Monoamine oxidase inhibitory activity of 3,5-biaryl-4,5-dihydro-1H- pyrazole-1-carboxylate derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.09.010

Ethyl and phenyl carbamate derivatives of pyrazoline (3a-3h) were synthesized and tested for their MAO inhibitory activity. All the compounds were found to be selective towards MAO-A. Phenyl carbamates (3e-3h) were better than ethyl carbamates (3a-3d) and

Full text of DOI:10.1016/j.ejmech.2013.09.010

Accepted Manuscript
Monoamine oxidase inhibitory activity of 3,5-biaryl-4,5-dihydro-1H-pyrazole-1-
carboxylate derivatives
B. Vishnu Nayak, S. Ciftci-Yabanoglu, Surender Singh Jadav, Monika Jagrat, Barij N.
Sinha, G. Ucar, Venkatesan Jayaprakash
PII:
S0223-5234(13)00582-5
10.1016/j.ejmech.2013.09.010
EJMECH 6412
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 March 2013
Revised Date: 3 September 2013
Accepted Date: 5 September 2013
Please cite this article as: B Vishnu Nayak, S Ciftci-Yabanoglu , S.S. Jadav, M. Jagrat, B.N. Sinha,
G Ucar , V. Jayaprakash, Monoamine oxidase inhibitory activity of 3,5-biaryl-4,5-dihydro-1H-
pyrazole-1-carboxylate derivatives, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.09.010.
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ACCEPTED MANUSCRIPT
Graphical abstract
Amongst the eight compounds (3a-3h), compound 3f was found to be potent and
selective towards hMAO-A and is competitive and reversible in nature.
3f: KihMAO-A: 0.005±2x10-4 µM
KihMAO-B: 56.00±3.05 µM

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Monoamine oxidase inhibitory activity of 3,5-biaryl-4,5-dihydro-1H-pyrazole-1-
carboxylate derivatives
1B Vishnu Nayak, 2Ciftci-Yabanoglu S, 1Surender Singh Jadav, 1Monika Jagrat, 1Barij N. Sinha,
2,*Ucar G, 1,3,*Venkatesan Jayaprakash
1Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi,
Jharkhand 835 215, India
2Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, Sıhhiye 06100,
Ankara, Turkey
3Valens Pharma Services, Regus Citi Centre, Level 6, Chennai Citi Centre, 10/11,
Dr.Radhakrishnan Salai, Chennai, Tamil Nadu 600 004, India
*Corresponding Author
Venkatesan Jayaprakash; Tel: +91-94701372264; E-mail: venkatesanj@bitmesra.ac.in
Gulberk Ucar, Tel: +90 312 3052553; E-mail: gulberk@hacettepe.edu.tr
A R T I C L E I N F O
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Human monoamine oxidase, inhibitors, pyrazolines, carbamates, molecular docking
MAO: Monoamine oxidase, hMAO: human MAO, rMAO: rat MAO, SAR: Structure-activity-
relationship, bb: back-bone, sc: side-chain
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ABSTRACT
Ethyl and phenyl carbamate derivatives of pyrazoline (3a-3h) were synthesized and tested for
their MAO inhibitory activity. All the compounds were found to be selective towards MAO-A.
Phenyl carbamates (3e-3h) were better than ethyl carbamates (3a-3d) and displayed the best
selectivity index. Compound 3f (KiMAO-A; 4.96±0.21 nM) was found to be equally potent as that
of standard drug, Moclobemide (KiMAO-A; 5.01±0.13 nM) but with best selectivity index (8.86 x
10-5). Molecular docking studies with R & S conformer of 3f revealed S-enantiomer is better than
R-enantiomer as reported earlier by other groups. It is proposed that VdW’s radii of the
substitution (bulkiness) in ring B determine the potency of phenyl carbamates.
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1.Introduction
Monoamine oxidase exists in two different isoform, MAO-A & MAO-B, classified based on
their specificity towards their substrate and their selective inhibitors.[1, 2]Iproniazide was the
first drug approved for the treatment of depressive illness during 1950s and that was followed by
imipramine, phenelzine, isocarboxazid and tranylcypromine. All are not selective towards MAO-
A and MAO-B isoform as well as not specific towards peripheral & brain MAO. Tyramine-
induced hypertensive crisis (Cheese-effect) reported with tranylcypromine during mid 1960s,
lead to the establishment of strict dietary restrictions by US-FDA.[3, 4] Gradually this factor
restricted the use of MAO-inhibitors and practitioner’s switched to the newer drugs, acting on
different mechanism, that did not required dietary restrictions (tricyclic antidepressants and
Selective Serotonin Reuptake Inhibitors).[5]“Cheese effect” was mainly due to the non-specific
inhibition of MAO-A in gut (peripheral) that warranted brain-specific MAO inhibitors. While
brain specific inhibitor reduces “cheese effect”, isoform selective inhibitors are to be developed
for their therapeutic potential in treatment of depression (MAO-A selective) and
neurodegenerative disorders (MAO-B selective).[6]Development of new generation of MAO
inhibitors that specifically inhibits brain-MAO in isoform selective and reversible mode has
renewed the research interest on MAO-inhibitors.[7, 8]
Chemical class ∆2-pyrazolines have been explored for the development of newer MAO-inhibitor
class assuming the structure as a cyclic analog of Iproniazide. Many ∆2-pyrazoline derivatives
were synthesized and tested for their antidepressant activity[9-17] and for their MAO-inhibitory
activity[18-28]. Our group has also reported few derivatives with MAO-inhibitory activity in rat-
MAO[29-31] and in human-MAO[32].
