Journal of Inorganic Biochemistry 250 (2024) 112418

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Synthesis, characterization, antiproliferative activity and DNA binding

calculation of substituted-phenyl-terpyridine copper(II) nitrate complexes
Kejuan Lin a, Xinjie Jia a, Xueying Zhang a, Weikeduo Li a, Benwei Wang a, Zhiyuan Wang a,
Xingyong Xue b, *, Xiaosu Fan c, *, Zhen Ma a, *
a
School of Chemistry and Chemical Engineering, Guangxi University, 530004 Nanning, Guangxi, People's Republic of China
b
School of Chemistry and Chemical Engineering, Guangxi Minzu University, 530006 Nanning, Guangxi, People's Republic of China
c
School of Agriculture, Guangxi University, 530004 Nanning, Guangxi, People's Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: Ten 4′- (R-phenyl) -2,2′: 6′, 2′ – terpyridine ligands (R = hydrogen (L1), hydroxyl (L2), methoxyl (L3), methyl­
Copper(II) nitrate complexes sulfonyl (L4), methyl (L5), phenyl (L6), fluoro (L7), chloro (L8), bromo (L9), and iodo (L10)) were synthesized.
Substituted-phenyl-terpyridine The reaction of these ligands with copper(II) nitrate led to complexes 1–10. The characterization of 1–10 was
Antiproliferative activity
carried out by means of mass spectrometry, elemental analysis, infrared spectroscopy and X-ray single crystal
DNA binding calculation
diffraction. Four cell lines including esophageal cancer cell line (Eca-109), human liver cancer cell line (Bel-
Molecular docking
7402), human breast cancer cell line (SIHa) and human normal liver cell line (HL-7702) were selected to carry
out antiproliferation and cytotoxicity experiments in vitro. The results showed that the complexes have strong
inhibitory ability on the growth of tumor cells. In order to study the anticancer mechanism of the complexes, the
binding mode and binding ability of the complexes with DNA were further determined and discussed with
UV–Vis spectroscopy and circular dichroism. The effects of the lowest binding energy and hydrogen bond on the
binding were studied using molecular docking calculation.

1. Introduction have various advantages in anti-cancer biological studies. Copper ion is

one of the essential trace elements in human body. It participates in
Cancer is a common and highly threatening disease, and is one of the many physiological processes, including gene expression, redox, im­
main causes of death in today's society. Its etiology is complicated and mune regulation [20,21], etc. Therefore, copper complexes have good
symptoms are difficult to detect, causing treatment is extremely difficult biocompatibility for human health. Secondly, copper complexes can
[1,2]. According to GLOBOCAN 2020 data, there were 19.29 million exert anti-tumor effects on cancer cells through various mechanisms. For
new cancer cases and 9.95 million new deaths worldwide [3]. In the example, copper complexes can interfere with DNA replication and
research and development process of anticancer drugs, using complexes repair, leading to tumor cell death [22,23]. In addition, copper com­
as anticancer drugs has always been a hot study field for researchers. plexes can also exert anticancer effects by inhibiting angiogenesis [24],
Since Rosenberg found that cisplatin has the effect of inhibiting cell inducing tumor cell apoptosis [25], and enhancing the immune system
growth in 1969 [4], platinum complexes such as carboplatin and oxa­ [26]. Copper complexes can adjust their drug metabolism and distri­
liplatin have entered medical clinical trials and gradually been widely bution by changing their structures and ligands, thereby improving their
used [5–8]. In addition, the complexes of metal ions such as zinc [9,10], efficacy and reducing side effects. This provides better prospects for the
palladium [11,12], copper [13,14] and gold [15,16] have also been clinical application of copper complexes.
found to have excellent anti-cancer capabilities. Substituted terpyridines are excellent ligands with good planarity
New drugs that bind to biological macromolecules, such as thymosin and stability for metal ions. Three N atoms can coordinate with metal
β4, exhibits sensitivity when coordinating with metal ions, and such salts in a tridentate manner, and substituents can be added at different
complexes actively participate in oxidative stress reactions and metal positions at the phenyl ring, thereby changing the chemical properties of
ion metabolism in the body [17–19]. Therefore, copper metal complexes complexes and affecting their biological activity. Based on the above

* Corresponding author.
E-mail addresses: 315254042@qq.com (X. Xue), fanxs@gxu.edu.cn (X. Fan), mzmz2009@sohu.com (Z. Ma).

