Hybrids of [TSAO-T]-[foscarnet]: The first conjugate of foscarnet with a non-nucleoside reverse transcriptase inhibitor through a labile covalent ester bond

Article abstract of DOI:10.1021/jm031045o

This paper describes the first example of combination of non-nucleoside reverse transcriptase inhibitors such as TSAO derivatives and foscarnet (PFA) in a single molecule through a labile covalent ester bond. The essential criteria in the design of these hybrids [TSAO-T]-[PFA] was to explore if the conjugation of foscarnet with the highly lipophilic TSAO derivative may facilitate the penetration of the conjugates through the cell membrane and if the hybrids escape extracellular hydrolysis and regenerate the parent inhibitors intracellulary. Several [TSAO-T]-[PFA] conjugates proved markedly inhibitory to HIV-1. Some of them also showed potent activity against PFA-resistant HIV-1 strains but fewer had detectable inhibitory activity against TSAO-resistant HIV-1 strains. These results indicated a pivotal role of the TSAO component of the hybrid but not the PFA component in the activity of the conjugates. Moreover, stability studies of the [TSAO-T]-[PFA] conjugates demonstrated that the compounds were stable in PBS whereas some of the conjugates regenerated the parent inhibitors in extracts from CEM cells.

Full text of DOI:10.1021/jm031045o

3418
J. Med. Chem. 2004, 47, 3418-3426
Hybrids of [TSAO-T]-[Foscarnet]: The First Conjugate of Foscarnet with a
Non-nucleoside Reverse Transcriptase Inhibitor through a Labile Covalent
Ester Bond†
Sonsoles Vela´zquez,*,‡ Esther Lobato´n,‡ Erik De Clercq,§ Dianna L. Koontz,| John W. Mellors,|
Jan Balzarini,§ and Mar´ıa-Jose´ Camarasa‡
Instituto de Quı´mica Me´dica (C.S.I.C.), Juan de la Cierva 3, E-28006 Madrid, Spain, and Rega Institute for Medical
Research, K. U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium, and University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261
Received September 26, 2003
This paper describes the first example of combination of non-nucleoside reverse transcriptase
inhibitors such as TSAO derivatives and foscarnet (PFA) in a single molecule through a labile
covalent ester bond. The essential criteria in the design of these hybrids [TSAO-T]-[PFA] was
to explore if the conjugation of foscarnet with the highly lipophilic TSAO derivative may
facilitate the penetration of the conjugates through the cell membrane and if the hybrids escape
extracellular hydrolysis and regenerate the parent inhibitors intracellulary. Several [TSAO-
T]-[PFA] conjugates proved markedly inhibitory to HIV-1. Some of them also showed potent
activity against PFA-resistant HIV-1 strains but fewer had detectable inhibitory activity against
TSAO-resistant HIV-1 strains. These results indicated a pivotal role of the TSAO component
of the hybrid but not the PFA component in the activity of the conjugates. Moreover, stability
studies of the [TSAO-T]-[PFA] conjugates demonstrated that the compounds were stable in
PBS whereas some of the conjugates regenerated the parent inhibitors in extracts from CEM
cells.
Introduction
Emergence of HIV drug resistance and the need for
long-term antiretroviral treatment are currently the
main causes for the failure of antiretroviral therapy.
Combination of different anti-HIV agents has become
the standard clinical practice to keep the viral load at
undetectable levels and to prevent emergence of virus
drug resistance.1,2
Among the human immunodeficiency virus (HIV)
reverse transcriptase (RT) inhibitors, the so-called non-
nucleoside RT inhibitors (NNRTIs) have gained a
primary role in the treatment of HIV infection in
combination with nucleoside analogue RT inhibitors
(NRTIs) and HIV protease inhibitors (PIs).3,4 The virus
can be markedly suppressed for a relatively long period
of time when exposed to multiple drug combination
therapy (highly active antiretroviral therapy, HAART).
The [2′,5′-bis-O-(tert-butyldimethylsilyl)-â-d-ribofura-
nosyl]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-diox-
ide) nucleosides (TSAO) are a rather unique class of
NNRTIs5,6 that seem to interact at the interface between
the p51 and p66 subunits of HIV-1 RT.7 The prototype
compound of this family is the thymine derivative
designated as TSAO-T (1) and the most selective
compound is its 3-N-methyl substituted derivative
TSAO-m3T (2) (Figure 1). Biochemical studies have
revealed that both TSAO-T and its 3-N-ethyl derivative
Figure 1. Structures of TSAO-T (1), TSAO-m3T (2), and
phosphonoformic acid (PFA, 3).
are able to destabilize the p66/p51 RT heterodimer in a
concentration-dependent manner resulting in a loss of
its ability to bind DNA.8,9 This suggests a new and
different mechanism of inhibition of HIV-1 RT with
regard to the other known NNRTIs. HIV-1 resistance
to NNRTIs is primarily associated with substitution of
amino acids at the lipophilic NNRTIs binding pocket in
the p66 subunit.4 However, TSAO compounds select a
single mutation (Glu-138-Lys) in the p51 subunit of
HIV-1 RT.10
Phosphonoformate (PFA, foscarnet, 3) (Figure 1) is
an effective antiviral agent approved for intravenous
treatment of human cytomegalovirus (HCMV) retinitis
in patients with AIDS.11 Although primarily used in the
treatment of AIDS-related HCMV infection, PFA is also
effective against HIV replication.12 PFA is proposed to
inhibit HIV RT by blocking the pyrophosphate binding
site. Although PFA is a potent inhibitor of HIV RT, its
highly ionic nature at physiological pH is an impediment
to its cellular uptake.13
* To whom correspondence should be addressed. Telephone: +34-
91 5622900. Fax: +34-91 5644853. E-mail: iqmsv29@iqm.csic.es.
† Dedicated to Prof. Dr. Jan Balzarini with best wishes on the
occasion of his birthday.
‡ Instituto de Qu´ımica Me´dica (C.S.I.C.).
Numerous combination experiments have been per-
§ Rega Institute for Medical Research.
| University of Pittsburgh School of Medicine.
formed between different classes of RT inhibitors.1,2
10.1021/jm031045o CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/21/2004

Hybrids of [TSAO-T]-[Foscarnet]
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3419
Figure 3. Hybrids [TSAO-T]-[PFA] of general formula I.
Structures of TSAO derivative 4 and [TSAO-T]-[PFA] conju-
gates 5 and 6.
cleavage at physiological pH, or enzyme-mediated ca-
talysis.
Figure 2. Isobologram of the anti-HIV-1(IIIB) activity of
combinations of PFA and TSAO-m3T in CEM cell cultures.
