# 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

Authors: Shahul Hameed Sukkur Saleem; Murugesan Sankarganesh; Paul Raj Adwin Jose; Karunganathan Sakthikumar; Liviu Mitu; Jeyaraj Dhaveethu Raja
Journal: Journal of Chemistry
 (2017)

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## Abstract

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, 1H-NMR, FT-IR, UV-visible, and ESR techniques. The electronic and ESR spectral data suggested that complexes1–4 possess square planar geometry. Antimicrobial activities ofHL and complexes1–4 were tested against four bacteria(Staphylococcus aureus,Staphylococcus pneumonia,Salmonella enterica typhi, andHaemophilus influenzae) and two fungal strains(Aspergillus flavus andAspergillus niger). These results show that complexes1–4 have good antimicrobial activity compared toHL. The DNA cleavage activity ofHL and complexes1–4 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 ofHL and complexes1–4 have been investigated by electronic absorption technique and viscometric measurements.


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## Body

## 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 and1H-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 (Scheme1).Scheme 1
Synthetic route of ligandHL.


### 2.4. Synthesis of Complexes1–4

A solution ofHL (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).Scheme 2
Proposed structure of complexes1–4.


### 2.5. Antimicrobial Assay

Antimicrobial activities of theHL and complexes1–4 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) andAspergillus 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 ofHL and complexes1–4 were studied by DPPH scavenging method [14]. The % inhibition was calculated according to the following formula:(1)%Inhibition=A0-A1A1×100,where A0 is the absorbance control and A1 is the absorbance of sample or standard.


### 2.7. DNA Cleavage Study

DNA cleavage activities ofHL and complexes1–4 with CT-DNA were demonstrated by agarose gel electrophoresis method as reported earlier [15].


### 2.8. DNA Interaction Studies

DNA interaction studies ofHL and complexes1–4 with CT-DNA in Tris-HCl buffer were analyzed by electronic absorption spectral titration and viscometric measurements [16, 17].


## 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 and1H-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 (Scheme1).Scheme 1
Synthetic route of ligandHL.


## 2.4. Synthesis of Complexes1–4

A solution ofHL (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).Scheme 2
Proposed structure of complexes1–4.


## 2.5. Antimicrobial Assay

Antimicrobial activities of theHL and complexes1–4 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) andAspergillus 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 ofHL and complexes1–4 were studied by DPPH scavenging method [14]. The % inhibition was calculated according to the following formula:(1)%Inhibition=A0-A1A1×100,where A0 is the absorbance control and A1 is the absorbance of sample or standard.


## 2.7. DNA Cleavage Study

DNA cleavage activities ofHL and complexes1–4 with CT-DNA were demonstrated by agarose gel electrophoresis method as reported earlier [15].


## 2.8. DNA Interaction Studies

DNA interaction studies ofHL and complexes1–4 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 synthesizedHL and complexes1–4 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 complexes1–4 (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.Table 1
Analytical and physical data of the ligandHL and complexes 1–4.
Compounds
Molecular formula
Colour
Yield%
M.P.°C
Calc. (Found) %
C
H
N
M
HL
C13H13N3O4
brown
84
260
56.87(56.09)
4.71(4.65)
15.21(15.01)
—
1
CuC15H15N3O6
brown
78
282
45.40(45.38)
3.81(3.62)
10.59(10.54)
16.01(16.00)
2
ZnC15H15N3O6
brown
79
278
45.19(45.08)
3.79(3.71)
10.54(10.49)
16.40(16.38)
3
CoC15H15N3O6
red
82
295
45.93(45.89)
3.85(3.79)
10.71(10.68)
15.73(15.69)
4
NiC15H15N3O6
brown
74
271
45.96(45.89)
3.86(3.81)
10.72(10.68)
14.97(14.89)


### 3.1. Mass Spectra

Mass spectra of theHL and complexes1–4 recorded at room temperature were used to compare their stoichiometry composition. The ligandHL showed a molecular ion peak at (m/z 276) corresponding to C13H13N3O4. The molecular ion peaks for the complexes1–4 observed at m/z 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

