First synthesis and evaluation of the inhibitory effects of Aza analogues of TSAO on HIV-1 replication

Article abstract of DOI:10.1021/jm050091g

Aza TSAO-T derivatives bearing a dihydroisothiazole dioxide ring instead of an oxathiole dioxide ring at the C-3′ position on the sugar moiety were prepared. We have synthesized four families of compounds depending on substitution at both N-3 and N-2″. Bi

Full text of DOI:10.1021/jm050091g

4276
J. Med. Chem. 2005, 48, 4276-4284
First Synthesis and Evaluation of the Inhibitory Effects of Aza Analogues of
TSAO on HIV-1 Replication
Albert Nguyen Van Nhien,† Cyrille Tomassi,† Christophe Len,† Jose´ Luis Marco-Contelles,‡ Jan Balzarini,§
Christophe Pannecouque,§ Erik De Clercq,§ and Denis Postel*,†
Laboratoire des Glucides (FRE 2779), Faculte´ des Sciences, Universite´ de Picardie Jules Verne, 33 rue Saint Leu, 80039
Amiens, France, Laboratorio de Radicales Libres, Instituto de Qu´ımica Orga´nica General, CSIC, C/Juan de la Cierva, 3;
28006-Madrid, Spain, and Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received January 31, 2005
Aza TSAO-T derivatives bearing a dihydroisothiazole dioxide ring instead of an oxathiole dioxide
ring at the C-3′ position on the sugar moiety were prepared. We have synthesized four families
of compounds depending on substitution at both N-3 and N-2′′. Biological evaluation showed
that these compounds are HIV-1(IIIB)-specific and potent reverse transcriptase inhibitors with
EC50 values between 0.13 and 3.5 µM in cell culture.
Introduction
when they are incorporated into the viral DNA because
they lack a 3′-OH group. NNRTIs represent a particular
group of compounds which bind to a hydrophobic pocket
near, but not at, the polymerase active site. These
induce restrictions in the dynamics of the enzyme,
resulting in its being in an inactive conformation.
Despite substantial advances in anti-HIV chemo-
therapy, notably with new treatment combinations,
numerous mutations have been observed for therapies
using NRTIs and NNRTIs. For these reasons, the search
for new drugs is a priority in medicinal chemistry.
[2′,5′-Bis-O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-
3′-spiro-5′′-(4′′-amino-1′′,2′′-oxathiole-2′′,2′′-dioxide)thy-
mine (TSAO-T) was first described in 1992 by Camarasa
et al.1 and Pe´rez-Pe´rez et al.1 as a specific anti-HIV
agent (Figure 1). This compound was the prototype of a
new and unique class of nucleoside analogues with a
specific inhibition against HIV-1, acting through a
noncompetitive mechanism against substrate and tem-
plate/primer (for an overview, see ref 1c). In contrast,
no inhibitory effect was observed on HIV-2 strains,
simian immunodeficiency virus (SIV), Moloney murine
sarcoma virus, and a broad range of DNA and RNA
viruses at subtoxic concentrations. With the aim of
establishing structure-activity relationship studies
(SAR), Camarasa et al. synthesized various congeners
of TSAO compounds based on a variety of modifications
to the silyl groups, nucleic acid base, and the sultone
moiety. In addition, various heterodimers were synthe-
sized where TSAO compounds were linked to NRTIs
through various types of spacer groups, in an attempt
to combine the inhibitory properties of both types of
compounds.2 The resulting SAR studies, by Camarasa
and co-workers demonstrated that the sugar part played
a crucial role in the interaction of the TSAO series of
compounds with HIV-1 RT. Of particular importance
was the presence of the SO2 and 4′′-NH2 groups on the
3′-spiro sultone. Moreover, the oxathiole ring was shown
to be a requirement on the sugar moiety with a ribo
configuration and the tert-butyldimethylsilyl (TBDMS)
group at both O-2′ and O-5′ positions (Figure 2). Recent
molecular modeling studies have indicated that the
methyl group of the N-3 methylated TSAO-T (TSAO-
Among the various viral human diseases, the acquired
immune deficiency syndrome (AIDS) has been one of the
major targets for the World Health Organization and
the scientific community for the next decades. Since the
identification of the human immunodeficiency virus
(HIV) in 1983-84 as the etiological agent for AIDS,
many therapeutic strategies have been proposed involv-
ing the viral enzymes that have critical roles in the life
cycle of the virus as key targets in the search for
effective drugs. Accordingly, different types of HIV
inhibitors have been developed and studied with the aim
of limiting the introduction of the virus into the host-
cell (virus adsorption, virus-cell fusion, and virus
uncoating) and inhibition of enzymes such as reverse
transcriptase (RT) which is involved in the proviral DNA
synthesis from genomic viral RNA, HIV protease, and
HIV integrase (IN) which catalyzes the integration of
viral DNA into the host DNA. The finding that dideoxy-
nucleosides, such as ddC (2′,3′-dideoxycytidine), ddI
(2′,3′-dideoxyinosine), and AZT (3′-azido-3′-deoxythymi-
dine) are potentially effective therapeutic agents for the
treatment of the AIDS has triggered explosive develop-
ments in the chemistry of the RT inhibitors. This
heterodimeric enzyme consisting of p66 (560 amino
acids) and p51 (440 amino acids) subunits has three
enzymatic activities, a DNA-dependent and a RNA-
dependent DNA polymerase activity and RNase-H
activity, which play a direct role in the conversion of a
single-stranded RNA genome into a double-stranded
DNA. Because of the essential role of RT in viral
replication, the polymerase activity had been a major
target for anti-HIV drugs. Anti-RT drugs have been
categorized as either nucleoside analogue RT inhibitors
(NRTIs), nucleotide RT inhibitors (NtRTIs), or non-
nucleoside RT inhibitors (NNRTIs). NRTIs and NtRTIs
are analogues of natural nucleosides involved in viral
DNA synthesis. They also act as DNA chain terminators
* Corresponding author: Tel. +33 3 22 82 75 70; fax +3 33 22 82
75 68; e-mail: denis.postel@sc.u-picardie.fr.
† Universite´ de Picardie Jules Verne.
‡ Instituto de Qu´ımica Orga´nica General, CSIC.
§ Katholieke Universiteit Leuven.
10.1021/jm050091g CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/26/2005

Aza Analogues of TSAO
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4277
Chart 1. Key Intermediates for the Access to ATSAOs
Figure 1. TSAO derivatives.
TSAO compounds, abbreviated as ATSAOs. Herein we
report, in full, our studies on the synthetic routes for
an easy access to ATSAOs with free, partially, or fully
substituted N-3 and N-2′′ starting from D-xylose.
Results and Discussion
Retrosynthetic analysis demonstrates that glyco-R-
aminonitrile formation, N-glycosylation, and carbanion-
mediated sulfonamide intramolecular cyclization are
key steps in the synthesis of ATSAO nucleosides (Chart
1).
Several glycoaminocyanation routes have been pub-
lished. Most of them consist of catalytic (asymmetric)
Strecker-type reactions using a Lewis acid as catalyst
and various cyanogen reagents.7a,b Enantioselective
approaches to the reaction generally use a preformed
imine whereby the nitrogen atom bears a chiral inducer
or chiral metallic complex as catalyst. However, few
examples have been reported for the synthesis of R-ami-
nonitriles of sugars and rarely for uloses involving a
nonanomeric carbon atom of a sugar ring at C-R.
At the outset of our project we selected derivatives C
(Chart 1), where the choice of 5-O-benzyl and 5-O-
benzoyl protecting groups was made on the basis that
such groups are well-known to be readily cleaved under
mild conditions to facilitate introduction of the final
TBDMS group on the O-5′. Such intermediates can be
derived from the substrates D, which are readily avail-
able from either D-ribose or D-xylose (Chart 1).
