Synthesis and Screening of Human Monoamine Oxidase-A Inhibitor Effect of New 2-Pyrazoline and Hydrazone Derivatives

Article abstract of DOI:10.1002/ardp.201500212

A group of 3,5-diaryl-2-pyrazoline and hydrazone derivatives was prepared via the reaction of various chalcones with hydrazide compounds in ethanol. Twenty original compounds were synthesized. Ten of these original compounds have a pyrazoline structure, n

Full text of DOI:10.1002/ardp.201500212

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Arch. Pharm. Chem. Life Sci. 2015, 348, 743–756
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Full Paper
Synthesis and Screening of Human Monoamine Oxidase-A
Inhibitor Effect of New 2-Pyrazoline and Hydrazone Derivatives
1
2
2
3
3
_
€
€
ꢀ
Begum Evranos-Aksoz , Ipek Baysal , Samiye Yabanoglu-CS iftcSi , Teodora Djikic , Kemal YelekcSi ,
2
4
€
Gulberk UcSar , and Rahmiye Ertan
1
Analysis and Control Laboratories of General Directorate of Pharmaceuticals and Pharmacy, Ministry of
Health of Turkey, Sıhhiye, Ankara, Turkey
Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, Sıhhiye, Ankara, Turkey
Department of Bioinformatics and Genetics (Head) Cibali Campus, Faculty of Engineering and Natural
Sciences, Kadir Has University, Fatih, Istanbul, Turkey
2
3
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara,
Turkey
A group of 3,5-diaryl-2-pyrazoline and hydrazone derivatives was prepared via the reaction of various
chalcones with hydrazide compounds in ethanol. Twenty original compounds were synthesized. Ten
of these original compounds have a pyrazoline structure, nine of these original compounds have a
hydrazone structure, and one of these original compounds has a chalcone structure. Structural
1
elucidation of the compounds was performed by IR, H NMR, 13C NMR, mass spectral data, and
elemental analyses. These compounds were tested for their inhibitory activities toward the A and B
isoforms of human monoamine oxidase (MAO). Except for 3k and 6c, all compounds were found to
be competitive, reversible, and selective inhibitors for either one of the isoforms (hMAO-A or MAO-B).
Compounds 3k and 6c were found to be competitive, reversible, but non-selective MAO inhibitors.
Compound 6h showed hMAO-B inhibitory activity whereas the others potently inhibited hMAO-A.
Compound 5c showed higher selectivity than the standard drug moclobemide. According to the
experimental Ki values, compounds 6i, 6d, and 6a exhibited the highest inhibitory activity toward
hMAO-A. The AutoDock 4.2 program was employed to perform automated molecular docking. The
calculated results obtained computationally were in good agreement with the experimental values.
Keywords: 2-Pyrazoline / Hydrazone / MAO inhibitors / Molecular docking
Received: June 22, 2015; Revised: July 17, 2015; Accepted: July 21, 2015
DOI 10.1002/ardp.201500212
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Two isoforms of MAO exist as MAO-A and MAO-B. MAO
enzymes are responsible for the oxidative deamination of
Introduction
Monoamine oxidase (MAO) is an enzyme present in the outer
mitochondrial membrane of neuronal and non-neuronal cells.
endogenous and xenobiotic amines and have a different
substrate preference, inhibitor specificity, and tissue distribu-
tion [1]. MAO-A preferably metabolizes serotonin, adrena-
line, and noradrenaline [2] whereas b-phenylethylamine and
benzylamine are predominantly metabolized by MAO-B [3].
Tyramine, dopamine, and some other important amines are
common substrates for both the isoenzymes [4].
Although MAOs are widely distributed in various organs,
most of the studies concerning their functional properties and
involvement in pathological processes have been mainly
€
€
Correspondence: Dr. Begum Evranos-Aksoz, Analysis and Control
Laboratories of the General Directorate of Pharmaceuticals and
Pharmacy, Ministry of Health of Turkey, 06100 Sıhhiye, Ankara,
Turkey.
E-mail: begumevranos@gmail.com
Fax: þ90-312-5655257
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focused on the central nervous system. In the periphery, MAO-
A is predominant in the heart, adipose tissue, and skin
fibroblasts; MAO-B is the major form found in platelets and
lymphocytes, while both isoenzymes are expressed in the
kidney and liver [5]. MAO inhibition allows endogenous
and exogenous substrates to accumulate and may thereby
alter the dynamics of regular monoamine transmitters, such
as noradrenaline, serotonin, or dopamine. MAO inhibitors
(MAOIs) are mainly used in psychiatry for the treatment of
depressive disorders and in neurology for the treatment of
Parkinson’s and Alzheimer’s diseases. Although the classical
non-selective and irreversible MAOIs, such as phenelzine
and tranylcypromine, are characterized by the risk of
inducing a hypertensive crisis when dietary tyramine is
ingested, selegiline (L-deprenyl), the selective and irreversible
MAO-B inhibitor and moclobemide, the selective and revers-
ible inhibitor of MAO-A, are free from this potential
interaction [1].
The structure of synthesized hydrazone and 2-pyrazoline
derivatives was elucidated by IR, 1H NMR, 13C NMR, mass
spectral data, and elementary analysis findings. The IR data
provided evidence for the formation of the expected
–
–
structures. OH, C O, and C N functions absorbed strongly
–
–
in the expected regions: OH at 3450–3410 cmꢀ1, NH at 3420–
ꢀ1
ꢀ1
–
–
–
–
3348 cm , C O at 1694–1617 cm , and C N at 1607–
1505 cmꢀ1, respectively. In 1H NMR spectrum of compounds
5a–j, the CH2 protons of the pyrazoline ring resonated as a
pair of doublets of doublets at 2.83–3.02 and 3.32–3.62 ppm.
The CH proton appeared as a doublet of doublets at d 5.18–
1
5.43 ppm. In H NMR spectrum of compounds 6a–i, ethylenic
protons were observed at 6.34–7.80 ppm. The protons
belonging to the aromatic ring and the other aliphatic
groups were observed with the expected chemical shift
and integral values. 13C NMR spectra of compounds 3b, 3l,
5a, 5e, 5i, 6b, 6d, 6g, and 6i are given in the Experimental
section. Mass analysis of compounds was performed by
using ESI (þ) and ESI (ꢀ) method. The characteristic peaks
were observed in the mass spectra of the compounds.
Molecular ion peaks ([M]þ) provided the molecular formula
of all synthesized compounds 5a–j/6a–i. Characteristic [Mþ2]
isotope peaks were observed in the mass spectra of the
compounds having a Cl or a Br atom; [Mþ4] isotope peaks
were observed in the mass spectra of the compounds which
have two Cl atoms. All compounds provided satisfactory
elemental analysis.
Chalcones are an important class of natural compounds.
Chalcones, or 1,3-diaryl-2-propen-1-ones, belong to the
flavonoid family. They are precursors for flavonoid biosyn-
thesis, which plays an ecological role in relation to plant color.
Chemically they consist of open-chain flavonoids in which the
two aromatic rings are joined by
a three-carbon a,b-
unsaturated carbonyl system [6, 7]. They are commonly
synthesized via the Claisen–Schmidt condensation between
acetophenone and benzaldehyde. Chalcones are important
intermediates in the synthesis of many pharmaceuticals
including 2-pyrazolines and hydrazones [8], and they have
important biological activities such as anti-inflammatory [9],
antibacterial [10], antiviral [11], antioxidant [12], antineo-
plastic [13], and MAO inhibitor activities [14, 15].
Of chalcones, 2-pyrazoline derivatives have been previ-
ously reported to exhibit various pharmacological activities
such as antihypertensive [16], antimicrobial [17–19], anti-
inflammatory [20–22], antidepressant [23], and MAO inhibi-
tory activities [24]. Some hydrazone compounds also have
MAO inhibitory activities [25, 26]. The necessity of discover-
ing new, selective, and reversible hMAO inhibitors directed
us to synthesize novel compounds with more selective and
potent MAO inhibitory activity. Thus, in the current study,
we synthesized new compounds containing 2-pyrazoline
or hydrazone and investigated their hMAO inhibitory
potencies.
Biological activity
All the newly synthesized compounds were screened for
their potential hMAO inhibitory activity using recombinant
hMAO-A and hMAO-B. The results of hMAO-A and hMAO-B
inhibition studies with title compounds are reported in
Table 1 together with their selectivity indexes. It has been
previously reported that the natural and synthetic chalcone
derivatives exhibit hMAO inhibitory activity [27, 28]. Although
chalcones have been shown to inhibit hMAO-B more potently
than hMAO-A [14], there are some exceptions such as
isoliquiritigenin which inhibits MAO-A more potently than
MAO-B [28]. Kinetic studies indicate that chalcones are
competitive inhibitors of MAOs [27]. In general, reversibility
of the inhibition of MAO by chalcones is controversial. In a
study, chalcones have been shown to bind irreversibly to
hMAO-B, by a binding mode similar to those of rasagiline and
(R)-deprenyl, two known potent MAO-B inhibitors [14]. Since
the side effects of known MAO inhibitors are mostly
associated with the irreversible inhibition particularly of
MAO-A, design of potent, selective, and reversible new
MAO-A inhibitors is of value.
