Application of phosphoramidate pronucleotide technology to abacavir leads to a significant enhancement of antiviral potency

Article abstract of DOI:10.1021/jm0491400

We report the first application of pronucleotide (ProTide) technology to the antiviral agent abacavir (Ziagen), used for the treatment of HIV infection. The phenylmethoxyalaninyl phosphoramidate of abacavir was prepared in good yield in one step. Also prepared was the corresponding phosphoramidate of the guanine nucleoside analogue "carbovir". The antiviral profile of each of the parent nucleosides was compared to that of the phosphoramidate ProTides. A significant (28- to 60-fold) increase in anti-HIV potency was noted for the ProTide of abacavir but not for that of carbovir. These findings were in agreement with the markedly higher (ca. 37-fold) levels of carbovir triphosphate that are formed in CEM cells upon response to the abacavir ProTide compared with the parent abacavir compound. In contrast the anti-HBV potency of both abacavir and carbovir were improved (10- and 20-fold, respectively) by ProTide formation. As in CEM cells, the abacavir ProTide provided significantly enhanced carbovir triphosphate levels in HepG2 2.2.15 cells over that of the parent nucleoside. On the basis of these data, a series of phosphoramidate analogues with structural variation in the ester and amino acid regions were prepared and their antiviral profiles described. In addition, the pharmacokinetic disposition of the abacavir phenylethoxyalaninyl phosphoramidate was evaluated in Cynomolgus monkeys.

Full text of DOI:10.1021/jm0491400

3504
J. Med. Chem. 2005, 48, 3504-3515
Application of Phosphoramidate Pronucleotide Technology to Abacavir Leads
to a Significant Enhancement of Antiviral Potency
Christopher McGuigan,*,† Sarah A. Harris,† Susan M. Daluge,‡ Kristjan S. Gudmundsson,‡ Ed W. McLean,‡
Thimysta C. Burnette,§ Harry Marr,§ Richard Hazen,# Lynn D. Condreay,# Lance Johnson,# Erik De Clercq,| and
Jan Balzarini|
Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, U.K., Medicinal Chemistry, 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 October 26, 2004
We report the first application of pronucleotide (ProTide) technology to the antiviral agent
abacavir (Ziagen), used for the treatment of HIV infection. The phenylmethoxyalaninyl
phosphoramidate of abacavir was prepared in good yield in one step. Also prepared was the
corresponding phosphoramidate of the guanine nucleoside analogue “carbovir”. The antiviral
profile of each of the parent nucleosides was compared to that of the phosphoramidate ProTides.
A significant (28- to 60-fold) increase in anti-HIV potency was noted for the ProTide of abacavir
but not for that of carbovir. These findings were in agreement with the markedly higher (ca.
37-fold) levels of carbovir triphosphate that are formed in CEM cells upon response to the
abacavir ProTide compared with the parent abacavir compound. In contrast the anti-HBV
potency of both abacavir and carbovir were improved (10- and 20-fold, respectively) by ProTide
formation. As in CEM cells, the abacavir ProTide provided significantly enhanced carbovir
triphosphate levels in HepG2 2.2.15 cells over that of the parent nucleoside. On the basis of
these data, a series of phosphoramidate analogues with structural variation in the ester and
amino acid regions were prepared and their antiviral profiles described. In addition, the
pharmacokinetic disposition of the abacavir phenylethoxyalaninyl phosphoramidate was
evaluated in Cynomolgus monkeys.
Introduction
sible only after formation of the monophosphate of a
nucleoside, led to the understanding of the unique in
vivo properties of abacavir and closely related analogues
modified at the 6-position of the purine.8 The contrasting
in vivo profiles of abacavir and CBV are attributable to
an activation pathway by which abacavir is converted
to the triphosphate of CBV in lymphocytes. This path-
way bypasses CBV, suggesting that the in vivo contrast
is attributable to markedly differing exposures to the
nucleoside CBV. Abacavir has demonstrated potent,
selective anti-HIV activity in the clinic as a mono-
therapy and in combination with nucleoside reverse
transcriptase inhibitors,1-4 non-nucleoside reverse tran-
scriptase inhibitors,5 and protease inhibitors.6
Abacavir and a few other 6-modified purine analogues
have exhibited anti-HIV activity comparable to that of
carbovir against HIV-1 (IIIB) in MT-4 cells (IC50 ≈ 4
µM).8 This is approximately 4-fold more potent than ddI
in this assay but less potent than AZT, 3TC, and ddC.
The relative potency of the nucleoside RT inhibitors in
the clinic does not parallel ranking from in vitro assays
likely because of the many confounding variables such
as pharmacokinetics and distribution of the nucleoside
analogues and levels of active triphosphates in target
cells and tissues. Indeed, 3TC and abacavir were
selected for clinical evaluation largely because of their
excellent safety and pharmacokinetic profiles in animals
rather than superior in vitro potency relative to AZT.
Metabolic studies have shown that abacavir is anabo-
lized in infected and uninfected CD4+ CEM cells and
in uninfected peripheral blood lymphocytes (PBLs) to
Abacavir (1) (Scheme 1) is the first member of the
well-studied carbocyclic nucleosides to be approved for
use as a drug.1-8 Carbocyclic nucleosides, lacking the
labile glycosidic linkage between the heterocycle and
sugar, have long been proposed to offer an attractive in
vivo stability advantage over the 2′,3′-dideoxynucleo-
sides. However, carbocyclic versions of 2′,3′-dideoxyad-
enosine (ddA), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-
cytidine (ddC), 2′,3′-dideoxyguanosine, and 3′-
deoxythymidine have demonstrated only modest anti-
HIV activity.9 The anti-HIV activity of the racemic
guanine member of this series, “carbovir” (CBV), 2, was
described by Vince and colleagues.10 Although the
triphosphate of (-)-carbovir is a potent and selective
inhibitor of HIV reverse transcriptase (RT),7,11 in vivo
preclinical studies eliminated 2 as a candidate for anti-
HIV therapy because of dose-limiting kidney and cardiac
toxicities, poor oral bioavailability, and limited central
nervous system (CNS) penetration.8 The problems
encountered with CBV could not be solved with existing
prodrug strategies, e.g., 5′-aminoacylesters12 or 2,6-
diaminopurine prodrugs.13 The realization of an unex-
pected enzymatic purine conversion/modification, pos-
* To whom correspondence should be addressed. Phone/fax: (+44)
029 2087 4537. E-mail mcguigan@cardiff.ac.uk.
† Cardiff University.
‡ Medicinal Chemistry Department, GlaxoSmithKline.
§ Drug Metabolism and Pharmacokinetics Department, GlaxoSmith-
Kline.
# Virology Department, GlaxoSmithKline.
| Katholieke Universiteit Leuven.
10.1021/jm0491400 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/16/2005

Application of Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3505
Scheme 1. Synthetic Route to Antiviral ProTides of Carbovir and Abacavir
(-)-CBV-TP, which is the only triphosphate observed.8,14
Abacavir monophosphate was detected, but the di- and
triphosphates of the nucleoside were not detected. The
only radiolabeled di- and triphosphates detected were
those of CBV. The levels of CBV-TP produced from
abacavir were not significantly different from those
formed from equimolar CBV, suggesting that the ana-
bolic pathway from abacavir to CBV-TP is relatively
efficient, although indirect. (-)-CBV-TP is a potent
competitive inhibitor with respect to dGTP for RT-
catalyzed DNA synthesis, with a Ki of 0.02 µM, and is
selective over mammalian DNA polymerases.8 The
distinctive antiviral profile of abacavir, via its unique
activation pathway, is attributable to two new en-
zymes: adenosine phosphotransferase, which phospho-
rylates abacavir to its’ 5′-monophosphate (abacavir-MP),
and a novel, previously unidentified and cytosolic
enzyme “abacavir monophosphate deaminase”, which
deaminates abacavir-MP to CBV-MP.14,15
Since the phosphorylation of abacavir by adenosine
phosphotransferase is unique to abacavir and little has
been reported concerning the in vivo levels and distri-
bution of this kinase, it was of interest to determine
whether the potency of 1 against HIV could be improved
via a pronucleotide (ProTide) approach16 to achieve
“kinase-bypass”. In addition, it was of interest to
determine whether the micromolar in vitro anti-HBV
activity of abacavir, insufficient to provide any clinical
indication of anti-HBV efficacy, could be improved after
conversion to its ProTide forms.
challenge because of the highly polar nature of these
compounds that limits their solubility in organic sol-
vents and that also presents more than one possible site
at which phosphorylation can occur. Previous work
undertaken with the acyclic nucleoside analogue acy-
clovir investigated the use of N-protection as a means
to increase the solubility of acyclovir and also to prevent
N-phosphorylation on the guanine base.17 However,
these synthetic routes afford a lengthier synthesis, with
N-protection, phosphorylation, and then deprotection,
resulting in poor yields of the desired 5′-phosphorami-
date derivative. Phosphorylation of unprotected acyclo-
vir using phenylmethoxyalaninyl phosphorochloridate
and N-methylimidazole (NMI) in THF resulted in little/
no product being isolated. A variety of protecting groups
(DMF, pixyl) have been utilized to N-protect the base
and aid solubility in the phosphorylation reaction.17
While these protected derivatives yielded the desired
phosphoramidates, removal of protecting groups in the
presence of the phosphoramidate proved to be trouble-
some because of basic cleavage of the ester. Similar
problems were encountered in the synthesis of abacavir
and other diaminopurine 5′-O-phosphoramidates. Thus,
it was desirable to achieve a one-step conversion of
diaminopurine and guanine nucleoside to 5′-phospho-
ramidates without the use of purine protecting groups.
The method of Uchiyama,18 which claims to achieve
selective O-phosphorylation without the need for N-
protection of nucleosides, was investigated. This ap-
proach is based on the concept of functional group
activation rather than protection. Thus, when a nucleo-
side is treated with 1 equiv of a strong organometalic
base (such as tert-butylmagnesium chloride), an equi-
librium mixture of the metal alkoxide and amide is
Results and Discussion
Chemistry. The synthesis of phosphorylated deriva-
tives of diaminopurine and guanine nucleosides is a

3506 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
McGuigan et al.
