Design, synthesis, structure and biological evaluation of new palladium(II) hydrazone complexes

Accepted Manuscript Research paper Design, synthesis, structure and biological evaluation of new palladium(II) hydrazone complexes Ganesan Ayyannan, Maruthachalam Mohanraj, Gunasekaran Raja, Nanjan Bhuvanesh, Raju Nandhakumar, Chinnasamy Jayabalakrishnan PII: DOI: Reference:

S0020-1693(16)30529-1 http://dx.doi.org/10.1016/j.ica.2016.09.025 ICA 17270

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Inorganica Chimica Acta

Received Date: Revised Date: Accepted Date:

13 May 2016 10 September 2016 13 September 2016

Please cite this article as: G. Ayyannan, M. Mohanraj, G. Raja, N. Bhuvanesh, R. Nandhakumar, C. Jayabalakrishnan, Design, synthesis, structure and biological evaluation of new palladium(II) hydrazone complexes, Inorganica Chimica Acta (2016), doi: http://dx.doi.org/10.1016/j.ica.2016.09.025

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Design, synthesis, structure and biological evaluation of new palladium(II) hydrazone complexes Ganesan Ayyannana, Maruthachalam Mohanraja, Gunasekaran Rajaa, Nanjan Bhuvaneshb, Raju Nandhakumarb, Chinnasamy Jayabalakrishnana* a

Post-Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya

College of Arts and Science, Coimbatore - 641 020, Tamil Nadu, India. b

Department of Chemistry, Karunya University, Karunya nagar, Coimbatore 641 114, India.

*Corresponding

author

contact

details,

e-mail

address:

[email protected]

(C.Jayabalakrishnan), Tel.: +0422 2692461, Fax.: 0422 2693812.

Abstract New palladium(II) complexes {[Pd(L)(PPh3)] (2) and [Pd(L)(AsPh3)] (3)} have been synthesized by using 4-hydoxy benzoic acid (5-chloro-2-hydroxy-benzylidene)-hydrazide (H2L) (1) ligand. The structure of the ligand (H2L) (1), and complexes (2 and 3) were confirmed by using analytical, spectral and single crystal X-ray diffraction techniques. The structure of complexes (2 and 3) revealed a square planar geometry around the Pd ion in which the hydrazone is coordinated through ONO atoms. The interaction of the compounds with calf thymus DNA (CT-DNA) was investigated by absorption, emission titration, electrochemical and viscosity methods, indicating that the ligand and the complexes interacted with CT-DNA through intercalation. The good interaction of the ligand and the Pd(II) complexes with bovine serum albumin (BSA) protein was confirmed by UV-visible and fluorescence spectroscopic studies. The better free radical scavenging ability of Pd(II) complexes was evaluated by in vitro antioxidant assays involving DPPH radical, hydroxyl radical and nitric oxide radical. Furthermore, in vitro cytotoxicity studies of 2 and 3 showed higher activity with lower IC50 values even at low concentrations. Keywords: Palladium(II) complexes, X-ray crystallography, biomolecular interaction, antioxidant, cytotoxic activity

1. Introduction Cancer is one of the most fatal non-communicable diseases which cause significant number of deaths every year. Chemotherapy is currently the most-utilised treatment for cancer treatment. Transition metal complexes have received great attention as cancer therapy, due to the clinical sources of cisplatin and its derivatives [1, 2]. However, they have inherent limitations due to resistance over a period of time and serious side effects in causing nausea and the failure of kidney and liver which are typical of heavy metal toxicity and low degree selectivity [3, 4]. Therefore, considerable attempts are being made to replace cisplatin with suitable alternatives by synthesizing number of transition metal complexes and screened for their potential anticancer activities. Among the transition metals, Pd plays a peculiar role in chemotherapy since it accumulates in tumors due to selective permeability through cancer cell membranes [5-7]. Since, the structural analogy and thermodynamic properties of Pt(II) complexes are nearly similar with the Pd(II) complexes [8, 9] a number of palladium(II) complexes have been reported with their potential anticancer activities [10-14]. Palladium(II) complexes undergo aquation and ligand exchange reactions 105 times faster than the corresponding platinum(II) complexes [15]. In the past 2 decades, investigations in anticancer agents have resulted in the development of a large number of palladium complexes containing N and O ligands, which exhibited effective antitumor properties [16, 17]. Moreover palladium(II) complexes with nitrogen and oxygen containing ligands are the subject of intensive biological evaluation in the search for less toxic and more selective anticancer therapies. To date, several Pd(II) complexes with their potential anticancer properties are reported [18-21]. In addition, hydrazones are the important class of ligands with interesting ligation properties due to the presence of ONO coordination sites. Various important properties of hydrazones along with their applications in medicine and analytical chemistry have led to increased interest in their complexation characteristics with Pd ions. It is well established that the formation of Pd-complexes plays a very important role to enhance the biological activity of free hydrazones [22]. DNA is the primary intracellular target of anti-cancer drugs, the interaction between small molecules and DNA can cause DNA damage in cancer cells, hence blocking the division of cancer cells and resulting in cell death. A study on the interaction of small molecules with DNA on a molecular level is very important in the development of novel chemotherapeutics and highly sensitive diagnostic agents [23-25]. Small molecules can interact with DNA through the following three non-covalent modes: intercalation, groove