Covalent conjugate combination of TSAO-T and PFA
would differ substantially from simple noncovalent
combination of these two drugs in that the conjugate,
transported initially as such into the cells, could have
a different antiviral profile. For example, an enzyme-
labile conjugate could gradually release the anti-HIV
parent compounds upon hydrolysis resulting in different
kinetics of uptake/release/availability to the target than
that afforded by the TSAO-T and PFA mixture.
As TSAO molecule the N-3 hydroxypropyl derivative
4 (Figure 3) was chosen because attachment of the
(CH2)3OH substituent to the N3 atom of the thymine of
the prototype TSAO-T resulted in a 2-fold better inhibi-
tor.7 Moreover, from a chemical point of view, compound
4 allows ester formation between PFA and the OH
functionality of the N3 thymine substituent. Two types
of [TSAO-T]-[PFA] adducts are possible, depending on
whether attachment of PFA to the hydroxyl group of
the TSAO derivative is via the carboxyl (5) or the
phosphonyl moieties (6) (Figure 3).
Although PFA ester derivatives in which the carboxyl
or phosphonyl group was linked to a nucleoside16 or, in
particular, to anti-HIV nucleosides have been previously
described,17-19 [TSAO-T]-[PFA] conjugates are, to the
best of our knowledge, the first example of conjugates
between PFA and NNRTIs. This paper describes the
synthesis of [TSAO-T]-[PFA] conjugates and their in
vitro anti-HIV activity against wild-type HIV-1 virus
and TSAO-resistant and PFA-resistant HIV-1 variants.
Stability studies of the [TSAO-T]-[PFA] conjugates in
cell extracts have also been carried out.
However, combinations of PFA with NRTIs other than
AZT, or with NNRTIs, are yet to be reported. Different
combinations using TSAO derivatives with either NRTIs
or NNRTIs have been explored.14,15 Interestingly, in
preliminary experiments we found that PFA was a
potent inhibitor of TSAO-characteristic HIV-1 RT/138
Lys mutant strain. The activity against the 138K
mutant was 1 order of magnitude higher than that
against virus wild type [IC50 (µg/mL) ) 0.34 vs 2.59,
respectively]. This observation prompted us to combine
both inhibitors in an attempt to prevent or delay the
emergence of TSAO-resistant virus. A variety of con-
centrations of TSAO-m3T and PFA have been combined
and added to CEM cell cultures infected with a low
multiplicity of HIV-1 infection. It would be interesting
to reveal whether combinations of TSAO-m3T and PFA
at a variety of concentrations are synergistic, additive
or antagonistic in displaying their anti-HIV activity.
Analysis of the antiviral activity of TSAO-m3T and PFA
combinations through construction of an isobologram
revealed additivity of the antiviral activity of both drugs
(Figure 2).
These results led us to combine TSAO derivatives and
foscarnet in a single molecule through a labile covalent
ester bond (I, Figure 3), as an alternative approach to
combination therapy, taking into consideration the
highly lipophilic nature of the TSAO molecule and the
hydrophilic nature of PFA. The major consideration in
the design of these hybrids was to explore whether the
lipophilic TSAO molecule may serve as driving-force for
PFA into the cells thus resulting in improvement in the
cell membrane permeability of PFA. Regardless of
whether the PFA was linked to the TSAO molecule
through the PO(OH)2 or the COOH group, a decreased
negative charge on this molecule (conjugate) relative to
PFA ought to facilitate movement (permeability) across
the cell membrane. The [TSAO-T]-[PFA] conjugate may
also escape extracellular hydrolysis and once inside the
cell would liberate the parent compounds. Therefore, we
prepared [TSAO-T]-[PFA] conjugates containing the
parent drugs linked via ester bonds to permit hydrolytic
Results
Synthesis. Linkage by a carboxylic ester bond to
prepare the target [TSAO-T]-[PFA] conjugates 5 was
first attempted by condensation of TSAO derivative 4
with (diethylphosphono)formic acid chloride in the pres-
ence of NEt3 and DMAP according to the procedures
described by Charvet et al.18a Alternatively, nucleoside
4 was first reacted with triphosgene in pyridine followed
by addition of triethyl phosphite.18a However, all these
acylation attempts were unsuccessful. In these reactions

3420 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Vela´zquez et al.
Scheme 1
Scheme 2
only unreacted material (4) together with deprotected
TSAO derivatives were isolated.
Linkage by a phosphoric ester bond was achieved
following the synthetic strategies summarized in Schemes
1-4. We first addressed our attention to the synthesis
of PFA diester intermediate 10 (Scheme 1) and 14
(Scheme 2) which after deprotection would give the
target conjugate 6 with the free carboxylic acid. Two
methods were used to prepare these [TSAO-T]-[PFA]
conjugate intermediates. In the first (Scheme 1), we
used the described method for the synthesis of PFA
diesters17b,18b which involves formation of a PFA triester
by reaction of phosphonochloridate and the appro-
priated alcohol, in the presence of triethylamine, fol-
lowed by deesterification with NaI. Thus, as shown in
Scheme 1, when trimethyl phosphonoformate 7 was
treated with PCl5 in CCl4 at 50 °C, and the crude
reaction product 8, after removal of volatile materials,
was reacted with the N-3 hydroxypropyl TSAO deriva-
tive 4 in dry CH2Cl2, the PFA triester 9 was obtained
in 91% yield. Compound 9 was subsequently treated
with NaI in dry THF, under argon, to give selective
O-methyl cleavage on phosphorus20 yielding the PFA
diester 10 as a sodium salt in 80% yield. The PFA
diester 14 was prepared by coupling the TSAO deriva-
tive 4 with a dichlorophosphonylformate (13)18a (Scheme
2) as follows. Tris(trimethylsilyl) phosphite was treated
with ethyl chloroformate 11 to yield ethyl [bis(trimethyl-
silyl)phosphono]formate 12 (Scheme 2) in 75% yield.
Treatment of compound 12 with SOCl2, according to the
procedure described by Vaghefi et al.,16a gave the ethyl
(dichlorophosphonyl)formate 13. Finally, coupling be-
tween the TSAO derivative 4 and 13 in CH2Cl2 in the
presence of NEt3 at -20 °C yielded the PFA diester 14
in 31% yield.
