Absolute configuration and biological profile of pyrazoline enantiomers as MAO inhibitory activity

Article abstract of DOI:10.1002/chir.23027

A new racemic pyrazoline derivative was synthesized and resolved to its enantiomers using analytic and semipreparative high-pressure liquid chromatography. The absolute configuration of both fractions was established using vibrational circular dichroism.

Full text of DOI:10.1002/chir.23027

Received: 18 March 2018
DOI: 10.1002/chir.23027
Revised: 14 September 2018
Accepted: 18 September 2018
R E G U L A R A R T I C L E
Absolute configuration and biological profile of pyrazoline
enantiomers as MAO inhibitory activity
1,2
3
4
5
Umut Salgin Goksen | Sevgi Sarigul | Patrick Bultinck | Wouter Herrebout |
3
6
7
1
Ilknur Dogan | Kemal Yelekci | Gulberk Ucar | Nesrin Gokhan Kelekci
1
Department of Pharmaceutical
Abstract
Chemistry, Faculty of Pharmacy,
Hacettepe University, Ankara, Turkey
A new racemic pyrazoline derivative was synthesized and resolved to its enan-
tiomers using analytic and semipreparative high‐pressure liquid chromatogra-
phy. The absolute configuration of both fractions was established using
vibrational circular dichroism. The in vitro monoamine oxidase (MAO) inhib-
itory profiles were evaluated for the racemate and both enantiomers separately
for the two isoforms of the enzyme. The racemic compound and both enantio-
mers were found to inhibit hMAO‐A selectively and competitively. In particu-
lar, the R enantiomer was detected as an exceptionally potent and a selective
2
Analyses and Control Laboratories,
Turkish Medicines and Medical Devices
Agency, Ankara, Turkey
3
Chemistry Department, Boğaziçi
University, Istanbul, Turkey
4
Department of Chemistry, Ghent
University, Ghent, Belgium
5
Department of Chemistry, University of
Antwerp, Antwerp, Belgium
−
3
−3
−5
MAO‐A inhibitor (K = 0.85 × 10 ± 0.05 × 10 μM and SI: 2.35 × 10 ),
6
i
Department of Bioinformatics and
whereas S was determined as poorer compound than R in terms of K and SI
Genetics, Faculty of Engineering and
Natural Sciences, Kadir Has University,
Istanbul, Turkey
i
(0.184 ± 0.007 and 0.001). The selectivity of the enantiomers was explained
by molecular modeling docking studies based on the PDB enzymatic models
of MAO isoforms.
7
Department of Biochemistry, Faculty of
Pharmacy, Hacettepe University, Ankara,
Turkey
KEYWORDS
Correspondence
2‐pyrazoline, molecular modeling docking, monoamine oxidase inhibitory activity, specific rotation,
stereochemistry, vibrational circular dichroism
Nesrin Gokhan Kelekci, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy, Hacettepe University, 06100,
Sıhhiye, Ankara, Turkey.
Emails: onesrin@gmail.com;
onesrin@hacettepe.edu.tr
Funding information
Hacettepe University Scientific Research
Projects Coordination Unit, Grant/Award
Numbers: 1527 and 012D06301001; IOF;
BOF; FWO‐Vlaanderen; Scientific and
Technological Research Council of
Turkey, Grant/Award Number: 112T746
3
1
| INTRODUCTION
amines in the brain and the peripheral tissues, regulat-
4
ing their level. Monoamine oxidase exists in two forms,
4
Monoamine oxidase (MAO) is a flavin adenine dinucleo-
tide (FAD)‐containing enzyme that is located in the outer
mitochondrial membranes of neuronal, glial, and other
MAO‐A and MAO‐B. Monoamine oxidase‐A catalyzes
the oxidative deamination of serotonin (5‐HT), adrena-
line (A), and noradrenaline (NA) and is selectively
inhibited by the irreversible inhibitor clorgyline and the
1,2
cells. It catalyzes the oxidative deamination of biogenic
Chirality. 2018;1–13.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

2
GOKSEN ET AL.
reversible inhibitor moclobemide. Monoamine oxidase‐B
catalyzes the oxidative deamination of phenylethylamine
and benzylamine and is selectively inhibited by the irre-
configuration using VCD. To this end the R and S enantio-
mers of the biologically active 1‐[2‐(2‐benzoxazolinone‐3‐
yl)acetyl]‐3‐phenyl‐5‐(3,4‐dimethoxyphenyl)‐4,5‐dihydro‐
(1H)‐pyrazole resolved on a chiral stationary phase were
also submitted to in vitro biological evaluation. The
results revealed that the R‐enantiomer shows higher
MAO‐A inhibitory activity than the S‐enantiomer.
4,5
versible inhibitor selegiline.
Monoamine oxidase‐A and MAO‐B play essential
roles in vital physiological processes and are involved in
6,7
the pathogenesis of various diseases in human. Due to
their key role, MAO inhibitors represent a useful tool
for the treatment of several psychiatric and neurological
diseases. In particular, reversible and selective MAO‐A
inhibitors are used as antidepressant and antianxiety
drugs, while MAO‐B inhibitors have been found to be
useful as coadjuvants in the treatment of Parkinson's dis-
2
| MATERIALS AND METHODS
.1 | General information
2
All chemicals and solvents used were purchased from
Merck A.G. and Aldrich Chemical Co. The melting point
of the new compound 5 was determined using a Thomas
Hoover Capillary Melting Point Apparatus. The specific
rotations of the enantiomers dissolved in acetone were
determined using a Polarimeter Rudolph Autopol IV at
25°C operating at the sodium D line. The infrared (IR)
spectra were obtained using a Perkin Elmer Spectrum
8-15
ease and Alzheimer's disease (AD).
