Copper(II)-5-chloro-2-hydroxybenzophenone Complexes with - Donors: Structural Insights and Antitumor Activity against A2780 Human Ovarian Cancer Cells of a Bathophen Derivative.

1
Introduction
Cancer is a major clinical
condition, with the incidence increasing
every year due to a variety of factors, including genetic changes,
chronic inflammation, unhealthy diets, and even substance abuse. Consequently,
cancer cases have been rising rapidly, underscoring the continuous
need for novel therapeutic strategies.
−

Through the advancement
of cancer research over the years, significant progress has been made
in understanding its mechanisms. However, the development of pioneering
metal-based drugs with antitumor properties remains a contemporary
challenge in the field of metallodrug design.

Platinum complexes are the most commonly used chemotherapeutic
agents studied worldwide. These complexes bind covalently to nucleobases
of the DNA, triggering apoptotic cell death. Despite their efficiency, these compounds are associated with several
side effects and drug resistance.
,
To overcome
these challenges, several metal-based compounds have been investigated
for anticancer therapeutic purposes.
In this context, copper­(II)
complexes are well-known for their
broad range of biological properties and their noteworthy antineoplastic
activity.
,
In general, their anticancer activity is
often attributed to the synergistic interaction between copper­(II)
ions and ligands that already have antitumor activity.
−

The cytotoxic effect of copper­(II) compounds is primarily attributed
to the ability of this ion to generate reactive oxygen species (ROS),
thereby disrupting key cellular mechanisms related to the redox process.
In normal cells, ROS are predominantly produced during mitochondrial
respiration. However, in most cellular environments associated with
tumor development, ROS levels are markedly elevated. Excessive ROS
levels lead to impaired mitochondrial respiration and damage to proteins,
nucleic acids, and other cellular components, ultimately resulting
in apoptosis.
−

Building on these findings, recent research has focused on
the
design of heteroleptic copper­(II) complexes combining diimine ligands,
such as 1,10-phenanthroline or 2,2′-bipyridine with auxiliary
bidentate ligands bearing O–O or O–N donor atoms. This
structural motif has proven effective in promoting DNA intercalation,
ROS-mediated cytotoxicity, and selective apoptosis in tumor cells,
as exemplified by the Casiopein family compounds. With this framework, hydroxylated benzophenone derivatives
stand out as attractive ancillary ligands due to their ability to
stabilize metal centers via chelation through phenolic and carbonyl
groups, while also introducing photophysical properties and enhanced
lipophilicity. In particular, benzophenones
have been explored to modulate the electronic environment and biological
reactivity of metal complexes. The incorporation of electron-withdrawing
groups, such as chlorine, in the aromatic scaffold can influence the
ligand’s donor ability and fine-tune metal–ligand interactions.
Notably, copper­(II) complexes incorporating such ligands exhibit remarkable
antitumor activity against HepG2 and HCT116 tumor cells. Furthermore, Levin et al. described that 9-dimethyl-1,10-phenanthroline
and 1,10′-phenanthroline Cu­(II)-based compounds exhibit better
activity than the latter and cisplatin, against MG-63, A549, MCF-7,
and MBA-MB-231 cells.

DNA remains
the main target for metal-based compounds. To obtain
new insights about the cytotoxicity and DNA-interacting properties
of these species, Gamez et al. prepared and studied different copper
complexes.
,
Their binding modes were explored
using several techniques. The authors revealed that the complex [CuCl2(Cltpy)] (Cltpy is 4′-chloro-2,2′:6′,2″-terpyridine)
is as efficient a DNA cleaver at 20 μM concentration. Despite
the lack of activity after 24 h, this complex has increased cytotoxicity
ability after 72 h incubation (120-fold more potent) toward A2780
ovarian cancer cells. Recently, the authors
studied complexes with the general formula [Cu­(en2ampy)]2+ (en2ampy is, namely, 3,3′-((1E,1′E)-(ethane-1,2-diylbis­(azaneylylidene))­bis­(methaneylylidene))­bis­(pyridin-2-amine).
Despite their planar structure and IC50 values lower than
cisplatin, these complexes were not able to intercalate between DNA
base pairs, confirming that, as expected, geometric features are not
the only parameters involved in these interactions.

Herein, we explore the cytotoxicity of copper­(II)
complexes with
a 5-chloro-2-hydroxybenzophenone and N–N donors. Accordingly,
we report the synthesis of five Cu­(II)-5-chlorobenzophenone-based
compounds containing different N-heterocyclic ligands. These compounds,
namely, [Cu­(5-Cl2HBz)­(phen)­(NO3)]Cu­(1), [Cu­(5-Cl2HBz)­(bathophen)]·(NO3)·1.5 H2O·CH3OHCu­(2), [Cu­(5-Cl2HBz)­(bipy)­(NO3)­(H2O)]Cu­(3), [Cu­(5-Cl2HBz)­(5,5′-bipy)­(NO3)]Cu (4), and [Cu­(5-Cl2HBz)­(tert-bipy)­(NO3)]·2H2OCu (5), where (5-Cl2HBz = 5-chloro-2-hydroxybenzophenonephenone, phen
= 1,10-phenanthroline, bathophen = 4,7-diphenyl-1,10-phenanthroline,
bipy = 2,2′-bipyridine, 5,5′-bipy = 5,5′-bipyridine
and tert-bipy = 4,4′-bis­(tert-butyl)-2,2′-bipyridine). These complexes were fully characterized
by elemental analysis, thermogravimetry, infrared spectroscopy, single-crystal
X-ray diffraction, UV–vis spectroscopy, ESI-MS, and EPR.
Their cytotoxic potential was explored in different cancer cell
lines. Due to the promising results, complex Cu­(2) was
further studied in A2780 human ovarian cancer cells. A clonogenic
assay was also performed. Furthermore, metal complex–DNA interactions
were studied. Complex Cu­(2) was found to interact with
the biomolecule via different modes, making it a promising starting
point for further development of new cytotoxic copper-bathophen agents.

