Synthesis, molecular modelling and biological activity of some pyridazinone derivatives as selective human monoamine oxidase-B inhibitors

Article abstract of DOI:10.1007/s43440-020-00070-w

Background: Since brain neurotransmitter levels are associated with the pathology of various neurodegenerative diseases like Parkinson and Alzheimer, monoamineoxidase (MAO) plays a critical role in balancing these neurotransmitters in the brain. MAO isofo

Full text of DOI:10.1007/s43440-020-00070-w

Pharmacological Reports
https://doi.org/10.1007/s43440-020-00070-w
ARTICLE
Synthesis, molecular modelling and biological activity of some
pyridazinone derivatives as selective human monoamine oxidase‑B
inhibitors
Zeynep Özdemir1 · Mehmet Abdullah Alagöz1 · Harun Uslu2 · Arzu Karakurt1 · Acelya Erikci3 · Gulberk Ucar3 ·
Mehtap Uysal4
Received: 18 October 2019 / Revised: 3 January 2020 / Accepted: 8 January 2020
© Maj Institute of Pharmacology Polish Academy of Sciences 2020
Abstract
Background Since brain neurotransmitter levels are associated with the pathology of various neurodegenerative diseases
like Parkinson and Alzheimer, monoamineoxidase (MAO) plays a critical role in balancing these neurotransmitters in the
brain. MAO isoforms appear as promising drug targets for the development of central nervous system agents. Pyridazinones
have a broad array of biological activities. Here, six pyridazinone derivatives were synthesized and their human monoamine
oxidase inhibitory activities were evaluated by molecular docking studies, in silico ADME prediction and in vitro biological
screening tests.
Methods The compounds were synthesized by the reaction of diferent piperazine derivatives with 3 (2H)-pyridazinone ring
and MAO-inhibitory efects were investigated. Docking studies were conducted with Maestro11.8 software.
Results Most of the synthesized compounds inhibited hMAO-B selectively except compound 4f. Compounds 4a–4e
inhibited hMAO-B selectively and reversibly in a competitive mode. Compound 4b was found as the most potent
(ki =0.022 0.001 µM) and selective (SI (Ki hMAO-A/hMAO-B)=206.82) hMAO-B inhibitor in this series. The results of dock-
ing studies were found to be consistent with the results of the in vivo activity studies. Compounds 4a–4e were found to be
non-toxic to HepG2 cells at 25 μM concentration. In silico calculations of ADME properties indicated that the compounds
have good pharmacokinetic profles.
Conclusion It was concluded that 4b is possibly recommended as a promising nominee for the design and development of
new pyridazinones which can be used in the treatment of neurological diseases.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s43440-020-00070-w) contains
supplementary material, which is available to authorized users.
* Zeynep Özdemir
zeynep.bulut@inonu.edu.tr; zpozdmr@gmail.com
1
Department of Pharmaceutical Chemistry, Faculty
of Pharmacy, Inonu University, 44280 Malatya, Turkey
2
Department of Anesthesiology, Vocational School of Health
Services, Fırat University, 23119 Elazığ, Turkey
3
Department of Biochemistry, Faculty of Pharmacy, Lokman
Hekim University, 06510 Ankara, Turkey
4
Department of Pharmaceutical Chemistry, Faculty
of Pharmacy, Gazi University, 06100 Ankara, Turkey
Vol.:(0123456789)
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Z. Özdemir et al.
Graphic abstract
Keywords Pyridazinone · Monoamine oxidase inhibition · Molecular docking
psychological–neurological disorders [3]. And also, the
use of MAOIs is limited due to undesirable drug–food
and drug–drug interactions and also due to their adverse
efects. Therefore, extensive eforts have been devoted
to the discovery of novel selective and reversible MAO
inhibitors to avoid their undesirable side efects. Selec-
tive MAO‐A inhibitors are generally developed for the
management of depression and anxiety disorders whereas
selective MAO‐B inhibitors are designed for the treatment
of PD and AD with a better safety profle as compared to
nonselective MAO inhibitors [4–8].
Introduction
Monoamine oxidase (MAO) is a favin adenine dinucleo-
tide (FAD)-dependent enzyme located mainly in the mito-
chondrial membrane of CNS and peripheral tissues which
is responsible for the metabolism of neurotransmitters such
as dopamine (DA), serotonin (5-HT), adrenaline (E), and
noradrenaline (NE) and for the inactivation of exogenous
arylalkyl amines. MAO exists in two isoforms such as
MAO-A and -B, which show high sequence similarity, but
exhibit diferent tissue distribution as well as diferent sub-
strate and inhibitor specifcities. MAO-A displays a higher
afnity for 5-HT and NE, while MAO-B prefers phenylethyl
amine (PEA). Although the metabolisms of DA, tryptamine
and tyramine are generated by both isoforms, DA is mainly
degraded by MAO-A in the rodent brain, while MAO-B is
responsible for the metabolism of DA in humans. MAO-A
is selectively inhibited by clorgyline, whereas MAO-B is
inhibited by selegiline (l-deprenyl) [1, 2].