The development of MAO inhibitors started with
hydrazine derivatives. However, they were withdrawn
16
due to their toxic side effects. Subsequently, different
families of heterocycles containing 2 or 4 nitrogen atoms
were used as scaffolds for synthesizing reversible and
17-23
selective MAO inhibitors.
2‐Pyrazolines, which form
1
one of these families, can also be considered as a cyclic
hydrazine derivative. On the basis of the clinical profiles
of hydrazine and other heterocycles, researchers focused
on structural modifications of the pyrazoline to enhance
the pharmacological activity. Various pyrazoline deriva-
tives were synthesized and screened for their MAO,
bovine serum amine oxidase, and semicarbazide sensitive
amine oxidase activities. A considerable number of the
prepared compounds were found to have bovine serum
amine oxidase, semicarbazide sensitive amine oxidase,
and MAO inhibitory activities comparable with or higher
One and a Nicolet 520 FTIR spectrometer. H‐NMR spec-
tra in dimethylsulfoxide (DMSO‐d ) were recorded on a
6
Bruker 400‐MHz UltraShield spectrometer. Electron
impact mass spectrometry of the sample in methanol
was performed using a Waters 2695 Alliance Micromass
ZQ LC/MS spectrometer. Elemental analysis (C, H, N)
was performed using a LECO CHNS 932 analyzer. The
purity of the compound was assessed by TLC on silicagel
HF254 + 366
.
24-34
3 | SYNTHESIS AND STRUCTURAL
ELUCIDATION
than the reference compounds.
In the light of the aforementioned findings and con-
tinuing our study of pyrazoline derivatives as potential
inhibitors of MAO‐A and MAO‐B isoforms, we synthe-
sized a series of 30 new pyrazoline derivatives that pos-
sess a stereogenic center on the 5 position of the ring
and 9 new hydrazon derivatives. New compounds were
screened for their in vitro hMAO inhibitory activities
using recombinant hMAO isoforms. All compounds
inhibited hMAO‐A potently, selectively, and reversibly.
Five compounds of these series exhibiting highest inhibi-
tory potency and selectivity toward hMAO‐A were
assessed for acute and subchronic antidepressant effects
using Porsolt's forced swimming test on mice. Our data
elicited that they have an antidepressant‐like action in
mice by possibly interacting with the monoaminergic
and serotonergic system (Umut Salgın Gökşen, unpub-
lished results).
2
‐Benzoxazolinone (1), ethyl (2‐benzoxazolinone‐3‐yl)
acetate (2), and 2‐(2‐benzoxazolinone‐3‐yl) acetylhydrazine
3
5-37
(3) were synthesized according to previous methods.
3
.1 | 1‐Phenyl‐3‐(3,4‐dimethoxyphenyl)‐2‐
propen‐1‐one (4) (chalcone)
Preparation of 4 was done according to a previously
described method, providing the chalcone (2.36 g,
38
88%). mp 86.5–88.5°C (from ethanol : water [3:1])
39,40
(
lit., 88°C
).
3
3
.2 | 1‐[2‐(2‐Benzoxazolinone‐3‐yl)acetyl]‐
‐phenyl‐5‐(3,4‐dimethoxyphenyl)‐4,5‐
dihydro‐(1H)‐pyrazole (5)
Then, we decided to do semipreparative chromato-
graphic enantioseparation of the most potent and
selective compound and to see the effect of the enantio-
mers on the MAO activity. We established the absolute
2‐(2‐Benzoxazolinone‐3‐yl) acetylhydrazine 3 (1 mmol)
was dissolved in 2 mL of DMF and 20 mL of n‐propanol.
Chalcone 4 (1 mmol) and eight drops of hydrochloric acid

GOKSEN ET AL.
3
were added to this solution and the latter refluxed for
for the analyses had particle size 5 μm; column size
250 × 4.6 mm, the semipreparative column had particle
size 5 μm; column size 250 × 10 mm) as stationary
phase at 35°C. The chromatograms were recorded, and
the chromatographic peaks were integrated using the
Shimadzu C‐R6A Chromatopac software.
41
approximately 120 hours. The reaction mixture was
then cooled, and the precipitate was recrystallized to give
the 4,5‐dihydro‐(1H)‐pyrazole (0.18 g, 13%) as a white
crystal. mp 204–204.5°C (from acetone : water [3:1]).
The purity of the synthesized compound was checked
by elemental analysis. Its structure was determined using
1
13
a combination of IR, H‐NMR, C‐NMR, and ESI‐MS.
1
13
5 | VIBRATIONAL CIRCULAR
DICHROISM SPECTRA
Both enantiomers exhibited identical IR, H‐NMR, C‐
NMR, and mass spectra.
The stereochemistry of the separate enantiomers was
determined by vibrational circular dichroism (VCD).
Infrared and VCD spectra of the compounds were
obtained using a BioTools ChiralIR‐2X dual PEM spec-
trometer. Solutions containing up to 3.0 mg of the sam-
ples from both fractions from the analytical separation
were dissolved in CDCl . Spectra were recorded in a
3
1
13
The IR, H‐NMR, C‐NMR, and mass spectral data are
1
00‐μm liquid cell equipped with BaF windows. For both
2
−
1
found to be IR Vmax/cm : 3,069, 3,000, 2,944, 2,839
C─H), 1,769, 1,680 (C═O), 1,603, 1,520, 1,490, 1,439
C═C, C═N), 1,369, 1,237, 1,140, 1,020 (C─O─C, C─N);
−1
enantiomers, 40,000 scans were recorded at 4‐cm reso-
lution and averaged. The analysis reported below is based
on the experimental spectra of the first eluent. A virtual
racemate was used for baseline subtraction. Hence, spec-
tra for the two enantiomers are exact mirror images.
