Pyrazoline based MAO inhibitors: Synthesis, biological evaluation and SAR studies

Article abstract of DOI:10.1016/j.bmcl.2011.05.057

Twenty-two pyrazoline derivatives were synthesized and tested for their human MAO (hMAO) inhibitory activity. Twelve molecules with unsubstituted ring A and substituted ring C (5-16) were found to be potent inhibitors of hMAO-A isoform with SI_MAO-A

Full text of DOI:10.1016/j.bmcl.2011.05.057

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4296–4300
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazoline based MAO inhibitors: Synthesis, biological evaluation
and SAR studies
Monika Jagrat a, Jagannath Behera a, Samiye Yabanoglu b, Ayse Ercan b, Gulberk Ucar b,
Barij Nayan Sinha a, Vadivelan Sankaran c, Arijit Basu a, Venkatesan Jayaprakash a,
⇑
a Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India
b Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhıye, Ankara, Turkey
c Informatics, GVK Biosciences Private Limited, 37 Sterling Road, Chennai, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 March 2011
Revised 6 May 2011
Accepted 18 May 2011
Available online 25 May 2011
Twenty-two pyrazoline derivatives were synthesized and tested for their human MAO (hMAO) inhibitory
activity. Twelve molecules with unsubstituted ring A and substituted ring C (5–16) were found to be
potent inhibitors of hMAO-A isoform with SIMAO-A in the order 103 and 104. Ten molecules with unsub-
stituted ring A and without ring C (21–30), in which eight molecules (21, 23–26, and 28–30) were selec-
tive for hMAO-A, one for hMAO-B (22) and the other one non-selective (27). Presence of ring C increases
potency as well as SI towards hMAO-A; however its absence decreases both potency and SI towards
hMAO-A and hMAO-B.
Keywords:
Pyrazolines
MAO
Ó 2011 Elsevier Ltd. All rights reserved.
Selectivity
Docking
Selective MAO-B inhibitors gained greater attention due to their
multiple roles that they can play in the therapy of neurodegenera-
tive disorders.1,2 Our group had reported earlier a few 3,5-diaryl
carbothioamide pyrazolines with MAO inhibitory activity. Two
molecules (12 and 13) without any substitution in the ring A and
one molecule without ring C (11) were found to be selective
against rat liver MAO-B (Fig. 1).3 Recently Boppana et al. have pro-
posed few molecules with potential MAO-B selectivity through
pharmacophore modelling. They also proposed three molecules
with unsubstituted ring A and without ring C in pyrazoline carbox-
amides (Fig. 1).4 In order to explore the possibility of getting poten-
tial MAO-B selective compound we prepared 22 pyrazoline
derivatives with unsubstituted ring A, with or without ring C.
Twelve molecules (5–16) with substituted ring C carrying
unsubstituted ring A were prepared using the method reported
earlier3 by following the Scheme 1. Another 10 molecules
(21–30) were prepared without ring C carrying unsubstituted ring
A according to the method reported earlier5 by following the
Scheme 2. Chalcones 1–2 and 17–20 were prepared through
Claisen–Schmidt condensation. To an equimolar quantity of aceto-
phenone and appropriate benzaldehyde in ethanol was added an
aqueous solution of sodium hydroxide (60%) dropwise with contin-
uous stirring at 0 °C over a period of 30 min. The reaction mixture
was kept at room temperature for about 48 h with occasional shak-
ing. It was then poured into ice-cold water to obtain the 17–20.
