Synthesis of some novel hydrazone and 2-pyrazoline derivatives: Monoamine oxidase inhibitory activities and docking studies

Article abstract of DOI:10.1016/j.bmcl.2014.06.015

A novel series of 2-pyrazoline and hydrazone derivatives were synthesized and investigated for their human monoamine oxidase (hMAO) inhibitory activity. All compounds inhibited the hMAO isoforms (MAO-A or MAO-B) competitively and reversibly. With the exce

Full text of DOI:10.1016/j.bmcl.2014.06.015

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3278–3284
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of some novel hydrazone and 2-pyrazoline derivatives:
Monoamine oxidase inhibitory activities and docking studies
b
b
c
d
Begüm Evranos-Aksöz a, , Samiye Yabanog˘lu-Çiftçi , Gülberk Uçar , Kemal Yelekçi , Rahmiye Ertan
⇑
a Analysis and Control Laboratories of General Directorate of Pharmaceuticals and Pharmacy, Ministry of Health of Turkey, 06100 Sıhhiye, Ankara, Turkey
b Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhiye, Ankara, Turkey
c Faculty of Engineering and Natural Sciences, Department of Bioinformatics and Genetics (Head) Cibali Campus, Kadir Has University, 34083 Fatih, Istanbul, Turkey
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 2-pyrazoline and hydrazone derivatives were synthesized and investigated for their
human monoamine oxidase (hMAO) inhibitory activity. All compounds inhibited the hMAO isoforms
(MAO-A or MAO-B) competitively and reversibly. With the exception of 5i, which was a selective
MAO-B inhibitor, all derivatives inhibited hMAO-A potently and selectively. According to the experimen-
tal Ki values, compounds 6e and 6h exhibited the highest inhibitory activity towards the hMAO-A,
whereas compound 5j, which carries a bromine atom at R4 of the A ring of the pyrazoline, appeared to
be the most selective MAO-A inhibitor. Tested compounds were docked computationally into the active
site of the hMAO-A and hMAO-B isozymes. The computationally obtained results were in good agreement
with the corresponding experimental values.
Received 14 April 2014
Revised 4 June 2014
Accepted 5 June 2014
Available online 17 June 2014
Keywords:
2-Pyrazoline
Hydrazone
MAO inhibitors
Molecular docking
Ó 2014 Elsevier Ltd. All rights reserved.
Monoamine oxidases (MAOs) are flavoenzymes which play an
important role in the oxidative catabolism of amine neurotrans-
mitters and dietary amines.1,2 MAO contains flavin adenine
dinucleotide (FAD) as a cofactor and exist in two isoforms in
mammals, namely MAO-A and MAO-B.3 MAO-A preferentially
deaminates serotonin and norepinephrine and is selectively inhib-
ited by clorgyline, whereas MAO-B preferentially deaminates -
phenylethylamine and benzylamine and is selectively inhibited
by l-deprenil.4,5
Inhibitors of MAO-A are clinically used as antidepressants and
anxiolytics,6,7 while MAO-B inhibitors are used in the treatment
of Parkinson’s disease and in the management of symptoms
associated with Alzheimer’s disease.8 The availability of the crystal
structures of the two isoforms of human MAO facilitates the under-
standing of the selective interactions between these proteins and
their ligands, making it possible to investigate the catalytic mech-
anism and recognize the pharmacophoric requirements necessary
for the rational design of new inhibitors.8–12
cyclic hydrazine moiety,15 and it has been found that they confer
MAO inhibitory and antidepressant activity.1,15–23 Acetyl substitu-
tion of the 2-pyrazoline ring on N1 has been found to favor
inhibitory activity on MAO isoforms. This substitution increases
the positive charge of N1 of the heterocycle which strengthens
the charge-transfer bond with the isoalloxazine nucleus of FAD
and reduces the steric hindrance of the molecules.24–26
Most currently used MAO inhibitors produce side effects due to
a lack of affinity and selectivity towards one of the isoforms. For
this reason, it is necessary to design more potent, reversible and
selective inhibitors of MAO-A and MAO-B. A series of chalcones
have been found to exhibit MAO inhibitory activity.27 It is also
known that pyrazoline and hydrazone derivatives inhibit
MAO.28,29 In this study, we have synthesized new hydrazone and
2-pyrazoline derivatives and evaluated their MAO inhibitory
activities.
Chalcone derivatives were prepared by the reaction of aceto-
phenone and benzaldehyde derivatives, 1 and 2, in KOH/MeOH.
