Surfactant-copper(II) Schiff base complexes: synthesis, structural investigation, DNA interaction, docking studies, and cytotoxic activity.

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Surfactant–copper(II) Schiff base complexes: synthesis, structural investigation, DNA interaction, docking studies, and cytotoxic activity a

a

a

Jagadeesan Lakshmipraba , Sankaralingam Arunachalam , Rajadurai Vijay Solomon , a

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Ponnambalam Venuvanalingam , Anvarbatcha Riyasdeen , Rajakumar Dhivya & Mohammad Abdulkader Akbarsha a

b

School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India

b

Mahatma Gandhi-Doerenkamp Centre, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India c

Department of Biomedical Science, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India Published online: 23 May 2014.

To cite this article: Jagadeesan Lakshmipraba, Sankaralingam Arunachalam, Rajadurai Vijay Solomon, Ponnambalam Venuvanalingam, Anvarbatcha Riyasdeen, Rajakumar Dhivya & Mohammad Abdulkader Akbarsha (2014): Surfactant–copper(II) Schiff base complexes: synthesis, structural investigation, DNA interaction, docking studies, and cytotoxic activity, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2014.918523 To link to this article: http://dx.doi.org/10.1080/07391102.2014.918523

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Journal of Biomolecular Structure and Dynamics, 2014 http://dx.doi.org/10.1080/07391102.2014.918523

Surfactant–copper(II) Schiff base complexes: synthesis, structural investigation, DNA interaction, docking studies, and cytotoxic activity Jagadeesan Lakshmiprabaa, Sankaralingam Arunachalama*, Rajadurai Vijay Solomona, Ponnambalam Venuvanalingama, Anvarbatcha Riyasdeenb, Rajakumar Dhivyac and Mohammad Abdulkader Akbarshab a

School of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India; bMahatma Gandhi-Doerenkamp Centre, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India; cDepartment of Biomedical Science, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India Communicated by Ramaswamy H. Sarma

Downloaded by [Baylor University Libraries] at 04:11 18 June 2014

(Received 20 January 2014; accepted 23 April 2014) A series of surfactant–copper(II) Schiff base complexes (1–6) of the general formula, [Cu(sal-R2)2] and [Cu(5-OMe-sal-R2)2], {where, sal = salicylaldehyde, 5-OMe-sal = 5-methoxy- salicylaldehyde, and R2 = dodecylamine (DA), tetradecylamine (TA), or cetylamine (CA)} have been synthesized and characterized by spectroscopic, ESI-MS, and elemental analysis methods. For a special reason, the structure of one of the complexes (2) was resolved by single crystal X-ray diffraction analysis and it indicates the presence of a distorted square-planar geometry in the complex. Analysis of the binding of these complexes with DNA has been carried out adapting UV-visible-, fluorescence-, as well as circular dichroism spectroscopic methods and viscosity experiments. The results indicate that the complexes bind via minor groove mode involving the hydrophobic surfactant chain. Increase in the length of the aliphatic chain of the ligands facilitates the binding. Further, molecular docking calculations have been performed to understand the nature as well as order of binding of these complexes with DNA. This docking analysis also suggested that the complexes interact with DNA through the alkyl chain present in the Schiff base ligands via the minor groove. In addition, the cytotoxic property of the surfactant–copper(II) Schiff base complexes have been studied against a breast cancer cell line. All six complexes reduced the visibility of the cells but complexes 2, 3, 5, and 6 brought about this effect at fairly low concentrations. Analyzed further, but a small percentage of cells succumbed to necrosis. Of these complexes (6) proved to be the most efficient aptotoxic agent. Keywords: Surfactant–Schiff base; copper; DNA interaction; molecular docking; cytotoxicty

Introduction The interaction of transition metal complexes with DNA has long been a subject of extensive investigation with a view to development of new materials for application in medicine, molecular biology, and biotechnology (Barton & Lolis, 1985; Erkkila, Odom, & Barton, 1999; Guo & Sadler, 2000; Huppert, 1999; Lippert, 2000; Rosenberg, VanCamp, & Krigas, 1965). The transition metal complexes show unique spectral and electrochemical properties, as well as the ability of their ligands to be adjusted to DNA interaction abilities. These investigations have contributed to synthesis of many new metal complexes, which bind to DNA by non-covalent interactions such as electrostatic binding, groove binding, and intercalative binding (Lakshmipraba et al., 2013; Li, Chen, Tang, & Zhang, 2008; Sanchez-Delgado, Anzellotti, & Suarez, 2004). The mode of coordination depends on the nature of the central metal atom, type of the ligand, as well as on the presence of other species capable to competing for coordination pockets.

*Corresponding author. Email: [email protected] © 2014 Taylor & Francis

Copper is an essential element due to its bioessential activity, oxidative nature, and its involvement in complex formation in many biological processes. (Calvin & Wilson, 1945; Lakshmipraba & Arunachalam, 2010; Marcus & Eliezer, 1969; Sigel & Sigel, 1996). More than a dozen of enzymes that depend on copper for their activity have been identified; the metabolic conversions catalyzed by all of these enzymes are oxidative. Due to their importance in biological processes, the design and synthesis of new Cu(II) complexes are aspects of emphasis in bioinorganic chemistry (Easmon et al., 2001; Green & Reed, 1998; Lakshmipraba, Arunachalam, Gandi, & Thirunalasundari, 2011; Liang et al., 2003; Ma et al., 1998). Although a number of Cu(II) complexes with varieties of ligand systems which effectively bind to DNA have been reported (Komor & Barton, 2013), there is still scope to design and study new Cu(II) complexes with special modifications in the ligand system as new chemical nucleases.


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J. Lakshmipraba et al.

