New 2-Pyrazoline and Hydrazone Derivatives as Potent and Selective Monoamine Oxidase A Inhibitors

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New 2‑Pyrazoline and Hydrazone Derivatives as Potent and

Selective Monoamine Oxidase A Inhibitors

Umut Salgin-Goksen, Gokcen Telli, Acelya Erikci, Ezgi Dedecengiz, Banu Cahide Tel, F. Betul Kaynak,

Kemal Yelekci, Gulberk Ucar,* and Nesrin Gokhan-Kelekci *

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ABSTRACT: Thirty compounds having 1-[2-(5-substituted-2-benzoxazolinone-3-yl) acetyl]-3,5-disubstitutedphenyl-2-pyrazoline

structure and nine compounds having N′-(1,3-disubstitutedphenylallylidene)-2-(5-substituted-2-benzoxazolinone-3-yl)-

acetohydrazide skeleton were synthesized and evaluated as monoamine oxidase (MAO) inhibitors. All of the compounds exhibited

selective MAO-A inhibitor activity in the nanomolar or low micromolar range. The results of the molecular docking for hydrazone

derivatives supported the in vitro results. Five compounds, 6 (0.008 μM, Selectivity Index (SI): 9.70 × 10⁻⁴), 7 (0.009 μM, SI: 4.55

× 10⁻⁵), 14 (0.001 μM, SI: 8.00 × 10⁻⁴), 21 (0.009 μM, SI: 1.37 × 10⁻⁵), and 42 (0.010 μM, SI: 5.40 × 10⁻⁶), exhibiting the

highest inhibition and selectivity toward hMAO-A and nontoxic to hepatocytes were assessed for antidepressant activity as acute and

subchronic in mice. All of these ﬁve compounds showed signiﬁcant antidepressant activity with subchronic administration consistent

with the increase in the brain serotonin levels and the compounds crossed the blood−brain barrier according to parallel artiﬁcial

membrane permeation assay. Compounds 14, 21, and 42 exhibited an ex vivo MAO-A proﬁle, which is highly consistent with the in

vitro data.

metabolized mostly with MAO-A⁸and β-phenylethylamine,

INTRODUCTION

■

and benzylamine is metabolized with MAO-B.⁹Dopamine and

Depression is an important psychiatric disease and has a high

some essential amines like tyramine interact with both

incidence in the world. According to the World Health

Organization, 350 million people are diagnosed with

isoforms of MAO.¹⁰MAOIs are especially classiﬁed into two

main categories in the clinic. The antidepressant activity is

depression. It was predicted that between 2020 and 2030,

mostly associated with inhibition of MAO-A and the

depression will be the second important reason for disability in

the world.¹Depression can lead to the development of several

consequent ability to counter the decrement in brain

noradrenaline (NE), dopamine (DA), and especially serotonin

second-order diseases besides the stress that is caused to the

(5-HT) levels during the depression.²MAO-B inhibitors could

be preferred in Alzheimer’s and Parkinson’s diseases.^11,12The

use of MAOIs to treat major depressive disorders declined

patients and their families.²⁻⁵It was reported that anxiety

disorders are observed before or during depression in 10−21%

of children and adolescents.⁶

Monoamine oxidase inhibitors (MAOIs) were the ﬁrst class

of antidepressants to be developed. MAOIs are known for their

mood-enhancing eﬀect by virtue of blocking the breakdown of

neurotransmitters by monoamine oxidase (MAO) (EC

1.4.3.4). Two isoforms of MAO have been discovered, which

exhibit distinct interests to inhibitors and varying speciﬁcities

to substrates.⁷Adrenaline, noradrenalin, and serotonin are

Received: August 28, 2020

Published: February 3, 2021

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1989

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Figure 1. Aryloxazolidinone derivatives of MAOIs.

Figure 2. Overall design strategy for 2-pyrazoline and hydrazone derivatives (the color code indicates common chemical structures).

sharply after it was found that inhibition of MAO can lead to a

severe cardiovascular event called the “cheese eﬀect”, which is

observed after intake of tyramine-containing foods. Studies

focused on improving novel selective and reversible MAOIs

with low tyramine potentiation properties.¹³

Toloxatone (Humoryl) is the lead compound of arylox-

azolidinones that are the relatively new classes of MAOIs

(Figure 1). It is the ﬁrst selective and reversible inhibitor of

MAO-A and represented as an antidepressant to clinical use.²²

Chemical modiﬁcations of toloxatone generated more selective

cimoxatone and beﬂoxatone derivatives that are active at

nanomolar concentrations.^11,23

Since MAOIs can increase 5-HT, NE, and DA simulta-

neously in the brain, they have been considered for usage in

treatment resistance depression instead of combination

therapies such as combining an inhibitor of 5-HT-NE reuptake

inhibitor with a certain dopaminergic drug or pairing a

dopamine agonist with a selective serotonin reuptake inhibitor

(SSRI) and a tricyclic antidepressants (TCA).³The ﬁndings

also indicate that these inhibitors have remarkable neuro-

protective eﬀects.⁴The recently designed and synthesized

highly selective and reversible MAOIs oﬀer new opportunities

for the development of superior antidepressant and anti-

parkinsonian agents through the selective inhibition of MAO-A

and MAO-B, respectively.

Classical MAOIs are analogues of natural MAO substrates

and have a structure similar to that of phenylethylamines

containing a reactive group, such as propargylamine and

hydrazine, which allows the binding to the enzyme via covalent

adducts resulting from bioactivation of the inhibitor to an

electrophilic intermediate.¹⁴2-Pyrazoline derivatives are cyclic

hydrazine moieties that are formed by the cyclization of linear

hydrazine derivatives.¹⁵Derivatives obtained by substitutions

on the 2-pyrazoline nucleus preferably at the N1, C3, and C5

positions show a remarkable eﬀect on the central nervous

system (CNS).¹⁶⁻¹⁸Chimenti et al. demonstrated that either

the N1 acetyl group or N1-propanoyl substitution at position 1

increased potency and selectivity of these inhibitors.¹⁹⁻²¹

According to the speciﬁed ﬁnding mentioned and the intent

of developing new more selective and potent MAO-A

inhibitors, we designed and synthesized some novel hybrid

compounds bearing oxazolidinone and diazo cores (30 new 2-

pyrazoline and 9 new hydrazone derivatives) as selective,

potent MAO-A inhibitors with contribution of the docking

studies (Figure 2 and Scheme 1). They were screened in vitro

to determine their inhibitory actions on MAO isotypes. Since

the biological activity of MAOIs depends heavily on their

ability to cross the blood−brain barrier (BBB), the BBB

permeability of selected new compounds was determined with

the parallel artiﬁcial membrane permeation assay of BBB

(PAMPA−BBB). Additionally, the potential acute and

subchronic antidepressant activities of ﬁve compounds that

exhibited the highest inhibitory selectivity and potency toward

hMAO-A were determined. Since depression and its comorbid

disorders are related to serotonin and dopamine interactions in

the prefrontal cortex, the levels of 5-HT, DA, and 5-

hydroxyindoleacetic acid (5-HIAA) and MAO activity were

determined in brain tissues of experimental animals to evaluate

the status of related neurotransmitters, their metabolites, and

the MAO-metabolizing enzyme. Our data indicated that the

compounds had an antidepressant-like activity in mice by the

possible interaction with the serotonergic and monoaminergic

systems.

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Scheme 1. Synthesis and Structure of the Compounds

mass spectrometry (ESI-MS) techniques. The purity of

compounds was determined by elemental analyses; the results

were within 0.4% of the theoretical values. Due to limited

laboratory facilities, chiral separation of only one compound

was made, the conﬁgurations of the separated enantiomers

were determined by Vibrational Circular Dichroism, and their

activities were examined.³⁰The spectra of synthesized

compounds are provided in the Supporting Information.

X-ray Crystal Structure. The molecular structure of

compounds 10 and 43 consists of two discrete entities.

Table S1 summarizes their main geometrical characteristics

according to the numbering scheme (Figures S1 and S2). The

most signiﬁcant diﬀerences between these two compounds are

the presence of a central pyrazoline ring in compound 10 and

hydrazone moiety in compound 43 (The packing arrange-

ments of both compounds are shown in Figures S3 and S4.) In

the crystal lattices of both compounds, there are multiple

intermolecular interactions and strong intra- and intermolec-

ular hydrogen bonds are responsible for the packing of the

molecules (Tables S2 and S3).

Druglikeness and ADMET Predictions. Druglikeness of

the designed compounds were predicted using AdmetSAR 2.0

server (http://lmmd.ecust.edu.cn/admetsar1)a program

that estimates the ADMET properties based on substructure

pattern recognition and then uses a support vector machine

algorithm to build a model.³¹The chemical information of

each designed compound was input in “smiles” format and the

corresponding ADMET properties were estimated (Table S4,

RESULTS AND DISCUSSION

■

Chemistry. A novel series of 1-[2-((5-methyl/chloro)-2-

benzoxazolinone-3-yl)acetyl]-3,5-diaryl-2-pyrazoline 5−34 and

N′-[(1,3-diaryl)allylidene]-2-[(5-methyl/chloro)-2-benzoxazo-

linone-3-yl]acetohydrazide 35−43 derivatives were synthesized

according to the protocols summarized in Scheme 1. The

conditions of reactions and the characterization of compounds

are explained in the Experimental Section.

2-Benzoxaolinone 1a and 5-methyl-2-benzoxazolinone 1b

were synthesized according to literature methods using 2-

aminophenol/4-methyl-2-aminophenol and urea.²⁴5-Chloro-

2-benzoxazolinone 1c was commercially available. Ethyl ((5-

methyl/chloro)-2-benzoxazolinone-3-yl) acetate derivatives

2a−2c²⁵were carried out by heating (5-methyl/chloro)-2-

benzoxazolinone 1a−1c with ethyl chloroacetate in K₂CO₃/

acetone. The acid hydrazides 3a−3c were prepared by

nucleophilic substitution of 2a−2c with hydrazine hydrate in

ethanol.²⁶⁻²⁸Treatment of appropriate aldehydes with

acetophenone derivatives under basic conditions using the

Claisen−Schmidt condensation²⁹gave α,β-unsaturated car-

bonyl compounds (chalcones) 4a−4j.

The reaction of hydrazides 3a−3c with chalcones 4a−4j in

n-propanol under acidic condition yielded the corresponding

2-pyrazoline 5−34 and hydrazone derivatives 35−43 (Scheme

1). Due to the low stability of hydrazones and diﬃculties in

their isolation, not all hydrazone compounds could be reached.

The structures of the synthesized compounds were determined

1

using IR, H NMR, ¹³C NMR, and electrospray ionization

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Table 1A. Experimental K_iValues of hMAO-Isoform Inhibition for Novel Newly Synthesized 2-Pyrazoline Derivatives and

c

Reference Compounds

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Table 1A. continued

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Table 1A. continued

a

b

SI was calculated as K_i(MAO-A)/K_i(MAO-B). Data demonstrate the mean

standard error of the mean (SEM) of three independent

c

experiments. Moclobemide and all synthesized compounds were found as reversible, competitive MAO-A inhibitors, while Selegiline (irreversible)

and Lazabemide (reversible) were found as competitive MAO-B inhibitors.

Supporting Information). All designed compounds were found

Jorgensen’s “Rule of 3”.³³All of the physicochemical

parameters assessed were within the range of drug candidacy.

to be drug-like having obeyed Lipinski’s “Rule of 5”³²and

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Table 1B. Calculated and Experimental K_iValues of hMAO-Isoform Inhibition for Novel Newly Synthesized Hydrazone

c

Derivatives and Reference Compounds

a

b

c

SI was calculated as K_i(MAO-A)/K_i(MAO-B). Data demonstrate the mean SEM of three independent experiments. Moclobemide and all

synthesized compounds were found as reversible, competitive MAO-A inhibitors, while Selegiline (irreversible) and Lazabemide (reversible) were

found as competitive MAO-B inhibitors.

