INTRODUCTION:

The area of research including synthesis and characterization of mixed ligand complexes is gaining increased attention of researchers working in inorganic chemistry since last few years^1-4. The structure of mixed ligand complexes is different from that of traditional complexes in the sense that they have more than one type of ligand attached with the same metal. This has offered an advantage of preparing complexes with varying properties due to possible structural variations compared to traditional Werner complexes. This has prompted inorganic researchers to concentrate their research in this area and as a result a huge amount of literature is available today in this field and a lot more is expected to get added.

Present paper is continuation of systematic research work going on in the area of synthesis and characterization of mixed ligand complexes of transition metals in our research laboratory^5-8.

In present investigation we report synthesis and characterization of a series of seven mixed ligand complexes of transition metal ions such as Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) using 2-aminophenol and 2-Chloroaniline as primary and secondary ligands respectively. All the synthesized complexes are characterized using molar conductance, magnetic susceptibility measurements, IR and electronic spectra. ¹H-NMR spectra was recorded for Cr(III) complex as a sample to study the protonic arrangement present in the structure of the complexes. Based on the results obtained an octahedral geometry was proposed for all the synthesized complexes. All the synthesized complexes were screened for their in vitro antibacterial activities against bacterial pathogens such as Escherichia coli, Salmonella typhi, Staphylococcus aures, Bacillus subtilis and antifungal activities against fungal pathogens such as Aspergillus niger, Penicilium chrysogenum, Fusarium moneliforme and Aspergillus flavus.

MATERIAL AND METHODS:

Chemicals (including solvents) used in present study were purchased from S. D. Fine Chemicals, Spectrochem Private Limited, Qualigens Fine Chemicals and Merck Chemicals. All the chemicals used were of AR grade. The purification of solvents was done by distillation as per literature⁹.

Molar conductance measurements were performed by preparing 10^-3M solutions in DMF solvent using Equiptronics conductivity meter with inbuilt magnetic stirrer model (Eq-664) at room temperature. Magnetic susceptibilities were determined on the SES Instrument’s magnetic susceptibility Gouy’s balance (model EMU-50) at room temperature using copper (II) sulphate as a standard. These magnetic susceptibility values were utilized to calculate magnetic moments using spin only formula m_eff = [n(n+2)]^1/2 BM for all the synthesized mixed ligand complexes¹⁰. IR spectra were recorded as KBr pellets in the region of 4000-400 cm^-1on a Perkin Elmer Spectrophotometer. Electronic spectra were recorded in DMSO solvent on a Shimadzu UV-1600 spectrophotometer. ¹H-NMR spectrum was recorded on Bruker Avance II 400 NMR spectrometer using DMSO-d⁶ (spectroscopic grade) as a solvent. The chemical shifts are given in ppm relative to tetramethylsilane (TMS).

Synthesis of complexes:

The general procedure used for the synthesis of complexes was as follows:

A hot methanolic solution of respective transition metal salts (1 equivalent, 10 mmol) was mixed with a hot methanolic solution of 2-aminophenol (1 equivalent, 10 mmol) and 2-Chloroaniline (1equivalent, 10 mmol). The resulting mixture was then left under reflux for 8-10 hours. The progress of the reaction was checked by taking TLC in chloroform (90%) + methanol (10%) solvent system after every 30 minutes. After refluxing for 8-10 hours, appropriate complexes were precipitated out on cooling the reaction mixture. The obtained complexes then were filtered, washed with methanol and recrystallized from ethanol. The synthesized complexes were obtained in 60-70 % yield. Scheme representing synthesis of mixed ligand complexes is given below (Figure 1).

Figure 1: Synthesis of mixed ligand complexes of transition metal ions using 2-Aminophenol and 2-Chloroaniline ligands

Measurement of Antibacterial activity:

Antibacterial activity was measured by agar cup method¹¹. Nutrient agar (Himedia) was prepared and sterilized at 15 psi for 15 minutes in the autoclave. It was allowed to cool below 45 ⁰C and seeded with turbid suspension of test bacteria separately, prepared from 24 hours old slant cultures. 3% inocula were used every time. The bacterial cultures selected were, two gram negative cultures viz. E. coli, S. typhi and two gram positive cultures viz. S. aureus, B. subtilis. This seeded preparation was then poured in sterile petri plate under aseptic condition and allowed to solidify.

Cups of 10mm diameter were bored in the agar plate with sterile cork borer. 100 ml of compound solution prepared in DMSO (1%) was added in the cup under aseptic condition with the help of micropipette. 100 ml of DMSO was also placed in one of the cup as blank (negative control). A standard antibiotic disk impregnated with 10 units of penicillin was also placed on the seeded nutrient agar surface as standard reference antibiotic (positive control). The plates were kept in refrigerator for 15 minutes to allow diffusion of the compound from agar cup into the medium. Then the plates were shifted to incubator at 37 ⁰C and incubated for 24 hours. After incubation plates were observed for the zone of inhibition of bacterial growth around the agar cup. Results were recorded by measuring the zone of inhibition in millimeter (mm) using zone reader.