MAO inhibitors have diferent structures such as pyra-
zoline, carboline, cyclopropylamine, hydrazine, hydrazide,
indolalkylamine, oxazolidinone benzothiazinone, coumarin,
safnamide, chromone, anilide and propargylamine [9–12].
Among them, substituted hydrazines and hydrazides, par-
ticularly thiazolylhydrazine derivatives have previously been
described as efective MAOIs showing inhibitory efects at
diferent micromolar concentrations [11, 13]. Pyridazinone
is a six-membered heterocycle which has two contiguous
nitrogen atoms, one carbonyl within the ring, and one endo-
cyclic double bond. Due to this structure, it is considered as
cyclic hydrazine. Pyridazinones have a broad array of bio-
logical activities including antibacterial, analgesic, antino-
ciceptive, anticonvulsant, antiinfammatory, antianemic,
diuretic, antiasthmatic, antisecretory, insecticidal antidepres-
sant, antihypertensive, cardiotonic, anti-cancer, and antiulcer
properties [14–25].
Since brain neurotransmitter levels are associated
with the pathology of various neurological diseases
including Parkinson’s disease (PD) and Alzheimer’s dis-
ease (AD), and MAO plays a critical role in the regula-
tion of these neurotransmitters in brain, MAO isoforms
appear as promising drug targets for the development
of CNS agents. Reversible and irreversible monoamine
oxidase inhibitors (MAOIs) are previously developed,
and are currently in use clinically for the treatment of
1 3

Synthesis, molecular modelling and biological activity of some pyridazinone derivatives…
As the pyridazinone ring structure also has a cyclic
hydrazide group which has been shown earlier to have MAO-
inhibitory activity, some 6-substitute-3(2H)-pyridazinone
derivatives were designed, synthesized and potential MAO-
inhibitory activities were evaluated. The 6th position of
pyridazinone was taken into consideration when making
changes in cognitive structures. Phenyl, substituted phenyl,
benzyl, and other groups are added as substituents in difer-
ent positions of this the ring. The interactions of compounds
with the human MAO (hMAO) isoforms were performed
using molecular docking approach. MAO inhibition efects
on synthesized molecules were determined by recombinant
hMAO isoforms. Since ADMET properties of compounds
are important parameters for determining their druglike
properties, and it has been reported that early estimation of
ADMET in the discovery phase reduces dramatically the
fraction of pharmacokinetics-related failure in the clinical
phases and saves the time and cost, the designed compounds
(4a–4f) were submitted to the ADMET SAR web-server to
perform an in silico analysis of pharmacokinetic parameters
and determine the ADMET properties.
Chloroform phase is separated with sodium sulfate, the chlo-
roform is removed under reduced pressure. The residue was
purifed by crystallization in ethanol.
To obtain 3a–f, 0.01 mol of 6-substituted-3(2H)-pyri-
dazinone, 0.02 mol of ethyl bromoacetate, and 0.02 mol of
potassium carbonate in 40 ml of acetone were prepared. The
mixture was refuxed for about 12 h. The mixture was cooled
after refux. The organic salts were removed by fltration.
Acetone in the reaction medium was removed by evaporation.
The residue was crystallized in methanol. The melting points
of the synthesized compounds were compared with the melt-
ing points of the substances registered in the literature [18,
19, 26–29].
Synthesis of title compounds (4a–f)
0.01 mol of ethyl 6-substituted-3(2H)-pyridazinone-2-ylace-
tate was added to 25 ml of methanol. 3 ml of 99% hydrazine
hydrate solution was added to this mixture and stirred at room
temperature for 3 h. The reaction medium was fltered, the
solid obtained was washed with water and dried. The residue
was crystallized with ethanol to be purifed. The melting points
of the synthesized compounds were compared with the melt-
ing points of the substances registered in the literature [18, 19,
26–29]. The spectral data of the newly synthesized compound
4e are given below. The structure of the compound is in agree-
ment with the spectral data.
Materials and methods
Chemistry
Synthesis starting compounds were obtained from Aldrich
and E. Merck. Starting materials were synthesized accord-
ing to a previous method [17, 19, 26]. The impurities were
checked with TLC. Melting points of compounds were
determined using an electrothermal device. IR spectral
data were obtained by ATR technique using Perkin Elmer
6‑[4‑(Ethoxycarbonyl)
piperazine‑1‑yl]‑3(2H)‑pyridazinone‑2‑yl
acetohydrazide (4e)
White crystals; yield: 79.23%; mp: 253–4 °C; IR (cm–1,
ATR): 3489 (N–H), 3168 (C–H aromatic), 2864 (C–H), 1676
(C=O), 1590 (C=N) and 1241 (C–N);1H-NMR (CDCl3–d,
300 MHz): δ (ppm)1.29 (3H; t; –OCH2CH3), 3.27 (4H; t;
CH2–N), 3.57 (4H; t; CH2–N), 4.15–4.18 (2H; q; –OCH2CH3),
4.69 (2H; s; –N–CH2–C=O), 4.84 (2H; s; –NH2), 6.92 (1H;
d; pyridazinone H5), 7.19 (1H; d; pyridazinone H4) and
7.93 (1H; s; –NH–N). 13C-NMR (100 MHz), δ14.65 (1C;
–OCH2CH3), 43.00 (2C; CH2–N), 46.39 (2C; CH2–N), 54.62
(1C; –N–CH2–C=O), 61.68 (1C; –OCH2CH3), 126.06 (1C;
pyridazinone C5), 131.24 (1C; pyridazinone C4), 149.29 (1C;
pyridazinone C6), 155.41 (1C; CH2–N–C=O), 158.61 (1C;
–O–C=O), and 168.08 (1C; pyridazinone C3); LC/MSMS
(ESI+) m/z 325.1 [M+H]+; anal. Calcd. For C13H20FN6O4.
