Protease-mediated enzymatic hydrolysis and activation of aryl phosphoramidate derivatives of stavudine

Article abstract of DOI:10.1016/j.ejmech.2004.11.015

Several proteases are capable of hydrolyzing the aryl substituted phosphoramidate derivatives of stavudine resulting in the formation of the active metabolite, alaninyl d4T monophosphate. Subtilisin Protease A, Subtilisin Griseus, Subtilisin Carlsberg, Papaya, Bacillus were amongst the most effective proteases in hydrolyzing stavudine derivatives and specificity of their activity was confirmed using several protease inhibitors to block the hydrolysis of these phosphoramidate derivatives. We found that these proteases exhibit chiral selectivity at the phosphorus center of stavudine derivatives. Our results indicate that cellular proteases may be responsible for the activation of these phosphoramidate derivatives. In addition, we show that the enzymatic hydrolysis takes place at the carboxymethyl ester side chain of these pro-drugs and the direct attack on the phosphorus center by these enzymes does not occur. Finally, we describe a novel activation pathway hitherto unknown for the activation and viral inhibitory characteristic shown by these phosphoramidate derivatives of stavudine.

Full text of DOI:10.1016/j.ejmech.2004.11.015

European Journal of Medicinal Chemistry 40 (2005) 452–466
www.elsevier.com/locate/ejmech
Original article
Protease-mediated enzymatic hydrolysis and activation of aryl
phosphoramidate derivatives of stavudine
T.K. Venkatachalam *, P. Samuel, S. Qazi, F.M. Uckun
Departments of Chemistry, Pharmaceutical Sciences, Bioinformatics, Virology and Immunology, Parker Hughes Institute and Paradigm Pharmaceuticals,
2699, Patton Road, Roseville, MN 55113, USA
Received 17 June 2004; received in revised form 8 October 2004; accepted 2 November 2004
Available online 03 March 2005
Abstract
Several proteases are capable of hydrolyzing the aryl substituted phosphoramidate derivatives of stavudine resulting in the formation of the
active metabolite, alaninyl d4T monophosphate. Subtilisin ProteaseA, Subtilisin Griseus, Subtilisin Carlsberg, Papaya, Bacillus were amongst
the most effective proteases in hydrolyzing stavudine derivatives and specificity of their activity was confirmed using several protease inhibi-
tors to block the hydrolysis of these phosphoramidate derivatives. We found that these proteases exhibit chiral selectivity at the phosphorus
center of stavudine derivatives. Our results indicate that cellular proteases may be responsible for the activation of these phosphoramidate
derivatives. In addition, we show that the enzymatic hydrolysis takes place at the carboxymethyl ester side chain of these pro-drugs and the
direct attack on the phosphorus center by these enzymes does not occur. Finally, we describe a novel activation pathway hitherto unknown for
the activation and viral inhibitory characteristic shown by these phosphoramidate derivatives of stavudine.
© 2005 Elsevier SAS. All rights reserved.
Keywords: HIV; Stampidine; Proteases; Hydrolysis; Mechanism; Distereoisomers
1. Introduction
version of the nucleoside to its monophosphate derivative
[9–11]. Anti-HIV ddN derivatives primarily rely on nucleo-
side and nucleotide kinases to convert them into the corre-
sponding 5′-triphosphates as discussed before. However, such
compounds were found to act as poor substrates for nucleo-
side kinases. [9,12–15]. Consequently, development of pro-
drug strategies were sought to bypass the initial nucleoside
kinase activation. In an attempt to overcome the dependence
of ddN analogs on intracellular nucleoside kinase activation,
we and others have prepared aryl phosphate derivatives of
zidovudine and stavudine [10,11,16–25]. Some of these
derivatives were found to be potent anti-HIV agents with sub-
nanomolar IC50 values.
Our lead anti-HIV compound stampidine is a novel phos-
phoramidate derivative of stavudine [23]. Stampidine was
100-times more active than stavudine and twice as active as
zidovudine against nine clinical HIV-1 isolates of non-B enve-
lope subtypes (A, C, F and G) originating from South
America, Asia, and sub-SaharanAfrica [20]. Stampidine was
effective against 20 genotypically and phenotypically nucleo-
side analog reverse transcriptase inhibitor (NRTI)-resistant
and six non-nucleoside inhibitor (NNRTI)-resistant HIV-
The human immunodeficiency virus (HIV)/AIDS pan-
demic continues its spread at a rate of over 15,000 new infec-
tions every day. Three categories of anti-retroviral agents in
clinical use are nucleoside analogs, nucleoside reverse tran-
scriptase inhibitors (NRTI), such as zidovudine and stavu-
dine (d4T) [1,2] protease inhibitors and non-nucleoside
reverse transcriptase inhibitors (NNRTI) [3–6]. The 5′-
triphosphates of 2′,3′-dideoxynucleoside analogs (ddN) which
are generated by the action of nucleoside and nucleotide
kinases, are potent inhibitors of HIV reverse transcriptase
[7,8]. The rate limiting step for the conversion of 3′-azido-3′-
deoxy thymidine (AZT) to its bioactive metabolite AZT-
triphosphate seems to be the conversion of the monophos-
phate derivative to the di-phosphate derivative, whereas the
rate limiting step for the intracellular generation of the bio-
active 2′,3′-dideoxy-2′,3′-didehydro thymidine (stavudine)
metabolite stavudine-triphosphate was reported to be the con-
* Corresponding author.
E-mail address: jhumphrey@ih.org (T.K. Venkatachalam).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.11.015

