Synthesis and antiviral activity of azoles obtained from carbohydrates

Article abstract of DOI:10.1016/j.carres.2008.06.028

Herein we describe the synthesis of 1,2,4-triazolyl-3-thione;1,3,4-oxadiazole, and imidazo[2,1-b]thiazole derivatives from carbohydrates. The antiviral activity of these compounds was tested against Dengue and Junin virus (the etiological agent of Argenti

Full text of DOI:10.1016/j.carres.2008.06.028

Carbohydrate Research 343 (2008) 2468–2474
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis and antiviral activity of azoles obtained from carbohydrates
b
José Sebastián Barradas a, María Inés Errea a, Norma B. D’Accorso a, , Claudia S. Sepúlveda ,
*
Laura B. Talarico b, Elsa B. Damonte b,
a CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria,
1428 Buenos Aires, Argentina
b Laboratorio de Virología, Departamento de Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria,
1428 Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe the synthesis of 1,2,4-triazolyl-3-thione;1,3,4-oxadiazole, and imidazo[2,1-b]thiazole
derivatives from carbohydrates. The antiviral activity of these compounds was tested against Dengue and
Junin virus (the etiological agent of Argentine hemorrhagic fever). The 3-(p-bromobenzoyl)-5-(1,2-O-iso-
Received 21 November 2007
Received in revised form 1 June 2008
Accepted 28 June 2008
propylidene-3-O-methyl-
a-D-xylofuranos-5-ulos-5-yl)imidazo[2,1-b]thiazole was able to inhibit the
Available online 8 July 2008
replication of both viruses in Vero cells at concentration significantly lower than the CC50
.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
1,2,4-Triazolyl-3-thione
1,3,4-Oxadiazole
Imidazo[2,1-b]thiazole
Carbohydrates
Antiviral activity
Azoles are heterocyclic compounds containing at least two nitro-
gen atoms in their structure and are proved to be clinically useful
agents against different kinds of diseases. They have been shown to
possess a broad spectrum of biological activity depending on their
particular structure. In fact, azoles constitute the largest group of
antifungal compounds clinically in use. Antitumoral, antiviral, anti-
bacterial, anti-inflammatory, and antihelminthic properties have
also been reported.1–5
In an attempt to correlate structure with antiviral activity, here-
in we report the synthesis and antiviral evaluation (against Dengue
and Junin virus) of 1,3,4-oxadiazoles, 1,2,4-triazolyl-3-thiones and
imidazo[2,1-b]thiazoles substituted with a carbohydrate derivative
and a halophenyl group. The choice of heterocyclic ring substitu-
ents was based on literature reports where the increase of biolog-
ical activity in several compounds was related to the presence of
asymmetric centers6 and/or halogens in their structure.7,8 On the
other hand, according to the current tendency of using compounds
that come from the renewable biomass, syntheses were carried out
using D-glucose as starting material.
Selective replacement of primary hydroxyl group in 1,2-O-iso-
propylidene-3-O-methyl-a-D a bromine
-glucofuranose (1)9 by
atom, by using the N-bromosuccinimide/DMF system, led to 2 with
acceptable yield as was previously described for related
compounds.10 Furthermore, an excellent primary versus secondary
hydroxyl group selectivity was observed, since the dibromocarbo-
hydrate derivate was not detected. These results reinforce those
previously reported for pyranose forms, making this methodology
a simple and versatile way to introduce a bromine atom into a
monosaccharide structure.
Compound 3 was obtained after treating 2 with 1-hydroxy-1,2-
benziodoxol-3(1H)-one-1-oxide (IBX) in DMSO as oxidizing
agent.11 The lack of the signal corresponding to H-5 in the 1H
NMR spectrum of 3, and the observation of the carbonyl signal
(198.8 ppm) in the 13C NMR spectrum, allowed confirmation that
oxidation had taken place. The 5,6-anhydro-1,2-isopropylidene-
3-O-methyl-a-D-glucofuranose was detected by NMR as a second-
ary product (see Section 1).
The coupling between compounds 3 and 4a/b led to the
corresponding heterocyclic derivatives 5a/b. In the 1H NMR spectra
the signals of the heterocyclic protons appeared at 8.94 and
8.51 ppm for both derivatives (5a/b), while in their 13C NMR spec-
tra the resonance corresponding to the heterocyclic carbons was
observed in a range between 159 and 128 ppm. In addition, the
appearance of aromatic signals and the absence of the methylenic
ones confirmed that the transformation 3?5 had occurred (See
Scheme 1).
Substituted imidazo[2,1-b]thiazoles (5a/b) were obtained by a
convergent synthetic pathway (Scheme 1), the synthesis of 6-
bromo-6-deoxy-1,2-O-isopropylidene-3-O-methyl-a-D-xylo-hexo-
furanos-5-ulose (3) being the key step of this synthetic approach.
* Corresponding author. Tel./fax: +54 11 45763346.
E-mail address: norma@qo.fcen.uba.ar (N. B. D’Accorso).
Research members of the National Research Council of Argentina (CONICET).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.06.028

J. S. Barradas et al. / Carbohydrate Research 343 (2008) 2468–2474
2469
HO
HO
Br
HO
Br
O
a
b
OMe
OMe
OMe
O
O
O
O
O
O
O
O
O
Me
Me
Me
Me
Me
Me
1
3
2
c
S
S
N
R
R
O
N
N
N
N
4a/b
OMe
O
O
R = a -COC6H5
b -COC6H4Br-p
O
5a/b
Me
Me
Scheme 1. Reagents and conditions: (a) NBS, PPh3 (1:2 equiv)/DMF (anhyd), 2: 72%; (b) IBX/DMSO (anhyd), 3: 18%; (c) THF/Et, 5a: 27%, 5b: 30%.
In order to obtain other azole derivatives and taking into
in the 13C NMR spectrum was considered diagnostic of the conver-
sion, since the replacement of the azido by an isothiocyanate group
shifts the C-6 resonance upfield18 (see Section 1).
account our previous studies,12 a series of 5-halophenyl-2-(1,2-O-
isopropylidene-a-D-xylo-tetrafuranosyl)-1,3,4-oxadiazoles (8a–f)
were synthesized (Scheme 2). Compounds 7a–f were obtained by
Reaction of 10 with either 4-chloro- or 4-bromobenzoic hydra-
zide led to thiosemicarbazides 11a and 11b in quantitative yield,
which by a further intramolecular cyclization19 gave the corres-
ponding 1,2,4-triazoline-3-thiones (12a/b). The NMR spectra of
12a/b showed the lack of the NH and carbonyl signals. In addition,
in their 13C NMR spectra the signal characteristic of a C@N bond
was found at 152.9 ppm while that corresponding to the C@S bond
was moved from 182.4 ppm (11a/b) to 169 ppm (12a/b).