The ensuing chalcone derivatives 3a–3h were then reacted with
hydrazide compounds to furnish hydrazone and 2-pyrazoline
derivatives, 5a–5j and 6a–6i (Scheme 1). Structures, physicochem-
ical and spectral characterization of the synthesized compounds
are given in Supplementary data.
Substrates and inhibitors of MAO usually carry an amino or
imino group, which seems to play an essential role in the orienta-
tion and complex formation at the active site of the enzyme.
Numerous substituted hydrazines and hydrazides have been
studied as MAO inhibitors.13,14 2-Pyrazolines can be considered a
Hydrazone formation is dependent on the Schiff base reaction,
and thus the optimization of the pH value affects the product yield.
In these reactions, 2-pyrazoline and hydrazone derivatives are
⇑
Corresponding author. Tel.: + 90 312 565 52 69; fax: +90 312 565 52 57.
E-mail address: begumevranos@gmail.com (B. Evranos-Aksöz).
http://dx.doi.org/10.1016/j.bmcl.2014.06.015
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

B. Evranos-Aksöz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3278–3284
3279
R1
HC
R2
R5
R6
O
R2
R3
R1
COCH3
+
KOH
CH3OH
R4
R5
R3
R4
R6
O
3a-3h
1
2
O
C
3a R1=OH, R2=R3=R5=H, R4=Cl, R6=CH3
3b R1 =OH, R2=R3=R5=H, R4=Br, R6=CH3
3c R1= R2=R3=R5=H, R3=Br, R6=OCH3
3d R1= R2=R4=R5=H, R3=Cl, R6=CH3
3e R1= R2=R4=R6=H, R3=Cl, R5=CH3
NH2 NH
R7
EtOH
reflux
4
R7=Pyridine-4-yl, furan-2-yl,
thiophene-2-yl, phenyl,
4-methoxy-1-phenyl,
3f R1= R2=R4=R5=H, R3=Cl, R6=OCH3
3g R1= R2=R4=R5=H, R3=OH, R6=CH3
4-methyl-1,2,3-thiadiazole-5-yl
3h R1= R2=R4=R5=H, R3=OCH3, R6=CH3
R5
3
R2
R2
R5
R6
2
R3
R1
3
R1
N
3
R3
R4
3
4
5
2
2
1
H
1
A
4
4
R6
H
2
1
4
B
B
B
A
A
1
R4
6
5
5
6
5
6
6
x
H
N
N
NH
C
O
R7
(C ring)
R7 (C ring)
O
5a-5j
6a-6i
5a R1=OH, R2=R3=R5=H, R4=Cl, R6=CH3, R7=pyridine-4-yl
5b R1=OH, R2=R3=R5=H, R4=Cl, R6=CH3, R7=furan-2-yl
5c R1=OH, R2=R3=R5=H, R4=Cl, R6=CH3, R7=phenyl
6a R1=R2=R4=R5=H, R3=Br, R6=OCH3, R7=4-methoxy-1-phenyl
6b R1=R2=R4=R5=H, R3=Br, R6=OCH3, R7=furan-2-yl
6c R1=R2=R4=R5=H, R3=Cl, R6=CH3, R7=thiophene-2-yl
5d R1=OH, R2=R3=R5=H, R4=Cl, R6=CH3, R7=4-methoxy-1-phenyl
6d R1=R2=R4=R5=H, R3=Cl, R6=CH3, R7=4-methyl-1,2,3-thiadiazole-5-yl
6e R1=R2=R4=R6=H, R3=Cl, R5=CH3, R7=4-methyl-1,2,3-thiadiazole-5-yl
6f R1=R2=R4=R5=H, R3=Cl, R6=OCH3, R7=4-methyl-1,2,3-thiadiazole-5-yl
6g R1=R2=R4=R5=H, R3=Cl, R6=OCH3, R7=4-methoxy-1-phenyl
5e R1=OH, R2=R3=R5=H, R4=Cl, R6=CH3, R7=4-methyl-1,2,3-thiadiazole-5-yl
5f R1=OH, R2=R3=R5=H, R4=Br, R6=CH3, R7=pyridine-4-yl
5g R1=OH, R2=R3=R5=H, R4=Br, R6=CH3, R7=furan-2-yl
5h R1=OH, R2=R3=R5=H, R4=Br, R6=CH3, R7=phenyl
6h R1=R2=R4=R5=H, R3=OH, R6=CH3, R7=4-methyl-1,2,3-thiadiazole-5-yl
6i R1=R2=R4=R5=H, R3=OCH3, R6=CH3, R7=4-methyl-1,2,3-thiadiazole-5-yl
5i R1=OH, R2=R3=R5=H, R4=Br, R6=CH3, R7=4-methoxy-1-phenyl
5j R1=OH, R2=R3=R5=H, R4=Br, R6=CH3, R7=thiophene-2-yl
Scheme 1. General synthesis of compounds 3a–3h, 5a–5j, and 6a–6i.
