Synthesis, Physicochemical Characterization, Biological Evaluation, In Silico and Molecular Docking Studies of Pd(II) Complexes with P, S-Donor Ligands

One homoleptic (1) and three heteroleptic (2–4) palladium(II) complexes were synthesized and characterized by various physicochemical techniques, i.e., elemental analysis, FTIR, Raman spectroscopy, 1H, 13C, and 31P NMR. Compound 1 was also confirmed by single crystal XRD, showing a slightly distorted square planar geometry. The antibacterial results obtained via the agar-well diffusion method for compound 1 were maximum among the screen compounds. All the compounds have shown good to significant antibacterial results against the tested bacterial strains, Escherichia coli, Klebsiella pneumonia, and Staphylococcus aureus, except 2 against Klebsiella pneumonia. Similarly, the molecular docking study of compound 3 has shown the best affinity with binding energy scores of −8.6569, −6.5716, and −7.6966 kcal/mol against Escherichia coli, Klebsiella pneumonia, and Staphylococcus aureus, respectively. Compound 2 has exhibited the highest activity (3.67 µM), followed by compound 3 (4.57 µM), 1 (6.94 µM), and 4 (21.7 µM) against the DU145 human prostate cancer cell line using the sulforhodamine B (SRB) method as compared to cisplatin (>200 µM). The highest docking score was obtained for compounds 2 (−7.5148 kcal/mol) and 3 (−7.0343 kcal/mol). Compound 2 shows that the Cl atom of the compound acts as a chain side acceptor for the DR5 receptor residue Asp B218 and the pyridine ring is involved in interaction with the Tyr A50 residue via arene-H, while Compound 3 interacts with the Asp B218 residue via the Cl atom. The physicochemical parameters determined by the SwissADME webserver revealed that no blood-brain barrier (BBB) permeation is predicted for all four compounds, while gastrointestinal absorption is low for compound 1 and high for the rest of the compounds (2–4). As concluding remarks based on the obtained in vitro biological results, the evaluated compounds after in vivo studies might be a good choice for future antibiotics and anticancer agents.


Introduction
Cancer is a devastating disease, yet many types can be entirely treated if discovered early on, and for many others, patients' lives can be greatly prolonged. Surgery, radiation therapy, 128.4, 134.5, 134.6 (Ar-C). 31 P NMR (ppm): 17.1.

Multinuclear NMR ( 1 H, 13 C and 31 P)
The NMR data of the ligands and their palladium complexes is given in the Section 3. The 1 H NMR data is almost similar, with no conspicuous differentiation. The 13 C NMR of the characteristic SCS fragment is observed in the range of 207.3-208.2 ppm and provides an important indication for the complex's formation. A slight up field from the ligand due to the accumulation of electronic density over the carbon atom in SCS after the complexation with palladium metal was observed. The 31 P NMR of the synthesized compounds is observed in the range of 20.3-27.5 ppm, and it also depicts a slight up field from the ligand as a result of the transfer of electron density from the ligand to the Pd 2+ center. Figures S2-S4 of the Supplementary Materials show the 1 H, 13 C, and 31 P NMR spectra of complex 1.

