Synthesis, biological investigation and molecular docking study of N-malonyl-1,2-dihydroisoquinoline derivatives as brain specific and shelf-stable MAO inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.02.039

A group of N-malonyl-1,2-dihydroisoquinoline derivatives were synthesized and investigated as brain specific and shelf-stable MAO inhibitors. N-malonyl-1,2-dihydroisoquinoline redox carrier system was linked through amidic bond to 4-chloro and 4-nitrobenzylidenehydrazines (9a-b), as monoamine oxidase inhibitors (MAOIs), and 2-phenethylamine (14), as a model drug, to afford a novel group of N-malonyl-1,2-dihydroisoquinoline chemical delivery systems (DHIQCDSs) (13a-b and 18). These systems are expected to be stable against air oxidation due to the presence of the carbonyl group close to nitrogen of the dihydroisoquinoline. The synthesized DHIQCDS (18) was subjected to various chemical and biological investigations to evaluate its stability and prove its ability to cross the blood brain barrier and lock-in the brain. The in vitro chemical and enzymatic oxidation studies showed reasonable stability and adequate rate of conversion of DHIQCDS (18) to its corresponding quaternary metabolites. In vivo distribution study in rats revealed preferential concentration of the active moiety in the brain. Moreover, compounds (9a-b, 12a-b and 17) were screened for their in vitro MAO inhibitory activity compared to clorgyline as a reference compound. The inhibition profile was found to be competitive for both MAO-A and MAO-B isozymes with more selectivity toward MAO-A. Molecular docking study of compounds (9a-b, 12a-b and 17) and the suggested metabolites was carried out on both MAO-A and MAO-B isozymes. Observation of the docked poses revealed many interactions with many residues previously reported to have an effect on the inhibition of MAO enzyme.

Full text of DOI:10.1016/j.ejmech.2015.02.039

European Journal of Medicinal Chemistry 93 (2015) 481e491
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological investigation and molecular docking study of
N-malonyl-1,2-dihydroisoquinoline derivatives as brain specific and
shelf-stable MAO inhibitors
,
Mohammed K. Abd El-Gaber a *, Hoda Y. Hassan a, Nadia M. Mahfouz a, Hassan H. Farag a,
Adnan A. Bekhit b
a Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of N-malonyl-1,2-dihydroisoquinoline derivatives were synthesized and investigated as brain
specific and shelf-stable MAO inhibitors. N-malonyl-1,2-dihydroisoquinoline redox carrier system was
linked through amidic bond to 4-chloro and 4-nitrobenzylidenehydrazines (9a-b), as monoamine oxi-
Received 12 December 2014
Received in revised form
19 February 2015
Accepted 20 February 2015
Available online 24 February 2015
dase inhibitors (MAOIs), and b-phenethylamine (14), as a model drug, to afford a novel group of N-
malonyl-1,2-dihydroisoquinoline chemical delivery systems (DHIQCDSs) (13a-b and 18). These systems
are expected to be stable against air oxidation due to the presence of the carbonyl group close to nitrogen
of the dihydroisoquinoline. The synthesized DHIQCDS (18) was subjected to various chemical and bio-
logical investigations to evaluate its stability and prove its ability to cross the blood brain barrier and
“lock-in” the brain. The in vitro chemical and enzymatic oxidation studies showed reasonable stability
and adequate rate of conversion of DHIQCDS (18) to its corresponding quaternary metabolites. In vivo
distribution study in rats revealed preferential concentration of the active moiety in the brain. Moreover,
compounds (9a-b, 12a-b and 17) were screened for their in vitro MAO inhibitory activity compared to
clorgyline as a reference compound. The inhibition profile was found to be competitive for both MAO-A
and MAO-B isozymes with more selectivity toward MAO-A. Molecular docking study of compounds (9a-
b, 12a-b and 17) and the suggested metabolites was carried out on both MAO-A and MAO-B isozymes.
Observation of the docked poses revealed many interactions with many residues previously reported to
have an effect on the inhibition of MAO enzyme.
Keywords:
MAOIs
1,2-Dihydroisoquinoline
Brain-specific delivery
Chemical delivery system
Docking
© 2015 Published by Elsevier Masson SAS.
1. Introduction
are common with general treatments [6e9]. One of these attempts
was the linking of the active compound to a brain-specific carrier to
Targeted drug delivery, defined as optimization of the thera-
peutic index by localizing the pharmacological activity of the drug
to the site of action, is one of the most important goals of phar-
maceutical research and drug development [1]. The brain is prob-
ably one of the least accessible organs for the delivery of active
pharmacological compounds. Despite its relatively high blood flow,
the existence of protective blood brain barrier (BBB) restricts the
passage of foreign particles into the brain [2e5]. Various attempts
have been made to overcome the limited access of drugs to the
brain and, at the same time, reduce the systemic side effects that
give a chemical delivery system (CDS) which delivers the active
compound specifically into the brain after its cleavage from the
carrier at the site of action [10e14].
The 1,4-dihydropyridine4pyridinium salt chemical delivery
system (DHPCDS) was described as a general and flexible method
for site-specific and sustained delivery of drugs to the brain
[15e18]. The biologically active compound linked to a lipoidal
dihydropyridine carrier easily penetrates the blood brain barrier;
oxidation of the carrier moiety in vivo to the ionic pyridinium salt
prevents its elimination from the brain “locked-in”, while elimi-
nation from the general circulation is accelerated. By time, the
release of active drug occurs by hydrolysis in a sustained manner at
a rate which allows reaching active concentrations at the site of
action [19e22]. The main obstacle faced the industrial
* Corresponding author.
E-mail address: moh_khalifa_84@yahoo.com (M.K. Abd El-Gaber).
http://dx.doi.org/10.1016/j.ejmech.2015.02.039
0223-5234/© 2015 Published by Elsevier Masson SAS.

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development of the idea is the very short half-life of the dihy-
dropyridine carrier, mainly due to their fast air oxidation and/or
hydration of the 5,6-double bond, which is a common problem with
all the DHPCDSs [23e25]. Accordingly, 1,4-dihydroquinoline and
1,2-dihydroisoquinoline were suggested as alternative carriers for
brain specific delivery with increased lipophilicity and enhanced
stability against oxidation [26,27].
nitrogen at 0 ꢀC and in presence of triethylamine as acid scavenger
to afford the malonate derivatives (10a-b and 15), Scheme 2.
The bromomalonates (11a-b and 16) were prepared from
compounds (10a-b and 15) by bromination either by using mo-
lecular bromine or a mixture of potassium bromide, hydrogen
peroxide and hydrochloric acid. A plausible reaction mechanism is
that bromide ion is oxidized by hydrogen peroxide into the bromo
ion (Brþ) which reacts with the enol form of the active methylene
compound providing selectively the desired monobromo derivative
while only nontoxic potassium chloride and water are produced as
a waste [30].
The quaternary isoquinolinium compounds (12a-b and 17) were
prepared by refluxing the bromomalonates (11a-b and 16) with
isoquinoline in absolute ethanol for 24 h. The designed racemic
DHIQCDSs (13a-b and 18) were prepared in 60e73 % yield by the
reduction of the corresponding quaternaries (12a-b and 17) using
sodium dithionite in presence of aqueous sodium bicarbonate and
methylene chloride under a stream of nitrogen gas at 0 ꢀC, Scheme
2. Methylene chloride serves to extract the DHIQCDSs (13a-b and
18) as soon as they is formed to protect them from degradation in
the alkaline aqueous medium. The reduction of quaternary iso-
quinolinium salts with sodium dithionite at alkaline pH produces
selectively the 1,2-dihydroisoquinoline derivatives and does not
proceed to the formation of the 1,2,3,4-tetrahydroisoquinoline
derivatives whatever the molar ratio of the dithionite used [31].
