Identification of the stereochemical requirements in the 4-aryl-2-cycloalkylidenhydrazinylthiazole scaffold for the design of selective human monoamine oxidase B inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.03.042

Exploring the effect that substituents on the cycloaliphatic ring had on the inhibitory activity against human monoamine oxidase B of a series of 4-aryl-2-cycloalkylidenhydrazinylthiazoles led to the synthesis of a new series of 2-methylcyclopentyl and 3-methylcyclopentyl derivatives which were tested in vitro as mixtures of diastereoisomers. In fact, due to the presence of a chiral center on the cycloaliphatic ring and a trisubstituted CN bond, they exist as four diastereoisomers ((E)-(R), (E)-(S), (Z)-(R), (Z)-(S)). 4-(2,4- Difluorophenyl)-2-(2-(3-methylcyclopentylidene)hydrazinyl)thiazole was chosen as a model to investigate the influence of stereochemical requirements on the inhibitory activity against hMAO-B of these derivatives after a stereoconservative synthesis and semi-preparative HPLC diastereoseparation. (R)-(Z) isomer of this compound was endowed with a potent and selective hMAO-B inhibition higher than that of reference drugs as also corroborated by molecular modeling studies.

Full text of DOI:10.1016/j.bmc.2014.03.042

Bioorganic & Medicinal Chemistry 22 (2014) 2887–2895
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of the stereochemical requirements in the
4-aryl-2-cycloalkylidenhydrazinylthiazole scaffold for the design
of selective human monoamine oxidase B inhibitors
a
Melissa D’Ascenzio a, Simone Carradori a, , Daniela Secci , Luisa Mannina a,e, Anatoly P. Sobolev e,
⇑
Celeste De Monte a, Roberto Cirilli b, Matilde Yáñez c, Stefano Alcaro d, Francesco Ortuso d
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
b Istituto Superiore di Sanità, Dipartimento del Farmaco, V.le Regina Elena 299, 00161 Rome, Italy
c Departamento de Farmacología, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur, E-15782 Santiago de Compostela (La Coruña), Spain
d Dipartimento di Scienze della Salute, ‘Magna Graecia’ University of Catanzaro, Campus Universitario ‘S. Venuta’, Viale Europa Loc. Germaneto, 88100 Catanzaro, Italy
e Istituto di Metodologie Chimiche, Laboratorio di Risonanza Magnetica ‘Annalaura Segre’, CNR, via Salaria km 29.300, 00015 Monterotondo, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Exploring the effect that substituents on the cycloaliphatic ring had on the inhibitory activity against
human monoamine oxidase B of a series of 4-aryl-2-cycloalkylidenhydrazinylthiazoles led to the synthe-
sis of a new series of 2-methylcyclopentyl and 3-methylcyclopentyl derivatives which were tested
in vitro as mixtures of diastereoisomers. In fact, due to the presence of a chiral center on the cycloali-
phatic ring and a trisubstituted C@N bond, they exist as four diastereoisomers ((E)-(R), (E)-(S), (Z)-(R),
(Z)-(S)). 4-(2,4-Difluorophenyl)-2-(2-(3-methylcyclopentylidene)hydrazinyl)thiazole was chosen as a
model to investigate the influence of stereochemical requirements on the inhibitory activity against
hMAO-B of these derivatives after a stereoconservative synthesis and semi-preparative HPLC diastereo-
separation. (R)-(Z) isomer of this compound was endowed with a potent and selective hMAO-B inhibition
higher than that of reference drugs as also corroborated by molecular modeling studies.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 12 January 2014
Revised 21 March 2014
Accepted 27 March 2014
Available online 5 April 2014
Keywords:
Monoamine oxidase inhibitors
Methylcyclopentane
Diastereoseparation
Molecular modeling
Thiazole
1. Introduction
monoamine: p-tyramine. Although p-tyramine cannot cross the
blood brain barrier, it can behave as a peripheral adrenergic ago-
Human monoamine oxidases (hMAOs) are FAD-dependent en-
zymes which are involved in the oxidative deamination of endog-
enous and exogenous primary, secondary and tertiary amines.
Located on the outer mitochondrial membrane, hMAOs play a fun-
damental physiological role in the regulation of monoaminergic
neurotransmissions by controlling the amounts of dopamine
(DA), adrenaline (A) and noradrenaline (NA) inside the synaptic
cleft. In the brain, hMAOs are present in two isoforms, hMAO-A
and hMAO-B, which differ in distribution, substrate specificity,
and sensibility to inhibitors. Inhibition of hMAOs, in fact, repre-
sents the rationale for the treatment of those pathologies in which
there is an impairment of synaptic transmission, such as depres-
sive disorders and neurodegenerative diseases.1 However, one of
the main limitations to the use of non-selective MAO-inhibitors
is the high prevalence of hMAO-A in the gastrointestinal tract,
where it is mainly involved in the metabolism of an exogenous
nist and cause dangerous hypertensive crisis: the so called
cheese-effect. The safety of hMAO-inhibitors has been considerably
increased by the introduction of selective anti-hMAO-B agents,
which are endowed with high therapeutic potential against neuro-
degenerative disorders like Parkinson’s and Alzheimer’s disease,
but devoid of unwanted peripheral effects.2 In fact, hMAO-B is
highly expressed in astrocytes around senile plaques and its activ-
ity is notably increased in patients with age-related gliosis and Alz-
heimer’s disease. hMAO-B selective inhibition is expected to
enhance dopaminergic neurotransmission, which is beneficial for
PD, and to reduce the oxidative stress exerted on neurons by
hydrogen peroxide, a byproduct of MAO-catalyzed deamination
of neurotransmitters, resulting in a neuroprotective and neurore-
storative effects.3,4 Therefore, there is a considerable interest in
obtaining potent and selective MAO inhibitors.5
For several years our group has been working on hMAO-B inhibi-
tors as potential co-adjuvants for the treatment of neurodegenerative
diseases:6 we recently reported on the promising in vitro activity and
selectivity of a large number of 4-aryl-2-hydrazinylthiazoles.7–15
⇑
Corresponding author. Tel.: +39 06 49913149; fax: +39 06 49913923.
E-mail address: simone.carradori@uniroma1.it (S. Carradori).