T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
453
1 isolates at subnanomolar to low nanomolar concentrations
[20]. Orally or intraperitoneally administered stampidine
exhibited significant and dose-dependent in vivo anti-HIV
activity against an NRTI-resistant clinical HIV-1 isolate in
severe combined immunodeficient (SCID) mice reconsti-
tuted with peripheral blood (PBL) mononuclear cells from
seronegative human donors [21]. Orally administered stam-
pidine showed a dose-dependent anti-retroviral effect in
chronically FIV-infected cats [22]. Stampidine therapy was
not associated with any clinical or laboratory evidence of tox-
icity at dose levels as high as 500 mg kg–1 or at cumulative
dose levels as high as 8.4 g kg–1. Stampidine exhibited favor-
able pharmacokinetic behavior in mice, rats, dogs, and cats
following oral administration [22,24]. 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 devel-
opment as a new anti-HIV drug.
recorded on a Nicolet Protege 460 spectrometer. Mass spec-
tra were performed on a Hewlett Packard MALDI-TOF spec-
trometer (Model G2025A LD-TOF). Melting points were
determined using a Melt John’s apparatus and are uncor-
rected. HPLCs were performed using a Hewlett Packard
1100 series instrument consisting of an automatic sampler,
an electronic degasser, a thermostatic control unit, and a diode
array detector in conjunction with a Chemstation software
assembly. The column used was an analytical RP-18 Lichro-
spher column, 5 m (4.6 × 150 µm) and eluent was acetonitrile/
water mixture. The flow rate was maintained at 1.0 ml min–1
and the detection wavelength was set at 275 nm. The column
was maintained at room temperature throughout the analysis.
Column chromatography was performed using silica gel
obtained from the Baker Company. The solvents used for
elution varied depending on the compound and included
either one or a combination of the following: ethyl acetate,
methanol, chloroform, hexane, methylene chloride, THF and
ether.
Analytical thin-layer chromatography (TLC) was per-
The generation of the active metabolite of stampidine was
originally thought to require the esterase-mediated hydroly-
sis of the carbomethoxy group associated with the alanine
side chain of stampidine [10,11,18–20,26–28]. We hypoth-
esized that in various tissue microenvironments the metabo-
lism of stampidine may occur through the action of hydro-
lytic enzymes other than esterases as well. A recent study
implicated lipase-mediated stereoselective hydrolysis of stam-
pidine as an important step in the activation of this prodrug
[29]. The purpose of the present study was to evaluate the
potential role of proteases in the activation of stampidine. Our
experimental results provide unprecedented evidence that
stampidine as well as other halogen-substituted phosphora-
midate derivatives of stavudine can be metabolized by
protease-mediated hydrolysis. To our knowledge this study
is the first to provide experimental evidence for a protease-
mediated metabolism of a nucleoside phosphoramidate pro-
drug.
formed on Merck pre-coated glass plates (silica gel 60, F254
,
250-µm thick), and visualized under 254-nm UV light. Col-
umn chromatography was performed using EM silica gel 60,
230–400 mesh.
Synthesis of the phosphoramidate analogs of stavudine was
achieved starting from substituted phenols and phosphorous
oxychloride. The resulting phosphodichloridate was reacted
with L-alanine methylester hydrochloride followed by stavu-
dine to furnish the required compounds. The detailed syn-
thetic scheme and methods were previously reported in our
earlier publications [18]. Synthesis of chloroethyl substi-
tuted stavudine derivatives were accomplished as described
below.
2.2. General procedure for synthesis of chloroethyl
substituted phosphoramidate derivatives of stavudine
A RB flask was charged with 20 ml of anhydrous acetoni-
trile and triethylamine (1.2 eq.) followed by triazole (2.50 eq.).
To this mixture was added a solution of appropriately substi-
tuted phenol (1.0 eq.) and bis(2-chloroethyl)amine phosphod-
ichloridate (1.00 eq.) in anhydrous acetonitrile. The contents
were stirred at 0 °C by external cooling using ice bath. After
addition the contents were brought to room temperature and
stirred for 5 h. Upon completion of the reaction, d4T (1.0 eq.)
was added to the mixture and the contents of the flask were
brought to 50 °C and allowed to stir for 1 week. After this
period, the solvent was evaporated under vacuum and the resi-
due was subjected to column chromatography using chloro-
form and methanol (10:1) and the appropriate fractions con-
taining the product were pooled together. Evaporation of the
solvent and further purification by preparative thin layer chro-
matography furnished analytically pure products in 10% yield
as solids.
2. Chemistry
2.1. All chemicals were purchased from Aldrich
(Milwaukee, WI) or Sigma and were used without further
purification
Unless otherwise noted, each reaction vessel was secured
with a rubber septum, and the reaction was performed under
nitrogen atmosphere. H and 13C NMR were obtained on a
1
Varian Mercury 300 instrument at ambient temperature in
d6-DMSO. Chemical shifts are reported as d values in parts
per million downfield from tetramethylsilane (d0.0 ppm) as
an internal standard or from the residual dimethylsulfoxide
signal (d 2.49 ppm for 1H NMR or d 39.7 ppm for 13C NMR).
Splitting patterns are designated as follows: s, singlet; d, dou-
blet; t, triplet; m, multiplet; br, broad peak. FT-IR spectra were

