Synthesis, characterization of some transition-metal complexes of a new heptadentate N5S2 schiff-base ligand and the effects of these metal complexes on U2OS cells cytotoxicity and DNA cleavage activity

Article abstract of DOI:10.1080/10426500801907589

Metal complexes of a heptadentate N5S2 donor Schiff-base ligand were synthesized by the template reaction between 2,6-bis(2-aminothiophenoxymethyl) pyridine and 2,2 '-bipyridine-6,6 '-dicarboxaldehyde in the presence of Zn(II), Cd(II), Hg(II) and Pb(II) perchlorate salts. The complexes were characterized by IR, 1H NMR, MS-FAB+, and elemental analyses. The mitochondrial activity of U2OS cells after exposure to these complexes was determined by colorimetric assay, which detects the conversion of 3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide (MTT) to formazon. DNA-binding activity of all complexes was also investigated using plasmid DNA pUC18 purified from E. coli. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426500801907589

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Phosphorus, Sulfur, and Silicon
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Synthesis, Characterization
of Some Transition-Metal
Complexes of a New
Heptadentate N5S2 Schiff-Base
Ligand and the Effects of These
Metal Complexes on U2OS Cells
Cytotoxicity and DNA Cleavage
Activity
G. Uluçcam a , N. Beynek a , Z. Seller b , G. Akalın b
, G. Turan c & K. Benkli c
a Trakya University, Faculty of Art and Science,
Department of Chemistry, Edirne, Turkey
b Anadolu University, Faculty of Pharmacy,
Department of Biochemistry, Eskişehir, Turkey
c Anadolu University, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Eskişehir,
Turkey
Published online: 12 Aug 2008.
To cite this article: G. Uluçcam , N. Beynek , Z. Seller , G. Akalın , G. Turan & K.
Benkli (2008) Synthesis, Characterization of Some Transition-Metal Complexes of a
New Heptadentate N5S2 Schiff-Base Ligand and the Effects of These Metal Complexes
on U2OS Cells Cytotoxicity and DNA Cleavage Activity, Phosphorus, Sulfur, and Silicon
and the Related Elements, 183:9, 2237-2247, DOI: 10.1080/10426500801907589
To link to this article: http://dx.doi.org/10.1080/10426500801907589

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Phosphorus, Sulfur, and Silicon, 183:2237–2247, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500801907589
Synthesis, Characterization of Some Transition-Metal
Complexes of a New Heptadentate N5S2 Schiff-Base
Ligand and the Effects of These Metal Complexes on
U2OS Cells Cytotoxicity and DNA Cleavage Activity
G. Uluc¸am,1 N. Beynek,1 Z. Seller,2 G. Akalın,2 G. Turan,3
and K. Benkli3
1Trakya University, Faculty of Art and Science, Department of
Chemistry, Edirne, Turkey
2Anadolu University, Faculty of Pharmacy, Department of Biochemistry,
Eskis¸ehir, Turkey
3Anadolu University, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Eskis¸ehir, Turkey
Metal complexes of a heptadentate N5S2 donor Schiff-base ligand were synthesized
by the template reaction between 2,6-bis(2-aminothiophenoxymethyl)pyridine and
2,2 ꢀ-bipyridine-6,6 ꢀ-dicarboxaldehyde in the presence of Zn(II), Cd(II), Hg(II) and
1
Pb(II) perchlorate salts. The complexes were characterized by IR, H NMR, MS-
FAB+, and elemental analyses. The mitochondrial activity of U2OS cells after ex-
posure to these complexes was determined by colorimetric assay, which detects the
conversion of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT)
to formazon. DNA-binding activity of all complexes was also investigated using
plasmid DNA pUC18 purified from E. coli.
Keywords Mitocondrial activity; MTT; Schiff base; template synthesis
INTRODUCTION
The synthesis of Schiff-base macrocyclic metal complexes received con-
siderable attention during the last decade because of their antitumor
and cytotoxic activity.1,2 The potential biological activity of complexes
containing sulfur and nitrogen may be a reason for this increased
interest. In particular complexes with N and/or S donor ligands of
copper(II),3,4 cobalt(II),5 nickel(II),3,5 platinum(II),1–3,6 and iron(II/III)6
Received 26 December 2007; accepted 4 January 2008.
Authors are grateful to Trakya University and Anadolu University, Commission of
Scientific Research Projects.
Address correspondence to N. Beynek, Trakya University, Faculty of Art and Science,
Department of Chemistry, Edirne 22030, Turkey. E-mail: nesrinbeynek@yahoo.co.uk
2237

