Synthesis and anti-HIV1 biological activity of novel 5″-ATSAO compounds

Article abstract of DOI:10.1016/j.bmc.2008.02.026

Aza TSAO-T derivatives bearing a substituted dihydroisothiazole dioxide ring with a phenyl group at 5″ position were prepared. Biological evaluation showed that phenyl group gives rise to a dramatical decrease of the inhibitory effect.

Full text of DOI:10.1016/j.bmc.2008.02.026

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4733–4741
Synthesis and anti-HIV1 biological activity of novel
500-ATSAO compounds
a
a
b
c
´
Cyrille Tomassi, Albert Nguyen Van Nhien, Jose Marco-Contelles, Jan Balzarini,
Christophe Pannecouque,c Erik De Clercqc and Denis Postela,*
aLaboratoire des Glucides (UMR6219), Faculte´ des Sciences, Universite´ de Picardie Jules Verne, 33 rue Saint Leu,
80039 Amiens, France
bLaboratorio de Radicales Libres (Instituto de Quı´mica Orga´nica General, CSIC), C/Juan de la Cierva, 3, 28006 Madrid, Spain
cRega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 12 November 2007; revised 1 February 2008; accepted 8 February 2008
Available online 13 February 2008
Abstract—Aza TSAO-T derivatives bearing a substituted dihydroisothiazole dioxide ring with a phenyl group at 500 position were
prepared. Biological evaluation showed that phenyl group gives rise to a dramatical decrease of the inhibitory effect.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
The three contacts comprise an extensive dimer interface
that includes the fingers subdomain of p51 with the palm
of p66. In this sense, the b7–b8 loop in p51 subunit is
essential for catalytic function on the p66 subunit.3
Since the identification of the human immunodeficiency
virus (HIV) in 1983–1984 as the etiological agent for
AIDS, many therapeutic strategies have been proposed
involving the viral enzymes that have critical roles in
the life cycle of the virus as key targets in the search
for effective drugs.
Non-nucleosides reverse transcriptase inhibitors (NNR-
TIs) represent a particular group of compounds which
bind to a hydrophobic pocket near, but not at the poly-
merase active site of p66. These compounds induce
restrictions in the dynamics of the enzyme, resulting in
an inactive conformation. [20,50-Bis-O-(tert-butyldimeth-
ylsilyl)-b-D-ribofuranosyl]-30-spiro-500-(400-amino-100,200-
oxathiole-200,200-dioxide)thymine (TSAO-T) was firstly
described in 1992 by Camarasa et al.4 as a specific anti-
HIV agent (Fig. 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
Because of the essential role of reverse transcriptase
(RT) in the viral replication, this enzyme has been a
key target in the development of novel anti-HIV drugs.
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 plays a direct role in the conversion of
a single-stranded RNA genome into the double-
stranded proviral DNA. It has been recognized that
the RT p66/p51 heterodimer is the biologically active
form of the enzyme, while the monomeric subunits are
inactive.1 Structural analysis reveals three major con-
tacts between the p66 and p51 subunits, with most of
the interaction surfaces being largely hydrophobic.2
O
O
R
R
N
N
N
N
TBDMSO
H2N
TBDMSO
H2N
O
O
O
O
OTBDMS
OTBDMS
R1
O
O
N
O
S
S
O
O
ATSAO-T derivatives
R= H, Boc, CH3
R1= H, CH3
TSAO-T
, R= H
Keywords: Glycoaminonitrile; CSIC reaction; TSAO; ATSAO;
NNRTI.
TSAO-m3T
, R= CH3
*
Corresponding author. Tel.: +33 322827570; e-mail: denis.postel@u-
picardie.fr
Figure 1. Aza analogues of TSAO.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.026

4734
C. Tomassi et al. / Bioorg. Med. Chem. 16 (2008) 4733–4741
R1O
H2N
non-competitive mechanism against the substrate and
template/primer.4c Extensive structure–activity studies
have been carried out toassessthe structural requirements
for their optimal interaction with HIV-1 RT. The ribo su-
gar plays an essential role in the interaction with the en-
zyme and the simultaneous presence of a 30-spiro-500-(400-
O
T
ATSAO-T
derivatives
O
N
S
R2
O
O
A
amino-100,200-oxathiole-200,200-dioxide)
moiety
and
R1O
O
R1O
O
T
O
TBDMS groups at both the 20 and 50 positions of the su-
gar moiety is a prerequisite for antiviral activity.5 Modifi-
cations on the groups at 20 and 50 positions,6 on the 30-
spiro moiety,7 as well as on the base part,8 have been per-
formed and evaluated. In comparison with the NRTIs
and NNRTIs, resistance of HIV-1 RT to TSAO type of
compounds due to the Glu138 ! Lys mutation has been
observed, hence suggesting that they may interact with
Glu138 residue, located in the b7–b8 loop in p51. The phe-
nomenon of resistance and eagerness for complete eluci-
dation of the precise binding mode of drug to RT has
prompted the exploration of further structural modifica-
tions of TSAO analogues.
NC
NC
O
O
NH2
N
S
R2
O
O
C
(R1= Bz, Bn)
B
O
R1O
O
O
HO
HO
OH
OH
O
O
D-xylose or D-ribose
D
R1= Bn or Bz
R2= H or CH3
Chart 1. Key intermediates for the access to ATSAOs.
ysis for the synthesis of the new ATSAO nucleosides,
based on a simple synthetic sequence that includes: (i)
aminocyanation of the monosaccharidic substrate; (ii)
N-glycosylation to give the nucleosidic scaffold; and
(iii) the carbanion-mediated sulfonamide intramolecular
cyclization (CSIC reaction) to introduce the spiro-dihy-
droisothiazolic ring at the 30-position.12
Recently, Camarasa et al. reported the synthesis and
evaluation of several compounds bearing at the N-3 po-
sition a variety of polar, lipophilic or aromatic groups
linked to that position through flexible polymethylene
linkers. The TSAO derivative with
a
N-meth-
ylcarboxamidobutyl group at the N-3 position was 5-
to 6-fold more active than the TSAO-T.9
In our initial work, we selected derivatives C (Chart 1),
bearing a 5-O-benzyl and 5-O-benzoyl as protecting
groups, a choice based on such groups are well known
to be readily cleaved under mild conditions to facilitate
the incorporation of the TBDMS group at the oxygen at
C-50. Such intermediates can be derived from substrates
D, which are readily available from either D-ribose or D-
xylose (Chart 1). Starting from the 5-O-benzoyl deriva-
tive, the N-glycosylation step affords an epimeric mix-
ture of thymine derivatives resulting from the probable
anchimeric assistance of the benzoyl group. In order
to control the stereospecificity of the N-glycosylation,
our investigations targeted to accessing 500-phenyl deriv-
atives of ATSAO-T were successful starting from the 5-
O-benzyl derivatives.
Our interest in carbohydrate chemistry, especially that
pertaining to glyco-a-aminonitriles,10 and their deriva-
tives,11 encouraged us to extend our investigations into
the carbanion-mediated sulfonamide intramolecular
cyclizations (CSIC reaction)12 to the synthesis of a new
family of aza-TSAO compounds, abbreviated as ATS-
AOs. Recently, we reported the first synthesis of ATS-
AOs in which the 30-spiro-oxathiazole ring is
substituted with a spiro-isothiazole moiety (Fig. 1).13
The evaluation of the inhibitory effects on HIV-1 replica-
tion demonstrated that these ATSAO derivatives showed
the same HIV-1 specific activity spectrum as the TSAO
derivatives published by Camarasa et al.4 ATSAO deriv-
atives that did not contain a methyl on the nitrogen atom
in the spiro moiety were potent inhibitors of HIV-1 in
CEM and MT-4 cell cultures (EC50: 0.13–0.53 lM) but
showed no inhibition activity against HIV-2 at subtoxic
concentrations. The computational study revealed that
the ATSAO compounds adopt a similar conformation
at the p66–p51 interface than for the TSAO analogues.14
2.2. Synthesis of the 500-phenyl ATSAO nucleosides (13–
16, 20)
The 3-R glyco-a-aminonitrile 2 was obtained stereose-
lectively in 70%.10 Subsequent treatment with
BnSO2Cl–pyridine–DMAP gave the key benzylsulfo-
namidonitrile 3 in 98% yield (Scheme 1). Like the meth-
anesulfonamidonitrile derivatives,13 the introduction of
Continuing with our work in this area, and in order to
gain a deeper insight into the structure-activity relation-
ship of ATSAO derivatives, herein we report, in full, the
synthesis and HIV-inhibition of new 500-phenyl ATSAO-
T derivatives 13–20.
