Synthesis, antiviral and anticancer activity of some novel thioureas derivedfrom N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide

Article abstract of DOI:10.1016/j.ejmech.2009.02.030

Due to a continuing effort to develop new antiviral agents, a series of 1-[4-(methanesulfonamido)-3-phenoxyphenyl]-3-alkyl/aryl thioureas 3a-i have been synthesized by the reaction of alkyl/aryl isothiocyanates with 4-amino-2-phenoxymethanesulfonanilide.

Full text of DOI:10.1016/j.ejmech.2009.02.030

European Journal of Medicinal Chemistry 44 (2009) 3591–3595
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiviral and anticancer activity of some novel thioureas derived
from N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide
,
a
b
b
Sevgi Karakus¸ a, S¸. Gu¨niz Ku¨çu¨kgu¨zel a , Ilkay Ku¨çu¨kgu¨zel , Erik De Clercq , Christophe Pannecouque ,
_
*
b
b
c
c
¨
Graciela Andrei , Robert Snoeck , Fikrettin S¸ahin , Omer Faruk Bayrak
a
_
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Tibbiye Cad. No: 49, 34668 Haydarpas¸a, Istanbul, Turkey
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
c
_
Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, 34755 Istanbul, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Due to a continuing effort to develop new antiviral agents, a series of 1-[4-(methanesulfonamido)-3-
phenoxyphenyl]-3-alkyl/aryl thioureas 3a–i have been synthesized by the reaction of alkyl/aryl
isothiocyanates with 4-amino-2-phenoxymethanesulfonanilide. These derivatives were structurally
characterized by the use of spectral techniques and evaluated for their anticancer and antiviral activities.
None of the tested compounds showed significant anticancer properties on A549 and L929 cell lines. All
synthesized compounds 3a–i were evaluated in vitro against HIV-1 (IIIB) and HIV-2 (ROD) strains in MT-
4 cells, as well as other selected viruses such as HSV-1, HSV-2, Coxsackie virus B4, Sindbis virus and
varicella–zoster virus using HEL, HeLa and Vero cell cultures. Compound 3b was able to block HIV
Received 3 October 2008
Received in revised form
12 February 2009
Accepted 26 February 2009
Available online 9 March 2009
Keywords:
Nimesulide
Thiourea
Antiviral activity
Anticancer activity
replication with almost 100% maximum protection at 125
65.9 g/ml against HIV-1 and HIV-2, respectively.
mg/ml, and IC50 values of 54.9 mg/ml and
m
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
selective COX-2 inhibitors such as nimesulide suppress the telo-
merase activity of gastric cancer cells [5] and exhibit anti-
proliferative effects on colorectal cancer cell lines [6]. Su and
et al. reported some novel nimesulide analogs which were eval-
uated as selective aromatase expression regulator in breast cells
[7,8]. Some thiourea derivatives have been reported to possess
anticancer activity against Erlich carcinoma and K562 human
leukemia cells [9]. Moreover, some clinical studies indicated
HIV infection and AIDS have been implicated as the first diseases
for which the discovery of drugs has been performed entirely via
a rational drug design approach. Current treatment regimens are
based on the use of two or more drugs from different classes of
inhibitors termed highly active antiretroviral therapy (HAART).
Some thiourea compounds were reported to be non-nucleoside
inhibitors (NNIs) of the reverse transcriptase (RT) enzyme of the
human immunodeficiency virus (HIV) [1]. High throughput
screening studies revealed that certain bis(aryl)thioureas carrying
an amide functionality, as exemplified by the structure shown
below, could be considered as inhibitors of herpes viruses such as
HSV, CMV and VZV [2]. In addition, some thiourea derivatives were
reported to be potent inhibitors of influenza virus neuraminidase,
Coxsackie virus B4 and thymidine kinase positive varicella–zoster
virus (TKþ VZV, OKA strain) [3,4].
a
significant correlation between viral infections and some
proliferative disorders.
