Bioinorganic Chemistry and Applications
Volume 2014 (2014), Article ID 483282, 11 pages
http://dx.doi.org/10.1155/2014/483282

Research Article

Synthesis, Spectroscopic Characterization, and Biological Evaluation Studies of 5-Bromo-3-(((hydroxy-2-methylquinolin-7-yl)methylene)hydrazono)indolin-2-one and Its Metal (II) Complexes

Kuruba Siddappa and Nabiya Sultana Mayana

Department of Studies and Research in Chemistry, Gulbarga University, Gulbarga, Karnataka 585106, India

Received 21 May 2014; Revised 12 September 2014; Accepted 15 September 2014; Published 12 October 2014

Academic Editor: Viktor Brabec

Copyright © 2014 Kuruba Siddappa and Nabiya Sultana Mayana. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The Schiff base ligand 5-bromo-3-(((8-hydroxy-2-methylquinolin-7-yl)methylene)hydrazono)indolin-2-one (BHMQMHI) was prepared via condensation of 5-bromo-3-hydrazonoindolin-2-one and 7-formyl-8-hydroxy-2-methylquinoline and its Co(II), Ni(II), Cu(II), Zn(II), Cd(II), and Hg(II) complexes have been synthesized and characterized by elemental analysis, conductance data, magnetic susceptibility measurements, IR, UV-Vis, mass spectrometry, ¹H NMR, ESR, XRD, and thermal studies. By these spectral studies it is found that Co(II), Ni(II), and Cu(II) complexes have exhibited octahedral geometry whereas the Zn(II), Cd(II), and Hg(II) complexes have exhibited tetrahedral geometry. Potentiometric studies have been carried out on complexes of Schiff base (BHMQMHI) with Cu(II), Co(II), and Ni(II). Calvin-Bjerrum pH-titration technique as used by Irving and Rossotti has been applied to determine stability constants in mixed solvents at °C. The present study reports the protonation constants of this ligand and stability constants of its metal complexes in dioxane-water (50%, v/v) mixtures. Metal-ligand stability constants fall in the order of Cu(II) > Co(II) > Ni(II) which is in agreement with those reported by Irving stability order. The Schiff base (BHMQMHI) and its metal complexes have been screened for their in vitro antibacterial and antifungal activities by minimum inhibitory concentration (MIC) method. The DNA cleavage activities of all the complexes were studied by agarose gel electrophoresis method. In addition, the free ligand along with its complexes has been studied for their antioxidant activity.

1. Introduction

Schiff base complexes have undergone a phenomenal growth during the recent years because of the versatility offered by these complexes in the field of industries, catalysis and biological system, and so forth. In this way, the synthesis, structural investigation, and reaction of transition metal Schiff bases have received a special attention, because of their biological activities as antitumoral, antifungal, and antiviral activities [1].

Hydrazones possessing an azomethine proton (–NHN=CH–) constitute an important class of compounds for new drug development. Many researchers have synthesised these compounds as well as their metal complexes as target structures and evaluated their biological activities. These observations have guided the development of new hydrazones with varied biological activities. The biological activity of complexes derived from hydrazones has been studied and contrasted with regard to their antibacterial, antitumoral, antiviral, antimalarial, and antitubercular properties. It has also been shown that the azomethine N, which has a lone pair of electrons in an hybridised orbital, is biologically important [2]. The isatin molecule (1H-indole-2, 3-Dione) is a versatile moiety that displays diverse biological activities. In this paper, we report the synthesis, structural characterization, thermal decomposition, and biological studies of some transition metal complexes with the host of a bishydrazone derived from 5-bromo-3-hydrazonoindolin-2-one and 7-formyl-8-hydroxy-2-methylquinoline, namely, (5-bromo-3-(((8-hydroxy-2-methylquinolin-7-yl)methylene)hydrazono)indolin-2-one (BHMQMHI)).

2. Experimental

2.1. Materials and Reagents

All chemicals used were of analytical grade. 5-Bromo isatin and hydrazine hydrate (99%) were obtained from Fluka and Sisco chemicals.

2.2. Solutions

Dioxane was purified by the standard procedures [3]. Ligand solution was prepared in double distilled alcohol. Metal salt solutions were prepared by dissolving the corresponding metal salt in double distilled deionized water and standardized by standard volumetric methods [3].

Calvin-Bjerrum Technique. The experimental procedure involves the acid titration, ligand titration, and metal titration. The details of titrations are shown in Table 1. The total volume in all the cases was 50 mL.

