Application of phosphoramidate ProTide technology significantly improves antiviral potency of carbocyclic adenosine derivatives

Article abstract of DOI:10.1021/jm060776w

We report the application of phosphoramidate pronucleotide (ProTide) technology to the antiviral agent carbocyclic L-d4A (L-Cd4A). The phenyl methyl alaninyl parent ProTide of L-Cd4A was prepared by Grignard-mediated phosphorochloridate reaction and resulted in a compound with significantly improved anti-HIV (2600-fold) and HBV activity. We describe modifications of the aryl, ester, and amino acid regions of the ProTide and how these changes affect antiviral activity and metabolic stability. Separate and distinct SARs were noted for HIV and HBV. Additionally, ProTides were prepared from the D-nucleoside D-Cd4A and the dideoxy analogues L-CddA and D-CddA. These compounds showed more modest potency improvements over the parent drug. In conclusion, the ProTide approach is highly successful when applied to L-Cd4A with potency improvements in vitro as high as 9000-fold against HIV. With a view to preclinical candidate selection we carried out metabolic stability studies using cynomolgus monkey liver and intestinal S9 fractions.

Full text of DOI:10.1021/jm060776w

J. Med. Chem. 2006, 49, 7215-7226
7215
Application of Phosphoramidate ProTide Technology Significantly Improves Antiviral Potency
of Carbocyclic Adenosine Derivatives
Christopher McGuigan,*,† Alshaimaa Hassan-Abdallah,† Sheila Srinivasan,† Yikang Wang,† Adam Siddiqui,† Susan M. Daluge,‡
Kristjan S. Gudmundsson,‡ Huiqiang Zhou,‡ Ed W. McLean,‡ Jennifer P. Peckham,‡ Thimysta C. Burnette,‡ Harry Marr,‡
Richard Hazen,‡ Lynn D. Condreay,‡ Lance Johnson,‡ and Jan Balzarini§
Welsh School of Pharmacy, Cardiff UniVersity, King Edward VII AVenue, Cardiff CF10 3XF, UK, DiVision of Chemistry MV CEDD; Drug
Metabolism and Pharmacokinetics and Virology Departments, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, and Rega
Institute for Medical Research, Katholieke UniVersiteit LeuVen, B-3000 LeuVen, Belgium
ReceiVed June 30, 2006
We report the application of phosphoramidate pronucleotide (ProTide) technology to the antiviral agent
carbocyclic L-d4A (L-Cd4A). The phenyl methyl alaninyl parent ProTide of L-Cd4A was prepared by
Grignard-mediated phosphorochloridate reaction and resulted in a compound with significantly improved
anti-HIV (2600-fold) and HBV activity. We describe modifications of the aryl, ester, and amino acid regions
of the ProTide and how these changes affect antiviral activity and metabolic stability. Separate and distinct
SARs were noted for HIV and HBV. Additionally, ProTides were prepared from the D-nucleoside D-Cd4A
and the dideoxy analogues L-CddA and D-CddA. These compounds showed more modest potency
improvements over the parent drug. In conclusion, the ProTide approach is highly successful when applied
to L-Cd4A with potency improvements in vitro as high as 9000-fold against HIV. With a view to preclinical
candidate selection we carried out metabolic stability studies using cynomolgus monkey liver and intestinal
S9 fractions.
Introduction
phosphate analogues as potent, nucleoside kinase-independent
antiretrovirals.8,9 Thus, phenyl methyl alanine phosphoramidates
have emerged as general nucleotide delivery forms, known as
aryloxy phosphoramidate ProTides. We have applied this motif
successfully to d4T,10 ddU,11 3TC,12 ddA,13 and d4A.14 In the
case of d4A, a 100-4000-fold boost in vitro antiviral activity
was noted on application of phosphoramidate ProTide technol-
ogy. Other labs have also utilized this methodology, notably
Franchetti and co-workers15 on isoddA and 8-azaisoddA and
Zemlicka et al.16 on alkene and related nucleosides. Applying
our methodology to anti-herpetic BVDU gives unusual results;
we found a decrease in antiviral action,17 while the NewBiotics
group reported promising anticancer action for the same
compounds.18 We have recently reported the enhancement of
the in vitro profile of these agents by modifications in the
phosphoramidate structure.19
A further issue surrounding nucleosides as drugs is the lability
of the glycoside (base-sugar) bond toward phosphorylase-
induced cleavage. This frequently leads to inactivation of
nucleoside drugs. Moreover, as in the case of 5-fluorouracil and
E-5-(2-bromovinyl)arabinofuranosyluracil, for example, coad-
ministration can lead to serious toxic events.20 Efforts to address
this problem have largely led to carbocyclic nucleosides. The
first of these to enter clinical use is the carbocyclic purine
analogue abacavir (ABC) (1, Figure 1).21-23 We have recently
reported the application of phosphoramidate ProTide methods
to 1 and noted a ca. 50-fold boost in anti-HIV potency and
correlated this directly with a similar increase in the intracellular
levels of the bioactive carbovir triphosphate.24 A 10-20-fold
boost was also noted in antihepatitis B activity for ProTides of
1.
Nucleoside analogues continue to dominate antiviral therapy
and also make a significant contribution to the chemotherapy
of cancer, particularly leukemia. Without exception, nucleoside
analogues with such activity require phosphorylation in vivo
to their active nucleotide forms. In the case of antiviral
nucleosides this is almost always the 5′-triphosphate. Poor
phosphorylation can be a major cause of poor activity, with
several examples now known where nucleoside analogues are
inactive, despite the corresponding triphosphates being inhibitors
at their enzyme (polymerase, reverse transcriptase) target.1,2 The
triphosphates themselves cannot be considered to be useful drugs
due to their inherent hydrolytic instability and poor membrane
permeation. However, it appears that in most cases the first
phosphorylation to the 5′-monophosphate is the rate-limiting
step,3 leading to the consideration of the monophosphates as
chemotherapeutic agents. In fact, nucleoside monophosphates
suffer from similar qualitative problems as triphosphates;
instability (in this case to phosphatases and nucleotidases) and
poor membrane permeation. Given these problems, and the
perceived advantage of bypassing the nucleoside kinase depen-
dence of nucleoside analogues, many groups have worked on
phosphate prodrug (“ProTide”) strategies.4-6 Since 1990, we
have developed a phosphoramidate strategy; initial work was
on anti-retroviral AZTa derivatized with alkyl phosphates
carrying an esterified amino acid.7 Alanine quickly emerged as
a most effective amino acid. Subsequently, we discovered aryl
* To whom correspondence should be addressed. Tel: +44 29 20874537.
Fax: +44 29 20874537. E-mail: mcguigan@cardiff.ac.uk.
† Cardiff University.
‡ GlaxoSmithKline.
Given the very high ProTide potentiation noted for adenines
such as d4A,14 we were interested to examine the effect on
carbocyclic adenines and particularly Cd4A. In fact, the
“natural” D-form D-Cd4A (2) is approximately 3-fold less potent
than (1) versus HIV.25 The enantiomer, L-Cd4A (3), has modest
activity versus HBV (ca. 1 µM) but is poorly active versus HIV
§ Katholieke Universiteit Leuven.
a Abbreviations: HIV, human immunodeficiency virus; HBV, hepatitis
B virus; AZT, 3′-azido-3′-deoxythymidine; d4T/d4A, 2′,3′-dideoxy-2′,3′-
didehydrothymidine/adenosine; ddU/ddA, 2′,3′-dideoxyuridine/adenosine;
3TC, L-3′-thia-2′,3′-dideoxycytidine; BVDU, E-5-(2-bromovinyl)-2′-deox-
yuridine; L-Cd4A, (1R,cis)-4-(6-amino-9H-purin-9-yl)-2-cyclopentene-1-
methanol.
10.1021/jm060776w CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/09/2006

7216 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
McGuigan et al.
the effect of ester modification on the antiviral potency of
phosphoramidate ProTides, with a clear preference for benzyl.31-2
Indeed, our recent work on BVDU ProTides versus cancer
indicated a >100-fold improvement of in vitro potency on
replacement of the methyl ester present in NewBiotics’ lead
thymectacin18 by a benzyl ester.19 Similarly, we have reported
extensive SAR studies on the amino acid region, including
natural amino acid variation,33 un-natural R,R-dialkyls,34 ster-
eochemical variation,35 amino acid extensions,36 and replace-
ments.37 In general, alanine and the un-natural amino acid R,R-
dimethylglycine emerged as the amino acids of choice. Indeed,
we recently noted that dimethylglycine was a particularly
efficacious motif with regard to anti-HBV activity when applied
to abacavir (1).24 Thus, using similar methods (Scheme 2) we
prepared the glycine (4g), valine (4h), leucine (4i), isoleucine
(4j), methionine (4k), methyl aspartate (4l), phenylalanine (4m),
proline (4n), lysine (4o), tyrosine-O-tert-butyl ether (4p), and
dimethylglycine (4r) analogues, each as the methyl ester. The
tyrosine compound (4q) was prepared via TFA-mediated
hydrolysis of (4p), it being notable that the phosphoramidate
was stable to these conditions. As noted above, we have
previously found D-alanine to be less effective than L-alanine.35
However, this has not been extended to other amino acids and
not on L-nucleosides. Thus, we prepared a small panel of
D-amino acid analogues: D-alanine (4s), D-phenylalanine (4t),
D-leucine (4u), D-valine (4v), D-tryptophan (4w), D-methyl
aspartate (4x), D-proline (4y), and D-methionine (4z).
As long ago as 1992 we noted the effects on in vitro potency
of aryl substitution in phosphoramidate ProTides.8,9 We identi-
fied p-halogen systems as particularly effective, including the
p-chloro.38,39 Indeed, we subsequently published a rigorous
QSAR analysis of this effect.40 The group of Uckun have very
actively pursued the p-bromo derivative on d4T (“stampi-
dine”).41
Thus, by the above methodologies, and preparing the aryloxy
phosphorochloridate from the appropriate phenol where it was
not commercially available, we prepared methyl alanine ana-
logues with aryl substitution as follows: p-chloro (4aa), p-nitro
(4ab), p-CF3 (4ac), m-CF3 (4ad), 3,4-dichloro (4ae), p-CO2Me
(4af), m-CO2Et (4ag), and o-CO2Et (4ah).
Figure 1. Structures of some antiviral carbocyclic nucleoside ana-
logues.
(ca. 80 µM).26 We wondered to what extent this difference might
reflect the relative efficiency of phosphorylation, which might
be bypassed by ProTide methodologies. This paper describes
our initial attempts in this regard.
Results and Discussion
Chemistry. D-Cd4A (2) and L-Cd4A (3) were prepared as
outlined in Scheme 1 following published procedures. Briefly,
4-amino-2-cyclopentene-1-methanol (D) was prepared from
commercially available azabicyclo[2.2.2]hept-5-en-3-one (A) as
peviously described in the literature.27 Condensation of this
4-amino-2-cyclopentene-1-methanol (D) and 5-amino-4,6-
dichloropyrimidine (E) in butanol at elevated temperature
resulted in the formation of 4-[(5-amino-6-chloro-4-pyrimidi-
nyl)amino]-2-cyclopentene-1-methanol (F).28-29
The carbocyclic chloropurine (G) was formed by treatment
of F with triethylorthoformate in the presence of acid. Finally,
treatment with liquid ammonia in a Parr bomb gave the desired
carbocyclic L-Cd4A (3). Carbobocyclic L-ddA (6) was synthe-
sized by reducing the cyclopentene using 5% Pd/C under 40
psi of hydrogen.
The D-analogues 2 and 7 were prepared in a similar manner
as described for the L-analogues.
We followed the standard phosphorochloridate approach to
the synthesis of ProTides that we developed in the 1990s.9 This
involved the preparation of an aryloxy phosphorodichloridate
by reaction of an appropriate phenol with phosphoryl chloride,
followed by condensation with an esterfied amino acid hydro-
chloride to give the key phosphorochloridate reagent. Reaction
of these phosphorochloridates with nucleosides such as Cd4A
has two challenges. The first is poor solubility, and the second
is regiochemistry. It is important to restrict the phosphorylation
to the 5′-hydroxyl group and eliminate any base (amino)
phosphorylation. This was addressed very successfully by
Uchiyama30 using Grignard reagents of strong bases to generate
the 5′-alkoxide, which gives preferential reaction with electro-
philes. We have noted the efficacy of the Uchiyama method on
abacavir.24 Thus, we employed the same general method here
(Scheme 2).
Finally, for purposes of comparison, the parent phenyl methyl
alanine derivatives were prepared from enantiomeric D-Cd4A-
(2) and the corresponding L-CddA (6) and D-CddA(7) (com-
pounds 5, 8, and 9, respectively).
Antiviral Activity. All of the phosphoramidates described
above (4a-ah), 5, 8, and 9 were tested in vitro against HIV-1,
HIV-2, and HBV, with nucleosides 2, 3, 6, and 7 as controls.