THF to avoid deprotection of the labile 5′-tert-butyldi-
methylsilyl group (TBDMS) (Scheme 3) failed to give
the final conjugate 6. Instead, mixtures of the 5′-de-
protected hydrolysis product 15 and the decarboxylated
derivative 16 (obtained via P-C bond cleavage) were
obtained. The products were isolated as the di-
sodium (15) and monosodium (16) salts in 53% and 43%
yield, respectively. Several previous studies have high-
lighted the overall hydrolytic instability of phosphono-
formate triesters,21 and the significant problem of hy-
drolitic P-C bond cleavage which results in the de-
struction of the parent drug structure. In the previous
studies the rate and product distribution are dependent
on pH and ester leaving group abalities.21a Interestingly,
when the saponification of PFA diester 10 was at-
tempted with 0.45 N NaOH, only the hydrolysis product
15 was isolated (Scheme 3). However, although hydroly-
sis can be diverted away from abortive P-C bond cleav-
age, deprotection of the 5′-TBDMS group of the sugar
moiety could not be avoided in the hydrolysis step. Sim-
ilar results were observed with [TSAO-T]-[PFA] con-
jugate 14 when submitted to the same saponification
procedures. All the silylation attempts of the 5′-depro-
tected [TSAO-T]-[PFA] conjugate 15 to give the desired
target compound 6 (Scheme 3) failed whatever the
experimental standard conditions (TBDMSCl/pyridine,
DMAP or imidazole) were used. The starting material
To prepare the final [TSAO-T]-[PFA] conjugate 6, the
PFA diester 10 (obtained in higher yield) was saponified
with aqueous sodium hydroxide in THF. Initial attempts
of base-catalyzed hydrolysis of 10 with 0.1 N NaOH in

Hybrids of [TSAO-T]-[Foscarnet]
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3421
Scheme 3
Scheme 4
conjugate 6 was obtained in 60% yield, by catalytic
hydrogenation (H2/Pd/C) of the benzyl intermediate
[TSAO-T]-[PFA] conjugate 20.
Biological Evaluation. Several TSAO-PFA conju-
gates were evaluated for their inhibitory activity against
HIV-1 and HIV-2 in MT-4 and CEM cell cultures (Table
1). Foscarnet and the parent TSAO compound 4 were
included as controls. Foscarnet was equally active
against both HIV-1 and HIV-2 with EC50 values ranging
between 105 and 140 µM. Foscarnet proved cytotoxic
at a CC50 of 230 µM (MT-4 cells). The TSAO derivative
4 was highly inhibitory against HIV-1 in both cell lines
(EC50: 0.01-0.03 µM) but was inactive against HIV-2
and cytotoxic at ∼ 3.9 µM [selectivity index (ratio
CC50/EC50) ∼ 100-380). Compound 9, representing the
dimethyl triester TSAO-PFA conjugate was ∼8-9-fold
less potent against HIV-1 but also 5-fold less cytotoxic.
It still lacked antiviral activity against HIV-2 at non-
toxic concentrations (10 µM). The benzyl derivative and
also the ethyl and methyl ester derivatives lost ∼100-
to 200-fold antiviral potency compared with the parent
compound 4. Interestingly, the unsubstituted (depro-
tected) TSAO-PFA conjugate 6 was markedly active
(EC50: 0.47-0.82 µM) (∼25- to 50-fold less active than
4) but was also less cytotoxic (CC50: 111-178 µM),
resulting in a selectivity comparable with the parental
TSAO derivative 4. It is important to note that the
deprotected conjugate 6 still lacked any measurable
activity against HIV-2.
When evaluated against a TSAO-m3T resistant HIV-1
strain containing the E138K mutation in its reverse
transcriptase (Table 1), only 14, 20, and 6 showed in-
hibitory activity (EC50: 20, 25 and 50 µM, respectively).
PFA was active against the mutant virus strain with
an EC50 of 87 µM (comparable to wild-type virus). In
contrast to PFA (IC50 for HIV-1 RT and HIV-1/138K RT
of 24 and 56 µM, respectively), all TSAO derivatives lost
potency against purified mutant RT enzyme (IC50 wild-
type HIV-1 RT: 3.7-11 µM; IC50 mutant HIV-1/138K
RT: 293 f 500 µM) (Table 2). These findings indicate
that the protected alkyl- and benzyl-substituted deriva-
tives as well as the deprotected TSAO-PFA conjugate
was recovered unchanged and/or P-C bond cleavage
was also observed in some of the experiments.
These results promted us to try a different strategy
for the synthesis of the disilylated target conjugate 6.
The approach involved the use of a benzyl as protecting
group for the carboxylic moiety of PFA (Scheme 4),
which would be removed in the last step under mild
conditions by catalytic hydrogenation, to prevent the
desilylation unwanted side-reaction. The PFA diester
benzyl derivative 20 was prepared by reaction of the
TSAO derivative 4 and benzyl (dichlorophosphonyl)-
formate 19, following a similar procedure to that de-
scribed for the synthesis of PFA diester 14. First, it was
necessary to prepare the phosphorylating reagent 19.
Thus, reaction of tris(trimethylsilyl) phosphite with ben-
zyl chloroformate (17) gave benzyl [bis(trimethylsilyl)-
phosphono]formate 18. Treatment of 18 with SOCl2
gave the phosphorylating reagent 19 in moderate yield.
Reaction of TSAO compound 4 with 19, in the pres-
ence of NEt3, led to the formation of the PFA diester
20 in 54% yield. Finally, the target [TSAO-T]-[PFA]

3422 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Vela´zquez et al.
Table 1. Inhibitory Activity of [TSAO-T]-[PFA] Conjugates against HIV-1 (IIIB), HIV-2 and TSAO-Resistant Mutant HIV-1E138k in
MT-4 and CEM Cell Cultures
EC50a (µM)
CC50b (µM)
MT-4
CEM
MT-4
CEM
compound
HIV-1
0.03 ( 0.01
133 ( 40
HIV-2
HIV-1
HIV-2
HIV-1E138K
log Pc
4
PFA
9
10
14
20
6
>2
0.01 ( 0.01
121 ( 26
105 ( 31
>10
>2
87
3.86 ( 0.29
3.86 ( 0.29
2.26
-0.56
7.12
140 ( 35
>10
230 ( 7.3
-
0.282 ( 0.005
20.1 ( 3.0
0.08 ( 0.0
4.5 ( 0.7
>50
>10
>50
25 ( 7.1
20
18.4 ( 1.9
55.5 ( 26.7
84.3 ( 12.2
84.0 ( 37.4
111 ( 2.1
20.9( 0.85
146 ( 41.7
135 ( 1.4
138 ( 7.8
178 ( 62.4
>50
>50
4.01
4.23 ( 0.57
1.26 ( 0.37
0.82 ( 0.05
>50
2.13 ( 1.62
3.0 ( 0.0
>50
4.01
>50
>50
5.02
>50
0.47 ( 0.46
>50
g50
0.63
a 50% Effective concentration, or compound concentration required to inhibit HIV-induced cytopathicity in cell culture by 50%. b 50%
Cytostatic concentration, or compound concentration required to inhibit CEM cell proliferation by 50% or to reduce MT-4 cell viability in
mock-infected cell cultures by 50%. c Log P: Interactive prediction analysis using E-state atom indices with neural network algorithms.
Algorithm available at www.logp.com.
