uvikon 860 UV spectrometer.
the rate of enzyme-induced activation of the (p-nitro-
phenyloxy)phosphoramidate of isoddA.
Differences between meta- and para-substitution ap-
pear to be minimal in the present series. The partition
coefficients of the p- and m-acyl derivatives 8d and 8i
are similar (8d log Pa ) 0.90, 8i log Pa ) 0.92), as are
their respective rates of degradation in the presence and
absence of carboxyesterase. These similarities are re-
flected well in their respective in vitro antiviral poten-
cies (EC50(HIV-1): 8d ) 0.11, 8i ) 0.08; EC50(HIV-2):
8d ) 0.10, 8i ) 0.09).
Finally, it is worth comparing the p-Cl and p-Me
derivatives (8e and 8g) directly against each other.
Medicinally, the methyl group is considered to be a good
isostere for the chlorine atom. There is no discernible
difference between the two compounds’ respective rates
of degradation in the presence of enzyme. The ap-
proximately 10-fold greater activity of 8e over 8g may
be associated with the electron-withdrawing character-
istics of the former compared to the electron-donating
character of the latter and/or the superior lipophilicity
of 8e.
In conclusion, we have been able to demonstrate a
structure-activity relationship between substituted
(aryloxy)phosphoramidates and their in vitro antiviral
activity. Optimum activity appears to correlate with
aryloxy substituents which have the following proper-
ties: exhibit a mild electron-withdrawing effect corre-
sponding to values of σ between 0.15 and 0.48 and give
rise to high lipophilicity as evidenced by octanol/water
partition coefficients. To corroborate these conclusions
further, the synthesis of a series of compounds possess-
ing these attributes is currently underway in our
laboratories.
Gen er a l P r oced u r e. Aryl (methoxyalaninyl)phosphoro-
chloridate (5 mmol) was added to a stirred solution of d4T (1.7
mmol) and N-methylimidazole (5 mmol) in tetrahydrofuran
(THF) (20 mL) at ambient temperature. After 16 h, the solvent
was removed under reduced pressure. The residual gum was
dissolved in chloroform (50 mL) and washed with 1 M HCl
(50 mL), sodium bicarbonate solution (50 mL), and water (50
mL). The organic phase was dried (MgSO4) and the solvent
removed under reduced pressure. The residue was purified by
column chromatography on silica with elution by dichlo-
romethane-methanol (97:3). Pooling of appropriate fractions
followed by removal of solvent under reduced pressure gave
the product as a brittle white foam.
2′,3′-Didehydro-2′,3′-dideoxythymidine 5′-(4-nitrophenyl
(methoxyalaninyl)phosphate) (8a): yield 53%; δP 4.72, 4.06;
δH 1.40 (3H, m, Ala-Me), 1.88 (3H, d, 5-Me), 3.77 (3H, s, OMe),
4.12 (2H, m, Ala-NH, Ala-CH), 4.40 (2H, m, H-5′), 5.10 (1H,
m, H-4′), 5.96 (1H, m, H-3′), 6.42 (1H, m, H-2′), 7.07 (1H, m,
H-1′), 7.28 (1H, m, H-6), 7.61 (2H, m, ortho-Ph), 8.09 (2H, m,
meta-Ph), 9.10 (1H, d, NH); δC 12.78, 12.87 (5-Me), 21.33, 21.40
(d, J ) 5.2, Ala-Me), 50.55, 50.62 (Ala-CH), 53.24 (OMe), 67.36,
68.08 (d, J ) 4.5, J ) 6.0, C-5′), 84.81, 84.93 (C-4′), 90.11, 90.41
(C-1′), 113.52, 113.70 (C-5), 116.10, 116.17 (d, J ) 5.7, Ph),
120.51, 120.62 (Ph), 127.19, 127.26 (C-2′), 133.22, 133.57
(C-3′), 135.92, 136.11 (C-6), 137.78, 137.97 (Ph), 151.16 (C-2),
153.04, 153.19 (Ph), 165.95, 165.99 (C-4), 175.99, 176.17 (d, J
) 7.5, J ) 6.8, Ala-CO); MS m/e FAB 511.1224 (MH+,
C
20H24N4O10P requires 511.1230); HPLC tR 35.56, 36.13 min,
tR 39.72, 40.27 min (gradient II).
