Exploring new selective 3-benzylquinoxaline-based MAO-A inhibitors: Design, synthesis, biological evaluation and docking studies

European Journal of Medicinal Chemistry 93 (2015) 308e320

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Exploring new selective 3-benzylquinoxaline-based MAO-A inhibitors: Design, synthesis, biological evaluation and docking studies Sherine N. Khattab a, Shimaa A.H. Abdel Moneim a, Adnan A. Bekhit b, **, Abdel Moneim El Massry a, c, Seham Y. Hassan a, Ayman El-Faham a, d, Hany Emary Ali Ahmed e, f, Adel Amer a, g, * a

Department of Chemistry, Faculty of Science, Alexandria University, Alexandria 21321, Egypt Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt c Department of Chemistry, Rabigh College of Science and Art, King Abdulaziz University, Saudi Arabia d Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, 11451 Riyadh, Saudi Arabia e Pharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College, Taibah University, Al-Madinha Al-Munawaraha, Saudi Arabia f Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt g Department of Chemistry, College of Science, Taibah University, Al-Madinha Al-Munawaraha, Saudi Arabia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 September 2014 Received in revised form 11 February 2015 Accepted 12 February 2015 Available online 16 February 2015

In this investigation, we searched for novel MAO-A inhibitors using a 3-benzylquinoxaline scaffold based on our earlier findings. Series of N0 -(3-benzylquinoxalin-2-yl)acetohydrazide, 4a, N0 -(3benzylquinoxalin-2-yl)benzohydrazide derivatives 4bef, N0 -[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl) acetyl]benzohydrazide derivatives 7aed, (9H-fluoren-9-yl)methyl 1-[2-(2-(3-benzyl-2-oxoquinoxalin1(2H)-yl)acetyl)-hydrazinyl]-2-ylcarbamate derivatives 8aec, 2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)-N0 benzylidene acetohydrazide derivatives 9aeh, and ethyl 2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetate derivatives 10aee were synthesized and evaluated in vitro as inhibitors of the two monoamine oxidase isoforms, MAO-A and MAO-B. Most of the compounds showed a selective MAO-A inhibitory activity in the nanomolar or low micromolar range. Compounds 4e and 9g were the most potent derivatives with high MAO-A selectivity and their molecular docking studies were performed in order to rationalize the obtained biological result. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Quinoxalin-2(1H)-one N-Aroylhydrazine N0 -Acetylaroylhydrazine Schiff's base Amino acids Monoamine oxidase

1. Introduction Depression has been reported to be the fourth global burden of disease, with nearly 12% of the global disability adjusted life years [1]. In addition to the psychological stress on patients and families, depression contributes to the development and progression of systemic and organ diseases [2e5]. Anxiety disorders, which often precede and co-occur with depression, are found in 10e21% of children and adolescents [6]. In the Middle East (namely, Egypt and the Kingdom of Saudi Arabia (KSA)) changing in the socioeconomic status have been shown to be associated with increased chronic

* Corresponding author. Department of Chemistry, College of Science, Taibah University, Al-Madinha Al-Munawaraha, Saudi Arabia. ** Corresponding author. E-mail address: [email protected] (A. Amer). http://dx.doi.org/10.1016/j.ejmech.2015.02.020 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

diseases including chronic mental diseases like depression [7e9]. The monoamine oxidase inhibitors (MAOIs) were the first drugs used to treat depression. They work by blocking the breakdown of a number of neurotransmitters involved in depression via an enzyme, MAO. MAO (EC 1.4.3.4; MAO) is a flavoprotein localized in the outer mitochondrial membrane and present in practically all mammalian tissues. The primary role of MAO lies in the metabolism of amines and in the regulation of neurotransmitter levels and intracellular amine stores [10]. Two isoforms of MAO (MAO-A and MAO-B) have been found [11]. These two forms of MAO are characterized by their different affinities to inhibitors and their different specificities to substrates [12]. MAO-A preferably metabolizes serotonin, adrenaline, and noradrenaline [13], whereas bphenylethylamine and benzylamine are predominantly metabolized by MAO-B [14]. Tyramine, dopamine, and some other important amines are common substrates for both isoenzymes [15].

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Nowadays, the therapeutic interest of MAOIs falls into two major categories. MAO-A inhibitors have been used mostly in the treatment of mental disorders, in particular depression and anxiety [16e18], while MAO-B inhibitors could be used in the treatment of Parkinson's disease and Alzheimer's disease [19,20]. In our efforts to add to the development of novel selective MAOA inhibitors, we have recently focused on utilizing the 3benzylquinoxaline scaffold where compound such 3-benzyl-2-(2morpholin-4-yl-ethyl)amino-quinoxaline I showed potent and high selectivity MAO-A inhibition activity [21,22]. We have also showed that a structurally related pyridazinylacetic acid derivatives synthesized in our laboratory were able to inhibit MAO-A with high selectivity index (SI) values [23]. The main goal of the present study was to synthesize a new family of hybrid quinoxaline derivatives IIeIV based on the 3-benzylquinoxalin-2(1H)-one unit (Chart 1 and Chart 2). The rational design of the new compounds was based on the following considerations: (i) the possession of the hydrazido functionality (e.g. Iproniazid), (ii) the presence of the benzamido functionality (e.g. Moclobemide), and (iii) the keeping benzylquinoxalinyl group which seems to play a role in orientation and complex formation at the active site of the enzyme.

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Fig. 2. The Z- and E-forms of compound 9d using molecular mechanics MM2 and PM3 calculations.

2. Results and discussion 2.1. Chemistry In our synthesis, the quinoxaline scaffold was easily prepared according to the reported method in the literature (Scheme 1) [24]. The reaction of compound 1 with phosphoryl chloride afforded the rapid formation of the corresponding 2-benzyl-3chloroquinoxaline 2 after neutralization with saturated sodium bicarbonate (Scheme 1). Further reaction of 2 with hydrazine hydrate in ethanol as a solvent afforded the 2-benzyl-3hydrazinoquinoxaline 3 (Scheme 1) [24]. The syntheses of N0 -(3-benzylquinoxalin-2-yl)acetohydrazide, 4a and N0 -(3-benzylquinoxalin-2-yl)benzohydrazide derivatives 4bef were carried out in good yield via amide coupling of the acids with 2-benzyl-3-hydrazinoquinoxaline 3 using O-(7azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) as a coupling reagent in the presence of triethyl amine (TEA) in dimethylformamide (DMF) at 0  C (Scheme 2). The structure of compounds 4aef was confirmed by elemental analysis, IR, and NMR spectroscopy. The reaction of the 3-benzylquinoxalin-2(1H)-one 1 with ethyl bromoacetate in dimethylformamide in the presence of potassium iodide and sodium bicarbonate afforded ethyl 2-(3-benzyl-2oxoquinoxalin-1(2H)-yl)acetate 5 as a single product as showed by TLC using ethyl acetate/hexane (1:1) as an eluent (Scheme 3) [25]. The structure of the quinoxaline derivatives as N-alkylated A rather than O-alkylated products B (Fig. 1) [26] were secured by analyzing the HMBC spectrum of 5. Heteronuclear correlation was observed between the singlet at d 4.96 ppm of the methylene

hydrogens of the acetic acid function and the carbons at d 130.16 (C10Q), 154.4 (C-2Q) and 167.18 ppm (C]O ester), while the benzyl CH2 hydrogens at d 4.28 ppm showed correlation to carbons at 129.62 (C-20 Ph, 60 Ph), 136.96 (C-10 Ph), 154.43 (C-3Q) and 159.18 ppm (C-2Q). Hydrazinolysis of the ethyl ester group in 5 was carried out in methanol under reflux for 4 h to afford the corresponding hydrazide 6 in 74.4% yield (Scheme 3). Our initial studies proceed by reacting aromatic carboxylic acids and hydrazide 6 in the presence of various coupling conditions, such as 1-hydroxybenzotriazole (HOBt) in the presence of 1,1,3,3-tetramethyl-2fluoroformamidinium hexafluorophosphate (TFFH) or HATU at 0  C and TEA as base in DMF and recrystallize from methylene dichloride (MDC). Both methods gave satisfactory yields of the desired N0 -(2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl) acetyl) benzohydrazide derivatives 7 (Scheme 3). As reported earlier such structure of type 7 could exist in four possible conformational forms (namely; EE, EZ, ZE and ZZ) due to the hindering of rotation of the CeN(1) and the CeN(2) bond [27]. However, our 1H NMR spectra of compounds 7aed showed broad NH signals with no fine splitting indicated fast rate of rotation. As a prototype the 1H NMR spectra of 7b in DMSO-d6 shows a singlet peak at d 4.14 ppm corresponding to the benzyl CH2 protons. Two other peaks were observed equivalent to two protons at d 4.98 and 5.25 ppm, in ratio 2:1, corresponding to only one methylene group (CH2) and since no definite conformers could be assigned they will just be named A and B.

Fig. 1. The two expected structures of compound 5.

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b a

c Fig. 3. a) 2D binding mode of compound 4e in MAO-A active site. b) 2D binding mode of compound 9g in MAO-A active site. c) 2D binding mode of compound moclobemide in MAO-A active site.

