Synthesis,Characterization, biocidal, Anti-diabetic, Anti-inflammatory and Anti-Cancer(MCF-7) Studies of Schiff base ligand and its metal (II) complexes

Introduction

Schiff bases obtain by the condensation of amino and carbonyl compounds are intrinsic class of ligand that coordinate to metal ions via azomethine nitrogen and have been extensively for its wide range of medicinal applications.[1]

In the past years, many new antibiotics failed to meet the challenges posed by multi drug-resistant bugs, such drugs are needed which are enough to work against & control antibiotic resistance[2]. Over the years, Schiff base and  their metal complexes have amazing properties like catalysts in different biological systems, polymers, dyes, antimicrobial activities, anti tumor, cytotoxic and drug fields [3], [4], [5].

Quinolines are a class of nitrogen heterocycles, present in a large number of natural and synthetic compounds that exhibit antimicrobial, antibiotic, anti malarial, antitumor, immuno suppressive, analgesic, anti convulsant and anti hypertensive activities[6]. 

Schiffbase complexes are play an important role in biological, pharamecutical, agrochemical and organic industries and to design of new mixed ligand complexes greater potential application in this field.[7]

In the this work, mononuclear metal(II) complexes withL¹ and with a co-ligand (8-hydroxy quinoline) were prepared. The prepared ML¹& ML¹L² have been characterized by physicochemical properties and in-vitro antimicrobial activity, anti-inflammatory, anti-diabetic and anti-cancer activities of metal complexes were also studied.

Materials and Methods

The 3-aminoquinoline and 2,5-dimethoxy benzaldehyde utilized for the preparation of ligand were purchased from TCI chemicals. The Metal acetate salts were purchased from Sigma Aldrich. All the solvents were purchased from Loba and used without further purification. 8-Hydroxyquinoline used in the present work was acquired from S.D Fine chem. Limited.

The percentage of C, N, O, and metal was determined by a FEI Quanta-250 FEG elemental analysis apparatus. The absor bention spectra of the L¹, ML¹& ML¹L² dissolved in dimethylformamide were recorded on the JASCO V 650 in the range of 200 – 800 nm. Vibrational spectra of the compounds was recorded in Bruker Tensor 27 in the range of 4000- 400 cm^-1. ¹H &¹³C NMR spectra were recorded on the Bruker 300 Hz spectrometer using DMSO-d₆ as a solvent. Mass spectrum was recorded for the L¹, ML¹& ML¹L²using 410 Prostar Binary LC with 500MS IT PDA Detectors.  TG/DTA analysis was carried out under nitrogen atmosphere using Shimadzu TG-50 thermobalance. The surface nature of the compounds were determined by CAREL ZEISS EVO 18 Scanning electron microscope.

Synthesis of L¹

The ligand (E)-1-(2,5-dimethoxyphenyl)-N-(quinolin-3-yl) methanimine was prepared using 2,5-dimethoxybenaldehyde and 3-aminoquinoline. 1mmol of 2,5-dimethoxybenaldehyde (0.166 g) dissolved in 20 ml of methanol was mixed drop wise into 1mmolCH₃OH solution of 3-aminoquinoline (0.144 g). The mixture was stirred continuous by and refluxed for 3 h at 50 – 70 °C, product obtained was filtered and dried.

Synthesis of ML¹

Methanolic solution of ligand (E)-1-(2,5-dimethoxyphenyl)-N-(quinolin-3-yl)methanimine (2mmol, 0.584g) was added to the methanolic solution of Metal(II) acetate salts (1 mmol) of  Cu(II), Ni(II),Co(II) and Zn(II) in the molar ratio 2: 1. All complexes were stirred and refluxed at 70 °C for about 4 hours. The hot solution was cooled and dried at room temperature.

Synthesis of mixed ML¹L²

The methanolic solution of (1mmol) (E)-1-(2,5-dimethoxyphenyl)-N-(quinolin-3-yl) methanimine (L¹) was added to the methanolic solution of Metal(II) acetate salts (1mmol) of (Cu(II), Ni(II), Co(II), and Zn(II)) in the RB Flask. 8-Hydroxy quinoline (L²) (1mmol) dissolved in methanol was mixed dropwise into the above mixture. The ligand, metal salt and 8-hydroxyquinoline were taken in the ratio 1 : 1 : 1. The mixture was stirred and refluxed for 3 h then dried at room temperature.

