Design, synthesis, and in vitro hMAO-B inhibitory evaluation of some 1-methyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles

Article abstract of DOI:10.1016/j.bmcl.2013.07.035

A series of 1-methyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles (3a-k and 4a-u) were designed, synthesized, and evaluated for their inhibitory efficacy towards the two hMAO isoforms. Most of the derivatives were found to be potent and selective hMAO-B inhibitors. In particular, derivative 3g showed greater hMAO-B affinity than selective inhibitor selegiline coupled with high selectivity index (SI = 145). The most selective hMAO-B inhibitor was the 3-methyl analogue 3f with an SI higher than 909.

Full text of DOI:10.1016/j.bmcl.2013.07.035

Accepted Manuscript
Design, Synthesis, and in vitro hMAO-B Inhibitory Evaluation of Some 1-
Methyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles
Rossella Fioravanti, Nicoletta Desideri, Mariangela Biava, Luca Proietti
Monaco, Laura Grammatica, Matilde Yá ñez
PII:
S0960-894X(13)00880-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.07.035
BMCL 20700
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
14 June 2013
12 July 2013
15 July 2013
Please cite this article as: Fioravanti, R., Desideri, N., Biava, M., Monaco, L.P., Grammatica, L., Yá ñez, M., Design,
Synthesis, and in vitro hMAO-B Inhibitory Evaluation of Some 1-Methyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles,
Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.07.035
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Graphical Abstract
Design, Synthesis, and in vitro hMAO-B
Inhibitory Evaluation of Some 1-Methyl-3,5-
diphenyl-4,5-dihydro-1H-pyrazoles
R. Fioravanti, N. Desideri, M. Biava, L. Proietti Monaco, L. Grammatica, M. Yáñez.
R
R1
R2
N
N
CH3
R = H; OH;
R1 = H; OH;
R2 = H; 2-F; 3-F; 4-F; 2-CH3; 3-CH3; 4-CH3; 2-Cl; 3-Cl; 4-Cl.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, Synthesis, and in vitro hMAO-B Inhibitory Evaluation of Some 1-
Methyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles
Rossella Fioravantia,* , Nicoletta Desideria, Mariangela Biavaa, Luca Proietti Monacoa, Laura Grammaticaa,
Matilde Yáñezb
a'LSDUWLPHQWRꢀGLꢀ&KLPLFDꢀHꢀ7HFQRORJLHꢀGHOꢀ)DUPDFRꢁꢀ8QLYHUVLWjꢀ³/Dꢀ6DSLHQ]D´ꢀ3ꢂOHꢀ$OGRꢀ0RURꢁꢀꢃꢁꢀꢄꢄꢅꢆꢃꢀ5RPHꢁꢀ,WDO\
bDepartamento de Farmacología and Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario
Sur, E-15782 Santiago de Compostela, La Coruña, Spain
ARTICLE INFO
ABSTRACT
A series of 1-methyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles (3a-k and 4a-u) were designed,
synthesized, and evaluated for their inhibitory efficacy towards the two hMAO isoforms. Most
of the derivatives were found to be potent and selective hMAO-B inhibitors. In particular,
derivative 3g showed greater hMAO-B affinity than selective inhibitor selegiline coupled with
high selectivity index (SI = 145). The most selective hMAO-B inhibitor was the 3-methyl
analogue 3f with an SI higher than 909.
Article history:
Received
Revised
Accepted
Available online
Keywords:
