Synthesis and antiviral evaluation of some carbonucleoside analogues

Article abstract of DOI:10.1002/jhet.5570420536

1,2-O-Isopropylidene-α-L-threofuranosyl heterocyclic derivatives were synthesized from 1,2-O-isopropylidene-α-D-xilopentadialdo-1,4-furanose and tested for antiviral activity against herpes simplex virus type 1, dengue virus type 2 and Juni?n virus. For c

Full text of DOI:10.1002/jhet.5570420536

Synthesis and Antiviral Evaluation of Some Carbonucleoside Analogues
Jul-Aug 2005
979
Miriam A. Martins Alho, Maria I. Errea, Vanesa L. Sguerra, Norma B. D'Accorso*
CIHIDECAR (CONICET), Departamento de Química Orgánica,
Facultad de Ciencias Exactas y Naturales Argentina
Laura B. Talarico, Cybele C. García, Elsa B. Damonte
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales Argentina
Received October 25, 2004
1,2-O-Isopropylidene-α-L-threofuranosyl heterocyclic derivatives were synthesized from 1,2-O-iso-
propylidene-α-D-xilopentadialdo-1,4-furanose and tested for antiviral activity against herpes simplex virus
type 1, dengue virus type 2 and Junín virus. For comparative propose, the antiviral activity of some of their
pyranosyl analogues were also tested. The furanosyl derivatives showed to be moderate inhibitors of Junin
virus and, in general, proved to be more effective than the pyranosyl analogues.
J. Heterocyclic Chem., 42, 979 (2005).
The synthesis of heterocycles linked to a sugar residue has
received much attention, and this is due to the broad spec-
trum of biological properties of these compounds [1-4].
Most of the published work is referred to carbonucleo-
sides, in which the heterocycle is linked by a C-C bond to
the C-1 of the carbohydrate moiety [1, 5-7].
However, chemical and enzymatic stability under physi-
ological conditions could be increased if the heterocycle is
linked to a glycosidic carbon other than C-1 [1]. Besides,
the biological activity depends on the heterocycle nature as
well as the sugar identity.
Looking for new antiviral agents, some L-threofura-
nosyl and L-arabinopyranosyl residues linked to different
heterocycles were tested for antiviral activity against her-
pes simplex virus type 1, dengue virus type 2 and the etio-
logical agent of Argentine hemorrhagic fever [8], known
as Junin virus (JUNV).
For the biological test the following compounds were
synthesized: 2-[4-(1,2-O-isopropylidene-β-L-threofura-
nosyl)]-5-phenyl-1,3,4-oxadiazole (3), 5-[4-(3-O-benzoyl-
1,2-O-isopropylidene-β-L-threofuranosyl)]tetrazole (4)
and 2-[4-(3-O-benzoyl-1,2-O-isopropylidene-β-L-threofu-
ranosyl)]-5-methyl-1,3,4-oxadiazole (5).