formed together. At the end of the reaction, only one of the prod-
ucts—either the hydrazone or the 2-pyrazoline derivative—can be
isolated. For this Letter, hydrazones were obtained (15–25.7%
yield) in an ethanol solution with the reaction of chalcone and acy-
lhydrazines at 78 °C during a period of 40–50 h. When we used
chalcones having a 20-OH group as the starting compound, only
2-pyrazolines were produced, but with chalcones not having a
hydroxy group at position 20, only hydrazones were generated.
Generally, 2-pyrazoline derivatives were obtained with a higher
yield than hydrazones. The highest yield was achieved with
20-hydroxy-50-chloro chalcone derivatives (27.33–74.48%).
molecular formula of all synthesized compounds 5a–5j/6a–6i.
Characteristic [M+2] isotope peaks were observed in the mass
spectra of the compounds having a halogen atom. All compounds
provided satisfactory elemental analyses.
The MAO-A and MAO-B inhibitory activities of the newly syn-
thesized 2-pyrazoline and hydrazone derivatives were determined
using the respective hMAO isoforms. Except compound 5i, all
tested compounds were found to inhibit MAO-A selectively and
competitively (Table 1). These novel compounds were reversible
inhibitors of hMAO-A, since the enzyme activity was restored after
the centrifugation-ultrafiltration steps. Compound 5i showed
selectivity towards the MAO-B isoform.
Among compounds 5a–5j, which are 2-pyrazoline derivatives
carrying a chloride substitution on the A ring at position 5, com-
pound 5c, which carries a unsubstituted phenyl ring (C ring), was
found to be the most potent MAO-A inhibitor according to its lowest
Ki value for hMAO-A (Table 1). However, compound 5j, which car-
ries a bromide atom at R4 of the A ring of pyrazoline, appeared as
the most selective MAO-A inhibitor in the pyrazoline series accord-
ing to its highest selectivity index (SI) value. SI was calculated as Ki
(MAO-B)/Ki (MAO-A); the experimental SI value calculated for a
compound increases as the selectivity to MAO-A isoform also
increases whereas the experimental SI value calculated for a com-
pound decreases, the selectivity to MAO-B increases. For this group
of compounds, chloride substitution at R4 of the phenyl ring was
identified as favorable in terms of MAO-A inhibitory potency,
whereas bromide substitution at R4 of the phenyl ring increased
the selectivity towards hMAO-A. Compound 5i, which has a
Structures of the synthesized hydrazone and 2-pyrazoline
derivatives were elucidated by IR, 1H NMR, 13C NMR, mass spectral
data, and elemental analyses. The IR spectra of the compounds
showed OH bonds at 3178–3446 cmꢀ1, C@O stretching bonds at
1665–1626 cmꢀ1, and C@N stretching bonds at 1605–1546 cm–1
.
