Synthesis, structural characterization and anti-breast cancer activity evaluation of three new Schiff base metal (II) complexes and their nanoparticles

Article abstract of DOI:10.1016/j.molstruc.2019.126938

Two Schiff base ligands 2-((ethylamino)methyl)-6-methoxyphenol (HL₁) and (E)-2-(((2-(dimethylamino)ethyl)imino)methyl)-6-methoxyphenol (HL₂) with bi-nucleating or tri-nucleating mode have been successfully prepared via an one step or

Full text of DOI:10.1016/j.molstruc.2019.126938

Journal of Molecular Structure 1199 (2020) 126938
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, structural characterization and anti-breast cancer activity
evaluation of three new Schiff base metal (II) complexes and their
nanoparticles
,
*
Di Wu a, Liang Guo b, Si-Jie Li a
a Department of Breast Surgery, The First Hospital of Jilin University, Changchun, Jilin, China
b Department of Pathology, The First Hospital of Jilin University, Changchun, Jilin, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two Schiff base ligands 2-((ethylamino)methyl)-6-methoxyphenol (HL1) and (E)-2-(((2-(dimethylamino)
ethyl)imino)methyl)-6-methoxyphenol (HL2) with bi-nucleating or tri-nucleating mode have been suc-
cessfully prepared via an one step organic condensation reaction, which were further applied in the
synthesis of three new Cu(II) and Ni(II)-based coordination complexes [Cu(L1)2](EtOH) (1), [Ni(L1)2](E-
tOH) (2) and [Cu(L2)(HL2)(SCN)](MeOH)2 (3). Furthermore, a hand grinding technique has been used to
decrease the particle diameter of complexes 1e3, thus forming nano-complexes. In biological activity
research, we revealed the important function of nanoparticle 1 in the treatment of breast cancer.
Mitochondria is critical in generating intracellular energy, and play a vital role in regulating cells life and
death. Therefore, the apoptosis of MCF7 breast cancer cells after treated with nanoparticles was detected
by Annexin V-FITC/PI method. The results showed that nanoparticles 1 could significantly raise apoptotic
cells number, but not nanoparticles 2e3. Moreover, we assessed the mitochondrial membrane potential
and reactive oxygen species (ROS) in MCF7 cancer cells, finally we draw this conclusion, nanoparticle 1
could damage the mitochondrial membrane, leading increased ROS level, and cause the apoptotic cell
death.
Received 18 June 2019
Received in revised form
15 August 2019
Accepted 17 August 2019
Available online 19 August 2019
Keywords:
Coordination complexes
Cu(II) chelating
Ni(II) chelating
Nanoparticles
Breast cancer
Proliferation
Apoptosis
ROS
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
many diseases. During the pathogenesis of myocardial dysfunction,
the increased level of ROS accumulation in cells will lead to the cell
Metal compounds have attracted wide attention due to their
applications in bactericidal, anticancer and flame retardant fields
[1e5]. The success of coordination metal complexes in cancer
chemotherapy has been demonstrated by Cisplatin, which is still
one of the most effective and world's best-selling anticancer drug
[6e9]. However, the Cisplatin and its analogic drugs (such as Car-
boplatin and Oxaliplatin) are limited by the serious side effects,
general toxicity, and drug resistance, which restrict their high-dose
administration [10]. In recent years, drug and bioinorganic chem-
ists have designed novel metal-based anticancer agents due to
obvious clinical problems of chemotherapeutic drugs. These anti-
cancer agents have the characteristics of low toxicity, good selec-
tivity and biological activity [11]. Myocardial dysfunction is getting
more and more attention of researchers, which is related with
apoptosis via the caspase mediated singling pathway.
Schiff base compounds are usually regarded as “privileged li-
gands” due to they are easily prepared by the condensation reaction
between imines and aldehydes. Schiff base ligands can not only
coordinate with various metal ions, but also stabilize them in
different oxidation states. Schiff bases have the characteristics of
imino NCH, it is helpful to elucidate the mechanism of Schiff
racemization and transamination. In recent 20 years, the chemical
research of nitrogen-containing Schiff base metal complexes and
their donors has attracted wide attention because of their broad
application prospects, such as antituberculosis, antineoplastic,
antimalarial, anticancer, antioxidant, antifungal, and anticonvul-
sant, corrosion inhibition as well as anti-inflammatory activity
[12e16]. Bidentate Schiff and the tridentate Schiff ligands have
been widely used in the construction of coordination complexes
which has various clinical and biological applications [17,18]. On the
other hand, Cu(II)-based coordination complexes have cytotoxic,
antiproliferative, genotoxic and antitumour activities. Another
* Corresponding author.