2
Experimental Section
2.1
Materials
Solvents and all chemicals
used were of reagent grade or comparable purity and were supplied
and used as received from Sigma-Aldrich: copper­(II) nitrate trihydrate,
1,10-phenanthroline (phen), 4,7-diphenyl-1,10-phenanthroline (bathophen),
2,2′-bipyridine (bipy), 5,5′-bipyridine (5,5′-bipy),
4,4′-bis­(tert-butyl)-2,2′-bipyridine
(tert-bipy), 5-chloro-2-hydroxybenzophenone (5-Cl2HBz),
and calf-thymus DNA (CT-DNA).

2.2
Instrumentation
Partial elemental
analyses were performed on a CHNS Thermo Scientific Fisons Instruments
EA 1108 model CHNS analyzer. Conductivity data (present as Ω–1 cm2 mol–1) were obtained
in methanol by using a Bel Engineering conductivity meter with a cell
constant equal to 1.25 cm–1. Measurements were made
at room temperature (18 °C) using a 10 mM solution. Thermogravimetric
analyses were carried out using Shimadzu thermal analysis equipment,
model 60H, with an alumina sample holder, in a N2 atmosphere,
with a flow rate of 50 mL min–1 and a heating rate
of 10 °C min–1. A Bomem-Michelson FT-IR spectrophotometer
was employed to record infrared spectra (4000 cm–1–400 cm–1) using KBr plates. The Raman spectra
data collection was carried out using a HORIBA LabRAM HR Evolution
spectrometer equipped with a 532 nm laser, a grating of 1800 gr/mm,
and a CCD cooled detector. Laser power at the sample was 1 mW. The
spectra were collected with a resolution of 1 cm–1. The electronic absorption spectra were acquired in methanol and
DMSO/buffer pH = 7.4 solution using a Shimadzu 1800 UV–vis
spectrophotometer with cuvettes with an optical path of 1 cm containing
solutions of the complexes and ligands of known concentrations. Full
mass spectra in positive ion mode were acquired on a MicroTOF-Q Bruker
equipped with electrospray mass ionization and an ion trap analyzer.
Samples were dissolved in MeOH in a 10 mM solution and continuously
pumped by a syringe (Hamilton 500 μL) with a flow of 15 μL
min–1 into the mass spectrometer. The positive ion
mode electrospray was achieved by application of +4.5 kV and 180 °C
desolvation temperature. The EPR measurements were performed in a
X-band spectrometer from Bruker, frequency 9.8 GHz. Each complex spectrum
was recorded at room temperature by extracting 50 μL of a 10
mM solution through a glass capillary tube and placing it in a regular
X-band EPR quartz tube. The parameters used were: 100 kHz of modulation
frequency, 1 G of modulation amplitude, and 3.65 mW of microwave power.
Two software packages were used to analyze spectrometry data (a) mmass
(open-source mass spectrometry tool) and (b) Envipat.

