Lead optimization for promising monoamine oxidase inhibitor from eugenol for the treatment of neurological disorder: synthesis and in silico based study

Natural based inhibitors of monoamine oxidase are promising drug candidates for the treatment of several neurodegenerative and neuropsychological disorders including depression, anxiety, Parkinson’s disease and Alzheimer’s disease. In the present study we designed and synthesized the eugenol based derivatives and investigated them for human MAO inhibitory potential as promising candidates for therapeutics of neurological disorders. Moreover, radical scavenging activity of designed derivatives was tested by and H2O2 and DPPH scavenging methods. Eugenol based derivatives were designed and synthesized for human MAO inhibitory action. The in silico and in vitro models were utilized for the evaluation of hMAO inhibition. The insight into molecular interactions among the compounds and both hMAO-A and hMAO-B active site was achieved by molecular docking studies. The two spectrophotometric titrations techniques were used to evaluate antioxidant potential. Compounds 5b and 16 were found as most active hMAO-A inhibitors with IC50 values of 5.989 ± 0.007 µM and 7.348 ± 0.027 µM respectively, through an appreciable selectivity index value of 0.19 and 0.14 respectively. In case of hMAO-B inhibition compounds 13a and 13b were found as most active hMAO-B inhibitors with IC50 values of 7.494 ± 0.014 µM and 9.183 ± 0.034 µM receptively and outstanding value of selectivity index of 5.14 and 5.72 respectively. Radical scavenging assay showed that compounds 5b, 5a, 9b, 9a were active antioxidants. The findings of present study indicated excellent correlation among dry lab and wet lab hMAO inhibitory experiments. Interestingly, the compounds exhibiting better MAO inhibition activity was also appeared as good antioxidant agents.

