Development of selective and reversible pyrazoline based MAO-A inhibitors: Synthesis, biological evaluation and docking studies

Article abstract of DOI:10.1016/j.bmc.2010.01.043

3,5-Diaryl pyrazolines analogs were synthesized and evaluated for their monoamine oxidase (MAO) inhibitory activity. The compounds were found reversible and selective towards MAO-A with selectivity index in the magnitude of 10³-10⁵. The docking studies were carried out to gain further structural insights of the binding mode and possible interactions with the active site of MAO-A. Interestingly, the theoretical (K_i) values obtained by molecular docking studies were in congruence with their experimental (K_i) values.

Full text of DOI:10.1016/j.bmc.2010.01.043

Bioorganic & Medicinal Chemistry 18 (2010) 1875–1881
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of selective and reversible pyrazoline based MAO-A
inhibitors: Synthesis, biological evaluation and docking studies
Muthukumar Karuppasamy a, Manojkumar Mahapatra a, Samiye Yabanoglu b, Gulberk Ucar b,
Barij Nayan Sinha a, Arijit Basu a, Nibha Mishra a, Ashoke Sharon c,
Umasankar Kulandaivelu d, Venkatesan Jayaprakash a,
*
a Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835215, India
b Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhıye, Ankara, Turkey
c Department of Applied Chemistry, Birla Institute of Technology, Mesra, Ranchi 835215, India
d Medicinal Chemistry Research Division, Vaagdevi College of Pharmacy, Hanamkonda, Warangal 506001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
3,5-Diaryl pyrazolines analogs were synthesized and evaluated for their monoamine oxidase (MAO)
inhibitory activity. The compounds were found reversible and selective towards MAO-A with selectivity
index in the magnitude of 103–105. The docking studies were carried out to gain further structural
insights of the binding mode and possible interactions with the active site of MAO-A. Interestingly, the
theoretical (Ki) values obtained by molecular docking studies were in congruence with their experimental
(Ki) values.
Received 2 December 2009
Revised 15 January 2010
Accepted 16 January 2010
Available online 25 January 2010
Keywords:
Pyrazolines
Ó 2010 Elsevier Ltd. All rights reserved.
MAO inhibitors
Molecular docking
1. Introduction
tive inhibitors of MAO-A/MAO-B leading to a new generation of
compounds.
Monoamine oxidase inhibitors (MAOIs) have shown therapeutic
value in variety of diseases.1 Earlier, MAO inhibitors introduced
into clinical practice were abandoned due to adverse side-effects,
such as hepatotoxicity, orthostatic hypertension and the so called
‘cheese effect’ characterized by hypertensive crises.2 These side-ef-
fects were hypothesized to be related to nonselective and irrevers-
ible monoamine oxidase inhibition. The new generation inhibitors
are warranted to selectively inhibit either of one of the isoforms
(MAO-A and MAO-B) to increase the therapeutic efficacy. These
two forms of MAO are characterized by their different sensitivities
to inhibitors and their different specificities to substrates.3 MAO-A
preferably metabolizes serotonin, adrenaline, and noradrenaline,4
whereas b-phenylethylamine and benzylamine are predominantly
metabolized by MAO-B.5 Tyramine, dopamine, and some other
important amines are common substrates for both the isoen-
zymes.6 Nowadays, the therapeutic interest of MAOIs falls into
two major categories. MAO-A inhibitors have been used mostly
in the treatment of mental disorders, in particular depression
and anxiety,7–9 while MAO-B inhibitors could be used in the treat-
ment of Parkinson’s disease and Alzheimer’s disease.10,11 Efforts
have been oriented towards the discovery of reversible and selec-
The classical period of the MAO inhibitors started with hydra-
zine derivatives. Their prototype, Iproniazid, was originally pro-
posed as a tuberculostatic agent eventually became the first
modern antidepressant. 2-Pyrazolines can be considered as a cyclic
hydrazine moiety. For this reason, many authors investigated MAO
inhibitory and antidepressant activities of 1,3,5-triphenyl-2-pyraz-
olines.12–33
Recently, we have reported31–33 few N,3,5-triaryl-4,5-dihydro-
1H-pyrazole-1-carbothioamide as MAO inhibitors (Fig. 1). Their
SAR analysis revealed some important structural insights like: (a)
4-hydroxy substitution in ring A increases the potency towards
MAO-A31 as well as reduces the cytotoxicity,33 (b) approximately
10–15 fold increase in potency was observed with five membered
hetero aromatic groups as ring B in comparison with phenyl
group31 (c) selectivity towards MAO-A was observed with any sub-
stitution in ring C. In the present study molecules with 4-hydroxy
substitution in ring A, thiophen-2-yl/furan-2-yl group as ring B and
various substitutions in ring C phenyl group were explored for po-
tent MAO-A inhibitory activity.