Similarly, (9H-fluoren-9-yl)methyl-1-(2-(2-(3-benzyl-2oxoquinoxalin-1-(2H)yl) -acetyl)hydrazinyl) -2-ylcarbamate derivatives 8 (Scheme 3) were prepared by coupling of several Fmoceamino acids (Fmoc-Leu-OH, Fmoc-Phe-OH and FmocTyr(OtBu)-OH) with hydrazide 6 in the presence of two equivalent of DIEA (diisopropylethylamine) using HATU as coupling reagent, in DMF as solvent. As extension to our work the reaction of 2-(3-benzyl-2oxoquinoxalin-1(2H)-yl)acetohydrazide 6 with aromatic aldehydes and ketones in the presence of drops of glacial acetic acid resulted in the formation of the corresponding Schiff bases 9aeh, (Scheme 3). Usually, the condensation reaction between hydrazides and carbonyl compounds are reported to give a mixture of Z/E-isomers [28]. In our case the two isomers were detected by 1H and 13C NMR spectroscopy. As prototype the 1H NMR spectrum of the Schiff's base 9d in DMSO-d6 showed singlet peak at d 4.14 ppm corresponding to the benzyl CH2 protons. Two other peaks were observed equivalent to two protons at d 5.42 and 5.00 ppm, in ratio 74.7%:25.3%, corresponding to the methylene CH2 group. Also the imine proton appears as two singlet peaks at d 8.03 and 8.19 ppm which confirm the presence of the two isomers. Therefore, it is

considered worthwhile to model the compounds using molecular mechanics MM2 and PM3 calculations. The calculated energy minimization results were found to be 25.20 and 30.09 kcal mol1 for each of the Z- and E-structures, respectively, Fig. 2. Accordingly, one can conclude that the major isomer could be the E-form. Similar to our previously reported pyridazine chemistry [23], the quinoxaline derivatives 10 were prepared with the hope that 3benzylquinoxlinyl moiety may enhance their inhibition activity and their selectivity as well. Thus, ester 5 was hydrolyzed by alcoholic potassium hydroxide to give the corresponding acids 11. Its reaction with different L-amino acids t-butyl esters (Leu, Phe and Tyr) in DMF in presence of TEA and HATU as coupling reagent at 0  C gave smoothly the derivatives 10 in high yield (Scheme 4). The structures of compounds 10 were determined by spectroscopic methods (IR, 1 H NMR) and by elemental analysis. 2.2. Biology The newly synthesized compounds 4aef, 7aed, 8aec, 9aeh and 10aec were tested to determine their activity and selectivity against MAO-A and MAO-B isoenzymes in the presence of the

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reference drugs and target compounds were expressed as IC50 in Table 1. The selectivity index (SI) was also given in Table 1. The results revealed that all synthesized compounds exhibited nanomolar activity against MAO-A target in the 1.7e98 nM range and high selectivity over B type ranging from more than 1000foldse612,000-folds. Of these examples, 4e, 8b, 8c, 9a, 9b, and 9g derivatives (1.7, 2.9, 3.4, 3.1, 3.6, and 1.6 nM respectively) have showed MAO-A inhibition activity comparable to the standard clorgyline as irreversible and moclobemide as reversible inhibitors (IC50 ¼ 3.1, 11,000 nM respectively). These compounds showed higher MAO-A inhibitory activity than MAO-B inhibitory activity and high selectivity than standard drugs. Compounds 4e and 9g are the most potent and selective compounds as MAO-A inhibitors. 2.3. Structureeactivity relationship (SAR) analysis Structureeactivity relationship analysis revealed that series of 3-benzylquinoxaline of different derivatives exhibited a wide range of MAO-A inhibitory activity in nanomolar concentrations. The first scaffold 3-benzyl quinoxaline 4aef with hydrazide moiety on 2 positions showed potent activity (74e1.7 nM) with the most potent one being 4e analog. The above mentioned activity of 4d may be attributed to p-hydroxy substituent that could be considered optimal for activity through the formation of stable hydrogenbonding interaction. While remove the hydrazide moiety to position N1 namely7aed does not have significant change in the activity. In addition, coupling of large or small amino acids instead of aromatic acids affording hydrazides or amides 8aec and 10aec respectively, with good activity in the presence of aromatic rings. In contrast, replacing the benzylpridazinonyl by benzylquinoxalinyl moiety to represent 10aec (Chart 2) did not enhance the selectivity. The formation of Schiff bases represented in compounds 9aeg leads to dramatic change in activity and selectivity resulting in many derivatives with high potency and selectivity. This is due to the well tolerance of the side chain in the pocket and some of them have shown the ability to stabilize their fitting by either strong hydrogen bonding or hydrophobic aromatic interactions. Collectively, these observations strongly indicate that the MAO-A activity profile of the new synthesized compounds are governed by the good ligandetarget interaction. As observed from the data, the best results were obtained with compounds 4e and 9g which possess hydroxyl group on the para position of the phenyl ring (hydrogen bonding factor). Finally, the size of side chain on quinoxaline ring (hydrophobic factor) affects activity and could be controlled to get optimum MAO-A potent and selective inhibitor. 2.4. Molecular modeling

Fig. 4. a) Connolly surface map of compound 4e in the binding pocket. b) Connolly surface map of compound 9g in the binding pocket. c) Connolly surface map of compound moclobemide in the binding pocket.

specific substrate, serotonin or benzylamine respectively. Bovine brain mitochondria were isolated according to Basford [29]. The method involves the determination of MAO-A activity of rat liver mitochondria using clorgyline as irreversible time-dependent reference standard, and moclobemide as reversible inhibitor [30]. The test compounds or reference standard were preincubated for 60 min with enzymes before the addition of the corresponding substrate to ensure fair comparison and fully inhibit MAO [30]. The activity of MAO-A and MAO-B was determined by the fluorimetric method, according to Matsumoto et al. [31] and the results for both

In an attempt to understand the reason for the observed inhibitory activity, these compounds were submitted to computational analysis using Molecular Operating Environment (MOE) [32]. Furthermore, docking protocols were done in order to compare and confirm the affinity and hence selectivity of the new compounds against MAO-A isoform over MAO-B one. Graphical 2D representations were showed to propose the possible binding mode for the two most active MAO-A inhibitors 4e and 9g and moclobemide reference drug, Fig. 3e5. Primary steps were done for both protein target structures used in docking work (PDB codes are, 2BXR for MAO-A and 1GOS for MAO-B) through energy minimization, removing of internal ligands, and selection of the residues that surround the FAD molecule by 9 Å from the 3ry nitrogen of the isoalloxazine ring in order to keep the structure planarity of the isoalloxazine FAD ring. The resulting energy minimum structures, after removing covalent ligands (clorgyline for 2BXR and pargyline for 1GOS) were used as final models for further work. Among the

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b

a

Fig. 5. a) 2D binding mode of compound 9g in MAO-B active site. b) 2D binding mode of compound moclobemide in MAO-B active site.

highly active compounds, some of them show high degree of selectivity against MAO-A over B one. Compounds 4e and 9g exhibited high potency and selectivity towards MAO-A (1.7 and 1.6 nM respectively). Analysis of the interaction mode revealed that compound 9g could bind to the MAO-A enzyme strongly through orientation of benzyl moiety into flexible tolerated cavity formed of mainly Val210 and Phe208 by aromatic interaction. While the phenoxy ring is well accommodated by the catalytic cleft facing the FAD cofactor by formation of stable hydrogen bonding through Gln74 (1.57 Å). Two different polar amino acids, Ser209 and Gly214, share in the complex stabilization as hydrogen bond acceptors to the carbonyl of quinoxalinone ring (2.91 and 3.07 Å). Moreover, the amide nitrogen of the linker binds to the backbone carbonyl of Glu216 through strong hydrogen bonding (1.44 Å). In addition, different hydrophobic residues surround the benzyl quinoxalinone ring like Leu97, Ile328, Ile335, Met324, and Val70 could form extra hydrophobic interaction to the compound in the active site. The docking results of compound 4e showed similar binding except the hydrogen bonding involves the Gly71 instead of Gly214, Fig. 3aeb. The molecular docking of moclobemide showed specific binding behavior towards most of the amino acid residues that are in general similar to the above potent ligands, Fig. 3c. The comparative computational study on the binding modes of 4e and 9g in the MAO-A active site cavity with the reversible drug moclobemide was summarized in Table 2. The molecular surface of the two potent selective structures and moclobemide is represented in the pocket, Fig. 4, by describing the polar and low polar moieties. Terminal hydrophilic, terminal hydrophobic and middle mild polar parts formed are responsible to strong pocket tolerance and share high degree of mapping in the binding cavity. In attempt to confirm selectivity behavior of the target compounds, we docked 9g as a prototype and moclobemide as a reference into MAO-B isoform to compare the different behavior against the isoenzymes, Fig. 5. The lowest energy minimum conformation of both ligands (9g and moclobemide) failed to show the same interactions with the corresponding residues as done in MAO-A. Based on our computational data, the experimental IC50 values could be compared to reference drug moclobemide and our new compounds might follow the noncovalent mechanistic pathway.

