Syntheses, structure, DNA-binding and DFT studies of a Cu(II) complex based on a pyrazolone derivative

Article abstract of DOI:10.1080/00958972.2019.1680835

A Cu(II) complex based on a pyrazolone derivative, 2-hydroxy-N′-((1-(4-methoxyphenyl)-3methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)(phenyl)methylene)benzohydrazide (H₂L), has been prepared. IR spectra, UV-vis spectra, elemental analysis and single-crystal X-ray diffraction indicate that the Cu(II) complex was mononuclear with the chemical composition of [Cu(HL)Cl]·CH₃OH. The Cu(II) compound presented herein exhibits interesting supramolecular characteristics and a novel 3D supramolecular architecture resulted due to the appropriate synergy of multiple intermolecular hydrogen bonds. The interaction of the Cu(II) complex with calf-thymus DNA was investigated by electronic absorption titration as well as EB-DNA competition experiment, and the results indicate that the Cu(II) compound which has a strong affinity for binding DNA is combined with DNA in an embedded manner. Furthermore, Time-Dependent Density Functional Theory calculations (TD-DFT) have been performed on optimized geometries for a better understanding of the electronic transitions in the UV-vis spectra of H₂L and Cu(II) complex.

Full text of DOI:10.1080/00958972.2019.1680835

Journal of Coordination Chemistry
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Syntheses, structure, DNA-binding and DFT
studies of a Cu(II) complex based on a pyrazolone
derivative
Wei Xi, Cai-Yun Wang, Huan-Huan Meng & Xue-Qin Song
To cite this article: Wei Xi, Cai-Yun Wang, Huan-Huan Meng & Xue-Qin Song (2019): Syntheses,
structure, DNA-binding and DFT studies of a Cu(II) complex based on a pyrazolone derivative,
Journal of Coordination Chemistry, DOI: 10.1080/00958972.2019.1680835
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JOURNAL OF COORDINATION CHEMISTRY
https://doi.org/10.1080/00958972.2019.1680835
Syntheses, structure, DNA-binding and DFT studies
of a Cu(II) complex based on a pyrazolone derivative
Wei Xi, Cai-Yun Wang, Huan-Huan Meng and Xue-Qin Song
School of Chemical and Biological Engineering, Lanzhou Jiaotong University, Lanzhou, China
ABSTRACT
ARTICLE HISTORY
A Cu(II) complex based on a pyrazolone derivative, 2-hydroxy-N0-
((1-(4-methoxyphenyl)-3methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)
(phenyl)methylene)benzohydrazide (H2L), has been prepared. IR
spectra, UV-vis spectra, elemental analysis and single-crystal X-
ray diffraction indicate that the Cu(II) complex was mononuclear
with the chemical composition of [Cu(HL)Cl]ꢀCH3OH. The Cu(II)
compound presented herein exhibits interesting supramolecular
Received 1 August 2019
Accepted 23 September 2019
KEYWORDS
Pyrazolone; Cu(II)
compound; crystal structure;
DNA binding; DFT
calculations
characteristics and
a novel 3D supramolecular architecture
resulted due to the appropriate synergy of multiple intermo-
lecular hydrogen bonds. The interaction of the Cu(II) complex
with calf-thymus DNA was investigated by electronic absorption
titration as well as EB-DNA competition experiment, and the
results indicate that the Cu(II) compound which has a strong affinity
for binding DNA is combined with DNA in an embedded manner.
Furthermore, Time-Dependent Density Functional Theory calcula-
tions (TD-DFT) have been performed on optimized geometries for a
better understanding of the electronic transitions in the UV-vis
spectra of H2L and Cu(II) complex.
1. Introduction
Cancer is a major chronic disease that seriously endangers human health and it is
documented that many cancers, such as lung cancer, skin cancer or breast cancer,
CONTACT Xue-Qin Song
songxq@mail.lzjtu.cn
School of Chemical and Biological Engineering, Lanzhou
Jiaotong University, Lanzhou 730070, China
Supplemental data for this article is available online at https://doi.org/10.1080/00958972.2019.1680835.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
W. XI ET AL.
have been found to have different symptoms and causes, but all are caused by abnor-
malities in DNA sequences [1]. Meanwhile, numerous biological experiments have
demonstrated that DNA is the main target for anticancer drugs [2, 3]. For example,
the mechanism of carboplatin for treating cancer is that it mainly crosslinked with the
DNA chain of cells to destroy DNA and inhibit tumor growth [4]. Therefore, studies on
the interaction of compounds with DNA is attracting attention and as a result, huge
quantities of such compounds, including metal compounds have been reported [5, 6].
For instance, Pt(II) complex displays an efficient DNA-binding ability in an intercalative
mode and certain Pt(II) complexes could efficiently cleave DNA with a micromolar
concentration without any external reagents [7, 8]. In addition, the Mn(II), Pd(II), and
Co(II) complexes all could bind calf-thymus DNA (CT-DNA) via intercalation in-between
DNA-bases [9–12]. The action effect of Cu(II) complex on DNA have been studied for
developing novel anticancer drugs [13]since the mixture of copper chloride and
lecithin was utilized to treat facial cancer patients in Germany in 1912. As one of the
indispensable trace elements in the human body, copper plays important roles in life
activities, such as energy metabolism, respiration and DNA synthesis. Copper exists in
all organs and tissues of people, and is usually in combination with proteins or other
organic compounds, rather than as free copper ions [14, 15]. Copper has the effect of
cracking nucleic acids directly due to its variable coordination structure and catalytic
activity of activated small molecules [16], which renders it special biological activity
and catalytic effect on the life system.
4-Acylpyrazolone is a kind of heterocyclic b-diketone type chelating agent, which can
form a large number of complexes with various metal ions due to its multiple potential
chelating sites including oxygen and nitrogen atoms. In addition, pyrazolone compounds
have a variety of biological activities, including anti-inflammatory, antibacterial, and antitu-
mor [17, 18], which makes them hot spots in the field for developing new antitumor drugs
[19]. Therefore, it is of great significance to develop new pyrazolone compounds and apply
them to prepare coordination compounds for the development of metal antitumor drugs.
Based on the above consideration and extend our study on coordination chemistry of
pyrazolone derivatives [20], a new pyrazolone derivative, 2-hydroxy-N0-((1-(4-methoxy-
phenyl)3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)(phenyl)methylene)benzohydrazide
(H2L), was prepared to construct new coordination compounds for biological activity
study. The salicylyl group was introduced in H2L herein to obtain more stable metal com-
pounds due to the increased chelating sites. Hence, a Cu(II) compound was afforded and
well characterized by IR spectra, UV-vis spectra, elemental analysis and X-ray diffraction.
The DNA-binding effect of the H2L and Cu(II) complex was studied by UV-vis and fluores-
cence spectra. In addition, Time-Dependent Density Functional Theory calculations (TD-
DFT) have been performed on optimized geometries for a better understanding of the
electronic transitions in the UV-vis spectra of H2L and Cu(II) complex.
2. Materials and methods
2.1. Materials and physical measurements
P-methoxyphenyl hydrazine hydrochloride, ethyl acetoacetate, glacial acetic acid,
methyl salicylate, hydrazine hydrate (80%), NaOH, NH4Cl, methanol and ethanol were

