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

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC 4.0 https://creativecommons.org/licenses/by-nc/4.0/ 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 © 2022 The Author(s). Published by the Royal Society of Chemistry Published version Ragab, Mona S.; Shehata, Mohamed R.; Shoukry, Mohamed M.; Haukka, Matti; Ragheb, Mohamed A. Ragab, M. S., Shehata, M. R., Shoukry, M. M., Haukka, M., & Ragheb, M. A. (2022). Oxidative DNA cleavage mediated by a new unexpected [Pd(BAPP)][PdCl4] complex (BAPP = 1,4-bis(3aminopropyl)piperazine) : crystal structure, DNA binding and cytotoxic behavior. RSC Advances, 12(3), 1871-1884. https://doi.org/10.1039/d1ra07793g 2022 Oxidative DNA cleavage mediated by a new unexpected [Pd(BAPP)][PdCl 4 ] complex (BAPP ¼ 1,4-bis(3-aminopropyl)piperazine): crystal structure, DNA binding and cytotoxic behavior† Mona S. Ragab, * a Mohamed R. Shehata, a Mohamed M. Shoukry, a Matti Haukka b and Mohamed A. Ragheb c A novel Pd(II) double complex, [Pd(BAPP)][PdCl 4 ], 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)] [PdCl 4 ] 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)][PdCl 4 ] 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)][PdCl 4 ] 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)][PdCl 4 ] complex could selectively inhibit the tested cancer cells in a safe way to the nontumorigenic cell (WI-38). From the DNA binding studies with ultraviolet-visible spectrophotometry, the [Pd(BAPP)][PdCl 4 ] 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)][PdCl 4 ] complex cleaved the intact supercoiled pBR322 DNA under physiological conditions in a concentrationdependent manner. Additionally, electrophoretic experiments were performed in the presence of different radical scavengers, namely DMSO, NaN 3 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)][PdCl 4 ] 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. 1. 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 a Department of Chemistry, Faculty of Science, Cairo University, Giza, 12613, Egypt. E-mail: [email protected]; [email protected]; [email protected] b Department of Chemistry, University of Jyv¨ askyl¨ a, P.O. Box 35, FI-40014 Jyv¨ askyl¨ a, Finland c Department of Chemistry (Biochemistry Division), Faculty of Science, Cairo University, Giza, Egypt †Electronic supplementary information (ESI) available. CCDC 2078974. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d1ra07793g Cite this: RSC Adv., 2022, 12,1871 Received 21st October 2021 Accepted 17th December 2021 DOI: 10.1039/d1ra07793g rsc.li/rsc-advances © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12,1871–1884 | 1871 RSC Advances PAPER Open Access Article. Published on 12 January 2022. Downloaded on 1/18/2022 10:26:20 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue 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 )](NO 3 ), [Cu(L)(ClO 4 )(- N 3 )] and [Cu 2 (m-1,3-NCS) 2 (L)](ClO 4 ) 2 $2H 2 O have been evaluated for anti-cancerous effect against human breast cancer cell line by Pait et al. 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. 21–23 In the light of these facts and in continuation of our previous work on palladium amine complexes, 24–29 novel Pd(II) complex, [Pd(BAPP)][PdCl 4 ], based on 1,4-bis(3aminopropyl)piperazine ligand (BAPP) has been synthesized. The unexpected geometrical features were successfully identied by single-crystal X-ray structure analysis and different spectroscopic tools. In this article, we present the antimicrobial activity of the [Pd(BAPP)][PdCl 4 ]complex compared to its free BAPP ligand. In vitro cytotoxic activity of the complex compared to its parental ligand, BAPP, was evaluated against three human tumorigenic cell lines, breast carcinoma (MCF7), hepatocellular carcinoma (HepG2) and epithelial colorectal adenocarcinoma cells (CaCo-2) in addition to the human fetal lung broblast normal cell (WI-38). The binding studies with calf thymus DNA (CT-DNA) were performed using UV-vis absorption. DNA cleavage propensities of [Pd(BAPP)][PdCl 4 ] complex and BAPP ligand were evaluated. A molecular docking study was carried out to assess the potential binding mode of the BAPP ligand and the [Pd(BAPP)][PdCl 4 ] complex to the double-stranded DNA. In addition, in silico ADME expectations were assessed to follow their drug-likeness parameters. 