2.5. Acute toxicity The test compounds 4aef, 7aed, 8aec, 9aeh and 10aec were further evaluated for their oral acute toxicity in male mice using a literature method [33e35] (Approval of animal care and use committee (ACUC), project number 22, 22/5/2013). The results indicated that test compounds proved to be non-toxic and well tolerated by the experimental animals up to 200 mg/kg, although no mortality was recorded at 300 mg/kg. Moreover, these compounds were tested for their parenteral toxicity and the results revealed that all the test compounds were non-toxic up to 100 mg/ kg. We could conclude that the synthesis and biochemical evaluation of the newly synthesized compounds led to the design of a novel class of MAO-A inhibitors with good safety margins. 3. Conclusion A new class of quinoxaline highly potent and selective MAO-A inhibitors was identified. All the compounds exhibited good inhibition in nanomolar range especially 4e and 9g showed high potency (1.7 and 1.6 nM) combined with high selectivity comparable to the standard drugs clorgyline and moclobemide. This work introduces novel benzylquinoxaline-based analogs for synthesis of MAO-A inhibitors displaying good activity and selectivity profiles. Molecular modeling studies were done to better understanding of the novel compounds' mechanism of interaction and selectivity analysis and might give a rational clue for the design of a potential lead for the treatment of psychological disorders. 4. Experimental 4.1. Chemistry Melting points were determined with a Mel-Temp apparatus and are uncorrected. Magnetic resonance spectra (1H NMR and 13C NMR spectra) were recorded using a JEOL 500 MHz spectrometer with the chemical shift values reported in d units (part per million). Infrared data were obtained using a PerkineElmer 1600 series Fourier transform instrument as KBr pellets. The compounds were named using Chem. Draw Ultra version 12, Cambridge soft

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Chart 1. Rational modification of 3-benzylquinoxaline scaffold to targets II and III.

Chart 2. Rational modification of pyridazinylacetic acid derivatives to benzylquinoxaline derivatives IV.

Corporation. Elemental analyses were performed on PerkineElmer 2400 elemental analyzer, and the obtained values were within ±0.3% of the theoretical values. Follow up of the reactions and checking the purity of the compounds were made by TLC on silica

gel-protected aluminum sheets (Type 60 GF254, Merck) and the spots were detected by exposure to UV-lamp at l 254 nm for few seconds.

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(DMSO-d6): d 62.00, 125.55, 126.57, 126.97, 128.06, 128.56, 128.88, 129.07, 129.65, 129.79, 132.29, 133.54, 137.69, 137.82, 140.74, 147.37, 151.20, 166.68. Elemental analysis Calcd for C22H18N4O: C, 74.56; H, 5.12; N, 15.81. Found: C, 74.76; H, 5.35; N, 15.66.

Scheme 1. Synthetic routes to obtain compounds 1 and 3.

4.1.1.3. N0 -(3-benzylquinoxalin-2-yl)-4-chlorobenzohydrazide 4c. The product was obtained as a yellow solid, mp 180e181  C, in yield 0.25 g (64.4%). IR (KBr): 3448 (NH), 1665 (CO, amide) cm1. 1H NMR (DMSO-d6): d 4.37 (s, 2H, CH2), 7.18 (t, H, AreH, J ¼ 7.7 Hz), 7.26 (t, 2H, 2AreH, J ¼ 7.7 Hz), 7.38 (d, 3H, 3AreH, J ¼ 7.7 Hz), 7.48e7.52 (m, 2H, 2 AreH), 7.58 (d, 2H, 2AreH, J ¼ 8.4 Hz), 7.81 (d, H, AreH, J ¼ 7.7 Hz), 7.96 (d, 2H, 2AreH, J ¼ 9.2 Hz), 9.37 (s, H, NH,D2O exchangeable), 10.65 (s, H, NH,D2O exchangeable). 13C NMR (DMSOd6): d 47.9, 125.55, 126.57, 126.97, 128.57, 128.87, 129.22, 129.65, 129.82, 129.99, 132.18, 137.16, 137.77, 140.66, 147.18, 151.03, 165.74. Elemental analysis Calcd for C22H17ClN4O: C, 67.95; H, 4.41; N, 14.41. Found: C, 68.15; H, 4.67; N, 14.23.

Scheme 2. General procedure for preparation of compounds 4aef.

4.1.1.4. N0 -(3-benzylquinoxalin-2-yl)-4-methoxybenzohydrazide 4d. The product was obtained as a yellow solid, mp 185e186  C, in yield 0.35 g (92.1%). IR (KBr): 3446 (NH), 1623 (CO, amide) cm1. 1H NMR (DMSO-d6): d 3.80 (s, 3H, CH3) 4.36 (s, 2H, CH2), 7.00e7.06 (m,2H, 2 AreH), 7.15e7.19 (m, H, AreH), 7.24e7.28 (m, 2H, 2AreH), 7.36e7.40 (m, 3H, 3AreH), 7.46e7.50 (m, 2H, 2 AreH), 7.77e7.80 (m, H, AreH), 7.94 (d, 2H, 2 AreH, J ¼ 7.9 Hz), 9.35 (br s, H, NH,D2O exchangeable), 10.40 (br s, H, NH,D2O exchangeable). 13C NMR (DMSO-d6): d 43.50, 55.93, 114.27, 125.69, 126.95, 128.54, 128.86, 129.66, 129.74, 129.95, 137.88, 162.49, 165.99. Elemental analysis Calcd for C23H20N4O2: C, 71.86; H, 5.24; N, 14.57. Found: C, 72.13; H, 5.45; N, 14.76.

4.1.1. General method for preparation of N0 -(3-benzylquinoxalin-2yl)acetohydrazide, 4a and N0 -(3-benzylquinoxalin-2-yl) benzohydrazide derivatives 4bef A solution of (1 mmol) of selected acid, 0.38 g (1 mmol) of HATU and 0.28 mL (2 mmol) of triethylamine (TEA) in 2 mL dimethylformamide (DMF) was stirred at 0  C for 3 min. Then 2-benzyl3-hydrazinylquinoxaline 3 (0.25 g, 1 mmol) was added. The reaction mixture was stirred at 0  C for 1 h and left overnight at room temperature. The reaction mixture was poured into ice water, filtered, washed with 5% aqueous citric acid solution (2  10 mL), saturated sodium bicarbonate solution (2  10 mL) and water. The crude product recrystallized from dichloromethane.

4.1.1.5. N0 -(3-benzylquinoxalin-2-yl)-4-hydroxybenzohydrazide 4e. The product was obtained as a yellow solid, mp 260e261  C, in yield 0.25 g (76.5%). IR (KBr): 3500e3400 (br, OH), 3442 (NH), 1740 (CO, amide) cm1. 1H NMR (DMSO-d6): d 2.05 (s, H, OH, D2O exchangeable), 4.56 (s, 2H, CH2), 6.98 (d, 2H, 2 AreH, J ¼ 8.4 Hz), 7.19 (t, H, AreH, J ¼ 7.7 Hz), 7.28 (t, 2H, 2 AreH, J ¼ 7.7 Hz), 7.43e7.58 (m, 6H, 6 AreH), 7.95e8.02 (m, 2H, 2 AreH), 10.21 (br s, H, NH, D2O exchangeable), 10.63 (br s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 47.5, 115.67, 116.00, 116.26, 116.44, 118.82, 122.80, 123.30, 126.32, 127.25, 128.03, 129.02, 129.13, 129.86, 130.36, 132.06, 144.42, 150.53, 154.18, 160.09, 167.22. Elemental analysis Calcd for C22H18N4O2: C, 71.34; H, 4.90; N, 15.13. Found: C, 71.56; H, 5.11; N, 14.89.

4.1.1.1. N0 -(3-benzylquinoxalin-2-yl)acetohydrazide 4a. The product was obtained as a yellow solid, mp 122e123  C, in yield 0.25 g (85.6%). IR (KBr): 3434, 3358 (NH), 1680 (CO, amide) cm1. 1H NMR (DMSO-d6): d 1.94 (s, 3H, CH3) 4.27 (s, 2H, CH2), 7.16 (t, H, AreH, J¼7.7 Hz), 7.22e7.25 (m, 2H, 2 AreH), 7.32 (d, 2H, 2AreH, J ¼ 7.7 Hz), 7.38e7.46 (m, H, AreH), 7.53e7.55 (m, 2H, AreH), 7.75e7.77 (m, H, AreH), 9.09 (s, H, NH, D2O exchangeable), 9.89 (s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 23.08, 47.85, 100.00, 126.92, 128.51, 128.83, 129.45, 129.63, 129.79, 149.00. Elemental analysis Calcd for C17H16N4O: C, 69.85; H, 5.52; N, 19.17. Found: C, 70.08; H, 5.34; N, 18.96. 4.1.1.2. N0 -(3-benzylquinoxalin-2-yl)benzohydrazide 4b. The product was obtained as a yellow solid, mp 123e124  C, in yield 0.26 g (73.4%). IR (KBr): 3417 (NH), 1630 (CO, amide) cm1. 1H NMR (DMSO-d6): d 4.38 (s, 2H, CH2), 7.18 (d, H, CH, AreH, J ¼ 6.9 Hz), 7.24e7.28 (m, 3H, 3 AreH), 7.37e7.41 (m, 2H, 2 AreH), 7.49e7.53 (m, 4H, 4 AreH), 7.55e7.59 (m, H, AreH), 7.80 (d, H, AreH, J ¼ 7.6 Hz), 7.95 (d, 2H, 2 AreH, J ¼ 7.6 Hz), 9.35 (s, H, NH, D2O exchangeable), 10.56 (s, H, NH, D2O exchangeable). 13C NMR