In the 1H NMR spectrum of compounds 5a–5j, the CH2 protons of
the pyrazoline ring resonated as a pair of doublets of doublets at
dH 2.88–2.92 ppm and dH 3.43–3.52 ppm. The CH proton appeared
as a doublet of doublets at dH 5.24–5.30 ppm. In the 1H NMR spec-
trum of compounds 6a–6i, ethylenic protons were observed at dH
6.51–7.90 ppm. The protons belonging to the aromatic ring and
the other aliphatic groups were observed with the expected chem-
ical shifts and integral values. 13C NMR spectrum of compounds 3b,
5a, 5d, 5g, 5i, 6d, 6e, 6i were given in Supplementary data. Mass
spectral analysis of the compounds was performed using the ESI
(+) or ESI (ꢀ) method, and the characteristic peaks were observed
in the mass spectra. Molecular ion peaks ([M]+) provided the

Table 1
Calculated and experimental values (Ki values, SI,
D
Gb, free energy binding) of the newly synthesized 2-pyrazoline and hydrazone derivatives for hMAO isoforms A and B
Calculated
Experimental
Compound
D
Gb (MAO-A)
Ki (MAO-A)
M]
Calculated energy
value for MAO-B
Ki (MAO-B) [lM]
SIa
Selectivity
Ki (MAO-A) [
l
M]b
Ki (MAO-B) [
l
M]b
SIa
Selectivity
[kcal/mol]
[l
3a
3b
3c
3d
3e
3f
3g
3h
5a
ꢀ9.20
ꢀ9.51
ꢀ9.31
ꢀ8.91
ꢀ9.18
ꢀ9.0
0.18
0.11
0.15
0.30
0.19
0.25
0.52
0.48
0.018
1.77
0.035
6.48
0.009
15.80
0.25
21.07
0.012
0.62
0.004
0.52
0.27
0.93
0.20
ꢀ6.94
ꢀ7.18
6.99
6.93
6.98
6.62
6.21
6.57
8.14
5.44
7.50
8.38
45.22
49.45
50.00
27.93
40.42
56.00
54.17
32.10
82.78
7.25
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
Non selective
MAO-A
MAO-A
MAO-A
MAO-A
MAO-B
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-B
MAO-A
0.14 0.01
7.22 0.24
4.22 0.20
5.55 0.33
7.70 0.40
7.00 0.34
13.90 1.05
27.00 1.97
14.90 1.19
5.23 0.27
51.57
32.46
55.50
32.08
29.17
57.91
45.00
26.61
8.72
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
0.13 0.009
0.10 0.009
0.24 0.01
0.16 0.01
0.24 0.01
0.60 0.23
0.56 0.03
0.60 0.04
7.68
14.00
28.17
15.41
1.49
12.84
0.15
25.47
0.63
105.39
5.34
441.84
0.09
107.00
0.23
4.63
0.26
30.89
8.46
84.37
2.53
66.63
2.11
2050.00
0.89
0.36
0.41
0.57
0.65
ꢀ8.57
ꢀ8.62
ꢀ10.57
ꢀ7.85
ꢀ10.17
ꢀ7.08
ꢀ10.95
ꢀ6.55
ꢀ9.02
ꢀ6.38
ꢀ10.81
ꢀ8.47
ꢀ11.42
ꢀ8.57
ꢀ8.97
ꢀ8.23
ꢀ9.13
ꢀ10.76
ꢀ9.53
ꢀ5.19
ꢀ9.21
ꢀ10.26
ꢀ10.19
ꢀ9.92
ꢀ10.29
ꢀ11.16
ꢀ11.32
ꢀ10.86
ꢀ10.33
ꢀ11.76
ꢀ10.32
ꢀ6.55
ꢀ8.00
R
S
R
S
R
S
R
S
R
S
R
S
R
S
R
S
R
S
R
S
ꢀ7.95
ꢀ6.67
ꢀ9.31
ꢀ6.27
ꢀ8.46
ꢀ5.43
ꢀ7.19
-4.58
ꢀ9.64
ꢀ5.42
ꢀ9.05
ꢀ7.28
ꢀ8.98
ꢀ6.15
ꢀ6.92
ꢀ5.56
ꢀ7.64
ꢀ5.70
ꢀ7.74
ꢀ3.67
ꢀ8.25
ꢀ8.78
ꢀ8.72
ꢀ8.52
ꢀ8.44
ꢀ8.22
ꢀ8.33
ꢀ8.25
ꢀ8.54
ꢀ5.33
ꢀ6.04
5b
5c
5d
5e
5f
4.29
3.93
0.11 0.002
0.012 0.002
4.67 0.03
0.99 0.04
0.75 0.03
9.00
62.50
4.28
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-B
MAO-A
70.00
6.67
21.36
20.97
7.50
172.58
57.50
8.90
20.00 1.23
72.00 4.55
2.00 0.13
0.24 0.011
0.18 0.008
0.45 0.02
300.00
11.11
31.11
5g
5h
5i
0.96
14.00 1.07
60.00 3.24
29.66 1.90
1640.00 65.00
33.22
42.30
6490
25.30
0.4222
11.72
68333
26.18
6.67
14.14
81.42
130.00
95.00
28.89
445.00
20.37
7.72
0.040 0.002
79.22 5.03
0.13 0.007
1500.00
0.3744
0.013
0.10
157.80
0.18
5j
12615.38
0.030
0.034
0.054
0.029
0.007
0.005
0.010
0.027
0.002
0.027
15.93
1.37
6a
6b
6c
6d
6e
6f
6g
6h
0.045 0.002
0.091 0.005
0.034 0.001
0.012 0.001
0.010 0.001
0.012 0.001
0.034 0.001
0.010 0.001
0.041 0.002
13.55 1.08
0.014 0.007
0.98 0.05
0.70 0.04
0.52 0.03
0.76 0.04
0.99 0.008
1.05 0.009
0.87 0.04
1.48 0.10
0.90 0.05
0.22 0.01
1.34 0.08
21.78
7.69
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-B
MAO-A
15.29
63.33
99.00
87.50
25.59
148.00
21.95
0.016
95.71
0.95
0.78
0.89
0.55
122.93
37.38
6i
Selegiline
Moclobemide
27.28
a
The selectivity index (SI) was calculated as Ki(MAO-B)/Ki(MAO-A).
