Chapter IV. Reactions of the tridentate chelating hydrazone Schiff bases benzoic acid (2-hydroxybenzylidene)-hydrazide

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1 104 Chapter IV Bivalent complexes of benzoic acid(2-hydroxy-benzylidene)-hydrazide ligand: Synthesis, structure and comparative in vitro evaluations of biological perspectives Abstract Reactions of the tridentate chelating hydrazone Schiff bases benzoic acid (2-hydroxybenzylidene)-hydrazide (H 2 L) (1) obtained from salicylaldehyde and benzhydrazide with [NiCl 2 (PPh 3 ) 2 ] and [CoCl 2 (PPh 3 ) 2 ] afforded respective metal hydrazone complexes of the composition [Ni(L)(PPh 3 )] (2) and [Co1(L) 2 ][Co2(H 2 O) 4 (OPPh 3 ) 2 ] (3). The molecular structure of both complexes 2 and 3 determined by single crystal X-ray diffraction revealed that the complex 2 is neutral in charge with distorted square planar geometry. However, complex 3 was found to be distorted octahedral geometry. All the synthesised compounds 1-3 were studied by interaction with calf thymus DNA (CT DNA) and bovine serum albumin (BSA). In addition in vitro free radical scavenging and cytotoxic potential of all the synthesised compounds were also investigated. Transition metal ions with variable oxidation states possess an important role in bioinorganic chemistry and redox enzyme systems and may provide the basis of models for active sites of biological systems. Artificial metallonucleases are of high demand as cellular regulators of DNA for therapeutic or biochemical purposes. 1 Metal complexes containing site-specific substructures and multiple reactive sites constitute a group of promising candidates for nuclease because of their electronic and structural advantages. 2 Moreover, these complexes generate highly cationic species that favor the electrostatic attraction to the anionic phosphate backbone of DNA. Incorporation of DNA-targeting moiety into the ligands can improve the selectivity of metallonucleases. 3 Therefore, there is an exigency to identify effective metal-based therapeutics particularly those overcome both inherent and acquired resistance to drug therapy and show improved therapeutic properties, stimulating the ongoing investigations of alternative molecular targeted metalbased drugs. 4 Anticancer drugs targeting biomolecule represent a rational advancement in the modern drug discovery. It is a general fact that considerable achievement is realized in enhancing the targeting and efficiency of metal-chelators through ligand design that can

2 105 significantly alter the biological properties by modifying reactivity or substitution related inertness and play a major role in their binding to DNA. Large planar ligands promote intercalative binding of the metal complexes to DNA, 5-10 whereas non-planar ligands or ligands that do not have extended planarity promote groove binding, particularly with octahedral metal complexes Since, nucleic acids are very important genetic substances, they provide different binding sites and binding modes for covalent and noncovalent interactions Interest in the binding of metal complexes to DNA has been motivated not only by a desire to understand the basics of these interaction modes but also for the development of metal complexes as antiinflammatory, antifungal, antibacterial and anticancer agents. Hence, much attention has been devoted on the design of metal-based complexes that bind to DNA Serum albumins, the most abundant proteins in blood plasma, have many physiological functions. Since most of the administered drugs bind extensively and reversibly to serum albumin to form protein-drug complexes, the biological activity of the drug such as the overall distribution, metabolism and efficacy in the body are correlated with their affinities towards serum albumin. Among serum albumins, bovine serum albumin (BSA) is an appropriate protein model for studying the interaction between serum albumins and drugs because of its medically important, unusual ligandbinding properties, ease of availability, low cost and structural homology with human serum albumin (HSA). 24,25 Therefore, the interaction between serum albumins and drugs not only provide useful information of the structural features that determine the therapeutic effectiveness of drugs, but also is crucial to study the pharmacological response of drugs and design of dosage forms. Hence, it has become an interesting research field that attracted the attention of biologists, chemists, pharmacists and therapists A great many hydrazones and their metal complexes have displayed diverse spectra of biological and pharmaceutical activities, such as anticancer, antitumor and antioxidative activities, as well as the inhibition of lipid peroxidation etc Hence, our present investigation focuses on the design and synthesis of Ni and Co complexes containing the hydrazone ligand obtained from salicylaldehyde and benzhydrazide as an anchoring group that can provide well defined binding environment as well as increases

3 106 the stability of the resultant metal complexes. All the synthesized compounds have been subjected to various experiments to assess their interacting ability with DNA/BSA and free radical scavenging and cytotoxic potentials. Experimental section Materials Reagent grade chemicals were used without further purification in all the synthetic work. All solvents were purified by standard methods. The compounds NiCl 2 6H 2 O, CoCl 2 6H 2 O, triphenylphosphine, benzhydrazide and salicylaldehyde were purchased from Sigma-Aldrich Chemie and Alfa Aesar, respectively and used as received. Calf thymus (CT DNA) and bovine serum albumin (BSA) were purchased from Himedia. The plasmid supercoiled (SC) puc19 DNA was purchased from Bangalore GeNei, Bangalore, India. The human cervical cancer cell line (HeLa), human liver hepatocellular carcinoma cells (HepG-2), human skin cancer cell line (A431) and normal NIH 3T3 mouse embryonic fibroblasts was obtained from National Centre for Cell Science (NCCS), Pune, India. All other chemicals and reagents used for DNA binding/cleavage, protein binding, antioxidant and cytotoxicity assays were of high quality. Physical measurements Microanalyses (% C, H and N) of the ligand (H 2 L) 1 and metal complexes 2 and 3 were performed on a Vario EL III CHNS analyzer. The infrared spectra of the ligand and the complexes were recorded using KBr pellets on a Nicolet Avatar spectrometer in the range of cm -1. The electronic absorption and emission spectra of the ligand and its complexes were recorded in DMSO:buffer solution using a Jasco V-630 spectrophotometer and Jasco FP 6600 spectrofluorometer, respectively. 1 H NMR spectrum of the ligand in CDCl 3 was recorded on a Bruker AMX 500 MHz spectrometer using tetramethylsilane as an internal standard. The X-ray diffraction data of the complexes 2 and 3 were collected at 293 K with MoKα radiation (λ = Å) using a Bruker Smart APEX II CCD diffractometer equipped with graphite monochromator. The structure was solved by direct method and

