Jebiti Haribabu1, Swaminathan Srividya1, Dharmasivam Mahendiran2, Dasararaju Gayathri3, Vemula Venkatramu4, Nattamai Bhuvanesh5, Ramasamy Karvembu1. 1. Department of Chemistry, National Institute of Technology, Tiruchirappalli 620015, India. 2. Department of Pathology, Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia. 3. Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600025, India. 4. Department of Physics, Krishna University Dr. MRAR PG Centre, Nuzvid 521201, India. 5. Department of Chemistry, Texas A & M University, College Station, Texas 77842, United States.
Abstract
Metal complexes have numerous applications in the current era, particularly in the field of pharmaceutical chemistry and catalysis. A novel synthetic approach for the same is always a beneficial addition to the literature. Henceforth, for the first time, we report the formation of three new Pd(II) complexes through the Michael addition pathway. Three chromone-based thiosemicarbazone ligands (SVSL1-SVSL3) and Pd(II) complexes (1-3) were synthesized and characterized by analytical and spectroscopic tools. The Michael addition pathway for the formation of complexes was confirmed by spectroscopic studies. Distorted square planar structure of complex 2 was confirmed by single-crystal X-ray diffraction. Complexes 1-3 were subjected to DNA- and BSA-binding studies. The complex with cyclohexyl substituent on the terminal N of thiosemicarbazone (3) showed the highest binding efficacy toward these biomolecules, which was further understood through molecular docking studies. The anticancer potential of these complexes was studied preliminarily by using MTT assay in cancer and normal cell lines along with the benchmark drugs (cisplatin, carboplatin, and gemcitabine). It was found that complex 3 was highly toxic toward MDA-MB-231 and AsPC-1 cancer cells with IC50 values of 0.5 and 0.9 μM, respectively, and was more efficient than the standard drugs. The programmed cell death mechanism of the complexes in MDA-MB-231 cancer cells was confirmed. Furthermore, the complexes induced apoptosis via ROS-mediated mitochondrial signaling pathway. Conveniently, all the complexes showed less toxicity (≥50 μM) against MCF-10a normal cell line. Molecular docking studies were performed with VEGFR2, EGFR, and SARS-CoV-2 main protease to illustrate the binding efficiency of the complexes with these receptors. To our surprise, binding potential of the complexes with SARS-CoV-2 main protease was higher than that with chloroquine and hydroxychloroquine.
Metalcomplexes have numerous applications in the current era, particularly in the field of pharmaceutical chemistry and catalysis. A novel synthetic approach for the same is always a beneficial addition to the literature. Henceforth, for the first time, we report the formation of three new Pd(II)complexes through the Michael addition pathway. Three chromone-based thiosemicarbazone ligands (SVSL1-SVSL3) and Pd(II)complexes (1-3) were synthesized and characterized by analytical and spectroscopic tools. The Michael addition pathway for the formation of complexes was confirmed by spectroscopic studies. Distorted square planar structure of complex 2 was confirmed by single-crystal X-ray diffraction. Complexes 1-3 were subjected to DNA- and BSA-binding studies. The complex with cyclohexyl substituent on the terminal N of thiosemicarbazone (3) showed the highest binding efficacy toward these biomolecules, which was further understood through molecular docking studies. The anticancer potential of these complexes was studied preliminarily by using MTT assay in cancer and normal cell lines along with the benchmark drugs (cisplatin, carboplatin, and gemcitabine). It was found that complex 3 was highly toxic toward MDA-MB-231 and AsPC-1cancercells with IC50 values of 0.5 and 0.9 μM, respectively, and was more efficient than the standard drugs. The programmed cell death mechanism of the complexes in MDA-MB-231cancercells was confirmed. Furthermore, the complexes induced apoptosis via ROS-mediated mitochondrial signaling pathway. Conveniently, all the complexes showed less toxicity (≥50 μM) against MCF-10a normal cell line. Molecular docking studies were performed with VEGFR2, EGFR, and SARS-CoV-2 main protease to illustrate the binding efficiency of the complexes with these receptors. To our surprise, binding potential of the complexes with SARS-CoV-2 main protease was higher than that with chloroquine and hydroxychloroquine.
Cancer, since its discovery,
has been a life-threatening disease globally. It marks the unmanageable
growth of abnormal cells that can colonize and disrupt the tissues.[1] The usage of metalcomplexes as drugs lingers
strongly, either unaided or in combination with the other drugs, even
though the side effects and development of drug resistance are highly
likely.[2] Currently, Pt therapeutics (cisplatin,
carboplatin, oxaliplatin, etc.) are in use for almost 50–70%
of cancer therapy. However, their dosage is restrained due to their
unsympathetic side effects such as nausea, vomiting, loss of sensation
in the extremities, and nephrotoxicity.[3,4] Pd(II)complexes
are noteworthy alternatives for the treatment of cancer because of
their structural and thermodynamic similarities to Pt(II)complexes.[5−7] There have been numerous reports on Pd(II)complexes with potential
anticancer properties against various cancercell lines (lung, prostate,
etc.); some are showing better activity (predominantly in in vitro system) than their Pt(II)counterparts.[8−11] Pd(II)complexes have shown remarkable antiproliferative effects
on humanbreast cancer including tumorcell lines that are resistant
to cisplatin, and also displayed good antimycobacterial effects.[12−15]Thiosemicarbazones (>C=N–NH–C(S)–N<)
(TSCs) have evolved as an imperative class of Schiff base ligands
that are structurally appealing and biologically active candidates.
