Synthesis of new 3-aryl-4,5-dihydropyrazole-1-carbothioamide derivatives. An investigation on their ability to inhibit monoamine oxidase

Article abstract of DOI:10.1016/j.ejmech.2010.07.009

Some differently substituted 3-aryl-4,5-dihydropyrazoles-1-carbothioamides have been synthesised with the aim to investigate their monoamine oxidase inhibitory activity. The chemical structures of the compounds have been characterized by means of their IR, ¹H NMR, ¹³C NMR spectroscopic data and elemental analyses. All the active compounds showed a selective activity towards the B isoform of the enzyme, regardless of the substitution on the heterocyclic ring. The inhibition of the enzymatic activity was measured on human recombinant MAO isoforms, expressed in baculovirus infected BTI insect cells. Docking experiments were carried out with the aim to rationalize the mechanism of inhibition of the most active and selective compound.

Full text of DOI:10.1016/j.ejmech.2010.07.009

European Journal of Medicinal Chemistry 45 (2010) 4490e4498
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of new 3-aryl-4,5-dihydropyrazole-1-carbothioamide derivatives.
An investigation on their ability to inhibit monoamine oxidase
,
c
a
b
b
c
E. Maccioni a, S. Alcaro b , F. Orallo , M.C. Cardia , S. Distinto , G. Costa , M. Yanez ,
*
M.L. Sanna a, S. Vigo a, R. Meleddu a, D. Secci d
a Dipartimento Farmaco Chimico Tecnologico, Via Ospedale 72 e 09124 Cagliari, Italy
b Dipartimento di Scienze Farmacobiologiche, Università “Magna Græcia” di Catanzaro, Complesso “Ninì Barbieri”, 88021, Roccelletta di Borgia, Catanzaro, Italy
c Department of Pharmacology, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain
d Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P. le A. Moro 5, 00185 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Some differently substituted 3-aryl-4,5-dihydropyrazoles-1-carbothioamides have been synthesised
with the aim to investigate their monoamine oxidase inhibitory activity. The chemical structures of the
compounds have been characterized by means of their IR, 1H NMR, 13C NMR spectroscopic data and
elemental analyses. All the active compounds showed a selective activity towards the B isoform of the
enzyme, regardless of the substitution on the heterocyclic ring. The inhibition of the enzymatic activity
was measured on human recombinant MAO isoforms, expressed in baculovirus infected BTI insect cells.
Docking experiments were carried out with the aim to rationalize the mechanism of inhibition of the
most active and selective compound.
Received 30 March 2010
Received in revised form
2 June 2010
Accepted 5 July 2010
Available online 17 July 2010
Keywords:
Dihydropyrazole synthesis and
characterization
Ó 2010 Elsevier Masson SAS. All rights reserved.
Monoamine oxidase selective inhibition
Parkinson disease
Docking
1. Introduction
Furthermore,theactivityofMAO-BisenhancedbyagingandinAD
patients [3,5]. In addition to this, the deamination reaction, promoted
Monoamine oxidase are key role enzymes in the catabolism of
amines like dopamine (DA), norepinephrine (NE), epinephrine,
serotonin (5HT), and 2-phenylethylamine (PEA) [1].
Two isoforms of the enzyme, differing for substrates and
selectivity of inhibitors [2], are known, named MAO-A and MAO-B.
5HT and NE are preferentially deaminated by the A isoform, while
by MAO-B, leads to the production of hydrogen peroxide and to other
reactive oxygen species responsible for neurological damaging [7e9].
Also, in the case of PD, a correlation between free radical production
and development of the pathology has been observed [10].
Thus, neurodegenerative disorders can be associated with the
production of oxidative stress, with an increased MAO-B activity,
and with a decrease in the elimination rate of free radical species
[5e14]. On the contrary, MAO-A does not increase with age, sug-
gesting that a totally independent mechanism regulates the
expression of the two enzymatic isoforms [12,14].
We have recently designed and synthesised some 1-thio-
carbamoyl-3,5-diaryl-4,5-dihydro-(1H)-pyrazole (I) and 2-thiazolyl
hydrazones (II), both, highly active and selective towards MAO-B
isoform (Fig. 1) [15,16]. The synthesis, the MAO inhibition, and the
pharmacophoric features of a series of 1-acetyl-3-aryl-4,5-dihydro-
1(H)-pyrazoles have been recently reported by some of us [17]. In
particular, it has been observed that the absence of substituents in
position 5 of the heterocyclic ring, generally leads to a decrease of
MAO-A inhibition potency.
b-phenylethylamine and benzylamine are MAO-B substrates.
Moreover, in mammals the inhibition of MAO-B leads to an
increase of the DA and 5HT levels as well as to a neuroprotective
effect [3]. Both MAO isoforms are important in the metabolism of
monoamine neurotransmitters and, as a result, MAO inhibitors
(MAOi) are studied for the treatment of several psychiatric and
neurological disorders. In particular, MAO-B inhibitors are coad-
juvant in the treatment of both Parkinson’s (PD) [4] and Alz-
heimer’s diseases (AD) [5], while MAO-A inhibitors are used as
antidepressant and anxiolytic drugs [6].
In order to observe whether this effect is also measurable in
series of 1-thiocarbamoyl-3-aryl-4,5-dihydro-1(H)-pyrazoles
* Corresponding author. Tel.: þ39 0961 3694197.
E-mail address: alcaro@unicz.it (S. Alcaro).
a
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.009

E. Maccioni et al. / European Journal of Medicinal Chemistry 45 (2010) 4490e4498
4491
heterocycle, a multiplet in the region 3.58e3.81 ppm and a set of
doublet of doublets, ranging between 4.07 and 4.40 ppm (JAX ¼ 4.6,
JBX ¼ 10.4, JAB ¼ 11.9). The methyl group appears as a doublet
in the region 1.25e1.33 ppm (J ¼ 7.3).
In order to have a better characterization of these compounds,
we performed 1H NMR (400 MHz), 13C NMR and heteronuclear
single quantum correlation (HSQC) of compounds 21 and 25.
The HSQC was particularly useful to confirm the assigned
structure. In fact, the correlation between the C4 of the dihy-
dropyrazole ring and its proton (multiplet in the region around
3.81 ppm) and the C5 of the heterocyclic ring and the methylene
protons (doublet of doublets in the region around 4.20 ppm) has
been evidenced.
All the synthesised compounds show an intense band in the
region 1057e1120 cmꢀ1, due to the C]S stretching. The C]N
stretching band in the region 1513e1595 cmꢀ1 is detectable and
confirms the formation of the pyrazoline ring. A medium intensity
band in the region between 1127 and 1248 cmꢀ1 can be assigned to
the CeN stretching. Compounds 1e8, 17, 19, 21, 23, 25 show two
adsorption bands, around 3120 cmꢀ1 and 3350 cmꢀ1 respectively,
due to the symmetric and asymmetric stretching of the NH2 group.
Compounds 9e16, 18, 20, 22, 24 show a sharp band around
3240 cmꢀ1 due to the NH stretching of the monosubstituted thio-
carbamoyl group.
Fig. 1. Comparison between compounds 1e25 and 1-thiocarbamoyl-3,5-diaryl-4,5-
dihydro-(1H)-pyrazole (I) and 2-thiazolyl hydrazones (II).
analogues, we have just synthesised a set of compounds whose
activity towards the two isoforms of MAO has been tested.
2. Results and discussion
The tested compounds (new compounds and reference inhibi-
tors) themselves were unable to directly react with the AmplexÒ
Red reagent, which indicates that these molecules do not interfere
with the measurements. On the other hand, the control activity of
hMAO-A and hMAO-B (using p-tyramine as common substrate for
both isoforms) was 165 ꢁ 2 p mol of p-tyramine oxidized to
p-hydroxyphenylacetaldehyde/min (n ¼ 20).
To achieve a better understanding of the structural requirements
for both inhibition and selectivity towards the two MAO isoforms,
we have synthesised a series of 1-(N-methyl)thiocarbamoyl-3-aryl-
4,5-dihydro-1(H)-pyrazoles and 1-thiocarbamoyl-3-aryl-4,5-dihy-
dro-1(H)-pyrazoles indicated as compounds 1e25 (Table 1).
All the synthesised compounds bear a differently substituted
aromatic group in position 3 of the heterocyclic ring. In particular,
the substituents which demonstrated as leading to the best
activity/selectivity in previously synthesised analogues were
introduced, namely thiophene-2-yl, 4-methoxyphenyl, 4-methyl-
phenyl, 4-fluorophenyl, and 4-chlorophenyl. The effect of methyl
group at 4th position (16e25) of heterocyclic ring and methyl group
in thiocarbamoyl-N at first position (9e16, 18, 20, 22, and 24) of
heterocyclic ring has also been investigated.
As shown in Table 1, the new compounds (7e10, 12, 14, 15, 18, 19
and 24) were selective hMAO-B inhibitors, although less effective
than the selective MAO-B reference inhibitor R-(ꢀ)-deprenyl.
The IC50 values ranged between 13.70
ꢁ
0.95 mM and
53.38 ꢁ 0.92
mM.
All the other new compounds had no significant effects on
hMAO-A and hMAO-B enzymatic activity.
Nevertheless, it is noteworthy that the presence of
a methoxyphenyl moiety in position 3 of the pyrazoline ring
(compounds 7, 12, 14, 18, and 19) always leads to active
compounds regardless of the introduction of methyl groups both
The synthesis of compounds 1e25 was performed by slightly
modifying the methods reported in literature [18], and is reported
in Scheme 1.
on the carbothioamide function and in position
4 of the
The procedure consists of two steps. The first step is the
synthesis of the Mannich bases by reaction of the appropriate
acetophenones (compounds 1e15) or propiophenones (compounds
16e25) with paraformaldehyde and dimethylamine hydrochloride.
Higher yields can be achieved by carefully measuring the amounts
of hydrochloric acid and ethanol. Best results are observed
when the minimum quantity of both ethanol and hydrochloric acid
are used. Generally methyl aryl ketones give higher yields (75e85%)
in comparison with ethyl aryl ketones (45e65%).
The second step of the synthetic route is the condensation of the
Mannich base with either thiosemicarbazide or methyl-
thiosemicarbazide to generate the required pyrazolines (1e25).
The structure of compounds 1e25 has been determined by
means of analytical and spectroscopical methods. NMR spectros-
copy was particularly efficient in the characterization of all the
compounds.
heterocyclic nucleus. Interestingly, the introduction of a 4-
methylphenyl moiety in position 3 of the pyrazoline ring leads to
active compounds only when position 4 of the heterocycle is
unsubstituted. A similar behaviour could be observed in the case
of compound 9, bearing a 4-fluorophenyl moiety in position 3 of
the pyrazoline, while 3-(4-chlorophenyl) pyrazoles are active
only when both position 4 and the thiocarbamoyl group are
methylated.
On the basis of the above, the methoxyphenyl moiety appears to
be the most efficient substitution for position 3 of the pyrazoline,
while the influence on the biological activity of both position 4 and
the thiocarbamoyl moiety is less predictable and related to the
nature of the substituent in position 3 of the pyrazoline ring.
In order to explore the nature of the ligandereceptor interac-
tions, we have carried out docking experiments into the active site
of both MAO A and B isoforms with AutoDock Vina [19]. We have
focused on the compound 14, which shows the highest selective
MAO-B inhibitory activity. The study confirmed the selectivity of
compound 14 for MAO-B isoform with a consistent difference in
the predicted affinity estimated to be ꢀ7.4 and ꢀ8.3 kcal/mol
respectively for MAO-A and B. According to Vina best scored
poses, the most stable complex configurations are depicted in
In the case of compounds 1e15 two sets of signals, in the region
3.26e3.46 ppm respectively have been detected. These signals are
false triplets (“t”), due to the similarity of the coupling constants
and are diagnostic of the formation of the dihydropyrazole ring.
As regards compounds 16e25, the 1H NMR showed three sets of
signals, corresponding to the protons in the position 4 and 5 of the

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E. Maccioni et al. / European Journal of Medicinal Chemistry 45 (2010) 4490e4498
Table 1
Structures and biological activity of compounds 1e25.
Compound
Ar
R
R0
H
MAO-A (IC50
)
MAO-B (IC50
**
)
SI A/B
/
1
H
**
2
3
4
5
H
H
H
H
H
H
H
H
**
**
**
**
**
**
**
/
/
/
/
**
**
6
7
8
9
H
H
H
H
H
**
**
**
**
/
H
15.47 ꢁ 0.61
18.14 ꢁ 0.73
50.95 ꢁ 2.38
m
m
m
M
M
M
>6.46b
>5.51b
>1.96
H
CH3
10
11
12
H
H
H
CH3
CH3
CH3
**
**
**
53.38 ꢁ 0.92
m
M
>1.87
**
/
71.30 ꢁ 2.26
m
M
>1.40
13
14
15
16
H
CH3
CH3
CH3
CH3
**
**
/
H
**
13.70 ꢁ 0.95
16.77 ꢁ 0.81
***
m
m
M
M
>7.30
>5.96
/
H
***
**
CH3

E. Maccioni et al. / European Journal of Medicinal Chemistry 45 (2010) 4490e4498
4493
Table 1 (continued)
Compound
Ar
R
R0
H
MAO-A (IC50
***
)
MAO-B (IC50
)
SI A/B
/
17
18
19
20
21
22
23
CH3
***
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
**
49.44 ꢁ 2.70
m
m
M
M
>2.02b
***
***
**
84.64 ꢁ 1.64
>1.18b
CH3
H
***
***
***
/
/
/
/
CH3
H
**
**
***
24
25
CH3
CH3
CH3
H
**
34.81 ꢁ 1.91
m
m
M
M
>2.87b
***
***
/
Clorgyline
4.46 ꢁ 0.32 nMa
61.35 ꢁ 1.13
0.000073
3431.12
0.87
R-(ꢀ)-deprenyl
67.25 ꢁ 1.02
m
M
Ma
19.60 ꢁ 0.86 nM
Iproniazide
6.56 ꢁ 0.76
m
7.54 ꢁ 0.36
mM
Moclobemide
361.38 ꢁ 19.37
m
M
*
<0.36b
Results are mean ꢁ S.E.M. from five experiments. Level of statistical significance: aP < 0.01 versus the corresponding IC50 values obtained against MAO-B, as determined by
ANOVA/Dunnett’s.
*Inactive at 1 mM (highest concentration tested).
**Inactive at 100
mM (highest concentration tested). At higher concentration the compounds precipitate.
***100 M inhibits the corresponding MAO activity by approximately (around) 40 or 50%. At higher concentration the compounds precipitate.
m
a
IC50 values and MAO-B selectivity ratios (SI A/B) [IC50 (MAO-A)]/[IC50 (MAO-B)] for the inhibitory effects of test drugs (new compounds and reference inhibitors) on the
enzymatic activity of human recombinant MAO isoforms expressed in baculovirus infected BTI insect cells.
b
Values obtained under the assumption that the corresponding IC50 against MAO-A is the highest concentration tested (100 mM).
Fig. 2. The complexes are stabilized by hydrogen bonds with
a tyrosine residue of the “aromatic cage” [20], water molecules
adjacent to FAD cofactor, and some hydrophobic interactions with
the residues in the binding pocket. The complex [MAO-A:14] is
stabilized by hydrophobic interaction with Ile180, Phe208, Ile325,
Leu337, Ile335 and hydrogen bonds between N pyrazole moiety
and HOH746, the S and Tyr444, NH and HOH805. While, in the
isoform B, the aryl moiety of compound 14 is located between the
“entrance” and “catalytic” cavities, separated by the residues
Ile199-Tyr326 [21]. This complex resulted stabilized by hydro-
phobic contacts with Ile199, Tyr326 and Leu171, instead, the thi-
ocarbamoyl portion is involved into two hydrogen bonds
respectively between the S and HOH2166, NH and Tyr435 of the
“aromatic cage”. So, we could observe that compound 14 is better
accommodated in the MAO-B cavity. As it is highlighted in Fig. 2,
the major differences in the isoforms binding pockets are that
Ile335 in MAO-A becomes Tyr326 in MAO-B, while Phe208 in
MAO-A corresponds to Ile199 in MAO-B. The residue 199 in MAO-
B, changing conformation, opens the access to a second cavity
[21]. This structural difference between the two isoforms enzymes
is the basis for selective MAO-B inhibition of many compounds
reported in literature [22] and also for compound 14. Neverthe-
less, MAO-B cavity is rather narrow, therefore, small changes
either in the aryl or pyrazoline moiety are reflected in the bio-
logical activity of this series. The differences on size and shape of
the binding sites influence the putative bioactive conformation
adopted by compound 14. Internal energies of the best ligand
Scheme 1. Synthetic pathway to compounds 1e25.

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E. Maccioni et al. / European Journal of Medicinal Chemistry 45 (2010) 4490e4498
Fig. 2. Docking results of compound 14 with MAO-A (top) and MAO-B (bottom). a and d) Differences of isoforms binding cavity. Ligand and residues are represented in licorice, FAD in
CPK style. Part of the enzyme in the background is visualized in new ribbon and surface style with VMD program [23]. b and e) Ligandscout [24] 3D visualization of compound
interactions with the enzyme: green and red arrows represent respectively donor and acceptor hydrogen bond, yellow spheres show the hydrophobic contacts. Binding pocket surfaces
are drawn as wireframe and colored in accordance to lipophilicity: pale yellow indicates lipophilic and light blue hydrophilic residues. c and f) 2D depiction of complex interactions,
including water molecules directly interacting with 14. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
poses show huge differences: 63.33 against 56.74 kcal/mol
respectively in MAO-A and MAO-B. Therefore, the binding into
MAO-A results entropically unfavoured. This can help to under-
stand the selectivity of this ligand toward MAO-B isoform. Finally,
the comparison of the results obtained with the best diary-
lthiocarbamoylpirazoline derivative [15] with respect to
compound 14 (Fig. 2) indicates the lack of an important double
stacking interaction with the aromatic residues, corresponding to
Tyr398 and Tyr435 of MAO-B isoform due to the absence of
a secondary aryl moiety.
nature of the other substituents. The analysis of the structure-based
study can help for the evaluation of the most appropriate substitu-
tion pattern in order to drive better MAO-B inhibitory activity
and selectivity for this series of compounds.
4. Experimental section
4.1. Chemistry
Melting points are uncorrected and were determined on an
Electrothermal 9100 apparatus. Infrared (IR) spectra were recorded
on a PerkineElmer 1640 FT spectrophotometer (KBr discs or nujol,
in cmꢀ1). 1H NMR spectra were recorded on a Varian 300 MHz or
a Varian 400 MHz using tetramethylsilane (TMS) as internal stan-
3. Conclusions
We have synthesised and characterised some differently
substituted 3-aryl-4,5-dihydropyrazoles-1-carbothioamides. More-
over, we have investigated their ability to selectively inhibit the two
isoforms of the monoamine oxidase. None of the active compounds
showed any activity towards the A isoform, thus indicating that the
3-aryl-4,5-dihydropyrazoles-1-carbothioamide moiety could be an
interesting scaffold for the design of selective MAO-B inhibitors. The
introduction of a methoxyphenyl moiety in position 3 of the 4,5-
dihydropyrazole revealed to be the most effective, regardless of the
dard (chemical shifts in
d values), and dimethylsulfoxide-d6 or
deuterochloroform as solvents. Electron ionisation (EI) mass
spectra were obtained on a Fisons QMD 1000 mass spectrometer
(70 eV, 200 mA, ion source temperature 200 ꢂC). The samples were
introduced directly into the ion source. Elemental analyses were
obtained on a PerkineElmer 240 B microanalyser. TLC chroma-
tography was performed using silica gel plates (Merck F 254).

E. Maccioni et al. / European Journal of Medicinal Chemistry 45 (2010) 4490e4498
4495
The structures of all compounds were assigned on the basis of
IR, NMR, mass spectra, and elemental analysis.
4.34 (“t”, 2H, CH2, J ¼ 9.5, 10.1); 6.06 (br.s, 1H, NH2, D2O-exch.); 6.92
(br.s, 1H, NH2, D2O-exch.); 6.97 (dd, 1H, 3-OCH3-phenyl, J ¼ 7.9,
J ¼ 1.5); 7.24 (d, 2H, 3-OCH3-phenyl, J ¼ 7.9); 7.32 (t, 1H, 3-OCH3-
phenyl, J ¼ 7.9). Anal. calcd. for C11H13N3OS %: C, 56.15; H, 5.57;
N, 17.86. Found %: C, 56.98; H, 5.55; N, 17.92.
4.2. General method for the preparation of the 1-aryl-3-
dimethylamino propenones
To a suspension of the appropriate ketone (10 mmol), para-
formaldehyde (13 mmol) and dimethylamine hydrochloride
(13 mmol), ethanol (5 ml) was added. The suspension was main-
tained under vigorous stirring and nitrogen flux, and conc. HCl
(0.20 ml) was added. The reaction was maintained under reflux
overnight. After cooling, in some cases a precipitate was obtained,
which was purified by treatment with petroleum ether. In most
cases aftercooling the solventwas removedunder reducedpressure,
a few drops of HCl were added, and an extraction using water and
dichloromethane was performed. To the aqueous layer, NaHCO3 was
added, in ordertoadjust the pH tobasic and a further extractionwith
dichloromethane was performed. The desired Mannich base was
obtained by collecting the organic layers, removing the solvent in
vacuo and treating the obtained product with petroleum ether.
4.3.5. 1-Thiocarbamoyl-3-(2,4-dichlorophenyl)-4,5-dihydropyrazole (5)
White solid; Yield: 78%; MS (m/z): 273, 275; Mp: 176e179 ꢂC; 1H
NMR (CDCl3)
d
: 3.46 (“t”, 2H, CH2, J ¼ 9.8, 10.1); 4.37 (“t”, 2H, CH2,
J ¼ 9.8, 10.1); 6.23 (br.s, 1H, NH2, D2O-exch.); 6.91 (br.s, 1H, NH2,
D2O-exch.); 7.30 (dd, 1H, 2,4-di Cl-phenyl, J ¼ 8.6, J ¼ 1.8); 7.48
(d, 1H, 2,4-di Cl-phenyl, J ¼ 1.8); 7.62 (d, 1H, 2,4-di Cl-phenyl,
J ¼ 8.6). Anal. calcd. for C10H9Cl2N3S %: C, 43.81; H, 3.31; N, 15.33.
Found %: C, 43.99; H, 3.29; N, 15.27.
4.3.6. 1-Thiocarbamoyl-3-(thiophen-2-yl)-4,5-dihydropyrazole (6)
Yellow crystals; Yield: 75%; MS (m/z): 211; Mp: 177e178 ꢂC; 1H
NMR (CDCl3)
d
: 3.30 (“t”, 2H, CH2, J ¼ 9.5, 10.4); 4.34 (“t”, 2H, CH2,
J ¼ 9.5, 10.4); 6.05 (br.s, 1H, NH2, D2O-exch.); 6.98 (br.s, 1H, NH2,
D2O-exch.); 7.08 (dd, 1H, H4-thiophenyl, J3 e4 ¼ 3.7, J4 e5 ¼ 4.9);
0
0
0
0
7.29 (dd, 1H, H3-thiophenyl, J3 e4 ¼ 3.7; J3 e5 ¼ 0.9); 7.45 (dd, 1H,
0
0
0
0
H5-thiophenyl, J4 e5 ¼ 4.9; J3 e5 ¼ 0.9). Anal. calcd. for C8H9N3S2%:
0
0
0
0
4.3. General method for the synthesis of the dihydropyrazole ring
C, 45.48; H, 4.29; N, 19.88. Found %: C, 45.67; H, 4.31; N, 19.79.
A warm solution of the appropriate Mannich base (10 mmol) in
2-propanol (12 ml) was dropwise added to a mixture of thio-
semicarbazide or methylthiosemicarbazide (10 mmol) dissolved in
2-propanol under nitrogen, until complete dissolution.
To the obtained limpid solution, KOH/ethanol was added
dropwise, and immediately the formation of a white precipitate
(KCl) insoluble in 2-propanol was observed which was filtered and
eliminated. The solution was concentrated to half of the original
volume and after cooling to room temperature, a precipitate was
formed which was filtered. The desired derivatives were dried
under vacuum and crystallized from methanol/ethanol to give
compounds 1e25.
4.3.7. 1-Thiocarbamoyl-3-(4-methoxyphenyl)-4,5-dihydropyrazole (7)
White solid; Yield: 83%; MS (m/z): 235; Mp: 209 ꢂC; 1H NMR
(CDCl3)
d
: 3.28 (t, 2H, CH2, J ¼ 9.8); 3.85 (s, 3H, OCH3); 4.34 (t, 2H,
CH2, J ¼ 9.8); 6.04 (br.s, 1H, NH2, D2O-exch.); 6.85 (br.s, 1H,
NH2, D2O-exch.); 6.93 (d, 2H, 4-OCH3-phenyl, J ¼ 8.9); 7.65
(d, 2H, 4-OCH3-phenyl, J ¼ 8.9). Anal. calcd. for C11H13N3OS %: C,
56.15; H, 5.37; N, 17.86. Found %: C, 55.94; H, 5.59; N, 17.81.
4.3.8. 1-Thiocarbamoyl-3-(4-methylphenyl)-4,5-dihydropyrazole (8)
Pale yellow crystalline solid; Yield: 80%; MS (m/z): 219; Mp:
185e186 ꢂC; 1H NMR (CDCl3)
d: 2.38 (s, 3H, CH3); 3.27 (“t”, 2H, CH2,
J ¼ 9.7, 10.1); 4.33 (“t”, 2H, CH2, J ¼ 9.7, 10.1); 6.10 (br.s, 1H, NH2, D2O-
exch.); 6.94 (br.s, 1H, NH2, D2O-exch.); 7.22 (d, 2H, 4-CH3-phenyl,
J ¼ 7.9); 7.59 (d, 2H, 4-CH3-phenyl, J ¼ 7.9). Anal. calcd. for C11H139N3S
%: C, 60.25; H, 5.97; N, 19.16. Found %: C, 60.47; H, 5.99; N, 19.09.
4.3.1. 1-Thiocarbamoyl-3-(4-fluorophenyl)-4,5-dihydropyrazole (1)
Pale yellow solid, yield: 79%; MS (m/z): 223; Mp: 153e156 ꢂC. 1H
NMR (CDCl3)
d
:3.28(“t”, 2H, CH2, J¼ 9.8,10.1); 4.36 (“t”, 2H, CH2, J¼ 9.8,
10.1); 6.21 (br.s, 1H, NH2, D2O-exch.); 6.91 (br.s, 1H, NH2, D2O-exch.);
7.11 (t, 2H, 4-F-phenyl, J ¼ 8.6); 7.71 (dt, 2H, 4-F-phenyl, J ¼ 8.6, 5.2).
Anal. calcd. for C10H10FN3S %: C, 53.80; H, 4.50; N, 18.82. Found
%: C, 54.00; H, 4.48; N, 18.75.
4.3.9. 1-(N-Methylthiocarbamoyl)-3-(4-fluorophenyl)-4,
5-dihydropyrazole (9)
Orange crystalline solid; Yield: 85%; MS (m/z): 237; Mp:
157e158 ꢂC; 1H NMR (CDCl3)
d
: 3.16e3.23 (m, 5H, CH2 þ NHeCH3);
4.3.2. 1-Thiocarbamoyl-3-(3-methylphenyl)-4,5-dihydropyrazole (2)
Yellow solid, yield: 76%; MS: (m/z): 219; Mp: 188e190 ꢂC; 1H
4.33 (t, 2H, CH2); 7.08 (t, 2H, 4-F-phenyl, J ¼ 8.5); 7.27 (d, 1H,
NHeCH3, D2O-exch., J ¼ 4.9); 7.68 (dd, 4H, 4-F-phenyl, J ¼ 8.5, 5.5).
Anal. calcd. for C11H12FN3S %: C, 55.68; H, 5.09; N, 17.71. Found %: C,
55.47; H, 5.10; N, 17.64.
NMR (CDCl3)
d
: 2.40 (s, 3H, CH3); 3.30 (“t”, 2H, CH2, J ¼ 9.5, 10.1);
4.36 (“t”, 2H, CH2, J ¼ 9.5, 10.1); 6.06 (br.s, 1H, NH2, D2O-exch.); 6.95
(br.s, 1H, NH2, D2O-exch.); 7.28 (d, 1H, 3-CH3-phenyl, J ¼ 7.3); 7.32
(t, 1H, 3-CH3-phenyl, J ¼ 7.3); 7.50 (d, 1H, 3-CH3-phenyl, J ¼ 7.3);
7.54 (s, 1H, 3-CH3-phenyl).
4.3.10. 1-(N-Methylthiocarbamoyl)-3-(3-methylphenyl)-4,
5-dihydropyrazole (10)
Anal. calcd. for C11H13N3S %: C, 60.25; H, 5.97; N, 19.16. Found %:
C, 60.07; H, 6.01; N, 19.08.
Orange solid; Yield: 77%; MS (m/z): 233; Mp: 139e140 ꢂC; 1H
NMR (CDCl3)
d
:
2.40 (s, 3H, CH3); 3.21e3.28 (m, 5H,
CH2 þ NHeCH3); 4.36 (t, 2H, CH2); 7.26 (t, 1H, 3-CH3-phenyl,
J ¼ 6.3); 7.32 (d, 2H, NHeCH3 e D2O-exch. þ 3-CH3-phenyl, J ¼ 7.5);
7.49 (d, 1H, 3-CH3-phenyl, J ¼ 7.5), 7.54 (s, 1H, 3-CH3-phenyl). Anal.
calcd. for C12H15N3S %: C, 61.77; H, 6.48; N, 18.00. Found %: C, 62.00;
H, 6.50; N, 18.05.
4.3.3. 1-Thiocarbamoyl-3-(2-methylphenyl)-4,5-dihydropyrazole (3)
White solid; Yield: 81%; MS (m/z): 219; Mp: 173e175 ꢂC; 1H
NMR (CDCl3)
d: 2.59 (s, 3H, CH3); 3.36 (“t”, 2H, CH2, J ¼ 9.5, 10.1);
4.31 (“t”, 2H, CH2, J ¼ 9.5, 10.1); 6.09 (br.s, 1H, NH2, D2O-exch.); 6.95
(br.s, 1H, NH2, D2O-exch.); 7.26e7.33 (m, 3H, 2-CH3-phenyl); 7.41
(d, 1H, 2-CH3-phenyl, J ¼ 7.6). Anal. calcd. for C11H13N3S %: C, 60.25;
H, 5.97; N, 19.16. Found %: C, 60.43; H, 5.95; N, 19.23.
4.3.11. 1-(N-Methylthiocarbamoyl)-3-(2-methylphenyl)-4,
5-dihydropyrazole (11)
White crystalline solid; Yield: 83%; MS (m/z): 233; Mp:
4.3.4. 1-Thiocarbamoyl-3-(3-methoxyphenyl)-4,5-dihydropyrazole (4)
White solid; Yield: 85%; MS (m/z): 235; Mp: 150e152 ꢂC; 1H
128e130 ꢂC; 1H NMR (CDCl3)
d: 2.58 (s, 3H, CH3); 3.21 (d, 3H,
NHeCH3); 3.29 (“t”, 2H, CH2, J ¼ 9.9, 10.3); 4.31 (“t”, 2H, CH2, J ¼ 9.9,
NMR (CDCl3)
d
: 3.26 (“t”, 2H, CH2, J ¼ 9.5, 10.1); 3.83 (s, 3H, OCH3);
10.3); 7.16 (s, 1H, NHeCH3, D2O-exch.); 7.24e7.34 (m, 3H, 2-CH3-

4496
E. Maccioni et al. / European Journal of Medicinal Chemistry 45 (2010) 4490e4498
phenyl), 7.40 (d,1H, 2-CH3-phenyl, J ¼ 7.0). Anal. calcd. for C12H15N3S
4.16 (dd,1H, JAx ¼ 4.4, JAB ¼ 11.7, HA); 4.32 (“t”,1H, JAB ¼ 11.4, HB); 6.93
(d, 2H, 4-OCH3-phenyl, J ¼ 8.8); 7.31 (br.s, 1H, NHeCH3, D2O-exch.);
7.67 (d, 2H, 4-OCH3-phenyl, J ¼ 8.8). Anal. calcd. for C13H17N3OS %: C,
59.29; H, 6.51; N, 15.95. Found %: C, 59.41; H, 6.49; N, 16.01.
%: C, 61.77; H, 6.48; N, 18.00. Found %: C, 61.98; H, 6.47; N, 17.96.
4.3.12. 1-(N-Methylthiocarbamoyl)-3-(3-methoxyphenyl)-4,
5-dihydropyrazole (12)
Orange solid; Yield: 76%; MS (m/z): 249; Mp: 128e130 ꢂC; 1H
4.3.19. 1-Thiocarbamoyl-3-(4-methoxyphenyl)-4-methyl-4,
5-dihydropyrazole (19)
NMR (CDCl3)
d
: 3.22e3.27 (m, 5H, CH2 þ NHeCH3); 3.85 (s, 3H,
OCH3); 4.37 (t, 2H, CH2); 6.99 (d, 1H, 3-OCH3-phenyl, J ¼ 7.9);
7.26e7.36 (m, 4H, 3-OCH3-phenyl þ NHeCH3, D2O-exch.). Anal.
calcd. for C12H15N3OS %: C, 57.81; H, 6.06; N, 16.85. Found %: C,
58.02; H, 6.03; N, 16.92.
White solid; Yield: 46%; MS (m/z): 249; Mp: 169e172 ꢂC; 1H
NMR (CDCl3)
d
: 1.31 (d, 3H, CH3, J ¼ 7.3); 3.60e3.68 (m., 1H,
CHxeCH3); 3.84 (s, 3H, OCH3); 4.13 (dd, 1H, JAx ¼ 4.6, JAB ¼ 11.9, HA);
4.34 (dd, 1H, JBx ¼ 10.4, JAB ¼ 11.9, HB); 6.93 (d, 2H, 4-OCH3-phenyl,
J ¼ 8.9); 7.7 (d, 2H, 4-OCH3-phenyl, J ¼ 8.9), NH2 not detected. Anal.
calcd. for C12H15N3OS %: C, 57.81; H, 6.06; N, 16.85. Found %: C,
57.63; H, 6.04; N, 16.91.
4.3.13. 1-(N-Methylthiocarbamoyl)-3-(thiophen-2-yl)-4,
5-dihydropyrazole (13)
Yellow-orange solid; Yield: 78%; MS (m/z): 225; Mp:
125e127 ꢂC; 1H NMR (CDCl3)
d
: 3.19 (d, 3H, NHeCH3, J ¼ 4.9); 3.25 (t,
4.3.20. 1-(N-Methylthiocarbamoyl)-3-(4-methylphenyl)-4-methyl-
4,5-dihydropyrazole (20)
2H, CH2, J ¼ 9.8); 4.37 (t, 2H, CH2, J ¼ 9.8); 7.07 (dd, 1H, H4-thio-
phenyl, J3 e4 ¼ 2.4, J4 e5 ¼ 5.2); 7.23 (s, 1H, NHeCH3, D2O-exch.);
White crystals; Yield: 51%; MS (m/z): 247; Mp: 182e184 ꢂC; 1H
0
0
0
0
7.26 (dd, 1H, H3-thiophenyl, J3 e4 ¼ 2.4; J3 e5 ¼ 1.2); 7.68 (dd, 1H,
NMR (CDCl3)
d: 1.30 (d, 3H, CH3, J ¼ 7.2); 2.39 (s, 3H, 4-CH3-phenyl);
0
0
0
0
H5-thiophenyl, J4 e5 ¼ 5.2; J3 e5 ¼ 1.2) Anal. calcd. for C9H11N3S2%:
3.21 (d, 3H, NHeCH3, J ¼ 4.9); 3.60e3.70 (m, 1H, CHxeCH3); 4.17
(dd, 1H, JAx ¼ 4.4, JAB ¼ 11.6, HA); 4.34 (“t”, 1H, JAB ¼ 11.5, HB); 7.32
(br.s, 1H, NHeCH3, D2O-exch.); 7.23 (d, 2H, 4-CH3-phenyl, J ¼ 8.0);
7.61 (d, 2H, 4-CH3-phenyl, J ¼ 8.2). Anal. calcd. for C13H17N3S %: C,
63.12; H, 6.93; N, 16.99. Found %: C, 62.93; H, 6.91; N, 17.04.
0
0
0
0
C, 47.97; H, 4. 92; N, 18.65. Found %: C, 48.15; H, 4.90; N, 18.73.
4.3.14. 1-(N-Methylthiocarbamoyl)-3-(4-methoxyphenyl)-4,
5-dihydropyrazole (14)
White crystalline solid; Yield: 77%; MS (m/z): 249; Mp:
190e191 ꢂC; 1H NMR (CDCl3)
d
: 3.18e3.25 (m, 5H, CH2 þ NHeCH3);
4.3.21. 1-Thiocarbamoyl-3-(4-methylphenyl)-4-methyl-4,
5-dihydropyrazole (21)
3.85 (s, 3H, OCH3); 4.34 (t, 2H, CH2); 6.92 (d, 2H, 4-OCH3-phenyl,
J ¼ 9.1); 7.27 (s, 1H, NH, D2O-exch.); 7.65 (d, 2H, 4-OCH3-phenyl,
J ¼ 8.9). Anal. calcd. for C12H15N3OS %: C, 57.81; H, 6.06 N, 16.85.
Found %: C, 57.98; H, 6.08; N, 16.80.
White solid; Yield: 47%; MS (m/z): 233; Mp: 150e152 ꢂC; 1H
NMR (CDCl3, 400 MHz)
d
1.25 (d, 3H, CH3, J ¼ 5.4); 2.34 (s, 3H, 4-
CH3-phenyl); 3.61e3.71 (m, 1H, CHxeCH3); 4.08 (dd, 1H, JAx ¼ 3.6,
JAB ¼ 9.0, HA); 4.30 (dd, 1H, JBx ¼ 7.8, JAB ¼ 9.0, HB); 6.19 (br.s, 1H,
NH2, D2O-exch.); 6.92 (br.s, 1H, NH2, D2O-exch.); 7.18 (d, 2H, 4-CH3-
phenyl, J ¼ 6.0); 7.56 (d, 2H, 4-CH3-phenyl, J ¼ 6.0). 13C NMR (CDCl3,
4.3.15. 1-(N-Methylthiocarbamoyl)-3-(4-methylphenyl)-4,
5-dihydropyrazole (15)
Yellow crystalline solid; Yield: 82%; MS (m/z): 249; Mp:
400 MHz): d 19.0 (CH3-pyr); 21.4 (phenyl-CH3); 39.5 (CH); 56.3
153e155 ꢂC; 1H NMR (CDCl3)
d
: 2.39 (s, 3H, CH3); 3.18e3.25 (m, 5H,
(CH2); 126.9, 127.0, 129.5, 141.1 (phenyl); 161.9 (C3-pyr); 176.1
(C]S). Anal. calcd. for C12H15N3S %: C, 61.77; H, 6.48; N, 18.00.
Found %: C, 61.89; H, 6.50; N, 17.95.
CH2 þ NHeCH3); 4.34 (t, 2H, CH2); 7.22(d, 2H, 4-methylphenyl,
J ¼ 8.1); 7.31 (s, 1H, NHeCH3, D2O-exch.); 7.49 (d, 1H, 4-methyl-
phenyl, J ¼ 7.5). Anal. calcd. for C12H15N3S %: C, 61.77; H, 6.49; N,
18.00. Found %: C, 62.02; H, 6.05; N, 17.93.
4.3.22. 1-(N-Methylthiocarbamoyl)-3-phenyl-4-methyl-4,
5-dihydropyrazole (22)
4.3.16. 1-(N-Methylthiocarbamoyl)-3-(4-fluorophenyl)-4-methyl-
4,5-dihydropyrazole (16)
White crystals; Yield: 52%; MS (m/z): 233; Mp: 174e176 ꢂC;
1H NMR (CDCl3)
d
1.32 (d, 3H, CH3, J ¼ 7.3); 3.22 (d, 3H,
Yellow crystals; Yield: 46%; MS (m/z): 251; Mp: 200e202 ꢂC; 1H
NHeCH3, J ¼ 4.9); 3.69e3.81 (m, 1H, CHxeC H3); 4.19 (dd,
1H, JAx ¼ 4.6, JAB ¼ 11.6, HA); 4.37 (“t”, 1H, JAB ¼ 11.3, HB); 7.35
(br.s, 1H, NHeCH3, D2O-exch); 7.42e7.44 (m, 3H, phenyl); 7.71e7.74
(m, 2H, phenyl). Anal. calcd. for C12H15N3S %: C, 61.77; H, 6.48; N,
18.00. Found %: C, 61.58; H, 6.47; N, 17.93.
NMR (CDCl3)
d
1.29 (d, 3H, CH3,
J
¼
7.0); 3.20 (d, 3H,
NHeCH3, J ¼ 4.9); 3.65e3.76 (m, 1H, CHxeCH3); 4.18 (dd, 1H,
JAx ¼ 4.6, JAB ¼ 11.6, HA); 4.36 (“t”, 1H, JAB ¼ 11.0, HB); 7.11 (t, 2H, 4-F-
phenyl, J ¼ 8.6); 7.31 (br.s, 1H, NHeCH3, D2O-exch.); 7.71 (dd, 2H,
4-F-phenyl, J ¼ 8.9, 5.2). Anal. calcd. for C12H14FN3S %: C, 57.35; H,
5.61; N, 16.72. Found %: C, 57.16; H, 5.59; N, 16.66.
4.3.23. 1-Thiocarbamoyl-3-phenyl-4-methyl-4,5-dihydropyrazole
(23)
4.3.17. 1-Thiocarbamoyl-3-(4-fluorophenyl))-4-methyl-4,
5-dihydropyrazole (17)
Pale brown solid; Yield: 47%; MS (m/z): 219; Mp: 145e147 ꢂC; 1H
NMR (CDCl3)
d
1.33 (d, 3H, CH3, J ¼ 7.3); 3.63e3.76 (m, 1H,
Pale brown solid; Yield: 51%; MS (m/z): 237; Mp: 178e180 ꢂC;
CHxeCH3); 4.16 (dd, 1H, JAx ¼ 4.6, JAB ¼ 11.9, HA); 4.40 (dd, 1H,
JBx ¼ 10.4, JAB ¼ 11.9, HB); 6.15 (br.s, 1H, NH2, D2O-exch.); 6.96
(br.s, 1H, NH2, D2O-exch.); 7.42e7.45 (m, 3H, phenyl); 7.71e7.75 (m,
2H, phenyl). Anal. calcd. for C11H13N3S %: C, 60.25; H, 5.97; N, 19.16.
Found %: C, 60.41; H, 5.95; N, 19.21.
1H NMR (CDCl3)
d
: 1.30 (d, 3H, CH3, J ¼ 7.2); 3.61e3.69 (m, 1H,
CHxeCH3); 4.14 (dd, 1H, JAx ¼ 4.6, JAB ¼ 11.9, HA); 4.37 (dd, 1H,
JBx ¼ 10.6, JAB ¼ 11.9, HB); 6.23 (br.s, 1H, NH2, D2O-exch.); 6.92
(br.s, 1H, NH2, D2O-exch.); 7.11 (t, 2H, 4-F-phenyl, J ¼ 8.8); 7.72 (dd,
2H, 4-F-phenyl, J ¼ 8.9, 5.4). Anal. calcd. for C11H12FN3S %: C, 55.68;
H, 5.09; N, 17.71. Found %: C, 55.87; H, 5.11; N, 17.77.
4.3.24. 1-(N-Methylthiocarbamoyl)-3-(4-chlorophenyl)-4-methyl-
4,5-dihydropyrazole (24)
4.3.18. 1-(N-Methylthiocarbamoyl)-3-(4-methoxyphenyl)-4-
methyl-4,5-dihydropyrazole (18)
Yellow solid; Yield: 51%; MS (m/z): 267, 269; Mp:
150e152 ꢂC; 1H NMR (CDCl3)
d: 1.30 (d, 3H, CH3, J ¼ 7.3); 3.21 (d, 3H,
White crystals; Yield: 49%; MS (m/z): 263; Mp: 158e160 ꢂC; 1H
NHeCH3, J ¼ 5.1) 3.58e3.70 (m, 1H, CHxeCH3); 4.19 (dd, 1H,
JAx ¼ 4.4, JAB ¼ 11.7, HA); 4.37 (“t”, 1H, JAB ¼ 11. 7, HB); 7.32
(br.s, 1H, NH-CH3, D2O-exch); 7.40 (d, 2H, 4-Cl-phenyl, J ¼ 8.8); 7.66
NMR (CDCl3)
d
1.29 (d, 3H, CH3,
J
¼
7.3); 3.20 (d, 3H,
NHeCH3, J ¼ 4.8); 3.63e3.75 (m, 1H, CHxeCH3); 3.85 (s, 3H, OCH3);

E. Maccioni et al. / European Journal of Medicinal Chemistry 45 (2010) 4490e4498
4497
(d, 2H, 4-Cl-phenyl, J ¼ 8.8). Anal. calcd. for C12H14ClN3S %: C, 53.83;
the MAO isoforms, which were replaced by a sodium phosphate
buffer solution.
H, 5.27; N, 15.69. Found %: C, 53.97; H, 5.24; N, 15.72.
4.3.25. 1-Thiocarbamoyl-3-(4-chlorophenyl)-4-methyl-4,
5-dihydropyrazole (25)
5.2. Data presentation and statistical analysis
White solid; Yield: 65%; MS (m/z): 253, 255; Mp: 182e184 ꢂC; 1H
Unless otherwise specified, results shown in the text and table
are expressed as mean ꢁ standard error of the mean (S.E.M.) from n
experiments. Significant differences between two means (P < 0.05
or P < 0.01) were determined by one-way analysis of variance
(ANOVA) followed by the Dunnett’s post-hoc test.
To study the possible effects of the test drugs (new compounds
or reference inhibitors) on MAO isoform enzymatic activity, the
variation of fluorescence per unit of time (fluorescence arbitrary U/
min) and, indirectly, the rate of H2O2 production and, therefore, the
p mol/min of resorufin produced in the reaction between H2O2 and
AmplexÒ Red reagent was evaluated. For this purpose, several
concentrations of resorufin were used to prepare a standard curve
with X ¼ p mol resorufin and Y ¼ fluorescence arbitrary U. Note
that the value of resorufin production is similar to the p mol of
p-tyramine oxidized to p-hydroxyphenylacetaldehyde per min
since the stoichiometry of the reaction (p-tyramine oxidized by
MAO isoforms/resorufin produced) is 1:1.
NMR (CDCl3, 400 MHz)
d
1.28 (d, 3H, CH3, J ¼ 5.4); 3.63e3.72 (m.,
1H, CHxeCH3); 4.12 (dd, 1H, JAx ¼ 3.6, JAB ¼ 9.0, HA); 4.35 (dd, 1H,
JBx ¼ 7.8, JAB ¼ 9.0, HB); 6.11 (br.s, 1H, NH2, D2O-exch.); 6.91 (br.s, 1H,
NH2, D2O-exch.); 7.37 (d, 2H, 4-Cl-phenyl, J ¼ 8.1); 7.62 (d, 2H, 4-Cl-
phenyl, J ¼ 8.1). 13C NMR (CDCl3, 400 MHz):
d
17.1 (CH3-pyr); 36.9
(CH); 67.2 (CH2); 128.6, 129.7, 135.58, 143.1 (phenyl); 157.3 (C3-pyr);
178.1 (C]S). Anal. calcd. for C11H12ClN3S %: C, 52.07; H, 4.77; N,
16.56. Found %: C, 51.91; H, 4.79; N, 16.61.
5. Pharmacological studies
5.1. Determination of MAO isoform activity
The potential effects of the test drugs on human monoamine
oxidase (hMAO) activity were investigated by measuring their
effects on the production of hydrogen peroxide H2O2 from p-tyra-
mine (a common substrate for both hMAO-A and hMAO-B), using
the AmplexÒ 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 con-
taining cDNA inserts for hMAO-A or hMAO-B (SigmaeAldrich
Química S.A., Alcobendas, Spain).
In these experiments, the inhibitory activity of the tested drugs
(new compounds and reference inhibitors) is expressed as IC50, i.e.
the concentration of these compounds required to reduce by 50%
the control MAO isoform enzymatic activity, estimated by least-
squares linear regression, using the OriginTM 5.0 program (Microcal
Software, Inc., Northampton, MA, USA), with X ¼ log of tested
compound molar concentration and
Y
¼
the corresponding
The production of H2O2 catalysed by MAO isoforms can be
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. In this study, hMAO
activity was evaluated using the above method following the
general procedure described previously by us [25].
percentage of inhibition of control resorufin production obtained
with each concentration. This regression was performed using data
obtained with 4e6 different concentrations of each tested
compound which inhibited the control MAO isoform enzymatic
activity by between 20 and 80%. In addition, the corresponding
MAO-B selectivity ratios (SI A/B) [IC50 (MAO-A)]/[IC50 (MAO-B)]
were calculated.
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 to oxidize (in the
control group) 165 p mol of p-tyramine/min were incubated for
15 min at 37 ꢂC in the corresponding wells from a 96-well flat-
bottom microtiter plate (BD, Franklin Lakes, NJ, USA) placed in
the dark fluorimeter chamber. After this incubation period, the
5.3. Drugs and chemicals
The drugs used in the experiments were the new compounds,
moclobemide (a generous gift from F. Hoffmann-La Roche Ltd.,
Basel, Switzerland), R-(ꢀ)-deprenyl hydrochloride, iproniazid
phosphate (purchased from SigmaeAldrich, Spain), resorufin
sodium salt, clorgyline hydrochloride, p-tyramine hydrochloride
and horseradish peroxidase (supplied in the AmplexÒ Red MAO
assay kit from Molecular Probes).
reaction was started by adding (final concentrations) 200
mM
AmplexÒ Red reagent,
1
U/ml horseradish peroxidase and
1 mM p-tyramine as a common substrate for both hMAO-A and
hMAO-B. The production of H2O2 and, consequently, of resor-
ufin was quantified at 37 ꢂC in a Multi-Detection microplate
fluorescence reader (FLX800TM, Bio-TekÒ Instruments, Inc.,
Winooski, VT, USA) based on the fluorescence generated
(excitation 545 nm, emission 590 nm) over a 15 min period,
a period in which the fluorescence increased linearly from the
beginning.
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
ability 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 determined by
adding these drugs to solutions containing only the AmplexÒ Red
reagent in a sodium phosphate buffer.
Appropriate dilutions of the above drugs were prepared every
day immediately before use in deionized water from the following
concentrated stock solutions kept at ꢀ20 ꢂC: the new compounds
(0.1 M) in dimethylsulfoxide (DMSO); R-(ꢀ)-deprenyl, moclobe-
mide, iproniazid, resorufin, clorgyline, p-tyramine and horseradish
peroxidase (0.1 M) in deionized water.
Due to the photosensitivity of some chemicals (e.g., AmplexÒ
Red reagent), all experiments were performed in the dark. In all
assays, neither deionized water (Milli-QÒ, Millipore Ibérica S.A.,
Madrid, Spain) nor appropriate dilutions of the vehicle used
(DMSO) had significant pharmacological effects.
5.4. Molecular modelling
The docking experiments were carried out using the software
program AutoDock Vina [19]. The graphic interface ADT (Auto-
DockTools) was used to manipulate the models and to perform
input/output procedures. The first step of the computational work
was to select from the Protein Data Bank (PDB) [26] appropriate
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

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E. Maccioni et al. / European Journal of Medicinal Chemistry 45 (2010) 4490e4498
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