2. Experimental section 2.1. 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 1 ) using a platinum crucible with a heating rate of 10 C min 1 . The Xray diffraction data of [Pd(BAPP)][PdCl 4 ] complex was collected on a Bruker Axs Kappa Apex diffractometer using Mo Karadiation. 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–H¼0.97 ˚ A, N–H ¼0.89 ˚ A and U iso ¼1.2U eq (parent atom). The crystallographic details are summarized in Table 1. The molecular modeling studies through the Density Functional Theory (DFT) calculations have been accomplished to investigate the equilibrium geometry of BAPP at the B3LYP/6-311G++(d,p) level of theory and that of the [Pd(BAPP)][PdCl 4 ] complex at the B3LYP/ GENCEP level of theory, 34 in which C, H, N and Cl atoms at 6311G++(d,p) and Pd atom at (LANL2DZ), 35 respectively, using Gaussian 09 program. 36 2.2. Synthesis of [Pd(BAPP)][PdCl 4 ] complex Mixture of PdCl 2 (177 mg; 1.0 mmol) and KCl (149 mg; 2.0 mmol) in the least amount of H 2 O was heated to about 50 C for 2 h with stirring till reaching a clear solution. Then, the aqueous solution of 1,4-bis(3-aminopropyl) piperazine (BAPP) (100 mg; 0.5 mmol) was added dropwise to the clear solution under hot condition with continuous stirring then the solution was le aside for crystallization at room temperature. Aer a few days, brown needle-like crystals, suitable for X-ray crystallographic study, were obtained. The analytical data indicate that the composition of the crystals corresponds to the compound having the formula [Pd(BAPP)][PdCl 4 ]. Yield: 74% (205 mg, 0.369 mmol). Elemental analysis calculated (%) for C 10 H 24 Cl 4 - N 4 Pd 2 (FW ¼554.97): C 21.64, H 4.36, N 10.10. Found: C 21.00, H 4.32, N 10.06. IR (KBr, nin cm 1 ): 3448 n a (NH 2 ), 2924 n a (CH 2 ), 2870 n s (CH 2 ); n a (C–N) 1458; n s (C–N) 1165. Molar cond. (DMF, 10 3 M, mScm 1 ): 9.5. UV-visible (DMSO, 10 4 M, nm): 236, 300, 421. FAB-MS (m/z, %): (M 2H) 552. 1 H NMR (DMSO, 300 MHz, ppm): 1.4–1.5 (m, 12H, (CH 2 ) Aliphatic ), 2.2–2.3 (m, 8H, (CH 2 – 1872 |RSC Adv., 2022, 12,1871–1884 © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper Open Access Article. Published on 12 January 2022. Downloaded on 1/18/2022 10:26:20 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online CH 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 ). 2.3. Bioactivity assay 2.3.1. In vitro antimicrobial activity. The antimicrobial activity of the compounds was tested using the agar well diffusion method. 37 The antimicrobial activity of the [Pd(BAPP)] [PdCl 4 ] complex and the BAPP ligand was assessed and repeated three times according to the procedure recommended through the National Committee for Clinical Laboratory Standards. 38 The bacterial strains of Staphylococcus aureus ATCC 13565 (Gram+), Bacillus subtilis ATCC 6051 (Gram+), Escherichia coli ATCC 10536 (Gram), Pseudomonas aeruginosa ATCC 27853 (Gram) in addition to two fungal strains of Candida albicans ATCC 10231 and Aspergillus avus ATCC 16872 were evaluated. 2.3.2. In vitro anticancer activity. The [Pd(BAPP)][PdCl 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 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 1 ), 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. 2.4. 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 (3¼ 6600 M 1 cm 1 ). 43,44 In the DNA binding experiments, the changes in the electronic absorption spectra of the [Pd(BAPP)] [PdCl 4 ]complex(90mM) 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 mMwere 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 (1) where [DNA] is the concentration of DNA, 3 A ¼A obsd / [compound], 3 f ¼the extinction coefficient for the free compound and 3 b ¼the extinction coefficient for the fully bound form compound. 