Promoting antihepatocellular carcinoma activity against human HepG2 cells via pyridine substituted palladium complexes: in vitro evaluation and QSAR studies

Bis(4-(4-nitrobenzyl)pyridine)dichloropalladium(II), [PdCl2L12], bis(2-amino-5-bromopyridine)dichloropalladium(II), [PdCl2L22], bis(2,4-dimethylpyridine)dichloropalladium(II), [PdCl2L32], bis(3,4-dimethylpyridine)dichloropalladium(II), [PdCl2L42] were prepared. The spectroscopic techniques (FT-IR and 1H-NMR, 13C-NMR) were used to characterize the compounds. Theoretical calculations were used to validate the experimental results. The LanL2DZ-based DFT/B3LYP method was used to define the most stable possible molecular structure for the complexes. Potential energy distribution analysis was performed to determine the theoretical vibration bands of the complexes. Molecular electrostatic potential maps, boundary molecular orbitals and Mulliken charge distribution were used to determine the active sites of the molecules. The interaction mechanisms between the complexes and liver cancer protein were investigated via molecular docking. The study on the antiproliferative effects of these complexes on hepatocellular carcinoma cells (HepG2) showed that they are potent candidates for use against this liver cancer cell line.


Introduction
Cancer, which is a complex disease characterized by uncontrolled cell proliferation, is the second largest cause of mortality in the world [1,2].Among other cancer types hepatocellular carcinoma (HCC), which is the most common type of primary liver cancer in adults, was the third most common cause of cancer deaths in 2020 in the world [3].The highest incidence and the lowest survival rate after treatment for HCC was observed in Asia and sub-Saharan Africa where hepatitis B infection is endemic [4].It is foreseen that new cancer cases will increase significantly in the next decades [4][5][6][7].There are many types of cancer treatment options which are usually very complex and they are developing gradually.Among them are chemotherapy, surgery, and radiation therapy.Chemotherapy entails use of special drugs to kill cancer cells and has been recently supported by new methods of treatment such as immunotherapy to give successful results.However, chemotherapeutics have many negative side effects which compromises effectiveness of this treatment method.Therefore, recently increasing importance and efforts are devoted to development of more effective chemotherapeutic drugs with fewer side effects [8,9].
Metal-based compounds have attracted much attention since cis-platin was discovered by Barnett Rosenberg in 1960 and they have been used widely in treatment of various cancer types, especially of head and neck, ovarian and colorectal cancers.Today, cis-platin and its derivatives (i.e.carboplatin and oxaliplatin) are still extensively used in cancer treatment [10].However, these compounds have major disadvantages such as the limitation of their efficacy to some cancer types as well as their extensive side effects [11].Hence, recently new studies on other metal complexes have increased.Among these metal complexes, cost effective palladium complexes have attracted much attention as they form compounds similar to platinum [12].Studies revealed that palladium (II) complexes have considerable anticancer activity on various cancer cell lines [13].The many different types of Pd (II) complexes (i.e.monomeric, dimeric, tetrameric, cyclo-palladated, palladacyclic, and heterobimetallic) were reported to have cytotoxic activity and the pyridine, pyrazole, quinoline and, Abstract: Bis(4-(4-nitrobenzyl)pyridine)dichloropalladium(II), [PdCl 2 L 1   2   ], bis(2-amino-5-bromopyridine)dichloropalladium(II), [PdCl 2 L 2 2 1,10-phenanthroline derivates of Pd (II) have great antiproliferative activity and all of them are promising anticancer agents as these complexes are more soluble than platinum complexes [14].Despite the large number of reports on pyridine derivative complexes (i.e.chloropyridine, bromopyridine [15], methylpyridine [16], (p-tolyl) pyridine [17], (2,4 dinitrobenzyl) and ((2,4,6 trinitrobenzyl) pyridine) [18] only a very few of them are related to their anticancer effect.Studies showed that these pyridine derivatives can be exploited as anticancer drugs [19] with the effective mechanism of action of Pd complexes that proceeds over inhibition of cell proliferation of cancer cells via DNA binding [14].Tabrizi et al. reported that the palladium complexes (II) with 2,2′-bipyridine (bpy) ligands have remarkable cytotoxic activity against the colorectal adenocarcinoma (HT-29), the breast (MCF-7), and the human squamous cervical adenocarcinoma (HeLa) cancer cell lines which was better than the effect of cisplatin [20].In a study by Kuduk-Jaworska et al., palladium(II) complexes with 2,6-dimethyl-4-nitropyridine complexes were observed to have strong antiproliferative effect against adenocarcinoma of the rectum (SW707), breast cancer (T47D), bladder cancer (HCV) and nonsmall cell lung carcinoma (A549) cancer cell lines [21].Franich et al. investigated the antiproliferative activity of palladium(II) with 4,4′-bipyridine ligands on murine lung cancer (LLC1 cells) and they reported that they had similar cytotoxic effect to cisplatin [22].
In this study, bis(4 4  2 ] were synthesized and characterized via spectroscopic techniques (FT-IR and 1 H-NMR, 13 C-NMR).The antiproliferative effects of these complexes on hepatocellular carcinoma cells (HepG2) were investigated and the interaction mechanisms of liver cancer protein and complexes were investigated by molecular docking studies.

