Dinuclear cobalt(II) complexes of Schiff-base compartmental ligands: Syntheses, crystal structure and bio-relevant catalytic activities

Polyhedron 60 (2013) 102–109

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Dinuclear cobalt(II) complexes of Schiff-base compartmental ligands: Syntheses, crystal structure and bio-relevant catalytic activities Arpita Banerjee a, Averi Guha a, Jaydeep Adhikary a, Amitava Khan b, Krishnendu Manna b, Sanjit Dey b, Ennio Zangrando c,⇑, Debasis Das a,⇑ a b c

Department of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India Department of Physiology, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India Dipartimento di Scienze Chimiche e Farmaceutiche, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy

a r t i c l e

i n f o

Article history: Received 25 September 2012 Accepted 1 May 2013 Available online 17 May 2013 Keywords: Cobalt(II) complexes DNA cleavage Cytotoxicity Catechol oxidase

a b s t r a c t Three dicobalt(II) complexes, namely [Co2(L1H)(H2O)2(OAc)2](OAc)2 (1), [Co2(L2)(H2O)2(OAc)2](OAc) (2) and [Co2(L3)(H2O)2(OAc)2](OAc) (3) of the p-cresol based ‘‘end-off’’ compartmental ligands 2,6-bis(R-iminomethyl)-4-methyl-phenolato, where R = N-ethylpiperazine for L1, 2-ethylpyridine for L2 and N-ethylpiperidine for L3, have been synthesized and characterized by common physicochemical techniques, and in the case of complex 1 also by single crystal X-ray diffraction analysis. All the complexes show excellent catecholase-like activity, monitored not only with 3,5-di-tert-butylcatechol but also with tetrachlorocatechol, a substrate reluctant to be oxidized. To the best of our knowledge, to date no cobalt complex has been found in the literature to manifest such activity. The complexes are observed to interact efficiently with CT-DNA and on incubation (employing plasmid pTZ57/R/T DNA) they exhibit concentration dependent DNA cleavage activity. The mechanisms related to the DNA cleavage and catecholase-like activities have been investigated. The cytotoxicity of the complexes has also been examined through an MTT assay. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cobalt(II) Schiff-base complexes are interesting compounds because of their capability to bind dioxygen reversibly [1–3] and their catalytic activity in oxidation reactions [4–7], in particular the oxidation of phenols, alcohols, flavonoides, nitroalkanes, hydrazines or olefins [8–10]. In addition, cobalt complexes have gained importance because of their application as potential hypoxia-activated pro-drugs [11–13]. Although cobalt is an essential trace element for humans, the metal is genotoxic and mutagenic at higher concentrations, especially to mammalian cells, and the reason for this genotoxicity is not yet very clear. Two probable mechanisms of toxicity have been indicated: (i) the inherent ability of cobalt to produce reactive oxygen species and (ii) the inactivation of the DNA repair pathway due to replacement of divalent metals in active sites by cobalt. The interaction of small molecules with DNA represents an important area of investigation since it is widely accepted that DNA is the major intracellular target of anticancer, antimicrobial and antiviral metallodrugs. Therefore metal complexes binding to specific nucleobases or more widely interacting with DNA can represent interesting models in the development of antitumor or antibacterial agents. Thus there has been consider⇑ Corresponding authors. Tel.: +91 33 24837031; fax: +91 33 23519755 (D. Das). E-mail address: [email protected] (D. Das). 0277-5387/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.poly.2013.05.014

able interest in DNA endonucleolytic cleavage reactions activated by metal ions and the interaction of metal complexes with DNA is now well documented [14,15]. We are continuously engaged in elucidating the functional mechanisms of catechol oxidase and DNA cleavage, exploiting small coordination compounds of transition and post transition metals. We report herein the synthesis and comprehensive characterization of three cationic cobalt(II) complexes using the p-cresol based ‘‘end-off’’ compartmental ligands 2,6-bis(R-iminomethyl)-4-methyl-phenolato, where R = N-ethylpiperazine for L1, 2-ethylpyridine for L2 and N-ethylpiperidine for L3 (Scheme 1), along with their bio-relevant catalytic activities. All the complexes, besides being model compounds for the met form of the active site of catechol oxidase, exhibit a broad spectrum of catalytic functions of biological significance. In particular they cause the cleavage in plasmid DNA and are found to be cytotoxic towards the liver carcinoma Hep G2 cell line.