Cytotoxicity was also evaluated in MT4 and CEM cells. All of
the data are presented in Tables 1 and 2 (in µM). Thus, the
parent phenyl methylalaninyl ProTide of L-Cd4A (4a) displayed
a ca. 2700-fold boost in anti-HIV potency, being active at 30
nM, vs 80 µM for the parent. The ProTide was ca. >15 times
more cytotoxic than the parent but still displays a selectivity
index (SI ) CC50/EC50) of >200. As expected, no significant
differences in potency were noted for HIV-2 vs HIV-1 and for
MT4 vs CEM cells. Versus hepatitis-B virus (HBV), where 3
is already quite active (EC50 ca. 1 µM), 4a is ca. 60-times more
potent at 17 nM and shows little toxicity (CC50 1280 µM; SI
ca. 75 000).
In the first instance, L-Cd4A (3) was converted into its phenyl
methylalaninyl phosphoramidate (4a) in 81% yield. As noted
for almost every nucleoside phosphoramidate ProTide, this was
isolated as a roughly 1:1 mixture of phosphate diastereomers,
as evidenced by two closely spaced 31P NMR signals (δP 3.8,
1
4.1). The isomeric mixture was also evident in the H NMR
As the ester was lengthened from methyl to ethyl (4b), there
was no significant change in antiviral potency, while the pattern
was variable for the secondary, ispropyl ester (4c) and tertiary
tert-butyl ester (4d). In general, the tert-butyl ester was less
active; this correlates with our previous conclusions29 and has
(e.g., OMe δH 3.70, 3.72) and the 13C NMR (e.g., CH3-Ala, δC
19.8, 20.0). Similarly prepared were the alanine analogues with
ester modification: ethyl (4b), isopropyl (4c), tert-butyl (4d),
tert-butyl-CH2 (4e), and benzyl (4f). We have previously noted

Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7217
Scheme 1. The Synthetic Route to L-Cd4A (3) and L-CddA (6)
Scheme 2. The Synthetic Route to ProTides of Carbocylic
proximately a log more active in each assay and thus rather
similar to alanine. The methionine (4k) and methyl aspartate
(4l) analogues were rather similar to the leucine compound,
while the phenylalanine analogue 4m was slightly more active,
particularly versus HBV, where it was the most potent amino
acid to date at 4.5 nM. The proline compound 4n was the least
active of the amino acids to date, an observation that we have
made previously of this rather unique amino acid.29 We report
in this paper our first successful ProTide example with lysine
as the amino acid. This was isolated and tested as its TFA salt
(4o) and found to be rather poorly active; in fact, it is rather
similar to parent 3 in several assays and 2-10-fold less active
than the proline analogue 4n. It is interesting to compare the
methionine (4k) and lysine (4o) cases, as they have side chains
with similar geometries. The lysine case is ca. 50-100 fold
less potent versus both HIV and HBV. Partly, this may
correspond with the higher polarity of the lysine compound
(particularly when protonated) and diminished membrane
permeability. The calculated ClogP values of 4k and 4o are
0.9 and 0.46 (Chemdraw Ultra 9.0), but these figures may not
fully reflect the likely protonation of the lysine side chain at
physiological pH, further diminishing its lipophilicity.
Nucleoside Analogues 2, 3, 6, and 7a
a For details of the structures, see Table 1.
been ascribed to the relative stability of tertiary esters to enzyme-
mediated hydrolysis. However, the isopropyl showed a slight
increase in potency versus HBV and variable results versus HIV
dependent on the cell line and assay. The extended system with
a tBuCH2 ester (4e) showed high activity versus both HIV and
HBV, being ca. 6-12-fold more potent than 4a. Finally,
regarding esters, the benzyl analogue 4f emerged as the most
potent ester versus HIV, being active at 9 nM and thus ca. 9000
times more active than 3. It was also rather nontoxic and
displayed an SI of >28 000. It was also highly active versus
HBV, with an EC50 of ca. 7 nM, although some cytotoxicity
was noted in this assay (at 5 µM).
The tyrosine analogue 4q was prepared via its protected tert-
butyl ether (4p), so we evaluated both the free and protected
versions in vitro. In fact, they were both rather similar in
antiviral profile and similar to valine. The boost in anti-HBV
activity seen for the Phe analogue (4m) was not seen for the
Tyr compounds.
Turning now to amino acid variations, leaving the ester as
methyl, we have previously noted a 60-70-fold reduction in
anti-HIV potency for d4T ProTides on alanine to glycine
replacement29 and a 20-40-fold reduction for the corresponding
abacavir ProTides.24 In this study with L-Cd4A we again note
a significant drop in anti-HIV potency on this substitution (4g),
giving, depending on the cell line, a 10-200-fold reduction.
However, HBV activity displays a different trend, showing only
a modest 5-fold drop in potency, thus retaining a log more
potency than the parent 3. Similarly, the valine compound 4h
showed a 10-20-fold reduction in anti-HIV potency as com-
pared to 4a but was equipotent to 4a against HBV, at 20 nM.
The data for the isoleucine analogue 4j parallel that of the valine
compound, as might be expected from their similar structure,
while the leucine analogue (4i), with the amino acid branch
one bond further out from the asymmetric center, is ap-
The dimethylglycine compound (4r) was not available for
evaluation versus the whole panel of assays, but initial data
indicate a slight reduction in potency vs HIV and slight increase
in potency against HBV. Thus, 4r emerged as the most potent
member of this series against HBV with activity at 2.5 nM,
thus being almost 400 times more potent than the parent
nucleoside.
We have previously found D-alanine to be significantly less
effective than natural L-alanine in phosphoramidate ProTides
of d4T.35 We recently noted the same trend for activity of
abacavir ProTides against HIV.24 In the present case the data
are clear and marked; the D-alanine system (4s) is ca. 100-fold
less potent than the L-alanine parent (4a) against HIV, but D
and L are equipotent against HBV. Indeed, comparison of the
D-alanine (4s) and dimethylglycine (4r) systems is instructive.

7218 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
McGuigan et al.
Table 1. Anti-HIV Activity and Cytotoxicity Data for Nucleoside and Nucleotide Analogues
MT4/µM Rega
CEM/µM Rega
HIV-1 HIV-2
MT4/µM GSK
compd
Ar
ester
AA
HIV-1
HIV-2
CC50
CC50
HIV-1
CC50
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Me
Et
iPr
tBu
tBuCH2
Bn
Ala
Ala
Ala
Ala
Ala
Ala
Gly
Val
Leu
Ile
0.045
ND
0.15
8.19
0.043
ND
4.35
1.09
0.19
0.96
0.16
0.32
ND
5.28
15.2
0.11
0.58
ND
0.043
ND
0.22
3.08
0.03
ND
5.73
1.05
0.13
1.13
0.21
0.26
ND
16.4
ND
91.4
84.9
20.2
ND
>250
152
23.2
99.6
34.7
53.5
ND
106
>250
19.8
26.9
ND
0.13
ND
0.3
2.93
0.067
ND
13.5
1
0.42
2.1
0.53
1.1
0.09
ND
0.25
3.67
0.083
ND
20
4.5
0.56
3.5
1
3.5
ND
7.5
150
5.5
18.1
ND
84
130
19.5
ND
g250
167
70
118
85.8
>50
ND
92.1
>250
79.7
110
ND
0.03
0.017
0.425
<0.26
>4
6.4
>25
100
<25
>10
255
.100
<25
>5
>5
>5
>16
16
.10
.25
<40
>32
20
>100
>40
<16
>16
>40
.10
ND
>25
2
0.009
7.3
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
-
0.64
0.64
1.3
1.4
>1
0.1
10
25
,1
0.95
0.14
2.05
2.8
<1
2.3
.16
2.1
ND
2.5
0.013
0.18
ND
0.06
0.077
0.12
-
<0.26
80
15
0.3
>10
1.8
50
0.20
Met
MeAsp
Phe
ND
3.5
25
Pro
21
Lys(TFA)
Tyr(OtBu)
Tyr
27.2
0.13
0.72
ND
3
5.33
ND
ND
9
1.6
7.5
20
5
110
7.67
0.015
0.1
0.06
0.15
0.09
0.13
0.065
0.053
ND
ND
ND
ND
ND
ND
ND
7
Me2Gly
D-Ala
D-Phe
D-Leu
D-Val
D-Trp
D-Asp(OMe)
D-Pro
D-Met
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
ND
ND
8.67
3.5
10
25
ND
ND
ND
71
ND
4t
13.3
1.24
6.93
13.3
3.9
5.93
0.93
14.2
21.9
3.71
105
3.48
0.018
0.17
0.046
0.04
0.076
0.056
0.033
ND
ND
ND
ND
ND
ND
ND
ND
108
88.5
230
93.4
g250
g250
211
3.11
17.4
6.4
16.3
13
12.9
8.39
4.17
ND
4u
4v
4w
4x
4y
4z
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
3
77.1
101
66.9
g250
227
133
3.43
21.6
5.87
22.4
15.6
11.5
4.39
ND
ND
ND
ND
ND
ND
ND
ND
15
117
105
7.87
0.047
0.1
0.06
0.1
0.11
0.13
0.08
0.057
ND
ND
ND
ND
ND
ND
ND
4.07
0.009
0.12
0.035
0.044
0.12
0.14
0.053
ND
ND
ND
ND
ND
p-ClPh
p-NO2Ph
p-CF3Ph
m-CF3Ph
m,p-Cl2Ph
p-CO2MePh
m-CO2EtPh
o-CO2EtPh
-
-
Ph
-
Ph
10
ND
4
>4
>4
-
<4
-
>100
>500
3.7
2
5
6
8
7
9
-
-
ND
ND
ND
ND
ND
ND
Me
-
Ala
-
Ala
-
Ala
>10
>12.5
>125
6.5
Me
-
ND
ND
ND
-
Ph
Me
The D-alanine analogue is ca. 10-fold less potent versus both
HIV and HBV. However, it is also less cytotoxic (>5 to >20
fold), leaving its selectivity index similar or slightly better.
Comparing the potency of the alanine (4a), D-alanine (4s),
and dimethylglycine (4r) systems indicates that substitution on
the “D-face” of the amino acid is beneficial for HBV and
detrimental for HIV. This may reflect cell to cell differences in
processing of the different phosphoramidates. Similar L to D
trends were observed for D-Phe (4t), D-Leu (4u), D-Val (4v),
D-Trp (4w), D-MeOAsp (4x), D-Pro (4y), and D-Met (4z), giving
reductions in anti-HIV potency for D-systems of 5-50-fold and
retention or only slight reduction for HBV. Exceptions were
the Val (4v) and Met (4z) cases, which did show significant
reductions in anti-HBV potency. Thus, versus HIV, in conclu-
sion alanine remained the most effective amino acid, although
dimethylglycine was of similar potency, as were Met and Leu
in some assays, and Val, Ile, and Tyr were also reasonably
effective. Glycine, proline, and lysine were poorly effective, as
were the D-amino acids in most cases. The amino acid could
be varied considerably with little reduction in potency against
HBV, and several amino acids were either equipotent or more
potent than alanine, notably dimethylglycine and D-alanine but
also phenylalanine.
boosted in d4T phosphoramidates39 and this has later been
picked up by the group of Uckun with their development of
stampidine, the p-bromo species.41 Thus, in the present case
we first prepared the p-chloro analogue, which we favor over
the p-bromo for toxicological reasons, given the mole for mole
release of p-halophenol on ProTide activation. Indeed, the
p-chlorophenyl methyl alanine compound (4aa) emerged as the
most potent methyl alanine to date with a ca. 3-10-fold potency
improvement against HIV as compared to 4a, with some cell
to cell variation, and a 5-fold greater potency against HBV than
4a. We have previously noted the poor anti-HIV efficacy of
ProTides with phenyl groups containing strongly electron
withdrawing substitution,40 and to some extent we found the
same to be the case here, with the p-nitro analogue (4ab) being
10-20 fold less active than the p-chloro lead (4aa). However,
it was not significantly less active than the unsubstituted phenyl
parent (4a) versus HIV. Moreover, 4ab was rather potent vs
HBV, being equipotent with 4aa and thus slightly more active
than 4a. This is in contrast to previous experience with d4T
ProTides and HIV.40 The CF3 group is not as electron
withdrawing as nitro and is more lipophilic; we previously noted
it to be more effective in the case of d4T ProTides vs HIV,40
and we noted the same trend here. Thus, 4ac was more active
than 4ab, and in general than 4a also, versus HIV and HBV.
Thus, 4ac is active versus HBV below 3 nM and is the most
active of the methyl alanine ProTides herein reported. However,
Finally, on the SAR of the L-Cd4A ProTides, we probed
several phenyl modifications. In 1995, we had highlighted
p-halogen substitution as a key area where activity could be

Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7219
Table 2. Anti-HBV Activity, Cytotoxicity, and Stability Data for
Nucleoside and Nucleotide Analogues
preparation of the ProTides 8 and 9. Thus, unlike several other
dideoxynucleosides, the carbocyclic analogues 6 and 7 are both
rather poorly active. Application of ProTides did give interesting
boosts in potency in both cases. Indeed, while the L-system (6)
was primarily enhanced (100-fold) versus HBV, the D-com-
pound (7) was only slightly enhanced vs HBV but significantly
so versus HIV (250-fold).
