Abasic analogues of TSAO-T as the first sugar derivatives that specifically inhibit HIV-1 reverse transcriptase

Article abstract of DOI:10.1021/jm980370m

With the aim of assessing the role that the thymine base of TSAO-T may play in the interaction of TSAO compounds with HIV-1 reverse transcriptase (RT), we have designed, synthesized, and evaluated for their anti-HIV-1 activity a series of 3-spiro sugar de

Full text of DOI:10.1021/jm980370m

4636
J . Med. Chem. 1998, 41, 4636-4647
Aba sic An a logu es of TSAO-T a s th e F ir st Su ga r Der iva tives Th a t Sp ecifica lly
In h ibit HIV-1 Rever se Tr a n scr ip ta se
Sonsoles Vela´zquez,§ Cristina Chamorro,§ Mar´ıa-J esu´s Pe´rez-Pe´rez,§ Rosa Alvarez,§ Mar´ıa-Luisa J imeno,§
Angel Mart´ın-Domenech,§ Carlos Pe´rez,† Federico Gago,† Erik De Clercq,‡ J an Balzarini,‡ Ana San-Fe´lix,§ and
Mar´ıa-J ose´ Camarasa*,§
Instituto de Quı´mica Me´dica, C.S.I.C., J uan de la Cierva 3, 28006 Madrid, Spain, Departamento de Farmacolog´ıa,
Universidad de Alcala´, 28871 Alcala´ de Henares, Madrid, Spain, and Rega Institute for Medical Research,
Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received J une 18, 1998
With the aim of assessing the role that the thymine base of TSAO-T may play in the interaction
of TSAO compounds with HIV-1 reverse transcriptase (RT), we have designed, synthesized,
and evaluated for their anti-HIV-1 activity a series of 3-spiro sugar derivatives substituted at
the anomeric position with nonaromatic rings or with amine, amide, urea, or thiourea moieties
that mimic parts or the whole thymine base of TSAO-T. Also, a dihydrouracil TSAO analogue
and O-glycosyl 3-spiro sugar derivatives substituted at the anomeric position with methyloxy
or benzyloxy groups have been prepared. Compounds substituted at the anomeric position
with an azido, amino, or methoxy group, respectively, were devoid of marked antiviral activity
(EC50: 10-200 µM). However, the substituted urea sugar derivatives led to an increase in
antiviral potency (EC50: 0.35-4 µM), among them those urea derivatives that mimic most
closely the intact TSAO-T molecule retained the highest antiviral activity. Also, the dihy-
drouracil TSAO derivative retained pronounced anti-HIV-1 activity. None of the compounds
showed any anti-HIV-2 activity. The results described herein represent the first examples of
sugar derivatives that interact in a specific manner with HIV-1 RT. Molecular modeling studies
carried out with a prototype urea derivative indicate that a heteroaromatic ring is not an
absolute requirement for a favorable interaction between TSAO-T and HIV-1 RT. Urea
derivatives, which can mimic to a large extent both the shape and the electrostatic potential
of a thymine ring, can effectively replace this nucleic acid base when incorporated into a TSAO
molecular framework with only moderate loss of activity.
Ch a r t 1. Chemical Structures of TSAO-T and TSAO-U
In tr od u ction
Key targets in the search for effective drugs useful
for AIDS therapy are the viral enzymes that have
critical roles in the life cycle of the human immuno-
deficiency virus type 1 (HIV-1). One such essential
enzyme is reverse transcriptase (RT).1,2 A number of
inhibitors of HIV RT have been developed.3-5 Among
them, the nonnucleoside RT inhibitors (NNRTI) repre-
sent a group of highly potent and specific inhibitors of
HIV-1 replication6,7 that interact noncompetitively with
the enzyme at an allosteric nonsubstrate binding site
that is distinct from, but functionally and also spatially
associated with, the substrate binding site.8-10 This
particular site corresponds to a flexible, highly hydro-
phobic pocket that is exclusively found in the reverse
transcriptase of HIV-1, and hence, NNRTIs are only
inhibitory to HIV-1 and not HIV-2 or other retro-
viruses.6,7
of TSAO molecules with both subunits at the same
time.12-15 Structurally TSAO are highly functionalized
nucleosides. The prototype compound is [1-[2′,5′-bis-O-
(tert-butyldimethylsilyl)-â-D-ribofuranosyl]thymine]-3′-
spiro-5′′-(4′′-amino-1′′,2′′-oxathiole 2′′,2′′-dioxide) desig-
nated as TSAO-T (1) (Chart 1).11b These compounds
exert their unique selectivity for HIV-1 through a
specific interaction with the HIV-1 RT that, unlike the
interaction of the nucleoside type of RT inhibitors, is
noncompetitive with regard to the normal substrates.16
Our experimental data strongly suggest a specific
interaction of the 3′-spiro moiety of TSAO molecules
with a glutamic acid residue at position 138 (Glu-138)
of the p51 subunit of HIV-1 RT.13,14,17
Among NNRTIs, TSAO derivatives represent a par-
ticular and peculiar group of specific RT inhibitors,
developed in our laboratories,11,12 that are able to
interfere at the interface between the p51 and p66 RT
subunits. Well-defined amino acids at both the p51 and
p66 RT subunits are needed for an optimum interaction
§ C.S.I.C.
† Universidad de Alcala´.
‡ Katholieke Universiteit Leuven.
Structure-activity relationship (SAR) studies within
10.1021/jm980370m CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/13/1998

Abasic Analogues of TSAO-T
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4637
Sch em e 1
this class of compounds have revealed that stringent
requirements exist with regard to the structural deter-
minants for optimum anti-HIV activity in cell culture.
The sugar part of the TSAO molecules plays a decisive
and crucial role in the interaction of TSAO compounds
with the target HIV-1 RT enzyme. However, the role
of the base part in this interaction is yet unclear.12
Thus, the presence of tert-butyldimethylsilyl (TBDMS)
groups at both the C-2′ and C-5′ positions and of the
unique 3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole 2′′,2′′-di-
oxide) in nucleosides with a D-ribo configuration is a
prerequisite for antiviral activity.11b,18-20 In contrast,
the nature of the heterocyclic base is less critical for
anti-HIV-1 activity. The thymine moiety of TSAO-T can
be replaced by a number of other pyrimidines, purines,
and 1,2,3-triazoles without a marked decrease in anti-
viral efficacy.11b,19-21
As part of our ongoing program to further explore the
effects of different substituents on the anti-HIV-1 activ-
ity of TSAO derivatives and, in particular, to determine
the role that the nucleobase may play in this interaction,
novel compounds were designed in which the sugar part
of the prototype compound TSAO-T was maintained but
the base part (thymine) was modified. A series of
3-spiro sugar derivatives substituted at the anomeric
position with nonaromatic rings or with amine, amide,
urea, or thiourea moieties derived from a systematic
disassemblage of the molecular architecture of the
thymine ring were prepared. The new compounds were
aimed at assessing (i) the role that aromaticity and (ii)
the role that different fragments mimicking parts or the
thymine base of TSAO-T may play in the interaction of
the TSAO functionality with the RT enzyme. Finally,
for comparative purposes we also prepared O-glycosyl
3-spiro sugar derivatives substituted at the anomeric
position with aromatic and nonaromatic lipophilic moi-
eties such as benzyl ether or methyl ether groups,
respectively.
1-amino sugar intermediate 5 (Scheme 2). This inter-
mediate was prepared by reduction of the ribosyl azide
derivative 4.21 Initial attempts to reduce 4 by catalytic
hydrogenation failed because reduction of 4 was slug-
gish and after 3 days only traces of the reduced product
5 were detected. However, compound 5 was obtained
in almost quantitative yield when 4 was reduced using
the method of Staudinger23 for the reduction of azides.
Thus, reaction of 4 with trimethylphosphine followed
by treatment with saturated methanolic ammonia (to
hydrolyze the phosphinimine intermediate formed) gave
the ribosylamine 5 as an anomeric mixture in which the
very major compound was the â anomer, as deduced
1
from its H NMR spectrum. Compound 5 was unstable
in solution and therefore was used immediately in the
next step without further purification. The instability
of anomeric glycosylamines is well-documented in the
literature.24 Acetylation of 5 with Ac2O/pyridine af-
forded a mixture of the two N-acetyl derivatives 6a and
6b in 20% and 23% yield, respectively. The yield
obtained for the R anomer (6a ) could be explained by
mutarotation of the starting â-amine 5 to the R com-
pound. This effect was also observed in the reaction of
5 with several isocyanates (vide infra). It should be
pointed out that, as mentioned above, reduction of the
â-ribofuranosylamine 4 yielded mainly the â-amine 5,
but on standing in solution, this compound mutarotates
very readily to the R compound. Therefore, even when
freshly prepared the anomeric amine 5 is a mixture of
R and â forms. The mutarotation of other furanosyl or
pyranosyl anomeric amines has been reported.24-26
Reaction of 5 with chlorosulfonyl isocyanate followed
by treatment with aqueous NaHCO3 gave the ribo-
furanosylurea derivatives 7a (45% yield) and 7b (24%
yield). Acetylation of urea derivative 7b with Ac2O/
pyridine gave the corresponding N-acetylurea derivative
8b (21%). Attachment of the acetyl residue to the N′H
of the urea and not to the 1-NH was established from
the 1H NMR spectrum by the disappearance of the broad
singlet at 6.98 ppm assigned to one of the N′H2 protons
and the downfield shift of the broad singlet correspond-
ing to the other N′H proton. The latter appeared at 9.55
ppm, due to the deshielding effect of the CdO of the
acetyl group attached to the N′H. No modification was
observed in the multiplicity of the signals corresponding
to the 1-NH or to the anomeric protons, with respect to
those observed for the starting 7b.