The antiviral activity of the new compounds was evaluated
against two viruses with an RNA genome, the Junin virus (JUNV)
and the dengue virus type 2 (DENV-2). These viruses were chosen
because they are human pathogenic agents of severe diseases such
as dengue fever, dengue hemorrhagic fever, and Argentine hemor-
rhagic fever and because there is currently no specific antiviral
chemotherapy for patient treatment. The toxicity for Vero cells
was first investigated and taking into account the cytotoxicity
results (Table 1), the antiviral activity of the azole derivatives
coupling 1,2-O-isopropylidene-a-D-xylopentadialdo-1,4-furanose
(6)13,14 with a series of benzoyl hidrazines. The intramolecular
cyclization of 7a–f gave the corresponding 1,3,4-oxadiazole deriv-
atives 8a–f.
In the 1H NMR spectra of compounds 7a–f the expected two
broad singlets around 8 and 11 ppm corresponding to the NH
groups were observed, while their 13C NMR spectra showed signals
of carbonyl groups around 170 ppm.
The 1H and 13C NMR spectra of 7a and 7d (o-halophenyl deriv-
atives) showed the presence of a diastereomeric mixture (syn and
anti isomers). Assignment of each isomer in the 1H NMR spectrum
was made taking into account data previously reported for related
compounds.15 In the NMR spectra of 7b, 7c, 7e, and 7f only one
isomer was observed, and the resonance of C-5 around 149 ppm
suggested the presence of the anti isomer.
In the 13C NMR spectra of 8a–f the signals of both heterocyclic
carbons were observed between 162 and 164 ppm, confirming that
the intramolecular cyclization of 7a–f had occurred.
was then evaluated at concentrations below 100
seen in Table 1, the thiazole 5b was able to inhibit the replication
of both viruses in Vero cells with EC50 values of 12.0 and 29.9
lM. As it can be
In order to extend the study to other azole derivates, substituted
1,2,4-triazoline-3-thiones were synthesized according to the syn-
thetic pathway shown in Scheme 3. The 6-deoxy-6-isothiocyanate
(10) was obtained from the 3,5-di-O-acetyl-6-deoxy-1,2-O-isopro-
lM
against JUNV and DENV-2, respectively. Thus, the selectivity index
(SI), that is, the relationship between antiviral activity and cytotox-
icity, expressed as the ratio CC50/EC50, for this compound was 10.2
and 4.1, respectively. Although these values of SI are not very high,
they are indicative of a specific inhibitory action of the compound
pylidene-6-azido-a-D
-glucofuranose (9)16,17 in quantitative yield.
The chemical shift of the signal corresponding to the C-6 observed
R
O
OH
O
a
O
b
N
C
OH
N
O
HN
O
O
N
X
O
OH
6
O
O
7(a-f)
O
Me
Me
8 (a-f)
O
Me
Me
O
a
d
e
-C6H4Cl -o
R = b -C6H4Cl -m
-C6H4Cl -p
-C6H4Br -o
Me
Me
-C6H4Br -m
-C6H4Br -p
c
f
Scheme 2. Reagents and conditions: (a) o, m, p-bromo or chlorobenzoic hydrazide/THF, 7a: 34%, 7b: 63%, 7c: 94%, 7d: 76%, 7e: 70%, 7f: 86%; (b) PIDA/MeOH, 8a: 40%, 8b: 50%,
8c: 40%, 8d: 58%, 8e: 54%, 8f: 34%.

2470
J. S. Barradas et al. / Carbohydrate Research 343 (2008) 2468–2474
O
S
RCHNHNCHN
AcO
N3
HO
SCN
AcO
OH
OAc
OAc
O
a
O
b
O
O
O
O
O
9
O
O
10
Me
11a/b
Me
Me
Me
Me
Me
c
N
NH
R = a -C6H4Cl-p
R = b -C6H4Br-p
S
R
N
OH
HO
O
O
12a/b
O
Me
Me
Scheme 3. Reagents and conditions: (a) (i) Ac2O/Py; (ii) CS2, (Ph)3P/dioxane, 10: 100%; (b) RCONHNH2/THF, 11a: 100%, 11b: 100%; (c) (i) NaOH 2N (aq); (ii) HCl (aq), 12a: 41%,
12b: 55%.
Table 1
mesh, Merck). Solvents were reagent grade and, in most cases,
dried, and distillated before use according to standard procedures.
Cytotoxicity and antiviral activity
Compound
CC50
(
l
M)a
EC50 (l
M)b
1.2. 6-Bromo-6-deoxy-1,2-O-isopropylidene-3-O-methyl-a-D-
glucofuranose (2)
JUNV
DENV-2
5a
5b
8a
8b
8c
8d
8e
8f
12a
12b
137.4
122.6
547.2
343.9
364.4
692.0
221.0
251.0
210.3
245.3
>100
12.0
>100
29.9
NBS (1.77 g, 10.08 mmol) in DMF was added to a stirred solu-
tion of triphenylphosphine (2.62 g, 9.97 mmol) and 19 (1.25 g,
5.34 mmol) in dry DMF under an argon atmosphere.10 The mixture
was stirred for 1 h at room temperature and quenched by addition
of MeOH. The solvent was evaporated, and the residue was
extracted with CH2Cl2/water and dried (Na2SO4). The crude prod-
uct was purified by column chromatography on silica gel (80:20
cyclohexane/acetone) affording the 6-bromo derivative (2) as
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
64.6
>100
>100
a
CC50 (cytotoxic concentration 50%): concentration required to reduce 50% of the
number of viable Vero cells.
pallid yellow oil (1.14 g, 72% yield); ½a D25
ꢁ44.5 (c 3.4, chloroform).
ꢀ
b
EC50 (effective concentration 50%): concentration required to reduce virus yield
Anal. Calcd for C10H17BrO5: C, 40.42; H, 5.77. Found: C, 40.45; H,
5.75. 1H NMR (500 MHz, CDCl3) d: 5.82 (d, 1H, J1,2 3.7 Hz, H-1),
4.54 (d, 1H, J2,1 3.7 Hz, H-2), 3.84 (d, 1H, J3,4 3.0. Hz, H-3), 4.06
(dd, 1H, J4,3 2.9 Hz, J4,5 8.3 Hz, H-4), 4.92 (ddd, 1H, J5,4 8.3 Hz, J5,6a
6.1 Hz; J5,6b 3.0 Hz, H-5), 3.53 (dd, 1H, J6a,5 6.1 Hz, J6a,6b 10.7 Hz,
H-6a), 3.67 (dd, 1H, J6b,5 3.0 Hz, J6b,6a 10.7 Hz, H-6b), 3.41 (s, 3H,
OMe), 1.43 (s, 3H, CMe2); 1.27 (s, 3H, CMe2); 13C NMR (125 MHz,
CDCl3) d: 105.1 (C-1), 81.5 (C-2), 83.8 (C-3), 80.7 (C-4), 68.0 (C-5),
38.3 (C-6), 111.9 (C Me2), 58.0 (OMe), 26.9 (CMe2), 26.3 (CMe2).
in Vero cells by 50%.
against virus multiplication at concentrations significantly lower
than the CC50. The oxadiazole 8f also exhibited antiviral action
against DENV-2 with an EC50 value of 64.6 lM, but with selectivity
lower than that of 5b (Table 1). No virucidal activity was detected
in any compound. When comparing the antiviral activity of 5a and
5b, we observed an increased activity in the latter that could only
be attributed to the presence of a bromine atom, showing the con-
venience of including halogens in the antiviral drugs designed.
