Synthesis, structure, protein binding, and cytotoxicity of zinc(II) complexes with tridentate NNO Schiff-base ligands

Article abstract of DOI:10.1080/00958972.2014.903328

Mononuclear and trinuclear zinc(II) complexes (1 and 2) with tridentate NNO Schiff-base ligands (HL¹ = N-2-pyridiylmethylidene-4-chloro-2- hydroxy-phenylamine, HL² = N-2-pyridiylmethylidene-2-hydroxy-5- chloro-phenylamine) have been

Full text of DOI:10.1080/00958972.2014.903328

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Synthesis, structure, protein binding,
and cytotoxicity of zinc(II) complexes
with tridentate NNO Schiff-base ligands
Mei Liab, Yi Goub, Yao Zhangb, Feng Yangb & Hong Liangab
a College of Chemistry and Chemical Engineering, Central South
University, Changsha, China
b State Key Laboratory Cultivation Base for the Chemistry and
Molecular Engineering of Medicinal Resources, Ministry of Science
and Technology of China, Guangxi Normal University, Guilin, China
Accepted author version posted online: 12 Mar 2014.Published
online: 08 Apr 2014.
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To cite this article: Mei Li, Yi Gou, Yao Zhang, Feng Yang & Hong Liang (2014) Synthesis, structure,
protein binding, and cytotoxicity of zinc(II) complexes with tridentate NNO Schiff-base ligands,
Journal of Coordination Chemistry, 67:6, 929-942, DOI: 10.1080/00958972.2014.903328
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Journal of Coordination Chemistry, 2014
Vol. 67, No. 6, 929–942, http://dx.doi.org/10.1080/00958972.2014.903328
Synthesis, structure, protein binding, and cytotoxicity of zinc(II)
complexes with tridentate NNO Schiff-base ligands
MEI LI†‡, YI GOU‡, YAO ZHANG‡, FENG YANG*‡ and HONG LIANG*†‡
†College of Chemistry and Chemical Engineering, Central South University, Changsha, China
‡State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal
Resources, Ministry of Science and Technology of China, Guangxi Normal University, Guilin, China
(Received 11 November 2013; accepted 18 February 2014)
Mononuclear and trinuclear zinc(II) complexes (1 and 2) with tridentate NNO Schiff-base ligands
(HL1 = N-2-pyridiylmethylidene-4-chloro-2-hydroxy-phenylamine, HL2 = N-2-pyridiylmethylidene-
2-hydroxy-5-chloro-phenylamine) have been synthesized and characterized by single-crystal X-ray
diffraction and elemental analysis. The binding properties of zinc(II) complexes with calf thymus
DNA (CT-DNA) and HSA were investigated by UV–visible, fluorescence, and circular dichroism
spectra. The zinc(II) complexes bind significantly to CT-DNA by intercalation and bind to protein
HSA through a static quenching mechanism. The in vitro cytotoxicity of the complexes on human
tumor cells lines was assessed by 3-(4,5-dimathylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide,
Hoechst 33258 staining experiments.
Keywords: Zinc(II) complexes; NNO Schiff-base ligand; DNA and HSA binding; Anticancer
activity
1. Introduction
Cisplatin and related platinum-based drugs are far from ideal anticancer agents due to their
serious side effects, such as general toxicity and acquired drug resistance [1–4]. The main
cellular target for platinum drugs is genomic DNA, in case of cisplatin the major antitumor
activity results from intrastrand cross-links and the formation of DNA kinks [5]. To circumvent
*Corresponding authors. Email: jxyangfeng@gmail.com (F. Yang); hliang@mailbox.gxnu.edu.cn (H. Liang)
© 2014 Taylor & Francis

