Transition metal complexes of novel ethyl pyruvate hydrazones as potential antitumor agents: Synthesis and physicochemical properties, DNA interactions and antiproliferative activity

Article abstract of DOI:10.1007/s00044-012-0111-1

A new series of ethyl pyruvate hydrazones and their later first row transition metal(II) complexes have been synthesized and characterized by means of elemental analyses, vibrational, electronic, ¹H and ¹³C NMR, electron paramagnetic resonance, and mass spectroscopic techniques along with thermal and magnetic studies. All complexes were found to be monomeric in nature and have octahedral geometry. The prepared compounds were evaluated for antiproliferative activity against the two human cancer cell lines (HeLa and HepG2). The Cu(II) complexes were found to have potential activity against the cell lines used than the ligand and other complexes. In addition, the DNA-binding/cleaving capacity of the compounds were analyzed by absorption spectroscopy, viscosity measurement, thermal denaturation studies and gel electrophoresis methods.

Full text of DOI:10.1007/s00044-012-0111-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1504–1516
DOI 10.1007/s00044-012-0111-1
ORIGINAL RESEARCH
Transition metal complexes of novel ethyl pyruvate hydrazones
as potential antitumor agents: synthesis and physicochemical
properties, DNA interactions and antiproliferative activity
•
Aishakhanam H. Pathan Kalagouda B. Gudasi
Received: 17 December 2011 / Accepted: 22 May 2012 / Published online: 22 June 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A new series of ethyl pyruvate hydrazones and
their later first row transition metal(II) complexes have
been synthesized and characterized by means of elemental
analyses, vibrational, electronic, 1H and 13C NMR, electron
paramagnetic resonance, and mass spectroscopic tech-
niques along with thermal and magnetic studies. All
complexes were found to be monomeric in nature and have
octahedral geometry. The prepared compounds were
evaluated for antiproliferative activity against the two
human cancer cell lines (HeLa and HepG2). The Cu(II)
complexes were found to have potential activity against the
cell lines used than the ligand and other complexes. In
addition, the DNA-binding/cleaving capacity of the com-
pounds were analyzed by absorption spectroscopy, vis-
cosity measurement, thermal denaturation studies and gel
electrophoresis methods.
based on titanium, copper, nickel, gold, cobalt, ruthenium
and tin are being intensively studied as platinum replace-
ments (Jakupec et al., 2008; Hadjikakou and Hadjiliadis,
2009; Strohfeldt and Tacke, 2008; Yang and Guo, 1999;
Hartinger and Dyson, 2009; Gust et al., 2004). Choice of
the ligands seems to be as important as the choice of
metals, because besides being the integral part of biologi-
cally active complexes these organic ligands can exert a
biological activity of their own (Ferrari et al., 2010).
In order to develop new antitumor drugs, which spe-
cifically target DNA, it is necessary to understand the
different binding mode of metal complex with DNA.
Basically, metal complexes interact with the double helix
of DNA in either a noncovalent or a covalent way. Among
these interactions, intercalation is one of the most impor-
tant DNA-binding modes, because it invariably leads to
cellular degradation. It was reported that the intercalating
ability increases with the planarity of ligands (Kumar et al.,
1985; Xu et al., 2003a, b). In addition, the coordination
geometry and ligand donor atom type also play key roles in
determining the binding mode of complexes to DNA
(Mahadvan and Palaniandavar, 1997; Xu et al., 2003a, b).
The metal ion type and its valence, which are responsible
for the geometry of the complexes, also affect the inter-
calating ability of metal complexes to DNA (Asadi et al.,
2004; Chaires, 1998).
Keywords Hydrazone ꢀ DNA binding ꢀ
Antiproliferative activity ꢀ HeLa ꢀ HepG2
Introduction
Even though platinum-based complexes had been in primary
focus of research on chemotherapy agents (Lippert, 1999;
Allardyce and Dyson, 2001; Abu-Surrah and Kettunen,
2006), the interests in this field have shifted to non-plati-
num-based agents as well, in order to find different metal
complexes with less side effects and similar or better
cytotoxicity. Thus, a wide variety of metal complexes
Notwithstanding the large amount of the literature about
ethyl pyruvate hydrazones, the interest in their chemistry
attracts more and more research groups. These compounds
present interesting chemical properties, both as organic
molecules and also as ligands in metal complexes (Pathan
et al., 2012; Hong et al., 2011; Affan et al., 2009a, b). The
main reason for which they are being studied is connected
with their biological and more specifically, pharmacologi-
cal properties since they present antiviral, antibacterial or
A. H. Pathan ꢀ K. B. Gudasi (&)
Department of Chemistry, Karnatak University,
Dharwad 580 003, Karnataka, India
e-mail: kbgudasi@gmail.com
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Med Chem Res (2013) 22:1504–1516
1505
Preparation of the Schiff base ligands
antitumor activities (Wiecek et al., 2009; Ferrari et al.,
2001; Affan et al., 2009a, b; Diaz et al., 1994). Their
chemical properties are also of remarkable interest because
of the presence of nitrogen and oxygen in the chelating
fragment, elements that are the main chemical species that
in biological systems such as metalloproteins and metal-
loenzymes coordinate and modulate the redox properties of
transition metal ions.
