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

Article abstract of DOI:10.1016/j.ejmech.2015.02.020

In this investigation, we searched for novel MAO-A inhibitors using a 3-benzylquinoxaline scaffold based on our earlier findings. Series of N′-(3-benzylquinoxalin-2-yl)acetohydrazide, 4a, N′-(3-benzylquinoxalin-2-yl)benzohydrazide derivatives 4b-f, N′-[2-

Full text of DOI:10.1016/j.ejmech.2015.02.020

European Journal of Medicinal Chemistry 93 (2015) 308e320
Contents lists available at ScienceDirect
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
,
,
, d
Abdel Moneim El Massry a c, Seham Y. Hassan a, Ayman El-Faham a
,
,
, g, *
Hany Emary Ali Ahmed e f, Adel Amer a
a Department of Chemistry, Faculty of Science, Alexandria University, Alexandria 21321, Egypt
b 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
a r t i c l e i n f o
a b s t r a c t
Article history:
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-(3-
benzylquinoxalin-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-oxoquinoxalin-
1(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.
Received 17 September 2014
Received in revised form
11 February 2015
Accepted 12 February 2015
Available online 16 February 2015
Keywords:
Quinoxalin-2(1H)-one
N-Aroylhydrazine
N0-Acetylaroylhydrazine
Schiff's base
Amino acids
Monoamine oxidase
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
diseases including chronic mental diseases like depression [7e9].
The monoamine oxidase inhibitors (MAOIs) were the first drugs
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
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 meta-
bolism 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 metab-
olizes serotonin, adrenaline, and noradrenaline [13], whereas
b-
* Corresponding author. Department of Chemistry, College of Science, Taibah
University, Al-Madinha Al-Munawaraha, Saudi Arabia.
** Corresponding author.
phenylethylamine and benzylamine are predominantly metabo-
lized by MAO-B [14]. Tyramine, dopamine, and some other
important amines are common substrates for both isoenzymes [15].
E-mail address: adel.amer@alex-sci.edu.eg (A. Amer).
http://dx.doi.org/10.1016/j.ejmech.2015.02.020
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
309
Nowadays, the therapeutic interest of MAOIs falls into two major
categories. MAO-A inhibitors have been used mostly in the treat-
ment 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 MAO-
A
inhibitors, we have recently focused on utilizing the 3-
benzylquinoxaline scaffold where compound such 3-benzyl-2-(2-
morpholin-4-yl-ethyl)amino-quinoxaline showed potent and
I
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 benzylqui-
noxalinyl group which seems to play a role in orientation and
complex formation at the active site of the enzyme.
Fig. 2. The Z- and E-forms of compound 9d using molecular mechanics MM2 and PM3
calculations.
2. Results and discussion
2.1. Chemistry
hydrogens of the acetic acid function and the carbons at
d 130.16 (C-
10Q), 154.4 (C-2Q) and 167.18 ppm (C]O ester), while the benzyl
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
CH2 hydrogens at
d 4.28 ppm showed correlation to carbons at
129.62 (C-20Ph, 60Ph), 136.96 (C-10Ph), 154.43 (C-3Q) and
159.18 ppm (C-2Q).
rapid
formation
of
the
corresponding
2-benzyl-3-
Hydrazinolysis of the ethyl ester group in 5 was carried out in
methanol under reflux for 4 h to afford the corresponding hydra-
zide 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
chloroquinoxaline 2 after neutralization with saturated sodium
bicarbonate (Scheme 1). Further reaction of 2 with hydrazine hy-
drate in ethanol as
a
solvent afforded the 2-benzyl-3-
hydrazinoquinoxaline 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
(HOBt)
fluoroformamidinium hexafluorophosphate (TFFH) or HATU at
ꢀC and TEA as base in DMF and recrystallize from methylene
in
the
presence
of
1,1,3,3-tetramethyl-2-
0
with
2-benzyl-3-hydrazinoquinoxaline
3
using
O-(7-
dichloride (MDC). Both methods gave satisfactory yields of the
desired N0-(2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl) acetyl) benzo-
hydrazide 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
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexa-
fluorophosphate (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-2-
oxoquinoxalin-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
peak at
d
4.14 ppm corresponding to the benzyl CH2 protons. Two
4.98 and
other peaks were observed equivalent to two protons at
d
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.
observed between the singlet at
d 4.96 ppm of the methylene
Fig. 1. The two expected structures of compound 5.