930
M. Li et al.
these problems, developing more effective, less toxic, target specific, and preferably non-cova-
lently bound anticancer drugs would be of importance. Zinc is the second most abundant
transition metal in living organisms after iron and the only metal that appears in all enzyme
classes and is essential for growth and development of various biological systems due to its
redox inertness, low toxicity, hard Lewis acid properties, and bioavailability. Hence, efficient
zinc(II)-based hydrolytic cleavage agents may be candidates with better applications at the
cellular level and constitute a remarkable achievement particularly for biomedical applications
[6, 7]. However, it is a pity that very little data exist on the cytotoxicity of zinc-based
compounds [8–13].
NNO Schiff-base ligands derived from pyridine-2-aldehyde and substituted 2-aminophe-
nol have rich coordination chemistry and their metal complexes show unique properties as
solid materials [14–16]. Few studies on biological activities of their metal complexes have
been reported [17]. To widen the scope of investigations on new biologically active pharma-
ceuticals of zinc(II) complexes, in this work, two new zinc complexes containing tridentate
NNO Schiff-base ligands have been obtained, and the complex-DNA binding, complex-
HSA binding, and cytotoxicity in vitro have been studied.
2. Experimental
2.1. Materials and physical measurements
All chemicals were commercially available and used without purification. 2-Amino-4-chloro-
phenol, 2-amino-5-chlorophenol, and pyridine-2-aldehyde were purchased from Alfa Aesar
Chemicals Co. (USA). Ethidium bromide (EB), HSA, calf thymus DNA (CT-DNA),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and Hoechst 33258
were purchased from Sigma (USA). Fetal bovine serum (FBS) and RPMI 1640 were obtained
from Hyclone (USA). The Tris–HCl buffer solution was prepared with triply-distilled water.
Elemental analyses (C, H, and N) were carried out on a Perkin-Elmer Series II CHNS/O
2400 elemental analyzer. Infrared spectra were obtained on a Perkin-Elmer FT-IR spectrom-
eter. Electrospray ionization mass spectrum (ESI-MS) was recorded on a Bruker HCT Elec-
trospray Ionization Mass Spectrometer. UV–visible (UV–vis) absorption spectra were
performed on a Varian Cary100 UV–vis spectrophotometer. Fluorescence measurements
were obtained using a Shimadzu RF-5301/PC spectrofluorophotometer. The circular
dichroic spectra of CT-DNA were performed on a JASCO J-810 automatic recording spec-
tropolarimeter operating at 25 °C.
2.2. Synthesis of HL1 and HL2
2.2.1. HL1. N-2-pyridiylmethylidene-4-chloro-2-hydroxy-phenylamine was prepared by
refluxing equimolar amounts of pyridine-2-carbaldehyde (0.96 mL, 10 mM) and 2-amino-5-
chlorophenol (1.44 g, 10 mM) in anhydrous methanol (60 mL) for 4 h with stirring. After
solvent was removed under reduced pressure, the product was recrystallized from 30 mL
n-hexane-CH2Cl2 (v : v = 1 : 1) and yellow crystals were collected from the filtrate. Yield:
1.65 g, 70.21%. FT-IR bands (KBr, cm−1): 1628, (C=N); 3100–3000, (–OH). ESI-MS (in
DMSO): m/z 231.7 (Calcd 232.67). Elemental analysis (%): Calcd for C12H9ClN2O
(Mr = 232.67): C, 61.89; H, 3.87; N, 12.03. Found: C, 61.70; H, 3.81; N, 11.93.