Results and discussion
Chemistry
Chalcone derivatives were prepared with the reaction of some
acetophenone and benzaldehyde derivatives in KOH/MeOH.
The ensuing chalcone derivatives were then reacted with
hydrazide compounds to furnish hydrazone and 2-pyrazoline
derivatives (Scheme 1). Structures, physicochemical, and
spectral characterization of the synthesized compounds are
given in the Experimental section.
In our study, newly synthesized chalcones (compounds 3a–l)
inhibited hMAO-A selectively and reversibly in a competitive
mode except for compound 3k. Compound 3k inhibited both
hMAO-A and hMAO-B potently, but with similar Ki values
(Table 1) suggesting that this compound is a non-selective
hMAO inhibitor. The most potent hMAO-A inhibitor in this
series was found to be compound 3b which carries two
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Scheme 1. General synthesis of 5a–j and 6a–i.
chloride atoms at the third and fifth positions of A ring and
is substituted with a methyl group at the third position of B
ring. Compound 3b inhibited the hMAO-A isoform reversibly
and selectively with a Ki value of 0.056 ꢁ 0.004 mM. Since
chalcones 3a and 3b, which have 3,5-dichloro substitution
on the A ring inhibited hMAO-A potently, chloride substitu-
tion at the third and fifth positions of A ring is suggested
to improve the MAO-A inhibitory activity of the chalcone.
The additional hydrophobic interaction between the
chloride at the substitution third position and PHE352,
ILE180, ILE335, and LEU337 residues and the chloride at the
substitution fifth position and PHE208, ILE335, and LEU337
residues causes high potency. According to the selectivity
index, compound 3f which has a chloride substitution at the
fifth position of A ring and carries a methoxy group at
the fourth position of the B ring appeared to be the
most selective hMAO-A inhibitor (SIKi hMAO-A/Ki hMAO-B ¼ 0.060)
among the synthesized chalcones. Methoxy substitution on
B ring seems to enhance the selectivity of the compound
toward the hMAO-A isoform. These enhancements can be
rationalized based on the observation of molecular docking
results such that the chloride substitution at the fifth
position of
A
ring makes two important hydrophobic
strong
interactions with TYR197 and TYR407. Also
a
hydrogen bond occurs between the oxygen of the methoxy
substitution on B ring and the side chain hydrogen and
LYS305. Experimental data concerning the hMAO inhibitory
activities of chalcones were found to be in good agreement
with the calculated data obtained from the molecular
docking studies (Table 2).
Newly synthesized pyrazolines (compounds 5a–j) were
found to inhibit human MAO potently and reversibly in a
competitive mode (Table 1). All compounds inhibited hMAO-
A selectively with Ki values ranging between 0.005 ꢁ 0.001
and 14.445 ꢁ 1.087 mM. Data showed that compounds 5a, 5e,
and 5g exhibited high activity toward hMAO-A with Ki values
of 0.005 ꢁ 0.001, 0.010 ꢁ 0.001, and 0.013 ꢁ 0.001 mM, respec-
tively. Among them, compound 5a which carries two chloride
atoms at the third and fifth positions of A ring and bears a
4-methoxy-1-phenyl as C ring showed the best activity for
hMAO-A. It is striking that compound 5a inhibited hMAO-A
more potently than moclobemide, a known potent MAO-A
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Table 1. Experimentally determined Ki values of the newly synthesized hydrazone and 2-pyrazoline derivatives for the
inhibition of hMAO isoforms A and Ba).
Ki(MAO-A)
[mM]b)
Ki(MAO-B)
[mM]b)
Inhibition
type
Compounds
SIc)
0.188
Reversibility
Selectivity
3a
0.060 ꢁ 0.004
0.056 ꢁ 0.004
0.070 ꢁ 0.005
0.390 ꢁ 0.025
0.175 ꢁ 0.010
0.078 ꢁ 0.008
0.488 ꢁ 0.027
0.755 ꢁ 0.058
0.748 ꢁ 0.055
0.628 ꢁ 0.042
0.387 ꢁ 0.030
0.201 ꢁ 0.018
0.005 ꢁ 0.001
0.088 ꢁ 0.005
0.123 ꢁ 0.012
0.120 ꢁ 0.009
0.010 ꢁ 0.001
0.655 ꢁ 0.039
0.013 ꢁ 0.001
12.250 ꢁ 1.060
14.445 ꢁ 1.087
0.377 ꢁ 0.020
0.010 ꢁ 0.001
0.078 ꢁ 0.005
0.032 ꢁ 0.002
0.008 ꢁ 0.001
0.016 ꢁ 0.002
0.035 ꢁ 0.002
0.075 ꢁ 0.004
0.874 ꢁ 0.051
0.008 ꢁ 0.10 ꢂ 10ꢀ3
17.112 ꢁ 1.334
0.012 ꢁ 0.001
0.320 ꢁ 0.013
0.328 ꢁ 0.019
0.560 ꢁ 0.033
1.960 ꢁ 0.134
1.115 ꢁ 0.090
1.280 ꢁ 0.099
0.621 ꢁ 0.038
2.154 ꢁ 0.194
2.005 ꢁ 0.152
1.302 ꢁ 0.105
0.389 ꢁ 0.035
0.278 ꢁ 0.020
0.100 ꢁ 0.009
1.300 ꢁ 0.097
2200.00 ꢁ 90.365
0.290 ꢁ 0.016
0.097 ꢁ 0.002
1.020 ꢁ 0.098
0.102 ꢁ 0.009
36.880 ꢁ 2.450
857.000 ꢁ 29.700
1.752 ꢁ 0.110
0.103 ꢁ 0.009
0.102 ꢁ 0.009
0.349 ꢁ 0.021
0.103 ꢁ 0.010
0.159 ꢁ 0.011
0.099 ꢁ 0.006
0.108 ꢁ 0.009
0.501 ꢁ 0.033
0.500 ꢁ 0.026
0.301 ꢁ 0.020
1.466 ꢁ 0.07
Competitive
Competetive
Competetive
Competetive
Competetive
Competitive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Competetive
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Irreversible
Reversible
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-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-B
MAO-A
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
6a
6b
6c
6d
6e
6f
6g
6h
6i
0.233
0.125
0.199
0.157
0.060
0.785
0.351
0.373
0.482
0.995
0.723
0.050
0.068
5.591 ꢂ 10ꢀ5
0.414
0.103
0.642
0.127
0.332
0.016
0.215
0.097
0.764
0.082
0.077
0.100
0.354
0.416
1.745
0.016
56.850
0.008
Selegiline
Moclobemide
a) Racemic compounds were used for the experiments. Compounds 3a–l are chalcones, compounds 5a–j are pyrazoline
derivatives, and compounds 6a–i are hydrazone derivatives.
b) Each value represents the mean ꢁ SEM of three independent experiments.
c) The selectivity index (SI) was calculated as Ki(MAO-A)/Ki(MAO-B).
inhibitor (Ki values are 0.005 ꢁ 0.001 and 0.012 ꢁ 0.001 mM,
respectively). From an inspection of the chemical structures of
these two compounds, it can be concluded that chloride
substitution at the third and fifth positions of A ring and a
methoxy substitution at the fourth position of C ring improve
the MAO-A inhibitory activity of the pyrazolines. These results
are also supported by the computational modeling studies
such that a methoxy group at the fourth position of C ring
with the side chain of TYR197, and a chloride atom at the fifth
position of A ring and TYR69 make hydrophobic interactions.
Compound 5c which carries two chloride atoms at the third
and fifth positions of A ring and bears a furane ring as a C ring
appeared to be the most selective hMAO-A inhibitor of this
series with a SI of 5.60 ꢁ 10ꢀ5 (Table 1). Since the replacement
of C ring with furane in the pyrazoline nucleus enhanced the
selectivity of pyrazolines for hMAO-A (Tables 1 and 2) and
docking studies also endorse this finding (Fig. 1), it is
suggested that the furane ring improves the selectivity of
pyrazolines toward hMAO-A. Experimental data concerning
the hMAO inhibitory activities of novel pyrazolines were
found to be in good agreement with the calculated data
obtained from the molecular docking studies (Table 2).
Newly synthesized hydrazones (compounds 6a–i) inhibited
hMAO-A or -B selectively and reversibly in a competitive mode
except for compound 6c. Compound 6h inhibited hMAO-B
selectively, but not as potently as selegiline. Compound 6c
inhibited both hMAO-A and -B potently, but with similar Ki
values (Table 1) suggesting that this compound is a non-
selective hMAO inhibitor. Among hydrazones, compound 6i
which carries a bromide at the fourth position of A ring, and
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Arch. Pharm. Chem. Life Sci. 2015, 348, 743–756
Monoamine Oxidase Inhibitors
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Table 2. Calculated Ki values of the newly synthesized hydrazone and 2-pyrazoline derivatives for the inhibition of
hMAO isoforms A and B.
