New 1-indanone thiosemicarbazone derivatives active against BVDV

Article abstract of DOI:10.1016/j.ejmech.2007.10.023

Identification of new therapeutic agents for the treatment of viral diseases represents an area of active investigation. In an effort to develop new antiviral compounds, a series of 1-indanone thiosemicarbazone derivatives were synthesized. These derivatives were structurally characterized using several spectroscopic techniques and evaluated against bovine viral diarrhoea virus as a surrogate model for hepatitis C virus. Thiosemicarbazone 2m showed potent anti-bovine viral diarrhoea virus activity with a higher selectivity index (SI = 80.29) than that of ribavirin (SI = 11.64). This result determines the potentiality of these thiosemicarbazones as antiviral agents for the treatment of infections caused by other highly related members of Flaviviridae family, as hepatitis C virus.

Full text of DOI:10.1016/j.ejmech.2007.10.023

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1767e1773
http://www.elsevier.com/locate/ejmech
Short communication
New 1-indanone thiosemicarbazone derivatives active against BVDV
a
c
b
Liliana M. Finkielsztein , Eliana F. Castro , Lucas E. Fabian , Graciela Y. Moltrasio
a,
*
,
´
Rodolfo H. Campos , Lucıa V. Cavallaro , Albertina G. Moglioni
c
c
b
´
a Ca´tedra de Qu´ımica Orga´nica III, Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires, Jun´ın 956 (1113),
Ciudad Auto´noma de Buenos Aires, Argentina
b Ca´tedra de Qu´ımica Medicinal, Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires, Jun´ın 956 (1113),
Ciudad Auto´noma de Buenos Aires, Argentina
c Ca´tedra de Virolog´ıa, Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires, Jun´ın 956 (1113), Ciudad Auto´noma de Buenos Aires, Argentina
Received 25 July 2007; received in revised form 18 October 2007; accepted 19 October 2007
Available online 25 October 2007
Abstract
Identification of new therapeutic agents for the treatment of viral diseases represents an area of active investigation. In an effort to develop
new antiviral compounds, a series of 1-indanone thiosemicarbazone derivatives were synthesized. These derivatives were structurally character-
ized using several spectroscopic techniques and evaluated against bovine viral diarrhoea virus as a surrogate model for hepatitis C virus. Thi-
osemicarbazone 2m showed potent anti-bovine viral diarrhoea virus activity with a higher selectivity index (SI ¼ 80.29) than that of ribavirin
(SI ¼ 11.64). This result determines the potentiality of these thiosemicarbazones as antiviral agents for the treatment of infections caused by
other highly related members of Flaviviridae family, as hepatitis C virus.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Thiosemicarbazones derived from 1-indanones; Antiviral activity
1. Introduction
family Flaviviridae. The member of Pestivirus genus bovine
viral diarrhoea virus (BVDV), a single-stranded RNA virus
of positive polarity, is one of the best characterized and has
one of the largest RNA genomes (12.5 Kb) in this family
[3]. The genome organization of the pestiviruses closely re-
semble those of hepatitis C virus. Because of this close rela-
tion, BVDV may provide a surrogate model for HCV [4],
both for the molecular study of viral proteins [5] and for the
evaluation of antiviral compounds [6].
Biological properties of thiosemicarbazone derivatives have
been studied since 1946 [7], when their activity against Myco-
bacterium tuberculosis was reported. Since then, this and other
biological properties of thiosemicarbazone derivatives such as
antibacterial [8], antitumoral [9], antiprotozoal [10] and cyto-
toxic activity [11] have been described. In 1950, Hamre et al.
[12] found that thiosemicarbazone derivatives from several
benzaldehydes were active against neurovaccinial infection
in mice when given orally. It was the first study on the antiviral
activity of thiosemicarbazone derivatives that prompted
further investigation of their properties in this area.
Hepatitis C virus (HCV) infection remains a main public
health problem with 175 million people infected in the world.
It is a major cause of cirrhosis and primary hepatocellular car-
cinoma and the main reason for liver transplant among adults
in western countries [1].