Zinc(II) Schiff-base complexes
931
2.2.2. HL2. N-2-pyridiylmethylidene-2-hydroxy-5-chlorophenylamine was prepared accord-
ing to a previously reported procedure [18] and yellow crystals were collected from the filtrate.
2.3. Synthesis of 1 and 2
2.3.1. Complex 1. To a yellow solution of HL1 for 1 (0.046 g, 0.2 mM) in MeOH (12 mL)
was added a methanolic solution (8 mL) of Zn(OAc)2·H2O (0.037 g, 0.2 mM) dropwise
with stirring for 2 h. The red reaction mixture was filtered and left to stand at room tempera-
ture for slow evaporation, and the red crystals of complexes suitable for X-ray analysis
were harvested after several days. Yield: 0.056 g, 68.29%. Elemental analysis (%): Calcd
for C24H16Cl2N4O2Zn (Mr = 528.70): C, 54.47; H, 3.03; N, 10.59. Found: C, 54.59; H,
3.01; N, 10.43.
2.3.2. Complex 2. Complex 2 was synthesized according to the above-described procedure
using HL2 instead of HL1. The red crystals suitable for X-ray analysis were harvested after
several days. Yield: 0.043 g, 52%. Elemental analysis (%): Calcd for C32H32Cl2N4O12Zn3
(Mr = 899.87): C, 42.67; H, 3.56; N, 6.22. Found: C, 42.59; H, 3.51; N, 6.23.
The outline of the synthesis of the two complexes is given in scheme 1.
2.4. X-ray crystallography
X-ray diffraction data for the complexes were collected on a Bruker Smart Apex II CCD
diffractometer equipped with graphite-monochromated Mo Kα radiation (λ = 0.71073) at
room temperature. The structures were solved with direct methods and refined using
SHELX-97 [19, 20]. The non-hydrogen atoms were located in successive difference Fourier
synthesis. The final refinement was performed by full-matrix least squares methods with
anisotropic thermal parameters for non-hydrogen atoms on F2. The hydrogens were added
theoretically, riding on the concerned atoms.
2.5. DNA-binding experiments
DNA absorbs at 260 nm; protein absorbs at 280 nm. The concentration of CT-DNA was
determined from its absorption intensity at 260 nm, taking the molar extinction coefficient
of 6600 M−1 cm−1 [21]. The absorbance at 260 nm (A260) and at 280 nm (A280) for CT-
DNA was measured to check its purity. The ratio A260/A280 was 1.8–1.9 in Tris–HCl/
NaCl buffer (pH 7.2, 5 mM Tris–HCl, 50 mM NaCl), indicating that the DNA was suffi-
ciently free of protein [22]. The 2 × 10−3 M CT-DNA stock solution was stored at 4 °C for
no more than 4 days before use. Complexes 1 and 2 were prepared as 2 × 10−3 M DMSO
stock solutions and diluted by Tris–HCl/NaCl buffer for DNA-binding studies. Absorption
titration experiments were carried out with fixed concentrations of 1 and 2 (2 × 10−5 M)
with varying concentrations of CT-DNA (0–140 μM). After the mixture was incubated for
5 min, absorption spectra were recorded.
EB displacement experiments were determined with an EB-bound CT-DNA solution by
the fluorescence spectral method. Thus, aliquots of a solution of 1 and 2 were added to a
solution of EB-bound CT-DNA ([EB] = 2 × 10−5 M, [CT-DNA] = 8 × 10−5 M) in Tris–HCl/

932
M. Li et al.
NaCl buffer. The corresponding fluorescence intensities at 602 nm (excitation at 350 nm)
were obtained.
The circular dichroism (CD) spectra of CT-DNA in the absence and presence of 1 and 2
were collected in Tris–HCl buffer (pH 7.2) containing 50 mM NaCl from 350 to 230 nm at
25 °C.
2.6. Cell culture
The tumor cell line HepG2 (human liver hepatocellular carcinoma cells) was obtained from
the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in a
humidified atmosphere containing 5% CO2 at 37 °C in RPMI-1640 medium supplemented
with 100 μg mL−1 streptomycin and 100 units mL−1 penicillin, 10% FBS.
2.7. Cytotoxicity evaluation
Cell viability was calculated in growth inhibition assays with exposure to drugs [23]. Cells
were seeded at a density of 1 × 104 cells per well in 96-well plates and cultured for 24 h.
Different concentrations of the complexes between 0 and 160 μM were added and the plates
were incubated in a 5% CO2 humidified atmosphere for 48 h. Then, 10 μL of 5 mg mL−1
MTT in phosphate buffered saline (PBS; pH 7.40) was added to each well, and cells were
incubated at 37 °C for an additional 4 h. The addition of 100 μL of DMSO dissolved formed
formazan crystals and the absorbance at 570 nm was recorded by an enzyme-linked immu-
nosorbent assay reader. The cytotoxicities of the complexes against tumor cell lines were
determined according to IC50 values which were expressed by plotting the percentage via-
bility versus concentration on a logarithmic graph and reading off the concentration at
which 50% of cells were viable relative to the control.
2.8. Hoechst 33258 staining
The nuclear morphology of cells was employed to detect early apoptosis, late apopto-
sis, and necrosis. HepG2 cells were seeded in a 6-well plate at a density of 5 × 105
well and incubated for 24 h. Then, various concentrations of complexes (0, 10, 20, and
40 μM) were added to the media and cells were cultured for another 24 h. Then, cells
were washed with PBS and stained with Hoechst 33258 for 5 min at 25 °C. After a
final wash in PBS, samples were measured with the aid of EPI fluorescence
microscopy (Nikon MF30 LED, Japan).
2.9. Interaction with HSA
The interactions of 1 and 2 with HSA were carried out using fluorescence spectra. Var-
ious concentrations of complexes (0–12 × 10−5 M) were added to a solution containing
1 × 10−6 M HSA and 20 mM NaCl/Tris–HCl buffer (pH 7.4). Fluorescence spectra were
obtained by recording the emission spectra (260–500 nm) corresponding to excitation at
280 nm.

