Palladium (II) complexes containing substituted thiosemicarbazones. Synthesis, spectral characterization, X-ray crystallography, biomolecular interactions and in vitro cytotoxicity

Journal Pre-proof Palladium (II) complexes containing substituted thiosemicarbazones. Synthesis, spectral characterization, X-ray crystallography, biomolecular interactions and in vitro cytotoxicity D. Anu, P. Naveen, Nigam P. Rath, M.V. Kaveri PII:

S0022-2860(20)30026-0

DOI:

https://doi.org/10.1016/j.molstruc.2020.127703

Reference:

MOLSTR 127703

To appear in:

Journal of Molecular Structure

Received Date: 9 September 2019 Revised Date:

3 November 2019

Accepted Date: 7 January 2020

Please cite this article as: D. Anu, P. Naveen, N.P. Rath, M.V. Kaveri, Palladium (II) complexes containing substituted thiosemicarbazones. Synthesis, spectral characterization, X-ray crystallography, biomolecular interactions and in vitro cytotoxicity, Journal of Molecular Structure (2020), doi: https:// doi.org/10.1016/j.molstruc.2020.127703. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Palladium

(II)

complexes

containing

substituted

thiosemicarbazones.

Synthesis,

characterization, X-ray crystallography, biomolecular interactions and in vitro cytotoxicity. D. Anu, a P. Naveen, a Nigam P. Rath b and M. V. Kaveri a*

Spectral

Palladium (II) complexes containing substituted thiosemicarbazones. Synthesis, Spectral characterization, X-ray crystallography, biomolecular interactions and in vitro cytotoxicity. D. Anu, a P. Naveen, a Nigam P. Rath b and M. V. Kaveri a* a

Department of Chemistry, Bharathiar University, Coimbatore- 641046, India

b

Department of chemistry and biochemistry, University of Missouri, St. Louis.

Abstract Palladium (II) complexes were synthesised from 4(N)-substituted thosemicarbazones schiff base ligands. The new complexes were confirmed by analytical, various spectroscopic techniques and single crystal X-ray crystallography. Crystallographic studies exhibited that the complexes 2 and 3 distorted square planer geometry around palladium metal ion. All the complexes are proved their DNA/protein binding ability by using absorption and emission titrations. Investigations of antioxidant properties showed that all the complexes have significant radical scavenging properties. The anticancer activity of Pd(II) complexes was probed in vitro cytotoxicity against human breast (MCF7) and lung (A549) cancer cell lines by MTT assay. Further, AO/EB and DAPI staining methods were carried out to detect the cell death induced by the complexes. Among all the complexes, complex 1 exhibited better cytotoxic activity.

Keywords: Pd (II) complexes; Spectroscopy; Single crystal X-ray; DNA/ protein binding; anti-oxidant and cytotoxicity.

*

Corresponding author: Tel.: +91-422-2428313

E-mail address: [email protected] (M.V. Kaveri)

1

1. Introduction Heterocyclic thiosemicarbazones (TSCs) possess biological activity depending on their parent aldehyde and ketone. Schiff bases of thiosemicarbazones have been extensively investigated owing to their potential antibacterial, antiviral, antimalarial, antitumour activities [1,2]. Ligand plays an important role in the anticancer activity of metallodrugs because they can modify reactivity and lipophilicity [3]. Usually thiosemicarbazones possess two nucleophilic centres namely N-H and S-H in their thione or thiolate form and a polar double bond (C=N) [4]. Because of the similar coordination modes and chemical properties of palladium (II) complexes and platinum (II) complexes exhibit square planar geometry [5]. In general, platinum(II) complexes are thermodynamically and kinetically more stable than their analogues palladium(II) complexes. Palladium(II) complexes undergo aquation and ligand exchange reactions 105 times faster than the corresponding platinum(II) complexes [6]. A range of palladium(II) complexes have been investigated as potential antitumor drugs and some of the palladium(II) thiosemicarbazone complexes are tested and proved to be efficient compounds of pharmaceutical interest exerting cytotoxicity against the second most dangerous type of cancer called breast cancer, anti-mycobacterium tuberculosis activity. [79]. The interaction between DNA and transition-metal complexes is studied for the development of new drugs [10]. The mechanism of action of TSCs is due to their ability to inhibit the biosynthesis of DNA, possibly by blocking the enzyme ribonucleotide diphosphate reductase, binding to the nitrogen bases of DNA, hindering base replication or creating lesions in DNA strands by oxidative rupture [11,12]. Furthermore, binding of drugs with proteins have also attracted enormous research interest. The magnitude of albumin interactions with drug is essential, because it plays a dominant role in drug disposition and efficacy [13]. The bound drug can act as a depot, whereas unbound drug can produce the desired pharmacological effect. The interaction of proteins plays an important role in absorption, transportation and deposition of a variety of endogenous and exogenous substances [14,15]. Therefore, the interaction study of metal complexes with biomolecules like DNA and BSA becomes very important to develop new drugs. Cisplatin plays a major landmark in the history of metal-based anticancer drugs. Recently several palladium and platinum based thiosemicarbazone complexes have been reported [15-19].