Triapine, which is presently in phase I clinical trial against cancercell lines, and thiacetazone, which has proved beneficial for the
treatment of tuberculosis, are some drugs that have highlighted the
biological activities of thisclass of compounds.[16,17] Heterocyclic TSCs have been portrayed as compounds that not only
target the ribonucleotide reductase but also other intracellular molecules
such as N-myc downstream-regulated gene-1 (NDRG1) and DNA topoisomerase
IIa (top2a).[18] Such properties of TSCs
fascinated the researchers to explore their coordination behavior
with metal ions.[18−21] In many cases, coordination of these ligands to metal ions increases
their biological activity, proposing that complexation could be a
significant approach for dose reduction. The coordination behavior
of TSCs and medicinal applications of their metalcomplexes have been
established briefly in the literature.[22−27]Chromones are compounds which contain a γ-pyrone nucleus
fused to a phenyl ring at the fifth and sixth positions. Chromones
and flavones have played a vital role in the human diet. Numerous
biologically active molecules derived from natural sources contain
a chromone moiety as their core.[28−31] These compounds have attracted
the pharmaceutical industries to a large extent owing to their antioxidant,
antitumor and antibacterial activities.[32−34] Besides these, several
chromone derivatives have been used as antimicrobial, DNA-binding,
DNA-cleavage, anticancer, antiallergic, neuroprotective, and pesticidal
agents.[28−44] Although the appeal of chromones and their derivatives is widespread,
the literature still lacks knowledge about their importance in coordination
chemistry. Our group has been meticulously involved in studying the
coordination behavior of heterocyclic TSCs with Ni(II), Cu(II), Zn(II),
Pd(II), and Ru(II) and its influence on the biomolecular interactions
and anticancer activity against various cancer and normal cell lines.[45−56] Most of these complexes induced cell deaththrough apoptosis.The broadcast of severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2), i.e., COVID-19, has been declared as pandemic by the
World Health Organization (WHO) and has affected hundreds of countries
in a matter of weeks. A global response to prepare health systems
is a priority.[57] Hence, we have extended
our research to understand the interactions between the present Pd(II)complexes and the SARS-CoV-2 main protease through molecular docking
(MD) studies. MD studies with DNA, BSA protein, vascular epithelial
growth factor receptor (VEGFR2), and epithelial growth factor receptor
(EGFR) have also been carried out to learn the mechanism of action
of the complexes through which they exhibit a cytotoxic effect.The biological properties of Pd, heterocyclic TSCs, and functionalized
chromones have motivated us to develop better anticancer agents than
the ones obtained earlier. Herein, we report the synthesis of the
novel Pd(II)complexes bearing chromone TSCthrough Michael addition.
The compounds were well-characterized by analytical, spectroscopic,
and single-crystal X-ray diffraction methods and were evaluated to
know their biomolecular (DNA, protein, VEGFR2, EGFR, and SARS-CoV-2
main protease) interactions and anticancer potential. To the best
of our knowledge, this is the first report on the synthesis of Pd(II)complexes through Michael addition.
Experimental
Section
Materials and Methods
Analytical-grade chemicals and
solvents were purchased from commercial suppliers (Sigma-Aldrich,
Promega, Biolegend, BD Biosciences, and Alfa Aesar) and used as received.
The melting points were determined on a Lab India instrument and are
uncorrected. Elemental analyses were carried out using a PerkinElmer
instrument. FT-IR spectra were obtained as KBr pellets using a Nicolet-iS5
spectrophotometer. UV–visible (UV–vis) spectra were
recorded using a Shimadzu-2600 spectrophotometer. Emission spectra
were recorded on a Jasco V-630 spectrophotometer using 5% DMSO in
buffer as solvent. NMR spectra were recorded in CDCl3/DMSO-d6 solvent by using TMS as an internal standard
on a Bruker 500/400 MHz spectrometer. ESI-MS spectra were obtained
using a Waters ZQ 4000 or Bruker maXis impact mass spectrometer. MALDI
mass spectra were recorded on a 4800 Applied Bio System mass spectrometer.
Single-crystal X-ray data collections for the ligand (SVSL1) and Pd(II)complex (2) were done at 110 K with a APEX Kα diffractometer
using graphite monochromated Mo Kα (λ = 0.70173 Å)
radiation. The structural solution was obtained readily using XT/XS
in APEX2[58,59] and refined by full matrix least-squares
on F2 using Olex2.[60] Stability of the Pd(II)complexes was determined in 1%
aqueous DMSO by recording their UV–vis spectra over a period
of 24 h at room temperature.[61] The ROS
level was calculated in MDA-MB-231cancercells using fluorescent
probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA).
Hoechst and acridine orange (AO)/ethidium bromide (EB) staining assays
were performed to analyze the morphological changes in MDA-MB-231cancercells.
Synthesis of the Chromone TSCs (SVSL1–SVSL3)
Chromone-appended TSC ligands (SVSL1–SVSL3) were synthesized
from the reactions between 3-formylchromone (1.74 g, 10 mmol) and
thiosemicarbazide or 4-ethylthiosemicarbazide or 4-cyclohexylthiosemicarbazide
(0.91–1.73 g, 10 mmol) in ethanol in the presence of acetic
acid (2–4 drops). The mixture was refluxed for 4 h. Upon cooling
the solution in deep freezer, a white precipitate was formed, which
was then filtered. The solid was washed with cold ethanol and dried
in vacuum. The white crystals of SVSL1 suitable for
X-ray diffraction were obtained from slow evaporation of the ligand
solution (1:3 DMF–CHCl3 mixture).
[PdCl2(PPh3)2] (350 mg, 0.5 mmol)
was dissolved in methanol (20 mL), and solution of the corresponding
chromone-based TSC ligand (123–164 mg, 0.5 mmol) in methanol
(10 mL) was added. The mixture was stirred at room temperature for
10 min under N2 atmosphere to form an orange precipitate,
which was separated from the solution by filtration, washed with cold
methanol and hexane, and dried in vacuum. The orange
crystals of complex 2 suitable for single-crystal X-ray
diffraction were obtained by recrystallization from its CH2Cl2–DMF (3:1) solution.