significant way to treat neurodegenerative and depressive disorders.
Monoamine oxidase (MAO; EC1.4.3.4) are mitochondrial membranous enzymes that catalyze the oxidative deamination of neurological catecholamines in the peripheral tissues and brain. The targeting of MAO is implicated in numerous mental disorders and renders psychological effects of some neurological drugs [5]. The metabolic by-products of reaction catalyze by MAO generates the corresponding aldehyde and ammonia along with hydrogen peroxide H 2 O 2 which are highly neurotoxic [6] (Fig. 1). The overexpression of MAO or excess action by MAO initiates the neurodegenerative process, anxiety and depression. The resulted aldehyde molecule from deamination is associated with α-synuclein aggregation [7]. That leads to the Parkinson's disease pathogenesis, however the H 2 O 2 overproduction cause the oxidative damage and aggravates the apoptotic signaling actions. The MAO-A inhibition is associated to antidepressant activity, whereas inhibitors of MAO-B are related to the Parkinson's disease treatment [8].
Moreover, the presently used MAO inhibitors are associated with numerous adverse effects, as hypertensive crises and liver toxicity [9]. Thus, the development of safe and effective MAO inhibitors is a challenge for the medicinal chemists to serve some potent and selective MAO inhibitors for the treatment of the neuropsychological and neurodegenerative disorders. The development of 3D crystallographic structure by Binda and coworkers has increased the enthusiasm of medicinal chemists to design more effective and targeted leads as MAO inhibitors [10]. By taking the advantage of modern computational methods such as molecular docking, absorption, distribution, metabolism, excretion and toxicity (ADMET), and QSAR along with synthetic medicinal chemistry might provide the target selective and effective MAO inhibitors.
Being the member of phenylpropanoids eugenol is chemically a substituted guaiacol via allyl chain. Eugenol is generally seeming as colorless to light yellow, oily liquid as aromatic essential oils extracted from basil, nutmeg, bay leaf, clove oil and cinnamon and its name is originated from Eugenia caryophyllata [11]. The general biosynthesis of eugenol occurs via amino acid tyrosine through sinapyl-alcohol dehydrogenase (SAD) forming coniferyl acetate and ultimately eugenol synthase to eugenol [12]. It is also worth to state the enormous work has been reported on natural phenols on MAO inhibition along with synthetic modifications and computational studies. Eugenol has attracted considerable attention because of its potential anticonvulsive, neuroprotective, anti-neurodegenerative activities and antidepressant. Moreover, many of these quoted the role of MAO for the neurological effects produced by eugenol [13]. Several reports also indicated the profound anti-oxidative effects of eugenol to reduce oxidative stress which is major cause of neurodegenerative disorders. Kong and coworkers evaluated the four phenols paeonol, honokiol, magnolol and eugenol for the MAO inhibitory potential from mitochondrial rat brain. Among all evaluated natural phenols eugenol showed significant MAO inhibitory potential [14] (Fig. 2).
Moreover, Clarke and coworkers evaluated eugenol from different biological sources as clove, oregano, nutmeg and cinnamon and administered to mice for 14 days. Eugenol significantly increased the forced swim test (FST) score. These observations show that eugenol cross the activity in a dose dependent manner bloodbrain barrier in a small amount and act as a competitive human MAO-A inhibitor (Ki = 25 µM) and MAO B (Ki = 211 µM) [15]. Stafford et al. [16] further showed that the eugenol as a major active constituent within Mentha aquatica has remarkably inhibited MAO-B isoform by leaf extract within ethyl acetate at an IC 50 value of 6 ± 5 μg/mL and within petroleum ether = 2 ± 1 μg/ mL).
In another report by Klein-Júnior et al. [17] essential oils extracted from Eryngium species having eugenol as principle constituent showed notable monoamine oxidase inhibitory activity with IC 50 value of 5.65 mg/mL. This study supported the fact that the natural species of Eryngium has eugenol as a chief bioactive secondary metabolite that potentially act on the central nervous system and could be promising drug agent for the treatment of neurodegenerative disorders. A comparative study conducted by Iriea and coworkers showed that eugenol display antidepressant-like activity via tail suspension test and forced swim test (FST) in mice better than imipramine (tricyclic antidepressant). The extracted eugenol from Rhizoma acori graminei stimulated hippocampus Fig. 1 Graphical illustration of the monoaminergic neurotransmitter's oxidative deamination to hydrogen peroxide and corresponding aldehyde brain-derived neurotrophic factor (BDNF) and showed antidepressant-like activity which was not observed as with imipramine [18]. Similarly, Sousa and coworkers demonstrated the eugenol form S. aromaticum (L.) Merr. renders the antidepressant-like actions via inhibiting monoamine neurotransmission (MAO-A) at the lethal dose (LD50 = 4.5 g/kg) in mice. Moreover, repeated administration of eugenol decreased immobility in the FST and tail suspension test (TST) in a dose-dependent mode in mice [19] (Table 1).
Furthermore, some studies have postulated the mechanism of its neurological actions for e.g. Dubey et al. [20], explored the effect of eugenol on the amyloid formation of selected globular proteins. They revealed the neuroprotective nature of eugenol against toxic amyloids by stabilizing native proteins and to delay the conversion  of protein species of native conformation into β-sheet assembled mature fibrils, which seems to be crucial for its inhibitory effect. Survey of compounds structurally related to eugenol has identified a few that inhibit MAOs more potently. Together, the activity of eugenol Mechan et al. [21] measured the MAO-A inhibition of oregano extract containing carvacrol. Both of the constituents exhibited selective MAO-A inhibitory activity in a dose dependent manner. Tao and coworkers evaluated the eugenol from Rhizoma acori graminei for MAO inhibitory action and established the docking poses [22]. Eugenol showed inhibition constant for hMAO-A as Ki = 26 µM and Ki = 15 µM for hMAO-B. Docking simulation studies showed that eugenol interacted mainly with Tyr197, Tyr407, Asn181, Tyr444, Tyr69, and Gly443 active residues within the dynamic site. The aromatic phenolic ring was sandwiched between aromatic cases formed by Tyr444 and Tyr407 and this geometrical position lead to π-π stacking. Moreover, within the MAO-B active site, the aromatic phenolic ring found as embedded within Leu171 and Gln206 side chain.
Inspired by above-mentioned studies and their outcomes we designed and synthesized the eugenol based  (Fig. 3). Moreover, their radical scavenging activity was also evaluated to overcome oxidative stress. The molecular docking studies were performed to rationalize the selectivity for both MAO isoforms.

