Novel [2′,5′-Bis-O-(tert-butyldimethylsily)-β-D- ribofuranosyl]-3′-spiro-5″-(4″-amino-1″, 2″-oxathiole-2″,2″-diozide) derivatives with anti-HIV-1 and anti-human-cytomegalovirus activity

Article abstract of DOI:10.1021/jm040868q

New [2′,5′-bis-O-(tert-butyldimethylsilyl)-β-D- ribofuranosyl]-3′-spiro-5″-(4″-amino-1″, 2″-oxathiole-2″,2″-dioxide) (TSAO) derivatives substituted at the 4″-amino group of the spiro moiety with different carbonyl functionalities have been designed and sy

Full text of DOI:10.1021/jm040868q

1158
J. Med. Chem. 2005, 48, 1158-1168
Novel [2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-
3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide) Derivatives with Anti-HIV-1
and Anti-Human-Cytomegalovirus Activity
Sonia de Castro,† Esther Lobato´n,† Mar´ıa-Jesu´s Pe´rez-Pe´rez,† Ana San-Fe´lix,† Alessandra Cordeiro,†
Graciela Andrei,‡ Robert Snoeck,‡ Erik De Clercq,‡ Jan Balzarini,‡ Mar´ıa-Jose´ Camarasa,*,† and
Sonsoles Vela´zquez*,†
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
Received August 2, 2004
New [2′,5′-bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxa-
thiole-2′′,2′′-dioxide) (TSAO) derivatives substituted at the 4′′-amino group of the spiro moiety
with different carbonyl functionalities have been designed and synthesized. Various synthetic
procedures, on the scarcely studied reactivity of the 3′-spiroaminooxathioledioxide moiety, have
been explored. The compounds were evaluated for their inhibitory effect on both wild-type and
TSAO-resistant HIV-1 strains, in cell culture. The presence of a methyl ester (10) or amide
groups (12) at the 4′′-position conferred the highest anti-HIV-1 activity, while the free oxalyl
acid derivative (11) was 10- to 20-fold less active against the virus. In contrast, the presence
at this position of (un)substituted ureido or acyl groups markedly diminished or annihilated
the anti-HIV-1 activity. Surprisingly, some of the target compounds also showed inhibition of
human cytomegalovirus (HCMV) replication at subtoxic concentrations. This has never been
observed previously for TSAO derivatives. In particular, compound 26 represents the first TSAO
derivative with dual anti-HIV-1 and -HCMV activity.
Introduction
Reverse transcriptase (RT) is a key enzyme that plays
an essential and multifunctional role in the replication
of the human immunodeficiency virus (HIV) and thus
represents an attractive target for the development of
new drugs useful in AIDS therapy.1
A class of inhibitors targeted at the viral RT, the so-
called nonnucleoside RT inhibitors (NNRTIs), are com-
pounds used in the treatment of HIV infections in
combination with nucleoside analogue RT inhibitors
(NRTIs) and/or HIV protease inhibitors (PIs) and/or the
fusion inhibitor enfuvirtide.2-4 Currently, only three
NNRTIs (namely, nevirapine, delarvidine, and efavirenz)
are available in clinical practice. 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).5 Al-
though NNRTIs generally exhibit low toxicity and
favorable pharmacokinetic properties, their clinical util-
ity is adversely affected by the emergence of drug-
resistant HIV-1 variants and by the problem of cross-
resistance to other NNRTIs.6 Therefore, the synthesis
of additional NNRTIs for use in the multidrug (cocktail)
approaches that may also be effective against mutant
HIV strains remains a high priority for medical re-
search.
Figure 1. Chemical structures of TSAO-T (1) and TSAO-m3T
(2).
thiole-2′′,2′′-dioxide) nucleosides (TSAOs), developed in
the past decade in our research group, represent a
rather unique class of compounds7,8 that seem to
interact at the interface between the p51 and p66
subunits of HIV-1 RT.9 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). More
than 700 TSAO derivatives have been prepared, and
extensive structure activity (SAR), resistance, and
metabolic studies have been performed. These studies
have revealed the structural requirements of this unique
family of compounds for their optimal interaction with
the enzyme (HIV-1 RT). A comprehensive review of all
the work performed within this family of compounds has
been recently published.10 Although the pharmacological
profile of TSAO molecules was unfavorable to their
clinical development,10,12 further biochemical studies
have shown that both TSAO-T and its 3-N-ethyl deriva-
tive are able to destabilize the p66/p51 RT heterodimer
in a concentration-dependent manner resulting in a loss
in the ability of the RT to bind to DNA.12,13 This suggests
Among the NNRTIs, [2′,5′-bis-O-(tert-butyldimethyl-
silyl)-â-D-ribofuranosyl]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxa-
* To whom correspondence should be addressed. For M.-J.C.:
telephone, (+34)-915622900; fax, (+34)-915644853; e-mail,
mj.camarasa@iqm.csic.es. For S.V.: (+34)-915622900; fax, (+34)-91
5644853; e-mail, iqmsv29@iqm.csic.es.
† Instituto de Qu´ımica Me´dica.
‡ Rega Institute for Medical Research.
10.1021/jm040868q CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/21/2005

TSAO Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1159
Scheme 1. Synthesis of Bicyclic Nucleosides 5-8 and 3′-C-branched Nucleoside 9
a new and different mechanism of inhibition of HIV-1
RT with regard to the other known NNRTIs, which
deserves further exploration.
HIV-1 resistance to NNRTIs is primarily associated
with mutations of amino acids at the lipophilic NNRTIs
binding pocket in the p66 subunit.4,14 However, resis-
tance to TSAO compounds is determined by a single
mutation (Glu-138-Lys) located in the p51 subunit of
HIV-1 RT, which does not generally result in cross-
resistance to other NNRTIs.15
Our experimental data strongly suggest a specific
interaction of the 4′′-amino group of the 3′-spiro-
aminooxathioledioxide moiety of TSAO molecules with
the carboxylic group of the glutamic acid residue at
position 138 (Glu-138) of the p51 subunit of HIV-1
RT.8-10
Aimed at obtaining novel derivatives more resilient
to drug resistance development by the virus than the
“first generation” of TSAO molecules, we designed a
series of TSAO compounds bearing at the 4′′-position
different carbonyl functionalities that may interact with
the amino group of the Lys-138 in the TSAO-resistant
viral strains. Moreover, the presence of amide or urea
groups at this 4"-position may stabilize interactions with
highly conserved residues of the enzyme close to the
Glu-B138/Lys-B138 of the p51 subunit such as the Asn-
B136. This paper reports on the synthesis and in vitro
anti-HIV-1 activity of these novel compounds against
both wild-type HIV-1 and TSAO-resistant HIV-1 strains.
To get compounds able to interact with the amino
group of the Lys-B138 (TSAO-resistant strains), the
direct replacement of the enamino group by a ketone
through acid hydrolysis (compound 6 in Scheme 1) was
envisaged. In view of the known acid lability of the 5′-
tert-butyldimethylsilyl group (TBDMS), the suitable
TSAO-deprotected nucleoside precursor 318 was used as
starting material. However, although compound 3 showed
high reactivity toward acid hydrolysis (1 N HCl in
methanol), the corresponding keto derivative 4 exhibited
a strong tendency toward its 5′-cyclic hemiacetal form,
and therefore, the isolated compound was identified as
5 (70% yield) (Scheme 1). Further attempts of silylation
of 5 to give the target compound 6 failed in all the
experimental conditions investigated [a large escess of
TBDMSCl either in pyridine or in the presence of
(dimethylamino)pyridine (DMAP) or of imidazole in
acetonitrile or dimethylformamide]. No bis-silylated
derivative 6 was observed in any of the conditions
tested, and only the monosilylated 5′-cyclic hemiacetal
7 was isolated (62% yield). Interestingly, when the 5′-
cyclic hemiacetal compound 5 was reacted with DMAP
in dry acetonitrile at 60 °C, the novel highly function-
alized bicyclic nucleoside 8 was obtained in a 49% yield
together with minor quantities of the 3′-C-branched
nucleoside 9 (9%). Further work performed in this
reaction clarified that compound 9 was obtained from
8 during the purification step that involved the use of
methanol. Therefore, the formation of 9 can be avoided
if methanol is excluded as solvent in the purification
step. The structure of compounds 5, 7, and 8 were
unequivocally assigned on the basis of their correspond-
ing spectroscopic data, using a combination of mono- and
bidimensional 1H and 13C NMR (g-HMBC, g-HSQC) and
IR techniques. The 1H NMR spectra for 5 and 7 showed
the disappearance of the characteristic signals of the
spiroaminooxathioledioxide moiety (a singlet at δ 5.56
ppm assigned to H-3′′ and a broad singlet at δ 6.95 ppm
assigned to NH2-4′′) and the presence of an AB system
at δ 3.62-4.05 ppm corresponding to the new H-3′′
protons and a new broad singlet at δ 8.41 and 7.44 ppm
corresponding to the hemiacetalic OH. In the g-HMBC
Results and Discussion
Synthesis. The preparation of the target compounds
represents a synthetic challenge because the chemical
reactivity of the 3′-spiroaminooxathioledioxide moiety
(aminosultone heterocyclic system) of TSAO compounds
has been scarcely studied.16,17 In this paper three types
of modifications of the 4′′-amino group of the 3′-spiro
moiety were addressed: (i) acid hydrolysis of this
enamino type system, (ii) acylation reactions of the
amino group with different electrophiles, and (iii)
substitution of this amino group by other conveniently
functionalized amines via transamination reactions.