E-mail address: sijie_li666@126.com (S.-J. Li).
https://doi.org/10.1016/j.molstruc.2019.126938
0022-2860/© 2019 Elsevier B.V. All rights reserved.

2
D. Wu et al. / Journal of Molecular Structure 1199 (2020) 126938
driving force for the employment of Cu (II) ion is its low toxicity,
which can be further reduced by formation of the coordination
complexes. In addition, Cu (II) complexes have been used as anti-
cancer agents because of their permeability selective to cancer cell
membranes, and many of these complexes are active against
platinum-resistant cancer cell lines [19,20]. Besides, recent litera-
tures have shown that some Ni(ll)-based complexes also reveal
potential anticancer activities [21e23]. In the design of metal-
organic anticancer drugs, the effects of the metal ions and the
organic ligands are two key factors which contribute to the anti-
cancer activity of the targeted anticancer drugs. To study the co-
ordination surrounding along with the metal type effects on the
anticancer activity of the resulting metal-organic complexes, in this
research, two Schiff base ligands 2-((ethylamino)methyl)-6-
methoxyphenol (HL1) and (E)-2-(((2-(dimethylamino)ethyl)
imino)methyl)-6-methoxyphenol (HL2) with bi-nucleating or tri-
nucleating mode have been successfully prepared via an one step
organic condensation reaction, which were further applied in the
synthesis of three new Ni(II) and Cu(II)-containing coordination
complexes [Cu(L1)2](EtOH) (1), [Ni(L1)2](EtOH) (2) and
[Cu(L2)(HL2)(SCN)](MeOH)2 (3) (Scheme 1). Furthermore, the green
grinding technique has been used to decrease the particle diameter
of complexes 1e3, thus forming nano-complexes. We also evalu-
ated the cell viability and proliferation of MCF7 human breast
cancer cells after treated with nanoparticles 1e3. Firstly, we eval-
uated the anticancer effect of nanoparticles 1e3 on MCF7 cells with
CCK-8 assay, results indicated only nanoparticle 1 could inhibit the
proliferation and activity of MCF7 cells, and had no cytotoxicity to
normal human cells. Further, we convinced that nanoparticle 1
could induce apoptosis and ROS accumulation in MCF7 cells by
triggering mitochondrial fragmentation. In general, these findings
mainly reflect that the compound has good anticancer activity in
the treatment of human breast cancer.
thermogravimetric analyzer was used for thermogravimetric
analysis in nitrogen atmosphere with heating rate of 10 C/min. On a
Rigaku Dmax 2500 diffractometer which has Cu-K
a radiation
(
l
¼ 1.5418 Å), we carried out the powder X-ray diffraction ana-
lyses. We determined the morphology of the nanostructured
compound (S-4200, Hitachi, Japan) via scanning electron micro-
scopy. Ultrasound was produced via a multi-wave KQ2200DE with
40 kHz frequency. Fourier transform infrared data were obtained
from KBr pellets in the range of 4000ꢀ400 cmꢀ1 on a 2000 spec-
trometer (Fig. S1). On
a PerkinElmer lambda which is 35
ultravioletevisible spectrophotometer we recorded the electronic
spectra (Fig. S2).
The normal lung cell line BEAS-2B and human breast cancer cell
line MCF7 was purchased from American Type Culture Collection
(ATCC, Rockville, MD), then cultured in ATCC-formulated RPMI-
1640 (Gibco, NY, USA) and Dulbecco's modified Eagle's medium
(DMEM; Gibco, NY, USA), respectively. 2% L-glutamine, 10% (V/V)
heat inactivated fetal bovine serum (FBS) as well as 100 U/mL
penicillin Streptomycin Solution were added to the culture me-
dium. The cells were cultured at 5% CO2, 37 ꢁC. On the basis of the
cell states, the culture medium was replaced, followed by passage
when 80% of them were fused.
