SYNTHESIS, CHARACTERIZATION, DNA CLEAVAGE AND ANTIMICROBIAL ACTIVITIES OF SCHIFF BASE LIGAND DERIVED FROM 5-CHLORO-3-PHENYL-1H-INDOLE-2-CARBOXYHYDRAZIDE AND O-VANILLIN AND ITS METAL (II) COMPLEXES

MAHADEV DHANRAJ UDAYAGIRI1, 2, NAGESH GUNVANTHRAO YERNALE1, BENNIKALLU HIRE MATHADA MRUTHYUNJAYASWAMY*

1Department of Studies and Research in Chemistry, Gulbarga University, Kalaburagi, Karnataka, India, 2Government College, Sedam Road, Kalaburagi, Karnataka, India
Email: [email protected]

Received: 10 Dec 2015 Revised and Accepted: 03 Feb 2016


ABSTRACT

Objective: The main objective of the present work was to synthesize a new Schiff base ligand 5-chloro-N'-(2-hydroxy-3-methoxybenzylidene)-3-phenyl-1H-indole-2-carbohydrazide and its Cu (II), Co (II), Ni (II) and Zn (II) complexes and study their biological activity.

Methods: The compounds are characterized by elemental analysis and various physicochemical techniques like IR, 1H NMR, ESI-mass, UV-Visible, TGA analyzes and molar conductance data. All the compounds were screened for their antibacterial and antifungal activity by MIC method. Further, antioxidant activity was performed by using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and DNA cleavage activity by Agarose Gel Electrophoresis (AGE) method.

Results: Spectral investigations suggested octahedral coordination geometrical arrangement for all the metal (II) complexes, having 1:2 stoichiometric ratio of the type [M(L)2]. The antimicrobial activity results revealed that the metal complexes were found to be more active than the free ligand. Furthermore, the DNA cleavage activity of the compounds on plasmid DNA pBR322 molecule showed moderate activity.

Conclusion: The newly synthesized ligand acts as ONO donor tridentate chelate and coordinated through an oxygen atom of carbonyl function, azomethine nitrogen and phenolic oxygen atom to the Cu, Co, Ni and Zn metal ions and form octahedral geometrical arrangement. All the complexes are found to be non-electrolytic in nature. Further, all the newly prepared compounds showed moderate biological activity.

Keywords: Indole, o-Vanillin, Transition metal complexes, Powder XRD, ESR, DNA cleavage Antimicrobial, Antioxidant


INTRODUCTION

Schiff base ligands and their transition metal complexes have gained a great deal of attention to the researchers worldwide in view of their potential biological importance viz., antibacterial, antifungal, antioxidant and anticancer activities [1]. Metal complexes of Schiff base ligand have revealed preferable enhancement in their activities after complexation as compared to uncoordinated ligand [2, 3]. The biological relevance of the metal complexes has been rationalized and found to be generally dependent on the nature of the metal ions, its oxidation state, nature and the type of the coordinated ligand and isomers [4-6]. Transition metal complexes surrounded by oxygen and nitrogen donor Schiff base ligands, which can be modified by choosing the suitable amine precursors and the ring may serve in coordination field [7]. Also, Schiff bases and their metal complexes have gained interest owing to their structural diversity and a wide range of potential biological activities such as anticancer, antitumor, antibacterial, antifungal, antitubercular, antioxidant, anti-inflammatory and corrosion inhibition [8-12].

The o-vanillin is the most prominent principle flavor and aroma compound in vanilla which is used as a food flavoring agent in foods, beverages, and pharmaceuticals [13]. Also, it possesses numerous biological activities such as anti-inflammatory, analgesic and antiviral activities. It is extensively studied in the medicinal field [14, 15]. In addition to this, it can also be used as an efficient herbicide, pesticide, bactericide [16] and the Schiff bases derived from o-vanillin moiety form stable complexes with metal ions [17, 18]. Hence, o-vanillin is an optimal candidate for the synthesis of various Schiff base ligands and their metal complexes with significant biological activities. Accordingly, we intend to report here the synthesis, spectral characterization, DNA cleavage and antimicrobial activities of novel Schiff base ligand derived from the reaction between 5-chloro-3-phenyl-1H-indole-2-carboxyhydrazide and o-vanillin and its metal complexes. The newly prepared compounds showed moderate biological activities.

MATERIALS AND METHODS

All the chemicals used were of high purity grade; solvents were dried and distilled before use. Melting points were determined by electrothermal apparatus using open capillary tubes. Metal and chloride contents were determined as per standard procedures [19]. The precursor 5-chloro-3-phenyl-1H-indole-2-carboxyhydrazide was prepared by as per literature method [20] and o-vanillin procured from Sigma-Aldrich Chemical Company India.

The IR Spectra were recorded as KBr pellets on a Perkin Elmer-Spectrum RX-I FTIR instrument (4000-400 cm-1). Elemental analysis was obtained from Perkin Elmer 2400 CHN Elemental Analyser. 1H NMR spectra of the ligand and its Zn complex were recorded on FT NMR Spectrometer model Avance-II (Bruker), 400 MHz instrument using d6-DMSO as a solvent. ESI-mass spectra were recorded on mass spectrometer equipped with electrospray ionization (ESI) source having a mass range of 4000 amu in quadruple and 20000 amu in ToF. UV-Visible spectra of Cu, Co and Ni complexes were recorded on Elico-SL 164 double beam UV-Visible spectrophotometer in the range 200-1000 nm in DMF solution at 1×10-3M concentration. The ESR spectra of the Cu complex in the polycrystalline state were recorded on BRUKER Bio-Spin Gmbh spectrometer at a microwave frequency of 9.1 GHz. The experiment was carried out by using DPPH as a reference with the field set at 3000 Gauss using tetracyanoethylene as the “g” marker (g = 2.0023). Powder XRD of the complexes was recorded in Bruker AXS D8 Advance (Cu, Wavelength 1.5406 Å sources). Molar conductivity measurements were recorded on an ELICO CM-180 conductivity bridge in dry DMF (10-3 M) solution using a dip-type conductivity cell fitted with a platinum electrode, and the magnetic susceptibility measurements were made at room temperature on a Gouy balance using Hg[Co(NCS)4] as the calibrant.