The presented study explores the MAO-inhibitory activity of ethyl and phenyl carbamate
derivatives of ∆2-pyrazolines. The compounds (3a-3h) were synthesized according to the
reactions outlined in Scheme 1. Chalcones (1a–1d) were prepared through Claisen–Schmidt
condensation of appropriate benzaldehydes with 2’-hydroxyacetophenone in 60% ethanolic
sodium hydroxide solution.[33] The pyrazoline intermediates (2a–2d) were obtained by the
condensation of (1a–1d) with excess hydrazine hydrate (99%) in methanol.[33] The final
products
3a-3h
were
obtained
by the
reaction
of
2a–2d
with
ethyl
chloroformate/phenylchloroformate with an addition of equimolar quantity of potassium
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carbonate.[34] All the compounds were tested for their MAO-inhibitory activity using hMAO-A
and hMAO-B.
2. Experimental section
2.1. Materials and methods
All the chemicals and solvents for synthesis were purchased from Aldrich. Unless otherwise
mentioned the solvents were used without purification. Melting points were determined using
Optimelt (Stanford Research Systems, Sunnyvale, CA 94089) by capillary method and are
uncorrected. Infrared (IR) spectra were taken on a FT-IR Spectrophotometer IR-Prestige 21
1
(Shimatzu Corporation, Japan) from 4000–400 cm-1 using KBr discs. H-NMR spectra were
recorded at 300 MHz in CDCl3 and 13C-NMR at 400 MHz in DMSO using a Bruker Avance 400
instrument (Bruker Instruments Inc., USA) and Varian 400 (Varian Inc., USA), respectively.
Chemical shifts were measured at δ units (ppm) relative to tetramethyl silane (TMS). Fast-atom
bombardment (FAB) mass spectra were recorded on a Jeol SX 102/DA-6000 mass spectrometer
(Jeol Ltd Akishima, Tokyo, Japan) using argon/xenon (6 kV, 10 mA) as FAB gas, m-
nitrobenzylalcohol as matrix, and 10 kV as accelerating voltage at room temperature. Reactions
were monitored by thin-layer chromatography on silica gel plates in either iodine or UV
chambers.
Monoamine oxidase A human (recombinant, expressed inbaculovirus infected BTI insect cells),
monoamine oxidase-B human (recombinant, expressed in baculovirus infected BTI insect cells),
R-(-)-selegiline hydrochloride, resorufin, dimethyl sulfoxide (DMSO) and some other chemicals
were purchased from Sigma–Aldrich™ (Germany). The Amplex®-Red MAO Assay
Kitcontained benzylamine, p-tyramine, Moclobemide (MAO-A inhibitor), Selegiline (MAO-B
inhibitor) and horseradish peroxidase.
All computational works were carried out in a single machine with RHEL5 operating system
running on 2.93 GHz Pentium4 processor with 2GB RAM. Maestro-8.4, AutoDock-4.2,
MGLTools-1.4.6 and openbabel were used for simulation studies.
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2.2. Chemistry
2.2.1.General procedure for synthesis of pyrazoline-carboxamide (3a-3h): -
To the solution of pyrazoline (2a-2d, 0.0008 M) in methanol (10 ml), was added an equimolar
quantity of ethylchloroformate/ phenylchloroformate drop-wise at <10 °C with stirring. An
equimolar quantity of potassium carbonate was added and stirring continued for another 15-20
min. The reaction mixture was then filtered and the filtrate upon evaporation provided compound
3a-3h. [34]
2.2.1.1. Ethyl 3-(2-hydroxyphenyl)- 5-(4-methoxyphenyl)- 4,5-dihydro-1H-pyrazole- 1-
carboxylate (3a)
1
Yield: 46%; mp: 116-118 °C; H-NMR (300 MHz, CDCl3): δ (ppm) 1.25 (appeared as doublet,
J=3.00, 3H, -CH3); 3.28 (dd, JAM= 17.7 Hz, JAX=5.10 Hz, HA); 3.79 (S, 3H, -OCH3); 3.84 (dd,
JMX= 17.10 Hz, JMA=10.80 Hz, HM); 4.19 (appeared as triplet, J=3.45 Hz, 2H, -CH2-); 5.34 (dd,
JXA= 11.85 Hz, JXM=5.40 Hz, HX); 6.85-7.36 (m, 8H, Ar-H); 10.70 (s, 1H, -OH); 13C-NMR (400
MHz, DMSO-d6): δ (ppm) 14.74 (-CH2-CH3), 43.52 (-CH2-CH3), 55.47 (OCH3), 59.74 (pyr-C4),
61.76 (pyr-C5), 114.43-156.17 (Ar-C), 157.34 (-C=O), 159.03 (pyr-C3); FAB-MS: 340 (M)+,
341 (M+1)+
2.2.1.2. Ethyl 3-(2-hydroxyphenyl)- 5-(4-methylphenyl)- 4,5-dihydro-1H-pyrazole- 1-
carboxylate (3b)
Yield: 42%; mp: 194-196 °C; 1H-NMR (CDCl3): δ (ppm) 1.24 (appeared as doublet, J=3.00, 3H,
-CH3); 2.33 (s, 3H, -CH3); 3.27 (dd, JAM= 17.55 Hz, JAX=5.10 Hz, HA); 3.84 (dd, JMX= 17.55 Hz,