2′,3′-Dideh ydr o-2′,3′-dideoxyth ym idin e 5′-(4-cyan oph en -
yl (m eth oxya la n in yl)p h osp h a te) (8b): yield 61%; δP 4.31,
3.71; δH 1.38 (3H, m, Ala-Me), 1.88 (3H, d, 5-Me), 3.73 (3H, s,
OMe), 4.02 (2H, m, Ala-NH, Ala-CH), 4.30 (2H, m, H-5′), 5.08
(1H, m, H-4′), 5.96 (1H, m, H-3′), 6.35 (1H, m, H-2′), 7.08 (1H,
m, H-1′), 7.33 (3H, m, H-6, ortho Ph), 7.69 (2H, m, meta-Ph),
9.56 (1H, d, NH); δC 12.84, 12.88 (5-Me), 21.18, 21.26 (Ala-
Me), 50.52, 50.62 (Ala-CH), 53.18 (OMe), 67.32, 68.04 (C-5′),
84.81, 84.93 (C-4′), 90.05, 90.34 (C-1′), 109.34, 109.46 (C-5),
111.64, 111.80 (C-5), 118.51 (CN), 121.49, 121.69 (d, J ) 5.3,
Ph), 128.02, 128.13 (C-2′), 133.23, 133.53 (C-3′), 134.50, 134.54
(Ph), 135.93, 136.16 (C-6), 151.34 (Ph), 154.05 (C-2), 160.64
(Ph), 164.31, 164.36 (C-4), 174.11, 174.33 (d, J ) 7.5, J ) 6.8,
Ala-CO); MS m/e FAB 491.1320 (MH+, C21H24N4O8P requires
491.1331); HPLC tR 35.02, 35.44 min, tR 37.39, 37.83 min
(gradient II).
2′,3′-Did eh yd r o-2′,3′-d id eoxyth ym id in e 5′-(4-(m eth yl-
ca r boxy)p h en yl (m eth oxya la n in yl)p h osp h a te) (8c): yield
91%; δP 3.40, 4.04; δH 1.28 (3H, m, Ala-Me), 1.75 (3H, d, 5-Me),
3.60 (3H, s, CH3CO), 3.83 (3H, s, ester-OCH3), 3.92 (1H, m,
Ala-CH), 4.29 (3H, m, H-5′, Ala-NH), 4.97 (1H, m, H-4′), 5.83
(1H, m, H-3′), 6.26 (1H, m, H-2′), 6.94 (1H, m, H-1′), 7.22 (3H,
m, H-6, ortho-Ph), 7.92 (2H, m, meta-Ph), 9.86 (1H, d, NH);
δC 13.51, 13.55 (5-Me), 21.86, 21.92 (d, J ) 3.6, J ) 2.9, Ala-
Me), 51.22, 51.33 (Ala-CH), 53.39 (ester-OMe), 53.78 (Ala-
OMe), 67.82, 67.88 (C-5′), 85.64, 85.76 (d, J ) 2.5, C-4′), 90.70,
90.97 (C-1′), 112.40, 112.54 (C-5), 121.07, 121.26 (d, J ) 5.2,
J ) 5.1, Ph), 128.06, 128.12 (C-2′), 128.62, 132.67, 132.73 (Ph),
134.08 (C-3′), 136.75, 136.97 (C-6), 152.25 (Ph), 155.10, 155.18
(C-2), 165.29, 165.30 (C-4), 175.03, 175.16 (d, J ) 7.1, J ) 6.3,
Ala-CO); MS m/e FAB 524.1421 (MH+, C22H27N3O10P requires
524.1434); HPLC tR 37.84, 38.10 min, tR 41.38, 41.65 min
(gradient II).