Chalcones 1–2 were obtained by adjusting the pH to 2 using 6 N
hydrochloric acid. The pyrazoline intermediate (3–4) were
obtained by the condensation of 1–2 with excess hydrazine
hydrate (99%) in ethanol. The reaction mixture was refluxed for
about 3 h and upon concentration and cooling provided 3–4. The
final pyrazoline thiocarboxamide derivatives (5–16) were obtained
by the reaction of 3–4 with appropriately substituted phenylisoth-
iocyanates. The reaction mixture in ethanol was refluxed for a
period of 30–45 min and upon cooling or concentration provided
5–16. Pyrazoline derivatives (21–30) were prepared by the con-
densation of 17–20 with 2 Molar equiv of hydrazine derivatives
(semicarbazide hydrochloride/thiosemicarbazide/aminoguanidine
bicarbonate). The reaction mixture in ethanol was refluxed for a
period of 12–18 h. The product (21–30) precipitated in hot solution
or upon concentration and cooling. All the intermediates were
characterised by IR spectroscopic and elemental analysis for CHNS.
In the elemental analysis, the observed values were within 0.4% of
the calculated values. Final compounds were characterised by 1H
NMR and FAB-MS (Supplementary data).
Monoamine oxidase A human (recombinant, expressed in
baculovirus infected BTI insect cells), monoamine oxidase B human
(recombinant, expressed in baculovirus infected BTI insect cells),
R-(À)-deprenyl hydrochloride, resorufin, dimethyl sulfoxide
(DMSO) and some other chemicals were purchased from
⇑
Corresponding author. Tel.: +91 9470137264.
E-mail address: venkatesanj@bitmesra.ac.in (V. Jayaprakash).
Sigma–Aldrich™ (Germany). Moclobemide was
a gift (Roche
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.057

M. Jagrat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4296–4300
4297
Figure 1. Selective MAO-B inhibitors reported3 and predicted4 earlier.
Scheme 1. Reagents and conditions: (a) NH2NH2ÁH2O (99%), C2H5OH, reflux, 3 h; (b) R2-C6H4–NCS, C2H5OH, reflux, 30–45 min.
methoxy (favourable at para > meta position) and methyl (favour-
able at meta > para position) group at either para or meta positions.
The compounds without ring C (21–30) were found to be selec-
tive towards MAO-A (except 22, 23 and 27) but the potency and
selectivity index are poor when compared with 5–16. Compound
22 is slightly selective towards MAO-B with Ki of 4.79 0.24 lM
and SIMAO-B of 1.46. The experimental Ki values of 21, 22 and 23
were found to be 25-, 8- and 10-fold higher than the predicted val-
ues (Fig. 1).4 This may be due to the fact that Boppanna et al.4 has
developed the pharmacophore model by including the ligands in
training set that could able to interact covalently with the receptor.
Moreover these three molecules were proposed by Boppana et al.4
as potent MAO-B selective inhibitors. But, only 22 was found to be
slightly selective towards MAO-B. They have predicted the data-
base only with the model developed for MAO-B inhibitors. No
model for MAO-A inhibitors was developed and molecules were
predicted against the model by Boppana et al.4 This may be the rea-
son why these molecules were predicted and reported as potent
MAO-B inhibitors. Possibly prediction of these molecules against
model for MAO-A inhibitors and MAO-B inhibitors might have pro-
vided a true picture of selectivity towards isoforms.
Further compound 7, 11, 22 and 27 were selected for molecular
docking simulations studies. Both R and S conformers were docked
to understand the impact of configuration at C5 carbon of pyrazo-
line towards activity and selectivity. It has been reported already
that enantiomer exhibited improved selectivity than racemates
through chiral separation of racemates8–10 and also through mod-
elling studies.11,12 Compounds 7 and 11 were studied against h
MAO-A isoform to understand the factors contributing towards po-
tency, while compounds 22 and 27 were studied against both
hMAO-A and hMAO-B to understand the factors determining the
Scheme 2. Reagents and conditions: (a) NH2–NH–C(=Y)–NH2 (where X = O, S, NH),
C2H5OH, reflux, 12–18 h.