Among the various types of ligands known in the literature, Schiff base ligands and their metal complexes have attracted considerable attention due to their importance in catalytic, analytical, pharmacological, and clinical applications (Karabach, Kirrillov, Haukka, Kopylovich, & Pombeiro, 2008; Patra, Bhowmick, Roy, Ramakumar, & Chakravarty, 2009; Prabhu & Ramesh, 2012a, 2013; Schuchardt, Carvalho, & Spinace, 1993; Todorovic et al., 2009; Wang, Yang, Wang, Cai, & Crewdson, 2006). Transition metal complexes containing Schiff base ligands have the ability to stabilize different metals in various oxidation states and the complexes are studied in view of their synthetic flexibility, selectivity, and sensitivity towards a variety of metal ions (Maria et al., 2004; Prabhu & Ramesh, 2012b; Spinu & Kriza, 2000). The electronic, steric, and conformational effects imparted by the coordinated ligands play an essential role in stabilizing the metal center, thereby improving the chemical, physical, and biological properties of the copper complexes (Barone, 2013). Surfactants are amphiphiles and are generally characterized as materials having hydrophilic head group and hydrophobic chain (tail) and are able to interact with both polar and non-polar compounds (Kumar & Arunachalam, 2008). They are the major building blocks in many physical, chemical, and biological systems (Kumar et al., 2009) and are used in antiseptic agents, cosmetics, detergents, germicides, and pharmaceutical industries (Schramm, Stasiuk, & Marangoni, 2003; Tadros, 2005). Surfactant–Schiff base metal complexes are a special class of surfactants having hydrophilic head part with metal center and a hydrophobic tail part. The incorporation of metal complexes into amphiphilic structures is of significant attraction due to their potential applications in magnetic, catalytic, and biological activities (Prabhu & Ramesh, 2012c; Udhayavani & Subramani, 2012; Ziessel et al., 2004). Though there are a few reports on the synthesis and characterization of surfactant–Schiff base copper(II) complexes (Binnemans, 2005; Paschke, Liebsch, Tschierske, Oakley, & Sinn, 2003), to the best of our knowledge, there is no report where the interaction of surfactant– Schiff base copper complexes with DNA has been investigated. In this background, we herein describe the synthesis of a series of new copper(II) complexes bearing surfactant–Schiff base ligands. All the complexes have been characterized by analytical and spectral methods. The structure of one of the complexes has been probed with the help of single crystal X-ray diffraction analysis. The binding of these complexes with DNA has been studied using UV–visible absorption, emission, circular dichroism, and viscosity measurements. In addition, molecular docking calculations have also been performed to gain further insights into the nature of DNA binding of the new surfactant–copper(II) Schiff base complexes.

Further, we chose to screen the cytotoxic effect of the DNA binding copper(II) complexes on MCF-7 cancer cell line. The complexes 2, 3, 5, and 6 killed the cancer cells at low concentrations compared to 1 and 4 and, hence, these four complex treated cells were subjected to morphological assessment and DNA damage analysis. Experimental Materials Calf thymus DNA and tetradecylamine (TA) were obtained from Sigma-Aldrich, Germany and were used as such. Copper(II) acetate monohydrate, dodecylamine (DA), and hexadecylamine (CA) were used as purchased from Merck, India. The ligands were prepared based on available literature (Garnovskii et al., 2009) by the condensation of salicylaldehyde derivatives with long chain primary aliphatic amines (ESI†). A solution of calf thymus DNA in the buffer (5 mM Tris–HCl/50 mM NaCl at pH 7.1) gave a ratio of UV absorbance at 260 and 280 nm of ~1.8~1.9:1, indicating that the DNA was sufficiently free of protein. Stock solutions of DNA were stored at 4 °C and used after no more than four days. The concentration of CT DNA in terms of base pairs was determined by UV absorbance at 260 nm by taking the molar extinction coefficient value 13,200 M−1 cm−1 for DNA at 260 nm (Lundback & Hard, 1996; Marty, N’soukpoé-Kossi, Charbonneau, Kreplak, & Tajmir-Riahi, 2009). Concentrated solutions of metal complexes were prepared by dissolving the complexes in ethanol and the solutions were diluted for the experiments with twice distilled water in buffer containing 5 mM Tris–HCl/50 mM NaCl at pH 7.1. The breast carcinoma MCF-7 cells were obtained from National Centre For Cell Science (NCCS), Pune.

Physical measurements and instrumentation The carbon, hydrogen, and nitrogen contents of samples were determined at Sophisticated Analytical Instrument Facility(SAIF), Lucknow, India. FT-IR spectra were recorded on a FT-IR JASCO 460 PLUS spectrophotometer with samples prepared as KBr pellets. ESI-mass data were recorded in Thermo Scientific LCQ Fleet Mass Spectrometer. Absorption spectra were done on a UV– vis–NIR Cary 300 Spectrophotometer using cuvettes of 1 cm path length, and emission spectra were recorded on a JASCO FP 770 spectrofluorimeter. EPR spectra were recorded on a JEOL-FA200 EPR spectrometer in methanol at 77 K and in solid form at room temperature. Circular dichroism spectra were recorded on a JASCO J-716 spectropolarimeter. Absorption titration experiments of surfactant– copper(II) Schiff base complexes in solution of buffer


Surfactant-copper(II) Schiff base complexes; DNA interaction, cytotoxic activity and ethanol (10:1 tris-buffer: ethanol) were performed by using a fixed complex concentration ([cpx] = 50 μM) to which increments of the DNA stock solution ([DNA] = 0–250 μM) were added. Then the complex DNA solutions were incubated for 10 min before the absorption spectra were recorded. Equal volume of DNA solution was added to both the complex and reference solutions to eliminate the absorbance of DNA itself, if any. For fluorescence quenching experiments DNA was pretreated with ethidium bromide (EB) in the ratio [DNA]/[EB] = 1 for 30 min. Surfactant–copper(II) Schiff base complexes were then added to this mixture and their effect on the emission intensity was measured. Samples were excited at 450 nm and emission was observed between 500 and 700 nm. Circular dichroic spectra were recorded at room temperature in the same tris buffer. Viscosity experiments were carried out using an Ubbelohde viscometer maintained at a constant temperature of 25.0 ± 0.2 °C. DNA solution was prepared by sonication in order to minimize complications arising from DNA flexibility. Conductivity studies were done in aqueous solutions of the complexes with an Elico conductivity bridge type CM 82 and a dip-type cell with a cell constant of 1.0. Synthesis of surfactant–copper(II) Schiff base complexes A solution of copper acetate dihydrate (1.25 mmol) was prepared in hot ethanol (10 ml) containing equal volume of water. To this solution the appropriate Schiff base ligand (2.5 mmol) in warm ethanol (30 ml) was added slowly with constant stirring. Then the mixture was refluxed for 30 min. The surfactant–copper(II) complex containing the Schiff base ligand was slowly precipitated out from the solution. The compound was filtered and recrystallized using hot methanol/chloroform (Scheme 1). The complexes were characterized by analytical and spectral methods.