2. Experimental 2.1. Materials The chemicals N-(2-aminoethyl)piperazine, 2-(2-aminoethyl)pyridine, N-(2-aminoethyl)piperidine and cobalt acetate dihydrate were obtained from commercial sources and used as received. Solvents were dried according to standard procedures and

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observed reflections [18]. Beside an uncoordinated acetate anion, the DFourier map revealed the presence of two independent lattice water molecules. Hydrogen atoms were fixed at idealized positions, except those of the water molecules, which were located on the DFourier map and refined, constraining the O–H bond distances to 0.85 Å. The Squeeze utility [19] has been applied to the data, due to the voids (of ca 210 Å3) present in the unit cell and likely filled by disordered solvent molecules: the final high R factor is likely attributable to this feature. Crystallographic data (1): C29H58Co2N6O15, M = 848.67, monoclinic, space group P2/c, a = 13.016(3), b = 13.662(3), c = 12.122(3) Å, b = 95.290(4)°, V = 2146.4(9) Å3, Z = 2, Dc = 1.313 g/cm3, l(Mo Ka) = 0.838 mm 1, F(0 0 0) = 896, h range = 1.49–25.00°. Final R1 = 0.0813, wR2 = 0.1770, S = 1.174 for 258 parameters and 10367 reflections, 3785 unique [Rint = 0.0659], of which 3106 with I > 2r(I), max positive and negative peaks in DF map 1.037 and 0.827 e Å 3.

2.3. Syntheses

Scheme 1. Synthetic outline of the Schiff-base ligands and their cobalt(II) complexes.

distilled prior to use. 2,6-Diformyl-4-methylphenol was prepared according to the literature method [16]. Calf Thymus DNA was purchased from Sigma Chemical Co. (USA) and the purity was determined by measuring the ratio of absorbance at 260 nm to that at 280 nm, which was found to be P1.8. pET28a supercoiled plasmid DNA, agarose, and buffers were used as received. Elemental analyses (carbon, hydrogen and nitrogen) were performed using a Perkin Elmer 24 °C elemental analyzer. Infrared spectra were recorded on KBr disks (400–4000 cm 1) with a Shimadzu FTIR8400S. Electronic spectra (800–200 nm) were recorded at 27 °C using a Shimadzu UV-3101PC in dry methanol. The magnetic susceptibilities were measured at 300 K using a Magway MSB Mk magnetic susceptibility balance made by Sherwood Scientific Ltd. The electrospray mass spectra were recorded on a MICROMASS Q-TOF mass spectrometer. 1H NMR spectra (300 MHz) were recorded in (CD3)2SO solvent on a Bruker AV300 Supercon NMR spectrometer using the solvent signal as the internal standard in a 5 mm BBO probe.

2.3.1. [Co2(L1H)(H2O)2(CH3CO2)2](CH3CO2)2 (1) A methanolic solution (5 mL) of N-(2-aminoethyl)piperazine (0.258 g, 2 mmol) was added dropwise to a heated methanolic solution (10 mL) of 2,6-diformyl-4-methylphenol (0.164 g, 1 mmol) and the resulting solution was refluxed for half an hour. Then, a methanolic solution (10 mL) of Co(CH3COO)2
2H2O (0.498 g, 2 mmol) was added and the resulting solution was stirred overnight. The resulting dark red solution was kept in a CaCl2 desiccator in the dark and after a few days crystals suitable for X-ray data analysis were obtained from this solution. (Yield 71%). Anal. Calc. for C29H58Co2N6O15: C, 41.04; H, 6.89; N, 9.90. Found: C, 41.01; H, 6.85; N, 9.88%. leff = 5.56 lB at 300 K. UV–Vis data (kmax, nm (e, M 1cm 1)): 377 (5408), 548 (76). 2.3.2. [Co2(L2)(H2O)2(CH3CO2)2](CH3CO2) (2) This was prepared by adopting a similar procedure as for complex 1, but 2-(2-aminoethyl)pyridine (0.258 g, 2 mmol) was used instead of N-(2-aminoethyl)piperazine.