Exp er im en ta l Section
All experiments involving water-sensitive compounds were
conducted under scrupulously dry conditions. Triethylamine
was dried by refluxing over calcium hydride. Anhydrous
tetrahydrofuran and dichloromethane were purchased from
Aldrich. N-Methylimidazole was purified by distillation. Nu-
cleosides were dried by storage at elevated temperature over
P2O5 in vacuo. Proton, carbon, and phosphorus nuclear mag-
netic resonance (1H, 13C, and 31P NMR) spectra were recorded
on a Bruker Avance DPX spectrometer operating at 300, 75.5,
and 121.5 MHz, respectively. All 13C and 31P NMR spectra were
recorded proton-decoupled. All NMR spectra were recorded in
CDCl3 at room temperature (20 ( 3 °C). Chemical shifts for
1H and 13C NMR spectra are quoted in parts per million
downfield from tetramethylsilane. Coupling constants are
referred to as J values in hertz (Hz). Signal splitting patterns
are described as singlet (s), doublet (d), triplet (t), quartet (q),
or multiplet (m). Chemical shifts for 31P NMR spectra 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 spectrometry
was fast atom bombardment (FAB) using MNOBA as matrix.
Column chromatography refers to flash column chromatogra-
phy carried out using Merck silica gel 60 (40-60 µm) as
stationary phase. HPLC (Shimadzu) was conducted on an
SSODS2 reverse-phase column using a water/acetonitrile
(Fisher, HPLC grade) eluent; gradient I (standard gradient):
0-80% CH3CN (0-60 min), 80-0% CH3CN (60-65 min), flow
rate 1 mL/min, UV detection at 265 nm; gradient II: 0-10%
CH3CN (0-5 min), 10-70% CH3CN (5-55 min), 70-0%
CH3CN (55-60 min), flow rate 1 mL/min, UV detection at 265
nm. Final products showed purities exceeding 99% with
undetectable levels (<0.02) of parent nucleosides in every case.
2′,3′-Dideh ydr o-2′,3′-dideoxyth ym idin e 5′-(4-acetylph en -
yl (m eth oxya la n in yl)p h osp h a te) (8d ): yield 91%; δP 3.56,
4.20; δH 1.38 (3H, m, Ala-Me), 1.89 (3H, d, 5-Me), 2.60 (3H, s,
CH3CO), 3.77 (3H, s, OMe), 4.02 (2H, m, Ala-NH, Ala-CH),
4.41 (2H, m, H-5′), 5.10 (1H, m, H-4′), 5.98 (1H, m, H-3′), 6.39
(1H, m, H-2′), 7.08 (1H, m, H-1′), 7.32 (3H, m, H-6, ortho-Ph),
7.98 (2H, m, meta-Ph), 9.11 (1H, d, NH); δC 12.79, 12.84
(5-Me), 21.32 (Ala-Me), 27.00 (COMe), 50.49, 50.61 (Ala-CH),
53.14 (OMe), 66.16, 67.78 (C-5′), 84.88, 84.99 (d, J ) 5.3, C-4′),

398 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Siddiqui et al.
90.03, 90.30 (C-1′), 111.65, 111.81 (C-5), 120.43, 120.65 (d, J
) 5.3, J ) 3.8, Ph), 127.93, 128.08 (C-2′), 130.78, 130.84 (Ph),
133.32, 133.65 (C-3′), 135.96, 136.20 (C-6), 151.23 (Ph), 153.34,
153.50 (C-2), 164.17 (C-4), 174.84 (Ala-CO), 197.15 (CO); MS
m/e FAB 508.1495 (MH+, C22H27N3O9P requires 508.1485);
HPLC tR 35.42, 35.65 min, tR 39.54, 40.28 min (gradient II).