Classical Strecker conditions applied to the protected
erythro-pentofuranos-3-ulose derivative 18a,b and 1a,9a,b
respectively, which were each obtained from D-xylose
using either PDC or a modified Swern oxidation, af-
forded the corresponding cyanohydrin. Several reaction
conditions were examined, but the best results were
obtained using NH3-MeOH and Ti(OiPr)4 as the Lewis
acid. The 3-R glyco-R-aminonitriles 2 and 2a were
obtained stereoselectively in 70% and 82% yield, re-
spectively. It should be noted that these conditions
represent a convenient and versatile method for the
formation of a large variety of N-substituted glyco-R-
aminonitriles from either alkyl- or arylamines. More-
over, Ti(OiPr)4 is a mild catalyst compatible with a large
variety of acid-sensitive functional groups such as
lactams, tert-butyldimethylsilyl ethers, and acetonides.
Figure 2. Aza analogues of TSAO.
m3T) is relatively hydrophobic which induces a confor-
mation with severely reduced flexibility which is pre-
shaped to the geometry of its putative binding site,
namely, the interface between the p66 and p51 subunits
of RT.3 In comparison with the other NRTIs and
NNRTIs, resistance of HIV-1 RT to TSAO type com-
pounds has also been observed, mainly due to the 138-
GlufLys mutation. The phenomenon of resistance and
eagerness for complete elucidation of the precise binding
mode of drug to RT has prompted the exploration of
further structural modifications of TSAO analogues.
Among more than 600 analogues described by Cama-
rasa and co-workers, the 3′-spiro ring moiety of such
compounds has been most studied and modified by
introduction of various substituents at C-3′′ (nitro,
halogen, alkenyl, alkynyl, allyl, and aromatic groups)
and N-4′′ (carbonyl, carboxylic acid, and ester). Ana-
logues modified at the 3′-spiro moiety including the xylo
and ribo form of 3′-spiro 4-amino-2-oxazolone and
4-amino-1,2,3-oxathiole-2,2-dioxide have also been syn-
thesized. Surprisingly, no derivatives have been inves-
tigated in which the O-1′′ has been substituted by a
nitrogen to give the corresponding [2′,5′-bis-O-(tert-
butyldimethylsilyl)-â-D-ribofuranosyl]-3′-spiro-3′′-(4′′-
amino-1′′,2′′-dihydroisothiazole-1′′,1′′-dioxide)thymine
analogues (Figure 2).
Our interest in carbohydrate chemistry, especially
that pertaining to glyco-R-aminonitriles4 and their
derivatives,5 encouraged us to extend our results ob-
tained as part of our investigations into the carbanion-
mediated sulfonamide intramolecular cyclization (CSIC
reaction)6 for the synthesis of the new family of aza-

4278 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Nguyen Van Nhien et al.
Chart 2. NMR Shift of Sulfite Derivatives (δ)
Scheme 1a
a Reagents and conditions: (a) i. Ti(OiPr)4 (1.2 equiv), NH3,
MeOH; ii. TMSCN (1.1 equiv); (b) CH3SO2Cl (3 equiv), C5H5N,
DMAP (0.5 equiv).
Scheme 2a
Scheme 3a
a Reagents and conditions: (a) TFA;H2O (9:1); (b) Ac2O, C5H5N;
(c) SOIm2, THF.
a Reagents and conditions: (a) i: Thymine, HMDS, (NH4)2SO4,
135 °C; ii: MeOH, H2O, reflux.
The compounds 2 and 2a failed to react readily with
RSO2Cl-pyridine alone; however, upon addition of
DMAP the key methanesulfonamidonitriles 3 and 3a
were each obtained in good yields (Scheme 1).
literature10 and their formation was found to be de-
pendent upon the reaction temperature. In our case,
several and various experimental conditions seemed to
correlate this formation with the endo/exo ratio result-
ing in the cyclic sulfite synthesis. 8ar could also be
explained by anchimeric assistance of the benzoyl group.
To control the stereospecificity of the N-glycosylation,
our next investigations will only concern the 5-O-benzyl
derivatives.
The tert-butyldimethylsilyl group was introduced in
the 2′- position using TBDMSCl and imidazole in DMF
to afford 9 in 89% (Scheme 4). Unfortunately, the CSIC
reaction6 of compound 9, using LDA base, gave the
dihydroisothiazolic derivative 10 in only 17% yield
(Scheme 4). The poor yields obtained could be due to
lithiation of the nucleic base. Therefore, to avoid such
a side reaction, the N-3 of 9 was protected and isolated
as the N-Boc derivative 11 in 88% yield (Scheme 4).
Next, the dihydroisothiazolic derivative 12 was obtained
efficiently using LDA in THF in 67% yield.
To achieve the N-glycosylation step for the introduc-
tion of the nucleic base, compounds 3 and 3a were each
deprotected in the anomeric position under classical
acidic conditions (TFA:H2O; 9:1) to give 4 and 4a, which
were converted into the corresponding diacetylated
derivatives 5 and 5a in 58% and 53%, respectively
(Scheme 2). According to the TSAO synthesis reported
by Camarasa, several glycosylations were attempted
using the Vorbru¨ggen method to introduce the thymine
moiety. However, it was demonstrated that such a
methodology was ineffective for free sulfonamido de-
rivatives 5 and 5a. As a alternative approach, we chose
to investigate the fusion procedure10 based on the cyclic
sulfite formation between C-1 and C-2 to effect substitu-
tion at the anomeric position. Thus, treatment of 4 and
4a with SO(Im)2 in THF (from thionyl chloride and
imidazole) gave the corresponding sulfite derivatives 6
and 6a (80 and 90%) as an endo and exo mixtures (endo/
exo: 1/1 to 3/2), which were characterized by 1H and 13
NMR spectroscopy (Chart 2).
C
Several routes were followed to deprotect both O-5′
and N-3′ positions (Scheme 5).
N-Glycosylation of 6 and 6a was achieved at 125 °C
using silylated thymine in dry conditions (Scheme 3) to
give a mixture of 2′-O-silylated (7, 7a) and 2′-hydroxy-
5-methyluridine derivatives (8, 8aâ) which, in turn, gave
after desilylation (TBAF; MeOH) exclusively 8 (82%)
and 8aâ (54.5%), respectively. In addition, the R N-
glycosylated epimer 8ar was obtained as the minor
compound (13.5%) starting from the 5-O-benzoylated
compound 6a. The formation of R-substituted com-
pounds via cyclic sulfites have been described in the
First, 12 was treated with Pd(OH)2-cyclohexene in
refluxing EtOH to give the debenzylated compounds 15
and 16, the latter being the result of a decarbamoylation
at N-3. Moreover, we also observed the polycyclic
compounds 13 and 14 which resulted from a Michael
intramolecular cyclization between the O-5′ and the
corresponding enamine. Finally, introduction of the 5′-
O-TBDMS group was achieved by treatment of the
mixture of compounds 13-16 with TBDMSCl in DMF
and imidazole as base to afford a mixture of 2′,5′-bis-

Aza Analogues of TSAO
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4279
Scheme 4a
Scheme 6a
a Reagents and conditions: (a) TBDMSCl, imidazole, DMF; (b)
Boc2O, CH2Cl2, C5H5N; (c) LDA, THF, -78 °C.
a Reagents and conditions: (a) CH3I, K2CO3, acetone; (b)
Cs2CO3, acetonitrile, 80 °C; (c). Pd(OH)2, cyclohexene, EtOH,
reflux; (d) TBDMSCl, imidazole, DMF, rt.