Synthesis of the complexes
The complexes were synthesized by reacting PdCl 2 with the pyridine derivatives in ethanol (Scheme).A solution of PdCl 2 (1.2 mol) was prepared in 50 mL of distilled water.The ethanolic solutions of the ligands (30 mL) were prepared by dissolving 2.4 mol of the ligands (L 1 :4-(4-nitrobenzyl)pyridine, L 2 :2-amino-5-bromopyridine,L 3 :2,4-dimethylpyridine,L 4 :3,4dimethylpyridine).These solutions were added to the palladium solution under constant stirring and reflux for 2 h.Then thus obtained precipitates were filtered and dried.

Instrumentation
FTIR spectra (4000 to 400 cm -1 ) were recorded using the Perkin Elmer Frontier FTIR spectrometer.Samples were taken in KBr pellets.NMR spectra were recorded using the Bruker Ultrashield Plus Biospin Avance III 400 MHz NanoBay FT-NMR instrument.d6-DMSO was used as solvent.Full mass analysis was performed by scanning in the positive (ES+) 50 -1000 Da range via High Resolution Mass Spectrometry using Waters SYNAPT G1 MS system.

Cell proliferation/viability assay
One hundred microliters of culture medium, 96-well flat-bottom cell culture plates (Greiner Bio One, Frickenhausen, Germany) at a density of 1 × 105 cells/well were used for seeding HepG2 cells.After 24 h of incubation, both unbound and dead cells were removed by washing twice with buffer solution (PBS, Irvine Scientific, Santa Ana, California, USA) prior to all assays.The cytotoxic effect of the complexes was determined by measuring mitochondrial dehydrogenase activity in HepG2 cells using methyl thiazolyl tetrazolium (MTT) as substrate.Different concentrations of the complexes were dissolved in RPMI-1640 medium (3.12, 6.25, 12.5, 25, 50, 100, and 200 µM) and were added to the cells which were then treated for 24 h.After incubation, cells were washed with fresh medium.One hundred microliters of MTT (5 mg mL -1 ) solution was added to all control and experimental cell groups.After 4 h of incubation, sodium dodecyl sulfate solution (10% (w/v), 100 μL) was added to each well to dissolve the formazan salt.The amount of formazan salts was quantified by measuring the absorbance value sat 570 nm in a microtiter plate reader (Sunrise, Tecan GmbH, Austria).IC50 values were calculated according to the MTT Assay results.Cell viability analysis was performed via one-way ANOVA followed by unpaired Student t-test.The IC50 values were calculated according to nonlinear quadratic model.p values <0.05 were considered to be statistically significant.Each experiment was performed in triplicates.

Evaluation of cell injury
Cell damage was assessed by measuring the release of lactate dehydrogenase (LDH) from the cells to the bath medium consisting of LDH.Cells were taken into 96-well plates and were incubated for 24 h after adding the complexes at different Scheme.Synthesis reaction of the complexes.concentrations (3.12, 6.25, 12.5, 25, 50, 100, and 200 µmol L -1 ).LDH concentration in the Supernatant was determined by using Clinical Chemistry Analyzer (ERBA XL 600, Meinheim, Germany) and a commercial colorimetric assay kit (TML Medical, Ankara, Turkey).Total LDH release, which caused HepG2 cell death, was determined.Each experiment was performed in triplicates.

