Synthesis and in vitro activity of novel N-3 acylated TSAO-T compounds against HIV-1 and HCV

Article abstract of DOI:10.1016/j.ejmech.2011.08.017

Preparation of a small library of derivatives of the potent HIV-1 Reverse Transcriptase inhibitor TSAO-T bearing mono or di-carbonyl substituents (designed after docking analysis) at position N-3 is reported. A one-pot synthetic methodology has been devel

Full text of DOI:10.1016/j.ejmech.2011.08.017

European Journal of Medicinal Chemistry 46 (2011) 5046e5056
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro activity of novel N-3 acylated TSAO-T compounds against
HIV-1 and HCV
,
,c
Marina Moura a, Solen Josse a, Albert Nguyen Van Nhien a, Carole Fournier b c, Gilles Duverlie b
,
Sandrine Castelain b c, Elena Soriano d, José Marco-Contelles d, Jan Balzarini e, Denis Postel a
,
,
*
a Laboratoire des Glucides (UMR 6219), Université de Picardie Jules Verne, 33 rue Saint Leu, 80039 Amiens, France
b Laboratoire de Virologie, Centre Hospitalo-Universitaire d0Amiens, 80054 Amiens, France
c EA4294, Université Picardie- Jules Verne, 80036 Amiens, France
d Laboratorio de Radicales Libres y Química Computacional (IQOG, CSIC), C/Juan de la Cierva, 3, 28006 Madrid, Spain
e Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Preparation of a small library of derivatives of the potent HIV-1 Reverse Transcriptase inhibitor TSAO-T
bearing mono or di-carbonyl substituents (designed after docking analysis) at position N-3 is reported.
A one-pot synthetic methodology has been developed that involves: (i) mono-reaction of TSAO-T with
glutaryl dichloride under phase transfer conditions and (ii) in situ acyclic substitution of the remaining
chloro atom by oxygen or nitrogen nucleophiles. The method is compatible with the polyfunctionality of
the TSAO-T molecule, proceeds with high conversion yields and allows introducing molecular diversity.
The anti-HIV-1 and -HCV activity was studied in cell culture. The new N-3 acylated TSAO-T derivatives
are active against HIV-1 (nanomolar range). Anti-HCV activity was observed in the micromolar range,
that is at compound concentrations that were found cytostatic against human T-lymphocytes.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 17 May 2011
Received in revised form
6 August 2011
Accepted 12 August 2011
Available online 22 August 2011
Keywords:
TSAO-T
N-3 acylated nucleoside
HIV-1
HCV
RT inhibitor
1. Introduction
order to form covalent bonds between the novel TSAO-T deriva-
tives and the HIV-1 RT amino acids in the NNRTI binding pocket,
[20,50-Bis-O-(tert-butyldimethylsilyl)-
b
-D-ribofuranosyl]-30-spiro-
series of compounds which incorporate a photoreactive moiety,
a strong electrophilic functional group [7,9] or amino acids [10] on
this nitrogen have also been described. Among all the N-3
modifications carried out on TSAO-T derivatives, preparation of
the N-methylcarbamoyl derivative 1 was the more efficient as it
turned out to be 6-fold more effective in comparison with TSAO-T
500-(400-amino-100,200-oxathiole-200,200-dioxide) thymine (TSAO-T) [1,2]
represents the prototype of a unique class of nucleoside analogues
with human immunodeficiency virus type-1 (HIV-1) Reverse Tran-
scriptase (RT) inhibitory activity, acting through a non competitive
mechanism of enzyme inhibition [3e5]. Resistanceof HIV-1 toTSAO-T
type compounds has been observed by selection of the E138K muta-
tion in the virus-encoded RT [6]. The phenomenon of resistance and
the interestinthe complete elucidationof the precise binding mode of
this family of compounds to HIV-1 RT has strongly stimulated the
exploration of structural modifications of TSAO-T analogues.
(EC50 ¼ 0.01
mM). This gain of activity has been explained after
docking studies by the formation of two hydrogen bonds with the
enzyme, one with Pro-B140 and one with Lys-B49. Previous
research carried out in our group indicated that acylation at the
N-3 position of TSAO-T was also a valid strategy in the search of
new and more active HIV-1 RT inhibitors. Thus, N-3-Boc deriva-
tive 2 turned to be 3-fold more potent than TSAO-T as a result
from a presumed additional hydrogen bond between the Boc
carbonyl oxygen and Lys-A172 [11e13]. So a carbonyl group at the
N-3 position of the base seems to be favourable for establishing
new binding interactions with the enzyme. Docking studies
suggested that associated bulky substituents restrict the proper
adjustment of the ligand inside the binding site. On the contrary,
we may speculate that compounds bearing an unhindered
Introduction of functionnal groups at the N-3 position has
been studied by Camarasa and colleagues. Thus, a variety of polar,
lipophilic, or aromatic groups linked to the N-3 position through
flexible polymethylene linkers of different length [7,8] or
unflexible unsaturated bonds [7,9] have been synthesised. In
* Corresponding author.
E-mail address: denis.postel@u-picardie.fr (D. Postel).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.017

M. Moura et al. / European Journal of Medicinal Chemistry 46 (2011) 5046e5056
5047
carbonyl group on this position could improve activity or resis-
2. Results and discussion
tance profiles by optimizing the H-bond interaction and reducing
the unfavourable steric hindrance. Thus, we hypothesize that the
2.1. Computational chemistry approach
presence of
a N-3 acylated not-hindered bulky substituent
(compound A) or a N-3 substituent which combined simulta-
neously all the functional groups present in compounds 1 and 2
(compound B) might provide additional interactions with the
enzyme, thus destabilizing the dimer interface and improving
inhibitory activity.
In order to rationalize our starting hypothesis presuming addi-
tional bindings with derivatives with two carbonyls (Fig. 1, B, C),
and before the recently reported crystal structure of the complex
TSAO-T//HIV-1 RT [20], we first performed a docking analysis using
compound B as a model. Besides the presumedH-bond interactions
with Lys-A101, Lys-A103 and Glu-B138, compound B may establish
four additional hydrogen bonds, as shown in Fig. 2: between the
On the other hand, the single-stranded RNA flavivirus hepatitis
C virus (HCV) affects about 170 million people is a major contrib-
utor to cirrhosis and hepatocellular carcinoma (HCC), and is one of
the most common indications for liver transplantation [14]. HCV
and HIV share routes of transmission, and thus, the prevalence of
HCV infection is 15e30% among HIV-infected patients [15]. In HIV-
positive patients, longer survival may permit the progression of
HCV-related liver disease and increased mortality due to its
complications. Some studies have described the faster progression
of HCV-related liver disease and higher HCV viral loads in HIV/HCV-
coinfected individuals [16]. Thus, discovery of compounds that
could be inhibitors of HIV and HCV is a great challenge [17,18].
Therefore, the compounds were first tested against HIV-1 in CEM
cell cultures, but, in addition, also to HCV in human Huh-7 cell
cultures.
The HCV polymerase structure shares the same general right-
handed configuration consisting of finger, thumb, and palm
domains observed in HIV RT. Despite this analogy, four different
allosteric binding sites have been discovered in the NS5B poly-
merase, whereas only one binding site for non-competitive inhib-
itors has been described for HIV-RT. Thus, a large structural variety
of non-competitive inhibitors can be used to potentially interact
with NS5B polymerase for HCV [19].
(carbamate) carbonyl oxygen and the N
z of Lys-A172 (in analogy
with the Boc derivatives), between the (carbamate) carboxylic
oxygen (not sterically impeded by a bulky neighbour) and the
hydroxyl group of Thr-B139, between the (amide) carbonyl and the
Nz of Lys-B49 and, finally, between the amide NH and the hydroxyl
group of Thr-A165 instead of Pro-B140.
2.2. Synthesis
The reported synthesis of 2 implies incorporation of the Boc
substituent in a step prior to the generation of the 400-amino-
oxathiol ring, and is not well-suited for molecular diversity-
oriented strategies [11e13]. In this context, we first endeavored in
the development of an efficient synthetic methodology for the
direct introduction of an acyl substituent at the N-3 position of the
preformed TSAO-T molecule compatible with the polyfunctionality
of the molecule, particularly with the presence of an amino group
at C-400. Attempts to prepare the corresponding 3-N-benzoyl
derivative 3 (Scheme 1) by reaction with benzoyl chloride using
a battery of base catalysts (pyridine, dimethylaminopyridine, trie-
thylamine, sodium hydride, potassium carbonate) were unsuc-
cessful, the starting material being recovered unreacted. Longer
reaction times or high temperatures resulted in hydrolysis of the
silyl ether groups, known to be essential for TSAO-T biological
activity.
In this context, we report an efficient methodology for direct
acylation of TSAO-T, using phase transfer conditions, suitable for
library generation of N-3 acylated compounds bearing one or two
carbonyl groups linked by a C3 alkyl or aryl chain as a model as well
as their biological evaluation as anti-HIV inhibitors and also their
biological evaluation as potential HCV inhibitors.
The lack of reactivity of the imide nitrogen in TSAO-T towards
acylation under the above conditions was probably due to a low
Fig. 1. Structures of TSAO-T, N-3 substituted analogues 1 and 2, new derivatives reported in this work (A and C), and the model compound used for docking analysis (B). The
suggested interactions of the substituent with amino acids in HIV-1 RT are indicated with hash bonds.