2.2. Preparation of complexes 1e3
For complex 1, we added Cu(NO3)2$3H2O which is 0.24 g and
1 mmol and Schiff base ligand HL1 which is 0.181 g and 1 mmol into
ethanol solution of 20 mL and mixed them fully by magnetic stir-
ring. The mixture was cooled to room temperature after reflux for
2 h. We filtered the blue precipitation of Cu(II) complex which we
obtained, then washed it in cold ethanol of 15 mL, and dried by
anhydrous CaCl2 in vacuum. The obtained solid was then dissolved
in the acetonitrile and then kept silence for two weeks to afford the
single crystals of complex 1 suitable for X-ray diffraction. Yield:
160 mg (41%, based on Cu(NO3)2$3H2O). Anal. Calc for
1
2. Experimental
(C22H30CuN2O5): C, 56.70; H, 6.49; N, 6.01%. Found: C, 56.59; H,
6.36; N, 5.94%. UVeVis: lmax (nm) (ε, Mꢀ1, cmꢀ1) (CH3OH): 233
(6.7 ꢂ 103), 268 (3.6 ꢂ 103), 333 (2.4 ꢂ 103), 410 (1.4 ꢂ 103).
The synthesis method of complex 2 was similar to that of
complex 1 except Ni(NO3)2$6H2O which is 0.29 g and 1 mmol has
been used to replace the Cu(NO3)2$3H2O. Yield: 201 mg (53%, based
on Ni(NO3)2$3H2O). Anal. Calc for 2 (C22H30N2NiO5): C, 57.30; H,
6.56; N, 6.07%. Found: C, 56.68; H, 6.62; N, 6.01%. UVeVis: lmax
(nm) (ε, Mꢀ1, cmꢀ1) (CH3OH): 231 (1.2 ꢂ 104), 270 (6.6 ꢂ 103), 340
(2.8 ꢂ 103), 430 (1.4 ꢂ 103), 540 (9.5 ꢂ 102).
2.1. Chemicals and measurements
All chemicals were available on the market and were utilized
with no further purified. DMSO was applied as the solvent medium
in the biological studies. We prepared the two organic ligands by
utilizing the literature approach [24]. The elements of N, H and C
were analyzed by Perkinelemer 240C analyzer. PE diamond
For complex 3, we added NaSCN which is 0.08 g and 1 mmol as
well as HL2 which is 0.448 g and 2.0 mmol to CH3OH of 20 ml and
dissolved them. Then we put the required metal salt
Cu(NO3)2$3H2O which is 0.24 g and 1 mmol) to CH3OH of 20 mL.
The solution was refluxed for about 3 h. After the methanol solution
was cooled to room temperature, the blue bulk single crystal of 3
was acquired by slow evaporation. Yield: 201 mg (53%, based on
Cu(NO3)2$3H2O). Anal. Calc for C27H42CuN5O6S: C, 51.62; H, 6.74; N,
11.15%. Found: C, 51.26; H, 6.26; N, 11.23%. UVeVis: lmax (nm) (ε,
Mꢀ1
,
cmꢀ1
)
(CH3OH): 230 (1.3 ꢂ 104), 270 (6.8 ꢂ 103), 342
(2.4 ꢂ 103), 426 (1.5 ꢂ 103), 570 (1.9 ꢂ 103).
The prepared complexes 1e3 were dissolved in 1% DMSO for
further experiment.
2.3. Crystal structure and refinement
Using a Xcalibur, Eos Gemini CCD diffractometer (Agilent
Technologies Inc) equipping graphite-monochromatized Enhance
(Mo) X-ray Source (
collect the single-crystal data for compounds 1e3. The data was
l
¼ 0.71073 Å) and 4ꢀ
u scan technology to
Scheme 1. The synthesis routes for the complexes 1e3.

D. Wu et al. / Journal of Molecular Structure 1199 (2020) 126938
3
compressed by the bruker-saint package to generate hkl file.
SADABS program is used for absorption correction. The structure of
anisotropic non-hydrogen atoms was further refined by utilizing
the SHELXL software (version_number: 2018/3) on F2 after solving
the direct method on the basis of the SHELXS software [25]. By
utilizing the riding model, H atoms were added to the calculations.
For the highly disordered nature of the lattice solvents, they could
not be figured out from the electron density map via the structural
refinement, and their detailed information has been determined
via the elemental analysis and TGA data [26,27]. The refinement
results and crystallographic parameters are summarized in Table 1.
well culture plate at the concentration of 2 ꢂ 105 cells per well.