In the present proposal, a novel carrier was investigated keeping
in mind all the requirements for a safe and useful brain-specific
chemical delivery system including; reasonable shelf-life stability
and a good rate of oxidation and “lock-in” the brain. The suggested
carrier is N-malonyl-1,2-dihydroisoquinoline derivative (1) [28],
such carrier is expected to be stable against air oxidation due to the
presence of the carbonyl group close to nitrogen of the dihy-
droisoquinoline. Such group will act as electron withdrawing,
whether by resonance or induction, which will result in decreasing
the electron density on the ring nitrogen. It is well known that the
lone pair of electrons on the ring nitrogen is the initiator of
oxidation and/or hydration of the dihydropyridine [23,24].
Accordingly, reduction of the electron density on the ring nitrogen
may result in a shelf-stable carrier system. On administration, the
suggested carrier (1) will be subjected to several enzymatic hy-
drolytic and oxidative processes as shown in Scheme 1. The carrier
(1) is designed so that, the rate of hydrolysis of the alkyl ester of
malonyl moiety is faster than that of amide bond (K1 > K6). Once
hydrolyzed, compound (1) gives the corresponding anion deriva-
tive (2). The rate of oxidation of the anion (2) is expected to be
accelerated, firstly by the negative charge on the acid anion func-
tion which reduces the energy of the transition state of the enzy-
matic oxidation to the corresponding quaternary acid (3), and
secondly by stabilization of the final quaternary acid (3) with the
2.2. Biological investigation
2.2.1. In vitro stability studies
The stability of DHIQCDS (18) was determined in the presence of
the oxidizing agents (10% methanolic silver nitrate and 30%
aqueous hydrogen peroxide), phosphate buffer solutions (0.2 M,
pH: 7.4 and 5.8) and biological fluids (80% mouse plasma and 20%
rabbit brain homogenate). To carry out these investigations, a
reverse-phase high performance liquid chromatographic (HPLC)
method for analysis of compound (18) and its metabolites in so-
lutions, plasma and brain homogenate was developed and vali-
dated before use.
close location of opposite charges, resembles the zwitterions of a-
amino acids. Once the quaternary acid (3) is formed it will be
“locked-in” the brain and hydrolyzed by time progress to release
the parent drug (6).
4-Chloro and 4-nitrobenzylidenehydrazines (9a-b), as MAOIs,
and
linked to the N-malonyl-1,2-dihydroisoquinoline carrier through
amidic bond to afford novel group of N-malonyl-1,2-
b-phenethylamine (14), as a model drug, were chosen to be
a
dihydroisoquinoline chemical delivery systems (DHIQCDSs) (13a-
b and 18).
2.2.1.1. Chemical oxidation with 10% silver nitrate and 30% hydrogen
peroxide. A freshly prepared DHIQCDS (18) in a concentration of
(2 ꢁ 10ꢂ4 M) was added to 10% methanolic silver nitrate or 30%
aqueous hydrogen peroxide. Either the decrease of absorption of
the DHIQCDS (18) or the increase of absorption of the oxidized
quaternary derivative (17) was monitored spectrophotometrically
in the UV region at their respective lmax. 311 nm for DHIQCDS (18)
and 342 nm for the quaternary derivative (17). The kinetics of
oxidation with 10% silver nitrate or 30% hydrogen peroxide was
found to obey pseudo first order reaction kinetics. The apparent
rates of oxidation (Kdissapp.) of DHIQCDS (18) and the t1/2 were
calculated by linear regression of Ln absorbance against time; the
results are shown in Table 1. DHIQCDS (18) was found to be rela-
tively stable in both 10% methanolic silver nitrate and 30% aqueous
hydrogen peroxide with t1/2 of 31.9 and 38.2 min respectively.
In addition to shelf stability and efficiency of brain-specific de-
livery, the target DHIQCDS (13a-b and 18) are designated to have
two pharmacokinetic properties which are difficult to combine
them together; immediate onset and long duration of action. The
immediate onset is achieved through the action of the “locked-in”
quaternary forms (12a-b and 17), carrying the same essential
structural features of MAOIs, while long duration of action is
maintained through sustained release of compounds (9a-b).
2. Results and discussion
2.1. Chemistry
The synthesis of racemic DHIQCDSs (13a-b and 18) has been
achieved as shown in Scheme 2. 4-Chloro and 4-
nitrobenzylidenehydrazines (9a-b) were prepared from 4-chloro
and 4-nitrobenzaldehyde (8a-b) either by stirring at room tem-
perature with ten equivalents of hydrazine hydrate over night or
reflux with five equivalents of hydrazine hydrate for 1 h using p-
toluenesulfonic acid as a catalyst [29]. By using p-toluenesulfonic
acid as a catalyst, the reaction proceeds selectively in a relatively
higher yield (~95%), higher product purity and shorter time. Ethyl
malonyl chloride was added dropwise to a solution of compounds
(9a-b) or phenethylamine (14) in dry methylene chloride under
2.2.1.2. Stability in buffer systems (shelf-life stability). Stability of
redox-carrier system in buffers of different pH is usually carried out
to give an idea about the best pH for their formulations, to help in
selecting the buffer system to be used as solvent and to expect their
shelf-life stability. The stability of DHIQCDS (18) was investigated in
different phosphate buffers (0.2 M, pH: 7.4 and 5.8) at temperature
(~37 ꢀC). Solutions of DHIQCDS (18) in phosphate buffers of
different pH values (2 ꢁ 10ꢂ4 M) were monitored for three days
using HPLC for both concentration of the DHIQCDS (18) and for
their oxidation or degradation products. The pseudo-first-order

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483
Scheme 1. The expected metabolic pathways of the proposed N-malonyl-1,2-dihydroisoquinoline CDS.
rate constants of disappearance of DHIQCDS (18) were calculated
by linear regression of Ln of its peak areas against time, t1/2 was
calculated and the results are listed in Table 2.
pathways of the carrier system in these media and their in vivo site-
specific conversion into the corresponding quaternary derivatives.
In this investigation, the model DHIQCDS (18) was subjected to
in vitro stability studies in 80% mouse plasma and 20% rabbit brain
homogenate. Scheme 3 illustrates the expected metabolic path-
ways of the tested DHIQCDS (18).
The study of the stability of compound (18) in plasma gives an
idea about its stability against enzymatic hydrolysis (K1 and K4) by
esterase predominant in plasma, since minimal oxidation is ex-
pected. On the other hand, investigation of its stability in brain
homogenate demonstrates the susceptibility to oxidation by flavin
dehydrogenases (K2 and K3). From the obtained results, it should be
possible to determine which comes first in vivo in the brain, hy-
drolysis of the DHIQCDS (18) or its oxidation to the corresponding
quaternary ester (17).
The results of the stability of DHIQCDS (18) in phosphate buffers
of different pH values indicated that compound (18) was stable in
both phosphate buffers at pH 7.4 and 5.8 but more stable in
phosphate buffer at pH 5.8. This is in contrast to most DHPCDSs,
which show much lower stability in acidic buffers due to acid-
catalyzed hydration. Hydrolysis was observed in both buffers, the
main hydrolytic product of the tested compound (18) was the
quaternary acid (20). The results showed that these DHIQCDS
should be stored in solid forms to be reconstituted in slightly acidic
buffer prior to administration.