Chemistry
The key starting material, 2-(4-allyl-2-methoxyphenoxy) acetohydrazide (3) was prepared according to published procedures by Al-Amiery et al. [23]. The synthesis was initiated from eugenol (1) which is commercially available from Hi-media. The synthesis of ethyl 2-(4-allyl-2-methoxyphenoxy) acetate (2) was carried out by refluxing ethyl chloroacetate with eugenol in the presence of anhydrous K 2 CO 3 in DMF at 80 °C for 24 h (Scheme 1). The confirmation of the formation of ethyl 2-(4-allyl-2-methoxyphenoxy)acetate (2) was evaluated by single spot TLC under UV lamp and IR as the phenolic 3400 cm −1 has been disappeared and the peak at 1735 cm −1 appeared to indicated formation of ester (  Finally, 5(a-b) and compound 7 were prepared with compound (3) and different acid chlorides in the presence of ammonium thiocyanide by refluxing at 100 °C for 4-6 h ( Table 3). The reaction completion was confirmed by TLC using the solvent systems methanol-ethyl acetate-benzene (30:30:40 v/v/v). And the single spots were visualized under UV lamp (255 and 360 nm). The 1 H-NMR spectrum exhibited a doublet at 8.72-7.95 indicating the presence of -CO-NH-NH-SO-proton for e.g. compound 5a. For compound 7, the IR spectrum has the following characteristic absorption bands: 3377 (C=H str., Ar), 1614 (C=O str., 2 0 amide), 1177 (C-O-C assym. str., Ar), 1027 (C=S str., thiosemicarbazide), 945 (N-N str., N-NH). The 13 CNMR peak at 178.9 indicated the presence of C=S bond formation in compound 7. The 1 H-NMR spectrum exhibited 7.65-7.59 multiplet showed the presence of secondary amide and at 3.51 doublet specified the amine group formation.
The compounds 11 and 9(a-b) were synthesized by (3) in the presence of nicotinoyl chloride and other natural based acid chlorides via reflux at 80 °C for 8 h in acetone. The IR peaks at 3440 indicated: phenolic O-H, 2960 cm −1 aromatic C=H, 1688 cm −1 secondary amide C=O, 1205 cm −1 aromatic C-O-C group, 1064 cm −1 thiosemicarbazide C=S, 917 cm −1 N-NH as N-N in compound 9a. In the 1 H-NMR spectrum doublet peaks at around 7.70 indicated the two secondary amide groups in compound 9a. In case of compound 11 the IR peaks found as 3388 cm −1 for aromatic C=H bond, 1636 cm −1 N=CH for C=N group, 1614 cm −1 for secondary amide C=O, 1229 cm −1 for aromatic C-O-C group, 913 cm −1 N-NH for N-N groups. The ester derivatives 16, 17, 13(a-d) were synthesized by refluxing eugenol with different natural based acid chlorides in the presence of pyridine at 80-90 °C for 8 h in diethyl ether. The confirmation for etherification reaction was done by single spot TLC using the solvent systems acetone-toluene (30:70, v/v). The IR spectra for compound 11 was found as 3077 cm −1 indicating aromatic C=H, 1700 cm −1 as ester formation as carbonyl group and 1206 cm −1 shown the aromatic C-O-C group. Moreover, the elemental analysis and mass spectrometric data were also found in agreement with compounds characterization.

In vitro human MAO-A and human MAO-B inhibition
The results of the enzymatic experiments of the eugenol based derivatives compounds 5b and 16 were found as most active hMAO-A inhibitors with IC 50 values of 5.989 ± 0.007 µM and 7.348 ± 0.027 µM respectively, through an appreciable selectivity index value of 0.19 and 0.14 respectively (Table 4). 3,4,5-trihydroxybenzoythiosemicarbazide unit within the 5b structure. Along with the location of carboxylic moiety in the 2-(4-allylphenoxy) acetyl nucleus the role of semicarbazides towards MAO inhibition has been observed. Moreover, the compound 16 exhibiting 4-hydroxy-3-methoxyphenyl-acrylate on 4-allyl-2-methoxyphenyl moiety has proven the importance of esteric linkage for h-MAOA active site. However, compound 17 with the absence of 2-methoxyphenyl groups at 3-phenylacrylate has appeared as a considerable hMAO-A inhibitor with an IC 50 value 13.92 ± 0.051 µM and selectivity index of 0.40. Compound 7 showed significant hMAO-A inhibition with IC 50 value 10.46 ± 0.072 µM and a selectivity index of 0.24. Presence of 4-cinnamoylthiosemicarbazide linked with 2-(4-allylphenoxy)acetyl ring in compound 7 shown that the compounds with acrylate group were more hMAO-A active than that of non acrylate unit. The reference compound chlorgyline showed hMAO-A inhibition with IC 50 value 18.74 ± 0.096 µM. It has been reported that the active site of MAO-A is highly hydrophobic in nature which facilitates the large sized structures within it feasibly. So there has been a rational point for design the specific hMAO-A inhibitors with large aromatic structures.
In the case of hMAO-B inhibition compounds 13a and 13b were found as most active hMAO-B inhibitors with IC 50 values of 7.494 ± 0.014 µM and 9.183 ± 0.034 µM receptively and outstanding value of selectivity index of 5.14 and 5.72 respectively. Presence of 4-hydroxy-3-methoxybenzoate within the 13a structure and small size of compound 13a contributed considerable hMAO-B inhibitory potential. In the case of compound 13b the 4-hydroxybenzoate linkage with eugenol enhanced the hMAO-B inhibition. It was observed that methoxy group slightly reduced the hMAO-B inhibitory efficacy as compared with 13a (having a single hydroxyl group at the aromatic ring). Moreover, the reference compound pargyline exhibited the value of 7.494 ± 0.014 µM for hMAO-B inhibition. As it is documented that hMAO-B enzyme composes of two cavities one is entrance cavity whereas another is substrate cavity, the amino acid residue Ile199 act as "gate keeper" for the substrate/inhibitor, so it depends upon the conformation of Ile199 (closed or open) for the interaction of the inhibitor compound. Moreover, the compounds 13a and 13b are smaller than other structures so they might be easily assessed the entrance cavity for inhibition.

