Synthesis, Characterization and in Vitro Antimicrobial Studies of Ternary Mn(II) Complexes with Isatinphenylhydrazone, Glycine and
8-hydroxyquinoline

Introduction

According to present scenario, ternary complexes¹ have great interest of researchers owing to their importance in biological, medicinal, pharmaceutical and agricultural fields. IPH is a Schiff base², derivative of isatin and phenylhydrazine and possesses a variety of biological³ potency such as; antibacterial efficiency against some gram-positive bacteria like B. subtilis and gram-negative bacteria like P. aeruginosa, E. coli etc. and promising activity against fungal strain like C. albicans and anti-HIV⁴ activities. Hence, IPH is very useful in prospect to synthesize biologically active mixed ligand complexes with selective amino acids⁵ like- glycine⁶, histidine, phenylalanine etc. and 8-hydroxyquinoline⁷. Such type of mixed ligand complexes^8-9 possess broad spectrum of pharmaceutical, medicinal, and biological activities such as- antimicrobial^10-12, anti-tumour^13-14, antioxidant¹⁵,

antimalarial¹⁶, and anti-inflammatory¹⁷ etc. These Schiff base derivatives and ternary complexes act as potent agents in drug^18-19 design process. Compounds from transition metal ions and amino acids can be applied as models to study the pharmacy dynamic effects²⁰ from drugs or with increasing the biocompatibility as well to reduce poisonous effects. 8-hydroxy quinoline is an important compound which has the ability to coordinate with the central metal ion and form five membered chelate ring that increases the stability of the complexes²¹, as well as the biological activity.

Experimental

Materials and methodology

In our experimental work, all solvents and chemicals used in their original form, purchased from Sigma Aldrich and TCI chemicals. Infrared spectra (FTIR) for the complexes were recorded employing a model PerkinElmer FT-IR (4000-400 cm^-1) spectrophotometer. Electronic spectra of complexes were recorded with PerkinElmer Lambda UV 750 ultraviolet-visible Spectrometer in ethanol within a wavelength range 200 – 950 nm. ¹H Nuclear Magnetic Resonance spectra of both prepared complexes were noted in DMSO-d₆ and CDCl₃ solutions using an internal standard (TMS) with a Jeol Resonance ECS-400 Spectrometer at 400 MHz. Magnetic moment measurements of complexes were made on the Gouy balance having model no: HO-ED-EM-08. For conductance measurements, compounds were dissolved in DMSO and measured using Systronics Direct Reading Conductivity Meter-304 (with cell constant 1.0 cm^-1).  Purification test of prepared compounds were monitored by TLC on Silica Gel-G plates. The plates were developed selecting suitable solvent and visualisation in UV-chamber.

Methodology

Synthesis of Ligand L(IPH)

Isatin-3-phenylhydrazone (IPH) was synthesized according to the earlier proposed research process²². This ligand was used for further complexation with metal ion.

Synthesis of complex-I [Mn(L)(Gly)(Cl)(H₂O)]

The ternary metal complex-I of Mn (II) was prepared by the mixing 5ml of ethanolic solution of IPH (10mmol; 0.0237 g), 5ml of aqueous solution of glycine (10mmol, 0.0075g) and 5ml of aqueous solution of metal salt MnCl₂.4H₂O (10mmol; 0.0197g) with constant stirring. Yellowish precipitate formed was further refluxed for ~3-4 hrs at 55^oC for reaction completion. Purification test of synthesized compound was checked using TLC on Silica Gel-G plates in appropriate developing solvent (benzene: chloroform in 1:4 ratio). Coloured precipitate was obtained that was further filtered, washed, recrystallized using ethanol, dried properly in vacuum and weighed. Dark brown coloured compound formed with 48.5% yield and M.P. 245.5^oC. (Scheme I)

Synthesis of ternary complex-II [Mn(L)(Q)(Cl)(H₂O)]

