Synthesis and antiviral activity of some imidazo[1,2-b][1,3,4]thiadiazole carbohydrate derivatives

Article abstract of DOI:10.1016/j.carres.2019.05.003

Herein we describe the synthesis of imidazo[2,1-b][1,3,4]thiadiazoles from carbohydrates with D-ribo and D-xylo configuration. The antiviral activity of these compounds was tested against Junín virus (the etiological agent of Argentine hemorrhagic fever). The p-chlorophenyl derivatives showed antiviral activity in a range of micromolar concentration.

Full text of DOI:10.1016/j.carres.2019.05.003

Accepted Manuscript
Synthesis and antiviral activity of some imidazo[1,2-b][1,3,4]thiadiazole carbohydrate
derivatives
Mirta L. Fascio, Claudia S. Sepúlveda, Elsa B. Damonte, Norma B. D'Accorso
PII:
S0008-6215(19)30176-4
https://doi.org/10.1016/j.carres.2019.05.003
CAR 7710
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 27 March 2019
Revised Date: 10 May 2019
Accepted Date: 15 May 2019
Please cite this article as: M.L. Fascio, C.S. Sepúlveda, E.B. Damonte, N.B. D'Accorso, Synthesis
and antiviral activity of some imidazo[1,2-b][1,3,4]thiadiazole carbohydrate derivatives, Carbohydrate
Research (2019), doi: https://doi.org/10.1016/j.carres.2019.05.003.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and antiviral activity of some imidazo[1,2-b][1,3,4]thiadiazole carbohydrate
derivatives.
Mirta L. Fascio*a,c, Claudia S. Sepúlvedab, Elsa B. Damonteb,d, Norma B. D’Accorso*a,c
.
a CONICET- Universidad de Buenos Aires. Centro de Investigaciones en Hidratos de
Carbono (CIHIDECAR).Buenos Aires, Argentina.
b CONICET- Universidad de Buenos Aires. Instituto de Química Biológica de la Facultad
de Ciencias Exactas y Naturales (IQUIBICEN).Buenos Aires, Argentina.
c Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de
Química Orgánica, Buenos Aires, Argentina
d Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de
Química Biológica, Buenos Aires, Argentina
Abstract
Herein we describe the synthesis of imidazo[2,1-b][1,3,4]thiadiazoles from carbohydrates
with D-ribo and D-xylo configuration. The antiviral activity of these compounds was tested
against Junín virus (the etiological agent of Argentine hemorrhagic fever). The p-
chlorophenyl derivatives showed antiviral activity in a range of micromolar concentration.
Keywords: Imidazo[2,1-b][1,3,4]thiadiazole; D-Xilo derivatives; D-Ribo derivatives; Junín
virus; Antiviral activity.
* Corresponding author: CIHIDECAR - CONICET- UBA, Departamento de Química
Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires.
Intendente Güiraldes 2160, Ciudad Universitaria, Pabellón II, Tercer Piso, C.P: 1428.
Buenos Aires. Argentina. E-mail address: norma@qo.fcen.uba.ar (N. B. D.),
mfascio@qo.fcen.uba.ar (M. L. F).

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1. Introduction
Diverse classes of heterocyclic represent very attractive compounds in medicinal chemistry
due to their wide spectrum of biological properties with therapeutic potential. In particular,
our previous studies described the synthesis and selective antiviral activity against Junín
virus (JUNV) of imidazo[2,1-b]thiazole carbohydrate derivatives [1]. JUNV is a member of
Arenaviridae, a family of enveloped RNA viruses including several human pathogens that
cause hemorrhagic fever (HF) in humans. Lassa virus (LASV) in West Africa as well as
Junín (JUNV), Machupo (MACV), Guanarito (GTOV) and Sabiá viruses (SABV) in South
America are etiological agents of HF with significant level of morbidity and mortality [2,3].
The Argentine HF caused by JUNV is a severe viral disease with significant level of
morbidity and mortality in infected patients. Although different types of compounds have
been studied for inhibitory anti-arenavirus activity, no specific and safe chemotherapy is
available for any member of the family. Treatment is limited to the use of immune
convalescent plasma or the guanosine analog ribavirin (RIB) (1-(β-D-ribofuranosyl)-1H-
1,2,4-triazole-3-carboxamide), but several drawbacks are associated to both therapies [3].
RIB is not equally effective against all HF-causing arenaviruses and its therapy,
recommended only within the early phase of clinical disease, can produce undesirable
secondary reaction [4–7]. Furthermore, since new viral populations emerge periodically,
the discovery and development of new compounds with antiviral activity is essential for
human health worldwide and has become a permanent challenge.
Based on our previous results with imidazothiazole as JUNV inhibitors [1], we have
focused in the search of new active compounds synthesized from a carbohydrate unit. In
this case, we selected imidazo[2,1-b][1,3,4]thiadiazole as heterocyclic molecule due to its
proven relevance in medicinal chemistry. This heterocyclic is part of a broad variety of
compounds that exhibit interesting and diverse biological activities such as anti-
inflammatory properties [8], tubulin polymerization inhibitors [9], antiproliferative activity
[10], staphylococcal biofilm inhibitors [11], anti-tuberculosis activity [12], among others
[13,14]. Therefore, the aim of this work was the synthesis and antiviral evaluation of some
imidazo[2,1-b][1,3,4]thiadiazole carbohydrate derivatives with
configuration.
D
-xilo and
D-ribo