Extensive conformational analyses were performed
(
(
1
H‐NMR (400 mHz, DMSO‐d ) δ (ppm) J (Hz); 3.24 (1H,
6
dd, H‐13a, JAB:18.3 Hz, JAX:5.0 Hz), 3.716 (3H, s, H‐
7a), 3.722 (3H, s, H‐18a), 3.91 (1H, dd, H‐13b,
JAB:18.2 Hz, JBX:11.7 Hz), 5.10 (1H, d, H‐8a, J:17.6 Hz),
.26 (1H, d, H‐8b, J:17.6 Hz), 5.54 (1H, dd, H‐14,
JBX:11.6 Hz, JAX:5.0 Hz), 6.75 (1H, dd, H‐20, J :8.3 Hz,
J2:1.9 Hz), 6.82 (1H, d, H‐16, J:1.9 Hz), 6.89 (1H, d, H‐
9, J:8.4 Hz), 7.13 (1H, t, H‐5), 7.19 (1H, t, H‐6), 7.28
1H, d, H‐4, J :7.6 Hz), 7.36 (1H, d, H‐7, J :7.7 Hz),
.49–7.52 (3H, m, H‐23 and H‐24), 7.86–7.88 (2H, m, H‐
2); C‐NMR (400 mHz, DMSO‐d ) δ (ppm); 42.0 (C‐
3), 43.8 (C‐8), 55.4 (C‐17a), 55.5 (C‐18a), 59.9 (C‐14),
09.4 (C‐7), 109.5 (C‐5 ve C‐6), 111.9 (C‐4), 117.3 (C‐16),
22.2 (C‐19), 123.8 (C‐20), 126.9 (C‐23), 128.7 (C‐22),
30.61 (C‐24), 130.65 (C‐3a), 131.4 (C‐21), 133.9 (C‐15),
41.8 (C‐7a), 148.0 (C‐18), 148.8 (C‐17), 154.0 (C‐12),
56.0 (C‐2), 163.1 (C‐9); ESI‐MS m/z: 496 ([M + K] ,
%), 481 ([M + H + Na] , %31), 480 ([M + Na] , %100),
58 ([M + H] ), %17,) 320 ([M ‐C H (OCH ) ] , %17),
76 ([2‐benzoks.‐CH ‐CO] , %13), 148 (2‐benzoks.‐CH ]
1
42
43
44
using the MMFF94S, MMF, and SYBYL force fields
5
using the Monte Carlo and reservoir‐filling algorithms, as
45
46,47
1
implemented in the Spartan08 and Conflex
software
packages, respectively. The geometries derived from the
molecular mechanics simulations were optimized at the
1
(
48
45
67
density functional theory level using Gaussian 09 using
7
2
1
1
1
1
1
1
3
4
the B3LYP/6‐31G* and B3LYP/cc‐pVTZ combinations of
density functional theory functional and basis set and a
SCRF model to account for solvent interactions. Boltzmann
weighted IR and VCD spectra were obtained by assuming
13
6
−1
Lorentzian band profiles with a FWHH of 10 cm . The
Boltzmann populations used were based on the standard
enthalpies obtained. Inspection of the data shows that
enlarging the basis set from 6‐31G* to cc‐pVTZ only has a
minor influence on the IR and VCD spectra. Both the exper-
imental and theoretical spectra are shown in Figure 1.
+
+
+
+
+
+
6
3
3 2
+
1
2
2
+
,
%16). Found: C, 68.18; H, 5.06; N, 9.15. Calculated for
C H N O : C, 68.26; H, 5.07; N, 9.19.
6 | BIOCHEMISTRY
26
23 3 5
6.1 | Material
4
| ANALYTIC SEPARATION AND
STEREOCHEMISTRY‐HPLC
Recombinant hMAOs and other chemicals were pur-
chased from Sigma‐Aldrich. The Amplex®‐Red MAO
assay kit was obtained from Molecular Probes (Invitrogen
Detection Technologies), USA.
Small amounts of compounds (approximately 5 mg)
were used to prepare the stock solutions, and several dilu-
tions obtained from these stocks were used in tests.
ANALYSIS
Liquid chromatography was performed using an ultravio-
let detector Shimadzu SPD‐6A (λ = 254 nm) in combina-
tion with a CHIRALPAK AD‐H column packed with
amylose tris (3,5‐dimethylphenylcarbamate) (the column

4
GOKSEN ET AL.
FIGURE 1 Experimental and calculated infrared and vibrational circular dichroism (VCD) spectra obtained for the R enantiomer of 1‐[2‐
2‐benzoxazolinone‐3‐yl)acetyl]‐3‐phenyl‐5‐(3,4‐dimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazole. For the experimental VCD spectra, the noise
(
was added as a solid line in red. Due to the use of a virtual racemate for baseline subtractions, the experimental spectra for the R and S
enantiomers are perfect mirror images
6
.2 | Determination of MAO activity
time points to follow the kinetics of the reactions. Back-
ground fluorescence was corrected by subtracting the
values derived from the control (no enzyme). The possible
capacity of the new compounds to modify the fluorescence
generated in the reaction mixture due to nonenzymatic
inhibition was determined by adding these compounds to
solutions containing only the Amplex Red reagent in a
sodium phosphate buffer. The newly synthesized com-
pounds were tested for their possible interactions with
The Amplex® Red MAO Assay Kit provides a one‐step fluo-
rometric method for the continuous measurement of MAO
activity using a fluorescence microplate reader.
assay is based on the detection of H O in a horse radish per-
oxidase coupled reaction using the Amplex Red reagent, a
highly sensitive and stable probe for H O . Because
resorufin, the reaction product, has absorption and fluores-
cence emission maxima of 571 and 585 nm, respectively,
there is no significant interference from auto fluorescence.
