Imidazo[2,1-b]thiazole carbohydrate derivatives: Synthesis and antiviral activity against Junin virus, agent of Argentine hemorrhagic fever

Article abstract of DOI:10.1016/j.ejmech.2010.11.012

Herein, we describe the syntheses of 3,5-disubstituted imidazo[2,1-b] thiazole. The cyclization step was performed in two different conditions by using either thermal or microwave heating. Comparing the results of both methodologies, we found that the mic

Full text of DOI:10.1016/j.ejmech.2010.11.012

European Journal of Medicinal Chemistry 46 (2011) 259e264
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Imidazo[2,1-b]thiazole carbohydrate derivatives: Synthesis and antiviral activity
against Junin virus, agent of Argentine hemorrhagic fever
,
,
c
José Sebastián Barradas a, María Inés Errea a b, Norma B. D’Accorso a , Claudia Soledad Sepúlveda ,
*
Elsa Beatriz Damonte c
a CIHIDECAR-CONICET - Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II - Ciudad Universitaria,
1428 Buenos Aires, Argentina
b Instituto Tecnológico de Buenos Aires, Avda. Eduardo Madero 399, 1106, Buenos Aires, Argentina
c Laboratorio de Virología - Departamento de Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II - 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 syntheses of 3,5-disubstituted imidazo[2,1-b]thiazole. The cyclization step was
performed in two different conditions by using either thermal or microwave heating. Comparing the
results of both methodologies, we found that the microwave assistance is an improved alternative to
obtain this family of heterocyclic compound. Compounds were first evaluated for cytotoxicity in Vero
cells by MTT method and then, the antiviral activity was assayed by a virus yield inhibition assay in the
range of concentrations lower than the corresponding CC50, using JUNV strain IV4454 as the model
system. The most active compounds (3 and 4), showed a level of antiviral activity against JUNV in
monkey Vero cells better than the reference substance ribavirin. Then, they are promising lead
compound for further analysis and characterization to establish their therapeutic potential against
hemorrhagic fever viruses.
Received 4 August 2010
Received in revised form
1 November 2010
Accepted 8 November 2010
Available online 27 November 2010
Keywords:
Imidazo[2,1-b]thiazole
Carbohydrates
Antiviral activity
Junin virus (JUNV)
Microwave irradiation
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
problem in the richest agricultural zones of Argentina [8].
Although the evaluation of different types of compounds for
Imidazo[2,1-b][1,3]-thiazole [1,2] derivatives occupy a pro-
minent place in medicinal chemistry because of their therapeutic
properties. Compounds containing this heterocyclic system have
been reported as potential acetylcholinesterase and butyryl-
cholinesterase inhibitors [3] as well as antihelmintic, fungicide,
herbicide, antitumor and cardiotonic agents [4,5].Anti-hyperten-
sive, antiinflammatory, and immunosuppressive properties of this
class of compounds were also reported together with their potent
and balanced enzyme and cellular activity against insulin-like
growth factor receptor and members of the epidermal growth
factor family of receptor tyrosine kinases [6].
In a previous report, we described the synthesis and the
antiviral activity against two hemorrhagic fever causing viruses,
dengue virus type 2 (DENV-2) and Junin virus (JUNV) [7] of
a series of nitrogenated heterocyclic compounds, including imi-
dazothiazoles. JUNV belongs to the Arenaviridae family and is the
etiological agent of Argentine hemorrhagic fever (AHF), an
endemoepidemic disease recognized as a major public health
inhibitory activity against JUNV has been reported [9], no specific
and safe chemotherapy is currently available. Ribavirin is the only
drug that has shown partial efficacy in the treatment of arenavirus
hemorrhagic fever, but with a high level of undesirable adverse
reactions [10]. The current therapy for AHF patients is the early
administration of standardized doses of convalescent plasma, but
this therapy is not effective when initiated after a week of illness
and 10% of treated patients develop late neurological syndrome
[10]. Other disadvantages of immune plasma therapy are the
difficulties in maintaining adequate stocks of plasma and the risk
of transfusion-borne diseases. Thus, there is a real need for active
drugs against JUNV and other viruses producing hemorrhagic
fever in humans.
In agreement with the potent bioactivities reported for
compounds possessing the imidazothiazole core in their structure, in
our earlier report antiviral activity against JUNV of the compound
3-(p-bromobenzoyl)-5-(1,2-O-isopropylidene-3-O-methyl-
a-D-xylo-
furanos-5-ulo-5-yl)imidazo[2,1-b]thiazole was detected at concen-
trations not affecting cell viability [7]. These results encourage us to
extend the study of the antiviral properties of imidazothiazoles.
New derivatives were synthesized by changing the substituents and
* Corresponding author. Tel.: þ54 11 4576 3346.
E-mail address: norma@qo.fcen.uba.ar (N.B. D’Accorso).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.012

260
J.S. Barradas et al. / European Journal of Medicinal Chemistry 46 (2011) 259e264
S
R2
S
N
N
R2
N
Br
O
N
N
OR1
OR1
O
O
O
1. i) or ii)
O
O
2. Et3N r.t.
O
O
Me
Me
Me
Me
2. R1 = CH3; R2 = -COC6H5
1a/b
1a R1= CH3
1b R1= COCH3
3. R1 = CH3; R2 = -COC6H4Br-p
4. R2 = CH3; R2 = -COC6H4Cl-p
5. R1 = CH3; R2 = -COC6H4F-p
6. R1 = COCH3; R2 = -COC6H5
7. R1 = COCH3; R2 = -COC6H4Br-p
8. R1 = COCH3; R2 = -COC6H4Cl-p
9. R1 = COCH3; R2 = -COC6H4F-p
i) Thermal condition: dioxane (anh.) reflux: 16h, 2. 65 % [6]; 3. 41 % [6]; 4. 39 %;
5. 37 %; 6. 34 %; 7. 19 %; 8. 20 %; 9. 32 %.
ii) Microwave condition: THF (anh.) 300W, 105°C, 90-150 min., 2. 61 %; 3. 39 %;
4. 44 %; 5. 53 %; 6. 34 %; 7. 37 %; 8. 37 %; 9. 25 %.
