Synthesis of 3' '-substituted TSAO derivatives with anti-HIV-1 and anti-HIV-2 activity through an efficient palladium-catalyzed cross-coupling approach.

Article abstract of DOI:10.1021/jm020820h

Various synthetic studies for the introduction of several functional groups at position 3' ' of the spiro moiety of TSAO derivatives have been explored. Among them, Stille cross-coupling of 3' '-iodo-TSAO derivatives with different stannanes provided an e

Full text of DOI:10.1021/jm020820h

3934
J . Med. Chem. 2002, 45, 3934-3945
Syn th esis of 3′′-Su bstitu ted TSAO Der iva tives w ith An ti-HIV-1 a n d An ti-HIV-2
Activity th r ou gh a n Efficien t P a lla d iu m -Ca ta lyzed Cr oss-Cou p lin g Ap p r oa ch
Esther Lobato´n,† Fa´tima Rodr´ıguez-Barrios,‡ Federico Gago,‡ Mar´ıa-J esu´s Pe´rez-Pe´rez,† Erik De Clercq,§
J an Balzarini,§ Mar´ıa-J ose´ Camarasa,† and Sonsoles Vela´zquez*,†
Instituto de Quı´mica Me´dica (C.S.I.C.), J uan de la Cierva 3, E-28006 Madrid, Spain, Departamento de Farmacolog´ıa,
Universidad de Alcala´, E-28871 Alcala´ de Henares, Madrid, Spain, and Rega Institute for Medical Research,
K. U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received J anuary 15, 2002
Various synthetic studies for the introduction of several functional groups at position 3′′ of the
spiro moiety of TSAO derivatives have been explored. Among them, Stille cross-coupling of
3′′-iodo-TSAO derivatives with different stannanes provided an efficient and straightforward
route for the direct and selective functionalization of the 3′′-position of the sultone spiro moiety
via carbon-carbon bond formation. The compounds synthesized were evaluated for their
inhibitory effect on HIV-1 and HIV-2 replication in cell culture. The introduction of a bromine
and particularly an iodine at the 3′′-position conferred the highest anti-HIV-1 activity. In
contrast, the presence at this position of (un)substituted vinyl, alkynyl, phenyl, or thienyl groups
markedly diminished the anti-HIV-1 activity. Surprisingly, several of the 3′′-alkenyl-substituted
TSAO derivatives also gained anti-HIV-2 activity at subtoxic concentrations, an observation
that is very unusual for NNRTIs and never observed before for TSAO derivatives. Finally, the
anti-HIV-1 activity of some of the 3′′-substituted TSAO derivatives is discussed in light of our
recently proposed molecular model of interaction of TSAO derivatives with the interphase
between the two subunits of HIV-1 reverse transcriptase.
In tr od u ction
Among the different families of specific nonnucleoside
reverse transcriptase inhibitors (NNRTIs) described so
far, [2′,5′-bis-O-(tert-butyldimethylsilyl)-â-D-ribofuran-
osyl]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-diox-
ide) nucleosides (TSAO) represent a rather unique class
of nucleoside analogues that have been identified as
highly specific noncompetitive inhibitors of the RNA-
dependent DNA polymerase function of reverse tran-
scriptase.1,2 The prototype compound is the thymine
derivative designated as TSAO-T (1). The most selective
F igu r e 1. Chemical structures of TSAO-T and TSAO-m3T.
compound is the 3-N-methyl-substituted derivative
TSAO-m3T (2) (Figure 1). There are several features of
are able to destabilize the p66/p51 RT heterodimer in a
concentration-dependent manner, leading to a loss in
its ability to bind DNA.4,9 This suggests a completely
new and different mechanism of inhibition of HIV-1 RT
with regard to the other known NNRTIs.
the “unique” character of the TSAO family of com-
pounds. Despite their nucleosidic structure, TSAO
analogues, like the other NNRTIs, are targeted at a non-
substrate-binding site of the human inmunodeficiency
virus type 1 (HIV-1) reverse transcriptase (RT).1,2b,3
TSAO derivatives are, so far, the only HIV-1 RT
inhibitors that seem to interfere at the interface be-
tween the p51 and the p66 subunits of the RT het-
erodimer.4 Well-defined amino acids at both the p51 and
the p66 RT subunits are needed for optimum interaction
of TSAO compounds with the HIV-1 RT.5,6 Our experi-
mental data strongly suggest a specific interaction of
the 3′-spiro moiety of TSAO molecules with the glutamic
acid residue at position 138 (Glu-138) of the p51 subunit
of HIV-1 RT.5a,6b,7,8 Recent biochemical studies have
revealed that both TSAO-T and its 3-N-ethyl derivative
Structure-activity relationship (SAR) studies within
the TSAO family have revealed that the sugar part
plays a crucial role in the interaction of the TSAO
compounds with their target enzyme.1-3 In particular,
the presence of the unique 3′-spiro ring of 4-amino-1,2-
oxathiole-2,2-dioxide in nucleosides having a ribo con-
figuration is crucial for antiviral activity, and replace-
ment of this amino sultone spiro ring by closely related
analogues results in a 100-fold decrease of the anti-
HIV-1 activity.7 Moreover, the presence of bulky tert-
butyldimethylsilyl (TBDMS) groups at both C-2′ and
C-5′ positions is also a prerequisite for antiviral activ-
ity.1,3,10
* To whom correspondence should be addressed. Telephone: +34-
91 5622900. Fax: +34-91 5644853. E-mail: iqmsv29@iqm.csic.es.
† Instituto de Qu´ımica Me´dica (C.S.I.C.).
‡ Universidad de Alcala´.
As part of our program to further explore the impor-
tance of substituent effects on the anti-HIV-1 activity/
toxicity of TSAO derivatives, we focused our attention
§ K. U. Leuven.
10.1021/jm020820h CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/31/2002

Synthesis of TSAO Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3935
Sch em e 1. Synthesis of Compounds 3-7 by Alkylation of TSAO-m3T
on modifications of the 3′-spiro moiety. This represents
the closest part of the molecule to the interface between
the p51 and p66 subunit domains. On the basis of this
hypothesis, we have now introduced different functional
groups at position 3′′ of the spiro ring of TSAO-T (1)
and TSAO-m3T (2) that may give additional interactions
with amino acids adjacent to the Glu-B138 of the p51
subunit. In these novel analogues, the 4′′-amino group
of the sultone spiro moiety was maintained to allow the
crucial interaction with the Glu-B138.
For the synthesis of the target compounds, two
important issues have been considered. First, the chemi-
cal reactivity of the amino sultone heterocyclic system,
first reported by us in 1988,11 has been scarcely studied
and poses a synthetic challenge.12 Second, the presence
of TBDMS at positions 2′ and 5′, sensitive to basic and
acid media, respectively, but essential for the antiviral
efficacy of the TSAO compounds, implies the selection
of smooth reaction conditions compatible with such
groups.
In this manuscript, we describe in detail a mild,
efficient, and general route13 for the synthesis of 3′′-
substituted TSAO analogues carrying differently sub-
stituted alkenyl, alkynyl, allyl, aromatic, and heteroar-
omatic groups. They were prepared from 3′′-iodo
compounds, as key intermediates, via Pd-catalyzed
Stille cross-coupling reactions. To the best of our
knowledge, there is no report in the literature for
palladium cross-coupling reactions in this amino sultone
heterocyclic system. Furthermore, the anti-HIV activity
of these novel compounds is also reported and the
results are discussed in the light of our recently
proposed molecular model for the TSAO-HIV-1 RT
interaction.14
procedures described in the literature for alkylation of
enamines.14 However, when we assayed the reaction of
TSAO-m3T (2) with alkyl halides, we noticed that the
presence of strong bases (KOH or NaH) was required,
in contrast to the alkylation of “classical” enamines
where no base is needed.15 Thus, reaction of 2 with CH3I
and KOH in dry dioxane followed by “in situ” silylation
with an excess of tert-butyldimethylsilyl chloride (TB-
DMSCl) in the presence of DMAP afforded the corre-
sponding 3′′-C-methylated derivative 4, together with
the N,C-dialkylated compound 3 in low yields (Scheme
1). On the other hand, reaction of 2 with allyl bromide
and NaH yielded the 4′′-N- and 3′′-C-allylated deriva-
tives 5 and 6 in a 1:1 ratio; under these reaction
conditions no deprotection of the TBDMS groups was
observed. Similarly, reaction of 2 with ICH2CONH2 in
the presence of NaH afforded, exclusively, the N,C-
dialkylated derivative 7. In all these reactions, mixtures
of N,C-alkylated products were obtained together with
considerable amounts of unreacted starting material.
Longer reaction times or increasing amounts of NaH
(additional 1 or 2 equiv) led to complex reaction mix-
tures from which only 2′- and /or 5′-deprotected com-
pounds were isolated.
In contrast to the lack of selectivity of the alkylation
reaction, halogenation occurs exclusively at the C-3′′
position. Thus, chloro, bromo, and iodo substituents
were easily introduced into the 3′′-position of the spiro
moiety by using the mild ceric ammonium nitrate (CAN)
mediated halogenation method,16 which avoids the use
of acidic and (or) high reaction temperature conditions
that are not compatible with the labile TBDMS groups.
When 1 was treated with lithium halides (LiCl or LiBr)
and CAN in acetonitrile at 80 °C, 3′′-chloro and 3′′-
bromo derivatives 8 and 9 were obtained in 50% and
80% yields, respectively (Scheme 2). Similarly, reaction
of 1 or 2 with I2 and CAN afforded the iodo derivatives
10 and 11 in excellent yields (93% and 90%, respec-
tively). Addition of NEt3 was always required to avoid
deprotection of the TBDMS group at the 5′-position.
Resu lts
Syn th esis. To perform modifications at the C-3′′
position of the 4-amino-1,2-oxathiole-2,2-dioxide spiro
ring of TSAO compounds, we first investigated alkyla-
tion reactions on the 3′-spiro moiety, following the

3936 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Lobato´n et al.
Sch em e 2. Synthesis of 3′′-Halo-TSAO Nucleosides
8-12
8-11 showed an upfield shift of the signal correspond-
ing to C-3′′ (∆δ ) 7-37 ppm) with respect to the same
signal in the starting TSAO derivatives 1 and 2.
At this point, taking into account the easy availability
of 3′′-halo-TSAO derivatives, we decided to use them as
key intermediates for the synthesis of the target 3′′-
substituted TSAO compounds. The palladium-catalyzed
cross-coupling reaction between organic electrophiles
and alkenyl or alkynyl donors, among others, provides
an extremely powerful synthetic tool for generating a
carbon-carbon bond.17 This versatile and mild reaction
is tolerant to a wide variety of functional groups on each
coupling partner, is stereospecific and regioselective,
and gives high yields of the coupling products. Initial
attempts of the cross-coupling of iodo derivative 10 and
activated alkenes (i.e., methyl acrylate) using catalytic
quantities of Pd(OAc)2 and Ph3P in dioxane in the
presence of NEt3 according to the original method of
Heck et al.,18 failed to give the coupled product. In the
reaction only unreacted starting material 10 together
with the corresponding deiodinated derivative 1 were
isolated (Scheme 3). On the other hand, palladium
catalyzed cross-coupling reactions of iodo derivative 10
with terminal alkynes (i.e., 5-chloro-1-pentyne) using
the Sonogashira modification19 in the presence of cata-
lytic quantities of (Ph3P)2PdCl2 and CuI in degassed
NEt3 at 50 °C also failed, and only 5′-deprotected
derivatives 12 and 13 (Scheme 3) were observed.