2.3
General Procedure to Synthesize
the Heteroleptic
Copper­(II) Complexes
The general synthetic route used to
synthesize the heteroleptic copper­(II) complexes with the general
formula [Cu­(5-Cl2HBz)­(N–N)­(NO3)], where N–N
= phenCu­(1), bathophenCu­(2), bipyCu­(3), 5,5′-bipyCu­(4), and tert-bipyCu­(5), is described in Scheme
. In a round-bottomed flask (50 mL), the Cu­(NO3)2 · 2.5 H2O (58 mg, 0.25 mmol) was dissolved
in methanol (10 mL). A methanolic solution of 5-Cl2HBz (58 mg, 0.25
mmol) was deprotonated by using KOH (20 mg, 0.35 mmol). The mixture
was stirred for 20 min and, afterward, a methanolic solution containing
an N-heterocycle (0.25 mmol) was added dropwise, and the resulting
solution was kept under reflux with stirring for 24 h. The crystals
were obtained by slow evaporation after 2 weeks. The obtained green
crystals were washed with ultrapure cold water to remove impurities
and dried under pressure.
[Cu­(5-Cl2HBz)­(phen)­(NO3)]Cu­(1).
Yield: 78%. Green crystal (FW = 537.4 g mol–1).
Elemental analysis for (CuC25H16ClN3O5) Calcd: 55.87%; H, 3.00%; N, 7.82%. Found: C, 55.49%;
H, 2.81%; N, 7.78%. Molar conductivity: (1 × 10–3 molL–1, MeOH): 108 Ω–1 cm2 mol–1. ESI-MS (CuC25H16N3O5Cl [M]+) calcd,
474.0196; found, 474.0339. IR (KBr, cm–1): 3060,
2990, 1645, 1592, 1505, 1417, 1338, 1143, 1090, 857, 737. UV–vis
(methanol), λmax/nm (ε/M–1 cm–1): 272 (17,000), 294 (16,000), 419 (146).
[Cu­(5-Cl2HBz)­(bathophen)]·(NO3)·1.5 H2O·CH3OHCu­(2). Yield:
68%. Dark green crystal (FW = 707.62 g mol–1). Elemental
analysis for (C76H62Cl2Cu2N6O15): C, 58.84%; H, 4.42%; N, 5.42%. Found:
C, 59.29%; H, 4.12%; N, 5.42%. Molar conductivity: (1 × 10–3 molL–1, MeOH): 97.5 Ω–1 cm2 mol–1. ESI-MS (CuH24ClN2O2 [M+]) calcd, 628.0973;
found, 628.1232. IR (KBr, cm–1): 3050, 3023, 1606,
1553, 1509, 1487, 1404, 1078, 924, 845, 765, 704. UV–vis (methanol),
λmax/nm (ε/M–1 cm–1): 220 (15,600), 285 (1470), 420 (102).
[Cu­(5-Cl2HBz)­(bipy)­(NO3)­(H2O)]Cu­(3). Yield: 82%.
Green crystal (FW = 531.40 g mol–1). Elemental analysis
for (CuC23H18ClN3O6):
C, 51.98%; H, 3.41%; N, 7.91%. Found: C, 52.35%;
H, 3.19%; N, 7.94. Molar conductivity: (1 × 10–3 molL–1, MeOH): 57.2 Ω–1 cm2 mol–1. ESI-MS: (CuC23H16ClCuN2O2 [M+]) calcd,
452.0347; found, 452.0311. IR (KBr, cm–1): 3088,
3055, 1577, 1549, 1451, 1413, 1258, 1080, 982, 762, UV–vis
(methanol) λmax/nm (ε/M–1 cm–1): 238 (15,600), 297 (1010).
[Cu­(5-Cl2HBz)­(5,5′-bipy)­(NO3)]Cu­(4). Yield: 77%. Green crystal (FW
= 541.43 g mol–1). Elemental analysis for (CuC25H20N3O5Cl): C, 55.46%; H,
3.72%; N, 7.33%. Found: C, 56.31%;
H, 3.36%; N, 7.13. Molar conductivity: (1 × 10–3 molL–1, MeOH): 183.2 Ω–1 cm2 mol–1. ESI-MS: (CuC25H20ClN2O2 [M+]) calcd,
480.066; found, 480.0884. IR (KBr, cm–1): 3005,
2950, 1596, 1548, 1496, 1373, 1271, 1218, 1120, 1024, 820, 732, 660.
UV–vis (methanol) λmax/nm (ε/M–1 cm–1): 251 (9300), 308 (8300), 413 (400).
[Cu­(5-Cl2HBz)­(tert-bipy)­(NO3)]·2H2OCu­(5). Yield: 75%. Green crystal (FW
= 661.62 g mol–1): Elemental analysis for (C31H36ClCuN3O7): C, 56.27%;
H, 5.48%; N, 6.35%. Found: C, 57.23%; H, 4.49%; N, 6.07%. Molar conductivity:
(1 × 10–3 mol L–1, MeOH):
42.2 Ω–1 cm2 mol–1. ESI: (CuC31H32ClN2O2 [M+]) calcd, 562.1448; found, 564.1911 m/z. IR (KBr, cm–1): 3066, 2972,
1623, 1604, 1468, 1379, 1280, 1158, 951, 824. UV–vis (methanol):
λmax/nm (ε/M–1 cm–1): 284 (2699), 296 (2682), 307 (2269), 415 (488).

2.4
Single-Crystal X-ray Diffraction
X-ray diffraction
data were collected on a Rigaku XtaLAB Synergy-S
Dualflex diffractometer equipped with a HyPix 6000HE detector, using
Cu Kα radiation (1.54184 Å). The crystal was kept at a
steady T = 100.0(2) K during data collection using
an Oxford Cryosystems 800 Series Cryostream Cooler. CrysAlisPro was
used for data collection and reduction, cell refinement, and absorption
correction. The solution of the structures
was performed using the Intrinsic Phasing method from SHELXT-2018/2
program, while the refinement was conducted
using the full matrix least-squares on F
2 using the SHELXL-2019/2 program with
both hosted on Olex2 system. Non-hydrogen
atoms were refined by considering anisotropic displacement parameters,
while the hydrogen atoms were refined isotropically at idealized positions
using the riding model. Structures were deposited in the Cambridge
Structural Database under CCDC numbers
2475351, 2475352, 2475353, 2475354, and 2475355, for complexes Cu­(1), Cu­(2), Cu­(3), Cu­(4), and Cu­(5), respectively.