2.2.2. In vitro stability in biological fluids
The in vitro stability studies of the redox chemical delivery
systems in biological fluids as plasma, blood and brain homogenate
are routinely carried out to investigate the transformation
2.2.2.1. Stability in 80% mouse plasma. The stability of the DHIQCDS
(18) was studied in 80% mouse plasma at 37 ꢀC. The pseudo first

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M.K. Abd El-Gaber et al. / European Journal of Medicinal Chemistry 93 (2015) 481e491
Scheme 2. Synthesis of the target racemic DHIQCDSs (13a-b and 18). Reagents and conditions: i, hydrazine hydrate stirring overnight; ii, reflux with hydrazine hydrate and p-
toluenesulfonic acid for 1 h; iii, ethyl malonyl chloride and triethyl amine stirring under nitrogen at 0 ꢀC for 4 h; iv, bromine in methylene chloride stirring for 6 h; v, stirring to a
mixture of potassium bromide, 1 M hydrochloric acid and 30% hydrogen peroxide in toluene at room temperature for 3 h; vi, reflux in ethanol for 24 h; vii, stirring with sodium
dithionite, sodium carbonate in methylene chloride/water mixture under a stream of nitrogen gas at 0 ꢀC for 3 h.
Table 1
both hydrolysis and oxidation at the same time but hydrolysis is the
major pathway.
Kinetics of oxidation of DHIQCDS (18) with 10% methanolic silver nitrate and 30%
aqueous hydrogen peroxide.
Oxidation medium
na
Kdissapp. (minꢂ1
)
t1/2 (min)
rb
2.2.2.2. Stability in 20% rabbit brain homogenate. DHIQCDS (18) was
investigated for its stability in 20% rabbit brain homogenate at
37 ꢀC, the reaction was found to obey pseudo first order kinetics.
The pseudo first order rate constant of disappearance of compound
(18) was calculated by linear regression of Ln peak area against
time, t1/2 was also calculated and the results are listed in Table 3.
With respect to the stability of DHIQCDS (18), it was interesting
to observe that the system was more labile in 80% mouse plasma
than in 20% rabbit brain homogenate (t1/2 ¼ 181.4 and 101.9 min,
respectively). This may be attributed to the higher concentration of
esterase enzyme in plasma than in brain. Species differences may
also be an added factor of that variation. The in vitro stability study
in biological fluid revealed that the investigated DHIQCDS (18) has
enough time to be distributed and enter the brain before complete
hydrolysis and oxidation to its quaternary metabolites. In spite of
the occurrence of oxidation and hydrolysis processes, the hydro-
lytic pathway was the major route. Both processes will result in
formation of the quaternary forms which will be “locked-in” the
brain followed by sequential release of the drug in the brain.
Accordingly, the sequential distribution, hydrolysis, and oxidation
scenario may be accomplished.
10% methanolic silver nitrate
30% aqueous hydrogen peroxide
4
4
0.0217
0.0181
31.93
38.22
0.991
0.985
a
n ¼ Number of trials.
b
r ¼ Correlation coefficient.
order rate constants of disappearance of compound (18) was
calculated by linear regression of Ln peak area against time, the
corresponding half-life t1/2 in 80% mouse plasma was calculated
and the results are listed in Table 3. The obtained data clearly
revealed that the rate of hydrolysis of DHIQCDS (18) in 80% mouse
plasma was about six times faster than the rate of hydrolysis in
phosphate buffer pH 7.4, which illustrates the importance of
enzymatic hydrolysis studies on DHIQCDS. Quaternary acid (20)
(major) and quaternary ester (17) (minor) were detected as hy-
drolysis and oxidation products which indicates the occurrence of
Table 2
Shelf-life stability of DHIQCDS (18) in phosphate buffers (0.2 M, pHs 7.4 and 5.8).
Phosphate buffer
(0.2 M, pHs)
n
K
dissapp. (minꢂ1) ꢁ 10ꢂ4 SD
t1/2 (min)
r
2.2.3. In vivo distribution study
The purpose of this study is to prove that DHIQCDS (18) will
deliver the corresponding quaternary forms to the brain in good
7.4
5.8
4
4
1.139 0.73
0.561 0.55
607.9
1237.5
0.998
0.991

M.K. Abd El-Gaber et al. / European Journal of Medicinal Chemistry 93 (2015) 481e491
485
Scheme 3. The expected metabolic pathways of the tested DHIQCDS (18).
concentration and due to their hydrophilic characters they will be
“locked-in” there. A freshly prepared solution of compound (18) in
DMSO was injected through the jugular vein to groups of female
Sprague Dawley rats at a dose of 20 mg/kg of body weight. At
specific time intervals, the injected animals were sacrificed and
their blood samples and brains were collected and analyzed by
HPLC. The results showed that the quaternary acid (20) was
detected at higher concentration in the blood within the first
15 min and decreased quickly to be undetectable within the first
hour, while its concentration in the brain increased gradually to be
the highest within 1 h. This trend is typical with the “lock-in”
property of the brain specific chemical delivery systems, Fig. 1.
The presence of the quaternary acid (20) as the major product in
brain proves the main idea of the new DHIQCDS (18). The target
compound (18) penetrates the BBB and rapidly hydrolyzed by brain
esterase to the corresponding acid (19), present as anion at pH 7.4.
Oxidation of the resultant anion (19) by brain flavin
Fig. 1. In vivo distribution of DHIQCDS (18) in rat blood and brain homogenate.
dehydrogenases to the corresponding quaternary acid (20) is
accelerated due to the negative charge on the carboxylate anion.
To test the efficiency of brain specific delivery of DHIQCDS (18),
the obtained results were compared with reported results of
selected efficient DHPCDSs including dopamine [32], phenethyl-
amine and tryptamine-DHPCDSs [33], in which the concentration
of the quaternary end products in brain was determined. The
overall efficiency of the carrier system can be measured by
Table 3
Stability of DHIQCDS (18) in 80% mouse plasma and 20% rabbit brain homogenate.
Biological enzyme
system
n
K
dissapp. (minꢂ1) ꢁ 10ꢂ3 SD
t1/2 (min)
r
80% mouse plasma
20% rabbit brain
homogenate
5
5
6.81 0.64
3.82 0.36
101.9
181.4
0.992
0.981

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Table 5
calculating the ratio of highest concentration of the quaternary
derivative in brain to the dose administered, considering the body
weight as the theoretical volume of distribution. Table 4 illustrates
the results of these calculations. The results showed that DHIQCDS
(18) is about 15e24 folds more efficient in brain specific delivery
than the other DHPCDSs. Without the involvement of active
transport systems or the use of the redox-CDSs, no drug can reach
significantly higher concentrations in the brain than in the blood.
Inhibitory activity of compounds (9a-b, 12a-b and 17) against MAO-A and MAO-B
isozymes.
Compound
MAO-A IC50 (M)a
MAO-B IC50 (M)a
Selectivity inhibition
index (SI)b
9a
9b
4.9 ꢁ 10ꢂ8 0.13
3.9 ꢁ 10ꢂ9 0.13
3.8 ꢁ 10ꢂ9 0.13
6.6 ꢁ 10ꢂ8 0.14
6.2 ꢁ 10ꢂ9 0.22
3.1 ꢁ 10ꢂ9 0.13
3.8 ꢁ 10ꢂ4 0.34
7.4 ꢁ 10ꢂ3 0.14
7.4 ꢁ 10ꢂ3 0.14
3.6 ꢁ 10ꢂ4 0.32
7.7 ꢁ 10ꢂ5 0.52
9.4 ꢁ 10ꢂ3 0.16
0.95 ꢁ 104
1.89 ꢁ 106
1.94 ꢁ 106
0.54 ꢁ 104
1.24 ꢁ 104
3.032 ꢁ 106
12ac
12bc
17c
Clorgyline
2.2.4. In vitro MAO inhibition evaluation
a
Compounds (9a-b, 12a-b and 17) were tested to determine their
inhibitory activity and selectivity toward MAO-A and MAO-B in the
presence of the specific substrate, serotonin or benzylamine,
respectively. Bovine brain mitochondria were isolated according to
Basford [34]. The activity of MAO-A and MAO-B was determined
using a fluorimetric method described by Matsumoto et al. [35].
