Introduction : Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate that has gained considerable attention due to its broad spectrum of biological and pharmaceutical activities. As an indole derivative, isatin contains an indole nucleus along with keto and lactam functional groups, making it a highly reactive and flexible scaffold for chemical modification. Its unique structural features allow it to serve as a starting material for the synthesis of a wide variety of biologically active molecules. Over the years, the synthesis and investigation of inorganic compounds containing biologically relevant ligands have been encouraged by the central role of metal ions in numerous biochemical processes. The therapeutic relevance of metal complexes in modern medicine can be traced back to the discovery of the anticancer properties of cisplatin, which remains a cornerstone in chemotherapy. Cisplatin, along with its derivatives carboplatin and oxaliplatin, continues to be among the most effective and widely used anticancer drugs globally.
Among essential transition metals, copper holds a prominent position due to its indispensable role in biological systems. Copper is critical for processes such as photosynthesis, mitochondrial respiration, carbon and nitrogen metabolism, oxidative stress protection, and cell wall biosynthesis. Beyond its physiological functions, copper has historically been employed to control microbial growth in wood preservation, aquaculture, agriculture, and medicine. The multifaceted biological roles of copper are well-documented, particularly its involvement in human diseases, which has been extensively studied from both medicinal-chemical and biochemical perspectives, focusing on the molecular physiology of copper transport. Numerous Cu(II) chelate complexes have demonstrated cytotoxic activity, often through the induction of apoptosis or inhibition of key enzymes. Complexes containing bi-Schiff base ligands have shown notable efficacy in reducing tumor size, delaying metastasis, and improving survival outcomes in preclinical models. Current research interest in copper complexes stems from their potential as antimicrobial, antiviral, anti-inflammatory, antitumor agents, enzyme inhibitors, and chemical nucleases. Several studies have highlighted the antiviral and antibacterial potential of Cu(II) complexes, with findings indicating that copper surfaces can reduce the infectivity of viruses such as influenza A. These complexes are increasingly regarded as promising alternatives to cisplatin due to their DNA-binding capabilities, cytotoxicity, and favorable bioactivity profiles.
Isatin and its derivatives occupy a unique position within the Schiff base family. Simple isatin-based Schiff bases, including acyl, aroyl, and heteroacroyl derivatives, possess additional donor sites such as >C=O and >C=N–, which enhance their chelating flexibility. This versatility allows them to form a wide range of complexes with transition and inner transition metals, attracting significant research interest. The resulting metal–isatin complexes often exhibit enhanced biological activities compared to the free ligand, including anticancer, antiviral, antimicrobial, and enzyme inhibitory effects. The isatin-based Schiff base copper(II) complexes, for instance, are structurally related to the antiviral drug methisazone and exemplify the principles of medicinal inorganic chemistry. Electroanalytical techniques have further enabled the elucidation of reaction mechanisms and interaction pathways of these complexes with biomolecules, particularly DNA, underscoring their therapeutic potential.
The remarkable bioactivity of isatin derivatives has motivated the synthesis of a variety of analogues, which have been screened for diverse biological activities such as anticancer, anti-HIV, anthelmintic, antimycobacterial, anti-inflammatory, antidiabetic, antimicrobial, trypanocidal, and antimalarial effects. Studies involving both free ligands and their metal complexes have employed in vitro assays, including minimum inhibitory concentration (MIC) tests against multiple bacterial and fungal strains, to evaluate antimicrobial efficacy. Insights from these investigations contribute to understanding the molecular mechanisms of isatin derivatives’ interactions with DNA and other biomolecules, facilitating the rational design of novel therapeutic agents.
Nickel, though not universally recognized as essential for higher plants, has demonstrated several beneficial effects on plant growth under specific conditions. In certain microorganisms, including bacteria (e.g., Alcaligenes eutrophus), cyanobacteria (e.g., Oscillatoria), and algae (e.g., Chlorella vulgaris), nickel is required for growth, although its precise biological role remains unclear. In higher plants, nickel is crucial for the optimal growth of specific species, including some pine trees and Ni-accumulator species of Alyssum. Low concentrations of nickel can stimulate seed germination and early growth, suggesting a nuanced role in plant development.
At the biochemical level, nickel is an essential component of the enzyme urease, which has been isolated from leguminous seeds. While urease is primarily associated with urea hydrolysis, evidence suggests it may also participate in the mobilization of stored seed nitrogen through compounds such as ureides or arginine during early seedling growth. Consequently, nickel may play a pivotal role in nitrogen metabolism and seedling development in higher plants, highlighting its significance in both plant and microbial systems.
EXPERIMENTAL
All chemicals used were of analytical reagent (AR) grade, obtained from Aldrich and SD Fine Chemicals, and were used as received without further purification. Elemental (CHN) analysis was performed on a Perkin-Elmer analyzer. IR spectra were recorded on a Perkin-Elmer FTIR using KBr discs. ^1H NMR spectra were obtained on a Bruker 400 MHz instrument using DMSO-d6 as solvent. Powder X-ray diffraction (PXRD) measurements were carried out using a PANalytical Empyrean diffractometer.