2

In this present article, we describe new palladium(II) complexes with substituted thiosemicarbazones. The interaction of palladium(II) complexes with CT-DNA and BSA has also been studied. Further, in vitro free radical scavenging activity and cytotoxicity activity have been carried out. 2. EXPERIMENTAL 2.1 Materials and methods All the reagents used were analytical grade. They were purified and dried according to the standard procedure [20]. Melting points were measured in a Lab India apparatus. Infrared spectra were measured as KBr pellets on Jasco FT-IR (400- 4100 cm-1 range) at the Department of Chemistry, Bharathiar University, Coimbatore and India. Electronic absorption spectra of the compounds were recorded using JASCO V-630 spectrophotometer and emission measurements were carried out by using a JASCO FP-6600 spectrofluorometer. 1

H-NMR spectra were recorded in DMSO at room temperature by using Bruker 400 MHz

instrument and with chemical shift relative to tetramethylsilane. The chemical shifts are expressed in parts per million (ppm). CT-DNA, BSA and ethidium bromide (EB) were obtained from Himedia. (DPPH)1-diphenyl-2-picrylhydrazyl radical, sulfuric acid, sodium phosphate, ammonium molybdate, trichloroacetic acid were obtained from Sigma Aldrich. Anhydrous ferric chloride, potassium, ferricyanide, anhydrous sodium carbonate, phosphate buffer, ascorbic acid and all other chemicals were obtained from BDH Chemical Laboratory (England, UK). 2.2 Synthesis of the new palladium complexes 2.2.1 Synthesis of [Pd(DMsal-tsc)(AsPh3)2] (1) To a solution of [PdCl2(AsPh3)2] (0.050 g, 0.06 mmol) in toluene 40 mL, the ligand 4,6-dimethoxysalicyaldehyde-thiosemicarbazone [H2-DMsal-tsc](H2-L1) (0.01g, 0.06 mmol) and two drops of triethylamine were added. The mixture was heated under reflux for 6 h and kept at room temperature for crystallization. Yield: 76%, M.p. 218 °C, Anal. Calcd for C28H26AsN3O3PdS: C, 50.50; H, 3.94; N, 6.31; S, 4.82. Found: C, 50.46; H, 3.99; N, 6.38; S, 4.85%. FT-IR (cm-1) in KBr: 1592 (νC=N), 751 (νC-S), 1431, 1085, 680 cm-1 (for AsPh3) ; UVVis (DMSO), λmax: 262 nm (intra-ligand transition); 321 nm and 360 nm (LMCT s→d); 1H NMR (DMSO-d6, ppm): 8.23 (s, CH=N), 3.31-3.37 (d, OCH3), 6.20–6.67 (m, aromatic protons). ESI-MS, (m/z); 666.14. The above procedure was used to prepare other palladium arsine complexes. 3

2.2.2 Synthesis of [Pd(DMsal-mtsc)(AsPh3)2] (2) The complex 2 was prepared by the procedure used for 1, using [PdCl2(AsPh3)2] and 4,6dimethoxysalicyaldehyde-4(N)-methylthiosemicarbazone [H2-DMsal-mtsc](H2-L2) (0.02g, 0.06 mmol). Dark orange coloured crystals suitable for X-ray studies were obtained on slow evaporation. Yield: 69 %. M.p. 221 °C. Anal. Calcd for C29H28AsN3O3PdS: C, 51.23; H, 4.15; N, 6.18; S, 4.72. Found: C, 51.28; H, 4.20; N, 6.21; S, 4.70%. FT-IR (cm-1) in KBr: 1588 (νC=N), 783 (νC-S), 1446, 1101, 662 cm-1 (for AsPh3); UV- Vis (DMSO), λmax: 274 nm, (intra-ligand transition); 371 nm (LMCT s→d); 1H- NMR (DMSO-d6, ppm): 8.56 (s, CH=N), 7.11-8.33 (m, aromatic protons) 2.91 (s, CH3), 3.73 (d, OCH3). 2.2.3 Synthesis of [Pd(DMsal-etsc)(AsPh3)2] (3) Complex 3 was prepared by using [PdCl2(AsPh3)2] (0.050g, 0.06 mmol) and 4,6dimethoxysalicyaldehyde-4(N)- ethylthiosemicarbazone [H2-DMsal-etsc](H2-L3) (0.02g, 0.06 mmol) as describes earlier. Dark orange crystals suitable for X-ray studies were obtained on slow evaporation. Yield: 61 %, M.p. 256 °C. Anal. Calcd for C30H31AsClN3O3PdS: C, 49.33; H, 4.28; N, 5.75; S, 4.39. Found: C, 49.39; H, 4.21; N, 5.77; S, 4.42%. FT-IR (cm-1) in KBr: 3412 (νOH), 1597, (νC=N), 745 (νC-S), 1402, 1099, 671 cm-1 (for AsPh3) ;UV- Vis (DMSO), λmax: 268 nm (intra-ligand transition), 310 nm and 376 nm (LMCT s→d);1H NMR (DMSOd6, ppm): 11.57 (s, OH), 8.54 (s, CH=N), 3.34-3.68 (d, OCH3), 6.68-8.30 (m, aromatic protons), 2.32-2.730 (d, CH2). 2.2.4 Synthesis of [Pd(DMsal-ptsc)(AsPh3)2] (4) Complex 4 was prepared by the reaction of [PdCl2(AsPh3)2] (0.050g, 0.06 mmol) and 4,6dimethoxysalicyaldehyde-4(N)-phenylthiosemicarbazone

[H2-DMsal-ptsc](H2-L4)

0.06 mmol) as described earlier. Yield: 63 %, M.p. 262 °C.