1.3. 6-Bromo-6-deoxy-1,2-O-isopropylidene-3-O-methyl-a-D-
xylo-hexofuranos-5-ulose (3)
1-Hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide (IBX) (1.55 g,
5.53 mmol) was added to a solution of 2 (1.09 g, 3.69 mmol) in
DMSO, and the mixture was stirred for 72 h at room temperature
and extracted with NaOH (aq)/CHCl3. The organic layer was dried
(Na2SO4) and evaporated. The residue was purified by column
chromatography on silica gel (90:10 cyclohexane/acetone).
Although TLC analyses of the product showed one spot, a second-
ary product was detected in the sample by NMR. Further purifica-
tion of the mixture on silica gel (98:2 CH2Cl2/acetone) led to pure
1. Experimental
1.1. General methods
NMR spectra were recorded on either a Bruker AC-200 or a Bru-
ker AMX-500 spectrometer. Assignments were confirmed with the
aid of two-dimensional techniques 1H, 1H (COSY 45) and 1H, 13C
(HSQC). For the assignment of the 13C NMR spectra, DEPT experi-
ments were also conducted. Analytical TLC was conducted on Silica
Gel 60G (Merck) on precoated plates. Optical rotations were mea-
sured with a Perkin–Elmer Model 343 Polarimeter. Column-chro-
matographic separations were performed on Silica Gel (240–400
compound 3 as a pallid yellow oil (143.56 mg, 18% yield); ½a D25
ꢀ
ꢁ76.9 (c 3.6, chloroform). Anal. Calcd for C10H15BrO5: C, 40.70; H,
5.12. Found: C, 40.72; H, 5.09. 1H NMR (500 MHz, CDCl3) d: 6.05
(d, 1H, J1,2 3.5 Hz, H-1), 4.60 (d, 1H, J2,1 3.5 Hz, H-2), 4.08 (d, 1H,

J. S. Barradas et al. / Carbohydrate Research 343 (2008) 2468–2474
2471
J3,4 3.7 Hz, H-3), 4.96 (d, 1H, J4,3 3.7 Hz, H-4), 4.36 (d, 1H, J6a,6b
15.7 Hz, H-6a), 4.23 (d, 1H, J6b,6a 15.7 Hz, H-6b), 3.50 (s, 3H,
OMe), 1.51 (s, 3H, CMe2), 1.31 (s, 3H, CMe2); 13C NMR (125 MHz,
CDCl3) d: 106.6 (C-1), 81.0 (C-2), 85.9 (C-3), 84.7 (C-4), 198.8 (C-
5), 35.5 (C-6), 112.6 (CMe2), 58.03 (OMe), 26.9 (CMe2), 26.3 (CMe2).
The secondary product was identified as 5,6-anhydro-1,2-O-iso-
d: 106.0 (C-1), 80.9 (C-2), 86.3 (C-3), 85.0 (C-4), 140.1, 130.4,
129.6, 127.2 (aromatic carbons), 156.5, 147.2, 134.9, 129.0, 128.4
(imidazo[2,1-b] carbons), 186.1 (CO), 185.1 (CO), 112.8 (CMe2),
58.5 (OMe), 27.0 (CMe2), 26.4 (CMe2).
1.6. General procedure for the synthesis of benzoylhidrazones
(7a–f)
propylidene-3-O-methyl-a-D
-glucofuranose; 1H NMR (500 MHz,
CDCl3) d: 5.92 (d, 1H, J1,2 3.7 Hz, H-1), 4.62 (d, 1H, J2,1 3.6 Hz,
H-2), 3.86 (d, 1H, J3,4 3.2 Hz, H-3), 3.72 (dd, 1H, J4,3 3.2 Hz, J4,5
7.1 Hz, H-4), 3.24 (ddd, 1H, J5,4 6.9 Hz, J5,6a 3.9 Hz, J5,6b 2.6 Hz,
H-5), 2.91 (dd, 1H, J6a,5 3.9 Hz, J6a,6b 5.1 Hz, H-6a), 2.77 (dd, 1H,
J6b,5 3.2 Hz, J6b,6a 5.1 Hz, H-6b), 3.45 (s, 3H, OMe), 1.47 (s, 3H,
CMe2), 1.32 (s, 3H, CMe2); 13C NMR (125 MHz, CDCl3) d: 105.2
(C-1), 81.9 (C-2), 84.3 (C-3), 81.5 (C-4), 47.9 (C-5), 46.8 (C6),
111.8 (CMe2), 58.2 (OMe), 26.7 (CMe2), 26.1 (CMe2).
A solution of 613,14 and the corresponding benzoylhydrazide in
dry THF (1:1 molar ratio) was refluxed with continuous stirring for
2 h. The mixtures were concentrated, and from these solutions
compounds 7(a–f) crystallized.
1.6.1. 1,2-O-Isopropylidene-a-D-xylopentadialdo-1,4-furanose
(o-chlorobenzoyl)hydrazone (7a)
34% yield as a mixture of anti and syn isomers (anti:syn 58:42).
Anal. Calcd for C15H17ClN2O5: C, 52.87; H, 5.03. Found: C, 52.90; H,
4.98. 1H NMR (500 MHz, CD3COCD3) Anti isomer d: 5.93 (d, 1H, J1,2
3.5 Hz, H-1), 4.56 (d, 1H, J2,1 3.5 Hz, H-2), 4.32 (d, 1H, J3,4 2.9, H-3),
4.67 (dd, 1H, J4,3 3.0, J4,5 6.4, H-4), 7.52–7.36 (m, 5H, H-5 + aromatic
protons), 1.44 (s, 3H, CMe2), 1.26 (s, 3H, CMe2), 10.82 (s, 1H, NH)
3.01, (s, 1H, OH); Syn isomer d: 5.88 (d, 1H, J1,2 3.3 Hz, H-1), 4.50
(d, 1H, J2,1 3.5 Hz, H-2), 4.11 (d, 1H, J3,4 2.9 Hz, H-3), 4.35 (dd, 1H,
J4,3 3.0 Hz, J4,5 7.1, H-4), 7.71 (d, 1H J5,4 6.4 Hz, H-5), 7.52–7.36
(m, 4H, aromatic protons), 1.31 (s, 3H, CMe2), 1.21 (s, 3H, CMe2),
10.73 (s, 1H, NH), 3.01, (s, 1H, OH); 13C NMR (125 MHz, CD3COCD3)
Anti isomer d: 107.0 (C-1), 87.3 (C-2), 78.2 (C-3), 82.1 (C-4), 149.4
(C-5), 137.5–128.2 (aromatic carbons), 164.3 (CO), 112.9 (CMe2),
27.9 (CMe2), 27.2 (CMe2); d: Syn isomer d 106.9 (C-1), 87.1 (C-2),
78.1 (C-3), 81.8 (C-4), 145.6 (C-5), 137.5–128.2 (aromatic carbons),
164.3 (CO), 112.7 (CMe2), 27.8 (CMe2), 26.9 (CMe2).