454
T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
2.3. Physical constants
2.3.6. 5′-[4-Bromophenyl methoxy alaninylphosphate]-
2′,3′-didehydro-3′-deoxylthymidine isomer 1(S)
1
2.3.1. 5′-(2′,3′-Didehydro-3′-deoxythymidine) phenyl
N-bis(2-chloroethyl) phosphoramidate
TLC: Rf: 0.51 [(CHCl3/MeOH) (9:1)], m.p. 59 °C; H
NMR (CDCl3) d 8.15(bs), 7.45–7.42(m), 7.42–7.27(m), 7.26–
7.19(m), 7.08–7.03(m), 6.36–6.35(d), 5.91–5.89(d), 5.04(s),
4.36–4.32(m), 4.0–3.92(m), 3.71–3.65(m), 3.61–3.54(m),
1.82(s), 1.58(bs), 1.34–1.32(m); 31P NMR (CDCl3): 3.11; IR
m 3228, 3064, 2918, 2848, 1743, 1689, 1485, 1385. 1246,
1223, 1153, 1113, 1088, 1036, 928, 837 cm–1; HPLC RT:
11.23 min, [␣]D = –30.
IR (KBr): 3184, 3061, 2365, 2329, 1690, 1490, 1455, 1249,
1087, 934, 774, 699 cm–1
.
1H NMR (CDCl3) d 1.86 (d, 3H, 5-CH3), 3.39–3.61 (m,
8H, NCH2CH2Cl), 4.23–4.40 (m, 2H, 5′-H), 5.03 (s, 1H,
4′-H), 5.87–5.96 (m, 1H, 1′-H), 6.28–6.37 (m, 1H, 2′-H), 7.00
(m, 1H, 3′-H), 7.17–7.20 (m, 2H, Ar-2, 6), 7.21 (s, 1H, 6-H),
7.31–7.34 (m, 2H, Ar-3, 5), 8.55 (s, 1H, 3-NH); 13C NMR
(CD3OD) d 12.90, 42.19, 49.67, 67.95, 84.79, 90.13, 111.61,
120.24, 125.66, 127.70, 130.10, 133.25, 135.71, 150.79,
163.60; 31P NMR d 4.66, 5.27; HPLC: RT 12.4, 14.1 min.
2.3.7. 5′-[4-Bromophenyl methoxy alaninylphosphate]-
2′,3′-didehydro-3′-deoxylthymidine isomer 2 (R)
1
TLC: Rf: 0.51 [(CHCl3/MeOH) (9:1)], m.p. 160 °C; H
NMR (CDCl3) d 8.21(bs), 7.45–7.43(m), 7.42–7.27(m), 7.26–
7.11(m), 7.11–7.09(m), 7.01–6.99(m), 6.31–6.28(d), 5.94–
5.91(d), 5.01(s), 4.30–4.26(m), 3.96–3.62(m), 1.88(s),
1.59(bs), 1.40–1.35(m); 31P NMR (CDCl3): 2.56; IR m 3423,
3242, 2924, 2852, 1741, 1693, 1589, 1485, 1385, 1248, 1223,
1153, 1112, 1090, 1038, 1018, 930 cm–1; HPLC RT: 12.1 min,
[␣]D = –27.5.
2.3.2. 5′-(2′,3′-Didehydro-3′-deoxythymidine)
4-methylphenyl N-bis(2-chloroethyl) phosphoramidate
IR (KBr): 3037, 2959, 2360, 1692, 1507, 1465, 1248, 1089,
941, 818, 756 cm–1.
1H NMR (CDCl3) d 1.85 (s, 3H, 5-CH3), 2.32 (s, 3H, CH3),
3.37–3.60 (m, 8H, NCH2CH2Cl), 4.22–4.41 (m, 2H, 5′-H),
5.03 (s, 1H, 4′-H), 5.92 (m, 1H, 1′-H), 6.33 (m, 1H, 2′-H),
6.97–7.14 (m, 5H, Ar-2, 3, 5, 6; 6-H; 3′-H), 8.23 (br, 1H,
3-NH); 13C NMR (CD3OD) d 12.89, 21.11, 42.21, 49.71,
67.32, 84.79, 90.11, 111.59, 119.93, 127.67, 130.53, 133.32,
135.41, 135.77, 150.70, 163.45; 31P NMR (CDCl3) d 4.79,
5.42; HPLC: RT: 21.9, 25.3 min.
2.4. General procedure for synthesis of substituted
phosphoramidate derivatives of stavudine (preparation
of ’D’ isomers)
Triethylamine (4.2 ml, 30 mmol) was added drop wise to
a stirred solution of POCl3 (1.53 g, 10 mmol) and substituted
phenol (10 mmol) in anhydrous chloroform (40 ml) at 0 °C.
The reaction was allowed to warm to room temperature and
stirred for 15 h. Then reaction 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 above reaction
flask. After stirring at room temperature for 6 h, d4T (0.45 g,
2.0 mmol) was added to above reaction flask and the reaction
solution was stirred for 4 days. The solvent 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.
2.3.3. 5′-(2′,3′-Didehydro-3′-deoxythymidine)
4-methoxyphenyl N-bis(2-chloroethyl) phosphoramidate
IR (KBr): 3066, 2958, 2360, 2339, 1695, 1506, 1462, 1249,
1091, 1034, 936, 838 cm–1.
1H NMR (CDCl3) d 1.86 (s, 3H, 5-CH3), 3.37–3.60 (m,
8H, NCH2CH2Cl), 3.79 (s, 3H, OCH3), 4.22–4.39 (m, 2H,
5′-H), 5.03 (s, 1H, 4′-H), 5.92 (m, 1H, 1′-H), 6.33 (m, 1H,
2′-H), 6.83–6.87 (m, 2H, Ar-2, 6), 7.09–7.14 (m, 2H, Ar-3,
5), 7.34 (s, 1H, 6-H), 7.27 (m, 1H, 3′-H), 8.14 (s, 1H, 3-NH);
13C NMR (CD3OD) d 12.90, 42.22, 49.69, 55.95, 67.83,
84.79, 90.11, 114.99, 121.14, 127.68, 133.31, 135.36, 135.76,
150.70, 163.80; 31P NMR (CDCl3) d 5.09, 5.69; HPLC: RT:
14.4, 16.6 min.
2.3.4. 5′-(2′,3′-Didehydro-3′-deoxythymidine)
4-bromophenyl N-bis(2-chloroethyl) phosphoramidate
IR (KBr): 3171, 3046, 2361, 2339, 1693, 1485, 1467, 1251,
1090, 930, 837, 778 cm–1.
2.4.1. d4T-5′-p-Bromophenyl methyl-D-alaninyl phosphate
(10)
1H NMR (CDCl3) d 1.90 (s, 3H, 5-CH3), 3.42–3.84 (m,
8H, NCH2CH2Cl), 4.18–4.40 (m, 2H, 5′-H), 5.04 (s, 1H,
4′-H), 5.93–5.96 (m, 1H, 1′-H), 6.29–6.37 (m, 1H, 2′-H), 7.00
(m, 1H, 3′-H), 7.07–7.12 (m, 2H, Ar-2, 6), 7.34 (s, 1H, 6-H),
7.44–7.47 (m, 2H, Ar-3, 5), 8.36 (s, 1H, 3-NH); 13C NMR
(CD3OD) d 12.90, 42.19, 49.62, 67.83, 84.87, 90.18, 122.02,
127.71, 133.12, 134.47, 135.27, 135.58, 136.13, 150.67,
163.80; 31P NMR(CDCl3) d 4.71, 5.22; HPLC: RT: 31.8,
35.8 min.
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. [␣]D
{MeOH} –25.2°: HPLC: 12.72, 12.92; % purity: >98.