HepG2-2.2.15
GSK
% S9
remaining
compd
HBV
CC50
int
liver
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
0.017
0.02
0.006
0.25
0.003
0.0075
0.086
0.02
0.01
0.06
0.03
0.05
0.0045
0.51
2.0
0.026
0.05
0.0025
0.0265
0.7
0.13
0.6
0.2
0.02
1280
22
>2
170
13
74
39
90
96
57
0
85
91
75
69
66
8
-
-
17
-
0
70
94
-
-
-
-
-
-
-
-
-
-
92
-
48
42
80
-
-
-
-
-
-
-
24
15
8
1
0
0
46
2
0
0
In conclusion, ProTide methods have been shown to signifi-
cantly enhance the antiviral profile of a series of carbocyclic
nucleosides, primarily L-Cd4A but also its D-analogue, and the
dd analogues in both L- and D-series. Very significant differences
were noted for HIV and HBV, with separate leads emerging
for each virus, with quite separate and distinct SARs noted for
each. Effects were noted for variations in the ester, amino acid,
and aryl regions. In general, the HBV system was more tolerant
of structural modifications.
5
>2
120
20
76
16
0
4
>2
9
-
-
95
-
0
16
41
-
-
-
-
-
-
-
>2
.100
>2
>2
10
>200
200
>2
>200
>2
>2
>2
>2
>2
>2
Several nanomolar compounds emerged, representing an
almost 4-log improvement in potency of several ProTides
versus the parent L-Cd4A nucleoside. With this background
we were keen to seek to perform some preclinical analysis
of the most promising compounds that would allow rational
choices regarding further evaluation. In particular, the stability
of the ProTides to metabolic deactivation prior to reaching their
target site was of particular interest, given our experience with
abacavir ProTides.24 Thus, we employed a cynomolgus monkey
liver and intestinal S9 stability assay to gain an initial
understanding of the stability issues in this family and to probe
any stability-structure correlations. Data are reported for
selected compounds in Table 2. Under the conditions of the
assay 4a underwent some decomposition (ca. 25% over 1 h) in
the intestinal fraction, but the majority of ProTide remained.
The position was reversed in the liver fraction, where only 30%
remained. The most striking of the ester variations is the benzyl
(4f), which showed complete disappearance in both assays over
1 h. Thus, although 4f was rather potent in the in vitro antiviral
assays, the S9 data may be predictive of a limited in vivo
exposure. Interestingly, the tyrosine compound (4q) revealed a
similar instability.
4t
4u
4v
4w
4x
4y
4z
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
3
>2
0.22
0.003
0.007
<0.0032
<2
0.72
>2
45
-
7
0.005
0.004
0.004
0.004
0.98
52
4
61
0.60
33
5.0
>2
>2
>2
19
25
-
-
-
-
-
-
-
12
>200
>200
2
5
6
8
7
9
5.4
>200
>2
>200
38
In terms of ester variation; the ethyl ester (4b) was less stable
than the methyl parent (4a) in both media, whereas branching
to isopropyl (4c) and tBu (4d) stabilized it in intestine but
destabilized it in liver. This would suggest that such esters may
be beneficial to consider for delivery to the liver, in hepatitis
or liver cancer.
it is also notably toxic in the HBV assay, being the only ProTide
in the family that is toxic at submicromolar concentrations.
Interestingly, the meta analogue 4ad is rather less cytotoxic but
also apparently slightly less active. Several other aryl substituted
compounds (4ae-4ah) are also noted in Table 2. The meta-
substituted ester is the most active against HIV, while several
compounds are highly active vs HBV. As noted above, the anti-
HBV activity appears less sensitive to aryl substitution than the
anti-HIV activity.
The D-enantiomer of 3, D-Cd4A (2), is slightly more active
than 3 against HIV and rather poorly active vs HBV. It was
interesting to see whether ProTides would have a similar impact
here and we report in Table 1 data on the parent phenyl methyl
alanine parent (5). Thus, a 50-fold boost in anti-HIV potency
is noted. This is much less of an improvement (ca. 2600-fold)
than noted above for the analogous compounds in the L-series
(3 and 4a), and thus the D-ProTide (5) is about a log less potent
than the L-ProTide analogue (4a). Similarly, versus HBV, the
D-ProTide (5) is only 13-fold more potent than the nucleoside,
whereas in the L-family the boost was 60-fold. More dramatic,
the D-ProTide (5) is cytotoxic at its effective concentration, with
a SI barely above unity, while the L-compound (4a) has an anti-
HBV SI of >75 000.
Several amino acid substitutions for alanine gave a similar
profile of increased or maintained intestinal stability but
diminished liver stability, e.g., Val (4h), Leu (4i), Ile (4j), and
dimethylglycine (4r). The only compounds with enhanced liver
stability over the parent 4a were the glycine (4g), Lys (4o) and
D-alanine (4s) amino acid variants and the m-CF3 aryl substitu-
tion (4ad). Interestingly, the lysine stabilization in liver was
uniquely accompanied by a significant labilization in intestine.
In terms of overall maximal stability, a property which might
be anticipated to be useful with regard to systemic drug delivery,
the D-alanine and glycine compounds emerge as the most
notable. Both retained good antiviral potency in the HBV assays
but were only moderate in the HIV assay. The m-CF3 compound
(4ad) also looked rather stable in both S9 assays and showed
good anti-HIV potency. This indicates the potential merit of
extensive aryl modification to tune stability and improve
pharmacokinetic properties; however, the relevance of the S9
stability data to the disposition of these ProTides in monkey
and/or across species has not been demonstrated.
Finally, we briefly pursued the application of the technology
to the dideoxy analogues L-CddA (6) and D-CddA (7) with the

7220 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
McGuigan et al.
Standard Procedure: Preparation of Phosphorodichloridates.
Dry triethylamine (1.0 mol equiv) and the appropriate substituted
phenol (1.0 mol equiv) in dry ether/THF (40-60 mL) were added
dropwise to a stirred solution of phosphorus oxychloride (1.0 mol
equiv) in dry ether/THF (40-60 mL) at -78 °C under nitrogen.
Following the addition, the reaction mixture was allowed to slowly
warm to room temperature and stirred overnight. The mixture was
filtered under nitrogen and the solvent removed under reduced
pressure to give the crude product as an oil.
Standard Procedure: Preparation of Phosphorochloridates.
Dry triethylamine (2.0 mol equiv) in dry dichloromethane (20-40
mL) was added dropwise to a stirred solution of dry dichlo-
romethane (40-60 mL) containing the appropriate phosphorod-
ichloridate (1.0 mol equiv) and the appropriate amino acid salt (1.0
mol equiv), at -78 °C under nitrogen. Following the addition, the
reaction mixture was allowed to slowly warm to room temperature
and stirred overnight. The solvent was removed under reduced
pressure and the crude residue was resuspended in dry diethyl ether
(or THF) and filtered under nitrogen. The solvent was removed
under reduced pressure to leave the crude product as an oil. All
crude phosphorochloridates were used as solutions in dry solvents
in subsequent reactions.
Conclusion
A range of phosphoramidate ProTides of L-Cd4A were
prepared and evaluated against HIV and HBV in vitro. Boosts
in anti-HIV potency as high as 9000-fold were noted, with ca.
250-fold enhancements for anti-HBV activity. Differing SARs
emerged for each virus. The parent ProTide for enantiomeric
D-Cd4A showed only modest potency boosts (10-50 fold) with
parallel increases in cytotoxicity. Similar conclusions were
reached with regard to the dideoxy analogues L-CddA and
D-CddA. In conclusion, the ProTide approach is highly suc-
cessful when applied to L-Cd4A with potency boosts in vitro as
high as 9000-fold vs HIV. With a view to preclinical candidate
selection, we carried out metabolic stability studies using
cynomolgus monkey liver and intestinal S9 fractions; this
revealed interesting differences in stability between the two
enzyme systems, suggestive of a complex metabolic SAR.
Experimental Section
Chemistry. General Procedures. 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. Nucleosides were dried by storage at
elevated temperature over P2O5 in vacuo. 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. All 13C and 31P spectra
were recorded proton-decoupled. All NMR spectra were recorded
in CDCl3 at room temperature (20 ( 3 °C). Chemical shifts for 1H
and 13C spectra are quoted in parts per million downfield from
tetramethylsilane. Coupling constants are referred to as J values.
Signal splitting patterns are described as singlet (s), doublet (d),
triplet (t), quartet (q), or multiplet (m). Chemical shifts for 31P
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 spectroscopy was
fast atom bombardment (FAB) using MNOBA as matrix. Column
chromatography refers to flash column chromatography 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 (containing 0.1% TFA)/acetonitrile (Fisher: HPLC
grade) eluent. Method 1: 0% CH3CN (0 min), 80% CH3CN (35
min), 80% CH3CN (45 min), 0% CH3CN (55 min); flow rate, 1
mL/min; UV detection at 254 nm. Method 2: 0% CH3CN (0 min),
80% CH3CN (15 min), 80% CH3CN (25 min), 0% CH3CN (35
min); flow rate, 1 mL/min; UV detection at 254 nm. Final products
showed purities exceeding 99% with undetectable levels (<0.02)
of parent nucleosides in every case. UV absorptions were deter-
mined using a Kontron Uvikon 860 UV spectrometer.
Standard Procedure: Preparation of Amino Acid Ester Salts.
Thionyl chloride (2.0 mol equiv) was added dropwise to a stirred
solution of the appropriate alcohol (15.0 mol equiv) at 0 °C under
nitrogen. The mixture was stirred at 0 °C for 30 min and then slowly
allowed to warm to room temperature. The appropriate amino acid
(1.0 mol equiv) was added and the mixture was heated at reflux
overnight. The solvent was removed under reduced pressure (last
traces of solvent removed by coevaporation with increasingly more
volatile solvents) to give the crude product as the solid hydrochlo-
ride salt.
Standard Procedure: Preparation of L-Alanine Ester Salts.
A mixture of L-alanine (1.0 mol equiv), the appropriate alcohol
(1.0 mol equiv), and p-toluenesulfonic acid (p-TSA) monohydrate
(1.1 mol equiv) in toluene (or benzene) was heated at reflux (>6
h), using a Dean-Stark apparatus. The solvent was removed under
reduced pressure (last traces of solvent removed by coevaporation
with increasingly more volatile solvents) to give the crude product
as the p-toluenesulfonate salt.
Standard Procedure: Preparation of L-Cd4A ProTides.
L-Cd4A (1.0 mol equiv) was dried by coevaporation with dry
toluene (3 × 5 mL) and then resuspended in dry acetonitrile
(MeCN). A 1.0 M solution of tBuMgCl in dry THF (1.5-2.0 mol
equiv) was added and the reaction mixture was stirred under
nitrogen at room temperature for 15 min. A solution of the
appropriate phosphorochloridate in dry MeCN (3.0 mol equiv) was
added and the reaction mixture was stirred at room temperature.
On completion of the reaction (as determined by TLC), the solvent
was removed under reduced pressure and the crude residue was
purified by column chromatography (see the main text for exact
purification methods).
L-Cd4A 5′-O-[phenyl methyl-L-alaninyl]phosphate (4a) was
prepared according to the standard procedure, from L-C-d4A (0.2
g, 0.9 mmol), tBuMgCl (1.0 M in THF, 1.8 mL, 1.8 mmol), phenyl-
(methyl-L-alaninyl)phosphorochloridate (2.2 mL of 0.324 g/mL
solution, 2.6 mmol), and dry MeCN (15 mL). TLC (8% MeOH in
DCM) showed the reaction to be complete after 1.5 h. The crude
residue was purified by column chromatography, using MeOH/
CHCl3 (5:95) as eluent, to give the product as a clear, colorless
oil, which solidified to an off-white foam after trituration and
coevaporation with diethyl ether (0.333 g, 0.7 mmol, 81%): δP
(CDCl3) 3.85, 4.09; δH (CDCl3) 1.37 (3H, m, CH3-Ala), 1.73 (1H,
m, 6′Ha), 2.89 (1H, m, 6′Hb), 3.22 (1H, m, 1′H), 3.70, 3.72 (3H, s,
OCH3), 4.17 (4H, m, CH-Ala, 5′H and NH-Ala), 5.76 (1H, m, 4′H),
5.97 (1H, m, 3′H), 6.18 (1H, m, 2′H), 6.29 (2H, bs, NH2), 7.27
(5H, m, ArH), 7.88, 7.93 (1H, s, purine-H), 8.39 (1H, s, purine-
H); δC (CDCl3) 19.81-20.02 (CH3-Ala), 33.75, 33.80 (6′C), 44.60-
44.78 (1′C), 49.03, 49.22 (CH-Ala), 51.45 (OCH3), 58.15, 58.25
(4′C), 67.45-67.73 (5′C), 118.74 (purine-5C), 119.00-119.12
(ArC), 123.88 (ArC), 128.61, 128.66 (ArC), 129.49, 129.57 (3′C),
136.24 (purine-8C), 137.68, 137.74 (2′C), 148.67 (ipso-ArC),
149.57, 149.66 (purine-C), 151.82 (purine-C), 154.68 (purine-C),
173.01-173.20 (CdO); MS [ES(+ve)] found 495.1519 (MNa+),
C21H25N6O5NaP requires 495.1522. Anal. (C21H25N6O5P·0.5H2O)
C, H, N.