Synthesis of Schiff base ligand (L)

An equimolar mixture of 5-chloro-3-phenyl-1H-indole-2-carboxy-hydrazide (0.001 mol) and o-vanillin (0.001 mol) in methanol (25 ml) with 1-2 drops of glacial acetic acid as a catalyst was refluxed on a water bath for about 4-5 h. Yellow colored solid separated in hot, was filtered, washed with hot ethanol, dried and recrystallized from 1, 4-dioxane (Scheme 1). The reaction was monitored by TLC.



Scheme 1: Synthesis of Schiff base ligand (L)

Preparation of metal (II) complexes

To the hot solution of 5-chloro-N'-(2-hydroxy-3-methoxy-benzylidene)-3-phenyl-1H-indole-2-carbohydrazide (0.002 mol) in ethanol (30 ml) was added a hot ethanolic solution (15 ml) of respective metal chlorides (0.002 mol). The reaction mixture was refluxed on a water bath for about 5 h. Sodium acetate (0.5 g) was added to the reaction mixture to maintain a neutral pH and to reflux was continued for 1 h more. The reaction mixture was poured into distilled water. The solid colored complexes separated were collected by filtration, washed with sufficient quantity of distilled water, then with hot ethanol to apparent dryness and dried in a vacuum over anhydrous calcium chloride in a desiccator.

Antibacterial and antifungal assay

The newly synthesized ligand and its Cu (II), Co (II), Ni (II) and Zn (II) complexes were screened for their antibacterial and antifungal activities by using Muller-Hinton agar and potato dextrose agar (PDA) diffusion methods respectively [21]. These activities were carried out in four different concentrations (100, 50, 25 and 12.5 µg/ml in DMSO solvent) against, Escherichia coli (MTCC 46), Salmonella typhi (MTCC 98) and Bacillus subtilis (MTCC 736). The antifungal activities were carried out against Candida albicans (MTCC 227), Cladosporium oxysporum (MTCC 1777) and Aspergillus niger (MTCC 1881) by a minimum inhibitory concentration (MIC) method. The above-mentioned organisms were obtained from the Department of Microbiology, Gulbarga University, Kalaburagi, Karnataka, India, which were previously procured from Institute of Microbial Technology Chandigarh (IMTC), India. The lowest concentration of each tested compound where the growth of bacteria/fungi was clearly inhibited is reported as MIC. The results were compared with the Gentamycin and Fluconazole, a broad-spectrum antibiotic for bacterial and fungal strains respectively. The experiment was done in triplicate, and the average values were presented.

DNA cleavage experiment

The extent to which the newly synthesized ligand and its metal complexes could function as DNA cleavage agents was examined using Calf-thymus DNA (Cat. No. 105850) as a target molecule. The electrophoresis method was employed to study the efficiency of cleavage by the synthesized compounds. Each test compound (100 µg) was added separately to the 225ng DNA sample, and these sample mixtures were incubated at 37 °C for 2 h. The electrophoresis of the test compounds was done according to the literature method [22]. Agarose (600 mg) was dissolved in hot tris-acetate-EDTA (TAE) buffer (60 ml) (4.84 g Tris base, pH-8.0, 0.5 M EDTA L-1) and heated to boil for a few minutes. When the gel attains approximately 55 °C, it was then poured into the gas cassette fitted with a comb. Slowly the gel was allowed to solidify by cooling to room temperature and then carefully the comb was removed. The solidified gel was placed in the electrophoresis chamber containing TAE buffer. The DNA sample (20 µL) initially treated with the test compounds were mixed with bromophenol blue dye in equimolar ratios along with standard DNA marker containing TAE buffer was loaded carefully into the wells and the constant 50 V of electricity was supplied for about 30 min. Later, the gel was removed, stained with ethidium bromide solution (10 µg ml-1) for 15-20 min and then the bands were observed and photographed under UV-illuminator.

Antioxidant assay (Free radical scavenging activity)

The Free radical scavenging activity of the test samples was determined by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) method [23]. Different concentrations of test compounds (12.5 µg, 25 µg, 50 µg and 100 µg) and standard butylated hydroxyanisole (BHA) were taken in different test tubes, and the volume of each test tube was adjusted to 100 µL by adding distilled DMF. To the tubes containing sample solutions in DMF, 5 ml methanolic solution of DPPH (0.1 mM) was added to these tubes. The tubes were allowed to stand for 30 min. The control experiment was carried out as above without the test samples. The absorbance of test solutions was measured at 517 nm. The reduction of DPPH was calculated relative to the measured absorbance of the control. Radical scavenging activity was calculated using the following formula:

RESULTS AND DISCUSSION

All the newly synthesized metal complexes are colored solids, amorphous and non-hygroscopic in nature and possess high melting points (>300 °C). The complexes are insoluble in water and common organic solvents but are soluble in DMF and DMSO. Elemental analysis and analytical data (table 1) of the complexes suggest that the metal to ligand ratio of all the complexes were, 1:2 stoichiometry of the type [M (L)2]. The molar conductance values are too low to account for any dissociation of the complexes in DMF (17-32 ohm-1 cm2 mole-1), indicating their non-electrolytic nature.