thymine or uracil using the N-glycosylation Vorbruggen
¨
method on the substrate 3 was inefficient. Therefore, we
investigated the fusion procedure,15 by using silylated
thymine. Thus, treatment of 4 with SO(Im)2 in THF,
prepared from thionyle chloride and imidazole, yielded
the corresponding sulfite derivatives 5 in 75% as an
endo/exo mixture, as was firmly established after de-
tailed inspection of the NMR spectroscopic data (Chart
2). Subsequent N-glycosylation at 125 ꢁC using silylated
base in dry conditions, gave the 20-O-silylated derivative
2. Results and discussion
2.1. Synthesis of the nucleosides
Based on the previous work from our laboratory,13 in
Chart 1 we have shown our planned retrosynthetic anal-

C. Tomassi et al. / Bioorg. Med. Chem. 16 (2008) 4733–4741
4735
Several routes were followed to deprotect both O-50 and
N-30 positions (Scheme 1).
OBn
OBn
O
OBn
O
O
O
O
O
a
c
b
NC
NC
O
O
NH
Bn
O
Unfortunately, mixtures of the debenzylated com-
pounds 11 and 12 were obtained from 10 with
Pd(OH)2-cyclohexene in reffluxing ethanol, in yields
ranging from [11 (80%), 12 (16%)] to [11 (9%), 12
(89%)], depending on the reaction time, after 2 h
40 min or after 17 h, respectively. As shown, in the best
reaction conditions, a small amount of the deprotected-
Boc nucleoside 12 was isolated, but we also found con-
ditions to obtain compound 12 as the major reaction
product, a fact that allowed us to prepare aldo 3-N-al-
kyl, substituted derivatives (see below). Contrary to
the alkylsulfonamide derivatives,13,16 no polycyclic
products were observed from Michael intramolecular
cyclization between the O-50 and the corresponding en-
amine. Finally, introduction of the 50-O-TBDMS phar-
macophore was achieved by treatment of the isolated
derivatives 11 and 12 with TBDMSCl in DMF and
imidazole to afford [1-(20,50-bis-O-tert-butyldimethylsi-
lyl-b-D-ribofuranosyl)-3-N-tert-butoxycarbonylthymine]-
30-spiro-300-(400-amino-200,300-dihydro-500-phenyl-100,100-dioxo-
isothiazole) (13) (80%) and [1-(20,50-bis-O-tert-butyldi-
methylsilyl-b-D-ribofuranosyl)]-30-spiro-300-(400-amino-200,
300-dihydro-500-phenyl-100,100-dioxo-isothiazole) (14) (74%),
respectively (Scheme 1).
NH2
O
O
S
O
1
2
3
O
OBn
OBn
NH
OBn
O
O
O
OH
N
e
O
g
O
NC
NC
d
S
O
NC
OH
O
NH
NH
Bn
O
Bn
O
S
S
OR
NH
Bn
O
O
S
O
O
5
4
6
7
R= TMS +
7
R= H
f
O
O
O
Boc
Boc
N
N
NH
OBn
O
OBn
OBn
O
N
N
N
O
O
O
O
h
H2N
Ph
i
NC
NC
OTBDMS
OTBDMS
NH
NH
OTBDMS
Bn
O
NH
Bn
O
S
S
S
O
O
O
O
10
9
8
j
O
O
Boc
H
N
N
N
N
RO
H2N
RO
H2N
O
O
O
O
+
The regiospecificity of the N-3 alkylation step as ob-
served previously13 was applied to the synthesis of 500-
phenyl aza TSAO-m3T and TSAO-et3T.
Ph
Ph
OTBDMS
OTBDMS
NH
O
NH
O
S
S
O
O
11 : R=H
13 : R=TBDMS (80%)
12
14
: R=H
: R=TBDMS (74%)
k
k
The introduction of the methyl and ethyl groups on 14
was achieved efficiently by a classical alkylation [MeI
(2 equiv) or EtI (2 equiv) and K2CO3 (0.5 equiv)] in
refluxing acetone to afford 15 (84%) and 16 (80%),
respectively (Scheme 2).
Scheme 1. Reagents and conditions: (a) i—Ti(OiPr)4 (1.2 equiv), NH3,
MeOH; (ii) TMSCN (1.1 equiv) (70%); (b) BnSO2Cl (3 equiv), py,
DMAP (0.5 equiv) (98%); (c) TFA/H2O (9:1) (95%); (d) SOIm2, THF
(75%); (e) i—thymine, HMDS, (NH4)2SO4, 135 ꢁC; ii—MeOH, H2O,
reflux; (f) TBAF, MeOH (71%, for two steps); (g) TBDMSCl,
imidazole, DMF (84%); (h) Boc2O, CH2Cl2, py (75%); (i) LDA,
THF, ꢀ78 ꢁC (76%); (j) Pd(OH)2, cyclohexene, EtOH, reflux (see the
text); (k) TBDMSCl, imidazole, DMF, rt.
The lack of reactivity previously observed in compounds
with free sulfonamido groups at N-200, towards methy-
lating agents,13 and the simultaneous one-pot CSIC
reaction which we have also observed during the N-ally-
lation, N-methylation and N-benzylation of monosac-
charidic substrates,16 encouraged us to investigate the
synthesis of 500-phenyl ATSAO derivatives having both
N-3 and N-200 methylated starting from the acyclic meth-
anesulfonamido derivative 7.
H1
BnO
H4
H1
BnO
H4
H2
O
H2
O
O
S
O
NC
O
NC
O
O
Bn(O)2SHN
endo
Bn(O)2SHN
S
exo
J1,2 endo (Hz) J1,2 exo (Hz)
4.6 3.6
O
C-1 endo C-1 exo H-1 endo H-1 ex
o
Compound 7 was permethylated using the system MeI/
K2CO3, in acetonitrile to give 17 in 65% yield after
111.0 108.6 6.52 6.63
Chart 2. NMR shift of sulfite derivative (d).
O
O
6 which upon treatment with TBAF in MeOH gave
exclusively 7 in 71% yield.
R
NH
N
N
N
TBDMSO
H2N
TBDMSO
H2N
O
O
O
O
a
The tert-butyldimethylsilyl group was introduced in the
20-position using TBDMSCl and imidazole in DMF to
afford 8 in 84% (Scheme 1). To avoid base lithiation dur-
ing the CSIC step, the N-3 of 8 was protected and iso-
lated as the N-Boc derivative 9 in 75% yield. Next, the
dihydroisothiazolic derivative 10 was obtained effi-
ciently using LDA in THF in 76% yield (Scheme 1).
Ph
Ph
OTBDMS
OTBDMS
NH
NH
S
S
O
O
O
O
14
15
R= Me (84%)
R= Et (80%)
16
Scheme 2. Reagents: (a) CH3I or C2H5I, K2CO3, acetone.

4736
C. Tomassi et al. / Bioorg. Med. Chem. 16 (2008) 4733–4741
O
O
4. Conclusion
Me
N
NH
In conclusion, various 500-substituted ATSAO have been
synthesized. Compounds 13–16, 20 have been evaluated
against HIV-1 and HIV-2 replication in CEM cells.
According to the biological results, introduction of a
phenyl group at the 500-position clearly decreased the
antiviral activity as previously described in the TSAO
series.17
OBn
OBn
N
N
O
O
O
O
b
a
NC
NC
Bn
OH
OTBDMS
O
N
NH
Bn
O
S
S
Me
O
O
O
7
17
O
Me
Me
N
N
N
OR
OBn
5. Experimental
N
O
O
O
O
H2N
Ph
c
H2N
Ph
5.1. Materials and methods
OTBDMS
Me
N
OTBDMS
Me
N
S
S
O
O
O
O
Melting points were determined on a digital melting-
point apparatus (Electrothermal) and are uncorrected.