Table 1: The experimental procedure involves the acid titration, ligand titration, and metal titration. = 25 ± 1°C  50% Dioxane-water  µ = 0.1 M/0.2 M NaClO₄.

Solution
(initial concentration)
Acid
titration
Ligand titration
Metal titration

HClO₄ (0.01 M)
5.0 mL
5.0 mL
5.0 mL

NaClO₄ (1 M/2 M)
5.0 mL
5.0 mL
5.0 mL

Dioxane
25.0 mL
25.0 mL
25.0 mL

Water
15.0 mL
10.0 mL
5.0 mL

Ligand (0.01 M)
—
5.0 mL
5.0 mL

Metal (0.01 M)
—
—
5.0 mL

2.3. Physical Measurements

pH measurements were carried out using Elico pH-meter model L1-122. Elemental analysis was carried out using a Heracus Carlo Erba 1108 CHN analyzer at STIC, Cochin. The IR spectra of the Schiff base (BHMQMHI) and its Co(II), Ni(II), Cu(II), Zn(II), Cd(II), and Hg(II) complexes were recorded in the region of 4000–250 cm⁻¹ on a Perkin Elmer Spectrum RX-IFTIR spectrophotometer. The electronic spectra of the Co(II), Ni(II), and Cu(II) complexes were recorded on an ELICO SL-164 double beam UV-visible spectrophotometer in the range of 200–1200 nm in DMF (10⁻³ M) solution. Magnetic susceptibility measurements were made at room temperature on a Gouy balance using Hg[Co(NCS)₄] as the calibrant. Molar conductivity measurements were recorded on an ELICO CM-180 conductivity bridge in DMF solution (10⁻³ M) using a dip-type conductivity cell fitted with a platinum electrode. The ¹H NMR spectra were recorded in DMSO-d₆ on a Bruker 500 MHz spectrophotometer using TMS as an internal standard. The mass spectra were recorded on a JEOL GC mate mass spectrophotometer. The ESR spectrum of the Cu(II) complex in the polycrystalline state was recorded on a Varian-E-4X band EPR spectrophotometer using TCNE as the “” marker () at room temperature. Thermal analyses were measured from room temperature to 1000°C in N₂ on a perkin Elmer, Diamond TG/DTA model thermal analyzer at STIC, Cochin, with a heating rate of 10°C min⁻¹. The XRD patterns of the Cu(II) complex were recorded on a Bruker AXS D8 Advance X-ray diffractometer using Cu Kα = 1.5404 radiation (λ Å).

2.4. Procedures

2.4.1. Potentiometric Measurements

The potentiometric measurements were carried out at °C and at constant ionic strengths (0.1 M and 0.2 M). The pH-meter was calibrated before each titration using standard buffers. The ionization constants of the investigated Schiff base (BHMQMHI) and the stability constants of its metal complexes with Co(II), Ni(II), and Cu(II) ions were determined potentiometrically using the technique of Calvin-Bjerrum Technique. The ionization constants of the ligand are calculated using the equation used by Irving and Rossotti while metal-ligand stability constants were calculated using methods applied for computing successive stability constants.

2.4.2. Synthesis of Schiff Base Ligand

5-Bromo-3-(((8-hydroxy-2-methylquinolin-7-yl)methylene)hydrazono)indolin-2-one (BHMQMHI): the Schiff base has been synthesized by refluxing the equimolar mixture of hot methanolic solution of 7-formyl-8-hydroxy-2-methylquinoline (0.01 mol, 30 mL) and hot methanolic solution of 5-bromo-3-hydrazonoindolin-2-one (0.01 mol, 30 mL) for 6-7 h in presence of catalytical amount of hydrochloric acid. The product obtained after the evaporation of the solvent was filtered, washed with cold methanol, and finally recrystallized from methanol to afford Schiff base (BHMQMHI) as shown in Figure 1. The purity of the compound has been checked by thin layer chromatography (TLC).

Figure 1: Schematic representation of Schiff base (BHMQMHI).

2.4.3. Synthesis of Metal Complexes

The metal complexes were prepared using metal chlorides and the Schiff base (BHMQMHI) by the general method. An ethanolic solution (40 mL) of Schiff base and Co(II), Ni(II), and Cu(II) chlorides in 1 : 2 molar ratio and 1 : 1 molar ratio for Zn(II), Cd(II), and Hg(II) complexes were refluxed on water bath for about 4 h. An aqueous solution of sodium acetate was added to the reaction mixture to adjust the pH to 6.0-7.0 and reflux was further continued for about an hour. The separated solid complexes were filtered off and washed thoroughly with water and then with little warm ethanol. The complexes obtained were finally dried under vacuum desiccator over fused CaCl₂. Elemental analysis data are shown in Table 2. The complexes were analyzed for their metal and chloride contents by standard methods [3].