Similarly, when this reaction was carried out in the
presence of CuI and tetrakis(triphenylphosphine)-
palladium(0) as catalyst in DMF at room temperature
Finally, selective deprotection of iodide 10 with 0.1 N
methanolic HCl yielded the 5′-deprotected iodo deriva-
tive 12 in 81% yield (Scheme 2). The position of
halogenation was established by 1H NMR and 13C NMR
spectroscopy. Thus, the vinylic proton (H-3′′) of 1 and 2
at 5.75 ppm (singlet) disappeared after halogenation.
On the other hand, 13C NMR spectra of halonucleosides
Sch em e 3. Palladium Catalyzed Cross-Coupling Reactions of Iodo Compound 10 with Alkenes, Alkynes, and
Trialkyltin Derivativesa

Synthesis of TSAO Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3937
Ta ble 1. Palladium-Catalyzed Cross-Coupling Reactions of Iodo Nucleosides 10-12 with Different n-Bu3SnR2 14a -h under
Optimized Conditionsa
ratio
yield of
entry
coupling product
R2
coupling/reductionb
coupling product (%)c
1
2
3
4
5
6
7
8
9
15a
15b
15c
15d
15e
17a
17d
17e
17f
-CHdCHCO2CH3 (E)
-CHdCHCO2CH2CH3 (E)
-CHdCH2
3:1
3:1
7:1
9:1
2:1
3:1
10:1
2:1
3:2
72
65
79
82
60
66
82
60
50
-CHdCHCH2OH (E)
-Ph
-CHdCHCO2CH3 (E)
-CHdCHCH2OH (E)
-Ph
10
11
12
17g
6
18c
-CtCCH3
-CH2CHdCH2
-CHdCH2
1:1
1:1
7:1
30
30
70
a
b
Most reactions were >1/2 complete after 1 h but were allowed to run for 12 h for convenience. HPLC ratio. c Isolated yields after
several chromatographic purifications.
with 2 equiv of NEt3,20 only unreacted starting material
10 and the corresponding dehalogenated derivative 1
were observed (Scheme 3).
ing: N-methylpyrrolidinone (NMP) as solvent at 60 °C,
4 equivs of stannane and 2 mol % of Pd2(dba)3, 8 mol %
of AsPh3 and 4 mol % of CuI as catalyst systems.13 It
should be noted that under these optimal reaction
conditions, the rate and overall yield of coupling,
compared to the initially used Pd(PPh3)4, were signifi-
cantly improved (<10% vs 72%) (Scheme 3). Thus, these
results represent yet one more example of the beneficial
effect of these palladium catalytic system [Pd2(dba)3/
AsPh3/Cu] in Stille cross-coupling reactions.23,24
Finally, the coupling process of the iodo 10 with
organotin reagents under classical Stille coupling condi-
tions21 was studied. The readily available methyl-(E)-
3-(tri-n-butylstannyl) acrylate 14a 22 was chosen as a
model reagent to couple with iodide 10 (Scheme 3)
because of the ease of monitoring of the reaction by
HPLC compared with commercially available vinylstan-
nane. Initial attempts of coupling iodide 10 with stan-
nane 14a in DMF and in the presence of Pd(PPh3)4 as
catalyst produced the desired cross-coupling product 15a
in low yield (<10%) together with small amounts of 1,
resulting by the reduction of the iodo precursor 10, and
compound 16 obtained by oxidative homocoupling of
stannane 14a . Homocoupling of stannanes and electro-
phile reductions are often common side reactions,
described in the literature for Stille couplings.21 It
should be also noted that in the reaction conditions
described above, 70% of the starting compound 10 was
recovered unchanged. Thus, our system appears difficult
to couple and required carefully optimized conditions.
After several endeavors for optimizacion the reaction
parameters, described in our preliminary communica-
tion,13 an optimized procedure for the Stille coupling was
elaborated. It was found that the yield and rate of the
reaction were significantly affected by the addition of a
catalyst obtained from the weakly coordinated Pd-
dibenzylidene acetone complex (Pd2(dba)3) and triphen-
ylarsine, as the ligand, and copper iodide as cocatalyst.
It should be pointed out that when an increased number
of equivalents of the stannane were used, the ratio
coupling/reduction products was higher. Thus, the best
conditions for coupling in our system were the follow-
Thus encouraged, we considered it to be of interest
to study the scope and limitations of the cross-coupling
reaction of iodide 10 with a broader range of stannanes,
under the optimized reaction conditions. Moreover, to
study the effect on the activity/toxicity of the resulting
compounds, the method was also further extended to
other electrophiles such as iodo-TSAO-m3T 11 and the
corresponding 5′-deprotected iodo-TSAO-T derivative
12. The results are summarized in Table 1. The reaction
tolerates TBDMS groups on the sugar moiety (entries
1-11), as well as an unprotected 5′-hydroxyl group
(entry 12). In all cases moderate to good yields of the
coupling products and variable amounts of the reduction
products (1, 2, and 13) were obtained. It should be noted
that separation of the desired coupling products from
the reduction side products was rather laborious, and
repeated chromatographic purifications were required
to give the coupling products in the yields shown in
Table 1. The ratio coupling/reduction products depends
on the nature of the stannane. In general, the higher
the transfer rate of the different groups from tin the
higher the ratio coupling/reduction products.20 Substi-
tuted or unsubstituted alkenyl, phenyl, and heteroaryl
groups on tin were all transferred in moderate to good
yields (entries 1-9). Among them, Stille coupling with

3938 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Lobato´n et al.
Ta ble 2. Anti-HIV Activity of 3′′-Substituted TSAO Derivatives in MT-4 and CEM Cell Cultures
a
EC50 (µM)
b
MT-4
CEM
CC50 (µM)
compd
HIV-1
HIV-2
>10
HIV-1
HIV-2
>10
MT-4
CEM
1 (TSAO-T)
2 (TSAO-m3T)
0.06 ( 0.03
0.06 ( 0.09
>250
0.05 ( 0.01
0.04 ( 0.01
g 100
12 ( 2.4
230 ( 7.3
>250
>250
>250
>250
>50
>250
>250
>50
3
4
5
6
7
8
9
10
11
15a
15b
15c
15d
15e
17a
17d
17e
17f
17g
18c
234 ( 23
10-250
126 ( 12
45 ( 53
3.9 ( 0.2
7.0 ( 1.2
17 ( 2.0
4.0 ( 0.04
5.6 ( 2.0
39 ( 5.6
174 ( 95
36 ( 7.6
18 ( 1.9
37 ( 23
37 ( 23
18 ( 2.1
65 ( 36
33 ( 14
5.0 ( 0.6
76 ( 11
0.25 ( 0.09
>50
2.2 ( 1.7
g250
>50
>50
25 ( 20
82 ( 11
3.2 ( 1.1
5.4 ( 1.3
18 ( 0.5
4.3 ( 0.2
3.9 ( 0.13
29 ( 7.8
40 ( 5.7
24 ( 3.0
17 ( 0.5
55 ( 45
55 ( 45
13 ( 19
24 ( 12
3.9 ( 2.5
3.3 ( 2.3
47 ( 18
>50
>50
>50
>50
>2
>2
>2
>2
>10
>10
>10
>10
0.93 ( 0.23
0.12 ( 0.02
0.11 ( 0.06
3.7 ( 0.01
10 ( 8.9
3.2 ( 2.2
6.6 ( 2.9
6.7 ( 1.6
2.9 ( 1.2
5.9 ( 1.8
>10
0.85 ( 0.21
0.06 ( 0.02
0.05 ( 0.03
3.0 ( 1.4
3.5 ( 0.7
4.0 ( 1.4
g2
>10
>2
>2
>10
>2
9.9 ( 6.4
>50
7.3 ( 2.3
>10
3.3 ( 3.5
5.4 ( 0.2
>50
4.0 ( 0.0
1.2 ( 0.0
>2
2.5 ( 0.7
0.98 ( 0.87
2.8 ( 2.7
1.7 ( 1.1
>2
>2
15 ( 5
>10
>10
>2
5.7 ( 0.58
>50
2.3 ( 2.0
>2
23 ( 0.9
>2
>2
>2
>2
>50
>50
a
b
50% effective concentration or compound concentration required to inhibit HIV-induced cytopathicity in cell culture by 50%. 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%.
vinyl stannane 14c (entries 3 and 12) or (E)-3-(tri-n-
butylstannyl)-2-propen-1-ol22 14d (entries 4 and 7) gave
the highest yields (70-82%). The unexpected low yield
observed in the coupling of iodide 11 with the highly
reactive alkynyl stannane 14g (entry 10) could be
explained by the higher reactivity of this stannane,
which leads to increasing amounts of the homocoupling
side product. Under the optimized conditions described
above, successful coupling was observed even in the
more demanding couplings (i.e., allyl derivatives, entry
11). However, the above methodology did not work with
tributylcyanostannane and tetramethylstannane. In the
latter case, it is generally accepted that an sp3 carbon
directly attached to the metal is less reactive than
carbons with lower hybridization in Pd-catalyzed reac-
tions,21 which likely accounts for the lack of reactivity
observed.