Table 1. Stability and Solubility of Salts of 6
formed. Selective O-phosphorylation depends on this
equilibrium and the enhanced nucleophilicity of the
oxygen function. Preferential formation of the magne-
sium iminoxide is observed with guanosine, but the
magnesium alkoxide is more reactive, and the subse-
quent phosphorylated species equilibrate to form a
single phosphorylated product, showing the desired 5′-
O-phosphorylation.18
We chose to investigate if the Uchiyama method
would be suitable for formation of the 5′-O-phosphora-
midates of abacavir. Thus, treatment of abacavir with
tert-butylmagnesium chloride in THF, followed by ad-
dition of phenylmethoxy-L-alaninyl phosphorochloridate,
was observed to be very rapid and gave a good yield of
desired products following silica gel purification. The
reaction yielded a 1:1 mixture of diastereomers (3a,b)
because of the chiral phosphorus. Other abacavir phos-
phoramidates (4-9) were prepared in a similar fashion.
The phosphorylation of carbovir (2) as a suspension in
THF under similar conditions was found to be more
problematic, requiring greater excess of phenylmethoxy-
alaninyl phosphorochloridate and longer reaction times
to yield 10 (Scheme 1).
The chirality at the phosphorus center results in the
formation of two diastereomeric phosphoramidate nu-
cleosides in an approximately 1:1 ratio that were
inseparable by silica gel chromatography. Because of the
very potent antiviral activity of these mixtures (see
below), we became interested in separating the diaste-
reoisomers for comparison of antiviral activities. The
diastereomeric mixtures of representative derivatives
3 and 6 were separated using supercritical fluid chro-
matography with a Chiralpak AS column and 22%
methanol in carbon dioxide as eluent to give 3a,b and
6a,b. Data on these separated isomers are discussed
below.
The metabolic intermediate (11) was efficiently pre-
pared by hydrolysis of both phosphate and carboxylate
esters of abacavir 5′-[phenyl(methoxy-L-alaninyl)]phos-
phate (3) in an aqueous solution of triethylamine.
Purification of the concentrated reaction was conve-
niently carried out using a QMA solid-phase extraction
cartridge (Sep-Pak Vac Accell Plus QMA cartridge,
Waters Corp.) as an anion exchange chromatography
column. Lyophilization of the eluent gave a diammo-
nium salt that was unstable on storage for a few days
at room temperature. However, passing the concen-
trated eluent through a Sep-Pak Vac Accell Plus CM
cartridge (Na+ form) followed by lyophilization gave a
more stable disodium salt that could be stored at room
temperature for several weeks.
solubility solid-state
(mg/mL)a stabilityb
salt form of 6
solid type
free base
glutarate
fumarate
succinate
amorphous (hygroscopic)
crystalline
0.054
0.084
0.086
0.069
93.7
98.9
97.1
99.6
crystalline
crystalline
a Solubility in phosphate buffered saline (pH 7.4) at room
temperature. b Solid-state stability is determined as the % of
parent (AUC) after 2 weeks at 60 °C and ambient humidity
normalized to initial AUC.
Thus, succinic acid, fumaric acid, and glutaric acid all
gave crystalline products. Data on these salts are
presented in Table 1. On the basis of the relative
stability of these products, the succinate salt was chosen
for pharmacokinetic evaluation. This abacavir phospho-
ramidate succinate salt was not deliquescent and was
stable at 60 °C for over 2 weeks.
Antiviral Activity. The phenylmethoxy-L-alaninyl
phosphoramidates of abacavir (3) and (-)-carbovir (10)
were assessed for their antiviral activity with 1 and 2
as positive controls. Both carbovir and abacavir exhib-
ited similar potencies and selectivities against HIV-1
and HIV-2, with EC50 values of 1.9-3 µM and CC50
values of 78-160 µM in CEM cell cultures (Table 2).
The phenylmethoxy-L-alaninyl phosphoramidate deriva-
tive of carbovir (10) was similar in potency to carbovir
with EC50 values approximately 2-fold lower than for
carbovir in CEM cell cultures and 7-fold less potency
in PBL. In contrast, the phenyl phosphoramidate de-
rivative of abacavir (3) showed a 27-fold increase in
potency against HIV-1 in the CEM cell line, a 25-fold
increase in potency against HIV-1 in PBLs, and a 33-
fold increase in potency against HIV-2 in the CEM cell
cultures compared to abacavir. While compound 3 did
show a decrease in its CC50, the selectivity index (SI)
was still more favorable compared to the parent drug.
The differential behavior of carbovir and abacavir in
human leukemic T cells upon derivatization is notable
and surprising. This difference may be cell- or virus-
dependent because ProTides 3 and 10 show similar
enhancements of potency (ca. 10- to 20-fold) against
HBV in Hep G2.2.15 cells. These data indicated to us
that the SARs we have thoroughly established for d4T
(stavudine) anti-HIV activity in lymphocytes may not
fully apply to the guanine carbocyclic nucleosides,
especially against HBV in hepatoma cells but also
against MSV in C3H/3T3 cells and herpesviruses in
HEL cell cultures (Table 3), where the ProTide 3
increased the antiviral activity of ABC (1) but where
the ProTide 10 significantly decreased the antiviral
activity of CBV (2).
For profiling in animals we chose to scale-up the ethyl
ester derivative 6. The procedure used to make multi-
gram amounts 6 was modified from the process used to
make research quantities, by using pyridine in addition
to THF as a cosolvent. Use of pyridine allowed the
reactions to be run more concentrated and reduced
losses due to salt precipitation. Purification of the
reaction by extraction followed by filtration through
silica gel gave 6 as a white hygroscopic solid foam.
Crystalline salt forms were sought, since the solid foams
formed by the free bases trapped solvents and were
difficult to formulate. As with abacavir, dibasic acids
were found to crystallize the ProTides in a 1:1 complex.
Table 4 presents the anti-HIV profile of the amino
acid and ester-modified series of abacavir phosphora-
midates (3-9), compared to the parent abacavir (1).
Against HIV, the amino acid dependence is strikingly
similar to that we have noted for phosphoramidates of
stavudine (d4T).19 Thus, (L-)alanine emerges as the
preferred amino acid. There is a clear stereochemical
preference for L-amino acids, with a ca. 50-fold reduction
in potency for D-alanine (4), this being rather similar
to glycine (5) in potency. The effect of variation in the
ester region (Table 4) also shows a close parallel to d4T
phosphoramidate data.20 Thus, the primary esters
methyl and ethyl (3, 6) were roughly equipotent. The

Application of Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3507
Table 2. Antiviral Activity for Abacavir, Carbovir, and Derivatives in Cell Culturea
EC50 (µM)
SI
EC50 (µM)
MSV
C3H/3T3
EC50 (µM)
HIV-1 (IIIB) HIV-1 HIV-2 (ROD) CC50 (µM) HIV-1 HIV-2
CC50 (µM)
HBV
CC50 (µM)
CEM
PBL
CEM
CEM
CEM
CEM
C3H/3T3 HepG2 2.2.15 HepG22.215
3
0.07
1.9
0.008
0.20
0.20
0.03
0.09
3.0
0.85
2.3
13
78
120
160
190
41
150
26
145
68
1.6
8.8
>20
6.0
>4
>20
>20
>20
0.55
5.6
17
110
200
150
ABC (1)
10
1.3
95
0.51
CBV (2)
2.0
79
10
a PBL refers to peripheral blood lymphocytes. MSV: murine moloney sarcoma virus. EC50: 50% effective concentration required to
inhibit HSV-induced cytopathicity by 50%. CC50: 50% cytostatic concentration required to inhibit HEL cell proliferation by 50%.
Table 3. Anti-HSV Activity for Carbovir and Abacavir
in the first step of conversion of the ProTide to mono-
phosphate inside the cells (Scheme 2). Thus, on the basis
of earlier metabolic studies with the analogues stavu-
dine (d4T) phosphoramidates,21 we reasoned that the
activation intracellular route for the current compounds
was likely to involve initial carboxyl ester hydrolysis to
give a short-lived carboxylate intermediate, which would
rapidly cyclize with loss of phenol to give another
transient intermediate, a highly energetic five-mem-
bered anhydride, which would spontaneously hydrolyze
to give the stable phosphate diester (11). In all prior
metabolic studies, compounds of the general structure
of 11 were frequently observed as major intracellular
metabolites. A phosphoramidase-type activity is then
implicated in the cleavage of the amino acid from (11)
to generate abacavir monophosphate (ABCMP). This
would then be processed as for abacavir via carbovir
monophosphate (CBVMP) to the bioactive triphosphate.
To further support this notion, we carried out some
preliminary metabolism studies on radiolabeled 3.21
Anabolism in CEM and HepG2 2.215 Cells. Ra-
diolabeled [3H]abacavir and the aryloxymethylalaninyl
ProTide of [3H]abacavir monophosphate (3) were ad-
ministered to CEM cell cultures at 10 µM. The highest
levels of formed metabolites were recorded between 6
and 24 h of incubation.22 ABC (1) was metabolized to
its 5′-monophosphate ABCMP, and reasonable levels of
carbovir 5′-phosphates, CBVMP, CBVDP, and CBVTP,
were also detected in the cell extracts (Table 6). In fact,
82 pmol/109 CEM cells of CBVTP was formed after 24
h, which represents an ∼0.1 µM CBVTP intracellular
concentration (assuming that 109 CEM cells represent
0.8 µL of packed volume).14 These levels were compa-
rable with those reported earlier by Faletto et al. who
found 160 pmol of CBVTP/109 CEM cells.14 When
radiolabeled 3 was administered to CEM cell cultures
for 24 h, pronounced levels of alaninyl ABCMP (11) and
detectable levels of ABCMP, CBVMP, CBVDP, and
CBVTP were formed (Table 6). Interestingly, at 10 µM
Derivatives in HEL Cell Culturesa
EC50 (µM) for
HSV TK+
EC50 (µM) for
HSV-1 TK-
HSV-1
(KOS)
HSV-2
(G)
CC50
B2006
VMW-1837
(µM)
3
20
900
480
5
20
900
480
8
20
900
>800
50
20
320
480
13
110
200
160
15
ABC (1)
10
CBV (2)
a TK+ refers to thymidine kinase-competent virus. TK- refers
to thymidine kinase-deficient virus.
secondary/isopropyl (7) was slightly less potent and the
tert-butyl (8) least potent, being ca. 100-fold less potent
than the methyl parent compound (3). Against HBV, the
most potent ProTide is that derived from the unusual
achiral amino acid dimethylglycine (9), which was 295
times more potent than abacavir. The primary and
secondary esters of the L-alanine analogues have similar
activity and are approximately 10- to 15-fold more
potent than abacavir. The tert-butyl ester of L-alanine
has activity comparable to that of 1. Dimethylglycine
compound 9 is also very active against HIV, equivalent
to the most potent L-Ala esters. The potency of the
dimethylglycine analogue against HIV and HBV is
interesting and unexpected, given the poor activity of
the D-alanine analogues.