Table 2: Physical and analytical data of the ligand (BHMQMHI) and its metal (II) complexes.

Compound
Molecular formula
Yield (%)
Found (calculated) %
m
( cm² mol⁻¹)

(BM)

C
H
N
M
Cl

Ligand
[C₁₉H₁₃N₄O₂Br]
81
55.76 (55.54)
3.20 (3.16)
13.69 (13.53)
—
—
—
  —

Co(II) complex
[Co(C₃₈H₂₄N₈O₄Br₂)]
79
52.14 (52.01)
2.76 (2.68)
12.80 (12.78)
6.73 (6.68)
—
27.23
 4.88

Ni(II) complex
[Ni(C₃₈H₂₄N₈O₄Br₂)]
70
52.15 (52.08)
2.76 (2.63)
12.80 (12.76)
6.70 (6.63)
—
20.43
 3.00

Cu(II) complex
[Cu(C₃₈H₂₄N₈O₄Br₂)]
65
51.86 (51.78)
2.75 (2.68)
12.73 (12.63)
7.22 (7.12)
—
18.45
 1.94

Zn(II) complex
[Zn(C₁₉H₁₂N₄O₂Br)Cl]
80
44.83 (44.77)
2.38 (2.28)
11.01 (10.08)
12.84 (12.70)
6.96 (6.76)
22.81
  —

Cd(II) complex
[Cd(C₁₉H₁₂N₄O₂Br)Cl]
77
41.04 (39.93)
2.18 (2.00)
10.08 (9.93)
20.21 (20.19)
6.37 (6.26)
23.76
  —

Hg(II) complex
[Hg(C₁₉H₁₂N₄O₂Br)Cl]
66
35.42 (34.54)
1.88 (1.78)
8.70 (8.65)
—
—
29.76
  —

3. Pharmacology

3.1. Antibacterial and Antifungal Activities

Antibacterial and antifungal activity of ligand and their complexes has been determined by serial tube dilution technique [4]. The stock solution of each compound was prepared by dissolving 10 mg of each test compound in 10 mL of freshly distilled DMSO. The various concentrations of the test compounds (100, 75, 50, 25, and 12.5 μg/mL⁻¹) were prepared by diluting the stock solution with the required volume of freshly distilled DMSO. The MIC was measured after 24 h in case of antibacterial activity and 48 h for antifungal activity.

The in vitro biological screening effects of the investigated compounds were tested against the bacteria S. aureus, B. subtilis, and P. aeruginosa cultured on Mueller Hinton agar media. The antifungal activity of the Schiff base and its metal complexes was tested against A. flavus, A. niger, and C. albicans on Czapek-Dox agar media. The control containing only DMSO and the standard antibiotic (Gentamycin and Amphotericin) was also kept for comparison [5].

3.2. Antioxidant Activity: Radical Scavenging Activity

The radical scavenging activity ligand (BHMQMHI) and its complexes were determined by using 1, 1-diphenyl-2, 2-picryl hydrazyl-free radical (DPPH) assay method [6]. DPPH is a stable free-radical molecule, accept an electrons or hydrogen radicals to become a stable diamagnetic molecule and also contained an odd electron in its structure that is frequently used for detection of the radical scavenging activity in chemical analysis. The reduction capability of DPPH radicals was determined by a decrease in its absorbance at 517 nm induced by antioxidants. The absorption maximum of a stable DPPH radical in ethanol was at 517 nm. The absorbance decreased when the DPPH is scavenged by an antioxidant, through donation of hydrogen to form a stable DPPH molecule. In the radical form, those molecules show an absorbance at 517 nm, which disappeared after acceptance of an electron or hydrogen radical from an antioxidant compound to become a stable diamagnetic spin-paired molecule. The stock solutions (1 mg/mL) of the ligand and its complexes were diluted to a final concentration of 25, 50, 75, and 100 μg/mL in methanol. DPPH solution (1 mL, 0.1 mmol) was added to 2.5 mL of test solution of different concentration and allowed to react at room temperature. After 30 min the absorbance was measured at 517 nm. A graph was plotted with percentage scavenging effects on the -axis and concentration (μg/mL) on the -axis. Radical scavenging activity was expressed as a percentage and was calculated using the following formula:

The scavenging capability of the ligand and its complexes were compared with standard drugs, namely, butylated hydroxyl anisole (BHA) and tertiary butylated hydroxyl quinoline (TBHQ) and ascorbic acid.

