Synthesis, Characterization and Biological Evaluation of New Schiff Base Ligand derived from 3-hydroxy-benzaldehyde and p-toluidine and its Divalent Metal Ions

A new Schiff base ligand (L) and its transition divalent metal complexes were prepared by the condensation reaction; Reaction was carried out at 70-80 C by refluxing equimolar ratio of toluidine and aldehyde by continuous stirring for 5-6 hours. Synthesized ligand and Ni(II), Fe(II), Co(II), Mn(II) and Zn(II) bimetallic complexes were characterized by using FT-IR, UV-visible spectroscopy, AAS, Single crystal X-ray analysis, H-NMR, molar conductance. Compounds were screened against two fungus Candida glabrata and Candida albicans by agar tube dilution protocol. In vivo anti-inflammatory activity via induced paw edema method and in vitro results by heat induced protein denaturation method were checked. Synthesized compounds were also showed antioxidant activity by using DPPH (diphenylpicrylhydrazyl) and Trolox was used as standard. These studies show that ligand and almost all metal complexes are reactive towards biological assays against reported standard drugs. Zn-L indicates more activeness for antioxidant activity and free ligand while Co-L recognized as more effective anti-inflammatory drug.


1.Introduction
Schiff bases were discovered first time by the chemist Hugo Schiff in 1864 [1]. These are condensation products of carbonyl compounds and amines [2], also called as imine or azomethine due to (-HC=N-) functional group [3], here nitrogen atom serves as bonding molecule for complexation.In azomethine derivatives, the C‚N linkage is essential for biological activity [4,5], several azomethine have been reported to possess remarkable antibacterial, antifungal, anticancer and antimalarial activities [6][7][8][9].
The derived compounds of Schiff base are of great importance nowadays. Mostly Schiff bases are crystalline and basic in nature. These are less expensive [10][11][12]. Ligand and its charged or neutral metal complexes can be easily synthesized. These metal complexes can be effectively used against cancer than the standard anti cancerous drug like cis-platin. Zn(II) complexes helps in faster healing of the wounds [13,14]. Schiff bases are soluble in variety of organic solvents except aqueous media at room temperature [15].
Biological activity probably conferred to them by the strong aromaticity of this ring system, which leads to great in vivo stability and generally, a lack of toxicity for higher vertebrates, including humans. Aside from these activities, Schiff bases also have other tremendous applications, they serves as dyes, agrochemicals, chemo sensor, catalyst, chemotherapeutic, anticorrosive, polymer [16][17][18][19][20][21]. Schiff bases have major concern in organic, inorganic, coordination, biochemistry and other material sciences [22].
As the continuation interest of our study of transition metal complexes, here we synthesis and characterization of new Schiff base ligand 3-hydroxybenzaldehyde with p-toluidine, and its complexes with Ni(II), Fe(II), Co(II), Mn(II), Zn (II) divalent metal ions. Structural confirmation of the  synthesized Schiff base ligand characterization was done with different analytical techniques like  single crystal X-ray analysis, 1 H-NMR, FT-IR, AAS, UV-visible spectroscopy and molar conductance. Biological evaluation of Schiff base ligand (L) and its transition metal complexes were also done by antifungal, antioxidant, and anti-inflammatory activities. Synthesized ligand and related complexes are said to demonstrate dynamic applications in the field of material sciences and medicine.

Material and methods
3-hydroxybenzaldehyde and p-toluidine were obtained from Sigma Aldrich. All the reagents, starting materials as well as solvents were purchased commercially and used without any further purification. Pure solvent like ethyl alcohol, n-hexane, ethyl acetate, chloroform, DMSO, DCM were used to check solubility of compounds at room temperature. Melting points were obtained from Gallenkamp apparatus. FT-IR was done on Perkin Elmer 1650 spectrophotometer at 4000-400 cm -1 in KBr pellets. AAS was done on AA320N atomic absorption spectrometer having 190-900 nm range. 6000 PC UV-visible spectrophotometer (190-1100 nm) was used for absorption measurements. Molar conductance measurements were calculated for 10 -3 M solution in DMSO at room temperature on METROHM 644 conductometer. Aluminum sheets layered with silica gel G-25-UV254 plate was used for TLC. Single crystal XRD method was used for structure determination of the Schiff base ligand (L) by suing Bruker SMART APEX II Diffractometer at 100(2) K temperature. BIOBASE oven (1150 W) was used for incubation and drying of crystals. ME-LEC-04P centrifuge apparatus of 4000 RPM, ME-JA5003N digital weight balance having 0.001 g minimum reading and Raecho RVIS-1 spectrophotometer (325-1000 nm) was used for biological activities.