4.1.1.6. N0 -(3-benzylquinoxalin-2-yl)-4-bromobenzohydrazide 4f. The product was obtained as a yellow solid, mp 175e176  C, in yield 0.41 g (93.8%). IR (KBr): 3443 (NH), 1628 (CO, amide) cm1. 1H NMR (DMSO-d6):d 4.38 (s, 2H, CH2), 7.21 (d, H, AreH, J ¼ 7.2 Hz), 7.29 (t, 2H, 2 AreH, J ¼ 7.8 Hz) 7.38 (d, 3H, 3 AreH, J ¼ 7.2 Hz), 7.49e7.52 (m, H, AreH), 7.58 (d, 2H, 2 AreH, J ¼ 9 Hz), 7.74e7.89 (m, 3H, AreH), 7.90e7.93 (m, H, CH, AreH), 10.45 (br s, H, NH, D2O exchangeable), 10.63 (br s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 45.00, 126.32, 127.90, 128.22, 129.00, 129.49, 131.49, 137.17, 142.9. Elemental analysis Calcd for C22H17BrN4O: C, 60.98; H, 3.95; N, 12.93. Found: C, 61.24; H, 4.16; N, 13.22. 4.1.2. Ethyl 2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetate 5 [17] (2 mmol) of 3-Benzyl-1H-quinoxalin-2-one derivatives 4 was dissolved in 5 mL dry DMF. 3.02 g (36 mmol) NaHCO3 and 0.432 g (2.6 mmol) KI were added to the reaction mixture and were stirred. Excess of ethyl bormoacetate, 1.33 gm (8 mmol), was then added to the reaction mixture. The reaction mixture was stirred at room temperature for 84 h and DMF was removed under reduced pressure. The residue was suspended in water (150 mL) and extracted with methylene chloride (100 mL). The organic layer was dried over

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315

Scheme 3. General procedure for preparation of compounds 7aed.

anhydrous sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was crystallized from ethanol as colorless crystals, mp 105e106  C, in yield 0.5 g (77.6%). IR (KBr): 1742 (CO, ester), 1651 (CO, amide) cm1. 1H NMR (CDCl3): d 1.24 (t, 3H, CH3, J ¼ 6.9 Hz), 4.22 (q, 2H, CH2, J ¼ 6.9 Hz), 4.28 (s, 2H, CH2), 4.98 (s, 2H, CH2), 7.00 (d, 1H, AreH, J ¼ 8.4 Hz), 7.20 (t, 1H, AreH, J ¼ 7.7 Hz), 7.25e7.35 (m, 3H, AreH), 7.40e7.50 (m, 3H, AreH), 7.87 (d, 1H, AreH, J ¼ 7.7 Hz). HMBC spectrum: The singlet at d 4.96 showed correlation to the carbons at d 130.16, 154.4 and 167.18 (C]O ester), while peak at d 4.28 showed correlation to carbons at 129.62, 136.96, 154.43 and 159.18. Elemental analysis Calcd for C19H18N2O3: C, 70.79; H, 5.63; N, 8.69. Found: C, 70.96; H, 5.44; N, 8.87. 4.1.3. Synthesis of 2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl) acetohydrazide 6 To a solution of ethyl 2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl) acetate 5 (1.04 g, 3.23 mmol) in 10 mL methanol, 1 mL hydrazine hydrate (80%) was added. The reaction mixture was refluxed for

about 4 h then cooled to room temperature. The excess of hydrazine was evaporated under reduced pressure. The product that precipitated was filtrated off and washed several time by ethanol. The product was obtained as white crystal, mp. 197e199  C, in yield 0.74 g (74.4%). IR (KBr): 3426, 3297 (NH), 1650 (CO, amide) cm1. 1 HNMR (DMSO-d6): d 4.13 (s, 2H, CH2), 4.825 (s, 2H, CH2), 4.26 (br s, 2H, NH2, D2O exchangeable), 7.17 (t, H, AreH), 7.22e7.32 (m, 6H, 6 AreH), 7.51 (t, H, AreH), 7.73 (d, H, AreH), 9.38 (s, H, NH, D2O exchangeable). 4.1.4. General method for preparation of N0 -[2-(3-benzyl-2oxoquinoxalin-1(2H)-yl)acetyl] benzohydrazide derivatives 7aed A solution of (1 mmol) of the selected acid, 0.38 g (1 mmol) of HATU and 0.35 mL (2 mmol) DIEA in 2 mL DMF was stirred at 0  C. Then 0.31 g (1 mmol) 2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetohydrazide 6 was added. The reaction mixture was stirred at 0  C for 1 h and left overnight at room temperature. The reaction mixture was poured into ice water, filtered, washed with 5%

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S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320 Table 2 Comparative docking analysis of prominent selective ligands against enzyme (MAOA). Compound 4e

Interaction residuesa

Ser209, Glu215 Leu97, Ile325, Phe208, Phe382 Gly71, Glu74 9g Ser209, Gly214 Glu215 Phe208 Glu74 Moclobemide Ser209 Ile207, Val210, Leu97 Phe208 Tyr407

a b

Interaction type

Ligand fragment

H-bonding NHeNH Hydrophobic Benzyl H-bonding H-bonding H-bonding Hydrophobic H-bonding H-bonding Hydrophobic

peOHePh C]OeQ NHeC]O Benzyl peOHePh C]O ClePh

Binding energy (dG, kcal/mol)b 11.8

12.5

8.9

Hydrophobic Ethyl H-bonding Morpholine

Residues are abbreviated as in the MOE software. Binding energy values are stated without any corrections.

aqueous citric acid solution (2  10 mL), saturated sodium bicarbonate solution (2  10 mL) and water. The crude product was recrystallized from dichloromethane.

Scheme 4. General procedure for preparation of compounds 10aec.

Table 1 Effect of some quinoxaline derivatives on the activity of MAO-A and B isoforms. Compound

4a 4b 4c 4d 4e 4f 7a 7b 7c 7d 8a 8b 8c 9a 9b 9c 9d 9e 9f 9g 9h 10a 10b 10c Clorgyline Moclobemide

IC50 (nM)a

Selectivity index (SI)b

MAO-A

MAO-B

66 78 34 86 1.7 74 52 64 82 56 82 2.9 3.4 3.1 3.6 6.6 8.4 22 98 1.6 7.3 56 88 36 3.1 11  103

620,000 360,000 730,000 920,000 940,000 980,000 680,000 880,000 980,000 640,000 840,000 440,000 280,000 880,000 840,000 82,000 960,000 26,000 84,000 980,000 96,000 87,000 760,000 890,000 940,000 145  104

9393 4615 21,471 10,698 552,941 13,243 13,077 13,750 11,951 11,429 10,244 151,724 8235 283,871 233,333 1242 11,428 1182 857 612,500 13,151 1554 8636 24,722 30,323 132

The most significant values are given in bold. a Values were determined from the kinetic experiments in which serotonin and benzylamine were used as substrates at 125 and 8 mM for measuring MAO-A and B activity respectively. The substrate concentrations are 1.25 fold of Km (MichaeliseMenten Constant). The IC50 value was calculated and analyzed using the fourparameter logistic function in SigmaPlot software (SigmaPlot 12.3, Systat Software. Inc., Richmond, CA, USA) using six independent experiments that were measured in duplicate and the average was taken for statistical analysis. b Selectivity Index (SI) was calculated by SI ¼ MAO-B IC50/MAO-A IC50.

4.1.4.1. N 0 -[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetyl]-4methylbenzohydrazide 7a. The product was obtained as a white solid, mp 210e213  C (dec), in yield 0.29 g (68%). IR (KBr): 3454, 3270 (NH), 1707, 1659 (CO, amide) cm1. 1H NMR (DMSO-d6): Isomer (A, 66.3%): d 2.26 (s, 3H, CH3), 4.93 (s, 2H, CH2), 5.30 (s, 2H, CH2), 6.97e7.31 (m, 3H, 3 AreH), 7.33e7.44 (m, 2H, 2 AreH), 7.53e7.70 (m, 5H, 5 AreH), 7.77e7.94 (m, 3H, 3 AreH), 9.88 (br s, 2H, 2 NH, D2O exchangeable). Isomer (B, 33.7%): d 2.26 (s, 3H, CH3), 4.93 (s, 2H, CH2), 5.70 (s, 2H, CH2),6.97e7.31 (m, 3H, 3 AreH), 7.33e7.44 (m, 2H, 2 AreH), 7.53e7.70 (m, 5H, 5 AreH), 7.77e7.94 (m, 3H, 3 AreH), 9.88 (br s, 2H, 2 NH, D2O exchangeable). 13C NMR (DMSO-d6): d 21.40, 45.02, 116.10, 124.59, 127.33, 128,35, 129.41, 129.66, 130.14, 130.23, 130.35, 132.22, 132.54, 132.75, 134.17, 134.90, 135.32, 140.85, 153.50, 155.30, 162.29, 192.96. Elemental analysis Calcd for C25H22N4O3: C, 70.41; H, 5.20; N, 13.14. Found: C, 71.65; H, 4.96; N, 13.35. 4.1.4.2. N 0 -[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetyl]-4chlorobenzohydrazide 7b. The product was obtained as a white solid, mp 130e131  C, in yield 0.30 g (67%). IR (KBr): 3233 (NH), 1708, 1660 (CO, amide) cm1. 1HNMR (DMSO-d6): Isomer (A, 66.3%): d 4.14 (s, 2H, CH2), 4.98 (s, 2H, CH2), 7.16e7.33 (m, 7H, 7 AreH), 7.38e7.41 (m, 2H, 2 AreH), 7.48e7.53 (m, H, AreH), 7.74 (t, H, AreH, J ¼ 8.4 Hz), 7.81 (d, H, AreH, J ¼ 8.4 Hz), 7.92 (d, H, AreH, J ¼ 7.7 Hz), 9.52 (br s, H, NH, D2O exchangeable), 10.48 (br s, H, NH, D2O exchangeable). Isomer (B, 33.7%): d 4.14 (s, 2H, CH2), 5.25 (s, 2H, CH2), 7.16e7.33 (m, 7H, 7 AreH), 7.38e7.41 (m, 2H, 2 AreH), 7.48e7.53 (m, H, AreH), 7.74 (t, H, AreH, J ¼ 8.4 Hz), 7.81 (d, H, AreH, J ¼ 8.4 Hz), 7.92 (d, H, AreH, J ¼ 7.7 Hz), 9.52 (br s, H, NH, D2O exchangeable), 10.48 (br s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 43.68, 44.07, 115.25, 124.12, 126.98, 129.09, 129.63, 129.73, 129.78, 130.56, 132.29, 132.41, 132.51, 133.28, 133.48, 136.79, 137.80, 154.53, 159.25, 164.52, 165.51. Elemental analysis Calcd for C24H19ClN4O3: C, 64.50; H, 4.29; N, 12.54. Found: C, 64.78; H, 4.67; N, 12.80. 4.1.4.3. N 0 -[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetyl]-4bromobenzo hydrazide 7c. The product was obtained as a white solid, mp 169e170  C, in yield 0.36 g (75.4%). IR (KBr): 3457, 3268 (NH), 1660 (CO, amide) cm1. 1HNMR (DMSO-d6): Isomer (A, 66.3%): d 4.14 (s, 2H, CH2), 4.96 (s, 2H, CH2) 7.16e7.33 (m, 7H, 7