2238
G. Uluc¸am et al.
are known sometimes to act as antitumor or therapeutic agents. Many
efforts have been made to establish further metal-containing cytostatics
however, without great success. Only little is known about the antitu-
mor efficacy of some transition metals such as zinc(II),7 cadmium(II),
lead(II), and mercury(II). For this reason, we synthesised some new
N5S2 Schiff-base complexes of these transition metals. Recently, we
also synthesized and characterized some new similar macrocyclic and
acyclic Schiff-base complexes using these metals.8–10
Here, we describe the complexes of a N5S2 donor ligand, which
were obtained from the reaction of the Schiff base, resulting from the
condensations of 2,6-bis(2-aminothiophenoxymethyl)pyridine and 2,2ꢀ-
bipyridine-6,6ꢀ-dicarboxaldehyde with Zn(II), Cd(II), Hg(II) and Pb(II)
1
perchlorate in methanol. The complexes were characterized by IR, H
NMR, FAB mass spectrometry, and elemental analyses.
The mitocondrial activity of U2OS cells after exposure to these com-
plexes was determined by colorimetric assay, which detects the conver-
sion of 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyltetrazolium bromide
(MTT) to formazon.11 DNA cleavage activity of all these complexes was
also investigated by using plasmid DNA pUC18 purified from E. Coli.12
RESULTS AND DISCUSSION
Chemistry
All the complexes were prepared by a template synthesis, in which
the Schiff base macrocyclic ligand resulted from the condensation
of 2,2ꢀ-bipyridine-6,6ꢀ-dicarboxaldehyde with 2,6-bis(2- aminothio-
phenoxymethyl)pyridine in the presence of Zn(II), Cd(II), Hg(II) and
Pb(II) ions (Scheme 1). For the synthesis of the dialdehyde compound,
2,2ꢀ-bipyridine-6,6ꢀ-dicarboxaldehyde,
6-amino-2-methylpyridine
(commercially available) was brominated with concentrated aqueous
hydrobromic acid and bromine.13 Then 6,6ꢀ-dimethyl-2,2ꢀ-bipyridine
was prepared using 6-bromo-2-methylpyridine by a modification of the
method described by Iyoda et al.14 and was oxidized to the dialdehyde
by selenium dioxide in 1,4-dioxane. The precursor diamino compound,
2,6-bis(2-aminothiophenoxymethyl)pyridine,15 was prepared starting
from 2,6-bis(hydroxymethyl)pyridine, which was converted to 2,6-
bis(bromomethyl)pyridine16 by reaction with 47% hydrobromic acid.
Then, one equivalent of 2,6-bis(bromomethyl)pyridine was reacted
with two equivalents of 2-aminothiophenol in ethanol under an
inert atmosphere. Physical data for all compounds are given in the
Experimental Section.
The complexes were obtained with 40–80% yields. The infrared spec-
tra of all metal complexes in the region of 400–4000 cm−1 show a strong

Metal Complexes of Heptadentate N S Schiff Base
2239
5
2
SCHEME 1 Synthetic route to the title complexes.
absorption band around 1616–1638 cm−1, which is assigned to the C N
streching vibration, indicating the formation of the Schiff-base prod-
ucts. Furthermore, the absence of C O and N H streching vibrations
in the spectra of the complexes, as compared to the aldehyde and di-
amine, respectively, further indicate the formation of the Schiff base.
For the metal complexes absorbtions at 1091–1094 and 624 cm−1 were
assigned to the ν3 and ν4 streching modes of ionic perchlorate.17 The
proton NMR spectrum of the soluble complex [PbL](ClO4)2 shows 23
peaks, which is consistent with the given structure. The composition
of the complexes was also confirmed by elemental analyses and by fast
atom bombardment mass spectrometry. In the mass spectrum of all
complexes two fragmentation patterns were observed corresponding to
the sequential loss of the counterion.
Cytotoxicity
Cytotoxic effects of metal complexes were tested using MTT assay as
described in Experimental. MTT is commonly employed as an indicator

