Physicochemical Studies of Benzocaine Bearing Heterocycles as Potential Antioxidant Agents

Abstract Series of new thirteen annulated heterocyclic were prepared and screened for their antioxidant activities. The ethyl-4(2-cyanoacetamido)benzoate as a multifunctional component which possess both electrophilic and nucleophilic centers that were utilized in organic synthesis of different candidates. These new compounds studied as antioxidants at different concentrations based on DPPH and ABTS assays using ascorbic acid as a standard antioxidant. The most potent compound was 5 that recorded as the best antioxidant compound with IC50 equal 0.006 mg/ml for DPPH results. While for ABTS study, compound 4 anticipated the best result with IC50 equal 27.46 µM. Different reactive and thermodynamic descriptors were calculated as a preliminary step for a comparative thermodynamic study for antioxidant property. The results clarified that the potent compounds showed dependence on SET–PT mechanism than HAT or SPLET mechanism. These mechanisms depended on different parameters as proton affinity and electron transfer energy that were studied. SET–PT mechanism was dependent on two descriptors: IP and BDE which were studied and observed that were less-valued in site A for two potent compounds 4 and 5. Graphical Abstract


Introduction
Therapeutic chemistry contribution to launch clinically advisable antioxidants to our world becomes strongly vital demand, after several environmental and clinical conditions surrounding humanity caused serious diseases as inflammation and cancer. 1,2 Different disorders such as pollution, radiation, pressure, smoking, and oxidative stress seriously affect humanity by rising the content of reactive species on the body (RS). Then RS can go through several chain reactions and gradually destruct our lipids, proteins, as well as DNA. 3 So it is advisable to receipt regular a natural balanced diet to rise the content of antioxidants in humans' body. 4,5 In this regard, some chemicals play important role as antioxidant as the benzocaine. Benzocaine is a chemical compound characterized by treatment of anesthesia, 6 in addition to possessing anti-oxidant, 7 antibacterial, 8 antifungal, and antitumor activity. Highly biologically active compounds such as leteprinim that could be obtained by the modification of benzocaine. The former compound enhances neuron survival in the brain, even more recent studies have discussed the use of leteprinim for the treatment of neurodegenerative disorders such as Alzheimer's, Parkinson's disease, and stroke. In order to get modification to benzocaine, cyano-acetanilide derivative is a key precursor for five-and six-membered heterocyclic compounds as well as other acyclic compounds. 9 We herein report the synthesis of new benzocaine derivatives as hybrid molecules possessing antioxidant activity operating low-cost cyanoacetanilide intermediate as the starting material. Then, structural characterization of compounds and antioxidant mechanism thermodynamically were discussed with the aid of the density functional theory (DFT) method in the gas and liquid phases. [10][11][12][13][14][15] A large number of natural occurring compounds have nitrile and amide systems in their structural system. Cyanoacetanilide derivatives possess nitrile and amide entities give rise to high potential bioactive pharmacological applications to those compounds. 16 Some cyanoacetanilide compounds reported as anti-oxidant, anti-inflammatory, anticancer, and antimicrobial agents in initial pre-clinical trials. Potent cores as benzothiazole moieties, 17 benzimidazole, 18 pyrazole, 19 thiophene, 20 and coumarin derivatives recently reported as antioxidant species. 21 Three particularly important mechanisms for the free radical scavenging activity were discussed later, the hydrogen atom transfer, single-electron transfer followed by proton transfer (SET-PT), and the sequential proton loss electron transfer (SPLET). 12

Organic synthesis
Gallenkamp electric melting point apparatus were used to measure the melting point and were uncorrected. The IR spectra V/cm À1 (KBr) were recorded on a ThermoFisher Scientific Nicolet IS10 FTIR spectrometer (USA), spectral analyses unit, Mansoura University. The 1 H NMR and 13 C NMR spectra were run on JNM-ECA 500 II (Jeol, Japan), Bruker WP spectrometer at 500, 400, 125, and 100 MHz, respectively, using tetramethylsilane (TMS) as an internal reference and DMSO-d6, as solvents.

General procedure for compounds 3-6
Ethyl-4-(2-cyanoacetamido) benzoate(1) (0.46 g, 2 mmol) was dissolved in DMF containing an equivalent amount of potassium hydroxide and cooled to 0-5 C then carbon disulfide (2 mmol, 0.152 ml) was added. The mixture was left stirring for 6 hours and then appreciate reagent (chloroacetyl chloride, ethyl bromoacetate, or methyl iodide) was added to mixture and left stirred for additional 6 hours. The mixture was treated with ice-cold water, filtered, dried, and recrystallized from ethanol in case of two compounds [3][4]. While the addition of methyl iodide was completed by in situ addition of orthophenylenediamine or 2-aminothiophenol. The latter mixtures were stirred overnight to complete the reactions to yield compounds 5-6 after treating with ice-cold water, filtered, dried, and recrystallized from ethanol.

