The anti-tumor activity of novel oxovanadium complexes derived from thiosemicarbazones and fluoro-phenanthroline derivatives

Article abstract of DOI:10.1016/j.poly.2016.07.021

Three oxovanadium complexes incorporating thiosemicarbazones and fluoro-phenanthroline derivatives [VO(hntdtsc)(CF₃PIP)] (1) (hntdtsc?=?2-hydroxyl-1-naphthaldehyde thiosemicarbazone, CF₃PIP?=?2-(2-trifluoromethyl phenyl)imidazole[4,5

Full text of DOI:10.1016/j.poly.2016.07.021

Polyhedron 117 (2016) 803–816
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
The anti-tumor activity of novel oxovanadium complexes derived from
thiosemicarbazones and fluoro-phenanthroline derivatives
Wen Rui a, Xuyan Tian b, Pengfei Zeng c, Weimin Liu d, Peng Ying c, Hongce Chen b, Jiazheng Lu c,
,
⇑
Ning Yang e, Hongyuan Chen b,
⇑
a Centre Laboratory, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China
b Department of Pathogen Biology and Immunology, School of Basic Course, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China
c School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China
d Department of Radiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, People’s Republic of China
e Department of Clinical pathology, the First Affiliated Hospital, Guangdong Pharmaceutical University, Guangzhou 510060, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three oxovanadium complexes incorporating thiosemicarbazones and fluoro-phenanthroline derivatives
[VO(hntdtsc)(CF3PIP)] (1) (hntdtsc = 2-hydroxyl-1-naphthaldehyde thiosemicarbazone, CF3PIP = 2-(2-tri-
fluoromethyl phenyl)imidazole[4,5-f][1,10]phenanthroline [VO(hntdtsc)(m-CF3PIP)] (2) (m-CF3PIP =
2-(3-trifluoromethyl phenyl)imidazole[4,5-f][1,10]phenanthroline), [VO(hntdtsc)(p-CF3PIP)] (3) (p-
CF3PIP = 2-(4-trifluoromethyl phenyl)imidazole[4,5-f][1,10]phenanthroline) were newly synthesized
and characterized by elemental analysis and spectroscopic techniques. Their interactions with calf-thy-
mus DNA (CT-DNA) and photocleavage properties with plasmid pBR322 DNA were investigated by a host
of analytical methods. The results suggest that these three complexes interact with CT-DNA through an
non-classical intercalative mode and can efficiently cleavage plasmid pBR322 DNA upon exposure to
ultraviolet light. In addition, they all exhibited considerable anti-proliferative activity in vitro against
human Hela, CaSki, SiHa, ECa9706, ECa109, MDA-MB-231 and MCF-7 tumor cell lines, to have an IC50 val-
Received 29 April 2016
Accepted 20 July 2016
Available online 30 July 2016
Keywords:
Oxovanadium complexes
Cytotoxicity
Apoptosis
DNA-binding
Photocleavage
ues for cytotoxicity in the low micromole range 0.31–6.15
lM, which is very close to that of cisplatin
(0.52–2.49 M). Furthermore, their antitumor mechanism has been analyzed by the cell cycle arrest,
l
and apoptosis analysis. The results showed that complexes 2 and 3 caused G0/G1 phase arrest in
ECa9706 cells, but differentiatedly induced G0/G1 and S phase arrest in ECa109 cells. And significant
apoptosis were observed in both the two tumor cell lines, which indicate these oxovanadium complexes
induce proliferative suppression of ECa9706 and ECa109 cells via the induction of apoptosis.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
diversity of some transition metal complexes bearing various kinds
of ligands such as ruthenium(II), nickel(II), cobalt(III), copper(II)
Since cisplatin has been discovered to be one of most widely
prescribed as well as a first and effective treatment for many can-
cer diagnoses, especially for testicular, pvarian, bladder, lung, and
stomach cancers [1–3]. For decades of years, more and more bio-
chemists and pharmacologists have paid their attention to a large
and zinc(II) due to their wide range of biological and physiological
activities [4–8]. Wherein, vanadium compounds are described for
participating in some biogenic vanadium systems such as vana-
dium-ionophore interaction, vanadate-dependent haloperoxidases,
vanadium-nitrogenase, vanadium-thiolate redox interaction, and
alkyne-reductase as well as isonitrile-reductase/ligase activities
of nitrogenases [9–13]. In recent years, some researchers have
been focused on interaction of vanadium compounds with DNA
[14–16]. DNA is generally regarded as the primary intracellular tar-
get of anticancer agents and hence the interaction between vana-
dium complexes with DNA can cause DNA damage in cancer
cells, blocking the division of cancer cells, and resulting in cell
death, which is related to the antitumor activity [17,18]. In this
Abbreviations: CT-DNA, calf thymus DNA; DMF, N, N-dimethylformamide;
DMSO, dimethyl sulfoxide; EDTA, N, N0-1,2-ethanediylbis[N-(carboxymethyl)]
glycine; ESI-MS, electrospray ionization mass spectra; IC50, 50% inhibition concen-
trations; MTT, 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide; PBS,
phosphate-buffered saline; PI, propidium iodide; Tm, DNA melting temperature
where half base pairs are unbound; Tris, tris(hydroxymethyl)aminomethane.
⇑
Corresponding authors. Fax: +86 20 39352186 (H. Chen).
E-mail addresses: lujia6812@163.com (J. Lu), hychen@gdpu.edu.cn (H. Chen).
http://dx.doi.org/10.1016/j.poly.2016.07.021
0277-5387/Ó 2016 Elsevier Ltd. All rights reserved.