Recombinant enzymes were diluted in a reaction
buffer (containing 0.25 M of sodium phosphate, pH 7.4).
One hundred microliters of hMAO‐A or MAO‐B solution
was incubated with 0.2 μL of inhibitor stock solution
49,50
The
2
2
2
2
Amplex Red reagent according to the method previously
49-51
decribed,
and it was found that our compounds did
not directly react with Amplex Red reagent.
6.3 | Kinetic studies
(clorgyline or pargyline, 0.5 mM) at room temperature
for 30 minutes. Enzyme and inhibitor concentrations
were kept as twofold lower in the final reaction volume.
The positive control solution was prepared by diluting the
The synthesized derivatives were dissolved in dimethyl
sulfoxide (DMSO), with a maximum concentration of 1%
and used in a wide concentration range of 0.10 to
200.00 μM. The reference inhibitors were also dissolved
in DMSO in a concentration range of 0.001 to 20.00 μM.
The mode of MAO inhibition was examined using
Lineweaver‐Burk plotting. The slopes of the Lineweaver‐
Burk plots were plotted versus the inhibitor concentration,
2
0‐mM H O working solution to the final concentration
2
2
of 10 μM in the reaction buffer, whereas the reaction buffer
without H O was prepared as the negative control. The
2
2
reaction was started by adding 100 μL of the Amplex Red
reagent to each microplate well containing the samples
and controls. The mixtures were incubated for 30 minutes
at room temperature. The fluorescence was measured using
excitation at 530 nm and emission at 590 nm at multiple
and the K values were determined from the x‐axis intercept
i
as −K . Each K value is the representative of single determi-
i
i
2
nation where the correlation coefficient (R ) of the replot of

GOKSEN ET AL.
5
the slopes versus the inhibitor concentrations was at least
as the target structure. Using a fast Dreiding‐like force
58
0
.98. The selectivity index (SI) was calculated as Ki
field, each protein's geometry was first optimized and
then submitted to the “Clean Geometry” toolkit of the
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 con-
(
hMAO‐A)/K (hMAO‐B). The protein concentration was
i
52
determined according to the Bradford method.
6.4 | Reversibility studies
5
9
stant was set to 10. The AutoDock Tools (vv. 1.5.4)
The reversibility of the hMAO inhibition with the new
compounds was determined by dialysis as previously
graphical user interface program was employed to setup
the enzymes for molecular docking.
53
described. Dialysis tubing 16 × 25 mm (SIGMA) with
a molecular weight cut‐off of 12,000 Da and a sample capac-
ity of 0.5 to 10 mL was used. Adequate amounts of the
recombinant enzymes (hMAO‐A or B; 1 U/mL) and the
inhibitors at a concentration equal to fourfold the IC50
values for the inhibition of hMAO‐A and hMAO‐B were
incubated in a potassium phosphate buffer (0.05 M, pH
The 3D structures of the ligand molecules were built,
optimized at PM3 level, and saved in pdb format. The
AutoDock Tools package was also employed to generate
the docking input files of the ligands. AutoDock 4.2.6
was used for all dockings; the detailed docking procedure
60
has been given elsewhere, and the detailed procedure
61
was reported in our earlier work.
7
3
.4, 5% sucrose containing 1% DMSO) for 15 minutes. at
7°C. Another set was prepared by preincubation of the
same amount of hMAO‐A and hMAO‐B with the reference
inhibitors. The enzyme‐inhibitor mixtures were subse-
quently dialyzed at 4°C in 80 mL of the dialysis buffer
8
| RESULTS AND DISCUSSION
.1 | Synthesis and characterization
8
(100‐mM potassium phosphate, pH 7.4, 5% sucrose). The
The synthesis pathway of 1‐[2‐(2‐benzoxazolinone‐3‐
yl)acetyl]‐3‐phenyl‐5‐(3,4‐dimethoxyphenyl)‐4,5‐dihydro‐
dialysis buffer was replaced with fresh buffer twice during
the 24 hours of dialysis. After dialysis, residual MAO activ-
ities were measured and expressed as mean ± SEM. For
comparison, undialyzed mixtures of the MAOs and the
inhibitors were included in the study.
1H‐pyrazole is given in Scheme 1. Treatment of 2‐
benzoxazolinone 1 with ethyl chloroacetate in K CO /
acetone gave the N‐alkylated product ethyl (2‐benzoxa-
zolinone‐3‐yl) acetate 2. The acid hydrazide 3 was pre-
pared by the reaction of ethyl (2‐benzoxazolinone‐3‐yl)
2
3
35
36,37
6.5 | Cytotoxicity studies
acetate and hydrazine hydrate in ethanol.
the other hand, α,β‐unsaturated carbonyl compound
chalcone) 4 was prepared by reacting 3,4‐dimethoxy-
benzaldehyde and acetophenone under basic condition
On
54
The cell viability was determined by a MTT assay.
(
Human hepatoma cell line HepG2 (Invitrogen) was used.
The cells were exposed to 5R and 5S at the concentrations
of 1, 5, and 25 μM, and 0.1% DMSO as a vehicle control
for 24 hours. Control cells treated with 0.1% DMSO
3
8
according to the Claisen‐Schmidt condensation. The
reaction of hydrazide 3 with chalcone 4 in n‐propanol
under acidic condition gave compound 1‐[2‐(2‐benzoxa-
zolinone‐3‐yl)acetyl]‐3‐phenyl‐5‐(3,4‐dimethoxyphenyl)‐
55
were used as 100% viability. Results were expressed as
mean ± SEM. Differences are considered statistically sig-
nificant at P < .05.
4,5‐dihydro‐1H‐pyrazole 5 (Scheme 1).