https://doi.org/10.1016/j.jinorgbio.2023.112418
Received 11 September 2023; Received in revised form 13 October 2023; Accepted 21 October 2023
Available online 21 October 2023
0162-0134/© 2023 Elsevier Inc. All rights reserved.
K. Lin et al. Journal of Inorganic Biochemistry 250 (2024) 112418

advantages, substituted terpyridine complexes may become excellent (1) and N(3) atoms at the ligand are located at both ends of the axis, and
potential anticancer drugs. According to research findings, substituted the N(2) atoms and the two O atoms of the two nitrate ions are located in
terpyridine ligands have excellent biological activity when combined the central plane. The average bond length of the Cu–N bond in the
with metal ions such as zinc [27–31], palladium [32] and silver [33]. Li structure is 1.974 Å, and the average bond length of the Cu–O bond is
and Guan's research works on copper (II) chloride terpyridine complexes 2.152 Å.
have proved the anti-tumor activity of copper complexes that the change There is no classical hydrogen bonding in the structure, but several
of the anions of metal salts may have a greater impact on the biological intermolecular π - π interactions exist in its structure. The centroid dis­
activity of the complexes [34,35]. tance of the intermolecular π - π interaction formed between the ter­
Based on this observation, complexes 1–10 were designed and syn­ minal pyridine ring of the complex: N(1)-C(1)-C(2)-C(3)- C(4)-C(5) and
thesized by using the substituted terpyridine ligands L1–10 and copper the central pyridine ring N(2)-C(6)-C(7)-C(8)-C(9)-C(10) is 3.692 Å. The
(II) nitrate. Their structural formula is [Cu(NO3)2LX (LX ¼ L1–10)], and crystal diagram and cell stacking diagram of other complexes are shown
their structural diagram is shown in Scheme 1. Their structures were in the Figs. S21 and S22.
characterized and confirmed by mass spectrometry, elemental analysis,
infrared spectroscopy and X-ray diffraction method. Their anticancer 2.3. Anti-cancer activities
activities were investigated by in vitro antiproliferative activity mea­
surement. The binding ability of the complexes with DNA was investi­ In order to evaluate the anti-cancer ability of complexes 1–10, four
gated by circular dichroism and ultraviolet spectroscopy. In addition, cell lines, including human esophageal cancer cells (Eca-109), human
the binding modes of these complexes with biological macromolecules, liver cancer cells (Bel-7402), human breast cancer cells (SIHa) and
such as DNA and DNA Topoisomerase, were calculated by molecular human normal liver cells (HL-7702) were selected for in vitro anti-
docking simulation. Through the above research, we aim to investigate proliferation experiments. The IC50 values of the complexes were
whether they have the potential to become a new generation of anti­ calculated according to the dose survival curve of the growth inhibition
cancer drugs. of the four cell lines, and the authenticity and effectiveness of the
complexes were judged by cell morphology and survival number.
2. Results and discussion Meanwhile, the commercially available cisplatin was used as a com­
parison to evaluate the potential value of the complex as an anticancer
2.1. Synthesis and characterization drug.
Fig. 2 shows the 72 h inhibitory effect of complexes 1–10 against
Substituted terpyridine ligands L1-L10 used in the experiment were liver cancer cell Bel-7402. The results of these complexes on Eca-109,
prepared by intermediate condensation of 2-acetylpyridine with a SIHa, and HL-7702 cells are shown in Figs. S23-S25. It can be seen
benzaldehyde with different groups like hydrogen, hydroxy, methoxy, from the figure that the cancer cells have obvious dose-dependent effect
methyl sulfonyl, methyl, phenyl, fluorine, chlorine, bromine, and iodo, on copper(II) terpyridine complexes: when the complex concentration is
respectively. Dissolve the obtained ligands and copper(II) nitrate tri­ high, the cell survival rate is low. It is worth noting that from the dose-
hydrate in dichloromethane and methanol at a ratio of 1:1, and add the dependent results, in normal cells HL-7702, complex 9 exhibits weak
ligand solution dropwise into the metal ion solution. After stirring, stand toxicity at low concentrations, and the drug with the lowest concen­
and slowly vaporize to obtain the crude products. After washed with tration in normal cells even exhibits cell promoting effects, while this
methanol and dichloromethane solvents, complexes1–10 can be phenomenon is not observed in Eca-109 and SIHa cells. Through this
obtained. phenomenon, we can control the drug concentration to make it less toxic
to the normal cells while having an inhibitory effect on the cancer cells.
2.2. Elucidation of the structures of the complexes The Figs. S26-S29 show the morphology of cells under different con­
centrations of the complexes. From the figures, it can be seen that there
The crystals of complexes 2–5, 8, 10 are obtained by hydrothermal or are significantly more cells at low concentrations, and the contour is
evaporation methods (See Table 1). The crystal structures of this series clearly visible; at high concentrations, the number of the cells sharply
of complexes are similar, consisting of a ligand molecule, a copper metal decreases, and a large number of necrotic cells were found, and there
ion, and two nitrate ions. The coordination mode of the copper ion is Cu were also phenomena such as blurred boundaries of the surviving cells.
(NO3)2. Complex 2 is used as an example for illustration of their struc­ However, in SIHa cells, although there was a difference between the
tures (Fig. 1). Three N atoms at the ligand molecule and two O atoms at number of the cells at the highest concentration and that at the lowest
two nitrate ions together form a triangular bipyramidal geometry. The N concentration, it was relatively not obvious, which is consistent with the
results shown in the dose-dependent graph, to indicate that the inhibi­
tory effect of copper(II) nitrate complexes on SIHa cells is not obvious.
Table 2 and Fig. 3 list the IC50 values of complexes 1–10 and cisplatin
on the four test cells. From a numerical perspective, the IC50 values of
the complexes are all lower than those of cisplatin and the difference is
significant. This means that the inhibitory effect of the complexes on the
cancer cells is more significant than that of cisplatin. From the
perspective of the different cell lines, the inhibitory effects of the com­
plexes on Eca-109 and Bel-7402 cell lines are significantly higher than
that of SIHa cell line, which is consistent with the results shown by the
dose-dependent curve. Among them, it is worth noting that complex 2
exhibits extremely strong inhibitory ability on all the three cancer cell
lines, and has a series of lowest IC50 values on Eca-109 cell line, with
extremely significant effects. In the cell morphology diagram, it can also
be observed that there is almost no cell survival in the Eca-109 cell line
corresponding to complex 2 at high concentrations, which may be
caused by hydrogen bonds generated by the hydroxyl substituent. At the
same time, the inhibitory ability of complex 10 against Eca-109 cell line
Scheme 1. The structures of complexes 1–10. has a large gap with the other cell lines, close to one tenth of the IC50

