Synthesis of Schiff Bases Containing Phenol Rings and Investigation of Their Antioxidant Capacity, Anticholinesterase, Butyrylcholinesterase, and Carbonic Anhydrase Inhibition Properties

The widespread usage of Schiff bases in chemistry, industry, medicine, and pharmacy has increased interest in these compounds. Schiff bases and derivative compounds have important bioactive properties. Heterocyclic compounds containing phenol derivative groups in their structure have the potential to capture free radicals that can cause diseases. In this study, we designed and synthesized eight Schiff bases (10–15) and hydrazineylidene derivatives (16–17), which contain phenol moieties and have the potential to be used as synthetic antioxidants, for the first time using microwave energy. Additionally, the antioxidant effects of Schiff bases (10–15) and hydrazineylidene derivatives (16–17) were studied using by the bioanalytical methods of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (ABTS•+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH•) scavenging activities, and Fe3+, Cu2+, and Fe3+-TPTZ complex reducing capacities. In the context of studies on antioxidants, Schiff bases (10–15) and hydrazineylidene derivatives (16–17) were found to be as powerful DPPH (IC50: 12.15–99.01 μg/mL) and ABTS•+ (IC50: 4.30–34.65 μg/mL). Additionally, the inhibition abilities of Schiff bases (10–15) and hydrazineylidene derivatives (16–17) were determined towards some metabolic enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and human carbonic anhydrase I and II (hCAs I and II), enzymes that are linked to some global disorders including Alzheimer’s disease (AD), epilepsy, and glaucoma. In the context of studies on enzyme inhibition, it was observed that the synthesized Schiff bases (10–15) and hydrazineylidene derivatives (16–17) inhibited AChE, BChE, hCAs I, and hCA II enzymes with IC50 values in ranges of 16.11–57.75 nM, 19.80–53.31 nM, 26.08 ± 8.53 nM, and 85.79 ± 24.80 nM, respectively. In addition, in light of the results obtained, we hope that this study will be useful and guiding for the evaluation of biological activities in the fields of the food, medical, and pharmaceutical industries in the future.