1160 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
de Castro et al.
Scheme 2. Synthesis of 4′′-N-Oxalyl Derivatives 10-14 and 4′′-N-Acyl TSAO Compounds 15-18
experiments of 5 and 7, a correlation peak was observed
between C-4′′ and H-5′, which is only compatible with
the hemiacetal structure. Compounds 5 and 7 exist
exclusively as a single 5′-cyclic hemiacetal diastereomer.
Selective NOE experiments showed that irradiation of
the hemiacetal-OH-4′′ caused enhancements of the
signals for H-2′ and one of the H-3′′ protons. This
observation allowed us to unequivocally assign the C-4′′
configuration of the only isolated diastereomer as S. On
the other hand, the IR spectra of the lactone bicyclic
nucleoside 8 showed a band at 1770 cm-1 characteristic
However, when the reaction of 2 and methyloxalyl
chloride was performed at room temperature in the
presence of stoichiometric amounts of AlCl3 and 4 Å
molecular sieves, the N-acylated compound 10 was
obtained in higher yield (66%). In contrast to the lack
of selectivity observed in previous alkylation reactions
of TSAO-m3T,17 acylation of 2 with methyloxalyl chlo-
ride occurs exclusively at the amino of the 4′′-position.
Attachment of the acyl residue to the amino group of
the sultone moiety and not to the 3′′-C-position in
compound 10 was established from their 1H NMR
spectra by the disappearance of the broad singlet at 6.45
ppm assigned to the 4′′-NH2 and by the appearance of
one 4′′-NH proton at 10.60 ppm, due to the deshielding
effect of the CdO of the acyl group attached to this NH.
This deshielding effect was also observed in the signal
corresponding to H-3′′, which showed a downfield shift
(∆δ) with respect to the same signal in the starting
TSAO derivative 2. The 13C NMR spectra of the com-
pound also confirmed the structural assignments.
The nucleoside 10 was used as a starting compound
to synthesize other N-oxalyl-substituted derivatives
(Scheme 2) bearing either carboxylic acid groups that
may interact with the Lys-B138 (TSAO-resistant strains)
or amido groups that may stabilize interactions with
highly conserved residues close to Glu-B138/Lys-B138
such as Asn-B136. Thus, the methyl ester group of
oxalyl nucleoside 10 was further transformed into the
free acid 11 (72% yield) by treatment with 1 N NaOH.
Alternatively, reaction of the methyl ester 10 with
appropriate amides afforded the amides 12-14 in 52%,
50%, and 60% yield, respectively (Scheme 2). The methyl
ester derivative 10 and the amides 12-14 were endowed
with potent anti-HIV-1 activity similar to or even
slightly higher than that of TSAO-m3T as will be
discussed later.
1
of five-membered ring lactones. The H NMR spectra
for this compound showed the disappearance of the
signals corresponding to the AB system of the sultone
ring and to the hemiacetalic OH and the presence of a
singlet at δ 3.38 ppm corresponding to the methyl of
the mesyl group. The 13C NMR spectra showed a
downfield shift of the C-4′′ (δ 170.92 ppm) corresponding
to the carbonyl of the lactone moiety. In the g-HMBC
experiments, the H-4′ and the H-5′ protons showed long-
range correlations with the carbonyl of the lactone
moiety, which confirmed the proposed structure.
Next, we focused on the synthesis of compounds
bearing carbonyl groups on the 4′′-amino of the spiro-
oxathioledioxide moiety. This was performed by reaction
of TSAO-m3T (2) with differently substituted acid
chlorides (Scheme 2). Ab initio theoretical calculations
of the amino sultone system19 showed that the nitrogen
atom of the amino sultone ring has mainly sp2 character
and the HOMO energy (EHOMO) of the amine was very
low (less than -9.73 eV), which corresponds to amines
with very low reactivity toward electrophiles.20 Because
of this expected low reactivity of the 4′′-amino group,
reaction of TSAO-m3T (2) with the highly reactive
methyloxalyl chloride reagent was first attempted.
Thus, when TSAO-m3T (2) was treated with methyl-
oxalyl chloride in the presence of DMAP as an acid
scavenger at room temperature, compound 10 was
isolated in 35% yield together with unreacted starting
material. A higher temperature in the reaction led to
complex reaction mixtures of unidentified products.
These results prompted us to explore the role that
each or both of the two carbonyl groups of the N-oxalyl
derivatives may play in the anti-HIV-1 activity. To
determine the importance of the carbonyl directly at-
tached to the 4′′-amino group, novel acyl TSAO com-

TSAO Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1161
Scheme 3. Synthesis of of (Un)Substituted 4′′-Ureido TSAO Compounds 19-25 and the Cyclic Compound 26
pounds 15-17 (Scheme 2) bearing one carbonyl group
were designed. Also, compound 18 with two carbonyl
groups linked through a methylene spacer was prepared
(Scheme 2). Thus, treatment of 2 with acetyl chloride
in the presence of AlCl3 and 4 Å molecular sieves
(optimized acylation conditions to get compound 10 from
2) or in the presence of different bases (pyridine,
triethylamine, or DMAP) and solvents (acetonitrile or
THF) at room temperature was unsuccessful. When the
reaction was performed in dichloroethane at 80 °C in
the presence of an excess of DMAP in a pressure
reaction vessel, the N-acyl derivative 15 was obtained
in moderate yield (42%). A similar treatment of 2 with
the corresponding acyl chlorides yielded the N-acyl
derivatives 16-18 (44%, 11%, and 51% yield, respec-
tively).
Next, 4′′-ureido-(un)substituted TSAO-m3T deriva-
tives bearing only one carbonyl group at the 4′′-position
(20, 21) or two carbonyl groups separated by different
spacers (22-25) were prepared by reaction of 2 with
differently substituted isocyanates (Scheme 3). Reaction
of 2 with chlorosulfonyl isocyanate, followed by treat-
ment with aqueous NaHCO3, gave the 5′-O-deprotected
4′′-unsubstituted ureido derivative 19 that was silylated
“in situ” (TBDMSCl/DMAP) to give the bisilylated 4′′-
ureido compound 20 in 40% overall yield. Similarly,
reaction of 2 with an excess of ethyl, benzoyl, ethoxy-
carbonyl, or methacryloyl isocyanate, in dry acetonitrile
at 80 °C, afforded the corresponding 4′′-N-alkyl- and
acyl-substituted ureido derivatives 21 (50%), 22 (90%),
23 (80%), and 24 (75%). When ethoxycarbonylmethyl
isocyanate was reacted with TSAO-m3T (2) under
similar reaction conditions, only the starting material
was recovered. However, when this reaction was carried
out in a sealed pressure tube in the presence of a
catalytic amount of triethylamine at 100 °C, the corre-
sponding N-substituted ureido derivative 25 was iso-
lated in 52% yield together with the unexpected cyclic
compound 26 (30%). Formation of this side product
could be explained by attack of a second molecule of the
isocyanate on the initially formed N-ureido derivative
25 and subsequent cyclization to give the dioxoimida-
zoline ring. Scarce reports on this type of cyclization
have been previously described.21
Finally, compounds 29a,b (Scheme 4), 31a,b, and
32a,b (Scheme 5), in which a carboxylic ester, carboxylic
acid, or amide, respectively, were attached to the 4′′-
amino group of the sultone ring through one or two
methylenes, were prepared. Compounds 29a, 31a, and
32a would allow us to study the influence of the
â-carbonyl group of the N-oxalyl derivatives 10-12 on
the anti-HIV-1 activity. Compounds 29a,b were ob-
tained from the TSAO-deprotected derivative 318 in a
20% and 28% overall yield by a three-step protocol
(Scheme 4), which is transamination reaction of 3 with
the corresponding amines, followed by “in situ” silylation
of the 2′- and 5′-hydroxyl groups and finally N-3
selective methylation of the thymine ring using stan-
dard conditions.18 The transamination reactions were
carried out by treatment of 3 with excess corresponding
methoxycarbonylalkylamine hydrochlorides in refluxing
methanol in a sealed tube for several days.
We also prepared the corresponding acid (31a,b) and
amide (32a,b) derivatives (Scheme 5). Thus, saponifica-
tion of 29a,b with 1 N NaOH to obtain 31a,b led to
complex reaction mixtures from which only 2′- and/or
5′-deprotected compounds were isolated. A milder al-
ternative that consisted of transesterification to the
benzyl esters (30a,b) followed by hydrogenolysis of the
benzyl moiety was attempted. Thus, transesterification
of 29a,b was readily achieved (Scheme 5) by heating
the corresponding nucleosides and benzyl alcohol in the
presence of a catalytic amount of 1,3-disubstituted
tetrabutyldistannoxane in toluene.22,23 Benzyl esters
thus obtained (30a,b) were hydrogenated (H2, Pd/C) to
afford the corresponding free acids 31a,b in 84% and
83% yield, respectively. Finally, treatment of 31a,b with
ammonia (2 M in methanol) in the presence of BOP as
coupling reagent and triethylamine as a base gave the
amide derivatives 32a,b in 44% and 82% yield, respec-
tively.
Biological Evaluation. The compounds were evalu-
ated for their inhibitory activity against HIV-1 and

1162 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
de Castro et al.
