Synthesis, characterization, and anti-amoebic activity of N-(pyrimidin-2-yl)benzenesulfonamide derivatives

Article abstract of DOI:10.1002/cbdv.201300009

A new series of N-(pyrimidin-2-yl)benzenesulfonamide derivatives, 3a-3i and 4a-4i, was synthesized from pyrimidin-2-amines, 2a-2i, with the aim to explore their effects on in vitro growth of Entamoeba histolytica. The chemical structures of the compounds

Full text of DOI:10.1002/cbdv.201300009

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Synthesis, Characterization, and Anti-Amoebic Activity of N-(Pyrimidin-2-
yl)benzenesulfonamide Derivatives
by Abdul Roouf Bhat1)a), Mohammad Arshad1)b), Eun Ju Leea), Smritee Pokharela), Inho Choi*a),
and Fareeda Athar*b)
a) School of Biotechnology, Yeungnam University, Gyeongsan 712–749, Republic of Korea
(phone: þ82-53-810-3024; fax: þ82-53-810-4769; e-mail: inhochoi@ynu.ac.kr)
b) Center for Interdisciplinary research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025
(phoneþ91-11-26981717 Ext. 4492; fax: þ91-11-26980164; e-mail: fathar@jmi.ac.in,
fareeda_athar@yahoo.co.in)
A
new series of N-(pyrimidin-2-yl)benzenesulfonamide derivatives, 3a–3i and 4a–4i, was
synthesized from pyrimidin-2-amines, 2a–2i, with the aim to explore their effects on in vitro growth of
Entamoeba histolytica. The chemical structures of the compounds were elucidated by elemental analysis,
FT-IR, 1H- and 13C-NMR, and ESI mass-spectral data. In vitro anti-amoebic activity was evaluated
against HM1:IMSS strain of Entamoeba histolytica. The IC50 values were calculated by using the double
dilution method. The results were compared with the IC50 value of the standard drug ꢀmetronidazoleꢁ.
The selected compounds were tested for their cytotoxic activities by cell-viability assay using H9C2
cardiac myoblasts cell line, and the results indicated that all the compounds displayed remarkable >80%
viabilities to a concentration of 100 mg/ml.
1. Introduction. – Amoebiasis, a disease caused by protozoan parasite Entamoeba
histolytica [1], is the third leading cause of death worldwide, surpassed by malaria and
schistosomiasis only [2][3]. Occasionally, E. histolytica trophozoites penetrate the
intestinal mucosa, causing amoebic colitis and spread via portal circulation to other
organs. Amoebic liver abscess is the most common extraintestinal manifestation of
invasive amoebiasis [4]. Amoebiasis can be efficiently treated by nitroimidazole
derivatives (metronidazole, tinidazole, and ornidazole), but recent studies revealed
that several toxic effects such as genotoxicity, gastric mucus irritation, spermatozoid
damage etc. are associated with their use [5–7]. In addition to the toxic nature, several
intestinal protozoan parasites show resistance to these medicines [8][9].
Moiety has been incorporated in many chemotherapeutically important
sulfa drugs [10], which have been reported to exhibit antibacterial activities [11][12],
HIV protease inhibitors [13], carbonic anhydrase inhibitors [14–16], antiepileptic
agents [17], anticonvulsant agents [18][19], anti-amoebic agents [20] and are used as
endothelin receptor ETA, being a selective antagonist [21][22]. Pyrimidine ring is
present in many biologically significant molecules such as alloxane, barbitone, nucleic
acids, such as uracil, thymine, and cytosine, and vitamins, such as thiamine, riboflavin,
and folic acids [23–26]. During the last decade, several pyrimidine derivatives have
1
)
These authors contributed equally to this work.
ꢂ 2013 Verlag Helvetica Chimica Acta AG, Zꢃrich

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
been developed as chemotherapeutic agents, which have been found wide clinical
applications [27]. A large number of pyrimidine-based molecules are known for their
antimetabolite properties, and are used as antineoplastic and anticancer agents [28].
There are many reports of compounds with pyrimidine moiety which act as antifolates,
antibacterials, antiprotozoals, antitubercular, antihistaminic, and analgesics [29–33].
Pyrimidine derivatives of sulfa drugs such as sulfadimidine and sulfamerazine are used
in acute urinary tract infections (UTI) and cerebrospinal meningitis [27]. Sulfonami-
deꢀtrimethoprim combinations are used extensively for opportunistic infections in
patients with AIDS [34]. These observations prompted the incorporation of a
sulfonamide moiety into the pyrimidine ring to make use of both functionalities to
enhance pharmacological activities. In this work, a new series of N-(pyrimidine-2-
yl)benzenesulfonamide, 3a–3i and 4a–4i, was synthesized and screened in vitro for
their ability to inhibit the growth of E. histolytica. The selected compounds were tested
for toxicity profile using H9C2 cardiac myoblasts cell line. This study is an additional
effort to develop new chemotherapeutic agents which are anti-amoebic and non-toxic
to human cells.
2. Results and Discussion. – 2.1. Chemistry. N-(4,6-Substituted pyrimidin-2-
yl)sulfonamide derivatives 3a–3i and 4a–4i were synthesized by a multi-step reaction
process starting from simple chemical entities. ClaisenꢀSchmidt condensation reaction
furnish 1a–1i from substituted acetophenone and benzaldehydes/chlorobenzaldehydes
in high yields [35]. The cyclization of 1a–1i in the presence of guanidine hydrochloride
and isopropoxide, obtained in situ by adding Na metal in i-PrOH, yielded 4,6-
disubstituted phenylpyrimidin-2-amines 2a–2i, respectively. Sulfonamide derivatives
3a–3i and 4a–4i (Scheme) were synthesized by condensation of PhSO2Cl and TsCl
with corresponding pyrimidine derivatives 2a–2i, respectively. The compounds 2a–2i,
3a–3i, and 4a–4i were obtained in good yields and were found stable in solid states.