2.5
Cell
Culture
The complexes were tested
against A2780 human ovarian carcinoma cells (ECACC 93112519), A549
human lung cancer (ATCC CCL-185), MCF-7 human breast cancer cells
(ATCC HTB-22), and noncancer lung cells MRC5 (ATCC CCL-171). The cells
were routinely maintained with Dulbecco’s modified Eagle medium
(DMEM; A549 and MRC-5) or Roswell Park Memorial Institute 1640 medium
(RPMI 1640; A2780 and MCF-7) supplemented with 10% fetal bovine serum
(FBS), at 37 °C in a humidified 5% CO2 atmosphere.
Cells were obtained from the Rio de Janeiro Cell Bank (BCRJ). Cell
culture media and FBS were obtained from Vitrocell and Gibco, respectively.

2.6
In Vitro Cytotoxicity Assay
The cytotoxic
of complexes Cu­(1)–Cu­(5) was investigated
via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Cells were seeded in 150 μL
of an appropriate medium in 96-well plates and then incubated at 37
°C in 5% CO2 for 24 h. The compounds were dissolved
in DMSO, and 0.75 μL was added to wells (final concentration
of 0.5% DMSO/well). Cells were incubated with the compounds for 48
h at 37 °C in 5% CO2. Then, 50 μL of MTT (1
mg mL–1 in PBS pH 7.4) was added to each well. Cells
were incubated again for 4 h, the medium was removed, and formazan
crystals were solubilized in DMSO (150 μL). The absorbance was
measured by using a BioTek Epoch microplate spectrophotometer at 540
nm. All compounds were tested in three independent experiments performed
in triplicate. DMSO was used as the negative control. The cell viability
(IC50) was determined using GraphPad Prism 8.0.2 software.

2.7
Clonogenic Assay
Cells (0.8 ×
103 cells per well) were seeded in a 6-well plate and then
incubated at 37 °C in 5% CO2 for 24 h. After this
period, the cells were treated with complex Cu­(2) at
1 × IC50, 1/2 × IC50, IC50, and 2 × IC50 concentrations (0.06–0.48 μM)
and incubated for an additional 48 h. After this period, RPMI medium
was replaced with fresh medium, and the plates were incubated for
an additional 10 days. Then, the culture medium was removed, and the
colonies formed were washed with PBS, fixed with a methanol/acetic
acid (3:1) solution, and stained with violet crystal 0.5% in methanol
for 30 min. Further, the plates were washed with water and dried.
The images were taken using an Invitrogen iBright 1500 Imaging System
(Thermo Fisher). The experiment was performed in triplicate. The number
of colonies was obtained using ImageJ software, as previously reported.

2.8
Cell Morphology and Double
Staining Assays
The morphological changes induced on A2780
were studied after treatment
with complex Cu­(2). Cells (1 × 104 cells
per well) were seeded in a 96-well plate and incubated at 37 °C
in a humidified atmosphere containing 5% CO2 for 24 h.
After this period, complex Cu­(2) was added at 1/2 ×
IC50, IC50, and 2 × IC50 concentrations
(0.12–0.48 μM). For the fluorescence images, the cells
were incubated with Hoechst 33258 and propidium iodide (PI) for 30
min in the dark. All images were obtained using a CELENA S Digital
Imaging System (Logos Biosystems).

2.9
DNA-Interacting
Properties
For the
DNA-binding studies, the concentration of calf thymus DNA (CT-DNA,
Sigma-Aldrich) was determined spectrophotometrically at 260 nm using
the nucleobase molar absorptivity of 6600 L mol–1 cm–1.

2.10
Viscosity Assay
The viscosity experiments
were carried out by using an Ostwald viscosimeter maintained in a
thermostatic bath at 25 °C. The samples (2.0 mL) were prepared
in Tris-HCl buffer (pH 7.4) containing 10% DMSO. The CT-DNA concentration
was kept constant at 100 μM, and the concentrations of complexes
were varied to obtain different molar ratios, [complex]/[CT-DNA] (0.10–0.75).
The final mixture complex/DNA was incubated at 37 °C for 1 h.
The flow times were recorded with a digital stopwatch in five replicates.
The specific viscosity values (η/η0)1/3 were plotted versus [complex]/[CT-DNA], where η and η0
correspond to the relative viscosity of DNA in the presence and the
absence of the complex, respectively. The equation η0 = (t – t
0)/t
0 was used to calculate the relative viscosity
of DNA (η0) values from the flow time of the DNA
solution (t) corrected for the flow time of the buffer
(t
0). Thiazole orange and cisplatin were
used as controls.

2.11
Circular Dichroism
The CD titrations
were carried out using a JASCO J-815 spectropolarimeter at 25 °C.
Solutions of CT-DNA (100 μM) in Tris-HCl buffer (pH 7.4) containing
10% DMSO with different molar ratios [complex]/[CT-DNA] (0.25–1.0)
were incubated at 37 °C for 24 h. A total of 4 accumulations
of the spectra were recorded from 230 to 500 nm using a quartz cuvette
with an optical path length of 0.5 cm, and a scanning rate of 200
nm min–1. The measurements were performed at the
University of São Paulo (USP), São CarlosSP,
Brazil.