The structural comparison of human MAO-A and MAO-B37,38 re-
veals high sequence similarity of 72% and the overall structure of
human MAO-A is quite similar to that of MAO-B (rms deviation
of 1.2 Å, 488 equiv C-a atom). The significant conformational
* Corresponding author. Tel.: +91 9470137264.
difference observed in the cavity-shaping loop of MAO-A from
E-mail address: venkatesanj@bitmesra.ac.in (V. Jayaprakash).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.043

1876
M. Karuppasamy et al. / Bioorg. Med. Chem. 18 (2010) 1875–1881
Figure 1. Structures of some potent N,3,5-triaryl-4,5-dihhydro-1H-pyrazole-1-carbothioamide from earlier work are shown. This forms a basis for synthesizing the current
molecules. The most potent analog 11 identified in the current work is also shown.
residues 210–216, which results in C
a
movements up to 6 Å in
2.1. Chemistry
comparison to MAO-B (residues 201–207). This makes the aro-
matic cage of MAO-A wider than that of MAO-B accommodating
bulkier aromatic groups. The previous docking studies31,32 also re-
vealed the importance of ring C substitution (Fig. 1) which was
found to be positioned in the aromatic cage (FAD, TYR407 and
TYR444, Pocket1). These observations formed a strong basis for
designing selective and potent MAO-A inhibitor.
The compounds were synthesized as shown in Scheme 1
according to previously reported method.31–33 4-Hydroxy chal-
cones 1–2 were prepared through Claisen–Schmidt condensation
of 4-hydroxy acetophenone with either furfuraldehyde or thio-
phene-2-carboxaldehyde. Compounds 3–4 were synthesized by
the reaction of excess hydrazine hydrate (80%) with 1–2, respec-
tively in ethanol. Compounds 5–18 were afforded by the reaction
of 3–4 with respective substituted phenyl isothiocyanates in dry
methanol under reflux condition. The structure, physicochemical,
and spectral data of these analogs were given in Table1.
2. Results and discussion
In continuation to our previous work fourteen novel pyrazolines
(N,3,5-triaryl-4,5-dihydro-1H-pyrazole-1-carbothioamide)
were
2.2. Biochemistry
synthesized and evaluated for their MAO inhibitory property. The
synthesized compounds were found to be competitive, reversible
and selective inhibitor of MAO-A. In addition, the molecular dock-
ing studies were carried out to reveal the binding mode of synthe-
sized molecules with MAO-A and MAO-B.
The synthesized compounds were subjected to MAO inhibitory
assay. MAO was purified from the rat liver and total MAO activity
was measured spectrophotometrically according to the method of
Holt.36 Assay mixture contained a chromogenic solution consisted
Scheme 1. Reagents and condition: (a) 80% NH2NH2ÁH2O, ethanol, reflux 3–6 h; (b) R-C6H4-NCS, dry methanol, 15–30 min.