In this paper we report the synthesis and anti-HIV-1
activity of these novel compounds.22 The compounds
described herein represent the first examples of sugar
derivatives that interact in a specific manner with
HIV-1 RT. To rationalize the results obtained, a
theoretical investigation was undertaken to gain insight
into the conformational preferences of a protoype urea
derivative and its possible mode of binding to the
nonnucleoside binding pocket.
Resu lts a n d Discu ssion
Ch em istr y. To determine the importance of aroma-
ticity of the base for the interaction of TSAO derivatives
with HIV-1 RT, we prepared the dihydrouracil TSAO
analogue 3. Thus, treatment of TSAO uracil 211b
(Scheme 1) with hydrogen in the presence of 10% Pd/C
gave the dihydrouracil derivative 3 in 52% yield.
On the other hand, to assess which is the minimum
fragment of the thymine moiety of the prototype com-
pound (TSAO-T, 1) necessary for the interaction with
HIV-1 RT, we designed 3-spiro sugar derivatives bear-
ing at the anomeric position different fragments that
mimic (parts of) or the whole thymine base of TSAO-T.
The 1-urea- or thiourea-substituted 3-spiro sugar de-
rivatives were prepared by reaction of the corresponding
isocyanates or isothiocyanates with the appropriate
Reaction of the amino sugar intermediate 5 with other
isocyanates such as ethyl, methacryloyl, or benzoyl
isocyanate in dry acetonitrile at room temperature
afforded the corresponding mixtures of the R and â N′-
substituted urea derivatives 9a and 9b (34%), 10a (16%)
and 10b (62%), or 11a (10%) and 11b (54%), respec-

4638 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Vela´zquez et al.
Sch em e 2
tively. The mixtures of anomers could be separated by
chromatography except in the case of N′-ethylurea
derivatives 9a and 9b.
therefore their anomeric configuration is â. A similar
NOE experiment with compounds 8b and 13 also
indicated the â anomeric configuration.
Two additional points should be highlighted regarding
Similarly, reaction of 5 with phenyl isocyanate or
phenyl isothiocyanate gave the â isomers 12b (62.5%)
or 13 (50%). In the latter reaction the corresponding R
anomer was not detected, whereas only traces of 12a
1
1
the H NMR data of these compounds. In the H NMR
spectra, N′-acylureas 8b, 10a , 10b, 11a , and 11b
showed a strong downfield shift of the anomeric NH
proton, which appeared at δNH ) 9 ppm. This suggests
the existence of an intramolecular hydrogen bond
between this NH proton and the carbonyl anchoring a
Z,E,Z-conformation for the acylurea moiety (Chart 2).
This strong downfield shift is in agreement with the
values reported in the literature for other N′-acyl-
substituted urea derivatives.29
In compound 13 the presence of a CdS group instead
of the CdO group as in 12b causes a significant
difference in the chemical shift of H-1 (∆δ ∼ 1 ppm) and
H-2 (∆δ ∼ 0.4 ppm) with respect to these values in 12b.
It has been reported in the literature that, in general,
the protons in the sulfur-containing compounds resonate
further downfield than the corresponding protons in the
oxygen-containing analogues.30-32
For comparative purposes we also prepared O-glycosyl
3-spiro sugar derivatives substituted at the anomeric
position with methyloxy or benzyloxy groups. The
methyl spiroribofuranoside 20 (Scheme 3) was prepared
from 3-C-cyano-3-O-mesylribofuranoside 1619 in six
steps. The 1,2-O-isopropylidene protecting group of 16
was cleaved by hydrolysis in (9:1) TFA-H2O solution.
Subsequent methylation (MeOH/H2SO4) and silylation
(TBDMSCl/DMAP) converted the intermediate 1,2-diols
to the methyl 2-silylated R- and â-(mesyloxy)nitriles 18
(57%). Treatment of 18 with DBU in acetonitrile
followed by treatment with saturated methanolic am-
monia afforded 19.
1
were detected by H NMR. Finally, reaction of 5 with
2-(methoxycarbonyl)phenyl isocyanate or 4-(methyl-
thio)phenyl isocyanate gave the corresponding N′-
substituted urea derivatives 14a (31%) and 14b (33%)
or 15a (4%) and 15b (31%).
The structures of new compounds were assigned on
the basis of the corresponding analytical and spectro-
scopic data. Assignment of the anomeric configuration
in this series was not trivial. Chemical shifts of the
nuclei at the anomeric site, ∆(H-1) and ∆(C-1), will be
sensitive to the configuration, but variation in the
nature of the substituent at C-1 can produce overlapping
ranges for both these parameters. It has been reported
in the literature that for N-acyl-D-ribosylamines, the R
anomeric proton appears at a downfield chemical shift
with respect to that of the one corresponding to the â
anomer.27 In general, our compounds fulfilled this
criterion, and we observed a generally larger downfield
shift of the anomeric proton corresponding to the R
isomer (δH-1R > δH-1â) except in the case of compounds
11a , 11b, 12a , and 12b (see Table 1). However, this
criterion must be used with caution especially when only
one anomer is available as in the case of 13.
Other parameters that have been used to establish
the anomeric configuration of N-acylribofuranosyl-
3
amines are the values of coupling constants J 1,2 and
3J 1,NH. It has been reported that the J 1,2 and J 1,NH
values for the R anomers are larger than the corre-
sponding coupling constant values in the â anomers.27,28
In our compounds the similar values observed between
the R and â anomers (see Table 1) precluded the use of
these parameters. Therefore, the anomeric configura-
tion in this series of compounds was unequivocally
determined by NOE experiments carried out on the
major isomers 6b, 7b, 10b, 11b, 12b, 14b, and 15b.
Thus, for these putative â anomers irradiation of H-1
caused enhancement of the signals for both H-2 and H-4.
The NOE observed between H-1 and H-4 indicated that
for all these compounds both protons H-1 and H-4 are
at the lower face (R face) of the furanose ring, and
Treatment of 19 with tert-butyldimethylsilyl chloride
1
(TBDMSCl) gave a 1:14 mixture (deduced from H NMR
spectrum) of the two anomeric forms (R and â) of the
2,5-bis-O-silylated methyl ribofuranoside 20. Only the
major â isomer of 20 could be isolated in a pure form
from the isomeric mixture. The â anomeric configura-
tion of the major isomer was unequivocally determined
by NOE experiments.