R2 N 2 R 1

OH

[Cu(sal-DA)2] (R1 = H, R2 = DA) (1): Color: Brown; Yield: 0.72 g, 90%; Anal. Calc. for C38H60CuN2O2 (640.44 g mol−1): C, 71.21; H, 9.44; N, 4.37%; Found: C, 71.17; H, 9.40; N, 4.31%; IR (KBr, cm−1): 2917 υ(C–H), 2850 υ(–CH2), 1623 υ(C=N),1328 υ(C–O); UV–vis (water, λmax, nm; ε, dm3 mol−1 cm−1): 396 (8890), 332 (11,290), 286, (16,210), 254 (18,120); EPR: gǁ = 2.245 g┴ = 2.019 (77 K, methanol), giso = 2.050 (room temperature); ESI-MS Pos. in MeOH: m/z (100%) = 640.58. [Cu(sal-TA)2] (R1 = H, R2 = TA) (2): Color: Brown; Yield: 0.69 g, 80%; Anal. Calc.: Anal. Calc. for C42H68CuN2O2 (696.55 g mol−1): C, 72.42; H, 9.84; N, 4.02%; Found: C, 72.34; H, 9.65; N, 3.95%; IR (KBr, cm−1): 2919 υ(C–H), 2849 υ(-CH2), 1622 υ(C=N) and 1331 υ(C–O); UV–vis (water, λmax, nm; ε, dm3 mol−1 cm−1): 389 (8950), 315 (11,450), 279 (16,470), 252 (18,330); EPR: gǁ = 2.241 g┴ = 2.020 (77 K, methanol), giso = 2.077 (room temperature); ESI-MS Pos. in MeOH: m/z (100%) = 696.83. [Cu(sal-CA)2] (R1 = H, R2 = CA) (3): Color: Brown; Yield: 0.80 g, 86%; Anal. Calc. for C46H76CuN2O2 (752.65 g mol−1): (Anal. Calc.: C, 73.41; H, 10.18; N, 3.72%; Found: C, 73.37; H, 10.12; N, 3.68%; IR (KBr, cm−1): 2918 υ(C–H), 2849 tð–CH2 Þ , 1627 υ(C=N), 1331 υ(C– 3 −1 cm−1): 388 O); UV–vis (water, λmax, nm; ε, dm mol (8870), 312 (11,340), 279 (16,390), 252 (18,260); EPR: gǁ = 2.243 g┴ = 2.015 (77 K, methanol), giso = 2.074 (room temperature); ESI-MS Pos. in MeOH: m/z (100%) = 752.83. [Cu(5-OMe-sal-DA)2] (R1 = OMe, R2 = DA) (4): Color: Brown; Yield: 0.74 g 85%; Anal. Calc.: C40H64CuN2O4 (700.49 g−1 mol): (Anal. Calc.: C, 68.58; H, 9.21; N, 4.00%; Found: C, 68.52; H, 9.16; N, 3.94%; IR (KBr, cm−1): 2916 υ(C–H), 2850 tð–CH2 Þ , 1627 υ(C=N), 1329 υ(C–O); UV–vis (water, λmax, nm; ε, dm3 mol−1 cm−1): 418 (9360), 312 (12,140), 284 (16,910), 260 (18,720); EPR: gǁ = 2.218 g┴ = 2.051 (77 K, methanol); giso = 2.048 (room temperature); ESI-MS Pos. in MeOH: (100%) m/z = 700.37.

R2 N

MeOH + Cu(OAc)2

Reflux, ~40 min

O Cu

R1

O

N R2

1 = [Cu(sal-DA)2]; R1 = H, R2 = Dodecylamine(DA) 2 = [Cu(sal-TA)2]; R1 = H, R2 = Tetradecylamine(TA) 3 = [Cu(sal-CA)2]; R1 = H, R2 = Hexadecylamine(CA) 4 = [Cu(5-OMe-sal-DA)2]; R1 = OMe, R2 = Dodecylamine(DA) 5 = [Cu(5-OMe-sal-TA)2]; R1 = OMe, R2 = Tetradecylamine(TA) 6 = [Cu(5-OMe-sal-CA)2]; R1 = OMe, R2 = Hexadecylamine(CA)

Scheme 1.

Formation of surfactant–copper(II) Schiff base complex.

3

R1


4


[Cu(5-OMe-sal-TA)2] (R1 = OMe, R2 = TA) (5): Color: Brown; Yield: 0.86 g, 91%; Anal. Calc.: C44H72CuN2O4 (756.60 g−1 mol): (Anal. Calc.: C, 67.06; H, 9.59; N, 3.70%; Found: C, 67.01; H, 9.52; N, 3.67%; IR (KBr, cm−1): 2921 υ(C–H), 2851 tð–CH2 Þ , 1621 υ(C=N), 1325 υ(C–O); UV-vis (water, λmax, nm; ε, dm3 mol−1 cm−1): 425 (9640), 315 (12,310), 283 (17,220), 259 (18,930); EPR: gǁ = 2.220 g┴ = 2.057 (77 K, methanol); giso = 2.052 (room temperature); ESI-MS Pos. in MeOH: m/z (100%) = 756.54. [Cu(5-OMe-sal-CA)2] (R1 = OMe, R2 = CA) (6): Color: Brown; Yield: 0.90 g, 89%; Anal. Calc.: C48H80CuN2O4 (812.71 g−1 mol): (Anal. Calc.: C, 70.94; H, 9.92; N, 3.45%; Found: C, 70.90; H, 9.87; N, 3.39%; IR (KBr, cm−1): 2921 υ(C–H), 2851 tð–CH2 Þ , 1619 υ(C=N), 1325 υ(C–O); UV–vis (water, λmax, nm; ε, dm3 mol−1 cm−1): 421 (9480), 306 (12,280), 282 (17,140), 261 (18,830); EPR : gǁ = 2.229 g┴ = 2.060 (77 K, methanol); giso = 2.049 (room temperature); ESI-MS Pos. in MeOH: m/z (100%) = 812.61. X-ray crystallography Single crystal of [Cu(sal-TA)2] (2) was obtained by the slow evaporation of ethanol solution of the complex at room temperature. The data collections were carried out using Bruker AXS Kappa APEX II single crystal X-ray diffractometer using monochromated Mo Kα radiation (kI = 0.71073 Å). The structures were solved by SHELXS-97 and refined by full-matrix least squares on F2 using SHELXL-97 (Sheldrick, 2008). All non-hydrogen atoms were refined anisotropically and the hydrogen atoms in these structures were located based on the difference Fourier map and constrained to the ideal positions in the refinement procedure. The unit cell parameters were determined by the method of difference vectors using reflections scanned from three different zones of the reciprocal lattice. The intensity data were measured using ω and φ scan with a frame width of 0.5°. Frame integration and data reduction were performed using the Bruker SAINT-Plus (Version 7.06a) software (Bruker-Nonius, 2004). CMC determination The critical micelle concentration (CMC) values of the surfactant–copper(II) complexes were measured conductometrically using a specific conductivity meter. Various concentrations of surfactant–copper (II) complexes were prepared in the range of 1 × 10−5–1 × 10−1 M in aqueous solutions. The conductivities of these solutions were measured at 30.0 °C. The conductivity cell

