Investigation of Antimicrobial , Antioxidant , and DNA Binding Studies of Bioactive Cu ( II ) , Zn ( II ) , Co ( II ) , and Ni ( II ) Complexes of Pyrimidine Derivative Schiff Base Ligand Shahul

A new pyrimidine based Schiff base ligand (HL) and its four complexes of type [MLOAc]⋅nH2O (Cu(II), 1; Zn(II), 2; Co(II), 3; and Ni(II), 4) have been synthesized and characterized by elemental analysis, MS, H-NMR, FT-IR, UV-visible, and ESR techniques. The electronic and ESR spectral data suggested that complexes 1–4 possess square planar geometry. Antimicrobial activities of HL and complexes 1–4 were tested against four bacteria (Staphylococcus aureus, Staphylococcus pneumonia, Salmonella enterica typhi, and Haemophilus influenzae) and two fungal strains (Aspergillus flavus and Aspergillus niger). These results show that complexes 1–4 have good antimicrobial activity compared toHL. The DNA cleavage activity of HL and complexes 1–4 was monitored by the agarose gel electrophoresis method. The antioxidant property of the prepared compounds was assessed by using 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging method. DNA binding properties of HL and complexes 1–4 have been investigated by electronic absorption technique and viscometric measurements.


Introduction
Schiff base ligand is considered as a trendiest base because of its various applications like catalytic, optical, electronic, antibacterial, antifungal, antiviral, anti-inflammatory, and antitumor activities [1][2][3][4][5].Transition metals play a more important role in drug design because of their biologically active metal ions and ligands [6].When they are chelated with Schiff base ligand, the biological activity of the metal ions was significantly increased based on the geometry, reactivity, and functional group present in the ligand.Coordination of such ligands with metal ions likewise, copper, zinc, cobalt, and nickel, has antimicrobial, antioxidant, and DNA interaction properties [7,8].Moreover, Schiff base ligand is synthesized from pyrimidine derivatives that are much more of interest, because the pyrimidine is a heterocyclic compound, which also is present in nucleic acids [9].These pyrimidine derivatives drugs are used in antimicrobial and anticancer related diseases [10,11].Similarly, salicylaldehyde derived Schiff base ligand exhibits better biological properties and its transition metal complexes increased biological activities [12].
In this research framework, we have synthesized the Cu(II), Zn(II), Co(II), and Ni(II) complexes with Schiff base ligand from pyrimidine and salicylaldehyde derivatives.They are characterized by different spectral and analytical methods and also their antimicrobial, antioxidant, and DNA binding properties were studied.

Zn(CH
, deoxyribose nucleic acid from calf thymus CT DNA, agarose gel, Tris-HCl, Tris-buffer, sodium chloride, bromophenol blue, and ethidium bromide were procured form Sigma Aldrich and Alfa Aesar company.

Instruments.
The electronic spectra and absorption spectral titration were recorded on a UV-Visible-1800 (Shimadzu) spectrophotometer and the IR spectra were done in KBr pellets on a FTIR (Shimadzu, IR Affinity-1) spectrometer.
The mass and 1 H-NMR spectra were recorded on an ESI-MS spectrometer, IIT Bombay, and Bruker Avance DRX 300 FT-NMR spectrometer, IISC, Bangalore.The DNA cleavage studies were carried out in DMSO solution using UVtransilluminator.

Synthesis of Ligand
HL.An ethanolic solution (10 mL) of 2,5-dihydroxybenzaldehyde (2 mmol) was added to the ethanolic solution (15 mL) of 2-amino-4,6-dimethoxypyrimidine (2 mmol) and next the mixture was refluxed for one hour.After solution was evaporated slowly on a water bath and finally reddish-brown solid was obtained and washed with ethanol and dried in vacuo (Scheme 1).niger) strains by the well diffusion method [13].Sparfloxacin (antibacterial) and Ketoconazole (antifungal) were used as standard drugs.

Synthesis of Complexes
2.6.Antioxidant Study.Antioxidant activity of HL and complexes 1-4 were studied by DPPH scavenging method [14].The % inhibition was calculated according to the following formula: where  0 is the absorbance control and  1 is the absorbance of sample or standard.

Results and Discussions
The newly synthesized HL and complexes 1-4 were found to be intensely coloured.The analytical data and physical properties of the prepared compounds are listed in Table 1.