2.2. X-ray crystal structure determination A crystal of suitable size of 1 was selected from the mother liquor and protected by paratone oil, then mounted on a glass fiber using epoxy resin. Intensity data were collected at 293 K using Mo Ka radiation (k = 0.7107 Å) on a Bruker SMART APEX diffractometer equipped with a CCD area detector. Integration and reduction of the data set were performed with SAINT software [17a], and an empirical absorption correction was also applied with the program SADABS [17b] The structure was solved by direct methods [18] and refined by the full-matrix least-squares method on F2 with all

Fig. 1. ORTEP diagram (ellipsoids at 50% probability) depicting the dinuclear complex cation with the atom numbering scheme of the crystallographic independent part.

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leff = 5.53 lB at 300 K.

Table 1 Selected bond lengths (Å) and angles (°) for complex 1. Co–O(1) Co–O(2)i Co–O(3)

2.038(4) 2.093(4) 2.068(3)

Co–O(4) Co–N(2) Co–N(3)

2.117(4) 2.357(4) 2.060(4)

O(1)–Co–O(2)i O(1)–Co–O(3) O(1)–Co–O(4) O(1)–Co–N(2) O(1)–Co–N(3) O(2)i–Co–O(3) O(2)i–Co–O(4) O(2)i–Co–N(2)

89.60(17) 100.21(14) 85.83(17) 93.81(15) 168.80(18) 94.87(14) 174.61(17) 82.83(16)

O(2)i–Co–N(3) O(3)–Co–O(4) O(3)–Co–N(2) O(3)–Co–N(3) O(4)–Co–N(2) O(4)–Co–N(3) N(2)–Co–N(2) Co–O(3)–Coi

97.07(17) 88.75(15) 165.79(13) 88.21(16) 94.61(17) 87.00(17) 78.20(17) 109.8(2)

Primed atoms at

x + 1, y,

z + 1/2.

(Yield 75%). Anal. Calc. for C29H36Co2N4O9: C, 49.58; H, 5.16; N, 7.97. Found: C, 49.52; H, 5.13; N, 7.95%. leff = 5.55 lB at 300 K. UV–Vis data (kmax, nm (e, M 1cm 1)): 379 (5421), 553 (82). ESI-MS m/z: 609.03 a.m.u. (calc. for {[Co2(L2)(CH3CO2)2] + H}+ m/z: 608.4230 a.m.u.)

2.3.3. [Co2(L3)(H2O)2(CH3CO2)2](CH3CO2) (3) This was synthesized by following a similar procedure as for complex 1, but N-(2-aminoethyl)piperidine (0.258 g, 2 mmol) was used instead of N-(2-aminoethyl)piperazine. (Yield 73%). Anal. Calc. for C29H48Co2N4O9: C, 48.74; H, 6.77; N, 7.84. Found: C, 48.72; H, 6.75; N, 7.81%.

UV–Vis data (kmax, nm (e, M 1cm 1)): 389 (5477), 556 (93). ESI-MS m/z: 621.78 a.m.u. (calc. for {[Co2(L3)(CH3CO2)2] + H}+ m/z: 620.5178 a.m.u.) 2.4. DNA cleavage activity To assess the DNA cleavage activity of the complexes, supercoiled pET28a plasmid DNA (300 ng for each set) was incubated with different concentrations of the complexes in Tris EDTA buffer (pH 8.0) solution at 37 °C for 45 min. The reaction in each case was stopped with 1 loading dye and resolved in 1% agarose gel. Upon gel electrophoresis of the reaction mixture, a concentration dependent DNA cleavage was observed. The band intensities of the supercoiled (SC) (Form I), nicked circular (NC) (Form II), and linear (L) (Form III) forms were estimated through densitometric analysis (Quantity one, BIORAD). 2.5. Bio activity on tumor cells by an MTT assay Human hepatocellular carcinoma cells (Hep G2) were cultured as a monolayer, where they were grown in a suspension in the nutrient medium, at 37 °C under a humidified air atmosphere with 5% CO2. Hep G2 (2  105 cells per well) were seeded into 96-well microtiter plates. Twenty-four hours later, after the cell adherence, six different concentrations of the investigated complex were added to the wells, except for the control cells to which a nutrient medium only was added. Nutrient medium RPMI-1640 was supplemented with L-glutamine (3 mM), streptomycin (100 lg/mL),

Fig. 2. Crystal packing down the c axis showing the H-bonding scheme, giving rise to a 3D network (big red spheres indicate lattice water molecules). (Color online.)