Pyrazole derivatives
Monoamine oxidases
hMAO inhibitors
2013 Elsevier Ltd. All rights reserved.
The monoamine oxidases (MAOs) are flavoenzymes located
at the outer membrane of mitochondria in the human brain and
peripheral tissues. MAOs appear as two isozymes, MAO-A and
B, distinguished by substrate and inhibitor selectivities. MAOs
catalyze the oxidative deamination of xenobiotic amines and
neurotransmitters and play an important role in the central
nervous system and peripheral organs. MAO-A metabolizes
serotonin, epinephrine and norepinephrine and inhibitors of this
isoform are used in the treatment of anxiety disorders and
depressive illness.1 MAO-B is considered to be a major
dopamine metabolizing enzyme and its inhibitors reduce the
catabolism of dopamine in the basal ganglia and are employed in
WKHꢀV\PSWRPDWLFꢀWKHUDS\ꢀRIꢀ3DUNLQVRQ¶VꢀGLVHDVHꢁ2,3 ,Qꢀ3DUNLQVRQ¶Vꢀ
disease, MAO-B inhibitors not only prolong the action of
dopamine in the brain, but also enhance dopamine levels after
treatment with levodopa, the metabolic precursor of dopamine.4,5
Furthermore, MAO-B inhibitors protect against the
QHXURGHJHQHUDWLYHꢀ SURFHVVHVꢀ DVVRFLDWHGꢀ ZLWKꢀ 3DUNLQVRQ¶Vꢀ
disease,6 decreasing the formation of dopanal and H2O2 that arise
from the MAO-B catalyzed oxidation of dopamine.7±11
possess biological activities such as antimicrobial,12 analgesic,13
DQWLLQÀDPPDWRU\14 and anticancer15 but also potent anti-MAO
efficacy.16
In the context of our search aimed at identifying potent MAO
inhibitors,17-19 we hereby report the synthesis and biological
evaluation of some 1-methyl-3,5-diphenyl-4,5-dihydro-1H-
pyrazoles (Figure 1).
R
R1
R2
N
N
CH3
R = H; OH;
R1 = H; OH;
R2 = H; 2-F; 3-F; 4-F; 2-CH3; 3-CH3; 4-CH3; 2-Cl; 3-Cl; 4-Cl.
Figure 1. 1-Methyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles
Based on the therapeutic implications of MAO inhibitors, the
search for novel and selective molecules continues to be an active
area of research in medicinal chemistry. Several nitrogen
containing heterocycles have been employed in the design of
MAO inhibitors. In particular, pyrazole derivatives not only
Derivatives 3a-k and 4a-u were synthesized as reported in
Scheme 1. The intermediate chalcones 1a-k and 2a-u were
prepared by base-catalyzed condensation of substituted
benzaldehydes with the appropriate acetophenone, eventually
followed by acid deprotection of the p-hydroxyl group (2a-u).
Further reaction of the chalcones 1a-k and 2a-u with
methylhydrazine in refluxing ethyl alcohol afforded the
*Corresponding author. Tel.: +39 06 49693259; fax: +39 06 49693259.
E.mail address rossella.fioravanti@uniroma1.it

R1
CH3
corresponding 4,5-dihydro-1H-pyrazoles 3a-k and 4a-u (see
Supplementary material).
HO
R1
O
CH3
All synthesized pyrazolines were screened for their potential
effects towards the two human MAO isoforms. (physicochemical
data, and anti-MAO efficacies are reported in Table 1).
O
(ii)
(i)
R1
O
O
R1
The inhibitory activities were investigated by measuring the
effects of each derivative on the production of hydrogen peroxide
(H2O2) from p-tyramine, using the Amplex“ Red MAO assay kit
(Molecular Probes, Inc., Eugene, Oregon, USA) and microsomal
MAO isoforms prepared from insect cells (BTI-TN-5B1-4)
infected with recombinant baculovirus containing cDNA inserts
for human MAO-A or MAO-B (Sigma±Aldrich Química S.A.,
Alcobendas, Spain). The production of H2O2 catalysed by MAO
R2
CH3
O
O
1a-k
(i)
(iii)
R1
O
O
R1
R2
R2
N
N
CH3
O
(iv)
isoforms
can
be
detected
using
10-acetyl-3,7-
non-
3a-k
dihydroxyphenoxazine (Amplex“ Red reagent),
a
HO
R1
HO
R1
fluorescent and highly sensitive probe that reacts with H2O2 in
the presence of horseradish peroxidase to produce a fluorescent
product, resorufin. In this study, MAO inhibition was evaluated
using the above mentioned method, following the general
procedure previously described by us.20
R2
(iii)
R2
N
N
O
CH3
2a-u
4a-u
Scheme 1. Reagents and conditions: (i) substituted benzaldehyde, EtOH, Ba(OH)2 x 8H2O, 30°C, 24h. (ii)
3,4-dihydro-2-H-pyran, CH2Cl2, 12h. (iii) methylhydrazine, KOH, EtOH, 80°C, 24h. (iv) 3N HCl , EtOH.