804
W. Rui et al. / Polyhedron 117 (2016) 803–816
regard, some oxovanadium complexes containing different types of
ligands showing considerable antiproliferative activities against
various kinds of cancer cell lines [19,20].
purification unless otherwise noted. VO(acac)2 (acac = acetylaceto-
nate) and 1,10-phenanthroline were obtained from Alfa Aesar
(Beijing, China). DMSO and CHCl3 were purchased from Shanghai
Jingchun Company (Shanghai, China). Cisplatin, CT-DNA and
pBR322 DNA were purchased from Aldrich (USA). Ethidium
bromide (EB), agarose, thiosemicarbazide, salicylaldehyde and
2-Hydroxy-1-naphthaldehyde were purchased from Sigma (USA).
Tris buffer A (Tris = tris(hydroxyl-methyl) aminomethane) contain-
ing 5 mM Tris–HCl and 50 mM NaCl (pH = 7.2) was used for
absorption titration and viscosity measurements. Tris buffer B con-
taining 50 mM Tris–HCl and 18 mM NaCl (pH = 7.2) was used for
the gel electrophoresis experiments. Buffer C containing 1.5 mM
Na2HPO4, 0.5 mM NaH2PO4 and 0.25 mM Na2H2EDTA (H4EDTA =
N,N0-ethane-1,2-diylbis[N-(carboxymethyl) glycine]) (pH = 7.0)
was used for thermal denaturation. A solution of CT-DNA in buffer
A gave a ratio of UV absorbance at 260 and 280 nm of 1.8–1.9:1,
indicating that the DNA was sufficiently free of protein [35].
The DNA concentration per nucleotide was determined by
absorption spectroscopy using the molar absorption coefficient
(6600 Mꢀ1 cmꢀ1) at 260 nm [36]. RPMI 1640 medium was pur-
chased from Hyclone (Logan, USA) for the tumor cells culture.
Trypsin, fetal calf serum and Annexin V-FITC/PI apoptosis detection
Kit were purchased from GIBCO Company (USA). Hoechst 33342
staining solution was purchased from Beyotime Institute of
Biotechnology (China). MTT (3-(4,5-dimethylthiazoyl-2-yl)2,
5-diphenyltetrazoliumbromide) and Rhodamine 123 (2-(6-
Amino-3-imino-3H-xanthen-9-yl)benzoic acid methyl ester) were
purchased from Sigma Company (USA). Double distilled water
was used for preparing various buffers, and solutions of com-
pounds were freshly prepared 2 h prior to biochemical evaluation.
In addition, there are large amounts of reports on transition
metal complexes bearing Schiff base ligands exhibiting some fan-
tastic pharmacological effects especially its application as potential
clinical therapeutics agents towards cancer [21,22]. On one hand,
thiosemicarbazones were usually acted as primary ligands for
many metals owing to its general physiological and pharmacolog-
ical activities including antibacterial, antifungal, antitumor, and
antiviral activities [23–25]. On the other hand, some oxovanadium
complexes containing thiosemicarbazones were capable to demon-
strate both in vitro antibacterial and antiproliferative properties
[26,27] as well as intense interaction with DNA, thus causing
DNA damage in cancer cells, which leading it as new potential
DNA-targeted antitumor drugs [28,29]. Moreover, 1,10-phenan-
throline derivatives were often served as auxiliary ligands due to
their idiosyncratic capacity of coordinating with metal ions by
N-alkyl and N-benzyl group as well as a wide variety of flexible
applications such as in fields of photovoltaic devices and solar
fuels, selective Typanosoma cruzi growth inhibitors, the treatment
of cancer diseases [30–32], and sequence-selective DNA-binding
and cleavage agents for DNA itself [16,27,33].
In our previous work, we have investigated the DNA-binding
affinities of three oxovanadium complexes, [VO(hntdtsc)(HPIP)],
[VO(hntdtsc)(m-HPIP)] and [VO(hntdtsc)(p-HPIP)] towards calf-
thymus DNA(CT-DNA) as well as their antitumor activities against
human cervical cancer Hela, CaSki and SiHa, leukemia ECa9706 and
ECa109, esophagus carcinoma MDA-MB-231, and MCF-7 cell lines
in vitro. They exhibited intense interaction with CT-DNA through a
non-classical intercalation mode, and exerted a certain antiprolif-
erative and cytotoxic activities against selected cultured tumor
cells [34]. Interestingly, previously we reported several oxovana-
dium complexes incorporating Schiff base substituted with elec-
tro-withdrawing group (–Cl) presented stronger DNA-binding
affinities and higher cytotoxic activities than that of substituted
with electron-donating group (–N(Et)2) [16]. It occurs to us that
the introduction of electro-withdrawing group in ligands probably
enhance DNA-binding capacity and antitumor properties.
2.2. Physical measurements
Microanalysis (C, H, and N) was carried out with a PerkinElmer
240Q elemental analyzer. UV–Visible spectra were recorded on a
Shimadzu UV-3101 PC spectrophotometer at room temperature.
Fluorescence measurements were performed on a Perkin–Elmer
Lambda 55 spectrofluorophotometer. Infrared spectra (IR) were
recorded on KBr disks a on a Bomem FTIR model MB102 instru-
ment. Electrospray Ionization Mass Spectra (ESI-MS) were
recorded on an LCQ system (Finnigan MAT, USA) using methanol
as mobile phase. 1H and 13C NMR spectra were recorded on a
Varian-500 spectrometer. All chemical shifts are given relative to
tetramethylsilane (TMS). Molar conductivities in DMF (10ꢀ3 M)
solution at room temperature were measured using a DDS-307
digital direct reading conductivity meter.
Thus, in the present work, we have designed and synthesized
three oxovanadium complexes incorporating thiosemicarbazones
and fluoro-phenanthroline derivatives [VO(hntdtsc)(CF3PIP)] (1)
(hntdtsc = 2-hydroxyl-1-naphthaldehyde thiosemicarbazone, CF3-
PIP = 2-(2-trifluoromethyl phenyl)imidazolen[1,10]phenanthroline
[VO(hntdtsc)(m-CF3PIP)] (2) (m-CF3PIP = 2-(3-trifluoromethyl phe-
nyl)imidazole[4,5-f][1,10]phenanthroline),
[VO(hntdtsc)(p-CF3-
PIP)] (3) (p-CF3PIP = 2-(4-trifluoromethyl phenyl)imidazole[4,5-f]
[1,10]phenanthroline) (see Scheme 1), and characterized by ele-
mental analysis, IR, ESI-MS, 1H NMR and 13C NMR. Their interac-
tions with calf-thymus DNA (CT-DNA) were studied by UV–Vis
absorption titration, fluorescence spectra, viscosity measurements
and thermal denaturation. The photocleavage properties with plas-
mid pBR322 DNA were investigated and in vitro cytotoxicity
against human Hela, CaSki, SiHa, ECa9706, ECa109, MDA-MB-231
and MCF-7 tumor cell lines were evaluated by MTT assay. In addi-
tion, their antitumor mechanism has been analyzed by cell cycle
arrest, apoptosis, and Annexin V-FITC/PI assay.
2.3. Synthesis and characterization
2.3.1. Synthesis of phenanthroline-based ligands: CF3PIP, m-CF3PIP
and p-CF3PIP
CF3PIP was synthesized through a modification of a previously
reported procedure [37]. A mixture of 2-(Trifluoromethyl)ben-
zaldehyde (0.69 mL, 5 mmol) and ammonium acetate (7.70 g,
0.1 mol) was added into a stirring solution of 1,10-phenanthro-
line-5,6-dione (0.99 g, 5 mmol) in 60 mL of glacial acetic acid,
and the mixture was continuously stirred at 60 °C for 6 h. Then
the deep red solution was cooled to room temperature and diluted
with 100 mL distilled water. A grayish yellow precipitate was
obtained by neutralization with ammonium hydroxide. Then the
mixture was filtered and washed with water for three times. The
crude solid power was purified by chromatography over 60–80
mesh SiO2 using absolute ethanol as eluent. The solvent was
removed and the products were collected, and dried in vacuo.
2. Materials and methods
2.1. Materials
All reagents and solvents used in the synthesis and physical
measurements were commercially available and without further