Scheme 1.
their relative position in the heterocyclic ring and the anti-JUNV
activity was analyzed.
low levels of cytotoxicity with CC50 values higher than 100
mM, with
compound 8 the only exception displaying CC50 of 70 M.
m
Then, the antiviral activity was assayed by a virus yield inhibition
assay in the range of concentrations lower than the corresponding
CC50, using JUNV strain IV4454 as the model system. All derivatives
were inhibitors of JUNV replication, but exhibited a wide range of
antiviral effective concentrations (Table 1). Compounds 4 and 3
showed the highest inhibitory effect against JUNV with EC50 values
2. Results and discussion
2.1. Chemistry
Substituted imidazo[2,1-b]thiazoles (2e7) were obtained by a
convergent synthetic pathway (Scheme 1), from either 6-bromo-
6-deoxy-1,2-O-isopropylidene-3-O-methyl-
ulose (1a) [7] or 6-bromo-6-deoxy-1,2-O-isopropylidene-3-O-acetyl-
around 1 mM, and, given thelowtoxicityof both azolesfor uninfected
cells, the selectivity index (SI:ratio CC50/EC50) was 463.7 and 268.2,
respectively. Furthermore, both derivatives possessed greater anti-
viral activity and selectivity than ribavirin, the only drug in clinical
use for arenavirus treatment which was evaluated in parallel as
a reference substance.
a-D-xylo-hexofuranos-5-
a-D-xylo-hexofuranos-5-ulose (1b).
Compound 1b was obtained from 1,2-O-isopropylidene-3-O-
acetyl-a-D-glucofuranose [11e13]. Selective replacement of the
primary hydroxyl group by a bromine atom [14] following by the
oxidation of the hydroxyl on C-5 using 1-hydroxy-1,2-benziodoxol-
3(1H)-one-1-oxide (IBX) as oxidizing agent, led us to obtained
compound 1b in quantitative yield [15].
In order to improve the coupling step between compounds
1a/b and N0-(5-arylthiazol-2-yl)-N,N-dimethylformamidine (see
Scheme 1), it was performed in two different conditions: i) by
thermal heating, as reported in the literature and ii) by microwave
assistance.
O
S
Br
OMe
N
O
+
N
O
N
O
O
R
10
Me
Me
1. i) or ii)
2. Et3N r.t.
Although there were no significant differences in the yields of
the final products between the two conditions, reactions times
dropped from 16 h to 90 min by changing thermal for microwave
condition. The drastic reduction in reaction time observed with the
microwave assisted method provides an improved alternative for
the synthesis of substituted imidazo[2,1-b]thiazoles.
O
S
OMe
N
O
N
O
O
O
In order to study the influence of the relative position of the
substituent in the heterocyclic ring in the antiviral activity of
the final products, the coupled position of the carbohydrate and the
heterocylic moieties were inverted as shown in Scheme 2.
All products were characterized by physical and spectroscopic
analysis and the results are shown in Section 4.
Me
Me
R
11. R = H
12. R = Br
13. R = Cl
14. R = F
Reagents : 1. i) Thermal condition: dioxane (anh.) reflux,16 h, 11. 70 %;
12. 40 %; 13. 33 %; 14. 59 %.
2.2. Biological studies
1. ii) Microwave condition: THF (anh.) 300W, 105°C, 90 min.,
11. 83 %; 12. 43 %; 13. 40 %; 14. 77 %.
All compounds were first evaluated for cytotoxicity in Vero cells
by MTT method. As seen in Table 1, most compounds exhibited very
Scheme 2.

J.S. Barradas et al. / European Journal of Medicinal Chemistry 46 (2011) 259e264
261
with compound 3 during 2 h at 37 ꢁC and then it was removed before
JUNV infection, it was not effective in reducing virus yield at 48 h p.i.
(Fig. 1). Similarly, following incubation of virus with compound for
2 h at 37 ꢁC before cell infection reduction in remaining JUNV
infectivity was only around 10% at the highest concentration tested
Table 1
Cytotoxicity and antiviral activity against JUNV of imidazothiazoles.
Compound
CC50
[
m
M]a
EC50 ꢀ SD[
m
M]b
SIc
2
3
4
5
6
7
8
9
11
12
13
14
>400
295
371
>400
>400
139
18.5 ꢀ 6.8
1.1 ꢀ 0.7
0.8 ꢀ 0.2
78.3 ꢀ 2.0
7.9 ꢀ 1.9
5.6 ꢀ 2.1
>21.6
268.2
463.7
>5.1
>50.6
24.8
of 50
mM. By contrast, when the imidazothiazole derivative was
added to cells simultaneously with virus inoculum (as in the
screening assay shown in Table 1), virus titters in cell supernatants at
48 h p.i. was significantly reduced at concentrations 15e20-fold
lower and in a dose dependent-manner (Fig. 1). Thus, the inhibitory
effect against JUNV was entirely exerted through a blockade in virus
multiplication during the infection process.
70
7.5 ꢀ 0.08
4.9 ꢀ 0.4
9.3
>400
>400
351
>400
>400
>400
>81.6
>32.5
81.6
>33.6
>137.9
>21.6
12.3 ꢀ 0.01
4.3 ꢀ 0.7
11.9 ꢀ 0.2
2.9 ꢀ 0.01
18.5 ꢀ 1.7
Ribavirin
3. Conclusions
Values are the mean of three determinations ꢀ standard deviation.
a
Cytotoxic concentration 50%: compound concentration required to reduce cell
viability by 50%, as determined by MTT assay.
In conclusion, a series of imidazo[2,1-b]thiazole substituted in
position 3 and 5 with a carbohydrate moiety and different hal-
obenzoyl groups was synthesized. All the new compounds were
characterized by NMR spectra, optical rotation as well as elemental
analyses.
b
Effective concentration 50%: compound concentration required to reduce virus
yield by 50%.
c
Selectivity index: ratio CC50/EC50
.
The most active imidazothiazole derivatives evaluated in the
present study, compounds 4 and 3, showed a level of antiviral
activity against JUNV in monkey Vero cells better than the reference
substance ribavirin. Compound 3, taken as model of this type of
derivatives, exerted the antiviral effect only during the intracellular
multiplication cycle of JUNV. Furthermore, it was also an effective
JUNV inhibitor in human cells. Then, they are promising lead
compounds for further analysis and characterization to establish
their therapeutic potential against hemorrhagic fever viruses.