Table 1. Chemical data, physical data, and biological activity of 1-methyl-3,5-diphenyl-4,5-dihydro-1H-pyrazoles
(3a-k and 4a-u) and reference inhibitors.
Purification
method
A
hMAO-A (IC50)
µM
hMAO-B (IC50)
µM
Compd
R1
R2
m.p. (°C)
Yield%
SIc
3a
3b
3c
3d
3e
3f
3g
3h
3j
3k
4a
4b
4c
4d
4e
4f
4g
4h
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2F
3F
Oil
164-166
Oil
32-35
Oil
Oil
30-33
Oil
63
28
41
61
46
46
42
83
13
40
37
29
33
15
29
43
35
20
40
37
36
36
20
31
36
61
12
25
19
33
1.68 0.03a
0.12 0.01
14
10
30
21
30
B
A
B
A
A
B
A
A
B
C
C
D
C
C
C
C
B
C
C
B
B
B
D
D
D
D
D
D
D
2.84 0.02a
1.82 0.05a
3.00 0.06a
3.26 0.08a
***
0.28 0.03
0.06 0.01
0.14 0.01
0.11 0.01
0.11 0.01
0.0096 0.0003
3.65 0.29
0.84 0.06
3.95 0.15
26.80 1.95
3.04 0.17
3.17 0.21
3.65 0.16
20.08 1.01
5.78 0.27
1.91 0.06
10.44 0.02
2.07 0.13
2.64 0.19
***
4F
2CH3
3CH3
4CH3
2Cl
3Cl
4Cl
H
2F
3F
4F
2CH3
3CH3
4CH3
2Cl
3Cl
4Cl
H
2F
3F
4F
> 909#
145
> 27#
61
1.39 0.07a
**
Oil
40-44
50.99 1.33a
**
> 25#
> 3.7#
> 33#
> 31#
> 27#
1.1
2.8
4.8
1.1
9
195-198
172-175
115-118
175-177
105-107
155-157
150-152
128-131
130-133
184-185
136-139
78-80
123-126
72-75
66-69
63-65
145-148
55-56
***
***
***
***
21.87 0.99
16.33 0.83b
9.13 0.52a
11.82 0.97
18.69 1.36a
12.73 0.8a
***
H
H
H
4.8
-
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
***
**
3.87 0.27
**
0.49 0.03
2.04 0.15
***
> 26#
-
0.81 0.04b
3.98 0.12b
***
1.6
1.9
-
-
3.7
2.3
4.1
0.000073
3362
0.87
< 0.36
2CH3
3CH3
4CH3
2Cl
3Cl
4Cl
***
***
1.10 0.12 a
0.42 0.06b
1.39 0.09a
0.0045 0.32 a
67.25 1.02a
6.56 0.76
361 19.37
0.30 0.02
0.18 0.02
0.34 0.03
61.35 1.13
0.0196 0.86
7.54 0.36
*
60-62
65-67
4u
Clorgyline
Selegiline
Iproniazid
Moclobemide
A= Chromatography on SiO2/CHCl3; B = crystallized from methanol; C = crystallized from acetonitrile; D = crystallized from n-hexane
a
All IC50 values shown in this table are the mean S.E.M. from five experiments. Level of statistical significance: P < 0.01 or bP < 0.05
versus the corresponding IC50 values obtained against hMAO-B, as determined by ANOVA/Dunnett´s. ** Inactive at 100 µM (highest
concentration tested). *** 100 µM inhibits the corresponding MAO activity by approximately 40-50%. At higher concentration the

compounds precipitate. cSI: hMAO-B selectivity index = IC50 (hMAO-A)/ IC50 (hMAO-B). # Values obtained under the assumption that
the corresponding IC50 against MAO-$ꢀLVꢀWKHꢀKLJKHVWꢀFRQFHQWUDWLRQꢀWHVWHGꢀꢂꢃꢄꢄꢀȝ0ꢅꢁ
All the new synthesized compounds (3a-k and 4a-u) and
reference inhibitors were tested towards hMAO isoforms. Results
reported in Table 1 show that most derivatives inhibited the
hMAO-B isoform with IC50 values in the low micromolar or
nanomolar range. Moreover, several compounds proved to be
able to inhibit hMAO-A with lower affinity. Therefore, the new
compounds are in general selective hMAO-B inhibitors.
both WKHꢀꢆ¶ꢀDQGꢀꢇ¶ꢀSRVLWLRQVꢀRIꢀWKH 3-phenyl ring, enhances both
hMAO-A and hMAO-B inhibition efficacies.