Table 1: Physical, analytical and molar conductance data

Compounds

M. W.

M. P.

( °C)

Color

(Yield %)

Elemental Analysis, Calc. (Found) [%]

λm

µeff

(BM)

C

H

N

M

C23H18N3O3Cl (L)

419

269

Yellow

(71)

65.87

(65.82)

4.29

(4.22)

10.02

(10.01)

--

--

--

[Cu(C23H17N3O3Cl)2]

[Cu(L)2]

899.5

>300

Green

(58)

 61.36

(61.33)

 3.77

(3.74)

9.33

(9.31)

7.06

(7.00)

32

1.86

[Co(C23H17N3O3Cl)2]

[Co(L)2]

894.9

>300

Brown

(55)

61.68

(61.63)

3.79

(3.72)

9.38

(9.40)

6.58

(6.40)

21

5.01

[Ni(C23H17N3O3Cl)2]

[Ni(L)2]

894.6

>300

Brown

(53)

61.70

(61.75)

3.80

(3.79)

9.38

(9.36)

6.55

(6.49)

17

2.93

[Zn(C23H17N3O3Cl)2]

[Zn(L)2]

901.3

>300

Orange

(55)

61.23

(61.22)

3.77

(3.75)

9.31

(9.37)

7.25

(7.30)

31

Dia.

M. W.: Molecular weight; M. P. Melting point


IR spectral studies

The important IR bands of the ligand and its metal complexes are represented in table 2. In the IR spectrum of ligand, absorption due to NH of indole displayed band at 3276 cm-1 and absorption due to NH of CONH function at 3061 cm-1. Two sharp peaks observed at 1657 and 1604 cm-1 are due to carbonyl and azomethine functions respectively.

In the IR spectra of all the metal complexes, it was observed that the absence of absorption band due to phenolic OH at 3309 cm-1 of ligand indicates the formation of a coordination bond between the metal ion and phenolic oxygen atom via deprotonation. This is further confirmed by the increase in absorption frequency about 72-83 cm-1 of phenolic v(C-O) which appeared in the region 1303-1314 cm-1 in all the complexes indicating the participation of oxygen atom of phenolic oxygen in coordination. The absorption bands due to NH of indole and NH of CONH function displayed the bands in the region 3201-3220 cm-1 and 3051-3054 cm-1, which have appeared at about the same region as in the case of ligand, thus confirming their non-involvement in coordination. The absorption frequency of carbonyl and azomethine functions which have appeared at 1657 and 1604 cm-1 in case of the ligand, have been shifted to lower frequency by 38-55 and 20-60 cm-1 respectively in all the metal complexes and appeared in the region 1619-1602 cm-1 and 1544-1584 cm-1 indicating the involvement of oxygen atom of carbonyl function as such without undergoing enolization [24] and nitrogen atom of azomethine [25] function in complexation with the metal ions. This is further confirmed by the appearance of new bands in the region 522-508 cm-1, and 462-429 cm-1 are due to M-O and M-N stretching vibrations [26] in all the complexes.


Table 2: IR spectral data (cm-1) of Schiff base ligand (L) and its metal complexes

Ligand/

Complexes

vOH

(phenolic)

vNH

(amide)

vNH

(indole)

vC=O

(carbonyl)

vC=N

(azomethine)

vC-O (phenolic)

vM-O

vM-N

L

3309

3276

3061

1657

1604

1231

--

--

[Cu(L)2]

--

3201

3054

1610

1564

1314

522

462

[Co(L)2]

--

3220

3053

1608

1544

1303

509

432

[Ni(L)2]

--

3209

3052

1619

1584

1305

510

430

[Zn(L)2]

--

3218

3051

1602

1546

1304

508

429


1H NMR spectral data

The 1H NMR spectrum of ligand (L) displayed three singles each at 12.160, 11.627 and 11.486 ppm are due to the proton of phenolic OH, amide NH and NH attached to indole moiety respectively. Azomethine proton (CH=N) has resonated as a singlet at 8.399 ppm. The signals due to eleven aromatic protons (ArH) have appeared as multiplets in the region 6.854-7.534 ppm and single appeared at 3.695 ppm is due to three protons of the methoxy group of o-vanillin moiety respectively. TheSchiff base ligand (L) upon complexation with Zn (II) ion showed the disappearance of signal due to the proton of phenolic OH confirms the involvement of bonding of phenolic oxygen to metal ion via deprotonation. The signals due to amide NH and indole NH have appeared at 11.814 ppm and 11.624 ppm respectively. The signal due to azomethine proton has (CH=N) resonated at 8.699 ppm. The signals due to eleven aromatic protons (ArH) have resonated as multiplets in the region 6.834-7.390 ppm and signal at 3.813 ppm are due to three protons of the methoxy group of o-vanillin moiety respectively. When compared to the NMR spectral data of the ligand and its [Zn (L)2] complex, all the signals due to protons have been shifted towards down field strength confirming the complexation.


Table 3: 1H NMR spectral data (ppm) of Schiff base ligand (L) and its [Zn(L)2] complex

Ligand/ complex

1H NMR data (ppm)

L

12.160 (s, 1H, Phenolic OH), 11.627 (s, 1H, CONH), 11.486 (s, 1H, indole NH), 8.399 (s, 1H, HC=N), 6.854-7.374 (m, 11H, ArH), 3.695 (s, 3H, OCH3).

[Zn(L)2]

11.814 (s, 1H, CONH), 11.624 (s, 1H, indole NH), 8.699 (s, 1H, HC=N), 6.834-7.390 (m, 11H, ArH), 3.813 (s, 3H, OCH3).



Fig.1: ESI mass spectrum of [Cu (L)2] complex

ESI-mass spectral data

The mass spectra of Ligand (L), [Cu (L)2], [Co(L)2] and [Ni(L)2] complexes showed a molecular ion peak recorded at m/z 420, 422 (100%, 33%), M+1 900, 902 (9%, 3%) (fig. 1), 894, 896 (60%, 20%) and M+1 895, 897 (6%, 2%) respectively which are equivalent to their molecular weighs.