L-Cd4A 5′-[phenyl ethyl-L-alaninyl]phosphate (4b) was pre-
pared as above and purified on a silica column using 3% methanol/
DCM and isolated as a white solid foam (3.54 g, 49%): δP (DMSO-
d6) 4.23, 3.88; δH (DMSO-d6) δ 8.16 (s, 1H), 8.05 and 8.01 (both
s, total 1H), 7.36 (m, 2H), 7.24 (br s, 2H), 7.19 (m, 3H), 6.13 (m,
1H), 6.02 (m, 2H), 5.63 (m, 1H), 4.03 (m overlapping q, J ) 7.0
Hz, 4H), 3.77 (m, 1H), 3.15 (m, 1H), 2.75 (m, 1H), 1.70 (m, 1H),
1.21 (d, J ) 7.1 Hz, 3H), 1.14 and 1.12 (2t, J ) 7.0, 7.0 Hz, 3H);
MS found 487.1841; C22H28N6O5P (MH)+ requires m/z 487.1859.
Anal. (C22H27N6O5P·0.7H2O) C, H, N.
L-Cd4A 5′-O-[phenyl isopropyl-L-alaninyl]phosphate (4c) was
prepared as above, and the crude residue was purified by column
chromatography, using MeOH/CHCl3 (5:95) as eluent, to give the

Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7221
product as a clear, colorless oil, which solidified to a white foam
after trituration and coevaporation with diethyl ether (0.39 g, 0.8
mmol, 89%): δP (CDCl3) 3.97, 4.20; δH (CDCl3) 1.14 (6H, m,
CH3), 1.31 (3H, m, CH3-Ala), 1.63 (1H, m, 6′Ha), 2.79 (1H, m,
6′Hb), 3.12 (1H, m, 1′H), 4.05 (4H, m, 5′H, CH-Ala and NH-Ala),
4.92 (1H, m, OCH), 5.66 (1H, m, 4′H), 5.87 (1H, m, 3′H), 6.08
(1H, m, 2′H), 6.22 (2H, bs, NH2), 7.06 (2H, m, ArH), 7.19 (3H,
m, ArH), 7.78, 7.82 (1H, s, purine-H), 8.29 (1H, s, purine-H); δC
(CDCl3) 21.45 (CH3-Ala), 22.04, 22.10 (CH3), 35.29 (6′C), 46.06
(1′C), 50.68, 50.83 (CH-Ala), 59.57, 59.67 (4′C), 69.58 (5′C and
OCH), 120.16 (purine-5C), 120.45-120.60 (ArC), 125.29 (ArC),
130.05, 130.09 (ArC), 130.99 (3′C), 137.61 (purine-8C), 139.10
(2′C), 150.11 (ipso-ArC), 151.13 (purine-C), 153.27 (purine-C),
156.14 (purine-C), 173.49-173.65 (CdO); MS [ES(+ve)] found
523.1832 (MNa+), C23H29N6O5NaP requires 523.1835. Anal.
(C23H29N6O5P) C, H, N.
L-Cd4A 5′-O-[phenyl tert-butyl-L-alaninyl]phosphate (4d) was
prepared as above, and the crude residue was purified by column
chromatography, using MeOH/CHCl3 (5:95) as eluent, to give the
product as a clear, colorless oil, which solidified to a white foam
after trituration and coevaporation with diethyl ether (0.262 g, 0.5
mmol, 59%): δP (CDCl3) 4.10, 4.27; δH (CDCl3) 1.24 (3H, m,
CH3-Ala), 1.35 (9H, s, CH3), 1.54 (1H, m, 6′Ha), 2.79 (1H, m, 6′Hb),
3.13 (1H, m, 1′H), 3.80 (1H, m, NH-Ala), 4.09 (3H, m, CH-Ala
and 5′H), 5.66 (1H, m, 4′H), 5.87 (1H, m, 3′H), 6.08 (1H, m, 2′H),
6.22 (2H, bs, NH2), 7.15 (5H, m, ArH), 7.78, 7.82 (1H, s, purine-
H), 8.29 (1H, s, purine-H); δC (CDCl3) 21.54 (CH3-Ala), 28.33
(CH3), 35.34 (6′C), 46.10 (1′C), 51.09, 51.21 (CH-Ala), 59.59, 59.67
(4′C), 69.01 (5′C), 82.33, 82.37 [C(CH3)3], 120.46 (purine-5C),
120.53-120.61 (ArC), 125.26 (ArC), 130.04, 130.07 (ArC), 130.96,
131.01 (3′C), 137.65 (purine-8C), 139.10 (2′C), 149.99 (ipso-ArC),
151.15 (purine-C), 153.24 (purine-C), 156.12 (purine-C), 173.13
(CdO); MS [ES(+ve)] found 537.2001 (MNa+), C24H31N6O5NaP
requires 537.1991. Anal. (C24H31N6O5P) C, H, N.
L-Cd4A 5′-O-[Phenyl 2,2-dimethyl-1-propyl-L-alaninyl]phos-
phate (4e). The crude residue was purified by column chromatog-
raphy, using MeOH/CHCl3 (5:95) as eluent, to give the product as
a clear, colorless oil, which solidified to a white foam after
trituration and coevaporation with diethyl ether (0.30 g, 0.6 mmol,
66%): δP (CDCl3) 3.89, 4.18; δH (CDCl3) 0.94 (9H, d, CH3), 1.41
(3H, m, CH3-Ala), 1.73 (1H, m, 6′Ha), 2.89 (1H, m, 6′Hb), 3.23
(1H, m, 1′H), 3.83 (2H, m, CH-Ala and NH-Ala), 4.14 (4H, m,
5′H and OCH2), 5.77 (1H, m, 4′H), 5.98 (1H, m, 3′H), 6.20 (3H,
m, 2′H and NH2), 7.18 (2H, m, ArH), 7.32 (3H, m, ArH), 7.88,
7.94 (1H, s, purine-H), 8.38 (1H, s, purine-H); δC (CDCl3) 21.59-
21.85 (CH3-Ala), 26.71, 26.74 (CH3), 31.83 [C(CH3)3], 35.26 (6′C),
46.07-46.24 (1′C), 50.63, 50.78 (CH-Ala), 59.60, 59.71 (4′C),
69.03-69.19 (5′C), 75.11, 75.16 (OCH2), 120.08 (purine-5C),
120.47-120.62 (ArC), 125.25 (ArC), 130.07, 130.12 (ArC), 130.97
(3′C), 137.69, 137.73 (purine-8C), 139.13, 139.23 (2′C), 150.10
(ipso-ArC), 151.00, 151.19 (purine-C), 153.19 (purine-C), 156.03
(purine-C), 174.02-174.25 (CdO); MS [ES(+ve)] found 551.2145
(MNa+), C25H33N6O5NaP requires 551.2148. Anal. (C25H33N6O5P)
C, H, N.
L-Cd4A 5′-[Phenyl benzyl-L-alaninyl]phosphate (4f). The
crude product was purified by silica column eluted with 5%
methanol/chloroform, as a white solid foam (270 mg, 57%); δP
(DMSO-d6) 4.00, 3.55; δH (DMSO-d6) δ 8.13 (s, 1H), 8.01 and
7.985 (both s, total 1H), 7.2-7.4 (m, 12H), 6.02 (m, 3H), 5.59 (m,
1H), 5.06 (m, 2H), 4.03 (m, 2H), 3.83 (m, 1H), 3.05 (m, 1H), 2.65
(m, 1H), 1.62 (m, 1H), 1.22 (d, J ) 7.0 Hz, 3H). Anal.
(C27H29N6O5P·0.5H2O) C, H, N.
139.87 (C8), 149.71 (C4), 150.99, 151.08 (ipso-Ph), 152.06 (C2),
155.34 (C6), 171.60, 171.69 (CO-Ala). Anal. (C20H23N6O5P·
1.5H2O) C, H, N.
L-Cd4A [phenyl methyl valinyl]phosphate (4h) was purified
as the compound above using 5% methanol/DCM and isolated as
a brittle white foam (86%): δP 4.64, 4.84; δH 0.83 (6H, m, CH-
(CH3)2), 1.65 (1H, m, H6′a), 1.95 (1H, m, CH(CH3)2), 2.82 (1H,
m, H6′b), 3.15 (1H, m, H4′), 3.60, 3.65 (3H, s, OCH3), 3.78 (1H,
m, CHCOO), 4.11 (2H, m, H5′), 5.68 (1H, m, H1′), 5.89 (1H, m,
H3′), 6.12 (1H, m, H2′), 7.10 (2H, m, Ar), 7.22 (3H, m, Ar), 7.84,
7.92 (1H, s, H8), 8.29 (1H, s, H2); δC (CDCl3) 17.91 (CH3CH),
19.36 (CH3CH), 32.48, 32.57 (CH(CH3)2), 35.14, 35.25 (C6′), 46.09
(C4′), 52.53, 52.57 (OCH3), 59.86 (C1′), 60.34, 60.51 (NHCHCO),
69.17 (C5′), 120.36 (C5), 120.53, 120.78 (o-Ph), 125.25, 125.29
(p-Ph), 129.43 (C3′), 130.02 (m-Ph), 137.93 (C2′), 141.02 (C8),
150.85 (C2), 151.12 (ipso-Ph), 150.01 (C4), 173.77 (CO-Ala); MS
(E/I found) 523.1855 C23H29N6O5P requires 523.1835. Anal.
(C23H29N6O5P·H2O) C, H, N.
L-Cd4A 5′-O-[phenyl methyl-L-leucinyl)]phosphate (4i) was
prepared as above and purified by column chromatography, using
MeOH/CHCl3 (5:95) as eluent, to give the product as a clear,
colorless oil, which solidified to a white foam after trituration and
coevaporation with diethyl ether (0.35 g, 0.7 mmol, 79%): δP
(CDCl3) 4.14, 4.37; δH (CDCl3) 0.78 (6H, m, CH3), 1.39 (2H, m,
CH2-Leu), 1.58 (2H, m, CH and 6′Ha), 2.77 (1H, m, 6′Hb), 3.10
(1H, m, 4′H), 3.56, 3.59 (3H, s, OCH3), 4.02 (4H, m, NH-Leu,
CH-Leu and 5′H), 5.65 (1H, m, 1′H), 5.86 (1H, m, 3′H), 6.00 (1H,
m, 2′H), 6.26 (2H, bs, NH2), 7.06 (2H, m, ArH), 7.21 (3H, m, ArH),
7.77, 7.84 (1H, s, purine-H), 8.28 (1H, s, purine-H); δC (CDCl3)
20.62, 20.75 (CH3), 21.64, 21.70 (CH3), 23.27, 23.37 (CH), 33.73,
33.85 (6′C), 42.58, 42.68 (CH2-Leu), 44.61, 44.81 (1′C), 51.21,
51.24 (CH-Leu, 51.93, 52.17 (OCH3), 58.13, 58.23 (4′C), 67.49,
67.79 (5′C), 118.74-119.10 (purine-5C and ArC), 123.81 (ArC),
128.56, 128.62 (ArC), 129.43, 129.50 (3′C), 136.27 (purine-8C),
137.74, 137.86 (2′C), 148.66 (ipso-ArC), 149.65-149.80 (purine-
C), 151.75 (purine-C), 154.60 (purine-C), 173.27-173.46 (CdO);
MS [ES(+ve)] found 537.2000 (MNa+), C24H31N6O5NaP requires
537.1991. Anal. (C24H31N6O5P) C, H, N.
L-Cd4A 5′-[phenyl methyl isoleucinyl]phosphate (4j) was
prepared as above and purified by column chromatography using
MeOH/CHCl3 (5:95) as eluent (45%): δP 4.55, 4.72; δH 0.92 (6H,
m, CH3CH2, CH3CH), 1.16 (1H, m, CH3CHaHb), 1.42 (1H, m, CH3-
CHaHb), 1.77 (2H, m, CH3CH, 6′-HaHb), 2.99 (1H, m, 6′-HaHb),
3.22 (1H, m, 4′-H), 3.62, 3.66 (3H, s, OCH3), 3.84 (2H, m, NH
(Ile), 5′-HaHb), 4.22 (2H, m, NCH (Ile), 5′-HaHb), 5.74 (1H, m,
1′-H), 5.99 (3H, m, 3′-H, NH2), 6.17 (1H, m, 2′-H), 7.11 (2H, m-Ar),
7.29 (3H, m, p-Ar, o-Ar), 7.86, 7.92 (1H, s, 2-H), 8.36 (1H, 2s,
8-H); δC 11.86 (CH3CH2), 15.70, 15.75 (CH3-CH), 25.06 (CH3-
CH2), 35.23, 35.34 (CHCH2), 36.97, 37.05 (6′-C), 46.16 (4′-C),
52.71 (OCH3), 54.58, 54.80 (1′-C), 59.59, 59.69 (CH-Gly), 68.93
(5′-C), 120.30 (5-C), 120.38, 120.45, 120.49, 120.55 (o-Ar), 125.31
(p-Ar), 130.89, 130.98 (m-Ar), 130.89, 130.98 (3′-C), 137.67 (8-
C), 139.20, 139.28 (2′-C), 150.20 (ipso-Ar), 151.10 (4-C), 153.29
(2-C), 155.98 (6-C), 173.67 (CdO); ES+ m/e found 537.2004
([MNa]+, C24H31N6O5NaP requires 537.1991). Anal. (C24H31N6O5P)
C, H, N.