Mass Spectra.
Mass spectra of the HL and complexes 1-4 recorded at room temperature were used to compare their stoichiometry composition.The ligand HL showed a molecular ion peak at (/ 276) corresponding to C 13 H 13 N 3 O 4 .The molecular ion peaks for the complexes 1-4 observed at /  396, 1; 398, 2; 392, 3; and 391, 4 confirms the stoichiometry of metal chelates as [ML] type and 1 : 1 ratio.

1 H-NMR Spectra.
The 1 H-NMR spectra of the HL and complex 2 show the signals and are summarized in Table 2 and Figure 1.In free ligand HL, the azomethine proton at 8.2 (s) ppm, pyrimidine proton at 6.45 (s) ppm, aromatic -CH protons at 6.8-6.23 (m) ppm, -OCH 3 protons at 3.73 (s), and phenolic -OH protons (C 2 and C 5 ) appeared at 9.58 (s) and 9.12 (s).After the complexation, the azomethine proton signal was shifted toward the downfield region at 8.5 (s) and -OH proton (C 2 ) is disappeared.These results suggest that the phenolic oxygen (C 2 ), azomethine, and pyrimidine nitrogen atoms are taking part in the complexation and there is no appreciable change in all other signals in this complex.In complex 2, a new peak is observed at 1.83 (s) due to acetate molecule involved in the complexation.

IR Spectra.
IR spectral data of HL and complexes 1-4 are shown in Table 3. IR spectrum of HL showed that a strong sharp band observed at 1564 cm −1 [18] is assigned to the azomethine group (-HC=N-), which was shifted to lower frequencies in the spectra of complexes 1-4 indicating that the involvement of azomethine nitrogen in coordination with the central metal ion and pyrimidine (C=N) band appeared at 1465 cm −1 which was shifted towards lower frequencies and in the range of 1465-1442 cm −1 due to the fact that pyrimidine nitrogen atom is one of the coordination sites around the central metal ion.In all metal complexes, carboxylate of the acetate group (CH 3 COO − ) is strongly absorbed (symmetry) in the range of 1640-1668 cm −1 and (asymmetry) more weakly at 1402-1428 cm −1 .The bands appeared in the region of 450-434 cm −1 were assigned to ] (M-N) for complexes 1-4, indicating that the imine and pyrimidine nitrogen atoms are involved coordination with central metal ions [19].The bands observed in the region of 497-502 cm −1 were assigned to ] (M-O) for complexes 1-4, indicating that the phenolic oxygen atom was involved in coordination with central metal ions [20].
3.4.Electronic Spectra.Electronic absorption data of HL and complexes 1-4 are depicted in Table 4.In the absorption spectra of HL, intense absorption bands at 292 and 370 nm were attributed to - * and - * transitions (Table 1) [21].
For complex 1, the bands appeared at 482 and 620 nm, which were assigned to the 2 B 1g → 2 E g transition and this indicates the square planar geometry [22].Complex 2 has d 10 configuration, which suggests the absence of d-d transition bands, so complex 2 reveals that INCT bands shift at 310 and 380 nm, respectively; this indicates the formation of zinc complex [14].For complex 3, the bands appeared at 428 and 465 nm which were attributable to the 1 A 1g → 1 B 1g transition in a square planar geometry [23].For complex 4, the bands are examined at 432 and 446 cm −1 , which were attributed to 1 A 1g → 1 A 2g transition and this shows square planar geometry around the central metal atom [24].

ESR Spectra.
The X-band ESR spectra of complex 1 were recorded in DMSO at room and liquid nitrogen temperature (Figure 2).The frozen solution spectrum shows well resolved four-line spectral lines.The results are summarized in Table 5.
The spin Hamiltonian parameters have been calculated by Kivelson's method.The observed -values are in the order  ‖ (2.11) >  ⊥ (2.04) >   (2.0027) indicating that the unpaired electron lies predominantly in d 2-2 orbital of Cu(II) ion [25].These results also supported the square planar geometry around the central metal(II) ion.The interaction coupling constant  value is calculated from the following: The observed  value of 4.184 shows that no interaction between Cu-Cu is centred in the solid state of the Cu(II) complex [26].If the  value is greater than 4, the exchange interaction is negligible.good antifungal activity compared to HL and complexes 2 and 4.Moreover, zone of inhibitory effect of newly prepared compounds is higher in A. niger fungi as compared to A. flavus.