Prepration of Schiff base ligand (L)
Schiff base ligand (L) was synthesized by the reflux condensation reaction of 3-hydroxybenzaldehyde (0.122 g, 1 mmole) along with amine component p-toluidine (0.107 g, 1 mmole) using ethanol as solvent (Scheme 1) in addition of few drops of dimethylamine for pH stabilization and to prevent zwitterion formation. The reaction was carried out at 70-80 o C temperature with continuous stirring for about 5-6 hours into a 250 mL of round bottom flask. Brown pellet like crystals were obtained after 48 hours dried at room temperature. Distilled water was used for washing and product was recrystallized and dried at 25-27 o C giving yield of about 85 % Shiny hazel brown needle like precipitates having melting point 113 o C and yield 85 % were obtained.

Figure 1. Structures of metal complexes
Biological Activities 1. Antifungal assay Compounds were selected for antifungal activity (in vitro) by using agar tube dilution method [23] to find out the reactiveness of ligand and metal complexes against Miconazole used as standard drug. Compounds were tested against Candida albicans and Candida glabrata fungi, (Fig. 2). Each sample was prepared 200 mg/mL in DMSO solvent. Activity was done on 96 welled plate which were incubated at 27 o C (28 ± 1 o C) for about 168 hours and percentage inhibition of the standard and synthesized compounds were calculated [24].

In vivo anti-inflammatory activity a) Experimental animal
Albino male SD rats having weight about 100-150 g were used to carried out the in vivo antiinflammatory screening of ligand (L) and metal ions complexes using Carrageenan as standard drug. Animals were kept in a lab having free contact with pathogens, at room temperature (25 ± 5 o C), moisture (50 ± 10 %) with accessibility of distilled water to the animals during the experimental handling. All the measures were ratified by the IEC (Institutional ethical committee) PUIC (Punjab University, Institute of Chemistry, Lahore) and international precautions were also kept in mind to create healthy atmosphere for rats.

b) Paw Edema Method (Procedure)
Rats weighing 150-200 g were used for the experimental work and they were distributed in three sections and all rats were deprived of food overnight. Dose of 100 µL of the test drug (synthesized) was given orally to the test group rats and in case of control group 100 µL distilled water and in case of standard group diclofenac sodium 1 mg was given orally. After 30 min of the orally given dose about 0.1 mL of Carrageenan injected into sub planter surface of rat's hind paw. Paw volume was note dinstantly after administration of Carrageenan at 0.0, 60, 120, 180 and 240 min time period, respectively ( Fig. 3), (Fig. 4). Row 1, 3, 5 shows sub planter sites of rat's paw indicating redness or inflammation which gradually decreases from left to right site from 0h to 3h time. Similarly row 2 (Schiff base ligand), 4(Co-L complex), 6(Diclofenac Sodium; standard drug) shows level of swelling decreased with passage of time, while series 4 in case of Co(II) complex shows maximum antiinflammatory activity [25]. Percentage protection [26]    where: Vt is for absorbance of the test sample and Vc shows absorbance of control moiety, (Fig. 5), (Table 1).

Antioxidant activity (in vitro) DDPH assay
Trolox stock solution was prepared by dissolving 0.005 g of Trolox in ethanol and raised the volume up to 10mL. The concentration of this solution was 2000 mM. From this stock solution 1000 mM, 500 mM, 250 mM, 125 mM, 62.5 mM, 31.25 mM, 15.62 mM, 7.81 mM, 3.9 mM and 1.95 mM Trolox dilutions were made by taking one ml of ethanol this will give 1000 mM solution. Then take one ml of 1000 mM solution and add 1 mL of ethanol this will give 500 mL then diluted further solution by applying same rule.
Trolox was used as standard for in vitro antioxidant assay [28]. Now add 1.85 mL of the DPPH (0.05 mM) solution in every bottle. Note that for every experiment freshly prepared and preserved in dark solution of Trolox was used.
In the exactly same way prepare synthesized sample (0.005 g) stock solutions ranging from 2000 -1.95 mM by using ethanol further dilute them in a brown bottle by adding 1 mL of stock solution and https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (4)