310
S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
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-2-
considered worthwhile to model the compounds using molecular
mechanics MM2 and PM3 calculations. The calculated energy
oxoquinoxalin-1-(2H)yl) -acetyl)hydrazinyl) -2-ylcarbamate de-
rivatives 8 (Scheme 3) were prepared by coupling of several
Fmoceamino acids (Fmoc-Leu-OH, Fmoc-Phe-OH and Fmoc-
Tyr(OtBu)-OH) with hydrazide 6 in the presence of two equivalent
of DIEA (diisopropylethylamine) using HATU as coupling reagent, in
DMF as solvent.
minimization
results
were
found
to
be
ꢁ25.20
and ꢁ30.09 kcal molꢁ1 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 3-
benzylquinoxlinyl 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
As extension to our work the reaction of 2-(3-benzyl-2-
oxoquinoxalin-1(2H)-yl)acetohydrazide
6 with aromatic alde-
hydes 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
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,
1H NMR) and by elemental analysis.
base 9d in DMSO-d6 showed singlet peak at
sponding to the benzyl CH2 protons. Two other peaks were
observed equivalent to two protons at 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 8.03 and 8.19 ppm
which confirm the presence of the two isomers. Therefore, it is
d 4.14 ppm corre-
2.2. Biology
d
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
d

S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
311
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 1000-
foldse612,000-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 hydrogen-
bonding interaction. While remove the hydrazide moiety to posi-
tion N1 namely7aed does not have significant change in the ac-
tivity. 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. Collec-
tively, 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
In an attempt to understand the reason for the observed
inhibitory activity, these compounds were submitted to computa-
tional 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 represen-
tations 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
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

312
S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
a
b
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.
2.5. Acute toxicity
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 accommo-
dated 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 sta-
bilization as hydrogen bond acceptors to the carbonyl of quinox-
alinone 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 interac-
tion 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 mo-
lecular 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 struc-
tures 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 resi-
dues 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 non-
covalent mechanistic pathway.
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 com-
mittee (ACUC), project number 22, 22/5/2013). The results indi-
cated 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 com-
pounds 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 evalua-
tion 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 inhi-
bition in nanomolar range especially 4e and 9g showed high po-
tency (1.7 and 1.6 nM) combined with high selectivity comparable
to the standard drugs clorgyline and moclobemide. This work in-
troduces 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 13
C
NMR spectra) were recorded using a JEOL 500 MHz spectrometer
with the chemical shift values reported in units (part per million).
d
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

S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
313
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
seconds.
l 254 nm for few