Table 1
Physicochemical and spectral characteristics of N,3,5-triaryl-4,5-dihydro-1H-pyrazole-1-carbothioamides
Compound
R
X
S
MF
MW
379
Y
MP
1H NMR (d ppm)
FAB-MS (m/z)
(%) (°C)
5
–H
C20H17N3OS2
66
47
85
45
49
41
47
93
43
52
55
86
78
47
185– 3.37 (HA, appeared as doublet, JAM = 16.5 Hz), 3.87 (HM, dd, JMA = 17.0 Hz, JMX = 11.1 Hz), 6.32 (HX, appeared as doublet,
187 JMX = 9.9 Hz), 6.85–7.87 (12H, m, Ar-H), 9.99 (1H, s, Ar-OH), 10.1 (1H, s, Ar-NH–)
379 (M+, bp)
380 (M+1)
381 (M+2)
409 (M+)
410 (M+1, bp)
411 (M+2)
393 (M+)
6
7
4-OCH3
4-CH3
2-OCH3
2-CH3
3-OCH3
3-CH3
-H
S
C21H19N3O2S2 409
178– 3.35 (HA, appeared as doublet, JAM = 8.9 Hz), 3.84 (3H, s, Ar-OCH3) 3.89 (HM, dd, JMA = 17.9 Hz, JMX = 11.1 Hz), 6.31 (HX,
180 appeared as doublet, JMX = 9.53 Hz), 6.83–7.83 (11H, m, Ar-H), 9.90 (1H, s, Ar-OH), 10.01 (1H, s, Ar-NH-)
S
C21H19N3OS2
393
170– 3.36 (HA, appeared as doublet, JAM = 15.3 Hz), 2.29 (3H, s, Ar-CH3) 3.86 (HM, dd, JMA = 17.4 Hz, JMX = 11.4 Hz), 6.31 (HX,
172 appeared as doublet, JMX = 9.9 Hz), 6.84–7.86 (11H, m, Ar-H), 9.91 (1H, s, Ar-OH), 10.09 (1H, s, Ar-NH–)
394 (M+1, bp)
395 (M+2)
8
S
C21H19N3O2S2 409
138– 3.53 (HA, appeared as doublet, JAM = 15.7 Hz), 3.88 (3H, s, Ar-OCH3) 3.92 (HM, appeared as doublet, JMA = 23.4 Hz), 6.31 (HX, 409 (M+)
139
appeared as doublet, JMX = 10.5 Hz), 6.63–8.23 (11H, m, Ar-H), 9.76 (1H, s, Ar-OH), 10.15 (1H, s, Ar-NH–)
410 (M+1, bp)
411 (M+2)
9
S
C21H19N3OS2
393
164– 3.34 (HA, appeared as triplet), 2.22 (3H, s, Ar-CH3) 3.87 (HM, dd, JMA = 17.55 Hz, JMX = 11.1 Hz), 6.27 (HX, appeared as doublet, 393 (M+)
165
JMX = 9.6 Hz), 6.83–7.84 (11H, m, Ar-H), 9.81 (1H, s, Ar-OH), 10.07 (1H, s, Ar-NH–)
394 (M+1, bp)
395 (M+2)
10
11
12
13
14
15
16
17
18
S
C21H19N3O2S2 409
139– 3.38 (HA, appeared as triplet), 3.74 (3H, s, Ar-OCH3), 3.87 (HM, dd, JMA = 16.95 Hz, JMX = 11.4 Hz), 6.32 (HX, appeared as
140 doublet, JMX = 9.6 Hz), 6.72–7.87 (11H, m, Ar-H), 9.94 (1H, s, Ar-OH), 10.10 (1H, s, Ar-NH–)
409 (M+)
410 (M+1, bp)
411 (M+2)
S
C21H19N3OS2
C20H17N3O2S
C21H19N3O3S
C21H19N3O2S
C21H19N3O3S
C21H19N3O2S
C21H19N3O3S
C21H19N3O2S
393
363
393
377
393
377
393
377
162– 3.33 (HA, merged with solvent peak), 2.29 (3H, s, Ar-CH3), 3.78 (HM, dd, JMA = 17.7 Hz, JMX = 11.4 Hz), 6.08 (HX, appeared as 393 (M+)
164
doublet, JMX = 10.2 Hz), 6.33–7.86 (11H, m, Ar-H), 9.90 (1H, s, Ar-OH), 10.08 (1H, s, Ar-OH)
394 (M+1, bp)
395 (M+2)
O
O
O
O
O
O
O
197– 3.39 (HA, dd, JAM = 7.2 Hz, JAX = 3.3 Hz), 3.79 (HM, dd, JMA = 17.85 Hz, JMX = 11.9 Hz), 6.09 (HX, dd, JMX = 11.1 Hz, JAX = 2.7 Hz), 363 (M+)
199
6.33–7.86 (12H, m, Ar-H), 9.97 (1H, s, Ar-OH), 10.08 (1H, s, Ar-NH–)
364 (M+1, bp)
365 (M+2)
393 (M+)
394 (M+1, bp)
395 (M+2)
377 (M+)
378 (M+1, bp)
379 (M+2)
393 (M+)
394 (M+1, bp)
395 (M+2)
377 (M+)
378 (M+1, bp)
379 (M+2)