compounds TSAO-T and TSAO-m3T. The novel TSAO
derivatives described herein invariably contained a
thymine moiety (as in TSAO-T) or a N3-methyl-
substituted thymine moiety (as in TSAO-m3T). Cellular
toxicity of the 3′′-iodo-substituted TSAO-T and TSAO-
m3T derivatives (CC50 ) 3.9-5.6 µM) was comparable
with that of TSAO-T but substantially more pronounced
than for TSAO-m3T (CC50 g 100 µM). The most active
anti-HIV-1 3′′-halogen derivatives 9, 10, and 11 and the
3′′-methyl derivative 4 proved to be inactive (>2-10 µM
for 9-11 and >500 µM for 4) against TSAO-resistant
HIV-1/138Lys mutant virus strains.25 Substitution of
the 3′′-spiro position by alkenyl derivatives containing
allyl (6) or an acetamide (7) moiety annihilates the
antiviral activity. In contrast, a variety of other alkenyl-
substituted TSAO derivatives showed anti-HIV-1 activ-
ity at EC50 concentrations between 1 and 10 µM, which
are 20- to 200-fold higher than those of the unsubsti-
tuted prototype compounds. Surprisingly, several of
these 3′′-substituted TSAO derivatives also gained anti-
HIV-2 activity at subtoxic concentrations, an observa-
tion that is very unusual for NNRTIs and never
observed before for TSAO derivatives. Usually, the anti-
HIV-1 activity was of the same order of magnitude as
their anti-HIV-2 activity. The structure-activity rela-
tionship for having both anti-HIV-1 and anti-HIV-2
activity is currently unclear. For example, the methyl
ester of the propionic acid derivative (15a ) shows both
anti-HIV-1 and anti-HIV-2 activity, whereas the ethyl
ester (15b) has only anti-HIV-1 activity. Also, the 3′′-
vinyl (15c) and hydroxypropenyl (15d , 17d ) derivatives
show activity against both viruses, but the 3′′-phenyl-
substituted compounds again show selectivity for only
HIV-1. 3′′-Propynyl-TSAO (17g) is inactive at subtoxic
concentrations. To exclude HIV-2 RT as a potential
target for those TSAO derivatives that gained antiviral
activity against HIV-2 in addition to anti-HIV-1 activity,
compounds 15a , 15c, and 15d were evaluated on their
The structures of compounds 15a -e, 17a ,d ,e-g, and
18c were assigned on the basis of their corresponding
analytical and spectroscopic data. The trans stereo-
chemistry around the double bond of the vinyl-substi-
tuted tin reagents 14a ,b and 14d was retained during
the coupling reaction as shown by the values of 3J H,H
)
15.6-15.9 Hz for the olefinic protons of the correspond-
ing coupling products 15a ,b,d and 17a ,d . Stille cou-
plings reaction of organic electrophiles with substituted
vinyl tin reagent usually proceeds with retention of
configuration.21
Biologica l Resu lts. The anti-HIV activity of the 3′′-
substituted TSAO derivatives was evaluated in MT-4
and CEM cell cultures and compared with the prototype
compounds TSAO-T and TSAO-m3T (Table 2). Among
the 3′′-halogen-substituted TSAO derivatives, the chloro
derivative (8) was devoid of antiviral activity whereas
the bromo (9) and particularly the iodo derivatives (10
and 11) were endowed with pronounced anti-HIV-1
activity. The iodo derivatives 10 and 11 were virtually
equally potent anti-HIV-1 agents as the prototype

Synthesis of TSAO Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3939
Ta ble 3. Electrostatic Component of the Interaction Energy
a
(kcal mol-1
)
between TSAO-m3T and Its Halogenated
Analogues with Selected RT Residuesb during the Molecular
Dynamics Simulations Calculated Using a Continuum Method14
Glu-B138 Lys-A101
Lys-A103
Arg-A172
TSAO-m3T (2)
-4.7 ( 0.2 -0.4 ( 0.2
0.1 ( 0.1 -0.05 ( 0.1
Cl-TSAO-m3T (8) -2.9 ( 0.6
1.2 ( 0.2
0.1 ( 0.03
1.6 ( 0.1
0.9 ( 0.04
0.4 ( 0.4
Br-TSAO-m3T (9) -3.8 ( 0.2 -0.7 ( 0.1 -0.9 ( 0.1
I-TSAO-m3T (10) -5.0 ( 0.5 -0.4 ( 0.2
0.6 ( 0.3
a
b
Average ( root-mean-square deviation. Differences in inter-
action of the different TSAO derivatives with other protein
residues fell within the standard errors of our measurements.
F igu r e 2. Effect of replacing hydrogen by halogen or methyl
changes in the orientation of the thymine ring that are
translated into differential interactions with Arg-A172,
which turned out to be particularly unfavorable for 8,
the least potent compound in this series.
on the calculated electrostatic solvation energies of the spiro
solv
ele
systems (∆∆G
in kcal mol-1) vs hydrophobic constants (π)
of the halogen substituents experimentally determined for
halobenzenes.28
The increase in hydrophobicity in going from 3′′-H to
methyl, Cl, Br, and I, as assessed from the incremental
π constants derived from log Po/w values for benzene
derivatives,28 was not directly correlated with the
decrease in activity of the present TSAO derivatives,
although it is in very good agreement with the differ-
ences in solvation free energies calculated for these
compounds (Figure 2). Therefore, this physicochemical
property alone is unlikely to be responsible for the
observed changes in activity. No correlation with activi-
ties was found either when the cost in electrostatic
desolvation energy upon complex formation was calcu-
lated (data not shown).
When the dynamic behavior of the complexes of the
Br, Cl, and I derivatives of TSAO-T, taken as represen-
tative of the series, in the putative binding site of HIV-1
RT was simulated and the interaction energies were
calculated, the largest differences with respect to the
complex with TSAO-m3T were detected in their interac-
tion with Glu-B138 in the p51 subunit and Arg-A172
in the p66 subunit (Table 3). These differences could
account for the observed differences in activity (Table
2). The presence of the halogen atom in the vicinity of
the exocyclic amino group also appeared to have an
influence on the interaction with the lysine residues that
are proposed to face the sulfoxide group providing
additional electrostatic steering for TSAO binding.14 The
net electrostatic interaction with Lys-A101 and Lys-
A103, however, is rather variable along the simulation
time (Table 3) because the side chains of these two more
distant residues are very exposed to the solvent. As a
result, their position relative to the amino sultone spiro
ring is not permanently fixed as is the case with the
buried carboxylate group of Glu-B138, and the overall
interaction fluctuates over time. This electrostatic in-
teraction appears to be comparatively more favorable
for 9 and 10, which could also account for the greater
inhibitory potency of these two compounds relative to
8.
inhibitory activity against recombinant HIV-2 RT and
found to be inactive at 500 µM. In addition, the most
active anti-HIV-2 compounds 15a , 15c, and 15d but also
17a and 17d were also shown to be inactive (>500 µM)
against a mutant HIV-1 RT that contains the TSAO-
characteristic Glu138Lys resistance mutation. Taken
together, our data point to a different mechanism of
action of some of the 3′′-substituted TSAO derivatives.
Recently, Buckheit26 reported on the NNRTI SJ -3366
that inhibits both HIV-1 and HIV-2 replication and that
inhibits HIV-1 but not HIV-2 RT. This compound was
found to interfere with entry of HIV as a second
mechanism of action. Investigations on the mechanism
of action of the TSAO derivatives against HIV-2 is
currently ongoing.
Com p u ta tion a l Resu lts a n d Discu ssion
To rationalize the biological results obtained in cell
culture assays,27 a theoretical investigation was under-
taken with the 3′′-halogenated and methyl-substituted
TSAO compounds. Halogen or methyl substitution at
the 3′′ position of the spiro moiety of 1 or 2 brings about
an increase in the electrostatic desolvation energy that
closely parallels the hydrophobicity increments expected
from the π constant values calculated from differences
in octanol-water partition coefficients for toluene and
halobenzenes with respect to benzene (Figure 2).
In our recently proposed model for 1 and 2 binding
to heterodimeric RT,14 the binding site is found at the
subunit interface in the vicinity of the â7-â8 loop of
p51. The spiro group of TSAO appears to play a
dominant role in aligning the molecule in the electro-
static field created by Lys-A101 and Lys-A103 in the
p66 subunit and Glu-B138 in the p51 subunit. In fact,
the most prominent interaction detected in our analysis
was between TSAO-T and Glu-138B, the residue that
is found mutated to lysine under the selective pressure
of TSAO derivatives.25 When the electrostatic compo-
nent of the interaction energy between the halogenated
analogues and these protein residues in the equilibrated
complexes was calculated, the favorable interaction
energy with Glu-138B was similar for compounds 2 (H)
and 10 (I) but steadily decreased for 9 (Br) and 8 (Cl).
On the other hand, the interaction with Lys-A101 and
Lys-A103 fluctuated more because of variations in the
orientation of the side chain with respect to the amino-
oxathiol-dioxide (Table 3). Adaptation of the binding site
to the presence of the halogen also brings about slight
Con clu sion s
In conclusion, various synthetic studies toward 3′′-
substituted TSAO nucleosides have been explored.
Among them, Stille cross-coupling of 3′′-iodo TSAO
derivatives with different stannanes provides an ef-
ficient and straightforward route for the direct and
selective functionalization of the 3′′-position of the
sultone spiro moiety via carbon-carbon bond formation.
Documented herein is an efficient method for the direct

3940 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Lobato´n et al.
The intermediate moving band gave 0.035 g (35%) of
unreacted starting material (TSAO-m3T).
attachment of (un)substituted vinyl, alkynyl, phenyl,
thienyl, and allyl groups to the sultone core. Surpris-
ingly, several of these 3′′-substituted TSAO derivatives
showed anti-HIV-1 and anti-HIV-2 activity at subtoxic
concentrations, an observation that is very unusual for
NNRTIs and never observed for TSAO derivatives.
Furthermore, among the 3′′-halogen-substituted TSAO
derivatives, particularly the 3′′-iodo derivatives showed
a potent anti-HIV-1 activity, which proved comparable
to that of the prototype compound TSAO-T. However,
there was a clear trend toward decreased antiviral
potency in going from iodine to bromine to chlorine.
Theoretical studies revealed that differences in the
electrostatic component of the interaction energy be-
tween the halogenated analogues and the HIV-1 RT
could account for the observed changes in activity.
The slowest moving band gave 0.018 g (18%) of 4 as a white
foam. 1H NMR [CDCl3, 200 MHz] δ: 0.78, 0.97 (s, 18H, 2t-
Bu), 1.91 (s, 3H, CH3-3′′), 1.98 (d, 3H, J )1.1 Hz, CH3-5), 3.34
(s, 3H, CH3-3), 3.84 (dd, 1H, J 4′,5′a ) 2.1 Hz, J 5′a,5′b ) 12.4 Hz,
H-5′a), 3.97 (dd, 1H, J 4′,5′b ) 2.9 Hz, H-5′b), 4.30 (dd, 1H, H-4′),
4.45 (d, 1H, J 1′,2′ ) 8.2 Hz, H-2′), 5.59 (bs, 2H, NH2), 6.09 (d,
1H, H-1′), 7.22 (d, 1H, H-6). Anal. (C26H47N3O8SSi2) C, H, N,
S.
1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-3-N-(m eth yl)th ym in e]-3′-spir o-5′′-(4′′-allylam in o-1′′,2′′-
oxa th iole-2′′,2′′-d ioxid e) a n d [1-[2′,5′-Bis-O-(ter t-bu tyld i-
m et h ylsilyl)-â-D-r ib ofu r a n osyl]-3-N-(m et h yl)t h ym in e]-
3′-sp ir o-5′′-(4′′-a m in o-3′′-a llyl-1′′,2′′-oxa th iole-2′′,2′′-d iox-
id e) (5 a n d 6). To a solution of TSAO-m3T (0.1 g, 0.17 mmol)
in dry THF (5 mL), NaH 60% (0.25 mmol) was added, and the
mixture was stirred for 15 min at room temperature. Then,
allyl bromide (30 µL, 0.34 mmol) was added and the reaction
mixture was stirred at room temperature for 24 h. The mixture
was neutralized with AcOH, filtered through silica gel, and
evaporated to dryness. The residue was purified by CCTLC
on the Chromatotron (hexane/ethyl acetate, 5:1). From the
fastest running fractions 0.02 g (19%) of 5 were isolated as a
white foam. 1H NMR [(CD3)2CO, 200 MHz] δ: 0.80, 0.96 (s,
18H, 2t-Bu), 2.01 (s, 3H, CH3-5), 3.26 (s, 3H, CH3-3), 3.82-
3.88 (m, 2H, CH2-CHdCH2), 4.00 (dd, 1H, J 4′,5′a ) 4.2 Hz,
J 5′a,5′b ) 12.4 Hz, H-5′a), 4.11 (dd, 1H, J 4′,5′b ) 3.6 Hz, H-5′b),
4.30 (dd, 1H, H-4′), 4.69 (d, 1H, J 1′,2′ ) 8.0 Hz, H-2′), 5.28 (dd,
1H, J ) 1.4 Hz, J cis ) 10.2 Hz, CH2-CHdCH2), 5.40 (dd, 1H,
J trans ) 17.2 Hz, CH2-CHdCH2), 5.79 (s, 1H, H-3′′), 5.91 (ddd,
1H, CH2-CHdCH2), 5.99 (d, 1H, H-1′), 6.60 (m, 1H, NH-4′′),
7.46 (d, 1H, H-6). Anal. (C28H49N3O8SSi2) C, H, N, S.