Scheme 5a
Scheme 7a
a Reagents and conditions: (a) CH3I or C2H5I, K2CO3, acetone.
best resulted in 20, being obtained in 55% yield using
sonochemistry in MeCN as solvent. As previously
reported, hydrogenolysis (Pd(OH)2-cyclohexene; reflux-
ing EtOH) gave a mixture of protected (21) and unpro-
tected (22) N-3 hemiaminal derivatives in 40% and 26%
yield, respectively. Finally, 23 and 24 were each ob-
tained by treatment with TBDMSCl and imidazole in
DMF from the isolated compounds 21 and 22, in 68%
and 71%, respectively (Scheme 6).
a Reagents and conditions: (a) Pd(OH)2, cyclohexene, EtOH,
Reflux; (b) TBDMSCl, imidazole, DMF, rt; (c) TFA, CH2Cl2, rt.
Access to the N-3 Methylated and Free N-2′′-
ATSAO-T Derivatives. Synthesis of the aza TSAO-
m3T analogues was investigated by a shorter route
which consisted of treating ATSAO-T derivative 18 with
the classical methylation reagents, MeI and K2CO3 in
acetone. Several attempts were made using various
amounts of MeI (1 to 3 equiv) but in each case a
regioselective introduction of the Me group occurred at
the N-3 position. This regiospecificity might be ex-
plained by the steric effect of the TBDMS group at O-2′
and the restricted conformation of the spiro ring leading
to an unfavorable orientation of the N-2′′ for effective
alkylation. Thus an efficient synthesis of N-3-alkylated
ATSAO derivatives was effected yielding the compounds
25 (87%) and 26 (81%), which are the aza analogues of
TSAO-m3T and TSAO-et3T, respectively (Scheme 7).
O-(tert-butyldimethylsilyl)-â-D-ribofuranosyl]-3′-spiro-
3′′-(4′′-amino-2′′,3′′-dihydroisothiazole-1′′,1′′-dioxide)-
thymine (18), which is the first aza analogue of TSAO-
T, and the corresponding N-3-Boc derivative 17, which
were separated by silica gel column chromatography
and obtained in 35.5% and 11.5% yield, respectively.
Compound 18 was also be obtained selectively from 12
in three steps involving acidic hydrolysis with TFA in
CH2Cl2, to give 10 (80%), followed by hydrogenolysis to
give a mixture of compounds 14 and 16 (80%) and finally
silylation to give the product 18 in 74% yield.
Access to the Deprotected N-3- and N-2′′-Methy-
lated ATSAO-T Derivatives. Compound 11 was the
most appropriate choice of intermediate to proceed to
the N-2′′-methylated ATSAO derivative (19), which was
achieved using MeI and K2CO3 in acetone with 95%
yield. The cyclization step was performed using Cs2CO3
in accordance with our earlier work on N-alkylated glyco
derivatives.11 Among the several attempts made, the
Access to the N-3- and N-2′′-Methylated AT-
SAO-T Derivatives. The lack of reactivity observed at
N-2′′ of 18 toward methylating reagents encouraged us
to investigate the synthesis of derivatives ATSAO

4280 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Nguyen Van Nhien et al.
Scheme 8a
strated HIV-1 specific reverse transcriptase inhibitory
activity. Moreover, ATSAO-Boc3T with the unsubsti-
tuted isothiazolic ring proved to be only 2- to 7-fold less
active against HIV-1 replication in MT-4 and CEM cells
than TSAO-T. Computational modeling studies are now
in progress and will be reported in due course.
Experimental Section
Materials and Methods. Melting points were determined
on a digital melting-point apparatus (Electrothermal) and are
uncorrected. Optical rotations were recorded in CHCl3, MeOH,
acetone, or DMSO with a digital polarimeter DIP-370 (JASCO)
using a 1 dm cell. 1H NMR and 13C NMR spectra were recorded
in CDCl3, acetone-d6, Me2SO-d6, or MeOD-d3 (internal Me4-
Si), respectively, at 300.13 MHz and at 75.47 MHz (Bruker
Avance-300). TLC was performed on Silica F254 (Merck) and
detection by UV light at 254 nm or by charring with phospho-
molybdic acid-H2SO4 reagent. Column chromatography was
effected on Silica Gel 60 (Merck, 230 mesh). Acetone, hexane,
ethyl acetate, and diethyl ether were distilled before use. Bases
and solvents were used as supplied. MeOH-NH3 is methanol
saturated (7 N) with ammonia gas at room temperature.
Elemental analyses have been carried out in Madrid (IQOG,
CSIC). 13C NMR resonances have been assigned by using
standard NMR (DEPT, COSY, HSQC) experiments. FTIR
spectra were obtained on a AVATAR 320 neat using ATR and
are reported in cm-1. Mass spectral data were acquiered on a
WATERS Micromass ZQ spectrometer or a WATERS Micro-
mass Q-TOFF spectrometer.
a Reagents and conditions: (a) Mel, K2CO3, acetonitrile; (b)
Cs2CO3, acetonitrile; (c) Pd(OH)2, cyclohexene, EtOH, reflux; (d)
TBDMSCl, imidazole, DMF, rt.
having both N-3 and N-2′′ methylated starting from the
acyclic methanesulfonamido derivative 9.
Compound 9 was permethylated using MeI and K2-
CO3 in acetonitrile to give 27 in 87% yield. The depro-
tection step was achieved using hydrogenolysis to give
the corresponding hemiaminal 29 in 50% yield. Finally,
the silylation step, performed as described earlier,
afforded the compound 30 in 74% (Scheme 8).
General Method for Silylations (A). To a solution of
nucleoside and imidazole (3 equiv) in DMF was added TBDM-
SCl (2.5 equiv). The reaction mixture was stirred at room-
temperature overnight then evaporated to dryness. The resi-
due was purified by flash chromatography.
Biological Activity. Several aza derivatives of TSAO-
m3T (30), TSAO-T (18 and 24), and two ATSAO-Boc3T
derivatives (17 and 23) were evaluated for their inhibi-
tory activity against HIV-1(IIIB) and HIV-2(ROD) in
human T-Lymphocyte MT-4 and CEM cell cultures
(Table 1). Those ATSAO derivatives that did not contain
a substituent (methyl) on the nitrogen atom in the spiro
moiety were most inhibitory to HIV-1 in CEM and MT-4
cell cultures (EC50: 0.13-0.53 µM). They were not
inhibitory against HIV-2 at subtoxic concentrations. The
methyl-substituted ATSAO derivatives (23, 24, 30) were
∼10-fold less inhibitory against HIV-1 than the unsub-
stituted ATSAO derivatives and also lacked any inhibi-
tory activity against HIV-2. The ATSAO-T derivatives
and also the ATSAO-Boc3T derivatives were cytostatic
at a CC50 that ranged between 19 and 105 µM. The
ATSAO-m3T and ATSAO-e3T derivatives 25 and 26
were more cytotoxic (CC50: 4.9-5.8 µM). Compound 30
(the only ATSAO-m3T derivative of the series having a
methyl group at both N-3 and N-2′′) lacked significant
cytostatic activity at 200-250 µM. The ATSAO deriva-
tives showed the same HIV-1-specific activity spectrum
as the TSAO derivatives published before.1a,b TSAO
derivatives were found to be markedly less inhibitory
against HIV-1 strains that contain NNRTI-specific
mutations in the RT such as Lys103 f Asn, Glu138 f
Lys, and Tyr181 f Cys.13,14 None of the ATSAO deriva-
tives were inhibitory against these mutant virus strains
in CEM cell culture (Table 1), pointing to a similar
mechanism of antiviral action as that of the prototype
TSAO-T and TSAO-m3T derivatives.