W. Rui et al. / Polyhedron 117 (2016) 803–816
805
OH
N
6
1
5
4
HN
S
R
1
2'
3'
O
O
N
10
9
N
1'
4'
2
V
NH
VO(acac)2
5
7
2
R
N
N
3
15
11
N
6
N
H
MeOH, 5h, reflux
+
8
1
5
4
S
6' 5'
R
2
R
1
3'
12
14
NH
N
2'
2
10
2
N
13
1'
4'
15
3
7
R
3
11
N
H
9
N
8
6' 5'
R
2
12
14
13
Scheme 1. Synthesis and structure of three kinds of oxidovanadium(IV) complexes [VO(hntdtsc) (o-CF3PIP) (1), R1 = CF3, R2 = H, R3 = H; VO(hntdtsc) (m-CF3PIP) (2), R1 = H,
R2 = CF3, R3 = H; VO(hntdtsc) (p-CF3PIP) (3), R1 = H, R2 = H, R3 = CF3].
m-CF3PIP and p-CF3PIP were prepared by a similar procedure
with the complex CF3PIP, with 3-(Trifluoromethyl)benzaldehyde
(0.69 mL, 5 mmol) and 4-(Trifluoromethyl)benzaldehyde (0.69 mL,
5 mmol) in place of 2-(Trifluoromethyl) benzaldehyde (0.69 mL,
5 mmol) respectively, and a pink-yellow precipitate were obtained.
CF3PIP: Yield: 61%. Anal. Found: C, 65.48; H, 3.14; N, 15.35; Calc.
for C20H11F3N4: C, 65.93; H, 3.04; N, 15.38. 1H NMR (DMSO-d6,
500 MHz) d: 9.03 (s, 2H, J = 8.7 Hz, ArH), 9.02 (s, 2H, J = 8.6 Hz,
ArH), 8.47 (m, 1H, J = 8.3 Hz, ArH), 8.45 (m, 1H, J = 8.2 Hz, ArH),
7.96 (m, 1H, J = 8.1 Hz, ArH), 7.82 (br, 3H, J = 7.6 Hz, ArH), 3.32 (s,
1H, –NH). 13C NMR (DMSO-d6, 500 MHz) d: 148.9 C(15), 148.0 C
(1,12), 143.7 C(5,14), 133.8 C(30,50), 129.7 C(10), 129.5 C(3), 129.1
C(7), 126.6 C(20,60), 125.9 C(40), 125.8 C(–CF3), 124.8 C(8),
124.7 C(4), 123.4 C(9), 123.3 C(10), 122.6 C(6,13). ESI-MS:
(CH3OH): m/z 365.0 ([M+H]+), 387.0 ([M+Na]+). IR (KBr disk):
2.3.2. Synthesis of [VO(hntdtsc)(CF3PIP)] (1)
This complex was synthesized by a mixture of CF3PIP (0.364 g,
1 mmol) and hntdtsc (0.245 g, 1 mmol) in absolute CH3OH
(60 mL) was heated at 80 °C under argon for 0.5 h. After dissolu-
tion, an aqueous solution (10 mL) of 1.0 mmol (0.265 g) quantity
of VO(acac)2 was added dropwise to this mixture and reflux for
another 4 h, then poured with equal-volume cold distilled water,
yellow-brown precipitate was formed. The solid power was puri-
fied by recrystallization in CH3OH/CHCl3 (3:1), and dried in vacuo.
Yield: 62%. Anal. Found: C, 56.89; H, 3.02; N, 14.60; Calc. for
C
32H20F3N7O2SV: C, 56.98; H, 2.99; N, 14.54. 1H NMR (DMSO-d6,
500 MHz) d: 12.65 (s, 1H, –NH), 9.40 (s, 1H, CH@N), 8.49 (m, 2H,
–ArH), 8.35 (m, 4H, J = 8.5 Hz, ArH), 8.38–8.42 (br m, 6H,
J = 8.4 Hz, ArH), 8.18 and 7.84 (2br s, 1H each NH2), 8.12 (d, 2H,
J = 8.1 Hz, ArH), 7.64 (d, 2H, ArH). ESI-MS (CH3OH): m/z: 675.0
m
(cmꢀ1) = 3406.8 (s, N–H), 3078.2 (m, C–H), 1606.7 (s),
([M+H]+), 673.0 ([MꢀH]ꢀ). IR (KBr disk):
m
(cmꢀ1) = 3338.4(s),
C@CAH),
CAO), 1086.1
CAS), 731.6 (m,
1551.8 (m), 1483.0 (s, C@C), 1315.3 (vs, C–F). Conductance
3275.1 (s, –NH2), 3031.9 (m), 1573.2 (s), 1493.8 (s, m
(X
ꢀ1 cm2 molꢀ1): 9.2.
1612.9 (vs,
(m, CAN), 954.0 (s,
VAN). Conductance (X
m
HC@N), 1310.5 cmꢀ1
V@O), 821.8 (s), 750.9 (m,
ꢀ1 cm2 molꢀ1): 11.3.
(mCAF), 1193.7 (m, m
m-CF3PIP: Yield: 58%. Anal. Found: C, 65.67; H, 3.13; N, 15.42;
m
m
m
Calc. for
C
20H11F3N4: C, 65.93; H, 3.04; N, 15.38. 1H NMR
m
(DMSO-d6, 500 MHz) d: 8.99 (m, 2H, J = 8.8 Hz, ArH), 8.83 (d, 2H,
J = 8.7 Hz, ArH), 8.56 (m, 2H, J = 8.2 Hz, ArH), 7.78 (m, 1H,
J = 8.0 Hz, ArH), 7.75 (m, 3H, J = 7.5 Hz, ArH). 3.38 (s, 1H, –NH).
13C NMR (DMSO-d6, 500 MHz) d: 148.2 C(15), 148.0 C(1,12),
143.6 C(5,14), 135.3 C(30,50), 132.5 C(10), 130.3 C(3), 129.8 C(7),
128.2 C(2’,6’), 127.9 C(40), 127.0 C(-CF3), 126.8 C(8), 126.7 C(4),
126.0 C(9), 124.8 C(10), 123.8 C(6,13). ESI-MS: (CH3OH): m/z
2.3.3. Synthesis of [VO(hntdtsc)(m-CF3PIP)] (2)
This complex was obtained by a similar procedure as for the
complex 1, with m-CF3PIP (0.364 g, 1 mmol) in place of CF3PIP.
Yield: 68%. Anal. Found: C, 56.94; H, 2.98; N, 14.62; Calcd for
C
32H20F3N7O2SV: C, 56.98; H, 2.99; N, 14.54. 1H NMR (DMSO-d6,
500 MHz) d: 11.89 (s, 1H, –NH), 9.24 (s, 1H, CH@N), 8.59 (m, 2H,
–ArH), 8.36 (m, 4H, J = 8.5 Hz, ArH), 8.36–8.44 (br m, 6H,
J = 8.4 Hz, ArH), 8.15 and 7.87 (2br s, 1H each NH2), 8.10 (d, 2H,
J = 8.1 Hz, ArH), 7.84 (d, 2H, ArH). ESI-MS (CH3OH): m/z: 675.0
365.0 ([M+H]+), 387.0 ([M+Na]+). IR (KBr disk):
(s, N–H), 3065.8, (m, C–H), 1607.2 (m), 1566.7 (s), 1548.5 (m,
C@C), 1312.2 (vs, C–F). Conductance (
ꢀ1 cm2 molꢀ1): 9.6.
m
(cmꢀ1) = 3241.6
X
([M+H]+), 673.0 ([MꢀH]ꢀ). IR (KBr disk):
m
(cmꢀ1) = 3441.4(s),
CACAH),
CAO), 1088.0
CAS), 733.2 (m,
p-CF3PIP: Yield: 66%. Anal. Found: C, 65.78; H, 3.24; N, 15.32;
Calc. for C20H11F3N4: C, 65.93; H, 3.04; N, 15.38. 1H NMR (DMSO-
d6, 500 MHz) d: 9.07 (d, 2H, J = 8.6 Hz, ArH), 8.84 (d, 2H,
J = 8.4 Hz, ArH), 8.04 (d, 1H, J = 8.1 Hz, ArH), 7.97 (d, 1H,
J = 7.8 Hz, ArH), 7.91 (d, 1H, J = 7.9 Hz, ArH), 7.83 (t, 3H,
J = 7.7 Hz, ArH). 3.40 (s, 1H, –NH). 13C NMR (DMSO-d6, 500 MHz)
d: 149.3 C(15), 147.7 C(1,12), 143.6 C(5,14), 131.6 C(30,50), 131.4
C(10), 130.1 C(3), 129.9 C(7), 129.6 C(20,60), 128.4 C(40), 125.5
C(–CF3), 125.2 C(8), 123.2 C(40), 123.1 C(4), 122.4 C(9), 122.3 C
(10), 121.6 C(6,13). ESI-MS: (CH3OH): m/z 365.0 ([M+H]+), 387.0
3275.1 (s, –NH2), 3030.9 (m), 1575.4 (s), 1493.6 (s,
m
1615.9 (vs,
(m, CAN), 950.4 (s,
VAN). Conductance (
m
HCAN), 1312.3 cmꢀ1
VAO), 821.8 (s), 754.5 (m,
ꢀ1 cm2 molꢀ1): 10.2.
(mCAF), 1190.7 (m, m
m
m
X
m
m
2.3.4. Synthesis of [VO(hntdtsc)(p-CF3PIP)] (3)
This complex was synthesized by a similar procedure as for the
complex 1, with p-CH3PIP (0.364 g, 1 mmol) in place of CF3PIP.
Yield: 53%. Anal. Found: C, 57.05; H, 3.03; N, 14.55; Calc. for
([M+Na]+). IR (KBr disk):
(w, C–H), 1608.2 (s), 1565.5 (m, C@C), 1326.5 (vs, C–F). Conduc-
tance (
ꢀ1 cm2 molꢀ1): 8.6.
m
(cmꢀ1) = 3371.2 (s, N–H), 3065.8
C
32H20F3N7O2SV: C, 56.98; H, 2.99; N, 14.54. 1H NMR (DMSO-d6,
500 MHz) d: 12.35 (s, 1H, –NH), 9.54 (s, 1H, CH@N), 8.39 (m, 2H,
–ArH), 8.30 (m, 4H, J = 8.0 Hz, ArH), 8.34–8.40 (br m, 6H,
X