4 107 refined using full-matrix least squares (on F 2 ) SHELXS97 and SHELXL97 37 and the graphics were produced using PLATON All the non-hydrogen atoms were refined anisotropically and the hydrogen atoms were positioned geometrically and refined as riding model. Synthesis of starting precursor complexes The precursor metal complexes [NiCl 2 (PPh 3 ) 2 ] and [CoCl 2 (PPh 3 ) 2 ] were prepared according to literature procedures. 39,40 Preparation of dichlorobis(triphenylphosphine)nickel(ii), [NiCl 2 (PPh 3 ) 2 ] A solution of NiCl 2 6H 2 O (2.38 g; 0.01 mol) in water (2 cm 3 ) was diluted with glacial acetic acid (50 cm 3 ) and a solution of triphenylphosphine (5.25 g; 0.02 mol) in glacial acetic acid was added. An olive green microcrystalline precipitate was formed. This when kept in contact with mother liquor for 24 hours to yield blue crystals, which were filtered, washed with glacial acetic acid and then dried in a vaccum desiccator. The complex was further washed with petroleum ether (60-80 o C) to remove traces of free triphenylphosphine. Yield: 76%. Colour: Green, mp: 257 C. Preparation of dichlorobis(triphenylphosphine)cobalt(ii), [CoCl 2 (PPh 3 ) 2 ] A solution of CoCl 2.6H 2 O (2.38g; 0.01mol) in water (2 cm 3 ) was diluted with glacial acetic acid (50 cm 3 ) and a solution of triphenylphosphine (5.25g; 0.02 mol) in glacial acetic acid was added. A sky blue microcrystalline precipitate was formed. This when kept in contact with mother liquor for 24 hours to yield the sky blue crystals, which were filtered, washed with glacial acetic acid and then dried in a vaccum desiccator. The complex was further washed with petroleum ether (60-80 o C) to remove traces of free triphenylphosphine. Yield: 79%. Colour: Blue; mp: 225 C.

5 108 Synthesis of hydrazone ligand The reaction involved in the synthesis of hydrazone ligand is given in scheme 4.1. H OH O H 2 N H N O EtOH + Reflux, 5h H OH N H N O Ligand (H 2 L) 1 Scheme 4.1 Synthesis of hydrazone ligand (H 2 L) 1. Synthesis of benzoic acid-(2-hydroxy-benzylidene)-hydrazide ligand H 2 L (1) The ligand benzoic acid (2-hydroxy-benzylidene)-hydrazide (H 2 L) (1) was prepared by refluxing a mixture of salicylaldehyde (0.122 g; 1 mm) and benzhydrazide (0.136 g; 1 mm) in 50 ml of absolute ethanol as given in scheme 4.1. After 5h, the reaction mixture was cooled to room temperature and the solid obtained was filtered, washed several times with distilled water and recrystallized from EtOH to afford the ligand 1 in pure form. Yield: 85%. Colour: Pale yellow; mp: 133 C. Anal. Found (%) for C 14 H 12 N 2 O 2 (Mol wt = ): C, 69.09; H, 4.62; N, Calculated (%): C, 69.99; H, 5.03; N, Selected IR bands (ν max in cm -1 ): 3389 (OH); 3218 (NH); 1624 (C=O); 1571(C=N); 1074 (N N); 1203 (phenolic, C OH). UV-visible (DMSO-buffer): λ max (nm): 286, 295 & 322 (ILCT). 1 H NMR (500 MHz, CDCl 3 ): δ (ppm) = (s, 1H, OH); (s, 1H, NH); 8.24 (s, 1H, CH=N); (m, 9H, Ar H). Synthesis of metal(ii) hydrazone complexes in scheme 4.2. The reactions involved in the synthesis of metal hydrazone complexes are given

6 109 [NiCl 2 (PPh 3 ) 2 ] MeOH, KOH, Reflux 5 h H N N O O Ni H OH N H N (H 2 L) 1 O [CoCl 2 (PPh 3 ) 2 ] MeOH, KOH, Reflux 5 h P O O P PPh 3 2 H 2 O OH Co 2 H 2 O OH 2 H. N N O O Co. O P O O. H2 O N N H Scheme 4.2 Synthesis of metal hydrazone complexes 2 and 3. 3 Synthesis of [Ni(L)(PPh 3 )] (2) Complex 2 was synthesised by refluxing equimolar amount of appropriate starting precursor [NiCl 2 (PPh 3 ) 2 ] (0.653 g; 1 mm) with the ligand 1 (0.226 g; 1 mm) in methanol (40 ml) in the presence of KOH (scheme 4.2) for 5h. After cooling the reaction mixture to room temperature, the precipitate formed was filtered, washed with methanol and dried in vacuum. Purity of the complex was checked by TLC following which tetragonal shaped red crystals of complex 2 suitable for single crystal X-ray diffraction studies were obtained by recrystallisation in dichloromethane. Yield: 65%. Colour: Red; mp: 184 C. Anal. Found (%) for NiC 32 H 25 N 2 O 2 P (Mol wt = ): C, 68.02; H, 4.12; N, Calculated (%): C, 68.73; H, 4.51; N, Selected IR bands (ν max in cm -1 ): 1598 & 1520 (C=N N=C); 1340 (phenolic C O); 1298 (enolic C O); 1096 (N N). UV-visible (DMSO-buffer): λ max (nm): 304 (ILCT); 374 (LMCT). 1 H NMR (500 MHz, CDCl 3 ): δ (ppm) = 8.36 (d, 1H, HC=N); (m, 24H, Ar H).