45 [DNA]/(3 a 3 f )¼[DNA]/(3 b 3 f ) + 1/[K b (3 b 3 f )] (1) 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 ¼7.1) 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. Table 1 Crystal data and structure refinement for [Pd(BAPP)][PdCl 4 ] CCDC 2078974 V(˚ A 3 ) 914.64(6) Empirical formula C 10 H 24 Cl 4 N 4 Pd 2 Z2 FW 554.93 m(Mo Ka) (mm 1 ) 2.545 Temp. (K) 298(2) No. rens 8524 l(˚ A) 0.7107 Unique rens 4148 Cryst. syst. Triclinic Completeness to q¼25.24299.6% Space group P 1 GOOF (F 2 ) 1.030 a(˚ A) 8.5750(2) R int 0.050 b(˚ A) 9.0820(4) R 1a (I$2s) 0.0358 c(˚ A) 12.9300(4) wR 2b (I$2s) 0.0762 a(deg) 76.030(2) b(deg) 83.160(2) g(deg) 69.500(2) a R 1 ¼PkF o jjF c k/PjF o j. b wR 2 ¼[P[w(F o2 F c2 ) 2 ]/P[w(F o2 ) 2 ]] 1/2 . © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12,1871–1884 | 1873 Paper RSC Advances Open Access Article. Published on 12 January 2022. Downloaded on 1/18/2022 10:26:20 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 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. 2.4.3. Molecular docking study. The binding energies concerning the DNA–ligand/complex interactions were computed theoretically MOE2019 soware. The structures of the BAPP ligand and the corresponding [Pd(BAPP)][PdCl 4 ] complexwereconvertedintothePDBformatle from the output of Gaussian 09 soware. The crystal structure of B-DNA (1BNA: 50-D(*CP*GP*CP*GP*AP*TP*TP*CP*GP*CP*G)-30)was downloaded from Protein Data Bank (http://www.rcsb.org/ pdb). 3. Results and discussion 3.1. 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 mScm 1 at 25 C, which presumably owing to ion pair formation. Similar behavior was previously reported in [Pd(PNHP)Cl][CuCl 2 ] 49 in which (PNHP) ¼aminophosphine-bis [2-(diphenylphosphino)ethyl]amine. The UV-Vis spectra of [Pd(BAPP)][PdCl 4 ] complex and its free parental ligand were recorded in DMSO. The stability of the [Pd(BAPP)][PdCl 4 ] complex was investigated via the observation of the change of the electronic absorption spectra over time (48 h). Slight or no change (Fig. S7†) was monitored. Both BAPP ligand and [Pd(BAPP)][PdCl 4 ] complex exhibited a sharp peak at 236 nm and 300 nm which could be assignable to the transitions of an electron from s(bonding orbital)/s*(antibonding) orbital and the nonbonding orbital/s*of the amine in the BAPP ligand. 50 The similarity between the absorption bands of the BAPP ligand and the [Pd(BAPP)][PdCl 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/z552 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 1 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 1 and 2939 cm 1 of medium intensity, respectively. 54,55 The bands noted at 1465 and 1157 cm 1 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 1 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 1 , respectively, under incorporation of nitrogen atomsintheformationof[Pd(BAPP)] 2+ complex cation. The proton NMR spectrum (Fig. S3†)oftheinvestigatedcomplex 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 CNMR 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 curvewasshowninFig.S8.†The TGA data imply that the complex starts the decomposition at 224 Ccon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. Scheme 1 Synthesis of [Pd(BAPP)][PdCl 4 ] complex. 1874 |RSC Adv., 2022, 12,1871–1884 © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper Open Access Article. Published on 12 January 2022. Downloaded on 1/18/2022 10:26:20 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 3.2. 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 demonstrates the structural renement details and the corresponding crystallographic data. The structural data have been deposited at the Cambridge Crystallographic Data Centre: CCDC No. 2078974.†The crystal structure data of the double salt of [Pd(BAPP)][PdCl 4 ] reveals that the asymmetric unit incorporates one [Pd(BAPP)] 2+ complex cation and two independenthalvesof[PdCl 4 ] 2 (acting as a counter ion or a complex anion). The anionic parts [PdCl 4 ] 2 reside on in the middle of the faces of the unit cell, so that half of each belongs 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 Z¼2. 