http://dx.doi.org/10.1016/j.bmc.2014.03.042
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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M. D’Ascenzio et al. / Bioorg. Med. Chem. 22 (2014) 2887–2895
3D-QSAR studies helped to rationalize experimental results and to
provide robust structure-activity relationships: (i) the aryl group at
C4 of the thiazole is responsible for the correct orientation of the
inhibitor inside the active site of hMAO-B and directly interacts with
two tyrosine residues (the so called ‘aromatic cage’) close to the cat-
alytic FAD;10 (ii) in order to obtain productive interactions within the
aromatic cage, the aryl moiety has to be ortho and/or para substituted
with an electron-withdrawing group (NO2, CN, F), (iii) the introduc-
tion of substituents on the C5 of the thiazole and/or highly hindered
substituents on the C4 (naphthalene, coumarin) determines a dra-
matic loss of activity and selectivity;12 (iv) 5- or 6-membered cyclo-
aliphatic and small heteroaromatic rings (furan, thiophene and
pyridine) are preferred on the N1 of the hydrazine.8,13,14
Accurate analyses of 1H NMR spectra (CDCl3, 400 MHz) revealed
that the obtained compounds were a mixture of E and Z geometric
isomers, the ratio of which could be measured by integrating the
area of the corresponding NH signal (see 1H NMR characterization
data), whereas CH3 signals were often overlapped and did not al-
low an accurate discrimination between the two isomers. Accord-
ing to previous theoretical and chromatographic study for similar
compounds, the choice of a polar protic solvent as reaction med-
ium (methanol) should favor the E-configuration and limit the
interconversion between the two isomers.16–18
3. HPLC diastereoseparation
Moreover, we discovered that (R)-enantiomers of some re-
ported chiral derivatives (2-methyl- and 3-methylcyclohexylidene
derivatives) were generally responsible for the higher activity and
selectivity of racemates against hMAO-B.8–13 Following this ratio-
nale, we designed a new series of 4-aryl-2-cyclopentylidenhydraz-
inylthiazole derivatives in which a methyl group was introduced
on the C2 or the C3 of the cycloaliphatic nucleus. The effects that
this chemical modification had on activity and selectivity of the
obtained derivatives were evaluated by determining the IC50 of
tested compounds against both hMAO-A and hMAO-B. Biological
results showed that these compounds were all able to inhibit
hMAO-B at nanomolar concentrations when tested as mixtures
of diastereoisomers. In order to further investigate the stereo-
chemical requirements for optimal hMAO-B inhibition, one of
the synthesized derivatives was chosen as a model: (E)-(R) and
(Z)-(R) diastereoisomers of 4-(2,4-difluorophenyl)-2-(2-(3-methyl-
The first step of our chromatographic experiments was carried
out with the aim to find a chiral HPLC system capable of simulta-
neously separating the four stereoisomers of rac-8: ((E)-(R), (Z)-
(R), (E)-(S), and (Z)-(S). As shown in Figure 1a, a clear resolution
of all the peaks was not possible in any of the conditions screened.
However, diasteroseparation of E and Z stereoisomers was possible
when enantiopure (R)-8 was injected in a column charged with
Chiralpak AD CSP under normal phase conditions (Figure 1b). The
absolute configuration of the thiazole derivatives in homochiral
form was assigned by chemical correlation.
The two peaks of (R)-8 were characterized by the same specific
rotation sign and similar area. It was found that the diastereomeric
ratio (Z/E), measured at the isosbestic point of 254 nm, was 46/54
similar to that obtained by 1H NMR spectroscopy (43/57). This va-
lue indicated that the stability of the second eluting diastereomer
((E)-(R)-8) was only slightly higher than the first eluting one ((Z)-
(R)-8).
cyclopentylidene)hydrazinyl)thiazole were synthesized via
a
stereoconservative route, separated by chiral semi-preparative
HPLC on polysaccharide-based stationary phase, and individually
tested against hMAO-A and hMAO-B.
The same HPLC separation conditions were used on a semi-pre-
parative scale to isolate mg-amounts of each (R)-8 isomer with
high degree of diastereomeric purity (Fig. 1c and d). In this way,
tens of a milligram of pure stereoisomers (E)-(R)-8 and (Z)-(R)-8
were made available for separate biological evaluation (Scheme 3).
2. Chemistry
Compounds 1–8 were synthesized in high yields (81–99%) as
mixtures of diastereoisomers according to an improved MW-as-
sisted protocol developed in our laboratory (Scheme 1).
2-Methyl-/3-methylcyclopentanone was reacted with thiosem-
icarbazide under MW irradiation (103 °C, 5 min, 300 W) in the
presence of catalytic amounts of acetic acid. The obtained thio-
semicarbazones were subsequently converted into the correspond-
ing 4-aryl-2-hydrazinylthiazole derivatives by reaction with the
4. NMR assignment of eluted stereoisomers
In order to clarify the stereochemistry of the products eluted
[(Z)-(R)-8 and (E)-(R)-8] from the chromatographic separation
regarding the C@N double bond, an NMR study was performed
using 1D and 2D NMR (1H–1H COSY and 1H–1H NOESY) techniques.
More in detail, the 1H–1H NOESY experiment allows the detection
of spatial proximities between 1H spins by taking advantage of the
dipolar coupling interaction. It can bring valuable information such
as double-bond configuration and spatial organization which
would be otherwise very difficult—if not impossible—to obtain.
The 1H–1H NOESY map of the first eluted product (data not
shown), shows a NOE contact between NH at 10.58 ppm and
CH2-a protons at 1.88 and 2.66 ppm indicating their spatial prox-
imity and therefore suggesting a (Z)-configuration. In the case of
the second eluted product, NOE contacts between NH at
corresponding a-halo-2-/2,4-substituted-acetophenones in metha-
nol under MW irradiation (90 °C, 10 min, 300 W). All synthesized
products were washed with petroleum and diethyl ether, purified
by column chromatography (ethyl acetate/n-hexane; 1:1), and
characterized by spectroscopic methods and elemental analysis.
The use of commercially available (R)-3-methylcyclopentanone
as starting material allowed synthesizing compound 8 as a single
enantiomer ((R)-8) via a stereoconservative pathway (Scheme 2).
O
R1
S
X
H2N
S
R1
N
H
NH2
N
H3C
N
H3C
H3C
O
N
N
H
NH2
N
H
S
MeOH, MW,
90 °C, 10 min,
300 W
CH3COOH, abs. EtOH,
MW, 103 °C, 5 min,
300 W
1-8
X
Cl Br
,
=
R
1 = 4-F, 4-CN, 4-NO2, 2,4-F
Scheme 1. General synthetic pattern of synthesized derivatives.

M. D’Ascenzio et al. / Bioorg. Med. Chem. 22 (2014) 2887–2895
2889
F
E/Z GEOMETRIC ISOMERS
N
HN
N
CHIRAL CENTER
F
S
H3C
(E)-(R)-8; (Z)-(R)-8; (E)-(S)-8; (Z)-(S)-8
IC50 hMAO-B = 9.34 0.29 nM
1,806
SI hMAO-B =
STEREOCONSERVATIVE
SYNTHESIS
F
F
N
HN
N
N
S
HN
N
H3C
F
F
+
S
H3C
(E)-(R)-8
(Z)-(R)-8
IC50 hMAO-B = 5.32 0.19 nM
2,915
SI hMAO-B =
Scheme 2. Stereoconservative synthesis of E and Z isomers of compound (R)-8.
10.60 ppm and CH2-d protons at 2.30 and 2.50 ppm were observed
implying the (E)-configuration of C@N double bond (Scheme 3).
(R)
(S)
900
600
300
0
5. Pharmacology
rac-8
(R)-8
(a)
Compounds 1–8 were evaluated for their ability to inhibit
hMAOs as mixtures of diastereomers, while the E and Z isomers
of (R)-8 were tested both individually and as a mixture. The biolog-
ical activity of tested compounds was investigated using the Am-
plexÒ Red MAO assay kit and microsomal MAO isoforms
prepared from insect cells (BTI-TN-5B1-4) infected with recombi-
nant baculovirus containing cDNA inserts for hMAO-A or hMAO-
B.19 p-Tyramine was chosen as common substrate for both
hMAO-A and hMAO-B. The reduction of the amount of hydrogen
peroxide (H2O2) produced by hMAOs catabolic activity was mea-
sured using 10-acetyl-3,7-dihydroxyphenoxazine (AmplexÒ Red
reagent), a non-fluorescent and highly sensitive probe that reacts
with H2O2 in the presence of horseradish peroxidase to produce
the fluorescent product resorufin. Both tested compounds and ref-
erence inhibitors were unable to directly react with AmplexÒ Red
reagent, indicating that these drugs were not interfering with the
measurements.