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2. Results and discussion
In order to obtain the imidazo[2,1-b][1,3,4]thiadiazole derivatives the synthetic route
developed is shown in Scheme 1.
O
O
Br
HO
HO
Br
O
HO
O
O
O
OH
O
OR1
c
a,b,c,d
d,e,a,b
H
H
Ref [16]
O
O
Ref [15]
O
O
OMe
OMe
O
O
H
O
H
O
5
1
4
d
2 R1 = -Me [1]
3 R1 = -p-OMePh [14]
Br
O
O
H
N
N
O
N N
NH2
R2
S
OMe
O
EtOH, reflux or
DMF, 90 °C
NH2
R2
O
S
6
R2
N
R2
S
EtOH, reflux or
DMF, 90 °C
N
S
N
7
8
9
R1 = -Me; R2 = -Et
R1 = -Me; R2 = p-ClPh
R1 = -Me; R2 = 3,4,5-triOMePh
N
N
H
N
O
OR1
10 R1 = -Me; R2 = p-NO2Ph
11 R1 = -p-OMePh; R2 = p-ClPh
O
OMe
O
14 R2 = -Et
O
12 R1 = -p-OMePh; R2 = 3,4,5-triOMePh
13 R1 = -p-OMePh; R2 = p-NO2Ph
15 R2 = p-ClPh
16 R2 = p-NO2Ph
H
O
Scheme 1. Synthetic pathway to obtain imidazo[2,1-b][1,3,4]thiadiazole derivatives.
Reagents and conditions: (a) NaH, DMF and then MeI; (b) CH3COOH 80%, r.t.; (c) Ph3P,
NBS, DMF, r.t.; (d) IBX, CH3CN, reflux; (e) NaBH4, EtOH/H2O, -20 °C.
Compounds with
D-xilo configuration (7-10) were obtained from 3-O-methyl-6-bromo-6-
deoxy-1,2-O-isopropylidene-α-
D
-xilo-hexofuranos-5-ulose [1] and the corresponding 2-
amino-5-substituted thiadiazoles. Meanwhile compounds 11-13 were performed from 3-p-
methoxyphenyl carbohydrate derivative 3[15].
Derivatives with
D-ribo configuration were obtained from 1,2;5,6-di-O-isopropylidene-α-
D
-glucofuranose (1) by inversion of C-3 configuration, using 1-hydroxy-1,2-benziodoxol-
3(1H)-one-1-oxide (IBX) in acetonitrile as oxidizing agent and sodium borohydride as
stereoselective reduction agent. The following steps were carried out using a similar
procedure to that described for
D-glucofuranose derivatives. The hydroxyl group of C-3
was methylated and then hydroxyls of C-5 and C-6 were deprotected in aqueous acetic acid

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(60%) to give 4 [16]. The selective bromination of the primary alcohol group was realized
with NBS-PPh3 [17]. The oxidation of hydroxyl group of C-5 to obtain 5 was performed
using IBX-CH3CN giving the corresponding α-bromoketone 6.
The imidazo[2,1- b][1,3,4]thiadiazole ring was obtained from a α-bromoketone derivative
(2, 3, or 6) and a 2-amino-5-substituted-1,3,4-thiadiazole by heating in the presence of
ethanol [14]. Compounds 7- 9, 11, 12, 14 and 15 were obtained with moderate yields. In the
case of the p-nitrophenyl derivatives the yields obtained using ethanol as solvent were low
and, in some cases, the reaction did not take place even after 24 h of heating. These
derivatives could only be synthesized when the reaction temperature exceeded 90 °C.
Therefore, the reaction between 2 and 2-amino-5-(p-nitrophenyl)thiadiazole was tested in
several solvents by heating at 90 °C. In dioxane or butanol the 2-amino-5-(p-nitrophenyl)
thiadiazole is very slightly soluble even at high temperatures; therefore the yields to obtain
10 were low (23% and 19% respectively). When the reaction was carried out in DMF the
heterocyclic turned out to be soluble and the yield was better than those obtained with
others solvents (41%). Decomposition of reagents was observed when the reaction
temperature exceeded 100 °C.
Imidazo[2,1-b][1,3,4]thiadiazole carbohydrate derivatives synthesized are present in Figure
1.