3.3. DNA Cleavage Experiment

Preparation of culture media and DNA isolation of Calf-thymus were done according to the literature procedure [7]. Nutrient broth (10 g L⁻¹ of peptone, 5 g L⁻¹ of yeast extract and 10 g L⁻¹ of NaCl) was used for culturing of Calf-thymus.

The cleavage of Calf-thymus DNA was determined by agarose gel electrophoresis [5]. Calf-thymus DNA was cultured, isolated, and used as DNA for the experiment. The precipitated DNA was separated by centrifugation and the pellet was dried and dissolved in tris buffer (10 mM tris pH 8.0) and stored in cold condition. 25 μg of the test samples was added to the isolated Calf-thymus DNA and incubated for 2 h at 37°C. After incubation, the samples were electrophoresed for 45 min at 50 V on agarose gel using TAE buffer (4.84 g tris base, pH 8.0, 0.5 M EDTA/L). After the run, gel was removed and stained with 10 μg/mL ethidium bromide (ETBR) for 10–15 min and the image was taken in UV transilluminator and photographed to determine the extent of DNA cleavage. The results are compared with standard DNA marker [8–10].

4. Results and Discussion

The Schiff base ligand and its transition metal complexes have been synthesized and characterized by spectral and elemental analytical data. All the complexes are freely soluble in acetonitrile, DMF, and DMSO and insoluble in water. The analytical data (Table 2) indicates that the stoichiometry of the complexes is 1 : 2 (metal to ligand ratio) for Co(II), Ni(II), Cu(II), and 1 : 1 (metal to ligand ratio) for Zn(II), Cd(II), and Hg(II) complexes. The lower conductance values (18–29 Ohm⁻¹ cm² mol⁻¹) of the complexes support their nonelectrolytic nature of the compounds [11, 12].

4.1. IR Spectral Studies

The prominent infrared spectral data with the tentative assignments of the Schiff base (BHMQMHI) and its Co(II), Ni(II), Cu(II), Zn(II), Cd(II), and Hg(II) complexes are presented in Table 3.

Table 3: IR spectral bands of the ligand (BHMQMHI) and its metal complexes (cm⁻¹).

Tentative assignments
L
Co(II) complex
Ni(II) complex
Cu(II) complex
Zn(II) complex
Cd(II) complex
Hg(II) complex

Hydrogen bonded OH group
 3364
—
—
—
—
—
—

Indole ring NH
 3203
3203
3203
3203
3023
3203
3203

ν(C=O) ring
 1696
1685
1686
1679
1647
1672
1667

ν(C=N) ring
 1603
1603
1603
1603
1603
1603
1603

ν(C=N) aldimine
 1583
1565
1525
1530
1533
1505
1549

ν(C–O)
 1289
1300
1307
1306
1314
1344
1360

ν(N–N)
 991
1067
1082
1035
1099
1059
1033

ν(M–N)
 —
479
496
468
477
489
489

ν(M–O)
 —
533
578
500
500
523
598

ν(M–Cl)
 —
—
—
—
344
358
366

The broad peak observed at 3364 cm⁻¹ in the IR spectra of the ligand assigned to (OH), which was found to have disappeared in all the complexes, thereby indicating deprotonation and formation of metal-oxygen bond [13]. This is further supported by the shifting of phenolic (C–O) towards higher frequency, indicating the coordination of the phenolate oxygen to metal ion. The (HC=N) vibration of the ligand occurs at 1583 cm⁻¹, which is shifted to a lower frequency in the complexes, indicating the involvement of the azomethine nitrogen in chelation with the metal ion [14]. The band corresponding to (C=O) at 1696 cm⁻¹ is shifted to a lower frequency, supporting the coordination of the carbonyl oxygen [15]. However vibrational characteristics of the ring (N–H) and (C=N) of the ketimine moiety remain almost unaffected, indicating the nonparticipation of these groups in coordination [16]. The bands in the regions 500–598 and 468–496 cm⁻¹ are ascribed to (M–O) and (M–N) vibrations, respectively [17]. The bands observed in the region 344–366 cm⁻¹ are due to the formation of (M–Cl) bond, which was characteristic of the involvement of chloride atom in coordination with Zn(II), Cd(II) and Hg(II) ions. But in Co(II), Ni(II), and Cu(II) complexes (M–Cl) bands are absent. From the above observations, it can be concluded that the ligand binds to the metal ion in a tridentate fashion through the deprotonated phenolate oxygen, Schiff base nitrogen, and the carbonyl oxygen of the isatin moiety.