Each value represents the mean SEM of three independent experiments. Racemic compounds were used for the experiments. All compounds inhibited hMAO isoforms competitively and reversibly.
b

B. Evranos-Aksöz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3278–3284
3281
bromide substitution at R4 and 4-methoxy-1-phenyl at R7, was
found to be a weak MAO-B inhibitor.
of a new series of potent, selective, and reversible MAO-A inhibi-
tors in the future.
The hydrazone derivatives 6a–6i were also found to be potent
MAO-A inhibitors. Among this series, compounds 6e and 6h, hav-
ing a 4-methyl-1,2,3-thiadiazole-5-yl substitution at the R7 posi-
tion of the C ring, were the most potent MAO-A inhibitors. Thus,
this substitution is suggested to play a favorable role in terms of
MAO-A inhibition for the novel hydrazone derivatives. However,
the selectivities of the newly synthesized hydrazones for hMAO-
A were found weaker than those of the 2-pyrazolines (Table 1).
In the present Letter, we successfully identified new com-
pounds which are reversible and selective inhibitors of hMAO-A.
The combination of chalcones (compounds 3a–3h) with the hydra-
zide moiety caused a remarkable increase in selectivity to MAO-A
in novel series (Table 1). For compounds 6a–6i, the 4-methyl-1,2,3-
thiadiazole-5-yl substitution on the C ring favors MAO-A inhibi-
tion. For compounds 5a–5j, the phenyl substitution on the C ring
favors MAO-A inhibition, whereas the 4-methoxy-1-phenyl substi-
tution at the R7 position (compound 5i) favors MAO-B inhibition.
The results of this study provide useful information for the design
In order to gain more insight on the binding mode of the com-
pounds with MAO-A and MAO-B, docking studies were employed
(Supplementary data). Molecular modeling approaches were per-
formed for compounds 5a–5j and 6a–6i for which Ki values had
been experimentally obtained (Table 1). The calculated inhibition
constants and free energies of the binding of these inhibitors to
MAO-A and MAO-B are presented in Table 1. Inhibitors 5a–5j were
tested experimentally only as their racemates, due to the difficulty
of separating the enantiomers, whereas calculations were done
separately for each enantiomer of these inhibitors for both iso-
zymes. As to be expected, for the isomeric compounds 5a–5j differ-
ent inhibition patterns were calculated for the respective
enantiomers for both MAO-A and MAO-B. According to the molec-
ular docking data, 2-pyrazolines (compounds 5a–5j) appeared as
MAO-A inhibitors, and their (R)-isomers, with the exception of
5h and 5j are predicted to inhibit MAO-A more effectively than
the (S)-isomers. The (R)-isomer of compound 5g was computed
to be non-selective, whereas the (S)-isomer of the same compound
Figure 1. (a) Three-dimensional orientation of 5c (R) in the active site of MAO-A. (b) Two-dimensional picture of 5c (R) in the active site of MAO-A. (c)Three-dimensional
orientation of 5c (R) in the active site of MAO-B. (d) Two-dimensional picture of 5c (R) in the active site of MAO-B.

3282
B. Evranos-Aksöz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3278–3284
is MAO-A selective. The (R)-isomer of 5i is predicted to be a
better MAO-A inhibitor than the (S)-isomer, and conversely, the
(S)-isomer a better MAO-B inhibitor. The calculated Ki values of
compounds 6a–6i for MAO-A were in good agreement with the
experimental values. The reference compound, selegiline, is a
well-known irreversible MAO-B inhibitor30 and the experimentally
For visualization of the enzyme inhibitor complexes, the pyraz-
oline 5c and the hydrazone 6h were chosen, based on their high
inhibitory potency and selectivity for one or the other of the two
MOA isoenzymes.
