Novel palladium(II) complexes of N-(5-nitro-salicylidene)-Schiff bases: Synthesis, spectroscopic characterization and cytotoxicity investigation

Journal of Molecular Structure 1207 (2020) 127852

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Novel palladium(II) complexes of N-(5-nitro-salicylidene)-Schiff bases: Synthesis, spectroscopic characterization and cytotoxicity investigation a, * € € , Perihan Gürkan a, Yaprak Dilber S¸imay Demir b, Mustafa Ark c Ozlem Ozdemir a b c

Department of Chemistry, Faculty of Science, Gazi University, 06500, Ankara, Turkey Department of Pharmacology, Faculty of Medicine, Hitit University, 19040, Çorum, Turkey Department of Pharmacology, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 November 2019 Received in revised form 3 February 2020 Accepted 4 February 2020 Available online 5 February 2020

A series of palladium(II) complexes (1ee3e) with the general formulae [Pd(L)(H2O)]$xH2O were newly synthesized by the interaction of palladium(II) chloride and the monosodium salts of N-(5-nitro-salicylidene)-Schiff base ligands (1ae3a) in aqueous DMF solution. The identities of all the complexes were proven by elemental analysis, FTIR, 1H, 13C NMR, LCeMS, UVevis, XPS, powder XRD spectra, thermal analysis, conductivity and magnetic susceptibility measurements. The obtained analytical and physicochemical results exhibited a square-planar coordination of the palladium(II) ion having a double deprotonated ligand and a coordinated water molecule. In vitro cytotoxicity of these Pd(II) complexes was screened against tumor cell lines (HeLa and MCF-7), and a normal human cell line (HEK-293). The complexes 1e and 2e exhibited a moderate antitumor activity against HeLa cell lines, while 3e had better activity than standard anticancer drug, doxorubicin. All three complexes showed the best active cytotoxicity than doxorubicin against MCF-7 cancer lines. For HEK-293 lines, a decrease in concentration of the complexes significantly decreased their toxicity. © 2020 Elsevier B.V. All rights reserved.

Keywords: Schiff bases Palladium(II) complexes Cytotoxicity HeLa MCF-7 HEK-293

1. Introduction Cancer is a group of diseases characterized by uncontrolled cell proliferation and disruption of vital tissues [1]. Platinum complexes cisplatin and its analogues such as lobaplatin, nedaplatin, oxaliplatin, carboplatin and heptaplatin have a unique place in the chemoterapeutic drugs [2]. Cisplatin (cis-diamminedichloroplatinum(II)) is effectively used in the chemotherapy of cancer types as ovarian, testicles, breast and melanoma [3]. Despite its high anticancer activity, cisplatin has exhibited several toxic side effects such as dose-limiting toxicity, neurotoxicity, nephrotoxicity, cardiotoxicity and ototoxicity [4]. These collateral effects and also the occurrence of cellular resistance limit the use of cisplatin in high dose [5]. In this direction, the development of new coordination compounds including different metal centers has become the

* Correspondence. € Ozdemir), € E-mail addresses: [email protected] (O. gurkanper@gmail. com (P. Gürkan), [email protected] (Y.D. S¸imay Demir), mark@gazi. edu.tr (M. Ark). https://doi.org/10.1016/j.molstruc.2020.127852 0022-2860/© 2020 Elsevier B.V. All rights reserved.

main aim of research in medicinal chemistry. Based on the significant similarities between thermodynamic parameters, complex geometry and coordination ability of platinum(II) and palladium(II) ions, Pd(II) seems to be a good alternative. Therefore, researchers have been focused on the synthesis of new palladium-based compounds. Many complexes of palladium with different bioligands such as amino acids [6,7], peptides [8], amino sugars [9] and drugs [10] have been reported in the literature. The pharmacological screening of these complexes has indicated that the model complex cispalladium (cis-[PdCl2(NH3)2]) does not possess any antitumor properties as compared to cisplatin [11]. But, the complex [Pd(phen)(Tyr)]þ bearing tryptophan as an amino acid has been shown to exhibit good anticancer activity against P388 lymphocytic leukaemia cells [12]. On the other hand, some palladium complexes of various Schiff bases containing biomolecules such as alkaloids [13], sulfa drugs [14] and chitosan [15,16] have been synthesized. As well, numerous papers on Pd(II) complexes of Schiff bases derived from amino acids or amino acid esters have been reported [17,18]. The Pd(II) complexes with ovanillin and amino acids (where amino acids are L-glutamic acid and L-tyrosine) have been found to be no cytotoxic against