These findings encouraged us to go further with our ongoing
studies on thiourea derivatives. Therefore, a novel series of 1-[4-
(methanesulfonamido)-3-phenoxyphenyl]-3-alkyl/aryl thioureas
were designed by the modification of the reported [2] bis(aryl)th-
iourea earlier. The intentional modifications were: i. introduction of
alkyl and aryl groups at R position; trying several linkers (L); ii.
introduction of a phenoxy ring; iii. replacement of acylamido group
with methanesulfonamido functionality (Fig. 1). All synthesized
compounds were screened in vitro against HIV-1 (IIIB) and HIV-2
(ROD) in MT-4 cells, as well as other selected viral strains (such as
HSV, CMV, VZV) using HeLa, Vero, HEL cells cytomegalovirus
(CMV), varicella–zoster virus (VZV) in human embryonic lung (HEL)
cells and anticancer activity together with cytotoxicity on A 549
and L 929 cell lines.
Nimesulide is a representative of the methanesulfonanilide
class of inhibitors, which display an interesting degree of COX-2
selectivity. COX-2 plays a pathogenic role in carcinogenesis and
supports tumor cell growth. Literature results demonstrate that
* Corresponding author. Tel.: þ90 216 349 12 16; fax: þ90 216 345 29 52.
E-mail address: gkucukguzel@marmara.edu.tr (S¸. Gu¨ niz Ku¨çu¨kgu¨zel).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.02.030

3592
S. Karakus¸ et al. / European Journal of Medicinal Chemistry 44 (2009) 3591–3595
H
N
O
H
N
O
S
R
CH3
S
S
O
Ar
L
O
R
L
N
H
N
H
N
H
N
H
Bis(aryl)thioureas
Compounds 3a-i
Fig. 1. The newly designed thiourea derivatives 3a–i.
O
H
O
O
H
N
O
S
O
H
N
O
S
a
b
N
S
CH3
CH3
CH3
S
O
O
O
O2N
H2N
N
H
N
H
R
1
2
3a-i
Scheme 1. Synthetic route to compounds 3a–i. Reagents and conditions: (a) SnCl2 þ HCl/MeOH, reflux; (b) R-NCS/dry acetone, reflux.
2. Result and discussion
3a–i, with less than 8 mmu bias between calculated and experi-
mental m/z values of either molecular or fragment ions (Table 1).
Ionization mode was electron impact (EI) in most cases, whereas
some compounds (3e, 3g and 3i) did not give molecular ion peaks
by using this technique. These compounds were analyzed using fast
atomic bombardment (FAB) procedure giving exact MHþ peaks
instead of Mþ in 3-nitrobenzyl alcohol matrix.
2.1. Chemistry
4-Amino-2-phenoxymethanesulfonanilide 2 was prepared by
the reaction of 4-nitro-2-phenoxymethanesulfonanilide (Nimesu-
lide) with SnCl2 solution in 5.4 ml of concentrated HCl in boiling
methanol [10]. This solution was refluxed and the resulting product
was obtained after evaporation of methanol and subsequent
neutralization with NaHCO3 (5%). 1-[4-(Methanesulfonamido)-3-
phenoxyphenyl]-3-alkyl/aryl thioureas 3a–i were synthesized by
the reaction of substituted alkyl/aryl isothiocyanates with
compound 2 in dry acetone (Scheme 1). This type of reactions were
reported to be performed in certain dry solvents or mixtures
[11,12]. In the present study, dry acetone was tried and found to be
useful. The physical and spectral data of thioureas 3a–i are given in
Tables 1 and 2. FTIR spectra exhibited N–H and C]S streching
vibrations at 3228–3371 and 1325–1392 cm–1, respectively. 1H
NMR spectral findings provide evidence for the formation of
thiourea function by displaying resonances at 7.67–9.4 and
9.19–10.23 ppm due to thiourea R–NH–CS and CS–NH–Ar function,
respectively [13] as well as the lack of resonances attributable to
NH2 function [10]. High resolution mass spectra (HRMS) confirmed
the molecular weights and empirical formulae of the compounds
2.2. Biological activity
2.2.1. Antiviral activity
Compounds 3a–i were tested for antiviral activity and cytotox-
icity in various viral test systems, according to previously published
procedures [14–18]. Thiourea derivatives 3a–i were evaluated for
their anti-HIV activity. With one exception, none of the synthesized
compounds showed any specific activity against HIV-1 (IIIB) or
HIV-2 (strain ROD) in MT-4 cells. Compound 3b had IC50 values of
54.9 mg/ml and 65.9 mg/ml against HIV-1 and HIV-2, respectively.