In addition, an improvement methodology to obtain these
promising compounds was also reported.
The antiviral activity of the analyzed compounds decreased
drastically when the methyl group of the carbohydrate residue was
replaced by the more polar acetyl group. Beside, the higher values
were observed when the carbohydrate residue was on position 5 of
the heterocycle (Table 1).
To better evaluate the perspectives of the imidazothiazoles as
inhibitors of a human hemorrhagic fever, the ability of compound
3 to inhibit JUNV replication in human cells was also analyzed. The
values of CC50 and EC50 determined by MTT and JUNV yield inhi-
bition assays, respectively, in the lung human cell line A549 were
274
m
M and 2.8 ꢀ 0.5
mM, respectively, with similar effectiveness as
in the monkey Vero cells (Table 1).
As a first approach to characterize the action of these compounds
on arenaviruses, the possibility that they acted directly either on the
virus particles or on the cells to be infected was investigated. To this
end, virus suspensions or cell monolayers were pre-incubated with
different compound concentrations before infection, as explained in
Experimental Protocols. When cell monolayers were pre-incubated
4. Experimental protocols
4.1. Chemistry
4.1.1. General remarks
Research chemicals were purchased from SigmaeAldrich Co and
used without further purification in the reactions or were prepared
according to procedures described in the literature [7]. For micro-
wave reactions a CEM Discover with 300 Watts of power was used,
using the mode Power Max ON and constant agitation. Reactions
were monitored by thin layer chromatography (TLC) on silica gel
plated (60 F254; Merck) visualizing with ultraviolet light or by
spraying with sulphuric acid in ethanolic solution. Column chro-
matographic separations were performed on silica gel 60 (240e400
mesh, Merck). Melting points were measured on a Thomas Hoover
melting point apparatus and are uncorrected, and the optical
100
80
60
40
20
0
rotation, [
Polarimeter. Solvents were reagent grade and, in most cases, dried
and distillated before use according to standard procedures. 1H, 13
a]D, measurements were made using a 343 Perkin Elmer
C
NMR spectra were recorded on a Bruker AC-200 spectrometer,
operating at 200, 50 MHz respectively; or a Bruker AMX-500
spectrometer, operating at 500, 125 MHz respectively. Assignments
of the 1H and 13C NMR spectra were confirmed with the aid of two
0
50
100
150
200
dimensional techniques 1H, 13C (HSQC, HMBC). Chemical shifts (
d)
Concentration [µM]
are reported in parts per million downfield from tetramethyl silane
as internal standard. Elemental analyses were performed on a Carlo
Erba EA 1108 CHN elemental analyzer.
Fig. 1. Effect of pre-treatment of cells or virus with compound 3. Pretreatment of virus
(:): suspensions of JUNV were incubated with different concentrations of compound
for 1.5 h at 37 ꢁC and then remaining infectivity was titrated by PFU. Pretreatment of
cells (-): Vero cells were pre-incubated with different concentrations of compound
during 2 h at 37 ꢁC; then, supernatants were removed and cells were infected with
JUNV in absence of compound. Virus yields were determined at 48 h p.i. by PFU.
Treatment during virus infection (C): Vero cells were infected with JUNV and
compound was added and maintained during all period of incubation. Virus yields
were measured at 48 h p.i. by PFU. Each value is the mean of duplicate assays.
4.1.2. 3-O-Acetyl-6-bromo-6-deoxy-1,2-O-isopropylidene-a-D-xylo-
hexofuranos-5-ulose (1b)
IBX (38.4 mmol) was added to a solution of 3-O-acetyl-6-bromo-
6-deoxy-1,2-O-isopropylidene- -glucofuranose (7.68 mmol) in
a-D

262
J.S. Barradas et al. / European Journal of Medicinal Chemistry 46 (2011) 259e264
acetonitrile (50 mL). The reaction mixture was refluxed during 16 h
and then cooled, filtered, concentrated and purified by flash column
chromatography, using cyclohexane: acetone as eluent (90:10),
Calcd. for C21H19FN2O6S: C, 56.50; H, 4.29; N, 6.27. Found: C, 56.85; H,
4.45; N, 5.92.
affording pure 1b (89.2% yield) as a syrup; [
a]
D - 72.6 (c 1, CHCl3); 1H
4.1.3.3. 3-(Benzoyl)-5-(3-O-acetyl-1,2-O-isopropylidene-
anos-5-ulo-5-yl)imidazo[2,1-b]thiazole (6). Compound
obtained as white amorphous solid: m.p. 147e149 ꢁC; [
1, CHCl3); 1H NMR (CDCl3):
1.39 (s, 3H, CH3), 1.59 (s, 3H, CH3), 1.90
a
-
D
-xylofur-
was
a]D - 75.9 (c
NMR (CDCl3): 1.33 (s, 3H, CH3),1.51 (s, 3H, CH3), 2.04 (s, 3H, CH3CO),
d
6
4.26 (s, 2H, H-6), 4.57 (d, 1H, H-2, J2,1 ¼ 3.6 Hz), 4.96 (d, 1H, H-4,
J4,3 ¼ 3.5 Hz), 5.47 (d, 1H, H-3, J3,4 ¼ 3.5 Hz), 6.06 (d, 1H, H-1,
d
J1,2 ¼ 3.6 Hz); 13C NMR (CDCl3):
d
20.7 (CH3CO), 26.2 [C(CH3)2], 26.8
(s, 3H, CH3CO), 4.64 (d, 1H, H-2, J2,1 ¼ 3.6 Hz), 5.17 (d, 1H, H-4,
J4,3 ¼ 3.3 Hz), 5.63 (d, 1H, H-3, J3,4 ¼ 3.3 Hz), 6.20 (d, 1H, H-1,
J1,2 ¼ 3.6 Hz), 7.59e7.92 (m, 5H, aromatic protons), 8.55 (s, 1H,
imidazo[2,1-b]thiazole proton), 8.91 (s, 1H, imidazo[2,1-b]thiazole
[C(CH3)2], 33.8 (C-6), 77.5 (C-3), 82.6 (C-2), 82.9 (C-4), 105.6 (C-1),
113.1 [C(CH3)2],157.4 (C-5),169.2 (C]O). Anal. Calcd. for C11H15BrO6:
C, 40.89; H, 4.68. Found: C, 40.39; H, 4.91.
proton); 13C NMR (CDCl3):
d 20.7 (CH3CO), 26.2 [C(CH3)2], 26.8 [C
(CH3)2], 77.5 (C-3), 82.4 (C-2), 83.2 (C-4), 105.5 (C-1), 113.0 [C
(CH3)2], 128.9, 129.1, 133.5, 136.3 (aromatic carbons), 126.6, 128.0,
135.1, 147.0, 156.8 (imidazo[2,1-b]thiazole carbons), 169.3, 183.4,
187.1 (C]O). Anal. Calcd. for C22H20N2O7S: C, 57.89; H, 4.42; N, 6.14.
