Therapeutic Switching of Metformin Using Heteroleptic Cu(II) and Zn(II) Complexes: A Combined Experimental and Computational Study.

1
Introduction
The development of a new
drug is a lengthy process that takes up
to 12 years to reach the market and requires a huge amount of funding.
,
Hence, finding new ways to use approved drugs is an alternative
strategy for the pharmaceutical industry. In this regard, drug repurposing
is an appealing technique due to its potential to accelerate the drug
development process, lower costs, and meet ever-increasing medical
needs.
,
It has recently gained popularity as a drug
discovery method to identify new therapeutic opportunities for existing
drugs. Examples include the repurposing
of sildenafil from its original indication; angina, to erectile dysfunction,
thalidomide from nausea to leprosy and multiple myeloma, and aspirin
from analgesia to colorectal cancer.
−

Cellular and preclinical
studies have also encouraged repurposing an inexpensive licensed drugs
for the treatment of cancer.

Metformin
(N,N-dimethylbiguanide)
is a first-line antidiabetic drug that has been in clinical use for
over half a century to treat type II diabetes mellitus.
,
Pharmaceutically, it exists in the form of metformin hydrochloride,
which is soluble in water, acetone, chloroform, and ether. Its ability to participate in the activation
of adenosine monophosphate-induced protein kinase (AMPK), a key enzyme
in the regulation of cellular metabolism, and its interaction with
other protein targets have caught the attention of researchers seeking
to repurpose it for cancer, Alzheimer’s disease, inflammatory
diseases, and COVID-19 treatments.
−

From a coordination chemistry perspective, metformin
is a good candidate for metallodrug coordination, as it can bind to
metal ions in a bidentate coordination manner via its two amino groups.
−

Enhancement of the antidiabetic and insulin-mimetic activity of
metformin–metal complexes relative to the metformin ligand
alone has been reported in previous works.
,,
However, the potential of metformin–metal
complexes in breast cancer treatment has not yet been well explored.
This study aims to repurpose metformin by synthesizing metal complexes
and evaluating their cytotoxic effects against breast cancer cell
lines.
In our previous work, we reported the computational screening
strategy
of the bidentate ligand chrysin, as well
as the synthesis, characterization, and cytotoxicity potentials of
Cr­(III), Cu­(II), and Zn­(II) complexes of metformin, 1,10-phenanthroline,
and chrysin, where the geometry and metal center showed structure–activity
relationships. The reported metal complexes showed promising potential
for cytotoxicity against the MCF-7 cell line. Moreover, the complexes
were characterized for their intraligand, ligand-to-metal, and metal-to-ligand
charge-transfer characteristics.
−

Inspired by the cytotoxic
potential of the reported metal complexes, we hereby report the design
and synthesis of a repurposed metformin-based compounds through Cu­(II)/Zn­(II)
complexation, yielding cytotoxic metal complexes effective against
the MCF-7 cell line. Furthermore, to the best of our knowledge, this
is the first report exploring metformin–heteroleptic Cu­(II)
and Zn­(II) complexes for cytotoxic applications supported by both
experimental and computational validation.

2
Materials and Methods
2.1
Chemicals
and Reagents
All chemicals
and reagents used in this work were of analytical grade and were used
without modification. Chrysin (Sigma-Aldrich, Burlington, MA, USA),
metal salts: Cu­(O2C2H3)·H2O and Zn­(O2C2H3)·2H2O were obtained from Loba Chemie Pvt. Ltd. Triethylamine,
NaHCO3 (Alpha Chemika, India), Mueller–Hinton agar,
Methanol, Ethanol, DMSO, ethyl acetate, dichloromethane (DCM), and
diethyl ether were purchased from Loba Chemie Pvt. Ltd. (India). Metformin
hydrogen chloride (Met·HCl) was kindly donated by Cadila Pharmaceuticals
PLC, Ethiopia.

