Design, synthesis and biological evaluation of heterocyclic methyl substituted pyridine Schiff base transition metal complexes

In recent years heterocyclic Schiff base metal complexes attract more attention in biological application and also showing interesting co-ordination chemistry. In this research article a novel heterocyclic methyl-substituted pyridine Schiff base transition metal complexes of Fe(III), Co(III), Cu(II), and Ni(II) have been design and synthesized by reacting metal acetate or metal salts (FeCl3, CoOAc, CuOAc, NiOAc), with substituted heterocyclic ligand. All newly synthesized metalcomplexes were characterized by spectroscopic data and screened for elemental analysis, FT-IR, ESR, Magnetic susceptibility and TGA. The Electronic spectra and magnetic susceptibility measurements indicates that square planer and octahedral geometry of these complexes also suggest their structure in which (N, O) group acts as bidentate ligand. The thermal stability, decomposition rate and thermodynamic parameters of synthesized metal complexes were calculated by Freeman Carroll method. Also the biostatistical data of antimicrobial and anti-oxidant activities of synthesized metal complexes indicates moderate to good results.


Introduction
A large number of heterocyclic Schiff base ligand and their transition metal complexes have been dynamically investigated not only fortheir biological application, thermal analysis, spectral analysis but also for their interesting coordination chemistry such as Cu(II), Co(II), Ni(II), Zn(II) and Ru(II)complexes with the Schiff base ligand derived from 5-amino-4H-1,2,4-triazole-3-thiol and 3-hydroxy-4-methoxybenzaldehyde, benzothiazoleimino-benzoic acid ligands and their Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) complexes. Again synthesis of bidentatetriazole ligand and its monomeric metal complexes in the company relating to Cd(II), Co(II), Fe(III), Cu(II), and Ni(II) ions is demonstrated and their antibacterial study relating to Ni(II), Cu(II), Fe(III), Cd(II), and Co(II) complexes of 4-amino-5-stearyl-1,2,4-triazole-3-thiol [1][2][3][4]. Mostly Schiff base with multiple and different coordination sites their metal complexes and different oxidation property. In general aromatic aldehyde reacts faster than ketones in condensation reaction leading to the formation of ligand. Schiff base ligand of aliphatic aldehyde and ketone are relatively unstable compared to that of aromatic aldehyde because the presence of effective conjugation present in ring system. The presence donating substituent at ortho position helpful for coordination also presence of lone pair of electron in the sp 2 hybidized orbital of nitrogen atom of the azomethine group is play significant role in coordination and offer good chelating ability. Schiff base ligands are easily synthesized from condensation of aliphatic or aromatic aldehyde with primary aliphatic or aromatic amine in ethanolic solvent the reaction is accelerated by adding the catalytic amount of acid [5]. The common structural feature of these compounds is the azomethine group with a general formula (R-CH = N-R,) Where R is any alkyl group which may be variously substituted. These compounds are also known as anils, imines, azomethine [6]. Schiff base are well known for their wide application and are useful intermediates in organic synthesis [7]. These compound has intrinsic biological activities, including anticancer [8], antitumor [9], antitubercular [10], antibacterial [11], antifungal and antifertility [12], herbicidal [13], antioxidant [14], antiproliferative [15], activities. Moreover Schiff bases also exhibit fluorescence [16], photoluminescence [17] and potentiometric titration [18], aggregation [19] and anthelmintic activities [20].
Schiff base ligands are easily synthesized and form complexes with almost all metal ions [21]. However the incorporation of transition metal ions in to these compounds have enormous wide application in the fields of the food industry, dye industry, analytical chemistry, catalysis, fungicidal, agrochemicals along with biological activities and decrease in the cytotoxicity of both metal ion and Schiff base [22,23]. The chemistry of coordination compound with heterocyclic shiff base ligand containing Oxygen, Nitrogen, Sulfur as donor atoms [24][25][26]. The Schiff base ligand containing these hetero atoms Coordinates to the metal atoms in different ways [27].These chelating properties of Schiff base ligand display application in many fields such as pharmaceutical, Industrial, and Agricultural field [28]. Schiff base complexes plat a vital role in designing metal complexes related to synthetic and natural oxygen carrier [29]. Metal complexes make the compound effectives as stereo specific catalyst towards oxidation, reduction, hydrolysis, biological activity and inorganic chemistry. In organic compound the presence of N=C along with other functional group form stable complexes compared to compound with only N=C coordinating moiety. Similarly pyridine derivatives have been of great interest because of their role in natural and synthetic organic chemistry. Many products which contain a pyridine sub unit exhibit biological activity such as antimicrobial [30] and antituberculosis activity [31].Tetra dentate Schiff base complexes have been shown to form stable complexes, with coordination taking place through the dinitrogen-dioxygen donor atoms [32,33]. Ligational, density functional theory, and biological studies on some new Schiff base metal complexes such as N 2 O 2 Tetradentate Schiff Base Metal Complexes [34,35].Also recently new schifff base metal complexes of Zr(II), Ni(II), La(III) and Th(IV) introduce which derived from Dithranol and heterocyclic ligand 8-hydroxyquinoline [36].
In present research paper heterocyclic N-substituted pyridine Schiff base ligand and their transition metals complexes were synthesized by condensing stoichiometric ratio 1:1 of substituted aromatic aldehyde and methyl derivative of heterocyclic primary amine in anhydrous ethanol along with catalytic amount of dil.HCl. Finally yellow colored precipitate of Schiff base ligand obtained. The methanolic solution of obtained ligand mixed and reflux with solution of Metal acetate or different metallic salt with constant stirring. The solvent was degassed through rotary evaporator, after cooling colored solid of metal complexes were separate out. The colored precipitate of different metal complexes washing by petroleum ether and dried under desiccators.