Pharmaceuticals, Germany). AmplexÒ-Red MAO Assay Kit
(Molecular Probes, USA) contained benzylamine, p-tyramine,
Clorgyline (MAO-A inhibitor), Pargyline (MAO-B inhibitor) and
horse radish peroxidase. The interactions of the synthesized com-
pounds (5–16 and 21–30) with hMAO isoforms were determined
by a fluorimetric method described and modified previously.6,7
The production of H2O2 catalysed by MAO isoforms was detected
using 10-acetyl-3,7-dihydroxyphenoxazine (AmplexÒ-Red reagent),
a non-fluorescent, highly sensitive and stable probe that reacts
with H2O2 in the presence of horseradish peroxidase to produce a
fluorescent product, resorufin. The results are presented in
Table 1(a and b).
All the compounds were found to inhibit MAO-A selectively and
reversibly, except 22, 23 and 27 those were non-selective towards
either MAO isoforms. The compounds with ring C (5–16) were po-
tent MAO-A inhibitors with Ki in nM range (except 15) and SIMAO-A
is in the order of 103–104. Compounds 7 and 11 were found to be
the most potent MAO-A inhibitors within this series with Ki values
99.55 9.07 and 90.45 4.73 nM, respectively. SAR within this ser-
ies reveals (i) 2-hydroxy substitution in ring B is better than 4-hy-
droxy (except 11 and 14), (ii) methoxy substitution in ring C is
better than methyl substitution (except 15) and (iii) meta substitu-
tion found to be favourable when above two factors exists. Ortho
hydroxy substitution in ring B increases potency and selectivity in-
dex towards MAO-A when there is methoxy substitution in meta
position of ring C. Whereas para hydroxy substitution in ring B
increases potency and selectivity towards MAO-A, with both
selectivity. The Estimated Ki (EKi in
lM) and Selectivity Index
(SI) for R, S and average of R and S were presented in Table 2. Dock-
ing protocol reported already by our group has been followed.3,5,13
Docking reports revealed that S-conformers are slightly better than
R-conformer (except 11 for MAO-A and 27 for MAO-B).
Since racemates were screened for MAO inhibitory activity, the
average Estimated Ki (EKi in lM) for S and R conformers was also
calculated (EKiav) for comparison. From the EKiav, it is clear that
7 and 11 were selective towards MAO-A, 22 was slightly selective

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M. Jagrat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4296–4300
Table 1
Experimental Ki values corresponding to the inhibition of human MAO isoforms by the newly synthesized pyrazoline derivatives
Code
Ki values for MAO-A (
lM)
Ki values for MAO-B (
l
M)
SIa
Inhibition type
Reversibility
MAO inhibitory selectivity
(a)
5
6
7
8
9
10
11
12
13
14
15
16
0.17 0.01
0.29 0.02
0.10 0.01
0.30 0.02
0.31 0.06
0.25 0.02
0.09 0.00
0.36 0.02
0.35 0.02
0.13 0.01
2.15 0.10
0.31 0.02
9.060 440
0.005 0.13
60.22 2.80
69.90 3.90
979.00 41.00
250.00 11.77
60.00 3.79
0.001
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
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
0.127
1.02 Â 10À4
0.001
0.005
22.00 1.50
0.001
400.90 21.00
300.01 20.00
355.00 16.08
255.60 10.50
2130 200
309.22 10.00
0.091 4.26
1.080 300
2.26 10À4
0.001
9.88 Â 10À4
5.09 Â 10À4
0.001
0.001
99.92
0.005
Selegiline
Moclobemide
(b)
21
22
23
24
25
26
27
28
0.490 0.03
7.00 0.53
3.20 0.18
4.79 0.24
1.88 0.09
1.66 0.10
1.80 0.11
4.00 0.20
0.805 0.04
4.11 0.25
4.80 0.21
4.22 0.19
0.091 4.26
1.080 300
0.153
1.461
0.521
0.181
0.185
0.114
1.000
0.099
0.140
0.035
99.92
0.005
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Selective for MAO-A
Selective for MAO-B
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Non-selective
Selective for MAO-A
Selective for MAO-A
Selective for MAO-A
Selective for MAO-B
Selective for MAO-A
0.980 0.04
0.300 0.02
0.333 0.02
0.456 0.02
0.805 0.04
0.400 0.02
0.670 0.03
0.150 0.01
9.060 440
0.005 0.13
29
30
Selegiline
Moclobemide
Ki 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 preincubated with the homoganates
l
for 60 min at 37 °C. Each value represents the mean SEM of three independent experiments.