Calculated DG
for MAO-A
(kcal/mol)
Calculated DG
for MAO-B
(kcal/mol)
Ki(MAO-A)
[mM]
Ki(MAO-B)
[mM]
Compoundsa)
SIb)
Selectivity
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
5a (R)
5a (S)
5b (R)
5b (S)
5c (R)
5c (S)
5d (R)
5d (S)
5e (R)
5e (S)
5f (R)
5f (S)
5g (R)
5g (S)
5h (R)
5h (S)
5i (R)
5i (S)
5j (R)
5j (S)
6a
ꢀ9.99
ꢀ10.36
ꢀ9.81
ꢀ9.02
ꢀ9.27
ꢀ9.55
ꢀ8.82
ꢀ8.41
ꢀ8.48
ꢀ8.58
ꢀ8.97
ꢀ9.24
ꢀ11.72
11.20
ꢀ9.76
ꢀ8.57
ꢀ9.47
ꢀ8.16
ꢀ10.09
ꢀ8.78
ꢀ12.22
10.94
ꢀ9.68
ꢀ8.31
ꢀ11.94
ꢀ11.17
ꢀ7.75
ꢀ6.52
ꢀ6.54
ꢀ6.48
ꢀ8.83
ꢀ8.80
ꢀ11.10
ꢀ9.93
ꢀ10.41
ꢀ11.19
ꢀ10.77
ꢀ10.80
ꢀ10.02
ꢀ10.10
ꢀ11.20
0.048
0.026
0.064
0.245
0.161
0.100
0.345
0.686
0.609
0.511
0.268
0.169
0.003
0.006
0.070
0.520
0.115
1.050
0.040
0.364
0.001
0.010
0.080
0.812
0.002
0.006
2.080
16.61
16.15
17.70
0.337
0.353
0.007
0.053
0.023
0.006
0.013
0.012
0.045
0.040
0.006
ꢀ8.98
ꢀ9.03
ꢀ8.58
ꢀ7.84
ꢀ8.17
ꢀ8.15
ꢀ8.54
ꢀ7.75
ꢀ7.97
ꢀ8.05
ꢀ8.88
ꢀ9.00
ꢀ9.28
ꢀ6.50
ꢀ8.05
ꢀ6.50
ꢀ9.40
ꢀ2.00
ꢀ8.99
ꢀ5.33
ꢀ10.58
ꢀ9.76
ꢀ8.06
ꢀ8.20
ꢀ10.20
ꢀ9.99
ꢀ7.03
ꢀ1.83
ꢀ7.46
ꢀ3.13
ꢀ7.77
ꢀ4.79
ꢀ9.58
ꢀ9.57
ꢀ9.12
ꢀ9.65
ꢀ9.33
ꢀ9.88
ꢀ9.60
ꢀ10.34
ꢀ10.72
0.261
0.241
0.508
1.800
1.030
1.070
0.549
2.070
1.430
1.260
0.307
0.251
0.157
0.033
1.260
17.180
0.130
34300.000
0.257
0.124
0.017
0.070
1.230
0.968
0.033
0.048
7.000
45390.000
3.41
5050.000
2.030
308.140
0.095
0.096
0.207
0.084
0.145
0.057
0.092
0.026
0.014
0.184
0.108
0.126
0.136
0.156
0.093
0.628
0.341
0.426
0.406
0.873
0.673
0.019
0.182
0.055
0.030
0.885
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-B
MAO-A
MAO-A
MAO-A
MAO-A
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-B
MAO-A
3.060 ꢂ 10ꢀ5
0.155
2.940
0.059
0.143
0.065
0.839
0.060
0.125
0.298
3.66 ꢂ 10ꢀ4
4.740
0.004
0.166
0.001
0.074
0.552
0.111
0.071
0.090
0.211
0.489
1.540
0.043
6b
6c
6d
6e
6f
6g
6h
6i
a) Compounds 3a–l are chalcones, compounds 5a–j are pyrazoline derivatives, and compounds 6a–i are hydrazone derivatives.
b) The selectivity index (SI) was calculated as Ki(MAO-A)/Ki(MAO-B).
bears a 4-methyl-1,2,3-thiadiazole-5-yl ring as C ring was
found to be the most potent and selective hMAO-A inhibitor.
Compound 6i inhibited hMAO-A more potently than moclo-
bemide, a known potent MAO-A inhibitor (Ki values are
0.008 ꢁ 0.10 ꢂ 10ꢀ3 and 0.012 ꢁ 0.001 mM, respectively). It is
suggested that 4-methyl-1,2,3-thiadiazole-5-yl ring increases
the inhibitory potency and selectivity of hydrazones toward
hMAO-A since docking studies showed that this moiety
interacts with the FAD co-factor located in the active site
cavity of hMAO-A (Fig. 2). Experimental data concerning the
hMAO inhibitory activities of novel hydrazones were found to
be in good agreement with the calculated data obtained from
the molecular docking studies (Table 2).
Palaska et al. [29] synthesized a series of 1-thiocarbamoyl-
3,5-diphenyl-4,5-dihydro-1H-pyrazole derivatives, screened
their MAO inhibitory activities and suggested that chloride
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Figure 1. Three-dimensional and two-dimensional orientations of 5c (R) in the active site of hMAO-A (a and b) and hMAO-B (c and d).
Amino acid side chains are shown as sticks, the inhibitor is shown as a ball and stick (green), and the co-factor FAD is depicted as a
yellow stick. In 2D pictures, purple color shows electrostatic and green color shows van der Waals attractions.
substitution on the phenyl ring located at the third position of
2-pyrazoline increased the inhibitory potency toward MAO-A.
They also indicated that replacement of chloride with
bromide significantly decreased the inhibitory potency. Our
data showing that the MAO-A inhibitory potency of
compound 5i in our series which is having a bromide atom
at R4 position significantly reduced when compared with
those of having a chloride atom at the same position of
the molecule is in good agreement with that of Palaska
group. Sahoo et al. [30] suggested that substitution on
the nitrogen atom at the first position of pyrazoline ring
increases the MAO-A inhibitory potency. Likewise, almost all
of our new compounds having a substitution on the same
position of pyrazoline moiety inhibited hMAO-A selectively
and potently.
the compounds 3f, 5c, and 6i which possessed the most
selective and potent inhibitory activities toward the hMAO-A
isoform. Reversibility of hMAO-A inhibition with the com-
pounds was compared with that of moclobemide, and the
reversibility of hMAO-A inhibition with compound 5c was
found to be better than that of moclobemide.
Modeling studies
In order to see the binding pose of these types of compounds
in detail, two representative ligands 5c and 6i were chosen.
To render visible the detailed interactions of the docked
poses of the inhibitor 5c and 6i were chosen. In order to
generate pictures, Accelrys Visualization 4.5 program was
used. Analysis of the optimal binding mode for 5c
(Fig. 1a and b) in the hMAO-A active site cavity revealed
that the furane ring of the compound is inserted between
TYR444 and TRY407 from the re face of the FAD co-factor. 5c
All tested compounds inhibited the hMAO reversibly.
Table 3 presents the reversibility of hMAO-A inhibition with
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Figure 2. Three-dimensional and two-dimensional orientations of 6i in the active site of hMAO-A (a and b, respectively) and hMAO-B
(c and d, respectively). Amino acid side chains are shown as sticks, the inhibitor is shown as a ball and stick (green), and the co-factor
FAD is depicted as a yellow stick. In 2D pictures, purple color shows electrostatic and green color shows van der Waals attractions.
makes various electrostatic and van der Waals interactions
with active site residues lining the cavity. A hydrogen bond
occurs between the carbonyl group of the inhibitor and the
hydroxy moiety of TYR444. The second hydrogen bond forms
between the hydroxyl moiety of the 5c and TYR407 side chain
hydroxyl group. The other strong electrostatic interactions
occur between GLN215 and the chloride atom on the phenyl
ring of 5c and the hydroxyl group of the 5c and backbone
carbonyl of ILE180. The binding mode adopted by 5c fits
snugly within a cavity lined with hydrophobic amino acid
residues. This hydrophobic pocket includes PHE352, ILE180,
ILE335, LEU337, GLY214, PHE208, and ILE207 amino acids.
Figure 1c and d shows the binding pose (3D and 2D) of 5c in
the active site of the MAO-B isoform. 5c occupies a space far
from the hydrophobic cage, surrounded by TYR398, TYR435,
and FAD co-factor, near the entrance cavity. The major
interaction is the hydrogen bond that forms between the
hydroxyl group of 5c and the side chain residue of CYS172.
The other hydrophobic amino acid residues are PHE168,
LEU171, TYR326, ILE198, PHE103, GLN206, ILE198, ILE199,
PRO102, TRP119, and LEU164 amino acids.
Table 3. Reversibility of hMAO inhibition with the
compounds 3f, 5c, 6i, and moclobemide.
hMAO-A inhibition (%)
Before
washing
After
washing
Compounds
Moclobemide (0.30 mM)
Compound 3f (0.30 mM)
Compound 5c (0.30 mM)
Compound 6i (0.30 mM)
90.00 ꢁ 5.02
81.80 ꢁ 0.45
89.95 ꢁ 4.55
83.26 ꢁ 4.11
8.22 ꢁ 0.50
12.00 ꢁ 1.10
8.14 ꢁ 0.61
10.96 ꢁ 0.95
Each value represents the mean ꢁ SEM of three experiments.