The1H-NMR spectra of theHL and complex2 show the signals and are summarized in Table 2 and Figure 1. In free ligandHL, 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 complex2, a new peak is observed at 1.83 (s) due to acetate molecule involved in the complexation.Table 2
1H-NMR spectral data of the ligand HL and complex 2.
Compounds
-O-CH3  (δ)
Aromatic-CH (δ)
Pyrimidine-CH (δ)
Phenolic-OH (δ)
CH=N(δ)
CH3COO−  (δ)
HL
3.73 (s)
6.8–6.23 (m)
6.45
9.12 (C5)9.58 (C2)
8.2
—
Complex2
3.73 (s)
6.8–6.23 (m)
6.5
9.14
8.5
1.83Figure 1
1H-NMR spectra of (a) ligandHL and (b) complex2.
(a)
(b)


### 3.3. IR Spectra

IR spectral data ofHL and complexes1–4 are shown in Table 3. IR spectrum ofHL 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 complexes1–4 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 ν(M-N) for complexes1–4, 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 ν(M-O) for complexes1–4, indicating that the phenolic oxygen atom was involved in coordination with central metal ions [20].Table 3
Infrared spectral data of the ligandHL and complexes 1–4 (cm−1).
Compounds
CH=N
Pyrimidine C=N
-OAc
-OH
M-N
M-O
HL
1564
1465
—
3368
—
—
1
1540
1442
1668
3364
434
497
2
1542
1445
1648
3360
447
498
3
1544
1448
1652
3363
460
504
4
1548
1446
1665
3371
450
502


### 3.4. Electronic Spectra

Electronic absorption data ofHL and complexes1–4 are depicted in Table 4. In the absorption spectra ofHL, intense absorption bands at 292 and 370 nm were attributed to π-π∗ and n-π∗ transitions (Table 1) [21]. For complex1, the bands appeared at 482 and 620 nm, which were assigned to the B1g2→Eg2 transition and this indicates the square planar geometry [22]. Complex2 has d10 configuration, which suggests the absence of d-d transition bands, so complex2 reveals that INCT bands shift at 310 and 380 nm, respectively; this indicates the formation of zinc complex [14]. For complex3, the bands appeared at 428 and 465 nm which were attributable to the A1g1→B1g1 transition in a square planar geometry [23]. For complex4, the bands are examined at 432 and 446 cm−1, which were attributed to A1g1→A2g1 transition and this shows square planar geometry around the central metal atom [24].Table 4
Electronic spectral data of the synthesized compounds.
Compounds
λ
m
a
x, cm−1
Band assignment
Suggested geometry
HL
292370
INCT∗
—
1
482620
B
1
g
2
→
E
g
2
Square planar
2
310380
INCT∗
Square planar
3
428465
A
1
g
1
→
B
1
g
1
Square planar
4
432446
A
1
g
1
→
A
2
g
1
Square planar
∗Intraligand charge transfer.


### 3.5. ESR Spectra

The X-band ESR spectra of complex1 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 g-values are in the order g∥(2.11)>g⊥2.04>ge(2.0027) indicating that the unpaired electron lies predominantly in dx2-y2 orbital of Cu(II) ion [25]. These results also supported the square planar geometry around the central metal(II) ion. The interaction coupling constant G value is calculated from the following: (2)G=g∥-2.00277g⊥-2.00277.The observed G 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 G value is greater than 4, the exchange interaction is negligible.Table 5
The ESR spectral data for complex1.
Compound
g
t
e
n
s
o
r
Hyperfine constant × 104 cm−1
G
g
∥
g
⊥
g
a
v
A
∥
A
⊥
A
i
s
o
1
2.11
2.04
2.06
80
25
43.3
4.184Figure 2
ESR spectra of complex1: (a) RT and (b) LNT.
(a)
(b)


### 3.6. Antimicrobial

Antibacterial activities ofHL and complexes1–4 were screened against the four different bacteria,Staphylococcus aureus (S. aureus),Staphylococcus pneumonia (S. pneumonia),Salmonella typhi (S. typhi), andHaemophilus 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, complexes1 and3 have good antibacterial activity compared toHL and complexes2 and4. Moreover, zone of inhibitory efficiency of synthesized compounds is higher inS. aureus bacteria as compared to other bacterial strains.Figure 3
Antimicrobial activity of ligandHL and complexes1–4: (a) antibacterial; (b) antifungal.
(a)
(b)Figure 4
Minimum inhibitory concentration of ligandHL and complexes1–4 (10−2 M): (a) antibacterial; (b) antifungal.
(a)
(b)Antifungal activities ofHL and complexes1–4 were screened against two different fungi,Aspergillus flavus (A. flavus) andAspergillus 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, complexes1 and3 have good antifungal activity compared toHL and complexes2 and4. Moreover, zone of inhibitory effect of newly prepared compounds is higher inA. niger fungi as compared toA. flavus.