Exp er im en ta l Section
Ch em ica l P r oced u r es. Microanalyses were obtained with
a Heraeus CHN-O-RAPID instrument. 1H NMR spectra were
recorded with a Varian Gemini, a Varian XL-300, and a Bruker
AM-200 spectrometer operating at 300 and 200 MHz with Me4-
Si as the internal standard. 13C NMR spectra were recorded
with a Varian XL-300 and a Bruker AM-200 spectrometer
operating at 75 and 50 MHz with Me4Si as 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 (Kiesegel
60 PF254 gipshaltig, Merck) (layer thickness 1 mm or 2 mm,
flow rate 5 mL/min). Preparative TLC was performed on 20
cm × 20 cm glass plates coated with a 2 mm layer of silica gel
PF254 (Merck). Analytical HPLC was carried out on a Waters
484 system using a µBondapak C18 (3.9 mm × 300 mm, 10
mm). Eluent: CH3CN/H2O (0.05% TFA). Flow rate: 1 mL/min.
Detection: UV (214 nm).
The intermediate moving band gave 0.03 g (28%) of 6 as a
white foam. 1H NMR [(CD3)2CO, 200 MHz] δ: 0.81, 0.98 (s,
18H, 2t-Bu), 1.95 (d, 3H, J )1.2 Hz, CH3-5), 3.13-3.17 (m,
2H, CH2-CHdCH2), 3.27 (s, 3H, CH3-3), 4.03 (dd, 1H, J 4′,5′a
)
3.6 Hz, J 5′a,5′b ) 12.2 Hz, H-5′a), 4.11 (dd, 1H, J 4′,5′b ) 3.4 Hz,
H-5′b), 4.34 (t, 1H, H-4′), 4.65 (d, 1H, J 1′,2′ ) 8.2 Hz, H-2′),
5.08 (dd, 1H, J ) 1.6 Hz, J cis ) 8.3 Hz, CH2-CHdCH2), 5.25
(dd, 1H, J trans ) 17.0 Hz, CH2-CHdCH2), 5.80-5.91 (m, 1H,
CH2-CHdCH2), 6.11 (bs, 2H, NH2), 6.13 (d, 1H, H-1′), 7.50
(d, 1H, H-6). Anal. (C28H49N3O8SSi2) C, H, N, S.
Triethylamine, dioxane, and acetonitrile were dried by
refluxing over calcium hydride. Tetrahydrofuran was dried by
refluxing over sodium/benzophenone. Anhydrous N-meth-
ylpyrrolidinone (NMP) was purchased from Aldrich.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-3-N-(m eth yl)th ym in e]-3′-sp ir o-5′′-(4′′-m eth yla m in o-
3′′-m eth yl-1′′,2′′-oxa th iole-2′′,2′′-d ioxid e) a n d [1-[2′,5′-Bis-
O -(t er t -b u t y ld im e t h y ls ily l)-â-D -r ib o fu r a n o s y l]-3-N -
(m eth yl)th ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-m eth yl-1′′,2′′-
oxa th iole-2′′,2′′-d ioxid e) (3 a n d 4). To a solution of TSAO-
m3T (2) (0.1 g, 0.17 mmol) in dry dioxane (5 mL), KOH (0.014
g, 0.25 mmol) was added and the mixture was stirred for 15
min at room temperature. Then, methyl iodide (48 µL, 0.34
mmol) was added and the reaction mixture was stirred at room
temperature for 1 h. The mixture was neutralized with AcOH,
filtered through silica gel, and evaporated to dryness. The
residue was dissolved in dry acetonitrile (5 mL), and then
4-(dimethylamino)pyridine (0. 83 g, 0.68 mmol) and TBDMSCl
(0.10 g, 0.68 mmol) were added. The mixture was stirred at
room temperature for 24 h and evaporated to dryness. The
residue was treated with ethyl acetate (20 mL) and water (20
mL). The organic phase was separated, and the aqueous phase
was extracted with ethyl acetate (2 × 20 mL). The combined
organics were succesively washed with cold (4 °C) 1 N HCl
(1 × 20 mL), water (1 × 20 mL), and brine (1 × 20 mL) and
finally were dried (Na2SO4), filtered, and evaporated to dry-
ness. The final residue was purified by CCTLC on the
Chromatotron (hexane/ethyl acetate, 2:1). From the fastest
running fractions, 0.02 g (20%) of 3 was isolated as a white
foam. 1H NMR [CDCl3, 200 MHz] δ: 0.81, 0.98 (s, 18H, 2t-
Bu), 1.98 (d, 3H, J ) 1.1 Hz, CH3-5), 2.23 (s, 3H, CH3-3′′), 3.08
(d, 3H, J ) 5.1 Hz, CH3-4′′), 3.35 (s, 3H, CH3-3), 3.80 (dd, 1H,
The slowest moving band afforded 0.040 g (40%) of unre-
acted starting material (TSAO-m3T).
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-3-N-(m eth yl)th ym in e]-3′-spir o-5′′-(4′′-car bam oylm eth -
yla m in o-3′′-ca r ba m oylm eth yl-1′′,2′′-oxa th iole-2′′,2′′-d iox-
id e) (7). Following the procedure described for the synthesis
of derivatives 5 and 6, TSAO-m3T (0.1 g, 0.17 mmol) was
reacted with iodoacetamide (0.062 g, 0.34 mmol) in THF (5
mL) at room temperature for 2 h. The final residue was
purified by CCTLC on the Chromatotron first with hexane/
ethyl acetate, 5:1, followed by a second purification on the
Chromatotron using dichloromethane/methanol, 20:1, as the
eluent. The fastest moving band gave compound 7 (0.047 g,
40%) as a white foam. 1H NMR [(CD3)2CO, 200 MHz] δ: 0.78,
0.97 (s, 18H, 2t-Bu), 1.95 (d, 3H, J ) 1.2 Hz, CH3-5), 3.22-
3.25 (m, 7H, CH3-3, 2CH2), 4.08 (dd, 1H, J 4′,5′a ) 4.6 Hz, J 5′a,5′b
) 12.0 Hz, H-5′a), 4.11 (dd, 1H, J 4′,5′b ) 4.0 Hz, H-5′b), 4.32
(dd, 1H, H-4′), 4.67 (d, 1H, J 1′,2′ ) 8.2 Hz, H-2′), 6.12 (d, 1H,
H-1′), 6.55 (bs, 4H, 2CONH2), 7.31 (s, 1H, NH-4′′), 7.54 (d, 1H,
H-6). Anal. (C29H51N5O10SSi2) C, H, N, S.
The slowest moving band gave 0.048 g (48%) of unreacted
starting material (TSAO-m3T).
Gen er a l P r oced u r e for th e Syn th esis of 3′′-Ha lo-TSAO
a n d Ha lo-TSAO-m 3T Der iva tives (8-11). To a solution of
TSAO-T (1) (1 mmol) or TSAO-m3T (2) (1 mmol) in dry
acetonitrile (12 mL) under argon, LiCl (3.6 mmol), LiBr (2
mmol), or I2 (0.6 mmol), CAN (0.5 mmol), and NEt3 (0.5 mmol)
were added. The reaction mixture was heated at 80 °C until
there was complete reaction of the starting material (1-48 h).
The final residue obtained after the appropiate workup was
J 4′,5′a ) 2.4 Hz, J 5′a,5′b ) 12.2 Hz, H-5′a), 3.96 (dd, 1H, J 4′,5′b
)
2.8 Hz, H-5′b), 4.26 (t, 1H, H-4′), 4.41 (d, 1H, J 1′,2′ ) 8.0 Hz,
H-2′), 5.97 (m, 1H, NH-4′′), 6.01 (d, 1H, H-1′), 7.26 (d, 1H, H-6).
Anal. (C27H49N3O8SSi2) C, H, N, S.

Synthesis of TSAO Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3941
purified by CCTLC on the Chromatotron. The workup condi-
tions, chromatography eluent, yield of the isolated products,
and analytical and spectroscopic data are indicated below for
each reaction.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-3-N-(m eth yl)th ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-iod o-
1′′,2′′-oxa th iole-2′′,2′′-d ioxid e) (11). According to the general
procedure, TSAO-m3T (0.20 g, 0.34 mmol) was reacted with I2
(0.05 g, 0.20 mmol) for 1 h and then concentrated to dryness.
The residue was treated with cold (4 °C) ethyl acetate (30 mL),
cold (4 °C) brine (15 mL), and cold (4 °C) NaHSO3 5% (10 mL).
The organic phase was separated, and the aqueous phase was
extracted with cold (4 °C) ethyl acetate (3 × 30 mL). The
combined organics were washed with cold (4 °C) brine (30 mL),
dried (Na2SO4), filtered, and evaporated to dryness. The final
residue was purified by CCTLC on the Chromatotron (hexane/
ethyl acetate, 1:1) to give compound 10 (0.22 g, 90%) as a white
amorphous solid. 1H NMR [(CD3)2CO, 300 MHz] δ: 0.79, 0.98
(s, 18H, 2t-Bu), 1.94 (d, 3H, J )1.2 Hz, CH3-5), 3.26 (s, 3H,
CH3-3), 4.06 (dd, 1H, J 4′,5′a ) 3.8 Hz, J 5′a,5′b ) 10.2 Hz, H-5′a),
4.10 (dd, 1H, J 4′,5′b ) 4.0 Hz, H-5′b), 4.37 (t, 1H, H-4′), 4.67 (d,
1H, J 1′,2′ ) 8.0 Hz, H-2′), 6.13 (d, 1H, H-1′), 6.55 (bs, 2H, NH2),
7.48 (d, 1H, H-6). 13C NMR [(CD3)2CO, 75 MHz] δ: 12.41 (CH3-
5), 18.41, 18.95 [(CH3)3-C-Si], 25.76, 26.46 [(CH3)3-C-Si],
28.10 (CH3-3), 47.84 (C-3′′), 62.89 (C-5′), 75.55 (C-2′), 86.71,
84.77 (C-1′, C-4′), 94.39 (C-3′), 112.34 (C-5′), 135.86 (C-6),
151.60 (C-4′′), 152.53 (C-2), 163.63 (C-4). Anal. (C25H44IN3O8-
SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]th ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-ch lor o-1′′,2′′-oxa th i-
ole-2′′,2′′-d ioxid e) (8). Via the general halogenation proce-
dure, TSAO-T (0.20 g, 0.34 mmol) was treated with LiCl (0.03
g, 0.68 mmol) for 48 h and then concentrated to dryness. The
residue was treated with cold (4 °C) ethyl acetate (30 mL) and
cold (4 °C) brine (15 mL). The organic phase was separated,
and the aqueous phase was extracted with cold (4 °C) ethyl
acetate (3 × 30 mL). The combined organics were washed with
cold (4 °C) brine (30 mL), dried (Na2SO4), filtered, and
evaporated to dryness. The final residue was purified by
CCTLC on the Chromatotron (hexane.ethyl acetate, 2:1) to give
1
compound 8 (0.11 g, 50%) as a white foam. H NMR [(CD3)2-
CO, 300 MHz] δ: 0.81, 0.97 (s, 18H, 2t-Bu), 1.90 (d, 3H, J )1.3
Hz, CH3-5), 4.08 (dd, 1H, J 4′,5′a ) 3.3 Hz, J 5′a,5′b ) 9.5 Hz, H-5′a),
4.13 (dd, 1H, J 4′,5′b ) 4.3 Hz, H-5′b), 4.39 (dd, 1H, H-4′), 4.69
(d, 1H, J 1′,2′ ) 8.2 Hz, H-2′), 6.09 (d, 1H, H-1′), 6.67 (bs, 2H,
NH2), 7,45 (d, 1H, H-6), 10.30 (bs, 1H, NH-3). 13C NMR [(CD3)2-
CO, 75 MHz] δ: 12.41 (CH3-5), 18.44, 18.96 [(CH3)3-C-Si],
25.74, 26.43 [(CH3)3-C-Si], 62.83 (C-5′), 75.44 (C-2′), 84.82,
86.63 (C-4′, C-1′), 92.50 (C-3′), 93.92 (C-3′′), 112.44 (C-5′),
135.75 (C-6), 145.57 (C-4′′), 151.62 (C-2), 163.66 (C-4). Anal.