1.4. N0-(5-Benzoylthiazol-2-yl)-N,N-dimethylformamidine and
N0-(5-p-bromobenzoylthiazol-2-yl)-N,N-dimethylformamidine
(4a/b)
Compounds 4a/b were synthesized from N,N0-bis(dimethylami-
nomethylene)thiourea as was previously described for 4b.20
Compound 4a: (light yellow crystals, 451.2 mg, 65% yield); mp:
114 °C. Anal. Calcd for C13H13N3OS: C, 60.21; H, 5.05; N, 16.20; S,
12.37. Found: C, 60.10; H, 5.22; N, 16.01; S, 12.30. 1H NMR
(200 MHz, CDCl3) d: 8.3 (s, 1H), 7.79 (s, 1H), 7.75–7.43 (m, 5H, aro-
matic protons), 3.11 (s, 3H), 3.08 (s, 3H); 13C NMR (50 MHz, CDCl3)
d: 187.3, 180.9, 156.5, 149.2, 138.4–128.5, 41.2, 45.3.
1.5. General procedure for the synthesis of imidazo[2,1-b]-
thiazoles (5a,b)
Compound 3 (140.71, 0.48 mmol) in THF was added to a solu-
tion of 4a (125.43 mg, 0.48 mmol) or 4b (163.34 mg, 0.48 mmol)
in dry THF. The reaction mixture was heated at reflux for 48 h,
2 equiv of Et3N were added, and the mixture was stirred for 24 h
at room temperature and evaporated. The crude product (5a or
5b) was purified by column chromatography on silica gel (75:25
cyclohexane/acetone).
1.6.2. 1,2-O-Isopropylidene-a-D-xylopentadialdo-1,4-furanose
(m-chlorobenzoyl)hydrazone (7b)
63% yield; ½a 2D5
ꢁ48.6 (c 0.9, chloroform). Anal. Calcd for
ꢀ
C
15H17ClN2O5: C, 52.87, H, 5.03. Found: C, 52.76; H, 5.08. 1H NMR
(500 MHz, CD3COCD3) d: 5.98 (d, 1H, J1,2 3.5 Hz, H-1), 4.60 (d, 1H,
J2,1 3.5 Hz, H-2), 4.35 (d, 1H, J3,4 2.7 Hz, H-3), 4.68 (dd, 1H, J4,3
2.7 Hz, H-4), 7.71 (d, 1H J5,4 6.4 Hz, H-5), 7.93–7.78 (m, 3H,
H-5 + aromatic protons), 7.63–7.48 (m, 2H, aromatic protons),
1.42 (s, 3H, CMe2), 1.32 (s, 3H, CMe2), 11.16 (s, 1H, NH), 2.99 (s,
1H, OH); 13C NMR (125 MHz, CD3COCD3) d: 106.3 (C-1), 86.7
(C-2), 77.4 (C-3), 81.4 (C-4), 149.4 (C-5), 136.9–128.7 (aromatic
carbons), 163.0 (CO), 112.2 (CMe2), 27.1 (CMe2), 26.4 (CMe2).
1.5.1. 3-Benzoyl-5-(1,2-O-isopropylidene-3-O-methyl-
a-D-
xylofuranos-5-ulo-5-yl)imidazo[2,1-b]thiazole (5a)
White waxy appearance, 55.52 mg, 27% yield, ½a D25
ꢁ99.2 (c 2.8,
ꢀ
chloroform). Anal. Calcd for C21H20N2O6S: C, 58.87; H, 4.70; N,
6.54; S, 7.48. Found: C, 58.70; H, 4.60; N, 6.60; S, 7.50. 1H NMR
(500 MHz, CDCl3) d: 6.18 (d, 1H, J1,2 3.60 Hz, H-1), 4.67 (d, 1H, J2,1
3.60 Hz, H-2), 4.19 (d, 1H, J3,4 3.60 Hz, H-3), 5.06 (d, 1H, J4,3
3.60 Hz, H-4), 7.91–7.55 (m, 5H, aromatic protons), 8.94 (s, 1H,
imidazo[2,1-b] proton), 8.51 (s, 1H, imidazo[2,1-b] proton), 3.31
(s, 3H, OMe), 1.54 (s, 3H, CMe2), 1.38 (s, 3H, CMe2); 13C NMR
(125 MHz, CDCl3) d: 105.8 (C-1), 80.7 (C-2), 86.1 (C-3), 84.8 (C-4),
136.4–128.8 (aromatic carbons), 156.5, 146.9, 134.6, 128.3 (imi-
dazo[2,1-b] carbons), 187.2 (CO), 184.8 (CO), 112.5 (CMe2), 58.3
(OMe), 26.9 (CMe2), 26.2 (CMe2).
1.6.3. 1,2-O-Isopropylidene-a-D-xylopentadialdo-1,4-furanose
(p-chlorobenzoyl)hydrazone (7c)
94% yield; ½a 2D5
ꢁ56.5 (c 0.6, chloroform). Anal. Calcd for
ꢀ
C
15H17ClN2O5: C, 52.87; H, 5.03. Found: C, 52.77; H, 5.15. 1H
NMR (500 MHz, CD3COCD3) d: 5.96 (d, 1H, J1,2 3.7 Hz, H-1), 4.57
(d, 1H, J2,1 3.6 Hz, H-2), 4.31 (d, 1H, J3,4 2.7 Hz, H-3), 4.68 (dd, 1H,
J4,3 2.7 Hz, H-4), 7.81 (d, 1H, J 6.4 Hz, H-5), 7.96 (d, 2H, J 7.9 Hz, aro-
matic protons), 7.48 (d, 2H, J 8.2 Hz, aromatic protons), 1.42 (s, 3H,
CMe2), 1.26 (s, 3H, CMe2), 11.3 (s, 1H, NH), 3.09 (s, 1H, OH); 13C
NMR (125 MHz, CD3COCD3) d: 106.9 (C-1), 87.2 (C-2), 78.2 (C-3),
82.1 (C-4), 149.8 (C-5), 138.9, 133.3, 131.0, 130.1 (aromatic car-
bons), 164.1 (CO), 112.9 (CMe2), 27.9 (CMe2), 27.1 (CMe2).
1.5.2. 3-(p-Bromobenzoyl)-5-(1,2-O-isopropylidene-3-O-
methyl-a-D-xylofuranos-5-ulo-5-yl)imidazo[2,1-b]thiazole (5b)
White waxy appearance, 73.07 mg, 30% yield, ½a D25
ꢁ120.5 (c
ꢀ
1.2, chloroform). Anal. Calcd for C21H19BrN2O6S: C, 49.71; H,
3.77; N, 5.52; S, 6.32. Found: C, 49.80; H, 3.70; N, 5.60; S, 6.40.