Similar method was employed as above. Equimolar solutions of IPH, 8-HQ and MnCl₂.4H₂O (5ml of each) were allowed to mix with continuous stirring. No precipitation was obtained, this reaction content was allowed to reflux for ~4-5 hrs at constant temperature (55⁰C). Reaction accomplishment was monitored by TLC using developing solvent benzene: CHCl₃ in 1:4 ratio, then the resultant solution was cooled and set overnight at room temperature. Coloured solid product was obtained that was further filtered and thoroughly washed and recrystallized using alcohol and completely dried in vacuum and weighed. Brownish green coloured compound formed with 45.4% yield and M.P. 254.4^oC. (Scheme II)

Plausible reaction scheme for ternary Complex-I and II

Scheme 1: Proposed synthesis of Complex-I Click here to View Scheme

Scheme 2: Plausible synthesis of Complex-II Click here to View scheme

Antimicrobial study

Antibacterial Evaluation-

The in vitro determination of antibacterial activityof both the complexes was done against two gram positive bacteria (B. subtilis and S. aureus) and two gram negative (E. coli and P. aeruginosa) by agar well diffusion method²³. The test compounds (20-80 μg/ml) were injected in the well. For calculation of zone of inhibition, subtraction of control zones from the test zones is made and finally the measurement of resultant zone diameter done with antibiotic zone reader (in mm). For antimicrobial spectrum, zone of inhibition diameters of compounds was compared with the standard i.e. Streptomycin.

Antifungal Evaluation-

Antifungal activity determination of the complexes was done with agar well diffusion method²⁴ (Cup plate method) against three fungal strains, T. reesei, A. niger and C. albicans.  Subculture of yeasts and saprophytic fungi were done onto dextrose agar, after then incubation for 24 hours at 37°C and  2 to 5 days at 25°C respectively.  Fungal spores suspensions were formed in sterile PBS and adjustment of concentration upto 106 cells/ml. Plates were incubated for 48 h at 37°C then after determination of bioactivities was performed by measurement of diameter of inhibition zone (in mm) and compared with standard i.e. Ketoconazole.

Results and Discussions

Physicochemical and analytical data

reveal that both the complexes are coloured, stable and soluble in ethanol, DMSO, DMF, and CHCl₃. The metal complexes are characterized by physicochemical, elemental analysis, molar conductivities, magnetic moment measurements and spectral techniques (FTIR, UV, ¹HNMR etc.). For both complexes, analytical data are in good agreement with calculated values (Table-1 and 2). In synthesized complexes, stoichiometry revealed 1:1:1 metal ligand ratio.

Table 1: Physicochemical analysis of synthesized complexes

Compound Empirical formula	Colour	M.P.	Mol. Weight found (cal.)	% Elemental analysis found (cal.)
				C	H	N	O	Cl	Mn
1. Complex-I C16H17N4O4ClMn	Dark Brown	245.5oC	417.8 (418.4)	45.86 (45.88)	4.05 (4.06)	13.40 (13.38)	15.27 (15.29)	8.50 (8.48)	13.10 (13.12)
2. Complex-II C23H19N4O3ClMn	Brownish green	254.4oC	488.6 (489.1)	56.40 (56.43)	3.90 (3.88)	11.42 (11.44)	9.80 (9.78)	7.27 (7.25)	11.20 (11.22)

 

Table 2: Molar conductivities and magnetic moment of compounds

Compound	Molar cond. ohm-1 cm2 mol-1 appr.	µeff BM appr.
[Mn(L)(Gly)(Cl)(H2O)]	11.9	5.92
[Mn(L)(Q)(Cl)(H2O)]	12.4	5.91

 

Spectral characterization

Spectral characterization of both the complexes was done employing different techniques.

