Stereochemical influence on lipase-mediated hydrolysis and biological activity of stampidine and other stavudine phosphoramidates

Article abstract of DOI:10.1016/j.bmc.2004.12.024

Stampidine and other halogen substituted stavudine phosphoramidates can be activated by lipase-mediated hydrolysis. The target site for the lipase appears to be the methyl ester group of the L-alanine side chain. Accordingly, the D-amino acid substituted

Full text of DOI:10.1016/j.bmc.2004.12.024

Bioorganic & Medicinal Chemistry 13 (2005) 1763–1773
Stereochemical influence on lipase-mediated hydrolysis
and biological activity of stampidine and other
stavudine phosphoramidates
T.K. Venkatachalam,a,b P. Samuela,b and F.M. Uckuna,b,
*
aDepartment of Chemistry, Department of Structural Biology, Department of Pharmaceutical Sciences and Paradigm
Pharmaceuticals, LLC, 2685 Patton Road, St. Paul, MN 55113, USA
bDepartment of Cell Biology and Department of Virology, Parker Hughes Institute, 2699 Patton Road, Roseville, MN 55113, USA
Received 30 April 2004; accepted 1 December 2004
Available online 21 January 2005
Abstract—Stampidine and other halogen substituted stavudine phosphoramidates can be activated by lipase-mediated hydrolysis.
The target site for the lipase appears to be the methyl ester group of the L-alanine side chain. Accordingly, the D-amino acid substi-
tuted isomers {Rp or Sp}are resistant to lipase-mediated hydrolysis and exhibit substantially less anti-HIV activity. Molecular
modeling results indicate that the L-amino acid configured isomers {Rp or Sp} are preferred in the lipase binding pocket.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
or laboratory evidence of toxicity at dose levels as high
as 500 mg/kg or at cumulative dose levels as high as
8.4 g/kg. Stampidine exhibited favorable pharmaco-
kinetic behavior in mice, rats, dogs, and cats following
oral administration.6,7 The documented in vitro potency
of stampidine against primary clinical HIV-1 isolates
with genotypic and/or phenotypic NRTI- or NNRTI-
resistance as well as non-B envelope subtypes together
with its in vivo antiretroviral activity in HIV-infected
Hu-PBL SCID mice and FIV-infected cats warrants its
further development as a new anti-HIV drug.
Stampidine is a novel phosphoramidate derivative of
stavudine that is being developed as a potential anti-
HIV agent.1–5 Stampidine was 100-times more active
than stavudine and twice as active as zidovudine against
nine clinical HIV-1 isolates of non-B envelope subtypes
(A, C, F, and G) originating from South America, Asia,
and sub-Saharan Africa.1 Stampidine was effective
against twenty genotypically and phenotypically nucleo-
side analog reverse transcriptase inhibitor (NRTI)-resis-
tant and six non-nucleoside inhibitor (NNRTI)-resistant
HIV-1 isolates at subnanomolar to low nanomolar
concentrations.1 Stampidine was active against HIV-1
isolates with five thymidine analogue mutations at
subnanomolar concentrations. Orally or intraperitone-
ally administered stampidine exhibited significant and
dose-dependent in vivo anti-HIV activity against an
NRTI-resistant clinical HIV-1 isolate in severe com-
bined immunodeficient (SCID) mice reconstituted with
peripheral blood (PBL) mononuclear cells from seroneg-
ative human donors.4 In the feline immunodeficiency
virus (FIV)-infected domestic cat model for AIDS, oral-
ly administered stampidine showed a dose-dependent
anti-retroviral effect in chronically FIV-infected cats.5
Stampidine therapy was not associated with any clinical
2. Results and discussion
The generation of ala-d4T-MP as the active metabolite
of stampidine was originally proposed to require the
esterase-mediated hydrolysis of the carbomethoxy group
associated with the alanine side chain of stampidine.7–33
We hypothesized that in various tissue microenviron-
ments the metabolism of stampidine may occur through
the action of hydrolytic enzymes other than esterases as
well. In a recent study, we found that lipase-mediated
stereoselective hydrolysis of stampidine and other phos-
phoramidates derivatives of stavudine play a key role
for their metabolism.34
Modeling studies and comparison of the hydrolysis rate
constants revealed a chiral preference of the lipase active
site for the putative Sp-stereoisomer. The in vitro
*
Corresponding author. Fax: +1 651 796 5493; e-mail: fatih_uckun@
ih.org
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.024

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T.K. Venkatachalam et al. / Bioorg. Med. Chem. 13 (2005) 1763–1773
anti-HIV activity of these compounds correlated with
their susceptibility to lipase- (but not esterase-) mediated
hydrolysis. Saboulard et al.14 McGuigan et al.10–13
Balzarini and co-workers,23–26 Wagner and co-work-
ers,30,31,8 Meier et al.45–48 have suggested an esterase-
dependent biochemical pathway for the hydrolysis of
phosphoramidate derivatives. Our recent studies pro-
vided experimental evidence that other enzymes includ-
ing lipases and proteases35 are also involved in the
activation of phosphoramidate derivatives of nucleoside
analogs. We propose that stampidine undergoes rapid
enzymatic hydrolysis in the presence of lipase according
to the following biochemical pathway: during the first
step, hydrolysis of the ester group results in the forma-
tion of carboxylic acid. A subsequent step involves an
intramolecular cyclization at the phosphorous center
with simultaneous elimination of the phenoxy group to
form a cyclic intermediate. In the presence of water, this
intermediate is converted into the active metabolite ala-
d4T-MP. We postulated that the lipase hydrolyzes the
methyl ester group of the L-alanine side chain to form
the cyclic intermediate in a stereoselective fashion. This
hypothesis was supported by experimental data showing
that chloroethylsubstituted derivatives of stampidine,
which possess a chloroethyl linker unit instead of a
methyl ester side chain, were resistant to lipase-mediated
hydrolysis, which excludes the possibility of a direct
hydrolysis of stampidine at the phosphorous center.
