Design, synthesis, and biological evaluation of pyridazinones containing the (2-fluorophenyl) piperazine moiety as selective MAO-B inhibitors

Article abstract of DOI:10.3390/molecules25225371

Twelve pyridazinones (T1–T12) containing the (2-fluorophenyl) piperazine moiety were designed, synthesized, and evaluated for monoamine oxidase (MAO) -A and -B inhibitory activities. T6 was found to be the most potent MAO-B inhibitor with an IC₅₀

Full text of DOI:10.3390/molecules25225371

molecules
Article
Design, Synthesis, and Biological Evaluation of
Pyridazinones Containing the (2-Fluorophenyl)
Piperazine Moiety as Selective MAO-B Inhibitors
Muhammed Çeçen 1,†, Jong Min Oh 2,†, Zeynep Özdemir 1,
*
, Saliha Ebru Büyüktuncel 3,
Mehtap Uysal 4, Mohamed A. Abdelgawad 5,6, Arafa Musa 7,8, Nicola Gambacorta 9,
Orazio Nicolotti 9 , Bijo Mathew 10,* and Hoon Kim 2,
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Inonu University, Malatya 44280, Turkey;
mcecen2344@gmail.com
2
3
4
5
6
7
Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences,
Sunchon National University, Suncheon 57922, Korea; ddazzo005@naver.com
Department of Analytical Chemistry, Faculty of Pharmacy, Inonu University, Malatya 44280, Turkey;
saliha.buyuktuncel@inonu.edu.tr
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Ankara 06100, Turkey;
mgokce99@gmail.com
Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University,
Sakaka 72341, Al Jouf, Saudi Arabia; mohamedabdelwahab976@yahoo.com
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef University,
Beni Suef 62514, Egypt
Department of Pharmacognosy, College of Pharmacy, Jouf University, Sakaka 72341, Al Jouf, Saudi Arabia;
akmusa@ju.edu.sa
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Cairo 11371, Egypt
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, Via E.
Orabona, 4, I-70125 Bari, Italy; nicola.gambacorta1@uniba.it (N.G.); orazio.nicolotti@uniba.it (O.N.)
Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham,
Amrita Health Science Campus, Kochi 682 041, India
8
9
10
*
Correspondence: zeynep.bulut@inonu.edu.tr (Z.Ö.);
bijovilaventgu@gmail.com or bijomathew@aims.amrita.edu (B.M.);
hoon@sunchon.ac.kr or hoon@scnu.ac.kr (H.K.)
†
These authors contributed equally to this work.
Academic Editor: Barbara De Filippis and Gunter Peter Eckert
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 30 October 2020; Accepted: 13 November 2020; Published: 17 November 2020
Abstract: Twelve pyridazinones (T1
designed, synthesized, and evaluated for monoamine oxidase (MAO) -A and -B inhibitory activities.
T6 was found to be the most potent MAO-B inhibitor with an IC50 value of 0.013 M, followed by T3
(IC50 = 0.039 M). Inhibitory potency for MAO-B was more enhanced by meta bromo substitution (T6
–T12) containing the (2-fluorophenyl) piperazine moiety were
µ
µ
)
than by para bromo substitution (T7). For para substitution, inhibitory potencies for MAO-B were as
follows: -Cl (T3) > -N(CH3)2 (T12) > -OCH3 (T9) > Br (T7) > F (T5) > -CH3 (T11) > -H (T1). T6 and
T3 efficiently inhibited MAO-A with IC50 values of 1.57 and 4.19 µM and had the highest selectivity
indices (SIs) for MAO-B (120.8 and 107.4, respectively). T3 and T6 were found to be reversible and
competitive inhibitors of MAO-B with Ki values of 0.014 and 0.0071, respectively. Moreover, T6 was
less toxic to healthy fibroblast cells (L929) than T3. Molecular docking simulations with MAO binding
sites returned higher docking scores for T6 and T3 with MAO-B than with MAO-A. These results
suggest that T3 and T6 are selective, reversible, and competitive inhibitors of MAO-B and should be
considered lead candidates for the treatment of neurodegenerative disorders like Alzheimer’s disease.
Keywords: monoamine oxidase; pyridazinone; kinetics; reversibility; ADME; molecular docking
Molecules 2020, 25, 5371; doi:10.3390/molecules25225371
www.mdpi.com/journal/molecules

Molecules 2020, 25, 5371
2 of 15
1. Introduction
Alzheimer’s disease (AD) is a fatal neurodegenerative disease characterized by progressive
cognitive dysfunctions, an inability to perform daily activities, and by the various neuropsychiatric
symptoms of late-stage AD [1]. AD is also associated with a reduction in the number of neurons in the
limbic system, which manages memory processes [
2
]. Furthermore, the levels of many neurotransmitters,
especially acetylcholine, are diminished in cerebral cortex in concert with neuronal loss, and the
levels of other neurotransmitters, such as cholinergic, noncholinergic, serotonergic, dopaminergic,
and neuropeptidergic neurotransmitters are altered in AD [3].
It has been reported that monoamine oxidase-B (MAO-B) activity is enhanced in AD, particularly
in reactive astrocytes near
that the MAO-B levels were elevated in astrocytes and in pyramidal neurons in the frontal cortex,
hippocampus CA1 region, and entorhinal cortex [ ]. MAO-B inhibitors can reduce neuronal damage
by inhibiting oxidative deamination, and it is believed that MAO-B inhibition reduces oxidative stress
and neurodegeneration, and thereby, delays AD progression [ ]. Many scaffolds, such as chalcones,
chromones, coumarins, thiazolidinones, isatins, and pyrazolines, have been reported to inhibit MAO-B
selectively and to exhibit promising neuroprotective properties [ 22]. In addition, MAO-B inhibitors
β-amyloid (Aβ) plaques [4]. In a more recent study, it was reported
5
6–8
9–
have been recently designed with two aryl or heteroaryl hydrophobic rings separated by short enamide,
unsaturated ketone, multi-conjugated ketone, amides, or hydrazone spacers [23–27].
Pyridazinones are pyridazine-containing heterocycles with two adjacent ring nitrogen atoms and
keto functionality. Several FDA-approved drugs with a pyridazinone nucleus, such as pimobendan,
bemoradan, indolidan, and levosimendan, have been commercialized over the past 30 years
as antihypertensive, and azanrinone and emorfazone are now marketed as cardiotonic and
anti-inflammatory agents, respectively [28]. Recently, our group explored a novel class of pyridazinone
hydrazides with pendant piperazine moieties and investigated their abilities to competitively and
reversibly inhibit MAO-B [29].
Here, we report the MAO-A and MAO-B inhibitory effects of a series of novel hydrazone
derivatives (T1
the anticancer activities of seven of these derivatives (T1
–
T12) bearing a phenyl ring with different substituents. We previously reported
T7) [30]. The other five (T8 T12) were
–
–
synthesized during the present study. Lead compounds were further examined by investigating
enzyme inhibition kinetics, reversibility and cytotoxicity to normal cell lines. In addition, binding
modes to MAO-A and MAO-B and interactions with their active sites were determined by molecular
docking simulations. Structure-activity relationships were investigated by comparing in vitro enzyme
inhibition and molecular docking results.
2. Results and Discussion
2.1. Chemistry
The synthetic routes used to produce the 12 derivatives are shown in Scheme 1. Compounds
were synthesized by reacting commercial 3,6-dichloropyridazine with (2-fluorophenyl)piperazine in
ethanol to afford 3-chloro-6-[4-(2-fluorophenyl)piperazine-1-yl]pyridazine. The physical and spectral
properties of 3-chloro-6-[4-(2-fluorophenyl)piperazine-1-yl]pyridazine concurred with literature data [30].
Compound 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone was obtained by hydrolyzing
3-chloro-6-substituted pyridazine in hot glacial acetic acid. Ethyl 6-(4-(2-fluorophenyl)piperazine-1-yl)-
3(2H)-pyridazinone-2-yl-acetate was obtained by reacting 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-
pyridazinone with ethyl bromoacetate in acetone in the presence of K2CO3. Then, 6-(4-(2-fluorophenyl)
piperazine-1-yl)-3(2H)-pyridazinone-2-yl-acetohydrazide was synthesized by condensing ethyl
6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-yl-acetate with hydrazine hydrate (yield
99%). The title compounds T1
6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-yl-acetohydrazidewithsubstituted/non-substituted
benzaldehydes, as we previously described [31]. The other five compounds (T8 12) were synthesized
–T7, which contain benzalhydrazone, were obtained by condensing
–