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AreH), 7.40e7.57 (m, 3H, 3 AreH), 7.72e7.76 (m, 2H, 2 AreH), 7.85 (d, H, AreH, J ¼ 8.4 Hz), 9.45 (br s, H, NH, D2O exchangeable), 10.38 (br s, H, NH, D2O exchangeable). Isomer (B, 33.7%): d 4.14 (s, 2H, CH2), 5.27 (s, 2H, CH2), 7.16e7.33 (m, 7H, 7 AreH), 7.40e7.57 (m, 3H, 3 AreH), 7.72e7.76 (m, 2H, 2 AreH), 7.85 (d, H, AreH, J ¼ 8.4 Hz),9.45 (br s, H, NH, D2O exchangeable), 10.38 (br s, H, NH, D2O exchangeable). Elemental analysis Calcd for C24H19BrN4O3: C, 58.67; H,3.90; N, 11.40. Found: C, 58.49; H, 4.15; N, 11.68. 4.1.4.4. N 0 -[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetyl]-4nitrobenzohydrazide 7d. The product was obtained as a yellow solid, mp 190e192  C (dec), in yield 0.32 g (70%). IR (KBr): 3461, 3273 (NH), 1647 (CO, amide) cm1. 1H NMR (DMSO-d6): Isomer (A, 72.1%): d 4.14 (s, 2H, CH2), 5.04 (s, 2H, CH2), 7.16e7.19 (m, H, AreH), 7.26 (t, 2H, 2 AreH, J ¼ 7.7 Hz), 7.31e7.38 (m, 4H, 4 AreH), 7.50e7.57 (m, 2H, 2 AreH), 7.76 (d, H, AreH, J ¼ 7.7 Hz), 8.04 (d, H, AreH, J ¼ 8.4 Hz), 8.14e8.19 (m, H, AreH), 8.25 (d, H, AreH, J ¼ 8.4 Hz), 9.41 (s, H, NH, D2O exchangeable), 10.77 (br, H, NH, D2O exchangeable). Isomer (B, 27.9%): d 4.14 (s, 2H, CH2), 5.24 (s, 2H, CH2), 7.16e7.19 (m, H, AreH), 7.26 (t, 2H, 2 AreH, J ¼ 7.7 Hz), 7.31e7.38 (m, 4H, 4 AreH), 7.50e7.57 (m, 2H, 2 AreH), 7.76 (d, H, AreH, J ¼ 7.7 Hz), 8.04 (d, H, AreH, J ¼ 8.4 Hz), 8.14e8.19 (m, H, AreH), 8.25 (d, H, AreH, J ¼ 8.4 Hz), 9.63 (s, H, NH, D2O exchangeable), 10.77 (br, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 43.68, 115.19, 124.27, 126.96, 128.88, 129.52, 129.74, 130.59, 132.55, 133.24, 137.72, 138.35, 149.92, 154.53, 159.24, 164.45, 166.09, 166.21. Elemental analysis Calcd for C24H19N5O5: C, 63.02; H, 4.19; N, 15.31. Found: C, 63.29; H, 4.37; N, 15.22. 4.1.5. General method for preparation of (9H-fluoren-9-yl)methyl 1-[2-(2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetyl)hydrazinyl]-2ylcarbamate derivatives 8aec A mixture of (1 mmol) of Fmoc-amino acids, 0.38 g (1 mmol) of HATU and 0.35 mL (2 mmol) diisopropylethylamine (DIEA) was stirred in 2 mL dimethylformamide at 0  C. Then 0.31 g (1 mmol) 2(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetohydrazide 6 was added. The reaction mixture was stirred at 0  C for 1 h and left overnight at room temperature. In case of Fmoc-L-leucine the reaction mixture was diluted with 80 mL ethyl acetate, and the mixture was washed with 5% aqueous citric acid solution (2  10 mL), saturated sodium bicarbonate solution (2  10 mL) and saturated sodium chloride solution (2  10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified by column (ethyl acetate/hexane, 1:2). In case of Fmoc-L-tyrosine-(O-t-Bu) and Fmoc-L-phenylalanine, the reaction mixture was poured into ice water, filtered, washed with 5% aqueous citric acid solution, saturated sodium bicarbonate solution, water, dried and recrystallized from dichloromethane. 4.1.5.1. (9H-fluoren-9-yl)methyl 1-[2-(2-(3-benzyl-2-oxoquinoxalin1(2H)-yl)acetyl)hydrazinyl]-4-methyl-1-oxopentan-2-ylcarbamate 8a. The product was obtained as a white solid, mp 203e205  C, in yield 0.48 g (74.6%). IR (KBr): 3445, 3281 (NH), 1655 (CO, amide) cm1. 1H NMR (DMSO-d6): d 0.79e0.89 (m, 6H, 2 CH3), 1.34e1.63 (m, 3H, CH2 and CH), 4.05e4.23 (m, 6H, 3 CH2), 4.94 (s, 2H, 2 CH), 7.34e7.86 (m, 17H, 17 AreH), 9.66 (s, H, NH, D2O exchangeable), 10.07 (br s, H,NH, D2O exchangeable), 10.48 (br s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 21.94, 23.49, 24.58, 41.29, 43.80, 47.16, 52.00, 66.14, 110.32, 115.17, 120.57, 120.62, 121.92, 124.10, 125.87, 126.94, 127.57, 127.83, 128.17, 128.86, 129.47, 129.75, 130.53, 132.53, 133.24, 137.73, 137.95, 139.95, 141.22, 144.26, 144.44, 154.49, 156.40, 159.24, 165.66, 169.68, 171.92, 174.86. Elemental analysis Calcd for C38H37N5O5: C, 70.90; H, 5.79; N, 10.88 Found: C, 71.18; H, 5.99; N, 11.10.