5048
M. Moura et al. / European Journal of Medicinal Chemistry 46 (2011) 5046e5056
Scheme 2. Cyanation of 5.
Fig. 2. Predicted binding mode for compound B. Residues relevant to the discussion
are displayed as sticks with carbon atoms colored according to their subunit. Ligand B
is displayed as sticks with carbon atoms in cyan.
Scheme 3. Acylation using glutaric acid route.
reactivity of the ion pair formed after generation of the corre-
sponding imidure anion. To overcome this problem, acylation with
benzoyl chloride (1.2 equivalents)-sodium carbonate (8 equiva-
lents; 0.23 M in water) in dichloromethane-water (1:2), using tet-
rabutylammonium bromide as phase transfer catalyst, was
attempted [21]. The reaction proceeded cleanly at room tempera-
ture. The desired benzimide 3 was the sole reaction product when
short reaction times (<1 h) were applied. Longer reaction times
resulted in concomitant formation of the 3-N, 400-N diacylated
derivative as a side-product, as detected by mass spectrometry of
the crude reaction product. The 3-N-methoxycarbonyl TSAO-T
derivative 4 was likewise obtained as a closely related analogue
of 3 for comparative purposes (Scheme 1).
In this case the reaction was found to be sensitive to the base
concentration. Thus, using sodium carbonate at 0.23 M, a mixture
of the desired 4-bromobutanoyl derivative 5 and of the cyclo-
propylcarbonyl product 6, arising from 5 by intramolecular cycli-
zation, was obtained (Scheme 1). This side-reaction could be
avoided by decreasing the base concentration to 0.11 M. However,
attempts to further elaborate compound 5 by replacement of the
bromo atom by a nitrile group (compound 7), a direct precursor of
the target compounds, was ineffective. Reaction with sodium
cyanide [22] afforded exclusively the cyclopropyl derivative 6,
while the trimethylsilyl cyanide-methanol system provoked
hydrolysis of the acyl group to give the starting compound TSAO-T
(Scheme 2).
In view of the above-commented difficulties, an alternative
convergent approach involving the coupling reaction of TSAO-T
with glutaric acid monomethyl ester chloride, as an example of
acyl chloride already bearing the desired di-carbonyl motif, was
considered (Scheme 3). However, in this case, a fast hydrolysis of
the acylating reagent was observed under phase transfer condi-
tions. Nevertheless, formation of di-carbonyl derivative 9 was
detected by mass spectrometry when high molar ratios of the acyl
chloride were used, though in low yields. Most probably, the
carbonyl oxygen of the ester group assists the nucleophilic
displacement of the chloro group by water intramolecularly
through a six-membered ring intermediate (Scheme 4).
In order to integrate a second carbonyl functionality according
to our molecular design (structure C in Fig. 1), acylation of TSAO-T
with 4-bromobutanoyl chloride using the above optimized phase
transfer methodology was next undertaken.
We reasoned that the use of the corresponding diacyl dichloride
would slow this side reaction, thereby favouring the intermolecular
process. Thus, reaction of TSAO-T with 3.5 equivalents of glutaryl
dichloride in phase transfer conditions at 8 ꢀC allowed the forma-
tion of the mono-imidation product 8. In situ displacement of the
Scheme 4. Nucleophilic displacement of the chloro group by water on 4-chloro glu-
Scheme 1. Acylation route using 4-bromobutanoyl chloride.
taric acid monomethyl ester.

M. Moura et al. / European Journal of Medicinal Chemistry 46 (2011) 5046e5056
5049
In the same way, the regioselectivity of the N-3 rather than in N-
400 acylation was determined by 13C NMR analysis. The chemical
shift of C-400 of TSAO-T and compounds 3, 4, 5 and 6 are similar
(150.3 ppm versus 150.2, 150.4, 150.1 and 150.5 ppm) which
confirms the acylation taken place in N-3 rather than in N-400
position. Indeed in the case of N-400 acylation, the chemical shift of
C-400 is modified. For example after acylation of N-400 with an ureido
or a methyloxamoylamino group the chemical shift of C-400 is
145.58 ppm or 140.48 ppm (between approximately 5 and 10 ppm
lower than TSAO-T) [19].
Fig. 3. Methyl isophtaloyl ester derivative 17.
remaining chloro group by nucleophiles like methanol, ammonia,
methylamine, dimethylamine sodium methanthiolate, tert-
butanol, benzylalcohol and benzylamine afforded the correspond-
ing N-3 substituted di-carbonyl TSAO-T derivatives 9e16 in 36e76%
yields after HPLC purification (conversion yields monitored by 1H
NMR were all in the range of 85e95%) (Scheme 5).
The same acylation condition reaction was performed with
isophtaloyl chloride to obtain, after displacement of the remaining
chloro group by methanol, the TSAO-T derivative 17 (Fig. 3). This
analogue could deliver us information about the importance of the
flexibility of methylene groups between the two carbonyls.
2.4. Biological activity
Novel N-3 acylated TSAO-T compounds 3e6 and 9e17 were
evaluated for their inhibitory activity against HIV-1(IIIB) and HIV-
2(ROD) in human T-lymphocyte CEM cell cultures and against
HCV in human Huh-7 hepatoma cell cultures (Table 1).
The stability of compounds 3-6 and 9e17 has been studied. After
72 h in PBS at 37 ꢀC no degradation has been observed. As an
example of this study, LCMS spectra obtained with compound 10
before, and after incubation are reported (Fig. 4).
2.3. Chemical structure analysis
2.4.1. Anti-HIV activity
Despite the variety of functional groups of the test compounds,
EC50 values were all comprised between 0.032 and 0.070 mM. No
gain of activity has been observed in comparison with compounds
1 and 2.
The 1H and 13C NMR data supported the integrity of the samples
and the structural assignment. Thus, the methylene protons in the
N-3 substituent for compounds 9e16 were observed between 3.0
and 2.0 ppm, whereas the carbonyl carbons resonated at
172e176 ppm. The rest of signals were consistent with the reported
data for TSAO-T derivatives.
Unfortunately with an EC50 of 0.070
mM (compound 4), the
presence of a methyl, less bulky group, does not improve anti-HIV
activity compared to TSAO-Boc3T (compound 2, EC50 ¼ 0.023
mM).
Because of the existing enolate forms of nucleosidic bases,
a competition between N-3 and O-4 acylation could be observed
(even if such a reaction has never been described in literature after
N-3 functionalism of TSAO-T) [7e10]. The 13C NMR data demon-
strated the regioselectivity of the acylation reaction in favour of N-3
position rather than O-4. Sekine and coll [21]. carried out a 13C NMR
study of O-4 and N-3 substituted nucleoside derivatives. In case of
a O-substitution the chemical shift in 13C of C-2 is higher
(approximately 4 ppm) than that of the starting product, whereas
during a N-substitution the chemical shift of C-2 carbon is lower
(approximately 2 ppm) than that of the starting product. The NMR
data for our compounds are in good agreement with these obser-
vations as the chemical shifts of C-2 (149.4, 148.3, 148.9 and
149.0 ppm for compounds 3, 4, 5 and 6 versus 152.0 ppm for the
TSAO-T) are lower by approximately 3 ppm. Moreover, the UV
spectra analysis was also in favour of N-3 acylation rather than O-4
So the reduction of the steric constraint does not support the
interaction with Thr139 as suggested in a previous work [13].
Nevertheless, compound 4 was less cytostatic (CC50 > 100 mM).
However if we compare the results obtained for the acylated
TSAO-T 3 and 6 with their methylene analogs 18 and 19 synthesized
by Camarasa and collaborators [8], anti-HIV activity is 4 times
higher for the new compounds (Fig. 5). This outcome is fully
consistent with our previous work which has demonstrated that
the carbonyl of the Boc group acts as a potential novel pharmaco-
phore for TSAO-T derivatives.
Taking into account the EC50 of compound 11 (EC50 ¼ 0.059 ꢁ
0.003
m
M) and compound 2 (EC50 ¼ 0.023 ꢁ 0.001
mM), introduction
of a second carbonyl does not improve the antiviral activity (Fig. 6).
None of the compounds in Table 1 showed activity against HIV-
2, nor against a mutant HIV-1 strain that contains the TSAO-T-
characteristic E138K mutation in its RT (data not shown). These
findings confirm HIV-1 RT as the target for the new TSAO-T
derivatives.
acylation. (TSAO-T:
264 nm).
l
¼ 222 and 264 nm; compound 5: ¼ 224 and
l
Scheme 5. Glutaryl dichloride route.

5050
M. Moura et al. / European Journal of Medicinal Chemistry 46 (2011) 5046e5056
Table 1
binding pocket and consequential significant rearrangements of RT
sub domains upon binding of the inhibitor. It shows that the
inhibitor binds inside the NNRTI-binding pocket, assuming
a “dragon” shape, and interacts extensively with almost all the
pocket residues. A relevant result was that aromatic side chains of
Tyr181 and Tyr188 are arranged in a different conformation
compared to the commonly observed NNRTI-bound conformation
in which both of the aromatic side chains are on one side, forming
a wall of the hydrophobic pocket. The hydrophobic valley created
by the rearrangement of the two aromatic side chains accommo-
dates the 50-TBDMS moiety, which establishes hydrophobic inter-
actions with Trp229, Pro95, Tyr183, and Glu138 (p51). The 20-
TBDMS group interacts with Phe227, Tyr318, His235, and Pro236.