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L929 cells by Muche et al. [18]. Previously we investigated the antiproliferative activity of a series of Schiff bases as monosodium salts (1a-3a) and neutral forms (1b-3b) against carcinoma cell lines (HeLa, MCF-7), and a normal human cell line (HEK-293). In this paper, we firstly reported the synthesis and the characterization of three new Pd(II) complexes of these Schiff base ligands. We evaluated their cytotoxicity activity against HeLa and MCF-7, and HEK-293 cell lines. 2. Experimental 2.1. Materials and instrumentation All starting chemicals and solvents were purchased from Sigma Aldrich and Merck Chemical Co. They were of reagent and spectroscopic grade, and used without any purification. Melting points were obtained on a Barnstead Electrothermal BI 9200 apparatus (Gazi University), and were uncorrected. Elemental analyses were measured on LECO CHNS-932 elemental analyzer (Ankara University). IR spectra were recorded on a Mattson 1000 FTIR spectrophotometer (Gazi University) in 4000e400 cm1 range using KBr discs. 1H and 13C NMR spectra (in DMSO‑d6 solution) were determined on a BrukerUltrashield 300 MHz spectrometer (Gazi University). The chemical shifts (d, ppm) are relative to an internal standard, tetramethylsilane (TMS). The conductivity measurements were carried out using a WTW Series Inolab Cond. 730 with a WTW Tetracon 325 electrode (Gazi University) at room temperature. The mass spectra were recorded using Waters 2695 Alliance Micromass ZQ massspectrometer (Ankara University). UVevis spectra (in DMF) were measured on an Analytik Jena UV200 spectrophotometer (Gazi University). Magnetic susceptibilities of complexes were obtained at room temperature using a Sheerwood Scientific MKI model Evans magnetic balance (Gazi University), Hg[Co(SCN)4] as a calibrant. Powder X-ray diffractograms were collected on a GNR APD 2000 Pro XRD (Gazi University) (40 kVe30 mA) with Cu Ka radiation (l ¼ 1.5406 Å) in the 2q ¼ 2e80 range. TGAeDTA curves were recorded using a SII 7300 EXSTAR (Gazi University) between 25 and 720  C at a heating rate of 10  C min1 in a N2 atmosphere. XPS determination was carried out with a Thermo K-Alpha spectrometer (Bilkent University). The X-ray source is Al Ka X-rays (monochromatic), carried out at 90 electron take-off angle. The experiments were conducted at pressures below 107 mbar. 2.2. Synthesis of Pd(II) complexes (1ee3e): general method The monosodium-Schiff bases (1ae3a) were synthesized in our previous work [19]. 3.00 mmol of the monosodium-Schiff base ligand was dissolved in water (40 mL). Solid PdCl2 (3 mmol, 0.532 g) was dissolved in 15 mL of N,N-dimethylformamide (DMF) with heating. Dark red solution of PdCl2 was added dropwise into the flask containing orange solution of ligand. The mixture was stirred at room temperature for 3 days, until its color turned to greenish. At the end of this time, the obtained precipitate was filtered, washed with water, ethyl alcohol and ether, and dried in a vacuum desiccator over anhydrous CaCl2. 2.3. Cytotoxicity assay 2.3.1. Cell culture Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco), fetal bovine serum (FBS) (Gibco), penisilin-streptomisin (PAA) and doksorubicin (Tocris) were used without purification. HeLa cell lines (human cervical cancer) and MCF-7 cell lines (human breast adenocancer) were obtained from Bilkent University (Molecular

Biology and Genetic Department) and Middle East Technical University (Biochemistry Department), respectively. Normal HEK293 cell lines (human embryonic kidney cell) were obtained from Ankara University (Biotechnology Institute). Three different human cell lines were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin. Cells were maintained at 37  C in a humidified atmosphere of 5% CO2 (Sanyo, Gazi University) in air. The culture medium was changed every 2 days. When the cell culture reached 70e80% confluence. they were treated with the complexes and doxorubicin at varying concentrations. At the end of two days, three different areas were chosen randomly, and an image of the cells was taken using phase-contrast microscope (Leica, Gazi University). Blebbing and normal cells were counted in the photographs by microscope camera (Leica DFC 420 C, Gazi University). The percentage of cells with blebs was calculated by dividing the number of cells with blebs by the total number of cells. 2.3.2. Real time monitoring of cytotoxicity An xCELLigence Real-Time Cell Analyzer (RTCA) DP system (Roche, Gazi University) was used to monitor the viability and migration of cells (HeLa, MCF-7 and HEK293), as previously described [20]. Briefly, 8  103e1  104 cells were plated per well of an E-plate. Cell growth and/or viability was monitored every 15 min for 20 h. After 20 h, the existing medium was replaced with fresh medium (DMEM þ 10% FBS þ 1% penisilin-streptomisin). When Cell Index was in the range of 1e1.5, the cells were incubated with the complexes and doxorubicin at varying concentrations. As an indicator of cell detachment, the cell index was recorded continuously for 48 h and analyzed by RTCA DP system. HeLa, MCF-7 and HEK293 cells were monitored for another 48 h, as a control. 2.3.3. Statistical analysis All values were expressed as the mean ± s. e.m and analyzed by Student’s t-test, and ANOVA. P < 0.05 was considered statistically significant. 3. Results 3.1. Chemistry The monosodium salts of Schiff bases (1ae3a) were derived from 5-nitro-salicylaldehyde and 4-aminobutyric acid, 5aminopentanoic acid and 6-aminohexanoic acid in our previous work [19]. Treatment of palladium(II) chloride with these ligands in 1:1 ratio afforded the neutral palladium(II) complexes. The new complexes (1ee3e) were isolated in yields between 56% and 69% (Table 1). They were obtained as a green powder, which are stable at room temperature, and soluble in DMSO and DMF. All the three Pd(II) complexes were fully characterized by elemental analysis, spectroscopic studies (FTIR, 1H, 13C NMR, LCeMS, UVevis, XPS and powder XRD), and thermal, conductivity and magnetic susceptibility measurements. Their melting points were found to be above 320  C. Elemental analyses showed that the ligand to the metal stoichiometric ratio is 1:1. IR data revealed that Schiff bases (1ae3a) act as a doubly deprotonated tridentate (O2N) ligand and coordinate to Pd(II) ion via imine nitrogen, hydroxyl and carboylate oxygen atoms. Also, NMR data proved the participation of the phenoliceO and imineeN atoms in chelation. The oxidation state of palladium was determined by XPS techniques, which is depent on the difference between the binding energies and the peak positions of Pd3d3/2 and Pd3d5/2 species. The molar conductivity measurement indicated that the complexes (1ee3e) are nonelectrolytic in nature. TGA and DTA curves confirmed the existence of one coordinated water molecule in all the complexes, and extra

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3

Table 1 Analytical, physical and UVevis data of new Pd(II) complexes. Comp.