(0.02g,

Anal. Calcd for

C34H31AsClN3O3PdS: C, 52.46; H, 4.01; N, 5.40; S, 4.12. Found: C, 52.49; H, 4.09; N, 5.46; S, 4.19%. FT-IR (cm-1) in KBr: 3446 (νOH), 1599 (νC=N), 768 (νC-S), 1438, 1088, 647 cm-1 (for AsPh3);UV- Vis (DMSO), λmax: 285 nm (intra-ligand transition); 375 nm and 392 nm (LMCT s→d),; 1H NMR (DMSO-d6, ppm): 11.69 (s, OH), 8.07 (s, CH=N), 3.15-3.18 and 3.32-3.39 (2d, OCH3), 6.01-7.67 (m, aromatic protons). ESI-MS, (m/z); 778.20. 2. 3 X-ray Crystallography: Single crystal data collection was performed using a Bruker X8 kappa Apex II charge Coupled Device (CCD) Detector single crystal X-Ray diffractometer equipped with an Oxford Cryostream LT device. All the data were collected using graphite mono chromated Mo-Kα (λ = 0.71073 Å) from a fine focus sealed tube X-Ray source [21]. Apex II and

4

SAINT software packages were used for data collection and data integration [22]. Structure solution and refinement were carried out using the SHELXTL-PLUS software package [23]. 2.4 Binding studies 2.4.1 DNA binding study The CT DNA binding studies were carried out in deionised water with the buffer tris(hydroxymethyl)-aminomethane (Tris, 5 mM) and sodium chloride (50 mM) and pH adjusted to 7.2 with hydrochloric acid at room temperature. The concentration of CT-DNA was determined by UV absorbance at 260 nm. Solutions of CT-DNA in Tris-HCl buffer gave a ratio of UV absorbance at 260 and 280 nm, A260/ A280, of approximately 1.9, indicating that the DNA was sufficiently free of protein. The molar absorption coefficient, ℇ260, was taken as 6600 M−1 cm−1. Various concentrations of CT-DNA were added to the ligand and complexes (25 µM dissolved in DMSO/Tris HCl buffer, 1 % DMSO in the final solution). While measuring the absorption spectra, an equal amount of DNA was added to both the test solution and the reference solution to eliminate the absorbance of DNA itself. Control experiments with DMSO were performed and no changes in the spectra of CT-DNA were observed. Absorption spectra were recorded after equilibrium at 20 °C for 5 min. The intrinsic binding constant Kb was determined by using following equation (1), [24-26]. [DNA]/[εa-εf]) = [DNA]/[εb-εf]+1/Kb[εb-εf]

(1)

The absorption coefficients εa, εf and εb correspond to Aobsd /[DNA], the extinction coefficient for the free compound and the extinction coefficient for the compound in the fully bound form respectively. The intrinsic binding constant Kb can be obtained from the ratio of the slope to the intercept from the plot of [DNA]/[εa −εf] versus [DNA]. In order to find out the mode of attachment of CT DNA to the compounds, fluorescence quenching experiments with EB-DNA were carried out by adding the complexes to Tris-HCl buffer solution of EB-DNA. The change in the fluorescence intensity was recorded. Before measurements, the system was shaken well and incubated at room temperature for 5 min. 2.4.2 Serum albumin binding study Bovine Serum Albumin (BSA) was purchased from HiMedia, The protein binding study was performed by tryptophan fluorescence quenching experiments using bovine serum albumin (BSA, 10 µM) as the substrate in phosphate buffer (pH = 7.2). Quenching of the emission intensity of tryptophan residues of BSA at 346 nm (excitation wavelength at 280 nm) was monitored using ligand and complexes as quenchers with increasing concentration (10-100µM) [27]. Synchronous fluorescence spectra of BSA with various concentrations of 5