Inhibitory activity of tested compounds against HIV-1 and HIV-2 in CEM cell cultures
and HCV.
Compounds HIV
EC50
HCV
(
m
M)a
CC50
(
m
M)b EC50 M)a CC50 M)b
(m (m
HIV-1
0.049 ꢁ 0.003
HIV-2
3
4
5
6
ꢂ4
54 ꢁ 8.5
> 100
9.8 ꢁ 1.0
>100
>100
0.070 ꢁ 0.023 ꢂ100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.052 ꢁ 0.028
0.042 ꢁ 0.014
0.054 ꢁ 0.005
0.038 ꢁ 0.025
0.059 ꢁ 0.003
0.032 ꢁ 0.016
0.037 ꢁ 0.028
0.033 ꢁ 0.017
0.040 ꢁ 0.029
0.052 ꢁ 0.014
0.041 ꢁ 0.028
ꢂ4
ꢂ4
ꢂ4
ꢂ4
ꢂ4
ꢂ4
ꢂ4
ꢂ4
ꢂ4
ꢂ4
ꢂ4
11 ꢁ 0.71
11 ꢁ 0.0
9.9 ꢁ 0.2
8.7 ꢁ 0.3
9.1 ꢁ 0.2
9.5 ꢁ 0.3
9.9 ꢁ 0.1
11 ꢁ 0.7
9.2 ꢁ 0.8
8.8 ꢁ 0.3
14 ꢁ 0.7
43 ꢁ 2
8.5 ꢁ 0.6
11 ꢁ 1
9
10
11
12
13
14
15
16
17
9 ꢁ 1
12 ꢁ 0.8
4.4 ꢁ 0.6
7.0 ꢁ 0.9
11 ꢁ 0.9
10 ꢁ 0.8
7.6 ꢁ 0.7
8.2 ꢁ 0.9
The ribose and spiro rings are positioned over the b6_b10_b9 sheet.
One of the sulfonyl oxygens is hydrogen bonded to with the main
chain amino group of Lys103, whereas the other S]O group is
involved in
a water-mediated interaction network with the
enzyme. Finally, the thymine group is positioned between the
aromatic side chains of Tyr188 and Phe227, and points toward the
flexible loop (residues 214e224) outside the binding pocket.
To shed light into our results, we have performed a docking
analysis with the crystal structure of the enzyme in this complex
(PDB code 3QO9) as target. As long as a crystallographic water
molecule appears near the thymine ring, and hence it could be
displaced by a N-3 substituent, we carried out the analysis with and
without it. While in the first case, the docking was unsuccessful, in
the second case the largest and lowest-energy cluster shows that
TSAO-Tc
TSAO-m3Tc
1c
0.060 ꢁ 0.030
>20
12 ꢁ 3
14 ꢁ 1.8
> 100
>100
>100
0.037 ꢁ 0.001 >250
115 ꢁ 14
10 / 50
26 ꢁ 1.1
0.01 ꢁ 0.01
>10
2d
0.023 ꢁ 0.001
20-C-Me-Ce
2.4 ꢁ 0.8
>33
a
50% Effective concentration.
50% Cytostatic concentration.
Data taken from ref [8].
Data taken from ref [13].
Data taken from ref [23].
b
c
d
e
Fig. 4. LCMS spectra of compound 10 before incubation (A) and after incubation (B) in PBS at 37 ꢀC for 72 h.
2.4.2. Molecular docking
molecule B is positioned analogously to TSAO-T in the crystal, and
the same interactions are observed (Fig. 7). The N-3 substituent is
accommodated by the space around the 214e224 loop that is
outside the NNRTI binding pocket (Fig. 7).
While the elaboration of this manuscript, the crystal structure of
the HIV-1 RT-TSAO-T complex was reported by Das et al. [20]. This
structure has revealed an unexpected expansion of the NNRTI
Fig. 5. Importance of carbonyl of the Boc group as a potential novel pharmacophore for TSAO-T derivatives (Initial interaction were predicted with Lys 172. The actual interactions
with the crystal is with Lys220).

M. Moura et al. / European Journal of Medicinal Chemistry 46 (2011) 5046e5056
5051
Fig. 6. Relative effect of a second carbonyl on the side chain (Initial interaction were predicted with Lys 172. The actual interactions with the crystal is with Lys220).
Fig. 7. Two views of the predicted binding mode for compound B (pdb 3QO9). Residues relevant to the discussion are displayed as sticks with carbon atoms colored according to
their subunit. Water molecules are shown as red asterisks. Ligand B is displayed as sticks with carbon atoms in cyan. For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article. (i.e., if the figure is black and white in the printed version and colour in the web version).
The key hydrophobic interactions with the aromatic side chains
(Tyr181, Tyr183 and Tyr188) were not observed in our previous
docking study with other crystal structures, due to the expansion of
the NNRTI binding pocket and rearrangements of RT subdomains
upon binding with this bulky class of inhibitors. Indeed, the new
model explains better our current results. The improved activity of
3 and 6 (carbonylic substituents) versus non-carbonylic groups can
be accounted by an additional H-bond with Lys220 side chain.
Larger N-3 substituents (or a second carbonylic group) marginally
enhance the potency of the TSAO by gaining interaction outside
NNRTI binding pocket, probably only by residual hydrophobic
interactions in the large open space around a flexible loop (p66,
residues 210e224).
and noticed that the EC50 values are very close (4.4
2.4
M for 20-C-Me-C) [23]. Moreover the lack of cytotoxicity of the
compounds (CC50 > 100 M) is particularly interesting. However, it
is currently unclear whether the anti-HCV activity observed in the
virus-infected Huh-7 cell cultures represent a specific antiviral
activity of the test compounds or is due to an underlying cytostatic
potential of the compounds. Nevertheless, we believe that our
findings may represent the starting point of a study to further
investigate the potential anti-HCV activity of TSAO-T derivatives.
mM for 4 and
m
m
3. Conclusion
In summary, we developed an efficient protocol for the regio-
selective acylation of TSAO-T at position N-3 using acyl chlorides as
acylating agents under phase transfer conditions. This method-
ology has been extended to the preparation of derivatives bearing
di-carbonyl motifs designed for cooperative interactions with
specific amino acids in the HIV-1 RT. The synthetic strategy involves
a two-step, one-pot transformation of TSAO-T by sequential reac-
tion with glutaryl dichloride and a nucleophile and is very well
suited for combinatorial approaches.
A study of in-depth modeling is running and will be reported in
due course.
2.4.3. HCV results
Taking into account the EC50 values for compounds 3, 6, 10, 13,
16, 17 and especially the EC50 of compound 12 we can conclude that
some TSAO-T derivatives are inhibitory to HCV. Results obtained for
compounds 9, 11, 14 and 15 were also interesting since their EC500s
against HCV were still <15
a weak inhibitor with an EC50 of 43
only one to have no activity (EC50 > 100
unlikely that the derivatives would act through a competitive
mechanism against a nucleotide substrate because of their struc-
ture (presence of the TBDMS groups in O-20 and O-50) we compared
the EC50 values of the derivatives with a known HCV inhibitor
targeting HCV RNA polymerase, the 20-C-Methyl-Cytidine [24e28],
m
M. Compound 5 can be considered as
M and compound 4 was the
M). Although it may be
For HIV, the increased inhibitory activity related to the presence
of a N-3 acylated not hindered bulky substituent or a N-3 di-
carbonyl substituent was not observed. The differences in activity
demonstrated by Camarasa according to the nature of the substit-
uents were not obtained with our compounds which suggest
a more important role of the first carbonyl. These results under-
lined the fact that only the synthesis of compounds C as model of
derivatives B was performed.
m
m

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M. Moura et al. / European Journal of Medicinal Chemistry 46 (2011) 5046e5056
For HCV, biological evaluation revealed that several compounds
Accordingly, the stability of each complex was estimated from the
addition of the internal energy (including the contributions due to
both inhibitor and enzyme and their interaction energy) by means
of the AMBER force field, and the solvation free energy by using
a PoissoneBoltzmann calculation for the electrostatic component
acted as micromolar inhibitors of HCV JFH1 replication with no
visible toxicity in the confluent Huh-7 cell cultures. The compounds
prove to have micromolar cytostatic activity in human T-lympho-
cyte cell cultures. Other biological evaluations are currently sought
in our laboratory in view to demonstrate anti-HCV RNA polymerase
activity of the novel TSAO derivatives.
and
a surface-area dependent term for the nonelectrostatic
contribution. The solute was assigned a dielectric of 1, the solvent
dielectric was set to 78.4, and the dielectric boundary was defined
using a 1.4 probe sphere and van der Waals radii taken from the
AMBER force field. A lattice spacing of 0.67 point/Å was employed.
To note that we have not estimate the entropy contribution to
binding to save computational time, and because both systems are
analogous and directly comparable.
4. Experimental
4.1. Computational methods
4.1.1. Molecular docking
All docking studies were performed with the program AutoDock
(version 3.0.5) [29]. As protein target, we selected the crystallo-
graphic structure of HIV1-RT in a complex with the non-nucleoside
4.2. General
inhibitor a-APA R 95845 (PDB code: 1HNI [30]). The ligand struc-
Materials and methods. Melting points are uncorrected. Optical
rotations were recorded in CH2Cl2 solution. 1H NMR (300.13 MHz)
and 13C NMR (75.47 MHz) spectra were recorded in CDCl3,
respectively. TLC were performed on Silica F254 and detection by
UV light at 254 nm or by charring with cerium molybdate reagent.
