Oxovanadium phenanthroimidazole derivatives: synthesis, DNA binding and antitumor activities

Article abstract of DOI:10.1007/s11243-018-0205-9

Four unsymmetrical oxovanadium phenanthroimidazole complexes, [VO(hntdtsc)(NPIP)] (1), [VO(hntdtsc)(CPIP)] (2), [VO(hntdtsc)(MEPIP)] (3) and [VO(hntdtsc)(HPIP)] (4) (hntdtsc?=?2-hydroxy-1-naphthaldehyde thiosemicarbazone, NPIP?=?2-(4-nitrophenyl)-imidazo[

Full text of DOI:10.1007/s11243-018-0205-9

Transition Metal Chemistry (2018) 43:171–183
https://doi.org/10.1007/s11243-018-0205-9
Oxovanadium phenanthroimidazole derivatives: synthesis, DNA
binding and antitumor activities
1
2
1
3
3
4
2
Yin‑Liang Bai · Ya‑Wu Zhang · Ji‑Yuan Xiao · Hai‑Wei Guo · Xiang‑Wen Liao · Wen‑Jie Li · You‑Cheng Zhang
Received: 28 September 2017 / Accepted: 5 January 2018 / Published online: 24 January 2018
©
Springer International Publishing AG, part of Springer Nature 2018
Abstract
Four unsymmetrical oxovanadium phenanthroimidazole complexes, [VO(hntdtsc)(NPIP)] (1), [VO(hntdtsc)(CPIP)] (2),
[
VO(hntdtsc)(MEPIP)] (3) and [VO(hntdtsc)(HPIP)] (4) (hntdtsc = 2-hydroxy-1-naphthaldehyde thiosemicarbazone,
NPIP = 2-(4-nitrophenyl)-imidazo[4,5-f]1,10-phenanthroline, CPIP = 2-(4-chlorphenyl)-imidazo[4,5-f]1,10-phenanthroline),
MEPIP = 2-(4-methylphenyl)-imidazo[4,5-f]1,10-phenanthroline), HPIP = 2-(4-hydroxylphenyl)-imidazo[4,5-f] 1,10-phen-
anthroline), have been synthesized and characterized. Their DNA binding and antitumor activities were determined by bio-
chemical methods. All four oxovanadium complexes can bind with CT-DNA by an intercalation model and can also cleave
supercoiled plasmid DNA in the presence of H O . The antitumor properties and mechanism of the complexes have been
2
2
analyzed by MTT assay, cell cycle analysis, apoptosis assay and Western blot analysis. The results showed that the free ligands
and their corresponding complexes all possess antiproliferative activities with very low IC values against Hela, BIU-87 and
50
SPC-A-1 cell lines. Complex 1, which has a strongly electron-withdrawing nitro group, exhibited the best antiproliferative
activities. Complex 1 caused G /G phase arrest of the cell cycle and induced apoptosis in Hela cells. Additionally, complex
0
1
1
attenuated the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2).This indicates that inhibition
of the ERK1/2 signaling pathway may contribute to the antitumor eꢀects of these complexes.
Introduction
subsequent release of cytochrome c, resulting in cancer
cell apoptosis [11, 12]. In addition, vanadium compounds
can enhance uptake of redox-active essential metal ions,
primarily Cu(II), from extracellular media into cells, caus-
ing disruption of cellular thiol levels. Vanadium complexes
can also have insulin-mimetic and antidiabetic properties
by increasing the cellular uptake of glucose by the GLUT4
transporter [13].
Vanadium has attracted considerable attention, because its
complexes with organic ligands can possess a variety of bio-
logical functions such as antibacterial, antitumor, insulin-
enhancing and antiparasitic eꢀects [1–6]. Vanadium com-
plexes have been investigated in tumor therapy due to their
considerable antiproliferation role in certain cancers [7–9].
The general mechanisms for antitumor action by vanadium
complexes involve inhibition of DNA repair processes [10],
opening of mitochondrial permeability transition pores,
In previous work, it was found that vanadium complexes
of thiosemicarbazones and 1,10-phenanthroline ligands can
possess excellent antiproliferative activities. Organic ligands,
especially with aromatic substituents, can enhance their bio-
logical activities [14]. In this paper, we report the preparation
of four new oxovanadium complexes of thiosemicarbazones,
namely 2-(4-nitrophenyl)-imidazo[4,5-f]1,10-phenanthroline
(NPIP), 2-(4-chlorphenyl)-imidazo[4,5-f]1,10-phenanthro-
line (CPIP), 2-(4-methylphenyl)-imidazo[4,5-f]1,10-phen-
anthroline (MEPIP) and 2-(4-hydroxylphenyl)-imidazo[4,5-
f]1,10-phenanthroline (HPIP), together with an investigation
of their antitumor activities. NPIP and CPIP possess strongly
electron-withdrawing groups, while MEPIP and HPIP pos-
sess electron-donating groups.
*
You-Cheng Zhang
lzuzhangych@163.com
1
2
3
4
Department of Pharmacy, Lanzhou University Second
Hospital, Lanzhou 730030, China
Department of General Surgery, Lanzhou University Second
Hospital, Cuiying Men Road 82, Lanzhou 730030, China
School of Pharmacy, Guangdong Pharmaceutical University,
Guangzhou 510006, China
Department of Clinical Pharmacology, School
of Pharmaceutical Sciences, Zhengzhou University,
Zhengzhou 450001, China
Vol.:(0
1
123456789)
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Transition Metal Chemistry (2018) 43:171–183
Oxovanadium complexes of these ligands, [VO(hntdtsc)
The position of the azomethine peak was reduced in fre-
quency compared the corresponding free ligands, consistent
with coordination of the azomethine nitrogen to vanadium.
The strong VO signal could also be clearly identiꢂed at
(
(
NPIP)] (1), [VO(hntdtsc)(CPIP)] (2), [VO(hntdtsc)
MEPIP)] (3) and [VO(hntdtsc)(HPIP)] (4), were prepared
and fully characterized by elemental analysis, UV–Vis, MS,
IR and NMR. The interactions of these complexes with calf-
thymus DNA (CT-DNA) were investigated by UV–Vis and
−1
945–955 cm [14]. In their ES-MS spectra, the free ligands
+
showed molecular peaks of m/z 246.0 ([M + 1] ) (hntdtsc),
+
+
ꢁ
uorescence spectroscopy, and by viscosity measurements.