Figure 1: Synthesis of Schiff base ligand[L1], [ML1] &[ML1L2]complexes. Click here to View figure

Results and Discussion

The analytical data of the L¹, ML¹& ML¹L² are given in Supplementary Table 1. ML¹& ML¹L² are coloured and sparingly soluble in ethanol, methanol, acetone, chloroform and soluble in DMSO and DMF but insoluble in water.

Molar Conductance

Molar conductance was measured at 1×10^-3 M for ML¹& ML¹L² in DMSO at room temperature.The mononuclearmetalcomplexes exhibit low value inthe range of 11-23 ohm^-1 cm² mol^-1[8] indicates that ML¹& ML¹L²are non-electrolytic as given in Supplementary Table 1.

FT – IR spectral studies

The IR spectra of complexes are studied with L¹ to determine the co-ordination sites that may be involved in chelation. The FT-IR data with the assignments of band frequencies of L¹, ML¹& ML¹L² complexes are presented Supplementary Table 2 and the spectrum, are shown in Supplementary Figure 1.

The sharp peak observed at 1581cm^-1 in the IR spectra of L¹ assigned to υ(CH=N) is shifted to lower frequency for ML¹& ML¹L², indicating the involvement of azomethine nitrogen in chelation with the metal ion [9]. However, stretching frequency of methoxy (-OCH₃) group appeared in the range of 2828-2833cm^-1 as observed in L¹, [ML¹] and [ML¹L²] complexes,  indicates the non-participation of methoxy oxygen in co-ordination [9]. The new bands in IR spectra of ML¹& ML¹L² in regions 493-533 cm ^-1 and 472- 497 cm ^-1 are ascribed to υ(M-O) and υ(M-N) respectively, which indicates the co-ordination of L¹, co-ligand with the metal ion[10].

Electronic spectral studies and magnetic moment

The Absorption spectrum of the L¹, ML¹ and ML¹L² were recorded in DMSO solution over the range of 200-800 nm and the spectral data are listed in Supplementary Table 3 and shown in  Figure 2.The UV spectra of L¹ reveals high intense absorption peaks at 270 nm (37037 cm^-1) and 369 nm (27100 cm^-1) which are due to π-π* and n-π* transitions respectively. But in metal complexes, azomethine peak was shifted to shorter wavelength, this shift is attributed to the coordination of the nitrogen atom of the imine group with the metal ion.

The Absorption spectra of  Cu(II) complexes [CuL¹] and  [CuL¹L²] exhibit two bands, the charge transfer region at 439 nm for CuL¹ complex and another band in the region 594 nm assigned to  ²T_2g→   ²E_g transition indicating the square planar geometry and the magnetic value is 1.95 B.M. Another Cu(II) complex CuL¹L²exhibits intense broadband at 440 nm which maybe reasonably assigned to²B_1g   →    ²A_1g transition, corresponds to a distorted squareplanar geometry for Cu(II) ion and the observed magnetic moment value is 1.81 B.M.[11].

The visible spectra of Ni(II) complexes,  [NiL¹] and  [NiL¹L²] show three bands. One intense peak show 381-392 nm due to charge transfer transition and the d-d transition exhibits in the range of 470-550 nm are assigned to ³ A_2g (F)  →³T_1g(P) and³A_2g(F)  → ³T_1g(F) transitions respectively, consistent with a square planar geometry around Ni(II) ion, which are further supported by the magnetic value range between 3.81-3.05 B.M.[12]

The UV spectrum of cobalt(II) complex [CoL¹] shows three intense peaks and [CoL¹L²] complex shows two intense peaks. One peak at 382-393 nm for complexes CoL¹ and CoL¹L²  is due to charge transfer transition and another two peaks exhibited transition in the visible region at 450-457 nm and 510-511 nm corresponding to tetrahedral geometry for Co(II) ion.The magnetic moment value of Co(II) complexes are in the range of 4.41 – 4.83 B.M.[13]

Figure 2: UV Spectra of L1, ML1 and ML1L2 Click here to View figure

The UVspectrum of Zn(II) complexes  [ZnL¹] and  [ZnL¹L²] shows bands in the range 392-482 nm may be due to L→ M charge transfer which indicates square planar geometry and it is diamagnetic nature[14].