Material method
The entire reagent used for the synthesis heterocyclic Schiff bases and their transition metal complexes Such as 2, 4-Dihydroxybenzaldehyde, 2-amino 4-methyl pyridine. (Heterocyclic primary amine), Cobalt acetate tetrahydrate, Nickel acetate, Cupric acetate monohydrate, Ferric chloride, hexane, methanol, ethyl acetate, glacial acetic acid, petroleum ether, were purchase from loba chemie and sigma Aldrich A.R grade and solvent like anhydrous ethanol were purified by standard distillation method and good purity. The reaction was monitored by thin layer chromatography by using pre-coated silica gel aluminum plates and ratio of n-hexane and ethyl acetate 7:3 proportion was used as mobile phase and spot is visualizing in U.V chamber.

Physical measurement
Different kinds of physico-chemicals techniques are used to characterize the structure of organic Schiff base ligand transition metal complexes. The melting points were taken in to open capillaries and are uncorrected. The FTIR characterization was recorded on model no Shimadzu FTIR 8400 S. using KBr disc method. And 1 H NMR spectra were obtained in CDCl 3 a solvent and Mass spectrum was done by using Bruker Compass Data analysis 4.2 having scanning capacity 600 m/z.The X-band of ESR spectra of copper complexes was recorded in the solid state at liquid nitrogen temperature 77 k in DMF as solvents using DPPH as standard on ESR-JEOL. ESR Spectrometer with X band. The synthesized heterocyclic Schiff bases their transition metal complexes were screened for antimicrobial activity and antioxidant activity by Disc diffusion method Disc size 6 mm. TGA analyses of all metal complexes were carried out at 25 °C to 500 °C in nitrogen atmosphere having heating rate 10 0 C per min using ShimadzuTGA-50.

Synthesis of Fe(III) complex. [DH-4M] Fe(III)
The gradually addition of (0.01 mol) hot methanolic solution of FeCl 3 to (0.03 mol) methanolic solution of Schiff base ligand [DH-4M] solution with constant stirring having stoichiometric ratio (1:3). The reflux condition was maintained for 4-5 h after completion of reaction the reaction mixture was filtered and concentrated precipitate was filtered and washes with cold methanol.

Analytical and spectral data of [DH-4M] Fe(III)
Practical

Synthesis of Co(III) complex. [DH-4M] Co(III)
The transition metal complexes were prepared by mixing a stoichiometric ratio (1:3) by dissolving in methanol. The hot methanolic solution of CoOAc (0.01 mol) and ligand [DH-4M] (0.03 mol) are mixed in hot condition with continuous stirring. The mixture were reflux for about 4-5 h. After cooling the volume of reaction mixture is reduced to half, and then colored solid metal complex is formed. Thus obtained solid metal complexes were purified by petroleum ether. The elemental analysis, spectral data and chemical analysis of synthesized metals complexes assigned geometry of Fe(III) and Co(III) metal complexes exhibit Octahedral structure.

