Synthesis and biological evaluation of new pyrazolone Schiff bases as monoamine oxidase and cholinesterase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.11.016

In the current work, Schiff base derivatives of antipyrine were synthesized. The chemical characterization of the compounds was confirmed using IR, ¹H NMR, ¹³C NMR and mass spectroscopies. The inhibitory potency of synthesized compounds was investigated towards acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and monoamine oxidases A and B (MAO-A and MAO-B) enzymes. Some of the compounds displayed significant inhibitory activity against AChE and MAO-B enzymes, respectively. According to AChE enzyme inhibition assay, compounds 3e and 3g were found as the most potent derivatives with IC₅₀ values of 0.285 μM and 0.057 μM, respectively. Also, compounds 3a (IC₅₀ = 0.114 μM), 3h (IC₅₀ = 0.049 μM), and 3i (IC₅₀ = 0.054 μM) were the most active derivatives against MAO-B enzyme activity. So as to understand inhibition type, enzyme kinetics studies were carried out. Furthermore, molecular docking studies were performed to define and evaluate the interaction mechanism between compounds 3g and 3h and related enzymes. ADME (Absorption, Distribution, Metabolism, and Excretion) and BBB (Blood, Brain, Barier) permeability predictions were applied to estimate pharmacokinetic profiles of synthesized compounds.

Full text of DOI:10.1016/j.bioorg.2018.11.016

Accepted Manuscript
Synthesis and Biological Evaluation of New Pyrazolone Schiff Bases as Mono-
amine Oxidase and Cholinesterase Inhibitors
Fatih Tok, Bedia Koçyiğit-Kaymakç ıoğlu, Begüm Nurpelin Sağlık, Serkan
Levent, Yusuf Özkay, Zafer Asım Kaplancıklı
PII:
S0045-2068(18)30873-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.016
YBIOO 2624
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 August 2018
12 November 2018
13 November 2018
Please cite this article as: F. Tok, B. Koçyiğit-Kaymakç ıoğlu, B. Nurpelin Sağlık, S. Levent, Y. Özkay, Z. Asım
Kaplancıklı, Synthesis and Biological Evaluation of New Pyrazolone Schiff Bases as Monoamine Oxidase and
Cholinesterase Inhibitors, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.11.016
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Synthesis and Biological Evaluation of New Pyrazolone Schiff Bases as Monoamine
Oxidase and Cholinesterase Inhibitors
Fatih Tok1, Bedia Koçyiğit-Kaymakçıoğlu1, Begüm Nurpelin Sağlık2,3, Serkan Levent2,3,
Yusuf Özkay2,3* and Zafer Asım Kaplancıklı2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, İstanbul,
Turkey
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskişehir,
Turkey
3Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu Universty,
Eskişehir, Turkey
*Corresponding author.
E-mail address: yozkay@anadolu.edu.tr (Y. Özkay).
Tel: +90-222-3350580/3779 Fax: +90-222-3350750.
Address: Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470,
Eskişehir, Turkey.

Abstract
In the current work, Schiff base derivatives of antipyrine were synthesized. The chemical
1
characterization of the compounds was confirmed using IR, H-NMR, 13C-NMR and mass
spectroscopies. The inhibitory potency of synthesized compounds was investigated towards
acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and monoamine oxidases A and
B (MAO-A and MAO-B) enzymes. Some of the compounds displayed significant inhibitory
activity against AChE and MAO-B enzymes, respectively. According to AChE enzyme
inhibition assay, compounds 3e and 3g were found as the most potent derivatives with IC50
values of 0.285 µM and 0.057 µM, respectively. Also, compounds 3a (IC50 = 0.114 µM), 3h
(IC50 = 0.049 µM), and 3i (IC50 =0.054 µM) were the most active derivatives against MAO-B
enzyme activity. So as to understand inhibition type, enzyme kinetics studies were carried out.
Furthermore, molecular docking studies were performed to define and evaluate the interaction
mechanism between compounds 3g and 3h and related enzymes. ADME (Absorption,
Distribution, Metabolism, and Excretion) and BBB (Blood, Brain, Barier) permeability
predictions were applied to estimate pharmacokinetic profiles of synthesized compounds.
Keywords: Schiff base; Acetylcholinesterase; Butyrylcholinesterase; Monoamine oxidase A;
Monoamine oxidase B; Docking.

1. Introduction
Alzheimer's disease (AD) is a degenerative disease of the central nervous system (CNS),
which is characterized by mental deterioration, especially in the elderly. The "cholinergic
hypothesis" means the inadequate cholinergic transmission in the synapse and constitutes the
basis of treatment in the AD. According to this hypothesis, cholinesterase (ChE) inhibitors
increase the acetylcholine level in the synapse and help reverse the symptoms of the disease
[1,2]. There are two ChE enzymes. Acetylcholinesterase (AChE) is the main enzyme
responsible for acetylcholine (ACh) hydrolysis in the cholinergic synapses, whereas
butyrylcholinesterase (BuChE) serves as a co-regulator of AChE activity. While ACh is the
substrate of AChE, butyrylcholine (BuCh) is the substrate of BChE and this issue is the main
difference between these two enzymes [3,4]. Therapeutic agents which are capable of
inhibiting both enzymes may provide additional benefits for AD. Current treatments for AD
mainly focus on the use of AChE inhibitors, namely donepezil, rivastigmin, galantamine,
which are accepted by the FDA. These drugs have only symptomatic effects, and therefore,
there is a need for the discovery of more effective agents to stop the progression of the disease
[5-7].
In the regulation of different biological processes, monoamine oxidase (MAO) enzymes play
a considerable role and, thereby, they are crucial targets in drug design for the treatment of
psychiatric and neurological disorders [8]. MAO-A inhibitors are clinically used
predominantly as antidepressant, anxiolytic and against autism [9,10], whereas MAO-B
inhibitors are generally used to treat symptoms related to Parkinson's and Alzheimer's disease
[11]. Despite their long clinical success, first-generation MAO inhibitors have several
problems including interactions with other drugs and tyramine, which is a dietary amine and
may cause a fatal hypertensive crisis. In view of these adverse actions and restrictions, the
clinical use of first generation of MAO inhibitors have been decreased, whereas second
generation MAO inhibitors such as selegiline and moclobemide have come into prominence
due to lack of above problems [12, 13]. In light of the above-mentioned information, the
interest of medical chemists in the development of new and potent MAO inhibitors has
increased.
In recent years, investigations on physical, chemical, and biological properties of pyrazoles
have given promising information. With their structural multiplicity and degree of nature, the
pyrazole derivatives exhibit various pharmacological properties [14]. The change of their
structures provides a high degree of diversity, which has proven useful in the investigation of