Currently, the best treatment available consists in the com-
bination of pegylated alpha interferon and the nucleoside ana-
logue ribavirin, but its effectiveness is only of about 50e60%
in patients suffering from chronic HCV infection and is asso-
ciated with important side effects [2]. Consequently, there is
an urgent need for highly effective and selective inhibitors
of HCV replication to improve the current antiviral strategies
against chronic HCV infection.
HCV is the sole member of the Hepacivirus genus, which
along with the flaviviruses and pestiviruses belong to the
* Corresponding author. Tel./fax: þ54 11 49648252.
E-mail address: gmoltra@ffyb.uba.ar (G.Y. Moltrasio).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.10.023

1768
L.M. Finkielsztein et al. / European Journal of Medicinal Chemistry 43 (2008) 1767e1773
Several studies made with thiosemicarbazone derivatives of
2.2. Evaluation of antiviral activity
N-methylisatine and N-allylisatine showed that these
compounds were capable of inhibiting structural protein
synthesis in human immunodeficiency virus (HIV) [13]. The
thiosemicarbazone of isatin was also found strongly active
as anti-poxvirus [14].
The synthesized compounds were screened for antiviral ac-
tivity on the replication of BVDV type-1 NADL strain in the
MadineDarby bovine kidney (MDBK) cell line by reduction
of cytopathic effect, as it was previously described (Table 2)
[18]. The cytotoxicity of all compounds evaluated was deter-
mined by means of the MTS/PMS method (Promega).
Among the TSCs assayed, six of them exhibited a selectiv-
ity index (SI ¼ 12.24e80.29) higher than that of the reference
ribavirin value (SI ¼ 11.64). Compound 2m showed the high-
est antiviral activity with an EC50 value of 1.75 mM. Moreover,
compound 2m presented a SI of 80.29, this value is around
seven times higher than that of ribavirin value.
The EC50 value of compound 2m lies in the micromolar
range and hence it is in accordance with that required for
new leads suitable for pharmaceutical development. The activ-
ity of this compound appears to be pestivirus specific and was
essentially inactive against others positive sense single-
stranded RNA viruses, such as polio virus type-1 Sabin strain
(EC50 ¼ 64 mM) and human immunodeficiency virus type-1
(EC50 > 38 mM). Moreover, 2m had no activity against the
negative stranded RNA vesicular stomatitis virus type-1 Indi-
ana strain (EC50 > 75.5 mM) and the double stranded DNA
herpes simplex virus type-1 F strain (EC50 > 64 mM).
Previously we have reported the inhibitory activity of sev-
eral thiosemicarbazone derivatives synthesized from aromatic
ketones and terpenones against Junin virus (JUNV), a RNA vi-
rus member of the Arenaviridae family and causative agent of
argentine hemorrhagic fever (AHF) [15]. The results obtained
showed that from 25 compounds tested, six of them having the
thiosemicarbazone group had a selective antiviral effect, with
SI values over 10. Among the thiosemicarbazone derivatives
the highest activity was found in those derived from 1-
indanone, particularly 5-methoxy-1-indanone (2k). This initi-
ated our interest to evaluate the antiviral activity of a series
of 1-indanone thiosemicarbazone derivatives (TSCs), with dif-
ferent patterns of substitution in the aromatic ring. In this way,
some new TSCs were synthesized and evaluated against
BVDV, a surrogate model for evaluation of anti-HCV activity.
2. Results and discussion
2.1. Synthesis
Despite the interesting antiviral properties of some of these
molecules, it is difficult to highlight in detail their structuree
activity relationship. Nevertheless, some general aspects merit
to be commented. Compound 2m, with two methoxy groups in
the aromatic ring, was identified as highly selective and potent
in vitro inhibitor of the replication of BVDV. The absence of
substituents (2a) or the presence of only one methoxy group
in the aromatic ring (2j, 2k and 2l), reduced markedly the
anti-BVDV activity. The incorporation of a halogen atom in
the aromatic ring (2oep), a lipophilic but electron-withdraw-
ing group, leads to very toxic compounds but with an interest-
ing activity. Further studies, in order to determine the
mechanism of action of these compounds, could explain the
influence of the type and position of the substituents in the ob-
served antiviral action. However, it is important to note that
the presence of the thiosemicarbazone group is essential for
the antiviral activity; 1-indanones, thiosemicarbazide and
semicarbazone derivatives of 1-indanones showed to be inac-
tive against BVDV.