Enzyme kinetic for MAO inhibition
On the basis of the finding that compounds 5b and 13a are most potent hMAO-A and hMAO-B inhibitors, Lineweaver-Burk plots were constructed to understand the mechanism of inhibition MAO-A and MAO-B by these compounds. The Michaelis-Menten kinetics for both isozymes at their predetermined initial rates of reaction is presented as Lineweaver-Burk plot. Meanwhile, For each inhibitor, a set of six plots was constructed by

Statistical analysis
The outcomes of all studies are examined as mean ± SEM (standard error mean). Investigational results were calculated statistically by applying the analysis of one-way variance (ANOVA). Though, ANOVA showed significant variation as computed by ANOVA/Dunnett's test. The p < 0.05 was observed as to be statistically considerable. The valuation of statistical parameters was performed by Graph Pad Prism 5.0 edition designed for Windows (San Diego, CA, USA).

Docking studies
Molecular docking study was performed to insight the enzyme-inhibitor interactions for structural requirements for MAO activity and selectivity of eugenol based derivatives. Most active compound 5b exhibited backbone hydrogen bonds Val93 via phenolic OH of 3,4,5-trihydroxybenzoyl unit. Moreover, one edge to face π-π stacking interaction (inter planer distance of ~ 5.04 Å) of 4-allyl-2-methoxyphenoxy and Phe112 was also appeared to support the ligand within the hMAO-A active site.
The docking pose of compound 16 within the hMAO-A active site revealed a side chain hydrogen bond interaction with polar amino acid residue Asn181 through the hydroxyl group of the 4-hydroxy-3-methoxyphenylacrylate unit at inter planer distance of ~ 2.71 Å. Formation of an edge to face π-π stacking interaction between Phe108 and eugenol aromatic ring supported the ligand within the substrate cavity by inter planer distance of ~ 5.05 Å. Moreover, the ligand 16 was found as embedded within the active site through hydrophobic residues Cys323, Ala111, Val210, Phe108, Leu97, Ile180, and Tyr197 via eugenol aromatic ring. Some polar residues such as Gln215, Ser209, and Thr336 were found as engaged towards ester linkage of 16. The 4-hydroxy-3-methoxyphenyl-acrylate unit was surrounded by an aromatic ring formed by Tyr407 and Tyr444, though the hydrophobic residues Leu337, Ile35, Tyr69, Phe352, and Tyr197 were also appeared near to compound 16.
In case of hMAO-B docking insight of compounds 13a and 13b showed two edges to face π-π stacking interaction between Tyr326 and both aromatic rings of compounds 13a and 13b through inter planer distance of ~ 5.24 Å and ~ 5.36 Å for 13a and ~ 5.29 Å and ~ 5.37 Å for 13b respectively. This outstanding behavior of 13a and 13b was noticed due to the small size of the compounds 13a and 13b as it has been reported that the active site of hMAO-B composes of two bipartite cavities. The inhibitor/substrate has to cross through two hydrophobic sub-cavities; the entrance cavity through dimensions of ~ 290 Å as well as the substrate cavity through dimensions of ~ 400 Å. Moreover both compounds 13a and 13b were surrounded by hydrophobic residues such as Phe168, Trp119, Leu167, Phe103, Pro104, Leu88, Pro102, Cys172, and Leu171. The aromatic case formed by residues Tyr188, Tyr398, Tyr435, Phe343; Tyr60 enclosed the 4-hydroxy-3-methoxybenzoate and 4-hydroxybenzoate units of 13a and 13b respectively. Most of the compound including eugenol showed hydrogen bonding with Pro102, and some of them interacted with gate keeper residue Ile199. The role of this gate keeper residue cannot be excluded from the discussion. The expedition of a substrate or inhibitor begin in the course of bendable loop (residue 99-112) which works as a gating residue at the outer mitochondrial membrane surface; the side chain isoleucine (Ile199) could exhibit two diverse conformations, closed conformation (separate both cavities) or open conformation (fuse both cavities) based on the character of the docked ligand. Maximum ligands showed edges to face π-π stacking with Trp119 and Tyr326.