Zinc(II) Schiff-base complexes
933
3. Results and discussion
3.1. Description of the structures
Complexes 1 and 2 were prepared by the same procedure (scheme 1) except using
2-amino-4-chlorophenol instead of 2-amino-5-chlorophenol. Complexes 1 and 2 are red,
but the yields are different, 1 (68.29%) > 2 (52%). The replacement of 4-chloro by 5-chloro
in the ligands resulted in change in reactivity of the ligands and led to different yields [24].
To characterize the bis-Schiff-base Zn(II) complexes and study their geometries with differ-
ent anions, X-ray crystal structures of the bis-Schiff-base complexes were determined. The
X-ray crystal structures of 1 and 2 are shown in figures 1 and 2. The details of crystallo-
graphic data and structure refinement parameters are summarized in table 1. Selected bond
distances and angles for the structures of 1 and 2 are listed in table 2. X-ray diffraction
reveals that 1 crystallizes in a triclinic system with space group P-1, while 2 crystallizes in
a monoclinic system with space group P21/n.
Complex 1 has two meridional Schiff-base ligands with pseudo-octahedral coordination
geometry [figure 1(A)]. The coordination geometry about Zn(II) is rather irregular, and the
relative deviations of bond lengths and angles from the ideal values (table 2) also support
the distorted geometry of the zinc(II) center. The two tridentate Schiff-base ligands are
almost planar and perpendicular (ca. 88.95°) to one another [figure 1(B)]. The bond lengths
with average Zn1–O at 2.086 Å and Zn1–N at 2.221 Å are consistent with other complexes
[25, 26]. Trans angles (within the meridional ligands) for the Zn(II) complex are O2–Zn1–
N6 = 151.774° and O4–Zn1–N7 = 152.624(2)° with an interligand trans angle of 165.349°
for N3–Zn1–N5.
In contrast to 1, 2 displays a trinuclear molecule with a central Zn(II) lying on a center of
inversion. The trinuclear complex is built up of two mononuclear ZnL moieties, which are
linked through bridging carboxylates and μ2-groups to the central Zn [figure 2(A)]. The
coordination geometry around the terminal Zn centers may be regarded as distorted square
pyramidal, described by the τ value 0.34 [27], and at the lower end of the range (0.32–0.45)
for similar Zn(II) complexes [28–30]. However, the central zinc resides on a center of sym-
metry and is surrounded by O6 donor set composed of four oxygens (Zn2–O3/O3i,
2.218 Å; Zn2–O5/O5i, 2.118 Å) of four bridging OAc- (syn–syn mode) and two phenolic
oxygens (Zn2–O1/O1i, 2.071 Å) of two Schiff bases to form an octahedral structure (Zn1–
Zn2–Zn1i = 180.00°). The distance between the central zinc ion (Zn2) and the two terminal
zincs is 3.3555 Å, which is in the expected range for a carboxylate bridged metal–metal
Figure 1. (A) Crystal structure of 1. Hydrogens are omitted for clarity. (B) The angle of two planar tridentate
Schiff-base ligands in 1.