Finally, we prepared the O-benzyl spirofuranosides
27 and 28 (Scheme 4). Attempts to obtain the benzyl
sugar derivatives by a reaction sequence similar to that
described for 20 was unsuccessful. For this reason

Abasic Analogues of TSAO-T
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4639
Ta ble 1. Selected 1H NMR Spectral Data of Compounds 6-15: Chemical Shifts (ppm), Multiplicity, and Coupling Constants (Hz)a
compd H-1 (J 1,2
)
H-2
H-4 (J 4,5
)
H-5
NH-1 (J 1,NH
)
H-3′
NH2-4′
others
6a
5.98 dd
(6.9)
5.11 dd
(6.7)
5.85 dd
(6.8)
5.43 t
(7.9)
5.56 dd
(8.0)
5.30 dd
(5.2)
5.21 t
(5.0)
5.95 dd
(6.8)
5.61 dd
(8.0)
5.26 t
(9.5)
6.01 dd
(6.8)
5.27 dd
(7.1)
6.01 dd
(6.9)
6.73 dd
(6.8)
6.04 dd
(6.8)
5.29 dd
(6.8)
4.71 d
4.22 t
(2.1)
4.03 m
3.89 m
7.25 bd
(10)
7.85 bd
(8.3)
6.68 bd
(7.8)
6.30 bd
(7.9)
9.02 bd
(10.4)
6.36 bd
(9.7)
6.42 bd
(5.0)
9.54 bd
(9.2)
9.25 bd
(10)
9.17 bd
(9.5)
9.76 bd
(9.46)
6.40 bd
(7.4)
6.33 bd
(9.9)
7.53 bd
(9.2)
7.02 bd
(9.6)
7.42 bd
(8.8)
6.31 bd
(9.8)
6.36 bd
(8.9)
5.72 s 6.33 bs 1.98 s, COCH3
5.59 s 6.45 bs 1.97 s, COCH3
6b
4.60 d
4.72 d
4.33 d
4.34 d
4.50 d
4.23 d
4.76 d
4.40 d
4.08 d
4.79 d
4.54 d
4.70 d
4.80 d
4.78 d
4.63 d
4.75 d
4.56 d
4.00 m
3.90 m
3.93 m
3.92 m
3.85 m
3.85 m
3.92 m
3.96 m
3.78 m
3.94 m
3.96 m
3.90 m
3.92 m
3.92 m
3.97 m
3.90 d
3.93 m
7a
4.28 m
4.14 t
5.72 s 6.40 bs 8.80 bs, 9.40 bs, CONH2
7b
5.67 s 6.37 bs 6.98 bs, 8.60 bs, CONH2
8b
3.16 t
(2.3)
4.31 t
(2.5)
4.13 t
(2.9)
4.26 m
5.70 s 6.38 bs 2.10 s, COCH3; 9.55 bs, CON′HCO
5.70 s 6.28 bs 0.90 m, CH2CH3; 3.90 m, N′HCH2
5.63 s 6.36 bs 0.90 m, CH2CH3; 3.90 m, N′HCH2
5.74 s 6.32 bs 5.62 m, 5.99 m, dCH2; 9.25 bs, CON′HCO
5.76 s 6.46 bs 5.75 m, 6.11 m, dCH2; 9.46 bs, CON′HCO
5.50 s 6.31 bs 7.57 m, 8.04 m, Ph; 9.82 bs, CON′HCO
5.77 s 6.33 bs 7.55 m, 8.04 m, Ph; 9.76 bs, CON′HCO
5.63 s 6.50 bs 7.00 m, 7.35 m, 7.50 m, Ph; 8.40 bs, CON′HPh
5.72 s 6.33 bs 7.00 m, Ph; 8.67 bs, CON′HPh
9a b
9bb
10a
10b
11a
11b
12a
12b
13
4.22 t
(2.3)
4.23 dd
(5.4, 3.6)
4.32 m
4.06 m
4.23 dd
(1.8, 2.5)
4.24 m
5.76 s 6.41 bs 7.4 m, Ph; 9.52 bs, CSN′HPh
14a
14b
15a
15b
4.27 t
5.73 s 6.30 bs 3.88 s, CO2CH3; 7.75 m, Ph; 10.07 bs, CON′HPh
5.62 s 6.33 bs 3.91 s, CO2CH3; 7.75 m, Ph; 10.27 bs, CONHPh
5.73 s 6.35 bs 2.42 s, SCH3; 7.30 m, Ph; 8.68 bs, CON′HPh
5.62 s 6.44 bs 2.43 s, SCH3; 7.30 m, Ph, 8.35 bs, CON′HPh
4.10 m
(2.8, 5.2)
4.23 t
(2.1)
4.06 m
(2.7, 4.6)
6.00 dd
(6.9)
5.26 dd
(7.1)
a
b
(CD3)2CO at 200 MHz. CDCl3 at 200 MHz.
Ch a r t 2
Sch em e 3
compounds 27 and 28 were prepared following our
reported procedure for the synthesis of the ribo- and
xylofuranosyl TSAO pyrimidine nucleosides.11b Treat-
ment of the ulose 22, prepared by oxidation of 2133
(pyridinium dichromate/Ac2O/CH2Cl2),34 with sodium
cyanide in a diethyl ether/water two-phase system in
the presence of NaHCO3 yielded, after mesylation
(mesyl chloride/pyridine) of the two epimeric cyano-
hydrins (23 and 24), a mixture of the respective O-
benzyl ribo and xylo cyanomesylates 25 (15%) and 26
(60%). Treatment of 25 or 26 with DBU in acetonitrile
gave the 3-spiro derivatives 27 (50%) and 28 (70%).
Assignment of the structures of these ribo and xylo
derivatives was made by comparison of their spectro-
scopic data with those of other ribo and xylo 3-cyano-
mesylates and spiro derivatives of this series
whose structures have been unequivocally deter-
mined.11b,19-21,35,36
Str u ctu r a l F ea tu r es. The acyclic substituent at the
anomeric position of the most active acylureas 8b and
10b (vide infra) could exist in different conformations
(Chart 3). The spectroscopic data were only compatible
with six-membered hydrogen-bonded pseudocycle con-
former II but not with conformers I or III. In addition,
in compound 10b conformer II can have two orientations

4640 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Vela´zquez et al.
Sch em e 4
between the NH anomeric and H-1 and H-2 protons
suggests the existence, for compound 10b, in solution
of a syn-anti equilibrium38 of the six-membered
pseudocycle.
Biologica l Activity. The inhibitory activity of the
test compounds was explored against HIV-1 (IIIB) and
HIV-2 (ROD) replication in MT-4 and CEM cell cultures
(Table 2). TSAO-T was included as a reference com-
pound.
Compounds 4, 5, and 20, containing the silylated
sugar part of TSAO but entirely lacking the base part
(replaced by an azido, amino, or methoxy group, respec-
tively), were devoid of marked antiviral activity (EC50:
>200, >10, and 21 µM). However, extending the amino
group in the C-1 â anomeric position of the sugar part
with additional functional groups led to an increase in
antiviral potency. Whereas 6b showed an EC50 of 2.6
and 2.8 µM against HIV-1 (CC50: 13 µM in MT-4 cells),
8b was active at 0.89 and 0.65 µM (CC50: 15-26 µM).
Compound 10b had a slightly further improved antiviral
activity (EC50: 0.59 and 0.56 µM) but also increased
toxicity (CC50: 6.1-8.6 µM).
F igu r e 1. NOEs observed for compound 10b upon irradiation
of anomeric NH and N′H protons.
Ch a r t 3. Conformations for acylureas 8b and 10b
As a rule, the corresponding R anomeric derivatives
were virtually devoid of significant antiviral activity
among those compounds that had an aliphatic extension
at the C-1 carbon of the sugar part. However, among
the aromatic extensions of the sugar moiety, this
property proved only to be the case when 11b was
compared with 11a . Surprisingly, the â anomeric 12b
proved to be 10-12-fold less active than its correspond-
ing R anomeric derivative. Also, 14a , 14b and 15a , 15b
were comparably active against HIV-1 replication in
both CEM and MT-4 cell lines. Their EC50 values
ranged between g2 and 5.3 µM, but the CC50 ranged
from 3.8 to 29 µM. Finally, both ribo and xylo deriva-
tives of the benzyl-substituted compounds 27 and 28
were endowed with antiviral activity (EC50: 4.6 to g6.4
µM) and cytotoxicity (CC50: 11-13 µM).
As a rule, none of the compounds had any anti-HIV-2
activity, which is consistent with the complete lack of
anti-HIV-2 activity of the TSAO derivatives (i.e., TSAO-
T). Also, akin to TSAO-T, 8b, 10b, 12a , 15a , and 15b
were not inhibitory to Glu-B138 Lys RT mutant virus
replication indicating that the interaction with Glu-138
of the enzyme is most likely preserved in the TSAO
of the R substituent (Figure 1). The predominant
orientation (in acetone) for this compound was unequivo-
cally determined by a selective NOE experiment.37
Thus, irradiation of the NH anomeric caused enhance-
ments of the signals for H-1 and H-2 sugar protons.
Likewise, irradiation of the N′H proton influenced the
signals for dCH2. These results were only compatible
with conformer IIa. Moreover, the NOE observed

Abasic Analogues of TSAO-T
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4641
Ta ble 2. Antiviral Activity of TSAO Derivatives against HIV-1 and HIV-2 in CEM and MT-4 Cell Cultures
EC50 (µM)a
MT-4
CEM
CC50 (µM)b
compd
2c
3
4
5
6a
6b
7a
HIV-1
HIV-2
HIV-1
HIV-2
MT-4
CEM
0.18 ( 0.04
0.92 ( 0.10
>20
>2
13 ( 0.9
21 ( 0.9
>200
0.31 ( 0.2
19 ( 0.2
>200
>200
>10
>8
>200
>10
g8
>200
>10
>8
>200
>10
>8
21 ( 0.4
16 ( 1.4
13 ( 1.4
16 ( 9.7
g8
>8
2.6 ( 0.8
>8
>250
124 ( 9.2
>250
>250
>10
>10
>1.3
>6.5
>250
>50
>2
57 ( 11
>50
>50
>10
>10
>6.5
>6.5
>250
>50
>2
>250
g250
26 ( 21
19 ( 2.7
2.7 ( 0.02
8.6 ( 4
g250
203 ( 66
g250
7b
8b
90 ( 35
0.89 ( 0.13
>10
>1.3
0.59 ( 0.02
>250
16 ( 9.3
0.65 ( 0.07
15 ( 3.7
20 ( 1.1
6.4 ( 2.2
6.1 ( 1.1
24 ( 4.6
48 ( 0.8
4.2 ( 0.4
10 ( 1.1
4.1 ( 0.4
22 ( 1.7
3.8 ( 1.4
6.4 ( 2.5
22 ( 0.9
9a + 9b
10a
10b
11a
11b
12a
12b
13
14a
14b
15a
15b
20
>10
>6.5
0.56 ( 0.34
>250
7.0 ( 2.6
0.65 ( 0.02
90 ( 6.7
4.3 ( 0.1
14 ( 1.3
3.7 ( 0.1
26 ( 2.5
17 ( 1.0
17 ( 0.4
29 ( 3.5
0.59 ( 0.2
5.1 ( 0.7
>2
>10
>2
>10
>2
>10
>10
>200
>6.4
>6.4
>20
g10
g2
>10
>2
5.3 ( 2.7
3.1 ( 2.3
4.2 ( 0.8
21 ( 5.4
4.5 ( 0.7
>10
>2
g2
>2
>2
5.0 ( 0.0
>10
21 ( 8.5
>200
27
28
TSAO-T
4.9 ( 2.1
4.6 ( 2.4
0.06 ( 0.03
g6.4
g6.4
0.06 ( 0.01
>6.4
>6.4
>20
12 ( 1.9
13 ( 1.8
14 ( 2
11 ( 0.64
11 ( 0.96
a
50% effective concentration, or concentration required to protect 50% of the virus-infected cells against destruction by the virus.