was calibrated with KCl solutions in the appropriate concentration range. Computational details The geometries of all the complexes were optimized at B3LYP/LANL2DZ level using G09 W program (Frisch et al., 2009). These optimized structures of the complexes were further considered for molecular docking analysis using HEX 6.3 i.e. an interactive protein docking and molecular superposition program and is usually used for feasible docking of protein–protein, ligands with proteins, enzymes, and DNA (Mustard & Ritchie, 2005). The docking parameters were set to include ligand–DNA interactions and various non-covalent interactions as implemented in the program. The crystal structure of B-DNA (the duplex DNA d(CGCGAATTCGCG)2 dodecamer (PDB ID: 355D)) was taken from Protein Data Bank (PDB) and used for docking studies. All possible poses have been considered as starting points and the docking analysis was performed. Cell culture The MCF-7 breast cancer cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (Sigma, USA) and 10,000 IU of penicillin and 100 μg ml−1 of streptomycin as antibiotics (Himedia, Mumbai, India), in 96-well culture plates, at 37 °C, in a humidified atmosphere of 5% CO2, in a CO2 incubator (Forma, Thermo Scientific, USA). All experiments were performed using cells from passage 15 or less. Cytotoxicity assay (MTT assay) The surfactant–copper(II) Schiff base complexes were dissolved in DMSO, diluted in culture medium and used to treat the MCF-7 cell over a complex concentration range of 3 to 30 μg ml−1 for a period of 24 and 48 h. DMSO solution was used as the solvent control. A miniaturized viability assay using 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was carried out according to the method described by Mosmann (1983). To each well, 20 μL of 5 mg ml−1 MTT in phosphate-buffer (PBS) was added. The plates were wrapped with aluminum foil and incubated for 4 h at 37 °C. The purple formazan product was dissolved by addition of 100 μL of 100% DMSO to each well. The absorbance was monitored at 570 nm (measurement) and 630 nm (reference) using a 96-well plate reader (Bio-Rad, Hercules, CA, USA). Data were collected for four replicates each and used to calculate the respective means. The percentage of inhibition was calculated, from this data, using the formula:

Surfactant-copper(II) Schiff base complexes; DNA interaction, cytotoxic activity

5

½Mean absorbance of untreated cells ðcontrolÞ Mean absorbance of treated cells ðtestÞ 100 Mean absorbance of untreated cells ðcontrolÞ The IC50 value was determined as concentration of the complex that was required to reduce the absorbance to half that of the control, i.e. to kill 50% of the cells.


Hoechst 33258 staining The pathology of complex treated cell was detected by staining trypsinized cells (4.0 × 104 ml−1) with 1 μl of Hoechst 33258 (1 mg ml−1, aqueous) for 10 min at 37 °C. A drop of cell suspension was placed on a glass slide and a cover slip was laid over to reduce light diffraction. At random 300 cells were observed in a fluorescent microscope (Carl Zeiss, Germany) fitted with a 377–355 nm filter, at 400× magnification and the percentage of cells reflecting pathological changes was calculated. Data were collected for four replicates each and used to calculate the mean and the standard deviation. Comet assay DNA damage was detected by performing comet assay of Singh, MacCoy, Tice, and Schneider (1988). Cells were suspended in low-melting-point agarose in PBS and pipetted out to microscope slides pre-coated with a layer of normal-melting-point agarose. Slides were chilled on ice for 10 min and then immersed in lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 0.2 mM NaOH, pH 10.01, and Triton X-100), and the solution was kept for 4 h at 4 °C in order to lyse the cells and to permit DNA unfolding. Thereafter, the slides were exposed to alkaline buffer (300 mM NaOH, 1 mM Na2EDTA, pH > 13) for 20 min to allow DNA unwinding. The slides were washed with buffer (0.4 M Tris, pH 7.5) to neutralize excess alkali and to remove detergents before staining with ethidium bromide (5 μL in 10 mg ml−1). Photographs were obtained is a fluorescent microscope. One hundred and fifty cells from each treatment group were digitalized and analyzed using CASP software. The images were used to estimate the DNA content of individual nuclei and to evaluate the degree of DNA damage in the tail(comet) representing the fraction of total DNA in the tail. Results and discussion Spectral characterization The FT-IR spectra of free ligands displayed absorption in the region of 1635–1642 cm−1 which is attributed to the υC=N absorptions (Sinn & Harris, 1969). Coordination of the surfactant–Schiff base ligand to the copper(II) ion