References and notes
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All the compounds unsubstituted on the 3-phenyl ring (3a-k)
were associated with potent and selective hMAO-B inihibitory
activity. 1-Methyl-3-phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazole
3g was the most potent hMAO-B inhibitor identified in this study
with an IC50 of 9.6 nM coupled with high selectivity (SI = 145).
In comparison with the reference inhibitors, 3g was at least 2-
fold more active. The displacement of the para methyl group to
the meta position on the 5-phenyl ring resulted in compound 3f,
approximately 11-fold less potent as a hMAO-B inhibitor than
the corresponding para methyl analogue 3g. However, 3f
exhibited the highest hMAO-B selectivity due to its poor hMAO-
A affinity up to the highest concentration tested (100 PM).
Generally, the introduction of a hydroxyl group at the para
position of the 3-phenyl ring (4a-k) led to a reduction of efficacy
against both isoforms with respect to unsubstituted analogues 3a-
k. In fact, all the 3-(4-hydroxyphenyl)-1-methyl-5-phenyl-4,5-
dihydro-1H-pyrazoles (4a-k) exhibited hMAO inhibition potency
in the micromolar range. In this series of compounds, the
presence of a fluorine substituent on the 3-phenyl ring led to
selective hMAO-B inhibitors (4b-d), while the introduction of
either a chlorine or a methyl group resulted in compounds 4e-k
endowed with low or modest hMAO-B selectivity.
Moreover, the introduction of a hydroxyl substituent at the
ortho position of 3-phenyl ring, gave access to compounds 4l-u,
that showed different results. While compounds 4l, 4n, 4q and 4r
are essentially inactive against both hMAO isoforms, 4o and 4t
displayed potency in the submicromolar range against both
hMAO-A and hMAO-B resulting in compounds endowed with
poor selectivity. Moreover, the chloro-substituted derivatives 4s-
u exhibited enhanced inhibitory potency with respect to the
corresponding 3-phenyl and 3-(4-hydroxyphenyl) analogues (3h-
k and 4 h-k, respectively).
By comparing the hMAO-B inhibitory activity of the new N1-
methyl derivatives (3a-k and 4a-u) with that of recently reported
N1-thiocarbamoyl analogues,16g,h it is possible to point out that
the introduction of a smaller group, such as the methyl one, led
to compounds more active and selective towards hMAO-B
isoform. Moreover, most of the previously studied 3,5-diphenyl-
4,5-dihydro-1H-pyrazole derivatives substituted at N1 position
of the pyrazoline ring with phenyl, acyl-, or thiocarbamoyl-
groups were evaluated using rat or bovine instead of human
MAOs. Since species-dependent differences in substrate
specificity and inhibitor selectivity have been reported,16a-c,e no
further comparison is possible.
17. Bolasco, A.; Carradori, S.; Fioravanti, R.; Expert Opinion on
Therapeutic Patents 2010, 20 (7), 909.
18. Desideri, N.; Bolasco, A.; Fioravanti, R.; Proietti Monaco, L.;
Orallo, F.; Yáñez, M.; Ortuso, F.; Alcaro, S.; J. Med. Chem.
2011, 54 (7), 2155.
19. Desideri, N.; Fioravanti, R.; Monaco Proietti, L.; Biava M.;
Yáñez, M.; Ortuso, F.; Alcaro, S.; Eur. J. Med. Chem. 2013, 59,
91.
20. Yáñez, M.; Fraiz, N.; Cano, E.; Orallo, F.; Biochem. Biophys.
Res. Commun. 2006, 344, 688.
We can conclude that in this series of inhibitors, an
unsubstituted 3-phenyl ring seems to be an important chemical
feature to obtain potent and selective hMAO-B inhibitors.
Furthemore, the presence of the chlorine substituent at the 5-
phenyl ring, coupled with the presence of two hydroxyl groups at
Supplementary data
Supplementary data associated with this article can be found,
in the online version,