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K. Lin et al. Journal of Inorganic Biochemistry 250 (2024) 112418

Table 1
Crystal data of complexes 2–5, 8, 10.
Complex 2 3 4 5 8 10

Empirical formula C21H15CuN5O7 C23H21CuN5O8 C22H17CuN5O9S C22H17CuN5O6 C21H15ClCuN5O7 C21H16CuIN5O7

Formula weight 512.92 558.99 591.01 510.95 548.37 640.83
Temperature(K) 296(2) 296(2) 296(2) 296(2) 296(2) 293(2)
Crystal system Monoclinic Triclinic Triclinic Monoclinic Monoclinic Monoclinic
space group P21/n P1 P-1 P21/n P21/c P21/c
a(Å) 12.8189(12) 8.4996(17) 7.8985(6) 12.9361(5) 14.6832(10) 14.7432(9)
b(Å) 9.4406(9) 9.4694(19) 10.6711(8) 9.9836(4) 15.0806(8) 15.2295(8)
c(Å) 18.033(2) 15.604(3) 14.7587(11) 17.2048(8) 20.3977(14) 20.7436(13)
α(◦ ) 90 83.48(3) 95.493(6) 90 90 90
β(◦ ) 109.766(12) 76.09(3) 105.314(7) 107.492(5) 105.160(7) 105.329(6)
γ(◦ ) 2053.7(4) 75.70(3) 101.785(7) 90 90 90
Volume (Å3) 3761.8(3) 1179.3(4) 1159.65(16) 2119.23(15) 4359.5(5) 4491.9(5)
Z 4 2 2 4 8 8
Calculated density (Mg/m3) 1.659 1.571 1.693 1.601 1.674 1.895
Absorption coefficient (mm− 1) 1.121 0.986 1.098 1.083 1.181 2.402
F(000) 1044 572 602 1044 2232 2520
Crystal size (mm− 1) 0.49 × 0.46 × 0.32 0.49 × 0.48 × 0.31 0.20 × 0.13 × 0.12 0.49 × 0.38 × 0.28 0.43 × 0.38 × 0.21 0.41 × 0.32 × 0.26
θmax,θmin (◦ ) 2.752–29.437 2.96 to 29.44 2.90 to 29.40 3.11 to 29.60 2.811–29.525 2.862–29.492
Index range h − 15-17 − 11 -10 − 9 -10 − 13-17 − 19-18 − 19-20
k − 12-9 − 12- 11 − 14 - 11 − 13- 10 − 20 − 20 -20-20
l − 24-21 − 17 -19 − 20 - 20 − 19- 23 − 26-26 − 27-25
Reflections collected/unique 12,628/4849 10,329 / 5429 9709 / 5346 11,463 / 5054 28,521/10295 30,142/10845
R(int) 0.0236 0.0253 0.0404 0.0241 0.0242 0.0289
Data/restraints/parameters 4849/0/309 5429 / 0 / 334 5346 / 0 / 349 5054 / 0 / 307 10,295/75/664 10,845/21/664
Goodness-of-fit on F2 1.036 1.033 1.050 1.055 1.023 1.038
Final R indices [I > 2σ(I)] R1 = 0.0349 R1 = 0.0424 R1 = 0.0717 R1 = 0.0581 R1 = 0.0356 R1 = 0.0313
wR2 = 0.0883 wR2 = 0.1129 wR2 = 0.2043 wR2 = 0.1490 wR2 = 0.0842 wR2 = 0.0715
R indices (all data) R1 = 0.0470 R1 = 0.0525 R1 = 0.0868, R1 = 0.0854 R1 = 0.0496 R1 = 0.0448
wR2 = 0.0957 wR2 = 0.1222 wR2 = 0.2298 wR2 = 0.1708 wR2 = 0.0926 wR2 = 0.0801
Largest diff. Peak and hole (e'Å− 3) 0.297and − 0.382 0.564 and − 0.467 0.911 and − 0.957 1.059 and − 1.187 0.318 and − 0.460 0.498 and − 0.776
CCDC 2,287,322 2,287,323 2,287,324 2,287,325 2,287,321 2,287,326