through the azomethine nitrogen is expected to reduce the electron density on the azomethine nitrogen and thus lowers the υC=N absorption frequency after complexation (1628–1619 cm−1). The free ligand displayed absorption due to υO–H at 3100–3400 cm−1 which was absent in all the complexes indicating coordination of the phenolate oxygen of the ligand to the metal center. This is also supported by the appearance of new bands in the range 1331–1332 cm−1 which may be attributed to the C–O of the coordinated ligand and is at a higher frequency when compared to that of the free ligand (1270–1276 cm−1). All the complexes exhibited bands in the regions of 2913–2931 cm−1 and 2849–2851 cm−1 can be assigned to C–H asymmetric and symmetric stretching frequency of aliphatic CH2 chain of the amine (Kumar & Arunachalam, 2008) The electronic absorption spectra of all complexes in ethanol at room temperature showed two bands in the region of 200–300 nm and one band in the region of 310–330 nm which were, respectively, assigned to the π→π* and n→π* transitions. In all the complexes, the band observed in the region of 400–450 nm was attributed to the metal-to-ligand charge-transfer (MLCT) transitions. EPR spectra of all the complexes at room temperature showed single isotropic feature near giso = 2.04–2.07 and in frozen solution (77 K, in methanol) exhibited a typical four-line spectral pattern, g|| in the range of 2.25–2.30, g┴ = 2.08–2.03. The trend observed, g|| > g┴ > 2.02, for the present copper complexes is typical of a copper(II) (d9) ion in axial symmetry with the unpaired electron present in the dx2−y2 orbital. The molecular structure of the complex (2) was determined by adopting single crystal X-ray diffraction technique to confirm the coordination mode of the surfactant–Schiff base ligand in the complex and the geometry of the complex. The ORTEP view of the complex is shown in Figure 1. The summary of the data collection and the refinement parameters are given in Table S1 (ESI†) and selected bond lengths and bond angles are presented in Table S2 (ESI†). The crystal structure shows that the copper(II) ion is tetra-coordinated in a squareplanar geometry by two ligand molecules acting as monoanionic bidentate N, O donor. The surfactant–Schiff base ligand coordinates to the copper(II) ion via the azomethine nitrogen and the phenolate oxygen of salicylaldehyde unit in the ligand forming six-membered chelate ring. The bond lengths and bond angles are in good agreement with reported data on related Cu(II) Schiff base complexes bearing O, N donor (Sabarinathan, 2010). This complex was found to exist in the trans-planar configuration.

6



Figure 1.

ORTEP diagram of [Cu(sal-TA)2] (2) with the atoms represented as 30% anisotropic ellipsoids.

In the centrosymmetric molecule of copper Schiff base of surfactant, the copper atom has a square-planar coordination. The six-membered chelate ring exists in the sofa conformation. Determination of critical micelle concentration The CMC values of the complexes were determined conductometrically using a specific conductivity meter. The conductivity cell was calibrated with KCl solutions in the appropriate concentration range. The cell constant was calculated using molar conductivity data for KCl (Barthel, Feuerlein, Neueder, & Wachter, 1980). Various concentrations of surfactant–copper(II) complexes were prepared in the concentration range 10−7–10−2 M in aqueous solution. The conductivity of these solutions was measured at 298 K. The temperature of the thermostat was maintained constant to within ±0.01 K. The conductance was measured after thorough mixing and temperature equilibration at each dilution. The establishment of equilibrium was checked by taking a series of readings since 15-min until no significant change occurred. The CMC values were computed from the slopes of [complex] vs. specific conductance data. The complex concentration at which the micellization starts is evident from the change in the slope of the plot and this particular concentration is the CMC under the experimental conditions. The CMC values were determined by fitting the data points above and below the break to two equations of the form y = mx + c and solving the two Table 1.

The intrinsic binding constants (Kb), M−1 and the Stern–Volmer constant (Ksv) of the complexes (1–6) with DNA.

Complexes 1 2 3 4 5 6

equations simultaneously to obtain the point of interaction. Least-squares analysis was employed, and the correlation coefficients were greater than 0.98 in all the cases. The conductivity measurements at four different temperatures were repeated three times and the accuracy of CMC values (Table 2) was found to be within ±2% error. The CMC values of the surfactant–copper(II) complexes thus obtained are given in Table 1. These values are very low compared to those of the simple organic surfactant, dodecylammonium chloride (CMC = 1.5 × 10−2 M). It was found that CMC value of surfactant– copper(II)-hexadecylamine Schiff base complexes was lower than that of the corresponding surfactant–copper (II)-dodecylamine Schiff base complexes. This may be due to an increase in hydrophobic character of the molecule in the coordination sphere in the case of hexadecylamine. The same trend was noticed in the case of substituted salicylaldehyde surfactant–copper(II) Schiff base complexes also. Increasing of the side chain length of the synthesized surfactant-copper(II) Schiff base complexes caused decrease in their critical micelle concentration (CMC) values significantly. The lowest CMC value obtained for surfactant–copper(II)-hexacylamine and its derivative Schiff base complexes (3 and 6) while the highest value was obtained for surfactant–copper(II)dodecylamine derivative Schiff base complexes (1 and 4). On complexation the CMC values of the surfactant– Schiff base copper(II) complexes decreases when compared to that of surfactant alone. The complexes of hexadecylamine copper(II) Schiff base complex and

Kb (M−1) (±0.32) 3

4.32 × 10 5.44 × 103 8.04 × 103 5.12 × 103 5.81 × 103 1.55 × 104

Red shift wavelength for DNA (nm) 0 0 1 4 5 7

Percentage of hypochromism 15 23 30 22 25 32

Ksv (±0.11) 3

1.85 × 10 1.99 × 103 3.45 × 103 2.00 × 103 2.53 × 103 2.76 × 103

CMC (M) 1.87 × 10−4 1.43 × 10−4 1.19 × 10−4 1.98 × 10−4 1.62 × 10−4 1.34 × 10−4


1999), which implies that these complexes bind to DNA relatively less strongly than classical intercalators and partial intercalators. Also these values are less when compared to that of salen bimetallic copper(II) complex, [Cu2(salen)2] (Reddy & Shilpa, 2011), but the binding constant observed for these complexes are comparable with the other copper salicylaldehyde schiff base complexes reported in the literature (Dong, Li, Liu, Xu, & Wang, 2011; Zhang, Wang, Zhang, & Yang, 2010). All the surfactant–copper(II) Schiff base complexes can bind with DNA in different binding modes depending on the

1.5 9.50E-008 9.00E-008

1.2

8.50E-008

Abs.

