Synthesis, characterization, photoluminescence, molecular docking and bioactivity of zinc (II) compounds based on different substituents

Article abstract of DOI:10.3390/molecules25153459

Six new zinc(II) complexes were prepared by the reaction of ZnBr₂ or ZnI₂ with 4⁰-(substituted-phenyl)-2,2⁰:6⁰,2⁰⁰-terpyridine compounds, bearing p-methylsulfonyl (L¹), p-methoxy (L²) and p-methyl (L³), which were characterized by elemental analysis, FT-IR, NMR and single crystal X-ray diffraction. The antiproliferative properties against Eca-109, A549 and Bel-7402 cell lines and the cytotoxicity test on RAW-264.7 of these compounds were monitored using a CCK-8 assay, and the studies indicate that the complexes show higher antiproliferative activities than cisplatin. The interactions of these complexes with CT-DNA and proteins (BSA) were studied by UV-Vis, circular dichroism (CD) and fluorescent spectroscopy, respectively. The results indicate that the interaction of these zinc(II) complexes with CT-DNA is achieved through intercalative binding, and their strong binding affinity to BSA is fulfilled through a static quenching mechanism. The simulation of the complexes with the CT-DNA fragment and BSA was studied by using molecular docking software. It further validates that the complexes interact with DNA through intercalative binding mode and that they have a strong interaction with BSA.

Full text of DOI:10.3390/molecules25153459

molecules
Article
Synthesis, Characterization, Photoluminescence,
Molecular Docking and Bioactivity of Zinc (II)
Compounds Based on Different Substituents
Rongping Liu 1,2, Hao Yan 3, Jinzhang Jiang 1, Jiahe Li 1, Xing Liang 1, Dengfeng Yang 4
,
Lixia Pan 2,*, Tisan Xie 3,* and Zhen Ma 1,
*
1
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, Guangxi, China;
18877571207@163.com (R.L.); jjz19941028@gmail.com (J.J.); lijiahe006@163.com (J.L.);
liangxing587@126.com (X.L.)
2
3
4
National Engineering Research Center for Non-Food Biorefinery, State Key Laboratory of Non-Food Biomass
and Enzyme Technology, Guangxi Academy of Sciences, Nanning 530004, Guangxi, China
School of Animal Science and Technology, Guangxi University, Nanning 530004, Guangxi, China;
yanhao_day@163.com
Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi
Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning 530004, Guangxi, China;
dengfengyang@163.com
*
Correspondence: panlixia@gxas.cn (L.P.); xietisan@gxu.edu.cn (T.X.); mzmz2009@sohu.com (Z.M.);
Tel.: +86-0771-250-3980 (L.P.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Simona Collina and Mariarosaria Miloso
Received: 1 July 2020; Accepted: 24 July 2020; Published: 29 July 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Six new zinc(II) complexes were prepared by the reaction of ZnBr2 or ZnI2
with 40-(substituted-phenyl)-2,20:60,200-terpyridine compounds, bearing p-methylsulfonyl (L1),
p-methoxy (L2) and p-methyl (L3), which were characterized by elemental analysis, FT-IR, NMR and
single crystal X-ray diffraction. The antiproliferative properties against Eca-109, A549 and Bel-7402
cell lines and the cytotoxicity test on RAW-264.7 of these compounds were monitored using a CCK-8
assay, and the studies indicate that the complexes show higher antiproliferative activities than
cisplatin. The interactions of these complexes with CT-DNA and proteins (BSA) were studied by
UV-Vis, circular dichroism (CD) and fluorescent spectroscopy, respectively. The results indicate
that the interaction of these zinc(II) complexes with CT-DNA is achieved through intercalative
binding, and their strong binding affinity to BSA is fulfilled through a static quenching mechanism.
The simulation of the complexes with the CT-DNA fragment and BSA was studied by using molecular
docking software. It further validates that the complexes interact with DNA through intercalative
binding mode and that they have a strong interaction with BSA.
Keywords: zinc (II) complexes; antiproliferative activity; CT-DNA binding; fluorescence quenching;
molecular docking
1. Introduction
Cancer is one of the leading causes of death worldwide, with more than 10 million people suffering
from the disease yearly [
1–
3]. Since its introduction in medical practice, cisplatin has been one of
the most effective drugs for the treatment of cancers [4–7
]. The biological activity of cisplatin comes
from the fact that its covalent binding with DNA leads to cell death or apoptosis. However, there are
also serious side effects such as nephrotoxicity, neurotoxicity, gastrointestinal toxicity, bone marrow
suppression and drug resistance [
platinum drugs and promote the development of alternative therapeutics [11
8
–
10], among others. These side effects limit the application of
13]. The production of
–
Molecules 2020, 25, 3459; doi:10.3390/molecules25153459
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3459
2 of 20
novel anticarcinogen medicines, which are less toxic, are more effective and target specific but show
high efficacy in chemotherapy, is an active area of research [14 15]. The transition metal complexes have
become candidate molecules for cancer treatment [16 19], and Zn(II) is of interest due to endogenous
compatibility with the living system. Zinc is the second most abundant essential trace element in the
human body, being involved in the synthesis of DNA and proteins [20 21]. Substituted terpyridine
compounds are effective ligands for transition metal complexes [22 25]. Therefore, Zn(II) terpyridine
,
–
,
–
complexes are worth exploring in molecular biology and related fields.
DNA and protein are two important biological targets in the research of anticancer drugs [26–28].
Studies have shown that one of the reasons for the cytotoxicity of cisplatin is its perfect non-specific
combination with serum protein [29 30]. Therefore, the interaction between complexes and proteins
,
plays a key role in the distribution and transport of small molecules into the biological system,
which affects the concentration and overall efficacy of drug molecules [31]. In our previous studies,
it has been found that zinc complexes have strong antitumor activity [22,23,32–34]. It is noteworthy that
the antitumor properties of these complexes can be regulated by the substituents at their 40-position.
Hence, the exploration of the biological activity of different substituted zinc terpyridine complexes has
a biological value for the design of the drug molecules.
Six new zinc complexes 1–6
of substituted terpyridine compounds at the 40-position with three
chemical groups, p-methylsulfonyl (L1), p-methoxy (L2) and p-methyl (L3), were synthesized and
characterized (Scheme 1). Their biological properties were studied as an extension of our previous
work. The antiproliferative properties against Eca-109, A549 and Bel-7402 cell lines and the cytotoxicity
test on RAW-264.7 of these compounds in vitro were studied using a CCK-8 assay. The interactions
between the complexes with CT-DNA and a protein (BSA) were investigated by UV-Vis titration and
CD spectroscopy (for the DNA) and fluorescence titration (for the protein). The binding constants (Kb)
and quenching constants (Kq) for the interaction between these complexes and CT-DNA and BSA were
determined. In addition, molecular docking was also carried out to verify their binding properties.
Scheme 1. The compounds used in the experiment.
2. Results and Discussion
2.1. Syntheses and Characterization
Ligands L1–L3 were prepared according to reported procedures [23
synthesized by reaction of the ligands with ZnBr2 or ZnI2 in stoichiometric amounts, respectively.
Complexes
were characterized by 1H-NMR spectroscopy (Figures S1–S6, Supplementary Materials),
IR (Figures S7–S12), elemental analysis and single crystal X-ray diffraction (Figure 1 and Figure S13–S17).
IR spectra of all the compounds show the pyridyl and phenyl characteristic bands. Compounds and
appear at the methylsulfonyl characteristic band at 1305 cm−1 and 1145 cm−1. The bands at 1235 cm−1
and 1184 cm−1 belong to the absorption of methoxy ( ) and those at 1380 cm−1 and 1442 cm−1
and
belong to the absorption of methyl ( and ). As shown in Figures S1–S6 (Supplementary Materials),
the hydrogen atoms at the pyridyl rings of all the iodide complexes ( and ) are affected by the
,33]. All the complexes were
1–6
1
2
3
4
5
6
2,
4
6
spatial position of the iodine atom, leading to the different chemical environments of the hydrogen
atoms and the different chemical shift values in the nuclear magnetic spectrum. It is interesting that