binding and external static electronic effects [26]. Among the different types of DNA binding compounds, intercalating and groove binding compounds are more promising, most widely studied as they form an important class of compounds for new anticancer drug development. Recently, there has been great interest on the binding of transition metal complexes with DNA, owing to their possible applications as new cancer therapeutic agents [27]. Therefore, the interaction of metal complexes towards DNA is useful in the design and synthesis of metal-based anticancer therapeutics. Serum albumins are the most abundant plasma proteins and their interaction with drug molecules has attracted great interest in recent years [28]. The interactions with various biomolecules proteins play an important role in the transportation and deposition of a variety of endogenous and exogenous substances such as fatty acid drugs and metal ions in blood stream. The binding affinity of drug to serum albumin is pivotal in the design of new drugs [29, 30]. The interaction of drug-protein may react in the formation of a stable protein complex which has important effect on the distribution, absorption, metabolism and properties of drugs. Such an interactions of drug compounds have aromatic rings are very important for protein sterilization and different regulation processes. Recently, it has been demonstrated that, some metal complexes have high affinity for binding to human serum albumin under physiological conditions to exhibit a variety of pharmacological properties. In this study, we have used bovine serum albumin (BSA) as the protein model due to its medical importance, low cost, easy availability, intrinsic fluorescence emission, and structural homology with human serum albumin (HSA). Therefore, the activity of metal complexes towards DNA and protein BSA is useful in the design and synthesis of metal-based anticancer therapeutics. Based on the above facts, we have been intense in studying the role of presence of aromatic rings, planarity and molecular composition of various transition metal complexes on DNA and protein binding and cytotoxicity studies. Hence, our present investigation focuses on the synthesis of two new palladium complexes containing hydrazone ligand obtained from the reaction of 5-chloro salicylaldehyde and 4-hydroxy benzhydrazide. The structure of the ligand and new complexes has been characterized by various spectro-analytical and crystallographic techniques. All the synthesised compounds have been subjected to different experiments to assess their interacting ability with DNA/protien and antioxidant and cytotoxicity potential.

2. Experimental section 2.1. Materials and methods All the reagents used were of analytical or chemically pure grade. Solvents were purified and dried according to standard procedures [31]. Doubly distilled water was used to prepare buffers. Ethidium bromide (EB), bovine serum albumin (BSA), calf thymus DNA (CT-DNA) were purchased from Sigma-Aldrich and used as received. The ligand and the starting complexes, [PdCl2(PPh3)2] and [PdCl2(AsPh3)2] were prepared according to the previous reports [32, 33]. Microanalyses (C, H and N) were performed on a Vario EL III CHNS analyser. IR spectra were recorded as KBr pellets in the 400-4000 cm-1 region using a Perkin Elmer FT-IR 8000 spectrophotometer. Electronic spectra were recorded in DMSO solution with a Systronics double beam UV-vis spectrophotometer 2202 in the range 200-800 nm. Fluorescence spectral data were performed on a JASCO FP-8200 fluorescence spectrophotometer at room temperature. 1H and 31P NMR spectra were recorded on a Bruker AV III 500 MHZ instrument using TMS and ortho phosphoric acid as an internal standard. Melting points were recorded with Veego VMP-DS heating table. 2.2. Synthesis of hydrazone ligand (H2L) (1) The ligand 4-hydoxy-benzoic acid (5-chloro-2-hydroxy-benzylidene)-hydrazide (H2L) (1) was prepared by refluxing an equimolar mixture of 4-hydroxy benzoic acid hydrazide (0.152 g; 1 mM) and 5-chloro-2-hydroxy benzaldehyde (0.156g; 1 mM) in 50 mL methanol for 6 h (Scheme 1). The reaction mixture was cooled to room temperature and the solid obtained was filtered, washed several times and recrystallized from methanol. White coloured crystals suitable for X-ray diffraction analysis were obtained by slow evaporation of its methanolic solution. Yield: 84%; M.P: 263°C. Anal. calcd. for C14H11ClN2O3 (%): C, 57.84; H, 3.81; N, 9.64. Found (%): C, 57.81; H, 3.83; N, 9.64. IR (KBr, cm-1): 3421 ν(OH); 3234 ν(NH); 1605 ν(C=O); 1598 ν(C=N); 1106 ν(N-N);. UV-vis (DMSO), λmax (nm) (ε M-1 cm-1): 305 (15538), 360 (12728) (π → π*, n→ π*). 1H NMR (DMSO-d6): δ 12.01 (s, 1H, hydrazine NH), δ 8.58 (s, 1H, H-C=N), δ 11.41 (s, 1H, o-OH), δ 10.19 (s, 1H, p-OH), δ 6.87-7.84 (m, 7H, aromatic). 2.3. Synthesis of [Pd(L)(PPh3)] (2) For the preparation of 2, a methanolic solution (20 cm3) of H2L (1) (0.0290g; 0.1mM) was mixed with dichloromethane (20 cm3) solution of [PdCl2(PPh3)2] (0.0701 g; 0.1mM) and two drops of triethylamine. The mixture was refluxed for 1 h. An orange coloured precipitate

was formed during this period. The reaction mixture was then cooled to room temperature, and the solid compound was filtered. It was washed with methanol and dried under vacuum. The single crystals of 2, grown from methanol/DMF were found to be suitable for X-ray diffraction. C32H24ClN2O3PPd (2): Yield: 81%, MP: 255 °C, Anal. calcd. (%): C, 58.46; H, 3.68; N, 4.26; Found (%): C, 58.51; H, 3.69; N, 4.25. Selected IR bands (cm-1): 1590 ν(C=N); 1356 ν(enolic C-O); 1434, 1049, 694 (for PPh3). UV-vis (DMSO), λmax(nm) (ε M-1 cm-1): 275 (11303), 313 (10393) (intra-ligand transitions); 412 (7848) (LMCT). 1H NMR (DMSO-d6): δ 8.53 (s, 1H, H-C=N), δ 9.92 (s, 1H, p-OH), δ 6.62-7.76 (m, 22H, aromatic). 2.4. Synthesis of [Pd(L)(AsPh3)] (3) It was prepared as described for 2 by the reaction of [PdCl2(AsPh3)2] (0.0791 g, 0.1mM) with ligand (0.0290g; 0.1mM). Dark orange coloured crystals obtained were found to be suitable for X-ray diffraction. C32H24ClN2O3AsPd (3): Yield: 79%, MP: 221 °C. Anal. calcd. (%): C, 54.80; H, 3.45; N, 3.99.Found (%): C, 54.69; H, 3.49; N, 4.06. Selected IR bands (cm-1): 1589 ν(C=N); 1355 ν(enolic C–O); 1435, 1080, 693 (for AsPh3). UV-vis (DMSO), λmax(nm) (ε M-1 cm-1): 270 (3848), 323 (3041) (intra-ligand transitions); 416 (2070) (LMCT). 1H NMR (DMSO-d6): δ 8.52 (s, 1H, H-C=N), δ 9.97 (s, 1H, p-OH), δ 6.74-7.76 (m, 22H, aromatic). 2.3. Crystal structure determination Single crystal X-ray diffraction data of 1, 2 and 3 were collected at room temperature on a Bruker AXS KAPPA APEX2 CCD diffractometer equipped with a fine focused sealed tube. The unit cell parameters were determined and the data collections of 1, 2 and 3 were performed using a graphite-mono chromate Mo Kα(k = 0.71073 Å) radiation by u and x scans. The data collected were reduced SAINT program [34] and the empirical absorption corrections were carried out using the SADABS program [35]. The structure of the ligand and complexes was solved by direct methods [36] using SHELXS-97, which revealed the position of all non-hydrogen atoms, and was refined by full-matrix least squares on F2 (SHELXL-97) [37]. All non-hydrogen atoms were refined anisotropically, while the hydrogen atoms were placed in calculated positions and refined as riding atoms. 2.4. DNA binding studies Experiments involving the interaction of ligands and complexes with CT-DNA were carried out in double distilled water with tris(hydroxymethyl)-aminomethane (Tris, 5 mM) and sodium chloride (50 mM). The pH 7.2 was adjusted using hydrochloric acid. A solution