General Method for Debenzylations (B). To a solution
of spiranic derivative in absolute ethanol was added Pd(OH)2/C
(0.3 equiv) and cyclohexene (34 equiv). The reaction mixture
was refluxed then filtered and evaporated to dryness. The
residue was purified by flash chromatography or used in the
next step without further purification.
General Method for the N-Alkylation of Sulfonamides
(C). To a solution of sulfonamide and K2CO3 (1.5 equiv) in
acetone was added MeI or EtI (2 equiv). The mixture was
refluxed until complete reaction then filtered through a silica
pad and evaporated to dryness. The residue was purified by
flash chromatography.
General Method for the CSIC Reaction Using Cs2CO3
(D). Cs2CO3 (1 equiv) was added to a solution of the sulfon-
amidonitrile in CH3CN. The mixture was refluxed until
complete reaction and then was filtered through Celite and
evaporated to dryness. The residue was purified by flash
chromatography.
3-Amino-5-O-benzyl-3-C-cyano-3-deoxy-N-mesyl-D-ri-
bofuranose (4). A 10 mL mixture of TFA and water (9/1; v/v)
was added to the compound 3 (1.0 g, 2.92 mmol). The reaction
mixture was stirred at room temperature for 2 h. Then, the
solvent was evaporated to dryness and the residue was purified
by flash chromatography (EtOAc/petroleum ether, 70:30) to
yield compound 4 (0.85 g, 95%) as a mixture of the two
anomeric epimers: IR (ATR) ν 3255, 1425, 1379, 1316, 1138,
929, 777, 750, 701 cm-1; MS (ES): 365.4 [M + Na]+. Anal.
(C14H18N2O6S) C, H, N, S.
3-Amino-5-O-benzoyl-3-C-cyano-3-deoxy-N-mesyl-D-ri-
bofuranose (4a). a 10 mL mixture of TFA and water (9/1;
v/v) was added to the compound 3a (4.048 g, 10.2 mmol). The
reaction mixture was stirred at room temperature for 2 h.
Then, the solvent was evaporated to dryness and the residue
was purified by flash chromatography (EtOAc/petroleum ether,
70:30) to yield compound 4a (3.607 g, 99%) as a mixture of
the two anomeric epimers: IR (ATR) ν 1706, 1451, 1394, 1339,
1276, 1133, 1065, 983, 713, 666 cm-1; MS (ES): 379.5 [M +
Na]+. Anal. (C14H16N2O7S) C, H, N, S.
In conclusion, we have synthezised a new family of
aza analogues of TSAO with diverse substitution on
both N-3 and N-2′′ positions. Some of them demon-

Aza Analogues of TSAO
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4281
Table 1. Inhibitory Activity of Test Compounds against HIV-1 and HIV-2 in CEM and MT-4 Cell Cultures
EC50a (µM)
group in
CEM
MT-4
HIV-1
(IIIB)
CC50b (µM)
HIV-2
compound N-2′′ N-3 HIV-1 (IIIB) Lys103f Asn Glu138f Lys Tyr181f Cys (ROD)
HIV-2
(ROD)
CEM
MT-4
>200
30
CH3 CH3 2.5 ( 0.7
CH3 Boc
>250
>10
>250
>10
>250
>10
>250 0.66 ( 0.1 >200 g250
23
3.5 ( 0.7
0.33 ( 0.11
3.5 ( 0.7
>10 1.8 ( 0.71 >20 20.6 ( 1.9 20 ( 1.3
g10 0.53 ( 0.04 >100 19.1 ( 0.4 105 ( 6.9
>10 1.6 ( 0.40 >20 21.6 ( 0.14 19 ( 0.71
5.8 ( 0.1
18
H
H
H
5.5 ( 0.7
g10
>10
g10
24
CH3
H
>10
>10
25
CH3 0.22 ( 0.0
C2H5 0.31 ( 0.13
Boc
H
4.0 ( 2.8
>2
5.0 ( 0.0
0.4
>10
g10
26
H
>2
>2
4.9 ( 0.1
17
H
0.43 ( 0.25
0.06 ( 0.01
>10
g10
>10 0.13 ( 0.03 21.2 ( 1.3 19 ( 0.73
>4 0.06 ( 0.03 >20 16 ( 2.0 14 ( 2.0
TSAO-T (1)c
-
4.6 ( 2.6
3.0 ( 0.0
a 50% Effective concentration. b 50% Cytostatic concentration. c Data taken from refs 12, 13.
1,2-Di-O-acetyl-3-amino-5-O-benzyl-3-C-cyano-3-deoxy-
3-N-mesyl-D-ribofuranose (5). A 125 mL mixture of pyridine
and Ac2O (4:1; v/v) was added to the compound 4 (0.95 g, 2.4
mmol) at 0 °C. The reaction mixture was stirred overnight at
room temperature. Then, 50 mL of MeOH was added and
stirred for an additional 1 h. The solvent was evaporated to
dryness, and the residue was purified by flash chromatography
(EtOAc/petroleum ether, 35:65) to give successively 5â (1.03
g, 18%) as a pale yellow solid and 5r (2.22 g, 40%) as a white
solid. 5r: mp 40-41 °C; [R]25D +67.88 (c 0.99, CHCl3); IR (ATR)
hexamethyldisilazane (12 mL) was refluxed overnight. The
excess of HMDS was removed under reduced pressure and
then added to compound 6 (0.46 g, 1.36 mmol). The reaction
mixture was heated at 125 °C. After 2 h, MeOH (10 mL) was
added and stirred at room temperature for 10 min, followed
by addition of water (1 mL), stirred at 65 °C for 5 min, and
filtered through Celite to afford a mixture of silylated deriva-
tives 7 and 8. The solvent was evaporated under reduced
pressure and the crude product solubilized with THF (10 mL)
and TBAF‚3H2O (514 mg, 1.63 mmol) for 30 min and filtered
through Celite. After flash chromatography (EtOAc/petroleum
ether, 50:50), compound 8 (0.5 g, 82%) was isolated as a white
solid: mp 194-199 °C; [R]25D -5.68 (c 1.01, CH3OH); IR (ATR)
ν 1682, 1330, 1258, 1136, 978, 768, 699 cm-1; MS (ES): 473.3
[M + Na]+, 489.4 [M + K]+. Anal. (C19H22N4O7S) C, H, N, S.
1-(3′-Amino-5′-O-benzoyl-3′-C-cyano-3′-deoxy-3′-N-me-
syl-â-D-ribofuranosyl)thymine (8aâ). A solution of thymine
(0.37 g, 2.98 mmol) and ammonium sulfate (catalytic amount)
in hexamethyldisilazane (14 mL) was refluxed overnight. The
excess of HMDS was removed under reduced pressure and
then added to compound 6a (0.60 g, 1.49 mmol). The reaction
mixture was heated at 125 °C. After 2 h, MeOH (10 mL) was
added and stirred at room temperature for 10 min, followed
by addition of water (1 mL), stirred at 65 °C for 5 min, and
filtered through Celite to afford a mixture of silylated deriva-
tive 7a, 8ar, and 8aâ. The solvent was evaporated under
reduced pressure and the crude solubilized with THF (10 mL)
and TBAF‚3H2O (514 mg, 1.63 mmol) for 30 min and filtered
through Celite. After flash chromatography (EtOAc/petroleum
ether, 60:40), compound 8aâ (0.47 g, 68%) was isolated as a
ν 1759, 1375, 1329, 1208, 1045, 977, 933, 891, 738, 700 cm-1
;
MS (ES): 449.6 [M + Na]+, 465.6 [M + K]+. Anal. (C18H22N2O8S)
calcd, C, 50.70; H, 5.20; N, 6.57; S,7.52 found, C, 49.13; H,
5.16; N, 6.41; S, 7.42. 5â: mp 114-116 °C; [R]25 -11.96 (c
D
1.18, CHCl3); IR (ATR) ν 1768, 1730, 1341, 1248, 1203, 1155,
1099, 964, 891, 749, 703 cm-1; MS (ES): 449.6 [M + Na]+,
465.6 [M + K]+. Anal. (C18H22N2O8S) C, H, N, S.