1
Spectral data such as FT-IR, H- and 13C-NMR, and ESI-MS, and elemental analyses
were in agreement with the proposed structures of the compounds. The purities were
established by sharp melting points and the elemental analysis. Selected diagnostic
bands of the FT-IR spectra of 2a–2i, 3a–3i, and 4a–4i provided further support for the
formation of the expected compounds. In addition to common bands which arise due to
n(C¼C) of aromatic region and benzene, bands which provide a primary information
about the conversion of reactants were present in FT-IR spectra. The compounds 2a–2i
showed stronger peaks in the regions near 1561–1511 and 1486–1446 cmꢀ1, due to
n(C¼N) vibration in aromatic system of the pyrimidine ring. A strong band in the range
of 3486–3310 cmꢀ1, attributed to NH2, rendered support to cyclization reaction. For
sulfonamide compounds, 3a–3i and 4a–4i, two bands in the range of 1334–1632 and
1017–1178 cmꢀ1 were observed which were ascribed to nasym(SO2) and nsym(SO2)
stretching vibrations, respectively. Besides, the shift of the band frequency to 3368–
3209 cmꢀ1 is attributed to n(NꢀH), since the NH2 functional group indicated a
conversion reaction.
The structures of the compounds were further elucidated by 1H-NMR spectroscopy.
For 2a–2i, a singlet between 5.47 and 5.12 ppm appeared due to the presence of NH2
group. In 3a–3i and 4a–4i, a singlet between 12.30 and 10.19 ppm for NH H-atom
suggested the conversion of NH2 !NH. The J values of some doublets which arise due

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2269
Scheme. Synthesis of Sulfonamide Derivatives 3a–3i and 4a–4i
a) MeOH, NaOH. b) Guanidine hydrochloride, iPrOH, reflux, 8 h. c) NaOH soln., PhSO2Cl or TsCl.
to spin-spin coupling in para-substituted Ph rings were informative about the formation
of the compounds. In the 13C-NMR spectra, the C¼N and C¼C C-atoms of pyrimidine
ring in 2a–2i resonated in the range of 163.1–159.2 and 105–98.0 ppm, respectively,
indicating the presence of the pyrimidine ring. In addition to these, the signals
attributed to aromatic and aliphatic C-atoms were also in favor of 2a–2i. In compounds
3a–3i and 4a–4i, a shift in the C¼N resonance were found, which can be considered as
caused by the induction of electron-withdrawing sulfonyl group. The C¼N signals
resonated in the range of 176.6–161.6 ppm. Besides, all these compounds were
subjected to ESI-MS analysis for further characterization. In the mass spectra of 2a–2i,
3a–3i, and 4a–4i, the characteristic peaks were observed as molecular ion ([Mþ1]þ )
peaks that confirmed the molecular weight of compounds.
2.2. Pharmacology. 2.2.1. Anti-Amoebic Activity. To combine the properties of two
different classes of biologically active compounds, we have synthesized a series of
pyrimidine sulfa analogs, 3a–3i and 4a–4i and compared their activities with those of
the corresponding precursors 2a–2i. All the compounds were screened in vitro for their
anti-amoebic activities against HM1:IMSS strain of E. histolytica cultured in TYIS-33

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Table. In vitro Anti-Amoebic Activities of Pyrimidine Derivatives 2a–2i, N-(Pyrimidin-2-yl)benzene-
sulfonamide Derivatives 3a–3i and 4a–4i against HM1 :IMSS Strain of E. histolytica, and Cytotoxicity
Profile of N-(Pyrimidin-2-yl)benzenesulfonamide which Showed 2–13-Fold Enhancement of in vitro
Anti-Amoebic Activity
Compound
Antiamoebic Activity
Toxicity IC50 [mm]
Safety Index (SI)*
IC50 [mm]
S.D.
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4d
4e
4f
6.15
2.5
0.06
0.01
0.05
0.03
0.06
0.05
0.03
0.06
0.03
0.04
0.02
0.05
0.01
0.01
0.06
0.01
0.04
0.05
0.05
0.04
0.01
1.06
0.06
0.07
0.04
0.06
0.01
0.06
–
–
–
–
–
–
–
–
–
–
–
428
357
–
–
424
–
330
–
–
–
–
–
–
–
–
–
–
–
1.72
4.47
7.21
2.63
1.66
5.74
1.59
2.34
3.04
0.44
0.7
5.32
1.57
0.82
3.62
0.13
1.95
2.68
0.9
–
–
974
510
–
–
518
–
2543
–
–
496
–
–
–
368
–
958
308
446
–
–
–
343
–
1.35
2.7
1.86
0.93
4.01
0.39
1.8
4g
4h
4i
373
555
MNZa)
a) Used as standard.
growth medium. The results are compiled in the Table. Sulfonamide derivatives showed
higher activities than their corresponding pyrimidine precursors. In the pyrimidine
series, compounds 2c (1.72 mm), 2g (1.66 mm), and 2i (1.59 mm) exhibited IC50 values
closer to that of the metronidazole (1.8 mm). However, the anti-amoebic activity was
enhanced by the insertion of sulfonamide group. In the sulfonamide series, the
compounds 3i (0.13 mm), 4i (0.39 mm), and 3c (0.45 mm) were found 3–13-fold more
active. Compounds 3d (0.7 mm), 3g (0.82 mm), 4c (0.9 mm), and 4g (0.93 mm) displayed a
1.9–2.5-fold difference, while compounds 3f (1.57 mm), 4d (1.35 mm), and 4f (1.86 mm)
had IC50 values approximately similar to that of the metronidazole. The differences in
IC50 values between pyrimidine-sulfonamides and their pyrimidine precursors indicate
that SO2NH moiety enhanced the activity. The compound 3i showed an excellent

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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activity in terms of its IC50 value and is the most active compound in the series. The
results were statistically evaluated by variance analyses. The null hypothesis was tested
using t test, and the significance of the differences between the IC50 value(s) of
metronidazole vs. 3c, 3d, 3f, 3g, 3i, 4c, 4d, 4f, 4g, and 4i were evaluated. The calculated t
values were higher than the table values at the 4% level. Hence, the character under
study was significantly influenced by the treatment [36].
2.2.2. Cell-Viability Assay. Compounds 3c, 3d, 3g, 3i, 4c, 4g, and 4i with IC50 value
less or near to that of metronidazole were screened to evaluate the toxic effects on
H9C2 cardiac myoblasts. The results were compared with a negative control DMSO
and a positive control (metronidazole). A subconfluent population of cardiac myoblast
cells was treated with increasing concentrations (1.57–200 mm) of test compounds and
positive control, and the number of viable cells was determined after 48 h by MTT (¼
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) cell viability test.