(C24H42ClN3O8SSi2) C, H, N, S.
[1-[2′-O-(ter t-Bu t yld im et h ylsilyl)-â-D-r ib ofu r a n osyl]-
t h ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-iod o-1′′,2′′-oxa t h iole-
2′′,2′′-d ioxid e) (12). Iodo compound 10 (0.10 g, 0.14 mmol)
was stirred with methanolic 0.1 N HCl (8 mL) at room
temperature for 45 min. The solution was neutralized with 1
N NaOH/MeOH, and the solvent was evaporated to dryness.
The residue was purified by CCTLC on the Chromatotron
(hexane/ethyl acetate, 1:2) and afforded compound 12 (0.075
g, 90%) as a white foam. 1H NMR [(CD3)2CO, 300 MHz] δ: 0.79
(s, 9H, t-Bu), 1.89 (d, 3H, J )1.2 Hz, CH3-5), 3.84 (m, 1H,
H-5′a), 4.01 (m, 1H, H-5′b), 4.39 (m, 1H, H-4′), 4.96 (d, 1H,
J 1′,2′ ) 8.2 Hz, H-2′), 5.96 (m, 2H, H-1′, 5′-OH), 6.72 (bs, 2H,
NH2), 7.91 (d, 1H, H-6), 10.20 (bs, 1H, NH-3). 13C NMR [(CD3)2-
CO, 75 MHz] δ: 12.48 (CH3-5), 17.69 [(CH3)3-C-Si], 25.09
[(CH3)3-C-Si], 27.36 (CH3-3), 47.40 (C-3′′), 61.03 (C-5′), 74.87
(C-2′), 84.84 (C-4′), 89.04 (C-3′), 95.69 (C-1′), 110.72 (C-5′),
135.77 (C-6), 151.32 (C-4′′), 151.84 (C-2), 162.75 (C-4). Anal.
(C18H28IN3O8SSi) C, H, N, S.
Gen er a l P r oced u r e for th e Syn th esis of 3′′-Su bstitu ted
TSAO a n d TSAO-m 3T Der iva tives (15a -e, 17a ,d -g, 18c,
6) via Stille Cou p lin g Con d ition s. A solution of the iodo-
nucleoside 10, 11, or 12 (1 mmol) in dry NMP (5 mL) was
treated with AsPh3 (0.08 mmol), Pd2(dba)3 (0.02 mmol), CuI
(0.04 mmol), and after 10 min the corresponding stannanne
(2 mmol). The resulting solution was stirred under argon at
60 °C. After 30 min and 1 h, two additional portions of the
corresponding stannane (2 × 1 mmol) were added and the
reaction was continued for 12 h at 60 °C. The reaction mixture
was allowed to cool to room temperature, and water (15 mL)
and ethyl acetate (25 mL) were added. The organic phase was
separated and washed several times with water (4 × 15 mL)
to remove NMP completely, dried (Na2SO4), filtered, and
evaporated under reduced pressure. The residue was redis-
solved in acetonitrile (20 mL) and washed with hot hexane
(4 × 15 mL) to remove the tin iodide. The final residue was
purified by CCTLC on a Chromatotron or by preparative TLC.
In general, several chromatographic purifications were re-
quired to give pure compounds in the yields shown below. The
chromatography eluents, yield, and analytical and spectro-
scopic data of the isolated products are indicated below for each
reaction.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]th ym in e]-3′-spir o-5′′-(4′′-a m in o-3′′-br om o-1′′,2′′-oxa th i-
ole-2′′,2′′-d ioxid e) (9). TSAO-T (0.20 g, 0.34 mmol) was
reacted with LiBr (0.06 g, 0.68 mmol) for 2 h according to the
general halogenation procedure, and then the mixture was
concentrated to dryness. The residue was treated with cold (4
°C) ethyl acetate (30 mL) and cold (4 °C) brine (15 mL). The
organic phase was separated, and the aqueous phase was
extracted with cold (4 °C) ethyl acetate (3 × 30 mL). The
combined organics were washed with cold (4 °C) brine (30 mL),
dried (Na2SO4), filtered, and evaporated to dryness. Purifica-
tion of the residue by CCTLC on the Chromatotron (hexane/
ethyl acetate, 3:1) afforded compound 9 (0.17 g, 75%) as a white
foam. 1H NMR [(CD3)2CO, 300 MHz] δ: 0.81, 0.97 (s, 18H, 2t-
Bu), 1.90 (d, 3H, J ) 1.1 Hz, CH3-5), 4.07 (dd, 1H, J 4′,5′a ) 3.4
Hz, J 5′a,5′b ) 10.5 Hz, H-5′a), 4.10 (dd, 1H, J 4′,5′b ) 4.1 Hz,
H-5′b), 4.37 (dd, 1H, H-4′), 4.68 (d, 1H, J 1′,2′ ) 8.2 Hz, H-2′),
6.06 (d, 1H, H-1′), 6.61 (bs, 2H, NH2), 7,45 (d, 1H, H-6), 10.30
(bs, 1H, NH-3). 13C NMR [(CD3)2CO, 75 MHz] δ: 12.40 (CH3-
5), 18.42, 18.96 [(CH3)3-C-Si], 25.73, 26.43 [(CH3)3-C-Si],
62.83 (C-5′), 75.47 (C-2′), 79.92 (C-3′′), 86.52, 86.72 (C-4′, C-1′),
93.47 (C-3′), 112.40 (C-5′), 135.79 (C-6), 148.00 (C-4′′), 151.60
(C-2), 163.61 (C-4). Anal. (C24H42BrN3O8SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]th ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-iod o-1′′,2′′-oxa th i-
ole-2′′,2′′-d ioxid e) (10). Following the general procedure,
TSAO-T (0.20 g, 0.34 mmol) was treated with I2 (0.05 g, 0.20
mmol) for 1 h and then concentrated to dryness. The residue
was treated with cold (4 °C) ethyl acetate (30 mL), cold (4 °C)
brine (15 mL), and cold (4 °C) NaHSO3 5% (10 mL). The
organic phase was separated, and the aqueous phase was
extracted with cold (4 °C) ethyl acetate (3 × 30 mL). The
combined organics were washed with cold (4 °C) brine (30 mL),
dried (Na2SO4), filtered, and evaporated to dryness. The
residue was purified by CCTLC on the Chromatotron (hexane/
ethyl acetate, 1:1) to give compound 10 (0.23 g, 93%) as a white
amorphous solid. 1H NMR [(CD3)2CO, 300 MHz] δ: 0.81, 0.96
(s, 18H, 2t-Bu), 1.90 (d, 3H, J )1.0 Hz, CH3-5), 4.06 (dd, 1H,
J 4′,5′a ) 3.6 Hz, J 5′a,5′b ) 12.2 Hz, H-5′a), 4.09 (dd, 1H, J 4′,5′b
)
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]th ym in e]-3′-spir o-5′′-[4′′-am in o-3′′-(E)-(2-m eth oxyacr yl-
oil)-1′′,2′′-oxa th iole-2′′,2′′-d ioxid e] (15a ). According to the
general procedure, iodo nucleoside 10 (0.1 g, 0.14 mmol) was
treated with methyl (E)-3-(tri-n-butylstannyl)acrylate 14a 20
(0.16 g, 0.56 mmol). The final residue was purified first by
CCTLC (dichloromethane/methanol, 50:1) followed by prepara-
tive TLC (dichloromethane/methanol, 20:1) developed three
times. The fastest moving fractions gave 0.016 g (20%) of the
3.7 Hz, H-5′b), 4.37 (t, 1H, H-4′), 4.67 (d, 1H, J 1′,2′ ) 8.2 Hz,
H-2′), 6.06 (d, 1H, H-1′), 6.56 (bs, 2H, NH2), 7.46 (d, 1H, H-6),
10.30 (bs, 1H, NH-3). 13C NMR [(CD3)2CO, 75 MHz] δ: 12.41
(CH3-5), 18.41, 18.95 [(CH3)-C-Si], 25.76, 26.46 [(CH3)3-C-
Si], 47.84 (C-3′′), 62.89 (C-5′), 75.56 (C-2′), 84.77, 86.71 (C-1′,
C-4′), 94.39 (C-3′), 112.34 (C-5′), 135.86 (C-6), 151.60 (C-4′′),
152.53 (C-2), 163.63 (C-4). Anal. (C24H42IN3O8SSi2) C, H, N,
S.

3942 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Lobato´n et al.
reduction product TSAO-T (1). The slowest moving band gave
compound 15a (0.068 g, 72%) as a white foam. 1H NMR [(CD3)2-
CO, 300 MHz] δ: 0.80, 0.95 (s, 18H, 2t-Bu), 1.90 (d, 3H, J )
1.2 Hz, CH3-5), 3.70 (s, 3H, OCH3), 4.01 (dd, 1H, J 4′,5′a ) 4.2
Hz, J 5′a,5′b ) 8.6 Hz, H-5′a), 4.13 (dd, 1H, J 4′,5′b ) 4.1 Hz, H-5′b),
4.39 (t, 1H, H-4′), 4.78 (d, 1H, J 1′,2′ ) 8.1 Hz, H-2′), 6.03 (d,
1H, H-1′), 6.08 (d, 1H, J trans ) 15.6 Hz, CHdCH-CO2Me),
7.49-7.57 (m, 4H, CHdCH-CO2Me, NH2, H-6), 10.40 (bs, 1H,
NH-3). 13C NMR [(CD3)2CO, 75 MHz] δ: 12.93 (CH3-5), 18.35,
18.92 [(CH3) 3-C-Si], 25.68, 26.23 [(CH3)3-C-Si], 54.3 (OCH3),
62.92 (C-5′), 74.80 (C-2′), 83.23, 88.90 (C-1′, C-4′), 92.42 (C-
3′), 110.31 (C-3′′), 111.28 (C-5), 129.22 (CHdCH-CO2Me),
132.12 (CHdCH-CO2Me), 134.2 (C-6), 151.11 (C-4′′), 152.11
(C-2), 160.11(CO2Me), 163.33 (C-4). Anal. (C28H47N3O10SSi2)
C, H, N, S.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]th ym in e]-3′-sp ir o-5′′-[4′′-a m in o-3′′-(E)-(2-eth oxya cr yl-
oil)-1′′,2′′-oxa th iole-2′′,2′′-d ioxid e] (15b). The general pro-
cedure was followed with iodo derivative 10 (0.1 g, 0.14 mmol)
and ethyl (E)-3-(tri-n-butylstannyl)acrylate 14b21 (0.21 g, 0.56
mmol). After the workup, the final residue was purified twice
by CCTLC on the Chromatotron (dichloromethane/methanol,
20:1). From the fastest moving band, TSAO-T (1) was isolated
(0.019 g, 23%). From the slowest moving band, compound 15b
(0.063 g, 65%) was isolated as a white foam. 1H NMR [(CD3)2-
CO, 300 MHz] δ: 0.80, 0.95 (s, 18H, 2t-Bu), 1.24 (t, 3H, J )
7.1 Hz, OCH2CH3), 1.90 (d, 3H, J ) 1.1 Hz, CH3-5), 3.06 (s,
3H, OCH3), 4.09-4.22 (m, 4H, 2H-5′, OCH2CH3), 4.39 (t, 1H,
J 4′,5′ ) 4.0 Hz, H-4′), 4.76 (d, 1H, J 1′,2′ ) 8.1 Hz, H-2′), 6.04 (d,
1H, H-1′), 6.06 (d, 1H, J trans ) 15.9 Hz, CHdCH-CO2Et), 7.46
(bs, 2H, NH2), 7.50 (m, 2H, H-6, CHdCH-CO2Et), 10.47 (bs,
1H, NH-3). Anal. (C29H49N3O10SSi2) C, H, N, S.
