INTRODUCTION:

The benzimidazole core is a versatile heterocyclic substructure present in a number of biomolecules including the purine bases of DNA and vitamin B₁₂ as well as in a range of therapeutic agents¹. This is also a found in potent antimicrobial, anti-HIV, antioxidant and antitumor agents^2-5. The metal complexes of benzimidazole and its derivatives have been evaluated for medicinal applications such as anticancer agents, antioxidant and enzyme inhibitors^6-9. Isoniazid has been used as a front-line drug in the treatment of tuberculosis (TB) however; resistant TB strains have limited its use.

The major route of isoniazid resistance relies on KatG enzyme disruption, which does not promote an electron transfer reaction, reactivity of isoniazid metal complexes as prototypes for novel self-activating metallodrugs against TB. It is well known from the literature that benzimidazole compounds containing the amine moiety have a strong ability to form metal complexes. Therefore, it was thought worthwhile to synthesize some metal complexes of this type of Mannich base and investigate its bonding characteristics. Moreover, heterocyclic Mannich bases that are derived from the corresponding heterocyclic intermediates also possess various useful properties, Metal complexes of mannich bases have been studied extensively in recent years due to selectivity and sensitivity of the ligands toward various metal ions^10,11.

In the present study we report the synthesis Co(II), Ni(II) and Cu(II) complexes derived from new mannich base ligand N'-(1H-benzimidazol-1-ylmethyl) pyridine-4-carbohydrazide (BI), and their characterization by elemental, spectral and XRD analysis were performed. We aimed to evaluate the antioxidant features of metal complexes In-vivo and docking interactions In-vitro.

EXPERIMENTAL:

MATERIALS:

The chemicals benzimidazole, Isoniazid and formaldehyde were Sigma-Aldrich Co. The chlorides of Co (II), Ni (II) and Cu (II) were of S.R.L. grade. All other reagents and solvents were purchased from commercial sources and were of analytical grade.

Physical measurements:

Melting points had been recorded on an electro-thermal melting factor apparatus and are uncorrected. ¹H NMR spectra were recorded on Bruker four hundred MHz spectrometer at IISc, Bangalore, Karnataka, India. The chemical shifts have been proven in δ values (ppm) with tetramethylsilane(TMS) as an internal standard. LC-MS changed into acquired the usage of a C-18 column on Shimadzu, LCMS 2010A, Japan. The FT-IR spectra of the compounds were taken as KBr pellet (a hundred mg) the usage of Shimadzu Fourier Transform Infrared (FTIR) spectrometer. Magnetic susceptibility have been measured at 35 °C through the Gouy technique, Silica gel GF254 thin plates from Merck were used for TLC and spots were positioned either by UV or dipping in potassium permanganate solution. The powder X-ray studies was carried out by using Rigaku MiniFlex instrument with Cu-K_αradiation (wavelength 0.154nm).

Synthesis of ligand and complexes:

Synthesis of ligand (BI):

Isoniazid (0.01mol, 2.742g) was dissolved in 35 mL of anhydrous ethanol, benzimidazole (0.01mol, 2.362g) and formaldehyde solution (37%, 0.05mol) were added, the mixture was stirred at 70°C for 10 h. the completion of the reaction was monitored by TLC, the reaction mixture was cooled and poured into crushed ice. The precipitate was collected by filtration, the precipitate dried and recrystallized from ethanol.

Scheme 1 Synthetic route of ligand (BI) via mannich base

Synthesis of metal complexes:

The Ethanolic solution of metal (II) chlorides (0.02mol) was added in drops of ethanolic of ligand BI (0.04mol) and the mixture was refluxed on water bath at 70 °C 4-5h. The solid complex obtained was filtered, washed with hot ethanol and dried in vacuum over anhydrous calcium chloride.

Figure 1 Proposed structures of Metal complexes (M=Co, Ni, & Cu)

Antioxidant activity:

The free radical scavenging activity of the ligand BI and complexes was measured In vitro by 2, 20- diphenyl-1-picrylhydrazyl (DPPH) assay. The stock solution was prepared by dissolving 24 mg DPPH with 100 ml methanol and stored at 20°C until required. The working solution was obtained by diluting DPPH solution with methanol to attain an absorbance of about 0.98±0.02 at 517 nm using the spectrophotometer. All the tested samples in various concentrations (50,75 and 100 μg/mL) were prepared in methanol and the homogeneous solutions were achieved by stirring. A liquot of test sample (1 mL) was added to 4 mL of 0.004% (w/v) methanol solution of DPPH and then reaction mixture was vortexed for 1 min and kept at room temperature for 30 min in the dark to complete the reaction. The absorbance was read against blank at 517 nm. The synthetic antioxidant BHT was used as positive control ^12,13. The ability of the tested samples at tested concentration to scavenge DPPH radicals was calculated using equation.