### 3.7. Antioxidant

The antioxidant activities ofHL and complexes1–4 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 complexes1 and3 have good scavenging abilityHL and complexes2 and4.Figure 5
% inhibition ofHL and complexes1–4 with DPPPH free radical.


### 3.8. DNA Cleavage

DNA cleavage studies ofHL and complexes1–4 with CT-DNA in the presence of hydrogen peroxide (H2O2) were analyzed by agarose gel electrophoresis techniques (Figure 6). From Figure 6, complex1 has good DNA damage activity compared to ligandHL and complexes2–4. These results reveal that complex1 is involved in the formation of hydroxyl radicals which may damage DNA via Fenton-type mechanism.Figure 6
Agarose gel diagram showing cleavage of CT-DNA by ligandHL and complexes1–4 at RT. Lane 1: DNA control + H2O2; Lane 2: DNA +HL + H2O2; Lane 3: DNA +1 + H2O2; Lane 4: DNA +2 + H2O2; Lane 5: DNA +3 + H2O2; Lane 6: DNA +4 + H2O2.


### 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 complex1 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 complexes1–4, the hypochromism, and slight red shift have been observed. Clearly, these obtained results are coinciding with the previously reported results [27] which suggest that theHL and complexes1–4 interact with CT-DNA via intercalation mode. The intrinsic binding constant (Kb) ofHL and complexes1–4 was determined by using the following formula (Table 6): DNA/εa-εf=DNA/εb-εf+Kbεb-εf-1, where [DNA] is the concentration of base pairs of DNA. The apparent absorption coefficients εa, εf, and εb correspond to Aobs./[M], the extinction coefficient for the free complex, and extinction coefficient for the complex in the fully bound form, respectively. The Kb values ofHL and complexes1–4 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).Table 6
Absorption spectral properties ofHL and complexes 1–4with CT-DNA.
Compounds
Freeλmax (nm)
Boundλmax (nm)
Red shiftΔλ
Type of chromism
Hypochromism(%)
Binding constant (Kb)
HL
293
294
01
Hypochromism and red shift
22.45
1.06 × 104
1
298
304
06
Hypochromism and red shift
40.82
4.76 × 105
2
300
305
05
Hypochromism and red shift
29.38
9.28 × 104
3
302
307
05
Hypochromism and red shift
38.74
1.27 × 105
4
301
306
05
Hypochromism and red shift
35.42
1.02 × 105Figure 7
Absorption spectra of complex1 with CT-DNA in Tris-HCl buffer.


#### 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] (Figure8) show that the viscous flow of DNA increases when increasing the concentration ofHL and complexes1–4. These results have suggested the interaction of compounds with DNA via intercalation binding mode.Figure 8
Effect of increasing concentration of complexes1–4 on the relative viscosity of CT-DNA.


## 3.1. Mass Spectra

Mass spectra of theHL and complexes1–4 recorded at room temperature were used to compare their stoichiometry composition. The ligandHL showed a molecular ion peak at (m/z 276) corresponding to C13H13N3O4. The molecular ion peaks for the complexes1–4 observed at m/z 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

The1H-NMR spectra of theHL and complex2 show the signals and are summarized in Table 2 and Figure 1. In free ligandHL, 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 complex2, a new peak is observed at 1.83 (s) due to acetate molecule involved in the complexation.Table 2
1H-NMR spectral data of the ligand HL and complex 2.
Compounds
-O-CH3  (δ)
Aromatic-CH (δ)
Pyrimidine-CH (δ)
Phenolic-OH (δ)
CH=N(δ)
CH3COO−  (δ)
HL
3.73 (s)
6.8–6.23 (m)
6.45
9.12 (C5)9.58 (C2)
8.2
—
Complex2
3.73 (s)
6.8–6.23 (m)
6.5
9.14
8.5
1.83Figure 1
1H-NMR spectra of (a) ligandHL and (b) complex2.
(a)
(b)