The MAO-A and MAO-B inhibition results are expressed as IC50. The
selectivity inhibition index (SI) is also calculated and the results are
listed in Table 5 in comparison with clorgyline as a reference
standard.
The results revealed that all the tested compounds showed
inhibitory activity for both MAO-A and MAO-B isozymes with more
selectivity toward MAO-A. Compounds (9b and 12a) showed
potent MAO-A inhibition comparable to that of clorgyline. In
addition, compounds (9b and 12a) were more selective toward
MAO-A than other compounds.
The results were expressed as mean SE, no. of experiments was 6.
b
c
SI ¼ MAO-B IC50/MAO-A IC50
.
Compounds (12a-b and 17) were tested as racemic mixture.
and Tyr-444.
The docking pose of compound (12a) into MAO-A active site
(Fig. 3) showed three Hydrogen bond interactions as follows: ester
C]O of the ligand and Gly-110 back bone, amide C]O and hy-
droxyl group in side chain of Thr-211 and C]N and Val-93 back-
bone. Also, Arene cation interaction was observed between the
positively charged quaternary nitrogen of isoquinoline ring and
Phe-208. In addition, several Van der Waals and hydrophobic in-
teractions were observed with Thr-205, Asn-212, Ile-335, Ile-325,
Ala-111, Leu-97 and Val-210.
Molecular docking simulation of compound (17) into MAO-B
active site (Fig. 4) showed two Hydrogen bond interactions as fol-
lows: ester C]O of the ligand with ser-59 and Tyr-60 back bone.
Arene cation interaction was also observed between the positively
charged quaternary nitrogen of isoquinoline ring and Tyr-398. In
addition, several Van der Waals and hydrophobic interactions were
observed with Tyr-398, Phe-343, Cys-397, Lys-296, Trp-388 Tyr-
326, Gln-206, Leu-56, Met-436, Leu-171, Ile-198 and Ile199.
The molecular docking study revealed that the inhibitory profile
of these compounds could be competitive. The hydrogen bonding
calculations and docking scores support the obtained experimental
in vitro MAO inhibitory activities for these compounds.
2.3. Molecular docking study
In an attempt to rationalize the obtained in vitro biological re-
sults and obtain insights into the possible binding modes of com-
pounds (9a-b, 12a-b and 17) within the active sites of MAO-A and
MAO-B, molecular docking study was performed. The X-ray crystal
structures of recombinant human MAO-A and MAO-B were ob-
tained from the Protein Data Bank. The structures of MAO-A co-
crystallized with clorgyline (PDB ID: 2BXR) [36] and MAO-B co-
crystallized with safinamide (PDB ID: 2V5Z) [37] were selected. The
molecular docking simulations were performed by MOE dock tool
of the modeling software Molecular Operating Environment (MOE
2013.08) [38] using Triangle matcher as placement scheme and
London dG as a scoring function. LigX tool was used to allow flex-
ible ligand and active site docking simulations and to explore the
binding modes of compounds (9a-b, 12a-b and 17) within the
active sites of MAO-A and MAO-B.
Molecular docking simulation of compound (9b) into MAO-A
active site (Fig. 2) revealed that the ligand was oriented in vicin-
ity of flavin adenine dinucleotide (FAD) cofactor and showed
several molecular interactions considered to be responsible for the
observed affinity. Three Hydrogen bond interactions were
observed; two of them between amino group of the ligand and C4]
O of FAD and Gly-443 backbone and the other between hydroxyl
group in side chain of Tyr-407 and C]N of the ligand. In addition,
Van der Waals interactions were observed with Gln-215, Phe-352
3. Conclusion
This investigation is concerned with the design of new brain-
specific MAOIs with reasonable shelf life stability and a good rate
of oxidation and hence “lock-in” the brain. The different In vitro
stability studies showed that the new MAOIs had enhanced shelf-
life stability compared with DHPCDS. In vivo distribution study in
rats proved the brain specific delivery and “lock-in” properties of
the new MAOIs which would alleviate peripheral MAOIs side ef-
fects and food incompatibilities. In vitro MAO inhibition evaluation
revealed that the designed MAOIs had inhibiting activity against
both MAO isozymes with greater selectivity towards MAO-A.
4. Experimental
4.1. Chemistry
Reactions were monitored by TLC analysis using Merck 9385
pre-coated aluminum plate Silica Gel (Kieselgel 60) with F254 in-
dicator thin layer plates and the spots were detected by exposure to
UV-lamp at 254 nm. Melting points were determined on Stuart
electrothermal melting point apparatus and were uncorrected. IR
spectra were recorded as KBr disks or CCl4 solutions on Thermo-
912AO683 FT-IR spectrophotometer. 1H NMR spectra were carried
out on 60 MHz Varian EM-360L NMR Spectrophotometer, 400 MHz
Jeol-JNM-LA400 FT-NMR Spectrometer and 500 MHz Varian
VNMRS-PS54 spectrophotometer using TMS as internal reference.
Table 4
Comparison of the results of in vivo distribution studies in rats for DHIQCDS (18) and
different DHPCDSs.
Compound
Dose
(mg/kg) max. conc. max. conc.
g/g) g/g)
Theoretical Experimental Ratio
(m
(m
Dopamine-DHPCDS [32].
Phenethylamine-DHPCDS [33]. 20
Tryptamine-DHPCDS [33].
DHIQCDS (18)
50
50
20
20
20
22
0.44
0.48
0.3
9.6
6
145.1
20
20
7.25

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487
Fig. 2. (A)Three-dimensional stereoview for the docking pose of compound (9b) in the MAO-A binding site. 9b is depicted in ball and stick. FAD and active site residues are depicted
in line. Atom coloration (Blue, nitrogens; gray, carbons; red, oxygens; white, hydrogen; green, chlorine). (B) Two-dimensional representation for the same pose. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. (A) The docked pose of compound (12a) into MAO-A active site, created using Connolly surface and maps tool colored by H-Bonding (purple), Hydrophobicity (green) and
Mild Polar (blue) regions. (B) Two-dimensional representation for the same pose. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
Fig. 4. (A) Three-dimensional representation of the docking pose of compound (17) inside the MAO-B represented in tubes backbone. (B) Two-dimensional representation for the
same pose.
Chemical shifts (
d) values are given in parts per million (ppm) using
4.1.1. General procedures for synthesis of 4-substituted
CDCl3 (7.29) or DMSO-d6 (2.5) as solvents and coupling constants (J)
in Hertz. Splitting patterns are designated as follows: s, singlet; br.s,
broad singlet; d, doublet; t, triplet; q, quartet; dd, doublet of
doublet; m, multiplet. ESI-MS Spectra were obtained on Micromass
Platform LCZ mass spectrometer. FAB-MS Spectra were recorded on
Jeol-JMS-600H mass spectrometer. HR-MS Spectra were performed
on a Fison/VG Autospec-TOF spectrometer at 70 eV with a source
temperature of 250 ꢀC. Ion mass to charge (m/z) values of molecular
ions (M) were stated with their relative abundances. Elemental
analysis was performed on AnalysenSysteme GmbH-D-63452-
HANAU apparatus and Perkin Elmer 2400 CHN elemental analyzer.
benzylidenehydrazines (9a-b)
Method A: An ethanolic solution of the respective benzaldehyde
(8a-b, 0.03 mol) was added in a dropwise manner with stirring over
1 h to a solution of hydrazine hydrate (14.8 mL, 0.3 mol) in 20 mL
ethanol at room temperature. After the addition was completed,
the mixture was stirred over night until the disappearances of
starting benzaldehyde derivatives as monitored by TLC. Upon
cooling in ice, compounds (9a-b) were precipitated, collected by
filtration, washed with diethyl ether and dried under vacuum.