50% cytostatic concentration, or concentration required to reduce the viability of mock-infected cells by 50%. c Data taken from ref 12b.
b
derivatives modified at the base part. It may not be
surprising that 8b and 10b, which mimic mostly the
intact TSAO molecule [with base ring opening between
N-1 and C-6 (for 10b) or C-6 and C-5 (lacking the C-6
carbon atom) (for 8b)], retained the highest antiviral
activity. This may perhaps indicate that the functional
groups present in the intact base and in the ring-opened
forms of TSAO (8b, 10b) are required for interaction
with RT. Also, the comparable anti-HIV-1 activity of
the dihydrouracil TSAO derivative 3 and the uracil
TSAO derivative 2 suggests that an aromatic entity on
C-1 of the sugar part is not an absolute requirement
for the interaction with the enzyme and is in agreement
with the findings for the open-ring TSAO structures.
Mod elin g. To rationalize the biological results ob-
tained, a theoretical investigation was undertaken to
gain insight into the conformational preferences of a
prototype urea derivative (compound 10b) and its mode
of interaction with the nonnucleoside binding pocket
that may reveal potential interaction points between the
NNRTIs binding pocket of the enzyme and the different
active molecules.
The intramolecular hydrogen bond (Chart 2) stabilizes
conformer I of compound 10b in vacuo over all the other
possible conformers (Table 3). The degree of similarity
with the thymine ring, however, is maximal (62%) for
conformer III, in which there is no such hydrogen bond.
The AutoDock calculations consistently placed the
thymine ring immediately above the plane of Tyr-A318,
in a cavity delineated by the side chains of residues Leu-
A100, Lys-A103, Val-A106, and Leu-A234 and the
carbonyl groups of Lys-A101, His-A235, and Pro-A236.
This is in agreement with the common finding of an
aromatic ring filling this part of the cavity in the
complexes of RT with other inhibitors.8-10 The orienta-
tions suggested by the docking program (Figure 2a),
however, were incompatible with what is known from
previous structure-activity11,12,16,18-21 and modeling
studies.39 Most importantly, since the N-1 atom of
thymine is attached to the C-1′ atom of the ribose, this
ring atom must be positioned such that it allows location
of the rest of the structure in the binding site so as to
place the exocyclic amino group on the spiro ring in a
suitable orientation for establishing an intermolecular
hydrogen bond with the side chain of Glu-B138. Indi-
rect support for this specific interaction has been
recently gained from work on TSAO analogues modified
on the spiro system.17,39 Besides, the fact that the N-3
position has been linked to rather long substituents
(AZT, etc.)19,40 without a great loss of potency may
indicate that this ring atom is facing the proposed
entrance to the pocket, namely, the channel between
Pro-A236 and the A225-A226 and A105-A106 loops.41
Consequently, we favor the view that the thymine ring
of TSAO-T is situated in this binding pocket in the
orientation shown in Figure 2b. Calculation and display
of the molecular electrostatic potential in this region of
the enzyme binding site (data not shown) reveal a highly
negative potential above the phenyl ring of Tyr-A318
as a result of electrostatic focusing.42 On the other
hand, the positive electrostatic potential of many bound
inhibitors is most prominent around the edges of the
heteroaromatic rings that point toward the faces of the
aromatic rings of both Tyr-A318 and Trp-A229. We
therefore believe that the ensuing electrostatic comple-
mentarity is important for ligand orientation and for
enhanced binding affinity. In the case of TSAO-T, the
carbonyl oxygen at the C-4 position of the thymine ring
gives rise to a globular region of negative potential in
the midst of an otherwise positive region. This may be
the reason AutoDock does not select for the binding
orientation that is most in consonance with our current

4642 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Vela´zquez et al.
Ta ble 3. Optimized Geometries of N1-Methylthymine and
Selected Conformers of the Urea Part of Compound 10b
molecular framework with only a moderate loss of
activity. As shown in this work, the abasic TSAO
analogue 10b is just 1 order of magnitude less active
than the prototype TSAO-T, and our calculations sug-
gest that this may be due to the energy penalty involved
in breaking the intramolecular hydrogen bond in order
to adopt a conformation suitable for binding.
The differences in activity found among the TSAO
analogues reported in this series can be explained by
taking into account their distinct capability to interact
with the hydrophobic pocket bounded by Tyr-A318. The
most active compounds of this series (compounds 10b,
8b, and 12a ) are those large enough to fill this pocket
and interact with hydrophobic residues Leu-A100,
Val-A106, Pro-A236, and Tyr-A318. Smaller compounds
(6, 7) cannot give rise to all of these interactions and
larger ones (11, 14, and 15) may gain limited access to
the bottom of this cavity because of occupancy of part
of the proposed entrance44 between Pro-A225 and Pro-
A236. In both cases the net result would be a decrease
in activity.
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 and a
Varian Unity 500 spectrometer operating at 498.84 MHz with
Me4Si as internal standard. 13C NMR spectra were recorded
with a Bruker AM-200 spectrometer operating at 50 MHz, with
Me4Si as internal standard. IR spectra were recorded with a
Shimadzu IR-435 spectrometer. Analytical TLC was per-
formed 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 PF
254 gipshaltig (Merck)), layer thickness 1 mm, flow rate 5 mL/
min. Flash column chromatography was performed with silica
gel 60 (230-400 mesh) (Merck).
[1-[2′,5′-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n -
osyl]-5,6-d ih yd r ou r a cil]-3′-sp ir o-5′′-(4′′-a m in o-1′′,2′′-oxa -
th iole 2′′,2′′-Dioxid e) (3). Compound 211b (0.11 g, 0.20 mmol)
was dissolved in MeOH (1 mL) and hydrogenated in the
presence of 10% Pd/C (0.06 g) at 30 psi at 30 °C until
disappearance of the starting material. The mixture was
filtered and evaporated. The residue was purified by CCTLC
on the chromatotron (CH2Cl2-ethyl acetate, 7:1) to give 3 (0.06
g, 52%) as an amorphous solid: 1H NMR (CDCl3, 300 MHz) δ
2.68 (m, 2H, H-5), 3.59 (m, 1H, H-6a), 3.67 (m, 1H, H-6b), 3.91
(m, 2H, H-5′), 4.23 (pt, 1H, H-4′, J 4′,5′ ) 3.18 Hz), 4.53 (d, 1H,
H-2′), 5.57 (s, 1H, H-3′′), 5.59 (d, 1H, H-1′, J 1′,2′ ) 7.6 Hz), 5.59
(bs, 2H, NH2-4′′), 8.22 (bs, 1H, NH-3). Anal. (C23H43N3O8SSi2)
C, H, N, S.