314
S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
(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.
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) cmꢁ1. 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 (DMSO-
d6):
d 47.9, 125.55, 126.57, 126.97, 128.57, 128.87, 129.22, 129.65,
Scheme 1. Synthetic routes to obtain compounds 1 and 3.
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.
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) cmꢁ1. 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.
Scheme 2. General procedure for preparation of compounds 4aef.
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,
4.1.1. General method for preparation of N0-(3-benzylquinoxalin-2-
yl)acetohydrazide, 4a and N0-(3-benzylquinoxalin-2-yl)
benzohydrazide derivatives 4bef
amide) cmꢁ1. 1H NMR (DMSO-d6):
d 2.05 (s, H, OH, D2O exchange-
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 dime-
thylformamide (DMF) was stirred at 0 ꢀC for 3 min. Then 2-benzyl-
3-hydrazinylquinoxaline 3 (0.25 g, 1 mmol) was added. The reac-
tion 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 ꢂ 10 mL),
saturated sodium bicarbonate solution (2 ꢂ 10 mL) and water. The
crude product recrystallized from dichloromethane.
able), 4.56(s, 2H,CH2), 6.98(d, 2H,2AreH, J¼8.4Hz), 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.
4.1.1.6. N0-(3-benzylquinoxalin-2-yl)-4-bromobenzohydrazide
4f.
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) cmꢁ1. 1H NMR
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) cmꢁ1. 1H NMR
(DMSO-d6):
d
4.38 (s, 2H, CH2), 7.21 (d, H, AreH, J ¼ 7.2 Hz), 7.29 (t,
(DMSO-d6):
d 1.94 (s, 3H, CH3) 4.27 (s, 2H, CH2), 7.16 (t, H, AreH,
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
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,
(DMSO-d6):
d 45.00, 126.32, 127.90, 128.22, 129.00, 129.49, 131.49,
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.
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 pres-
sure. The residue was suspended in water (150 mL) and extracted
with methylene chloride (100 mL). The organic layer was dried over
4.1.1.2. N0-(3-benzylquinoxalin-2-yl)benzohydrazide 4b. The prod-
uct was obtained as a yellow solid, mp 123e124 ꢀC, in yield 0.26 g
(73.4%). IR (KBr): 3417 (NH), 1630 (CO, amide) cmꢁ1 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

S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
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,
about 4 h then cooled to room temperature. The excess of hydrazine
was evaporated under reduced pressure. The product that precip-
itated was filtrated off and washed several time by ethanol. The
product was obtained as white crystal, mp. 197e199 ꢀC, in yield
ester), 1651 (CO, amide) cmꢁ1. 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,
0.74 g (74.4%). IR (KBr): 3426, 3297 (NH), 1650 (CO, amide) cmꢁ1
.
1HNMR (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).
AreH, J ¼ 7.7 Hz). HMBC spectrum: The singlet at
correlation to the carbons at 130.16, 154.4 and 167.18 (C]O ester),
while peak at 4.28 showed correlation to carbons at 129.62,
d 4.96 showed
d
d
4.1.4. General method for preparation of N0-[2-(3-benzyl-2-
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.
oxoquinoxalin-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)ace-
tohydrazide 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%
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