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Figure 1. Molecular structure of the new imidazo[2,1-b][1,3,4]thiadiazole derivatives.
The final compounds were tested against JUNV in two cell lines. The cytotoxicities as well
as their antiviral activity against JUNV are shown in Table 1.
Table 1. Cytotoxicity and antiviral activity against JUNV strain IV4454
A549
Vero
EC50 (µM)
>1000
Compound
CC50 (µM)
>1000
715
EC50 (µM)
>1000
SI
CC50 (µM)
>1000
830
SI
I
I
3.79
1.54
I
7
236.97
3.50
1.12
I
8
188.42 ± 7.10
178.74 ± 8.12
>500
275
380
339.67
9
589
700
>700
10
11
>1000
>1000
I
>1000
>6.22
160.77 ± 0.74

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546
<50
>500
>50
I
925
60.25
>1000
710
1.74
I
12
861.19 ± 25.8
>50
I
13
>1000
659
>1000
>500
>1000
I
>1000
I
14
I
I
141.68
5.01
>4.54
>21.6
15
>1000
307
>1000
>400
16
220.48 ± 9.30
18.5 ± 1.7
RIB
15.9
19.2 ± 1.5
CC50: cytotoxicity concentration 50%, compound concentration required to reduce the cellular
viability by 50% compared to untreated controls.
EC50: effective concentration 50%, compound concentration required to reduce virus yield by 50%
compared with untreated controls.
SI: selectivity index, ratio CC50/EC50.
I: Inactive.
As seen in Table 1, most compounds exhibited low or null levels of antiviral activity
compared to rivabirin. Compounds with electron with drawing groups showed better
antiviral activity than those derivatives with electron donor substituent. The p-chloro
derivatives (8, 11 and 15) resulted with moderate and selective antiviral activity against
JUNV infection of Vero cells. The effectiveness of an antiviral compound was evaluated by
the selectivity index (SI) which is determined as CC50 / EC50 ratio and evidences how safe
and effective is an antiviral (see Table 1).The values of SI for derivatives 8, 11 and 15
indicate that the compound concentration able to inhibit virus multiplication is at least 3.5-
6.22-fold lower than the concentration required to affect cell viability. It is important to
note that the imidatiazo[2,1-b]thiazole previously synthesized [1] have a carbonyl group
between the heterocyclic ring and carbohydrate moiety and this fact may be responsible of
their higher effectiveness and SI against JUNV infection.
3. Conclusion
We were able to synthesize a series of new carbohydrate derivatives bearing a unit of 2-
substituted imidazo[2,1-b][1,3,4]thiadiazoles. Both
derivatives were obtained. In the case of the -xilo derivatives, two different protective
groups for the hydroxyl in position 3 were synthesized. Aryl or akyl group were
D-ribo and D-xylo configuration
D

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incorporated to the imidazo[2,1- b][1,3,4]thiadiazole ring in position 2. These compounds
were tested against JUNV infection. Most of them were inactive, except the p-chlorophenyl
derivatives that showed an EC50 close to 200 micromolar although they were less effective
than the reference compound RIB.
4. Experimental
4.1. General remarks
Research chemicals were purchased from Sigma-Aldrich Co and used without further
purification in the reactions. Reactions were monitored by thin layer chromatography
(TLC) on silica gel plates (60 F254; Merck) visualizing with ultraviolet light or by dipping
the TLC plates in a cerium molybdate solution and then heating them. Column
chromatographic separations were performed on silica gel 60 (240-400 mesh, Merck).
Melting points were measured on a Thomas Hoover melting point apparatus (Philadelphia,
USA) and are uncorrected, and the optical rotation, [α]D, measurements were made using a
343 Perkin Elmer Polarimeter. Solvents were reagent grade and, in most cases, dried and
distillated before use according to standard procedures. 1H, 13C NMR spectra were recorded
on a Bruker AC-200 spectrometer, operating at 200, 50 MHz respectively; Bruker Fourier
300, operating at 300, 75 MHz respectively or a Bruker Advance Neo 500 spectrometer,
operating at 500, 125 MHz respectively. Chemical shifts (δ) are reported in parts per
million. FT-IR spectra were realized in ATR mode using a Nicolet IS50 spectrometer.
HRMS were realized in a Bruker MicrOTOF Q-II - ESI-Qq-TOF mass spectrometer.
4.2. Chemistry
Compound 2, 3, 4, 5 were obtained from 1 by a synthetic sequence procedure previously
described in literature [7], [15], [16], [17].
4.2.1.
3-O-Methyl-6-bromo-6-deoxy-1,2-O-isopropylidene-α-D-ribo-hexofuranos-5-
ulose (6)