However, caution should be exercised in that the inactiv-
ity of the chloroethyl analogs may be due to other rea-
sons such as extra cellular instability or involvement of
other enzymes. Thus, our model implies that the lipase-
mediated formation of the cyclic intermediate is a key
step in the metabolism of stampidine and relies on the
initial configuration of the stereoisomers [Rp or Sp].
This is in accordance with the earlier notion by
Subbord et al.,14 and Balzarini and co-workers.23–26 In
an attempt to further confirm that the carboxymethyl
ester side chain is the target site for the lipase-mediated
hydrolysis, we replaced the L-alanine side chain with a
D-alanine side chain, that does not fit the binding pocket
of the lipase without spatial constraints. ÔLÕ and ÔDÕ-con-
figured amino acid modification of phosphoramidate
derivatives have been reported by McGuigan et al.36
In their work these authors have prepared the unsubsti-
tuted phenyl phosphoramidate derivative of stavudine
and examined its antiviral activity. Naesens et al.37,38
have studied the metabolism and anti-HIV activity of
phosphoramidate derivatives of d4T-MP with variations
in the amino acid moiety and came to the conclusion
that enzymes involved in the formation of the amino
acyl d4T MP metabolite have specificity for L-alanine
as the amino acid moiety. In a recent study, Siccardin
et al.39 have examined the stereospecific chemical and
lysis at the methyl ester side chain of the stampidine
molecule plays an important role for the metabolic
activation of this anti-HIV pro-drugs, which is consis-
tent with and extends the work previously
reported.36,8,10,20–33
Earlier studies on phosphoramidate derivatives of stavu-
dine demonstrated that these compounds can be acti-
vated by esterases.7–33 Recently we found that lipases
as well as proteases can also activate these deriva-
tives.34,35 However, the position of enzymatic attack
has not been deciphered. In order to establish the site
of enzymatic attack we prepared the ÔDÕ-amino acid
ester substituted phosphoramidate derivatives39,40 and
compared their activation by a lipase. During our syn-
thesis of the phosphoramidate derivatives we obtained
two isomers due to the chirality at phosphorus center
of the molecule [Sp and Rp], consistent with previous
reports.8,10,23–33,35,38–47 Using HPLC, we were able to
determine the ratios of each isomer. Table 1 shows the
enantiomeric excess ratios obtained for four of these
stavudine compounds having both ÔLÕ and ÔDÕ configura-
tion at the amino acid terminal. It is interesting to note
that in the case of the ÔLÕ-amino acid methylester isomer
we were able to obtain a ratio of approximately 40% of
isomer 1, [Rp or Sp] and 60% of isomer 2, [Rp or Sp] but
the ÔDÕ-amino acid methylester substituted phosphorus
isomers [Rp or Sp] were obtained almost in equal pro-
portions in the majority of the compounds. This points
out that the approach of nucleoside in the final segment
of synthesis is affected by the configuration at the amino
acid terminal of the phosphorochloridate. We have also
observed similar results when the amino acid is changed
in the structure of arylphosphoramidate derivatives of
zidovudine (Venkatachalam, unpublished results).