8.00E-008

0.9

7.50E-008 0.00000 0.00005 0.00010 0.00015 0.00020 0.00025

[DNA] (M)

0.6

0.3

0.0 200

300

400

500

600

700

7

400

800

Figure 2. Absorption spectra of [Cu(5-OMe-sal-DA)2] (4) [cpx] = 50 μM in the absence and in the presence of increasing amount of CT-DNA, [DNA] = 0–250 μM, inset: plot of [DNA] vs [DNA]/(εa − εb).

derivatives (3 and 6) produced the lowest values which attributed to their long hydrophobic chains. Electronic absorption studies Electronic absorption spectra of our surfactant–copper(II) Schiff base complexes (1–6) in the presence of DNA clearly indicate hypochromism and red shift upon increasing the concentration of DNA (Figure 2). If a metal complex exhibits hypochromism along with red shift, it generally indicates intercalation of metal complex with DNA. In our case, we have observed only hypochromism without red shift and this extent of hypochromism is commonly associated with the strength of DNA interaction. The observed increase in the hypochromism is in the order (6) > (5) > (4) and (3) > (2) > (1). This binding strength indicated through binding constants was calculated using the equation, [DNA]/(εa − εf) = [DNA]/ (εb − εf) + 1/Kb (εb – εf) (Quiroga et al., 1998), where [DNA] is the concentration of DNA expressed in base pairs, εa, εf, and εb are the extinction coefficients for the apparent, free, and fully bound complex. Using this procedure plots of [DNA]/(εb − εf) vs. [DNA] were made and the ratio of slope to intercept gave the binding constant (Kb). The intrinsic binding constant value for these surfactant–copper(II) Schiff base complexes were calculated at below CMC (Table 1). However, the binding constant values are very much lower than the potential intercalators like ethidium bromide (Kb, 7.0 × 107 M−1 in 40 mM Tris/HCl, pH 7.9) (Waring, 1965) and the partially intercalating complexes like [Co(phen)2(dppz)]3+ (Kb = 9.09 × 105 M−1) (Arounagiri & Maiya, 1996) and [Ru(imp)2(dppz)]2+ (Kb = 2.19 × 107 M−1) (Liu et al.,

300

Intensity


Wavelength(nm)

200

100

0 500

550

600

650

700

Wavelength (nm)

Figure 3. Emission spectra of [DNA] = 150 μM in the absence and in the presence of increasing amount of [Cu(5OMe-sal-DA)2] [cpx] = 0−720 μM (4).

Figure 4. Circular dichroism spectra of CT-DNA (20 μM) at 25 °C in the absence and presence of the complexes; [cpx] = 15 μM (1–3), 30 μM (4–6).

8

J. Lakshmipraba et al. 2.0

copper(II) complexes (4–6) containing methoxy substituted salicylaldehyde ring showed higher binding constants than unsubstituted salicylaldehyde complexes (1–3) due to the hydrophobic nature of the methoxy salicylaldehyde-copper(II) center.

1 2 3 4 5 6

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

0.0

0.2

0.4

0.6

0.8

1.0


[cpx]/DNA]

Figure 5. Effect of the complexes (1–6) on the relative viscosities of CT-DNA at 30 °C [DNA] = 50 μM.

salicylaldehyde and substitution on salicylaldehyde ring and the aliphatic chain length. The methylene groups in the long chain amine part can bind through groove in the hydrophobic interior of DNA. Generally, salicylaldehyde metal complexes bind via major groove and are not expected to be classical intercalators (Reddy & Shilpa, 2011; Zhou, Li, & Pin, 2007). But in our complexes, the copper(II) salicylaldehyde head part is expected to bind via the major groove and the aliphatic long chain stack between the base pairs of DNA. On increasing the surfactant chain length (1–3) in salicylaldehyde, the hydrophobicity is expected to increase. The surfactant–

Figure 6.

Competitive ethidium bromide binding studies All the surfactant–copper(II) Schiff base complexes are non-emissive. In order to further investigate the binding modes of the copper(II) complexes (1–6) with DNA, competitive ethidium bromide (EB) binding studies were conducted. On adding the complexes (1–6) to EB-bound DNA the emission intensity was quenched by the complexes (Figure 3). To quantify the displacement, the concentration of the complex at which EB fluorescence decreases by 50% is calculated. In order to understand quantitatively the magnitude of the binding strength of all the complexes with DNA, the Stern–Volmer equation Table 2. IC50 value for surfactant–copper(II) Schiff base complexes (1–6). IC50 (μM) MCF-7 cell line 1 2 3 4 5 6 Cisplatin

Docked pose of complexes (2) (left) and (5) (right) with DNA.

24 h

48 h

46.2 ± 3.2 32.2 ± 2.2 24.0 ± 1.6 43.5 ± 3.6 28.4 ± 2.3 20.2 ± 1.1 45.7 ± 1.0

41.3 ± 2.3 25.3 ± 2.1 18.4 ± 1.4 35.4 ± 3.1 21.2 ± 1.9 14.3 ± 1.3 1.89 ± 0.06


ethidium ion. This must be due to the cationic surfactant complexes-induced perturbation of the organization of the DNA, leading to dissociation of the ethidium-bound DNA.

Circular dichroism absorption spectra CD spectroscopy is very sensitive to conformational changes in DNA. Positive CD band centered at 275 nm


was employed (Lakowicz & Weber, 1973). The Stern–Volmer equation is given by I0/I = 1 + Ksv[Q], where I0 and I represent the fluorescence intensities in the absence and in the presence of the quencher, respectively, [Q] is the concentration of quencher, Ksv is the Stern–Volmer quenching constant. The plot of I0/I vs. [Q] gives Ksv and the values are given in Table 1. The aliphatic tail chain length and size of head group with salicylaldehyde play vital roles in binding in these complexes. So, the complexes (1–6) are able to displace

9

Figure 7. Control and complex 2, 3, 5, and 6 treated (24 h, 48 h) MCF-7 cells stained with Hoechst 33528. Arrows point to cells with apoptotic morphology. Scale bar: 35 μm.

