Oxidative DNA cleavage mediated by a new unexpected [Pd(BAPP)][PdCl4] complex (BAPP = 1,4-bis(3-aminopropyl)piperazine): crystal structure, DNA binding and cytotoxic behavior

A novel Pd(ii) double complex, [Pd(BAPP)][PdCl4], containing the 1,4-bis(3-aminopropyl)piperazine (BAPP) ligand is investigated. X-ray crystallography of a single crystal confirmed the structure of the [Pd(BAPP)][PdCl4] complex. The spectroscopic behavior was also elucidated using elemental analysis, nuclear magnetic resonance and Fourier-transform infrared spectroscopy, and mass spectrometry. The antimicrobial susceptibility of the [Pd(BAPP)][PdCl4] complex against all tested microbial strains was lower than that of the BAPP ligand except for C. albicans. The cytotoxic impacts of the BAPP ligand and its [Pd(BAPP)][PdCl4] complex were evaluated in vitro for HepG2, CaCo-2 and MCF7 cell lines as well as the WI-38 normal cell line. The anticancer activity was markedly improved by the complexation. The [Pd(BAPP)][PdCl4] complex could selectively inhibit the tested cancer cells in a safe way to the non-tumorigenic cell (WI-38). From the DNA binding studies with ultraviolet-visible spectrophotometry, the [Pd(BAPP)][PdCl4] complex interacts more efficiently with the calf thymus DNA than its BAPP ligand through the intercalative binding mode. In the absence of an external reductant, the [Pd(BAPP)][PdCl4] complex cleaved the intact supercoiled pBR322 DNA under physiological conditions in a concentration-dependent manner. Additionally, electrophoretic experiments were performed in the presence of different radical scavengers, namely DMSO, NaN3 and KI, and ruled out the hydrolytic mechanistic pathway of the reaction and suggested that the oxidative mechanism is the preferred one. The results of the binding affinity of the [Pd(BAPP)][PdCl4] complex to human DNA were modeled using a molecular docking study showing that the complex interacts more strongly with human DNA than the ligand. Finally, an in vitro pharmacokinetic study was assessed through in silico ADME predictions.


Introduction
Cancer is one of the major causes of the increase in morbidity and mortality rates, second to cardiovascular disease. 1 As reported, 9.6 million cancer-related deaths have been enumerated in 2018. 2 It is forecasted to increase up to 22.7 million in 2030 worldwide. 3 Medicinal inorganic chemistry is a prosperous area, in which most scientists focused their efforts in the design of metal-based drugs which could be considered as a potential chemotherapeutic agent for cancer treatment. 4,5 Cisplatin, carboplatin, and oxaliplatin succeeded to play the role of Ptbased anticancer agents, but their unwelcome interaction with non-cancerous cells leads to some unwanted toxic effects and hinder their clinical application 6 such as, neurotoxicity, nephrotoxicity, ototoxicity, myelosuppression 7 and limited usage for different cancer cells 8 in addition to the acquired cellular resistance towards platinum-based drugs. 9 Thus, the development of non-Pt(II)-based drugs including the Pd(II) complexes have been devoted special attention owing to the structural similarities between Pt(II) and Pd(II) with the higher kinetic lability of Pd(II) versus Pt(II) analogues. 10 It is worth mentioning that judicious choice of the inert chelating ligands can relatively decrease these kinetic labilities to the extent to allow the complex for reaching to target, DNA, without dissociation in passing through the cytoplasm and accordingly enhance their cytotoxic activity. 11,12 Hence, the desire to explore more potent and less hazardous Pd(II)-based anticancer agent become a thriving area for drug discoverers. Piperazine is a crucial building block owing to the structural resemblance to glucose and therefore to the cyclodextrins. So, metal-based macrocyclic drugs including nitrogen-containing heterocyclic piperazine derivatives exhibit many biological features such as the antitumor activity, in which a series of clinical anticancer drugs such as entrectinib, palbociclib and olmutinib are convinced to be used in cancer treatment clinically. 13 There are some previously reported copper complexes based on piperazine derived ligand 2-((2-piperazin-1-yl-ethylimino)-methyl)phenol (H 2 L) namely [Cu(L)(H 2 O) 2 (NO 3 14 Some reported complexes exhibited better in vitro antiproliferative activity against the tested cancer cell lines than cisplatin. 13 Moreover, they may act as inhibitors for HIV-1 replication, 15 anti-inammatory effect 16 and antimicrobial activity against drug-resistant strains like pathogenic Staphylococcus. 17 Additionally, a wide range of medicinal applications of compounds bearing piperazine scaffold such as cardioprotective, antidiabetic, relieving pain agent, antituberculosis and antimalarial activities. 18,19 In addition, generally the presence of a pair of cis-primary amines situated in the precursor ligand is the primary requirement for successful synthesis of the complex. Thus, structural studies and the evaluation of the antimicrobial activity of copper(II), cobalt(II) and nickel(II) metal complexes of a hexadentate ligand 1,4-bis((2-hydroxybenzaldehyde)propyl)piperazine were also reported. 20 The super-helical DNA is the primary cellular target for most clinically important antitumoral and antimicrobial drugs. So, in the scope of rationally understanding the cytotoxic behavior of various drugs at the molecular level, DNA binding/cleavage propensities are probed. As reported before, under physiological conditions, the metal complexes possessing efficient DNA binding and cleavage propensities are mostly considered as drugs for cancer treatment and genomic research. [21][22][23] In the light of these facts and in continuation of our previous work on palladium amine complexes, 24