L-Cd4A 5′-[phenyl methyl methioninyl]phosphate (4k) was
prepared as above and purified using MeOH/CHCl3 5:95 on a silica
column and isolated as a brittle white foam (96%); δP 3.79, 4.19;
δH 1.70 (1H, m, H6′a), 1.85 (2H, m, CH2CHCO), 1.97 (3H, s,
SCH3), 2.39 (2H, t, CH2SCH3), 2.81 (1H, m, H6′b), 3.15 (1H, m,
H4′), 3.64, 3.67 (3H, s, OCH3), 3.91 (1H, m, NHCHCO), 4.13 (2H,
m, H5′), 5.68 (1H, m, H1′), 5.89 (1H, m, H3′), 6.12 (1H, m, H2′),
7.12 (2H, m, Ar), 7.28 (3H, m, Ar), 7.90, 8.00 (1H, s, H8), 8.29
(1H, s, H2); δC (CDCl3) 15.74 (SCH3), 29.97, 30.09 (CH2CH),
33.93 (C6′), 35.12 (CH2SCH3), 46.18 (C4′), 53.00 (OCH3), 53.79,
54.00 (NHCHCO), 59.98 (C1′), 69.40 (C5′), 120.35 (C5), 120.50,
120.56 (o-Ph), 125.38, 125.46 (p-Ph), 129.52 (C3′), 130.12, 130.15
(m-Ph), 137.96 (C2′), 139.87 (C8), 151.07 (C4), 172.82 (CO); MS
(E/I) found 555.1567 (MNa+), C23H29N6O5PSNa requires 555.1554.
Anal. (C23H29N6O5PS·2H2O) C, H, N.
L-Cd4A [phenyl methyl glycinyl]phosphate (4g) was purified
as the compound above using 5% methanol/DCM and isolated as
a brittle white foam (38%): δP 4.51; δH 1.72 (1H, m, H6′a), 2.82
(1H, m, H6′b), 3.20 (1H, m, H4′), 3.70 (3H, s, OCH3), 4.00 (2H,
m, H5′), 4.20 (2H, m, CH2CO) 5.68 (1H, m), 5.90 (1H, m), 6.15
(1H, m), 7.20 (5H, m), 7.99 (1H, s), 8.31 (1H, s); δC (CDCl3) 34.97
(C6′), 43.22, 43.29 (CH2CO), 46.04, 46.14 (C4′), 52.79 (OCH3),
59.72, 59.81 (C1′), 69.10 (C5′), 119.74 (C5), 120.46, 120.52 (o-
Ph), 125.35 (p-Ph), 129.51 (C3′), 130.10 (m-Ph), 137.79 (C2′),

7222 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
McGuigan et al.
L-Cd4A 5′-[phenyl dimethyl aspartinyl]phosphate (4l) was
prepared as above and isolated after silica column chromatography
using MeOH/CHCl3 8:92, as a brittle white foam (34%): δP 3.78,
4.38; δH 1.74 (1H, m, H6′a), 2.85 (1H, m, H6′b), 3.22 (1H, m,
H4′), 3.50 (2H, s, CHCH2CO), 3.65 (3H, s, OCH3), 3.75 (3H, s,
OCH3), 4.13 (1H, m, NHCHCO), 4.26 (2H, m, H5′), 5.71 (1H, m,
H1′), 5.92 (1H, m, H3′), 6.23 (1H, m, H2′), 7.16 (5H, m, Ar), 8.00
(1H, m, H8), 8.13, 8.21 (1H, s, H2); δC (CDCl3) 34.98 (C6′), 46.18
(C4′), 51.43, 51.66 (CHCO), 52.05 (OCH3), 53.26 (CH3O), 59.70
(C1′), 68.41 (CH2CO), 69.21 (C5′); MS (E/I) found 553.1597
(MNa+), C23H27N6O7PNa requires 553.1577. Anal. (C23H27N6O7P‚
H2O) C, H, N.
L-Cd4A 5′-[phenyl methyl-L-phenylalaninyl]phosphate (4m)
was prepared as above and purified on a silica column eluted with
5% methanol/chloroform. Evaporation of a methanol solution left
the product as a white solid foam (500 mg, 42%): δP (DMSO-d6)
3.61, 3.25; δH (DMSO-d6) δ 8.14 and 8.13 (both s, total 1H), 8.01
and 7.985 (s, total 1H), 7.2-7.4 (m, 5H), 6.95-7.05 (m, 5H), 7.00
and 6.98 (both br s, total 2H), 6.1 (m, 1H), 5.975 (m, 2H), 5.58
(m, 1H), 3.55-4.0 (m, 3H), 3.57 (s, 3H), 2.95 (m, 2H), 2.7 (m,
2H), 1.55 (m, 1H). Anal. (C27H29N6O5P·0.4H2O) C, H, N.
L-Cd4A 5′-[phenyl methyl prolinyl]phosphate (4n) was pre-
pared as above and purified on a silica column with elution using
MeOH/CHCl3 5:95 (19% yield): δP 2.89, 3.04; δH 1.68-2.19 (5H,
br m, NCH2CH2, NCH2CH2CH2, 6′-HaHb), 2.94 (1H, m, 6′-HaHb),
3.32 (2H, m, 4′-H, NCHaCHb), 3.42 (1H, m, NCHaCHb), 3.61, 3.74
(3H, s, OCH3), 4.12, 4.30 (m, 5′-H, prolinyl CH), 5.70 (2H, br s,
NH2) 5.81 (1H, m, 1′-H), 5.98 (1H, m, 3′-H), 6.19, 6.25 (1H, m,
2′-H), 7.21 (2H, m, m-Ar), 7.35 (3H, m, p-Ar, o-Ar), 7.88, 8.02
(1H, s, 2-H), 8.43 (1H, s, 8-H); δC 25.30, 25.58, 25.70 (NCH2-
CH2), 31.41, 31.86, 31.97 (NCH2CH2CH2), 35.11, 35.19 (6′-C),
46.10, 46.18, 46.28 (4′-C), 47.21, 48.54 (NCH2), 52.51, 52.67
(OCH3), 59.61 (1′-C), 60.13, 61.06, 61.12 (CH-Pro), 68.81, 68.88
(5′-C), 120.29, 120.36, 120.49, 120.55 (5-C, o-Ar), 125.14, 125.21
(p-Ar), 130.7 (m-Ar), 130.82, 130.94 (3′-C), 137.83, 137.90 (8-C),
139.20, 139.28 (2′-C), 151.13, 151.23 (ipso-Ar, 4-C), 153.16 (2-
C), 155.91 (6-C), 174.14, 174.70 (CdO); ES+ m/e found 521.1682
(MNa+, C23H27N6O5PNa requires 521.1678. Anal. (C23H27N6O5P·
H2O) C, H, N.
L-Cd4A 5′-[phenyl methyl lysinyl]phosphate, TFA salt (4o)
was prepared as above and purified on a silica column with elution
using MeOH/CHCl3 5:95 to give a brittle white foam (40%): δP
4.83, 5.45; δH 1.33 (2H, m, CH2-Lys), 1.61 (4H, m, (CH2)2-Lys),
1.75 (1H, m, H6′a), 2.84 (3H, m, CH2 (Lys), H6′b), 3.24 (1H, m,
H4′), 3.65 (3H, d, CH3O), 3.87 (1H, m, CHNH-Lys), 4.24 (2H, m,
H5′), 5.72 (1H, m, H1′), 6.03 (1H, m, H3′), 6.22 (1H, m, H2′),
7.21 (3H, m, Ar), 7.35 (2H, m, Ar), 8.12 (1H, d, H8), 8.28 (1H, s,
H2); δC (CDCl3) 23.86, 24.01 (CH2-Lys), 28.24, 28.38 (CH2-Lys),
34.58, 34.62, 34.71 (CH2NH2-Lys), 35.81, 35.87 (C6′), 40.86 (CH2-
CHN), 47.45, 47.49, 47.56, 47.60 (C4′), 53.22 (CH-Lys), 55.95,
56.10 (OCH3), 61.73, 61.76 (C1′), 70.28, 70.35, 70.40, 70.48 (C5′),
120.58, 120.62 (C5), 120.69, 121.56, 121.63, 121.92, 121.98 (o-
Ph), 126.48, 126.65 (m-Ph), 130.85, 131.21 (p-Ph), 131.51, 131.57
(C3′), 141.92 (C2′), 150.64 (C4), 151.94, 152.06 (ipso-Ph), 152.51,
152.58 (C2), 156.39, 156.47 (C4), 175.15, 175.19, 175.42, 175.47
(CO); MS (E/I) found 530.2275 (MH+), C24H33N7O5P requires
530.2281. Anal. (C28H34N7O9PF6·0.5H2O) C, H, N.
L-C-d4A 5′-O-[phenyl methyl-L-(O-tert-butyl)tyrosinyl]phos-
phate (4p) was prepared as above and purified on a silica column
with elution using MeOH/CHCl3 5:95 to give the product as a clear,
colorless oil, which solidified to a white foam after trituration and
coevaporation with diethyl ether (0.32 g, 60%). δP (CDCl3) 3.98,
4.04; δH (CDCl3) 1.32 (9H, s, CH3), 1.64 (1H, m, 6′Ha), 2.83 (1H,
m, 6′Hb), 2.95 (2H, m, CH2-Tyr), 3.08 (1H, m, 1′H), 3.61, 3.66
(3H, s, OCH3), 4.07 (4H, m, NH (Tyr), CH (Tyr) and 5′H), 5.73
(1H, m, 4′H), 5.94 (1H, m, 3′H), 6.11 (1H, m, 2′H), 6.39 (2H, bs,
NH2), 6.88 (2H, d, ArH-Tyr), 6.98 (2H, d, ArH-Tyr), 7.16 (2H, m,
ArH), 7.29 (3H, m ArH), 7.84, 7.88 (1H, s, purine-H), 8.37, 8.38
(1H, s, purine-H); δC (CDCl3) 29.23 (CH3), 35.25 (6′C), 40.21 (CH2-
Tyr), 46.05, 46.15 (1′C), 52.63, 52.69 (OCH3), 55.84, 56.27 (CH-
Tyr), 59.64 (4′C), 67.49, 67.79 (5′C), 78.89 [C(CH3)3], 120.25
(purine-5C), 120.45, 120.51 (ArC), 124.57 (ArC), 125.38 (ArC),
130.09-130.38 (ArC), 130.56, 130.94 (3′C), 137.66, 137.72
(purine-8C), 139.32 (2′C), 150.20 (ipso-ArC), 151.50 (purine-C),
153.28 (purine-C), 154.96 (ipso-ArC), 155.87 (purine-C), 173.22
(CdO); MS [ES(+ve)] found 643.2405 (MNa+), C31H37N6O6NaP
requires 643.2410. Anal. (C31H37N6O6P) C, H, N.
L-Cd4A 5′-O-[Phenyl(methyl-L-tyrosinyl)]phosphate (4q).
L-Cd4A 5′-O-[phenyl methyl-L-(O-tert-butyl)tyrosinyl)]phosphate
(4p) (0.554 g, 0.9 mmol) was dissolved in anhydrous DCM (3 mL).