Molecules 2020, 25, 3459
3 of 20
the maximum and minimum chemical shifts of the pyridyl hydrogens decrease accordingly with the
substituent groups’ sequence of methylsulfonyl (–SO2CH3)→methoxy (–OCH3)→methyl (–CH3).
Figure 1. Thermal ellipsoid plot, drawn at the 50% probability level, of [ZnBr2L1] (1) with atomic
numbering scheme. Selected bond lengths (Å) and angles (◦): Zn(1)-N(2) 2.099(2), Zn(1)-N(1)
2.187(2), Zn(1)-N(3) 2.215(2), Zn(1)-Br(2) 2.4065(4), Zn(1)-Br(1) 2.4143(4), N(2)-Zn(1)-N(1) 74.22(8)
N(2)-Zn(1)-N(3) 74.27(8), N(1)-Zn(1)-N(3) 146.24(8), N(2)-Zn(1)-Br(2) 138.77(6), N(1)-Zn(1)-Br(2) 98.90(6)
N(3)-Zn(1)-Br(2) 96.78(5), N(2)-Zn(1)-Br(1) 108.69(6), N(1)-Zn(1)-Br(1) 97.22(6) N(3)-Zn(1)-Br(1) 104.06(6),
Br(2)-Zn(1)-Br(1) 112.512(17).
2.2. Elucidation of the Structures of the Compounds
Crystals of all the complexes were obtained by evaporation of their DMF solutions, which were
suitable for X-ray analysis.
Compound
1 is a mononuclear complex that crystallized in a centrosymmetric space group C2/c
with one molecule per asymmetric unit, as shown in Figure 1. The zinc ion is coordinated by three
N atoms of the 400-methylsulfonyl-40-phenyl-2,20:60,200-terpyridine ligand and two bromide anions.
In order to better understand the geometry of the complex, its value for the tau parameter was calculated
as 0.56 [35]. The result shows that its coordination environment is distorted trigonal-bipyramidal.
The average bond length of the Zn–N bond is 2.167 Å, and that of the Zn–Br bond is 2.4104 Å. The crystal
structure shows that the three pyridine groups are basically planar with the root-mean-square (RMS)
deviation being 0.1267 Å. The phenyl with methylsulfonyl substituent forms a dihedral angle between
the central pyridine ring of 26.79(9)◦ and the plane determined by Zn–N1–N2–N3 of 25.22(10)◦ Å.
There are no classical hydrogen bonds in the structure but several hydrogen bonds between the
hydrogen of carbon atoms (C1, C18, C20 and C22) and neighboring atoms (O atom and Br atom) in the
structure. The distance for the hydrogen bonds of C1–H . . . O1 is 3.2878(1) Å, and it is 2.8903(1) for
C18–H . . . O2, 3.6487(1) for C18–H . . . Br2 and 3.8044(1) Å for C22–H . . . Br1. An intermolecular
interaction is detected, which relates to a middle pyridyl and a plane defined by Zn1–N2–C10–C11–N3,
with a centroid distance of 3.6348(1) Å. There is a –ring interaction in the form of Y–X . . . Cg in the
π–π
π
structure with the distance (X . . . Cg) of 3.9082(1) Å, involved in the oxygen atom at the phenyl group
and the middle pyridine ring of the neighboring ligand.
Like
3, 5 and 6 and P21/n for 4 (Figures S13–S17, Supplementary Materials). The selected bond angles and
lengths of are listed in the captions of Figures S13–S17. In order to better understand the geometry
of the complexes, the values of the tau parameter were also calculated ( = 0.48, 0.60, 0.54, 0.56 and
0.59 for compounds , respectively) [35]. The results show that the coordination environment for
compounds is distorted trigonal-bipyramidal, whereas it is a distorted square-pyramidal for
1, 2–6 are also mononuclear species crystallized in space groups, i.e., P212121 for 2, P21/c for
2–6
τ
2–6
3–6

Molecules 2020, 25, 3459
4 of 20
compound
2. All the complexes present a
π
–ring interaction in the structures. In
2
, there are two
π–ring
(Y–X . . . Cg) interactions with atom-centroid distances (X . . . Cg) being 3.9525(7) Å and (Y . . . Cg
being 4.7005(8) Å. In and , there are two kinds of –ring (X–H . . . Cg) interactions, giving the
atom-centroid distance (X . . . Cg) of 3.9183(1) for , 3.7288(1) for
3.814(5) Å for as well as (H . . . Cg) of 2.960 for , 2.990 for , 2.910 and 2.820 for
respectively. In , there is one kind of
)
3,
4,
5
6
π
3
4, 3.7543(1) and 3.7348(1) for
5
and
6
3
4
5
and 2.870 Å for 6,
4
π–π interaction, showing a distance of 3.5953(1) Å between one
terminal pyridyl N1–C1–C2–C3–C4–C5 and one plane defined by Zn1–N1–C5–C6–N2.
2.3. Photoluminescent Properties
The emission spectra of all the compounds are shown in Figure 2. Due to the difference between the
substituted groups and the anionic ligands, the solid photoluminescent properties of these complexes
at room temperature are interesting. The zinc bromide complexes
(388 nm for , 426 nm for and 412 nm for ). As the substituent groups vary from electron absorption
to donor electron (1→5→3), the emission spectra were redshifted. Compounds and show one or
two low strength bands (596.5 nm for , 380 nm and 512.5 nm for ). However, zinc iodide complexes
and show five bands in the emission spectra and there is an obvious strength band at 428.5 nm,
427.5 nm and 428 nm, respectively. Four other low strength bands show their positions at 391 nm,
490.5 nm, 534.5 nm and 597 nm for , 370.5 nm, 491 nm, 535.5 nm and 601 nm for and 383 nm, 491 nm,
534.5 nm and 595.5 nm for . It is interesting that the strong peaks of the zinc iodide compounds do
1, 3 and 5 have only one strong peak
1
3
5
1
5
1
5
2
,
4
6
2
4
6
not change visibly and that the peaks have redshifted in comparison with the strong peaks of the zinc
bromide complexes. According to the reported photoluminescent results of 40-phenyl-terpyridine and
other substituted terpyridine compounds, the peaks around 380 nm are assigned tentatively to the
π–π*/MLCT mechanism [22,23]. The other peaks of the iodide anion compounds (2, 4 and 6) in the
low-energy region are assigned temporarily to n–π*/LMCT [22].
412
428
383
491
534.5
595.5
380
6
426
512.5
5
427.5
601
370.5
388
491
535.5
4
1000
800
600
400
200
0
428.5
3
391
490.5
534.5
596.5
597
2
1
400
500
600
700
800
Wavelength(nm)
Figure 2. Solid state emission spectra (excitation at 300 nm) of complexes 1–6 at room temperature.
2.4. Solution Stability
The stability of complexes
by UV-Vis spectroscopy. Stock concentrations of complexes
with PBS buffer solution. The stability analysis was performed at 0, 24, 48 and 72 h. As shown in
Figure 3 for and and are shown in Figure S18, Supplementary Materials), there are no
1–
6
under physiological conditions (PBS buffer, pH = 7.4) was monitored
were prepared in DMSO and diluted
1–6
1
3
(
2
,
4,
5
6
obvious changes in the UV-Vis spectra of the compounds, indicating that the complexes are stable
under physiological conditions.

Molecules 2020, 25, 3459
5 of 20
2
1
0
2
0
0
24
48
72
24
48
72
1
0
250
300
350
400
450
250
300
350
400
450
Wavelength(nm)
Wavelength(nm)
(a)
(b)
Figure 3. The UV-vis spectra of complexes (a) 1 and (b) 3 in PBS buffer solution over a period of 72 h.
2.5. Antiproliferative Activity and Toxicity Studies
The in vitro antiproliferative properties of complexes 1–6 against Eca-109, A549 and Bel-7402
tumor lines were examined by employing the CCK-8 assay with cisplatin as a positive control. As seen
from Figure 4, all the complexes display a dose-dependent manner on the viability of the test cells.
Table 1 shows the IC50 values of the compounds in the range of 0.066–0.945
lower than that of cisplatin (5.66–11.45 M). It is interesting that the different cells respond distinctively
to these complexes. The complexes display remarkable cytotoxicity against Eca-109 in comparison to
A549 and Bel-7402. Notably, the antiproliferative activity of and is better than that of the other
µM, which is significantly
µ
1
2
compounds towards the three cell lines and, when the para-substituent is the same, the activity of
the iodide anion complexes is generally better than that of the bromide anion ones. Compared with
that of zinc chloride complexes [34], the antiproliferative activity of these complexes is stronger.
This activity of the bromide complexes follows the order of
1 > 3 > 5 and that of the iodide complexes
is 2 > 4 > 6. These results reveal that both the substituents and counterion have effects on the activity.
It is worth mentioning that, among these compounds, the order of the activity is consistent with the
electronegativity values (methylsulfonyl (
The viability of RAW264.7 cells treated with compounds
methods used on the tumor cell lines, and the plots of cell viability of compounds
concentration are shown in Figure 5b. The results reveal that the compounds promote the growth of
RAW264.7 cells at low concentrations (<1 M) and inhibit the growth of the cells at high concentrations
(>1 M). As seen by the CCK-8 results, complexes show significant cytotoxicity at 0.066–0.945
−
2.96) < methoxy (
−
2.86) < methyl (
was tested following the same
in increasing
−
2.28) of the substituents.
1–6
1–6
µ
µ
1
–6
µM
towards the cancer cells with no apparent cytotoxicity towards the normal cells.
Table 1. IC50 of complexes against human cancer cells in vitro.
Eca-109
95% Confidence
A549
95% Confidence
Bel-7042
Compound
95% Confidence
IC50 (µM)
IC50 (µM)
IC50 (µM)
Intervals (µM)
Intervals (µM)
Intervals (µM)
1
2
3
4
5
6
0.079
0.066
0.169
0.075
0.197
0.146
5.465
0.0533–0.1188
0.0464–0.0946
0.1246–0.2311
0.0503–0.1144
0.1449–0.2693
0.1208–0.1780
4.982–5.994
0.309
0.290
0.482
0.307
0.748
0.945
11.99
0.6419–0.8715
0.3822–0.6072
0.2465–0.3824
0.2501–0.3819
0.2072–0.4068
0.6751–0.9521
10.79–13.32
0.358
0.331
0.414
0.397
0.616
0.522
3.088
0.3468–0.7850
0.2434–0.5271
0.2817–0.6089
0.2408–0.6553
0.3681–1.032
0.2533–0.4325
2.856–3.340
cisplatin