[[2,5-Bis-O-(ter t-bu tyld im eth ylsilyl)-D-r ibofu r a n osyl]-
a m in e]-3-sp ir o-5′-(4′-a m in o-1′,2′-oxa th iole 2′,2′-Dioxid e)
(5). To a solution of azide 421 (0.2 g, 0.4 mmol) in dry THF (5
mL) was added a 1 M solution of trimethylphosphine in THF
(0.6 mL, 0.6 mmol). The mixture was stirred at room tem-
perature for 20 min and then evaporated to dryness. The
residue was dissolved in methanol (6 mL) and treated with
saturated methanolic ammonia (6 mL) at room temperature
for 3 h. The solvent was evaporated to dryness, and the
residue was coevaporated with dry acetonitrile (2 × 6 mL) to
give the amino sugar 5 (quantitative yield) which was used
inmediately in the next step without further purification. 5
was a mixture of R and â anomers in which the â anomer was
the very major compound. 1H NMR ((CD3)2CO, 500 MHz)
â-anomer δ 3.90 (m, H-5), 4.1 (t, H-4, J 4,5 ) 2.5 Hz), 4.25 (d,
H-2), 5.25 (t, H-1, J 1,2 ) 6.8 Hz), 5.62 (s, H-3′), 6.35 (bt, NH2-
1), 6.42 (bs, NH2-4′′); R anomer δ 2.25 (bd, NH2-1, J 1,NH ) 2.2
Hz), 3.86 (m, H-5), 4.01 (d, H-2), 4.07 (t, H-4, J 4,5 ) 2.8 Hz),
4.54 (m, H-1, J 1,2 ) 7.2 Hz), 5.62 (s, H-3′), 6.22 (bs, NH2-4′′).
a
b
Energy values are given with respect to conformer I. Simi-
larity with respect to N1-methylthymine, as assessed with ASP.48
working model.43 On the other hand, of all the possible
conformers of 10b, the one that AutoDock places in this
binding pocket is that resembling most closely the
thymine ring (conformer III, Table 3), as assessed by a
similarity index of 0.62. In this conformation, the
intramolecular hydrogen bond is broken, and the ac-
companying energy penalty (∼12 kcal mol-1 in vacuo)
may account for the loss of affinity of this compound,
in comparison with TSAO-T, for the NNRTIs binding
pocket. Further modifications of the thymine ring are
currently in progress to test these hypotheses.
Con clu sion s
A close interaction with Tyr-A318 at the bottom of
the hydrophobic binding site is maintained in all the
X-ray complexes reported to date between RT and
NNRTIs, in most cases involving an aromatic moiety.
From the present results it would appear that a het-
eroaromatic ring is not an absolute requirement in order
to get a favorable interaction in this region of the
binding pocket. Urea derivatives, which can mimic to
a large extent both the shape and the electrostatic
potential of a thymine ring, can effectively replace this
nucleic acid base when incorporated into the TSAO

Abasic Analogues of TSAO-T
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4643
F igu r e 2. (a) Stereoview of the different binding orientations suggested by AutoDock for a thymine ring in part of the nonnucleoside
binding pocket. Amino acid residues shown are Leu-100, Lys-101, Lys-102, Lys-103, Val-106, Leu-234, His-235, Pro-236, and
Tyr-318 of the p66 palm domain of HIV-1 reverse transcriptase. Hydrogen atoms are not displayed for clarity. (b) Stereoview of
the binding orientation proposed for the thymine ring of TSAO-T based on the available structure-activity relationship data.
[N-Acetyl-[2,5-bis-O-(ter t-bu tyld im eth ylsilyl)-r- a n d -â-
D-r ib ofu r a n osyl]a m in e ]-3-sp ir o-5′-(4′-a m in o-1′,2′-oxa -
th iole 2′,2′-Dioxid e) (6a a n d 6b). To a solution of 5 (0.2 g,
0.4 mmol) in pyridine (3 mL) was added acetic anhydride (0.26
mL). The reaction mixture was kept for 17 h at room
temperature and then evaporated to dryness. The residue was
dissolved in dichloromethane (50 mL) and washed successively
with 0.1 N HCl (10 mL) and water (2 × 10 mL). The organic
layer was dried (Na2SO4), filtered, and evaporated to dryness.
The residue was purified twice by CCTLC on the chromatotron
(hexane-ethyl acetate, 3:1; then CH2Cl2-ethyl acetate, 4:1).
The fastest moving band gave 6a as a white foam (0.04 g,
20%): [R]D +44.60 (c 1, chloroform). Anal. (C21H42N2O7SSi2)
C, H, N, S.
The slowest moving band gave 6b (0.05 g, 23%) as a white
foam: [R]D +8.80 (c 0.2, chloroform); 13C NMR ((CD3)2CO, 50
MHz) δ 25.93, 26.17 (tBu), 63.57 (C-5), 73.77 (C-2), 79.51 (C-
3′), 84.3 (C-4), 92.37 (C-1), 95.13 (C-3), 152.36 (C-4′), 170.35
(CdO). Anal. (C21H42N2O7SSi2) C, H, N, S.
[N-[2,5-Bis-O-(ter t-bu tyld im eth ylsilyl)-r- a n d -â-D-r i-
bofu r an osyl]u r ea]-3-spir o-5′-(4′-am in o-1′,2′-oxath iole 2′,2′-
Dioxid e) (7a a n d 7b). To a cooled (-15 to -20 °C) solution

4644 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Vela´zquez et al.
of ribofuranosylamine 5 (0.2 g, 0.4 mmol) in dry acetonitrile
(12 mL) was added chlorosulfonyl isocyanate (0.035 mL, 0.4
mmol) in the absence of humidity. The resulting mixture was
stirred at -20 °C until 5 disappeared, then neutralized
(NaHCO3), and evaporated to dryness. The residue was
dissolved in isobutyl alcohol (20 mL) and washed with water
(2 × 10 mL). The organic layer was dried (Na2SO4), filtered,
and evaporated to dryness. The residue was purified by
CCTLC on the chromatotron (CH2Cl2-acetone, 60:2) to give
0.15 g (71.5%) of the ribofuranosylureas 7a and 7b. This
isomeric mixture was purified by CCTLC on the chromatotron
(CH2Cl2-MeOH, 5:1). The fastest moving band gave 7a (0.095
g, 45%) as a white foam: [R]D +24 (c 0.2, methanol). Anal.
(C20H41N3O7SSi2) C, H, N, S.
The slowest moving band gave 7b (0.051 g, 24%) as an
amorphous solid: [R]D +7.33 (c 0.7, methanol); 13C NMR
[(CD3)2CO, 50 MHz] δ 26.09, 26.45 (tBu), 65.14 (C-5), 76.86
(C-2), 84.17, 84.34 (C-3′, C-4), 92.21 (C-1), 93.81 (C-3), 152.85,
156.97 (CONH2, C-4′). Anal. (C20H41N3O7SSi2) C, H, N, S.
[N′-Acetyl-N-[2,5-bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r i-
bofu r an osyl]u r ea]-3-spir o-5′-(4′-am in o-1′,2′-oxath iole 2′,2′-
Dioxid e) (8b). Compound 7b (0.1 g, 0.19 mmol) was acety-
lated as described for 6a and 6b. Purification of the residue
by CCTLC on the chromatotron (hexane-ethyl acetate, 1:1)
gave 8b (0.023 g, 21%) as a white foam. Anal. (C22H43N3O8-
SSi2) C, H, N, S.
Rea ction of Ribofu r a n osyla m in e 5 w ith Isocya n a tes
a n d Isoth iocya n a tes. Gen er a l P r oced u r e for th e Syn -
th esis of N′-Su bstitu ted Ur ea s a n d Th iou r ea s 9-15. A
mixture of ribofuranosylamine 5 (0.2 g, 0.4 mmol), dry aceto-
nitrile (5 mL), and the appropiate isocyanate or isothiocyanate
(0.06 mmol) was stirred at room temperature overnight. After
evaporation of the solvent, the residue was purified by CCTLC
on the chromatotron. The chromatography eluent, yield, and
[R]D values of the isolated products (9-15) are indicated below
for each reaction.
[N-[2,5-Bis-O-(ter t-bu tyld im eth ylsilyl)-r- a n d -â-D-r i-
bofu r a n osyl]-N′-et h ylu r ea ]-3-sp ir o-5′-(4′-a m in o-1′,2′-ox-
a th iole 2′,2′-Dioxid e) (9a a n d 9b). The general procedure
was followed with ethyl isocyanate. Chromatography with
hexane-ethyl acetate (4:1) afforded 0.076 g (34%) of a 1:1
mixture of 9a and 9b. Anal. (C22H45N3O7SSi2) C, H, N, S.
[N-[2,5-Bis-O-(ter t-bu tyld im eth ylsilyl)-r- a n d -â-D-r i-
bofu r a n osyl]-N′-m eth a cr yloylu r ea ]-3-sp ir o-5′-(4′-a m in o-
1′,2′-oxa th iole 2′,2′-Dioxid e) (10a a n d 10b). The general
procedure was followed with methacryloyl isocyanate. Chro-
matography first with CH2Cl2-MeOH (100:1) and then with
hexane-ethyl acetate (2:1) afforded from the fastest moving
band 0.15 g (62%) of 10b as a foam: [R]D -17.8 (c 0.4,
chloroform). Anal. (C24H45N3O8SSi2) C, H, N, S.
[N-[2,5-Bis-O-(ter t-bu tyld im eth ylsilyl)-â-D-r ibofu r a n o-
syl]-N′-p h en ylth iou r ea ]-3-sp ir o-5′-(4′-a m in o-1′,2′-oxa th i-
ole 2′,2′-Dioxid e) (13). The general procedure was followed
with phenyl isothiocyanate. Chromatography with CH2Cl2-
acetone (200:1) gave 13 (0.13 g, 50%) as an amorphous solid:
[R]D +0.5 (c 0.5, chloroform). Anal. (C26H45N3O6S2Si2) C, H,
N, S.