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 ¼171.4(3)and N 2 –Pd 2 –N 3 ¼ 170.5(3)).ThesumofbiteanglearoundthePd 2 atom is 360.3.AsreportedrecentlybyHouserandco-workers,the geometry of four coordinate complexes could be ascertained Fig. 1 ORTEP view for [Pd(BAPP)][PdCl 4 ] complex showing 50% probability ellipsoids and the atom-numbering scheme. Fig. 2 The optimized structure of BAPP and the natural charges on atoms of BAPP by density function B3LYP/6-311G++(dp). © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12,1871–1884 | 1875 Paper RSC Advances Open Access Article. Published on 12 January 2022. Downloaded on 1/18/2022 10:26:20 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online quantitatively through the new parameter, s 4 ¼[360(a+ b)]/141where (a¼:N 1 –Pd 2 –N 2 ¼171.4(3)and b¼:N 2 – Pd 2 –N 3 ¼170.5(3)). The value of s 4 ¼0.13 conrms the distortion of square planar geometry of the complex cation. 60 Despite the distortion, the environment around the palladium center is planar. The large bending in the bond angle of N 1 – Pd 2 –N 2 was forced to be 73.4(2)by the restraint of the vemembered ring. The bond lengths of the four Pd–NarePd 2 – N 1 ¼2.052(6), Pd 2 –N 2 ¼2.054(6), Pd 2 –N 3 ¼2.053(7) and Pd 2 – N 4 ¼2.058(7), which compare well with the previously reported [Pd(BAPP)](ClO 4 ) 2 61 analogue and lies in the range of many other reported palladium amine complexes. 34,62,63 3.3. Molecular DFT calculation of BAPP ligand and [Pd(BAPP)][PdCl 4 ] complex Fig. 2 and 3 show the optimized structures of the BAPP ligand and the [Pd(BAPP)][PdCl 4 ] complex, respectively, in the form of the lowest energy congurations. The natural charges resulted from Natural Bond Orbital Analysis (NBO) were also shown. The two palladium atoms are four-coordinate in square planar geometry. The atoms N 1 ,N 2 ,N 4 and N 3 are almost in one plane deviated by 7.236. Also, the atoms Cl 1 ,Cl 2 ,Cl 3 and Cl 4 are almost in one plane deviated by 0.365. The bite angles in the distorted square planar geometry are ranging from 72.48(for N 1 –Pd 2 –N 2 bite angle) to 94.69for N 3 –Pd 2 –N 4 bite angle as shown in 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 2.885 ˚ Ain the BAPP ligand decreased by 0.385 ˚ A to 2.500 ˚ A in the complex. Also, the N 3 –N 4 bond length was drastically decreased from 11.413 ˚ A to 3.085 ˚ A 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 (0.564), N 2 (0.567), N 3 (0.945), N 4 (0.944). 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 )¼E LUMO 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 Fig. 3 The optimized structure, the vector of the dipole moment, and the natural charges on active centers of [Pd(BAPP)][PdCl 4 ] complex. Table 2 Comparison of the important X-ray and DFT optimized bond lengths (˚ A) and bond angles () of [Pd(BAPP)][PdCl 4 ] complex Type of bond Bond length (˚ A) Type of Angle Angle () X-ray DFT X-ray DFT Pd 2 –N 1 2.047 2.102 N 1 –Pd 2 –N 2 73.44 72.48 Pd 2 –N 2 2.054 2.126 N 1 –Pd 2 –N 3 98.60 93.18 Pd 2 –N 3 2.053 2.095 N 2 –Pd 2 –N 4 98.97 99.06 Pd 2 –N 4 2.058 2.100 N 3 –Pd 2 –N 4 89.30 94.69 Pd 1 –Cl 1 2.313 2.473 N 1 –Pd 2 –N 4 171.5 169.8 Pd 1 –Cl 2 2.301 2.473 N 2 –Pd 2 –N 3 170.6 164.9 Pd 1 –Cl 3 2.313 2.421 Cl 1 –Pd 1 –Cl 2 89.73 91.49 Pd 1 –Cl 4 2.301 2.426 Cl 1 –Pd 1 –Cl 4 90.27 88.24 Cl 2 –Pd 1 –Cl 3 90.27 89.73 Cl 3 –Pd 1 –Cl 4 89.73 90.07 Cl 1 –Pd 1 –Cl 3 180.0 175.1 Cl 2 –Pd 1 –Cl 4 180.0 174.3 N 1 –N 2 –N 4 –N 3 6.520 a 7.236 a Cl 1 –Cl 2 –Cl 3 –Cl 4 0.000 a 0.365 a a Dihedral angle. 