New Tetra-dentate Schiff Base Ligand N2O2 and Its Complexes with Some of Metal Ions: Preparation, Identification, and Studying Their Enzymatic and Biological Activities

In present work, new tetra-dentate ligand, titled 3,5-bis ((E)-5-Bromo-2-hydroxy benzylidene amino) benzoic acid (H3L), was prepared via an acid-catalyzed condensation process. New four metallic ligand complexes with Co(II), Ni(II), Cu(II) and Zn(II) ions, were also prepared from the refluxing of equivalent moles. Ligand's structure and its complexes; were confirmed by numerous characterization methods, including Ultraviolet-Visible, Infrared, Mass Spectrometer, 1 H and 13 C Nuclear Magnetic Resonance spectra, atomic absorption, magnetic moments, and molar conductivity measurements. The results of the spectroscopic analyzes proved that the prepared ligand acts as tetradentate bi-ionic ligand and it was bonded to the metal ions by two nitrogen atoms of the two azomethine groups and by two oxygen atoms of the two phenolic hydroxyl groups after losing their two protons. Octahedral structure proposed to all prepared complexes. The (anti-bacterial) and (anti-fungal) activities of these compound were screened against (E. coli, S. aureus, Klebsiella spp., S. epidermidis,), and (Candida albicans). The results indicated that these compounds have moderated inhibition behavior. The activity of the prepared compounds against Acetyl Choline Esterase Enzyme (AChE) have also studied and the obtained data indicated the presence of different inhibition behavior.


Introduction:
Generally, the ease of preparation of organic Schiff bases compounds (from the condensation among an aldehydes and a primary amines), and its ability to form stable coordination complexes (via chelation by azomethine group) with a different and huge number of metal ions in diverse oxidation states and coordination numbers, motivated the researchers in different scientific fields to prepare and use these privileged organic compounds [1][2][3][4][5][6] . The poly or tetra-dentate Schiff base ligands with N 2 O 2 coordination system and their metallic complexes have been also gained the attention of scientific researchers due to excellent complexation ability, so it is used in analytical field to remove of some heavy pollutant metals (such as Hg(II) and Ag(I)) from liquid media, in selective separation and purification of Cu(II) ions from mixtures, used as optical sensors for the determination of copper ion, and used as modified electrodes sensor of the aliphatic alcohols [7][8][9][10][11] . In biological fields, the complexes of N 2 O 2 Schiff base ligands were used as anti-bacterial against gram negative and gram-positive types, antifungal, anticancer, antioxidant, as enzyme inhibitors, antiinflammatory, and DNA-cleavage in presence of hydrogen peroxide [12][13][14][15][16][17][18][19][20] . In catalytic reactions the tetra-dentate Schiff base ligands have been used for controlling the radical polymerization, as catalysts for oxidation of phenol, a good catalytic for increasing the chain length of the imine compartment, used as active catalyst for Suzuki and Heck reactions of Iodobenzene, used to catalyze the aerobic oxidation of catechol derivative, and used as catalyst for conversing of Naphthalene derivative to derivative of Naphthoquinone (K 3 vitamin), in green synthesis process [21][22][23][24][25][26] . Also some chelated complexes N 2 O 2 Schiff base ligands reveals its coefficients as photoactive materials due to their high intensity of fluorescence 27 .

Materials and Methods:
All chemicals were acquired from suppliers companies and used as received. The IR spectra of all prepared compounds have been obtained (as a discs of KBr) in the range of 400-4000 cm -1 by using Shimadzu spectrophotometer (FTIR) model (4800S). The electronic spectra registered by using (Cary 100 con.) spectrophotometer. A (digital) SMP30 Stuart apparatus utilized for detecting melting points. Mass analysis of ligand has been done with the (Shimadzu) GC-MS QP-2010. NMR spectra related to ( 1 H and 13 C) recorded with Bruker DMX-500 spectrophotometer (300 MHz) by using (DMSO-d 6 ) as a solvent.

Preparation of Complexes
We mixed (0.001 mol, 5 mL ethanol) from each metallic salt, with (0.001 mol, 25 mL ethanol) from ligand. Few drops of Triethylamine were added to ligand solution before mixing to liberate the phenolic oxygen atom. The reaction mixtures were refluxed for 3 hrs., and then the obtained colored products, were filtered, washed with diethyl ether, and chloroform (Scheme 2).

Mass Spectrum of Ligand
The Fig. 1, represents mass spectrum of ligand, which showed a mother ion peak at (m/z=518), corresponding to [M+H]. The suggested pathways of ligand fragmentation and the structural assignments of each observed fragment are described in (Scheme 3).

NMR Spectra of Zn(II) Complex:
In 1 HNMR spectrum of Zn(II) complex, (Fig.4), the two phenolic protons disappeared due to coordination. Signals of two azomethinic protons shifted to downfield region (appeared at = 8.51 ppm), this confirmed the involvement of this characteristic group in coordination 1 . The signals of triethylamine molecule appeared at (=0.842 ppm, t, 3H) and (=2.53 ppm, q, 2H), respectively 24 . The ligand spectrum also revealed another new signal at (=5.16 ppm, s, 2H), related to coordinated H 2 O 30 . The 13 C signals of Zn(II) complex did not appear clearly due to poor solubility. The infrared bands assignments of ligand and its metallic complexes have been listed in Table 2. The FTIR spectrum of ligand, displayed the stretching frequency of carboxylic (O-H) group as medium intensity broadband at the frequency of (2500-3100) cm -1 31 . Stretching frequency of the carboxylic (C=O) group appeared as strong band at (1697) cm -1 for free ligand, this group disappeared in spectra of all complexes due to formation of carboxylate ion since we added triethylamine (as deprotonating agent) to their solutions 31,32 . The stretching frequency of the (CH=N) group appeared as a strong band in (1620) cm -1 for ligand, this band has been shifted to lower frequencies in the spectra of prepared complexes, this shifting proofs the bonded of it with metal ions 29 . The ligand phenolic (C-O) bonds revealed stretching band at (1279) cm -1 , which blue shifted in spectra of complexes, because it coordinated with metal ions. The additional and new bands noticed in complexes spectra at the frequency range (532-523 and 463-449) cm -1 , were attributed to ν(M-O) and ν(M-N), respectively 33 . The spectra of all prepared complexes also revealed new bands at (3438-3388) cm -1 assigned to stretching vibrational of coordinated water molecules 33 . The new bands appeared at (879-877) cm -1 in complexes spectra, also proofs the presence of coordinated (H 2 O) molecules 33