Evaluation of In Vitro Biological Screening Results

and Molecular Modeling Data. hMAO activities were

determined using Amplex Red MAO assay kit. The synthesized

compounds were screened for their hMAO inhibitory activities

using moclobemide (A-selective, reversible), selegiline (B-

selective, irreversible), and lazabemide (B-selective, reversible)

as reference inhibitors (Tables 1A and 1B). Speciﬁc enzyme

activities were calculated as 0.175 0.012 nmol/mg/min (n =

3) for hMAO-A and 0.135 0.009 nmol/mg/min (n = 3) for

hMAO-B. Selectivity indexes (SI) were expressed as K_i(MAO-

A)/K_i(MAO-B).

Docking calculations were performed using AutoDock to

determine binding aﬃnities of the synthesized hydrazone

derivatives (compounds 35−43) with MAO-A and MAO-B.

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Figure 3. (A−C) 3D representations of compound 38 docked in the MAO-A (2Z5X) (A) active site. Magniﬁed view of the active site (B). 2D

interaction diagrams of compound 38 with amino acid residues lining the MAO-A active site (C).

Accelrys’ (Biovia) Discovery Studio Protocols and visualization

programs were used to render the poses of docked inhibitors in

the active site of MAO-A and MAO-B isozymes. However, the

computational inhibition constants obtained for each enan-

tiomer of compounds 5−34 were not compared with the

experimental inhibition constant measurements which were

obtained for racemic forms. Thus, a correlation analysis would

be insigniﬁcant for these compounds in the present study. All

two-dimensional (2D) and three-dimensional (3D) pictures

were produced using Biovia 4.6 visualization program. Two

eﬀective and selective compounds from hydrazone (38 and

42) group interacting with MAO-A and MAO-B were chosen.

Most of the newly synthesized pyrazolines (compounds 5−

34) and hydrazones (compounds 35−43) inhibited hMAO-A

potently and selectively. Among pyrazoline derivatives,

compound 14 having 4-methoxy group at R₂and 2-chlorine

at R₃exhibited the highest inhibitory activity toward hMAO-A

with a K_ivalue of 0.001 μM. This compound was more potent

than moclobemide, the known selective and reversible MAO-A

inhibitor inhibiting hMAO-A with the K_ivalue of 0.010

0.001 μM.

Experimental data showed that the compound 21, bearing 4-

methoxy group at R₂and 2,3-dimethoxy groups at R₃inhibited

hMAO-A potently with a K_ivalue of 0.009 μM (Table 1A).

In vitro screening data demonstrating that hydrazone

derivative compounds 35, 36, 38, 41, and 43 showed the

most inhibitory potency toward hMAO-A with the same K_i

value of 0.001 μM indicated that the hMAO-A inhibitory

ability of newly synthesized hydrazones is remarkably better

than that of moclobemide (K_i= 0.10 μM) (Table 1B).

Molecular modeling studies of compound 38, which is a

hydrazone derivative of pyrazoline derivative, and compound

14 were also carried out for MAO-A and MAO-B isozymes

(Figures 3 and 4). Figure 3A shows the orientation of

compound 38 in the active site of MAO-A (K_i= 0.394 × 10⁻³

μM). The benzoxazolinone ring of the inhibitor is inserted

between TYR407 and TYR444 making a π−π stacked

interaction with TYR444 (Figure 3B). Another hydrogen

bond forms between the carbonyl groups of the inhibitor and

TYR407. PHE352 forms a strong π−π interaction with the

chlorophenyl ring of the inhibitor. CYS323 residue forms a π−

sulfur interaction with the same ring. A hydrogen bond is

formed between the NH moiety of the inhibitor and ILE207

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Figure 4. (A−C) 3D representations of compound 38 docked in the MAO-B (2V5Z) (B) active site. Magniﬁed view of the active site (B). 2D

interaction diagrams of compound 38 with amino acid residues lining the MAO-B active site (C).

backbone carbonyl group. In addition to these eﬀective

interactions, various hydrophobic and van der Waals

interactions contribute to generating a better MAO-A inhibitor

compared to MAO-B isozyme (Figure 3C).

Figure 4A shows compound 38 in the active site of MAO-B.

The benzoxazolinone ring of the inhibitor is oriented away

from the hydrophobic cage, making only one π-lone pair

interaction with GLN206 (Figure 4B). The carbonyl group of

the benzoxazolinone ring and double-bonded nitrogen atom of

the inhibitor make two hydrogen bonds with CYS172 and

TYR326, respectively. Various hydrophobic and van der Waals

interactions occur between compound 38 and the active-site

residues of MAO-B (K_i= 23.69 μM) (Figure 4C).

Among the hydrazones, compound 42 appeared the most

selective hMAO-A inhibitor with an SI value of 5.40 × 10⁻⁶

(calculated SI: 4.30 × 10⁻⁷). The selectivity of compound 42

toward hMAO-A isoform (K_i= 0.010 μM) was much higher

than that of moclobemide (SI = 0.013) (Table 1B). According

to the docking data, the benzoxazolinone ring of compound 42

was sandwiched between the TYR444 and TYR407 residues in

the active site of hMAO-A making two strong π−π interactions

with these residues while hMAO-B has only one π−π

interaction between phenyl and TYR435, making compound

42 more potent hMAO-A inhibitor (K_i= 0.004 μM) than that

of hMAO-B (K_i= 9300.00 μM) (Figure S5).

As a result, we found a good correlation between the

experimental and calculated data for the hydrazones since

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a

Table 2. Reversibility of hMAO Inhibition by the Selected Novel Derivatives

test compounds incubated

with hMAO

hMAO-A activity before

dialysis (%)

hMAO-A activity after

dialysis (%)

hMAO-B activity before

dialysis (%)

hMAO-B activity after

dialysis (%)

reversibility

with no inhibitor

selegiline

lazabemide

moclobemide

6

7

14

21

41

42

43

100 0.00

89.96 2.25

11.00 0.01

36.00 1.19

24.55 1.09

23.11 2.47

18.11 1.05

22.16 1.00

19.55 1.84

20.16 1.06

21.91 2.09

100 0.00

90.01 2.00

97.00 1.16

93.77 2.16

91.74 1.93

94.00 3.21

90.16 1.88

92.55 1.39

92.24 2.60

93.57 2.46

93.26 3.00

100 0.00

51.80 2.30

89.00 1.05

88.00 1.53

85.00 1.61

78.90 1.35

78.20 1.60

74.19 3.00

77.29 2.90

82.37 2.31

77.22 1.64

100 0.00

52.40 2.03

99.00 1.70

89.55 1.90

92.25 1.66

94.80 2.07

91.50 2.64

94.16 3.71

97.00 2.33

95.21 3.27

93.41 2.11

irreversible

reversible

a

Data demonstrate mean SEM (n = 3).

a b

,

Table 3. In Vitro Cytotoxicity of the Selected Novel Derivatives

viability (%)

compounds

7.5 μM

15 μM

30 μM

selegiline

lazabemide

moclobemide

6

90.00 3.55

92.40 2.00

90.80 2.87

96.25 2.05

90.39 3.00

91.23 4.54

100.80 5.60

91.35 4.17

118.30 4.50

92.11 3.36

87.23 2.90

89.66 3.00

88.50 2.90

88.29 1.00

88.25 1.13

89.97 2.03

99.60 4.00

89.00 2.66

99.12 4.55

90.61 3.73

84.00 2.70

85.60 2.76

86.00 2.39

77.33 1.97*

7

85.27 3.37

85.00 1.16

84.93 8.59

87.67 1.61

88.55 2.39

87.40 1.35

14

21

41

42

43

a

b

Data demonstrate mean SEM (n = 3). The viability of cell was calculated as a percentage of the control. *p < 0.05 vs control.

these compounds do not have stereoisomers, and the

correlation between the calculated and experimental data for

these compounds can be successfully demonstrated.

Reversibility. The reversibility tests were performed for the

most potent and selective hMAO-A inhibitors in this series

(compounds 6, 7, 14, 21, 41, 42, and 43) using the dialysis

method (Table 2). The compounds are found to be reversible

hMAO inhibitors that have remarkable advantages compared

to irreversible inhibitors with important side eﬀects.

Cytotoxicity. The cytotoxicities of the most potent and

selective compounds (compounds 6, 7, 14, 21, 41, 42, and 43)

were tested in HepG2 cells at three concentrations (7.5, 15,

and 30 μM). Data indicated that the selected compounds,

except compound 6, which was slightly toxic to the cells at a

concentration of 30 μM and at the title concentrations, were

not toxic to hepatic cells (Table 3).

Table 4. Permeability of the Commercial Drugs Utilized for

Assay Validation and of the Selected Novel Derivatives

Determined Using the PAMPA−BBB Assay

c

permeability (10⁻⁶cm s⁻¹

)

a

b

compounds

bibliography

experimental

prediction

testosterone

verapamil

β-estradiol

progesterone

corticosterone

piroxicam

hydrocortisone

lomeﬂoxacin

dopamine

17.0

16.0

12.0

9.3

5.1

2.5

1.8

1.1

0.2

16.00 1.54

15.03 1.23

9.99 0.67

7.55 0.56

4.11 0.35

2.20 0.11

1.58 0.12

0.99 0.06

0.32 0.02

8.90 0.76

9.44 1.04

11.00 0.97

8.05 0.78

14.55 1.10

15.60 1.34

14.02 1.35

CNS+

CNS+/−

CNS−

CNS+

6

7

BBB Permeation. Since neuroactive drugs are required to

cross the BBB to function,³⁶PAMPA−BBB was performed to

assess the capability of newly synthesized derivatives to cross

the BBB. The assay was validated by comparing the

permeabilities of nine commercial drugs with presented values

(Table 4). A linear correlation was obtained with a plot of

experimental vs bibliographic data (R²= 0.9934). According to

the limits evaluated by Di et al.,³⁷the novel compounds were

classiﬁed as follows: Pe (10⁻⁶cm s⁻¹) > 4.00: CNS+ (high

BBB permeation anticipated)

14

21

41

42

43

a

Dimethylsulfoxide (DMSO, 1% w/v) at 5 mg/mL was used to

dissolve the compounds. They are diluted with PBS/EtOH (70:30).

The compounds reached a 50 μg/mL ﬁnal concentration.

b

c

Bibliography is from ref 37. Data demonstrate mean

three independent experiments.