## 3.3. IR Spectra

IR spectral data ofHL and complexes1–4 are shown in Table 3. IR spectrum ofHL 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 complexes1–4 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 ν(M-N) for complexes1–4, 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 ν(M-O) for complexes1–4, indicating that the phenolic oxygen atom was involved in coordination with central metal ions [20].Table 3
Infrared spectral data of the ligandHL and complexes 1–4 (cm−1).
Compounds
CH=N
Pyrimidine C=N
-OAc
-OH
M-N
M-O
HL
1564
1465
—
3368
—
—
1
1540
1442
1668
3364
434
497
2
1542
1445
1648
3360
447
498
3
1544
1448
1652
3363
460
504
4
1548
1446
1665
3371
450
502


## 3.4. Electronic Spectra

Electronic absorption data ofHL and complexes1–4 are depicted in Table 4. In the absorption spectra ofHL, intense absorption bands at 292 and 370 nm were attributed to π-π∗ and n-π∗ transitions (Table 1) [21]. For complex1, the bands appeared at 482 and 620 nm, which were assigned to the B1g2→Eg2 transition and this indicates the square planar geometry [22]. Complex2 has d10 configuration, which suggests the absence of d-d transition bands, so complex2 reveals that INCT bands shift at 310 and 380 nm, respectively; this indicates the formation of zinc complex [14]. For complex3, the bands appeared at 428 and 465 nm which were attributable to the A1g1→B1g1 transition in a square planar geometry [23]. For complex4, the bands are examined at 432 and 446 cm−1, which were attributed to A1g1→A2g1 transition and this shows square planar geometry around the central metal atom [24].Table 4
Electronic spectral data of the synthesized compounds.
Compounds
λ
m
a
x, cm−1
Band assignment
Suggested geometry
HL
292370
INCT∗
—
1
482620
B
1
g
2
→
E
g
2
Square planar
2
310380
INCT∗
Square planar
3
428465
A
1
g
1
→
B
1
g
1
Square planar
4
432446
A
1
g
1
→
A
2
g
1
Square planar
∗Intraligand charge transfer.


## 3.5. ESR Spectra

The X-band ESR spectra of complex1 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 g-values are in the order g∥(2.11)>g⊥2.04>ge(2.0027) indicating that the unpaired electron lies predominantly in dx2-y2 orbital of Cu(II) ion [25]. These results also supported the square planar geometry around the central metal(II) ion. The interaction coupling constant G value is calculated from the following: (2)G=g∥-2.00277g⊥-2.00277.The observed G 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 G value is greater than 4, the exchange interaction is negligible.Table 5
The ESR spectral data for complex1.
Compound
g
t
e
n
s
o
r
Hyperfine constant × 104 cm−1
G
g
∥
g
⊥
g
a
v
A
∥
A
⊥
A
i
s
o
1
2.11
2.04
2.06
80
25
43.3
4.184Figure 2
ESR spectra of complex1: (a) RT and (b) LNT.
(a)
(b)


## 3.6. Antimicrobial

Antibacterial activities ofHL and complexes1–4 were screened against the four different bacteria,Staphylococcus aureus (S. aureus),Staphylococcus pneumonia (S. pneumonia),Salmonella typhi (S. typhi), andHaemophilus 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, complexes1 and3 have good antibacterial activity compared toHL and complexes2 and4. Moreover, zone of inhibitory efficiency of synthesized compounds is higher inS. aureus bacteria as compared to other bacterial strains.Figure 3
Antimicrobial activity of ligandHL and complexes1–4: (a) antibacterial; (b) antifungal.
(a)
(b)Figure 4
Minimum inhibitory concentration of ligandHL and complexes1–4 (10−2 M): (a) antibacterial; (b) antifungal.
(a)
(b)Antifungal activities ofHL and complexes1–4 were screened against two different fungi,Aspergillus flavus (A. flavus) andAspergillus 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, complexes1 and3 have good antifungal activity compared toHL and complexes2 and4. Moreover, zone of inhibitory effect of newly prepared compounds is higher inA. niger fungi as compared toA. flavus.


## 3.7. Antioxidant

The antioxidant activities ofHL and complexes1–4 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 complexes1 and3 have good scavenging abilityHL and complexes2 and4.Figure 5
% inhibition ofHL and complexes1–4 with DPPPH free radical.