Instruments and programs
Elemental analysis of the complex was realized through Vario EL III (CHN) analyzer at the micro-analytical center, Cairo University. JASCO spectrometer FT/IR-460 plus was used to accomplish the Fourier transform infrared spectra of the complex. The electron impact mass spectrum of the complex was recorded at 70 eV with the aid of a SHIMADZU QP-2010 plus mass spectrometer. The absorption titration measurements of CT-DNA were determined with a Shimadzu UV 1800 spectrophotometer. A Shimadzu DTG-60H apparatus synchronized with DTG/TG was used to analyze the thermal behavior of the complex under nitrogen atmosphere (20 mL min À1 ) using a platinum crucible with a heating rate of 10 C min À1 . The Xray diffraction data of [Pd(BAPP)][PdCl 4 ] complex was collected on a Bruker Axs Kappa Apex diffractometer using Mo Ka radiation. The Denzo-Scalepack 30 soware package was used for cell renement and data reduction. A multi-scan absorption correction (Sortav 31 ) was applied to the intensities before structure solution. The structure was solved by the intrinsic phasing (SHELXT 32 ) method. Structural renement was carried out using SHELXL 32 soware with SHELXLE 33 graphical user interface. The NH hydrogen atom was located from the difference Fourier map and rened isotropically. All other hydrogen atoms were positioned geometrically and constrained to ride on their parent atoms, with C-H ¼ 0.97Å, N-H ¼ 0.89Å and U iso ¼ 1.2U eq (parent atom). The crystallographic details are summarized in Table 1   2 ) Piperazine ), 2.5 (s, 4H, NH 2 ). 13 C NMR (DMSO, 300 MHz, ppm) 30.4 (C 2 , C 5 ), 40.1 (C 1 , C 4 ), 53.0 (C 7 , C 8 , C 9 , C 10 ), 55.9 (C 3 , C 6 ).

Bioactivity assay
2.3.1. In vitro antimicrobial activity. The antimicrobial activity of the compounds was tested using the agar well diffusion method. 37 4 ] complex and the BAPP ligand were tested for the hepatocellular carcinoma (HepG2), breast cancer (MCF7), and human epithelial colorectal adenocarcinoma (CaCo-2) cell lines, in addition to human fetal lung broblast normal cell (WI-38) to evaluate their cytotoxic behavior through using MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] standard assay. 39,40 Human cell lines were obtained from VACSERA (Giza, Egypt) and maintained in DMEM (Gibco, Life Technologies Inc., UK) supplemented with 10% FBS (Gibco) and 1% penicillin/ streptomycin (Gibco) in a humidied incubator containing 5% CO 2 at 37 C. According to the MTT system, the measurement of the level of mitochondrial dehydrogenases reects well with the cellular metabolic activity. 41 The cells (1 Â 10 4 cells per well) were seeded on the 96-multiwell plate for 1 day before the treatment with the tested compounds to allow the attachment of the cells. The untreated cells played the role of the negative control. The cells were treated by [Pd(BAPP)][PdCl 4 ] complex or its BAPP ligand at different concentrations (5, 10, 25, 50 mM) and incubated for 2 days. Each concentration treatment was performed in triplicate, repeated 3 times and the mean values were recorded. Upon the addition of MTT (100 mL, 0.5 mg mL À1 ), water-soluble yellow dye on the living cell, the tetrazolium solution was reduced by the mitochondrial dehydrogenases, and then the insoluble reduced formazan was dissolved by DMSO to yield the purple solution. The optical density was measured spectrophotometrically by ELISA reader at 490 nm. The relation between the relative cell viability versus concentration was plotted, and IC 50 values were evaluated.