1
Both enantiomers exhibited identical IR, H‐NMR,
C‐NMR, and mass spectra. The IR spectrum of 5
13
−
1
7
| MOLECULAR MODELING
revealed two stretching bands at 1,769 and 1,680 cm
due to the carbonyl function of the lactam and acetyl
groups, respectively. C═C, C═N, and C─O─C, C─N
stretching bands were found near 1,603 to 1,439 cm
and 1,369 to 1,020 cm , respectively. The existence of
the C═N stretching bands and disappearance of the
N─H stretching bands prove the closure of the 4,5‐
dihydro‐(1H)‐pyrazole ring. Aromatic and aliphatic
C─H stretching bands were observed near 3,069 to
STUDIES
−
1
The crystal structures of MAO‐A and MAO‐B were
extracted from the PDB (http://www.rcsb.org, for MAO‐
A pdb code: 2Z5X; human MAO in complex with harmine,
−
1
56
resolution 2.2 Å and for MAO‐B pdb code: 2V5Z; human
MAO‐B in complex with inhibitor safinamide, resolution
57
1
.6 Å ). Each structure was cleaned of all water molecules
−
1
and inhibitors as well as all noninteracting ions before
being used in the docking studies. The initial oxidized
form of the FAD was used in all docking studies. For
MAO‐A and MAO‐B, one of the two subunits was taken
2,839 cm .
1
In the H‐NMR spectrum of 5, multiplet or doublet
peaks belonging to the protons of aromatic rings were
observed at 7.88 to 6.75 ppm. Three distinct doublet of

6
GOKSEN ET AL.
SCHEME 1 Synthesis pathway of 1‐[2‐(2‐benzoxazolinone‐3‐yl)acetyl]‐3‐phenyl‐5‐(3,4‐dimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazole 5
doublets of the ABX system in the 4,5‐dihydro‐(1H)‐
pyrazole ring (a CH proton and two anisochronous pro-
choice of the eluent was done as follows: First, different
ratios of hexane : ethanol mixtures were tried as the
mobile phase; however, none of them could resolve the
compound into its enantiomers. Therefore, it was decided
to use a more polar mobile phase. When an etha-
nol : methanol mixture with 80:20 ratio was used, the res-
olution was again not achieved. The enantiomers were
finally resolved analytically with the separation factor
(α) of 1.36 when a 50:50 ethanol : methanol mixture
was used as the eluent (flow rate 0.4 mL/min). The enan-
tiomers were then collected by semipreparative HPLC
using an CHIRALPAK AD‐H semipreparative column
under the same conditions. One hundred microliters of
multiple injections of the racemate afforded pure enantio-
mers (Figure 2).
tons of a CH ) appeared at 5.54 to 3.24 ppm. The CH
2
(
H ) proton appeared at 5.54 ppm due to vicinal coupling
x
with the two magnetically nonequivalent protons of the
methylene group at position 4 of the 4,5‐dihydro‐(1H)‐
pyrazole ring. The signals of HA and HB of the 4,5‐
dihydro‐(1H)‐pyrazole ring were observed as doublet
of doublets at 3.24 and 3.91 ppm, respectively. The
CH2 protons between the 2‐benzoxazolinone and 4,5‐
dihydro‐(1H)‐pyrazole ring resonated as a pair of doublet
of doublets at 5.10 and 5.26 ppm. The signals for methoxy
groups appeared at 3.72 ppm as two separate peaks. Addi-
13
tional support for the structure of 5 was provided by C‐
13
NMR spectra. In the C‐NMR spectra, the lactam and ace-
tyl C═O groups gave two peaks at around 156 and
13
1
63 ppm. The peak at 154 ppm in the C‐NMR spectrum
assigned to the C═N moiety confirms the closure of 4,5‐
dihydro‐(1H)‐pyrazole ring). The ions produced under
electrospray ionization showed a characteristic [M + Na]
+
ion peak as the base signal in the mass spectrum.
8.2 | Analytic separation and absolute
configuration
Because it is known that the two enantiomers of a chiral
62-65
molecule may exhibit different biological activities,
we have decided to separate both enantiomers to evaluate
their separate selective human MAO inhibitory activities.
This separation was achieved for both enantiomers of 5
using analytic and semipreparative HPLC. In this way,
FIGURE
chromatogram of 5 showing the first eluted (retention time
3.29 min) and the second eluted (retention time 56.25 min)
2
High‐performance
liquid
chromatography
5
mg of each enantiomer was resolved and used further
in VCD and MAO inhibition studies.
4
During the separation, the compound was dissolved
in methanol and 30 μL was injected to the column. The
enantiomer at 35°C. Column: Chiralpak AD‐H, eluent (vol/vol)
50:50 ethanol : methanol flow rate: 0.4 mL/min (α):1.36

GOKSEN ET AL.
7
2
5
1
. Eluent (R‐enantiomer): [α]
= +[25 ± 3])
365
computed the similarity between this second experimen-
tal spectrum and the theoretical spectrum for a computed
R enantiomer to be 19.6%. The similarity between this
second experimental spectrum and that for a computed
S spectrum amounts 68.5%, showing that the second elu-
ent corresponds to the S enantiomer.
(
c 0.0008 g/mL in acetone).
8
[
.3 | Eluent (S‐enantiomer):
25
α]
= −(25 ± 3) (c 0.0009 g/mL in
65
3
acetone)
The similarity index for IR is substantially lower than
the values obtained in many other studies. This deviation,
most probably, is related to the appearance of a broad
intense spectral feature in the experimental spectrum
The measurement of circular dichroism associated
with molecular vibrational transitions is referred to as
66-68
VCD.
The assignment of the absolute configuration
−
1
of the collected enantiomers was performed using VCD.