Empirical formula color

Mol. weight (g/mol)

Yield %

M.p. ( C)

Found (Calcd.) % C

H

N

Pda

1e

[Pd(C11H10N2O5)H2O] Light green

374.65

56

>320

39.59 (35.27)

3.268 (3.23)

8.450 (7.48)

29.67 (28.42)

2e

[Pd(C12H12N2O5)H2O] Green

388.67

69

>320

38.12 (37.08)

3.121 (3.63)

7.406 (7.21)

28.60 (27.40)

3e

[Pd(C13H14N2O5)H2O].H2O Dark green

420.71

66

>320

37.44 (37.14)

3.404 (4.29)

6.878 (6.67)

24.24 (25.31)

lmax (nm)b(logε)

Lb(S cm2 mol1)

mef

289 377 440 291 378 439 292 372 441

4.3

dia

4.2

dia

2.8

dia

(4.01) (4.52) (sh) (3.97) (4.00) (4.57) (sh) (4.02) (4.00) (4.52) (sh) (3.96)

M DMF solution (LDMF ¼ 1.0 mS cm1). a Thermal analysis. b In 103.

one water molecule in 3e. On the basis of these results, the structures of the complexes (1ee3e) were formulated as [Pd(L)(H2O)]$ xH2O. As seen in Scheme 1, the square-planar geometry was formed by four-coordinate palladium as the center, O(8), O(9) and N(1) atoms of ligand as the corner, and a water molecule as the other corner. 3.1.1. FTIR spectra Infrared spectrum of the new palladium(II) complexes (1ee3e) was given in Fig. 1 and Fig. S1, and the important bands were scheduled in Table 2. When IR data of the complexes (1e-3e) were compared to the free ligands (1ae3a), there were seen some significant changes for the stretching vibration bands of the functional groups: i Phenolic (CeO) band of the corresponding ligands (1231e1233 cm1) shifted to lower wavenumber for the complexes and observed at 1100 cm1. ii. n(C]N) wavenumber of the ligands shifted from 1657 to 1663 cm1 to 1603e1605 cm1. iii The asymmetric n(COO) vibration of the ligands (1611e1613 cm1) gave shifts to higher wavenumber, and it appeared at 1700e1708 cm1. The difference (Dn ¼ 297e304 cm1) between the asymmetric and symmetric n(COO) wavenumbers indicated a monodentate nature of carboxylate group in the complexes [21]. The greatest negative shift (Dn ¼ 54e58 cm1 and 131e133 cm1 for iminic band and phenolic band, respectively) [22] and the positive shift (Dn ¼ 89e95 cm1 for carboxylate band) [23] supported strong binding of these functional groups to metal(II) ion. These results were an evidence for the dianionic ONO

Fig. 1. FTIR spectrum of 1e.

tridentate coordination mode of the ligands in the solid state. For the complexes, a broad band at 3300e3420 cm1 was assigned to stretching vibration of the OeH bond of the water in coordination sphere and uncoordinated water. The bands at 1550e1553 cm1 and 1315e1317 cm1 were attributed to the asymmetric and symmetric n(ONO) stretching vibrations, respectively. And, the new weak bands at 527e529 cm1 and 459e464 cm1 may be due to n(PdeO) and n(PdeN) [24]. 3.1.2. 1H NMR spectra NMR spectra of diamagnetic palladium(II) complexes (1ee3e)

Scheme 1. The synthesis of the new Pd(II) complexes (1ee3e).

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4 Table 2 FTIR data of new Pd(II) complexes.

nH2O

Comp.

nCeH

nCH/CH2

nC ¼ N

nC ¼ C benzene

nCOO as nCOO s

nONO as nONO s

dCeH

nCeO

n(PdeO) n(PdeN)

1700.3 1403.3 1708.1 1404.5 1705.4 1406.4

1550.7 1315.3 1551.5 1317.4 1553.0 1317.2

1467.3

1100.3

1467.7 1444.5 1466.7 1444.6

1100.6

528.6 463.8 527.2 459.8 528.5 461.6

benzene 1e

3418.5

3065.2

2929.1

1603.4

Overlap.

2e

3416.8

3067.5

2926.9

1604.7

Overlap.

3e

3348.5

3065.1

2927.9

1604.9

1495.3

1099.7

amino acid ligands [27]. The signal at d 167.29 ppm was due to the phenolic (C-3) carbon atom [28]. The peak at d 163.51 ppm was attributed to the imine (C-1) carbon atom [29]. According to these assigments, it could be said that 13C signals of (C-9), (C-3) and (C-1) carbon atoms shift to upfield as compared to the free ligand, which is an evidence for the complexation through these atoms. The aromatic carbon atoms were observed in the range of d 120e136 ppm. The alkyl (C-11), (C-10), (C-12) carbon atoms appeared at d 26.86 ppm, 36.27 ppm and 58.95 ppm as expected, respectively. In 13C NMR spectrum of 2e and 3e (Figs. S4e5), the carboxylate (C-9) carbon atom gave signal in the range of d 174e181 ppm. The phenolic (C-3) carbon atom was seen at d 167e168 ppm. The imine (C-1) carbon atom appeared at d 163e164 ppm. The aromatic carbon atoms were observed within d 117e137 ppm. The alkyl carbon atoms resonated between d 22 ppm and 57 ppm for 2e; d 24 ppm and 58 ppm for 3e.