Introduction
Excessive formation of reactive oxygen species (ROS) and free radicals in living metabolism can cause cell death by damaging many cellular biomolecules, especially nucleic acids, membrane lipids, and proteins. On the other hand, antioxidants neutralize free radicals and effectively terminate radical chain reactions [1,2]. Hydroxyl radicals (OH•), superoxide anion radicals (O 2 • − ), hydrogen peroxide (H 2 O 2 ), and singlet oxygen ( 1 O 2 ) can be considered the most common ROS. They are formed as a result of normal metabolic functions of the body or as a result of physical and mental stress. Additionally, radiations, organic solvents, pesticides, and cigarette smoke are thought to be exogenous sources of ROS and free radical sources [3].
They are commonly represented by the R-CH=N-Ar formula [28,29]. Schiff bases are commonly found in the structure of naturally occurring products. They have an important role in synthesis and pharmaceutical research. The chemical structures of some pharmacologically active Schiff bases are given in Figure 1. Imine structures have drug potential for diseases caused by free radical damage due to their antioxidant abilities [30]. When the literature is reviewed, it is seen that Schiff bases exhibit antioxidant ability by removing ROS [31]. Additionally, the connection between the chemical structures of Schiff bases and their antioxidant ability is well established [32]. Additionally, Schiff bases and derivatives containing the azomethine group (-N=CH-) have different applications. Therefore, they are of great interest due to their potential biological properties, including their antioxidant effects.
Heterocyclic structures containing imine and phenol groups have ROS and free radical scavenging abilities. Therefore, they have potential to be used as a medicine against some diseases induced by oxidative stress [30]. Among the chemicals that can be utilized as synthetic antioxidants, Schiff bases have a significant role and potentials. By reviewing the literature, we observed that many studies have been carried out on the antioxidant ability of Schiff bases. They are conventionally obtained through the heat treatment of ketones or aldehydes with amine compounds under acidic conditions. With the latest developments in chemistry, alternative assays have been searched and utilized for the synthesis of Schiff bases [33]. Scientists have synthesized many new and chemical compounds using the heating method. However, this method generally takes a long time and increases chemical consumption and costs [34]. The use of microwave energy has become an appreciated and popular topic in synthetic organic chemistry. The use of this energy in experiments after the 1980s has brought great benefits in organic synthesis [35]. This technique has many advantages over the classical techniques. It reduces the formation of by-products and the evaporation of solvents, shortens the reaction time, and the reactions result in higher yields [36].
Recently, increasing studies on Schiff bases and the antioxidant activity of these bases encouraged us to carry out this study. In this work, we synthesized eight Schiff bases and hydrazineylidene derivatives (10)(11)(12)(13)(14)(15)(16)(17) containing phenol rings using microwave irradiation (Scheme 1). Another important goal of this study is to examine the antioxidant abilities of these compounds. For this purpose, isovanillin (1) and different amine compounds Imine structures have drug potential for diseases caused by free radical damage due to their antioxidant abilities [30]. When the literature is reviewed, it is seen that Schiff bases exhibit antioxidant ability by removing ROS [31]. Additionally, the connection between the chemical structures of Schiff bases and their antioxidant ability is well established [32]. Additionally, Schiff bases and derivatives containing the azomethine group (-N=CH-) have different applications. Therefore, they are of great interest due to their potential biological properties, including their antioxidant effects.
Heterocyclic structures containing imine and phenol groups have ROS and free radical scavenging abilities. Therefore, they have potential to be used as a medicine against some diseases induced by oxidative stress [30]. Among the chemicals that can be utilized as synthetic antioxidants, Schiff bases have a significant role and potentials. By reviewing the literature, we observed that many studies have been carried out on the antioxidant ability of Schiff bases. They are conventionally obtained through the heat treatment of ketones or aldehydes with amine compounds under acidic conditions. With the latest developments in chemistry, alternative assays have been searched and utilized for the synthesis of Schiff bases [33]. Scientists have synthesized many new and chemical compounds using the heating method. However, this method generally takes a long time and increases chemical consumption and costs [34]. The use of microwave energy has become an appreciated and popular topic in synthetic organic chemistry. The use of this energy in experiments after the 1980s has brought great benefits in organic synthesis [35]. This technique has many advantages over the classical techniques. It reduces the formation of by-products and the evaporation of solvents, shortens the reaction time, and the reactions result in higher yields [36].
Except for compounds 11 and 16, the compounds were synthesized for the first time. It is known from the literature that compounds 11 and 16 were synthesized [37] through the condensation method using methanol or ethanol at rt or reflux. However, in this study, for the re-synthesis of compound 11 and 16, the microwave method was also used for the first time. The structures of these compounds were determined using 1 H-NMR, 13 C-NMR, FT-IR spectroscopies, and HR-MS. Then, their antioxidant properties were investigated using ABTS •+ and DPPH· scavenging abilities, and Fe 3+ , Cu 2+ , and Fe 3+ -TPTZ complex reducing capacities. Additionally, the inhibition effects of Schiff bases (10-15) and hydrazineylidene derivatives (16 and 17) were determined against some metabolic enzymes including AChE, BChE, hCA I, and hCA II enzymes, which are linked to some global disorders including Alzheimer's disease (AD), epilepsy, and glaucoma.

Materials and Apparatus
The reactions were visualized via thin-layer chromatography (TLC, 60-mesh, Darmstadt, Germany). 1 H NMR and 13 C NMR spectra were taken at 400 MHz and 100 MHz using CDCl3 (Varian spectrometer, Danbury, CT, USA). The melting points of Schiff bases were determined on a capillary melting apparatus and were uncorrected (BUCHI 530).  Except for compounds 11 and 16, the compounds were synthesized for the first time. It is known from the literature that compounds 11 and 16 were synthesized [37] through the condensation method using methanol or ethanol at rt or reflux. However, in this study, for the re-synthesis of compound 11 and 16, the microwave method was also used for the first time. The structures of these compounds were determined using 1 H-NMR, 13 C-NMR, FT-IR spectroscopies, and HR-MS. Then, their antioxidant properties were investigated using ABTS •+ and DPPH· scavenging abilities, and Fe 3+ , Cu 2+ , and Fe 3+ -TPTZ complex reducing capacities. Additionally, the inhibition effects of Schiff bases (10-15) and hydrazineylidene derivatives (16 and 17) were determined against some metabolic enzymes including AChE, BChE, hCA I, and hCA II enzymes, which are linked to some global disorders including Alzheimer's disease (AD), epilepsy, and glaucoma.