342.0 ([M + 1] ) (NPIP), 331.1 ([M + 1] ) (CPIP), 327.1
+
+
Their cleavage reactions with pBR322 supercoiled plasmid
DNA were studied by gel electrophoresis. In addition, their
antitumor properties and mechanism of action have been
analyzed by MTT assay, cell cycle analysis, apoptosis assay
and Western blot analysis. The results showed that the free
ligands and their corresponding complexes all possess anti-
tumor activities with very low IC values and complex 1
([M + 1] ) (MEPIP) and 313.0 ([M + 1] ) (HPIP). The
oxovanadium complexes gave molecular peaks at m/z 652.1
+
+
+
([M + 1] ) (1), 639.1 ([M + 1] ) (2), 619.1 ([M + 1] ) (3)
+
and 623.0 ([M + 1] ) (4).
1
13
In the H and C-NMR spectra of the free ligands
recorded in DMSO-d , all the protons due to heteroaromatic
6
groups were found in their expected regions. In addition,
5
0
exhibited the highest activity against tumor cells.
for the four complexes, the peaks of hydroxyl (OH), amine
(
NH ) and (NHCO) were absent, indicating that these ligand
2
groups were coordinated to vanadium.
Results and discussion
Synthesis and characterization
DNA‑binding studies
[
[
VO(hntdtsc)(NPIP)] (1), [VO(hntdtsc)(CPIP)] (2),
VO(hntdtsc)(MEPIP)] (3) and [VO(hntdtsc)(HPIP)] (4)
Absorption titrations
were prepared by reꢁuxing hntdtsc and vo(acac) with NPIP,
Absorption titrations in the UV–Vis range were carried
out in order to investigate the interactions of these com-
plexes with CT-DNA. Hypochromism and bathochromism
can occur with the intercalation of complexes into the base
pairs of DNA [15, 16], which is correlated with the stacking
interactions between the planar aromatic chromophore of the
complexes and the base pairs of DNA. For metal interaction,
DNA binding is associated with hypochromism and showed
a redshift in the MLCT and ligand bands.
2
CPIP, MEPIP and HPIP, respectively, in absolute methanol.
Elemental analysis, MS, IR and NMR spectroscopy were
implemented to characterize the complexes and the free
ligands. The structures of these complexes and free ligands
are shown in Scheme 1.
The IR spectra show peaks from the Schiꢀ bases(–NH–)
−
1
−1
at 3450–3160 cm , aromatic (C–H) at 2987–3050 cm ,
−1
azomethine group (–CH=N) and (C=C) at 1592–1626 cm .
Scheme 1 Structure of the ligands and complexes
1
3

Transition Metal Chemistry (2018) 43:171–183
173
Figure 1 shows the electronic spectra of the four oxova-
nadium complexes obtained in the presence of CT-DNA.
Appreciable hypochromism and bathochromism can be
observed for all four complexes with increasing amounts
of DNA. The specific values of hypochromism and
bathochromism were 41.6% and 3.5 nm (complex 1), 38.8%
and 2.5 nm (complex 2), 32.4% and 2 nm (complex 3),
DNA is subject to electrophoresis, relatively fast migration
will be observed for the intact supercoiled form (Form I). If
scission occurs on one strand, the supercoil will relax to gen-
erate a slower moving open circular form (Form II) [26, 27].
The results of our experiments are shown in Figs. 4 and
5. No obvious cleavage of DNA was observed in the absence
of any complex (lanes 11 and 12 in Figs. 4, 5, respectively).
However, the open circular form (Form II) was observed in
the presence of diꢀerent concentrations of complexes 1, 2,
3 and 4 (Figs. 4, 5). Hence, all four complexes possess DNA
cleavage ability. To explore the DNA cleavage mechanism,
control experiments were carried out in the presence of
l-histamine or dimethyl sulfoxide (DMSO). No signiꢂcant
change was observed in the presence of l-histamine, which
is a singlet oxygen quencher. However, in the presence of the
hydroxyl radical scavenger DMSO, inhibition of DNA cleav-
age occurred, suggesting that the ∙OH free radical is likely to
be the reactive species for the cleavage reaction. This can be
explained by the generation of ∙OH radicals obtained from
3
1.3% and 2 nm (complex 4). In addition, the intrinsic bind-
ing constant K was calculated by monitoring the change in
b
absorbance with increasing amounts of CT-DNA; the K
b
5
−1
values of complexes 1, 2, 3 and 4 were 1.53 × 10 M ,
5
−1
5
−1
5
−1
1
.41 × 10 M , 1.05 × 10 M and 0.95 × 10 M , respec-
tively. Complex 1 has the highest K , and the values suggest
b
that these oxovanadium complexes may bind to DNA in an
intercalative mode.
Fluorescence spectroscopic studies
Fluorescence spectroscopic titration experiments were
performed in Tris buꢀer A. The emission spectra of the
four complexes in the absence and presence of CT-DNA
are shown in Fig. 2 and clearly increase the intensity of
the luminescence emitted by the complexes steadily upon
increasing the amount of CT-DNA, suggesting that these
four oxovanadium complexes may intercalate into the base
pairs of the DNA. The hydrophobic environment inside the
DNA helix reduces the accessibility of water molecules to
the complexes, thus lengthening their luminescence lifetimes
and increasing their emission intensity [17–19].
2
+
the oxidation of VO in the presence of H O [28].
2
2
Antitumor activities
Cytotoxicity assays
The complexes and the corresponding free ligands were
evaluated for their ability to inhibit the growth of Hela, BIU-
87 and SPC-A-1 cell lines using MTT assays [29, 30]. The
IC values were calculated after 24 h of incubation with
5
0
the test compound at diꢀerent concentrations using cispl-
atin as a control. The results are listed in Table 1. All four
oxovanadium complexes as well as the free ligands showed
signiꢂcant cytotoxicities against all three tumor cells, and
the antitumor activities were also concentration depend-
ent. Complex 1 showed the largest inhibitory eꢀects against
the Hela, BIU-87 and SPC-A-1 cell lines with IC values
Viscosity measurements
In the absence of X-ray structure data, viscosity measure-
ments, which are sensitive to the change in the length of
DNA, are considered to be the most critical test of the clas-
sical intercalation model and provide the most deꢂnitive
means of inferring the binding mode of DNA in solution.
The viscosity of the DNA solution is expected to increase
significantly if the intercalation mode is in operation,
whereas partial, non-classical intercalation will have a small
eꢀect on the viscosity [20–25].