C-Si], 63.22 (C-5′), 75.40 (C-2′), 85.23, 88.20 (C-1′, C-4′), 92.42
(C-3′), 111.31 (C-3′′), 111.48 (C-5′), 129.32 (CHdCH-CH2OH),
134.42 (CHdCH-CH2OH, C-6), 152.11 (C-4′′), 152.31 (C-2),
163.03 (C-4). Anal. (C27H47N3O9SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]t h ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-p h en yl-1′′,2′′-oxa -
th iole-2′′,2′′-d ioxid e) (15e). Via the general procedure, iodo
nucleoside 10 (0.1 g, 0.14 mmol) was treated with tri-n-
butylphenylstannane 14e (0.18 g, 0.56 mmol). The final
residue was purified by CCTLC on the Chromatotron (hexane/
ethyl acetate, 2:1) twice using the same eluent. The fastest
moving fractions afforded 0.057 g (60%) of 15e as a white foam.
1H NMR [(CD3)2CO, 300 MHz] δ: 0.83, 0.96 (s, 18H, 2t-Bu),
1.91 (d, 3H, J )1.3 Hz, CH3-5), 4.13 (dd, 1H, J 4′,5′a ) 3.5 Hz,
J 5′a,5′b ) 12.3 Hz, H-5′a), 4.15 (dd, 1H, J 4′,5′b ) 3.7 Hz, H-5′b),
4.45 (dd, 1H, H-4′), 4.78 (d, 1H, J 1′,2′ ) 8.2 Hz, H-2′), 6.11 (d,
1H, H-1′), 6.45 (bs, 2H, NH2), 7.37 (m, 1H, Ph), 7.45 (m, 2H,
Ph), 7.52 (d, 1H, H-6), 7.57 (m, 1H, Ph), 7.59 (m, 1H, Ph), 10.32
(bs, 1H, NH). 13C NMR [(CD3)2CO, 75 MHz] δ: 12.43 (CH3-5),
18.43, 18.93 [(CH3)3-C-Si], 25.72, 26.41 [(CH3)3-C-Si], 63.11
(C-5′), 75.35 (C-2′), 85.20, (C-4′), 86.80 (C-1′), 91.35 (C-3′),
105.40 (C-3′′), 112.26 (C-5), 128.28, 128.94, 129.13, 129.97 (Ph),
135.77 (C-6), 145.48 (C-4′′) 151.62 (C-2), 163.64 (C-4). Anal.
(C30H47N3O8SSi2) C, H, N, S.
The slowest moving fractions gave 0.041 g (20%) of TSAO-T
(1).
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-3-N-(m eth yl)th ym in e]-3′-sp ir o-5′′-[4′′-a m in o-3′′-(E)-
(2-m eth oxya cr yloil)-1′′,2′′-oxa th iole-2′′,2′′-d ioxid e] (17a ).
Following the general procedure, iodo nucleoside 11 (0.1 g, 0.14
mmol) was reacted with (E)-methyl 3-(tri-n-butylstannyl)-
acrylate 14a 21 (0.16 g, 0.55 mmol). The residue was purified
first by CCTLC (dichloromethane/methanol, 25:1) followed by
preparative TLC (dichloromethane/methanol, 25:1), developed
three times. From the fastest moving band, TSAO-m3T (2)
(0.015 g, 16%) was isolated. From the slowest moving band,
compound 17a (0.063 g, 66%) was isolated as a white foam.
1H NMR [(CD3)2CO, 300 MHz] δ: 0.82, 0.91 (s, 18H, 2t-Bu),
1.90 (s, 3H, CH3-5), 3.27 (s, 3H, CH3-3), 3.71 (s, 3H, OCH3),
4.01 (dd, 1H, J 4′,5′a ) 3.8 Hz, J 5′a,5′b ) 12.0 Hz, H-5′a), 4.13
(dd, 1H, J 4′,5′b ) 4.0 Hz, H-5′b), 4.42 (t, 1H, H-4′), 4.76 (d, 1H,
J 1′,2′ ) 8.2 Hz, H-2′), 6.08 (d, 1H, J trans ) 15.8 Hz, CHdCH-
CO2Me), 6.13 (d, 1H, H-1′), 6.42 (bs, 2H, NH2), 7.51 (s, 1H,
H-6), 7.54 (d, 1H, CHdCH-CO2Me). 13C NMR [(CD3)2CO, 75
MHz] δ: 12.93 (CH3-5), 18.38 (CH3), 18.95, 18.92 [(CH3)3-C-
Si], 25.68, 26.23 [(CH3)3-C-Si], 54.0 (OCH3), 62.72 (C-5′),
74.80 (C-2′), 83.23, 88.90 (C-1′, C-4′), 92.44 (C-3′), 110.33 (C-
3′′), 111.18 (C-5′), 129.23 (CHdCH-CO2Me), 132.12 (CHd
CH-CO2Me), 134.2 (C-6), 151.11, 152.11 (C-4′′, C-2), 160.13
(CO2Me), 163.35 (C-4). Anal. (C29H49N3O10SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]th ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-vin yl-1′′,2′′-oxa th i-
ole-2′′,2′′-d ioxid e) (15c). The general procedure was followed
with iodo derivative 10 (0.1 g, 0.14 mmol) and tri-n-butylvi-
nylstannane 14c (0.18 g, 0.56 mmol). The residue obtained
after the workup was purified twice by CCTLC on the
Chromatotron (hexane/ethyl acetate, 2:1). From the fastest
moving fractions, compound 15c (0.068 g, 79%) was identified
1
as a white foam. H NMR [(CD3)2CO, 300 MHz] δ: 0.79, 0.80
(s, 18H, 2t-Bu), 1.97 (d, 3H, J ) 1.2 Hz, CH3-5), 4.05 (dd, 1H,
J 4′,5′a ) 3.5 Hz, J 5′a,5′b ) 12.5 Hz, H-5′a), 4.10 (dd, 1H, J 4′,5′b
)
3.8 Hz, H-5′b), 4.32 (dd, 1H, H-4′), 4.67 (d, 1H, J 1′,2′ ) 8.2 Hz,
H-2′), 5.17 (dd, 1H, J cis ) 11.4 Hz, J ) 0.6 Hz, CHdCH2), 5.45
(dd, 1H, J trans ) 17.5 Hz, CHdCH2), 6.05 (d, 1H, H-1′), 6.49
(bs, 2H, NH2), 6.53 (dd, 1H, CHdCH2), 7,49 (d, 1H, H-6), 10.32
(bs, 1H, NH-3). 13C NMR [(CD3)2CO, 75 MHz] δ: 12.53 (CH3-
5), 18.38, 18.93 [(CH3)3-C-Si], 25.68, 26.37 [(CH3)3-C-Si],
63.02 (C-5′), 75.94 (C-2′), 84.92, 85.33 (C-1′, C-4′), 91.24 (C-
3′), 111.01 (C-3′′), 112.32 (C-5), 121.94 (CHdCH2), 133.70,
136.42 (CHdCH2, C-6), 145.51 (C-4′′), 151.61 (C-2), 163.73 (C-
4). Anal. (C26H45N3O8SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-d -r ibofu r a n o-
syl]-3-N-(m eth yl)th ym in e]-3′-sp ir o-5′′-[4′′-a m in o-3′′-(E)-(1-
h yd r oxy-2-p r op en ylen o)-1′′,2′′-oxa t h iole-2′′,2′′-d ioxid e]
(17d ). Iodo nucleoside 11 (0.1 g, 0.14 mmol) was treated with
(E)-3-(tri-n-butylstannyl)-2-propen-1-ol 14d 21 (0.19 g, 0.56
mmol) according to the general procedure. The final residue
was purified twice by CCTLC on the Chromatotron (dichlo-
romethane/methanol, 20:1). The fastest moving fractions gave
0.009 g (10%) of TSAO-m3T (2). The slowest moving band
afforded compound 17d (0.075 g, 82%) as a white foam. 1H
NMR [(CD3)2CO, 300 MHz] δ: 0.79, 0.27 (s, 18H, 2t-Bu), 1.95
(d, 3H, J ) 1.4 Hz, CH3-5), 3.27 (s, 3H, CH3-3), 3.99 (m, 1H,
OH), 4.07 (dd, 1H, J 4′,5′a ) 3.3 Hz, J 5′a,5′b ) 8.0 Hz, H-5′a), 4.13
(dd, 1H, J 4′,5′b ) 3.3 Hz, H-5′b), 4.20 (m, 2H, CH2), 4.36 (t, 1H,
From the slowest moving band, TSAO-T (1) (0.008 g, 10%)
was isolated.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]t h ym in e]-3′-sp ir o-5′′-[4′′-a m in o-3′′-(E)-(1-h yd r oxy-2-
p r op en ylen o)-1′′,2′′-oxa th iole-2′′,2′′-d ioxid e] (15d ). Iodo
compound 10 (0.10 g, 0.14 mmol) was reacted with (E)-3-(tri-
n-butylstannyl)-2-propen-1-ol 14d 21 (0.19 g, 0.56 mmol) ac-
cording to the general procedure. Two consecutive purifications
of the residue by CCTLC on the Chromatotron (dichloro-
methane/methanol, 25:1) gave TSAO-T (1) (0.008 g, 10%) from
the fastest moving fractions. The slowest moving fractions
1
afforded 0.074 g (82%) of compound 15d as a white foam. H
H-4′), 4.65 (d, 1H, J 1′,2′ ) 8.2 Hz, H-2′), 6.13 (dt, 1H, J trans )
NMR [(CD3)2CO, 300 MHz] δ: 0.81, 0.97 (s, 18H, 2t-Bu), 1.90
(d, 3H, J ) 1.2 Hz, CH3-5), 4.01 (dd, 1H, J 4′,5′a ) 3.5 Hz, J 5′a,5′b
) 8.9 Hz, H-5′a), 4.08 (dd, 2H, J ) 1.6 Hz, J CH2,OH ) 4.7 Hz,
CH2), 4.10 (dd, 1H, J 4′,5′b ) 3.4 Hz, H-5′b), 4.32 (t, 1H, H-4′),
4.70 (d, 1H, J 1′,2′ ) 8.2 Hz, H-2′), 6.06 (d, 1H, H-1′), 6.12 (dt,
1H, J trans ) 16.0, J CH,CH2 ) 4.6 Hz, CHdCH-CH2OH), 6.35
(dt, 1H, CHdCH-CH2OH), 6.48 (bs, 2H, NH2), 7,49 (d, 1H,
H-6), 10.31 (bs, 1H, NH-3). 13C NMR [(CD3)2CO, 75 MHz] δ:
13.03 (CH3-5), 18.38, 18.95 [(CH3)3-C-Si], 25.68, 26.33 [(CH3)3-
16.0 Hz, J CH,CH2 ) 4.6 Hz, CHdCH-CH2OH), 6.16 (d, 1H,
H-1′), 6.35 (dt, 1H, J ) 1.8 Hz, CHdCH-CH2OH), 6.54 (bs,
2H, NH2), 7.52 (d, 1H, H-6). 13C NMR [(CD3)2CO, 75 MHz] δ:
13.09 (CH3-5), 18.41, 18.98 [(CH3)3-C-Si], 26.34, 26.38 [(CH3)3-
C-Si], 28.06 (CH3-3), 61.7 (CH2), 63.13 (C-5′), 75.40 (C-2′),
85.17, 88.23 (C-1′, C-4′), 92.42 (C-3′), 111.31 (C-3′′), 112.48 (C-
5), 114.77 (CHdCH-CH2OH), 129.27 (CHdCH-CH2OH),
134.45 (C-6), 152.14, 152.22 (C-2, C-4′′), 163.34 (C-4). Anal.