H2O: C, 45.61; H, 6.48; N, 24.55. Found: C, 45.21; H, 6.164;
N, 24.25%.
1
Spectrometer H-NMR. 13C-NMR spectra were obtained
using Brucker Avonce Ultrashield FT-NMR spectrometer at
300 MHz and 75 MHz, respectively, in DMSO. LC/MSMS
spectra were recorded on Agilent 6400 Series. Triple quad-
rupole elemental analysis of the compounds was carried out
using Leco CHNS-932. All analyses were conducted in the
central research laboratory of Inonu University.
Synthesis of starting compounds (1a–f, 2a–f, 3a–f)
To obtain 1a–f, 0.01 mol of substituted piperazine/piperi-
dine and 0.01 mol of 3,6-dichloropyridazine were dissolved
in 15 ml EtOH, it was stirred under refux for 6 h. And then,
the medium was transferred to ice water. The solid formed
was fltered and purifed by crystallization with ethanol.
To obtain 2a–f, 0.05 mol of 6-substituted-3-chloropyri-
dazinone derivative is dissolved in 30 ml of glacial acetic acid
and was refuxed for 6 h. Acetic acid is removed from the reac-
tion medium with a rotary evaporator. Residue was added to
water and then the extraction is carried out with chloroform.
1 3

Z. Özdemir et al.
drug development. The PAMPA-BBB system mimics the
rate of transcellular passive difusion of drugs across the
BBB by measuring the efective permeability (Pe, cm/s) of
an artifcial lipid membrane [33]. The assay was carried out
in accordance with the instructions. (Pion Inc., USA) [34].
Biological activity
MAO‑inhibitory activity screening
The synthesized derivatives were investigated for their
hMAO-inhibitory effects with recombinant hMAO-A
and hMAO-B enzymes and Amplex Red MAO assay kit
according to the manufacturer’s instructions. Recombinant
enzymes and the other chemicals were purchased from
Sigma-Aldrich (Germany).
In vitro cytotoxicity
Cell viability was measured by a quantitative colorimet-
ric assay with 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide (MTT) (Sigma-Aldrich) [35]. Human
hepatoma cell line HepG2 (Invitrogen) was used. MTT
reduction was measured at 590 nm. Control cells treated
with 0.1% DMSO were used at 100% viability [34]. Signif-
cance was determined using student’s t test. Results were
expressed as mean SEM. Diferences are considered sta-
tistically signifcant at p<0.05.
In the assay, MAO activity is measured continuously.
Tyramine (0.05–0.50 mM) was used as the common sub-
strate for MAO isoforms. Selegiline, lazabemide and
moclobemide were used as specifc MAO inhibitors. Fluo-
rescence was measured using excitation at 530 nm and emis-
sion at 590 nm [30, 31].
Kinetic studies
To understand the mode of MAO inhibition, the synthesized
molecules and reference inhibitors (selegiline: selective and
irreversible MAO-B inhibitor, lazabemide: selective and
reversible MAO-B inhibitor and moclobemide as selective
and reversible MAO-A inhibitor) were dissolved inDMSO
and prepared at concentrations of 0.001–20.00 µM. Results
are shown with double reciprocal Lineweaver–Burk plots.
SI values were calculated as Ki. Protein content of the sam-
ples was determined by Bradford’s method (SIGMA Total
Protein kit, Micro TP0100).
Molecular docking
The three-dimensional structures of compounds were consti-
tuted with Maestro11.8 software with the aid of MacroModel
(Schrödinger, LLC, NY) software and the OPLS_2005 force
feld parameters, and were optimized by conjugated gradi-
ent method [36]. The ligand models were then ligated with
LigPrep (Schrödinger, LLC, NY) software to determine
the appropriate tautomeric and ionization states. The struc-
tures of MAO proteins retrieved from RCSB (www.rcsb.
org) using the protein preparation wizard of Maestro for
hMAO-A and 2Z5X, hMAO-B 4A79 PDB proteins [37].
Within this context, undesirable solvent molecules, ligands
and segments in the crystal structures were deleted using
Prime (Schrödinger, LLC, NY), Impact (Schrödinger, LLC,
NY), Epic (Schrödinger, LLC, NY) and Propka softwares.
The orientations of the polarized hydrogens and the water
have been adjusted; hydrogen molecules were added and
charges were assigned [38]. Grid maps of the binding sites
of proteins were generated using Maestro’s receptor grid-
forming panel and the remaining ligands were ligated 40
times in standard precision mode using Glide (Schrödinger,
LLC, NY) software [39–41]. The mean of the MAO’s scores
were calculated with visually examining anchor poses and
taking into account the ft score of each of the ligaments
determined for each ligand.