Found: C, 57.74; H, 4.40; N, 5.91.
4.1.3. General procedure for the synthesis of compounds 2e9
Procedure 1 (Thermal Heating): A mixture of 6-bromo-6-deoxy-
1,2-O-isopropylidene-3-O-methyl-
(6 mmol) [7] (to obtain compounds 2e5) or 6-bromo-6-deoxy-
1,2-O-isopropylidene-3-O-acetyl- -xylo-hexofuranos-5-ulose (1b)
a-D-xylo-hexofuranos-5-ulose (1a)
a-D
(6 mmol) (to prepare products 6e9) and N0-(5-benzoylthiazol-2-yl)-
N,N-dimethylformamidine(0.75 mmol)inanhydrous dioxane(20mL)
was refluxed for 16 h under argon atmosphere. Et3N (1.2 mmol) was
added and the mixture was stirred for 12 h at room temperature. The
crude product was purified by column chromatography on silica gel G
(cyclohexane: acetone 95:5 and then 80:20) affording the products
2e9.
4.1.3.4. 3-(p-Bromobenzoyl)-5-(3-O-acetyl-1,2-O-isopropylidene-
xylofuranos-5-ulo-5-yl)imidazo[2,1-b]thiazole (7). Compound 7 was
obtained as yellow amorphous solid: m.p. 164e165 ꢁC; [
D - 54.4 (c
1, CHCl3); 1H NMR (CDCl3):
1.39 (s, 3H, CH3), 1.58 (s, 3H, CH3), 1.90
(s, 3H, CH3CO), 4.63 (d, 1H, H-2, J2,1 ¼ 3.6 Hz), 5.16 (d, 1H, H-4,
J4,3 ¼ 3.3 Hz), 5.62 (d, 1H, H-3, J3,4 ¼ 3.3 Hz), 6.20 (d, 1H, H-1,
J1,2 ¼ 3.6 Hz), 7.74 (d, 2H, J 8.5 Hz, aromatic protons), 7.78 (d, 2H,
J 8.5 Hz, aromatic protons), 8.55 (s, 1H, imidazo[2,1-b]thiazole
proton), 8.89 (s, 1H, imidazo[2,1-b]thiazole proton); 13C NMR
a-D-
a]
d
Procedure 2 (Microwave Heating): A mixture of 6-bromo-6-
deoxy-1,2-O-isopropylidene-3-O-methyl-
ulose (1a) (6 mmol) [7] or 6-bromo-6-deoxy-1,2-O-isopropylidene-
3-O-acetyl-
-xylo-hexofuranos-5-ulose (1b)(6 mmol) and N0-(5-
a-D-xylo-hexofuranos-5-
(CDCl3):
d 20.7 (CH3CO), 26.2 [C(CH3)2], 26.8 [C(CH3)2], 77.5 (C-3),
a-D
82.4 (C-2), 83.2 (C-4), 105.5 (C-1), 113.1 [C(CH3)2], 128.8, 130.3, 132.5,
135.0 (aromatic carbons), 126.6, 127.9, 134.7, 146.8, 156.6 (imidazo
[2,1-b]thiazole carbons), 169.3, 183.5, 186.0 (C]O). Anal. Calcd. for
C22H19BrN2O7S: C, 49.36; H, 3.58; N, 5.23. Found: C, 49.32; H, 3.69;
N, 4.84.
benzoylthiazol-2-yl)-N,N-dimethylformamidine (0.75 mmol) in
anhydrous THF (6 mL) was heated under microwave irradiation
(300 W, 105 ꢁC), for 90 min (compounds 2e5) or 150 min
(Compounds 6e9). The reaction mixture was diluted with anhy-
drous THF (20 mL) and Et3N (1.2 mmol) was added under argon
atmosphere. After the addition of triethylamine the procedure was
the same as described for thermal conditions.
4.1.3.5. 3-(p-Chlorobenzoyl)-5-(3-O-acetyl-1,2-O-isopropylidene-
xylofuranos-5-ulo-5-yl)imidazo[2,1-b]thiazole (8). Compound 8 was
obtained as yellow amorphous solid: m.p. 166e168 ꢁC; [
D - 89.2 (c
1, CHCl3); 1H NMR (CDCl3):
1.30 (s, 3H, CH3), 1.50 (s, 3H, CH3), 1.81
a-D-
a]
4.1.3.1. 3-(p-Chlorobenzoyl)-5-(1,2-O-isopropylidene-3-O-methyl-
xylofuranos-5-ulo-5-yl)imidazo[2,1-b]thiazole (4). Compound 4 was
obtained as yellow amorphous solid: m.p. 77e80 ꢁC; [
D - 115.6 (c
1, CHCl3); 1H NMR (CDCl3):
1.40 (s, 3H, CH3), 1.56 (s, 3H, CH3), 3.33
a-D-
d
(s, 3H, CH3CO), 4.55 (d, 1H, H-2, J2,1 ¼ 3.6 Hz), 5.10 (d, 1H, H-4,
J4,3 ¼ 3.3 Hz), 5.53 (d, 1H, H-3, J3,4 ¼ 3.3 Hz), 6.11 (d, 1H, H-1,
J1,2 ¼ 3.6 Hz), 7.48 (d, 2H, J 8.6 Hz, aromatic protons), 7.78 (d, 2H,
J 8.6 Hz, aromatic protons), 8.46 (s, 1H, imidazo[2,1-b]thiazole
proton), 8.80 (s, 1H, imidazo[2,1-b]thiazole proton); 13C NMR
a]
d
(s, 3H, CH3O), 4.20 (d, 1H, H-3, J3,4 ¼ 3.6 Hz), 4.68 (d, 1H, H-2,
J2,1 ¼ 3.6 Hz), 5.06 (d, 1H, H-4, J4,3 ¼ 3.6 Hz), 6.19 (d, 1H, H-1,
J1,2 ¼ 3.6 Hz), 7.57 (d, 2H, J 8.6 Hz, aromatic protons), 7.86 (d, 2H,
J 8.6 Hz, aromatic protons), 8.52 (s, 1H, imidazo[2,1-b]thiazole
proton), 8.94 (s, 1H, imidazo[2,1-b]thiazole proton); 13C NMR
(CDCl3):
d 20.6 (CH3CO), 26.2 [C(CH3)2], 26.8 [C(CH3)2], 77.7 (C-3),
82.4 (C-2), 83.2 (C-4), 105.5 (C-1), 113.1 [C(CH3)2], 129.6, 130.2, 134.7,
140.1 (aromatic carbons), 126.6, 127.9, 134.6, 147.0, 156.7 (imidazo
[2,1-b]thiazole carbons), 169.3, 183.4, 185.8 (C]O). Anal. Calcd. for
C22H19ClN2O7S: C, 53.83; H, 3.90; N, 5.71. Found: C, 54.27; H, 3.93; N,
5.44.