Biological activities
2.2.1. DPPH free radical scavenging activity assay The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of the benzocaine derivatives were determined according to the reported method by Blois et al. 22 The conversion of stable DPPH free radical from purple to yellow colored diphenylpicryl hydrazine was the indication to electron donating ability of a test compound. DPPH radical (0.004%) was dissolved in methanol and then 4 ml of the solution was mixed with 1 ml of a serial dilution of the tested compounds. After incubation in the dark for 30 minutes at room temperature, the absorbance of the samples was measured against a control at 517 nm. The radical scavenging activity (RSA) % of the DPPH radicals was calculated from (Equation 1): where A control is defined as the absorbance of the control, A sample is defined as the absorbance of benzocaine derivatives. The scavenging activity of free radicals in the sample is due to the presence of molecules which is known as antioxidants. The inhibitive concentration (IC50) of the sample required to scavenge DPPH radical by 50% was obtained by a linear regression analysis curve plotting the different concentrations of a sample against the percentage of remaining DPPH. IC50 (half maximal inhibitory concentration) value is the concentration of the sample that can scavenge 50% of DPPH free radical in DPPH free radical scavenging method. The IC50 value is inversely proportional to the free radical scavenging activity/antioxidant property of the sample. Thus, the sample will require less amount in scavenging the free radical if the IC50 value is less or vice versa.

ABTS radical cation decolorization assay
This assay was done according to the original idea of Re et al., 23 absorbance was measured at a wavelength of 734 nm. ABTS [(2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid))diammonium salt] produces stable free radical that could be decolorized in its non-radical form. The ABTSÁþ working solution was prepared by diluting the ABTSÁþ stock solution in pure methanol, a blank was adjusted in this present assay to be exactly 0.6 before measuring the absorbance for all the test compounds. Free radical scavenging activity was evaluated by mixing 900 ml of the blue-green ABTSÁþ working solution with 10 ml of the solutions of the target test compounds at various concentrations. The percent reduction in absorbance (which represents the ABTSÁþ radical cation scavenging activity of the test compound) was calculated according to the following equation: ABTS Áþ radical cation scavenging activity of test compound % ð Þ ¼ 100ðA blank À AtestÞ=A blank, where, A blank is the absorbance of ABTSÁþ radical cation in H 2 O and MeOH directly before adding the test compound to the ABTSÁþ radical cation (A blank was adjusted to be 0.60), while A test is the absorbance of the ABTSÁþ radical cation (or of the reaction mixture) in H 2 O and MeOH after 15 minutes of adding the test compound to the ABTSÁþ radical cation. For each of the test compounds, the IC50 (the inhibitory concentration 50%, it is the concentration of any test compound needed to decrease the absorption or the amount of ABTSÁþ radical cations by 50% at a wavelength of 734 nm) was compared to that of L-ascorbic acid. The calculation of ABTSÁþ IC50 value (for each test compound along with the reference L-ascorbic acid) achieved using GraphPad Prism 6 software and the more powerful compound as antioxidant possessed lower IC50 value.