1,2-Di-O-acetyl-3-amino-5-O-benzoyl-3-C-cyano-3-deoxy-
3-N-mesyl-D-ribofuranose (5a). A 27 mL mixture of pyridine
and Ac2O (3:1; v/v) was added to the compound 4a (0.95 g, 2.4
mmol) at 0 °C. The reaction mixture was stirred overnight at
room temperature. Then, 20 mL of MeOH was added and
stirred for an additional 1 h. The solvent was evaporated to
dryness, and the residue was purified by flash chromatography
(EtOAc/petroleum ether, 40:60) to give successively 5aâ (0.11
g, 10%) and 5ar (0.47 g, 43%) as white solids. 5aâ: mp 60-
62 °C; [R]25 +19.2° (c 1.25, CHCl3); IR (ATR) ν 1760, 1728,
D
1372, 1336, 1207, 1098, 972, 889, 713 cm-1; MS (ES): 463.5
[M + Na]+, 479.5 [M + K]+. Anal. (C18H20N2O9S) C, H, N, S.
5ar: mp 68-70 °C; [R]25 +70.06 (c 4.66, CHCl3); IR (ATR) ν
D
1764, 1725, 1333, 1272, 1228, 1108, 972, 893, 712 cm-1; MS
(ES): 463.5 [M + Na]+, 479.5 [M + K]+. Anal. (C18H20N2O9S)
C, H, N, S.
white solid: mp 170-175 °C; [R]25 +8.7 (c 1.21, CHCl3); IR
D
(ATR) ν 1701, 1383, 1331, 1272, 1136, 977, 786, 713 cm-1; MS
(ES): 487 [M + Na]+, 503 [M + K]+. Anal. (C19H20N4O8S) C,
H, N, S.
3-Amino-5-O-benzyl-3-C-cyano-3-deoxy-3-N-mesyl-1,2-
O-sulfinyl-r-D-ribofuranose (6). To a solution of imidazole
(2.13 g, 31.36 mmol) in THF (15 mL) was added SOCl2 (0.57
mL, 7.84 mmol) at 0 °C. After 1 h, the reaction mixture was
filtered and additionned to a solution of compound 4 (1.34 g,
3.92 mmol) in THF (15 mL) at -15 °C and stirred for 45 min.
The solvent was evaporated to dryness, and the residue was
purified by flash chromatography (EtOAc/petroleum ether, 50:
50) to give 6 (1.42 g, 93%) as an endo/exo mixture which was
used in the next step without further purification. exo-6: mp
1-(3′-Amino-5′-O-benzyl-2′-O-tert-butyldimethylsilyl-
3′-C-cyano-3′-deoxy-3′-N-mesyl-â-D-ribofuranosyl)thy-
mine (9). Following the general method (A), imidazole (0.17
g, 2.46 mmol) and TBDMSCl (0.31 g, 2.05 mmol) were added
to the compound 8 (0.31 g, 2.05 mmol) in DMF (15 mL)
overnight. After flash chromatography (EtOAc/petroleum ether,
40:60), compound 9 (0.36 g, 89%) was isolated as a white
solid: mp 189-191 °C; [R]25D -10.82 (c 1.44, CHCl3); IR (ATR)
ν 1694, 1382, 1259, 1134, 842, 779 cm-1; MS (ES): 587.3 [M
+ Na]+, 603.2 [M + K]+. Anal. (C25H36N4O7SSi) C, H, N, S.
[1-[5′-O-Benzyl-2′-O-tert-butyldimethylsilyl-â-D-ribo-
furanosyl]thymine]-3′-spiro-3′′-(2′′-H-4′′-amino-2′′,3′′-di-
hydro-1′′,1′′-dioxo-isothiazole) (10). Method 1. To a solu-
tion of LDA (freshly prepared from 0.76 mmol of nBuLi and
0.78 mmol of diisopropylamine) in THF (2.5 mL) was added
at -78 °C a solution of sulfonamidonitrile 9 (106 mg, 0.19
mmol) in dry THF (1.5 mL). After 1 h, water (5 mL) was added
and the reaction mixture slightly acidified with aqueous HCl
and extracted with EtOAc. The extracts were dried (Na2SO4)
and evaporated. The residue was purified by flash chroma-
tography (EtOAc/petroleum ether, 50:50) to give compound 10
(17 mg, 16%) as a white solid: mp 139-141 °C; [R]25D -8.3 (c
0.44, CHCl3); IR (ATR) ν 1690, 1641, 1464, 1378, 1278, 1255,
1129, 843, 775, 739 cm-1; MS (ES): 587.20 [M + Na]+, 603.17
58-60 °C; [R]25 +3.1 (c 1.33, CHCl3); endo-6: mp 127-129
D
°C; [R]25 +72.5 (c 1.50, CHCl3).
D
3-Amino-5-O-benzoyl-3-C-cyano-3-deoxy-3-N-mesyl-1,2-
O-sulfinyl-r-D-ribofuranose (6a). To a solution of imidazole
(1.27 g, 18.64 mmol) in THF (10 mL) was added SOCl2 (0.34
mL, 4.66 mmol) at 0 °C. After 1 h, the reaction mixture was
filtered and added to a solution of compound 4a (0.83 g, 2.33
mmol) in THF (10 mL) at -15 °C and stirred for 45 min. The
solvent was evaporated to dryness, and the residue was
purified by flash chromatography (EtOAc/petroleum ether, 50:
50) to give 6a (0.78 g, 83%) as an endo/exo mixture which was
used in the next step without further purification.
1-(3′-Amino-5′-O-benzyl-3′-C-cyano-3′-deoxy-3′-N-mesyl-
â-D-ribofuranosyl)thymine (8). A solution of thymine (0.33
g, 2.72 mmol) and ammonium sulfate (catalytic amount) in

4282 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Nguyen Van Nhien et al.
[M + K]+. Anal. (C25H36N4O7SSi) calcd. C, 53.17; H, 6.43; N,
purified by flash chromatography (EtOAc/petroleum ether, 40:
60) to give product 20 (20 mg, 32%) as a white solid: mp 121-
122 °C; [R]25D +15 (c 0.2, acetone); IR (ATR) ν 1797, 1715, 1671,
1458, 1370, 1249, 1140, 833 cm-1; MS (ES): 679.34 [M + 1]+,
701.24 [M + Na]+, 717.23 [M + K]+. Anal. (C31H46N4O9SSi) C,
H, N, S.
Method 2. Compound 19 (117 mg, 0.17 mmol) and Cs2CO3
(56 mg, 0.17 mmol) in CH3CN (50 mL) were subjected to
ultrasonic waves (10 s every 5 s at 60 °C) for 1 h 45 min. The
mixture was filtered and evaporated. After flash chromatog-
raphy, compound 20 (60 mg, 51%) was isolated.
9.92; S, 5.68, found: C, 52.65; H, 6.28; N, 8.99; S, 5.04.
Method 2. A 3 mL mixture of TFA and CH2Cl2 (2/3; v/v)
was added to compound 12 (68 mg, 0.1 mmol). The reaction
mixture was stirred at room temperature for 3 h 30 min. Then,
the solvent was evaporated to dryness, and the residue was
purified by flash chromatography (EtOAc/petroleum ether, 50:
50) to yield compound 10 (45 mg, 80%) as a white solid.