Synthesis of [DH-4M] Ni(II) and [DH-4M] Cu(II)
Schiff base metal complexes of heterocyclic Schiff base ligand were synthesized by gradual addition of ethanolic solution of ligand [DH-4M] to solution of NiOAc and CuOAc in ethanol. The mixture was stirred for 2-3 h at room temperature having ligand-metal stoichiometric ratio (2:1). Then mixture was heated at reflux for about 4 to 5 h on cooling the volume of reaction mixture is reduced to half, and then colored solid metal complex is formed, washed with ethanol twice.The elemental analysis, spectral data and chemical analysis of synthesized metals complexes assigned geometry of Ni(II) and Cu(II) metal complexes exhibit square planer structure.

Analytical and spectral data of [DH-4M] Ni(II)
Practical

FT-IR spectra
In this investigation FT-IR is effective tool to detect functional group form in ligand. The observed frequency of carbonyl and primary amine was disappearing. This clearly indicate that formation of azomethine (CH=N) group in Schiff base ligand. The frequency observed at 1600 cm −1 were shifted to lower frequency in metal complexes clearly indicates that azomethine nitrogen coordinate to metal ion. Here was an important variation in the position of these bands in case of metal complexes as compared to chelates. The new weak bands appear in the region 415-445 cm −1 and 510-535 cm −1 can be assigned to the metal nitrogen (M-N) and metal oxygen (M-O) vibration modes in co-ordination complexes respectively.

Mass spectra
Mass spectrometry has been successfully used to determine the molecular ion peak for heterocyclic Schiff base ligand. The peak obtained are in good agreement with proposed structure of heterocyclic Schiff base ligandas shown in Fig. 1.

Electronic spectra
The electronics spectra of heterocyclic Schiff base ligand and their transition metals complexes were recorded in DMSO solution between 200 and 800 nm at ambient temperature. In free ligand two band were observed at 278, 335 nm which can be assigned to л-л* transition of the aromatic ring and for imines groups respectively [38]. These bands were also observed at similar position in the U.V spectra of complexes.

1 H NMR spectra of ligand
In  .175, G = 5.71, ƒ = 136.01, were derived. It was practical that gll > g ┴ > g avg and ƒ value are in span 105-135 cm-1 this brand of arrangement has been reported that square planner complex [42]. Similarly fact point to that unpaired electron lies in the dx 2 -y 2 orbital giving 2 B1g as the ground state. In addition value of gll = 2.39 is nearer to 2.3-2.4 indicating the existence of mixed copper-Nitrogen and Copper-Oxygen bands in these chelates [43][44][45][46]. The value of α 2 = 0.5 indicate complete covalent bonding while that of value of α 2 = 1.0 suggest perfectly ionic bonding. The obtained value of α 2 = 0.65 is less than one which indicates that the complex has some covalent character in the ligand environment [47].

Anti-microbial activities
The Synthesized transition metal complexes were screening for their antimicrobial activity particularly Antibacterial and Antifungal activity and this is done with the help of disc diffusion method. Microorganism like gram positive bacteria Staphylococcus aureus, Gram negative bacteria Escherichia coli. The microorganism fungi used for these activities are Candida albicans Aspergillus niger and Fusarium moniliforme.  Fig. 4 and Table 1 2

.8 Antioxidant activity
The free radical scavenging activity of the Schiff bases and their transition metal complexes. Its test sample was determined using 2, 2 diphenyl-1 picrylhydrazyl radical (DPPH) method. Different concentration of test compound 50, 100, 200, 300, 400 mg/ml and its standard is Ascorbic acid as well as Gallic acid. The antioxidant activity of the sample was done by using DPPH radical scavenging method. The assay was carried out in a 96 well microlitre plate to 200 μl of DPPH solution, 10 μl of each of the test sample or standard solution was added separately in wells of the microlitre plates. The plates were incubated at 37 0 C for 20 min and the observance of each well was at 517 nm, using ELISA reader against corresponding test and standard blanks and the remaining DPPH was calculated. The IC 50 (Inhibitory Concentration) is concentration of the sample required to Scavenge 50% DPPH free radicals.
The IC 50 calculated based on regression equation generated by plotting the graph of Conc. Vs % inhibition.
where Y = intercept i.e., 50; C = Concentration required to inhibit the oxidative radical to 50%; M and C are the %Inhibition = Controll − Sample Controll * 100  Table 2.
From the conclusion of a biostatical data shows synthesized transition metal complex Fig. 5