For all compounds, ca. 80% viable cells were found at 100 mm concentration (Fig.). To
investigate the selectivity of the compounds, the safety index (SI) was calculated and
defined as toxicity IC50/protozoal IC50, where toxicity IC50 is defined as the
concentration of compound that kills 50% of the human (kidney epithelial) cell line,
and protozoal IC50 is the concentration that kills 50% of amoeba protozoa. This allows
us to estimate which compound might be potentially efincacious or toxic against human
cells in vivo. The numerical results for each compound are collected in the Table. An
ELISA plate reader (Labsystems Multiskan RC, FI-Helsinki) at 570 nm with a
reference wavelength of 655 nm was used.
Figure. Effect of different concentrations of substituted benzene sulfonamide on percent viability of H9C2
cardiac myoblast cell line
2.2.3. StructureꢀActivity Relationships. Sulfa drugs are synthetic antimicrobial
agents with a wide spectrum of microorganisms, and pyrimidine molecules possess a
defined place in the world of medicine. The metronidazole is a well-known drug for
amoebiasis. The mechanism follows the reduction of NO2 group by pyruvate :ferre-
doxin oxidoreductase. Reduced intermediate particle interacts with intracellular

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targets. Cytotoxic intermediate particles interact with host cell DNA, resulting in
cleavage of the DNA strand and fatal destabilization of the DNA helix [37][38]. In this
study, a new series of N-(4,6-disubstituted pyrimidin-2-yl)benzenesulfonamide deriv-
atives, 3a–3i and 4a–4i, were compared in vitro. Compound 3i and 4i were found 4–13
times better than metronidazole. In both compounds, the core pyrimidine ring is
substituted with 4-nitrophenyl and 2-chlorophenyl rings. It can be assumed that the
NO2 group is mainly responsible for the activity. However, further studies are needed to
disclose the mechanism of action. The compound 4c which is substituted with Ph and 2-
chlorophenyl groups also showed three times higher activity than metronidazole,
whereas the compounds with tolyl groups were found least active in the series,
indicating that the electron-donating and electron-withdrawing nature of the sub-
stituents plays an important role. However, this study is not sufficient to reach a
conclusion.
3. Conclusions. – A novel library of N-(pyrimidin-2-yl)benzenesulfonamides was
obtained, with few reaction steps and low-molecular-weight starting materials, to
achieve chemicals that can be used as drug to target the protozoa that cause amoebic
infection. Considering the individual drug activities of sulfonamides and pyrimidin-2-
amine, structure modifications were performed to obtain products that possess both a
pyrimidine moiety and a benzene sulfonamide. All the pyrimidines, i.e., 2a–2i, and
benzene sulfonamides, i.e., 3a–3i and 4a–4i, were screened for their anti-amoebic
activities against HM1:IMSS strain of E. histolytica. Several compounds showed
excellent activities higher than that of metronidazole in terms of IC50 values. Among
them, 3i and 4i were the most potent with better inhibitory effects and higher SI values.
Experimental Part
General. All chemicals were purchased from SigmaꢀAldrich Chemical Company (USA) and were
used as such. Reactions were monitored on Merck pre-coated aluminum plate silica gel 60 F254 TLC
plates. M.p.: KSW melting-point apparatus; uncorrected. IR Spectra: KBr disks; Perkin Elmer model
1620 FT-IR spectrophotometer. 1H- and 13C-NMR spectra: at r.t.; Brucker Spectroscopic DPX-300 MHz
spectrometer in DMSO with TMS as an internal standard; chemical-shift values in ppm. ESI-MS:
Micromass Quattro II triple quadrupole mass spectrometer. Elemental analysis (C, H, N): Heraeus Vario
EL III analyzer by Central Drug Research Instituted, Lucknow, India; results within ꢁ0.3 of the
theoretical values.
1. Synthesis of Chalcones. Chalcones 1a–1i were prepared as described in [35].
2. General Procedure for the Synthesis of Pyrimidine Derivatives 2a–2i. To a soln. of guanidine
hydrochloride (1.1 equiv.) in 50 ml of iPrOH, Na metal (1.1 equiv.) was added. The mixture was refluxed
for 2 h, and the appropriate chalcone (1.0 equiv.) was added, and the mixture was further refluxed for 8 h.
The solvent was removed under reduced pressure. H2O was added, and the aq. phase was extracted with
CHCl3. Org. phases were dried (Na2SO4), filtered, and concentrated. The crude product was purified by
crystallization from MeOH and EtOH, or sometimes by CC (SiO2; 2% MeOH in CHCl3) to afford the
pure compounds.
4,6-Diphenylpyrimidin-2-amine (2a). Yield: 80%. M.p. 828. IR: 745, 729 (benzene), 1536, 1480
1
(C¼N), 3393 (NH2). H-NMR (CDCl3): 7.36–7.21 (m, 10 arom. H); 7.09 (s, 1 arom. H); 5.12 (s, NH2).
13C-NMR (CDCl3): 163.6, 163.2 (C¼N); 162 (NꢀC¼C); 133.2, 131.2, 129.5, 129.1, 128.8, 127.6, 126.9,
126.8, 103.2 (arom. C). ESI-MS: 248.34 ([Mþ1]þ ). Anal. calc. for C16H13N3 (247.29): C 77.71, H 5.30, N
16.99; found: C 77.72, H 5.32, N 16.97.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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4-(4-Methylphenyl)-6-phenylpyrimidin-2-amine (2b). Yield: 76%. M.p. 988. IR: 713, 742 (benzene),
1519, 1461 (C¼N), 2933 (Me), 3389 (NH2). 1H-NMR (CDCl3): 7.62–7.78 (m, 7 arom. H); 7.52 (d, J¼7.1,
2 arom. H); 6.98 (s, 1 arom. H); 5.16 (s, NH2); 2.13 (s, Me). 13C-NMR (CDCl3): 163.6, 162.1 (C¼N); 160.4
(NꢀC¼C); 135.1, 129.3, 131.2, 133.6, 126.5, 129.3, 128.8, 101.3 (arom. C); 23.7 (alk. C). ESI-MS: 262.76
([Mþ1]þ ). Anal. calc. for C17H15N3 (261.32): C 78.13, H 5.79, N 16.08; found: C 78.36, H 5.68, N 16.39.