SEM of

Pe (10⁻⁶cm s⁻¹) < 2.00: CNS− (low BBB permeation

anticipated)

Pe (10⁻⁶cm s⁻¹) from 4.00 to 2.00: CNS (uncertain BBB

permeation)

Of the most selective and potent compounds exhibiting

MAO-A activity, 6, 7, 14, 21, 41, 42, and 43 were included in

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Table 5. Brain-Tissue MAO-A, 5-HT, DA, 5-HIAA, and 3,4-dihydroxyphenylacetic acid (DOPAC) Levels after the Acute and

a

Subchronic Administrations of Test Compounds in Mice (n = 6)

compounds

MAO-A (nmol/min/mg)

5-HT (μg/g tissue)

DA (μg/g tissue)

5-HIAA (μg/g tissue)

DOPAC (μg/g tissue)

Acute Administration

control

0.40 0.012

0.35 0.011**

0.80 0.006

0.83 0.012 *

7.08 0.58

7.10 0.09

7.08 0.21

7.08 0.37

7.10 0.42

7.13 0.12

7.16 0.38

0.28 0.011

0.32 0.012*

1.22 0.20

1.24 0.05

1.23 0.27

1.23 0.10

1.24 0.23

1.25 0.46

1.25 0.24

moclobemide

compound 6

compound 7

compound 14

compound 21

compound 42

0.31 0.007** ^ψψψ

0.28 0.007*** ^ψψψ

0.25 0.009*** ^ψψψ

0.23 0.014*** ^ψψψ

0.20 0.014*** ^ψψψ

0.88 0.011 ***^ψψ

0.91 0.012 *** ^ψψ

0.98 0.009 *** ^ψψψ

1.10 0.026 ***^ψψψ

1.24 0.114*** ^ψψψ

0.32 0.009 *

0.32 0.007*

0.35 0.009* ^ψψ

0.35 0.012** ^{ψ ψ}

0.45 0.011*** ^ψψψ

Subchronic Administration

control

0.40 0.005

0.33 0.014*

0.29 0.005**

0.27 0.006***^ψψ

0.24 0.004*** ^ψψψ

0.21 0.005*** ^ψψψ

0.16 0.009*** ^ψψψ

0.78 0.01

7.45 0.22

7.40 0.12

7.30 0.42

7.31 0.55

7.28 0.65

7.43 0.51

7.43 0.48

0.18 0.01

0.23 0.01**

0.24 0.05 *

0.24 0.01 **

0.31 0.01 ***^ψψ

0.30 0.01 ***^ψψψ

0.35 0.01***^ψψψ

1.54 0.12

1.52 0.11

1.59 0.11

1.58 0.08

1.57 0.11

1.52 0.11

1.52 0.18

moclobemide

compound 6

compound 7

compound 14

compound 21

compound 42

0.91 0.01 ***

1.01 0.03***^ψ

1.04 0.04 *** ^ψ

1.19 0.04 ***^ψψψ

1.15 0.02 *** ^ψψψ

1.28 0.01 *** ^ψψψ

a

*** p < 0.001, ** p < 0.01, * p < 0.05 vs control; ψψψ p < 0.001, ψψ p < 0.01, ψ p < 0.05 vs moclobemide.

the assay. As seen in Table 5, they can successfully pass the

BBB. Compounds 41, 42, and 43 exhibited the highest

permeability considering that they may easily pass the BBB and

achieve the targets in the CNS, which were in accordance with

our design strategy. This result supports us to perform more

detailed studies with these novel prodrugs.

Structure−Activity Relationship. The structure−activity

relationships of the newly synthesized 2-pyrazoline derivatives

were evaluated by considering the substitutions on the

benzoxazolinone ring at the ﬁrst position (R₁) and the phenyl

rings at the third (R₂) and ﬁfth positions (R₃). Although it is

hard to make a deﬁnitive structure−activity relationship

discussion, some generalizations could be postulated (Table

1A).

When compounds contain hydrogen, methyl, or chlorine at

R₁; no substitution at R₂; and 4-methyl, 3-methoxy, or 3,4-

dimethoxy substitutions at R₃(compounds 5, 6, 7, 15, 16, 17,

25, 26, and 27) were compared, compounds 5, 6, and 7 with

no substitution at R₁appeared to be more potent than the ones

with methyl or chlorine at R₁. Compounds 15, 16, and 17,

which have a methyl group at R1, inhibited hMAO-A more

potently than those having a chlorine at R₁(compounds 25,

26, and 27). Among the compounds with 4-methyl groups at

R₃(5, 15, and 25), the inhibitory potency of compounds 5 (R₁

= H) and 15 (R₁:CH₃) toward hMAO-A was found to be

remarkably higher, about 10 times, than the inhibitory potency

of compound 25, which has a chlorine at R₁. Among the

compounds carrying a 3-methoxy group at R₃(compounds 6,

16, and 26), compound 6 with no substitution at R₁and

compound 16 with a methyl group at R₁strongly inhibited

hMAO-A, whereas the inhibitory potency of compound 26

with a chlorine at R₁was relatively lower. Among the

compounds with a 3,4-dimethoxy substitution at R₃(com-

pounds 7, 17, and 27), compound 7 with no substitution at R₁

and R₂appeared as a potent hMAO-A inhibitor, whereas the

inhibitory potency of compound 17 with a methyl at R₁was

found to be lower than that of compound 7. Compound 27

with a chlorine at R₁showed the lowest inhibitory activity

toward hMAO-A in this series.

R₂(compounds 8, 9, 18, 19, 28, and 29), the hMAO-A

inhibitory potency of compounds with no substitution at R₃

(compounds 8, 18, and 28) was higher than those having a

2,3-dimethoxy substitution at R₃(compounds 9, 19, and 29).

Compounds 8 and 9 that have no substitution at R₁showed

more potent inhibitory activity toward hMAO-A than the

others in this group.

When compounds carrying 4-methoxy at R₂and having no

substitution or having 2,3-dimethoxy or 2-chlorine substitu-

tions at R₃(compounds 10, 11, 14, 20, 21, 24, 30, 31, and 34)

were compared, it was found that the compounds having 2,3-

dimethoxy at R₃(compounds 11, 21, and 31) possessed lower

inhibitory activity toward hMAO-A than the other compounds

in this group. Among the compounds having 2,3-dimethoxy at

R₃(compounds 11, 21, and 31), compound 21 which is

bearing methyl at R₁appeared as the most potent hMAO-A

inhibitor. Among the compounds having no substitution at R₃

(compounds 10, 20, and 30), compound 30, which carries

chlorine at R₁, appeared as the most potent hMAO-A inhibitor.

Among the compounds having 2-chlorine at R₃(compounds

14, 24, and 34), compound 14, which has no substitution at

R₁, appeared as the most potent hMAO-A inhibitor.

Compound 14 was found to be the most potent hMAO-A

inhibitor in the novel 2-pyrazolines presented here.

When compounds having 2-chlorine at R₃and 2-methoxy or

3-methoxy or 4-methoxy at R₂(compounds 12, 13, 14, 22, 23,

24, 32, 33, and 34) were compared, compounds with no

substitution at R₁(compounds 12, 13, and 14) were found to

inhibit hMAO-A more potently than the others. Compounds

carrying 4-methoxy at R₂(compounds 14, 24, and 34)

inhibited hMAO-A more potently.

When compounds carrying no substitution at R₃and having

4-methyl or 4-methoxy substitutions at R₂(compounds 8, 10,

18, 20, 28, and 30) were compared, it was noted that

compounds 8 and 10 that have no substitution at R₁inhibited

hMAO-A more potently than the others. Compounds 28 and

30 with chlorine at R₁showed better inhibitory potency

toward hMAO-A compared to those carrying methyl at R₁.

Hydrazones having 2-methoxy at R₂and 2-chlorine at R₃

(compounds 37, 40, and 43) were also evaluated. Compounds

37 with no substitution and compound 43 with chlorine

In comparison to compounds with no substitution or 2,3-

dimethoxy substitution at R₃carrying 4-methyl substitution at

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Figure 5. (A, B). Eﬀects of a single-dose (A) and subchronic administrations (B) of the compounds and moclobemide on the immobility time in

the PFST; n = 8−12, *p < 0.05. The immobility times of mice administrated with moclobemide or the test compounds were compared with the

control.

Table 6. Dose−Response Study: Brain-Tissue MAO-A Activity; 5-HT, DA, 5-HIAA, and DOPAC Levels after the Subchronic

a

Administration of Test Compounds 21 and 42 and Moclebemide in Mice (n = 6)

compounds

dose (mg/kg) MAO-A (nmol/min/mg)

0.39 0.04

5-HT (μg/g tissue)

DA (μg/g tissue) 5-HIAA (μg/g tissue) DOPAC (μg/g tissue)

control

moclobemide

0.78 0.01

0.78 0.004

0.79 0.08

0.82 0.09 **

0.91 0.01 ***

0.80 0.05 ** ^ψψψ

0.85 0.05 *** ^ψψψ

0.99 0.05 *** ^ψψψ

1.10 0.04 *** ^ψψψ

0.81 0.06 ** ^ψψψ

0.83 0.01 *** ^ψψ

0.92 0.02 *** ^ψψψ

1.19 0.04 *** ^ψψψ

7.45 0.22

7.48 0.06

7.51 0.14

7.54 0.05

7.58 0.12

7.35 0.61

7.44 0.67

7.44 0.41

7.43 0.51

7.45 1.00

7.44 0.89

7.43 0.65

7.43 0.48

0.33 0.02

0.33 0.04

0.32 0.01

0.30 0.05

0.29 0.01 **

0.32 0.04 ** ^ψ

0.30 0.03 *** ^ψψψ

0.29 0.01 ***

0.22 0.01 *** ^ψψψ

0.32 0.03 *

0.30 0.05 ** ^ψψ

0.26 0.01 *** ^ψψψ

0.18 0.01 *** ^ψψψ

1.54 0.12

1.54 0.05

1.53 0.05

1.53 0.07

1.53 0.11

1.46 0.47

1.53 0.28

1.53 0.11

1.52 0.11

1.53 0.47

1.53 0.24

1.52 0.30

1.52 0.18

1

0.41 0.05

3

0.41 0.05

10

20

1

0.40 0.06 *

0.33 0.01 **

compound 21

compound 42

0.39 0.08 *** ^ψψ

0.34 0.05 *** ^ψψψ

0.27 0.06 *** ^ψψψ

0.21 0.04 *** ^ψψψ

0.36 0.03 *** ^ψψψ

0.30 0.05 *** ^ψψψ

0.25 0.07 *** ^ψψψ

0.16 0.09 *** ^ψψψ

3

10

30

1

3

10

30

a

*** p < 0.001, ** p < 0.01, * p < 0.05 vs control; ψψψ p < 0.001, ψψ p < 0.01, ψ p < 0.05 vs moclobemide.

substitution at R₁were found to be more potent and selective

inhibitors than compound 40 with a methyl group at R₁(Table

1B).

were also performed for 1 h after 7 days of subchronic

administration of the compounds.^46,47

Acute, oral administration of compounds 14, 21, and 42

decreased the immobility time similar to the moclobemide.

However, the decrease was not statistically signiﬁcant

compared to control. Compounds 6 and 7 did not reduce

the immobility time compared to the control (Figure 5A).

These data were from previous reports indicating that no

behavioral changes are expected with a single-dose admin-

istration of antidepressants including moclobemide.⁴⁸The

immobility time was decreased after subchronic administration

of compounds 6, 7, 14, 21, 42, and moclobemide (Figure 5B).

No statistically signiﬁcant diﬀerences were obtained between

the groups.

The compounds 14, 21, and 42 and moclobemide decreased

immobility time with acute administration, and the increasing

antidepressant activity reached statistical signiﬁcance after

subchronic administration. On the other hand, compounds 6

and 7 did not provide any reduction in the immobility time

with a single dose and caused an important decrease in

immobility time with recurring doses. Therefore, it can be

suggested that the pharmacokinetics of compounds 6 and 7

may be undergoing some problems such as ﬁrst-pass eﬀects

that prevent their activity after a single-dose administration.

Behavioral Evaluation. Porsolt’s forced swim test (PFST)

is based on behavioral despair, and it is a very commonly used

method to evaluate antidepressant activity in drug develop-

ment studies.³⁸⁻⁴⁰An increase in the escape-directed behavior

and the reduction of the immobility time indicate the

antidepressant eﬀect of tested compounds.⁴¹⁻⁴³The acute

and subchronic antidepressant eﬀects of selected compounds

6, 7, 14, 21, and 42 exhibiting the highest activity in vitro were

assessed using PFST; moclobemide was used as the reference

drug. To avoid the extra stress that may be caused by long-

term oral gavage, 7-day subchronic application was preferred

for the screening test. The in vivo oral dose of 30 mg/kg

typically used in screening tests was used for testing the new

compounds, while 20 mg/kg oral dose was used for

moclobemide according to the previous data^44,45and the

daily therapeutic dosage for a 70 kg adult human is 300−600

mg. Pharmacological analyses were performed 1 h after a

single-dose (30 mg/kg) administration to evaluate the acute

eﬀects of the compounds and to observe the acute alterations

in basal monoamine levels. Since the therapeutic response of

antidepressant therapy generally begins in weeks, experiments

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Following the behavioral assessments, the eﬀects of acute

and subchronic administrations of the compounds 6, 7, 14, 21,

and 42 and moclobemide on the brain-tissue MAO-A

selectivity were determined ex vivo by biochemical analysis.