Measurement of Antifungal activity:

Antifungal activity was measured by Poison Plate Method¹¹. The medium used was potato Dextrose agar (Himedia). The medium was prepared and sterilized at 10psi in autoclave for 15 minutes. Then the compound to be tested is added to the sterile medium in aseptic condition so as to get final concentration as 1%. A plate with DMSO was prepared as blank (negative control). Similarly a plate with 1% Gresiofulvin was prepared as standard reference plate (positive control). Aspergilllus niger, Peniclilium chrysogenum, Fusarium moneliforme and Aspergillus flavus were selected as test fungal cultures. They were allowed to grow on slant for 48 hours so as to get profuse speculation. 5 mL of 1:100 aqueous solution of Tween 80 was added to the slant and spores were scraped with the help of nicrome wire loop to form suspension.

The fungal suspension was spot inoculated on the plates prepared using compound with the help of nicrome wire loop. The plates were incubated at room temperature for 48 hours. After incubation plates were observed for the growth of inoculated fungi. Results were recorded as growth of fungi (no antifungal activity), reduced growth of fungi (moderate antifungal activity) and no growth of inoculated fungi (antifungal activity).

RESULTS AND DISCUSSION:

The synthesized metal complexes can be represented with general formula [M(C₁₂H₁₂Cl₃N₂O)] for divalent transition metal ions, where M = Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) and with [M(C₁₂H₁₂Cl₃N₂O)].X for trivalent transition metal ions, where M = Cr(III), Fe(III) and X = Cl^-.

Thin layer chromatography (TLC):

TLC was recorded after a regular interval of 30 minutes during the progress of the reaction to find out exact end point of the reaction. Observations noted such as absence of reactants and appearance of a new single spot at the bottom on TLC plate after completion of reaction indicated towards completion of reaction and purity of synthesized mixed ligand complexes.

Physicochemical data:

Observations such as color, melting point and percentage yield were recorded for all the synthesized complexes. Theoretical molecular weights and molecular formula are estimated for all the synthesized complexes using ‘Chem Draw ultra 11.0’ programme. Melting points recorded for all the synthesized complexes are in the range 120-260 ⁰C which indicated that all the synthesized complexes are thermally stable. All the synthesized complexes were recorded to have dark colours. Percentage yield for each complex forming reaction was calculated with the help of theoretical and practical yield and was found to be in the range of 70-80 % which seems to be fairly good. The physicochemical data recorded for all the synthesized complexes is represented in Table 1.

Table 1: Physicochemical data of the synthesized mixed ligand complexes

Proposed Molecular formula	Calculated Molecular Weight (gms)	Colour	Percentage Yield (%)
C₁₃H₁₅Cl₄CrN₂O	409.08	Greenish	70
C₁₃H₁₅Cl₃MnN₂O	376.57	Black	72
C₁₃H₁₅Cl₄FeN₂O	412.93	Black	72
C₁₃H₁₅Cl₃CoN₂O	380.56	Black	74
C₁₃H₁₅Cl₃N₂NiO	380.32	Black	72
C₁₃H₁₅Cl₃CuN₂O	385.18	Greenish Black	78
C₁₃H₁₅Cl₃ZnN₂O	387.04	Black	210

Solubility behavior:

Solubility behavior of all the synthesized mixed ligand complexes was checked using different solvents available in the laboratory such as water, methanol, ethanol, chloroform, dichloromethane, acetone, ethyl acetate, N, N-Dimethylformamide (DMF) and Dimethylsulphoxide (DMSO). The synthesized complexes were found to be completely soluble in DMF and DMSO whereas partially soluble in chloroform and insoluble in all the remaining solvents.

IR spectra:

IR spectra of all the synthesized mixed ligand complexes have been recorded in the range 4000-400 cm^-1. Interpretation of the spectra is done by considering few main peaks observed.

The close observation of structures of complexes confirmed the presence of primary amine groups (–NH₂) in the structure of complexes. Thus peaks in the region of 3200-3500 cm^-1 due to symmetric and asymmetric stretching vibrations of n(N-H) can be expected. In present investigation the presence of peaks in the range 3000-3300 cm^-1 can be assigned due to such symmetric and asymmetric stretching vibrations which indicated towards presence of -NH₂ groups¹². Presence of bands around 1480 and 2854 cm^-1 region can be assigned to n(C=C) and n(C-H) aromatic stretching respectively. In present case peaks due n(C=C) and n(C-H) due to aromatic stretching vibrations in the region 1400-1600 cm^-1 and around 2850 cm^-1 respectively are also present¹².