(CDCl3):
d 26.3 [C(CH3)2], 26.9 [C(CH3)2], 58.3 (CH3O), 80.8 (C-2),
84.9 (C-4), 86.1 (C-3), 105.8 (C-1), 112.6 [C(CH3)2], 129.4, 130.2, 134.7,
140.0 (aromatic carbons), 127.0, 128.2, 134.3, 146.8, 156.3 (imidazo
[2,1-b]thiazole carbons), 185.0, 186.0 (C]O). Anal. Calcd. for
C21H19ClN2O6S: C, 54.49; H, 4.14; N, 6.05. Found: C, 54.87; H, 4.25;
N, 5.77.
4.1.3.6. 3-(p-Fluorobenzoyl)-5-(3-O-acetyl-1,2-O-isopropylidene-
xylofuranos-5-ulo-5-yl)imidazo[2,1-b]thiazole (9). Compound 9 was
obtained as white amorphous solid: m.p. 152e153 ꢁC; [
D - 88.6 (c
1, CHCl3); 1H NMR (CDCl3):
1.39 (s, 3H, CH3), 1.59 (s, 3H, CH3), 1.90
a-D-
a]
4.1.3.2. 3-(p-Fluorobenzoyl)-5-(1,2-O-isopropylidene-3-O-methyl-
xylofuranos-5-ulo-5-yl)imidazo[2,1-b]thiazole (5). Compound 5 was
obtained as white amorphous solid: m.p. 88e91 ꢁC; [
D - 125.9 (c 1,
CHCl3); 1H NMR (CDCl3):
1.39 (s, 3H, CH3), 1.55 (s, 3H, CH3), 3.32 (s,
a-D-
d
(s, 3H, CH3CO), 4.64 (d, 1H, H-2, J2,1 ¼ 3.6 Hz), 5.17 (d, 1H, H-4,
J4,3 ¼ 3.3 Hz), 5.63 (d, 1H, H-3, J3,4 ¼ 3.3 Hz), 6.20 (d, 1H, H-1,
J1,2 ¼ 3.6 Hz), 7.28 (m, 2H, aromatic protons), 7.96 (m, 2H, aromatic
protons), 8.56 (s, 1H, imidazo[2,1-b]thiazole proton), 8.90 (s, 1H,
a]
d
3H, CH3O), 4.20 (d, 1H, H-3, J3,4 ¼ 3.6 Hz), 4.68 (d, 1H, H-2,
J2,1 ¼ 3.6 Hz), 5.06 (d, 1H, H-4, J4,3 ¼ 3.6 Hz), 6.18 (d, 1H, H-1,
J1,2 ¼ 3,6 Hz), 7.26 (m, 2H, aromatic protons), 7.95 (m, 2H, aromatic
protons), 8.51 (s, 1H, imidazo [2,1-b] proton), 8.93 (s, 1H, imidazo
imidazo[2,1-b]thiazole proton); 13C NMR (CDCl3):
d 20.6 (CH3CO),
26.2 [C(CH3)2], 26.8 [C(CH3)2], 77.5 (C-3), 82.4 (C-2), 83.2 (C-4),
105.5 (C-1), 113.1 [C(CH3)2], 116.4, 116.6, 131.5, 131.6, 132.5, 132.6,
165.0, 167.0 (aromatic carbons), 126.6, 127.7, 134.9, 146.8, 156.6
(imidazo[2,1-b]thiazole carbons), 169.3, 183.5, 185.5 (C]O). Anal.
Calcd. for C22H19FN2O7S: C, 55.69; H, 4.04; N, 5.90. Found: C, 55.46;
H, 3.98; N, 5.64.
[2,1-b] proton); 13C NMR (CDCl3):
d 26.3 [C(CH3)2], 26.9 [C(CH3)2],
58.3 (CH3O), 80.7 (C-2), 84.8 (C-4), 86.1 (C-3), 105.8 (C-1), 112.6 [C
(CH3)2], 116.3, 131.5, 132.7, 165.5 (aromatic carbons), 127.0, 128.1,
134.4, 147.0, 156.4 (imidazo [2,1-b] carbons), 184.9, 185.6 (C]O). Anal.