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Figure 2(a and b) shows the 3D and 2D pictures of 6i in the
active site of the hMAO-A isoform. The bromophenyl ring was
inserted between the TYR444 and TYR407 residues, which
constitute the hydrophobic cage of the active site, making
one p–p interaction with TYR407 and TYR444. The methyl-
phenyl ring of 6i makes two more p–p interactions, one with
PHE208 and the other with LEU337. The 4-methyl-1,2,3-
thiadiazole-5-yl ring also makes one p–s with FAD and two
electrostatic interactions with LYS305. Tyr69, Phe352, Ile335,
Gln215, and ASP339 are the other active site residues involved
in hydrophobic and polar interactions with the inhibitor.
Figure 2c and d shows the binding pattern of 6i to hMAO-B.
The inhibitor is located near the hydrophobic cage sur-
rounded by Tyr398, Tyr435, and FAD. Ile171, ILE316, LEU88,
THR314, CYS172, PRO102, THR201, ILE198, ILE199, GLN201,
TYR326, and THR201 are the other active site residues
interacting with the inhibitor. The potency of the both 5c
and 6i molecules on hMAO-A compared to hMAO-B can be
evaluated from the above data, indicating that 5c is stabilized
by various p–p interactions in hMAO-A compared to hMAO-B.
All of these data may suggest why the hMAO-A inhibitory
potency of 5c (Ki ¼ 0.115 mM) is better than that for hMAO-B
(0.13 mM). Inhibitor 6i also more effectively binds to the
hMAO-A isoform than to the MAO-B isoform [Selectivity
Index (SI) ¼ Ki(hMAO-B)/Ki(hMAO-A), 5c ¼ 1.13 and 6i ¼ 2.33].
Compounds 6h and 6i are positional isomers in which a
methyl group is at different positions (6h has it on R7 while 6i
has it on R6). This single variation could be able to make the
molecules behave differently (Table 1); 6h is selective toward
MAO-B while 6i is selective toward MAO-A. Compound 6h has
a methyl group at the p-position of the phenyl ring, which
makes favorable hydrophobic interactions with Lys305,
Phe353 residues as well as three more interactions with the
FAD of MAO-A. These interactions force the phenyl ring to go
into the hydrophobic pocket exactly between the Phe352 and
Tyr69 residues. In the same manner, in MAO-B, the methyl
group interacts with Phe343, Tyr60 residues, and co-factor
FAD. On the other hand, when the methyl group is in m-
position of compound 6i, a completely different binding pose
is observed. In the MAO-A binding site, 6i is flipped, forcing
the methyl group to interact with Phe208, Ile335, and Leu337.
In MAO-B m-methyl group makes another interaction with
Leu88 and Tyr326 located in the entrance cavity hindering
interaction with the FAD co-factor in the hydrophobic pocket.
That might be the reason for 6h to be selective MAO-B and 6i
selective MAO-A inhibitors. Details of the interactions of
the compounds 6h and 6i are given in the Supporting
Information.
chalcone derivatives have been previously reported to exhibit
various pharmacological activities including MAO inhibitory
activities [16–26]. The necessity of discovering new, selective,
and reversible hMAO inhibitors directed us to synthesize
novel chalcone derivatives as more selective, potent, and
reversible MAO inhibitors. Thus, in the present study, we
synthesized new compounds containing 2-pyrazoline or
hydrazone and investigated their hMAO inhibitory potencies.
Conventional methods were used for the synthesis of
chalcones, pyrazolines, and hydrazones in our study. These
methods have been previously reported by different
researches and shown to give satisfactory results [31, 32].
MAO activities were determined by a fluorimetric method
previously reported using recombinant hMAO-A and -B as
enzyme sources [33–35]. We used p-tyramine as a substrate
in the activity measurements as it is a common substrate for
both MAO-A and -B which caused no interference in our
experimental set. The possible capacity of the new com-
pounds to quench the fluorescence generated in the reaction
mixture was determined by adding these compounds to
solutions containing only the Amplex Red reagent or
resorufin in a sodium phosphate buffer [36]. The new
compounds and reference inhibitors themselves did not
react directly with Amplex1 Red reagent or did not effect
the fluorescence generated in the reaction mixture. Newly
synthesized compounds did not cause any inhibition on the
activity of HRP in the test medium.
AutoDock 4.2 docking program which was the most used
program among the community was used to investigate the
molecular mechanisms of the interaction of newly synthesized
compounds with hMAO. AutoDock 4.2 has semiempirical
scoring function, which was calibrated against experimentally
dependable inhibition values and it was shown to produce the
least error and artifacts comparing the other programs [37].
The newly synthesized compounds were found to be mostly
competitive, reversible, and selective inhibitors of hMAO-A.
2-Pyrazoline compounds showed higher selectivity than
hydrazones. The data indicated that 2-hydroxy-3,5-dichloro
phenyl and 4-methoxy-1-phenyl rings designed as A and C
rings, respectively, increased the inhibitory potency for
hMAO-A in the series of 5a–j. Compounds 5a and 5e in this
series exhibited higher inhibition potency for hMAO-A than
moclobemide. hMAO-A inhibitory potency of compound 5a
was found to be higher than that of compound 5e suggesting
that methyl substitution on the R6 position of B ring decreases
the inhibition potency and selectivity for hMAO-A.
Replacement of A ring by 2-hydroxy-3,5-dichlorophenyl
ring and addition of pyridine, phenyl, or 4-methyl-1,2,3-
thiadiazole-5-yl rings to the scaffold as a C ring decreased
the potency of inhibition. Addition of 2-furyl to the scaffold
as the C ring decreased the potency, but increased the
selectivity. Among the hydrazones, compounds 6a, 6d, and 6i
appeared to be the most potent compounds. These deriva-
tives showed higher inhibitory potency but lower selectivity
for hMAO-A than moclobemide. Data indicated that a
4-methyl-1,2,3-thiadiazole-5-yl ring as the C ring is essential
Conclusion
Chalcones are intermediates for synthesizing various hetero-
cyclic compounds which have been reported to possess
various biological activities [6–15]. 2-Pyrazoline and hydrazone
derivatives which have been synthesized by starting from
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for inhibitory potency and selectivity for hMAO-A in the
hydrazone series. Replacement of the thiophene-2-yl or
furan-2-yl ring by 4-methyl-1,2,3-thiadiazole-5-yl ring in-
creased the potency. When A ring has a 2,4-dimethoxy
scaffold, replacement of 2-furyl by the thiophene-2-yl
increased the potency and selectivity. Replacement of the
fluorine by a bromine atom on the third position of A ring
decreased the potency and the selectivity of inhibition.
Biological experiments were carried out using only racemic
mixtures of compounds 5a–j. The calculated Ki values (Table 2),
at least for one of the enantiomers, agreed with the
experimentally determined values (Table 1). Overall, the
docking studies provided valuable data for the rationalization
of the experimental results, and allowed us to estimate the
binding mode, the inhibition constant, and the free energy of
binding, suggesting that molecular docking is one of the
promising tools for use in the discovery of novel, potent, and
selective MAO inhibitors potentially useful as pharmacologi-
cal agents.
(E)-1-(3,5-Dichloro-2-hydroxyphenyl)-3-p-tolylprop-2-en-
1-one (3a)
Orange powder (ethyl alcohol). Yield: 57.5%, m.p. 138°C
[lit. 115°C] [39]. C16H12Cl2O2.
(E)-1-(3,5-Dichloro-2-hydroxyphenyl)-3-m-tolylprop-2-en-
1-one (3b)
Yellow-orange powder (ethyl alcohol). Yield: 73.80%, m.p.
ꢀ1
–
–
103°C. IR (KBr): n ¼ 3430 (OH), 1711 (C O) cm
.
1H NMR
(CDCl3, 400 MHz): d ¼ 2.42 (s, 3H, –CH3), 7.26–7.50 (4H,
aromatic-H), 7.52 (d, 1H, J ¼ 15.2 Hz, a-ethylenic-H), 7.58 (d,
1H, J ¼ 2 Hz, 40-H), 7.82 (d, 1H, J ¼ 2 Hz, 60-H), 7.96 (d, 1H,
J ¼ 15.2 Hz, b-ethylenic-H), 13.4 (s, 1H, –OH). 13C NMR (DMSO-
d6, 100 MHz): d ¼ 20.80 (–CH3), 120.57, 122.03, 122.54, 122.83,
127.09, 128.82, 128.85, 129.83, 132.19, 134.09, 135.17, 138.26,
þ
–
147.09, 156.63, 192.86 (C O). MS, ESI: m/z ¼ 307 [MþH]
–
(100%), 309 [MþHþþ2]þ (66%), 311 [MþHþþ4]þ (12%).