Scavenging ratio (%) = [(A_i‒A_o) ⁄ (A_c‒A_o)] × 100%

Where A_i is the absorbance in the presence of the test compound; A₀ is absorbance of the blank in the absence of the test compound; A_c is the absorbance in the absence of the test compound.

Molecular docking:

Molecular modeling studies were performed by using Hex 8.0.0 protein-ligand docking in PDB formats. The parameters used for docking include: correlation type-shape only, FFT mode -3D, grid dimension –0.6, receptor range –180, ligand range –180, twist range –360, distance range –40. The starting coordinates of the human antioxidant enzyme in complexes with the competitive inhibitor DTT (PDB: 3MNG) were taken from the Protein Data Bank (http://www.rcsb.org/pdb)^12,14.The selected ligands were docked against the lead competitive inhibitor ligand DTT at the crystal enzyme structure of the target protein and the best energy conformations of receptor ligand were studied, and the energy of binding was calculated as the difference between the energy of the complex and the individual energies of enzyme and ligand.

RESULTS AND DISCUSSION:

The analytical data of the metal complexes reveals that while reactions with metal (II) chlorides and ligand performed in 1:2 (M:L) molar ratio, the BI behaves as a bidentate ligand and coordinate through metal nitrogen and metal oxygen of carbonyl group giving octahedral geometry for Co(II) and Cu(II) complexes and distorted square planar geometry for Ni(II) complexes. The observed molar conductance of the complexes in DMSO (10^-2 M solution) are consistent with Co(II), Ni(II) and Cu(II)complexes are non electrolytic in nature [17].

¹H NMR and Mass spectra:

NMR spectrum of BI showed a doublet signals at 10.12 to 9.83 ppm attributed to two –NH protons [24], and for –CH=N a singlet peak appears at the range of 8.76 ppm. The multiple signals of 7.77 to 7.22 ppm attributed to aromatic protons for imidazole and Isoniazid rings respectively which reveals the structural evidences for BI. In the mass spectrum showed molecular ion peak [M⁺] at m/z 268.3 (267.2), that equivalent to the molecular weight of BI ligand respectively.

IR Spectra:

The BI showed a sharp band at 1704 cm⁻¹for ν(C=O) and the position of ν(C=O) band is shifted to the lower frequencies by 1605-1612 cm^-1in the spectra of the CO(II), Ni(II) and Cu(II) metal complexes, indicating that the coordination occurs through the oxygen atom to the metal ion¹⁵. The ν(Ar CH) stretching vibrations appeared at 2945 cm⁻¹ and medium band appears at 1304 cm^-1 for ν_b(N-H), The band ν(C=N) observed around 1535 cm^-1 was shifted to lower frequencies in the spectra of all the complexes 1500-1509 cm^-1 indicating that coordination of ν(C=N) via nitrogen atom of the ligand. For the coordination to the metal atom new peaks observed at the range of 474 cm⁻¹,475 cm⁻¹ and 469 cm⁻¹ are due to ν(M-N) bonding and followed 510 cm^-1,510 cm^-1, and 511 cm^-1 , are assigned as ν(M-O) mode of bonding ^16,17.

Electronic spectra and magnetic moments:

The absorption band observed at 32051 cm^-1and 245 cm^-1 in the spectrum of BI ligand is predominantly due to n→π* and π→π* transitions ¹⁸. These transitions are shifted slightly in the formation of metal complexes. The d-d bands observed at 21367 cm^-1 and 10131 cm^-1 for Co(II) complex are assigned as ⁴T_1g(F) → ⁴T_2g(F) (ν₁) and → ⁴A_2g(F) (ν₂) transitions, and magnetic moment is

1.94 B.M, represents octahedral geometry¹⁹, the electronic spectra of Ni(II) complex showed the bands at

15873 cm^-1 and 10266 cm^-1 corresponding to ¹A_1g → ¹B_1g(ν₁) and → ¹B_2g(ν₂) respectively and magnetic moment is 3.63 B.M leads to square planar geometry²⁰. The copper complexes shows one board band at 15151 cm^-12B_1g → ²A_1g(ν₁) and → ²B_2g(ν₂) transitions and magnetic moment is 1.83 B.M, indicating octahedral geometry ²¹.