of CT-DNA in the buffer gave a ratio of UV absorbance of about 1.9 at 260 and 280 nm, indicating that the DNA was sufficiently free of protein [38, 39]. The concentration of CTDNA was calculated from its known extinction coefficient at 260 nm (6600 M-1 cm-1). A stock solution of CT-DNA was stored at 277 K and used after no more than 4 days. Absorption titration experiments were performed by using a fixed concentration of test compounds to which increments of the DNA stock solution were added. An equal concentration solution of CT-DNA was added to the compound solution and reference solution to eliminate the absorbance of CT-DNA itself. The same experimental procedure was followed for emission studies also. Further support for the complexes binding to DNA via intercalation is given through emission quenching experiments. DNA was pretreated with ethidium bromide (EB) for 30 min. Then the test solutions were added to the mixture of EBDNA, and the change in the fluorescence intensity was measured. The excitation and the emission wavelength were 515 nm and 604-606 nm, respectively. The electrochemical titration experiments were performed by keeping the concentration of the test compounds constant while varying the CT-DNA concentration using Tris-HCl buffer as solvent. The relative viscosities for DNA in the absence (ηo) and presence (η) of the compounds was calculated using the relation η = (t-to)/to, where t and to are the observed flow time for each sample and buffer. The values of relative viscosity (η/ ηo) 1/3 were plotted against R, where R= [DNA]/[compound]. 2.5. Protein binding studies The excitation wavelength of BSA at 280 nm and the emission at 344 nm were monitored for the protein binding studies using fluorescence spectra recorded with synthesized compounds (1-3). The excitation wavelength of BSA at 280 nm and the quenching of the emission intensity of tryptophan residues of BSA at 346 nm were monitored using the complexes as quenchers with increasing concentrations [40]. The excitation and emission slit widths, and scan rates were constantly maintained for all the experiments. Samples were carefully degassed using pure nitrogen gas for 15 min. Quartz cells (4 × 1 × 1 cm) with high vacuum Teflon stopcocks were used for degassing. Stock solution of BSA was prepared in 50 mM phosphate buffer (pH=7.2) and stored in the dark at 4°C for further use. Concentrated stock solution of the complexes prepared as mentioned for the DNA binding experiments except that the phosphate buffer was used instead of Tris-HCl buffer for all the experiments. BSA solution (1mM) was titrated by successive additions of test solutions 1, 2, and 3 using micropipettes for all of the experiments.

2.6. Antioxidant assays The DPPH, OH and NO radical scavenging activities of the ligand and Pd-complexes were determined by the methods described by Blois, Nash and Green et al., respectively [41-43]. For each of the above assay, tests were done in triplicate by varying the concentration of the compounds ranging from 10-50 µM. The percentage activity was calculated by using the formula, % activity = [(A0–Ac)/A0] × 100, where A0 and Ac represent the absorbance in the absence and presence of the test compounds, respectively. The 50% activities (IC50) were calculated from the results of percentage activity. 2.7. Cytotoxicity studies Cytotoxicity of the compounds was carried out on human cervical (HeLa) and human breast

(MCF-7)

cancer

cell

lines.

Cell

viability

was

carried

out

using

the

3-[4, 5-dimethylthiazol-2-yl] 2, 5-diphenyltetrazolium bromide (MTT) assay method [44]. The HeLa and MCF-7 cells were grown in eagles minimum essential medium (EMEM) containing 10% fetal bovine serum (FBS). For the screening experiment, the cells were seeded onto 96-well plates at plating density of 10,000 cells/ well and incubated to allow for cell attachment at 37 ºC, 5% CO2, 95% air and 100% relative humidity for 24 h prior to the addition of the compounds. The compounds were dissolved in DMSO and diluted in the respective medium containing 1% FBS. After 24 h the medium was replaced with the respective medium with 1% FBS containing the compounds at various concentrations and incubated at 37 ºC under conditions of 5% CO2, 95% air and 100% relative humidity for 48 h. Triplication was maintained and the medium not containing the compounds served as control. After 48 h, 15 µL of MTT (5 mg mL-1) in phosphate buffered saline (PBS) was added to each well and incubated at 37 ºC for 4 h. The medium with MTT was then removed and the formed formazan crystals were dissolved in 100 µL of DMSO. The absorbance was then measured at 570 nm using micro plate reader. The % cell inhibition was determined using the following formula, % cell inhibition=100-Abs (sample)/Abs (control) × 100. The IC50 values were determined from the graph plotted between % cell inhibition and concentration. 3. RESULTS & DISCUSSION 3.1. Synthesis The reaction between [PdCl2 (EPh3)2] (where E=P or As) and 1 yielded [Pd(L)(PPh3)] (2) and [Pd(L)(AsPh3)] (3) (Scheme 2). The analytical data of the above synthesized complexes are in good agreement with the proposed molecular formulae of 1:1 metal to ligand stoichiometry (given under the experimental part). All of the three synthesized

compounds are quite stable in air and light and soluble in most of the organic solvents, such as methanol, ethanol, CH2Cl2, CHCl3, DMF and DMSO. OH OH Methanol