3-O-Benzoyl-1,2-O-isopropylidene-α -D-xilofura-
nurononitrile (1) was obtained by dehydration of the corre-
sponding oxime (which was generated in situ by reaction of
the aldehyde with hydroxylamine hydrochloride in pyridine),
using benzoyl chloride. Treatment of 1,2-O-isopropylidene-
α-D-xilopentadialdo-1,4-furanose with a benzoylhydrazide
yielded 1,2-O-isopropylidene-β-D-xilopentadialdo-1,4-fura-
nose benzoylhydrazone (2). The oxidative cyclization of 2,
using phenyliodide diacetate (PIDA), led to 2-[4-(1,2-O-iso-
propylidene-β-L-threofuranosyl)-5-phenyl-1,3,4-oxadiazole
(3). 5-[4-(3-O-Benzoyl-1,2-O-isopropylidene-β-L-threofura-
nosyl)]tetrazole (4) was prepared by treatment of compound
1 with NH N in tetrahydrofurane and 2-[4-(3-O-benzoyl-1,2-
4
3
O-isopropylidene-β-L-threofuranosyl)]-5-methyl-1,3,4-oxadi-
azole (5) was synthesized by treatment of 4 with acetic anhy-
dride (Scheme 1). We successfully applied this transformation
on other carbohydrate derivatives [9], and the mechanism in
Scheme 1

980
M. A. Martins Alho, M. I. Errea, V. L. Sguerra, N. B. D'Accorso,
L. Talarico, C. C. García, and E. B. Damonte
Vol. 42
gas phase using the mass spectrometer as reactor was stud-
ied [10]. Compounds 6 - 11 were synthesized as we previ-
ously described [9, 11] (Figure 1). Compounds 1 - 5 were
Besides, compounds were studied by electron ioniza-
tion mass spectrometry (EI - MS). Even the molecular
+•
ion (M ) was only observed in the spectra of com-
1
13
+•
•
characterized by H nmr and C nmr. The chemical shift
pounds 3 and 5, signals corresponding to (M - CH )
3
Figure 1
1
and coupling constants obtained from the H nmr spectra
together with others expected by fragmentation of this
kind of compounds were observed in all spectra (see
Experimental).
13
are listed in Table 1. The assignment of the C nmr spectra
is shown in Table 2.
Table 1
H nmr Chemical Shifts (δ/ppm) and H-H Coupling Constant (J / Hz) for Compounds 1 – 5 [a]
1
1 [b]
2 [c]
3 [b]
4[b]
5 [b]
H-1
6.02 (d, 1H)
5.96 (d, 1H)
6.14 6 (d, 1H)
6.12 (d, 1H)
6.21 (d, 1H)
J
= 3.3
J
= 3.3
J
= 3.7
J
= 3.6
J
= 3.3
1,2
1,2
1,2
1,2
1,2
H-2
H-3
4.70 (d, 1H)
5.58 (d, 1H)
4.59 (d, 1H)
4.70 (d, 1H)
4.74 (d, 1H)
4.72 (d, 1H)
4.78 (d, 1H)
5.72 (d, 1H)
4.84 (d, 1H)
5.69 (s, 1H)
J
= 3.3
J
= 4.8
J
= 2.6
J
= 3.3
3,4
3,4
3,4
3,4
H-4
5.04 (d, 1H)
4.31 (dd, 1H)
= 3.7
5.36 (d, 1H)
5.88 (d, 1H)
5.69 (s, 1H)
J
4,5
H-5
N-H
-C(CH )
-
-
7.79 (d, 1H)
10.93 (s, 1H)
1.47 (s, 3H)
1.30 (s, 3H)
7.95-7.40 (m, 5H)
-
-
-
-
-
-
-
1.54 (s, 3H)
1.34 (s, 3H)
7.30-7.65 (m, 5H)
-
-
1.52 (s, 3H)
1.38 (s, 3H)
8.15-7.40 (m, 5H)
-
1.55 (s, 3H)
1.32 (s, 3H)
7.66-7.21 (m, 5H)
-
-
1.61 (s, 3H)
1.38 (s, 3H)
7.97-7.39 (m, 5H)
2.38 (s, 3H)
-
3 2
Aromatics
-C-5'(oxadiazol)-CH
-OH
3
4.67 (s, 1H)
4.01 (s, 1H)
[a] Chemical shifts are referred to TMS. Multiplicity of coupling and number of protons are given in brackets: s = singlet, d = doublet, dd = double
doublet, m = complex multiplet. [b] Chloroform-d [c] dimethylsulfoxide-d
1;
6

Jul-Aug 2005
Synthesis and Antiviral Evaluation of Some Carbonucleoside Analogues
981
Table 2
13
C Nmr Chemical Shifts (δ / ppm) for Compounds 1 – 5
1 [a]
2 [b]
3 [a]
4 [a]
5 [a]
C-1
C-2
C-3
C-4
C-5
C-2'
C-5'
105.6
82.6
76.5
68.6
113.8 [c]
-
-
113.8 [c]
27.0 and 26.2
-
165.0
134.1-128.5
105.5
85.8
76.8
80.7
147.4
-
-
111.4
26.4 and 25.7
-
160.3
131.8-127.6
105.9
84.4
75.9
73.8
105.4
83.6
77.6
74.0
-
-
153.1
113.3
26.8 and 25.3
-
164.9
105.4
83.2
77.5
73.4
-
-
162.3
165.6
112.6
26.4 and 25.7
-
-
161.4
164.9 [c]
113.3
26.8 and 26.3
10.8
164.9 [c]
133.8-128.6
-C(CH )
-C(CH )
-C-5'(oxadiazol)-CH
C=O
Aromatics
3 2
3 2
3
132.2-123.1
133.7-127.6
[a] Chloroform-d ; [b] Dimethylsulfoxide-d ; [c] overlapped signals.
