The electronic spectra and magnetic moment of OVABA and its metal complexes have been measured at room temperature

The electronic spectra and magnetic moment of OVABA and its metal complexes have been measured at room temperature. OVABA exhibited the absorption bands at 35,714 and 29,940 cm-1 corresponds to ???????and n ?????transitions respectively. The complex 1 is paramagnetic with magnetic moment of 1.81BM which is in good agreement with the presence of unpaired electrons. The electronic spectra (S: 6) of complex 1 showed absorption in the region 16,630 cm-1 corresponding to the transition 2B1g ?2A1g and the bands in the region 30,211 and 36,211cm-1 corresponds to charge transfer bands which is consistent with square planar geometry in complex 172-74 . The complex 2 showed the magnetic moment of 4.19BM which is consistent with the presence of three unpaired electrons. The electronic spectra of the complex 2 exhibited bands at 11,876; 15,313 and 19,696 cm-1 and these bands corresponds to 4T1g(F)? 4T2g(F)(?1); 4T1g(F)? 4A2g(F)(?2) and 4T1g(F)? 4T2g(P)(?3) transitions respectively The position of these bands are consistent with octahedral geometry of complex 2. The complex 3 showed the magnetic moment of 3.17BM which is consistent with the presence of three unpaired electrons. The electronic spectra of the complex 3 exhibited three absorption bands in the region 17,605; 25,794 and 38,461 cm-1 and these bands corresponds to 4A2g(F)? 4T1g(P)(?1); 4A2g(F)? 4T1g(F)(?2) and 4A2g(F)? 4T2g(P)(?3) respectively. The ligand field parameters (Dq, B, b) have also been calculated (Table 3) for the complexes 2 and 3 by using Konig’s method. The calculated value of B for the complexes 2 and 3 shows that M-L bond is appreciably covalent. The value of B, which is lower than the free ion value of 971cm-1 for complex 2 and 918cm-1 for complex 3, indicates overlapping of ligand metal orbitals. These values signifies covalent character of the metal-ligand bonds and an overlapping of metal-ligand orbitals. The value of b lies in the range of 0.32-0.66, which suggests that the complexes 2 and 3 exhibit appreciable covalent character. The complex 4 is found to be diamagnetic which is consistent with the d10 configuration and electronic spectrum showed an absorption band at 24,635 cm-1 assigned to ligand to metal charge transfer transition, which is compatible to an octahedral geometry.

Powder diffraction technique uses X-ray, neutron or electron diffraction on microcrystalline samples for structural characterization of materials. This technique explains that each solid represents a definite compound of a definite structure which does not possess any starting materials. In the present investigation single crystals of the complexes 1, 2, 3 and 4 could not be prepared to get the single crystal XRD and hence the powder XRD studies were carried out for structural elucidation. The X-ray diffractogram of OVABA and its complexes were carried in the range of 50 to 800 2? values, which are shown in the S: 7. The spectra indicates that OVABA and its complexes have well resolved crystalline patterns, with various degrees of crystallinity. The presence of extra peaks in the complexes compared to OVABA indicates that metal ions are coordinated with the ligand to form complexes. The complexes were identified using a known standard method. The results indicates that the synthesized OVABA and its metal complexes are crystalline.

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Electronic absorption studies is one of the most useful technique to show the binding modes of the complexes with DNA. In general, complexes with aromatic character when binds to DNA through intercalation results in bathochromism and hypochromism which is due to the stacking interaction between aromatic chromophore of the complexes and the base pair units of DNA. The absorption spectra of the complexes 1, 2, 3 and 4 in the absence and presence of calf thymus DNA are illustrated in (Fig 2). In the present experiment, with increase in the amount of CT- DNA to the complexes causes a subsequent decrease in peak intensities. Hypochromic and bathochromic shifts of the intra ligand absorptions were observed upon the addition of varying amounts of DNA to the complexes. Hypochromism was proposed due to the interaction between the electronic state of the intercalating chromophore of the metal complexes and that of the DNA base pairs. Furthermore with the decrease in intensity of peaks, a small red shift (bathochromism) was also observed in the spectra. These absorptional changes shows that the complexes bind to DNA bases through intercalation The strength of binding of synthesized complexes (1, 2, 3 and 4) with DNA was quantitatively evaluated by determining intrinsic binding constants (Kb). In plot of DNA/ ?a-?f versus DNA, kb the ratio of slope to the intercept is calculated. The calculated Kb values of complexes were found to be 5.12±0.03×104 M-1, 4.67±0.05 x 104 M-1, 3.59±0.02×104 M-1 for the complexes 1, 2, 3 and 4 respectively. The calculated Kb values of the complexes are comparable with ethidium bromide. The intrinsic binding constant of the synthesized complexes follows the order as 1 ; 2 ; 3 ; 4.