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penicillin (100 IU/mL), 10% heat inactivated (56 °C) FBS and 25 mM HEPES, adjusting the pH to 7.2 with a bicarbonate solution. The assay was performed as follows: 20 lL of MTT solution (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL in phosphate buffered saline) were added to each well, incubating the samples for further 4 h. Then, 100 lL of 10% SDS were added to extract the insoluble product formazan, resulting from the conversion of the MTT dye by viable cells. The number of viable cells in each well was proportional to the intensity of the absorbance of light, which was estimated in an ELISA plate reader at 595 nm.

primary amine. A methanolic solution (5 mL) of N-(2-aminoethyl)piperazine (0.258 g, 2 mmol) was added dropwise to a heated methanolic solution (10 mL) of 2,6-diformyl-4-methylphenol (0.164 g, 1 mmol) with constant stirring. The resulting mixture was refluxed for an hour and after cooling the solvent was reduced by a rotary evaporator. The solid mass thus obtained was recentralized several times from dry methanol to get the pure ligand. 1H NMR (300 MHz, DMSO-d6, 25 °C) d: 14.72 (br, 1H, OH), 9.14 (s, 2H, C@N), 7.62 (s, 2H, Ar), 3.78–3.54 (t, 4H, CH2), 2.89–2.467 (m, 20H, CH2), 2.35 (s, 3H, CH3). The Schiff-base ligand L2 was prepared using the same procedure as for L1, where 2-(2-aminoethyl)pyridine was used instead of N-(2-aminoethyl)piperazine (0.258 g, 2 mmol). 1 H NMR (300 MHz, DMSO-d6, 25 °C) d: 14.68 (br, 1H, OH), 9.09 (s, 2H, C@N), 7.61–7.12 (m, 10H, Ar), 3.65 (t, 4H, CH2), 2.35 (s, 3H, CH3), 1.69 (t, 4H, CH2). The Schiff-base ligand L3 was prepared using the same procedure as for L1 where N-(2-aminoethyl)piperidine was used instead

3. Results and discussion 3.1. Synthesis and characterization The Schiff-base ligands were synthesized and characterized by H NMR as shown in the S.I. file. The Schiff-base ligand L1 was prepared by a condensation reaction between an aldehyde and a

1

3,5-DTBQ

4.0

2.8 2.6

3.5

3,5-DTBQ

2.4 2.2

3.0

Absorbance

1.8

2.0 1.5

1.6 1.4 1.2 1.0 0.8

Complex 1

1.0

Complex 2

0.6 0.4

0.5

0.2

3,5-DTBC

0.0 300

400

3,5-DTBC

0.0

500

600

700

300

800

400

500

600

700

Wavelength(nm)

Wavelength(nm)

(b)

(a) 3,5-DTBQ

1.8 1.6 1.4 1.2

Absorbance

Absorbance

2.0

2.5

1.0 0.8

Complex 3

0.6 0.4 0.2

3,5-DTBC

0.0 300

400

500

600

700

800

Wavelength(nm)

(c) Fig. 3. UV–Vis spectral changes of complexes 1 (a), 2 (b) and 3 (c) in methanol upon addition of 100 fold of 3,5-DTBC, observed at fixed intervals of time.

800

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of N-(2-aminoethyl)piperazine (0.258 g, 2 mmol). 1H NMR (300 MHz, DMSO-d6, 25 °C) d: 14.43 (br, 1H, OH), 8.92 (s, 2H, C@N), 7.36 (s, 2H, Ar), 3.93–3.68 (t, 4H, CH2), 2.81–2.48 (m, 24H, CH2), 2.21 (s, 3H, CH3). The complexes have been synthesized by adopting the template synthesis technique, treating a methanolic solution of Co(CH3COO)2
2H2O with the Schiff-base formed in situ between 2,6-diformyl-4-methylphenol and the diamines. The complexes show bands due to the C@N stretch in the range 1635–1649 cm 1 and a skeletal vibration in the range 1549–1560 cm 1. Additionally broad bands centred in the range 1375–1390 cm 1 are attributable to the acetate groups. The electronic spectra of the three complexes are similar. They exhibit an intense absorption band at 380 nm assigned to the p–p⁄ transition associated with the azomethine group and a