T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
455
2.4.2. d4T-5′-p-fluorophenyl methoxy-D-alaninyl phosphate
(11)
1H NMR (300 MHz, CDCl3) d 1.33 (q, 3H, J = 4.2 Hz),
3. Results and discussion
Due to the stereochemistry of its phosphorous chiral cen-
ter, stampidine exists as a mixture of two possible diastere-
omers (Fig. 1). The TLC profile of the diastereomeric mix-
ture of stampidine shows a single spot with an Rf value of
0.5 using a mixture of chloroform/methanol in the ratio of
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.
1
9:1. Fig. 2A depicts the H NMR spectrum of stampidine
31P NMR (121 MHz, CDCl3) d 2.96, 3.34; UV (MeOH) kmax
:
with chemical shifts consistent with its structure. Fig. 2B
shows the 31P NMR spectrum of stampidine with two sharp
peaks confirming the presence of two diastereomers. The
HPLC analysis of stampidine shows two distinct peaks cor-
responding to its putative steroisomers (Fig. 2C). The indi-
vidual stereoisomers were separated by preparative HPLC due
to their distinct retention times of 20.0 and 21.2 min, respec-
tively, lyophilized, and examined by IR, polariometry, HPLC,
NMR, and melting point analysis. The IR spectra of the puri-
fied diastereomers were identical with a characteristic band
at 1690 cm–1 due to the C=O unit in their structure. No sig-
nificant differences in optical rotation were observed (–30°
vs. –27.5°) by polarimetric analysis and their individual 1H
NMR spectra were identical (Fig. 2D and G). Fig. 2E shows
the 31P NMR spectrum of diastereomer #1 with a sharp sig-
nal at 3.20 ppm and Fig. 2H shows the 31P NMR spectrum of
diastereomer #2 with a sharp signal at 2.56 ppm in agree-
ment with the two peaks observed in the 31P NMR spectrum
of the diastereomeric mixture of stampidine. The melting point
analysis yielded melting points of 59–60 °C for the mixture,
60 °C for diastereomer #1 and 160 °C for diastereomer #2.
The HPLC profiles of individual isomers of stampidine is
shown in Fig. 2F and I. Repetitive HPLC and 31P NMR analy-
ses during a 36 h kinetics experiment confirmed that the sepa-
rated diastereomers are stable and do not undergo intercon-
version. The in vitro anti-HIV potency of the purified
diastereomers (IC50: 0.001 µM for both) was identical to the
in vitro anti-HIV potency of the diastereomeric mixture of
stampidine (IC50: 0.001 µM).
266 nm. [␣]D {MeOH} –22.2°: HPLC: 10.25, 10.35 min. %
purity: 98.0.
2.4.3. d4T-5′-p-Methylphenyl methoxy-D-alaninyl
phosphate (12)
1H NMR (300 MHz, CDCl3) d 1.32 (t, 3H, J = 7.2 Hz),
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. [␣]D {MeOH} –24.6°: HPLC: 11.33,
11.44 min. % purity: >98.
2.4.4. d4T-5′-p-Methoxyphenyl methoxy-D-alaninyl
phosphate (13)
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),
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.
2.4.5. d4T-5′-Phenyl methoxy-D-alaninyl phosphate (14)
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), 7.19 (d, 2H, 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) kmax: 266 nm. HPLC: 12.71, 12.88 min.
We first set out to evaluate the ability of the serine pro-
tease Subtilisin Carlsberg to hydrolyze nine different phos-
phoramidate derivatives of stavudine, including stampidine.
The chemical reactions of Subtilisin Carlsberg with phospho-
ramidate derivatives were monitored using HPLC and the rate
of hydrolysis at room temperature was calculated for the two
diastereomers of each compound using a first order rate equa-
tion. The rate constants are given in Table 1. The selectivity
2.4.6. d4T-5′-p-Chlorophenyl methoxy-D-alaninyl
phosphate (15)
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), 7.02
(s, 1H), 7.14 (d, 2H, J = 8.1 Hz), 7.24 (s, 1H), 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 2.77, 3.16; UV (MeOH) kmax: 266 nm. HPLC:
10.00 min.
Fig. 1
. Structures of two individual isomers of 5′-[4-bromophenyl methoxy
alaninylphosphate]-2′,3′-didehydro-3′-deoxylthymidine.