934
M. Li et al.
Figure 2. (A) Crystal structure of 2. Hydrogens and H2O are omitted for clarity (symmetry code: 1 − x, 1 − y, 1 − z).
(B) Schematic representation of the 6-connected α–Po topological net of 2.
Table 1. Crystallographic data and structure refinement parameters for 1 and 2.
1
2
Empirical formula
Formula weight
C
24H16Cl2N4O2Zn
C32H32Cl2N4O12Zn3
899.87
528.70
Temperature/K
296(2)
293(2)
Crystal system
Space group
Triclinic
P-1
Monoclinic
P21/n
a/Å
b/Å
c/Å
α/°
8.008(4)
12.084(7)
13.133(7)
115.113(8)
100.271(9)
99.708(9)
1088.9(10)
2
8.9794(9)
20.3939(16)
10.2034(12)
90.00
106.109(11)
90.00
1795.1(3)
2
1.665
β/°
γ/°
Volume/Å3
Z
ρ
Calcd/mg mm−3
1.612
1.404
μ/mm−1
2.203
F (0 0 0)
552
881
Crystal size/mm3
0.35 × 0.25 × 0.11
1.78–26.37°
0.20 × 0.08 × 0.05
5.72–52.74°
2Θ range for data collection
Index ranges
−10 ≤ h ≤ 10, −15 ≤ k ≤ 15, −16 ≤ l ≤ 16 −11 ≤ h ≤ 11, −25 ≤ k ≤ 24, −12 ≤ l ≤ 10
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I > 2σ (I)]
Final R indexes [all data]
12,422
4429 [R(int) = 0.0915]
4429/0/298
9606
3670 [R(int) = 0.0844]
3670/3/249
1.015
R1 = 0.0702, wR2 = 0.1134
R1 = 0.1393, wR2 = 0.1431
0.709/−0.679
1.150
R1 = 0.0781, wR2 = 0.1931
R1 = 0.1300, wR2 = 0.2226
Largest diff. peak/hole/e Å−3 1.404/−1.665
entity. These Zn–Zn distances are longer than that (3.3420 Å) observed for the linear trinu-
clear zinc(II) complex, [Zn3L2(CH3COO)4] [30], where L denotes [C5H4NC(CH3)
=NC6H4(O–)]. There exist three weak interactions: O–H···O hydrogen bond interactions
involving the uncoordinated H2O and a coordinated carboxyl oxygen (O3) [O6···O3 =
3.089 Å]; O–H···Cl hydrogen bond interactions between the adjacent molecules (O6···Cl1
= 3.198 Å and the O6–H6A···Cl1 angle is 112.4°); and O···O interactions (O6···O6i =
2.854 Å). When above weak interactions are taken into account, the resulting structure