new therapeutic agents. Pyrazole ring system and its hydrogenated analogues pyrazoline and
pyrazolidine can be considered as a cyclic hydrazine, which is a pharmacophore in several
MAO-inhibitors and also displays high affinity for various enzymes including cholinesterases
[15,16]. Thus pyrazole derivatives fit well to design strategy of new effective compounds
targeting AChE and/or MAO [17]. In literature there are several studies including enzyme
inhibitory activities of pyrazole derivatives against MAO [18-22], AChE [23-25] and both
enzymes [26-28].
Above observations encouraged us to perform a study targeting the development of new
AChE and/or MAO inhibitors that may be beneficial in the treatment of AD. Thus, herein we
present the synthesis and enzyme inhibition studies of new pyrazolone derivatives.
2. Materials and methods
2.1. Chemistry
2.1.1. General
Sigma Aldrich (St. Louis, Mo., USA), S.D Fine Chemicals (India) and Merck (Darmstadt,
Germany) are suppliers of chemicals, reagents and solvents used in the study. All solvents and
reagents were of reagent grade and used directly without purification. The monitoring of the
reaction progress was accomplished by thin layer chromatography (TLC) on commercially
available silica gel (Kieselgel 60, F254) coated aluminum layers (Merck) with the solvent
system petroleum ether:ethyl acetate (10:90). Electrothermal 9100 digital melting point
apparatus (Electrothermal, Essex, UK) and Shimadzu FTIR 8400 S (Kyoto, Japan) were used
for melting point and infrared spectroscopy analysis, respectively. Proton and carbon NMR
spectra of compounds in DMSO-d6 were obtained by DPX 300 NMR spectrometry (Bruker
Bioscience, Billerica, Mass., USA) and Bruker DPX 75 NMR spectrometry, respectively. The
mass measurements were carried out by High Resolution Mass Spectroscopy system
Shimadzu LCMS-IT-TOF instrument equipped with SPD-M20A PDA detector (Kyoto,
Japan) which was also used to control purity indexes.
2.1.2. Synthesis of 1,5-dimethyl-4-nitroso-2-phenyl-1H-pyrazol-3(2H)-one (1)
Antipyrine (50 mmol, 9.41 g) was dissolved in acetic acid (50 mL). The solution was cooled
o
to 0-5 C and sodium nitrite (60 mmol, 4.14 g) in water (25 mL). The final solution was
stirred for 20 minutes and then diluted with water. The precipitated product was filtered and
dried [29].

2.1.3. Synthesis of 4-amino-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (2)
Acetic acid (10 mL) was added on 4-nitrosoantipyrine (23 mmol, 5 g) in water-ethanol
solution. The mixture was cooled to 0-5 ° C and stirred continuously on a magnetic stirrer.
Zinc dust was added in portions until color of the solution changed. The precipitated product
was filtered and dried [30].
2.1.4. Synthesis of 4-Substituted Antipyrine Derivatives (3a-3p)
A mixture of 4-aminoantipyrine (1 mmol, 0.23 g) and appropriate substituted aldehyde (1
mmol) in ethanol (10 ml) was refluxed for 4 hours with the presence of a few drops of glacial
acetic acid. The reaction was completed by thin layer chromatography control. The precipitate
was filtered, dried and purified with methanol [31].
2.1.4.1. 4-(4-Hydroxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3a)
Yield 72%, m.p. 232 ºC. FTIR (ATR, cm-1): 3072 (aromatic C-H), 1654 (C=O), 1579-1425
1
(C=C, C=N), 1311 (C-O), 1157 (C-N). H-NMR (300 MHz, DMSO-d6, ppm)  2.41 (s, 3H,
C-CH3), 3.12 (s, 3H, N-CH3), 6.82-7.66 (m, 9H, Ar-H), 9.46 (s, 1H, =CH-), 9.90 (s, 1H, OH).
13C-NMR (75 MHz, DMSO-d6, ppm)  10.26, 36.09, 116.06, 117.51, 124.70, 127.09, 129.30,
129.55, 129.56, 135.23, 152.16, 155.33, 160.13, 160.41 HRMS (m/z): [M+H]+ calcd for
C18H17N3O: 308.1394; found 308.1396.
2.1.4.2. 4-(2,4-Dichlorobenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3b)
Yield 72%, m.p. 211 ºC. FTIR (ATR, cm-1): 3049 (aromatic C-H), 2924 (C-H), 1643 (C=O),
1
1581-1487 (C=C, C=N), 1128 (C-N), 1049 (C-Cl). H-NMR (300 MHz, DMSO-d6, ppm) 
2.48 (s, 3H, C-CH3), 3.24 (s, 3H, N-CH3), 7.36-8.18 (m, 8H, Ar-H), 9.92 (s, 1H, =CH-). 13C-
NMR (75 MHz, DMSO-d6, ppm)  10.14, 35.49, 116.41, 125.51, 127.70, 128.26, 128.32,
129.64, 129.77, 133.95, 134.74, 134.90, 135.37, 148.80, 152.82, 159.65. HRMS (m/z):
[M+H]+ calcd for C18H15N3OCl2: 360.0665; found 360.0682.
2.1.4.3.
1,5-Dimethyl-2-phenyl-4-(4-(piperidin-1-yl)benzylideneamino)-1H-pyrazol-3(2H)-
one (3c)
Yield 62%, m.p. 217 ºC. FTIR (ATR, cm-1): 3052 (aromatic C-H), 2918-2848 (C-H), 1647
(C=O), 1591-1454 (C=C, C=N), 1180 (C-N). 1H-NMR (300 MHz, DMSO-d6, ppm)  1.58 (s,
6H, piperidine –CH2-), 2.41 (s, 3H, C-CH3), 3.11 (s, 3H, N-CH3), 3.32 (s, 4H, piperidine N-
CH2-), 6.94-7.64 (m, 9H, Ar-H), 9.45 (s, 1H, =CH-). 13C-NMR (75 MHz, DMSO-d6, ppm) 