Materials and Apparatus
The reactions were visualized via thin-layer chromatography (TLC, 60-mesh, Darmstadt, Germany). 1 H NMR and 13 C NMR spectra were taken at 400 MHz and 100 MHz using CDCl 3 (Varian spectrometer, Danbury, CT, USA). The melting points of Schiff bases were determined on a capillary melting apparatus and were uncorrected (BUCHI 530).
The Cu 2+ reducing ability of Schiff bases (10-15) and hydrazineylidene derivatives (16 and 17) were detected according to a prior study [41]. For this purpose, 0.25 mL of CuCl 2 solution (10 mM), 0.25 mL of ethanolic neocuproine solution (7.5 × 10 −3 M), and 250 µL of NH 4 Ac buffer solution (1.0 M) in different concentrations (10-30 µg/mL) were transferred to test tubes containing Schiff base (10-15) and hydrazineylidene derivative (16 and 17) samples. The total volume was made up to 2 mL with distilled water, and their absorbance values were recorded at 450 nm after 30 min of incubation.
The Fe 3+ -TPTZ complex reducing ability of Schiff bases (10-15) and hydrazineylidene derivatives (16 and 17) was realized according to a previous study [42]. spectrophotometrically measured at 593 nm. All experiments of reducing abilities were repeated three times and the results are given as the arithmetic mean of these repetitions.

Radical Scavenging Capacities
DPPH· and ABTS •+ scavenging methods are the most widely used spectrophotometric methods to determine the antioxidant capacity of newly synthesized compounds. The DPPH•scavenging effect of Schiff bases (10-15) and hydrazineylidene derivatives (16 and 17) was realized according to the Blois method [43]. Briefly, 1 mL of DPPH • solution (0.1 mM), which was prepared in ethanol and was a violet/purple color depending on the concentration of the antioxidant, was added to the Schiff base (10-15) and hydrazineylidene derivative (16 and 17) samples at different concentrations (10-30 µg/mL). Then, they were incubated at room temperature for 30 min and their absorbance values were recorded at 517 nm.
The ABTS radical cation scavenging assay was used as a way to calculate antioxidant capacity based on this radical scavenging ability. Firstly, an aqueous solution of ABTS (7.0 mM) was oxidized by oxidants such as K 2 S 2 O 8 (2.5 mM) for the production of its radical cation (ABTS •+ ) [44]. The ABTS •+ solution was diluted with a phosphate buffer (0.1 M, pH 7.4) prior to use, adjusting the absorbance value of the control to 0.750 ± 0.025 at 734 nm. Then, 1 mL of ABTS •+ solution was added to 3 mL Schiff base (10-15) and hydrazineylidene derivative (16 and 17) solutions at different concentrations (10-30 µg/mL). After 30 min, the remaining absorbance of ABTS •+ measured at 734 nm.

AChE and BChE Inhibition Assay
The AChE and BChE inhibition of Schiff bases (10-15) and hydrazineylidene derivatives (16 and 17) was realized according to a putative Ellman's assay as given in previous studies [45]. Acetylthiocholine iodide/butyrylthiocholine iodide (AChI)/BChI) and 5,5 -dithiobis(2-nitro-benzoic acid) (DTNB) were used as the substrate pattern for both cholinergic reactions. Briefly, 1 mL of Tris/HCl buffer (1.0 M, pH 8.0), 10 µL of different concentrations of Schiff bases (10-15) and hydrazineylidene derivatives (16 and 17), and 50 µL AChE/BChE enzymes were mixed in a test tube. Then, the samples were incubated at 25 • C for 15 min, and 50 µL of DTNB solution (0.5 mM,) was transferred. Then, the reaction was started by adding 50 µL of AChI/BChI solutions (10 mM), and absorbances were recorded at 412 nm. All experiments were repeated three times and the results are given as the arithmetic mean of these repetitions.