393 (M+)
394 (M+1, bp)
395 (M+2)
377 (M+)
4-OCH3
4-CH3
2-OCH3
2-CH3
3-OCH3
3-CH3
158– 3.36 (HA, appeared as doublet, JAM = 8.85 Hz), 3.82 (3H, s, Ar-OCH3) 3.87 (HM, dd, JMA = 17.85 Hz, JMX = 10.8 Hz), 6.31 (HX,
160 appeared as doublet, JMX = 4.35 Hz), 6.84–7.87 (11H, m, Ar-H), 9.89 (1H, s, Ar-OH), 9.92 (1H, s, Ar-NH–)
149– 3.34 (HA, dd, JAM = 18.9 Hz, JAX = 3.0 Hz), 2.29 (3H, s, Ar-CH3) 3.77 (HM, dd, JMA = 17.7 Hz, JMX = 11.4 Hz), 6.07 (HX, dd,
151 JMX = 11.4 Hz, JAX = 2.7 Hz), 6.32–7.85 (11H, m, Ar-H), 9.89 (1H, s, Ar-OH), 10.06 (1H, s, Ar-NH–)
121– 3.37 (HA, dd, JAM = 13.65 Hz, JAX = 2.4 Hz), 3.87 (3H, s, Ar-OCH3) 3.82 (HM, dd, JMA = 19.8 Hz, JMX = 8.7 Hz), 6.08 (HX, dd,
124 JMX = 11.1 Hz, JAX = 2.4 Hz), 6.35–8.24 (11H, m, Ar-H), 9.75 (1H, s, Ar-OH), 10.15 (1H, s, Ar-NH–)
123– 3.34 (HA, dd, JAM = 17.7 Hz, JAX = 2.7 Hz), 2.21 (3H, s, Ar-CH3) 3.79 (HM, dd, JMA = 17.7 Hz, JMX = 11.7 Hz), 6.05 (HX, dd,
125
JMX = 11.1 Hz, JAX = 2.4 Hz), 6.30–7.83 (11H, m, Ar-H), 9.79 (1H, s, Ar-OH), 10.07 (1H, s, Ar-NH–)
73–
75
3.38 (HA, dd, JAM = 17.8 Hz, JAX = 2.7 Hz), 3.68 (3H, s, Ar-OCH3), 3.73 (HM, dd, JMA = 17.5 Hz, JMX = 11.1 Hz), 6.32 (HX, dd,
JMX = 11.4 Hz, JAX = 2.7 Hz), 6.72–7.87 (11H, m, Ar-H), 9.93 (1H, s, Ar-OH), 10.07 (1H, s, Ar-NH–)
168– 3.34 (HA, dd, JAM = 18.3 Hz, JAX = 3.3 Hz), 2.29 (3H, s, Ar-CH3), 3.78 (HM, dd, JMA = 17.85 Hz, JMX = 11.4 Hz), 6.08 (HX, dd,
169 JMX = 11.1 Hz, JAX = 3.0 Hz), 6.33–7.86 (11H, m, Ar-H), 9.90 (1H, s, Ar-OH), 10.09 (1H, s, Ar-OH)
378 (M+1, bp)
379 (M+2)

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M. Karuppasamy et al. / Bioorg. Med. Chem. 18 (2010) 1875–1881
of vanillinic acid, 4-aminoantipyrin and peroxidase type II in potas-
sium phosphate buffer (pH 7.6). Assay mixture was pre-incubated
with substrate p-tyramine before addition of enzyme. The reaction
was initiated by the addition of homogenate and increase in absor-
bance was monitored at 498 nm at 37 °C for 60 min. Results were
expressed as nmol hÀ1 mgÀ1. Newly synthesized compounds were
dissolved in DMSO and used in the concentration range of 1–
positioned in the aromatic cage (FAD, TYR407, and TYR444, Pock-
et1. As discussed earlier amongst compounds 5–11 methyl substi-
tution (7, 9, and 11) in ring C was found better in terms of potency
than methoxy (6, 8, and 10) substitution. Also 3-methyl derivative
(11) was found superior than 2-methyl or 4-methyl derivative (7,
9). The superior potency of the 3-methyl derivative (11) can be
attributed to the
p–p stacking that was observed with TYR444
1000
lM. Inhibitors were then incubated with purified MAO at
and ring C (Figure 2b). Whereas, for compounds 7 and 9 (4-methyl
37 °C for 0–60 min prior to adding to the assay mixture. Reversibil-
ity of the inhibition of MAO by these compounds was assessed by