Mass spectral data were acquired on a WATERS Micromass ZQ
spectrometer or a WATERS Micromass Q-TOFF spectrometer. HPLC
analysis was performed on Waters-Breeze (2487 Dual l Absorbance
Detector and 1525 Binary HPLC Pump) with Prevail C18 column.
Column chromatography was effected on Silica Gel 60 (230 mesh).
Cyclohexane and ethyl acetate were distilled before use. TSAO-T
was synthesized according reference [36].
ture were deleted, and polar hydrogens were added. Affinity grid
files were generated with AutoGrid (version 3.0). The carboxylic
carbon atom of the residue GluB138 was chosen as the center of the
grids, and the dimensions of the cubic grid were 90 ꢃ 90 ꢃ 90 Å3
with grid points separated by 0.300 Å. At each grid point, the
receptor0s atomic affinity potentials for aliphatic carbon, aromatic
carbon, oxygen, nitrogen, sulfur, and hydrogen atoms were
computed for intra- and intermolecular energy evaluation of each
docked configuration. For simplicity, the Si atoms were assimilated
to aliphatic C atoms. Amber united-atom charges and solvation
parameters were assigned to the protein with the program ADT
(AutoDock Tools, version 1.3a.1).
The ligand coordinates were obtained from ab initio geometry
optimizations with Gaussian 98 [31], from structures with E4
envelope puckering for the sugar ring. A common set of atom-
centered RHF/6-31G*//RHF/3-21G* charges was then derived by
using the RESP methodology [32].
4.3. General methods
4.3.1. General procedure for the synthesis of 3-N-monocarbonyl
TSAO-T derivatives (3e6)
The genetic algorithm implemented in AutoDock was used to
generate different RT-bound ligand conformers by randomly
changing the torsion angles and overall orientation of the molecule.
After docking, the 100 solutions were clustered, and the clusters
were ranked by the lowest energy representative of each cluster
(see Supporting Information).
TSAO-T (1 mmol), Na2CO3 (8 mmol) and Bu4NBr (0.1 mmol)
were dissolved in a mixture of CH2Cl2 and water (1/2 V/V). Acylated
reactant (1.2 mmol) was added to the mixture with vigorous stir-
ring at room temperature until disappearance of the starting
material (TLC monitoring). Water and CH2Cl2 were added; the
organic phase was separated, washed with water (3 times), dried
(Na2SO4) and concentrated. The crude product was purified by flash
chromatography followed by crystallization from water to give the
final product.
4.1.2. Molecular dynamics
The complex obtained from the docking experiments was used
as starting structure for the MD simulation. This system was placed
into a 25 Å radius spherical cap of 652 TIP3 [33] water molecules
centered on C10 of the ligand. The AMBER (amber99 parm file) all-
atom force field [34] was used for the enzyme. For the ligand,
bonded and nonbonded parameters were derived, by analogy or
through interpolation, from those already present in the AMBER
force field, whereas HF/6-31G* RESP charges were used. After
minimization, the whole system was partitioned into a mobile and
a frozen region. The former consisted of the ligand, all the residues
within a radius of 20 Å from the ligand, and the water molecules.
After equilibration (300 ps) where positional restraints on both the
ligand and the protein were progressively reduced and finally
removed, a 2 ns MD simulation (at 298 K) was carried out. A cutoff
of 12 Å for nonbonded interactions was used, and SHAKE [35] was
applied to all bonds involving hydrogens, and an integration step of
2 fs was used throughout.
4.3.1.1. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b-d-ribofuranosyl]-3-
N-benzoylthymine]-30-spiro-500-(400-amino-100,200-oxathiole-200,200-
dioxide) (3). According to the general procedure; TSAO-T (50 mg,
8.48 ꢃ 10ꢄ2 mmol), Na2CO3 (72 mg, 0.679 mmol) and Bu4NBr
(2.7 mg, 8.48 ꢃ 10ꢄ3 mmol) in CH2Cl2 (1 mL) and water (2 mL) were
treated with benzoyl chloride (11.8
mL, 0.101 mmol). The crude
product was purified by flash chromatography (EtOAc/cyclohexane,
3/7) followed by crystallization from water to give 3 (45 mg, 76%)
as a white solid. Rf ¼ 0.62 (EtOAc/Cyclohexane : 5/5); Mp ¼
171e172 ꢀC; ½a 2D1
ꢅ
¼ ꢄ38 (c 0.1, CH2Cl2); 1H NMR (CDCl3; 300 MHz) :
d
7.94 (m, 2H, COC6H5), 7.69 (m, 1H, COC6H5), 7.52 (m, 2H, COC6H5),
7.36 (s, 1H, H-6), 5.96 (d, 1H, J 1 ,2 ¼ 7.9 Hz, H-10), 5.63 (s, 2H, NH2),
0
0
5.60 (s, 1H, H-30), 4.62 (d, 1H, H-20), 4.31 (m, 1H, H-40), 4.02 (dd,
J5 a,4 ¼ 2.6 Hz, J5 a,5 b ¼ 12.4 Hz, 1H, H-50a), 3.90 (dd, J5 b,4 ¼ 2.6 Hz,
1H, H-50b), 2.01 (s, 3H, CH3 base), 0.99 (s, 9H, SiC(CH3)3), 0.88 (s, 9H,
SiC(CH3)3), 0.22 (s, 3H, Si(CH3)2), 0.11 (s, 3H, Si(CH3)2), 0.10 (s, 3H,
0
0
0
0
0
0
The structural stability of the complexes was examined by
monitoring the rmsd profiles for the subset of residues that form
the walls of the binding site with regard to the crystallographic
structure. To examine the energetic stability of the complexes, an
MM/PB-SA analysis was performed by using 100 snapshots
collected regularly along the last 500 ps of the MD simulation.
Si(CH3)2), 0.04 (s, 3H, Si(CH3)2); 13C NMR (CDCl3; 75 MHz) :
d 168.2
(CO), 162.5 (C-4), 150.5 (C-400), 149.4 (C-2), 135.2 (C-6), 131.4 (CIV
C6H5), 135.9, 130.5, 129.1 (C6H5), 111.9 (C-5), 92.9 (C-300), 91.9 (C-30),
87.9 (C-10), 83.7 (C-40), 74.4 (C-20), 62.0 (C-50), 26.0, 25.4
(2x SiC(CH3)3), 18.4, 17.9 (2x SiC(CH3)3), 12.3 (CH3

M. Moura et al. / European Journal of Medicinal Chemistry 46 (2011) 5046e5056
5053
base), ꢄ4.4, ꢄ4.8, ꢄ5.0, ꢄ5.6 (2x Si(CH3)2). HRMS: C31H47N3O9S-
NMR (CDCl3; 75 MHz) : d
176.4 (CO), 161.7 (C-4), 150.4 (C-400), 149.0
Si2Na calcd. 716.2469, found 716.2484.
(C-2), 135.4 (C-6), 111.9 (C-5), 93.3 (C-300), 92.3 (C-30), 87.2 (C-10),
83.7 (C-40), 74.7 (C-20), 62.1 (C-50), 26.0, 25.3 (2x SiC(CH3)3), 20.1
(CH), 18.4, 17.9 (2x SiC(CH3)3), 13.0, 13.1 (2x CH2), 12.3 (CH3
base), ꢄ4.5, ꢄ4.8, ꢄ5.2, ꢄ5.7 (2x Si(CH3)2). HRMS: C28H47N3O9S-
Si2Na calcd. 680.2469, found 680.2485.
4.3.1.2. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b-d-ribofuranosyl]-3-
N-(methoxycarbonyl)thymine]-30-spiro-500-(400-amino-100,200-oxa-
thiole-200,200-dioxide) (4). According to the general procedure TSAO-
T (50 mg, 8.48 ꢃ 10ꢄ2 mmol), Na2CO3 (72 mg, 0.679 mmol) and
Bu4NBr (2.7 mg, 8.48 ꢃ 10ꢄ3 mmol) in CH2Cl2 (1 mL) and water
4.3.2. General procedure for the synthesis of 3-N-dicarbonyl TSAO-T
derivatives (9e16)
(2 mL) were treated with methyl chloroformate (10.4
mL,
0.110 mmol). The crude product was purified by HPLC (CH3CN/H2O
0.1%TFA) followed by crystallization from water to give 4 (23 mg,
TSAO-T (1 mmol), Na2CO3 (8 mmol) and Bu4NBr (0.1 mmol)
were dissolved in a mixture of CH2Cl2 and water (1/2 V/V). Glutaryl
dichloride (3.5 mmol) was added to the mixture with vigorous
stirring at 8 ꢀC until disappearance of the starting material (mass
spectrometry monitoring). After 15 min the reaction mixture was
quenched with appropriate nucleophile. Vigorous stirring was
continued at room temperature between 5 and 15 min. Water and
CH2Cl2 were added; the organic phase was separated, washed with
water (3 times), dried (Na2SO4) and concentrated. The crude
product was purified by HPLC followed by crystallization from
water to give the final product.