10.28, 24.41, 25.45, 36.19, 48.92, 114.95, 117.89, 124.59, 127.00, 127.46, 129.11, 129.53,
135.30, 151.95, 153.07, 155.51, 160.53. HRMS (m/z): [M+H]+ calcd for C23H26N4O:
375.2179; found 375.2182.
2.1.4.4. 4-(3-Ethoxy-4-hydroxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-
one (3d)
Yield 72%, m.p. 218 ºC. FTIR (ATR, cm-1): 3115 (aromatic C-H), 2972-2918 (C-H), 1620
1
(C=O), 1575-1415 (C=C, C=N), 1375 (C-O), 1126 (C-N). H-NMR (300 MHz, DMSO-d6,
ppm)  1.34 (t, 3H, CH3), 2.43 (s, 3H, C-CH3), 3.13 (s, 3H, N-CH3), 4.05 (q, 2H, -OCH2-),
6.84-7.55 (m, 8H, Ar-H), 9.42 (s, 1H, =CH-). 13C-NMR (75 MHz, DMSO-d6, ppm)  10.30,
15.20, 36.10, 64.27, 111.31, 115.97, 124.70, 127.09, 129.30, 129.55, 129.56, 135.23, 152.16,
155.33, 160.13, 160.41. HRMS (m/z): [M+H]+ calcd for C20H21N3O3: 352.1656; found
352.1662.
2.1.4.5. 4-(2,6-Dimethylbenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3e)
Yield 65%, m.p. 119 ºC. FTIR (ATR, cm-1): 3062 (aromatic C-H), 2914 (C-H), 1651 (C=O),
1593-1402 (C=C, C=N), 1134 (C-N). 1H-NMR (300 MHz, DMSO-d6, ppm)  2.41 (s, 3H, C-
CH3), 2.48 (s, 6H, Ar-CH3), 3.17 (s, 3H, N-CH3), 7.07-7.55 (m, 8H, Ar-H), 9.94 (s, 1H, =CH-
). 13C-NMR (75 MHz, DMSO-d6, ppm)  10.26, 21.52, 35.84, 117.72, 124.99, 127.28, 129.10,
129.33, 129.56, 134.44, 135.05, 137.82, 152.69, 156.38, 159.97. HRMS (m/z): [M+H]+ calcd
for C20H21N3O: 320.1757; found 320.1764.
2.1.4.6. 4-(2,6-Dichlorobenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3f)
Yield 72%, m.p. 199 ºC. FTIR (ATR, cm-1): 3047 (aromatic C-H), 2929 (C-H), 1647 (C=O),
1579-1413 (C=C, C=N), 1307 (C-O), 1192 (C-N), 1087 (C-Cl). 1H-NMR (300 MHz, DMSO-
d6, ppm)  2.42 (s, 3H, C-CH3), 3.23 (s, 3H, N-CH3), 7.37-7.56 (m, 8H, Ar-H), 9.77 (s, 1H,
=CH-). 13C-NMR (75 MHz, DMSO-d6, ppm)  10.12, 35.42, 116.06, 125.58, 127.72, 129.64,
129.83, 131.14, 133.38, 134.29, 134.71, 150.19, 153.03, 159.46. HRMS (m/z): [M+H]+ calcd
for C18H15N3OCl2: 360.0665; found 360.0680.
2.1.4.7.
4-(4-(3-(Dimethylamino)propoxy)benzylideneamino)-1,5-dimethyl-2-phenyl-1H-
pyrazol-3(2H)-one (3g)

Yield 79%, m.p. 146 ºC. FTIR (ATR, cm-1): 3072 (aromatic C-H), 2924-2802 (C-H), 1647
1
(C=O), 1606-1423 (C=C, C=N), 1311 (C-O), 1157 (C-N). H-NMR (300 MHz, DMSO-d6,
ppm)  1.80 (m, 2H, CH2), 2.13 (s, 6H, N-CH3), 2.31 (t, 2H, NCH2), 2.42 (s, 3H, C-CH3),
3.13 (s, 3H, N-CH3), 4.01 (t, 2H, OCH2), 6.97-7.75 (m, 9H, Ar-H), 9.51 (s, 1H, =CH-). 13C-
NMR (75 MHz, DMSO-d6, ppm)  10.23, 27.31, 35.99, 45.66, 56.08, 66.42, 115.10, 117.25,
124.80, 127.18, 129.35, 129.58, 130.66, 135.13, 152.31, 154.81, 160.29, 160.92. HRMS
(m/z): [M+H]+ calcd for C23H28N4O: 393.2285; found 393.2286.
2.1.4.8. 1,5-Dimethyl-4-(4-nitrobenzylideneamino)-2-phenyl-1H-pyrazol-3(2H)-one (3h)
Yield 72%, m.p. 259 ºC. FTIR (ATR, cm-1): 3078 (aromatic C-H), 2960 (C-H), 1637 (C=O),
1
1570-1487 (C=C, C=N, NO2), 1128 (C-N). H-NMR (300 MHz, DMSO-d6, ppm)  2.48 (s,
3H, C-CH3), 3.25 (s, 3H, N-CH3), 7.36-8.30 (m, 9H, Ar-H), 9.66 (s, 1H, =CH-). 13C-NMR (75
MHz, DMSO-d6, ppm)  10.16, 35.42, 124.48, 125.74, 127.86, 128.37, 129.70, 134.70,
144.05, 148.24, 151.27, 152.85, 159.45, 164.07. HRMS (m/z): [M+H]+ calcd for C18H16N4O3:
337.1295; found 337.1308.
2.1.4.9. 1,5-Dimethyl-4-(3-nitrobenzylideneamino)-2-phenyl-1H-pyrazol-3(2H)-one (3i)
Yield 72%, m.p. 218 ºC. FTIR (ATR, cm-1): 3061 (aromatic C-H), 2945-2877 (C-H), 1620
1
(C=O), 1593-1413 (C=C, C=N, NO2), 1136 (C-N). H-NMR (300 MHz, DMSO-d6, ppm) 
2.48 (s, 3H, C-CH3), 3.22 (s, 3H, N-CH3), 7.36-8.58 (m, 9H, Ar-H), 9.65 (s, 1H, =CH-). 13C-
NMR (75 MHz, DMSO-d6, ppm)  10.21, 35.51, 115.94, 121.00, 124.62, 125.49, 127.70,
129.68, 130.84, 134.09, 134.74, 139.74, 148.80, 151.65, 152.73, 159.62. HRMS (m/z):
[M+H]+ calcd for C18H16N4O3: 337.1295; found 337.1310.
2.1.4.10. 4-(4-Chlorobenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3j)
Yield 72%, m.p. 254 ºC. FTIR (ATR, cm-1): 3059 (aromatic C-H), 2941 (C-H), 1647 (C=O),
1
1591-1481 (C=C, C=N), 1132 (C-N), 1070 (C-Cl). H-NMR (300 MHz, DMSO-d6, ppm) 
2.45 (s, 3H, C-CH3), 3.19 (s, 3H, N-CH3), 7.35-7.84 (m, 9H, Ar-H), 9.56 (s, 1H, =CH-). 13C-
NMR (75 MHz, DMSO-d6, ppm)  10.19, 35.71, 116.46, 125.22, 127.49, 129.24, 129.29,
129.63, 134.98, 136.90, 152.68, 153.13, 159.93. HRMS (m/z): [M+H]+ calcd for
C18H16N3OCl: 326.1055; found 326.1061.
2.1.4.11.
4-(3,4-Dichlorobenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
(3k)