316
S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
Table 2
Comparative docking analysis of prominent selective ligands against enzyme (MAO-
A).
Compound
Interaction
residuesa
Interaction Ligand
Binding energy
(dG, kcal/mol)b
type
fragment
4e
Ser209, Glu215
Leu97, Ile325,
Phe208, Phe382
Gly71, Glu74
Ser209, Gly214
Glu215
H-bonding NHeNH
Hydrophobic Benzyl
ꢁ11.8
ꢁ12.5
ꢁ8.9
H-bonding peOHePh
H-bonding C]OeQ
H-bonding NHeC]O
Hydrophobic Benzyl
H-bonding peOHePh
H-bonding C]O
9g
Phe208
Glu74
Moclobemide Ser209
Ile207, Val210,
Hydrophobic ClePh
Leu97
Phe208
Tyr407
Hydrophobic Ethyl
H-bonding Morpholine
a
Residues are abbreviated as in the MOE software.
Binding energy values are stated without any corrections.
b
aqueous citric acid solution (2 ꢂ 10 mL), saturated sodium bicar-
bonate solution (2 ꢂ 10 mL) and water. The crude product was
recrystallized from dichloromethane.
4.1.4.1. N0-[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetyl]-4-
methylbenzohydrazide 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) cmꢁ1. 1H NMR (DMSO-d6): Iso-
Scheme 4. General procedure for preparation of compounds 10aec.
mer (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),
Table 1
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
Effect of some quinoxaline derivatives on the activity of MAO-A and B isoforms.
Compound
IC50 (nM)a
MAO-A
Selectivity index (SI)b
MAO-B
(DMSO-d6):
d 21.40, 45.02, 116.10, 124.59, 127.33, 128,35, 129.41,
4a
4b
4c
4d
4e
4f
7a
7b
7c
7d
8a
8b
8c
9a
9b
9c
9d
9e
9f
9g
9h
10a
10b
10c
66
78
34
86
1.7
74
52
64
82
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 ꢂ 104
9393
4615
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.
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
4.1.4.2. N0-[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetyl]-4-
chlorobenzohydrazide 7b. The product was obtained as a white
solid, mp 130e131 ꢀC, in yield 0.30 g (67%). IR (KBr): 3233 (NH),
56
82
.
1708, 1660 (CO, amide) cmꢁ1 1HNMR (DMSO-d6): Isomer (A,
2.9
3.4
3.1
3.6
6.6
8.4
22
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
98
857
1.6
7.3
56
88
36
612,500
13,151
1554
8636
(DMSO-d6):
d 43.68, 44.07, 115.25, 124.12, 126.98, 129.09, 129.63,
24,722
30,323
132
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
Clorgyline
Moclobemide
3.1
11 ꢂ 103
C24H19ClN4O3: C, 64.50; H, 4.29; N, 12.54. Found: C, 64.78; H, 4.67;
The most significant values are given in bold.
N, 12.80.
a
Values were determined from the kinetic experiments in which serotonin and
benzylamine were used as substrates at 125 and 8 M for measuring MAO-A and B
m
4.1.4.3. N0-[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetyl]-4-
bromobenzo hydrazide 7c. The product was obtained as a white
solid, mp 169e170 ꢀC, in yield 0.36 g (75.4%). IR (KBr): 3457, 3268
activity respectively. The substrate concentrations are 1.25 fold of Km (Michae-
liseMenten Constant). The IC50 value was calculated and analyzed using the four-
parameter logistic function in SigmaPlot software (SigmaPlot 12.3, Systat Soft-
ware. Inc., Richmond, CA, USA) using six independent experiments that were
measured in duplicate and the average was taken for statistical analysis.
(NH), 1660 (CO, amide) cmꢁ1
.
1HNMR (DMSO-d6): Isomer (A,
b
Selectivity Index (SI) was calculated by SI ¼ MAO-B IC50/MAO-A IC50.
66.3%): d 4.14 (s, 2H, CH2), 4.96 (s, 2H, CH2) 7.16e7.33 (m, 7H, 7

S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
317
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
4.1.5.2. (9H-fluoren-9-yl)methyl 1-[2-(2-(3-benzyl-2-oxoquinoxalin-
1(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
(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,
AreH), 7.72e7.76 (m, 2H, AreH), 7.85 (d, H, AreH,
3
2
(CO, amide) cmꢁ1. 1H NMR (DMSO-d6):
d 2.50e3.36 (m, 4H, 2 CH2),
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.
4.10e4.14 (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.
4.1.4.4. N0-[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetyl]-4-
nitrobenzohydrazide 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) cmꢁ1. 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),
4.1.5.3. (9H-fluoren-9-yl)methyl 1-[2-(2-(3-benzyl-2-oxoquinoxalin-
1(2H)-yl)acetyl) hydrazinyl]-3-(4-tert-butoxyphenyl)-1-oxopropan-
2-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
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
(CO, urethane), 1655 (CO, amide) cmꢁ1 1H NMR (DMSO-d6):
.
exchangeable). Isomer (B, 27.9%):
d 4.14 (s, 2H, CH2), 5.24 (s, 2H,
d
0.99e1.18 (m, 9H, 3 CH3), 2.69e3.02 (m, 2H, CH2), 4.06e4.14 (m,
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
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,
(DMSO-d6):
d 43.68, 115.19, 124.27, 126.96, 128.88, 129.52, 129.74,
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.
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]-2-
ylcarbamate 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
4.1.6. General method for preparation of 2-(3-benzyl-2-
oxoquinoxalin-1(2H)-yl)-N0-benzylidene acetohydrazide derivatives
9aeh
A solution of 2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acetohy-
drazide 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.
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 ꢂ 10 mL), saturated sodium
bicarbonate solution (2 ꢂ 10 mL) and saturated sodium chloride
solution (2 ꢂ 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).
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) cmꢁ1 1H NMR (DMSO-d6): Isomer (A,
In case of Fmoc-L-tyrosine-(O-t-Bu) and Fmoc-L-phenylalanine, the
77.6%):
d
4.15 (s, 2H, CH2), 5.42 (s, 2H, CH2), 7.17 (t, H, AreH,
reaction mixture was poured into ice water, filtered, washed with
5% aqueous citric acid solution, saturated sodium bicarbonate so-
lution, water, dried and recrystallized from dichloromethane.
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
4.1.5.1. (9H-fluoren-9-yl)methyl 1-[2-(2-(3-benzyl-2-oxoquinoxalin-
1(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)
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,
cmꢁ1 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
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,
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.
14.35.
4.1.6.2. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl)-N0-(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,