Computational details
Theoretical calculations were performed using Gaussian 09 software [23].Quantum chemical calculations were performed using the B3LYP functional and LanL2DZ basis set and the density functional theory (DFT) method.B3LYP vibrational wave-numbers were found to be higher than experimental wave-numbers.Therefore, the scaling factor was used for the wave numbers and the mismatch effects were not taken into consideration.The wave-numbers calculated by B3LYP/ Lanl2DZ bases set were scaled in the infrared spectra by 0.96, 1, 0.98, and 0.85 in the ranges [4000-2001] cm -1 , [2000-1407] cm -1 , [1406-341] cm -1 and [340-0] cm -1 , respectively [24].In general, the scaled wave-numbers were calculated and were found to be in good agreement with the experimental ones.The geometry was optimized after performing the frequency calculations by using the same basis set.The results were visualized by using Gauss View software [25].Total energy distribution was calculated using VEDA software [26].The 3D crystal structure of the targeted protein (PDB ID: 2OH4) was obtained using the RSBC PDB format.The optimized structures of the complexes were determined using the DFT/B3LYP/LanL2DZ basis set.Molecular docking studies were performed using Hex (version 8.0.0) software [27].In molecular docking studies FFT mode: 3D fast lifetime; distance range: 40; twist range: 360; correlation type: shape + electro + DARS; grid size: 0.6; receptor spacing: 180 and ligand spacing: 180 parameters were used.PyMOL molecular graphics software was used to visualize the data obtained from the HEX 8.00 software [28].

Synthesis of the complexes
The complexes were synthesized according to the method given elsewhere [19].The pyridine derivatives were reacted with the palladium chloride solution prepared in ethanol by refluxing.The formation of these complexes is pH-dependent and there were obtained in the pH range 2-3.Therefore, the pH of the media was adjusted to 2-3 via ammonia and the brown colored complexes were separated after precipitating.After washing with ethanol a few times they were dried in the oven at 70 °C.In our previous work [19] the complexes were not further purified since they could have been precipitated in the pure form.

Structural analysis of the complexes
The FT-IR spectra of the complexes were obtained in the range 4000-400 cm -1 .The experimental FT-IR data of the complexes are presented in Table 1 and the vibrational bands conferred to them are presented in Table S1 (in supporting information) in detail.
For all the complexes, while the vibrational band observed at 3030-3080 cm -1 indicates the Ar-H streching in the pyridine (Py) ring, the weak bands observed at 2990-2806 cm -1 indicates the C-H stretching vibrations of the aliphatic C-H groups.The intense bands observed in the range 1622-1402 cm -1 belong to the aromatic C=C and C=N stretching vibrations in the pyridine (Py) ring.The bands observed in the range 1265-1016 cm -1 show the in-plane C-H bending vibrations.The bands observed in the range 880-503 cm -1 indicate the C-C bending vibrations (in-plane).The vibrational band at 503-433 cm -1 indicates the Pd-N stretching vibrations.Beside the common stretching vibrations of the synthesized complexes the intense peaks observed with the [PdCl 2 L 1  2 ] complex at 1350 cm -1 indicate the N-O stretching vibrations of the nitro group.For the [PdCl 2 L 2  2 ] complex, the intense bands observed in the range 3427-3180 cm -1 indicate the N-H stretching vibrations of the NH 2 group bound to the Py ring at the ortho position.The weak bands observed at 889 cm -1 indicate the C-Br stretching vibrations.These experimental findings were supported with the theoretical calculations.The Table 1.The selected stretching and bending vibrations of the complexes.] complex, the calculation showed that the C-H stretching vibrations were to appear in the range 3363-2965 cm -1 .These bands were observed as medium and weak intensity bands in the FTIR spectra.The vibrational bands conferred to the in-plane C-C and C-H bending vibrations are presented in Table S1 in detail.For the [PdCl 2 L 4   2   ] complex, the C-H stretching vibrations of [PdCl 2 L 4  2 ]-a structural isomer of the [PdCl 2 L 3 2 ] complex-were calculated to be in the range 3141-2918 cm -1 .The results of the calculations showed that while the inplane C-H stretching bands would appear in the range 1520-1448 cm -1 , the C-H bending vibrations were to be observed in the range 1073-852 cm -1 .The Pd-Cl stretching vibration, which is characteristic for this complex, was calculated to appear at 415 and 409 cm -1 .The obtained experimental and theoretical results are compatible with each other.

