A. K. Mishra et al. / Bioorg. Med. Chem. 14 (2006) 6333–6340
6339
Table 8. Thermal data of the complexes
Complexes
Step no.
TG (Coats–Redfern method)
DH (kJ gꢀ1
)
Temperature range (K)
n
Ea (kJ molꢀ1
)
S# (JKꢀ1 molꢀ1
)
[Pt(L4)2Cl2]
I
302–670
670–1149
1
1
18.26
17.76
5.66
4.18
89.54
165.19
II
bacterial activity of the drug, is to add it in known con-
centrations to the cultures of the test organisms.
40–4 lL of compound solutions added to 160–196 lL,
respectively, of fresh medium in wells to give final con-
centrations of 100–1 lM. All assays were performed in
two independent sets of quadruplicate tests. Control
group containing no drug as well as equivalent amounts
of DMSO was run in each assay.
4.10. Disc diffusion assay
The disc diffusion assay (Rasoanaivo and Ratsimaman-
ga-Urverg, 1993) was used to determine antibacterial
activity of the drug using Gram-positive and Gram-neg-
ative strains of bacteria namely S. aureus and E. coli.
Base plates were prepared by pouring 10 mL of auto-
claved Muller–Hinton agar (Biolab) into sterile Petri
dishes (9 cm) and allowing them to settle. Molten auto-
claved Muller–Hinton agar that had been kept at 48 ꢁC
was inoculated with a broth culture (106–108 mLꢀ1) of
the test organism and then poured over the base plate.
The discs were air-dried and placed on the top of the
agar layer. Four replicants of each drug were tested
(four disc per plate) with a gentamycin disc (0.5 lg/disc)
as a reference. The plates were then incubated for 18 h at
room temperature. Antibacterial activity is expressed as
a ratio of the inhibition zone produced by the drug to
the inhibition zone produced by the gentamycin
standard.
Following 48 h of exposure of cells to drug, each well
was carefully rinsed with 200 lL PBS buffer. Cytotoxic-
ity was assessed using MTT (3-[4,5-dimethylthiazol-2yl]-
2,5-diphenyltetrazolium bromide). MTT solutions 20 lL
(5 mg mLꢀ1 dd H2O) along with 200 lL of fresh, com-
plete media were added to each well and plates were
incubated for 4 h. Following incubation, the medium
was removed and the purple formazan precipitate in
each well was sterilized in 200 lL DMSO. Absorbance
was measured using Techman Magellan microplate
reader (molecular device) at 570 nm and the percentage
(%) cytotoxicity was calculated as
OD in sample well
% Cytotoxicity ¼ 1 ꢀ
ꢂ 100
OD in control well
FCS, foetal calf serum; PBS, phosphate-buffered saline.
(FCS was obtained from Genetix, DMSO from cell
culture tested, MTT from SRL and DMEM was
purchased from Sigma, USA.)
4.11. Micro dilution antibacterial assay
The serial dilution technique described by Eloff (1998),
using 96-well micro plates, to determine the minimum
inhibitory concentration (MIC) of the drugs for antibac-
terial activity was used. Two milliliter cultures of four
bacterial strains of S. aureus and E. coli were prepared
and placed in a water bath overnight at 37 ꢁC. The over-
night cultures were diluted with sterile Muller–Hinton
broth. The drugs were resuspended to a concentration
of 60 lg/disc (in DMSO) with sterile distilled water in
a 96 well micro plate. A similar 2-fold serial dilution
of gentamycin (Sigma) was used as positive control
against each bacterium. One hundred microliters of each
bacterial culture was added to each well. The plates were
covered and incubated overnight at 37 ꢁC. To indicate
bacterial growth, p-iodonitrotetrazolium violet was add-
ed to each well and the plates were incubated at 37 ꢁC
for 30 min. Bacterial growth in the wells was indicated
by a red colour, whereas clear wells indicated inhibition.
References and notes
1. Wheate, N. J.; Collins, J. G. Coord. Chem. Rev. 2003, 133,
241.
2. Mqano, C.; Treviasan, A.; Giovagnini, L.; Fregona, D.
Toxicol. In Vitro 2002, 16, 413.
3. Rosenberg, B.; Van Camp, L.; Trosko, J. E.; Mansour, V.
H. Nature 1969, 222, 385.
4. Einhorn, L. H.; Donohue, J. J. Am. Intern. Med. 1977, 87,
293.
5. Ozols, R. F.; Young, R. C. Semin. Oncol. 1984, 11, 251.
6. Soloway, M. S. J. Urol. 1978, 120, 716.
7. Fiorentino, M. V.; Ghiotto, C. In Platinum and Other
Meatl Coordination Compounds in Cancer Chemotherapy;
Nicolini, M., Ed.; Martinus Nijhoff: Boston, 1988; p 415.
8. Wagener, D. J.; Yap, S. H.; Wobbes, T.; Burghouts, J. T.;
van Dam, F. E.; Hillen, H. F.; Hogendoorn, G. J.;
Scheerder, H.; van der, V. Cancer Chemother. Pharmacol.
1985, 15, 86.
4.12. In vitro cell growth inhibition assay
9. Von Hoff, D. D.; Schilsky, R.; Reichert, C. M.; Reddick,
R. L.; Rozencweig, M.; Young, R. C.; Muggia, F. M.
Cancer Treat. Rep. 1979, 63, 1527.
10. Krakoff, I. H. Cancer Treat. Rep. 1979, 63, 1523.
11. Manav, N.; Mishra, A. K.; Kaushik, N. K. Spectrochim.
Acta, Part A 2004, 60, 3087.
12. Hall, M. D.; Hambley, T. W. Coord. Chem. Rev. 2002,
232, 49.
13. Hambley, T. W.; Jones, A. R. Coord. Chem. Rev. 2001,
212, 35.
Cells were seeded in 96-well plates at a concentration of
0.1–1.0 · 104 cells/well in 200 lL of complete media and
incubated for 24 h at 37 ꢁC in 5% CO2 atmosphere to
allow for cell adhesion. Stock solutions (4 mM) of the
compounds made in DMSO were filter-sterilized, then
diluted to 1 mM in incomplete media. The 1 mM solu-
tions were further diluted to 500 and 50 lM incomplete
media for treatment against HeLa cell lines, where