Scheme 4. Synthesis of 4′′-N-Alkyl TSAO Compounds 27-29
Scheme 5. Synthesis of 4′′-N-Alkyl TSAO Compounds
30-32
pressure of escalating drug regimens of 11 and 12. Two
virus strains independently selected in the presence of
compound 11 were found to have the E138K mutation
in its reverse transcriptase. This mutation is a charac-
teristic amino acid change in the RT of TSAO-resistant
virus strains. When virus strains were selected in the
presence of 12, one strain acquired the V106A mutation
in its RT whereas another strain also contained, besides
E138K, L228V in its RT. There is only one article in
the literature in which the latter mutation has been
reported to appear under NRTI drug pressure.24 This
mutation is due to a transversion mutation of the first
nucleotide in codon 228 (CTT f GTT). Compound 11
was 12- to 15-fold resistant to the E138K and E138K +
L228V RT HIV-1 strains, whereas compound 12 was
∼40-fold resistant to E138K and g50-fold resistant to
the double mutant virus strains. These levels of resis-
tance are less pronounced than the resistance level
observed for TSAO-m3T against the mutant virus
strains (>1000-fold). The NNRTIs nevirapine and UC-
781 and the NtRTI tenofovir virtually fully kept their
inhibitory potential against both E138K and E138K +
L228V RT virus mutants (data not shown).
HIV-2 in MT-4 and CEM cell cultures and compared
with the prototype TSAO-T and TSAO-m3T compounds
(Table 1).
The 5′-cyclic hemiacetal and bicyclic lactone deriva-
tives 5, 7, and 8 were devoid of anti-HIV activity but
also lacked any significant cytostatic activity at 100 µM
in CEM and MT-4 cell cultures. Whereas the methyl
ester nucleoside 10 and the amide 12 proved exquisitely
inhibitory against HIV-1 in both MT-4 and CEM cell
cultures, the free acid derivative 11 was 10- to 20-fold
less active against the virus. Also, the azetidine deriva-
tive 14 showed decreased antiviral activity compared
to the amide 12. In contrast, compounds containing
N-acyl groups on the 4′′-amino group of the spiro moiety
of TSAO (15-18) were devoid of antiviral activity at
subtoxic concentrations. Also, the ureido derivatives (20,
21) or those nucleosides containing two carbonyl groups
separated by different spacers (22-25) were inactive.
Compound 26, in which the two carbonyl functions were
separated by a cyclic entity, showed surprising antiviral
activity (EC50 ) 0.2-0.9 µM) although it was very close
to its cytostatic activity (CC50 ) 1.4 µM). Interestingly,
whereas 29a was markedly inhibitory against HIV-1
and relatively nontoxic, 29b, in which the 4′′-NH2 group
was separated from the methyl ester by two methylenes
instead of one methylene, lacked any antiviral efficacy.
Drug-resistant HIV-1 strains were selected under
When the compounds were evaluated against replica-
tion of human CMV in cell culture, several compounds
showed pronounced antiviral activity at concentrations
in the lower micromolar range (i.e., 0.29-2.0 µM for
compounds 16, 20, 22, 23, 26, that is, at a concentration
well below their toxicity threshold). In particular,
compound 22 showed the highest anti-human-cytome-
galovirus (anti-HCMV) activity lacking marked cyto-
toxicity or antiproliferative activity against HEL cell
cultures (selectivity index CC50/EC50 or MCC/EC50
g
100). It should be mentioned, however, that this com-
pound was rather cytostatic against the lymphocytic
CEM and MT-4 cell cultures. It should also be noted
that compound 26 showed marked inhibitory activity
against HCMV at 0.29-0.32 µM, 10-fold lower than the
cytotoxicity threshold. Interestingly, this compound was
also very active against HIV-1. Therefore, it could be
considered as the first TSAO derivative with dual
activity against HIV-1 and HCMV. The antiviral activity
profile of this molecule deserves further investigation.
The highly modified structures of these TSAO mol-
ecules point to a different mechanism of inhibition of
HCMV than that of currently used anti-HCMV nucleo-

TSAO Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1163
Table 1. Antiviral Activity of TSAO Derivatives in Cell Culture
HCMV
HIV
in human embryonic lung (HEL) cells
EC50 a (µM)
cytotoxicity (µM)
EC50 c (µM)
MT-4
CEM
cell
cell
CC50 b (µM)
MT-4 CEM
HIV-1
(IIIB)
HIV-2
(ROD)
HIV-1
(IIIB)
HIV-2
(ROD)
HCMV
HCMV morphology growth
e
compd
(AD-169) (Davis)
(MCC)d
(CC50)
1 (TSAO-T)
0.06 ( 0.03
>10
0.05 ( 0.01
0.04 ( 0.01
>100
>10
>250
>100
>100
>50
>4
12 ( 2
2 (TSAO-m3T) 0.06 ( 0.09
>250
>100
>100
>250
>4
230 ( 7
>100
5
>100
>100
7
>100
>100
>100
g250
>100
8
>50
>50
160 ( 39
5.57 ( 2.21
179
253
>80
6.5
164
179
36
g400
>400
400
g16
65
g80
g3.2
g50
400
16
g3.2
g80
16
20
16
g3.2
g400
g400
g400
g400
300
80
36
10
0.08 ( 0.006
1.30 ( 0.45
0.06 ( 0.02
0.23 ( 0.04
10.3 ( 2.6
31.1 ( 20.4 72.6 ( 6.5
9.59 ( 0.87 9.96 ( 1.48
17.4 ( 3.1
10.7 ( 0.6
4.9 ( 1.5
29.9 ( 7.3
11.9 ( 0.3
3.7 ( 0.4
>200
146
14.5
84.4
>200
9.7
11
>10
>50
12
0.057 ( 0.008 >2
0.028 ( 0.017 >10
6.5
14
0.8 ( 0.1
>5
>5
>20
>50
0.7 ( 0.1
>25
>10
>25
>5
21.6 ( 3.1
25.7 ( 5.0
4.91 ( 1.54
30.7 ( 0.3
29.7 ( 3.5
4.36 ( 0.13
4.26 ( 0.30
10
10
15
>5
40
1.3
g20
9
>3.2
1.8
1.2
2.3
>20
>3.2
0.29
>400
>400
>400
>400
g50
>16
>3.2
>16
>16
1.2
16
>5
>5
17
>20
>5
>20
>20
>25
>2
25
>200
29
11
10
18
19.5 ( 7.8
>2
g5
10
20
>2
>2
>2
2.0
21
>2
>2
>2
2.0
22
>2
>10
>2
>2
>2
3.33 ( 0.04 2.29 ( 0.07
3.0 ( 0.35
0.8
>200
14
23
>2
>2
>2
1.8
24
>0.8
>0.8
>2
>0.8
>2
>0.8
>2
1.19 ( 0.59 1.20 ( 0.52
3.78 ( 1.60 5.02 ( 0.62
1.43 ( 0.50 1.42 ( 0.51
>4
1.2
25
>2
>3.2
0.32
>400
>400
>400
>400
32
13
26
0.88 ( 0.02
1.61 ( 0.89
>250
>2
0.24 ( 0.02
0.6 ( 0.0
>250
6.7 ( 3.1
>250
>10
>2
2.5
29a
29b
30a
30b
31a
31b
32a
32b
>125
>250
>250
>250
>10
>50
>10
>10
>125
>250
>250
>250
>10
>10
>2
105 ( 29
>250
96 ( 41
>250
>250
>250
65 ( 16
19.4 ( 0.5
14.6 ( 2.0
21.0 ( 1.2
160
94
>250
>200
>200
136
34
13.5
14.8
>250
>10
>50
>10
>10
>250
30 ( 1
>10
11.5 ( 3.2
14.9 ( 3.5
20.0 ( 4.5
8.0
>3.2
29
>2
16
g80
5
>10
a 50% effective concentration required to inhibit HIV-induced cytopathogenicity by 50%. b 50% cytostatic concentration to inhibit MT-4
or CEM cell proliferation by 50%. c 50% effective concentration to inhibit HCMV-induced plaque formation by 50%. d Minimal cytotoxic
concentration required to cause a microscopically visible alteration of cell morphology. e 50% cytostatic concentration required to inhibit
HEL cell proliferation by 50%.
1H NMR spectra were recorded with a Varian Gemini, a Varian
side analogues. However, although it has been shown
XL-300, and a Bruker AM-200 spectrometer operating at 300
that TSAO derivatives in general interact in a specific
and 200 MHz with Me4Si as the internal standard. 13C NMR
manner with the virus-encoded reverse transcriptase,
spectra were recorded with a Varian XL-300 and a Bruker AM-
it is currently unclear whether the TSAO derivatives
200 spectrometer operating at 75 and 50 MHz with Me4Si as
that are inhibitory to HCMV are also targeted against
HCMV DNA polymerase (acting as a nonnucleosidic
inhibitor) or inhibit another viral (or cellular) function
necessary for virus replication.
the internal standard. Analytical thin-layer chromatography
(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 (Kie-
segel 60 PF254 gipshaltig (Merck), layer thickness of 1 mm,
flow rate of 5 mL/min). Flash column chromatography was
performed with silica gel 60 (230-400 mesh) (Merck). Analyti-
cal HPLC was carried out on a Waters 484 system using a
µBondapak C18 (3.9 mm × 300 mm, 10 mm). Isocratic condi-
tions were used: mobile phase CH3CN/H2O (0.05% TFA); flow
rate, 1 mL/min; detection, UV (254 nm). All retention times
are quoted in minutes.