XRD analysis:

The powder XRD diffraction patterns is carried out for all the complexes, and by evaluating the diffraction patterns of complexes Co(II), Ni(II), and Cu(II) showed crystalline in nature representing in figure 2.

(A)

(B)

(c)

Figure 1 XRD patterns of [A]. [CoCl₂(BI)₂], [B]. [Ni (BI)₂] Cl₂ and [C]. [CuCl₂(BI)₂] complexes

The Miller indices (hkl) along with observed and calculated d angles, 2θ, sin2θ values, and relative intensities, along with cell parameters are given in tables 2, 3 and 4. For [CoCl₂(BI)₂] complex lattice parameters and angle calculated are a = 9.5496 Å, b = 15.4706 Å, c = 7.3935, β = 98.0241°, unit cell volume of the complex 1.378×10^-8 cm. [Ni(BI)₂]Cl₂ complex lattice parameters and angle calculated are a = 11.9487 Å, b = 14.3386 Å, c = 13.4797, β = 94.8014°, unit cell volume of the complex 2.1261×10^-8 cm. and [CuCl₂(BI)₂] complex lattice parameters and angle calculated are a = 13.4747 Å, b = 11.2711 Å, c = 8.6961, β = 90.245 °, unit cell volume of the complex 1.378×10^-8 cm. The average crystallite sizes of the complexes dxrd were calculated using Debye Scherrer equation (D =Kƛ/βCos θ) Where D = Particle size, K = Dimensionless shape factor, ƛ = Xray wavelength (0.15406Å) β = Line broadening at half the maximum intensity, θ = Diffraction angle. [CoCl₂(BI)₂], [Ni (BI)₂]Cl₂ and [CuCl₂(BI)₂] crystallite size of 93.28nm (monoclinic), 33.54nm (orthorhombic) and 63.31nm (monoclinic) respectively suggesting that the complexes are in a nanocrystalline phase^{22, 23}.

Table 2 XRD data of [CoCl₂(BI)₂] H₂O complex

Peak No	2θ	θ	Sinθ	h k l	d		Intensity
Peak No	2θ	θ	Sinθ	h k l	Cal	Obs	Intensity
1	19.86	9.93	0.484001751	200	4.53	4.53	10.43
2	22.25	11.125	0.991636177	113	4.06	4.12	46.49
4	23.78	11.89	0.625966778	040	3.81	3.81	100
5	25.71	12.855	0.284638574	140	3.54	3.51	21.29
6	27.97	13.985	0.988444933	042	3.28	3.38	6.57
7	28.67	14.335	0.980494781	133	3.20	3.27	55.90
8	30.08	15.04	0.619388265	140	3.07	3.51	4.90
9	31.98	15.99	0.278312486	320	2.90	2.80	7.92
10	34.76	17.38	0.994879566	025	2.70	2.72	14.79
11	39.16	19.58	0.667200485	006	2.43	2.43	4.59

Table 3 XRD data of [Ni (BI)₂] Cl₂ H₂O complex

Peak No	2θ	θ	Sinθ	h k l	d		Intensity
Peak No	2θ	θ	Sinθ	h k l	Cal	Obs	Intensity
1	10.97	5.485	0.716090602	110	8.05	8.00	27.38
2	14.87	7.435	0.91350372	120	5.94	5.94	10.07
4	20.76	10.38	0.816441972	102	4.27	4.24	22.82
5	21.99	10.995	0.99999984	220	4.03	4.00	31.25
6	23.75	11.875	0.63759366	201	3.74	4.20	10.01
7	24.74	12.37	0.19511099	140	3.59	3.58	15.36
8	27.97	13.985	0.988444933	003	3.18	3.18	10.12
9	29.05	14.525	0.92573073	202	3.06	3.34	10.54
10	3046	1523	0.62289431	103	2.14	3.01	8.14
11	32.30	16.15	0.42778132	212	2.76	3.28	17.48

Table 5 XRD data of [CuCl₂(BI)₂] H₂O complex

Peak No	2θ	θ	Sinθ	h k l	d		Intensity
Peak No	2θ	θ	Sinθ	h k l	Cal	Obs	Intensity
1	19.79	9.895	0.453084237	210	4.48	4.46	98.96
2	20.58	10.29	0.761239268	023	4.31	4.14	73.17
4	22.37	11.185	0.982112532	221	3.96	4.00	43.61
5	24.19	12.095	0.45410786	004	3.67	3.25	84.83
6	25.53	12.765	0.197325851	141	3.48	3.48	37.33
7	26.59	13.295	0.665847662	043	3.34	3.04	50.69
8	27.79	13.895	0.970820607	133	3.20	3.32	100
9	29.17	14.585	0.90138753	144	3.05	3.311	19.97