¹ H NMR

The structure of the Schiffbase ligand is fairly supported by ¹H NMR spectra recorded in CDCl₃ as a solvent. On examining the ¹H NMR spectrum of the ligand it exhibited a multiplet signal at δ (8.51 – 6.93) ppm for aromatic protons. The presence of azomethine(CH=N) group is indicated by a singlet at δ (8.92) ppm. Which confirms the formation of imine ligand. A signal at δ (3.81) ppm indicates the presence of methoxy  ( O-CH₃) protonsas shown in Supplementary Figure 2.

¹³C NMR

In ¹³C NMR, the number of signals represents the number of carbons of the synthesized compound which are non-equivalent. The signal that appeared at δ (158.34) ppm is attributed to azomethine carbon. Signals observed at δ = 110 – 129 ppm are assigned for aromatic carbons. Phenyl ring of ligand showed the following signals 146.61(CH=N), 147.15,145.73 (C-N). The methoxy carbons show peaks at 56.25 and 55.94 ppm as shown in Supplementary Figure 3.

Mass Spectra

Mass spectral data gives valuable information on molecular weights, the synthesized ligands and its metal(II) complexes were characterized by ESI–MS spectrometry in positive ion mode [15]. The ESI mass spectra of the L¹, ML¹& ML¹L² were recorded and the obtained molecular peaks confirmed the proposed structures. The ESI mass spectrum of the Schiffbase ligand shows an intense peak at 294.05. The mass spectrum of the CuL¹, NiL¹, CoL¹, ZnL¹, CuL¹L², NiL¹L², CoL¹L² and ZnL¹L² complexes gives a molecular ion peak at 781.99, 833.28, 835.03, 802.84, 636.14, 678.48, 683.43 and 647.24 m/z respectively which indicate the stoichiometry composition of complexesas shown in Supplementary Table 4 and Supplementary Figure 4.

EPR Spectra

EPR spectrum of Cu(II) complexes were recorded in the solid-state at room temperature as shown inSupplementary Figure 5.The CuL¹ & Cu L¹L² complexes gives g_ǁ = 2.0610, 2.06145 and g﬩ = 2.00975, 2.02272 respectively.The g_ǁvalues of these complexes lie below 2.3 (g_ǁ< 2.3)which indicates the covalent environment around the Cu(II) ion [16]. The calculated G (G = g‖ -2.0023/g┴-2.0023) value is 7.88 for CuL¹ complex and 2.86 for CuL¹L² complex which indicates squareplanar geometry for CuL¹ complex and distorted square planar geometry for CuL¹L² complex.

SEM

The surface morphology of the synthesized L¹, ML¹& ML¹L² was done by scanning electron microscopy. The synthesized L¹, ML¹& ML¹L² were carried out and the obtained images are shown in Figure 3. The SEM images indicates that the L¹, ML¹& ML¹L² have different distinctive surface morphology[15].

Figure 3: SEM analysis of Schiff base ligand, ML1 and ML1L2 Click here to View figure

The powder XRD for the L¹, ML¹ and ML¹L² were performed. The synthesized compounds show well explicatescrystalline peaks indicating that the samples are crystalline.The ligand and all synthesized complexes have specific‘d’ values which can be used for its characterization. The crystalline size of the ligand and their complexes d_XRD could be estimated from XRD patterns by the Scherrer’s formula[17]. The value of crystalline size of the ligand and all complexes are (Ligand=100.74, CuL¹=49.87, NiL¹=74.45, CoL¹=78.02, ZnL¹=64.44, CuL¹L²= 64.92, NiL¹L²= 47.72, CoL¹L²= 29.65, ZnL¹L²= 41.03) respectively as shown in Supplementary Table 5 and Supplementary Figure 6.

Antimicrobial activity

The ligand,ML¹& ML¹L² were screened for antimicrobial activity by disc diffusion method and are compared with standards tetracycline and fluconazoleas shown in Figure 4.The results are presented inSupplementary Table 6.

The antibacterial and antifungal actions of the Schiff base ligand, ML¹& ML¹L² were tested against Gram(+) bacteria (Staphylococcus-aureus), Gram(-) bacteria (Escherichia-coli) and one fungus (Candida-albicans). All metal(II) complexes show maximum inhibition zone than the free ligand. ML¹L² complexes show good activity compared to the ML¹ complexes because L²(8-Hydroxyquinoline) acted as a co-ligand in ML¹L²complexes. From the above results, it is concluded that CuL¹L²complex exhibits the highest antimicrobial activity.