Carbonic Anhydrase Purification and Inhibition Studies
Both hCA I and II isoforms were purified using the affinity chromatography technique including Sepharose-4B-L-Tyrosine-sulfanilamide affinity material [46]. Both isoforms' activity was spectrophotometrically determined according to the Verpoorte assay [47]. One CA unit is defined as the CA quantity, which catalyzes p-Nitrophenylacetate substrate to p-nitrophenolate in 3 min at 348 nm (25 • C). The protein quantity was spectrophotometrically measured at 595 nm according to the Bradford method as bovine serum albumin equivalent [48]. SDS-PAGE was realized according to the Laemmle procedure, which includes 3 and 10% acrylamide concentrations used to control the enzyme purity [49]. All experiments of the CA inhibition assay were repeated three times and the results are given as the arithmetic mean of these repetitions.

Antioxidant Results
The excessive formation of free radicals and ROS in living organisms disrupts the structure of many cellular biomolecules and may cause some degenerative diseases [51]. Therefore, oxidative stress and ROS pose significant risks for many chronic diseases such as cancer, cardiovascular diseases, immunodeficiency syndrome, age-related pathologies, arteriosclerosis, diabetes mellitus, and obesity [52]. Antioxidants eliminate undesirable harmful effects of ROS and free radicals even at low concentrations. Additionally, they reduce oxidative stresses in the human body or food systems and prevent harmful effects of ROS [53].
All the synthesized Schiff bases (10-15) and hydrazineylidene derivatives (16 and 17) displayed in vitro inhibition effects against cytosolic hCA I, which is associated with cerebral and retinal edema, hCA II, which is associated with edema, glaucoma, epilepsy, and mountain sickness, and AChE and BChE, which have been linked with AD for their inhibition efficacy. Mountain sickness is a disease that affects mountaineers or travelers at high altitudes who do not have enough time to acclimatize to altitudes above 2400 m [56,57]. They often develop symptoms including headaches, appetite loss, nausea, poor sleep, gastrointestinal distress, and general malaise due to low oxygen levels. In some cases, mountain sickness can cause brain edema and even death. The CA inhibitory effects of the Schiff bases (10-15) and hydrazineylidene derivatives (16 and 17) were determined using an esterase assay [53] and compared to acetazolamide (AZA). For the determination of the synthesized Schiff bases' (10-15) and hydrazineylidene derivatives' (16 and 17) action towards AChE and BChE, Ellman's procedure [45] was employed, and compared to the standard inhibitor of Tacrine. Further, the following insights can be gleaned from the studied enzyme inhibition results given in Tables 3 and 4. The synthesized Schiff bases (10)(11)(12)(13)(14)(15) and hydrazineylidene derivatives (16 and 17) exhibited effective inhibition profiles against widespread cytosolic hCA I isozyme with Ki values ranging from 26.08 ± 8.53 nM to 85.79 ± 24.80 nM. Isovanilin (1), as our starting material, showed a lesser inhibition profile (Ki: 112.30 ± 25.75 nM) when compared to the synthesized Schiff bases (10)(11)(12)(13)(14)(15) and hydrazineylidene derivatives (16 and 17). However, within this series, the compounds (E/Z)-5-(((4-aminophenyl)imino)methyl)-2-methoxyphenol (14) were found to be the best inhibitor (Ki: 26.08 ± 8.53 nM) towards cytosolic hCA I isozyme in comparison with AZA (K i : 35.39 ± 9.01 nM). However, the compounds 17, 13, and 14 showed stronger inhibition ability than AZA. Common to all hCA isomers of the α-CA family is a highly conserved active site, which a Zn 2+ ion coordinated by His94, His96, and His119 residues and an H 2 O molecule. Most hCA inhibitors have been identified as Zn 2+ -binding molecules. Overexpression of the hCA I isozyme has been associated with cerebral and retinal edema, while the hCA II isoform has been associated with altitude sickness, glaucoma, and epilepsy [58].