2.12
Fluorescence Dye Displacement Assay
A solution of CT-DNA (100 μM) was preincubated with EB (100
μM) in Tris-HCl buffer (pH 7.4) for 1 h at 37 °C to allow
full interaction of the dye with the biomolecule. Increasing amounts
of Cu­(2) (10–100 μM) were subsequently added
to the DNA samples, followed by incubation for 24 h. Fluorescence
spectra were registered from 370 to 700 nm at 25 °C upon excitation
at 510 nm by using a Synergy/H1-Biotek fluorimeter. For data analysis,
the classical Stern–Volmer equation was used: F
0/F = 1 + K
sv [Q], where F
0 and F represent the fluorescence intensities of the DNA–dye
complex in the absence and presence of a quencher, respectively. K
sv is the linear Stern–Volmer quenching
constant and [Q] is the concentration of the added
complex.
The experiment was performed in triplicate, and the K
sv was obtained via linear regression and reported
as means ± SD.

2.13
Agarose Gel Electrophoresis
Agarose
gel electrophoresis using pBR322 plasmid DNA was performed to obtain
insights into the cleavage potential of Cu­(2). A stock
solution of the complex in DMSO was prepared and diluted in the Tris-HCl
buffer (pH 7.4). The plasmid pBR322 (100 μM) was treated with
different concentrations of Cu­(2) (10, 20, 40, 60, 80,
and 100 μM), and the samples were incubated at 298 K for 1 h.
Samples of free DNA and cisplatin (100 μM) were used as the
controls. A gel was prepared using agarose (1%) in TAE buffer 1×
(Tris-acetate-EDTA), and the samples were loaded with 10 μL
of loading buffer (30% glycerol, 5 mM xylene cyanol). The gel was
run in a TAE 1× at 50 V and 40 mA for 2 h in a Bio-Rad horizontal
tank. Furthermore, the gel was stained with ethidium bromide (EB)
during 1 h, and the image was obtained using a Gel Doc EZ Imager instrument
(Bio-Rad).

3
Results and Discussion
3.1
Synthesis and Characterization
Complexes
Cu­(1)–Cu­(5) were obtained by the
reaction of Cu­(NO3)2 · 2.5 H2O with the ligands 1,10-phenanthroline (phen), 4,7-diphenyl-1,10-phenanthroline
(bathophen), 2,2′-bipyridine (bipy), 5,5′-bipyridine
(5,5′-bipy), 4,4′-bis­(tert-butyl)-2,2′-bipyridine
(tert-bipy), and 5-chloro-2-hydroxybenzophenone (5-Cl2HBz)
(see Scheme
).
The copper­(II) complexes Cu­(1)–Cu­(5) were obtained as air-stable green crystals with satisfactory yields
(70 to 80%). Molar conductivity measurements indicated 1:1 electrolyte
behavior for all compounds, with NO3
– as the counterion. Furthermore, % of
C, H, and N elemental analysis results for complexes Cu­(1)–Cu­(5) agree with the proposed structures. Suitable
crystals of all complexes grew up by slow diffusion of a methanol-dichloromethane
solution, and their structures were determined by single-crystal X-ray
diffraction analysis. The presence of crystallization water molecules
in Cu­(2), Cu­(3), and Cu­(5)
were confirmed by the thermogravimetric curves, which show a weight
loss of 3.81% (calcd, 3.81%) for Cu­(2), 3.11% (calcd,
3.38%) for Cu­(3), and 5.21% (calcd, 5.44%) for Cu­(5) (Figures S1–S3, Supporting Information).
The FT-IR spectra for Cu­(1)–Cu­(5) exhibited bands at approximately 3069 cm–1, corresponding
to aromatic C–H stretching vibrations from the N–N heterocyclic
derivatives and 5-Cl2HBz ligands. The CO stretching vibrations
were shifted from the observed 1625 cm–1 in the
free 5-Cl2HBz ligand to lower wavenumbers at 1606 cm–1 for Cu­(2) and 1596 cm–1 for Cu­(4) upon coordination, suggesting binding to the copper­(II)
metallic center. The nitrate molecule presents two bands, which may
indicate coordination to the metallic center. These bands are observed
in regions from 765 to 799 cm–1 and from 1370 to
1390 cm–1, which are references to the stretching
υ­(ONO) and the deformation of structure δ­(ONO), respectively
(see Figures S4–S8, Supporting Information). In a copper­(II) nitrate complex where the nitrate ion adopts a
monodentate coordination mode, the Cu–O stretching vibration
in Raman spectra typically appears in the range of 310–350
cm–1. This vibrational
mode is attributed to the bond between the copper­(II) center and an
oxygen atom of the nitrate group. In the case of complexes Cu­(1)–Cu­(5), the Cu–O stretching vibration
of nitrate is observed between 300 and 400 cm–1 (see
Figures S9–S13, Supporting Information).
The UV–vis absorption spectra of complexes Cu­(1)–Cu­(5) in methanol exhibited a distinct
absorption
band with a maximum ranging from 222 to 273 nm, corresponding to π–π*
transitions associated with intraligand (IL) electronic excitations.
Additionally, absorption features observed between 355 and 423 nm
were assigned to metal-to-ligand charge transfer (MLCT) (see Figures
S14–S18, Supporting Information).
The presence of complexes in a solution of methanol was investigated
by ESI–MS in positive ion mode. The mass spectra of complexes
Cu­(1)–Cu­(5) (Figure S19–S23, Supporting Information) show the main [M]+ experimental (theoretical) monoisotopic signals at m/z 474.0199 (474.0196); 626.0953 (626.0822);
450.0215 (450.0186); 478.0539 (478.0509); and 562.1414 (562.1448)
Da, respectively.