the complexes were obtained from 300 to 400 nm when ∆λ = 60 nm and from 290 to 500 nm when ∆λ = 15 nm. For synchronous fluorescence spectra, the same concentrations of BSA and the compounds were used and the spectra were measured at two different ∆λ values such as 15 and 60 nm [28,29]. 2.5 Antioxidant assay The antioxidant activity of the complexes were determined by 2,2'-diphenyl-1picrylhydrazyl (DPPH) radical scavenging activity, ABTS•+ scavenging activity and total antioxidant activity. 2.5.1 (DPPH) 2,2'-diphenyl-1-picrylhydrazyl radical scavenging activity DPPH radical scavenging activity of the complexes were measured according to the method of Blois [30]. While scavenging free radical, the antioxidants donate hydrogen and form a stable DPPH-H molecule. Complexes in 1ml of 10% DMSO were added to 5 ml of DPPH (0.1mM in MeOH) and were mixed rapidly. The solution was incubated at 37°C for 30 min in the dark. The decrease in the absorbance of DPPH was measured at 517 nm. BHT and ascorbic acid were used as a positive control. 2.5.2 ABTS•+ scavenging activity The ABTS•+ scavenging activity of the samples were determined according to the method reported in the literature [31]. ABTS•+ was produced by reacting 7 mM ABTS aqueous solution with 2.4 mM potassium persulphate in the dark for 12-16 h at room temperature. Prior to assay, this solution was diluted in ethanol (1:89, v/v) and equilibrated at 25 °C for the absorbance (0.700±0.02) at 734 nm. The stock solution of the samples were diluted such that after introduction of 100 µL of sample or trolox aliquots into the assay, they produced between 20 and 80% inhibition of the blank absorbance. About 1 mL of diluted ABTS solution was added to 30-50 µL of sample or trolox (final concentration 0.1-5 mM) and incubated at 30 °C exactly for 30 min. Triplicate determinations were made at each dilution of the standard, at 734 nm and the graph was plotted as a function of trolox concentration. The total antioxidant activity (TAA) was defined as the concentration of trolox having equivalent antioxidant activity expressed as mM TE/g of samples. 2.5.3 Total antioxidant activity Total antioxidant activity was determined by -green phosphomolybdenum method [32]. Triplicates of 100 µl of sample and different concentrations of standard (ascorbic acid in 1 mM dimethyl sulphoxide) were added with 3 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) in a test tube. The test tubes 6

were incubated in a water bath at 95 °C for 90 min. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695 nm against the reagent blank. A typical blank solution contained 3 mL of reagent solution and water in place of sample, and it was incubated under the same conditions as the rest of the sample. The synthetic antioxidant (BHT) and natural antioxidant (ascorbic acid) were used as standards. The results were reported as mean value of percentage of inhibition using the formula (Eqn 2), I% = (Ablank – Asample)/(Ablank)*100 … … …(2) Where Ablank is absorbance of control reaction, Asample is absorbance of the test sample. 2.6 Cytotoxicity The procedure for evaluation of Cytotoxicity (MTT assay) and fluorescence microscopic analysis of apoptotic cell death are given in supporting information. 3. Result and Discussion 3.1 Synthesis and characterization The stoichiometric reactions of [PdCl2(AsPh3)2] with dimethoxy thiosemicarbazones ligands in 1:1 toluene resulted in the formation of new complexes, where the ligand acted as a bidentate NS and also tridentate ONS donor. The analytical data confirm the stoichiometry of the complexes. The structure of complexes 2 and 3 were confirmed through single crystal Xray crystallographic studies. All the complexes are stable and soluble in organic solvents such as

methanol,

ethanol,

dichloromethane,

dimethylsulfoxide.

7

chloroform,

dimethylformamide

and

Scheme 1 Preparation of new Pd(II) Complexes 3.2 Spectroscopic Studies IR Spectroscopy The IR spectra of the ligands H2L1-H2L4 and their corresponding complexes (1-4) provided information about the metal ligand bonding. The ligands (H2L1-H2L4) showed a strong absorption at 1611 -1633 cm-1 corresponding to ν(C=N), which was shifted to lower frequency (1588-1599 cm-1) in the complexes indicating that the azomethine nitrogen is involved in coordination with palladium metal ion [33,34]. Further, a broad band around in the spectra of the ligands 3415-3434 cm-1 indicates the presence νOH. This band was observed at 3412 and 3446 cm-1 in complex 3 and 4 indicating the non-participation of phenolic oxygen in metal ion coordination. Whereas, in complex 1 and 2 νOH completely disappeared indicating the coordination to palladium ion after deprotonation [35]. A band at 812-820 cm-1 assigned to ν(C=S) in free ligands was completely disappeared in all the complexes and a new band appeared at 745-783 cm-1 due to ν(C-S) showing the enolisation of NH-C=S group and subsequent coordination through the deprotonated sulphur atom [36,37]. In addition, the characteristic absorption bands corresponding to triphenylarsine were also present in the expected region [38]. 8

Electronic Spectroscopy The UV-visible spectra of the palladium complexes have been recorded in DMSO solvent. All the complexes displayed two to three absorptions bands in the region 262-392 nm. The bands around 262-295 nm correspond to the intraligand transition [39,40]. However, the absorption bands around 310-392 nm correspond to LMCT (s→d) transition.[41] 1

H-NMR Spectroscopy The 1H NMR spectra of free ligands H2L1-4 and the corresponding complexes 1-4