Trifluoroacetic acid (TFA, 1 mL) was added and the solution was
stirred for 30 min, after which time TLC (10% MeOH in DCM)
showed no starting material left. The solvent was removed under
reduced pressure and the crude residue was purified three times by
column chromatography using MeOH/DCM (8:92) as eluent. The
product was obtained as a clear, colorless oil which solidified to a
white foam after trituration and coevaporation with diethyl ether
(0.358 g, 0.6 mmol, 71%): δP (MeOH-d4) 4.62, 4.92; δH (MeOH-
d4) 1.60 (1H, m, 6′Ha), 2.83 (4H, m, 6′Hb, CH2 and 1′H), 3.57,
3.61 (3H, s, OCH3), 3.84 (3H, m, 5′H and CH-Tyr), 5.65 (1H, m,
4′H), 5.96 (1H, m, 3′H), 6.08 (1H, m, 2′H), 6.67 (2H, m, ArH
(Tyr), 7.05 (2H, m, ArH), 7.25 (2H, m, ArH-Tyr), 7.99, 8.03 (1H,
s, purine-H), 8.21, 8.22 (1H, s, purine-H); δC (MeOH-d4) 35.86
(6′C), 40.42, 40.52 (CH2-Tyr), 47.34, 47.45 (1′C), 53.01, 53.04
(OCH3), 58.32, 58.62 (4′C), 61.41, 61.46 (CH-Tyr), 69.99 (5′C),
116.71 (purine-5C), 120.68 (ipso-ArC), 121.52-121.79 (ArC),
126.32 (ArC), 129.26, 129.34 (ArC), 131.06 (ArC), 131.53 (ipso-
ArC), 132.00, 132.03 (3′C), 139.05 (purine-8C), 141.02 (2′C),
150.68 (purine-C), 152.50 (ipso-ArC), 154.01 (purine-C), 157.70,
157.90 (purine-C), 175.10 (CdO); MS [ES(+ve)] found 587.1790
(MNa+), C27H29N6O6NaP requires 587.1784. Anal. (C27H29N6O6P·
0.5H2O) C, H, N.
L-Cd4A 5′-[phenyl methyl dimethylglycinyl]phosphate (4r)
was prepared using the standard methods above and purified on a
silica column with elution using MeOH/CHCl3 5:95. Evaporation
of a methanol solution left as a white solid foam (200 mg, 48%):
δP (DMSO-d6) δ 2.43, 2.39; δH (DMSO-d6) δ 8.15 (s, 1H), 8.03
and 8.01 (both s, total 1H), 7.1-7.4 (m, 7H), 6.10 (m, 1H), 6.00
(m, 1H), 5.89 (m, 1H), 5.625 (m, 1H), 4.08 (m, 2H), 3.55 (s, 3H),
3.12 (m, 1H), 2.74 (m, 1H), 1.70 (m, 1H), 1.35 (m, 6H). Anal.
(C22H27N6O5P·0.5H2O) C, H, N.
L-Cd4A 5′-[phenyl methyl D-alaninyl]phosphate (4s) was
prepared as above and purified on a silica column with elution using
MeOH/CHCl3 5:95. Evaporation of a methanol solution left the
title compound as a white solid foam (290 mg, 71%): δP (DMSO-
d6) 3.96, 3.63; δH (DMSO-d6) δ 8.13 (s, 1H), 8.02 and 8.01 (both
s, total 1H), 7.36 (m, 1H), 7.34 (m, 1H), 7.19 (m, 5H), 6.13 (m,
1H), 6.02 (m, 2H), 5.63 (m, 1H), 4.1 (m, 2H), 3.8 (m, 1H), 3.57
(s, 3H), 3.12 (m, 1H), 2.72 (m, 1H), 1.68 (m, 1H), 1.20 (m, 3H).
Anal. (C21H25N6O5P·0.5H2O) C, H, N.
L-Cd4A 5′-[phenyl methyl D-phenylalaninyl]phosphate (4t)
was prepared using the standard methods above and purified on a
silica column with elution using MeOH/CHCl3 5:95 to give the
product as a clear, colorless oil, which solidified to a white foam
on trituration and coevaporation with diethyl ether (0.20 g, 24%):
δP (CDCl3) 3.92, 4.04; δH (CDCl3) 1.7 (H6′, 1H, m), 2.8 (H6′, 1H,
m), 3.1 (H4′, 1H, m), 3.2 (CH2, 1H, m), 3.7 (CH3O, 3H, d, J )
12.04 Hz), 4.1 (H5′, 2H, m), 4.6 (CHAA, 1H, q), 5.7 (H1′, 1H,
m), 5.9 (H3′, 1H, m), 6.1 (H2′, 1H, m), 6.5 (NH2, 2H, bs), 7.2 (Ar,
10H, m), 7.85 (H8, 1H, d, J ) 13 Hz), 8.4 (H2, 1H, s); δC (CDCl3)
28.66 (CH2Ar), 33.7 (C6′), 39.45 (CH3AA), 44.65 (C4′), 51.25
(CH3O), 55.0 (CH-NH), 58.2 (C1′), 67.5 (C5′), 118.11 (C5), 118.98
(Ar), 123.8 (Ar), 126.3 (Ar), 127.5 (Ar), 128.4 (m), 128.6 (C3′),
129.5 (Ar), 134.6 (C2′), 134.9 (Ar), 137.5 (C8), 148.5 (C4), 149.5
(Ar), 151.7 (C2), 154.7 (C6), 171.8 (CO). Anal. (C27H29N6O5P‚
H2O) C, H, N.
L-Cd4A 5′-[phenyl methyl D-leucinyl] phosphate (4u) was
prepared using the standard methods above and purified on a silica
column with elution using MeOH/CHCl3 5:95 to give the product
as a clear, colorless oil, which solidified to a white foam on
trituration and coevaporation with diethyl ether (0.10 g, 0.25 mmol,
28%): δp (CDCl3) 4.27, 4.36; δH (CDCl3) 0.8 ((CH3)2CH, 6H, m),

Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7223
1.5 (CH2iPr, 2H, m), 1.7 (CH(CH3)2, H6′, 2H, m),2.75 (H6′, 1H,
m), 3.1 (H4′, 1H, m),3.6 (CH3, 3H, d, J ) 12.8 Hz), 4.05 (H5′,
2H, m), 4.55 (CHAA, 1H, t), 5.65 (H1′ ,1H, m), 5.9 (H3′, 1H, m),
6.05 (H2′, 1H, m), 6.55 (NH2, 2H, bs), 7.2 (Ar, 5H, m), 7.8 (H8,
1H, s), 8.23 (H2, 1H, s); δC (CDCl3) 22.05 (CH(CH3)2), 23.1
((CH3)2C), 24.7 (CH2iPr), 35.19 (C6′), 43.9 (C4′), 46.01 (CH3O),
53.59 (CHNH), 59.61 (C1′), 59.61 (C5′), 120,046 (C5), 120.4 (Ar),
125.213 (Ar), 129.9 (C3′), 130.95 (Ar), 137.57 (C2′), 138.94 (C8),
149.9 (C4), 151.0 (Ar), 153.19 (C2), 156.24 (C6), 174 (CO); MS
[(ES(+ve)]) found 537.1991 (MNa+), C24H31N6O5PNa requires
537.2001. Anal. (C24H31N6O5P·2.5H2O) C, H, N.
L-Cd4A 5′-[phenyl methyl D-valinyl]phosphate (4v) was
prepared using the standard methods above and purified on a silica
column with elution using MeOH/CHCl3 5:95 to give the product
as a clear, colorless oil, which solidified to a white foam on
trituration and coevaporation with diethyl ether (0.105 g, 0.209
mmol, 26%): δP (CDCl3) 4.76, 4.85; δH (CDCl3) 0.9 ((CH3)2CH,
6H, m), 1.8 (H6′, 1H, m), 2.1 (CH(CH3)2, 1H, m), 3.25 (H4′, 1H,
m), 3.7 (CH3O, 3H, d, J ) 12.6 Hz), 3.9 (1H, CHAA, m), 4.2
(H5′, 2H, m), 5.8 (H1′, 1H, m), 6 (H3′, 1H, m), 6.2 (H2′, 1H, m),
6.3 (NH2, 2H, bs), 7.25 (Ar, 5H, m), 7.9 (H8, 1H, d, J ) 12.00
Hz), 8.4 (H2, 1H, s); δC (CDCl3) 7.8((CH3)2C), 9.3 ((CH3)2C), C6′
(32.4), C4′ (35.2), 46.2 (CH3O), 52.5 (C1′), 60.4 (CH-NH), 69.2
(C5′), 120.3 (C5), 120.5 (Ar), 125.2 (Ar), 130.03 (C3′), 130.9 (Ar),
137.6 (C2′), 139.1(C8), 150.1 (C4), 151.0 (ipsoPh), 153.2 (C2),
156.0 (C6), 173.69 (CO); MS [(ES(+ve)]) found 523.1835 (MNa+),
C23H29N6O5PNa requires 523.1821. Anal. (C23H29N6O5P‚H2O) C,
H, N.
L-Cd4A 5′-[phenyl methyl-D-tryptophan] phosphate (4w) was
prepared using the standard methods above and purified on a silica
column with elution using MeOH/CHCl3 5:95 to give the product
as a clear, colorless oil, which solidified to a white foam on
trituration and coevaporation with diethyl ether (0.054 g, 0.209
mmol, 23%): δP (CDCl3) 4.13, 4.23; δH (CDCl3) 1.5 (H6′, 1H,
m), 2.65 (H6′, 1H, m), 3.2 (CH2, 1H, m), 3.62 (CH3O, 3H, d, J )
12.0 Hz), 3.95 (H5′, 2H, m), 4.35 (CHAA, 1H, m), 5.67 (H1′, 1H,
m), 5.9 (H3′, 1H, m), 6.0 (H2′, 1H, m), 6.4 (NH2, 2H, bs), 7.2 (Ar,
9H, m), 7.5 (H (Trp), 1H, s), 7.73 (H8, 1H, s), 8.35 (H2, 1H, s),
9.05 (NH (Trp), 1H, s); δC (CDCl3) 30.5 (CH2-Trp), 34.9 (C6′),
45.9 (C4′), 52.7 (CH3), 55.6 (CHAA), 59.6 (C1′), 68.85 (C5′), 109.7
(C′3), 111.79 (C′6), 118.8 (C′9), 119.8 (C5), 120.5 (Ar), 122.3 (C′8),
123.8 (C′7), 125 (C′2), 125.3 (m), 127.7 (C3′), 130.0 (C′4), 151.1
(Ar), 156.13 (C2), 160.6 (C6), 173.79 (CO); MS [(ES(+ve)]) found
610.1944 (MNa+), C29H30N7O5PNa requires 610.1957. Anal.
(C29H30N7O5P‚2H2O) C, H, N.
L-Cd4A 5′-[phenyl methyl D-aspartyl methyl ester]phosphate
(4x) was prepared using the standard methods above and purified
on a silica column with elution using MeOH/CHCl3 5:95 to give
the product as a clear, colorless oil, which solidified to a white
foam on trituration and coevaporation with diethyl ether (0.12 g,
0.21 mmol, 28%): δP (CDCl3) 3.79, 4.25; δH (CDCl3) 1.7 (H6′,
1H, m), 2.8 (CH2OMe, H6′, 3H, m), 3.1 (H4′, 1H, m), 3.6 (CH3O,
3H, d, J ) 13.0 Hz), 3.7 (CH3O, 3H, s), 4.1 (H5′, 2H, m), 4.4
(CH3AA, 1H, m), 5.5 (H1′, 1H, m), 5.9 (H3′, 1H, m), 6.1 (H2′,
1H, m), 6.3 (NH2, 2H, bs), 7.15 (Ar, 5H, m), 7.8 (H8, 1H, s), 8.3
(H2, 1H, s); δC (CDCl3) 35.097 (CH2-Asp), 38.6 (C6′), 46.1 (C4′),
51.5 (OCH3-Asp), 52.4 (OCH3), 53.26 (CHNH), 59.65 (C1′), 69.23
(C5′), 120.45 (C5), 120.6 (Ar), 125.45 (Ar), 130.9 (Ar), 137.7 (C2′),
139.2 (C8), 149.8 (C4), 150.9 (Ar), 152.9 (C2), 155.9 (C6), 171.3
(CO-Asp), 172.3 (COMe); MS [(ES(+ve)]) found 553.1577
(MNa+), C23H27N6O7PNa requires 553.1599. Anal. (C23H27N6O7P‚
H2O) C, H, N.
7.8 (H2, 1H, d, J ) 11.99 Hz), 8.2 (H8, 1H, s); δC (CDCl3) 25.22
(CH2-Pro), 31.79 (CH2CH), 35.043 (C6′), 45.99 (C4′), 48.2 (CH2-
NH), 52,54 (CH3O), 59.46 (C1′), 60.2 (CH-Pro), 68.855 (C5′), 119.9
(C5), 120.48 (o-Ar), 125.07 (p-Ar), 129.8 (C3′), 130.8 (m-Ar), 137.5
(C2′), 138.9(C8), 149.8 (ipso-Ar), 151.12 (C4), 153 (C2), 156 (C6),
174.5 (CO); MS [(ES(+ve)]) found 521.1678 (MNa+) C23H27N6O5-
PNa requires 521.1678. Anal. (C23H27N6O5P) C, H, N.