FTIR spectra

FTIR spectra (Table-3) give important information regarding all the functional groups. The –OH group of carboxyl group in free glycine appears at ~ 3100 cm^-1 disappears in complex-I specify that COOH group getting deprotonated during complexation with metal ion and the broad peak at 3124 cm^-1 recommend the water molecule presence in complex-I. A broad peak for – NH₂ appears at 2900 cm^-1  in glycine shift to 3028 cm^-1 in complex-I. When spectrum of free ligand HQ is observed, the peak seen at 3182 cm^-1  assigned for υ(OH) stretching frequency disappears due to deprotonation of HQ to form anion in complex-II and a broad peak
at 3434 cm-1 indicating presence of water molecule. Azomethine group (C=N) shows a sharp peak at 1597 cm^-1 and 1616 cm^-1 in complex-I and II respectively, lower than that of in free ligand appearing at 1622 cm^-1 support complexation of N of azomethine group with metal ion. Formation of ternary complexes was further evidenced by existence of four additional bands 900 cm^-1, 744 cm^-1, 676 cm^-1, 503 cm^-1 for complex-I and 788 cm^-1, 745 cm^-1, 688 cm^-1, 520 cm^-1 for complex-II assigned to M-Cl, M–OH₂, M-O and M-N bonds, respectively. (Fig 1,2)

Table 3: IR spectral vibrations υ (cm^-1) for ligands and complexes

Compound	-OH	NH2	C=N	M-Cl	M-OH2	M-O	M-N
Glycine	3093	2900	–	–	–	–	–
8-HQ	3182	–	–	–	–	–	–
IPH (NH)	3438	–	1622	–	–	–	–
[Mn(L)(Gly)(Cl)(H2O)]	3124	3028	1597	900	744	676	503
[Mn(L)(Q)(Cl)(H2O)]	3434	–	1616	788	745	688	520

Figure 1: FTIR spectrum of Complex-I [Mn(L)(Gly)(Cl)(H2O)] Click here to View figure

Figure 2: FTIR spectrum of Complex-II [Mn(L)(Q)(Cl)(H2O)] Click here to View figure

In UV-Visible spectra

λ_max (nm) and ε_max values recorded in ethanol for both ternary complexes (Table-4). Electronic spectra comprise of two absorption maxima at 295nm assigning π-π∗ transition and 375nm assigned for n-π∗ transition for isatinphenylhydrazone³. However, spectra of both synthesized complexes exhibit π-π∗ transition at 300nm, 301nm and n-π∗ transition at 404nm, 406nm respectively, showing bathochromic shift and support metal complex formation. (Fig 3,4)

Table 4: UV Spectral Bands of IPH and Complexes

Compound	λmax in nm (εmax)
	π-π∗	n-π∗
IPH	295 (0.346)	375(0.260)
[Mn(L)(Gly)(Cl)(H2O)]	300 (0.415)	404 (0.327)
[Mn(L)(Q)(Cl)(H2O)]	301 (0.585)	406 (0.548)

 

Figure 3: UV spectrum of Complex-I [Mn(L)(Gly)(Cl)(H2O)] Click here to View figure

Figure 4: UV spectrum of Complex-II [Mn(L)(Q)(Cl)(H2O)] Click here to View figure

¹H-NMR spectrum

¹H-NMR spectrum of complex-I recorded in DMSO-d₆and that ofcomplex-II recorded inCDCl₃. Results of¹H-NMR spectraare tabulated (Table-5) and the chemical shift values expressed in δ (ppm), it can be concluded that each complex shows a broad singlet for amide hydrogen (CO-NH) at δ = 11.1 ppm and 12.4 ppm for complex-I and II_, respectively. Disappearance of a singlet at   δ =12 ppm in complex-I shows that COOH group of glycine present as COO- with loss of hydrogen. This group can show amido-iminol tautomerism. Aromatic hydrogens appeared as multiplet within the range of δ = 6.9-7.5 and 6.6-7.7 ppm. The NH₂ group of glycine obtained at δ =3.8 ppm (broad, singlet). H₂O and NH₂ peak collapse in complex- I, hence, it is getting broad. While in complex-II, H₂O peak observed at δ = 4.0ppm, this supports that both complexes fulfil their secondary valency with a water molecule. The DMSO signal²⁵ also appeared at δ = 2.5 ppm in spectra. (Fig 5,6)