Although only weakly active, this compound was able to block the
HIV replication effectively with almost 100% maximum protection
at 125 mg/ml (from virus-induced cytopathogeneity). It would be
worthwhile to design several analogues of this compound to
optimize its anti-HIV potency and this will be the subject of our
future research. Based on the experience with this type of
Table 1
Some physicochemical characteristics of thiourea derivatives 3a–i.
Compounds
R
Formula
M.p. (ꢀC)
N–H, C]S
Log Pa
HR MS (m/z)
Calculated
Found
3a
3b
3c
3d
3e
3f
CH3
C2H5
C3H7
CH2CH]CH2
CH2C6H5
CH2CH2C6H5
CH2CH2C6H4 Cl (4)
C15H17N3O3S2
C16H19N3O3S2
C17H21N3O3S2
C17H19N3O3S2
C21H21N3O3S2
C22H23N3O3S2
C22H22ClN3O3S2
205-9
145-51
120-4
193-4
129-30
138-9
183-7
3350, 3267, 1338
3356, 1336
3333, 1392
3335, 1386
3306, 1323
2.73
3.19
3.51
3.20
3.94
4.33
4.88
352.0784 (FAB)
365.0868 (EI)
379.1024 (EI)
377.0868 (EI)
428.1097 (FAB)
441.1181 (EI)
352.0791 (MHþ
365.0881 (Mþ)
)
379.1054 (Mþ
)
377.0855 (Mþ
428.1122 (MHþ
)
)
3323,3228,1325
3356, 1336
441.1143 (Mþ
)
3g
476.0864 (FAB)
476.0870 (MHþ)
478.0829 (MH þ 2)
3h
3i
C6H11
C6H4 NO2 (4)
C20H25N3O3S2
C20H18N4O5S2
204-5
109-14 (dec.)
3371, 3234, 1338
3321, 1371
4.23
4.10
420.1410 (FAB)
459.0791 (FAB)
420.1417 (MHþ
)
459.0792 (MHþ
)
a
Calculation of log P values were performed using ALOGPS 2.102 log P/log S calculation software http://www.vcclab.org.

S. Karakus¸ et al. / European Journal of Medicinal Chemistry 44 (2009) 3591–3595
3593
Table 2
1H NMR spectral data of 3a–i.
Compounds 1H NMR (DMSO-d6, ppm)
3a
2.86 (d, 3H, NH–CH3); 2.94 (s, 3H, SO2–CH3); 7.04–7.42 (m, 8H, Ar–H);
7.66 (bs, 1H, NH–CH3); 9.22 (s, 1H, NH); 9.51 (bs, 1H, NH).
3b
1.04 (t, 3H, N–CH2–CH3); 2.94 (s, 3H, SO2–CH3);3.30 (q, 2H, N–CH2–
CH3); 7.03–7.42 (m, 8H, Ar–H); 7.69 (bs, 1H, NH–CH 2–CH3); 9.20 (bs,
1H, NH); 9.41 (bs, 1H, NH).
3c
0.81 (t, 3H, N–CH2–CH3); 1.46 (m, 2H, N–CH2–CH3); 2.93 (s, 3H, SO2–
CH3); 3.35 (q, 2H, N–CH2–CH2–CH3); 7.05–7.42 (m, 8H, Ar–H); 7.70 (bs,
1H, NH–CH2); 9.20 (bs, 1H, NH); 9.42 (bs, 1H, NH).
3d
2.93 (s, 3H, SO2–CH3); 4.05 (t, 2H, N–CH2–CH]); 5.03 (d, 1H,
–CH]CH2, J ¼ 17.2 Hz, trans); 5.08 (d, 1H, –CH]CH2, J ¼ 10.3 Hz, cis);
5.76–5.85 (m, 1H, –CH]CH2), 7.05–7.41 (m, 8H, Ar–H); 7.80 (bs, 1H,
NH–CH2); 9.22 (bs, 1H, NH); 9.52 (bs, 1H, NH).
3e
3f
2.93 (s, 3H, SO2–CH3); 4.66 (d, 2H, HN–CH2–C6H5); 7.06–7.40 (m, 13H,
Ar–H); 8.13 (bs, 1H, NH–CH2); 9.22 (bs, 1H, NH); 9.59 (s, 1H, NH).