(1H, m, H-2′), 7.10 (1H, m, H-1′), 7.32 (1H, m, H-6), 7.50 (2H,
m, Ph), 7.80 (2H, m, Ph) 8.83 (1H, d, NH); δC 12.70, 12.80
(5-Me), 21.28, 21.35 (d, J ) 4.5, Ala-Me), 27.13 (COMe), 50.50,
50.59 (Ala-CH), 53.10 (OMe), 67.08, 67.75 (d, J ) 4.5, C-5′),
84.91, 85.02 (d J ) 2.3, J ) 3.0, C-4′), 90.01, 90.27 (C-1′),
111.65, 111.80 (C-5), 120.18, 120.26 (d, J ) 5.2, Ph), 125.27,
125.53 (d, J ) 4.5, Ph) 127.92, 128.02 (C-2′), 130.49 (Ph),
133.34, 133.73 (C-3′), 136.02, 136.28 (C-6), 139.12 (Ph), 150.96
(Ph), 151.32, 151.07 (C-2), 164.32 (C-4), 174.32, 174.22 (Ala-
CO), 197.35, 197.45 (CO); MS m/e FAB 508.1477 (MH+,
2′,3′-Dideh ydr o-2′,3′-dideoxyth ym idin e 5′-(4-ch lor oph en -
yl (m eth oxya la n in yl)p h osp h a te) (8e): yield 72%; δP 3.97,
4.59; δH 1.39 (3H, m, Ala-Me), 1.91 (3H, d, 5-Me), 3.77 (3H, s,
OMe), 3.98 (2H, m, Ala-NH, Ala-CH), 4.36 (2H, m, H-5′), 5.09
(1H, m, H-4′), 5.93 (1H, m, H-3′), 6.40 (1H, m, H-2′), 7.10 (1H,
m, H-1′), 7.26 (5H, m, H-6, Ph), 9.11 (1H, d, NH); δC 12.79,
12.84 (5-Me), 21.34, 21.41 (d, J ) 5.3, Ala-Me), 50.49, 50.61
(Ala-CH), 53.11 (OMe), 67.04, 67.72 (d, J ) 0, J ) 5.3, C-5′),
84.92, 85.01 (C-4′), 90.01, 90.27 (C-1′), 111.68, 111.83 (C-5),
121.88, 122.05 (d, J ) 4.5, Ph), 127.89, 128.03 (C-2′), 130.78,
130.84 (Ph), 133.35, 133.69 (C-3′), 135.95, 136.24 (C-6), 149.19,
149.34 (d, J ) 6.8, J ) 6.0, Ph), 151.23, 151.25 (C-2), 164.15,
164.20 (C-4), 174.21, 174.40 (d, J ) 7.5, J ) 6.8, Ala-CO); MS
m/e FAB 500.1003 (MH+, C20H24N3O8PCl requires 500.0990);
HPLC tR 44.43, 44.79 min, tR 41.82, 42.32 min (gradient II).
2′,3′-Dideh ydr o-2′,3′-dideoxyth ym idin e 5′-(4-flu or oph en -
yl (m eth oxya la n in yl)p h osp h a te) (8f): yield 68%; δP 4.15,
4.77; δH 1.36 (3H, m, Ala-Me), 1.91 (3H, d, 5-Me), 3.79 (3H, s,
OMe), 4.05 (2H, m, Ala-NH, Ala-CH), 4.37 (2H, m, H-5′), 5.10
(1H, m, H-4′), 5.99 (1H, m, H-3′), 6.37 (1H, m, H-2′), 7.08 (3H,
m, H-1′, ortho-Ph), 7.29 (3H, m, H-6, meta-Ph), 8.88 (1H, d,
NH); δC 12.78, 12.84 (5-Me), 21.40, 21.47 (d, J ) 5.3, Ala-Me),
50.48, 50.62 (Ala-CH), 53.12 (OMe), 66.95, 67.70 (d, J ) 4.5,
C-5′), 84.92, 85.06 (C-4′), 90.01, 90.27 (C-1′), 111.68, 111.83
(C-5), 116.65, 116.94 (Ph), 121.99, 122.10 (d, J ) 4.5, J ) 3.7,
Ph), 127.83, 128.00 (C-2′), 133.42, 133.76 (C-3′), 135.99, 136.29
(C-6), 146.50 (Ph), 151.15 (C-2), 163.99, 164.06 (C-4), 174.17,
174.22, 174.40 (d, J ) 7.5, J ) 6.8, Ala-CO); MS m/e FAB
484.1285 (MH+, C20H24N3O8FP requires 484.1285); HPLC tR
39.15 min, tR 42.05 min (gradient II).