Optical rotations were recorded in CHCl3, MeOH, ace-
tone, or DMSO with a digital polarimeter DIP-370
19 : R=H
20 : R=TBDMS
18
d
Scheme 3. Reagents and conditions: (a) CH3I (2 equiv), K2CO3 (1.5
equiv), acetone, reflux, 24 h (65%); (b) Cs2CO3, (1 equiv), CH3CN,
reflux (90%); (c) Pd(OH)2, cyclohexene, EtOH, reflux (99%); (d)
TBDMSCl, imidazole, DMF, rt (75%).
1
(JASCO) using a 1 dm cell. H NMR and 13C NMR
spectra were recorded in CDCl3, acetone-d6, Me2SO-
d6, or MeOD-d3 (internal Me4Si), respectively, at
300.13 MHz and at 75.47 MHz (Bruker Avance-300).
TLC was performed on Silica F254 (Merck) and detec-
tion by UV light at 254 nm or by charring with phos-
24 h (Scheme 3). Improvements in the cyclization step
from 17 using Cs2CO3 in refluxing acetonitrile resulted
in the formation of 18 in 90% yield. It was interesting
to note that, according to the results previously ob-
served for the one-pot-N-alkylation plus CSIC reaction
on phenylsulfonamido compounds,16a increasing the
reaction time to 72 h afforded the cyclic derivative 18
in 40% overall yield from precursor 7. It should be noted
that the CSIC reaction provided a higher yield for the
benzylsulfonamide derivative than for the methylated
derivative.13 Next, hydrogenolysis gave 19 quantita-
tively, which was silylated as described earlier, to afford
20 in 75% (Scheme 3).
phomolybdic
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. Elemen-
tal analyses have been carried out in Madrid (IQOG,
CSIC). 13C NMR resonances have been assigned by
using standard NMR (DEPT, COSY, HSQC) experi-
ments. FTIR spectra were obtained on a AVATARTM
320 neat using ATR and are reported in cmꢀ1. Mass
spectral data were acquired on a WATERS Micromass
ZQ spectrometer or a WATERS Micromass Q-TOFF
spectrometer.
3. Biological activity
5.1.1. 3-Amino-5-O-benzyl-3-C-cyano-3-deoxy-3-N-phen-
ylmethanesulfonyl-D-ribofuranose (4). TFA and water
The inhibitory activity against RT of HIV-1(IIIB) and
HIV-2(ROD) of compounds 13–16, 20 was evaluated
in CEM cell cultures (see Section 5). The results are
shown in Table 1.
(100 mL, 9/1; v/v) were added to compound
3
(15.5 g, 34 mmol). The reaction mixture was stirred
at room temperature for 1 h 30 min. Then, the solvent
was evaporated to dryness and the residue was puri-
fied by flash chromatography (EtOAc/petroleum ether,
35:65) to yield compound 4 (12 g, 84%) as a mixture
of the two anomeric epimers; IR (ATR) m 1695,
Table 1. Inhibitory activity of test compounds against HIV-1 and
HIV-2 in CEM cell cultures
1319, 1128, 1068, 696 cmꢀ1
;
1H NMR (CDCl3,
Compound
EC50 (lM)
CC50 (lM)
300 MHz) d 7.41–7.28 (m, OCH2C6H5, SO2CH2C6H5),
5.34 (d, 1H, J1,2 = 4.3 Hz, H-1b), 5.16 (s, 1H, H-1a),
HIV-1
HIV-2
13
14
>50
P2
>50
>50
>2
30 1.0
P2
>50
>50
>2
P10
P10
P10
P10
10.9 4.53
19.1 0.4
16 1.0
4.61–4.36
SO2CH2C6H5), 3.76 (m, H-5a,b); 13C NMR (CDCl3,
75 MHz) 131.6, 131.5 (2C, Cip), 129.5–128.4
(m,
H-2a,b,
H-4a,b,
OCH2C6H5,
15
16
d
20
ATSAO-Ta
TSAO-T
(OCH2C6H5, SO2CH2C6H5), 117.6, 117.4 (2 CN),
102.7 (C-1a), 96.1 (C-1b), 81.0 (C-4a), 82.1 (C-4b),
81.4 (C-2a), 76.3 (C-2b), 74.6, 74.3 (OCH2C6H5a,b),
70.2, 69.6 (C-5a,b), 60.9, 60.8 (C-3a,b), 60.3, 60.1
(SO2CH2C6H5a,b); MS (ES): 459.1 [M+1]+. Anal.
(C20H22N2O6S) C, H, N, S. Calcd C, 57.40; H, 5.30;
N, 6.69; S, 7.66. Found: C, 55.83; H, 5.44; N, 6.52;
S, 7.26.
0.33 0.11
0.06 0.010
>100
>4
EC50, effective concentration or concentration required to protect
CEM cells against the cytopathogenicity of HIV by 50%. CC50, cyto-
toxic concentration or concentration required to reduce CEM cell
viability by 50%.
a (Fig. 1, R = R1 = H).

C. Tomassi et al. / Bioorg. Med. Chem. 16 (2008) 4733–4741
4737
5.1.2. 3-Amino-5-O-benzyl-3-C-cyano-3-deoxy-3-N-phen-
ylmethanesulfonyl-1,2-O-sulfinyl-a-D-ribofuranose (5).
5.1.4. 1-(30-Amino-50-O-benzyl-20-O-tert-butyldimethylsi-
lyl-30-C-cyano-30-deoxy-30-N-phenylmethanesulfonyl-b-
D-ribofuranosyl)thymine (8). To a solution of 7 (3.66 g,
6.95 mmol) and imidazole (1.42 g, 20.85 mmol) in
DMF (55 mL) was added TBDMSCl (1.42 g,
20.85 mmol). The reaction mixture was stirred at room
temperature overnight then evaporated to dryness. After
flash chromatography (EtOAc/petroleum ether, 30:70)
To a solution of imidazole (3.58 g, 52.64 mmol) in
THF (35 mL) was added SOCl2 (0.85 mL,
13.16 mmol) at 0 ꢁC. After 1 h, the reaction mixture
was filtered and added to a solution of compound 4
(2.75 g, 6.58 mmol) in THF (30 mL) at ꢀ15 ꢁC and
stirred for 50 min. The solvent was evaporated to dry-
ness and the residue was purified by flash chromatog-
compound 8 (3.73 g, 84%) was isolated as a white solid:
25
D
raphy (EtOAc/petroleum ether, 30:70) to give
5
mp 148–151 ꢁC; ½aꢁ ꢀ25 (c 0.18, CHCl3); IR (ATR) m
(1.92 g, 63%, isolated as a mixture of endo/exo form
which are used in the next step without further purifi-
cation); IR (ATR) m 1456, 1353, 1199, 1095, 898,
1710, 1683, 1656, 1397, 1264, 1152, 1072, 840 cmꢀ1
;
1H NMR (CDCl3, 300 MHz) d 9.81 (s, 1H, NH), 7.37
0
0
(m, 11H, H-6, C6H5), 6.23 (d, J1 ,2 = 7.9 Hz, 1H, H-
10), 5.91 (s, 1H, NH), 4.70 (d, 1H, OCH2C6H5-A),
4.67 (d, 1H, H-20), 4.57 (d, 1H, SO2CH2C6H5-A), 4.48
(d, 1H, SO2CH2C6H5-B), 4.45 (d, 1H, OCH2C6H5-B),
4.02 (s, 1H, H-40), 3.78 (s, 2H, H50), 1.52 (s, 3H,
CH3), 0.78 (s, 9H, [SiC(CH3)3]), 0.1 (s, 3H, SiCH3),
ꢀ0.16 (s, 3H, SiCH3); 13C NMR (CDCl3, 75 MHz) d
163.8 (C-4), 151.4 (C-2), 137.2 (Cip), 135.0 (C-6),
131.4, 129.5, 129.1, 129.0 (9C, C6H5), 117.5 (CN),
113.1 (C-5), 85.1 (C-10), 83.5 (C-40), 78.1 (C-20), 74.8
(OCH2C6H5), 70.3 (C-50), 61.1 5 (SO2CH2C6H5), 59.6
(C-30), 25.7 ([SiC(CH3)3]), 18.0 ([SiC(CH3)3]), 12.3
(CH3), ꢀ4.2 (SiCH3), ꢀ4.6 (SiCH3); MS (ES): 663.4
[M+Na]+, 679.3 [M+K]+. Anal. (C31H40N4O7SSi) C,
H, N, S. Calcd C, 58.10; H, 6.29; N, 8.74; S, 5.00.