4-(2-Chlorophenyl)-6-phenylpyrimidin-2-amine (2c). Yield: 78%. M.p. 618. IR: 762, 717 (benzene),
1549, 1446 (C¼N), 3397 (NH2). 1H-NMR (CDCl3): 7.43–7.35 (m, 5 arom. H); 7.27–7.19 (m, 4 arom. H);
6.96 (s, 1 arom. H); 5.21 (s, NH2). 13C-NMR (CDCl3): 162.2, 161.9 (C¼N); 161.3 (NꢀC¼C); 132.3, 130.2,
129.3, 129.2, 128.7, 128.4, 127.6, 127.4, 104.3 (arom. C). ESI-MS: 282.81 ([Mþ1]þ ). Anal. calc. for
C16H12ClN3 (281.74): C 68.21, H 4.29, N 14.91; found: C 68.1, H 4.39, N 14.7.
4-(4-Chlorophenyl)-6-phenylpyrimidin-2-amine (2d). Yield: 68%. M.p. 728. IR: 789, 721 (benzene),
1525, 1459 (C¼N), 3394 (NH2). 1H-NMR (CDCl3): 7.31 (d, J¼7.3, 2 arom. H); 7.28–7.19 (m, 5 arom. H);
7.14 (d, J¼7.3, 2 arom. H); 7.11 (s, 1 arom. H); 5.28 (s, NH2). 13C-NMR (CDCl3): 164.6, 163.9 (C¼N);
163.1 (NꢀC¼C); 133.1, 132.1, 130.7, 129.2, 128.1, 127.3, 126.9, 125.6, 102.6 (arom. C). ESI-MS: 282.96
([Mþ1]þ ). Anal. calc. for C16H12ClN3 (281.74): C 68.21, H 4.29, N 14.91; found: C 68.2, H 4.32, N 14.3.
4-(4-Chlorophenyl)-6-(4-methylphenyl)pyrimidin-2-amine (2e). Yield: 88%. M.p. 808. IR: 781, 739
1
(benzene), 1535, 1486 (C¼N), 2963 (Me), 3310 (NH2). H-NMR (CDCl3): 7.23 (d, J¼7.2, 2 arom. H);
7.18 (d, J¼7.4, 1 arom. H); 7.18 (d, J¼7.1, 1 arom. H); 7.14 (d, J¼7.2, 2 arom. H); 7.11 (d, J¼7.1, 2 arom.
H); 6.89 (s, 1 arom. H); 5.39 (s, NH2); 2.15 (s, Me). 13C-NMR (CDCl3): 162.9, 160.4 (C¼N); 161.1
(NꢀC¼C); 136.1, 134.7, 134.3, 131.3, 130.7, 128.3, 129.7, 128.7, 127.4, 125.6, 103.1 (arom. C); 24.1 (alk. C).
ESI-MS: 318.57 ([MþNa]þ ). Anal. calc. for C17H14ClN3 (295.77): C 69.03, H 4.77, N 14.21; found: C
68.97, H 4.88, N 14.39.
4-(2-Chlorophenyl)-6-(4-methylphenyl)pyrimidin-2-amine (2f). Yield: 53%. M.p. 988. IR: 709, 827
(benzene), 1511, 1457 (C¼N), 2911 (Me), 3328 (NH2). 1H-NMR (CDCl3): 7.42–7.32 (m, 4 arom. H); 7.21
(d, J¼7.4, 2 arom. H); 7.04 (d, J¼7.4, 2 arom. H); 6.93 (s, 1 arom. H); 5.26 (s, NH2); 2.09 (s, Me).
13C-NMR (CDCl3): 163.1, 162.9 (C¼N); 159.2 (NꢀC¼C); 134.3, 134.1, 129.4, 128.9, 128.8, 128.6, 127.6,
105.2 (arom. C); 23.7 (alk. C). ESI-MS: 286.73 ([Mþ1]þ ). Anal. calc. for C17H14ClN3 (295.77): C 69.03,
H 4.77, N 14.21; found: C 68.35, H 4.36, N 14.91.
4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-amine (2g). Yield: 65%. M.p. 1028. IR: 827, 819
(benzene), 1240 (NO2(sym.)), 1533, 1462 (C¼N), 1610 (NO2(asym.)), 3409 (NH2). 1H-NMR (CDCl3):
7.26 (d, J¼7.4, 2 arom. H); 7.21 (d, J¼7.4, 2 arom. H); 7.15 (d, J¼7.2, 2 arom. H); 7.11 (d, J¼7.2, 2 arom.
H); 6.91 (s, 1 arom. H); 5.47 (s, NH2). 13C-NMR (CDCl3): 163.2, 162.9 (C¼N); 162.1 (NꢀC¼C); 134.2,
133.9, 131.2, 131.2, 129.4, 129.2, 128.9, 128.6, 103.5 (arom. C). ESI-MS: 327.75 ([Mþ1]þ ). Anal. calc. for
C16H11ClN4O2 (326.74): C 58.82, H 3.39, N 17.15; found: C 58.76, H 3.49, N 17.31.
4-(4-Nitrophenyl)-6-phenylpyrimidin-2-amine (2h). Yield: 61%. M.p. 1018. IR: 817, 743 (benzene),
1245 (NO2(sym.)), 1524, 1443 (C¼N), 1621, (NO2(asym.)), 3328 (NH2). 1H-NMR (CDCl3): 7.93 (d, J¼
7.3, 2 arom. H); 7.34–7.21 (m, 5 arom. H); 7.28 (d, J¼7.3, 2 arom. H); 7.09 (s, 1 arom. H); 5.22 (s, NH2).
13C-NMR (CDCl3): 163.7, 163.6 (NꢀC¼C); 161.1 (C¼N); 133.9, 133.4, 132.5, 132.3, 130.7, 129.1, 128.7,
127.9, 127.6, 98.0 (arom. C). ESI-MS: 293.41 ([Mþ1]þ ). Anal. calc. for C16H12N4O2 (292.29): C 65.75, H
4.14, N 19.17; found: C 65.77, H 4.29, N 19.31.
