Abstract
A new pyrimidine based Schiff base ligand (HL) and its four complexes of type [MLOAc]·H2O (Cu(II), 1; Zn(II), 2; Co(II), 3; and Ni(II), 4) have been synthesized and characterized by elemental analysis, MS, 1H-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 to HL. 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-1-picrylhydrazyl (DPPH) free radical scavenging method. DNA binding properties of HL and complexes 1–4 have been investigated by electronic absorption technique and viscometric measurements.
1. 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 [1–5]. 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.
2. Experimental
2.1. Materials and Methods
2,5-Dihydroxybenzaldehyde, 2-amino-4,6-dimethoxypyrimidine, Cu(CH3COO)2·H2O, Zn(CH3COO)2·2H2O, Co(CH3COO)2·2H2O, Ni(CH3COO)2·2H2O, 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.
2.2. 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 1H-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 UV-transilluminator.
2.3. 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).
2.4. Synthesis of Complexes 1–4
A solution of HL (1 mmol) in methanol (40 mL) was added slowly to a solution of metal(II) acetate salts (1 mmol) in methanol (30 mL) with constant stirring. The reaction mixture was refluxed for 2 hours. Then, the resultant solution was evaporated slowly on a water bath and finally a solid product was obtained and washed with cold ethanol and dried in vacuo (Scheme 2).
2.5. Antimicrobial Assay
Antimicrobial activities of the HL and complexes 1–4 were screened against the four different bacteria, Staphylococcus aureus (S. aureus), Staphylococcus pneumonia (S. pneumonia), Salmonella enterica typhi (S. typhi), and Haemophilus influenzae (H. influenzae), and two fungi, Aspergillus flavus (A. flavus) and Aspergillus niger (A. niger) strains by the well diffusion method [13]. Sparfloxacin (antibacterial) and Ketoconazole (antifungal) were used as standard drugs.
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 is the absorbance control and is the absorbance of sample or standard.
2.7. DNA Cleavage Study
DNA cleavage activities of HL and complexes 1–4 with CT-DNA were demonstrated by agarose gel electrophoresis method as reported earlier [15].
2.8. DNA Interaction Studies
DNA interaction studies of HL and complexes 1–4 with CT-DNA in Tris-HCl buffer were analyzed by electronic absorption spectral titration and viscometric measurements [16, 17].
3. 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. The low molar conductivity of the complexes 1–4 (9.72, 1; 10.80, 2; 11.60, 3; and 12.4, 4 ohm−1 cm2 mol−1) shows that they is nonelectrolytic nature due to lack of dissociation.
3.1. 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 C13H13N3O4. 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.
3.2. 1H-NMR Spectra
The 1H-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, -OCH3 protons at 3.73 (s), and phenolic -OH protons (C2 and C5) 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 (C2) is disappeared. These results suggest that the phenolic oxygen (C2), 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.
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3.3. 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 (CH3COO−) 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 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 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 transition and this indicates the square planar geometry [22]. Complex 2 has d10 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 transition in a square planar geometry [23]. For complex 4, the bands are examined at 432 and 446 cm−1, which were attributed to transition and this shows square planar geometry around the central metal atom [24].
3.5. 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 indicating that the unpaired electron lies predominantly in 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.
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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 Figures 3(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.
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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 Figures 3(b) and 4(b). From the above data, complexes 1 and 3 have 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.
3.7. 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 (H2O2) 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.
3.9. DNA Interaction
3.9.1. Absorption Spectral Titration
Absorption spectroscopy is one of the most common methods for determining the binding mode of CT-DNA with complexes. The absorption spectra of complex 1 in the presence and absence of CT-DNA with different concentration in Tris-HCl buffer are depicted in Figure 7. The increasing concentration of CT-DNA to the fixed concentration of complexes 1–4, the hypochromism, 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 , 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 × 105) > 3 (1.27 × 105) > 4 (1.02 × 105) > 2 (9.28 × 104) > HL (1.06 × 104).
3.9.2. 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.
4. Conclusions
In 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.
Conflicts of Interest
The authors declare that they have no conflicts of interest regarding the publication of this paper.
Acknowledgments
The authors express their heartfelt gratitude to the Department of Science and Technology (DST), Science and Engineering Research Board (SERB-Ref. no. SR/FT/CS-117/2011, dated 29.06.2012), New Delhi, for financial assistance and also express deepest gratitude to the Managing Board, Principal, and Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai, for providing research facilities and constant encouragements.