7 110 Synthesis of [Co 1 (L) 2 ][Co 2 (H 2 O) 4 (OPPh 3 ) 2 ] (3) Complex 3 was synthesised by refluxing equimolar amount of appropriate starting precursor [CoCl 2 (PPh 3 ) 2 ] (0.653 g; 1 mm) with the ligand 1 (0.226 g; 1 mm) in methanol (40 ml) in the presence of KOH (scheme 4.2) for 5h. After cooling the reaction mixture to room temperature, the precipitate formed was filtered, washed with methanol and dried in vacuum. Purity of the complex was checked by TLC following which brown coloured sugar like crystals of complex 3 suitable for single crystal X-ray diffraction studies were obtained by recrystallisation in dichloromethane. Yield: 29%. Colour: Reddish brown; mp: 198 C. Anal. Found (%) for Co 2 C 82 H 75 N 4 O 12 P 3 (Mol wt = ): C, 64.93; H, 4.68; N, Calculated (%): C, 64.83; H, 4.98; N, Selected IR bands (ν max in cm -1 ): 1600 & 1519 (C=N N=C); 1339 (phenolic C O); 1267 (enolic C O); 1094 (N N). UV-visible (DMSO-buffer): λ max (nm): 327 (ILCT); 384 (LMCT). DNA binding studies Electronic absorption experiments Absorption titration experiments were performed by maintaining a constant concentration of the test compound (25 μm) but with varying nucleotide concentration (0-25 μm) in buffer. After each addition of DNA to the test solutions, the absorption readings were noted. The data were then fit to the following equation to obtain intrinsic 41 binding constant K b [DNA]/[ε a -ε f ] = [DNA]/[ε b -ε f ] + 1/K b [ε b -ε f ] where, [DNA] is the concentration of DNA in base pairs, ε a is the extinction coefficient observed at a given DNA concentration, ε f is the extinction coefficient of the free complex in solution, and ε b is the extinction coefficient of the complex when fully bound to DNA. A plot of [DNA]/[ε a -ε f ] versus [DNA] gave a slope 1/[ε a -ε f ] and Y intercept equal to 1/K b [ε b -ε f ], respectively. The intrinsic binding constant K b is the ratio of the slope to intercept.

8 111 Competitive binding experiments The apparent binding constant (K app ) of ligand 1 and complexes 2 and 3 with DNA was determined by a fluorescence spectral technique using ethidiumbromide (EB)- bound CT DNA solution in Tris buffer (ph, 7.2). The changes in fluorescence intensities at 601 nm (546 nm, excitation) of EB bound to DNA were recorded with respect to increasing concentration of the test compounds. EB was non-emissive in Tris buffer (ph, 7.2) due to fluorescence quenching of the free EB by the solvent molecules. In the presence of DNA, EB showed enhanced emission intensity due to its intercalative binding to DNA. A competitive binding of the ligand and its corresponding complexes to CT DNA resulted in the displacement of the bound EB thereby decreasing its emission intensity. The quenching constant (K q ) was calculated using the classical Stern-Volmer equation, 42 I 0 /I = K q [Q] + 1 where, I 0 and I are the respective emission intensities in the absence and presence of quencher, K q is the quenching constant, [Q] is the quencher concentration. K q is the slope, obtained from the plot of I 0 /I vs [Q]. The apparent binding constant (K app ) has been calculated from the equation, K EB [EB] = K app [complex] The complex concentration was obtained from the value at a 50% reduction of the fluorescence intensity of EB, K EB = M -1 and [EB] = 5 μm. DNA cleavage experiments The extent of DNA cleavage induced by the test compounds was monitored by agarose gel electrophoresis. A solution containing 25 µl of puc19 DNA (1 µg), HCl (50 mm, ph 7.5), NaCl (50 mm), the metal complex (35 μm), and H 2 O 2 (60 μm) was incubated at 37 C for 1 h. Subsequently, 4 µl of 6X DNA loading buffer containing 0.25% bromophenol blue, 0.25% xylene cyanol and 60% glycerol was added to the test solution and then mixed with 1% agarose gel containing 1.0 μg/ml of ethidium bromide. Electrophoresis was performed at 5 V/cm for 2 h in a TBE buffer and the bands were visualized under UV light and photographed. The cleavage efficiencies were measured by determination of the ability of each complex to convert the super coiled DNA (SC) to the

9 112 open circular form (OC). After electrophoresis, the proportion of both the cleaved and uncleaved DNA in each fraction was quantitatively estimated on the basis of the band intensities using the BIORAD Gel Documentation System. The intensity of each band relative to that of the plasmid supercoiled form was multiplied by 1.43 to take account of the reduced affinity for ethidium bromide. 43 Protein binding studies Binding of the free hydrazone ligand 1 and metal complexes 2 and 3 with bovine serum albumin (BSA) was studied using fluorescence spectra recorded by excitation at 280 nm and corresponding emission at 345 nm assignable to that of free bovine serum albumin (BSA). The excitation and emission slit widths and scan rates were constantly maintained for all the experiments. Samples were carefully degassed using pure nitrogen gas for 15 minutes by using quartz cells (4 1 1 cm) with high vacuum Teflon stopcocks. Stock solution of BSA was prepared in 50 mm phosphate buffer (ph, 7.2) and stored in dark at 4 C for further use. Concentrated stock solutions of the test compounds were prepared by dissolving them in DMSO:phosphate buffer (5:95) and diluted suitably with phosphate buffer to get required concentrations. 2.5 ml of BSA solution (1 μm) was titrated by successive additions of a 2 μl stock solution of compounds 1, 2 and 3 (10-3 M) using a micropipette. Synchronous fluorescence spectra was also recorded using the same concentration of BSA and the synthesised compounds as mentioned above with two different λ (difference between the excitation and emission wavelengths of BSA) values such as 15 and 60 nm. Antioxidant studies ABTS cationic radical scavenging activity Total antioxidant activity assay using ABTS cationic radical was studied according to the following procedure. 44 ABTS (2,2'-Azino-3-ethylbenzthiazoline-6- sulfonic acid diammonium salt) was dissolved in water to a 5 mm concentration and its cationic radical was produced by reacting with 5 mm potassium persulfate. The resulting mixture was kept in dark at room temperature for h before use. Prior to assay, the solution was diluted in ethanol (about 1:79 v/v) and equilibrated to 30 ºC to give an