Yield 72%, m.p. 239 ºC. FTIR (ATR, cm-1): 3084 (aromatic C-H), 2964 (C-H), 1643 (C=O),
1
1589-1411 (C=C, C=N), 1132 (C-N), 1022 (C-Cl). H-NMR (300 MHz, DMSO-d6, ppm) 
2.47 (s, 3H, C-CH3), 3.21 (s, 3H, N-CH3), 7.35-8.04 (m, 8H, Ar-H), 9.52 (s, 1H, =CH-). 13C-
NMR (75 MHz, DMSO-d6, ppm)  10.19, 35.57, 116.10, 125.40, 127.64, 128.66, 129.65,
129.66, 131.45, 132.22, 132.59, 134.84, 138.79, 151.42, 152.77, 159.71. HRMS (m/z):
[M+H]+ calcd for C18H15N3OCl2: 360.0665; found 360.0672.
2.1.4.12. 4-(2-Hydroxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3l)
Yield 72%, m.p. 202 ºC. FTIR (ATR, cm-1): 3064 (aromatic C-H), 2937 (C-H), 1651 (C=O),
1
1589-1411 (C=C, C=N), 1303 (C-O), 1199 (C-N). H-NMR (300 MHz, DMSO-d6, ppm) 
2.40 (s, 3H, C-CH3), 3.20 (s, 3H, N-CH3), 6.89-7.57 (m, 9H, Ar-H), 9.70 (s, 1H, =CH-), 12.94
(s, 1H, OH). 13C-NMR (75 MHz, DMSO-d6, ppm)  10.31, 35.61, 114.52, 116.86, 119.64,
120.68, 125.43, 127.72, 129.68, 131.54, 132.20, 134.69, 150.67, 157.97, 159.58, 159.90.
HRMS (m/z): [M+H]+ calcd for C18H17N3O2: 308.1394; found 308.1404.
2.1.4.13.
4-(4-(4-Chlorophenoxy)benzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-
3(2H)-one (3m)
Yield 72%, m.p. 134 ºC. FTIR (ATR, cm-1): 3061 (aromatic C-H), 2962 (C-H), 1641 (C=O),
1571-1438 (C=C, C=N), 1305 (C-O), 1130 (C-N), 1008 (C-Cl). 1H-NMR (300 MHz, DMSO-
d6, ppm)  2.41 (s, 3H, C-CH3), 3.18 (s, 3H, N-CH3), 7.06-7.58 (m, 13H, Ar-H), 9.54 (s, 1H,
=CH-). 13C-NMR (75 MHz, DMSO-d6, ppm)  10.17, 35.71, 116.68, 120.89, 123.76, 125.18,
127.46, 127.86, 129.62, 130.41, 131.03, 134.96, 140.25, 152.69, 153.65, 155.95, 157.23,
159.91. HRMS (m/z): [M+H]+ calcd for C24H20N3O2Cl: 418.1317; found 418.1324.
2.1.4.14.
Methyl
4-((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
ylimino)methyl)benzoate (3n)
Yield 72%, m.p. 207 ºC. FTIR (ATR, cm-1): 3042 (aromatic C-H), 2989-2953 (C-H), 1708
1
(C=O ester), 1647 (C=O), 1556-1408 (C=C, C=N), 1305 (C-O), 1190 (C-N). H-NMR (300
MHz, DMSO-d6, ppm)  2.41 (s, 3H, C-CH3), 3.22 (s, 3H, N-CH3), 3.87 (s, 3H, O-CH3),
7.36-8.03 (m, 9H, Ar-H), 9.63 (s, 1H, =CH-). 13C-NMR (75 MHz, DMSO-d6, ppm)  10.19,
35.57, 52.67, 116.34, 125.45, 127.65, 127.69, 129.66, 130.04, 130.82, 134.84, 142.27, 152.80,
152.87, 159.72, 166.40. HRMS (m/z): [M+H]+ calcd for C20H19N3O3: 350.1499; found
350.1498.

2.1.4.15.
4-(4-(benzyloxy)benzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
(3o)
Yield 72%, m.p. 188 ºC. FTIR (ATR, cm-1): 3047 (aromatic C-H), 2943-2879 (C-H), 1633
1
(C=O), 1600-1413 (C=C, C=N), 1301 (C-O), 1130 (C-N). H-NMR (300 MHz, DMSO-d6,
ppm)  2.42 (s, 3H, C-CH3), 3.14 (s, 3H, N-CH3), 5.16 (s, 2H, O-CH2), 7.07-7.77 (m, 14H,
Ar-H), 9.51 (s, 1H, =CH-). 13C-NMR (75 MHz, DMSO-d6, ppm)  10.25, 35.97, 69.81,
115.50, 117.19, 124.84, 127.21, 128.25, 128.40, 128.94, 129.34, 129.59, 130.98, 135.12,
137.24, 152.35, 154.68, 160.26, 160.57. HRMS (m/z): [M+H]+ calcd for C25H23N3O2:
398.1863; found 398.1868.
2.1.4.16. 4-(Benzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3p)
Yield 72%, m.p. 179 ºC. FTIR (ATR, cm-1): 3045 (aromatic C-H), 2945 (C-H), 1647 (C=O),
1
1595-1415 (C=C, C=N), 1303 (C-O), 1157 (C-N). H-NMR (300 MHz, DMSO-d6, ppm) 
2.45 (s, 3H, C-CH3), 3.17 (s, 3H, N-CH3), 7.35-7.82 (m, 10H, Ar-H), 9.59 (s, 1H, =CH). 13C-
NMR (75 MHz, DMSO-d6, ppm)  10.21, 35.78, 116.73, 125.08, 127.38, 127.68, 129.20,
129.62, 130.63, 134.99, 137.97, 152.66, 154.79, 160.05. HRMS (m/z): [M+H]+ calcd for
C18H17N3O: 292.1444; found 292.1448.
2.2. Biological evaluation
2.2.1. Cholinesterase Inhibition Assay
AChE and BChE inhibitory activity of all synthesized compounds were evaluated by
modified Ellman’s spectrophotometric method [32]. All chemicals, reagents and reference
drugs (AChE (E.C.3.1.1.7, from electric eel), BChE (from equine serum), 5,5'-dithiobis-(2-
nitrobenzoic acide) (DTNB), acetylthiocholine iodide (ATC), butyrylthiocholine iodide
(BTC), donepezil hydrochloride and tacrine) used in the method were supplied from Sigma-
Aldrich (Germany) and Fluka (Germany). AChE and BChE enzyme inhibition assay were
applied as previously described [3, 33].
2.2.2. AChE Kinetics Assay
AChE enzyme kinetics study was performed for compound 3g to determine the inhibition
type. The compound was prepared at three different concentrations (IC50, 2xIC50 and IC50/2).
The substrate (ATC) was used at various concentrations (600, 300, 150, 75, 37.5 and 18.75
µM). The enzyme kinetics assay was carried out as in our previous publications [3, 33].