-300
900
600
300
0
(b)
ee>99%
-300
400
200
0
(c)
de>99%
(Z)-(R)-(+)-8
20 +33 (c 0.1, EtOH)
[ ]
α
D
Under our experimental conditions, hMAO-A displayed
Michaelis constant (Km) equal to 457.17 38.62 M and a maxi-
mum reaction velocity (Vmax in the control group of
185.67 12.06 nanomol p-tyramine/min/mg protein, whereas
hMAO-B showed a Km of 220.33 32.80 M and a Vmax equal to
24.32 1.97 nanomol p-tyramine/min/mg protein (n = 5). Most
tested drugs concentration-dependently inhibited this enzymatic
control activity (Table 1).
a
l
400
200
0
)
(d)
(E)-(R)-(+)-8
20
l
[
α
]
de=98%
+39 (c 0.1, EtOH)
D
0
3
6
9
Elution time (min)
6. Results and discussion
Figure 1. HPLC traces of rac-8 (a) and (R)-8 (b), and semi-preparative isolation of Z
and E isomers of (R)-8 (c and d). Column: Chiralpak AD (250 mm  4.6 mm I.D.);
mobile phase: n-hexane/2-propanol 90:10 (v/v); flow rate: 1 mL/min; detector: UV
at 254 nm; temperature: 25 °C.
Inhibition data, including selectivity index (ratio), against
hMAO-A and -B are presented in Table 1. All compounds bearing

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M. D’Ascenzio et al. / Bioorg. Med. Chem. 22 (2014) 2887–2895
F
c d
N
HN
N
S
b
a
H3C
F
(Z)-(R)-8
F
IC50 hMAO-B = 2.91 0.08 nM
SI hMAO-B = 4,735
N
S
HN
N
F
HPLC DIASTEREOSEPARATION
H3C
(E)-(R)-8 + (Z)-(R)-8
F
IC50 hMAO-B = 5.32 0.19 nM
SI hMAO-B = 2,915
N
HN
N
d
a
F
c
b
S
H3C
(E)-(R)-8
IC50 hMAO-B = 5.06 0.07 nM
SI hMAO-B = 2,581
Ratio (IC50 hMAO-B (E,R)-8 /IC50 hMAO-B (Z,R)-8)= 1.74
Scheme 3. Chemical approach to the determination of the relationship between structure and activity in the 4-aryl-2-hydrazinylthiazole scaffold.
Table 1
Structure and biological activity of new derivatives and reference compounds after a 15 min preincubation period with human MAO isoforms
Compound
R
R1
IC50 hMAO-A
IC50 hMAO-B
Ratio
1
2
3
4
5
6
7
8
2-CH3
2-CH3
2-CH3
2-CH3
3-CH3
3-CH3
3-CH3
3-CH3
3-CH3
3-CH3
3-CH3
4-F
4.58 0.32
19.23 0.96
4.31 0.21
10.93 0.67
l
l
l
l
l
Ma
Ma
Ma
Ma
Ma
9.13 0.32 nM
8.31 0.21 nM
38.03 0.96 nM
6.16 0.15 nM
65.49 4.35 nM
375.71 12.45 nM
227.48 11.85 nM
9.34 0.29 nM
5.32 0.19 nM
2.91 0.08 nM
5.06 0.07 nM
502
2314
113
4-NO2
4-CN
2,4-F
4-F
4-NO2
4-CN
2,4-F
2,4-F
2,4-F
2,4-F
1774
30
1.89 0.01
>266b
49
**
11.09 0.59
16.87 0.64
15.51 0.75
13.78 0.05
13.06 0.08
l
l
l
l
l
Ma
Ma
Ma
Ma
Ma
1806
2915
4735
2581
0.000073
3431
0.87
(R)-8
(Z)-(R)-8
(E)-(R)-8
Clorgyline
(R)-(À)-Deprenyl
Iproniazid
Moclobemide
Isatin
4.46 0.32 nMa
61.35 1.13
lM
67.25 1.02
6.56 0.76
l
l
Ma
M
19.60 0.86 nM
7.54 0.36
l
M
M
*
361.38 19.37
l
M
<0.36c
>5.3b
**
18.75 1.24
l
Ratio: hMAO-B selectivity index = IC50(hMAO-A)/IC50(hMAO-B). Each IC50 value is the mean S.E.M. from five experiments (n = 5).
a
Level of statistical significance: P <0.01 versus the corresponding IC50 values obtained against hMAO-B, as determined by ANOVA/Dunnett’s.
*
Inactive at 1 mM (highest concentration tested).
**
Inactive at 100
l
M (highest concentration tested). At higher concentrations the compounds precipitate.
Value obtained under the assumption that the corresponding IC50 against hMAO-A is the highest concentration tested (100
Value obtained under the assumption that the corresponding IC50 against hMAO-B is the highest concentration tested (1 mM).
b
c
lM).
2-/3-methylcyclopentylidene substituent at N1-hydrazine inhibit
hMAO-B in the nanomolar range, as expected on the basis of our
previous works on structurally similar derivatives (2- and 3-meth-
ylcyclohexylidene derivatives).8–13
(5–8) showed a slightly lower inhibitory activity against hMAO-B
(see compound 8, IC50 = 9.34 0.29 nM and ratio >1806) their
IC50 were, however, comparable to the reference drug (R)-(À)-
deprenyl 19.60 0.86 nM), with the only exception of compounds
6 and 7. As in the previously studied 2- and 3-methylcyclohexylid-
ene analogues, where the stereochemistry of the methyl substitu-
ent on the cycloaliphatic ring was an important modulator of
biological activity, we were prompted to study this phenomenon
with regards to the newly synthesized 2- and 3-methylcyclopenty-
lidene derivatives. For this purpose, compound 8 was synthesized
as single enantiomer starting from commercially available (R)-3-
methylcyclopentanone, and its E and Z stereoisomers were tested
both as a mixture and as single isomers after HPLC semi-prepara-
tive resolution. As expected, the enantiopure E and Z isomers
In general, 2-methylcyclopentylidene derivatives (1–4) were
better inhibitors towards human MAO-B (see compound 2,
IC50 = 8.31 0.21 nM and ratio >2314) than the corresponding 3-
methylcyclopentylidene counterparts (5–8). The inhibitory activity
and selectivity towards the B isoform is favored by ortho/para
disubstitution of the phenyl ring with electron-withdrawing
groups (especially with the presence of fluorine atoms), thus con-
firming the fundamental role that the dipolar moment of the inhib-
itor plays in the interaction with the aromatic cage in the active
site of the enzyme. Although 3-methylcyclopentylidene derivatives

M. D’Ascenzio et al. / Bioorg. Med. Chem. 22 (2014) 2887–2895
2891
Table 2
reported bumps among our inhibitors and some residues of former
isozyme.
Docking descriptors of (Z)-(R)-8 and (E)-(R)-8 hMAOs recognition. GScoreXP and
data are reported in kcal/mol
DEint
Compound
hMAO-A
GScoreXP
hMAO-B
GScoreXP DEint
7. Conclusion
D
Eint
We designed and synthesized new 4-aryl-2-cycloalkylidenhyd-
azinylthiazoles with the aim of investigating the stereochemical
requirements in this scaffold regarding the hMAO inhibition.