Molecules 2020, 25, 5371
3 of 15
using the same methods and are reported for the first time. The molecular structures of these compounds
1
were confirmed by H-NMR, 13C-NMR, and HRMS (Supplementary Materials). The molecular
structures, yields, melting points, molecular weights, and molecular formulas of T1–T12 are provided
in Table 1.
Scheme 1. Synthetic pathway for the production of 6-(4-(2-fluorophenyl) piperazine-1-yl)-3(2H)-
pyridazinone-2-acetyl-2-(substituted/non-substituted benzal)hydrazone derivatives (T1–T12).
Table 1. Molecular structures, yields, melting points, molecular weights, and molecular formulae
of T1-12.
Compound No.
R
Yield (%)
P
MM
Molecular Formula
T1
T2
T3
T4
T5
T6
T7
T8
T9
H
4-CH3
2-Cl
4-Cl
4-Br
2-CH3O
4-N(CH3)2
3-Br
2-F
4-F
4- OCH3
2-CH3
79.90
78.15
73.19
91.21
85.69
77.45
68.77
90.08
88.36
57.70
75.32
72.21
226–228
198–200
203–205
192–194
209–211
239–241
237–239
207–208
225–226
178–180
148–150
244–245
434.48
448.49
468.91
468.91
513.36
464.49
477.53
513.36
452.47
452.47
464.49
448.49
C23H23FN6O2
C24H25FN6O2
C23H22ClFN6O2
C23H22ClFN6O2
C23H22BrFN6O2
C24H25FN6O3
C25H28FN7O2
C23H22BrFN6O2
C23H22 F2N6O2
C23H22 F2N6O2
C24H25FN6O3
C24H25FN6O2
T10
T11
T12
It was observed that these compounds were in two stereoisomeric forms by interpretation of
NMR spectra. For example, T10 existed as two stereoisomeric forms in 1H-NMR spectrum (Figure 1A),
indicating the resonance of methylene attached to the carbonyl group at 5.30 and 4.87 ppm (2H;
s; CH2CO) and two pairs of singlet (one pair at 8.0 and 8.4 ppm and another one at 9.5 and 10.6 ppm)
(Figure 1B). In addition, the carbons of pyridazinone C6, pyridazinone C4, and 2-fluorophenyl C5 were
double in 13C-NMR spectrum at low MHz (Figure 1C). These pairs of signals clearly show that these
compounds exist in two stereoisomeric forms, as other isomers described previously [32,33].

Molecules 2020, 25, 5371
4 of 15
(A)
(B)
(C)
Figure 1. Structures of two conformational isomers (
of T10.
A B C) spectra
), and 1H-NMR ( ) and 13C-NMR (
2.2. MAO Inhibitory Activities
Of the 12 derivatives, six potently inhibited MAO-B with residual activities of <50% at 1.0
(Table 2). T6 most potently inhibited MAO-B, with an IC50 value of 0.013 M, and was followed by
T3 and T4 (IC50 = 0.039 and 0.099 M, respectively). T2 T12, and T9 also potently inhibited MAO-B
(IC50 = 0.10, 0.15, and 0.20 M, respectively). Bromine at the meta position (T6) increased MAO-B
inhibitory activity more so than at the para position (T7). Regarding substituents at the para position,
MAO-B inhibitory activities increased in the order: –Cl (T3) > –N(CH3)2 (T12) > -OCH3 (T9) > -Br (T7
µM
µ
µ
,
µ
)

Molecules 2020, 25, 5371
5 of 15
> -F (T5) > -CH3 (T11) > -H (T1). In addition, T6 efficiently inhibited MAO-A with an IC50 value of
1.57 M, followed by T3 (IC50 = 4.19 M) (Table 2), and T6 also had the highest selectivity index (SI)
value (120.8) for MAO-B, followed by T3 (SI = 107.4).
µ
µ
Table 2. Inhibitions of recombinant human MAO-A and MAO-B by T1 to T12 a.
Residual Activity (%) IC50 (µM)
SI b
Compounds
MAO-A
MAO-B
MAO-A
MAO-B
(10 µM)
(1.0 µM)
T1
T2
T3
T4
T5
T6
T7
T8
T9
96.6 ± 1.20
24.6 ± 2.86
24.6 ± 0.97
75.2 ± 4.70
84.0 ± 0.68
20.7 ± 0.62
25.4 ± 6.92
73.3 ± 2.86
35.2 ± 0.56
33.7 ± 4.70
70.4 ± 8.50
61.8 ± 1.65
99.4 ± 0.85
4.22 ± 0.85
3.01 ± 0.85
6.00 ± 0.94
54.0 ± 2.83
−15.3 ± 0.94
57.8 ± 9.58
67.2 ± 9.58
2.60 ± 0.74
95.3 ± 6.70
61.1 ± 2.98
17.9 ± 5.95
-
7.68 ± 0.14
0.10 ± 0.035
0.039 ± 0.0028
0.099 ± 0.0069
1.68 ± 0.11
0.013 ± 0.0016
0.87 ± 0.056
2.67 ± 0.051
0.20 ± 0.0017
35.3 ± 0.39
6.86 ± 0.37
0.15 ± 0.021
-
-
4.29 ± 0.12
42.9
107.4
-
-
120.8
-
-
33.8
0.21
-
4.19 ± 0.27
-
-
1.57 ± 0.80
4.43 ± 0.29
-
6.76 ± 0.46
T10
T11
T12
7.35 ± 0.059
-
-
-
Toloxatone
Lazabemide
Clorgyline
Pargyline
1.08 ± 0.025
-
0.14 ± 0.011
0.0070 ± 0.00070
-
-
0.030 ± 0.00083
a
Results are expressed as the means
(SI) values shown are expressed as IC50 of MAO-A/IC50 of MAO-B; IC50 values for reference compounds were
±
standard errors of duplicate or triplicate experiments; b Selectivity index
determined after preincubating them for 30 min with MAO-A or MAO-B.
The presence of different substituents on the phenyl hydrazone ring did not appreciably impact
MAO selectivity, whereas the introduction of halogens, methoxy, methyl, or dimethylamino groups
reduced MAO-B inhibition versus non-substituted hydrazone. The lead compounds T6 and T3
which had methoxy or chlorine groups at the ortho position of the phenyl system, were 11 and 3 times
more potent, respectively, than the reference reversible MAO-B inhibitor lazabemide. Furthermore,
T6 was twice as potent as the reference irreversible MAO-B inhibitor pargyline. Shifting methoxy group
from the ortho to the para position of the phenyl ring (T11) dramatically reduced MAO-B inhibition
,
(IC50 = 6.86
±
0.37 µM) and shifting chlorine from the ortho to the para (T4) reduced MAO-B inhibition.
These observations indicate that ortho is preferred over para in terms of MAO-B inhibition. Interestingly,
recent studies have reported that chlorine and methoxy groups have considerable impacts on MAO-B
inhibition [34,35].
2.3. Kinetics of MAO-B Inhibitions
Lineweaver-Burk plots and secondary plots obtained for MAO-B inhibition showed that T3 and
T6 were competitive inhibitors (Figure 2A,C), with Ki values of 0.014
respectively (Figure 2B,D). These results suggest that T3 and T6 are competitive inhibitors of MAO-B.
±
0.0014 and 0.0071
±
0.0033 µM,
2.4. Reversibility Studies
Reversibility studies were performed using a dialysis-based method. Dialysis recovered MAO-B
inhibition by T3 from 22.2% (AU) to 79.4% (AD) (Figure 3). T6 was also recovered from 36.9% (AU)
to 79.0% (AD) (Figure 3). These degrees of recovery were similar to that observed for lazabemide
(the reversible reference MAO-B inhibitor) (from 36.9% to 83.0%). However, inhibition by pargyline
(irreversible MAO-B inhibitor) was not recovered. These experiments showed that inhibitions of