5
0
of 1.09 ± 0.16, 4.51 ± 0.68 and 7.61 ± 0.55 μM, respec-
tively. Overall, the antitumor activities follow the order
1 > 2 > 3 > 4, consistent with their DNA-binding abilities.
It should be noted that under the same conditions, the
IC of complex 1 was lower than that of cisplatin, which
5
0
Figure 3 shows the eꢀect of the complexes on the vis-
cosity of CT-DNA. The viscosity is signiꢂcantly increased
with increasing complex concentration, in the order
gave IC values against the Hela, BIU-87 and SPC-A-1
50
cell lines of 5.54 ± 0.81, 7.82 ± 0.64 and 8.65 ± 1.01 μM,
respectively. The potency of complex 1 and its high aꢃnity
for DNA may be associated with the presence of an electron-
withdrawing nitro group in this complex.
1
> 2 > 3 > 4. This result also supports an intercalation
mode for the binding of all four complexes to DNA.
DNA cleavage
Cell cycle analysis
To further elucidate the interaction of DNA with these com-
plexes, DNA cleavage experiments were carried out in order
to monitor the cleavage reaction on supercoiled plasmid
DNA by agarose gel electrophoresis. When circular plasmid
The cell cycle distribution was analyzed by ꢁow cytometry
with PI staining, in order to further elucidate the mechanism
of the antiproliferative eꢀect of these complexes on cancer
cells. Hela cells were treated with complex 1 at diꢀerent
1
3

174
Transition Metal Chemistry (2018) 43:171–183
Fig. 1 Absorption spectral trace
of the complexes 1 (a), 2 (b),
3
(c) and 4 (d) on the addition
of CT-DNA in Tris–HCl buꢀer
A. [V] = 20 μM. Arrow shows
the decreasing absorbance with
the increasing amounts of CT-
DNA. Plots of [DNA]/(ε − ε )
a
f
versus [DNA] for the titration of
[
V] with CT-DNA
1
3

Transition Metal Chemistry (2018) 43:171–183
175
Fig. 2 Emission spectra of
complexes 1 (a), 2 (b), 3 (c)
and 4 (d) in Tris–HCl buꢀer A
in the absence and presence of
CT-DNA. [V] = 20 μM. Arrows
show the increasing intensity
with the increasing concentra-
tion of DNA
concentrations (0.5, 1.0 and 2.0 μM) for 24 h. This experi-
ment signiꢂcantly increased the proportion of cells at G /G
0
1
phase and decreased the proportion of cells at S and G /M
2
phases (Fig. 6). This indicates that the complex inhibits the
growth of Hela cells by disrupting the G /G phase of the
0
1
cell cycle.
The occurrence of apoptosis was further investigated
with Hoechst 33258 staining to investigate whether the
inhibition of cell growth in the Hela cells caused by
complex 1 was associated with apoptosis. Representa-
tive staining fluorescence photomicrographs of cultured
Hela cells treated with or without complex 1 are shown
in Fig. 7. As can be readily observed by comparison with
the control, the cells treated with complex 1 exhibited
typical apoptotic features after 24 h, including cellular
Fig. 3 Eꢀects of increasing amounts of complexes 1 (ꢂlled square), 2
(
ꢂlled circle), 3 (ꢂlled triangle) and 4 (ꢂlled inverted triangle) on the
relative viscosity of CT-DNA at 28(± 0.1) ꢄC [DNA] = 0.40 mM
Fig. 4 Cleavage of pBR322 DNA by oxidovanadium com-
plexes 1 and 2 (15–60 μM) in the absence and presence of H O
DNA + 1 (30 μM) + H O + l-histidine (0.02 M); lane 6, DNA + 1
2
2
(30 μM) + H O + DMSO (2 μL); lane 5, DNA + 2 (15 μM) + H O ;
2
2
2
2
2
2
(
30 mM) in buꢀer B (pH 7.2). Lane 12, DNA control; lane 11,
lane 2, DNA
+
2
2
(30 μM)
+
H O ; lane 3, DNA
+
2
2
2
DNA + H O ; lane 10, DNA + 1 (15 μM) + H O ; lane 9, DNA + 1
(60 μM) + H O ; lane 2, DNA + 2 (30 μM) + H O + l-histidine
2
2
2
2
2
2
2
(
30 μM) + H2O2; lane 8, DNA + 1 (60 μM) + H O ; lane 7,
(0.02 M); lane 1, DNA + 2 (30 μM) + H O + DMSO (2 μL)
2 2
2
2
1
3

176
Transition Metal Chemistry (2018) 43:171–183
Fig. 5 Cleavage of pBR322 DNA by oxidovanadium com-
plexes 3 and 4 (15–60 μM) in the absence and presence of H O
DNA + 3 (30 μM) + H O + l-histidine (0.02 M); lane 8, DNA + 3
2
2
(30 μM) + H O + DMSO (2 μL); lane 5, DNA + 4 (15 μM) + H O ;
2
2
2
2
2
2
(
30 mM) in buꢀer B (pH 7.2). Lane 12, DNA control; lane 11,
lane 2, DNA
+
2
4
(30 μM)
+
H O ; lane 3, DNA
+
4
2
2
DNA + H O ; lane 10, DNA + 3 (15 μM) + H O ; lane 9, DNA + 3
(60 μM) + H O ; lane 2, DNA + 4 (30 μM) + H O + l-histidine
2
2
2
2
2
2
2
(
30 μM) + H O ; lane 7, DNA + 3 (60 μM) + H O ; lane 6,
(0.02 M); lane 1, DNA + 4(30 μM) + H O + DMSO (2 μL)
2 2
2
2
2
2
Phosphorylation of ERK1/2
Table 1 The IC50 values for complexes and ligands against Hela,
As shown in Fig. 9, Western blot analysis showed that both
complex 1 (2 µM) and U0126 (ERK inhibitor) attenuated
the phosphorylation of ERK1/2 in Hela cells treated for 24 h
BIU-87 and SPC-A-1 cell lines
Compounds
IC50 (μM)
Hela
(
P < 0.05; P < 0.05, respectively). The total ERK1/2 levels
BIU-87
SPC-A-1
were not signiꢂcantly diꢀerent among the groups.