Table 5 contains a comparison of the two sets of
purified phosphate isomers, compared to the succinate
salts of the ∼1:1 diastereomeric mixtures (3a,b and
6a,b). In each case, the isomer that eluted from a
reverse-phase column with liquid carbon dioxide/
methanol with the shorter retention time proved to be
more potent against HIV-1 (4- and 9-fold). However,
against HBV the isomers were less differentiated. Since
the diastereomeric mixtures were equiactive (within
experimental deviation) with the more potent of the
purified isomers, we decided to pursue the mixtures. We
reasoned that the chirality at phosphorus is removed
Table 4. Antiviral Activity against HIV and HBV for a Series of Abacavir Phosphoramidatesa with Varying Amino Acids and Esters
EC50 (µM)
EC50 (µM)
CC50 (µM) EC50 (µM) HBV CC50 (µM)
CC50 (µM)
MT4
amino acid/ester HIV-1 CEM HIV-2 CEM
CEM
HepG2 2.2.15
HepG2
HIV1 MT4 HIV2 MT4
3a,b L-Ala/Me
0.050
0.070
0.48
3.7
0.050
0.061
0.85
6.0
13
12
17
0.55
0.35
0.33
5.0
17
18
0.20
0.20
0.48
6.6
0.26
0.21
0.60
3.4
4.6
8.6
5.2
2.1
6
L-Ala/Ethyl
L-Ala /i-Pr
L-Ala/t-Bu
L-Ala “X”
D-Ala/Me
Gly/Me
DiMeGly/Me
7
10
8
8.6
11
11
4
1.2
115
46
1.9
110
52
1.4
1.8
0.067
1.9
2.0
4.5
2
0.064
3.0
2.3
1.3
1.0
0.019
5.6
10.5
3.4
7.2
0.14
5.9
4.1
5.4
0.11
7.3
4.5
8.5
8.1
5
>56
>130
78
58
9
5.5
110
150
1
192
2
160
a All compounds are ∼1:1 mixtures of diastereomers (epimeric at phosphate). EC50: 50% effective concentration required to inhibit
HSV-induced cytopathicity by 50%. CC50: 50% cytostatic concentration required to inhibit HEL cell proliferation by 50%.

3508 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
McGuigan et al.
Scheme 2. Proposed Metabolic Activation Route for Abacavir Phosphoramididatesa
a (i) Esterase-type activity; (ii) spontaneous, probably via OPh displacement from the carboxylate (NGP); (iii) phosphoramidase. The
ABCMP thus formed is processed via CBVMP to the bioactive CBVTP.
Table 5. Comparison of Succinate Salts of Separated Isomers
to ∼1:1 Mixtures of Diastereomersa
HIV 1
CC50
HBV
EC50 (µM) (µM)
EC50 (µM)
CC50 (µM)
compd
MT4
MT4 HepG2 2.2.15 HepG2 2.2.15
3a,b (mixture)
3a (fast)
3b (slow)
6a,b (mixture)
6a (fast)
6b (slow)
1
0.037
0.027
0.11
4.3
1
0.55
0.4
17
10
35
18
4
1.5
0.022
0.0155
0.14
4.6
0.95
5
0.35
0.2
0.27
5.6
7.5
35
110
2.15
89
a EC50: 50% effective concentration required to inhibit HSV-
induced cytopathicity by 50%. CC50: 50% cytostatic concentration
required to inhibit HEL cell proliferation by 50%.
ProTide, as much as 3020 pmol of CBVTP/109 CEM cells
were found after 24 h of drug exposure, i.e., a 37-fold
higher CBV triphosphate concentration than observed
to appear in the presence of parental ABC. These
markedly increased levels of active metabolite in the
presence of ABC ProTide correspond well with a ∼30-
fold higher antiviral potency of the ProTide of ABC in
CEM cell cultures than ABC. However, the highest
metabolite levels were those afforded by the alaninyl
ABC-MP derivative (11) together with CBV-MP, which
were together 4-fold higher than those of CBV-TP. This
is in sharp contrast with the alaninyl phosphoramidate
d4T metabolite, which markedly accumulated (up to
100-fold) in aryloxymethylalaninyl d4TMP ProTide-
exposed cell cultures compared with the d4TTP levels.21
Thus, exposure of CEM cell cultures by the ProTide of
ABC resulted in the eventual appearance of markedly
Figure 1. Intracellular levels of carbovir triphospate (CBV-
TP) in HepG2 2.2.15 cells incubated with [8-3H]abacavir (ABC,
1) (10 µM, 2 mCi/µmol) or [8-3H]abacavir 5′-[phenyl(methoxy-
L-alaninyl)]phosphate (ABCpro, 3) (1 µM, 2 mCi/µmol) for up
to 48 h. Values are the mean ( standard deviation of triplicate
determinations at each time point.
higher intracellular CBV-TP levels, resulting in a more
pronounced antiviral efficacy of the ProTide against
HIV.
Metabolic profiles of [3H]ABC and its ProTide 3 in
HepG2 2.2.15 cells were qualitatively similar to those
observed in CEM cells, with CBV-TP being the pre-
dominant radiolabeled anabolite. Peak CBV-TP levels
in HepG2 2.2.15 cells incubated with [3H]ABC (10 µM)
or 3 (1 µM) were observed at 6-12 h (Figure 1).
Table 6. Metabolites of [3H]ABC and the Aryloxymethylalaninyl ABC-MP ProTide of [3H]ABC (3) in CEM Cell Cultures Exposed for
24 h to the Drugs
metabolites of ABC and proABC (pmol/109 cells)
extracellular
drug concn (10 µM)
ABC + proABC +
(CBV) fr 2-3
ABC +
(CBV) fr 2-3
ABCMP
fr 6-8
CBV-MP
fr 11-13
CBVMP +
alaninyl-ABCMP fr 11-13
CBVDP
fr 19-21
CBVTP
fr 35-38
1
3
762
5.8
280
108
42
3494
82
3020
5004
12410

Application of Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3509
Figure 2. Intracellular levels of carbovir triphosphate (CBV-
TP) in HepG2 2.2.15 cells incubated with various concentra-
tions of [8-3H]abacavir (ABC, 1) (0.1, 1, 10, and 100 µM; 2 mCi/
Figure 3. Plasma levels of abacavir 5′-[phenyl(ethoxy-L-
alaninyl)]phosphate (ABCpro, 6) and its metabolites, alaninyl
ABC-MP (11), abacavir monophospate (ABC-MP), abacavir
(ABC, 1), and carbovir (CBV, 2), in female cynomolgus
monkeys following intravenous administration of ABCpro
succinate salt (6 succinate) at 11.5 mg/kg (17.3 µmol/kg).
µmol)
or
[8-3H]abacavir
5′-[phenyl(methoxy-L-
alaninyl)]phosphate (ABCpro, 3) (0.01, 0.1, 1, and 10 µM; 2
mCi/µmol) for 6 h. Values are the mean ( standard deviation
of triplicate determinations at each concentration.
higher than those provided by an equivalent concentra-
tion of ABC. As seen in the time course experiment, the
difference in peak CBV-TP levels produced by 3 versus
ABC was greater than that predicted by its 10-fold
improvement in anti-HBV activity (Table 2). This dif-
ference, along with the time-dependent CBV-TP profiles
observed in the time course experiment (Figure 1),
suggests that the kinetics of CBV-TP formation and/or
clearance play a key role in determining in vitro potency
and ultimately in vivo efficacy of such a ProTide.
Pharmacokinetics and Oral Bioavailability in
Cynomolgus Monkeys. Following intravenous admin-
istration of its succinate salt (6 succinate) (11.5 mg/kg
or 17.3 µmol/kg) in cynomolgus monkeys, abacavir 5′-
[phenyl(ethoxy-L-alaninyl)]phosphate (6) was rapidly
cleared from plasma at a mean rate of 6.8 L h-1 kg-1
(∼2.6-fold greater than hepatic blood flow in monkey23)
with an elimination half-life of approximately 7 min
(Table 7). Systemic exposure to ProTide (6) in the
monkey was short-lived following either intravenous or
oral administration, being at or below the limit of
detection generally by 1-2 h postdose (Figures 3 and
4). Metabolism contributed significantly to the clearance
Although the extracellular drug concentrations in this
time course experiment were normalized relative to
their potencies in the HBV assay (i.e., 5.6 or 0.55 µM,
respectively; Table 2), the peak CBV-TP level produced
by the ProTide (3) was approximately 2-fold higher than
that produced by the parent compound (1). However,
after 24- and 48-h incubations, CBV-TP levels in
3-treated cells were below those in ABC-treated cells
(Figure 1). Relative amounts of CBV-MP, -DP, and -TP
produced by 3 and ABC at these time points were
comparable, suggesting that the difference in anabolic
efficiency over time may be attributable to a decrease
in cellular uptake of 3 and/or its anabolism up to CBV-
MP, but this could not be determined from these
analyses because of coelution of 3, ABC, and the
upstream anabolites, i.e., alaninyl-ABC-MP and ABC-
MP, in the chromatographic method used. In a separate
dose response experiment, anabolism of ProTide to CBV-
TP in HepG2 2.2.15 cells was shown to be quite efficient
compared to that of ABC (Figure 2). Over a 100-fold
drug concentration range (0.1-10 µM), peak CBV-TP
levels provided by 3 were approximately 30- to 80-fold
Table 7. Pharmacokinetic Parameter Value Estimates for Abacavir 5′-[Phenyl(ethoxy-L-alaninyl)]phosphate (ABCpro, 6) and Its
Metabolites, Alaninyl ABC-MP (11), Abacavir Monophospate (ABC-MP), Abacavir (ABC, 1), and Carbovir (CBV, 2), in Female
Cynomolgus Monkeys Following Intravenous or Oral Administration of ABVpro Succinate Salt (6 Succinate) at 11.5 mg/kg (17.3
µmol/kg)a
pharmacokinetic parameter valuesb
intravenous
oral
analyte
ABCpro
CLp (L h-1 kg-1
)
t1/2 (hr)
AUCIV (µM‚h)
Cmax (µM)
Tmax (h)
t1/2app (h)
AUCPO (µM‚h)
F (%)
22 ( 7c
6.8 ( 2.0
NAd
NA
NA
NA
0.11 ( 0.02
NA
2.7 ( 0.8
9.2 ( 1.4
0.4 ( 0.1
6.0 ( 2.9
0.7 ( 0.2
0.6 ( 0.4
1.8 ( 1.1
0.1 ( 0.1
1.0 ( 0.8
< 0.1
0.8 ( 0.3
1.3 ( 0.6
1.3 ( 0.6
3.0 ( 2.6
4.7 ( 1.2
0.4 ( 0.2c
0.8 ( 0.1
NCe
2.6 ( 1.0
6.0 ( 2.6
0.7 ( 0.4c
2.6 ( 0.1
NC
6.0 ( 4.8
0.4 ( 0.1
Ala-ABC-MP
ABC-MP
ABC
NA
NA
CBV
NA
total
19.1 ( 1.5
9.5 ( 3.2
50 ( 15
a Values are the mean ( standard deviation for n ) 3 except where noted. b CLp, total plasma clearance of an intravenous dose; t1/2
,
terminal elimination half-life following intravenous administration; t1/2app, apparent elimination half-life following oral administration;
AUC, area under the plasma concentration-time curve for an intravenous (iv) or oral (po) dose, extrapolated to infinity; Cmax, maximum
concentration observed after an oral dose; Tmax, time of Cmax; F, oral bioavailability. c Mean for n ) 2. Limited data precluded calculation
of these parameter values for one animal. d NA, not applicable. e NC, not calculable because of limited data for all three animals.