[N-[2,5-Bis-O-(ter t-bu tyld im eth ylsilyl)-r- a n d -â-D-r i-
b ofu r a n osyl]-N′-(2-(m et h oxyca r b on yl)p h en yl)u r ea ]-3-
sp ir o-5′-(4′-a m in o-1′,2′-oxa th iole 2′,2′-Dioxid e) (14a a n d
14b ). Isocyanate: 2-(methoxycarbonyl)phenyl isocyanate.
Chromatography twice with hexane-ethyl acetate (2:1) gave
from the fastest moving band 0.09 g (33%) of 14b as a foam:
[R]D -9.53 (c 0.8, chloroform). Anal. (C28H47N3O9SSi2) C, H,
N, S.
The slowest moving band gave 14a (0.085 g, 31.5%) as a
foam: [R]D +53.22 (c 0.4, chloroform). Anal. (C28H47N3O9SSi2)
C, H, N, S.
[N-[2,5-Bis-O-(ter t-bu tyld im eth ylsilyl)-r- a n d -â-D-r i-
bofu r a n osyl]-N′-(4-(m eth ylth io)p h en yl)u r ea ]-3-sp ir o-5′-
(4′-a m in o-1′,2′-oxa t h iole 2′,2′-Dioxid e) (15a a n d 15b ).
Isocyanate: 4-(methylthio)phenyl isocyanate. Chromatogra-
phy with CH2Cl2-MeOH (20:1) gave from the fastest moving
band 15a (0.01 g, 4%) as a foam: [R]D +46.5 (c 0.2, chloroform).
Anal. (C27H47N3O7S2Si2) C, H, N, S.
The slowest moving band gave 15b (0.08 g, 31%) as a foam:
[R]D -24.5 (c 0.5, chloroform). Anal. (C27H47N3O7S2Si2) C, H,
N, S.
Meth yl 5-O-Ben zoyl-2-O-(ter t-bu tyld im eth ylsilyl)-3-C-
cya n o-3-O-m esyl-D-r ibofu r a n osid e (18). A solution of cya-
nomesylate 1619 (0.5 g, 1.26 mmol) in a 9:1 mixture of
trifluoracetic acid and H2O (5 mL) was stirred at room
temperature for 4 h. Volatiles were removed, and the residue
was coevaporated with methanol (3 × 5 mL). The crude
residue was then dissolved in methanol (10 mL), treated with
concentrated H2SO4 (0.15 mL), and stirred at room tempera-
ture for 3 days. The solution was neutralized (pyridine),
evaporated, and coevaporated with pyridine (2 × 5 mL) to
afford 17 that was used in the next step without further
purification. The crude containing 17 (0.42 g) was dissolved
in dry acetonitrile (8 mL), and tert-butyldimethylsilyl chloride
(TBDMSCl) (0.14 g) and DMAP (0.13 g) were added. The
reaction mixture was stirred for 8 h at room temperature.
Volatiles were removed, and the residue was treated with ethyl
acetate and filtered. Purification by flash column chromatog-
raphy (hexane-ethyl acetate, 4:1) gave 18 as a syrup (0.35 g,
57% from 16): 1H NMR (CDCl3, 200 MHz) δ 3.03 (s, 3H, SO2-
CH3), 3.28 (s, 3H, OCH3), 4.55 (m, 4H, H-2, 2H-5), 4.80 (d, 1H,
H-1, J 1,2 ) 5.4 Hz), 7.60 (m, 5H, Ph). Anal. (C21H31NO8SSi)
C, H, N, S.
[Met h yl 2,5-Bis-O-(ter t-b u t yld im et h ylsilyl)-â-D-r ib o-
fu r a n osid e]-3-sp ir o-5′-(4′-a m in o-1′,2′-oxa t h iole 2′,2′-Di-
oxid e) (20). To a solution of 18 (0.3 g, 0.62 mmol) in dry
acetonitrile (6 mL) was added DBU (0.092 mL, 0.62 mmol).
The reaction was stirred at room temperature for 90 min. The
solution was neutralized (acetic acid), and volatiles were
removed. The residue was stirred in a saturated methanolic
ammonia solution (20 mL) for 6 h. The solvent was evaporated
to dryness. The crude was dissolved in dry acetonitrile (6 mL),
and TBDMSCl (0.19 g) and DMAP (0.15 g) were added. The
mixture was stirred at room temperature for 24 h. Volatiles
were removed; the residue was treated with ethyl acetate (20
mL) and filtered. The residue was purified by flash column
chromatography (hexane-ethyl acetate, 5:1) to give 20 (0.14
g, 45%, from 18) as an amorphous solid: [R]D -17.5 (c 0.28,
chloroform); 1H NMR [(CD3)2CO, 300 MHz] δ 3.51 (s, 3H,
OCH3), 3.92 (m, 2H, H-5), 4.22 (t, 1H, H-4, J 4,5 ) 2.3 Hz), 4.37
(d, 1H, H-2), 4.84 (d, 1H, H-1, J 1,2 ) 5.3 Hz), 5.67 (s, 1H, H-3′),
6.29 (bs, 1H, NH2-4′). Anal. (C20H41NO7SSi2) C, H, N, S.
Ben zyl 2,5-Bis-O-(ter t-bu tyld im eth ylsilyl)-3-C-cya n o-
3-O-m esyl-â-D-r ibofu r a n osid e (25 a n d 26). A solution of
2132 (1.43 g, 3 mmol) in CH2Cl2 (50 mL) containing pyridinium
dichromate (0.8 g, 2.14 mmol) and acetic anhydride (1 mL, 9.41
mmol) was refluxed for 3 h. The solvent was evaporated, and
The slowest moving band gave 10a (0.039 g, 16%) as a
foam: [R]D +57.8 (c 0.6, chloroform). Anal. (C24H45N3O8SSi2)
C, H, N, S.
[N′-Ben zoyl-N-[2,5-bis-O-(ter t-bu tyldim eth ylsilyl)-r- an d
-â-D-r ibofu r a n osyl]u r ea ]-3-sp ir o-5′-(4′-a m in o-1′,2′-oxa th i-
ole 2′,2′-Dioxid e) (11a a n d 11b). Isocyanate: benzoyl iso-
cyanate. Chromatography first with hexane-ethyl acetate
(1:1) and then with CH2Cl2-MeOH (20:1) gave from the fastest
moving band 0.14 g (54%) of 11b as an amorphous solid: [R]D
+62.3 (c 0.1, chloroform). Anal. (C27H45N3O8SSi2) C, H, N, S.
The slowest moving band gave 11a (0.025 g, 10%) as an
amorphous solid: [R]D -40.1 (c 0.1, chloroform). Anal.
(C27H45N3O8SSi2) C, H, N, S.
[N-[2,5-Bis-O-(ter t-bu tyld im eth ylsilyl)-r- a n d -â-D-r i-
bofu r a n osyl]-N′-p h en ylu r ea ]-3-sp ir o-5′-(4′-a m in o-1′,2′-ox-
a th iole 2′,2′-Dioxid e) (12a a n d 12b). Isocyanate: phenyl
isocyanate. Chromatography with CH2Cl2-acetone (60:1)
afforded from the fastest moving band 0.15 g (62.5%) of 12b
as a foam: [R]D +0.5 (c 0.5, chloroform). Anal. (C26H45N3O7-
SSi2) C, H, N, S.
The slowest moving band gave 12a (0.015 g, 6.2%) as a
foam: [R]D -0.06 (c 0.45, chloroform). Anal. (C26H45N3O7SSi2)
C, H, N, S.