3.10.2Fluorescence studies:
The fluorescence studies experiments were carried out further to know the binding affinities of the complexes to CT-DNA with respect to EB. In general, EB in Tris-HCl buffer solution show a low emission intensity due to fluorescence quenching of the free EB by the solvent molecule. However when EB interactively binds with adjacent DNA base pairs, its emission intensity dramatically increases. This high emission intensity is reversed by successive addition of complexing agents (complexes) to DNA-EB system. In the present work, with increase in the concentration of the complexes 1, 2, 3 and 4 to DNA-EB complex showed a substantial reduction in fluorescence emission intensity (Fig 3). This results also suggests that the complexes could disturb DNA-bound EB and bind to CT-DNA through intercalation with almost the same affinity. This result also supports intercalation of metal complexes to CT-DNA. The above data were evaluated using Stern–Volmer equation. The quenching plots illustrate that the quenching of EB bound to DNA by the complexes is in good agreement with the linear Stern Volmer equation, which proves that the four complexes effectively bind to DNA. The Ksq values for 1, 2, 3 and 4 are 0.489, 0.312, .287 and 0.212 respectively.

3.10.3. Viscosity measurements:
For further establishment of the interactions between the complexes and DNA, viscosity measurements were carried out. A classical intercalation model demands that the DNA helix must lengthen as base pairs are separated to accommodate the binding ligand, which leads to the increase of DNA viscosity. A well-known DNA intercalator is Ethidium bromide which increases the relative viscosity strongly by lengthening the DNA double helix through intercalation. In the present study, with increase in the concentration of complexes the relative viscosity increases steadily similar to the behaviour of Ethidium bromide and the binding affinity to DNA follows the order EB ; 1 ; 2 ; 3 ; 4 (Fig 4). This further confirms intercalation mode of binding with DNA molecule.
3.10.4.DNA cleavage:
DNA cleavage efficiency of the synthesized metal complexes were performed using pUC-19 DNA in the presence and absence of H2O2 as an oxidant. When circular plasmid DNA is exposed to electrophoresis, the fast migration will be observed for the super coiled (SC) form-I. If one strand is cleaved, the SC form will relax to produce a slow-moving open circular (OC) form-II or nicked form. If both strands are cleaved, a linear form III will be generated that migrates at a rate in between form-I and form-II. The nuclease activity was greatly increased by the incorporation of metal ion in the respective copolymer. From the Fig 5, it is evident that the complexes can cleave DNA more efficiently in the presence of oxidant, which may be due to the formation of hydroxyl free radicals and the control DNA does not shown effective cleavage. All the complexes 1, 2, 3 and 4 exhibit efficient DNA cleavage activity which may be attributed to its higher DNA-binding affinity. The complex 1 has good cleavage activity than the complexes 2, 3 and 4.

The ground state configuration of Cu (II) ion with an unpaired electron can be conveniently explained by ESR studies. The X-band ESR spectrum of complex 1 was recorded in DMSO at 300K and 77K (LNT). The spectrum (S: 5a and 5b) Shows a well-resolved four-line spectrum. The spin Hamiltonian parameters were calculated from the spectra, are given in Table 2. The g tensor values can be used to derive the ground state of the copper complex. In square planar complexes, the unpaired electron lies in the dx2-y2 orbital. For the complex 1, the g tensor values obtained are gll = 2.23 ; g?= 2.05 ; 2.0023 respectively, which suggests that the complex is square planar. This also supports the fact that the unpaired electron predominantly lies in the dx2-y2 orbital 66, as evident from the value of the exchange interaction term G, estimated from the expression Eq. (4).