JMA=12.00 Hz, HM); 4.19 (appeared as doublet, J=6.90 Hz, 2H, -CH2-); 5.35 (dd, JXA= 11.85 Hz,
JXM=5.4 Hz, HX); 6.87-7.35 (m, 8H, Ar-H); 10.70 (s, 1H, -OH); FAB-MS: 324 (M)+, 325 (M+1)+
2.2.1.3. Ethyl 3-(2-hydroxyphenyl)- 5-(4-chlorophenyl)- 4,5-dihydro-1H-pyrazole- 1-
carboxylate (3c)
1
Yield: 58%; mp: 180-184 °C; H-NMR (CDCl3): δ (ppm) 1.25 (appeared as singlet, 3H, -CH3);
3.25 (dd, JAM= 17.70 Hz, JAX=5.40 Hz, HA); 3.87 (dd, JMX= 17.70 Hz, JMA=12.00 Hz, HM); 4.21
(appeared as doublet, J=5.70 Hz, 2H, -CH2-); 5.36 (dd, JXA= 11.70 Hz, JXM=5.40 Hz, HX); 6.88-
7.37 (m, 8H, Ar-H); 10.61 (s, 1H, -OH); 13C-NMR (400 MHz, DMSO-d6): δ (ppm) 14.72 (-CH2-
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CH3), 43.53 (-CH2-CH3), 59.67 (pyr-C4), 61.91 (pyr-C5), 116.08-152.18 (Ar-C), 156.10 (pyr-
C3), 157.34 (-C=O); FAB-MS: 344 (M)+, 345 (M+1)+, 346 (M+2)+
2.2.1.4.Ethyl 3-(2-hydroxyphenyl)- 5-phenyl- 4,5-dihydro-1H-pyrazole- 1-carboxylate (3d)
1
Yield: 51%; mp: 159-162 °C; H-NMR (CDCl3): δ (ppm) 1.23 (appeared as singlet, 3H, -CH3);
3.30 (dd, JAM= 17.70 Hz, JAX=5.40 Hz, HA); 3.87 (dd, JMX= 17.55 Hz, JMA=11.70 Hz, HM);
4.19(appeared as triplet, J=3.30 Hz, 2H, -CH2-); 5.39 (dd, JXA= 11.85 Hz, JXM=5.40 Hz, HX);
6.87-7.61 (m, 9H, Ar-H); 10.69 (s, 1H, -OH); 13C-NMR (400 MHz, DMSO-d6): δ (ppm) 14.71 (-
CH2-CH3), 43.66 (-CH2-CH3), 60.25 (pyr-C4), 61.82 (pyr-C5), 116.10-152.21 (Ar-C), 156.13
(pyr-C3), 157.34 (-C=O); FAB-MS: 310 (M)+, 311 (M+1)+
2.2.1.5. Phenyl 3-(2-hydroxyphenyl)- 5-(4-methoxyphenyl)- 4,5-dihydro-1H-pyrazole- 1-
carboxylate (3e)
Yield: 71%; mp: 161-165 °C; 1H-NMR (CDCl3): δ (ppm) 3.39 (dd, JAM= 17.70 Hz, JAX=4.80 Hz,
HA); 3.80 (S, 3H, -OCH3); 3.93 (dd, JMX= 17.55 Hz, JMA=12.00 Hz, HM); 5.47 (appeared as
doublet, JXM=5.40 Hz, HX); 6.87-7.39 (m, 12H, Ar-H); 10.64 (s, 1H, -OH); FAB-MS: 388 (M)+,
389 (M+1)+
2.2.1.6. Phenyl 3-(2-hydroxyphenyl)- 5-(4-methylphenyl)- 4,5-dihydro-1H-pyrazole- 1-
carboxylate (3f)
1
Yield: 63%; mp: 147-149 °C; H-NMR (CDCl3): δ (ppm) 2.34 (s, 3H, -CH3); 3.38 (dd, JAM
=
17.70 Hz, JAX=4.80 Hz, HA); 3.94 (dd, JMX= 17.70 Hz, JMA=11.70 Hz, HM); 5.47 (appeared as
doublet, JXM=7.50 Hz, HX); 6.90-7.39 (m, 12H, Ar-H); 10.65 (s, 1H, -OH); 13C-NMR (400 MHz,
DMSO-d6): δ (ppm) 21.09 (–CH3), (pyr-C4 peak masked by solvent peak); 60.50 (pyr-C5),
116.14-137.32 (Ar-C), 150.47 (pyr-C3), 157.42 (-C=O); FAB-MS: 372 (M)+, 373 (M+1)+
2.2.1.7.Phenyl
3-(2-hydroxyphenyl)-
5-(4-chlorophenyl)-
4,5-dihydro-1H-pyrazole-1-
carboxylate (3g)
Yield: 59%; mp: 135-137 °C; 1H-NMR (CDCl3): δ (ppm) 3.36 (dd, JAM= 17.70 Hz, JAX=5.40 Hz,
HA); 3.96 (dd, JMX= 17.70 Hz, JMA=11.70 Hz, HM); 5.50 (appeared as triplet, JXM=5.40 Hz, HX);
6.91-7.40 (m, 12H, Ar-H); 10.56 (s, 1H, -OH); FAB-MS: 392 (M)+, 393 (M+1)+, 394 (M+2)+
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2.2.1.8. Phenyl 3-(2-hydroxyphenyl)- 5-phenyl- 4,5-dihydro-1H-pyrazole- 1-carboxylate (3h)
Yield: 61%; mp: 151-153 °C; 1H-NMR (CDCl3): δ (ppm) 3.41 (dd, JAM= 17.25 Hz, JAX=5.70 Hz,
HA); 3.96 (dd, JMX= 17.55 Hz, JMA=12.00 Hz, HM); 5.52 (dd, JXA= 11.55 Hz, JXM=4.80 Hz, HX);
6.90-7.38 (m, 13H, Ar-H); 10.63 (s, 1H, -OH); FAB-MS: 358 (M)+, 359 (M+1)+
2.3. Biochemistry
2.3.1 Determination of hMAO-A and -B activities
The activities of hMAO-A and hMAO-B were determined using p-tyramine as common
substrate and calculated as 180.20 ± 7.33 pmol/mg/min (n=3) and 149.50±8.11 pmol/mg/min
(n=3), respectively. The interactions of the synthesized compounds with hMAO isoforms were
determined by a fluorimetric method described and modified previously. [20, 35, 36] The
production of H2O2 catalyzed by MAO isoforms was detected using Amplex®-Red reagent, a
non-fluorescent, highly sensitive, and stable probe that reacts with H2O2 in the presence of
horseradish peroxidase to produce the fluorescent product resorufin. The reaction was started by
adding (final concentrations) 200 µM Amplex Red reagent, 1 U/mL horseradish peroxidase, and
p-tyramine (concentration range 0.1-1 mM). Control experiments were carried out by replacing
the synthesized compound and reference inhibitors. The possible capacity of novel 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.