318
S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
amide) cmꢁ1. 1H NMR (DMSO-d6): Isomer (A, 69.9%):
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
d
4.15 (s, 2H,
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%): 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 (DMSO-
d
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
d6):
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.
(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
C
24H20N4O3: C, 69.89; H, 4.89; N, 13.58. Found: C, 70.12; H, 5.15; N,
4.1.6.7. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl)-N0-[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,
13.33.
4.1.6.3. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl)-N0-(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) cmꢁ1. 1H NMR
(DMSO-d6): Isomer (A, 80.5%):
5.43 (s, 2H, CH2), 6.99e7.83 (m, 13H, 13 AreH), 8.01 (s, H, CH, ald-
hyde), 11.71 (s, H, NH, D2O exchangeable). Isomer (B, 19.5%): 3.80
amide) cmꢁ1. 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,
d 3.80 (s, 3H, CH3), 4.18 (s, 2H, CH2),
d
NH, D2O exchangeable). Isomer (B, 21.1%): d 2.24 (s, 3H, CH3) 4.15 (s,
(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 exchange-
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
able). 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
C
s, H, OH, D2O exchangeable), 10.74 (s, H, NH, D2O exchangeable). 13
C
NMR (DMSO-d6):
d 14.08, 43.68, 115.47, 115.65, 124.00, 126.96,
25H22N4O3: C, 70.41; H, 5.20; N, 13.14. Found: C, 70.66; H, 5.48; N,
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.
13.37.
4.1.6.4. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl)-N0-(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) cmꢁ1 1H NMR (DMSO-
.
4.1.6.8. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl)-N0-(2-oxoindolin-
3ylidene) 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,
d6): Isomer (A, 74.7%):
(m, 10H, 10 AreH), 7.68e7.79 (m, 3H, 3 AreH), 8.03 (s, H, CH, ald-
hyde), 11.87 (s, H, NH, D2O exchangeable). Isomer (B, 25.3%): 4.14
d 4.14 (s, 2H, CH2), 5.42 (s, 2H, CH2) 7.18e7.52
d
amide) cmꢁ1. 1H NMR (DMSO-d6): Isomer (A):
d 4.15 (s, 2H, CH2),
(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,
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
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.
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).
4.1.6.5. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl)-N0-(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) cmꢁ1.1H NMR
(DMSO-d6): Isomer (A, 81.4%):
7.11e7.79 (m, 13H, 13 AreH), 8.02 (s, H, CH, aldhyde), 11.87 (s, H, NH,
D2O exchangeable). Isomer (B,18.6%): 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):
44.22, 44.65,
13C 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.
d 4.14 (s, 2H, CH2), 5.42 (s, 2H, CH2)
d
d
4.1.7. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl) acetic acid 11
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.
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),
4.1.6.6. 2-(3-Benzyl-2-oxoquinoxalin-1(2H)-yl)-N0-(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
1744 (CO, acid), 1625 (CO, amide) cmꢁ1. 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
(NH), 1684 (CO, amide), 1652 (CO, amide) cmꢁ1 1H NMR (DMSO-
.
d6): 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
C
9.27.
17H14N2O3: C, 69.38; H, 4.79; N, 9.52. Found: C, 69.15; H, 5.03; N,
AreH), 7.77 (d, H, AreH, J ¼ 8.4 Hz), 7.98 (d, 2H, 2 AreH, J ¼ 7.7 Hz),