Identification of bands due to n(M-O) and n(M-N) in the spectra of complexes is an important task as these are absent in the spectra of ligands and hence can be used as an evidence for completion of the complexation process. The position of these peaks is not fixed though and many researchers have reported presence of these peaks at various values^13-16. In present investigation the peaks observed in the range 590-620 cm^-1 and 500-550 cm^-1 can be assigned for n(M-O) and n(M-N) present in the complexes. IR spectroscopic data recorded for all the synthesized mixed ligand complexes is represented in Table 2.

Table 2: IR spectroscopic data for all the synthesized mixed ligand complexes

Complexes	n(N-H) /cm^-1	n(C=C) /cm^-1	n(M-O) /cm^-1	n(M-N) /cm^-1
C₁₃H₁₅Cl₄CrN₂O	3028.24	1492.90	682.80	513.07
C₁₃H₁₅Cl₃MnN₂O	3267.41	1480.50	698.23	518.85
C₁₃H₁₅Cl₄FeN₂O	3356.14	1479.40	682.80	551.64
C₁₃H₁₅Cl₃CoN₂O	3350.35	1487.12	678.94	555.50
C₁₃H₁₅Cl₃N₂NiO	3332.99	1487.12	584.43	491.85
C₁₃H₁₅Cl₃CuN₂O	3167.12	1469.76	694.37	594.08
C₁₃H₁₅Cl₃ZnN₂O	3226.91	1485.19	682.80	586.36

Electronic spectra:

Electronic spectra for all the synthesized mixed ligand complexes were recorded in DMF solvent in the range 400-1000 nm. The results obtained indicated towards octahedral geometry for all the synthesized mixed ligand complexes¹⁷. The observations noted from recorded electronic spectra of all the synthesized mixed ligand complexes along with molar conductance and magnetic moment values recorded are represented in Table 3.

Table 3: Electronic spectral data, molar conductance and magnetic moment values recorded for all the synthesized mixed ligand complexes

Complexes	Peaks assigned (nm)	Electronic transitions	Molar conductance (ohm^-1 cm² mol^-1)	Magnetic moment (B. M.)
C₁₃H₁₅Cl₄CrN₂O	440 560 660	⁴A_2g® ⁴T_1g(F) ⁴A_2g ®⁴T_2g(F) ⁴A_2g ®⁴T_1g(P)	58	3.98
C₁₃H₁₅Cl₃MnN₂O	600 780	⁶A_1g ® ⁴E_g ⁶A_1g ® ⁴T_2g(G)	22	5.95
C₁₃H₁₅Cl₄FeN₂O	500 700	⁶A_1g ® ⁴E_g ⁶A_1g ® ⁴T_2g(G)	60	5.98
C₁₃H₁₅Cl₃CoN₂O	440 520 630	⁴T_1g® ⁴A_2g(F) ⁴T_1g® ⁴T_2g(F) ⁴T_1g® ⁴T_1g(P)	25	4.85
C₁₃H₁₅Cl₃N₂NiO	580 680 780	³A_2g(F) ® ³T_1g(F) ³A_2g(F) ® ³T_2g(F) ³A_2g(F) ® ³T_1g(P)	27	2.92
C₁₃H₁₅Cl₃CuN₂O	620	²E_g ® ²T_2g	22	1.85
C₁₃H₁₅Cl₃ZnN₂O	-------	-----------	28	Diamagnetic

Molar conductance:

Molar conductance values for all the synthesized complexes were measured by preparing 10^-3 M solutions in DMF solvent. The observed molar conductance values (22-28 ohm^-1cm^-2mol^-1) for complexes with divalent metal ions Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) indicated towards their non-electrolytic behavior and the higher values (i.e. 58 and 60 ohm^-1cm^-2mol^-1) recorded for trivalent metal ion complexes of Cr(III) and Fe(III) respectively indicated towards 1:1 electrolytic nature of these complexes^18-19.

Magnetic properties:

Magnetic susceptibility values for all the synthesized mixed ligand complexes were recorded at room temperature using copper (II) sulphate as an internal standard. These were then converted into magnetic moment values using spin only formula. Magnetic moment value of 3.98, 5.95 and 5.98 B. M. recorded for Cr(III), Mn(II) and Fe(III) complexes corresponds to the presence of three, five and five unpaired electrons in these complexes respectively. Thus high spin nature and octahedral environment present in these complexes can be predicted²⁰. Magnetic moment values of 4.85, 2.92 and 1.85 B. M. were recorded for Co(II), Ni(II) and Cu(II) complexes respectively which corresponds to the presence of three, two and one unpaired electrons respectively in these complexes. An octahedral geometry of these complexes can be predicted²⁰. Zn(II) complex was found to be diamagnetic in nature consistent with the (d¹⁰) configuration of Zn in this complex²⁰.