10 113 absorbance of 0.70±0.02 at 734 nm. After the addition of 2 ml of diluted ABTS cationic radical solution to different concentration (10-50 µm) of the compounds, the absorbance was taken at 30 ºC exactly 30 min after the initial mixing and the reaction mixture without test sample was used as control. Superoxide radical scavenging activity The superoxide (O - 2 ) radical scavenging assay was done based on the capacity of the compounds to inhibit formazan formation by scavenging the superoxide radicals generated in riboflavin-light-nbt system. 45 Each 3 ml reaction mixture contained 50 mm sodium phosphate buffer (ph, 7.6), 20 µg riboflavin, 12 mm EDTA and 0.1 mg NBT. Reaction was started by illuminating the reaction mixture with different concentration of the test compounds (10-50 µm) for 90 seconds. Immediately after illumination, the absorbance was measured at 590 nm. The entire reaction assembly was enclosed in a box lined with aluminum foil. The above reaction mixture without test sample was used as control. Hydroxyl radical scavenging activity The hydroxyl (OH) radical scavenging activity of compounds 1-3 have been investigated using the Nash method. 46 In vitro hydroxyl radicals were generated by Fe 3+ / ascorbic acid system. The detection of hydroxyl radicals was carried out by measuring the amount of formaldehyde formed from the oxidation reaction with DMSO. The formaldehyde produced was detected spectrophotometrically at 412 nm. A mixture of 1.0 ml of iron-edta solution (ferrous ammonium sulphate (0.331 mm) and EDTA (0.698 mm), 0.5 ml of EDTA solution (0.048 mm) and 1.0 ml of DMSO (10.83 mm) DMSO (v/v) in 0.1 M phosphate buffer, ph 7.4) were sequentially added to the test tubes containing the test compounds with different concentrations in the range of µm. The reaction was initiated by adding 0.5 ml of ascorbic acid (1.25 mm) and incubated at C for 15 min in a water bath. After incubation, the reaction was terminated by the addition of 1.0 ml of ice cold TCA (107 mm). Subsequently, 3.0 ml of Nash reagent was added to each tube and left at room temperature for 15 min. The reaction mixture

11 114 without sample was used as control. The intensity of the colour formed was measured spectrophotometrically at 412 nm against reagent blank. In the case of above three assays, all the tests were run in triplicate. The percentage of activity was calculated using the formula, % of activity = [(A 0 - A C ) / A 0 ] 100 where, A 0 and A C are the absorbance in the absence and presence of the tested compounds respectively. The IC 50 can be calculated using the percentage of activity results. Cytotoxicity The in vitro cytotoxicity assay (IC 50 ) was performed on the human cervical cancer cell line (HeLa), human liver hepatocellular carcinoma cells (HepG-2), human skin cancer cell line (A431) and normal NIH 3T3 mouse embryonic cell line. The tumor cell lines used in this work were grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS) and the NIH 3T3 fibroblasts were grown in Dulbeccos Modified Eagles Medium (DMEM) containing 10% FBS. For the screening experiments, the cells were seeded into 96 well plates in 100 ml of the respective medium containing 10% FBS, at a plating density of 10,000 cells/well. The cells were incubated at 37 C in 5% CO 2 and 95% air at a relative humidity of 100% for 24 h prior to the addition of the complexes. The complexes were solubilized in dimethylsulfoxide and diluted in the respective serum free medium. After 24 h, 100 ml of the medium containing the test compounds with various concentrations (e.g. 15, 30, 60, 120, 250 and 500 μm) was added and incubated at 37 C in an atmosphere of 5% CO 2 and 95% air with 100% relative humidity for 48 h. All measurements were made in triplicate and the medium containing no test complexes served as the control. After 48 h, 15 ml of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37 C for 4 h. The medium with MTT was then flicked off and the formazan crystals that had formed were solubilized in 100 ml of DMSO and the absorbance at 570 nm was measured using a micro plate reader. The % cell inhibition was determined using the following formula, % Cell inhibition = Abs (sample) / Abs (control) 100.

12 115 The IC 50 values were calculated from the graph plotted between % cell inhibition and concentration of the test compounds. Results and discussion The reactions between [MCl 2 (PPh 3 ) 2 ] (where, M = Ni(II) (or) Co(II)) and the hydrazone ligand benzoic acid-(2-hydroxy-benzylidene)-hydrazide (1) in 1:1 molar ratio yielded complexes of the type [Ni(L)(PPh 3 )] (2) and [Co1(L) 2 ][Co2(H 2 O) 4 (OPPh 3 ) 2 ] (3) (scheme 4.1). The analytical data of the above said complexes are in good agreement with the proposed molecular formulae with 1:1 and 1:2 metal to ligand stoichiometries (presented under the experimental part), respectively. All the three synthesised compounds are quite stable in air and light and soluble in most of the organic solvents such as MeOH, EtOH, CH 2 Cl 2, CHCl 3, DMF and DMSO and are well characterised using several physico-chemical techniques. Infrared spectra The IR spectra of the metal hydrazone complexes were compared with those of respective free hydrazone ligand in the region cm -1. The spectrum of the ligand 1displayed characteristic absorption bands in the range of 3389, 3218, 1624, 1571, 1074 and 1203 cm -1 due to ν (O H), ν (N H), ν (C=O), ν (C=N), ν (N N) and ν (C OH) vibrations, respectively. The bands due to ν (N H) and ν (C=O) vibrations of the free ligand was absent for complexes 2 and 3, thus indicating that enolisation and deprotonation had taken place prior to coordination. This fact was further confirmed by the appearance of two new bands in the spectra of complexes around cm -1 and cm -1 that corresponds to ν (C=N N=C) and ν (C O) stretching vibrations, respectively. The bands attributed to ν (C=N) stretching was shifted to higher frequencies while a positive shift of about ~20-22 cm -1 was observed for ν (N N) stretching vibration in comparison with that of their respective free ligands, thus implying that the nitrogen atom of the azomethine group is coordinated to the metal in these complexes. All these facts suggested that the hydrazone ligand behaves as a binegative tridentate (ONO) chelating ligand in all the three complexes 2 and 3.

13 116 Electronic spectra The electronic absorption spectrum of ligand 1 exhibited three absorption bands respectively at 286, 295 and 322 nm that are assigned to the intra ligand charge transfer (ILCT) transitions of the type π π* and n π*. 47 But, the spectrum of complex 2 showed only two absorptions at 304 and 374 nm corresponding to π π* and ligand to metal charge transfer (LMCT) transitions, respectively, whereas complex 3 exhibited absorptions at 327 and 384 nm, assigned as due to n π* and LMCT transitions. 48,49 1 H NMR spectra The 1 H NMR spectra of the hydrazone ligand 1 exhibited a sharp and broad singlet at and ppm is attributed to the phenolic OH and NH protons. A sharp singlet appeared at 8.24 ppm is assigned to azomethine proton (CH=N) and a multiplet in the range of ppm region are assigned to the aromatic protons of the phenyl [Ni(L)(PPh 3 )] (2) Fig H NMR spectrum of complex 2.