Cisplatin
5.54 ± 0.81
7.82 ± 0.64
8.65 ± 1.01
hntdtsc
VO(acac)2
CPIP
NPIP
MEPIP
100.04 ± 7.56 98.16 ± 6.78 74.56 ± 5.63
87.21 ± 7.24 76.87 ± 6.12 84.14 ± 5.89
39.69 ± 7.12 60.23 ± 5.12 41.02 ± 3.45
24.35 ± 1.63 39.65 ± 5.56 31.01 ± 5.01
41.02 ± 4.63 56.45 ± 7.36 61.02 ± 6.32
78.62 ± 7.02 47.89 ± 4.12 54.36 ± 2.13
Conclusion
In this study, four oxovanadium complexes of mixed thio-
semicarbazone and phenanthroimidazole ligands have been
prepared and characterized. All four of the complexes can
bind with CT-DNA by intercalation and also show eꢃcient
cleavage of supercoiled plasmid DNA in the presence of
H O . Both the free ligands and their corresponding com-
HPIP
VO(hntdtsc)CPIP
VO(hntdtsc)NPIP
VO(hntdtsc)MEPIP
VO(hntdtsc)HPIP
10.36 ± 1.23
1.09 ± 0.16
20.11 ± 2.68 34.52 ± 3.67 24.63 ± 4.37
40.26 ± 4.01 19.86 ± 2.47 26.32 ± 4.75
8.69 ± 1.05 21.43 ± 3.24
4.51 ± 0.68 7.61 ± 0.55
2
2
plexes all possess cytotoxic activities against Hela, BIU-87
and SPC-A-1 cell lines, and complex 1 exhibited the highest
activity against tumor cells. The proliferative suppression of
Hela cells by complex 1 is predominantly via the induction
of apoptosis and inhibition of tumor cell growth by induc-
ing a block in the G /G phase of the cell cycle. Complex
The IC50 values for complexes and ligands against Hela, BIU-87 and
SPC-A-1 cell lines. All data were presented as mean ± SE
0
1
1
which has an electron-withdrawing nitro substituent and
morphological change, fragmentation, apoptotic bodies,
condensation of chromatin (brightly stained) and mem-
brane blebbing. Furthermore, the proportion of apoptotic
cells increased in a dose-dependent manner.
larger DNA-binding aꢃnity also exhibited highest cytotoxic
activity against tumor cells; taken together with the results
of a previous study [14], this suggests that the introduction
of electron-withdrawing groups to phenanthroimidazole
ligands may enhance the biological activity of their vana-
dium complexes.
Annexin V-FITC/PI staining was implemented to
determine early, late apoptotic and necrotic cells with
the results shown in Fig. 8. The proportion of Annexin
V-FITC-stained cells (both early and late apoptotic cells)
increased with increasing concentrations of complex 1;
the proportions were 25.8% (0.5 µM), 53.9% (1.0 µM) and
Mitogen-activated protein kinase (MAPK) cascades are
key signaling pathways involved in the regulation of cell pro-
liferation, apoptosis, survival and diꢀerentiation. Aberrant
regulation of MAPK cascades can contribute to cancer. In
particular, the extracellular signal-regulated kinase (ERK), as
one of MAPKs subtypes, is widely involved in human onco-
genesis [45–47]. ERK is a downstream component of the
Ras–Raf–MEK–ERK–MAPK signaling pathway. Activated
Raf phosphorylates and activates the kinase MEK, which
in turn phosphorylates and activates ERK kinase. Activated
ERK regulates a number of cellular events, including cell cycle
8
1.4% (2.0 µM), respectively. Also, complex 1 was found
to predominantly cause early apoptotic cells, such that the
proportions of Annexin V-FITC-stained early apoptotic
cells were 18.3% (0.5 µM), 47.5% (1.0 µM) and 75.2%
(
2.0 µM). In conclusion, the results suggest that complex
1
induces proliferative suppression of Hela cells predomi-
nantly via the induction of apoptosis.
1
3

Transition Metal Chemistry (2018) 43:171–183
177
Fig. 6 DNA content and cell
cycle analysis of Hela cells after
complex 1 treatment. Hela cells
were cultured with complex
a
b
G
0
/G
1
: 54.99%
0 1
G /G : 57.58%
S: 30.69%
G /M: 9.13%
2
S: 31.29%
G
2
/M: 11.82%
1
0
2
(a 0.1% DMSO (control); b
.5 μM; c 1 μM; d 2 μM) for
4 h. The percentage of non-
apoptotic cells within each cell
cycle was determined by ꢁow
cytometry
d
c
G
0
/G
1
: 63.79%
G /G : 70.42%
0
1
S: 23.76%
/M: 7.95%
S: 18.61%
G /M: 8.22%
2
G
2
progression, apoptosis, diꢀerentiation and evasion from cell
death [48–50]. Because of its important role, the ERK signal-
ing pathway has been the subject of intense research leading
to the discovery and development of pharmacologic inhibitors
for the treatment of cancer [45–48]. So it is worth noting that
vanadium species have been closely linked with deregulation
of cellular pathways through tyrosine phosphorylation. The
Western blot analysis also showed that complex 1 attenuated
the phosphorylation of ERK1/2 in Hela cells, similar to the
U0126 (ERK pathway inhibitor), while having no eꢀect on
the total ERK1/2 levels. In conclusion, our study demonstrated
that complex 1 inhibits cell proliferation by inducing apoptosis
and G /G phase arrest, and inhibition of the ERK1/2 signaling
company. Calf-thymus DNA (CT-DNA) and pBR 322
DNA were obtained from Sigma. Other materials were
obtained from commercial sources and used as received
(analytical reagents). Tris–HCl buffer A (5 mM
Tris(hydroxymetylaminomethane)–HCl, 50 mM NaCl,
pH = 7.2) was used for absorption titrations, luminescence
titrations, and viscosity experiments. Tris–HCl buꢀer B
(50 mM Tris–HCl and 18 mM NaCl, pH = 7.2) was used
for DNA cleavage experiments. All buꢀers were prepared
using double-distilled water. A solution of CT-DNA in the
buꢀer gave a ratio of UV absorbance at 260 and 280 nm
of ca. 1.8–1.9, indicating that the DNA was suꢃciently
free of protein [31]. The DNA concentration per nucleo-
tide was determined by absorption spectroscopy using the
0
1
pathway may also contribute to the antitumor eꢀects of these
−
1
−1
complexes.
molar absorption coeꢃcient (6600 M cm ) at 260 nm
[
32]. Hela, BIU-87 and SPC-A-1 cell lines were purchased
from The Cell Bank of Type Culture Collection of the
Chinese Academy of Sciences (Shanghai, China). RPMI
Experimental
1
640 medium was purchased from Hyclone (Logan, USA);
Materials and methods
trypsin, fetal calf serum and Annexin V-FITC/PI apoptosis
detection Kit were purchased from GIBCO company (USA).