Ethyl 2-(2-(2-(phenylamino) acetyl) hydrazono)
propanoate [L1H]
To a solution of 2-(phenylamino)acetohydrazide (1.65 g,
0.01 mmol) (Narang et al., 1994) in methanol (25 mL) was
added a solution of ethyl pyruvate (1.16 g, 0.01 mmol) in
methanol (10 mL), and the mixture was stirred for 6 h at
room temperature. The colorless precipitate which formed
was set aside for 1 h, then filtered, washed with methanol.
The solid product was dried in vacuo for 4 h (Scheme 1).
Yield; 78 %. M. P. 138–140 °C.
Thus, in this article we describe the preparation, char-
acterization, and DNA-binding/cleavage studies of the
transition metal complexes derived from ethyl pyruvate
phenyl glycine hydrazones. In addition their in vitro anti-
proliferative studies against two human cancer cell lines
(human liver carcinoma cell line [HeLa] and human cervix
carcinoma cell line [HepG2]) were also carried out.
Ethyl 2-(2-(2-(4-bromophenylamino)acetyl)hydrazono)
propanoate [L2H]
The same procedure was followed for the preparation of
the ligand L2H by using 2-(4-bromophenylamino)acet-
ohydrazide (2.81 g, 0.01 mmol).
Experimental
Materials and methods
Yield; 76 % M. P. 162–165 °C.
All reagents were commercially available (Aldrich or
Merck) and used as supplied. Solvents were purified and
dried according to standard procedures (Vogel, 1968). The
metal chlorides used were in the hydrated form.
Preparation of complexes
To the hot solution of ligands in ethanol (0.004 mol in
25 mL), an ethanolic solution of the metal chloride
(0.004 mol in 25 mL) was added drop wise and stirred for
an hour. The mixture was heated at reflux for 5–6 h. The
product formed was filtered off, washed several times with
ethanol and dried in vacuum over P2O5. Tentatively pro-
posed structures of the complexes are shown in Scheme 2.
Analysis and physical measurements
CHN was determined using Thermo quest elemental ana-
lyzer. The metal contents of the complexes were determined
according to the standard methods [Vogel, 1968]. Melting
points were determined in open capillaries and are uncor-
rected. The IR spectra were obtained in KBr Discs on a
Nicolet 170 SX FT-IR spectrometer in the 4,000–400 cm-1
region. 1H and 13C NMR spectra were recorded on a
BRUKER 500 MHz spectrometer in DMSO-d6 with tetra-
methylsilane (TMS) as an internal standard. Conductivity
measurements were performed in DMF solution with
ELICO-CM-82 Conductivity Bridge at 25.0 °C. Electron
paramagnetic resonance (EPR) spectra of Cu(II) complexes
were recorded at room temperature on a Varian E-4 X-band
spectrometer using tetracyanoethylene (TCNE) as g-mar-
ker. The UV–Vis spectra were recorded on a Varian Cary 50
Bio UV–Visible spectrophotometer. The cyclic voltam-
metric experiments were carried out with a three electrode
apparatus using a CHI1110A electrochemical analyzer
(USA). Magnetic measurements were made on Johnson
Matthey magnetic susceptibility balance at room tempera-
ture by using Hg[Co(CNS)4] as calibrant. Thermal analysis
of the metal complexes were carried out on Universal V2.4F
TA Instrument keeping final temperature at 1,000 °C and
heating rate maintained at 10 °C/min. The Ostwald vis-
cometer is used for hydrodynamic measurements.
Biological study
DNA-binding studies using absorption spectroscopy
An absorption titration was carried out by keeping the
concentration of the complex constant while varying the
DNA concentration and measuring the absorbance at kmax
of the complex after each addition of DNA. The
UV–Visible spectra were recorded after equilibration at
26.0 0.1 °C for 10 min after each addition. The intrinsic
binding constant, Kb, was determined according to Eq. (1)
where [DNA] is the concentration of DNA in base pairs,
the apparent absorption coefficients ea, ef, and eb corre-
spond to Aobs/[complex], extinction coefficient for the free
complex and the extinction coefficient of the complex in
the totally bound form, respectively.
½DNAꢁ=ðea ꢂ efÞ ¼ ½DNAꢁ=ðeb ꢂ efÞ þ 1=½Kbðeb ꢂ efÞꢁ
ð1Þ
The data were fitted to above equation and graph was
obtained with a slope equal to 1/(eb - ef) and intercept
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Med Chem Res (2013) 22:1504–1516
Scheme 1 Synthetic route for
the preparation of ligand
O
H
NH2
N
Cl
CH3
O
CH3
reflux 8hrs
O
+
EtOH
X
X
O
H
O
Ester
Ethylchloroacetate
Aniline
Et
/
O
2
H
.