2.2
Instruments and Experimental
Conditions
The electronic absorption spectra of the ligands
and their metal
complexes were recorded using a UV–vis spectrophotometer (SM-1600
Spectrophotometer) in the 200–800 nm range at room temperature.
Both the ligands (chrysin and metformin) and the synthesized metal
complexes were dissolved in DMSO. A quartz cell of 1.0 cm thickness
was used to hold the diluted solutions of 1.0 × 10–4 M. Finally, the absorbance and wavelength (nm) readings were plotted
using the Origin 18 software, and the changes made to the characteristic
maximum absorption wavelength (λmax) of the ligands
were used to characterize the synthesized metal complexes.
In
order to analyze the changes in the vibrational energy levels of the
ligands following complexation, both ligands and synthesized metal
complexes were subjected to FTIR analysis. A 0.8 g potassium bromide
(KBr) tablet was pressed with 1 mg of the test samples. The spectra
of the ligands and their complexes were measured in the mid-infrared
region (400–4000 cm–1) using a PerkinElmer
BX FTIR spectrometer (Shimadzu Corporation, Japan). The wavenumber
(cm–1) and transmittance were processed in Origin
18.
Thermogravimetric/differential thermal analysis (TGA/DTA)
of the
synthesized complexes was conducted using a thermal analyzer (DTG-60H,
Shimadzu Corporation). Briefly, 10 mg of the test sample was loaded
into a high-temperature crucible suspended in a computer-controlled
furnace and an electronic balance with microgram sensitivity. The
sample mass and furnace temperature were continuously recorded from
25 to 800 °C with a heating rate of 10 °C/min under a nitrogen
atmosphere (20 mL/min). Finally, temperature, TGA, and DTA data were
obtained. The TGA data were treated to calculate the mass, weight
loss percent, and weight percent of the samples. A multicurve graph
was plotted using Origin 18 and used to assign the associated fragments
and thermal stability of the complexes.
The high-resolution
mass spectra of the synthesized Cu­(II) and
Zn­(II) metal complexes were obtained with a Waters-LCT-Premier mass
spectrometer. Briefly, we used a high-resolution mass spectrometer
that operates with a sample concentration of 2 ng/μL, a capillary
voltage of 2500 V, a desolvation temperature of 250 °C using
nitrogen gas at 250 L/h, electrospray positive ionization mode, a
Bruker APEX II CCD area detector diffractometer with graphite monochromated
Mo Kα radiation (50 kV, 30 mA), and a measurement temperature
of 173 (2) K coupled with APEX 2 data collection software. The test
sample was dissolved in methanol to a concentration of 2 ng/μL
and introduced by direct infusion. The collection method involved
4 scans with a width of 0.5 and 512 × 512-bit data frames. Data
reduction was achieved using the program SAINT+, and face-indexed
absorption corrections were made using XPRE.
−

The morphology
and elemental composition of the synthesized metal complexes were
determined by using scanning electron microscopy (JSM-6500F system,
JEOL, Tokyo, Japan) coupled with energy-dispersive X-ray spectroscopy
(EDS). Briefly, 50 mg of the powder for each sample was mounted on
a copper stub with carbon tape. All samples were analyzed at a 9.7
mm working distance with an accelerating voltage of 15.0 kV and magnification
ranging from 350 to 1200×. The irradiated samples emitted X-rays
with energies characteristic of the elements present in the synthesized
metal complexes. The X-ray intensities were directly proportional
to the concentrations of the elements in the metal complexes.
X-ray diffraction was used to identify the crystalline and polycrystalline
natures and properties of the synthesized metal complexes. An X-ray
diffractometer (XRD-7000, Shimadzu Co., Japan), operating with diffraction
angles (2θ) from 5 to 80° was used. The X-ray wavelength
of CuKα (λ = 1.5406 Å) operated at 40 kV and 30 mA
was employed. The obtained diffraction
angles and associated peak intensities were used to identify the phases
of the synthesized metal complexes. Moreover, the average crystallite
size of the complexes was calculated using the Debye–Scherrer
equation. Hence, phase identification
of the synthesized metal complexes was accomplished by comparing the
experimental XRD pattern with a reference pattern obtained from the
Crystallography Open Database (COD) for inorganic materials. The comparison
between the experimental XRD pattern and the reference was conducted
using QUALX2.0. Accordingly, the crystal
system of the complexes, the space group of the complexes, the interplanar
spacing (d), the Miller indices (hkl), and the lattice parameters (a, b, c, α, β, and γ) of the synthesized
complexes were determined using the same protocol.
The molar
conductance of the metal complexes was recorded at room
temperature by using an electrical conductometer (AD8000). The complexes
were prepared in 1.0 × 10–3 M DMSO and measured
in triplicate. The same molarity and temperature conditions were used
to measure the electrical conductivity of the complexes.