2′,3′-Dideh ydr o-2′,3′-dideoxyth ym idin e 5′-(4-m eth ylph en -
yl (m eth oxya la n in yl)p h osp h a te) (8g): yield 65%; δP 4.02,
4.67; δH 1.36 (3H, m, Ala-Me), 1.87 (3H, d, 5-Me), 2.34 (3H, s,
Ph-Me), 3.75 (3H, d, OMe), 3.95 (2H, m, Ala-NH, Ala-CH), 4.36
(2H, m, H-5′), 5.05 (1H, m, H-4′), 5.92 (1H, m, H-3′), 6.36 (1H,
m, H-2′), 7.12 (4H, m, H-1′, H-6, meta-Ph), 7.35 (2H, m, ortho-
Ph), 9.40 (1H, d, NH); δC 12.75, 12.79 (5-Me), 21.13, 21.29
(Ph-Me), 21.20, 21.38 (d, J ) 9.8, J ) 3.8, Ala-Me), 50.45, 50.58
(Ala-CH), 53.01 (OMe), 66.80, 67.45 (d, J ) 4.5, J ) 5.3, C-5′),
85.02, 85.05 (d, J ) 5.3, C-4′), 89.93, 90.18 (C-1′), 111.75
(C-5), 120.19, 120.37 (d, J ) 5.3, J ) 4.5, Ph), 127.72, 127.90
(C-2′), 130.57, 130.63 (Ph), 133.48, 133.83 (C-3′), 136.07, 136.38
(C-6), 148.39 (Ph), 151.37 (C-2), 164.37 (C-4), 174.52 (Ala-CO);
MS m/e FAB 480.1541 (MH+, C21H27N3O8P requires 480.1536);
HPLC tR 40.10, 40.62 min, tR 43.53, 44.05 min (gradient II).
2′,3′-Did eh yd r o-2′,3′-d id eoxyth ym id in e 5′-(4-m eth oxy-
p h en yl (m eth oxya la n in yl)p h osp h a te) (8h ): yield 64%; δP
4.34, 4.97; δH 1.39 (3H, m, Ala-Me), 1.89 (3H, d, 5-Me), 3.76
(3H, d, OMe), 3.82 (3H, d, OMe), 3.89 (1H, m, Ala-NH), 4.02
(1H, m, Ala-CH), 4.39 (2H, m, H-5′), 5.07 (1H, m, H-4′), 5.97
(1H, m, H-3′), 6.38 (1H, m, H-2′), 6.87 (2H, m, H-1′, H-6, meta-
Ph), 7.12 (3H, m, H-1′, ortho-Ph), 7.37 (1H, m, H-6), 9.23 (1H,
d, NH); δC 12.75, 12.80 (5-Me), 21.36, 21.43 (d, J ) 4.5, Ala-
Me), 50.46, 50.61 (Ala-CH), 53.04 (OMe), 56.03 (Ph-OMe),
66.81, 67.48 (d, J ) 4.5, J ) 5.3, C-5′), 85.02, 85.13 (d, J )
3.0, J ) 4.5, C-4′), 89.95, 90.21 (C-1′), 111.69, 111.82 (C-5),
115.03, 115.10 (Ph), 121.40, 121.59 (d, J ) 5.3, J ) 4.5, Ph),
127.71, 127.90 (C-2′), 133.50, 133.86 (C-3′), 136.10, 136.43
(C-6), 144.09, 144.28 (d, J ) 6.0, J ) 6.8, Ph), 151.23, 151.28
(C-2), 157.21 (Ph), 164.16, 164.24 (C-4), 174.34, 174.52 (d, J
) 7.5, Ala-CO); MS m/e FAB 496.1480 (MH+, C21H27N3O9P
requires 496.1485); HPLC tR 36.67, 37.03 min, tR 40.94 min
(gradient II).
C
22H27N3O9P requires 508.1485); HPLC tR 35.74, 36.01 min,
tR 39.59, 39.83 min (gradient II).
Deter m in a tion of Com p ou n d P a r tition Coefficien ts.
The following method was used to determine the Pa values
for compounds 8a -8i and 2. A sample of each compound (2
µM) was dissolved in 1-octanol (10 mL) by rapid magnetic
stirring for 10 min. Aqueous buffer (pH 7.0) (Fisons) (5 mL)
was added to an aliquot of the octanol solution (5 mL), and
the two phases were mixed by rapid magnetic stirring for 5
min at 25 °C. Following separation of the two layers by
standing for a further 5 min, aliquots of the octanol layer and
of the original octanol solution were removed and their UV
absorptions determined at 265 nm. The Pa values were
calculated from the ratio of the absorptions of the two octanol
aliquots. Each experiment was repeated at least three times.