Computational details
Since computer aided chemistry could facilitate the selection of target moiety among different novel synthetized compounds and study the effects of different parameters on oxidative process. All information regarding the thermodynamic parameters has been used to get conclusions about their possible oxidation mechanism, hence the conclusion was the consideration of water resources as active components in pharmaceutical formulations. Material studio and Guass view 5.0.9 programs, have been used for DFT study. A B3LYP functional have been used with the basis sets, 6-311 G(d, p) have been used, the solvent was taken into account by the simple point charge (SPC) model. 13,15,24
Interestingly, the condensation reactions with different carbonyl compounds as p-bromobenzaldehyde, piperonal, furfural, and/or isatin were a facile strategy to construct compounds 8-12. Due to the reactivity of isatin, the reaction went feasibly without any catalyst while the other condensation reactions required piperidine as a catalyst. 1 H NMR spectrum of compound 9 as an example, showed aliphatic protons at d 1.29, 4.27 ppm, furan protons at d 6.86, 7.44, and 8.20 ppm, aromatic protons at d 7.81, 7.94 ppm, CH olefinic and NH at d 8.14, 10.56 ppm, respectively. 1H-1H NOESY of compound 8c as shown in Figure 2 showed correlation peak between CH olefinic and NH group that confirmed trans-configuration of the compound (Scheme 2).
Furthermore, the attack of activated methylene of compound 1 on other carbonyl compounds as indan-1-one (Scheme 3) in basic condition followed by treatment with elemental sulfur furnished thiophene derivative 13. Novel 2-iminopyran derivatives 15, 17 were synthesized when ethyl-4(2-cyanoacetamido)benzoate(1) reacted with enamine derivatives as 2-((dimethylamino)methylene)-2,3-dihydro-1H-inden-1-one (14) and/or (1-(4-chlorophenyl)-3-(dimethylamino)prop-2-en-1-one (16). Moreover, the condensation reaction with DMFDMA furnished enamine 18 that turned into iminopyrazole derivative 19 when reacted with hydrazine monohydrate in ethanol containing triethylamine as a catalyst. Catalytic dependence of triethylamine or piperidine when thiourea was used as a reagent did not produce any products. Interestingly, the latter reaction underwent using pyridine as a catalyst. The structural features of synthesized compounds were confirmed by experimental data as IR, 1 H NMR, and 13 C NMR tools. The disappearance of activated methylene protons of starting material 1 in 1 H NMR spectra was the driving force of these different reactions. In addition, disappearance of nitrile group in IR spectra confirmed the cyclization in some compounds as 13,15,17,19, and 20. 1 H NMR spectrum of compound 15, showed

Antioxidant activity
Benzocaine turns iron (II) into iron (III) causing Methemoglobinemia, this main side effect limited local anethethetic of benzocaine and stopped its utilization by dentists to relief pain of teeth. 26 Hence studying the modified benzocaine derivatives as antioxidant species is another point of view, although there is urgent demand for studying the main anesthesia function of our targets within modified skeletons and it will be checked in another future research.

DPPH free radical scavenging activity
The synthesized compounds were evaluated as antioxidant agents using DPPH assay at different concentrations of each tested sample, Ascorbic acid was used as a standard antioxidant. The radical scavenging activity was determined for the synthesized compounds using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) as a stable radical organic compound and its oxidative method widely used in the determination of the capacity of free radical scavengers or the capacity of hydrogen donors following in vitro assay (Figure 3). 27,28 Table 1 showed IC50 values (mg/ml) for benzocaine derivatives, the results showed that compounds 5, 6 with IC50 equal (0.006, 0.007 mg/ml, respectively) were more potent than ascorbic acid with IC50(0.022 mg/ml). The compound 3 showed results (IC50 ¼ 0.021 mg/ml) bit similar comparing with ascorbic acid (IC50 ¼ 0.022 mg/ ml). While the compounds 8c, 8d, 9,12,13,17,18,20, and 21 showed moderate results <1 mg/ ml. The compounds 4, 8a, 8b, 8e, 8f, 11, 15, and 18 showed little activity as antioxidant species.

ABTS radical cation decolorization assay
The radical ABTS was obtained by mixing ABTS þ (2,2 0 -azinobis(3-ethylbenzothiazoline6-sulfonic acid) radical cation), potassium persulfate in the dark, a compound that can give a hydrogen atom or an electron (Figure 4), with decolorization of dark green color. 29 Results of the antioxidant capacities of the target benzocaine derivatives (expressed as IC50 values) in a comparison with ascorbic acid as a reference antioxidant. Table 2 showed IC50 values (mM) for benzocaine derivatives, the results showed that compounds 4 with IC50 equal (27.46 mM) were more potent than ascorbic acid with IC50(27.57 mM). The compounds 5, 9, and 17 showed good results, while the compounds 8d, 11, 18, and 19 showed moderate results. The compounds 6, 8a, 8b, 8c, 8f, 12, and 13 showed less activity as antioxidant species.