Conclusions
In conclusion, several TSAO derivatives substituted,
at the 4′′-amino group of the spiromoiety, with different
carbonyl functionalities have been synthesized. Com-
pounds 10 and 12 were among the more potent and
selective inhibitors of HIV-1. Surprisingly, several of
these 4′′-substituted TSAO derivatives also gained
activity against anti-HCMV at subtoxic concentrations.
To date, all TSAO compounds reported by us showed
complete selectivity for HIV-1, with no antiviral activity
against a range of other (DNA or RNA) viruses. Com-
pound 26 represents the first example of an NNRTI with
dual anti-HIV-1 and -HCMV activity. This dual activity
seems interesting because opportunistic infections caused
by HCMV are common among AIDS patients.
Triethylamine, dioxane, 1,2-dichloromethane, 1,2-dichloro-
ethane, toluene, and acetonitrile were dried by refluxing over
calcium hydride.
The name of the bicyclic nucleoside 8 is given according to
the von Baeyer nomenclature. However, for easy comparison,
the assignments of the signals of the NMR spectra follow
standard carbohydrate/nucleoside numbering (i.e., the fura-
nose skeleton numbered 1′-5′) with the thymine moiety having
the highest priority.
(4′′S)-4′′,O-5′-Cyclic Hemiacetal of [1-(â-D-Ribofurano-
syl)thymine]-3′-spiro-5′′-(4′′-oxo-1′′,2′′-oxathiolane-2′′,2′′-
dio´xide) (5). To a solution of nucleoside 3 (0.2 g, 0.55 mmol)
in methanol (5 mL), a 0.1 N HCl solution in methanol (30 mL)
was added, and the mixture was stirred at room temperature
for 24 h. The mixture was neutralized to pH 5 with a solution
of 1 N NaOH in methanol, and the solvent was evaporated to
dryness. The residue was treated with isobutanol (30 mL) and
Experimental Section
Chemical Procedures. Microanalyses were obtained with
a Heraeus CHN-O-RAPID instrument. Mass spectra were
measured on a quadropole mass spectrometer equipped with
an electrospray source (Hewlett-Packard, LC/MS HP 1100).

1164 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
de Castro et al.
1
water (30 mL). The organic phase was separated, and the
aqueous phase was extracted several times with isobutanol
(3 × 20 mL). The combined organics were dried over Na2SO4,
filtered, and evaporated to dryness. The final residue was
purified by column chromatography (dicloromethane/methanol,
6:1) to give 0.15 g (70%) of 5 as an amorphous white solid. 1H
NMR spectral analysis of compound 5 proved that it exists as
a single 5′-cyclic hemiacetal diastereomer in (CD3)2SO solution.
1H NMR [200 MHz, (CD3)2SO] δ: 1.80 (s, 3H, CH3-5), 3.65 (d,
1H, J3“a,3”b ) 14.0 Hz, H-3′′a), 4.03 (dd, 1H, J4′,5′a ) 3.6 Hz,
J5′a,5′b ) 10.4 Hz, H-5′a), 4.05 (d, 1H, H-3′′b), 4.13 (dd, 1H, J4′,5′b
) 5.2 Hz, H-5′b), 4.64 (dd, 1H, J2′,OH ) 6.8 Hz, J1′,2′ ) 7.2 Hz,
H-2′), 4.58 (dd, 1H, H-4′), 5.87 (d, 1H, H-1′), 6.03 (d, 1H, OH),
7.59 (m, 1H, H-6), 8.41 (bs, 1H, OH), 11.47 (bs, 1H, NH-3).
13C NMR [50 MHz, (CD3)2SO] δ: 11.87 (CH3-5), 54.85 (CH2-
3′′), 68.92 (C-5′), 69.39 (C-2′), 82.72 (C-4′), 89.75 (C-1′), 96.36
(C-4′′), 107.67 (C-3′), 110.14 (C-5), 135.71 (C-6), 150.40 (C-2),
163.35 (C-4). MS (ES+) m/z 363.1 (M + 1)+. Anal. (C12H14N2O9S)
C, H, N, S. Long-range correlations observed in a 1H-13C
g-HMBC experiment were hemiacetal-C-4′′/5′-CH2 for com-
pound 5. Selective NOE experiments showed that irradiation
of the hemiacetal-OH-4“ caused enhancements of the signals
for H-2 and one of the H-3” protons.
an amorphous white solid was isolated. H NMR [300 MHz,
(CD3)2CO] δ: 1.83 (d, 3H, J ) 1.3 Hz, CH3-5), 3.33 (s, 3H,
OSO2CH3), 3.73 (m, 1H, H-5′a), 3.81 (s, 3H, OCH3), 3.85 (m,
1H, H-5′b), 4.29 (t, 1H, J4′,5a′ ) J4′,5b′ ) 3.2 Hz, H-4′), 4.42 (bt,
1H, OH-5′), 4.99 (t, 1H, J1′,2′ ) J2′,OH ) 6.6 Hz, H-2′), 5.56 (bd,
1H, OH-2′), 6.04 (d, 1H, H-1′), 7.83 (d, 1H, H-6), 10.18 (bs,
1H, NH-3). MS (ES+) m/z 395.1 (M + 1)+. Anal. (C13H18N2O10S)
C, H, N, S.
When chromatographic purification of the final residue by
CCTLC on the Chromatotron was carried out with dichlo-
romethane/ethyl acetate (2:1) as eluent, 0.082 g of compound
8 (62%) was the only isolated product in this reaction.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofurano-
syl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-methoxalylami-
no-1′′,2′′-oxathiole-2′′,2′′-dioxide) (10). Method A. To a
solution of TSAO-m3T (2) (0.2 g. 0.33 mmol) in dry 1,2-
dichloroethane (3 mL), DMAP (0.121 g, 0.99 mmol) and
methyloxalyl chloride (0.022 mL, 0.47 mmol) were added. The
reaction mixture was stirred at room temperature for 12 h.
After the mixture was cooled, the solvent was removed and
the residue was treated with dichloromethane (20 mL) and
water (20 mL). The organic phase was successively washed
with with 1 N HCl (1 × 20 mL), water (1 × 20 mL), and brine
(1 × 20 mL). The organic phase was dried (Na2SO4), filtered,
and evaporated to dryness. The final residue was purified by
CCTLC on the Chromatotron (dichloromethane/ethyl acetate,
10:1). The fastest moving fractions gave 0.08 g (35%) of 10 as
(4′′S)-4′′,O-5′-Cyclic Hemiacetal of [1-[2′-O-(tert-But-
yldimethylsilyl)-â-D-ribofuranosyl]thymine]-3′-spiro-5′′-
(4′′-oxo-1′′,2′′-oxathiolane-2′′,2′′-dio´xide) (7). Compound 5
(0.1 g, 0.28 mmol) was dissolved in dry acetonitrile (5 mL) and
then 4-(dimethylamino)pyridine (DMAP) (0.17 g, 1.4 mmol)
and tert-butyldimethylsilyl chloride (TBDMSCl) (0.21 g, 1.4
mmol) were added. The mixture was stirred at room temper-
ature for 24 h and evaporated to dryness. The residue was
treated with ethyl acetate (20 mL), filtered over silica gel and
evaporated to dryness. The final residue was purified by
CCTLC on the Chromatotron (dichloromethane:methanol, 20:
1) to give 0.082 g (62%) of 7 as an amorphous white solid.1H
NMR [200 MHz, (CD3)2CO] δ: 0.88 (s, 9H, t-Bu), 1.85 (d, 3H,
J ) 1.2 Hz, CH3-5), 3.62 (d, 1H, J3′′a,3′′b ) 13.9 Hz, H-3′′a), 4.03
(d, 1H, H-3′′b), 4.20 (dd, 1H, J4′,5′a ) 2.9, J5′a,5′b ) 10.7 Hz,
H-5′a), 4.15 (dd, 1H, J4′,5′b ) 4.9 Hz, H-5′b), 4.79 (dd, 1H, H-4′),
4.93 (d, 1H, J1′,2′ ) 6.1 Hz, H-2′), 6.02 (d, 1H, H-1′), 7.44 (bs,
1H, OH), 7.58 (d, 1H, H-6), 10.25 (bs, 1H, NH-3). 13C NMR
[50 MHz, CDCl3] δ: 12.27 (CH3-5), 18.04 [(CH3)3-C-Si], 25.55
[(CH3)3-C-Si], 56.08 (CH2-3′′), 70.09 (C-5′), 71.91 (C-2′), 83.48
(C-4′), 95.38 (C-1′), 98.17 (C-4′′), 109.8 (C-5), 111.93 (C-3′),
130.93 (C-6), 150.35 (C-2), 163.76 (C-4). MS (ES+) m/z 477.2
(M + 1)+. Anal. (C18H28N2O9SSi) C, H, N, S. Long-range
1
a white amorphous solid. HPLC: tR ) 12.29 min (65:35). H
NMR [300 MHz, (CD3)2CO] δ: 0.70, 0.79 (2s, 18H, 2t-Bu), 1.97
(d, 3H, J ) 1.1 Hz, CH3-5), 3.28 (s, 3H, CH3-3), 3.92 (s, 3H,
CO2CH3), 4.01 (dd, 1H, J4′,5′a ) 2.3, J5′a,5′b ) 13.0 Hz, H-5′a),
4.11 (dd, 1H, J4′,5′b ) 2.2 Hz, H-5′b), 4.48 (t, 1H, H-4′), 4.97 (d,
1H, J1′,2′ ) 8.0 Hz, H-2′), 6.07 (d, 1H, H-1′), 7.77 (s, 1H, H-3′′),
7.94 (d, 1H, H-6), 10.60 (bs, 1H, NH-4′′). 13C NMR [50 MHz,
(CD3)2CO] δ: 12.92 (CH3-5), 18.33, 19.05 [(CH3)3-C-Si], 25.68,
26.23 [(CH3)3-C-Si], 28.11 (CH3-3), 54.26 (OCH3), 61.86 (C-
5′), 73.78 (C-2′), 83.23, 88.92 (C-1′, C-4′), 92.43 (C-3′), 110.26
(C-3′′), 111.19 (C-5), 134.01 (C-6), 139.43 (C-4′′), 151.12 (C-2),
155.59 (CO-CO2CH3), 159.12 (CO-CO2CH3), 163.01 (C-4).