3510 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
McGuigan et al.
dimethylglycine emerged as a useful derivative, par-
ticularly in the anti-HBV assay.
Preliminary pharmacokinetic evaluation of one of the
simpler abacavir ProTides, the phenylethoxyalaninyl
phosphoramidate, in cynomolgus monkeys suggests that
further optimization of these prodrugs for application
to peripheral or systemic disease indications, e.g., HIV
therapy, is required. Thus far, our attempts to enhance
their metabolic stability, that is, their ability to survive
first-pass (gut and liver) metabolism, while maintaining
antiviral potency have indicated that this will be quite
challenging within this class of compounds. However,
sufficient delivery of intact ProTide to the liver may be
achievable, but further studies are needed to explore
this possibility.
Experimental Section
High-performance liquid chromatography (HPLC) was per-
formed on an SSODS2 reverse-phase column with an eluent
of water/acetonitrile. Method 1 involves the following param-
eters: 100% water (0 min), 20% water (35 min), 20% water
(45 min), 100% water (55 min), with a flow rate of 1 mL/min
Figure 4. Plasma levels of abacavir 5′-[phenyl(ethoxy-L-
alaninyl)]phosphate (ABCpro, 6) and its metabolites, alaninyl
ABC-MP (11), abacavir monophosphate (ABC-MP), abacavir
(ABC, 1), and carbovir (CBV, 2), in female Cynomolgus
monkeys following oral administration of ABCpro succinate
salt (6 succinate) at 11.5 mg/kg (17.3 µmol/kg).
and detection by UV at 254 nm. Standards are acetone (tR
)
4.54 min) and toluene (tR ) 10.21 min). Final product showed
purities greater than 99%, with undetectable amounts of the
parent nucleoside. For practical purposes, standard procedures
are given where applicable.
of ProTide (6). This metabolism produced several active
metabolites, albeit less potent than the ProTide itself
(Table 4). Alaninyl-ABC-MP (11) was the predominant
metabolite of intravenously administered ProTide, ac-
counting for approximately 48% of the total area under
the concentration-time curve (AUC), followed by ABC
(1), which accounted for approximately 31% (Table 7).
This order was reversed when the ProTide was admin-
istered orally, with ABC being the predominant me-
tabolite and accounting for 63% of the total AUC (Table
7). ABC was the predominant active species in the
plasma from 4 to 10 h after either dose (Figures 3 and
4). ABC-MP and CBV were also detected in plasma, but
exposure to these metabolites was low, and all analytes
were below detection at 24 h postdose. While exposure
to the orally administered ProTide itself was low, total
exposure to the ProTide and its active metabolites (9.5
( 3.2 µM‚h) (Table 7) approached that estimated for an
equivalent dose of ABC (16 µM‚h at 5 mg/kg or 17.5
µmol/kg ABC24). Perhaps even more noteworthy was the
prolonged half-life of ABC derived from orally admin-
istered ProTide (2.6 ( 1.0 h) compared to orally admin-
istered parent (1.1 h 24). These data are suggestive of a
delayed release of ABC from the ProTide, with metabo-
lism of the alaninyl-ABC-MP intermediate most likely
being the rate-limiting step.
In conclusion, we herein report the application of the
aryloxy phosphoramidate ProTide technology to the
carbocyclic nucleoside analogues carbovir and abacavir.
A significant (28- to 60-fold) increase in the in vitro anti-
HIV potency of abacavir was noted on parent ProTide
formation, with little enhancement in potency for car-
bovir. These data correlated well with observed en-
hancements in intracellular carbovir triphosphate levels
from the abacavir ProTide. By contrast, in the anti-HBV
assay, ProTide formation enhanced the potency of both
abacavir and carbovir.
Procedure A. Phenyl dichlorophosphate (1 mol equiv) and
the appropriate amino acid ester hydrochloride salt (1 mol
equiv) were suspended in anhydrous CH2Cl2 (30-60 mL).
Anhydrous triethylamine (2 mol equiv) in CH2Cl2 (30 mL) was
added dropwise at -80 °C, and the reaction was allowed to
warm to room temperature overnight. The solvent was re-
moved under reduced pressure and under nitrogen to give
white solids. This solid was suspended in Et2O (2 × 25 mL)
and filtered, and the filtrate was concentrated in vacuo to give
the products as crude oils. These were dissolved in anhydrous
THF and used without further purification.
Procedure B. The nucleoside (1 mol equiv) was dried by
azeotroping with anhydrous pyridine (3 × 5 mL) and then
suspended in anhydrous THF (5-30 mL). To the suspension
was added t-BuMgCl (1-2 mol equiv, 1.0 M solution in THF)
dropwise, and the resulting suspension was stirred for 10 min.
The phosphorochloridate (3 mol equiv, solution in THF) was
then added dropwise, and the resulting solution was stirred
at room temperature for 24-96 h. The reaction was quenched
by the addition of saturated aqueous ammonium chloride (0.1
mL), and after 10 min the solvent was removed under reduced
pressure. The crude product was purified by silica column
chromatography.
Phenyl(methoxy-L-alaninyl)
Phosphorochloridate
(Methyl N-[Chloro(phenoxy)phosphoryl]-L-alaninate).
This was synthesized according to procedure A, using L-alanine
methyl ester hydrochloride (2.0 g, 14.34 mmol), phenyl phos-
phorodichloridate (3.02 g, 2.14 mL, 14.34 mmol), and anhy-
drous triethylamine (2.90 g, 4.0 mL, 28.68 mmol). The product
1
(3.91 g, 98.2%) was isolated as a colorless crude oil. H NMR
(CDCl3): δ 7.39-7.34 (2H, m), 7.29-7.20 (3H, m), 4.49-4.37
(1H, q), 4.27-4.09 (1H, m), 3.78 (3H, two s), 1.52-1.49 (3H,
two d). 31P NMR (CDCl3): δ 9.28, 8.97 (1:1). 13C NMR
(CDCl3): δ 173.6, 150.1, 130.25 (2C), 126.4, 120.9 (2C), 53.2,
51.0, 20.9.
(-)-Carbovir 5′-[Phenyl(methoxy-L-alaninyl)]phos-
phate (Methyl N-[{[(1S,4R)-4-(2-Amino-6-oxo-1,6-dihy-
dro-9H-purin-9-yl)cyclopent-2-en-1-yl]methoxy}(phen-
oxy)phosphoryl]-L-alaninate) (10). (-)-Carbovir (300 mg,
1.21 mmol) was treated with t-BuMgCl (2.43 mL of a 1.0 M
solution in THF, 2.43 mmol) and phenyl(methoxy-L-alaninyl)
phosphorochloridate (5.32 mL of a 0.68 M solution in THF,
3.64 mmol) for 1 week according to procedure B to give after
purification by column chromatography (3-5% MeOH in
CHCl3) the desired product as a white solid (136.5 mg, 23%).
A small series of ester modified and amino acid
modified analogues of the lead abacavir ProTide were
prepared and studied, and initial SARs were estab-
lished. It is notable that the unusual, achiral amino acid

Application of Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3511
1H NMR(CDCl3): δ 7.67 (1H, two s), 7.37-7.30 (2H, m), 7.21-
7.14 (3H, m), 6.17-6.10 (1H, m), 5.97-5.94 (1H, m), 5.53-
5.48 (1H, m), 4.28-4.15 (2H, m), 4.00-3.87 (1H, m), 3.66 (3H,
two s), 3.18 (1H, d), 2.83-2.71 (1H, m), 1.82-1.66 (1H, m),
1.36-1.29 (3H, two d). 31P NMR (MeOH-d4): δ 5.18, 4.86 (1:
1). 13C NMR (CDCl3): δ 174.4, 158.5, 154.1, 151.7, 151.1, 136.9,
136.5, 130.7, 129.7 (2C), 125.0, 120.4 (2C), 116.8, 68.9, 59.8,
51.7, 50.5, 46.0, 34.2, 19.3. MS: m/z 489 (M + H). FAB: for
C21H26O6N6P, requires 489.165 146, found 489.164 677.
HPLC: tR ) 22.13, 22.51 (100%), method 1.
7.36 (3H, becomes 2H on D2O exchange), 7.20 (3H, m), 5.9-
6.1 (3H, m), 5.88 (2H, bs, D2O exchangeable), 5.44 (1H, m),
4.0-4.2 (2H, m), 3.85 (1H, m), 3.60 (3H, s), 3.05 (2H, m), 2.65
(1H, m), 2.44 (4H, s), 1.64 (1H, m), 1.23 (3H, m), 0.5-0.7 (4H,
m). 31P NMR (DMSO-d6): δ 3.99 and 3.66. Anal. (C24H30N7O5P‚
C4H6O4‚0.5H2O) C, H, N.
Phenyl(methoxy-D-alaninyl)phosphorochloridate
(Methyl N-[Chloro(phenoxy)phosphoryl]-D-alaninate).
This was synthesized according to procedure A, using D-alanine
methyl ester hydrochloride (1.0 g, 7.17 mol), phenyl phospho-
rodichloridate (1.51 g, 1.07 mL, 7.17 mmol), and anhydrous
triethylamine (1.45 g, 2.0 mL, 14.0 mmol) to yield 1.66 g
(83.4%) of product. 1H NMR (CDCl3): δ 7.39-7.30 (2H, t), 7.29-
7.09 (3H, m), 4.85-4.80 (1H, d), 4.19-4.11 (1H, m), 3.75 (3H,
two s), 1.52-1.49 (3H, two d). 31P NMR (CDCl3): δ 9.38, 9.18
(1:1). 13C NMR (CDCl3): δ 173.6, 150.1, 130.3 (2C), 126.4 (2C),
120.9, 53.2, 50.9, 21.0.