3.2
Crystal Structure Determination
of Cu­(1)–Cu­(5)
Single crystals
of all
complexes were obtained by slow evaporation from a methanolic/dichloromethane
solution (1:2 ratio). The complexes Cu­(1), Cu­(2), Cu­(4), and Cu­(5) crystallize in the
triclinic system (space group ), while
the complex Cu­(3)
crystallizes in the orthorhombic space group Pbca. The crystallographic parameters of complexes Cu­(1)–Cu­(5) are summarized in Table
.
The crystal structure of complex Cu­(1) is shown in Figure
and crystallographic
structures for Cu­(2), Cu­(3), Cu­(4), and Cu­(5) are presented in Supporting Information (Figure S24). The X-ray diffraction studies confirm
the bidentate coordination of the diimine and 2-hydroxybenzophenone
derivatives in all complexes. The structural data are consistent with
results obtained for previously reported compounds. For complex Cu­(2), the solvation sites are
occupied by a nitrate ion, 1.5 water molecules, and one methanol molecule.
The coordination geometries of the copper­(II) complexes were analyzed
using τ parameter introduced by Addison et al., which provides
a quantitative measure for distinguishing between square-pyramidal
and trigonal-bipyramidal arrangements in five-coordinated species.
The bond distances and angles of Cu­(1)–Cu­(5) are summarized in Table S1 (Supporting Information). The τ parameter was calculated as τ
= (β – α)/60, where β and α represent
the largest and second-largest coordination angles, respectively. As shown in Table
, the Cu­(1) and Cu­(5) complexes present τ values of 0.03 and 0.04, indicating nearly
ideal square-pyramidal geometries. Complexes Cu­(3) and
Cu­(4) display slightly higher τ values (0.14 and
0.12), consistent with moderately distorted square-pyramidal structures.
The complex Cu­(2), with a coordination number of four,
was not analyzed by this method, but its bond angles are characteristic
of a distorted square planar environment, as indicated by two nearly
linear trans and cis axes close
to 90°.
Notably, the complex Cu­(3) exhibits a
coordinated
water molecule instead of a nitrate ion. The Cu–O1 bond distances
range from 1.8808(12) Å to 1.9255(14) Å across the complexes.
The C7O2 bond length in the coordinated ligands (1.250(2)
Å to 1.260(19) Å) is slightly elongated compared to the
free ligand 1.2392(16) Å, indicating bond lengthening upon coordination.
Similarly, the C2–O1 bond distance in the coordination ligands
1.295(19) Å to 1.301(2) Å is shorter than in the free ligand
1.3506(16) Å, suggesting electron delocalization within the structural
moiety. All copper­(II) complexes feature
a 5-chloro-2-hydroxybenzophenone ligand forming a six-membered chelate
ring on the one side and an N-heterocyclic ligand forming a five-membered
ring on the other side. In the axial positions of Cu­(1), Cu­(4), and Cu­(5), a nitrate ion coordinates
in a monodentate mode. The Cu–O3 bond distances 2.2352(16)
Å to 2.4220(14) Å are significantly longer than the equatorial
bonds, consistent with the Jahn–Teller effect. This elongated
axial bonding weakens the Cu–O3 interaction, explaining the
observed ionic labilization of the nitrate group in solution, which
dissociates to form an electrolyte species. Table S1 summarizes the geometrical parameters for Cu­(1)–Cu­(5).
To better understand the noncovalent
interactions within the complex
structures, the Hirshfeld surface (HS) analysis, a key method for
investigating intermolecular interactions in crystalline solids, was
performed. The HS and two-dimensional fingerprint plots for Cu­(1), Cu­(3), and Cu­(4) complexes HS
were obtained from the crystallographic information files (CIFs) generated
by SCXRD analyses using the CrystalExplorer 17.5 program package. The HS of Cu­(2) and Cu­(5) could not be generated due to the application of solvent mask use.
The d
norm surfaces were mapped over the
color scale from −0.7 (red) to 1.3 (blue), and the shape index
surfaces were obtained in the range of −1.0 (red) to 1.0 (blue)
The bidimensional fingerprint plots were generated with the combination
of the di and de distances in the scale of 0.4 to 2.8 Å. One
of the important characteristics of high-efficient stability of complexes
is the presence of π–π interactions in the crystalline
structure due to the presence of several rings in the ligands used
aligned by the intermolecular interactions, as exemplified by complex
Cu­(1) in Figure S25 (Supporting Information). In Figure
, the
strongest contacts (C–H···O, C···C,
O–H···O, C– H···C, and
C–H···N) are highlighted.
The Cu­(1) and Cu­(3) cation
surfaces were
obtained by mapping them using the d
norm function, which is obtained through the combination of the normalized
distances from the nearest atom outside (de) and inside (di) the surface.
The d
norm surface shows regions in red,
white, and blue to indicate contacts with distances shorter and longer
than the sum of the vdW radii of the involved atoms, respectively.
For Cu­(1) and Cu­(4), it is possible to observe
the presence of non-classical C–H···O interactions,
only in Cu­(1) in the structure, as the C···C
contacts are related to the π–π interactions between
the rings, as verified in the shape index surfaces.
The 2D-fingerprint
plots of the complexes (Figure S26–28, Supporting Information) clearly show the intermolecular
contacts present in the structures, and its decomposition allows the
identification of the contribution for each contact to the crystal
packing, showing that the major contributions for the complexes were
H···H and O···H contacts. The H···H
contact comprises 28.7%, 36.0%, and 32.7% for Cu­(1),
Cu­(3), and Cu­(4), respectively. Meanwhile
the O···H contact represented 11.2% Cu­(1), 14.7% Cu­(3), and 11.6% Cu­(4). Furthermore,
the C···C contact is more prominent in the structures
with strong π–π interactions, as in the Cu­(1) complex where these contact comprise 11.4%, in contrast
with the other two complexes (Cu­(3) and Cu (4)) where they represent only 5.5% and 4.2%, respectively (Figure
S29, Supporting Information).