Structural Study of Complex 1
Complex 1 single crystal XRD data was collected at 200 K using a STOE IPDS image plate detector using MoK α radiation, as depicted in Table 1. The STOE X-AREA application was used to get cell parameters, gather data, and integrate data. With hydrogen atoms in optimal locations, all non-H atoms were refined anisotropically after the structure was solved and refined using SHELXT [36] and SHELXL-2015 [37].
The ORTEP view of complex 1 in a monoclynic crystla system with the P21 space group is given in Figure 1. The dimensions of the unit cell are: a = 16.3128(4)Å, b = 10.2144(2)Å, and c = 18.7283(4)Å, while the α and γ = 90 • and β = 115.793(1) • . The crystal unit contains two independent molecules. The environment around the palladium atom is square planar with slight distortion, having bond angles of Cl1-Pd1-Cl2 = 178.40 • and P(2)-Pd1-P(1) = 179.39 • , as given in Table 2. Similarly, the geometry around the P1 atom is distorted tetrahedral, with bond angles of C(21)-P(1)-C(31) = 102.4 • , C(21)-P(1)-C(11) = 106.8 • , and C(21)-P(1)-Pd1 = 117.5 • . The geometrical arrangement around the P2 atom is also distorted tetrahedral, with the largest distortion being caused by the palladium atom due to its large size, and the bond angles are: C(51)-P(2)-Pd1 = 118.7 • , C(51)-P(2)-C(61) = 101.0 • , and C(51)-P(2)-C(41) = 105.9 • . The hydrogen bonding and other short contacts in a molecule affect the phyiscochemical properties of the molecule. The synthesized compound of palladium shows no hydrogen bonding, but it exhibits short contacts around some outer hydrogen and carbon atoms present in the propyl and benzene rings. The torsion angle for the atoms Cl1-Pd1-P(1)-C(21) is −30.7(5) • , which is synclinal; Cl2-Pd1-P(1)-C(21) is 147.7(5) • , which is anticlinal; and P(2)-Pd1-P(1)-C(21) is −74(11) • , which is also anticlinal.  Table 3 describes the anticancer efficacy of the Pd(II) complexes against cisplatin-re-   Table 3 describes the anticancer efficacy of the Pd(II) complexes against cisplatinresistant DU145 human prostate carcinoma cells. The screened compounds have shown activity in the order of 2 > 3 > 4 > 1. The higher activity of the heteroleptic compounds 2, 3, and 4 containing different organophosphine ligands despite sharing a dithiocarbamate group is due to the electronegative nitrogen atom included in the organophosphine ligand, which can interact with the DNA strands of the carcinoma cells in a variety of ways'. This may be responsible for the greater activity of compound 2.  Table 4 describes the antibacterial potentials of the Pd(II) complexes against various Gram-positive and negative bacterial strains. Compound 1 is the most active, while complex 3 is the least active compound. The higher activity of complex 1 may be attributed to the fact that it has no dithiocarbamate ligand, while the other three complexes (2-4) have dithiocarbamate ligands, which can react with other active compounds inside the cell, so they may not have reached the target moiety.

In Silico Assessment
In order to make a crucial decision on a daily basis about which compound should be synthesized, tested, and promoted as a drug candidate, the study of pharmacokinetics has utmost importance. Not only the efficacy and toxicity but also other parameters such as absorption, distribution, metabolism, and excretion are thoroughly studied before the processing of a molecule to be marketed as medicine for the benefit of patients.
The pharmacokinetic parameters of the synthesized complexes were studied by Swis-sADME webserver. The physicochemical properties show that the complexes 1, 2, 3, and 4 have 10, 5, 7, and 5 rotatable bonds, respectively, as shown in Table 5. The lipophilicity LogP o/w (iLOGP) is zero for all four complexes, while LogP o/w (XLOGP3) is 9.49, 6.46, 7.54, and 7.56 for complexes 1, 2, 3, and 4, respectively. Poor solubility in water is predicted, which is in agreement with the experimental observation. No blood-brain barrier (BBB) permeation is predicted for all four complexes, while gastrointestinal (GI) absorption is low for complex 1 and high for the rest of the three complexes. No drug likeness for complexes 1, 3, and 4 and yes for complex 2 is predicted by the Lipinsky model, while the Veber model has predicted positive drug likeness for all four complexes. The medicinal chemistry parameter has predicted zero alerts for all four complexes by PAINS, one alert for complexes 1, 2, and 4, and two alerts for complex 3 by Brenk.

Molecular Docking Results
The compounds docking results with the selected antibacterial targets are provided in Table 6. Lower binding energies indicate a stronger binding affinity of the complexes with the target proteins. Among the screened compounds, 3 have shown the highest binding affinity of −8.6569, −6.5716, and −7.6966 kcal/mol against E. coli, K. pneumonia, and S. aureus, respectively. The next highest binding energy is shown by compound 4 against all three bacterial strains, which is evident from the binding energy value given in Table 6. The docking confirmation and 2D interaction diagram of complex 3 against the three bacterial proteins are given in Figures 2-4. Figure 2 shows that the Cl atoms of Compound 3 interact with the His 203 and Lys 162 residues of E. coli. Figure 3 shows that the benzene ring of Compound 3 interacts with the Asn B220 residues of K. pneumonia via arene-H. a MW: molecular weight; RT: rotatable bonds; S: solubility; PS: poorly soluble; GI A: gastrointe absorption; vio: violation; BBB P: blood brain barrier permeant.