prominent band at 550 nm corresponding to the charge transfer bands from the filled pp orbital of the phenolic oxygen to the vacant orbital of Co(II), as observed by other researchers [20,21] with similar systems. The magnetic moments of the complexes at 300 K, reported in the experimental section, are very close to the spin only value of 5.48 lB per Co(II) (evaluated on the basis of l2 = 2lCo2 using lCo = 3.88 lB), which indicates the high spin state of the cobalt ions in the present complexes [22]. We were successful in synthesizing single crystals of complex 1 (vide infra), but failed for complexes 2 and 3. We have performed an ESI-MS study for further conformation of molecular compositions of 2 and 3. The ESI-MS study of 2 and 3 in methanol shows that the most abundant peak, called the base peak, is at m/z = 609.03 and 621.78 a.m.u. which corroborate well with their respective

2.0

TCQ

2.0

1.5

Absorbance

Absorbance

1.5

1.0

Complex 1

TCQ 1.0

Complex 2 0.5

0.5

TCC

TCC 0.0

0.0

300

400

500

600

700

800

300

400

500

600

700

800

Wavelength(nm)

Wavelength(nm)

(a)

(b) 2.0

Absorbance

1.5

1.0

TCQ

0.5

Complex 3 TCC

0.0 300

400

500

600

700

800

Wavelength(nm)

(c) Fig. 4. UV–Vis spectral changes of complexes 1 (a), 2 (b) and 3 (c) in methanol upon addition of 100 fold of TCC, observed at fixed intervals of time.

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monopositive species of the composition {[Co2(L2)(CH3CO2)2] + H}+ (calc. m/z = 608.4230 a.m.u.) and {[Co2(L3)(CH3CO2)2] + H}+ (calc. m/z = : 620.5178 a.m.u.), respectively. 3.2. Description of the crystal structure The X-ray structure determination of complex 1 reveals that the crystallographic independent part includes half of the dicobalt complex, an acetate anion and two lattice water molecules. An ORTEP view of the cationic l-phenoxo-di-l-acetato-dicobalt(II) complex with the atom labelling scheme is shown in Fig. 1. The complex is located on a crystallographic two fold axis passing between the cobalt atoms and bisecting the phenolato ring. The metal ions exhibit a distorted octahedral coordination sphere comprising in the equatorial plane the phenoxido-bridging oxygen, the oxygen of one bridging acetato group, the imine and amine nitrogen donors, the latter being from the piperazine fragment. An aqua and an acetato oxygen complete the coordination sphere at the axial positions. Thus the two carboxylate groups bridge the cobalt(II) ions in a syn–syn coordination mode and form a dihedral angle of 81.0(3)°. This arrangement, having a Co–O(3)–Coi bond angle of 109.8(2)°, leads to an intermetallic separation of 3.384(2) Å. The Co–N(amine) bond distance (2.357(4) Å) is significantly longer than the Co–N(imine) one (2.060(4) Å), justified not only by the different N hybridization, but likely to avoid steric clashes between the piperazine and the bridging acetate groups. The Co–O bond lengths vary from 2.038(4) to 2.117(4) Å, the longest value being related to the axial aqua ligand (Table 1). The me-

tal ions are significantly displaced by 0.75 Å from the phenolato mean plane. The piperazine moieties, with the expected chair conformation, are protonated, as confirmed by the H bonds involving the NH2 groups with uncoordinated carboxylate oxygens (N


O distances of 2.71 and 2.85 Å). Actually for the sake of charge balance, only one of the two amines should be protonated but, due to the imposed crystallographic symmetry, one H atom on the protonated piperazine was fixed at half occupancy. The crystal packing shows an extended H-bonding scheme (Fig. 2), leading to a three dimensional (3D) supramolecular arrangement that outlines channels oriented down the c axis, likely filled by solvent molecules.