2.3
Synthesis and Characterization
of the Synthesized
Metal Complexes
The Cu­(II)–metformin–chrysin
complex (1) was synthesized according to a reported method
on the synthesis of a similar chrysin complex with minor modifications. Unlike the reported homoleptic chrysin-based
complex, our synthesis includes metformin as a coordinating ligand.
Additionally, triethylamine was added to enhance the basicity of the
reaction medium, a step absent in the original method. While the reported
synthesis employed a mixture of DMF and ethanol, the present synthesis
utilized a 1:3 ratio mixture of methanol and ethanol as a solvent.
Briefly, 1 mmol of Cu­(O2C2H3)·H2O (0.199 g) was added slowly to 1 mmol of chrysin (0.254 g)
in a hot (40 °C) 1:3 methanol–ethanol solution in the
presence of the deprotonating agent NaOH (1 mmol). The resulting yellow-green
solution was refluxed and stirred for 50 min, after which 1 mmol of
a methanolic solution of metformin (0.165 g) was slowly added, followed
by the addition of 1 mmol of triethylamine (140 μL). The reaction
mixture was refluxed for an additional 8 h at 80 °C. The progress
of the reaction was monitored with TLC. The reaction mixture was filtered
and treated with diethyl ether and stored at 4 °C for 2 weeks,
after which gray-brown powders were collected using slow evaporation.
Complex 2, which is the same complex but with a Zn­(II)
metal center, was synthesized using a similar procedure, except for
the refluxing step that was performed for 6 h at 60 °C. This
procedure yielded a yellow-orange powder with visible grain size.
The schematic representation of the synthesis procedure is presented
in Figure
.

2.4
Biological
Activity
2.4.1
Cytotoxicity: Cells and Cell Maintenance
MCF-7 breast cancer cells, previously stored in liquid nitrogen
at −190 °C, were cultured at 37 °C under a humidified
5% CO2 atmosphere. The cells were cultured in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10% Fetal Bovine Serum
(FBS), 2 mM l-glutamine, 50 IU/mL penicillin, and 50 μg/mL
streptomycin.

2.4.2
Cytotoxicity
Assay
The cytotoxicity
of the synthesized complexes (1 and 2) was
evaluated using the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay, as per the manufacturer’s instructions
(Cell Proliferation Kit I (MTT, Roche Diagnostics Gmb, Germany).
Briefly, MCF-7 breast cancer cells were seeded in a clear, flat-bottomed
96-well plate at a density of 5 × 103 cells per well
in 100 μL of growth medium. The cells were allowed to attach
for 24 h. The synthesized compounds were dissolved in phosphate-buffered
saline (PBS, pH ∼ 7.4) and serially diluted with the growth
medium to prepare different concentrations ranging from 100 to 3.125
μM. Spent growth medium was discarded, and the cells were then
treated with the compounds diluted with the growth medium for 24 h
at 37.5 °C. Cells treated with a mixed solvent (PBS) at 215 concentrations
matching those of the compounds were reserved as control group. Cisplatin
was included as a positive control. After the 24-h incubation period,
10 μL of the MTT labeling reagent (final concentration of 0.5
mg/mL) was added to each well and incubated at 37.5 °C for 4
h in a humidified 5% CO2 atmosphere. DMSO was added to
each well to dissolve the formazan crystals. Finally, the optical
density was measured at 570 nm by using a Multiskan FC Microplate
photometer (ThermoFisher Scientific). Photomicrographs of treated
and untreated MCF-7 cells were taken using a Primovert inverted microscope
equipped with Axiocam 208 color camera (Carl Zeiss).
,
The experiments were conducted independently three times and results
presented as mean ± standard error of the mean (SEM).