Molecular Docking Results
The compounds docking results with the selected antibacterial targets are prov in Table 6. Lower binding energies indicate a stronger binding affinity of the comp with the target proteins. Among the screened compounds, 3 have shown the highest b ing affinity of −8.6569, −6.5716, and −7.6966 kcal/mol against E. coli, K. pneumonia, a aureus, respectively. The next highest binding energy is shown by compound 4 again three bacterial strains, which is evident from the binding energy value given in Tab The docking confirmation and 2D interaction diagram of complex 3 against the three terial proteins are given in Figures 2-4. Figure 2 shows that the Cl atoms of Compou interact with the His 203 and Lys 162 residues of E. coli. Figure 3 shows that the ben ring of Compound 3 interacts with the Asn B220 residues of K. pneumonia via arene−     The interaction of the compounds 1-4 with DR5 (1BU3) shows that the maximum binding affinity was observed for compounds 2 (−7.5148 kcal/mol) and then for compound 3 (−7.0343 kcal/mol). The maximum in vitro antitumor activity was also observed for compound 2. The docking results reinforced the in vitro results. The interaction with the DR5 receptor of representative compounds 2 and 3 is given in Figures 5 and 6, respectively. The 2D diagram of compound 2 shows that the Cl atom of the compound acts as a chain side The interaction of the compounds 1-4 with DR5 (1BU3) shows that the maximum binding affinity was observed for compounds 2 (−7.5148 kcal/mol) and then for compound 3 (−7.0343 kcal/mol). The maximum in vitro antitumor activity was also observed for compound 2. The docking results reinforced the in vitro results. The interaction with the DR5 receptor of representative compounds 2 and 3 is given in Figures 5 and 6, respectively. The 2D diagram of compound 2 shows that the Cl atom of the compound acts as a chain side acceptor for the DR5 receptor residue Asp B218 while the pyridine ring is involved in interaction with the Tyr A50 residue via arene-H. Similarly, compound 3 interacts with the Asp B218 residue via the Cl atom.    Against E. coli, the best affinity was shown by the Pd(II) organophosphine precursor having a diphenyl-p-tolylphosphine moiety (−6.8335 kcal/mol), followed by a diphenyl-2- pyridylphosphine moiety (−6.3463 kcal/mol). Similarly, against K. pneumonia and S. aureus, the maximum binding affinity was observed for the Pd(II) organophosphine precursor having a diphenyl-2-ethoxyphenyl phosphine moiety (−5.3247, −6.1572 kcal/mol), followed by a diphenyl-p-tolylphosphine moiety (−5.0306, −6.0522 kcal/mol), respectively.
The best binding affinity against DR5 (1DU3) was observed for the Pd(II) organophosphine precursor having a diphenyl-2-ethoxyphenyl phosphine moiety (−7.1002 kcal/mol), followed by a diphenyl-p-tolylphosphine moiety (−6.9220 kcal/mol). From the comparison of binding energy, we can say that the starting Pd(II) organophosphine precursors have low binding affinity as compared to their corresponding Pd dithiocarbamate complexes. The results are shown in Table S1 and Figures S5-S8 of the Supplementary Materials.
NMR spectra were recorded on Mercury 200 MHz and Bruker 300 MHz spectrometers. 1 H NMR (300.13 MHz): CDCl 3 (7.26 from SiMe4). 13 C NMR (75.47 MHz), internal standard TMS; 31 P NMR (121.49 MHz): CDCl 3 . IR spectra were recorded on a Nicolet 6700 FT-IR instrument in the range of 400-4000 cm −1 and Raman spectra (±1 cm −1 ) were measured with an InVia Renishaw spectrometer, using argon ion (514.5 nm) and near infrared diode (785 nm) lasers. Wire 2.0 software was used for the Raman data acquisition and spectra manipulations. The elemental analyses were conducted on a LECO-183 CHNS analyzer. Melting points were measured on the Stuart SMP10 apparatus and are uncorrected. GraphPad Prism was applied for statistical analysis.