10


and negative CD band centered at 240 nm are the characteristics of B-DNA in solution (Figure 4). In the literature it is reported that the binding of surfactants with DNA changes the CD intensity due to conformational changes as a consequence of surfactant binding (Sathyaraj, Weyhermüller, & Nair, 2010). Addition of complexes (1) to (6) to CT-DNA, the CD spectra of DNA showed small increase in CD intensity of both positive and negative bands. The small change in intensity indicates minor groove mode of binding of our surfactant–copper(II) Schiff base complexes with DNA.


Viscosity measurement For the complexes (1–6), the plot of the relative specific viscosity (η−ηsurf/ηo)⅓ vs. [cpx]/[DNA] ratio shows a significant changes in the viscosity (Figure 5). The plot shows the capability of the complexes to decrease the viscosity of DNA depending upon the surfactant chain length and aromatic substitution. The increased degree of viscosity follows the order (6) > (5) > (4) and (3) > (2) > (1). The complexes (4–6) show higher viscosity compared to complexes (1–3) due to hydrophobicity of methoxy salicyladehyde– copper(II) complexes with the long chain amine. The results from viscosity measurements confirm the groove mode of binding of DNA. Molecular modeling The optimized geometries of the complexes are given in Figure S1 (ESI†) and the selected bond parameters with the crystal structure data are given in Table S2 (ESI†).

Figure 8.

The data presented in the Table S2 indicate that computed bond parameters are in good agreement with the crystal structure. To gain further insight into the nature of DNA binding of these complexes, molecular docking calculations were performed and the most probable docked poses are given in Figure 6 (more figures are given in Figure S2 (ESI†)). This figure clearly shows that the complex (2) approaches the DNA via minor groove, and the availability of the extended alkyl chain facilitates binding of the complex to the hydrophobic interior of DNA. The complex (5) interacts with DNA through minor groove and gains additional stabilization via hydrogen bonding of methoxy group with the minor groove. Thus, the complex (5) is found to have higher binding affinity than its analog {complex (2)}. In both the cases, the copper-salicylidine moiety was observed to be locked in the minor groove, thus allowing their extended alkyl chain to interact with the DNA bases in minor groove mode. It is interesting to note that the binding energy of complex (3) was 4.4 kcal mol−1 less than that of complex (1) and the order of binding efficiency is as follows: (3) > (2) > (1). Similarly, the complex (6) was found to have higher binding energy than complex (5) by 1.2 kcal mol−1, and the order of binding ability of these complexes is (6) > (5) > (4). It is worth noting that the methoxy complexes are found to have better binding ability than the corresponding unsubstituted analogs. This convincingly agrees with the outcome of other experiments (binding constant and spectroscopic studies). Thus, our molecular modeling studies throw light on the binding modes by which these complexes interact with DNA and complement the experimental observations.

Bar diagram showing percentage of normal, apoptotic, and necrotic cells as revealed in Hoechst 33258-staining.

11



Figure 9. Comet images of DNA double-strand breaks at 24 and 48 h treatment of surfactant–copper(II) Schiff base complexes (2, 3, 5 and 6). Scale bar: 35 μm.

Cytotoxicity studies MTT assay The cytotoxic effect of the surfactant–copper(II) Schiff base complexes was examined on cultured MCF-7 human breast carcinoma cells by exposing cells for 24 and 48 h to medium containing the respective complexes at 3–30 μg ml−1 concentration. The surfactant–copper(II) Schiff base complexes inhibited the growth of the cancer

cells significantly, in a dose- and duration-dependent manner. The cytotoxic activity was determined according to the dose values of the exposure of the complex required to reduce survival of the cells to 50% (IC50). The surfactant–copper(II) Schiff base complexes are highly cytotoxic to MCF-7 cancer cells, and the IC50 values of the complexes (Table 2) were lesser for 48 h treatment group than for 24 h treatment. The complexes (2–5) are found to be very active against cancer cells


12


Figure 10.

Extent of DNA damage in MCF-7 cancer cell population as defined by the percentage of DNA in the tail.

and their IC50 values (Table 2) obtained by plotting the cell viability against concentrations of the complexes reveal that all the complexes exhibit cytotoxicity higher than that of cisplatin (Jaividhya, Dhivy, Akbarsha, & Palaniandavar, 2012) for 24 h incubations. Also, the IC50 values at 48 h are lower than those at 24 h clearly indicating that they are dose- and time- dependent. The cytotoxic effect of the complex may be interpreted as due to its amphiphilic nature and, hence, would reduce the energy status in tumors and also alter hypoxia status in the cancer cell microenvironment, which are factors that would influence the antitumor activity. Assessment of cell death based on morphological features After treatment with the respective IC50 concentrations of the surfactant–copper(II) complexes for 24 and 48 h the cells were observed for cytological changes adopting Hoechst 33,258 staining (Kasibhatla, Finucane, Brunner, Wetzel, & Green, 2000). The observations revealed that the complexes brought about marginalization and/or fragmentation of chromatin, bi-nucleation, cytoplasmic vacuolation, nuclear shrinkage, cytoplasmic blebbing, and late apoptosis indication of dot-like chromatin and condensation in the MCF-7 breast cancer cells (Figure 7). These cytological changes indicate that the cells were committed to cell death, mostly apoptosis, and rarely necrosis. Data collected from the manual counting of cells with abnormal nuclear features are shown in Figure 8. Single-cell gel electrophoresis (comet assay) Among the different techniques used for measuring and analyzing DNA strand breaks in mammalian cells, singlecell gel electrophoresis (comet assay) is a rapid, simple,

visual, and sensitive technique to asses DNA fragmentation typical of DNA damage and of an early stage of apoptosis (Maire, Rast, Landkocz, & Vasseur, 2007). As shown in Figure 9, the images were used to estimate the DNA content of individual nuclei and to evaluate the degree of DNA damage representing the fraction of total DNA in the tail. Cells were assigned to five groups: 0–20% (intact), 20–40% (slightly damaged), 40–60% (damaged), 60–80% (highly damaged), and >80% (dead). The results revealed that DNA damage was induced in MCF-7 cancer cells by the copper(II) complexes, and the incidence was greater at 48 h of treatment than at 24 h (Figure 10). Overall, although all four complexes tested on breast cancer cell proved to be cytotoxic, the performance of complex (6) was highly efficient compared to the other three. Relying on the results of the DNA binding analysis, it is concluded that the cytotoxic effect is brought about by binding of minor groove and intercalating between the base pairs, DNA damage trigger the apoptosis machines, which is revealed in this study. Conclusion A series of six square-planar copper(II)–Schiff base complexes containing long aliphatic chain were synthesized. The characterization of the complexes was accomplished by analytical (elemental analysis, ESI-MS) and spectral (FT-IR, UV–vis, EPR) methods. X-ray diffraction study of the complex (2) confirms the N and O coordination mode of long chain Schiff base ligand and the geometry of the complex. DNA binding interaction was assessed for all these complexes using absorption, emission, and circular dichroism spectral methods as well as viscosity experiments. In all the complexes, copper(II) salicylaldehyde part binds via minor groove and the surfactant chain intercalates between the base