S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
319
4.1.8. General method for preparation of ethyl 2-(3-benzyl-2-
oxoquinoxalin-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
and MAO-B selectivity in the presence of the specific substrate,
serotonin (MAO-A) at concentration 125 M or benzylamine (MAO-
B) at concentration 8 M, respectively.
m
m
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 prote-
ase inhibitor) using a Teflon piston homogenizer. The homogenate
was centrifuged at 1000 ꢂ g for 10 min to remove cell debris and
unseparated cells. The supernatant was carefully decanted, re-
suspended in the homogenization buffer and centrifuged at
1000 ꢂ g for 10 min. The resultant supernatant was carefully dec-
anted and centrifuged at 10,000 ꢂ 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.
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 ꢂ 10 mL), saturated sodium bicarbonate solution
(2 ꢂ 10 mL) and saturated sodium chloride solution (2 ꢂ 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)-acety-
lamino]-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,
4.2.2. Activity assay
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.
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 com-
pounds at five different concentrations ranging from 0.5 nM to
4.1.8.2. tert-Butyl
2-[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acet-
amido]-3-phenylpropanoate 10b. The crude compound was recyr-
stallized (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,
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 water-
bath 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 intraperi-
toneal injection in doses of 10, 25, 50, 75, 100 mg/kg. The per-
centage survival was followed up to 7 days [33].
amide) cmꢁ1. 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).
4.1.8.3. tert-Butyl
2-[2-(3-benzyl-2-oxoquinoxalin-1(2H)-yl)acet-
amido]-3-(4-tert-butoxyphenyl)propanoate 10c. The crude com-
pound 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) cmꢁ1 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. 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