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A mixture of IBX (1.30 g, 3.81 mmol) and 3-O-methyl-6-bromo-6-deoxy-1,2-O-
isopropylidene-α-D-allofuranose 5 (0.28 g, 0.93 mmol) in acetonitrile (30 mL) was refluxed
during 16 h. The reaction mixture was allowed to reach room temperature and the solid was
filtered. The solution was evaporated and purified by flash column chromatography, using
cyclohexane: acetone (80:20) as eluent, affording pure 6 (0.25 mg, 91% yield) as an
amorphous solid; mp: 67–68 °C. IR (neat cm-1): 2983, 2938, 1731, 1376, 1025, 989, 855,
871, 800. 1H NMR (200 MHz, CDCl3) δ: 5.88 (d, 1H, J1,2 3.4 Hz, H-1), 4.73 (t, 1H, J2,1
=
J2,3 3.8 Hz, H-2), 4.65 (d, 1H, J4,3 9.1 Hz, H-4), 4.19 (d, 1H, J6a,6b 13.3 Hz, H-6a), 4.08 (d,
1H, J6b,6a 13.3 Hz, H-6b), 3.80 (dd, 1H, J3,2 4.2, J3,2 9.0 Hz, H-3), 3.52 (s, 3H, OMe), 1.59
13
(s, 3H, CMe2), 1.38 (s, 3H, CMe2); C NMR (50 MHz, CDCl3) δ: 198.8 (C=O), 114.0 (C
Me2), 104.6 (C-1), 82.8, 80.9 (C-2, C-4), 77.5 (C-3), 58.9 (OMe), 31.9 (CH2Br), 27.0
(CMe2), 26.6 (CMe2).
4.2.2. General procedure for preparing 5-substituted-1,3,4-thiadiazol-2-amine.
The 5-substituted-1,3,4-thiadiazol-2-amine was synthesized as previously described in
literature [18]. General procedure: A stirring mixture of benzoic acid (7.32 g, 60 mmol), N-
aminothiourea (6.38 g, 70 mmol) and POCl3 (20 ml) was heated at 75 °C for 0.5 h. After
cooling down to room temperature, water was added. The reaction mixture was refluxed for
4 h. After cooling, the mixture was basified to pH 8 by the drop-wise addition of 50 percent
NaOH. The precipitate was filtered and recrystallized from ethanol.
5-(p-Nitrophenyl)-1,3,4-thiadiazol-2-amine: mp 249-252 °C (in lit 250-252 °C)[18].
5-(p-Chlorophenyl)-1,3,4-thiadiazol-2-amine: mp 226-228 °C (in lit 227-229°C)[19].
5-(3, 4, 5-trimethoxyphenyl)-1,3,4-thiadiazol-2-amine: mp 225-227 °C (in lit 220°C)[20].
4.2.3. General procedure for preparing imidazo[2,1- b][1,3,4]thiadiazole.
Method A: A mixture of 2-amino-5-(substituted)-1,3,4-thiadiazole (0,755 mmol) and the β-
bromoketo derivative (0.51 mmol) in ethanol (4 mL) was refluxed overnight. The mixture

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was then cooled and the solvent evaporated. The solid was purified by column
chromatography.
Method B: A mixture of 2-amino-5-(substituted)-1,3,4-thiadiazole (0.755 mmol) and the β-
bromoketo derivative (0.51 mmol) in DMF (4 mL) was heated at 90°C overnight. The
mixture was then cooled and the solvent evaporated. The solid was purified by column
chromatography.
4.2.3.1.
6-(1,2-O-Isopropylidene-3-methyl-α-D-xilofuranos-4-yl)-2-ethylimidazo[2,1-
b][1,3,4]thiadiazole (7).
Elution solvent for column chromatograpy: cyclohexane:acetone 85:15. Yield 85% (method
A); mp: 67–68 °C. [α]D -47.8 (c 1, CHCl3). IR (neat cm-1) : 2990, 2929, 1468, 1087, 1019,
1
858, 765. H NMR (200 MHz, CDCl3) δ: 7.67 (s, 1H, heterocyclic proton), 5.93 (d, 1H,
J1,2 3.8 Hz, H-1), 5.25 (d, 1H, J4,3 2.8 Hz, H-3), 4.61 (d, 1H, J2,1 3.8 Hz, H-2), 3.86 (d, 1H,
J3,4 3.1 Hz, H-4), 3.19 (s, 3H, OMe), 2.92 (q, 2H, JCH
7.6 Hz, CH2CH3), 1.46 (s, 3H,
,CH3
2
CMe2), 1.32 (t, 3H, JCH
7.6 Hz, CH2CH3), 1.27 (s, 3H, CMe2); 13C NMR (75 MHz,
,CH2
3
CDCl3) δ: 165.5, 144.0, 141.2, 112.5 (heterocyclic carbons), 111.7 (C Me2), 104.5 (C-1),
84.8 (C-2), 78.3 (C-3), 82.2 (C-4), 58.2 (OMe), 26.7 (CMe2), 26.2 (CMe2), 25,5 (CH2CH3),
12,9 (CH2CH3). HRMS (ESI) calcd for [M+H]+: 326.1169. Found: 326,1161.
4.2.3.2. 6-(1,2-O-Isopropylidene-3-methyl-α-D-xylofuranos-4-yl)-2-(p-chlorophenyl)
imidazo[2,1- b][1,3,4]thiadiazole (8).
Elution solvent for column chromatograpy: cyclohexane:acetone 9:1. Yield 41% (method
A); mp: 118–120 °C; [α]D -30.1 (c 1, CHCl3). IR (neat cm-1): 2990, 1467, 1120, 1076,
1
1024, 823, 591. H NMR (300 MHz, CDCl3) δ: 7.84 (s, 1H, heterocyclic proton), 7.79 (d,
2H, J 8.5 Hz, aromatic protons), 7.47 (d, 2H, J 8.5 Hz, aromatic protons), 6.04 (d, 1H, J1,2
3.8 Hz, H-1), 5.37 (d, 1H, J3,4 3.0 Hz, H-3), 4.70 (d, 1H, J2,1 3.8 Hz, H-2), 3.98 (d, 1H, J4,3
13
3.1 Hz, H-4), 3.29 (s, 3H, OMe), 1.56 (s, 3H, CMe2), 1.37 (s, 3H, CMe2); C NMR (75
MHz, CDCl3) δ: 160.2, 144.0, 142.5, 113.1 (heterocyclic carbons), 137.9, 128.9, 129.7,
128.1 (aromatic carbons), 112.0 (C Me2), 104.8 (C-1), 85.1 (C-2), 78.6 (C-3), 82.5 (C-4),
58.5(OMe), 27.0 (CMe2), 26.5 (CMe2). HRMS (ESI) calcd for [M+H]+: 408.0779. Found:
408.0786.