Method B: The respective benzaldehyde (8a-b, 0.03 mol) was
added to a stirred solution of hydrazine hydrate (7.4 mL, 0.15 mol)
and p-toluenesulfonic acid (0.2 g, 0.0012 mol) in 20 mL ethanol. The
mixture was heated at 70e80 ꢀC until compounds (8a-b) were
completely consumed, about 1 h, as monitored by TLC. The reaction

488
M.K. Abd El-Gaber et al. / European Journal of Medicinal Chemistry 93 (2015) 481e491
mixture was cooled to 5e10 ꢀC and ice-cold water was added
dropwise until the mass became completely thickened. The latter
was filtered, washed with cold water and dried under vacuum.
5.83.
4.1.3. General procedures for synthesis of ethyl 2-bromo-3-
(substituted)-3-oxopropanoates (11 a-b and 16)
4.1.1.1. 4-Nitrobenzylidenehydrazine (9a). Yellow crystals, method
A: 4.48 g (89.9%), method B: 4.74 g (95.9%); mp(ꢀC) 136e137; IR
(KBr, cmꢂ1): 3450 (NH2), 1624(CH]N); 1H NMR (60 MHz, CDCl3)
Method A:
d
(ppm) 5.9 (br.s, 2H, NH2, D2O exchangeable), 7.6 (d, 2H, J ¼ 9,
Bromine (0.5 mL, 0.0093 mol, diluted with 20 mL methylene
chloride) was added dropwise to a stirred solution of compounds
(10a-b or 15) (0.0085 mol) in 100 mL dry methylene chloride. The
reaction mixture was stirred for 6 h at room temperature and then
quenched with saturated aqueous solutions of sodium thiosulfate
and 5% sodium bicarbonate. The organic layer was separated and
the aqueous layer was extracted three times with methylene
chloride. The combined organic layers were washed with brine and
dried over anhydrous sodium sulfate, filtered and evaporated under
vacuum. The residues were chromatographed using eluent (hex-
ane/ethyl acetate, 90:10) and recrystallized from appropriate sol-
vents to afford products (11a-b and 16).
AreH), 8.3 (d, 2H, J ¼ 9, AreH), 7.7 (s, 1H, CH]N).
4.1.1.2. 4-Chlorobenzylidenehydrazine (9b). White crystals, method
A: 4.16 g (90.6%), method B: 4.36 g (94.3%); mp(ꢀC) 55e57; IR(KBr,
cmꢂ1): 3424 (NH2), 1578(CH]N); 1H NMR (60 MHz, CDCl3)
d (ppm)
5.7 (br.s, 2H, NH2, D2O exchangeable), 8.1 (d, 2H, J ¼ 8.5, AreH), 8.65
(d, 2H, J ¼ 8.5, AreH), 9.5 (s, 1H, CH]N).
4.1.2. General procedure for synthesis of ethyl 3-(substituted)-3-
oxopropanoates (10a-b and 15)
Ethyl malonyl chloride (2.6 mL, 0.02 mol) was added in a
dropwise manner to a stirred solution of the respective benzyli-
denehydrazine derivative (9a-b) or phenethylamine (14) (0.02 mol)
and triethylamine (3 mL, 0.022 mol) in 150 mL dry methylene
chloride under nitrogen at 0 ꢀC. The reaction mixture was stirred
for 4 h at room temperature and then quenched with 5% sodium
bicarbonate and brine. The organic layer was separated and the
aqueous layer was extracted three times with methylene chloride.
The combined organic layers were washed with brine and dried
over anhydrous sodium sulfate, filtered and evaporated under
vacuum. The residues were chromatographed using eluent (hex-
ane/ethyl acetate, 80:20) and recrystallized from appropriate sol-
vents to afford products (10a-b and 15).
Method B:
Each of compounds (10a-b and 15, 0.0075 mol) was added with
stirring to a mixture of potassium bromide (417.5 mg, 0.0375 mol),
1 M hydrochloric acid (3.75 mL, 0.0375 mol) and 30% hydrogen
peroxide (1.7 mL, 0.15 mol) in 20 mL toluene. The mixture was
stirred at room temperature for 3 h. The reaction is quenched by
addition of saturated aqueous solutions of sodium thiosulfate and
5% sodium bicarbonate NaHCO3. The organic layer was separated
and the aqueous phase was extracted three times with ethyl ace-
tate. The combined organic layers were washed with brine, dried
over anhydrous magnesium sulfate, filtered and evaporated under
vacuum. The residues were chromatographed using eluent (hex-
ane/ethyl acetate, 90:10) and recrystallized from appropriate sol-
vents to afford products (11a-b and 16).
4.1.2.1. Ethyl 3-(3-(2-(4-nitrobenzylidene)hydrazinyl)-3-
oxopropanoate (10a). Yellow crystals (methanol), 4.36 g (78.2%);
mp(ꢀC) 166e168; IR (KBr, cmꢂ1): 3188 (NH), 1731 (OeC]O), 1678
(NHeC]O), 1599 (CH]N); 1H NMR (400 MHz, DMSO-d6)
d (ppm)
1.16 (t, 3H, J ¼ 7.2, CH2CH3); 3.36 (s, 2H, CH2eCO); 4.06 (q, 2H,
J ¼ 7.3, OCH2CH3); 7.86 (d, 2H, J ¼ 7.2, AreH); 8.26 (d, 2H, J ¼ 7.2,
AreH); 8.05 (s,1H, CH]N); 11.87 (s, 1H, NH, D2O exchangeable); MS
(ES-): m/z 278.0 [100%, MꢂH], 279.2 [9.7%, M]; HRMS: m/z Calcd for
4.1.3.1. Ethyl 2-bromo-3-(3-(2-(4-nitrobenzylidene)hydrazinyl)-3-
oxopropanoate (11a). Yellow crystals (carbon tetrachloride), Yel-
low crystals (carbon tetrachloride), method A 1.64 g (54.2%),
method B 1.7 (63.3%); mp(ꢀC) 154e156; IR (KBr, cmꢂ1): 3184 (NH),
1734 (OeC]O), 1670 (NHeC]O), 1589 (CH]N); 1H NMR
C
C
12H13N3O5: 280.0933 Found: 280.0945; Anal. Calcd for
12H13N3O5: C, 51.61; H, 4.69; N, 15.05. Found: C, 51.20; H, 4.87; N,
(400 MHz, DMSO-d6)
d
(ppm) 1.12 (t, 3H, J ¼ 7.2, CH2CH3); 4.12 (q,
15.04.
2H, J ¼ 7.2, OCH2CH3); 5.9 (s,1H, CHeBr); 7.84 (d, 2H, J ¼ 8.4, AreH);
8.24 (d, 2H, J ¼ 8.4, AreH); 8.06 (s,1H, CH]N); 12.21 (s, 1H, NH, D2O
exchangeable); MS (ES-): m/z 357.9 [100%, M]; HRMS: m/z Calcd for
4.1.2.2. Ethyl 3-(3-(2-(4-chlorobenzylidene)hydrazinyl)-3-
oxopropanoate (10b). White crystals (methylene chloride), 4.39 g
(93.6%); mp(ꢀC) 56e57; IR (KBr, cmꢂ1): 3186 (NH), 1733 (OeC]O),
C12H12BrN3O5: 358.0039. Found: 358.0059.