2.78 (t, 2H, NH–CH2 –CH2–); 2.93 (s, 3H, SO2–CH3); 3.63 (q, 2H, NH–CH2
–CH2–); 7.04–7.41 (m, 13H, Ar–H); 7.70 (bs, 1H, NH–CH2); 9.21 (s, 1H,
NH); 9.53 (bs, 1H, NH).
3g
3h
3i
2.81 (m, 2H, NH–CH2 –CH2–); 2.96 (s, 3H, SO2–CH3); 3.34 (m, 2H, NH–
CH2–CH2–); 7.05–7.44 (m, 12H, Ar–H); 7.75 (bs, 1H, NH–C6H4–); 9.29 (s,
1H, NH); 9.58 (bs, 1H, NH).
1.10–1.25 (m, 10H, cyclohexyl CH2); 1.80 (s, 1H, N–CH-cyclohexyl); 2.93
(s, 3H, SO2–CH3); 7.06–7.41 (m, 8H, Ar–H); 7.57, 7.59 (2b, 1H, NH-
cyclohexyl); 9.19 (bs, 1H, NH); 9.33 (s, 1H, NH).
2.96 (s, 3H, SO2–CH3); 7.06–8.12 (m, 12H, Ar–H); 9.2–9.4 (bs, 1H, NH–
C6H4–); 10.23 and 10.29 (bs, 2H, NH).
molecules, thioureas are considered to act on the allosteric site of
HIV-1 RT, and a certain degree of flexibility might be required for
binding to HIV-1 RT. The absence of anti-HIV potency in most of the
compounds was probably due to their inability to exist in butterfly-
like conformation.
The compounds were also evaluated for in vitro antiviral activity
against herpes simplex virus [HSV-1 (strain KOS), thymidine
kinase-deficient (TK–) strain of HSV-1 resistant to acyclovir (ACVR),
HSV-2 (G)], varicella–zoster virus (VZV) [OKA strain and a thymi-
dine kinase deficient (TK–) VZV (07/1 strain)], cytomegalovirus
(CMV) [AD-169 strain and Davis strain], vaccinia virus (VV), vesic-
ular stomatitis virus (VSV) in HEL cell cultures; VSV, Coxsackie virus
B4, respiratory syncytial virus (RSV) in HeLa cell cultures and par-
ainfluenza-3 virus, Reo virus-1, Sindbis virus, Coxsackie virus B4
and Punta Toro virus Vero cell cultures. Results are presented in
Tables 3–5. As a result of broad spectrum antiviral screening of 3a–i,
the derivatives, which had minimal antivirally effective concen-
tration (MIC) less than one-fifth of minimal cytotoxic concentration
(if 5 ꢁ MIC < MCC), were considered active. There was only one
derivative (compound 3i) which showed marginal activity towards
several viruses in HEL cell cultures. This derivative with 4-nitro-
phenyl moiety showed inhibitory potency against vaccinia virus
and vesicular stomatitis virus by an MIC value of 8
mg/ml whilst
affecting the HEL cells with an MCC of 40 g/ml, which led up to
m
a selectivity index (SI ¼ MCC/MIC) of 5. It is currently unclear
whether the activity is due to its toxicity in the cell culture or to
a real antiviral effect. Compound 3i was found to be inactive against
HSV-1 (KOS) and HSV-2 (G) strains.
No activity was observed against the strains which were tested
with compounds 3a–i in HeLa cell cultures.
The test compounds were also screened for their antiviral
potency against varicella–zoster virus (VZV) [OKA strain and
a thymidine kinase deficient (TK–) VZV (07/1 strain)], cytomegalo-
virus (CMV) [AD-169 and Davis strains]. However, no activity was
observed with any of the screened compounds, except for
compounds 3c and 3d which had an inhibitory effect on CMV with an
EC50 value of approximately 55 ug/ml, and on VZV (3d) with an EC50
value of 49 to 59
mg/ml, and a MCC >100 mg/ml. These derivatives are
less potent than the standard drugs cidofovir and ganciclovir.

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S. Karakus¸ et al. / European Journal of Medicinal Chemistry 44 (2009) 3591–3595
Table 4
Cytotoxicity and antiviral activity of compounds 3a–i against cytomegalovirus (CMV), varicella–zoster virus (VZV) in human embryonic lung (HEL) cells.