Molecules 2020, 25, 5371
6 of 15
MAO-B by T3 and T6 were recovered by dialysis to the reversible reference level, thus showing that
T3 and T6 reversibly inhibit MAO-B.
(A)
(B)
3000
2500
2000
1500
1000
500
100
90
80
70
60
50
40
30
20
10
0
Control
0.010 uM
0.020 uM
0.040 uM
0
-10
0
10
20
30
-0.02
0
0.02
0.04
0.06
1/[S], mM-1
[T3], uM
(C)
(D)
2500
2000
1500
1000
500
80
Control
0.25 uM
0.50 uM
1.0 uM
70
60
50
40
30
20
10
0
0
-10
0
10
20
30
-0.015
-0.005
0.005
0.015
0.025
1/[S], mM-1
[T6], uM
Figure 2. Lineweaver-Burk plots for MAO-B inhibition by T3 or T6
plots (B,D) of slopes vs. inhibitor concentrations.
(A,C), and respective secondary
Undialyzed
Dialyzed
100
80
60
40
20
0
Figure 3. Recoveries of MAO-B inhibitions by T3 and T6 by dialysis.
2.5. Cytotoxic Effects of the Active Pyridazinone Derivatives
The cytotoxic effects of T3 and T6 were investigated using L929 cells (a healthy fibroblast cell line).
T3 caused complete cell death at 50 and 100 M), but no significant death at 1, 10, or 20 M (Figure 4).
µ
µ

Molecules 2020, 25, 5371
7 of 15
T6 did not have a cytotoxic effect at any dose (Figure 4). The IC50 values of T3 and T6 were 27.05 and
120.6 µM, respectively. These results suggested that T6 is a more appropriate drug candidate [30].
(A)
(B)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
1
10 20 50 100 nc
1
10 20 50 100 nc
Concentration (µM)
Concentration (µM)
Figure 4. L929 cell viabilities after treatments with T3
(A
) or T6
(B). nc, normal control. The experiments
were performed with three sets at each concentration.
2.6. Molecular Docking Studies
Docking simulations were used to investigate molecular interactions and binding modes of T3 or
T6 with MAO-A or MAO-B. Simulations docking scores are provided in Table 3. The ortho-fluorine
phenyl rings of T3 and T6 interacted with F352 of MAO-A by π-π contact and by hydrophobic
interaction with Y444, Y407, and the FAD of MAO-A (Figure 5). Moreover, the pyridazinone ring of T6
was well oriented towards F208 of MAO-A, and established another hydrophobic interaction. The ortho
positioned fluorine on the phenyl ring of T3 or T6 faced a hydrophobic cage consisting of FAD, Y435,
and Y398 of MAO-B, similar to MAO-A binding mode (Figure 6). Pyridazinone rings participated in
hydrophobic interactions with the MAO-B selective residue Y326, and the hydrazone moieties of T3
and T6 formed hydrogen bonds with the E84 side-chain and the T201 backbone of MAO-B.
Table 3. Docking score values for T3 and T6 interactions with MAO-A or MAO-B.
Docking Score (kcal/mol)
Compounds
MAO-A
MAO-B
T3
T6
−8.20
−8.43
−9.45
−9.56
Figure 5. Top scored poses of T3
(
A
, green sticks) and T6
(
B, magenta sticks) towards MAO-A. Green
lines show π-π interactions. Water molecules are rendered as cyan spheres.

Molecules 2020, 25, 5371
8 of 15
Figure 6. Top scored poses of T3
(A, green sticks) and T6 (B, magenta sticks) towards MAO-B. Red arrows
indicate hydrogen bonds. Water molecules are rendered as cyan spheres.
Computational studies of T3 or T6 interactions with MAO-A or MAO-B provided satisfactory
explanations for the experimental biological data obtained. Docking scores of T3 and T6 agreed
with IC50 experimental values, which indicated T6 is better than T3. Regarding binding modes,
the ortho-fluorine phenyl rings of T3 and T6 face the hydrophobic cages of MAO-A and MAO-B.
Interestingly, the main difference between MAO-A and MAO-B interactions centers on the presence
of two hydrogen bonds between the hydrazone moieties of T3 and T6 and E84 and T201 of MAO-B,
due to no hydrogen bond with their corresponding sites V93 and V210 in MAO-A, thus supporting
that T3 and T6 are selective MAO-B inhibitors.
2.7. ADME Prediction
The physicochemical parameters and likenesses of T3 and T6 were further investigated by
BOILED-Egg construction and using the SwissADME online platform. These online programs determine
whether compounds are suitable for oral administration based on the bioavailability radar panel
containing physicochemical properties such as flexibility, insolubility, lipophilicity, saturation, size,
and polarity, and provide rapid appraisals of the drug-likenesses of drug candidates. The BOILED-Egg
constructions of T3 and T6 predicted that both are well absorbed in the GI tract but in outside of
blood-brain barrier (BBB) permeation range (Figure 7). T3 and T6 were shown to lie within the pink
regions (optimal ranges) (Figure 8). Bioavailability radar predicted both compounds had acceptable
pharmacokinetics and satisfied drug-likeness rules. These results suggest that T3 and T6 should be
considered potential drug candidates.
Figure 7. The BOILED-Egg construction of T3 and T6.