solution of a complex can analyze the changes in UV spectra at different

DNA concentrations. Fig. 4 and Fig. S30 show the corresponding spectra
of these ten complexes. The spectrum shows a clear absorption peak at
260 nm, and with the increase of DNA concentration, the absorption
peak exhibits a significant color increasing effect, i.e. the absorbance
gradually increases. This indicates that during the process of adding
DNA, the complex interacts with DNA, and this binding becomes
stronger with the increase of concentration [37–39]. The absorption
peak appearing at 270–300 nm in the UV spectrum is the self UV peak of
CT-DNA, and the peak gradually increases with the gradual addition of
DNA; the characteristic peaks that appear between 320 and 360 nm are
mainly attributed to charge transfer in the ligand (ILCT).
In order to determine the binding strength of these complexes with
DNA, the Benesi Hildebrand equation is often introduced to calculate the
binding constant as follows [40,41]:
A0 εf εf 1
= + (1)
A − A0 εb − εf εb − εf Kb [DNA]
In this Eqution, A0 and A are the absorbance of a complex and the
absorbance after binding with DNA, respectively; εf and εb are the
extinction coefficients of the free form and a complex after binding with
DNA, respectively. Linear fitting was performed on A0/(A-A0) and 1/
[DNA], with an intercept of εf/ (εb- εf). The slope is εf/Kb(εb- εf).
Fig. 1. Crystal structure of complex 2. Therefore, the fitting value of Kb can be calculated based on the ratio of
intercept to slope. The Kb values of complexes 1–10 are shown in
value of the normal cells, which indicates that complex 10 has a strong Table 3. The binding constants of the complexes are on the same order,
inhibitory ability on the esophageal cancer cells (Eca-109) and a small indicating that the complexes have the same binding mode. However,
toxicity to the normal cells, showing the potential to be developed as a the difference of the substituent groups leads to different binding con­
specific therapy drug. stants. Among them, the binding constants of complexes 2, 6 and 10 are
higher, which indicates that the hydrogen bond generated by the hy­
droxyl, π - π stacking of the phenyl ring and the increase of electro­
2.4. DNA targeting of complexes study
negativity of the halogenated elements may have a greater impact on the
binding ability. Interestingly, the strength of the binding ability is
2.4.1. UV–vis absorption titrations
basically consistent with the antiproliferation activity of the complexes
UV–visible absorption titration can be used to study whether com­
on the cancer cells, which further proves the possibility of the
plexes effectively bind to DNA [36]. To add DNA dropwise to the DMSO

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K. Lin et al. Journal of Inorganic Biochemistry 250 (2024) 112418

Fig. 2. The plots of cell viability vs the concentration of complexes 1–10 against Bel-7402 cells.

Table 2
IC50 values (μM) of complexes 1–10 and the reference complex cisplatin against Eca109, SIHa, Bel-7402 and HL-7702 cells obtained from the dose–response assay.
Complexes Eca-109 SIHa Bel-7402 HL-7702

IC50 95%Confdence IC50 95%Confdence IC50 95%Confdence IC50 95%Confdence

(μM) intervals (μM) (μM) intervals (μM) (μM) intervals (μM) (μM) intervals (μM)

1 1.196 0.706–2.039 4.278 1.946–21.29 1.87 0.746–5.833 1.874 1.199–2.923

2 0.542 0.211–0.917 1.757 1.431–2.160 0.918 0.560–1.340 0.127 0.076–0.188
3 1.513 0.806–2.892 4.273 2.103–15.14 1.705 1.208–2.411 0.84 0.620–1.12
4 0.585 0.025–1.368 3.187 1.427–14.27 0.691 0.175–1.331 0.517 0.306–0.838
5 0.817 0.523–1.206 4.132 1.513–153.2 2.365 1.387–4.401 0.611 0.522–0.711
6 1.184 0.289–3.117 4.716 1.814–123.7 5.982 3.030–25.92 1.31 0.864–1.972
7 1.074 0.373–2.357 3.881 1.830–14.74 2.71 1.476–6.001 2.046 0.769–5.665
8 0.708 0.322–1.265 1.905 0.829–5.111 1.696 1.159–2.495 1.07 0.627–1.80
9 1.248 0.902–1.707 1.934 0.643–8.625 2.213 1.143–4.932 1.113 0.782–1.57
10 0.656 0.552–0.782 3.442 2.447–5.170 2.652 2.003–3.589 5.802 1.666–33.29
Cisplatin 12.25 10.70–14.02 9.085 7.102–11.65 13.07 2.304–74.12 18.02 12.79–25.38