Hoechst 33258 staining solution was purchased from Beyo-
time Institute of Biotechnology (China). MTT, rhodamine
VO(acac) (acac = acetylacetonate) and 1,10-phen-
2
anthroline were purchased from Shanghai Jingchun
1
3

178
Transition Metal Chemistry (2018) 43:171–183
Fig. 7 Eꢀects of complex 1 on the morphology of Hela cells were
assayed by Hoechst 33258 staining. After treatment with complex 1
totic cells were detected by Hoechst 33258 staining and examined by
ꢁuorescence microscopy
[
a 0.1% DMSO (control); b 0.5 μM; c 1 μM; d 2 μM] for 24 h, apop-
1
23 and U0126 (ERK1/2 inhibitor) were purchased from
FACSCalibur™, USA). Fluorescence microscopy of apoptosis
assays was carried out with a ꢁuorescence microscope (Olym-
pus OX31, Olympus Corporation, Japan).
Sigma (USA). Anti-p-ERK1/2, anti-ERK1/2 and β-actin
antibodies were purchased from Santa Cruz Biotechnology
(
USA).
Microanalyses (C, H, S and N) were obtained out with a
DNA binding and cleavage
PerkinElmer 240Q elemental analyzer. Electrospray mass
spectra (ES-MS) were recorded on an LCQ system (Finnigan
MAT, USA) using methanol as mobile phase. Infrared spectra
were recorded on a PerkinElmer Lambda 35 instrument using
Absorption titration experiments were performed with ꢂxed
concentration of the complexes (20 µM), while gradually
increasing the concentration of CT-DNA. V-DNA solutions
were allowed to incubate for 5 min before the absorption spec-
tra were recorded. In order to compare the binding strength
1
13
KBr pellets. H and C-NMR spectra were recorded on a Var-
ian-500 spectrometer. All chemical shifts are given relative
to tetramethylsilane (TMS). UV–Vis spectra were recorded
on a Shimadzu UV-3101 PC spectrophotometer at room tem-
perature. Emission spectra were recorded on a PerkinElmer
Lambda 55 spectroꢁuorophotometer. Molar conductivities in
DMF (1 mM/l) solution at room temperature were measured
using a DDS-307 digital direct reading conductivity meter.
Cell cycle analysis and Annexin V-FITC/PI assay of apop-
totic cells were recorded on a FACScan ꢁow cytometer (BD
of the complexes, their intrinsic binding constants (K ) were
b
determined by monitoring the changes of absorbance in the
ligand transfer band with increasing concentration of CT-
DNA. K was then calculated using the following equation
b
[33–41]:
[
DNA] [DNA]
=
1
+
ꢀ
ꢁ
ꢀa − ꢀf
ꢀb − ꢀf
K ꢀ − ꢀ
b b f
1
3

Transition Metal Chemistry (2018) 43:171–183
179
Fig. 8 Distribution map of cell
apoptosis. Hela cells were incu-
bated with diꢀerent concentra-
tions of the complex 1 [a 0.1%
DMSO (control); b 0.5 μM; c
1
μM; d 2 μM] for 24 h, and
subjected to Annexin V-FITC/
PI staining, and analyzed by
ꢁ
ow cytometry
where [DNA] is the concentration of DNA in the base pairs,
IS-5500 fluorescence chemiluminescence and visible
imaging system [18].
ε is the extinction coeꢃcient observed for A
/[V], ε is
b
a
obsed
the extinction coeꢃcient of the complex when fully bound
to DNA and ε is the extinction coeꢃcient of the complex
Cytotoxicity assays
f
in free solution.
Viscosity measurements were taken with an Ubbe-
lohde viscometer maintained at a constant temperature of
The eꢀects of the test compounds on the growth of Hela,
BIU-87 and SPC-A-1 cell lines were determined with the
aid of MTT dye assays. The compounds were dissolved in
DMSO (0.1%) and diluted with RPMI 1640 to the required
concentrations prior to use. The control was prepared by
addition of culture medium (100 μL). Wells containing cul-
ture medium without cells were used as blanks. Hela, BIU-
(
28.0 ± 0.1) ꢄC in a thermostatic bath. A digital stopwatch
was used for ꢁow time, and each sample was measured ꢂve
times to obtain the average ꢁow time. Data are presented
1
/3
as (η/η ) versus binding ratio [42], where η is the viscos-
0
ity of DNA in the presence of the complex and η is the
0
4
viscosity of DNA alone [41].
87 and SPC-A-1 with a density 2 × 10 cells per well were
The cleavage of supercoiled pBR322 DNA by the com-
plexes was studied by gel electrophoresis; pBR322 DNA
precultured in 96-well microtiter plates for 48 h at 37 ꢄC, 5%
CO . Upon completion of the incubation, stock MTT dye
2
(
0.1 μg) was treated with the required complex in buꢀer
solution was added to each well. After 4 h incubation, a solu-
tion containing N,N-dimethylformamide (50%) plus sodium
dodecyl sulfate (20%) was added to solubilize the MTT
formazan. The cell viability was determined by measuring
the absorbance of each well at 490 nm using a Multiskan
ASCENT microplate reader. IC values were determined by
B, and the solution was incubated at 37 ꢄC. The samples
were analyzed by electrophoresis for 1.5 h at 85 V on a
0
.8% agarose gel in TBE (89 mM Tris–borate acid, 2 mM
EDTA, pH = 8.3). The gel was stained with 1 µg/ml eth-
idium bromide and photographed on an Alpha Innotech
50
1
3

180
Transition Metal Chemistry (2018) 43:171–183
Annexin V‑FITC/PI assay of apoptotic cells
Hela cells treated with the complexes for 24 h were deter-
mined by ꢁow cytometry using a commercially available
Annexin V-FITC/PI apoptosis detection kit. After treat-
ment, cells were harvested, washed twice in ice-cold PBS,
5
and resuspended in 500 μl of binding buꢀer at 1–5 × 10
cells/ml. The samples were incubated with 5 μl of Annexin
V-FITC and 5 μl propidium iodide in the dark for 15 min
at room temperature. Finally, samples were analyzed by
ꢁ
ow cytometry and evaluated based on the percentage of
cells for Annexin V positive.