DNA-interaction assays
2.4.1. DNA binding affinity. Calf thymus DNA (CT-DNA) was used in the DNA binding experiments. The CT-DNA was dissolved in Tris-NaCl (5 mM Tris-HCl/50 mM NaCl (pH 6.9-7.1)) buffer. The DNA purity was conrmed by measuring the UV absorbance ratio value of (A 260 /A 280 ) and it was found to be more than 1.8. 42 The concentration of DNA per nucleotide was measured from the absorbance value at A 260 (3 ¼ 6600 M À1 cm À1 ). 43,44 In the DNA binding experiments, the changes in the electronic absorption spectra of the [Pd(BAPP)] [PdCl 4 ] complex (90 mM) or its BAPP ligand (450 mM) (in 5% DMSO solution) upon the titration with increasing amounts of the buffered CT-DNA over a range of 10-100 mM were monitored at room temperature and each experiment was repeated twice and the mean values were recorded. Aer each titration, the reaction mixture had been agitated for 2 min before measuring the absorption spectrum which in turn reaching equilibrium. The calculation of the DNA-binding constants (K b ) was performed by applying Wolfe-Shimer eqn 2.4.2. DNA cleavage ability. DNA cleavage ability was monitored using agarose gel electrophoresis assay. The plasmid pBR322 ($0.4 mg) in 5 mM Tris buffer (pH ¼ 7.1) and 37 C were subjected to the treatment with the BAPP ligand or the [Pd(BAPP)][PdCl 4 ] complex (100 mM), then incubated for 2.5 h. The incubation time was followed by electrophoresis for 2 h at 70 V on 1% agarose gel using a Tris-boric acid-EDTA buffer.
Aer electrophoresis, the bands were visualized using UV light and photographed. ImageJ soware was utilized to identify the % of the cleavage products. 46,47 The nuclease activity of the tested compounds was evaluated through the degree of transformation of the supercoiled form (SC) to the nicked (N) and linear (L) form of pBR322 DNA plasmid. The cleavage mechanism was studied in the presence of different scavengers as DMSO, NaN 3 , KI or the chelating agent EDTA.

Synthesis and structural characterization of the [Pd(BAPP)][PdCl 4 ] complex
[Pd(BAPP)][PdCl 4 ] complex was afforded through the reaction of BAPP ligand with the aqueous solution of K 2 PdCl 4 (1 : 2 molar ratio) at 50 C (Scheme 1). The coordination behavior of BAPP ligand towards Pd(II) ion was investigated via the X-ray crystallography, molar conductivity, IR, mass spectrometry, 1 H NMR, 13 C NMR and thermal studies. The complex behaves as a nonconductor species in DMF solution, 48 as the molar conductance value is 9.5 mS cm À1 at 25 C, which presumably owing to ion pair formation. Similar behavior was previously reported in  4 ] complex reects that the spectrum of the complex was predominated mainly by ligand transitions. Besides, only the complex displayed one broadband as a shoulder in the region of 389-489 nm due to a combination of nitrogen-Pd(II) charge transfer (L-M) and Pd(II) d-d bands 51 as expected for the square planar geometry through the involvement of the BAPP ligand in the coordination to the Pd(II) ion. 52 In the mass spectrum (Fig. S1 †), the detected molecular ion peak at m/z 552 is convenient with the molecular formula C 10 -H 24 Cl 4 N 4 Pd 2 of the complex. IR spectrum of the BAPP ligand (Fig. S2 †) shows a broad medium band at 3356 cm À1 belonging to the asymmetric stretching vibration mode of terminal amino groups of the diamine BAPP ligand. 53 An asymmetric and symmetric stretching vibration mode of methylene group was noted at 2823 cm À1 and 2939 cm À1 of medium intensity, respectively. 54,55 The bands noted at 1465 and 1157 cm À1 assigned for (C-N) asymmetric and symmetric stretching vibrations, respectively. 56 Upon complexation, the band assigned for the amino group is shied to a higher wavenumber 3449 cm À1 ascribing the coordination through the lone pair of the nitrogen atom. 57 Similarly, the shi of the (C-N) asymmetric and symmetric vibrations to 1458 and 1165 cm À1 , respectively, under incorporation of nitrogen atoms in the formation of [Pd(BAPP)] 2+ complex cation. The proton NMR spectrum (Fig. S3 †) of the investigated complex recorded in DMSO, showed a multiplet signal in the range 1.4-1.5 ppm is attributable to aliphatic -CH 2 . 58 The signal at 2.5 ppm is particularly for the protons of -NH 2 . The 13 C NMR showed four strong important signals which are consistent with the expected conformational symmetry of the complex. The thermogravimetric prole of the [Pd(BAPP)][PdCl 4 ] was recorded under atmospheric condition and the obtained TG curve was shown in Fig. S8. † The TGA data imply that the complex starts the decomposition at 224 C conrming that neither water nor solvent molecules were present in the complex. 59 The thermal degradation proceeds via two stages, the rst one in the temperature range 30-292 C, derived from the mass loss of 36.70% (calcd 36.04%) corresponds to C 10 H 24 N 4 moiety of the BAPP ligand. The second one in the temperature range 292-705 C, derived from the mass loss of 24.31% (calcd 25.55%) corresponds to the loss of two Cl 2 leaving two Pd metals as a thermally stable nal decomposition residue.