Anal. (C28H47N3O11SSi2) C, H, N, S. From the slowest moving
fractions 0.108 g (36%) of starting material was recovered.
Method B. A solution of methyloxalyl chloride (0.13 mL,
4.2 mmol) and aluminum chloride (0.19 g, 4.2 mmol) in dry
1,2-dichloroethane (1 mL) was added dropwise to a solution
of TSAO-m3T (0.7 g, 1.19 mmol) in dry 1,2- dichloroethane (2
mL) in the presence of 4 Å molecular sieves (0.9 g). The
reaction mixture was stirred for 24 h at room temperature and
then was quenched with ice/water (10 mL). The mixture was
stirred for an additional hour and filtered through a Celite
pad. The organic layer was separated, washed several times
with water (3 × 10 mL), dried (Na2SO4), filtered, and evapo-
rated to dryness. The residue was purified by CCTLC on the
Chromatotron (hexane/ethyl acetate, 2:1) to give 0.50 g (66%)
of 10 as a white amorphous solid. From the slowest moving
fractions 0.054 g (18%) of starting material was recovered.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofurano-
syl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-oxaloamino-
1′′,2′′-oxathiole-2′′,2′′-dioxide) (11). To a solution of the
oxalyl nucleoside 10 (0.1 g, 0.14 mmol) in 1,4-dioxane (2 mL),
a solution of 1 N NaOH in dioxane (0.16 mL, 0.16 mmol) was
added, and the mixture was stirred for 1 h at room tempera-
ture. Then the mixture was neutralized with 0.1 N HCl. The
resulting mixture was dissolved in ethyl acetate and was
successively washed with water (2 × 10 mL) and brine (2 ×
10 mL). Finally, the organic phase was dried (Na2SO4), filtered,
and evaporated to dryness. The final residue was purified by
CCTLC on the Chromatotron (dichloromethane/methanol,
15:1) to give 0.10 g (72%) of 11 as a white amorphous solid.
Anal. (C27H45N3O11SSi2) C, H, N, S.
1
correlations observed in a H-13C gHMBC experiment were
hemiacetal-C-4′′/5′-CH2 for compound 7. Selective NOE experi-
ments showed that irradiation of the hemiacetal-OH-4′′ caused
enhancements of the signals for H-2 and one of the H-3′′
protons.
1-([1′S,3′R,4′R,5′R]-4′-Hydroxyl-5′mesyl-6′-oxo-2′,7′-
dioxabicyclo[3.3.0]octan-3′-yl)thymine (8). To a suspension
of the cyclic hemiacetal 5 (0.1 g, 0.28 mol) in dry acetonitrile
(10 mL), DMAP (0.068 g, 0.56 mmol) was added. The resulting
mixture was stirred at 60 °C for 24 h and then acetonitrile
(25 mL) was removed by rotary evaporation under reduced
pressure. The residue was treated with ethyl acetate (25 mL)
and filtered through silica gel. The final residue was purified
by CCTLC on the Chromatotron using dichloromethane/
methanol, 20:1, as eluent. The fastest moving fractions af-
forded 0.050 g (49%) of 8 as an amorphous white solid. IR
(KBr) ) 1770 cm-1. 1H NMR [300 MHz, (CD3)2CO] δ: 1.82 (d,
3H, J ) 1.3 Hz, CH3-5), 3.38 (s, 3H, OSO2CH3), 4.45 (dd, 1H,
J4′,5′a ) 2.4 Hz, J5′a,5′b ) 10.5 Hz, H-5′a), 4.73 (m, 2H, H-2′,
H-5′b), 5.21 (dd, 1H, J4′,5′a ) 2.4 Hz, J4′,5′b ) 6.3 Hz, H-4′), 5.80
(bs, 1H, 4′′-OH), 6.04 (d, 1H, J1′,2′ ) 8.1 Hz, H-1′), 7.70 (d, 1H,
H-6), 10.21 (bs, 1H, NH-3). 13C NMR [100 MHz, (CD3)2CO] δ:
11.59 (CH3-5), 40.54 (OSO2CH3), 71.27 (C-5′), 74.91 (C-2′),
79.90 (C-4′), 84.81 (C-3′), 88.14 (C-1′), 111.36 (C-5), 135.74 (C-
6), 150.95 (C-2), 163.24 (C-4), 170.92 (C-4′′). MS (ES+) m/z
363.4 (M + 1)+. Anal. (C12H14N2O9S) C, H, N, S.
General Procedure for the Synthesis of 4′′-Oxamoyl
TSAO-m3T Derivatives (12-14). A solution of oxalyl nucleo-
side 10 (0.1 g, 0.14 mmol) and the corresponding amine was
stirred at room temperature until complete reaction of the
starting material (45 min to 4 h). Then the reaction mixture
From the slowest moving fractions 0.01 g (9%) of [1-[3′-O-
mesyl-3′-C-methoxycarbonyl-â-D-ribofuranosyl]thymine] (9) as

TSAO Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1165
was neutralized with AcOH and evaporated to dryness to give
the crude product. The product was redissolved in dichloro-
methane (20 mL), and the solution was washed with water (2
× 20 mL) and brine (1 × 20 mL), dried (Na2SO4), filtered, and
evaporated to dryness. The final residue was purified by
CCTLC on the Chromatotron. The chromatography eluent and
yield are indicated below for each reaction.
CH3-5), 2.25 (s, 3H, CH3-CO), 3.31 (s, 3H, CH3-3), 3.95 (dd,
1H, J4′,5′a ) 6.3 Hz, J5′a,5′b ) 12.2 Hz, H-5′a), 4.07 (dd, 1H, J4′,5′b
) 3.7 Hz, J5′a,5′b ) 12.2 Hz, H-5′b), 4.21 (dd, 1H, H-4′), 5.01 (d,
1H, J1′,2′ ) 6.7 Hz, H-2′), 5.60 (d, 1H, H-1′), 7.44 (s, 1H, H-3′′),
7.63 (s, 1H, H-6), 9.95 (s, 1H, NH). MS (ESI+): m/z 646.3 (M+).
Anal. (C27H47N3O9SSi2) C, H, N, S. Recovered starting material
was 0.028 g (28%).
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofurano-
syl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-oxamoylamino-
1′′,2′′-oxathiole-2′′,2′′-dioxide) (12). According to the general
procedure, nucleoside 10 (0.1 g, 0.14 mmol) was reacted with
methanol saturated with ammonia (5 mL) at room tempera-
ture for 45 min. The residue was purified by CCTLC (dichlo-
romethane/ethyl acetate, 10:1) to give 0.049 g (52%) of 12 as
a white amorphous solid. 1H NMR [300 MHz, (CD3)2CO] δ:
0.78, 0.97 (2s, 18H, 2t-Bu), 1.97 (s, 3H, CH3-5), 3.28 (s, 3H,
CH3-3), 4.10 (dd, 1H, J4′,5′a ) 2.2 Hz, J5′a,5′b ) 12.9 Hz, H-5′a),
4.31 (dd, 1H, J4′,5′b ) 3.7 Hz, H-5′b), 4.43 (dd, 1H, H-4′), 4.72
(d, 1H, J1′,2′ ) 8.3 Hz, H-2′), 6.25 (d, 1H, H-1′), 7.64 (bs, 1H,
NH), 7.50 (s, 1H, H-6), 7.78 (s, 1H, H-3′′), 8.15 (bs, 1H, NH),
10.65 (bs, 1H, NH). 13C NMR [75 MHz, (CD3)2CO] δ: 13.01
(CH3-5), 18.30, 19.73 [(CH3)3-C-Si], 25.57, 26.89 [(CH3)3-C-
Si, 28.64 (CH3-3), 62.70 (C-5′), 74.98 (C-2′), 84.69, 86.95 (C-1′,
C-4′), 94.49 (C-3′), 110.71, 111.59 (C-3′′, C-5), 133.76 (C-6),
140.56 (C-4′′), 152.19 (C-2), 160.58 (COCONH2), 160.81 (CO-
CONH2), 163.31 (C-4). Anal. (C27H46N4O10SSi2) C, H, N, S.
Recovered starting material was 0.021 g (21%).