806
W. Rui et al. / Polyhedron 117 (2016) 803–816
J = 8.3 Hz, ArH), 8.18 and 7.84 (2br s, 1H each NH2), 8.06 (d, 2H,
J = 8.1 Hz, ArH), 7.68 (d, 2H, ArH). ESI-MS (CH3OH): m/z: 675.0
The data are presented as (A ꢀ A0)/(Af ꢀ A0) versus T [41], where A,
A0 and Af are the observed, the initial, and the final absorbance at
260 nm, respectively.
([M+H]+), 673.0 ([MꢀH]ꢀ). IR (KBr disk):
m
(cmꢀ1) = 3335.4(s),
CACAH),
CAO), 1086.1
CAS), 731.6 (m,
3278.1 (s, –NH2), 3031.6 (m), 15703.2 (s), 1491.8 (s,
m
1618.5 (vs,
(m, CAN), 955.3 (s,
VAN). Conductance (
m
HCAN), 1323.0 cmꢀ1
VAO), 820.8 (s), 755.9 (m,
ꢀ1 cm2 molꢀ1): 11.8.
(m
CAF), 1193.7 (m,
m
2.5. DNA cleavage assays
m
m
X
m
m
The cleavage activity of supercoiled pBR322 DNA by the oxo-
vanadium complexes was investigated by adopting agarose gel
2.4. DNA-binding experiments
electrophoresis experiment. pBR322 DNA (0.1 lg) was treated with
the complexes in Tris buffer B in different concentrations by gradi-
ent, and the solutions were exposure to ultraviolet light at 365 nm
for 1 h. The samples were analyzed by electrophoresis for 2 h at
80 V in TBE (89 mM Tris-borate acid, 2 mM EDTA, pH 8.3) contain-
Interaction of the oxovanadium complexes with CT-DNA was
studied in Tris buffer A. The absorption titration experiment was
performed at room temperature to determine the DNA binding
affinity between the DNA and the complexes. A mixed concentra-
ing 0.8% agarose gel. The gel was stained with 0.1 lg/mL DuRed
tion of the oxovanadium complexes 3.0 mL (20
l
M) was taken in
nucleic acid gel stain and photographed under UV light on an Alpha
Innotech IS-5500 fluorescence chemiluminescence and visible
imaging system.
a cuvette to which the DNA stock solutions were added. The oxo-
vanadium-DNA solutions were incubated at room temperature
for 5 min before the absorption spectra were recorded. Titrations
were continued until there was no further change noticed in the
spectra. In order to further elucidate the binding strength of the
complex, the intrinsic binding constant Kb with CT-DNA was
obtained by monitoring the change in the absorbance of the ligand
transfer band with increasing amounts of DNA. Kb was then calcu-
lated using the following equation [38].
2.6. Cell culture
The cell lines Hela, CaSki, SiHa (human cervical carcinoma),
ECa9706, ECa109 (human esophageal carcinoma), and MDA-MB-
231, MCF-7 (human breast carcinoma) were purchased from Amer-
ican Type Culture Collection. Cells were grown in a humidified
atmosphere containing 5% CO2 at 37 °C in RPMI-1640 medium sup-
½DNAꢁ
½DNAꢁ
1
¼
þ
ð1Þ
plemented with 10% fetal bovine serum, 100 lg/mL streptomycin
ea
ꢀ
ef eb
ꢀ
ef Kbðeb
ꢀ
ef
Þ
and 100 units/mL penicillin. And subcultures were performed with
0.05% trypsin-0.02% EDTA in phosphate-buffered saline (PBS;
Gibco BRL Co., USA).
where [DNA] is the concentration of DNA in the base pairs, and ea, ef
and eb refer to the corresponding apparent absorption coefficient
Aobsd/[Vanadium], the extinction coefficient for the free oxovana-
dium complex and the extinction coefficient for the oxovanadium
complex in the fully bound form, respectively. In plots of [DNA]/
(ea – ef) versus [DNA], Kb is obtained by the ratio of the slope to
the intercept.
Fluorescence titration experiments were also conducted by
adopting a procedure analogous to that used in UV–Vis absorbtion
titration experiment. Constant concentration of oxovanadium
2.7. Cytotoxicity evaluation
Cell viability was determined by using standard MTT (3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-terazolium bromide) dye
assay as described previously, which is based on the ability of
the viable cells to reduce a soluble yellow tetrazolium salt to blue
formazan crystals [42]. Cells were seeded in 96-well microassay
culture plates at a density of 2 ꢂ 104 cells per well and incubated
at 37 °C in a 5% CO2 atmosphere for 24 h. Various concentrations
of the complexes tested ranging 10ꢀ6–10ꢀ4 mol/L were then added
to the wells to achieve final concentration and incubated for
another 48 h under the same conditions. Control wells were pre-
complexes (20 lM) was employed to which increments of DNA
solution was added. Meanwhile, Emission quenching experiments
were carried out at room temperature by the addition of micro liter
aliquots of oxovanadium complexes (0.01 mM) to the 3 mL of
325 lM calf thymus DNA-bound ethidium bromide (1.3 lM) in
pared by addition of culture medium (100
culture medium without cells were used as blanks. Upon comple-
tion of the incubation, then stock MTT dye solution (20 L, 5 mg/
lL). Wells containing
Tris buffer A. The quenching constant Ksv was determined by using
Stern–Volmer equation [39].
l
F0=F ¼ 1 þ Ksv½Qꢁ
ð2Þ
mL) was added to each well. After 4 h incubation, a solution con-
taining N,N-dimethylformanmide (50%) and sodium dodecyl sul-
fate (20%) was added to solubilize the MTT formazan.
Absorbance at 490 nm of the dissolved solution was measured on
a Multiskan SSCENT microplate reader. The IC50 values were deter-
mined by plotting the percentage viability versus concentration on
a logarithmic graph and reading off the concentration at which 50%
of cells remain viable relative to the control. Each experiment was
repeated at least three times to get the mean values.
where F0 and F are the fluorescence intensities in the absence and
presence of the quencher respectively, Q is the concentration of
the quencher, Ksv is a linear Stern–Volmer quenching constant
and is obtained from the slope.
Viscosity measurements were carried out with an Ubbelohde
viscometer maintained at
a
constant temperature of
(28.0 0.1) °C in a thermostatic bath. Flow time was measured
with a digital stopwatch, and each sample was measured at least
thrice to calculate the average flow time. Date are presented as
1/3
(g/g0)
versus binding ratio [40], where
g
is the viscosity of
2.8. Measurement of apoptosis by A-VFIT/PI analysis
DNA in the presence of complex while g0 is the viscosity of DNA
alone.
ECa9706 and ECa109 cell lines treated with complexes 2 or 3
were determined by flow cytometry using a commercially avail-
able Annexin V-FITC/PI apoptosis detection Kit for 24 h and 48 h
respectively [43]. Briefly, cells were harvested after the target 1
Thermal denaturation studies were carried out with Shimadzu
UV-3101 PC spectrophotometer equipped with a Peltier tempera-
ture-controlling programmer ( 0.1 °C). The melting temperature
(Tm) was taken as the mid-point of the hyperchromic transition.
The melting curves were obtained by measuring the absorbance
treatment with different concentrations (0, 5, 10 and 20
24 h, and washed with PBS, then resuspended in 100
L 1ꢂ binding
buffer. Then 5 L of annexin V-FITC and 5 L of propidium iodide
(PI) were added to the cells (100 L) and incubated for 1 h at room
temperature in the dark. After 400
L 1ꢂ binding buffer was added
lM) for
l
at 260 nm for solutions of CT-DNA (80
presence of oxovanadium complex [20
temperature was scanned from 50 to 90 °C at a speed of 5 °C minꢀ1
l
M) in the absence and
l
l
l
M] as a function of the
l
.
l