were run in DMSO-d6 solution, and data were listed in Table 3. The atom numbering scheme of the compounds was presented in Scheme 2. In 1H NMR spectrum of 1e, the singlet peak at d 12.20 ppm was assigned to the proton of coordinated water. As illustrated in Fig. 2, OeH (H-8) signal of the free ligand disappeared. This disappearence gave information on the deprotonation of hydroxyl proton and the chelation of phenolic oxygen atom to Pd(II) ion. The imine (H-1) proton of ligand shifted from d 8.80 ppm to d 10.40 ppm indicating the binding of imine-N atom to metal center [25]. 1e had three signals in the aromatic region d 7.10 ppm, 8.10 ppm and 8.30e8.60 ppm as a doublet or multiplet, which were corresponded to (H-4), (H-5) and (H-7) protons, respectively. The doubling and broadening of these signals were a result of the complex formation [26]. As well, the methylene protons (H-11), (H-10) and (H-12) were observed as multiplets at d 1.90 ppm, 3.05 ppm and 3.70 ppm. Similar chemical shift values were recorded in 1H NMR spectrum of 2e and 3e (Figs. S2e3). The proton of water appeared at d 12.00e12.10 ppm as a singlet. The chemical shift of this signal was confirmed by D2O-exchangeable spectrum for 2e. In D2O-added spectrum (Fig. S2a), the peak at d 12.10 ppm was absent. The imine (H-1) proton was seen at d 10.30 ppm as a singlet. The disappearence of H-8 signal and the downfield shift (D d ¼ 1.5e1.6 ppm) of the peak due to imine group supported the coordination to palladium(II) ion, which are consistent with FTIR spectral data. The complexes displayed three signals in the aromatic region. The H-4, H-5 and H-7 protons of phenyl ring resonated between d 6.95 ppm and 8.60 ppm, and slightly broadened compared with the free ligands. The methylene protons were signalized in the range of d 1.50e3.70 ppm for 2e; d 1.20e3.65 ppm for 3e.

3.1.4. Mass spectra The liquid chromatographyemass spectrometry (LCeMS, ESI positive) analyses of the new palladium(II) complexes (1e-3e) were presented in experimental part. In case of 1e (Fig. S6), the fragment ion was observed at m/z 239.4 (RI% ¼ 17%) by the loss of H2O$ (m/z 18.01), NO2$ (m/z 45.99) and C3H4O2$ (m/z 72.02) radicals from the molecular ion. This fragment was followed by the loss of C4H4$ (m/z 52.03) and C2H4O$ (m/z 44.02) radicals; showing the peaks at m/z 191.3 (RI% ¼ 18.2%) and 147.3 (RI% ¼ 70%). The mass spectral pattern of 2e (Fig. S7) exhibited the similar fragment ion at m/z 239.4 (RI % ¼ 61.2%) which was related to the loss of H2O$ (m/z 18.01), NO2$ (m/z 45.99) and C4H6O2$ (m/z 86.03) radicals. The relative abundance of this peak was due to mass of isotope contributions of carbon (1213C) and palladium (105108Pd) atoms. The loss of C4H4$ (m/z 52.03) radical from this fragment ion produced the peak at 191.4 (RI% ¼ 53.5%). The base peak was seen at m/z 147.4 through the loss of C2H4O$ radical (m/z 44.02). As depicted in Fig. S8, mass spectrum of 3e had the fragment ion at m/z 401.9, which is

3.1.3. 13C NMR spectra In proton-decoupled 13C NMR spectrum of 1e (Fig. 3), the peak at d 176.93 ppm was ascribed to the carboxylate (C-9) carbon atom, which is a typical signal for complexes with monocoordinated

Table 3 1 H and13C chemical shifts of new Pd(II) complexes in DMSO-d6. Number Comp.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

H2O

1e

10.40 (s, 1H)a 163.51 10.30 (s, 1H) 163.67

e

e

e

3.70 (s, 2H) 57.55

e

12.10 (s, 2H)

168.36

3.70 (m, 2H) 58.95 1.70 (s, 2H) 31.43

12.20 (s, 2H)

176.93 e

1.90 (m, 2H) 26.86 1.50 (s, 2H) 22.14

e

e e

3.05 (m, 2H) 36.27 2.20 (s, 2H) 33.77

e

118.98 e

e

overlapped

24.50

26.06

1.20e1.75 (m, 6H) 31.08

3.65 (s, 2H) 58.00

12.00 (s, 2H)

168.00

2.20 (m, 2H) 33.94

overlapped

117.47

8.30e8.60 (d-d, 1H) 129.53 8.40e8.60 (d, 1H) 129.35 129.79 8.30e8.55 (d, 1H) 129.50

e

167.29 e

8.10 (m, 1H) 133.19 8.10 (s, 1H) 131.18 132.77 8.10 (s, 1H) 131.00 133.00

e

120.03 e

7.10 (d, 1H) 121.07 7.00 (d, 1H) 120.27 120.94 6.95 (d, 1H) 121.04

2e

3e

a

10.30 (s, 1H) 163.00

s, singlet; d, doublet; t, triplet; m, multiplet.