Binding ability of the Pd(II)complexes with calf-thymus DNA (CT
DNA) was evaluated using electronic absorption titration, ethidium
bromide (EB) displacement and viscosity experiments. Binding of the
complexes with bovineserum albumin (BSA) protein was studied by UV–vis
and fluorescence spectroscopy. The detailed experimental procedures
are provided in the Supporting Information.
Molecular Docking Studies
Molecular docking was performed
for the Pd(II)complexes using AutoDock Tools (ADT) version 1.5.6
and AutoDock version 4.2.5 software[62] with
the receptor binding sites of DNA hexamer d(CGATCG)2 (Protein
Data Bank (PDB) ID: 1Z3F),[63] BSA (PDB ID: 3V03),[64] and SARS-CoV-2 main protease (PDB ID: 6Y2F)[65] obtained from the Protein Data Bank (http://www.rcsb.org/pdb).[66] Schrodinger Glide[67] was used for MD studies of the complexes with VEGFR2 and EGFR enzymes.[68,69]
Antiproliferative Activity
Complexes 1–3 and positive controls (cisplatin, carboplatin,
and gemcitabine) were dissolved in DMSO (10 mM) to prepare stock solutions
for antiproliferative assay. Human pancreatic (PANC-1), human breast
adenocarcinoma (MCF-7), epithelial human breast (MDA-MB-231) and nontumorigenic
epithelial breast (MCF-10a) cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM), and pancreatic carcinoma (AsPC-1)
cells were maintained in Roswell Park Memorial Institute (RPMI-1640)
medium, with 10% fetal bovineserum (FBS). The amount of DMSO was
maintained below 0.5% (v/v) so that
it does not have any influence on the cancercell proliferation results.
The cancer and normal cells (1.5 × 104) were treated
with the test compounds (0–100 μM) under 5–6%
CO2 atmosphere at 37 °C and incubated for 72 h. A
blank was also conducted and labeled as control. MTT reagent [5 mg/mL,
10 μL] was then added to each of the cell lines. After 2 h of
incubation, 100 μL of DMSO was added and the absorbance of the
formazan product was measured at 570 nm using a multi-well ELISA plate
reader. The formazan product formation is directly proportional to
the viability of the cells measured by Trypan blue staining.[70]
Nuclear Morphological Changes
MDA-MB-231cancercells in DMEM (5 × 105) were seeded in dishes
(35 mm diameter), attached overnight, and incubated at 37 °C
under 5% CO2. The corresponding complex (25 μM) was
then added to each well. Then, the dishes were incubated at 37 °C
under 5% CO2 for 48 h. Upon completion of the incubation,
the wells were washed 3 times with PBS after removing the culture
media. The cells were then stained with 4′,6-diamidino-2-phenylindole
(DAPI) and imaged by fluorescence microscopy.
Cell Cycle
Analysis
The detailed procedure adopted for the study is
provided in the Supporting Information.
Cell cycle analysis was carried out using a fluorescence-activated
cell sorter (FACS) [Becton Dickinson (BD) cell analyzer] at the FL2
channel (595 nm), and the distributions of cells in various cell cycle
phases were determined from the histogram generated by the Cell Quest
Pro software (BD Biosciences, San Jose, CA).
Western
Blot Analysis
MDA-MB-231cancercells (3 × 105) were seeded into 6-well plates for 48 h and incubated with the
complexes (25 μM) in the presence of 10% FBS. Then, the cells
were collected using trypsinization and washed 3 times with PBS, lysed
in cell lysis buffer and 2 μg/mL leupeptin, and then centrifuged
at 10000 rpm for 10 min at 4 °C. The protein concentration of
the supernatant was determined by bicinchoninic acid (BCA) assay.
The samples were subjected to SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) with 10% resolving gel. Gels were then exchanged by poly(vinylidene
difluoride) membranes (Millipore) and obstructed with 5% non-fat milk
in Tris-buffered saline (TBST, 0.1% Tween 20) buffer for 1 h. After
the membranes were incubated with primary antibodies at 1:2000 dilutions
in 5% non-fat milk overnight at 37 °C, the membranes were washed
4 times with TBST buffer. Then, the secondary antibodies were conjugated
with horseradish peroxidase at 1:2000 dilutions for 1 h at room temperature
and afterward washed 4 times with TBST buffer. To evaluate the presence
of comparable quantity of proteins in every lane, the membranes were
stripped finally to detect β-actin followed by visualization
with the enhanced chemiluminescence (ECL) PLUS detection reagents
following the manufacturer’s procedure (Amersham Biosciences).
Results and Discussion
Synthesis of the Ligands and Complexes
The chromone TSC ligands (SVSL1–SVSL3) were synthesized
by reacting 3-formylchromone with (un)substituted thiosemicarbazide
in ethanol in the presence of acetic acid (Scheme ). The ligands were air-stable, soluble in
dimethylformamide and dimethyl sulfoxide, and insoluble in methanol,
chloroform, acetonitrile, benzene, and water.
Scheme 1
Synthesis of the
Chromone TSC Ligands
The Pd(II)complexes
of the general formula [Pd(ONS-SVSL-OCH3)(PPh3)] were synthesized from the reactions of ligands SVSL1–SVSL3
with [PdCl2(PPh3)2] in methanol under
inert condition (Scheme ). Complexes 1–3 were obtained in
good yields as orange solids. They were air- and light-stable and
soluble in most of the organic solvents. The analytical data of ligands
and their complexes were in good agreement with the molecular formulas
suggested.