2.3.2. Kinetic experiments
Synthesized compounds were dissolved in dimethyl sulfoxide, with a maximum concentration of
1%, and used in the concentration range of 1-100 ꢀM. Kinetic data for interaction of the enzyme
isoforms with the compounds were determined using the Microsoft Excel package program. The
slopes of the Lineweaver–Burk plots were plotted versus the inhibitor concentration and the Ki
values were determined from the x-axis intercept as −Ki. Each Ki value 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 (Ki (MAO-A) / Ki (MAO-B)) was also calculated.
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The protein was determined according to the Bradford method,[37] in which bovine serum
albumin was used as a standard.
2.3.4. Reversibility experiments
Reversibility of the MAO inhibition with synthesized compounds was evaluated by a
centrifugation-ultrafiltration method.[38] In brief, adequate amounts of the recombinant hMAO-
A or B were incubated together with a single concentration of the newly synthesized compounds
or the reference inhibitors in a sodium phosphate buffer (0.05 M, pH 7.4) for 1 hour at 37°C.
After this incubation period, an aliquot was stored at 4°C and used for the measurement of
MAO-A and -B activity. The remaining incubated sample was placed in an Ultrafree-0.5
centrifugal tube with a 30 kDa Biomax membrane and centrifuged at 9000x g for 20 min at 4°C.
The enzyme retained in the 30 kDa membrane was resuspended in a sodium phosphate buffer at
4°C and centrifuged again two successive times. After the third centrifugation, the enzyme
retained in the membrane was resuspended in sodium phosphate buffer (300 mL) and an aliquot
of this suspension was used for MAO-A and -B activity determination. Control experiments were
performed simultaneously (to define 100% MAO activity) by replacing the test drugs with
appropriate dilutions of the vehicles. The corresponding values of percent (%) MAO isoform
inhibition was separately calculated for samples with and without repeated washing.
2.4 Molecular docking simulation
In order to understand the interaction of the potent molecule (6) with MAO-A and MAO-B
molecular docking studies were carried out using AutoDock4.2. Docking protocol reported
earlier by our group has been followed with X-ray crystal structure of hMAO-A (2BXR) and
hMAO-B (2BYB). Protein preparation: Protein Preparation Wizard of Maestro-8.4 (Schrodinger
LLC) has been used to prepare protein. Crystallographic models 2BXR (hMAO-A) and 2BYB
(hMAO-B) were downloaded from www.rcsb.org. Water and covalently linked ligands were
deleted and bond order was corrected for FAD. After assigning charge and protonation state
finally energy minimization was done using OPLS2005 force field. PDB written by Maestro for
prepared proteins were rewritten by Open Babel for AutoDock compatible atom type. Ligand
preparation: Ligand structures were drawn using build panel & prepared using Ligprep module
implemented Maestro-8.4 (Schrodinger LLC). Energy minimization is carried out using MMFF
force field. Structures were saved in .pdb format and rewritten using openbable for AutoDock
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compatible atom type. Docking: Grid parameter file (.gpf) and Docking parameter files (.dpf)
were written using MGLTools-1.4.6. Receptor grids were generated using 60x60x60 grid points
in xyz with grid spacing of 0.375 Å. Grid box was centered on N5 atom of FAD. Map types
were generated using autogrid4. Docking was carried out with following parameters with
number of runs: 50, population size: 150, number of evaluations: 2500000 and number of
generations: 27000 numbers of generations, using autodock4. Analysis: Analysis of docking
results was done using MGLTools-1.4.6. Top scoring molecule in the largest cluster was
analyzed for its interaction with the protein.
3. Results & Discussion
3.1. Chemistry
The intermediate chalcones (1a-1d) and pyrazolines (2a-2d) were reported earlier and confirmed
by their melting point. The final compounds were confirmed by the 1H-NMR and FAB-MS. 1H-
NMR spectra displayed the characteristic AMX pattern for pyrazolines as three double-doublets
(dd) in the range δ 3.252-3.406 ppm (JAM: 17.25-17.70 Hz & JAX 4.80-5.70 Hz), δ 3.837-3.963
ppm (JMX: 17.10-17.70 Hz & JMA 10.80-12.00 Hz) and δ 5.343-5.498 ppm (3a-3d & 3h; JAM
:
11.55-11.85 Hz & JAX: 4.80-5.40 Hz) for HA, HM and HX, respectively. Only in case of
compounds 3e-3g, HX signal appeared either as doublet or triplet. The phenolic-OH proton
appeared in the range of δ 10.559-10.701 ppm as a singlet. Methyl &methylene proton of ethyl
group in compounds 3a-3d appeared in the range of δ 1.226-1.252 ppm & δ 4.191-4.207,
respectively. Methoxy group in ring B of 3a & 3e appeared in the range of δ 3.789-3.799, while
13
methyl group in ring B of 3b & 3f in the range of δ 2.328-2.340 ppm. In C-NMR spectra the
peaks for pyr-C3, C4 and C5 are in the range of δ 150.47-159.03 ppm, δ 59.67-60.25 ppm and δ
60.50-61.91 ppm, respectively. The peaks for –C=O, -CH2-CH3, -CH2-CH3, Ar-OCH3 and Ar–
CH3 are in the range of δ 157.34-157.42 ppm (3a, 3c, 3d & 3f), δ 43.52-43.66 ppm (3a, 3c &
3d), δ 14.71-14.73 ppm (3a, 3c & 3d), δ 55.47 ppm (3a) and δ 21.09 ppm (3f), respectively.
FAB-MS of all the compounds displayed [M]+ and [M+1]+ peaks.