Table 2. Inhibitory Activity of Test Compounds against HIV-1
RT Wild-Type and Mutant HIV-1/138Lys RT
IC50a (µM)
compound
HIV-1 WT RT
HIV-1/138Lys RT
TSAO-m3T
3.1 ( 1.2
24
>100
PFA
4
56 ( 15
>100
8.1 ( 4.6
11 ( 4.0
4.7 ( 0.2
6.3 ( 3.1
3.8 ( 4.0
3.7 ( 0.3
9
>500
10
14
20
6
394 ( 6.0
293 ( 14
312 ( 20
437 ( 66
Figure 4. Stability of [TSAO-T]-[PFA] conjugate 6 in ex-
tracts from CEM cells.
than the more hydrophilic diester 10, 14, and 20
analogues, but the deprotected conjugate 6, being the
most hydrophilic derivative among all tested com-
pounds, was more inhibitory against HIV-1 than 10 and
20 but less inhibitory than 9 and 4. Therefore, we
conclude that differences in efficiency of cellular uptake
(due to lipophilicity of the drugs) is probably not the
only contributing factor to the antiviral activity of the
compounds.
When the stability of the compounds was evaluated
in phosphate-buffered saline (PBS, pH 7.4), all the
conjugates proved fully stable within 3 h of incubation.
No traces of any degradation products could be detected
within this time period. Interestingly, when concen-
trated CEM cell extracts were exposed to the test
compounds, only the deprotected TSAO-PFA conjugate
6 released the parent TSAO compound 4. In fact, g 80%
of drug was converted to the parent compound within
3 h of incubation, reflecting an intracellular half-life of
∼30 min in the concentrated cell extract (Figure 4).
Some 5′-deprotected 4 was also observed when exposed
to CEM cell extracts (data not shown). These findings
suggest that in the deprotected TSAO-PFA conjugate
the P-O bond can be intracellularly cleaved regenerat-
ing the parent TSAO and the intact PFA molecule at
equimolar concentrations. It should be kept in mind,
however, that the cell extracts that were used to
examine the stability of the conjugates are derived from
highly concentrated CEM cell suspensions, whose cell
number markedly exceed the number of CEM cells used
in the virus-infected cell cultures.
a 50% Inhibitory concentration or drug concentration required
to inhibit the RT-catalyzed reaction using poly rC.dG and [3H]dGTP
as the template/primer and radiolabeled substrate, respectively.
Table 3. Anti-HIV Activity of TSAO Derivative 4, PFA, and
[TSAO-T]-[PFA] Conjugate 6 against the PFA-Resistant
Mutant HIVW88G in P4/R5 Cells
EC50a (µM)
virus
4b
PFA
6
HIV-1(LAI)
HIV-1W88G
Fold-Rc
0.043 ( 0.024
0.027 ( 0.006
0.63
44 ( 21
> 300 ( 99
> 6.9
0.30 ( 0.25
0.16 ( 0.06
0.53
a 50% Effective concentration (mean ( standard deviation).
Results are the average of five independent experiments. b In this
particular case, results are the average of three experiments.
c Calculated by taking the average EC50 for mutant HIV-1W88G
divided by the average EC50 for HIV-1(LAI).
inhibit HIV-1 RT predominantly by the TSAO part of
the molecule and not by the PFA part. Otherwise, a
more pronounced inhibitory activity of the conjugates
against TSAO-resistant mutant HIV-1 RT should have
been observed.
The TSAO derivative 4, the TSAO-PFA conjugate 6,
and PFA were evaluated for their inhibitory activity in
P4/R5 against a mutant PFA-resistant HIV-1 encoding
the W88G mutation in RT (Table 3). Whereas PFA was
>7-fold less active against the HIV-1W88G strain, both
TSAO and TSAO-PFA conjugate 6 did not lose potency
against HIV-1W88G compared with wild-type HIV-1
(Table 3). Thus, the PFA-resistant virus did not show
cross-resistance with the TSAO-PFA conjugate 6, again
highlighting the key role of the TSAO portion of the
molecule and not the PFA part in the antiviral activity
of TSAO-PFA conjugate.
Given the pronounced antiviral activity of 4, the cell
extract study, the rather weak activity of PFA against
HIV-1, and the fact that compound 6 has a comparable
anti-RT activity as compound 4, we may conclude that
the activity of the TSAO-PFA conjugate 6 seen in cell
culture could be probably due either to the conjugate
as such and, to a minor extent, to the release of the
The log P values were calculated for each of the
compounds.22 There was no correlation between lipohi-
licity of the test compounds and their antiviral activity.
The most lipophilic compound (9) proved more active

Hybrids of [TSAO-T]-[Foscarnet]
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3423
THF (1.5 mL), and the solution was left to stir under argon at
room temperature. After 6 h, the reaction mixture was
evaporated to dryness and the residue was purified by CCTLC
on the chromatotron (ethyl acetate:methanol, 5:1) to give
compound 10 (0.079 g, 80%) as a white amorphous solid. IR
(KBr) ) 1710 cm-1. 1H NMR [400 MHz, CD3OD] δ: 1.00, 1.16
(2s, 18H, 2t-Bu), 2.12 (m, 2H, CH2), 2.17 (d, 3H, J ) 0.9 Hz,
CH3-5), 3.94 (s, 3H, CO2CH3), 4.19 (m, 6H, NCH2CH2CH2O,
2H-5′), 4.53 (t, 1H, J ) 3.8 Hz, H-4′), 4.74 (d, 1H, J1′,2′ ) 8.2
Hz, H-2′), 5.83 (s, 1H, H-3′′), 6.27 (d, 1H, H-1′), 7.75 (d, 1H,
H-6). 13C NMR [100 MHz, CD3OD] δ: 9.30 (CH2), 13.16
(CH3-5), 18.73, 19.18 [(CH3)3CSi], 25.95, 26.53 [(CH3)3CSi],
29.94 (d, JP,CH ) 6.9 Hz, CH2), 40.16 (NCH2), 51.73 (d, JP,CH
) 3.8 Hz, CO2CH3), 63.33 (C-5′), 65.33 (d, JP,CH ) 6.1 Hz, CH2-
OP), 76.10 (C-2′), 85.79 (C-4′), 87.54 (C-1′), 91.216 (C-3′′), 93.54
(C-3′), 112.22 (C-5), 135.26 (C-6), 150.48 (C-2),153.17 (C-4′′),
164.55 (C-4), 174.61 (d, JP,C ) 248 Hz, CO2CH3). 31P NMR
[161.88 MHz, CD3OD] δ: -7.41. Anal. (C29H51N3NaO13PSSi2)
C, H, N, S.
parent drug 4 if the conjugate would have been ef-
ficiently taken up by the cells. However, in case of poor
uptake of the TSAO-PFA conjugate 6, it is possible that
the antiviral activity seen is mainly due to either the
intact conjugate or to the released parental TSAO
molecule 4 after intracellular hydrolysis of the conju-
gate. The antiviral efficacy of conjugated compounds
depends on many factors, such as enzyme inhibition,
cell membrane permeability, extracellular stability,
intracellullar hydrolysis, and the interactions between
them which are very complex. Additional research is
required to reveal the exact mechanism of antiviral
action of the deprotected TSAO-PFA conjugate deriva-
tive.