Description of the crystal structure
The crystal structure of the [Pd(BAPP)][PdCl 4 ] complex was presented by the ORTEP diagram in Fig. 1. Table 1  to a different unit cell, Fig. 1. The cationic complex carrying the +2 charges requires two negative charges for electroneutrality. The title complex was crystallized in the triclinic lattice with space group P 1 and the crystal packing with Z ¼ 2. In the cationic complex [Pd(BAPP)] 2+ , the palladium atom was situated in a slightly distorted square planar geometry, surrounded by four nitrogen atoms of the tetradentate ligand (BAPP). This distortion is indicated through the deviation from the linearity of the subtended angles around the palladium center (N 1 -Pd 2 -N 4 ¼ 171. 4(3) and N 2 -Pd 2 -N 3 ¼ 170.5 (3) ). The sum of bite angle around the Pd 2 atom is 360.3 . As reported recently by Houser and co-workers, the geometry of four coordinate complexes could be ascertained     Table 2. The optimization implies the conformational changes of the piperazine ring from chair conformation to boat one upon complexation. This was evidenced by the calculated distance between N 1 -N 2 of the piperazine ring to be 2.885Å in the BAPP ligand decreased by 0.385Å to 2.500Å in the complex. Also, the N 3 -N 4 bond length was drastically decreased from 11.413Å to 3.085Å upon complex formation as given in Table  S2. † The natural charges computed from the NBO-analysis for the BAPP ligand and the [Pd(BAPP)][PdCl 4 ] complex were given in Fig. 2 and 3. The more negative natural charges on the nitrogen atoms coordination centers in the case of uncoordinated BAPP are N 1 (À0.564), N 2 (À0.567), N 3 (À0.945), N 4 (À0.944). These charges are decreased upon complexation as shown in Fig. 2 and 3. Moreover, the complex shows more positive natural charges of Pd 2 (+0.559) coordinated to nitrogen atoms relative to Pd 1 (+0.260) coordinated to chloride atoms implying the Lewis acidity-basicity variation or back donation from the two different coordinating atoms.
The calculated total energies, energies of highest occupied molecular orbital (HOMO), energies of lowest unoccupied molecular orbital (LUMO) and dipole moments are reported in Table 3. The more negative value of total energy of the [Pd(BAPP)][PdCl 4 ] complex relative to that of BAPP ligand refers to higher stability. Also, the energy gap (E g ) ¼ E LUMO À E HOMO , is also reported in Table 3 and Fig. 4. Fig. 5 shows the electrostatic potential maps (MEP) which were generated through a new cubic contour to allocate the electron cloud distribution over the molecule functional groups. The electrophilic (blue zone) and the nucleophilic (red zone) areas were claried. The BAPP ligand map shows red spots in the contour of the amine group    1 mM) and CaCo-2 (IC 50 ¼ 14.9 AE 1.5 mM) than the BAPP ligand which shows no cytotoxic effect towards the tested carcinogenic cell lines as presented in Fig. 6.
On the other hand, the complex and the BAPP ligand induce less damage to the human fetal lung broblast normal cell (WI-38). To the best of our knowledge few palladium complexes containing [PdCl 2 ] as a counter ion were encountered and their cytotoxic behavior towards different cell line did not previously discussed. 70 However, in light of these results, in vitro studies are necessary to validate the in vivo results in future trends.
3.6. DNA-interaction assays 3.6.1. CT-DNA binding affinity. In cancer therapy, the mechanism of antitumor activity of the metal-based anticancer therapeutics could be initially considered through the evaluation of their DNA binding affinities. 71,72 A wide varieties of different compounds could interact covalently or non-covalently with DNA. The covalent mode of interaction includes the formation of a covalent bond between the compound and the nucleic acid. While in the non-covalent mode, the compounds bind reversibly with the DNA helix through different modes including the electrostatic interaction with the external    Besides, an isosbestic point was noticed around 244 nm. The negative value of the standard Gibbs free energy change (DG ¼ À7.118 kcal mol À1 ) for the [Pd(BAPP)][PdCl 4 ] complex-DNA binding affinity derived from the following equation DG ¼ ÀRT ln K b conrms that the association between the complex-DNA to form an adduct is spontaneous.
3.6.2. Plasmid DNA cleaving ability. Metallonucleases are an alternative to the bio-nucleases, which can cleave the phosphodiester bonds of the DNA nucleotides. 82 The main advantages of chemical nucleases are their relatively smaller size and their higher stability towards the temperature and over wide pH range. 83 Some clinically reported articial nucleases were approved to treat cancer, 84,85 in which the cell death happens by DNA cleavage. The nuclease activity of the [Pd(BAPP)][PdCl 4 ] complex and its BAPP ligand was studied under the physiological pH and temperature. The DNA cleavage propensity was measured through inspecting the gradual degradation of the intact supercoiled form (SC (form I)) to nicked circular (NC (form II)) and linear (L (form III)) forms. 86 Upon electrophoresis of the pBR322 plasmid DNA, the supercoiled form will immigrate with the highest speed in the gel. If any scission occurs on one strand of the double-stranded plasmid DNA, the supercoiled form will be relaxed to the slowest moving nicked circular form.    Fig. 10. These facts dictate the involvement of (O 2 2À ) and ( 1 O 2 ) as reactive oxygen species in the cleavage process. In addition, the EDTA chelating   An insight into the interaction of the titled ligand and its corresponding complex with macromolecules could be provided through a docking study. Moreover, the relative binding free energy and the different possible binding modes of the studied compounds to the B-DNA (PDB ID: 1BNA) active site could be well estimated. In the present work, the binding free energy of the BAPP ligand and [Pd(BAPP)][PdCl 4 ] complex to the human DNA receptor were found to be À1.5 and À20.0 kcal mol À1 , respectively, Table 4, Fig. 11 and 12. The more negative binding energy of the complex indicates the stronger DNA interaction. The molecular docking data are well correlated with the DNA binding/cleavage studies.