Preparation of Schiff Base Ligands
Schiff base ligands were synthesized by reacting 5-bromo isatin (2.26 g, 10 mmol) with 4-methyl-2-nitroaniline (1.57 g, 10 mmol) in a 1:1 molar ratio in ethanol, with a few drops of acetic acid as catalyst. The reaction mixture was refluxed for 2 hours. After evaporation of excess solvent, the solid product was collected by filtration, washed with a small amount of ethanol, recrystallized from ethanol, and dried to yield ligand L1 (85%, m.p. 145 °C).
A similar procedure was used to prepare another Schiff base ligand, L2, by reacting salicylaldehyde (1 mL, 10 mmol) with 4-methyl-2-nitroaniline (1.57 g, 10 mmol) under identical conditions (yield 90%, m.p. 86 °C).
Preparation of Metal Acetylacetonates
Nickel acetylacetonate (Ni(acac)₂): Nickel chloride (1.2 g, 0.02 mol) was dissolved in 50 mL of distilled water. Separately, acetylacetone (10 mL, 10 g, 0.10 mol) was mixed with 50 mL of 10% NaOH solution and stirred until fully dissolved. The solution was cooled in an ice bath and filtered, then added slowly with stirring to the nickel chloride solution. Upon cooling, green Ni(acac)₂ crystals formed, which were collected by filtration, washed with cold water, and dried (yield 65%, m.p. 220 °C).
Copper acetylacetonate (Cu(acac)₂): Prepared similarly using copper sulfate pentahydrate (1.7 g, 0.02 mol), yielding a blue powder (62%, m.p. 280 °C).
Preparation of Schiff Base Mixed-Ligand Complexes
Nickel complexes: To a dry ethanolic solution of L1 (3.9 g, 0.01 mol), Ni(acac)₂ (4.87 g, 0.01 mol) dissolved in 20 mL of dry ethanol was added dropwise under constant stirring. The mixture was refluxed for 4 hours. The resulting dark green precipitate was filtered, washed with ethanol, and dried over fused CaCl₂ (yield 75%, m.p. 187 °C).
L2–Ni complexes were prepared similarly, yielding orange crystals (75%, m.p. ~120 °C).
Copper complexes: L1 (3.9 g, 0.01 mol) was dissolved in methanol, and Cu(acac)₂ (2.65 g, 0.01 mol) in 20 mL ethanol was added. The mixture was refluxed for 4 hours. The dark blue precipitate was collected by suction filtration, washed with a small amount of methanol, and dried over fused CaCl₂ (yield 65%, m.p. 205 °C).
L2–Cu complexes were prepared under the same conditions, producing dark orange crystals (70%, m.p. ~200 °C).
Tentative Structures: The tentative structures of the Schiff base ligands (L1 and L2) and their corresponding mixed-ligand metal complexes were proposed based on spectroscopic and elemental analyses.
(2Z)-5-bromo-3-[(4-methyl-2-nitrophyl)imino]-1,2-dihydro-3H-indol-3-one
Biological Activities of the Ligands and Metal Complexes
The Schiff base ligands and their transition metal mixed-ligand complexes were evaluated for their biological activities against common microbes, including Escherichia coli and Staphylococcus aureus. Additionally, their antifungal potential was assessed against Aspergillus niger and Candida albicans. The observed activities were compared with standard drugs and controls, and the results are discussed in detail.
Antibacterial Activity Assay
The antibacterial activities of all isolated Schiff base ligands and their mixed-ligand complexes were determined using the agar well diffusion method. Bacterial cultures were subcultured in nutrient broth for in vitro testing, prepared by dissolving 24 g of nutrient broth in distilled water and autoclaving the mixture at 120 °C for 15 minutes.
For the assay, stock solutions were prepared by dissolving 5 mg of each compound in 9 mL of DMSO to achieve a concentration of 100 μg/mL. Additionally, 1.15 mL of liquid nutrient agar was separately prepared for the activation of the target microorganisms, and 1 mL of nutrient broth was used for the antibacterial testing.
Inoculation was carried out using micropipettes with sterilized tips: 100 μL of the activated microbial strain was evenly spread over the surface of the agar plates. Two wells, each 8 mm in diameter, were then created in the media. The plates were incubated at 37 °C for 48 hours.
The antibacterial activity was determined by measuring the diameter of the zone of inhibition, expressed in millimeters (mm). The results were compared against a standard antibiotic (gentamicin) and a control (DMSO).
Antifungal activity assay17
For in vitro antifungal activity the moulds were grown on sabouraud dextrose agar (SDA) at 250C for 7 days and determined by using agar well diffusion method and fungal growth were subcultured on nutrient broth for their in vitro testing. 15 mL of molten SDA (450C) was added to 100 µL volume of each compound having concentration of 100 µL/mL in the DMSO and pored into a sterile Petri plate. The solid appeared at the petri plate which poisoned agar plates were inoculated at the centre with fungal plugs (8 mm) obtained from activity growing colony and incubated at 250C for 7 days. Diameter of the fungal colonies was measured and expressed as present mycelial inhibition.