C
16H12Cl2O2: C 62.20, H 3.93; calcd. C 62.56, H 3.94.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(4-methoxyphenyl)-
prop-2-en-1-one (3c)
Orange powder (ethyl alcohol). Yield: 90.75%, m.p. 113°C
[lit. 104°C] [40]. C16H13BrO3.
Experimental
Chemistry
Melting points were determined with an Electrothermal
9100 type melting point apparatus (Engineering Ltd., Essex,
UK), and are uncorrected. All instrumental analyses were
performed at the Central Laboratory of the Faculty of
Pharmaceuticals of Ankara University, Ankara, Turkey. IR
spectra were recorded on a Jasco FT/IR-420 spectrometer
(Jasco, Easton, MD, USA). 1H NMR and 13C NMR spectra
were measured with a Varian Mercury 400 MHz FT-NMR
spectrometer (Varian Inc., Palo Alto, CA, USA) in DMSO-d6.
All chemical shifts were reported as d (in ppm) values.
Mass spectra were recorded on a Waters Micromass ZQ
instrument (Waters Corporation, Milford, MA, USA). Ele-
mentary analyses were performed on a Leco CHNS 932
analyzer (Leco, St. Joseph, MI, USA), and satisfactory results
ꢁ 0.4% of calculated values (C, H, N) were obtained.
Reactions were followed and purity of compounds was
assessed by thin-layer chromatography (TLC) on silica
gel-protected aluminum sheets (Type 60 GF254; Merck,
Darmstadt, Germany), and spots were detected by exposure
to a UV lamp at 254 nm for a few seconds. The chemical
reagents used in syntheses were purchased from Merck and
Aldrich (Milwaukee, MI, USA).
(E)-1-(2-Hydroxy-4-methoxyphenyl)-3-(4-methoxyphenyl)-
prop-2-en-1-one (3d)
Yellow powder (ethyl alcohol). Yield: 76%, m.p. 90.1°C
[lit. 106–108°C] [41]. C17H16O4.
(E)-1-(2-Hydroxy-4-methoxyphenyl)-3-(2-methoxyphenyl)-
prop-2-en-1-one (3e)
Yellow powder (ethyl alcohol). Yield: 94.63%, m.p. 95.40°C
[lit. 92°C] [42]. C17H16O4.
(E)-1-(5-Chloro-2-hydroxyphenyl)-3-(4-methoxyphenyl)-
prop-2-en-1-one (3f)
Yellow powder (ethyl alcohol). Yield: 84.20%, m.p. 106.50°C
[lit.104°C] [43]. C16H13ClO3.
(E)-1-(2,4-Dimethoxyphenyl)-3-p-tolylprop-2-en-1-one
(3g)
Yellow powder (ethyl alcohol). Yield: 55.60%, m.p. 118°C
[lit. 117°C] [44]. C18H18O3.
(E)-1-(4-Fluorophenyl)-3-(4-methoxyphenyl)prop-2-en-1-
one (3h)
White crystals (ethyl alcohol). Yield: 97.70%, m.p. 107.2°C
General procedure for the preparation of the chalcone
derivatives 3a–l
[lit. 98°C] [45]. C16H13FO2.
To a stirred solution of KOH (50%) in water (5 mL) cooled to
0°C in an ice bath was added dropwise a solution of
acetophenone (4.99 mmol) and aldehyde (6.018 mmol) in
ethanol (20 mL). The reaction mixture was stirred at room
temperature through night. Then the mixture was poured
into ice, adjusted to pH 3–4 with 1N HCl, and then filtered.
The resultant was crystallized from ethanol [38].
(E)-1-(4-Fluorophenyl)-3-p-tolylprop-2-en-1-one (3i)
White crystals (ethyl alcohol). Yield: 98.00%, m.p. 142.4°C
[lit. 135–137°C] [46]. C16H13FO.
(E)-1-(4-Fluorophenyl)-3-m-tolylprop-2-en-1-one (3j)
White crystals (ethyl alcohol). Yield: 88.10%, m.p. 86°C.
1H NMR d ppm (CDCl3, 400 MHz): d ¼ 2.41 (s, 3H, –CH3),
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7.16–7.34 (m, 4H, 2,4,5,6-H), 7.44–7.51 (3H, 30,50-H; J ¼ 15.6 Hz,
a-ethylenic-H), 7.80 (d, 1H, J ¼ 16 Hz, b-ethylenic-H), 8.06 (t,
2H, 20,60-H). MS (ESI): m/z ¼ 241 [MþH]þ (100%). C16H13FO.
(3-(3,5-Dichloro-2-hydroxyphenyl)-5-m-tolyl-4,5-
dihydropyrazole-1-yl)(furane-2-yl)methanone (5c)
White powder (ethyl alcohol). Yield: 36.74%, m.p. 235.2°C.
ꢀ1
–
–
–
IR (KBr): n ¼ 3420 (OH), 1646 (amide C O), 1558 (C N) cm
.
–
(E)-1-(4-Bromophenyl)-3-p-tolylprop-2-en-1-one (3k)
Yellow powder (ethyl alcohol). Yield: 94.70%, m.p. 167.6°C
[lit. 163–164] [47]. C16H13FO.
1H NMR (DMSO-d6, 400 MHz): d ¼ 2.36 (s, 3H, –CH3), 2.95 (dd,
1H, J1 ¼ 12.8 Hz, J2 ¼ 16.8 Hz, HA), 3.53 (dd, 1H, HB), 5.37 (dd,
1H, J1 ¼ 2.8 Hz, J2 ¼ 12 Hz, HX), 6.71–7.95 (9H, aromatic-H),
10.97 (s, 1H, –OH). MS (ESI): m/z ¼ 415 [MþH]þ (100%), 417
[MþHþ2]þ (66%), 419 [MþHþ4]þ (12%). C21H16Cl2N2O3: C
60.70, H 3.63, N 6.98; calcd. C 60.74, H 3.88, N 6.75.
(E)-1-(4-Bromophenyl)-3-m-tolylprop-2-en-1-one (3l)
Cream powder (ethyl alcohol). Yield: 97.90%, m.p. 129.5°C.
1H NMR (CDCl3, 400 MHz): d ¼ 2.40 (s, 3H, –CH3), 7.23–7.48
(5H, 2,4,5,6-H; J ¼ 15.6 Hz, a-ethylenic-H), 7.64 (d, 2H, A2X2,
30,50-H), 7.79 (d, 1H, J ¼ 16 Hz, b-ethylenic-H), 7.90 (d, 2H,
A2X2, 20,60-H). 13C NMR (DMSO-d6, 100 MHz): d ¼ 20.84 (–CH3),
121.46, 126.44, 127.28, 128.80, 129.21, 130.54, 131.49,
(3-(3,5-Dichloro-2-hydroxyphenyl)-5-m-tolyl-4,5-
dihydropyrazole-1-yl)(phenyl)methanone (5d)
Yellow powder (ethyl alcohol). Yield: 26%. M.p. 250.2°C. IR (KBr):
n¼ 3424 (OH), 1642 (amide C¼O), 1574 (C¼N) cmꢀ1
.
1H NMR
–
131.83, 134.46, 136.53, 138.17, 144.68, 188.28 (C O). MS
(DMSO-d6, 400 MHz): d¼ 2.33 (s, 3H, –CH3), 2.93 (dd, 1H, J1 ¼ 11.6
Hz, J2 ¼ 16.8 Hz, HA), 3.55(dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 17.2 Hz, HB),5.34
(dd, 1H, J1 ¼ 2.8 Hz, J2 ¼ 12.4 Hz, HX), 7.19–7.94 (11H, aromatic-H),
11.00 (s, 1H, OH). MS (ESI): m/z ¼ 447 [MþNa]þ (100%), 449
[MþNaþ2] (66%), 451 [MþNaþ4] (12%). C23H18Cl2N2O2 ꢃ 0.25
H2O: C 64.34, H 4.37, N 6.78; calcd. C 64.41, H 4.32, N 6.53.
–
(ESI): m/z ¼ 301 [MþH]þ (97%), 303 [MþHþ2]þ (100%).
C
16H13BrO.
General procedure for the preparation of compounds 5a–j
and 6a–i
A mixture of chalcone (1.42 mmol) and hydrazide derivatives
(1.42 mmol) was refluxed in ethanol (20 mL) for 36–80 h. The
precipitate was filtered and recrystallized from ethanol.
(3-(3,5-Dichloro-2-hydroxyphenyl)-5-m-tolyl-4,5-
dihydropyrazole-1-yl)(4-methoxyphenyl)methanone (5e)
Yellow powder (ethyl alcohol). Yield: 37.95%, m.p. 220°C.ꢀI1R
–
–
–
(3-(3,5-Dichloro-2-hydroxyphenyl)-5-p-tolyl-4,5-
dihydropyrazole-1-yl)(4-methoxyphenyl)methanone (5a)
Yellow powder (ethyl alcohol). Yield: 67%, m.p. 238.9°C.
(KBr): n ¼ 3420 (OH), 1637 (amide C O), 1567 (C N) cm
.