G = (gll- 2.0023) / (g?- 2.0023) (4)
According to Hathaway 67, if the value of G is greater than 4.0, the local tetragonal axes are aligned parallel or only slightly misaligned. If G is lesser than 4.0, significant exchange coupling is present and the misalignment is appreciable. For complex 1, the value of G = 4.77 which indicates that the local tetragonal axes aligned parallel or slightly misaligned with the presence of unpaired electron in dx2-y2 orbital. This result also suggests that the exchange coupling effects are not operative in the present complex.
The isotropic ESR parameters giso = 2.11 and Aiso = 83.0 are calculated from the position spacing of the resonance lines from room temperature solution spectrum of the complex. The spectrum showed typical eight-line pattern which indicates that a single copper is present in the molecule which is a monomer. This is also supported by the magnetic moment of complex 1 (1.81 BM) which confirms the mononuclear nature of the complex. In the frozen solid state, two types of resonance components have been observed in the spectrum, one set due to parallel features and the other due to perpendicular features, which suggests axially symmetric anisotropy with well-resolved sixteen-line hyperfine splitting, characteristic of an interaction between the electron and copper nuclear spin. From the anisotropic ESR spectrum, the anisotropic parameters were calculated and the order of values are All = 160.8 ; A? = 44.9; gll = 2.23 ; g? = 2.05 indicating that the unpaired electron is present in the dx2-y2 orbital with square- planar geometry of the complex 169.
The bonding parameters 70 ?2, ?2, ?2 of the complex 1 was calculated. These bonding parameters may be considered as measure of covalency of in-plane ? bonding, out of-plane ??bonding, in-plane ?-bonding and out-of-plane ?-bonding.
?2, ?2, ?2 parameters were calculated using following equations. Eq. (5).

?2= – (All/0.036) + (gll – 2.0036) + 3/7 (g? – 2.0036) + 0.04 (5)
?????????????????????????2 = (gll – 2.0036) E/ -8? ?2(6)
?2 = (gll – 2.0036) E / -2? ?2(7)
Here ? = 828cm-1 for free Cu (II) ion and E is the electronic energy for 2BIg ?2A1g transition which is 16,630cm-1 for the complex 1. The ? value is calculated by using the following equation.
gav = 1/3gll +2g? and gav = 21-2?/10Dq
The calculated ? for the complex 1 is 275cm-1 which if smaller than the free Cu (II) ion. This
reduction in ? value than compared to that of free ion indicates the covalent character in M-L bond.**evi PV and the value alsc indicates considerable mixing of ground and excited state terms.
The bonding parameter (?2 = 0.81-0.99) considerable covalent character between metal-ligand. If the ?2 value is 0.5 then it implies complete covalent bonding, while that of 1.0 implies complete ionic bonding. For the complex 1, the calculated the calculated ?2 value is
0.732 which indicates that the complex has some covalent character. The observed ?2 value (1.59) and ?2 value (1.34) shows that there is interaction in the out-of-plane ?-bonding, whereas the in-plane ?? bonding is completely ionic.
This is also confirmed by orbital reduction factor 71 K which can be estimated using Eq. 8 ; 9.

Kll = ?2?2(8)
K? = ?2?2(9)
Significant information about the nature of bonding in the complex 1 can be derived from the relative magnitudes of K|| and K?. In the case of pure ?-bonding. K||?K? = 0.77, whereas K|| ;K? implies considerable in-plane ?-bonding, while for out-of plane ? -bonding, K|| ; K?. For the present complex, the observed order K|| (1.16) ; K? (0.98) implies a greater contribution from out of plane ?-bonding than for in-plane ?-bonding in metal ligand ?-bonding. Thus, the ESR study of the copper complex has provided supporting evidence for the optical results.

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2.3 DNA binding
2.3.1 Preparation of stock solutions

The CT-DNA stock solution was prepared by mixing up of 5 mM Tris-HCl and 50 mM NaCl in distilled water (pH=7.5). The concentration of stock solution was measured by UV absorbance at 260 nm, taking molar absorption coefficient as 6600 M-1cm-16. UV absorption at 260 and 280 nm gave a ratio (A260/A280) of 1.83 (usually between 1.8-1.9), which suggests that the prepared CT-DNA solution was sufficiently free of protein 7. The stock solution was stored at 4? and used within 4 days. The concentration of ethidium bromide (EB) was determined at 480 nm (?=5860 M-1cm-1) 8. Binding experiments were carried out with 10-3M DMSO solutions of complexes (1, 2, 3 and 4) in 5mM Tris-HCl/50mM NaCl solution (Tris-buffer, pH=7.5).