The TSCs 2aep (Table 1) were obtained from the corre-
sponding 1-indanones (1aep) by treatment with thiosemicar-
bazide (Scheme 1).
Ketones (1-indanones) 1aec and 1jep are of commercial
source; 1d, e, h, i were synthesized following the described
procedures [16,17], and 1h and 1i were obtained as a mixture,
as they cannot be separated they were transformed into the
corresponding TSCs in this way.
Finally, compounds 1f and 1g are new compounds and were
obtained following the protocol presented in Scheme 2.
All the compounds were identified by spectral data (1H, 13
C
NMR and IR). The synthesized TSCs and their spectroscopic
characteristics are shown in Table 1.
The products obtained were isolated by conventional work-
up and purified by crystallization in ethanol. Melting point de-
termination was carried out to check the purity of compounds.
Both analytical and spectral data (IR and NMR) of all the
compounds are in full agreement with the proposed structures.
The IR spectra show bands in the region 3419e3435 cmꢀ1 and
3198e3263 cmꢀ1 due to stretching frequencies for NeH. The
absence of SeH stretch at 2500e2600 cmꢀ1 and the presence
of NeH stretch at 3118e3153 cmꢀ1 in the spectra, suggest
that all compounds are in the thione form and it is confirmed
by the presence of a band in the region 1083e1096 cmꢀ1 for
C]S. Finally a band for C]N appears in the region 1582e
The thiosemicarbazones possess the ability to form metallic
complexes and hence they can remove metals from biological
systems and in this way they are capable of inhibiting the ac-
tivity of metal-requiring proteins [19]. Further studies in the
action mechanism of this kind of compounds represent a chal-
lenge of our investigation.
1
1595 cmꢀ1 in all the TSCs. In all the H NMR were observed
3. Conclusion
three broad singlets for the three hydrogen atoms bonded to
the nitrogen atoms (d between 7.00 and 10.30 ppm). In the
13C NMR spectra were observed the corresponding signals
to C]N (d between 155.3 and 160.5 ppm) and to C]S (d be-
tween 176.7 and 179.0 ppm).
In the present study, the most potent evaluated TSC (2m)
showed a level of in vitro activity against BVDV better than
that of ribavirin (Table 2) and consequently, it is a promising
lead compound to pursue further analysis of other chemical

Table 1
Synthesized TSCs and their physical, analytical and spectral data
H
N
R7
R4
N
NH2
R6