According to Edmondson et al.31 the human MAO-B enzyme has
two cavities connected by the amino acid ILE199 that serves as a
‘gate’. The entrance cavity has a volume of 290 Å3 and is very
hydrophobic in nature. The second cavity, with a volume of
390 Å3, harbors the substrate binding site. At the far end of the sub-
strate cavity, the coenzyme FAD is located. Computer analysis of
this cavity indicated that the amino acid side chains lining the
cavity are very hydrophobic and favorable for amine binding. The
FAD and nearly two parallel tyrosyl residues (398 and 435) form
an ‘aromatic cage’. On the other hand, human MAO-A has only a
single cavity of 550 Å3, in which FAD and two nearly parallel
tyrosyl residues (407 and 444) also form an ‘aromatic cage’. The
substrate binding sites of both MAO-A and MAO-B are quite
hydrophobic in nature.
determined inhibition constants were 13.55
MAO-A and MAO-B, respectively (Table 1). However, the calculated
values were 15.93 M (MAO-A) and 122.93 M (MAO-B). In the
lM and 0.22 lM for
l
l
molecular docking calculations, we only simulated the best dock-
ing conformation and the maximum interactions between the
ligand and active site residues of the enzyme. If the ligand binds
irreversibly or exhibits suicide-type inhibition, then it is hard to
calculate the resulting interaction with docking simulations. In
other words, for docking simulation, we only took the initial
enzyme-inhibitor complex formation into consideration. Moclobe-
mide is a well-known reversible MAO-A inhibitor30 and the
calculated values are in agreement with this fact, even though
the experimental and calculated values for moclobemide differ
more than those for our new inhibitors.
The (R)-enantiomer of 5c (calculated Ki = 0.009
lM) was docked
in the active site of MAO-A as shown in Figure 1a and b. The phenyl
Figure 2. (e) Three-dimensional orientation of 6h in the active site of MAO-A. (f) Two-dimensional picture of 6h in the active site of MAO-A. (g) Three-dimensional
orientation of 6h in the active site of MAO-B. (h) Two-dimensional picture of 6h in the active site of MAO-B.

B. Evranos-Aksöz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3278–3284
3283
ring is oriented horizontally between the phenolic side chains of
Tyr444 and Tyr407 residues, and it approached from the re face
stationary and the inhibitors are flexible. Depending on the
inhibitors 6–7 single rotatable bonds are selected as flexible.
The docking process generated the best conformer by rotating
these single bonds which snuggly fits the active site of the enzyme.
These final conformers may not be the same as started minimized
structures in the active site. The structure of the compound 6h was
assigned and their stereoisomers were estimated based on QM cal-
culations: Both isomers were optimized and their energies were
calculated. According to B3LYP/6-31G⁄ optimization E isomer
(Fig. 4) is only 0.54 kcal/mol more stable than Z (Fig. 3) isomer.
The synthesized compounds were found to be mostly competi-
tive, reversible, and selective inhibitors of hMAO-A. 2-Pyrazoline
compounds showed higher selectivity towards hMAO-A than chal-
cones and hydrazones while the combination of 2-pyrazoline and
hydrazone increased the inhibitory potency.
The data indicate that the 2-hydroxy-5-bromo phenyl ring (A
ring) is essential both for selectivity and potency of hMAO-A
inhibition by compounds 5a–5j. Selectivity decreased, when the
2-hydroxy-5-bromo phenyl ring was replaced by the 2-hydroxy-
5-chloro phenyl ring. Addition of 4-methoxyphenyl or 2-furyl to
the scaffold as the C ring decreased the potency and selectivity of
inhibition, while the addition of a phenyl ring as the C ring to
the scaffold increased this effect. Addition of thiophene-2-yl as a
C ring to 2-pyrazoline compounds increased their selectivity, as
evidenced by the SI value of compound 5j, which carries such a C
ring, is almost 130-fold higher than that of the reference MOA-A
inhibitor moclobemide. On the contrary, addition of thiophene-
2-yl as a C ring to hydrazone compounds decreased their potency
and selectivity. The addition of a pyridine moiety as the C ring to
2-pyrazoline compounds reduced their potency and selectivity
towards hMAO-A. For hydrazone compounds, replacement of the
chlorine atom at R3 of the A ring by a hydroxy group increased
the potency and selectivity towards hMAO-A, while a methoxy
group in this position decreased both these parameters.