Molecules 2020, 25, 3459
6 of 20
120
100
80
120
100
80
120
100
80
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
60
60
60
40
40
40
20
20
20
-1.2
-0.8
-0.4
0.0
0.4
-1.2
-0.8
-0.4
0.0
0.4
0.8
0.0
0.4
0.8
1.2
1.6
Log[C]
Log[C]
Log[C]
(a)
(b)
(c)
Figure 4. The curves of antitumor activities of compounds
(c) Bel-7402 cell line.
1–
6
against (
a) Eca-109, (b) A549 and
Compound 1
Compound 3
120
100
l)
o
tr
n
o
80
c
f
o
60
(%
y
it
40
il
b
ia
20
v
ll
e
C
0
5
2
6
6
0
.
0
5
2
1
.
5
0
5
2
.
0
4
5
.
0
x
e
[
C
l
1
p
3
o
m
m
o
p
2
C
l
e
2
x
]
4
1
8
(a)
(b)
Figure 5.
compound
(
a
1
) Microscopic photographs of Eca-109 cancer cells treated with different concentrations of
and control photographs (C for blank control group, C1 for 2.5 M cisplatin, C2 for 20 µM
µ
cisplatin). (b) The cell viability of compounds 1–6 against RAW264.7.
2.6. Protein Binding Analysis
2.6.1. Fluorescence Quenching Analysis
The structure and transmission characteristics of BSA are similar to HSA [36]. BSA was used
instead of HSA for drug delivery due to its ease to purify and stability. The binding of metal
complexes to proteins was studied by fluorescence spectrum titration. Endogenous fluorescence of BSA
comes from residues as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe), and conformational
changes of BSA-binding metal complexes are obtained by observing the fluorescence of Trp residues
at 350 nm [27]. Figure 6 shows the fluorescence emission spectra of BSA (1.6
different concentrations of complexes (0–20 M) and (0–20 M). Interestingly, with the increase of
concentration of at 350 nm, BSA shows strong fluorescence quenching, accompanied by fluorescence
µM) incubated with
4
µ
6
µ
4
enhancement at 450 nm. The same phenomenon occurs for the other compounds (shown in Figure S22,
Supplementary Materials). It is worth mentioning that, unlike other complexes, low concentration
of
1 and 2 significantly enhances fluorescence intensity at 380 nm when added into BSA, while the
quenching is relatively weak. This appearance may be due to a resonant energy transfer. The strong
quenching effect indicates that the metal complexes are bound to BSA and change the microenvironment

Molecules 2020, 25, 3459
7 of 20
around Trp residues. The Stern–Volmer equation [37] (Equation (1)) was used to study the quenching
mechanism of BSA and the complexes:
I0/I = 1 + Kqτ0[Q] = 1 + Ksv[Q]
(1)
where the fluorescence intensity of the protein is expressed by I0 and I in absence and in presence
of quencher, respectively; Ksv is the Stern–Volmer quenching rate constant; Kq is the quenching
rate constant; and
(valued 10−8 s) and quencher concentration, respectively. The maximum causing a value of dynamic
quenching in biopolymers is 2 23]. In Table 2, it can be observed that the range
1010 M−1 S−1
of Kq is 4.6
1012 to 1.8 1013, indicating that the interaction of the complexes and BSA is a static
quenching mechanism.
τ0 and [Q] represent the lifetime of fluorescent molecule in absence of quencher
×
[
×
×
Table 2. Quenching constants and binding constants of compounds 1–6 with BSA.
Ksv (M−1
)
Kq (M−1
)
Kb (M−1
)
Compound
n
Ra
1.76 × 105
1.76 × 105
1.15 × 105
1.63 × 105
4.63 × 104
7.67 × 104
1.76 × 1013
1.76 × 1013
1.15 × 1013
1.63 × 1013
4.63 × 1012
7.67 × 1012
4.47 × 103
1.80 × 104
2.95 × 105
3.80 × 105
1.07 × 104
2.24 × 104
1
2
3
4
5
6
0.72
0.83
1.07
1.08
0.87
0.89
0.9783
0.9989
0.9919
0.9943
0.9946
0.9960
The Ra is the linear correlated coefficient for the Ksv values.
4
3
2
（complex 4）
y = 1+163140x
R=0.99428
0.000005
0.000010
[Complex]
1.8
(complex 6)
1.6
1.4
1.2
y = 1+76675x
R=0.99596
0.000000
0.000005
0.000010
[Complex]
(a)
(b)
Figure 6.
(a
) Fluorescence emission spectra of 1.6 M BSA in a Tris-HCl buffer (pH = 7.2) solution with
µ
series concentration of compounds
4
and , and (b) curves for the Stern–Volmer equation as the insets.
6

Molecules 2020, 25, 3459
8 of 20
2.6.2. Binding Constant and Site Analysis
Calculation of the binding constants (Kb) was achieved according to the Scatchard equation [38
(Equation (2)):
]
log [(I0 − I)/I] = logKb + nlog[Q]
(2)
where Kb and n represent the binding constant and the binding site number, respectively. The binding
constants Kb and the binding site number were obtained from the intercept and slope of the double
logarithm regression plot between log [(I0
constant (>104 L mol−1) was obtained under the condition of high binding affinity [39]. The Kb range is
4.5
103 to 3.8 105 L mol−1 and the binding site number is equivalent to 1. The experimental results
show that complexes 1–6 can effectively bind to BSA, affecting the microenvironment of Trp residues
and bringing drugs into the target cells to promote apoptosis [40 42]. It can be seen in Figure 7 that the
−
I)/I] and log [Q]. The data are shown in Table 2. The binding
×
×
·
–
absorption spectra of the complexes partially overlap with the excitation spectrum of BSA.
Figure 7. Absorption spectra of 20 µM of compounds 1–6 in Tris-HCl buffer.
2.6.3. Protein Docking
Molecular docking is helpful to know the interaction of complexes and BSA [43
in Figure 8 for (Figure S23, Supplementary Materials, for the other compounds), it enters and is
embedded in the hydrophobic cavity of subdomain IIA of BSA at binding site I [45 47]. The complex
interacts with BSA protein primarily through a hydrogen bond and van der Waals force. It is seen that
,44]. As shown
1
–
1
is surrounded by amino acid residues such as TRP-213, ARG-194, AlA-341, AlA-209, ASP-450, VAL-342,
ARG-217, LEU-480, SER-201, and LEU-210. The binding energies for all the compounds are listed in
Table 3, namely −7.69 (1), −8.28 (2), −7.00 (3), −6.99 (4), −7.26 (5) and −7.79 (6). The results show that
the other complexes can also spontaneously enter into the hydrophobic region of the subdomain IIA of
BSA like 1.
Table 3. Docking energy of compounds
compounds 1–6 to BSA.
1
–6
to DNA. Quenching constants and binding constants of
BSA
n
Compound
DNA(4JD8)
1
2
3
4
5
6
−7.69
−8.28
−7.00
−6.99
−7.26
−7.79
−7.90
−8.65
−8.42
−8.76
−8.51
−8.78

Molecules 2020, 25, 3459
9 of 20
(a)
(b)
Figure 8. Docking conformation of complex 1 to (a) BSA and (b) amino acid residues surrounding 1.
2.7. DNA Binding Studies
2.7.1. UV-Vis Spectroscopy
UV-Vis titration absorption spectrometry is one of the effective methods to study the binding pattern
of metal complexes to DNA [48 49]. When the complexes are inserted into the DNA base interlayer
and the aromatic group of the complexes and the DNA base generate a interaction, the absorption
spectrum shows hypochromism and redshift [50 51]. While the complexes are inserted into the DNA
,
π–π
,
groove, the absorption band (LMCT) shows hyperchromicity and blueshift [52]. The binding pattern
was determined by the change observed in the absorption spectrum. The binding affinity was evaluated
by calculating the binding constant (Kb). The equation [27] (Equation (3)) is as follows:
A0/A − A0 = 1/fa + 1/faKb[DNA]
(3)
and
The absorption spectra of the metal complexes and CT-DNA are shown in Figure 9 (
1
3
)
and Figure S24, Supplementary Materials (the other compounds). With an increase of CT-DNA
concentrations, the LMCT absorption bands of the complexes give the results of a significant
hypochromism, which shows that the interaction between the complexes and DNA is consistent with
the embedding mode. This mode is confirmed by molecular docking calculation and CD spectroscopy.
The binding constants (Kb) of the metal complexes and CT-DNA are shown in Table 4, indicating that all
the metal complexes have strong interactions with DNA. The results show that the binding constants
of the compounds with DNA have a tendency of increase, which has a relationship with the 4-position
substituents of the compounds to give a spatial effect that the binding constants increase as the
electronegativity of the substituents decreases. This tendency is also contrary to the order of the
antiproliferative ability of these compounds against the cells.
Table 4. Binding constants of compounds 1–6 with CT-DNA.
Kb (M−1
)
Compound
Ra
n
2.79 × 103
2.47 × 103
1.90 × 104
2.15 × 104
6.63 × 104
6.69 × 104
1
2
3
4
5
6
0.9940
0.9906
0.9900
0.9924
0.9931
0.9974