R5
S
2a-p
2 (TSC)
R4
H
R5
H
R6
H
R7 MWa
Experimental
elemental analysisb
M.p.c (ꢁC) Yield (%) 1H NMR (d, ppm; JHz)
13C NMR (d, ppm)
IR (cmꢀ1
)
a (Ref. [15])
H
H
205.2852
175e177 61
2.88 (2H, m); 3.07 (2H, m);
7.34e7.87 (4H, m); 7.90 (1H, s);
8.17 (1H, s); 10.21 (s, 1H)
27.3; 28.4; 157.1; 178.7;
among others
3395.9; 3253.4; 3171.7;
3408.0; 1602.6; 1485.4;
among others
b
c
H
CH3
H
H
H
H
219.3122 H: 5.96%; C: 60.31%; 201e203 59
N: 19.13%; S: 14.64%
2.33 (3H, s); 2.85 (2H, m); 3.01
22.0; 28.0; 28.8; 122.4;
3224.3; 3199.2; 3118.2;
1583.4; 1512.7; 1303.1;
1090.2; 858.8
(2H, m); 7.10 (1H, d, J ¼ 7.8); 7.18 126.8, 128.8; 135.6; 141.6;
(1H, s); 7.74 (1H, d, J ¼ 7.8); 7.87 149.9; 158.4; 178.8
(1H, s); 8.12 (1H, s); 10.08 (1H, s)
CH3
H
H
219.3122 H: 5.98%; C: 60.33%; 223e224 60
N: 19.12%; S: 14.60%
2.21 (3H, s); 2.83 (2H, m); 2.93
(2H, m); 7.18 (2H, d, J ¼ 4.3);
7.63 (1H, t, J ¼ 4.3); 7.86 (1H, s);
8.03 (1H, s); 10.08 (1H, s)
18.3; 27.4; 119.6; 127.6,
131.5; 135.0; 137.6; 148.2;
158.4; 178.5
3387.2; 3173.1; 3128.92,
1583.7; 1640.0; 1583.7;
1515.5; 1296.6; 1096.2;
779.8; 711.6
d
H
CH3
CH3
CH3 233.3392 H: 6.46%; C: 61.82%; 258e259 83
N: 18.00%; S: 13.73%
2.30 (3H, s); 2.80 (2H, m); 2.98
(2H, m); 6.92 (1H, s); 7.02 (1H, s); 130.0; 132.1; 135.1; 139.8;
20.7; 21.0; 27.6; 27.7; 123.4; 3434.6; 3198.5; 3127.4;
2999.8; 1582.9; 1512.9;
1504.1; 1298.8; 1083.4;
855.8
7.20 (1H, s); 8.26 (1H, s); 10.14
(1H, s)e
2.34 (3H, s); 2.48 (3H, s); 2.94
150.0; 160.3; 178.3
e
f
Br
H
CH3 312.2354 H: 4.52%; C: 46.13%; dd
N: 13.44%; S:
10.26%; Br: 25.62%
82
21.9; 27.9; 28.6; 122.2;
3417.7; 3263.2; 3198.6;
3153.5; 1594.8; 1505.1;
1298.9; 1090.9; 859.8
(4H, s); 7.11 (1H, s); 7.23 (1H, s); 126.6; 128.6; 135.5; 141.4;
8.30 (1H, s); 10.30 (1H, s) 149.8; 158.3; 178.6
CH3 Cl
CH3 267.7841 H: 5.28%; C: 53.87%; 267e269 66
N: 15.66%; S:
11.96%; Cl: 13.26%
2.36 (3H, s); 2.67 (3H, s); 2.92e2.97 17.5; 21.7; 27.8; 28.7; 123.0; 3419.2; 3247.8; 3200.2;
(4H, m); 7.23 (1H, s); 7.28 (1H, s); 125.8; 133.7; 134.4; 138.4;
3141.1; 1595.0; 1514.1;
1299.8; 1092.8; 860.8
16.1; 20.6; 27.2; 27.6; 121.1; 3394.7; 3253.6; 3209.0;
8.30 (1H, s); 10.17 (1H, s)
2.28 (3H, s); 2.35 (3H, s); 2.88
148.8; 160.5; 179.0
g
CH3 Cl
CH3
H
267.7841 H: 5.27%; C: 53.89%; 260e262 65
N: 15.67%; S:
11.95%; Cl: 13.25%
(2H, m); 2.98 (2H, m); 7.74 (1H, s); 132.2; 134.5; 135.3; 135.9;
146.7; 156.0; 178.4
3143.1; 3003.9; 1584.1;