According to what we demonstrated with 2- and 3-methylcyclo-
hexylidene derivatives, these 2- and 3-methylcyclopentylidene
compounds displayed good inhibitory activity and high selectivity
against hMAO-B. Moreover, (R)-8 stereoisomer, obtained by a ste-
reoconservative synthesis, was a two-fold selective hMAO-B inhib-
itor than the racemic mixture. With respect to E/Z isomers, we
performed a semi-preparative HPLC to separate and test each geo-
metric isomer of this potent derivative. Biological assays stated the
best and more selective hMAO-B inhibition (two-fold) by (Z)-(R)-
(8) proposing a stereospecific enzyme recognition of the inhibitor.
These results were also corroborated by computational studies.
Molecular modeling addressed to steric hindrance the different
affinity of both E and Z isomers of (R)-8 with respect to hMAO-A
and -B. Actually, the recognition was driven by the known larger
volume of the hMAO-B active site with respect to the hMAO-A one.
(Z)-(R)-8
(E)-(R)-8
À0.643
À2.012
À18.47
À19.03
À10.004
À9.620
À49.66
À48.82
showed higher activity and selectivity against hMAO-B than the
racemic one (Scheme 2) and the (Z)-(R)-8 isomer, in particular,
displayed a surprising inhibitory activity (IC50 hMAO-B = 2.91
0.08 nM) and an excellent hMAO-B selectivity (ratio = 4735), which
was higher than that of the most potent reference drug and about
two-fold than that of the parent compound ((R)-8). As a matter
of this, a molecular modeling approach confirmed the essential
stereochemical requirements for this heterocyclic scaffold to
obtain potent and selective hMAO-B inhibitors.
The hMAO-A and -B recognition of both E and Z isomers of (R)-8
was evaluated by means of docking techniques. According to
experimental IC50 data, docking scoring function (GScoreXP) and
theoretical interaction energy (DEint) strongly remarked the better
accommodation of both E and Z isomers of (R)-8 into the hMAO-B
active site with respect to the hMAO-A one (Table 2).
8. Experimental section
8.1. Chemistry
The docking poses analysis addressed the previously reported
isozyme preference to the known broader binding site of hMAO-
B with respect to hMAO-A. A per residue binding energy evaluation
revealed a larger number of amino acids involved in the hMAO-B
recognition of both (R)-8 isomers. Moreover, while in hMAO-B all
interacting residues reported productive interaction energy to
our inhibitors, in hMAO-A four residues, Lys305, Phe352, Tyr407
and Tyr444 showed disfavoring contribution, due to steric clashes,
to the ligands recognition (Fig. 2).
The configuration of the double bond did not remarkably influ-
ence the binding modes of (R)-8, actually E and Z isomers showed
quite similar recognition in both hMAOs. In all cases, the difluoro-
phenyl ring was oriented toward the FAD cofactor and the chiral 3-
methylcyclopentyl was located at the binding cleft entrance site. In
the hMAO-B, the methylcyclopentyl moiety found a better accom-
modation into a lipophilic cage whereas in hMAO-A such an area is
hidden by Phe208. The substitution of hMAO-A Ile180, Asn181,
Phe208 and Ile335 with the corresponding hMAO-B Leu171,
Cys172, Ile199 and Tyr326 respectively, strongly modify the shape
of the active site and, as a consequence, the orientation of the thi-
azole ring (Fig. 3). The different shape and the hMAO-A active site
volume smaller than hMAO-B, could also explain the previously
Starting materials and reagents were obtained from commercial
suppliers and were used without further purification. Microwave-
assisted reactions were performed in a Biotage Initiator™ 2.0
(Uppsala, Sweden). Melting points (mp) were determined by the
capillary method on an FP62 apparatus (Mettler-Toledo) and are
uncorrected. 1H and 13C NMR spectra were recorded at 400 or
600 MHz on a Bruker spectrometer using DMSO-d6 or CDCl3 as sol-
vents. Chemical shifts are expressed as d units (ppm) relative to
TMS. Coupling constants J are expressed in hertz (Hz). IR spectra
were registered on a Perkin–Elmer FTIR Spectrometer Spectrum
1000 in anhydrous potassium bromide. Elemental analyses for C,
H, and N were determined with a Perkin–Elmer 240 B microanalyz-
er and the analytical results were within 0.4% for all compounds.
All reactions were monitored by TLC on 0.2 mm thick silica gel
plates (60 F254 Merck). Preparative flash column chromatography
was carried out on silica gel (230–400 mesh, G60 Merck). Organic
solutions were dried over anhydrous sodium sulfate. Concentra-
tion and evaporation of the solvent after reaction or extraction
Figure 2. hMAO-A and -B per residue interaction energy contribution to (E)-(R)-8 and (Z)-(R)-8 recognition.

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M. D’Ascenzio et al. / Bioorg. Med. Chem. 22 (2014) 2887–2895
Figure 3. Docking best poses of both E and Z isomers of (R)-8 into the (a) hMAO-A and (b) hMAO-B. Binding site shapes are depicted in transparent plum surface, the inhibitor
E and Z isomers are in polytube purple or green carbon atoms respectively, hMAO-A and -B mutated residues are in polytube white carbon atoms, FAD cofactor is reported in
spacefill CPK colored.
were carried out on a rotary evaporator (Büchi Rotavapor) operat-
ing at reduced pressure.
8.3. General procedure for the MW-assisted synthesis of
derivatives (1–8 and (R)-8)
8.2. General procedure for the MW-assisted synthesis of
thiosemicarbazone intermediates
The thiosemicarbazone intermediates (1.5 mmol) were added
to a solution of the correspondent
a-halo-2-/2,4-substituted-ace-
tophenone in 2 mL of methanol. The mixture was pre-stirred in a
sealed vessel for 1 min and then heated up to 90 °C by microwave
irradiation for 10 min. The reaction was cooled down with pressur-
ized air, filtered, and the obtained solid washed with n-hexane and
diethyl ether. The crude mixture was purified by column chroma-
tography (SiO2, ethyl acetate/n-hexane 1:1) to give desired com-
pounds in high yields (81–99%).
In a 5 mL vessel suitable for microwave reactor (2.45 GHz high-
frequency microwaves, power range 0–300 W) 2- or 3-methylcy-
clopentanone (3.4 mmol) and acetic acid (2–3 drops) were added
to a suspension of thiosemicarbazide (0.30 g, 3.4 mmol) in 2 mL
of absolute ethanol. After the vessel was securely sealed, the mix-
ture was pre-stirred for 30 s and then heated up to 103 °C by
microwave irradiation for 5 min. If not set, the irradiation power
reaches its maximum at the beginning of reaction and then it de-
creases to lower and constant values. The vial internal temperature
was controlled by an equipped IR sensor. After cooling the reaction
down to room temperature using a stream of pressurized air, the
obtained suspension was filtered and the solid washed with petro-
leum ether, n-hexane, and diethyl ether. The crude mixture was
purified by column chromatography (SiO2, ethyl acetate/n-hexane)
to give the desired thiosemicarbazone derivatives.