Reversibility
Reversibility was calculated with the dialysis method for-
merly reported [32]. Briefy, 16×25 mm of dialysis tubings
with a molecular weight cut-of of 12 was used. Enzyme
samples and the reference inhibitors were incubated with
the compounds at a concentration fvefold of their Ki values
for the inhibition of hMAO-A and –B at 37 °C. Samples
were dialyzed two times in 24 h at 4 °C in dialysis bufer of
100 mM potassium phosphate, pH 7.4, 5% sucrose. Residual
MAO activities were measured following dialysis.
In vitro blood–brain barrier permeation assay
(PAMPA‑BBB)
Since the blood–brain barrier (BBB) plays a major role in
the difusion of the drugs to the CNS, examination of drug
transport through BBB is highly needed for the development
of potential neuropharmaceuticals. Endothelial cell culture
models of BBB are submitted as in vitro systems suitable
for the procedures. Parallel artifcial membrane permeability
assay (PAMPA) is considered to be one of the most pow-
erful physicochemical screening methods in CNS-targeted
The receptor–ligand interactions were simulated and
scored. Theoretical Ki value for each ligand was calculated
with AutoDock 4.2 software [42, 43]. AutoDock4.2 soft-
ware was used to clarify the interactions of molecules with
hMAOs at molecular level. The compounds were docked to
active sites of X-ray crystal structures of hMAO-A (PDB:
2Z5X) and hMAO-B (PDB:4A79) [43].
1 3

Synthesis, molecular modelling and biological activity of some pyridazinone derivatives…
In silico ADMET
Results
Screening of pharmacokinetic parameters
and drug‑likeness
Chemistry
4a–f were synthesized according to the Scheme 1. Com-
pound 4e has not yet been published. Molecular structure of
this new compound was proved with 1H-NMR, 13C-NMR,
LC/MSMS and elementary analyses. Molecular features of
4a–f are given in Table 1.
The compounds were submitted for an in silico ADMET
screening test to indicate the ADME properties of the syn-
thesized derivatives. To have knowledge about whether the
compounds planned to be synthesized can be pharmaceu-
ticals, chemical bioavailability and optimum physical and
chemical properties required for these compounds, Lipin-
ski’s rule of fve, drug-likeness model score, total polar
surface area (TPSA), miLog P, molecular volume (MV),
number of rotatable bonds, number of hydrogen donor
and acceptor atoms and aqueous solubility were calcu-
lated with software. The SMILES codes of the compounds
were obtained for use in the Molinspiration software. Then
some parameters were calculated by this software (https://
www.molinspiration.com).
Biological activity
MAO‑inhibitory screening
Specific enzyme activities were calculated as
0.147 0.009 nmol/mg/min (n = 3) for hMAO-A and
0.134 0.007 nmol/mg/min (n=3) for hMAO-B. All syn-
thesized pyridazinones except compound 4f, which inhib-
ited the MAO isoforms non-selectively, inhibited hMAO-
B selectively at low micromolar concentration ranges
(Table 2).
In silico ADMET prediction
Kinetic studies
The synthesized compounds were prepared in 2D
Sketcher (Maestro, Schrodinger 11.8). The features (a
set of ADMET-related properties which is a total of 46
molecular descriptors) were calculated using the QikProp
program (Maestro, Schrodinger 11.8) running in normal
mode. QikProp is an ADME estimation program that theo-
retically calculates the various parameters of molecules
and uses them to compare them with those of 95% of
known drugs.
Figure 1 shows inhibition of hMAO-B by compound 4b,
the most efective molecule in the series. The slopes of the
Lineweaver–Burk plots were plotted versus the inhibitor
concentration and the Ki value was determined from the
x-axis intercept as−Ki. It has been shown that the lines are
linear and intersect on the y-axis, which suggests that com-
pound 4b is a competitive inhibitor of hMAO-B (Fig. 1). Ki
value was thought to be 0.023 µM.
Scheme 1 Synthesis of the compounds
1 3