317

4.1.5.2. (9H-fluoren-9-yl)methyl 1-[2-(2-(3-benzyl-2-oxoquinoxalin1(2H)yl)acetyl)hydrazinyl]-1-oxo-3-phenylpropan-2-ylcarbamate 8b. The product was obtained as a white solid, mp 193e195  C, in yield 0.67 g (98.7%). IR (KBr): 3446, 3283 (NH), 1695 (CO, urethane), 1654 (CO, amide) cm1. 1H NMR (DMSO-d6): d 2.50e3.36 (m, 4H, 2 CH2), 4.10e4.14 (m, 4H, 2 CH2), 5.00e5.10 (m, 2H, 2 CH), 7.11e8.08 (m, 22H, 22 AreH), 9.74 (s, H, NH, D2O exchangeable), 10.31 (br s, H, NH, D2O exchangeable), 10.60 (br s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 41.50, 54.80, 109.71, 114.56, 119.94, 121.30, 123.49, 126.31, 127.20, 127.52, 127.96, 128.24, 128.83, 129.11, 129.15, 129.91, 131.87, 132.63, 137.10, 137.31, 139.30, 142.44, 158.60, 164.79. Elemental analysis Calcd for C41H35N5O5: C, 72.66; H, 5.21; N, 10.33. Found: C, 72.49; H, 4.89; N, 10.56. 4.1.5.3. (9H-fluoren-9-yl)methyl 1-[2-(2-(3-benzyl-2-oxoquinoxalin1(2H)-yl)acetyl) hydrazinyl]-3-(4-tert-butoxyphenyl)-1-oxopropan2-ylcarbamate 8c. The product was obtained as a white solid, mp 188e190  C, in yield 0.62 g (82.7%). IR (KBr): 3445, 3279 (NH), 1695 (CO, urethane), 1655 (CO, amide) cm1. 1H NMR (DMSO-d6): d 0.99e1.18 (m, 9H, 3 CH3), 2.69e3.02 (m, 2H, CH2), 4.06e4.14 (m, 6H, 3 CH2), 4.25e4.31 (m, H, CH), 4.96e4.98 (m, H, CH), 6.67e6.86 (m, 2H, 2 AreH), 7.17e7.45 (m, 13H, 13 AreH), 7.52e7.84 (m, 6H, 6 AreH), 10.25 (s, H, NH, D2O exchangeable), 10.43 (br s, H, NH, D2O exchangeable), 10.50 (s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 28.79, 28.99, 43.69, 46.97, 55.23, 66.22, 78.08, 115.39, 120.59, 123.81, 124.11, 125.88, 126.95, 127.57, 128.15, 128.86, 129.75, 130.26, 130.37, 130.53, 132.54, 132.94, 133.27, 137.73, 141.20, 144.26, 153.97, 154.45, 156.33, 159.24, 165.68, 171.23. Elemental analysis Calcd for C45H43N5O6: C, 72.08; H, 5.78; N, 9.34 Found: C, 71.87; H, 6.02; N, 9.61. 4.1.6. General method for preparation of 2-(3-benzyl-2oxoquinoxalin-1(2H)-yl)-N0 -benzylidene acetohydrazide derivatives 9aeh A solution of 2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetohydrazide 6 (1 mmol) in ethanol (10 mL) was added to substituted aldehydes or ketones (1 mmol) which were dissolved in ethanol (10 mL), and glacial acetic acid (2 drops) and the reaction mixture was then refluxed for 3 h. The product was separated out on cooling, filtered off, recrystallized from ethanol and dried. 4.1.6.1. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl)-N0 -benzylideneaceto hydrazide 9a. The product was obtained as a white solid, mp 275e276  C, in yield 0.29 g (73.2%). IR (KBr): 3446 (NH), 1683 (CO, amide), 1655 (CO, amide) cm1. 1H NMR (DMSO-d6): Isomer (A, 77.6%): d 4.15 (s, 2H, CH2), 5.42 (s, 2H, CH2), 7.17 (t, H, AreH, J ¼ 7.7 Hz), 7.26 (t, 2H, 2 AreH, J ¼ 7.7 Hz), 7.31e7.35 (m, 3H, 3 AreH), 7.40e7.46 (m, 4H, 4 AreH), 7.50e7.57 (m, H, AreH), 7.66e7.67 (m, H, AreH), 7.71e7.73 (m, H, AreH), 7.78 (d, H, CH, AreH, J ¼ 7.6 Hz), 8.05 (s, H, CH, aldhyde), 11.80 (s, H, NH, D2O exchangeable). Isomer (B, 22.4%): d 4.15 (s, 2H, CH2), 5.01 (s, 2H, CH2), 7.17 (t, H, AreH, J ¼ 7.7 Hz), 7.26 (t, 2H, 2 AreH, J ¼ 7.7 Hz), 7.31e7.35 (m, 3H, 3 AreH), 7.40e7.46 (m, 4H, 4 AreH), 7.50e7.57 (m, H, AreH), 7.66e7.67 (m, H, AreH), 7.71e7.73 (m, H, AreH), 7.78 (d, H, CH, AreH, J ¼ 7.6 Hz), 8.20 (s, H, CH, aldhyde), 11.80 (s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 43.68, 115.47, 124.04, 126.97, 127.55, 128.90, 129.38, 129.69, 130.67, 132.49, 133.56, 134.46, 137.77, 144.82, 154.57, 168.00. Elemental analysis Calcd for C24H20N4O2: C, 72.71; H, 5.08; N, 14.13. Found: C, 72.54; H, 4.87; N, 14.35. 4 .1. 6 . 2 . 2 - ( 3 - B e n z y l - 2 - o x o q u i n o x a l i n - 1 ( 2 H ) - y l ) - N 0 - ( 4 hydroxybenzylidene)-acetohydrazide 9b. The product was obtained as a white solid, mp 250e251  C, in yield 0.40 g (97.1%). IR (KBr): 3500e3400 (OH), 3447 (NH), 1679 (CO, amide), 1655 (CO,

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amide) cm1. 1H NMR (DMSO-d6): Isomer (A, 69.9%): d 4.15 (s, 2H, CH2), 5.37 (s, 2H, CH2), 6.78e6.80 (m, 2H, 2 AreH), 7.16e7.55 (m, 10H, 10 AreH), 7.77 (d, H, AreH, J ¼ 7.7 Hz), 7.93 (s, H, CH, aldhyde), 10.06 (br s, H, OH, D2O exchangeable),11.80 (s, H, NH, D2O exchangeable). Isomer (B, 30.1%): d 4.15 (s, 2H, CH2), 4.97 (s, 2H, CH2), 6.78e6.80 (m, 2H, 2 AreH), 7.16e7.55 (m, 10H, 10 AreH), 7.77 (d, H, AreH, J ¼ 7.7 Hz), 8.07 (s, H, CH, aldhyde), 10.06 (br s, H, OH, D2O exchangeable), 11.80 (s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 43.68, 115.34, 115.47, 116.24, 124.00, 125.39, 126.96, 128.89, 129.28, 129.69, 130.66, 133.57, 137.78, 145.11, 154.55, 159.05, 160.03, 162.03, 166.36, 167.57, 177.31. Elemental analysis Calcd for C24H20N4O3: C, 69.89; H, 4.89; N, 13.58. Found: C, 70.12; H, 5.15; N, 13.33. 4 .1. 6 . 3 . 2 - ( 3 - B e n z y l - 2 - o x o q u i n o x a l i n - 1 ( 2 H ) - y l ) - N 0 - ( 4 methoxybenzylidene)-acetohydrazide 9c. The product was obtained as a white solid, mp. 250e251  C (dec), in yield 0.39 g (91.6%). IR (KBr): 3453 (NH), 1680 (CO, amide), 1652 (CO, amide) cm1. 1H NMR (DMSO-d6): Isomer (A, 80.5%): d 3.80 (s, 3H, CH3), 4.18 (s, 2H, CH2), 5.43 (s, 2H, CH2), 6.99e7.83 (m, 13H, 13 AreH), 8.01 (s, H, CH, aldhyde), 11.71 (s, H, NH, D2O exchangeable). Isomer (B, 19.5%): d 3.80 (s, 3H, CH3), 4.18 (s, 2H, CH2), 5.02 (s, 2H, CH2), 6.99e7.83 (m, 13H, 13 AreH), 8.16 (s, H, CH, aldhyde), 11.74 (s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 43.59, 55.24, 114.22, 123.40, 126.34, 126.40, 128.26, 128.52, 129.07, 130.02, 131.83, 132.92, 137.13, 144.02, 153.90, 158.39, 160.68, 167.07. Elemental analysis Calcd for C25H22N4O3: C, 70.41; H, 5.20; N, 13.14. Found: C, 70.66; H, 5.48; N, 13.37. 4 .1. 6 . 4 . 2 - ( 3 - B e n z y l - 2 - o x o q u i n o x a l i n - 1 ( 2 H ) - y l ) - N 0 - ( 4 chlorobenzylidene) acetohydrazide 9d. The product was obtained as a white solid, mp 254e255  C, in yield 0.37 g (85.9%). IR (KBr): 3448 (NH), 1683 (CO, amide), 1654 (CO, amide) cm1. 1H NMR (DMSOd6): Isomer (A, 74.7%): d 4.14 (s, 2H, CH2), 5.42 (s, 2H, CH2) 7.18e7.52 (m, 10H, 10 AreH), 7.68e7.79 (m, 3H, 3 AreH), 8.03 (s, H, CH, aldhyde), 11.87 (s, H, NH, D2O exchangeable). Isomer (B, 25.3%): d 4.14 (s, 2H, CH2), 5.00 (s, 2H, CH2), 7.18e7.52 (m, 10H, 10 AreH), 7.68e7.79 (m, 3H, 3 AreH), 8.19 (s, H, CH, aldhyde), 11.94 (s, H,NH, D2O exchangeable). 13C NMR (DMSO-d6): d 44.22, 44.62, 61.82, 71.41, 115.42, 124.17, 126.97, 128.89, 129.21, 129.32, 129.44, 129.67, 130.68, 132.47, 133.40, 133.51, 135.04, 137.73, 142.56, 146.54, 154.55, 159.14, 163.45, 168.08. Elemental analysis Calcd for C24H19ClN4O2: C, 66.90; H, 4.44; N, 13.00. Found: C, 67.16; H, 4.75; N, 13.24. 4 .1. 6 . 5 . 2 - ( 3 - B e n z y l - 2 - o x o q u i n o x a l i n - 1 ( 2 H ) - y l ) - N 0 - ( 4 bromobenzylidene) acetohydrazide 9e. The product was obtained as a yellow solid, mp 244e246  C, in yield 0.39 g (82.1%). IR (KBr): 3447 (NH), 1680 (CO, amide), 1654 (CO, amide) cm1.1H NMR (DMSO-d6): Isomer (A, 81.4%): d 4.14 (s, 2H, CH2), 5.42 (s, 2H, CH2) 7.11e7.79 (m, 13H, 13 AreH), 8.02 (s, H, CH, aldhyde), 11.87 (s, H, NH, D2O exchangeable). Isomer (B, 18.6%): d 4.14 (s, 2H, CH2), 5.00 (s, 2H, CH2), 7.11e7.79 (m, 13H, 13 AreH), 8.19 (s, H, CH, aldhyde), 11.93 (s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 44.22, 44.65, 115.47, 123.85, 124.04, 124.14, 126.96, 128.89, 129.44, 129.54, 129.69, 130.65, 132.34, 132.49, 133.54, 133.77, 137.76, 143.60, 154.55, 159.03, 163.45, 168.09. Elemental analysis Calcd for C24H19BrN4O2: C, 60.64; H, 4.03; N, 11.79. Found: C, 60.89; H, 4.32; N, 11.58. 4 .1. 6 . 6 . 2 - ( 3 - B e n z y l - 2 - o x o q u i n o x a l i n - 1 ( 2 H ) - y l ) - N 0 - ( 4 nitrobenzylidene) acetohydrazide 9f. The product was obtained as a white solid, mp 260e261  C, in yield 0.36 g (81.6%). IR (KBr): 3434 (NH), 1684 (CO, amide), 1652 (CO, amide) cm1. 1H NMR (DMSOd6): Isomer (A, 78.8%): d 4.14 (s, 2H, CH2), 5.46 (s, 2H, CH2), 7.17 (t, H, AreH, J ¼ 6.9 Hz), 7.24e7.34 (m, 5H, 5 AreH), 7.41e7.55 (m, 2H, 2 AreH), 7.77 (d, H, AreH, J ¼ 8.4 Hz), 7.98 (d, 2H, 2 AreH, J ¼ 7.7 Hz),