Molecules 2020, 25, 5371
9 of 15
B
A
Figure 8. The bioavailability radar of T3 (A) and T6 (B).
3. Materials and Methods
3.1. Chemical Studies
All chemicals used in this study were purchased from Aldrich, Fluka AG, or E. Merck.
3-Chloro-6-substituted pyridazines, 3-chloro-6-(4-(2-fluorophenyl)piperazine-1-yl)pyridazine, 6-(4-(2-
fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone, ethyl 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-
pyridazinone-2-ylacetate, and6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-ylacetohydrazide
were synthesized as previously described [20–24]. The purities of these compounds were checked by
TLC using Merck Kieselgel F254 plates. Melting points were determined using an Electrothermal 9200
(Cole-Parmer Instrument Co. Vernon Hills, IL, USA), and IR spectra were recorded on a Perkin Elmer
1
Spectrometer using the ATR technique. H-NMR and 13C-NMR spectra were recorded on a Brucker
Avonce Ultrashield FT-NMR spectrometer in DMSO at 600 and 75 MHz, respectively. Mass spectra
(HRMS) were obtained using a combined Waters Acquity Ultra Performance Liquid Chromatography
Micromass and an LCT Premier XE UPLC/MS TOFF spectrophotometer (Waters Corp, Milford, MA,
USA) in ESI+ and ESI− modes.
3.2. Synthesis of Intermediates
3.2.1. Synthesis of 3-chloro-6-[4-(2-fluorophenyl)piperazine-1-yl]pyridazine (1)
A solution containing 0.01 mol of 3,6-dichloropyridazine and 0.01 mol of (2-fluorophenyl)
piperazine in 15 mL of ethanol was stirred under reflux at 120 ◦C for 6 h with the cooler adjusted to
15 ◦C as previously described [36]. The reaction mix was poured into ice water, and the precipitate
obtained by filtration was purified by crystallization from ethanol. Melting points of non-original
compounds were in accord with previously reported values [36].
3.2.2. Synthesis of 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone (2)
A solution of 0.05 mol of 3-chloro-6-[4-(2-fluorophenyl)piperazine-1-yl]pyridazine in 30 mL glacial
acetic acid was refluxed for 6 h. The acetic acid was then removed under reduced pressure, and the
residue obtained was dissolved in water and extracted with chloroform. The organic phase was
dried over sodium sulfate and evaporated under reduced pressure, and the residue was purified
by recrystallization from ethanol. Melting points of non-original compounds were in accord with
previously reported values [36].

Molecules 2020, 25, 5371
10 of 15
3.2.3. Synthesis of ethyl 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-yl-acetate (3)
A mixture of 0.01 mol 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone, 0.02 mol ethyl
bromoacetate, and 0.02 mol potassium carbonate in acetone (40 mL) was refluxed overnight. After the
mixture had cooled, organic salts were filtered off, the solvent was evaporated, and the residue
was purified by recrystallization from n-hexane to give the esters. Melting points of non-original
compounds were in accord with previously reported values [36].
3.2.4. Synthesis of 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-yl-acetohydrazide (4)
Hydrazine hydrate (99%, 3 mL) was added to the solution of 0.01 mol ethyl 6-(4-(2-fluorophenyl)
piperazine-1-yl)-3(2H)-pyridazinone-2-yl-acetate in 25 mL methanol and stirred for 3 h at room
temperature. The precipitate obtained was filtered off, washed with water, dried, and recrystallized
from ethanol. Melting points of non-original compounds were in accord with previously reported
values [36].
3.2.5. General Procedure for the Synthesis of T1–T12
A solution containing 0.01 mol 6-(4-(2-fluorophenyl)piperazine-1-yl)-3(2H)-pyridazinone-2-yl-
acetohydrazide and 0.01 mol substituted/non-substituted benzaldehyde was stirred in ethanol (15 mL),
refluxed for 6 h, and poured into ice water. The precipitate was filtered, dried, and crystallized from
methanol:water (8:2, v/v) [34]. All spectral data of the five new compounds (T8–T12) were in accord
with assigned structures.
3.2.6. 6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(3-bromobenzal)
hydrazon (T8)
White crystals (methanol/water); yield 90.08%; m.p. 207–208 ◦C; 1H-NMR (DMSO-d6, 300 MHz):
δ
3.18 (4H; t; CH2N; b+b0), 3.45 (4H; t; CH2N; a+a0), 5.31 (2H; s; CH2CO), 6.96 (1H; d; J = 4.14 Hz,
pyridazinone H5), 6.97–7.87 (9H; m; phenyl protons, pyridazinone H4), 8.58 (1H; s; -N=CH-) and 10.80
(1H; s; -NH-N). 13C-NMR (DMSO-d6, 300 MHz), 46.82 (2C, CH2-N; b+b0), 50.05 (2C, CH2-N; a+a0),
53.14 (1C; -N-
H2-C=O), 115.13 (1C; =CH), 119.10 (1C; pyridazinone C5), 122.96 (1C; 2-fluorophenyl
δ
C
C4), 124.55 (1C; 3-bromophenyl C5), 125.90 (1C; 2-fluorophenyl C5), 126.05 (1C; 3-bromophenyl
C2), 129.72 (1C; 3-bromophenyl C6), 130.26 (1C; pyridazinone C4), 131.08 (1C; 2-fluorophenyl C6),
134.22 (1C; 3-bromophenyl C3), 142.99 (1C; 2-fluorophenyl C3), 147.39 (1C; 3-bromophenyl C1), 149.13
(1C; 2-fluorophenyl C1), 154.40 (1C; 3-bromophenyl C4), 156.56 (1C; pyridazinone C6), 158.53 (1C;
C
2-fluorophenyl C2), 161.02 (1C; CH2-N- =O), 168.75 (1C; pyridazinone C3); C23H22BrFN6O2 MS (ESI+)
calculated: 513.3714, found: m/e 513.10526 (M+; 100.0%).
3.2.7. 6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(2-fluorobenzal)
hydrazon (T9)
White crystals (methanol/water); yield 88.36%; m.p. 225–226 ◦C; 1H-NMR (DMSO-d6, 300 MHz):
δ
; 3.19 (4H; t; CH2N; b+b0), 3.47 (4H; t; CH2N; a+a0), 5.31 (2H; s; CH2CO), 6.94 (1H; d; J = 4.15 Hz,
pyridazinone H5), 6.95–7.49 (9H; m; phenyl protons, pyridazinone H4), 8.96 (1H; s; -N=CH-) and 10.69
(1H; s; -NH-N). 13C-NMR (DMSO-d6, 300 MHz), 46.63 (2C, CH2N; b+b0), 50.07 (2C, CH2N; a+a0),
53.29 (1C; -N-
C
H2-C=O), 115.83 (1C; =CH), 116.14 (1C; pyridazinone C5), 119.06 (1C; 2-fluorophenyl
0
0
0
C6 ), 122.90 (1C; 2-fluorophenyl C5 ), 124.51 (1C; 2-fluorophenyl C4 ), 125.84 (1C; 2-fluorophenyl
C5), 129.07 (1C; 2-fluorophenyl C4), 129.15 (1C; pyridazinone C4), 129.62 (1C; 2-fluorophenyl C6),
0
129.70 (1C; 2-fluorophenyl C3), 129.78 (1C; 2-fluorophenyl C3 ), 131.09 (1C; 2-fluorophenyl C1), 143.43
0
0
(1C; 2-fluorophenyl C1 ), 147.80 (1C; 2-fluorophenyl C2 ), 149.09 (1C; pyridazinone C6), 154.54 (1C;
2-fluorophenyl C2), 158.79 (1C; CH2-N-
C
=O) and 168.73 (1C; pyridazinone C3); C23H22F2N6O2 MS
(ESI+) calculated: 452.47, found: 453.18594 m/e (M+; 100.0%).