Synthesis
The synthesized compounds were prepared in three steps. In step-1, dithiocarbamate was prepared; in step-2, palladium(II) organophosphine complexes were prepared; and in step-3, heteroleptic palladium complexes containing both organophosphine and dithiocarbamate ligands were prepared [17,18].
A carbon disulfide solution (30 mL) in dry methanol was added dropwise to dimethyl amine and potassium hydroxide dissolved in methanol (30 mL) in an equimolar ratio. The reaction mixture was stirred for 4 h at 0 • C (Scheme 1). A white-colored product was precipitated, followed by filtration and washing with methanol, and finally dried in an open atmosphere.

Synthesis
The synthesized compounds were prepared in three steps. In step-1, dithiocarbamate was prepared; in step-2, palladium(II) organophosphine complexes were prepared; and in step-3, heteroleptic palladium complexes containing both organophosphine and dithiocarbamate ligands were prepared [17,18].
A carbon disulfide solution (30 mL) in dry methanol was added dropwise to dimethyl amine and potassium hydroxide dissolved in methanol (30 mL) in an equimolar ratio. The reaction mixture was stirred for 4 h at 0 °C (Scheme 1). A white-colored product was precipitated, followed by filtration and washing with methanol, and finally dried in an open atmosphere.
Heteroleptic or mixed ligand palladium(II) complexes (2)(3)(4) were prepared by the reaction of potassium salt of dithiocarbamate dissolved in dichloromethane (25 mL) with palladium phosphine complex dissolved in dichloromethane (30 mL), Scheme 1. The reaction mixture was kept on reflux with vigorous stirring until the completion of the reaction. Thin-layer chromatography was used to monitor the development of the reaction.
Upon the completion of the reaction, the solid by-product was filtered, the solvent was evaporated by a rotary evaporator, and the product was dissolved in a suitable mixture of solvents for recrystallization.

Chlorido-(dimethyldithiocarbamato-κ 2 S,Sʹ)(diphenyl-2-pyridylphosphine) palladium(II) (2)
A total of 0.05 g (0.41 mmol) of potassium salt of dimethyldithiocarbamate was reacted with 0. Heteroleptic or mixed ligand palladium(II) complexes (2)(3)(4) were prepared by the reaction of potassium salt of dithiocarbamate dissolved in dichloromethane (25 mL) with palladium phosphine complex dissolved in dichloromethane (30 mL), Scheme 1. The reaction mixture was kept on reflux with vigorous stirring until the completion of the reaction. Thin-layer chromatography was used to monitor the development of the reaction. Upon the completion of the reaction, the solid by-product was filtered, the solvent was evaporated by a rotary evaporator, and the product was dissolved in a suitable mixture of solvents for recrystallization. for 3 h (Scheme 1). The product was filtered, washed with methanol several times, a dried in a vacuum. Heteroleptic or mixed ligand palladium(II) complexes (2)(3)(4) were prepared by t reaction of potassium salt of dithiocarbamate dissolved in dichloromethane (25 mL) w palladium phosphine complex dissolved in dichloromethane (30 mL), Scheme 1. The action mixture was kept on reflux with vigorous stirring until the completion of the re tion. Thin-layer chromatography was used to monitor the development of the reactio Upon the completion of the reaction, the solid by-product was filtered, the solvent w evaporated by a rotary evaporator, and the product was dissolved in a suitable mixture solvents for recrystallization.

Antibacterial Activity Assay
The antimicrobial potential of the prepared compounds was evaluated using the agar-well diffusion method against five bacterial strains: two Gram-negative and three Gram-positive strains: E. coli, K. pneumoniae, S. epidermidis, S. aureus, and B. subtilis [39]. Using a sterile cotton swab, about 10 4 -10 6 colony-forming units (CFU)/mL of bacterial inoculums were dispersed on top of nutritional agar. The tested compounds having a concentration of 2 mg/mL in DMSO were transferred to each well, and then the plates were incubated for 20 h at 37 • C, and finally the inhibition zone (in mm) was measured to determine the antibacterial activity. Streptomycin was used as the standard drug and DMSO as a positive control for the determination of growth inhibition, and the experiment was performed three times [39].