The culture method is described above. The cells were separated
into different groups, then treated them utilizing nanoparticles 1e3
(1 ꢂ IC50) for 24 h, respectively. Apoptosis inducer kit and dimethyl
sulfoxide were utilized as the positive and negative contrast. After
treatment, the cells were gained with 0.25% w/v trypsin and
washed three times with phosphate buffer at 4 ꢁC. Then, Annexin
V-FITC and PI were stained in 100
mL 1 ꢂ Binding Buffer and
cultured in dark at room temperature for 15 min. After washing
with PBS twice, the stained cells were determined by flow cytom-
etry (BD, NJ, USA) at 488 nm excitation wavelength and 525 and
625 nm emission wavelength utilizing the flow cytometry.
Apoptotic analysis was performed in three independent
experiments.
2.4. Anti-proliferation assay by CCK-8
To examine whether nanoparticles 1e3 could cause the death of
MCF7 cancer cells, we used serious doses of nanos to treat cells for
24 h. The cell Counting Kit-8 (CCK-8) used in this experiment was
acquired from Japan Dojindo. According to the scheme, we tested
the survival rate and proliferation rate of normal lung cell line
BEAS-2B and human breast cancer cell line MCF7 after 24 h treat-
ment by nanoparticles 1e3 [28]. Briefly, MCF7 and BEAS-2B cells at
logarithmic growth stage were planted on 96-well plate at
1 ꢂ 104 cells per well, and cultured in 37 ꢁC and 5% humidified CO2
condition. When the cells achieved 70%e80% confluence, the cells
were treated with 1e3 (1, 2, 4, 8, 10, 20, 40, 80, 100 mM) consistence
of nanoparticles for 24 h. Then, we removed the culture medium,
and the cells were washed three times with pre-sealed PBS. CCK8
(Sigma) was cultured in 100 mL medium without FBS. The cells were
cultured in darkness for 2 h. The cells number in the three holes
was measured by absorption spectrophotometry of monosodium
2.6. Cell cycle assay
Apoptotic cells often combined with cell cycle arrest. So, we
detect the influence of nano 1e3 on the cancer cell cycle of MCF7.
Propidium iodide (PI) (BD Bioscience, USA) was used for cell cycle
analysis in this experiment following the instructions [30]. In brief,
the MCF7 cancer cells were fostered and treated with an indicated
concentration of nanoparticles 1e3 as described above. After that,
the cells were digested, washed with the cold PBS, and fixed
overnight with 75% ethanol at ꢀ20 ꢁC. 500
mL PI solution was added
for incubation for 15 min and collected for flow cytometric analysis.
All experiments needed to be performed at least three times.
salts
with
WST-8
(2-(2-methoxy-4-nitrobenzene)-3-(4-
nitrobenzene)-5-(2,4-disulfonophenyl) -2H tetrazole reduced at
450 nm. The cell proliferation rate was calculated according to the
measured optical density (OD) value. All experiments needed to be
repeated three times. Used SPSS version 22.0 to calculate IC50 value.
2.7. Mitochondrial membrane potential assay
As mentioned above, we utilize JC-1 dye to analyze the changes
in mitochondrial membrane potential [31]. MCF7 cancer cells were
cultured overnight at 5% humidified CO2 in a 6-well culture plate
with a concentration of 1 ꢂ 106 cells/pore at 37ꢁ Celsius. The cells
were then treated with 1e3 nanoparticles at a specified concen-
2.5. Cell apoptosis detection
On the basis of producer' instructions, apoptotic MCF7 cancer
cells percentage was determined with the Annexin V-FITC/PI
apoptosis detection Kit (BioVision, Mountain View, CA, USA) [29]. In
brief, we collected MCF7 cancer cells and inoculated them on a 6-
tration for 24 h. After treatment, cells were dyed with 1 mg/ml JC-1
dye at 37 ꢁC for 20 min to avoid illumination. Then trypsinized,
harvested, and used PBS to wash the cells, and subjected to flow
cytometry analysis.
Table 1
Refinement and Structural parameters indexes for complex 1e3.