Western blot analysis
The ERK1/2 phosphorylation assay was performed as pre-
viously described [44]. The total proteins were extracted
from cultured Hela cells treated with complex 1 (2 µM) or
U0126 (ERK pathway inhibitor) for 24 h. Western blots
were performed according to the manufacturer’s proce-
dures. Equal samples from each group were separated by
SDS polyacrylamide gel electrophoresis for 1 h and trans-
ferred onto a PVDF membrane. The membranes were
blocked in 5% BSA for 1 h at room temperature and then
incubated with primary antibody (p-ERK1/2, t-ERK1/2,
β-actin) overnight at 4 ꢄC, followed by secondary antibody
conjugation to horseradish peroxidase for 2 h. Immunob-
lots were visualized with ECL Western-blotting detection
reagents and analyzed with Image pro plus V7.0 software.
Fig. 9 Complex 1 attenuated the phosphorylation of ERK1/2. a: con-
trol; b: complex 1 (2 µM); c:U0126. Hela cells treated with the com-
plex 1 (2 µM) or U0126 (ERK pathway inhibitor) for 24 h. All data
were presented as mean ± SE. ꢅP < 0.05, versus control
plotting the percentage viability versus concentration on a
logarithmic graph and reading oꢀ the concentration at which
5
0% of cells remained viable relative to the control [29, 30].
Cell cycle analysis
Analysis of the cell cycle of both control and treated cancer
cells was determined. Using standard methods, the DNA of
cells was stained with PI, and the proportions of non-apop-
totic cells in diꢀerent phases of the cell cycle were recorded.
The cancer cells were treated with the complexes, harvested
by centrifugation at 1000g for 5 min, and then washed with
ice-cold PBS. The collected cells were ꢂxed overnight with
cold 70% ethanol and then stained with PI solution consist-
ing of 50 μg/ml PI plus 10 μg/ml RNase. After incubating
for 10 min at room temperature in the dark, ꢁuorescence-
activated cells were sorted with a FACScan ꢁow cytometer
using CellQuest 3.0.1 software.
Synthesis of 2‑(4‑nitrophenyl)‑imidazo[4,5‑f]1,10‑
phenanthroline (NPIP)
A solution of phenanthraquinone (2.5 mmol, 0.45 g),
ammonium acetate (50 mmol, 3.85 g) and p-nitrobenzal-
dehyde (3.5 mmol, 0.53 g) in glacial acetic acid 10 ml was
reꢁuxed for 6 h. The cooled deep red solution was diluted
with water (25 ml) and neutralized with ammonium
hydroxide. The mixture was ꢂltered, and the precipitate
was washed with water. The crude product was puriꢂed
by chromatography over 60–80 mesh SiO using abso-
2
lute ethanol as eluent. The solvent was removed, and the
product was collected and dried at 50 ꢄC in vacuo. Yield:
88%. Anal. Calcd.: C, 66.86; H, 3.25; N, 20.52%; Found:
Fluorescence microscopy of apoptosis assays
C, 66.64; H, 3.33; N, 20.47%. ES-MS (CH OH, m/z):
3
+
−1
This method was modiꢂed from a previous report [43].
Brieꢁy, after exposure to the complexes for 24 h, Hela cells
were washed twice with PBS and then stained with 10 μg/ml
Hoechst 33258 staining solution at 37 ꢄC for 30 min accord-
ing to the manufacturer’s instructions. Finally, the cells were
observed under a ꢁuorescence microscope.
342.0 ([M + 1] ). IR (KBr) (ν /cm ): 3096 s, 1601 m,
max
1564 m, 1516 s, 1476 s, 1455 s, 1421 s, 1397 s, 1344 s,
1296 m, 1109 m, 857 m, 806 m, 739 s, 707 m, 617 m.
1
H-NMR (500 MHz, DMSO-d6): 9.02 (d, 2H), 8.85 (d,
1
3
2H), 8.45 (d, 2H), 8.40 (d, 2H), 7.80 (q, 2H). C-NMR
1
3

Transition Metal Chemistry (2018) 43:171–183
181
(
600 MHz, DMSO-d6): 172.17, 148.33, 147.07, 147.28,
43.86, 135.93, 129.71, 126.83, 124.30, 123.30.
Synthesis of 2‑hydroxy‑1‑naphthaldehyde thiosemi‑
carbazone (hntdtsc)
1
A solution of 2-hydroxy-1-naphthaldehyde (5 mmol, 0.86 g)
in absolute ethanol (10 ml) was added dropwise to a stirred
solution of thiosemicarbazide (5 mmol, 0.455 g) in absolute
ethanol (10 ml). The mixture was stirred at 50 ꢄC for 3 h to
give a white precipitate, which was used without further
puriꢂcation. Yield: 1.03 g, 84%. Anal. Calcd.: C, 58.76; H,
Synthesis of 2‑(4‑chlorphenyl)‑imidazo[4,5‑f]1,10‑
phenanthroline) (CPIP)
CPIP was synthesized by a similar procedure as for NPIP,
but with p-chlorobenzaldehyde (3.5 mmol, 0.49 g) in place
of p-nitrobenzaldehyde. Yield: 80%. Anal. Calcd.: C, 68.99;
4.52; N, 17.13; S, 13.07%; Found: C, 58.70; H, 4.61; N,
+
H, 3.35; N, 16.94%; Found: C, 68.87; H, 3.42; N, 16.94%.
17.08; S, 13.01%. ES-MS (CH OH, m/z): 246.0 ([M + 1] ).
3
+
−1
ES-MS (CH OH, m/z): 331.1([M + 1] ). IR (KBr) (ν
/
IR (KBr) (ν /cm ): 3450 s, 3253 s, 3167 m, 3053 m,
3
max
max
−
1
cm ): 3079 s, 1686 m, 1604 m, 1563 m, 1514 m, 1475 s,
1625 s, 1593 m,1572 s, 1509 m, 1472 m, 1452 m, 1033 m,
1
1
451 s, 1420 m, 1397 m, 1352 m, 1191 m, 1102 m, 1071 m,
014 m, 955 m, 836 m, 803 m, 738 s, 704 m, 687 m, 547 m.