4.2. ¹H-NMR Spectral Studies

¹H-NMR spectra of the ligand (BHMQMHI) and its Zn(II), Cd(II), and Hg(II) complexes were recorded in DMSO-d⁶ using TMS as an internal standard and the integrated intensities of the signals are well agreed with the number of protons of each type. The signal at δ (10.3) (s, 1H) was assigned to phenolic −OH in the ligand. The signal is disappear in Zn(II), Cd(II) and Hg(II) complexes, which indicates the involvement of phenolic oxygen atom in the coordination via deprotonation [18]. The signal at δ (8.4) (s, 1H) is due to the azomethine group in the ligand (BHMQMHI). These signals shifts downfield in the regions δ (9.1) (s, 1H), δ (8.8) (s, 1H), and δ (9.0) (s, 1H) in the spectra of Zn(II), Cd(II), and Hg(II) complexes, respectively, and confirms the coordination of “N” of (–C=N–) group in bonding with the metal ions [19]. The peak that appeared at δ (9.7) (s, 1H) is due to the hydrogen of –NH in the ligand, but in case of Zn(II), Cd(II), and Hg(II) complexes the peak was observed at δ (9.7) (s, 1H), and this signal remains unaltered in the spectra of Zn(II), Cd(II), and Hg(II) complexes confirming the noncoordination (–NH) in bonding with metal ions [20]. In ligand seven aromatic protons have been observed in the region δ (6.9–7.5) (m, 7H) as a multiplet, and it is shifted downfield in the Zn(II), Cd(II), and Hg(II) complexes. The signals observed at δ (2.5) are due to methyl protons that remain almost unaffected in Zn(II), Cd(II), and Hg(II) complexes indicating the nonparticipation of these groups in coordination [21].

4.3. Mass Spectral Studies

The mass spectrum of the Schiff base (BHMQMHI) shows a molecular ion peak at m/z 409.24, which is equivalent to its molecular weight. The mass spectra of the Co(II), Ni(II), Cu(II), Zn(II), Cd(II), and Hg(II) complexes showed a molecular ion peak at m/z 875.39, 875.15, 880.00, 509.09, 556.09, and 644.27 which is the same as that of the molecular weight of the complexes. This confirms the proposed structure for the complexes.

4.4. Electronic Spectral and Magnetic Studies

The electronic spectral data of metal (II) complexes were recorded in DMF as shown in Table 4. They have been studied with the view to obtain more information on stereochemistry of the complexes and to procedure more support for the conclusion, deduced with the help of magnetic data. The Co(II) complex of the electronic absorption bands appears at 15384 and 20000 cm⁻¹ due to ⁴T1g (F) ⁴A2g (F) () and ⁴T1g (F) ⁴T2g (P) () transitions, respectively, in an octahedral environment [22]. The band could not be observed because of its very low intensity. However the position of the band has been computed (7148 cm⁻¹) by the equation Dq. The ligand field parameters such as Dq, B ′, , and have been calculated by using band-fitting equation given by Underhill and Billing [23]. The crystal field splitting (Dq) energy value was at 823 cm⁻¹. These values are well within the range and also reported by most of the octahedral Co(II) complexes. Co(II) complex under present investigation process interelectronic repulsion parameter (B ′) 929 cm⁻¹. The Racah parameter is less than free ion value suggesting a considerable orbital overlap and delocalization of electrons on the metal ion. The nephelauxetic ratio () for the Co(II) complex is 0.89. This is less than one, suggesting partial covalency in the metal ligand bond. The values Dq, , , , and LFSE suggest the octahedral geometry for Co(II) complex [24]. The Co(II) complex displays a magnetic moment value of 4.88 BM, which is within the range of 4.46–5.53 BM.

Table 4: Electronic spectral bands and ligand field parameters of the Co(II), Ni(II), and Cu(II) complexes in DMF (10⁻³ M) solution.