3.3. Catechol oxidase activity The catecholase activity of the complexes was evaluated using 3,5-DTBC (3,5-di-tert-butylcatechol) and TCC (tetrachlorocatechol) as substrates. The reactions were carried out in methanol at 25 °C under aerobic conditions and were monitored by means of UV–Vis spectroscopy, following the same technique as we reported earlier [23]. The time dependent spectral changes of the complexes upon addition of 3,5-DTBC and TCC in methanol are shown in Figs. 3 and 4 respectively, which clearly demonstrate the gradual increment of the concentration of 3,5-DTBQ (3,5-di-tert-butylbenzoquinone, kmax = 400 nm) and TCQ (tetrachlorobenzoquinone, kmax = 430 nm) with time. These results suggest that all the complexes are active in catalyzing the oxidation of both 3,5-DTBC and TCC. Here it is

300000 250000 Complex 1

1/V

200000

Complex 2 Complex 3

150000

Linear (Complex 1) Linear (Complex 2)

100000

Linear (Complex 3)

50000 0 0

200

400

600

800

1000

1200

1/[S] Fig. 5. The Lineweaver–Burk plots (double reciprocal plot) for complexes 1–3 with 3,5-DTBC in methanol medium.

4500000

1/V

4000000 3500000

Complex 1

3000000

Complex 2

2500000

Complex 3

2000000

Linear (Complex 1)

1500000

Linear (Complex 2)

1000000

Linear (Complex 3)

500000 0 0

200

400

600

800

1000

1200

1/[S] Fig. 6. The Lineweaver–Burk plots (double reciprocal plot) for complexes 1–3 with TCC in methanol medium.

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The kinetic study indicated that complex 1 exhibits the highest activity amongst the three catalysts. From this observation we can state that the positively charged piperazine moiety present in the ligand system of complex 1 is creating a favourable path to facilitate the catalyst (Co-center) substrate (catechol) interaction, a prerequisite for the catalytic activity. A similar proposal has now been well accepted, especially to explain the activity of CuZn-superoxide dismutase (SOD), where Cu(II) lies at the bottom of a narrow channel and the positively charged arginine and lysine residues are supposed to play a role in attracting the anions and guiding them into the channel [24–26].

Table 2 Enzyme kinetics data. Wavelength (nm)

Vmax (M s

1

With 3,5-DTBC as the substrate 1 382 2 392 3 397

1.24  10 1.28  10 1.19  10

4

With TCC as the substrate 1 425 2 421 3 422

1.01  10 1.14  10 1.09  10

5

Complex

)

5 5

6 6

KM (M)

kcat (h

2.45  10 1.78  10 2.39  10

3

4.04  10 1.54  10 3.36  10

3

1

)

4.47  102 4.59  101 4.29  101

3 3

3.64  101 4.11 3.93

3 3

3.4. DNA cleavage activity

worth noting that the oxidation of TCC to TCQ catalyzed by cobalt complexes has never been reported in the literature to date. The kinetics for the oxidation of the substrates 3,5-DTBC and TCC were determined by the initial rate method at 25 °C. The concentration of the substrates 3,5-DTBC and TCC was always kept at least 10 times larger than that of the complex and the increase of the respective quinones concentration was determined at 400 and 430 nm, respectively for each complex. Solutions of the substrate with concentrations ranging from 0.001 to 0.05 mol dm 3 were prepared from a concentrated stock solution in methanol. 2 mL of the substrate solution were poured into a 1 cm spectrophotometer quartz cell thermostated at 25 °C. Then 0.04 mL of 0.005 mol dm 3 complex solution was quickly added to it so that the ultimate concentration of the complex became 1  10–4 3 mol dm . The dependence of the initial rate on the concentration of the substrates, monitored spectrophotometrically at the respective wavelengths, is given in the S.I. file. The initial rates method follows a first-order dependence on complex concentration since it showed saturation kinetics at higher substrate concentrations. For this reason, a treatment based on the Michaelis–Menten model was seemed to be appropriate. The values of the Michaelis binding constant (KM), maximum velocity (Vmax) and rate constant for the dissociation of the substrate (i.e., turnover number, kcat) were calculated for each complex from the Lineweaver–Burk graph 1/V vs 1/[S] (Figs. 5 and 6) using the equation 1/V = {KM/Vmax}{1/[S]} + 1/Vmax. The enzyme kinetics data are listed in Table 2.