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-benzoylamino-
1′′,2′′-oxathiole-2′′,2′′-dioxide) (16). According to the general
procedure, TSAO-m3T (0.1 g, 0.17 mmol) was reacted with
benzoyl chloride (0.28 mL, 0.24 mmol) and DMAP (0.13 g, 1.02
mmol) for 2 h. The final residue was purified by CCTLC on
the Chromatotron to give 0.053 g (44%) of 16 as a white
amorphous solid. HPLC: tR ) 27.24 min (65:35). MS (ESI+):
m/z 708.3 (M+). Anal. (C32H49N3O9SSi2) C, H, N, S. Recovered
starting material was 0.035 g (35%)
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-methoxy-
carbonylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (17). The
general procedure was followed with TSAO-m3T (0.1 g, 0.17
mmol), methyl chloroformate (0.037 mL, 0.48 mmol), and
DMAP (0.124 g, 1.02 mmol) for 1 h. Purification of the final
residue by CCTLC on the Chromatotron gave compound 17
(0.012 g, 11%) as a white amorphous solid. HPLC: tR ) 13.93
min (65:35). Anal. (C27H47N3O10SSi2) C, H, N, S. Recovered
starting material: 0.07 g (70%).
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-methyloxamoyl-
amino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (13). Via the general
procedure, nucleoside 10 (0.1 g, 0.14 mmol) was treated with
methylamine (8 M solution in ethanol, 10 mL) at room
temperature for 4 h. After the workup, the residue was purified
by CCTLC (hexane/ethyl acetate 3:1) to yield 0.048 g (50%) of
compound 13 as a white amorphous solid. HPLC: tR ) 12.26
min (65:35). MS (ESI+): m/z 689.1 (M+). Anal. (C28H48N4O10-
SSi2) C, H, N, S. From the slowest moving fractions 0.016 g
(19%) of TSAO-m3T was isolated.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-methoxy-
malonylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (18). Follow-
ing the general procedure, TSAO-m3T (0.1 g, 0.17 mmol) was
treated with methoxymalonyl chloride (0.069 mL, 0.65 mmol)
and DMAP (0.25 g, 2.04 mmol) for 24 h. Purification of the
final residue by CCTLC on the Chromatotron gave compound
18 (0.060 g, 51%) as a white amorphous solid. HPLC:
tR ) 11.43 min (65:35). MS (ESI+): m/z 704.3 (M+). Anal.
(C29H49N3O11SSi2) C, H, N, S. Recovered starting material: 9
mg (9%)
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-[4′′-(1-azetidinyl)-
oxalylamino-1′′2′′-oxathiole-2′′,2′′-dioxide] (14). To a solu-
tion of compound 10 (0.1 g, 0.14 mmol) in MeOH (2 mL)
azetidine (0.01 mL, 0.15 mmol) was added. The reaction
mixture was stirred at 0 °C for 4 h. After the workup according
to the general procedure, the residue was purified by CCTLC
(hexane/ethyl acetate, 2.1) to give 14 (0.060 g, 60%) as a white
foam. HPLC: tR ) 15.91 min (65:35). MS (ESI+): m/z 715.2
(M+). Anal. (C29H50N4O10SSi2) C, H, N, S. From the slowest
moving fractions 0.014 g (17%) of TSAO-m3T was isolated.
General Acylation Procedure for the Synthesis of 4′′-
Substituted TSAO-m3T Derivatives (15-18) by Reaction
with Acid Chlorides. To a solution of TSAO-m3T (2) (1 mmol)
in dry 1,2-dichloroethane (14 mL), the corresponding acid
chloride (1.5-4 equiv) and DMAP (6-12 equiv) were added.
The mixture was stirred in an Ace pressure tube for 0.5-24 h
at 80 °C. The reaction mixture was allowed to cool to room
temperature. Then dichloromethane was added and the solu-
tion was successively washed with 1 N HCl (1 × 20 mL), water
(1 × 20 mL), and brine (1 × 20 mL). The organic phase was
dried (Na2SO4), filtered, and evaporated to dryness. The final
residue was purified by CCTLC on the Chromatotron (hexane/
ethyl acetate, 2:1).
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-ureido-1′′,2′′-
oxathiole-2′′,2′′-dioxide) (20). To a cooled (-30 °C) solution
of TSAO-m3T (2) (0.2 g, 0.33 mmol) in dry dichloromethane (5
mL), previously degassed under an argon atmosphere, chlo-
rosulfonyl isocyanate (0.19 g, 1.32 mmol) was added. The
resulting mixture was stirred at -30 °C for 15 min. Then the
reaction was quenched (NaHCO3), and the organic layer was
separated. The aqueous layer was extracted several times with
ethyl acetate (3 × 20 mL). The combined organic layers were
washed with water (20 mL) and brine (20 mL), dried (Na2-
SO4), filtered, and evaporated to dryness. The residue (com-
pound 19) was dissolved in dry acetonitrile (2 mL) and treated
with TBDMSCl (0.098 g, 0.66 mmol) and DMAP (0.08 g, 0.66
mmol). The resulting reaction mixture was stirred at room
temperature for 5 h, and the solvent was removed. The residue
was redissolved in dichloromethane (20 mL) and was succe-
sively washed with 1 N HCl (1 × 20 mL), water (1 × 20 mL),
and brine (1 × 20 mL). The organic phase was dried (Na2SO4),
filtered, and evaporated to dryness. The final residue was
purified by CCTLC on the Chromatotron (hexane/ethyl acetate,
3:1), yielding 0.085 g (40%) of compound 20 as a white foam.
MS (ESI+) m/z 669.3 (M + Na)+. Anal. (C26H46N4O9SSi2) C, H,
N, S.
The yields of the isolated products are indicated below for
each reaction. In all cases, unreacted starting compound was
isolated.
General Acylation Procedure for the Synthesis of 4′′-
Ureido Substituted TSAO-m3T Derivatives (21-24) by
Reaction with Isocyanates. To a solution of TSAO-m3T (0.1
g, 0.17 mmol) in dry acetonitrile (2 mL) the appropiate
isocyanate (4-20 equivalents) was added. The reaction mix-
ture was stirred at 80 °C for 3-96 h. After evaporation of the
solvent, the residue was purified by CCTLC on the Chroma-
totron. The chromatography eluent, yield, and analytical and
spectroscopic data of the isolated products are indicated below
for each reaction.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofurano-
syl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-acetylamino-
1′′,2′′-oxathiole-2′′,2′′-dioxide) (15). According to the general
procedure, TSAO-m3T (2) (0.1 g, 0.17 mmol) was reacted with
acetyl chloride (0.048 mL, 0.68 mmol) and DMAP (0.25 g, 2.04
mmol) for 24 h. The final residue was purified by CCTLC on
the Chromatotron to give 0.046 g (42%) of 15 as a white
amorphous solid. HPLC: tR ) 10.53 min (65:35). 1H NMR [300
MHz, (CD3)2CO] δ: 0.80, 0.81 (2s, 18H, 2t-Bu), 1.92 (s, 3H,
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-[4′′-(3-ethyl-

1166 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
de Castro et al.
ureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide] (21). The general
procedure was followed with TSAO-m3T (2) (0.1 g, 0.17 mmol)
and ethyl isocyanate (0.26 mL, 3.30 mmol) for 96 h. Chroma-
tography of the final residue with dicloromethane/methanol
(200:1) afforded 0.058 g (50%) of 21 as a white foam. 1H NMR
[300 MHz, (CD3)2CO] δ: 0.83, 0.89 (2s, 18H, 2t-Bu), 1.15 (t,
3H, J ) 7.2 Hz, CH2CH3), 1.93 (d, 3H, J ) 1.2 Hz, CH3-5),
3.25 (m, 2H, JCH2,NH ) 5.6 Hz, CH2CH3), 3.32 (s, 3H, CH3-3),
3.88 (dd, 1H, J4′,5′a ) 6.7 Hz, J5′a,5′b ) 11.9 Hz, H-5′a), 4.08
(dd, 1H, J4′,5′b ) 4.5 Hz, H-5′b), 4.18 (dd, 1H, H-4′), 5.04 (d,
1H, J1′,2′ ) 6.6 Hz, H-2′), 5.50 (d, 1H, H-1′), 6.18 (bt, 1H, NH),
H-5′b), 4.22 (m, 2H, J ) 1.6, J ) 7.0 Hz, CH2CH3), 4.44 (m,
3H, H-4′, CH2N), 4.60 (m, 2H, J ) 17.9 Hz, J ) 15.6 Hz, CH2),
4.84 (d, 1H, J1′2′ ) 7.8 Hz, H-2′), 6.02 (d, 1H, H-1′), 7.43 (s,
1H, H-3′′), 7.55 (d, 1H, H-6), 10.25 (bs, 1H, NH-4′′). 13C NMR
[100 MHz, (CD3)2CO] δ: 13.01 (CH3-5), 14.39 (CH3CH2O),
18.34, 19.01 [(CH3)3-C-Si], 25.69, 26.22 [(CH3)3-C-Si], 28.05
(CH3-3), 40.61 (CH2-N), 48.70 (CH2), 62.03 (C-5′), 62.67 (CH2-
OOC), 74.70 (C-2′), 85.01 (C-4′), 89.34 (C-1′), 91.37 (C-3′),
107.23 (C-3′′), 111.49 (C-5), 135.09 (C-6), 142.38 (C-4′′), 148.30
(CH2CON), 151.96 (C-2), 156.64 (NCON), 163.34 (C-4), 167.09
(COOCH2CH3), 167.50 (NHCON). MS (ESI-) m/z 814.5 (M -
1)-. Anal. (C33H53N5O13SSi2) C, H, N, S. The slowest moving
fractions yielded 0.01 g (10%) of starting material.