J.S. Barradas et al. / European Journal of Medicinal Chemistry 46 (2011) 259e264
263
4.1.4. General procedure for the synthesis of 3-(1,2-O-isopropylidene-
3-O-methyl- -xylofuranos-5-ulo-5-yl)-5-benzoylimidazo[2,1-b]
thiazole and 3-(1,2-O-isopropylidene-3-O-methyl- -xylofuranos-
5-ulo-5-yl)-5-(p-halobenzoyl)-imidazo[2,1-b]thiazole (11e14)
4.1.4.1. N,N-dimethylformamidyl-5-(1,2-O-isopropylidene-3-O-
proton), 9.46 (s, 1H, imidazo[2,1-b]thiazole proton); 13C NMR
(CDCl3): 26.3 [C(CH3)2], 26.9 [C(CH3)2], 58.6 (CH3O), 80.9 (C-2), 85.7
(C-4), 86.5 (C-3), 106.1 (C-1), 112.5 [C(CH3)2], 127.7, 130.1, 132.1, 136.4
(aromatic carbons), 126.8, 129.4, 133.2, 145.5, 156.9 (imidazo[2,1-b]
thiazole carbons),181.8,190.0 (C]O). Anal. Calcd. for C21H19BrN2O6S:
C, 49.71; H, 3.77; N, 5.52. Found: C, 49.32; H, 3.69; N, 4.84.
a-D
d
a-D
methyl-
a-
D-xylofuranos-5-ulo-5-yl)-1,3-thiazole (10). 6-Bromo-6-
deoxy-1,2-O-isopropylidene-3-O-methyl-
a-D
-xylo-hexofuranos-5-
ulose (1.85 mmol) was added to a solution of N,N0-bis(dimethyla-
minomethtlene)thiourea (2.77 mmol) in anhydrous dicloro-
methane (25 mL), and refluxed for 3 h under argon atmosphere.
Et3N (3.7 mmol) was added and the mixture was stirred for 12 h at
room temperature. The crude product was purified by flash column
chromatography, using cyclohexane: acetone as eluent (90:10 to
4.1.4.4. 3-(1,2-O-Isopropylidene-3-O-methyl-
yl)-5-(p-chlorobenzoyl)-imidazo[2,1-b]thiazole (13). Compound 10
was obtained as yellowamorphous solid: m.p. 73e76 ꢁC; [
]D - 72.7 (c
1, CHCl3); 1H NMR (CDCl3):
1.40 (s, 3H, CH3),1.55 (s, 3H, CH3), 3.35 (s,
a-D-xylofuranos-5-ulo-5-
a
d
3H, CH3O), 4.24 (d,1H, H-3, J3,4 ¼ 3.7 Hz), 4.69(d,1H, H-2,J2,1 ¼3.6Hz),
5.11 (d,1H, H-4, J4,3 ¼ 3.7 Hz), 6.27 (d,1H, H-1, J1,2 ¼ 3.6 Hz), 7.53 (d, 2H,
J8.6Hz, aromaticprotons), 7.86(d, 2H, J8.6Hz, aromaticprotons), 7.98
(s, 1H, imidazo[2,1-b]thiazole proton), 9.46 (s, 1H, imidazo[2,1-b]
75:25) to obtain compound 10 as a waxy solid; [
a] - 24.3 (c 1,
D
CHCl3); 1H NMR (CDCl3):
d
1.37 (s, 3H, CH3C),1.52 (s, 3H, CH3C), 3.13
(s, 3H, CH3N), 3.17 (s, 3H, CH3N), 3.32 (s, 3H, CH3O), 4.18 (d,1H, H-3,
J3,4 ¼ 3.6 Hz), 4.62 (d, 1H, H-2, J4,3 ¼ 3.7 Hz), 5.05 (d, 1H, H-4,
J3,4 ¼ 3.6 Hz), 6.14 (d, 1H, H-1, J1,2 ¼ 3.6 Hz), 8.35 (s, 1H, thiazole
thiazole proton); 13C NMR (CDCl3):
d 26.3 [C(CH3)2], 26.9 [C(CH3)2],
58.5 (CH3O), 80.9 (C-2), 85.7 (C-4), 86.5 (C-3), 106.1 (C-1), 112.5 [C
(CH3)2], 129.2, 130.1, 135.9, 139.2 (aromatic carbons), 126.8, 129.4,
133.2,145.4, 156.9 (imidazo[2,1-b]thiazole carbons), 181.6, 190.0 (C]
O). Anal. Calcd. for C21H19ClN2O6S: C, 54.49; H, 4.14; N, 6.05. Found: C,
54.96; H, 4.30; N, 5.92.
proton), 8.39 (s, 1H, eHC]N); 13C NMR (CDCl3):
d 26.9 [C(CH3)2],
27.0 [C(CH3)2], 35.2 [(CH3)2N], 41.2 [(CH3N], 58.6 (CH3O), 81.3 (C-2),
85.1 (C-4), 86.3 (C-3),105.7 (C-1),112.5 [C(CH3)2],130.7,148.8,181.0
(thiazole carbons), 156.6 (C]N), 187.8 (C]O). Anal. Calcd. for
C15H21N3O5S: C, 50.69; H, 5.96 N, 11.82. Found: C, 51.01; H, 5.70 N,
11.24.
4.1.4.5. 3-(1,2-O-Isopropylidene-3-O-methyl-
yl)-5-(p-fluorobenzoyl)-imidazo[2,1-b]thiazole (14). Compound 14
was obtained as yellow amorphous solid : m.p. 72e74 ꢁC; [
D - 92.5
(c 1, CHCl3); 1H NMR (CDCl3):
1.31 (s, 3H, CH3),1.47 (s, 3H, CH3), 3.27
a-D-xylofuranos-5-ulo-5-
Procedure 1 (Thermal heating): A mixture of N,N-dimethylforma-
a]
midyl-5-(1,2-O-isopropylidene-3-O-methyl-a-d-xylofuranos-5-ulo-5-
d
yl)-1,3-thiazole (0.45 mmol) and the corresponding
a
-carbonylated
(s, 3H, CH3O), 4.16 (d, 1H, H-3, J3,4 ¼ 3.6 Hz), 4.61 (d, 1H, H-2,
J2,1 ¼ 3.6 Hz), 5.03 (d, 1H, H-4, J4,3 ¼ 3.7 Hz), 6.19 (d, 1H, H-1,
J1,2 ¼ 3.6 Hz), 7.14e7.20 (m, 2H, J 8.6 Hz, aromatic protons), 7.85e7.88
(m, 2H, J 8.6 Hz, aromatic protons), 7.87 (s,1H, imidazo[2,1-b]thiazole
proton), 9.39 (s, 1H, imidazo[2,1-b]thiazole proton); 13C NMR
bromide compound (0.68 mmol) in anhydrous dioxane (20 mL) was
refluxed for 16 h under argon atmosphere. Et3N (0.9 mmol) was
added and the mixture was stirred for 12 h at room temperature.
Compounds 11e14 were obtained and, after being purified as was
described for compounds 4e9, pure compounds were afforded.