have been recorded in DMSO solvent and displayed all the expected signals (Fig. S1-S8). In the spectra of the free ligands H2L1-4, a singlet appeared around δ 9.20-δ 10.22 ppm is corresponding to N(2)HCS proton [42]. However, in the spectra of the complexes (1-4), there was no resonance attributable to N(2)H, indicating the coordination of ligands in the anionic form after deprotonation at N(2). In the free ligands a sharp singlet at δ 11.19–δ 11.29 ppm is assigned to the phenolic (-OH) proton. This was completely disappeared in the spectra of complexes 1 and 2 confirming the coordination of phenolic oxygen to palladium metal ion. But, in the spectra of complexes 3 and 4, a singlet appeared at δ 11.57 and δ 11.69 ppm showed the non-participation of phenolic oxygen in metal ion coordination [43]. A multiplet appeared around at δ 6.10-δ 7.70 ppm are corresponding to aromatic protons of free ligands, complexes and also triphenylarsine protons. A doublet corresponding to the –OCH3 protons was also found around δ 3.15-δ 3.73 ppm for both ligands and complexes. The singlets due to azomethine (-CH=N-) proton observed in the range δ 8.50-δ 8.53 ppm in the free ligands were found in the range δ 8.07-δ 8.56 ppm in all the complexes. This supports the coordination of azomethine nitrogen to the palladium ion [44]. In addition, a doublet was observed δ 2.90 and δ 2.91 ppm in ligand H2L2 and the complex 2 due to the terminal methyl protons. Further, a multiplet around δ 2.32-δ 2.73 ppm in H2L3 and 3 correspond to the methylene protons of ethyl group. The ESI-MS data for complexes 1 and 4 are shown in Fig. S9 and S10. The complexes displayed their molecular-ion peaks at m/z 666.14 and 778.20 respectively. 3.3 X-ray crystallography The structures of the newly synthesized palladium (II) complexes 2 and 3 were determined by the single crystal X-ray diffraction studies (Fig. 1 and Fig. 2). The summary of data collection and the refinement parameters are given in Table 1. The selected bond

9

lengths and bond angles are given in Table 2 and hydrogen bonding parameters are given in Table S1. Complex 2 crystallized in the monoclinic space group P21/n whereas, complex 3 crystallized in the triclinic space group P-1. In complex 2, the palladium (II) ion is coordinated to the tridentate ligand through the sulphur atom with Pd(1)-S(1) bond distance of 2.238 (14) Å, phenolic oxygen with Pd(1)-O(1) bond distance of 2.009 (3) Å and the nitrogen atom with Pd(1)-N(1) bond distance of 1.999 (4) Å respectively. The remaining binding site is occupied by triphenylarsine with Pd(1)-As(1) bond distance of 2.378 (7) Å. The triphenylarsine in the complex, N(1) nitrogen atom and thiolate sulphur (S1) phenolic oxygen (O1) are mutually trans to each other. The trans angles [O(1)-Pd(1)-S(1) is 176.66 (10)°] and [N(1)-Pd(1)-As(1) is 175.25 (12)°] deviate considerably from the ideal angle of 180° causing distortion in the square planar geometry.[45] In complex 3 the palladium (II) ion is coordinated through the thiolate sulphur atom with Pd(1)-S(1) bond distance of 2.246 (5) Å and nitrogen atom with Pd(1)-N(1) bond distance of 2.063 (12) Å. The remaining sites are occupied by a chlorine atom with Pd(1)Cl(1) bond distance of 2.337 (4) Å and triphenylarsine with Pd(1)-As(1) bond distance of 2.355 (2) Å respectively. The triphenylarsine in the complex N(1) nitrogen atom and thiolate sulphur (S1) chlorine atom (Cl) are mutually trans to each other. The trans angles [S(1)Pd(1)-Cl(1) is 173.29 (17)°] and [N(1)-Pd(1)-As(1) is 175.8 (4)°] deviate considerably from the ideal angle of 180° causing distortion in the square planar geometry of the complex. The bond distances and angles between palladium and the coordinated atoms are similar to the reported Pd(II) complexes.[46,47]. In complex 3 hydrogen bonding interaction is found with the donor-acceptor distance of 2.580 (17)° corresponding to the N(2)-O(1).

10

Fig. 1 ORTEP diagram of complex 2

Fig. 2 ORTEP diagram of complex 3

Table 1.Crystallographic data for the complexes (2) and (3) Complex 2

11

Complex 3

CCDC No

1947910

1947911

Empirical formula

C29 H28 As N3 O3Pd S

C30 H31 As Cl N3 O3Pd S

Formula weight

679.92

730.41

Temperature

100K

100 K

Wavelength

0.71073

0.71073

Crystal system

monoclinic

Triclinic

Space group

P21/n

P-1

A

13.718(4) Å

9.265(5) Å

B

7.890(2) Å

9.827(6) Å

C

26.136(7) Å

17.361(10) Å

α

90 °

98.068(14)°

β

92.450 (7)°

102.426(14)°

γ

90 °

105.056(13)°

Volume

2826.3(13) Å3

1458.0(15) Å3

Z

4

2

Calculated density

1.598 Mg/m3

1.664 Mg/m3

Absorption coefficient

1.928 mm-1

1.963 mm-1

F(000)

1368

736

Crystal size

0.246 x 0.179 x 0.082 mm

0.258 x 0.166 x 0.042 mm

Theta range for data collection

1.560 to 26.411°

2.194 to 24.996°

-11≤h≤17, -9≤k≤9, -32≤l≤32

-10≤h≤6, -11≤k≤11, -

Limiting indices

20≤l≤20

Reflections collected / unique

20301 / 349

8827/ 358

Absorption correction Max. and min. transmission

0.7454 and 0.6499

0.6937 and 0.4683

Full-matrix least-squares on F2

Full-matrix least-squares on

Refinement method

F2

Data / restraints / parameters

5789 / 1/ 349

4935 / 0 / 358

Goodness-of-fit on F

1.019

0.960

Final R indices [I>2sigma(I)]