Z. Özdemir et al.
Table 1 Molecular structures, yields, melting points and molecular weights of 4a–f
Compounds
A
R
Yield (%)
MP (oC)
MW (g/mol)
Molecular formula
4a
4b
4c
4d
4e
4f
N
N
N
N
N
C
Phenyl
82
81
78
79
60
75
251–252
228–229
207–208
253–254
258–259
218–219
328.37
362.81
397.26
346.36
324.34
341.41
C16H20N6O2
C16H19ClN6O2
C16H18Cl2N6O2
C16H19FN6O2
C13H20N6O4
C18H23N5O2
4-chlorophenyl
3,4-dichlorophenyl
2-fuorophenyl
Ethoxy carbonyl
Benzyl
Table 2 Calculated and experimental Ki values for the inhibition of hMAO isoforms by the synthesized pyridazinone derivatives
Compounds Calculated Calculated
SI
Selectivity Experimental Ki
Experimental SI
Ki (hMAO-B)
[µM]
Inhibition type
Selectivity
Ki hMAO-
A)
Ki (hMAO-
B)
(hMAO-A)
[µM]
[µM]
[µM]
4a
4b
4c
4d
4e
4f
1.95
2.24
1.53
1.18
4.13
1.27
29.34
0.106
0.071
0.108
0.117
0.800
0.633
23.73
21.23
2.64
18.40 MAO-B
31.55 MAO-B
14.17 MAO-B
10.09 MAO-B
5.16 MAO-A
2.01 MAO-B
1.24 MAO-B
12.44 MAO-B
0.71 MAO-A
2.200 0.015
4.550 0.241
1.820 0.110
2.167 0.140
4.220 0.147
1.045 0.002
17.000 1.125
3200.00 55.000
0.013 0.001
0.055 0.002
0.022 0.001 206.82 Competitive
0.050 0.002
0.061 0.003
0.744 0.030
0.997 0.043
0.220 0.013 77.27 Suicide inhibitor MAO-B
0.004 0.001 800.00 Competitive
1.95 0.12 0.007 Competitive
40.00 Competitive
MAO-B
MAO-B
MAO-B
MAO-B
MAO-B
Non-selective
36.40 Competitive
35.52 Competitive
5.67 Competitive
1.05 Competitive
Selegiline
Lazabemide 264.11
Moclobemide 1.87
MAO-B
MAO-A
Each value represents the mean SEM of three independent experiments. The selectivity index (SI) was calculated as Ki(MAO-A)/Ki (MAO-B).
Selectivity towards MAO-A increases as the corresponding SI decreases while selectivity towards MAO-B isoform increases as the correspond-
ing SI increases
To investigate whether the present compounds could pass
this barrier, PAMPA-BBB was used. Assay validation was
made by comparing experimental permeabilities of nine com-
mercial drugs with reported values (Table 4). A plot of experi-
mental data versus reference values gave a good linear correla-
tion (R2=0.997). According to the limits reported earlier [34]
for BBB permeation, compounds were classifed as follows:
Reversibility
The present test showed that selegiline, a wellknown sui-
cide inhibitor of MAO-B appeared as irreversible inhibi-
tor since the residual hMAO-B activity was not recovered
after dialysis (Table 3). On incubation with lazabemide, the
remaining hMAO-B activity was found to be13.00 1.01%.
The enzyme activity was recovered up to 96.87 2.97% after
dialysis indicating that lazabemide is a reversible inhibitor
of hMAO-B.
(
)
CNS + (high BBB permeation predicted): Pe 10−6cm s−1 > 4.00,
(
)
CNS − (low BBB permeation predicted): Pe 10−6cm s−1 < 2.00,
Blood–brain barrier permeation
CNS (BBB permeation uncertain)
(
)
The synthesized derivatives were screened for their ability
to cross the blood–brain barrier (BBB) since getting drugs
across BBB is a key for the design and development of more
successful therapies to treat CNS disorders, like Alzheimer’s
disease and depression.
: Pe 10−6cm s−1 from 4.00 to 2.00.
The data showed that all of the novel pyridazinone
derivatives can cross the BBB successfully. Compounds 4b
1 3

Synthesis, molecular modelling and biological activity of some pyridazinone derivatives…
Fig. 1 The frst graph shows
Lineweaver–Burk plots for
the inhibition of hMAO-B by
compound 4b. Inhibitor concen-
trations are shown at the left. v
velocity (nmol/h/mg protein).
S p-Tyramine (mM). From the
second graph, Ki was found to
be 0.023 µM
showed the highest permeability suggesting that it may cross
the BBB easily and reach the biological targets located in the
CNS, which is highly consistent with our design strategy.
Compounds were dissolved in DMSO at a concentration
of 5 mg/ml and diluted with phosphate bufer solution/etha-
nol (70:30) to give a 25 µg/ml concentration. Final concen-
tration was 100 µg/ml. Results are expressed as the SEM of
three separate experiments. Values for the standard drugs
were obtained from the reference by Di et al. [34].
(Table 5). The results showed that these compounds were
not toxic to hepatic cells at 1 µM and 5 µM concentrations.
Compounds, except 4f, did not have any cytotoxicity at
25 µM concentration.
Molecular docking
The phenyl ring in the structure of the highest active com-
pound 4b interacted with π–π cation and with TRP 119 and
PHE 103 in the active site of the enzyme. The hydrazine
(NH2–NH–) group of compound 4b also interacted via
hydrogen bonds with PRO102 and GLU84 and carbonyl
(C=O) groups with TYR 326 (Fig. 2).
Cytotoxicity
In vitro cytotoxicity of the synthesized pyridazinone deriva-
tives were tested at three diferent concentrations (1–25 µM)
1 3