L-Cd4A 5′-[phenyl methyl D-methioninyl]phosphate (4z) was
prepared using the standard methods above and purified on a silica
column with elution using MeOH/CHCl3 5:95 to give the product
as a clear, colorless oil, which solidified to a white foam on
trituration and coevaporation with diethyl ether (0.15 g, 0.23 mmol,
34%): δP (CDCl3) 4.23, 4.40; δH (CDCl3) 1.65 (CH2, 2H, m), 1.9
(CH2S, m, 2H), 1.95 (CH3S, 3H, m), 2.4 (H6′, 1H, m), 2.8 (H4′,
1H, m), 3.1 (H5′, m, 2H), 3.6 (CH3O, 3H, d, J ) 12.01 Hz), 4.2
(CHAA, 1H, m), 5.7 (H1′, 1H, m), 5.85 (H3′, 1H, m), 6.1 (H2′,
1H, m), 7.1 (Ar, 6H, m), 7.8 (H8, 1H, d, J ) 12.01 Hz), 8.3 (H2,
1H, s); δC (CDCl3) 15.5 (CH3S), 29.9 (CH2S), 33.6 (CH2CH), 35.14
(C6′), 46.1 (C4′), 50.38 (CH3), 53.8 (CHAA), 59.6 (C1′), 69.22
(C5′), 119.855 (C5), 120.3 (Ar), 125.2 (Ar), 130.031 (Ar), 130.94
(C3′), 137.54 (C8), 138.9 (C2′), 149.8 (C4), 151.05 (Ar), 153.14
(C2), 156.3 (C6), 173.814 (CO); MS [(ES(+ve)]) found 555.1544
(MNa+), C23H29N6O5PSNa requires 555.1555. Anal. (C23H29N6O5-
PS·2H2O) C, H, N.
L-Cd4A 5′-[4-chlorophenyl methyl L-alaninyl]phosphate (4aa)
was prepared using the standard methods above and purified on a
silica column with elution using MeOH/CHCl3 5:95 to give the
product as a clear, colorless oil, which solidified to a white foam
on trituration and coevaporation with diethyl ether (0.19 g, 0.47
mmol, 53%): δP (CDCl3) 3.88, 4.16; δH (CDCl3) 1.3 (CH3CH, 3H,
m), 1.7 (H6′, 1H, m), 2.9 (H6′, 1H, m), 3.2 (H4′, 1H, m), 3.7 (OCH3,
3H, d, J ) 12.01 Hz), 4.2 (H5′+NH, 3H, m), 4.2 (CHAA, 1H, m),
5.7 (H1′, 1H, m), 6 (H3′, 1H, m), 6.2 (H2′+NH2, 3H, m), 7.3 (Ar,
4H, m), 7.95 (H8, 1H, d, J ) 12.00 Hz), 8.4 (H2, 1H, d, J ) 6.03
Hz); δC (CDCl3) 15.1702 (CH3AA), 35.1 (C6′), 46.1 (C4′), 50.5
(CHNH), 59.6 (C1′), 69.2 (C5′), 120.133 (C5), 121.8 (Ar), 130.0
(C3′), 130.59 (Ar), 131.0 (Ar), 137.6 (C2′), 139 (C8), 149.5 (C4),
150.07 (Ar), 153.0 (C2), 155 (C6), 174.4 (CO); MS [(ES(+ve)])
found 529.1781 (MNa+), C21H24ClN6O5PNa requires 529.1782.
Anal. (C21H24ClN6O5P·0.5H2O) C, H, N.
L-Cd4A 5′-[4-nitrophenyl (methyl-L-alaninyl)]phosphate (4ab)
was prepared using the standard methods above and purified on a
silica column with elution using MeOH/CHCl3 5:95 to give the
product as a clear, colorless oil, which solidified to a white foam
on trituration and coevaporation with diethyl ether (0.12 g, 0.29
mmol, 28%): δP (CDCl3) 3.71, 3.97; δH (CDCl3) 1.3 (CH3CH, 3H,
m), 1.7 (H6′, 1H, m), 2.9 (H6′, 1H, m), 3.2 (H4′, 1H, m), 3.7 (OCH3,
3H, d, J ) 12.40 Hz), 4.1 (H5′+NH, 3H, m), 4.3 (CHAA, 1H, m),
5.65 (H1′, 1H, m), 6 (H3′, 1H, m), 6.2 (H2′, 1H, m), 6.7 (NH2,
2H, bs), 7.3 (Ar, 2H, m), 7.95 (H8, 1H, d, J ) 11.89 Hz), 8.2(Ar,
2H, m), 8.4 (H2, 1H, s); δC (CDCl3) 15.69 (CH3AA), 35.09 (C6′),
46.1 (C4′), 46.05 (CHNH), C1′ (59.6), 69.2 (C5′), 120.11 (C5),
121.00 (Ar), 125.9 (Ar), 131.2 (C3′), 131.0 (Ar), 137.6 (C2′), 139
(C8), 144.8 (C4), 150.07 (Ar), 153.0 (C2), 155 (C6), 174.4 (CO);
MS [(ES(+ve)]) found 539.2532 (MNa+), C21H24N7O7PNa requires
539.2531. Anal. (C21H24N7O7P) C, H, N.
L-Cd4A 5′-[4-(trifluoromethyl)phenyl methyl L-alaninyl]phos-
phate (4ac) was prepared using the standard methods above and
purified on a silica column with elution using MeOH/CHCl3 5:95
to give the product as a clear, colorless oil, which solidified to a
white foam on trituration and coevaporation with diethyl ether (0.13
g, 0.39 mmol, 31%): δP (CDCl3) 3.62, 3.93; δH (CDCl3) 1.3 (CH3-
CH, 3H, m), 1.7 (H6′, 1H, m), 2.9 (H6′, 1H, m), 3.2 (H4′, 1H, m),
3.7 (OCH3, 3H, d, J ) 12.3 Hz), 4.2 (H5′+NH, 3H, m), 4.2 (CHAA,
1H, m), 5.7 (H1′, 1H, m), 6 (H3′, 1H, m), 6.2 (H2′+NH2, 3H, m),
7.3 (Ar, 4H, m), 7.95 (H8, 1H, d, J ) 11.98 Hz), 8.4 (H2, 1H, s);
δC (CDCl3) 15.1702 (CH3AA), 35.1 (C6′), 46.1 (C4′), 50.5
(CHNH), C1′ (59.6), 69.2 (C5′), 120.133 (C5), 121.8 (Ar), 130.0
(C3′), 130.59 (Ar), 131.0 (Ar), 137.6 (C2′), 139 (C8), 149.5 (C4),
150.07 (Ar), 153.0 (C2), 155 (C6), 174.4 (CO); MS [(ES(+ve)])
found 563.1398 (MNa+), C22H24N6O5PF3Na requires 563.1396.
L-Cd4A 5′-[phenyl methyl D-prolinyl]phosphate (4y) was
prepared using the standard methods above and purified on a silica
column with elution using MeOH/CHCl3 5:95 to give the product
as a clear, colorless oil, which solidified to a white foam on
trituration and coevaporation with diethyl ether (0.23 g, 0.60 mmol,
66%): δP (CDCl3) 2.63, 2.76; δH (CDCl3)1.8 (CH2-Pro, CH2CH,
H6′, 5H, m), 2.8 (H6′, 1H, m), 3.1 (H4, 1H, m), 3.25 (CH2N, 2H,
m), 3.5 (CH3O, 3H, s), 4.05 (CH,1H, m), 4.25 (H5′, 2H, m), 5.6
(H1′, 1H, m), 5.8 (H3′, 1H, m), 5.9 (H2′, 1H, m), 7.1 (Ar, 5H, m),

7224 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
McGuigan et al.
Anal. (C22H24N6O5PF3·2H2O·MeCN) C, H, N. H: calcd, 5.06, found
3.98. N: calcd, 15.88, found 15.38.
and purified on a silica column with elution using MeOH/CHCl3
5:95 to give the title compound isolated as a brittle white foam
(39%): δP (CDCl3) 3.95, 4.05; δH (CDCl3) 1.28 (6H, m, CH3-Ala,
CH3CH2), 1.69 (1H, m, H6′a), 2.80 (1H, m, H6′b), 3.15 (1H, m,
H4′), 3.52, 3.60 (3H, s, OCH3-Ala), 4.18 (5H, m, CH2CH3, H5′,
CH-Ala), 5.70 (1H, m, H1′), 5.88 (1H, m, H3′), 6.12 (1H, m, H2′),
7.16 (1H, m, Ar), 7.44 (2H, m, Ar), 7.80 (1H, m, Ar), 7.87, 7.92
(1H, s, H8), 8.28 (1H, s, H2); δC (CDCl3) 14.43, 14.64 (CH3CH2O),
21.29, 21.36, 21.60, 21.66 (CH3-Ala), 35.15 (C6′), 46.07, 46.13,
46.18, 46.23 (C4′), 50.44 (CH-Ala), 52.75, 52.80 (OCH3), 59.57
(C1′), 61.75, 61.80 (CH3CH2O), 69.31, 69.40 (C5′), 120.07 (C5),
122.84, 122.87, 123.10, 123.14, 123.47, 123.54, 123.60 (o-Ph),
125.23, 125.34 (p-Ph), 130.98 (C3′), 131.74, 131.86 (m-Ph), 137.68,
137.81 (C2′), 139.30, 139.49 (C8), 150.09, 150.20 (C4), 150.28
(ipso-Ph), 153.19 (C2), 156.02 (C6), 165.58, 165.66 (CO(Ar)),
174.32, 174.43, 174.61, 174.70 (CO-Ala); MS (E/I) 567.1722
(MNa+), C24H28N6O7PNa requires 567.1733. Anal. (C24H29N6O7P‚
H2O) C, H, N.
L-C-d4A-5′-[3-(trifluoromethyl)phenyl methyl L-alaninyl] phos-
phate (4ad) was prepared using the standard methods above and
purified on a silica column with elution using MeOH/CHCl3 5:95
to give the product as a clear, colorless oil, which solidified to a
white foam on trituration and coevaporation with diethyl ether (0.13
g, 0.39 mmol, 34%). δP (CDCl3) 3.77, 4.09; δH (CDCl3) 1.3 (CH3-
CH, 3H, m), 1.7 (H6′, 1H, m), 2.9 (H6′, 1H, m), 3.2 (H4′, 1H, m),
3.7 (OCH3, 3H, d, J ) 11.09 Hz), 4.2 (H5′+NH, 3H, m), 4.2
(CHAA, 1H, m), 5.7 (H1′, 1H, m), 6 (H3′, 1H, m), 6.2 (H2′+NH2,
3H, m), 7.3 (Ar, 4H, m), 7.95 (H8, 1H, d, J ) 12.09 Hz), 8.4 (H2,
1H, s); δC (CDCl3) 15.1702 (CH3AA), 35.1 (C6′), 46.1 (C4′), 50.5
(CHNH), C1′ (59.6), 69.2 (C5′), 120.133 (C5), 121.8 (Ar), 130.0
(C3′), 130.59 (Ar), 131.0 (Ar), 137.6 (C2′), 139 (C8), 149.5 (C4),
150.07 (Ar), 153.0 (C2), 155 (C6), 174.4 (CO); MS [(ES(+ve)])
found 563.1392 (MNa+), C22H24N6O5PF3Na requires 563.1396.
Anal. (C22H24N6O5PF3) C, H, N.
D-Cd4A 5′-[Phenyl methyl L-alaninyl]phosphate (5). (1S,cis)-
4-(6-Amino-9H-purin-9-yl)-2-cyclopentene-1-methanol (290 mg,
1.25 mmol) was dissolved in a mixture of dry pyridine (3 mL) and
THF (3 mL). tert-Butyl magnesium chloride (1 M in THF, 1.25
mL) was added. After 10 min, a solution of phenyl(methyl-L-
alaninyl)phosphorochloridate (0.70 g, 2.5 mmol) in THF (5 mL)
was added. Volatiles were removed and the residual gummy solid
was partitioned between CH2Cl2 (50 mL) and water (50 mL). The
organic layer was dried (magnesium sulfate), filtered, and concen-
trated to a syrup. Column chromatography (5% MeOH/CHCl3) gave
the title compound as a white solid foam (0.38 g, 65%): δP
(CDCl3): 3.12, 2.80; δH (CDCl3) 8.37 (s, 1H), 7.84 and 7.81 (both
s, 1H), 7.28-7.11 (m, 5H), 6.15 (m, 1H), 5.96 (m, 1H), 5.72 (m,
1H), 5.58 (m, 1H), 4.2-3.9 (m, 3H), 3.7 (s, 3H), 3.2 (m, 1H), 2.85
(m, 1H), 1.70 (m, 1H), 1.2-1.4 (m, 3H); MS m/z 473 (M + H).
Anal. (C21H25N6O5P·0.2CHCl3) C, H, N.
L-CddA 5′-O-[Phenyl methyl-L-alaninyl]phosphate (8). Com-
pound 4a 0.53 g, 1.1 mmol) in absolute EtOH (45 mL) was shaken
with 10% Pd/C (170 mg) at 40 psi H2 for 12 h. Filtration through
Celite and concentration gave a solid that was purified by column
chromatography (3% MeOH/CHCl3) to give the title compound as
a white foam (0.26 g, 49%): δP (CDCl3) 3.05, 2.90; δH (CDCl3)
8.38 (s, 1H), 7.92 (s, 1H), 7.28 (m, 5H), 5.87 (s, 2H), 4.90 (m,
1H), 4.24-4.03 (m, 3H), 3.89-3.70 (m, 4H), 2.49 (m, 1H), 2.33
(m, 1H), 2.11-1.78 (m, 4H), 1.42 (m, 4H); MS m/z 475 (M + H).