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4.2.3.4.
6-(1,2-O-Isopropylidene-3-methyl-α-D-xylofuranos-4-yl)-2-(3´,4´,5´-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (9).
Elution solvent for column chromatography: cyclohexane:acetone 85:15. Yield : 45%
(method A), 71 % (method B); mp: 95–98 °C; [α]D -30.7 (c 1, CHCl3). IR (neat cm-1):
2990, 2925, 1585, 1467, 1127, 1077, 1023, 997, 855, 834, 730.
1H NMR (300 MHz,
CDCl3) δ: 7.85 (s, 1H, heterocyclic proton), 7.07 (s, 1H, trimethoxyphenyl proton), 6.05
(d, 1H, J1,2 3.8 Hz, H-1), 5.37 (d, 1H, J4,3 3.0 Hz, H-4), 4.71 (d, 1H, J2,1 3.8 Hz, H-2), 3.99
(d, 1H, J3,4 3.1 Hz, H-3), 3.96 (s, 6H, OMe of trimetoxyphenyl), 3.92 (s, 3H, OMe of
trimetoxyphenyl), 3.30 (s, 3H, OMe), 1.57 (s, 3H, CMe2), 1.38 (s, 3H, CMe2); 13C NMR (75
MHz, CDCl3) δ: 161.4, 144.0, 142.0, 113.0 (heterocyclic carbons), 153.9, 141.1, 125.6,
104.1 (aromatic carbons), 111.9 (C Me2), 104.7 (C-1), 85.0 (C-2), 82.4 (C-4), 78.5 (C-3),
61.1, 58.4, 56.5 (OMe), 26.9 (CMe2), 26.4 (CMe2). HRMS (ESI) calcd for [M+H]+:
464.1486. Found: 464.1491.
4.2.3.3 6-(1,2-O-Isopropylidene-3-methyl-α-D-xylofuranos-4-yl)-2-(p-nitrophenyl)
imidazo[2,1- b][1,3,4]thiadiazole (10).
Elution solvent for column chromatography: cyclohexane:acetone 9:1. Yield 41% (method
B); mp: 170–172 °C; [α]D -35.0 (c 1, CHCl3). IR (neat cm-1): 2983, 1514, 1469, 1346,
1084, 1021, 714, 687, 851. 1H NMR (300 MHz, CDCl3) δ: 8.36 (d, 2H, J 8.8 Hz,
aromatic protons), 8.05 (d, 2H, J 8.8 Hz, aromatic protons), 7.90 (s, 1H, heterocyclic
proton), 6.05 (d, 1H, J1,2 3.7 Hz, H-1), 5.38 (d, 1H, J3,4 3.0 Hz, H-3), 4.71 (d, 1H, J2,1 3.8
Hz, H-2), 4.00 (d, 1H, J4,3 3.2 Hz, H-4), 3.30 (s, 3H, OMe), 1.56 (s, 3H, CMe2), 1.37 (s,
3H, CMe2); 13C NMR (75 MHz, CDCl3) δ: 158.6, 144.1, 143.4, 113.3 (heterocyclic
carbons), 149.5, 136.1, 127.7, 124.7 (aromatic carbons), 112.1 (C Me2), 104.9 (C-1), 85.1
(C-2), 82.4 (C-4), 78.6 (C-3), 58.5 (OMe), 27.0 (CMe2), 26.4 (CMe2). HRMS (ESI) calcd
for [M+H]+: 419.1020; Found: 419.1022.
4.2.3.5.
6-(1,2-O-Isopropylidene-3-(p-methoxyphenyl)-α-D-xylofuranos-4-yl)-2-(p-
chlorophenyl)imidazo[2,1- b][1,3,4]thiadiazole (11).