H N

CHO

N

H 2N

Reflux 6 h OH

O

OH

H N O

Cl

Cl

Scheme 1. Synthesis of hydrazone ligand. OH OH

N

H N

OH

N

[PdCl2 (EPh3) 2]

N O

O

CH2Cl2/MeOH Reflux 1 h

Cl

O Cl

Pd E

Where E = P (or) As

Scheme 2. Synthesis of the palladium(II) complexes 3.2. Spectroscopic studies The electronic absorption spectra of 1, 2 and 3 were recorded in the UV-Visible region using DMSO as a solvent. Electronic spectra of 1 showed two strong absorption bands at 235-245 nm and 260-305 nm, which are assigned to π-π* and n-π* transitions respectively. In the electronic spectra of all the complexes, three to four bands were observed at 240-410 nm. The bands appearing with high intensity at 240-310 nm corresponds to intra-ligand transitions. A less intense band at 356-410 nm has been assigned to the ligand to metal charge transfer transition (LMCT) [45]. FT-IR spectroscopy is a preliminary tool to find out the coordination mode of the ligand to central metal ions in complexes. The FT-IR spectrum of ligand H2L shows peaks at 3421, 3234, 1650 and 1598 cm-1 due to O-H, N-H, C=O and C=N vibrations, respectively. In spectra of complexes the strong band OH did not registered due to deprotonation of phenolic oxygen and its coordination to the palladium(II) ion. The NH and C=O stretching vibrations were absent in complexes 2 and 3, which reveals the enolization of the NH proton followed by the deprotonation prior to coordination with the palladium(II) ion [46]. In addition, a azomethine C=N peak is showed towards lower frequency in the complexes, due to the participation of azomethine nitrogen for coordination with the central palladium(II) ion. In addition the bands confirming the presence of triphenyl phosphine and arsine in were 2 and 3 observed in the expected region [47]. All these facts suggested that the ligand H2L were coordinated to palladium(II) ion via the phenolate oxygen, the azomethine nitrogen and the imidolate oxygen in complexes.

1

H NMR spectra of 1, 2 and 3 were recorded and assigned on the basis of observed

chemical shifts. The spectrum of 1 showed a singlet at 11.41 ppm due to phenolic OH, which completely disappeared in that of complexes confirming the involvement of phenolic oxygen in coordination. Also, a weak singlet appearing at 12.01 ppm has been assigned due to the –NH proton. However, the NMR spectra of 2 and 3 did not register any signals corresponding to -NH proton which indicates the enolization of N(2) imine nitrogen followed by deprotonation during the coordination of ligand with Pd ion [48]. The spectrum of H2L showed a sharp singlet at 8.58 ppm corresponding to azomethine proton. However, in case of the complexes (2 and 3), the singlet at around 8.58 ppm gets shifted (2 and 3 are 8.53 and 8.52 ppm, respectively) due to the coordination of azomethine N with Pd(II) ion [49]. The spectra of 1, 2 and 3 show signals at 6.62-7.84 ppm due to the presence of aromatic protons. The

31

P NMR spectrum of the 2 showed a singlet at 21 ppm, which confirm that one

triphenylphosphine group is coordinated to palladium in the complex.

3.3. X-ray crystallography The molecular structure of the ligand (1) and two complexes have been determined by single crystal XRD to confirm the coordination mode of the hydrazone ligand in the complexes and the stereochemistry of the complexes. The summary of the data collected and the refinement parameters are given in Table 1. The selected bond lengths and bond angles are given in Table 2. An ORTEP representation of 1 is shown in Figure 1. The compound crystallizes in a triclinic space group Pna21. The azomethine bond length, 1.278(3) Å is in conformity with the formed C=N double bond length. The oxygen O(3) atom and the hydrazine nitrogen N(1) are in a trans position with respect to the C(8)-N(2) bond. And the bond distances for N(1)-N(2) at 1.379(2), and for N(2)-C(8) at 1.352(3)Å are closer to N-N and N-C normal single bonds respectively The bond distances in the hydrazone side chain agree well with the values observed for other hydrazone where the C(8)-O(3) group is present in the keto form (1.246(2)Å). The molecular structure of the complexes 2 and 3 along with the atomic numbering scheme are given in Fig. 2 and 3. The selected bond lengths and bond angles are summarised in Table 3. The single crystal X-ray study reveals that the complexes 2 and 3 crystallized in monoclinic space group P21/c with four molecules per unit cell. In both the complexes, coordination geometry around palladium ion can be described as a distorted square planar. The palladium is coordinated through phenolic oxygen, azomethine nitrogen N(2) and the enolate oxygen atoms of the ligand and the remaining site is occupied by triphenylphosphine

in complex 2 and triphenylarsine in complex 3 respectively. The square planar geometry is slightly distorted as shown by the bond angles around the Pd ion. The distortion in the complexes from the ideal square planar geometry is due to the angle of the ONO chelate of hydrazone ligand and the bending of the ligand towards the chelate which is evident from the respective angles. The trans angles of complex 2 are N(2)-Pd(1)-P; 171.70(7)° and O(2)Pd(1)-O(1); 172.82(8)° and complex 3 are N(1)-Pd(1)-As(1); 171.41(8)° and O(2)-Pd(1)O(1); 174.08(10)°, which shows a slight deviation from the expected linear trans geometry, suggesting distortion in the square planar coordination geometry [50, 51]. The C=O bond distance in C(8)-O(3) of the free ligand is found to be 1.246(2) Å. Whereas, upon complexation, the value of C(7)-O(1) increases to 1.298(3) Å and 1.303(4) Å for complexes 2 and 3 respectively. Further, the C(7)-N(1) of (2) and C(7)-N(2) of (3) bond length substantiates the formation of a double bond upon keto-enol tautomerization of the hydrazinic nitrogen followed by the deprotonation of enolate oxygen. There is an increase in the C=N bond lengths C(8)-N(2); 1.281(3) Å for 2 and C(8)-N(1); 1.285(4) Å for 3 compared to ligand 1 C(7)-N(1); 1.278(3) Å due to azomethine nitrogen involved in coordination. The Pd(1)-P(1) and Pd(1)-As(1) bond lengths 2.2830(7) and 2.3906(5) Å are in agreement with other structurally characterized palladium phosphine and arsine complexes. The selected bond lengths and bond angles agree very well with those that are reported for other palladium(II) complexes [52].