Abacavir 5′-[Phenyl(methoxy-L-alaninyl)]phosphate
(Methyl N-[({(1S,4R)-4-[2-Amino-6-(cyclopropylamino)-
9H-purin-9-yl]cyclopent-2-en-1-yl}methoxy)(phenoxy)-
phosphoryl]-L-alaninate) (3a,b). Separation of Isomers.
This was synthesized according to procedure B, using abacavir
(500 mg, 1.75 mmol), t-BuMgCl (1.75 mL of 1.0 M solution in
THF, 1.75 mmol), and phenyl(methoxy-L-alaninyl) phospho-
rochloridate (11.17 mL of 0.47 M solution in THF, 5.24 mmol)
in THF (30 mL) and stirring at room temperature for 70 h.
The crude product was purified by column chromatography
(3% MeOH in CH2Cl2 and then 2% MeOH in CH2Cl2) to give
3 as a white foam after trituration with dichloromethane (442
Abacavir 5′-[Phenyl(methoxy-D-alaninyl)]phosphate
(Methyl N-[({(1S,4R)-4-[2-Amino-6-(cyclopropylamino)-
9H-purin-9-yl]cyclopent-2-en-1-yl}methoxy)(phenoxy)-
phosphoryl]-D-alaninate) (4). This was synthesized accord-
ing to procedure B, using abacavir (400 mg, 1.4 mmol),
t-BuMgCl (2.1 mL of 1.0 M solution in THF, 2.1 mmol), and
phenyl(methoxy-D-alaninyl) phosphorochloridate (7.0 mL of 0.6
M solution in THF, 4.19 mmol) in THF (25 mL) stirring at
room temperature for 36 h. The crude product was purified
by column chromatography (3% MeOH in CHCl3 and then
2.5% MeOH in CHCl3) to give 4 as a white foam (318.6 mg,
1
mg, 48%). H NMR (CDCl3): δ 7.5 (1H, two s), 7.1-7.4 (5H,
m), 6.1 (1H, m), 5.9 (2H, m), 5.5-5.6 (1H, m), 4.9 (2H, bs), 4.2
(2H, m), 4.05 (1H, m), 3.7 (3H, s), 3.6-3.8 (1H, m), 3.17 (1H,
m), 3.0 (1H, m), 2.8 (1H, m), 1.7 (1H, m), 1.4 (3H, two d), 0.9
(2H, m), 0.6 (2H, m). 31P NMR (CDCl3): δ 3.07, 3.02. 31P NMR
(MeOH-d4): δ 3.97, 3.88. 13C NMR (CDCl3): δ 174.6, 160.3,
156.6, 151.3, 151.1, 136.8, 135.9, 131.5, 130.0 (2C), 125.2, 120.5
(2C), 115.0, 69.2, 59.2, 52.8, 50.5, 46.0, 34.9, 24.2, 21.2, 7.7
(2C). MS: m/z 528 (M + H). MS FAB: C24H31O5N7P, requires
528.214 231, found 528.213 848. HPLC: tR ) 30.33 (100%),
method 1. IR: 3328.6 (N-H str), 2922.1, 2862.9 (C-H str),
1734.4 (CdO str), 1590.9 (aromatic C-C str), 1462.9 (C-H
def), 1376.8 (-CH3 sym def), 1207.1 (P-O-aryl), 1154.0 (C-O
str), 1027.7 (P-O-alkyl), 933.4 (olefinic C-H def), 721.8
(monosub aromatic C-H def). Anal. (C24H30O5N7P‚0.4CH2Cl2)
C, H, N.
1
43.2%). H NMR (CDCl3): δ 7.53 (1H, two s), 7.37-7.32 (2H,
m), 7.29 (1H, d), 7.25-7.15 (2H, m), 6.10 (1H, t, J ) 5.28 Hz),
6.03 (1H, bs, NHcPr), 5.94-5.89 (1H, m), 5.54 (1H, m), 5.01
(2H, bs, NH2), 4.26-3.83 (4H, m), 3.72 (3H, two s), 3.18 (1H,
s), 3.02 (1H, bs), 2.86-2.75 (1H, m), 1.78-1.64 (1H, m), 1.39-
1.36 (3H, two d), 0.90-0.83 (2H, q, J ) 6.13 Hz), 0.63 (2H,
bs). 31P NMR (CDCl3): δ 3.93, 3.70. 13C NMR (CDCl3): δ 174.5,
160.3, 156.6, 151.2, 151.0, 136.8, 136.1, 131.5, 130.0 (2C),
125.3, 120.5 (2C), 115.2, 69.3, 59.3, 52.9, 50.5, 46.0, 34.9, 24.1,
21.4, 7.8 (2C). MS: m/z 528 (M + H). MS FAB: for C24H31O5N7P
requires 528.212 431, found 528.211 505. HPLC: tR ) 29.807
(100%), method 1. IR: 3333.6 (N-H str), 2923.4, 2853.4 (C-H
str), 1734.1 (CdO str), 1591.1 (aromatic C-C str), 1458.3 (C-H
def), 1376.7 (-CH3 sym def), 1208.3 (P-O-aryl), 1153.3 (C-O
str), 1026.9 (P-O-alkyl), 931.9 (olefinic C-H def), 721.6
(monosub aromatic C-H def). Anal. (C24H30O5N7P) C, H, N.
The phosphate isomers were separated with supercritical
fluid chromatography using a Chiralpak AS column and 25%
methanol in carbon dioxide as the eluent. The first isomer to
elute with a tR of 2.9 min from a Chiralpak AS column (25%
methanol in carbon dioxide, flow rate of 2 mL/min, tempera-
ture of 40 °C, 3000 psi, enantiopure) and upon evaporation of
solvents gave the isomer 3a as a white foam after trituration
with chloroform (recovery of >95%). 1H NMR (CDCl3): δ 7.50
(1H, s), 7.3-7.4 (2H, m), 7.15-7.25 (3H, m), 6.11 (1H, m), 5.91
(1H, m), 5.86 (1H, s), 5.55 (1H, m), 4.89 (2H, s), 4.24 (2H, m),
4.05 (1H, m), 3.72 (3H, s), 3.65 (1H, m), 3.20 (1H, m), 3.02
(1H, m), 2.83 (1H, m), 1.72 (1H, m), 1.37 (3H, d), 0.89 (2H, m),
0.62 (2H, m). 31P NMR (CDCl3): δ 3.07. Anal. (C24H30N7O5P‚
0.14CHCl3) C, H, N.
The second isomer to elute with a tR of 6.7 min from a
Chiralpak AS column (25% methanol in carbon dioxide, flow
rate of 2 mL/min, temperature of 40 °C, 3000 psi, enantiopure)
and upon evaporation of solvents gave the isomer 3b as a white
foam after trituration with chloroform. 1H NMR (CDCl3): δ
7.52 (1H, s), 7.25-7.4 (2H, m), 7.15-7.22 (3H, m), 6.11 (1H,
m), 5.94 (1H, m), 5.85 (1H, s), 5.55 (1H, m), 4.88 (2H, s), 4.22
(2H, m), 4.04 (1H, m), 3.75 (3H, s), 3.7-3.75 (1H, m), 3.17 (1H,
m), 3.04 (1H, m), 2.80 (1H, m), 1.73 (1H, m), 1.42 (3H, d), 0.89
(2H, m), 0.67 (2H, m). 31P NMR (CDCl3): δ 3.0. Anal.
(C24H30N7O5P‚0.2CHCl3) C, H, N.
Abacavir 5′-[Phenyl(methoxy-L-alaninyl)]phosphate
Succinate Salt (3 Succinate). Abacavir 5′-[phenyl(methoxy-
L-alaninyl)]phosphate (1:1 mixture of isomers of 3a,b, 100 mg,
0.19 mmol) was dissolved in methanol. To this solution was
added succinic acid (22 mg, 0.19 mmol), and the resulting
solution was evaporated to dryness. The residue was dissolved
in acetonitrile (10 mL) with heating. Precipitate formed upon
cooling. The mixture was stored in the refrigerator overnight
and the solid was collected by filtration to give 70 mg (57%) of
a 1:1 mixture of 3 succinate as a crystalline solid. 1H NMR
(DMSO-d6): δ 12.15 (2H, s, D2O exchangeable), 7.61 (1H, s),
Phenyl(methoxyglycinyl) Phosphorochloridate (Meth-
yl N-[Chloro(phenoxy)phosphoryl]glycinate). This was
synthesized according to procedure A, using glycine methyl
ester (1.5 g, 11.9 mmol), phenyl phosphorodichloridate (2.52
g, 1.79 mL, 11.9 mmol), and anhydrous triethylamine (2.42 g,
3.33 mL, 23.9 mmol) to yield 3.07 g (97.15%) of the product as
an oil. 1H NMR (CDCl3): δ 7.43-7.38 (2H, m), 7.31-7.25 (3H,
m), 4.67 (1H, bs, NHala), 3.94 (2H, dd), 3.83 (3H, s). 31P NMR
(CDCl3): δ 10.43. 13C NMR (CDCl3): δ 170.4, 150.1, 130.2 (2C),
126.4, 120.8 (2C), 53.1, 43.4.
Abacavir5′-[Phenyl(methoxyglycinyl)]phosphate(Meth-
yl N-[({(1S,4R)-4-[2-Amino-6-(cyclopropylamino)-9H-pu-
rin-9-yl]cyclopent-2-en-1-yl}methoxy)(phenoxy)-
phosphoryl]glycinate) (5). This was synthesized according
to procedure B, using abacavir (300 mg, 1.05 mmol), t-BuMgCl
(1.57 mL of 1.0 M solution in THF, 1.57 mmol), and phenyl-
(methoxyglycinyl) phosphorochloridate (4.06 mL of 0.774 M
solution in THF, 3.14 mmol) in THF (20 mL) stirring at room
temperature for 96 h. The crude product was purified by
column chromatography (3% MeOH in CHCl3 and then with
2.5% MeOH in CHCl3) to give 5 as a white foam (82.6 mg,
1
15.4%). H NMR (CDCl3): δ 7.38 (1H, two s), 7.24-7.19 (2H,
t), 7.15-7.10 (2H, t), 7.07-7.02 (1H, t), 6.00-5.96 (2H, m),
5.80-5.76 (1H, m), 5.45-5.41 (1H, t), 4.99 (2H, bs, NH2), 4.14-
4.00 (3H, m), 3.62 (3H, s), 3.03 (1H, d), 2.91 (1H, d), 2.73-
2.62 (1H, m), 1.62-1.51 (1H, m), 1.45-1.43 (2H, m), 0.78-
0.71 (2H, q), 0.54-0.49 (2H, t). 31P NMR (CDCl3): δ 4.79, 4.67
(1:1). 13C NMR(CDCl3): δ 172.1, 160.2, 156.6, 152.0, 151.7,
137.7, 137.1, 132.0, 130.8 (2C), 126.0, 121.2 (2C), 115.5, 69.9,
60.0, 53.5, 46.7, 43.9, 35.4, 25.0, 8.5 (2C). MS: m/z 514 (M +

3512 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
McGuigan et al.