In silico ADMET profile of eugenol derivatives
The hypothetical evaluation of adsorption, distribution, metabolism, excretion and toxicity (ADMET) descriptors prediction was carried out by quick prop graphical user interface of Mastro-11.1 Schrödinger (Table 5) [24]. Evaluation of lipophilicity (logP) indicated the octanol-water partition coefficient that must be within limit 5. It could be predicted from the table that all of the eugenol based derivatives shown logP values within the limit. Lesser blood-brain barrier (BBB) access and poor membrane permeability is associated with a larger rate of tPSA (> 145 Å 2 ) [32]. Nearly all of the synthesized derivatives exhibited acceptable values of tPSA within a range of 35-140 (Table 4) [25], below 5 hydrogen bond donors must be in lead, and the number of hydrogen bond acceptors (HBA) must be under 10. The aforesaid standard meets as well, because except 5b (with 6 HBD) complete series of derivatives exhibited 0-4 HBD and 1-9 HBA. But this criterion is not fully reliable and has been challenged in case of natural potent well known therapeutic agents. All of the derivatives could be considered to exhibits fine oral bioavailability, examined as confirmed by Veber rule [26]. This states that the rotatable bonds magnitude should be correspondent to 10 and tPSA ≤ 140 Å, or else the whole no. of hydrogen bond donor and acceptor (HBA + HBD) must be ≤ 12. It is considerable that not any of derivatives was discarded, and each one of the derivatives proved promising ADMET rank ( Table 4).

Determination of DPPH scavenging activity
Results of DPPH scavenging assay showed that compounds 5b, 5a, 9b, 9a were most active antioxidants with IC 50 values of 8.304 ± 0.073 µM, 9.152 ± 0.063 µM, 9.293 ± 0.028 µM, 9.514 ± 0.017 µM respectively (Table 6). It was observed that these compounds were possessing a large number of hydrogen donors as NH and OH groups within their structures. Presence of these groups generally considered to be contributors for potential antioxidant effect by polyphenolic compounds. However, the eugenol and reference compound l-ascorbic acid exhibited the IC 50 value of 10.29 ± 0.011 and 8.58 ± 0.009 respectively. Other compounds such as compounds 7 and 11 were also appeared to be considerable antioxidants with IC 50 values of 9.786 ± 0.012 µM and 11.21 ± 0.044 µM respectively (Fig. 7). It is notable that the compound 7 was observed as a poor agent for DPPH scavenging, the reason for this might be the absence of any OH or NH group with the compound 7 structure.

H 2 O 2 scavenging activity
The H 2 O 2 scavenging assay was carried out for further evaluate the antioxidant potential of synthesized compounds. In cases of H 2 O 2 scavenging the compounds    (Table 7). Surprisingly, the compounds with good DPPH scavenging profile were observed as considerable H 2 O 2 scavengers. Thus, the presence of the OH and NH groups is essential for the antioxidant activity. However, eugenol and reference compound l-ascorbic acid exhibited the IC 50 values of 9.980 ± 0.012 µM and 8.121 ± 0.082 µM respectively (Fig. 8). Compounds 7 and 11 were showing similar behavior towards H 2 O 2 and the IC 50 values of them were 9.755 ± 0.021 µM and 10.33 ± 0.022 µM respectively.