136.00 e 136.62 e 136.50

174.71 e

e 175.00 181.50

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5

Scheme 2. Numbering of proton and carbon atoms within Pd(II) complexes (1ee3e).

responsible of the loss of a neutral crystallization H2O molecule from its molecular formula. This fragment gave the peak at m/z 239.1 (RI% ¼ 75.9%) through the loss of H2O$ (m/z 18.01), NO2$ (m/z 45.99) and C5H8O2$ (m/z 100.05) radicals. The following peak at m/z 210.5 (17.5%) may be resulted from the elimination of O$ (m/z 15.99) and CH3$ (m/z 15.02) radicals. The base peak appeared at m/z 147.2 corresponding to the loss of C5H3$ (m/z 63.02) radical. 3.1.5. Magnetic, conductivity measurements and UVevisible spectra Magnetic susceptibility of the new solid complexes (1ee3e) was determined at 298 K. This confirmed their diamagnetic structure supporting low-spin 3 d8 electronic configuration of square-planar geometry. The molar conductivity of palladium(II) complexes was measured at room temperature in DMF solution (1  103 M). The low conductance was found to be about 4.3e2.8 S cm2 mol1 showing their non-electrolytic nature [30]. In electronic absorption spectrum of 1ee3e, two strong band and a shoulder band appeared in DMF (Fig. 4). The first maxima at 289e292 nm was corresponded to p/p* transition of phenolic chromophore. The second maxima at 372e378 nm may be due to the admixture of p/p* and n/p* transitions of phenolic, iminic and nitro chromophores. This band exhibited small red-shifting (Dlmax~ 6e12 nm) with respect to that of the corresponding ligands, that indicates the coordination of Neimine atom to metal

ion. The third shoulder band at 439e441 nm may be belonged to a combination of a ligand-to-metal charge transfer transition [31], and spin allowed ded transition (1A1g / 1E1g) [32] for the squareplanar geometry of d8 configuration [33]. 3.1.6. Thermal analyses TGA and DTA thermograms of the new palladium(II) complexes (1ee3e) were recorded under nitrogen gas atmosphere in the temperature range from ambient to 720  C at a heating rate of 10  C min1. The thermoanalytical data were summarized in Table 4. As depicted in Fig. 5, 1e decomposed in a three steps upon heating. The first mass loss (5.00%) within the temperature range of 42.59e180.59  C corresponded to the loss of one coordinated water molecule in its structure. The second mass loss (22.00%) between 180.59  C and 292.47  C was due to the decomposition of a part of the ligand molecule and evolution of CO/CO2 gas. This thermal dehydration step was accompanied by the exothermic effect (DTAmax: 290.40  C). The third step started at 292.47  C and continued to 711.92  C. In this step, the strong exothermic DTA peak was observed at 353.06  C, which was compatible with the burning of the organic residue. The total mass loss of these steps was 65.00%. This indicated that the remaining part with the carbon, palladium oxide (PdO) and metallic palladium (Pd) are present in the final residue. According to this decomposition product, the palladium content of 1e was found to be 29.67% (%calc ¼ 28.42).

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6

Fig. 2. 1H NMR spectrum of 1e in DMSOed6.

Thermal decomposition of the complexes 2e and 3e takes place in three steps (Figs. S9e10). The first thermal reaction was related to the loss of the water molecules in the temperature range of 38.03e163.14  C and 43.34e233.77  C. The mass loss was 4.62% for 2e and 8.59% for 3e, which was attributed to the loss of one coordinated water molecule for 2e and two water molecules for 3e. This step was accompanied by exothermic effect (DTAmax: 206.23  C). The second thermal reaction was observed between 163.14  C and 270.01  C for 2e; 233.77  C and 280.02  C for 3e. This step was related to the decomposition of a part of the ligand molecule and evolution of CO/CO2 gas, which was accompanied by exothermic effect (DTAmax: 211.36  C for 2e and 247.42  C for 3e). At temperatures higher than 270  C, 2e fully decomposed with having the broad exothermic peak at 382.44  C in DTA. The palladium content

Fig. 3.

13

C NMR spectrum of 1e in DMSOed6.

of 2e was calculated from PdO and Pd percentages, and it was found to be 28.60% (%calc ¼ 27.40). For 3e, third thermal reaction occurred in the range of 280.02e711.92  C, accompanied by exothermic effects (DTAmax: 309.73  C and 387.58  C). The total mass loss of three steps was only 50.66%. This incomplete decomposition may be associated with the formation of the final product, namely hydroxy(phenoxy)palladium ([Pd(C6H5O)OH]). The palladium content of 3e was determined as 24.24% (%calc ¼ 25.31), that agreed well with the given complex structure. 3.1.7. XPS spectra The general X-ray photoelectron spectra of the new palladium(II) complexes (1e-3e) were given in Fig. 6a and Figs. S11ae12a. The regions of C1s, N1s, O1s, and Pd3d were fitted by using Origin 5 programme (100% Gaussian). The electron binding energies (BE, eV) of species were listed in Table 5. XPS spectrum of C1s region was deconvoluted to four peak components for 1e, three peak components for 2e and 3e (Fig. 6b, Figs. S11be12b). The peak with BE in the range of

Fig. 4. UVevis spectrum of Pd(II) complexes in DMF solution (1  103 M).

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Table 4 Thermal decomposition of new Pd(II) complexes. Comp.