Identification code
1
2
3
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a/Å
C20H24CuN2O4
419.95
150.15
monoclinic
P21/c
10.9632(7)
10.4428(3)
9.6135(3)
90
96.892(5)
90
1092.66(8)
2
1.276
1.024
C20H24N2NiO4
415.12
150.15
orthorhombic
P21212
9.8663(3)
23.424(2)
4.912(3)
90
90
90
1135.2(7)
2
1.214
C25H34CuN5O4S
564.17
293(2)
triclinic
P-1
9.6870(9)
10.3590(10)
17.3597(15)
101.621(8)
96.025(8)
107.117(8)
1605.4(3)
2
b/Å
c/Å
ꢁ
ꢁ
a
/
b
/
ꢁ
g
/
Volume/Å3
Z
rcalcg/cm3
1.167
0.778
m
/mmꢀ1
0.878
Data/restraints/parameters
2714/1/126
1.096
2526/0/125
1.000
8913/2/331
1.026
Goodness-of-fit on F2
Final R indexes [I ꢃ 2
s
(I)]
R1 ¼ 0.0517,
1.08/-1.01
1906552
u
R2 ¼ 0.1590
R1 ¼ 0.0597,
1.12/-0.87
1906553
u
R2 ¼ 0.1341
R1 ¼ 0.1072,
1.67/-1.07
1906554
u
R2 ¼ 0.2786
Largest diff. peak/hole e Åꢀ3
CCDC

4
D. Wu et al. / Journal of Molecular Structure 1199 (2020) 126938
2.8. Intracellular ROS detection
acquired by slowly evaporating the mixture of HL1 and
Cu(NO3)2$3H2O. The prepared metal-organic complexes are soluble
in dimethyl sulfoxide, ethanol, methanol, acetone, acetonitrile and
other common solvents. Elemental analysis of metal complexes
1e2 showed that the ratio of M(II) ion to Schiff base ligand HL1 is
1:2. The phase purity of the two as-prepared crystalline products is
confirmed via the PXRD measurements and their atomic arrange-
ments are established via the structural solution and refinements
and structural solution on the basis of the corresponding crystal
data. The single crystal X-ray diffraction study reveal that complex
1 belongs to the monoclinic crystal system with the space group
P21/c and demonstrates a mononuclear structure. The data collec-
tion and refinement parameters are listed in Table 1. As shown in
Fig.1a, the basic molecular repeating unit of 1 is composed of one L1-
ligand and a half of crystallographically independent Cu (II) ion. The
coordinate geometry of the central Cu (II) ion is completed by two N
atoms (N1 and N1A) as well as two O atoms (O1 and O1A) from two
different L-1 ligands, which forms a completely square plane coor-
dinate geometry (Fig. 1b). The distances of all the Cu(II)eO as well
as Cu(II)eN bond are in the normal ranges, and are comparable
with those of similar compounds in the literature [13,14]. As for the
L-1 ligand, its azomethine (N1eC8) bond length is 1.265(3) Å, which
is in the range characteristic of C]N values. C9eH9A$$$O1 forms
an intramolecular hydrogen bond interaction which could further
stabilize the whole network. The packing of the structure of 1 re-
sults in a 3D supremolecular structure with a solvent accessible free
void of 19.1% as showed by the software PLATON, which is stabi-
The accumulation of ROS in cells was measured by fluorescent
probe 2,7-Dichlorofluorescein diacetate. In the present of ROS. The
20,70-dichloro fluorescin diacetate could be translated into 20,70-
dichloro fluorescin, and usually as the index of intracellular ROS
production [29]. In this study, 5 mmol/L DCFH-DA was pre-loaded in
37 ꢁC alkaline medium for 30 min, using serum-free medium to
wash 3 times, and treated with various nanoparticles 1e3. Flow
cytometry was used to detect the fluorescence of ROS in each group
at 488/530 nm excitation wavelength, and the fluorescence was
analyzed by FlowJo 7.6 software. All experiments need to be done
three or more times.
2.9. Statistical analysis
In this study, all required experiments were conducted in trip-
licate form, and the data were exhibited in the form of
mean standard deviation (SD) in three independent experiments.
Utilizing student's t-test to compare the two groups, and using one-
way ANOVA to compare Prism Software with more than three
groups. When ***p < 0.005, **p < 0.01 as well as * p < 0.05, we
considered the differences were remarkable.
3. Results and discussion
3.1. Molecular structure and characterization for complexes 1-3
lized by CH$$$p and CH/O weak interactions (Fig. 1c).
Complex 2 is a mononuclear complex and its structure is char-
acterized by single crystal X-ray crystallography, which reveals that
it shows a similar atomic arrangement with complex 1 but with
different coordination surrounding for the centered metal ion. The
Refluxing ethylamine and 2-hydroxy-3-methoxybenzaldehyde
in the ethanol solution for 3 h to afford the HL1 ligand, which is
white solid after the removal of the solvents via the rotary evapo-
ration. In CH3CN solution, single crystals of complex 1e2 were
Fig. 1. (a) 1's asymmetric unit view (generated using the OLEX 2). (b) View around the plane coordinates of Cu(II) ions in 1 (generated using the Diamond 3.2k). (c) 1's 3D packing
diagram view (generated using the Diamond 3.2k).