Nitric oxide scavenging activity
To validate the results of above mentioned antioxidant activity assays we carried out the nitric oxide scavenging activity of the titled compounds. Results of nitric oxide scavenging activity showed that the compound 5b was found as most active NO· scavenger with IC 50 value of 6.456 ± 0.023 µM as compared with eugenol (IC 50 value 11.724 ± 0.003 µM) and reference compound l-ascorbic acid with IC 50 values of 7.523 ± 0.005 µM (Table 8). Moreover compounds 5a, 9b, 9a were also observed as mild active NO· scavengers with IC 50 values of 7.156 ± 0.002 µM, 8.095 ± 0.018 µM, 9.007 ± 0.014 µM respectively (Fig. 9). The result of the nitric oxide scavenging activity showed similar routine for the antioxidant action as observed with DPPH and H 2 O 2 scavenging profile.

Structure-activity relationship (SAR) studies
The structure-activity relationship of the evaluated eugenol based derivatives by means of their antioxidant activity and MAO inhibitory activity and results is summarized in Fig. 11.

Materials and methods
Unless otherwise noted, the analytical grade chemicals required for synthesis and antioxidant activity procured from Hi-media Laboratories. The biological evaluation of the test drugs on hMAO activity was investigated by measuring their effects on the production of hydrogen peroxide (H 2 O 2 ) from p-tyramine (a common substrate for hMAO-A and hMAO-B), using the Amplex Red MAO assay kit (Sigma USA) and microsomal MAO isoforms prepared from insect cells (BTI-TN-5B14) infected with recombinant baculovirus containing cDNA inserts for hMAO-A or hMAO-B (Sigma-Aldrich USA) were used as source for the two microsomal MAO isoforms. Reactions were monitored by thin layer chromatography TLC performed on 0.25 mm precoated silica gel plates procured from Merck, and the spots were visualized in   (Hz). Infrared (IR) spectra were recorded on Perkin Elmer FTIR spectrophotometer by using KBr pellets technique.

General procedure for the synthesis of acid chlorides
A mixture of different aromatic carboxylic acids and thionyl chloride (10 mL) was stirred and refluxed 80 °C for 2-4 h in the presence of pyridine (1-2 drops) till the completion of reaction. Distillation of the reaction mixture removed the excess of thionyl chloride. Finally the purity of resulting product was appropriate to use it directly for the following synthesis.

General procedure for preparation of eugenol esters (13a-17)
The target esters derivatives derived from eugenol, which were prepared by gently refluxing a solution of eugenol in ether (50 mL) with a solution of different aromatic acid chlorides (0.05 mol) in diethyl ether (50 mL).
The reaction mixture was heated on a water bath (80 °C) until the complete removal of hydrogen chloride and completion of reaction was checked by single spot TLC. The shiny white precipitates so obtained were filtered using suction and rendered free from chloride by repeated washing with water and finally purified by recrystallization with acetone.

Spectral data
Presence of 4-hydroxy-3-methoxybenzoate within the 13a structure and small size of compound 13a contributed remarkable hMAO-B inhibitory potential.
The 4-hydroxybenzoate linkage with eugenol enhanced the hMAO-B inhibition in compound 13b and the methoxy group slightly reduced the hMAO-B inhibitory efficacy as compared with 13a (having single hydroxyl group at aromatic ring). Important for the derivatization and contribute antioxidant activity Fig. 11 Through insight into an examination of the antioxidant MAO screening assay explicated of some SARs J = 6.2, 1.0 Hz, 4H). 13 3.78 (s, 3H), 3.29 (dp, J = 6.2, 1.0 Hz, 2H). 13