Z. Özdemir et al.
Table 3 Reversibility of the inhibition of hMAO isoforms by synthesized pyridazinone derivatives
Compounds incubated with hMAO-A activity
hMAO-A activity after hMAO-B activity
hMAO-B activity after Reversibility
dialysis (%)
hMAO isoforms
before dialysis (%)
dialysis (%)
before dialysis (%)
4a
4b
4c
4d
4e
4f
85.01 3.00
87.00 2.25
80.26 2.59
85.22 2.33
79.90 2.61
79.90 3.77
31.11 1.90
81.26 1.79
91.26 1.67
100 0.00
95.45 2.35
97.60 3.06
97.00 2.85
96.89 2.90
95.05 3.07
94.79 2.66
98.54 2.61
97.00 2.04
97.50 2.55
100 0.00
20.14 1.06
12.66 1.00
31.22 2.70
29.60 1.87
39.0 2.48
39.75 1.96
81.55 1.96
42.90 1.87
13.00 1.01
100 0.00
98.00 2.63
97.22 2.85
98.14 2.44
98.06 2.37
97.56 2.13
95.77 2.29
97.00 2.62
45.55 1.74
96.87 2.97
100 0.00
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Irreversible
Reversible
–
Moclobemide
Selegiline
Lazabemide
Control
*Each value represents the mean SEM (n=3). Control: without inhibitor
Table 4 Experimental permeability (Pe 10–6 cm s–1)) in the PAMPA-
BBB assay for standard drugs used in the validation of the assay and
for the synthesized pyridazinone derivatives
Table 5 In vitro cytotoxicity of synthesized pyridazinone derivatives
Code % Viability
0.25 μM
0.5 μM
1 μM
Compounds
Bibliography
Pe (×10–
Experimental
Prediction
Pe (×10–6 cm s–1)
4a
4b
4c
4d
4e
4f
77.36 1.20
83.28 2.72
76.42 3.18
80.14 1.15
79.66 0.88
74.02 2.08
71.06 1.15
80.26 1.45
75.00 1.73
79.18 2.84
76.26 2.96
70.24 1.45
69.30 1.45
79.04 0.68
73.46 1.33
80.05 0.57
69.36 2.18
62.00 1.52 *
6 cm s–1)
Testosterone
Verapamil
β-estradiol
Progesterone
Corticosterone
Piroxicam
Hydrocortisone
Lomefoxacin
Dopamine
4a
4b
4c
4d
4e
17.0
16.0
12.0
9.3
16.11 1.02
15.55 1.11
11.33 0.98
9.13 0.29
4.78 0.30
2.40 0.12
1.78 0.09
1.07 0.08
0.22 0.01
12.88 0.99
15.27 0.75
10.55 0.96
11.27 0.84
9.55 0.68
7.11 0.43
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+/–
CNS–
5.1
Data are expressed as mean SEM (n=3). Cell viability is expressed
as a percentage of the control value. p<0.05 is considered as statisti-
cally signifcant (*p<0.05 vs. control)
2.5
1.8
1.1
CNS–
CNS–
0.2
Table S1–S5). QPlogPo/w values showed that the synthesized
compounds have an ideal lipophilicity other than compound
4e (0.9). The QPlogPo/w value of Compound 4e was quite
low due to the polar groups it carries unlike other compounds.
According to the results of the intestinal barrier (QPPCaco)
and blood–brain barrier (QPPMDCK) calculated for the 4a–4f
compounds, other than 4e, especially compounds 4b and 4c,
showed very good permeability. A very low blood–brain bar-
rier passage is envisaged for compound 4e with a QPPMDCK
of 35.8. This also coincides with the experimentally calculated
blood–brain barrier transition. Oral absorption values of the
compounds were also predicted to be quite good except for
compound 4e (67%) with values of 80% or more. As a result,
according to the estimated ADMET study results, all com-
pounds comply with Lipinski’s fve rules and Jorgensen’s 3 s
rule.
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
4f
To interpret the interactions, the interaction of the inhibitor
with the active site of the enzyme is shown by compound 4b.
Docking studies have shown that compound 4b is near the
FAD cofactor in the active binding site of the enzyme (Fig. 3).
Docking studies showed that 4b is bound to the catalytic
site of 4A79 with high afnity (Table 6).
In Silico ADMET prediction
All calculated descriptive properties of 4a–4f were calcu-
lated within the recommended value ranges (Table 7 and
1 3

Synthesis, molecular modelling and biological activity of some pyridazinone derivatives…
Table 6 Molecular docking
Compound Docking score
Glide AutoDock
–8.156 –9.52
binding scores and binding
interactions of 4a–f within the
MAO-B (4A79) active site
4a
4b
4c
4d
4e
4f
–8.54 –9.75
–7.970 –9.5
–8.073 –9.46
–7.383 –8.32
–8.192 –7.87
phenyl, substituted phenyl, ethoxy carbonyl and benzyl
groups were attached to the fourth position of piperazine
or piperidine rings located at the sixth position of the pyri-
dazinone structure.
MAO isoforms are described earlier to difer in their
oligomerization architecture and structures of their active
sites. The active sites of the MAOs consist of a hydrophobic
cavity located in front of the favin cofactor. The only hydro-
philic section is near the favin and is required for recogni-
tion and directionality of the substrate amine functionality.
This hydrophilic region plays a key role in substrate binding
[44]. The binding of substrates or inhibitors to hMAO-B
involves an initial negotiation of a protein loop located near
the membrane surface and two hydrophobic cavities called
as “entrance cavity” and “active site cavity” [45, 46].
