5996
S. Yadav et al. / European Journal of Medicinal Chemistry 45 (2010) 5985e5997
Compound 14: Mp (ꢂC) 102e104; Yield-33.5%; 1H NMR (DMSO)
ppm: 6.90e7.84 (m, 8H, AreH of ArSO3H and benzimidazole),
of the performance is obtained from cross validated q2) method
which is defined as
d
3.66e3.72 (d, 2H, CH2 of C]CH2; Jtrans ¼ 18 Hz); IR (KBr pellets,
cmꢀ1): 1613.57 (C]O str., tertiary amide), 755.49 (OCN bending),
1450.12 (C]C str., skeletal vibration of phenyl nucleus), 1233.56
(SO3 asymmetric stretch), 1004.74 (CeH out of plane bending of C
(CH3) ¼ CH2), 1384.75 (CH in plane bending of C(CH3) ¼ CH2),
1531.84 (C]C str.), 1262.16 (CH in plane bending of phenyl ring).
Compound 15: Mp (ꢂC) 110e112; Yield-44.0%; 1H NMR (DMSO)
X
X
q2 ¼ 1 ꢀ
Ypredicted ꢀ Ya2ctual
=
Yactual ꢀ Ym2 ean
where, Ypredicted, Yactual and Ymean are predicted, actual and mean
2
values of target property (pMIC) respectively. S (Ypredicted ꢀ Yactual
)
is predictive residual error sum of squares [51].
4.4. Evaluation of antiviral activity
4.4.1. Antiviral assays
d
ppm: 7.27e8.12 (m, 8H, AreH of ArSO3H and benzimidazole), 2.50
(s, 1H, OH of SO3H), 8.46e9.66 (m, 4H, H of pyridine ring); IR (KBr,
pellets, cmꢀ1): 1617.65 (C]O str., tertiary amide), 751.40 (OCN
bending), 1450.12 (C]C str., skeletal vibration of phenyl nucleus),
1233.56 (SO3 asymmetric stretch), 1531.84 (C]C str.), 1266.25 (CH
in plane bending of phenyl ring), 1392.92 (CeN str. of tertiary
amide), 829.04 (CeH out of plane bending of 3-substituted
pyridine).
The antiviral screening of the 4-[1-(substituted aryl/alkyl
carbonyl)-benzoimidazol-2-yl]-benzenesulfonic acids (1-20) was
performed against Feline Corona virus (FIPV), Feline Herpes virus in
CRFK cell cultures; Herpes simplex virus-1 (KOS) [HSV-1 KOS],
Herpes simplex virus-2 (G) [HSV-2G], Vaccinia virus [VV], Vesicular
stomatitis virus [VSV], Herpes simplex virus-1 TKꢀ KOS ACVr [HSV-
1 TKꢀ KOS ACVr] in HEL cell cultures; Vesicular stomatitis virus
(VSV), Coxsackie virus B4, Respiratory syncytial virus in HeLa cell
cultures; Para-influenza-3 virus, Reovirus-1, Sindbis virus, Cox-
sackie virus B4, Punta Toro virus in Vero cell cultures; Influenza A
virus H1N1 subtype, Influenza A virus H3N2 subtype and influenza
B virus in MDCK cell cultures, human immunodeficiency virus type
1 (HIV-1)(IIIB) and HIV-2(ROD) in MT-4 cell cultures and the results
were expressed as the 50% effective concentration (EC50) or drug
concentration required to inhibit virus-induced cytopathicity by
50%. Read-out was through microscopical inspection or the MTS
viability staining method. Cells, grown in 96-well plates, were
infected with 100 CCID50 of virus, one CCID50 being the 50% cell
culture infective dose in the presence of serial dilutions of the
compounds. The cultures were further incubated at 37 ꢂC for
several (2e4) days, until complete cytopathicity was observed in
the infected and untreated virus control.
Compound 20: Mp (ꢂC) 156e158; Yield-35.8%; 1H NMR (DMSO)
d
ppm: 7.12e7.88 (m, 12H, AreH of ArCH3, ArSO3H and benzimid-
azole), 2.50 (s, 3H, CH3 of ArCH3); IR (KBr pellets, cmꢀ1): 1621.74
(C]O str., tertiary amide), 1450.12 (C]C str., skeletal vibration of
phenyl nucleus),1192.7 (SO3 asymmetric stretch), 808.61 (SeN str.),
1033.34 (SO2 symmetric str.), 747.32 (OCN bending).
4.2. Evaluation of antimicrobial activity
4.2.1. Determination of minimum inhibitory concentration
The antimicrobial activity was performed against Gram-positive
bacteria: S. aureus, Bacillus sublitis, Gram-negative bacterium: E. coli
and fungal strains: C. albicans and A. niger by tube dilution method.
Dilutions of test and standard compounds [ciprofloxacin (antibac-
terial) and fluconazole (antifungal)] were prepared in double
strength nutrient broth e I.P. (bacteria) and Sabouraud dextrose
broth I.P. (fungi) [46]. The samples were incubated at 37 ꢂC for 24 h
(bacteria), at 25 ꢂC for 7 d (A. niger) and at 37 ꢂC for 48 h (C. albi-
cans), respectively, and the results were recorded in terms of MIC
(the lowest concentration of test substance which inhibited the
growth of microorganisms).
4.4.2. Cytotoxic assays
The cytotoxicity of the compounds was evaluated in parallel
with their antiviral activity in uninfected cell cultures, and is
expressed as the minimum cytotoxic concentration (MCC) that
causes a microscopically detectable alteration of normal cell
morphology (HEL, HeLa, CRFK, MDCK and Vero cells).
4.2.2. Determination of minimum bactericidal/fungicidal
concentration (MBC/MFC)
The minimum bactericidal concentration (MBC) and fungicidal
concentration (MFC) were determined by subculturing 100 mL of
Acknowledgements
culture from each tube that remained clear in the MIC determina-
tion into fresh medium. MBC and MFC values represent the lowest
concentration of compound that produces a 99.9% end point
reduction [47].
We would like to thank Mrs. Leentje Persoons, Frieda De Meyer,
Kristien Erven and Kris Uyttersprot, Rega Institute for Medical
Research, Belgium for their excellent technical assistance in the
evaluation of antiviral activity. The antiviral evaluations were
supported by the K.U. Leuven (GOA no. 10/014).
4.3. QSAR studies
References
The structures of substituted benzimidazole derivatives are first
pre-optimized with the Molecular Mechanics Force Field (MMþ)
procedure included in Hyperchem 6.03 [48] and the resulting
geometries are further refined by means of the semiempirical
method PM3 (parametric Method-3). We chose a gradient norm
limit of 0.01 kcal/Aꢂ for the geometry optimization. The lowest
energy structure was used for each molecule to calculate physico-
chemical properties using TSAR 3.3 software for Windows [49].
Further, the regression analysis was performed using the SPSS
software package [50].
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The predictive powers of the equations were validated by leave
one out (LOO) cross validation method, where a model is built with
N-1 compounds and Nth compound is predicted. Each compound is
left out of the model derivation and predicted in turn. An indication