Zinc(II) Schiff-base complexes
Table 2. Selected bond lengths (Å) and angles (°) for 1 and 2.
935
1
Zn1–O2
2.097(4)
Zn1–N5
2.134(5)
Zn1–N3
2.133(5)
Zn1–N6
2.315(5)
Zn1–O4
2.075(4)
Zn1–N7
2.263(6)
O2–Zn1–N3
O2–Zn1–N5
O2–Zn1–N6
O2–Zn1–N7
O2–C9–C14
O2–C9–C20
N3–Zn1–N5
O4–Zn1–N6
O4–Zn1–N7
115.05(18)
78.63(18)
151.76(19)
91.17(19)
121.9(5)
N3–Zn1–N6
N3–Zn1–N7
N3–C11–C8
N3–C12–C13
O4–Zn1–O2
O4–Zn1–N3
O4–Zn1–N5
N5–Zn1–N6
N5–Zn1–N7
91.68(18)
74.00(18)
113.4(5)
118.3(6)
99.03(19)
78.68(17)
105.26(18)
74.03(19)
101.62(18)
121.4(6)
165.35(18)
94.88(18)
152.63(18)
2
Zn1–O1
Zn1–O4
Zn1–N2
2.087(4)
1.930(5)
2.073(5)
153.96(19)
103.23(18)
90.33(19)
113.6(2)
99.87(19)
112.2(2)
95.2(2)
Zn2–O1
Zn2–O3
Zn2–O5
2.070(4)
2.128(4)
2.119(5)
75.9(2)
O1–Zn1–N1
O2–Zn1–O1
O2–Zn1–N1
O2–Zn1–N2
O4–Zn1–O1
O4–Zn1–O2
O4–Zn1–N1
O4–Zn1–N2
N2–Zn1–O1
N2–Zn1–N1
O5–Zn2–O3
Zn2–O1–Zn1
C1–N1–C5
C5–N1–Zn1
C6–N2–Zn1
C6–N2–C7
C7–N2–Zn1
O1–Zn2–O5
93.8(2)
107.65(19)
118.1(6)
112.8(4)
118.5(4)
127.5(6)
113.9(4)
88.31(19)
133.3(2)
78.30(18)
displays a 3D 6-connected net with α–Po (41263) topology [figure 2(B)]. A distorted cube-
like unit of the resulting α–Po net shows dimensions of 11.724 × 11.724 × 10.203 Å.
3.2. Interaction with CT-DNA
3.2.1. Absorption spectral studies. DNA binding is the critical step for DNA cleavage in
most cases [31–33]. Therefore, the binding ability of complexes to CT-DNA was studied by
using UV–vis absorption and fluorescence spectroscopy. The typical titration curves for 1
and 2 are shown in figure 3. The absorption peaks of both complexes at 268 and 370 nm
are attributed to intraligand π–π* transition; with increasing the CT-DNA concentrations,
hypochromism of 11.0% at 268 nm and 12.6% at 370 nm for 1, hypochromism of 10.3% at
268 nm and 11.9% at 370 nm for 2, and a slight redshift for both complexes are observed.
These results suggest intercalation between the complexes and DNA, because intercalation
would give hypochromism and bathochromism in UV absorption spectra due to the interca-
lative mode involving a strong stacking interaction between an aromatic chromophore and
the base pairs of DNA [34]. The extent of the hypochromism commonly reflects the
strength of intercalative interaction [35]. The intrinsic binding constant Kb was determined
from a plot of [DNA]/(εa − εf) versus [DNA] using the equation: [DNA]/(εa − εf) = [DNA]/
(εb − εf) + 1/Kb(εb − εf), where [DNA] is the concentration of DNA in base pairs. The appar-
ent absorption coefficients εa, εf, and εb correspond to Aobsd./[complex], the extinction coef-
ficient for the free complex and extinction coefficient for the complex in the fully bound
form, respectively [36]. Kb is given by the ratio of slope to intercept. From absorption data,
the binding constant Kb for 1 is 1.4 × 103 M−1 M−1 and for 2 is 1.2 × 103 M−1, lower than

936
M. Li et al.
Figure 3. Absorption spectra of 1 and 2 ([complex] = 2.0 × 10–5 M) in the absence (dashed line) and presence
(solid line) of increasing amounts of CT-DNA from 1 : 1 to 7 : 1.
those observed [37–41] for the classical intercalator EB (Kb, 4.94 × 105 M−1 in a 25 mM
Tris–HCl/40 mM NaCl buffer, pH 7.9). The results suggest that interaction of the two com-
plexes with DNA is weak intercalation; the lower Kb of 2 than 1 indicates steric hindrance
has an effect on the interaction with DNA.
3.2.2. Fluorescence quenching studies. Competitive binding was carried out to further
clarify the interaction of the complex with DNA. EB emission fluorescence intensity at
600 nm in the presence of DNA is due to its strong intercalation between adjacent DNA
base pairs [42, 43]. It was previously reported that addition of another molecule could
quench the enhanced fluorescence. This reduction in the emission intensity may be ascribed
to replacement of molecular fluorophores and/or electron transfer [44, 45]. The relative
binding of 1 and 2 to CT-DNA was studied with an EB-bound CT-DNA solution in 5 mM
Tris–HCl/50 mM NaCl buffer (pH 7.2). The fluorescence spectra of EB-bound CT-DNA are
similar in 1 and 2. The result of 2 is shown in figure 4(A). In fluorescence spectra, the
Figure 4. (A) Fluorescence quenching curves of EB bound to DNA by 2 [complex] = 0–14 × 10–5 M). The arrow
shows the intensity changes on increasing the complex concentration. (B) Plot of I0/I vs. [complex] for the titration
of complexes to EB-bound CT-DNA.