The MAO-A activity was reduced by both the acute and

subchronic administrations of the test compounds in the brain

(Table 5), suggesting that the compounds crossed the BBB.

Subchronic administration of the new compounds and

moclobemide caused more reduction in tissue MAO-A levels

compared to acute administration. In agreement with the

compounds’ relatively higher inhibitory potency and better

selectivity (Tables 1A and 1B), the compounds inhibited

brain-tissue MAO-A activity more potently than that of

moclobemide (Table 5). It is known that MAO catalyzes the

deamination of biogenic amines including 5-HT and DA and

produces 5-HIAA and DOPAC metabolites, respectively.¹³5-

HT is more eﬃciently oxidized by MAO-A, while DA is a

substrate for both subtypes. To examine the eﬀect of the

MAO-A inhibition on the brain levels of monoamines, brain-

tissue levels of 5-HT, DA, and their main metabolites following

the acute and subchronic administration of new compounds to

mice were determined (Table 5). Brain-tissue 5-HT levels

were signiﬁcantly elevated, and 5-HIAA levels were reduced

compared with those of the control group following both the

acute and subchronic administrations of new compounds and

moclobemide (Tables 5 and 6). It is known that the activity of

MAO-A increases in depression and involves depletion of 5-

HT in the brain; inhibition of MAO-A activity prevents the

breakdown of 5-HT and increases 5-HT level in the synaptic

cleft.¹³On the other hand, the brain DA level was not aﬀected

by treatment with the new compounds and moclobemide. It

was postulated that inhibition of MAO caused increased only

in 5-HT concentrations and did not alter the DA level.

Our overall data demonstrate that new compounds strongly

and selectively inhibit MAO-A and produce an antidepressant-

like eﬀect in mice possibly due to the increase in 5-HT brain

levels caused by MAO-A inhibition. Although the compounds

increased 5-HT levels in ex vivo analysis more than that of

moclobemide, no diﬀerence was found in the antidepressant

activity between the compounds and moclobemide. It can be

thought that a 7-day subchronic treatment is not long enough

for the compounds to show these marked changes in behavior.

It was previously postulated that MAO-inhibitor treatments

can alter the motor functions,⁴⁹which may aﬀect the reliability

of the PFST. Motor activity was assessed by open-ﬁeld test

(OFT) to eliminate any possible changes in the motor

function. The distance moved in the box was measured

following the administration to assess the locomotor activity.

The new compounds and moclobemide caused no alteration in

locomotor activity of mice (Figure 6).

Figure 6. Total distance traveled in the open-ﬁeld test by mice

administrated subchronically 30 mg/kg of the test compounds and 20

mg/kg moclobemide. n = 8−10.

21 and 42 started to decrease the immobility time only at the

30 mg/kg dose (Figure 7A). However, the dose−response

curves indicate that moclobemide at 20 mg/kg dose reached

E_maxvalue (i.e., it reached its maximum antidepressant-like

activity), while compounds 21 and 42 did not reach such a

plateau or the maximum activity. Therefore, these compounds

may provide better antidepressant activity than moclobemide

at higher doses (Figure 7B).

CONCLUSIONS

■

Depression is a life-threatening and highly debilitating

neuropsychiatric disease. Reduced activity of serotonin-related

pathways plays a causal role in the pathophysiology of

depression. MAOIs, particularly MAO-A inhibitors, are found

eﬃcient in atypical and treatment resistance depression,

anxiety, and bipolar depression. However, MAOIs are rarely

used as a consequence of safety and intolerability problems and

dietary restrictions. Selective reversible inhibitors of MAO-A

(RIMAs) were introduced in the 1980s. In clinics,

moclobemide is the only RIMA available; therefore, new

potent RIMAs with fewer side eﬀects are needed.

We have designed, prepared, and tested a new series of

MAO-A-selective inhibitors based on the 2-pyrazoline and

hydrazone scaﬀold. All of the compounds showed high

inhibition in the nanomolar concentration and selectivity; in

particular, compounds 14, 21, and 42 showed important

potency and selectivity compared to the reference drugs

clorgyline and moclobemide. Examination of the structure of

the compounds indicated that the hydrazone derivatives were

more selective and potent inhibitors of the MAO-A enzyme

than 2-pyrazolines. To better understand the mechanism of

selectivity and interaction of the novel compound, molecular

modeling studies were performed for providing rational

guidance for the design of novel potential leads for drugs.

The results of the molecular docking supported the in vitro

results.

Together with biochemical data, these ﬁndings indicate that

the brain-tissue DA and DOPAC levels did not change

following the administration of the new compounds,

suggesting that the compounds did not have any signiﬁcant

side eﬀects on the motor functions.

We also performed dose−response studies with the two

most eﬃcient compounds to investigate minimum eﬀective

doses of compounds 21 (a 2-pyrazoline derivative) and 42 (a

hydrazone derivative) in mice. These compounds and

moclobemide were administered to mice at 1, 3, 10, and 30

mg/kg doses for 7 days of subchronic oral administration. In

PFST, moclobemide decreased the immobility time at 10 mg/

kg dose compared with the control group, while compounds

The in vitro ﬁndings were conﬁrmed in vivo by the PFST and

ex vivo by measuring the alterations in the brain levels of 5-HT,

DA, and their metabolites (5-HIAA, DOPAC) in mice. After

acute administration, all compounds decreased 5-HIAA levels

with a concomitant increase in 5-HT levels. However, these

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Figure 7. (A, B). Dose−response data with the PFST at 1, 3,10, and 30 mg/kg doses of the test compounds and 1, 3, 10, and 20 mg/kg doses of

moclobemide (A); n = 8−12, *p < 0.05. The immobility times of mice treated with test compounds or moclobemide were compared to those of

the control mice. Dose−response curves of moclobemide and compounds 21 and 42. Increase in the activity % of the most eﬀective compounds

and moclobemide with increasing doses (B).

(TMS). ¹³C NMR spectra were recorded on a Varian Mercury 400

MHz high-performance digital FT-NMR spectrometer utilizing

dimethylsulfoxide or acetone with chemical shifts being reported as

δ (ppm). Mass spectra were recorded using a Waters 2695 Alliance

acute changes in brain monoamine levels were not reﬂected in

behavior. On the other hand, all compounds exhibited

statistically signiﬁcant antidepressant activity after subchronic

administration consistent with the increase in brain 5-HT and

Micromass ZQ LC/MS spectrometer in methanol according to the

MAO-A levels. Ex vivo analysis showed that despite the high

ESI⁺technique. Melting points were determined using a Thomas

doses of test compounds being administered, the DA levels did

Hoover Capillary melting point apparatus and were uncorrected.

not change compared to the control group, indicating a high

MAO-A selectivity. Compounds 14, 21, and 42, the most

potent and selective inhibitors of MAO-A, exhibited an ex vivo

MAO-A proﬁle, which is highly consistent with the in vitro

data, indicating an increase in 5-HT and decrease in 5-HIAA

brain levels.

It is not surprising that the compounds are less eﬀective in

vivo than in vitro, as antidepressants are generally known to

show greater activity with prolonged administration. It was

suggested that the 7-day subchronic treatment is not long

enough to observe that apparent behavioral alterations in mice

resulted from the depleted brain monoamine levels, and a

longer administration time may be needed to provide higher

antidepressant activity. It should also be noted that the

compounds may undergo some physiological changes in the

body due to their pharmacokinetic properties.

The dose−response curves (compounds 21 and 42)

suggested that the eﬃcacy of the compounds might be higher

than that of moclobemide and that the compounds may show

higher activity with increasing doses. However, the potency of

the compounds was lower than that of moclobemide at lower

doses.

The selected seven compounds showed no toxicity on

hepatic cells. PAMPA−BBB showed that these compounds can

cross the BBB, indicating that it might be possible to develop

novel selective MAO-A inhibitors, based on this class of

compounds, with a potential for generating improved treat-

ment options for depression and mood disorders.

Thin-layer chromatography (TLC) was performed using Merck 60

F254 silica gel plates (E. Merck, Darmstadt, Germany). The purity of

the compounds was checked by elemental analyses (C, H, N)

performed on a LECO CHNS 932 analyzer in the laboratory of the

Ankara University. All compounds were obtained with a purity of

1

>95%. H NMR, ¹³C NMR, and mass interpretations and all spectral

data of the synthesized compounds are provided in the Supporting

Information.

General Procedure for the Preparation of 1,3-Di(substituted)-

phenyl-2-propen-1-ones (4) (Chalcones). Chalcone derivatives were

synthesized by condensing acetophenone (10 mmol) and benzalde-

hyde (10 mmol) derivatives in the presence of sodium hydroxide

(12.5 mmol) in water and ethanol (5/3 mL) at 0 °C for 1 h. The solid

mass separated out was ﬁltered, dried, and crystallized from

methanol.²⁹4a: m.p. 95−97 °C:⁵⁰95−96 °C (ethanol). 4b: m.p.

62−63 °C:⁵¹61−62 °C (ethanol). 4c: m.p. 86,5−88,5 °C:⁵²90−92

°C. 4d: m.p. 55−57 °C:⁵³(57−58 °C). 4e: This compound has been

synthesized previously,^54,55but its spectral properties have not been

elucidated. It is a yellow solid; yield 96% (2.699 g); mp 75,5−77,5 °C.

IR cm⁻¹: 3007, 2933, 2838 (C−H), 1660 (CO), 1609, 1574, 1475,

1428 (CC), 1266, 1227, 1180, 1069 (C−O−C). ¹H NMR

(DMSO-d₆, 400 mHz) δ (ppm): 2.42 (s, 3H, −CH₃), 3.87 (s, 3H,

−OCH₃), 3.88 (s, 3H, −OCH₃), 6.96 (dd, 1H, phenyl-H, J₁:8.4 Hz,

J₂:1.2 Hz), 7.08 (t, 1H, phenyl-H), 7.25−7.26 (m, 1H, phenyl-H),

7.29 (d, 2H, 4-methylphenyl-2H, J: 8.0 Hz), 7.59 (d, 1H, −CO−

CH=, J: 15.6 Hz), 7.93 (d, 2H, 4-methylphenyl-2H, J: 8.0 Hz), 8.08

(d, 1H, phenyl−CH=, J: 15.6 Hz). MS (ESI) m/z (%): 283 [M+H]⁺

(100%). Elemental analysis calculated (%) for C₁₈H₁₈O₃: C 76.57, H

6.43. Found: C 76.84, H 6.52. 4f: m.p. 103−105 °C:⁵⁶103−105 °C

(ethanol), 4g: m.p. 102−103 °C:⁵⁷102−103 °C, 4h: m.p. 56−58

°C:⁵⁸65−67 °C (methanol:water), 4i: This compound has been

synthesized previously,⁵⁹but its spectral properties have not been

elucidated. It is a yellow solid; yield 95% (2.581 g); m.p. 55−57 °C.