2
H
N
2
H
N
O
H
N
NH2
N
H
X
Hydrazide
X= H, Br
Step 2
O
O
O
H
N
O
H
N
NH2
N
N
H
Methanol
N
+
O
O
H
X
Stirred at
room temp
Hydrazide
X
O
X= H, Br
Scheme 2 Tentatively
proposed structures of the
complexes
X
O
N
N
H
N
N
O
HN
N
O
OH2
O
M
O
O
M
O
O
H
N
H2O
Cl
N
N
O
X
X
M= Co(II), Ni(II), X= H
M= Co(II), X= Br
M= Cu(II), Zn(II) X= H
M= Ni(II), Cu(II), Zn(II) X= Br
equal to 1/[K (eb - ef)]. Hence Kb was obtained from the
ratio of the intercept to the slope (Li et al., 2005).
viscosity of DNA in the presence and absence of the
complex respectively. The values of g and g0 were calcu-
lated by using the Eq. (2)
Viscosity measurements
g ¼ ðt ꢂ t0Þ=t0;
ð2Þ
where ‘‘t’’ is the observed flow time of solution containing
DNA and complexes and ‘‘t0’’ is the flow time of DNA
solution alone. Relative viscosities for DNA were calcu-
lated from the relation (g/g0) (Satyanarayana et al., 1992).
Viscosity measurements were carried out using Ostwald
micro-viscometer maintained at a constant temperature of
26.0 0.1 °C in a thermostat bath. The rate of flow of the
buffer (10 mM Tris HCl), DNA (100 lM), and DNA with
the complexes at various concentrations (50–200 lM) were
measured with a digital stopwatch; each sample was
measured three times and an average flow time was cal-
culated. Data are presented in plot of (g/g0)1/3 versus the
ratio [complex]/[DNA], where g and g0 are the specific
Thermal denaturation studies
These were carried out on UV-spectrometer, equipped with
temperature controlling thermostat. The melting curves
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Med Chem Res (2013) 22:1504–1516
1507
(Tm) of both free E. coli DNA and DNA-bound complexes
were obtained by measuring the hyperchromicity of E. coli
DNA at 260 nm as a function of temperature. The melting
temperatures were measured with 45-lM DNA in phos-
phate buffer at pH 6.8 (l = 0.2 M NaCl). The temperature
was scanned from 20 to 80 °C at a speed of 5 °C/min. The
melting temperature (Tm) was taken as the mid-point of the
hyperchromic transition.
After 24 h incubation, the cells were treated with the
newly synthesized compounds and then incubated for fur-
ther 24 h. 100 mL of PBS solution of MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide,
5 mg/mL) was added to each well, and incubation was
continued for another 4 h. The medium was discarded and
the cells were washed with sterile PBS and then the
resultant formazan blue crystals were dissolved in 500-lL
DMSO and the optical density (OD) was measured at
570 nm (reference filter 690 nm) using an automatic
microplate reader. Triplicate readings were obtained for
each sample, and optical density values of the three wells
were averaged for one sample. The results were recorded
and expressed as % inhibition in cell growth of HepG2 and
HeLa compared with the control.
DNA cleavage studies using gel electrophoresis
For the gel electrophoresis experiments (Arslantas et al.,
2007) the solution of complexes in DMSO (1 mg/mL) were
prepared and these test samples (1 lg) were added to the
genomic DNA samples of E. coli and incubated for 2 h at
37 °C. Agarose gel was prepared in TAE buffer (4.84 g
Tris base, pH 8.0, 0.5 M EDTA/L. pH 7.3), the solidified
gel obtained at *55 °C was placed in electrophoresis
chamber flooded with TAE buffer. Then 20 lL each of the
incubated complex-DNA mixtures (mixed with bromo-
phenol blue dye at 1:1 ratio) was loaded on the gel along
with standard DNA marker and electrophoresis was carried
out under TAE buffer system at 50 V for 2 h. At the end of
electrophoresis, the gel was carefully stained with ethidium
bromide (EtBr) solution (10 lg/mL) for 10–15 min and
visualized under UV light using a Bio-Rad Trans illumi-
nator. The illuminated gel was photographed by using a
Polaroid camera (a red filter and Polaroid film were used).
Results and discussion
The analytical and physicochemical data of the ligands and
their metal complexes are summarized in Table 1. This
indicates that the CoL1, CoL2, and NiL2 complexes possess
1:2 metal to ligand ratio, whereas the other complexes have
1:1 stoichiometry. These complexes are soluble in solvents
like DMF, DMSO and acetonitrile. The molar conductance
values (Table 1) indicate the non-electrolytic nature of the
complexes (Geary, 1971).