dilution.
and 2-methyl substitution, respectively) the particular interaction
(p–p
stacking) is not observed (Figure 2c). 3-Methyl substitution in
ring C orients the phenyl ring in the aromatic cage in such a way, so
as to make the stacking possible. Low selectivity of these com-
All the synthesized compounds were found to be selective and
reversible inhibitor of MAO-A with outstanding selectivity index
in the magnitude of 103–105. Compounds 5–11 with thiophen-2-
yl group as ring B were superior in potency in comparison to those
with furan-2-yl group as ring B, compounds 12–18. Amongst com-
pounds 5–11 methyl substitution (7, 9, and 11) in ring C was found
better in terms of potency than methoxy (6, 8, and 10). The order of
activity for methyl substituted compounds (7, 9, and 11) is 3-CH3
(11) > 2-CH3 (9) > 4-CH3 (7). Similarly, for methoxy substitution
in ring C (6, 8, and 10) the order of activity is 3-CH3 (6) > 2-CH3
(8) > 4-CH3 (10). Compound (5) where ring C is unsubstituted
was found least active.
p–p
pounds towards MAO-B can be attributed to the following: In case
of MAO-B pockets 2 and 3 were reduced and merged to narrow
cavity primarily due to different orientation of the cavity forming
loop.34,35 Therefore, compounds 5–18 with additional ring C expe-
rience a significant steric hindrance. Interestingly, the results ob-
tained computationally are in good agreement with their
experimental values. The calculated and experimental pKi were
plotted and presented in Figure 3. The outstanding co-relation be-
tween the predicted and the experimental values ensures that the
docking and scoring function adopted here, appropriately depicts
the molecular recognition process of these compounds with either
isoform of MAO.
2.3. Molecular docking
From the above study we have successfully identified few com-
pounds which are reversible and selective inhibitor of MAO-A. A
concise idea about their structure–activity relationship was also
drawn and importance of different substitutions especially at ring
B and ring C was understood. Presence of five membered heterocy-
clic ring B and substitution at 1N position of the pyrazoline ring
shifts the selectivity spectrum towards MAO-A. Therefore the pres-
ent finding reveals some significant novel molecules along with
structural insights using molecular docking calculation. Further it
initiates detail investigation to optimize the molecules and to dis-
cover a significant drug candidate as reversible MAO inhibitors.
The structural and binding mode studies of the compounds with
MAO-A and MAO-B were carried out using molecular docking by
AutoDock 4.0. The best scoring molecules from the largest cluster
were considered for further structural and interaction studies.