2.3.2 Absorption spectral studies
Absorption titrations were performed on a SHIMADZU 160A UV/Vis spectrophotometer using 1-cm quartz micro-cuvettes. Titrations were carried out by keeping concentration of complexes (1, 2, 3 and 4) constant at 10 µM and by varying DNA concentration from 0-100 µM. In the reference cell a DNA blank was placed to offset any absorbance due to DNA at measured wavelength. The following equation was used to calculate the intrinsic binding constants of complexes 9.

DNA/(?a-?f) = DNA/(?b-?f)+1/Kb(?b-?f) (1)
where ?a, ?f and ?b represents apparent extinction coefficient, extinction coefficient of free (unbound) complex and extinction coefficient of fully bound complex respectively. ?a was calculated from Aobs/complex. A plot of DNA/(?a-?f) vs. DNA gives a slope 1/(?b-?f) and intercept 1/Kb (?b-?f). The intrinsic binding constant (Kb) was determined from the ratio of slope and intercept.

2.5.3 Fluorescence titration:
Fluorescence quenching studies were carried out using 5mM Tris-HCI/50mM NaCl (pH= 7.5). A solution containing ethidium bromide (EB) and DNA was titrated with different concentrations of the complexes. The concentration of DNA was always 130µM DNA-phosphate. The concentration of the complexes were in the range of 10-100 µM
and the concentration of EB was 40 µM. The solutions were excited at 520nm and the emission range was set between 540-700nm. The spectra were analysed using Stern-Volmer equation given as Eq (2)
I0/I = 1 + ksqr (2)
Where I0 and I are the fluorescence intensities in the absence and the presence of complexes respectively, ksq is the linear stern-Volmer quenching constant, r is the concentration of complex to DNA.
2.5.4 Viscosity studies:
Flow times of viscosity experiment will provide information to predict the binding mode of complexes with CT-DNA. Viscometric titrations were carried out at room temperature using an Ostwald viscometer. Concentration of each complex was varied from 0-120 µM and DNA concentration was fixed at 20 µM. Flow times were measured using a digital timer and each sample solution was measured in triplicate and an average value for flow time was calculated. Data from this experiment was presented as (?/?0)1/3 versus complex/ CT-DNA where ? represents the viscosity of DNA in the presence of complexes (1, 2, 3 and 4) and ?0 represents viscosities of DNA alone. Viscosity values were calculated from the observed flow time of CT-DNA containing solutions (t) corrected for that tris-buffer alone (5 mM Tris-HCl/50 mM NaCl) (t0), ? = (t-t0).

2.5.5. DNA cleavage:
DNA cleavage studies were performed using gel electrophoresis technique. TE buffer (10mM
Tris-HCl and 1mM Na2EDTA) and Tris-acetate-EDTA (TAE) buffer (pH=8.0; 40 mM Tris-base, 20 mM acetic acid, 1 mM EDTA) solutions were used in the experiment. TE buffer was used for dilution of pUC-19 DNA. Supercoiled pUC-19 DNA (0.1 g µl-1) was treated with 30 µM concentration of the complexes (1, 2, 3 and 4) and the volume was made up to 16µL using 5mM of Tris-HCl /5mM NaCl buffer solution. The resulting solution was incubated at 370C for 2h after the addition of DNA. 2µL of 25% bromo phenol blue was added to quench the reaction. Samples were electrophoresed for 2h at 50V in Tris-acetate-EDTA (TAE) buffer using 1% agarose gel containing 1.01g/mL ethidium bromide and photographed under UV light.

2.4 Cytotoxic studies
Human cervival adenocarcinoma cells (HeLa) were maintained in DMEM (with 1000 mg/L glucose) supplemented with 0.584 gm/L L-glutamine (HiMedia), 3.7 gm/L sodium bicarbonate and 0.004 gm/L folic acid (Sigma-Aldrich, Germany). Human pancreatic carcinoma (MiaPaca2) and Mouse melanoma (B16F10) cells were maintained in RPMI-1640 (with sodium bicarbonate) supplemented with 0.3 gm/L L-glutamine. All the cell cultures were supplied with 10% FBS (fetal bovine serum) and 5000 U/mL penicillin-streptomycin (Thermo Fisher Scientific) and incubated at 37? in an atmosphere of 5% Carbon dioxide and 95% air. In 96 well plates, 5000 cells per well were seeded and incubated overnight in 37? incubator for 24 hr. Then, six concentrations of synthesized compounds (1 and 2) were treated for 24 hr in HeLa, MiaPaca2 and B16F10 cells. After the incubation period 20 µL of MTT reagent (5 mg/mL) was added to each well and incubated for another 4 hr 11-13. Reduction of MTT to its insoluble form (formazan) was evident from the purple colored precipitates being formed after incubation. 100 µL of DMSO was added to each well to solubilize the precipitates and optical density of the resulting solutions was measured after overnight at 560 nm on BioTek Synergy microtiter plate reader. IC50 values were calculated in µg/ml using origin software and represent concentration of test compound to inhibit cell viability by 50%. Every concentration for each compound was tested in triplicate and mean values ± SD were presented.