Antitumor Activity
Using the technique described in the referred articles, the anticancer activity of the synthesized compounds was assessed against DU145 human prostate cancer (HTB-81) cells [40][41][42]. Cisplatin was used as a reference drug. Compounds with a concentration of 50 mmol were dissolved in DMSO and diluted into nine consecutive concentrations, with the final concentration of DMSO on the cells not exceeding 0.05 percent. In 96-well flat-bottom microtiter plates, DU145 prostate cancer cells were planted at a density of 5000 cells per well for the growth inhibition test. Following a 24-h incubation period, cells were treated for four days with varying doses of each treatment. The remaining viable cells were fixed with 50% cold trichloroacetic acid for 60 min at 4 • C, stained with 0.4% sulforhodamine B (SRB) for four hours at 25 • C, washed with 1% acetic acid, and allowed to dry overnight. After dissolving the colored residue in 10 mM Tris base (pH = 10) and at 490 nm using an ELx808 microplate reader, the optical density was recorded. Graph Pad Prism was applied for data analysis, and the sigmoidal dose response curve was used to compute the IC 50 . The test for growth inhibition was repeated three times [40][41][42].

In Silico Studies
The attrition rate for clinical trials used to develop new drugs has increased to 90% during the last ten years. Over a five-year period, on average, 26.8 small molecules were approved as FDA drugs. Only 12 innovative small-molecule medicines were approved by the FDA in 2016-the fewest such approvals over the previous fifty years [43,44]. Pharmaceutical firms invest millions of dollars to push a new treatment through clinical trials; therefore, failure in the latter stages of drug development often results in large financial losses [45]. The major reasons why drug candidates fail in clinical trials are undesirable pharmacokinetic characteristics and unacceptable toxicity [46]. Therefore, it is crucial for science to select candidates with the right balance of potency along with absorption, distribution, metabolism, excretion, and toxicity (ADMET). Parameters for ADMET and drug-like properties are given in Table 5.

Molecular Docking Analysis
The binding mode and affinity of a small molecule or ligand with a macromolecule, such as a protein or DNA, can be predicted using molecular docking. In the case of drug discovery, molecular docking is used to identify potential drug candidates that can bind to a target protein with high affinity and specificity. Here we have treated Palladium (II) complexes against three bacterial strains: K. pneumoniae, S. aureus, and E. coli, as well as trial receptor DR5, for which the PDB files were obtained from the RCSB PDB homepage.
MOE-Dock software version 2015 was used to perform docking studies so as to identify the binding interactions of the screened compounds in the active site of three antibacterial targets, such as PDB_ID: 4EXS from Klebsiella pneumonia [47], PDB_ID: 5ZH8 from Staphylococcus aureus [48,49], and PDB_ID: 6G9S from Escherichia coli [50], as well as death receptor (DR5) PDB_ID: 1DU3. The compounds were built in MOE, and energy was minimized by using the MOE's default settings parameters, Placement: Triangle Matcher and Rescoring: London dG, for the docking study [51]. Two conformations were generated for each ligand. For further molecular interaction analysis, the highestranked conformation of each compound with the lowest binding energy score was used. The interactions between receptor-solvent and ligand-solvent were excluded during the generation of the 2D interaction diagram.

Conclusions
A total of four Pd(II) complexes, including one homoleptic and three heteroleptic, have been prepared quantitatively. The single crystal analysis of complex 1 shows that the geometry around the palladium atom is square planar with slight distortion. The synthesized compounds have shown significant antibacterial activity against the selected targets. Compound 2 has shown the maximum antitumor activity against DU145 human prostate carcinoma cells. The lower binding energy or higher inhibition values indicate a stronger binding affinity of complex 3 with the target proteins. The molecular docking study of the evaluated compounds with DR5 (1BU3) shows the highest binding affinity for compound 2 (−7.5148 kcal/mol) and then for compound 3 (−7.0343 kcal/mol). In silico studies have been performed by the SwissADME webserver, and the compounds generally have shown one or two violations of Lipinski's rule of five.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ph16060806/s1, Figure S1: FTIR spectrum of complex 2; Figure S2  Data Availability Statement: Data will be available on request to corresponding authors.