Table 1. Enantiomeric excess ratios of phosphorus isomers [Rp or Sp]
obtained during synthesis of phosphoramidate derivatives of stavudine
with L- and D-amino acid ester substitution
O
HN
O
O
O
N
X
O P
O
NH
H
MeOOC
H
Me
Compd
X
Amino acid
configuration
Isomer #1 (%) Isomer #2(%)
(1)stamp Br
ÔLÕ
ÔDÕ
ÔLÕ
ÔDÕ
ÔDÕ
40.0
50.0
38.9
50.0
43.0
51.0
41.5
50.0
36.0
52.0
46.0
60.0
50.0
61.1
50.0
57.0
49.0
58.5
50.0
64.0
48.0
54.0
(2)
(3)
Br
F
(4)
(5)
F
Me
(7)
(8)
OMe ÔDÕ
OMe ÔLÕ
enzymatic stability of
a phosphoramidate triester
pro-drug in human plasma and came to the conclusion
that there was no stereoselective preference for phos-
phate diastereoisomers. In our study, the D-amino acid
substituted phosphoramidate isomers [Rp or Sp} were
resistant to hydrolysis and they were substantially less
active against HIV than corresponding L-amino acid
substituted phosphoramidate isomers {Rp or Sp}. These
experimental results suggest that lipase-mediated hydro-
(9)
H
ÔDÕ
ÔLÕ
ÔDÕ
ÔLÕ
(10)
(11)
(12)
H
Cl
Cl
Isomers 1 and 2represents the chirality at the phosphorus center of the
compound [Rp or Sp]. Ratios are measured using a HPLC assay for all
the phosphoramidate derivatives under identical experimental
conditions.

T.K. Venkatachalam et al. / Bioorg. Med. Chem. 13 (2005) 1763–1773
1765
D-amino acid substituted phosphoramidates under iden-
tical conditions to obtain a relative rate constant value.
In the past we have calculated the rate constant values of
individual isomers {Rp or Sp} and found that one of the
isomers undergoes hydrolysis at a 4-times faster rate.34
These results support our hypothesis that the hydrolysis
occurs at the methyl ester side chain of stavudine
phosphoramidates (Fig. 3). Likewise, both stereo-
isomers {Rp or Sp} of ÔDÕ-amino acid configured com-
pounds (compound #2) were analyzed for potential
interaction with lipase. The conformational difference
occurs at the C-alpha position of the amino acid, which
is closer to the active site than that at the phosphate po-
sition, thus creating a greater energy barrier for stereoi-
somers {Rp or Sp}. Additionally our docking and
subsequent energy analyses indicated that neither stereo-
isomer for the phosphate [Rp or Sp} of ÔDÕ-amino acid
configured compounds (compound #2) favorably fits
the active site because of steric clash with the protein.
With the chiral ÔDÕ-amino acid configured compounds,
the C-beta methyl group unfavorably stacks with the
phenol group in an attempt to fit the active site. Overall
the energy cost is high for both Rp- and Sp-isomers of
ÔDÕ-amino acid substituted compound #2 (Fig. 4). As a
result this model suggests that the ÔDÕ-amino acid substi-
tuted derivatives {both Rp or SP] (compound #2) are
poor substrates for lipase when compared to the ÔLÕ-
amino acid configured compound #1, which is consis-
tent with our experimental data. Previously, by compar-
ing this lipase active site model with a homologous
model of the esterase active site, we observed a larger
spatial opening near the entrance of the active site in
the esterase. The larger opening corresponds to the
roomier binding sites for both stavudine and the phen-
oxy group. The esterase thus appears to be more forgiv-
ing in accommodating the different structure of both
enantiomers and thus less discriminative. These observa-
tions hold true for ÔDÕ-amino acid configured compound
#2. The overall result is that our model is in agreement
with the hypothesis suggesting that the hydrolysis rate is
determined by the stereo-selection by the active site of
the enzyme. The observed differences in the rate of
lipase-mediated hydrolysis of L- versus D-amino acid
substituted isomers [Rp or Sp] of stampidine and other
stavudine phosphoramidates indicate that the lipase
ÔprefersÕ the L-amino acid configured [Rp or Sp] isomers.
Consequently, D-amino acid substituted isomers [Rp or
Sp] are poor substrates for the lipase, which is in agree-
ment with our experimental data.