8.13 (s, H, CH, aldhyde), 8.25 (d, 2H, 2 AreH, J ¼ 6.9 Hz), 12.10 (s, H, NH, D2O exchangeable). Isomer (B, 21.2%): d 4.14 (s, 2H, CH2), 5.04 (s, 2H, CH2), 7.17 (t, H, CH, AreH, J ¼ 6.9 Hz), 7.24e7.34 (m, 5H, 5 AreH), 7.41e7.55 (m, 2H, 2 AreH), 7.77 (d, H, AreH, J ¼ 8.4 Hz), 7.92 (d, 2H, 2 AreH, J ¼ 7.7 Hz), 8.25 (d, 2H, 2 AreH, J ¼ 6.9 Hz), 8.30 (s, H, CH, aldhyde), 12.10 (s, H, NH, D2O exchangeable). 13C NMR (DMSOd6): d 43.68, 115.41, 124.08, 124.52, 126.97, 128.48, 128.62, 128.89, 129.69, 130.68, 132.49, 133.48, 137.73, 140.72, 142.46, 148.33, 154.54, 159.00, 168.46. Elemental analysis Calcd for C24H19N5O4: C, 65.30; H, 4.34; N, 15.86. Found: C, 65.58; H, 4.41; N, 15.99. 4 .1. 6 . 7 . 2 - ( 3 - B e n z yl - 2 - o xo q u i n o x al i n - 1 ( 2 H ) - yl ) - N 0 - [ 1 - ( 4 hydroxyphenyl) ethylidene] acetohydrazide 9g. The product was obtained as a white solid, mp 250e252  C, in yield 0.41 g (96.1%). IR (KBr): 3500e3400 (OH), 3248 (NH), 1664 (CO, amide), 1637 (CO, amide) cm1. 1H NMR (DMSO-d6): Isomer (A, 78.9%): d 2.21 (s, 3H, CH3) 4.15 (s, 2H, CH2), 5.41 (s, 2H, CH2), 6.75 (t, 2H, 2 AreH, J ¼ 7.7 Hz), 7.17 (t, H, AreH, J ¼ 7.7 Hz) 7.24e7.43 (m, 6H, 6 AreH), 7.50e7.56 (m, 2H, 2 AreH), 7.68 (d, H, AreH, J ¼ 7.7 Hz), 7.77 (d, H, AreH, J ¼ 8.4 Hz), 9.78 (br s, H, OH, D2O exchangeable), 10.90 (s, H, NH, D2O exchangeable). Isomer (B, 21.1%): d 2.24 (s, 3H, CH3) 4.15 (s, 2H, CH2), 5.10 (s, 2H, CH2), 6.98 (t, H, AreH, J ¼ 7.7 Hz), 7.10 (t, 2H, 2 AreH, J ¼ 7.7 Hz), 7.24e7.43 (m, 6H, 6 AreH), 7.56e7.60 (m, 2H, 2 AreH), 7.68 (d, H, AreH, J ¼ 7.7 Hz), 7.89e7.91 (m, H, AreH), 9.78 (br s, H, OH, D2O exchangeable), 10.74 (s, H, NH, D2O exchangeable). 13C NMR (DMSO-d6): d 14.08, 43.68, 115.47, 115.65, 124.00, 126.96, 128.35, 128.48, 128.89, 129.31, 129.70, 130.64, 132.47, 133.56, 137.80, 149.62, 154.59, 159.08, 159.21, 168.21, 168.51. Elemental analysis Calcd for C25H22N4O3: C, 70.41; H, 5.20; N, 13.14. Found: C, 70.72; H, 5.01; N, 13.41. 4.1.6.8. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl)-N0 -(2-oxoindolin3ylidene) acetohydrazide 9h. The product was obtained as a yellow solid, mp 255e256  C (dec), in yield 0.31 g (70.9%). IR (KBr): 3439, 3254 (NH), 1732 (CO, amide), 1694 (CO, amide), 1648 (CO, amide) cm1. 1H NMR (DMSO-d6): Isomer (A): d 4.15 (s, 2H, CH2), 5.55 (s, 2H, CH2), 6.88e6.94 (m, H, AreH), 7.00e7.08 (m, H, AreH), 7.17 (t, H, AreH, J ¼ 7.7 Hz), 7.24e7.36 (m, 6H, 6 AreH), 7.45e7.57 (m, 2H, 2 AreH), 7.78 (d, H, AreH, J ¼ 8.4 Hz), 8.11 (d, H, AreH, J ¼ 6.9 Hz), 10.84 (s, H, NH, D2O exchangeable), 11.60 (s, H, NH, D2O exchangeable). Isomer (B): d 4.14 (s, 2H, CH2), 5.44 (s, 2H, CH2), 6.88e6.94 (m, H, AreH), 7.00e7.08 (m, H, AreH), 7.17 (t, H, AreH, J ¼ 7.7 Hz), 7.24e7.36 (m, 6H, 6 AreH), 7.45e7.57 (m, 2H, 2 AreH), 7.78 (d, H, AreH, J ¼ 8.4 Hz), 8.11 (d, H, AreH, J ¼ 6.9 Hz), 11.30 (br s, H, NH, D2O exchangeable), 12.69 (br s, H, NH, D2O exchangeable). 13 C NMR (DMSO-d6): d 44.68, 44.79, 111.25, 115.34, 115.69, 122.33, 124.19, 126.98, 128.90, 129.69, 130.70, 132.52, 133.39, 137.72, 144.38, 154.58, 159.06, 165.11. Elemental analysis Calcd for C25H19N5O3: C, 68.64; H, 4.38; N, 16.01. Found: C, 68.46; H, 4.76; N,15.88. 4.1.7. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl) acetic acid 11 Dissolve (1 mmol) of the ester 5 in 20 mL of 10% KOH in ethanol. The reaction mixture was refluxed for 3 h. The reaction mixture was neutralized with 1 N HCl solution. The precipitate formed was filtered off and washed with water to give a white solid, mp 232e233  C, in yield 0.27 g (91.74%). IR (KBr): 3200e2800 (OH), 1744 (CO, acid), 1625 (CO, amide) cm1. 1H NMR (DMSO-d6): d 4.13 (s, 2H, CH2), 4.96 (s, 2H, CH2), 7.17 (t, H, AreH, J ¼ 7.7 Hz), 7.25 (t, 2H, 2AreH, J ¼ 7.7 Hz), 7.29e7.35 (m, 3H, 3AreH), 7.43 (d, H, AreH, J ¼ 8.5 Hz), 7.54 (t, H, AreH, J ¼ 7.7 Hz), 7.77 (d, H, AreH, J ¼ 6.9 Hz), 12.2 (s, 1H, COOH, D2O exchangeable). Elemental analysis Calcd for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52. Found: C, 69.15; H, 5.03; N, 9.27.

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4.1.8. General method for preparation of ethyl 2-(3-benzyl-2oxoquinoxalin-1(2H)-yl)acetate derivatives 10aee A mixture of (0.294 g, 1 mmol) of acid 6, 0.38 g (1 mmol) of HATU and 0.28 mL (2 mmol) of triethylamine (TEA) was stirred at 0  C for 3 min in 2 mL dimethylformamide. Then amino acid t-butyl ester (1 mmol) was added. The reaction mixture was stirred at 0  C for 1 h and left overnight at room temperature. In case of L-leucine t-butyl ester the reaction mixture was diluted with 80 mL ethyl acetate, and the mixture was washed with 5% aqueous citric acid solution (2  10 mL), saturated sodium bicarbonate solution (2  10 mL) and saturated sodium chloride solution (2  10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo. The crude product was purified by column. In case of L-(O-t-Bu)-tyrosine t-butyl ester and L-phenylalanine t-butyl ester the reaction mixture was poured into ice water, filtered, washed with 5% aqueous citric acid solution, saturated sodium bicarbonate solution and finally with water, dried and recrystallized. 4.1.8.1. tert-Butyl 2-[2-(3-benzyl-2-oxo-2H-quinoxalin-1-yl)-acetylamino]-4-methyl-pentanoate 10a. The product was purified by column (1:1, ethyl acetate: hexane) to obtain a pale yellow oily product, 0.35 g (75.6%) yield. 1H NMR (CDCl3): d 0.83 (d, 6H, 2 CH3, J ¼ 3.9 Hz), 1.34 (s, 9H, 3 CH3), 1.37e1.54 (m, 2H, CH2), 1.69 (m, 1H, CH), 4.29 (s, 2H, CH2), 4.38 (m, 1H, CH), 4.83, 4.88 (2d, 2H, CH2,J ¼ 15.4 Hz), 6.61 (d, 1H, NH), 7.18e7.22 (m, H, AreH), 7.26e7.30 (m, 2H, 2 AreH), 7.33e7.36 (m, H, AreH), 7.40e7.45 (m, 3H, 3 AreH), 7.51 (td, H, AreH, J ¼ 6.9 Hz, J ¼ 1.5 Hz), 7.86 (dd, H, ArH, J ¼ 7.7 Hz, J ¼ 1.5 Hz). Elemental analysis Calcd for C27H33N3O4: C, 69.95; H, 7.18; N, 9.06. Found: C, 70.19; H, 6.99; N, 9.34.