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3.2. Enzyme Inhibitory Assay (hMAO-A & hMAO-B)
All the compounds (3a-3h) tested were found to inhibit hMAO-A competitively and reversibly
(Table 1). A Lineweaver–Burk reciprocal plot (Figure 1a) showed that the inhibition of hMAO-
A with compound 3f was competitive with an experimentally-derived Ki value of 0.004 ꢀM
obtained from y-intercept replots (Figure 1b) exhibiting increase in Km. It seems that the novel
compounds may interact within the catalytic site of hMAO-A.
In the reversibility and irreversibility tests, hMAO-A inhibition was found to be reversible in
presence of the compounds as shown by the recovery enzyme activity after repeated washing
(Table 2).
Compounds 3e-3h with phenyloxy-carbonyl substitution at N1 were many-fold potent than their
counterparts with ethyloxy-carbonyl substitution, 3a-3d. Substitution in ring B of 3a-3d has a
little effect on potency towards MAO-A/MAO-B as well as selectivity. While the same in
compounds 3e-3h has pronounced effect on potency and selectivity towards MAO-A and MAO-
B. One with methyl substitution (3f) has shown potent inhibition that is followed by chloro (3g),
unsubstituted (3h) and methoxy (3e). It seems rather than electronic property of the substitution,
its VdW volume is responsible for increasing potency. For crucial interaction with protein ring B
should have substitution with appropriate VdW volume (bulkiness) that increases potency.
Compound 3f (KiMAO-A; 0.005±2x10-4 µM) was found to be equally potent as that of standard
drug, Moclobemide (KiMAO-A; 0.005±2x10-4 µM) but with best selectivity index (8.86 x 10-5).
3.3. Molecular docking simulation
Molecular level interaction was studied through molecular docking simulation carried out with
AutoDock4.2. As racemic mixture was screened for its enzyme inhibitory assay we docked
individual enantiomer (R&S) for interaction studies as reported earlier.[18, 38-42] Docking
results suggested that S-enantiomer is better than R-enantiomer that is in agreement with all the
previous studies reported till date.[39-41]
Analysis of S-enantiomer of 3f revealed that 2-hydroxyl functional group in the phenyl ring at
C3 position of pyrazoline establishes two H-bonding interaction with SER209 [O---HN (bb)-
SER209& H---OH (sc)-SER209]. This helps in positioning the ring-A in P2 (lined by GLY71,
GLN74, ARG206, ILE207, PHE208, GLU216 and TRP441) ring-B in P3 (lined by ILE180,
ILE335, LEU337, MET350 and PHE352) and ring-C in P1 (Aromatic cage, FAD, TYR407,
10

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TYR444). The position also favors pi-pi interaction of ring-A with TRP441 and ring-C with
TYR444 (Figure 2a). Analysis of R-enantiomer revealed H-bonding interaction of carbonyl
oxygen of 3f with Hydrogen of phenolic hydroxyl functional group of TYR444. This interaction
favors the positioning of ring-B in P1 but both ring-A and ring-C are not fully accommodated in
other two pockets. This conformer also exhibited pi-pi interaction of ring-B with PHE352 and
ring-C with TYR407. All these interactions made ring-A to occupy/share the space at the center
along with pyrazoline ring and potentially keeping P2 vacant. This reduces the VdW interaction
and preventing the H-bond interaction of phenolic hydroxyl group of 3f leading to many folds
decreased activity of R-enantiomer compared with S-enantiomer (Figure 2b). The component
energies of these two enantiomers are presented in Table 3.
Acknowledgement
DBT, Govt. of India and UGC, Govt. of India are acknowledged for availing JRF for first &
third and fourth author respectively. (i) CIF, BIT, Mesra, India; (ii) SAIF, CDRI, Lucknow, India
and (ii) Dr. Reddy’s ILS, Hyderabad, India are acknowledged for spectral characterization of
intermediates and final compounds, respectively. The biochemical part of the study was
supported by a grant from Hacettepe University Scientific Research Projects Coordination Unit
(Project No: HUBAB 010 D06 301 001).
References.
[1] G.G.S. Collins, M. Sandler, E.D. Williams, M.B.H. Youdim, Multiple Forms of Human Brain
Mitochondrial Monoamine Oxidase, Nature, 225 (1970) 817-820.
[2] M.B.H. Youdim, G.G.S. Collins, M. Sandler, A.B.B. Jones, C.M.B. Pare, W.J. Nicholson, Biological
Sciences: Human Brain Monoamine Oxidase: Multiple Forms and Selective Inhibitors, Nature, 236
(1972) 225-228.
[3] B. Blackwell, Hypertensive crisis due to monoamine-oxidase inhibitors, Lancet, 2 (1963) 849-850.
[4] R.D. Knapp, Cheese and monoamine oxidase inhibitors, Headache, 4 (1964) 157-158.
[5] M. Yamada, H. Yasuhara, Clinical pharmacology of MAO inhibitors: safety and future.,
Neurotoxicology, 25 (2004) 215-221.
11

ACCEPTED MANUSCRIPT
[6] M.B. Youdim, M. Weinstock, Therapeutic applications of selective and non-selective inhibitors of
monoamine oxidase A and B that do not cause significant tyramine potentiation., Neurotoxicology, 25
(2004) 243-250.
[7] M. Da Prada, H.H. Keller, R. Kettler, [Comparison of the new MAO-A inhibitors moclobemide,
brofaromine and toloxatone with tranylcypromine in an animal experiment: significance for clinical
practice]. Psychiatr Prax, 16 Suppl 1 (1989) 18-24.
[8] G. Lavian, J.P. Finberg, M.B. Youdim, The advent of a new generation of monoamine oxidase
inhibitor antidepressants: pharmacologic studies with moclobemide and brofaromine., Clin
Neuropharmacol, 16 Suppl 2 (1993) S1-7.