Molecular geometry studies
The optimized bond lengths and bond angles of the complexes are given in Table S2.Mainly five different bond lengths were observed in common for the C-C, C-H, C-N, N-Pd, and Pd-Cl bonds for these complexes.The length of the Pd-Cl and Pd-N bonds for all of the four complexes were calculated to be the same and are 2.27 and 2.05 Å, respectively.When all the bond lengths were examined, the longest bond length was determined to be the one between the palladium and the chlorine atoms.For the [PdCl 2 L 1  2 ] complex, the shortest bond length, which was calculated to be 0.96 Å, was observed to belong to the O-H bond.In other complexes, the length of the bond between the carbon and hydrogen atoms was calculated to be 1.07 Å.It can be seen in Table S2, the bonding angles between the Cl-Pd-Cl, Cl-Pd-N and N-Pd-N atoms are 90°.These bonding angles are the smallest for all the four complexes.

NMR studies
The 1 H-NMR and 13 C-NMR spectra of the complexes obtained in DMSO, which was used as the solvent, are presented in Table 2.The results of the theoretical calculations are presented in Table S3 in detail.
In the 1 H-NMR spectrum of the [PdCl 2 L 1 2 ] complex, the multiple peaks observed in the range 8.64-7.42ppm can be attributed to the Ar-H (16 H) peaks in the Py ring.The peaks observed at 4.24 ppm indicate the Al-H protons (4 H).The theoretical 1 H-NMR values for this complex were calculated to be in the range 9.48-4.07ppm.
In the 13 C-NMR spectrum, the peak observed at 152.5 ppm shows the two C atoms of the complex in the o-position.The peak observed at 149.4 ppm shows the two C atoms in the p-position and the peak at 146.0 ppm shows the two C atoms to which the nitro group in the phenyl ring is bound.The other peak observed at 146.2 ppm indicates the two C atoms via which the phenyl ring is bound to the -CH 2 bridge.The peak observed at 130.1 ppm indicates the four C atoms of the aromatic carbon in the phenyl ring.The peak at 125.3 ppm indicates the four C atoms in the metaposition.The peak at 123.6 ppm shows the four C atoms in the other aromatic carbon in the phenyl ring.The peak at 40.0 ppm indicates the two C atoms in the aliphatic groups of the complex.When these results are compared with the theoretical data it was seen that these values were distributed in the range 163-50 ppm and the chemical shifts observed in the 13 C-NMR spectra of the structures were >100 ppm as expected according to the calculations.
The multiple peaks observed in the range 8.76-6.14ppm in the 1 H-NMR spectrum of the [PdCl 2 L 2   2   ] complex show the Ar-H (6 H) peaks in the Py ring.The singlet peak observed at 7.64 ppm shows the protons (4 H) in the amine group.In the 13 C-NMR spectrum of the complex, the peak belonging to the two C atoms in the amine group at the o-position of the ring was observed at 158.1 ppm.The peak for the two C atoms in the other o-position was observed at 149.3 ppm.The peak for the two C atoms in the p-position was observed at 140.5 ppm.The peak for the two C atoms in the m-position was observed at 112.3 ppm.The peak observed at 103.5 ppm shows the two C atoms in the halogen groups of the complex.The multiple peaks observed in the range 8.40-7.65 ppm in the 1 H-NMR spectrum of the [PdCl 2 L 3  2 ] belong to the Ar-H (6 H) in the Py ring.These values were calculated to be in the range 9.76-7.99ppm theoretically.The peaks observed in the range 3.31-2.26ppm indicate the protons (12 H) in the methyl group.Theoretically they were found to be in the range 5.96-1.37ppm.
In the 13 C-NMR spectrum, the peak observed at 150.2 ppm shows the four C atoms in the o-position.The peak of the two C atoms in the para position was observed at 140.6 ppm.The peak observed at 134.9 ppm indicates the four C atoms in the m-position.The peak at 17.9 ppm shows the four C atoms in the methyl groups.Theoretically they were calculated to be in the 174.51-26.77ppm range.
In the 1 H-NMR spectrum of the [PdCl 2 L 4  2 ] complex, the multiple peaks observed in the range 8.45-7.30ppm indicate the Ar-H (6 H) peaks in the Py ring.The peaks observed in the range 3.31-2.24ppm indicate the protons (12 H) in the methyl group.
In the 13 C-NMR spectrum, the peak observed at 151.5 ppm shows the four C atoms in the o-position.The peak at 149.9 ppm shows the two C atoms in the p-position.The peak at 134.2 ppm indicates the four C atoms in the m-position.The peak at 18.5 ppm shows the four C atoms in the methyl group.