1
6
The compounds were initially evaluated for their cyto-
toxicity in Vero cell cultures. None of them were cyto-
toxic in the range of concentrations tested. The antiviral
activity of all the compounds was assayed against three
human viruses: herpes simplex virus type 1 (HSV-1),
Junín virus (JUNV) and dengue virus type 2 (DEN-2).
As shown in Table 3, most of the compounds were
totally inactive against HSV-1 and DEN-2, but showed
moderate activity against JUNV. The most effective
inhibition was observed with the compounds 3, 5 and 10
antiviral potential and selectivity against JUNV than the
most active carbonucleoside analogue tested here.
Table 3
Antiviral Activity and Cytotoxicity of Compounds 1-11
Compound CC (mmolar) [a]
IC (mmolar) [b]
50
50
HSV-1
DEN-2
JUNV
1
2
3
4
5
6
7
8
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
>0.50
0.50
0.25
0.43
0.26
0.46
0.36
>0.50
>0.50
0.23
with inhibitory concentration 50% (IC ) values in the
50
range 0.23 - 0.26 mmolar.
All the compounds did not show significant cytotoxicity
for Vero cells up to 0.5 mmolar, the maximum concentra-
tion possible to be evaluated due to their water solubility.
Most of the compounds were inactive against HSV-1
and DEN-2. Only compound 1 was able to inhibit DEN-2,
but the value of the antiviral concentration was very near
to the cytotoxic dose (Table 3). By contrast, a more potent
activity of several products was observed against JUNV,
an arenavirus etiological agent of Argentine hemorrhagic
fever [8]. In general, the furanosyl derivatives proved to
be more effective than the pyranosyl derivatives: com-
pounds 2 - 7 inhibited JUNV multiplication, and the 3, 5
and 7 products were found the most selective agents with
9
10
11
>0.50
[a] Cytotoxic concentration 50%: Compound concentration required to
reduce cell viability by 50 %. Cytotoxicity was determined by the MTT
assay; [b] Inhibitory concentration 50%: Compound concentration required
to inhibit virus yield (JUNV and DEN-2) or virus plaques (HSV-1) by 50%.
Results are presented as the mean from duplicate independent tests.
In conclusion, seven of the derivatives tested showed
a moderate degree of antiviral activity, meanwhile
three of them (3, 5, and 7) proved to be moderate
inhibitors of the arenavirus JUNV at non cytotoxic con-
centrations while no significant activity was observed
against HSV-1 and DEN-2. Particularly, when the hete-
rocycle was an oxadiazole ring (3 and 5) the activity of
the compounds was slightly higher than when a thia-
diazole was present (7).
These results showed that future syntheses, in this par-
ticular field, must be oriented to the generation of L-threo-
furanosyl oxadiazole derivatives, with improved capabili-
ties against JUNV. Actually, we are already working on the
synthesis of new compounds including these particular
groups.
IC values in the range 0.25 - 0.36 mmoles. By contrast
50
with the furanosic compounds, among the four L-ara-
binopyranosyl derivatives only compound 10 showed
selective activity against JUNV (IC = 0.23 mmolar); it
50
is surprising that the analog of compound 10 in the L-thre-
ofuranosyl group of derivatives, compound 4, was very
weakly active. For comparative purposes, the carboxam-
ide nucleoside ribavirin (1-β-D-ribofuranosyl-1,2,4-tria-
zole-3-carboxamide), the only compound that has shown
partial efficacy against JUNV infections [12], was taken
as a reference substance. The IC of ribavirin against
50
JUNV was determined to be 18.3 µmolar and the CC
50
was >400 µmolar, exhibiting a very significantly higher

982
M. A. Martins Alho, M. I. Errea, V. L. Sguerra, N. B. D'Accorso,
L. Talarico, C. C. García, and E. B. Damonte
Vol. 42
EXPERIMENTAL
PIDA (0.32 g, 1 mmol) were added. The mixture was stirred at
room temperature and the reaction was followed by tlc (cyclo-
hexane/ethyl acetate 4:1 v/v) until no changes was observed. The
reaction medium was evaporated in vacuo and the residue
extracted with dichloromethane-water. The combined organic
layers were dried over sodium sulfate, and evaporated in vacuo.
The residue was purified by flash chromatography, using mix-
tures of cyclohexane/ethyl acetate as eluent. Compound 3 was
General Methods.
Melting points were determined on a Thomas Hoover capillary
apparatus and are uncorrected. Precoated Merck silica gel 60F-
254 plates were used for thin layer chromatography (tlc). Flash
chromatography was performed using silica gel 60G (mean parti-
cle size 15 µm) purchased from Merck, Inc. H nmr (200 MHz)
and C nmr (50 MHz) spectra were measured with a 200 MHz
Bruker spectrometer in deuteriochloroform or hexadeuterated
dimethylsulfoxide, as is indicated in each case. Heteronuclear
correlated spectra were obtained using the standard Bruker pro-
gram (HETCOR; program name: XHCORRDC.AUR) at 500
MHz, with a spectral width of 2551.02 Hz in the H direction
(resolution 128 Hz per point) and 151151.52 Hz in the C direc-
1
isolated as syrup (0.27 g, 88%); [α] +1.96 (c 0.8, chloroform);
D
13
+•
+•
•
+•
•
ms: m/z 304 (M ), 289 (M -CH ), 245 (M -CH -CH CO-
3
3
2
+•
•
+•
•
H ), 229 (M - CH -CH COOH), 187 (M - CH -CH COOH-
2
3
3
3
3
C H O).
2
2
Anal. Calcd. for 2(C
H N O )•C H O : C, 58.62; H, 5.75;
15 16 2 5 4 8 2
N, 8.05. Found: C, 58.27; H, 5.48; N, 8.33.
1
13
5-[4-(3-O-Benzoyl-1,2-O-isopropylidene-β-L-threofuranosyl)]-
tetrazole (4).
tion (resolution 2048 Hz per point).
Mass spectra were recorded at 70 eV on a Shimadzu QP-5000
spectrometer. Elemental analyses were within ±0.4% of the theo-
retical values.
Compound 1 (2.02 g; 7 mmoles) and sublimated ammonium
azide (0.60 g, 10 mmoles) were suspended in tetrahydrofurane
(25 mL), and then allowed react at room temperature. The reac-
tion was followed by tlc (cyclohexane/ethyl acetate 4:1 v/v) and
after 21 days the conversion was not complete. The mixture was
evaporated in vacuo to give a syrup, which was purified by flash
chromatography, using mixtures of cyclohexane/ethyl acetate as
Synthesis of Carbonucleosides and Related Compounds.
3-O-Benzoyl-1,2-O-isopropylidene-α-D-xilofuranurononitrile
(1).
eluent. Compound 4 was isolated as syrup (1.49 g, 64%); [α]
-
To a stirred solution of 1,2-O-Isopropylidene-α-D-xilopenta-
dialdo-1,4-furanose [13] (0.19 g; 1 mmol) in pyridine, hydroxy-
lamine hydrochloride (0.076 g, 1.1 mmoles) was added. The mix-
ture was heated in a boiling water bath for 0.5 hour and the reac-
tion was followed by tlc (cyclohexane/ethyl acetate 4:1 v/v), then
cooled using an ice bath and, after that, 0.7 mL of benzoyl chlo-
ride were slowly added. After heating the mixture for 0.5 hour in
a water bath at 60°, the reaction was completed; then the reaction
medium was dissolved in dichloromethane and successively
extracted with water, hydrochloride acid (1 N), water, sodium
hydroxide (10%) and water. The organic layer was treated with
charcoal, filtered through celite and concentrated. The residue
was separated by flash column chromatography using mixtures
of cyclohexane/ethyl acetate as eluent. Compound 1 was isolated
D
+•
•
+•
59.4 (c 1, chloroform); ms: m/z 317 (M -CH ), 274 (M -ace-
3
+•
•
+•
•
+•
tone or M -CH -N H), 214 (M -CH -CH COOH), 195 (M -
CH -C H COOH), 153 (M -CH -C H COOH-CH CO), 125
3
3
3
3
•
+•
•
3
6
5
3
6
5
2
+•
•
(M -CH -C H COOH-CH CO-CO).
3
6
5
2
Anal. Calcd. for C
H N O •C H O : C, 54.28; H, 5.71; N,
15 16 4 5 4 8 2
13.33. Found: C, 54.47; H, 5.65; N, 13.01.
2-[4-(3-O-Benzoyl-1,2-O-isopropylidene-β-L-threofuranosyl)]-
5-methyl-1,3,4-oxadiazole (5).
Compound 4, (0.83 g; 2.5 mmoles) was suspended in 25 mL of
acetic anhydride, and then heated at reflux for 4.5 hours, follow-
ing the reaction by tlc (cyclohexane/ethyl acetate 9:1 v/v). The
reaction was stopped by addition of ethanol and the medium
evaporated. The residue was purified by flash chromatography,
using mixtures of cyclohexane/ethyl acetate as eluent.
as a solid (0.22 g, 76%) of mp 115-117° (ethanol); [α] -44.3 (c
D
+•
•
+•
1, chloroform); ms: m/z 274 (M -CH ); 231 (M -CO(CH ) ),
214 (M -CH -CH COOH), 152 (M -CH -C H COOH).
3
3 2
Compound 5 was isolated as syrup in (0.53 g, 61%); [α] -58.4
+•
•
+•
•
D
3
3
3
6 5
+•
+•
•
+•
(c 1, chloroform); ms: m/z 346 (M ), 331 (M -CH ), 209 (M -
CH -C H COOH), 167 (M -CH -C H COOH-CH CO), 139
3
Anal. Calcd. for C
H NO : C, 62.28; H, 5.19; N, 4.84.
15 15 5
•
+•
•
3
6
5
3
6
5
2
Found: C, 62.09; H, 5.32; N, 4.80.
+•
•
(M -CH - C H COOH-CH CO-CO).
3
6
5
2
1,2-O-Isopropylidene-α -D-xilopentadialdo-1,4-furanose
Benzoylhydrazone (2).
Anal. Calcd. for 2(C
7.22. Found: C, 61.17; H, 6.42; N, 7.18.
H N O )•C H : C, 61.18; H, 6.19; N,
17 18 2 6 6 12
To stirred solution of 1,2-O-isopropylidene-α-D-xilopentadi-
aldo-1,4-furanose (1.75 g; 9.3 mmoles) in ethanol, benzoyl
hydrazide (1.29 g, 9.5 mmoles) was added. The reaction mixture
was heated at reflux with continuous stirring for 0.5 hour. After
that, the mixture was concentrated and from this solution crystal-
Biological Tests.
Cells and Viruses.
Vero (African green monkey kidney) cells were grown as
monolayers in Eagle's minimum essential medium (MEM) sup-
plemented with 5% inactivated calf serum. For maintenance
medium (MM), the serum concentration was reduced to 1.5%.
The C6/36 mosquito cell line from Aedes albopictus was cultured
at 28° in L-15 Medium (Leibovitz) supplemented with 0.3% tryp-
tose phosphate broth, 0.02% glutamine, 1% MEM non-essential
amino acids solution and 5% fetal calf serum.
The virus strains used were as follows: JUNV strain IV4454,
DEN-2 strain NGC, and HSV-1 strain F. DEN-2 stocks were pre-
pared in C6/36 cells and titrated by plaque formation in Vero cells.
JUNV and HSV-1 stocks were prepared and titrated in Vero cells.
lized compound 2 (1.14 g, 40%); mp 153° (ethanol); [α] -67.3
D
+•
•
+•
(c 1, ethanol); ms: m/z: 291 (M -CH ); 218 (M -CO(CH ) -
3
3 2
+•
+
CO-H ), 176 (C H N O ), 147 (C H N O ).
2
9
8
2
2
8 7 2
Anal. Calcd. for C
H N O : C, 58.82; H, 5.88; N, 9.15.
15 18 2 5
Found: C, 58.91; H, 5.67; N, 8.93.
2-[4-(1,2-O-Isopropylidene-β-L-threofuranosyl)]-5-phenyl-
1,3,4-oxadiazole (3).
To a stirred solution of compound 2 (0.31 g; 1 mmol) in 8 mL
of methanol, sodium acetate trihydrate (0.27 g, 2 mmoles) and

Jul-Aug 2005
Synthesis and Antiviral Evaluation of Some Carbonucleoside Analogues
983
Cytotoxicity Assay.
Científicas y Técnicas (CONICET), Agencia Nacional para la
Promoción Científica y Tecnológica (ANPCyT), and
Ministerio de Salud, Argentina. NBD and EBD are members of
the Research Career from CONICET and CCG is fellow of the
same institution. VLS is fellow of UBA. We are indebted to
Prof. Dr. A. Monge (Universidad de Navarra - España) for the
microanalyses.
Stock solutions of the compounds for the biological assays
were prepared at a concentration of 50 mmolar in dimethylsul-
foxide. Cytotoxicity was measured by 3-(4,5-dimethylthiazol-
2yl)-2,5-diphenyl tetrazolium bromide (MTT, supplied by
Sigma-Aldrich, USA) method in Vero cells. Confluent cultures in
96-well plates were exposed to two-fold dilutions of the com-
pounds, with three wells for each dilution, during 48 hours of
incubation at 37°. Then, 10 µl of MM containing MTT (final con-
centration 0.5 mg/ml) was added to each well. After 2 hours of
incubation, the supernatant was decanted and 200 µl ethanol was
added to each well to solubilize the formazan crystals. After vig-
orous shaking, absorbance was measured in a microplate reader
and cytotoxicity was calculated as the cytotoxic concentration
REFERENCES AND NOTES
*
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were determined by a plaque assay. The IC was calculated as
50
the concentration required to reduce virus yield by 50% in the
compound-treated cultures compared with untreated ones.
The anti-HSV-1 activity was determined by a plaque reduction
assay. Vero cells grown in 24-well microplates were infected with
about 50 PFU/well. After 1 hour adsorption at 37º, residual inocu-
lum was replaced by MM containing 0.7% methylcellulose and the
corresponding dose of each compound. Plaques were counted after
2 days of incubation at 37º and IC was calculated as the com-
50
pound concentration required to reduce virus plaques by 50%.
Acknowledgements.
This work was supported by grants from Universidad de
Buenos Aires (UBA), Consejo Nacional de Investigaciones
[13] M. L Fascio, V. J Montesano and N. B. D'Accorso, J.
Heterocyclic Chem., 35, 103 (1998) and references therein.