Synthesis, Crystal Structure, DFT Studies and Evaluation of the Antioxidant Activity of 3,4-Dimethoxybenzenamine Schiff Bases

Schiff bases of 3,4-dimethoxybenzenamine 1–25 were synthesized and evaluated for their antioxidant activity. All the synthesized compounds were characterized by various spectroscopic techniques. In addition, the characterizations of compounds 13, 15 and 16 were supported by crystal X-ray determinations and their geometrical parameters were compared with theoretical DFT calculations at the B3LYP level of theory. Furthermore, the X-ray crystal data of two non-crystalline compounds 8 and 18 were theoretically calculated and compared with the practical values of compounds 13, 15, 16 and found a good agreement. The compounds showed good DPPH scavenging activity ranging from 10.12 to 84.34 μM where compounds 1–4 and 6 showed stronger activity than the standard n-propyl gallate. For the superoxide anion radical assay, compounds 1–3 showed better activity than the standard.


(iii) Sequential Proton Loss Electron Transfer (SPLET)
The above mechanism consists of three steps. In the first step, a heterolytic bond dissociation of a phenolic hydroxyl group leads to the formation of a phenoxyl anion and the release of a proton. In the second step, an electron transfer from the phenoxyl anion to the free radical leads to the formation of a phenoxyl radical and an anion (R − ). In the end, the protonation of R − leads to the formation of RH. This mechanism is strongly favored under alkaline conditions (e.g., high pH), which may help in the proton of the first step [39,40].

(iv) Adduct formation (AF)
The AF mechanism is more specific and is observed between (a) carbon centered radicals and double bonds; or (b) hydroxyl radicals and aromatic rings. Numerous side reactions may occur that lead to stable adducts from [ArOH-R] • .
In continuation of our research on the synthesis of bioactive small molecules [41][42][43], we synthesized a series of 3,4-dimethoxybenzenamine Schiff bases (Scheme 1) and evaluated their antioxidant potential in the search of the potential antioxidant leads.

DPPH Scavenging Activity
The synthesized compounds 1-25 showed activity in the range of 10.12-84.34 μM (Table 1). Compound 1 (IC 50 = 10.12 ± 0.54 μM) showed highest activity, three times more active than the standard (IC 50 = 30.30 ± 0.2 μM). This is due to the ortho-trihydroxyl group which is known to show very good activity [51,52]. Compound 2 is a meta-trihydroxyl analogue but showed slightly less activity than compound 1. This may be due to the ortho-trihydroxyl groups of compound 1 which is similar to the catecholic moeity known to exhibit good antioxidant activities [53][54][55][56]. Incidently, compounds 1 and 2 which have an additional hydroxyl group as compared to compounds 4 and 6, showed stronger antioxidant activities than the latter two compounds. Among the five dihydroxyl analogues, compound 3, 4 and 6 showed better activity than standard. The activity of compound 3 is due to the 2', 5' positions of the dihydroxyl groups, favorable for stabilization of the free radical. The catecholic moeity in compounds 4 and 6 is well known structural feature for good activity [53][54][55][56]. Other meta-dihydroxyl analogues 5 and 7 also showed very close activity as compared to the standard. Compound 8 showed good activity due to adjacent 3-methoxyl and 4-hydroxyl positions. However, its other analogue 11 with reversed arrangement showed moderate activity.
For the mono-hydroxyl series, compound 9 having hydroxyl at 4' position showed good activity while its other analogues 10 and 15 showed no activity, due to the lack of free radical stabilizing capability. The presence of a bromo substituent further increases the capability to stabilize radicals as illustrated by compound 12. meta-arranged methoxyl and hydroxyl groups contributed to the good activity of compound 14. The remaining compounds do not possess functional groups to help stabilize free radicals and are therefore inactive.

Superoxide Scavenging Activity
Compounds 1, 2 and 3 showed better activity than the standard drug n-propylgallate (Table 2). Compound 4 showed good activity. Compounds 5, 6, 7, 8, 9 and 12 showed moderate activities, while compound 14, 20 and 21 showed weak activities. The good activity of compounds 1-3 may be due to more stabilizing potential of these compounds to stabilize free radicals generated during bioassay. DPPH scavenging activity and superoxide scavenging activity mainly depend on the hydroxyl position as well as number of hydroxyl groups present in the molecule [57,58]. SEM a is the standard error of the mean, NA b = Not active, n-propyl gallate c was the standard drug for the superoxide anionradical scavenging assays.

Compound 13
The structure of compound 13 is composed of a dimethoxybenzene moiety link with methoxyphenol moiety via azomethin bridge which adopts an E configuration ( Figure 1). The dimethoxy-substituted planar benzene moiety (C1-C6) is oriented at a dihedral angle of 29.33(9)° with respect to the methoxy-substituted planar phenol moiety (C1'-C6') with standard deviation of 0.016(2)° for C2' atom from root mean square plane. In the crystal lattice ( Figure 2), molecules are linked via C-H···O hydrogen bonding and form three dimensional consolidated network of mirror imaged sets running along the b axis where each set contains four molecule.

