One new series of Cu(II), Co(II), and Ni(II) Schiff base complexes was prepared through the condensation reaction between 1-phenylindoline-2,3-dione with isonicotinohydrazide followed by metalation, respectively. The Schiff base ligand(L),
Isatin(1-H indole-2,3-dione) Schiff bases are significant in therapeutic and pharmaceutical compounds in the field [
All chemicals were purchased from Sigma-Aldrich, Merck-(A.R) and used as received without further purification. The isatin Schiff base was prepared according to the literature procedure [
C, H, and N analyses of free Schiff base ligands and their metal complexes were performed in C, H, and N analyzer Elementar Vario EL III. Metal contents were analyzed by the standard procedures. Hand-Held Meter LF330 was used to measure the molar conductance of free Schiff base ligands and metal complexes in DMSO (
DNA-binding experiments were performed by UV-visible spectroscopy in Tris-HCl/NaCl buffer (5 mmol L−1 Tris–HCl/50 mmol L−1 NaCl buffer, pH 7.2) and used DMSO (10%) solution of metal complexes. The concentration of CT-DNA was determined from the absorption intensity at 260 nm with a value of 6600 (mol L−1)−1 cm−1. Absorption titration experiments were made using different concentrations of CT-DNA, while keeping the complex concentration constant. Correction was made for the absorbance of the CT-DNA itself. Samples were equilibrated before recording each spectrum. For metal complexes, the intrinsic binding constant (
Circular dichroism spectra were registered in a JASCO J-810 spectropolarimeter, using a quartz cuvette of 0.2 cm path length, at room temperature, in the range 230–330 nm. The initial experimental DNA concentration was 800
Cyclic voltammetry analysis was carried out in a Bio-Analytical System (BAS) model CV-50W electrochemical analyzer. All voltammetric experiments were performed in a single compartment cell of volume 10–15 mL containing a three electrode system comprising a carbon working electrode, Pt-wire as auxiliary electrode, and reference electrode as an Ag/AgCl.
pUC19 DNA at pH 7.5 in Tris-HCL buffered solution was used to perform Agarose gel electrophoresis. Oxidative cleavage of DNA was examined by keeping the concentration of the 30
Cytotoxicity studies were carried out using human gastric cancer cell line (designated AGS) which were obtained from National Centre for Cell Science (NCCS), Pune, India. Cell viability was carried out using the MTT assay method. The AGS cells were grown in Dulbecco’s Modified Eagle’s Medium (DME) and Ham’s F-12 Nutrient Mixture containing 10% fetal bovine serum (FBS), 1% Glutamine, 1% antibiotic, 1% sodium bicarbonate, and 1% nonessential amino acids. For screening experiment, AGS cells were seeded into 96-well plates in 100
1-Phenyl isatin (1 mMol) and isonicotinohydrazide (1 mMol) were dissolved in 50 mL of absolute ethanol; three drops of glacial acetic acid were added and the resulting solution was refluxed for 5 h. The results compounds were precipitated upon cooling to room temperature, isolated by filtration, and recrystallized from EtOH. Yellow colored crystalline compounds were obtained (Scheme
Synthesis of Schiff base ligand.
The metal(II) complexes in this study were prepared by mixing of 1 mMol of corresponding metal(II) chloride in ethanol with 2 mM of the Schiff base in the molar ratio 1 : 2. The reaction mixture was refluxed at 60°C for 4 hrs [
Synthesis of Schiff base complexes.
The Schiff base ligand (L) and their complexes with Cu(II), Co(II), and Ni(II) were found to be air stable, amorphous, moisture free, and soluble only in DMF and DMSO solvents and kept in vacuum desiccators under nitrogen atmosphere and used for chemical and biological studies. The experimental results are discussed under various subheadings as detailed below.
Physicochemical characteristics such as melting point (m.p.), color, yield, elemental analysis, and conductivity of the ligand(L) and complexes were determined and the data shown in Table
Composition and physical characteristics of ligand and their complexes.
Ligand/ |
Molecular |
Color | Found (calculated) % |
M.P |
Yield |
|
|||
---|---|---|---|---|---|---|---|---|---|
M | C | H | N | ||||||
L | C20H14N4O2 | Crystalline yellow | 70.01 |
3.97 |
16.52 |
180 | 95 | — | |
L–Cu | C41H31N8O4Cl2Cu | Green | 7.51 |
59.37 |
3.71 |
13.11 |
>300 | 83 | 22.000 |
L–Co | C41H31N8O4Cl2Co | Dark green | 6.58 |
59.32 |
3.63 |
13.47 |
>285 | 80 | 34.40 |
L–Ni | C41H31N8O4Cl2Ni | Yellow | 7.31 |
59.35 |
3.68 |
13.56 |
>285 | 80 | 28.50 |
The 1H-NMR (300 MHz, CDCl3,
(a) 1H NMR spectrum and (b) 13C NMR of ligand.
Mass spectrometry (MS) an analytical technique that measures the mass-to-charge ratio of charged particles. ESI mass spectra for ligand and complexes were recorded and are shown in Figure
Mass spectrum of ligand (a) and copper complex (b).
In order to study the bonding mode of ligand moiety to metal ion in the complexes, IR spectra of the free ligand were compared with those of the metal complexes. The FT-IR spectral data are summarized in Table
Infrared spectral data for the free ligand and their complexes in KBr disc (cm−1).