W. Rui et al. / Polyhedron 117 (2016) 803–816
807
Table 1
into each tube, the stained cells were analyzed by flow cytometry.
Molar conductivity data of oxovanadium complexes and the ligands.
The rate of cell apoptosis was analyzed.
Compounds
X
ꢀ1 cm2 molꢀ1
CF3PIP
m-CF3PIP
9.2
9.6
2.9. Cell cycle analysis
p-CF3PIP
8.6
VO(hntdtsc)(CF3PIP)
VO(hntdtsc)(m-CF3PIP)
VO(hntdtsc)(p-CF3PIP)
11.3
10.2
11.8
Flow cytometry was generally used to evaluate DNA contents
and cell analysis [44]. Using standard methods, the DNA of cells
were stained with PI, and the proportion of non-apoptotic cells
in different phases of the cell cycle was recorded. The human eso-
phageal carcinoma cell lines ECa9706 and ECa109 were treated
with the complexes 2 and 3 for 48 h, respectively. Cells were then
harvested and washed with ice-cold PBS, centrifuged at 1000 ꢂ g
for 5 min, resuspended in 1 mL of PBS and fixed by dropwise addi-
tion of 9 mL precooled 70% ethanol overnight at ꢀ20 °C, and then
analysis, ESI-MS, IR and 1H NMR data of all the complexes are in
good agreement with the expected structures.
The molar conductance values of phenanthroline-based ligands
and complexes 1–3 in DMF (1 mM) are shown in Table 1. The con-
ductive properties indicate that both oxovanadium complexes and
the corresponding ligands are nonelectrolytes.
stained with PI solution consisting of 50 lg/mL PI, 10 lg/mL RNase
in the dark at room temperature for 10 min. The distribution of the
cell cycle was analyzed by using a FACSCan flow cytometer and
evaluated based on the percentage of cells for Annexin V positive.
3.2. DNA-binding properties
3.2.1. UV absorption spectroscopy studies
3. Results and disscussion
It is generally accepted that DNA is the primary pharmacologi-
cal target of many metal based antitumor agents. The binding
affinities of DNA-metal complexes have been a clue of paramount
importance for understanding the mechanism of effective metal-
based chemotherapeutic drugs [46]. Herein, the interaction
between complexes 1–3 and CT-DNA was investigated by UV–Vis
absorption spectroscopy. The electronic absorption spectra of com-
3.1. Synthesis and characterization
The ligands CF3PIP, m-CF3PIP, and p-CF3PIP were synthesized by
condensing 1,10-phenanthroline-5,6-dionea and trifluoromethyl
benzaldehyde in the presence of ammonium acetate using glacial
acetic acid as solvent. The oxovanadium complexes were prepared
by refluxing corresponding ligands with VO(acac)2 in the appropri-
ate mole ratios in absolute methanol. The desired complexes were
purified to crystallize out yellowish-brown crystals in CH3OH/
CHCl3 (3:1). The complexes are soluble in DMSO, DMF, MeOH
and EtOH.
plexes 1–3 are characterized by intense p–
p⁄ transition of aromatic
rings in the absence and presence of increasing concentration of
CT-DNA, which are shown in Fig. 1. The maximal peak at ca.
265 nm is described to intraligand
p–
p⁄ charge transfer. As the
concentration of CT-DNA increasing, obvious hypochromism and
apparent bathochromism are observed in the ultraviolet titration
process. The percentage hypochromism of complex 1 at 268 nm,
2 at 265 nm and 3 at 270 nm upon binding to DNA was 52.4%,
68.6%, and 74.3% respectively. The appreciable bathochromism
for complexes 1–3 reached at 3 nm, 6.5 nm and 5.5 nm, respec-
tively. These spectral characteristics obviously suggest that these
complexes interact with DNA most likely through a mode that
involves a stacking interaction between the aromatic chromophore
and the base pairs of DNA.
In order to compare quantitatively the DNA-binding affinities of
these complexes, the intrinsic binding constant Kb was determined
from the changes in absorbance in the ligand change transfer
bands with increasing amounts of CT-DNA. The values of Kb were
calculated as well using the Eq. (1), reaching (4.68 0.01) ꢂ
105 Mꢀ1, (7.60 0.01) ꢂ 105 Mꢀ1 and (8.39 0.01) ꢂ 105 Mꢀ1 for
complexes 1, 2 and 3, respectively. The Kb values are proximate
to the classical intercalator (e.g. EB-DNA ꢃ106) [47] as well as
higher than those of analogous DNA-intercalative oxovanadium
complexes reported previously [48,49]. The results suggest that
the interaction of complexes 1–3 with DNA is a strong intercalative
mode.
Interestingly, from a comparison of the DNA-binding activities
of complexes 1, 2 and 3, Kb values increase in the order 1 < 2 < 3,
this may due to the substituents of trifluoromethyl phenyl intro-
duced on the phenanthroline ring of complexes 1–3, which acts
as an electron-drawing substituent on the intercalative ligand
and thus gives stronger stacking interactions with DNA for com-
plex 1–3, leading to the intercalation into base pairs of CT-DNA
of the complexes may be altered, thus resulting in higher Kb. Mean-
while, the electronic effects of substituted group at the different
locations introduced on the aromatic ring of phenanthroline-based
ligands may make differences in the DNA-binding affinities.
The results also indicates an –CF3 group substituted on the
In the region of IR spectra of phenanthroline-based ligands
show absorption at ca. 3300 cmꢀ1 (imidazole N–H), 3030 cmꢀ1
(m (m ,
C@CAH), 1460–1618 cmꢀ1 C@C), 740 cmꢀ1 700 cmꢀ1 (dCAH),
1285–1323 cmꢀ1
(mCAF), respectively. In parallel, IR spectra absorp-
tion of complexes 1–3 observed at ca. 3310 cmꢀ1 (imidazole N–H),
3280 cmꢀ1 and 3340 cmꢀ1 is assigned to (–NH2), 3030 cmꢀ1
(
m
C@CAH), 1490–1580 cmꢀ1
(m
C@C), 1612–16,196 cmꢀ1
(m
ACH@N),
CAS), 731–737 cmꢀ1 for
VAN), which indicates both two mixed ligands are coordinated
to vanadium. The strong (
V@O) band at 950–956 cmꢀ1 observed
1310–1323 cmꢀ1 CAF), 810–313 cmꢀ1
(m (m
(m
m
at oxovanadium complexes, which is not present in the spectrum
of free ligands could be cleared identified for the formulation of
the complex [45].
Ultraviolet absorption spectrum of complexes 1–3 show an
intense band at ca. 265 nm assignable to
matic rings of phenanthroline-based ligands, while the less intense
band at ca. 340 nm is typical of charge transfer between ligand and
metal (ligand-to-metal charge transfer) [27]. The obtained spectral
data indicates that the Schiff base bonded through the phenolate
oxygen, imine nitrogen and thiolate sulfur atoms leaving the thio-
methyl as the pendant group. This implies that these complexes
bear the central V(IV) atom with coordination numbers 5 in a
square-pyramidal geometry [16,27,45].
p–
p⁄ transition of aro-
In the 1H NMR spectra of phenanthroline-based ligands showed
peaks of aromatic hydrogen (ArH), imidazole secondary amine
(NH) proton. However, in the 1H NMR spectra of complexes, the
peaks of imine (CH@N) proton and primary amine (–NH2) were
observed, which affirmed that the Schiff base ligand was coordi-
nated to vanadium.
The ESI-MS spectrum of three complexes 1–3 in DMSO show
the molecular ion peaks at m/z 675.0, which is assigned to
[M+H]+, and at m/z 643.0 is assigned to ([MꢀH]ꢀ). Elemental