Procedure 2 (Microwave heating): A mixture of N,N-dime-
(CDCl3): d 26.3 [C(CH3)2], 26.9 [C(CH3)2], 58.6 (CH3O), 80.9 (C-2), 85.7
(C-4), 86.5 (C-3), 106.1 (C-1), 112.5 [C(CH3)2], 116.0, 116.1, 131.2, 134.0,
164.5, 166.5 (aromatic carbons), 129.4, 133.1, 131.2, 145.3, 156.9
(imidazo[2,1-b]thiazole carbons), 181.5, 189.8 (C]O). Anal. Calcd. for
C21H19FN2O6S: C, 56.50; H, 4.29; N, 6.27. Found: C, 56.39; H, 4.32; N,
6.04.
thylformamidyl-5-(1,2-O-isopropylidene-3-O-methyl-
anos-5-ulo-5-yl)-1,3-thiazole (0.45 mmol) and the corresponding
-carbonylated bromide compound (0.68 mmol) in anhydrous THF
a-d-xylofur-
a
(6 mL) was heated under microwave irradiation (300 W, 100 ꢁC), for
90 min. The reaction mixture was diluted with anhydrous THF
(20 mL) and Et3N (0.9 mmol) was added under argon atmosphere,
continuing with the procedure as was described for thermal
conditions.
4.2. Biological activity
4.2.1. Cells and viruses
4.1.4.2. 3-(1,2-O-Isopropylidene-3-O-methyl-
5-yl)-5-benzoyl-imidazo[2,1-b]thiazole) (11). Compound 11 was
obtained as white amorphous solid: m.p. 84e87 ꢁC; [
D - 80.8 (c 1,
CHCl3); 1H NMR (CDCl3):
1.39 (s, 3H, CH3), 1.55 (s, 3H, CH3), 3.35
a
-
D
-xylofuranos-5-ulo-
The cell lines Vero (African green monkey kidney) and A549
(lung carcinoma human cells) were grown in Eagle’s minimum
essential medium (MEM) (GIBCO, USA) supplemented with 5% and
a
]
d
10% inactivated calf serum, respectively, and 50 mg/mL gentamycin.
(s, 3H, CH3O), 4.24 (d, 1H, H-3, J3,4 ¼ 3.7 Hz), 4.69 (d, 1H, H-2,
J2,1 ¼ 3.7 Hz), 5.11 (d, 1H, H-4, J4,3 ¼ 3.7 Hz), 6.28 (d, 1H, H-1,
J1,2 ¼ 3.7 Hz), 7.54e7.91 (m, 5H, aromatic protons), 8.00 (s, 1H,
imidazo[2,1-b]thiazole proton), 9.49 (s, 1H, imidazo[2,1-b]thiazole
For maintenance medium (MM), the serum concentration was
reduced to 1.5%.
The attenuated strain IV4454 of JUNV was used. Virus stocks
were propagated and titrated by plaque forming units (PFU)
method in Vero cells.
proton); 13C NMR (CDCl3):
d 26.3 [C(CH3)2], 26.9 [C(CH3)2], 58.5
(CH3O), 80.9 (C-2), 85.7 (C-4), 86.5 (C-3), 106.1 (C-1), 112.8 [C
(CH3)2], 128.7, 128.8, 132.7, 137.7 (aromatic carbons), 127.1, 129.5,
133.0, 145.8, 156.7 (imidazo[2,1-b]thiazole carbons), 183.0, 189.8
(C]O). Anal. Calcd. for C21H20N2O6S: C, 58.87; H, 4.70; N, 6.54.
Found: C, 59.02; H, 4.76; N, 6.65.
4.2.2. Cytotoxicity assay
Cytotoxicity was measured by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT, SigmaeAldrich, USA)
method [16] in Vero cells. Confluent cultures in 96-well plates were
exposed to different concentrations of the compounds, with three
wells for each concentration, and incubated for 48 h at 37 ꢁC. Then
4.1.4.3. 3-(1,2-O-Isopropylidene-3-O-methyl-
yl)-5-(p-bromobenzoyl)-imidazo[2,1-b]thiazole (12). Compound 11
was obtained as yellow amorphous solid: m.p. 77e79 ꢁC; [
D - 42.5
(c 1, CHCl3); 1H NMR (CDCl3):
1.40 (s, 3H, CH3), 1.55 (s, 3H, CH3),
a-D-xylofuranos-5-ulo-5-
10
added to each well. After 2 h of incubation at 37 ꢁC, the supernatant
was removed and 200 l of ethanol was added to each well to
ml of MM containing MTT (final concentration 0.5 mg/mL) was
a]
d
m
3.35 (s, 3H, CH3O), 4.24 (d, 1H, H-3, J3,4 ¼ 3.6 Hz), 4.69 (d, 1H, H-2,
J2,1 ¼ 3.6 Hz), 5.11 (d, 1H, H-4, J4,3 ¼ 3.7 Hz), 6.27 (d, 1H, H-1,
J1,2 ¼ 3.6 Hz), 7.70 (d, 2H, J 8.6 Hz, aromatic protons), 7.78 (d, 2H,
J 8.6 Hz, aromatic protons), 7.98 (s, 1H, imidazo[2,1-b]thiazole
solubilize the formazan crystals. After vigorous shaking, absor-
bance was measured in a microplate reader at 595 nm. The cyto-
toxic concentration 50% (CC50) was calculated as the compound
concentration required to reduce the MTT signal by 50% compared

264
J.S. Barradas et al. / European Journal of Medicinal Chemistry 46 (2011) 259e264
to untreated controls. All determinations were performed twice
and each in duplicate.
References
[1] A. Andreani, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi,
G. Lenaz, R. Fato, C. Bergamini, G. Farruggia, Synthesis and antitumor activity
of guanylhydrazones from imidazo[2,1-b]thiazoles and from the new
heterocyclic system thiazolo[20,30:2,3]imidazo[4,5-c]quinoline, J. Med. Chem.
48 (2005) 3085e3089.
[2] A. Andreani, A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi, M. Recanatini,
V. Garaliene, Potential antitumor agents. Part 291: synthesis and potential
coanthracyclinic activity of imidazo[2,1-b]thiazole guanylhydrazones, Bioorg.
Med. Chem. 8 (2000) 2359e2366.