JOURNAL OF COORDINATION CHEMISTRY
3
Scheme 1. The synthetic route of H2L.
from commercial sources and not further purified. All chemicals were analytical grade.
4-Benzoyl-2-(4-methoxy-phenyl)-5-methyl-2,4-dihydro-pyrazol-3-one and salicylhydra-
zide were prepared according to the literature [21]. CT-DNA was purchased from
Yuanye Biotechnology Company (Shanghai, China). The UV absorption ratio of CT-DNA
solution at 260 and 280 nm was about 1.8 ꢁ 1.9, indicating that CT-DNA contained no
protein [22]. The CT-DNA concentration of each nucleotide was determined by spec-
trophotometry using the extinction coefficient of 6600 cmꢂ1 at 260 nm [23].
Carbon, nitrogen, and hydrogen analyses were performed using an EL elemental ana-
lyzer. Infrared spectra were obtained with KBr discs on a Thermo Mattson FTIR spectrom-
eter from 4000 ꢁ 400 cmꢂ1 with an average of 128 scans and 4 cmꢂ1 of spectral
resolution. UV-vis absorption spectra were determined with a UV-1800 Shimadzu spec-
trophotometer. Emission fluorescence spectra were recorded on an F-7000 fluorescence
spectrophotometer (Japan Hitachi company) at room temperature. The width of the exci-
1
tation and emission slits are both 5.0 nm. H and 13C NMR spectra were recorded in
CDCl3 solution at room temperature on a Bruker 500 instrument operating at a frequency
of 500 MHz and referenced to tetramethylsilane (0.00 ppm) as an internal standard.
2.2. Synthesis of the ligand
The synthetic route for H2L is shown in Scheme 1. To an ethanolic solution (10 mL)
containing 2.28 g (3 mmol) 4-benzoyl-2-(4-methoxy-phenyl)-5-methyl-2,4-dihydro-
pyrazol-3-one, an ethanolic solution of 0.46 g (3 mmol) salicylhydrazide and 0.5 mL
acetic acid was added dropwise within 30 min. The reaction mixture was continuously
stirred and refluxed at 80 ꢃC for 4 h. After the resulted solution was cooled to room
temperature, 10 mL water were added and the mixture was further stirred to obtain
precipitates which was filtered and recrystallized from ethanol to afford yellow solids.
1
H2L: 0.93 g, Yield: 70%. H NMR (CDCl3, 500 MHz), d (ppm): 1.54 (s, 3H), 3.78 (s, 1H),
3.84 (s, 3H), 6.97 (dd, 4H), 7.47 (m, 1H), 7.68 (m, 6H), 7.84 (t, 3H), 11.70(s, 1H). 13C NMR
(CDCl3, 400 MHz), d (ppm): 166.61, 157.50, 149.04, 135.00, 132.69, 129.75, 129.46,
129.07, 127.45, 122.43, 119.55, 118.22, 114.06, 113.96, 55.52, 55.45, 16.11. Anal. Calcd
for C25H22N4O4: C, 67.86; H, 5.01; N, 12.66. Found: C, 66.69; H, 5.04; N, 12.62.
2.3. Synthesis of the Cu(II) complex
Seventeen milligrams of (0.1 mmol) CuCl2ꢀ2H2O in 4 mL methanol was added dropwise
to a 2 mL methanol solution of 44 mg (0.1 mmol) H2L at room temperature, and the