Investigation of the MEP surface and Mulliken charge distribution
Molecular electrostatic potential surface maps (MEP), which allow us to observe the variable charge region, show the charge distributions of molecules in three dimensions.
Mulliken atomic charge distributions of the complexes are given in Table 3. Figure 1 shows that the MEP surfaces of the complexes range from the darkest red to the darkest blue.Blue, red, and green colors indicate nucleophilicity, electrophilicity, and hydrogen bond interactions (regions of neutral or zero electrostatic potential), respectively [29,30].
In the MEP map, it is observed that the negative potential regions in the molecule are concentrated on oxygen and chlorine atoms.Complexes with such active sites generally show good biological activity.The Mulliken population analysis was performed in detail and the relevant results are given in Figure 1.

Investigation of the frontier molecular orbitals (FMOs) and the chemical reactivity
The difference between HOMO and LUMO energies determines the chemical reactivity and kinetic stability of a molecule.Moreover, with the calculated HOMO-LUMO values, the polarization, electronegativity, hardness, and reactivity of the energy gap molecule can be determined.The surface images are presented in Figure 2. The chemical reactivity indices are given in Table 4.A smaller HOMO-LUMO energy deficiency can indicate greater biological activity [31,32].For the 4  2 ] complexes while the HOMO energies were calculated to be -5.83,-6.43, -5.60 and -5.77 eV; the LUMO energies were calculated to be -2.08,-2.55, -1.88, and -1.99 eV, respectively.Also, the energy gap between the HOMO and LUMO orbitals were calculated to be 3.75, 3.88, 3.72 and 3.78, respectively.

Investigation of the cytotoxicity
The effect of the complexes on cell proliferation of the HepG2 cells was investigated via MTT Assay.Half maximal inhibitory concentrations (IC50) of complexes were determined according to the MTT Assay results.IC50 values of [PdCl 2 L 1   2   ], [PdCl 2 L 2  2 ], [PdCl 2 L 3  2 ] and [PdCl 2 L 4 2 ] complexes were found to be 498.69µmol L -1 , 157.21 µmol L -1 , 216.5 µmol L -1 and 61.04 µmol L -1 , respectively.The change in cell viability was observed to be concentration dependent and also was affected from the structure of the complexes.HepG2 cells were interacted with complex solutions of different concentrations (3.12, 6.25, 12.5, 25, 50, 100, and 200 µmol L -1 ).The proliferation of the HepG2 cell line is suppressed depending on the added complex dose.Moreover, as can be seen in Figure 3, the data obtained from the cell viability study indicated that bis(dichlorobis(3,4-dimethylpyridine) palladium(II), [PdCl 2 L 4   2   ] was the most cytotoxic complex and bis(4-(4-nitrobenzyl)pyridine)dichloropalladium(II), [PdCl 2 L 1  2 ] was the least cytotoxic.In recent years, the cytotoxic activities of many types of complexes against various cancer cell lines have been studied.Among them palladium complexes (II) with 2,2′-bipyridine (bpy) ligands were found to be cytotoxic against colorectal adenocarcinoma and breast cancer cell lines [20].In addition cytotoxic activity of palladium(II) complexes with 2,6-dimethyl-4-nitropyridine complexes and palladium(II) with 4,4′-bipyridine ligands were observed against several cancer cell lines in a dose-dependent manner [21,22].In this research, each complex suppressed the proliferation of HepG2 cell line in a dose-dependent manner with parallel to similar studies in the literature.