Found: C, 54.87; H, 5.84; N, 7.92; S, 4.62.
697 cmꢀ1 1H NMR (CDCl3, 300 MHz) d 7.42–7.24
;
(m, 20H, OCH2C6H5, SO2CH2C6H5), 6.63 (d,
J1,2 = 3.6 Hz, H-1exo), 6.52 (5 d, J1,2 = 4.6 Hz, H-1endo),
5.82 (d, 1H, H-2exo), 5.56 (d, 1H, H-2endo), 4.52 (m,
9H, H-4, OCH2C6H5, SO2CH2C6H5), 4.10 (m, 1H,
H-4), 3.91 (m, 4H, H-5); 13C NMR (CDCl3,
75 MHz)
d
136.5–128.1
(12C,
OCH2C6H5,
SO2CH2C6H5), 15.5, 115.1 (2CN), 110.9 (C-1endo),
108.6 (C-1exo), 88.1 (C-2endo), 84.7 (C-2exo), 78.8, 78.6
(2C-4), 74.7, 74.6 (2 OCH2C6H5), 68.2, 67.6 (2C-5),
63.2, 63.1 (2C-3), 61.3, 60.9 (2 SO2CH2C6H5); MS
(ES): 487.1 [M+Na]+.
5.1.3. 1-(30-Amino-50-O-benzyl-30-C-cyano-30-deoxy-30-
N-phenylmethanesulfonyl-b-D-ribofuranosyl)thymine (7).
A solution of thymine (1.08 g, 8.54 mmol) and ammo-
nium sulfate (catalytic amount) in hexamethyldisilaz-
ane (25 mL) was refluxed overnight. The excess of
HMDS was removed under reduced pressure then
added to compound 5 (1.98 g, 4.27 mmol). The reac-
tion 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), stir-
red at 65 ꢁC for 5 min and filtered through Celite to
afford a mixture of silylated derivative 6 and 7. The
solvent was evaporated under reduced pressure and
the crude solubilized with MeOH (50 mL) and
NH4FÆ3H2O (2 g, 54 mmol) for 2 h and filtered
through Celite. After flash chromatography (EtOAc/
5.1.5. 1-(30-Amino-50-O-benzyl-20-O-tert-butyldimethylsi-
lyl-30-C-cyano-30-deoxy-30-N-phenylmethanesulfonyl-b-
D-ribofuranosyl)-3-N-tert-butoxycarbonylthymine (9). A
solution of compound 8 (0.73 g, 1.15 mmol) and Boc2O
(0.53 g, 2.3 mmol) in 15 mL of a (4:1) mixture of CH2Cl2
and pyridine was stirred at room temperature for 15 h.
The solvent was removed and the residue flash chro-
matographed (EtOAc/petroleum ether, 25:75) to give 9
25
D
(0.64 g, 75%) as a white solid: mp 91–92 ꢁC; ½aꢁ ꢀ9 (c
0.13, acetone); IR (ATR) m 1792, 1723, 1674, 1455,
1371, 1268, 1134, 840 cmꢀ1
;
1H NMR (CDCl3,
300 MHz) d 7.40 (m, 11H, H-6, C6H5), 6.07 (d,
J1 ,2 = 7.7 Hz, 1H, H-10), 5.40 (s, 1H, NH), 4.71 (d,
JA,B = 9.9 Hz, 1H, OCH2C6H5-A), 4.63 (d, 1H, H-20),
4.50 (s, 2H, SO2CH2C6H5), 4.48 (d, 1H, OCH2C6H5-
0
0
petroleum ether, 40:60) compound 7 (1.6 g, 71%)
was isolated as a white solid: mp 131–132 ꢁC; ½aꢁ
25
D
B), 4.25 (s, 1H, H-40), 3.85 (dd, J4 ,5 A = 1.9 Hz,
0 0
ꢀ33 (c 0.17, acetone); IR (ATR) m 1690, 1667, 1644,
1472, 1384, 1244, 1142, 910, 752, 692 cmꢀ1
;
1H
J5 A,5 B = 11.2 Hz, 1H, H-50A), 3.79 (dd, J4 ,5 B = 1.3 Hz,
1H, H-50B), 1.57 (s, 9H, [OC(CH3)3]), 1.49 (s, 3H, CH3),
0.83 (s, 9H, [SiC(CH3)3]), 0.1 (s, 3H, SiCH3), ꢀ0.14 (s,
3H, SiCH3); 13C NMR (CDCl3, 75 MHz) d 161.2 (C-
4), 149.0 (C-2), 147.8 (CO), 137.1 (Cip), 134.4 (C-6),
131.4, 129.7, 129.6, 129.2, 129.0 (8C, C6H5), 128.0
(Cip), 117.5 (CN), 112.4 (C-5), 87.1 [OC(CH3)3], 85.6
(C-10), 83.4 (C-40), 78.6 (C-20), 74.7 (OCH2C6H5), 70.3
(C-50), 61.2 (SO2CH2C6H5), 59.6 (C-30), 27.8
[OC(CH3)3], 25.8 [SiC(CH3)3], 18.0 [SiC(CH3)3], 12.3
(CH3), ꢀ4.1 (SiCH3), ꢀ4.9 (SiCH3); MS (ES): 763.3
[M+Na]+, 779.3 [M+K]+. Anal. (C36H48N4O9SSi) C,
H, N, S. Calcd C, 58.36; H, 6.53; N, 7.56; S, 4.33.
Found: C, 57.93; H, 6.46; N, 7.28; S, 4.12.
0
0
0
0
NMR (CDCl3, 300 MHz) d 10.12 (br s, 1H, NH),
7.43 (m, 11H, H-6, 2C6H5), 6.50 (s, 1H, NH), 6.16
(d, J1 ,2 = 7.9 Hz, 1H, H-10), 4.88 (d, 1H, H-20), 4.80
(d, 1H, JA,B = 13.8 Hz, SO2CH2C6H5-A), 4.72 (d,
1H, JA,B = 10.8 Hz, OCH2C6H5-A), 4.67 (d, 1H,
SO2CH2C6H5-B), 4.62 (d, 1H, OCH2C6H5-B), 4.27
0
0
(t, 1H, H-40), 3.86 (dd, J4 ,5 A = 2.3 Hz, 1H, H50-A),
0
0
3.76 (dd, J4 ,5 B = 1.7 Hz, J5 A,5 B = 11.2 Hz, 1H, H50-
B), 1.46 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz)
d 163.4 (C-4), 151.2 (C-2), 138.0 (Cip), 135.1 (C-6),
131.6, 129.6, 128.9, 128.6, 128.5 (9C, C6H5), 117.9
(CN), 111.5 (C-5), 85.4 (C-10), 83.7 (C-40), 77.4 (C-
0
0
0
0
20),
74.1
(OCH2C6H5),
70.2
(C-50),
60.3
(SO2CH2C6H5), 59.6 (C-30), 11.7 (CH3); MS (ES):
549.1 [M+Na]+, 565.1 [M+K]+. Anal. (C25H26N4O7S)
C, H, N, S. Calcd C, 57.02; H, 4.98; N, 10.61; S,
6.09. Found: C, 53.58; H, 5.27; N, 9.84; S, 5.68.
5.1.6. [1-[50-O-Benzyl-20-O-tert-butyldimethylsilyl-b-D-
ribofuranosyl]-3-N-tert-butoxycarbonylthymine]-30-spiro-
300-(200-H-400-amino-200,300-dihydro-100,100-dioxo-isothiazole-

4738
C. Tomassi et al. / Bioorg. Med. Chem. 16 (2008) 4733–4741
500-phenyl) (10). To a solution of LDA (freshly prepared
from 4 mmol of nBuLi and 4.4 mmol of diisopropyl-
amine) in THF (24 mL) was added at ꢀ78 ꢁC a solution
of sulfonamidonitrile 9 (0.76 g, 1 mmol) in dry THF
(10 mL). After 15 min, water (10 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, 30:70)
(CO), 139.9 (C-6), 130.0, 128.9, 128.3, (4C, C6H5),
128.0 (Cip), 112.3 (C-5), 112.2 (C-500), 95.5 (C-10), 87.7
[OC(CH3)3], 87.1 (C-40), 71.3 (C-30), 70.5 (C-20), 63.3
(C-50), 27.8 [OC(CH3)3], 25.7 [SiC(CH3)3], 18.1
[SiC(CH3)3], 12.6 (CH3), ꢀ4.4 (SiCH3), ꢀ4.7 (SiCH3);
MS (ES): 673.4 [M+Na]+, 689.3 [M+K]+. Anal.