4
W. XI ET AL.
Table 1. Crystallographic data and data collection parameters for the Cu(II) complex.
Empirical formula
CuC26H25ClN4O5
Temperature (K), M
296(2), 572.5
Crystal system, Space group
Unit cell dimensions
Monoclinic, P21/c
a ¼ 9.8688(12) Å, a¼ 90.00ꢃ b ¼ 15.217(3) Å,
b¼103.072(3)ꢃ c ¼ 18.178(2) Å, c ¼ 90.00ꢃ
Volume (Å3), Z
2667.8(6), 4
Absorption coefficient (mm–1
F(000)
)
0.962
1180
h Range for data collection (ꢃ)
Index ranges
3.45 ꢁ 25.00
–11 ꢄ h ꢄ 11; –18 ꢄ k ꢄ 16; –21 ꢄ l ꢄ 19
Reflections collected/unique
13211/4684
Rint
0.0289
4784 / 1 / 343
1.029
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I >2r(I)]
R indices (all data)
R1 ¼ 0.0441, wR2 ¼ 0.1260
R1 ¼ 0.0594, wR2 ¼ 0.1398
reaction mixture was stirred subsequently for 4 h at room temperature to obtain a
large amount of precipitate. Then, 30 mL methanol were added to dissolve the precipi-
tation, and the resulted solution was filtered into several 10-mL glass vials for evapor-
ation. Single-crystals suitable for crystal analysis were obtained after three weeks when
solvent was partially evaporated. Yield: 24 mg, 42% based on H2L. Anal. Calcd for
CuClC26H25N4O5: C, 54.55; H, 4.40; 9.79. Found: C, 54.74; H, 4.38; N, 9.75.
2.4. X-ray single-crystal diffraction analysis
Single-crystal X-ray diffraction data were recorded on an Oxford Diffraction Gemini R
Ultra diffractometer with graphite-monochromated Mo Ka radiation (k ¼ 0.71073 Å) at
293 K. Absorption corrections were applied using a multi-scan technique. No crystal
decay was noted during the data collections. The frames were integrated with the
Bruker SAINT software package using a narrow-frame algorithm. Data were corrected
for absorption effects using the empirical multi-scan method (SADABS) [24]. The struc-
tures were solved by direct methods and refined on F2 by a full-matrix least-squares
procedure. SHELXL-2014 was used for both structural solutions and refinements [25].
Non-hydrogen atoms of the compounds were refined with anisotropic temperature
parameters. All hydrogens were placed in geometrically idealized positions
(C–H ¼ 0.96 Å for methyl groups, C–H ¼ 0.97 Å for CH2 groups, C–H ¼ 0.93 Å for phenyl
groups and O–H ¼ 0.82 Å for hydroxyl groups) and constrained to ride on their parent
atoms with Uiso (H) ¼ 1.5 Ueq(C) for methyl groups, 1.2 Ueq(C) for aromatic and CH2
groups, and 1.5 Ueq(O) for hydroxyl groups. The crystal structure parameters of the
Cu(II) complex are given in Table 1. Graphics were drawn with DIAMOND (Version
3.2) [26].
3. Results and discussion
3.1. Synthesis and characterization
H2L was prepared as yellow solid in four steps from the commercially available p-
methoxyphenyl hydrazine hydrochloride, methyl salicylate and hydrazine hydrate as