2.5
Computational Studies
Density functional
theory (DFT) calculations were performed using the B3LYP
−

hybrid functional together with the 6-311++G­(d, p) basis set for the light atoms (H, C, N, and O) and Los
Alamos National Laboratory 2-Double-Zeta (LANL2DZ) pseudopotentials
for the metal atoms (Cu­(II) and Zn­(II)) to account for relativistic effects. The nonbonding interactions
during the calculations were corrected using Grimme’s dispersion
correction. This is because such a combination
of functional and basis sets has been used to lower the computational
cost and has been reported in previous studies to give a good agreement
with the experimental results.
−

Vibrational frequency calculations
were performed at the same level of theory to confirm that the optimized
geometries corresponded to real energy minima without any imaginary
vibrational frequencies.
The quantum-chemical descriptors: energy
gap (ΔE = E
LUMO
–
E
HOMO), electronegativity (χ = −0.5­(E
HOMO + E
LUMO)),
electronic chemical potential (μ = 0.5­(E
HOMO + E
LUMO) = −χ),
global chemical hardness (η = 0.5­(E
LUMO – E
HOMO)), global softness (σ
= 0.5η), global electrophilicity index (ω = μ2
/2η), and nucleophilicity index (Nu
= 1/ω) were calculated and analyzed at the same level of theory.
,
Such quantum-chemical descriptors were used to establish how the
structure, stability, and reactivity of the compounds relate to their
biological activity.

2.6
Molecular Docking Studies
The molecular
docking analysis of the synthesized complexes with the estrogen receptor
alpha (ERα; PDB: 5GS4) was conducted following
the same protocols as reported in our previous work.
−

To account for conformational flexibility, 100 conformers were generated
and analyzed for each compound. The conformers with the lowest binding
free energy were used to visualize the interactions between the active
amino acids and the molecules using the Discovery Studio software.

2.7
Statistical Analysis
Data were analyzed
using GraphPad Prism 10, Version 10.5.0. One-way analysis of variance
(ANOVA) and two-way ANOVA, followed by Tukey’s multiple comparisons
test, were used to compare the means of two groups and three or more
groups, respectively. A p-value equal to or less
than 0.05 was considered statistically significant.

3
Results and Discussion
3.1
Physicochemical Properties
The synthesized
metal complexes (1 and 2) were subjected
to a solubility test using solvents of varying polarity. The complexes
were soluble only in DMF and DMSO solvents. The recorded melting point
values showed that the complexes decompose at temperatures greater
than 180 °C (Table
). The molar conductance values were found to be 34.80 and 126.60
Ω–1 mol–1 cm2 for complexes 1 and 2, respectively, confirming
the coordinative mode of acetate in complex 1. In contrast,
complex 2 exhibits an ionic mode of coordination, indicating
its electrolytic nature, whereas complex 1 is nonelectrolytic.
This analysis agrees with previous studies.

3.2
FTIR Analysis
The FTIR spectra of
the synthesized complexes are presented in Figures S1 and S2 of the Supporting Information (SI). Infrared spectroscopic spectral
analysis indicated that metformin can be characterized by its N–H
stretching vibration, asymmetrical and symmetrical C–N vibrations,
and NH2 deformation, in the ranges of 3100–3490,
1583–1626, and 1470–1540 cm–1, respectively,
in addition to other stretching and bending modes.
,
These vibrational frequencies were found to appear at 3429, 1637,
and 1483–1409 cm–1. In complex 1, the N–H stretching vibration, C–N vibrations, and
NH2 deformation peaks clearly appeared at 3330, 1555, and
1409 cm–1, respectively. Similarly, these vibrational
frequencies were recorded at 3395, 1566, and 1444 cm–1 for complex 2. The M–O and M–N vibrational
frequencies were observed at 622 and 611 cm–1 for
complex 1 and at 641 and 612 cm–1 for
complex 2, respectively, confirming the presence of a
similar coordination environment for both metal centers.

3.3
UV–vis/TD-DFT Analysis
According
to the UV–vis and TD-DFT calculated absorption spectra of chrysin
in DMSO (Figure S4), the π →
π* transitions of the benzoyl and cinnamoyl groups appeared
at 267/261 nm and 320/325 nm (exp/calc), respectively, in line with
previously reported results.
,
The bathochromic and
hypochromic shifts in the characteristic absorption spectra of chrysin
observed for complexes 1 and 2 are indicated
in Figure
. For complex 1, the characteristic peak for the benzoyl system of chrysin
underwent a bathochromic shift to 271/268 nm with an observable change
in the spectral width. Specifically, a small and broad peak associated
with the cinnamoyl system of chrysin (320/325 nm, π →
π*) was found to be red-shifted to 316/363 nm, confirming the
presence of a new electronic distribution and coordination of the
ligands to the Cu­(II) center. New broad and less intense peaks (Figure
, top panel) that
appeared at 403/473 nm are ascribed to charge transfer between the
two ligands (ligand–ligand charge transfer) or metal-to-ligand
charge transfer (MLCT), which also confirms the presence of a new
electronic phenomenon due to metal–ligand coordination.
In complex 2, the
benzoyl group also underwent a bathochromic
shift (to 270/271 nm), while the main absorption peak of the cinnamoyl
system of chrysin appeared at 296/313 nm, indicating a hypsochromic
shift. The absence of any peak above 400 nm in the experimental UV–vis
spectrum (Figure
,
bottom panel) confirms the absence of d–d electronic transitions
due to the completely filled d-orbital of the Zn­(II)-centered complex
(2). However, the tendency of the Zn­(II) metal center
to undergo MLCT is clearly observed in the TD-DFT-calculated HOMO–LUMO
distribution (Figure
).