Step

DT ( C)

DTAmax( C)

Mass loss (%)

Residue (mass ratio) calcd./found

%Pd calcd./found

1e

1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd

42.59e180.59 180.59e292.47 292.47e711.92 38.03e163.14 163.14e270.01 270.01e711.92 43.34e233.77 233.77e280.02 280.02e711.92

e 290.40 353.06 e 211.36 382.44 206.23 247.42 309.73 387.58

5.00 22.00 38.00 4.62 29.06 33.11 8.59 14.48 27.61

C/PdO/Pd (1/4:2/3:1/2) 36.79/35.00 C/PdO/Pd (1/4:2/3:1/2) 35.46/33.21 [Pd(C6H5O)OH]

28.42/29.67

2e

3e

(exo) (exo) (exo) (exo) (exo) (exo) (exo) (exo)

27.40/28.60

25.31/24.24

51.44/49.32

decreased after upon complexation with Pd(II) ion, and thus 1ee3e had lower crystallinity in phase than that of 1ae3a by comparing their diffractograms. For 1e, the maximum was observed at 13.6 , which was related to a d-spacing of 6.51 Å. For the other complexes, a new maximum was recorded at 39e40 belonginging to interplanar distance d ¼ 2.26e2.31 Å. 3.2. Cytotoxicity assay

Fig. 5. TGAeDTA thermograms of 1e.

285.56e284.58 eV was belonged to the carbons of alkyl (CeC) and aromatic (C]C). The peak between 285.76 eV and 285.94 eV was assigned to the carbons of alkyl (CeN) and (CeO). The imine (C]N) carbon was seen at 287.83 eV for only 1e. The carboylate (COO) carbon was at 288 eV [34]. XPS spectrum of N1s region was deconvoluted to three peak components for all the complexes (Fig. 6c, Figs. S11ce12c). The peak with BE at 399e400 eV was associated to the alkyl (CeN) and imine (C]N) nitrogens [35]. The peak at 400e401 eV was attributed to the nitrogen in a lower oxidation state (AreN]O). And the peak >405 eV was related to the nitrogen of nitro group (AreNO2). XPS spectrum of O1s region was deconvoluted to three peak components for all the complexes (Fig. 6d, Figs. S11de12d). The first peak with BE at 531 eV was corresponded to the oxygen of phenolic (CeO) group [36]. The second peak at ca 532e534 eV was due to the oxygen of carboxylate group. The third peak at 534e536 eV was assigned to the oxygen of (AreN]O) group. XPS spectrum of Pd3d region was deconvoluted to two peak components for all the complexes (Fig. 6e, Figs. S11ee12e). The peaks with BE at 338e339 eV and 343e344 eV were assigned to 3d5/2 and 3d3/2, respectively [37]. These energies corresponded to the presence of Pd2þ chemical state in the complexes, which are as an indication for complex formation. 3.1.8. Powder X-ray diffractogram The powder X-ray diffractograms of the new palladium(II) complexes (1ee3e) were recorded over the 2q ¼ 0e70 range, and presented in Fig. 7 and Figs. S13e14. The ‘d’ values and 2q angles were collected in Table 6. XRD patterns of 1ee3e showed less intense and broaden reflections indicating their amorphous nature. From these data, it could be said that the crystallinity of the free ligands (1ae3a)

In vitro cytotoxicities of palladium(II) complexes (1ee3e) were evaluated against human HeLa cervical cancer and MCF-7 breast adenocancer cell lines, and a normal human cell line HEK-293 embryonic kidney cell. The results were compared to the solvent control (untreated cell in DMSO) and to the positive control (treated cell with doxorubicin). As could be seen in Fig. 8a, the complexes 1ee3e exerted dosedependent antiproliferative effect against HeLa cell line. At concentration of 1 mM, 1ee3e achieved to kill 10%, 20% and 90% percentages of HeLa cell, respectively. Their cytotoxicities followed the order: 3e > 2e > 1e. This suggested that there is an obvious correlation between cytotoxicity and increasement of lipophilic character of the complexes, which is due to a number of an alkyl group in structure. Lipophilicity has an important role in the passage of drug across the cell membrane [38]. In the concentration range of 0.3 mMe0.1 mM, especially 3e failed to cause antiproliferation on HeLa cell line. The cell-death value of malignant cell was found to be within 8e10% for 1e; 7e10% for 2e; 17e9% for 3e. At concentrations lower than 0.1 mM, 2e and 3e killed ~12% percentage of HeLa cell. Although this, 1e could not inhibit the growth of carcinoma cell, and caused an increase in the cell proliferation. In comparison to doxorubicin, it was observed that 3e is the more active against selected cell line. Microphotographs of HeLa cells, which were pretreated with the complexes and standard for 48 h were depicted in Fig. 8b. It was determined that doxorubicin, 2e and 3e increased the number of cells exhibiting bleb formation, which was absent in control cells. But, not significant changes were detected in the cell morphology for 1e. Blebs may be formed in the cytoplasm or nuclei/nuclear membranes of cancer cells in various situations like as cell spreading, cytokinesis and apoptosis [20]. Consistent with the phase-contrast observations, it may be suggested that 3e has antiproliferative effect on HeLa cells by a different mechanism of action from doxorubicin. This effect may be explained by the formation of the covalent bond between Pd(II) ion and double helix structure of tumor cell DNA. In this way, the complexes seemed to inhibit DNA replication through intercalation. The cellular antitumor mechanism of the complexes needs to be investigated by a series of in vitro and in vivo studies like as apoptotic staining fluorescence microscopic analysis [39]. It has been reported that Pd(II) complexes of tridentate Schiff bases can specially cleave DNA, and this leads to induce their antitumor activity [40]. Cell structure studies have demonstrated that platinum-

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Fig. 6. XPS spectrum of 1e: (a) general spectrum, (b) C1s region, (c) N1s region, (d) O1s region and (e) Pd3d region.