interaction between the complexes and DNA. complexes 1, 2, 3and 5 first weakens and then increases, while the other
complexes show a continuous weakening trend, indicating that the
2.4.2. Circular dichroism (CD) measurements complexes have an impact on the accumulation of DNA bases, The
Circular dichroism is a common method for exploring the structural different manifestations of strength may be due to the significant in­
changes of chiral substances, used to study changes in DNA structure fluence of the substituent structure caused by changes in substituents on
[42–44]. The interaction between the copper(II) nitrate complexes and the π - π stacking and hydrogen bonding interactions between DNA base
DNA was investigated by high-precision circular dichroism. Fig. 5 and pairs and complexes, thereby altering the interaction generated by their
the Fig. S31 show the CD spectra of complexes 1–10 binding to different binding. Of course, both the weakening of the negative peak intensity
amounts of CT-DNA. The CD spectra of DNA after the addition of all the and the different changes in the positive peak are important manifes­
complexes exhibit characteristic positive absorption peaks near 276 nm tations of the embedding binding between the complex and DNA, as the
and characteristic negative absorption peaks near 247 nm. other two no-covalent binding do not cause changes in right-handed
According to the analysis of the circular dichroism of the binding helicity and base stacking. It is worth noting that the red shift of the
between the complex and CT-DNA, the negative peaks at 245-250 nm of positive peak in the spectrum of copper based complexes is extremely
all complexes show a trend of gradually increasing color with the in­ significant. The main reason for the red shift in the spectrum is that
crease of the concentration of the complex. This indicates that as the when the complex is embedded into the DNA structure, it changes the
concentration of the complex increases, the right-handed helicity of structure and geometric configuration of the DNA. The magnitude of the
DNA gradually weakens; The positive peak changes at 275-300 nm vary red shift represents the strength of the configuration change. Such a
with the substituents of the complexes. The positive peak intensity of clear red shift indicates that complexes 1–10 have a significant impact

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K. Lin et al. Journal of Inorganic Biochemistry 250 (2024) 112418

Fig. 3. IC50 values (μM) from the dose-response assay of complexes 1–10 and cisplatin against the Eca-109、SIHa、Bel-7402 and HL-7702 cell lines, after an in­
cubation time of 48 h.

Fig. 5. Circular dichroism spectra of CT-DNA (4.0 × 10 mol/L) in the presence

or absence of complex 1 in Tris-HCl buffer (pH 7.2) at 20 ◦ C.
Fig. 4. UV–vis spectrum for complex 10 in Tris-HCl buffer (1 × 10− 3) with CT-
DNA (pH = 7.2). dichroism peaks (ICD) [45–47]. The ICD peaks of complexes 1–10 are
concentrated at 325-335 nm. The main reason for this result may be that
the ligand itself undergoes certain chiral changes when the complex is
Table 3 embedded in DNA, resulting in changes in the spectrum. The generation
Binding constants Kb values of complexes 1–10 interacting with CT-DNA.
of ICD further proves the influence of complexes on DNA structure.
Complex Kb (M− 1) lg Kb Ra

1 3.017 × 105 5.479 0.99624 2.4.3. Molecular docking studies

2 2.638 × 106 6.421 0.99254 Macromolecular docking calculation plays an important role in drug
3 4.902 × 105 5.690 0.98084 development by simulating the interaction between small molecules and
4 2.405 × 105 5.381 0.99963
biological targets [48,49]. In this experimental study, the interactive
5 1.255 × 105 4.098 0.99413
6 2.770 × 106 6.443 0.95355 molecular graphics program AutoDock [50,51] was used to simulate
7 8.347 × 105 5.921 0.98662 Macromolecular docking based on the structure of the synthesized
8 6.941 × 105 5.841 0.99743 complexes 1–10, respectively to investigate their interactions with the
9 7.545 × 105 5.877 0.98207 DNA structure d (CGCGAATTCGCG) 2 dodecamer (PDB ID: 1BNA) [52],
10 1.119 × 106 6.048 0.98308
ds (ATGCAT) 2 DNA double strand (PDB ID: 4JD8) [53] and the DNA
Topoisomerase structure (PDB ID: 1SC7) [54], and study their potential
on the DNA structure, which is consistent with the anticancer results of binding mode and Binding energy.
the complex. The molecular interaction between complexes 2–5, 8 and 10 and
The ligand in the complex is subjected to the reverse action of DNA, 1BNAand 4JD8 was simulated in detail. The binding free energy is
resulting in a certain chiral effect in the ligand structure that did not shown in the Table 4, and the mode of action effect diagram is shown in
previously have chiral effect, or when metal ions have a special binding Fig. 6 and the Figs. S32-S42. It can be clearly seen that the small
method with DNA, it will lead to the generation of induced circular molecule of the complexes interacts with DNA in the embedded binding

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K. Lin et al. Journal of Inorganic Biochemistry 250 (2024) 112418

Table 4 the theoretical basis is sufficient.