Molecules 2020, 25, 5371
11 of 15
3.2.8. 6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(4-florobenzal)
hydrazon (T10)
White crystals (methanol/water); yield 57.70%; m.p. 178–180 ◦C; 1H-NMR (DMSO-d6, 300 MHz):
; 3.17 (4H; t; CH2N; b+b0), 3.46 (4H; t; CH2N; a+a0), 5.30 (2H; s; CH2CO), 6.84 (1H; d; J = 4.15 Hz,
δ
pyridazinone H5), 6.85–7.83 (9H; m; phenyl protons, pyridazinone H4), 8.08 (1H; s; -N=CH-) and 10.68
(1H; s; -NH-N). 13C-NMR (DMSO-d6, 300 MHz), 46.84 (2C, CH2N; b+b0), 50.07 (2C, CH2N; a+a0),
53.29 (1C; -N-C
H2-C=O), 115.83 (1C; =CH), 116.05 (1C; pyridazinone C5), 116.35 (1C; 4-fluorophenyl
C6), 122.90 (1C; 4-fluorophenyl C5), 124.51 (1C; 4-fluorophenyl C4), 125.84 (1C; 2-fluorophenyl
C5), 129.07 (1C; 2-fluorophenyl C4), 129.62 (1C; pyridazinone C4), 129.70 (1C; 2-fluorophenyl C6),
130.68 (1C; 2-fluorophenyl C3), 139.71 (1C; 4-fluorophenyl C3), 147.80 (1C; 2-fluorophenyl C1), 149.09
(1C; 4-fluorophenyl C1), 154.54 (1C; 4-fluorophenyl C2), 158.79 (1C; pyridazinone C6), 162.52 (1C;
C
2-fluorophenyl C2), 165.16 (1C; CH2-N- =O) and 168.73 (1C; pyridazinone C3); C23H22F2N6O2 MS
(ESI+) calculated: 452.47, found: 453.18583 m/e (M+; 100.0%).
3.2.9. 6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(4-methoxybenzal)
hydrazon (T11)
White crystals (methanol/water); yield 75.32%; m.p. 148–150 ◦C. 1H-NMR (DMSO-d6, 300 MHz):
δ
3.17 (4H; t; CH2N; b+b0), 3.45 (4H; t; CH2N; a+a0), 3.85 (3H; s; CH3O), 5.30 (2H; s; CH2CO), 6.88
(1H; d; J = 4.15 Hz, pyridazinone H5), 6.89–7.79 (9H; m; phenyl protons, pyridazinone H4), 8.64 (1H;
s; -N=CH-) and 10.55 (1H; s; -NH-N). 13C-NMR (DMSO-d6, 300 MHz), 46.83 (2C, CH2N; b+b0), 50.07 (2C,
CH2N; a+a0), 53.03 (1C; -N-
CH2-C=O), 55.38 (1C; -OCH3), 114.07 (1C; =CH), 116.12 (1C; pyridazinone
C5), 119.07 (1C; 4-methoxyphenyl C6), 124.50 (1C; 4-methoxyphenyl C5), 125.77 (1C; 4-methoxyphenyl
C4), 126.30 (1C; 2-fluorophenyl C5), 128.82 (1C; 2-fluorophenyl C4), 129.43 (1C; pyridazinone C4),
130.14 (1C; 2-fluorophenyl C6), 131.10 (1C; 2-fluorophenyl C3), 139.63 (1C; 4-methoxyphenyl C3),
144.42 (1C; 2-fluorophenyl C1), 149.04 (1C; 4-methoxyphenyl C1), 154.39 (1C; 4-methoxyphenyl C2),
156.83 (1C; pyridazinone C6), 158.82 (1C; 2-fluorophenyl C2), 161.35 (1C; CH2-N-
C=O) and 168.56 (1C;
pyridazinone C3); C24H25FN6O3 MS (ESI+) calculated: 464.49, found: 465.20506 m/e (M+; 100.0%).
3.2.10. 6-[4-(2-Fluorophenyl)piperazine-1-yl]-3(2H)-pyridazinone-2-acetyl-2-(2-methylbenzal)
hydrazon (T12)
White crystals (methanol/water); yield 72.21%; m.p. 244–245 ◦C; 1H-NMR (DMSO-d6, 300 MHz):
δ
2.50 (3H; s; -CH3), 3.17 (4H; t; CH2N; b+b0), 3.45 (4H; t; CH2N; a+a0), 5.30 (2H; s; CH2CO), 6.92 (1H;
d; J = 4.19 Hz, pyridazinone H5), 6.92–7.77 (9H; m; phenyl protons, pyridazinone H4), 8.37 (1H; s;
-N=CH-) and 10.58 (1H; s; -NH-N). 13C-NMR (DMSO-d6, 300 MHz),
20.16 (1C; -CH3), 46.69 (2C; CH2-N;
b+b0), 50.10 (2C, CH2-N; a+a0), 53.36 (1C; -N-
H2-C=O), 116.13 (1C; =CH), 119.07 (1C; pyridazinone
δ
C
C5), 122.87 (1C; 4-methylphenyl C6), 124.54 (1C; 2-methylphenyl C2), 125.78 (1C; 2-methylphenyl
C3), 126.24 (1C; 4-methylphenyl C5), 127.47 (1C; 4-methylphenyl C4), 129.96 (1C; pyridazinone C4),
130.85 (1C; 2-fluorophenyl C3), 131.48 (1C; 2-fluorophenyl C4), 137.13 (1C; 2-fluorophenyl C5), 143.70
(1C; 2-methylphenyl C1), 147.49 (1C; 2-fluorophenyl C6), 149.71 (1C; 2-fluorophenyl C1), 156.98 (1C;
2-fluorophenyl C2), 158.81 (1C; pyridazinone C6) 163.96 (1C; CH2-N-
C=O) and 168.57 (1C; pyridazinone
C3); C24H25FN6O2 MS (ESI+) calculated: 448.49, found: 449.21034 m/e (M+; 100.0%).
3.3. Enzyme Assays
Recombinant human MAO-A and MAO-B inhibitory activities of were continuously assayed
as described previously [37], using kynuramine (0.06 mM) and benzylamine (0.3 mM), respectively.
Substrate concentrations were 1.5
×
and 2.3
×
Km, respectively, and their Km values were 0.041 mM and
0.13 mM, respectively. MAO-A and MAO-B inhibitory activities were measured after preincubating
enzymes and inhibitors for 15 min. Enzymes and chemicals were obtained from Sigma-Aldrich
(St. Louis, MO, USA).