Complexes
Transitions in cm⁻¹
Dq (cm⁻¹)
(cm⁻¹)

%

LFSE (k cal)

Co(II) complex
7148
15384
20000
823
929
0.89
10.64
2.15
14.11

Ni(II) complex
9614
15384
25000
961
769
0.73
26.02
1.60
32.96

Cu(II) complex

14285–17391

1583
—
—
—
—
27.15

The electronic spectrum of Ni(II) complex shows two bands at 15384 and 25000 cm⁻¹ assignable to ³A2g (F) ³T1g (F) () and ³A2g (F) ³T1g (P) () transitions, respectively, in an octahedral environment [25]. The lowest band was not observed due to limited range of the instrument used. However, it is calculated by using equation suggested by Billing and Underhill. Racah parameter B ′ is less than the free ion value of 1040 cm⁻¹ indicating the covalent character of the complex. The ratios and further support the octahedral geometry around the Ni(II) ion [26]. The Ni(II) complex showed the magnetic moment value of 3.00 BM, which is within the range of 2.7–3.3 BM.

The Cu(II) complex exhibits a broad asymmetric band in the region 14285–17391 cm⁻¹. The broadness of the band may be due to dynamic Jahn-Teller distortion and is assigned to ²B1g²A2g (), ²B1g²Eg (), and ²B1g²B2g () transitions. The Cu(II) complexes showed magnetic moment value of 1.94 BM, which is within the range of 1.75–2.20 BM which is consistent with octahedral geometry [27].

4.5. Thermogravimetric Analysis

The Copper (II) complex is subjected to thermogravimetric analysis in dynamic air in 40–750°C temperature range, at a heat ingrate of 10°C/min. The complex is stable up to 200°C and exhibits a single stage decomposition pattern, as is evident from the TG-DTA profile. The mass loss in the single stage decomposition occurred at 221.00°C which can be attributed to the loss of ligand moiety further leaving behind the metal oxide residue. The complex shows gradual degradation up to 709.3°C. The single stage decomposition of the metal complex usually occurs when there is a high degree of electron delocalization along a conjugated system which leads to uniformity in bond strength [28].

4.6. ESR Spectrum of Cu(II) Complex

The ESR spectrum of the Cu(II) complex in a polycrystalline state was recorded at room temperature. The and values were found to be 2.405 and 2.041, respectively. The was calculated to be 2.057. The broadening of this signal might be due to dipolar interactions, indicating lowered site symmetry suggesting that the unpaired electron resides mainly in the orbital [29]. The axial symmetry parameter “” was determined as , suggesting that there is no exchange interaction in the Cu(II) complex [30].

4.7. Powder X-Ray Diffraction

The Cu(II) complex has been characterized by powder XRD studies with a view to find the type of crystal system. The XRD data of Cu(II) complex is given in Table 5. There are 11 reflections (2) between 15.804 and 68.526 with maxima at corresponding to the value of . The interplanar spacing () has been calculated from the positions of intense peaks using Bragg’s equation (where  Å). The observed and calculated values of are quite consistent. The experimental values of sin²/common factor are recorded for each peak in Figure 2. The values of the complex were found to be 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, and 17. The presence of forbidden number 7 indicates the Cu(II) complex belongs to hexagonal system [31].

Table 5: X-ray diffraction data of Cu(II) complex.

2

h  k  l
value
in Å

Cal
Abs

15.804
7.902
0.1374
0.0189
1
1 0 0
5.6030
5.6008
5.600

24.445
12.222
0.2117
0.0448
2.37234 (2)
1 1 0
3.6384
3.6363
5.600

26.887
13.444
0.2324
0.0540
2.85970 (3)
1 1 1
3.3132
3.3120
5.600

30.581
15.290
0.2637
0.0695
3.67509 (4)
2 2 0
2.9256
2.9198
5.600

35.523
17.761
0.3050
0.0930
4.92361 (5)
2 1 0
2.5251
2.5241
5.600

40.680
20.340
0.3475
0.1208
6.39237 (6)
2 1 1
2.2161
2.2152
5.600

42.960
21.480
0.3661
0.1340
7.09426 (7)
—
2.1036
2.1028
5.600

49.915
24.957
0.4219
0.1780
9.41976 (9)
2 2 1
1.8255
1.8248
5.600

53.767
26.883
0.4521
0.2044
10.8179 (11)
3 1 1
1.7035
1.7028
5.600

56.129
28.064
0.4704
0.2213
11.7106 (12)
2 2 2
1.6373
1.6366
5.600

68.526
34.263
0.5629
0.3169
16.7699 (17)
3 2 2
1.3682
1.3676
5.600

Figure 2: Powder XRD pattern of Cu(II) complex.