Discussion
In this study, the synthesis of Schiff bases using the microwave method, and their antioxidant capacity and some metabolic enzyme inhibition properties were reported for the first time. Here, six Schiff bases (10-15) and two hydrazineylidene derivatives (16 and 17) were synthesized using an environmentally friendly methodology. A simple, efficient, and fast method was applied for synthesis that does not include solvents or catalysts. Compared to other methods, microwave irradiation is the simplest and cheapest way to synthesize novel Schiff bases.
Schiff bases are an important class of organic compounds that are commonly used and researched due to their unique structural properties and biological activities. Additionally, they have numerous applications, especially in biochemistry and medicine [59]. It is known that Schiff bases have received great attention due to their many biological and pharmaceutical activities [60] such as antifungal, antiviral, antitumor, antibacterial, antimalarial, anticancer, and anti-inflammatory activities and enzyme inhibition properties [61][62][63][64]. The biological activity of Schiff bases originates from the imine or azomethine (-C=N-) func-tional groups, as well as hydrophobic aromatic groups, and they can coordinate easily with metals to form versatile functional complexes [65]. The enzyme inhibition properties of Schiff bases were tested against very important metabolic enzymes such as CA, AChE, and BChE, which are associated with some global disorders, and it was observed that they effectively inhibited them. Recently, three series of symmetrical Schiff bases and their amine derivatives were tested towards AChE and hCA I and II isoenzymes, and demonstrated nanomolar inhibition profiles against the indicated metabolic enzymes, which have a significant role in drug discovery and design as well as in toxicology and medicine [59]. It was reported that Schiff bases, as kind of compounds containing azomethine groups, exhibited antioxidant activity, especially O 2 •scavenging activity. In this context, it has been observed that Schiff base complexes containing copper can almost completely remove the existing O 2 •even at low concentrations [66]. In another study, the antioxidant activity of some resveratrol analogues including 4hydroxyphenyl-benzo[d]thiazole, p-(N,N-dimethyl)aminobenzylidene-2-aminothiophenol, and p-nitrobenzylidene-2-aminothiophenol were synthesized, characterized, and their antioxidant activity was evaluated [67]. Better antioxidant and antifungal activity of chitosan derivatives bearing Schiff bases were reported. In a recent study, many chitosan derivatives containing Schiff bases were synthesized. In this study, the structural characterization of chloracetyl chitosan oligosaccharide derivatives grafted with pyridine-4-aldehyde Schiff bases was performed, and their antioxidant activities against DPPH•radical and O 2 •were determined [68]. Similarly, it was indicated that some chiral selenite ligands and their palladium complexes have antioxidant activity, which increased with concentration [69]. In a recent study, it was shown that the Co 2+ and Fe 2+ complexes of Schiff bases demonstrated effective antioxidant ability using different methods including the FRAP and CUPRAC reducing methods, and ABTS and DPPH radical scavenging methods [60]. Similarly, some Schiff base ligands ((E)-6-methyl-2-(2,3,4-trimethoxybenzylideneamino)-4,5,6,7-tetrahydrobenzo[b]-thiophene-3-carbonitrile) and their Co 2+ and Pd 2+ complexes exhibited powerful antioxidant abilities [10]. The transition metal complexes from bidentate Schiff base ligands containing both amine (-NH 2 ) and -OH groups have been extensively studied. In particular, Schiff base-metal complexes containing nitrogen and oxygen donor atoms show many application areas such as catalytic and biological activities [10]. Antioxidants containing such Schiff bases significantly prevent the formation and accumulation of free radicals, and protect the body from oxidative damage. In a recent study, Schiff bases and new secondary amine derivatives of p-vanillin demonstrated powerful antioxidant abilities by strongly scavenging ABTS (IC 50 : 1.25-464.38 mM) and DPPH radicals (IC 50 : 2.20-870.78 mM) and by exhibiting strong Fe[(CN) 6 ] 3+ reducing ability [70].
In conclusion, the newly synthesized Schiff bases are promising potential antioxidant agent candidates for the scavenging of ROS, which cause damage in humans. Additionally, we believe that these results may be useful for the synthesis of new hCA I and II isoenzymes, AChE and BChE inhibitors, and in the development of drugs for the treatment of some common and global diseases including edema, epilepsy, glaucoma, mountain sickness, and AD.