4-(2-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-amine (2i). Yield: 83%. M.p. 1118. IR: 864, 887
(benzene), 1235 (NO2(sym.)), 1561, 1461 (C¼N), 1641 (NO2(asym.)), 3486 ( NH2). 1H-NMR (CDCl3):
7.92 (d, J¼7.3, 2 arom. H); 7.56 (d, J¼7.3, 2 arom. H); 7.38 (d, J¼7.2, 1 arom. H); 7.32 (d, J¼7.2, 1 arom.
H); 7.17 (m, 2 arom. H); 7.11 (s, 1 arom. H); 5.31 (s, NH2). 13C-NMR (CDCl3): 164.4, 163.3 (C¼N); 161.4
(NꢀC¼C); 134.3, 133.8, 133.5, 132.5, 131.2, 130.8, 129.3, 128.9, 128.7, 127.4, 103.5 (arom. C). ESI-MS:
326.77 ([Mþ1]þ ). Anal. calc. for C16H11ClN4O2 (326.74): C 58.82, H 3.39, N 17.15; found: C 58.76, H
3.41, N 16.98.
3. General Procedure for the Synthesis of Compounds 3a–3i and 4a–4i. The amines 2a–2i
(2.0 equiv.) were dissolved in NaOH soln. (10%, 40 ml), followed by addition of PhSO2Cl or TsCl
(1 equiv.) in small portions. The mixture was heated on H2O bath to complete the reaction. Dil. HCl was
added to precipitate the sulfonamides of the primary amines. Solids obtained were filtered and washed
with little cold H2O.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
N-(4,6-Diphenylpyrimidin-2-yl)benzenesulfonamide (3a). Yield: 71%. M.p. 1198. IR: 1025 (SO2-
(sym.)), 1570 (SO2(asym.)), 3249 (NꢀH). 1H-NMR (CDCl3): 10.71 (s, NH); 7.64–7.51 (m, 5 arom. SO2-
H), 7.29–7.22 (m, 10 arom. H); 6.99 (s, 1 arom. H). 13C-NMR (CDCl3): 172.6, 169.2 (C¼N); 161.3
(NꢀC¼C); 133.2, 132.1, 131.3, 131.1, 129.7 129.5, 129.1, 128.8, 127.6, 126.9, 126.8, 124.6, 103.2 (arom. C).
ESI-MS: 388.56 ([Mþ1]þ ). Anal. calc. for C22H17N3O2S (387.45): C 68.20, H 4.42, N 10.85; found: C
68.34, H 4.48, N 10.85.
N-[4-(4-Methylphenyl)-6-phenylpyrimidin-2-yl]benzenesulfonamide (3b). Yield: 53%. M.p. 1208.
IR: 1102 (SO2(sym.)), 1472 (SO2(asym.)), 2931 (Me), 3350 (NꢀH). 1H-NMR (CDCl3): 10.19 (s, NH);
7.91 (d, J¼6.5, 2 arom. H); 7.74–7.63 (m, 8 arom. H); 7.52 (d, J¼7.3, 2 arom. H); 7.43 (d, J¼7.3, 2 arom.
H); 6.89 (s, 1 arom. H); 2.18 (s, Me). 13C-NMR (CDCl3): 176.4, 168.1 (C¼N); 161.2 (NꢀC¼C); 135.3
(arom. C, Ts); 135.1, 132.7, 129.3, 128.4, 127.5, 127.3, 101.3 (arom. C); 24.7 (alk. C). ESI-MS: 402.73 ([Mþ
1]þ ). Anal. calc. for C23H19N3O2S (401.48): C 68.81, H 4.77, N 10.47; found: C 68.73, H 4.91, N 10.36.
N-[4-(2-Chlorophenyl)-6-phenylpyrimidin-2-yl]benzenesulfonamide (3c). Yield: 59%. M.p. 1068.
IR: 1086 (SO2(sym.)), 1434 (SO2(asym.)), 3324 (NꢀH). 1H-NMR (CDCl3): 10.67 (s, NH); 7.67–7.54 (m,
5 arom. H); 7.23–7.05 (m, 9 arom. H) 6.89 (s, 1 arom. H). 13C-NMR (CDCl3): 166.3, 161.9 (C¼N); 157.8
(NꢀC¼C); 139.7, 133.3, 132.4, 131.1, 129.8, 129.3, 128.6, 127.4, 123.6, 124.3 103.8 (arom. C). ESI-MS:
422.88 ([Mþ1]þ ). Anal. calc. for C22H16ClN3O2S (421.90): C 62.63, H 3.82, N 9.96; found: C 62.71, H
3.82, N 9.78.
N-[4-(4-Chlorophenyl)-6-phenylpyrimidin-2-yl]benzenesulfonamide (3d). Yield: 55%. M.p. 798. IR:
1
3263 (NꢀH), 1578 (SO2(asym.)), 1026 (SO2(sym.)). H-NMR ((D6)DMSO): 10.37 (s, NH); 7.48–7.58
(m, 10 arom. H); 6.91 (s, 1 arom. H); 7.32 (d, J¼7.21, 2 arom. H); 7.21 (d, J¼7.21, 2 arom. H). 13C-NMR:
164.3, 172.3 (C¼N); 161.3 (NꢀC¼C); 135.9 (arom. C, Ts); 132.5, 131.5,130.7, 128.4, 126.4. ESI-MS: 422.95
([Mþ1]þ ). Anal. calc. for C22H16ClN3O2S (421.90): C 62.63, H 3.82, N 9.96; found: C 62.65, H 3.74, N
9.81.
N-[4-(4-Chlorophenyl)-6-(4-methylphenyl)pyrimidin-2-yl]benzenesulfonamide (3e). Yield: 79%.
M.p. 1408. IR: 1124 (SO2(sym.)), 1532 (SO2(asym.)), 2955 (Me), 3367 (NꢀH). 1H-NMR (CDCl3):
10.93 (s, NH); 7.62 (d, J¼7.43, 2 arom. H); 7.47–7.32 (m, 9 arom. H); 7.21 (d, J¼7.23, 2 arom. H); 6.83 (s,
1 arom. H); 2.13 (s, Me). 13C-NMR (CDCl3): 171.1, 165.1 (C¼N); 159.4 (NꢀC¼C); 36.4 (arom. C, Ts);
132.8, 131.3, 129.4, 127.3, 126.8 (arom. C); 23.3 (alk. C). ESI-MS: 458.45 ([MþNa]þ ). Anal. calc. for
C23H18ClN3O2S (435.92): C 63.37, H 4.16, N 9.64; found: C 63.21, H 4.19, N 9.73.