3. Lipase-mediated hydrolysis of D-amino acid substituted
phosphoramidate isomers [Rp or Sp} of stampidine and
other phosphoramidate derivatives of stavudine
ÔDÕ-Amino acid substituted phosphoramidate isomers
[Rp or Sp} were prepared for stampidine and five other
phosphoramidate derivatives of stavudine by replacing
the L-alanine side chain with a D-alanine side chain
(Table 2).18,19 The compounds were further characterized
using routine techniques and the results are presented in
the experimental section. We compared the rate of
lipase-mediated hydrolysis of these phosphoramidates
using Candida B lipase as a model enzyme. The first
order rate constants for disappearance of starting mate-
rial during lipase-mediated hydrolysis were dependent
on the configuration of the amino acid. Since multiple
steps are involved for the formation of the metabolite
and intermediates we were unable to determine the ki-
netic order for this process. However for simplicity we
assumed that this process follows first order rate early
in the kinetic cycle. The D-amino acid substituted iso-
mers {Rp or Sp] were substantially more resistant to
lipase-mediated hydrolysis, as reflected by markedly
lower first order rate constants (Table 2). For example,
the rate constants were 3.99 for the L-amino acid substi-
tuted isomer of stampidine, but only 0.41 for the corre-
sponding D-amino acid substituted isomer (Figs. 1 and
2). The rate constants were calculated by integration
of both peaks in the chromatogram. Our interest was
not focused on the absolute rate constant values of Rp
or Sp-isomers, but instead of a comparison with L- or
Table 2. First order rate constants for disappearance of starting
material using lipase and arylphosphoramidate derivatives of stavu-
dine at room temperature
O
HN
O
O P
O
O
N
X
O
NH
H
MeOOC
H
Me
Compound
X
Amino acid
Rate constant HTL VIIIB
configuration for lipase-
mediated
hydrolysis
(nM)
1(stamp)
2
Br
Br
F
ÔLÕ18,19
ÔDÕ
ÔLÕ18,19
ÔDÕ
3.99
0.41
2.59
0.27
0.16
2.2
1
0*
212 155
3
1
0
4
F
Me
851 720
427 107
6
ÔDÕ
3.1. General mechanism of hydrolysis by lipase or esterase
enzymes
7
OMe ÔLÕ18,19
OMe ÔDÕ
6
3
8
0.18
2.20
0.14
5.80
0.18
—
1290 313
0.6
1787 215
0.3
5837 2304
9
H
ÔLÕ18,19
ÔDÕ
ÔLÕ18,19
ÔDÕ
—
—
2
After having established that enzymes are responsible
for the hydrolysis of this series of phosphoramidate
derivatives of stavudine, we propose the following mech-
anism of hydrolysis, which is consistent with the earlier
reports.8,10,14,15,17,27–33 In the first step, the ester side
chain is hydrolyzed by the enzyme to the carboxylic acid
that subsequently cyclizes and eliminates the phenoxy
moiety resulting in a cyclic intermediate (Fig. 3). This
unstable intermediate is further hydrolyzed by a water
10
11
12
d4T
AZT
H
Cl
Cl
—
—
1
18
4
2
1
—
Rate constants are expressed per hour. Repeated trials gave rate
constant values within 5%.
* Prep HPLC separated individual isomers {Rp or Sp] showed identical
activity of 1 nM (Venkatachalam et al., to be published).

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T.K. Venkatachalam et al. / Bioorg. Med. Chem. 13 (2005) 1763–1773
Figure 1. Lipase-mediated hydrolysis profile of compound 1 at various time intervals. The two peaks represent the phosphorus diastereoisomers {Rp
and Sp}.
molecule to yield the active metabolite. In the case of
phosphoramidate derivatives containing the ÔLÕ-config-
ured amino acid terminal, the initial step of hydrolysis
of the carboxy ester side chain is faster as compared to
that of the ÔDÕ-amino acid configured derivatives. This
result supports our hypothesis that these enzymes attack
at the methylester side chain of the phosphoramidate
derivatives and not at the phosphorus center or another
site. Since we have only altered the configuration at the
methylester side chain, one would conclude that any
changes in the enzyme activity must be associated with
this alteration. Based on these experimental results, we
have demonstrated the site at which these enzymes act
during hydrolysis of these phosphoramidate derivatives
of stavudine.
3.2. Proof of hydrolysis of phosphoramidate derivatives
with lipase
In our earlier discussion we postulated that lipases are
also involved in hydrolysis of these phosphoramidate
analogs and proposed a mechanism for the hydrolysis.

T.K. Venkatachalam et al. / Bioorg. Med. Chem. 13 (2005) 1763–1773
1767
Figure 2. Lipase-mediated hydrolysis profile of compound 2 at various time intervals. The two peaks represent the phosphorus diastereoisomers {Rp
and Sp}.
(Fig. 3). There are three pathways through which the
hydrolysis product could result from the starting materi-
als. In the first pathway, the attack of the enzyme takes
place at the phosphorus center eliminating the phenoxy
group in the structure, yielding d4T alaninyl methylester
monophosphate. However, the final product of the reac-
tion was d4T alaninyl monophosphate indicating that
the ester side chain has been hydrolyzed. Based on this
evidence one can rule out the first pathway.