1240 m, 821 m, 753 m. H NMR (500 MHz, DMSO-d6):
1
11.42 (s, 1H), 10.51 (s, 1H), 9.06 (s, 1H), 7.85 and 8.25
(2br s, 1H), 8.53 (d, 1H), 7.89 (d, 1H), 7.86 (d, 1H), 7.57 (t,
1
H NMR (500 MHz, DMSO-d6):9.04 (d, 2H), 8.90 (d,
1
3
13
2
H), 8.50 (d, 2H), 8.45 (d, 2H), 7.84 (q, 2H). C-NMR
500 MHz, DMSO-d6, ppm): 149.44, 147.88, 143.13,
29.59, 129.12, 128.91, 127.85, 123.29.
1H),7.38 (t, 1H), 7.21 (d, 1H). C NMR (500 MHz, DMSO-
(
d6, ppm): 156.61, 143.03, 132.48, 131.51, 128.68, 128.06,
127.88, 123.45, 122.86, 118.33, 109.76.
1
Synthesis of [VO(hntdtsc)(NPIP)] (1)
Synthesis of 2‑(4‑methylphenyl)‑imidazo[4,5‑f]1,10‑
phenanthroline) (MEPIP)
A mixture of hntdtsc (0.5 mmol, 0.123 g) and NPIP
(
0.5 mmol, 0.171 g) in absolute methanol (100 ml) was
MEPIP was synthesized by a similar procedure as for NPIP
but with p-methyl benzaldehyde (3.5 mmol, 0.42 g) in place
of p-nitrobenzaldehyde. Yield: 83%. Anal. Calcd.: C, 73.61;
heated at 80 ꢄC under argon for 2 h. After dissolution, a solu-
tion of VO(acac) (0.5 mmol, 0.132 g) methanol (10 ml) was
2
added dropwise to this mixture. The mixture was reꢁuxed
for another 4 h to give a reddish-brown precipitate. The solid
powder was isolated from the hot solution, washed with
absolute methanol and dried in vacuo. Yield: 76%. Anal.
Calcd.: C, 57.15; H, 3.09; N, 17.20; S, 4.92%; Found: C,
H, 4.32; N, 17.17%; Found: C, 73.57; H, 4.38; N, 17.11%.
+
ES-MS (CH OH, m/z): 327.1([M + 1] ). IR (KBr) (ν
/
max
3
−
1
cm ):3081 s, 1612 m, 1561 s, 1523 m, 1483 s, 1453 m,
1
8
397 m, 1353 m, 1189 m, 1070 m, 1030 m, 957 m, 824 m,
1
03 m, 739 s, 723 m, 687 m, 646 m. H NMR (500 MHz,
57.09; H, 3.14; N, 17.16; S, 4.89%. ES-MS (CH OH, m/z):
3
+
−1
DMSO-d6): 9.05 (d, 2H), 8.94 (d, 2H), 8.19 (d, 2H), 7.84
652.1 ([M + 1] ). IR (KBr) (ν /cm ): 3355 m. 3180 m.
max
1
3
(
q, 2H), 7.44 (d, 2H), 1.913 (s, 3H). C NMR (500 MHz,
DMSO-d6, ppm): 172.05, 150.72, 147.74, 143.55, 139.31,
29.58, 127.32, 123.26, 21.01.
1615 m, 1599 s, 1538 m, 1514 s, 1454 m, 1340 s, 1193 m,
1
955 m, 856 m, 825 m, 731 m, 709 m, 498 m. H NMR
1
(500 MHz, DMSO-d6): 9.39 (s, H),9.04 (d, 2H),8.88 (m,
2
1
H),8.49 (br s, 3H), 8.19 (s, 1H),7.82 (m, 3H), 7.56 (br m,
H), 7.37 (br m, 1H), 7.31 (s, 1H), 7.19 (d, 2H), 7.09 (d,
Synthesis of 2‑(4‑hydroxylphenyl)‑imidazo[4,5‑f]
,10‑phenanthroline) (HPIP)
2H), 6.66 (s, H).
1
Synthesis of [VO(hntdtsc)(CPIP)] (2)
HPIP was synthesized by a similar procedure as for NPIP
but with p-hydroxybenzaldehyde (3.5 mmol, 0.48 g) in place
of p-nitrobenzaldehyde. Yield: 80%. Anal. Calcd.: C, 73.07;
Complex 2 was synthesized by a similar procedure as for
complex 1, but with CPIP (0.5 mmol, 0.166 g) in place of
NPIP. Yield: 70%. Anal. Calcd.: C, 58.09; H, 3.14; N, 15.30;
H, 3.87; N, 17.94%; Found: C, 72.97; H, 3.95; N, 17.86%.
+
ES-MS (CH OH, m/z): 313.0 ([M + 1] ). IR (KBr)(ν
/
max
S, 5.00%; Found: C, 57.97; H, 3.23; N, 15.22; S, 4.82%.
3
−
1
+
cm ): 3396 m, 3160 s, 1614 s, 1593 m, 1566 m, 1522 m,
ES-MS (CH OH, m/z): 639.1([M + 1] ). IR (KBr) (ν
/
max
3
−
1
1
1
6
482 s, 1455 m, 1419 m, 1401 m, 1355 m, 1279 m, 1231 m,
184 m, 1073 m, 1032 m, 960 m, 835 m, 801 m, 738 s,
cm ): 3318 m, 3175 m, 1615 m, 1598 s, 1575 m, 1538 s,
1504 s, 1454 s, 1427 m, 1334 m, 1193 m, 945 m, 821 m,
1
1
92 m. H NMR (500 MHz, DMSO-d6):9.04 (d, 2H), 8.92
738 m, 730 m, 498 m. H NMR (500 MHz, DMSO-d ): 9.36
6
(
d, 2H), 8.32 (d, 2H), 7.84 (d, 2H), 7.47 (d, 2H), 3.28 (s,
(s, H), 9.04 (d, 2H), 8.89 (m, 2H), 8.52 (br m, 3H), 8.28 (br
s,1H), 7.71-7.82(d, 3H), 7.56 (br m, 1H), 7.38 (br m, 1H),
7.326 (s, 1H), 7.20 (d, 2H), 7.01 (d, 2H), 6.649 (s, H).
1
3
H). C NMR (500 MHz, DMSO-d6, ppm): 150.63, 147.68,
42.87, 129.66, 128.12, 123.28.
1
1
3

182
Transition Metal Chemistry (2018) 43:171–183
4
.