8.4. Characterization data for new compounds (obtained as a
mixture of E/Z isomers)
8.4.1. 1-(4-(4-Fluorophenyl)thiazol-2-yl)-2-(2-methyl-
cyclopentylidene)hydrazine (1)
E/Z ratio: 56/44. Yield 98%; mp 174–176 °C; IR cmÀ1 (KBr): 1612
(C@N), 1229 (Ar-F); 1H NMR (CDCl3, 600 MHz) d 1.20–1.22 (d,
J = 6.8 Hz, 3H, CH3), 1.35–1.36 (m, 1H, cyclopentyl), 1.54–1.55 (m,

M. D’Ascenzio et al. / Bioorg. Med. Chem. 22 (2014) 2887–2895
2893
1H, cyclopentyl), 1.77–1.78 (m, 1H, cyclopentyl), 2.01–2.03 (m, 1H,
cyclopentyl), 2.12–2.14 (m, 1H, cyclopentyl), 2.55–2.57 (m, 1H,
cyclopentyl), 2.74–2.76 (m, 1H, cyclopentyl), 6.63 (s, 1H, C5H-thia-
zole), 7.18–7.20 (dd, JHH = 8.6 Hz, JHF = 8.6 Hz, 2H, Ar), 7.70–7.72
(dd, JHH = 8.6 Hz, JHF = 5.0 Hz, 2H, Ar), 12.35 (br s, 1H, (E)-NH, D2O
exch.), 14.25 (br s, 1H, (Z)-NH, D2O exch.). 13C NMR (CDCl3,
150 MHz) d 16.70 (CH3), 22.71 (CH2, cyclopentyl), 30.50 (CH, cyclo-
pentyl), 33.95 (CH2, cyclopentyl), 40.15 (CH2, cyclopentyl), 100.21
(CH, thiazole), 116.93 (d, JC–F = 22 Hz, CH, Ar), 123.99 (d, JC–
F = 3.5 Hz, C, Ar), 127.82 (d, JC–F = 6.7 Hz, CH, Ar), 139.54 (C, thia-
zole), 163.80 (d, JC–F = 252 Hz, CF, Ar), 169.65 (C, thiazole), 174.85
(C@N). Anal. Calcd for C15H16FN3S: C, 62.26; H, 5.57; N, 14.52.
Found: C, 62.57; H, 5.39; N, 14.39.
3.4 Hz, CH, Ar), 129.26 (d, JC–F = 10 Hz, CH, Ar), 133.48 (C, thiazole),
160.30 (dd, JC–F = 255 Hz, 12 Hz, CF, Ar), 163.58 (dd, JC–F = 255 Hz,
12 Hz, CF, Ar), 169.08 (C, thiazole), 175.00 (C@N). Anal. Calcd for
C15H15F2N3S: C, 58.62; H, 4.92; N, 13.67. Found: C, 58.89; H,
5.12; N, 13.49.
8.4.5. 1-(4-(4-Fluorophenyl)thiazol-2-yl)-2-(3-methylcyclopen-
tylidene)hydrazine (5)
Yield 89%; mp 225–227 °C; IR cmÀ1 (KBr): 1615 (C@N), 1235
(Ar-F); 1H NMR (CDCl3, 600 MHz) d 1.09–1.11 (d, J = 6.3 Hz, 3H,
CH3), 1.38–1.40 (m, 1H, cyclopentyl), 1.50–1.51 (m, 1H, cyclopen-
tyl), 2.01–2.03 (m, 1H, cyclopentyl), 2.17–2.19 (m, 1H, cyclopen-
tyl), 2.23–2.25 (m, 1H, cyclopentyl), 2.57–2.58 (m, 1H,
cyclopentyl), 2.71–2.73 (m, 1H, cyclopentyl), 6.63 (s, 1H, C5H-thia-
zole), 7.18–7.20 (dd, JHF = 8.5 Hz, JHH = 8.5 Hz, 2H, Ar), 7.70–7.72
(dd, JHH = 8.6 Hz, JHF = 5.0 Hz, 2H, Ar), 12.23 (br s, 1H, NH, D2O
exch.). 13C NMR (CDCl3, 150 MHz) d 19.56, 19.96 (CH3, cyclopen-
tyl), 30.41, 32.74, 33.07, 33.08, 33.56, 33.75, 38.34, 41.56 (CH,
8.4.2. 1-(2-Methylcyclopentylidene)-2-(4-(4-nitrophenyl)thiazol-
2-yl)hydrazine (2)
Yield 97%; mp 170–172 °C; cmÀ1 (KBr): 1629 (C@N), 1510 and
1339 (NO2); 1H NMR (DMSO-d6, 400 MHz) d 1.09 (br s, 3H, CH3),
1.28–1.29 (m, 1H, cyclopentyl), 1.64–1.65 (m, 1H, cyclopentyl),
1.87–1.88 (m, 1H, cyclopentyl), 1.97–1.98 (m, 1H, cyclopentyl), 2.23–
2.25 (m, 1H, cyclopentyl), 2.58–2.60 (m, 1H, cyclopentyl), 2.61–
2.63 (m, 1H, C2H), 7.63 (s, 1H, C5H-thiazole), 8.09–8.11 (d,
J = 7.8 Hz, 2H, Ar), 8.26–8.28 (d, J = 7.8 Hz, 2H, Ar), 10.75 (br
s, 1H, NH, D2O exch.); 13C NMR (DMSO-d6, 101 MHz) d 17.33 (CH3),
22.72 (CH2, cyclopentyl), 29.28 (CH, cyclopentyl), 33.88 (CH2,
cyclopentyl), 108.71 (C, thiazole), 124.48 (2 x CH, Ar), 126.76
(2 Â CH, Ar), 129.09 (C, Ar), 140.85 (C, thiazole), 146.59 (C, Ar),
165.49 (C, thiazole), 170.71 (C@N), (CH2-cyclopentyl signal missing
due to overlap with DMSO-d6). Anal. Calcd for C15H16N4O2S: C,
56.94; H, 5.10; N, 17.71. Found: C, 57.13; H, 4.98; N, 17.99.
CH2, cyclopentyl), 100.10, 100.14 (CH, thiazole), 116.95 (d, JC–F
=
22 Hz, CH, Ar), 123.98 (d, JC–F = 3.5 Hz, C, Ar), 127.84 (d, JC–F = 8.7
Hz, CH, Ar), 139.55, 139.60 (C, thiazole),163.84 (d, JC–F = 252 Hz,
CF, Ar), 169.41, 169.47 (C, thiazole), 172.14, 172.24 (C@N). Anal.
Calcd for C15H16FN3S: C, 62.26; H, 5.57; N, 14.52. Found: C,
61.99; H, 5.41; N, 14.29.
8.4.6. 1-(3-Methylcyclopentylidene)-2-(4-(4-nitrophenyl)thiazol-
2-yl) hydrazine (6)
Yield 96%; mp 176–177 °C; IR cmÀ1 (KBr): 1620 (C@N), 1517
and 1346 (NO2); 1H NMR (DMSO-d6) d 1.03 (s, 3H, CH3), 1.70–1.72
(m, 1H, cyclopentyl), 1.89–1.91 (m, 1H, cyclopentyl), 1.93–1.96
(m, 1H, cyclopentyl), 2.02–2.04 (m, 1H, cyclopentyl), 2.35–2.37 (m,
1H, cyclopentyl), 2.47–2.49 (m, 1H, cyclopentyl), 2.75–2.77
(m, cyclopentyl), 7.62 (s, 1H, C5H-thiazole), 8.08–8.10 (d,
J = 8.9 Hz, 2H, Ar), 8.25–8.27 (d, J = 8.9 Hz, 1H, Ar), 10.74 (br s,
1H, NH, D2O exch.); 13C NMR (DMSO-d6, 101 MHz) d 19.83
(CH3), 20.34 (CH2, cyclopentyl), 29.21 (CH, cyclopentyl), 32.93
(CH2, cyclopentyl), 41.36 (CH2, cyclopentyl), 108.66 (C, thiazole),
124.51 (2 Â CH, Ar), 126.85 (2 Â CH, Ar), 140.65 (C, Ar), 146.61
(C, thiazole), 147.76 (C, Ar), 163.27 (C, thiazole), 170.30 (C@N).