Electronic spectral studies

The green colored [Cu (L)2] complex displayed a low-intensity single broad band in the region 15462-17924 cm-1. The broadness of the band designates the three transitions 2B1g2A1g (v1), 2B1g 2B2g(v2) and 2B1g 2Eg(v3), which are similar in energy and give rise to only one broadband and the broadness of the band may be due to dynamic Jahn-Teller distortion. The obtained data suggest the distorted octahedral geometry around the Cu (II) ion [27].

The [Co (L)2] complex under present study displayed two absorption bands at 16889 cm-1 and 20103 cm-1. These bands are assigned to be 4T1g (F) → 4A2g (F) (v2) and 4T1g (F) → 4T2g (P)(v3) transitions, respectively, which are in good agreement with the literature values for octahedral geometry [28, 29]. The lowest band, v1 could not be observed due to the limited range of the instrument used, but it could be calculated using the band fitting procedure suggested by Underhill and Billing [30]. The calculated v1 value is presented in table 4. These transition values suggest the octahedral geometry of the Co (II) complex.

The [Ni(L)2] complex under present investigation exhibited two absorption bands in the region 15098 cm-1and 25092 cm-1, which are assigned to 3A2g3T1g (F) (v2) and 3A2g (F) → 3T1g (P) (v3) transitions respectively in an octahedral environment [31]. The transition value of band v1 was calculated by using a band fitting procedure [31]. The proposed octahedral geometry for the complexes was further supported by the calculated values of ligand field parameters, such as Racah interelectronic repulsion parameter (B’), the nephelauxetic parameter (ß), ligand field splitting energy (10 Dq) and ligand field stabilization energy (LFSE) [31].

The calculated B’ values for the [Co (L)2] and [Ni(L)2] complexes are lower than the free ion values, which is due to the orbital overlap and delocalization of d-orbitals. The ß values are important in determining the covalency for the metal-ligand bond, and they were found to be less than unity, suggesting a considerable amount of covalency for the metal-ligand bonds. The ß value for the [Ni (L)2] complexes was less than that of the [Co (L)2] complexes, indicating the greater covalency of the Metal-Ligand (M-L) bond.


Table 4: Electronic spectral data

Complexes

Transitions in cm-1

Dq

(cm-1)

B'

(cm-1)

ß

ß%

v2/v1

LFSE

(k cal.)

v1*

v2

v3

[Cu(L)2]

15462-17924

--

--

--

--

--

28.61

[Co(L)2]

7879

16889

20103

901

890

0.916

8.34

2.14

15.44

[Ni(L)2]

9310

15098

25092

931

817

0.785

21.44

1.62

31.92

*Calculated values


Magnetic susceptibility studies

The room temperature magnetic measurements were obtained for paramagnetic Co (II), Ni (II) and Cu (II) complexes The observed magnetic moment for Cu (II) complex is 1.86 BM which attributes to one unpaired electron with a slight orbital contribution to the spin only a value of 1.73 BM and the absence of spin-spin interactions in the complex accounting for the possibility of a distorted octahedral geometry [32].

In octahedral Co (II) complex the ground state is 4T1g and the orbital contribution to the singlet state lowers the magnetic moment values for the various Co (II) complexes which are in the range 4.12-4.70 and 4.70-5.20 BM for tetrahedral and octahedral complexes respectively [33]. In the present study, the observed magnetic moment values for Co (II) complexes are 5.01 BM indicates octahedral geometry for Co (II) complex. For Ni (II) complex, the observed magnetic moment value is 2.93 BM which is well within the expected range of Ni (II) complex with octahedral geometry, i.e. 2.83-3.50 BM [34].

Thermal studies

The thermal stabilities for Cu (II), Co (II), Ni (II) and Zn (II) complexes been studied as a function of temperature. The proposed stepwise thermal degradation of the complexes with respect to temperature and the formation of respective metal oxides are depicted in table 5.

TG-DTA curve of Cu (II) complex showed that the first stage of decomposition represents a weight loss of two OCH3 groups of vanillin moieties at 249 °C with a practical weight loss of 7.26% (Cald. 6.89%). The resultant complex underwent the second stage of degradation and gave a break at 345 °C with a practical weight loss of 36.26% (Cald. 36.17%), which corresponds to the loss of C14HNCl species of indole moiety and a C6H5 group. Thereafter, the compound showed decomposition in a gradual manner rather than with the sharp decomposition up to 627 °C and onwards due to the loss of the remaining organic moiety. The weight of the residue corresponds to cupric oxide.

The thermogram of Co (II) complex showed the first stage of decomposition at 388 °C with a practical weight loss of 7.26% (Cald. 6.92%), which corresponds to weight loss due to two OCH3 groups of vanillin moieties. Further, the complex underwent decomposition and gave a break at 400 °C with a practical weight loss of 73.70% (Cald. 73.59%), corresponds to weight loss of a deprotonated ligand and C14H9NCl species of indole moiety. Thereafter the complex showed gradual decomposition up to 715 °C with a weight loss of the remaining organic moiety, the weight of the residue corresponds to cobalt oxide.

In the thermogram of the Ni (II) complex, the first stage of decomposition represents the weight loss due to OCH3 groups of vanillin moieties at 378 °C with a practical weight loss of 5.70% (Cald. 6.93%). The resultant complex underwent further degradation and gave a break at 405 °C with a practical weight loss of 31.13% (Cald. 31.46%), which corresponds to the loss due to a C14H10NCl species of indole moiety and a chlorine atom. Thereafter, the compound showed a gradual decomposition up to 715 °C with a weight loss of remaining organic moiety. The weight of the residue corresponds to nickel oxide.

In the case of Zn (II) complex, the first stage of decomposition occurs at 364 °C with a practical weight loss of 5.91% (Cald. 6.87%), which represents the loss due to two OCH3 groups of vanillin moieties. Further, the complex underwent the second stage of decomposition and gave a break at 395 °C with a practical weight loss of 44.56% (Cald. 45.27%), the loss due to a C14H10NCl molecule of indole moiety and two phenyl groups. Thereafter, the compound showed a gradual decomposition up to 716 °C with the weight loss of the remaining organic moiety. The weight of the residue corresponds to zinc oxide.