Table 6 Antimicrobial datas – Zone inhibition

Compounds	Antibacterial zone inhibition in mm (mean ± SD)			Antifungal zone inhibition in mm (mean ± SD)
Compounds	*S. aureus*	*B. subtilis*	*E. coli*	*S. coccus*	*C. albicans*	*A. niger*
Ligand (BI)	03±0.3	05±0.2	05±0.7	04±0.4	03±0.1	-
Co(II)	06±0.3	-	07±0.3	08±0.2	-	06±0.3
Ni(II)	14±0.2	12±0.4	10±0.2	10±0.6	09±0.2	11±0.3
Cu(II)	13±0.1	15±0.1	12±0.3	08±0.4	10±0.1	10±0.3
Chloramphenicol	15±0.2	16±0.3	13±0.3	12±0.2	11±0.4	13±0.3
Fluconazole	-	-	-	12±0.2	10±0.1	12±0.3
DMSO	0	0	0	0	0	0

Table 7 Antimicrobial datas – minimal inhibitory concentration

Compounds	MIC of the compounds in 25µg/mL
Compounds	*S. aureus*	*B. subtilis*	*E. coli*	*S. coccus*	*C. albicans*	*A. niger*
Ligand (BI)	10	-	12	09	08	-
Co(II)	11	10	-	08	10	11
Ni(II)	18	21	17	19	21	15
Cu(II)	19	20	22	17	13	22

Antimicrobial activity:

All the synthesized complexes and ligand (BI) have been examined towards three bacterial and fungal strains the use of agar well diffusion method ²⁶. All bacterial traces were maintained on nutrient agar medium at ±37 °C, and fungal strains were maintained on potato dextrose agar (PDA) at ±25 °C. The test compounds had been dissolved in DMSO. Sample-loaded plates were inoculated with the microorganism incubated at 37 °C for 24 h, and culture was incubated at 25 °C for 60 h. DMSO as control and chloramphenicol and fluconazole is used as standards for bactericide and fungicide. (Table 6) All the synthesized complexes and ligand (BI) showed inhibition property, among them Ni(II) and Cu(II) complexes shows excellent when compared to standards. The compounds were also tested for minimal inhibitory concentration (MIC) values ¹³. MIC values of less than 25µg/mL are shown in table 7, it observed that ligand (BI) and Co(II) complex showed least activity, Ni(II) and Cu(II) complexes showed promising activity.

CONCLUSION:

The new mannich base ligand synthesized N'-(1H-benzimidazol-1-ylmethyl) pyridine-4-carbohydrazide (BI) and characterized. The spectral data showed that ligand coordinated through carbonyl oxygen and imidazole nitrogen which imply that bidentate in nature, electronic spectra suggest that octahedral geometry for Co(II) and Cu(II) complexes, Ni(II) complexes exhibited square planar geometry, From XRD datas it was found that Co(II), Cu(II), possessing monoclinic and Ni(II) complex showed orthorhombic crystallite phase. Antioxidant activity enhanced on complexation with metal ions, resulting promising scavenging activity for all complexes, Cu(II) complex gives highest and prominent activity near to that standard BHT. Molecular docking studies of synthesized compounds are carried out, which reveals excellent binding energy interactions with protein enzyme DTT (PDB: 3MNG).

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Received on 07.05.2017 Modified on 10.07.2017

Asian J. Research Chem. 2017; 10(4):470-476.

DOI:10.5958/0974-4150.2017.00076.1

Compounds	Colour	Mol.Wt	Yield (%)	Calcd. (found) (%)			Molar conductance (ohm^–1cm² mol^–1)
Compounds	Colour	Mol.Wt	Yield (%)	C	H	N	Molar conductance (ohm^–1cm² mol^–1)
C₁₄H₁₃N₅O (BI)	Creamy white	267.2	70	62.91(61.91)	4.90(3.96)	26.20(25.31)	-
[CoCl₂(BI)₂] H₂O (1)	Dark green	591.48	65	56.86(55.12)	4.09(3.91)	23.68(22.51)	14.20
[Ni (BI)₂] Cl₂ H₂O (2)	Light blue	591.24	60	56.88(55.81)	4.09(3.97)	23.69(22.51)	38.55
[CuCl₂(BI)₂] H₂O (3)	Light brown	600.13	63	56.42(56.65)	4.06(3.87)	10.66(09.25)	17.81