456
T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
Fig. 2
.
1HNMR, 31P NMR and HPLC profiles of distereoisomers as well as separated individual isomers of 4- bromophenyl phosphoramidate (stampdidine).
index indicates the relative rates of hydrolysis for the two
diastereomers. Notably, the diastereomers corresponding to
the peak#2 in the HPLC chromatograms of these compounds
were found to undergo hydrolysis much faster than those cor-
responding to peak#1 (Fig. 3). Subtilisin Carlsberg was able
to differentiate between the two diastereomers of the unsub-
stituted control phosphoramidate derivative of stavudine
showing an almost 10-fold difference in the rate of hydroly-
sis (0.7 vs. 7.0).
Hydrolysis with Substilisin Carlsberg was not signifi-
cantly affected by electron donating or electron withdrawing
substituents (Hammett’s Sigma) (Spearman’s Rank Correla-
tion, Rho = +0.3, P = 0.4 for peak 1; Spearman’s Rank Cor-
relation, Rho = 0.0, P = 1 for peak 2) demonstrating that elec-
tronic factors play a minor role during enzymatic hydrolysis
with this particular serine protease, unlike chemical hydroly-
sis. [25] Comparison of the rates of hydrolysis between iso-
mer 1 and isomer 2 among the stavudine derivatives studied
showed that most of the compounds had a selectivity index
value ranging from 0.1 to 0.5 for Subtilisin Carlsberg, 0.8–
26.0 for Carcia Papaya and 0.02–0.22 for alanine racemase.
Marked variation in the selectivity index was observed for
Carcia Papaya demonstrating that this enzyme discriminated
the phosphorus isomers preferentially as compared to other
enzymes used in the present study.
Table 1
Rate Constants for Hydrolysis of Phosphoramidate Derivatives of Stavudine
using Enzyme Protease Subtilisin Carlsberg and Carcia Papaya at Room tem-
perature
Compound#
X
Subtilisin Carlsberg
Carcia Papaya
We next examined the ability of the cysteine protease, Car-
cia Papaya to hydrolyze these phosphoramidate derivatives.
Table 1 presents the rates of hydrolysis and selectivity index
obtained for the substituted phosphoramidate derivatives at
room temperature. The rates of hydrolysis of stavudine deriva-
tives were much slower for Carcia Papaya than Subtilisin
Carlsberg. The most striking difference is for the preferential
hydrolysis of isomer 1 by Carcia Papaya. Fig. 4 shows rep-
resentative HPLC chromatogram profiles for compound 4
treated with enzyme Carcia Papaya for various time intervals
Alanine racemase also hydrolyzed these derivatives and
showed a preferential hydrolysis of one isomer over the other
(Table 2 and Fig. 5). However, the rate of hydrolysis was much
#1* #2*
SI
#1* #2* SI
(#1/#2)
0.5 0.21 0.26
(#1/#2)
0.8
1
2
3
4-OMe 2.76
5.87
3.83
4.62
3Br
2Cl-
4Br
4-F
1.43
2.07
0.4
2.70 0.07 38.6
0.52 0.02 26.0
0.5
4
5
6
7
8
1.34
3.02
0.4
0.6
0.3
0.1
0.2
0.4
0.68 0.13
5.2
2-Br
2-Cl
H
9.81 16.78
0.72 0.06 12.0
0.29 0.02 14.5
1.36
0.74
0.78
2.73
4.34
6.97
3.68
6.37
0.73 0.12
0.86 0.42
1.09 0.77
6.1
2.0
1.4
4-Cl
9 Stampidine 4-Br
The rate constants for isomer (#1) and isomer (#2) in h–1. SI indicates the
selectivity index calculated as the ratio of the rate constants for #1 and #2.