Anal. Calcd for C15H16N4O2S: C, 56.94; H, 5.10; N, 17.71. Found:
C, 57.20; H, 4.90; N, 18.01.
8.4.3. 1-(4-(4-Cyanophenyl)thiazol-2-yl)-2-(2-
methylcyclopentylidene)hydrazine (3)
E/Z ratio: 54/46. Yield 98%; mp 178–180 °C; IR cmÀ1 (KBr): 2231
(C„N), 1619 (C@N); 1H NMR (CDCl3, 400 MHz) d 1.20–1.22
(d, J = 6.7 Hz, 3H, CH3), 1.22–1.23 (m, 1H, C3H), 1.81–1.82 (m,
1H, cyclopentyl), 2.02–2.04 (m, 1H, cyclopentyl), 2.11–2.13 (m, 1H,
cyclopentyl), 2.60–2.62 (m, 1H, cyclopentyl), 2.63–2.65 (m, 1H,
cyclopentyl), 2.73–2.75 (m, 1H, cyclopentyl), 6.88 (s, 1H, C5H-thia-
zole), 7.78–7.80 (d, J = 6.2 Hz, 2H, Ar), 7.85–7.87 (d, J = 6.2 Hz, 2H,
Ar), 12.18 (br s, 1H, (E)-NH, D2O exch.), 14.15 (br s, 1H, (Z)-NH,
D2O exch.); 13C NMR (DMSO-d6, 101 MHz) d 17.36 (CH3), 22.72
(CH2, cyclopentyl), 29.29 (CH, cyclopentyl), 33.87 (CH2, cyclopen-
tyl), 107.76 (C, thiazole), 110.06 (C, Ar), 119.40 (C„N), 126.61
(2 Â CH, Ar), 133.12 (2 Â CH, Ar), 138.74 (C, Ar), 147.94 (C, thia-
zole), 165.83 (C, thiazole), 170.61 (C@N), (CH2-cyclopentyl signal
missing due to overlap with DMSO-d6). Anal. Calcd for
8.4.7. 1-(4-(4-Cyanophenyl)thiazol-2-yl)-2-(3-methylcyclopen-
tylidene)hydrazine (7)
E/Z ratio: 54/46. Yield 98%; mp 180–182 °C; IR cmÀ1 (KBr): 2229
(C„N), 1620 (C@N); 1H NMR (CDCl3, 400 MHz) d 1.09–1.11 (br s,
3H, CH3), 1.54–1.55 (m, 1H, cyclopentyl), 1.56–1.58 (m, 1H, cyclo-
pentyl), 1.59–1.61 (m, 1H, cyclopentyl), 2.36–2.38 (m, 1H, cyclo-
pentyl), 2.52–2.54 (m, 1H, cyclopentyl), 2.66–2.68 (m, 1H,
cyclopentyl), 2.83–2.85 (m, 1H, cyclopentyl), 6.87 (s, 1H, C5H-thia-
zole), 7.77–7.78 (d, Jo = 8.2 Hz, 2H, Ar), 7.84–7.87 (d, Jo = 8.2 Hz, 2H,
Ar), 12.15 (br s, 1H, (E)-NH, D2O exch.), 14.21 (br s, 1H, (Z)-NH, D2O
exch.); 13C NMR (DMSO-d6, 101 MHz) d 19.82 (CH3), 20.32 (CH2,
cyclopentyl), 29.32 (CH, cyclopentyl), 32.93 (CH2, cyclopentyl),
41.37 (CH2, cyclopentyl), 107.74 (C, thiazole), 110.20 (C, Ar),
119.35 (CꢀN), 126.70 (2 Â CH, Ar), 133.12 (2 Â CH, Ar), 138.32
(C, Ar), 147.32 (C, thiazole), 163.94 (C, thiazole), 170.14 (C@N).
Anal. Calcd for C16H16N4S: C, 64.84; H, 5.44; N, 18.90. Found: C,
65.04; H, 5.29; N, 19.10.
C16H16N4S: C, 64.84; H, 5.44; N, 18.90. Found: C, 65.01; H, 5.23;
N, 19.19.
8.4.4. 1-(4-(2,4-Difluorophenyl)thiazol-2-yl)-2-(2-methylcyclop-
entylidene)hydrazine (4)
E/Z ratio: 56/44. Yield 99%; mp 160–163 °C; IR cmÀ1 (KBr): 3490
(N–H), 1618 (C@N), 1235 (Ar-F); 1H NMR (CDCl3, 600 MHz) d
1.20–1.22 (d, J = 6.7 Hz, 3H, CH3), 1.27–1.28 (m, 1H, cyclopentyl),
1.66–1.67 (m, 1H, cyclopentyl), 1.77–1.78 (m, 1H, cyclopentyl), 2.60–
2.62 (m, 1H, cyclopentyl), 2.09–2.10 (m, 1H, cyclopentyl), 2.60–
2.62 (m, 1H, cyclopentyl), 2.71–2.73 (m, cyclopentyl), 6.95 (s, 1H,
C5H-thiazole), 6.98–6.99 (ddd, JHF = 11.4, 8.4 Hz, JHH = 2.1 Hz,1H,
Ar), 7.07–7.08 (dd, JHH = 8.5 Hz, JHF = 8.5 Hz, 1H, Ar), 7.85–7.87
(ddd, JHH = 8.2 Hz, JHF = 8.2, 6.2 Hz, 1H, Ar), 12.41 (br s, 1H, (E)-
NH, D2O exch.), 14.44 (br s, 1H, (Z)-NH, D2O exch.). 13C NMR (CDCl3,
150 MHz) d 16.71 (CH3), 22.73 (CH2, cyclopentyl), 30.61 (CH, cyclo-
pentyl), 33.97 (CH2, cyclopentyl), 40.18 (CH2, cyclopentyl), 104.78
(d, JC–F = 15.7 Hz, CH, thiazole), 105.45 (t, JC–F = 26 Hz, CH, Ar),
112.69 (dd, JC–F = 12 Hz, 3.6 Hz, C, Ar), 113.17 (dd, JC–F = 22 Hz,
8.4.8. 1-(4-(2,4-Difluorophenyl)thiazol-2-yl)-2-(3-methylcyclop-
entylidene)hydrazine (8)
E/Z ratio: 53/47. Yield 88%; mp 131–134 °C; IR cmÀ1 (KBr): 3498
(N–H), 1598 (C@N), 1238 (Ar-F); 1H NMR (CDCl3, 600 MHz) d 1.11–
1.12 (br s, 3H, CH3), 1.42–1.45 (m, 1H, cyclopentyl), 1.47–1.51 (m,
1H, cyclopentyl), 2.43–2.46 (m, 1H, cyclopentyl), 2.61–2.64 (m, 1H,