Zinc(II) Schiff-base complexes
937
addition of 1 and 2 to solution of EB-DNA induced a decrease in the fluorescence intensity
at 590 nm (350 nm excitation). The extent of reduction in the emission intensity measures
the binding propensity of the complex to DNA [figure 4(A)]. The relative binding intensity
of 1 and 2 to CT-DNA was determined by the classical Stern–Volmer equation: I0/I = 1 + K
[Q], where I0 and I are the fluorescence intensities in the absence and presence of complex,
respectively, K is a linear Stern–Volmer constant, and [Q] is the concentration of complex
to that of DNA. K was calculated from the slope of a plot of I0/I versus [Q] [46]. The
apparent binding constants K of 1 and 2 at 25 °C were calculated to be 6.20 × 104 M−1 and
4.85 × 104 M−1, respectively [figure 4(B)]. The binding constants of the classical intercala-
tors and metallointercalators were in the order of 107 M−1 [47]. The results suggest that 1
and 2 interact with DNA through intercalation, releasing some free EB from the EB-DNA
complex, consistent with the above absorption spectral results.
3.2.3. CD spectral studies. CD spectroscopy is useful in monitoring the conformational
variations of DNA in solution. The CD spectra of CT-DNA exhibit a positive band at 275
nm due to base stacking and a negative band at 245 nm due to the helicity of B-type DNA
[48]. While groove binding interaction of small molecules with DNA shows little or no per-
turbations on the base stacking and helicity bands, intercalation enhances the intensities of
both bands, stabilizing the right-handed B conformation of CT-DNA. Figure 5 displays the
CD spectra of CT-DNA with and without 1 and 2. In the presence of complexes, both the
positive (~275 nm) and negative (~245 nm) bands decreased in intensity, clearly indicating
interactions between the complexes and DNA. The decreased intensity in the negative band
suggests the complex can unwind the DNA helix and lead to loss of helicity [49].
3.3. MTT assay
The cytotoxicity of zinc complexes against HepG2 was estimated by MTT assay. The
growth inhibition of HepG2 cells with the chemicals is IC50 = 40 1 μM (1), 20 2 μM (2),
Figure 5. CD spectra of CT-DNA (3 mL solution, 2.0 × 10–4 M) in the absence (dashed line) and presence of
complexes (solid line), with [compound]/[CT-DNA] = 0.25 in Tris–HCl buffer, pH 7.2.

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M. Li et al.
102 2 μM (HL2), and 1000.97 μM (Zn(OAc)2), respectively. The results showed that 2
derived from pyridine-2-aldehyde and 2-amino-4-chlorophenol was essential for anticancer
activities and Zn(OAc)2 could not inhibit proliferation of HepG2 cells. Cell growth inhibi-
tion activity by zinc complex must be derived from biological activities of this zinc com-
plex, not simply from free zinc chaperoned into the cells by the ligand.
3.4. Apoptosis evaluation
Apoptosis can be identified by visualizing nuclear changes and apoptotic body formation.
From the results of MTT assay, 2 was selected for this apoptosis evaluation. HepG2 cells
were treated with 2 at various concentrations for 24 h, stained with Hoechst 33258 [50, 51].
As shown in figure 6, Hoechst 33258 staining showed blue apoptotic cells with apoptotic
features that exhibit nuclear pyknosis, showing round shapes and an enhanced fluorescent
signal. The results show that 2 can induce apoptosis of HepG2 cells.
3.5. Interaction with HSA
Interaction between serum albumin and compounds is important in drug transport and drug
metabolism functions of HSA [52, 53]. The fluorescence of HSA comes from tryptophan,
tyrosine, and phenylalanine residues. Phenylalanine has a very low quantum yield and fluo-
rescence of a tyrosine ionized is almost completely quenched; the intrinsic fluorescence of
many proteins is mainly contributed by tryptophan alone.
The fluorescence emission of HSA tryptophan residues was similar in the presence of 1
and 2. The result of 2 is shown in figure 7. With increasing concentrations of the com-
plexes, the intensity of the characteristic fluorescence emission band at 342 nm decreases
regularly, which indicates that interaction has occurred between the complex and HSA. The
fluorescence quenching is described according to the Stern–Volmer equation, F0/F = 1 + Kq
τ0[Q] = 1 + KSV[Q], where F0 and F represent the fluorescence intensities in the absence and
in the presence of quencher, Kq is the quenching rate constant, τ0 is the average lifetime of
the biomolecule without quencher (about 10−8 s) [53], KSV is the Stern–Volmer quenching
constant, and [Q] is the concentration of quencher. Figure 8(A) displays the Stern–Volmer
plots of the quenching of HSA fluorescence by the complexes, and KSV can be obtained by
a slope from the plot of F0/F versus [Q]. The calculated values of KSV and Kq for interac-
tion of the complexes with HSA are 1.02 × 105 M−1 and 1.02 × 1013 M−1 s−1 for 1, 0.84 ×
105 M−1 and 0.84 × 1013 M−1 s−1 for 2, respectively. The results indicate that 1 and 2
quench the fluorescence intensity of HSA.
Figure 6. Apoptosis of HepG2 cells induced by the complex. Morphological changes of HepG2 cells treated with
various concentrations of 2 (10, 20, and 30 μM) for 24 h. Hoechst 33258 staining.