4.8. Potentiometric Determination of the Ionization Constants

The ionization constants of the ionizable group in Schiff base under investigation are determined by a method similar to that described by Calvin-Bjerrum [32]. The average of protons associated with the ligand at different pH values is calculated utilizing acid and ligand titration curves (Figures 3(a) and 3(b)). The pKa values can be calculated from the curves obtained by plotting versus pH. The formation curves are found between 0 and 1. This indicates that the ligands have one dissociable proton. Ligand exhibits only one pKa value in the range of 10.0 in 0.1 M ionic strength and 9.4 in 0.2 M ionic strengths, respectively, in 50% dioxane-water, this can be attributed to the ionization of phenolic –OH of the Schiff base (BHMQMHI).

(a)

(b)

Figure 3: Plot of pH versus volume of NaOH added.

4.9. Potentiometric Determination of the Stability Constants

The stability constants of the Co(II), Ni(II), and Cu(II) complexes with (BHMQMHI) are determined potentiometrically using the method described by Calvin-Bjerrum. The formation curves of the investigated complexes are obtained by plotting a graph between the average number of ligands attached per metal ion and the free ligand exponent (pL). Values of and pL are calculated. The maximum values calculated for metal-ligand system are found not to exceed two indicating the formation of 1 : 1 and 1 : 2 (metal : ligand) complexes. The mean and values of the Co(II), Ni(II), and Cu(II) complexes with Schiff base (BHMQMHI) are listed in Table 6. The order of stability constants is found to be Cu(II) > Co(II) > Ni(II) in accordance with the Irving and Williams order [33] for divalent metal ions of the 3d series. It is clear from Table 6 that the stability of Cu(II) complexes is considerably larger as compared to other metals of the 3d series. Under the influence of the ligand field, Cu(II) (3d ⁹) will receive some extra stabilization [34] due to tetragonal distortion of octahedral symmetry in their complexes. The Cu(II) complexes will be further stabilized due to the Jahn-Teller effect [35].

Table 6: Cumulative data of and values for Schiff base (BHMQMHI) complexes.

Solvent
Ionic strength = 0.1 M
Ionic strength = 0.2 M

pKa
pKa

10.0
9.4

Copper
Cobalt
Nickel
Copper
Cobalt
Nickel

50% Dioxane-water
5.644
3.900
5.554
5.025
4.5199
4.2300
5.499
5.0325
5.476
3.536
4.442
3.835

5. Pharmacology Results

5.1. Antimicrobial Evaluation of Ligand (BHMQMHI) and Its Metal (II) Complexes

The ligand and metal complexes were screened for antibacterial activity and the results are presented in Table 7. The synthesized Schiff base (BHMQMHI) has an inhibitory effect (MIC values of 75–100 μg/mL⁻¹) on growth of the tested bacterial strains. All complexes showed greater bactericidal activities against S. aureus (MIC 12.5–50 μg/mL⁻¹), B. subtilis (MIC 12.5–50 μg/mL⁻¹), and P. aeruginosa (MIC 12.5–50 μg/mL⁻¹) than the ligand. In the fungal studies, the ligand had an inhibitory effect (MIC values in range 50–100 μg/mL⁻¹) on the growth of the tested strains and complexes again showed greater fungicidal activities against A. flavus (MIC 12.5–75 μg/mL⁻¹), A. niger (MIC 12.5–25 μg/mL⁻¹), and C. albicans (MIC 12.5–50 μg/mL⁻¹). Co(II) and Zn(II) complexes had greater bacterial and fungal activities than the ligand (BHMQMHI).

Table 7: The antimicrobial activity of ligand and its metal (II) complexes evaluated by MIC (μg/mL⁻¹).

Schiff base/complexes
S. aureus
B. subtilis
P. aeruginosa
A. flavus
A. niger
C. albicans

Ligand
100
75
75
100
50
75

Co(II) complex
12.50
12.50
12.50
12.50
12.50
12.50

Ni(II) complex
12.50
25
25
25
25
12.50

Cu(II) complex
25
50
12.50
75
12.50
25

Zn(II) complex
12.50
12.50
12.50
12.50
12.50
12.50

Cd(II) complex
50
50
25
50
25
50

Hg(II) complex
25
12.50
50
12.50
25
12.50

Gentamycine
12.50
12.50
12.50
—
—
—

Amphotericin
—
—
—
12.50
12.50
12.50

All the metal complexes individually exhibited varying degrees of inhibitory effect on the growth of the tested bacterial/fungal species. Table 7 shows the activity of the metal complexes became more pronounced when coordinated with the metal ions [36]. This enhancement in the activity may be due to the structure of Schiff base ligand by possessing an azomethine (C=N) linkage. The toxic activity of the complexes with the ligand can be ascribed to the increase in the lipophilic nature of the complexes arising from chelation. The mode of action of complexes involves the formation of hydrogen bonds with the imino group by the active sites leading to interference with the cell wall synthesis. This hydrogen bond formation damages the cytoplasmic membrane and the cell permeability may also be altered leading to cell death [37].