ESR spectral studies of the Cu (II) complex

The ESR spectrum of the Cu (II) complex in a polycrystalline state was recorded at room temperature to elucidate the geometry and the degree of covalency of the metal-ligand bond or environment around the metal ion. The spin Hamiltonian parameters for the Cu(II) complex is used to derive the ground state. In octahedral geometry the g-tensor parameter with g1>g ||>2.0023, the unpaired electron lies in the dz2orbital and g1g||>>2.0023, the unpaired electron lies in the dx2-y2 orbital in the ground state [35]. In the present case, the observed measurements of Cu(II) complex is g1 (2.161)g||> (2.0352)>2.0023 indicating that the complex are axially symmetric, and the copper site has a dx2-y2 ground state characteristic of octahedral geometry [36].

The g|| value is an important function for indicating the metal-ligand bond character, for covalent character g||<2.3 and for ionic g||>2.3 respectively [37]. In the present case Cu (II) complex has the g|| values were less than 2.3, indicating an appreciable covalent character of the metal-ligand bond. The geometric parameter (G), which is the measure of the extent of exchange interaction and is calculated by using g-tensor values by the expression G = g||-2.0023/g1-2.0023. According to Hathaway [38, 39], if the G value is less than 4, the exchange interaction between the copper centers is noticed, whereas if its value is greater than 4, the exchange interaction is negligible. The calculated G-value for the present Cu (II) complex is 4.879 indicating that the exchange coupling effects are not operating in the present complexes.


Table 5: Thermal degradation pattern of metal complexes

Metal

Complexes

Temp. ( °C)

Weight loss (%)

Metal oxide (%)

Inference

Obs.

Calc.

Obs.

Calc.

[Cu(L)2]

249

7.26

6.89

--

--

Loss due to two OCH3 groups of vanillin moieties.

 

345

36.26

36.17

--

--

Loss due to C14HNCl species of indole moiety and a phenyl group.

Up to 627

--

--

8.84

8.91

Loss due to remaining organic moiety

[Co(L)2]

388

7.26

6.92

--

--

Loss due to two OCH3 groups of vanillin moieties.

 

400

73.70

73.59

--

--

Loss due to a deprotonated ligand and C14H9NCl species of indole moiety.

Up to 715

--

--

8.37

8.45

Loss due to remaining organic moieties.

[Ni(L)2]

378

5.70

6.93

--

--

Loss due to two OCH3 groups of vanillin moieties.

 

405

31.13

31.46

--

--

Loss due to a C14H10NCl molecule of indole moiety and a chlorine atom.

Up to 715

--

--

8.34

8.99

Loss due to remaining organic moiety.

[Zn(L)2]

364

5.91

6.87

--

--

Loss due to two OCH3 groups of vanillin moieties.

 

395

44.56

45.27

--

--

Loss due to a C14H10NCl molecule of indole moiety and two phenyl groups.

Up to 716

--

--

9.02

10.21

Loss due to remaining organic moiety.


Powder X-ray diffraction studies (Powder-XRD)

Crystals that are suitable for single-crystal studies were not obtained since all the metal complexes are not soluble in common solvents, but soluble in some polar solvents like DMF and DMSO. Hence powder-XRD pattern of all the metal complexes has been studied in order to test the degree of crystallinity of the complexes. Powder X-ray diffraction pattern for Cu(II) complex (fig. 2) showed 5 reflections in the range of 3-80 ° (2q), which arise from diffraction of X-ray by the planes of the complex. The interplanar spacing (d) has been calculated by using Bragg’s equation, (nl=2d sinq). The calculated inter-planar d-spacing together with relative intensities with respect to the most intense peak have been recorded and depicted in table 6. The unit cell calculations have been calculated for cubic symmetry from the entire important peaks, and h2+k2+l2 values were determined. The observed inter-planar d-spacing values have been compared with the calculated ones, and it was found to be in good agreement. The h2+k2+l2 values are 1, 2, 27, 44 and 49 for Cu (II) complex. It was observed that the absence of forbidden numbers (7, 15, 23, 71 etc.) indicates that complexes have cubic symmetry.

Similar calculations were performed for Co (II), Ni (II) and Zn(II) complexes, they showed 5, 6 and 5 reflections each in the range 3-80 ° respectively, which are raised from the diffraction of X-ray by the planes of these complexes. All the important peaks of the complexes have been indexed, and observed values of interplanar distances (d) have been compared with the calculated ones, and it was found to be in good agreement. The unit cell calculations were performed for the cubic system, and the h2+k2+l2 values were determined for the above complexes. The h2+k2+l2 values were 1, 19, 26, 34 and 54 for Co(II) complex, and 1, 24, 29, 34, 38 and 56 for Ni(II) complex 1,1,1, 2 and 4 for Zn(II) complex respectively. It was observed that the absence of forbidden numbers (7, 15, 23, 71 etc.) indicates that complexes have cubic symmetry.



Fig.2: Powder X-ray spectrum of [Cu (L)2] complex


Table 6: Powder X-ray data of [Cu (L)2] complex

S. No.

2q

q

Sinq

Sin2q

1000 Sin2q

1000 Sin2q/CF

(h2+k2+l2)

h k l

d

a in A

Obs.

Calc.