Infrared spectral studies
Antiproliferative activity
The important IR frequencies of the ligands L1H and L2H
and their metal complexes are given in Table 2. The sharp
bands of medium intensity at *3,281 and *3,189 cm-1
are assigned to stretching frequencies of hydrazine NH and
phenyl NH, respectively, for the ligands. In the spectra of
Cu(II) and Zn(II) complexes of both the ligands the band
due to phenyl NH is not observed and might have been
obscured by the broad band at 3,412–3,423 cm-1 due to the
presence of coordinated water molecules, which was further
confirmed by thermal studies, whereas in the other com-
plexes this band was observed at 3,170 15 cm-1. The
v(NH) stretching frequency at 3,281 cm-1 is absent in the
spectra of all the complexes, indicating the deprotonation of
hydrazine NH and subsequent coordination through the
enolate functionality, which was further confirmed by dis-
appearance of adjacent t(C=O) band (hydrazone) at
1696 cm-1. A strong band appearing at 1,716 cm-1 in L1H
and 1,720 cm-1 in L2H are assigned to t(C=O) group of
pyruvate moiety. In the spectra of all the complexes, it has
shifted to lower wave number by 80–90 cm-1 indicating
the coordination of carbonyl oxygen via breakdown of the
intermolecular hydrogen bond (Sahni et al., 1982). The
absorption due to azomethine t(C=N) in the free ligands is
Under a sterile condition, HepG2 and HeLa were grown in
RPMI 1640 supplemented with 10 % fetal bovine serum
(FBS). Antiproliferative activity of both the ligands and
their Co(II), Ni(II), and Cu(II) at 100-lM concentration
was tested against HepG2 and HeLa cell lines using MTT
assay (Mosmann 1983, Betancur-Galvis et al., 1999). Each
compound was initially solubilized in dimethyl sulfoxide
(DMSO); however, each final dilution contained less than
1 % DMSO.
The cells at *80 % confluence were selected for tryp-
sinization. The cells were harvested by removing the
medium and then 1 mL of trypsin–EDTA (200 mg/L for
EDTA, 500 mg/L for trypsin in a ratio (1:250) is added
and incubated at 37 °C for about 5 min. The cells were
detached from the plate and collected in a centrifuge tube
and centrifuged at 1,000 rpm for 5 min. Supernatant
solution was removed and the cells were resuspended in
10-mL RPMI-1640 culture medium. Cell number was
determined using hemocytometer. The cell suspension was
diluted to required concentration of 5 9 104 cells/mL.
24-well microplates were seeded with 500 lL of cell sus-
pension and incubated at 37 °C and 5 % CO2 for 24 h.
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Med Chem Res (2013) 22:1504–1516
Conductivity Magnetic
Table 1 Analytical, conductivity and magnetic data for the ligands and their complexes
Compounds
abbreviations
Compounds
Elemental analysis (%) found (calculated)
ohm-1 cm2
mol-1
moment
leff BM.
C
H
N
M
–
Cl
L1H
C1
C2
C3
C4
L2H
C5
C6
C7
C8
C13H17N3O3
[Co(L1)2]
[Ni(L1)2]
59.29 (59.30) 6.53 (6.51) 16.03 (15.96)
–
–
–
–
–
53.55 (53.52) 5.51 (5.53) 14.46 (14.43) 10.26 (10.10)
52.62 (52.66) 5.50 (5.48) 14.75 (14.74) 10.43 (10.29)
10.03
12.32
4.19
2.91
[Cu(L1)Cl(H2O)2] 39.37 (39.30) 5.10 (5.07) 10.59 (10.58) 16.20 (15.99) 8.96 (8.92) 22.34
[Zn(L1)Cl(H2O)2] 39.07 (39.12) 5.10 (5.05) 10.51 (10.53) 16.43 (16.38) 8.90 (8.88) 18.97
1.82
Diamagnetic
–
C13H16BrN3O3
[Co(L2)2]
45.67 (45.63) 4.73 (4.71) 12.27 (12.28)
–
–
–
–
41.16 (41.13) 4.05 (4.08) 11.35 (11.34) 8.03 (7.95)
13.79
4.23
[Ni(L2)Cl(H2O)2] 33.19 (33.13) 4.08 (4.06) 8.95 (8.91)
[Cu(L2)Cl(H2O)2] 32.73 (32.79) 4.02 (4.02) 8.86 (8.82)
[Zn(L2)Cl(H2O)2] 32.63 (32.66) 4.05 (4.01) 8.78 (8.79)
12.66 (12.45) 7.56 (7.52) 20.63
13.40 (13.34) 7.49 (7.44) 25.37
13.58 (13.68) 7.46 (7.42) 15.98
3.01
1.88
Diamagnetic
Table 2 IR spectral data of the ligands and their complexes
Compounds
Amide
m(C=O)
Phenyl m(NH)
Hydrazine m(NH)
Azomethine m(C=N)
Ester m(C=O)
m(N–N)
m(C–O)
L1H
C1
C2
C3
C4
L2H
C5
C6
C7
C8
1696.6
3179.4
3162.1
3176.7
3196.9
3176.2
3183.7
3163.8
3154.6
3230.2
3239.2
3202.0
1610.3
1602.2
1603.9
1598.5
1602.9
1627.3
1596.5
1596.6
1598.3
1595.9
1716.8
1624.9
1629.2
1634.9
1627.3
1720.9
1631.1
1623.7
1620.7