Table 1: in vitro antibacterial and antifungal activities of the Mixed ligand metal complexes
| Sl. No. | Compounds | Concentration | Bacteria | Fungus | ||
| E.coli | S.aureus | A.niger | C.albicans | |||
| 1. | L1 | 100 mg | 12 mm | 13 mm | 13 mm | 14 mm |
| 2. | L1 Ni(acac) | 100 mg | 18 mm | 19 mm | 19 mm | 20 mm |
| 3. | L1 Cu(acac) | 100 mg | 19 mm | 18 mm | 18 mm | 18 mm |
| 4. | L2 | 100 mg | 11 mm | 14 mm | 13 mm | 13 mm |
| 5. | L2 Ni(acac) | 100 mg | 16 mm | 17 mm | 17 mm | 18 mm |
| 6. | L2 Cu(acac) | 100 mg | 18 mm | 18 mm | 19 mm | 19 mm |
| 7. | Control (DMSO) | 100 mg | 8 mm | 8 mm | 8 mm | 8 mm |
| 8. | Standard (Gentamycene) | 100 mg | 20 mm | 20 mm | — | — |
| 9. | Standard (Nystatine) | 100 mg | — | — | 20 mm | 20 mm |
RESULTS AND DISCUSSIONS
Schiff bases and their mixed-ligand transition metal complexes were synthesized using isatin and 4-methyl-2-nitroaniline as ligands with Ni(acac)₂ and Cu(acac)₂. The resulting compounds were characterized using various spectroscopic techniques, including IR, UV–Vis, ¹H NMR, and TGA/DTA, as well as through analytical methods. The spectroscopic data indicated that the complexes are hexacoordinated, with coordination occurring via the azomethine nitrogen, the carbonyl group of isatin, and the hydroxyl oxygen of 4-methyl-2-nitroaniline. In this reaction, 4-methyl-2-nitroaniline acts as a bidentate ligand, combining with the metal acetylacetonates to form stable mixed-ligand complexes..
CONCLUSION
Schiff base ligands and their mixed-ligand transition metal complexes were successfully synthesized and characterized using various analytical and spectroscopic methods. All the synthesized compounds demonstrated notable antimicrobial activity against both bacterial and fungal strains. Comparative analysis of the antimicrobial data indicated that the metal complexes possess higher activity than the corresponding free ligands. Additionally, ligands containing a nitro group exhibited superior antimicrobial effects compared to those with a bromo group. Therefore, these compounds show promise as potential pharmacophores for the development of new antimicrobial agents.
REFERENCES
1. J Devi; N Batara, Spectrochem Acta A Mol Biomol Spectrosc, 2015, 135, 710-719.
2. J Devi; N Batara, R Mallhotra Spectrochem Acta A Mol Biomol Spectrosc, 2012, 97, 397-405.
3. Jai Devi A, Nisha Batra and Jyothi Yadav, JCPR, 2018, 10(5), 121-125.
4. S. Puig and D.J. Thiele, “Molecular mechanisms of Copper uptake and distribution,” Currrent Opinion in Chemical Biology, vol.6, no.2. pp.171-180, 2002.
5. L. Tripathi, P. Kumar and A.K. Singhai, Role of Chelates in treatment of cancer. Indian Journal of Cancer, vol.44, pp. 62-71, 2007.
6. Jane.E.Weder, Carolyn.T.Dillona, Trevor.W.Hambleya, Brendan.J.Kennedya, Peter.A.Laya, J. Ray Biffinb, Hubert.L, Regtopb and Neal M.Davies, Copper complexes of non-stereoidal anti-inflammatory drus an opportunity yet to be realized, Coordination chemistry Reviews vol. 232, pp 95-126, 2002.
7. V K Sharma, S Srivastava, A Srivastava, Polish J Chem, 2006, 80, 387-396.
8. P Selvam, M Chandramohan, E De Clercq, M Witvrouw, C Pannecouque, Euro J Pharm Sci, 2001,
14, 313-316.
9. S N Pandeya, S Smitha, M Jyothi, S K Sridhar, Acta Pharma, 2005, 55, 27-46
10. A.Moustatih, M.M.L.Fiallo, A.Garnier-Suillerot, J. Med. Chem. 1989, 32, 336.
11. L.Muslin, W.Roth and H.Erlenmeyer, Helv.Chim.Acta. 1953, 36, 886.
12. W.M.Farror, H.Calvin and F.H.Schucler, J.Am.Pharm.Assoc. 1954, 43, 370.
13. Shin, Ichiro, Takase, J.Chem.Soc. (Japan), 1948, 61, 157
14. H.Köpf, Eur.J.Med.Chem., Chimica Therapeutica. 1981, 16B, 275.
15. G.P.Pez and J.N.Armor, Adv in Organometallic Chem. 1981, 19, 1.
16. C.S.Bajgur, W.R.Tikkanen and J.L.Petersen, Inorg.Chem. 1985, 24, 2539.
17. M.V.Gururaj, M.Phil Thesis, Gulbarga University, 1993.
18. Indian Pharmacopeia, Government of India, New Delhi, 1985, Appendix, 4, 90.