Computational study
Compounds possessing reactive nitrogen, oxygen, and carbon atom species usually achieved good results through antioxidant tests. The antioxidant is a compound capable of breaking the chain of free radicals and donation of hydrogen atoms. The mechanism of action of antioxidant tests has a sequence initiated by the transfer of hydrogen atom, single electron, and followed by proton transfer. So counting some descriptors as number of donor atoms, acceptor atoms, sulfur, nitrogen, and oxygen species could reflect number of atoms capable to undergo antioxidant behavior. As number of atoms capable of antioxidant behavior was increased, the antioxidant activity should be increased. Table 3 reflected some descriptors as number of donor atoms, acceptor atoms, sulfur, nitrogen, and oxygen species, theses descriptors were calculated by MOE software 2014. Compounds 5,13,19, and 20 possess high numbers of donor atoms although antioxidant potential of these compounds was dissimilar. Studying theses structural features of compounds did not match parallel with experimental results. In order to correlate the structure with antioxidant activity, studying some 3D-descriptors as HOMO, LUMO frontier molecular orbitals were interesting. The chemical reactivity was reflected through the energy band gap (E LUMO ÀE HOMO ), electronegativity (v), chemical potential (m), global hardness (g), global softness (r), and global electrophilicity index (x) that were calculated from energies of frontier molecular orbitals (E HOMO , E LUMO ), by Equations (1) to (6) as follow: 30 l ¼ Àv g ¼ 1=r For all the compounds, the geometry optimizations was performed using the DFT by the DNP basis set in Materials Studio package. 31 The solvation effects were considered, the dielectric constant (80.4) was used in order to simulate the solvent (water). DFT calculations have been performed to confirm that all the optimized structures as actual minima.
As shown in Table 4, compounds 18 and 20 possessed high energy gap equal 3.25 and 3.05 ev, respectively, however two compounds showed little activity as antioxidant species. As the compounds have high energy gap were called hard molecules, while those with low energy gap were called soft molecules. 32 But compounds 5, 6 showed energy gap equal to 2.98 and 2.76 ev as shown in Figure 5, respectively, and these compounds recorded best results using DPPH assay. Also, compounds 3 and 4 manifested energy gap equal to 1.67 and 1.68 ev as shown in Figure 6, respectively, and compound 4 recorded high activity using ABTS assay. A good nucleophile is characterized by low values of (m) and (x); while a good electrophile is characterized by high values of (m) and (x). From Table 5, compounds 5, 13, and 19 had low m while compounds 3, 8a, 8b, and 20 had low values of x. As the value of m was decreased, the ability of compound to accept electrons increased. 33 So the ability of compound 5 to gain electrons matched parallel with the value of m. Unfortunately, the compound 19 that manifested low value of m, and showed less activity as antioxidant species. Furthermore, it is found that the stability of molecules is related to hardness. Chemical hardness is the resistance of the chemical potential to change in the number of the electron; which could be correlated to the stability and reactivity of a chemical system. Compounds 5,18, and 20 had high value of g which reflected their reactivity. Pauling presented the concept of electronegativity as the power of an atom in a molecule to attract an electron to itself. 34 According to Parr et al., the electrophilicity index has been introduced to measure the energy lowering due to the highest electron transfer between donor and acceptor. Compound 5   Code  a_don  a_donacc  a_nN  a_nO  a_nS  Code  a_don  a_donacc  a_nN  a_nO  a_nS  5  3  6  4  3  0  11  1  5  4  3  0  6  2  5  3  3  1  9  1  4  2  4  0  3  1  5  2  4  2  12  2  6  3  4  0  4  1  6  2  7  2  13  3  4  2  3  1  8a  1  4  2  3   showed moderate value of electrophilicity, compound 8b had least value, and compound 19 had highest value.