JOURNAL OF COORDINATION CHEMISTRY
5
Figure 1. IR spectra of H2L and Cu(II) complex in solid state.
1
shown in Scheme 1. The H and 13C NMR spectra (Supplementary material, Figures S1
and S2) confirmed the identity of H2L. The formation of Cu(II) complex was first ascer-
tained by the IR spectra determination. As shown in Figure 1, a wide characteristic
absorption band at 3385 cmꢂ1 characteristic of enolic group on pyrazolone and the
absence of band from ketone carbonyl ascertained that H2L exists in the form of
enolic in the solid. The band at 3025 cmꢂ1 is assigned to the phenolic hydroxyl group
of ligand. The strong band at 1639 cmꢂ1 from the stretching vibrations of the acylhy-
drazone C ¼ O with the acylhydrazone C ¼ N and the pyrazole ring C ¼ N stretching
vibrations appeared at 1607 and 1512 cmꢂ1, respectively. By contrast, in the IR spectra
of the complex [Cu(HL)Cl]ꢀCH3OH, the characteristic peak of enolic group disappeared,
indicating that this oxygen participated in coordination. Meanwhile, the stretching
vibrations of acylhydrazone C ¼ O shifted approximately 30 cmꢂ1 towards the low
wavenumber, indicating coordination also via acylhydrazone C ¼ O [27]. Similar
changes were found on the stretching vibrations of the acylhydrazone C ¼ N which
shifted approximately 35 cmꢂ1 towards low wavenumber, indicating the coordination
of this nitrogen. The stretching vibrations of pyrazole ring C ¼ N remained at
1512 cmꢂ1, suggesting that the ring nitrogen does not take part in coordination. In
the far-IR region, new bands at 603 and 527 cmꢂ1 in the complexes are assigned to
v(M-O) and v(M-N) [28] of stretching vibrations. These results could be ascertained by
the crystal analysis as follows.
3.2. X-ray crystal structure of [Cu(HL)Cl]ꢀCH3OH
X-ray structural analysis reveals that the mononuclear Cu(II) complex crystallized in the
monoclinic system with P21/c space group. The crystal structure of the complex with

6
W. XI ET AL.
Figure 2. (a) View of the coordination environment of Cu1 center with thermal ellipsoids at 30%
probability (all hydrogens have been omitted for clarity). (b) The coordination polyhedron of Cu1.
atomic numbering scheme is shown in Figure 2(a). Its asymmetric unit contains one
crystallographically independent Cu(II) ion, one ligand anion HL– and one Cl– ion. Each
Cu(II) atom is tetra-coordinated by atoms O2, O3 and N2 from one anionic ligand HL-
and one Cl– ion (Cl1) to form a planar quadrilateral geometry as shown in Figure 2(b).
The atoms O2, O5, N4 and Cl1 constitute the quadrilateral plane and the Cu(II) atom
strays from the quadrilateral plane of 0.004 Å. The Cu–O2/O3 bond distances in quadri-
lateral plane are 1.950(3) Å/1.896(3) Å, the Cu–N2 bond distances are 1.954(3) Å, and
the Cu–Cl1 bond distances are 2.221(3) Å. The angle of O2–Cu1–O3 is 174.26(11)ꢃ and
the angle of Cl1–Cu1–N2 is 173.32(9)ꢃ which is slightly deviated from the theoretical
value of 180ꢃ. Therefore, the local coordination geometry around the Cu(II) center can
be described as a slightly distorted quadrilateral plane. Selected bond distances and
angles for the Cu(II) complex are listed in Table S1 (Supplementary material). In
addition, the bond lengths of O3–C16 (1.287(4) Å) and C15–C16 (1.412(5) Å) prove
that oxygen O3 of the carbonyls participates in coordination by the enolic form, and
their active hydrogen is replaced by the Cu(II) ion [29]. To test the purity of the bulk
samples of the Cu(II) compound, the PXRD measurements was determined. As shown
in Figure S3 (Supplementary material), the PXRD patterns of the bulk samples were
similar to that of simulated, demonstrating the phase purity of the bulk samples of
the Cu(II) compound.
To further analyze the crystal structure of the complex, significant hydrogen bond-
ing interactions were found between the molecules. As shown in Figure 3(a), two
mononuclear Cu(II) units are first connected by two crystalline methanol molecules in
aid of hydrogen bonding where two crystalline methanol molecules serve as both
donors and acceptors. The phenolic hydroxyl group of the ligand, the C–H on the ben-
zene ring of the salicylhydrazide and pyrazolone acted as hydrogen bonding donor.
The oxygen atom on the carbonyl group and the coordinated Cl– ion acted as hydro-
gen bond acceptor. Thus, a supramolecular macrocycle is formed through O1–H1ꢀꢀꢀO5,
O5–H5AꢀꢀꢀCl1 and C10–H10ꢀꢀꢀO2 hydrogen bonds between two Cu(II) molecules. With
the help of the C14–H14ꢀꢀꢀO3 hydrogen bond interaction, adjacent dimers can also be
connected together, forming an infinite 1D chain along the a-axis, as shown in Figure
3(b). The distance of C14ꢀꢀꢀO3 is 3.469 Å and the angle of C14–H14ꢀꢀꢀO3 is 158.79ꢃ.