The experimental IR and VCD spectra for the first eluent
and the calculated IR and VCD spectra for the R enantio-
mer obtained at the B3LYP/cc‐pvTZ level are shown in
Figure 1. Due to the limited amount of sample available,
the absorbances in IR are rather small and the corre-
sponding VCD spectrum is characterized by a substantial
noise level. However, in general, good agreement is found
in which most, if not all, of the characteristic features
observed are reproduced by the calculations. The agree-
ment between experiment and theory allows the charac-
terization of the absolute configuration of the first
eluent as R and that of the second eluent as S.
near 1,000 cm , while no such band is observed in the
calculated spectra. The origin of this band could not
be established, but all spectroscopic data together
clearly establish the (absolute) chemical structure of the
compound.
8.4 | Biochemistry
The newly synthesized compound 5 (in racemic form)
was screened for its inhibitory activity toward human
recombinant MAO‐A and MAO‐B. Specific enzyme activ-
ities were calculated as 0.171 ± 0.008 nmol/(mg.min)
(n = 3) for hMAO‐A and 0.133 ± 0.009 nmol/(mg.min)
Numerical data confirming the assignment of the
absolute configuration of the first eluent as the R enantio-
mer and the second eluent as the S enantiomer were
obtained using the CompareVOA algorithm described
in Debie et al.69 Enantiomers have exactly the same
IR spectrum and hence the same similarity measure
between the theoretical and experimental IR spectra.
The B3LYP/cc‐pVTZ IR similarity index, using a scaling
factor for the calculated frequencies of 0.985, and based
upon the 1,000 to 1,800 cm spectral range studied, is
determined to be 65.1%. Vibrational circular dichroism
spectra, however, show a mirror image relationship
between enantiomers. Using the experimental spectrum
of the first eluent, we computed the similarity index
between this experimental spectrum and the theoretical
spectra of the R and S enantiomers. The similarity
between this first experimental spectrum and the theoret-
ical spectrum for a computed R enantiomer amounts
(n = 3) for hMAO‐B. The SI was expressed as K (MAO‐
i
A)/K (MAO‐B). 5 was found to inhibit hMAO‐A selec-
i
tively and competitively because the IC50 value for
hMAO‐A inhibition was calculated as 1.20 ± 0.02 μM,
whereas the IC50 value for hMAO‐B inhibition was calcu-
lated as 425.88 ± 16.45 μM. Its experimental SI (K [MAO‐
i
A]/K [MAO‐B]) was found to be 0.003.
i
For the kinetic experiments, the catalytic rates of
hMAO‐A and MAO‐B at different p‐tyramine concentra-
tions were measured. Lineweaver‐Burk graphs were
constructed in the absence of the inhibitor, and in the
presence of compound 5 or 5R or 5S and reference inhib-
itors. Because the lines are linear and intersect on the
y‐axis, compounds 5, 5R, and 5S are suggested to be com-
petitive inhibitors of hMAO‐A that may interact within
−
1
the catalytic site of hMAO‐A. K values for hMAO‐A
i
and hMAO‐B inhibition for 5 (racemic form) were
−
3
−3
determined as 0.91 × 10
± 0.01 × 10
μM and
6
8.5%. The similarity between the first experimental spec-
20.00 ± 1.55 μM, respectively (Table 1). The experimental
−
5
trum and that for a computed S spectrum amounts only
1
4
SI for 5 was found as 4.55 × 10 , showing that it is a
remarkably selective and potent MAO‐A inhibitor.
Turning to the individual isomers, isolated enantio-
mer 5R inhibited both hMAO‐A and hMAO‐B potently.
However, this compound was found to inhibit hMAO‐A
9.6%. This leads to an enantiomeric similarity index of
8.9%. This establishes that the first eluent corresponds
to the R enantiomer. The confidence level for the assign-
ment, based on the localization of the current assign-
ments with respect to the database of correct and
incorrect assignments contained in the CompareVOA
algorithm, is 99%. All calculations and analysis were
repeated at the B3LYP/ 6‐31G* level of theory and
resulted in very similar results albeit slightly worse. Using
the experimental spectrum of the second eluent, we
−
3
−3
more potently (K = 0.85 × 10 ± 0.05 × 10 μM for
i
hMAO‐A). The experimental SI value of 5R was found
−
5
as 2.35 × 10 , showing that this derivative is highly
selective toward hMAO‐A when compared with the
kinetic data of moclobemide, a known selective MAO‐A
inhibitor (Table 1). The kinetic behavior of 5R is

8
GOKSEN ET AL.
i
TABLE 1 Calculated and experimental K values corresponding to the inhibition of hMAO isoforms by 1‐[2‐(2‐benzoxazolinone‐3‐yl)
acetyl]‐3‐phenyl‐5‐(3,4‐dimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazole 5
Calc. K Value Calc. K Value Exp K
i
i
i
Value
Exp K
i
Value
Inhibition Type,
Selectivity, and
Reversibility
for MAO‐A for MAO‐B
for MAO‐A
for MAO‐B
a
b
b
a
Compound
(μM)
(μM)
Calc. SI
(μM)
(μM)
Exp. SI
−3
−4
−5
5
(racemic)
‐
‐
‐
0.91 × 10 ± 10
20.00 ± 1.55 4.55 × 10
MAO‐A,
competitive,
reversible
−
3
−5
−3
−3
−5
5
R
0.74 × 10
0.229
29.47
193.25
2.51 × 10
0.001
0.85 × 10 ± 0.05 × 10
0.184 ± 0.007
36.20 ± 1.86 2.35 × 10
MAO‐A,
competitive,
reversible
5S
175.00 ± 9.30
0.09 ± 0.004
0.001
Selegiline
MAO‐B
22.02
5.71
34.07
0.646
0.023
9.060 ± 0.44
0.005 ± 0.001
100.67
MAO‐B, suicide
inhibitor
irreversible
(
inhibitor)
Moclobemide
250.74
1.22 ± 0.08
0.004
MAO‐A,
(
MAO‐A
competitive,
reversible
inhibitor)
a
i i
Selectivity index calculated as K (MAO‐A)/K (MAO‐B). The selectivity toward MAO‐A increases as the corresponding SI decreases, while selectivity toward
MAO‐B isoform increases as the corresponding SI increases.
b
Each value represents the mean ± standard error of mean (SEM) of three independent experiments.
indicated in Figure 3. 5S also inhibited hMAO‐A and
hMAO‐B potently. However, the SI of 5S was calculated
as 0.001, which is poorer than that of compound 5R.
