Novel Schiff Base Transition Metal Complexes: Synthesis and In Vitro Bioassay

Introduction: The chemistry of Schiff base ligands and their metal complexes has grown tremendously, covering a wide range of organometallic compounds as well as diverse aspects of bioinorganic chemistry [4]. Schiff base ligands are often referred to as “privileged ligands” because they are typically synthesized through the condensation of aldehydes with primary amines [3]. These ligands can coordinate with a variety of metals, stabilizing them in multiple oxidation states. In addition, they find applications as pigments and dyes, catalysts, intermediates in organic synthesis, and polymer stabilizers [5,9]. Numerous Schiff bases have also demonstrated a broad spectrum of biological and chemical properties. Building on these findings, and as part of our ongoing research into the coordination chemistry of multidentate ligands [19], this study further explores their potential.

MATERIALS AND PHYSICAL MEASUREMENTS

Materials and Instrumentation
Cardanol was obtained from M/S Satya Cashew, Chennai, India. Formaldehyde (37% solution), hydrochloric acid, epichlorohydrin, glutamic acid, sodium hydroxide, and other chemicals used were of GR/AR grade quality from Merck Chemicals. All solvents were purified using standard methods. Microanalytical data (C, H, N) were recorded on a Perkin Elmer 2400 instrument. IR spectra were obtained using a PE IR Spectrum Instrument, Model System 2000. ^1H NMR spectra were recorded on an AMX–300 MHz FT NMR Spectrometer. Conductivity measurements were carried out using a Systronics–305 conductivity meter. Electronic spectra of the ligands and their complexes were measured using a Perkin Elmer Lambda–25 UV-Visible Spectrophotometer in the range of 200–1100 nm. Surface morphology was examined using a JSM–5610 Scanning Electron Microscope. The HT-29 (colon cancer) cell line was initially procured from the National Centre for Cell Sciences.

Synthesis of Schiff Base Ligand
The Schiff base ligand was synthesized according to a reported method [18]. Ethanolic solutions of DFMPM and glutamic acid were mixed in a 1:2 molar ratio in a round-bottom flask and refluxed for 1 hour. The reaction mixture was then poured into ice, yielding a yellow precipitate of the Schiff base ligand. The solid was filtered, washed with water, and dried over anhydrous calcium chloride. The crude product was recrystallized from 50% absolute ethanol. Yield: 58%; Melting point: 225 °C [6].

Preparation of Schiff Base Metal Complexes
The metal complexes were prepared by adding aqueous solutions of Cu(II) nitrate, Ni(II) nitrate, and Co(II) nitrate to the Schiff base ligand in ethanol in a 1:2 molar ratio, followed by refluxing at 80 °C for approximately 12 hours [2]. The resulting precipitates were filtered, washed with ethanol, diethyl ether, and hot water, and finally dried under vacuum at 90 °C. The yields of the complexes ranged from 60% to 64%.

RESULTS AND DISCUSSION

        All the metal complexes are coloured solids, stable towards air and  have high melting points (above 250^oC).  The complexes are insoluble in water and common organic solvents, but are soluble in DMF, CDCl₃ and DMSO.

Elemental analysis :

      The analytical data suggest that all the complexes are mono nuclear with the ligands coordinated to the central metal atom.  The metal to ligand ratio in all complexes was 1:2 and their formulae have been computed and given in table 1. Conductivities of solution of the complexes in DMF showed that all the complexes are non – electrolytes because their conductivity values  were low.   However, the conductivity value is higher than expected for non- electrolytes probably due to partial solvolysis of complexes in DMF medium.

Table : 1 Physical Characteristics and analytical data of the complexes

Compound	Yield %	Colour	Mol. Formula	Mol.wt	M. point	Elemental Analysis,                 found (calcd) %
						C	H	N
C57H86N2O10	58	Brown	C57H86N2O10	958	225	71.42 (71.39)	8.70 (8.97)	2.10 (2.92)
[Co(LV)]	60	Purple	CoC57H82N2O10	1012.93	>250	67.43 (67.52)	8.18 (8.09)	2.54 (2.76)
[Ni(LV)]	64	Pale green	NiC57H82N2O10	1012.71	>250	67.32 (67.54)	8.90 (8.09)	2.45 (2.76)
[Cu(LV)]	64	Green	CuC57H84N2O10	1019.55	>250	67.67 (67.08)	8.12 (8.23)	2.72 (2.74)

IR Spectrum

Selected IR spectral bands for the ligand and its metal complexes are summarized in Table 2. The IR spectrum of the free ligand is primarily characterized by strong bands at 2925 cm⁻¹, 2855 cm⁻¹, 1690 cm⁻¹, 1595 cm⁻¹, and 1452 cm⁻¹, corresponding to C–H stretching, O–C stretching, azomethine (C=N) stretching, asymmetric COO⁻ stretching, and symmetric COO⁻ stretching, respectively (Fig. 8.1).