Investigation of the effect of complexes on cell injury
Cell injury was investigated via LDH assay conducted in culture media since LDH is a stable cytosolic enzyme in normal cells and when membrane damage occurs it leaks into the extracellular fluid.According to the cell viability assay bis(dichlorobis(3,4-dimethylpyridine) palladium(II), [PdCl 2 L 4   2   ] was the most cytotoxic complex and this complex increased LDH release-a cell damage marker-remarkably.The LDH release caused by bis(2-amino-5-bromopyridine)  2 ] complex was the second highest among all the complexes studied.The LDH concentration (IU/mL) in the supernatant is given in Figure 4.

Molecular docking studies
The molecular docking technique describes the interactions between drug and enzyme and is therefore used in drug design studies [33].Pd-based drugs are used in treatment of various cancers [34,35].In this study, the interaction mechanism between the synthesized palladium complexes and the 2OH4 encoded HepG2 protein was investigated.The 3D crystal structure of the protein (PDB ID: 2OH4) targeted for HepG2 was observed using the RSBC PDB format.
The DFT/B3LYP/LanL2DZ basis set was used to optimize the structures of the synthesized complexes.The interaction energies between the complexes and the protein arise from the hydrogen bonds and van der Waals interactions and confer stability to the complex.According to the results of the molecular docking studies, which are given in Figure 5, the proteinligand interactions for the synthesized complexes were between the active residues in the 2OH4 protein and the H and Cl atoms of the ligands.It was observed that the synthesized complexes interacted with a few residue of the 2OH4 protein.

Conclusion
Currently, drug resistance is developing in cancer treatment and side effects of chemotherapy are frequently observed.Therefore, synthesis of new and effective chemotherapy agents has profound importance.In this study, novel bis(4- ] complexes were prepared.The spectroscopic techniques were used to characterization studies.In addition to experimental studies, theoretical calculations (molecular structures, potential energy distribution analysis, MEP, HOMO-LUMO orbitals and Mulliken charge distribution) were also made.Experimental results were supported by theoretical calculations.The study on the antiproliferative effects of these complexes on hepatocellular carcinoma cells (HepG2) was carried out.In our study, it was observed that the synthesized complexes had a different antiproliferative effect.According to the MTT results, the [PdCl 2 L 4   2   ] and [PdCl 2 L 2  2 ] complexes were found to be very effective even at low concentrations.The data obtained in the cell injury study were compatible with these results.This indicated that palladium complexes with pyridine ligands have antiproliferative activities on HepG2 cells.The cell based studies conducted indicated that the synthesized novel palladium complexes had cytotoxic effects on HepG2 cell line and the [PdCl 2 L 4   2   ] complex was found to be the most effective among all other complexes studied.
The interaction mechanisms between the complexes and the liver cancer protein were investigated via molecular docking.The study on the antiproliferative effects of these complexes on hepatocellular carcinoma cells (HepG2) showed that they are potent candidates for use against this liver cancer cell line.In vivo studies would allow better understanding of the metabolic effects of these compounds.
Table S1.Detailed assignments of experimental and theoretical wavenumbers (cm -1 ) of the complexes along with potential energy distribution (PED).

Figure 3 .
Figure 3. Viability of HepG2 cells treated with different doses of the complexes for 24 h.(*p < 0.05 indicates the significance of difference as compared with that of the control).

Figure 4 .
Figure 4. Cell culture supernatant lactate dehydrogenase (LDH) levels after 24-h incubation period.(*p < 0.05 indicates the significance of difference as compared with that of the control).

Figure 5 .
Figure 5. Docking diagram of the 2HQ6 target protein and the complex-protein interactions studies were performed via the LanL2DZ based DFT/B3LYP method.The results of the theoretical calculations indicate that the N-H and C-H stretching vibrations should appear in the ranges 3570-3357 and 3135-3092 cm -1 , respectively.The vibrational bands for C-Br, Pd-Cl and Pd-N, which are characteristic for this complex, were observed to lie in the fingerprint region.The theoretical potential energy distribution (%PED) showed that the vibrational bands of C=C, C=N and C-C overlapped.For the [PdCl 2 L 3 2 theoretical

Table 2 .
The 1 H-NMR and13C-NMR data for the complexes.

Table 3 .
Mulliken atomic charge of the complexes.

Table 4 .
Some global reactivity properties of the complexes.

Table S2 .
Optimized parameters for the complexes (bond length and bond angles)

Table S3 .
Theoretical 1 H NMR and13C-NMR results for the complexes