2240
G. Uluc¸am et al.
FIGURE 1 Toxicity of metal complexes for U2OS cells. Cells were incubated
with various concentrations of the complexes. At each time point, MTT assay
was performed. The results are the mean of triplicate wells.
of cell number and viability, since it is converted to a coloured formazan
derivative via mitocondrial dehyrogenase activity only by viable cells.
The results are shown in Figure 1 and the IC50 value of each com-
pounds is given in Table I. It was observed that cytotoxic activity
of [HgL](ClO4)2 and [PbL](ClO4)2 increased considerably as time col-
lapsed. The cells were exposed to 0.0256–40 µg/mL of compounds
[HgL](ClO4)2 and [PbL](ClO4)2 for 24 h, and these two compounds
showed no cytotoxicity for this cell line (Figures 1b and 1d). However,
the cells treated with 80 µg/mL of either [HgL](ClO4)2 or [PbL](ClO4)2
showed 26
5% and 41
0.9% of cell viability, respectively. These
TABLE I IC50 Values of the Complexes with Ligand (L)
Compounds
[ZnL](ClO4)2
10
[CdL](ClO4)2
0,7
[HgL](ClO4)2
70
[PbL](ClO4)2
70
U2OS cell lines IC50
(µg/mL)

Metal Complexes of Heptadentate N S Schiff Base
2241
5
2
two compounds have IC50 values of 70 µg/mL as showed in Table I.
However compounds [ZnL](ClO4)2 and [CdL](ClO4)2 increased the cy-
totoxic function, demonstrating that these two complexes give cytotoxic
derivatives. The IC50 value was found 10
0.4 for [ZnL](ClO4)2 and
0.7 0.04 µg/mL for [CdL](ClO4)2. The marked increase in cytotoxicity
of [CdL](ClO4)2 (IC50 value was 0.7 µg/mL) suggests that this complex
is very important for the cytotoxic activity in this particular cell line
(Figure 1a).
The ability of the metal complexes to interact with plasmid DNA
pUC18 purified from E.coli in vitro was investigated by electrophoretic
mobility method. The electrophoretic mobility of pUC18 DNA from the
supercoiled forms to circular form was compared with plasmid DNA
after incubation with the derivatives for 24 h at 37◦C in the dark
(Figure 2). [ZnL](ClO4)2 and [PbL](ClO4)2 allowed the change in mo-
bility of Form I band to Form II band. The increase in concentrations
FIGURE 2 Effects of metal complexes on the electrophoretic mobility of su-
percoiled and open forms of the DNA plasmid pUC18. Electrophoresis was
carried out after incubation of the plasmid pUC18 with (a) [ZnL](ClO4)2,
(b) [PbL](ClO4)2, (c) [CdL](ClO4)2, and (d) [HgL](ClO4)2 at concentrations of
the complexes ranging from 0,1–60 µM; Lane 1 applies to pUC18 plasmid DNA
only, lanes 2–6 apply to plasmid interacted with increasing concentrations of
the compounds.