The reversibility tests were carried out using the dialysis
method. Compound 5 and its enantiomers 5R and 5S
appeared as reversible inhibitors of hMAO‐A (Table 2).
As seen in Table 2, moclobemide, a known selective
MAO‐A inhibitor, strongly inhibited hMAO‐A. Following
incubation of the enzyme with moclobemide, the remain-
ing MAO‐A activity was determined as 34.3 ± 1.4%.
hMAO‐A activity was recovered up to 91.2 ± 2.9% after
dialysis indicating that the inhibition is reversible. 5R
70
FIGURE 3 A, Lineweaver‐Burk plots for the inhibition of hMAO‐A by 5R. [S]: substrate concentration (mM), [V]: reaction velocity (nmol/
hour/mg). Inhibitor concentrations are shown at the left. B, The second graph represents the plots of the slopes of the Lineweaver‐Burk plots
−
3
−3
i
versus inhibitor concentration [I]. K was calculated as 0.85 × 10 ± 0.05 × 10 nM

GOKSEN ET AL.
9
TABLE 2 Reversibility of the inhibition of hMAOs by the novel compounds
Test Compound
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
Moclobemide
Selegiline
100 ± 0.00
34.28 ± 1.37
91.31 ± 2.84
39.22 ± 1.13
10.46 ± 0.54
30.55 ± 1.68
100 ± 0.00
91.22 ± 2.88
92.16 ± 3.14
92.45 ± 2.34
95.21 ± 1.33
93.00 ± 2.11
100 ± 0.00
75.29 ± 3.01
50.29 ± 1.66
87.26 ± 2.60
79.50 ± 2.46
80.60 ± 2.16
100 ± 0.00
92.00 ± 4.00
51.02 ± 2.30
94.11 ± 3.27
94.00 ± 2.14
91.33 ± 2.00
Reversible
Irreversible
Reversible
Reversible
Reversible
5
5
R
S
5
Each value represents the mean ± SEM (n = 3).
inhibited hMAO‐A more potently than moclobemide
10.5 ± 0.6%). Monoamine oxidase‐A activity was recov-
significant interactions, four π‐π interactions were identi-
fied between the side chain of Tyr407, Tyr444, and the
benzoxazolinone ring of the inhibitor (pink dashed‐line in
Figure 4C). Also, in the 2D picture (Figure 4C), 5R enters
various Van der Waals interactions with Ile180, Asn181,
Lys305, Ileu335, Phe352, Tyr69, Gln215, Tyr197, Asp339,
Lys218, Ala68, Gly67, Leu337, Phe208, and Gly443 amino
acids.
(
ered to 95.2 ± 1.3% after dialysis, indicating that the inhi-
bition of hMAO‐A with 5R is almost totally reversible.
In vitro cytotoxicity of the new inhibitors was tested at
three different concentrations (1, 5, and 25 μM; Table 3).
The results showed that they are not toxic to hepatic cells
at the test concentrations.
As shown in Figure 4B, enantiomer 5S is located at
the active site of MAO‐A (K = 229.44 nM). The 2‐
i
8
.5 | Molecular modeling docking studies
benzoxazolinone ring cannot make effective π‐π bonds
with Tyr407 and Tyr444 because it cannot approach
to FAD ring as in the case of 5R isomer. The 3,4‐
dimethoxyphenyl ring of 5S makes one π‐σ interaction
with Tyr197 and one π‐π with Tyr407. The phenyl ring
at position 3 of the 2‐pyrazoline ring makes two π‐alkyl
interactions with Leu335 and Ile180 and π‐π interaction
with Phe208. Figure 4B and D shows that the inhibitor
enters several Van der Waals interactions with Asn181,
Lys305, Ileu335, Phe352, Tyr69, Gly443, and Thr408
amino acids.
To rationalize the mode of interaction and the impact of
the absolute configuration, molecular modeling docking
studies were performed. Figure 4A to D shows the result
of the molecular modeling docking of 5R and 5S within
the MAO‐A active site. Analysis of the binding modes
for the R isomer in the MAO‐A active site cavity revealed
that 5R is located in front of the FAD cofactor from re
face. 5R interacts with the active site residues lining the
cavity as well as the FAD cofactor effectively (Figure 4A;
Ki = 738.93 pM). Two hydrogen bonds occur between
the side chain amine hydrogens of Lys 305 and the
methoxy moieties of the phenyl group of the inhibitor.