Lineweaver-Burk plots were formed by using Microsoft Office Excel 2013. The Ki values of
the compound were easily calculated from second plot with a common intercept on the x-axis
(corresponding to −Ki).
2.2.3. MAO Inhibition Assay
MAO inhibition assay including fluorometric measurement of Ampliflu™ Red (10-Acetyl-
3,7-dihydroxyphenoxazine) was performed. Inhibitory activity of synthesized compounds
(3a-3p) against MAO-A and MAO-B enzymes was measured as reported earlier [12, 13, 34].
2.2.4. MAO Kinetics Assay
MAO enzyme kinetics assay was carried out with the same materials used in the MAO
inhibitory activity. Tyramine was used as a substrate at the following concentrations: 20, 10, 5,
2.5, 1.25, and 0.625 μM. In order to specify the inhibition type on MAO-B enzyme, compound
3h was prepared at the concentrations of IC50/2, IC50 and 2 x IC50. Enzyme kinetics assay was
applied according to previous publications [12, 13, 34].
2.2.5. Prediction of ADME parameters and BBB permeability
QikProp 4.8 software [35] was used to calculate ADME parameters and BBB permeability of
all final compounds (3a-3p).
2.2.6. Molecular Docking Studies
Molecular docking studies were performed to discover the binding modes of compounds 3g
and 3h to active sites of AChE and MAO-B enzymes, respectively. The crystal structures of
AChE (PDB code: 4EY7) [36] and MAO-B (PDB code: 2V5Z) [37] were retrieved from the
Protein Data Bank server (www.pdb.org). Docking procedures were followed as reported
previously [3, 12, 13, 34].
3. Results and discussion
3.1. Chemistry
The synthesis scheme of the target compounds (3a-3p) is outlined in Scheme 1. Schiff base
derivatives were prepared by reacting various aldehydes and 4-aminoantipyrine. Structure
determination and biological evaluation of the final compounds have been made firstly in this
1
study. The structures of synthesized compounds (3a-3p) were confirmed by IR, H-NMR,
13C-NMR and mass spectroscopy. IR spectra of the compounds afforded C-H stretching bands

of aromatic rings (3115-3030 cm-1) and C=O stretching bands of hydrazide moiety (1681-
1
1620 bands. In the H-NMR spectra, C-H proton of Schiff base derivatives resonated as a
singlet peak at 9.94-9.42 ppm. Methyl groups attached to 1st and 5th positions of pyrazolone
ring were observed at 3.11-3.25 ppm and 2.40-2.48 ppm as two singlets, respectively. The
protons on the aromatic ring were recorded between 6.82 and 8.58 ppm. The other aliphatic
groups were observed at the expected shifting values. In the 13C-NMR spectra, methyl
carbons on the pyrazolone ring had peaks at 10 and 36 ppm. The signal due to the hydrazide
carbonyl carbons appeared at 169.15-159.46 ppm. The signal due to the Schiff base carbons
were observed at 162.32-152.73 ppm. Other aromatic carbons were recorded between 111
ppm and 158 ppm. The results of the MS analysis agreed well with the calculated mass of the
compounds. The purity of all compounds was controlled by a PDA detector and found to be
greater than 97%.
*
R
4-hidroxyphenyl
*
3i
R
3-nitrophenyl
3a
3b
3c
3d
3e
3f
2,4-dichlorophenyl
4-chlorophenyl
3j
4-(piperidino)phenyl
3-ethoxy-4-hidroxyphenyl
2,6-dimethylphenyl
2,6-dichlorophenyl
3,4-dichlorophenyl
2-hidroxyphenyl
3k
3l
4-[4-(chlorophenoxy)]phenyl
4-(methoxycarbonyl)phenyl
4-(benzyloxy)phenyl
phenyl
3m
3n
3o
3p
4-[3-(dimethylamino)propoxy]phenyl
4-nitrophenyl
3g
3h
Scheme 1. The synthetic route of the compounds (3a-3p).
3.2. Biological evaluation
3.2.1. Enzyme Inhibition
Synthesized compounds were evaluated for their anticholinesterase and MAO inhibitory
activities by using in vitro spectrophotometric and fluorimetric methods, respectively [3, 12,
13, 21, 34]. Both assays were carried out in two steps. Firstly, compounds 3a-3p were tested
at 10−3 and 10-4 M concentrations. Selected compounds that indicate promising inhibitory
activity at initial concentrations were taken into second step at concentrations of 10−5-10−9 M.