All three complexes are observed to interact with DNA in a similar fashion and Fig. 7 shows the electrophoresis results for the interaction of complex 1 with DNA as a representative example. From the assay it is clear that 1 shows a differential DNA cleavage pattern in agarose gel. There is a dose dependent rise in forms II and III. From 100 lM concentration upwards the DNA cleavage activity is very prominent, and at 200 lM form II is prominent. However, we found a decrease in the fluorescence intensity of the supercoiled (SC) (Form I), the nicked circular (NC) (Form II), and the linear (L) (Form III) forms by increasing the complex concentration with respect to the control. It is well established that two mechanisms are likely involved in DNA cleavage: (i) hydrolytic cleavage and (ii) free radical mechanism. In our case, the DNA cleavage was observed to be inhibited to a significant extent when the reaction was carried out at complex concentration of 100 lM (Fig. 8) in the presence of increasing concentrations of NaN3 (a singlet oxygen scavenger). On the other hand, the use of DMSO, as hydroxyl free radical scavenger, did not inhibit the DNA cleavage. The above facts reveal that the ability of the complex to generate singlet oxygen is most likely responsible for the cleavage of DNA. 3.5. Bioactivity Cell survival was determined by an MTT assay treating HepG2 cancerous cells with the complexes for 72 h. From this assay we

Complex 1 Con

20

50

70

100

150

200 µM

Fig. 7. Gel electrophoresis study showing the cleavage of supercoiled pET28a plasmid DNA (300 ng in each lane) with different concentrations of complex 1, in Tris EDTA buffer (pH 8.0) at 37 °C for 45 min.

Complex1+ DMSO Con

Com

50

75

100

Complex1+Sodium azide 50

75

100 µM

Fig. 8. Lane 1: pET28a plasmid DNA (300 ng in lane). Lane 2: +100 lM of the complex 1. Lanes 3–5: +100 lM of the complex 1 in presence of radical scavenger DMSO. Lanes 6–8: +100 lM of the complex in presence of NaN3 with their densitometric analysis of the Gel photograph.

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Acknowledgement We thank CSIR, New Delhi for financial support (01/(2464)/11/ EMR-II dt 16-05-2011 to DD). We are also thankful to Dr. (Mrs.) Krishna Das Saha, Indian Institute of Chemical Biology for help on the MTT assays. Appendix A. Supplementary data CCDC 902477 contains the supplementary crystallographic data for complex 1. These data can be obtained free of charge via http:// www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: [email protected]. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.poly.2013.05.014. References Fig. 9. Cell cytotoxicity estimated by an MTT assay and the effects of the complexes on Hep G2 cells viability (data expressed as% of cell death with respect to untreated controls).

Table 3 Half maximal inhibitory concentration (IC50) in lM of the complex. Complex

Half maximal inhibitory concentration (IC50) in (lM)

1 2 3 cis-Platin

20.69 18.55 22.68 24.1

[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

can derive that the complexes inhibited the proliferation of HepG2 cancerous cell (Fig. 9): (%) = (OD of the control group OD of the treatment group)/OD of the control group  100%, where OD is that observed at 595 nm. Table 3 displays the IC50 values (in lM) of complexes 1–3 and that of cis-platin [27] towards the Hep G2 cell line. The data clearly suggest that the Co(II) based complexes, especially complexes 2 and 1, are more effective than cis-platin on Hep G2 cancerous cells. From the MTT assay it is apparent that the complexes are able to inhibit the proliferation of the Hep G2 cell since they inhibit the proliferation of liver carcinoma cells by more than 50% at 100 lM concentration. At this concentration the supercoiled DNA was nicked. Thus the complexes might be inducted as an effective agent for antitumor activity. 4. Conclusions

[14] [15] [16] [17]

Three dinuclear Co(II) complexes of p-cresol based ‘‘end-off’’ compartmental ligands, reported here, are observed to exhibit catecholase-like activity, not only with 3,5-DTBC (model substrate) but also with TCC, a species which is very hard to oxidize. To the best of our knowledge this report demonstrates for the first time the oxidation of TCC catalyzed by a dinuclear Co(II) complex. Amongst the three complexes, the one having the 2,6-bis(N-ethylpiperazine-iminomethyl)-4-methyl-phenol ligand system (1) shows the highest catalytic activity. The positive charge centers generated on the piperazine nitrogen of the ligand might be instrumental in its extraordinary efficiency. Moreover the complexes are observed to display concentration dependent DNA cleavage and complex 1 shows the highest activity. The active oxygen species generated by the complex are most probably responsible for this behavior. Based on these results complex 1 may be regarded as a promising antitumor agent.

[18] [19] [20] [21] [22] [23] [24] [25]

[26] [27]

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