JOURNAL OF COORDINATION CHEMISTRY
7
Figure 3. (a) The supramolecular dimer of the Cu(II) compound constructed by O–HꢀꢀꢀO, O–HꢀꢀꢀCl
and C–HꢀꢀꢀO hydrogen bonds; (b) The 1D supramolecular chain of the Cu(II) compound constructed
by C–HꢀꢀꢀO hydrogen bonds; (c) The 3D supramolecular architecture constructed by C–HꢀꢀꢀO hydro-
gen bonds by two directions.

8
W. XI ET AL.
Figure 4. (a) UV–vis absorption spectra of H2L (5.0 ꢆ 10ꢂ5 M) and its Cu(II) compound (5.0 ꢆ 10ꢂ5 M)
in CH3OH solution; (b) The simulated UV–vis absorption spectra of H2L in methanol with the oscillator
strength of main excited states (strength > 0.02) shown in red; (c) The simulated UV–Vis absorption
spectra of the Cu(II) compound in methanol with the oscillator strength of main excited states
(strength > 0.02) shown in red.
Finally, the 1-D chain further forms an infinite 3D supramolecular framework by the
interaction of C5–H5ꢀꢀꢀO4 hydrogen bonds in two directions as shown Figure 3(c). The
distance of C5ꢀꢀꢀO4 is 3.430 Å and the angle of C5–H5ꢀꢀꢀO4 is 147.38ꢃ. Selected hydro-
gen-bonding distances (Å) and angles (ꢃ) for Cu(II) complex are listed in Table S2
(Supplementary material), both within the ranges of those reported hydrogen
bonds [30].
3.3. Absorption spectra study
In order to shed some light on the geometrical structure of the complex, the absorp-
tion spectra of H2L and [Cu(HL)Cl]ꢀCH3OH were recorded in methanol solution at room
temperature (Figure 4(a)). The absorption spectrum of H2L consists of two bands at
ꢅ
242 and 302 nm, which are attributed to the p!p transitions of H2L. The molar
absorption coefficient value (e) for H2L was calculated as 2.48 ꢆ 104 Mꢂ1cmꢂ1, indicat-
ing that H2L has a strong ability to absorb light. A slight blue-shift was observed in
the absorption spectrum after ligand coordination with copper, which is evidence of
coordination of H2L. To shed some light on the electronic spectra of H2L and its Cu(II)
complex, TD-DFT calculations were performed on the optimized geometries of H2L
and its Cu(II) compound in methanol with theoretical simulation of UV-vis absorption
spectra of H2L and the Cu(II) complexes shown in Figures 4(b,c). The calculated vertical
electronic excitation energy (eV) and oscillator strength (f) are summarized in Table 2.
The experimental absorption bands of the ligand and complex have been explained
in aid of TD-DFT calculations. The intense bands of the ligand at 242 and 302 nm are
intra-ligand charge-transfer (ILCT) characters in H2L, while the corresponding theoret-
ical bands are at 246 and 303 nm, in agreement with experimental results. As for the
Cu(II) complex, the simulated peak at 246 nm and that of experiment at 236 nm is
attributed to a-HOMO–1 ! a-LUMO þ 1 (53%) and b-HOMO–1 ! b-LUMO þ 2 transi-
tion (65%). Meanwhile, the simulated peak at 304 nm and the experimental one
observed at 295 nm is corresponding to a-HOMO–1 ! a-LUMO (49%) and b-HOMO–1
! b-LUMO transitions (63%). Clearly, the theoretical simulation results are in agree-
ment with that of the experiment.