3.4
Frontier Molecular Orbital Analysis of the
Complexes
Frontier molecular orbitals (FMOs) play a very
important role in the reactivity and stability of chemical compounds. To understand the distribution of FMOs, we calculated
the HOMO and LUMO of the ligands and resulting complexes together
with their band gap energies, as shown in Figure
. The isodensity distribution of the HOMO–LUMO
in metformin and chrysin molecules confirmed the presence of intramolecular
charge transfer. This effect is more pronounced in the presence of
metal centers. The isodensity surfaces of HOMO and LUMO of complex 1 were found to reside on the metformin–Cu­(II) part
of the molecule and the Cu­(II)–chrysin part of the complexes,
respectively, confirming the presence of a d–d electronic transition
and MLCT character. In complex 2, the HOMO is distributed
over the entire complex, while the LUMO is localized on the chrysin
part of the complex, confirming the presence of intraligand (ligand–ligand)
charge transfer and MLCT, in line with the experimental findings.

3.5
Quantum Chemical Descriptor Analysis of the
Complexes
The quantum chemical descriptors calculated in
this study are presented in Table
. The energy gap, E
g, between
the HOMO and LUMO energies, E
HOMO and E
LUMO, respectively, is a significant stability
index and is associated with structural and kinetic stability. A molecule with a large band gap is described
as a hard molecule with a high kinetic stability, low chemical reactivity,
and high molecular stability.
−

The E
g values for the ligands and the synthesized complexes were found
to be 5.988, 4.403, 2.964, and 3.648 eV for metformin, chrysin, complex 1, and complex 2, respectively, confirming the
reduction of hardness in the ligands during complexation and the improvement
in the biological activity following the formation of the resulting
soft metal complexes (1 and 2). Complex 2 showed improved stability over complex 1. On
the other hand, compounds with a small band gap energy are soft and
easily interact with soft molecules (DNA, proteins, receptors, etc.).
,
The high biological activity observed in the cytotoxicity of complex 2 (vide infra) can be associated with its
large dipole moment.
The chemical potential (μ) of the metal complexes,
which
determines their chemical reactivity ranking and has a direct relationship
with their Gibbs free energy, is shown in Table
. The chemical reactivity increases with
decreasing chemical potential, indicating
that the chemical reactivity of complex 1 (−4.401
eV) is higher than that of complex 2 (−4.125 eV).
Relative to the ligands, the metal complexes showed higher nucleophilicity
(Nu) because of the reduced band gap energy, which allows better interaction
with the biomolecules for biological activity. In line with this,
the increased dipole moment values, the decreased hardness (η)
and increased softness (δ) of the metal complexes relative to
the ligands could suggest enhanced biological activity of the metal
complexes.

3.6
Molecular Electrostatic
Potential Analysis
Molecular electrostatic potential (ESP)
is an important quantum
chemical descriptor for understanding intermolecular interactions
in three dimensions. The molecular structures
and ESP of the metal complexes are presented in Figure
. The metformin-coordinated side of the metal
complexes showed positive ESP. Complex 1 has both electron-rich
(red) and electron-deficient regions (blue) susceptible to nucleophilic
and electrophilic attack, respectively, while most parts of complex 2 showed potential electrophilic sites, with the metformin-coordinated
side being the most susceptible region. These findings suggest that
structure–activity relationship strategies targeting specific
electrostatic interactions could be a valuable strategy for enhanced
biological activity. The higher positive ESP distribution of complex 2 may be one of the reasons for its higher binding affinity
and smaller inhibition constant against estrogen receptor alpha (ERα;
PDB: 5GS4) in
the molecular docking study (vide infra).