P ig Liver Ester a se Hyd r olysis: Sta n d a r d Assa y. The
enzymatic reactions were performed in all cases using pig liver
esterase (PLE) (E.C. 3.1.1.1; Sigma) (19 units/mg). Typically
9 µmol of the substrate and 10 mg of PLE were dissolved in a
mixture of acetone (0.1 mL) and 0.05 M pH 7.6 Trizma buffer
(1 mL, made up in D2O). The mixture was transferred to an
NMR tube and maintained at 37 °C. The reactions were
monitored periodically by 31P NMR spectroscopy over 48 h. The
reactions were followed by measuring the decrease of starting
material, and the data were graphically visualized by plotting
the percent of starting material against time. From the
logarithmic plot, half-lives were determined for comparison of
substrate activity.
Ch em ica l Hyd r olysis: Sta n d a r d Assa y. Chemical hy-
drolyses were evaluated under the same conditions used for
the PLE hydrolysis: 9 µmol of substrate was dissolved in a
mixture of acetone (0.1 mL) and 0.05 M pH 7.6 Trizma buffer
(1 mL, made up in D2O) and maintained at 37 °C. The rate of
hydrolysis was determined periodically by monitoring the
decrease of starting material by 31P NMR spectroscopy for 6
days. From a logarithmic plot, half-lives were determined for
comparison of substrate stability.
An tivir a l Activity Deter m in a tion s. 1. Vir u ses. HIV-
1(strain IIIB) was kindly provided by Dr. R. C. Gallo (at that
time at the National Institutes of Health, Bethesda, MD).
Virus stocks were prepared from the supernatants of HIV-1-
infected MT-4 cells. HIV-2 (strain ROD) was a gift from Dr.
L. Montagnier (Pasteur Institute, Paris, France), and virus
stocks were prepared from the supernatants of HIV-2-infected
MT-4 cells. MSV was prepared from tumors induced following
intramuscular inoculation of 3-day-old NMRI mice with MSV.
2. An ti-r etr ovir u s Assa ys. CEM/0 and CEM/TK- cells
were suspended at 250 000 cells/mL of culture medium and
infected with HIV-1 or HIV-2 at ∼20 and ∼100 CCID50/mL,
respectively. Then, 100 µL of infected cell suspension was
added to 200-µL microtiter plate wells containing 100 µL of
an appropriate dilution of the test compound. After 4 days of
incubation at 37 °C, cell cultures were examined for giant cell
formation.
C3H/3T3 cells were seeded at 20 000 cells/mL into wells of
tissue culture cluster plates (48 wells/plate). Following a 24-h
incubation period, cell cultures were infected with 80 focus
forming units of MSV during 120 min, whereafter the culture
medium was replaced by 1 mL of fresh medium containing
appropriate concentrations of the test compound. After 6 days,
transformation of the cells was examined microscopically.
The EC50 was defined as the compound concentration
required to inhibit HIV-induced cytopathicity (giant cell
formation) in CEM cell cultures by 50% or as the compound
2′,3′-Dideh ydr o-2′,3′-dideoxyth ym idin e 5′-(3-acetylph en -
yl (m eth oxya la n in yl)(p h osp h a te) (8i): yield 72%; δP 4.05,
4.70; δH 1.42 (3H, m, Ala-Me), 1.88 (3H, d, 5-Me), 2.67 (3H, s,
CH3CO), 3.79 (3H, s, OMe), 4.01 (2H, m, Ala-NH, Ala-CH),
4.40 (2H, m, H-5′), 5.10 (1H, m, H-4′), 5.99 (1H, m, H-3′), 6.40

Substituted-Aryl Phosphoramidate Derivatives of d4T
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concentration required to inhibit CEM cell proliferation by
50%. The MCC was defined as the compound concentration
required to cause a microscopically visible morphological
alteration of the C3H/T3T3 cell cultures.
Ack n ow led gm en t. We thank the AIDS Directed
Programme of the MRC, the Biomedical Research
Programme of the European Commision, and the Bel-
gian Geconcerteerde Onderzoeksacties (Project 95/5) for
financial support. We would also like to thank Mrs. A.
Absillis for excellent technical assistance, Ms. Helen
Murphy for secretarial support, and Stephen Moses for
the synthesis of 8c.
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