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and MAO-B selectivity in the presence of the specific substrate, serotonin (MAO-A) at concentration 125 mM or benzylamine (MAOB) at concentration 8 mM, respectively. 4.2.1. Mitochondria preparation Bovine brain mitochondria were isolated according to Basford [21]. Briefly, fresh bovine brain was obtained from a slaughterhouse and placed in an ice-cold buffered sucrose (0.32 mol/l sucrose, 4 mmol/l HEPES; pH 7.4) and transported to laboratory. The brain cortex was homogenized in ten volumes (w/v) of ice-cold buffered sucrose containing aprotinin (0.15 U/ml, competitive serine protease inhibitor) using a Teflon piston homogenizer. The homogenate was centrifuged at 1000  g for 10 min to remove cell debris and unseparated cells. The supernatant was carefully decanted, resuspended in the homogenization buffer and centrifuged at 1000  g for 10 min. The resultant supernatant was carefully decanted and centrifuged at 10,000  g for 15 min. The obtained mitochondria pellet was rinsed twice with the homogenization buffer by repeat of the resuspension and centrifugation steps. The protein concentration of the resuspended pellet adjusted to 20 mg/ ml, prepared in 1 ml aliquots and stored at 80  C.

4.1.8.3. tert-Butyl 2-[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetamido]-3-(4-tert-butoxyphenyl)propanoate 10c. The crude compound was recyrstallized (methylene chloride/hexane) and the product was obtained as a white powder, 0.52 g (91.3%) yield, mp 65e66  C. IR (KBr): 3313 (NH), 1732 (CO, ester), 1691 (CO, amide), 1659 (CO, amide) cm1. 1H NMR (CDCl3): d 1.28, 1.31 (2s, 18H, 6 CH3), 2.89e2.98 (m, 2H, CH2), 4.23, 4.28 (2d, 2H, CH2, J ¼ 13.8 Hz), 4.66 (m, H, CH), 4.72, 4.92 (2d, 2H, CH2, J ¼ 15.3 Hz), 6.71 (m, 3H, 2 AreH, NH), 6.78e6.81 (m, 2H, 2 AreH), 7.18e7.22 (m, H, AreH), 7.27e7.30 (m, 2H, 2 AreH), 7.33e7.38 (m, 2H, 2 AreH), 7.44 (d, 2H, AreH, J ¼ 7.7 Hz), 7.49e7.53 (m, H, AreH), 7.86 (d, H, AreH, J ¼ 8.4 Hz). Elemental analysis Calcd for C34H39N3O5: C, 71.68; H, 6.90; N, 7.38. Found: C, 71.43; H, 7.08; N, 7.54.

4.2.2. Activity assay The activity of MAO-A and MAO-B was determined using a fluorimetric method described by Matsumoto et al. [31]. The mitochondrial fractions were preincubated at 38  C for 30 min with the substrate following the inhibition of one of the MAO isoforms with the specific inhibitor, L-deprenyl (l0.5 mM) to determine MAO-A activity and clorgyline (10.5 mM) to determine MAO-B activity. The incubation mixture contained 0.1 ml phosphate buffer (0.25 M, pH 7.4), mitochondrial suspension (6 mg/ml), the specific substrate for MAO-A or MAO-B (0.1 mM) and test compounds at five different concentrations ranging from 0.5 nM to 0.1 M (0, 0.5 nM, 5 nM, 5 mM, 5 mM and 100 mM) dissolved in propylene glycol. The mixture was incubated in a shaking waterbath at 37  C for 60 min. The reaction was quenched by adding perchloric acid. The samples were centrifuged at 10,000 g for 5 min and the supernatant was completed to 2.7 ml using 1 N NaOH and measured with a PerkineElmer Lf 45 Spectrofluorimeter. The values were from 6 independent samples that were measured in duplicate. The average value of the duplicate measurements was used for the statistical analysis. The IC50 value was calculated and analyzed using the four-parameter logistic function in SigmaPlot software (SigmaPlot 12.3, Systat Software. Inc., Richmond, CA, USA). Protein concentration was determined according to a previously reported method [36]. The MAO-A and MAO-B results are expressed as IC50 (Table 1). Propylene glycol was used as negative control and did not show any effect on the enzyme activity. The test compounds were further evaluated for their oral acute toxicity in male mice (20 g each obtained from Medical Research Institute, Alexandria University) according to previously reported methods [34,35]. The animals were divided into groups of six mice each. The compounds were suspended in 1% gum acacia and given orally in doses of 1, 10, 100, 200, 250, 300 mg/kg. The mortality percentage in each group was recorded after 24 h. Moreover, the test compounds were investigated for their parenteral acute toxicity in groups of mice of six animals each. The compounds or their vehicle, propylene glycol (control), were given by intraperitoneal injection in doses of 10, 25, 50, 75, 100 mg/kg. The percentage survival was followed up to 7 days [33].

4.2. Biology

4.3. Modeling studies

The newly synthesized compounds 3ae3f, 7ae7e, 9ae9g, and 10ae10h were tested to determine their activity toward MAO-A

Molecular docking studies were carried out to understand the molecular binding modes of the active synthesized compounds

4.1.8.2. tert-Butyl 2-[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetamido]-3-phenylpropanoate 10b. The crude compound was recyrstallized (methylene chloride/hexane) and the product was obtained as a white powder, 0.43 g (86.5%) yield, mp 100e101  C. IR (KBr): 3310 (NH), 1731 (CO, ester), 1690 (CO, amide), 1659 (CO, amide) cm1. 1H NMR (CDCl3): d 1.37 (s, 9H, 3 CH3), 2.87 (dd, H, CH2, J ¼ 13.8 Hz, J ¼ 6.2 Hz), 3.03 (dd, H, CH2, J ¼ 13.8 Hz, J ¼ 6.2 Hz), 4.21, 4.26 (2d, 2H, CH2, J ¼ 13.8 Hz), 4.59, 4.95 (2d, 2H, CH2, J ¼ 14.6 Hz), 4.65 (m, H, CH), 6.73 (d, H, NH), 6.81 (d, 2H, 2 AreH, J ¼ 7.7 Hz), 6.93e7.02 (m, 3H, 3 AreH), 7.18e7.22 (m, H, AreH), 7.26e7.31 (m, 2H, 2 AreH), 7.33e7.38 (m, 2H, 2 AreH), 7.45 (d, 2H, AreH, J ¼ 6.9 Hz), 7.45e7.51 (m, H, AreH), 7.87 (dd, H, AreH, J ¼ 8.4 Hz, J ¼ 1.5 Hz). Elemental analysis Calcd for C30H31N3O4: C, 72.41; H, 6.28; N, 8.44. Found: C, 72.66; H, 6.03; N, 8.21. MS: Neg: 497 (M), (496, M-H); Pos: 464, 520 (Na þ M).

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towards two different biological targets belonging for human monoamine oxidases enzymes. The Protein Data Bank [37] (PDB) crystallographic structures of human MAO-A (PDB code 2BXR) and human MAO-B (PDB code 1GOS) were considered as the enzyme targets for docking simulations. The proteins were considered without co-crystallized ligands for the purpose of the docking simulations. Before screening the active compounds, the docking protocol was validated by running the simulation only using the bound ligands and low RMSD between docked and crystal conformations. AutoDock 3.0 [38] and MOE [32] softwares were used for all docking calculations. The AutoDockTools [38] package was employed to generate the docking input files and to analyze the docking results. A grid box size of 90  90  90 points with a spacing of 0.375 Å between the grid points was generated that covered almost the entire protein surface. Ligands were fully flexibly docked. All non-polar hydrogens and crystallographic water molecules were removed prior to the calculations. The docking grid was centered on the mass center of the bound TSA. In each case, 100 docked structures were generated using genetic algorithm searches. A default protocol was applied with an initial population of 50 randomly placed conformations, a maximum number of 2.5  105 energy evaluations, and a maximum number of 2.7  104 generations. Heavy atom comparison root mean square deviations (RMSD values) were calculated and initial ligand binding modes were plotted. Protein-ligand interaction plots were generated using MOE software, 2012. Acknowledgment The authors would like to acknowledge the Chemical Computing Group for using MOE software. References [1] T.B. Ustun, J.L. Ayuso-Mateos, S. Chatterji, C. Mathers, C.J. Murray, Br. J. Psychiatry 184 (2004) 386e392. [2] C.J. Murray, A.D. Lopez, Lancet 1997 (349) (1997) 1436e1442. [3] D.L. Musselman, D.L. Evans, C.B. Nemeroff, Arch. Gen. Psychiatry 55 (1998) 580e592. [4] D. Michelson, C. Stratakis, L. Hill, J. Reynolds, E. Galliven, G. Chrousos, P. Gold, N. Engl. J. Med. 335 (1996) 1176e1181. [5] R. Schulz, S.R. Beach, D.G. Ives, L.M. Martir, A.A. Ariyo, W.J. Kop, Arch. Intern.

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