Compounds Antiviral activity vs. CMV Antiviral activity vs. VZV EC50 g/ml) Cytotoxicity (VZV) CC50 (mg/ml)
(
m
g/ml)a Cytotoxicity (CMV) CC50
(m
EC50
g/ml)a
(m
c
c
AD-169 strain Davis strain TKþ VZV OKA strain TK- VZV 07/1 strain Cell morphology (MCC)b Cell growth (CC50
) )
Cell morphology (MCC)b Cell growth (CC50
3b
3c
3d
3e
3f
>100
54.7
54.7
>4
>20
>4
>100
54.7
54.7
>4
>20
>4
54.7
1.7
0.37
>100
>20
58.5
>4
>20
>20
>20
1.0
>100
>20
49
>100
>100
>100
20
100
20
>100
400
400
>100
56
82.4
45.2
48
33.6
>100
80
>100
100
>100
20
>100
56
82.4
45.2
48
82.4
>100
190
>4
>20
>0.8
>20
15
ꢂ100
3h
3i
ꢂ4
54.7
100
Ganciclovir 1.4
Cidofovir 0.24
>50
>50
0.0095
12.6
57
244
a
Effective concentration required to reduce virus plaque formation by 50%. Virus input was 20 plaque forming units (PFU).
Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.
Cytotoxic concentration required to reduce cell growth by 50%.
b
c
2.2.2. Anticancer activity
a Jeol JMS-700 instrument. Merck silica gel 60 F254 plates were
used for analytical TLC.
Cytotoxic and anticancer effects of synthesized compounds
were tested at four different concentrations. Compound 3a, as
shown in Fig. 2, caused 10–20% cytotoxic effect at the highest
concentration after a 4 day incubation period. The anticancer
activity results of 1-[4-(methanesulfonamido)-3-phenoxyphenyl]-
3-alkyl/aryl thioureas 3a–i on A549 lung cell growth, showed that
three compounds (3b, 3c and 3h) derived from ethyl, propyl and
cyclohexyl isothiocyanates inhibited proliferation of A549 lung cell
SMILES were generated from the structures using the ACD/
ChemSketch version 8.0 molecular editor (http://www.acdlabs.
com) and then log P values were calculated using ALOGPS 2.102
log P/log S calculation software [20,21]. The calculated log P values
for all the compounds are given in Table 1.
3.1.2. Preparation of 4-amino-2-phenoxymethanesulfonanilide 2
Nimesulide (0.0032 mole) was dissolved in boiling methanol
and SnCl2 (0.016 mole) solution in 5.4 ml of concentrated HCl was
carefully added. This solution was refluxed for 30 min. and the
resulting product was obtained after evaporation of methanol
followed by neutralization with NaHCO3 (5%). Crystals from
ethanol; m.p. 159–160 ꢀC (159–160 ꢀC [10])
by inhibiting 10–20% of cells at 10 mM. (Fig. 2) [19]. As known, the
lipophilicity of the compounds plays an important role during
the penetration of these compounds into the cells and increases the
activity. Log P values of the synthesized compounds were calcu-
lated using ALOGPS 2.102 log P/log S calculation software (Table 1).
These results demonstrated that there were no direct correlations
between the inhibitory effect of three compounds (3b, 3c and 3h)
against A549 lung cell and the lipophilicity of these compounds.
3.1.3. Synthesis of 1-[4-(methanesulfonamido)-3-phenoxyphenyl]-
3-alkyl/aryl thioureas 3a–i
A
dry acetone solution of 4-amino-2-phenoxymethanesu-
lfonanilide 2 and equimolar substituted alkyl/aryl isothiocyanates in
dry acetone was heated under reflux for 5 h. The completion of
reaction was checked by TLC (benzene:acetone, 70:30, v/v) . The
precipitate obtained was filtered off and recrystallized twice with
dry methanol/ethanol.
3. Experimental
3.1. Chemistry
3.1.1. Instrumentation and chemicals
All chemical compounds were purchased from Fluka. Nimesu-
lide was generously donated by Sanovel Pharmaceuticals (Turkey).