General Procedure for the Synthesis of 4′′-Substituted
TSAO-m3T Derivatives 29a,b via Transamination Reac-
tion. TSAO deprotected nucleoside 318 (1 equiv) and the
corresponding amine hydrochloride (3 equiv) in methanol was
refluxed for 2-10 days. The solvent was removed, and the
residue was filtered through silica gel using ethyl acetate/
methanol, 8:1, as eluent. The residue (compounds 27a,b) was
dissolved in dry acetonitrile (4 mL) and treated with TBDMSCl
(0.15 g, 1 mmol) and DMAP (0.12 g, 1 mmol). The resulting
reaction mixture was heated at 80 °C for 5 h, and the solvent
was removed. The residue was redissolved in ethyl acetate (20
mL) and was successively washed with 1 N HCl (1 × 20 mL),
water (1 × 20 mL), and brine (1 × 20 mL). The organic phase
was dried (Na2SO4), filtered, and evaporated to dryness. The
residue (compounds 28a,b) was dissolved in dry acetone (4 mL)
and K2CO3 (0.028 g, 0.2 mmol), and methyl iodide (0.05 mL,
0.8 mmol) was added. The mixture was heated to 50 °C
overnight. The solvent was removed, and the final residue was
purified by CCTLC on the Chromatotron.
7.12 (s, 1H, H-3′′), 7.67 (d, 1H, H-6), 8.87 (bs, 1H, NH-4′′). 13
C
NMR [75 MHz, (CD3)2CO] δ: 12.91 (CH3-5), 15.36 (NH-CH2-
CH3), 18.39, 18.81 [(CH3)3-C-Si], 25.72, 26.18 [(CH3)3-C-
Si, 28.03 (CH3-3), 35.61 (NH-CH2-CH3), 61.60 (C-5′), 73.55
(C-2′), 84.38 (C-4′), 89.25 (C-3′), 96.45 (C-1′), 100.06 (C-3′′),
111.63 (C-5), 138.89 (C-6), 145.41 (C-4′′), 152.34 (C-2), 153.67
(NHCONH), 163.17 (C-4). Anal. (C28H50N4O9SSi2) C, H, N, S.
Recovered starting material was 0.048 g (48%).
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-[4′′-(3-benzoyl-
ureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide] (22). TSAO-m3T (2)
(0.1 g, 0.17 mmol) was reacted with benzoyl isocyanate (0.12
mL, 1.02 mmol) for 26 h. Chromatography of the final residue
on the Chromatotron (hexane/ethyl acetate 3:1) yielded 0.12
g (90%) of 22 as a white foam. Anal. (C33H50N4O10SSi2) C, H,
N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-[4′′-(3-ethoxy-
carbonylureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide] (23). Ac-
cording to the general procedure, TSAO-m3T (2) (0.1 g, 0.17
mmol) was treated with ethoxycarbonyl isocyanate (0.041 mL,
0.51 mmol) for 3 h. The final residue was purified on the
Chromatotron (dicloromethane/methanol, 200:1) to give 0.095
g (80%) of 23 as a white foam. Anal. (C29H50N4O11SSi2) C, H,
N, S. Recovered starting material was 0.005 g (5%).
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-[4′′-(3-meth-
acryloylureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide] (24). Fol-
lowing the general procedure, TSAO-m3T (2) (0.1 g, 0.17 mmol)
was reacted with methacryloyl isocyanate (0.12 g, 1.02 mmol)
for 72 h. Purification of the final residue on the Chromatotron
(dicloromethane/methanol, 60:1) afforded 0.09 g (75%) of 24
as a white foam. Anal. (C30H50N4O10SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-[4′′-(3-ethoxy-
carbonylmethylureido)-1′′,2′′-oxathiole-2′′,2′′-dioxide] (25).
TSAO-m3T(2)(0.10g,0.17mmol)wastreatedwithethoxycarbonyl-
methyl isocyanate (0.034 mL, 0.34 mmol) in the presence of
NEt3 (0.010 mL, 0.07 mmol. The mixture was stirred in an
Ace pressure tube for 12 h at 100 °C. After the reaction mixture
was allowed to cool to room temperature, the solvent was
evaporated to dryness. The residue was purified by CCTLC
on the Chromatotron (hexane/ethyl acetate, 4:1). The fastest
moving fractions afforded 0.06 g (52%) of 25 as a white foam.
1H NMR [300 MHz, (CD3)2CO] δ: 0.84, 0.88 (2s, 18H, 2t-Bu),
1.25 (t, 3H, J ) 7.1 Hz, CH2CH3), 1.93 (d, 3H, J ) 1.2 Hz,
CH3-5), 3.33 (s, 3H, CH3-3), 3.92 (dd, 1H, J4′,5′a ) 7.0 Hz, J5′a,5′b
) 11.9 Hz, H-5′a), 4.24-4.40 (m, 6H, CH2CO2CH2CH3, H-4′,
H-5′b), 5.07 (d, 1H, J1′,2′ ) 6.5 Hz, H-2′), 5.53 (d, 1H, H-1′),
6.53 (m, 1H, NH), 7.13 (s, 1H, H-3′′), 7.69 (d, 1H, H-6), 9.30
(bs, 1H, NH-4′′). 13C NMR [75 MHz, (CD3)2CO] δ: 14.35, 14.56
(CH3-CH2, CH3-5), 18.55, 18.79 [(CH3)3-C-Si], 25.84, 26.23
[(CH3)3-C-Si], 29.03 (CH3-3), 42.55 (CH2CO2CH2CH3), 61.62
(C-5′), 73.50 (C-2′), 84.45 (C-4′), 89.21 (C-3′) 97.44 (C-1′), 100.67
(C-3′′), 111.75 (C-5), 139.01 (C-6), 145.51 (C-4′′), 152.52 (C-2),
153.95 (NHCONH), 163.18 (C-4), 170.42 (CO2CH2CH3). MS
(ESI+) m/z 733.3 (M + 1)+. Anal. (C30H52N4O11SSi2) C, H, N,
S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-methoxy-
carbonylmethylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (29a).
According to the general procedure, compound 3 (0.5 g, 1
mmol) was treated with H-Gly-OMe‚HCl (0.414 g, 3 mmol) in
methanol (20 mL) at reflux for 10 days. After silylation and
N-3 methylation, the final residue was purified on the chro-
matotron with hexane/ethyl acetate (4:1), which afforded 0.146
g (20%) of 29a as a white foam. HPLC: tR ) 10.53 min (65:
1
35). H NMR [300 MHz, (CD3)2CO] δ: 0.80, 0.96 (2s, 18H, 2
t-Bu), 1.96 (s, 3H, CH3-5), 3.27 (s, 3H, CH3-3), 3.79 (s, 3H,
OCH3), 4.02-4.26 (m, 4H, H-5′, CH2-N), 4.35 (dd, 1H, J4′,5′a
) 3.2 Hz, J4′,5′b ) 2.93 Hz, H-4′), 4.65 (d, 1H, J1′,2′ ) 8.1, Hz,
H-2′), 5.92 (s, 1H, H-3′′), 6.10 (d, 1H, H-1′), 6.85 (bt, 1H,
JNH-CH2 ) 5.7 Hz, NH-CH2), 7.49 (s, 1H, H-6).13C NMR [75
MHz, (CD3)2CO] δ: 13.05 (CH3-5), 18.38, 19.16 [(CH3)2-C-
Si], 25.62, 26.43 [(CH3)2-C-Si], 28.09 (CH3-3), 46.78 (OCH3),
52.78 (CH2), 62.97 (C-5′), 75.64 (C-2′), 85.09 (C-4′), 87.89 (C-
3′′), 91.98 (C-1′), 92.52 (C-3′), 111.32 (C-5), 134.23 (C-6), 151.94,
152.11 (C-4′′, C-2), 163.34 (COCH2), 170.10 (C-4). MS (ESI+)
m/z 676.3 (M+). Anal. (C28H49N3O10SSi) C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-methoxy-
carbonylethylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (29b).
Compound 3 (0.50 g, 1 mmol) was reacted with H-â-Ala-
OMe.HCl (4.2 g, 3 mmol) in methanol (20 mL) at reflux for 10
days. After silylation and N-3 methylation, the final residue
was purified on the Chromatotron with hexane/ethyl acetate
(4:1), which afforded 0.212 g (28%) of 29b as a white foam.
HPLC: tR ) 10.21 min (65:35). MS (ESI+): m/z 690.1 (M+).
Anal. (C29H51N3O10SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-benzyloxy-
carbonylmethylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (30a).
A toluene solution (20 mL) of 1-hydroxy-3-chlorotetrabutyl-
distannoxane22 (0.164 g, 0.16 mmol), benzyl alcohol (0.208 mL,
2.0 mmol), and 29a (0.143 g, 0.20 mmol) was refluxed for 24
h. The toluene and excess benzyl alcohol were evaporated in
vacuo. The residue was purified by flash column chromatog-
raphy (hexane/ethyl acetate, 3:1) to give 0.125 g (82%) of 30a
as a white foam. MS (ESI+): m/z 752.2 (M+). Anal. (C34H53N3O10-
SSi2) C, H, N, S.