4.Results and discussions FT-IR
The metal-ligand bond was verified by comparing the IR spectrum of the Schiff base ligand with metal (II) complexes. Absence of band characteristics at 1550 cm -1 for primary amine and 1720 cm -1 for aldehyde and appearance of sharp band at 1620 cm -1 indicates the azomethine group (-CH=N-). Spectral values of the prepared samples are given below (Table 3) [30,31]. The FT-IR spectra predicted all the absorption bands of the Schiff base ligand (L) and some new bands at specific frequency confirmed the modes of absorption and the coordination of the ligand (L) with the metal ions through azomethine nitrogen and oxygen (carboxyl, sulfate). The phenolic oxygen is not involved in coordination. In the complexes Mn(II), Co(II), Zn(II) two bands appeared in the region 1658-1651 cm -1 (ν-asymmetry) and 1419-1396 cm -1 (ν-symmetry) which attribute the carboxylate part in the acetate group and also suggest that they are responsible for the consisting of unidentate coordination site with the metal ion due to the value of differences between asymmetry and symmetry was greater than 200 cm -1 (Δν ≥ 200 cm -1 ). The far IR spectra of the complexes show weak bands in the region 466-434 cm -1 and 512-475 cm -1 corresponding to ν(M-N) and ν(M-O) vibrations, respectively and other absorption bands were no appreciable change in the ligand (L) and metal complexes [32]. Schiff base ligand (L) C14H13NO structure was confirmed by the proton 1 H-NMR spectroscopy. Peaks were detailed on BRUKER 300 MHz instrument by using CDCl3 as solvent. One singlet peak was observed for CH3 group, one broad peak at δ 5.025 ppm shows residual NH2, aromatic benzene ring protons were shown in the form of multiplet at δ 7.562-6.743 ppm, one more deshielded singlet was recorded at δ 8.404 ppm for (-CH=N-) proton, one singlet was observed at δ 9.890 ppm for OH group [33,34].

Single crystal X-ray analysis
For single crystal X-ray analysis, Bruker SMART APEX II Diffractometer fortified with CCD detector was used [35]. Single crystal of complex was grown by evaporation method with a volume ratio of 3:1 of CH3CN:CH3OH. The room temperature single crystal X-ray experiments were conducted on a (Bruker Smart APEX) Single Crystal Diffractometer equipped with graphite monochromatized Mo-Kα radiation. Single Orange plate like ligand having formula of C14H13NO with single crystal was measured with λ = 1.54178 Å for X-ray intensity data by using Bruker SAINT Software package [36], (Table 4), (Fig. 6).  UV-visible spectroscopy UV-visible spectra for synthesized Schiff base ligand (L) and complexes with Ni(II), Co(II), Mn(II), Zn(II), Fe(II) metal ions were determined in the presence of the ethanol. λmax for ligand (L) was observed at 380 nm that predicts the intramolecular charge transfer interaction and π-π* electronic transitions for (-CH=N-) azomethine group. Ligand λmax absorption peak moves to the lower wavelength, complexes range was from 360-335 nm.
The elemental analysis proposes the stoichiometry to be 1:2 (metal / ligand). The geometry of metal complexes was also confirmed on the basis of data collected through electronic absorption spectra and magnetic moment [37]. The electronic spectra and magnetic moment values suggested their tetrahedral geometry (Table 5).

Molar conductance
Molar conductance values of the synthesized ligand (L) and its metal ions complexes were calculated in the DMSO (10 -3 M solutions) at 27 o C by conductometer. Values of the measured molar conductance were in the range of 16-24 Ω -1 .cm 2 .mol -1 . From the obtained data non-electrolyte nature of the synthesized compounds was predicted (Table 5) [38,39].

4.Conclusions
In this piece of work a new Schiff base ligand (L) with molecular formula C14H13NO was prepared; its purity confirms by 1 H-NMR and its divalent metal ions complexes that show biological activities. Single crystal X-ray analysis shows orange plate like monoclinic crystal structure of ligand. Molar conductance values show non-electrolyte behavior of the complexes. Compounds are A B https://doi.org /10.37358/Rev. Chim.1949 comparatively active against antifungal assay, in-vivo and in-vitro results of anti-inflammatory activity that metal complexes and ligand (L) are more reactive than standard. In-vitro antioxidant out comes reveals that compounds are active at lower concentration. The activities of all the complexes obtained were found to be moderate even though higher concentrations were applied. This may be due to the bulkiness of the molecule with a complicated structure which in turn restricts their mobility to the target cell or active site although all the complexes were obtained as a monomeric and fourcoordinated metal(II).