Table 5: ¹H NMR spectral values in δ (ppm) of synthesized complexes

Compound	CO-NH	Ar-H	NH2	HO-H
[Mn(L)(Gly)(Cl)(H2O)]	11.1	6.9-7.5 (multiplet)	3.8 ( broad, singlet)	3.9
[Mn(L)(Q)(Cl)(H2O)]	12.4	6.6-7.7 (multiplet)	–	4.0

 

Figure 5: 1H NMR spectrum of Complex-I [Mn(L)(Gly)(Cl)(H2O)] Click here to View figure

Figure 6: 1H NMR spectrum of Complex-II [Mn(L)(Q)(Cl)(H2O)] Click here to View figure

Biological Assay

The in vitro biological evaluation of above synthesized compounds carried out against selective strains of bacteria and fungi using agar well diffusion method. Complex-I screened against bacteria- E. coli (G^–) and B. subtilis (G⁺) whether Complex-II screened against S. aureus (G⁺) and P. aeruginosa (G^–) and results compared with Streptomycin. For antifungal studies, three fungal pathogens- T. reesei, A. niger and C. albicans were taken for evaluation. Results showed that both the complexes have significant antimicrobial activity and more activity obtained for complexes than the free schiff base ligand. Results revealed that complex-II have more zone of inhibition than the complex-I and also found more effective for fungal strain rather than bacterial strain. Complex-I is almost inactive against B. subtilis. The results of antimicrobial study are tabulated in Table-6.(Fig 7)

Table 6: Antimicrobial evaluation of synthesised compounds

Compound	Conc. (μg/mL)	Zone of inhibition (mm)
		Antibacterial Activity				Antifungal Activity
		B. subtilis	E. coli	S. aureus	P. aeruginosa	T. reesei	A. niger	C. Albicans
IPH	500 mg/cm3	45	100	140	240	NT	NT	160
Complex-I	20 40 60 80	NS	0 0 0 9	NT	NT	0 0 0 10	NT	0 9 11 13
Complex-II	20 40 60 80	NT	NT	10 12 15 22	11 13 15 16	NT	10 17 21 24	12 15 18 19
Standard	80	51	46	35	42	28	30	35

NS- Not Seen, NT- Not Tested

Figure 7: Antimicrobial activity at different conc. of complex-I (a) and complex-II (b) show inhibition zone against selected bacteria and fungi Click here to View figure

Conclusion

On the basis of all spectral and analytical evidences, distorted Octahedral geometry proposed for both synthesized complexes of Mn(II). Conductivity measurement reveals the non-electrolytic behaviour of both complexes and magnetic moment measurements support the paramagnetic nature of the synthesized complexes of Mn(II) with five unpaired electrons. In complex-I, ligand L (IPH) coordinates with metal with two donor sites i.e. N of azomethine (C=N) group and O of carbonyl group of isatin ring, whereas glycine acts as monoanionic bidentate ligand after deprotonation, coordinate through N of NH₂ group and O^– of carboxylategroup. Similarly, in complex-II the secondary ligand is deprotonated hydroxyquinoline which coordinates through N of quinoline ring and O^– of hydroxyl group. Remaining valances of Mn(II) are satisfied with chloride ion and one water molecule confirmed by presence of corresponding absorption band in FTIR spectra. Antimicrobial results indicate both the complexes to be more effective against fungal pathogens but less active for bacterial strains. It can be concluded that complex-I exhibits more zone of inhibition against C. albicans as compared to others, However, complex-II shows significant antifungal activity against A. niger. These results confirm both the complexes to possess more penetration effect through the cell wall of microbes. Further, other complexes of Mn(II) are to be synthesized and studied against other microbial strains to recognize the pharmacophore to develop and explore for drug designing with minimal side effects on physiological and biological systems.

Acknowledgment

Authors are obliged to Department of Chemistry, UOR, Jaipur for providing all the essential and required lab facilities, equipment and chemicals required. Further, authors are also grateful to MNIT, Jaipur for providing spectral analysis facilities and Seminal Applied Science Pvt. Ltd., Jaipur for antimicrobial studies.

Conflict of interest

No conflict of interest is declared by authors.

Funding Sources

There are no Funding source for this aritcle.

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