(C29H42N4O9SSi) C, H, N, S. Calcd C, 53.52; H, 6.50;
N, 8.61; S, 4.93. Found: C, 53.44; H, 6.48; N, 8.35; S,
25
D
4.78. 12: mp 158–159 ꢁC; ½aꢁ +24 (c 0.15, CH3OH);
to give compound 10 (0.562 g, 76%) as a white solid:
IR (ATR) m 2922, 1685, 1643, 1471, 1257, 1150, 1043,
25
D
mp 132–133 ꢁC; ½aꢁ +68 (c 0.17, acetone); IR (ATR)
840, 783 cmꢀ1 1H NMR ((CD3)2CO, 300 MHz) d
;
7.57–7.35 (m, 6H, H-6, C6H5), 6.39 (s, 2H, NH2), 5.92
m 1778, 1723, 1681, 1370, 1258, 1151, 1091, 840,
1
696 cmꢀ1; H NMR (CDCl3, 300 MHz) d 7.53 (s, 1H,
(d, J1 ,2 = 7.7 Hz, 1H, H-10), 5.70 (q, 1H, OH50), 5.60
0
0
H-6), 7.40 (m, 10H, 2C6H5), 5.94 (d, J1 ,2 = 7.4 Hz,
1H, H-10), 5.38 (s, 1H, NH), 5.29 (s, 2H, NH2), 4.73
(d, 1H, H-20), 4.66 (d, 2H, CH2C6H5), 4.57 (m, 1H, H-
(s, 1H, NH), 5.23 (d, 1H, H-20), 4.43 (m, 1H, H-40),
0
0
0
0
0
0
0
4.01 (dt, J5 A,5 B = 12.7 Hz, J4 ,5 A = 2.8 Hz, JOH5 ,5 A
0
=
3.2 Hz, 1H, H-50A), 3.82 (ddd, J4 ,5 A = 1.6 Hz,
0
0
40), 3.95 (dd, J4 ,5 A = 2.8 Hz, J5 A,5 B = 11.4 Hz, 1H,
JOH5 ,5 B = 5.1 Hz, 1H, H-50B), 1.88 (s, 3H, CH3), 0.89
(s, 9H, [SiC(CH3)3]), 0.17 (s, 3H, SiCH3), 0.05 (s, 3H,
SiCH3); 13C NMR ((CD3)2CO, 75 MHz) d 163.2 (C-4),
151.6 (C-2), 148.1 (C-400), 137.9 (C-6), 129.4, 128.4,
128.1 (6C, C6H5), 111.6 (C-5), 112.2 (C-500), 89.8 (C-
10), 86.5 (C-40), 73.3 (C-20), 70.7 (C-30), 61.8 (C-50),
25.3 [SiC(CH3)3], 17.9 [SiC(CH3)3], 11.9 (CH3), ꢀ5.1
(SiCH3), ꢀ5.4 (SiCH3); MS (ES): 573.5 [M+Na]+,
589.4 [M+K]+. Anal. (C24H34N4O7SSi) C, H, N, S.
Calcd C, 52.34; H, 6.22; N, 10.17; S, 5.82. Found: C,
56.40; H, 6.98; N, 8.66; S, 4.97.
0
0 0
0
0
0
0
H-50A), 3.77 (dd, J4 ,5 B = 2.0 Hz, 1H, H-50B), 1.61 (s,
3H, CH3), 1.58 (s, 9H, [OC(CH3)3]), 0.88 (s, 9H,
[SiC(CH3)3]), 0.1 (s, 3H, SiCH3), 0.01 (s, 3H, SiCH3);
13C NMR (CDCl3, 75 MHz) d 161.2 (C-4), 149.0 (C-
2), 147.7 (CO), 146.7 (C-400), 136.2 (Cip), 135.4 (C-6),
129.9, 129.5, 129.2, 129.1, 128.3, 127.8 (9C, C6H5),
112.6 (C-500), 112.2 (C-5), 88.5 (C-10), 87.2 [OC(CH3)3],
84.3 (C-40), 74.4 (C-20), 74.3 (CH2C6H5), 70.2 (C-50),
69.9 (C-30), 27.8 [OC(CH3)3], 25.7 [SiC(CH3)3], 18.2
[SiC(CH3)3], 12.5 (CH3), ꢀ4.5 (SiCH3), ꢀ4.6 (SiCH3);
MS (ES): 763.3 [M+Na]+. Anal. (C36H48N4O9SSi) C,
H, N, S. Calcd C, 58.36; H, 6.53; N, 7.56; S, 4.33.
Found: C, 57.70; H, 6.41; N, 7.38; S, 4.18.
0
0
5.1.8. [1-[20,50-Bis-O-tert-butyldimethylsilyl-b-D-ribofur-
anosyl]-3-N-tert-butoxycarbonylthymine]-30-spiro-300-(200-
H-400-amino-200,300-dihydro-100,100-dioxo-isothiazole-500-phe-
nyl) (13). To a solution of 11 (158 mg, 0.24 mmol) and
imidazole (50 mg, 0.72 mmol) in DMF (5 mL) was
added TBDMSCl (91 mg, 0.6 mmol). The reaction mix-
ture was stirred at room temperature overnight then
evaporated to dryness. After flash chromatography
(EtOAc/petroleum ether, 25:65) compound 13 (149 mg,
5.1.7. [1-[20-O-tert-Butyldimethylsilyl-b-D-ribofurano-
syl]-3-N-tert-butoxycarbonylthymine]-30-spiro-300-(200-H-
400-amino-200,300-dihydro-100,100-dioxo-isothiazole-500-phenyl)
(11) and [1-[20-O-tert-butyldimethylsilyl-b-D-ribofurano-
syl]-30-spiro-300-(200-H-400-amino-200,300-dihydro-100,100-dioxo-
isothiazole-500-phenyl) (12). Method A. To a solution of
10 (283 mg, 0.38 mmol) in absolute ethanol (7 mL) were
added Pd(OH)2/C (71 mg, 0.1 mmol) and cyclohexene
(0.87 mL, 8.3 mmol). The reaction mixture was refluxed
for 2 h 30 min to give a mixture of compounds 11 and
12. The solvent was evaporated and the residue chro-
matographed (EtOAc/petroleum ether, 60:40) to give
successively 11 (198 mg, 80%) and 12 (33 mg, 16%) as
white solids. Method B. To a solution of 10 (502 mg,
0.68 mmol) in absolute ethanol (12.5 mL) were added
Pd(OH)2/C (127 mg, 0.18 mmol) and cyclohexene
(1.55 mL, 14.8 mmol). The reaction mixture was re-
fluxed for 17 h to give a mixture of compounds 11 and
12. The solvent was evaporated and the residue chro-
matographed (EtOAc/petroleum ether, 60:40) to give
80%) was isolated as a white solid: mp 145–148 ꢁC;
25
½aꢁ +43 (c 0.15, acetone); IR (ATR) m 1783, 1718,
D
1
1676, 1370, 1254, 1147, 836 cmꢀ1; H NMR (CDCl3,
300 MHz) d 7.48 (m, 5H, C6H5), 7.26 (s, 1H, H-6),
5.60 (d, J1 ,2 = 7.3 Hz, 1H, H-10), 5.38 (s, 3H, NH,
0
0
NH2), 4.86 (d, 1H, H-20), 4.36 (s, 1H, H-40), 4.05 (dd,
J5 A,5 B = 12.3 Hz, J4 ,5 A = 2.0 Hz 1H, H-50A), 3.93
0
0
0
0
(dd, J4 ,5 B = 3.6 Hz 1H, H-50B), 2.00 (s, 3H, CH3),
1.60 (s, 9H, [OC(CH3)3]), 0.95 (s, 9H, [SiC(CH3)3]),
0.89 (s, 9H, [SiC(CH3)3]), 0.18 (s, 3H, SiCH3), 0.16 (s,
3H, SiCH3), 0.13 (s, 3H, SiCH3), 0.02 (s, 3H, SiCH3);
13C NMR (CDCl3, 75 MHz) d 161.2 (C-4), 148.9 (C-
2), 147.5 (2C, CO, C-400), 136.5 (C-6), 130.0, 129.00,
128.5, 127.9 (6C, C6H5), 112.2 (C-5), 111.3 (C-500), 91.1
(C-10), 87.5 [OC(CH3)3], 86.2 (C-40), 73.9 (C-20), 68.4
(C-30), 62.7 (C-50), 27.8 [OC(CH3)3], 26.5 [SiC(CH3)3],
25.8 [SiC(CH3)3], 18.8 [SiC(CH3)3], 18.2 [SiC(CH3)3],
12.8 (CH3), ꢀ4.4 (SiCH3), ꢀ4.5 (SiCH3), ꢀ4.6 (SiCH3),
ꢀ5.1 (SiCH3); MS (ES): 787.2 [M+Na]+, 803.1 [M+K]+.