14 117 moieties of the ligand. Upon complexation complex 2 shows the following: the disappearance of the signals at and ppm due to the OH and NH protons indicated that these protons underwent deprotonation prior to the coordination with the metal ion. In addition, the azomethine proton also deshielded and appeared at 8.36 ppm as a doublet for 2, suggesting the participation of azomethine nitrogen (CH=N) in coordination with the nickel ion (Fig. 4.1). The appearance of doublet may be due to the nuclear quadrupolar effect of the nitrogen atom. The aromatic protons of 2 were found in the range of ppm. X-ray crystallography From the elemental analyses, IR, electronic and 1 H NMR spectroscopic studies it is understood that both the complexes 2 and 3 are not structurally similar. Hence, the exact structure of both the complexes has been studied by single crystal X-ray diffraction method. Crystallographic study of [Ni(L)(PPh 3 )] (2) and [Co1(L) 2 ][Co2(H 2 O) 4 (OPPh 3 ) 2 ] (3) An attempt to synthesis 1:1 metal ligand stoichiometric hydrazone complexes led to an interesting observation that the hydrazone showed a variable behaviour between nickel and cobalt systems encouraged us to carry out a systematic study on the structure of the complexes. It was found that the reaction of H 2 L with [NiCl 2 (PPh 3 ) 2 ] yielded the complex [Ni(L)(PPh 3 )] (2) with distorted square planar geometry whereas a similar reactions with [CoCl 2 (PPh 3 ) 2 ] produced a novel complex of the composition [Co(L) 2 ][Co(H 2 O) 4 (OPPh 3 ) 2 ] (3). The molecular structure and its corresponding atom numbering scheme for complexes 2 and 3 are shown in Figs. 4.2 and 4.3, respectively with the relevant bond distances and angles collected in Tables 4.1 and 4.2. The crystals of complex 2 belong to the monoclinic crystal system with P21/c space group. The coordination geometry around nickel was distorted square planar in [Ni(L)(PPh 3 )], in which the dianionic ligand acted as a planar tridentate forming a five membered and a six membered metallocycles involving the nickel ion. As expected, the hydrazone ligand is bonded to the nickel ion via phenolate oxygen, the enolate oxygen and the imine nitrogen atom, the corresponding bite angles being 95.8(1) and 83.6(1) for [O1 Ni1 N1] and

15 118 [N1 Ni1 O2]. The ONO donor set of atoms of the ligand occupies three coordination sites of the square planar geometry and the fourth coordination site is occupied by the phosphorus atom of the triphenylphosphine. The unequal bond lengths of [Ni1 N1] 1.847(2) Å, [Ni1 O1] 1.814(2) Å, [Ni1 O2]1.812 (2) Å and [Ni P1] (7) Å as well as unequal bond angles generated by these bonds at the Ni-acceptor center indicate a distortion from the perfect square planar geometry. These values are similar to those reported earlier for similar type of metal(ii) hydrazones. 50 Fig. 4.2 Molecular structure of complex 2, with displacement ellipsoids drawn at the 25% probability level. The molecular structure of 3 shown in Fig. 4.3 is quite different from that of 2 in all its parameters and nature. Complex 3, consists of an anionic complex [Co(L) 2 ] in which the cobalt metal center (Co is labeled as Co1) is coordinated by two units of binegative tridentate hydrazone ligand (H 2 L) lead to a distorted octahedral environment with 1:2 metal to ligand stoichiometry. The complex was formed with a charge of 2- by preserving the cobalt in its +2 oxidation state. To compensate the charge 2- on the [Co1(L) 2 ], a tetra aqua Co(II) cationic complex [Co(H 2 O) 4 (OPPh 3 ) 2 ] in which case Co is labeled as Co2 was formed and present in the unit cell. Out of four coordinated water molecules two of them showed missing hydrogen atoms. Further, a molecule of phosphonium oxide as well as water with missing hydrogen was also found in the same

16 119 unit cell. The four equatorial positions of cobalt atom in the anionic complex is occupied by phenolate -O and the enolate -O atoms and the two axial positions by imine -N of the deprotonated H 2 L ligand in its enol form. However, in the cationic complex part, four equatorial positions were occupied by water molecules and the two axial positions by phosphonium oxide through the oxygen atoms in a neutral manner form an octahedral geometry. In the above said dianionic part [Co1(L) 2 ], the coordination behaviour of the two ligands are very similar and indicate that the ligand adopt the enol form and gets deprotonated prior to the coordination with the metal ion. However, the crystallographic dimensions of the ligands were non-equivalent and hence they are only chemically equivalent. The two oxygen and nitrogen atoms exhibited ligand-metal-ligand bite angles of 83.2(3) [O1 Co1 N2], 90.1(3) [N2 Co1 O4], 88.3(3) [O2 Co1 N4] and 83.0(3) [N4 Co1 O3]. The bond length of [Co1 N4] 1.887(7)Å, [Co1 O4] 1.886(6)Å, [Co1 O2] 1.876(6)Å, [Co1 N2] 1.890(6)Å, [Co1 O1] 1.911(5)Å, [Co1 O3] 1.854(7)Å are comparable in length to those of the earlier reports. 51 The trans angles, namely [N4 Co1 N2] 172.5(3), [O2 Co1 O1] 178.8(3) and [O4 Co1 O3] 178.6(3) are Fig. 4.3 Molecular structure of complex 3, with displacement ellipsoids drawn at the 25% probability level. (Hydrogen atoms and water molecule was omitted for clarity).

17 120 constrained from the ideal value of 180. This showed that the complex [Co1(L) 2 ] possess distorted octahedral geometry. But in the case of cationic cobalt part, the coordinated ligands (triphenylphosphonium oxide and water) were chemically and crystallographically equivalent. Hence, the symmetry was generated with in the molecule and the corresponding bond length [Co2 O3W] 2.153(6)Å, [Co2 O5] 2.057(4)Å, [Co2 O2W] 2.117(7)Å, [P1 O5] 1.492(4)Å and bond angles of [O5 Co2 O2W] 91.1(2), [O5 Co2 O3W] 93.0(2), [O2W Co2 O3W] 92.26(2), [P1 O5 Co2] 144.7(3). In addition, there exist intermolecular hydrogen bonding between the above discussed cationic cobalt complex with the respective anionic cobalt hydrazone complexes those were present on either side of the former species as presented in the Fig Similarly an independent hydrogen bonding was also observed between the free triphenylphosphonium oxide and water molecules occupying the crystal lattice that were not shown in Fig Fig.4.4 Molecular structure of complex 3 with intermolecular hydrogen bonding at the 25% probability level. (Hydrogen atoms, water and phosphoniumoxide molecules was omitted for clarity).