Surfactant-copper(II) Schiff base complexes; DNA interaction, cytotoxic activity pairs of DNA. On increasing the chain length of the long chain amine the binding affinity increased due to hydrophobicity of the long chain. Further, the binding mode of these complexes and the binding ability were confirmed the observations from the experiments by docking analysis. Our docking results confirm that complexes with methoxy salicylaldehyde show better binding ability than the unsubstituted analogs and, moreover, all these complexes use their extended alkyl chain to interact with DNA via minor groove. As the length of the alkyl chain increases, the binding ability also increases. Thus, the computed results corroborate the experimental findings. The complex (6) displays remarkable cytotoxicity against MCF-7 cancer cell line than its analogs, which is consistent with its ability to bind strongly with DNA. The cytotoxicity of these cationic surfactant complexes might be due to their ability to bind the minor groove of DNA through hydrophobic interaction of the extended alkyl chain, which would result in DNA strand breaks. This DNA damage, if not repaired, would lead to cell death accounting for the cytotoxic potential of the complexes. The efficient cytotoxicity is due to the hydrophobicity of the complex with long chain surfactant and substitution in the salicylaldehyde. Thus, suitable hydrophobic ligands can be used as elements in designing efficient metal-based anticancer agents. Supplementary material Electronic supplementary information (ESI) available: Experimental section; Spectral data of the ligand; Hydrogen bonding, optimized geometries of 2; docking image for all the complexes with DNA, crystal data, structure refinement, selected bond parameters for 2. CCDC reference number 885973 (for 2). The supplementary material for this paper is available online at http://dx.doi.10.1080/07391102.2014.918523. Funding We are grateful to the UGC-SAP and DST-FIST programmes of the Department of Chemistry, Bharathidasan University. Council of Scientific and Industrial Research (CSIR), New Delhi is acknowledged for the financial support [Scheme. No. 09/475 (0154)/2010-EMR-I dated. 09/02/2011] for Senior Research Fellowship to JLP. One of the authors, SA., thanks the DST [grant number SR/S1/IC-13/2009], CSIR [grant number 41-223/2012 (SR)], and UGC [grant number 01(2461)/11/EMR-II].

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Cleavage, Antimicrobial and Molecular Docking Studies.

Synthesis and Characterization with Antineoplastic, Biochemical, Cytotoxic, and Antimicrobial Studies of Schiff Base Cu(II) Ion Complexes.

Synthesis, characterization, DNA binding, DNA cleavage and antimicrobial studies of Schiff base ligand and its metal complexes.

DNA Cleavage, Cytotoxic Activities, and Antimicrobial Studies of Ternary Copper(II) Complexes of Isoxazole Schiff Base and Heterocyclic Compounds.

Novel nanohybrids of cobalt(III) Schiff base complexes and clay: synthesis and structural determinations.

Synthesis and characterization of Schiff base octaazamacrocyclic complexes and their biological studies.

Immobilization of cobalt(III) Schiff base complexes onto Montmorillonite-K10: Synthesis, experimental and theoretical structural determination.

Synthesis, interaction with DNA and antiproliferative activities of two novel Cu(II) complexes with Schiff base of benzimidazole.

Synthesis, characterization, antimicrobial, DNA cleavage, and in vitro cytotoxic studies of some metal complexes of schiff base ligand derived from thiazole and quinoline moiety.

Transition Metal(II) Complexes with Cefotaxime-Derived Schiff Base: Synthesis, Characterization, and Antimicrobial Studies.

Effects of copper ions on DNA binding and cytotoxic activity of a chiral salicylidene Schiff base.

Two new heterodinuclear Schiff base complexes: synthesis, crystal structure and thermal studies.

Synthesis, spectral characterization and DNA bindings of tridentate N2O donor Schiff base metal(II) complexes.

Synthesis, characterization and biological activity of some unsymmetrical Schiff base transition metal complexes.

A novel Schiff base: Synthesis, structural characterisation and comparative sensor studies for metal ion detections.

Schiff base hybrids as antiproliferative and phosphodiesterase 4 inhibitors: design, synthesis, and docking studies.

Synthesis, characterization, DNA interaction, antioxidant and anticancer activity studies of ruthenium(II) polypyridyl complexes.

Metal (II) Complexes Derived from Naphthofuran-2-carbohydrazide and Diacetylmonoxime Schiff Base: Synthesis, Spectroscopic, Electrochemical, and Biological Investigation.

Design, synthesis and SAR studies of novel 1,2-bis(aminomethyl)cyclohexane platinum(II) complexes with cytotoxic activity. Studies of interaction with DNA of iodinated seven-membered 1,4-diaminoplatinocycles.

Synthesis, spectroscopic studies, antimicrobial activities and antitumor of a new monodentate V-shaped Schiff base and its transition metal complexes.

Evaluation of DNA Binding, Cleavage, and Cytotoxic Activity of Cu(II), Co(II), and Ni(II) Schiff Base Complexes of 1-Phenylindoline-2,3-dione with Isonicotinohydrazide.

Crystal Structure, Cytotoxicity and Interaction with DNA of Zinc (II) Complexes with o-Vanillin Schiff Base Ligands.

protein of five transition metal complexes with Schiff base ligands.

Synthesis, Spectroscopic, Molecular Structure, and Antibacterial Studies of Dibutyltin(IV) Schiff Base Complexes Derived from Phenylalanine, Isoleucine, and Glycine.