a
Selectivity index. It was calculated as Ki (MAO-A)/Ki (MAO-B).
Table 2
Molecular docking results of compound 7, 11, 22 and 27
Code
MAO-A (2BXR)
MAO-B (2BYB)
SIMAO-A
rac/rac
R
S
Average
EKiav
R
S
Average
EKiav
EFEB
EKi
EFEB
EKi
EFEB
EKi
EFEB
EKi
R/R
S/S
7
11
22
27
À9.43
À8.44
À7.56
À8.80
0.13
0.65
2.88
0.35
À9.77
À7.87
À7.67
À8.81
0.07
1.70
2.39
0.35
0.10
1.18
2.64
0.35
À3.48
À2.86
À7.74
À8.39
2830
8020
À4.78
À4.54
À7.78
À7.77
315.00
468.90
1.99
1572.50
4244.45
2.05
15,725
3612.3
0.8
21769.2
12338.5
0.7
4500
275.8
0.8
2.10
0.71
2.00
1.36
3.9
2.0
5.7
EFEB—Estimated Free Energy of Binding in Kcal/mol; Eki—Estimated Ki in lM; SI—Selectivity Index.
towards MAO-B, very much in agreement with experimental
result. The compound 27 has been found to have ꢀ4-fold selectiv-
ity towards MAO-A, Experimental results suggest the molecule as
non-selective.
interaction and accommodates ring C in Pocket 2 (P2, delimited by
GLY71, GLN74, ARG206, ILE207, PHE208, GLU216 and TRP441). Both
ring A (with PHE208 and TYR444) and ring C (TRP441) displayed
p?p interaction. The S-conformer did not display any p?p interac-
The compounds 7 and 11, Estimated Ki of R-conformers (EKiR)
were ꢀ2-fold better than its S-counterpart, but exhibits ꢀ17,000-
and ꢀ12,000-fold increased selectivity towards MAO-A, respec-
tively. Compounds 22, EKiR was almost equal to EKiS against
MAO-A as well as for MAO-B. The selectivity towards MAO-B is
marginal and the S-conformer exhibits only ꢀ0.1-fold better selec-
tivity compared with its R-counterpart/racemate. Compound 27,
EKiR was almost equal to EKiS against MAO-A, while against
MAO-B EKiR was ꢀ3-fold better than EKiS. Due to this the molecule
displayed ꢀ4-, ꢀ2- and ꢀ6-fold selectivity towards MAO-A for
racemate, R-conformers and S-conformer, respectively. Simulation
results suggested that for compounds 7 and 11 the R-isomer exhib-
ited improved selectivity but compounds 22 and 27 displayed a
marginal improvement.
tion, but ring C is very well accommodated in P1, ring B in P2 and ring
A in P3. The o-hydroxy group in ring B could able establishes an
H-bond with SER209. In R-conformer of compound 11, the ring A
is accommodated in P3 exhibiting
p?p interaction with PHE208
and TYR444 similar to 7. But the p-substitution in ring B is not
tolerated by P1, a rotation around the single bond between C3 of
pyrazoline and ring A, moves ring B to P2 and kept ring C away from
P1. This allows ring B to exhibit
factors contribute towards reduced potency of 11 compared with 7.