–
1H NMR (DMSO-d6, 400MHz): d¼ 2.36 (s, 3H, –CH3), 2.95 (dd, 1H,
J1 ¼ 12 Hz, J2 ¼ 16.8 Hz, HA),3.57(dd, 1H,J1 ¼ 2.4 Hz, J2 ¼ 17.2 Hz,
HB),3.82(s,3H,–OCH3),5.36(dd,1H,J1 ¼ 2.8 Hz, J2 ¼ 12.4 Hz,HX),
7.03 (d, 2H, J ¼ 8.8 Hz, A2X2, C ring H3, H5), 7.23 (s, 1H, A ring H4)
7.34–7.39 (3H, B ring-H), 7.70 (s, 1H, J ¼ 2.4 Hz, A ring H6), 7.87 (d,
2H, J ¼ 8.8 Hz, A2X2, C ring H2, H6), 7.90 (s, 1H, B ring H), 10.91 (s,
1H, –OH). 13C NMR (DMSO-d6, 100 MHz): d ¼ 20.99 (–CH3), 31.48,
55.35, 77.37, 113.39, 122.46, 122.78, 123.26, 123.52, 125.09,
125.57, 126.91, 128.41, 129.07, 130.13, 137.68, 138.83, 151.16,
161.94.MS(ESI):m/z ¼ 455 [MþH]þ (30%), 477 [MþNa]þ (100%),
479 [MþNaþ2]þ (66%), 481 [MþNaþ4]þ (12%). C24H20Cl2N2O3:
C, 63.20, H 4.47, N 6.41; calcd. C 63.31, H4.43, N 6.15.
ꢀ1
–
–
IR (KBr): n ¼ 3440 (OH), 1636 (amide C O), 1600 (C N) cm
.
–
–
1H NMR (DMSO-d6, 400 MHz): d ¼ 2.34 (s, 3H, –CH3), 2.98 (dd,
1H, J1 ¼ 11.6 Hz, J2 ¼ 17.2 Hz, HA), 3.55 (dd, 1H, J1 ¼ 2.8 Hz,
J2 ¼ 16.8 Hz, HB), 3.82(s, 3H, –OCH3), 5.37 (dd, 1H, J1 ¼ 2.8 Hz,
J2 ¼ 11.6 Hz, HX), 7.03 (d, 2H, J ¼ 8.8 Hz, A2X2, C ring H3, H5),
7.27 (d, 2H, J ¼ 7.6 Hz, A2X2, B ring H2, H6), 7.45 (d, 2H, B ring
H3, H5), 7.69 (s, 1H, A ring H4), 7.87 (d, 2H, J ¼ 8.4 Hz, A2X2,
C ring H2, H6), 7.93 (s, 1H, A ring H6), 10.92 (s, 1H, –OH).
13C NMR (DMSO-d6, 100 MHz): d ¼ 20.68 (–CH3), 31.24,
55.36, 77.16, 113.41, 122.45, 122.78, 123.28, 125.07,
125.58, 126.36, 129.01, 130.13, 135.88, 137.85, 151.13,
161.95. MS (ESI): m/z ¼ 455 [MþH]þ (20%), 477 [MþNa]þ
(100%), 479 [MþNaþ2]þ (66%), 481 [MþNaþ4]þ (12%).
(3-(3,5-Dichloro-2-hydroxyphenyl)-5-m-tolyl-4,5-
dihydropyrazole-1-yl)(4-methyl-1,2,3-thiadiazole-5-yl)-
methanone (5f)
C
24H20Cl2N2O3 ꢃ 0.25 H2O: C 62.79, H 4.25, N 6.33; calcd. C
62.81, H 4.47.
Yellow powder (ethyl alcohol). Yield: 45.10%, m.p. 250°C.ꢀI1R
–
–
–
(KBr): n ¼ 3410 (OH), 1652 (amide C O), 1576 (C N) cm
.
–
(3-(3,5-Dichloro-2-hydroxyphenyl)-5-m-tolyl-4,5-
dihydropyrazole-1-yl)(pyridine-4-yl)methanone (5b)
White powder (ethyl alcohol). Yield: 38.6%, m.p. 219.1°C.
1H NMR (DMSO-d6, 400 MHz): d ¼ 2.36 (s, 3H, –CH3), 2.95–3.02
(4H, C ring CH3 and HA), 3.62 (dd, 1H, J1 ¼ 3.2 Hz, J2 ¼ 17.2 Hz,
HB), 5.42 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 11.6 Hz, HX), 7.22–7.36 (4H,
B ring-H), 7.77 (d, 2H, A ring-H), 11.78 (s, 1H, –OH). MS (ESI):
m/z ¼ 445 [MꢀH]þ (99%), 447 [M–Hþ2]þ (66%), 449 [M–Hþ4]
(12%). C20H16Cl2N4O2S: C 53.51, H 3.38, N 12.63, S 7.21; calcd.
C 53.70, H 3.61, N 12.52, S 7.17.
ꢀ1
–
–
IR (KBr): n ¼ 3446 (OH), 1652 (amide C O), 1558 (C N) cm
.
–
–
1H NMR (DMSO-d6, 400 MHz): d ¼ 2.35 (s, 3H, –CH3),
2.96 (dd, 1H, J1 ¼ 12.4 Hz, J2 ¼ 16.8 Hz, HA), 3.57 (dd, 1H,
J1 ¼ 2.8 Hz, J2 ¼ 17.2 Hz, HB), 5.37 (dd, 1H, J1 ¼ 2.8 Hz,
J2 ¼ 12 Hz, HX), 7.22–7.96 (8H, aromatic-H), 8.75 (d, 2H,
pyridine H2, H6), 11.24 (s, 1H, –OH). MS (ESI): m/z ¼ 426
[MþH]þ (100%), 428 [MþHþ2]þ (66%), 430 [MþHþ4]þ
(12%). C22H17Cl2N3O2 ꢃ 0.25 H2O: C 61.53,H 4.22, N 9.90;
calcd. C 61.47, H 4.07, N 9.78.
(3-(2-Hydroxy-4-methoxyphenyl)-5-(2-methoxyphenyl)-
4,5-dihydropyrazole-1-yl)(phenyl)methanone (5g)
White powder (ethyl alcohol). Yield: 18%, m.p. 220°C. IR (KBr):
ꢀ1
n ¼ 3450 (OH), 1663 (amide C O), 1599 (C N) cm
.
1H NMR
–
–
–
–
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Arch. Pharm. Chem. Life Sci. 2015, 348, 743–756
Monoamine Oxidase Inhibitors
Archiv der Pharmazie
(DMSO-d6, 400 MH): d ¼ 2.83 (dd, 1H, HA), 3.32 (dd, 1H, HB),
3.78 (d, 3H, –OCH3), 3.82 (s, 3H, –OCH3), 5.43 (dd, 1H,
J1 ¼ 2.4 Hz, J2 ¼ 12.8 Hz, HX), 6.56–7.98 (12H, aromatic-H),
10.81 (s, 1H, –OH). MS (ESI): m/z ¼ 403 [MþH]þ (100%).
–OCH3), 3.87 (s, 3H, –OCH3), 6.36 (d, 1H, J ¼16 Hz, a-ethylenic-
H), 6.73 (d, 1H, A ring H5), 6.78 (s, 1H, A ring H3), 7.10–7.43 (7H,
B ring H3 and H5, B ring H2 and H6, A ring H6, b-ethylenic-H,
C ring-H), 7.93 (s, 2H, C ring –H), 9.51 (s, 1H, –NH). MS (ESI):
m/z ¼ 407 [MþH]þ (100%). C23H22N2O3S ꢃ 0.25 H2O: C 67.12,
H 5.38, N 7.03, S 7.74; calcd. C 67.24, H 5.48, N 6.82, S 7.79.
C
24H22N2O4: C 71.28, H 5.31, N 7.14; calcd. C 71.63, H 5.51,
N 6.96.
(3-(2-Hydroxy-4-methoxyphenyl)-5-(4-methoxyphenyl)-
4,5-dihydropyrazole-1-yl)(4-methoxyphenyl)methanone
(5h)
N0-(1-(2,4-Dimethoxyphenyl)-3-p-tolylallylidene)furane-2-
carbohydrazide (6b)
White powder (ethyl alcohol). Yield: 51.85%, m.p. 188ꢀ.51.
–
–
–
White powder (ethyl alcohol). Yield: 21%, m.p. 207.6°C.
IR (KBr): n ¼ 3348 (NH), 1694 (amide C O), 1607 (C N) cm
.
–
ꢀ1
–
–
IR (KBr): n ¼ 3439 (OH), 1630 (amide C O), 1604 (C N) cm
.