Table 1 displays the anticholinesterase and MAO inhibitory activities of compounds 3a-3p at
10−3 and 10−4 M concentrations. None of the compounds displayed noteworthy activity
against BChE enzyme. MAO-A inhibition potency of the compounds was almost at low
levels. Although most of the compounds were not able to inhibit AChE, compounds 3c and 3g
differed from the series with their intrinsic inhibitory potential against AChE. Thus, these
compounds were diluted and tested in further concentrations (10−5-10−9 M) against AChE
along with a reference drug donepezil (Table 2). The IC50 values on AChE were recorded as
0.285 μM, 0.057 μM and 0.029 μM for 3c, 3g and donepezil, respectively. MAO-B were the
most sensitive enzyme against the synthesized compounds. All compounds indicated more
than 50% inhibition potency at a concentration of 10-3 M. Furthermore, compounds 3a, 3h
and 3i showed significant inhibition and thus they were assessed in the second step test. IC50
values of compounds 3a, 3h, 3i and selegiline against MAO-B were calculated as 0.114 μM,
0.049 μM, 0.054 μM and 0.040 μM, respectively (Table 3).
Results of enzyme inhibition studies revealed that substituent variation strongly affects the
inhibition potency of compounds. Basic nitrogen atom in the substituents of 3c and 3g caused
a significant increase in AChE activity. On the other hand, incorporation of a strong electron
withdrawing nitro group enhanced the MAO-B activity of 3h and 3i.
Table 1. % Inhibition of the synthesized compounds 3a-3p and reference agents against AChE, BChE, MAO-A
and MAO-B enzymes at concentrations of 10-3 and 10-4 M.
AChE Inhibition %
10-3 M 10-4 M
BChE Inhibition %
10-3 M 10-4 M
MAO-A Inhibition %
10-3 M 10-4 M
MAO-B Inhibition %
10-3 M 10-4 M
Comp.
30.88±0.58 16.28±0.41 30.88±0.58 16.28±0.41 55.28±0.75 25.11±0.60 94.27±1.25 85.13±1.08
28.15±0.61 14.10±0.50 28.15±0.61 14.10±0.50 44.13±0.62 20.48±0.50 68.25±1.05 39.30±0.97
85.26±1.56 78.20±1.20 85.26±1.56 78.20±1.20 58.21±0.74 30.22±0.48 62.19±1.08 34.75±0.88
44.71±0.94 25.99±0.74 44.71±0.94 25.99±0.74 51.03±0.43 24.77±0.40 75.29±0.96 40.17±0.71
21.30±0.63 14.79±0.47 21.30±0.63 14.79±0.47 48.23±0.58 22.42±0.37 68.26±1.00 42.49±0.84
40.79±0.48 28.39±0.41 40.79±0.48 28.39±0.41 33.18±0.66 27.88±0.40 65.29±0.87 44.30±0.52
96.28±1.36 88.12±1.07 96.28±1.36 88.12±1.07 42.27±0.59 19.39±0.41 52.28±0.63 34.36±0.51
35.29±0.63 30.20±0.52 35.29±0.63 30.20±0.52 38.17±0.52 18.26±0.30 98.20±1.19 89.10±1.24
37.18±0.55 31.66±0.40 37.18±0.55 31.66±0.40 42.16±0.58 28.51±0.41 93.28±1.29 87.22±1.18
25.08±0.75 20.60±0.69 25.08±0.75 20.60±0.69 49.33±0.63 30.08±0.47 70.29±0.95 42.46±0.81
36.79±0.67 31.22±0.54 36.79±0.67 31.22±0.54 51.80±0.44 38.15±0.31 66.24±0.63 45.29±0.48
43.81±0.76 24.08±0.81 43.81±0.76 24.08±0.81 42.29±0.67 21.74±0.42 52.46±0.38 34.19±0.27
31.12±0.85 19.25±0.70 31.12±0.85 19.25±0.70 39.26±0.43 18.14±0.33 53.28±0.62 45.49±0.54
50.49±1.25 31.23±0.73 50.49±1.25 31.23±0.73 46.34±0.56 40.77±0.27 61.29±0.75 40.29±0.62
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n

30.28±0.82 24.67±0.76 30.28±0.82 24.67±0.76 48.26±0.55 27.54±0.31 54.16±0.75 35.19±0.52
49.16±0.89 40.28±0.82 49.16±0.89 40.28±0.82 35.61±0.41 24.62±0.30 58.22±0.55 28.16±0.39
3o
3p
99.48±1.92 98.56±1.76
-
-
-
-
-
-
-
-
-
-
-
-
Ref-1
Ref-2
Ref-3
Ref-4
-
-
-
-
-
-
94.12±2.76 82.14±2.69
-
-
-
-
98.91±1.28 96.88±1.31
-
-
98.91±1.28 96.88±1.31
Ref-1: Donepezil, Ref-2: Tacrine, Ref-3: Moclobemide, Ref-4: Selegiline
Table 2. Evaluation of 3c, 3g and donepezil against AChE at further concentartions and IC50 values.
Compound
10-5 M
10-6 M
10-7 M
10-8 M
10-9 M
IC50 (µM)
65.79±1.13
85.17±1.28
95.30±1.64
58.97±0.98
77.39±1.02
92.15±1.88
46.77±0.85
63.44±1.18
81.36±1.41
35.60±0.76
40.89±0.94
41.78±0.84
20.08±0.54
21.63±0.52
25.62±0.58
0.285±0.009
0.057±0.002
0.029±0.001
3c
3g
Donepezil
Table 3. IC50 values of 3a, 3h, 3i and selegiline against MAO-B.
Compound
10-5 M
10-6 M
10-7 M
10-8 M
10-9 M
IC50 (µM)
74.29±1.11
81.00±1.05
79.17±1.06
86.96±1.82
67.19±0.87
76.46±1.14
72.63±0.97
79.23±1.72
54.22±0.76
67.19±0.97
62.29±0.84
65.37±1.13
34.75±0.79
40.29±0.82
39.97±0.76
43.28±1.03
20.30±0.54
25.74±0.76
22.11±0.62
14.70±0.26
0.114±0.003
0.049±0.002
0.054±0.002
0.040±0.002
3a
3h
3i
Selegiline
3.2.2. Enzyme Kinetics
The mechanisms of AChE and MAO-B inhibition were investigated by enzyme kinetics. The
type of inhibition was estimated by the linear Lineweaver-Burk graphics. The substrate
velocity curves in the absence and presence of the most potent compounds (3g for AChE and
3h for MAO-B) were recorded in the enzyme kinetics analysis. These compounds were
prepared at concentrations of IC50/2, IC50 and 2 x IC50. The different substrate concentrations
ranging from 600 µM to 18.75 µM for AChE and from 20 µM to 0.625 µM for MAO-B were
used to gain the initial velocity measurements. The Ki (intercept on the x-axis) values of
compounds were determined from the secondary plot of the Km/Vmax (slope) versus varying
concentrations [34]. The graphical analysis of steady-state inhibition data for AChE and
MAO-B enzymes are shown in Figures 1 and 2, respectively.
The type of inhibition can be determined as mixed-type, competitive, uncompetitive, or
noncompetitive by the Lineweaver-Burk plot. In an uncompetitive type inhibition graphic,
there are non-diagonal parallel lines. If the lines do not cross x- or y-axis at the same point, it