3.7
Mass
Spectrometric Analysis
The high-resolution
mass spectrometry (HRMS) data of the synthesized metal complexes were
used to study the fragmentation patterns of the complexes. The HRMS
results are presented in Figures S6 and S7 of the Supporting Information. In complex 1, the molecular ion peak was not observed due to the increased
intensity of the remaining peaks. A peak appeared at m/z = 393.992 (calc = 395.030), which can be attributed
to the complex cation [C14H13CuN5O5]+. Another peak supporting the proposed
mass fragmentation appeared at m/z = 353.265 and corresponds to the C13H14CuN5O3 fragment. A peak at m/z = 321.131 corresponds to the chemical fragment C13H9CuN4O4. The fragments for the
coordinating ligands, chrysin and metformin, appeared as intense peaks
at 255.065 and 130.109, respectively. Similarly, in complex 2, a molecular ion peak at the expected m/z ∼ 503 was not observed due to intensity
differences between the base peaks and the rest of the peaks. It is
important to note that such behavior has been reported in the literature
for related complexes.
−

Moreover, heteroleptic Cu­(II)/Zn­(II) complexes can also frequently
undergo ligand loss, acetate dissociation, or metal-centered fragmentation
during ionization, resulting in stable fragment ions rather than a
molecular ion. The fragments obtained for the Zn­(II) complex were
found at m/z = 473.005, which corresponds
to the fragment C20H17N5O5Zn, confirming the presence of parts of chrysin, an acetate ion,
and metformin. Moreover, the presence of the coordinated ligands was
confirmed based on the presence of their characteristic mass spectra
at m/z = 255.065 and 130.109 for
chrysin and metformin, respectively. Therefore, the fragment ions
observed are consistent with the expected fragmentation pathways and
support the proposed compositions and coordination environments.

3.8
SEM-EDX Analysis
The morphology and
atomic composition of complexes 1 and 2 were
studied using SEM-EDX. The SEM micrographs presented in Figure
reveal that the complexes
consist of uniformly aggregated grains. Complex 1 did
not yield smooth surfaces but showed the presence of uniformly sized
small grains, which is in line with the polycrystalline phase reported
in the XRD results. A relatively more uniform and smoother surface
was observed in 2, which might be due to its high percent
crystallinity (Figure
b). The EDX spectra of 1 and 2 showed characteristic
signals corresponding to carbon, oxygen, and nitrogen, together with
the respective metals, further confirming the successful synthesis
of the metal complexes.

3.9
Thermogravimetric
Analysis
The TGA/DTA
analyses of the synthesized metformin–chrysin mixed-ligand-based
complexes are presented in Figure
and Table
. The thermal degradation pattern of complex 1 proceeded in four main degradation steps, and the thermograph data
revealed that: (i) from 55 to 207 °C (DTG
max = 103 °C), the weight loss was 24.65%, corresponding
to the release of 3H2O, C2H3O2, and OH chemical entities, which is in good accordance with
the calculated value (24.60%); (ii) from 209 to 326 °C, the weight
loss was due to the C13H7O moiety, with the
total weight loss being 32.34% (calculated value: 32.31%); (iii) from
335 to 449 °C, the weight loss was 10.12% (calculated value:
10.01%), corresponding to the decomposition of the CH3N3 moiety; and (iv) from 500 to 557 °C, the weight loss
was 15.25% (calculated value: 15.20%), corresponding to the decomposition
of the C5H8N2 moiety. The total weight
loss was 82.36% (calculated: 82.20%), with the residue related to
copper oxide (CuO). The organic moiety decomposition and the percent
of CuO residue are similar to the previously reported metal complexes.
,

The TGA and DTA analyses of complex 2 showed that
it thermally decomposed in two main degradation steps: (i) from 205
to 493 °C, the weight loss was 25.6%, corresponding to the release
of C4H9N5 molecules, which is in
good accordance with the calculated value (25.4%); and (ii) from 501
to 758 °C, the loss due to C15H11O2 groups gave rise to a weight loss of 43.81% (calculated value:
43.60%). The final step showed a total weight loss of 69.41%, which
corresponds to the remaining zinc oxide, in line with reported studies.
,