D. Wu et al. / Journal of Molecular Structure 1199 (2020) 126938
5
Fig. 2. (a) 20 s asymmetric unit view (generated using the OLEX 2). (b) View of coordination mode for Ni(II) ion in 2 (generated using the Diamond 3.2k). (c) View of H-bond
interaction in 2 (generated using the Diamond 3.2k). (d)20 s 3D packing diagram view (generated using the Diamond 3.2k).
structural solution and the refinement results demonstrate that
complex 2 locate in the orthogonal space group P21212 and behave
as a discrete structure. The asymmetric unit of 2 consists of a Schiff
base ligand and a Ni (II) ion (Fig. 2a). The coordination environment
of Ni(II) ion is finished by two phenoxido-O-donors of O1 and O1A
as well as two imine N-donors of N1 and N1A from two Schiff base
ligands, resulting in a tetrahedral geometry (Fig. 2b). All the dis-
tances of Ni(II)eN as well as Ni(II)eO bond are in normal ranges and
are comparable with those of similar compounds on the basis of
N,O-donor Schiff base ligands in the literatures [8e10]. Two phe-
noxy oxygen atoms satisfy the double positive charge of Ni (II) ion.
Compound 2 has a C2 axis in which Ni (II) ions are located in the
symmetric center. The planes angle N1eNieO1 with
N1AeNi1eO1A is 89.12ꢁ, which means the flat tetrahedral geom-
etry. As for L-1 ligand, its azomethine (N1eC8) bond length is
1.271(3) Å, which is in the range characteristic of C]N values.
Complex 2 forms 1D H-bonding network along c axis, involving the
C9eH9A$$$O1 interactions with the distance of donor-acceptor is
2.152 Å (Fig. 2c). The packing of the structure of 2 results in a 3D
supremolecular structure with a solvent accessible free void of
environment is usually found for the Cu ion with þ2 oxidation
state, while the Cu ion with þ1 oxidation state usually has the
coordination numbers less than five [33e35]. The analysis results of
single crystal X-ray show that the ligand to metal stoichiometry for
complex 3 is 2:1. The X-ray single crystal diffraction study reveals
that complex 3 belongs to the triclinic system of space group P-1.
The perspective and selective atomic numbering scheme of the
complexes are shown in Fig. 3a. It shows that there is one L-2 ligand,
one HL2 ligand, one Cu (II) ion as well as one coordinated SCNꢀ
group in the molecular unit. The structure of the complex composes
of a distorted octahedral geometry of hexacoordinated Cu(II) ion,
whose coordination environment is formed by two phenoxy oxy-
gen, two imine nitrogen atoms and a amine nitrogen atom from
two demodified Schiff base ligands. Thiocyanate nitrogen atoms
occupy the sixth coordination position of Cu (II), thus completing
the distorted octahedral geometry of Cu (II). The octahedron
equatorial plane is composed of phenoxy atom, imine-nitrogen
atom as well as amine-nitrogen atom, while the thiocyanate-
nitrogen atom and phenoxy-oxygen atom are the axial co-
ordinates. An interesting finding is that the bond length of the
equatorial part is longer than its axial bond length. This may be due
to the steric hindrance around the metal center. Bidentate binding
model of tridentate Schiff base is the most interesting structural
characteristic of the complex. This keeps the Schiff base still
pendant. There is a difference between the ideal value and the bond
angle (90, 180), which clearly shows the deformation of octahedral
geometry. The bond length of the Cu(II)eN amine is longer than
that of the Cu(II)eN imine, which was also found in previously
reported analogous complexes on the basis of N, O - donor Schiff
base ligands [13e16]. The bond length Cu(II)eN imine in tridentate
bonded Schiff bases is slightly shorter than that in bidentate
25.1% as showed by software PLATON, which is stabilized by CH$$$
p
and CH/O weak interactions (Fig. 2d).
A yellow solid ligand (E)-2-(((2-(dimethylamino)ethyl)imino)
methyl)-6-methoxyphenol (HL2) was obtained by refluxing N, N-
dimethyl-1, 2-diaminoethane and 3-methoxysalicylaldehyde in the
methanol solution for 1 h. The acquired ligand were directly uti-
lized for preparing targeted Cu(II) coordination complexes.