1675 (NHeC]O), 1612 (CH]N); 1H NMR (500 MHz, CDCl3)
d
(ppm)
4.1.3.2. Ethyl 2-bromo-3-(3-(2-(4-chlorobenzylidene)hydrazinyl)-3-
oxopropanoate (11b). White crystals (methylene chloride), White
crystals (methylene chloride), method A 1.66 g (56.4%), method B
1.8 g (69.1%); mp (ꢀC) 141e144; IR (KBr, cmꢂ1): 3192 (NH), 1734
(OeC]O), 1682 (NHeC]O), 1611 (CH]N); 1H NMR (400 MHz,
1.26 (t, 3H, J ¼ 7.1, CH2CH3); 3.77 (s, 2H, CH2eCO); 4.20 (q, 2H,
J ¼ 7.3, OCH2CH3); 7.35 (d, 2H, J ¼ 7.2, AreH); 7.56 (d, 2H, J ¼ 7.2,
AreH); 7.82 (s,1H, CH]N); 10.48 (s, 1H, NH, D2O exchangeable); MS
(ES-): m/z 267.0 [100%, MꢂH], 268.2 [8.75%, M]; HRMS: m/z Calcd
for C12H13ClN2O3: 269.0693 Found: 269.0705; Anal. Calcd for
DMSO-d6)
d
(ppm) 1.11 (t, 3H, J ¼ 6.8, CH2CH3); 4.08 (q, 2H, J ¼ 7.2,
C
12H13ClN2O3: C, 53.64; H, 4.88; N, 10.43. Found: C, 53.17; H, 5.42;
OCH2CH3); 5.82 (s, 1H, CHeBr); 7.49 (d, 2H, J ¼ 8.4, AreH); 7.72 (d,
2H, J ¼ 8.4, AreH); 7.94 (s,1H, CH]N); 11.97 (s, 1H, NH, D2O
exchangeable); MS (ES-): m/z 346.9 [100%, MꢂH]; Anal. Calcd for
N, 10.38.
4.1.2.3. Ethyl 3-oxo-3-(phenethylamino)propanoate (15). White
crystals (diethyl ether/hexane), 4.03 g (75.3%); mp(ꢀC) 155e157; IR
(KBr, cmꢂ1): 3301 (NH), 1740 (OeC]O), 1636 (NHeC]O); 1H NMR
C12H12BrN2O3: C, 41.46; H, 3.48; N, 8.06. Found: C, 41.61; H, 3.58; N,
8.13.
(400 MHz, DMSO-d6)
d
(ppm) 1.16 (t, 3H, J ¼ 7.3, CH2CH3); 2.69 (t,
4.1.3.3. Ethyl 2-bromo-3-oxo-3-(phenethylamino)propanoate (16).
White crystals (methylene chloride/hexane), method A 1.81 g
(68.2%), method B 1.68 g (71.6%); mp (ꢀC) 53e54; IR (KBr, cmꢂ1):
3270 (NH), 1682 (OeC]O), 1652 (NHeC]O); 1H NMR (400 MHz,
2H, J ¼ 7.3, CH2CH2NH); 3.17 (s, 2H, CH2eCO); 3.27 (m, 2H,
CH2CH2NH); 4.06 (q, 2H, J ¼ 7.3, OCH2CH3); 7.19 (m, 3H, AreH); 7.29
(m, 2H, AreH); 8.17 (t, 1H, J ¼ 4.8, NH, D2O exchangeable); MS
(FABþ): m/z 236.31 [92.9%, M þ H], 258.9 [6%, M þ Na]; Anal. Calcd
for C13H17NO3: C, 66.36; H, 7.28; N, 5.95. Found: C, 66.14; H, 7.47; N,
DMSO-d6)
d
(ppm) 1.16 (t, 3H, J ¼ 6.8, CH2CH3); 2.71 (t, 2H, J ¼ 6.8,
CH2CH2NH); 3.29 (m, 2H, CH2CH2NH); 4.14 (q, 2H, J ¼ 6.9,

M.K. Abd El-Gaber et al. / European Journal of Medicinal Chemistry 93 (2015) 481e491
489
OCH2CH3); 5.1 (s, 1H, CHeBr) 7.19 (m, 3H, AreH); 7.30 (m, 2H,
AreH); 8.54 (t, 1H, J ¼ 4.9 NH, D2O exchangeable); MS (FABþ): m/z
314.30 [32.6%, M þ H] Anal. Calcd for C13H17NO3: C, 49.70; H, 5.13;
N, 4.46. Found: C, 49.93; H, 5.39; N, 4.39.
protected from light and stored in freezer and their purities were
checked by HPLC analysis.
4.1.5.1. Ethyl
2-(isoquinolin-2(1H)-yl)-3-(2-(4-nitrobenzylidene)
hydrazinyl)-3-oxopropanoate (13a). Yellow oil, Yellow oil, 0.53 g
4.1.4. General procedure for synthesis of 2-(1-ethoxy-1,3-dioxo-3-
(substiuted)-isoquinolinium bromides (12a-b and 17)
(66.1%); IR (CCl4, cmꢂ1): 3183 (NH), 1734 (OeC]O), 1668 (NHeC]
O); 1H NMR (60 MHz, CDCl3)
d (ppm) 1.3 (t, 3H, CH2CH3); 4.5 (q, 2H,
To a stirred solution of isoquinoline (0.7 mL, 0.0057 mol) in
absolute ethanol (100 mL), a solution of the respective ethyl bro-
mopropionate derivative (11a-b or 16, 0.0063 mol) in absolute
ethanol (40 mL) was added dropwise while stirring. The reaction
mixture was refluxed for 24 h and the solvent was evaporated
under reduced pressure. The residues were triturated with ether to
afford products (12a-b and 17). The purities of compounds (12a-b
and 17) were checked by HPLC analysis.
OCH2CH3); 4.9 (s, 1H, NeCH); 6.1 (s, 2H, C1 (2H) isoquinoline);
7.8e8.9 (m, 10H, AreH isoquinoline & phenyl); 9.1 (s, 1H, CH]N);
12.5 (brs, 1H, NH, D2O exchangeable); HPLC purity (97.81%).
4.1.5.2. Ethyl 3-(2-(4-chlorobenzylidene)hydrazinyl)-2-(isoquinolin-
2(1H)-yl)-3-oxopropanoate (13b). Faint yellow oil, 0.47 g (60.2%); IR
(CCl4, cmꢂ1): 3193 (NH), 1734 (OeC]O), 1682 (NHeC]O); 1H NMR
(60 MHz, CDCl3) d (ppm) 1.3 (t, 3H, CH2CH3); 4.4 (q, 2H, OCH2CH3);
4.7 (s, 1H, NeCH); 5.6 (s, 2H, C1 (2H) isoquinoline); 7.6e8.4 (m, 10H,
AreH isoquinoline & phenyl); 8.5 (s, 1H, CH]N); 11.7 (brs, 1H, NH,
D2O exchangeable); HPLC purity (98.12%).