R1 = 0.0484, wR2 = 0.0793

R1 = 0.0894, wR2 = 0.1465

R indices (all data)

R1 = 0.0913, wR2 = 0.0914

R1 = 0.2314, wR2 = 0.1956

Largest diff. peak and hole

0.683 and -1.039 e.Å-3

1.393 and -1.419 e.Å-3

2

Table 2 Bond lengths [Å] and angles [°] of the complexes 2 and 3 Atoms

Complex 2 12

Complex 3

Pd(1)-N(1)

1.999(4)

2.063(12)

Pd(1)-O(1)

2.009(3)

-

Pd(1)-S(1)

2.2383(14)

2.246(5)

Pd(1)-As(1)

2.3782(7)

2.355(2)

Pd(1)-Cl(1)

-

2.337(4)

N(1)-Pd(1)-O(1)

92.79 (15)

-

N(1)-Pd(1)-S(1)

85.04 (12)

84.0 (4)

O(1)-Pd(1)-S(1)

176.66 (10)

-

N(1)-Pd(1)-As(1)

175.25 (12)

175.8 (4)

O(1)-Pd(1)-As(1)

90.05 (10)

-

S(1)-Pd(1)-As(1)

92.28 (4)

91.88 (13)

N(1)-Pd(1)-Cl(1)

-

95.7 (4)

S(1)-Pd(1)-Cl(1)

-

173.29 (17)

Cl(1)-Pd(1)-As(1)

-

88.44 (12)

3.4 DNA binding studies Electronic absorption spectroscopy is used to determine the binding ability of metal complexes with DNA. Compounds that bound to DNA through intercalation mode show bathochromic shift. The absorption spectra of complexes (1-4), in the presence and absence of CT-DNA are shown in Fig. 3. While increasing the concentration of DNA, the absorption bands of complex 1 show hypochromism of about 24.8% and 6.5% with red shifts of 1 and 0 nm, respectively at 252 and 331 nm. The absorption bands of complex 2 show hypochromism of about 14%, 18% and 14.6% with red shifts of 4, 0 and 1 nm, respectively at 248, 343 and 389 nm. However, the absorption bands of complex 3 show hypochromism of about 20.6% and 34.6% with red shift of 4 and 0 nm, respectively at 249 and 332 nm. In complex 4 the absorption bands show hypochromism of about 16.5% and 24.4% with red shifts of 2 and 3 nm respectively, at 250 and 361 nm. These results suggest that test compounds bind to the DNA helix via intercalation. [48,49] The intrinsic binding constant Kb was calculated by the changes monitored in absorption bands with increasing concentration of DNA using the equation, [DNA]/[Ԑa-Ԑf] =[DNA]/ [Ԑa-Ԑf] + 1/Kb [Ԑa-Ԑf].

(3)

Kb is obtained from the ratio of slope to the intercept of [DNA] versus [DNA]/[Ԑa-Ԑf] plots (Fig. S11). The binding constant value clearly shows that complex 1 binds strongly with CT13

DNA than the rest of the complexes.

Fig. 3 Electronic spectra of the new complexes (1-4) upon addition (5-50 µM) of CT-DNA (trisHCl buffer, pH 7.2). Arrows show the absorption intensity decrease upon increasing DNA concentration. Table 3. The Kb and Ksv values for the interaction of complexes (1-4) Kb/M-1

Ksv/M-1

1

5.02±0.209×105

3.26±0.721×103

2

1.26±0.825×105

1.91±0.680×103

3

1.26±0.825×105

1.97±0.716×103

4

3.00±0.200×104

2.54±1.056×103

Complex

3. 5 Competitive binding studies (EB) EB-DNA complex is used to study the binding mode of DNA and only measures their ability to influence the EB luminescence intensities in the EB-DNA complex. EB emits intense fluorescent light in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs. The fluorescence intensity of EB-DNA will be reduced by addition

14

of second molecule (complexes) that could replace EB from the bound EB-DNA complex. The fluorescence emission spectra of EB bound to DNA in the absence and presence of the complexes are shown in Fig. 4. The figure clearly shows that reduction in fluorescence intensity of about 25.6%, 24.1%, 22.9% and 27.3% with hypsochromic shifts of 1, 1, 0 and 0 nm for complexes 1-4 respectively. By using Stern- Volmer equation Ksv values can be obtained. Io/Icorr = Ksv[Q] +1

(4),

Where Io is the emission intensity in the absence of compounds, Icorr is the corrected emission intensity in the presence of compound, Ksv is the quenching constant, and [Q] is the concentration of the compound. In order to correct the inner filter effect, the following Eqn.(5) is used Icorr = Iobs * 10(Aexc+Aem)/2

(5),

Where Icorr is the corrected fluorescence value, Iobs is the measured fluorescence value, Aexc the absorption value at the excitation wavelength and Aem is the absorption value at the emission wavelength. The Stern-Volmer quenching constant (Ksv) were calculated for the complexes 1-4 from the slope of Io-Icorr versus [Q] shown in Fig. 5 and values are given in Table 3. From the results, we can notice that the complexes can replace EB from the EB-DNA complex. The binding of palladium complexes to CT-DNA leads to a marked increase in the emission intensity, which is in conformity with those observed for other intercalators. [50,51] The higher binding affinity of the Pd(II) complexes is due to the extension of the π system of the intercalated ligand on coordination.