Theoretical mechanism
Subsequently, two of the potent compounds with best antioxidant activities 4 and 5 were thermodynamically studied. The relationship between the activity and the structural characteristics of antioxidant compounds can be interpreted based on three main mechanistic pathways of radical scavenging. Firstly, the formal hydrogen transfer (FHT) mechanism where the main step is the dissociation of a hydrogen atom from the antioxidant molecule. Therefore this mechanism is defined energetically by the bond dissociation enthalpy (BDE).
Secondly, the SET-PT mechanism that is characterized by two thermodynamic parameters: ionization potential (IE) (for the electron transfer step) and proton dissociation enthalpy (PDE) (proton transfer from the ionized molecule).
Third, sequential proton loss electron transfer (SPLET) mechanism where the first step is proton dissociation that was characterized energetically by proton affinity (PA) and electron transfer enthalpy (ETE) which is the coming step in the mechanism.
The total enthalpies of the species X is calculated from the following equation: where E 0 is the calculated total electronic energy, ZPE stands for zero-point energy, DH trans , DH rot , and DH vib are the translational, rotational, and vibrational contributions to the enthalpy. Finally, RT represents pV-work term and is added to convert the energy to the enthalpy. Total enthalpies were calculated at T ¼ 298 K (Table 6).
4.3.1.1. Analysis of the FHT mechanism. FHT mechanism shows dependence on BDE value which reflects a mechanism in which H atom directly shifted to free radical from the antioxidant species by NH homolytic bond cleavage. The stability of the corresponding NH group was a reflection to BDE value. The easier N À H bond could be broken, the lower BDE value of a compound, the higher antioxidant activity of the compound presented. For compound 5, three possible sites were preferred to carry radical as shown in Figure 7, site A had the lower BDE in vacuum equal 8.51 kJ/mol compared with site B, C: 5.61, 4.44 kJ/mol, respectively. For compound 4, the BDE values in vacuum comparing different sites were nearly similar, site A>B>C, which presented that site C is more favorable for radical abstract. In water, BDE value for compound 5 had the following order: site C<B<A, so site C was more favorable for radical abstract. Interestingly, compound 4 BDE value of site A had negative value which reflect that site A was thermodynamically favorable through this mechanism in contract to compound 5 that did not thermodynamically undergo through the same mechanism.
4.3.1.2. Analysis of the SET-PT mechanism. As redox reaction was accompanied by formation of charged species, studying SET-PT and SPLET in a nonpolar environment is not accurate. Therefore, thermodynamic functions in polar condition were taking into account. The participation of investigated compounds in the SET-PT mechanism was dependent on two descriptors: IP and BDE as the following: 4.3.1.2.1. Ionic potential. The ionization potential (IP) was estimated as presented in Equation (10). IP describes the process of electron transference by the antioxidant to the free radical. The lower the IP value for a given molecule, the stronger the antioxidant properties. For the compound 5, site A had lower IP in hydrated condition, hence this site was more favorable than C. Interestingly, sites A and B had nearly the same values. For compound 4, site A retained less value and could transfer electron easily. PA represents the reaction enthalpy of production of anion from an antioxidant (the first step in the SPLET mechanism). A lower PA value favored the deprotonating of the benzocaine antioxidant to give the corresponding anion. Site B of two compounds 4 and 5 was the more acidic isomer (Table 7).

Electron transfer enthalpies.
ETEs demonstrate the tendency of the corresponding anions to donate electrons. The withdrawing of an electron from the anion of potent compound is the process with the lowest energetic cost. The lower the ETE value, the more active is the resulting anion for a given molecule. Removal of an electron from potent compound were favored for site C of compounds 4, 5.
As mentioned, multiple step mechanisms such as SPLET, the first step which reflected the deprotonating of the benzocaine antioxidant molecule was the most significant. So, the proton dissociation is the step with low energetic cost in SPLET mechanism, the values of PA of all sites is thermodynamic less favorable, so electron transfer step is not going on. However, the values of IP were thermodynamically favor in SET-PT mechanism The IP value at each molecular site is observed to be lower than that of ETE (Table 7), this showed that single electron transfer from the anionic form is less favorable than that from the neutral form. According to IP values, site A of compound 4, site C of compound 5 were more favorable. Hence SET-PT mechanism was more important than HAT, and/or SPLET mechanism.

Molecular docking
Molecular docking studies using MOE software was introduced to give virtual screening of molecular binding modes of the prepared compounds 4 and 5 inside the pocket of (3aj7) loaded from PDB. 35 3AJ7 was a template with the sequence of the isomaltase from Saccharomyces downloaded in pdb format from Protein Data Bank First, removal of water molecules accomplished from the complex, then, the structural preparation of macromolecule as preliminary steps for docking. Docking visualization for compound 4 showed some interactions as donor and acceptors bonds with phenyl ring, nitrile group and site A as shown in Figure 7. These bond distance showed 3.44, 3.10, and 3.04 (Å) with GLU, GLY, and HIS amino acids. The docking score was À4.06 (kcal/mol) and RMSD was 1.88 (Å). While compound 5 showed docking score À4.88 (kcal/mol) and RMSD was 1.31 (Å). Site A in compound 5 showed donor bonds with distance 3.03 (Å). Molecular docking results were in complete agreement with thermodynamic study (Figure 8).

Conclusion
Novel heterocycles were constructed, and characterized by different spectral tools. Evaluation of antioxidant potential of compounds was conducted using ABTS and DPPH methods. Compounds 4 and 5 were more potent with IC50 lower than ascorbic acid in two tested assay. Studying antioxidant mechanism thermodynamically by DFT theory presented that two potent compounds were dependent on SET-PT mechanism. It was north mentioned that incorporation of benzocaine with imidazole nucleus and alkylated mercapto derivatives was promising route in exploiting of potent antioxidant species. This research is a gateway toward an efficient exploitation of antioxidant property of benzocaine derivatives in the fields of food chemistry and pharmacy ( Figure 8).

Disclosure statement
No potential conflict of interest was reported by the author(s).