[9] M. Abdel-Aziz, G.l.-D. Abuo-Rahma, A.A. Hassan, Synthesis of novel pyrazole derivatives and
evaluation of their antidepressant and anticonvulsant activities, Eur J Med Chem, 44 (2009) 3480-
3487.
[10] A.A. Bilgin, E. Palaska, R. Sunal, Studies on the synthesis and antidepressant activity of some 1-
thiocarbamoyl-3,5-diphenyl-2-pyrazolines, Arzneimittelforschung, 43 (1993) 1041-1044.
[11] O.D. Can, U.D. Ozkay, Z.A. Kaplancikli, Y. Oztürk, Effects of some 1,3,5-trisubstitued-2-pyrazoline
derivatives on depression and anxiety parameters of mice, Arch Pharm Res, 32 (2009) 1293-1299.
[12] Z. Ozdemir, H.B. Kandilci, B. Gümüşel, U. Caliş, A.A. Bilgin, Synthesis and studies on
antidepressant and anticonvulsant activities of some 3-(2-furyl)-pyrazoline derivatives, Eur J Med
Chem, 42 (2007) 373-379.
[13] Z. Ozdemir, H.B. Kandilci, B. Gumusel, U. Calis, A.A. Bilgin, Synthesis and studies on
antidepressant and anticonvulsant activities of some 3-(2-thienyl)pyrazoline derivatives, Arch Pharm
(Weinheim), 341 (2008) 701-707.
[14] E. Palaska, M. Aytemir, I.T. Uzbay, D. Erol, Synthesis and antidepressant activities of some 3,5-
diphenyl-2-pyrazolines, Eur J Med Chem, 36 (2001) 539-543.
[15] Y. Rajendra Prasad, A. Lakshmana Rao, L. Prasoona, K. Murali, P. Ravi Kumar, Synthesis and
antidepressant activity of some 1,3,5-triphenyl-2-pyrazolines and 3-(2''-hydroxy naphthalen-1''-yl)-1,5-
diphenyl-2-pyrazolines, Bioorg Med Chem Lett, 15 (2005) 5030-5034.
[16] O. Ruhoğlu, Z. Ozdemir, U. Caliş, B. Gümüşel, A.A. Bilgin, Synthesis of and pharmacological studies
on the antidepressant and anticonvulsant activities of some 1,3,5-trisubstituted pyrazolines,
Arzneimittelforschung, 55 (2005) 431-436.
[17] D. Secci, A. Bolasco, P. Chimenti, S. Carradori, The state of the art of pyrazole derivatives as
monoamine oxidase inhibitors and antidepressant/anticonvulsant agents, Curr Med Chem, 18 (2011)
5114-5144.
[18] F. Chimenti, A. Bolasco, F. Manna, D. Secci, P. Chimenti, A. Granese, O. Befani, P. Turini, S. Alcaro,
F. Ortuso, Synthesis and molecular modelling of novel substituted-4,5-dihydro-(1H)-pyrazole
12

ACCEPTED MANUSCRIPT
derivatives as potent and highly selective monoamine oxidase-A inhibitors, Chem Biol Drug Des, 67
(2006) 206-214.
[19] F. Chimenti, A. Bolasco, F. Manna, D. Secci, P. Chimenti, A. Granese, O. Befani, P. Turini, R. Cirilli,
F. La Torre, S. Alcaro, F. Ortuso, T. Langer, Synthesis, biological evaluation and 3D-QSAR of 1,3,5-
trisubstituted-4,5-dihydro-(1H)-pyrazole derivatives as potent and highly selective monoamine
oxidase A inhibitors, Curr Med Chem, 13 (2006) 1411-1428.
[20] F. Chimenti, E. Maccioni, D. Secci, A. Bolasco, P. Chimenti, A. Granese, S. Carradori, S. Alcaro, F.
Ortuso, M. Yáñez, F. Orallo, R. Cirilli, R. Ferretti, F. La Torre, Synthesis, stereochemical identification,
and selective inhibitory activity against human monoamine oxidase-B of 2-methylcyclohexylidene-(4-
arylthiazol-2-yl)hydrazones., J Med Chem, 51 (2008) 4874-4880.
[21] F. Chimenti, S. Carradori, D. Secci, A. Bolasco, B. Bizzarri, P. Chimenti, A. Granese, M. Yáñez, F.
Orallo, Synthesis and inhibitory activity against human monoamine oxidase of N1-thiocarbamoyl-3,5-
di(hetero)aryl-4,5-dihydro-(1H)-pyrazole derivatives., Eur J Med Chem, 45 (2010) 800-804.
[22] F. Chimenti, B. Bizzarri, A. Bolasco, D. Secci, P. Chimenti, A. Granese, S. Carradori, M. D'Ascenzio,
D. Lilli, D. Rivanera, Synthesis and biological evaluation of novel 2,4-disubstituted-1,3-thiazoles as
anti-Candida spp. agents., Eur J Med Chem, 46 (2011) 378-382.
[23] D. Secci, S. Carradori, A. Bolasco, B. Bizzarri, M. D'Ascenzio, E. Maccioni, Discovery and
optimization of pyrazoline derivatives as promising monoamine oxidase inhibitors, Curr Top Med
Chem, 12 (2012) 2240-2257.
[24] N. Gökhan, A. Yeşilada, G. Uçar, K. Erol, A.A. Bilgin, 1-N-substituted thiocarbamoyl-3-phenyl-5-
thienyl-2-pyrazolines: synthesis and evaluation as MAO inhibitors, Arch Pharm (Weinheim), 336
(2003) 362-371.
[25] N. Gökhan-Kelekçi, S. Koyunoğlu, S. Yabanoğlu, K. Yelekçi, O. Ozgen, G. Uçar, K. Erol, E. Kendi, A.
Yeşilada, New pyrazoline bearing 4(3H)-quinazolinone inhibitors of monoamine oxidase: synthesis,
biological evaluation, and structural determinants of MAO-A and MAO-B selectivity, Bioorg Med
Chem, 17 (2009) 675-689.