3.3
EPR Spectroscopy for Cu­(1)–Cu­(5)
Given that Cu­(II) is paramagnetic (S = 1/2), complexes
Cu­(1)–Cu­(5) were investigated by
EPR spectroscopy. All compounds exhibit similar effective g values
(g
eff ∼ 2.07), while distinct spectral
features are observed in their respective transitions. Complexes Cu­(1), Cu­(2), Cu­(3), and Cu­(5) exhibit four well-defined main resonant lines, which can be attributed
to the interaction of the unpaired electron with the nuclear spin
of copper (63Cu and 65Cu, I = 3/2), due to the n = 2I + 1
rule. These signals have different intensities
because of the anisotropy arising from the long correlation time (τ),
which is mainly associated with the size of the ligands since methanol
is not a solvent with sufficient viscosity to restrict the complex
motion. In the case of complexes Cu­(2), Cu­(3), and Cu­(5), the transitions related to the interaction
of the unpaired electron with the 14N nucleus of the nitrogen
ligands (I = 1) are also evident. The complex Cu­(4) presented the most distinct
signal of the entire series, showing a broad spectrum that may be
associated with exchange interactions. Since all EPR spectra were
recorded at the same molarity and temperature, the authors hypothesize
that in solution this species underwent dimerization between its units,
favoring a more effective interaction between the Cu­(II) centers,
contributing to line broadening (Figures S30–S34, Supporting Information).

3.4
Stability
in Solution
The stability
of the complexes was studied prior to biological investigations. The
experiments were conducted by monitoring the spectra of the complexes
in aqueous solutions. The spectra of complexes Cu­(1)–Cu­(5) were recorded in Tris buffer containing 1% of DMSO. No
changes were observed for up to 72 h in all cases indicating that
the complexes were stable under these conditions (Figures S19–S23). It is important to emphasize that
no significant changes were observed in the spectra during stability
experiments, reaffirming that they remain stable (Figure S35–S39).

3.5
Biological
Investigations
The anticancer
activity of copper complexes Cu­(1)–Cu­(5) was investigated in three human cancer cell lines: A2780 (ovarian),
A549 (lung), and MCF-7 (breast), as well in the nontumorigenic MRC-5
lung cells following 48 h of incubation time. The cytotoxicity data,
expressed as IC50 ± SD (μM), are presented in Table
. Overall, these complexes
were cytotoxic in all cancer cells tested. In general, complexes Cu­(1)–Cu­(5) exhibited increased cytotoxicity
in comparison to the Cu­(NO3)2 starting material
and all free ligands. Moreover, complex Cu (2) presented
a high antitumor effect in all cell lines, particularly in ovarian
cancer, showing IC50 equal to 0.24 μM, which is 36-fold
more potent than the cisplatin drug (Figure
A). It should be mentioned that bathophen
was the only cytotoxic ligand in all cell lines tested, showing IC50 values ranging from 1.10 to 2.38 μM. Among the tested
cell lines, A2780 ovarian cells were particularly sensitive to Cu­(1)–Cu­(5). This result is particularly
noteworthy with respect to the cytotoxicity activity against the A2780
cell line, especially in contrast with previously reported related
systems, such as the Cu­(phen)2(OH)2(ClO4)2 complex. In this context, the complex Cu (2) exhibits approximately a 2-fold increase in cytotoxic potency. Lower selectivity indexes were obtained (SI:
0.86–2.26) since they also affected noncancerous MRC-5 cells
(Figure
B). Due to
these promising results, the complex Cu­(2) was selected
for detailed biological studies.
The morphology of the
A2780 cells was studied upon treatment with
Cu­(2) at different concentrations (0.12–0.48 μM).
As presented in Figure
, a decrease in cell density and adhesion was observed in the presence
of the complex. Significant changes were detected mainly at higher
concentrations. Hoechst/PI double staining was used to confirm cell
damage. In the absence of Cu­(2), only Hoechst fluorescence
is detected and, as expected, no cell death was observed. On the other
hand, in the presence of the complex, the population of damaged cells
increased in a dependent manner, indicating cell death (Figure
A).
In the next step, the antiproliferative potential of Cu­(2) was investigated. For this purpose, the clonogenic formation assay
was conducted. The cells were treated
with the copper complex at different concentrations (0.06–0.48
μM), and the colony formation was examined over 10 days. Our
results revealed that Cu­(2) was able to drastically reduce
the number of colonies at a concentration of 0.06 and 0.12 μM,
which is lower than its IC50 value (Figure
B). These results are in accordance with
those reported for different metal-based compounds in ovarian cancer
cells.
−