Abasic Analogues of TSAO-T
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4645
ethyl acetate (50 mL) was added, at which point the chromium
complex precipitated. The mixture was passed quickly through
a flash silica gel column with more ethyl acetate, and the
colorless eluate was evaporated. The residues were coevapo-
rated with ethanol (4 × 10 mL) and then with toluene (2 × 10
2. Sim ila r ity Ca lcu la tion s. The ASP (Automated Simi-
larity Package) program48 was used to compare the molecular
electrostatic potentials (MEP) of N1-methylthymine (T) and
the four different conformers of the acyclic analogue present
in 10b (U). A quantitative measure of the similarity (S)
between the shapes of the charge distributions of two super-
imposed molecules, T and U, can be computed according to
the following equation:49
1
mL). The product (1.1 g, 95-98% purity, from H NMR), ulose
22, was used inmediately in the next step without further
purification. Thus, crude ulose 22 (1.1 g, 2.3 mmol) was added
to a rapidly stirred mixture containing NaCN (0.22 g, 4.6
mmol) and NaHCO3 (0.39 g, 4.69 mmol) in H2O (6 mL) and
diethyl ether (12 mL). After 4 days at room temperature, the
two phases were separated and extracted with diethyl ether
(2 × 25 mL). The combined ethereal extracts were dried (Na2-
SO4), filtered, and evaporated to dryness. The residue, a
mixture of the two epimeric cyanohydrins (23 and 24), was
treated with mesyl chloride (0.86 mL, 11 mmol) in dry pyridine
(26 mL) at 4 °C for 48 h. The mixture was poured into ice
and water and extracted with CH2Cl2 (2 × 25 mL). The
combined extracts were washed successively with 1 N HCl (25
mL), aqueous NaHCO3 (25 mL), and brine (25 mL) and dried
(NaSO4). After evaporation of the solvent, the residue was
purified by column chromatography (hexane-ethyl acetate, 18:
1). The fastest moving fractions afforded 26 as a syrup (0.76
g, 60%): [R]D -46.2 (c 1, chloroform); 1H NMR (CDCl3, 200
MHz) δ 3.14 (s, 3H, SO2CH3), 3.90 (d, 2H, 2H-5), 4.49 (d, 1H,
CHPh, J ) 11.8 Hz), 4.57 (t, 1H, H-4, J 4,5 ) 6.5 Hz), 4.76 (d,
1H, CHPh), 4.82 (s, 1H, H-2), 4.99 (s, 1H, H-1), 7.32 (m, 5H,
Ph); 13C NMR (CDCl3, 50 MHz) δ 17.91, 18.22 (tBu), 40.19
(CH3SO2), 60.39 (C-5), 70.14 (OCH2Ph), 80.91, 84.52 (C-2, C-4),
82.25 (C-3), 107.49 (C-1), 113.77 (CN), 128.04 (CH-Ar), 136.76
(C-Ar). Anal. (C26H45NO7SSi2) C, H, N, S.
F F dv
∫
T
U
STU
)
F2 dv
F2 dv
∫
x
∫
x
T
U
where FT and FU are the electron densities of molecules T and
U, respectively. The numerator is a measure of the charge
density for the two molecules, and the denominator is a
normalization factor so that the function takes a range of
values from 0 to 1, where 1 indicates identical electron
densities. Calculated indices range from +1.0 (perfect similar-
ity) to -1.0.
The similarity index was calculated by numerical integra-
tion of the MEPs over a three-dimensional grid of points
separated by 0.25 Å and extending 10 Å beyond the bounds of
the molecules. The MEPs were computed at each grid point
(r) outside the van der Waals surface of the molecules by
means of the following equation:
n
qi
MEP(r) )
∑
i)1 r - ri
The slowest moving fractions afforded 25 (0.19 g, 15%) as a
syrup: [R]D +5 (c 1, chloroform); H NMR (CDCl3, 200 MHz)
δ 3.12 (s, 3H, SO2CH3), 3.95 (dd, 2H, 2H-5), 4.00 (d, 1H, CHPh,
J ) 11.9 Hz), 4.53 (d, 1H, H-2), 4.56 (d, 1H, CHPh), 4.65 (m,
1H, H-4), 5.02 (d, 1H, H-1, J 1,2 ) 4.2 Hz), 7.35 (m, 5H, Ph).
Anal. (C26H45NO7SSi2) C, H, N, S.
where qi is the point charge on atom i, ri is the position of
atom i, and n is the number of charges in the molecule.
3. Au tom a ted Dock in g P r oced u r e. To produce a rela-
tively unbiased docking of both the thymine ring of TSAO-T
and the abasic thymine analogue present in compound 10b,
the Monte Carlo simulated annealing technique implemented
in AutoDock50 was used. A reduced molecular model of the
protein containing the binding site for nonnucleoside inhibitors
was obtained from the X-ray coordinates of HIV-1 RT in its
complex with nevirapine.44 Rapid energy evaluation of each
configuration explored was achieved by precalculating atomic
affinity potentials for carbon, oxygen, nitrogen, and hydrogen
atoms in the nonnucleoside binding site using a three-
dimensional grid51 centered on Leu-A100. An additional grid
of electrostatic potential was calculated using a Poisson-
Boltzmann finite difference method, as described below.
4. Con tin u u m Electr osta tics Ca lcu la tion s. Finite dif-
ference solutions to the linearized Poisson-Boltzmann equa-
tion,52 as implemented in the DelPhi module of Insight-II,45
were used to calculate the electrostatic potential in and around
the nonnucleoside binding site of HIV-1 RT. Dielectric con-
stant values assigned were 80.0 for the solvent (0.145 M) and
4.0 for the protein. The dielectric boundary was positioned
at the solvent-accessible molecular surface,53 calculated with
a spherical probe of 1.4-Å radius, and a 2-Å thick ion exclusion
layer was considered. Van der Waals parameters and point
charges were taken from the AMBER54 database.55 To improve
the accuracy of the calculated electrostatic potentials in the
binding site, focusing56 was done in three consecutive steps.
A first cubic lattice was defined with 1.0-Å spacing and
dimensions of 82 × 84 × 80 Å3 centered on the nonnucleoside
binding pocket so as to leave a minimum separation of 35 Å
between any protein atom and the borders of the box. The
potentials at the grid points delimiting the first box were
calculated analytically by treating each protein atom as a
Debye-Hu¨ckel sphere.42 A smaller (52 × 54 × 51 Å3) and finer
grid (0.75-Å spacing) was then defined, the boundary potentials
of which were linearly interpolated from those calculated in
the previous run. This procedure was repeated in order to
obtain the final grid (31 × 33 × 30 Å3) with 0.5-Å spacing.
Biologica l Meth od s. 1. Cells a n d Vir u ses. MT-4 cells
were kindly provided by Dr. N. Yamamoto (Tokyo, J apan);
CEM cells were obtained from the American Tissue Culture
1
[Ben zyl 2,5-Bis-O-(ter t-b u t yld im et h ylsilyl)-â-D-r ib o-
fu r a n osid e]-3-sp ir o-5′-(4′-a m in o-1′,2′-oxa t h iole 2′,2′-Di-
oxid e) (27). To a solution of 25 (0.1 g, 0.17 mmol) in dry
acetonitrile (5 mL) was added DBU (0.029 mL, 0.19 mmol).
The mixture was stirred at room temperature for 2 h, neutral-
ized (acetic acid), and evaporated to dryness. The residue was
purified by CCTLC on the chromatotron (hexane-ethyl ac-
etate, 8:1) to give 27 (0.05 g, 50%) as a syrup: [R]D -61.6 (c 1,
1
chloroform); H NMR (CDCl3, 200 MHz) δ 3.80 (m, 3H, H-4,
2H-5), 4.30 (d, 1H, H-2), 4.57 (d, 1H, CHPh, J ) 11.3 Hz), 4.81
(d, 1H, CHPh), 5.08 (d, 1H, H-1, J 1,2 ) 3.8 Hz), 5.49 (bs, 2H,
NH2-4′), 5.50 (s, 1H, H-3′), 7.24 (m, 5H, Ph); 13C NMR (CDCl3,
50 MHz) δ 25.98 (tBu), 65.11 (C-5), 72.00 (CH2Ph) 76.61, 83.98,
92.39 (C-2, C-4, C-3′), 106.22 (C-1), 128.06 (CH-Ph), 136.61
(C-Ph), 151.03 (C-4′). Anal. (C26H45NO7SSi2) C, H, N.
[Ben zyl 2,5-Bis-O-(ter t-b u t yld im et h ylsilyl)-â-D-xylo-
fu r a n osid e]-3-sp ir o-5′-(4′-a m in o-1′,2′-oxa t h iole 2′,2′-Di-
oxid e) (28). Following the method described for the synthesis
of 27, cyanomesyl derivative 26 (0.1 g, 0.17 mmol) was treated
with DBU for 2 h. After the workup, the residue was purified
by CCTLC on the chromatotron (hexane-ethyl acetate, 8:1)
to give 28 (0.07 g, 70%) as a syrup: [R]D -8.8 (c 1, chloroform);
1H NMR (CDCl3, 200 MHz) δ 3.87 (d, 2H, H-5), 4.47 (d, 1H,
H-2), 4.58 (m, 2H, H-4, CHPh, J 4,5 ) 4.5 Hz), 4.78 (d, 1H,
CHPh, J ) 11.9 Hz), 5.06 (d, 1H, H-1, J 1,2 ) 1.7 Hz), 5.58 (s,
1H, H-3′), 6.05 (bs, 1H, NH2-4′), 7.35 (m, 5H, Ph); 13C NMR
(CDCl3, 50 MHz) δ 25.66, 25.84 (tBu), 62.01 (C-5), 69.63 (CH2-
Ph), 83.42, 83.92 (C-2, C-4), 91.66 (C-3), 91.93 (C-3′), 107.27
(C-1), 128.21 (CH-Ph), 136.62 (C-Ph), 152.81 (C-4′). Anal.
(C26H45NO7SSi2) C, H, N.
Mod elin g Meth od s. 1. Ab In itio Ca lcu la tion s. Both
N1-methylthymine and the urea moiety present in 10b in four
alternative conformations were model-built in Insight-II.45
Each molecule was fully optimized with the ab initio quantum
mechanical program Gaussian 9446 using the 3-21G* basis set.