1H NMR (500 MHz, CDCl3) d: 6.18 (d, 1H, J1,2 3.60 Hz, H-1), 4.67
(d, 1H, J2,1 3.60 Hz, H-2), 4.19 (d, 1H, J3,4 3.60 Hz, H-3), 5.06 (d,
1H, J4,3 3.60 Hz, H-4), 7.85 (d, 2H, J 8.6 Hz, aromatic protons),
7.56 (d, 2H, J 8.6 Hz, aromatic protons), 8.94 (s, 1H, imidazo[2,1-b]
proton), 8.51 (s, 1H, imidazo[2,1-b] proton), 3.33 (s, 3H, OMe),
1.54 (s, 3H, CMe2), 1.39 (s, 3H, CMe2); 13C NMR (125 MHz, CDCl3)
1.6.4. 1,2-O-Isopropylidene-a-D-xylopentadialdo-1,4-furanose
(o-bromobenzoyl)hydrazone (7d)
76% yield as a mixture of anti and syn isomers (anti:syn 61:49).
Anal. Calcd for C15H17BrN2O5: C, 46.87; H, 4.45. Found: C, 47.01; H,
4.47. 1H NMR (500 MHz, CD3COCD3) Anti isomer d: 5.93 (d, 1H, J1,2
3.3 Hz, H-1), 4.55 (d, 1H, J2,1 3.3 Hz, H-2), 4.29 (d, 1H, J3,4 2.9 Hz,
H-3), 4.66 (dd, 1H, J4,3 2.8 Hz, J4,5.6.3 Hz, H-4), 7.63–7.33 (m, 5H,
H-5 + aromatic protons), 1.42 (s, 3H, CMe2), 1.24 (s, 3H, CMe2),

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J. S. Barradas et al. / Carbohydrate Research 343 (2008) 2468–2474
10.90 (s, 1H, NH) 3.10, (s, 1H, OH); Syn isomer d: 5.87 (d, 1H, J1,2
3.3 Hz, H-1), 4.49 (d, 1H, J2,1 3.5 Hz, H-2), 4.10 (d, 1H, J3,4 2.6 Hz,
H-3), 4.29 (m, 1H, H-4), 7.70 (d, 1H, J5,4 6.3 Hz, H-5), 7.63–7.33
(m, 4H, aromatic protons), 1.35 (s, 3H, CMe2), 1.20 (s, 3H, CMe2),
10.80 (s, 1H, NH), 3.10, (s, 1H, OH); 13C NMR (125 MHz, CD3COCD3)
Anti isomer d: 106.4 (C-1), 86.7 (C-2), 77.5 (C-3), 81.5 (C-4); 148.9
(C-5), 134.1–128.6 (aromatic carbons), 164.7 (CO), 112.4 (CMe2),
27.3 (CMe2), 26.5 (CMe2); d: Syn isomer d 106.3 (C-1), 86.5 (C-2),
77.5 (C-3), 81.2 (C-4), 145.2 (C-5), 134.1–128.6 (aromatic carbons),
164.7 (CO), 112.1 (CMe2), 27.2 (CMe2), 26.4 (CMe2).
2H, H-2 + H-3), 5.43 (d, 1H, J3,4 2.4 Hz, H-4), 7.85–7.24 (m, 4H,
aromatic protons), 1.57 (s, 3H, CMe2), 1.38 (s, 3H, CMe2), 4.61 (s,
1H, –OH); 13C NMR (125 MHz, CDCl3) d: 105.7 (C-1), 84.6 (C-2),
74.7 (C-3), 76.1 (C-4), 135.1–124.4 (aromatic carbons), 164.3,
162.7 (oxadiazole carbons), 112.6 (CMe2), 26.9 (CMe2), 26.2 (CMe2).
1.7.3. 2-(1,2-O-Isopropylidene-a-D-xylo-tetrafuranos-4-yl)-5-
(p-chlorophenyl)-l,3,4-oxadiazole (8c)
40% yield; mp: 193–195 °C; ½a D25
ꢀ
+3.2 (c 3.1, chloroform). Anal.
Calcd for C15H15ClN2O5: C, 53.19; H, 4.46. Found: C, 52.87; H, 4.44.
1H NMR (500 MHz, CDCl3) d: 6.15 (d, 1H, J1,2 3.5 Hz, H-1), 4.74 (d,
2H, H-2 + H-3), 5.39 (1H, J3,4 2.6 Hz, H-4), 7.91 (d, 2H, J 8.6 Hz, aro-
matic protons), 7.43 (d, J 8.8 Hz, 2H, aromatic protons), 1.58 (s, 3H,
CMe2), 1.38 (s, 3H, CMe2), 4.31 (s, 1H, –OH); 13C NMR (125 MHz,
CDCl3) d: 105.7 (C-1), 84.4 (C-2), 74.4 (C-3), 76.1 (C-4), 138.6–
121.4 (aromatic carbons), 164.7, 162.5 (oxadiazole carbons),
112.6 (CMe2), 26.9 (CMe2), 26.1 (CMe2).
1.6.5. 1,2-O-Isopropylidene-a-D-xylopentadialdo-1,4-furanose
(m-bromobenzoyl)hydrazone (7e)
70% yield; ½a 2D5
ꢁ59.3 (c 1.1, chloroform). Anal. Calcd for
ꢀ
C
15H17BrN2O5: C, 46.87; H, 4.45. Found: C, 46.90; H, 4.40. 1H
NMR (500 MHz, CD3COCD3) d: 5.98 (d, 1H, J1,2 3.5 Hz, H-1), 4.60
(d, 1H, J2,1 3.5 Hz, H-2), 4.35 (d, 1H, J3,4 2.7 Hz, H-3), 4.68 (dd, 1H,
J4,3 2.7 Hz, J4,5 6.4 Hz, H-4), 7.81 (d, 1H, J5,4 6.5 Hz, H-5), 8.09 (s,
1H, aromatic proton), 7.92 (d, 1H, J 7.5 Hz, aromatic proton), 7.74
(d, 1H, J 7.9 Hz, aromatic proton), 7.43 (t, 1H, J 7.8, aromatic pro-
ton), 1.44 (s, 3H, CMe2), 1.27 (s, 3H, CMe2), 11.32 (s, 1H, NH),
4.93 (s, 1H, OH); 13C NMR (125 MHz, CD3COCD3) d: 106.3 (C-1),
86.5 (C-2), 77.5 (C-3), 81.4 (C-4), 149.3 (C-5), 136.2–122.8 (aro-
matic carbons), 163.0 (CO), 112.2 (CMe2), 27.2 (CMe2), 26.5 (CMe2).