1

3.915

1.9575

0.0341

0.001

1.162

1.00 (1)

1 0 0

22.55

22.58

22.58

2

60228

3.114

0.0543

0.002

2.950

2.50 (2)

1 1 0

14.17

14.18

22.41

3

20.513

10.2565

0.1780

0.031

31.684

27.2 (27)

3 3 3

4.32

4.32

22.58

4

26.141

13.0705

0.2261

0.051

51.12

43.9 (44)

6 2 2

3.40

3.40

22.56

5

27.689

13.8445

0.2392

0.057

57.21

49.2 (49)

7 0 0

3.21

3.22

22.61


Antimicrobial activity results

In most of the cases, the metal complexes exhibited promising antibacterial and antifungal activity greater than the free ligand. This activity was found to be enhanced on coordination with metal ions. This enhancement in the antimicrobial activity of the complexes over the free ligand can be explained on the basis of chelation theory [40, 41]. The enhancement in the activity may be rationalized on the basis that ligands possess azomethine (C=N) bond. Moreover, in the metal complex, the positive charge of the metal ion is partially shared with the hetero donor atoms (N and O) present in the ligand, and there may be p-electron delocalization over the whole chelating system [42-44]. Hence, there will be an increase in the lipophilic character of the metal complexes which favores its permeation through the lipid layer of the bacterial membranes and blocking the metal binding sites in the enzymes of microorganisms. The minimum inhibitory concentration (MIC) values of the compounds against the respective bacterial and fungal strains are summarized in table 7.


Table 7: Size of inhibition zone (mm) formed at different concentrations (12.5, 25, 50 and 100 µg/ml in DMSO solvent) against various Bacteria and fungi

Compounds

Bacteria

Fungi

B. Subtilis

E. coli

S. typhi

C. albicans

C. oxysporum

A. niger

L

10.29±0.19

10.12±0.32

10.31±0.8

10.71±0.62

10.91±0.10

11.48±0.52

[Cu(L)2]

13.24±0.57

13.10±0.20

11.15±0.14

11.49±0.67

12.63±0.22

12.41±0.35

[Co(L)2]

13.62±0.33

12.19±0.34

13.62±0.91

12.17±0.34

12.66±0.47

14.28±0.10

[Ni(L)2]

12.41±0.28

11.42±0.75

13.28±0.16

13.52±0.36

13.77±0.53

13.16±0.11

[Zn(L)2]

13.68±0.11

11.45±0.61

11.37±0.23

13.14±0.35

13.99±0.12

12.33±0.38

Gentamicin

18.34±0.23

18.13±0.12

18.91±0.27

--

--

--

Fluconazole

--

--

--

20.10±0.30

21.16±0.13

19.31±0.29

Note: The stock solutions of the test compounds were prepared by dissolving 10 mg of the test compound in 10 ml of freshly distilled DMSO (1 mg/ml)., The experiment was done in triplicate (n = 3), and the average values were calculated., The values presented in the above table are in±SEM


Antioxidant assay (DPPH free radical scavenging activity)

The free radical scavenging activity of the ligand and its metal complexes was done by DPPH method. The antioxidant activity of the test compounds was examined by measuring the radical scavenging effect of DPPH radicals. The results of the free radical scavenging activity of the compounds at different concentrations are shown in fig. 3. It was observed that the free radical scavenging activity of these compounds was concentration dependent. Among the examined compounds, ligand (L), Cu and Co complexes have exhibited good scavenging activity, whereas Ni and Zn complex showed moderate activity. The marked antioxidant activity of metal complexes is due to the coordination of metal with azomethine nitrogen and carbonyl oxygen of amide function attached to the 2-position of indole. In the case of metal complexes, the hydrogen of azomethine is more acidic. Hence, hydrogen of azomethine could be easily donated to the DPPH free radical and convert itself into the stable free radical. Moreover, the acidic nature of hydrogen atom attached to azomethine nitrogen increases on complexation with metal ions thereby making that hydrogen atom more liable.



Fig.3: Antioxidant activity results

The experiment was done in triplicate (n = 3), and the average values were calculated. The values presented in the above fig. are in±SEM, Error bars were omitted for simple presentation. On X-axis: Newly prepared compounds and Y-axis: Percentage of scavenging activity

DNA cleavage activity

The ligand and its Cu, Co, Ni and Zn complexes were studied for their DNA cleavage activity by agarose gel electrophoresis method against Calf-thymus DNA (Cat. No-105850) as a target molecule and the gel picture showing cleavage is depicted in fig. 4. Treatment of DNA on the ligand and complexes revealed that all the complexes have acted on DNA as there was a molecular weight difference between the treated DNA samples and the control. The difference was observed in bands of lanes compared to the control Calf-thymus DNA. The results indicate the important role of nitrogen and oxygen atoms to the metal ions in these isolated DNA cleavage reactions [45]. On the basis of the cleavage of DNA observed in case of ligand and its Cu, Co, Ni and Zn complexes, it can be concluded that all the compounds under present study inhibited the growth of pathogenic organism by DNA cleavage as has been observed on the DNA cleavage of Calf-thymus DNA.



Fig.4: DNA cleavage: M: Standard DNA, C: Control DNA (untreated pBR 322), L1: Ligand (L), L1M1: Cu (II) complex, L1M2: Co (II) complex, LIM3: Ni (II) complex and L1M4: Zn (II) complex

CONCLUSION

The spectral data revealed that the coordinating ability of the ligand has been proved in complexation reaction with Cu, Co, Ni and Zn ions. The newly synthesized ligand acts as ONO donor tridentate chelate. Metal ions are coordinated through an oxygen atom of carbonyl function, azo methine nitrogen and phenolic oxygen atom to the Cu, Co, Ni and Zn metal ions and form octahedral geometrical arrangement. The antimicrobial activity results showed that all the complexes have exhibited moderate activity when compared to its ligand. The electrophoretic studies indicated that Cu (II) and Co(II) complexes have good efficiency towards DNA cleavage. Based on the analytical data and spectral studies, proposed structure of all the complexes is depicted in fig. 5.



Fig.5: Proposed structures of the metal (II) complexes

ACKNOWLEDGEMENT

Authors are thankful to Professor and Chairman, Department of Chemistry, Gulbarga University, Kalaburagi. We are grateful to the SAIF Punjab University, Chandigarh, STIC Cochin University, for providing spectral data, and also, we are thankful for BioGenics Research and Training Centre in Biotechnology, Hubli for biological activities.