Furthermore, the þ2 oxidation state of the Cu(II) ion has been
confirmed via the bond valence sum (BVS) calculation, which af-
fords a total value of 2.04 for the Cu(II) ion [32]. According to the
literature,
the
six-coordinated
octahedral
coordination

6
D. Wu et al. / Journal of Molecular Structure 1199 (2020) 126938
Fig. 3. (a) 3's asymmetric view (generated using the OLEX 2). (b) View for the CeH$$$
p
interactions in 3 (generated using the Diamond 3.2k). (c) 3's 3D packing diagram view
(generated using the Diamond 3.2k).
bonded Schiff bases. The complex exhibits obvious CeH$$$
teractions (Fig. 3b). The H atom on the methoxyl group participates
in inter-molecular CeH$$$ interaction with the benzene ring
(C1eC2eC3eC4eC5eC6). Owing to the forms of inter-molecular
CeH$$$ interaction, in the complex crystal packing, one-
p
in-
complexes 1e3 prepared, their PXRD patterns were collected using
their freshly prepared crystalline samples at room temperature.
According to the information in Fig. 4a, the resulting PXRD curves
are all consistent with those calculated from their corresponding
crystal data, confirming the bulk phase purity of as-prepared three
complexes. Thermogravimetric analysis (TGA) was carried out to
probe their thermodynamic stability along with the information of
the lattice guests (Fig. 4b). For complex 1, 9.46% weight loss could
be found between the temperature of 25 and 140 ꢁC, which could
be regarded as the result of the removal of one lattice EtOH
p
p
a
dimensional array is formed (Fig. 3c).
3.2. PXRD, TGA and SEM characterizations
In order to examine the phase purity of the Schiff base
Fig. 4. (a) Complexes 1e3's PXRD patterns; (b) Complexes 1e3's TGA curves.

D. Wu et al. / Journal of Molecular Structure 1199 (2020) 126938
7
Fig. 5. The SEM images of nanoparticles 1 (a), 2 (b) and 3 (c).
Fig. 6. Nanoparticle 1 restrains the proliferation and viability of MCF7 cells. (A) The survival curves of MCF7 cells treated with 1e3 nanoparticles and ligands (1,2,4,8,10,20,40,80,100
M) for 24 h were plotted by CCK8 method.(B) normal lung cell line BEAS-2B were treated with the same consistence of ligands and nanoparticles 1e3, CCK-8 method was used to
m
measure cell viability. (C) The MCF7 cell viability curves after treated with complexes 1e3 determined by CCK-8. The data were expressed as three independent experiments
mean SD.
molecule (calcd 9.87%). After 142 ꢁC, 20% of the weight loss could be
found to drop sharply owing to the decomposition of the discrete
structure. Complex 2 TGA profile shows that a weight loss rate of
10.2% from room temperature to 167 ꢁC, this is related to the escape
of one lattice EtOH molecule. The framework could be stable up to
the temperature of 170 ꢁC. The thermogravimetric data plot of
complex 3 shows a 10.8% weight loss started from the temperature
of 25 ꢁC till the temperature of 195 ꢁC, it could be considered that

8
D. Wu et al. / Journal of Molecular Structure 1199 (2020) 126938
Table 2
IC50 value of nanoparticles 1e3, complexes 1e3 and ligands (mM).
Cell/drug
Oxaliplatin
Nanoparticle 1
Nanoparticles 2
Nanoparticles 3
Complex 1
Complex 2
Complex 3
HL1
HL2
MCF7
BEAS-2B
9.11 0.21
>70
2.02 0.08
>70
>70
>70
>70
>70
43.63 2.01
e
39.53 1.64
e
22.21 1.31
e
>70
>70
>70
>70
Fig. 7. Nano 1 promotes the MCF7 cell apoptosis and cell cycle arrest. Using 1e3 (1 ꢂ IC50) nanoparticles specified doses to treat the MCF7 cancer cells for 24 h. (A) the cell apoptosis
was detected by Annexin V-FITC apoptosis detection kit in flow cytometry. (B) Flow cytometry was used to quantitatively evaluate and analyze the distribution of cell cycle. Each
group was repeated three times. The results were represented as three independent experiments means SD. Compared with the control group, *p < 0.05 was obviously different.
this is due to the weightlessness of two lattice MeOH molecules.
The extra part is due to the other parts of the molecule are dis-
integrated at such high temperatures. It need to be pointed out that
the decomposition temperature of complex 3 is higher than that of
complex 1, which is due to the stronger chelating ability of tri-
nucleating HL2 ligand.