Anal. (C35H56N4O9SSi2) C, H, N, S. Calcd C, 54.95; H,
7.38; N, 7.32; S, 4.19. Found: C, 55.01; H, 7.19; N, 7.11;
S, 3.99.
0
0
successively 11 (38 mg, 9%) and 12 (334 mg, 89%) as
25
D
white solids. 11: mp 142 ꢁC; ½aꢁ +49 (c 0.11, acetone);
IR (ATR) m 1788, 1713, 1672, 1370, 1254, 1147, 1086,
1045, 836 cmꢀ1 1H NMR (CDCl3, 300 MHz) d 7.46
;
(m, 5H, C6H5), 7.26 (s, 1H, H-6), 5.78 (s, 2H, NH2),
5.40 (s, 1H, NH), 5.38 (d, J1 ,2 = 7.7 Hz, 1H, H-10),
0
0
5.29 (d, 1H, H-20), 5.05 (d, 1H, OH-50), 4.55 (s, 1H,
H-40), 3.94 (d, J5 A,50B = 10.1 Hz, 1H, H-50A), 3.60
0
(dd, 1H, H-50B), 1.95 (s, 3H, CH3), 1.60 (s, 9H,
[OC(CH3)3]), 0.86 (s, 9H, [SiC(CH3)3]), 0.14 (s, 3H,
SiCH3), 0.05 (s, 3H, SiCH3); 13C NMR (CDCl3,
75 MHz) d 161.1 (C-4), 149.5 (C-2), 148.0 (C-400), 147.4
5.1.9. [1-[20,50-Bis-O-tert-butyldimethylsilyl-b-D-ribofur-
anosyl]thymine]-30-spiro-300-(200-H-400-amino-200,300-dihy-
dro-100,100-dioxo-isothiazole-500-phenyl) (14). To a solution

C. Tomassi et al. / Bioorg. Med. Chem. 16 (2008) 4733–4741
4739
of 12 (313 mg, 0.57 mmol) and imidazole (117 mg,
1.71 mmol) in DMF (7 mL) was added TBDMSCl
(217 mg, 1.42 mmol). The reaction mixture was stirred
at room temperature overnight then evaporated to dry-
ness. After flash chromatography (EtOAc/petroleum
(13 mg, 0.1 mmol) in acetone (5 mL) was added EtI
(0.030 mL, 0.38 mmol). The mixture was refluxed until
complete reaction then filtered through a silica pad
and evaporated to dryness. After flash chromatography
(EtOAc/petroleum ether, 15:85) compound 16 (108 mg,
25
ether, 30:70) compound 14 (284 mg, 74%) was isolated
80%) was isolated as a white solid: mp 93–95 ꢁC; ½aꢁ
D
25
D
as a white solid: mp 139–141 ꢁC; ½aꢁ +28 (c 0.25,
+41 (c 0.165, acetone); IR (ATR) m 1705, 1699, 1643,
1473, 1255, 1157, 831 cmꢀ1
;
1H NMR (CDCl3,
MeOH); IR (ATR) m 1695, 1644, 1249, 1170, 1040,
1
840 cmꢀ1; H NMR (CDCl3, 300 MHz) d 10.40 (s, 1H,
NH-3), 7.68 (s, 1H, H-6), 7.50 (m, 2H, C6H5), 7.45 (m,
300 MHz) d 7.50 (m, 5H, C6H5), 7.21 (s, 1H, H-6),
5.50 (s, 3H, H-10, NH2), 5.37 (s, 1H, NH), 4.93 (d,
J1 ,2 = 7.1 Hz, 1H, H-20), 4.28 (q, J4 ,5 A = 2.3 Hz,
2H, C6H5), 7.35 (m, 1H, C6H5), 6.19 (s, 2H, NH2),
0
0
0
0
5.70 (d, J1 ,2 = 7.2 Hz, 1H, H-10), 5.63 (s, 1H, NH),
5.12 (d, 1H, H-20), 4.30 (t, 1H, H-40), 4.12 (d, 2H, H-
50), 1.90 (s, 3H, CH3), 0.93 (s, 9H, [SiC(CH3)3]), 0.91
(s, 9H, [SiC(CH3)3]), 0.17 (s, 3H, SiCH3), 0.14 (s, 3H,
SiCH3), 0.12 (s, 3H, SiCH3), 0.07 (s, 3H, SiCH3); 13C
NMR (CDCl3, 75 MHz) d 163.3 (C-4), 151.3 (C-2),
148.3 (C-400), 138.1 (C-6), 129.4, 128.9, 128.6, 128.2 (6
C, C6H5), 111.7 (C-5), 107.9 (C-500), 91.6 (C-10), 86.9
(C-40), 73.6 (C-20), 67.6 (C-30), 62.8 (C-50), 25.9
[SiC(CH3)3], 25.4 [SiC(CH3)3], 18.5 [SiC(CH3)3], 18.0
[SiC(CH3)3], 11.9 (CH3), ꢀ5.1 (SiCH3), ꢀ5.4 (SiCH3),
ꢀ5.5 (SiCH3), ꢀ5.6 (SiCH3); MS (ES): 687.3
[M+Na]+, 703.3 [M+K]+. Anal. (C30H48N4O7SSi2) C,
H, N, S. Calcd C, 54.19; H, 7.28; N, 8.43; S, 4.82.
Found: C, 55.86; H, 7.54; N, 7.72; S, 4.33.
J4 ,5 B = 4.7 Hz, 1H, H-40), 4.06 (dd, J5 A,5 B = 12.2 Hz,
1H, H-50-A), 4.00 (q, JCH2, CH3 = 7 Hz, 2H, CH2CH3),
3.95 (dd, 1H, H-50B), 1.99 (s, 3H, CH3), 1.20 (t, 3H,
CH2CH3), 0.93 (s, 9H, [SiC(CH3)3]), 0.87 (s, 9H,
[SiC(CH3)3]), 0.16 (s, 3H, SiCH3), 0.14 (s, 3 H, SiCH3),
0.12 (s, 3H, SiCH3), ꢀ0.04 (s, 3H, SiCH3); 13C NMR
(CDCl3, 75 MHz) d 163.1 (C-4), 151.2 (C-2), 147.8 (C-
400), 136.0 (C-6), 129.9, 128.9, 128.5 (5C, C6H5), 127.9
(Cip), 111.7 (C-5), 110.0 (C-500), 92.8 (C-10), 86.3 (C-
40), 73.7 (C-20), 67.9 (C-30), 62.5 (C-50), 37.1 (CH2CH3),
26.4 [SiC(CH3)3], 25.8 [SiC(CH3)3], 18.8 [SiC(CH3)3],
18.2 [SiC(CH3)3], 13.4 (CH3), 13.1 (CH2CH3), ꢀ4.4
(SiCH3), ꢀ4.5 (SiCH3), ꢀ4.7 (SiCH3), ꢀ5.0 (SiCH3);
MS (ES): 715.3 [M+Na]+, 732.3 [M+K]+. Anal.