3.6
DNA Interacting Properties
DNA is
a crucial target for several metal-based compounds. Considering that copper-based compounds can interact with
DNA and the promising results obtained for Cu(2), we decided to investigate
its ability to interact with this biomolecule.
−

Several techniques
can be used to study different DNA structures.
,
Here, viscosity, circular dichroism (CD), fluorescence, and agarose
gel electrophoresis experiments were performed.
First, a viscosity
assay was conducted by measuring the relative viscosity of the DNA
solution after incubation with the complex. Thiazole orange (TO) and
cisplatin were also used as controls. While TO is an intercalating
agent that increases the viscosity of the biomolecule, cisplatin acts
via covalent bonding, promoting the distortion of the double helix
and causing a decrease in viscosity. As
presented in Figure
A, Cu­(2) causes a decrease in the viscosity upon titration
with CT-DNA, leading to an analogous trend than cisplatin. In contrast
to cisplatin, which is an alkylating agent that binds to the purine
bases on the DNA, Cu­(2) can act via several ways, and
the observed results could be a consequence of different effects,
including condensation.
,

Subsequently, CD was
used to obtain more insights into the DNA/complex
interaction. The CD technique is suitable to investigate the structural
changes in biomolecules, such as DNA.
−

B-DNA is the most common
and studied DNA form, and its CD spectra present two main bands at
275 and 245 nm, which arise from base stacking and right-handed helicity
of DNA, respectively. Following an increase in the complex/DNA ratio,
the intensity of both bands is affected in a concentration-dependent
manner, suggesting significant alterations in the secondary structure
of DNA. It is worth noting that such behavior may be associated with
DNA condensation and, therefore, should be treated with caution (Figure
B).
−

Negative band appears to be more sensitive to the complex, and its
intensity nearly disappeared at a ratio higher than 0.50. Furthermore,
a weak positive signal around 325 nm was observed, which was assigned
as an induced CD (ICD) signal arising due to the coupling between
the electric transition moments of the complex and the DNA base pairs.
These changes are typical of strong interactions between the complex
and the biomolecule.
,

Considering the extended
aromatic rings of the bathophen ligand,
eventually the complex Cu­(2) can potentially intercalate
into DNA base pairs. We therefore investigated whether complex Cu­(2) can interact with DNA through additional modes in addition
to covalent ones. For this, we performed a fluorescence competition
assay with EB. EB is a DNA-intercalating dye that bounded to DNA emits
fluorescence at 610 nm upon excitation at 541 nm. The binding displacement
of EB by an external agent causes fluorescence quenching, which may
be indicative of an intercalative behavior.
−

Upon an increase
in the complex/DNA ratio, the emission decreased as a consequence
of fluorescence quenching (Figure
C). These observations suggest an intercalative contribution
to the binding mode.
A quantitative analysis using a Stern–Volmer
equation was
conducted to evaluate the affinity of complex Cu (2)
toward CT-DNA. The K
SV value obtained
for Cu (2) was 1.01 ± 0.40 × 104 M–1, which is lower than those reported for classical
intercalators such as EB (106 M–1). However, this value is consistent with results
reported for copper­(II)-based compounds and suggests that eventually
Cu (2) can displace EB and intercalate between the DNA
base pairs.
It is worth noting that the displacement leading
to fluorescence
quenching does not necessarily confirm pure intercalation, since EB
can be displaced by various DNA binders.
,
As reported, different binding modes can take place, and due to
their structural diversity, several compounds interact through a multimodal
binding mechanism 67. Using the pBR322 plasmid DNA, the
gel electrophoresis experiments were used to investigate the DNA cleavage
properties of Cu­(2). Three distinct plasmid forms were
observed, whose mobility depends on their topology: (i) supercoiled
DNA (SC) that migrates faster on the gel, (ii) open circular DNA form
(OC), resulting from single-strand scission, which exhibits a slower
migration, and (iii) linear form (L), arising from double-strand cleavage,
which migrates between SC and OC forms. As expected, two bands are
observed for the plasmid (Figure S40, lane 1, see Supporting Information), corresponding to the SC and OC forms.
The altered plasmid mobility behavior observed in lane 2 may be attributed
to cisplatin-induced DNA cross-linking. In contrast to other copper-based
compounds, no plasmid cleavage was detected after incubation with
Cu­(2) at the maximum concentration tested (lanes 3–8).

4
Conclusions
Five
novel heteroleptic Cu­(II) complexes with 5-chloro-2-hydroxybenzophenone
and N–N donor ligands were synthesized and characterized. Single-crystal
X-ray diffraction confirmed distinct coordination geometries, while
spectroscopic studies demonstrated stability in solution.
All
complexes exhibited cytotoxic activity; however, complex Cu
(2) displayed potent cytotoxicity against A2780 ovarian
cancer cells (IC50 = 0.24 μM), surpassing that of
cisplatin by 36-fold. The studies carried out here revealed that the
DNA–Cu­(2) interaction occurs via multimodal interaction
modes, affecting the structure of the double-helix. These findings
highlight the potential of Cu­(II)/2-hydroxybenzophenone complexes
as tunable anticancer agents, with Cu­(2) containing bathophen
emerging as a particularly promising candidate for further development.

Supplementary Material