The calculated free energy of binding of complexes 2–5, 8 and 10 with B-DNA In order to understand the mechanism of the antiproliferative ac­
(1BNA), oligonucleotide (4JD8) and DNA Topo-I (1SC7). tivity of the complexes and further clarify their structure–activity rela­
Complex Affinity (kcal/mol) tionship, the molecular docking of the complexes with DNA Topo I
1BNA 4JD8 1SC7
complex was also studied. In general, human Topoisomerase I cleaves a
DNA strand, resulting in double strand breakage and ultimately cell
2 6.67 7.52 4.97
− − −
death. Through molecular docking studies, it can predict the binding
3 − 6.89 − 7.06 − 8.18
4 − 7.43 − 8.94 − 8.53 sites of the complexes and DNA Topo I complexes, the size of binding
5 − 6.89 − 8.04 − 9.53 affinity and various interactions between ligands and receptors. The best
8 − 8.02 − 8.62 − 6.29 binding conformation and its binding free energy are shown in SI.
10 − 6.01 − 8.86 − 3.75 Fig. 7 and the Figs. S43-S47. The best binding energy is between
− 3.75 - 6.29 kcal/mol. It is worth noting that in the optimal binding
mode, and its forces mainly include van der Waals interaction, hydro­ conformation, complexes 2 and 10 do not directly bind to DNA, while
phobic interaction, and π - π superposition. According to the data in the complex 8 is directly embedded in the DNA structure. This indicates that
table, the optimal conformational binding energy of the complexes is the complexes not only interact with DNA, but may also have certain
between − 7.52 - -8.86 kcal/mol. With the increase of the electronega­ interactions with proteins in the process of producing anticancer effects.
tivity of the substituent of the complexes, the optimal conformational Moreover, the complexes also bind to DNA Topo I complexes through π -
binding energy gradually increases, and this rule is basically consistent π stacking, Van der Waals force, hydrophobic interaction and hydrogen
with the anticancer ability of the complexes, which also shows that the bonding and the hydrogen bonding is mainly generated between the
results of the UV titration are consistent with the simulation results, and residues of the small molecules of the complexes and the biological

Fig. 6. The most favorable conformation of complex 2 bound with ds(ATGCAT)2(4JD8).

Fig. 7. The most favorable conformation of complex 10 bound with ds(ATGCAT)2(1SC7).

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K. Lin et al. Journal of Inorganic Biochemistry 250 (2024) 112418