For the second pathway we propose a simultaneous at-
tack on the phosphorus site as well as at the methyl ester
side chain. In this mechanism, formation of d4T alanine
monophosphate can result as a final product. However,
we can rule out the attack on the phosphorus center as
discussed previously based on the experimental results
with ÔDÕ-amino acid substituted stavudine derivatives,
which showed resistant towards hydrolysis. Additionally
if the attack occurred at the phosphorus center the

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T.K. Venkatachalam et al. / Bioorg. Med. Chem. 13 (2005) 1763–1773
A. L- isomer
O
O
HN
HN
O
O
N
Lipase
O
X
O
P
O
N
O
X
O
O
P
O
NH
O
H
NH
O
MeOOC
H
Me
"L"
CH3OH
H
HO
Me
A
O
B
O
N
HN
HN
O
P
O
N
O
P
O
O
O
-O
O
O
O
NH
NH
O
O
H
X
O-
Me
H
HO
Me
D
C
E
X-C6H4OH
B. D- isomer
O
O
HN
HN
O
O
N
Lipase
O
P
X
O
P
O
O
N
O
X
O
O
NH
O
H
NH
MeOOC
H
Me
CH3OH
"D"
HO
H
Me
A
{Very slow reaction)
O
B
Figure 3. Enzyme mediated hydrolysis of both ÔLÕ- and ÔDÕ-amino acid substituted isomers of [Rp or Sp] phosphoramidate derivatives of stavudine.
ÔDÕ-amino acid configured isomer of the phosphoramidate
{Rp or SP} should have reacted and formed the hydro-
lysis product. From our results, we found that a ÔDÕ-
amino acid substituted compound is not hydrolyzed by
lipase. Therefore, we can rule out the second pathway.
we treated Cos 7 cells with ÔDÕ- versus ÔLÕ-amino acid
substituted isomers [Rp or Sp] of stampidine. The cellu-
lar extracts were subjected to HPLC analysis to compare
the metabolic rates of the compounds (Fig. 5A and B).
The ÔDÕ-amino acid substituted isomer [Rp or Sp] did
not hydrolyze even after 3 h demonstrating that a
change of stereochemistry at the amino acid side chain
of these phosphoramidate derivatives of stavudine
results in poor intracellular metabolism.
In the third pathway, the ester side chain of the phos-
phoramidate is hydrolyzed to form the carboxylic acid,
which then eliminates the phenoxy moiety yielding a
unstable cyclic intermediate. This intermediate is then
hydrolyzed in the presence of water to yield d4T alaninyl
monophosphate as the final product. This is further sup-
ported by the experimental data as only the ÔLÕ-amino
acid substituted phosphoramidate isomers {Rp or Sp},
but not ÔDÕ-amino acid substituted isomers {Rp or Sp}
were susceptible to hydrolysis by lipase. This may
be owing to the change in the binding characteristics
as well as the stereochemical geometry of the binding
pocket. Hence we conclude that similar to the ester-
ase8,10,14,17,27–33,45–50 activated mechanism, these phos-
phoramidate derivatives undergo hydrolysis by lipase
through the ester side chain of the molecule.
3.4. Antiviral activity of phosphoramidate derivatives
of stavudine
We examined the antiviral activity of stavudine phos-
phoramidate derivatives with ÔLÕ- versus ÔDÕ-configura-
tions at the amino acid side chain. In general, D-amino
acid substituted phosphoramidate isomers [Rp or Sp]
were substantially less active than the corresponding
ÔLÕ-amino acid substituted phosphoramidate isomers
[Rp or Sp]. This is in accordance with and extends the
results obtained by McGuigan et al.36 for ÔDÕ-amino acid
substituted phenyl phosphoramidate derivatives. Pre-
parative HPLC separated individual isomers of com-
pound #1 that showed identical anti-HIV activity of
1 nM. This result demonstrates that chirality change at
the phosphorus center did not alter the antiviral potency
of the compound (Venkatachalam et al., to be pub-
lished). This experimental finding further supports the
3.3. Intracellular metabolism of ‘L’- versus ‘D’-amino acid
substituted isomers of stampidine {Rp or Sp}
In order to evaluate whether stereochemistry at the
amino acid chain imparts a differential hydrolytic profile

T.K. Venkatachalam et al. / Bioorg. Med. Chem. 13 (2005) 1763–1773
1769
(40 mL) at 0 ꢁC. The reaction was allowed to warm to
room temperature and stirred for 15 h. Then the reac-
tion mixture was cooled to ꢀ70 ꢁC and D-alanine methyl
ester hydrochloride (1.39 g, 10 mmol) was added to the
reaction flask; the reaction was allowed to warm to
room temperature and stirred overnight. 1,2,4- Triazole
(1.80 g, 25 mmol) was then added to the above reaction
flask. After stirring at room temperature for 6 h, d4T
(0.45 g, 2.0 mmol) was added to the above reaction flask
and the reaction solution was stirred for 4 days. The sol-
vent was removed under reduced pressure. The crude
product was purified by column chromatography
(100% CHCl3 followed by 5% MeOH in CHCl3) and it
was further purified using preparative TLC to obtain
analytically pure compound.