2.5 Molecular docking
2.5.1 Protein preparation
The ligands (1 and 2) were docked into the active pocket of human DNA topoisomerase I. The required 3D crystal structure for receptor protein (DNA topo I) was retrieved from protein data bank (PDB: 1T8I). Following the retrieval, the protein structure was imported into Accelrys Discovery Studio 2.5, where in all the essential steps for clean protein preparation were undertaken. Water molecules co-crystallized with receptor protein were removed and all hydrogen atoms, including those to define correct ionization and tautomeric states of amino acid residues were added. Energy minimization of protein was done by applying CHARMm force field at RMS gradient of 0.01 with a maximum number of 1000 steps. This process was stopped as soon as the protein reached a convergence gradient of 0.001 kcal mol-114.

2.6.Molecular docking Studies:
A primary objective in molecular docking was the ability to estimate the scoring function and evaluate protein ligand interactions in order to predict the binding affinity and activity of the ligand molecule. The docking program GOLD has been employed to generate the bioactive binding poses of hetero cyclic derivatives in the active site of protein human DNA topoisomerase I. GOLD 3.1 (Genetic Optimization for Ligand Docking. Cambridge Crystallographic Data Centre) uses a genetic algorithm to explore the conformational space of the ligands in addition to some flexibility of active site residues. Protein coordinates from the bound ligand of DNA topoisomerase I were used to define the active site. Docking calculations were performed using the default GOLD fitness function and default GOLD parameters were used to produce the set of optimal conformations of both the ligand and the protein. Each simulation was performed 10 times: yielding 10 docked conformations unless three of the 10 poses were within 1.5A0 RMSD of each other. The lowest energy conformations were regarded as the binding conformations between ligands and the protein. Greater the GOLD fitness score better the binding affinity. Hit molecules which showed the expected interactions with the critical amino acids present in the active site of the protein, may show potent antagonist properties towards DNA topoisomerase I.
2.7.1.Anti-microbial Activity:
In vitro anti-microbial activity of OVABA and its complexes (1, 2, 3 and 4) in DMSO medium were screened against few bacterial and fungal strains using standard agar as the nutrient medium by a well diffusion method 42-44. Fresh bacterial strains having 5 x10-5 colonies were added to nutrient agar medium and poured in to petri plates. Wells were made in the cooled agar plates (1cm). 10mg of each compound were dissolved in 2 mL DMSO and 100µL was loaded in the well. The sensitivity was observed after 24 – 48h incubation at 370C. The zone of inhibition was recorded in centimetres 45, 46. The observed zone of inhibition (in cm) of the OVABA and its complexes were compared with streptomycin (antibiotic) and mancozeb (anti-fungal drug) which are the commercially available controls.

Cytotoxic studies:
Human cervical adenocarcinoma cells (HeLa) were maintained in DMEM (with 1000 mg/L glucose) supplemented with 0.584gm/L L-glutamine (HiMedia), 3.7gm/L sodium bicarbonate and 0.004gm/L folic acid (Sigma-Aldrich, Germany). Human pancreatic carcinoma (MiaPaca2) and Mouse melanoma (B16F10) cells were maintained in RPMI-1640 (with sodium bicarbonate) supplemented with 0.3 gm/L L-glutamine. All the cell cultures were supplied with 10% FBS (fetal bovine serum) and 5000 U/mL penicillin-streptomycin (thermo Fisher Scientific) and incubated at 370C for 24h. Then, six concentrations (5, 10, 25, 50, 75, 100 µg) of synthesized complexes (1, 2, 3 and 4) were treated for 24h in HeLa, MiaPaca2 and B16F10 cells. After the incubation period 30 µL of MTT reagent (2 mg/mL) was added to each well and incubated for another 4h. Reduction of MTT to its insoluble form (formazan) was evident from the purple coloured precipitates being formed after incubation. 100 µL of DMSO was added to each well to solubilize the precipitates and optical density of the resulting solutions was measured after overnight at 560 nm on BioTek Synergy microtiter plate reader. IC50 values were calculated in µg/mL using origin software and represent concentration of test compound to inhibit cell viability by 50%. Every concentration for each compound was tested in triplicate and mean values ± SD were presented. The % cell viability was calculated using the following Eq. (3).