1627.7
1141.6
1119.1
1109.9
1112.2
1109.1
1140.2
1113.3
1106.9
1107.1
1117.1
1271.2
1208.5
1212.5
1207.7
1215.3
1268.6
1211.1
1213.3
1255.0
1215.0
–
–
–
–
–
–
–
–
1693.7
3201.8
–
–
–
–
–
–
–
–
observed at 1,610 and 1,627 cm-1, respectively, and has
shifted to lower wave number (1,596–1,602 cm-1) in the
complexes, indicating the coordination of azomethine
nitrogen (Bellamy, 1958). The stretching frequencies of
coordinated/lattice held water molecules in the complexes
were observed in the range 3,350–3,410 cm-1. The com-
plexes show a non ligand band in the region 430–455 cm-1
ascribable to the m(M–N) (Thomas et al., 1995). Thus IR
spectra of the complexes show the involvement of enolic
oxygen, carbonyl oxygen, and azomethine nitrogen in
coordination.
methylene protons (–CH2–C=O) resonate at 3.95 ppm and
the aromatic protons appeared as two multiplet in the region
6.56–7.09 ppm. The triplet for –CH3 methyl protons of ester
appears at 1.27 ppm and the quartet for methylene protons at
4.20 ppm. The singlets at 11.06, 6.06, and 3.95 ppm
observed in the spectrum of L2H were assigned to hydrazine
NH, phenyl NH, and the methylene protons, respectively
and while the aromatic protons resonate in the region
6.55–7.22 ppm. In the 13C NMR spectrum of the L1H and
L2H, the signals at 172.91 and 172.52 correspond to the
respective carbonyl carbon (HN–C=O). The signals at
164.02, 139.96, and 163.96, 140.07 are assigned to C=N and
C=O, respectively for L1H and L2H, while the corresponding
methylene and methyl carbon signals of ester group are
observed in the region 60.98 0.03 and 12.45 0.23.
1H and 13C NMR spectral studies
1H and 13C NMR spectra of the ligands and 1H NMR spectra
of zinc complexes were recorded in DMSO-d6 solution. The
representative spectra are displayed in Fig. 1a–c.
The 1H NMR spectrum of L1H shows a singlet at
11.03 ppm and was assigned to hydrazine NH, whereas the
singlet at 5.76 ppm was assigned to phenyl NH proton. The
1
Disappearance of the hydrazine NH signal in the H NMR
spectra of Zn complexes confirms the involvement of car-
bonyl oxygen in coordination via deprotonation. A slight
downfield shift observed for the CH3–C=N– protons, indi-
cate the coordination of nitrogen to the metal ion. The other
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Med Chem Res (2013) 22:1504–1516
1509
1
Fig. 1 a H NMR spectrum
of L2H. b 13C NMR spectrum of
1
L2H. c H NMR spectrum of
[Zn(L2)Cl(H2O)2]
signals remained unaltered in the corresponding zinc
complexes.
band at *340 nm in the spectra of ligands with a shoulder
on low energy side is observed due to n ? p* transition
associated with azomethine linkage (Marchetti et al., 1999).
The peak observed near 360–370 nm (*25,000), in all the
complexes is assigned to L ? M charge transfer transi-
tions. This peak is observed in higher energy region with
high intensity in the case of [Co(L2)2], [Ni((L2)2], and
[Co(L1)2] complexes as compared to the other com-
plexes. This may be attributed to the combined effect of
Electronic spectral studies
Figure 2 shows the absorption spectra of L1H and its metal
complexes in the range 275–500 nm. All the complexes
exhibit electronic transitions around 260–315 nm (*5100),
assigned to the intra-ligand p ? p* transitions. A broad
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Med Chem Res (2013) 22:1504–1516
intra-ligand transition and L ? M charge transfer transi-
tions (Sharma and Mishra, 1985; Antolini et al., 1984).
Electronic spectra of cobalt complexes show d–d transitions
around 430, 610, and 689 nm (e * 250 lcm-1 mol-1
)
4
4
Fig. 2b assignable to 4T1g (F) ? T1g(P), 4T1g (F) ? A2g
4
and 4T1g(F) ? T2g transitions which is consistent with the
octahedral geometry (Bailar et al., 1975). In Ni(II) com-
plex, the lowest energy band observed around 870 nm
3
3
(e * 100 lcm-1 mol-1) was due to A2g ? T2g (m1) and
bands at 680 and 440 nm (e * 200 lcm-1 mol-1) were
3
3
3
3
assigned to A2g ? T1g (m2) and A2g ? T1g(P)(m3),
respectively. Appearance of these three spin allowed d–d
bands indicates an octahedral geometry (Bailar et al., 1975).