Molecules 2020, 25, 5371
12 of 15
3.4. Enzyme Inhibitions and Kinetics
The inhibitory activities of the twelve compounds against MAO-A and MAO-B were first
investigated at an inhibitor concentration of 10 M. For the compounds showing residual activities of
µ
<50%, IC50 values for MAO-A and MAO-B inhibitions were determined. Kinetic experiments were
carried out at five substrate and three inhibitor concentrations [38].
3.5. Analysis of Inhibitor Reversibilities
The reversibilities of T3 and T6 inhibitions were investigated by dialysis after preincubating them
with MAO-B for 30 min, as previously described [39]. The concentrations used were T3 0.080
T6 0.020 M, lazabemide (reference reversible MAO-B inhibitor) 0.30 M, and pargyline (reference
irreversible MAO-B inhibitor) 0.060 M. Reversibilities were determined using the relative activities of
µM,
µ
µ
µ
undialyzed (AU) and dialyzed (AD) samples [40].
3.6. Cytotoxicity
An MTS (5-(3-carboxymethoxyphenyl)-2-(4,5-dimethyl-thiazolyl)-3-(4-sulfophenyl)) tetrazolium
assay was used to evaluate the cytotoxic activities of T3 and T6 by measuring formazan absorbance.
MTS is converted to formazan by mitochondria in metabolically active cells via NAD(P)H-dependent
dehydrogenase enzymes. L929 cells (a mouse fibroblast cell line) were used to evaluate the cytotoxicities.
Briefly, cells (5
serum and 1% penicillin/streptomycin, and incubated for 24 h. After supernatants were removed, the
cells were treated with the compounds dissolved in DMSO (at 1, 10, 20, 50, or 100 M) in triplicate for
×
104 cells/well) were seeded into 96-well plates in DMEM containing 10% fetal bovine
µ
48 h at 37 ◦C in a 5% CO2 atmosphere. MTS reagent (Uptima, Interchim, Montluçon, France) was then
added to each well and incubated for 3 h. Absorbances were measured at 490 nm using a microplate
reader (BioTek, Winooski, VT, USA), and cell viabilities were calculated as percentage of untreated
controls [30].
3.7. Molecular Docking Studies
T3 and T6 were docked using X-ray solved structures of MAO-A and MAO-B obtained from the
Protein Data Bank (PDB) by retrieving entries 2Z5X and 2V5Z, respectively [41,42]. Protein targets
were prepared using the Protein Preparation Wizard Schrödinger Suite 2018-4 (New York, NY, USA)
for refining and optimizing crystal structures after correcting for protonation states and adding missing
hydrogen atoms [43,44]. Nine water molecules within MAO-A and eight water molecules within
MAO-B were not deleted for docking simulations, as was previously performed [45]. To generate
all possible tautomers and protonation states at physiological pH, T3 and T6 were treated using the
LigPrep tool [46]. The obtained files were used for docking simulations by employing Grid-based
Ligand Docking with Energetics (GLIDE) [47]. Enclosing boxes were automatically centered on the
related cognate ligands, T3 and T6. The default force Field OPLS_20058 and the standard precision
(SP) docking protocol with default settings were employed for the in silico analyses. To confirm the
validity of docking studies, re-docking simulations were performed on T3 and T6 at their binding sites.
The cognate ligands are flagged as HRM and SAG for MAO-A and MAO-B, respectively, and moved
back to the original positions with Root Mean Square Deviations (RMSD) accounting for all heavy
atoms equal to 0.760 Å and 0.395 Å for HRM and SAG, respectively [48].
3.8. ADME Prediction
ADME parameters were predicted in silico to determine a range of physical-chemical properties,
lipophilicity, water solubility, and pharmacokinetic data, using the login-free website http://www.
swissadme.ch/ [49].

Molecules 2020, 25, 5371
13 of 15
4. Conclusions
The present work describes the synthesis of twelve pyridazinones (T1
–
T12) containing the
2-fluorophenyl substituted piperazine moiety. All synthesized compounds were evaluated for MAO-A
and MAO-B inhibitory activities. Most of the compounds were selective MAO-B inhibitors, and T3
and T6 were found to be selective, competitive, and reversible MAO-B inhibitors. T3 and T6 potently
and reversibly inhibited MAO-B in the sub-micromolar range, and were relatively non-toxic to
healthy fibroblasts. We concluded that T3 and T6 offer new developmental leads for the treatment of
neurodegenerative disorders.
Supplementary Materials: 1H- and 13C-NMR spectral data are available in the Supplementary Materials.
Author Contributions: Conceptualization: Z.Ö., B.M. and H.K.; biological activity: J.M.O.; kinetics: J.M.O.;
docking analysis: N.G., O.N.; synthesis: M.C., Z.Ö.; data curation: B.M., J.M.O.; writing—original draft preparation:
B.M., S.E.B., M.U., M.A.A., A.M.; writing—review and editing: B.M. and K.H.; supervision: H.K.; funding
acquisition: H.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the
Republic of Korea government (NRF-2019R1A2C1088967) (to H.K.).
Conflicts of Interest: The authors have no conflict of interest to declare.
References
1.
Harilal, S.; Jose, J.; Parambi, D.G.T.; Kumar, R.; Mathew, G.E.; Uddin, M.S.; Kim, H.; Mathew, B. Advancements
in nanotherapeutics for Alzheimer’s disease: Current perspectives. J. Pharm. Pharmacol. 2019, 71, 1370–1383.
[CrossRef] [PubMed]
2.
3.
4.
Geldenhuys, W.J.; Darvesh, A.S. Pharmacotherapy of Alzheimer’s disease: Current and future trends.
Expert. Rev. Neurother. 2015, 5, 3–5. [CrossRef] [PubMed]
Chiu, P.Y.; Wei, C.Y. Donepezil in the one-year treatment of dementia with Lewy bodies and Alzheimer’s
disease. J. Neurol. Sci. 2017, 81, 22. [CrossRef]
Mathew, B.; Parambi, D.G.T.; Mathew, G.E.; Uddin, M.S.; Inasu, S.T.; Kim, H.; Marathakam, A.;
Unnikrishnan, M.K.; Carradori, S. Emerging therapeutic potentials of dual-acting MAO and AChE inhibitors
in Alzheimer’s and Parkinson’s diseases. Arch. Pharm. 2019, 352, e1900177. [CrossRef] [PubMed]
Kim, D.; Baik, S.H.; Kang, S.; Cho, S.W.; Bae, J.; Cha, M.Y.; Sailor, M.J.; Jung, I.M.; Ahn, K.H. Close correlation
of monoamine oxidase activity with progress of Alzheimer’s disease in mice, observed by in vivo two-photon
imaging. ACS Cent. Sci. 2016, 2, 967–975. [CrossRef] [PubMed]
5.
6.
7.
8.
9.
Kumar, B.; Prakash, V.; Kumar, V. A perspective on monoamine oxidase enzyme as drug target: Challenges
and opportunities. Curr. Drug Targets. 2017, 18, 87–97. [CrossRef] [PubMed]
Finberg, J.P.M.; Rabey, J.M. Inhibitors of MAO-A and MAO-B in psychiatry and neurology. Front. Pharmacol.
2016, 7, 340. [CrossRef]
Guglielmi, P.; Mathew, B.; Secci, D.; Carradori, S. Chalcones: Unearthing their therapeutic possibility as
monoamine oxidase B inhibitors. Eur. J. Med. Chem. 2020, 112650. [CrossRef]
Carradori, S.; Silvestri, R. New frontiers in selective human MAO-B inhibitors. J. Med. Chem. 2015, 58, 6717–6732.
[CrossRef]
10. Tripathi, R.K.P.; Ayyannan, S.R. Monoamine oxidase-B inhibitors as potential neurotherapeutic agents:
An overview and update. Med. Res. Rev. 2019, 39, 1603–1706. [CrossRef]
11. Guglielmi, P.; Carradori, S.; Ammazzalorso, A.; Secci, D. Novel approaches to the discovery of selective human
monoamine oxidase-B inhibitors: Is there room for improvement? Expert Opin. Drug Discov. 2019, 14, 995–1035.
[CrossRef] [PubMed]
12. Kumar, B.; Sheetal, S.; Mantha, A.K.; Kumar, V. Recent developments on the structure-activity relationship
studies of MAO inhibitors and their role in different neurological disorders. RSC Adv. 2016, 6, 42660–42683.
[CrossRef]
13. Mathew, B.; Haridas, A.; Suresh, J.; Mathew, G.E.; Ucar, G.; Jayaprakash, V. Monoamine oxidase inhibitory
actions of chalcones. A mini review. Cent. Nerv. Syst. Agents Med. Chem. 2016, 16, 120–136. [CrossRef]
[PubMed]