Figure 4: Anti-microbial activity of the ligand, ML1&ML1L2complexes. Click here to View figure

Anti-inflammatory activity

The synthesized Schiff base-ligand and its Copper(II) complexes [CuL¹& CuL¹L²] were examined for anti-inflammatory activity by HRBC stabilization method at various concentrations (20, 40, 60, 80, 100 μg / ml). Diclofenac sodium was used as a standard. The percentage inhibition of hemolysis by the ligand, ML¹&ML¹L²are presented in Supplementary Table 7. In all the concentration of CuL¹ and CuL¹L² complexes show a maximum % inhibition of hemolysis compared to the free ligand. The CuL¹L² complex shows a minimum IC₅₀ value of 28.50μg/ml and the complexes stabilized human blood cell membrane in a dose-dependent manner[18]. Thus CuL¹L² complex shows the best anti-inflammatory activity.

Anti diabetic Studies

The antidiabetic activity was examined by the standard amylase inhibition assay. The inhibition activity of the synthesized compounds against α-amylase is given in Supplementary  Table 8. Both copper(II) complexes exhibit more inhibition activity than the free Schiff base ligand. CuL¹L² complex shows excellent amylase inhibition activity. CuL¹ complex shows fine activity as compared to the schiffbase ligand. The IC₅₀ value of the free Schiffbase, CuL¹ and CuL¹L² complexes are 54.80, 11.91 and 3.86μg/ml respectively.

Anticancer activity

Anti-cancer activity of the (E)-1-(2,5-dimethoxyphenyl)-N-(quinolin-3-yl)methanimine, CuL¹ and CuL¹L² metal complexes were tested against MCF-7 (breast cancer) at different concentrations (3.125, 6.25, 12.5, 25, 50μg/ml)as shown in Figure 5. The viability of the cancer cells by ligand and its metal complexes are 100% at zero concentration. (97.85, 90.40, 71.81, 46.72, 33.49 %) for ligand, (97.09, 87.39, 70.84, 48.98, 26.93 %) for CuL and (89.91, 74.46, 59.78, 37.58, 24.34%) for CuL¹L²  when the concentration increases the cell viability of cancer cells decreases, that mean the cytotoxicity of the cells decreases and the complex is potential which is due to less number of live cells[19]. The in-vitro anticancer studies reveal that the CuL¹ and CuL¹L² have moderate activity than the ligand. Particularly CuL¹L² complex are highly active with IC₅₀ value 24.92 μg/ml compared to the free ligand and CuL¹ complex.

Figure 5: Anti-Cancer activity of synthesized ligand,CuL1and CuL1L2complexes. Click here to View figure

Conclusions

Schiff base complexes of (b-e) obtained from (E)-1-(2,5-dimethoxyphenyl)-N-(quinolin-3-yl)methanimine in a ratio (1:2) and  co-ligand complex with 8-hydroxy quinoline at ratio (1:1:1) have been synthesized and characterized. The stoichiometry of all complexes are confirmed by elemental analysis. All the metal complexes are non-electrolyte nature dependent upon molar conductance value. The physicochemical data reveals that CuL¹, NiL¹, ZnL¹, NiL¹L², ZnL¹L², complexes have square planar geometry, CoL¹& CoL¹L² complexes have tetrahedral geometry, CuL¹L² complex have distorted square planar geometry. The powder XRD pattern showed the crystalline nature of the L, ML¹&ML¹L². By using the SEM analysis, morphology of all complexes is have been studied. The synthesized ligand and their ML¹ and ML¹L² metal complexes were tested against gram(+), gram(-) and one fungus strain. The data showed that CuL¹ and CuL¹L² exhibits better activity than other compounds. CuL¹L² shows high zone of inhibition compared to the CuL¹ complex. The anti-inflammatory, anti-diabetic and anticancer studies of ligand, CuL¹and CuL¹L² were done. From the studies, it is suggested that CuL¹L²shows better activity than the free ligand and CuL¹. Finally, it is concluded that CuL¹L² may be used as effective biological agents with reduced toxicity and higher efficiency.

Supplementary Information

Additional files : Table S1- S9. Additional Figure S1 and S6.pdf

Acknowledgement

None

Conflict of Interests

None

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