It seems that pyridazinone, the main skeleton which is
present in the structure of this series and the existence of
the piperazine or piperidine ring linked to the pyridazinone
seems to be necessary and important for the interaction of
molecules with hMAO isoforms. Compounds 4a, 4b, 4c and
4d which are bearing phenyl substitution at piperazine ring
inhibited hMAO-B potently via low Ki values (0.055 0.020,
0.022 0.001, 0.050 0.002 and 0.061 0.003 µM, respec-
tively). Compound 4e, which is carrying ethoxy carbonyl
group substituted to piperazine ring also inhibited hMAO-
B (Ki=0.744 0.030 µM), however, inhibitory potency of
compound 4e was found to be lower than that of compounds
4a, 4b, 4c and 4d suggesting that phenyl substitution at
piperazine ring enhances the hMAO-B inhibitory potency.
Piperazine ring linked to pyridazinone skeleton appears to
be essential for better hMAO-B inhibitory activity in this
series since compound 4f, which is having piperidine ring
linked to pyridazinone, inhibited hMAO-B less potently
(Ki=0.997 0.043 µM). Compound 4f exhibited no selec-
tivity towards hMAO isoforms whereas compounds 4a, 4b,
4c, 4d and 4e which are having piperazine ring linked to
pyridazinone inhibited hMAO-B selectively suggesting that
piperazine ring linked to pyridazinone increases the selectiv-
ity of the compound towards hMAO-B isoform (Table 2).
Among the compounds which possess piperazine ring
attached to the pyridazinone backbone, compound 4b was
found as the most effective MAO-B inhibitor, bearing
Fig. 2 Superimposition of the binding modes of 4b on MAO-B
(4A79) catalytic site obtained from Glide
Fig. 3 Binding of compound 4b to the MAO-B binding cavity
Discussion
Biological activity
In the present study, six pyridazinone derivatives were
synthesized to examine the relationship between the struc-
ture and activity of the compounds. For this purpose,
1 3

Z. Özdemir et al.
Table 7 Some pharmacokinetic
parameters important for
good oral bioavailability of
compound 4a–4f
Compounds Percent human QPPMDCK QPlogBB
oral absorption
MV
–
297.06 1.896
310.59 2.407
324.13 2.809
301.99 2.021
286.98 0.904
317.90 2.564
miLog P TPSA Drug like-
ness model
score
–
–
–3< <–1
–1.3
≤5
–
–
4a
4b
4c
4d
4e
4f
79.8
83.4
85.7
80.5
67.1
84.5
94.0
252.0
526.1
126.5
35.8
104.3
96.49 –0.17
96.49 –0.20
96.49 –0.21
96.49 –0.19
122.8 –0.12
93.26 –0.08
–1.2
–1.0
–1.3
–1.9
–1.3
Percent human oral absorption predicted human oral absorption on 0 to 100% scale. The prediction is
based on a quantitative multiple linear regression model.>80% is high<20% is poor. QPPMDCK pre-
dicted apparent MDCK cell permeability in nm/sec. MDCK cells are considered to be good mimics of the
blood–brain barrier
4-chlorophenyl ring as R group at the piperazine ring. This
compound (Ki = 0.022 0.001 µM) inhibited hMAO-B
which indicates that the compound 4b is more efective than
selegiline, (Ki=0.220 0.013 µM). The selectivity of com-
pound 4b towards hMAO-B also was found to be the high-
est one (SI=206.82) among the synthesized pyridazinone
derivatives. SI value of compound 4b appeared to be much
better than that of selegiline (SI=77.27). It has been postu-
lated that halogen substituents in the phenyl ring enhances
the MAO-B inhibition since halogen substituents improve
the steric packing of small-molecules through Vander Waals
interactions which facilitates binding of the derivatives to
the hydrophobic active site of hMAO-B [47].
potency and selectivity towards hMAO-B (SI=5.67) were
found to be worse than those of compounds carrying phenyl
ring as R group.
Reversibilty
The encouraging therapeutic efects of reversible, selective
and competitive MAO inhibitors has prompted the research-
ers in the last years to investigate novel, selective and revers-
ible drug candidates with no tyramine pressure response.
Since the irreversible clinical use of MAO inhibitors is
reported to be due to their serious cardiovascular or gastro-
intestinal side efects, current studies are mostly focused on
the discovery of highly selective and reversible ones [48].
We describe here a series of pyridazinone derivaties as
potent, selective and reversible inhibitors of hMAO-B which
may have considerable advantages compared to irreversible
inhibitors possessing serious pharmacological side efects.
Our experimental study showed that compound 4c, which
carries 3,4-dichlorophenyl substitution at R position of pyri-
dazinone moiety has also been found as a potent hMAO-B
inhibitor with a Ki value of 0.050 0.002 µM which makes it
as more potent than selegiline. However, the hMAO-B inhib-
itory potency of compound 4c appeared to be lower than that
of the compound 4b suggesting that the substitution of sec-
ond chloride in phenyl ring decreases the inhibitory potency
of the pyridazinones possibly causing the increase of the
distance between the compound and the residues located in
the region which is associated with activity. This substitution
also caused a decrease in the selectivity of the compound 4c
towards hMAO-B (SI=36.40).