(C32H52N4O7SSi2) C, H, N, S. Calcd C, 55.46; H, 7.56;
N, 8.08; S, 4.63. Found: C, 56.62; H, 7.61; N, 7.51; S,
4.25.
0
0
0
0
0
0
5.1.10. [1-[20,50-Bis-O-tert-butyldimethylsilyl-b-D-ribofur-
anosyl]-3-N-methylthymine]-30-spiro-300-(200-H-400-amino-
200,300-dihydro-100,100-dioxo-isothiazole-500-phenyl) (15). To
a solution of 14 (80 mg, 0.12 mmol) and K2CO3 (8 mg,
0.06 mmol) in acetone (3 mL) was added MeI
(0.015 mL, 0.24 mmol). The mixture was refluxed until
complete reaction then filtered through a silica pad
and evaporated to dryness. After flash chromatography
(EtOAc/petroleum ether, 20:80) compound 15 (70 mg,
5.1.12. 1-(30-Amino-50-O-benzyl-20-O-tert-butyldimethyl-
silyl-30-C-cyano-30-deoxy-30-N-benzylsulfonyl-30-N-methyl-
b-D-ribofuranosyl)-3-N-methylthymine (17). To a solu-
tion of 7 (600 mg, 0.94 mmol) and K2CO3 (195 mg,
0.47 mmol) in acetone (15 mL) was added MeI
(0.117 mL, 1.87 mmol). The mixture was refluxed for
24 h and evaporated to dryness. After flash chromatog-
raphy (EtOAc/petroleum ether, 20:80) compound 17
84%) was isolated as a white solid: mp 159–160 ꢁC;
25
½aꢁ +48 (c 0.12, acetone); IR (ATR) m 1709, 1672,
D
(408 mg, 65%) was isolated as a white solid: mp 71–
1644, 1254, 1156, 840, 775, 696 cmꢀ1; 1H NMR (CDCl3,
300 MHz) d 7.51 (m, 5H, C6H5), 7.22 (s, 1H, H-6), 5.50
(s, 3H, H-10, NH2), 5.39 (s, 1H, NH), 4.93 (d,
25
D
1662, 1644, 1463, 1351, 1151, 1128, 845, 780,
72 ꢁC; ½aꢁ +43 (c 0.24, acetone); IR (ATR) m 1709,
701 cmꢀ1 1H NMR (CDCl3, 300 MHz) d 7.40 (m,
;
J1 ,2 = 7.1 Hz, 1H, H-20), 4.30 (q, J4 ,5 A = 2.4 Hz,
0
0
0
0
11H, H-6, 2C6H5), 6.18 (d, J1 ,2 = 5.2 Hz, 1H, H-10),
4.76 (d, JA,B = 11.1 Hz, 1H, OCH2C6H5-A), 4.72 (d,
1H, H-20), 4.54 (d, 1H, OCH2C6H5-B), 4.53 (m, 1H,
H-40), 4.42 (m, 2H, SO2CH2C6H5), 4.00 (dd,
0
0
J4 ,5 B = 4.7 Hz, 1H, H-40), 4.05 (dd, J5 A,5 B = 12.2 Hz,
1H, H-50-A), 3.95 (dd, 1H, H-50B), 3.36 (s, 3H, N-
CH3), 2.01 (s, 3H, CH3), 0.95 {s, 9H, [SiC(CH3)3]},
0.88 (s, 9H, {SiC(CH3)3]}), 0.17 (s, 3H, SiCH3), 0.15
(s, 3H, SiCH3), 0.13 (s, 3H, SiCH3), ꢀ0.05 (s, 3H,
SiCH3); 13C NMR (CDCl3, 75 MHz) d 163.5 (C-4),
151.5 (C-2), 147.7 (C-400), 135.9 (C-6), 130.0, 128.9,
128.5 (5C, C6H5), 127.9 (Cip), 111.6 (C-5), 110.1 (C-
500), 93.1 (C-10), 86.4 (C-40), 73.8 (C-20), 67.9 (C-30),
62.5 (C-50), 28.5 (N-CH3), 26.5 [SiC(CH3)3], 25.8
[SiC(CH3)3], 18.8 [SiC(CH3)3], 18.2 [SiC(CH3)3], 13.4
(CH3), ꢀ4.4 (SiCH3), ꢀ4.5 (SiCH3), ꢀ4.7 (SiCH3),
ꢀ5.0 (SiCH3); MS (ES): 701.3 [M+Na]+, 717.3
[M+K]+. Anal. (C31H50N4O7SSi2) C, H, N, S. Calcd
C, 54.84; H, 7.42; N, 8.25; S, 4.72. Found: C, 55.98;
H, 7.55; N, 7.60; S, 4.36.
0
0
0
0
J4,5 A = 4.0 Hz, J5 A,5 B = 10.1 Hz, 1H, H-50A), 3.91
0
0
0
(dd, J4,5 B = 3.4 Hz, 1H, H-50B), 3.32 (s, 3H, CH3-N3),
0
2.85 (s, 3H, CH3NSO2), 1.74 (s, 3H, CH3), 0.87 (s, 9H,
[SiC(CH3)3]), 0.21 (s, 3H, SiCH3), ꢀ0.09 (s, 3H, SiCH3);
13C NMR (CDCl3, 75 MHz) d 163.6 (C-4), 151.6 (C-2),
137.2 (Cip), 133.2 (C-6), 131.5, 129.3, 128.0, (9C, C6H5),
117.5 (CN), 111.8 (C-5), 89.2 (C-10), 82.2 (C-40), 81.5 (C-
20), 74.6 (O-CH2C6H5), 69.5 (C-50), 64.8 (C-30), 60.5
(SO2CH2C6H5), 35.9 (CH3-N3), 28.5 (CH3-NSO2),
25.9 [SiC(CH3)3], 18.1 [SiC(CH3)3], 13.4 (CH3), ꢀ3.97
(SiCH3), ꢀ4.9 (SiCH3); MS (ES): 691.3 [M+Na]+,
707.2 [M+K]+. Anal. (C33H44N4O7SSi) C, H, N, S.
Calcd C, 59.26; H, 6.63; N, 8.38; S, 4.79. Found: C,
59.03; H, 6.49; N, 8.04; S, 4.67.
5.1.11. [1-[20,50-Bis-O-tert-butyldimethylsilyl-b-D-ribofur-
anosyl]-3-N-ethylthymine]-30-spiro-300-(200-H-400-amino-200,
300-dihydro-100,100-dioxo-isothiazole-500-phenyl) (16). To a
solution of 14 (127 mg, 0.19 mmol) and K2CO3
5.1.13. [1-[50-O-Benzyl-20-O-tert-butyldimethylsilyl-b-D-
ribofuranosyl]-3-N-methylthymine]-30-spiro-300-(400-ami-

4740
C. Tomassi et al. / Bioorg. Med. Chem. 16 (2008) 4733–4741
25
was isolated as a white solid: mp 129–131 ꢁC; ½aꢁ +22
no-200,300-dihydro-200-N-methyl-100,100-dioxo-isothiazole-500-
phenyl) (18). A solution of 17 (342 mg, 0.51 mmol) and
Cs2CO3 (189 mg, 0.51 mmol) in acetonitrile (5 mL),
was refluxed for 25 min and evaporated to dryness.