1.7.4. 2-(1,2-O-Isopropylidene-a-D-xylo-tetrafuranos-4-yl)-5-
(o-bromophenyl)-l,3,4-oxadiazole (8d)
58% yield; mp: 69–71 °C; ½a D25
ꢁ1.7 (c 1.8, chloroform). Anal.
ꢀ
Calcd for C15H15BrN2O5: C, 47.02; H, 3.90. Found: C, 46.99; H,
3.87. 1H NMR (500 MHz, CDCl3) d: 6.07 (d, 1H, J1,2 3.5 Hz, H-1),
4.65 (d, 2H, H-2 + H-3), 5.36 (d, 1H, J3,4 2.7 Hz, H-4), 7.79–7.21
(m, 4H, aromatic protons), 1.53 (s, 3H, CMe2), 1.39 (s, 3H, CMe2),
4.35 (s, 1H, –OH); 13C NMR (125 MHz, CDCl3) d: 105.6 (C-1), 84.5
(C-2), 74.5 (C-3), 76.0 (C-4), 134.4–121.6 (aromatic carbons),
164.1–162.7 (oxadiazole carbons), 112.4 (CMe2), 26.8 (CMe2),
26.0 (CMe2).
1.6.6. 1,2-O-Isopropylidene-a-D-xylopentadialdo-1,4-furanose
(p-bromobenzoyl)hydrazone (7f)
86% yield; ½a 2D5
ꢁ22.2 (c 1.5, chloroform). Anal. Calcd for
ꢀ
C
15H17BrN2O5: C, 46.87; H, 4.45. Found: C, 46.91; H, 4.46. 1H
NMR (500 MHz, CD3COCD3) d: 5.96 (d, 1H, J1,2 3.5 Hz, H-1), 4.59
(d, 1H, J2,1 3.6 Hz, H-2), 4.35 (d, 1H, J3,4 2.7 Hz, H-3), 4.66 (dd, 1H,
J4,3 2.7 Hz, J4,5 6.5, H-4), 7.78 (d, 1H, J5,4 6.6 Hz, H-5), 7.66 (d, 2H,
J 8.6 Hz, aromatic protons), 7.87 (d, 2H, J 8.1, aromatic protons),
1.45 (s, 3H, CMe2), 1.28 (s, 3H, CMe2), 11.21 (s, 1H, NH), 3.10 (s,
1H, OH); 13C NMR (125 MHz, CD3COCD3) d: 106.5 (C-1), 86.7
(C-2), 77.7 (C-3), 81.6 (C-4), 149.1 (C-5), 132.6–130.6 (aromatic
carbons), 163.4 (CO), 112.4 (CMe2), 27.4 (CMe2), 26.7 (CMe2).
1.7.5. 2-(1,2-O-Isopropylidene-a-D-xylo-tetrafuranos-4-yl)-5-
(m-bromophenyl)-l,3,4-oxadiazole (8e)
54% yield; mp: 84–85 °C; ½a D25
ꢀ
+3.1 (c 1.9, chloroform). Anal.
Calcd for C15H15BrN2O5: C, 47.02; H, 3.90. Found: C, 46.92; H,
3.88. 1H NMR (500 MHz, CDCl3) d: 6.16 (d, 1H, J1,2 3.5 Hz, H-1),
4.74 (d, 2H, H-2 + H-3), 5.39 (d, 1H, J3,4 2.2 Hz, H-4), 8.16–7.32 (m,
4H, aromatic protons), 1.58 (s, 3H, CMe2), 1.39 (s, 3H, CMe2), 4.54
(s, 1H, –OH); 13C NMR (125 MHz, CDCl3) d: 105.8 (C-1), 84.4 (C-2),
74.2 (C-3), 76.2 (C-4), 135.2–123.2 (aromatic carbons), 164.2–
162.7 (oxadiazole carbons), 112.7 CMe2, 26.9 (CMe2), 26.1 (CMe2).
1.7. General procedure for the synthesis of l,3,4-oxadiazoles
(8a–f)
Sodium acetate trihydrate and PIDA were added in a 1.2:1
molar proportion to a stirred solution of 7a–f in MeOH. The mix-
ture was stirred at room temperature, and the reaction was fol-
lowed by TLC (80:20 cyclohexane/ethyl acetate). The reaction
medium was evaporated and extracted with CH2Cl2/water the or-
ganic layers were evaporated and the residue was purified by flash
chromatography (80:20 cyclohexane/ethyl acetate), giving the
products as white crystals.
1.7.6. 2-(1,2-O-Isopropylidene-a-D-xylo-tetrafuranos-4-yl)-5-
(p-bromophenyl)-l,3,4-oxadiazole (8f)
34% yield; mp: 182–184 °C; ½a D25
ꢁ0.6 (c 1.9, chloroform). Anal.
ꢀ
Calcd for C15H15BrN2O5: C, 47.02; H, 3.90. Found: C, 46.89; H, 3.87.
1H NMR (500 MHz, CDCl3) d: 6.16 (d, 1H, J1,2 3.5 Hz, H-1), 4.73 (d,
2H, H-2 + H-3), 5.39 (d, 1H, J3,4 2.6 Hz, H-4), 7.84 (d, 2H, J 8.8 Hz,
aromatic protons), 7.60 (d, 2H, J 8.8 Hz, aromatic protons), 1.57
(s, 3H, CMe2), 1.37 (s, 3H, CMe2), 4.32 (s, 1H, –OH); 13C NMR
(125 MHz, CDCl3) d: 105.7 (C-1), 84.4 (C-2), 74.3 (C-3), 76.1 (C-4),
132.5–121.8 (aromatic carbons), 164.8, 162.6 (oxadiazole carbons),
112.6 (CMe2), 26.9 (CMe2), 26.1 (CMe2).
1.7.1. 2-(1,2-O-Isopropylidene-a-D-xylo-tetrafuranos-4-yl)-5-
(o-chlorophenyl)-l,3,4-oxadiazole (8a)
40% yield; mp: 110–112 °C; ½a D25
ꢀ
+1.5 (c 2.8, chloroform). Anal.
Calcd for C15H15ClN2O5: C, 53.19; H, 4.46. Found: C, 53.03; H, 4.44.
1H NMR (500 MHz, CDCl3) d: 6.16 (d, 1H, J1,2 3.5 Hz, H-1), 4.74 (d,
2H, H-2 + H-3), 5.43 (d, 1H, J3,4 2.7 Hz, H-4), 7.93–7.31 (m. 4H,
aromatic protons), 1.56 (s, 3H, CMe2), 1.36 (s, 3H, CMe2), 4.45 (s,
1H, –OH); 13C NMR (125 MHz, CDCl3) d: 105.6 (C-1), 84.4 (C-2),
74.6 (C-3), 76.1 (C-4), 133.1–122.1 (aromatic carbons), 163.6,
162.7 (oxadiazole carbons), 112.4 (CMe2), 26.9 (CMe2), 26.1 (CMe2).