Recombinant human MAO inhibition screening
To evaluate IC 50 values for the inhibition of human MAO-A and MAO-B by newly synthesized compounds the commercially available recombinant human enzymes expressed in insect cells were used (Sigma-Aldrich). The MAO inhibition assay was based on the method described by Anderson et al., 1993 and Chimenti et al. with some modifications [27,28]. The Amplex Red MAO Kit tyramine (Sigma-Aldrich) affords a suitable fluorimetric method to evaluate MAO enzyme action of synthetic and natural compounds. In the assay, MAO reacts with p-tyramine, a common substrate for both MAO isoforms, ensuing in the production of H 2 O 2 , which is identified by a fluorimetric method (λ ex = 530/λ em = 585 nm). Each test well contained 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4), a range of concentrations of the eugenol based derivatives or reference compounds along with recombinant hMAO-A or hMAO-B in appropriate quantity. After the preparation of this mixture the wells were subjected to incubation for 15 min at 37 °C, placed in the dark fluorimeter chamber. Once this incubation period over, the process was rendered by mixing 200 lM Amplex Red reagent (Sigma-Aldrich), 1 U/mL horseradish peroxidase (Sigma-Aldrich) and p-tyramine (Sigma-Aldrich). Subsequently, the formation of hydrogen peroxide exerted by MAO isoforms was quantified by using Amplex-Red reagent, a non-fluorescent probe that reacts with H 2 O 2 in the existence of horse radish peroxidase to generate the fluorescent component resorufin. Moreover, the control analysis was calculated in the absence of compound and reference inhibitors. The probability of compounds to change the fluorescence produced in the reaction mix due to non-enzymatic inhibition was assessed by mixing titled compounds to solutions of single Amplex Red reagent in a sodium phosphate buffer. Data were processed in Microsoft Excel, to calculate IC 50 values.

In silico ADME prediction
The in silico ADMET evaluation of the synthesized compounds was performed by QikProp software by Schrödinger suit. Several descriptors for determination of essential pharmacological properties were calculated for instance Lipinski rule, total polar surface area, MDCK, Caco-2, no. of rotatable bonds, logPoct and logBB. Caco-2 cell and Madin-Darby canine kidney (MDCK) cell models are considered crucially unfailing in vitro model to estimate the oral drug absorption. For a CNS acting drug the logBB is an important descriptor to identify blood-brain barrier (BBB) diffusion and it is estimated as the concentration range of compound concentration in peripheral blood (C blood ) and brain (C brain ) [29][30][31][32][33]. The ADMET score calculated by QikProp showed the crucial parameters for the drug-like properties of the synthesized compounds.

Enzyme kinetics
Lineweaver-Burk plots for the inhibition of MAO-A and MAO-B were constructed by using p-tyramine at eight different concentrations for each plot (15-250 μM). For each test inhibitor, six Lineweaver-Burk plots were constructed. Plotting of Michaelis-Menten curves was used to estimate inhibition data by global fitting of the to the Michaelis-Menten equation using the Prism 5 software package.

Preparation of ligands
The 3D structures for all the eugenol based derivatives were designed in MDL MOL format and then processed by LigPrep module of Schrödinger. The LigPrep utilized to optimize and assign the accurate protonation pose and torsions to all ligands. Finally the stereochemical structures around 32 was done at pH state 7.0 ± 2.0 via utilizing ionizer. The desalting of ligands and optimization and tautomerization was done by utilizing OPLS 2005 force field.

Preparation of proteins
The 3-D crystallographic protein structures for hMAO-A and hMAO-B at good resolution were taken from the Protein Data Bank with succession codes 2Z5X [34] and 2V5Z [35] respectively. Prior to molecular docking the removal of inhibitors as co-crystallized form, safinamide for 2V5Z and harmine for 2Z5X and structural water molecules was done from hMAO-A and hMAO-B proteins. The explicit active sites were precisely set at 1000 A3 standard grid box located near cofactor FAD's N5 atom. Consequently, the protonation and charge position were allocated and minimization of energy was done via OPLS2005 force field.

Docking methodology
Molecular docking was carried out by implementing flexible algorithm with XP approach of Glide. The grid box was generated frequently for assortment and formation of centroid of the dynamic site residues and the integral receptor protein was processed via energy minimization at RMSD rate of 0.17 Å. The side chains of B-factor in MAO protein were separated before docking experiments. All of the amino acid residues were refined and energy minimization and optimization of primary side chains. The aforementioned process exposed the correct site of ligand structure and conformation for induced fit docking. Therefore, with the XP Glide mode redocking was performed which afforded dock score for every ligand.