Molecules 2020, 25, 5371
14 of 15
14. Choi, J.W.; Jang, B.K.; Cho, N.; Park, J.H.; Yeon, S.K.; Ju, E.J.; Lee, Y.S.; Han, G.; Pae, A.M.; Kim, D.J.; et al.
Synthesis of a series of unsaturated ketone derivatives as selective and reversible monoamine oxidase
inhibitors. Bioorg. Med. Chem. 2015, 23, 6486–6496. [CrossRef] [PubMed]
15. Mathew, B.; Mathew, G.E.; Petzer, J.P.; Petzer, A. Structural exploration of synthetic chromones as selective
MAO-B inhibitors. A Mini Review. Comb. Chem. High Throughput Screen. 2017, 20, 522–532. [CrossRef]
[PubMed]
16. Gaspar, A.; Silva, T.; Yáñez, M.; Viña, D.; Orallo, F.; Ortuso, F.; Uriarte, E.; Alcaro, S.; Borges, F. Chromone, a
privileged scaffold for the development of monoamine oxidase inhibition. J. Med. Chem. 2011, 54, 5165–5173.
[CrossRef]
17. Reis, J.; Cagide, F.; Chavarria, D.; Silva, T.B.; Fernandes, C.; Gaspar, A.; Uriarte, E.; Remiao, F.; Alcaro, S.;
Ortuso, F.; et al. Discovery of new chemical entities of old targets. Insight on the lead optimization of
chromones based monoamine oxidase B inhibitors. J. Med. Chem. 2016, 59, 5879–5893. [CrossRef]
18. Badavath, V.N.; Baysal, I.; Ucar, G.; Sinha, B.N.; Jayaprakash, V. Monoamine oxidase inhibitory activity of
novel pyrazoline analogues: Curcumin based design and synthesis. ACS Med. Chem. Lett. 2016, 7, 56–61.
[CrossRef]
19. Secci, D.; Bolasco, A.; Chimenti, P.; Carradori, S. The state of the art of pyrazole derivatives as monoamine
oxidase inhibitors and antidepressant/anticonvulsant agents. Curr. Med. Chem. 2011, 18, 5114–5144.
[CrossRef]
20. Mathew, B.; Suresh, J.; Anbazhagan, S.; Mathew, G.E. Pyrazoline. A promising scaffold for the inhibition of
monoamine oxidase. Cent. Nerv. Syst. Agents Med. Chem. 2013, 13, 195–206. [CrossRef]
21. Matos, M.J.; Vina, D.; Vazquez-Rodriquez, S.; Uriarte, E.; Santana, L. Focusing on new monoamine oxidase
inhibitors. Differently substituted coumarins as an interesting scaffold. Curr. Top. Med. Chem. 2012, 12, 2210–2233.
[CrossRef] [PubMed]
22. Guglielmi, P.; Secci, D.; Petzer, A.; Bagetta, D.; Chimenti, P.; Rotondi, G.; Ferrante, C.; Recinella, L.; Leone, S.;
Alcaro, S.; et al. Benzo[b]tiophen-3-ol derivatives as effective inhibitors of human monoamine oxidase: Design,
synthesis, and biological activity. J. Enzym. Inhib. Med. Chem. 2019, 34, 1511–1525. [CrossRef] [PubMed]
23. Oh, J.M.; Rangarajan, T.M.; Chaudhary, R.; Singh, R.P.; Singh, M.; Singh, R.P.; Tondo, A.R.; Gambacorta, N.;
Nicolotti, O.; Mathew, B.; et al. Novel class of chalcone oxime ethers as potent monoamine oxidase-B and
acetylcholinesterase inhibitors. Molecules 2020, 25, 2356. [CrossRef] [PubMed]
24. Maliyakkal, N.; Eom, B.H.; Heo, J.H.; Almoyad, M.A.A.; Parambi, D.G.T.; Gambacorta, N.; Nicolotti, O.;
Beeran, A.A.; Kim, H.; Mathew, B. A new potent and selective monoamine oxidase-B inhibitor with
extended conjugation in chalcone framework. 1-[4-(morpholin-4-yl)phenyl]-5-phenylpenta-2,4-dien-1-one.
Chem. Med. Chem. 2020, 15, 1629–1633. [CrossRef] [PubMed]
25. Hagenow, J.; Hagenow, S.; Grau, K.; Khanfar, M.; Hefke, L.; Proschak, E.; Stark, H. Reversible small molecule
inhibitors of MAO A and MAO B with anilide motifs. Drug Des. Dev. Ther. 2020, 14, 371–393. [CrossRef] [PubMed]
26. Kavully, F.S.; Oh, J.M.; Dev, S.; Kaipakasseri, S.; Palakkathondi, A.; Vengamthodi, A.; Azeez, R.F.A.;
Tondo, A.R.; Nicolotti, O.; Kim, H.; et al. Design of enamides as new selective monoamine oxidase-B
inhibitors. J. Pharm. Pharmacol. 2020, 72, 916–926. [CrossRef]
27. Turan-Zitouni, G.; Hussein, W.; Saglık, B.N.; Tabbi, A.; Korkut, B. Design, synthesis and biological evaluation
of novel N-pyridyl-hydrazone derivatives as potential monoamine oxidase (MAO) inhibitors. Molecules
2018, 23, 113–124. [CrossRef] [PubMed]
28. Dubey, S.; Bhosle, P.A. Pyridazinone: An important element of pharmacophore possessing broad spectrum
of activity. Med. Chem. Res. 2015, 24, 3579–3598. [CrossRef]
29. Özdemir, Z.; Alagöz, M.A.; Uslu, H.; Karakurt, A.; Erikci, A.; Ucar, G.; Uysa, M. Synthesis, molecular
modelling and biological activity of some pyridazinone derivatives as selective human monoamine oxidase-B
inhibitors. Pharmacol. Rep. 2020, 72, 692–704. [CrossRef]
˙
30. Özdemir, Z.; Bas¸ak-Türkmen, N.; Ayhan, I.; Çiftçi, O.; Uysal, M. Synthesis of new 6-[4-(2-fluorophenylpiperazine
-1-yl)]-3(2h)-pyridazinone-2-acethyl-2-(substitutedbenzal)hydrazine derivatives and evaluation of their
cytotoxic effects in liver and colon cancer cell lines. Pharm. Chem. J. 2019, 52, 923–929. [CrossRef]
31. Özdemir, Z.; Utku, S.; Mathew, B.; Carradori, S.; Orlando, G.; Simone, S.D.; Alagöz, M.A.; Özçelik, A.B.;
Uysal, M.; Claudio Ferrante, F. Synthesis and biological evaluation of new 3(2H)-pyridazinone derivatives as
non-toxic anti-cancer compounds against colon carcinoma cells. J. Enzym. Inhib. Med. Chem. 2020, 35, 1100–1109.
[CrossRef] [PubMed]