15

Fig. 4 Emission spectra of DNA-EB system (λexi = 515 nm, λem = 530-750 nm) in the presence of complexes (1-4). [DNA] = 10 µM, [Compounds] = 10-100 µM, [EB] = 10 µM. Arrow shows the emission intensity changes upon addition of complex concentration. 1.3 1.2

Io-I

1.1 1.0

Complex 1 Complex 2 Complex 3 Complex 4

0.9 0.8 0

20

40

60

80

100

Q

Fig. 5 Stern-Volmer plots of EB-DNA fluorescence titration of the complexes (1-4)

16

3. 6 Protein binding studies The fluorescence quenching mechanisms are usually classified as static and dynamic quenching. UV- visible absorption spectroscopy is the simple way to find out the type of quenching. Dynamic quenching refers to the fluorophore and the quencher come into contact during the transient existence of the excited state. Static quenching refers to fluorophorequencher complex formation. In the presence and absence of BSA with all the complexes are shown in Fig. S12. This shows the possible quenching mechanism of BSA by the complex is static quenching. [52] In order to investigate the binding of complexes 1-4 with BSA, fluorescence spectra were recorded. A solution of BSA (10 µM) was titrated with various concentration of the compounds (10-100 µM). Fluorescence spectra were recorded in the range of 290-550 nm upon excitation at 280 nm. The effects of complexes on the fluorescence emission spectrum of BSA are shown in Fig. 6. While adding complexes to the solution of BSA, there is significant decrease of the fluorescence intensity of BSA at 350 nm up to 59%, 41.2%, 31.9% and 43.2% of the initial fluorescence intensity of BSA by a small red shift of 3, 0, 0, 1 nm for complexes 1-4 respectively. The results suggested the interaction of the compounds with the BSA protein.[53,54] According to the Stern-Volmer equation (2) quenching constant Ksv obtained from the plot of Io/I versus [Q] was found for all the complexes (Table S2 and Fig. 7). When small molecules bind with the active site of BSA, the binding constant and number of binding sites can be calculated by using Scatchard equation (6). log [(Fo-F)/F] = log Kb + n log [Q]

(6),

Where Kb is the binding constant of quencher with BSA, n is the number of binding sites, Fo is the fluorescence intensity in the absence of the quencher and F is the fluorescence intensity in the presence of the quencher. The Kb can be determined from the plot of log [(FoF)/F] versus log [Q] (Fig. S13) The values of “n” indicate the existence of just a single binding site of BSA is involved. The larger values of Kq and Kb indicate a strong interaction between the BSA protein and the complexes.[55]

17

Fig. 6 The emission spectra of BSA (10 µM; λexc = 280 nm; λemi = 346 nm) in the presence of increasing amounts of new complexes (1-4) (10 – 100 µM). The arrow shows the emission intensity changes upon addition of complexes concentration.

2.6

Complex 1 Complex 2 Complex 3 Complex 4

2.4 2.2 2.0

Io-I

1.8 1.6 1.4 1.2 1.0 0.8 0

20

40

60

80

100

Q

Fig. 7 Stern–Volmer plot of the BSA fluorescence titration of the complexes (1-4).

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Synchronous fluorescence spectra Synchronous fluorescence spectra provide information on the microenvironment, particularly in the vicinity of the fluorophore functional groups. The fluorescence of BSA is due to the presence of tyrosine and tryptophan residues. According to miller, in the synchronous fluorescence spectroscopy the difference between the excitation and emission wavelength (∆λ=λemi-λex) indicates the type of chromophores.[56] The ∆λ of 60 nm shows the characteristic of tryptophan residue while ∆λ of 15 nm shows tyrosine residue. The synchronous spectra of BSA with various concentration of complexes (1-4) are shown in Fig. S14. The synchronous fluorescence spectral study clearly indicates that the fluorescence intensity of both tryptophan and tyrosine was affected with increasing concentration of the complexes. The result shows that the interaction of complexes with BSA affects the micro region of both tyrosine and tryptophan.[57] The strong interaction of these complexes with BSA makes the complexes suitable for further biological study. Antioxidant activity The antioxidant activity of complexes (1-4) was evaluated in a series of in vitro assays involving ABTS•+, DPPH and total antioxidant activity. In humans, free radicals damage the DNA helix and lead to various diseases such as cataract, cancer, rheumatism etc. The radical scavenging activity of the palladium complexes along with standards such as ascorbic acid and BHT in a cell free system, has been examined with reference to DPPH, ABTS•+ and total antioxidant activity. The power of compounds to scavenge various radicals was found to be slightly higher than that of standards. DPPH radical scavenging assay The DPPH radical scavenging activity of complexes (1-4) is represented in Fig. 8. In DPPH assay, the violet colour DPPH solution produces an absorption band at 517 nm in the visible spectrum. Complex 3 has higher potential to reduce the stable DPPH radical, may be due to the ethyl substitution in the terminal nitrogen of the ligand, which is higher than that of standards (BHT and ascorbic acid). The DPPH radical scavenging of new palladium (II) complexes is better when compared to other similar reported palladium complexes [58].