is called as mix type as observed in Figure 1. Competitive inhibitors have the same
intersection point on the y-axis but have different slopes and intersection points on the x-axis
between the two sets of data, while noncompetitive inhibitors have plots with the same
intersection on x-axis but there are different slopes and intersection on y-axis as observed in
Figure 2. These results show that compounds 3g and 3h are mixed-type and non-competitive
inhibitors, respectively. Namely, both compounds can bind to the free enzyme or the enzyme-
substrate complex. If any type of the above graphics is observed in Lineweaver-Burk plots,
this mean that tested compound is a reversible inhibitor, which binds to enzyme by
noncovalent interactions as hydrophobic interactions, ionic bonds, and hydrogen bonds
without forming any chemical bonds or reactions with the enzyme. These interactions occur
quickly and can be easily removed. For this reason, the complex of the enzyme and inhibitor
is rapidly degraded just before the irreversible inhibition. Because of the ability to bind
reversibly to biomolecules, such inhibitors present a lower risk of side effects than irreversible
inhibitors. As a result, enzyme kinetics studies have displayed the biological significance of
compounds 3g and 3h owing to their mixed-type and noncompetitive inhibition potencies,
respectively, as opposed to irreversible enzyme inhibitors.

(A)
(B)
Figure 1. (A) Lineweaver–Burk plots for the inhibition of AChE by compound 3g. [S], substrate concentration
(μM); V, reaction velocity (abs/min)-1. Vmax values from 2 x IC50 to Control; 178.571, 243.902, 270.275 and
434.738 (abs/min)-1. Km values of the mixed type inhibition; 52.179, 48.537, 43.243, 50.348 (µM). (B)
Secondary plot for calculation of steady-state inhibition constant (Ki) of compound 3g. Ki was calculated as
0.0635 μM.

(A)
(B)
Figure 2. (A) Lineweaver–Burk plots for the inhibition of MAO-B by compound 3h. [S], substrate concentration
(μM); V, reaction velocity (nmol/min/mg protein). Inhibitor concentrations are shown at the left. Vmax values
from 2 x IC50 to Control; 169.492, 212.766, 250.000 and 526.316 (nmol/min/mg protein). Km value of the non-
competitive inhibition; 0.966±0.029 (µM). (B) Secondary plot for calculation of steady-state inhibition constant
(Ki) of compound 3h. Ki was calculated as 0.0471 μM.

3.2.3. ADME and BBB Permeability Predictions
Most of the clinical trials for new drug development are failing due to insufficient ADME
(absorption, distribution, metabolism and excretion) properties of the drug candidate
compound. Failures that occur during the last phases of these studies lead to an increase in the
cost of new drug development. Early identification of problematic issues can significantly
reduce the amount of wasted time and funds, and can rationalize the overall development
process. Therefore, the pharmacokinetic properties of new drug candidates are one of the
important issues that should be assessed as early as possible in the drug development process.
ADME estimation can be used to focus on lead optimization to improve the preferred
properties of a compound [38]. Hence, predictions of ADME parameters of synthesized
compounds (3a-3p) were carried out by QikProp 4.8 software [35], assesing the violations of
Lipinski's rule of five [39] and Jorgensen's rule of three [40]. These rules are good indicators
of the ADME properties of new drug candidates, and are necessary for the optimization of a
biologically active compound. Molecular weight (MW), number of rotatable bonds (RB),
dipole moment (DM), molecular volume (MV), number of hydrogen donors (DHB), number
of hydrogen acceptors (AHB), polar surface area (PSA), octanol/water partition coefficient
(log P), aqueous solubility (log S), apparent Caco-2 cell permeability (PCaco), number of
likely primer metabolic reactions (PM), and percent of human oral absorption (%HOA) are
given in Table 4 along with the violations of rules of three (VRT) and five (VRF). As seen in
Table 4 all compounds (3a-3p) are in agreement with both rules by causing no more than one
violation.
In particular, drugs targeting the CNS should pass the blood-brain barrier (BBB) first.
Although BBB has a protective nature, the non-penetration of molecules into the BBB is an
important obstacle for CNS drug candidates and should be addressed early in the drug
discovery process. Thus, the task of predicting the BBB permeability of new compounds is of
paramount importance [38]. In the present study the BBB permeability of the synthesized
compounds (3a-3p) was also estimated by QikProp 4.8 software [35]. Apparent MDCK cell
permeability (PMDCK), brain/blood partition coefficient (log BB), and central nervous
system activity scores (CNSAS) were calculated for this purpose. MDCK cells are considered
to be a good mimic for the blood brain barrier. According to software predictions, the
PMDCK values of <25 and >500 nm/s are recommended as poor and great for non-active
transport of compound. Besides, in order to pass BBB, logBB and CNSAS have

recommended values of -3 to 1.2 and -2 to 2, respectively. It is seen in Table 4 that PMDCK,
logBB and CNSAS are in the recommended ranges. Thus, it can be deduced that the
synthesized compounds have the ability to pass the BBB, which is extremely important for
CNS related drugs.
As a result of the ADME and BBB Permeability estimates, it can be suggested that all
synthesized compounds may have a good pharmacokinetic profile.