1876 |RSC Adv., 2022, 12,1871–1884 © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper Open Access Article. Published on 12 January 2022. Downloaded on 1/18/2022 10:26:20 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Table 3 Calculated energies of BAPP and [Pd (BAPP)][PdCl 4 ]complex at B3LYP/6-311G(dp) for all atoms except Pd at B3LYP/LANL2DZ E a HOMO b LUMO c E gd Dipole moment e BAPP 614.382 4.9307 2.5813 7.5120 1.6566 [Pd(BAPP)][PdCl 4 ]2709.059 5.8461 3.0585 2.7876 23.9199 a The total energy (a.u.). b Highest occupied molecular orbital (eV). c Lowest unoccupied molecular orbital (eV). d E g ¼E LUMO E HOMO (eV). e Dipole moment (debye). Fig. 4 HOMO and LUMO charge density maps of BAPP and its [Pd(BAPP)][PdCl 4 ] complex. Fig. 5 Molecular electrostatic potential (MEP) surface of BAPP (left) and [Pd(BAPP)][PdCl 4 ] complex (right). © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12,1871–1884 | 1877 Paper RSC Advances Open Access Article. Published on 12 January 2022. Downloaded on 1/18/2022 10:26:20 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online and nitrogen atoms coordinating sites which signify their nucleophilic tendency towards the Pd 2+ metal ion. For the complex [Pd(BAPP)][PdCl 4 ], the map is divided into two zones, the red zone is around the contour of the anionic complex [PdCl 4 ] 2 , while the blue one is around the contour of the cationic complex [Pd(BAPP)] 2+ . 3.4. Antimicrobial activity The antimicrobial activity of the BAPP ligand and its [Pd(BAPP)] [PdCl 4 ] complex has been tested against different microorganisms such as bacteria and fungi. The antimicrobial activity values for the [Pd(BAPP)][PdCl 4 ] complex are lower than those found for the BAPP ligand for all the tested species except for C. albicans is more susceptible to the [Pd(BAPP)][PdCl 4 ] complex than the BAPP ligand. Generally, it seems to be the coordination of Pd(II) ion to the tetra-dentate BAPP ligand reduced the antimicrobial activities of the corresponding BAPP ligand. Whereby, the activity could not be attributed to the presence of the metal center only, 64 as the free ligand exhibited a superior activity than the corresponding cationic complex of [Pd [BAPP]] 2+ . As reported before, in a large number of palladium complexes bearing N,N-chelate ligands, the free ligand shows an antimicrobial activity while their corresponding metal complexes were devoid to have an antibacterial and antifungal properties such as [Pd(L 1 ) 2 ] and [Pd(L 2 ) 2 ] complexes (where L l ¼ 3-(1-(phenylamino)ethylidene)-chroman-2,4-dione and L 2 ¼3- (1-(o-toluidino)ethylidene)-chroman-2,4-dione), L 1 and L 2 had higher activity towards Staphylococcus aureus and Salmonella enterica, respectively. In addition to other palladium(II) complexes based on quinolinylaminophosphonates ligands such as diethyl [a-anilino-(quinolin-3-ylmethyl)]phosphonate (L 3 ) and dibutyl [a-(quinolin-3-ylamino)-N-benzyl]phosphonates (L 4 ), only L 3 and L 4 act as bacteriostatic agents against B. thuringiensis var. kurstaki (for L 3 ), B. megaterium and S. epidermidis (for L 4 ). 65–68 This indicates that the activity is not depending solely on the existence of metal ions but rather a synergistic effect of many factors. 69 3.5. Cytotoxic activity In the cytotoxicity analysis, in comparison with the BAPP ligand, the [Pd(BAPP)][PdCl 4 ] complex shows more killing potential towards the tested cancer cell lines of MCF7 (IC 50 ¼49 3.7 mM), HepG2 (IC 50 ¼14.6 1.1 mM) and CaCo-2 (IC 50 ¼14.9  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 (WI38). 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 Fig. 6 Cellular growth inhibition of (A) BAPP ligand (B) [Pd(BAPP)] [PdCl 4 ] complex against normal cell (W138), breast carcinoma (MCF7), hepatocellular carcinoma (HepG2) and epithelial colorectal adenocarcinoma cells (CaCo-2). The cell viability was measured by MTT assay after incubation for 48 h. Fig. 7 Absorption spectrophotometric titration of BAPP ligand (4.5  10 4 M) against increasing concentrations of CT-DNA. [DNA] ¼0–100 mM, step 20 mM. Arrow ([) refers to the hyperchromic effect. The inset shows the linear fit of [DNA]/(3 a 3 f )vs. [DNA]. 1878 |RSC Adv., 2022, 12,1871–1884 © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Advances Paper Open Access Article. Published on 12 January 2022. Downloaded on 1/18/2022 10:26:20 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online