Molecules 2020, 25, 3459
10 of 20
(a)
(b)
Figure 9. Absorption spectra of 20
solution with series concentrations of CT-DNA. The plots of A0/(A
CT-DNA are shown as the insets.
µ
M of compounds (
a
)
1
and (
b
)
−
3
in a Tris-HCl buffer (pH = 7.2)
A0) versus the concentration of
2.7.2. Circular Dichroism Analysis
CD spectroscopy was further used to study the DNA interacting behavior with complexes 1–6.
CD study is an effective method to detect minor conformational change of DNA induced by metal drug
binding. The characteristic CD spectra of B-form of CT-DNA (calf thymus DNA) displays a positive
band at 275 nm (due to base stacking) and a negative band at 246 nm (related to right-handed helicity)
due to the coupling of the stacked planar bases arranged in chiral helices [53,54]. Simple groove binding
does not cause observable changes in the positive band at 275 nm, whereas intercalation impacts the
intensities of the negative and positive bands. As Figure 10 and Figure S25 (Supplementary Materials)
show, the CD spectrum of CT-DNA with different ratios of the complexes was detected in Tris-HCl
buffer solution systems. It can be clearly seen that obvious changes are observed with the participation
of the complexes in both positive and negative bands. The intensity of the negative bands decreases

Molecules 2020, 25, 3459
11 of 20
and that of the positive bands increases. The results indicate that the metal complexes with DNA
display an intercalation binding mode, which is consistent with the conclusion of the UV-Vis titration
study. The binding effect of the bromine compounds with DNA has a tendency of 1 > 5 > 3, which may
be determined by both electronegativity and spatial effect. However, differing from that of the bromide
complexes, the binding effect of the iodide compounds with DNA increases and this consequence has
a relationship with the effects of the 4-position substituents of the compounds, which displays a spatial
effect on the binding of the compounds with DNA.
8
6
8
6
native
r=0.5
r=1
native
r=0.5
r=1
4
4
2
2
0
0
250
300
350
400
250
300
350
400
Wavelength(nm)
Wavelength(nm)
-2
-4
-2
-4
(a)
(b)
Figure 10. CD spectra of compounds (a) 1 and (b) 3 to CT-DNA at different concentration ratios.
2.7.3. Molecular Docking Studies with CT-DNA
Molecular docking technology is an important tool to know the interaction between small
molecules of drugs and biological targets in medicinal research [55–57]. The molecular docking study
was carried out by the structure of the zinc (II) complexes with DNA, and the modes and energy of the
binding were calculated. As shown in Figure 11 and Figure S26 (Supplementary Materials), all the
complexes are bound primarily by intercalative mode in DNA. The small molecule inserts at the base
interlayer, to produce a
as shown in Table 3, is in the range of
that the intercalation of the compounds into DNA is spontaneous and effective. The results show that
the binding energy of the compounds with DNA has a tendency to decrease when the anionic ligands
are the same, which is related to the 4-position substituents of the compounds. This consequence is
different from the order of the antiproliferative ability of these compounds against the cancer cells.
π
–
π
interaction with the base. In addition, the binding energy of complexes 1–6,
7.90 to
8.78 kcal mol−1. It is clear from the binding energies
−
−
Figure 11. Docking conformation of complex 1 to DNA.
3. Materials and Methods
1H-NMR spectra were determined with an Agilent Technologies 800/54 premium (Rogowska,
Wroclaw, Poland). Elemental analyses were run on an Elementar Vario EL III Elemental Analyzer
(Langenselbold, Frankfurt, Germany). IR spectra were recorded on a Nicolet IS10 (Carlsbad, CA, USA).

Molecules 2020, 25, 3459
12 of 20
Intensity data of single crystals were collected using an Agilent SuperNova diffractometer (Santa Clara,
CA, USA). UV-Vis absorption spectra were performed using a Beckman coulter DU800 (Brea, CA,
USA). Fluorescence spectra were recorded on a PerkinElmer LS 55 (Waltham, MA, USA) luminescence
spectrometer with a red-sensitive photomultiplier type R928. Circular dichroism (CD) spectra carried
out on a Chirascan CD spectropolarimeter (Applied Photophysics, Leatherhead, Surrey, U.K.). All the
cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and
Tris-HCl buffer, BSA and CT-DNA were obtained from Beijing Solarbio Science & Technology Co., Ltd.,
Asbio and Solarbio (Beijing, China), respectively. BSA were dissolved in Tris-HCl buffer and stored
at 4 ◦C. The complexes were dissolved in DMSO to prepare stock solution and the solubility of the
complexes was 20 mM/mL in DMSO, while the solubility of
5 was 15 mM/mL. All reagents used in the
experiments were of analytical grade or purified by standard methods.
3.1. Synthesis
The ligands L1–L3 were prepared by following reported procedures [23
,33]. Compounds 1–6
were synthesized by reaction of the ligands with zinc salts (ZnBr2 or ZnI2). A 10 mL methanol solution
of zinc salt (9 × 10−4 mol) was slowly added into 10 mL dichloromethane solution of the ligands
(
5
9 × 10−4 mol). The mixture was stirred for 24 h at room temperature. The compounds were white (
1, 2,
and 6), yellow (3) or orange (4) powders and characterized by elemental analysis, IR spectroscopy
and NMR analysis. Suitable crystals for determination were obtained by slow evaporation of the
solutions of the compounds in DMF in different colors.
3.1.1. [ZnBr2L1] (1)
A white solid (yield: 80%) was obtained upon filtration of the solution and washed with
CH2Cl2/CH3OH and dried in a desiccator. Orange crystals were obtained by evaporation of its DMF
solution, which were suitable for X-ray analysis. Anal. calcd for C22H17Br2N3O2SZn: C, 43.13, H, 2.80,
1
N, 6.86%. Found: C, 43.24, H, 2.78, N, 6.90%. H-NMR (800 MHz, DMSO-d6)
δ 9.18 (s, 2H), 8.95 (d,
J = 7.9 Hz, 2H), 8.89 (s, 2H), 8.49 (d, J = 7.9 Hz, 2H), 8.36 (t, J = 7.9 Hz, 2H), 8.19 (d, J = 7.9 Hz, 2H),
7.90 (d, J = 6.5 Hz, 2H), 3.35 (s, 4H). IR (cm−1): 3060 (m), 1615 (s), 1598 (s), 1576 (s, Vpyridyl-H), 1548 (s),
1474 (vs, Vpyridyl-H), 1427 (s), 1391 (s), 1305 (vs), 1251 (m), 1145 (vs), 1092 (s), 1012 (s), 951 (m), 837 (m),
791 (vs, γaryl-H), 769 (s), 732 (s), 673 (s), 636 (m).
3.1.2. [ZnI2L1] (2)
A white solid (yield: 62%) was obtained upon filtration of the solution and washed with
CH2Cl2/CH3OH and dried in a desiccator. Brown crystals were obtained by evaporation of its DMF
solution, which were suitable for X-ray analysis. Anal. calcd for C22H17I2N3O2SZn: C, 37.39, H, 2.42,
1
N, 5.95%. Found: C, 37.52, H, 2.39, N, 5.97%. H-NMR (800 MHz, DMSO-d6)
δ 9.46 (s, 1H), 9.21 (s,
1H), 9.15 (d, J = 8.0 Hz, 1H), 9.03 (d, J = 7.9 Hz, 1H), 8.96–8.93 (m, 1H), 8.66 (d, J = 7.9 Hz, 1H), 8.52 (d,
J = 7.9 Hz, 1H), 8.43 (t, J = 7.9 Hz, 1H), 8.30 (t, J = 8.2 Hz, 2H), 8.21 (d, J = 7.9 Hz, 1H), 7.97 (dt, J = 24.2,
4.8 Hz, 2H), 7.51 (t, J = 6.2 Hz, 1H), 3.33 (d, J = 2.1 Hz, 3H). IR (cm−1): 3069 (m), 1613 (s, Vpyridyl-H),
1599 (s), 1570 (m, Vpyridyl-H), 1548 (s, Vpyridyl-H), 1475 (vs, Vpyridyl-H), 1427 (s), 1393 (s), 1313 (vs),
1248 (m), 1147 (vs), 1093 (s), 1012 (s), 960 (m), 832 (m), 792 (vs, γaryl-H), 768 (s), 731 (s), 657 (s), 637 (s).
3.1.3. [ZnBr2L2] (3)
A yellow solid (yield: 75%) was obtained upon filtration of the solution and washed with
CH2Cl2/CH3OH and dried in a desiccator. Orange crystals were obtained by evaporation of its DMF
solution, which were suitable for X-ray analysis. Anal. calcd for C22H17Br2N3OZn
·
H2O: C, 45.35 H,
1
3.29, N, 7.21%. Found: C, 45.25, H, 3.32, N, 7.02%. H-NMR (800 MHz, DMSO-d6)
δ 9.02 (s, 2H), 8.93 (d,
J = 7.9 Hz, 2H), 8.86 (d, J = 4.9 Hz, 2H), 8.32 (t, J = 7.7 Hz, 2H), 8.26 (d, J = 8.3 Hz, 2H), 7.89–7.86 (m,
2H), 7.17 (d, J = 8.2 Hz, 2H), 3.87 (s, 3H). IR (cm−1): 3049 (m), 2838 (m), 1592 (vs), 1572 (s, Vpyridyl-H),