Comparative analysis of the ligand and its metal complexes revealed additional absorption features. Broad bands observed at 3540 cm⁻¹, 3429 cm⁻¹, and 3412 cm⁻¹ were attributed to –OH groups of lattice or coordinated water molecules. The C–H stretching vibrations were observed in the range 2925–2986 cm⁻¹, while O–C stretching appeared at 2885–2854 cm⁻¹. Bands in the region 1625–1680 cm⁻¹ were assigned to C=N stretching vibrations. Notably, the imine (C=N) peak in the metal complexes exhibited a shift relative to the free ligand, indicating coordination of the imine nitrogen to the metal ion.

The asymmetric and symmetric COO⁻ stretching frequencies appeared at 1600–1594 cm⁻¹ and 1492–1383 cm⁻¹, respectively. Additional bands at 700–779 cm⁻¹ were assigned to M–N bonds, and those at 425–545 cm⁻¹ were attributed to M–O bonds, confirming coordination of the ligand to the metal center.

Table : 2 Selected UV and FTIR frequencies (cm^-1) of the ligand and complexes

Ligand/ Complex	nO -H	nC – H	nO – C	nC = N	nasym coo-	nsym coo-	M-N	M-O
L	–	2925	2855	1690	1595	1452	–	–
[CoL]	3429	2986	2885	1625	1594	1383	700	545
[NiL]	3412	2925	2855	1680	1600	1492	750	474
[CuL]	3540	2925	2854	1637	_	1424	779	425

Fig.1.FTIR Spectrum of Schiff base ligand

Electronic Spectra
The electronic spectrum of the ligand exhibits a broad band at 241 nm, attributed to the π → π* transition of the C=N chromophore [9]. In the octahedral ligand field of Co(II) complexes, the d-orbitals show a ⁴T₁g(F) → ⁴T₂g(F) transition with an absorption band at 908 nm, which is characteristic of octahedral geometry. The Ni(II) complex displays an intense band at 352 nm, corresponding to the ³A₂g(F) → ³T₂g(P) transition (400–300 nm), consistent with octahedral geometry. For the Cu(II) complex, an intense band appears at 448 nm, assigned to the ²B₁g(F) → ²E₁g(P) transition (470–450 nm), indicating a tetragonally distorted octahedral environment.

¹H NMR
The ¹H NMR spectrum of the ligand (Fig. 5) shows a multiplet at δ 7.135–7.174 ppm, corresponding to protons of the substituted aromatic ring. A singlet at δ 8.3 ppm confirms the presence of the H–C=N group. Signals observed at δ 1.263–1.529 ppm are assigned to –CH₂– protons. The multiplets at δ 6.731–6.751 ppm and δ 3.838–3.988 ppm correspond to the olefinic protons of the side chain and the O–CH₂ group of the ligand, respectively.

Based on the present study the structure of the ligand and complexes may be given as follows  (scheme 1-2)

SEM  Analysis

        Scanning  electron  micrography  is  used  to  evaluate  morphology  of  the  Schiff  base  metal  complexes [10] .  The  SEM  picture  of  Cu(II) complex  is  shown  in  fig. 6. From  the  fig. 6  pitted  and  rough  surface  is  observed  in  the  complex.  The  particle  size  of  the  Cu(II)  complex  were  in  the  diameter  range  of  few  microns.

XRD

        The  XRD  pattern  of  Cu(II)  complexes  show well defined  crystalline  peaks  indicating  that  the  samples  are  crystalline  in  nature .  The  above  complexes  have  specific   ‘d’  values  which  can  be  used  for  its  characterization .  The  crystallite  size  of  the  complexes  d_XRD  could  be  estimated  from  XRD  patterns  by  the  Scherre’s  formula [8]. XRD  shows  that  Co(II)  complex has  the  average  crystallite  size of 4.1 nm suggesting  the  complexes  to  be  nano  crystalline (Fig7).

Invitro biological assay

         The  biological  activities  of  synthesized  Schiff  base  and  its  metal  complexes  have  been  studied  for  their  antibacterial  and  antifungal  activities  by  disc  diffusion  method ,  and  the  stock  solution  (0.001 mol) was  prepared  by  dissolving  the  compounds  in  DMSO  and  the  antimicrobial  activity  was  estimated  based  on  the  size  of  inhibition   zone  in  the  discs [10,1 ]^. Four bacterial stains  Klebsiella sps , E.Coli, P.aeruginosa , S.aureus  were  incubated  for  24h  at 37^oC  and  Fungal  stains Candida sps , Aspergillus sps  were  incubated  for  48h  at 37^oC.