Zinc(II) Schiff-base complexes
939
Figure 7. Fluorescence emission spectra of the HSA (2 μM) system in the absence (dashed line) and presence
(solid lines) of 2 (2, 4, 6, 8, 10, and 12 μM).
Figure 8. (A) Plot of F0/F vs. [complex] for the titration of the complex to HSA. (B) Plot of log[(F0 − F)/F] vs.
log[complex] for the titration of the complex to HSA.
Quenching mechanisms are usually dynamic quenching or static quenching. The process
of dynamic quenching occurs when the fluorophore and the quencher collide during the
transient existence of the exited state. Static quenching refers to fluorophore–quencher
complex formation. Compared with the maximum scatter collision-quenching constant of
biopolymers fluorescence (2 × 1010 M−1 s−1), Kq values of the complexes are higher,
indicating the existence of a static quenching mechanism [54].
For static quenching interaction, the binding constant (K) and the number of binding sites
(n) can be determined according to the Scatchard equation [55]: log(F0 − F)/F = logK + n
log[Q]. The n and K (1.0 and 9.78 × 104 M−1 L for 1, 0.9 and 4.01 × 104 M−1 L for 2) can
be determined by the slope and intercept of the double logarithm regression curve of log
(F0 − F)/F versus log[Q] [figure 8(B)], which show 1 and 2 exhibit higher binding constants
for HSA.

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M. Li et al.
Scheme 1. The synthesis of complexes.
4. Conclusion
Two zinc(II) complexes have been prepared and characterized structurally. The reactivity
toward CT-DNA and protein HSA reveal that 1 and 2 can interact with CT-DNA by interca-
lation, and 1 and 2 bind to HSA which is responsible for quenching of tryptophan fluores-
cence by static quenching. The zinc(II) complexes are cytotoxic to HepG2 cells, the
mechanism of cell death appears to be apoptosis. Our results found that zinc(II) complexes
may prove to be anticancer drugs, possibly targeting DNA and inducing apoptosis in the
selected cancer cell lines.

Zinc(II) Schiff-base complexes
941
Supplementary material
Crystallographic data for the structures reported in this article have been deposited with the
Cambridge Crystallographic Data Center, CCDC No. 964990 for 1 and 959446 for 2. Cop-
ies of this information may be obtained free of charge from the Director, CCDC, 12 Union
Road, Cambridge, CB2 1EZ, UK (Fax: +44-1223-336033; E-mail: deposit@ccdc.cam.ac.uk
or http://www.ccdc.cam.ac.uk).
Funding
This work was supported by the Natural Science Foundation of China [grant number 31060121],
[grant number 21171043] and Natural Science Foundation of Guangxi [grant number
2012GXNSFCB053001].
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