Thermogravimertic analysis
TGA study find out the thermal stability of complexes and its degradation pattern in which the change in the weight of the substance is recorded as function of temperature. In TGA technique several kinetics parameter such as energy of activation (Ea), free energy change (∆G), Entropy change (∆S), Order of reaction (n). Thermogravimertic analysis measure weight loss curve gives information on change in sample composition, thermal stability, and kinetic parameters for chemical reaction in the samples. Also in TGA Pre-exponential factor decide the pathway of reaction mechanism. The Several methods have been reported for calculating the kinetics parameter of solid state reaction [48][49][50].Such as Freeman and Carroll method [51] Sharp-Wentworth [52] Coats-Redfern [53] Broido [54] and Horowitz-Metzger method [55]. The thermal decomposition kinetics was proposed by Flynn Freeman and Carrol published their method of kinetics analysis of thermo analytical data. TGA of the transition metal complexes were carried out from ambient temperature to 500 °C in Nitrogen atmosphere having heating rate 10 °C per minute. The kinetic parameters of all the transition metal complexes by Freeman and Carroll methods have been determined and computerized software is essential for processing the enormous amount of data involved in this analysis and represented graphically by Microsoft Excel. Procedure suggested by Nair et al. [56].

Freeman and Carroll method
where dw/dt = rate of change of weight with time; Wr = Wc-W; Wc = Weight loss at the completion of reaction; W = Total weight loss up to time t; Ea = Energy of activation; N = order of reaction.
In the thermogram of [DH-4M] Fe(III) metal complex Fig. 6 shows that there is no degradation pattern was observed and there is no significant change of mass was observed over the entire range of temperature so the complex was thermally stable. In the thermogram of [DH-4M] Co(III) metal complex its mass loss pattern exhibit in single step. The first phase of decomposition temperature range 70-130 °C with a mass loss 5.16% (Calculated 5.12%) indicate the loss of two crystal lattice water molecule. The thermogram of [DH-4M] Ni(II) complex Fig. 7 shows a two step decomposition the first step was observed in the range 58-123 °C with a mass loss 5.36% (Calculated 5.14%) indicate loss of two crystal lattice water molecule and no significant mass loss was observed in the range 160-325 °C. In the second phase of decomposition was shown in the range 330-375 °C with a mass loss 3.19% (Calculated 35.32%) indicate the loss of one acetate molecule after mass loss remain constant over the entire range of temperature. The TGA curve of [DH-4M] Cu(II) complex was studied from ambient temperature to 500 °C under Nitrogen atmosphere. Mass loss was not observed over the entire range of temperature so the copper complex thermally stable. The kinetic parameters of degradation of the metal complexes calculated by Freeman-Carroll (FC) method.
A negative change in entropy indicate that the disorder of an isolated system has decreases negative (∆S) value as shown in Table 3 indicates more ordered activated state that may be possible through chemisorptions

Conclusion
All the complexes are colored solid and physical and analytical data of ligand and their metal complexes are reported with the expected values. The heterocyclic Schiff ligand prepared in this way are formed nearly high   The ESR data [DH-4M] Cu(II) provide the information about the geometry of complexes. The sequence of gll > g ┴ > g avg indicates the complex is square planer geometryand the unpaired electron lies in the dx 2 -y 2 orbital. The 10Dq or Δo value for Fe +3 is − 2.0Δo + 2P, for Co +3 is − 0.4Δo + P, for Ni +2 is − 2.44Δo + 4P, for Cu +2 is 1.21 + 4P. Also the geometry of [DH-4M] Cu confirmed by additional supporting evidences of magnetic susceptibility and FT-IR data.TGA analysis results of[DH-4M] Fe(III) and [DH-4M] Cu(II) indicates there is degradation pattern was observe over the entire range of temperature and the complex [DH-4M] Co(III) mass loss pattern exhibits in single step, complex [DH-4M] Ni(II) shows two step decomposition. In TGA several kinetics parameter such as energy of activation (Ea), free energy change (∆G), Entropy change (∆S), Order of reaction (n) by Freeman Carroll Method. The Order of reaction is consistently one and negative value of entropy indicates that more ordered activated state possible through chemisorptions of oxygen, decomposition product and polarization of bond in activated state.