42%) as
a
white solid. Rf
¼
0.69 (CH2Cl2/MeOH : 98/2);
Mp ¼ 229e230 ꢀC; ½a D21
ꢅ
¼ ꢄ20 (c 0.1, CH2Cl2); 1H NMR (CDCl3;
0
0
0
300 MHz) :
d
7.26 (s, 1H, H-6), 5.94 (d, 1H, J 1 ,2 ¼ 8.0 Hz, H-1 ), 5.66
(s, 1H, H-300), 5.55 (s, 2H, NH2), 4.55 (d, 1H, H-20), 4.38 (m, 1H, H-40),
0
0
0
0
4.07 (s, 3H, OCH3), 4.04 (dd, J5 a,4 ¼ 2.7 Hz, J5 a,5 b ¼ 12.4 Hz, 1H, H-
50a), 3.92 (dd, J5 b,4 ¼ 2.3 Hz, 1H, H-5 b), 2.01 (s, 3H, CH3 base), 0.99
(s, 9H, SiC(CH3)3), 0.87 (s, 9H, SiC(CH3)3), 0.24 (s, 3H, Si(CH3)2), 0.22
(s, 3H, Si(CH3)2), 0.08 (s, 3H, Si(CH3)2), ꢄ0.03 (s, 3H, Si(CH3)2); 13C
0
0
0
NMR (CDCl3; 75 MHz) : d
160.8 (C-4), 150.2 (CO), 150.1 (C-40), 148.4
(C-2), 135.2 (C-6), 111.5 (C-5), 93.5 (C-30), 92.1 (C-30), 87.6 (C-10),
83.8 (C-40), 74.7 (C-20), 62.1 (C-50), 56.2 (OCH3), 26.0, 25.2 (2x
4.3.2.1. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b-d-ribofuranosyl]-3-
SiC(CH3)3),
18.4,
17.8
(2x
SiC(CH3)3),
12.3
(CH3
N-(O-methoxyglutaryl)thymine]-30spiro-500-(400-amino-100,200-oxa-
thiole-200,200-dioxide) (9). According to the general procedure TSAO-
T (64.7 mg, 0.109 mmol), Na2CO3 (93 mg, 0.878 mmol) and Bu4NBr
(3.5 mg, 1.09 ꢃ 10ꢄ2 mmol) in CH2Cl2 (2 mL) and water (4 mL) were
base), ꢄ4.5, ꢄ4.9, ꢄ5.3, ꢄ5.7 (2x Si(CH3)2). HRMS: C26H45N3O10S-
Si2Na calcd. 670.2262, found 670.2237.
4.3.1.3. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b-d-ribofuranosyl]-3-
treated with glutaryl dichloride (42 mL, 0.329 mmol) and quenched
N-(bromobutanoyl)thymine]-30-spiro-500-(400-amino-100,200-oxathiole-
200,200-dioxide) (5). According to the general procedure TSAO-T
(104 mg, 0.176 mmol), Na2CO3 (150 mg, 1.41 mmol) and Bu4NBr
(5.7 mg, 1.76 ꢃ 10ꢄ2 mmol) in CH2Cl2 (7 mL) and water (14 mL)
with MeOH (4 mL). The crude product was purified by HPLC
(MeOH/H2O) followed by crystallization from water to give 9
(51 mg, 36%) as a white solid. Rf ¼ 0.69 (EtOAc/Cyclohexane : 5/5);
Mp ¼ 165e167 ꢀC; ½a D21
ꢅ
¼ ꢄ19 (c 0.1, CH2Cl2); 1H NMR (CDCl3;
0
0
0
were treated with 4-bromobutanoyl chloride (23
mL, 0.196 mmol).
300 MHz) :
d
7.26 (s, 1H, H-6), 5.97 (d, 1H, J 1 ,2 ¼ 8.1 Hz, H-1 ), 5.67
The crude product was purified by HPLC (MeOH/H2O) followed by
crystallization from water to give 5 (91 mg, 70%) as a white solid.
(s, 1H, H-300), 5.60 (s, 2H, NH2), 4.52 (d, 1H, H-20), 4.37 (m, 1H, H-40),
4.03 (dd, J5 a,4 ¼ 2.6 Hz, J5 a,5 b ¼ 12.4 Hz, 1H, H-50a), 3.92 (dd,
0
0
0
0
Rf ¼ 0.67 (CH2Cl2/MeOH : 98/2); Mp ¼ 192e193 ꢀC; ½a D21
ꢅ
¼ ꢄ14 (c
J5 b,4 ¼ 2.1 Hz, 1H, H-50b), 3.71 (s, 3H, OCH3), 2.91 (t, Ja,b ¼ 6.9 Hz,
2H, CH2a), 2.49 (t, Jc,b ¼ 7.4 Hz, 2H, CH2c), 2.10 (m, 2H, CH2b), 2.00 (s,
3H, CH3 base), 1.00 (s, 9H, SiC(CH3)3), 0.87 (s, 9H, SiC(CH3)3), 0.24 (s,
3H, Si(CH3)2), 0.23 (s, 3H, Si(CH3)2), 0.09 (s, 3H, Si(CH3)2), ꢄ0.03 (s,
0
0
0.1, CH2Cl2); 1H NMR (CDCl3; 300 MHz) :
d
7.23 (s, 1H, H-6), 5.94 (d,
0
00
0
0
1H, J 1 ,2 ¼ 8.1 Hz, H-1 ), 5.63 (s, 2H, NH2), 5.56 (s, 1H, H-3 ), 4.48 (d,
1H, H-20), 4.34 (m, 1H, H-40), 3.98 (dd, J5 a,4
¼
2.6 Hz,
0
0
J5 a,5 b ¼ 12.4 Hz,1H, H-50a), 3.87 (dd, J5 b,4 ¼ 2.0 Hz,1H, H-5 b), 3.49
(t, Ja,b ¼ 6.4 Hz, 2H, CH2a), 2.98 (t, Jc,b ¼ 6.8 Hz, 2H, CH2c), 2.30 (m,
2H, CH2b), 1.96 (s, 3H, CH3 base), 0.95 (s, 9H, SiC(CH3)3), 0.83 (s, 9H,
SiC(CH3)3), 0.20 (s, 3H, Si(CH3)2), 0.19 (s, 3H, Si(CH3)2), 0.05 (s, 3H,
3H, Si(CH3)2); 13C NMR (CDCl3; 75 MHz) :
d 174.9 (CO), 173.2 (CO),
0
0
0
0
0
161.8 (C-4),150.2 (C-400),148.9 (C-2),135.0 (C-6),111.9 (C-5), 93.4 (C-
300), 92.2 (C-30), 86.9 (C-10), 83.7 (C-40), 74.7 (C-20), 62.1 (C-50), 51.6
(OCH3), 39.4, 32.3 (2x CH2), 26.0, 25.2 (2x SiC(CH3)3), 18.8 (CH2),
18.4, 17.8 (2x SiC(CH3)3), 12.2 (CH3 base), ꢄ4.5, ꢄ4.8, ꢄ5.2, ꢄ5.6 (2x
Si(CH3)2). HRMS: C30H51N3O11SSi2Na calcd.740.2681, found
740.2671.
Si(CH3)2), ꢄ0.07 (s, 3H, Si(CH3)2); 13C NMR (CDCl3; 75 MHz) :
d 174.7
(CO), 161.8 (C-4), 150.2 (C-400), 148.9 (C-2), 135.0 (C-6), 112.0 (C-5),
93.4 (C-300), 92.4 (C-30), 86.7(C-10), 83.7 (C-40), 74.8 (C-20), 62.1 (C-
50), 38.9, 31.8, 26.7 (3x CH2), 26.0, 25.3 (2x SiC(CH3)3), 18.4, 17.8 (2x
SiC(CH3)3), 12.2 (CH3 base), ꢄ4.5, ꢄ4.8, ꢄ5.2, ꢄ5.6 (2x Si(CH3)2).
HRMS: C28H48BrN3O9SSi2Na calcd. 760.1731, found 760.1719.