Determination of DPPH scavenging activity
The antioxidant activity of the newly synthesized compound was measured on the basis of the scavenging activity of the stable 1, 1-diphenyl 2-picrylhyorazyl (DPPH) free radical The evaluation of the antioxidant activity was based on free radical scavenging effect of stable DPPH· with spectrophotometric titration [36,37]. Solutions of synthesized compounds were set in ethanol/water (1:1) having 10 mM to 100 mM at a period of 10 mM. The 0.01% DPPH· stock solution was prepared in ethanol/ water (1:1). Samples were prepared as 5 mL DPPH· solution was mixed with 5 mL l-ascorbic acid solution then shaken vigorously and kept in the dark. Corresponding blank sample were prepared and l-ascorbic acid was used as reference standard. All samples were subjected for incubation at 37 °C for an hour in the dark chamber prior to evaluate absorbance. To evaluate the absorption spectra, firstly blank spectra for ethanol/water were observed afterward the spectrophotometric titration was carried out at various concentrations of titled compounds. The absorbance was calculated at 517 nm with UV/Vis Epoch ELISA reader, and radical scavenging activity was calculated as a reduction in absorbance of DPPH·. The actual decrease in absorption induced by the test was compared with the positive controls. The IC 50 values were calculated use the dose inhibition curve in linear range by plotting the extract concentration versus the corresponding scavenging effect through following equation [(A DR − A PR )/A O ] × 100. Where A DR is the absorbance prior to reaction and A PR is the absorbance after reaction with DPPH·.

Hydrogen peroxide scavenging (H 2 O 2 ) assay
Hydrogen peroxide generally turns into water and oxygen and produces highly toxic hydroxyl radical that renders the damage to DNA and peroxidation of lipid. H 2 O 2 scavenging ability of eugenol based derivatives was evaluated by method described by Nakano and colleagues with some alteration [38,39]. The hydrogen peroxide solution (40 mM) was prepared in phosphate buffer (40 mM pH 7.4) and addition of several ranges of synthesized derivatives (05-80 μg/mL) was done into H 2 O 2 solution (2 mL). The determination of H 2 O 2 concentration was done by UV/Vis Epoch ELISA reader at 230 nm upon incubation of 10 min. Solution of phosphate buffer without H 2 O 2 was noted as blank reading. Antioxidant potential of synthesized compounds is shown in Table 3. The hydrogen peroxide percentage inhibition was estimated through the formula [(A H − A D )/A O ] × 100, where A H is the absorbance of the control and A D is the absorbance of compounds/reference taken as 05-80 μg/mL l-ascorbic acid.

Nitric oxide scavenging activity
The generation of nitric oxide (NO·) on biological tissues is mediated by particular nitric oxide synthases enzyme that control the catabolism of the arginine to citrulline via creation of NO through a five electron oxidative reaction. At the physiological pH (7.2) in aqueous solution sodium nitroprusside decompose to produce NO·. Finally, NO· reacts with oxygen at underaerobic conditions and produces stable products (nitrate and nitrite), this amount can be determined by means of Griess reagent. The procedure for the nitric oxide scavenging activity was adopted from reports of Alam et al. [40].

Ferric reducing-antioxidant power (FRAP) assay
The capacity of antioxidants to decrease ferric iron is measured by this method. The reaction of 2,3,5-triphenyl-1,3,4-triaza-2-azoniacyclopenta-1,4-diene chloride (TPTZ) and complex ferric iron yields ferrous form at low pH. The monitoring of the above reduction reaction at 593 nm under ELISA reader reveals the antioxidant potential. This method was adopted from the reports of Benzie et al. [41].

Conclusion
All of the research findings facilitated to conclude the structural specificity rationale for the hMAO-B and hMAO-A of this new series of natural eugenol based inhibitors. The results of the study improved our confidence in our scheme and encouraged us to carry on our study to design more persuasive and selective inhibitors. The considerable hMAO-A inhibitory potential of compounds 5b with IC 50 value (5.989 ± 0.007 µM) might be due to presence of 3,4,5-trihydroxybenzoy-thiosemicarbazide unit within the 5b structure. Moreover the compound 16 exhibiting 4-hydroxy-3-methoxyphenyl-acrylate on 4-allyl-2-methoxyphenyl moiety has proven the importance of esteric linkage for h-MAOA active site. In case of compound 13b the 4-hydroxybenzoate linkage with eugenol enhanced the hMAO-B inhibition. I was observed that methoxy group slightly reduced the hMAO-B inhibitory efficacy as compared with 13a (having single hydroxyl group at aromatic ring). Together with significant MAO inhibitory activity of abovementioned derivatives the future trend is directed towards pre-clinical studies. The reduction of physiological antioxidants in the CNS causes excessive oxidative stress by overproduction of MAO in brain and is concerned with neurodegenerative disorders. We synthesized the natural eugenol based compounds which exhibits considerable MAO inhibitory potential along with antioxidant activity that might probably pay for foremost advantages to explore the fundamental neurological pathology.