In silico pharmacokinetic indexes
In silico ADMET (Absorption, Distribution, Metabolism, Elimination, Toxicity) assay is an approach to give an insight if a compound has acceptable pharmacokinetics and pharmacodynamics properties before administration to the body or not. 92 The penetrate brain-barrier (BBB), partition coefficient (log P o/ w ), gastrointestinal absorption (GI), topological polar surface area (TPSA), solubility and skin permeation (log K p ) of the BAPP ligand as well as the [Pd(BAPP)][PdCl 4 ] complex were evaluated (Table S4 †) by the aid of SwissADME server. 93 The results indicate that the [Pd(BAPP)][PdCl 4 ] complex has high human gastrointestinal intestinal absorption probability (GI) synchronized with no blood-brain barrier permeation which is claimed as an ideal drug candidate. The synthetic accessibility score exists in the range of (1-10) which implies the moderate synthetic accessibility of the [Pd(BAPP)][PdCl 4 ] complex.

Conclusion
In conclusion, a novel palladium(II) complex bearing 1,4-bis(3aminopropyl)piperazine ligand has been isolated and fully characterized using different spectroscopic tools. X-ray singlecrystal diffraction study points out that the palladium center in the [Pd(BAPP)] 2+ cationic complex is four coordinated with slight distortion in the square planar geometry (s 4 value ¼ 0.13). The thermal stability of the [Pd(BAPP)][PdCl 4 ] complex demonstrates the absence of water molecules either inside or outside the coordination sphere. The [Pd(BAPP)][PdCl 4 ] complex showed lower antimicrobial activity than those found for the BAPP ligand for all the tested species except for C. albicans. The in vitro cell viability test by the MTT assay indicates that the [Pd(BAPP)][PdCl 4 ] complex has anticancer activities against MCF7, HepG2 and CaCo-2 without any hazardous effect to the healthy cell (WI-38). The DNA binding study has been investigated using the electronic absorption spectrophotometric measurements. The results suggest that the interaction of the complex with the DNA occurred via an intercalative mode with K b value equal to 1.65 AE 0.08 Â 10 5 M À1 . In addition, the [Pd(BAPP)][PdCl 4 ] complex can cleave the pBR322 plasmid DNA efficiently without any external reducing agent in a concentration-dependent activity. To elucidate the mechanism of action by using different ROS scavengers such as DMSO, KI

Conflicts of interest
There are no conicts to declare.