18 121 Table 4.1 Crystal structure data of complexes 2 and 3. Description Complex 2 Complex 3 Empirical formula C 32 H 25 N 2 NiO 2 P Co 2 C 82 H 75 N 4 O 12 P 3 Formula weight Temperature (K) (Å) Crystal system Monoclinic Triclinic Space group P21/c P 1 Cell dimensions a (Å) b (Å) c (Å) ( ) ( ) ( ) (2) (1) (2) (1) 90 Z 4 1 hkl limits -21<=h=>21-15<=k=>8-23<=l=> (5) (6) (9) (3) (3) (3) -10<=h=>10-14<=k=>14-20<=l=>20 D calcd (Mg/m 3 ) F(000) Crystal size (mm 3 ) Independent reflections Data / restraints / parameters 9769 / 0 / / 5 / 743 Goodness-of-fit on F Final R indices [I>2sigma(I)] R1 = , wr2 = R1 = , wr2 = R indices (all data) R1 = , wr2 = R1 = , wr2 = Table 4.2 Selected bond lengths (Ǻ) and bond angles ( ). Complex 2 Complex 3 Bond lengths Bond angles Bond lengths Bond angles Ni1 N (2) N1 Ni1 O1 95.8(1) Co1 N (6) O1 Co1 N2 83.4(3) Ni1 O (2) N1 Ni1 O2 83.6(1) Co1 O (6) N2 Co1 O2 96.0(3) Ni1 O (2) O2 Ni1 P (7) Co1 O (5) O4 Co1 N4 95.8(3) Ni1 P (7) P1 Ni1 O (7) Co1 N (6) N4 Co1 O3 83.2(3) N1 N (3) O1 Ni1 O (1) Co1 O (5) O1 Co1 O3 88.5(2) N2 C (4) N1 Ni1 P (7) Co1 O (6) O3 Co1 O2 90.5(3) C8 O (4) C6 C7 N1 N (3) N1 N (8) O2 Co1 O4 90.4(2) C1 O (4) C9 C8 N2 N (2) N4 N3 1.38(1) O4 Co1 O1 90.7(2) C1 C (4) C9 C8 O2 Ni (2) C1 O (9) N4 Co1 O1 92.2(3) C6 C (4) C2 C1 O1 Ni (2) C15 O3 1.33(1) N4 Co1 O2 88.3(3) C7 N (4) Co2 O2W 2.117(7) N2 Co1 O3 90.5(3) Co2 O3W 2.153(6) N2 Co1 O4 90.1(3) Co2 O (4) N2 Co1 N (3) P1 O (4) O1 Co1 O (3) O3 Co1 O (3) O2W Co2 O3W 92.2(2) O5 Co2 O3W 93.03(2) O5 Co2 O2W 91.1(2) P1 O5 Co (3) C23 C22 N4 N (7) N4 N3 C15 C (7) C9 C8 N2 N (7) N2 N1 C1 C (7)

19 122 Pharmacological evaluations DNA binding studies Electronic absorption measurements The electronic absorption spectrum of ligand 1 exhibited three absorption bands (286, 295 and 322 nm) whereas its nickel (2) and cobalt (3) complexes displayed only two absorption bands namely at 304, 374, 327 and 384 nm respectively that were discussed in the previous section. Monitoring the changes in absorption spectra of the test compounds 1, 2 and 3 upon the incremental addition of DNA is one of the most widely used methods to determine overall binding constants. In general, a compound that binds to DNA through intercalation results in hypochromism and bathochromism involving strong stacking interaction between an aromatic chromophore and the base pairs of DNA. The electronic absorption spectra of 1, 2 and 3 in the absence and presence of CT DNA are shown in Fig Upon the addition of calf thymus DNA solution to the compounds 1, 2 and 3 a decrease in molar absorptivity (hypochromism, 13-61%) has been observed with respect to all the above said absorption bands without any shift in wavelength. Among them, the hydrazone ligand (1) showed hypochromism of 13.36, and 14.20% respectively at 286, 295 and 322 nm, whereas the bands exhibited by complex 2 showed hypochromism of about 60.42% (304 nm) and 60.56% (374 nm) and 3 displayed about and 19.08% at 327 and 384 nm respectively indicating the strong binding of them with DNA. In order to compare the DNA-binding affinity of these compounds quantitatively, their intrinsic binding constants were calculated by the changes monitored in absorption at higher energy band with increasing concentrations of DNA was calculated and are found to be M -1, M -1 and M -1 corresponding to the hydrazone ligand 1 and complexes 2 and 3, respectively indicating their strong binding to DNA, that are comparable with other reported intercalating complexes The magnitude of binding constant value clearly showed that the complex 2 bound more strongly with CT DNA than the free hydrazone ligand 1 and complex 3.

20 Absorbance [DNA]/( a - f ) x 10-9 M Absorbance Absorbance [DNA]/( a - f ) x 10-8 M [DNA]/( a - f ) x 10-9 M [DNA] x 10-6 M [DNA] x 10-6 M Wavelength (nm) Wavelength (nm) [DNA] x 10-6 M Wavelength (nm) Fig. 4.5 Electronic absorption spectra of ligand 1 and its nickel (2) and cobalt (3) complexes (25 μm) in the absence and presence of increasing amounts of CT DNA (2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5 and 20.0, 22.5 and 25 μm). Arrows show the changes in absorbance with respect to an increase in the DNA concentration (Inset: Plot of [DNA]/(ε a -ε f )) vs [DNA]. Competitive binding measurements In order to further prove the intercalating mode of binding, the competitive DNA binding of the test compounds 1, 2 and 3 have been studied by monitoring the changes in emission intensity of ethidium bromide (EB) bound to CT DNA as a function of concentration of the compound added. EB was non-emissive in Tris-HCl buffer solution (ph, 7.2) due to fluorescence quenching of free EB by the solvent molecules. In the presence of DNA, EB showed enhanced emission intensity due to its intercalative binding to DNA. When hydrazone ligand 1 and complexes 2 and 3 were added to DNA