N-[4-(2-Chlorophenyl)-6-(4-methylphenyl)pyrimidin-2-yl]benzenesulfonamide (3f). Yield: 87%.
M.p. 1368. IR: 1152 (SO2(sym.)), 1511 (SO2(asym.)), 2923 (Me), 3266 (NꢀH). 1H-NMR (CDCl3):
11.08 (s, NꢀH); 7.76–7.53 (m, 9 arom. H); 7.23–7.15 (m, 4 arom. H); 7.10 (s, 1 arom. H); 2.13 (s, Me).
13C-NMR (CDCl3): 168.9, 161.6 (C¼N); 158.9 (NꢀC¼C); 133.9 (arom. C, Ts); 133.1, 132.6, 130.7, 129.4,
125.7, 103.2 (arom. C); 22.4 (alk. C). ESI-MS: 497.12 ([Mþ1]þ ). Anal. calc. for C23H18ClN3O2S (435.93):
C 63.37, H 4.16, N 9.64; found: C 63.43, H 4.02, N 9.76.
N-[4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-yl]benzenesulfonamide (3g). Yield: 67%. M.p.
1208. IR: 1051 (SO2(sym.)), 1458 (SO2(asym.)), 3364 (NꢀH). 1H-NMR (CDCl3): 10.86 (s, NH); 7.68 (d,
J¼7.46, 2 arom. H); 7.26 (d, J¼7.4, 2 arom. H); 7.21 (d, J¼7.3, 2 arom. H); 7.15 (d, J¼7.46, 2 arom. H);
7.07–6.93 (m, 5 arom. H). 13C-NMR (CDCl3): 170.5, 164.7 (C¼N); 158.1 (NꢀC¼C); 135 (arom. C, Ts);
132.9, 131.8,129.6, 129.3, 128.1, 125.3, 106.3 (arom. C). ESI-MS: 467.49 ([Mþ1]þ ). Anal. calc. for
C22H15ClN4O4S (466.90): C 56.59, H 3.24, N 12.00; found: C 56.57, H 3.13, N 11.96.
N-[4-(4-Nitrophenyl)-6-phenylpyrimidin-2-yl]benzenesulfonamide (3h). Yield: 74%. M.p. 1058. IR:
1096 (SO2(sym.)), 1496 (SO2(asym.)), 3209 (NꢀH). 1H-NMR ((D6)DMSO): 12.06 (s, NH); 7.64 (d, J¼
7.3, 2 arom. H); 7.41–7.35 (m, 5 arom. H, Ts); 7.34–7.21 (m, 5 arom. H); 7.28 (d, J¼7.3, 2 arom. H); 7.09 (s,
1 arom. H). 13C-NMR ((D6)DMSO): 171.4, 163.0 (C¼N); 161.4 (NꢀC¼C); 136.2 (arom. C, Ts); 133.4,
132.5, 132.3, 130.7, 130.6, 129.1, 128.7,127.9, 127.6, 125.6, 99.3 (arom. C). ESI-MS: 433.41 ([Mþ1]þ ).
Anal. calc. for C22H16N4O4S (432.45): C 61.10, H 3.73, N 12.96; found: C 61.18, H 3.68, N 12.92.
N-[4-(2-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-yl]benzenesulfonamide (3i). Yield: 48%. M.p.
1
1128. IR: 1178 (SO2(sym.)), 1516 (SO2(asym.)), 3316 (NꢀH). H-NMR ((D6)DMSO): 10.81 (s, NH);
7.92 (d, J¼7.3, 2 arom. H); 7.56 (d, J¼7.3, 2 arom. H); 7.34 (d, J¼7.11, 2 arom. H); 7.38 (d, J¼7.2, 1 arom.
H); 7.32 (d, J¼7.2, 1 arom. H); 7.17 (m, 2 arom. H); 7.16–7.11 (m, 4 arom. H). 13C-NMR ((D6)DMSO):
169.5, 163.7, (C¼N); 163.2 (NꢀC¼C); 135.8 (arom. C, Ts); 134.3, 133.8, 133.5, 132.5, 131.2, 132.1, 129.8,

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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128.9, 128.7, 103.5 (arom. C). ESI-MS: 467.79 ([Mþ1]þ ). Anal. calc. for C22H15ClN4O4S (466.90): C
56.59, H 3.24, N 12.00; found: C 56.63, H 3.28, N 11.94.
N-(4,6-Diphenylpyrimidin-2-yl)-4-methylbenzenesulfonamide (4a). Yield: 74%. M.p. 958. IR: 1024
(SO2(sym.)), 1508 (SO2(asym.)), 3313 (NꢀH). 1H-NMR (CDCl3): 10.23 (s, NH); 7.61 (d, J¼7.4, 2 arom.
H, Ts); 7.45 (d, J¼7.4, 2 arom. H, Ts); 7.36–7.23 (m, 10 arom. H); 7.01 (s, 1 arom. H); 2.3 (s, Me).
13C-NMR (CDCl3): 169.9, 163.4 (C¼N); 159.3 (NꢀC¼C); 142.3, 133.2, 131.7, 131.2, 129.5, 129.1,
128.8,127.7 127.6, 126.9, 126.8, 123.7, 103.2 (arom. C); 24.3 (Me). ESI-MS: 402.56 ([Mþ1]þ ). Anal. calc.
for C23H19N3O2S (401.48): C 68.81, H 4.77, N 10.47; found: C 68.84, H 4.874, N 10.52.
4-Methyl-N-[4-(4-methylphenyl)-6-phenylpyrimidin-2-yl]benzenesulfonamide (4b). Yield: 58%.