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Elution solvent for column chromatography: cyclohexane:acetone 9:1. Yield 25% (method
A); mp: 77–80 °C. [α]D -22.3 (c 1, CHCl3). IR (neat cm-1): 2991, 2929, 1514, 1464, 1247,
1
1080, 1022, 828, 593. H NMR (300 MHz, CDCl3) δ: 7.85 (s, 1H, heterocyclic proton),
7.82 (d, 2H, J 8.5 Hz, p-chlorophenyl protons), 7.49 (d, 2H, J 8.5 Hz, p-chlorophenyl
protons), 7.04 (d, 2H, J 8.5 Hz, p-methoxyphenyl protons), 6.77 (d, 2H, J 8.6 Hz, p-
methoxyphenyl protons), 6.06 (d, 1H, J1,2 3.7 Hz, H-1), 5.39 (d, 1H, J3,4 2.8 Hz, H-3), 4.68
(d, 1H, J2,1 3.8 Hz, H-2), 4.40 (d, 1H, JCH2 11.6 Hz, CH2), 4.29 (d, 1H, JCH2 11.7 Hz, CH2),
4.18 (d, 1H, J4,3 2.9 Hz, H-4), 3.75 (s, 3H, OMe), 1.55 (s, 3H, CMe2), 1.35 (s, 3H, CMe2);
13C NMR (75 MHz, CDCl3) δ: 160.2, 143.8, 142.7, 113.4 (heterocyclic carbons), 137.9,
129.7, 129.0, 128.1 (p-chlorophenyl carbons), 159.4, 129.4, 113.8 (p-methoxyphenyl
carbons), 112.0 (CMe2), 104.9 (C-1), 83.6 (C-2), 82.7 (C-4), 78.9 (C-3), 72.5 (CH2), 55.4
(OMe), 27.0 (CMe2), 26.5 (CMe2). HRMS (ESI) calcd for [M+H]+: 514.1198; Found:
514.1200.
4.2.3.7 6-(1,2-O-Isopropylidene-3-(p-methoxyphenyl)-α-D-xylofuranos-4-yl)-2-(3’,4’,
5’-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (12).
Elution solvent for column chromatography: cyclohexane:acetone 95:5 and then 90:10.
Yield 40% (method A); mp: 61–63 °C. [α]D -12.3 (c 1, CHCl3). IR (neat cm-1): 2987, 2942,
1
1585, 1514, 1466, 1238, 1125, 1073, 1022, 998, 828, 730. H NMR (300 MHz, CDCl3) δ:
7.83 (s, 1H, heterocyclic proton), 7.08 (s, 1H, trimethoxyphenyl proton), 7.04 (d, 2H, J
8.6 Hz, aromatic protons), 6.77 (d, 2H, J 8.7 Hz, aromatic protons), 6.06 (d, 1H, J1,2 3.7 Hz,
H-1), 5.39 (d, 1H, J3,4 2.8 Hz, H-3), 4.68 (d, 1H, J2,1 3.7 Hz, H-2), 4.40 (d, 1H, JCH 11.7
2
Hz, CH2, 4.30 (d, 1H, JCH2 11.7 Hz, CH2), 4.18 (d, 1H, J4,3 3.0 Hz, H-4), 3.96 (s, 6H, OMe
of trimetoxyphenyl), 3.92 (s, 3H, OMe of trimetoxyphenyl), 3.75 (s, 3H, OMe), 1.55 (s,
13
3H, CMe2), 1.35 (s, 3H, CMe2); C NMR (75 MHz, CDCl3) δ: 161.3, 143.8, 142.3, 113.2
(heterocyclic carbons), 153.9, 141.3, 125.7, 104.2 (trimethoxy carbons), 159.4, 129.7,
129.4, 113.8 (p-methoxyphenyl carbons), 112.0 (C Me2), 104.8 (C-1), 83.6 (C-2), 82.7 (C-
4), 78.9 (C-3), 72.5 (CH2), 61.2, 56.6, 55.4 (OMe), 27.0 (CMe2), 26.5 (CMe2). HRMS (ESI)
calcd for [M+H]+: 570.1905. Found: 570.1905.