1-(3′-Amino-5′-O-benzyl-2′-O-tert-butyldimethylsilyl-3′-
C-cyano-3′-deoxy-3′-N-mesyl-â-D-ribofuranosyl)-3-N-tert-
butoxycarbonylthymine (11). A solution of compound 9
(2.79 g, 4.95 mmol) and Boc2O (2.16 g, 9.9 mmol) in 30 mL of
a (4:1) mixture of CH2Cl2 and pyridine was stirred at room
temperature for 8 h 30 min. The solvent was removed and the
residue flash chromatographed (EtOAc/petroleum ether, 20:
80) to give 11 (2.88 g, 88%) as a white solid: mp 88-90 °C;
[R]25D -1.3 (c 0.72, CHCl3); IR (ATR) ν 1786,1710, 1679, 1370,
1264, 1141, 841, 783 cm-1; MS (ES): 687.44 [M + Na]+. Anal.
(C30H44N4O9SSi) C, H, N, S.
[1-[5′-O-Benzyl-2′-O-tert-butyldimethylsilyl-â-D-ribo-
furanosyl]-3-N-tert-butoxycarbonylthymine]-3′-spiro-3′′-
(2′′-H-4′′-amino-2′′,3′′-dihydro-1′′,1′′-dioxo-isothiazole) (12).
To a solution of LDA (freshly prepared from 4.4 mmol of nBuLi
and 4.51 mmol of diisopropylamine) in THF (10 mL) was added
at -78 °C a solution of sulfonamidonitrile 11 (0.72 g, 1.1 mmol)
in dry THF (5 mL). After 1 h, water (5 mL) was added and
the reaction mixture slightly acidified with aqueous HCl and
extracted with EtOAc. The extracts were dried (Na2SO4) and
evaporated. The residue was purified by flash chromatography
(EtOAc/petroleum ether, 45:55) to give compound 12 (1.47 g,
67%) as a white solid: mp 145-146 °C; [R]25D -16.25 (c 0.61,
CHCl3); IR (ATR) ν 1786, 1715, 1666, 1370, 1255, 1140, 844,
644 cm-1; MS (ES): 665.35 [M + 1]+, 687.34 [M + Na]+, 703.25
[M + K]+. Anal. (C30H44N4O9SSi) C, H, N, S.
O5′,4′′-Cyclo-[1-[2′-O-tert-butyldimethylsilyl-â-D-ribo-
furanosyl]thymine]-3′-spiro-3′′-[4′′-amino-2′′-N-methyl-
1′′,1′′-dioxo-isothiazolidine] (22) and O5′,4′′-Cyclo-[1-[2′-
O-tert-butyldimethylsilyl-â-D-ribofuranosyl]-3-N-tert-
butoxycarbonylthymine]-3′-spiro-3′′-[4′′-amino-2′′-N-
methyl-1′′,1′′-dioxo-isothiazolidine] (21). Following the
general method (B), 20 (628 mg, 0.92 mmol), Pd(OH)2/C (170
mg, 0.23 mmol), and cyclohexene (2.11 mL, 31 mmol) in
absolute EtOH (10 mL) for 12 h gave successively, after flash
chromatography (EtOAc/petroleum ether, 45:55) compounds
21 (144 mg, 26%) and 22 (172 mg, 40%) as white solids. 21:
mp 126-128 °C; [R]25D +26 (c 0.15, acetone); IR (ATR) ν 1781,
1715, 1671, 1249, 1140, 778 cm-1; MS (ES): 589.33 [M + 1]+,
611.22 [M + Na]+. Anal. (C24H40N4O9SSi) C, H, N, S. 22: mp
108-109 °C; [R]25D +19 (c 0.1, acetone); IR (ATR) ν 1713, 1672,
1467, 1254, 1151, 836 cm-1; MS (ES): 511.17 [M + Na]+. Anal.
(C19H32N4O7SSi) C, H, N, S.
[1-[2′,5′-Bis-O-tert-butyldimethylsilyl-â-D-ribofurano-
syl]thymine]-3′-spiro-3′′-(4′′-amino-2′′,3′′-dihydro-2′′-N-
methyl-1′′,1′′-dioxo-isothiazole) (24). Following the general
method (A), imidazole (57 mg, 0.8 mmol) and TBDMSCl (106
mg, 0.70 mmol) were added to the compound 21 (137 mg, 0.28
mmol) in DMF (5 mL) for 15 h. After flash chromatography
(EtOAc/petroleum ether, 35:65), compound 24 (120 mg, 71%)
was isolated as a white solid: mp 167-168 °C; [R]25D +2.49 (c
0.7, CHCl3); IR (ATR) ν 1702, 1647, 1464, 1262,1140, 1047,
833 cm-1; MS (ES): 603.27 [M + 1]+, 625.25 [M + Na]+. Anal.
(C25H46N4O7SSi2) C, H, N, S.
[1-[2′,5′-Bis-O-tert-butyldimethylsilyl-â-D-ribofurano-
syl]-3-N-tert-butoxycarbonylthymine]-3′-spiro-3′′-(4′′-
amino-2′′,3′′-dihydro-2′′-N-methyl-1′′,1′′-dioxo-isothi-
azole) (23). Following the general method (A), imidazole (35
mg, 0.51 mmol) and TBDMSCl (64 mg, 0.42 mmol) were added
to the compound 22 (100 mg, 0.17 mmol) in DMF (5 mL) for
14 h. After flash chromatography (EtOAc/petroleum ether,
30:70), compound 23 (81 mg, 68%) was isolated as a white
solid: mp 146-148 °C; [R]25D -13.36 (c 0.87, CHCl3); IR (ATR)
ν 1786, 1715, 1671, 1260, 1140, 838, 773 cm-1; MS (ES): 703.32
[M + 1]+, 725.30 [M + Na]+. Anal. (C30H54N4O9SSi2) C, H, N,
S.
[1-[2′,5′-Bis-O-tert-butyldimethylsilyl-â-D-ribofurano-
syl]-3-N-methylthymine]-3′-spiro-3′′-(2′′-H-4′′-amino-2′′,3′′-
dihydro-1′′,1′′-dioxo-isothiazole) (25). Following the general
method (C), 18 (51 mg, 0.09 mmol), K2CO3 (6 mg, 0.045 mmol),
and MeI (0.011 mL, 0.18 mmol) in acetone (2 mL) for 6 h gave,
after flash chromatography (EtOAc/petroleum ether, 30:70),
product 25 (45 mg, 87%) as a white solid: mp 245-246 °C;
[R]25D +4.87 (c 0.5, CHCl3); IR (ATR) ν 2927, 2357, 1722, 1659,
1636, 1473, 1364, 1142, 839 cm-1; MS (ES): 603.4 [M + 1]+,
625.3 [M + Na]+, 641.2 [M + K]+. Anal. (C25H46N4O7SSi2) C,
H, N, S.
[1-[2′,5′-Bis-O-tert-butyldimethylsilyl-â-D-ribofurano-
syl]-3-N-tert-butoxycarbonylthymine]-3′-spiro-3′′-(2′′-H-
4′′-amino-2′′,3′′-dihydro-1′′,1′′-dioxo-isothiazole) (17) and
[1-[2′,5′-Bis-O-tert-butyldimethylsilyl-â-D-ribofuranosyl]-
thymine]-3′-spiro-3′′-(2′′-H-4′′-amino-2′′,3′′-dihydro-1′′,1′′-
dioxo-isothiazole) (18). Following the general method (B),
12 (468 mg, 0.7 mmol), Pd(OH)2/C (130 mg, 0.19 mmol), and
cyclohexene (1.62 mL, 24 mmol) in absolute EtOH (8 mL) were
refluxed for 14 h to give a complex mixture of compounds 13,
14, 15, and 16. The crude product was solubilized in DMF (5
mL), and TBDMSCl (264 mg, 1.75 mmol) and imidazole (143
mg, 2.1 mmol) were added and stirred at room temperature.