(C28H49N3O9SSi2) C, H, N, S.

Synthesis of TSAO Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3943
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-3-N-(m eth yl)th ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-p h en -
yl-1′′,2′′-oxa th iole-2′′,2′′-d ioxid e) (17e). According to the
general procedure, iodo nucleoside 11 (0.1 g, 0.14 mmol) was
reacted with tri-n-butylphenylstannane 14e (0.18 g, 0.56
mmol). Purification of the final residue by CCTLC on the
Chromatotron (hexane/ethyl acetate, 2:1) gave compound 17e
(0.057 g, 60%) as a white foam from the fastest moving
fractions. 1H NMR [(CD3)2CO, 300 MHz] δ: 0.81, 0.89 (s, 18H,
2t-Bu), 1.95 (d, 3H, J )1.2 Hz, CH3-5), 3.29 (s, 3H, CH3-3),
4.01 (dd, 1H, J 4′,5′a ) 4.3 Hz, J 5′a,5′b ) 12.3 Hz, H-5′a), 4.15
(dd, 1H, J 4′,5′b ) 4.8 Hz, H-5′b), 4.45 (t, 1H, H-4′), 4.78 (d, 1H,
J 1′,2′ ) 6.5 Hz, H-2′), 6.20 (d, 1H, H-1′), 6.54 (bs, 2H, NH2),
7.33-7.60 (m, 6H, Ph, H-6). 13C NMR [(CD3)2CO, 75 MHz] δ:
13.15 (CH3-5), 18.39, 18.93 [(CH3)3-C-Si], 25.65, 26.41 [(CH3)3-
C-Si], 29.03 (CH3-3), 63.13 (C-5′), 75.61 (C-2′), 85.29, 87.40
(C-1′, C-4′), 91.45 (C-3′), 108.2 (C-3′′), 111.33 (C-5), 128.93,
129.12, 129.96 (Ph), 134.01 (C-6), 145.44 (C-4′′), 152.12 (C-2),
163.30 (C-4). Anal. (C31H49N3O8SSi2) C, H, N, S.
preparative TLC (hexane/ethyl acetate, 4:1). From the fastest
moving band, compound 6 (0.026, 30%) was isolated as a white
amorphous solid. From the slowest moving fractions, 0.016 g
(20%) of TSAO-m3T (2) was isolated.
[1-[2′-O-(ter t-Bu t yld im et h ylsilyl)-â-D-r ib ofu r a n osyl]-
th ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-vin yl-1′′,2′′-oxa th iole-
2′′,2′′-d ioxid e) (18c). According to the general procedure, iodo
nucleoside 12 (0.1 g, 0.17 mmol) was reacted with tri-n-
butylvinylstannane 14c (0.21 g, 0.67 mmol). Purification of the
final residue by CCTLC on the Chromatotron (hexane/ethyl
acetate, 2:1) twice gave compound 18c (0.06 g, 70%) as a white
1
foam from the fastest moving fractions. H NMR [(CD3)2CO,
300 MHz] δ: 0.86 (s, 9H, t-Bu), 1.85 (d, 3H, J ) 1.2 Hz, CH3-
5), 3.87 (dd, 1H, J 4′,5′a ) 2.5 Hz, H-5′a), 4.01 (dd, 1H, J 4′,5′b
)
2.4 Hz, H-5′b), 4.37 (t, 1H, H-4′), 5.00 (d, 1H, J 1′,2′ ) 8.1 Hz,
H-2′), 5.19 (dd, 1H, J cis ) 11.4 Hz, J ) 0.8 Hz, CHdCH2), 5.49
(dd, 1H, J trans ) 17.6 Hz, CHdCH2), 5.87 (d, 1H, H-1′), 5.91
(m, 1H, OH), 6.56 (dd, 1H, CHdCH2), 6.79 (bs, 2H, NH2), 7,89
(d, 1H, H-6), 10.35 (bs, 1H, NH-3). Anal. (C20H31N3O8SSi) C,
H, N, S.
The slowest moving fractions afforded 0.027 g (15%) of
TSAO-m3T (2).
From the slowest moving fractions, 0.014 g (15%) of [1-[2′-
O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]thymine]-3′-spiro-
5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide)1b (13) was isolated.
Biologica l Meth od s. a . Cells a n d Vir u ses. 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).
b. Activity Assa y for Test Com p ou n d s a ga in st HIV-1
a n d HIV-2 in Cell Cu ltu r es. An amount of 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
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-3-N-(m et h yl)t h ym in e]-3′-sp ir o-5′′-[4′′-a m in o-3′′-(2-
th ien yl)-1′′,2′′-oxa th iole-2′′,2′′-d ioxid e] (17f). Iodo nucleo-
side 11 (0.1 g, 0.14 mmol) was treated with tri-n-butyl-2-
thienylstannane 14f (0.21 g, 0.56 mmol) according to the
general procedure. After the workup, the residue was purified
by CCTLC on the Chromatotron (hexane/ethyl acetate, 2:1).
From the fastest moving band, compound 17f (0.048 g, 50%)
was isolated as a white amorphous solid. 1H NMR [(CD3)2CO,
300 MHz] δ: 0.80, 0.96 (s, 18H, 2t-Bu), 1.94 (d, 3H, J )1.2
Hz, CH3-5), 3.26 (s, 3H, CH3-3), 4.03 (dd, 1H, J 4′,5′a ) 3.5 Hz,
J 5′a,5′b ) 12.3 Hz, H-5′a), 4.15 (dd, 1H, J 4′,5′b ) 3.7 Hz, H-5′b),
4.44 (t, 1H, H-4′), 4.73 (d, 1H, J 1′,2′ ) 8.2 Hz, H-2′), 6.19 (d,
1H, H-1′), 6.54 (bs, 2H, NH2), 7.16 (dd, 1H, J 3,4 ) 3.6 Hz,
J 4,5 ) 5.1 Hz, H-4thiopheno), 7.25 (dd, 1H, J 3,5) 2.3 Hz, J 3,4 ) 3.6
Hz, H-3thiopheno), 7.52 (s, 1H, H-6), 7.61 (dd, 1H, H-5thiopheno).
13C NMR [(CD3)2CO, 75 MHz] δ: 13.01 (CH3-5), 18.47, 19.02
[(CH3)3-C-Si], 26.51, 28.11 [(CH3)3-C-Si], 28.63 (CH3-3),
63.15 (C-5′), 75.80 (C-2′), 85.36 (C-4′), 87.02 (C-1′), 92.00 (C-
3′), 101.51 (C-3′′), 111.45 (C-5), 127.67, 128.08, 128.28, 128.49
(thiopheno), 134.92 (C-6), 146.34 (C-4′′), 152.24 (C-2), 163.40
(C-4). Anal. (C29H47N3O8S2Si2) C, H, N, S.
(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%.
From the slowest moving fractions, 0.047 g (26%) of TSAO-
m3T (2) was identified.
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-3-N-(m et h yl)t h ym in e]-3′-sp ir o-5′′-[4′′-a m in o-3′′-(1-
p r op yn yl)-1′′,2′′-oxa th iole-2′′,2′′-d ioxid e] (17g). Following
the general procedure, iodo derivative 11 (0.1 g, 0.14 mmol)
was reacted with tri-n-butyl-1-propynylstannane 14g (0.18 g,
0.56 mmol). The final residue was purified first by CCTLC
(hexane/ethyl acetate, 3:1) followed by preparative TLC (hex-
ane/ethyl acetate, 4:1). The fastest moving fractions afforded
0.025 g (30%) of TSAO-m3T (2). The slowest moving fractions
gave 0.027 g (30%) of compound 17g as a white foam. 1H NMR
[(CD3)2CO, 300 MHz] δ: 0.84, 0.95 (s, 18H, 2t-Bu), 1.92 (d,
3H, J )1.2 Hz, CH3-5), 2.11 (s, 3H, CH3-CtC), 3.28 (s, 3H,
CH3-3), 3.92 (dd, 1H, J 4′,5′a ) 4.2 Hz, J 5′a,5′b ) 12.4 Hz, H-5′a),
4.12 (dd, 1H, J 4′,5′b ) 4.3 Hz, H-5′b), 4.33 (t, 1H, H-4′), 4.67 (d,
1H, J 1′,2′ ) 8.3 Hz, H-2′), 6.15 (d, 1H, H-1′), 6.81 (bs, 2H, NH2),
7.49 (d, 1H, H-6). 13C NMR [(CD3)2CO, 75 MHz] δ: 5.07 (CH3-
CtC), 12.98 (CH3-5), 17.79, 18.39 [(CH3)3-C-Si], 25.14, 26.02
[(CH3)3-C-Si], 28.15 (CH3-3), 62.04 (C-5′), 75.25 (C-2′), 77.22
(CH3-CtC), 83.42 (C-4′), 87.55 (C-1′), 91.27 (C-3′), 93.71
(CH3-CtC), 98.83 (C-3′′), 110.89 (C-5′), 133.36 (C-6), 150.05
(C-2), 151.07 (C-4′′), 163.15 (C-4). Anal. (C28H47N3O8SSi2) C,
H, N, S.
c. Rever se Tr a n scr ip ta se Assa y. For determination of the
50% inhibitory concentration (IC50) of the test compounds
against HIV-2 RT, the RNA-dependent DNA polymerase assay
was performed as follows. The reaction mixture (50 µL)
contained 50 mM Tris-HCl (pH 7.8), 5 mM DTT, 300 µM
glutathione, 500 µM EDTA, 150 mM KCl, 5 mM MgCl2, 1.25
µg of bovine serum albumin, a fixed concentration of the
labeled substrate [3H]dGTP (5.6 µM, 1 µCi; specific radioactiv-
ity, 3.6 Ci/mmol; Amersham Pharmacia Biotech), a fixed
concentration of the template/primer poly(rC)‚oligo(dG)12-18
(0.1 mM; Amersham Pharmacia Biotech), 0.06% Triton X-100,
5 µL of inhibitor solution [containing various concentrations
(10-fold dilutions) of the compounds], and 5 µL of the RT
preparations. The reaction mixtures were incubated at 37 °C
for 30 min, at which time 200 µL of yeast RNA (2 mg/mL) and
1 mL of trichloroacetic acid (5% v/v) in saturated phosphate
buffer were added. The solutions were kept on ice for at least
15 min, after which the acid-insoluble material was filtered
over Whatman GF/C glass-fiber filters and washed with 5%
trichloroacetic acid in H2O and ethanol. The filters were then
analyzed for radioactivity in a liquid scintillation counter
(Canberra Packard, Zellik, Belgium).