2242
G. Uluc¸am et al.
(30 and 60 µM) is due to unwinding of the supercoiled Form I DNA to
the relaxed circular Form II DNA. On the other hand, the plasmid DNA
treated with high concentrations of [CdL](ClO4)2 and [HgL](ClO4)2 did
not appear on the agarose gel, which might be due to complete cleavage
of the plasmid DNA.
EXPERIMENTAL
Chemistry
Melting points were determined using an Gallenkamp MPD350.BM2.5
digital melting point apparatus and were uncorrected. The compounds
were checked for purity by TLC on silica gel 60 F254 (Merck). Elemental
analyses were performed on a CHNS-O Carlo Erba EA 1108 elemental
analyser; IR spectra were obtained with a Shimadzu 470 IR spectropho-
tometer using nujol mulls or KBr disc; 1H NMR spectra were recorded
with a Varian (300 MHz) or Bruker NMR spectrometer (250 MHz) in
CDCl3 or DMF-d7as solvent. 13C NMR spectra were recorded with a
Varian spectrometer at 75.5 MHz in CDCl3 as solvent; MS-FAB+ spec-
tra were obtained with a Finnigan Mat 95 mass spectrometer.
General Synthesis Procedure
THF was distilled from sodium metal in the presence of benzophenone
immediately prior to use. 1,4-Dioxane was distilled prior to use. Et4NI
was dried at 100◦C under reduced pressure. All other reagents were
used as purchased from commercial suppliers without further purifica-
tion. Preparation of NiCl2(PPh3)2 and purification of Zn powder both
followed a literature method.18
6-Bromo-2-methylpyridine13
6-Amino-2-methylpyridine (1.08 g, 0.1 mol) in 47% hydrobromic acid
(34.5 mL, 0.3 mol) was placed in an ice-salt-bath with stirring. Bromide
(15.4 mL, 0.3 mol) was added dropwise to this solution with stirring
while the temperature was kept at 0◦C. A solution of sodium nitrite
(17 g, 0.25 mol) in water (25 mL) was then introduced at such a rate
that the temperature of the reaction mixture did not rise above 5◦C.
After completion of the reaction the dark brown solution was made
alkaline with aqueous sodium hydroxide solution. The light yellow re-
action mixture was extracted with diethyl ether. The extract was dried
with sodium sulfate and the solvent was then removed in vacuo. Yield:
12.6 g (75.2%). 1H NMR (300 MHz, CDCl3): δ = 2.45 (s, 3H, CH3), 7.03
(d, J = 7.5 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H).

Metal Complexes of Heptadentate N S Schiff Base
2243
5
2
13C NMR (75.5 MHz, CDCl3): δ = 24.3 (CH3), 122.3, 125.2, 138.8, 141.5,
160.2.
6,6ꢀ-Dimethyl-2,2ꢀ-bipypridine14
NiCl2(PPh3)2(4.9 g, 7.5 mmol), Zn powder (1.47 g, 22.5 mmol), dry
Et4NI (3.86 g, 15 mmol) and dry THF (20 mL) were stirred at room
temperature under argon. 6-Bromo-2-methylpyridine (2.58 g, 15 mmol)
in dry THF (5 mL) was added to the reaction mixture. The mixture was
stirred at 50◦C for two days. The reaction solution was poured into
a mixture of 2 M aqueous ammonia (100 mL), diethyl ether (50 mL)
and hexane (50 mL). The precipitate was filtered and the organic layer
was separated. The aqueous layer was extracted with diethyl ether:
hexane (1:1). The organic layer was washed successively with water
and saturated aqueous NaCl solution, dried with anhydrous MgSO4,
and the solvent was evaporated in vacuo. The resulting white solid
was suspended in water and heated. The suspension was acidified with
concentrated HCl and filtered hot. The filtrate was cooled to ambient
temperature, neutralised with saturated aqueous NaOH solution, and
the solid formed was filtreted to give desired product as a cream solid
1
(0.94 g, 68%). M.p. 88–89◦C. H NMR (300 MHz; CDCl3): δ = 2.63 (s,
6H, CH3), 7.15 (d, J = 7.4 Hz, 2H), 7.69 (t, J = 7.6 Hz, 2H), 8.21 (d, J =
7.9 Hz, 2H). 13C NMR (75.5 MHz; CDCl3): δ = 24.8 (CH3), 118.5, 123.4,
137.3, 155.9, 158.1.
2,2 ꢀ-Bipyridine-6,6 ꢀ-dicarboxaldehyde19
A mixture of 6,6ꢀ-dimethyl-2,2ꢀ-bipyridine (2.0 g, 20 mmol) and se-
lenium dioxide (10 g, 90 mmol) in 1,4-dioxane containing 4% of water
(250 mL) was heated under reflux for 4 h. The reaction mixture was
filtered through celite while hot. The product separated from the cold
filtrate as cream crystals (1.91 g, 45%). IR (KBr): ν(cm−1) = 1699 (C O),
1
1577 (pyridine). H NMR (300 MHz, CDCl3): δ = 7.25 (d, J = 8.7 Hz,
2H), 8.05 (t, J = 6.6 Hz, 2H), 8.82 (d, J = 7.0 Hz, 2H), 10.18 (s, 2H,
HC O). 13C NMR (75.5 MHz, CDCl3): δ = 122.2, 125.6, 138.5, 152.8,
155.8, 193.6 (C O).
2,6-Bis(bromomethyl)pyridine16
2,6-Bis(hydroxymethyl)pyridine (20 g, 0.145 mol) and 47% aqueous
hybrobromic acid (33.3 mL, 0.29 mol) was refluxed for 2 h. The mixture
was then neutralised at 0◦C by the addition of a concentrated aqueous
solution of sodium hydroxide. The solid product was collected, washed
with water, and dried. Recrystallization from petroleum ether (b.p. 60–
80◦C) gave a white crystalline solid (21.9 g, 57%). M.p. 87–88◦C. IR