IR cm⁻¹: 3070, 2938, 2837 (C−H), 1661 (CO), 1589, 1466, 1430

EXPERIMENTAL SECTION

■

Chemistry. All chemicals and solvents used in the chemical part

were purchased from Merck A.G., Aldrich Chemical. Infrared (IR)

spectra were ensured with a PerkinElmer SpectrumOne, Nicolet 520

FT-IR spectrometer, and the results were expressed in wavenumber

(cm⁻¹). ¹H NMR spectra were recorded on a Bruker 400 MHz

UltraShield spectrometer utilizing dimethylsulfoxide or acetone with

chemical shifts being reported as δ (ppm) from tetramethylsilane

1

(CC), 1321, 1271, 1256, 1199, 1173, 1032 (C−O−C). H NMR

(DMSO-d₆, 400 mHz) δ (ppm): 3.86 (s, 3H, −OCH₃), 7.26 (dd, 1H,

phenyl-H, J₁:8.0 Hz, J₂:2.0 Hz), 7.44−7.49 (m, 2H, phenyl-2H), 7.52

(d, 1H, phenyl-H, J: 8.0 Hz), 7.58 (dd, 1H, phenyl-H, J₁:7.6 Hz, J₂:1.6

Hz), 7.63 (m, 1H, phenyl-H,), 7.78 (d, 1H, phenyl-H, J: 7.6 Hz), 7.97

(d, 1H, −CO−CH=, J: 16.0 Hz), 8.05 (d, 1H, phenyl-CH=, J: 15.6

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Hz), 8.24 (dd, 1H, phenyl-H, J₁:7.2 Hz, J₂:2.4 Hz). MS (ESI) m/z

(%): 297 [M + Na + 2]⁺(36%), 296 [M + H + Na]⁺, 295 [M + Na]⁺

(100%), 275 [M + H + 2]⁺(16%), 273 [M + H]⁺(49%), 237 [M⁺−

Cl]⁺, 167 [(2-Cl)C₆H₄-CHCH−CO+2]⁺, 165 [(2-Cl)C₆H₄−

CHCH−CO]⁺, 135 [(3-CH₃O)C₆H₄−CO]⁺. Elemental analysis

calculated (%) for C₁₆H₁₃ClO₂: C 70.46, H 4.80; Found: C 69.58, H

4.81. 4j: m.p 90−92 °C:⁶⁰(E isomer 87.8−89.3 °C, methanol:di-

chloromethane).

General Procedure for the Preparation of 1-[2-(5-Substituted-2-

benzoxazolinone-3-yl)acetyl]-3,5-di(substituted)phenyl-2-pyrazo-

lines (5−34) and N′-[1,3-Di(substitued)phenylallylidene]-2-(5-sub-

stituted-2-benzoxazolinone-3-yl)acetohydrazide (35−43). 2-(5-

Substituted-2-benzoxazolinone-3-yl)acetylhydrazine (3 mmol) was

dissolved in 2 mL of N,N-dimethylformamide (DMF) and 20 mL

of n-propanol. 1,3-Di(substituted)phenyl-2-propen-1-one (3 mmol)

and eight drops of hydrochloric acid were added to this solution and

reﬂuxed for approximately 120 h. At the end of the reaction,

pyrazoline, hydrazone, or a mixture of the two was obtained as the

product. The reaction mixture was then cooled, and the solid

precipitated was recrystallized. If solid was not precipitated or

precipitated clearly, the solution was puriﬁed by column chromatog-

raphy. Due to the low stability of hydrazones and diﬃculties in their

isolation, not all hydrazone compounds could be reached.

C₂₇H₂₅N₃O₆: C 66.52, H 5.17, N 8.62. Found: C 66.47, H 5.47, N

8.38.

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-(2-methoxylphenyl)-5-(2-

chlorophenyl)-2-pyrazoline (12). Cream solid; mp 200−201 °C. IR

cm⁻¹: 1759, 1670 (CO), 1599, 1492 (CC, CN), 1374, 1253,

1

1121, 1032 (C−O−C, C−N). For H NMR, ¹³C NMR, and mass

data, see the Supporting Information. Elemental analysis calculated

(%) for C₂₅H₂₀ClN₃O₄: C 65.01, H 4.36, N 9.10. Found: C 64.75, H

4.31, N 9.02.

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-(3-methoxylphenyl)-5-(2-

chlorophenyl)-2-pyrazoline (13). White solid; mp 220.5−221.5 °C.

IR cm⁻¹: 3060, 2931, 2835 (C−H), 1772, 1676 (CO), 1560, 1463

(CC, CN), 1307, 1271, 1151, 1118, 1035 (C−O−C, C−N). For

¹H NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₅H₂₀ClN₃O₄: C 65.01, H4.36,

N 9.10. Found: C 64.74, H 4.26, N 9.06.

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-(4-methoxylphenyl)-5-(2-

chlorophenyl)-2-pyrazoline (14). White solid; mp 237−238 °C. IR

cm⁻¹: 3054, 2934, 2828 (C−H), 1759, 1666 (CO), 1606, 1493,

1463 (CC, CN), 1398, 1248, 1175, 1039 (C−O−C, C−N). For

¹H NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₅H₂₀ClN₃O₄: C 65.01, H

4.36, N 9.10. Found: C 65.16, H 4.37, N 9.22.

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-phenyl-5-(4-methylphen-

yl)-2-pyrazoline (5). White solid; mp 214.5−215.5 °C. IR cm⁻¹:

3059, 2921 (C−H), 1778, 1668 (CO), 1601, 1487, 1440 (CC,

NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₅H₂₁N₃O₄: C 72.98, H 5.14,

N 10.21. Found: C 72.73, H 5.24, N 10.39.

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-phenyl-5-(4-

methylphenyl)-2-pyrazoline (15). White solid; mp 185−186 °C. IR

cm⁻¹: 2925 (C−H), 1768, 1677 (CO), 1495, 1440 (CC, C

1

N), 1387, 1238, 1114, 1018 (C−O−C, C−N). For H NMR, ¹³C

CN), 1365, 1314, 1231, 1148, 1018 (C−O−C, C−N). For H

NMR, and mass data, see the Supporting Information. Elemental

analysis calculated (%) for C₂₆H₂₃N₃O₃: C 73.39, H 5.45, N 9.88.

Found: C 73.20, H 5.18, N 9.81.

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-phenyl-5-(3-methoxy-

phenyl)-2-pyrazoline (6). White solid; mp 185.5−187 °C. IR cm⁻¹:

3067, 3037, 2844 (C−H), 1752, 1668 (CO), 1596, 1490, 1433

NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₅H₂₁N₃O₄: C 70.25, H 4.95,

N 9.83. Found: C 70.47, H 5.11, N 9.90.

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-phenyl-5-(3-

methoxylphenyl)-2-pyrazoline (16). White-cream solid; mp 186−

186.5 °C. IR cm⁻¹: 3055, 2933, 2830 (C−H), 1758, 1671 (CO),

1601, 1499, 1440 (CC, CN), 1389, 1258, 1241, 1136, 1042,

1022 (C−O−C, C−N). For ¹H NMR, ¹³C NMR, and mass data, see

the Supporting Information. Elemental analysis calculated (%) for

C₂₆H₂₃N₃O₄: C 70.74, H 5.25, N 9.52. Found: C 70.80, H 5.31, N

9.53.

1

(CC, CN), 1370, 1241, 1135, 1015 (C−O−C, C−N). For H

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-phenyl-5-(3,4-dimethox-

yphenyl)-2-pyrazoline (7).³⁰White solid; mp 204−204.5 °C. IR

cm⁻¹: 3069, 3000, 2944, 2839 (C−H), 1769, 1680 (CO), 1603,

1520,1490, 1439 (CC, CN), 1369, 1237, 1140, 1020 (C−O−C,

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-phenyl-5-(3,4-

dimethoxylphenyl)-2-pyrazoline (17).³⁴White solid; mp 175−176

°C. IR cm⁻¹: 3067, 2937, 2838 (C−H), 1767, 1672 (CO), 1515,

1499, 1448 (CC, CN), 1392, 1235, 1137, 1027 (C−O−C, C−

1

C−N). For H NMR, ¹³C NMR, and mass data, see the Supporting

1

N). For H NMR, ¹³C NMR, and mass data, see the Supporting

Information. Elemental analysis calculated (%) for C₂₄H₂₃N₃O₅: C

68.26, H 5.07, N 9.19. Found: C 68.18, H 5.06, N 9.15.

Information. Elemental analysis calculated (%) for C₂₇H₂₅N₃O₅: C

68.78, H 5.34, N 8.91. Found: C 68.40, H 5.31, N 8.84.

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-(4-methylphenyl)-5-phe-

nyl-2-pyrazoline (8). White solid; mp 200−201.5 °C. IR cm⁻¹: 3067,

3040 (C−H), 1769, 1659 (CO), 1603, 1483, 1440 (CC, CN),

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-(4-methyl-

phenyl)-5-phenyl-2-pyrazoline (18). Cream solid; mp 203−204.5

°C. IR cm⁻¹: 3049, 3025, 2916 (C−H gerilim), 1756, 1675 (CO),

1498, 1453 (CC, CN), 1393, 1240, 1135, 1026 (C−O−C, C−

1

1366, 1244, 1100, 1018 (C−O−C, C−N). For H NMR, ¹³C NMR,

and mass data, see the Supporting Information. Elemental analysis

calculated (%) for C₂₅H₂₁N₃O₃: C 72.98, H 5.14, N 10.21. Found: C

72.74, H 4.97, N 10.26.

1

N). For H NMR, ¹³C NMR, and mass data, see the Supporting

Information. Elemental analysis calculated (%) for C₂₆H₂₃N₃O₃: C

73.39, H 5.45, N 9.88. Found: C 72.98, H 5.69, N 9.81.

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-(4-methylphenyl)-5-(2,3-

dimethoxyphenyl-)-2-pyrazoline(9). Cream solid; mp 181−183 °C.

IR cm⁻¹: 2941, 2831 (C−H), 1762, 1673 (CO), 1609, 1477, 1447

(CC, CN), 1367, 1300, 1271, 1078, 1022 (C−O−C, C−N). For

¹H NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₇H₂₅N₃O₅: C 68.78, H 5.34,

N 8.91. Found C 68.81, H 5.03, N 9.11.

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-(4-methoxylphenyl)-5-

phenyl-2-pyrazoline (10). Cream solid; mp 219−219.5 °C. IR cm⁻¹:

2957, 2835 (C−H), 1762, 1656 (CO), 1606, 1518, 1494, 1453

(CC, CN), 1370, 1316, 1264, 1108, 1045, (C−O−C, C−N).

For ¹H NMR, ¹³C NMR, and mass data, see the Supporting

Information. Elemental analysis calculated (%) for C₂₅H₂₁N₃O₄: C

70.25, H 4.95, N 9.83. Found: C 69.79, H 4.81, N 9.83.

1-[2-(2-Benzoxazolinone-3-yl)acetyl]-3-(4-methoxylphenyl)-5-

(2,3-dimethoxy-phenyl)-2-pyrazoline (11). Cream solid; mp 194−

195 °C. IR cm⁻¹: 3011, 2928, 2838 (C−H), 1766, 1676 (CO),

1603, 1477, 1454 (CC, CN), 1390, 1251, 1172, 1072, 1026 (C−

O−C, C−N). For ¹H NMR, ¹³C NMR, and mass data, see the

Supporting Information. Elemental analysis calculated (%) for

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-(4-methyl-

phenyl)-5-(2,3-dimethoxyphenyl)-2-pyrazoline (19). Cream solid;

mp 191.5−192.5 °C. IR cm⁻¹: 2944, 2838 (C−H), 1778, 1672 (C

O), 1487, 1444 (CC, CN), 1377, 1266, 1085 (C−O−C, C−N).

For ¹H NMR, ¹³C NMR, and mass data, see the Supporting

Information. Elemental analysis calculated (%) for C₂₈H₂₇N₃O₅: C

69.26, H 5.60, N 8.65. Found: 69.30, H 5.56, N 8.73.