The calculated inhibition constants (Ki) as well as their experimen-
tal values for each enzyme–inhibitor complex are shown in Table
2. All the analogs share a common binding mode and occupy sim-
ilar position in the active site. Two compounds 11 (ring B = thio-
phenyl) and 17 (ring B = furyl) in complex with MAO-A, was
shown in Figure 2a. Ring A (p-hydroxy phenyl) is accommodated
well in pocket3 which is formed due to cavity-shaping loop, char-
acterized by GLY74, ARG206, ILE207, PHE208, GLU216, TRP441.
Hydrogen bonding was observed between the hydroxyl hydrogen
and the peptidyl oxygen of ILE207. Ring B (five membered hetero-
aromatic ring) is well accommodated in pocket2 (ILE180, ILE335,
LEU337, MET350, PHE352) and ring C (substituted phenyl) were
3. Experimental
3.1. Chemistry
Melting points were determined using Thermonik Melting Point
Apparatus (Campbell electronics, India) by capillary method and
Table 2
Calculated and experimental Ki values corresponding to the inhibition of MAO isoforms by the newly synthesized pyrazoline derivatives
Code
Experimentala (Ki)
Calculatedb (Ki)
MAO-A (nM) MAO-B (nM)
Inhibition type
Reversibility
MAO inhibitory Selectivity
HeLa cell culture
CC50 (lM)
MAO-A (nM)
MAO-B (nM)
5
6
7
8
480.10 33.00
500.55 36.70
350.55 22.00
300.55 21.00
280.60 12.22
180.55 12.09
150.10 10.05
700.76 38.00
950.23 60.07
480.22 29.00
755.22 40.33
230.70 17.00
175.90 11.80
591.00 37.50
105,660 9210
5.53 0.27
47,000 300
71,51,000 40,260
71,540 4300
44,000 2990
70,150 4560
11,00,000 770,000
22,10,000 110,000
778,000 50,000
10,60,000 100,000
50,10,000 300,000
173,500 10,040
28,840 1200
460,450 29,900
580,200 36,000
1350 120
395.12
326.52
260.36
277.24
150.85
124.14
155.78
414.15
402.47
305.08
284.80
129.05
152.05
222.38
—
38,300
67,65,900
55,570
41,190
73,060
718,200
21,60,000
604,000
10,64,345
30,60,000
160,440
212,700
449,380
218,100
—
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-A
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9
10
11
12
13
14
15
16
17
18
SEL
MOC
1080 300
—
—
SEL - Selegeline, MOC - Moclobemide.
a
Values were determined from the kinetic experiments in which p-tyramine (substrate) was used at 500
lM to measure MAO-A and 2.5 mM to measure MAO-B. Pargyline
or clorgyline were added at 0.50 M to determine the isoenzymes A and B. Newly synthesized compounds and the known inhibitors were pre-incubated with the
l
homoganates for 60 min. at 37 °C. Each value represents the mean SEM of three independent experiments.
b
Values obtained through AutoDock program.

M. Karuppasamy et al. / Bioorg. Med. Chem. 18 (2010) 1875–1881
1879
Figure 2. (a) Compounds 11 and 17 in complex with MAO-A, was shown. Ring A (p-hydroxy phenyl) is accommodated well in pocket3 which is formed due to cavity-shaping
loop, characterized by GLY74, ARG206, ILE207, PHE208, GLU216, TRP441 whereas, ring B (five membered heteroaromatic ring) is well accommodated in pocket2 (ILE180,
ILE335, LEU337, MET350, PHE352) and ring C (substituted phenyl) of these compounds were positioned in the aromatic cage (FAD, TYR407, and TYR444, pocket1). (b)
Compounds 7 and 9 in complex with MAO-A are shown. (c) Compound 11 in complex with MAO-A are shown. 3-Methyl substitution in ring C orients the phenyl ring in the
aromatic cage in such a way, so as to enable the pi–pi stacking.