macromolecules. νC=N), 1554(m, νC=C), 1504(m, νC=C),1471 (s, νC=C), 1427 (s,
νC=C), 1387(s, βC–H), 1326 (m, βC–H), 1286 (s, βC–H), 1149 (m,
3. Conclusion βC–H), 1095 (w, βC–H), 1012 (m, βC– H), 966(w, γC–H), 844 (w, γC–H),
798 (m, γC–H), 773 (m, γC–H), 727 (m, γC–H), 678 (w, γC–H), 652 (m),
In this paper, ten complexes 1–10 of the substituted terpyridines 557(m), 534(m). ESI-MS: [CuLB4OHCH3]+: (485.00, 94.47%),
were synthesized with copper(II) nitrate as metal salt, and characterized [CuLB4NO3]+: (512.02, 2.83%).
by elemental analysis, circular dichroism, mass spectrometry and X-ray [Cu(NO3)2L5] (5), Elemental analysis: Anal. Calcd for C22H17Cu­
diffraction. In vitro anticancer experiments have shown that complexes N5O6⋅3.6CH2Cl2⋅2.5H2O: C, 35.68; H, 3.42; N, 8.13, Found: C, 35.41; H,
1–10 have a high ability to kill the cancer cells, and some of them have 3.47; N, 8.46. IR (KBr disc, cm− 1): 3070 (m, νC–H), 1604 (s, νC=N),
low cytotoxicity to the normal cells, which indicate their potential as 1550(m, νC=C), 1477(s, νC=C),1402 (s, νC=C), 1384(s, βC–H), 1325(m,
anticancer drugs. The UV titration results show that the series of com­ βC–H), 1280 (s, βC–H), 1247 (w, βC–H), 1097 (w, βC–H), 1018 (m, βC–
plexes can effectively bind to CT-DNA and have excellent binding abil­ H),821 (w, γC–H), 792 (m, γC–H), 748 (m, γC–H), 727 (m, γC–H), 651
ity. The substituents at the ligand can affect the binding ability of the (w, γC–H),651(m), 580 (m). ESI-MS: [CuLB5OHCH3]+: (421.03,
complexes, and the results are consistent with the anticancer properties. 88.21%), [CuLB5NO3]+: (448.08, 10.60%).
Circular dichroism experiments have demonstrated that the complexes [Cu(NO3)2L6] (6), Elemental analysis: Anal. Calcd for C27H19Cu­
can alter the conformation of DNA, thereby affecting cell activity. The N5O6⋅4.2CH2Cl2⋅H2O: C, 39.54; H, 3.13; N, 7.39, Found: C, 39.81; H,
results of molecular docking calculation also corroborate the results of 3.37; N, 7.08. IR (KBr disc, cm− 1): 3062 (m, νC–H), 1608 (s, νC=N),
anti-cancer experiments, and the binding mode with Topoisomerase 1552 (m, νC=C), 1473 (s, νC=C),1427 (s, νC=C), 1384 (s, βC–H), 1296
proves that the ability of the complexes to affect cell activity may be (s, βC–H), 1247 (m, βC–H), 1163 (w, βC–H), 1012 (m, βC– H), 895 (w,
diverse. γC–H), 827 (w, γC–H), 794 (m, γC–H), 727 (m, γC–H), 698 (w, γC–H),
653 (m). ESI-MS: [LB6 + H+]+: (385.04, 49.96%), [CuLB6 + H+]+:
4. Experimental section (442.98, 77.68%).
[Cu(NO3)2L7] (7), Elemental analysis: Anal. Calcd for C21H14Cu­
Chemicals used in this study were of analytical grade and used as N5O6F⋅3.6CH2Cl2⋅4.5H2O: C, 32.77; H, 3.38; N, 7.77, Found: C, 32.47;
procured from commercial sources. Elemental analyses were run on an H, 3.02; N, 7.56. IR (KBr disc, cm− 1): 3062 (m, νC–H), 1602 (s, νC=N),
Elementar Vario EL III Elemental Analyzer. IR spectra were determined 1564 (m, νC=C), 1515 (m, νC=C), 1473 (s, νC=C), 1434 (m, νC=C),
with a Nicolet iS10 using potassium bromide tablet of complex. Single 1382 (s), 1296 (s, βC– H), 1228 (s, νC–F), 1162 (m, βC–H), 1105 (m,
crystal X-ray diffractions were measured using an Agilent SuperNova βC–H), 1016 (s, βC–H), 892 (w, γC–H), 844 (m, γC– H), 798 (s, γC–H),
diffractometer. Optical densities were recorded using a microplate 750 (s, γC–H), 727 (s, γC–H), 651 (m) and 580 (m). ESI-MS: [CuLB7 +
reader (Infnitem200 Pro). Circular dichroism spectrum of the complexes H+]+: (390.53, 83.91%), [CuLB7NO3]+: (452.03, 43.03%).
with DNA was performed on a Chiracan CD spectropolarimeter (Applied [Cu(NO3)2L8] (8), Elemental analysis: Anal. Calcd for C21H14Cu­
Photophysics, UK). UV–Vis absorption spectrum was performed using a N5O6Cl⋅1.5CH2Cl2⋅2.5H2O: C, 38.40; H, 3.15; N, 9.95, Found: C, 38.26;
Beckman coulter DU800 (Brea, CA, USA). MS spectra were recorded on H, 2.81; N, 9.87. IR (KBr, disc, cm− 1): 3064 (m, νC–H), 1608 (s, νC=N),
an Exactive Mass Spectrometer, followed by ESI ionization. The MS 1552 (m, νC=C), 1475 (s, νC=C), 1473 (s, νC=C), 1384 (s), 1247(w,
spectra can be found in the SI. βC–H), 1161 (w, βC–H), 1072 (w, βC–H), 1014 (m, βC–H), 895 (w,
γC–H), 827 (m, γC–H), 794 (s, γC–H), 725 (w, νC–Cl), 698 (m) and 651
4.1. Synthesis (m). ESI-MS: [CuLB8OH]+: (421.03, 88.21%), [CuLB8NO3OH]+: (484.99,
9.61%).
The ligands L1–L10 were synthesized according to the literature. A [Cu(NO3)2L9] (9), Elemental analysis: Anal. Calcd for C21H14Cu­
mixture of Cu(NO3)2 (2 mmol) and ligands (1 mmol) in 10 mL DMF was N5O6Br⋅2.6CH2Cl2⋅3H2O: C, 33.32; H, 2.99; N, 8.23, Found: C, 33.59; H,
stirred overnight at room temperature and washed with CH2Cl2/CH3OH 3.21; N, 8.07. IR (KBr, disc, cm− 1): 3062 (m, νC–H), 1608 (m, νC=N),
after fltering the solution to prepare complexes 1–10. High quality 1550 (w, νC=C), 1554 (m, νC=C), 1473 (s, νC=C), 1431 (s, νC=C), 1384
crystals of complexes 2, 3, 4, 5, 8 and 10 were obtained by slowly (s), 1247 (m, βC–H), 1161 (w, βC–H), 1072 (w, βC–H), 1024 (m, βC–H),
evaporating their DMF solution at room temperature. 1004 (m, βC–H), 896 (w, γC–H), 829 (m, γC–H), 794 (m, γC–H), 748 (w,
[Cu(NO3)2L1] (1) was synthesized according to the literature γC–H), 725 (m, γC–H), 653 (m, νC–Br). ESI-MS: [CuLB9 + H+]+: (451.11,
[55,56]. 86.26%), [CuLB9NO3]+: (513.95, 11.29%).
[Cu(NO3)2L2] (2), Elemental analysis: Anal. Calcd for C21H15Cu­ [Cu(NO3)2L10] (10), Elemental analysis: Anal. Calcd for C21H14Cu­
N5O7⋅3.2CH2Cl2⋅H2O: C, 36.21; H, 2.94; N, 8.72, Found: C, 35.87; H, N5O6I⋅4.1CH2Cl2⋅1.6H2O: C, 30.15; H, 2.56; N, 7.00, Found: C, 29.83; H,
2.46; N, 8.31. IR (KBr disc, cm− 1): 3437(m, νO–H), 3070 (m, νC–H), 2.26; N, 6.73. IR (KBr, disc, cm− 1): 3062(m, νC–H), 1602 (m, νC=N),
1596 (s, νC=N), 1521(m, νC=C), 1473 (s, νC=C), 1415 (s, νC=C), 1382 1564 (w, νC=C), 1515 (m, νC=C), 1473 (s, νC=C), 1434 (m, νC=C),
(s, βC–H), 1280 (m, βC–H), 1232 (s, βC–H), 1184 (m, βC–H), 1118 (w, 1382 (s), 1228 (m, βC–H), 1162 (m, βC– H), 1103 (w, βC–H), 1016 (m,
βC–H), 1062 (w, βC–H), 1024 (m, βC– H), 893 (w, γC–H), 842 (m, γC–H), βC–H), 893 (w, γC–H), 844 (m, γC–H), 798 (s, γC–H), 750 (w, γC–H),
792 (m, γC–H), 750 (m, γC–H), 725 (m, γC–H), 692 (w, γC–H), 652 (m), 651 (m) and 580(m, νC–I). ESI-MS: [CuLB10 + H+]+: (499.91, 93.15%),
568(m). ESI-MS: [CuLB2 + H+]+: (388.09, 30.42%), [CuLB2NO3]+: [CuLB10NO3]+: (559.91, 37.85%).
(451.86, 88.61%).
[Cu(NO3)2L3] (3), Elemental analysis: Anal. Calcd for C22H17Cu­ 4.2. X-ray determinations
N5O7⋅1.7CH2Cl2⋅2.6H2O: C, 39.64; H, 3.59; N, 9.75, Found: C, 39.27; H,
3.16; N, 9.38. IR (KBr disc, cm− 1): 3068 (m, νC–H), 1596 (s, νC=N), Single crystals 2–5,8,10 were installed on glass fibers for diffraction
1522(m, νC=C), 1473 (s, νC=C), 1433 (s, νC=C), 1383(s, βC–H), 1304 experiments. Single crystal X-ray diffraction intensity data was collected
(m, βC–H), 1238 (s, βC–H), 1186 (m, βC–H), 1161 (w, βC–H), 1062 (w, using an Agilent SuperNova diffractometer, using ω Scanning at 0.5◦ per
βC–H), 1026 (m, βC– H), 835 (w, γC–H), 792 (m, γC–H), 748 (m, γC–H), frame, monochromatic Mo-K α (λ = 0.71073 Å) radiation to obtain a
725 (m, γC–H), 692 (w, γC–H), 654 (m), 586(m). ESI-MS: complete data sphere. The cell parameters were retrieved by the Agilent
[CuLB3OCH3]+: (434.01, 95.71%), [CuLB3NO3]+: (464.05, 5.99%). CrysAlsPro software and all observed reflections were refined using
[Cu(NO3)2L4] (4), Elemental analysis: Anal. Calcd for C22H17Cu­ Agilent CrysAlsPro. The structure was solved directly using the SHELXS-
N5O8S⋅0.6CH2Cl2⋅1.8H2O: C, 41.23; H, 3.34; N, 10.64, Found: C, 40.87; 97 package, and refined using the SHELXS-97 package. It was calculated
H, 3.02; N, 10.33. IR (KBr disc, cm− 1): 2924 (m, νCH2–H), 1610 (s, with WinGX System-Version 1.80.03. The remaining hydrogen atoms