1H NMR (DMSO-d6, 400 MHz): d ¼ 2.28 (s, 3H, –CH3), 3.77 (s, 3H,
–OCH3), 3.87 (s, 3H, –OCH3), 6.34 (d, 1H, J ¼ 16 Hz, a-ethylenic-
H), 6.65 (s, 1H, A ring H3), 6.76 (d, 1H, A ring H5), 6.82 (1H,
C ring –H), 7.14–7.22 (5H, B ring H3 and H5, b-ethylenic-H,
A ring H6, C ring –H), 7.41 (d, 2H, B ring H2 and H6), 7.83 (1H,
C ring –H), 9.25 (s, 1H, –NH). 13C NMR (DMSO-d6, 100 MHz):
d ¼ 20.83, 55.43, 55.80, 99.33, 106.37, 109.51, 110.52, 112.33,
127.02, 127.85, 130.71, 133.05, 136.27, 138.29, 145.99, 157.30,
162.07. MS (ESI): m/z ¼ 391 [MþH]þ (30%), 413 [MþNa]þ
(100%). C23H22N2O4: C 70.74, H 5.27, N 7.21; calcd. C 70.75,
H 5.68, N 7.17.
–
–
1H NMR (DMSO-d6, 400 MHz): d ¼ 2.85 (dd, 1H, J1 ¼ 12 Hz,
J2 ¼ 16.8 Hz, HA), 3.32 (dd, 1H, HB), 3.77 (s, 6H, A and B ring
–OCH3), 3.81 (s, 3H, C ring –OCH3), 5.18 (dd, 1H, J1 ¼ 2.4 Hz,
J2 ¼ 12.4 Hz, HX), 6.55–7.93 (11H, aromatic-H), 10.66 (s, 1H,
–OH). MS (ESI): m/z ¼ 433 [MþH]þ (100%), 455 [MþNa]þ
(100%). C25H24N2O5 ꢃ 0.25 H2O: C 68.41, H 5.76, N 6.38; calcd. C
68.73, H 5.61, N 6.41.
(3-(5-Bromo-2-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-
dihydropyrazole-1-yl)(4-methoxyphenyl)methanone (5i)
White powder (ethyl alcohol). Yield: 24.19%, m.p. 258.6°C.
ꢀ1
IR (KBr): n ¼ 3410 (OH), 1626 (amide C O), 1515 (C N) cm
.
N0-(1-(4-Fluorophenyl)-3-(4-methoxyphenyl)allylidene)-
thiophene-2-carbohydrazide (6c)
–
–
–
–
1H NMR (DMSO-d6, 400 MHz): d ¼ 2.92 (dd, 1H, HA), 3.46 (dd,
1H, J1 ¼ 2.8 Hz, J2 ¼ 17.2 Hz, HB), 3.78 (s, 3H, B ring –OCH3), 3.82
(s, 3H, C ring –OCH3), 5.22 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 12 Hz, HX),
6.97–7.03 (m, 5H, A ring H3, B ring H3 and H5, C ring H3 and H5),
7.49 (d, 2H, B ring H2 and H6, s, 1H, A ring H4), 7.86 (d, 2H, C
ring H2 and H6), 8.11 (s, 1H, A ring H6), 10.83 (s, 1H, –OH).
13C NMR (DMSO-d6, 100 MHz): d ¼ 31.75, 37.28, 55.12, 55.32,
76.44, 112.96, 113.36, 113.75, 120.05, 122.29, 125.65, 126.48,
128.00, 129.99, 131.28, 133.48, 155.99, 159.28, 161.85. MS
(ESI): m/z ¼ 481 [MþH]þ (20%), 483 [MþHþ2]þ (30%), 503
[MþNa]þ (95%), 505 [MþNaþ2]þ (100%). C24H21BrN2O4 ꢃ 0.5
H2O: C 58.71, H 4.18, N 5.96; calcd. C 58.89, H 4.49, N 5.72.
Yellow powder (ethyl alcohol). Yield: 21%, m.p. 197.2.
ꢀ1
–
–
–
–
IR (KBr): n ¼ 1633 (amide C O), 1602 (C N) cm
.
1H NMR
(DMSO-d6, 400 MHz): d ¼ 3.80 (s, 3H, –OCH3), 6.76 (d, 1H,
J ¼ 16 Hz, a-ethylenic-H), 6.98 (d, 2H, B ring H3 and H5), 7.18
(broad t, 1H, C ring –H), 7.34 (t, 2H, A ring H3 and H5), 7.69
(t, 5H, A ring H2 and H6, B ring H2 and H6, b-ethylenic-H), 7.88
(d, 1H, C ring –H), 8.04 (d, 1H, C ring –H), 11.60 (s, 1H, –NH).
MS (ESI): m/z ¼ 381 [MþH]þ (100%). C21H17FN2O2S: C 66.42,
H 4.43, N 7.59, S 8.28; calcd. C 66.30, H 4.50, N 7.36, S 8.43.
N0-(1-(4-Fluorophenyl)-3-(4-methoxyphenyl)allylidene)-4-
methyl-1,2,3-thiadiazole-5-carbohydrazide (6d)
(3-(5-Chloro-2-hydroxyphenyl)-5-(4-methoxyphenyl)-4,5-
dihydropyrazole-1-yl)(thiophene-2-yl)methanone (5j)
Yellow powder (ethyl alcohol). Yield: 74.74%, m.p. 279.8°C.
Yellow powder (ethyl alcohol). Yield: 52.31%, ꢀm1 .p. 206.4.
.
1H NMR
–
–
–
–
IR (KBr): n ¼ 1677 (amide C O), 1597 (C N) cm
(DMSO-d6, 400 MHz): d ¼ 2.99 (s, 3H, –CH3), 3.81 (s, 3H, –OCH3),
6.81 (d, 1H, J ¼ 16 Hz, a-ethylenic-H), 7.00 (d, 2H, B ring H3 and
H5), 7.41 (t, 2H, A ring H3 and H5), 7.62–7.72 (m, 5H, B ring
H2 and H6, A ring H2 and H6, b-ethylenic-H), 12.25 (s, 1H, –NH).
13C NMR (DMSO-d6, 100 MHz): d ¼ 15.13, 55.24, 114.14,
115.31, 115.53, 115.71, 128.07, 129.79, 131.67, 131.76,
132.19, 132.23, 135.14, 140.97, 150.64, 160.60, 161.13,
161.51, 163.06, 163.95. MS (ESI): m/z ¼ 395 [MꢀH]þ (100%).
ꢀ1
–
–
IR (KBr): n ¼ 3445 (OH), 1617 (amide C O), 1558 (C N) cm
.
–
–
1H NMR (DMSO-d6, 400 MHz): d ¼ 2.92 (dd, 1H, J1 ¼ 12.4 Hz,
J2 ¼ 17.2 Hz, HA), 3.62 (dd, 1H, HB), 3.80 (s, 3H, –OCH3), 5.26
(dd, 1H, HX), 7.00 (d, 1H, A ring H3), 7.06 (d, 2H, B ring H3 and
H5), 7.24 (s, 1H, thiophene H4), 7.40 (dd, 1H, J1 ¼ 2.8 Hz,
J2 ¼ 8.4 Hz, A ring H4), 7.47 (d, 2H, B ring H2 and H6), 8.04 (s, 3H,
A ring H6, thiophene H3and H5), 11.20 (s, 1H, –OH). MS (ESI):
m/z¼ 413 [MþH]þ (20%), 435 [MþNa]þ (100%), 437 [MþNaþ2]
(33%). C21H17ClN2O3S: C 61.10, H 4.12, N 6.86, S 7.74; calcd. C
61.09, H 4.15, N 6.78, S 7.76.
C
20H17FN4O2S ꢃ 0.25 H2O: C 60.09, H 4.31, N 14.16, S 8.14; calcd.
C 59.93, H 4.37, N 13.98,S 7.99.
N0-(1-(4-Fluorophenyl)-3-p-tolylallylidene)thiophene-2-
carbohydrazide (6e)
N’-(1-(2,4-Dimethoxyphenyl)-3-p-tolylallylidene)-
thiophene-2-carbohydrazide (6a)
Yellow powder (ethyl alcohol). Yield: 28.81%, ꢀm1 .p. 198.3.
.
1H NMR
–
–
–
Yellow powder (ethyl alcohol). Yield: 23.00%, m.p. 178ꢀ.21.
IR (KBr): n ¼ 1652 (amide C O), 1558 (C N) cm
–
–
–
–
IR (KBr): n ¼ 3420 (NH), 1631 (amide C O), 1505 (C N) cm
.
(DMSO-d6, 400 MHz): d ¼ 3.32 (s, 3H, –CH3), 6.78 (d, 1H,
J ¼ 16 Hz, a-ethylenic-H), 7.18 (t, 1H, C ring –H), 7.23 (d, 2H,
–
1H NMR (DMSO-d6, 400 MHz): d ¼ 2.30 (s, 3H, –CH3), 3.77 (s, 3H,
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B. Evranos-Aksoz et al.
€
Archiv der Pharmazie
B ring H3 and H5), 7.34 (t, 2H, A ring H3 and H5), 7.62–
7.69 (m, 5H, B ring H2 and H6, b-ethylenic-H, A ring H2
and H6), 7.89 (d, 1H, C ring –H), 8.04 (d, 1H, C ring –H),
11.6 (s, 1H, –NH). MS (ESI): m/z ¼ 365 [MþH]þ (100%).
C 54.22, H 3.88, N 12.65, S 7.27; calcd. C 54.43, H 3.88, N 12.69,
S 7.26.