are uncorrected. Infrared (IR) spectra were taken on a Fourier
Transform Infrared Spectrophotometer IR-Prestige 21 (Shimatzu
Corporation, Japan) from 4000–400 cmÀ1 using KBr discs. 1H
NMR spectra were recorded at 400 MHz in DMSO-d6 using a Bruker
Avance 400 instrument (Bruker Instruments Inc., USA). Chemical
shifts were measured at d units (ppm) relative to tetramethylsilane
(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-nitro-
benzyl alcohol as matrix, and 10 kV as accelerating voltage at room
temperature. Elemental analysis was performed on a Vario EL III
Elemental Analyser (Elementar, Germany) using sulfanilamide as
standard. All chemicals were purchased from Merck, Spectrochem
or CDH, India. Solvents were of reagent grade and were purified
and dried by standard procedure. Reactions were monitored by
thin-layer chromatography on silica gel plates in either iodine or

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M. Karuppasamy et al. / Bioorg. Med. Chem. 18 (2010) 1875–1881
let was resuspended in 0.3 M sucrose and was layered onto 1.2 M
sucrose, centrifuged at 53,000g for 2 hours and resuspended in
potassium phosphate buffer, pH 7.4, kept at À70 °C until used.
maoBexp
maoBpred
maoAexp
maoApred
7
6
5
4
3
3.2.2. Measurement of MAO activity
Total MAO activity was measured spectrophotometrically
PKi
according to the Holt method.34 The assay mixture contained a
2
1
0
chromogenic solution consisted of 1 mM vanillic acid, 500 lM 4-
aminoantipyrine, and 4 U mlÀ1 peroxidase type II in 0.2 M potas-
sium phosphate buffer, pH 7.6. The assay mixture contained
maoApred
maoAexp
maoBpred
maoBexp
167
ll chromogenic solution, 667
ll substrate solution (500 lM
p-tyramine) and 133
ll potassium phosphate buffer, pH 7.6. The
compounds
mixture was pre-incubated at 37 °C for 10 min before the addition
of enzyme. The reaction was initiated by adding the homogenate
(100 ll), and an increase in absorbance was monitored at 498 nm
Figure 3. Experimental (exp) and predicted (pred) pKi values of the synthesized
compounds against MAO-A and MAO-B.
at 37 °C for 60 min. A molar absorption coefficient of 4654 MÀ1
cmÀ1 was used to calculate the initial velocity of the reaction. Re-
sults were expressed as nmol hÀ1 mgÀ1
.
UV chambers. Intermediates were characterized by IR spectro-
scopic analysis and elemental analysis for CHN. In the elemental
analysis, the observed values were within 0.4% of the calculated
values. Final compounds were characterized by 1H NMR and FAB
mass spectrometry (MS). The final yields and the physicochemical
and spectral data of the compounds 1–32 are presented in Table 1.
3.2.3. Selective measurement of MAO-A and MAO-B activities
Homogenates were incubated with the substrate p-tyramine
(500
lowing the inhibition of one of the MAO isoforms with selective
inhibitors. Aqueous solution of clorgyline (50 M), as selective
lM to measure MAO-A and 2.5 mM to measure MAO-B) fol-
l
MAO-A and -B inhibitors were added to homogenates at the ratio
3.1.1. General procedure for the synthesis of chalcones (1–2)
To a solution of 4-hydroxy acetophenone (0.01 M) and suitably
substituted aldehydes (0.01 M) in ethanol (10 ml) was added aque-
ous solution of potassium hydroxide (60%) drop wise with contin-
uous stirring at 0°C over a period of 15 minutes. The reaction
mixture was kept at room temperature for about 48 h with occa-
sional shaking. After 48 h it was poured into ice-cold water, and
then neutralized to pH 2 using 6 N hydrochloric acid. The yellow
precipitate obtained was filtered, washed, dried, and recrystallized
from dry methanol. The intermediates 1–2 were obtained.
of 1:100 (v/v), yielding the final inhibitor concentrations of
0.50 lM. Homogenates were incubated with these inhibitors at
37 °C for 60 min prior to activity measurement. After incubation
of homogenates with selective inhibitors, total MAO activity was
determined by the method described above.