4.3.2.2. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b-d-ribofuranosyl]-3-
N-(aminoglutaryl)thymine]-30-spiro-500-(400-amino-100,200-oxathiole-
200,200-dioxide) (10). According to the general procedure TSAO-T
(61.8 mg, 0.104 mmol), Na2CO3 (89 mg, 0.839 mmol) and Bu4NBr
(3.4 mg, 1.04 10ꢄ2 mmol) in CH2Cl2 (5 mL) and water (10 mL) were
4.3.1.4. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b-d-ribofuranosyl]-3-
N-(cyclopropanoyl)thymine]-30-spiro-500-(400-amino-100,200-oxathiole-
200,200-dioxide) (6). According procedure described in. 4.3.3 and
using a 0.23 M concentration of sodium carbonate, a mixture of the
treated with glutaryl dichloride (47
mL, 0.367 mmol) and quenched
with NH3 (7 N MeOH) (90 L, 0.629 mmol). The crude product was
m
desired 4-bromobutanoyl derivative
5
and of the cyclo-
purified by HPLC (MeOH/H2O) followed by crystallization from
water to give 10 (51 mg, 70%) as a white solid. Rf ¼ 0.10 (EtOAc/
propylcarbonyl product 6 was obtained in a ratio 76/24 determined
by NMR analysis on the crude mixture. A pure sample of 6 was
obtained after HPLC (MeOH/H2O) purification. Rf ¼ 0.53 (CH2Cl2/
Cyclohexane : 5/5); Mp ¼ 114e115 ꢀC; ½a D21
ꢅ
¼ ꢄ17 (c 0.1, CH2Cl2); 1H
NMR (CDCl3; 300 MHz) :
10,20
d 7.28 (s, 1H, H-6), 6.01 (d, 1H, J
MeOH : 98/2); Mp ¼ 199e201 ꢀC; ½a D21
ꢅ
¼ ꢄ40 (c 0.1, CH2Cl2); 1H
¼ 8.1 Hz, H-10), 5.83 (s, 1H, CONH2), 5.68 (s, 1H, H-300), 5.61 (s,
NMR (CDCl3; 300 MHz) :
10,20
d
7.27 (s, 1H, H-6), 5.98 (d, 1H, J
2H, NH2), 5.45 (s, 1H, CONH2), 4.48 (d, 1H, H-20), 4.39 (m, 1H, H-40),
¼ 8.0 Hz, H-10), 5.67 (s, 2H, NH2), 5.63 (s, 1H, H-300), 4.56 (d, 1H,
4.03 (dd, J5 a,4 ¼ 2.7 Hz, J5 a,5 b ¼ 12.5 Hz, 1H, H-50a), 3.92 (dd,
0
0
0
0
H-20), 4.37 (m, 1H, H-40), 4.03 (dd, J5 a,4 ¼ 2.7 Hz, J5 a,5 b ¼ 12.4 Hz,
J5 b,4 ¼ 1.9 Hz, 1H, H-50b), 2.93 (t, Ja,b ¼ 6.2 Hz, 2H, CH2a), 2.41 (t,
Jc,b ¼ 7.1 Hz, 2H, CH2c), 2.10 (m, 2H, CH2b), 2.00 (s, 3H, CH3 base),
1.00 (s, 9H, SiC(CH3)3), 0.83 (s, 9H, SiC(CH3)3), 0.25 (s, 3H, Si(CH3)2),
0.24 (s, 3H, Si(CH3)2), 0.09 (s, 3H, Si(CH3)2), ꢄ0.03 (s, 3H, Si(CH3)2);
0
0
0
0
0
0
1H, H-50a), 3.92 (dd, J5 b,4 ¼ 2.2 Hz, 1H, H-50b), 2.03 (s, 3H, CH3
base), 1.99 (m, 1H, CH), 1.42 (m, 1H, CH2), 1.22 (m, 1H, CH2), 1.00 (s,
9H, SiC(CH3)3), 0.85 (s, 9H, SiC(CH3)3), 0.24 (s, 3H, Si(CH3)2), 0.23 (s,
3H, Si(CH3)2), 0.09 (s, 3H, Si(CH3)2), ꢄ0.02 (s, 3H, Si(CH3)2); 13C
0
0
13C NMR (CDCl3; 75 MHz) :
d 175.3 (CO), 174.8 (CO), 161.9 (C-4),

5054
M. Moura et al. / European Journal of Medicinal Chemistry 46 (2011) 5046e5056
150.2 (C-400), 149.1 (C-2), 134.8 (C-6), 112.0 (C-5), 93.4 (C-300), 92.5
(C-30), 86.2 (C-10), 83.8 (C-40), 74.9 (C-20), 62.2 (C-50), 39.3, 33.8 (2x
CH2), 26.0, 25.2 (2x SiC(CH3)3), 19.4 (CH2), 18.4, 17.8 (2x SiC(CH3)3),
12.2 (CH3 base), ꢄ4.5, ꢄ4.8, ꢄ5.2, ꢄ5.7 (2x Si(CH3)2). HRMS:
C29H50N4O10SSi2Na calcd. 725.2684, found 725.2679.
was purified by HPLC to give 13 (80 mg, 76%) as a white solid.
Rf ¼ 0.73 (CH2Cl2/MeOH : 9.8/0.2); Mp ¼ 171e172 ꢀC; ½a D21
: ꢄ24 (c
ꢅ
0.1, CH2Cl2); 1H NMR (CDCl3; 300 MHz) :
d 7.25 (s, 1H, H-6), 5.95 (d, J
¼ 8.0 Hz, 1H, H-10), 5.66 (s, 1H, H-300), 5.57 (s, 2H, NH2), 4.53 (d,
10,20
1H, H-20), 4.37 (m, 1H, H-40), 4.03 (dd, J5 a,4
¼
2.7 Hz,
0
0
0
J5 a,5 b ¼ 12.5 Hz, 1H, H-50a), 3.92 (dd, J5 b,4 ¼ 2.2 Hz,1H, H-5 b), 2.90
(t, Ja,b ¼ 6.9 Hz, 2H, CH2a), 2.73 (t, Jc,b ¼ 7.3 Hz, 2H, CH2c), 2.33 (s, 3H,
SCH3), 2.13 (m, 2H, CH2b), 2.00 (s, 3H, CH3 base), 1.00 (s, 9H,
SiC(CH3)3), 0.88 (s, 9H, SiC(CH3)3), 0.24 (s, 3H, Si(CH3)2), 0.23 (s, 3H,
Si(CH3)2), 0.09 (s, 3H, Si(CH3)2), ꢄ0.02 (s, 3H, Si(CH3)2); 13C NMR
0
0
0
0
4.3.2.3. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b-d-ribofuranosyl]-3-
N-(N-methylaminoglutaryl)thymine]- 30-spiro-500-(400-amino-100,200-
oxathiole-200,200-dioxide) (11). According to the general procedure
TSAO-T (64.9 mg, 0.110 mmol), Na2CO3 (93 mg, 0.881 mmol) and
Bu4NBr (3.5 mg, 1.1 10ꢄ2 mmol) in CH2Cl2 (2 mL) and water (4 mL)
(CDCl3; 75 MHz) :
d 198.9 (COSCH3), 174.8 (COCH2), 161.8 (C-4),
were treated with glutaryl dichloride (56.2
m
L, 0.440 mmol) and
150.3 (C-400), 149.0 (C-2), 135.2 (C-6), 112.0 (C-5), 93.5 (C-300), 92.2
(C-30), 87.1 (C-10), 83.8 (C-40), 74.7 (C-20), 62.1 (C-50), 42.0, 39.4
(CH2), 26.0, 25.3 (SiC(CH3)3), 19.4 (CH2), 18.4, 17.9 (SiC(CH3)3), 12.2
(CH3 base), 11.6 (SCH3), ꢄ4.5, ꢄ4.8, ꢄ5.2, ꢄ5.7 (Si(CH3)2); HRMS :
C30H51N3O10S2Si2Na calcd. 756.2452, found 756.2444.
quenched with MeNH2 (33% EtOH) (108 L, 0.851 mmol). The crude
m
product was purified by flash chromatography (EtOAc/cyclohexane,
7/3) followed by crystallization from water to give 11 (55 mg, 70%)
as
a
white solid. Rf
¼
0.21 (EtOAc/Cyclohexane :5/5);
Mp ¼ 118e120 ꢀC; ½a D21
ꢅ
¼ ꢄ22 (c 0.1, CH2Cl2); 1H NMR (CDCl3;
0
300 MHz) :
d
7.30 (s, 1H, H-6), 6.04 (d, 1H, J 1 ,2 ¼ 8.2 Hz, H-1 ), 5.96
4.3.2.6. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b-d-ribofuranosyl]-3-
0
0
(s, 1H, NH), 5.82 (s, 2H, NH2), 5.69 (s, 1H, H-300), 4.44 (d, 1H, H-20),
N-(tert-butoxyglutaryl) thymine]-30-spiro-500-(400-amino-100,200-oxa-
thiole-200,200-dioxide) (14). According to the general procedure
TSAO-T (103 mg, 0.17 mmol), Na2CO3 (148 mg, 1.39 mmol) and
Bu4NBr (5.6 mg, 1.7 ꢃ 10ꢄ2 mmol) in CH2Cl2 (10 mL) and water
4.38 (m, 1H, H-40), 4.02 (dd, J5 a,4 ¼ 2.6 Hz, J5 a,5 b ¼ 12.6 Hz, 1H, H-
0
0
0
0
50a), 3.92 (dd, J5 b,4 ¼ 1.6 Hz, 1H, H-50b), 2.88 (t, Ja,b ¼ 6.3 Hz, 2H,
CH2a), 2.81 (d, JNH,CH3 ¼ 4.8 Hz, 3H, CH3NH), 2.34 (t, Jc,b ¼ 7.2 Hz, 2H,
CH2c), 2.10 (m, 2H, CH2b), 1.99 (s, 3H, CH3 base), 1.00 (s, 9H,
SiC(CH3)3), 0.85 (s, 9H, SiC(CH3)3), 0.24 (s, 3H, Si(CH3)2), 0.23 (s, 3H,
Si(CH3)2), 0.09 (s, 3H, Si(CH3)2), ꢄ0.04 (s, 3H, Si(CH3)2); 13C NMR
0
0
(20 mL) were treated with glutaryl dichloride (78 mL, 0.61 mmol)
and quenched with t-BuOH (1 mL, 0.01 mol). The crude product
was purified by HPLC to give 14 (82 mg, 62%) as a white solid.
(CDCl3; 75 MHz) :
d
175.4 (CO), 172.9 (CO), 161.9 (C-4), 150.3 (C-400),
Rf ¼ 0.80 (EtOAc/Cyclohexane : 5/5); Mp ¼ 179e180 ꢀC; ½a D21
: ꢄ19
ꢅ
149.1 (C-2), 134.6 (C-6), 112.0 (C-5), 93.1 (C-300), 92.8 (C-30), 86.7 (C-
10), 83.8 (C-40), 75.0 (C-20), 62.2 (C-50), 39.3, 34.7 (2x CH2), 26.3
(CH3NH), 26.0, 25.2 (2x SiC(CH3)3), 19.7 (CH2), 18.4, 17.8 (2x
SiC(CH3)3), 12.2 (CH3 base), ꢄ4.5, ꢄ4.8, ꢄ5.2, ꢄ5.6 (2x Si(CH3)2).