1.8. 3,5-Di-O-acetyl-6-deoxy-1,2-O-isopropylidene-6-
isothiocyanate-a-D-glucofuranose (10)
Compound 10 was obtained as a syrup (1.10 g, 3.18 mmol,
84.9%) from 916,17 (1.23 g, 3.74 mmol), as previously described
for similar derivatives;21
½
a 2D5
ꢀ
ꢁ50.2 (c 2.1, chloroform). Anal. Calcd
for C14H19NO7S: C, 48.69; H, 5.55; N, 4.06; S, 9.28. Found: C, 48.90;
H, 5.47; N, 3.74; S, 9.23. 1H NMR (200 MHz, CDCl3) d: 5.91 (d, 1H,
J1,2 3.5 Hz, H-1), 4.51 (d, 1H, J2,1 3.6 Hz, H-2), 5.37 (d, 1H, J3,4
2.8 Hz, H-3), 4.43 (dd, 1H, J4,3 2.9 Hz, J4,5 9.3 Hz, H-4), 5.13 (dt,
1H, J5,4 9.5 Hz, J5,6a 3.5 Hz, J5,6b 3.8 Hz, H-5), 3.84 (m, 2H,
H-6a + H-6b), 2.08 (s, 3H, MeCO), 2.06 (s, 3H, MeCO), 1.56 (s, 3H,
CMe2), 1.32 (s, 3H, CMe2); 13C NMR (50 MHz, CDCl3) d: 105.1
1.7.2. 2-(1,2-O-Isopropylidene-a-D-xylo-tetrafuranos-4-yl)-5-
(m-chlorophenyl)-l,3,4-oxadiazole (8b)
50% yield; mp: 135–136 °C; ½a D25
ꢀ
+4.7 (c 2.9, chloroform). Anal.
Calcd for C15H15ClN2O5: C, 53.19; H, 4.46. Found: C, 53.08; H, 4.43.
1H NMR (500 MHz, CDCl3) d: 6.17 (d, 1H, J1,2 3.5 Hz, H-1), 4.73 (d,

J. S. Barradas et al. / Carbohydrate Research 343 (2008) 2468–2474
2473
(C-1), 83.3 (C-2), 76.7 (C-3), 74.5 (C-4), 67.4 (C-5), 46.5 (C-6), 169.5,
169.3 (CO), 112.8 (CMe2), 20.7 (MeCO), 20.6 (MeCO), 26.8 (CMe2),
26.2 (CMe2).
J5,6b 9.8 Hz, H-5), 4.47 (dd, 1H, J6a,5 3.1 Hz, J6a,6b 14.2 Hz, H-6a),
4.15 (dd, 1H, J6b,5 9.8 Hz, J6b,6a 14.2 Hz, H-6b), 8.04 (d, 2H, J
8.5 Hz, aromatic protons), 7.58 (d, 2H, J 8.5 Hz, aromatic protons),
1.41 (s, 3H, CMe2), 1.27 (s, 3H, CMe2), 12.85 (s, 1H, NH); 13C NMR
(125 MHz, CD3COCD3) d: 106.1 (C-1), 86.0 (C-2), 75.3 (C-3), 83.0
(C-4), 66.5 (C-5), 49.5 (C-6), 152.9 (heterocyclic carbon), 136.9–
126.5 (aromatic carbons), 169.5 (CS), 111.8 (C Me2), 27.1 (CMe2),
26.4 (CMe2).
1.9. General procedure for the synthesis of compounds (11a,b)
A
solution of p-chloro- or p-bromobenzoic hydrazide
(2.84 mmol) and 10 (946.61 mg, 2.84 mmol) in dry THF was
refluxed for 2 h, and the solvent was evaporated, affording the
p-chloro and p-bromo substituted thiosemicarbazides (11a and
11b) in quantitative yield.
1.10.2. 5-(p-Clorophenyl)-4-(1,2-O-isopropylidene-a-D-
glucofuranos-6-yl)-1,2,4-triazoline-3-thione (12b)
142.11 mg, 55%; mp: 236 °C (dec); ½a D25
ꢀ
+5.0 (c 2.5, MeOH). Anal.
1.9.1. 3,5-di-O-acetyl-6-deoxy-1,2-isopropylidene-6-[(p-
Calcd for C16H18BrlN3O4S: C, 44.55; H, 4.40. Found: C, 44.95; H,
4.65. 1H NMR (500 MHz, CD3COCD3) d: 5.88 (d, 1H, J1,2 3.7 Hz,
H-1), 4.48 (d, 1H, J2,1 3.5 Hz, H-2), 4.23 (d, 1H, J3,4 2.8 Hz, H-3),
3.93 (dd, 1H, J4,3 2.8 Hz, J4,5 7.8, H-4), 4.65 (dt, 1H, J5,4 3.1 Hz, J5,6a
3.1 Hz, J5,6b 9.7 Hz, H-5), 4.45 (dd, 1H, J6a,5 3.1 Hz, J6a,6b 14.8 Hz,
H-6a), 4.13 (dd, 1H, J6b,5 9.7 Hz, J6b,6a 14.8 Hz, H-6b), 7.84 (d, 2H,
J 8.5 Hz, aromatic protons), 7.75 (d, 2H, J 8.5 Hz, aromatic protons),
1.39 (s, 3H, CMe2), 1.26 (s, 3H, CMe2), 12.81 (s, 1H, NH); 13C NMR
(125 MHz, CD3COCD3) d: 106.1 (C-1), 85.2 (C-2), 74.5 (C-3), 82.2
(C-4), 67.7 (C-5), 48.8 (C-6), 132.7–125.3 (aromatic carbons),
152.9 (heterocyclic carbon), 169.6 (CS), 111.8 (CMe2), 26.6
(CMe2), 26.3 (CMe2).
clorobenzohydrazinecarbothionyl)amino]-
(11a)
a-D-glucofuranose
White crystals, mp: 86 °C; ½a D25
ꢀ
+2.7 (c 2.6, chloroform). Anal.
Calcd for C21H26ClN3O8S: C, 48.88; H, 5.08; N, 8.14; S, 6.21. Found:
C, 49.20; H, 5.08; N, 8.04; S, 6.05. 1H NMR (500 MHz, CDCl3) d: 5.85
(d, 1H, J1,2 3.6 Hz, H-1), 4.44 (d, 1H, J2,1 3.7 Hz, H-2), 5.29 (d, 1H, J3,4
3.0 Hz, H-3), 4.31 (dd, 1H, J4,3 2.8 Hz, J4,5 8.9, H-4), 5.14 (ddd, 1H,
J5,4 8.8 Hz, J5,6a 3.4, J5,6b 7.8, H-5), 4.05 (d (w), 1H, J6a,6b 13.9 Hz,
H-6a), 3.84 (m, 1H, H-6b), 7.83 (d, 2H, J 8.5 Hz, aromatic protons),
7.38 (d, 2H, J 8.5 Hz, aromatic protons), 2.00 (s, 3H, MeCO), 1.90 (s,
3H, MeCO), 1.45 (s, 3H, CMe2), 1.27 (s, 3H, CMe2), 9.71 (s, 1H, NH),
8.57 (s, 1H, NH), 7.50 (s(w), 1H, NH); 13C NMR (125 MHz, CDCl3) d:
105.0 (C-1), 83.0 (C-2), 74.9 (C-3), 78.3 (C-4), 68.2 (C-5), 46.5 (C-6),
138.8–128.5 (aromatic carbons), 170.8 (CO), 169.6 (CO), 182.4 (CS),
112.4 (CMe2), 20.9 (MeCO), 20.7 (MeCO), 26.6 (CMe2), 26.1
(CMe2).