Molecules 2020, 25, 3459
13 of 20
1541 (m), 1519 (vs), 1476 (s, Vpyridyl-H), 1433 (s), 1408 (s), 1235 (vs), 1184 (s), 1066 (s), 1018 (s), 837 (s),
789 (vs, γaryl-H), 732 (s), 635 (s), 578 (s).
3.1.4. [ZnI2L2] (4)
An orange solid (yield: 70%) was obtained upon filtration of the solution and washed with
CH2Cl2/CH3OH and dried in a desiccator. Brown crystals were obtained by evaporation of its DMF
solution, which were suitable for X-ray analysis. Anal. calcd for C22H17I2N3OZn: C, 40.12, H, 2.60,
1
N, 6.38%. Found: C, 40.23, H, 2.07, N, 6.23%. H-NMR (800 MHz, DMSO-d6)
δ 9.33 (s, 1H), 9.15 (d,
J = 8.1 Hz, 1H), 9.07 (s, 1H), 9.03 (d, J = 7.9 Hz, 1H), 8.92 (d, J = 5.0 Hz, 1H), 8.46 (d, J = 8.1 Hz, 1H),
8.40 (t, J = 7.7 Hz, 1H), 8.31–8.25 (m, 2H), 7.93 (t, J = 5.6 Hz, 2H), 7.47 (dd, J = 7.3, 5.2 Hz, 1H), 7.28 (d,
J = 8.2 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 3.91 (d, J = 40.6 Hz, 3H). IR (cm−1): 3063 (m), 2838 (m), 1596(s),
1567 (s, Vpyridyl-H), 1541 (m), 1516 (s), 1472 (s, Vpyridyl-H), 1431 (s), 1431 (s), 1403 (s), 1241 (s), 1184 (s),
1069 (m), 1009 (s), 876 (m), 826 (s), 790 (vs, γaryl-H), 727(s), 731 (s), 638 (s), 578 (s).
3.1.5. [ZnBr2L3] (5)
A white solid (yield: 73%) was obtained upon filtration of the solution and washed with
CH2Cl2/CH3OH and dried in a desiccator. Orange crystals were obtained by evaporation of its DMF
solution, which were suitable for X-ray analysis. Anal. calcd for C22H17Br2N3Zn: C, 48.16, H, 3.12,
1
N, 7.66%. Found: C, 48.61, H, 2.74, N, 7.65%. H-NMR (800 MHz, DMSO-d6)
δ 9.07 (s, 2H), 8.96 (d,
J = 8.0 Hz, 2H), 8.87 (d, J = 4.8 Hz, 2H), 8.34 (t, J = 7.8 Hz, 2H), 8.19 (d, J = 7.7 Hz, 2H), 7.92–7.87 (m, 2H),
7.46 (d, J = 7.8 Hz, 2H), 2.43 (s, 3H). IR (cm−1): 3069 (m), 1611 (s), 1595 (s), 1573 (s, Vpyridyl-H), 1545 (s),
1473(s, Vpyridyl-H), 1425 (s), 1402 (s), 1251 (s), 1150 (m), 1012 (s), 879 (m), 829 (s), 792 (vs, γaryl-H), 732 (s),
689 (s), 657 (s), 634 (s).
3.1.6. [ZnI2L3] (6)
A white solid (yield: 61%) was obtained upon filtration of the solution and washed with
CH2Cl2/CH3OH and dried in a desiccator. Brown crystals were obtained by evaporation of its DMF
solution, which were suitable for X-ray analysis. Anal. calcd for C22H17I2N3Zn: C, 41.12, H, 2.66,
1
N, 6.54%. Found: C, 41.15, H, 2.50, N, 6.60%. H-NMR (800 MHz, DMSO-d6)
δ 9.36 (s, 1H), 9.15 (d,
J = 8.1 Hz, 1H), 9.11 (s, 1H), 9.04 (d, J = 8.0 Hz, 1H), 8.92 (d, J = 4.9 Hz, 1H), 8.41 (d, J = 7.8 Hz, 1H),
8.37 (d, J = 7.6 Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.20 (s, 1H), 7.94 (d, J = 4.3 Hz, 2H), 7.57 (s, 1H),
7.48 (d, J = 7.1 Hz, 2H), 2.43 (s, 3H). IR (cm−1): 3051 (m), 1609 (s), 1573 (s, Vpyridyl-H), 1544 (s), 1473 (s,
Vpyridyl-H), 1424 (s), 1400 (s), 1254 (m), 1155 (s), 1012 (s), 888 (m), 824 (s), 791 (vs, γaryl-H), 731 (s), 689 (s),
657 (s), 634 (s).
3.2. X-ray Determinations
Crystals of complexes
diffractometer (Santa Clara, CA, USA) with omega scans of 0.5◦ per frame with the monochromatic
Mo K = 0.71073 Å) radiation. Cell parameters were retrieved by Agilent CrysAlisPro (Santa Clara,
1–6 intensity data were collected using an Agilent SuperNova single crystal
α (λ
CA, USA) [58], and all the reflections refinement were performed using Agilent CrysAlisPro [59].
The direct method was used to solve structures using the SHELXS-97 (Göttingen, Niedersachsen,
Germany) [60] package and refined using SHELXL-97. Calculations were carried out by the WinGX
System-Version 1.80.03 (Glasgow, Scotland, U.K.) [61]. The remaining hydrogen atoms were inserted
in calculated positions. Least square refinements, with anisotropic thermal motion parameters for
all the nonhydrogen atoms and isotropic for the remaining atoms, were employed. These data are
available free of charge from the Cambridge Crystallographic Data Centre (Cambridge, Cambs, U.K.).
Crystal data of complexes 1–6 are listed in Table 5.

Molecules 2020, 25, 3459
14 of 20
Table 5. Crystal data for compounds 1–6.
Compound
1
2
3
4
5
6
Empirical formula
Formula weight
Temperature
C44H38Br4N6O6S2Zn2 C24H23I2N3O3S2Zn
C22H17Br2N3OZn
564.58
C22H17I2N3OZn
658.56
C22H17Br2N3Zn
548.58
571(2) K
Monoclinic
P21/c
C22H17I2N3Zn
642.56
571(2) K
Monoclinic
P21/c
1261.3
293(2) K
Monoclinic
C2/c
784.74
293(2) K
Orthorhombic
P212121
293(2) K
Monoclinic
P21/c
571(2) K
Monoclinic
P21/n
Crystal system
space group
´
13.2530(5)
14.4141(5)
11.748(2)
20.209(3)
8.2757(2)
8.8978(2)
8.23930(10)
14.9859(2)
8.1062(2)
a (Å)
´
14.8951(5)
15.9409(3)
15.5081(3)
b (Å)
´
24.6133(9)
90
95.604(3)
90
23.075(4)
17.1200(6)
90
98.939(2)
90
2084.71(11)
4
15.8601(3)
16.7323(3)
17.3032(4)
c (Å)
α (◦)
β (◦)
γ (◦)
90
90
90
90
105.1540(10)
90
2171.35(8)
4
90
97.2180(10)
90
2049.62(5)
4
90
96.9060(10)
90
2159.43(8)
4
´
Volume (Å3)
4679.4(3)
4
5478.7(16)
8
Z
Calculated density (Mg/m3)
Absorption coefficient (mm−1
F (000)
1.79
1.903
1.799
5.03
1112
2.015
3.993
1256
1.778
5.11
1080
1.976
4.009
1224
4.584
3.334
)
2496
3040
Crystal size (mm−1
)
0.36 × 0.30 × 0.22
2.95, 27.50
−16→17
0.49 × 0.11 × 0.09
2.80, 29.69
−16→17
0.39 × 0.36 × 0.30
2.73, 27.15
−10→10
0.48 × 0.43 × 0.40
1.84, 27.12
−11→11
0.39 × 0.30 × 0.21
5.52, 26.63
−9→10
0.26 × 0.25 × 0.18
2.37, 27.19
−10→10
θmax, θmin (◦)
Index range h
k
l
−18→15
−27→27
−19→15
−19→20
−13→18
−19→17
−31→30
−31→29
−14→21
−20→20
−20→21
−22→18
Reflections collected/unique
13958/5198
[R(int) = 0.0236]
5198/0/290
1.012
74225/14185
[R(int) = 0.0297]
14185/0/631
1.021
14640/4523
[R(int) = 0.0309]
4523/0/262
1.017
29741/4806
[R(int) = 0.0264]
4806/0/262
1.013
9325/4150
[R(int) = 0.0541]
4150/0/253
0.943
18433/4788
[R(int) = 0.0251]
4788/0/253
0.994
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R1 = 0.0320
wR2 = 0.0889
R1 = 0.0386
wR2 = 0.0931
R1 = 0.0317
wR2 = 0.0777
R1 = 0.0427
wR2 = 0.0852
R1 = 0.0280
wR2 = 0.0657
R1 = 0.0445
wR2 = 0.0716
R1 = 0.0250
wR2 = 0.0650
R1 = 0.0294
wR2 = 0.0677
R1 = 0.0344
wR2 = 0.0630
R1 = 0.0602
wR2 = 0.0660
R1 = 0.0309
wR2 = 0.0825
R1 = 0.0409
wR2 = 0.0880
R indices (all data)
´
Largest diff. peak and hole (e Å−3
)
0.939 and −0.562
1.580 and −0.806
0.703 and −0.448
0.395 and −0.920
0.965 and −0.565
0.807 and −0.822
CCDC number
1969820
1969821
1969822
1969823
1969824
1969825