7
K. Lin et al. Journal of Inorganic Biochemistry 250 (2024) 112418

were inserted into the calculated positions. Using least squares acquisition, Writing – review & editing.
improvement, the thermal motion parameters of all nonhydrogen atoms
were anisotropic, while the other atoms were isotropic.
Declaration of Competing Interest
4.3. Cytotoxicity and antiproliferative activities studies
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
The cytotoxicity and antiproliferative activities of the complexes
the work reported in this paper.
were determined to evaluate by CCK-8 assay in three human cancer cell
lines (Eca-109, SIHa and Bel-7402) and one normal cell line (HL-7702).
Data availability
All cells were cultured in DMEM medium supplemented with 10% fetal
bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37 ◦ C
All data are included in the Supporting Information of this article.
and 5% CO2. Cells were inoculated into 96 well plates with 3000 cells
per well. After 16 h, a series of concentrations of complexes 1–10 were
added to the predetermined wells and incubated for 48 h. The inverted Acknowledgements
microscope (Nikon Eclipse TS100) and Nikon digital camera (DXM
1200F) were used to observe the cell morphology and image. The cell This research was founded by the National Natural Science Foun­
survival rate was measured using the commercial cell counting kit-8 dation of China (Grant No. 21261002) , the College Students' Innovative
(CCK-8) (Beyotime Biotechnology, China). GraphPad Prism 5.0 Entrepreneurial Training Plan Program of Guangxi University
(GraphPad Software, San Diego, CA, USA) is used to calculate the 50% (S202210593279) and the Scientific Project of Guangxi Minzu Univer­
inhibition concentration (IC50). sity (Grant: 2020KJQD08).

4.4. UV–Vis spectroscopy measurements Appendix A. Supplementary data

Using 5 mM Tris-HCl and 50 mM NaCl (pH 7.2) as buffer, the UV–Vis Supplementary data to this article can be found online at https://doi.
spectra of the complexes were measured using a DU 800 UV–Vis spec­ org/10.1016/j.jinorgbio.2023.112418.
trophotometer (Beckman Coulter, Fullerton, CA). Dissolved the weighed
complex in 1 mL DMSO solution to prepare 2 mmol/L stock solution. References
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