2244
G. Uluc¸am et al.
(KBr): ν (cm−1) = 1590, 1571 (pyridine), 1206 (CH2-Br). 1H NMR (250
MHz, CDCl3): δ = 4.52 (s, 4H, CH2), 7.36 (d, J = 7.7 Hz, 2H), 7.69 (t,
J = 7.7 Hz, 1H).13C NMR (75.5 MHz, CDCl3): δ = 33.6 (CH2), 123.0,
138.3, 157.0.
2,6-Bis(2-aminothiophenoxymethyl)pyridine15
To a strirred solution of sodium ethoxide (sodium 2.3 g, 0.1 mol,
absolute ethanol 50 mL), 2-aminothiophenol (12.5 g, 0.1 mol) in
absolute ethanol (250 mL) was added dropwise over a period of
30 min at room temperature, under argon. Freshly prepared 2,6-
bis(bromomethyl)pyridine (13.2 g, 0.05 mol) in absolute ethanol
(100 mL) was added dropwise, and the reaction mixture was refluxed
for 4 h. On cooling, the reaction mixture was poured into an equal vol-
ume of water, where upon standing cream flocculent crystals were ob-
tained. The product was collected by filtration, washed with water, and
dried over phosphorus pentoxide (14.5 g, 82%). M.p. 80–82◦C. IR (KBr):
ν (cm−1) = 3408, 3312 (N H); 1606, 1587 (aromatic ring). 1H NMR
(250 MHz, CDCl3): δ = 3.99 (s, 4H, CH2), 4.37 (s, 4H, NH2), 6.59 (t, J =
7.4 Hz, 2H), 6.66 (d, J = 8.0 Hz, 2H), 6.81 (d, J = 7.7 Hz, 2H), 7.09
(t, J = 7.7 Hz, 2H), 7.22 (d, J = 7.7 Hz, 2H), 7.34 (t, J = 7.7 Hz, 1H).
13C NMR (75.5 MHz, CDCl3): δ = 41.5 (CH2), 115.1, 117.3, 118.4, 121.6,
130.3, 136.7, 136.8, 149.0, 158,0.
Metal Ion-Controlled Synthesis of L Complexes in the
Presence of Zn(II), Cd(II), Hg(II), and Pb(II) Perchlorate Salts
2,2ꢀ-Bipyridine-6,6ꢀ-dicarboxaldehyde (0.18 g, 1 mmol) and the appro-
priate metal perchlorate salt (1 mmol) were dissolved in hot MeOH
(25 mL) and 2,6-bis(2-aminothiophenoxymethyl)pyridine (0.36 g,
1 mmol) in methanol (25 mL) was added dropwise with stirring. The
mixture was refluxed for 3–4 h and filtered hot. The solvent of the re-
action mixture was reduced to half its original volume, and then the
mixture was placed in a refrigerator to induce crystallization. The crys-
tallized product was filtered and dried.
[ZnL](ClO4)2
Pale yellow solid, yield: 0.32 g (40%). IR (KBr): ν(cm−1) = 1628
(C N), 1587 (pyridine), 1094 and 624 (perchlorate anion). m/z: 594
[ZnL(ClO4)]+, 693 [ZnL]2+. Found: C, 46.6; H, 3.0; N, 8.5; S, 8.5%. Calcd.
for C31H23N5S2O8Cl2Zn: C, 46.9; H, 2.9; N, 8.8; S, 8.1%.
[CdL](ClO4)2
Yellow solid, yield: 0.67 g (80%). IR (KBr): ν(cm−1) = 1638 (C N),
1590 (pyridine), 1091 and 624 (perchlorate anion). m/z: 742