808
W. Rui et al. / Polyhedron 117 (2016) 803–816
Fig. 1. Absorption spectral of the complexes 1 (a), 2 (b) and 3 (c) in Tris–HCl buffer A upon increasing amounts of CT-DNA. [V] = 20
lM, [DNA] = (0–100)
lM. Arrow shows the
decreasing absorbance upon increasing CT-DNA concentrations. Inset: Plots of [DNA]/(ea-ef) versus [DNA] for the titration of [V] with CT-DNA.
para-position of the aromatic exhibits stronger DNA-binding affin-
ity than substituted on the ortho-position and meta-position.
were carried out as shown in Fig. 3. As can be seen in Fig. 3, the flu-
orescence emission intensities of complexes 1–3 at ca. 580 nm
(350 nm excitation) decreased with the increasing complex con-
centrations, which suggested that 1, 2 and 3 could displace DNA-
bound EB and bind to CT-DNA at the intercalation sites with almost
the same affinity [52]. The relative binding intensity to CT-DNA
was determined by the Eq. (2), the quchening constant Ksv at
25 °C was calculated to be 4.21 ꢂ 104 Mꢀ1 for 1, 5.61 ꢂ 104 Mꢀ1
for 2, and 1.07 ꢂ 105 Mꢀ1 for 3, respectively. It may be due to oxo-
vanadium complexes interacting with DNA through intercalation
binding, so releasing some free EB from the EB-DNA complex.
Moreover, the number of binding site of oxovanadium com-
plexes with CT-DNA were calculated by adopting the following
equation: lg(F0 ꢀ F)/F = lgKA + nlg[Q] [53], as shown in Fig. 4, where
F0 and F are the fluorescence intensities in the absence and pres-
ence of complex, respectively, KA is apparent binding constant, n
is the number of binding site of complex with DNA, and [Q] is
the concentration of complex to that of DNA. KA was calculated
from the slope of plot lg(F0 ꢀ F)/F versus lg[Q]. The apparent bind-
ing constant KA of 1, 2 and 3 were calculated to 6.68 ꢂ 104,
2.83 ꢂ 105 and 1.17 ꢂ 106 respectively. And the number of binding
site n were obtained as 1.06, 1.14 and 1.23 for complexes 1, 2 and 3
respectively.
3.2.2. Fluorescence spectroscopy studies
The interaction of the complexes (20 lM) with CT-DNA was
investigated using fluorescence emission titration experiment in
the Tris buffer A at room temperature. As shown in Fig. 2, upon
addition of CT-DNA, the fluorescence emission intensities of the
complexes 1–3 is found to depend on DNA concentration, their
emission intensities increase by about 41.67% at 688 nm, 48.55%
at 657 nm and 65.38% at 676 nm times larger than those in the
absence of CT-DNA and saturates at a [DNA]/[V] ratio of 20:1,
implying that complexes can strongly interact with DNA and be
protected by the DNA efficiently. Since the hydrophobic environ-
ments inside the DNA helix reduces the accessibility of solvent
water molecules to the complex and the complex mobility is
restricted at the binding site, it leads to decrease of relaxation
and hence fluorescence intensity increases.
In addition, fluorescence quenching experiments were also per-
formed to further investigate interaction strength of oxovanadium
complexes as well as the number of binding site with CT-DNA. EB
is one of the most sensitive fluorescence probes, its fluorescence
intensity can be greatly enhanced in the presence of DNA due to
its strong intercalation between the adjacent DNA base pairs
[50]. A complex’s competitive binding to EB-bound DNA can
reduce the fluorescent intensity due to displacement of bound EB
from DNA [51]. The interaction of complexes 1–3 with CT-DNA
3.2.3. Viscosity measurements
The measurement of viscosity of DNA is regarded as the most
critical and the least ambigious test of DNA-binding model in

W. Rui et al. / Polyhedron 117 (2016) 803–816
809
Fig. 2. Emission spectra of 1 (a), 2 (b) and 3 (c) in Tris–HCl buffer A in the absence and presence of CT-DNA. [V] = 20
lM. Arrows show the increasing intensity with increasing
concentrations of DNA.
solution in the absence of X-ray crystallographic structural data
[54]. It is well-known that a classical intercalation model leads to
an apparent increase in viscosity of DNA solution due to base-pairs
are pushed apart and hence an increase in overall DNA length. In
contrast, a partial, non-classical intercalation of complexes could
bend (or kink) the DNA helix and reduce its effective length and,
concomitantly, its viscosity [38,55].
To investigate further the DNA-binding mode of oxovanadium
complexes 1–3, viscosity measurements on solutions of CT-DNA
incubated with the complexes were performed. The effects of com-
plexes 1–3 on the viscosity of CT-DNA are shown in Fig. 5. With
amounts of the complexes increasing, the relative viscosity of
DNA increases continuously to some extend. The results thus
provide strong evidence for the interaction of complexes 1–3 with
CT-DNA by intercalation modes. Moreover, the large increase in the
relative viscosity revealed that 2 and 3 are better intercalators than
1, which is consistent with our foregoing hypothesis that electronic
effects of introduction of an –CF3 group substituted on the phenan-
throline-based ligands exhibits stronger DNA-binding affinity.
and offer information about the interaction strength of complexes
with DNA. With the temperature in the solution rising, the double-
stranded DNA will gradually dissociate to single strands and gener-
ate a hyperchromic effect on absorption spectra of DNA bases.
Thus, the melting temperature Tm, which is defined as the temper-
ature where half of the total base pairs are unbounded, is usually
introduced. Generally, Tm will increase considerable when inter-
calative binding occurs, since intercalation of the complex into
DNA base pairs causes stabilization of base stacking and hence
raises the melting temperature of the double-stranded DNA
[16,29,34].
The melting curves of CT-DNA in the absence and presence of
complexes 1, 2 and 3 are illustrated in Fig. 6. As shown in Fig. 6,
the Tm of CT-DNA in the absence of the complex is 60.8 0.2 °C,
simultaneously, when the concentration ratio [V]/[DNA] reached
at 1:4, Tm values in the presence of the complexes 1–3 are observed
at 69.5 0.2 °C, 67.9 0.2 °C and 64.6 0.2 °C, respectively. A com-
parison of the
DTm values of the three complexes (8.7 °C, 7.1 °C and
3.8 °C, respectively) was agreement with those classical intercala-
tors [15,16,28], which provided strong evidence for their binding
with DNA by intercalation modes.
3.2.4. Thermal denaturation studies
DNA melting experiments are generally applied to investigate
the extent of intercalation, which were carried out by monitoring
the intensity of DNA bases at 260 nm at different temperatures
in the absence and presence of oxovanadium complex. Thermal
behaviors of DNA in the presence of complexes can give insight
into their conformational changes when the temperature is raised
The results reveal that oxovanadium complexes in collaboration
with thiosemicarbazones and imidazole[4,5-f][1,10]phenanthro-
line-based ligands substituted with –CF3 group exhibit appreciable
DNA intercalative properties, and complex 3 exhibit strongest
DNA-binding affinity than 1 and 2, it is quite consistent with their
binding abilities with CT-DNA.