1.11. Antiviral studies
1.11.1. Cells and viruses
Vero (African green monkey kidney) cells were grown in Eagle’s
minimum essential medium (MEM) supplemented with 5% inacti-
1.9.2. 3,5-di-O-acetyl-6-deoxy-1,2-isopropylidene-6-[(p-
vated calf serum and 50 lg/ml gentamicin. For maintenance med-
bromobenzohydrazinecarbothionyl)amino]-
(11b)
a
-
D
-glucofuranose
ium (MM), the serum concentration was reduced to 1.5%.
The C6/36 HT mosquito cell line from Aedes albopictus was cul-
tured at 33 °C in L-15 medium (Leibovitz) supplemented with 0.3%
tryptose phosphate broth, 0.02% glutamine, 1% MEM non-essential
amino acids solution, and 5% fetal calf serum.
The IV4454 strain of virus Junín (JUNV) and the NGC strain of
dengue virus type 2 (DENV-2) were used. DENV-2 stocks were pre-
pared in C6/36 HT cells and titrated by plaque formation in Vero
cells. JUNV stocks were prepared and titrated by plaque formation
in Vero cells.
White crystals, mp: 83 °C; ½a D25
ꢀ
+3.1 (c 2.7, chloroform). Anal.
Calcd for C21H26BrN3O8S: C, 45.01; H, 4.68; N, 7.50; S, 5.72. Found:
C, 45.40; H, 5.10; N, 7.41; S, 5.52. 1H NMR (500 MHz, CDCl3) d: 5.86
(d, 1H, J1,2 3.6 Hz, H-1), 4.45 (d, 1H, J2,1 3.7 Hz, H-2), 5.30 (d, 1H, J3,4
3.0 Hz, H-3), 4.32 (dd, 1H, J4,3 2.9 Hz, J4,5 8.9, H-4), 5.15 (ddd, 1H,
J5,4 8.8 Hz, J5,6a 3.5, J5,6b 7.7, H-5), 4.06 (d (w), 1H, J6a,6b 13.9 Hz,
H-6a), 3.84 (m, 1H, H-6b), 7.75 (d, 2H, J 8.8 Hz, aromatic protons),
7.58 (d, 2H, J 8.8 Hz, aromatic protons), 2.00 (s, 3H, MeCO), 1.96 (s,
3H, MeCO), 1.47 (s, 3H, CMe2), 1.28 (s, 3H, CMe2), 9.50 (s, 1H, NH),
9.03 (s, 1H, NH), 7.45 (s(w), 1H, NH); 13C NMR (125 MHz,
CD3COCD3) d: 105.1 (C-1), 83.1 (C-2) 75.0 (C-3), 78.4 (C-4), 68.4
(C-5), 46.6 (C-6), 132.1–127.6 (aromatic carbons), 171.0 (CO),
169.6 (CO), 182.3 (CS), 112.5 (CMe2), 20.9 (MeCO), 20.7 (MeCO),
26.6 (CMe2), 26.1 (CMe2).
1.11.2. Biological assays
Vero cell viability was measured by the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma–Aldrich,
USA) method. Confluent cultures in 96-well plates were exposed
to twofold dilutions of the compounds, three wells for each con-
centration, and incubated for 48 h at 37 °C. Then, 10 ll of MM con-
1.10. General procedure for the synthesis of 1,2,4-triazoline-3-
thiones (12a,b)
taining MTT (final concentration, 0.5 mg/mL) was added to each
well. After 2 h of incubation at 37 °C, the supernatant was
removed, 200
the formazan crystals, and absorbance was measured in a micro-
plate reader at 595 nm. The cytotoxic concentration 50% (CC50
ll of ethanol was added to each well to solubilize
Thiosemicarbazides (11a and 11b) (0.60 mmol) were sus-
pended in 1 M NaOH and refluxed for 2 h. Solutions were neutral-
ized, evaporated, and dissolved in acetone, and residual mineral
salts were filtrated off. Evaporation of acetone yielded the crude
products, which were further purified by flash column chromato-
graphy (90:10 cyclohexane: acetone) affording 12a and 12b as
white crystals.
)
was calculated as the compound concentration required to reduce
cell viability by 50%. The antiviral activity was determined by a
virus yield inhibition assay. Vero cells grown in 24-well plates
were infected at a multiplicity of infection of 0.1 PFU/cell of JUNV
or DENV-2. After 1 h adsorption at 37 °C, cells were washed and
refed with MM containing different concentrations of the com-
pounds (two wells per concentration). After 48 h of incubation at
37 °C, supernatant cultures were harvested and extracellular virus
yields were determined by a plaque assay. The effective concentra-
tion 50% (EC50) was calculated as the concentration required to
reduce virus yield by 50% in the compound-treated cultures
compared with untreated ones. Finally, virucidal activity was
determined by incubating a virus suspension of 106 PFU of JUNV
1.10.1. 5-(p-Clorophenyl)-4-(1,2-O-isopropylidene-a-D-
glucofuranos-6-yl)-1,2,4-triazoline-3-thione (12a)
94.98 mg, 41%; mp: 224 °C (dec); ½a D25
ꢀ
+3.2 (c 1.4, MeOH). Anal.
Calcd for C16H18ClN3O4S: C, 49.34; H, 4.87. Found: C, 49.72; H, 4.88.
1H NMR (500 MHz, CD3COCD3) d: 5.89 (d, 1H, J1,2 3.7 Hz, H-1), 4.49
(d, 1H, J2,1 3.8 Hz, H-2), 4.25 (d, 1H, J3,4 2.8 Hz, H-3), 3.99 (dd, 1H,
J4,3 2.8 Hz, J4,5 7.8 Hz, H-4), 4.67 (ddd, 1H, J5,4 7.8 Hz, J5,6a 3.0 Hz,

2474
J. S. Barradas et al. / Carbohydrate Research 343 (2008) 2468–2474
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or DENV-2 with an equal volume of MM either with or without
different concentrations of each compound for 1.5 h at 37 °C. The
samples were then diluted in cold MM to determine residual
infectivity in a plaque formation assay using Vero cells. The ratios
of virus titer in compound-treated samples with respect to virus
titer in control samples were calculated.
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Acknowledgments
Financial support from CONICET (PIP 5011, 5513), ANPCyT (PICT
13922, 14124), and UBA is gratefully acknowledged.
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