3.10
Powder X-ray Diffraction Analysis
Attempts to grow single crystals were unsuccessful, as the products
consistently formed as polycrystalline or amorphous solids. The pXRD
patterns of the synthesized complexes further confirm their polycrystalline
nature, showing broad reflections with varying intensities in the
2θ range of 5°–80° (Figure
). Complex 1 is a monoclinic
crystal (COD = 00-702-2172) with a C 1 2/c 1 space group and lattice
parameters of 22.6900, 13.916, 20.317, 90°, 90°, and 98.975°
for a, b, c, α,
β, and γ, respectively. Complex 2 is a triclinic
crystal (COD = 00-600-0487) with a P −1 space group and lattice
parameters of 7.966, 9.082, 11.832, 81.421°, 71.301°, and
74.998° for a, b, c, α, β, and γ, respectively. The average crystalline
size of the complexes, determined from the Debye–Scherrer equation, was 19 and 18 nm for complexes 1 and 2, respectively. The percent crystalline index
of the complexes was calculated using eq
and found to exist as 23% and 94% crystallinity for 1 and 2, respectively, inferring the synthesis
of diverse crystalline metal complexes.where A
c is the
area of the crystal and A
a is the area
of the amorphous part of the materials obtained from the XRD data.
The number of dislocation lines per unit area of the crystal, the
dislocation density (δ) of the complexes, was determined from
its relation to the average crystallite size (D)
of the complexes, according to previously reported studies:
,

Accordingly,
the dislocation density values of complexes 1 and 2 were calculated to be 2.989 × 10–3 and 3.083 × 10–3 nm–2,
respectively, confirming that the synthesized crystalline metal
complexes have relatively less dislocation density and less irregularity
within the structure. This is in line with previously reported values
of dislocation density ranging from 3.00 × 10–4 to 2.10 × 10–3 nm–2 for
Cu­(II), Ni­(II), Mn­(II), and UO2(II) mixed-ligand polycrystalline
complexes.

3.11
Cytotoxicity
Study
The percentage
cell viability before treatment (0 μM) and after treatment with
complex 1 and complex 2 at various concentrations
(3.125–100 μM), along with the corresponding cellular
morphological changes at 100 μM are presented in Figures
and , respectively. Both synthesized complexes reduced the viability
of MCF-7 cells in a concentration-dependent manner. Notably, treatment
with complex 1 resulted in a significant decrease in
cell viability to 52.0% (p ≤ 0.01), 13.0%
(p ≤ 0.0001), and 5.4% (p ≤ 0.0001) at concentrations of 25 μM, 50 μM,
and 100 μM, respectively (Figure
a). In contrast, cells treated with complex 2 exhibited higher viabilities of 53.6% (p ≤
0.01), 48.9% (≤0.0001), and 34.3% (≤0.0001) at concentrations
of 25 μM, 50 μM, and 100 μM, respectively
(Figure
b). These
findings suggest complex 1 is more potent than complex 2, and the differences in cytotoxic activity between the two
complexes were statistically significant at concentrations of 50 μM
(p ≤ 0.01) and 100 μM (p ≤ 0.05) (Figure
c). According to previous studies,
−

a compound
is considered cytotoxic if it reduces cell viability below 70%. Based
on this criterion, both complexes 1 and 2 can be classified as cytotoxic. The IC50 values determined
in this study were 18.93 μM for complex 1 and 43.31
μM for complex 2, compared to the previously reported
IC50 of 18.62 μM for cisplatin in our earlier investigations.
,
The results support the conclusion that Cu­(II)–metformin–chrysin
and Zn­(II)–metformin–chrysin complexes are promising
candidates for cytotoxic activity against MCF-7 cell lines, consistent
with previously reported results.
,

The epithelial-like skeletons of the MCF-7 cells
with a polygonal
morphology were clearly observed in untreated MCF-7 cells (Figure
). However, after
treatment with 100 μM of the synthesized complexes and cisplatin
for 24 h, the cells’ morphology and density had changed and
decreased. This phenomenon was most clearly observed for the Cu­(II)-centered
complex 1, consistent with its significantly lower cell
viability (5.4%) compared to that of complex 2 (34.3%)
at 100 μM. Our microscopy and percent cell viability results
clearly demonstrate that the metal complexes in this study are cytotoxic
against MCF-7 cell lines.