Scheme 2
Synthesis of the Pd(II) Complexes
Spectroscopic Confirmation of the Structures
The ligands
displayed two bands in their UV–vis absorption spectra in the
regions 258–264 and 328–339 nm, which were characteristic
of π → π* and n → π* transitions,
respectively. In contrast, the complexes depicted four bands. In addition
to the intraligand transitions appeared around 259–274 and
330–341 nm, two new bands were seen in the regions 370–386
and 384–396 nm, which corresponded to ligand to metalcharge
transfer transitions (LMCT).[69,70]FT-IR spectra
of the ligands exhibited bands at 3354–3377 and 3230–3249
cm–1, which were assigned to terminal and thioamide
NH groups, respectively. Although the terminal NH stretching frequency
(3349–3360 cm–1) remained in the spectra
of the complexes, thioamide NH stretching frequency disappeared. In
the spectra of the complexes, the three characteristic stretching
frequencies (C=O, C=N, and C=S) decreased compared
to those of the ligands, which suggested that the ligands coordinated
to Pd(II) ion through carbonyl oxygen, imine nitrogen, and thiocarbonyl
sulfur atoms. A new band was observed around 1580–1604 cm–1, which was characteristic of C–O–C
stretching. The appearance of this band preliminarily indicated that
the complex formation might have happened through Michael addition.
The bands around 1434–1453, 1056–1096, and 745–759
cm–1 corresponded to the stretching frequencies
of triphenylphosphine moiety.The 1H NMR spectra
of SVSL1–SVSL3 mainly exhibited four sets of signals (Figures S1, S3, and S5). The first set of signals
appeared at 11.50–11.54 and 8.10–8.25 ppm corresponded
to thioamide and terminal NH protons, respectively. In the spectra
of the complexes (Figures S7, S11, and S15), a signal due to the thioamide NH proton disappeared and that of
terminal NH proton was shifted to a lower chemical shift value (4.23–4.49
ppm) when compared to the corresponding free ligands. Second, an azomethine
proton was observed around 9.08–9.80 ppm as a singlet in the
spectra of the ligands, whereas the same proton was observed at 7.73–7.79
ppm as a doublet in the spectra of the complexes. Third, a signal
located at 8.10–8.59 ppm in the spectra of the ligands was
due to the OCH proton of the chromone moiety, which was shifted upfield
(5.60–5.75 ppm) upon coordination of the ligands. Furthermore,
a new signal appeared at 3.40–3.47 ppm in the spectra of the
complexes was assigned to the OCH3 protons, which confirmed
that the Michael addition had occurred during the complex formation.
Finally, the last set of signals corresponding to the aromatic protons
were observed around 7.52–8.09 ppm in the spectra of the ligands,
whereas they were seen around 6.59–7.71 ppm after coordination
of the ligands. The ethyl protons of SVSL2 and 2 were
observed in the regions 2.50–1.14 and 3.21–1.08 ppm,
respectively. The cyclohexyl protons of SVSL3 and complex 3 were detected at 4.23–1.22 and 4.30–1.04 ppm, respectively.In the 13C NMR spectra of the ligands, the peaks due
to C=S, C=O, C–O, and C=N carbons appeared
at 176.0–178.4, 175.2–175.3, 156.1, and 155.4–155.7
ppm, respectively (Figures S2, S4, and S6). The same carbon signals were observed at lower chemical shift
values (165.1–166.9, 158.8–159.7, 153.7–153.8,
and 145.8–146.6 ppm) in the spectra of the complexes (Figures S8, S12, and S16). Two new signals were
observed in the spectra of the complexes at 97.4 and 55.2 ppm, which
were due to the O–CH and OCH3carbons, respectively,
confirming that the Michael addition had happened in the chromone
(C2) moiety. The 31P NMR spectra of the complexes showed
a signal at 26.0–26.1 ppm as a singlet, which was characteristic
of phosphorus atom of the coordinated triphenylphosphine (Figures S10, S14, and S18, Table S1). 1H and 13C NMR spectral evidence
for the formation of complex 3 from SVSL3 is shown in Figures and S19. All the ligands and complexes were also
characterized by mass spectrometry (Figures S20–25).
Figure 1
1H and 13C NMR spectral evidence for the transformation
of the chromone TSC (SVSL3) into 3.
1H and 13C NMR spectral evidence for the transformation
of the chromone TSC (SVSL3) into 3.
Molecular Structures and Validation of Michael Addition
Crystal structures of SVSL1 and complex 2 with the atomic
labeling scheme are displayed in Figures and 3, respectively.
The structural parameters and data collection details for SVSL1 and
complex 2 are provided in Table S2. Ligand SVSL1crystallized in the primitive monoclinic P21/c space group. Complex 2 crystallized in the monocliniccrystal system with the P21/n space group. The complex adopted
a distorted square planar geometry. The Pd(II) ion bound to O–, N, and S– donor atoms of the TSC
ligand in such way that six- and five-membered rings were formed,
and the fourth coordination site was occupied by triphenylphosphine.
In the structure of complex 2, increase in the C–O
and C–S bond lengths and decrease in the C=N bond length
were noted compared to those in the corresponding ligand.[71]
Figure 2
Molecular structure of SVSL1 showing the atomic labeling
scheme and thermal ellipsoids at the 50% probability level.
Figure 3
Molecular structure of 2 showing the
atomic labeling scheme and thermal ellipsoids at the 50% probability
level.