The S-conformer did not display any interaction similar to 7.
p?pinteraction with TRP441. These
p?p
Here too the ring C is kept away from P1, a rotation around the single
bond between thiocarbamoyl C and N1 of pyrazoline puts ring A in
P2 and ring B in P3. It clearly shows that p-substitution in either ring
B or ring C reduces potency towards MAO-A as well as have a great
impact on selectivity index also. The interaction of 7(R) and 7(S)
are shown in Figure 2.
In R-conformers of compound 7, the ring A is accommodated in
Pocket3 (P3, delimited by ILE180, ILE335, LEU337, MET350 and
PHE352). Ortho substitution in ring B (7) keeps it at the entrance of
the aromatic cage (P1, FAD, TYR407 and TYR444) with edge–edge
In MAO-A active site, the R-conformer of compound 22, ring A
interacts with P2 while ring B with P3. The carbamoyl group was

M. Jagrat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4296–4300
4299
Figure 2. Interaction of 7(R) and 7(S) with hMAO-A (PDB Code: 2BXR) and 22(R) and 22(S) with hMAO-B (PDB Code: 2BYB).
kept away from P1. Only ring B displays one
p
?
p
interaction with
positions were reported to have selective MAO-B inhibitory activ-
ity at
M concentration.12 N-Carbamoyl pyrazolines were not stud-
PHE208. Absence of third ring (non-polar) and exposed –CO–NH2
group (polar) in highly hydrophobic active site may be the reason
for its reduced potency. S-conformer too keeps ring A in P2 and
ring B in P3, but ring B was not positioned well to show any
l
ied for their MAO inhibitory activity and 22 with selective MAO-B
inhibitory activity provides an opportunity to further exploration.
The current study once again establishes the following facts: (i)
presence of third aryl ring (ring C) increases potency and selectiv-
ity towards MAO-A, (ii) absence of third aryl ring drastically
reduces the potency and selectivity towards MAO-A. Further
modifications may reveal factors determining nonselectivity and
selectivity towards MAO-B and (iii) carbamoyl substitution at N1
position of pyrazoline along with unsubstituted ring A and hetero-
cyclic ring B (4-pyridyl) provides selectivity towards hMAO-B
(though poor, this may serve as a starting point for further explo-
ration). Further derivatives should be prepared to explore the pos-
sibility of getting a potent and selective hMAO-B inhibitor of this
kind.
p?p interaction with PHE208, whereas ring A displays p?p inter-
action with PHE208. Moreover the pyrazoline N2 and carbamoyl
NH displays two H-bonding interaction with TYR444 hydroxy H
and O respectively. This additional H-bonding interaction keeps
S-conformer slightly better than its R-conformer.
In MAO-B active site, the R-conformer of compound 22, ring A
was very well accommodated in Aromatic Cage (AC, FAD, TYR398
and TYR435) and displays p?p interaction with TYR435. The ring
B and carbamoyl group were accommodated in Narrow Hydropho-
bic Cavity (NHC). In S-conformer the entire structure is accommo-
dated in NHC with carbamoyl-NH establishing H-bond interaction
with backbone carbonyl-O of ILE198. Both the conformers are
equally active with different interaction within the active site of
MAO-B. Compound 27, both R and S conformers, keep ring A in P2
Acknowledgment
of MAO-A and none displayed any
p
?
p
interaction. But the
Authors are thankful to SAIF, CDRI, Lucknow, India for providing
spectral data.
carbamoyl-NH establishes H-bond interaction with Hydroxyl-O of
SER209 (R-conformer) or TYR444 (S-conformer). This keeps ring C
at the entrance of P1 (R-conformer, edge–face interaction) or in P3
(S-conformer, hydrophobic interaction). In MAO-B active site, ring
A of both the conformers were well accommodated in AC and ring
B along with thiocarbamoyl group is accommodated in NHC. The
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.057.
ring B displays
p?p interaction with either FAD-adenine (R-con-
former) or TYR188 and TYR326 (S-conformer). The interaction of
References and notes
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