% cell viability = A570 of treated cells/ A570 of control cells × 100 (3)
Graphs are plotted considering the concentration of the sample in X-axis and % of Cell Viability at Y-axis. Cell control and sample control is included in each assay to compare the full cell viability in Cytotoxicity and anti-cancer activity assessments.
3.1. IR Spectra:
The IR spectra of OVABA and its complexes 1, 2, 3 and 4 when compared they showed some similarities and dissimilarities (S:1). A sharp band at 1610cm-1 assigned to the azomethine nitrogen of OVABA 47 has been shifted to 1624-1641 cm-1 in the complexes, which shows the coordination of the metal to the azomethine nitrogen 48, 49. The band observed at 1251 cm-1 in the OVABA spectra corresponds to ?(C-O) has shown a negative shift of 10-20cm-1 indicating coordination through phenolic oxygen 50. A sharp band at 1107cm-1 assigned to ? (-OCH3) in ligand has not shown considerable shift in metal complexes. This indicates the non-involvement of oxygen atom of terminal methoxy group in coordination with metal ions. The ligand showed a strong absorption band at 3375cm-1 which is assigned to ?(OH) remain unchanged in all the complexes. This also supports non-involvement of alcoholic –OH in coordination 51, 52. New bands observed at 474-594cm-1 and 291- 435cm-1 in the far infrared region were assigned to ?(M-O) and ? (M-N) modes. This indicates the formation of M-O bond in the complexes 53 and the lone pair of electron present on the nitrogen atom of the OVABA are involved in coordination with the metal ion. Thus from IR spectral studies it is concluded that OVABA acts as {uninegative} bidentate ligand and gets coordinated with the central metal ion via azomethine (N) and phenolic (O).

3.2.Molecular Modelling:
Geometrical structures of OVABA and its complexes 1, 2, 3 and 4 were calculated by optimizing their bond lengths and bond angles using Argus Lab software. Geometrical structures were presented in Fig 1. Selected geometrical parameters (bond lengths and bond angles) of the complexes are given in Table 1. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are the main orbitals that take part in chemical stability. The molecular structures (HOMO and LUMO) of OVABA are presented in the Fig 2. The 3-dimensiional structures of the metal complexes with possible configuration were evaluated using molecular Mehanics (UFF) calculations. The most stable structure with minimum energy among the possible ones is judged as the most possible structure for all the complexes as shown Table 1.

3.4. Thermo gravimetric Analysis:
Thermal degradation of the complexes under present investigation were shown in TGA thermo grams (S:3) which shows that for the complex 1 there is no weight loss below 1000C which suggest the absence of coordinated water molecule. For 2 and 4 complexes there is a weight loss of (~ 5-6%) indicating a loss of two coordinated water molecules 59, 60 whereas for 3 complex there is a weight loss of (~ 3%) indicating a loss one coordinated water molecule. The complexes exhibit thermal stability up to 3500C.

3.5.LCMS:
The LCMS spectra of OVABA and complexes 1, 2, 3 and 4 shows the molecular ion peak which are significantly more abundant than other fragment ions. The fragmentations proposed are equivalent with the proposed empirical formula of synthesized ligand and metal complexes. The spectrum resolves an M+1 peak (S: 4) at m/Z 257 which corresponds to the molecular weight of the OVABA 25661. The M+1 peak at m/z 665, 635, 615, 575 for complex 1, 2, 3 and 4 are consistent with the molecular weight of the synthesized 1, 2, 3 and 4 complexes respectively.
3.6.Molar conductivity:
The molar conductivity of the complexes 1, 2, 3 and 4 were measured in DMSO solvent at 370C. The conductivity values of the complexes are equal to 1.9 to 4.9 ? -1cm2 mol-1 at 10-3 M concentration shows that the complexes are non-electrolytes62-64 and chloride ion is present within the coordination sphere65 for complex 3.