320
S.N. Khattab et al. / European Journal of Medicinal Chemistry 93 (2015) 308e320
Med. 160 (2000) 1761e1768.
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 confor-
mations. 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 ꢂ 90 ꢂ 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 flex-
ibly 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 ꢂ 105 energy evaluations, and a maximum number of 2.7 ꢂ 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.
[6] M.L. Seney, E. Sibille, Biol. Sex Differ. 5 (2014) 17.
[7] M. Ghanem, M. Gadallah, F.A. Meky, S. Mourad, G. El-Kholy, East. Mediterr.
Health J. 15 (2009) 65e75.
[8] M.F. Abdul-Mohsen, J. Fam. Community Med. 11 (2004) 23e29.
[9] M.M. Bassiony, Depression Anxiety 21 (2005) 90e94.
[10] L.W. Elmer, J.M. Bertoni, Expert Opin. Pharmacother. 9 (2008) 2759e2772.
[11] G.G.S. Collins, M. Sandler, E.D. Williams, M.B.H. Youdim, Nature 225 (1970)
817e820.
[12] A.S. Kalgutkar, N. Castagnoli Jr., Med. Res. Rev. 15 (1995) 325e388.
[13] J.P. Johnston, Biochem. Pharmacol. 17 (1968) 1285e1297.
[14] J. Knoll, K. Magyar, Adv. Biochem. Psychophys. 5 (1972) 393e408.
[15] A. O'Carroll, C.J. Fowler, J.P. Phillips, I. Tobbia, K.F.N.-S. Tipton, Arch. Phar-
macol. 322 (1983) 198e202.
[16] M.V. Rudorfer, W.Z. Potter, Drugs 37 (1989) 713e738.
[17] P. Pacher, E. Kohegyi, V. Keckemeti, S. Furst, Curr. Med. Chem. 8 (2001)
89e100.
[18] P. Pacher, V. Keckemeti, Curr. Med. Chem. 11 (2004) 925e943.
[19] J. Wouters, Curr. Med. Chem. 5 (1998) 137e162.
[20] M.C. Carreiras, J.L. Marco, Curr. Pharm. Des. 10 (2004) 367e372.
[21] S.Y. Hassan, Sh N. Kattab, A. Bekhit, A. Amer, Bioorg. Med. Chem. Lett. 16
(2006) 1753e1756.
[22] Sh N. Khattab, S. Yassin, A. Bekhit, A.M. El Massry, V. Langer, A. Amer, Eur. J.
Med. Chem. 45 (2010) 4479e4489.
[23] Sh N. Khattab, A.A. Bekhit, A. El-Faham, A.M. El Massry, A. Amer, Chem. Pharm.
Bull. Jpn. 56 (2008) 1717e1721.
[24] a) A. Amer, A.M. El Massry, M. Badawi, B.H. Ali, A. Bashir, Sixteenth Interna-
tional Congress of Heterocyclic Chemistry POI-5, Aug. 10e15, Montana State
University, Bozeman, 1997;
b) A.M. El Massry, Heterocycl. Comm. 5 (1999) 555.
[25] Fujisawa Pharmaceutical Co, Jpn. Kokai Tokkyo Koho (1988). JP 63301874 A
19881208 [Chem. Abstr. 111 (1989) 78027u].
[26] D.S. Lawrence, J.E. Copper, C.D. Smith, J. Med. Chem. 44 (2001) 594e601.
[27] L.M. Kapkan, A. Yu Chervinskii, E.V. Titov, J. Mol. Struct. 60 (1980) 187e192.
[28] J.M.S. Filho, A.C.L. Leite, B.G. Oliveira, D.R.M. Moreira, M.S. Lima, M.B.P. Soares,
L.F.C.C. Leite, Bioorg. Med. Chem. 17 (2009) 6682e6691.
[29] R.E. Basford, Methods Enzymol. 10 (1967) 96e101.
[30] W.C. Schneider, J. Biol. Chem. 176 (1948) 259.
Acknowledgment
[31] T. Matsumoto, O. Suzuki, T. Furuta, M. Asai, Y. Kurokawa, Y. Rimura,
Y. Katsumata, I. Takahashi, Clin. Biochem. 18 (1985) 126e129.
[32] Molecular Operating Environment (MOE), Chemical Computing Group Inc,
2012. www.chemcomp.com.
The authors would like to acknowledge the Chemical
Computing Group for using MOE software.
[33] J.T. Litchfield, F. Wilcoxon, J. Pharmacol. Exp. Ther. 96 (1949) 99e113.
[34] M. Verma, M. Tripathi, A.K. Saxena, K. Shanker, Eur. J. Med. Chem. 29 (1994)
941e946.
[35] A.A. Bekhit, H.T.Y. Fahmy, Arch. Pharm. Pharm. Med. Chem. 336 (2003)
111e118.
[36] M.M. Bradford, Anal. Biochem. 72 (1976) 248e254.
[37] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig,
I.N. Shindyalov, P.E. Bourne, Nucleic Acids Res. 28 (2000) 235e242.
[38] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew,
A.J. Olson, J. Comput. Chem. 19 (1999) 1639e1662.
References
[1] T.B. Ustun, J.L. Ayuso-Mateos, S. Chatterji, C. Mathers, C.J. Murray, Br. J. Psy-
chiatry 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.