JOURNAL OF COORDINATION CHEMISTRY
9
Table 2. TD-DFT Calculated electronic excitations of H2L and its Cu(II) compound in CH3OH.
Excitation (eV)
kex (nm)
Osc. strength (f)
Key transitions
Character
kexpt
H2L
4.0860
4.1649
5.0400
ꢅ
303.27
297.64
246.00
244.15
0.2681
0.0491
0.2501
0.1496
(56%) HOMO–1 ! LUMO
(56%) HOMO–1 ! LUMO
(58%) HOMO–1 ! LUMO þ 2
(57%) HOMO–1 ! LUMO þ 3
H2L(p) ! H2L (p )
302
–
242
–
ꢅ
H2L(p)! H2L (p )
ꢅ
H2L(p)! H2L (p )
ꢅ
5.0664
H2L(p)! H2L (p )
[Cu(HL)Cl]ꢀCH3OH
ꢅ
2.9156
304.21
0.1649
(49%) a-HOMO–1 ! a-LUMO
(63%) b-HOMO–1 ! b-LUMO
(24%) a-HOMO–2 ! a-LUMO þ 1
(50%) a-HOMO–4 ! a-LUMO
(35%) b-HOMO–3 ! b-LUMO
(53%) a-HOMO–1 ! a-LUMO þ 1
(65%) b-HOMO–1 ! b-LUMO þ 2
M(p)! H2L (p )
295
ꢅ
M(p)! H2L (p )
ꢅ
2.9404
3.4826
3.6988
301.53
257.63
245.92
0.0311
0.1944
0.0322
M(p)! H2L (p )
–
–
236
ꢅ
M(p)! H2L (p )
ꢅ
H2L(p)! H2L (p )
ꢅ
M(p) ! H2L (p )
ꢅ
M(p) ! H2L (p )
Table 3. Experimental and calculated bond distances (Å) in the Cu(II) compound.
Bond lengths
Experimental
Calculated
Bond lengths
Experimental
Calculated
Cu1–O3
Cu1–N2
1.896 (2)
1.954 (3)
1.90909
1.98121
Cu1–O2
Cu1–Cl1
1.950 (3)
2.2206 (11)
1.98949
2.26264
3.4. Theoretical calculations
To better understand the electronic structure of the H2L and its Cu(II) complex, we
performed density functional theory (DFT) calculations. The molecular geometry was
optimized by the Gaussian 09 with the level of B3LYP. Nonmetal atoms were
described by B3LYP/6-31G and metal atoms were treated by B3LYP/LANL2DZ basis
sets. The optimized structures of H2L and the Cu(II) compound is displayed in Figure
S4 (Supplementary material). As shown in Table 3, the optimized bond lengths for the
Cu(II) complex are well replicated with the experimental one with minor deviations in
bond distances in the range of 0.017–0.039 Å in Cu–O, 0.027 Å and 0.042 Å for Cu-N
and Cu-Cl. The reason for the deviation is that the theoretical calculations were per-
formed on an isolated molecule in gas phase whereas X-ray measurements being car-
ried out in the solid state [31].
As shown in Figure 5(a), the calculated molecular orbital energies of the LUMO þ 2,
LUMO þ 1, LUMO, HOMO, HOMO–1, and HOMO–2 for H2L are –0.625, –0.711, –1.229,
–5.663, –6.065, and –6.133 eV, respectively. The energy gaps between HOMO–LUMO
are 4.434 eV for H2L. The calculated molecular a and b orbital energy of the HOMO
and LUMO for the Cu(II) compound are –5.1930, –5.1911 and –2.2916, –2.3422 eV,
respectively. The energy gaps between HOMO and LUMO are 2.9014 (a) and 2.8489
(b) for the Cu(II) compound as depicted in Figures 5(b,c). The plot of the highest
occupied molecular orbitals (HOMOs) of the Cu(II) compound indicate that the HOMOs
are comparatively less localized and reside mainly on the orbitals of the pyrazolone
fragment. The LUMOs are delocalized and spread over the orbitals of the
entire molecule.
As shown in Table 4, the HOMO–LUMO structures orbital energy occupying
the boundary are all negative, testifying the chemical stability of the molecule
[32, 33]. Compared with the experimental results, the calculated data are con-
sistent with the UV-vis absorption spectra obtained from methanol solution. All

10
W. XI ET AL.
Figure 5. (a) Surface plots of some selected molecular orbitals of the ligand H2L; (b) Surface plots
of some selected molecular a-orbitals of the Cu(II) compound; (c) Surface plots of some selected
molecular b-orbitals of the Cu(II) compound.