Melting points were determined with Bu¨chi-530 apparatus. The IR
spectra were obtained with a Shimadzu FTIR–8400. 1H NMR spectra
3.1.4. Antiviral activity
Compounds3a–i were tested forantiviralactivityand cytotoxicity
in various viral test systems, according to previously published
procedures [14–18]. The synthesized compounds were tested against
human immunodeficiency virus [HIV-1 (IIIB) and HIV-2 (ROD)],
vesicular stomatitis virus, Coxsackie virus B4, respiratory syncytical
virus, parainfluenza-3 virus, reovirus, Sindbis virus, PuntoToro virus,
herpes simplex virus type 1 and 2 and vaccinia virus-induced cyto-
pathicity at subtoxic concentrations in MT-4 cells, HeLa, Vero or Hel
cell culture. Brivudin, (S)-DHPA, ribavirin, acyclovir, cidofovir and
ganciclovir were used as the reference compounds.
in DMSO-d6 were recorded on
a Bru¨ker Avance-DPX-400
spectrometer (400 MHz). HREI–mass spectra were performed using
Table 5
Cytotoxicity and antiviral activity of compounds 3a–i against HIV-I(IIIB) and HIV-
II(ROD).
Compounds
HIV-1 (IIIB
)
HIV-2 (ROD)
IC50 g/ml)
IC50 g/ml)
(m
CC50
(
m
g/ml)
(
m
CC50 (mg/ml)
3.1.5. Anticancer activity
3a
3b
3c
3d
3e
3f
3g
3h
3i
>125
>125
>125
89.33
101.38
61.63
20.78
14.83
35.30
15.17
>125
>125
>125
89.33
101.38
61.63
20.78
14.83
35.30
15.17
54.85a
65.90a
The synthesized compounds were tested for their anticancer
activities and cytotoxicity properties. The CellTiter 96 Aqueous
ONE Solution (Promega, Madison, WI) was used to evaluate
cellular viability utilizing reduction of 3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo-
lium (MTS). A 549 and L 929 cell line were used to test both
anticancer effects and cytotoxicity. Cells were routinely grown in
a 75 mm flask in an environment containing 5% CO2 and passed
every 3 days. Cell viability was analyzed using the MTS assay.
>89.33
>87.20
20.15
>20.78
>14.83
>35.30
>15.17
>89.33
>12.80
>18.30
>20.78
>14.83
>35.30
>15.17
a
For compound 3b maximum protection (%) values at 125 mg/ml were 97% (HIV-1)
and 89.5% (HIV-2).

S. Karakus¸ et al. / European Journal of Medicinal Chemistry 44 (2009) 3591–3595
3595
3a
3b
140
120
100
80
60
40
120
100
80
60
40
20
0
10 nM
100 nM
1 µM
10 nM
100 nM
1 µM
20
0
10 µM
10 µM
1st day
2nd day
3rd day
4th day
1st day
2nd day
3rd day
4th day
3h
3c
120
100
80
60
40
20
0
120
100
80
60
40
20
0
10 nM
100 nM
1 µM
10 nM
100 nM
1 µM
10 µM
10 µM
1st day
2nd day
3rd day
4th day
1st day
2nd day
3rd day
4th day
Fig. 2. The growth inhibitory effect of compounds 3a–c, 3h against A549 lung cell.
5000 Cells were plated in each well of a 96-well tissue culture
plate. After 24 h of growth, the medium was replaced with fresh
medium containing compounds 3a–i at different concentration
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levels (10 nM, 100 nM, 1
for 4 days [19].
mM and 10 mM) and the cells were grown
The MTS assay was performed according to the protocol
provided by the Manufacturer. In short, 20 L of MTS solution was
m
[3] P.C. Nair, M.E. Sobhia, Eur. J. Med. Chem. 43 (2008) 293–299.
added to each well, and cells were incubated at 37 ꢀC for 1–3 h. The
absorbance (at 490 nm) of each well was determined. Data are
presented as a percentage of the values obtained from cells
cultured under the same conditions in the absence of test
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Acknowledgement
Nimesulide was supplied by Sanovel Pharmaceutical Industry
Inc. The authors are grateful to Dr. Ju¨rgen Gross from the Institute of
Organic Chemistry, University of Heidelberg, for his generous help
on obtaining HR-EI/FAB mass spectra of the synthesized
compounds. This work was supported by the Research Fund of
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Marmara University, project number: SAG.YYP.130206 /0002, and
the GOA nr. 05/19 of the KULeuven. We thank L. Persoons, L. Van
den Heurck, K. Erven and Anita Camps for excellent technical
assistance with (some of) the antiviral activity assays.