Molecular docking
Compound 4a carrying a non-substituted phenyl ring reacted
π–π cation and with TRP 119 and PHE 103 in regions asso-
ciated with activity, like compound 4b. NH group also forms
hydrogen bonds with TYR 326. Compound 4d bearing fuo-
rine substituent at second position instead of chlorine in the
phenyl ring reacted via π–π cation interaction with TRP 119
and PHE 103 and hydrogen bonds with NH2 group and TYR
326 in a similar manner to compound 4b. The fuorine in the
structure of 4d did not interact with any residues in region
associated with activity of the enzyme, but caused a change
in the conformation of the molecule. Due to this conforma-
tional change, the molecule could not form make hydrogen
bonds with PRO102 and GLU84. Decrease in activity of
compound 4d can be explained by loss of interaction with
PRO102 and GLU84 in the active site of the enzyme. The
structural diference of compound 4c from compound 4b is
Compound 4d, which is carrying 2-fuorophenyl ring as R
group at the piperazine ring also inhibited hMAO-B. How-
ever, inhibitory potency and the selectivity towards hMAO-
B of this compound (Ki = 0.061 0.003 µM; SI = 35.52)
were lower than those of the compounds 4b and 4c which
are having chloride substitutions at phenyl ring possibly due
to the relatively weak interaction of the fuoride atom with
the amino acids located in the region associated with activ-
ity or the steric hindrance of fuoride. Compound 4e, which
is bearing ethoxy carbonyl group as R group at the pipera-
zine ring inhibited hMAO-B selectively, but its inhibitory
1 3

Synthesis, molecular modelling and biological activity of some pyridazinone derivatives…
Fig. 4 Superimposition of the binding modes of Moclobemide and 4b on MAO-A (2Z5X) catalytic site obtained from Glide
that it contains an additional chlorine substituent in the third
position. The chlorine atoms in the structure of 4c did not
interact directly with the residues in the active site of the
enzyme. The interaction of TRP 119 and PHE 103, which
we think is related to activity along with the addition of
an extra chlorine atom to the structure, has not been lost;
however, the interaction of PRO102, GLU84 and TYR 326,
has disappeared. The activity is, therefore, thought to be
reduced.
VAL209 and hydrophobic interaction with SER209 and
GLN215 in the active site of the enzyme (Fig. 4).
Conclusion
In recent years pyridazinones has become the subject of
a large number of research due to their wide spectrum of
biological activities such as analgesic, antiinfammatory,
antidepressant, antihypertensive, antithrombotic, diuretics
and anti-HIV [49]. The pyridazinone derivatives bearing
hydrazide moiety were synthesized and their human mono-
amine oxidase (hMAO) inhibitory activities were evaluated
by molecular docking studies by in silico ADME prediction
and in vitro biological screening tests. Most of the synthe-
sized compounds inhibited hMAO-B selectively except com-
pound 4f. Compounds 4a–4e inhibited hMAO-B selectively
and reversibly in a competitive mode. Compound 4b, having
4-chlorophenyl ring as R group attached to piperazine moi-
ety was found to be the most potent (ki=0.022 0.001 µM)
and selective (SI (Ki hMAO-A/hMAO-B) = 206.82) hMAO-B
inhibitor in synthesized compounds.
In the compound 4e, unlike the other compounds, there
is no phenyl ring. Therefore, the interaction of TRP 119
and PHE 103 with π–π cation was eliminated and the activ-
ity was signifcantly decreased. In addition, as a result of
conformational changes, the hydrazide substituent of the
molecule bonded only with TYR 326 via hydrogen bond-
ing. Compound 4f, in contrast to other compounds, carries
piperidine instead of piperazine ring. Furthermore, the ben-
zyl group is attached to position four of the piperidine ring.
The introduction of a carbon atom between the piperidine
and the phenyl ring changed the planarity of the molecule.
As a result of this conformational change, the phenyl ring
was removed from the residues, TRP 119 and PHE 103,
which were important for activity and could not interact. In
addition, the interaction of PRO102, GLU84 and TYR 326
disappeared and activity was signifcantly reduced.
As a result of the docking studies, the MAO-B inhibi-
tor efect on the synthesized compounds was closely related
to the interaction of the enzyme with TRP 119, PHE 103,
PRO102, GLU84 and TYR 326 residues. The pyridazinone
ring and hydrazide group in the structure of the compounds
are important factors in the placement of the compounds in
the enzyme active site. In silico ADMET prediction stud-
ies showed that the compounds were drug like in terms of
most of the physicochemical parameters and they possessed
generally favourable pharmacokinetic properties except their
hERG inhibition potential.
The synthesized compounds showed no selective inhibi-
tory properties against MAO-A. To explain the reason for
this low selectivity, the interaction of moclobemide, which
is used as a reference compound in activity studies, with
the active site of the enzyme 2Z5X was also investigated by
docking studies. Figure 3 shows that Moclobemide inter-
acts with the hydrogen bonding and π-cation with PHE208
and π–π cation with TYR407. In addition, the molecule
was found to have polar interaction with TYR444, ILE207,
1 3

Z. Özdemir et al.
thiazolylhydrazine derivatives as selective and reversible hMAO-
A inhibitors. Eur J Med Chem. 2018;144:68–81.
In addition, the compliance with Lipinski’s fve rules is
very important for the compounds to be drugs, and all of the
compounds we synthesized comply with. Within the scope
of this information, the compounds are considered to be
potentially selective MAO-B inhibitors.
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Compliance with ethical standards
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