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-(4-methoxyl-

phenyl)-5-phenyl-2-pyrazoline (20). Cream solid; mp 188−189.5

°C. IR cm⁻¹: 1755, 1672 (CO), 1605, 1452 (CC, CN), 1392,

1

1262, 1172, 1026 (C−O−C, C−N). For H NMR, ¹³C NMR, and

mass data, see the Supporting Information. Elemental analysis

calculated (%) for C₂₆H₂₃N₃O₄: C 70.74, H 5.25, N 9.52. Found:

C 70.32, H 5.43, N 9.51.

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-(4-methoxyl-

phenyl)-5-(2,3-dimethoxyphenyl)-2-pyrazoline (21). White solid;

mp 211−212.5 °C. IR cm⁻¹: 2941, 2834 (C−H), 1759, 1672 (C

O), 1605, 1495, 1444 (CC, CN), 1392, 1310, 1243, 1176, 1081

1

(C−O−C, C−N). For H NMR, ¹³C NMR, and mass data, see the

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Supporting Information. Elemental analysis calculated (%) for

C₂₈H₂₇N₃O₆: C 67.06, H 5.43, N 8.38. Found: C 67.15, H 5.71, N

8.38.

(CO), 1613, 1520, 1490, 1463 (CC, CN), 1380, 1338, 1248,

1

1176, 1065, 1022, 1002 (C−O−C, C−N). For H NMR, ¹³C NMR,

and mass data, see the Supporting Information. Elemental analysis

calculated (%) for C₂₇H₂₄ClN₃O₆.0,9CHCl₃: C 53.24, H 3.99, N 6.68.

Found: C 53.10, H 3.78, N 6.93.

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-(2-methoxyl-

phenyl)-5-(2-chlorophenyl)-2-pyrazoline (22). Cream solid; mp

200−202 °C. IR cm⁻¹: 1767, 1668 (CO), 1605, 1471, 1450

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-(2-methoxyl-

phenyl)-5-(2-chlorophenyl)-2-pyrazoline (32). White solid; mp

217.5−219 °C. IR cm⁻¹: 3074, 2944, 2830 (C−H), 1763, 1668

(CO), 1593, 1491, 1436 (CC, CN), 1385, 1239, 1121, 1022

1

(CC, CN), 1393, 1240, 1027 (C−O−C, C−N). For H NMR,

¹³C NMR, and mass data, see the Supporting Information. Elemental

analysis calculated (%) for C₂₆H₂₂ClN₃O₄: C 65.62, H 4.66, N 8.83.

Found: C 65.21, H 4.68, N 8.97.

1

(C−O−C, C−N). For H NMR, ¹³C NMR, and mass data, see the

Supporting Information. Elemental analysis calculated (%) for

C₂₅H₁₉Cl₂N₃O₄: C 60.50, H 3.86, N 8.47. Found: C 60.48, H 3.93,

N 8.74.

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-(3-methoxyl-

phenyl)-5-(2-chlorophenyl)-2-pyrazoline (23). Cream solid; mp

232.5−233.5 °C. IR cm⁻¹: 1763, 1683 (CO), 1609, 1573, 1443

1

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-(3-methoxyl-

phenyl)-5-(2-chlorophenyl)-2-pyrazoline (33). White solid; mp

211−211.5 °C. IR cm⁻¹: 3070, 2948, 2834 (C−H), 1767, 1676

(CO), 1597, 1491, 1440 (CC, CN), 1381, 1306, 1235, 1117,

1018 (C−O−C, C−N). For ¹H NMR, ¹³C NMR, and mass data, see

the Supporting Information. Elemental analysis calculated (%) for

C₂₅H₁₉Cl₂N₃O₄: C 60.50, H 3.86, N 8.47. Found: C 60.21, H 3.70, N

8.53.

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-(4-methoxyl-

phenyl)-5-(2-chlorophenyl)-2-pyrazoline (34). Cream solid; mp

213−215 °C. IR cm⁻¹: 3067, 2941, 2838 (C−H), 1779, 1664 (C

O), 1605, 1491, 1459 (CC, CN), 1385, 1251, 1172, 1109, 1030,

1014 (C−O−C, C−N). For ¹H NMR, ¹³C NMR, and mass data, see

the Supporting Information. Elemental analysis calculated (%) for

C₂₅H₁₉Cl₂N₃O₄: C 60.50, H 3.86, N 8.47. Found: C 60.55, H 4.04, N

8.68.

(CC, CN), 1395, 1243, 1207, 1026 (C−O−C, C−N). For H

NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₆H₂₂ClN₃O₄: C 65.62, H

4.66, N 8.83. Found: C 65.46, H 4.60, N 8.88.

1-[2-(5-Methyl-2-benzoxazolinone-3-yl)acetyl]-3-(4-methoxyl-

phenyl)-5-(2-chlorophenyl)-2-pyrazoline (24). White solid; mp

221−222 °C. IR cm⁻¹: 1771, 1664 (CO), 1605, 1503, 1459

1

(CC, CN), 1392, 1255, 1172, 1014 (C−O−C, C−N). For H

NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₆H₂₂ClN₃O₄: C 65.62, H

4.66, N 8.83. Found: C 65.75, H 4.57, N 8.87.

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-phenyl-5-(4-

methylphenyl)-2-pyrazoline (25). White solid; mp 174.5−175 °C. IR

cm⁻¹: 2951, 2925 (C−H), 1782, 1679 (CO), 1610, 1489, 1441

(CC, CN), 1383, 1353, 1305, 1240, 1146, 1115, 1061, 1025

1

(C−O−C, C−N). For H NMR, ¹³C NMR, and mass data, see the

N′-(1,3-Diphenylallylidene)-2-(2-benzoxazolinone-3-yl)-

acetohydrazide (35). Yellow solid; mp 162−163 °C. IR cm⁻¹: 3201

(N−H), 3102, 3004, 2944 (C−H, aliphatic), 1756, 1678 (CO),

1617, 1487, 1430 (CC, CN), 1372, 1229, 1097, 1022 (C−O−C,

Supporting Information. Elemental analysis calculated (%) for

C₂₅H₂₀ClN₃O₃: C 67.34, H 4.52, N 9.42. Found: C 67.15, H 4.72,

N 9.59.

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-phenyl-5-(3-

methoxylphenyl)-2-pyrazoline (26).³⁵Cream solid; mp 212−214

°C. IR cm⁻¹: 3071, 3027, 2948, 2917, 2826 (C−H), 1763, 1669 (C

O), 1486, 1442 (CC, CN), 1384, 1257, 1240, 1020 (C−O−C,

1

C−N). For H NMR, ¹³C NMR, and mass data, see the Supporting

Information. Elemental analysis calculated (%) for C₂₄H₁₉N₃O₃: C

72.53, H 4.82, N 10.57. Found: C 72.39, H 4.73, N 10.57.

1

C−N). For H NMR, ¹³C NMR, and mass data, see the Supporting

N′-[1-Phenyl-3-(3-methoxyphenyl)allylidene]-2-(2-benzoxazoli-

none-3-yl)acetohydrazide (36). Cream solid; mp 150−152 °C. IR

cm⁻¹: 1781, 1687 (CO), 1604, 1488, 1435 (CC, CN), 1386,

Information. Elemental analysis calculated (%) for C₂₅H₂₀ClN₃O₄: C

65.01, H 4.36, N 9.10. Found: C 65.25, H 4.70, N 9.12.

1

1238, 1020 (C−O−C, C−N). For H NMR, ¹³C NMR, and mass

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-phenyl-5-(3,4-

methoxylphenyl)-2-pyrazoline (27).³⁵White solid; mp 197−199 °C.

IR cm⁻¹: 3070, 3000, 2937, 2842 (C−H), 1765, 1672 (CO), 1617,

1488, 1448 (CC, CN), 1389, 1235, 1148, 1029 (C−O−C, C−

data, see the Supporting Information. Elemental analysis calculated

(%) for C₂₅H₂₁N₃O₄: C 70.25, H 4.95, N 9.83. Found: C 70.00, H

4.84, N 9.80.

1

N). For H NMR, ¹³C NMR, and mass data, see the Supporting

N′-[1-(2-Methoxyphenyl)-3-(2-chlorophenyl)allylidene]-2-(2-

benzoxazolinone-3-yl)acetohydrazide (37). Yellow solid; mp 212−

213 °C. IR cm⁻¹: 3208 (N−H), 3114, 3067, 3004, 2972, 2952, 2838

(C−H), 1768, 1687 (CO), 1605, 1574, 1489, 1433 (CC, C

Information. Elemental analysis calculated (%) for C₂₆H₂₂ClN₃O₅: C

63.48, H 4.51, N 8.54. Found: C 63.10, H 4.44, N 8.70.

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-(4-methylphen-

yl)-5-phenyl-2-pyrazoline (28). White solid; mp 174−176 °C. IR

cm⁻¹: 3055, 2952 (C−H), 1775, 1676 (CO), 1605, 1487, 1444

1

N), 1364, 1241, 1107, 1085, 1020 (C−O−C, C−N). For H NMR,

¹³C NMR, and mass data, see the Supporting Information. Elemental

1

analysis calculated (%) for C₂₅H₂₀ClN₃O₄: C 65.01, H 4.36, N 9.10.

Found: C 65.12, H 4.22, N 9.26.

N′-[1-(4-Methoxyphenyl)-3-(2-chlorophenyl)allylidene]-2-(2-

benzoxazolinone-3-yl)acetohydrazide (38). Yellow solid; mp

184.5−185.5 °C. IR cm⁻¹: 1783, 1683 (CO), 1607, 1509, 1488

(CC, CN), 1365, 1252, 1174, 1102, 1021 (C−O−C, C−N). For

¹H NMR, ¹³C NMR, and mass data, see the Supporting Information.

(CC, CN), 1385, 1318, 1243, 1014 (C−O−C, C−N). For H

NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₅H₂₀ClN₃O₃: C 67.34, H

4.52, N 9.42. Found: C 67.17, H 4.73, N 9.60.

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-(4-methylphen-

yl)-5-(2,3-dimethoxyphenyl)-2-pyrazoline (29).³⁵White solid; mp

183−184 °C. IR cm⁻¹: 3019, 2960, 3944, 2838 (C−H), 1771, 1681

(CO), 1613, 1484, 1442 (CC, CN), 1378, 1267, 1216, 1073,

1015 (C−O−C, C−N). For ¹H NMR, ¹³C NMR, and mass data, see

the Supporting Information. Elemental analysis calculated (%) for

C₂₇H₂₄ClN₃O₅: C 64.10, H 4.78, N 8.31. Found: C 64.00, H 4.74, N

8.54.

Elemental analysis calculated (%) for C₂₅H₂₀ClN₃O₄: C 65.01, H

4.36, N 9.10. Found: C 65.14, H 4.45, N 9.23.

N′-[1-(4-Methoxyphenyl)-3-phenylallylidene]-2-(5-methyl-2-

benzoxazolinone-3-yl)acetohydrazide (39). Yellow solid; mp 139−

142 °C. IR cm⁻¹: 3192 (N−H), 2936, 2845 (C−H), 1775, 1668

(CO), 1605, 1494, 1456 (CC, CN), 1381, 1305, 1241, 1175,

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-(4-methoxyl-

phenyl)-5-phenyl-2-pyrazoline (30). Cream solid; mp 165−167 °C.

IR cm⁻¹: 2944, 2842 (C−H), 1775, 1668 (CO), 1601, 1487, 1455

1

1129, 1023 (C−O−C, C−N). For H NMR, ¹³C NMR, and mass

data, see the Supporting Information. Elemental analysis calculated

(%) for C₂₆H₂₃N₃O₄: C 70.74, H 5.25, N 9.52. Found: C 70.67, H

5.05, N 9.69.