3.2.4. Analysis of the kinetic data
Newly synthesized compounds were dissolved in dimethyl sulf-
oxide (DMSO), maximum concentration 1% and used in the con-
centration range of 1–1000 lM. Inhibitors were incubated with
the purified MAO at 37 °C for 0–60 min prior to adding them to
the assay mixture. The reversibility of the inhibition of the enzyme
by novel compounds was assessed by dialysis performed over 24 h
at 37 °C relative to a potassium phosphate buffer, pH 7.6 capable of
restoring 98–100% of the enzyme activity. Kinetic data for interac-
tion of the enzyme with the compounds were determined using
the Microsoft Excel package program. The inhibitory activities of
the novel compounds for MAO-A and MAO-B were determined at
37 °C after incubation of the homogenates (previously treated with
clorgyline for MAO-A or -B measurement) with the compounds for
60 min. Lineweaver–Burk plots were used to estimate the inhibi-
tion constant (Ki) of the inhibitors. IC50 values were determined
from plots of residual activity percentage, calculated in relation
to a sample of the enzyme treated under the same conditions with-
out inhibitor, versus inhibitor concentration [I]. IC50 values were
determined using non-linear regression analysis according to the
equation for a sigmoid plot. Logarithmic transformation was also
used for the determination of IC50 values for some compounds
which showed their inhibition in a large concentration range.
3.1.2. General procedure for the synthesis of 3,5-diaryl-4,5-
dihydro-1H-pyrazole (3–4)
Appropriate chalcone (1–2) was treated with 10 times excess of
hydrazine hydrate (80%) in dry ethanol and refluxed for 3–6 h. The
hot reaction mixture was then poured into ice-cold water. The so-
lid separated out was filtered, washed, dried and recrystallized
from ethanol to afford respective pyrazoline (3–4).
3.1.3. General procedure for the synthesis of N,3,5-triaryl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (5–18)
An equimolar quantity of appropriate pyrazoline (3–4) and suit-
ably substituted phenyl isothiocyanates in dry methanol was re-
fluxed for 15–30 min to afford compounds 5–18. The structures,
physicochemical and spectral characteristics of compounds 5–18
was presented in Table 1.
3.2. Biochemistry
All chemicals used were purchased from Sigma–Aldrich Co.
(Germany).
3.2.5. Protein determination
The protein was determined according to the Bradford meth-
3.2.1. Isolation of MAO from rat liver homogenates
od37 in which bovine serum albumin was used as a standard.
The ethics Committee of Laboratory Animals at Hacettepe Uni-
versity, Turkey (2001/25-4), approved the animal experimentation.
MAO was purified from the rat liver according to the Holt method
with some modifications.36 Liver tissue was homogenized 1:40 (w/
v) in 0.3 M sucrose. Following centrifugation at 1000g for 10 min,
the supernatant was centrifuged at 10,000g for 30 min to obtain
crude mitochondrial pellet. The pellet was incubated with CHAPS
of 1% at 37 °C for 60 min and centrifuged at 1000g for 15 min. Pel-
3.3. Molecular docking
3.3.1. Protein preparation
PDB structures (www.rcsb.org) were downloaded and were re-
fined using Schrödinger protein preparation wizard. Protonation
states were adjusted and finally a restrained energy minimization
using OPLS2005 force field was carried out.

M. Karuppasamy et al. / Bioorg. Med. Chem. 18 (2010) 1875–1881
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Structure of the ligands were sketched using built panel of Mae-
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Docking parameters: Number of Genetic Algorithm (GA) runs:
10, Population size: 150, Maximum number of evaluation:
2500,000, Maximum number of generation: 27,000.
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Acknowledgments
M.K. and M.M. thankful to AICTE for providing fellowship.
Authors are thankful to SAIF, CDRI, Lucknow for providing spectral
data.
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Supplementary data
Supplementary data associated with this article can be found, in
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