M.p. 948. IR: 1035 (SO2(sym.)), 1538 (SO2(asym.)), 2964 (Me), 3291 (NꢀH). 1H-NMR ((D6)DMSO):
10.47 (s, NH); 7.85 (d, J¼6.5, arom. H); 7.34 (d, J¼6.5, 2 arom. H); 7.46 (d, J¼7.31, 2 arom. H); 7.41 (d,
J¼7.31, 2 arom. H); 6.93 (s, 1 arom. H); 2.33 (s, Me), 2.14 (s, Me), 7.41–7.36 (m, 5 arom. H). 13C-NMR
((D6)DMSO): 174.4, 169.2 (C¼N); 160.9 (NꢀC¼C); 134.7, 133.6, 131.2, 129.5,129.3, 128.4, 127.2, 126.9
(arom. C); 134.3 (arom. C, Ts); 21.3(alk. C). ESI-MS: 416.88 ([Mþ1]þ ). Anal. calc. for C24H21N3O2S
(415.51): C 69.37, H 5.09, N 10.11; found: C 69.56, H 5.16, N 9.93.
N-[4-(2-Chlorophenyl)-6-phenylpyrimidin-2-yl]-4-methylbenzenesulfonamide (4c). Yield: 61%.
M.p. 988. IR: 1124 (SO2(sym.)), 1401 (SO2(asym.)), 3301 (NꢀH). 1H-NMR ((D6)DMSO): 12.30 (s,
NH); 7.68 (d, J¼7.5, 2 arom. H); 7.32 (d, J¼7.5, 2 arom. H, Ts); 7.25–7.11 (m, 9 arom. H); 6.93 (s, 1 arom.
H); 2.19 (s, Me). 13C-NMR ((D6)DMSO): 173.3, 161.7 (C¼N); 161.8 (NꢀC¼C); 142.1, 133.5, 132.4, 131.1,
129.8, 129.3, 128.6, 127.4, 126.7, 124.3 123.6, 124.1, 101.93 (arom. C); 25.6 (C). ESI-MS: 437.01 ([Mþ1]þ ).
Anal. calc. for C23H18ClN3O2S (435.93): C 63.37, H 4.16, N 9.64; found: C 63.46, H 4.13, N 9.66.
N-[4-(4-Chlorophenyl)-6-phenylpyrimidin-2-yl]-4-methylbenzenesulfonamide (4d). Yield: 80%.
1
M.p. 788. IR: 1017 (SO2(sym.)), 1469 (SO2(asym.)), 3285 (NꢀH). H-NMR (CDCl3): 10.47 (s, NH);
7.59 (d, J¼7.01, 2 arom. H); 7.46 (d, J¼7.01, 2 arom. H); 7.38–7.29 (m, 5 arom. H); 7.26 (d, J¼7.4, 2 arom.
H); 7.21 (d, J¼7.4, 2 arom. H); 6.91 (s, 1 arom. H); 2.23 (s, Me). 13C-NMR (CDCl3): 173.5, 164.7 (C¼N);
160.3 (NꢀC¼C); 135.4 (arom.; C, Ts); 131.5, 131.2, 129.4, 129.1, 128.6, 127.2 (arom. C); 23.01 (Me). ESI-
MS: 436.99 ([Mþ1]þ ). Anal. calc. for C23H18ClN3O2S (435.93): C 63.37, H 4.16, N 9.64; found: C 63.31, H
4.19, N 9.59.
N-[4-(4-Chlorophenyl)-6-(4-methylphenyl)pyrimidin-2-yl]-4-methylbenzenesulfonamide (4e).
1
Yield: 74%. M.p. 1108. IR: 1087 (SO2(sym.)), 1548 (SO2(asym.)), 2961 (Me), 3311 (NꢀH). H-NMR
(CDCl3): 12.05 (s, NH); 7.72 (d, J¼7.43, 2 arom. H); 7.47–7.36 (m, 6 arom. H); 7.29 (d, J¼6.9, 2 arom. H);
7.22 (d, J¼7.18, arom. H); 6.88 (s, 1 arom. H); 2.26 (s, Me); 2.05 (s, Me). 13C-NMR ((D6)DMSO): 169.5,
168.3 (C¼N); 161.4 (NꢀC¼C); 136.3 (arom. C, Ts); 132.6, 131.8. 129.9. 129.6, 128.9, 106.4 (arom. C); 23.6,
21.4 (Me). ESI-MS: 451.19 ([MþNa]þ ). Anal. calc. for C24H20ClN3O2S (449.95): C 64.06, H 4.48, N 9.34;
found: C 65.17, H 4.51, N 9.39.
N-[4-(2-Chlorophenyl)-6-(4-methylphenyl)pyrimidin-2-yl]-4-methylbenzenesulfonamide (4f). Yield:
46%. M.p. 958. IR: 1119 (SO2(sym.)), 1443 (SO2(asym.)), 3261 (NꢀH). 1H-NMR (CDCl3): 11.23 (s,
NH); 7.92 (d, J¼7.5, 2 arom. H); 7.21 (d, J¼7.31, 2 arom. H); 7.15–7.03 (m, 7 arom. H); 6.79 (d, J¼7.5, 2
arom. H, Ts); 2.2 (s, 2 Me). 13C-NMR (CDCl3): 168.6, 164.4 (C¼N); 159.4 (NꢀC¼C), 142.6 (arom. C, Ts);
133.1, 132.3, 131.4, 129.9, 129.3, 128, 127.6, 125.5, 106.6 (arom. C); 24.7 (C). ESI-MS: 450.76 ([Mþ1]þ ).
Anal. calc. for C24H20ClN3O2S (449.95): C 64.06, H 4.48, N 9.34; found: C 64.25, H 4.46, N 9.39.
N-[4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-yl]-4-methylbenzenesulfonamide (4g). Yield:
59%. M.p. 1188. IR: 1073 (SO2(sym.)), 1457 (SO2(asym.)), 3327 (NꢀH). 1H-NMR ((D6)DMSO): 11.64
(s, NH); 7.71 (d, J¼7.6, 2 arom. H); 7.26 (d, J¼7.4, 2 arom. H); 7.21 (d, J¼7.4, 2 arom. H); 7.15 (d, J¼7.2,
2 arom. H); 7.11 (d, J¼7.2, 2 arom. H); 7.09 (d, J¼7.6, 2 arom. H, Ts); 7.1 (s, 1 arom. H); 2.1 (s, Me).