810
W. Rui et al. / Polyhedron 117 (2016) 803–816
Fig. 3. Emission quenching spectral traces of complexes 1, 2 and 3 using 325 lM calf thymus DNA ethidium bromide (EB) 1.3 lM at different concentrations in 5 mM Tris–
HCl buffer (pH = 7.2) at 25 °C. The inset shows the plots for the complexes 1–3.
3.2.5. DNA photocleavage activities
plasmid DNA occurred in the presence of hydroxyl radical scav-
enger DMSO (lane 7, lane 12 in Figs. 7 and 8, respectively), suggest-
ing that ꢄOH radicals is most likely to be the reactive species on
account of the oxidation of VO2+ in the presence of H2O2 for the
cleavage reaction [16,34,48]. Under comparative conditions, the
cleavage ability follows the order of 3 > 2 ꢅ 1. The result indicates
that oxovanadium complexes 1–3 can efficiently cleavage plasmid
DNA, which is consistent with the degree of their intrinsic binding
constant (Kb) with DNA.
It is reported that many antitumor agents can interact with DNA
and cause DNA strand scission and photocleavage activity of
various metal complexes have been reported [48,49]. When
circular plasmid DNA is subject to electrophoresis, relatively fast
migration will be observed for the intact supercoiled form (form
I). If scission occurs on one strand (nicking), the supercoil will relax
to generate a slower moving open circular form (form II). If both
strands are cleaved, a linear form (form III) will be generated
[34,54]. For this reason, we have also studied the capacity of the
present oxovanadium complexes 1–3 to cleavage plasmid DNA
(pBR 322 DNA) by agarose gel electrophoresis experiments as
shown in Figs. 7 and 8, Gel electrophoresis separation of pBR322
DNA was studied after incubation with complexes and explosure
to ultraviolet light at 365 nm for 1 h.
3.2.6. In vitro cytotoxicity assays
The cytotoxicity of three oxovanadium complexes 1–3 in vitro
against tumor cells was evaluated by MTT assay with seven human
tumor cell lines: Hela, CaSki, SiHa, ECa7906, ECa109, MDA-MB-231
and MCF-7 cells, cisplatin was compared as positive control group
As is illustrated in Figs. 7 and 8, it is clear to figure out the cleav-
age activity of plasmid pBR322 DNA after incubation with different
concentrations of V-complexes at 37 °C after explosure to ultravi-
olet light at 365 nm for 1 h. The nicked form II can barely observed
for H2O2 alone in the absence of the complexes (lane 2 in Figs. 7
and 8). However, in the presence of complexes 1, 2 and 3 with
30 mM H2O2, obvious cleavage of pBR322 DNA were observed from
the formation of form II, and cleaving activity tends to increase
gradually with the concentration of the complexes increasing rang-
in various concentrations (0.1325–40 lM). The inhibitory percent-
age against growth of cancer cells was determined. The cell viabil-
ities (%) obtained with continuous exposure for 48 h are depicted
in Fig. 9. As can be seen in Fig. 9, cell viability is concentration-
dependent with increasing concentrations of complexes 1–3. The
corresponding IC50 values were calculated after 48 h incubation
and listed in Table 2.
It is evident from Table 2 that three oxovanadium complexes all
exhibit high cytotoxcities against tested cultured cell lines, espe-
cially towards leukemia (ECa9706 and ECa109) cells and esopha-
gus carcinoma (MDA-MB-231, MCF-7) cells, with IC50 values
ing from 15
tion, under the same experimental conditions with lane 4, 0.02 M
-Histidine was added thus works as a singlet oxygen quencher
lM to 60 lM (lane 3, 4 and 5 in Figs. 7 and 8). In addi-
L
ranging from 0.31 to 4.68 lM. Interestingly, these oxovanadium
(lane 6, lane 11 in Figs. 7 and 8, respectively), resulting in slightly
inhibition of the cleavage of DNA. Meanwhile, no cleavage of
complexes exhibited highly cytotoxic activities against selected
cultured cells compared with cisplatin. Among the tested cells,

W. Rui et al. / Polyhedron 117 (2016) 803–816
811
Fig. 4. Bilogarithmic graph of lg(F0 ꢀ F)/F vs. lg[Q], with concentrations of complexes 1–3 ranging from 2
lM to 20
lM at 25 °C. Linear fitting equation was obtained by Origin
softwave.
Fig. 5. Effect of increasing amounts of complexes 1 (N), 2 (d) and 3 (j) on the
relative viscosity of CT-DNA at (28.0 0.1) °C, [DNA] = 0.2 mM.
Fig. 6. Thermal denaturation of CT-DNA in the absence and presence of complexes
1 (d), 2 (N) and 3 (.), [V] = 20 M, [DNA] = 80 M.
l
l
to some degree. Previously, we reported oxovanadium complexes
incorporating ligands substituted with hydroxyl group (–OH)
showed a certain antiproliferative activities against ECa9706 and
ECa109 cell lines, which IC50 values ranging from 11.6 to
complex 2 showed similar cytotoxicities to complex 3, however,
comparing with 1, both 2 and 3 displayed higher inhibitory
effect than 1, especially against ECa9706 and ECa109 cells (IC50
0.31–2.55
lM). It is suggested that the oxovanadium complexes
89.2 lM. Thus, it is assumed that the cytotoxicity activities of
might selectively inhibit the growth of different tumor cell lines

812
W. Rui et al. / Polyhedron 117 (2016) 803–816
Fig. 7. Cleavage of pBR322 DNA by complexes 1 and 2 (15–60
l
M) in the absence and presence of H2O2 (30 mM) in buffer B (pH 7.2). Lane 1, DNA control; lane 2, DNA + H2O2;
lane 3, DNA + 1 (15
(30 M) + H2O2 + DMSO (2
Histidine (0.02 M); lane 12, DNA + 2 (30
l
M) + H2O2; lane 4, DNA + 1 (30
L); lane 8, DNA + 2 (15
M) + H2O2 + DMSO (2
l
l
M) + H2O2; lane 5, DNA + 1 (60
M) + H2O2; lane 9, DNA + 2 (30
L).
l
M) + H2O2; lane 6, DNA + 1 (30
l
l
M) + H2O2
M) + H2O2; lane 11, DNA + 2 (30
+
L
-Histidine (0.02 M); lane 7, DNA + 1
M) + H2O2
l
l
lM) + H2O2; lane 10, DNA + 2 (60
l
+ L-
l
l
oxovanadium complexes may be associated with electronic effects
of substituted groups in phenanthroline-based ligands, and could
be significantly enhanced by introduction of strong electronic-
withdrawing trifluoromethyl (–CF3). This is consistent with its
binding abilities with CT-DNA, indicating that the antitumor abili-
ties of the oxidovanadium complexes may be closely related to
their DNA binding mode.
Fig. 8. Cleavage of pBR322 DNA by complex 3 (15–60
presence of H2O2 (30 mM) in buffer B (pH 7.2). Lane 1, DNA control; lane 2, DNA
+ H2O2; lane 3, DNA + 3 (15 M) + H2O2; lane 4, DNA + 3 (30 M) + H2O2; lane 5,
DNA + 3 (60 M) + H2O2; lane 6, DNA + 3 (30 M) + H2O2 -Histidine (0.02 M);
lane 7, DNA + 3 (30 M) + H2O2 + DMSO (2 L).
lM) in the absence and
l
l
3.2.7. Apoptosis assement by Annexin A-VFIT/PI analysis
Cell apoptosis, also known as programmed cell death (PCD), is a
gene controlled active suicide process that is associated with
l
l
+ L
l
l
Fig. 9. Effects of complex 1, 2 and 3 on the growth of CaSki, Hela, SiHa, MDA-MB-231, MCF-7, ECa9706 and ECa109 cells after incubation for 48 h. The last two are effects of
Cisplatin on cells after incubation for 24 h and 48 h respectively. The percentage of cell inhibition was quantified by the MTT assay. Data represent the mean SD of at least
three independent experiments.