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4.2.3.6.
6-(1,2-O-Isopropylidene-3-(p-methoxyphenyl)-α-D-xylofuranos-4-yl)-2-(p-
nitrophenyl)imidazo[2,1- b][1,3,4]thiadiazole (13).
Elution solvent for column chromatography: cyclohexane:acetone 9:1. Yield 58% (method
B); mp: 141–143 °C ; [α]D -17.9 (c 1, CHCl3). IR (neat cm-1): 2929, 1520, 1345, 1244,
1079, 1024, 851, 814, 738, 684. 1H NMR (500 MHz, CDCl3) δ: 8.37 (d, 2H, J 8.8 Hz, p-
nitrophenyl protons), 8.07 (d, 2H, J 8.8 Hz, p-nitrophenyl protons), 7.89 (s, 1H,
heterocyclic proton), 7.04 (d, 2H, J 8.6 Hz, p-methoxyphenyl protons), 6.77 (d, 2H, J 8.6
Hz, p-methoxyphenyl protons), 6.06 (d, 1H, J1,2 3.7 Hz, H-1), 5.39 (d, 1H, J3,4 3.2 Hz, H-
3), 4.69 (d, 1H, J2,1 3.8 Hz, H-2), 4.41 (d, 1H, JCH 11.7 Hz, CH2), 4.30 (d, 1H, JCH 11.7
2
2
Hz, CH2), 4.20 (d, 1H, J4,3 3.1 Hz, H-4), 3.75 (s, 3H, OMe), 1.55 (s, 3H, CMe2), 1.36 (s,
3H, CMe2); 13C NMR (125 MHz, CDCl3) δ: 158.6, 143.8, 143.6, 113.5 (heterocyclic
carbons), 149.5, 136.1, 127.7, 124.7 (p-nitrophenyl carbons), 159.4, 129.6, 129.3, 113.8
(p-methoxyphenyl carbons), 112.1 (C Me2), 104.9 (C-1), 83.5 (C-2), 82.6 (C-4), 78.9 (C-3),
72.5 (CH2), 55.4 (OMe), 27.0 (CMe2), 26.5 (CMe2). HRMS (ESI) calcd for [M+H]+:
525.1438. Found: 525.1455.
4.2.3.8. 6-(1,2-O-Isopropylidene-3-methyl-α-D-ribofuranos-4-yl)-2-ethylimidazo[2,1-
b][1,3,4]thiadiazole (14).
Elution solvent for column chromatography: cyclohexane:acetone 9:1. Yield 42% (method
A); mp: 134–136 °C; [α]D + 37.6 (c 1, CHCl3). IR (neat cm-1) : 3129, 2978, 2942, 1472,
1
1063, 1012, 998, 953, 888, 862. H NMR (200 MHz, CDCl3) δ: 7.74 (s, 1H, heterocyclic
proton), 5.92 (d, 1H, J1,2 3.7 Hz, H-1), 4.96 (d, 1H, J4,3 8.9 Hz, H-4), 4.79 (t, 1H, J2,1 = J2,3
4.0 Hz, H-2), 4.18 (dd, 1H, J3,2 4.3, J3,2 8.9 Hz, H-3), 3.40 (s, 3H, OMe), 2.99 (q, J 7.6,
13
CH2CH3), 1.65 (s, 3H, CMe2), 1.40 (t, J 7.5, CH2CH3), 1.38 (s, 3H, CMe2); C NMR (50
MHz, CDCl3) δ: 166.2, 145.6, 142.3, 113.7 (heterocyclic carbons), 113.0 (C Me2), 103.9
(C-1), 83.8 (C-3), 77.4 (C-2), 74.7 (C-4), 58.8 (OMe), 26.9 (CMe2), 26.5 (CMe2), 25.8
(CH2CH3), 13.1 (CH2CH3). HRMS (ESI) calcd for [M+H]+: 326.1169. Found: 326.1160.
4.2.3.9 6-(1,2-O-Isopropylidene-3-methyl-α-D-ribofuranos-4-yl)-2-(p-chlorophenyl)
imidazo[2,1-b][1,3,4]thiadiazole (15).