T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
457
Fig. 3. HPLC profiles of fluoro substituted aryl phosphoramidate derivative compound #4 treated with enzyme Subtilisin Carlsberg for various time intervals.
slower as compared to Subtilisin Carlsberg. Interestingly, there
was a significant correlation between electron donating or
electron withdrawing substituents (Hammett’s Sigma) and the
rate of hydrolysis for peak 2 (Spearman’s Rank Correlation,
Rho = +0.77, P = 0.01) in the presence of alanine racemase,
suggesting a different mechanism of hydrolysis compared to
that catalyzed by the Subtisilin Carlsberg or Carcia Papaya.
Taken together, these findings prompt the hypothesis that
proteases may be involved in the enzymatic hydrolysis of sta-
vudine phosphoramidate derivatives necessary for the forma-
tion of the active metabolite ala-d4T-MP (Scheme 1). In the
above scheme, the hydrolysis of the carbomethoxy ester side
chain to form the carboxylic acid represents the first step. We
hypothesize that the phosphoramidate derivatives of stavu-
dine undergo enzymatic hydrolysis by the presence of sub-
tilisin or other proteases as follows: The water molecule
attacks the phosphorus center of these derivatives resulting a
pentacoordinated phosphorus complex having an oxyanion.
Similar types of pentacoordinated phosphorus intermediates
in enzyme catalyzed reactions has been proposed by Kubiak
et al. [30]. In the next step, the negative charge on the oxygen
is transferred to eliminate the phenoxy moiety forming d4T-
alaninyl methylester monophosphate (Scheme 2). Further
hydrolysis by the enzyme yields d4T alanine monophos-
phate.The scheme also depicts the aminoacid in the protein
chain involved in the process. In the case of these proteases,
we believe both histidine and serine are involved. This mecha-
nism is in agreement with the proposed hydrolysis of phos-
phonate esters, by serine proteases as disclosed by Sko-
rdalakes et al. [31].Alternatively, there may be a simultaneous

458
T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
Fig. 4
.
HPLC profiles of the fluoro- substituted aryl phosphoramidate derivative compound #4 treated with enzyme Carcia Papaya for various time intervals.
attack of two centers in the molecule. Unlike those phospho-
nate esters reported in the literature [31], these phosphorami-
date derivatives of stavudine posses another ester group in
their structure. Therefore, we hypothesize that hydrolysis by
serine protease enzymes similar to that reported in the previ-
ous discussion. However, in the first pathway, we propose the
formation of d4T alaninyl methylester attached monophos-
phate as an intermediate step preceding the formation of the
metabolite alanine-d4T-monophosphate.
quent step the intermediate is converted into the active
metabolite [E]. The conversion of [B] to [E] could occur either
stepwise, spontaneously or enzymatically.
Evidence from the literature suggests that the conversion
of [B] to [E] is likely to be spontaneous [32–36]. The cyclic
phosphoramidate immediately hydrolyzes to the active
metabolite. In order to confirm this hypothesis, we tried to
synthesize the cyclic phosphoramidate following a previ-
ously reported method [37–40]. Although we could prepare
the simple cyclic phosphoramidate derivatives derived from
glycine, several attempts to isolate the stavudine substituted
cyclic phosphoramidate failed under our experimental con-
As discussed above, we have shown that in protease-
mediated hydrolysis the methyl ester side chain is converted
into the carboxylic acid [B] (see Scheme 1). In the subse-

T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
459
Table 2
Table 3
Rate constants for hydrolysis of phosphoramidate derivatives of stavudine
using enzyme alanine racemase at room temperature
Area under curve (HPLC) observed for the chloroethyl substituted aryl phos-
phoramidate derivatives of stavudine with protease Subtilisin Carlsberg at
various time intervals
Compound#
X
Isomer 1a
0.070
0.02
Isomer 2 a SI (#1/#2)
X
0 min
#2
60 min
#2
140 min
#1 #2
Comments
1
2
3
4
5
6
7
8
4-OMe
3Br
0.67
0.85
2.39
0.73
1.52
1.25
0.65
1.02
1.09
0.10
0.02
0.22
0.12
0.11
0.08
0.02
0.23
0.20
#1
OMe 223
#1
210
176
186
209
226
180
168
220
203
222
180
184
218
217
176
186
210
No hydrolysis
2Cl-4Br
4-F
0.53
H
180
176
217
178
174
225
″
″
″
0.09
Br
Me
2-Br
2-Cl
H
0.17
0.10
0.01
#1 and #2 indicates each of the isomers peak observed in HPLC chromato-
gram.
4-Cl
0.23
9 = Stampidine 4-Br
0.21
Table 4
a The rate constants for isomer (#1) and isomer (#2) in h–1. SI indicates the
Data on protease inhibition by protease inhibitors during hydrolysis of phos-
selectivity index calculated as the ratio of the rate constants for #1 and #2.
phoramidate derivatives of stavudine a
ditions. This result suggests that the conversion of [B] to [E]
must be fast. At this time it is not known whether or not enzy-
matic influence is required for this step.
We next set out to experimentally determine if an attack
on the carboxymethylester side chain occurs during protease-
mediated hydrolysis. To this end, we synthesized chloroethyl
substituted stavudine phosphoramidate derivatives that lack
the carbomethoxy ester group in their structure (Scheme 3).
The rate of protease-mediated hydrolysis was examined for
these compounds using Subtilisin Carlsberg. No hydrolysis
was observed even after 140 min of incubation with this pro-
tease (Table 3). These results indicate that the hydrolysis of
these compounds by the protease requires the ester group and
does not involve an attack on the phosphorous chiral center
alone. We propose that stampidine undergoes rapid enzy-
matic hydrolysis in the presence of protease according to the
following putative pathway (Scheme 1). In the first step, com-
pound B is generated by protease -mediated hydrolysis of the
Compound#
X
Enzyme used
Protease
inhibitor
Inhibition
9
8
9
8
9
8
9
4-Br
4-Cl
4-Br
4-Cl
4-Br
4-Cl
4-Br
Subtilisin, Carlsberg
Subtilisin, Carlsberg
Carcia Papaya
Antipain HCl Yes
″
Yes
Yes
Yes
Yes
Yes
Yes
″
Carcia Papaya
″
Carcia Papaya
Leupeptin
Carcia Papaya
″
Carcia Papaya
EDTA free
tabs
8
2
4-Cl
3-Br
Carcia Papaya
Carcua Papaya
″
Yes
Yes
″
a No hydrolysis of substrates observed even after several hours at room
temperature in presence of protease inhibitors.
Scheme 1. Putative enzymatic hydrolysis pathway for phosphoramidate derivatives of stavudine in the presence of proteases.