Table 4. Calculated ADME parameters of compounds (3a-3p)
Comp
3a
MW
RB
4
DM
5.865
8.498
6.642
7.123
6.666
5.144
4.615
16.148
11.784
8.493
9.083
4.415
8.503
9.592
6.874
6.259
MV
DHB
AHB
5.75
5
PSA
logP
2.819
4.913
4.788
3.591
4.467
4.923
3.846
2.800
2.800
4.093
4.534
2.836
5.634
3.169
5.507
3.588
logS
PCaco
logBB
-0.641
0.313
-0.092
-0.809
0.099
0.258
-0.022
-1.167
-1.167
0.13
PM
2
PMDCK
583.079
CNSAS
%HOA
100
VRF
0
VRT
0
307.351
360.242
374.485
351.404
319.405
360.242
392.500
336.349
336.349
325.797
360.242
307.351
417.894
349.388
397.476
291.352
1026.605
1140.063
1277.142
1168.814
1136.613
1150.378
1342.549
1085.835
1085.835
1047.548
1087.846
1022.291
1298.972
1155.973
1327.105
1003.435
1
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
64.044
41.380
46.600
71.339
36.774
38.214
53.453
90.028
90.028
41.364
41.362
62.229
49.458
76.315
49.251
41.361
-3.985
-6.142
-6.303
-5.236
-5.188
-6.467
-4.056
-4.081
-4.081
-4.958
-5.597
-3.975
-6.696
-4.556
-6.586
-4.191
1164.223
4422.829
3783.479
1242.236
5254.316
4723.673
924.503
0
1
3
1
10000.000
2084.405
625.423
100
0
1
3b
3c
3
6
1
0
100
0
1
3d
6
6.5
5
3
-1
1
100
0
0
3
3
2972.643
9475.216
502.783
100
0
0
3e
3f
3
5
1
1
100
0
1
3g
3h
3i
8
7.75
6
3
1
100
0
0
4
397.230
2
182.374
-2
-2
1
89.857
89.857
100
0
0
4
6
397.230
2
182.374
0
0
3j
3
5
3864.616
3864.021
1638.697
3864.925
1217.414
3864.891
3864.891
1
5261.801
10000.000
843.719
0
0
3k
3l
3
5
0.266
-0.479
-0.026
-0.684
-0.282
-0.035
1
1
100
0
0
4
5.75
5.5
7
2
0
100
0
0
3m
3n
3o
3p
5
1
5262.255
611.926
0
100
1
1
4
1
0
100
0
0
6
5.75
5
3
2132.927
2132.926
0
100
1
1
3
1
0
100
0
0
MW: Molecular weight, RB: Number of rotatable bonds, DM: Computed dipole moment, MV: Total solvent-accessible volume, DHB: Estimated number of hydrogen bond
donors, AHB: Estimated number of hydrogen bond acceptors, PSA: Van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms, logP:
Predicted octanol/water partition coefficient, logS: Predicted aqueous solubility, PCaco: Predicted apparent Caco-2 cell permeability, logBB: Predicted brain/blood partition
coefficient, PMDCK: Predicted apparent MDCK cell permeability, PM: Number of likely metabolic reactions, CNSAS: Predicted central nervous system activity score,
%HOA: Predicted human oral absorption percent, VRF: Number of violations of Lipinski’s rule of five. The rules are: MW < 500, logP < 5, DHB ≤ 5, AHB≤ 10, Positive
PSA value. VRT: Number of violations of Jorgensen’s rule of three. The three rules are: logS
>
-5.7, PCaco
>
22 nm/s, PM
<
7.

3.2.3. Molecular Docking Studies
Docking studies were performed in order to gain more insight into the binding mode of
compounds 3g and 3h to AChE and MAO-B enzymes. Studies were carried out by using the
X-ray crystal structures of Homo sapiens AChE (hAChE PDB ID:4EY7) [36] and MAO-B
(PDB ID: 2V5Z) [37] obtained from Protein Data Bank server (www.pdb.org).
The docking poses of the compound 3g are presented in Figures 3 and 4. According to the
superimposition poses of this compound with donepezil, it is clearly understood that 3g binds
to AChE enzyme in a similar position with donepezil due to the dual binding sites. Lipophilic
part of the compound is consist of 1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one scaffold,
while dimethylaminoalkyl side chain constitutes a polar basic center. The docking poses
indicate that PAS region of AChE interact with lipophilic group in the structure, whereas the
polar and basic groups bind to the CAS region of the enzyme.
It is observed that carbonyl of pyrazole interacts with amino group of Phe295 by forming a
hydrogen bond. Another hydrogen bond is related to nitrogen of dimethyamino group. This
nitrogen atom forms a hydrogen bond with the carboxyl of Glu202. An efficient binding is
also provided by the formation of cation-π interaction between this nitrogen atom and Trp86.
The other important factor of binding to the active site is π-π interaction between 1,4-
disubstituted phenyl and Tyr337. Docking results also show that extended carbon chain
enhances the van der Waals interactions with the amino acids in the active site and intensifies
the proper bonding.

Figure 3. Three-dimensional pose of compound 3g (pink colored) and donepezil (green colored) in the enzyme
active site (AChE PDB Code: 4EY7).
Figure 4. The interacting mode of compound 3g in the active region of AChE. The inhibitor, colored with pink,
and the important residues, colored with turquoise, in the active site of the enzyme are presented by tube model.
Three-dimensional pose of compound 3h in the active site of MAO-B is presented in the
Figure 5. Intereactions between enzyme and inhibitor is displayed in the Figure 6. The
compound 3h adequately binds to amino acid residues, lining the cavity by overlapping at the

same site and, locating very near the FAD cofactor. The docking poses on MAO-B reveal that
the carbonyl of pyrazole establishes a hydrogen bond with amino of Cys172. There is a π- π
interaction between pyrazole and phenyl of Try32. The nitrogen atom of nitro group forms
cation-π interaction with phenyl of Tyr435. It is thought that this interaction is important to
explain why compound 3h is the most active derivative in the series. Thus, it may be
suggested that the presence of an electron withdrawing group such as nitro has a positive
contribution to the activity.
Figure 5. Three-dimensional pose of compound 3h in the enzyme active site (MAO-B PDB Code: 2V5Z).

Figure 6. The interacting mode of compound 3h in the active region of MAO-B. The inhibitor and the important
residues in the active site of the enzyme are presented by tube model. The inhibitor is colored with dark green.
The FAD molecule is colored red with ball and stick model.
4. Conclusion
In this study, we synthesized some Schiff bases as cholinesterase and MAO inhibitors.
According to activity results, compounds 3g and 3h displayed the highest inhibition towards
AChE and MAO-B, respectively. In an effort to explain their inhibitory effects, the enzyme
kinetic and docking studies were applied. ADME and BBB permeability predictions enhanced
the biological significance of these compounds. Consequently, this study presents important
results for developing new enzyme inhibitors, which may be beneficial for neurodegenerative
diseases such as Parkinson's and Alzheimer's diseases.
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Synthesis and Biological Evaluation of New Schiff Bases as Dual Monoamine Oxidase-
Cholinesterase Inhibitors

Selective AChE inhibitor
Selective MAO-B inhibitor
Compound 3g
Compound 3h

Highlights
 16 new Schiff base derivatives were synthesized.
 Synthesized compounds were evaluated for their acetylcholinesterase (AChE),
butyrylcholinesterase (BuChE), and monoamine oxidases A and B (MAO-A and
MAO-B) enzymes inhibitory activities.
 Compounds 3e and 3g were found as the most potent derivatives against AChE
enzyme.
 Compounds 3a (IC50 = 0.114 µM), 3h (IC50 = 0.049 µM), and 3i (IC50 =0.054 µM)
were the most active derivatives against MAO-B enzyme activity.
 A good ADME (Absorption, Distribution, Metabolism, and Excretion) and BBB
(Blood, Brain, Barier) profile was predicted for all synthesized compounds.
 Molecular docking studies were performed to define and evaluate the interaction
mechanism between compounds 3g and 3h and related enzymes.