HRMS: C30H52N4O10SSi2Na calcd. 739.2840, found 739.2837.
(c 0.1, CH2Cl2); 1H NMR (CDCl3; 300 MHz) 0:0 d 7.25 (s, 1H, H-6), 5.95
0
0
0
(d, J 1 ,2 ¼ 8.0 Hz, 1H, H-1 ), 5.66 (s, 1H, H-3 ), 5.59 (s, 2H, NH2), 4.53
(d, 1H, H-20), 4.36 (m, 1H, H-40), 4.03 (dd, J5 a,4 ¼ 2.7 Hz,
0
0
J5 a,5 b ¼ 12.5 Hz, 1H, H-50a), 3.92 (dd, J5 b,4 ¼ 2.3 Hz,1H, H-5 b), 2.89
(t, Ja,b ¼ 6.9 Hz, 2H, CH2a), 2.38 (t, Jc,b ¼ 7.4 Hz, 2H, CH2c), 2.05 (m,
2H, CH2b), 2.00 (s, 3H, CH3 base), 1.47 (s, 9H, OtBu), 0.99 (s, 9H,
SiC(CH3)3), 0.87 (s, 9H, SiC(CH3)3), 0.25 (s, 3H, Si(CH3)2), 0.23 (s, 3H,
Si(CH3)2), 0.09 (s, 3H, Si(CH3)2), ꢄ0.03 (s, 3H, Si(CH3)2); 13C NMR
0
0
0
0
0
4.3.2.4. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b-d-ribofuranosyl]-3-
N-(N,N- dimethylaminoglutaryl)thymine]-30-spiro-500-(400-amino-100,
200-oxathiole-200,200-dioxide) (12). According to the general proce-
dure TSAO-T (55.2 mg, 9.6 10ꢄ2 mmol), Na2CO3 (79 mg,
0.749 mmol) and Bu4NBr (3.0 mg, 9.6 10ꢄ3 mmol) in CH2Cl2 (5 mL)
(CDCl3; 75 MHz) : d
175.0 (CO), 172.1 (CO), 161.8 (C-4), 150.3 (C-400),
148.9 (C-2), 135.1 (C-6), 111.9 (C-5), 93.4 (C-300), 92.1 (C-30), 87.1 (C-
10), 83.7 (C-40), 74.7 (C-20), 62.1 (C-50), 39.5, 33.9 (CH2), 28.1 (Ot-Bu),
26.0, 25.2 (SiC(CH3)3), 18.9 (CH2), 18.4, 17.8 (SiC(CH3)3), 12.2 (CH3
base), ꢄ4.5, ꢄ4.8, ꢄ5.2, ꢄ5.7 (Si(CH3)2); HRMS : C33H57N3O11SSi2Na
calcd. 782.3150, found 782.3161.
and water (10 mL) were treated with glutaryl dichloride (41.8
0.327 mmol) and quenched with dimethylamine (40% in water)
(70.5 L, 0.562 mmol). The crude product was purified by HPLC
mL,
m
(MeOH/H2O) followed by crystallization from water to give 12
(45 mg, 66%) as a white solid. Rf ¼ 0.10 (EtOAc/Cyclohexane : 5/5);
4.3.2.7. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
N-(benzyloxyglutaryl)
b
-d-ribofuranosyl]-3-
Mp ¼ 168e170 ꢀC; ½a D21
ꢅ
¼ ꢄ23 (c 0.1, CH2Cl2); 1H NMR (0CDCl3;
thymine]-30-spiro-500-(400-amino-100,200-oxa-
300 MHz) :
d
7.28 (s, 1H, H-6), 6.02 (d, 1H, J 1 ,2 ¼ 8.2 Hz, H-1 ), 5.76
thiole-200,200-dioxide) (15). According to the general procedure
TSAO-T (51 mg, 8.6 ꢃ 10ꢄ2 mmol), Na2CO3 (73 mg, 0.68 mmol) and
Bu4NBr (2.8 mg, 8.6 ꢃ 10ꢄ3 mmol) in CH2Cl2 (5 mL) and water
0
0
(s, 2H, NH2), 5.67 (s, 1H, H-300), 4.47 (d, 1H, H-20), 4.37 (m, 1H, H-40),
4.02 (dd, J5 a,4 ¼ 2.70 Hz, J5 a,5 b ¼ 12.5 Hz, 1H, H-50a), 3.92 (dd,
0
0
0
0
0
0
J5 b,4 ¼ 1.7 Hz,1H, H-5 b), 3.04 (s, 3H, NCH3), 2.96 (s, 3H, NCH3), 2.92
(m, 2H, CH2a), 2.49 (t, Jc,b ¼ 7.1 Hz, 2H, CH2c), 2.09 (m, 2H, CH2b),1.99
(s, 3H, CH3 base), 0.99 (s, 9H, SiC(CH3)3), 0.86 (s, 9H, SiC(CH3)3), 0.24
(s, 3H, Si(CH3)2), 0.23 (s, 3H, Si(CH3)2), 0.07 (s, 3H, Si(CH3)2), ꢄ0.04
(10 mL) were treated with glutaryl dichloride (38
m
L, 0.30 mmol)
and quenched with BnOH (53
mL, 0.51 mmol). The crude product
was purified by HPLC to give 15 (35 mg, 51%). Rf ¼ 0.29 (EtOAc/
Cyclohexane : 5/5); Mp ¼ 185e187 ꢀC; ½a D21
¼ ꢄ33 (c 0.1, CH2Cl2);
ꢅ
(s, 3H, Si(CH3)2); 13C NMR (CDCl3; 75 MHz) :
d
175.6 (CO), 172.0
NMR 1H (CDCl3; 300 MHz) :
d
7.39e7.38 (m, 5H, C5H060), 7.25 (s, 1H,
(CO), 161.8 (C-4), 150.3 (C-400), 149.0 (C-2), 134.7 (C-6), 111.9 (C-5),
93.2 (C-300), 92.5 (C-30), 86.2 (C-10), 83.7 (C-40), 74.9 (C-20), 62.1 (C-
50), 39.6 (CH2), 37.2 (CH3N), 35.4 (CH3N), 31.4 (CH2), 26.0, 25.2 (2x
SiC(CH3)3), 19.1 (CH2), 18.4, 17.8 (2x SiC(CH3)3), 12.2 (CH3
base), ꢄ4.5, ꢄ4.8, ꢄ5.2, ꢄ5.6 (2x Si(CH3)2). HRMS: C31H54N4O10S-
Si2Na calcd. 753.2997, found 753.3003.
H-6), 5.95 (d, J 1 ,2 ¼ 8.0 Hz, 1H, H-1 ), 5.65 (s, 1H, H-3 ), 5.57 (s, 2H,
0
0
0
NH2), 5.16 (s, 2H, CH2C6H5), 4.53 (d, 1H, H-20), 4.36 (m, 1H, H-40),
4.03 (dd, J5 a,4 ¼ 2.7 Hz, J5 a,5 b ¼ 12.5 Hz, 1H, H-50a), 3.92 (dd,
0
0
0
0
J5 b,4 ¼ 2.2 Hz, 1H, H-50b), 2.92 (t, Ja,b ¼ 6.9 Hz, 2H, CH2a), 2.55 (t,
Jc,b ¼ 7.4 Hz, 2H, CH2c), 2.12 (m, 2H, CH2b), 2.00 (s, 3H, CH3 base),
0.99 (s, 9H, SiC(CH3)3), 0.87 (s, 9H, SiC(CH3)3), 0.24 (s, 3H, Si(CH3)2),
0.22 (s, 3H, Si(CH3)2), 0.09 (s, 3H, Si(CH3)2), ꢄ0.03 (s, 3H, Si(CH3)2);
0
0
4.3.2.5. [1-[20,50-Bis-O-tert-butyldimethylsilyl-
b
-d-ribofuranosyl]-3-
13C NMR (CDCl3; 75 MHz) :
d 174.9 (CO), 172.6 (CO), 161.8 (C-4),
N-(thiomethylglutaryl) thymine]-30-spiro-500-(400-amino-100,200-oxa-
thiole-200,200-dioxide) (13). According to the general procedure
TSAO-T (84 mg, 0.14 mmol), Na2CO3 (121 mg, 1.14 mmol) and
Bu4NBr (4.6 mg, 1.42 ꢃ 10ꢄ2 mmol) in CH2Cl2 (7 mL) and water
150.2 (C-400), 148.9 (C-2), 135.1 (C-6), 128.6, 128.2, 128.1 (C6H5), 111.9
(C-5), 93.4 (C-300), 92.1 (C-30), 87.1 (C-10), 83.7 (C-40), 74.7 (C-20), 66.3
(CH2C5H6), 62.1 (C-50), 39.4, 32.5 (CH2), 26.0, 25.2 (SiC(CH3)3), 18.8
(CH2), 18.4, 17.8 (SiC(CH3)3), 12.2 (CH3 base), ꢄ4.5, ꢄ4.8, ꢄ5.2, ꢄ5.6
(Si(CH3)2); HRMS : C36H55N3O11SSi2Na calcd. 816.2994, found
816.2969.