An additional hydrogen bond forms between the carbonyl
groups of the inhibitor and the cofactor FAD
The experimental data given in Table 1 agree with
these observations. All of the computational inhibition
results support that the MAO‐A inhibitory potency of
−
3
the R enantiomer (K = 0.74 × 10 μM) is much higher
i
(green dashed line in Figure 4C). In addition to these
and 310 times more selective in comparison to the S
enantiomer (K = 0.229 μM).
i
TABLE 3 In vitro cytotoxicity of 1‐[2‐(2‐benzoxazolinone‐3‐yl)
acetyl]‐3‐phenyl‐5‐(3,4‐dimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazole
derivatives
In Figure 5, 5R and 5S were superimposed within
the MAO‐A active site. As Figure 5 shows, the
benzoxazolinone ring of 5R orients itself in the hydropho-
bic cage surrounded by Tyr407, Tyr444, and the FAD
cofactor, making four π‐π interactions. In the case of
Viability (%)
Compound
5
1 μM
5 μM
25 μM
5S, the 3,4‐dimethoxyphenyl ring of the 5S occupies the
95.26 ± 1.52
97.45 ± 1.30
93.56 ± 2.13
92.25 ± 2.01
95.22 ± 1.85
90.55 ± 1.03
89.26 ± 1.77
90.22 ± 1.97
88.77 ± 2.01
same volume of the hydrophobic cage, making only one
π‐σ interaction. The other moieties of the 4,5‐dihydro‐
(1H)‐pyrazole ring of both isomers do not overlap in the
active site of the enzyme either. Having stereogenic cen-
ter in 5 results in a very different binding mode and
significant inhibition constant difference.
5
R
S
5
Data were expressed as mean ± SEM (n = 3). Cell viability was expressed as
a percentage of the control value. p < .05 was considered as statistically
significant.

10
GOKSEN ET AL.
FIGURE 4 Three‐dimensional representations of docked poses of the 5R isomer (A) and the 5S isomer (B) in the MAO‐A active site. Two‐
dimensional interaction diagrams of the 5R isomer (C) and the 5S isomer (D) with amino acid residues lining the MAO‐A active site
hydrophobic cage surrounded by Tyr407, Tyr444, and
the FAD cofactor, making four π‐π interactions. In the
case of 5S, the 3,4‐dimethoxyphenyl ring of the 5S
occupies the same volume of the hydrophobic cage, mak-
ing only one π‐σ interaction. The other moieties of the
4,5‐dihydro‐(1H)‐pyrazole ring of both isomers do not
overlap in the active site of the enzyme either. Figure 4
C and D shows the interaction diagram reflected in 2D
pictures. It was seen from Figure 5 that the hydrogen
attached to the chiral C‐14 in 5R enantiomer forms a
hydrogen bond with the Gln 215 amino acid residue; on
the other hand, this interaction is absent in 5S isomer.
Having stereogenic center in 5 results in a very different
binding mode and significant inhibition constant differ-
ence between R and S stereoisomer.
FIGURE
5
The 5R (magenta) and 5S (green) isomers
9
| CONCLUSION
superimposed within the active site of MAO‐A isozyme. The
hydrogen attached to the chiral C‐14 are shown in white color; they
are oriented differently in the active site of the enzyme
1‐[2‐(2‐Benzoxazolinone‐3‐yl)acetyl]‐3‐phenyl‐5‐(3,4‐
dimethoxyphenyl)‐4,5‐dihydro‐(1H)‐pyrazole (5) was syn-
thesized and separated by analytic and semipreparative
HPLC technique and evaluated for in vitro inhibitory
profiles of both enantiomers and the racemate toward
hMAOs. Racemic 5 was found to inhibit hMAO‐A
In Figure 5, 5R and 5S were superimposed within
the MAO‐A active site. As Figure 5 shows, the
benzoxazolinone ring of 5R orients itself in the

GOKSEN ET AL.
11
selectively and competitively. According to the expecta-
tion that the chiral center may lead to different ligand
enzyme interactions, the separate enantiomers were
resubmitted to in vitro biological evaluation. Isolated
enantiomers 5R and 5S were also found to be selective
MAO‐A inhibitors. The experimental SI was found to be
3. Ramsay RR, Albreht A. Kinetics, mechanism, and inhibition of
monoamine oxidase. J Neural Transm. 2018. https://doi.org/
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4. Bousquet P, Feldman J, Schwartz J. Central cardiovascular
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5
230(1):232‐236.
2
.35 × 10 for 5R, showing that this derivative is a highly
5. Grimsby J, Lan NC, Neve R, Chen K, Shih JC. Tissue distribu-
selective and potent MAO‐A inhibitor compared to
moclobemide. A molecular modeling docking study was
carried out using PDB enzymatic models in order to eval-
uate the molecular interactions of the enantiomers with
hMAO isoforms. 5R appeared as the more potent and
selective MAO‐A inhibitor when compared to 5S. This
indicates that 5R interacts more strongly with the active
site of MAO‐A than 5S. The key factor for the efficient
and selective activity is, most probably, the shorter
distance between 5R and the FAD ring at the active site
of MAO.
In summary, the results show that separated enantio-
mers of the newly synthesized 4,5‐dihydro‐(1H)‐pyrazole
derivative may be promising candidates as potent MAO‐
A inhibitor agents. These findings may assist medicinal
chemists working in this area, and in vivo studies on mice
have recently been initiated.
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ACKNOWLEDGMENTS
11. Carreiras MC, Marco JL. Recent approaches to novel anti‐
Alzheimer therapy. Curr Pharm Des. 2004;10(25):3167‐3175.
Funding by the Scientific and Technological Research
Council of Turkey (project number: 112T746) and by
the Hacettepe University Scientific Research Projects
Coordination Unit (project number: 012D06301001 and
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527) is acknowledged. W.A.H and P.B. acknowledge sup-
port by the fund for scientific research FWO‐Vlaanderen
and by BOF and IOF funding schemes.
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potential of monoamine oxidase inhibitors. Nat Rev Neurosci.
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CONFLICT OF INTEREST
5. Bortolato M, Chen K, Shih JC. Monoamine oxidase inactivation:
from pathophysiology to therapeutics. Adv Drug Deliv Rev.
There are no conflicts of interest to declare.
2008;60(13–14):1527‐1533.
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ORCID
monoamine oxidase inhibitors. In: Developments of drug and
modern medicines part I. Drug design.
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Nesrin Gokhan Kelekci
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