[26] U. Salgın-Gökşen, S. Yabanoğlu-Çiftçi, A. Ercan, K. Yelekçi, G. Uçar, N. Gökhan-Kelekçi, Evaluation
of selective human MAO inhibitory activities of some novel pyrazoline derivatives, J Neural Transm,
(2013) 1-11.
[27] G. Ucar, N. Gokhan, A. Yesilada, A.A. Bilgin, 1-N-Substituted thiocarbamoyl-3-phenyl-5-thienyl-2-
pyrazolines: a novel cholinesterase and selective monoamine oxidase B inhibitors for the treatment of
Parkinson's and Alzheimer's diseases, Neurosci Lett, 382 (2005) 327-331.
[28] S. Yabanoglu, G. Ucar, N. Gokhan, U. Salgin, A. Yesilada, A.A. Bilgin, Interaction of rat lung SSAO
with the novel 1-N-substituted thiocarbamoyl-3-substituted phenyl-5-(2-pyrolyl)-2-pyrazoline
derivatives, J Neural Transm, 114 (2007) 769-773.
13

ACCEPTED MANUSCRIPT
[29] V. Jayaprakash, B.N. Sinha, G. Ucar, A. Ercan, Pyrazoline-based mycobactin analogues as MAO-
inhibitors, Bioorg Med Chem Lett, 18 (2008) 6362-6368.
[30] M. Karuppasamy, M. Mahapatra, S. Yabanoglu, G. Ucar, B.N. Sinha, A. Basu, N. Mishra, A. Sharon,
U. Kulandaivelu, V. Jayaprakash, Development of selective and reversible pyrazoline based MAO-A
inhibitors: Synthesis, biological evaluation and docking studies, Bioorg Med Chem, 18 (2010) 1875-
1881.
[31] A. Sahoo, S. Yabanoglu, B.N. Sinha, G. Ucar, A. Basu, V. Jayaprakash, Towards development of
selective and reversible pyrazoline based MAO-inhibitors: Synthesis, biological evaluation and docking
studies, Bioorg Med Chem Lett, 20 (2010) 132-136.
[32] M. Jagrat, J. Behera, S. Yabanoglu, A. Ercan, G. Ucar, B.N. Sinha, V. Sankaran, A. Basu, V.
Jayaprakash, Pyrazoline based MAO inhibitors: synthesis, biological evaluation and SAR studies,
Bioorg Med Chem Lett, 21 (2011) 4296-4300.
[33] J.A. Ferreras, J.S. Ryu, F. Di Lello, D.S. Tan, L.E. Quadri, Small-molecule inhibition of siderophore
biosynthesis in Mycobacterium tuberculosis and Yersinia pestis., Nat Chem Biol, 1 (2005) 29-32.
[34] S. Chakrabarty, Jasmine, C. Bhadaliya, B.N. Sinha, A. Mahesh, H. Bai, S.Y. Blond, V. Jayaprakash,
Inhibitors of human histone deacetylase: synthesis and enzyme assay of hydroxamates with
piperazine linker, Arch Pharm (Weinheim), 343 (2010) 167-172.
[35] M.C. Anderson, F. Hasan, J.M. McCrodden, K.F. Tipton, Monoamine oxidase inhibitors and the
cheese effect, Neurochem Res, 18 (1993) 1145-1149.
[36] M. Yáñez, N. Fraiz, E. Cano, F. Orallo, Inhibitory effects of cis- and trans-resveratrol on
noradrenaline and 5-hydroxytryptamine uptake and on monoamine oxidase activity, Biochem Biophys
Res Commun, 344 (2006) 688-695.
[37] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding., Anal Biochem, 72 (1976) 248-254.
[38] F. Chimenti, S. Carradori, D. Secci, A. Bolasco, B. Bizzarri, P. Chimenti, A. Granese, M. Yáñez, F.
Orallo, Synthesis and inhibitory activity against human monoamine oxidase of N1-thiocarbamoyl-3,5-
di(hetero)aryl-4,5-dihydro-(1H)-pyrazole derivatives, Eur J Med Chem, 45 (2010) 800-804.
[39] R. Cirilli, R. Ferretti, B. Gallinella, L. Turchetto, A. Bolasco, D. Secci, P. Chimenti, M. Pierini, V.
Fares, O. Befani, F. La Torre, Enantiomers of C(5)-chiral 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-
pyrazole derivatives: Analytical and semipreparative HPLC separation, chiroptical properties, absolute
configuration, and inhibitory activity against monoamine oxidase, Chirality, 16 (2004) 625-636.
[40] F. Chimenti, A. Bolasco, F. Manna, D. Secci, P. Chimenti, O. Befani, P. Turini, V. Giovannini, B.
Mondovì, R. Cirilli, F. La Torre, Synthesis and selective inhibitory activity of 1-acetyl-3,5-diphenyl-4,5-
dihydro-(1H)-pyrazole derivatives against monoamine oxidase, J Med Chem, 47 (2004) 2071-2074.
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[41] F. Chimenti, E. Maccioni, D. Secci, A. Bolasco, P. Chimenti, A. Granese, O. Befani, P. Turini, S.
Alcaro, F. Ortuso, R. Cirilli, F. La Torre, M.C. Cardia, S. Distinto, 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, 48 (2005) 7113-7122.
[42] F. Chimenti, R. Fioravanti, A. Bolasco, F. Manna, P. Chimenti, D. Secci, F. Rossi, P. Turini, F. Ortuso,
S. Alcaro, M.C. Cardia, Synthesis, molecular modeling studies and selective inhibitory activity against
MAO of N1-propanoyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives, Eur J Med Chem, 43 (2008)
2262-2267.