1

(CC, CN), 1381, 1251, 1176, 1018 (C−O−C, C−N). For H

NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₅H₂₀ClN₃O₄: C 65.01, H

4.36, N 9.10. Found: C 64.66, H 4.64, N 9.28.

N′-[1-(2-Methoxyphenyl)-3-(2-chlorophenyl)allylidene]-2-(5-

methyl-2-benzoxazolinone-3-yl)acetohydrazide (40). Yellow solid;

mp 222−224 °C. IR cm⁻¹: 3212 (N−H), 2834 (C−H), 1768, 1688

(CO), 1597, 1492, 1433 (CC, CN), 1388, 1241, 1085, 1021

1-[2-(5-Chloro-2-benzoxazolinone-3-yl)acetyl]-3-(4-methoxyl-

phenyl)-5-(2,3-dimethoxyphenyl)-2-pyrazoline (31). Cream solid;

mp 132−134 °C. IR cm⁻¹: 3014, 2951, 2831 (C−H), 1769, 1653

1

(C−O−C, C−N). For H NMR, ¹³C NMR, and mass data, see the

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Supporting Information. Elemental analysis calculated (%) for

C₂₆H₂₂ClN₃O₄: C 65.62, H 4.66, N 8.83. Found: C 65.53, H 4.86,

N 9.03.

semiempirical force ﬁeld with an improved unbound state model as

well as its updated desolvation term, to estimate the free energy of

binding, was utilized for the search of optimal ligand binding

conformation. The detailed docking procedures are given elsewhere.⁷¹

Accelrys Visualization 4.5 program was used for rendering the 2D and

3D pictures.

N′-[1-Phenyl-3-(2-methoxyphenyl)allylidene]-2-(5-chloro-2-ben-

zoxazolinone-3-yl)acetohydrazide (41). Cream solid; mp 136−138

°C. IR cm⁻¹: 1782, 1666 (CO), 1619, 1528, 1490 (CC, CN),

1

1384, 1241, 1108, 1018 (C−O−C, C−N). For H NMR, ¹³C NMR,

Biological Activity. MAO Activity Determination. The chemicals

and recombinant hMAO enzymes were obtained from Sigma-Aldrich

(Munich, Germany). A ﬂuorimetric method, Amplex Red MAO assay

kit (Molecular Probes), was performed following the instructions of

the manufacturer. p-Tyramine (0.05−0.50 mM) was used as the

common substrate for both hMAO-A and -B. Recombinant enzymes

were diluted in reaction buﬀer (containing 0.25 M sodium phosphate,

pH 7.4). Enzyme solution (100 μL) was used for each reaction. H₂O₂

working solution (20 mM) was diluted to the ﬁnal concentration of

10 μM in reaction buﬀer and used as the positive control. The

negative control was prepared as the reaction buﬀer without H₂O₂.

The diluted enzyme samples and controls (100 μL) were pipetted

into separate wells of a microplate. The reaction was started by adding

100 μL of the Amplex Red working solution to each microplate well.

The mixtures were incubated for 30 min at room temperature in the

dark. Fluorescence was measured at multiple time points (excitation:

530 nm and emission: 590 nm). The obtained control values were

subtracted to correct the background ﬂuorescence. The possible

ﬂuorescence due to nonenzymatic inhibition was determined by

adding the compounds to solutions containing only the Amplex Red

reagent. HRP activity was not inhibited by newly synthesized

compounds in the test medium.^72,73MAO activity was expressed as

nmol/h/mg.

Kinetic Determination. Synthesized compounds with a max-

imum concentration of 1% were prepared in DMSO and used in a

concentration range of 0.01 μM to 10.00 mM. Selegiline was dissolved

also in DMSO in the concentration range of 0.01−100.00 μM. MAO

inhibition was examined using Lineweaver−Burk plotting. K_ivalues

were determined from the x-axis intercept as −K_i. Each K_ivalue is the

representative of single determination, where the correlation

coeﬃcient (R²) of the replot of the slopes versus the inhibitor

concentrations was at least 0.98. SI was calculated as K_i(hMAO-A)/

K_i(hMAO-B). The protein was determined using Pierce Coomassie

(Bradford) Protein Assay Kit (Cat no: 23200), which is based on the

Bradford method.⁷⁴

and mass data, see the Supporting Information. Elemental analysis

calculated (%) for C₂₅H₂₀ClN₃O₄: C 65.01, H 4.36, N 9.10. Found: C

65.37, H 4.43, N 9.30.

N′-[1-(4-Methoxyphenyl)-3-(2,3-dimethoxyphenyl)allylidene]-2-

(5-chloro-2-benzoxazolinone-3-yl)acetohydrazide (42). White

solid; mp 172−173.5 °C. IR cm⁻¹: 3328 (N−H), 3060, 2933, 2833

(C−H), 1788, 1686 (CO), 1608, 1572, 1493, 1441 (CC, C

N), 1382, 1338, 1249, 1172, 1066, 1016, 1003 (C−O−C, C−N). For

¹H NMR, ¹³C NMR, and mass data, see the Supporting Information.

Elemental analysis calculated (%) for C₂₇H₂₄ClN₃O₆: C 62.13, H

4.63, N 8.05. Found: C 62.08, H 4.69, N 8.25.

N′-[1-(2-Methoxyphenyl)-3-(2-chlorophenyl)allylidene]-2-(5-

chloro-2-benzoxazolinone-3-yl)acetohydrazide (43). Yellow solid;

mp 214−216 °C. IR cm⁻¹: 3204 (N−H), 3087, 3004, 2942, 2839

(C−H), 1781, 1686 (CO), 1604, 1572, 1490, 1434 (CC, C

1

N), 1384, 1252, 1110, 1084, 1019 (C−O−C, C−N). For H NMR,

¹³C NMR, and mass data, see the Supporting Information. Elemental

analysis calculated (%) for C₂₅H₁₉Cl₂N₃O₄: C 60.50, H 3.86, N 8.47.

Found: C 60.49, H 3.97, N 8.56.

X-ray Crystal Structure Determination. Data collection

measurements for cream color single crystal of compound 10 and

yellow color single crystal of compound 43 suitable for data collection

were selected and performed at 293 K using an Agilent Xcalibur Eos

diﬀractometer with CrysAlis PRO⁶¹and graphite-monochromated

Mo Kα radiation (λ = 0.71073 Å). The structure was solved by direct

methods by SHELXS-97⁶²within WINGX⁶³suite of software. The

reﬁnement was carried out by full-matrix least-squares method on F²

using SHELXL-97.⁶⁴All H atoms were constrained to ride on their

parent atoms, with C−H = 0.93−0.98 Å, N−H = 0.86 Å and U_iso(H)

=1.2U_eq(C,N,O). The crystallographic data as well as details of the

data collection and reﬁnement results for the compounds are listed in

Table S5, Supporting Information.⁶¹Molecular diagrams were created

using ORTEP-3.⁶⁵Geometric calculations were performed using

PLATON.⁶⁶

Molecular Modeling Studies. Enzyme Preparation. The crystal

structure of hMAO-A, which is co-crystallized with harmine (a

reversible inhibitor), and the crystal structure of hMAO-B, which is

complexed with saﬁnamide (a reversible inhibitor), were used for

protein setup (http://www.rcsb.org), for MAO-A pdb code: 2Z5X;

hMAO-A in complex with harmine, resolution 2.2 Å⁶⁷and for MAO-

B pdb code: 2V5Z; hMAO-B in complex with inhibitor saﬁnamide,

resolution 1.6 Å.⁶⁸All water molecules, inhibitors, and noninteracting

ions were cleared from each structure before being used in the

docking studies. The initial oxidized form of the FAD was used in all

docking studies. One of the two subunits was taken as the target

structure for MAO-A and MAO-B. Each protein’s geometry was ﬁrst

optimized by a fast Dreiding-like force ﬁeld and then submitted to the

“Clean Geometry” toolkit of Discovery Studio (Accelrys, Inc.) for a

more complete check. Missing hydrogen atoms were added based on

the protonation state of the titratable residues at a pH of 7.4. The

ionic strength was set to 0.145, and the dielectric constant was set to

10.

Ligand Setup. The 3D structures of ligand molecules were built,

optimized at the (PM3) level, and saved in pdb format. The

AutoDock Tools (vv.1.5.4) (ADT) 69 graphical user interface

program⁶⁹was employed to generate grid parameter ﬁle (gpf) and

docking parameter ﬁle (dpf) as docking input ﬁles.

Docking Calculations. Docking was carried out using AutoDock

4.2.6 program⁷⁰using HPxw8600_Work_Station. The coordinates of

the N5 atoms of the ﬂavin cofactor were chosen as the grid centers for

both MAO-A and MAO-B isozymes. The grid box sizes were adjusted

to cover the entire binding site: X, Y, Z; 70, 70, 70 Å dimensions,

respectively. Lamarckian genetic algorithm (LGA), which uses

Reversibility Assay. The dialysis method was performed as

previously described for measuring the MAO inhibition.⁷⁵Dialysis

tubings with a molecular weight cutoﬀ of 12 000 were used. Sample

capacity was 0.5−10 mL. hMAO-A or hMAO-B enzymes (1 U/mL)

and the inhibitors were incubated in potassium phosphate buﬀer

(0.05 M, pH 7.4, 5% sucrose containing 1% DMSO) for 15 min at 37

°C. The inhibitor concentrations used were 4-fold of the IC₅₀values

for the inhibition of hMAO-A and -B. Reaction mixtures having the

same amount of hMAO-A and -B were also preincubated with the

reference inhibitors. Enzyme−inhibitor mixtures were subsequently

dialyzed at 4 °C in dialysis buﬀer (100 mM potassium phosphate, pH

7.4, 5% sucrose). The dialysis buﬀer was replaced with fresh buﬀer

two times during 24 h of dialysis. After the dialysis, dialyzed and

undialyzed mixtures were simultaneously assayed for MAO-A activity

to evaluate the reversibility of the inhibition produced by the test

compounds. All reactions were carried out in triplicate, and the

residual enzyme activity was expressed as mean SEM.

In Vitro Cytotoxicity Assay. Cell viability was measured by MTT

assay using Human hepatoma cells HepG2 (Invitrogen).⁷⁶The novel

compounds (1, 5, and 25 μM) or 0.1% DMSO as a vehicle control are

used for the assay. Results were expressed as mean

SEM.

Diﬀerences are considered statistically signiﬁcant at p < 0.05.

In Vitro Blood−Brain Barrier Permeation Assay (PAMPA−

BBB). The brain penetration ability of the compounds was

determined using a PAMPA−BBB according to a previous method

(Cyprotex).³⁷Brieﬂy, compounds and the reference drugs dissolved

in DMSO were diluted with PBS/EtOH (70:30) to give a ﬁnal

concentration of 25 μg/mL. The diluted solution (200 μL) was added

to the donor wells, and PBS/EtOH (300 μL, 70:30) solution was

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J. Med. Chem. 2021, 64, 1989−2009

Journal of Medicinal Chemistry

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Article

added to the acceptor wells. The donor ﬁlter plate was placed on the

acceptor plate and kept at 25°C for 16 h. The donor plate was

removed, and the concentrations of the compounds in the wells were

measured using a UV plate reader.

Pharmacological Tests. Animals. Albino male CD-1 mice (20−

25 g) were used. The animals were kept in a room at a constant

temperature under 12 h day/12 h night period with ad libitum access

to food and water. All animal experiments were performed with the

permission of Hacettepe University Animal Experimentations Local

Ethics Board (2012/39, 2014/20).

Drug Administration. The compounds (30 mg/kg) and

moclobemide (20 mg/kg) were administered orally with gavage in

200 μL of 0.5% carboxymethylcellulose (CMC) solution. The control

group was only administrated with vehicle (0.5% CMC/200 μL/each

dose). All experiments were performed at the same time of the day, 1

h after the last administration of the drugs. Doses of 1, 3, 10, and 20

mg/kg/day for moclobemide and 1, 3, 10, and 30 mg/kg/day for

compounds were administered during 7 days for the dose−response

study.