H). MS FAB: C23H29O5N7P requires 514.196 781, found
514.195 321. HPLC: tR ) 28.419 (99.9%), method 1. IR: 3342.0
(N-H str), 1749.8 (CdO str), 1596.2, 1488.4 (aromatic C-C
str), 1451.9 (C-H def), 1394.7 (-CH3 sym def), 1259.6 (PdO),
1212.1 (P-O-aryl), 1151.6 (C-O str), 1026.8 (P-O-alkyl),
937.8 (olefinic C-H def), 760.7 (monosub aromatic C-H def).
(3H, m), 1.15 (3H, t), 0.5-0.7 (4H, m). 31P NMR (DMSO-d6):
δ 4.00 and 3.67. Anal. (C25H32N7O5P‚C4H4O4‚0.5H2O) C, H, N.
Abacavir 5′-[Phenyl(ethoxy-L-alaninyl)]phosphate Gl-
utarate Salt (6 Glutarate). Abacavir 5′-[phenyl(ethoxy-L-
alaninyl)]phosphate (1:1 mixture of isomers of 6, 200 mg, 0.38
mmol) was dissolved in ethanol. To this solution was added
glutaric acid (50 mg, 0.38 mmol), and the resulting solution
was evaporated to dryness. The residue was dissolved in
acetonitrile (10 mL) with heating. The mixture was stored in
the refrigerator overnight and the solid was collected by
filtration to give 130 mg (50%) of a 1:1 mixture of 6 glutarate
as a crystalline solid. 1H NMR (DMSO-d6): δ 7.6 (1H, s), 7.1-
7.4 (6H, m), 5.9-6.1 (3H, m), 5.87 (2H, bs), 5.44 (1H, m), 3.95-
4.2 (4H, m), 3.8 (1H, m), 3.1 (m, 2H), 2.65 (1H, m), 2.25 (4H,
Phenyl(ethoxy-L-alaninyl) Phosphorochloridate (Eth-
yl N-[Chloro(phenoxy)phosphoryl]-L-alaninate). This was
synthesized according to procedure A, using L-alanine ethyl
ester hydrochloride (1.0 g, 6.51 mmol), phenyl phosphorod-
ichloridate (1.37 g, 0.97 mL, 6.51 mmol), and anhydrous
triethylamine (1.32 g, 1.81 mL, 13.0 mmol) to yield 1.85 g
1
(97.4%) of the desired product as an oil. H NMR(CDCl3): δ
7.42-7.35 (2H, dd), 7.31-7.25 (3H, m), 4.71 (1H, d, NHala),
4.31-4.13 (3H, m), 1.55-1.52 (3H, m), 1.33-1.30 (3H, two d).
31P NMR (CDCl3): δ 9.41, 9.16 (1:1). 13C NMR: δ 173.1, 150.2,
130.3 (2C), 126.4, 120.9 (2C), 62.3, 51.0, 20.9, 14.5.
t), 1.7 (3H, m), 1.23 (3H, m), 1.15 (3H, t), 0.5-0.7 (4H, m). 31
P
NMR (DMSO-d6): δ 4.00 and 3.68. Anal. (C25H32N7O5P‚
C5H8O4‚0.5H2O) C, H, N.
Abacavir 5′-[Phenyl(ethoxy-L-alaninyl)]phosphate Suc-
cinate Salt (6 Succinate). Separation of Isomers and
Preparation of Succinates (6a,b Succinate). For scaling
up of abacavir 5′-[phenyl(ethoxy-L-alaninyl)]phosphate succi-
nate salt a modification of procedure B was used. Thus, to
abacavir (15 g, 52 mmol) was added anhydrous pyridine (200
mL). Subsequently, t-BuMgCl (60 mL, 1 M solution in THF,
60 mmol) was added slowly. The reaction mixture was stirred
for 20 min, and a solution of phenylethoxy-L-alaninyl phos-
phorochloridate (31 g, 107 mmol in 100 mL of THF, synthe-
sized according to procedure A) was added. The reaction
mixture was stirred at room temperature for 10 h and
subsequently concentrated in vacuo to a brown syrup. This
syrup was dissolved in CH2Cl2 (100 mL), and the CH2Cl2 was
extracted with water (2 × 100 mL) and saturated aqueous
sodium bicarbonate, dried (MgSO4), filtered, and concentrated
to a brown foam. This solid foam was purified by filtration
through a silica gel plug (100 g, silica, 5% methanol in
chloroform) to give 18.5 g (67%) of 6 as a white foam. This
foam (6, 18.5 g, 34 mmol) was dissolved in ethanol (150 mL).
To this was added succinic acid (4.0 g, 34 mmol), and the
resulting solution was stirred for 30 min, then concentrated
to dryness. The resulting solid was dissolved in refluxing
acetonitrile (100 mL), then allowed to crystallize upon cooling
to give 18.8 g (84%) of 6 succinate as a crystalline solid.
The phosphate isomers of 6 were separated with supercriti-
cal fluid chromatography using a Chiralpak AS column and
22% methanol in carbon dioxide as the eluent. The first isomer
to elute with a tR of 3.2 min from a Chiralpak AS column (25%
methanol in carbon dioxide, flow rate of 2 mL/min, tempera-
ture of 40 °C, 3000 psi, enantiopure) and upon evaporation of
solvents gave the isomer (6a) as a white foam (recovery of
>95%). Treatment of this foam with succinic acid as described
above gave the enantiopure faster-eluting isomer as a crystal-
line succinate salt (6a succinate). 1H NMR (DMSO-d6): δ 12.14
(2H, s), 7.57 (1H, s), 7.1-7.4 (6H, m), 5.9-6.1 (3H, m), 5.85
(2H, bs), 5.42 (1H, m), 3.95-4.15 (4H, m), 3.8 (1H, m), 3.05
(2H, m), 2.65 (1H, m), 2.4 (4H, s), 1.63 (1H, m), 1.4 (3H, two
d), 1.12 (3H, t), 0.5-0.7 (4H, m). 31P NMR (DMSO-d6): δ 4.00.
Anal. (C25H32N7O5P‚C4H6O4‚0.5H2O) C, H, N.
The second isomer to elute with a tR of 7.4 min from a
Chiralpak AS column (22% methanol in carbon dioxide, flow
rate of 2 mL/min, temperature of 40 °C, 3000 psi, enantiopure)
and upon evaporation of solvents gave the isomer 6b as a white
foam. Treatment of this foam with succinic acid as described
above gave the enantiopure slower-eluting isomer as a crystal-
line succinate salt (6b succinate). 1H NMR (DMSO-d6): δ 12.14
(2H, s), 7.58 (1H, s), 7.1-7.4 (6H, m), 5.9-6.1 (3H, m), 5.85
(2H, bs), 5.42 (1H, m), 3.95-4.15 (4H, m), 3.8 (1H, m), 3.05
(2H, m), 2.65 (1H, m), 2.4 (4H, s), 1.63 (1H, m), 1.4 (3H, two
d), 1.12 (3H, t), 0.5-0.7 (4H, m). 31P NMR (DMSO-d6): δ 3.68.
Anal. (C25H32N7O5P‚C4H6O4) C, H, N.
Phenyl(isopropoxy-L-alaninyl) Phosphorochloridate
(Isopropyl N-[Chloro(phenoxy)phosphoryl]-L-alaninate).
This was synthesized according to procedure A, using L-alanine
isopropyl ester hydrochloride salt (0.5 g, 2.98 mmol), phenyl
phosphorodichloridate (0.45 mL, 2.98 mmol), and triethyl-
Abacavir 5′-[Phenyl(ethoxy-L-alaninyl)]phosphate (Eth-
yl N-[({(1S,4R)-4-[2-Amino-6-(cyclopropylamino)-9H-pu-
rin-9-yl]cyclopent-2-en-1-yl}methoxy)(phenoxy)phos-
phoryl]-L-alaninate) (6). This was synthesized according to
procedure B, using abacavir (300 mg, 1.4 mmol), t-BuMgCl
(1.57 mL of 1.0 M solution in THF, 1.57 mmol), and phenyl-
(ethoxy-L-alaninyl) phosphorochloridate (6.45 mL of 0.49 M
solution in THF, 3.14 mmol) in anhydrous THF (20 mL) and
stirring at room temperature for 24 h. The crude product was
purified by column chromatography (2.5% MeOH in CHCl3)
to give 6 as a pale-yellow foam (290 mg, 51.1%). 1H NMR
(CDCl3): δ 7.47 (1H, two s), 7.10-7.46 (5H, m), 6.07 (1H, m),
5.9 (1H, m), 5.78 (1H, s), 5.5 (1H, m), 4.84 (2H, bs), 4.1 (4H,
m), 4.00 (1H, m), 3.64 (1H, m), 3.14 (1H, m), 3.0 (1H, m), 2.78
(1H, m), 1.68 (1H, m), 1.36 (3H, two d), 1.22 (3H, two t), 0.86
(2H, m), 0.6 (2H, m). 31P NMR (CDCl3): δ 3.05, 3.02. 31P
NMR: δ 4.04, 3.96 (1:1). 13C NMR: δ 173.35, 159.8, 156.0,
150.6, 150.4, 136.1, 135.1, 130.8, 129.3 (2C), 124.5, 119.8 (2C),
114.4, 68.6, 61.2, 58.5, 50.0, 45.3, 34.3, 23.4, 20.6, 13.8, 7.0
(2C). MS: m/z 542 (M + H). MS FAB: C25H33O5N7P, requires
542.228 081, found 542.228 131. HPLC: tR ) 31.76, 32.03
(100%), method 1. IR: 3334.1 (N-H str), 1734.5 (CdO str),
1595.9, 1488.0 (aromatic C-C str), 1450.3 (C-H def), 1394.2
(-CH3 sym def), 1252.8 (PdO), 1210.4 (P-O-aryl), 1153.3
(C-O str), 1026.0 (P-O-alkyl), 934.8 (olefinic C-H def), 759.0
(monosub aromatic C-H def). Anal. (C25H32O5N7P‚0.25CH2-
Cl2) C, H, N.