Atom-centered charges were then calculated on the optimized
geometries employing a 6-31G* basis set.47

4646 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Vela´zquez et al.
(6) De Clercq, E. HIV-1 specific RT inhibitors: Highly selective
inhibitors of human immunodeficiency virus type 1 that are
specifically targeted at the viral reverse transcriptase. Med. Res.
Rev. 1993, 13, 229-258.
(7) De Clercq, E. Nonnucleoside reverse transcriptase inhibitors
(NNRTIs) for the treatment of human immunodeficiency virus
type 1 (HIV-1) infections: strategies to overcome drug resistance
development. Med. Res. Rev. 1996, 16, 125-157.
(8) J acobo-Molina, A.; Ding, J .; Nanni, R. G.; Clark, A. D., J r.; Lu,
X.; Tantillo, C.; Williams, R. L.; Kamer, G.; Ferris, A. L.; Clark,
P.; Hizi, A.; Hughes, S. H.; Arnold, E. Crystal structure of human
immunodeficiency virus type 1 reverse transcriptase complexed
with double-stranded DNA at 3.0 Å resolution shows bent DNA.
Proc. Natl. Acad Sci. U.S.A. 1993, 90, 6320-6324. (b) Tantillo,
C.; Ding, J .; J acobo-Molina, A.; Nanni, R. G.; Boyer, P. L.;
Hughes, S. H.; Pauwels, R.; Andries, K.; J anssen, P. A. J .;
Arnold, E. Locations of anti-AIDS drug binding sites and
resistance mutations in the three-dimensional structure of HIV-1
reverse transcriptase. Implications for mechanisms of drug
inhibition and resistance. J . Mol. Biol. 1994, 243, 369-387.
(9) Nanni, R. G.; Ding, J .; J acobo-Molina, A.; Hughes, S. H.; Arnold,
E. Review of HIV-1 reverse transcriptase three-dimensional
structure: Implications for drug design. Perspect. Drug Discov.
Des. 1993, 1, 129-150.
Collection (Rockville, MD). HIV-1(IIIB) was originally obtained
from the culture supernatant of persistently HIV-1-infected
H9 cells and was provided by Dr. R. C. Gallo and Dr. M.
Popovic (National Institutes of Health, Bethesda, MD). HIV-
2(ROD) was a kind gift of Dr. L. Montagnier (Pasteur Institute,
Paris, France).
2. Sen sitivity of Sever a l HIV-1 Mu ta n t Str a in s to
Va r iou s Test Com p ou n d s in CEM a n d MT-4 Cells. CEM
cells were suspended at 250 000 cells/mL of culture medium
and infected with HIV-1(IIIB) or HIV-2(ROD) at 100 times the
50% cell culture infective dose (CCID50) (1 CCID50 being the
dose infective for 50% of the cell cultures)/mL of cell suspen-
sion. Then, 100 µL of the infected CEM cell suspension was
added to 200-µL microtiter plate wells containing 100 µL of
an appropriate dilution of the test compounds. After 4 days
of incubation at 37 °C, the CEM cell cultures were examined
for syncytium formation. The EC50 was determined as the
compound concentration required to inhibit syncytium forma-
tion by 50%. The CC50 was determined as the compound
concentration required to inhibit CEM cell proliferation by
50%.
MT-4 cells (5 × 105 cells/mL) were suspended in fresh
culture medium and infected with HIV-1 or HIV-2 at 100 times
the 50% CCID50/mL of cell suspension. Then, 100 µL of the
infected cell suspension was transferred to microtiter plate
wells, mixed with 100 µL of the appropriate dilutions of the
TSAO derivatives, and further incubated at 37 °C. After 5
days the number of viable cells was determined in a blood-
cell-counting chamber by Trypan blue staining for both virus-
infected and mock-infected cell cultures. The 50% effective
(10) Smerdon, S. J .; J a¨ger, J .; Wang, J .; Kohlstaedt, L. A.; Chirino,
A. J .; Friedman, J . M.; Rice, P. A.; Steitz, T. A. Structure of the
binding site for nonnucleoside inhibitors of the reverse tran-
scriptase of human immunodeficiency virus type 1. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 3911-3915.
(11) Balzarini, J .; Pe´rez-Pe´rez, M. J .; San-Fe´lix, A.; Schols, D.; Perno,
C. F.; Vandamme, A. M.; Camarasa, M. J .; De Clercq, E. 2′,5′-
Bis-O-(tert-butyldimethylsilyl)-3′-spiro-5′′-(4′′-amino-1′′,2′′-ox-
athiole-2′′,2′′-dioxide)pyrimidine (TSAO) nucleoside analogues:
Highly selective inhibitors of human immunodeficiency virus
type 1 that are targeted at the viral reverse transcriptase. Proc.
Natl. Acad. Sci. U.S.A. 1992, 89, 4392-4396. (b) Camarasa, M.
J .; Pe´rez-Pe´rez, M. J .; San-Fe´lix, A.; Balzarini, J .; De Clercq, E.
3′-Spironucleosides (TSAO derivatives), a new class of specific
human immunodeficiency virus type 1 inhibitors: Synthesis and
antiviral activity of 3′-spiro-5′′-[4′′-amino-1′′,2′′-oxathiole-2′′,2′′-
dioxide]pyrimidine nucleosides. J . Med. Chem. 1992, 35, 2721-
2727.
(12) Camarasa, M. J .; Pe´rez-Pe´rez, M. J .; Vela´zquez, S.; San-Fe´lix,
A.; Alvarez, A.; 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) Balzarini, J .;
Camarasa, M. J .; Karlsson, A. TSAO derivatives: Highly specific
human immunodeficiency virus type 1 (HIV-1) reverse tran-
scriptase inhibitors. Drugs Future 1993, 18, 1043-1055.
(13) J onckheere, H.; Taymans, J . M.; Balzarini, J .; Vela´zquez, S.;
Camarasa, M. J .; Desmyter, J .; De Clercq, E.; Anne´, J . Resis-
tance of HIV-1 reverse transcriptase against [2′,5′-bis-O-(tert-
butyldimethylsilyl)-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-
dioxide)] (TSAO) derivatives is determined by the mutation
Glu138 f Lys on the p51 subunit. J . Biol. Chem. 1994, 269,
25255-25258.
(14) 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 transcriptase
(RT) to HIV-1-specific RT inhibitors. Biochem. Biophys. Res.
Commun. 1994, 201, 1305-1312.
(15) Boyer, P. L.; Ding, J .; Arnold, E.; Hughes, S. H. Subunit
specificity of mutations that confer resistance to nonnucleoside
inhibitors in human immunodeficiency virus type 1 reverse
transcriptase. Antimicrob. Agents Chemother. 1994, 38, 1909-
1914.
(16) Balzarini, 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-(t-butyldimethylsilyl)-3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-
2′′,2′′-dioxide) thymine (TSAO-T). J . Biol. Chem. 1992, 267,
11831-11838.
(17) 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, 507-509.
concentration (EC50) and 50% cytotoxic concentration (CC50
)
were defined as the compound concentrations required to
reduce by 50% the number of viable cells in the virus-infected
and mock-infected cell cultures, respectively.
3. RT En zym e Assa y. The RT assays contained in a total
reaction mixture volume (50 µL) 50 mM Tris‚HCl (pH 7.8), 5
mM dithiothreitol, 300 mM glutathione, 500 µM EDTA, 150
mM KCl, 5 mM MgCl2, 1.25 µg of bovine serum albumin,
appropriate concentrations of labeled substrate [8-3H]dGTP
(specific radioactivity 15.6 Ci/mmol) (Moravek Biochemicals,
Brea, CA), a fixed concentration of the template/primer
poly(C)‚oligo(dG)12-18 (0.1 mM), 0.06% Triton X-100, 10 µL of
inhibitor at various concentrations, and 1 µL of the RT
preparation. The reaction mixtures were incubated at 37 °C
for 15 min, at which time 100 µL of calf thymus DNA (150
µg/mL), 2 mL of Na4P2O7 (0.1 M in 1 M HCl), and 2 mL of
trichloroacetic acid (10% v/v) were added. The solutions were
kept on ice for 30 min, after which the acid-insoluble material
was washed and analyzed for radioactivity. In the experi-
ments where the IC50 values of the test compounds were
determined with respect to [8-3H]dGTP, a fixed concentration
of the natural substrate [8-3H]dGTP (2.5 µM) was used.
Ack n ow led gm en t. We thank Francisco Caballero
for editorial assistance and Lizette van Berckelaer and
Ann Absillis for excellent technical assistance. This
research was supported in part by grants from the
Spanish CICYT (Project SAF97-0048-C02-01), the NATO
Collaborative Research (Grant No. CRG 920777), and
the Biomedical Research Programme of the European
Commission (Project BMH4-CT97-2161).
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