After flash chromatography (EtOAc/petroleum ether,
D
(c 0.2, acetone); IR (ATR) m 1706, 1676, 1649, 1466,
1261, 1152, 1046, 838 cmꢀ1
;
1H NMR (CDCl3,
300 MHz) d 7.56–7.36 (m, 5H, C6H5), 7.10 (s, 1H, H-
0
6), 5.71 (s, 2H, NH2), 5.13 (d, J1 ,2 = 6.5 Hz, 1H, H-
0
0 0
30:70) compound 18 (307 mg, 90%) was isolated as a
20), 4.93 (d, 1H, H-10), 4.31 (q, J4 ,5 A = 3.5 Hz,
25
D
0
0
white solid: mp 113–114 ꢁC; ½aꢁ +31 (c 0.1, acetone);
J4 ,5 B = 6.5 Hz, 1H, H-40), 4.04 (m, 2H, H-50), 3.32 (s,
3H, CH3NSO2), 2.99 (s, 3H, CH3N-3), 1.98 (s, 3H,
CH3), 0.90 (s, 9H, [SiC(CH3)3]), 0.81 (s, 9H,
[SiC(CH3)3]), 0.14 (s, 3 H, SiCH3), 0.06 (s, 6H, 2 SiCH3),
0.01 (s, 3H, SiCH3); 13C NMR (CDCl3, 75 MHz) d
163.4 (C-4), 151.3 (C-2), 147.6 (C-400), 139.1 (C-6),
129.9–128.9 (5C, C6H5), 127.7 (Cip), 111.5 (C-5), 104.0
(C-500), 99.8 (C-10), 80.0 (C-40), 75.0 (C-20), 68.4 (C-30),
62.2 (C-50), 28.3 (CH3NSO2), 27.3 (CH3N-3), 26.4
[SiC(CH3)3], 25.8 [SiC(CH3)3], 18.9 [SiC(CH3)3], 18.5
[SiC(CH3)3], 13.3 (CH3), ꢀ4.3 (SiCH3), ꢀ4.8 (SiCH3),
ꢀ4.9 (SiCH3), ꢀ5.6 (SiCH3); MS (ES): 715.4
[M+Na]+, 731.4 [M+K]+. Anal. (C32H52N4O7SSi2) C,
H, N, S. Calcd C, 55.46; H, 7.56; N, 8.08; S, 4.63.
Found: C, 56.43; H, 7.55; N, 7.61; S, 4.37.
IR (ATR) m 1705, 1650, 1469, 1261, 1154, 1047, 840,
766 cmꢀ1 1H NMR (CDCl3, 300 MHz) d 7.46 (m,
;
10H, 2C6H5), 7.10 (s, 1H, H-6), 5.71 (br s, 2H, NH2),
5.14 (d, J1 ,2 = 6.6 Hz, 1H, H-20), 4.96 (d, 1H, H-10),
0
0
0
0
4.57 (s, 2H, CH2C6H5), 4.50 (dd, J4 ,5 a = 3.1 Hz,
J4 ,5 b = 6.7 Hz, 1H, H-40), 3.93 (m, 2H, H-50), 3.33 (s,
3H, N-CH3), 2.99 (s, 3H, CH3NSO2), 1.96 (s, 3H,
CH3), 0.80 (s, 9H, SiC(CH3)3), 0.14 (s, 3H, SiCH3),
0.01 (s, 3H, SiCH3); 13C NMR (CDCl3, 75 MHz) d
163.4 (C-4), 151.4 (C-2), 147.4 (C-400), 139.0 (C-6),
137.7 (Cip), 129.9–127.6 (5C, C6H5), 111.6 (C-5), 103.9
(C-500), 99.4 (C-10), 78.1 (C-40), 74.9 (C-20), 74.4
(CH2C6H5), 68.9 (C-50), 68.7 (C-30), 28.3 (CH3N-3),
27.2 (CH3NSO2), 25.9 (SiC(CH3)3), 18.5 (SiC(CH3)3),
13.3 (CH3), ꢀ4.3 (SiCH3), ꢀ5.6 (SiCH3); MS (ES):
691.3 [M+Na]+, 707.2 [M+K]+. Anal. (C33H44N4O7SSi)
C, H, N, S. Calcd C, 59.26; H, 6.63; N, 8.38; S, 4.79.
Found: C, 60.22; H, 6.89; N, 7.78; S, 4.47.
0
0
5.1.16. 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).
5.1.14. [1-[20-O-tert-Butyldimethylsilyl-b-D-ribofurano-
syl]-3-N-ethylthymine]-30-spiro-300-(400-amino-200,300-dihy-
dro-200-N-methyl-100,100-dioxo-isothiazole-500-phenyl) (19).
To a solution of 18 (279 mg, 0.42 mmol) in absolute eth-
anol (7.7 mL) were added Pd(OH)2/C (78 mg,
0.11 mmol) and cyclohexene (0.95 mL, 9 mmol). The
reaction mixture was refluxed for 1 h 25 min, and the
solvent was evaporated. The residue was chromato-
5.1.17. Anti-HIV evaluation. 4 · 105 CEM cells per mil-
liliter were infected with HIV-1 or HIV-2 at ꢂ100
CCID50 (50% cell culture infective dose) per milliliter
of cell suspension. Then 100 ll of the infected cell sus-
pension was transferred to microtiter plate wells and
mixed with 100 ll of the appropriate dilutions of the test
compounds. After 4 days giant cell formation (CEM)
was recorded microscopically in the HIV-infected cell
cultures.13 The 50% effective concentration (EC50) and
50% cytotoxic concentration (CC50) of the test com-
pounds were defined as the compound concentrations
required to inhibit virus-induced cytopathicity (CEM)
by 50%, or to reduce by 50% the number of viable cells
in mock-infected CEM cell cultures.
graphed (EtOAc/petroleum ether, 70:30) to give 19
25
(240 mg, 99%) as a white solid: mp 130–132 ꢁC; ½aꢁ
D
+19 (c 0.15, acetone); IR (ATR) m 2925, 1704, 1644,
1463, 1258, 1151, 1035, 840, 761 cmꢀ1
;
1H NMR
((CD3)2CO, 300 MHz) d 7.79 (s, H-6), 7.61–7.35 (m,
0
0
5H, C6H5), 6.35 (s, 2H, NH2), 5.60 (d, J1 ,2 = 7.1 Hz,
1H, H-10), 5.16 (d, 1H, H-20), 4.45 (q, 1H, H-40), 3.95
(m, 2H, H-50), 3.28 (s, 3H, CH3NSO2), 2.99 (s, 3H,
CH3N-3), 1.92 (s, 3H, CH3), 0.83 (s, 9H, [SiC(CH3)3]),
0.12 (s, 3H, SiCH3), 0.01 (s, 3H, SiCH3); 13C NMR
(CDCl3, 75 MHz) d 162.9 (C-4), 151.7 (C-2), 148.0 (C-
400), 138.9 (C-6), 129.4–128.1 (5C, C6H5), 110.6 (C-5),
103.3 (C-500), 96.4 (C-10), 80.6 (C-40), 75.6 (C-20), 69.5
(C-30), 61.0 (C-50), 27.4 (CH3), 26.3 [SiC(CH3)3], 25.4
[SiC(CH3)3], 18.1 [SiC(CH3)3], 12.4 (CH3), ꢀ4.9
(SiCH3), ꢀ6.1 (SiCH3); MS (ES): 601.3 [M+Na]+. Anal.
(C26H38N4O7SSi) C, H, N, S. Calcd C, 53.96; H, 6.62;
N, 9.68; S, 5.54. Found: C, 57.92; H, 7.49; N, 7.72; S,
4.45.
Acknowledgments
`
C.T. thanks the Ministere Franc¸ais de la Recherche
(France) for a fellowship. D.P. thanks the Conseil
´
`
Regional de Picardie and the Ministere Franc¸ais de la
Recherche for financial support and G. Mackenzie for
helpful discussions. D.P. and J.M.C. thank Miss Laura
´
Domınguez for some preliminary experiments.
5.1.15. [1-[20,50-Bis-O-tert-butyldimethylsilyl-b-D-ribofur-
anosyl]-3-N-methylthymine]-30-spiro-300-(400-amino-200,300-
dihydro-200-N-methyl-100,100-dioxo-isothiazole-500-phenyl)
(20). To a solution of 19 (200 mg, 0.35 mmol) and imid-
azole (130 mg, 0.87 mmol) in DMF (5 mL) was added
TBDMSCl (71 mg, 1.05 mmol). The reaction mixture
was stirred at room temperature overnight then evapo-
rated to dryness. After flash chromatography (EtOAc/
petroleum ether, 30:70) compound 19 (180 mg, 75%)
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