2
2
Experimental Section
Chemical Procedures. Microanalyses were obtained with
a Heraeus CHN-O-RAPID instrument. IR spectra were ob-
tained on a Perkin-Elmer spectrum one spectrophotometer.
1H NMR spectra were recorded with a Varian Gemini, a Varian
XL-300, and a Bruker AM-200 spectrometer operating at 300
and at 200 MHz with Me4Si as internal standard. 13C NMR
spectra were recorded with a Varian XL-300 and a Bruker AM-
200 spectrometer operating at 75 MHz and at 50 MHz with
Me4Si as internal standard. 31P spectra were recorded on a
Varian INOVA 400 spectrometer operating at 161.89 MHz,
using acetone-d6 or CD3OD as solvent at 30 °C with phosphoric
acid as external standard. Analytical thin-layer chromatog-
raphy (TLC) was performed on silica gel 60 F254 (Merck).
Separations on silica gel were performed by preparative
centrifugal circular thin-layer chromatography (CCTLC) on a
chromatotron (Kiesegel 60 PF254 gipshaltig, Merck) layer
thickness 1 mm or 2 mm, flow rate 5 mL/min. Preparative
reverse phase purification was carried out using reverse phase
SPE cartridges.
Ethyl [Bis(trimethylsilyl)phosphono]formate (12).18a
Tris(trimethylsilyl) phosphite (25 mL, 67 mmol) was added
dropwise to ethyl chloroformate 11 (7.2 mL, 67 mmol) in an
ice bath under an argon atmosphere. The mixture was left
stirring overnight at room temperature and distilled under 10
mmHg. Trimethylsilyl chloride distilled first, then bis(trim-
ethylsilyl)phosphorous acid, and finally ethyl [bis(trimethyl-
silyl)phosphono]formate 12 (13 g, bp10 100-102 °C, bp10 68-
70 °C18a) was obtained in 75% yield. 1H NMR [200 MHz,
(CD3)2CO] δ: 0.28 (m, 27H, 9CH3), 1.29 (t, 3H, J ) 7.2 Hz,
CO2CH2CH3), 4.23 (q, 2H, J ) 7.1 Hz, CO2CH2CH3).
Ethyl (Dichlorophosphonyl)formate (13).18a Ethyl [bis-
(trimethylsilyl)phosphono]formate 12 (5 g, 18 mmol) was
dissolved in 20 mL of dry toluene, under an argon atmosphere.
Thionyl chloride (3.2 mL, 53 mmol) was added, and the
mixture was refluxed for 2 h. Toluene and thionyl chloride
were removed under reduced pressure, and the resulting
mixture was purified by vacuum distillation (10 mmHg) to give
2.7 g of a colorless oil (13) in 84% yield (bp10 84-86 °C, bp10
64 °C18a).1H NMR [200 MHz, (CD3)2CO] δ: 1.41 (t, 3H, J )
7.2 Hz, CO2CH2CH3), 4.47 (q, 2H, J ) 7.2 Hz, CO2CH2CH3).
Triethylamine, dichloromethane and toluene were dried by
refluxing over calcium hydride. Tetrahydrofuran was dried by
refluxing over sodium/benzophenone.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofurano-
syl]-3-N-[(3-[[[(ethyloxycarbonyl)hydroxyphosphonyl]-
oxy]propyl]thymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide) (14). Ethyl (dichlorophosphonyl)formate (13)
(0.047 g, 0.25 mmol) was dissolved in 2.5 mL of dry CH2Cl2,
under an argon atmosphere. The mixture was cooled to -20
°C, and a solution of the alcohol 4 (0.1 g, 0.15 mmol) and NEt3
(70 µL, 0.45 mmol) in dry CH2Cl2 (3.5 mL), also precooled to
-20 °C, was added dropwise. The mixture was stirred for 5
min at -20 °C, and then the solvent was removed under
reduced pressure. The remaining residue was dissolved in
EtOAc (20 mL) and succesively washed with H2O (4 × 10 mL)
and brine (10 mL). The organic phase was dried (Na2SO4),
filtered, and evaporated to dryness. The final residue was
purified by CCTLC on the chromatotron (ethyl acetate:
methanol, 5:1) to give compound 14 (0.036 g, 31%) as a white
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofurano-
syl]-3-N-[3-[[[(methoxycarbonyl)methoxyphosphonyl]-
oxy]propyl]thymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide) (9). PCl5 (0.102 g, 0.496 mmol) was added to
a solution of trimethylphosphonoformate 7 (72 µL, 0.54 mmol)
in dry CCl4 (4.5 mL), and the suspension was warmed to 50
°C for 1.5 h. The reaction mixture was evaporated to dryness
under reduced pressure. The residue 8 was cooled to -50 °C
under an argon atmosphere, and a solution of 4 (0.1 g, 0.15
mmol) and Et3N (75 µL, 2.56 mmol) in dry CH2Cl2 (1.1 mL),
also precooled to -50 °C, was added. Once the reaction was
completed, the solvent was evaporated to dryness and the
residue was purified by CCTLC on the chromatotron (hexane:
ethyl acetate, 1:1) to give compound 9 (0.12 g, 91%) as a white
amorphous solid. IR (KBr) 1710 cm-1 1H NMR [300 MHz,
.
(CD3)2CO] δ: 0.83, 0.98 (2s, 18H, 2t-Bu), 1.95 (d, 3H, J ) 1.2
Hz, CH3-5), 2.01 (m, 2H, CH2), 3.87 (m, 3H, OCH3), 4.10 (m,
5H, CH2, OCH3), 4.30 (m, 2H, H-5′), 4.28 (m, 2H, CH2O), 4.35
(m, 1H, H-4′), 4.67 (d, 1H, J1′,2′ ) 8.2 Hz, H-2′), 5.78 (s, 1H,
H-3′′), 6.11 (d, 1H, H-1′), 6.50 (bs, 2H, NH2), 7.53 (d, 1H, H-6).