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-3-N-(m eth yl)th ym in e]-3′-sp ir o-5′′-(4′′-a m in o-3′′-a llyl-
1′′,2′′-oxa th iole-2′′,2′′-d ioxid e) (6). Iodo nucleoside 11 (0.1
g, 0.14 mmol) was reacted with tri-n-butylallylstannane 14h
(0.18 g, 0.56 mmol) according to the general procedure. After
the workup, the residue was purified by CCTLC on the
Chromatotron twice (hexane/ethyl acetate, 5:1) followed by
The IC50 for each test compound was determined as the
compound concentration that inhibited HIV RT activity by
50%.
Com p u ta tion a l Meth od s. The spiro systems of 1 and 2,
their 3′′-methylated derivative 4, and the corresponding
halogenated analogues 8 (Cl-), 9 (Br-), and 10 (I-) were

3944 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Lobato´n et al.
model-built in Insight-II.29 Their geometries were fully opti-
mized with the ab initio quantum mechanical program Gauss-
ian 9430 using the 6-31+G(d) basis set except for the iodo
derivative for which a polarized electric property 3-21+G basis
set31 was employed. Atomic point charges were derived by
fitting the molecular electrostatic potential calculated on each
optimized geometry to a monopole-monopole expression.32 The
electrostatic contribution to the solvation free energy of each
spiro system was obtained by solving the linearized Poisson
equation by means of a finite difference method,33 as imple-
mented in the DelPhi module of Insight II. The total electro-
static free energy in vacuo (exterior dielectric ) 1) was
subtracted from the total electrostatic free energy in water
(exterior dielectric ) 80) using the same interior dielectric for
the solute (ꢀ ) 4) and the same grid definitions in both cases.34
A solvent-accessible surface,35 calculated using a probe radius
of 1.4 Å, defined the boundary between each solute molecule
and the exterior. Cubic grids with a resolution of 1.0 Å were
centered on the molecular systems considered, leaving a
separation of 15 Å between any solute atom and the borders
of the box. The potentials at the grid points delimiting the box
were calculated analytically by treating each charge atom as
selective inhibitors of human immunodeficiency virus type 1.
Antimicrob. Agents Chemother. 1992, 36, 1073-1080. (b) Balzari-
ni, J .; Pe´rez-Pe´rez, M. J .; San-Fe´lix, A.; Camarasa, M. J .;
Bathurst, I. C.; Barr, P. J .; De Clercq, E. Kinetics of inhibition
of human immunodeficiency virus type 1 (HIV-1) reverse tran-
scriptase by the novel HIV-1-specific nucleoside analogue [2′,5′-
Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3′-spiro-5′′-(4′′-
amino-1′′,2′′-oxathiole-2′′,2′′-dioxide) thymine (TSAO-T). J . Biol.
Chem. 1992, 267, 11831-11838.
(3) Camarasa, M. J .; San-Fe´lix, A.; Pe´rez-Pe´rez, M. J .; Vela´zquez,
S.; Alvarez, R.; Chamorro, C.; J imeno, M. L.; Pe´rez, C.; Gago,
F.; De Clercq, E.; Balzarini, J . HIV-1 specific reverse tran-
scriptase inhibitors: why are TSAO-nucleosides so unique? J .
Carbohydr. Chem. 2000, 19, 451-469.
(4) Harris, D.; Lee, R.; Misra, H. S.; Pandey, P. K.; Pandey, V. N.
The p51 subunit of human immunodeficiency virus type 1
reverse transcriptase is essential in loading the p66 subunit on
the template primer. Biochemistry 1998, 37, 5903-5908.
(5) (a) Balzarini, J .; Kleim, J . P.; Riess, G.; Camarasa, M. J .; De
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) Boyer, P. L.; Ding, J .;
Arnold, E.; Hughes, S. H. Subunit specificity of mutations that
confer resistance to nonnucleoside inhibitors in human immu-
nodeficiency virus type 1 reverse transcriptase. Antimicrob.
Agents Chemother. 1994, 38, 1909-1914.
(6) (a) Camarasa, M. J .; Pe´rez-Pe´rez, M. J .; Vela´zquez, S.; San-Fe´lix,
A.; Alvarez, R.; Ingate, S.; J imeno, M. L.; Karlsson, A.; De Clercq,
E.; Balzarini, J . TSAO derivatives: Highly specific inhibitors of
human immunodeficiency virus type-1 (HIV-1) replication.
Nucleosides Nucleotides 1995, 14, 585-594. (b) J onckheere, H.;
Taymans, J . M.; Balzarini, J .; Vela´zquez, S.; Camarasa, M. J .;
Desmyter, J .; De Clercq, E.; Anne´, J . Resistance of HIV-1 reverse
transcriptase against [2′,5′-Bis-O-(tert-butyldimethylsilyl)-3′-
spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide)] (TSAO) deriva-
tives is determined by the mutation Glu138 f Lys on the p51
subunit. J . Biol. Chem. 1994, 269, 25255-25258.
(7) (a) Alvarez, R.; J imeno, M. L.; Pe´rez-Pe´rez, M. J .; De Clercq, E.;
Balzarini, J .; Camarasa, M. J . Synthesis and anti-human
immunodeficiency virus type 1 activity of novel 3′-spiro nucleo-
side analogues of TSAO-T. Antiviral Chem. Chemother. 1997,
8, 507-517. (b) Alvarez, R.; J imeno, M. L.; Gago, F.; Balzarini,
J .; Pe´rez-Pe´rez, M. J .; Camarasa, M. J . Novel 3′-spiro nucleoside
analogues of TSAO-T. Part II. A comparative study based on
NMR conformational analysis in solution and theoretical calcu-
lations. Antiviral Chem. Chemother. 1998, 9, 333-340.
(8) Balzarini, J .; Karlsson, A.; Vandamme, A. M.; Pe´rez-Pe´rez, M.
J .; Zhang, H.; Vrang, L.; Oberg, B.; Backbro, K.; Unge, T.; San-
Fe´lix, A.; Vela´zquez, S.; Camarasa, M. J .; De Clercq, E. Human
immunodeficiency virus type 1 (HIV-1) strains selected for
resistance against the HIV-1-specific [2′,5′-bis-O-(tert-butyldi-
methylsilyl)-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide)]-
â-D-pentofuranosyl (TSAO) nucleoside analogues retain sensi-
tivity to HIV-1-specific nonnucleoside inhibitors. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 6952-6956.
a
Debye-Hu¨ckel sphere. The accuracy of the calculated
electrostatic potentials was subsequently improved by defining
a smaller box (10 Å separation) with a lower grid resolution
(0.5 Å spacing) so that the new boundary potentials were
linearly interpolated from those calculated in the previous run
(focusing).
To explore the effect of halogen substitution on the binding
affinity of TSAO-T, our previously reported reduced model of
RT comprising the NNRTI binding site and all those residues
within 30 Å of residue 138 of the p51 subunit was used, which
included a spherical “drop” of about 370 TIP3P water mol-
ecules centered on the C_ of Glu-B138.14 A molecular dynamics
trajectory spanning 1 ns was then simulated for the complexes
of RT with 2, 8, 9, and 10 under the same conditions reported
previously, and the electrostatic interactions between each of
these ligands and individual RT amino acids were calculated
by solving the Poisson-Boltzmann equation as recently de-
scribed.14 Coordinates from the last 300 ps were used for the
analysis.
Ack n ow led gm en t. We thank Ann Absillis and
Lizette van Berckelaer for excellent technical assistance.
We also thank the Ministery of Education of Spain for
a grant to E.L. and J anssen-Cilag, S.A. for an award to
E.L. The Spanish CICYT (Project SAF2000-0153-C02-
01), the Comunidad de Madrid (Project 08.2/0044/2000),
and the European Commission (Project QLK2-CT-2000-
00291) are also acknowledged for financial support.
(9) Sluis-Cremer, N.; Dmitrienko, G. I.; Balzarini, J .; Camarasa, M.
J .; Parniak, M. A. Human immunodeficiency virus type 1 reverse
transcriptase dimer destabilization by 1-{spiro-[4′′-amino-2′′,2′′-
dioxo-1′′,2′′-oxathiole-5′′,3′-[2′,5′-bis-O-(tert-butyldimethylsilyl)-
â-D-ribofuranosyl]]}-3-ethylthymine. Biochemistry 2000, 39, 1427-
1433.
(10) Ingate, S.; Pe´rez-Pe´rez, M. J .; De Clercq, E.; Balzarini, J .;
Camarasa, M. J . Synthesis and anti-HIV-1 activity of novel
TSAO-T derivatives modified at the 2′- and 5′-positions of the
sugar moiety. Antiviral Res. 1995, 27, 281-299.
(11) Calvo-Mateo, A.; Camarasa, M. J .; D´ıaz-Ortiz, A.; Go´mez de las
Heras, F. Novel aldol-type cyclocondensation of O-mesyl (meth-
ylsulphonyl) cyanohydrins. Application to the stereospecific
synthesis of branched-chain sugars. J . Chem. Soc., Chem.
Commun. 1988, 1114-1115.
(12) Camarasa, M. J .; J imeno, M. L.; Pe´rez-Pe´rez, M. J .; Alvarez,
R.; Vela´zquez, S.; Lozano, A. Synthesis, NMR studies and
theoretical calculations of novel 3-spiro-branched ribofuranoses.
Tetrahedron 1999, 55, 12187-12200.
(13) Lobato´n, E.; Camarasa, M. J .; Vela´zquez, S. An efficient
synthesis of 3′-spiro sultone nucleosides functionalized on the
sultone moiety via Pd-catalyzed cross-coupling reaction. Synlett
2000, 9, 1312-1314.
(14) Rodr´ıguez-Barrios, F.; Pe´rez, C.; Lobato´n, E.; Vela´zquez, S.;
Chamorro, C.; San-Fe´lix, A.; Pe´rez-Pe´rez, M. J .; Camarasa, M.
J .; Pelemans, H.; Balzarini, J .; Gago, F. Identification of a
putative binding site for [2′,5′-bis-O-(tert-butyldimethylsilyl)-â-
d-ribofuranosyl]-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-di-
oxide) thymine (TSAO) derivatives at the p51-p66 interface of
HIV-1 reverse transcriptase. J . Med. Chem. 2001, 44, 1853-
1865.
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