21 Intensity I 0 /I Intensity Intensity I 0 /I I 0 /I 124 pretreated with EB {[DNA]:[EB] = 2:1}, the DNA-induced emission intensity of EB was decreased with shift in wavelength (Fig. 4.6). It is a well established phenomenon that addition of a second DNA binding molecule would quench the EB emission by either replacing the EB molecules that were originally bound to DNA (if it binds to DNA more strongly than EB) or accepting an excited state electron from EB. Since, the complexes 2 and 3 possess Ni and Co ions coordinated with the ligand, they efficiently compete with EB for intercalative binding sites on DNA by replacing them, which was evidenced through quenching of emission intensity of DNA-bound EB. The apparent binding constant, K app have been calculated from the following equation, K EB [EB] = K app [compound] [Q] x 10-6 M [Q] x 10-6 M Wavelength (nm) Wavelength (nm) [Q] x 10-6 M Wavelength (nm) Fig. 4.6 Emission spectra of DNA-EB in the presence of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150 µm of ligand 1 and its nickel (2) and cobalt complexes (3). Arrow indicates the changes in the emission intensity as a function of ligand concentrations. Inset: Stern-Volmer plot of the fluorescence titration data corresponding to ligand 1 and its nickel (2) and cobalt complexes (3).

22 125 The fluorescence spectra with Stern-Volmer plot (I 0 /I versus [Q]) of EB bound- DNA quenched by free ligand 1 and corresponding metal complexes 2 and 3 are shown in Fig. 4.6 as insets. The observed linearity in the plot supported the fact that the quenching of EB bound to DNA by the test compounds are in good agreement with the linear Stern-Volmer equation. The calculated apparent binding constant values, K app of compounds 1, 2 and 3 were M -1, M -1 and M -1, whereas, the quenching constant K q was determined to be M 1, M -1 and M -1. These data revealed the higher quenching efficiency of complex 2 than that of both free ligand 1 and complex 3 reflecting a strong binding between the nickel square planar complex 2 with DNA and support the outcome of absorption spectral study discussed in the preceding section. The results of binding experiments clearly proved that among the free hydrazone ligand and its metal complexes, the later showed strong binding with DNA than the former that may be due to the chelation of the ligand with the metal ion that resulted in an alteration of electronic properties of the former. In general, the presence of an extended planarity in the chelated ligand would increase the strength of the interaction of the complexes with DNA. Changing the metal ions and the geometry of the complex (square planar, tetrahedral and octahedral) can also modify the binding properties and this may show variations upon interaction with nucleic acids. Among the complexes 2 and 3, complex 2 was identified to bind more strongly with DNA than the complex 3 that could be explained in terms of the planarity and geometry of the metal complexes. In this case, more binding affinity shown by complex 2 is due to the fact that it has square planar geometry whereas the complex 3 possesses octahedral geometry. The ability of more planar structure of the former to penetrate deep into the DNA helix made complex 2 as a vivid candidate for effective binding. But, the octahedral complex 3 with axial ligands finds it difficult to intercalate with DNA due to the steric inhibition. Moreover, in complex 3 a set of bulky triphenylphosponium oxide ligand also prevented the intercalation of it with DNA. The overall order of interaction of compounds 1, 2 and 3 with CT DNA as well as BSA decreased in the order 2 > 3 > 1.

23 DNA form (%) 126 DNA cleavage activity The interaction of puc19 plasmid DNA with the complexes 2 and 3 were evaluated in order to determine the efficiency with which they sensitize DNA cleavage by monitoring the transition from the naturally occurring, covalently closed circular form (Form I) to the open circular relaxed form (Form II). This transition normally occurs when one of the strands of the plasmid is nicked, and can be determined by gel electrophoresis of the plasmid. The tests were performed under aerobic conditions with H 2 O 2 as a co-oxidant using a complex concentration of 35 μm. Neither H 2 O 2 nor the metal complexes 2 and 3 yielded significant strand scission when applied individually (figure not shown) demonstrating that the combinations of both of them are required to cause effective cleavage of plasmid DNA. Fig. 4.7 reveals that cleavage of puc19 DNA Form I Form II Lane 1 Lane 2 Lane 3 Lane 4 Test complexes Fig. 4.7 Gel-electrophoresis pictures for the metal hydrazone complexes. Photograph showing the effects of transition metal hydrazones on DNA of puc19: Lane 1: SC puc19 DNA (0.5 μg) alone; Lane 2: SC puc19 DNA (0.5 μg) + H 2 O 2 (60 μm); Lane 3: SC puc19 DNA (0.5 μg) + H 2 O 2 (60 μm) + complex 2(35 μm); Lane 4: SC puc19 DNA (0.5 μg) + H 2 O 2 (60 μm) + complex 3 (35 μm).

24 127 induced by complexes 2 and 3 in the presence of H 2 O 2 results in the conversion of Form I into Form II. The extent of Form I diminished gradually upon partial conversion to Form II with simultaneous increase in the intensity of the later form. However, the free hydrazone ligand completely failed to induce any cleavage (not shown in Figure 4.6). Based upon the ability of complexes 2 and 3 to convert the supercoiled form (Form I SC) to the open circular form (Form II OC) given in Fig. 4.7 (bar diagram), it is obvious that the complex 2 with more planar square planar geometry has strong capability to cleave the supercoiled plasmid DNA when compared to that of complex 3 that possess highly crowded octahedral geometry around the metal ion that hinders the scissoring of DNA into the above said forms. Protein binding studies Fluorescence quenching measurements Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions, such as excited-state reactions, energy transfer, ground-state complex formation and collisional quenching. 56 The quenching mechanisms are usually classified as either dynamic or static quenching. Dynamic quenching results from collision between the quencher and fluorophore those are present in the excited state. Static quenching is due to the formation of a ground-state complex between the fluorophore and quencher. Fig. 4.8 shows the fluorescence emission spectra of BSA after the addition of compounds 1, 2 and 3 in various concentrations. When increasing amount of test compounds 1-3 was titrated against a fixed concentration of BSA, the fluorescence intensity of BSA decreased gradually with a hypsochromic shift of 4, 7 and 5 nm respectively. These results indicated that the free ligand 1 and corresponding metal hydrazone complexes 2 and 3 did interact with BSA and quenched the intrinsic fluorescence of the same. The fluorescence quenching is described by Stern-Volmer relation, I 0 /I = 1 + K SV [Q] where, I 0 and I are the fluorescence intensities of the fluorophore in the absence and presence of quencher, K SV is the Stern-Volmer quenching constant and [Q] is the quencher concentration. The K SV value obtained as slope from the plot of I 0 /I versus [Q]