Porsolt’s Forced Swim Test (PFST). A modiﬁed Porsolt’s forced

swim test was used for the evaluation of antidepressant activity of

synthesized compounds.^77,78Cylindrical glass tanks (19 cm height x

12 cm diameter) were used in the experiments. The cylinders were

ﬁlled with water at 25 1 °C, and the water depth was adjusted to 15

cm. The mice were individually forced to swim. The duration of

immobility or struggling with only small limb movements in a period

of 6 min was scored. Scoring was performed every 60 s for 6 min.

Immobility was judged when the mice remained ﬂoating in the water,

whereas struggling was judged when the mice made active motions

necessary to maintain its head above water. Immobility was regarded

as depression-like behavior (behavioral despair).

D1 (+0.05), D2 (−0.39), and the guard cell (+0.40). Results were

expressed as μg/g tissue.

Statistical Analysis. Statistical analysis of biochemical tests was

conducted using GraphPad Prism 4.03 (GraphPad Software, San

Diego, CA, ABD) compatible with Windows. Student’s t test was used

to achieve comparison between study groups. Results were expressed

as mean SD, and p < 0.05 was accepted statistically signiﬁcant. Data

for pharmacological tests were analyzed by one-way analysis of

variance (ANOVA) followed by post hoc Dunnet’s test. Data were

expressed as mean

SEM. p < 0.05 was accepted statistically

signiﬁcant.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01504.

■

sı

Spectral data of the newly synthesized compounds; X-

ray crystal structure; druglikeness and ADMET

predictions; and additional molecular modeling data of

selected compounds (PDF)

Molecular formula strings for the newly synthesized

compounds (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Gulberk Ucar − Department of Biochemistry, Faculty of

Pharmacy, Hacettepe University, 06100 Ankara, Turkey;

Email: gulberk.ucar@gmail.com

Open-Field Test (OFT). Open-ﬁeld test was used for assessing the

changes of locomotor activity of the mice. The test was performed

according to Painsipp, Wultsch et al. (2008)⁷⁹and Brunner, Farzi et

al. (2014).⁸⁰The compounds and moclobemide were given

subchronically as described above. Brieﬂy, the mice were put in the

center of a 50 × 50 × 30 cm³black plastic box individually and

tracked for 5 min with a video camera system (VideoMot2, TSE).

The total distance traveled in the box was measured in 5 min. After

the behavioral tests, the mice were sacriﬁced by cervical dislocation

and their brain tissue was isolated for biochemical experiments.

Determination of Monoamine Levels in Brain Tissue. Determi-

nation of MAO Activity. Brain tissue was homogenized in 10 mM

phosphate buﬀer pH 7.4 containing 0.25 M sucrose, following

diﬀerential centrifugation to obtain the mitochondrial fraction.⁸¹

Tissue protein content was determined using the Bradford method.⁷⁴

Tissue MAO-A and B activities were determined using monoamine

oxidase assay kit (Sigma-Aldrich, MAK 136) according to the

manufacturer’s instructions. The MAO activity was expressed as

nmol/min/mg.

Determination of Serotonin (5-HT) Level. The brain tissue 5-HT

content was determined after homogenization in phosphate buﬀer

(pH 7.4) using Mouse 5-HT Elisa kit based on the competitive

enzyme-linked immunosorbent assay (ELISA) using a monoclonal

anti-5-HT antibody and a 5-HT-HRP conjugate. Catalog Number:

MBS723181 (MyBioSource) The 5-HT concentration in each sample

is interpolated from a prepared standard curve and expressed as μg/g

tissue.

Nesrin Gokhan-Kelekci − Department of Pharmaceutical

Chemistry, Faculty of Pharmacy, Hacettepe University,

06100 Ankara, Turkey; orcid.org/0000-0001-7383-

8443; Email: onesrin@gmail.com

Authors

Umut Salgin-Goksen − Department of Pharmaceutical

Chemistry, Faculty of Pharmacy, Hacettepe University,

06100 Ankara, Turkey; Turkish Medicines and Medical

Devices Agency, Analyses and Control Laboratories, 06100

Ankara, Turkey

Gokcen Telli − Department of Pharmacology, Faculty of

Pharmacy, Hacettepe University, 06100 Ankara, Turkey

Acelya Erikci − Department of Biochemistry, Faculty of

Pharmacy, Lokman Hekim University, 06510 Ankara,

Turkey

Ezgi Dedecengiz − Department of Physics Engineering,

Faculty of Engineering, Hacettepe University, 06800 Ankara,

Turkey

Banu Cahide Tel − Department of Pharmacology, Faculty of

Pharmacy, Hacettepe University, 06100 Ankara, Turkey

F. Betul Kaynak − Department of Physics Engineering, Faculty

of Engineering, Hacettepe University, 06800 Ankara, Turkey

Kemal Yelekci − Department of Bioinformatics and Genetics,

Faculty of Engineering and Natural Sciences, Kadir Has

University, 34083 Fatih, Istanbul, Turkey; orcid.org/

0000-0002-0052-4926

Determination of Dopamine Level. Dopamine concentration in

brain tissue was determined after tissue homogenization in PBS using

Mouse Dopamine ELISA Kit (Cat. no: MBS035672) (MyBioSource)

according to the manufacturer’s manual. A standard curve was

prepared using the professional curve ﬁtting software, and results were

expressed as μg/g tissue.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01504

Determination of Transmitter Metabolite Levels. DOPAC and 5-

HIAA were measured according to a previous method based on

electrochemical detection by Nucleosil 5 C18 (Dionex) column.⁸²

The mobile phase consisted of a 0.1 M citrate/acetate buﬀer pH 3.9

with 10% methanol, 1 M ethylenediamine tetraacetic acid (EDTA),

and 1.2 heptane sulfonic acid. The detector voltage conditions were

Author Contributions

N.G.K. designed the study; U.S.G. and N.G.K. synthesized the

new compounds and evaluated their structures; E.D. and

F.B.K. provided the X-ray crystal structure of the compounds;

A.E. and G.U. carried out and evaluated the in vitro and ex vivo

2006

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J. Med. Chem. 2021, 64, 1989−2009

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(6) Seney, M. L.; Sibille, E. Sex Differences in Mood Disorders:

Perspectives from Humans and Rodent Models. Biol. Sex Differ. 2014,

5, No. 17.

biochemical tests; G.T. and B.C.T. accomplished the

pharmacological part; K.Y. performed the computational

studies; G.T. and A.E. conducted the statistical analyses;

N.G.K., G.U., and B.C.T. interpreted the data; N.G.K. and

G.U. edited the manuscript and provided critical revisions; and

N.G.K. is the guarantor.

(7) Collins, G. G.; Sandler, M.; Williams, E. D.; Youdim, M. B.

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Notes

The authors declare no competing ﬁnancial interest.

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ACS includes X-ray crystallographic data for compounds 10

and 43; druglikeness and ADMET properties, docking results

1

of compounds 14S, 21R, and 42; H NMR, ¹³C NMR, and

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mass interpretations; and all spectral data and molecular

formula strings of the synthesized compounds. Crystallo-

graphic data for the structures reported in this paper have been

deposited with the Cambridge Crystallographic Data Centre

(The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ,

U.K.; E-mail: deposit@ccdc.cam.ac.uk; www: http://www.

ccdc.cam.ac.uk; fax: +44 1223 336033) and are available free of

charge on request, quoting the Deposition No. CCDC

1411742 for (10) and 1411743 for (43).

(11) Wouters, J. Structural Aspects Of Monoamine Oxidase And Its

Reversible Inhibition. Curr Med. Chem. 1998, 5, 137−162.

(12) Carreiras, C.; Marco, J. L. Recent Approaches To Novel Anti-

Alzheimer Therapy. Curr. Pharm. Des. 2004, 10, 3167−3175.

(13) Finberg, J. P. M. Update On The Pharmacology Of Selective

Inhibitors Of Mao-A And Mao-B: Focus On Modulation Of Cns

Monoamine Neurotransmitter Release. Pharmacol. Ther. 2014, 143,

133−152.

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Laguna. History And Therapeutic Use Of MAO-A Inhibitors: A

Historical Perspective Of MAO-A Inhibitors As Antidepressant Drug.

Curr. Med. Chem. 2012, 12, 2275−2282.

(15) Chimenti, F.; Maccioni, E.; Secci, D.; Bolasco, A.; Chimenti, P.;

Granese, A.; Befani, O.; Turini, P.; Alcaro, S.; Ortuso, F.; Cirilli, R.; La

Torre, F.; Cardia, M. C.; Distinto, S. Synthesis, Molecular Modeling

Studies, And Selective Inhibitory Activity Against Monoamine

Oxidase Of 1-Thiocarbamoyl-3,5-Diaryl-4,5-Dihydro-(1H)- Pyrazole

Derivatives. J. Med. Chem. 2005, 48, 7113−7122.

ACKNOWLEDGMENTS

■

This study was supported by grants from The Scientiﬁc and

Technological Research Council of Turkey (TUBITAK)

(112T746) and the Hacettepe University Scientiﬁc Research

Projects Coordination Unit (012D06301001 and 1527). The

authors are thankful to Prof. Dr. Erhan Palaska for providing

MS spectra. They thank Hacettepe University Technology

Transfer Center for providing funding and English-language

editing service.

(16) Manna, F.; Chimenti, F.; Bolasco, A.; Secci, D.; Bizzarri, B.;

Befani, O.; Turini, P.; Mondovi, B.; Alcaro, S.; Tafi, A. Inhibition Of

Amine Oxidases Activity By 1-Acetyl-3,5-Diphenyl-4,5-Dihydro-

(1H)-Pyrazole Derivatives. Bioorg. Med. Chem. Lett. 2002, 12,

3629−3633.

(17) 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.

(18) Mathew, B.; Suresh, J.; Anbazhagan, S. Synthesis, Preclinical

Evaluation And Antidepressant Activity Of 5-Substituted Phenyl-3-

(Thiophen-2-Yl)-4, 5-Dihydro-1H-Pyrazole-1-Carbothioamides.

EXCLI J. 2014, 13, 437−445.

(19) Chimenti, F.; Bolasco, A.; Manna, F.; Secci, D.; Chimenti, P.;

Befani, O.; Turini, P.; Giovannini, V.; Mondovi, B.; Cirilli, R.; La

Torre, F. Synthesis And Selective Inhibitory Activity Of 1-Acetyl-3,5-

Diphenyl-4,5-Dihydro-(1H)-Pyrazole Derivatives Against Mono-

amine Oxidase. J. Med. Chem. 2004, 47, 2071−2074.

ABBREVIATIONS USED

■

PFST, Porsolt’s forced swim test; MAOIs, monoamine oxidase

inhibitors; SSRI, selective serotonin reuptake inhibitors; TCA,

tricyclic antidepressants; CNS, central nervous system; BBB,

blood−brain barrier; PAMPA, parallel artiﬁcial membrane

permeation assay; OFT, open-ﬁeld test; 5-HIAA, 5-hydrox-

yindoleacetic acid; DOPAC, 3,4-dihydroxyphenylacetic acid;

RIMAs, selective reversible inhibitors of MAO-A; MTT, 3-[4,5

dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide;

DMEM, Dulbecco’s modiﬁed Eagle’s medium; FBS, fetal

bovine serum; DMSO, dimethylsulfoxide; PBL, polar brain

lipid; CMC, carboxymethylcellulose

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New 2-Pyrazoline and Hydrazone Derivatives as Potent and Selective Monoamine Oxidase A Inhibitors

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