Anal. (C21H27N6O5P·0.5H2O) C, H, N. N: calcd 17.38; found 16.94.
D-CddA 5′-[Phenyl methyl L-alaninyl] phosphate (9). (1R,cis)-
3-(6-Amino-9H-purin-9-yl)-1-cyclopentyl-1-methanol (174 mg, 0.75
mmol) was dissolved in a mixture of dry pyridine (3 mL) and THF
(3 mL). tert-Butyl magnesium chloride (1 M in THF, 0.75 mL)
was added. After 10 min, a solution of phenyl(methyl-L-alaninyl)-
phosphorochloridate (0.41 g) in THF (5 mL) was added. Volatiles
were removed, and the residual solid was partitioned between CH2-
Cl2 (50 mL) and water (50 mL). The organic layer was dried
(magnesium sulfate), filtered, and concentrated to a syrup. Column
chromatography (10% MeOH/CHCl3) gave the product as a white
solid foam (0.25 g, 71%): δH (CDCl3) 8.37 (s, 1H), 7.90 and 7.89
(both s, 1H), 7. 35-7.18 (m, 5H), 6.01 (m, 2H), 4.90 (m, 1H),4.22-
3.80 (m, 5H), 3.74 and 3.73 (both s, 3H), 2.50 (m, 2H), 2.35 (m,
1H), 2.2-1.8 (m, 3H), 1.43 (q, 3H); MS m/z 475 (M + H). Anal.
(C21H27N6O5P‚H2O) C, H, N.
L-Cd4A 5′-[3,4-dichlorophenyl methyl L-alaninyl]phosphate
(4ae) was prepared using the standard methods above and purified
on a silica column with elution using MeOH/CHCl3 5:95 to give
the product as a clear, colorless oil, which solidified to a white
foam on trituration and coevaporation with diethyl ether (0.22 g,
0.13 mmol, 32%): δP (CDCl3) 3.77, 4.09; δH (CDCl3) 1.3 (CH3-
CH, 3H, m), 1.7 (H6′, 1H, m), 2.9 (H6′, 1H, m), 3.2 (H4′, 1H, m),
3.7 (OCH3, 3H, d, J ) 12.19 Hz), 4.2 (H5′+NH, 3H, m), 4.2
(CHAA, 1H, m), 5.7 (H1′, 1H, m), 6 (H3′, 1H, m), 6.2 (H2′+NH2,
3H, m), 7.3 (Ar, 3H, m), 7.95 (H8, 1H, d, J ) 12.10 Hz), 8.4 (H2,
1H, s); δC (CDCl3) 15.1702 (CH3AA), 35.1 (C6′), 46.1 (C4′), 50.5
(CHNH), C1′ (59.6), 69.2 (C5′), 120.133 (C5), 121.8 (Ar), 130.0
(C3′), 130.59 (Ar), 131.0 (Ar), 137.6 (C2′), 139 (C8), 149.5 (C4),
150.07 (Ar), 153.0 (C2), 155 (C6), 174.4 (CO); MS [(ES(+ve)])
found 563.0742 (MNa+), C21H23N6O5PCl2Na requires 563.0742.
Anal. (C21H23N6O5PCl2) C, H, N.
L-Cd4A 5′-[4-carboxymethylphenyl methyl L-alaninyl]phos-
phate (4af) was prepared using the standard methods above and
purified on a silica column with elution using MeOH/CHCl3 5:95
to give the product as a clear, colorless oil, which solidified to a
white foam on trituration and coevaporation with diethyl ether (0.20
g, 0.39 mmol, 45%): δp (CDCl3) 3.31, 3.67; δH (CDCl3) 1.3
(6H+CH3-CO+CH3-CH, m) 1.55 (H6′, 1H, m), 2.8 (H6′, 1H, m),
3.1 (H4′, 1H, m), 3.7 (CH3O, 3H, d, J ) 12.32 Hz), 4.0 (CHAA,
1H, m), 4.1 (H5′+NH, 3H, m), 5.8 (H1′, 1H, m), 5, 7 (H3′, 1H,
m), 6.1 (H2′, 1H, m), 6.8 (NH2, 2H, m), 7.2 (Ar, 4H, m), 7.8 (H8,
1H, d, J ) 12.23 Hz), 8.2 (H2, 1H, s); δC (CDCl3) 11.3 (CH3CO),
14.7 (CH3AA), 35.11 (C6′), 46.9 (C4′), 50.1 (CH3O), 52.4 (CHNH),
53.4 (CH3O), 59.7 (C1′), 68.8 (C5′), 119.5 (C5), 126 (Ar), 129.7
(C3′), 130 (Ar), 131.9 (Ar), 132 (Ar), 137.8 (C2′), 139.1 (C8), 148.7
(C4), 150 (Ar) 152.8 (C2), 154.8 (Ar), 156.0 (C6), 166.1 (COCH3),
174.9 (COOCH3); MS [(ES(+ve)]) found 553.4640 (MNa+),
C23H27N6O7PNa requires 553.4641. Anal. (C23H27N6O7P) C, H, N.
L-Cd4A 5′-[3-carboxylic ethyl ester phenyl methyl L-alaninyl]-
phosphate (4ag) was prepared using the standard methods above
and purified on a silica column with elution using MeOH/CHCl3
5:95 to give the product as a clear, colorless oil, which solidified
to a white foam on trituration and coevaporation with diethyl ether
(0.12 g, 0.39 mmol, 34%): δp 4.006, 4.214; δH 1.3 (6H+CH3-
CH2+CH3CH, m), 1.55 (1H, H6′, m), 2.8 (1H, H6′, m), 3.1 (1H,
H4′, m), 3.7 (3H, CH3O, d, J ) 12.09 Hz), 4.0 (CHAA, 1H, m),
4.1 (H5′+NH, 3H, m), 4.3 (CH3CH2), 5.8 (H1′, 1H, m), 5, 7 (H3′,
1H, m), 6.1 (H2′, 1H, m), 6.6 (NH2, 2H, m), 7.2 (Ar, 2H, m), 7.8
(Ar, 2H, m), 7.8 (H8, 1H, d, J ) 12.00 Hz), 8.2 (H2, 1H, s); δC
14.67 (CH3AA), 21.22 (CH3CH2), 35.11 (C6′), 46.85 (C4′), 50.1
(CH3O), 52.4 (CHNH), 53.38(CH3O), 59.7 (C1′), 61.6 (CH2O), 68.8
(C5′), 119.5 (C5), 126 (Ar), 129.7 (C3′), 130 (Ar), 131.9 (Ar), 132
(Ar), 137.8 (C2′), 139.1 (C8), 148.7 (C4), 150 (Ar) 152.8 (C2),
154.8 (Ar), 156.0 (C6), 166.059 (COEt), 174.9 (COCH3); MS [(ES-
(+ve)]) found 567.1721 (MNa+), C24H28N6O7PNa requires 567.1733.
Anal. (C24H28N6O7P) C, H, N.
Antiviral Assays. Cells. Human T-lymphocytic CEM cells were
obtained from the American Type Culture Collection (Rockville,
MD). MT-4 cells were a kind gift from Dr. L. Montagnier (Pasteur
Institute, Paris, France).
Viruses. HIV-1(IIIB) was provided by Dr. R.C. Gallo and Dr.
M. Popovic (at that time at the National Cancer Institute, NIH,
Bethesda, MD) and HIV-2(ROD) was from Dr. L. Montagnier (at
that time at the Pasteur Institute, Paris, France).
Antiretrovirus Assays. The methodology of the anti-HIV assays
has been described previously. Briefly, MT-4 or CEM cells (4.5 ×
105 cells per mL) were suspended in fresh culture medium and
infected with HIV-1 or HIV-2 at 100 CCID50 (1 CCID50 being the
L-Cd4A 5′[(2-carboxylic ethyl ester) phenyl methyl L-alaninyl]-
phosphate (4ah) was prepared using the standard methods above

Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7225
dose infective for 50% of the cell cultures) per milliliter of cell
suspension. Then, 100 µL of the infected cell suspension was
transferred to 96-well microplate wells, mixed with 100 µL of the
appropriate dilutions of the test compounds (i.e., final concentrations
of 200, 40, 8, 1.6, 0.32, and 0.062 µg/mL), and further incubated
at 37 °C. After 4-5 days, giant cell formation was recorded
microscopically in the CEM cell cultures. The MT-4 cell cultures
were treated with trypan blue and the number of viable cells was
determined. The 50% effective concentration (EC50) corresponded
to the compound concentrations required to prevent syncytium
formation by 50% in the virus-infected CEM cell cultures or the
compound concentrations required to reduce the amount of living
cells by 50% in the MT-4 cell cultures.
MT-4 Cell Assay. Antiviral HIV activity and compound-induced
cytotoxicity were measured in parallel by means of an MTS-based
procedure in MT-4 cells. Aliquots of the test compounds were
serially diluted in medium [Roswell Park Memorial Institute
medium (RPMI) 1640, 10% v/v FBS and 10 µg/mL gentamicin]
in 96-well plates. Exponentially growing MT-4 cells were harvested
and centrifuged at 192g for 10 min in a Jouan centrifuge. Cell pellets
were resuspended in fresh medium (RPMI 1640, 20% vol/vol FBS,
20% v/v Il-2, and 10 µg/mL gentamicin) to a density of 5 × 105
cells/mL. Cell aliquots were infected by the addition of HIV-1 IIIB
diluted to give a viral inoculum of 100 × TCID50 per well. A similar
cell aliquot was diluted with medium to provide a mock-infected
control. Cell infection was allowed to proceed for 1 h at 37 °C in
a tissue culture incubator with humidified 5% CO2 atmosphere.
After incubation, the virus-treated cell suspensions were diluted
6-fold with fresh medium, and 125 µL of the cell suspension was
added to each well of the plate containing prediluted compound.
Compound GW678248 was tested over final concentration ranges
of 0.5-500 nM. Plates were then placed in a tissue culture incubator
at 37 °C with humidified 5% CO2 for 5 days. HIV-induced
cytopathic effects were assessed with a CellTiter 96 MTS staining
method (cat. no. G3581; Promega, Madison, WI). Optical density
was measured at 492 nM using a microplate absorbance reader (cat.
no. 20-300; Tecan, RTP, NC).
HBV potency and growth inhibition potential of compounds were
evaluated in the assay reported by Jansen et al.42 Briefly, HepG2-
2.2.15 cells constitutively producing HBV43 were seeded at a density
of 5 × 103 per well into 96-well microtiter plates. Culture medium
containing drug was replaced every other day for 9 days. HBV
content was determined in culture supernatant by binding virus to
anti-HBsAg coated plates followed by PCR amplification and
quantitation of released virion DNA. Dilutions of a standardized
HBV-containing supernatant were included on every plate, and
HBV DNA concentrations of test wells were calculated from this
HBV standard curve. Samples were tested in conjunction with both
positive (0.448 fg/µL plasmid DNA) and negative (RPMI medium
supplemented with 2 mM L-glutamine and 10% fetal calf serum)
controls. Data were normalized to non-drug-treated cells and
expressed as a percent of control for analysis. Effects on cell growth
were performed by fixing monolayers with 70% ethanol with
subsequent staining with bisbenzimide H33342. Fluorescence values
of drug-treated cells were compared to non-drug-treated cells and
expressed as a percentage of control.
Metabolic Stability in Cynomolgus Monkey Liver and
Intestinal S9. Each compound (10 µM final concentration) was
incubated at 37 °C in pooled S9 postmitochondrial homogenates
prepared from cynomolgus livers or intestinal segments (Xenotech,
LLC, Kansas City, KS). Stock solutions of the test compounds were
prepared in DMSO such that the final DMSO concentration in the
reaction mixture (0.5 mL) was <1%. All reactions were initiated
by addition of NADPH to a final concentration of 2.5 mM as
described by Wring et al.44 At time 0 (preincubation) 10, 20, 30,
and 60 min, the reaction (200-µL aliquot) was stopped by addition
of cold acetonitrile (400 µL). The resulting samples were vortexed
and centrifuged (2600g, 15 min), and the supernatants were
analyzed for parent compound by LC/MS/MS. Metabolic stability,
expressed as the amount (%) of compound remaining, was
determined by comparison of peak areas in the pre- and postincu-
bation samples.
Acknowledgment. We thank Helen Murphy for excellent
secretarial assistance and Ann Absillis for dedicated experi-
mental work. This work was supported by grants from the
Concerted Actions of the KU Leuven (GOA Krediet 2005/19)
and the Flemish Foundation of Scientific Research (FWO
Krediet G-0267-04).
Supporting Information Available: Analytical data for the key
target compounds and experimental procedures for parent nucleo-
sides. This material is available free of charge via the Internet at
http://pubs.acs.org.
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