13C NMR [50 MHz, CDCl3] δ: 12.87 (CH3-5), 18.34, 19.01
[(CH3)3CSi], 25.69, 26.22 [(CH3)3CSi], 30.38 (d, JP,CH2 ) 6.7 Hz,
1
amorphous solid. IR (KBr) ) 1710 cm-1. H NMR [400 MHz,
CD3OD] δ: 0.67, 0.83 (2s, 18H, 2t-Bu), 1.16 (t, 3H, J ) 7.1
Hz, CH2CH3), 1.82 (m, 5H, CH3-5, CH2), 3.79-3.92 (m, 6H,
NCH2CH2CH2O, 2H-5′), 4.07 (q, 2H, CH2CH3), 4.19 (t, 1H, J
) 3.6 Hz, H-4′), 4.41 (d, 1H, J1′,2′ ) 8.2 Hz, H-2′), 5.48 (s, 1H,
H-3′′), 5.86 (d, 1H, H-1′), 7.36 (d, 1H, H-6). 13C NMR [100 MHz,
CD3OD] δ: 13.11 (CH3-5), 14.60 (CH2CH3), 18.79, 19.23
[(CH3)3CSi], 25.91, 26.49 [(CH3)3CSi], 30.07 (d, JP,CH2 ) 5.9 Hz,
CH2), 38.20 (NCH2), 51.72 (d, JP,CH ) 3.8 Hz, CO2CH3), 52.6
3
(d, JP,CH ) 6.2 Hz, OCH3), 62.91 (C-5′), 65.6 (d, JP,CH ) 6.1
3
2
CH2), 40.18 (NCH2), 61.28 (d, JP,CH ) 4.2 Hz, CO2CH2CH3)
Hz, CH2O), 76.22 (C-2′), 85.68, 87.54 (C-4′, C-1′), 91.15 (C-1′),
93.16 (C-3′), 111.99 (C-5), 134.31 (C-6), 152.42, 153.25 (C-2,
C-4′′), 163.85 (C-4), 174.20 (d, JP,C ) 248 Hz, CO2CH3). 31P
NMR [161.88 MHz, (CD3)2CO] δ: -2.53. Anal. (C30H54N3O13-
PSSi2) C, H, N, S.
2
63.21 (C-5′), 65.34 (d, JP,CH ) 6.2 Hz, CH2OP), 75.98 (C-2′),
2
85.70 (C-4′), 88.75 (C-1′), 90.88 (C-3′′), 93.16 (C-3′), 112.17
(C-5), 135.72 (C-6), 152.45 (C-2), 153.60 (C-4′′), 164.51 (C-4),
172.34 (d, JP,C ) 250 Hz, CO2CH2CH3). 31P NMR [161.88 MHz,
CD3OD] δ: -4.50. Anal. (C30H54N3O13PSSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofurano-
syl]-3-N-[3-[[[(methoxycarbonyl)hydroxyphosphonyl]oxy]-
propyl]thymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide), Sodium Salt (10). NaI (0.017 g, 0.11 mmol)
was added to a stirred solution of 9 (0.1 g, 0.13 mmol) in dry
[1-[2′-O-(tert-Butyldimethylsilyl)-â-D-ribofuranosyl]-3-
N-[(3-[[(hydroxycarbonyl)hydroxyphosphonyl]oxy]-
propyl]thymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide) Sodium Salt (15). Method A. Compound 10

3424 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Vela´zquez et al.
(0.1 g, 0.13 mmol) was dissolved in dry THF (1.5 mL), and an
aqueous solution of 0.1 N NaOH (0.75 mL, 0.2 mmol) was
added. The mixture was stirred for 2 h at room temperature.
The mixture was neutralized with DOWEX 50 Wx4 (H+ form,
previously washed with distilled water), filtered, and evapo-
rated to dryness. The residue was purified by reverse phase
chromatrography using SPE cartridges (acetonitrile: water,
95:5). The fastest moving fractions afforded 15 (0.035 g, 53%)
°C, and a solution of the alcohol 4 (0.1 g, 0.15 mmol) and NEt3
(70 µL, 0.45 mmol) in dry CH2Cl2 (3.5 mL), also precooled to
-20 °C, was added dropwise, according to the procedure
described for the synthesis of 14. The final residue after the
workup was purified by CCTLC on the chromatotron (ethyl
acetate:methanol, 5:1) to give compound 20 (0.07 g, 54%) as a
1
white amorphous solid. IR (KBr) ) 1710 cm-1. H NMR [400
MHz, CD3OD] δ: 0.99, 1.15 (2s, 18H, 2t-Bu), 2.12 (m, 2H, CH2),
2.15 (s, 3H, CH3-5,), 3.91-4.20 (m, 6H, NCH2CH2CH2O, 2H-
5′), 4.51 (t, 1H, J ) 3.6 Hz, H-4′), 4.77 (d, 1H, J1′,2′ ) 8.1 Hz,
H-2′), 5.39 (ABs, 2H, CH2Ph), 5.81 (s, 1H, H-3′′), 6.19 (d, 1H,
H-1′), 7.53-7.46 (m, 3H, Ph), 7.60 (m, 2H, Ph), 7.70 (s, 1H,
H-6). 13C NMR [100 MHz, CD3OD] δ: 9.30 (CH2), 13.06
1
as a white amorphous solid. H NMR [400 MHz, CD3OD] δ:
1.01 (s, 9H, t-Bu), 2.12 (m, 5H, CH2, CH3-5), 3.97 (dd, 1H, J5′a,5′b
) 12.3, J4′,5′a ) 2.5 Hz, H-5′a), 4.12 (dd, 1H, J4′,5′b ) 2.8 Hz,
H-5′b), 4.26 (m, 2H, NCH2CH2CH2O), 4.53 (t, 1H, H-4′), 4.90
(d, 1H, J1′,2′ ) 8.0 Hz, H-2′), 5.83 (s, 1H, H-3′′), 6.26 (d, 1H,
H-1′), 8.14 (s, 1H, H-6). 13C NMR [100 MHz, CD3OD] δ: 13.34
(CH
3-5), 18.76, 19.21 [(CH3)3CSi], 25.95, 26.48 [(CH3)3CSi],
(CH3-5), 18.88 [(CH3)3CSi], 26.10 [(CH3)3CSi], 30.93 (d, JP,CH
29.94 (d, JP,CH ) 6.8 Hz, CH2), 40.18 (NCH2), 62.01 (C-5′),
2
2
) 4.0 Hz, CH2), 40.42 (NCH2), 61.67 (C-5′), 63.92 (d, JP,CH
)
65.31 (d, JP,CH ) 6.1 Hz, CH2O), 66.71 (d, JP,CH ) 3.8 Hz,
6.1 Hz, CH2O), 76.75 (C-2′), 86.29 (C-4′), 89.52 (C-1′), 91.228
(C-3′′), 95.79 (C-3′), 112.13 (C-5), 137.11 (C-6), 153.05 (C-2),
153.17 (C-4′′), 164.77 (C-4), 199.27 (d, JP,C ) 230 Hz, CO2Na).
31P NMR [161.88 MHz, CD3OD] δ: -12.19. Anal. (C22H34N3-
Na2O13PSSi) C, H, N, S.