JOURNAL OF COORDINATION CHEMISTRY
11
Table 4. Absorption spectra and HOMO–LUMO structures orbital energy of the Cu(II) compound.
a
kedge (nm)
Eopt g (eV)
EHOMO (eV)
ELUMO (eV)
DEgap (eV)
425
2.917
–5.193 (a)
–5.191 (b)
–2.291 (a)
–2.342 (b)
2.902 (a)
2.849 (b)
aEoptg estimated from the UV–Vis absorption spectra.
these results indicate that the complex is stable and the DFT method with
B3LYP/6–31G and B3LYP/LANL2DZ basis group used in the calculations
is reasonable.
3.5. DNA-Binding experiments
3.5.1. Electronic absorption titration
Absorption spectrum titration is first utilized to characterize the binding mode and
binding affinity of metal complexes with DNA by increasing the concentration of CT-
DNA while the concentration of the test substance remained constant [34]. To obtain
the absorption spectrum, the required amount of CT-DNA is added to the test solution
as reference to eliminate the absorbance of CT-DNA itself. Each sample solution was
scanned at a range of 190 nm to 500 nm and the mixture was balanced for 5 min
before recording [35]. According to the absorption titration data, the following equa-
tion is used to obtain the DNA-binding constant [36]:
½DNAꢇ=ðea–efÞ ¼ ½DNAꢇ=ðeb–efÞ þ 1=Kbðeb–efÞ
(1)
where [DNA] is the concentration of DNA in base pairs, ea corresponds to the extinc-
tion coefficient observed (Aobsd/[M]), ef corresponds to the extinction coefficient of the
free compound, eb is the extinction coefficient of the compound fully bounded to
DNA, and Kb is the intrinsic binding constant. The ratio of slope to intercept in the
plot of [DNA]/(ea – ef) versus [DNA] gives the value of Kb.
The absorption spectra of H2L and the Cu(II) compound upon addition of CT-DNA
are given in Figure 6. The intense absorption bands at 355 ꢁ 380 nm observed in both
ꢅ
H2L and its Cu(II) compound are attributed to intra ligand p!p transitions. Upon
addition of CT-DNA, the absorption band of H2L shows a hypochromism of 4.86%. By
contrast, the absorption band of the Cu(II) compound exhibits a hypochromism of
11.49% with a red-shift. Therefore, the Cu(II) compound is most likely to bind to DNA
through an intercalative mode, because the hypochromic effect and red-shift of the
UV absorption spectrum of metal complex was a result of an intercalating manner of
DNA with metal compounds [37]. The binding constants Kb for H2L and the Cu(II) com-
pound has been determined from the plot of [DNA]/(ea – ef) versus [DNA] as shown in
Figures 6(c,d). The Kb of H2L and Cu(II) complex were 5.28 ꢆ 103 M–1 (R ¼ 0.992 for six
points) and 2.83 ꢆ 104 M–1 (R ¼ 0.995 for nine points), respectively. The results show
that the binding strength of the Cu(II) complex rises far above H2L. Notably, the Kb
value is higher than that of the classical intercalator ethidium bromide (EB) versus CT-
DNA [38], indicating that the binding strength between the Cu(II) complex and CT-
DNA is very strong [39, 40].
Based on the experimental and theoretical results, the reasons for the strong bind-
ing of the Cu(II) compound to DNA was speculated to be electronic and spatial effects