CONFLICT OF INTERESTS

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

REFERENCES

  1. Singh K, Barwa MS, Tyagi P. Synthesis, characterization and biological studies of Co(II), Ni(II), Cu(II) and Zn(II) complexes with bidentate schiff bases derived by heterocyclic ketone. Eur J Med Chem 2006;41:147-53.
  2. Bakir M, Green O, Mulder WH. Synthesis, characterization and molecular sensing behavior of [ZnCl 2 (? 3-N, N, O-dpkbh)](dpkbh= di-2-pyridyl ketone benzoyl hydrazone). J Mol Struct 2008;873:17-28.
  3. Nagesh GY, Mruthyunjayaswamy BHM. Synthesis, characterization and biological relevance of some metal (II) complexes with oxygen, nitrogen, and oxygen (ONO) donor schiff base ligand derived from thiazole and 2-hydroxy-1-naphthaldehyde. J Mol Struct 2015;1085:198-206.
  4. Guo Z, Sadler PJ. Medicinal inorganic chemistry. Adv Inorg Chem 2000;49:183-306.
  5. Arion VB, Reisner E, Fremuth M, Jakupec MA, Keppler BK, Kukushkin VY, et al. Synthesis, X-ray diffraction structures, spectroscopic properties, and in vitro antitumor activity of isomeric (1H-1,2,4-Triazole)Ru(III) complexes. Inorg Chem 2003;42:6024-31.
  6. Nagesh GY, Mahendra RK, Mruthyunjayaswamy BHM. Synthesis, characterization, thermal study and biological evaluation of Cu(II), Co(II), Ni(II) and Zn(II) complexes of Schiff base ligand containing thiazole moiety. J Mol Struct 2015;1079:423-32.
  7. Kozlyuk N, Lopez T, Roth P, Acquaye JH. Synthesis and the characterization of Schiff-base copper complexes: reactivity with DNA, 4-NPP, and BNPP. Inorg Chim Acta 2015;428:176-84.
  8. Vijesh AM, Isloor AM, Shetty P, Shetty P, Sudarshan S, Fun HK. New pyrazole derivatives containing 1,2,4-triazoles and benzoxazoles as potent antimicrobial and analgesic agents. Eur J Med Chem 2013;62:410-5.
  9. Keypour H, Khanmohammadi H, Wainwright, and KP, Taylor MR. Synthesis, crystal structure, NMR and ab initio molecular orbital studies of some magnesium(II) macrocyclic Schiff-base complexes, with two 2-aminoethyl pendant arms. Inorg Chim Acta 2004;357:1283-91.
  10. Bagihalli GB, Avaji PG, Patil SA, Badamiet PS. Synthesis, spectral characterization, in vitro antibacterial, antifungal and cytotoxic activities of Co(II), Ni(II) and Cu(II) complexes with 1,2,4-triazole Schiff bases. Eur J Med Chem 2008;43:2639-49.
  11. Bedfort RB, Bruce DW, Frost RM, Goodby JW, Hirdb M. Iron (III) salen-type catalysts for the cross-coupling of aryl Grignards with alkyl halides bearing ß-hydrogens. Chem Commun 2004;24:2822-3.
  12. Bryliakov KP, Talsi EP, Evgenii P. Evidence for the formation of an iodosylbenzene (salen) active iron intermediate in a (salen)iron (III)-catalyzed asymmetric sulfide oxidation. Angew Chem Int Ed 2004;43:5228-30.
  13. Gulcan M, Sonmez M. Synthesis and characterization of Cu(II), Ni(II), Co(II), Mn(II) and Cd(II) transition metal complexes of tridentate Schiff base derived from O-vanillin and N-aminopyrimidine-2-thione. Phosphorus Sulfur Silicon Relat Elem 2011;186:1962-71.
  14. Queffelec C, Bailly F, Mbemba G, Mouscadet JF, Hayes S, Debyser Z, et al. Synthesis and antiviral properties of some polyphenols related to Salvia genus. Bioorg Med Chem Lett 2008;18:4736-40.
  15. Lirdprapamongkol K, Kramb JP, Suthiphongchai T, Surarit R, Srisomsap C, Dannhardt G, et al. Vanillin suppresses metastatic potential of human cancer cells through PI3K inhibition and decreases angiogenesis in vivo. J Agric Food Chem 2009;57:3055-63.
  16. Xuan TD, Toyama T, Fukuta M, Khanh TD, Tawata S. Chemical interaction in the invasiveness of cogongrass (Imperatacylindrica (L.) Beauv.). J Agric Food Chem 2009;57:9448-53.
  17. Tabassum S, Amir S, Arjmand F, Pettinari C, Marchetti F, Masciocchi N, et al. Mixed-ligand Cu(II)–vanillin Schiff base complexes; effect of co-ligands on their DNA binding, DNA cleavage, SOD mimetic and anticancer activity. Eur J Med Chem 2013;60:216-32.
  18. Nagesh GY, Mahadev DU, Mruthyunjayaswamy BHM. Mononuclear metal (II) Schiff base complexes derived from thiazole and o-vanillin moieties: synthesis, characterization, thermal behavior and biological evaluation. Int J Pharm Sci Rev Res 2015;31:190-7.
  19. Mendham J, Denney RC, Barnes JD, Thomas MJK. Vogel’s quantitative chemical analysis. 6nd ed. Prentice Hall London; 2000.
  20. Hiremath SP, Mruthyunjayaswamy BHM, Purohit MG. Synthesis of substituted 2-aminoindolees and 2-(2?-Phenyl-1?, 3?, 4?-oxadiazolyl) amino indoles. Indian J Chem 1978;16B:789-92.
  21. Sadana AK, Miraza Y, Aneja KR, Prakash O. Hypervalent iodine mediated synthesis of 1-aryl/hetryl-1,2,4-triazolo[4,3-a] pyridines and 1-aryl/hetryl 5-methyl-1,2,4-triazolo[4,3-a]quinolines as antibacterial agents. Eur J Med Chem 2003;38:533-6.