The Cu(II) complexes exhibit absorptions around 430 and
680 nm (e * 290 lcm-1 mol-1) assigned to the d–d tran-
sitions Fig. 2c, whereas the bands around 360 and 320 are
due to ligand transitions. Zn(II) complexes show absorp-
tion around 400 nm, are corresponding to intra-ligand
transitions.
Mass, EPR spectral studies and magnetochemistry
The mass spectra of the L1H and L2H show molecular ion
peaks at 263 (M?) and 344 (M?), respectively, corre-
sponding to their respective molecular weights. The ESI
mass spectrum of CuL2 complex shows the molecular ion
peak at 474 (M?) and the corresponding isotopic peaks
(M?2) and (M?4) were observed at 476 and 478. The solid
state X-band EPR spectra of copper complexes exhibit
isotropic intense broad signal with giso values 2.04, 2.05,
respectively (representative EPR spectrum is shown in the
Fig. 2 a Absorption spectra of L1H and its metal complexes in the
range 275–500 nm. b Absorption spectrum of [Cu(L1)Cl(H2O)2]
showing d–d transition. c Absorption spectrum of [Co(L2)2] showing
d–d transitions
Fig. 3), with no hyperfine splitting in g or g\ region. The
k
experimentally determined room temperature magnetic
moments of all the complexes are given in Table 1. The
values suggest octahedral geometries for Co(II) and Ni(II)
complexes and presence of an unpaired electron in Cu(II)
complex (Cotton and Wilkinson 1988).
corresponds to the liberation of one chloride as HCl,
respectively. Further decrease of mass in higher tempera-
ture range is ascribed to the ligand decomposition and the
final product was analyzed to be respective metal oxides.
Thermograms of [Co(L1)2] and [Ni(L1)2] show no weight
loss in the region 30–250 °C indicating the absence of
coordinated or lattice held solvent molecules. Further
weight loss in the region 250–600 °C is due to the loss of
ligand molecules. The plateau obtained after heating the
complexes above 700 °C corresponds to the formation of
stable CoO and NiO. The metal content calculated (10.21
and 10.37 %, respectively) from this residue, tallies with
metal analysis (10.10 and 10.29 %, respectively).
Thermal studies
The thermal stability and decomposition pattern of the
complexes were analyzed by thermogravimetry (TG) and
differential thermal analysis (DTA). It is observed that
decomposition of [Ni(L2)Cl(H2O)2] and [Cu(L1)Cl(H2O)2]
complexes takes place in three stages. The first step of
decomposition corresponds to a mass loss of 7.91 % (cal-
culated 7.63 %) and 9.23 % (9.06 %) in the range
130–190 °C is attributed to the loss of two coordinated
water molecules in the respective complexes. The corre-
sponding differential peak (DTA) signifies the endothermic
process. Second stage of mass loss of 7.49 % (7.52 %)
and 8.84 % (8.92 %) in the range 200–280 °C with
respective DTA curve representing the exothermic process,
Electrochemistry
The electrochemical behavior of the complexes was
investigated at room temperature by cyclic voltammetric
experiments. All the complexes were scanned in the
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Med Chem Res (2013) 22:1504–1516
1511
Fig. 3 EPR spectrum of
[Cu(L2)Cl(H2O)2]
Biological studies
DNA-binding studies using absorption spectroscopy
Electronic absorption spectroscopy is universally employed
to determine the binding nature of complexes with DNA. The
interaction of complexes with DNA causes electronic per-
turbations in the transition metal complexes at the maxima.
The data obtained from this experiment shows a significant
hypochromism with an insignificant blue shift at the maxima
when increasing amounts of E. coli DNA were added to the
solution of complex. The extent of spectral change is related
to the strength of binding of the complexes to E. coli DNA.
Both the ligands have no effect on the absorption of DNA
solution. The representative absorption spectrum of the
[Cu(L2)Cl(H2O)2] is given in Fig. 5a. Broad bands around
430 nm and intra-ligand transition at 310–350 nm were
monitored as a function of added DNAs. The observations
reveal that increasing amount of DNA results in the signifi-
cant hypochromism (Pyle et al., 1989) as well as blue shift of
*2 nm for the band at 430 nm, whereas hypochromism and
red shift of 3 nm were seen for the band around 330 nm,
which indicate the intercalative mode of binding. The
intrinsic binding constant Kb = 4.9 9 104 obtained for this
complex agrees with the classical intercalators (Pyle et al.,
1989). In the absorption spectrum of [Co(L1)2] as shown in
Fig. 5b, hypochromism with red shift of bands at 430, 353
and 309 nm also indicate the intercalative mode of binding.