Molecules 2020, 25, 3459
15 of 20
3.3. Solution Chemistry
The stability of the zinc (II) complexes
1
–
6
was analyzed by UV-vis absorption spectra under
physiological conditions. Stock concentrations of complexes 1–6 prepared with DMSO were diluted
with PBS (pH = 7.4), and stability analysis was performed at 0, 24, 48 and 72 h at 37 ◦C.
3.4. Cell Study
In this assay, human esophageal cancer cell line (Eca-109), human lung cancer cell line (A549),
human hepatocellular carcinoma cell line (Bel-7402) and mouse monocyte macrophage (RAW264.7)
were used to evaluate the antiproliferative activities or cytotoxicity in vitro by CCK-8 assay for
determining cell viability with cisplatin as the positive control. All the cells are purchased from the
American Type Culture Collection (Manassas, VA, USA). The cells were cultured in 100
DMEM growth media containing 10% FBS and 1% PS. First, 3000 cells were added to 96-well plates
and were cultured overnight in a humidified atmosphere at 37 ◦C with 5% CO2. Then, complexes
µL complete
1–6
and cisplatin were diluted with complete DMEM growth media to obtain the working concentration,
which was added to the tissue culture plate and incubated for 72 h. The CCK-8 solution was added to
each well. After 3 h incubation, the absorbance of each well at 490 nm was measured using an enzyme
labeling instrument (Tecan, Grödig, Salzburg, Austrla). Cell morphology was measured and imaged
by an inverted microscope (Nikon eclipses TS100, Tokyo, Japan) and a Nikon digital camera (Tokyo,
Japan). The 50% inhibitive concentration (IC50) was calculated by Graph Pad Prism 5.0 (San Diego,
CA, USA).
3.5. Protein Binding Studies
Bovine serum albumin (BSA) was dissolved in a Tris-HCl buffer (pH = 7.4) solution to prepare a
1.6
×
10−6 M BSA solution. The protein concentration was measured by spectrophotometry at 280 nm
with the extinction coefficient of 44,300 M−1 cm−1. Stock solutions of complexes
1
–6
(1
×
10−2 M) were
prepared in DMSO. The emission spectrum (300–600 nm) of BSA was monitored at the excitation
wavelength of 280 nm.
3.6. DNA Binding Studies
3.6.1. UV-Vis Spectra Titration
The UV absorption of CT-DNA that gave the ratio at 260/280 nm was 1.92, indicating that
the DNA contained no protein. The molar extinction coefficient of CT-DNA was 6600 M−1 cm−1
.
Stock concentrations of complexes 1–6 prepared with DMSO were diluted with Tris-HCl (pH = 7.2)
buffer, and then incubated with different concentrations of CT-DNA. Absorption spectra of the
complexes incubated with different concentrations of CT-DNA (5 min) were recorded at 200–800 nm.
3.6.2. Circular Dichroism (CD) Measurements
CD spectra of compounds 1–6 with different concentrations for interacting with CT-DNA
(400 µM) were detected from 225 to 600 nm at 30 ◦C. Complexes 1–6 were dissolved in a DNA-buffer
(DNA dissolved in Tris-HCl, pH = 7.2) system containing 0.4% DMSO, incubating for 24 h.
3.7. Molecular Docking
The interaction between the complexes and the DNA was simulated by the molecular docking
technique and the docking study was carried out with the AutoDock4.2 program (San Diego, CA,
USA). The crystal data of DNA (ID: 4JD8) and BSA (ID: 3V03) were obtained from the Protein Data
Bank (PDB) and the PDF format of the zinc (II) complexes was acquired from the structures of their
crystals for blind docking and the output for further analysis.

Molecules 2020, 25, 3459
16 of 20
4. Conclusions
Six new zinc (II) complexes were prepared by the reaction of ZnBr2 or ZnI2 with 40-(substituted-
phenyl)-2,20:60,200-terpyridine, which were characterized by elemental analysis, FT-IR, NMR and single
crystal X-ray diffraction. The complexes exhibit interesting photoluminescence at room temperature
and solution stability under physiological conditions. The antiproliferative properties were monitored
using a CCK-8 assay, and the results indicate that the complexes show higher activities against Eca-109,
A549 and Bel-7402 cell lines than cisplatin and display low cytotoxicity towards the normal cell line
RAW264.7. In particular, complexes 1–2 display remarkable effects in all the cells. It is interesting that
the sequence of antiproliferative activity of these compounds is consistent with the electronegativity of
their substituents. Interaction of these complexes with CT-DNA and BSA was obtained by UV-Vis,
CD spectra and fluorescence spectroscopy. The results show that these zinc (II) complexes interact with
CT-DNA through an intercalative binding mode and have a strong binding affinity to BSA through
a static quenching mechanism. The docking of the complexes with the CT-DNA fragment and BSA
were simulated using molecular docking software. It is further validated that the complexes bind with
DNA through intercalative binding and that these complexes spontaneously bind to the active domain
of BSA, suggesting that the complexes may be candidates as potential BSA fluorophores. As seen by
these results, electronic effect, electronegativity and steric effects of the substituents and anions of 1–6
play important roles in bioactivity, which are significant for the design of new zinc complexes with
anticancer potential, and the compounds might be candidates as antitumor drugs.
Supplementary Materials: The following are available online, Figures S1–S6: 1H-NMR spectra of compounds
1
–
6
in DMSO, Figures S7–S12: IR spectra of compounds
the 50% probability level, of compounds
complexes and in PBS buffer solution over a period of 72 h, Figures S19: Microscopic photographs of
Eca-109 cancer cells treated with different concentrations of compound and , Figures S20–S21: Microscopic
photographs of the A549 and Bel-7402 cancer cells treated with different concentrations of compounds and
control photographs, Figures S22: Fluorescence emission spectra of 1.6 M BSA in a Tris-HCl buffer (pH = 7.2)
solution with series concentration of compounds and , and the curves for Stern–Volmer equation are shown
as the insets, Figure S23: Docking conformation of complexes to BSA and amino acid residues surrounding
compounds . Figures S24: Absorption spectra of 20 M of compounds and in a Tris-HCl buffer
pH = 7.2) solution with series concentration of CT-DNA. The plots of A0/(A A0) versus the concentration of
and to CT-DNA at different
1–6, Figure S13–S17: Thermal ellipsoid plot, drawn at
2–6 with atomic numbering scheme, Figures S18: The UV-vis spectra of
2
,
4
,
5
6
2,
4
,
5
6
1–6
µ
1,
2,
3
5
2–6
2
–
6
µ
2,
4,
5
6
(
−
CT-DNA are shown as the insets. Figure S25: CD spectra of compounds
2,
4
,
5
6
concentration ratios, Figures S26: Docking conformation of complexes 2–6 to DNA (PDB ID: 4JD8).
Author Contributions: Conceptualization, Z.M., L.P., T.X. and R.L.; formal analysis, R.L. and J.J.; investigation, R.L.,
H.Y., J.J., J.L., X.L. and D.Y.; resources, D.Y., L.P. and T.X.; writing—Original draft preparation, R.L.; writing—Review
and editing, Z.M., L.P. and T.X.; visualization, R.L.; project administration, Z.M.; funding acquisition, Z.M., L.P.
and D.Y. All authors have read and agreed to the published version of the manuscript.
Funding: This research is funded by the National Natural Science Foundation of China (Grant Nos.
21261002, 31660251, 31860245 and 31960203), the Natural Science Foundation of Guangxi Province (Grant Nos.
2018GXNSFAA138024, 2018GXNSFAA281019 and 2017GXNSFAA198010) and the Central Government Directs
Special Funds for Local Science and Technology Development Projects (ZY1949015).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Baig, M.H.; Adil, M.; Khan, R.; Dhadi, S.; Ahmad, K.; Rabbani, G.; Bashir, T.; Imran, M.A.; Husain, F.M.;
Lee, E.J.; et al. Enzyme targeting strategies for prevention and treatment of cancer: Implications for cancer
therapy. Semin. Cancer Boil. 2019, 56, 1–11. [CrossRef]
Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018:
GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA A Cancer
J. Clin. 2018, 68, 394–424. [CrossRef] [PubMed]
Ferlay, J.; Colombet, M.; Soerjomataram, I.; Mathers, C.; Parkin, D.M.; Piñeros, M.; Znaor, A.; Bray, F. Estimating
the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int. J. Cancer 2018
144, 1941–1953. [CrossRef] [PubMed]
,