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Elution solvent for column chromatography: cyclohexane:acetone 9:1. Yield 33% (method
A); 65% (method B); mp: 160–162 °C; [α]D +24.5 (c 1, CHCl3). IR (neat cm-1): 2925,
1
1464, 1120, 1082, 1012, 965, 834, 594. H NMR (200 MHz, CDCl3) δ: 7.84 (s, 1H,
heterocyclic proton), 7.79 (d, 2H, J 8.5 Hz, aromatic protons), 7.47 (d, 2H, J 8.5 Hz,
aromatic protons), 5.95 (d, 1H, J1,2 3.7 Hz, H-1), 5.00 (d, 1H, J4,3 8.9 Hz, H-4), 4.81 (t, 1H,
J2,1 = J2,3 4.0 Hz, H-2), 4.18 (dd, 1H, J3,2 4.3, J3,2 8.9 Hz, H-3), 3.42 (s, 3H, OMe), 1.67 (s,
13
3H, CMe2), 1.40 (s, 3H, CMe2); C NMR (50 MHz, CDCl3) δ: 160.8, 145.4, 143.3, 114.0
(heterocyclic carbons), 138.1, 129.7, 128.7, 128.1 (aromatic carbons), 113.1 (C Me2), 104.0
(C-1), 83.9 (C-3), 77.4 (C-2), 74.8 (C-4), 58.8 (OMe), 26.9 (CMe2), 26.5 (CMe2). HRMS
(ESI) calcd for [M+H]+: 408.0779. Found: 408.0789.
4.2.3.10. 6-(1,2-O-Isopropylidene-3-methyl-α-D-ribofuranos-4-yl)-2-(p-nitrophenyl)
imidazo[2,1- b][1,3,4]thiadiazole (16).
Elution solvent for column chromatography: cyclohexane:acetone 9:1. Yield 29% (method
B); mp: 181–184 °C; [α]D +24.7 (c 1, CHCl3). IR (neat cm-1): 2990, 2929, 1519, 1469,
1
1342, 1085, 1019, 855, 752, 687. H NMR (200 MHz, CDCl3) δ: 8.37 (d, 2H, J 8.8 Hz,
aromatic protons), 8.06 (d, 2H, J 8.8 Hz, aromatic protons),7.91 (s, 1H, heterocyclic
proton), 5.96 (d, 1H, J1,2 3.6 Hz, H-1), 5.03 (d, 1H, J4,3 8.9 Hz, H-4), 4.84 (t, 1H, J2,1 = J2,3
3.9 Hz, H-2), 4.19 (dd, 1H, J3,2 4.2, J3,2 8.9 Hz, H-3), 3.44 (s, 3H, OMe), 1.68 (s, 3H,
CMe2), 1.41 (s, 3H, CMe2); 13C NMR (125 MHz, CDCl3) δ: 159.3, 145.4, 144.1, 114.2
(heterocyclic carbons), 149.6, 135.8, 127.8, 124.7 (aromatic carbons), 113.2 (C Me2), 104.1
(C-1), 84.0 (C-3), 77.4 (C-2), 74.7 (C-4), 58.8 (OMe), 26.9 (CMe2), 26.5 (CMe2). HRMS
(ESI) calcd for [M+H]+: 419.1020. Found: 419.1024.
4.3. Biological assays
4.3.1. Cells and virus
The cell lines Vero (African green monkey kidney, ATCC CCL-81) and A549 (human lung
carcinoma, ATCC CCL-185) were grown in Eagle's minimum essential medium (MEM)
(Gibco, Thermo Fisher Scientific, USA) supplemented with 5 % inactivated bovine serum
and 50 µg mL-1 gentamycin. For maintenance medium (MM), the serum concentration was

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reduced to 1.5 %. The JUNV strain used was the naturally attenuated IV4454 strain
obtained from a mild human case [21]. Virus stocks were propagated in Vero cells and
titrated by plaque formation in the same cell line.
4.3.2. Cytotoxicity assay
Cytotoxicity 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 different concentrations of the compounds, with three wells for each
concentration, using incubation conditions equivalent to those used in the antiviral assays.
Then 10 µL of MM containing MTT (final concentration 0.5 mg mL-1) was added to each
well. After 2 h of incubation at 37 °C, the supernatant was removed and 200 µL of EtOH
was added to each well to solubilize the formazan crystals. After vigorous shaking,
absorbance was measured in a microplate reader at 595 nm. The cytotoxic concentration
50% (CC50) was calculated as the compound concentration required to reduce the MTT
signal by 50% compared to untreated controls. All determinations were performed in
triplicate.
3.2.3. Antiviral activity
Antiviral activity was determined by a virus yield inhibition assay. Vero or A549 cells
grown in 24-well plates were infected at a multiplicity of infection (m.o.i.) of 0.1 PFU/cell.
After 1 h adsorption at 37 ºC, cells were washed and reefed with MM containing or not
serial two-fold dilutions of each compound. After 48 h of incubation at 37 ºC, supernatant
cultures were harvested and extracellular virus yields were determined by a 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
control cultures. Ribavirin (RIB) (Sigma-Aldrich, USA) was used as a positive anti-
arenavirus control in a simultaneous antiviral assay. All determinations were performed in
triplicate.
Acknowledgments

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The authors thank University of Buenos Aires (UBACyT 20020170100090BA,
200201170100363BA), CONICET (PIP112-2015-0100443CO) and Agencia Nacional para
la Promoción Científica y Tecnológica (ANPCyT) (PICT 2015 3080), Argentina, for
financial support.
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Highlights:
•
New
glucose.
The nitro compounds only could be obtained at highest temperatures.
D
-xilo and
D
-ribo imidazothiazoles derivatives were synthesized from D-
•
•
Heterocyclization gave better yields when N,N-dimethylformamide is used as
solvents.