2894
M. D’Ascenzio et al. / Bioorg. Med. Chem. 22 (2014) 2887–2895
cyclopentyl), 2.67–2.69 (m, 1H, cyclopentyl), 2.78–2.80 (m, 1H,
cyclopentyl), 2.84–2.86 (m, 1H, cyclopentyl), 6.91 (s, 1H, C5H-thia-
zole), 6.94–6.99 (m, 1H, Ar) 7.07–7.11 (m, 1H, Ar), 7.88–7.91 (m,
1H, Ar), 12.36 (br s, 1H, (E)-NH, D2O exch.), 14.40 (br s, 1H, (Z)-
NH, D2O exch.). 13C NMR (DMSO-d6, 150 MHz) d 19.77, 20.30
(CH3, cyclopentyl), 29.04, 32.73, 32.81, 32.86, 33.00, 33.21, 37.44,
41.30 (CH, CH2 cyclopentyl), 104.89 (t, JC–F = 26 Hz, CH, Ar),
107.68 (d, JC–F = 13 Hz, CH, thiazole), 112.16 (dd, JC–F = 21 Hz,
3.6 Hz, CH, Ar), 119.21 (C, Ar), 130.77 (dd, JC–F = 10 Hz, 4.5 Hz, CH,
Ar), 142.66 (C, thiazole), 159.89 (dd, JC–F = 252 Hz, 12 Hz, CF, Ar),
161.61 (dd, JC–F = 247 Hz, 12 Hz, CF, Ar), 162.79 (C, thiazole),
169.40 (C@N). Anal. Calcd for C15H15F2N3S: C, 58.62; H, 4.92; N,
13.67. Found: C, 58.96; H, 5.19; N, 13.89.
carried out with a Perkin–Elmer (Norwalk, CT, USA) 200 LC pump
equipped with a Rheodyne (Cotati, CA, USA) injector, a 1 mL sam-
ple loop, a Perkin-Elmer LC 101 oven, and a Waters 484 detector
(Waters Corporation, Milford, MA, USA). The signal was acquired
and processed by Clarity software (DataApex, Prague, The Czech
Republic).
8.4.13. Polarimetry
Specific rotations of the distereomers of (R)-8 were measured at
589 nm by a Perkin-Elmer polarimeter model 241 equipped with a
Na/Hg lamp. The volume of the cell was 1 mL and the optical path
was 10 cm. The system was set to 20 °C.
8.4.14. E/Z Assignment of geometric isomers of compound (R)-8
Sample was dissolved in DMSO-d6. The NMR spectra were re-
corded at 27 °C on a Bruker AVANCE600 NMR spectrometer oper-
ating at the proton frequency of 600.13 MHz and equipped with
a Bruker multinuclear z-gradient inverse probehead. Chemical
shifts (d-scale) of proton spectra are reported with respect to the
solvent residual signal at 2.50 ppm. 1H–1H COSY experiment was
acquired using 2048 data points in f2, 256 increments in f1, spec-
tral width 6.6 kHz in both dimensions, 3 s recycle delay. 1H–1H
NOESY experiment was recorded in the phase sensitive mode
(TPPI) with a mixing time of 0.8 s and by using the following
parameters: a spectral width of 6.6 kHz in both dimensions, 1024
data points in f2, 512 increments in f1, a recycle delay of 3 s, 16
dummy scans and 16 scans; the data were processed with an
squared sine window function and a 1024 Â 1024 data matrix size.
8.4.9. (R)-1-(4-(2,4-Difluorophenyl)thiazol-2-yl)-2-(3-methylcyc-
lopentylidene)hydrazine ((R)-8)
E/Z ratio: 57/43. Yield 97%; mp 134–135 °C; IR cmÀ1 (KBr): 3474
(N–H), 1597 (C@N), 1234 (Ar-F); 1H NMR (CDCl3, 400 MHz) d 1.10–
1.16 (m, 3H, CH3), 1.45–1.47 (m, 1H, cyclopentyl), 1.55–1.57
(m, 1H, cyclopentyl), 2.12–2.13 (m, 1H, cyclopentyl), 2.15–2.17 (m,
1H, cyclopentyl), 2.22–2.24 (m, 1H, cyclopentyl), 2.52–2.53 (m,
1H, cyclopentyl), 2.67–2.68 (m, 1H, cyclopentyl), 6.94 (s, 1H, C5H-
thiazole), 7.00–7.03 (m, 1H, Ar), 7.06–7.08 (m, 1H, Ar), 7.85–7.87
(m, 1H, Ar), 12.37 (br s, 1H, (E)-NH, D2O exch.), 14.25 (br s, 1H,
(Z)-NH, D2O exch.). Anal. Calcd for C15H15F2N3S: C, 58.62; H, 4.92;
N, 13.67. Found: C, 58.34; H, 4.79; N, 13.89.
8.4.10. (Z)-(R)-1-(4-(2,4-Difluorophenyl)thiazol-2-yl)-2-(3-methyl-
cyclopentylidene)hydrazine ((Z)-(R)-8)
1H NMR (DMSO-d6, 600.13 MHz) d 1.04 (d, J = 6.6 Hz, 3H, CH3),
1.29 (m, 1H, CH2 (c)), 1.88 (m, 2H, CH2 (c), CH2 (a)), 2.15 (m, 1H,
CH (b)), 2.39 (dddd, J = 17.0; 9.8; 7.8; 2.0 Hz, 1H, CH2 (d)), 2.43
(ddd, J = 17.0; 7.8; 3.8 Hz, 1H, CH2 (d)), 2.66 (dd, J = 18.2; 7.8 Hz,
1H, CH2 (a)), 7.11 (d, JH–F = 2.5, 1H, C5H-thiazole), 7.16 (ddd,
8.4.15. Determination of hMAO isoform activity
The effects of the test compounds on the enzymatic activity of
hMAO isoform were evaluated by a fluorimetric method following
the experimental protocol previously described by us.19 Sodium
phosphate buffer (0.1 mL, 0.05 M, pH 7.4) containing different con-
centrations of the test drugs (new compounds or reference inhibi-
tors) and adequate amounts of recombinant hMAO-A or hMAO-B
required and adjusted to obtain the same reaction velocity in our
experimental conditions, that is, to oxidize (in the control group)
165 pmol of p-tyramine/min, were incubated for 15 min at 37 °C
in a flat-black-bottom 96-well microtest™ plate (BD Biosciences,
Franklin Lakes, NJ, USA) placed in a dark fluorimetric chamber.
After this incubation period, the reaction was started by adding (fi-
JH–H = 8.8; 2.7 Hz, JH–F = 8.8 Hz, 1H, C3H, Ar), 7.32 (ddd, JH–H
=
2.7 Hz, JH–F = 11.7; 9.3 Hz, 1H, C5H, Ar), 8.03 (ddd, JH–H = 8.8 Hz,
JH–F = 8.8; 6.9 Hz 1H, C2H, Ar), 10.58 (br s, 1H, (Z)-NH, D2O exch.).
13C NMR (DMSO-d6, 150 MHz) d 20.60 (CH3, cyclopentyl), 33.00,
33.10, 33.40, 37.50 (CH, CH2 cyclopentyl), 105.00 (t, JC–F = 26 Hz,
CH, Ar), 107.80 (d, JC–F = 13 Hz, CH, thiazole), 112.30 (dd, JC–F
=
21 Hz, 3.6 Hz, CH, Ar), 119.21 (C, Ar), 130.80 (dd, JC–F = 10 Hz,
4.5 Hz, CH, Ar), 159.89 (dd, JC–F = 252 Hz, 12 Hz, CF, Ar), 161.61
(dd, JC–F = 247 Hz, 12 Hz, CF, Ar), 162.79 (C, thiazole), 169.40 (C@N).
nal concentrations) 200 l
M AmplexÒ Red reagent, 1 U/mL horse-
radish peroxidase and 1 mM p-tyramine. The production of H2O2
and, consequently, of resorufin was quantified at 37 °C in a mul-
tidetection microplate fluorescence reader (Fluostar Optima, BMG
Labtech GmbH, Offenburg, Germany) based on the fluorescence
generated (excitation, 545 nm, emission, 590 nm) over a 15 min
period, in which the fluorescence increased linearly.
8.4.11. (E)-(R)-1-(4-(2,4-Difluorophenyl)thiazol-2-yl)-2-(3-
methylcyclopentylidene)hydrazine ((E)-(R)-8)
1H NMR (DMSO-d6; 600.13 MHz) d 1.02 (d, J = 6.6 Hz, 3H, CH3),
1.37 (m, 1H, CH2 (c)), 1.95 (m, 1H, CH2 (c)), 2.01 (m, 1H, CH2 (a)),
2.06 (m, 1H, CH (b)), 2.30 (m, 1H, CH2 (d)), 2.50 (m, 1H, CH2 (d),
CH2 (a)), 7.11 (d, JH–F = 2.5, 1H, C5H-thiazole), 7.16 (ddd, JH–H
=
Control experiments were carried out simultaneously by replac-
ing the test drugs with appropriate dilutions of the vehicles. In
addition, the potential ability of the above test drugs to modify
the fluorescence generated in the reaction mixture due to non-
enzymatic inhibition was determined by adding these drugs to
solutions containing only the AmplexÒ Red reagent in a sodium
phosphate buffer. To determine the kinetic parameters of hMAO-
A and hMAO-B (Km and Vmax), the corresponding enzymatic activ-
ity of both isoforms was evaluated in presence of a wide range of
p-tyramine concentrations. The specific fluorescence emission
was calculated after subtraction of the background activity, which
was determined from vials containing all components except the
hMAO isoforms.
8.8; 2.7 Hz, JH–F = 8.8 Hz, 1H, C3H, Ar), 7.32 (ddd, JH–H = 2.7 Hz,
JH–F = 11.7; 9.3 Hz, 1H, C5H, Ar), 8.03 (ddd, JH–H = 8.8 Hz, JH–F = 8.8;
6.9 Hz 1H, C2H, Ar), 10.60 (br s, 1H, (E)-NH, D2O exch.).
8.4.12. HPLC diastereoseparation
Analytical HPLC analysis of rac-8 was performed using commer-
cially available 250 mm  4.6 mm I.D. Chiralpak AD column (Chiral
Technologies Europe, Illkirch, France). HPLC grade solvents were
purchased from Aldrich (St. Louis, Missouri USA). Semi-preparative
HPLC diastereoseparation of (R)-8 was performed using commer-
cially available 250 mm  10 mm I.D. Chiralpak AD column. The
analytical HPLC apparatus consisted of a Perkin–Elmer (Norwalk,
CT, USA) 200 LC pump equipped with a Rheodyne (Cotati, CA,
USA) injector, a 20 mL sample loop, an HPLC Dionex CC-100 oven
(Sunnyvale, CA, USA) and a Jasco Model CD 2095 Plus UV/CD detec-
tor (Jasco, Tokyo, Japan). Semi-preparative separations were
The drugs, vehicle and chemicals used in the experiments were
the new compounds, moclobemide (F. Hoffmann-La Roche Ltd,
Basel, Switzerland), dimethyl sulfoxide, R-(À)-deprenyl hydrochlo-
ride, iproniazid phosphate and isatin (Sigma–Aldrich, Spain),

M. D’Ascenzio et al. / Bioorg. Med. Chem. 22 (2014) 2887–2895
2895
resorufin sodium salt, clorgyline hydrochloride, p-tyramine hydro-
References and notes
chloride, sodium phosphate and horseradish peroxidase (supplied
in the AmplexÒ Red MAO assay kit from Molecular Probes). Unless
otherwise specified, results shown in the text and tables are ex-
pressed 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 (AN-
OVA) followed by the Dunnett’s post-hoc test. Graph Pad Prism
Software (GraphPad Software, San Diego, California, USA) was used
to perform statistical analyses and to calculate IC50 values and ki-
netic parameters.
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8.4.16. Molecular modeling
The Scrodinger Suite 2013-1 was used for all molecular model-
ing tasks.20 The hMAO-A and hMAO-B enzyme models were ob-
tained from the Protein Data Bank21 (PDB) crystallographic
structures 2Z5X22 and 2V5Z,23 respectively. The selected PDB en-
tries represented the best crystallographic resolution structures
of human MAO isoforms in non-covalent complexes with known
inhibitors available. In both enzyme models the original co-crystal-
lized ligands and water molecules were removed, FAD double
bonds were fixed and hydrogen atoms were added. The conforma-
tional properties of both E and Z isomers of (R)-8 were explored by
means of 2000 steps of Monte Carlo (MC) search applied to the li-
gands rotatable bonds. The energy evaluation of the MC structures
was carried out with the OPLS-AA24 force field and solvent effects
were taken into using the implicit solvation model GB/SA water.25
Both (E)-(R)-8 and (Z)-(R)-8 highlighted 6 unique conformations
within 12 kcal/mol above the global minimum energy ones. The
energy difference between the most energy stable conformers of
E and Z isomers was equal to 0.89 kcal/mol in favor of the E form.
Boltzmann analysis, computed at 300 K, was applied to MC ensem-
bles. The global minimum energy structures, in both cases report-
ing a population larger than 86%, were submitted to Glide26
docking simulations with respect to hMAO-A and -B theoretical
models. A regular box, of about 110,000 Å3, centered onto the
FAD N5 atom, was considered as the enzyme active site for both
hMAO-A and -B. Induced fit phenomena were taken into account
using the ligand flexible algorithm. The binding affinity was esti-
mated by means of the Glide XP scoring function (GScoreXP). The
top ranked complexes were considered for the binding modes
graphical analysis. PyMol software27 was used for depicting molec-
ular modeling figures and for calculating the enzyme bind clefts
shape.
23. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.
E.; Mattevi, A. J. Med. Chem. 2007, 50, 5848.
24. Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. J. J. Phys. Chem. B
2001, 105, 6474.
25. Hasel, W.; Hendrickson, T. F.; Still, W. C. Tetrahedron Comput. Methodol. 1988, 1,
103.
26. (a) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.;
Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem. 2006, 49, 6177; (b)
Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W.
T.; Banks, J. L. J. Med. Chem. 2004, 47, 1750; (c) Friesner, R. A.; Banks, J. L.;
Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E.
H.; Shaw, D. E.; Shelley, M.; Perry, J. K.; Francis, P.; Shenkin, P. S. J. Med. Chem.
2004, 47, 1739.
Acknowledgments
This work was supported by ‘FILAS’ - ‘Italy’ (research project n°
ASR2, Regione Lazio, Italy).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.042.
27. The PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC. http://
www.schrodinger.com.