2
2
CH2Ph,), 75.94 (C-2′), 85.73 (C-4′), 88.69 (C-1′), 90.91 (C-3′′),
93.18 (C-3′), 112.15 (C-5), 129.14 (Ph), 129.48 (Ph), 135.70
(C-6), 137.41 (Ph), 152.43 (C-2), 153.62 (C-4′′), 164.46 (C-4),
175.62 (d, JP,C ) 230 Hz, CO2CH2Ph). 31P NMR [161.88 MHz,
CD3OD] δ: -4.24. Anal. (C35H56N3O13PSSi2) C, H, N, S.
The slowest moving fractions gave [1-[2′-O-(tert-butyldi-
methylsilyl)-â-d-ribofuranosyl]-3-N-[3-[[[(hydroxyphospho-
noyl)oxy]propyl]thymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide) sodium salt (16) (0.035 g, 43%) as a white
amorphous solid. 1H NMR [400 MHz, CD3OD] δ: 0.67 (s, 9H,
t-Bu), 1.79 (m, 5H, CH2, CH3-5), 3.59-4.01 (m, 6H, NCH2-
CH2CH2O, 2H-5′), 4.21 (m, 1H, H-4′), 4.57 (d, 1H, J1′,2′ ) 7.7
Hz, H-2′), 5.51 (s, 1H, H-3′′), 5.97 (d, 1H, H-1′), 6.59 (d, 1H,
JP,H ) 623 Hz, PH), 7.72 (s, 1H, H-6). 13C NMR [100 MHz,
CD3OD] δ: 13.34 (CH3-5), 18.75 [(CH3)3CSi], 26.09 [(CH3)3CSi],
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofurano-
syl]-3-N-[3-[[[(hydroxycarbonyl)hydroxyphosphonyl]oxy]-
propyl]thymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide) (6). Compound 20 (0.1 g, 0.12 mmol) was
dissolved in methanol (10 mL) and hydrogenated in the
presence of 10% Pd/C (0.01 g, 7.8 mmol) at 30 psi at 25 °C for
1 h. The mixture was filtered, washed with methanol (10 mL),
and evaporated to give 6 (0.06 g, 60%) as an amorphous solid.
1H NMR [400 MHz, CD3OD] δ: 0.81, 0.97 (2s, 18H, 2t-Bu),
1,92 (m, 2H, CH2), 1.96 (s, 3H, CH3-5,), 3.74-4.08 (m, 6H,
NCH2CH2CH2O, 2H-5′), 4.33 (t, 1H, J4′,5′a ) 3.8, J4′,5′b ) 3.7
Hz, H-4′), 4.54 (d, 1H, J1′,2′ ) 8.1 Hz, H-2′), 5.63 (s, 1H,
H-3′′), 6.03 (d, 1H, H-1′), 7.51 (s, 1H, H-6). 13C NMR [100 MHz,
CD3OD] δ: -5.47, -4.94, -4.91, -4.24 (CH3-Si), 9.30 (CH2),
13.12 (CH3-5), 18.78, 19.23 [(CH3)3CSi], 25.91, 26.48 [(CH3)3-
30.74 (d, JP,CH ) 6.1 Hz, CH2), 40.11 (NCH2), 61.65 (C-5′),
2
62.92 (d, JP,CH ) 5.5 Hz, CH2O), 76.75 (C-2′), 86.29 (C-4′), 89.52
2
(C-1′), 91.28 (C-3′′), 95.66 (C-3′), 112.23 (C-5), 137.11 (C-6),
153.05 (C-2), 153.56 (C-4′′), 163.47 (C-4).31P NMR (161.88 MHz,
CD3OD) δ: 6.83. Anal. (C21H35N3NaO11PSSi) C, H, N, S.
CSi], 30.07 (d, JP,CH ) 6.8 Hz, CH2), 40.16 (NCH2), 63.22
Method B. Compound 10 (0.1 g, 0.13 mmol) was dissolved
in dry THF (1.5 mL), and an aqueous solution of NaOH 0.45
N (0.75 mL, 0.2 mmol) was added. The mixture was stirred
for 30 min at room temperature. The mixture was neutralized
with DOWEX 50 Wx4 (H+ form, previously washed with
distilled water), filtered, and evaporated to dryness. The
residue was purified by reverse phase chromatrography using
SPE cartridges (acetonitrile: water, 95:5) to give 15 (0.063 g,
76%) as a white amorphous solid.
Benzyl [Bis(trimethylsilyl)phosphono]formate (18).
Tris(trimethylsilyl) phosphite (12 mL, 36 mmol) was added
dropwise to benzyl chloroformate 17 (5.4 mL, 36 mmol) in an
ice bath under an argon atmosphere. The mixture was left
stirring overnight at room temperature and distilled under 10
mmHg. Trimethylsilyl chloride distilled first, then bis(tri-
methylsilyl)phosphorous acid, and finally 11 g of a colorless
oil benzyl [bis(trimethylsilyl)phosphono]formate 18 was ob-
tained in 89% yield (bp10 154-156 °C). 1H NMR [200 MHz,
(CD3)2CO] δ: 0.02 (m, 27H, 9CH3), 5.21 (s, 2H, CH2), 7.4
(m, 5H, Ph). 13C NMR [100 MHz, (CD3)2CO] δ: 0.70, 1,96
(CH3-Si), 67.40 (CH2), 129,24, 129.34, 136.20, 167.45 (Ph),
170.20 (CO2Bn). 31P NMR [161.88 MHz, (CD3)2CO] δ: -21.81.
Benzyl (Dichlorophosphonyl)formate (19). Benzyl [bis-
(trimethylsilyl)phosphono]formate 18 (2 g, 5.5 mmol) was
dissolved in 10 mL of dry toluene, under an argon atmosphere.
Thionyl chloride (1.1 mL, 16 mmol) was added, and the
mixture was refluxed for 2 h. Toluene and thionyl chloride
were removed under reduced pressure, and the resulting
mixture was purified by vacuum distillation (10 mmHg) to give
0.4 g of a colorless oil (19) in 30% yield (bp10 154-120-122
°C).1H NMR [200 MHz, (CD3)2CO] δ: 5.25 (s, 2H, CH2), 7.4
(m, 5H, Ph). 31P NMR [161.88 MHz, (CD3)2CO] δ: 10.80.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofurano-
syl]-3-N-[(3-[[[(benzyloxycarbonyl)hydroxyphosphonyl]-
oxy]propyl]thymine]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide) (20). Benzyl (dichlorophosphonyl)formate (19)
(0.06 g, 0.25 mmol) was dissolved in 2.5 mL of dry CH2Cl2,
under an argon atmosphere. The mixture was cooled to -20
2
(C-5′), 64.30 (d, JP,CH ) 6.1 Hz, CH2O), 76.03 (C-2′), 85.71
(C-4′), 88.51 (C-1′), 920.96 (C-3′′), 93.22 (C-3′), 112.18 (C-5),
135.62 (C-6), 152.48 (C-2),153.53 (C-4′′), 164.55 (C-4), 182.01
(d, JP,C ) 235 Hz, CO2H). 31P NMR [161.88 MHz, CD3OD] δ:
-12.20. Anal. (C28H50N3O13PSSi2) C, H, N, S.