## 3.8. DNA Cleavage

DNA cleavage studies ofHL and complexes1–4 with CT-DNA in the presence of hydrogen peroxide (H2O2) were analyzed by agarose gel electrophoresis techniques (Figure 6). From Figure 6, complex1 has good DNA damage activity compared to ligandHL and complexes2–4. These results reveal that complex1 is involved in the formation of hydroxyl radicals which may damage DNA via Fenton-type mechanism.Figure 6
Agarose gel diagram showing cleavage of CT-DNA by ligandHL and complexes1–4 at RT. Lane 1: DNA control + H2O2; Lane 2: DNA +HL + H2O2; Lane 3: DNA +1 + H2O2; Lane 4: DNA +2 + H2O2; Lane 5: DNA +3 + H2O2; Lane 6: DNA +4 + H2O2.


## 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 complex1 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 complexes1–4, the hypochromism, and slight red shift have been observed. Clearly, these obtained results are coinciding with the previously reported results [27] which suggest that theHL and complexes1–4 interact with CT-DNA via intercalation mode. The intrinsic binding constant (Kb) ofHL and complexes1–4 was determined by using the following formula (Table 6): DNA/εa-εf=DNA/εb-εf+Kbεb-εf-1, where [DNA] is the concentration of base pairs of DNA. The apparent absorption coefficients εa, εf, and εb correspond to Aobs./[M], the extinction coefficient for the free complex, and extinction coefficient for the complex in the fully bound form, respectively. The Kb values ofHL and complexes1–4 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).Table 6
Absorption spectral properties ofHL and complexes 1–4with CT-DNA.
Compounds
Freeλmax (nm)
Boundλmax (nm)
Red shiftΔλ
Type of chromism
Hypochromism(%)
Binding constant (Kb)
HL
293
294
01
Hypochromism and red shift
22.45
1.06 × 104
1
298
304
06
Hypochromism and red shift
40.82
4.76 × 105
2
300
305
05
Hypochromism and red shift
29.38
9.28 × 104
3
302
307
05
Hypochromism and red shift
38.74
1.27 × 105
4
301
306
05
Hypochromism and red shift
35.42
1.02 × 105Figure 7
Absorption spectra of complex1 with CT-DNA in Tris-HCl buffer.


### 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] (Figure8) show that the viscous flow of DNA increases when increasing the concentration ofHL and complexes1–4. These results have suggested the interaction of compounds with DNA via intercalation binding mode.Figure 8
Effect of increasing concentration of complexes1–4 on the relative viscosity of CT-DNA.


## 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 complex1 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 complexes1–4, the hypochromism, and slight red shift have been observed. Clearly, these obtained results are coinciding with the previously reported results [27] which suggest that theHL and complexes1–4 interact with CT-DNA via intercalation mode. The intrinsic binding constant (Kb) ofHL and complexes1–4 was determined by using the following formula (Table 6): DNA/εa-εf=DNA/εb-εf+Kbεb-εf-1, where [DNA] is the concentration of base pairs of DNA. The apparent absorption coefficients εa, εf, and εb correspond to Aobs./[M], the extinction coefficient for the free complex, and extinction coefficient for the complex in the fully bound form, respectively. The Kb values ofHL and complexes1–4 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).Table 6
Absorption spectral properties ofHL and complexes 1–4with CT-DNA.
Compounds
Freeλmax (nm)
Boundλmax (nm)
Red shiftΔλ
Type of chromism
Hypochromism(%)
Binding constant (Kb)
HL
293
294
01
Hypochromism and red shift
22.45
1.06 × 104
1
298
304
06
Hypochromism and red shift
40.82
4.76 × 105
2
300
305
05
Hypochromism and red shift
29.38
9.28 × 104
3
302
307
05
Hypochromism and red shift
38.74
1.27 × 105
4
301
306
05
Hypochromism and red shift
35.42
1.02 × 105Figure 7
Absorption spectra of complex1 with CT-DNA in Tris-HCl buffer.


## 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] (Figure8) show that the viscous flow of DNA increases when increasing the concentration ofHL and complexes1–4. These results have suggested the interaction of compounds with DNA via intercalation binding mode.Figure 8
Effect of increasing concentration of complexes1–4 on the relative viscosity of CT-DNA.


## 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 complexes1–4 is of 1 : 1 ratio and the type is ML. All the spectral results suggest that complexes1–4 possess square planar geometry around the central metal atom. Antimicrobial and antioxidant results reveal that complexes1 and3 have good antimicrobial and the ability to scavenge DPPH radicals compared to ligandHL and complexes2 and4. Complex1 has good DNA cleavage ability compared toHL and complexes2–4. DNA interactions studies results have suggested that the newly prepared compounds interact with CT-DNA via intercalation mode.



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