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Scheme 1
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Figure 1a
Figure 1b
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2a
2b
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Table 1. MAO inhibitory activity of compounds 3a-3h
MAO-A
Ki (µM)
A
MAO-B
Ki (µM)
R
R1
SIc
MAO
selectivity
Compd
Inhibition type Reversibility
B
3a
3b
3c
3d
3e
3f
3g
-OCH3 -C2H5 0.61±0.03
5.00±0.18
6.00±0.34
15.55±1.03
11.00±1.05
0.121
0.147
0.049
0.073
Competitive
Competitive
Competitive
Competitive
Competitive
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
MAO-A Selective
MAO-A Selective
MAO-A Selective
MAO-A Selective
MAO-A Selective
MAO-A Selective
MAO-A Selective
MAO-A Selective
MAO-A Selective
MAO-A Selective
-CH3
-Cl
-H
-C2H5 0.88±0.04
-C2H5 0.76±0.04
-C2H5 0.81±0.03
-OCH3 -C6H5 0.31±0.02
305.00±21.55 0.001
-CH3
-Cl
-H
-C6H5 0.005±2x10-4
-C6H5 0.03±0.002
-C6H5 0.25±0.002
9.06±0.44
56.00±3.05
45.00±2.80
8.86x10-5 Competitive
6.56x10-4 Competitive
3h
530.00+30.25 4.68x10-4 Competitive
aSEL
bMOC
0.09±0.004
99.92
0.005
Competitive
Competitive
0.005±2X10-4 1.08±3.00
aSEL-Selegeline; bMOC-Moclobemide; cSelectivity Index for Ki (MAO-A) /Ki (MAO-B)
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Table 2. Reversibility of hMAO inhibition with the compound 3f and moclobemide
Compound
hMAO-A inhibition (%)
Before washing
After washing
89.00±4.55
94.80±0.45
9.56±0.44
Moclobemide (300 nM)
Compound 3f (300 nM)
8.00±0.39
Each value represents the mean ± S.E.M. of three experiments
Table 3. Component energies of R- and S-enantiomers of 3f with hMAO-A (2BXR)
Enantiomer Estimated
binding free
Estimated
Ki value
(nM)
Component energies
Electrostatic H-bond VdW
energy
Desolvation
(Kcal/mole)
R-
S-
-8.47
-9.38
620.30
133.22
-0.1406
-0.1439
-0.4693
-0.9407
-13.0428 2.2931
-13.1134 2.1125
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Scheme 1. Reagents & Conditions: (a) (i) NaOH (60%), MeOH, 72 h, rt; (ii) HCl (6N) upto pH
2; (b) NH2NH2.H2O, reflux, 3-6 h; (c) Cl-CO-OR1 (where R1= -C2H5 or -C6H5), K2CO3, stirring,
30 min.
Figure 1a. Inhibition of hMAO-A with compound 3f in the presence of various concentrations
of the inhibitor. 1b. Graph for Ki value was calculation. [S] = p-tyramine (mM), v = velocity
(nmol/hour/mg), Ki= 0.004 ꢀM.
Figure 2a. R-enantiomerof 3f in hMAO-A (PDB: 2BXR) active site; 2b. S-enantiomer of 3f in
hMAO-A (PDB: 2BXR) active site. H-bonds are shown as green dots and pi-pi interaction as
yellow cylinders.
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Highlights
ꢀ
ꢀ
ꢀ
ꢀ
3a-3f were competitive, reversible inhibitors & selective inhibitors of hMAO-
A
Phenyl carbamates (3e-3h) were potent with best selectivity index towards
hMAO-A
Molecular docking studies revealed that S-conformers were better than R-
conformers
Compound 3f, the best amongst the eight with SIhMAO-A=8.86x10-5

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Supplementary material
1. General synthetic procedure for the intermediates
1.1. General procedure for synthesis of benzylidineacetophtnone (1a-1d): -
To a solution of 2’-hydroxy acetophenone (0.01M) in methanol (10ml) was added an equimolar
concentration of appropriate benzaldehydes, which was maintained at <100C. To this mixture
was added an ice-cooled solution of sodium hydroxide (60%) with continuous stirring for a
period of 30-45 min. The reaction mixture was then left at room temperature for about 72h with
occasional shaking. After 72h, it was poured into ice-cold water, and was then adjusted to pH-2
using 6N hydrochloric acid. The yellow precipitate obtained was filtered, washed with water and
dried. The crude product was then recrystallized from methanol to give yellow product. (Yield-
85-91%)
1.2. General procedure for synthesis of pyrazoline (2a-2d): -
To a solution of chalcone (1a-1d, 0.01M) in methanol (20ml) was added hydrazine hydrate
(80%, 8ml). The reaction mixture was then refluxed for about 3-6h and kept aside for a while.
The solid separated out was filtered, washed with ice-cooled methanol, dried and recrystallized
from methanol. (Yield-71-76%)

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2. Docking Scores of R- and S-enantiomers of compounds 3a-3h
2BXR (hMAO-A)
2BYB (hMAO-B)
R
S
R
S
code
3a
3b
3c
3d
3e
3f
-8.36
-8.44
-8.53
-8.16
-9.25
-9.38
-9.14
-9.4
0.74
0.66
0.56
1.04
0.17
0.13
0.20
0.13
-8.54
-8.35
-8.82
-8.57
-7.93
-8.47
-8.16
-9.41
0.55
0.76
0.34
0.52
1.55
0.62
1.05
0.13
0.65
0.71
0.45
0.78
0.86
0.38
0.62
0.13
-6.87 9.23 -7.82
-6.79 10.46 -7.6
-6.61 14.19 -7.25
-6.49 17.45 -7.55
1.86
2.68
4.88
2.92
5.55
6.57
9.54
10.19
1310.13
-4.14 920.26 -3.78 1700
-3.5 2710
-0.1 850100 426405
3g
3h
-3.37 3400 -3.84 1520
-5.33 124.02 -3.43 3040
2460
1582.01

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