4.1.4.1. 2-(1-ethoxy-3-(2-(4-nitrobenzylidene)hydrazinyl)-1,3-
dioxopropan-2-yl) isoquinolinium bromide (12a). Yellow viscous oil,
Yellow viscous oil, 2.1 g (68.7%); IR (CCl4, cmꢂ1): 3083 (NH), 1732
(OeC]O), 1682 (NHeC]O); 1H NMR (400 MHz, DMSO-d6)
d
(ppm)
4.1.5.3. Ethyl 3-(2-(4-chlorobenzylidene)hydrazinyl)-2-(isoquinolin-
2(1H)-yl)-3-oxopropanoate (18). Colorless oil, 0.53 g (72.9%); IR
(CCl4, cmꢂ1): 3269 (NH), 1755 (OeC]O), 1652 (NHeC]O); 1H NMR
1.18 (t, 3H, J ¼ 6.8, CH2CH3); 4.26 (q, 2H, J ¼ 6.8, OCH2CH3); 6.25 (s,
1H, CHeNþ); 7.99 (d, 2H, J ¼ 7.8, AreH phenyl); 8.12 (d, 2H, J ¼ 7.8);
8.09 (s, 1H, CH]N); 8.24e9.04 (m, 6H, AreH isoquinoline); 10.15
(s,1H, C1(H)isoquinoline); 12.5 (s, 1H, NH, D2O exchangeable); MS
(ESþ): m/z 407.1 [31.2%, M]; HPLC purity (99.06%).
(60 MHz, CDCl3) d (ppm) 1.2 (t, 3H, CH2CH3); 2.8 (t, 2H, CH2CH2NH);
3.5 (m, 2H, CH2CH2NH); 4.1 (q, 2H, OCH2CH3); 4.4 (s, 1H, NeCH); 5.1
(s, 2H, C1 (2H) isoquinoline); 7.1e8.2 (m, 11H, AreH isoquinoline &
phenyl); 9.1 (brs, 1H, NH, D2O exchangeable); HPLC purity (98.43%).
4.1.4.2. 2-(1-ethoxy-3-(2-(4-chlorobenzylidene)hydrazinyl)-1,3-
dioxopropan-2-yl) isoquinolinium bromide (12b). Faint yellow
viscous oil, Faint yellow viscous oil, 1.9 g (63.4%); IR (CCl4, cmꢂ1):
3396 (NH), 1683 (OeC]O), 1645 (NHeC]O); 1H NMR (400 MHz,
4.2. Biological investigation
All Spectrophotometric measurements were determined on
Jenway 6365 UV/Visible Spectrophotometer, using 1.00 cm quartz
cells. Spectroflourimetric measurements were determined on Per-
kineElmer Lf 45 Spectrofluorimeter at wavelengths 315 nm for the
excitation and 380 nm for the emission. Samples are analyzed using
Younglin HPLC system composed of pump [Younglin 930D, Korea],
a degasser and mixer [Younglin SDV50A, Korea], a reverse phase
C18 column [Represil gold C18 250 ꢁ 4.6 mm, Germany], a variable
DMSO-d6)
d
(ppm) 1.12 (t, 3H, J ¼ 6.8, CH2CH3); 3.31 (q, 2H, J ¼ 6.8,
OCH2CH3); 7.07 (s, 1H, CHeNþ); 7.62 (d, 2H, J ¼ 7.8, AreH phenyl);
7.94 (d, 2H, J ¼ 7.8); 8.00 (m, 1H, CH]N); 8.24e9.04 (m, 6H, AreH
isoquinoline); 9.95 (s,1H, C1(H) isoquinoline); 12.57 (s, 1H, NH, D2O
exchangeable); MS (FABþ): m/z 397.98 [5.9%, M þ H]; HPLC purity
(98.93%).
4.1.4.3. 2-(1-ethoxy-1,3-dioxo-3-(phenethylamino)propan-2-yl)iso-
quinolin-2-ium bromide (17). White crystals (methanol), White
crystals (methanol), 2.1 g (74.9%); mp(ꢀC) 145e146; IR (KBr, cmꢂ1):
3163 (NH), 1758 (OeC]O), 1676 (NHeC]O); 1H NMR (400 MHz,
wave length detector [Younglin UV730D, Korea], a 20 mL injection
loop and Autochro-3000 chromatographic data software for
recording of data. Chromatographic separation were achieved using
a mobile phase of (methanol: acetonitrile: 0.1 N potassium dihy-
drogenphosphate: ethylamine, 30:30:40: 2) and the pH was
adjusted to 5.8 with few drops of HCl. The pH and ratio of the
mobile phase components were adjusted to attain complete sepa-
ration of the peaks. All measurements were carried out using a flow
rate of 1 mL/min and at 254 nm detection. DHIQCDS (18), the
quaternary ester (17) and the quaternary acid (20) were separated
at 26.4, 4.3 and 3.1 min respectively. The eluted metabolite fractions
were collected, evaporated under nitrogen and reconstituted in
methanol for identification by FAB-mass spectroscopy. The qua-
ternary ester (17) showed characteristic m/z 364.46 [13.9%, M þ H]
and 385.72 [4.4%, M þ Na]. The quaternary acid (20) revealed
(M þ H) at m/z 336.37 [5.7%, M þ H] and m/z 359.78 [100%, M þ Na].
The kinetic data were calculated with the average of at least three
experiments.
DMSO-d6)
d
(ppm) 1.23 (t, 3H, J ¼ 7.2, CH2CH3); 2.73 (t, 2H,
J ¼ 6.8,CH2CH2NH); 3.38 (2H, CH2CH2NH); 4.26 (q, 2H, J ¼ 7.2,
OCH2CH3); 6.7 (s, 1H, CHeNþ); 7.03 (m, 3H, AreH phenyl); 7.11 (m,
2H, AreH phenyl); 8.09e8.69 (m, 6H, AreH isoquinoline); 9.2 (t,1H,
J ¼ 6, NH, D2O exchangeable); 10.07 (s, 1H, C1(H) isoquinoline); MS
(FABþ): m/z 364.46 [13.9%, M þ H]; 385.72 [4.4%, M þ Na]; HPLC
purity (99.63%); Anal. Calcd for C22H23N2O3: C, 59.60; H, 5.23; N,
6.32. Found: C, 59.35; H, 5.50; N, 6.28.
4.1.5. General procedure for synthesis of ethyl 2-(1H-isoquinolin-2-
yl)-3-(substiuted)-3-oxopropanoates (13a-b and 18)
A solution of the respective quaternary bromide salt (12a-b or
170.002 mol) in methylene chloride (100 mL) was cooled in an ice
bath with stirring, A solution of the respective quaternary bromide
salt (12a-b or 17, 0.002 mol) in methylene chloride (100 mL) was
cooled in an ice bath with stirring. To this cooled solution, dea-
erated water (50 mL) was added followed by addition of sodium
bicarbonate (1 g, 0.012 mol), and sodium dithionite (1.4 g,
0.008 mol). The biphasic mixture was vigorously stirred under ni-
trogen in ice bath for 3 h. The organic layer was separated and the
aqueous phase was extracted three times with methylene chloride.
The combined organic layers were washed with brine, dried over
anhydrous magnesium sulfate, filtered and evaporated under
reduced pressure to dryness. The oily products (13a-b and 18) were
4.2.1. In vitro stability studies
4.2.1.1. Chemical oxidation with 10% silver nitrate and 30% hydrogen
peroxide. In four series, each of 3 test tubes, 1 mL of (2 ꢁ 10ꢂ4 M)
methanolic solution of the DHIQCDS (18) was added to either 5 mL
of 10% methanolic silver nitrate solution or 10 mL of a 30% hydrogen
peroxide solution at 37 ꢀC. The mixture was shaken and at specified
time intervals (5, 10, 20 and 30 min), 3 tubes were centrifuged and
the supernatant was monitored spectrophotometrically for the
disappearance of DHIQCDS (18) and the appearance of the

490
M.K. Abd El-Gaber et al. / European Journal of Medicinal Chemistry 93 (2015) 481e491
corresponding quaternary ester (17) at their specific lmax. Apparent
pseudo first order rate constant of oxidation (Kdissapp., minꢂ1) was
determined by linear regression of Ln absorbance against time in
minutes.
determine the amount of the blood added. The animal was then
decapitated, and the brain was removed quickly, washed with ice
cold saline solution, weighed, wrapped in aluminum foil and
immediately kept in the freezer together with the blood samples.