460
T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
Fig. 5
.
HPLC profiles of bromo substituted aryl phosphoramidate derivative (9) treated with enzyme alanine racemase at various intervals of time.
methylester side chain of stampidine. The subsequent step
involves an intramolecular cyclization step involving the phos-
phorus center with simultaneous elimination of the phenoxy
group to form the cyclic intermediate (D). In the presence of
water, this intermediate is converted into the active metabo-
lite (E). We believe that the protease hydrolyzes the methyl
ester group of the L-alanine side chain to form the cyclic inter-
mediate D in a stereoselective fashion. This notion is sup-
ported by experimental data showing that chloroethylsubsti-
tuted derivatives of stampidine which possess a chloroethyl
linker unit instead of a methyl ester side chain were resistant
to protease-mediated hydrolysis which excludes the possibil-
ity of a direct hydrolysis of stampidine at the phosphorous
center (Table 3). Thus, our model indicates that the protease-
mediated formation of the cyclic intermediate is a key step in
metabolism of stampidine and relies on the initial configura-
tion of the stereoisomers. We postulate that the stereoisomer
corresponding to peak#1 in the HPLC chromatogram enters
preferentially into the active site of the protease and under-
goes rapid hydrolysis to form the cyclic intermediate.At sub-
sequent steps of the metabolism, the chirality of the cyclic
intermediate at its phosphorous center becomes irrelevant due
to the fact that the active metabolite of stampidine which is
the final product of this pathway is not chiral. In order to
establish that indeed these protease enzymes are involved and
that these phosphoramdiate derivatives act as substrate com-
petitors, we examined the specificity of the interaction using
protease inhibitors to block the enzymatic hydrolysis.

T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
461
Table 4 shows the enzymes used and the protease inhibi-
tors selected for the said study. We chose only two com-
pounds from the series to illustrate the role of protease inhibi-
tors. Both 4-Cl and 4-Br substituted phosphoramidate
derivatives of stavudine were found to undergo hydrolysis in
the presence of serine proteases; however, introduction of anti-
pain, leupeptin and EDTA-free protease inhibitor cocktail
were found to completely block the protease-mediated
hydrolysis of these stavudine phosphoramidates.
Fig. 6
.
HPLC profiles of bromo substituted hosphoramidate derivative (9)
with Subtilisin Carlsberg in presence of protease inhibitor(antipain).A: Subs-
trate without enzyme, B: substrate with Enzyme C: Substrate with enzyme
and protease inhibitor.
We were able to show experimentally that protease inhibi-
tors block the hydrolysis of these phosphoramidate deriva-
tives and formation of their active metabolite ala-d4T-MP.
Table 5
Fig. 6 depicts the HPLC profile when a protease inhibitor
was added to the reaction medium. Panel A shows the HPLC
chromatogram of the 4-bromo-phenyl substituted phospho-
ramidate derivative of stavudine (=stampidine) before the
addition of Subtilisin Carlsberg. Panel B represents the HPLC
profile of stampidine 1 h after addition of Subtilisin Carls-
berg. Panel C depicts the HPLC profile of the reaction mix-
ture after addition of Subtilisin Carlsberg in the presence of
the protease inhibitor antipain.
First order rate constants for disappearance of starting material using various
protease enzymes with arylphosphoramidate derivatives of stavudine at room
temperature
Compound#
X
Amino acid Carlsberg Carcia
HTLVIIIB
configuration
Papaya (nM)
In an attempt to further confirm the methyl ester group of
the L-alanine side chain of the stavudine phosphoramidates
as the target site for the proteolytic hydrolysis, we replaced
the L-alanine side chain with a D-alanine side chain. As evi-
denced in Table 5 and Fig. 7, these “D-isomers” (Fig. 8) were
resistant to proteolytic hydrolysis by Subtilisin Carlsberg or
Carcia Papaya. Furthermore, the D-isomers were substan-
tially less active against HIV than the corresponding
L-isomers, which is consistent with the notion that pro-
teolytic hydrolysis at the methyl ester side chain of the sta-
9
10
4
11
12
1
13
7
Br
Br
F
F
Me
’L’
’D’
’L’
’D’
’D’
9.10
0.23
4.36
0.20
0.04
8.63
0.02
7.71
0.03
4.46
0.15
1.86
0.01
0.81
0.01
0.01
0.47
0.01
0.85
0.01
1.28
0.02
1
0
212 155
1
851 720
427 107
6
0
OMe ’L’
OMe ’D’
3
1290 313
0.6
1787 215
0.3
5837 2304
H
H
Cl
Cl
’L’
’D’
’L’
’D’
2
14
8
15
1

462
T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
Scheme 2
.
Proposed mechanism of enzyme catalysis during hydrolysis of phosphoramidate derivatives of stavudine.
vudine phosphoramidates plays an important role for the acti-
vation of these anti-HIV prodrugs.
4. Conclusions
Aryl substituted phosphoramidate derivatives of stavu-
dine were found to undergo hydrolysis in the presence of pro-
teases. We hypothesize that the methoxy ester group on the
side chain of these derivatives is first converted into the cor-
responding carboxylic acid derivative, which then undergoes
intramolecular cyclization to form the final active metabo-
Scheme 3. Synthetic scheme for chloroethyl substituted phosphoramidate
derivatives of stavudine.