A comparative study of the ligand and complexes (MIC values) indicated that the complexes exhibited higher antimicrobial activity than the free ligand. Such increased activity of the complexes can be explained on the basis of Overtone’s concept and Tweedy’s chelation theory [38]. These complexes also disturb the respiration process of the cell and thus block the synthesis of the proteins, restricting the further growth of the organism. Furthermore, the mode of action of the compound may involve the formation of a hydrogen bond through the azomethine group with the active center of the cell, resulting in interference with the normal cell processes. In general, metal complexes are more active than ligands because metal complexes may serve as a vehicle for the activation of ligands as the principle cytotoxic species [39].

5.2. Antioxidant Activity: Radical Scavenging Activity

The metal (II) complexes prepared from Schiff base (BHMQMHI) were subjected for free radical scavenging activity by DPPH method [5, 6]. From the investigation it was clearly observed that metal complexes scavenge DPPH effectively than Schiff base (BHMQMHI). The Co(II), Zn(II), Cu(II), and Ni(II) complexes show superior activity while Cd(II) and Hg(II) complexes are moderate activity as a radical scavenger compound with standards as shown in Figure 4.

Figure 4: Antioxidant results of Schiff base and its metal (II) complexes.

5.3. DNA Cleavage Efficiency

The Schiff base metal complexes were subjected to their DNA cleavage activity by agarose gel electrophoresis method [5]. From Figure 5, it was clearly indicated that the Co(II) (lane M1), Ni(II) (lane M2), Cu(II) (lane M3), Zn(II) (lane M4), and Cd(II) (lane M5) shows complete DNA cleavage activity and Hg(II) (lane M6) complex shows partial DNA cleavage activity. The Schiff base metal complexes have acted on DNA effectively, since as there was molecular weight difference between the control and the treated DNA samples. The completion of gel electrophoresis experiment clearly indicated that the intensity of the treated DNA samples has diminished due to the cleavage of DNA. These results indicated that the metal ions played an important role in the cleavage of DNA [40]. The information obtained in this study could be helpful in the understanding of the mechanism of interactions of metal (II) complexes with nucleic acids and should be useful in the development of potential probes for investigation of the structure and conformation of DNA or new therapeutic agents for some diseases [41].

Figure 5: Gel picture showing the cleavage analysis of samples.

6. Conclusion

This study shows that synthesized Schiff base (BHMQMHI) acts as tridentate ligand coordinating to metal ion through azomethine nitrogen, carbonyl oxygen, and phenolic oxygen atom via deprotonation. The synthesized new Schiff base and its metal complexes have been confirmed by the analytical data, IR, electronic, mass spectrometry, ¹H NMR, ESR spectral data, magnetic susceptibility, molar conductance, XRD, and thermal studies. The results of the potentiometric studies on complexes of Schiff base (BHMQMHI) with Cu(II), Co(II), and Ni(II) using Calvin-Bjerrum pH-titration technique as used by Irving and Rossotti indicate that the order of stability is Cu(II) > Co(II) > Ni(II). This order is in accordance with Irving-Williams order of stability and formation constants and values are decreases as the ionic strength increases. This observation is in agreement with Debye-Hukel equation. The synthesized Schiff base metal complexes show better antibacterial and antifungal activity than the ligand. The Co(II), Ni(II), Cu(II), Zn(II), Cd(II), and Hg(II) complexes were also found to show significant antioxidant and DNA cleavage activity. Based on the analytical and spectral studies, we propose octahedral geometry for the Co(II), Ni(II), and Cu(II) complexes and tetrahedral geometry for the Zn(II), Cd(II), and Hg(II) complexes. (Figures 6 and 7).

Figure 6: Proposed structure of Co(II), Ni(II), and Cu(II) metal complexes (octahedral).

Figure 7: Proposed structure of Zn(II), Cd(II), and Hg(II) metal complexes (tetrahedral).

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors are thankful to the Chairman, Department of Chemistry, Gulbarga University, Gulbarga, for encouragement and facilities. One of the authors (Nabiya Sultana Mayana) is thankful to UGC New Delhi for the grant of research fellowship in science for minority students under MANF Scheme. They are also thankful to IIT Chennai, IIT Bombay, and STIC Cochin for providing spectral data.

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