Molecular structure of SVSL1 showing the atomic labeling
scheme and thermal ellipsoids at the 50% probability level.Molecular structure of 2 showing the
atomic labeling scheme and thermal ellipsoids at the 50% probability
level.In addition, the methoxy group
present
in the C2 position of chromone moiety of the ligand clearly suggested
the involvement of in situ Michael addition during
the complex formation.[56,72] NMR and FT-IR spectra of the
complexes also showed the same. It is expected that the ligand initially
coordinates to the Pd(II) ion via azomethinenitrogen and thiolato
sulfur (after enolization and deprotonation). Then methanol undergoes
Michael addition at the C2carbon of chromone to form the Michael
adduct. Finally, anionicoxygen (after deprotonation of −OH)
binds with the Pd(II) ion to give the Pd(II)complex. Surprisingly,
there is no precedence for such synthesis of Pd(II)complexes, that
too under mild conditions. This might be a key scheme for developing
new anticancer drugs and catalysts in the future. With the similar
chromone TSC ligands containing a methyl or phenyl substituent at
the terminal N, a different transformation was observed with Pd(II);
however, this reaction was not carried out in an inert atmosphere.[56]
Spectroscopic Evidences for Michael Addition
An aliquot of the reaction mixture was taken immediately after
the addition of Pd precursor [PdCl2(PPh3)2] to SVSL3, and analyzed. Peaks were seen at m/z 263.0990 [PPh3, M + H]+, 696.1099 [3, M – CH3OH]+, and 728.1390 [3, M + H]+ in its mass spectrum.
The reaction was allowed to proceed further for 10 min, and an aliquot
was taken and analyzed after 5 and 10 min. No mass spectral changes
were observed. The comparison of these spectra is illustrated in Figure S26. Moreover, 1H NMR spectra
taken for these aliquots and SVSL3 were compared (Figure S19). All the spectral results indicated that the formation
of complex 3 took place via Michael addition in a very
short time. Scheme depicts the plausible mechanism for the formation of Pd(II)complexes
from the corresponding ligands.
Scheme 3
Plausible Mechanism for the Formation
of the Pd(II) Complexes
Stability in Water Medium
Stability of the complexes was
tested in 1% DMSO–water mixture using UV–vis spectroscopy.
There was almost negligible change in the spectra of the complexes,
which indicated that the complexes were stable in water (Figure S27).
Intercalation with DNA
Anticancer agents are complexes known to show activity by binding
to DNA either covalently or noncovalently. Noncovalent interactions
occur through an intercalative, an electrostatic, or a groove (minor
or major) mode of binding, while in covalent interactions, labile
ligand of complexes is exchanged by the N donor atom of the nucleotide
in the base pairs of DNA.[73] The intercalative
mode of binding of the present complexes with CT DNA was confirmed
by UV–vis absorption, emission, and viscosity techniques, and
the order of binding strength was found to be 3 > 1 > 2. Complex 3 showed greater
binding than the other two complexes. This may be due to the presence
of cyclohexyl group in complex 3 in addition to triphenylphosphine,
which shows hydrophobic interactions with DNA.[55,73] The results and detailed discussion are provided in the Supporting
Information (Figures S28–S30 and Table S3).In addition to UV–vis,
fluorescence, and viscosity studies, a thermal denaturation technique
was used to confirm the intercalation mode of binding of the complexes
with CT DNA. In general, a high ΔTm value represents an intercalative mode of binding of a compound
with DNA, while a low value (1–3 °C) refers to the groove
and/or electrostatic binding mode(s). The thermal denaturation experiment
carried out at a wavelength of 260 nm for CT DNA alone gave a Tm value of 61.0 °C (Figure S31). Upon addition of complexes 1–3, the Tm value increased to 67.1
(±0.5)–68.2 (±1.5) °C. The ΔTm (6.1–7.2 °C) values of CT DNA in the presence
of the complexes were comparable to those observed for the classical
intercalators, suggesting the significant DNA binding affinity of
the complexes.[74]Furthermore, to
probe the binding of the complexes with DNA hexamer d(CGATCG)2 (PDB ID: 1Z3F), MD studies were performed (Table S5). The intercalation mode of binding of the Pd(II)complexes with
DNA is shown in Figure S32. As expected,
complex 3 exhibited six hydrogen bonding interactions
with DNA. A terminal N–H formed two hydrogen bonding interactions
with the phosphateoxygen. The OCH3 group formed two hydrogen
bonding interactions with the NH2 of DC-5, and the other
oxygen atoms displayed hydrogen bonding interactions with the NH2 of DC-1 and DG-2. In addition, cyclohexyl ring exhibited
hydrophobic interactions with the backbone of DNA. The results of
docking studies offered proof that the complexes bound to DNA via
intercalation mode, which is in accordance with the experimental findings.[75,76]
Binding with Protein
The complexes quenched the inherent
fluorescence of BSA protein due to their binding with BSA. Solving
the Stern–Volmer and Scatchard equations using the plot of F°/F versus [Q] gave the quenching
constant (Kq) values [1.37 × 105 (1), 1.29 × 105 (2), and 2.24 × 105 (3)] which followed
the order 3 > 1 > 2 (Figures S33–S35 and Table S4). Complex 3 was a better quencher than
the other two complexes, which was due to the interactions of both
triphenylphosphine and cyclohexyl groups with the active site amino
acids of BSA. The possible interactions between complex 3 and BSA were investigated through MD studies.[52,55,77] UV–vis absorption spectra of BSA
recorded in the presence of complexes 1–3 predicted a static type of quenching.[78] Upon incremental addition of the complexes to BSA, the
fluorescence intensity of tyrosine residue (Δλ = 15 nm)
at 304 nm decreased in the magnitudes of 65.3 (1), 61.1
(2), and 75.4% (3), and that of tryptophan
residue (Δλ = 60 nm) at 345 nm decreased by 70.1, 69.5
and 78.6% for complexes 1, 2, and 3, respectively (Figure S36 and S37). The results from synchronous spectral study suggested that all
the complexes brought about changes in the microenvironment of both
tyrosine and tryptophan residues, but more so in tryptophan than in
tyrosine.[55]Complex 3 showed the highest binding energy (−7.90 kcal/mol) among
the three complexes docked with BSA (PDB ID: 3V03). The N–H
group in complex 3 formed hydrogen bonding (2.3 Å)
with C=O of ARG-208 (Figure S38 and Table S5). A polar interaction existed between
heterocyclicoxygen and ASP-323 residue. The phenyl rings of triphenylphosphine
exhibited hydrophobic interactions with VAL-215, PHE-227, THR-231,
and ALA-324 amino acid residues, while one of them also formed a π–cationic
interaction with LYS-211. The cyclohexyl ring fit into the hydrophobic
pocket that consisted of VAL-215, LEU-326, GLY-327, LEU-330, LEU-346,
and GLU-353 amino acid residues.[79]
Cytotoxic
to Cancer Cells
Cytotoxicity of the Pd(II)complexes was
assessed against PANC-1, AsPC-1, MCF-7, and MDA-MB-231cancercell
lines and MCF-10acell line along with standard anticancer therapeutics,
cisplatin, carboplatin, and gemcitabine (Table ).