In light of the following bioactivity evaluation, it is necessary to
make the above coordination complexes with micron dimension
(Fig. S3) into nano-scale, which could promote drug release to the
whole body and absorbed by specific tissues through intravenous

D. Wu et al. / Journal of Molecular Structure 1199 (2020) 126938
9
Fig. 8. Nano 1 triggers mitochondrial fragmentation and intracellular ROS accumulation. Using 1e3 (1 ꢂ IC50) nanoparticles to treat the MCF7 cancer cells with 70%e80% confluence
for 24 h. (A) The ratio of JC-1 aggregates to JC-1 monomers in MCF7 cancer cells treated with different nanoparticles 1e3 was studied. (B) Using ROS detection kit to quantitatively
analyzed ROS content in MCF7 cancer cells.

10
D. Wu et al. / Journal of Molecular Structure 1199 (2020) 126938
injection. Interestingly, for single crystals of complexes 1e3 when
subjected to a mechanical grinding in a mortar and pestle for about
30 min, crystalline nanoscale complexes were obtained. Powder X-
ray diffraction (PXRD) studies confirm the crystalline properties of
nanomaterials. It is also observed that PXRD patterns of these nano-
compounds completely match with bulk PXRD patterns and
simulation of corresponding complexes. The production of the
nanoparticles was further evidenced through SEM researches that
are acquired via dropping nanoparticles in DMSO dispersion solu-
tion on glass surface. The morphology of particles for complexes is
observed as spherical for nanoparticle 1, sheet form for the nano-
particles 2e3. The average size for the nanoparticle 1 is around
170 nm and the average thickness for the complexes 2 and 3 is
48 nm and 76 nm, respectively (Fig. 5).
We speculated the nano 1 might increase the level of ROS and ul-
timately induce apoptosis of MCF7 cells. According to the result
shown in Fig. 8B, the nano 1 significantly increased intracellular
ROS accumulation, and the ROS level was not changed in the
nanoparticles 2e3 treatment group. These results suggest that only
nanoparticle 1 can induce the death of MCF7 cells, combined with
ROS accumulation and mitochondrial death in human breast cancer
cells.
4. Conclusion
In summary, we have successfully prepared two Schiff base li-
gands 2-((ethylamino)methyl)-6-methoxyphenol (HL1) and (E)-2-
(((2-(dimethylamino)ethyl)imino)methyl)-6-methoxyphenol (HL2)
with different amount of chelating donors, which are further used
in the synthesis of three novel Ni(II)-based and Cu(II) coordination
complexes with various coordination surroundings for the center
metal ions. In addition, the green manual grinding technology has
been used to decrease the particle diameter of complexes 1e3, thus
forming nano-complexes. Besides, we also found that nanoparticles
1 had anticancer activity against human breast cancer cells using
CCK-8 detection kit with the IC50 value in the range of micromolar.
This study offers the evidence of nanoparticle 1 appear causes
obvious mitochondrial fragmentation in MCF7 cancer cells, which
then initiated the intracellular ROS accumulation and finally caused
the cancer cell apoptosis. In conclusion, nanoparticle 1 is likely to
be an anticancer agent for human breast cancer treatment.
3.3. Anti-viability activity of nano 1 in MCF7 cancer cells
In order to study the inhibition of nanoparticles 1e3 on the
activity and proliferation in MCF7 cancer cells, the CCK-8 cell pro-
liferation test was carried out. As results shown in Fig. 6A, the
compound reduced the proportion of viable MCF7 cells with a
dose-dependent method, Unexpectedly, nanoparticles 2e3 showed
no inhibitory effect on MCF7 cancer cells growth. All the three
nanos have no cytotoxicity on normal lung cell line BEAS-2B cells
(Fig. 6B). To exclude the influence of HL1 and HL2 ligand on the
inhibitory effect of nanoparticles 1e3 against cancer cells viability,
the cytotoxicity of HL1 and HL2 was also measured by CCK-8 assay,
as results showed in Fig. 6A and B, HL1 and HL2 have no effect on
cell growth. The anticancer activity of complexes 1e3 was
measured and showed in Fig. 6C, which reflected that the com-
plexes 1e3 also showed anticancer activity but they are obvious
lower than those of the nanoparticles 1e3, highlighting the ad-
vantages of the formation of the nanoparticles.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.126938.
The IC50 values of nanos, complexes and ligands were computed
by SPSS 22.0, the results were showed in Table 2. The IC50 value of
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