The intrinsic binding constant Kb of all the complexes is
given in the Table 3. The observations for these compounds
can be rationalized by the following reasons. When the
Fig. 4 Voltammogram of [Cu(L2)Cl(H2O)2]
potential range form ?1.0 to -1.0 V with different scan
rates (0.05, 0.1 and 0.15 V/s). Both the Cu(II) complexes
possess redox behavior (cyclic voltammogram of CuL2 is
shown in Fig. 4), while the other complexes did not show
any electrochemical response over the working potential
range indicating that electrochemical activity exhibited by
copper complexes is purely metal based (Rajendiran et al.,
2007). Both the copper complexes show a single peak in
the range 0.29–0.46 V, in the anodic potential scan, which
corresponds to Cu(II) ? Cu(III), oxidation reaction. Dur-
ing the backward scan, complexes exhibit a cathodic peak
in the range from -0.18 to -0.31 V corresponding to the
reduction of metal species, Cu(III) ? Cu(II). The redox
couple Cu(II)/Cu(III) shows a high value of DEp = (Epa
Epc) which is greater than 60 mV indicating the quasi-
reversible nature of the redox process (Bailey et al., 1986).
-
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Med Chem Res (2013) 22:1504–1516
Fig. 6 Effect of increasing amount of complexes of L1H series on the
relative viscosity of DNA at 26.0 0.1 °C
In classical intercalation, the DNA helix lengthens as base
pairs are separated to accommodate the bound ligand
leading to an increased DNA viscosity whereas a partial,
non-classical ligand intercalation causes a bend in DNA
helix reducing its effective length and thereby its viscosity.
Therefore, viscosity measurement is regarded as the least
ambiguous and the most critical means for studying the
binding mode of metal complexes with DNA in solution
and provides stronger arguments for intercalative binding
mode (Satyanarayana et al., 1992; Jin and Yang 1997). The
effects of the free ligand L1H and its complexes on the
viscosity of E. coli DNA are shown in Fig. 6. As illustrated
in this figure, with increasing amount of the compounds,
the relative viscosity of E. coli DNA increases steadily,
which suggest that the compounds are bound to DNA by
intercalation. The increased degree of viscosity, which may
depend on their affinity to DNA follows the order of
Cu(II) [ Co(II) [ Ni (II) [ L1H. Similar effect is also
seen in case of L2H and its complexes. The extent of
intercalation observed is more in the copper complexes of
both the ligands.
Fig. 5 a Absorption spectra of [Cu(L2)Cl(H2O)2] showing the
variation of absorption with respect to the increase in the DNA
concentration. b Absorption spectra of [Co(L1)2] showing the
variation of absorption with respect to the increase in the DNA
concentration
Table 3 DNA-binding constant of the complexes
Compounds
abbreviations
Complexes
Binding constants
Kb in M-1
C1
C2
C3
C4
C5
C6
C7
C8
[Co(L1)2]
[Ni(L1)2]
1.1 9 104
2.9 9 103
1.2 9 104
2.1 9 103
1.0 9 104
4.1 9 103
4.9 9 104
1.6 9 103
[Cu(L1)Cl(H2O)2]
[Zn(L1)Cl(H2O)2]
[Co(L2)2]
[Ni(L2)Cl(H2O)2]
[Cu(L2)Cl(H2O)2]
[Zn(L2)Cl(H2O)2]
compounds intercalate the base pairs of E. coli DNA, the
p*-orbital of the intercalated ligands can couple with the
p-orbital of the base pairs of E. coli DNA, thus decreasing
the p ? p* transition energy and resulting in the batho-
chromism (Wang et al., 2005; Li et al., 2011). Furthermore,
the coupling p-orbital is partially filled by electrons, thus
decreasing the transition probabilities and concomitantly
resulting in hypochromism. The intrinsic binding con-
stants of all the complexes suggest that the [Co(L1)2],
[Cu(L1)Cl(H2O)2], and [Cu(L2)Cl(H2O)2] complexes show
more binding property than the remaining complexes.
Thermal denaturation study
Thermal behaviors of DNA in the presence of complexes
can give insight into their conformational changes when
temperature is raised, and provide the information about
the interaction strength of complexes with DNA (Kelly
et al., 1985). The Tm of E. coli DNA in absence of the
complexes is found to be 58 1 °C (Tselepi-Kalouli and
Katsaros, 1989). On the bases of viscometric measurements
the complexes CuL1, CoL2, and CuL2 have chosen for the
thermal denaturation experiment, because of their stronger
binding capability with the E. coli DNA than any other
complexes. However, with addition of CuL2 and CoL2
complexes, the Tm of the DNA increases dramatically to
*65 1 °C. whereas the presence of other complex
Viscosity measurement study
To confirm further, the interaction mode of the compounds
and E. coli DNA, viscosity measurements were carried out.