based drugs interact with DNA covalently and form DNA adducts, and thus inhibit cell division through inhibiting nucleic acid reduplication and achieve cell-death/apoptosis [41]. Besides, the antiproliferative activity of doxorubicin is thought to be related to its ability to bind to DNA and inhibit both the synthesis of nucleic acid and the catalytic activity of topoisomeraseeII [42]. From the results in Fig. 9a, the highest activity of palladium(II) complexes was noted against MCF-7 cell lines. At concentration of 1 mM, they had very significant antiproliferative effect indicating to exhibit the cell-death value of 100%. At concentration of 0.3 mM, 1e showed very low toxicity and achieved to kill only 3% percentage of MCF-7 cells. Despite this, 2e and 3e killed 63% and 78% percentages

of cells, respectively. At concentrations lower than 0.3 mM, cytotoxicity activity of 2e decreased with decreasing its concentration. But, it still showed a weak antiproliferative effect in the all dose range used. In the concentration range of 0.3 mMe0.03 mM, 1e and 3e increased the cell proliferation of MCF-7 lines. Of all the studied complexes and standard, complex 2e may seem to be the most active against MCF-7. The morphological changes in MCF-7 cell line were confirmed by phase contrast microscopy, and given in Fig. 9b. All the Pd(II) complexes caused very noticeable modification in the morphology of cancer cell. It was observed that membrane blebbing induced by apoptotic cell-death was present in cells treated with the complexes. And, percentage of the blebbing cells reached

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Table 5 The data of XPS spectra of new Pd(II) complexes. Comp.

C1s

N1s

B.E. (eV) 1e

3e

a

b

CeC, C¼C CeN, CeO C¼N COO CeC, C]C CeN, CeO C¼N COO CeC, C]C CeN, CeO C¼N COO

285.56 285.76 287.83 288.48 284.71 285.94 -a 288.27 284.58 285.76 -a 288.32

2e

b

Species

O1s

Pd(II)

B.E. (eV)

Species

B.E. (eV)

Species

Pd3d5/2

Pd3d3/2

399.58 401.32 405.94

CeN, C]N AreN]O Ar-NO2

531.88 533.43 534.42

CeO C¼O AreN]O

338.59

343.95

399.25 400.80 405.56

CeN, C]N AreN]O Ar-NO2

531.61 532.64 534.61

CeO C¼O AreN]O

338.11

343.44

399.28 401.36 405.50

CeN, C]N AreN]O Ar-NO2

531.63 533.70 535.56

CeO C¼O AreN]O

338.02

343.27

Not observed. Aryl.

Fig. 7. Solid X-ray diffractogram of 3e.

to the maximum for all the complexes. These results showed that lipophilic character of the complexes 1e‒3e does not have any influences on their antiproliferative activity against MCF-7 lines. In order to find the side-effect of palladium(II) complexes on normal human cell line, HEK293 cell line was tested under the same conditions. According to the obtained results (Fig. 10a), 3e killed whole of the normal cells at concentration of 1 mM. The other complexes were also found to be highly cytotoxic, which their celldeath value (~94%) is very close to doxorubicin. At concentration of 0.3 mM, their antiproliferative effect was in the order: 3e > 2e > 1e. The death of cancerous cells was obtained 22%, 37% and 97%, respectively. The complex 1e did not show any toxicity at the concentration 0.1 mM. A decrease in concentration of the other complexes significantly decreased their toxicity within the concentration range of 0.1 mMe0.03 mM. The changes in investigated cells were shown in Fig. 10b. As depicted in here, treatment of HEK293 cell with the complexes induced the cell detachment and

formation of blebs collecting within clusters. These data confirmed that antiproliferative effect of the complexes on normal cell was similar to that of the carcinoma cell lines. Literature survey has exhibited that antiproliferative activities of the compounds are both structure and dose-dependent. In vitro cytotoxic studies showed that Pd(II) complexes of biopolymeric amphiphilic Schiff bases caused a decrease in cell viability of MCF7 cell lines, depending on increasing in their concentration [43]. Palladium(II) complexes of 3-formyl chromone Schiff bases were reported to be much less active against MCF-7 cells and human colon carcinoma (COLO 205) cell lines, when compared to cisplatin [44]. For Pd(II) complex of coumarin-thiazole based Schiff base, the cell viability decreased as a function of increasing concentration, and the most effective dose was found to be 100 mM against MCF-7, human prostate adenocarcinoma (LNCAP) and human colon carcinoma (LS174T) cell lines [45]. Besides, Pd(II) complexes of Schiff bases derived from aminoneocryptolepine and salicyaldehyde displayed mild antiproliferative activity against colorectal adenocarcinoma (HT-29) cells as compared with free ligands [46]. Palladium(II) triphenylphosphine complexes bearing tridentate Schiff bases revealed low activity against Agrobacterium tumefaciens (AT10) [47]. On the other hand, Pd(II) complex of 2-thiophene N(4)phenylthiosemicarbazone could not inhibit cell proliferation growth of HepG2 cells and normal QSG7701 cells [48]. From these results, it can be suggested that newly obtained Pd(II) complexes (1ee3e) will be good tumor inhibitors in literature. 4. Conclusions In this work, three new palladium(II) complexes (1ee3e) of N(5-nitro-salicylidene)-Schiff bases were prepared as potential

Table 6 Powder X-ray diffraction data of new Pd(II) complexes. Comp.

Peak

2q

d (Å)

Comp.

Peak

2q

d (Å)

Comp.