Molecules 2020, 25, 5371
15 of 15
32. Vergara, S.; Carda, M.; Agut, R.; Yepes, L.M.; Velez, I.D.; Robledo, S.M.; Galeano, W.C. Synthesis, antiprotozoal
activityandcytotoxicityinU-937macrophagesoftriclosan–hydrazonehybrids.Med. Chem. Res.2017, 26, 3262–3273
.
[CrossRef]
33. Govindasami, T.; Pandey, A.; Palanivelu, N.; Pandey, A. Synthesis, Characterization and antibacterial activity
of biologically important vanillin related hydrazone derivatives. Int. J. Org. Chem. 2011, 1, 71–77. [CrossRef]
34. Mathew, B.; Mathew, G.E.; Ucar, G.; Joy, M.; Nafna, E.K.; Suresh, J. Monoamine oxidase inhibitory activity of
methoxy-substituted chalcones. Int. J. Biol. Macromol. 2017, 104, 1321–1329. [CrossRef] [PubMed]
35. Mathew, B.; Ucar, G.; Mathew, G.E.; Mathew, S.; Purapurath, P.K.; Moolayil, F.; Mohan, S.; Gupta, S.V.
Monoamine oxidase inhibitory activity: Methyl- versus chloro-chalcone derivatives. Chem. Med. Chem.
2016, 11, 2649–2655. [CrossRef]
36. Özçelik, A.B.; Özdemir, Z.; Sari, S.; Utku, S.; Uysal, M. A new series of pyridazinone derivatives as
cholinesterases inhibitors: Synthesis, in vitro activity and molecular modeling studies. Pharmacol. Rep.
2019, 71, 1253–1263. [CrossRef]
37. Mathew, B.; Baek, S.C.; Parambi, D.G.T.; Lee, J.P.; Joy, M.; Annie Rilda, P.R.; Randev, R.V.; Nithyamol, P.;
Vijayan, V.; Inasu, S.T.; et al. Selected aryl thiosemicarbazones as a new class of multi-targeted monoamine
oxidase inhibitors. Med. Chem. Comm. 2018, 9, 1871–1881. [CrossRef]
38. Lee, H.W.; Ryu, H.W.; Kang, M.G.; Park, D.; Oh, S.R.; Kim, H. Potent selective monoamine oxidase B inhibition
by maackiain, a pterocarpan from the roots of Sophora flavescens. Bioorg. Med. Chem. Lett. 2016, 26, 4714–4719.
[CrossRef]
39. Baek, S.C.; Lee, H.W.; Ryu, H.W.; Kang, M.G.; Park, D.; Kim, S.H.; Cho, M.L.; Oh, S.R.; Kim, H. Selective
inhibition of monoamine oxidase A by hispidol. Bioorg. Med. Chem. Lett. 2018, 15, 584–588. [CrossRef]
40. Parambi, D.G.T.; Oh, J.M.; Baek, S.C.; Lee, J.P.; Tondo, A.R.; Nicolotti, O.; Kim, H.; Mathew, B. Design,
synthesis and biological evaluation of oxygenated chalcones as potent and selective MAO-B inhibitors.
Bioorg. Chem. 2019, 93, 103335. [CrossRef]
41. Son, S.Y.; Ma, J.; Kondou, Y.; Yoshimura, M.; Yamashita, E.; Tsukihara, T. Structure of human monoamine
oxidase A at 2.2-A resolution: The control of opening the entry for substrates/inhibitors. Proc. Natl. Acad.
Sci. USA 2008, 105, 5739–5744. [CrossRef] [PubMed]
42. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.E.; Mattevi, A. Structures of
human monoamine oxidase B complexes with selective noncovalent inhibitors: Safinamide and coumarin
analogs. J. Med. Chem. 2007, 50, 5848–5852. [CrossRef]
43. Schrödinger Release 2018-2. Prime; Schrödinger, LLC: New York, NY, USA, 2018.
44. Schrödinger Release 2018-2. Glide; Schrödinger, LLC: New York, NY, USA, 2018.
45. Mangiatordi, G.F.; Alberga, D.; Pisani, L.; Gadaleta, D.; Trisciuzzi, D.; Farina, R.; Carotti, A.; Lattanzi, G.;
Catto, M.; Nicolotti, O. A rational approach to elucidate human monoamine oxidase molecular selectivity.
Eur. J. Pharm. Sci. 2017, 101, 90–99. [CrossRef] [PubMed]
46. LigPrep; Schrödinger, LLC: New York, NY, USA, 2018.
47. Friesner, R.A.; Banks, J.L.; Murphy, R.B.; Halgren, T.A.; Klicic, J.J.; Mainz, D.T.; Repasky, M.P.; Knoll, E.H.;
Shelley, M.; Perry, J.K.; et al. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739–1749. [CrossRef] [PubMed]
48. Reeta; Baek, S.C.; Lee, J.P.; Rangarajan, T.M.; Ayushee; Singh, R.P.; Singh, M.; Mangiatordi, G.F.; Nicolotti, O.;
Kim, H.; et al. Ethyl acetohydroxamate incorporated chalcones: Unveiling a novel class of chalcones for
multitarget monoamine oxidase-B inhibitors against Alzheimer’s disease. CNS Neurol. Disord. Drug Targets
2019, 8, 643–654. [CrossRef] [PubMed]
49. Daina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness
and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are available from the authors.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional
affiliations.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).