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Med Chem Res (2013) 22:1504–1516
1513
electrophoresis photograph Fig. 8a of L1H series, only
Cu(II) complex (lane 3) has shown an increase in the
intensity and tailing compared to remaining compounds
this illustrates the higher binding ability of the said com-
plex with E. coli DNA, whereas, in case of L2H series
Fig. 8b except lane 4 all the compounds have shown
increase in the intensity and tailing indicating that the L2H
series have enhanced DNA-binding ability than the L1H
series.
In vitro antiproliferative activity
Ligands and their Co(II), Ni(II) and Cu(II) complexes were
tested for potential in vitro antiproliferative effects using
MTT test on a panel of two human cell lines, which were
derived from different cancer types i.e., HeLa (cervical
carcinoma) and HepG2 (liver carcinoma). Figures 9a, b
and 10a, b show effect of different concentrations of tested
compounds on two human cancer cell lines after 24 h
incubation time. All tested compounds showed noticeable
antiproliferative effect in the low micromolar, or submi-
cromolar range against HeLa cell line, whereas less active
against HepG2 cell line. The percentage inhibitions of
copper complexes are found to be *80 %, and the other
compounds show \ 50 %. A comparatively enhanced
antiproliferative effect was seen against the HepG2 with
bromide substituted compounds (L2H and its complexes)
than the L1H and its complexes as shown in Fig. 10b,
Fig. 7 Melting curves of E. coli DNA in the absence and presence of
complexes
shows the Tm of 63 1 °C. This is characteristic of an
´
intercalative binding behavior of the complexes (Lopez
et al., 1996). The graphs showing the effect of increase in
temperature on the stability of E. coli DNA are shown in
the Fig. 7.
DNA cleavage studies using gel electrophoresis
The cleavage efficiency of the metal complexes compared
to that of control is due to their efficient DNA-binding
ability. Both the ligands and complexes were allowed to
interact with the E. coli DNA for cleavage study. In the gel
Fig. 8 a Photograph of gel
electrophoresis experiment of
L1H-series on DNA of E. coli.
M DNA marker, C untreated
DNA, 1 ligand (L1H), 2
[Co(L1)2], 3 [Cu(L1)Cl(H2O)2],
4 [Ni(L1)2], 5
[Zn(L1)Cl(H2O)2].
b Photograph of gel
electrophoresis experiment of
L2H-series on DNA of E. coli.
M DNA marker, C untreated
DNA, 1 ligand (L2H), 2
[Co(L2)2], 3 [Cu(L2)Cl(H2O)2],
4 [Ni(L2)Cl(H2O)2], 5
[Zn(L2)Cl(H2O)2]
123

1514
Med Chem Res (2013) 22:1504–1516
Fig. 9 a Effect of test
compounds (L1H Series) on cell
growth of HeLa and b HepG2
incubated for 24 h in vitro
Fig. 10 a Effect of test
compounds (L2H Series) on cell
growth of HeLa and b HepG2
incubated for 24 h in vitro
indicates the presence of halogen makes the compound
more lipophilic in character to enter into the cell, hence
enhancing the antiproliferative effect. Cu(II) complexes are
found to have highest antiproliferative activity against both
the cell lines whereas Co(II) and Ni(II) complexes are
moderately active and ligands are least active among the
lot against the cancer cell lines used.
compounds with E. coli DNA. The complexes have shown
enhanced antiproliferative activity than the ligands. Both
the copper complexes have exhibited significant antipro-
liferative activity, which is correlated to the strong inter-
action of complexes with DNA. The enhancement of the
antiproliferative activity of copper complexes, with respect
to other compounds, might be related with their different
structural characteristics, where the coordinated chloride
and water molecules would have a lipophilic effect,
thereby enhancing the anti proliferative activity.
Conclusions
Acknowledgments The authors thank USIC, Karnatak University,
Dharwad for providing the spectral facilities. Recording of ESR
spectra from IIT Bombay is gratefully acknowledged. One of the
authors (Aishakhanam H. Pathan) is thankful to UGC for providing
financial assistance under its Research Fellowship in Science for
Meritorious Students.
All the complexes are found to be monomeric and octa-
hedral in nature. Both the ligands act as ONO tridentate
monobasic chelating ligands. The copper complexes
underwent redox reaction in the applied range of potential
(-1 to ?1 V) and exhibit quasi-reversible responses.
Subsequently both the prepared ligands and their metal
complexes have been evaluated for in vitro antiprolifera-
tive activity against the two human cancer cell lines, (Hela
and HepG2). In addition, an extensive DNA-binding/
cleaving capacity of the compounds was analyzed by
absorption spectroscopy, viscosity measurement, thermal
denaturation studies and gel electrophoresis methods.
The observations made in DNA-binding studies reveal
the classical intercalation mode of interaction of the
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