Table 1
IC50 (μM)
Values of the Pd(II) Complexes and the Benchmark Compounds[84−87] in PANC-1, AsPC-1, MCF-7, MDA-MB-231, and MCF-10a Cells as Determined
by MTT Assay after 72 h Incubations at 37°Ca
cancer cells
normal cells
IC50 (μM)
IC50 (μM)
compound
PANC-1
AsPC-1
MCF-7
MDA-MB-231
MCF-10a
cisplatin
33.03 ± 0.04
19.01 ± 0.15
19.14 ± 0.17
7.1 ± 0.02
39.33 ± 1.14
carboplatin
36.08 ± 0.14
21.18 ± 0.02
22.7 ± 0.09
7.9 ± 0.14
41.05 ± 0.41
gemcitabine
21.8 ± 0.02
15.4 ± 0.14
5.85 ± 0.17
≥50
≥50
1
2.1 ± 0.02
4.9 ± 0.14
14.6 ± 0.23
1.52 ± 0.65
≥50
2
1.46 ± 0.02
1.35 ± 0.15
9.34 ± 0.11
0.7 ± 0.58
≥50
3
1.1 ± 0.02
0.9 ± 0.06
7.56 ± 0.42
0.5 ± 0.12
≥50
Results are mean ± SD.
Results are mean ± SD.Interestingly, the Pd(II)complexes
showed very good activity, and lower toxicity than the positive controls
used in the normal cell line (MCF-10a). The cell viability was time-dependent,
and the results confirmed the potent antiproliferative activity of
the complexes against the tested cancercell lines (Figures S39–S43). The complexes showed the IC50 values of 1.1–2.1 ± 0.02 μM in PANC-1cancercell
line, which were 10 times lower than the ones observed for the standard
drugs. The complexes showed higher IC50 values in MCF-7
(7.56 ± 0.42 to 1.52 ± 0.65 μM) and AsPC-1 (4.9 ±
0.14 to 0.9 ± 0.06 μM) cancercell lines, but they were
still lower than those exhibited by the positive controls. Superior
activity of the complexes was seen in the MDA-MB-231cell line, with
the lowest IC50 value of 0.5 ± 0.12 μM exhibited
by complex 3. The higher activity of complex 3 can be attributed to the presence of a hydrophobiccyclohexyl substituent.[52] The IC50 values obtained for the
present complexes are comparable to the ones obtained previously for
the Pd(II)complexes incubated for 72 h.[80−83] The results proposed that the
prepared Pd(II)complexes can be potential candidates for application
in chemotherapy, given their selectivity toward cancercells over
normal cells.
Apoptotic Mode of Cell Death
Generally,
healthy cells have spherical nuclei wherein the DNA is evenly distributed.
During apoptosis, the natural mechanism adopted by living systems
to eradicate damaged cells, the DNA undergoes condensation which can
be detected via Hoechst staining.[88] Liu
et al. reported that the various stages of apoptoticcells could be
detected by AO/EB staining. A yellow-green AO nuclear stain marks
the early stage apoptoticcells, whereas the late stage ones are marked
by an asymmetric and localized orange nuclear EB stain.[89] In order to view these morphological changes
induced by the complexes, MDA-MB-231cancercells were stained with
Hoechst 33258 and AO/EB after treatment with the complexes for 48
h (Figures and 5).
Figure 4
Hoechst 33258 staining of MDA-MB-231 cells for 48 h: control
(a) and complexes 1 (b), 2 (c), and 3 (d).
Figure 5
AO/EB staining of MDA-MB-231 cells for 48 h:
control (a) and complexes 1 (b), 2 (c),
and 3 (d).
Hoechst 33258 staining of MDA-MB-231cells for 48 h: control
(a) and complexes 1 (b), 2 (c), and 3 (d).AO/EB staining of MDA-MB-231cells for 48 h:
control (a) and complexes 1 (b), 2 (c),
and 3 (d).The apoptotic features
such as nuclear swelling, cytoplasmic vacuolation, chromatin fragmentation,
and cytoplasmic blebbing were observed.[3] Figuratively, the viable cells (control) contained green nuclei
alongside an ordered structure, and early apoptoticcells contained
green nuclei besides perinuclear chromatin condensation as bright
green patches. Late apoptoticcells contained orange to red nuclei
alongside condensed chromatin, and necroticcells of homogeneously
larger size contained orange to red nuclei without condensed chromatin.[90] Altogether, these morphological variations proposed
that the complexes provoked cell deaththrough apoptosis, and complex 3 displayed the highest apoptotic activity than the other
two complexes in both the staining methods.
Nuclear Morphological Changes
in MDA-MB-231
DAPI visualizes nuclear DNA in both the living
and fixed cells. DAPI staining is also used to determine the number
of nuclei and assess gross cell morphology.[91] Prior to DAPI staining and observation under a fluorescence microscope,
the complexes were treated with MDA-MB-231cells for 48 h (Figure S44). The blue channel (λexcitation = 340 nm) showed the DAPI-stained nuclei, and the red channel (λexcitation = 456 nm) indicates the luminescence of the complexes.