16
Crystallographic data of compound 13 (CCDC 980015), can be obtained from Cambridge Crystallographic Data Center without any cost. Crystal and experimental data of compound 13 are presented in Table 3 and the hydrogen bonding data in Table 4.

Compound 16
Compound 16 is structurally similar to Compound 13 and Compound 15 with the only difference that all four substituents on the aromatic skeleton are methoxyl groups. In this molecule the two benzene rings and the azomethine group are practically coplanar and the molecule adopts an E configuration about the C8-N7 bond. The dihedral angle between the two planar benzene rings (C1'-C6' and C1-C6) was found to be 40.56(6)° with standard deviation of −0.019(1)° for C1 atom from root mean square plane. All the bond distances are within normal range comparable to those of similar compounds. No chemical intramolecular interaction was observed. However in the crystal structure, molecules were linked via C2-H2'A...O1 and C3-H3A...O1 intermolecular interactions to form R 2 2 (20) ring motive running along c-axis.
The crystallographic data of compound 16 (CCDC 980016), can be obtained from Cambridge Crystallographic Data Center. Crystal and experimental data of compound 16 presented in Table 3 and hydrogen bonding data in Table 6.

DFT Calculations
Crystal structures of compounds 13, 15 and 16 were compared to their optimized minima ( Figure 3). The initial geometrical structures were obtained from the molden files of the X-ray solved structures. The optimization has been carried out at the B3LYP/6-311+G(d,p) level of theory by using Gaussian 09 package [7]. The minima of the optimized structures were confirmed by the absence of imaginary frequencies. The experimental and calculated bond lengths, bond and dihedral angles of the compounds are presented in Table 7.
The experimental bond lengths and bond angles z-matrix coordinates are well reproduced theoretically. On the other hand, the dihedral angles are well reproduced, except for the dihedral angle between the 3,4-dimethoxybenzenyl and the azo group where a slight deviation was observed between the crystal structure compared to the optimized conformation. The hydrogen bonding between 2'-OH group and the azo group in Compound 13 is well reproduced theoretically, with a difference of 0.04 Å ( Figure 1). In order to generalize the comparison between structural X-ray and calculated results, the optimized structures of compounds 1, 2, 3, 8 and 18 were obtained at the same level of theory (Table 7 and Figure 3). The structural parameters (bond, angles, and torsion angles) for the optimized structures of 1, 2, 3, 8 and 18 are very similar to the optimized and X-ray parameters of 13, 15 and 16.

General Information
NMR experiments were performed in DMSO-d 6 on a Bruker Ultra Shield 500 MHz FT NMR (Wissembourg, Switzerland). CHN analysis was performed on a Carlo Erba Strumentazione-Mod-1106 (Milan, Italy). Electron impact mass spectra (EI-MS) were recorded on a Finnigan MAT-311A instrument (Bremen, Germany). Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Darmstadt, Germany). Chromatograms were visualized by UV at 254 and 365 nm.

DPPH (1,1-Diphenyl-2-picryl hydrazyl) Free Radical Scavenging Activity
The free radical scavenging activity was measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay using literature protocols. The reaction mixture contained test sample (5 μL, 1 mM in DMSO) and DPPH (Sigma, 95 μL, 300 μM) in ethanol. The reaction mixture was taken into a 96-well microtiter plate and incubated at 37 °C for 30 min. The absorbance was measured at 515 nm using microtitre plate reader (Molecular Devices, Sunnyvale, CA, USA). Percent radical scavenging activity was determined in comparison with DMSO containing control (Table 1). IC 50 values represent the concentration of compounds able to scavenge 50% of DPPH radicals. Propyl gallate was used as positive control. All chemicals used were of analytical grade (Sigma, Ronkonkoma, NY, USA).

In Vitro Assay for Superoxide Anion Radical Scavenging Activity
The superoxide producing system was set up by mixing phenazinemethosulfate (PMS), NADH, and oxygen (air), and the production of superoxide was estimated by the nitroblue tetrazolium method. Measurement of superoxide radical scavenging activity was carried out on the basis of the method described by the modified method used by Ferda. In aerobic reaction mixtures containing NADH, phenazine methosulphate and nitro blue tetrazolium, PMS is reduced by NADH and then gave rise to

Theoretical Calculations
The optimization of the synthesized Schiff bases were performed at the B3LYP/6-311++G(d,p) level of theory [59]. The minima were confirmed by vibrational frequency analysis (i.e., no imaginary frequency were found). All theoretical calculations were carried out using Gaussian09 package [60].

Conclusions
In conclusion, compounds having hydroxyl groups at suitable places as well as number of hydroxyl groups play a key role in the antioxidant activity. Three crystal structures along with its theoretical calculations are also reported with experimental value well correlated.