Antioxidant.
The antioxidant activities of HL and complexes 1-4 were analyzed by using DPPH stable free radical and are depicted in Figure 5.This experiment was carried out by using UV-visible spectroscopy; results suggest that the absorption peak intensity decreases and disappears because of the addition of newly prepared compounds.Hence, the synthesized compounds can donate the hydrogen atom to DPPH free radical and color of the compounds changes from purple to yellow.The results have suggested that complexes 1 and 3 have good scavenging ability HL and complexes 2 and 4.
3.8.DNA Cleavage.DNA cleavage studies of HL and complexes 1-4 with CT-DNA in the presence of hydrogen peroxide (H 2 O 2 ) were analyzed by agarose gel electrophoresis techniques (Figure 6).From Figure 6, complex 1 has good DNA damage activity compared to ligand HL and complexes  2-4.These results reveal that complex 1 is involved in the formation of hydroxyl radicals which may damage DNA via Fenton-type mechanism.and slight red shift have been observed.Clearly, these obtained results are coinciding with the previously reported results [27] which suggest that the HL and complexes  1-4 interact with CT-DNA via intercalation mode.The intrinsic binding constant (  ) of HL and complexes 1-4 was determined by using the following formula (Table 6): , where [DNA] is the concentration of base pairs of DNA.The apparent absorption coefficients   ,   , and   correspond to  obs./[M], the extinction coefficient for the free complex, and extinction coefficient for the complex in the fully bound form, respectively.The   values of HL and complexes 1-4 are in the following order: 1 (4.76 × 10 5 ) > 3 (1.27× 10 5 ) > 4 (1.02 × 10 5 ) > 2 (9.28 × 10 4 ) > HL (1.06 × 10 4 ).

Viscometric Measurements.
To clarify the binding mode of newly synthesized compounds with CT-DNA by using viscometric measurements, the plots of relative viscosity versus [complex]/[DNA] (Figure 8) show that the viscous flow of DNA increases when increasing the concentration of HL and complexes 1-4.These results have suggested the interaction of compounds with DNA via intercalation binding mode.

Conclusions
In

3. 6 .
Antimicrobial.Antibacterial activities of HL and complexes 1-4 were screened against the four different bacteria, Staphylococcus aureus (S. aureus), Staphylococcus pneumonia (S. pneumonia), Salmonella typhi (S. typhi), and Haemophilus influenzae (H.influenzae), and Sparfloxacin (standard drug).The zone of inhibition and minimum inhibitory concentration (MIC) values of synthesized compounds against bacteria are given in Figures3(a) and 4(a).From the above data, complexes 1 and 3 have good antibacterial activity compared to HL and complexes 2 and 4.Moreover, zone of inhibitory efficiency of synthesized compounds is higher in S. aureus bacteria as compared to other bacterial strains.Antifungal activities of HL and complexes 1-4 were screened against two different fungi, Aspergillus flavus (A.flavus) and Aspergillus niger (A.niger) strains.Ketoconazole was used as standard drug.The zone of inhibition and minimum inhibitory concentration (MIC) values of synthesized compounds against fungal strains are given in Figures3(b) and 4(b).From the above data, complexes 1 and 3 have

Figure 8 :
Figure 8: Effect of increasing concentration of complexes 1-4 on the relative viscosity of CT-DNA.

Table 1 :
Analytical and physical data of the ligand HL and complexes 1-4.

Table 2 :
1H-NMR spectral data of the ligand HL and complex 2.

Table 4 :
Electronic spectral data of the synthesized compounds.

Table 5 :
The ESR spectral data for complex 1.

Table 6 :
Absorption spectral properties of HL and complexes 1-4 with CT-DNA.
summary, we have successfully synthesized the Cu(II), Zn(II), Co(II), and Ni(II) complexes of Schiff base ligand bearing pyrimidine derivatives.The newly synthesized compounds were analyzed by various spectral and analytical techniques.The elemental and mass spectral results support that the stoichiometry of complexes 1-4 is of 1 : 1 ratio and the type is ML.All the spectral results suggest that complexes 1-4 possess square planar geometry around the central metal atom.Antimicrobial and antioxidant results reveal that complexes 1 and 3 have good antimicrobial and the ability to scavenge DPPH radicals compared to ligand HL and complexes 2 and 4. Complex 1 has good DNA cleavage ability compared to HL and complexes 2-4.DNA interactions studies results have suggested that the newly prepared compounds interact with CT-DNA via intercalation mode.