¹H-NMR spectra:

¹H-NMR spectrum was recorded for Cr(III) complex as a sample study to confirm the protonic arrangement in the skeleton of synthesized mixed ligand complexes. The spectrum recorded showed three major peaks. The singlet peak observed at d 2.6 ppm can be assigned due to the residual protons of DMSO-d⁶ solvent²¹. The well resolved multiplet in observed in the region of d 6.5-7.7 ppm corresponds to presence of aromatic protons in the complex. The singlet peak observed in the region of d 3.0-5.5 ppm corresponds to -NH₂ protons of amine groups of the ring.

Antibacterial activity:

The standard used for recording antibacterial activity, penicillin, have shown zone of inhibition of 11-30 mm against bacterial pathogens Escherichia coli, Salmonella typhi, Staphylococcus aureus and bacillus subtilis. Overall all the complexes (except chromium (III) complex in case of E. coli) show moderate to strong activity against all the bacterial pathogens. Hence can be potential candidate to be antibacterial agents against the bacterial pathogens studied in present investigation. Results obtained from antibacterial study are represented in Table 4

Antifungal activity:

The standard used in case of antifungal activity is griseofulvin. Compared with the standard compound complexes of Cr(III), Mn(II) and Zn(II) ions show good antifungal activity in few cases. Of all the remaining cases in most of the cases reduced growth is observed which indicated towards presence of moderate activity in these complexes. Ni(II) complex don’t show any antifungal activity against any of the fungal pathogens studied. Results obtained from antifungal activity study are represented in Table 5.

Table 4: Antibacterial activity recorded for all the synthesized mixed ligand complexes

Complex	Symbol used	Zone of inhibition (in mm)
Complex	Symbol used	*Escherichia coli*	*Salmonella typhi*	*Staphylococcus aureus*	*Bacillus subtilis*
C₁₂H₁₂Cl₄CrN₂O	B	Negative	14	19	13
C₁₂H₁₂Cl₃MnN₂O	C	12	16	19	16
C₁₂H₁₂Cl₄FeN₂O	F	14	19	20	19
C₁₂H₁₂Cl₃CoN₂O	G	22	20	30	19
C₁₂H₁₂Cl₃N₂NiO	D	15	15	13	11
C₁₂H₁₂Cl₃CuN₂O	A	17	20	17	30
C₁₂H₁₂Cl₃ZnN₂O	E	17	19	25	20
Penicillin		11	24	36	30
DMSO		Negative	Negative	Negative	Negative

*Negative = No activity

Table 5: Antifungal activity recorded for all the synthesized mixed ligand complexes

Complex	Symbol	*Aspergillus niger*	*Penicillium chrysogenum*	*Fusarium moneliforme*	*Aspergillus flavus*
C₁₂H₁₂Cl₄CrN₂O	B	RG	Negative	RG	RG
C₁₂H₁₂Cl₃MnN₂O	C	RG	Negative	RG	RG
C₁₂H₁₂Cl₄FeN₂O	F	Positive	RG	RG	Positive
C₁₂H₁₂Cl₃CoN₂O	G	RG	RG	RG	RG
C₁₂H₁₂Cl₃N₂NiO	D	Positive	Positive	Positive	Positive
C₁₂H₁₂Cl₃CuN₂O	A	RG	RG	RG	RG
C₁₂H₁₂Cl₃ZnN₂O	E	RG	Negative	Negative	RG
Griseofulvin		Negative	Negative	Negative	Negative
DMSO		Positive	Positive	Positive	Positive

*Negative = No growth (more than 90% reduction in growth means antifungal activity present)

**Positive = Growth observed (means antifungal activity absent)

***RG = reduced growth (More than 50 % and less than 90% reduction in growth)

Figure 2:Antibacterial activity recorded for all the synthesized mixed ligand complexes

Figure 3: Antifungal activity recorded for all the synthesized mixed ligand complexes.

CONCLUSIONS:

Synthesis of a series of seven mixed ligand complexes of transition metal ions such as Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) using 2-aminophenol and 2-Chloroaniline is reported. The synthesized complexes are characterized using techniques such as molar conductance, magnetic susceptibility measurements, IR and electronic spectra. Based on the results obtained a six coordinated octahedral geometry has been proposed for all the synthesized complexes.

ACKNOWLEDGEMENTS:

Corresponding author Dr. D. S. Wankhede is thankful to Director, SAIF, Punjab University, Chandigarh for providing spectral data. Thanks are also to Principal, Yeshwant Mahavidyalaya, Nanded for providing IR spectra and to Principal, NSB College, Nanded for helping in recording antimicrobial properties of synthesized complexes.

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Received on 03.07.2017 Modified on 07.08.2017

Asian J. Research Chem. 2017; 10(5): 639-645.

DOI: 10.5958/0974-4150.2017.00108.0