After 20 h, the solvent was evaporated and the residue
chromatographed (EtOAc/petroleum ether, 35:65) to give suc-
cessively 17 (55 mg, 11.5%) and 18 (146 mg, 35.5%) as white
solids. 17: mp 159-160 °C; [R]25 +18 (c 0.1, acetone); IR
D
(ATR) ν 2915, 1789,1721, 1681, 1371, 1256, 1146 839 cm-1
;
MS (ES): 711.29 [M + Na]+, 727.26 [M + K]+. Anal.
(C29H52N4O9SSi2) C, H, N, S. 18: mp 142-143 °C; [R]25 +16
D
(c 0.15, acetone); IR (ATR) ν 2967, 2891, 2356, 2338,1710, 1380,
1255, 1068, 840 cm-1; MS (ES): 589.25 [M + 1]+, 611.24 [M +
Na]+, 627.21 [M + K]+. Anal. (C24H44N4O7SSi2) C, H, N, S.
1-(3′-Amino-5′-O-benzyl-2′-O-tert-butyldimethylsilyl-3′-
C-cyano-3′-deoxy-3′-N-mesyl-3′-N-methyl-â-D-ribofurano-
syl)-3-N-tert-butoxycarbonylthymine (19). Following the
general method (C), 11 (82 mg, 0.12 mmol), K2CO3 (26 mg,
0.18 mmol), and MeI (0.015 mL, 0.24 mmol) in acetone (3 mL)
for 1 h 45 min gave, after flash chromatography (EtOAc/
petroleum ether, 20:80), product 19 (67 mg, 81%) as a white
[1-[2′,5′-Bis-O-tert-butyldimethylsilyl-â-D-ribofurano-
syl]-3-N-ethylthymine]-3′-spiro-3′′-(2′′-H-4′′-amino-2′′,3′′-
dihydro-1′′,1′′-dioxo-isothiazole) (26). Following the general
method (C), 18 (51 mg, 0.09 mmol), K2CO3 (6 mg, 0.045 mmol),
and EtI (0.014 mL, 0.18 mmol) in acetone (2 mL) for 7 h gave,
after flash chromatography (EtOAc/petroleum ether, 35:65),
product 26 (43 mg, 81%) as a white solid: mp 108-109 °C;
[R]25D +9 (c 0.065, acetone); IR (ATR) ν 2925, 1709, 1672, 1644,
1472, 1254, 1133, 836 cm-1; MS (ES): 617.5 [M + 1]+, 639.4
[M + Na]+, 655.4 [M + K]+. Anal. (C26H48N4O7SSi2) calcd. C,
50.62; H, 7.84; N, 9.08; S, 5.20, found: C, 51.09; H, 7.67; N,
8.71; S, 4.93.
solid: mp 67-69 °C; [R]25 +4.89 (c 0.63, CHCl3); IR (ATR) ν
D
1785, 1718, 1671, 1346, 1143, 841, 774 cm-1; MS (ES): 679.28
[M + 1]+, 701.27 [M + Na]+. Anal. (C31H46N4O9SSi) C, H, N,
S.
[1-[5′-O-Benzyl-2′-O-tert-butyldimethylsilyl-â-D-ribo-
furanosyl]-3-N-tert-butoxycarbonylthymine]-3′-spiro-3′′-
(4′′-amino-2′′,3′′-dihydro-2′′-N-methyl-1′′,1′′-dioxo-isothi-
azole) (20). Method 1. Following the general method (D),
Cs2CO3 (31 mg, 0.1 mmol) was added to a solution of 19 (64
mg, 0.1 mmol) in CH3CN. The reaction mixture was refluxed
for 5 h and then evaporated to dryness. The residue was

Aza Analogues of TSAO
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4283
1-(3′-Amino-5′-O-benzyl-2′-O-tert-butyldimethylsilyl-3′-
C-cyano-3′-deoxy-3′-N-mesyl-3′-N-methyl-â-D-ribofurano-
syl)-3-N-methylthymine (27). Following the general method
(C), 9 (0.2 g, 0.35 mmol), K2CO3 (0.03 g, 0.52 mmol), and MeI
(0.02 mL, 0.70 mmol) in acetone (5 mL) for 9 h gave, after
flash chromatography (EtOAc/petroleum ether, 35:65) product
iste`re Franc¸ais de la Recherche for financial support and
G. Mackenzie for helpful discussions. J.B., C.P., and
E.D.C. thank Mrs. Ann Absillis, Liesbet De Dier, and
Cindy Heens for excellent technical assistance. We also
thank the European Commission (QLK2-C.T.-2000-
00291) for financial support.
27 (0.18 g, 87%) as a white solid: mp 81-83 °C; [R]25 36 (c
D
0.1, acetone); IR (ATR) ν 1711, 1673, 1650, 1469, 1347, 1261,
1156, 1035, 840, 781, 658 cm-1; MS (ES): 615.4, [M + Na]+,
631.4 [M + K]+. Anal. (C27H40N4O7SSi) calcd. C, 54.71; H, 6.80;
N, 9.45; S, 4.74, found: C, 54.80; H, 6.68; N, 9.33; S, 5.31.
Supporting Information Available: 1H and 13C NMR
chemical shift assignments and elemental analysis data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
[1-[5′-O-Benzyl-2′-O-tert-butyldimethylsilyl-â-D-ribo-
furanosyl]-3-N-methylthymine]-3′-spiro-3′′-(4′′-amino-2′′,3′′-
dihydro-2′′-N-methyl-1′′,1′′-dioxo-isothiazole) (28). Fol-
lowing the general method (D), Cs2CO3 (0.54 g, 1.68 mmol)
was added to a solution of 27 (1.0 g, 1.68 mmol) in CH3CN (10
mL). The reaction mixture was refluxed for 15 h and then
evaporated to dryness. The residue was purified by flash
chromatography (EtOAc/petroleum ether, 30:70) to give prod-
uct 28 (0.30 g, 30%) as a slight yellow solid: mp 118-120 °C;
[R]25D +2.1 (c 2.51, CHCl3); IR (ATR) ν 1706, 1647, 1616, 1471,
1256, 1136, 1051, 838, 766 cm-1; MS (ES): 615.4 [M + Na]+,
631.3 [M + K]+. Anal. (C27H40N4O7SSi) C, H, N, S.
References
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furanosyl]-3-N-methylthymine]-3′-spiro-3′′-[4′′-amino-2′′-
N-methyl-1′′,1′′-dioxo-isothiazolidine] (29). Following the
general method (B), 28 (53 mg, 0.09 mmol), Pd(OH)2/C (14 mg,
0.02 mmol), and cyclohexene (0.2 mL, 2 mmol) in absolute
EtOH (1 mL) for 2 h 30 min gave, after flash chromatography
(EtOAc/petroleum ether, 60:40), product 29 (22 mg, 50%) as a
comprehensive story of
a unique family of HIV-1 specific
inhibitors of reverse transcriptase. Curr. Topics Med. Chem.
2004, 4, 945-963.
(2) Camarasa, M.-J.; San-Fe´lix, A.; Pe´rez-Pe´rez, M.-J.; Vela´zquez,
S.; Alvarez, R.; Chamorro, C.; Jimeno, 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. (b) Vela´zquez, S.; Lobaton,
E.; De Clercq, E.; Koontz, D. L.; Mellors, J. W.; Balzarini, J.;
Camarasa, M.-J. Hybrids of [TSAO-T]-[Foscarnet]: The First
Conjugate of Foscarnet with a Non-nucleoside Reverse Tran-
scriptase Inhibitor through a labile covalent ester bond. J. Med.