Metal Complexes of Heptadentate N S Schiff Base
2245
5
2
[CdL (ClO4)]+, 642 [CdL]2+. Found: C, 44.2; H, 2.8; N, 8.0; S, 7.5%.
Calcd. for C31H23N5S2O8Cl2Cd: C, 44.3; H, 2.7; N, 8.3; S, 7.6%.
[HgL](ClO4)2
Yellow solid, yield: 0.69 g (74%). IR (KBr): ν(cm−1) = 1628 (C N),
1587 (pyridine), 1094 and 624 (perchlorate anion). m/z: 830 [HgL
(ClO4)]+, 731 [HgL]2+. Found: C, 39.1; H, 2.5; N, 7.1; S, 7.5%. Calcd.
for C31H23N5S2O8Cl2Hg · H2O: C, 39.3; H, 2.6; N, 7.4; S, 6.8%.
[PbL](ClO4)2
Orange solid, yield: 0.374 g (40%). IR (KBr): ν(cm−1) = 1616 (C N),
1587 (pyridine), 1091 and 624 (perchlorate anion). m/z: 837 [PbL
1
(ClO4)]+, 736 [PbL]2+. H NMR (300 MHz, d7-DMF): δ = 4.36 (s, 4H,
CH2), 6.64 (t, J = 7.0 Hz, 2H), 6.95 (d, J = 7.6 Hz, 2H), 7.10–7.88 (m,
8H), 8.62 (d, J = 7.9 Hz, 2H), 8.80 (t, J = 7.6 Hz, 1H), 9.12 (d, J = 7.0
Hz, 2H), 9.60 (s, 2H, HC N).
Cell Culture
U2OS cells were obtained and cultured as recommended by the
American Type Culture Collection (ATCC, Rockville, MD, USA). Cells
were maintained in McCoy’s medium (Sigma) supplemented with 10%
(v/v) of Foetal Bovine Serum (FBS) (Gibco) as adherent monolayers.
Cells were incubated at 37◦C under 5% CO2 and 95% air in a humidi-
fied atmosphere.
Cytotoxicity Assays
U2OS cells in exponential growth phase were harvested and the cell
number was determined using a haemocytometer. Samples were resus-
pended in fresh medium to give a density of 2.5 × 104/mL and then
stock solutions of the complexes (2.57, 2.58, 2.60, and 2.61) were pre-
pared as 1 mg/mL in DMSO. Final concentrations (ranging from 0.0256
to 80 µg/mL) were prepared with fresh cell-culture medium (DMSO
concentration < 0.1%). 200 µL of cells was placed into each of a 96-well
microtitre plate and incubated for 24 and 48 h. At the end of the ex-
posure time, 20 µL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium bromide] (5 mg/mL, Sigma) stock solution was added to
each well, and the plates were incubated for a further 2 h. The medium
was removed and discarded and 200 µL of DMSO were added to each
well. Cells were incubated at 25◦C for 10 min further, and then the
absorbance was read on a Bio-Tek (ELX 808 IU) ELISA reader at a
wavelength of 540 nm. The signal generated is directly proportional to

2246
G. Uluc¸am et al.
the number of viable (metabolically active) cells in the wells. The values
of the blank wells were subtracted from each well of treated and control
cells, and the % viability was determined according to Kumi-Diaka20 as
given below:
(the absorbance of the treated cells) − (the absorbance of the blank)
% viable cells =
(the absorbance of the control) − (the absorbance of the blank)
× 100
(1)
Interaction with pUC18 Plasmid DNA
Interaction between the complexes and pUC18 plasmid DNA was
studied by agarose gel electrophoresis. pUC18 plasmid DNA aliquots
(0.5 µg/mL) were incubated in the presence of the increasing concentra-
tions of compounds ranging from 0.1 to 60 µM. Incubation was carried
out in dark at 37◦C for 24 h 15 µL of drug-DNA mixture containing
0.5 µg DNA was loaded onto the 1% agarose gel and electrophoresis
was carried under Tris buffer for 2 h at 5 V · cm−1. At the end of the
electrophoresis, the gel was stained in the Tris buffer containing ethid-
ium bromide (0.5 mg/mL) and visualized under UV light (Kodak).21
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