Molar Conductance, Magnetic Properties and Electronic Spectra of Complexes:
The conductivity values of complexes were recorded at temperature of laboratory, for DMSO solutions ( The discolored solution of (H 3 L) ligand, shows two peaks the first at (258 nm, 38759 cm -1 ), assigned to  * of benzene rings 34 . The second absorption peak was noticed at (337 nm, 29673 cm -1 ), assigned to n* transitions of nonbonding electrons related to (HC=N) groups 29 . In spectra of all complexes the second peak shifted to higher wavelength (red shift), where appeared at the range of (396-403 nm), this confirms the involvement of two (CH=N) groups in coordination with metallic ions. Spectrum of Cu(II) complex, showed new peak at (690 nm, 14492 cm -1 ) assigned to 2 Eg 2 T 2 g transition of octahedral geometry 29,35 . The Fig. 6 represents the electronic spectra of ligand and Co(II) complex Table 3. Both of (in-vitro) antimicrobial studies of prepared compounds were accomplished by following disc diffusion method. We prepared two concentrations [10 -3 & 10 -4 ] M, from each prepared compounds to evaluate the susceptibilities of bacteria and fungi, by measuring the diameter (mm) of inhibition zone (IZ), which surrounded the holes, after incubating the plate for 24 hrs., at (37 °C). (DMSO) solvent did not reveal any growth of inhibition, so it was used as negative control 29 Table 4. The results of both studies graphically are represented in Fig.7.  , then the type of inhibition constant Ki, maximum velocity Vmax and michalis-menton constant Km were detwrmined. At first, we examined the effect of solvent (DMSO), which no longer exhibited any inhibitory effect as observed and as Z. Nabeel observed too 37 . The tested complexes in the mixture at exceptional concentrations [10 -3 , 10 -5 , 10 -7 , 10 -9 , 10 -11 and 10 -13 M] (Fig.8). Figure 9 illustrates the characteristic effects of inhibitors concentrations [M] on acetylcholine (AChE) concentrations. As illustrated above, benzylidene amino benzoic acid (ligand) complexes caused noticeable inhibition effects on enzyme activity, if we make a comparison with the normal value of it (1.27 μmol/2min/mL). The second section of this study included an understanding of the type of inhibition and calculate the some kinetic parameters, such as; Km, Vmax, and Ki, at extraordinary of substrate concentrations and beneath of the same stipulations. The graph of Lineweaver-Burk showed that the complexes inhibited AChE by two types of inhibition (Mix and Non), and gave various values of Ki (Table 5)   From this analysis, the study showed that Km ranged from higher, same in the presence of complexes relative to the non-inhibiting model. A high value of (km) indicates a lower affinity of substrate [s] toward the enzyme and a higher affinity of inhibitors to attach with the (active-site) cleft of the enzyme, that is present in Co(II) complex (mix inhibition); while, the Cu(II) and Zn(II) does not quantify the substrate at the (activesite) of the enzyme, (non-competitive inhibition). AChE showed inhibition constant (Ki) in the range (10 -4 -10 -5 M) in presence of maximum inhibitors concentrations, which is probably due to variant type of inhibition from non and mix. (Table 5), clearly revealed that the (Vmax) value of control sample (2 μmol/mL/2min) in normal sample was larger than that of inhibited samples, so it is evident, that the amount of active enzyme (Vmax) is present in non-inhibited system.

Figure 6. Electronic spectra of ligand and Co(II) complex
Mesut et al. 38 found that the synthesized derivatives of sulfonamides show potential inhibitor properties for AChE with Ki constants in the range of 2.54 ± 0.22-299.60 ± 8.73 µM. The derivatives of sulfonamides exhibited different inhibition type. We determined that the derivatives (S1, S1i, S3, and S3i) showed a competitive inhibition effect, whereas others (S2, S2i, S4, and S4i) showed mixed-type inhibition. As a result, the sulfonamide derivatives can used as an alternative acetylcholinesterase inhibitor due to this effect. Inhibitors with fewer side effects are thought to be important in the treatment of AD.

Conclusions:
The newly synthesized Schiff bases ligand and its complexes with Co(II), Ni(II) Cu(II), and Zn(II), were characterized via various and different physical and analytical studies. The collected data proved that the ligand behaves as a dibasic N 2 O 2 tetra-dentate ligand; with forming thermally stable mononuclear metallic complexes. According to the results of different techniques, we suggest an octahedral environment around each metal ions. The synthesized compounds revealed moderate inhibition behavior against some of the chosen pathogens (gram-positive, gram-negative bacteria and Candida fungi) for solution of (10 -3 M), while the lowest concentration of prepared compounds (10 -4 M), did not exhibit any noticeable inhibition behaviors. The enzyme activity of complexes against Acetyl Choline Esterase Enzyme (AChE) were also studied and the obtained data indicated the presence of two different inhibition behaviors (mixed and non-competitive).