  22. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); 1989.
  23. Singh RP, Murthy KNC, Jayaprakasha GK. Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. J Agric Food Chem 2002;50:81-6.
  24. Geary WJ. The use of conductivity measurements in organic solvents for the characterization of coordination compounds. Coord Chem Rev 1971;7:81-122.
  25. Roy S, Mandal TN, Das K, Butcher RJ, Rheingold AL, Kar SK. Syntheses, characterization, and X-ray crystal structures of two cis-di oxovanadium (V) complexes of pyrazole-derived, Schiff-base ligands. J Coord Chem 2010;63:2146-57.
  26. Chandra S, Gupta LK. Electronic, EPR, Magnetic and mass spectral studies of mono and homo binuclear Co(II) and Cu(II) complexes with a novel macrocyclic ligand. Spectrochim Acta Part A 2005;62:1102-6.
  27. Dholakiya PP, Patel MN. Synthesis, spectroscopic studies, and antimicrobial activity of Mn(II), Co(II), Ni(II), Cu(II), And Cd(II) complexes with bidentate schiff bases and 2,2'-Bipyridylamine. Synth React Inorg Met–Org Chem 2002;32:753-62.
  28. Koji A, Kanako M, Ohba M, Okawa H. Site specificity of metal ions in heteronuclear complexes derived from an “End-Off” compartmental ligand. Inorg Chem 2002;41:4461-7.
  29. Rai RA. Metal complexes of 5-(o)hydroxyphenyl-1,3,4-oxadiazole-2-thione. J Inorg Nucl Chem 1980;42:450-3.
  30. Underhill AE, Billing DE. Calculations of the racah parameter b for nickel (II) and cobalt (II) compounds. Nature1966;210:834-5.
  31. Satyanarayana DN. Electronic absorption spectroscopy and related technique, University Press: India Limited, (New Delhi); 2001.
  32. Singh DP, Kumar R, Malik V, Tyagi P. Synthesis and characterization of complexes of Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) with macrocycle 3,4,11,12-tetraoxo-1,2,5,6,9,10,13,14-octaazacyclo-hexadeca-6,8,14,16-tetraene and their biological screening. Transition Met Chem 2007;32:1051-5.
  33. Baranwal BP, Gupta T. Synthesis and physicochemical studies on iron (II, III, III) and cobalt (II) thiocarboxylates. Synth React Inorg Met-Org Chem 2004:34:1737-54.
  34. Rao TR, Archana P. Synthesis and spectral studies on 3d metal complexes of mesogenic Schiff base ligands. Part 1. Complexes of N-(4-Butylphenyl) salicyl aldimine. Synth React Inorg Met-Org Chem 2005;35:299-304.
  35. Balasubramanian S, Krishnan CN. Synthesis and characterization of five-coordinate macrocyclic complexes of nickel(II) and copper(II). Polyhedron 1986;5:669-75.
  36. Thaker BT, Tandel PK, Patel AS, Vyas CJ, Jesani MS, Patel DM. Synthesis and mesomorphic characterization of Cu(II), Ni(II) and Pd(II) complex with azomethine and chalcone as a bridging group. Indian J Chem 205;44A:265-70.
  37. Kilveson D. Publications of daniel kivelson. J Phys Chem B 1997;101:8631-4.
  38. Hathaway BJ, Billing DE. The electronic properties and stereochemistry of mononuclear complexes of the copper (II) ion. Coord Chem Rev 1970;5:143-207.
  39. Mruthyunjayaswamy BHM, Nagesh GY, Ramesh M, Priyanka B, Heena B. Synthesis, characterization and antioxidant activity of Schiff base ligand and its metal complexes containing thiazole moiety. Der Pharm Chem 2015;7:556-62.
  40. Chohan ZH, Arif M, Akhtar MA, Supuran CT. Metal-based antibacterial and antifungal agents: synthesis, characterization, and in vitro biological evaluation of Co(II), Cu(II), Ni(II), and Zn(II) complexes with amino acid-derived compounds. Bioinorg Chem Appl 2006:1-13. doi.org/10.1155/ BCA/ 2006/83131. [Article in Press]
  41. Yernale NG, Mruthyunjayaswamy BHM. Synthesis, Characterization, Antimicrobial, DNA Cleavage, and in vitro cytotoxic studies of some metal complexes of schiff base ligand derived from thiazole and quinoline moiety. Bioinorg Chem Appl 2014;314963:1-17.
  42. Thimmaiah KN, Lioyd WD, Chandrappa GT. Stereochemistry and fungitoxicity of complexes of p-anisaldehyde thiosemicarbazone with Mn(II), Fe(II), Co(II) and Ni(II). Inorg Chem Acta 1985;106:81-5.
  43. El-Wahab ZHA, Mashaly MM, Salman AA, El-Shetary BA, Faheim AA. Co(II), Ce(III) and UO2(VI) bis-salicylate thiosemicarbazide complexes: binary and ternary complexes, thermal studies and antimicrobial activity. Spectrochim Acta A 2004;60:2861-73.
  44. Mahendra Raj K, Vivekanand B, Nagesh GY, Mruthyunjayaswamy BHM. Synthesis, spectroscopic characterization, electrochemistry and biological evaluation of some binuclear transition metal complexes of bicompartmental ONO donor ligands containing benzo[b]thiophene moiety. J Mol Struct 2014;1059:280-93.
  45. Yernale NG, Mruthyunjayaswamy BHM. Metal (II) Complexes of ONO donor Schiff base ligand as a new class of bioactive compounds containing indole core: Synthesis and characterization. Int J Pharm Pharm Sci 2016;8:197-204.