(14 mL) were treated with glutaryl dichloride (64 mL, 0.49 mmol)
and quenched with MeSNa (60 mg, 0.85 mmol). The crude product

M. Moura et al. / European Journal of Medicinal Chemistry 46 (2011) 5046e5056
5055
4.3.2.8. [1-[20,50-Bis-O-tert-butyldiméthylsilyl-
b
-d-ribofuranosyl]-3-
the test compounds. Syncytia formation was recorded microscop-
ically in the HIV-infected cell cultures after 4 days. The 50% effective
concentration (EC50) of the test compounds was defined as the
compound concentration required to inhibit virus-induced cyto-
pathicity (CEM) by 50%. The 50% cytostatic/cytotoxic concentration
(CC50) was defined as the compound concentration required to
inhibit CEM cell proliferation by 50%.
N-(N-benzylaminoglutaryl) thymine]-30-spiro-500-(400-amino-100,200-
oxathiole-200,200-dioxide) (16). According to the general procedure
TSAO-T (53 mg, 9.0 ꢃ 10ꢄ2 mmol), Na2CO3 (76 mg, 0.72 mmol) and
Bu4NBr (2.9 mg, 9.0 ꢃ 10ꢄ3 mmol) in CH2Cl2 (5 mL) and water
(10 mL) were treated with glutaryl dichloride (40
mL, 0.31 mmol)
and quenched with BnNH2 (59
mL, 0.54 mmol). The crude product
was purified by HPLC to give 16 (40 mg, 56%). Rf ¼ 0.23 (EtOAc/
Cyclohexane : 5/5); Mp ¼ 120e122 ꢀC; ½a D
ꢅ
21 : ꢄ21 (c 0.1, CH2Cl2); 1H
4.4.2. HCV
NMR (CDCl3; 300 MHz) :
d
7.36e7.29 (m, 6H, C5H6 et0H-6), 6.20 (t,
4.4.2.1. Cell culture. Huh-7 human hepatoma cells [37] were grown
in Dulbecco0s modified essential medium (Invitrogen) supple-
mented with 10% fetal bovine serum.
0
0
J ¼ 5.6 Hz, 1H, NHC5H6,), 6.01 (d, J 1 ,2 ¼ 8.1 Hz, 1H, H-1 ), 5.71 (s, 2H,
NH2), 5.67 (s, 1H, H-300), 4.47e4.43 (m, 3H, CH2C6H5 et H-20), 4.37
(m, 1H, H-40), 4.02 (dd, J5 a,4 ¼ 2.7 Hz, J5 a,5 b ¼ 12.5 Hz, 1H, H-50a),
0
0
0
0
3.91 (dd, J5 b,4 ¼ 1.8 Hz, 1H, H-50b), 2.89 (t, Ja,b ¼ 6.2 Hz, 2H, CH2a),
2.39 (t, Jc,b ¼ 7.2 Hz, 2H, CH2c), 2.09 (m, 2H, CH2b), 1.96 (s, 3H, CH3
base), 1.00 (s, 9H, SiC(CH3)3), 0.86 (s, 9H, SiC(CH3)3), 0.24 (s, 3H,
Si(CH3)2), 0.23 (s, 3H, Si(CH3)2), 0.08 (s, 3H, Si(CH3)2), ꢄ0.04 (s, 3H,
4.4.2.2. HCV replication assay. A modified assay based on a pub-
lished method [38] was used. In this work, we performed a modi-
fied version containing a Luciferase reporter gene of the plasmid
encoding JFH1 genome (genotype 2a), kindly provided by T. Wakita
[39] (National Institute of Infectious Diseases, Tokyo, Japan).
Plasmid encoding the wild-type HCV genome was linearized at
the 30 end of the HCV cDNA with the restriction enzyme XbaI and
treated with the Mung Bean Nuclease (New England Biolabs).
In vitro transcripts were generated using the Megascript kit
according to the manufacturer0s protocol (Ambion). The in vitro
reaction was set up and incubated at 37 ꢀC for 4 h and transcripts
were precipitated by addition of LiCl. Ten micrograms of RNA were
delivered into Huh-7 cells by electroporation as described previ-
ously [40]. Huh-7 electroporated cells were seeded at a density of
2 ꢃ 104 cells/well in 96-well plates. Compounds were dissolved in
DMSO, diluted in Dulbecco0s modified essential medium supple-
mented with 10% fetal bovine serum in a serial fashion to create an
appropriate range of concentrations, and added to cells approx-
imatively 24 h after plating. After 72 h exposure, the media was
discarded from the assay plate and cell monolayers were lysed by
0
0
Si(CH3)2); 13C NMR (CDCl3; 75 MHz) :
d 175.3 (CO), 172.0 (CO), 161.8
(C-4), 150.2 (C-400), 149.0 (C-2), 134.7 (C-6), 128.7, 127.8127.5 (C6H5),
112.0 (C-5), 93.4 (C-300), 92.6 (C-30), 86.0 (C-10), 83.8 (C-40), 74.9 (C-
20), 62.2 (C-50), 43.7 (CH2C5H6), 39.3, 34.7 (CH2), 26.0, 25.2
(SiC(CH3)3), 19.7 (CH2), 18.4, 17.8 (SiC(CH3)3), 12.2 (CH3
base), ꢄ4.5, ꢄ4.8, ꢄ5.1, ꢄ5.7 (Si(CH3)2); HRMS : C36H56N4O10SSi2Na
calcd. 815.3153, found 815.3151.
4.3.2.9. [1-[20,50-Bis-O-tert-butyldiméthylsilyl-
b-d-ribofuranosyl]-3-
N-(methoxyisophtaloyl) thymine]-30spiro-500-(400-aminoꢄ-1200,200-oxa-
thiole-200,200-dioxide) (17). TSAO-T (52 mg, 9.6 ꢃ 10
mmol),
Na2CO3 (74 mg, 0.70 mmol) and Bu4NBr (2.8 mg, 8.7 ꢃ 10ꢄ3 mmol)
were dissolved in a mixture of CH2Cl2 (3 mL) and water (6 mL).
Isophtaloyle dichloride (23 mg, 0.11 mmol) was added to the
mixture with vigorous stirring at 8 ꢀC until disappearance of the
starting material (mass spectrometry monitoring). After 15 min the
reaction mixture was quenched with MeOH (2 mL, 0.049 mol).
Vigorous stirring was continued at room temperature between 5
and 15 min. Water (10 mL) and CH2Cl2 (10 mL) were added; the
organic phase was separated, washed with water (3 ꢃ 10 mL), dried
(Na2SO4) and concentrated. The crude product was purified by
HPLC to give 17 (28 mg, 42%). Rf ¼ 0.48 (EtOAc/Cyclohexane : 5/5);
addition of 100 ml of Renilla Luciferase Assay Lysis Buffer (Promega)
with incubation at 20 ꢀC for 30 min on an orbital shaker. Following
incubation, luminescence was assessed as indicated by the manu-
facturer (Promega) on a CentroXS3 LB960 Berthold. EC50 and CC50
values were determined using plots of luminescence versus log
compound concentration.
Mp ¼ 99e100 ꢀC; ½a D
ꢅ
21: ꢄ26 (c 0.1, CH2Cl2); 1H NMR (CDCl3;
300 MHz) :
d
8.57 (s, 1H, H-1 C6H4), 8.35 (d, J
¼ 7.8 Hz, 1H, H-4
4,2
Acknowledgments
C6H4), 8.14 (d, J 2,3 ¼ 7.8 Hz, 1H, H-2 C6H4), 7.63 (m, 1H, H-3 C6H4),
7.35 (s, 1H, H-6), 5.89 (d, J 1 ,2 ¼ 7.8 Hz, 1H, H-10), 5.65 (s, 1H, H-300),
0
0
We thank the Conseil Régional de Picardie for financial support
and D.Lesur for his helpful analysis work. Mrs. Leen Ingels provided
excellent technical assistance for the HIV assays. The HIV research
of JB was supported by the K.U.Leuven (GOA no. 10/014).
5.51 (s, 2H, NH2), 4.65 (d, 1H, H-20), 4.37 (m, 1H, H-40), 4.02 (dd,
J5 a,4 ¼ 2.6 Hz, J5 a,5 b ¼ 12.4 Hz, 1H, H-50a), 3.97 (s, 3H, OCH3), 3.92
0
0
0
0
(dd, J5 b,4 ¼ 2.7 Hz, 1H, H-50b), 2.05 (s, 3H, CH3 base), 0.99 (s, 9H,
SiC(CH3)3), 0.89 (s, 9H, SiC(CH3)3), 0.23 (s, 3H, Si(CH3)2), 0.22 (s, 3H,
Si(CH3)2), 0.09 (s, 3H, Si(CH3)2), ꢄ0.04 (s, 3H, Si(CH3)2); 13C NMR
0
0
Appendix. Supplementary material
(CDCl3; 75 MHz) :
d 167.9 (COC6H4), 165.6 (COOMe), 162.5 (C-4),
150.4 (C-400), 149.4 (C-2), 135.9 (C-6), 135.8e129.4 (C6H4), 111.9 (C-
5), 93.1 (C-300), 92.2 (C-30), 87.6 (C-10), 83.8 (C-40), 74.6 (C-20), 62.1
(C-50), 52.5 (OMe), 26.0, 25.3 (SiC(CH3)3), 18.4, 17.9 (SiC(CH3)3), 12.3
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.08.017.
(CH3 base), ꢄ4.5, ꢄ4.9, ꢄ5.0, ꢄ5.7 (Si(CH3)2). HRMS
:
References
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