Abacavir 5′-[Phenyl(ethoxy-L-alaninyl)]phosphate Suc-
cinate Salt (6 Succinate). Abacavir 5′-[phenyl(ethoxy-L-
alaninyl)]phosphate (1:1 mixture of isomers of 6, 376 mg, 0.7
mmol) was dissolved in ethanol. To this solution was added
succinic acid (82 mg, 0.7 mmol), and the resulting solution was
evaporated to dryness. The residue was dissolved in acetoni-
trile (10-20 mL) with heating. Precipitate formed upon
cooling. The mixture was stored in the refrigerator overnight
and the solid was collected by filtration to give 330 mg (72%)
of a 1:1 mixture of 6 succinate as a crystalline solid. 1H NMR
(DMSO-d6): δ 12.14 (2H, s), 7.58 (1H, s), 7.1-7.4 (6H, m), 5.9-
6.1 (3H, m), 5.85 (2H, bs), 5.42 (1H, m), 3.95-4.15 (4H, m),
3.8 (1H, m), 3.05 (2H, m), 2.65 (1H, m), 2.4 (4H, s), 1.63 (1H,
m), 1.4 (3H, two d), 1.12 (3H, t), 0.5-0.7 (4H, m). 31P NMR
(DMSO-d6): δ 4.00 and 3.68. FAB MS: calcd for C25H32N7O5P
(M + H)+ (m/z) 542.2281, found 542.2282. Anal. (C25H32N7O5P‚
C4H6O4‚0.5H2O) C, H, N.
Abacavir 5′-[Phenyl(ethoxy-L-alaninyl)]phosphate Fu-
marate Salt (6 Fumarate). Abacavir 5′-[phenyl(ethoxy-L-
alaninyl)]phosphate (1:1 mixture of isomers of 6, 198 mg, 0.37
mmol) was dissolved in ethanol. To this solution was added
fumaric acid (43 mg, 0.37 mmol), and the resulting solution
was evaporated to dryness. The residue was dissolved in
acetonitrile (10 mL) with heating. Precipitate formed upon
cooling. The mixture was stored in the refrigerator overnight
and the solid was collected by filtration to give 185 mg (75%)
of a 1:1 mixture of 6 fumarate as a crystalline solid. 1H NMR
(DMSO-d6): δ 7.6 (1H, s), 7.1-7.4 (6H, m), 6.64 (2H, s), 5.9-
6.1 (3H, m), 5.87 (2H, bs), 5.44 (1H, m), 3.95-4.15 (4H, m),
3.84 (1H, m), 3.05 (2H, m), 2.65 (1H, m), 1.63 (1H, m), 1.23

Application of Phosphoramidate ProTide Technology
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3513
amine (0.83 mL 5.97 mmol) in CH2Cl2 (70 mL) to yield the
product (1.12 g, quant,) as a yellow oil. 31P NMR (CDCl3): δ
9.45, 9.17 (1:1). 13C NMR (CDCl3): δ 172.6, 150.2, 130.3 (2C),
126.4, 121.0 (2C), 70.1, 51.1, 22.1, 20.9.
7.10 (2H, t), 7.07-7.02 (1H, t), 6.00-5.96 (2H, m), 5.80-5.76
(1H), 5.45-5.41 (1H, t), 4.99 (2H, bs, NH2), 4.14-4.00 (3H,
m), 3.62 (3H, s), 3.03 (1H, d), 2.91 (1H, d), 2.73-2.62 (1H, m),
1.62-1.51 (1H, m, 1 of H6′), 1.45-1.43 (6H, t, 2 × CH3), 0.78-
0.71 (2H, q, 2H of CH2cPr), 0.54-0.49 (2H). 31P NMR
(CDCl3): δ 2.49. MS ES+: m/z 541.9 (100%, M+), 563.8 (30%,
M + Na+). MS FAB: for C25H33O5N7P requires 542.228 081,
found 542.228 428. HPLC: tR ) 28.347 (100%), method 1. IR:
3346.0 (N-H str), 2923.0, 2853.5 (C-H str), 1734.0 (CdO str),
1590.2 (aromatic C-C str), 1458.4 (C-H def), 1376.8 (-CH3
sym def), 1261.3 (PdO), 1152.7 (C-O str), 1028.0 (P-O-alkyl),
936.0 (olefinic C-H def), 721.7 (monosub aromatic C-H def).
Abacavir 5′-Phenyl(isopropoxy-L-alaninyl)phosphate
(Isopropyl N-[({(1S,4R)-4-[2-Amino-6-(cyclopropylamino)-
9H-purin-9-yl]cyclopent-2-en-1-yl}methoxy)(phenoxy)-
phosphoryl]-L-alaninate) (7). This was synthesized accord-
ing to procedure A, using abacavir (100 mg, 0.35 mmol),
t-BuMgCl (0.7 mL of a 1.0 M solution in THF, 0.7 mmol,) in
THF (3 mL), and phenyl(isopropyl-L-alaninyl) phosphorochlo-
ridate (1.76 mL of a 0.597 M solution in THF, 1.05 mmol) at
room temperature for 72 h. The crude product was purified
by column chromatography (3% MeOH in CHCl3) to give 7
(106.8 mg, 54.8%) as a pale-yellow foamy solid. 1H NMR
(CDCl3): δ 7.41 (1H, two s), 7.24-7.19 (2H, m), 7.13-7.03 (3H,
m), 6.37 (1H, bs, NHcPr), 5.98 (1H, t), 5.80-5.76 (1H, m), 5.43
(1H, bs), 5.21 (2H, bs, NH2), 4.94-4.86 (1H, m), 4.15-3.98 (2H,
m), 3.92-3.83 (1H, m), 3.59 (1H, bs, NHala), 3.06-2.98 (1H,
m), 2.93 (1H, bs), 2.74-2.63 (1H, m), 1.62-1.53 (1H, m), 1.34-
1.18 (3H, m), 1.15-1.11 (6H, m), 0.79-0.73 (2H, q), 0.53 (2H,
m). 31P NMR (CDCl3): δ 4.02, 3.98 (1:1). 13C NMR: δ 173.5,
159.8, 156.2, 151.1, 151.0, 136.9, 136.1, 131.3, 130.0 (2C),
125.3, 120.5 (2C), 115.0, 69.6, 69.2, 59.3, 50.7, 46.0, 34.9, 24.2,
22.0 (2C), 21.4, 7.8 (2C). MS: m/z 557 (M + H). MS MALD/I
Abacavir 5′-(L-Alaninyl)phosphate Disodium Salt. (N-
[({(1S,4R)-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-
yl]cyclopent-2-en-1-yl}methoxy)phosphoryl]-D-alani-
nate Disodium Salt) (11). Abacavir 5′-[phenyl(methoxy-L-
alaninyl)]phosphate (1:1 mixture of isomers of 3a and 3b, 500
mg, 0.95 mmol) was suspended in a solution of triethylamine
(30 mL) and deionized water (30 mL) and stirred at room
temperature for 18 h. The resulting solution was evaporated
to dryness in vacuo, and the residue was dissolved in water
(50 mL), extracted with dichloromethane (2 × 50 mL), and
purified by anion exchange chromatography using a Sep-Pak
Vac 35 cm3 Accell Plus QMA cartridge (Waters Corp., P/N
-
WAT054725) (HCO3 form) with an aqueous ammonium
TOF: found 555.6, C26H34O5N7P requires 555.2. HPLC: tR
35.85 (100%), method 1.
)
bicarbonate buffer (0-0.5 M linear gradient, 1 L). The ap-
propriate fractions were combined and evaporated to dryness
in vacuo. The residue was twice dissolved in deionized water
and evaporated to dryness in vacuo to give 11 as the am-
monium salt. This salt was dissolved in deionized water and
passed through a Sep-Pak Vac 20 cm3 Accell Plus CM cartridge
(Waters Corp., P/N WAT054675) (Na+ form) using deionized
water. The appropriate fractions were combined and lyophi-
lized to give 0.430 g (86% yield) of 11 disodium salt 2.5 hydrate
as a white solid. 1H NMR (D2O): δ 7.71 (1H, s), 6.11 (1H, m),
5.80 (1H, m), 5.32 (1H, m), 3.66 (2H, m), 3.41 (1H, m), 2.99
(1H, m), 2.68 (2H, m), 1.49 (1H, m), 1.12 (3H, d), 0.74 (2H, m),
0.54 (2H, m). 31P NMR (D2O): δ 8.68. MS (ES-) m/z 436 (M -
H). Anal. (C17H22N7Na2O5P‚2.5H2O) C, H, N.
Phenyl-tert-butyloxy-L-alaninylphosphorochlori-
date (tert-Butyl N-[Chloro(phenoxy)phosphoryl]-L-alan-
inate). This was synthesized according to procedure A, using
L-alanine tert-butyl ester hydrochloride (0.5 g, 2.75 mmol),
phenyl phosphorodichloridate (0.41 mL, 2.75 mmol), and
triethylamine (0.77 mL, 5.5 mmol) to yield 0.77 g (87.5%) of
product. 1H NMR(CDCl3): δ 7.44-7.39 (2H, t), 7.32-7.26 (3H,
m), 4.47-4.34 (1H, m, NHala), 4.17-4.04 (1H, m), 1.53 (9H,
s). 31P NMR (CDCl3): δ 9.53, 9.20 (1:1). 13C NMR (CDCl3): δ
170.7, 148.7, 128.9 (2C), 124.9, 119.5 (2C), 81.65, 50.0, 26.9
(3C).
Abacavir 5′-(Phenyl-tert-butyloxy-L-alaninyl)phosphate
(tert-Butyl N-[({(1S,4R)-4-[2-Amino-6-(cyclopropylamino)-
9H-purin-9-yl]cyclopent-2-en-1-yl}methoxy)(phenoxy)-
phosphoryl]-L-alaninate) (8). This was synthesized accord-
ing to procedure B, using abacavir (140 mg, 0.52 mmol),
t-BuMgCl (1.05 mL of a 1.0 M solution in THF, 1.05 mmol),
and phenyl(tert-butyloxy-L-alaninyl) phosphorochloridate (3.3
mL of a 0.48 M solution in THF, 1.57 mmol) in anhydrous THF
(4 mL) stirring at room temperature for 48 h. The crude
product was purified by column chromatography (3% MeOH
in CHCl3) to give 8 (192.3 mg, 69.0%) as a white foam. 1H NMR
(CDCl3): δ 7.40 (1H, two s), 7.23-7.18 (2H, t), 7.12 (2H, d),
7.06-7.02 (1H, t), 6.09 (1H, m), 5.97 (1H, m), 5.77 (1H, d,
NHcPr), 5.44 (1H, m), 5.10 (2H, bs, NH2), 4.14-4.05 (3H, m),
3.85-3.77 (1H, q), 3.04 (1H, bs), 2.93 (1H, m), 2.72-2.62 (1H,
m), 1.58-1.53 (1H, t), 1.34 (9H, two s), 1.27-1.23 (3H, t), 0.73
(2H, d), 0.51 (2H, m). 31P NMR (CDCl3): δ 4.15 (s). 13C NMR
(CDCl3): δ 173.2, 160.4, 156.7, 151.2 (2C), 136.8, 135.9, 131.5,
130.0 (2C), 125.2, 120.6 (2C), 115.2, 82.3, 69.3, 59.1, 46.0, 35.0,
28.3 (3C), 24.2, 21.5, 7.8 (2C). MS: m/z 571 (M + H). MS FAB:
For C27H37O5N7P requires 570.2594, found 570.2598. HPLC:
tR ) 36.158 (100%), method 1. Anal. (C27H36O5N7P‚0.33H2O)
C, H, N.