Wang G, Wang J, Fu Y, Bai L, He M, Li B, Fu Q (2013) Sys-
temic treatment with vanadium absorbed by Coprinus comatus
promotes femoral fracture healing in streptozotocin-diabetic
rats. Biol Trace Elem Res 151:424–433
Synthesis of [VO(hntdtsc)(MEPIP)] (3)
Complex 3 was synthesized by a similar procedure as for
complex 1, but with MEPIP (0.5 mmol, 0.164 g) in place
of NPIP. Yield: 67%. Anal. Calcd.: C, 61.39; H, 3.74;
5. Reddy GNR, Kondaiah S, Setty KN, Rao RM, Ramulu JS (2012)
Synthesis, structural characterization and anti-bacterial activity
of some novel Schiꢀ base ligands and their vanadium(IV) metal
complexes. Orient J Chem 28(4):1673–1683
N, 15.80; S, 5.17%; Found: C, 61.33; H, 3.86; N, 15.73;
+
S, 5.14. %. ES-MS (CH OH, m/z): 619.1 ([M + 1] ).
3
6. Park S-J, Youn C-K, Hyun JW, You HJ (2013) The anti-obesity
eꢀect of natural vanadium-containing Jeju ground water. Biol
Trace Elem Res 151(2):294–300
−
1
IR (KBr) (ν /cm ): 3367 m, 1614 s, 1598 s, 1575 m,
max
1
1
4
538 s, 1504 m, 1482 s, 1454 m, 1427 m, 1399 m, 1362 m,
7
. Holko P, Ligeza J, Kisielewska J, Kordowiak AM, Klein A
337 m, 1191 m, 1077 m, 961 m, 824 m, 737 m, 499 m,
(
2008) The eꢀect of vanadyl sulphate (VOSO ) on autocrine
4
1
27 m. H NMR (500 MHz, DMSO-d6): 9.39 (s, H), 9.04
growth of human epithelial cancer cell lines. Pol J Pathol 59:3–8
(
d, 2H), 8.92 (m, 2H), 8.51 (br m, 3H), 8.43 (br s,1H),
8. Klein A, Holko P, Ligeza J, Kordowiak AM (2008) Sodium
orthovanadate aꢀects growth of some human epithelial cancer
cells (A549, HTB44, DU145). Folia Biol (Krakow) 56:115–121
7
.83(d, 3H), 7.55 (br m, 1H), 7.38-7.43 (br m, 3H), 7.21
(
s, 1H), 7.20 (d, 2H), 6.99 (d, 2H), 6.61 (s, H),2.14 (s, 3H).
9
.
Molinuevo MS, Cortizo AM, Etcheverry SB (2008)
Vanadium(IV) complexes inhibit adhesion, migration and col-
ony formation of UMR106 osteosarcoma cells. Cancer Chem-
other Pharmacol 61:767–773
Synthesis of [VO(hntdtsc)(HPIP)] (4)
1
0. Rodríguez-Mercado JJ, Mateos-Nava RA, Altamirano-Lozano
MA (2011) DNA damage induction in human cells exposed to
vanadium oxides in vitro. Toxicol In Vitro 25:1996–2002
Complex 4 was synthesized by a similar procedure as for
complex 1, but with HPIP (0.5 mmol, 0.156 g) in place
of NPIP. Yield: 72%. Anal. Calcd.: C, 59.81; H, 3.40; N,
11. Soares SS, Henao F, Aureliano M, Gutiérrez-Merino C (2008)
Vana-date induces necrotic death in neonatal rat cardiomyo-
cytes through mitochondrial membrane depolarization. Chem
Res Toxicol 21:607–618
1
5
5.75; S, 5.15%; Found: C, 59.71; H, 3.53; N, 15.69; S,
+
.09%. ES-MS (CH OH, m/z): 623.0([M + 1] ). IR (KBr)
3
12. Zhao Y, Ye L, Liu H, Xia Q, Zhang Y, Yang X, Wang K (2010)
Vanadium compounds induced mitochondria permeability tran-
sition pore (PTP) opening related to oxidative stress. J Inorg
Biochem 104:371–378
−
1
(
ν
/cm ): 3320 m, 3191 m, 1614 s, 1597 s, 1597 m,
max
1
1
1
575 m, 1537 m, 1482 s, 1454 m, 1428 m, 1386 m,
362 m, 1336 m, 1278 m, 1247 m, 1176 m, 1193 m,
1
3. Pessoa JC, Etcheverry SB, Gambino D (2015) Vanadium com-
pounds in medicine. Coord Chem Rev 301:24–48
1
079 m, 947 m, 824 m, 742 m, 730 m, 499 m. H NMR
(
500 MHz, DMSO-d ): 9.40 (s, H), 9.05 (d, 2H), 8.95 (d,
14. Ying P, Zeng P, Jiazheng L, Chen H, Liao X, Yang N (2015)
New oxidovanadium complexes incorporating thiosemicarba-
zones and 1, 10-phenanthroline derivatives as DNA cleavage,
potential anticancer agents, and hydroxyl radical scavenger.
Chem Biol Drug Des 86:926–937
6
2
H), 8.50 (br m, 3H), 8.40 (br s, 1H), 7.83(d, 3H), 7.55 (br
m, 1H), 7.38-7.43 (br m, 3H), 7.18-32 (br m, 2H), 6.94-
.00 (br m, 2H),3.17 (s, 1H).
7
1
5. Hwang JH, Larson RK, Abu-Omar MM (2003) Kinetics and
mechanistic studies of anticarcinogenic bisperoxovanadium(V)
compounds: ligand substitution reactions at physiologi-
cal pH and relevance to DNA interactions. Inorg Chem
42(24):7967–7977
Acknowledgements This study was supported by Natural Science
Foundation of Gansu Province, China (Grant No. 145RJZA143) and
Administration of traditional Chinese medicine Scientiꢂc Research
Funds of Gansu Province (Grant No. GZK-2017-53). We thank Dr.
Zhi-Jian Han for language editing and improvement.
16. Wu H-L, Li K, Sun T, Kou F, Jia F, Yuan J-K, Liu B, Qi B-L
(
2011) Synthesis, structure, and DNA-binding properties of
Conflict of interest The authors declare that they have no conꢁict of
manganese(II) and zinc(II) complexes with tris(N-methylbenzimi-
dazol-2-ylmethyl)amine ligand. Transition Met Chem 36(1):21–28
7. Ji LN, Zhou XH, Liu JG (2001) DNA-binding activities and poten-
tial antitumor properties of some ruthenium complexes. Coord
Chem Rev 216–217(1):513–516
interest.
1
1
8. Leelavathy L, Anbu S, Kandaswamy M et al (2009) Antibac-
terium properties of some porphyrin derivatives. Polyhedron
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