3.12
Molecular Docking Studies
A molecular
docking study of the synthesized mixed-ligand complexes was conducted
against the estrogen receptor alpha (ERα; PDB: 5GS4), a clinical biomarker
for subtype breast cancers, to gain insight
into the interaction of the complexes with the active amino acids
of ERα. The interactions of the synthesized mixed-ligand–metal
complexes (1 and 2) with the prominent residual
amino acid interactions of ERα are presented in Figures
and . Complex 1 was found to bind to Glu 323 via a conventional
hydrogen bond to Arg 394, Trp 393, Glu 323, Phe 445, Met 357, Ile
386, Leu 387, and Leu 349 via van der Waals forces and to Lys 449,
Glu 353, His 356, and Arg 352 via π-alkyl/π-sigma interactions
with a binding affinity of −5.69 kcal mol–1. In complex 2, the same amino acid, Glu 323, is engaged
in a hydrogen bonding interaction, while new amino acids such as Lys
449, Trp 360, Met 357, His 356, and Leu 346 interact via van der Waals
forces, while Glu 323 showed additional π-alkyl/π-sigma
interaction with a binding affinity of −6.12 kcal mol–1. The involvement of amino acidsGlu 323 in a conventional
hydrogen bonding interaction; Trp 393, Phe 445, Met 357, Ile 386,
and Leu 387 in van der Waals forces; and Glu 353 and Arg 352 in the
π-alkyl/π-sigma interaction environmentconfirmed
that the metal complexes are fit into a similar binding pocket of
the receptor. The type of interactions, binding energy, inhibition
constant, and the root-mean-square deviation (RMSD) values for the
molecular docking of the metal complexes against ERα are shown
in Table
. A higher
binding affinity and smaller minimum inhibitory concentration obtained
for complex 2, contrary to the in vitro cytotoxicity results, could be associated with the geometric effect
of the metal center. However, it is important to note that Cu­(II)
and Zn­(II) complexes follow multiple mechanisms of action to treat
cancer cells, viz., oxidative damage to DNA, depletion of reduced
glutathione, and/or cell death by apoptotic and nonapoptotic dose-dependent
DNA binding mechanisms. Hence, this needs
further investigation through in vivo as well as
mechanistic studies.

4
Conclusions
In this work, we reported
the successful design and synthesis of
cytotoxic Cu­(II) (1) and Zn­(II) (2) mixed-ligand
complexes comprising the antidiabetic drug metformin and the naturally
available flavonoid chrysin as ligands. The powder XRD results confirmed
the polycrystalline nature of the complexes, with a high crystallinity
percentage (94%) for complex 2. Molar conductance measurements
indicated an electrolytic nature for complex 2 and a
nonelectrolytic nature for complex 1. The geometries
of the metal complexes were predicted from the molar conductance measurements
together with other techniques (UV–vis, FTIR, HRMS, SEM-EDX,
and XRD). Hence, the metal complexes were found to be square pyramidal
for complex 1 and tetrahedral for complex 2. This is further supported by the good agreement between the experimental
and TD-DFT calculated absorption spectra of both complexes. The IC50 values were found to be 18.93 and 43.31 μM, respectively,
for 1 and 2, compared to those of the positive
control cisplatin reported in our previous study (IC50 =
18.62 μM). The band gap energies of the metal complexes were
found to be 2.964 and 3.648 eV, respectively, for 1 and 2, compared to the ligands, metformin (5.988 eV) and chrysin
(4.403 eV). The molecular docking simulations against estrogen receptor
alpha (ERα; PDB: 5GS4) support the in vitro biological
activity results, with a binding energy of −5.69 kcalmol–1 and an inhibition constant of 51.37 μM for 1, and −6.12 kcalmol–1 and 30.12
μM for 2. Overall, this work presented the role
of metformin repurposing via heteroleptic metal complex design in
enhancing biological activity. The observed synergistic effects affirm
that metal coordination can significantly potentiate the therapeutic
efficacy of organic ligands and drug molecules. Moreover, the findings
of this study suggest that metformin can be therapeutically repurposed
for breast cancer treatment through metal complexation. The approach
demonstrated here highlights metal complexation as an alternative
strategy for the therapeutic switching and repurposing of approved
drugs. However, a comprehensive in vivo and pharmacological
investigations are strongly recommended to validate the observed promising in vitro cytotoxicity results. Moreover, further evaluation
of these complexes for their activity as antidiabetic agents and for
cytotoxicity against different cancer cell lines is recommended.

Supplementary Material