Abacavir 5′-(L-Alaninyl)phosphate Disodium Salt (N-
[({(1S,4R)-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-
yl]cyclopent-2-en-1-yl}methoxy)phosphoryl]-D-alani-
nate Disodium Salt) (11). Abacavir 5′-[phenyl(methoxy-L-
alaninyl)]phosphate (1:1 mixture of isomers of 3a and 3b, 500
mg, 0.95 mmol) was suspended in a solution of triethylamine
(30 mL) and deionized water (30 mL) and stirred at room
temperature for 18 h. The resulting solution was evaporated
to dryness in vacuo, and the residue was dissolved in water
(50 mL), extracted with CH2Cl2 (2 × 50 mL), and purified by
anion exchange chromatography using a Sep-Pak Vac 35 cm3
Accell Plus QMA cartridge (Waters Corp., P/N WAT054725)
(HCO3- form) with an aqueous ammonium bicarbonate buffer
(0-0.5 M linear gradient, 1 L). The appropriate fractions were
combined and evaporated to dryness in vacuo. The residue was
twice dissolved in deionized water and evaporated to dryness
in vacuo to give 11 as the ammonium salt. This salt was
dissolved in deionized water and passed through a Sep-Pak
Vac 20 cm3 Accell Plus CM cartridge (Waters Corp., P/N
WAT054675) (Na+ form) using deionized water. The appropri-
ate fractions were combined and lyophilized to give 0.430 g
(86% yield) of 11 disodium salt 2.5 hydrate as a white solid.
1H NMR (D2O): δ 7.71 (1H, s), 6.11 (1H, m), 5.80 (1H, m),
5.32 (1H, m), 3.66 (2H, m), 3.41 (1H, m), 2.99 (1H, m), 2.68
(2H, m), 1.49 (1H, m), 1.12 (3H, d), 0.74 (2H, m), 0.54 (2H, m).
31P NMR (D2O): δ 8.68. MS (ES-) m/z 436 (M - H). Anal.
(C17H22N7Na2O5P‚2.5H2O) C, H, N.
Abacavir
5′-(Phenylmethoxydimethylglycinyl)-
phosphate (tert-Butyl N-[({(1S,4R)-4-[2-Amino-6-(cyclo-
propylamino)-9H-purin-9-yl]cyclopent-2-en-1-yl}-
methoxy)(phenoxy)phosphoryl]dimethylglycinate) (9).
This was synthesized according to standard procedure 4, using
abacavir (300 mg, 1.05 mmol), t-BuMgCl (1.0M solution in
THF) (1.57 mL, 1.57 mmol), and phenyl(methoxydimethyl-
glycinyl) phosphorochloridate (0.59 M solution in THF) (5.3
mL, 3.14 mmol) in THF (20 mL) stirring at room temperature
for 96 h. The crude product was purified by eluting with 3%
MeOH in CHCl3 and then with 2.5% MeOH in CHCl3 to give
Antiviral Activity Assays. The activity of the test com-
pounds against HIV-1- and HIV-2 induced cytopathicity in
CEM cell cultures was measured at day 4 as previously
described.21 The antiviral activity of the test compounds was
estimated by microscopical examination of virus-induced giant
cell formation. HIV was added at approximately 100 CCID50
to the cell cultures (1 CCID50 ) 50% cell culture infective dose).
The anti-HSV-1 and anti-HSV-2 assays were based on inhibi-
1
the product 9 as a white foam (193.7 mg, 34.14%). H NMR-
(CDCl3): δ 7.40 + 7.36 (1H, 2 × S), 7.24 - 7.19 (2H, t), 7.15-

3514 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
McGuigan et al.
tion of virus-induced cytopathicity in human embryonic lung
(HEL) cell cultures. Confluent cell cultures in microtiter 96-
well plates were inoculated with 100 CCID50 of HSV-1 (KOS),
HSV-2 (G), and HSV-1 TK- (B2006 and VMW-1837) (1 CCID50
being the virus dose to infect 50% of the cell cultures). After a
1 h virus adsorption period, residual virus was removed and
the cell cultures were incubated in the presence of varying
concentrations of the test compounds. Virus cytopathicity was
recorded as soon as it reached completion in the control-virus-
infected cell cultures.
The activity of the test compounds against HBV and the
potential of these compounds to inhibit growth of human
hepatoma cells were determined as previously described26
using HepG2 2.2.15 cells, which constitutively produce HBV.
Radiolabeled Abacavir and Abacavir ProTide. [8-3H]-
Abacavir ([3H]ABC) and [8-3H]abacavir 5′-[phenyl(methoxy-
L-alaninyl)]phosphate ([3H]ABCpro) were prepared by Amer-
sham Pharmacia Biotech (Buckinghamshire, England) via
radiolabel exchange from tritiated water to ABC (1) and
ABCpro (3), respectively. The radiolabel purity of these
compounds was g95%. Their specific activities were ap-
proximately 7 and 9 Ci/mmol, respectively.
Metabolism of Abacavir and Its Aryloxymethoxy-
alaninylphosphoramidate Derivative in CEM Cells. The
metabolism of [3H]ABC and its radiolabeled ProTide ary-
loxymethylalaninyl abacavir monophosphate ([3H]ABCpro) (3)
in CEM cells was studied according to previously established
procedures.21 Briefly, human T4 lymphocyte CEM cells were
seeded at (2-4) × 105 cells/mL in RPMI-1640 culture medium
supplemented with 10% fetal calf serum, 2 mM l-glutamine,
and 0.075% NaHCO3. Five milliliter cell suspensions in 25 cm2
culture flasks were then incubated with 10 µM [3H]ABC or
1.1 µM pro[3H]ABC (5 µCi/5 mL). At 24 h, drug-treated cells
were centrifuged, washed twice with cold RPMI-1640 medium,
and precipitated with cold methanol/water (60:40). After
centrifugation, the supernatants were subjected to HPLC
analysis and separated on a Partisphere SAX column (What-
man, Clifton, NJ). A linear gradient of 0.005 M (NH4)H2PO4
(pH 5.0) (buffer A) to 0.30 M (NH4)H2PO4 (pH 5.0) (buffer B)
was used. The retention times of the metabolites were as
follows: ABC, carbovir (CBV), and pro-ABC, 2-3 min; ABCMP
(abacavir 5′-monophosphate), 7-8 min; CBVMP (carbovir 5′-
monophosphate) and alaninyl ABC-MP, 11-13 min; CBVDP
(carbovir 5′-diphosphate), 19-21 min; CBVTP (carbovir 5′-
triphosphate), 35-38 min.
overnight prior to and until approximately 4 h after each dose;
water was provided ad libitum. Serial blood samples were
collected at selected times from predose to 24 h postdose and
were centrifuged to obtain plasma within 30 min of collection.
The resulting plasma samples were frozen at -20 °C and
shipped on dry ice to GlaxoSmithKline for analysis.
Plasma samples (200 µL) were deproteinated by addition
of approximately 4 volumes of acetonitrile (750 µL) followed
by centrifugation (15800g, 10 min, 4 °C). The resulting
supernatants were dried under nitrogen, and the dried extracts
were reconstituted in 5% acetonitrile/0.1% acetic acid, pH 5.6
(200 µL). Plasma standards prepared by serial dilution of
analyte stock solutions with normal monkey plasma were
processed identically. The plasma extracts were analyzed by
LC/MS/MS using a Phenomenex Luna 3-µm C18 column (100
mm × 2 mm). The samples were eluted by 5% acetonitrile/
0.1% acetic acid, pH 5.6, for 3 min followed by a linear gradient
to 95% acetonitrile over 5 min. Analytes were detected by
positive ion MRM analysis, monitoring transitions m/z 542-
191 for the ProTide (6), m/z 438-191 for alaninyl ABC-MP
(11), m/z 367-191 for ABC-MP, m/z 287-191 for ABC (1), and
m/z 248-152 for CBV (2). Analyte concentrations were deter-
mined by reference to a corresponding standard curve con-
structed by linear regression analysis of weighted (1/x2) peak
areas as measured by LC/MS analysis. Pharmacokinetic
parameter values were determined by noncompartmental
analysis of the plasma concentration-time data.
Acknowledgment. We thank Manon Villeneuve for
separation of the ProTide diastereomers, Mark Patrick
and Paul Johnson for stability and solubility testing,
Brian Owens for bioanalysis (monkey pharmacokinetics
study), Ria Van Berwaer for the metabolic studies, and
Ann Absillis and Lizette van Berchelaer for the antiviral
assays.
Supporting Information Available: Analytical data for
target compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
Anabolism in HepG2 2.2.15 Cells. Anabolism studies with
[3H]ABC and [3H]ABCpro in HepG2 2.2.15 cells were con-
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2.2.15 cells were seeded into T-75 flasks at 107 cells/flask, and
after approximately 24 h of growth, the cultures were treated
with [3H]ABC or [3H]ABCpro at the desired final concentration
(0.01-100 µM, 2 mCi/µmol). At selected times following drug
addition (2-48 h), cells were washed twice with ice-cold PBS
(5 mL) and then extracted in situ by addition of ice-cold water
(0.6 mL) followed by ice-cold acetonitrile (2.4 mL). Cell counts
at collection were determined in representative flasks prior
to extraction. The cell extracts were centrifuged, the super-
natants were evaporated to dryness, and the dried extracts
were reconstituted in 100 µL of HPLC-grade water. The
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exchange HPLC according to the method described by Faletto
et al.14
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molgus monkeys (2.0-3.2 kg) at 11.5 mg/kg (5 mL/kg) with a
1-week washout period between doses (i.e., intravenous dose
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