The overlay showed the total superimposition of the blue channel with
the red channel, which indicated that the complexes induced nuclear
damage and thus exposed the dispersed cytoplasmic and nuclear fluorescence.[68,92]
Reactive Oxygen Species (ROS) Generation
Numerous anticancer
and chemotherapeutic agents stimulate apoptosis via ROS generation.
DCFH-DA dye is one among the most extensively used tools for detecting
ROS species. The dye is beneficial since it can permeate the cell
and gets hydrolyzed to DCFH carboxylate anion intracellularly, which
is retained by the cell. A fluorescent product, dichlorofluorescein
(DCF), is formed as a result of 2e– oxidation of
DCFH due to the presence of intracellular ROS species, which then
can be monitored.[93] The complexes were
examined for their ROS generation ability using fluorescent probe
DCFH-DA.[94] The complex-treated cells displayed
an enhancement in the DCF fluorescence intensity. The fluorescence
intensities were higher than that shown by the control (Figure ). The fluorescence intensity
of DCF was found to be 198.6 a.u. when MDA-MB-231cells were treated
with complex 3, which is approximately 4 times more than
that displayed by the cells treated with the control. The capability
of complex 3 to display higher cytotoxicity than the
other two complexes may be attributed to its ability to effectively
generate ROS which promote apoptosis.[3,95]
Figure 6
DCFH-DA stained
MDA-MB-231 cells: control (a) and complexes 1 (b), 2 (c), and 3 (d). The fluorescence intensities
of the complexes along with the control (e).
DCFH-DA stained
MDA-MB-231cells: control (a) and complexes 1 (b), 2 (c), and 3 (d). The fluorescence intensities
of the complexes along with the control (e).
Mitochondrial-Mediated Apoptosis
Western blotting technique
enables us to identify selected proteins in a mixture of proteins
that has been extracted from the cell. This helps in the monitoring
of the cellular upregulation of certain proteins during an apoptoticcycle.[96] To elucidate the mechanism adopted
by the complexes to bring about apoptosis, Western blot analysis was
applied to the anti- and pro-apoptotic bodies of proteins as well
as caspases (Figure ).
Figure 7
Western blot analysis of caspase-3, caspase-9, Bcl-2, Bcl-x, Bad,
and Bax in MDA-MB-231 cells treated with complexes 1, 2, and 3 for 48 h. β-Actin was used as
an internal control.
Western blot analysis of caspase-3, caspase-9, Bcl-2, Bcl-x, Bad,
and Bax in MDA-MB-231cells treated with complexes 1, 2, and 3 for 48 h. β-Actin was used as
an internal control.There was a downregulation
of anti-apoptotic protein and an upregulation of pro-apoptotic ones
which were characteristics for the induction of intrinsic pathway
of apoptosis. There was also an upregulation of caspase-3 and caspase-9,
which were involved in the final step of caspasecascade triggering
demise of the cell through apoptosis (Figure ). Complex 3, yet again showed
a better activity, which lead to the conclusion that the complexes
induced apoptosis through mitochondrial mediated pathway.
Figure 8
Percentage
expression levels of caspase-3, caspase-9, Bcl-2, Bcl-x, Bad, and
Bax in MDA-MB-231 cells treated with complexes 1, 2, and 3 for 48 h relative to the control (β-actin).
Results are the mean ± SD (n = 6). *, p < 0.05; **, p < 0.01.
Percentage
expression levels of caspase-3, caspase-9, Bcl-2, Bcl-x, Bad, and
Bax in MDA-MB-231cells treated with complexes 1, 2, and 3 for 48 h relative to the control (β-actin).
Results are the mean ± SD (n = 6). *, p < 0.05; **, p < 0.01.
Cell Cycle Arrest
A cell cycle can be divided into
four distinct phases: G1, S (DNA synthesis phase), G2, and M (mitosis).
However, G2 and M phases have indistinguishable DNA content and hence
could not be distinguished based on their dissimilarities in DNA content.[97] The cell viability assays exposed recognizable
alterations in the cellular morphology that were consistent with apoptosis.
Cell cycle analysis was carried out for the complexes in MDA-MB-231cells to quantify the apoptoticcells (Figure ). Propidium iodide (PI) is said to identify
the dead cells which are apoptotic in the sub-G1 region of the cell
cycle, although viable cell membranes are relatively impermeable to
it. On treatment of MDA-MB-231cells with the Pd(II)complexes at
their IC50 concentration for 48 h, the cell population
in sub-G0/G1 phase increased from 1.21% (control) to 14.6, 23.5, and
34.5%, respectively, for complexes 1–3, with slight changes in phases: G0/G1 (from 54.36% in the control
to 53.23, 54.35, and 55.71%, respectively, for complexes 1–3), S (from 24.5% in the control to 26.78, 25.93,
and 23.2%, respectively, for complexes 1–3), and G2/M (from 28.3% in the control to 31.2, 29.4, and
30.6%, respectively, for complexes 1–3), indicating apoptosis induction. Over 50% cells were observed in
the sub-G1 (apoptotic) region of the cell cycle (Figure ).[74,98] This substantial number of cells in the sub-G1 population lead us
to conclude that apoptosis was indeed the mechanism by which the Pd(II)complexes induced cell death. A possible cell death mechanism is shown
in Figure .
Figure 9
Cell cycle
analysis of MDA-MB-231 cells treated with the IC50 concentration
of complexes 1, 2, and 3 for
48 h.
Figure 10
Percentage cell population in various
phases of MDA-MB-231 cell cycle when treated with complexes 1, 2, and 3 for 48 h.
Figure 11
Proposed cell death mechanism for the Pd(II) complexes in MDA-MB-231
cancer cells.