13C-NMR ((D6)DMSO): 171.5, 164.2 (C¼N); 159.3 (NꢀC¼C); 137.3 ( arom. C, Ts); 132.3, 131.2, 130.4,
129.9, 129.6, 128.7, 125.2, 103.6 (arom. C); 21.8 (Me). ESI-MS: 481.45 ([Mþ1]þ ). Anal. calc. for
C23H17ClN4O4S (480.92): C 57.44, H 3.56, N 11.65; found: C 57.47, H 3.51, N 11.55.
4-Methyl-N-[4-(4-nitrophenyl)-6-phenylpyrimidin-2-yl]benzenesulfonamide (4h). Yield: 87%. M.p.
878. IR: 1145 (SO2(sym.)), 1583 (SO2(asym.)), 3224 (NꢀH). 1H-NMR ((D6)DMSO): 12.15 (s, NH); 7.88
(d, J¼7.7, 2 arom. H); 7.53 (d, J¼7.3, 2 arom. H); 7.34–7.21 (m, 5 arom. H); 7.18 (d, J¼7.3, 2 arom. H);
7.09 (s, 1 arom. H); 7.04 (d, J¼7.7, 2 arom. H, Ts); 2.7 (s, Me). 13C-NMR ((D6)DMSO): 173.4, 165.2
(C¼N); 161.3 (NꢀC¼C); 134.2 (arom. C, Ts); 143.8, 125.8, 130.2, 133.4, 133.9, 132.5, 132.3, 130.7, 127.9,

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
127.6, 98.0 (arom. C); 23.8 (C). ESI-MS: 447.56 ([Mþ1]þ ). Anal. calc. for C23H18N4O4S (446.48): C 61.87,
H 4.06, N 12.55; found: C 58.77, H 3.68, N 8.94.
N-[4-(2-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-yl]-4-methylbenzenesulfonamide (4i). Yield:
71%. M.p. 1078. IR: 1024 (SO2(sym.)), 1519 (SO2(asym.)), 3368 (NꢀH). 1H-NMR ((D6)DMSO):
11.45 (s, NH); 7.62 (d, J¼7.31, 2 arom. H); 7.43 (d, J¼7.37, 2 arom. H); 7.29 (d, J¼7.37, 2 arom. H); 7.24 (d,
J¼7.21, 2 arom. H); 7.19 (d, J¼7.21, 1 arom. H); 7.17 (d, J¼7.31, 2 arom. H); 1.9 (s, Me). 13C-NMR
((D6)DMSO): 173.6, 163.2 (C¼N); 161.7 (NꢀC¼C); 136.3 (arom. C, Ts); 134.2, 133.4, 133.2, 132.5, 131.2,
132.1, 129.8, 128.9, 128.2, 103.6 (arom. C); 26.8 (C). ESI-MS: 481.93 ([Mþ1]þ ). Anal. calc. for
C23H17ClN4O4S (480.92): C 57.44, H 3.56, N 11.65; found: C 57.55, H 3.44, N 11.58.
Biological Activity. Anti-Amoebic Activity. The compounds 2a–2i, 3a–3i, and 4a–4i were screened
in vitro for anti-amoebic activity against the HM1: IMSS strain of E. histolytica by the microdilution
method [39]. E. histolytica trophozoites were cultured in TYIS-33 growth medium [40] in wells of 96
microtiter plate (Costar).The test compounds were dissolved in DMSO (40 ml), at which level no
inhibition of the amoeba occurred [41][42]. The culture medium was added to obtain a concentration of
1 mg/ml. Twofold serial dilutions were made. Each test includes metronidazole as a standard amoebic
drug, control wells (culture medium plus amoeba), and a blank (culture medium only). The number of
amoeba per mm was estimated with a haemocytometer, and trypan blue exclusion was used to confirm
viability. The cell suspension was diluted to 105 organisms/ml by adding fresh medium, and 170 ml of this
suspension was added to the test and control wells in the plate. An ennoculum of 1.7ꢂ104 organisms/well
was chosen, so that confluent, but not excessive, growth took place. The plates were sealed, gassed for
10 min with N2, and incubated at 378 for 72 h. After incubation, the growth of the amoeba was checked
with a low-power microscope. The culture medium was removed by inverting the plate and shaking
gently. The plates were immediately washed with 0.9% aq. NaCl soln. at 378. This procedure was
performed quickly, and the plate was not allowed to cool to prevent the detachment of amoebae. The
plate was dried in at r.t., and the amoebae were fixed with chilled MeOH by keeping the plate in an ice
bath for 15 min, dried, and stained with 0.5% aq. eosin for 15 min. The stained plate was washed once
with tap H2O and then twice with dist. H2O and allowed to dry. Then, 0.1n aq. NaOH soln. (200 ml) was
added to each well to dissolve the protein and to release the dye (eosine). The optical density of the
resulting soln. in each well was determined at 490 nm with a microplate reader. The inhibition [%] of
amoebal growth was calculated from the optical densities of the control and test wells, and plotted vs. the
logarithm of the dose of the drug tested. Linear-regression analysis was used to determine the best-fitting
straight line from which IC50 values were determined. The experiments were performed thrice for each
compound tested.
Cell-Viability Assay. H9C2 Rat cardiac myoblasts were cultured and maintained as monolayer in
high-glucose DMEM (Dulbeccoꢁs modified Eagleꢁs medium), supplemented with 10% fetal bovine
serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin B, at 378 in a
humidified incubator with 5% CO2 [42]. Cells were incubated with different concentrations of
compounds 3c, 3d, 3g, 3i, 4c, 4g, and 4i in DMSO and metronidazole for 48 h at 378 in 5% CO2 humidified
incubator together with untreated control sample. At appropriate time points, cells were washed in PBS
(¼ phosphate-buffered saline), treated with 600 ml MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium bromide) soln. (5 mg/ml) and incubated for 45 min at 378. After 45 min of incubation at
378, the cell supernatants were discarded, MTT crystals were dissolved in iPrOH, and the absorbance was
measured at 570 nm. All assays were performed in triplicate. Percent viability was defined as the relative
absorbance of treated vs. untreated control cells.
This work was supported by the 2011 Yeungnam University Research grant. The authors are also
grateful to Higher Education Department, Jammu and Kashmir, for their cooperation and M. A. also
thanks UGC MANF (scheme wide letter No. F.40–65(C/M)/2009(SA-III/MANF)) for providing
financial assistance.
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Received January 13, 2013