19

Fig. 8 DPPH radical scavenging activity of Pd(II) complexes. ABTS•+ cation radical scavenging assay The antioxidant activities of the complexes were determined by reading the absorbance of the test solution at 734 nm and expressed in percentage of inhibition. [59] (Fig. 9). Complex 2 shows higher radical scavenging activity may be due to the methyl substitution in the terminal nitrogen of the ligand than ascorbic acid and lower than that of BHT.

Fig. 9 ABTS cation radical scavenging activity of Pd(II) complexes. Total antioxidant activity The antioxidant mechanism involves electron transfer, which is dependent on the structure of antioxidant agent. The complexes (1-4) were tested in 100 µM fixed concentration (Fig. 10). The data suggest that they may find themselves as promising antioxidant agents in future drug development strategy [60].

20

Fig. 10 Estimation of Total antioxidant capacity of new Pd(II) complexes. Cytotoxic assay The in vitro cytotoxic activity of the palladium complexes (1-4) was carried out against both human breast cancer (MCF-7) and human lung cancer (A-549) by using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [Fig. 11 and Table S3]. The new Palladium complexes (1-4) were analysed by means of cell inhibition expressed in IC50 values and were found as 36± 1.0, 27±1.0, 35±1.0 and 29±1.0 for MCF-7 cell lines. In case of A-549 cell line the IC50 values were found to 20± 1.5, 23± 1.5, 24± 1.5 and 23± 1 for complexes 1-4 respectively. All complexes shows moderate activity compared to the standard Cisplatin. [61,62] In addition, the coordination of the ligands to the Pd(II) ion enhances the anti-proliferative activity of the complexes against both the cell lines.

Fig. 11 MTT assay for new complexes (1-4) 21

Fluorescence microscopy analysis and nuclear fragmentation Acridine orange/Ethidium bromide (AO/EtBr) staining method The apoptotic cell death was analysed by fluorescence microscopic. Acridine orange (AO) stained apoptotic and viable cells emit green fluorescence when bound to double stranded DNA and red fluorescence to single stranded RNA.[63] Fluorescence microscopic analysis revealed morphological changes in both the cell lines MCF-7 and A-549, which confirmed that all the complexes (1-4) are able to induce apoptosis in MCF-7 and in A-549 cell lines. The observed morphological changes are due to the interaction of the palladium complexes with the tested cell lines. Control cells did not show any cell death.

Fig. 12 AO/EB stained A-549 and MCF-7 (control and cells treated with complexes (C1-C4) cells at 24 h incubation. The yellow or orange colour cells show early apoptotic cells. DAPI staining method DAPI is a blue fluorescent dye that preferentially stains DNA by strongly binding to adenine-thymine-rich regions, whose penetration in the live cells doesn’t cause much fluorescence, whereas its penetration through the damaged cell membrane results in effective staining. When bound to DNA, it has an absorption maxima at a wavelength of 358 nm and emission at 461 nm. Therefore for fluorescence microscopy, DAPI is excited with UV light

22

and is detected through a blue/cyan filter. Hence, we examined the effect of new palladium complexes (1-4) on both the cell lines (MCF-7 and A-549) by using DAPI staining method. Fluorescence microscopy images of both cancer cell lines in presence of all the complexes are shown in Fig. 13. From the figure, it is evident that blue fetches showed that all the complexes exhibited higher level of nuclear fragmentation.[64] MCF-7 cell line has higher level fragmentation compared to A-549 cell line.

Fig. 13 DAPI (MCF-7) and (A-549) (control and cells treated with complexes (C1-C4) after 24 h incubation. The blue fetches show nuclear fragmentation in the cancer cells. CONCLUSION The present article describes the synthesis of palladium(II) complexes of 4(N)substituted thiosemicarbazones. The synthesized ligands and complexes were characterised by using analytical and various spectroscopic studies. Complex 2 and 3 were structurally characterised and exhibited distortion square planar geometry. All the complexes were found through intercalation with CT-DNA. Static type of quenching mechanism has been established for all the complexes and bound well with BSA albumins. All the complexes proved their effectiveness in radical scavenging activity. MTT assay performed by using

23

MCF-7 and A-549 cells indicated the cytotoxic nature of all the. Further, AO-EB and DAPI staining assays recommended an apoptotic type of cell death when the cancer cells are treated with all the complexes.

Acknowledgement: The author D.A. greatly acknowledged UGC, New Delhi, India, for UGC-BSR fellowship (F.25-1/2014-15(BSR)7-26/2007/(BSR) Dated: 05.11.2015. Crystallographic data for complexes have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication (CCDC No. 1947910 (complex 2) and 1947911 (complex 3). The data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving html of from Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK [Fax : +44-1223/336-033; email : depositccdc.cam.ac.uk].

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Highlights


Four new Pd(II) complexes have been synthesized.




The structure of the complexes 2 and 3 were confirmed by X-ray crystallography.




The DNA/protein binding studies were carried with CT-DNA and BSA as models.




Complexes exhibited significant free radical scavenging activities.




The anticancer activity of the Pd(II) complexes was probed in vitro cytotoxicity against human breast (MCF7) and lung (A549) cancer cell lines by MTT assay and cytological changes observed in (AO/EB and DAPI) staining method.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper