Synthesis and inhibitory activity against human monoamine oxidase of N1-thiocarbamoyl-3,5-di(hetero)aryl-4,5-dihydro-(1H)-pyrazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.11.003

A series of N1-thiocarbamoyl-3,5-di(hetero)aryl-4,5-dihydro-(1H)-pyrazole derivatives has been synthesized and assayed for their ability to inhibit the activity of the A and B isoforms of human monoamine oxidase (hMAO). Some of these compounds were endowed with a selective inhibitory activity against hMAO-B in the micromolar range. The most active of the series is the compound 13, N1-thiocarbamoyl-3-(fur-2′-yl)-5-(4′-fluoro-phenyl)-4,5-dihydro-(1H)-pyrazole, with IC₅₀ 2.75 ± 0.81 μM value and selectivity ratio of 25, which is the best candidate for further investigations.

Full text of DOI:10.1016/j.ejmech.2009.11.003

European Journal of Medicinal Chemistry 45 (2010) 800–804
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and inhibitory activity against human monoamine oxidase
of N1-thiocarbamoyl-3,5-di(hetero)aryl-4,5-dihydro-(1H)-pyrazole derivatives
,
a
a
a
Franco Chimenti a, Simone Carradori a , Daniela Secci , Adriana Bolasco , Bruna Bizzarri ,
*
a
a
b
b
´ ˜
Paola Chimenti , Arianna Granese , Matilde Yanez , Francisco Orallo
a
`
Dipartimento di Chimica e Tecnologie del Farmaco, Universita di Roma, ‘‘La Sapienza’’ P.le Aldo Moro, 5, 00185 Rome, Italy
b Departamento de Farmacolog´ıa and Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur,
E-15782 Santiago de Compostela (La Corun˜a), Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of N1-thiocarbamoyl-3,5-di(hetero)aryl-4,5-dihydro-(1H)-pyrazole derivatives has been
Received 13 March 2009
Received in revised form
29 October 2009
Accepted 2 November 2009
Available online 6 November 2009
synthesized and assayed for their ability to inhibit the activity of the A and B isoforms of human
monoamine oxidase (hMAO). Some of these compounds were endowed with a selective inhibitory
activity against hMAO-B in the micromolar range. The most active of the series is the compound 13, N1-
thiocarbamoyl-3-(fur-20-yl)-5-(40-fluoro-phenyl)-4,5-dihydro-(1H)-pyrazole, with IC50 2.75 ꢀ 0.81
mM
value and selectivity ratio of 25, which is the best candidate for further investigations.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyrazoline
hMAO inhibitors
Parkinson’s disease
N-Thiocarbamoyl
1. Introduction
renewed due to the discovery of an age-related increase in hMAO-B
expression after the 60th year of life especially in glial cells [6,7].
The recent description of the crystal structure of the two iso-
forms of human MAO provides to elucidate the mechanism
underlying and allows investigation of the selective interactions
between these proteins and their ligands, to probe the catalytic
mechanism, and to gain a complete understanding of the phar-
macophoric requirements necessary for the rational design of new
inhibitors [8,9].
Monoamine oxidase (MAO; EC 1.4.3.4) is a flavoprotein located at
the outer membrane of mitochondriain neuronal, glial, and othercells.
It catalyzes oxidative deamination of monoamine and so is a target
enzyme for antidepressant drugs. In addition, it is also responsible for
the biotransformation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyr-
idine (MPTP) into 1-methyl-4-phenylpyridinium a Parkinsonian
producing neurotoxin [1].
MAO exists in two forms, namely MAO-A and MAO-B. Specific
substrates and inhibitors characterize both MAO subtypes. MAO-A
oxidises norepinephrine and serotonin [5-hydroxytryptamine, 5-
HT], whereas MAO-B preferentially deaminates 2-phenylethyl-
amine (2-PEA) and benzylamine. These properties determine the
clinical importance of MAO inhibitors. In fact, interest in selective
MAO-B inhibitors has increased in the last years due to their ther-
apeutic potential in aging related neurodegenerative diseases such
as Alzheimer’s disease (AD) and Parkinson’s disease (PD) and in
selective MAO-A inhibitors due to their therapeutic potential in the
treatment of neurological disorders such as depression [2–5].
Furthermore, interest in selective inhibitors of hMAO-B has
Several
N1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-(1H)-pyr-
azoles have been synthesized in these last years and assayed as
MAO inhibitors against monoamine oxidases isolated and purified
from the mitochondrial extracts of rat liver homogenates and
human platelets [10]. The results showed a good MAO inhibition
and a better selectivity toward isoform A of the enzyme. Further
some authors have also evaluated their antidepressant activity by
the predictive ‘‘forced swimming test’’ and other neurological tests
in mice [11–13]. In addition, other authors recently investigated
some N-alkyl-3,5-di(hetero)aryl-1-thiocarbamoyl-pyrazolines for
their monoamine oxidase inhibition [11,13–15], for their interaction
with rat lung semicarbazide-sensitive amine oxidases (SSAOs) [16]
and as dual agents (MAO-B inhibitors and anti-inflammatory
analgesics) in the treatment of Alzheimer’s disease [17].
On the basis of these studies, in the past we evaluated the
importance of the substitution at N1 of the pyrazoline nucleus with
* Corresponding author. Tel.: þ39 06 49913149; fax: þ39 06 49913772.
E-mail address: simone.carradori@uniroma1.it (S. Carradori).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.003

F. Chimenti et al. / European Journal of Medicinal Chemistry 45 (2010) 800–804
801
Table 1
Ar
Chemical and physical data of derivatives 1–20.
Ar'
Ar0
mp (ꢂC)
ClogP m/z
281.38 86
Yield (%)
N
Comp Ar
N
1
2
3
4
5
6
7
8
Ph
Ph
Ph
Ph
Ph
183–185 3.08
164–165 3.56
183–189 3.64
203–205 1.69
261–265 3.72
40-CH3–Ph
40-Cl–Ph
fur-20-yl
40-F–Ph
295.40 82
315.82 72
271.34 83
313.39 75
301.43 87
305.40 85
329.85 84
333.81 71
350.27 80
304.80 89
285.37 81
289.33 89
277.37 93
276.38 88
270.35 81
288.34 83
304.80 84
260.32 70
276.38 75
H2N
S
Fig. 1. General formula of derivatives 1–20.
40-CH3–Ph
40-CH3–Ph
40-F–Ph
40-Cl–Ph
40-Cl–Ph
40-Cl–Ph
40-Cl–Ph
fur-20-yl
fur-20-yl
fur-20-yl
thiophen-20-yl 196–200 3.55
thiophen-20-yl 203–205 3.22
40-CH3–Ph
40-F–Ph
40-Cl–Ph
an acetyl or a propanoyl group [18,19], or with a thiocarbamoyl one
[20]. This last substitution reduces the steric hindrance in the
catalytic site and strengthens the interaction with the isoalloxazine
nucleus of the cofactor flavin–adenine dinucleotide (FAD). So, new
derivatives with (hetero)aromatic moieties in the 3 and 5 positions
of the pharmacophore (Fig. 1) were designed and investigated for
their influence in modulating hMAO inhibitory activity.
Further we wished to compare our results obtained by using
hMAOs to those evaluated with rMAOs or established animal
models. In fact in the literature, human brain, liver and blood
platelets have been little used as hMAO sources to screen inhibitors,
because of the easy accessibility of rat and bovine brain or liver for
in vitro studies. But species-dependent differences (single amino
acid substitutions may induce deep changes in the secondary and
tertiary structures of these isoforms) have been reported by
different authors for several classes of compounds [21,22]. There-
fore the divergences found, after extrapolating data on MAO inhi-
bition from different species sources to the human enzyme, could
have a deep impact on the development of new and selective hMAO
inhibitors. For these reasons we prepared and assayed a new series
of N1-thiocarbamoyl-3,5-di(hetero)aryl-4,5-dihydro-(1H)-pyrazole
derivatives, which all showed suitable physical properties (calcu-
lated LogP <5), to verify the effects of structural modifications on
inhibition of both human isoforms.
147–149 4.12
264–267 3.79
238–240 4.19
230–238 2.18
144–156 2.18
190–192 1.85
9
10
11
12
13
14
15
16
17
18
19
20
pyrrol-20-yl
40-CH3–Ph
40-F–Ph
thiophen-20-yl 218–220 1.67
thiophen-20-yl pyrrol-20-yl
172–176 1.60
190–210 1.62
209–213 1.78
225–226 2.18
123–130 0.24
pyrrol-20-yl
pyrrol-20-yl
pyrrol-20-yl
pyrrol-20-yl
pyrrol-20-yl
Ph
40-F–Ph
40-Cl–Ph
fur-20-yl
thiophen-20-yl 220–222 1.60
and 2). In particular, the two methylene protons (Ha and Hb) and
methyne proton (Hc), in positions 4 and 5, respectively, of the
dihydro-(1H)-pyrazole ring, give rise to a well defined system of
three double doublets with different J values, indicating not only
the formation of the pyrazoline, but also the exact position of the
C]N double bond.
In MS spectra, the fragment peaks which correspond to loss of
–SH, –NH2, –CSNH2 from the molecular ion are consistent with the
postulated structure. Characteristic M þ 2 isotope peaks are
observed in the mass spectra of the compounds having a halogen or
a sulfur. The IR spectra generally presented C]N stretching bands
in the region around 1600 cmꢁ1 because of the ring closure.
The syntheses of some compounds (1–4, 8, 10) have been
described in previous references [11,13] and were performed with
slight modifications; their analytical and spectral data were in full
agreement with those reported in the literature.
2. Chemistry
The starting 1,3-di(hetero)aryl-2-propen-1-ones (chalcones) (a)
have been synthesized by Claisen–Schmidt condensation at reflux
between substituted aryl or heteroaryl ketones and appropriate
substituted aryl or heteroaryl aldehydes according to methods fully
optimized in our laboratory (KOH/EtOH) [20]. Then, as shown in
Scheme 1, these intermediates were treated with thiosemicarbazide
(molar ratio 1:2) in KOH/EtOH to afford the desired products
without further purifications (1–20) in good yields (70–93%).
The structures of the compounds were confirmed by elemental
analysis, mass spectrometry, IR and 1H NMR spectroscopy (Tables 1
3. Biochemical assay
The potential effects of the test drugs on hMAO activity were
investigated by measuring their effects on the production of
hydrogen peroxide H2O2 from p-tyramine, using the AmplexÒ Red
MAO assay kit (Molecular Probes, Inc., Eugene, Oregon, USA) and
human MAO isoforms in microsomes prepared from insect cells
(BTI-TN-5B1-4) infected with recombinant baculovirus containing
Ar
KOH/EtOH
H
´
H3C
cDNA inserts for hMAO-A or hMAO-B (Sigma–Aldrich Quımica S.A.,
Ar'
+
Alcobendas, Spain). The inhibition of hMAO activity was evaluated
using the above method following the general procedure described
previously by us [23]. The test drugs (new compounds and refer-
ence inhibitors) themselves were unable to directly react with
AmplexÒ Red reagent. In addition, these test drugs had no effects
on the resorufin standard fluorescence curve which clearly indicate
that these compounds do not react with resorufin and do not
quench the fluorescence generated by this product.
The control activity of hMAO-A and hMAO-B (using p-tyramine
as common substrate for both isoforms) was 165 ꢀ 2 pmol of p-
tyramine oxidized to p-hydroxyphenylacetaldehyde/min (n ¼ 20).
O
O
S
H2N
Ar
N
H
NH2
Ar'
Ar'
N
Ar
N
S
O
KOH/EtOH
H2N
4. Results and discussion
a
1-20
The hMAO-A and hMAO-B inhibition data are reported in Table 3
Scheme 1. General synthetic pathway of derivatives 1–20.
together with the selectivity index (SI ¼ hMAO-B selectivity ratios

802
F. Chimenti et al. / European Journal of Medicinal Chemistry 45 (2010) 800–804
Table 2
Table 3
1H NMR of new derivatives.
IC50 values and hMAO-B selectivity ratios ([IC50(hMAO-A)]/[IC50(hMAO-B)]) for the
inhibitory effects of tested drugs (new compounds and reference inhibitors) on the
enzymatic activity of human recombinant MAO isoforms expressed in baculovirus
infected BTI insect cells.
Ha
Hb
Hc
Ar
N
Ar'
Compound
IC50 mM hMAO-A
IC50
m
M hMAO-B
Ratio
N
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
***
***
***
48.31 ꢀ 2.86
32.81 ꢀ 1.43
33.29 ꢀ 1.25
32.48 ꢀ 1.64
7.18 ꢀ 0.55
18.26 ꢀ 0.98
23.21 ꢀ 1.07
12.84 ꢀ 0.61
16.33 ꢀ 2.22
39.37 ꢀ 1.65
29.38 ꢀ 2.87
54.51 ꢀ 4.56
2.75 ꢀ 0.11
**
>2.1c
>3c
S
H2N
>3c
54.65 ꢀ 3.12
1.7
Compound 1H NMR (
5a
d
ppm)
***
***
***
***
>14c
>5.5c
>4.3c
>7.8c
>6.1c
1.7
2.41 (s, 3H, ArCH3), 3.18–3.20 (dd, Jab ¼ 17.44 Hz, Jac ¼ 3.08 Hz, 1H,
Ha), 3.80–3.83 (dd, Jab ¼ 17.45 Hz, Jbc ¼ 11.13 Hz, 1H, Hb), 6.00–6.02
(dd, Jac ¼ 3.08 Hz, Jbc ¼ 11.12 Hz, 1H, Hc), 6.98–7.02 (m, 2H, Ar),
7.20–7.24 (m, 6H, Ar), 7.60 (s, 2H, NH2, D2O exch.).
2.34 (s, 3H, ArCH3), 3.06–3.08 (dd, Jab ¼ 17.34 Hz, Jac ¼ 3.00 Hz, 1H,
Ha), 3.79–3.86 (dd, Jab ¼ 17.36 Hz, Jbc ¼ 11.04 Hz, 1H, Hb), 6.19–6.22
(dd, Jac ¼ 3.01 Hz, Jbc ¼ 11.04 Hz, 1H, Hc), 6.91–6.98 (m, 2H,
thiophene), 7.26–7.28 (m, 1H, thiophene), 7.34–7.36 (m, 2H, Ar),
7.78–7.80 (m, 2H, Ar), 8.03 (s, 2H, NH2, D2O exch.).
3.34–3.39 (dd, Jab ¼ 17.48 Hz, Jac ¼ 3.08 Hz, 1H, Ha), 3.76–3.83 (dd,
Jab ¼ 17.48 Hz, Jbc ¼ 11.12 Hz, 1H, Hb), 6.36–6.39 (dd, Jac ¼ 3.09 Hz,
Jbc ¼ 11.12 Hz, 1H, Hc), 6.94–6.98 (m, 2H, thiophene), 7.05–7.06 (m,
1H, thiophene), 7.12–7.20 (m, 4H, Ar), 7.75 (s, 2H, NH2, D2O exch.).
3.18–3.20 (dd, Jab ¼ 17.52 Hz, Jac ¼ 3.02 Hz, 1H, Ha), 3.81–3.84 (dd,
Jab ¼ 17.52 Hz, Jbc ¼ 11.16 Hz, 1H, Hb), 6.02–6.04 (dd, Jac ¼ 3.04 Hz,
Jbc ¼ 11.16 Hz, 1H, Hc), 7.02–7.04 (m, 3H, Ar), 7.23–7.24 (m, 1H, Ar),
7.41–7.43 (m, 2H, Ar), 7.64–7.66 (m, 2H, Ar), 8.09 (s, 2H, NH2, D2O
exch.).
3.38–3.40 (dd, Jab ¼ 17.48 Hz, Jac ¼ 3.13 Hz, 1H, Ha), 3.69–3.74 (dd,
Jab ¼ 17.48 Hz, Jbc ¼ 11.18 Hz, 1H, Hb), 5.88–5.90 (dd, Jac ¼ 3.13 Hz,
Jbc ¼ 11.19 Hz, 1H, Hc), 6.54–6.57 (m, 1H, pyrrole), 7.53–7.55 (m, 2H,
pyrrole), 7.86–7.94 (m, 4H, Ar), 7.99 (s, 2H, NH2, D2O exch.), 10.64
(bs, 1H, NH, D2O exch.).
***
68.87 ꢀ 3.61b
6b
50.14 ꢀ 4.96b
1.7
1.2
25
nd
2.1
nd
65.84 ꢀ 3.14
69.45 ꢀ 3.25a
**
69.97 ꢀ 3.78b
33.26 ꢀ 1.99
7a
**
**
***
***
**
**
**
17
18
19
20
nd
nd
>1.4c
nd
9b
69.38 ꢀ 4.23
**
**
Clorgyline
R-(ꢁ)-deprenyl
Iproniazide
Moclobemide
Isatin
4.46 ꢀ 0.32 nMa
61.35 ꢀ 1.13
m
M
0.000073
3431
0.87
<0.36d
>5.3c
67.25 ꢀ 1.02
6.56 ꢀ 0.76
361.38 ꢀ 19.3
***
m
Ma
19.60 ꢀ 0.86 nM
mM
7.54 ꢀ 0.36
mM
m
M
*
11b
12b
18.75 ꢀ 1.24
mM
Each IC50 value is the mean ꢀ S.E.M. from five experiments (n ¼ 5).
*Inactive at 1 mM (highest concentration tested).
**Inactive at 100
compounds precipitate.
***100 M inhibits enzymatic activity around (by approximately) 40–45%. At higher
mM (highest concentration tested). At higher concentrations the
2.24 (s, 3H, ArCH3), 2.94–2.98 (dd, Jab ¼ 17.44 Hz, Jac ¼ 3.10 Hz, 1H,
Ha), 3.82–3.86 (dd, Jab ¼ 17.44 Hz, Jbc ¼ 11.12 Hz, 1H, Hb), 5.85–5.88
(dd, Jac ¼ 3.09 Hz, Jbc ¼ 11.12 Hz, 1H, Hc), 6.65–6.67 (m, 1H, furan),
6.97–7.11 (m, 5H, Ar and furan), 7.87–7.89 (m, 1H, Ar), 7.90 (s, 2H,
NH2, D2O exch.).
m
concentrations the compounds precipitate.
a
Level of statistical significance: P < 0.01 versus the corresponding IC50 values
obtained against hMAO-B, as determined by ANOVA/Dunnett’s.
13b
14b
15b
3.00–3.02 (dd, Jab ¼ 17.40 Hz, Jac ¼ 3.00 Hz, 1H, Ha), 3.84–3.89 (dd,
Jab ¼ 17.40 Hz, Jbc ¼ 11.19 Hz, 1H, Hb), 5.89–5.93 (dd, Jac ¼ 3.00 Hz,
Jbc ¼ 11.19 Hz, 1H, Hc), 6.66–6.67 (m, 1H, furyl), 7.03–7.15 (m, 5H, Ar
and furan), 7.91 (s, 1H, Ar), 8.01 (s, 2H, NH2, D2O exch.).
3.28–3.30 (dd, Jab ¼ 17.38 Hz, Jac ¼ 3.03 Hz, 1H, Ha), 4.01–4.03 (dd,
Jab ¼ 17.38 Hz, Jbc ¼ 11.29 Hz, 1H, Hb), 5.49–5.52 (dd, Jac ¼ 3.03 Hz,
Jbc ¼ 11.29 Hz, 1H, Hc), 6.12–6.17 (m, 2H, furan), 6.53–7.55 (m, 1H,
furan), 6.97–6.99 (m, 3H, thiophene), 7.70 (s, 2H, NH2, D2O exch.).
3.39–3.43 (dd, Jab ¼ 17.45 Hz, Jac ¼ 3.06 Hz, 1H, Ha), 3.75–3.81 (dd,
Jab ¼ 17.44 Hz, Jbc ¼ 11.10 Hz, 1H, Hb), 6.06–6.09 (dd, Jac ¼ 3.06 Hz,
Jbc ¼ 11.10 Hz, 1H, Hc), 6.66 (s, 1H, thiophene), 7.16–7.19 (m, 2H,
thiophene), 7.58–7.59 (m, 2H, pyrrole), 7.76–7.78 (m, 1H, pyrrole),
8.00 (bs, 2H, NH2, D2O exch.), 10.74 (bs, 1H, NH, D2O exch.).
3.06–3.11 (dd, Jab ¼ 17.42 Hz, Jac ¼ 3.10 Hz, 1H, Ha), 4.38–4.39 (dd,
Jab ¼ 17.40 Hz, Jbc ¼ 11.14 Hz, 1H, Hb), 6.11–6.13 (dd, Jac ¼ 3.10 Hz,
Jbc ¼ 11.15 Hz, 1H, Hc), 6.89–7.02 (m, 2H, pyrrole), 7.19–7.27 (m, 4H,
Ar and pyrrole), 7.39–7.43 (m, 2H, Ar), 7.75 (bs, 2H, NH2, D2O exch.),
11.85 (bs, 1H, NH, D2O exch.).
3.08–3.11 (dd, Jab ¼ 17.45 Hz, Jac ¼ 3.02 Hz, 1H, Ha), 4.25–4.29 (dd,
Jab ¼ 17.45 Hz, Jbc ¼ 11.10 Hz, 1H, Hb), 6.09–6.12 (dd, Jac ¼ 3.02 Hz,
Jbc ¼ 11.10 Hz, 1H, Hc), 6.92–7.03 (m, 4H, pyrrole and Ar), 7.40–7.50
(m, 3H, Ar), 7.70 (s, 2H, NH2, D2O exch.), 11.69 (bs, 1H, NH, D2O
exch.).
3.18–3.20 (dd, Jab ¼ 17.48 Hz, Jac ¼ 3.11 Hz, 1H, Ha), 4.20–4.24 (dd,
Jab ¼ 17.48 Hz, Jbc ¼ 11.13 Hz, 1H, Hb), 6.07–6.10 (dd, Jac ¼ 3.12 Hz,
Jbc ¼ 11.14 Hz, 1H, Hc), 6.94–7.00 (m, 2H, pyrrole), 7.30–7.32 (m, 2H,
Ar and pyrrole), 7.40–7.44 (m, 3H, Ar), 7.79 (s, 2H, NH2, D2O exch.),
11.70 (bs, 1H, NH, D2O exch.).
b
Level of statistical significance: P < 0.05 versus the corresponding IC50 values
obtained against hMAO-B, as determined by ANOVA/Dunnett’s.
c
Values obtained under the assumption that the corresponding IC50 against
hMAO-A or hMAO-B is the highest concentration tested (100 mM).
d
Value obtained under the assumption that the corresponding IC50 against
hMAO-B is the highest concentration tested (1 mM).
[IC50(hMAO-A)]/[IC50(hMAO-B)]). Enzymatic assays revealed that all
tested compounds were weak to moderate hMAO inhibitors at low
micromolar concentrations (Table 3).
We can observe that the presence of a fluorine atom in the 40-
position of the 5-phenyl substituent on the pyrazoline ring is
important for the activity. In fact, more potent active and B-
selective compounds are derivatives 5, 9, and 13 with IC50 values
16b
17b
18b
ranging between 2.75 ꢀ 0.81
selectivity ranging between >6.1 and 25. The most active of the
M and selectivity ratio 25)
m
M and 16.33 ꢀ 2.22
mM and B-
series is compound 13 (IC50 2.75 ꢀ 0.81
m
with a 40-fluorophenyl substituent in 5 position and a fur-20-yl
group in 3-position of the pyrazoline ring. The concurrent presence
of heteroaromatic substituent in 3- and 5-position of the pyrazo-
line ring in compounds 15 and 19 leads to a decrease in the
potency and selectivity for the hMAO-B activity, while the presence
of a 40-methylphenyl and a 40-fluorophenyl, in the same positions
of the ring, preserves the activity and a discrete B-selectivity
(compound 5).
19b
20b
3.08–3.11 (dd, Jab ¼ 17.45 Hz, Jac ¼ 3.02 Hz, 1H, Ha), 4.25–4.29 (dd,
Jab ¼ 17.45 Hz, Jbc ¼ 11.10 Hz, 1H, Hb), 6.09–6.12 (dd, Jac ¼ 3.02 Hz,
Jbc ¼ 11.10 Hz, 1H, Hc), 6.92–7.03 (m, 4H, pyrrole and Ar), 7.40–7.50
(m, 3H, Ar), 7.70 (s, 2H, NH2, D2O exch.), 11.69 (bs, 1H, NH, D2O exch.).
3.08–3.11 (dd, Jab ¼ 17.45 Hz, Jac ¼ 3.02 Hz, 1H, Ha), 4.25–4.29 (dd,
Jab ¼ 17.45 Hz, Jbc ¼ 11.10 Hz, 1H, Hb), 6.09–6.12 (dd, Jac ¼ 3.02 Hz,
Jbc ¼ 11.10 Hz, 1H, Hc), 6.92–7.03 (m, 4H, pyrrole and Ar), 7.40–7.50
(m, 3H, Ar), 7.70 (s, 2H, NH2, D2O exch.), 11.69 (bs, 1H, NH, D2O exch.).
Furthermore we can observe that for compounds 8, 9, 10, and 11,
which bear a 40-chlorophenyl substituent in 3 position, the hMAO-B
inhibitory activity increase with the presence of small substituent
in 5 position of the pyrazoline nucleus (compounds 8 and 9).
On the basis of these considerations we can indicate compound
13, N1-thiocarbamoyl-3-(fur-20-yl)-5-(40-fluoro-phenyl)-4,5-dihy-
dro-(1H)-pyrazole as the best candidate for further investigations.
a
CDCl3.
DMSO-d6.
b

F. Chimenti et al. / European Journal of Medicinal Chemistry 45 (2010) 800–804
803
Furthermore comparing some N1-acetyl or N1-propanoyl
derivatives, synthesized in our previous papers [18,19], with the
new N1-thiocarbamoyl derivatives bearing the same substituent
(H, H and Cl, Cl and F, Cl and CH3, CH3 and F, Cl and Cl) on the 3,5-
diaryl moiety, it is possible to highlight that this more polarizable
group (the N1-thiocarbamoyl) led to a slight decrease in MAO
inhibitory activity and to a better selectivity toward hMAO-B
isoform.
In the reversibility and irreversibility tests, hMAO-B inhibition
was irreversible in presence of the compounds more active (5 and
13) as shown by the lack enzyme activity restoration after repeated
washing. Similar results were obtained for R-(ꢁ)-deprenyl (Table
4). However, significant recovery of hMAO-B activity was observed
after repeated washing of isatin, indicating that this drug is
a reversible inhibitor of this hMAO isoform.
Although we have not investigated in this study the mechanism
by which the compounds 5 and 13 act as irreversible inhibitors of
hMAO-B activity, it is possible that these compounds, like most
MAO irreversible inhibitors, establish a covalent interaction with
the active center of the enzyme [24]. Other possibility is that the
compounds 5 and 13 are, like some MAO irreversible inhibitors
(e.g.: R-(ꢁ)-deprenyl), suicide inhibitors, i.e., act as a substrate for
the target enzyme, which finally generates a new compound that
irreversibly inhibits MAO activity [25].
reflux with thiosemicarbazide (100 mmol) for 24 h. The desired
product precipitated from reaction mixture after cooling, was
filtered off under vacuum and crystallized from suitable solvent
(ethanol or 2-propanol) and dried.
5.3. Determination of MAO isoform activity
The effects of the test compounds on hMAO isoform enzymatic
activity were evaluated by a fluorimetric method following the
experimental protocol previously described by us [23].
Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4)
containing the test drugs (new compounds or reference inhibitors)
in various concentrations and adequate amounts of recombinant
hMAO-A or hMAO-B required and adjusted to obtain in our
experimental conditions the same reaction velocity, i.e., to oxidize
(in the control group) the same concentration of substrate:
165 pmol of p-tyramine/min (hMAO-A: 1.1
activity: 150 nmol of p-tyramine oxidized to p-hydroxy-
phenylacetaldehyde/min/mg protein; hMAO-B: 7.5 protein;
mg protein; specific
mg
specific activity: 22 nmol of p-tyramine transformed/min/mg
protein) were incubated for 15 min at 37 ꢂC in a flat-black-bottom
96-well microtestÔ plate (BD Biosciences, Franklin Lakes, NJ, USA)
placed in the dark fluorimeter chamber. After this incubation
period, the reaction was started by adding (final concentrations)
200 m
M AmplexÒ Red reagent, 1 U/mL horseradish peroxidase and
1 mM p-tyramine. The production of H2O2 and, consequently, of
resorufin was quantified at 37 ꢂC in a multidetection microplate
fluorescence reader (FLX800Ô, Bio-TekÒ Instruments, Inc.,
Winooski, VT, USA) based on the fluorescence generated (excita-
tion, 545 nm, emission, 590 nm) over a 15 min period, in which the
fluorescence increased linearly.
Control experiments were carried out simultaneously by
replacing the test drugs (new compounds and reference inhibitors)
with appropriate dilutions of the vehicles. In addition, the possible
capacity of the above test drugs to modify the fluorescence
generated in the reaction mixture due to non-enzymatic inhibition
(e.g., for directly reacting with AmplexÒ Red reagent) was deter-
mined by adding these drugs to solutions containing only the
AmplexÒ Red reagent in a sodium phosphate buffer.
5. Experimental protocols
5.1. Chemistry
The chemicals, solvents for synthesis and spectral grade solvents
were purchased from Aldrich (Italy) without further purification.
Melting points (uncorrected) were determined automatically on an
FP62 apparatus (Mettler-Toledo). 1H NMR spectra were recorded at
400 MHz on a Bruker spectrometer using DMSO-d6 or CDCl3 as the
solvent. Chemical shifts are expressed as d units (parts per millions)
relative to the solvent peak. Coupling constants J are valued in Hertz
(Hz). IR spectra were registered on a Perkin Elmer FT-IR Spec-
trometer Spectrum 1000 in KBr. Elemental analysis for C, H, and N
were recorded on a Perkin–Elmer 240 B microanalyzer and the
analytical results were within ꢀ0.4% of the theoretical values for all
compounds. All reactions were monitored by TLC performed on
0.2 mm thick silica gel plates (60 F254 Merck). Electron ionization
(EI) mass spectra were obtained by a Fisons QMD 1000 mass
The specific fluorescence emission (used to obtain the final
results) was calculated after subtraction of the background activity,
which was determined from vials containing all components except
the hMAO isoforms, which were replaced by a sodium phosphate
buffer solution.
spectrometer (70 eV, 200 m
A, ion source temperature of 200 ꢂC).
The samples were introduced directly into the ion source. Lip-
ophilicity parameter, CLogP, was calculated for each molecule by
using ChemDraw ultra 8.0.
5.4. Reversibility and irreversibility experiments
To evaluate whether some of the tested compounds (5 and 13)
are reversible or irreversible hMAO-B inhibitors, an effective
centrifugation–ultrafiltration method (so-called repeated washing)
was used [23].
5.2. General procedure for the synthesis of derivatives 1–20
The appropriate chalcone (a) (50 mmol) was dissolved in
Briefly, adequate amounts of the recombinant hMAO-B were
incubated together with a single concentration (see Table 4) of the
test drugs or the reference inhibitor R-(ꢁ)-deprenyl in a sodium
phosphate buffer (0.05 M, pH 7.4) for 15 min at 37 ꢂC.
100 mL of a mixture of KOH in ethanol and vigorously stirred at
Table 4
Reversibility and irreversibility of hMAO-B inhibition.
After this incubation period, an aliquot was stored at 4 ꢂC and
used for subsequent measurement of hMAO-B activity (see the
subsection determination of MAO activity). The remaining incu-
Compound
% hMAO-B inhibition
Before washing
After repeated
washing
bated sample (300 ml) was placed in an Ultrafree-0.5 centrifugal
R-(ꢁ)-deprenyl (20 nM)
51.45 ꢀ 2.69
84.36 ꢀ 5.24
54.46 ꢀ 3.16
53.34 ꢀ 3.42
52.05 ꢀ 2.88
16.62 ꢀ 1.18a
57.58 ꢀ 3.49
47.83 ꢀ 3.14
tube (Millipore, Billerica, USA) with a 30KDa Biomax membrane in
the middle of the tube and centrifuged (9000g, 20 min, 4 ꢂC) in
a centrifuge (J2-MI, Beckman Instruments, Inc., Palo Alto, California,
USA). The enzyme retained in the 30 KDa membrane was resus-
pended in sodium phosphate buffer at 4 ꢂC and centrifuged again
two successive times. After the third centrifugation, the enzyme
Isatin (50 mM)
5 (10
13 (3
m
m
M)
M)
Each value is the mean ꢀ S.E.M. from five experiments (n ¼ 5).
a
Level of statistical significance: P < 0.01 versus the corresponding % hMAO-B
inhibition before washing, as determined by ANOVA/Dunnett’s.

804
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Acknowledgment
This work was supported by grants from MURST (Italy), Minis-
terio de Sanidad y Consumo (Spain; FISS PI061537) and Consellerıa
´
´
de Innovacion e Industria de la Xunta de Galicia (Spain; INCI-
TE07PXI203039ES, INCITE08E1R203054ES and 08CSA019203PR).
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´
´
Educacion y Ordenacion Universitaria de la Xunta de Galicia (Spain)
for giving him financial support to intensify his research activity
and to reduce his teaching during the academic year 2007–2008
¨ ¨
[17] N. Go¨khan, S. Yabanoglu, E. Ku¨peli, U. Salgin, G. OOzgenUçar, E. Yes¸ilada,
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(2006) 1411–1428.
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[Programa de promocion de intensification de la actividad inves-
tigadora en el sistema Universitario de Galicia (SUG)].
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F. Rossi, P. Turini, F. Ortuso, S. Alcaro, M.C. Cardia, Eur. J. Med. Chem. 43
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Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2009.11.003.
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