3.4. DNA Binding studies 3.4.1. Electronic absorption studies Electronic absorption spectroscopy is one of the most useful tools for the investigation of the mode of interaction of targeted compounds with DNA. The absorption spectrum of ligand and complexes at constant concentration (25 µM) in the absence and presence of CT-DNA with different concentration is shown in Fig. 4. In the presence of DNA, the absorption bands of the ligand 1 exhibited hypochromism of about 3.66% and 5.76% with blue shift of about 1 nm at 289 and 346 nm respectively. However, 2 exhibited the hypochromism of about 57.98%, 60.03% and 62.89% with blue shift of 3, 5 and 1 nm at 270, 317 and 410 nm respectively. On the other hand, 3 exhibited hypochromism of about 37.22%, 31.25% and 28.45% with blue shift of about 4, 5 and 2 nm at 272, 325 and 410 nm respectively. These results suggested an intimate association of the test compounds with CT-DNA, and it is also likely that these compounds bind to the DNA helix via intercalation. After the compounds intercalate to the base pairs of DNA, the π* orbital of the intercalated

compounds could couple with π orbitals of the base pairs, thus decreasing the π→π* transition energies, hence resulting in hypochromism. The two Pd(II) complex showed more hypochromicity than the ligand, indicating that the binding strength of the Pd(II) complexes is stronger than that of the free ligand. In order to further evaluate the binding strength of the compounds, the intrinsic binding constants (Kb) of them with CT-DNA were determined from the following equation, [DNA]/[εa- εf] = [DNA]/[εa- εf] + 1/Kb [εb- εf] where [DNA] is the concentration of DNA in base pairs, εa is the extinction coefficient observed at a given DNA concentration, εf is the extinction coefficient of the free complex in solution and εb is the extinction coefficient of the complex when fully bound to DNA. In plots of [DNA]/(εa-εf) versus [DNA] (Fig. 5), Kb is given by the ratio of slope to the intercept. The magnitudes of intrinsic binding constants (Kb) were calculated to be 4.3 × 103 M-1, 3.2 × 104 M-1 and 2.8 × 104 M-1 for the compounds 1, 2 and 3 respectively (Table 4). The observed values of Kb revealed that the ligand and the Pd(II) complexes bind to DNA via intercalative mode [53, 54, 55]. And also, complex 2 is strongly bound with CT-DNA than that of 3 and 1, and then the order of binding affinity is 2˃3˃1. Though it has been found that ligand and complexes can bind to DNA by intercalation, the binding mode needs to be proved through further experiments. 3.4.2. Ethidium bromide displacement assay In order to further confirm the intercalating mode of binding between the synthesized compounds and CT-DNA, an ethidium bromide displacement experiment was carried out. Ethidium bromide is a phenanthridine fluorescence dye, it was non-emissive in Tris-HCl buffer solution (PH 7.2) due to fluorescence quenching of free EB by the solvent molecule. In the presence of DNA, EB showed enhanced emission intensity due to its strong intercalation of the planar phenanthridine ring between the adjacent DNA base pairs [56]. If the test compounds can intercalate into DNA, the binding sites of DNA available for EB will be decreased, hence the fluorescence intensity of EB will be quenched. The emission spectra of the DNA-EB system with increasing concentration of the test compounds are shown in Fig. 6. Upon increasing the amounts of ligand and the palladium(II) complexes causes 61%, 74% and 70% for 1, 2 and 3 respectively. A significant decrease of the fluorescence emission intensity band of the bound DNA-EB system indicates that the strong binding affinity of the compounds with DNA over DNA-EB. This hypochromism of the ligand and the complexes could compete with EB in binding to DNA and complex 2 binds to DNA stronger than

complex 3. In general, the binding affinities of metal chelate towards DNA are better than that of the ligands due to chelate effect. The quenching parameter can be analyzed according to the Stern-Volmer equation F0 / F = Kq / [Q] + 1 where F0 is the emission intensity in the absence of complex, F is the emission intensity in the presence of complex, Kq is the quenching constant, and [Q] is the concentration of the compound. The Kq value has been obtained as a slope from the plot of F0 / F versus [Q] (Fig. 7), the quenching constants (Kq) to the three compounds as 4.49 × 10 4 M-1, 6.98 × 10 4 M-1 and 5.45 × 104 M-1 respectively. Further, the binding constant (Kapp) value obtained for the compounds using the following equation. KEB[EB] = Kapp[compound] where the compound concentration is the value at a 50% reduction in the fluorescence intensity of EB, KEB (1.0 ×107M−1) is the DNA binding constant of EB, [EB] is the concentration of EB = 5 µM), and they were found to be 3.3 × 10 5 M−1, 5.2 × 105 M−1 and 4.0 × 10 5 M−1 respectively for 1-3 (Table 4). These results suggest that the interaction of the Pd(II) complexes (2 and 3) with DNA is stronger than that of the free ligand 1, which is consistent with the electronic absorption spectral results. Moreover, complex 2 showed higher DNA binding affinity compared to complex 3. The quenching and binding constants of the ligand and palladium(II) complexes suggested that the interaction of the tested compounds with CT-DNA should be of intercalation [57, 58]. 3.4.3. Cyclic voltammetry The cyclic voltammetric technique was employed to study the interaction of the complexes with DNA in order to confirm the DNA binding modes suggested by the spectroscopic studies. Well known that the electrochemical potential of synthesized complexes will shift positively when it intercalates into DNA, and if it is bound to DNA by electrostatic interaction, the potential would shift in a negative direction [59, 60]. Study the binding mode between the Pd(II) complexes and CT-DNA, the cyclic voltammetric behaviours of complexes in the absence and presence of CT-DNA in Tris HCl buffer (pH 7.2) were studied using the scan rate of 0.10 Vs-1, and the results are shown in Fig. 8. Upon addition of CT-DNA to complex solution, new redox peaks did not appear and the current intensity decreased, suggesting the existence of an interaction between each complex and CT-DNA. The drop of the voltammetric current in the presence of CT-DNA may be attributed to slow diffusion of the palladium(II) complexes bound to CT-DNA [61]. For the