Peak

2q

d (Å)

1e

1 2 3 4 5 6 7 8 9

9.4 13.6 23.5 27.6 35.1 40.1 44.0 56.3

9.40 6.51 3.78 3.23 2.56 2.25 2.06 1.63

2e

1 2 3 4 5 6 7 8 9

5.2 21.1 23.7 27.8 38.8 39.9 41.7 46.1 48.0 55.7 60.0 67.9

16.98 4.21 3.75 3.21 2.32 2.26 2.16 1.97 1.89 1.65 1.54 1.38

3e

1 2 3 4 5 6 7 8 9

1.0 7.6 39.0 46.0 48.1 67.3

88.27 11.62 2.31 1.97 1.89 1.39

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Fig. 8. (a) The evaluation of cytotoxic activity of Pd(II) complexes (1ee3e) against HeLa carcinoma cell lines. * shows significant difference from control group. (One-way analysis of variance ANOVA, Tukey, p < 0.05, n ¼ 3). (b) Phase contrast photographs of HeLa cells at 48 h (  200).

Fig. 9. (a) The evaluation of cytotoxic activity of Pd(II) complexes (1ee3e) against MCF-7 carcinoma cell lines. * shows significant difference from control group. (One-way analysis of variance ANOVA, Tukey, p < 0.05, n ¼ 3). (b) Phase contrast photographs of MCF-7 cells at 48 h (  200).

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Fig. 10. (a) The evaluation of cytotoxic activity of Pd(II) complexes (1ee3e) against HEK-293 normal cell lines. * shows significant difference from control group. (One-way analysis of variance ANOVA, Tukey, p < 0.05, n ¼ 3). (b) Phase contrast photographs of HEK-293 cells at 48 h (  200).

anticancer agents. Their structures were characterized by various analytical and spectroscopic measurements. The physicochemical investigations supported the complexation between Pd(II) ion and the ligand through the nitrogen atom of the imine group and the oxygen atoms of hydroxyl and carboylate groups. In vitro antitumor activity of all three complexes was tested against HeLa, MCF-7 and HEK-293 cell lines. The obtained results showed that antiproliferative effect of 3e is better than that of the other complexes and doxorubicin against HeLa cell lines. This efficiency decreased in the complex series following the order: 3e > 2e > 1e. The complexes (1ee3e) demonstrated the best cytotoxicity against MCF-7 cells among investigated cancer cell lines. Their killing percentage was found to be 100% at concentration of 1 mM indicating that they are more effective than doxorubicin. For HEK-293 cell lines, the complexes displayed similar antiproliferative effect as doxorubicin at 1 mM dosage. But, their cytotoxicity decreased with a decrease in the concentration. Declaration of competing interest No conflict of interest was declared by the authors.

CRediT authorship contribution statement € € Ozlem Ozdemir: Conceptualization, Formal analysis, Methodology, Writing - original draft, Writing - review & editing. Perihan Gürkan: Funding acquisition, Project administration, Supervision. Yaprak Dilber S¸imay Demir: Investigation, Visualization. Mustafa Ark: Data curation, Investigation, Supervision, Validation.

Acknowledgements We are grateful to Research Foundation of Gazi University (Project ID: F.E.F.05/2013e08). And, we are thankful to Gazi University Photonics Application and Research Center for XRD mesurements. We also thank Bilkent University-UNAM for XPS measurements. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.molstruc.2020.127852. References [1] K.P. Rakesh, H.K. Kumara, H.M. Manukumar, D.C. Gowda, Bioorg. Chem. 87 (2019) 252e264, https://doi.org/10.1016/j.bioorg.2019.03.038. [2] W.X.C. Oliveira, M.M. da Costa, A.P.S. Fontes, C.B. Pinheiro, F.C.S. de Paula, E.H.L. Jaimes, E.F. Pedroso, P.P. de Souza, E.C. Pereira-Maia, C.L.M. Pereira, Polyhedron 76 (2014) 16e21, https://doi.org/10.1016/j.poly.2014.03.049. [3] I. Ott, R. Gust, Arch. Pharm. Chem. Life Sci. 340 (2007) 117e126, https:// doi.org/10.1002/ardp.200600151. [4] A. Bakalova, H. Varbanov, R. Buyukliev, S. Stanchev, G. Momekov, D. Ivanov, Inorg. Chim. Acta. 363 (2010) 1568e1576, https://doi.org/10.1016/ j.ica.2010.01.008. [5] L.-J. Li, B. Fu, Y. Qiao, C. Wang, Y.-Y. Huang, C.-C. Liu, C. Tian, J.-L. Du, Inorg. Chim. Acta. 419 (2014) 135e140, https://doi.org/10.1016/j.ica.2014.04.036. [6] S. Kasselouri, A. Garoufis, N. Hadjiliadis, Inorg. Chim. Acta. 135 (1987) L23eL25, https://doi.org/10.1016/S0020-1693(00)81294-3. [7] A.K. Paul, H. Mansuri-Torshizi, T.S. Srivastava, S.J. Chavan, M.P. Chitnis, J. Inorg. Biochem. 50 (1993) 9e20, https://doi.org/10.1016/0162-0134(93)80010-7. [8] M.R. Shehata, Arabian J. Chem. 12 (2019) 1395e1405, https://doi.org/10.1016/ j.arabjc.2014.11.017. [9] I. Brudzinska, Y. Mikata, M. Obata, C. Ohtsuki, S. Yano, Bioorg. Med. Chem. Lett 14 (2004) 2533e2536, https://doi.org/10.1016/j.bmcl.2004.02.095.

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