1516.2; 1294.6; 1094.9;
856.3
7.96 (1H, s); 8.21 (1H, s); 10.28
(1H, s)
h
i
H CH3 CH3
CH3 CH3 H
H
H
233.3392 H: 6.50%; C: 61.84%;
N: 17.99%; S: 13.72%
48
2.15 (3H, s, 2i); 2.23 (3H, s, 2h);
2.24 (3H, s, 2h); 2.26 (3H, s, 2i);
2.95e2.98 (m, 2h); 2.82e2.87 (m,
2h þ 2i); 7.10 (1H, d, J ¼ 7.8, 2i);
7.14 (1H, s, 2h); 7.58 (1H, d, J ¼ 7.8,
2i); 7.64 (1H, s, 2h); 7.86 (s,
2h þ 2i); 8.11 (s, 2h þ 2i); 10.12
(s, 2h þ 2i)
15.4; 20.0; 20.1; 20.5; 28.0; 3426.6; 3235.3; 3167.6;
28.3; 119.5; 122.8; 126.9;
129.6; 133.6; 135.9; 139.6;
140.4; 147.3; 158.5; 178.6
3131.9; 1586.8; 1511.9;
1297.8; 1093.2; 859.7
(continued on next page)

Table 1 (continued)
2 (TSC)
R4
R5
H
R6
H
R7
H
MWa
Experimental
elemental analysisb
M.p.c (ꢁC) Yield (%) 1H NMR (d, ppm; JHz)
13C NMR (d, ppm)
IR (cmꢀ1
)
j
OCH3
235.3117 H: 5.58%; C: 56.21%; 227e228 80
N: 17.84%; S: 13.62%
2.83 (2H, s); 2.91 (2H, s); 6.95
25.8; 27.7; 55.8; 112.4;
3431.6; 3247.7; 3179.1;
3140.4; 1637.7; 1587.4;
1515.1; 1264.5; 1094.3;
773.5; 699.3
(1H, d, J ¼ 7.7); 7.26 (1H, t, J ¼ 7.7); 114.5; 129.4; 137.4; 139.7;
7.41 (1H, d, J ¼ 7.7); 8.05 (1H, s);
156.6; 158.1; 178.8
10.11 (1H, s)e
k (Ref. [15])
H
H
H
OCH3
H
H
H
H
H
H
235.3117
235.3117
179e180 76
217e219 35
2.82 (2H, m); 3.04 (2H, m); 3.80
(3H, s); 6.85e7.80 (3H, s); 7.82
(1H, s); 8.07 (1H, s); 10.08 (1H, s)
2.87e2.89 (4H, m); 3.79 (3H, s);
6.92e7.48 (3H, s); 8.03 (1H, s);
8.18 (1H, s); 10.18 (1H, s)
27.6; 28.4; 156.9; 178.5;
among others
3400.8; 3275.2; 3181.5;
3041.0; 1602.8; 1526.3;
among others
l (Ref. [15])
OCH3
27.5; 27.9; 156.9; 178.5;
among others
3398.3; 3254.7; 3158.9;
3050.2; 1603.3; 1481.4;
among others
m
n
OCH3 OCH3
265.33.81 H: 5.71%; C: 54.41%; 246e247 75
N: 15.85%; S: 12.06%
2.83 (2H, m); 2.95 (2H, m); 6.93
(1H, s); 7.41 (1H, s); 7.97 (1H, s);
8.11 (1H, s); 10.08 (1H, s)e
28.1; 28.5; 56.1; 56.2; 104.3; 3373.3; 3259.4; 3151.9;
108.3; 130.0; 142.8; 149.3;
152.4; 158.5; 178.3
23.9; 26.2; 54.9; 58.4; 111.6; 3390.2; 3255.5; 3153.0;
1616.6; 1594.9; 1264,3;
1092.9; 853.0
OCH3 OCH3
H
H
265.3581 H: 5.70%; C: 54.31%; 212e213 75
N: 15.83%; S: 12.09%
2.82 (2H, m); 2.98 (2H, m); 7.01
(1H, d, J ¼ 8.5); 7.53 (1H, d,
116.4; 130.0; 140.2; 143.5;
1609.8; 1515.2; 1274.6;
1092.9; 802.7
J ¼ 8.5); 7.81 (1H, s); 7.98 (1H, s); 156.0; 176.7
10.02 (1H, s)e
o
H
H
Cl
Br
H
H
239.7301 H: 4.20%; C: 50.13%; 214e215 88
N: 17.51%; S:
13.39%; Cl: 14.81%
2.89 (2H, m); 3.06 (2H, m); 7.34
(1H, dd, J ¼ 8.2, J ¼ 1.2); 7.40
(1H, d, J ¼ 1.2); 7.91 (1H, d,
J ¼ 8.2); 8.01 (1H, s); 8.21 (1H, s);
10.30 (1H, s)
27.8; 28.6; 123.8; 126.2;
127.8; 135.7; 137.2; 151.3;
156.5; 178.9
3395.3; 3246.9; 3136.5;
1582.8; 1525.1; 1300.9;
1093.7; 865.9; 854.9; 706.5
p
H
284.1814 H: 3.55%; C: 42.31%; 213e215 89
N: 14.77%; S: 11.27
Br: 28.15%
2.88 (2H, m); 3.07 (2H, m); 7.48
(1H, d, J ¼ 8.3); 7.60 (1H, s); 7.84
(1H, d, J ¼ 8.3); 8.01 (1H, s); 8.21
(1H, s); 10.30 (1H, s)
27.2; 28.1; 123.6; 123.7;
128.5; 129.9; 137.3; 150.8;
155.3; 178.5
3430.0; 3253.6; 3145.9;
1594.7; 1501.0; 1463.6;
1288.2; 1096.4; 847.4; 798.4
a
Molecular weight.
b
c
d
e
Elemental analysis found was within ꢂ0.4% of the theoretical values.
Melting point.
Decompose before melting.
The signals corresponding to methyl and methoxy groups are overlapped by DMSO-d6/water.

L.M. Finkielsztein et al. / European Journal of Medicinal Chemistry 43 (2008) 1767e1773
1771
room temperature and carefully poured onto ice and was ex-
tracted with CH2Cl2 (20 mL), washed with water
(2 ꢃ 10 mL), dried over anhydrous Na2SO4, and evaporated
1
in vacuo to yield the crude product (1.87 g, 94%). H NMR
(CDCl3): d 2.42 (s, CH3Ar, compd. 3); 2.43 (s, 2 ꢃ CH3Ar,
compd. 4); 2.48 (s, CH3Ar, compd. 3); 3.32 (t, J ¼ 6.6 Hz,
CH2CO, compd. 3); 3.41 (t, J ¼ 7.0 Hz, CH2CO, compd. 4);
3.89 (m, CH2Cl, compds. 3 and 4); 7.15 (d, J ¼ 7.9 Hz,
HAr, compd. 3); 7.34 (d, J ¼ 7.9 Hz, HAr, compd. 3); 7.66
(s, HAr, compd. 4). Anal. Calc. for C11H12Cl2O (231.12) C:
57.16; H: 5.23; Cl: 30.68. Found C: 57.21; H: 5.21; Cl: 30.58.
Scheme 1. Reagents and conditions: (i) NH2NHCSNH2, EtOH, reflux.
modified derivatives to establish their therapeutic potential
against BVDV and HCV.
4.1.2. 6-Chloro-5,7-dimethylindan-1-one (1f) and
5echloro-4,6-dimethylindan-1-one (1g)
4. Experimental protocols
The mixture of compounds 3 and 4 (1.25 g, 5.4 mmol) was
added in small portions with swirling to concentrated H2SO4
(15 mL). The resulting solution was heated on a silicon bath
at 90 ꢁC. After 1 h, the reaction mixture was cautiously poured
onto ice and then extracted with CHCl3 (40 mL). The organic
layer was washed with a solution of 10% NaOH (2 ꢃ 15 mL),
water (15 mL), dried over anhydrous Na2SO4, and evaporated
in vacuo. The residue containing the mixture of indanones 1f
and 1g was separated by flash chromatography using hexanee
ethyl acetate (9:1) as eluent.
Compound 1f (0.47 g, 28%): m.p. 94e96 ꢁC. 1H NMR
(CDCl3): d 2.44 (3H, s, CH3Ar); 2.68 (2H, m, CH2CO);
2.71 (3H, s, CH3Ar); 2.99 (2H, m, CH2Ar); 7.19 (1H, s,
HAr). 13C NMR (CDCl3): d 15.8; 21.0; 25.0; 36.3; 122.4;
134.0; 134.7; 136.0; 141.4; 152.7; 206.4. MS m/z: 194 [M]þ
(100), 196 [M þ 2]þ (28), 159 [MꢀCl]þ (49), 131
[Mꢀ(Cl þ CO)]þ (91). Anal. Calc. for C11H11ClO (194.66)
C: 67.87; H: 5.70; Cl: 18.21. Found C: 67.84; H: 5.69; Cl
18.17. Compound 1g (0.60 g, 37%): m.p. 92e94 ꢁC. 1H
NMR (CDCl3): d 2.39 (3H, s, CH3Ar); 2.42 (3H, s, CH3Ar);
2.70 (2H, t, J ¼ 9.4 Hz, CH2CO); 3.02 (2H, t, J ¼ 9.4 Hz,
CH2Ar); 7.49 (1H, s, HAr). 13C NMR (CDCl3): d 14.4;
21.9; 24.5; 37.3; 125.8; 133.5; 134.6; 136.7; 142.8; 153.8;
206.7. MS m/z: 194 [M]þ (100), 196 [M þ 2]þ (37), 159
[MꢀCl]þ (24), 131 [Mꢀ(Cl þ CO)]þ (77). Anal. Calc. for
4.1. Chemistry
Melting points (uncorrected) were determined on a Thomas
Hoover apparatus. Thin layer chromatography (TLC) was used
to monitor reactions. Flash chromatography was performed
with silica gel (Merck silica gel 60, 230e400 mesh). IR spec-
tra were recorded as KBr pellets using a PerkineElmer Spec-
trum One FT-IR spectrophotometer. H NMR and 13C NMR
1
spectra were recorded on a Bruker 500 MHz spectrometer.
Mass spectra were recorded on a Shimadzu QP 5000 spec-
trometer. Micro-analyses (C, H, N) were carried out on a Carlo
Erba elemental analyser (Model 1106) and were within 0.4%
of the theoretical values.
4.1.1. 3-Chloro-1-(3-chloro-2,4-dimethylphenyl)propan-
1-one (3) and 3-chloro-1-(4-chloro-3,5-dimethylphenyl)-
propan-1-one (4).
AlCl3 (3.07 g, 23.0 mmol) was added to a magnetically
stirred solution of 2-chloro-1,3-dimethylbenzene (1.20 g,
8.5 mmol) and b-chloro-propionyl chloride (1.08 mL,
11.3 mmol) in CS2 (10 mL) at 0 ꢁC over a period of 30 min.
The reaction mixture was heated under reflux for a further
30 min. The resulting dark-brown solution was cooled at
Scheme 2. Reagents and conditions: (i) b-chloro-propionyl chloride, AlCl3, CS2, 0 ꢁC, 30 min; (ii) H2SO4 (c), 90 ꢁC, 1 h.

1772
L.M. Finkielsztein et al. / European Journal of Medicinal Chemistry 43 (2008) 1767e1773
Table 2
Antiviral activity of 1-indanone TSCs against BVDV strain NADL in MDBK
cells
4.2.2. Cytotoxic assay
MDBK cells were seeded in microplates at a density of
1 ꢃ 104 cells per well of a 96-well plate in Minimal Essential
Medium (Gibco) supplemented with Fetal Bovine Serum
(MEM-FBS); 24 h later, serial dilutions of the test compounds
were added. Cells were allowed to proliferate for 3 days at
37 ꢁC, after which the cell number was determined by means
of MTS/PMS method (Promega). The yield of formazan prod-
uct is proportional to the number of living cells. After 3 h at
37 ꢁC, the absorbance was determined at 490 nm. The percent
cell viability was calculated as follows:
2
R4
R5
R6
R7 CC50 (mM)a EC50 (mM)b SIc
a
H
H
H
H
156.30
130.00
164.80
51.70
18.10
6.48
8.13
8.60
b
H
CH3
H
H
H
20.06
20.27
ND
c
CH3
H
H
H
CH3
d
CH3
CH3
CH3
H
ND
8.94
e
Br
H
H
Cl
CH3 139.10
15.50
10.91
< 6.53
23.93d
f
CH3
H
80.70
72.50
161.30d
7.40
>11.10
6.74d
g
CH3 Cl
CH3
CH3
H
h
H
CH3 CH3
CH3
H
i
H
j
OCH3
H
H
H
H
822.50
149.00
106.30
140.50
140.80
>27.00
>15.00
54.00
242.40
18.80
26.00
1.75
3.39
7.93
Cell viability ¼ ðAbst ꢃ 100=AbscÞ
k
OCH3
H
OCH3 OCH3
H
OCH3
H
l
H
H
4.09
Absc ¼ absorbance of cells untreated; Abst ¼ absorbance of
cells treated with a certain dilution of compound.
The 50% cytotoxic concentration (CC50) was defined as the
concentration that inhibited the proliferation of exponentially
growing cells by 50% and was calculated using logarithmic in-
terpolation from dose-response curves.
m
H
H
H
80.29
12.24
3.07
n
OCH3 OCH3
H
H
H
11.50
8.80
1.81
o
H
H
Cl
Br
H
p
Ribavirin
H
8.29
11.64
4.62
a
50% cytotoxic concentration; concentration required to reduce MDBK
cells viability by 50%.
b
50% effective concentration; concentration required to inhibit BVDV cy-
topathic effect in MDBK cells by 50%.
4.2.3. Anti-BVDV assay
c
Selectivity index or ratio of CC50 to EC50.
Values corresponding to the mixture of compounds 2h and 2i.
MDBKcellswereseededinmicroplates(96wells)atadensity
of 1 ꢃ 104 cells per well, in MEM-FBS. Following 24 h of incu-
bation at 37 ꢁC and 5% CO2, medium was removed and a serial
dilution of the test compounds was added. After which, the
BVDVinoculum was added to each well. This inoculum resulted
in a greater than 80% of cytophatic effect after 3 days of incuba-
tion at 37 ꢁC. Uninfected cells and cells receiving virus without
compound were included in each assay plate. After 3 days
medium was removed and MEM-DHS (donor horse serum), sup-
plemented with MTS/PMS solution, was added to each well. The
absorbance of each well was read at 490 nm. The 50% effective
concentration (EC50) was defined as the concentration of
compound that offered 50% protection of the cells against
virus-induced cytopathic effect (CPE) and was calculated using
logarithmic interpolation from dose-response curves.
d
C11H11ClO (194.66) C: 67.87; H: 5.70; Cl: 18.21. Found C:
67.94; H: 5.68; Cl: 18.27.
4.1.3. General procedure for the synthesis of TSCs (2aep)
The TSC derivatives (Table 2) were obtained from the cor-
responding 1-indanones (1ae1p) by treatment with thiosemi-
carbazide (Scheme 2). A suspension of 1-indanone (1.2 mmol)
and thiosemicarbazide (2.7 mmol) in absolute ethanol (20 mL)
was heated under reflux for 30 min, then concentrated H2SO4
(0,10 mL) was added and the heating was continued until the
1-indanone was consumed. The conversion of 1-indanone in
the corresponding TSC was monitored by TLC on silica gel
60 F254 using chloroformeethanol (1:0.1) as eluent. The sol-
vent was removed in vacuo and the solid was suspended in wa-
ter (20 mL), filtered and washed with water (2 ꢃ 5 mL), EtOH
(5 mL), CH2Cl2 (5 mL) and finally hexane (2 ꢃ 5 mL).
The compounds were identified by spectral data (1H, 13C
NMR and IR). The synthesized TSCs, their yield, physical
and spectroscopic characteristics are summarized in Table 2.
Acknowledgments
This study was supported by grants from the University of
Buenos Aires UBACYT (2004e2007) B008, B016, B033 and
B036.
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