Biological Methods. Cells and Viruses. Human immu-
nodeficiency virus type 1 [HIV-1 (IIIB)] was obtained from Dr.
R. C. Gallo (when at the National Cancer Institute, Bethesda,
MD). HIV-2 (ROD) was provided by Dr. L. Montagnier (when
at the Pasteur Institute, Paris, France). The P4/R5 cells were
obtained from Ned Landau from the Salk Institute.
Activity Assay of Test Compounds against HIV-1 and
HIV-2 in Cell Culture. 4 × 105 CEM or 3 × 105 MT-4 cells
per milliliter were infected with HIV-1 or HIV-2 at ∼100
CCID50 (50% cell culture infective dose) per milliliter of cell
suspension. Then, 100 µL of the infected cell suspension was
transferred to microtiter plate wells and mixed with 100 µL
of the appropriate dilutions of the test compounds. Giant cell
formation (CEM) or HIV-induced cytopathicity (MT-4) was
recorded microscopically (CEM) or by trypan blue dye exclusion
(MT-4) in the HIV-infected cell cultures after 4 days (CEM)
or 5 days (MT-4). The 50% effective concentration (EC50) of
the test compounds was defined as the compound concentra-
tion required to inhibit virus-induced cytopathicity (CEM) or
to reduce cell viability (MT-4) by 50%. The 50% cytostatic or
cytotoxic concentration (CC50) was defined as the compound
concentration required to inhibit CEM cell proliferation by 50%
or to reduce the number of viable MT-4 cells in mock-infected
cell cultures by 50%.
Activity Assay of Test Compounds against TSAO-
Resistant HIV-1 Strains in Cell Culture. CEM cells were
suspended at 250 000 cells per milliliter of culture medium
and infected with TSAO-resistant mutant HIV-1 strains at 100
50% cell culture infective doses per milliliter. Then 100 µL of
the infected cell suspensions was added to 200-µL microtiter
plate wells containing 100 µL of an appropriate dilution of the
test compounds. After 4 days incubation at 37 °C, the cell

Hybrids of [TSAO-T]-[Foscarnet]
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3425
cultures were examined for syncytium formation. The EC50
was determined as the compound concentration required to
inhibit syncytium formation by 50%.
We thank the Ministery of Education of Spain for a
grant to E.L. and Janssen-Cilag, S.A. for an award to
E.L. The Spanish CICYT (projects SAF2000-0153-C02-
01 and SAF2003-07219-C02-01), the Comunidad de
Madrid (Project 08.2/0044/2000), the European Com-
mission (Project QLK2-CT-2000-00291), the U.S. Na-
tional Cancer Institute (Contract 20xS190A), and the
AIDS Clinical TRials Group, Division of AIDS, National
Institute of Allergy and Infectious Diseases [Contract
203VC001 and AI046383-04 (D.L.K. and J.W.M.)] are
also acknowledged for financial support.
Activity Assay of Test Compounds against HIV-1LAI
(WT) and PFA-Resistant HIV-1W88G Strain. Susceptibility
assays were conducted in P4/R5 cells. The P4/R5 reporter cell
line is a Hela cell line that had been stably transfected with a
Tat-activated â-galactosidase gene under the control of an
HIV-L TR promoter (provided by Ned Landau, Salk Institute).
Drug dilutions were made 2- or 3-fold and added to the plate
in triplicate. An amount of 5 × 104 cells/mL were infected with
HIVLAI(wild type) and HIVW88G at an MOI of 0.05 TCID50/mL.
At 48 h post-infection, a lysis buffer and a luminescent
substrate was added to each well. The amount of â-galactosi-
dase produced by the P4/R5 cells was then determined using
a luminometer.
Anti-Reverse Transcriptase Assays. The source of the
reverse transcriptases used were either recombinant HIV-1
RT (derived from HIV-1 IIIB) and Glu138Lys mutated recom-
binant HIV-1 RT, constructed and prepared as described
before.23 The RT assays contained in a total reaction mixture
volume (50 µL) 50 mM Tris-HCl (pH 7.8), 5 mM dithiothreitol,
300 mM glutathione, 500 µM EDTA, 150 mM KCl, 5 mM
MgCl2, 1.25 µg of bovine serum albumin, labeled substrate
[8-3H]dGTP (specific radioactivity, 15.6 Ci/mmole) (2.5 µM)
(Moravek Biochemicals, Brea, CA), a fixed concentration of the
template/primer poly(C).oligo(dG)12-18 (0.1 mM), 0.06% Triton
X-100, 10 µL of inhibitor at various concentrations, and 1 µL
of the RT preparation. The reaction mixtures were incubated
at 37 °C for 30 min, at which time 100 µL of calf thymus DNA
(150 µg/mL), 2 mL of Na4P2O7 (0.1 M in 1 M HCl), and 2 mL
of trichloroacetic acid (10% v/v) were added. The solutions were
kept on ice for 30 min, after which the acid-insoluble material
was washed and analyzed for radioactivity. The IC50 for each
test compound was determined as the compound concentration
that inhibited HIV RT activity by 50%.
Stability Assay of Test Compounds in PBS and Cell
Extracts. CEM cells were grown in RPMI-1640 medium
containing 10% newborn calf serum, 2 mM L-glutamine, and
0.075% NaHCO3. When reaching a cell density of ∼106 to 2 ×
106 cells/mL, the cell cultures were centrifuged at 1200 rpm
in a Megafuge 3.0R (Vanderheyden, Brussels, Belgium) and
washed twice with phosphate-buffered saline (PBS, pH 7.4).
Then, a concentrated cell suspension of ∼50 × 106 cells/mL
PBS was sonicated (3 × 10 s) on ice to lyse the cells. The crude
cell extract was then centrifuged at 100 000g in an ultracen-
trifuge (PMSE-65) (Beun-de-Ronde), and the supernatant
stored at -80 °C.
Test compound solutions were prepared in PBS and ana-
lyzed for chemical stability by HPLC/Reverse phase C-18
column (Waters 2690, Alliance, Brussels, Belgium). Also,
compound solutions were exposed to the crude CEM cell
extracts and analyzed for stability in the same assay system.
The potential breakdown products that may arise in function
of incubation time at 37 °C were separated by an acetonitrile/
H2O elution gradient.
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Lizette van Berckelaer for excellent technical assistance.

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