W. Rui et al. / Polyhedron 117 (2016) 803–816
813
Table 2
The IC50 values for complexes 1, 2 and 3 against selected tumor cell lines for 48 h.
Compounds
IC50 (lM)
CaSki
SiHa
Hela
MDA-MB-231
MCF-7
ECa9706
ECa109
Cisplatin
–
–
>10
>10
6.15 0.02
–
>10
3.76 0.03
3.32 0.02
2.49 0.01
0.52 0.04
<0.31
2.25 0.01
>10
3.58 0.01
0.47 0.02
0.53 0.01
>10
2.55 0.02
1.16 0.02
0.52 0.01
4.94 0.03
<0.31
Complex 1
Complex 2
Complex 3
1.45 0.03
<0.31
<0.31
<0.31
2.04 0.03
Fig. 10. Cell apoptosis analysis of ECa9706 (a) and ECa109 (b) cells after 48 h of treatment with complexes 2 and 3 respectively. Induction of apoptosis by complexes 2 and 3
in ECa9706 (a) and ECa109 cells evaluated by Annexin-V-FITC/PI staining. Data represent the mean SD of at least three independent experiments. Quadrants represent the
following populations: non-apoptotic viable cells in the lower left quadrant (Annexin-V-negative/PI-negative);early apoptotic cells in the lower right quadrant (Annexin-V-
positive/PI-negative); late apoptotic or necrotic cells in the upper right quadrant (Annexin-V-positive/PI-positive); and mechanically injured cells in the upper left quadrant
(Annexin-V-negative/PI-positive). Data shown are representative of three separate experiments. P < 0.01 vs. control.
evolution of organisms, maintenance of balance of interior envi-
ronment, which is generally regarded as a determining factor to
explore potential mechanisms towards cancer [56]. Annexin V-
FITC/PI staining was usually used to detect Apoptotic cells. The
staining single-positive for Annexin V-FITC mostly reflected early
apoptotic cells and the staining single positive for PI mostly
reflected necrotic cells, so cells that were stained doublepositive
could be either necrotic or apoptotic cells [57]. According to
Table 2, complexes 2 and 3 exhibit highest cytotoxicity against
ECa9706 and ECa109 cells, thus, to further investigate the whether
the antiproliferative effect of the oxovanadium(IV) complexes on
tumor cells is associated with cell apoptosis, apoptosis assessment
were inverstigated by Annexin A-VFIT/PI analysis.
37.7% 1.1% for 48 h, respectively. Analogously, in Fig. 10(b), the
percentage of early and apoptotic cells treated with complexes 2
and 3 towards ECa109 cells was 3.25% 0.8%, 2.29% 0.8% and
44.4% 1.5%, 32.1% 1.4% for 48 h, respectively. The results illus-
trated that complexes 2 and 3 could effectively induce apoptosis
in ECa9706 and ECa109 cells in a time-dependent manner. Analysis
of apoptosis cells rate by flow cytometry is presented as shown in
Fig. 11.
3.2.8. Cell cycle analysis by flow cytometry
The cell cycle of ECa9706 and ECa109 cells were determined by
flow cytometer in PI (propidium iodide) stained cells. The cell cycle
is a series of events that cells go through to reproduce and create
more cells, which consists of four distinct phases: G0/G1 phase, S
phase (synthesis), G2 phase (collectively known as interphase)
and M phase (mitosis). The G1 stage is the stage when accumula-
tions of energy and material for DNA replication occur. The S stage
is the stage of DNA replication. The G2 stage is the stage of
As shown in Fig. 10(a), Annexin V-FITC/PI staining data showed
that percentage of early treated with complexes 2 and 3 towards
ECa9706 cells was 1.92% 0.8%, 1.92% 0.8% for 24 h, and
7.12% 0.9%, 6.41% 0.9% for 48 h, respectively. The percentage
of and late apoptotic cells for 2 and 3 was 33.4% 1.1 % and

814
W. Rui et al. / Polyhedron 117 (2016) 803–816
Fig. 11. Cell cycle analysis of ECa9706 and ECa109 cells treatment with 10 lM of complexes 2 and 3 for 12 and 24 h, respectively. Percentages of cells in the G0/G1, S, and G2/
M phases are presented in a histogram (a, b) and (c, d) correspond to the following figure e and f). Data shown are representative of three separate experiments. P < 0.01 vs.
control.
preparation for the M stage. The M stage is ‘‘mitosis”, and is when
nuclear and cytoplasmic division occurs [58]. As shown in Fig. 11,
cytometrie profiles of the PI-stained DNA show an accumulation of
cells in the G0/G1 phase of the ECa9706 cell cycle treated with
against several tumor cells compared with that of cisplatin, indi-
cating that they are potential anticancer agents. The flow cytomet-
ric analysis showed that complexes 2 and 3 induced cell cycle
arrest of ECa9706 cells at G0/G1 phase, and induced cell cycle
arrest of ECa109 cells at G0/G1 and phases. And significant apopto-
sis were observed in both the two tumor cell lines, which indicate
that these oxovanadium complexes induce proliferative suppres-
sion of ECa9706 and ECa109 cells via the induction of apoptosis.
It is also assumed that the DNA-binding affinities of oxovanadium
complexes probably associated with electronic effects of substi-
tuted groups in phenanthroline-based ligands, and introduction
of strong electron-withdrawing group trifluoromethyl (–CF3) at
para-position and meta-position could enhance antitumor activi-
ties than at ortho-position to a greater degree.
complexes 2 and 3 at 10 lM for 12 h and 24 h respectively, accom-
panied by a decrease in the percentage at S and G2/M phases.
These date suggest that complexes 2 and 3 can inhibit the cell
growth in ECa9706 is G0/G1 arrest. In the G0/G1 region of
Fig. 11, the distribution of the cells increased from 66.12% to
83.23% for complex 2 and 79.95% for complex 3, which indicates
that complexes 2 and 3 can effectively induce apoptosis in
ECa9706 cells. Likewise, complexes 2 and 3 inhibit the cell growth
in ECa109 is both G0/G1 and S phases, the distribution of the cells
increased from 73.40% to 82.38% at G0/G1 phase and 4.85% to
11.36% at S phase for complex 2, 78.84% at G0/G1 phase and
12.30% at S phase for complex 3, respectively, which indicates that
the two complexes could delay or inhibit cell cycle progression
through both G0/G1 and S phases in ECa109 cells.
Conflict of interest
The authors have declared that there is no conflict of interest.
4. Conclusion
Acknowledgements
Three novel oxovanadium complexes [VO(hntdtsc)(CF3PIP)] (1),
[VO(hntdtsc)(m-CF3PIP)] (2) and [VO(hntdtsc)(p-CF3PIP)] (3) were
synthesized and characterized. The DNA-binding behaviors show
that the three complexes interact with CT-DNA through intercala-
tive mode and the DNA-binding affinity follows the order 3 > 2 > 1.
Meanwhile, they can efficiently cleavage pBR322 plasmid DNA in
the presence of H2O2 probably via generating hydroxyl radical. In
addition, they exhibit high antiproliferative activities in vitro
We gratefully acknowledge financial support for this work by
the Science and Technology Research Project of Guangdong Pro-
vince (No. 2012B031800431 and 2015A020211036), the Science
and Technology Research Project of Guangzhou City (No.
2014J4100058), P. R. China and Cultivation of Natural Science Joint
Fund of the First Affiliated Hospital and the Scientific & Technical
Department of Guangdong Pharmaceutical University (No. 2014-
36).

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