6. Physical constants of new compounds3
6.1. d4T-50-p-Bromophenyl methyl-D-alaninyl phosphate (2)
1H NMR (300 MHz, CDCl3) d 1.33 (t, 3H, J = 7.0 Hz),
1.81 (d, 3H, J = 6.6 Hz), 3.58–4.06 (m, 2H), 3.70 (s, 3H),
4.28 (s, 2H), 5.00 (s, 1H), 5.90 (m, 1H), 6.33 (s, 1H), 7.02
(s, 1H), 7.08 (d, 2H, J = 9.0 Hz), 7.24 (s, 1H), 7.43 (d,
2H, J = 8.1 Hz), 8.61 (s, 1H); 13C NMR (75 MHz,
CDCl3) d 12.8, 21.3, 50.3, 53.0, 66.7, 67.6, 84.8, 89.9,
111.5, 118.4, 122.1, 127.6, 133.0, 133.5, 136.0, 150.9,
163.6. 31P NMR (121 MHz, CDCl3) d 2.56, 2.97; UV
(MeOH) kmax: 266 nm. [a]D {MeOH} ꢀ25.2: HPLC:
12.72, 12.92; % purity: >98.
Figure 4. Model of ÔLÕ-amino acid substituted compound 1 and ÔDÕ-
amino acid substituted compound 2 in the active site of lipase. (A) A
ÔLÕ-amino acid substituted derivative is docked into the active site of
lipase with favorable conformation. (B) A ÔDÕ-amino acid substituted
compound, which is modeled in the same active site with unfavorable
conformation. Prepared with Insight II.
6.2. d4T-50-p-Fluorophenyl methoxy-D-alaninyl
phosphate (4)
1H NMR (300 MHz, CDCl3) d 1.33 (q, 3H, J = 4.2Hz),
1.86 (d, 3H, J = 7.5 Hz), 3.50–4.06 (m, 2H), 3.70 (s, 3H),
4.28 (s, 2H), 5.00 (s, 1H), 5.89 (m, 1H), 6.98–7.04 (m,
3H), 7.13–7.17 (m, 2H), 7.28 (s, 1H), 8.41 (s, 1H); 13C
NMR (75 MHz, CDCl3) d 12.8, 21.3, 50.3, 53.0, 66.6,
67.5, 84.8, 89.9, 111.5, 116.6, 121.7, 127.6, 133.4,
135.9, 150.8, 163.6. 31P NMR (121 MHz, CDCl3) d
2.96, 3.34; UV (MeOH) kmax: 266 nm. [a]D {MeOH}
ꢀ22.2: HPLC: 10.25, 10.35 min. % Purity: 98.0.
notion that the lipase-mediated hydrolysis at the methyl
ester side chain of stampidine and other stavudine phos-
phoramidates plays an important role for the activation
of these anti-HIV pro-drugs.
4. General experimental
NMR spectra were obtained at ambient temperature in
deuterated solvent. Chemical shifts are reported as d val-
ues in parts per million downfield and referenced to the
solvent. Coupling constants (J) are reported in Hz.
HPLCs were obtained using an analytical RP–18
Lichrospher column, (4.6 · 250 mm) and CH3CN/H2O
as the eluent. The flow rate was maintained at 1.0 mL/
min and the detection wavelength was set at 266 nm.
The column was maintained at room temperature
throughout the analysis. Column and TLC chromato-
graphy was performed using silica gel 60, 230–400 mesh.
6.3. d4T-50-p-Methylphenyl methoxy-D-alaninyl
phosphate (5)
1H NMR (300 MHz, CDCl3) d 1.32(t, 3H, J = 7.2Hz),
1.85 (d, 3H, J = 6.6 Hz), 2.30 (s, 3H), 3.44–4.05 (m, 2H),
3.69 (s, 3H), 4.28 (s, 2H), 5.00 (s, 1H), 5.89 (m, 1H), 6.33
(s, 1H), 7.00–7.12(m, 5H), 7.30 (s, 1H), 8.35 (s, 1H); 13C
NMR (75 MHz, CDCl3) d 12.7, 21.3, 30.0, 50.3, 53.0,
66.4, 67.3, 84.8, 89.9, 111.5, 120.0, 127.5, 133.4, 133.5,
136.1, 150.8, 163.6. 31P NMR (121 MHz, CDCl3) d
2.78, 3.27; UV (MeOH) kmax: 266 nm. [a]D {MeOH}
ꢀ24.6: HPLC: 11.33, 11.44 min. % Purity: >98.