Biochemistry
Chemicals
C
21H17FN2OS: C 68.97, H 4.74, N 7.80, S 8.73; calcd. C
69.21, H 4.70, N 7.69.
hMAO-A and hMAO-B (both recombinant, expressed in
baculovirus-infected BTI insect cells), R-(ꢀ)-deprenyl hydro-
chloride (selegiline), moclobemide, resorufin, dimethyl sulf-
oxide (DMSO), and other chemicals were purchased from
Sigma–Aldrich (Munich, Germany). The Amplex1-Red MAO
assay kit (Cell Technology Inc., Mountain View, CA, USA)
contained benzylamine, p-tyramine, clorgyline (MAO-A in-
hibitor), pargyline (MAO-B inhibitor), and horseradish
peroxidase.
N0-(1-(4-Fluorophenyl)-3-m-tolylallylidene)thiophene-2-
carbohydrazide (6f)
Yellow powder (ethyl alcohol). Yield: 33.17%, ꢀm1 .p. 176.2.
.
1H NMR
–
–
–
–
IR (KBr): n ¼ 1630 (amide C O), 1558 (C N) cm
(DMSO-d6, 400 MHz): 2.34 (s, 3H, –CH3), 6.78 (d, 1H, J ¼16 Hz,
a-ethylenic-H), 7.19–7.8 (m, 10H, aromatic and b-ethylenic-H),
7.90 (dd, 1H, C ring –H), 8.05 (dd, 1H, C ring –H), 11.69 (s, 1H,
–NH). MS (ESI): m/z ¼ 364 [MþH]þ (50%), 387 [MþNa]þ
(100%). C21H17FN2OS: C 69.12, H 4.58, N 7.76, S 8.80; calcd.
C 69.21, H 4.70, N 7.69, S 8.80.
Determination of hMAO-A and -B activities
The activities of hMAO-A and hMAO-B were determined
using p-tyramine as common substrate and calculated as
161.50 ꢁ 8.33 pmol/mg/min (n ¼ 3) and 140.00 ꢁ 7.55 pmol/
mg/min (n ¼ 3), respectively. The interactions of the synthe-
sized compounds with hMAO isoforms were determined by a
fluorimetric method described and modified previously [33–-
35]. The production of H2O2 catalyzed by MAO isoforms was
detected using Amplex1-Red reagent, a non-fluorescent
probe that reacts with H2O2 in the presence of horseradish
peroxidase to produce the fluorescent product resorufin. The
reaction was started by adding 200 mM Amplex Red reagent,
1 U/mL horseradish peroxidase, and p-tyramine (concentra-
tion range 0.1–1mM). Control experiments were carried out
by replacing the compound and reference inhibitors. The
possible capacity of compounds to modify the fluorescence
generated in the reaction mixture due to non-enzymatic
inhibition was determined by adding these compounds to
solutions containing only the Amplex Red reagent in a sodium
phosphate buffer.
N0-(1-(4-Bromophenyl)-3-p-tolylallylidene)thiophene-2-
carbohydrazide (6g)
Yellow powder (ethyl alcohol). Yield: 32.75%, ꢀm1 .p. 207.6.
–
–
–
–
IR (KBr): n ¼ 1628 (amide C O), 1575 (C N) cm
.
1H NMR
(DMSO-d6, 400 MHz): 2.33 (s, 3H, –CH3), 6.79 (d, 1H, J ¼16 Hz,
a-ethylenic-H), 7.18 (t, 1H, C ring –H), 7.23 (d, 2H, B ring H),
7.56–7.71 (7H, B ring –H, b-ethylenic-H, A ring –H), 7.88 (d, 1H,
C ring –H), 8.03 (d, 1H, C ring –H), 11.70 (s, 1H, –NH). 13C NMR
(DMSO-d6, 100 MHz): d ¼ 20.92, 116.77, 122.48, 127.95,
129.23, 130.11, 131.32, 132.96, 135.99, 139.22, 139.77. MS
(ESI): m/z ¼ 447 [MþNa]þ (100%), 449 [MþNaþ2]þ (97%).
C
21H17BrN2OS: C 59.11, H 3.70, N 6.73, S 7.59; calcd. C 59.30,
H 4.03, N 6.59, S 7.54.
N0-(1-(4-Bromophenyl)-3-p-tolylallylidene)-4-methyl-1,2,3-
thiadiazole-5-carbohydrazide (6h)
Yellow powder (ethyl alcohol). Yield: 19.90%, ꢀm1 .p. 209.4.
–
–
–
–
IR (KBr): n ¼ 1675 (amide C O), 1575 (C N) cm
.
1H NMR
(DMSO-d6, 400 MHz): 2.33(s, 3H, B ring –CH3), 2.99 (s, 3H, C
ring –CH3), 6.84 (d, 1H, J ¼16 Hz, a-ethylenic-H), 7.25 (d, 2H,
J ¼ 8.4 Hz, B ring H3 and H5, A2X2), 7.57–7.79 (m, 7H, J ¼ 8.4 Hz,
B ring H2 and H6, A2X2, J ¼ 20 Hz, b-ethylenic-H, A ring –H),
12.25 (s, 1H, –NH). MS (ESI): m/z ¼ 441 [MþH]þ (50%), 443
[MþHþ2] (45%), 463 [MþNa]þ (90%), 465 [MþNaþ2]þ
(100%). C20H17BrN4OS: C 54.83, H 3.96, N 12.43, S 7.19; calcd.
C 54.43, H 3.88, N 12.69, S 7.26.
Kinetic experiments
Synthesized compounds were dissolved in dimethyl sulfoxide,
with a maximum concentration of 1% and used in the
concentration range of 1–100 mM. Kinetic data for interaction
of the enzyme isoforms with the compounds were deter-
mined using the Microsoft Excel package program. The slopes
of the Lineweaver–Burk plots were plotted versus the
inhibitor concentration and the Ki values were determined
from the x-axis intercept as ꢀKi. Each Ki value is the
representative of single determination where the correlation
coefficient (R2) of the replot of the slopes versus the inhibitor
concentrations was at least 0.98. SI (Ki(MAO-A)/Ki(MAO-B))
was also calculated. The protein was determined according
to the Bradford method [48].
N0-(1-(4-Bromophenyl)-3-m-tolylallylidene)-4-methyl-
1,2,3-thiadiazole-5-carbohydrazide (6i)
Yellow powder (ethyl alcohol). Yield: 21.30%, ꢀm1 .p. 177.6.
–
–
–
–
IR (KBr): n ¼ 1640 (amide C O), 1558 (C N) cm
.
1H NMR
(DMSO-d6, 400 MHz): 2.34 (s, 3H, B ring –CH3), 2.99 (s, 3H,
C ring –CH3), 6.83 (d, 1H, J ¼ 16 Hz, a-ethylenic-H), 7.21–7.79
(9H, aromatic-H, b-ethylenic-H), 12.33 (s, 1H, –NH). 13C NMR
(DMSO-d6, 100 MHz): d ¼ 15.21, 20.92, 117.69, 123.09, 125.54,
128.54, 128.61, 130.50, 131.57, 131.63, 134.98, 135.13,
135.38, 137.91, 141.39, 150.26, 161.34, 163.23. MS (ESI):
m/z ¼ 439 [MꢀH]þ (100%), 441 [M–Hþ2]þ (97%). C20H17BrN4OS:
Reversibility experiments
Reversibility of the MAO inhibition with synthesized com-
pounds was evaluated by a centrifugation–ultrafiltration
method [49]. In brief, adequate amounts of the recombinant
hMAO-A or -B were incubated together with a single
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RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2015, 348, 743–756
Monoamine Oxidase Inhibitors
Archiv der Pharmazie
concentration of the synthesized compounds or the reference
inhibitors in a sodium phosphate buffer (0.05 M, pH 7.4) for
1 h at 37°C. After this incubation period, an aliquot was stored
at 4°C and used for the measurement of MAO-A and -B
activity. The remaining incubated sample was placed in an
Ultrafree-0.5 centrifugal tube with a 30 kDa Biomax mem-
brane and centrifuged at 9000g for 20 min at 4°C. The enzyme
retained in the 30 kDa membrane was resuspended in a
sodium phosphate buffer at 4°C and centrifuged again twice.
After the third centrifugation, the enzyme retained in the
membrane was resuspended in sodium phosphate buffer
(300 mL) and an aliquot of this suspension was used for MAO-
A and -B activity determination. Control experiments were
performed simultaneously (to determine the 100% MAO
activity) by replacing the test compounds with appropriate
dilutions of the vehicles. The corresponding values of percent
(%) MAO isoform inhibition were separately calculated for
samples with and without repeated washing.
References
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Molecular models of the inhibitors were built and optimized
using SPARTAN 10.0 at PM3 level on iMac 3.4 GHz Intel Core i7
processor. The crystal structure of hMAO-A complexed
with the reversible inhibitor harmine (coded 2Z5X, with a
̊
resolution of 2.20 A) [50] and the crystal structure of hMAO-B
co-crystallized with the reversible inhibitor safinamide
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