Molecules 2020, 25, 3459
17 of 20
4.
5.
Galluzzi, L.; Senovilla, L.; Vitale, I.; Michels, J.; Martins, I.; Kepp, O.; Castedo, M.; Kroemer, G.
Molecular mechanisms of cisplatin resistance. Oncogene 2011, 31, 1869–1883. [CrossRef] [PubMed]
Xing, S.; Qu, Y.; Li, C.; Huang, A.; Tong, S.; Wu, C.; Fan, K. Deregulation of lncRNA-AC078883.3 and
microRNA-19a is involved in the development of chemoresistance to cisplatin via modulating signaling
pathway of PTEN/AKT. J. Cell. Physiol. 2019, 234, 22657–22665. [CrossRef]
6.
7.
Dasari, S.; Tchounwou, P.B. Cisplatin in cancer therapy: Molecular mechanisms of action. Eur. J. Pharmacol.
2014, 740, 364–378. [CrossRef]
Karges, J.; Blacque, O.; Jakubaszek, M.; Goud, B.; Goldner, P.; Gasser, G. Systematic investigation of the
antiproliferative activity of a series of ruthenium terpyridine complexes. J. Inorg. Biochem. 2019, 198, 110752.
[CrossRef]
8.
9.
Ghosh, S. Cisplatin: The first metal based anticancer drug. Bioorganic Chem. 2019, 88, 102925. [CrossRef]
Hamers, F.; Gispen, W.; Neijt, J. Neurotoxic side-effects of cisplatin. Eur. J. Cancer Clin. Oncol. 1991
27, 372–376. [CrossRef]
,
10. Munteanu, A.-C.; Notaro, A.; Jakubaszek, M.; Cowell, J.; Tharaud, M.; Goud, B.; Uivarosi, V.; Gasser, G.
Synthesis, Characterization, Cytotoxic Activity, and Metabolic Studies of Ruthenium(II) Polypyridyl
Complexes Containing Flavonoid Ligands. Inorg. Chem. 2020, 59, 4424–4434. [CrossRef]
11. Huang, W.; Liu, Y.; Wang, J.; Yuan, X.; Jin, H.-W.; Zhang, L.-R.; Zhang, J.-T.; Liu, Z.-M.; Cui, J.-R. Small-molecule
compounds targeting the STAT3 DNA-binding domain suppress survival of cisplatin-resistant human ovarian
cancer cells by inducing apoptosis. Eur. J. Med. Chem. 2018, 157, 887–897. [CrossRef] [PubMed]
12. Bruijnincx, P.C.A.; Sadler, P.J. New trends for metal complexes with anticancer activity. Curr. Opin. Chem. Boil.
2008, 12, 197–206. [CrossRef] [PubMed]
13. Zhang, C.X.; Lippard, S.J. New metal complexes as potential therapeutics. Curr. Opin. Chem. Boil. 2003
7, 481–489. [CrossRef]
,
14. Gezici, S.;
S, ekerog˘lu, N. Current Perspectives in the Application of Medicinal Plants Against Cancer: Novel
Therapeutic Agents. Anti-Cancer Agents Med. Chem. 2019, 19, 101–111. [CrossRef] [PubMed]
15. Barabas, K.; Milner, R.J.; Lurie, D.; Adin, C. Cisplatin: A review of toxicities and therapeutic applications.
Vet. Comp. Oncol. 2008, 6, 1–18. [CrossRef] [PubMed]
16. Cleare, M. Transition metal complexes in cancer chemotherapy. Co-Ord. Chem. Rev. 1974, 12, 349–405.
[CrossRef]
17. Frezza, M.; Hindo, S.; Chen, D.; Davenport, A.; Schmitt, S.; Tomco, D.; Dou, Q.P. Novel metals and metal
complexes as platforms for cancer therapy. Curr. Pharm. Des. 2010, 16, 1813–1825. [CrossRef]
18. Farrell, N. Transition Metal Complexes as Drugs and Chemotherapeutic Agents; Springer Science and Business
Media LLC: Berlin, Germany; Kluwer Academic: Dordrecht, The Netherlands, 2012; Volume 11.
19. Liang, J.-X.; Zhong, H.-J.; Yang, G.; Vellaisamy, K.; Ma, D.-L.; Leung, C.-H. Recent development of transition
metal complexes with in vivo antitumor activity. J. Inorg. Biochem. 2017, 177, 276–286. [CrossRef]
20. Al-Fartusie, F.S.; Mohssan, S.N. Essential trace elements and their vital roles in human body. Indian J. Adv.
Chem. Sci. 2017, 5, 127–136. [CrossRef]
21. Stefanidou, M.E.; Maravelias, C.; Donà, A.; Spiliopoulou, C. Zinc: A multipurpose trace element. Arch. Toxicol.
2005, 80, 1–9. [CrossRef]
22. Liang, X.; Jiang, J.; Xue, X.; Huang, L.; Ding, X.; Nong, D.; Chen, H.; Pan, L.; Ma, Z. Synthesis, characterization,
photoluminescence, anti-tumor activity, DFT calculations and molecular docking with proteins of zinc(ii)
halogen substituted terpyridine compounds. Dalton Trans. 2019, 48, 10488–10504. [CrossRef]
23. Huang, L.; Liu, R.; Li, J.; Liang, X.; Lan, Q.; Shi, X.; Pan, L.; Chen, H.; Ma, Z. Synthesis,
characterization, anti-tumor activity, photo-luminescence and BHb/HHb/Hsp90 molecular docking of
zinc(II) hydroxyl-terpyridine complexes. J. Inorg. Biochem. 2019, 201, 110790. [CrossRef] [PubMed]
24. Lazarevic, T.; Rilak, A.; Bugarcˇic´, Ž.D. Platinum, palladium, gold and ruthenium complexes as anticancer
agents: Current clinical uses, cytotoxicity studies and future perspectives. Eur. J. Med. Chem. 2017, 142, 8–31.
[CrossRef] [PubMed]
25. Belicchi-Ferrari, M.; Bisceglie, F.; Buschini, A.; Franzoni, S.; Pelosi, G.; Pinelli, S.; Tarasconi, P.; Tavone, M.
Synthesis, structural characterization and antiproliferative and toxic bio-activities of copper(II) and nickel(II)
citronellal N4-ethylmorpholine thiosemicarbazonates. J. Inorg. Biochem. 2010, 104, 199–206. [CrossRef]
[PubMed]

Molecules 2020, 25, 3459
18 of 20
26. Adhireksan, Z.; Davey, G.E.; Campomanes, P.; Groessl, M.; Clavel, C.M.; Yu, H.; Nazarov, A.A.; Yeo, C.H.F.;
Ang, W.H.; Dröge, P.; et al. Ligand substitutions between ruthenium–cymene compounds can control protein
versus DNA targeting and anticancer activity. Nat. Commun. 2014, 5, 3462. [CrossRef]
27. Jain, S.; Khan, T.A.; Patil, Y.P.; Pagariya, D.; Kishore, N.; Tapryal, S.; Naik, A.D.; Naik, S.G. Bio-affinity of
copper(II) complexes with nitrogen and oxygen donor ligands: Synthesis, structural studies and in vitro DNA
and HSA interaction of copper(II) complexes. J. Photochem. Photobiol. B Boil. 2017, 174, 35–43. [CrossRef]
28. Farooqi, S.I.; Arshad, N.; Channar, P.A.; Perveen, F.; Saeed, A.; Larik, F.A.; Javeed, A. Synthesis, theoretical,
spectroscopic and electrochemical DNA binding investigations of 1, 3, 4-thiadiazole derivatives of ibuprofen
and ciprofloxacin: Cancer cell line studies. J. Photochem. Photobiol. B Boil. 2018, 189, 104–118. [CrossRef]
29. Liedtke, K.R.; Freund, E.; Hermes, M.; Oswald, S.; Heidecke, C.D.; Partecke, L.I.; Bekeschus, S. Gas
plasma-conditioned ringer’s lactate enhances the cytotoxic activity of cisplatin and gemcitabine in pancreatic
cancer in vitro and in ovo. Cancers 2020, 12, 123. [CrossRef]
30. Boulikas, T.; Vougiouka, M. Cisplatin and platinum drugs at the molecular level. (Review). Oncol. Rep. 2003
,
10, 1663–1682. [CrossRef]
31. Annaraj, B.; Neelakantan, M.A. Synthesis, crystal structure, spectral characterization and biological
exploration of water soluble Cu(II) complexes of vitamin B6 derivative. Eur. J. Med. Chem. 2015
102, 1–8. [CrossRef]
,
32. Ma, Z.; Cao, Y.; Li, Q.; Da Silva, M.F.C.G.; Da Silva, J.J.F.; Pombeiro, A.J. Synthesis, characterization, solid-state
photo-luminescence and anti-tumor activity of zinc (II) 40-phenyl-terpyridine compounds. J. Inorg. Biochem.
2010, 104, 704–711. [CrossRef] [PubMed]
33. Jiang, J.; Li, J.; Liu, C.; Liu, R.; Liang, X.; Zhou, Y.; Pan, L.; Chen, H.; Ma, Z. Study on the substitution effects
of zinc benzoate terpyridine complexes on photoluminescence, antiproliferative potential and DNA binding
properties. JBIC J. Boil. Inorg. Chem. 2020, 25, 311–324. [CrossRef]
34. Li, J.; Liu, R.; Jiang, J.; Liang, X.; Huang, L.; Huang, G.; Chen, H.; Pan, L.; Ma, Z. Zinc(II) Terpyridine
Complexes: Substituent Effect on Photoluminescence, Antiproliferative Activity, and DNA Interaction.
Molecules 2019, 24, 4519. [CrossRef] [PubMed]
35. Addison, A.W.; Rao, T.N.; Reedijk, J.; Van Rijn, J.; Verschoor, G.C. Synthesis, structure, and spectroscopic
properties of copper(II) compounds containing nitrogen-sulphur donor ligands; The crystal and molecular
structure of aqua[1,7-bis(N-methylbenzimidazol-2’-yl)-2,6-dithiaheptane]copper(II) perchlorate. J. Chem. Soc.
Dalton Trans. 1984, 11, 1349–1356. [CrossRef]
36. Sathyadevi, P.; Krishnamoorthy, P.; Bhuvanesh, N.S.; Kalaiselvi, P.; Padma, V.V.; Dharmaraj, N. Organometallic
ruthenium(II) complexes: Synthesis, structure and influence of substitution at azomethine carbon towards
DNA/BSA binding, radical scavenging and cytotoxicity. Eur. J. Med. Chem. 2012, 55, 420–431. [CrossRef]
37. Senthil Raja, D.; Bhuvanesh, N.S.P.; Natarajan, K. Effect of N(4)-Phenyl Substitution in
2-Oxo-1,2-dihydroquinoline-3-carbaldehyde Semicarbazones on the Structure, DNA/Protein Interaction,
and Antioxidative and Cytotoxic Activity of Cu(II) Complexes. Inorg. Chem. 2011, 50, 12852–12866.
[CrossRef]
38. Wani, T.A.; Bakheit, A.H.; Zargar, S.; Bhat, M.A.; Al-Majed, A.-R.A. Molecular docking and experimental
investigation of new indole derivative cyclooxygenase inhibitor to probe its binding mechanism with bovine
serum albumin. Bioorg. Chem. 2019, 89, 103010. [CrossRef]
39. Moghaddam, M.M.; Pirouzi, M.; Saberi, M.R.; Chamani, J. Comparison of the binding behavior of FCCP
with HSA and HTF as determined by spectroscopic and molecular modeling techniques. Luminescence 2013
,
29, 314–331. [CrossRef]
40. Ma, W.; Zhang, S.; Tian, Z.; Xu, Z.; Zhang, Y.; Xia, X.; Chen, X.; Liu, Z. Potential anticancer agent for selective
damage to mitochondria or lysosomes: Naphthalimide-modified fluorescent biomarker half-sandwich
iridium (III) and ruthenium (II) complexes. Eur. J. Med. Chem. 2019, 181, 111599. [CrossRef]
41. Anjomshoa, M.; Fatemi, S.J.; Torkzadeh-Mahani, M.; Hadadzadeh, H. DNA- and BSA-binding studies
and anticancer activity against human breast cancer cells (MCF-7) of the zinc(II) complex coordinated by
5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 2014, 127, 511–520.
[CrossRef]