[3] A. Andreani, S. Burnelli, M. Granaiola, M. Guardigli, A. Leoni, A. Locatelli,
R. Morigi, M. Rambaldi, M. Rizzoli, L. Varoli, A. Roda, Chemiluminescent high
throughput microassay applied to imidazo[2,1-b]thiazole derivatives as
potential acetylcholinesterase and butyrylcholinesterase inhibitors, Eur. J.
Med. Chem. 43 (2008) 657e661.
[4] A. Andreani, M. Rambaldi, A. Locatelli, R. Bossa, A. Fraccari, I. Galatulas,
Potential antitumor agents. XXI. Structure determination and antitumor
activity of imidazo[2,1-b]thiazole guanylhydrazones, J. Med. Chem. 35 (1992)
4634e4637.
4.2.3. Antiviral assay
Antiviral activity was determined by a virus yield inhibition
assay [17]. Vero and A549 cells grown in 24-well plates were
infected at a multiplicity of infection (MOI) of 0.1 PFU/cell. After
1 h adsorption at 37 ꢁC, cells were washed and re-fed with MM
containing or not serial two-fold dilutions of each compound.
Ribavirin (SigmaeAldrich, USA) was used as a reference anti-are-
navirus substance. After 48 h of incubation at 37 ꢁC, supernatant
cultures were harvested and extracellular virus yields were
determined by plaque assay. The effective concentration 50%
(EC50) was calculated as the concentration required to reduce virus
yield by 50% in the compound-treated cultures compared with
untreated ones. All determinations were performed twice and
each in duplicate.
[5] S. Jaguelin, A. Robert, P. Gayral, Réaction des cyanoépoxydes avec le 2-mer-
captobenzimidazole et la 2-aminothiazoline et evaluation biologique des
produits formés, J. Med. Chem. 26 (1991) 51e57.
[6] S.D. Fidanze, S.A. Erickson, G.T. Wang, R. Mantei, R.F. Clark, B.K. Sorensen,
N.Y. Bamaung, P. Kovar, E.F. Johnson, K.K. Swinger, K.D. Stewart, Q. Zhang,
L.A. Tucker, W.N. Pappano, J.L. Wilsbacher, J. Wang, G.S. Sheppard, R.L. Bell,
S.K. Davidsen, R.D. Hubbard, Imidazo[2,1-b]thiazoles: multitargeted inhibitors
of both the insulin-like growth factor receptor and members of the epidermal
growth factor family of receptor tyrosine kinases, Bioorg. Med. Chem. Lett. 20
(2010) 2452e2455.
[7] J.S. Barradas, M.I. Errea, N.B. D’Accorso, C.S. Sepúlveda, L.B. Talarico,
E.B. Damonte, Synthesis and antiviral activity of azoles obtained from carbo-
hydrates, Carbohydr. Res. 343 (2008) 2468e2474.
[8] C.J. Peters, Human infection with arenavirus in the Americas, Curr. Top.
Microbiol. Immunol. 262 (2002) 65e74.
[9] E.B. Damonte, C.E. Coto, Treatment of arenavirus infections: from basic studies
to the challenge of antiviral therapy, Adv. Virus Res. 58 (2002) 125e155.
[10] D.A. Enría, A.M. Briggiler, Z. Sánchez, Treatment of Argentine hemorrhagic
fever, Antiviral Res. 78 (2008) 132e139.
4.2.4. Effect of pre-treatment of virus or cells with compound prior
to infection
Virus and cell pre-treatment assays were performed as previ-
ously described [18]. For virus pre-treatment, equal volumes of
a virus suspension containing approximately 1 ꢂ 106 PFU of virus
and various concentrations of compound in MM were mixed and
incubated for 1.5 h at 37 ꢁC. A virus control was also performed by
incubation of the virus suspension with MM under the same
conditions. Then, samples were chilled and diluted further with
MM before being placed on Vero cell cultures for plaque assay, to
assess that titer reduction was only due to cell-free virion
inactivation.
[11] O.T. Schmidt, in: R.L. Whistler, M.L. Wolfrom (Eds.), Methods in Carbohydrate
Chemistry, vol. 2, Academic Press, New York, 1963, pp. 318e325.
[12] H. Weber, H.G. Khorana, Total synthesis of the structural gene for an alanine
transfer ribonucleic acid from yeast. Chemical synthesis of an icosadeox-
ynucleotide corresponding to the nucleotide sequence 21 to 40, J. Mol. Biol. 72
(1972) 219e249.
[13] R.I. Zhdanov, S.M. Zhenodarova, Chemical methods of oligonucleotide Síntesis,
Synthesis (1975) 222e245.
[14] M.M. Ponpipom, S. Hanessian, A method for the selective bromination of
primary alcohol groups, Carbohydr. Res. 18 (1971) 342e344.
In cell pre-treatment assay, Vero cell monolayers were pre-
incubated with MM containing different compound concentrations
during 2 h at 37 ꢁC. Then, supernatants were removed; cells were
thoroughly washed with PBS, and infected with JUNV at a MOI of
0.1 in the absence of compound. After 1 h incubation at 37 ꢁC,
inocula were discarded and MM was added. Virus yields were
determined at 48 h p.i. by plaque formation in Vero cells.
[15] J.D. More, N.S. Finney, A simple and advantageous protocol for the oxidation
of alcohols with o-iodobenzoic acid (IBX), Org. Lett. 4 (2002) 3001e3003.
[16] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. Methods 65
(1983) 55e63.
[17] N.A. Candurra, L. Maskin, E.B. Damonte, Inhibition of arenavirus multiplication
in vitro by phenotiazines, Antiviral Res. 31 (1996) 149e158.
[18] C.S. Sepúlveda, M.L. Fascio, M.B. Mazzucco, M.L. Docampo Palacios, R.F. Pellón,
C.C. García, N.B. D’Accorso, E.B. Damonte, Synthesis and evaluation of N-
substituted acridones as antiviral agents against hemorrhagic fever viruses,
Antivir. Chem. Chemother. 19 (2008) 41e47.
Acknowledgements
Research was supported by Agencia Nacional de Promoción
Científica y Tecnológica, Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET) and Universidad de Buenos Aires
(UBA), Argentina. E.B.D. and N.B.D. are members of Research Career
from CONICET and C.S.S. and J.S.B. are fellows from CONICET.