12
W. XI ET AL.
Figure 6. (a) Electronic spectra of H2L (3 ꢆ 10ꢂ3 M) in Tris-HCl buffer (pH ¼ 7.2) upon addition of CT-
DNA (0–220 lL, 2.5ꢆ 10ꢂ3 M) (arrow shows the emission intensity changes upon increasing DNA con-
centration); (b) Electronic spectra of the Cu(II) complex (3ꢆ 10ꢂ3 M) in Tris-HCl buffer (pH ¼ 7.2) upon
addition of CT-DNA (0–220 lL, 2.5ꢆ 10ꢂ3 M) (arrow shows the emission intensity changes upon
increasing DNA concentration); (c) Plots of [DNA]/(ea – ef) vs. [DNA] for H2L; d) Plots of [DNA]/(ea – ef)
vs. [DNA] for the Cu(II) complex.
caused by Cu(II)0s chelation as follows: (a) The increased co-planarity upon Cu(II)
coordination may result in high affinity for DNA; (b) The reduced electron density on
the ligand caused by the coordination of the central Cu(II) atom is conducive to
embedding the DNA base pair.
3.5.2. EB-DNA fluorescence competition experiment
In order to further study the binding properties of H2L and the Cu(II) complex with
DNA, competitive binding experiment was carried out. Commonly, the fluorescence of
EB is weak in solvent, but enhanced when embedded in DNA base pair. The addition
of a second molecule competes with the EB for binding of DNA, allowing the fluores-
cence to quench, which demonstrates that the second molecule is embedded in the
DNA base pair [41, 42]. Therefore, the binding degree between the second molecule
and CT-DNA can be determined according to the degree of fluorescence quenching.
For EB-DNA competition determination, 10 lL EB (2.2 ꢆ 10ꢂ3 M) solution and 10 lL
DNA (2.5 ꢆ 10ꢂ3 M) stock solution were added to 2.5 mL Tris-HCl buffer solution (pH ¼
7.2), and the fluorescence spectra was determined upon excited at 520 nm. The

JOURNAL OF COORDINATION CHEMISTRY
13
Figure 7. (a) Emission spectra of EB bound to CT-DNA excited at 520 nm in the presence of H2L
(3 ꢆ 10ꢂ3 M); (b) Emission spectra of EB bound to CT-DNA in the presence of the Cu(II) compound
(3 ꢆ 10ꢂ3 M); (c) Fluorescence quenching curves of EB bound to CT-DNA induced by H2L; (d)
Fluorescence quenching curves of EB bound to CT-DNA induced by the Cu(II) compound.
fluorescence quenching of EB-DNA bound induced by H2L and the Cu(II) complex are
shown in Figures 7(a,b). With H2L and its Cu(II) compound gradually introduced into
the EB-DNA solution (5 lL for each addition), respectively, the fluorescence intensities
of EB-DNA centered at 598 nm obviously weakened under the same determination
conditions. Based on the changes of fluorescence emission spectra, the influence of
compounds added to EB-DNA complex solution was obtained [43, 44]. The spectra
were analyzed according to the classical Stern-Volmer equation [45]:
I0=I ¼ 1 þ KSV½Qꢇ
(2)
where I0 and I are the fluorescence intensities at 598 nm in the absence and presence
of the quencher, respectively, KSV is the linear Stern-Volmer quenching constant, and
[Q] is the concentration of the quencher.
The behavior of H2L and Cu(II) complex are in good agreement with the Stern-
Volmer equation, which provides further evidence that the two compounds bind to
DNA by embedding as depicted in Figures 7(c,d). The KSV values for H2L and the Cu(II)
complex are 3.87 ꢆ 103 Mꢂ1 (R ¼ 0.992 for eight points) and 2.35 ꢆ 104 Mꢂ1 (R ¼ 0.974
for seven points), respectively. The higher quenching efficiency of the Cu(II) compound
in contrast to that of H2L indicates that the Cu(II) compound has stronger DNA bind-
ing than H2L [46, 47], consistent with the absorption study results.

14
W. XI ET AL.
4. Conclusion
A new Cu(II) compound constructed by a new pyrazolone derivative was prepared
and well characterized by IR, UV-vis and X-ray diffraction. Single-crystal X-ray analysis
reveals that the Cu(II) compound presented herein exhibits interesting supramolecu-
lar characteristics and a novel 3D supramolecular architecture was resulted due to
the appropriate synergy of multiple intermolecular hydrogen bonds. Furthermore,
TD-DFT have been performed on optimized geometries for a better understanding
of the electronic transitions in the UV-vis spectra of H2L and the Cu(II) complex.
Electronic absorption titration and EB-DNA competition experiment indicate that the
Cu(II) complex are combined with DNA in an embedded manner and has a strong
affinity for binding. This study clearly shows the effect of copper complexes on
DNA and provides a foundation for further research and development of new metal
anticancer drugs.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China
(Grant 21661019).
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