of FAD making one
p–p interaction. The hydrogen atom of the
hydroxy group of the 2-hydroxy phenyl ring of the inhibitor forms
a hydrogen bond with the hydroxy group of Tyr407. Ile207, Gly214,
Tyr197, Tyr69, Asn181, Phe352, Ile335, Gln215, Ile180, Trp441, and
Gln74 are the other active site residues interacting with the inhib-
itor. The same compound exhibits different binding patterns with
MAO-B (Ki = 0.63 lM), as can be seen in Figure 1c and d. The inhib-
itor is placed distant from the hydrophobic cage by Tyr398, Tyr435,
and FAD. In this case, the inhibitor is located close to the entrance
cavity. The hydroxy group hydrogen of the 2-hydroxy phenyl ring
of the inhibitor forms a hydrogen bond with the entrance site res-
idue Gln206, while the carbonyl group of the inhibitor is hydrogen-
bound to the side chain of Gln206. Ile171, Phe168, Tyr188, Tyr398,
Tyr435, Tyr326, Cys172, Ile198, Phe343, and Met341 are the other
active site residues interacting with the inhibitor.
Compound 6h (calculated Ki = 0.002 lM) was docked in
the active site of MAO-A as shown in Figure 2e and f. The
4-methyl-1,2,3-thiadiazole-5-yl ring is inserted into the hydropho-
bic cage which is comprised of Tyr444, Tyr407, and FAD. The inhib-
itor approached from the re face of FAD and makes a
p–p
interaction with Tyr444. The hydroxy group of the 4-hydroxy phe-
nyl ring forms a hydrogen bond with the hydrogen atom of the
Cys323 side chain. Tyr407, Ile207, Tyr197, Tyr69, Phe352, Ile335,
Gln215, Gly67, Gly66, Lys305, Phe305, and Met350 are the other
active site residues involved in hydrophobic and polar interactions
with the inhibitor. Figure 2g and h show the binding pattern of 6h
to MAO-B (calculated Ki = 0.89 lM). The inhibitor is located in the
hydrophobic cage surrounded by Tyr398, Tyr435, and FAD. The
methyl-1,2,3-thiadiazole-5-yl ring is sandwiched between Tyr435
and Tyr398 and makes one pp interaction with Tyr435. The hydro-
gen atom of the hydroxy group of the 4-hydroxy phenyl ring is
hydrogen-bound to Ile199. The azo group of the inhibitor makes
another polar attraction with the sulfhydryl group of Cys172. The
4-hydroxy phenyl ring makes the second pp interaction with
Tyr60. Ile171, Ile198, Tyr188, Tyr326, Phe343, and Gly57 are the
other active site residues interacting with the inhibitor.
All biological experiments were carried out using only racemic
mixtures of compounds 5a–5j. The calculated Ki values, at least for
one of the enantiomers, agreed with the experimentally deter-
mined values (Table 1).
The inhibitor, 6h, was optimized using SPARTAN 10 program at
PM3 level. The docking simulation was started from these lowest
energy conformations. The macromolecule (enzymes) was held
Overall, the docking method provided us with invaluable data
for the rationalization of the observed experimental results, and
allowed us to estimate the binding mode, the inhibition constant
Figure 3. Z isomer of compound 6h.
Figure 4. E isomer of compound 6h.

3284
B. Evranos-Aksöz et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3278–3284
12. Hubalek, F.; Binda, C.; Khalil, A.; Li, M.; Mattevi, A.; Castagnoli, N.; Edmondson,
D. E. J. Biol. Chem. 2005, 280, 15761.
13. Chimenti, F.; Bolasco, A.; Secci, D.; Chimenti, P.; Granese, A.; Carradori, S.;
Yáñez, M.; Orallo, F.; Ortuso, F.; Alcaro, S. Bioorg. Med. Chem. 2010, 18, 5715.
14. Secci, D.; Bolasco, A.; Carradori, S.; D’Ascenzio, M.; Nescatelli, R.; Yáñez, M. Eur.
J. Med. Chem. 2012, 58, 405.
and the free energy of binding, all of which are promising tools for
the discovery of novel, potent, and selective MAO inhibitors poten-
tially useful as pharmacological agents.
Acknowledgment
15. Sahoo, A.; Yabanoglu, S.; Sinha, B. N.; Ucar, G.; Basu, A.; Jayaprakash, V. Bioorg.
Med. Chem. Lett. 2010, 20, 132.
16. Bilgin, A. A.; Palaska, E.; Sunal, R. Arznei-Forschung 1993, 43, 1041.
17. Palaska, E.; Erol, D. D.; Demirdamar, R. Eur. J. Med. Chem. 1996, 31, 43.
18. Palaska, E.; Aytemir, M.; Uzbay, I. T.; Erol, D. Eur. J. Med. Chem. 2001, 36, 539.
19. Gökhan, N.; Yesßilada, A.; Uçar, G.; Erol, K.; Bilgin, A. A. Arch. Pharm. Pharm. Med.
Chem. 2003, 336, 362.