Table 3 : Antimicrobial Activity for Bacteria

S.No	Samples	Media	Zone of Inhibition (mm)
			Klebsiell a sps	E.coli	P.aeru ginosa	S.aur eus
1	C57H86N2O2	Mueller Hinton Agar	6.0	6.0	6.0	6.0
2	[CoL]		8.0	11.0	6.0	10.0
3	[NiL]		9.0	6.0	6.0	6.0
4	[CuL]		6.0	6.0	10.0	6.0
5	PC (Chloram phenicol)		25.0	26.0	24.0	25.0
6	NC		6.0	6.0	6.0	6.0

Table 4 : Antimicrobial Activity for Fungi

S.No	Samples	Media	Zone of Inhibition (mm)
			Candida sps	Aspergillus sps
1	C53H86N2O2	Mueller Hinton Agar	6.0	6.0
2	[CoL]		6.0	14.0
3	[NiL]		15.0	6.0
4	[CuL]		6.0	6.0
5	PC(Nystatin)		25	26
6	NC		6	6

The antimicrobial activity results (Tables 3 and 4) indicate that the enhanced activity of the metal complexes may be attributed to the influence of metal ions on normal cell membranes [7]. Metal chelates possess both polar and nonpolar characteristics, which facilitate their penetration into cells and tissues. Furthermore, chelation can either enhance or suppress the biochemical activity of bioactive organic molecules. Coordination also modifies lipophilicity, which governs the rate of cellular uptake, making the metal complexes more active than the free ligands. Consequently, the metal complexes exhibit greater antimicrobial activity than the uncoordinated ligands or free metal ions, consistent with previously reported literature [17]. The mode of action may involve hydrogen bonding between the azomethine group (>C=N) and active sites on cellular components [18], thereby interfering with normal cellular processes.DNA Cleavage Studies
The DNA cleavage ability of the complexes was evaluated using gel electrophoresis. All the metal complexes were capable of converting supercoiled DNA into open circular forms, as shown in Fig. 8. The higher cleavage efficiency of the complexes, compared to control experiments, is attributed to their strong DNA-binding ability. Control experiments with DNA alone showed no significant cleavage, even after extended exposure. This demonstrates that the DNA damage observed with Co(II), Ni(II), and Cu(II) complexes is due to the cleavage activity of the metal complexes. The observed DNA cleavage activity follows the order: Co(II) > Cu(II) > Ni(II) with λ-DNA. The oxidative DNA cleavage likely occurs through singlet oxygen-mediated oxidation of guanine nucleobases.

Lane 1 : DNA + L + H₂O₂     

Lane 2  : DNA + CoL₂ + H₂O₂

Lane  3 : DNA + [NiL₂(H₂O)₂]

Lane  4 : DNA + CuL₂  + H₂O₂

Lane  5 : Control DNA

INVITRO ANTICANCER ACTIVITY DETERMINATION BY MTT ASSAY

        Cultured cell lines were kept at 37^oC in a humidified 5% CO₂ incubator. The viability of cells were evaluated by direct observation of cells by Inverted phase constrast microscope and followed by MTT assay method [11,15,16].  

Anticancer Activity

                The result of anticancer activities are presented in table 5.  The colon carcinoma (HT-29) cells, were sensitive to the Co (II) complex with an IC₅₀ value of 72.07  mM.  The enhancement of cytotoxic activity may be assigned to that the positive charge of the metal increased  the acidity of coordinated ligand that bears protons, leading to stronger hydrogen bonds which enhanced the biological activity [14]. It seems that changing the anion, coordination sites, and the nature of the metal ion has a pronounced effect on the biological behavior by altering the binding ability of DNA[13].  Gaetke and Chow had reported  that metal has been suggested to facilitate oxidative tissue injury through a free radical mediated pathway analogous to the Fenton reaction. (fig 9)

Table  5 : % viability of Co(II) complex.

Sample  Concentration (µg/ml)	Average OD at  540nm	Percentage Viability
Control	0.4945
6.25	0.4654	94.11
12.5	0.4481	90.61
25	0.3603	72.86
50	0.2962	59.89
100	0.1491	30.15
IC50 Value = 72.07 µg/ml

CONCLUSION

                Schiff base complexes of Cu(II), Ni(II), and Co(II) with DFMPM and glutamic acid were synthesized and thoroughly characterized. UV-visible, IR, and NMR analyses indicated the geometries of the complexes, with Co(II) and Ni(II) forming hexacoordinated structures, whereas the Cu(II) complex is tetracoordinated. Antimicrobial studies demonstrated that the metal complexes exhibit greater activity than the free ligand. The DNA cleavage activity of the complexes followed the order: Co(II) > Cu(II) > Ni(II). Additionally, in vitro anticancer evaluation showed that the Co(II) complex exhibited moderate activity against HT-29 (Colon Carcinoma) cells, with an IC₅₀ value of 72.07 μM.

Conflict to interest

          The authors declare that they have no conflict of interest.

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