The whole brain, previously equilibrated at 37 ꢀC, was homoge-
nized in 1 mL of water, then 4 mL of 5% DMSO in acetonitrile was
added to the homogenate, and the mixture was homogenized again
and centrifuged at 4000 rpm for 10 min. The blood samples were
also centrifuged at 4000 rpm for 10 min and supernatants from the
brain and the blood samples were analyzed by HPLC.
4.2.1.2. Stability in buffer systems (shelf-life stability). Two series;
each of five screw capped tubes, to each tube, contains 5 mL
phosphate buffers of pH 5.8 or 7.4, 1 mL of freshly prepared
(2 ꢁ 10ꢂ4 M) methanolic solution of DHIQCDS (18) was added. The
mixture was kept in dark all the time of experiment (3 days). At
appropriate time intervals (5, 180, 1880, 2440 min), 20 mL of the
mixture was taken and analyzed by HPLC for the content of the
4.2.3.1. Control experiment. Six Sprague Dawley female rats were
weighed and injected with DMSO in a dose of 1 mL/kg body weight.
At different times, one rat was sacrificed by decapitation; its blood
was collected in heparinized tube. The brain was removed quickly,
washed with ice cold saline solution, weighed, wrapped in
aluminum foil, and kept in dry ice until homogenized.
DHIQCDS (18) and its corresponding metabolites. The apparent
pseudo first order rate constant of disappearance (Kdissapp., minꢂ1
)
of the DHIQCDS (18) was determined by linear regression of Ln
peak area of DHIQCDS (18) against time in minutes.
4.2.2. In vitro stability in biological fluids
4.2.2.1. Stability in 80% mouse plasma. A freshly collected hepa-
rinized blood samples, obtained from mice, were centrifuged at
4000 rpm for 20 min and the supernatants (plasma) were collected
by a Pasteur pipette. To 5 mL of freshly collected plasma, diluted
with phosphate buffer(0.2 M, pH 7.4) to 80%, prewarmed at 37 ꢀC in
A) Control blood
To 1 mL of the collected blood, 4 mL of ice cold 5% DMSO/
acetonitrile was added and shaken vigorously. The mixture was
then centrifuged at a rate of 4000 rpm for 10 min, and kept in the
freezer till use. The supernatant was analyzed by HPLC.
a water bath for 5 min, 300
m
L of (2 ꢁ 10ꢂ4 M) methanolic solution
of freshly prepared DHIQCDS (18) was added. The tested mixture
was kept at 37 ꢀC the whole time of the experiment. At suitable
time intervals (5, 60, 120, 180, 240 min) samples of 0.5 mL was
withdrawn from the tested mixture, added immediately to 2 mL of
ice cold methanol, vortexed, and placed in the freezer (ꢂ20 ꢀC).
When all the samples have been collected, they were centrifuged,
and the supernatants were analyzed by HPLC for their contents of
the DHIQCDS (18) and its corresponding metabolites. The apparent
B) Control brain
The brain was homogenized with 1 mL of water and then 4 mL of
ice cold 5% DMSO/acetonitrile was added, and the mixture was
homogenized again, centrifuged at 4000 rpm for 10 min and kept in
the freezer until use. The supernatant was analyzed by HPLC.
pseudo first order rate constant of disappearance (Kdisssapp., minꢂ1
)
4.2.4. In vitro MAO inhibition evaluation
of the DHIQCDS (18) was determined by linear regression of the Ln
peak area of compound (18) against time in minutes.
Bovine brain mitochondria were isolated according to Basford
[34]. A mixture of 0.1 mL phosphate buffer (0.25 M, pH 7.4), 0.1 mL
of the mitochondrial suspension (6 mg/mL), 0.5 mL (0.1 mM) of the
specific inhibitor for MAO-A (clorgyline) or MAO-B (L-deprenyl)
and 0.2 mL of the test compounds (9a-b, 15a-b and 17) at five
4.2.2.2. Stability in 20% rabbit brain homogenate. Rabbits were
sacrificed by decapitation and their brains were removed, washed
with ice cold saline solution, weighed (total weight ~ 8.5 g) and
homogenized in a tissue homogenizer with four times its weight of
aqueous ice cold isotonic phosphate buffer (0.2 M, pH 7.4), while
keeping the homogenizer tube in ice bath. To 5 mL of the freshly
prepared brain homogenate, previously equilibrated at 37 ꢀC in a
different concentrations (0, 0.5 nM, 5 nM, 5 mM, 5 mM and 100 mM)
dissolved in propylene glycol was incubated for 20 min. Then,
0.2 mL of kynuramine solution (2.5 mM) was added and the
mixture was incubated in a shaking water-bath at 37 ꢀC for 60 min.
The reaction was quenched by adding 1 mL 10% W/V perchloric
acid. The samples were centrifuged at 10 000 g for 5 min and the
supernatant was completed to 2.7 mL using 1N sodium hydroxide
and measured with a spectrofluorimeter. The average value of the
duplicate measurements was used for the statistical analysis. The
MAO-A and MAO-B inhibition results are expressed as IC50. Pro-
pylene glycol was used as negative control and did not show any
inhibition.
water bath for 5 min, 400
m
L of (2 ꢁ 10ꢂ4 M) methanolic solution of
freshly prepared DHIQCDS (18) was added. The tested mixture was
kept at 37 ꢀC the whole time of the experiment. Samples of 0.5 mL
were withdrawn from the tested mixture at different time intervals
(5, 60, 120, 180, 240 min) and immediately added to 3 mL of ice cold
methanol, vortexed, and placed in the freezer (ꢂ20 ꢀC). When all
the samples have been collected, they were centrifuged, and the
supernatants were analyzed by HPLC for their contents of the
DHIQCDS (18) and its corresponding metabolites. The apparent
4.3. Molecular docking study
pseudo first order rate constant of disappearance (Kdissapp., minꢂ1
)
of the DHIQCDS (18) was determined by linear regression the Ln
peak area of compound (18) against time in minutes.
All the molecular modeling calculations and docking simulation
studies were performed using Molecular Operating Environment
(MOE®) version 2013.08 [38]. The computational software operated
under “Windows XP” installed on an Intel Pentium IV PC with a
dual core 3.2 GHz processor and 4 GB memory. The X-ray crystal-
lographic structure of MAO-A complexed with clorgyline (PDB Id:
2BXR) and MAO-B complexed with safinamide (PDB Id: 2V5Z) were
obtained from the Protein Data Bank. The enzymes were prepared
for docking studies where: (i) the ligand molecule was removed
from the enzyme active site. (ii) hydrogen atoms were added to the
structure with their standard geometry. (iii) MOE Alpha Site Finder
was used for the active sites search in the enzyme structure and
4.2.3. In vivo distribution study
Six groups, each of three Sprague Dawley female rats of average
weight of (65e70 g), were anesthetized with 0.7 mL 25% aqueous
solution of urethane. The freshly prepared solution of DHIQCDS (18)
(25 mg/mL in DMSO) was injected through the external jugular
vein at a dose of 20 mg/kg of body weight. At appropriate time
intervals (5, 15, 30, 45, 60, 75 min) blood samples were withdrawn
from the eyeball and added immediately to a previously weighted
centrifuge tubes containing 4 mL of acetonitrile, then reweighed to

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This work was supported by Young Researchers' Fund from
Assiut University.The authors are also grateful to Dr. Mohammed K.
Abdel-Hamid and Dr. Paul Keller, School of Chemistry, University of
Wollongong, Australia for great assistance in performing different
spectroscopic analyses.
ꢀ
€
Appendix A. Supplementary data
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