25 Intensity I 0 /I Intensity Intensity I 0 /I I 0 /I 128 (Fig. 4.8 as insets) with respect to compounds 1, 2 and 3 are found to be M -1, M -1 and M [Q] x 10-6 M [Q] x 10-6 M Waqvelength (nm) Wavelength (nm) [Q] x 10-6 M Wavelength (nm) Fig. 4.8 Emission spectra of BSA ( M; λ exi = 280 nm; λ emi = 345 nm) as a function of concentration of the ligand (1) and its nickel (2) and cobalt complexes (3) (0, 0.8, 1.6, 2.4, 3.2 and M). Arrow indicates the effect of the test compounds on the fluorescence emission of BSA. In addition, the UV-visible absorption spectra of BSA and free ligand 1 or metal hydrazone complexes 2 or 3 were measured to find the type of quenching exists. Addition of the above compounds to BSA obviously leads to an increase in BSA absorption intensity without affecting the position of absorption band indicating that the type of interaction between the test compounds and BSA was mainly a static quenching process. 57 The UV absorption spectra of pure BSA and BSA-test compounds are shown in Fig. 4.9.

26 Absorbance Absorbance Absorbance BSA + Ligand 1 BSA 0.4 BSA + Complex 2 BSA Wavelength (nm) Wavelength (nm) BSA + Complex 3 BSA Wavelength (nm) Fig. 4.9 Absorption spectra of respective BSA ( M), BSA-ligand 1, BSA-complex 2 and BSA-complex 3 (BSA= M and ligand 1 / complex 2 / complex 3 = M). Binding analysis When small molecules bind independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules is represented by the Scatchard equation 58,59 log [F 0 -F/F] = log K + n log [Q] where, K and n are the binding constant and the number of binding sites, respectively. Thus, a plot of log [F 0 -F/F] versus log [Q] (Fig. 4.10) can be used to determine the values of both K and n and such values calculated for compounds 1, 2 and 3 are listed in Table 4.3.

27 log [F 0 -F/F] Ligand 1 Complex 2 Complex log [Q] Fig Plot of log [F 0 -F/F] versus log [Q] Table 4.3. Binding constant and number of binding sites of the interactions of free ligand 1 and its corresponding complexes 2 and 3 with BSA. Compound K (M -1 ) n BSA + ligand BSA + complex BSA + complex From the values of n, we inferred that there is only one independent class of binding sites on BSA was available for all the compounds under investigation and a linear relation between the binding constant and number of binding sites was identified. These data showed the higher binding efficiency of complex 2 compared to that of the respective ligand 1 and complex 3 under investigation reflecting a strong binding of the former with albumin than rest of the compounds, similar to the results observed in DNA binding study. Characteristics of synchronous fluorescence spectra Synchronous fluorescence spectrum usually provides information about the molecular environment in the vicinity of chromophore molecules in low concentrations under physiological conditions. It is a sensitive technique for detecting micro

28 Intensity Intensity Intensity Intensity Intensity Intensity A 200 1B Wavelength (nm) 2A Wavelength (nm) 2B Wavelength (nm) Wavelength (nm) 200 3A 200 3B Wavelength (nm) Wavelength (nm) Fig Synchronous spectra of BSA ( M) as a function of concentration of the ligand 1 and its complexes 2 and 3 (0, 0.8, 1.6, 2.4, 3.2 and M) with wavelength difference of Δλ = 15 nm (1A, 2A and 3A) and Δλ = 60 nm (1B, 2B and 3B) (a). Arrow indicates the change in emission intensity w.r.t various concentration of the test samples.

29 132 environmental changes of these chromophores with several advantages such as spectral simplification, bandwidth reduction and avoiding different perturbing effects. 60 It is well known that the fluorescence of BSA may be due to presence of tyrosine, tryptophan and phenylalanine residues and hence spectroscopic methods are usually applied to study the conformation of serum protein. According to Miller, in synchronous fluorescence spectroscopy, the difference between excitation and emission wavelength (Δλ = λ emi - λ exc ) reflects the spectra of a different nature of chromophores, with large Δλ values such as 60 nm, the synchronous fluorescence of BSA is characteristic of tryptophan residue and the small Δλ values such as 15 nm is characteristic of tyrosine. 61 To explore the structural changes of BSA due to the addition of the compounds 1, 2 and 3, we measured synchronous fluorescence spectra of BSA with added test compounds at both Δλ = 15 nm and 60 nm. Upon increasing the concentration of compound 1, the intensity of emission corresponding to tyrosine was increased very slightly whereas in the case of compounds 2 and 3 slight decrease in the intensity without any change in position of the wavelength was observed. However, the fluorescence emission of tryptophan showed significant decrease in the intensity without any change in the position of emission band by the addition of ligand and its complexes. These experimental results indicate that the compounds affect microenvironment of the tryptophan residues more effectively during the binding process. The spectra of compounds 1, 2 and 3 are given in Fig Antioxidant studies The antioxidant activity of free ligand 1 and metal complexes 2 and 3 were evaluated in a series of in vitro assays involving ABTS cationic radicals, superoxide and hydroxyl radicals in a dose dependent manner. IC 50 values of the ligand 1 in relevance to ABTS cationic, O - 2 and OH assays were 38.87, and >50 μm, respectively, The complexes 2 and 3 showed their IC 50 values at 20.08, 24.42, 31.26, 25.11, and μm, respectively. The results of these experiments are shown in Fig In general, the antioxidant activity of the ligand and its M(II) complexes [where M = Ni, and Co] against the free radicals i.e., ABTS cationic, O - 2 and OH was found to decrease in the order of 2 > 3 > 1. The results indicated that the metal complexes exhibited greater antioxidant activity than the free ligand. Among the tested compounds,