Cell cycle
analysis of MDA-MB-231cells treated with the IC50 concentration
of complexes 1, 2, and 3 for
48 h.Percentage cell population in various
phases of MDA-MB-231cell cycle when treated with complexes 1, 2, and 3 for 48 h.Proposed cell death mechanism for the Pd(II)complexes in MDA-MB-231cancercells.
Interaction with SARS-CoV-2
Main Protease and Other Receptors
Interactions at the active
sites of VEGFR2 and EGFR are tabulated (Table S6) and represented in Figures S45–S46. The COVID-19 drug pipeline has not been propagated as much as the
pandemic, but its rate of expansion is nevertheless cause for pause.
Since the beginning of this year, there have been more than 180 clinical
trials launched by researchers of everything from repurposed antivirals
and immuno-modulators to unproven cell therapies and vitamin C.[99] To date, several metalcomplexes were reported
to show good antiviral properties, and we have used an in
silico MD method to identify the binding potential of Pd(II)complexes 1–3 with the SARS-CoV-2
main protease. The main protease is the best characterized drug target
as this enzyme is highly essential in the replication of the virus.
Docking energy, an indication of the ligand binding efficiency, of
the enzyme–palladiumcomplexes (−8.70, −8.39,
and −9.38 kcal/mol for 1, 2, and 3, respectively) was comparable with that of cocrystal ligand
alpha-ketoamide (−8.41 kcal/mol) but much better than those
of chloroquine (−5.60 kcal/mol) and hydroxychloroquine (−6.01
kcal/mol). Complex 3 showed good binding energy among
the compounds used in the present study and had nonbonding interactions
with active site residues THR-25, THR-26, HIS-41, CYS-44, MET-49,
TYR-54, ASN-142, GLY-143, SER-144, CYS-145, HIS-163, GLU-166, ASP-187,
and GLN-189. Interactions of cocrystal ligand, chloroquine, hydroxychloroquine,
and complexes 1–3 at the active site
of the enzyme are represented in Figure .
Figure 12
Binding mode of cocrystal ligand alpha-ketoamide
(a), chloroquine (b), hydroxychloroquine (c), 1 (d), 2 (e), and 3 (f) at the active site of SARS-CoV-2
main protease.
Binding mode of cocrystal ligand alpha-ketoamide
(a), chloroquine (b), hydroxychloroquine (c), 1 (d), 2 (e), and 3 (f) at the active site of SARS-CoV-2
main protease.
Conclusions
Three
novel Pd(II) Michael adduct chromone TSCcomplexes (1–3) were synthesized and characterized. A distorted
square planar geometry was proposed for the complexes. The synthetic
route of the present complexes is novel and was proved by spectroscopic
and single-crystal XRD tools. Complex 3 showed the highest
binding efficacy toward CT DNA and BSA. Cytotoxicity of these complexes
was evaluated against a panel of four humancancercell lines (PANC-1,
AsPC-1, MCF-7, and MDA-MB-231) and one normal (MCF-10a) cell line
along with standard anticancer drugs (cisplatin, carboplatin, and
gemcitabine). Complex 3 (IC50 = 0.5 μM)
showed an exceptional activity in the MDA-MB-231cell line wherein
the IC50 value was nearly 7 or 50 times lower than that
of cisplatin (IC50 = 7.1 μM), carboplatin (IC50 = 7.9 μM), or gemcitabine (IC50 ≥
50). The same complex exhibited potential activity against PANC-1
and AsPC-1cells with IC50 values of 1.1 and 0.9 μM,
respectively. Cytotoxicity results of the complexes were compared
with those of cisplatin, carboplatin, and gemcitabine, and it was
inferred that all the complexes exhibited higher activity than the
standard drugs. Furthermore, to elucidate the inhibition mechanism
of the complexes, staining assays were employed. Hoechst, AO/EB, and
DAPI staining imparted that besides cellular uptake of the complexes,
there were apoptotic like morphological changes happening in MDA-MB-231cancercells. Additionally, DCFH-DA staining assay revealed the generation
of ROS species in the cancercells. Along with the results obtained
from cell cycle arrest and Western blot analysis, it was confirmed
that the complexes induced cell deaththrough the intrinsic pathway
of apoptosis. The complexes showed good in silico binding energies with VEGFR2, EGFR, and the SARS-CoV-2 main protease.
The docking energies of the SARS-CoV-2 main protease with the Pdcomplexes
(−8.70, −8.39, and −9.38 kcal/mol for 1, 2, and 3 respectively) were comparable
with that of cocrystal ligand (−8.41 kcal/mol) and much better
than those of chloroquine (−5.60 kcal/mol) and hydroxychloroquine
(−6.01 kcal/mol). In vitro studies will be
carried out for these complexes to establish their usefulness as clinical
antiviral agents.
Authors: Lisa C Crowley; Brooke J Marfell; Adrian P Scott; Jeanne A Boughaba; Grace Chojnowski; Melinda E Christensen; Nigel J Waterhouse Journal: Cold Spring Harb Protoc Date: 2016-12-01
Authors: Carleen Cullinane; Glen B Deacon; Penny R Drago; Anja P Erven; Peter C Junk; Jenny Luu; Gerd Meyer; Simon Schmitz; Ingo Ott; Julia Schur; Lorraine K Webster; Axel Klein Journal: Dalton Trans Date: 2018-02-06 Impact factor: 4.390
Authors: Sabina W Jaros; Agnieszka Krogul-Sobczak; Barbara Bażanów; Magdalena Florek; Dominik Poradowski; Dmytro S Nesterov; Urszula Śliwińska-Hill; Alexander M Kirillov; Piotr Smoleński Journal: Inorg Chem Date: 2021-09-21 Impact factor: 5.165
Scheme 1
Scheme 2
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Scheme 3
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