5. Synthetic procedure
6.4. d4T-50-p-Methoxyphenyl methoxy-D-alaninyl
phosphate (8)
Triethylamine (4.2mL, 30 mmol) was added dropwise
to a stirred solution of POCl3 (1.53 g, 10 mmol) and
substituted phenol (10 mmol) in anhydrous chloroform
1H NMR (300 MHz, CDCl3) d 1.33 (t, 3H, J = 8.7 Hz),
1.81 (d, 3H, J = 6.0 Hz), 3.44–4.06 (m, 2H), 3.70 (s, 3H),

1770
T.K. Venkatachalam et al. / Bioorg. Med. Chem. 13 (2005) 1763–1773
Figure 5. (A) HPLC profiles of stampidine (ÔLÕ-amino acid substituted compound 1) and its hydrolyzed products in Cos 7 cells at various time
intervals of exposure. The two HPLC peaks represent the phosphorus diastereoisomers [Rp and Sp]. (B) HPLC profiles of stampidine (ÔDÕ-amino acid
substituted compound 2) and its products in Cos 7 cells at various time intervals of exposure. The two peaks represent phosphorus diastereoisomers
{Rp and Sp].
3.78 (s, 3H), 4.28 (s, 2H), 4.99 (s, 1H), 5.88 (m, 1H), 6.34
(s, 1H), 6.83 (d, 2H, J = 9.0 Hz), 7.02(s, 1H), 7.10 (d,
2H, J = 10.5 Hz), 7.30 (d, 1H, J = 8.4 Hz), 8.41 (s,
1H); 13C NMR (75 MHz, CDCl3) d 12.8, 21.4, 50.3,
52.9, 55.9, 66.9, 84.8, 89.8, 111.5, 114.9, 121.3, 127.6,
133.6, 136.2, 150.8, 163.6. 31P NMR (121 MHz, CDCl3)
d 3.11, 3.55; UV (MeOH) kmax: 266 nm. HPLC: 10.20,
10.27 min.
7.02(s, 1H), 7.19 (d, H2 ,
J = 7.5 Hz), 7.28–7.36 (m,
6H), 8.27 (s, 1H); 13C NMR (75 MHz, CDCl3) d 12.8,
21.4, 50.4, 53.0, 66.5, 67.4, 84.8, 89.9, 111.5, 120.3,
125.4, 127.5, 133.0, 133.5, 136.0, 150.8, 163.5. 31P
NMR (121 MHz, CDCl3) d 2.17, 2.64; UV (MeOH)
k
max: 266 nm. HPLC: 12.71, 12.88 min.
6.6. d4T-50-p-Chlorophenyl methoxy-D-alaninyl
phosphate (12)
6.5. d4T-50-Phenyl methoxy-D-alaninyl phosphate (10)
1H NMR (300 MHz, CDCl3) d 1.33 (t, 3H, J = 7.5 Hz),
1.86 (d, 3H, J = 7.8 Hz), 3.55–4.06 (m, 2H), 3.70 (s, 3H),
4.28 (s, 2H), 5.00 (s, 1H), 5.91 (m, 1H), 6.33 (m, 1H),
1H NMR (300 MHz, CDCl3) d 1.33 (t, 3H, J = 7.6 Hz),
1.86 (d, 3H, J = 6.6 Hz), 3.44–4.06 (m, 2H), 3.70 (s, 3H),
4.30 (m, 2H), 5.00 (s, 1H), 5.89 (m, 1H), 6.33 (m, 1H),
7.02(s, 1H), 7.14 (d, H2 ,
J = 8.1 Hz), 7.24 (s, 1H),

T.K. Venkatachalam et al. / Bioorg. Med. Chem. 13 (2005) 1763–1773
1771
Figure 5. (continued)
7.29 (d, 2H, J = 8.7 Hz), 8.55 (s, 1H); 13C NMR
(75 MHz, CDCl3) d 12.8, 21.3, 50.3, 53.0, 66.7, 67.6,
84.8, 89.9, 111.5, 121.7, 127.6, 133.0, 133.4,
136.0, 150.8, 163.6. 31P NMR (121 MHz, CDCl3) d
a 20 mL scintillation glass vial. Using a pipetman,
3 mL of methanol was added and the contents were vor-
texed for 2min until a homogenous solution resulted.
Using another pipetman, 100 mL of this stock solution
was transferred into another scintillation vial, and to
this was added 900 mL of water and the contents vor-
texed. In the meantime, 5 mg of solid lipase powder
was weighed and transferred to a volumetric flask. To
this was added 8 mL of water and the contents were sha-
ken to dissolve the enzyme. From the stock solution of
the compound as mentioned above, 500 mL of the meth-
anolic solution of the phosphoramidate derivative was
pipetted out into another glass vial and to this 500 mL
2.77, 3.16; UV (MeOH) kmax
:
266 nm. HPLC:
10.00 min.