T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
463
Fig. 7. Protease-mediated hydrolysis profile of compound 9 at various time intervals.
lite. A direct attack of the protease on the phosphorus site of
these molecules was ruled out using phosphoramidate deriva-
tives devoid of a carboxymethylester group in their struc-
tures. We were able to show experimentally that protease
inhibitors block the hydrolysis of these phosphoramidate
derivatives and formation of their active metabolite. The
mechanism of protease-mediated hydrolysis of phosphorami-
date derivatives of stavudine is in agreement with the pro-
posed mechanism of hydrolysis of phosphonate esters. Our
model indicates that the protease-mediated formation of a
cyclic intermediate is a key step in metabolism of stampidine
and relies on the initial configuration of the stereoisomers.
5.1. Experimental conditions for protease enzymatic study
For the kinetic study, a known amount of the phosphora-
midate derivative was carefully weighed (5–7 mg) using a
Metller analytical balance and transferred into a scintillation
glass vial. Three milliliters of methanol was added to this
vial and the contents were vortexed for 2 min until a homog-
enous solution was obtained. Hundred microliters of this solu-
tion was transferred into another scintallation vial and to this
was added 950 µl of water and the contents vortexed. In par-
allel, 5–6 mg amounts of the respective proteases were
weighed and transferred to a volumetric flask. Eight millili-

464
T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
Fig. 8. Protease-mediated hydrolysis profile of compound 10 at various time intervals.
ters of water was added and the contents were shaken to dis-
solve the protease. The reaction mixtures for the kinetic study
were prepared as follows: From the stock solution of the com-
pound, 500 µl was pipetted out into another glass vial and to
this 500 µl of diluted solution of the enzyme was added and
the contents were shaken to form a homogenous solution.
From this reaction mixture 50 µl per time point was used for
HPLC analysis using a Lichrospher (RP) analytical column
of (4 × 250 mm). The eluent used for the HPLC was
water/TFA/TEA (0.1%) and CH3CN with a 65:35 ratio. The
column was maintained at room temperature. The flow rate
was maintained at 1 ml min–1, the detection wavelength was
adjusted to 265 nm and the reference wavelength was kept at
400 nm.
5.2. Statistical analysis
Hydrolysis rates were determined by fitting single expo-
nential decay equations to the disappearance of each isomer
substrate in the presence of enzyme. 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 per hour since some of
the reactions were too slow to obtain meaningful results. Ham-

T.K. Venkatachalam et al. / European Journal of Medicinal Chemistry 40 (2005) 452–466
465
[5] R. Vig, C. Mao, T.K. Venkatachalam, L. Tuel-Ahlgren, E.A. Sudbeck,
F.M. Uckun, Bioorg. Med. Chem. 8 (1998) 1789.
mett Sigma values were correlated to the log transformed
hydrolysis rate constants using a linear regression model (JMP
Software, SAS Institute Inc.).All P-values less than 0.05 were
considered significant.
[6] C. Mao, R. Vig, T.K. Venkatachalam, E.A. Sudbeck, F.M. Uckun,
Bioorg. Med. Chem. Lett. 8 (1998) 2213.
[7] P.A. Furman, J.A. Fyfe, M.H. St. Clair, K. Weinhold, J.L. Rideout,
G.A. Freeman, et al., Proc. Natl. Acad. Sci. USA 83 (1986) 8333.
5.3. Estimation of products
[8] Z. Hao, D.A. Cooney, N.R. Hartman, C.F. Perno, A. Fridland, A.L. De
Vico, et al., Mol. Pharmacol. 34 (1988) 431.
The amount of products observed during the reaction of
these phosphoramidates were estimated from the area ob-
tained from the HPLC profiles.Authentic samples of the prod-
ucts when possible were run to identify the peaks observed
during the reaction. Further confirmation of the product struc-
tures 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 per hour
as some of the reactions were too slow to obtain meaningful
results.
[9] J. Balzarini, D.A. Cooney, M. Dalal, G.I. Kang, J.E. Cupp, E. De
Clercq, et al., Mol. Pharmacol. 32 (1987) 798.
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5.4. In vitro assays of anti-HIV activity
[16] C. McGuigan, R.N. Pathirana, N. Mahmood, K.G. Devine, A.J. Hay,
Antiviral Res. 17 (1992) 311.
Normal human peripheral blood mononuclear cells (PB-
MNC) from HIV-negative donors were cultured 72 h in RPMI
1640 supplemented with 20% (v/v) heat-inactivated fetal
bovine serum (FBS), 3% interleukin-2, 2 mM L-glutamine,
25 mM HEPES, 2 g l–1 NaHCO3, 50 mg ml–1 gentamicin,
and 4 mg ml–1 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 atmo-
sphere. Subsequently, cells were cultured in 96-well micro-
titer plates (100 ml per well; 2 × 106 cells per ml) in the pres-
ence of various concentrations of d4T phosphoramidates and
aliquots of culture supernatants were removed from the wells
on the seventh day after infection for p24 antigen assays, as
previously described. The applied p24 enzyme immunoassay
(EIA) was unmodified kinetic assay commercially available
from Coulter Corporation/Immunotech. Inc. (Westbrooke,
ME), which utilizes a murine mAb to HIV core protein coated
on to microwell strips to which the antigen present in the test
culture supernatant samples binds. Percent viral inhibition was
calculated by comparing the p24 values from untreated
infected cells (i.e. virus controls).
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