Compounds | C=N |
C=O |
C=O |
NH | M–O | M–N |
---|---|---|---|---|---|---|
L | 1602 | 1695 | 1685 | 3269 | — | — |
L–Cu | 1612 | 1691 | 1673 | 3265 | 601 | 453 |
L–Co | 1612 | 1693 | 1676 | 3263 | 572 | 447 |
L–Ni | 1610 | 1693 | 1674 | 3268 | 574 | 449 |
The electronic spectra of the ligand and its Cu(II), Co(II), and Ni(II) complexes were recorded in DMSO and their probable assignments are given in Table
Electronic spectral parameters and magnetic moment with suggested geometry of the complexes.
Compound |
|
|
LMCT | d-d band | Assignment | Suggested structure |
|
---|---|---|---|---|---|---|---|
L | 36764 | 29239 | |||||
L–Cu | 35971 | 29069 | 22026 | 12345 | 2B1g→2B2g, Eg | Distorted octahedral | 4.52 |
L–Co | 36231 | 29068 | — | 16339, 14836 | 4T1g(F)→4T1g(P) | Octahedral | 4.13 |
L–Ni | 36496 | 29069 | 22132 | — | 3A2g→3T1g (F) | Octahedral | 3.91 |
The X-band EPR spectrum of the copper(II) complex was recorded in the solid state at room temperature and in DMSO solvents at liquid nitrogen temperature using the DPPH radical as the
EPR spectrum of copper complex at 77 K.
A cyclic voltammogram of Cu(II) complex presented in Table
Electrochemical parameters for Cu(II), Co(II), and Ni(II) complexes.
Compound | Redox couple | Epa |
Epc |
|
Ipa/Ipc |
---|---|---|---|---|---|
L–Cu | Cu(II)/Cu(I) | −214 | −103 | 111 | 1.03 |
L–Co | Co(II)/Co(I) | 790 | 610 | 180 | 0.82 |
L–Ni | Ni(II)/Ni(I) | 521 | 384 | 137 | 0.91 |
CV spectrum of Cu(II) complex.
DNA is a molecule of great biological significance and controls the structure and function of cells [
The change of the UV spectra of complexes in the presence of different concentrations of DNA was studied. Hypochromism and red shift in the UV absorption spectra were observed upon addition of DNA increasing concentrations to the complexes solution in the absorption intensity region 275–280 nm and 344–346 nm. These effects are particularly pronounced for intercalators. In the case of groove binders wavelength shift is usually correlated with a conformational change on binding or complex formation [
In particular,
Absorption properties of metal (II) complexes with CT-DNA.
Complexes |
|
|
Hypochromicity |
|
---|---|---|---|---|
L–Cu(II) | 275, 345 | 3 | 59.05, 65.45 | 10.50 |
L–Co(II) | 280, 346 | 5 | 37.53, 42.18 | 5.88 |
L–Ni(II) | 275, 344 | 2 | 37.53, 58.48 | 6.81 |
Absorption spectra of (a) Cu(II) and (b) Co(II) complexes in the absence and in the presence of the CT-DNA. [complex] = 30
CD spectra is a useful technique in diagnosing changes in DNA morphology during drug–DNA interactions, since CD signals are quite sensitive to the mode of DNA interactions with small molecules [
Circular dichroism spectra of DNA (80
Electrochemical investigations of metal-DNA interactions provide a useful complement to spectroscopic methods. Cyclic voltammogram of copper complex in the presence of CT-DNA in various concentrations is shown in Figure
Cyclic voltammogram of copper(II) complex in the absence and presence of increasing amounts CT-DNA at room temperature in DMSO: buffer (1 : 2) mixture (pH 7.2) (scan rate 0.1 Vs−1).
The ability of Cu(II), Co(II), and Ni(II) complexes to perform DNA cleavage was monitored by agarose gel electrophoresis with the pUC19 plasmid DNA. The experimental results were shown in Figure
Cleavage of super coiled pUC19 (10
The cytotoxicity assay for the new complexes was assessed using the method of MTT reduction. The market reference Mitomycin-C was used as a positive control. All the ligands and complexes were found to be cytotoxic to liver cancer cell line (HepG2). The IC50 values (50% inhibition of cell growth for 48 h) for complexes Cu, Co, and Ni are 5
Treatment of complexes that exert an antiproliferative effect on liver cancer cell line. HepG2 cells were treated with complexes (Cu, Co, and Ni) for 48 h. Control received appropriate carriers. Cell viability was assessed by MTT cell proliferation assay.
One of the most important goals of pharmacological research is the search for new molecular structures which exhibit effective antitumor activities. This has driven inorganic and organometallic chemists to look for new metal compounds with good activities, preferably against tumors that are responsible for high cancer mortality. In this study, new series of Schiff base (L) and its complexes Cu(II), Co(II), and Ni(II) showed octahedral geometry. The binding behaviors of the complexes toward CT-DNA were investigated by absorption spectroscopy, CD, and CV techniques. In conclusion competitive binding of complexes for DNA indicated that complexes could interact as a groove binder. It should be noted that the observed intrinsic binding constant (5.88–10.50 × 104 M−1) is comparable to other groove binders as well and complexes Cu and Ni have stronger binding affinity than Co. The complexes bind to super coiled plasmid pUC19 DNA and display efficient hydrolytic cleavage and are a specific groove binder. The cytotoxic studies showed that the complexes Cu and Ni exhibit good cytotoxic activity against AGS cell line. Furthermore, these complexes have potential practical applications to formulate into an efficient drug against cancer.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors sincerely acknowledge the financial support received from UGC-JRF (BSR), New Delhi. The authors express their sincere thanks to Dr. Kumaresan, Department of Genetics, School of Biological Sciences, MKU, for providing necessary research facilities.