7. Experimental conditions for lipase study
For the kinetic study, a known amount of the phosphor-
amidate derivative was carefully weighed (5–7 mg)
using a Metller analytical balance and transferred into

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T.K. Venkatachalam et al. / Bioorg. Med. Chem. 13 (2005) 1763–1773
of lipase solution was added and the contents were sha-
ken to form a homogenous solution. From this reaction
mixture 50 mL was used for HPLC analysis. The col-
umn used was a Lichrospher (RP-18) analytical column
of 4 · 250 mm. The eluent used for HPLC was water/
TFA and TEA (0.1%) and CH3CN in the ratio of
65:35. The column was maintained at room tempera-
ture. The flow rate was maintained at 1 mL/min, the
detection wavelength was adjusted to 265 nm and the
reference wavelength was kept at 400 nm. An aliquot
of the sample was drawn at various intervals of time
from the reaction vial and analyzed. For fast reactions
two HPLC instruments were used simultaneously to
obtain the rates.
bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and
1.0 mM sodium pyruvate, 90%; fetal bovine serum,
10%, at 37 ꢁC in a humidified atmosphere containing
5% CO2. The cell line was obtained from the Cell Bio-
logy Laboratory of the Parker Hughes Cancer Center.
140 million cells were used for the enzyme experiments.
To obtain these cells, 30 million frozen cells were plated
in a T-150 flask with 20 mL of medium and incubated
at 37 ꢁC for 48 h. The cells were collected in a 50 mL
conical tube and centrifuged at 1000 rpm for 5 min,
the supernatant decanted off and the cells were
resuspended in 90 mL of medium and grown further in
three T-150 flasks with 30 mL each at 37 ꢁC for another
48 h to give 140 million cells. The cells were collected by
centrifugation at 1000 rpm for 5 min, subsequently
washed thrice in 1 · PBS and then resuspended in
1 mL of 1 · PBS.
8. Estimation of products
The amount of products observed during the reaction of
these phosphoramidates were estimated from the area
obtained from the HPLC profiles. Authentic samples
of the products, when possible, were run to identify the
peaks observed during the reaction. Further confirma-
tion of the product structures were obtained using a
LC/mass instrument. The rate of reaction was computed
by using first order rate constants and an average of eight
to nine time points were used for this estimate. The rate
constants reported refers to rate/hr as some of the reac-
tions were too slow to obtain meaningful results.
For the HPLC measurements, approximately, 0.5 mL of
the suspended cell mixture in PBS buffer was taken and
treated with compound 1 (stampidine) or compound 2
and incubated at 37 ꢁC. At various time intervals, an ali-
quot (clear supernatent) was drawn and assayed using
the HPLC. Authentic samples were used to identify
the products during T-lymphocyte mediated hydrolysis
of stampidine. A lichrospher-RP-18 column was used
for this purpose and the eluent was a mixture consisting
of 65% water containing 0.1% TEA and TFA and 35%
of acetonitrile. The flow rate was maintained at 1 mL/
min and the column was maintained at room temper-
ature throughout the analysis. An aliquot of the samples
were drawn at various time intervals and assayed using a
diode-array detector.
9. In vitro assays of anti-HIV activity
Normal human peripheral blood mononuclear cells
(PBMNC) from HIV-negative donors were cultured
for 72h in RPMI 1640 supplemented with 20% (v/v)
heat-inactivated fetal bovine serum (FBS), 3% interleu-
kin-2, 2 mM L-glutamine, 25 mM HEPES, 2 g/L NaH-
11. Conclusions
Stampidine and other halogen substituted stavudine
phosphoramidates can be activated by lipase-mediated
hydrolysis. The target site for the lipase appears to be
the methyl ester group of the ÔLÕ-alanine side chain.
Molecular modeling results indicate that the ÔLÕ-amino
acid substituted isomer [Rp or Sp] is preferred in the
lipase binding pocket. Accordingly, the ÔDÕ-amino acid
substituted phosphoramidate isomers [Rp or Sp} are
resistant to lipase-mediated hydrolysis and exhibit sub-
stantially less anti-HIV activity.
CO3,
50 mg/mL
gentamicin,
and
4 mg/mL
phytohemagglutinin prior to exposure to HIV-1 at a
multiplicity of infection (MOI) of 0.1 during a 1 h
adsorption period at 37 ꢁC in a humidified 5% CO2
atmosphere. Subsequently, cells were cultured in 96-well
microtiter plates (100 mL/well; 2 · 106 cells/mL) in the
presence of various concentrations of d4T phosphoram-
idates and aliquots of culture supernatants were re-
moved from the wells on the 7th day after infection
for p24 antigen assays, as previously described.51 The
applied p24 enzyme immunoassay (EIA) was an unmod-
ified kinetic assay commercially available from Coulter
Corporation/Immunotech. Inc. (Westbrooke, ME),
which utilizes a murine mAb to an HIV core protein
coated on to microwell strips to which the antigen pres-
ent in the test culture supernatant samples binds. Per-
cent viral inhibition was calculated by comparing the
p24 values from untreated infected cells (i.e., virus
controls).
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