addition of CT-DNA to complex solution both the cathodic Epc and the anodic Epa potentials of the complexes 2 and 3 exhibit a positive shift. The results suggested that the Pd(II) complexes possess intercalative mode of binding between the DNA base pairs. Appreciable changes in peak potential and current intensity clearly indicated intercalation mode of interaction between palladium(II) complexes and CT-DNA [62]. 3.4.4. Viscosity measurements To further investigate the binding mode between the synthesized complexes and DNA, viscosity measurements were done. Fig. 9 shows the relative viscosity change of DNA in the presence of ligand 1 complexes 2 and 3. It can be seen that upon increasing the amount of the compounds, the relative viscosity of CT-DNA gradually increases. These results reveal that 1, 2 and 3 produce a relatively apparent increase in DNA viscosity, which is consistent with DNA intercalative binding modes as suggested above [63, 64]. These DNA binding experimental results suggested that 1, 2 and 3 bind to CT-DNA by the intercalation mode. In the present study, the intercalation mode is due to the insertion of the H2L and Pd(II) complexes in between the DNA base pairs. This phenomenon assists an increase in the separation of base pair at intercalation sites and thus an increase in overall DNA length [65]. 3.5. Protein binding studies 3.5.1. UV-Visible spectra The electronic absorption spectroscopic measurement is a simple and effective method to study whether the quenching mechanism follow static or dynamic mode of interaction. Dynamic quenching only affects the excited states of the fluorophores and hence there are no changes in the absorption spectra. But static quenching usually leads to ground state complex formation which results in perturbation of the absorption spectrum of the fluorophore. The absorption spectrum of BSA shows two bands; a strong band around 200 nm is related to the absorption of protein backbone and a weak band around 278 nm that is due to the absorption of aromatic amino acids (Trp, Tyr, and Phe). The absorption spectra of BSA in the absence and presence of compounds are shown in Fig. 10. It shows that the intensity of BSA increases with a small blue shift of 2 nm. The results demonstrate that an interaction of compounds with BSA through static quenching and it was involved in the formation of the ground state complex of the type BSA-complex [66, 67].

3.5.2. Fluorescence spectroscopic studies Proteins are important chemical substances and major targets for many types of medicines. Studies on binding of metal complexes with proteins are becoming increasingly important for interpreting the metabolism and transporting processes. There are three types of intrinsic fluorophores in BSA, viz. tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). The indole groups of the tryptophan residues are the dominant source of emission in proteins. Changes in the emission spectra of tryptophan are common in response to protein conformational transitions, subunit associations, substrate binding, or denaturation. Therefore, the intrinsic fluorescence of proteins can provide considerable information on their structure and dynamics and is often utilized in the study of protein folding and association reactions. The binding of BSA with the compounds (1-3) was studied by fluorescence measurement at room temperature. A solution of BSA (1µM) was titrated with various concentrations of the compounds (0-25µM). The fluorescence of BSA at around 345 nm with a little blue shift was gradually quenched upon increasing the concentration of ligand and complexes Fig. 11. Upon the addition of the synthesized compounds to the solution of BSA resulted in a significant decrease in the fluorescence intensity of BSA at 345 nm. Up to 48.49%, 82.54% and 68.67% of the initial fluorescence intensity of BSA accompanied by a hypsochromic shift of 1 nm for the 1, 2 and 3 respectively have been observed. The observed hypochromicity with blue shift has revealed that all the complexes bind with the BSA protein [68]. Fluorescence quenching data were further analyzed with the Stern-Volmer equation and the Scatchard equation. From the plot of Fo/F versus [Q], the quenching constant (Kq) can be calculated (Fig. 12). It is assumed that the binding of compounds with BSA occurs at equilibrium and the equilibrium binding constant can be analyzed according to the Scatchard equation. log[ (Fo – F) / F ] = log Kbin + n log[Q] where Kbin is the binding constant of the compound with BSA and n is the number of binding sites. The binding constant (Kbin) and the number of binding sites (n) have been calculated from the plot of log[(Fo−F)/F] versus log[Q] (Fig. 13). The calculated Kq, Kbin, and n values are listed in Table 5. The values of n at room temperature are approximately equal to 1, for all of the compounds, indicating the existence of just a single binding site in BSA for ligand (1) and the complexes (2, 3). Moreover the larger values of Kq and Kbin indicate a strong interaction between the BSA protein and the complex over the ligand [69]. The observed

strong interaction of these new compounds (1-3) with BSA suggests that the complexes may be fit for anticancer studies. 3.6. Antioxidative activity The results obtained in all the above experiments strongly supported that the new synthesized compounds exhibited good DNA and BSA binding affinity. Hence, it is considered worthwhile to study the free radical scavenging activity of all the compounds. The antioxidant properties of hydrazone ligand and its Pd complexes have attracted a lot of interests, mainly in the in vitro systems [70]. The IC50 values of the ligand and new Pd complexes for the selected free radicals show interesting results and are presented in Table.6. The IC50 values indicated that the compounds have antioxidant activity in the order of 2>3>1. The ligand 1 displayed almost comparable radical scavenging activity with respect to standard antioxidant ascorbic acid (Aca). But the two Pd complexes exhibited higher activity when compared to their corresponding ligand as well as the standards, which clearly indicated that the Pd(II) chelation plays a vital role in determining the antioxidative properties. It was noted that compound 2 has good radical scavenging activity compared to 1 and 3. From the above results, the scavenging effect of the free ligand is significantly less when compared to their corresponding Pd(II) complexes which is due to the chelation of the organic ligand with the central metal Pd ion and the antioxidant activities of the complexes are in agreement with the reported Palladium(II) complexes [71]. 3.7. Cytotoxicity assay The cytotoxicity of the present compounds (1, 2, and 3) against human cervical (HeLa) and human breast (MCF-7) cancer cell lines was investigated by using MTT assay method and IC50 values are given in Table 7. The IC50 values showed that the new complexes 2 and 3 are significantly active against HeLa and MCF-7 with less toxicity to normal cells. However, it is to be noted that ligand 1 did not show any significant activity on the above cancer cells which confirmed that the chelation of the ligand with the Pd(II) ion is the only responsible factor for the observed cytotoxic properties of the complexes. The results indicate that the Pd complexes exhibited better antitumor activities against the human cancer cell lines which may be attributed to the extended planar structure induced by the π→π* conjugation resulting from the chelating of the Pd(II) with ligand and the cationic nature of the complexes [72]. Moreover, the complex containing triphenylphosphine as a co-ligand showed better cytotoxic effects than the complexes containing triphenylarsine [73]. The significant activity of 2 might be due to the presence of the phosphine ligand, which is supposed to provide a

better cytotoxicity by enhancing lipophilicity and consequently permeability through the cell membrane. In general, the order of activity of the synthesized compounds may be assigned as 2>3>1. Though the synthesized complexes were active against tumor cell lines under in vitro cytotoxicity experiments, the IC50 values are comparable with standard drug cisplatin. The lower efficacy of the current complexes may be due to the distorted square planar geometry [74]. Overall, cytotoxic behaviour of the Pd(II) complexes is very similar to that of the DNA/BSA binding activity as discussed earlier, particularly, 2 has better activity. 4. Conclusion In summary, two new Pd(II) complexes containing 4-hydoxy-benzoic acid (5-chloro2-hydroxy-benzylidene)-hydrazide

(H2L)

have

been

successfully

synthesized

and

characterized by various spectral and analytical techniques. The molecular structure of the H2L and complexes were confirmed by single crystal X-ray diffraction studies. The single crystal XRD results of complexes confirmed the distorted square planar geometry around the Pd ion through ONO coordination of 1. It was found that the Pd-complexes interact with CT-DNA and BSA protein more strongly than the ligand. The intercalation mode of binding of ligand and complexes with DNA was confirmed by DNA binding study. The antioxidant activity revealed that the Pd(II) complexes can act as potential antioxidants against DPPH, NO and OH radical. The in vitro cytotoxicity assay demonstrated that the Pd(II) complexes possess higher activity against HeLa and MCF-7 cancer cells. Based on the experimental results, it is concluded that the binding ability, antioxidant properties and cytotoxicity of complex 2 is higher than that of complex 3 and 1. Hence, it is concluded that the triphenylphosphine as co ligand led to an increased interaction with DNA/BSA, free radical and tumor cell line than the rest of the ligand and complex, which is may the liphophilic effect of triphenylphosphine in the complexes could enhance the biological activity.

Acknowledgements One of the authors, G.Raja gratefully acknowledges University Grants Commission (UGC), Hyderabad, India for financial assistance (NO.F MRP-5925/15 (SERO/UGC)). Supporting information: Electronic supplementary information (ESI) available: Crystallographic data for the structures reported

in

this

paper

have

been

deposited

with

the Cambridge

Crystallographic Data Centre (CCDC) as supplementary publication numbers (CCDC

1446484 CCDC 1456688 and CCDC 1456689, for 1, 2 and 3 respectively). Copies of the data can be obtained free of charge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel.: + 44-1223-336408; Fax: + 44-1223-336003; e-mail: [email protected] ; Web site http://www.ccdc.cam.ac.uk).

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Figure 1. ORTEP diagram of 1 with thermal ellipsoid at 50% probability

Figure 2. ORTEP diagram of 2 with thermal ellipsoid at 50% probability

Figure 3. ORTEP diagram of 3 with thermal ellipsoid at 50% probability

Figure 4. Electronic spectra of the compounds (1), (2) and (3) in Tris-HCl buffer upon addition of CT-DNA. [Compound] = 25µM, [DNA] = 0-50µM. Arrow shows that the absorption intensities decrease upon increasing DNA concentration

Figure 5. Plots of [DNA]/(εa-εf) versus [DNA] for compounds1-3 with CT-DNA.

Figure 6. Fluorescence quenching curves of ethidium bromide bound to DNA: 1, 2 and 3. [DNA] = 10µM, [EB] = 10µM, and [complex] = 0-50 µM. Arrow shows the emission intensity changes upon increasing complex concentration.

Figure 7. Stern-Volmer plots of the EB-DNA fluorescence titration for compound 1-3.

Figure 8. Cyclic voltammogram of complex 2 and 3 in the absence and presence (inner line) of DNA (10 µM). Scan rate: 100 mV s-1

Figure 9. Effect of the compounds (1-3) on the viscosity of CT-DNA.

Figure 10. Absorbance titrations of 1-3 with BSA.

Figure 11. Fluorescence titrations of 1-3 (0-20µM) with BSA (1µM).

Figure 12. Stern-Volmer plots of the fluorescence titrations of 1-3 with BSA

Figure 13. Plot of log[Q] versus log[(F0-F)/F]

Table 1. Crystal and structure refinement data. Compound Empirical formula Formula weight Temperature Wavelength (Å) Crystal system Space group Unit cell dimensions a (Å) b (Å) c (Å) α ) β ) γ ) Volume (Å3) Z Density (calculated) Mg/m3 Absorption coefficient mm-1 F(000) Crystal size/mm3 Theta range for data collection (°) Index ranges

Reflections collected Independent reflections Refinement method Data/restraints/parameters 2 Goodness-of-fit on F Final R indices [I > 2 σ (I)] R indices (all data) Largest diff. peak and hole e.Å-3

1 C14H11ClN2O3 290.70 296(2) K 0.71073 Triclinic Pna21

2 C32H24ClN2O3PPd 661.83 296(2) K 0.71073 Monoclinic P 21/c

3 C32H24ClN2O3AsPd 701.30 296(2) K 0.71073 Monoclinic P21/c

9.4265(4) 9.8423(6) 13.7746(8) 90 90 90 1277.99(12) 4 1.511 0.308 600 0.50 x 0.35 x 0.30 2.54 to 28.15

15.3009(5) 9.0238(3) 21.4522(6) 90 108.836(1) 90 2803.33(15) 4 1.568 0.852 1336 0.35 x 0.30 x 0.25 1.41 to 25.00

15.6707(6) 9.0731(3) 21.2193(8) 90 109.225(2) 90 2848.75(18) 4 1.635 1.935 1400 0.25 x 0.20 x 0.20 1.38 to 28.34

-12 ≤ h ≤ 7, -5 ≤ k ≤ 13, -18 ≤ l ≤ 18 5549 3011 [R(int)=0.0223] Full-matrix least2 squares on F 3011/1/194 0.936 R1 = 0.0356, wR2 = 0.0997 R1 = 0.0405, wR2 = 0.1054 0.256 and -0.265

-18 ≤ h ≤ 18, -10 ≤ k ≤ 10, -25 ≤ l ≤ 25 25268 4939 [R(int) = 0.0260] Full-matrix least2 squares on F 4939/0/362 1.245 R1 = 0.0313, wR2 = 0.0851 R1 = 0.0391, wR2 = 0.0989 0.794 and -0.784

-20 ≤ h ≤ 20, -12 ≤ k ≤ 12, -28 ≤ l ≤ 25 22049 6971 [R(int) = 0.0371] Full-matrix least2 squares on F 6971/0/361 1.076 R1 = 0.0355, wR2 = 0.0863 R1 = 0.0676, wR2 = 0.1154 0.639 and -0.938

Table 2. Selected bond lengths [Å] and bond angles [°] for ligand 1. Bond lengths 1 C(1) Cl(1) 1.740(2) C(7) N(1) 1.278(3) N(1) N(2) 1.379(2) C(8) O(3) 1.246(2) C(8)-N(2) 1.352(3) C(4) O(1) 1.356(3) C(12)-O(2) 1.359(2) Bond angles N(1)-C(7)-C(3) 119.25(17) O(3)-C(8)-N(2) 121.41(17) O(3)-C(8)-C(9) 122.05(17) N(2)-C(8)-C(9) 116.51(16) C(7)-N(1)-N(2) 118.54(15) C(8)-N(2)-N(1) 117.91(15) C(8)-N(2)-H(2N2) 126.1(19) N(1)-N(2)-H(2N2) 114.8(19) Table 3. Selected bond lengths [Å] and angles [°] of palladium(II) complexes 2 and 3. Bond lengths 2 Pd(1)-N(2) 1.981(2) Pd(1)-N(1) Pd(1)-O(1) 1.9967(19) Pd(1)-O(1) Pd(1)-O(2) 1.9603(19) Pd(1)-O(2) Pd(1)-P(1) 2.2830(7) Pd(1)-As(1) Bond angles N(1)-N(2)-Pd(1) 114.31(16) N(2)-N(1)-Pd(1) C(7)-O(1)-Pd(1) 110.24(17) C(7)-O(1)-Pd(1) O(2)-Pd(1)-N(2) 94.30(9) O(2)-Pd(1)-N(1) O(2)-Pd(1)-O(1) 172.82(8) O(2)-Pd(1)-O(1) N(2)-Pd(1)-O(1) 79.93(8) N(1)-Pd(1)-O(1) O(2)-Pd(1)-P 90.55(6) O(2)-Pd(1)-As(1) N(2)-Pd(1)-P 171.70(7) N(1)-Pd(1)-As(1) O(1)-Pd(1)-P 95.69(6) O(1)-Pd(1)-As(1)

3 1.969(3) 1.995(2) 1.958(3) 2.3906(5) 114.4(2) 110.2(2) 95.03(11) 174.08(10) 80.16(11) 89.60(8) 171.41(8) 95.61(7)

Table 4. DNA binding constant (Kb), quenching constant (Kq) and apparent binding constant (Kapp) values Compounds

Kb (M−1)

Kq (M−1)

Kapp (M−1)

1

4.3 × 103

4.49 × 104

3.3 × 105

2

3.2 × 104

6.98 × 104

5.2 × 105

3

2.8 × 104

5.45 × 104

4.0 × 105

Table 5. Quenching constant (Kq), binding constant (Kbin), and number of binding sites (n) for the interactions of compounds (1-3) with BSA Compounds

Kq (M−1)

Kbin (M−1)

n

1

2.9 × 104

1.7 × 105

1.10

2

8.6 × 104

3.5 × 105

1.20

3

4

5

1.31

6.3 × 10

2.7× 10

Table.6. Antioxidant activity of the ligand and new palladium(II) complexes with standard ascorbic acid. IC50 (µM) Compounds

DPPH∙

OH∙

NO∙

1

82.03

31.62

75.23

2

37.43

6.95

43.33

3

45.15

10.42

51.96

Ascorbic acid (Asc)

27.71

5.12

37.52

Table 7. Cytotoxic activity of the ligand and palladium(II) complexes against the HeLa and MCF-7 cancer cell lines. IC50values (µM) Compounds

HeLa

MCF-7

1

98.36

96.23

2

18.92

18.65

3

24.63

31.74

Cisplatin

16.21

15.35

Graphical abstract Palladium(II) hydrazone complexes were synthesized and characterized by spectral and single crystal XRD. Their DNA/BSA binding capability, antioxidant and in vitro cytotoxic potential were evaluated

Graphical Abstract

Highlights
Molecular structure of ligand and complexes was elucidated by X- ray diffraction study.
The ligand and complexes interact with CT-DNA and protein.
The complexes possess significant antioxidative property against DPPH, OH and NO radical.
The complexes showed higher cytotoxicity than the ligand against tumor cells.