Molecules 2020, 25, 3459
19 of 20
42. Zhou, P.; Wu, S.; Hegazy, M.; Li, H.; Xu, X.; Lu, H.; Huang, X. Engineered borate ester conjugated
protein-polymer nanoconjugates for pH-responsive drug delivery. Mater. Sci. Eng. C 2019, 104, 109914.
[CrossRef] [PubMed]
43. Behnamfar, M.T.; Hadadzadeh, H.; Simpson, J.; Darabi, F.; Shahpiri, A.; Khayamian, T.; Ebrahimi, M.;
Rudbari, H.A.; Salimi, M. Experimental and molecular modeling studies of the interaction of the polypyridyl
Fe(II) and Fe(III) complexes with DNA and BSA. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 2015
134, 502–516. [CrossRef] [PubMed]
,
44. Heydari, A.; Mansouri-Torshizi, H. Design, synthesis, characterization, cytotoxicity, molecular docking and
analysis of binding interactions of novel acetylacetonatopalladium(ii) alanine and valine complexes with
CT-DNA and BSA. RSC Adv. 2016, 6, 96121–96137. [CrossRef]
45. Ali, M.S.; Al-Lohedan, H.A. Deciphering the interaction of procaine with bovine serum albumin and
elucidation of binding site: A multi spectroscopic and molecular docking study. J. Mol. Liq. 2017
236, 232–240. [CrossRef]
,
46. Shi, J.-H.; Pan, D.-Q.; Jiang, M.; Liu, T.-T.; Wang, Q. Binding interaction of ramipril with bovine serum
albumin (BSA): Insights from multi-spectroscopy and molecular docking methods. J. Photochem. Photobiol. B
Boil. 2016, 164, 103–111. [CrossRef] [PubMed]
47. Kaur, A.; Khan, I.A.; Banipal, P.K.; Banipal, T.S. Deciphering the complexation process of a fluoroquinolone
antibiotic, levofloxacin, with bovine serum albumin in the presence of additives. Spectrochim. Acta Part A
Mol. Biomol. Spectrosc. 2018, 191, 259–270. [CrossRef]
48. Alsaeedi, M.S.; Babgi, B.A.; Hussien, M.; Abdellattif, M.H.; Humphrey, M.G. DNA-Binding and Anticancer
Activity of Binuclear Gold(I) Alkynyl Complexes with a Phenanthrenyl Bridging Ligand. Molecules 2020
25, 1033. [CrossRef]
,
49. Esfahani, N.H.; Salami, F.; Saberi, Z.; Karami, K.; Lighvan, Z.M.; Ramezani, M.; Alibolandi, M.; Farzad, S.A.;
Khayamian, T. DNA G-quadruplexes binding and antitumor activity of palladium aryl oxime ligand
complexes encapsulated in either albumin or algal cellulose nanoparticles. Colloids Surf. B Biointerfaces 2019
176, 70–79. [CrossRef]
,
50. Sirajuddin, M.; Ali, S.; Badshah, A. Drug–DNA interactions and their study by UV–Visible, fluorescence
spectroscopies and cyclic voltametry. J. Photochem. Photobiol. B Boil. 2013, 124, 1–19. [CrossRef]
51. Kellett, A.; Molphy, Z.; Slator, C.; McKee, V.; Farrell, N.P. Molecular methods for assessment of non-covalent
metallodrug–DNA interactions. Chem. Soc. Rev. 2019, 48, 971–988. [CrossRef]
52. Draksharapu, A.; Boersma, A.J.; Leising, M.; Meetsma, A.; Browne, W.R.; Roelfes, G. Binding of copper(ii)
polypyridyl complexes to DNA and consequences for DNA-based asymmetric catalysis. Dalton Trans. 2015
44, 3647–3655. [CrossRef] [PubMed]
,
53. Ribeiro, A.G.; De Almeida, S.M.V.; De Oliveira, J.F.; Souza, T.R.C.D.L.; Dos Santos, K.L.;
Albuquerque, A.P.D.B.; Nogueira, M.C.D.B.L.; Junior, L.B.D.C.; De Moura, R.O.; Da Silva, A.C.; et al.
Novel 4-quinoline-thiosemicarbazone derivatives: Synthesis, antiproliferative activity, in vitro and in silico
biomacromolecule interaction studies and topoisomerase inhibition. Eur. J. Med. Chem. 2019, 182, 111592.
[CrossRef] [PubMed]
54. Meenongwa, A.; Brissos, R.F.; Soikum, C.; Chaveerach, P.; Gamez, P.; Trongpanich, Y.; Chaveerach, U.
Effects of N, N-heterocyclic ligands on the in vitro cytotoxicity and DNA interactions of copper (II) chloride
complexes from amidino-O-methylurea ligands. New. J. Chem. 2016, 40, 5861–5876. [CrossRef]
55. Pagadala, N.S.; Syed, K.; Tuszynski, J.A. Software for molecular docking: A review. Biophys. Rev. 2017
9, 91–102. [CrossRef] [PubMed]
,
56. Mariappan, M.; Alagarsamy, R.; Panneerselvam, A.P.; Anbazhagan, V.; Rajendran, S.; Arunachalam, J.
Synthesis, solvatochromism, photochemistry, DNA binding, photocleavage, cytotoxicity and molecular
docking studies of a ruthenium(II) complex bearing photoactive subunit. J. Photochem. Photobiol. A Chem.
2018, 356, 617–626. [CrossRef]
57. Ibezim, A.; Debnath, B.; Ntie-Kang, F.; Mbah, C.J.; Nwodo, N.J. Binding of anti-Trypanosoma natural products
from African flora against selected drug targets: A docking study. Med. Chem. Res. 2017, 26, 562–579.
[CrossRef]
58. CrysAlis PRO. Agilent; Agilent Technologies: Yarnton, UK, 2012.

Molecules 2020, 25, 3459
20 of 20
59. Sheldrick, G.M. Crystal structure refinement with SHELXL. Acta Crystallogr. Sect. C Struct. Chem. 2015
71, 3–8. [CrossRef]
,
60. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. Sect. A Found. Crystallogr. 2007, 64, 112–122.
[CrossRef]
61. Farrugia, L.J. WinGX suite for small-molecule single-crystal crystallography. J. Appl. Crystallogr. 1999
32, 837–838. [CrossRef]
,
Sample Availability: Samples of the compounds 1–6 are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).