20. Uçar, G.; Gokhan, N.; Yesßilada, A.; Bilgin, A. Neurosci. Lett. 2005, 382, 327.
21. Gökhan-Kelekçi, N.; Yabanog˘lu, S.; Küpeli, E.; Salgın, U.; Özgen, Ö.; Uçar, G.;
Yesßilada, E.; Kendi, E.; Yesßilada, A.; Bilgin, A. A. Bioorg. Med. Chem. 2007, 15,
5775.
We would like to express our deep gratitude to Professor Hakan
Göker for providing the spectroscopic data.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.06.
015.
22. Palaska, E.; Aydin, F.; Uçar, G.; Erol, D. Arch. Pharm. Chem. Life Sci. 2008, 341,
209.
23. Karuppasamy, M.; Mahapatra, M.; Yabanoglu, S.; Ucar, G.; Sinha, B. N.; Basu, A.;
Mishra, N.; Sharon, A.; Kulandaivelu, U.; Jayaprakash, V. Bioorg. Med. Chem.
1875, 2010, 18.
References and notes
24. Wouters, J.; Ooms, F.; Jegham, S.; Koenig, J. J.; George, P.; Durant, F. Eur. J. Med.
Chem. 1997, 32, 721.
25. Manna, F.; Chimenti, F.; Bolasco, A.; Secci, D.; Bruna Bizzarri, B.; Befani, O.;
Turini, P.; Mondovı, B.; Alcaro, S.; Tafi, A. Bioorg. Med. Chem. Lett. 2002, 12,
3629.
26. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.; Befani, O.; Turini,
P.; Alcaro, S.; Ortuso, F. Bioorg. Med. Chem. Lett. 2004, 14, 3697.
27. Chimenti, F.; Fioravanti, R.; Bolasco, A.; Chimenti, P.; Secci, D.; Rossi, F.; Yanez,
M.; Orallo, F.; Ortuso, F.; Alcaro, S. J. Med. Chem. 2009, 52, 2818.
28. Chimenti, F.; Bolasco, A.; Manna, F.; Secci, D.; Chimenti, P.; Befani, O.; Turini, P.;
Giovannini, V.; Mondovı, B.; Cirilli, R.; La Torre, F. J. Med. Chem. 2004, 47, 2071.
29. Chimenti, F.; Maccioni, E.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.;
Carradori, S.; Alcaro, S.; Ortuso, F.; Yanez, M.; Orallo, F.; Cirilli, R.; Ferretti, R.; La
Torre, F. J. Med. Chem. 2008, 51, 4874.
1. Chimenti, F.; Maccioni, E.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.;
Befanı, O.; Turını, P.; Alcaro, S.; Ortuso, F.; Cirilli, R.; La Torre, F.; Cardia, M. C.;
Distinti, S. J. Med. Chem. 2005, 48, 7113.
2. Westlund,K.N.;Denney, R.M.;Rose, R.M.;Abell,C.W. Neuroscience1988,25, 439.
3. Bach, A. W.; Lan, N. C.; Johnson, D. L.; Abell, C. W.; Bembenek, M. E.; Kwan, S.
W.; Seeburg, P. H.; Shih, J. C. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4934.
4. Johnston, J. P. Biochem. Pharmacol. 1968, 17, 1285.
5. Knoll, J.; Magyar, K. Adv. Biochem. Psychopharmacol. 1972, 5, 393.
6. Pare, C. M. Pharmakopsychiatr Neuropsychopharmakol 1976, 9, 187.
7. Shih, J.; Chen, K.; Ridd, M. J. Annu. Rev. Neurosci. 1999, 22, 197.
8. Binda, C.; Newton-Vinson, P.; Hubalek, F.; Edmondson, D. E.; Mattevi, A. Nat.
Struct. Biol. 2002, 9, 22.
9. Binda, C.; Hubalek, F.; Li, M.; Edmondson, D. E.; Mattevi, A. FEBS Lett. 2004, 564,
225.
10. Edmondson, D. E.; Binda, C.; Mattevi, A. Neurotoxicology 2004, 25, 63.
11. De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 12684.
30. Fioravanti, R.; Desideri, N.; Biava, M.; Monaco, L. P.; Grammatica, L.; Yáñez, M.
Bioorg. Med. Chem. Lett. 2013, 23, 5128.
31. Edmondson, D. E.; Binda, C.; Mattevi, A. Arch. Biochem. Biophys. 2007, 464, 269.