Chem. 2004, 47, 3418-3426.
(3) 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) (TSAO) derivatives at the p51-p66 interface of HIV-1
reverse transcriptase. J. Med. Chem. 2001, 44, 1853-1865.
(4) Postel, D.; Nguyen Van Nhien, A.; Pillon, M.; Villa, P.; Ronco,
G. Stereoselective synthesis of R-aminonitriles at nonanomeric
positions of monosaccharides. Tetrahedron Lett. 2000, 41, 6403-
6406.
white solid: mp 90-91 °C; [R]25 -14.2° (c 0.53, CHCl3); IR
D
(ATR) ν 1710, 1673, 1645, 1469, 1300, 1142, 841 cm-1
;
HRMS: calcd. for C20H35N4O7SSi [M + ] 503.2013; found
503.1996.
[1-[2′,5′-Bis-O-tert-butyldimethylsilyl-â-D-ribofurano-
syl]-3-N-methylthymine]-3′-spiro-3′′-(4′′-amino-2′′,3′′-di-
hydro-2′′-N-methyl-1′′,1′′-dioxo-isothiazole) (30). Following
the general method (A), imidazole (20 mg, 0.3 mmol) and
TBDMSCl (40 mg, 0.25 mmol) were added to the compound
29 (50 mg, 0.1 mmol) in DMF (4 mL) for 4 h. After flash
chromatography (EtOAc/petroleum ether, 30:70), compound 30
(40 mg, 65%) was isolated as a white solid: mp 126-127 °C;
[R]25D +14.5 (c 0.81, CHCl3); IR (ATR) ν 1702, 1644, 1621, 1471,
1255, 1138, 838, 785, 656 cm-1; MS (ES): 639.4 [M + Na]+.
Anal. (C26H48N4O7SSi2) calcd. C, 50.62; H, 7.84; N, 9.08; S, 5.20,
found: C, 50.61; H, 8.02; N, 8.26; S, 4.95.
Biological Methods. Human immunodeficiency virus type
1 [HIV-1(IIIB)] was obtained from Dr. R. C. Gallo (when at
the National Cancer Institute, Bethesda, MD). HIV-2(ROD)
was provided by Dr. L. Montagnier (when at the Pasteur
Institute, Paris, France). The selection and characterization
of the mutant HIV-1(IIIB) strains bearing the Lys103 f Asn,
Glu138 f Lys and Tyr181 f Cys mutations in their RT was
described previously.13
Anti-HIV Evaluation. 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. After 4
days, giant cell formation (CEM) or HIV-induced cytopathicity
(MT-4) was recorded microscopically (CEM) or by the MTT
method (MT-4) in the HIV-infected cell cultures.13 The 50%
effective concentration (EC50) and 50% cytotoxic concentration
(CC50) of the test compounds were defined as the compound
concentrations required to inhibit virus-induced cytopathicity
(CEM) or to reduce cell viability (MT-4) by 50%, or to reduce
by 50% the number of viable cells in mock-infected CEM and
MT-4 cell cultures, respectively.
(5) Postel, D.; Nguyen Van Nhien, A.; Villa, P.; Ronco, G. New
4-amino-5H-2,3-dihydroisothiazole 1,1-dioxides from sugar R-ami-
no nitriles using the CSIC reaction. Tetrahedron Lett. 2001, 42,
593-595.
(6) Postel, D.; Nguyen Van Nhien, A.; Marco, J. L. Chemistry of
sulfonate- and sulfonamide-stabilized carbanions - the CSIC
reactions. Eur. J. Org. Chem. 2003, 19, 3713-3726.
(7) Ishitani, H.; Komiyama, S.; Hasegawa, Y.; Kobayashi, S. Cata-
lytic, enantioselective synthesis of R-aminonitriles with a novel
zirconium catalyst. Ang. Chem., Int. Ed. 1998, 37, 3186-3188.
(b) Ishitani, H.; Komiyama, S.; Hasegawa, Y.; Kobayashi, S.
Catalytic asymmetric Strecker Synthesis. Preparation of enan-
tiomerically pure R-amino acid derivatives from aldimines and
tributyltin cyanide or achiral aldehydes, amines, and hydrogen
cyanide using a chiral zirconium catalyst. J. Am. Chem. Soc.
2000, 122, 762-766.
(8) Steiner, B.; Koo´s, M.; Langer, V.; Gyepesova´, D.; Smrcok, L.
4-Amino-4-cyano-4,6-dideoxy sugar derivatives from methyl
6-deoxy-2, 3-O-isopropylidene-R-L-lyxo-hexopyranosid-4-ulose via
Strecker-type reaction. Carbohydr. Res. 1998, 311, 1-9. (b) Len,
C.; Postel, D.; Mackenzie, G.; Villa, P.; Ronco, G. Spirohydantoins
from D-ribose as new potent enzymatic inhibitors. Pharm.
Pharmacol. Commun. 1999, 5, 165-168.
(9) Tong, G. L.; Lee, W. W.; Goodman, L. Synthesis of some 3′-O-
methyl purine ribonucleosides. J. Org. Chem. 1967, 32, 1984-
1986. (b) Maravcova, J.; Capkova, J.; Stanek, J. One-pot
synthesis of 1,2-O-isopropylidene-R-D-xylofuranose. Carbohydr.
Res. 1994, 263, 61-66.
(10) Gagnieu, C. H.; Guiller, A.; Pacheco, H. Synthesis, structure et
re´activite´ vis-a`-vis de nucle´ophiles du 1,2:3,4-disulfite de â-L-
arabinopyranose. Carbohydr. Res. 1988, 180, 223-231. (b)
Gagnieu, C. H.; Guiller, A.; Pacheco, H. Synthe`se de nucle´osides
Acknowledgment. C.T. thanks the Ministe`re
Franc¸ais de la Recherche (France) for a fellowship. D.P.
thanks the Conseil Re´gional de Picardie and the Min-

4284 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Nguyen Van Nhien et al.
pyrimidiques non prote´ge´s en O-2′ a` partir de sulfites cycliques
en C-1-C-2. Carbohydr. Res. 1988, 180, 233-242.
(12) Chamorro, C.; Pe´rez-Pe´rez, M.-J; Rodr´ıguez-Barrios, F.; Gago,
F.; De Clercq, E.; Balzarini, J.; San-Fe´lix, A. Synthesis and anti-
HIV evaluation of novel TSAO-T derivatives. Camarasa, M.-J.
Antiviral Res. 2001, 50, 207-222.
(13) Balzarini, J.; Karlsson, A.; Pe´rez-Pe´rez, M.-J.; Vrang, L.; Wal-
bers, J.; Zhang, H.; O¨ berg, B.; Vandamme, A.-M.; Camarasa, M.-
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show differential activity against HIV-1 mutant strains contain-
ing different amino acid substitutions in the reverse tran-
scriptase. Virology 1993, 192, 246-253.
(11) Dom´ınguez, L.; Nguyen Van Nhien, A.; Tomassi, C.; Len, C.;
Postel, D.; Marco-Contelles, J. Synthesis of 4-amino-5-H-2,3-
dihydroisothiazole-1,1-dioxide ring systems on sugar templates
via carbanion-mediated sulfonamide intramolecular cyclization
reactions (CSIC protocols) of glyco-alpha-sulfonamidonitriles. J.
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Tetrahedron 2004, 60 (21), 4709-4727.
JM050091G