The next moving fractions gave 0.042 g (30%) of 26 as a
1
white foam. H NMR [400 MHz, (CD3)2CO] δ: 0.79, 0.87 (2s,
18H, 2t-Bu), 1.27 (t, 3H, J ) 7.0 Hz, CH2CH3), 1.93 (d, 3H, J
) 1.1 Hz, CH3-5), 3.26 (s, 3H, CH3-3), 4.03 (dd, 1H, J4′,5′a
)
5.0 Hz, J5′a,5′b ) 12.3 Hz, H-5′a), 4.10 (dd, 1H, J4′,5′b ) 5.7 Hz,

TSAO Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1167
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-benzyloxy-
carbonylethylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (30b).
Compound 29b (0.212 g, 0.30 mmol) was reacted with 1-hy-
droxy-3-chlorotetrabutyldistannoxane22 (0.247 g, 0.45 mmol)
and benzyl alcohol (0.310 mL, 3 mmol) in toluene (40 mL), as
described for 30a. Purification of the residue by flash column
chromatography (hexane/ethyl acetate, 3:1) yielded 0.15 g
(65%) of 30b as a white foam. HPLC: tR ) 24.20 min (65:35).
MS (ESI+): m/z 766.3 (M+). Anal. (C35H55N3O10SSi2) C, H, N,
S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5"-(4′′-carboxymeth-
ylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (31a). A solution of
compound 30a (0.100 g, 0.125 mmol) in ethyl acetate (15 mL)
containing 0.036 g of 10 % Pd/C (40 wt %) was hydrogenated
at 30 psi at 30 °C for 2 h. The reaction mixture was filtered,
and the filtrate was evaporated to dryness under reduced
pressure to give 0.068 g (84%) of 31a as a white amorphous
solid. MS (ESI+): m/z 663.1 (M + 1)+. Anal. (C27H47N3O10SSi2)
C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-carboxyeth-
ylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (31b). Compound
30b (0.095 g, 0.12 mmol) was hydrogenated in ethyl acetate
(12 mL) in the presence of 0.038 g of 10% Pd/C (40 wt %) at
30 psi at 30 °C for 1 h. The reaction mixture was filtered, and
the filtrate was evaporated to dryness under reduced pressure
to give 0.070 g (83%) of 31b as a white amorphous solid.
HPLC: tR ) 4.39 min (65:35). MS (ESI+): m/z 676.3 (M+). Anal.
(C28H49N3O10SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-carbamoyl-
methylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (32a). To a
solution of the acid derivative 31a (0.06 g, 0.09 mmol) in dry
dichloromethane (6 mL) at -20 °C, NEt3 (0.013 mL, 0.09
mmol) and a 2 M solution of ammonia in methanol (0.112 mL,
0.225 mmol) were added. After 15 min at -20 °C, BOP (0.04
g, 0.09 mmol) was added and the reaction mixture was stirred
at room temperature overnight and the solvent was removed.
The residue was dissolved in ethyl acetate (20 mL) and was
successively washed with a 10% citric acid solution (1 × 20
mL), 10% sodium bicarbonate (1 × 20 mL), and brine (1 × 20
mL). The organic phase was dried (Na2SO4), filtered, and
evaporated to dryness. The final residue was purified by
CCTLC on the Chromatotron (dichloromethane/methanol, 20:
1) to give compound 32a (0.027 g, 44%) as a white amorphous
solid. HPLC: tR ) 4.06 min (65:35). MS (ESI+): m/z 661.3 (M+).
Anal. (C27H48N4O9SSi2) C, H, N, S.
or HIV-2 at ∼100 CCID50 (50% cell culture infective dose) per
milliliter of cell suspension. Then an amount of 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 concentration 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%.
c. Selection of Drug-Resistant Virus Strains and
Identification of Mutations in HIV-1 Reverse Tran-
scriptase. Drug resistance was selected for compounds 11 and
12 against HIV-I (IIIB) in 1 mL cell cultures (48-well plates)
under escalating drug regimens. The compound concentrations
that were added at initiation of the drug selection were about
2-3 times their EC50 value. Then, as soon as full cytopathicity
appeared, the concentrations were gradually increased by 2-
or 3-fold. The cultures were passaged every 3-4 days by
adding ∼900 µL of fresh cell culture medium to 50-100 µL
from the virus-infected cell cultures. The mutations that
appeared in the RT genes of the drug-exposed virus strains
were determined according to previously published proce-
dures.25
d. Activity Assay of Test Compounds against HCMV
in Cell Culture. Confluent HEL cells grown in 96-well
microtiter plates were inoculated with HCMV at an input of
100 PFU (plaque forming units) per well. After a 1-2 h
incubation period, residual virus was removed, and the
infected cells were further incubated with MEM (supplemented
with 2% inactivated FCS, 1% l-glutamine, and 0.3% sodium
bicarbonate) containing varying concentrations of the com-
pounds (in duplicate).
After 7 days of incubation at 37 °C in a 5% CO2 atmosphere,
the cells were fixed with ethanol and stained with 2.5% Giemsa
solution. Virus plaque formation or viral cytophatic effect
[virus input: 100 PFU] was monitored microscopically. The
antiviral activity is expressed as IC50, which represents the
compound concentration required to reduce virus plaque
formation or cytopathicity by 50%. IC50 values were estimated
from graphic plots of the number of plaques (percentage of
control) or percentage of cytopathicity as a function of the
concentration of the test compounds. A variety of test com-
pound concentrations were used and differed 5-fold from each
other. The IC50 was calculated from the graphic plots using
the compound concentrations that were just above and just
below the IC50 value. Data are the mean of two independent
experiments.
[1-[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3-N-(methyl)thymine]-3′-spiro-5′′-(4′′-carbamoyleth-
ylamino-1′′,2′′-oxathiole-2′′,2′′-dioxide) (32b). According to
the procedure described for 32a, the acid derivative 31b (0.06
g, 0.09 mmol) was reacted with triethylamine (0.013 mL, 0.09
mmol), 2 M solution of ammonia in methanol (0.112 mL, 0.225
mmol), and BOP (0.04 g, 0.09 mmol) in dry dichloromethane
(6 mL). Purification of the final residue by CCTLC on the
Chromatotron (dichloromethane/methanol, 20:1) yielded com-
pound 32b (0.051 g, 82%) as a white amorphous solid. HPLC:
tR ) 3.72 min (65:35). MS (ESI+): m/z 675.3 (M+). Anal.
(C28H50N4O9SSi2) C, H, N, S.
Biological Methods. a. Cells and Viruses. Human im-
munodeficiency 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. Montag-
nier (when at the Pasteur Institute, Paris, France).
Confluent HEL fibroblasts, grown in 96-well microtiter
plates, were infected with HCMV Davis strain at 100 PFU/
well. After a 2 h adsorption period, residual virus was removed
and the infected cells were further incubated with medium. A
concentration of the test compounds at 5 µg/mL was added at
different time points after infection: 2 h (control) or 6, 14, 24,
30, 36, 48, or 72 h. The cultures were further incubated at 37
°C in a 5% CO2 atmosphere and fixed at day 7. The percentage
of CPE was then calculated for each point. The last time point
for which an activity comparable to that of the control was
recorded corresponds to the stage of the virus cycle at which
the test compound interacts.
The laboratory HCMV strains Davis and AD-169 were used
in the CPE (cytopathic effect) reduction assays. Virus stocks
consisted of cell-free virus obtained from the supernatant of
infected cell cultures that had been clarified by low-speed
centrifugation. The virus stocks were stored at -80 °C.
b. Activity Assay of Test Compounds against HIV-1
and HIV-2 in Cell Cultures. A total number of 4 × 105 CEM
or 3 × 105 MT-4 cells per milliliter were infected with HIV-1
e. Cytotoxicity Assays. Cytotoxicity measurements were
based on the inhibition of HEL cell growth. HEL fibroblasts
were seeded at a rate of 5 × 103 cells/well in 96-well microtiter
plates and allowed to proliferate for 24 h. Different concentra-
tions of the test compounds were then added (in duplicate),
and after 3 days of incubation at 37 °C in 5% CO2 atmosphere,
the cell number was determined with a Coulter counter.
Cytotoxicity is expressed as CC50, which represents the

1168 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
de Castro et al.
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dioxo-1′′,2′′-oxathiole-5′′,3′-[2′,5′-bis-O-(tert-butyldimethylsilyl)-
â-D-ribofuranosyl]]}-3-ethylthymine. Biochemistry 2000, 39, 1427-
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Clercq, E.; Karlsson, A. Sensitivity of (138 GLU f LYS) mutated
human immunodeficiency virus type 1 (HIV-1) reverse tran-
scriptase (RT) to HIV-1-specific RT inhibitors. Biochem. Biophys.
Res. Commun. 1994, 201, 1305-1312. (b) Jonckheere, H.;
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compound concentration required to reduce cell growth by 50%.
As a second parameter of cytotoxicity, the minimum cytotoxic
concentration (MCC) to cause a microscopically detectable
change in morphology of normal cells treated with the com-
pounds was determined.
Acknowledgment. We thank Ann Absillis, Lies
Vandenheurck, and Steven Carmans for excellent tech-
nical assistance. 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 MEC (Project SAF2003-
07219-C02-01) and the European Commission (Projects
QLK2-CT-2000-0291 and HPAW-CT-2002-90001) are
also acknowledged for financial support.
Supporting Information Available: 1H and 13C NMR
chemical shift assignments of compounds 11, 13, 14, 16-18,
20, 22-24, 29b, and 30a,b-32a,b and elemental analysis
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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