Synthesis, semipreparative HPLC separation, biological evaluation, and 3D-QSAR of hydrazothiazole derivatives as human monoamine oxidase B inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.05.070

The present study reports on synthesis in high yields (70-99%), HPLC enantioseparation, inhibitory activity against human monoamino oxidases, and molecular modeling including 3D-QSAR studies, of a large series of (4-aryl-thiazol-2-yl)hydrazones (1-45). Most of the synthesized compounds proved to be potent and selective inhibitors of hMAO-B isoform in the micromolar or nanomolar range, thus demonstrating that hydrazothiazole could be considered a good pharmacophore to design new hMAO-B inhibitors. Due to the presence in some derivatives of a chiral center, we also performed a semipreparative chromatographic enantioseparation of these compounds obtained by a stereoconservative pattern. The separated enantiomers were submitted to in vitro biological evaluation to point out the stereorecognition of the active site of the enzyme towards these structures. Finally, a 3D-QSAR study was carried out using Comparative Molecular Field Analysis (CoMFA), aiming to deduce rational guidelines for the further structural modification of these lead compounds.

Full text of DOI:10.1016/j.bmc.2010.05.070

Bioorganic & Medicinal Chemistry 18 (2010) 5063–5070
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, semipreparative HPLC separation, biological evaluation, and
3D-QSAR of hydrazothiazole derivatives as human monoamine oxidase
B inhibitors
a
a
a
Franco Chimenti a, Daniela Secci a, , Adriana Bolasco , Paola Chimenti , Arianna Granese ,
Simone Carradori a, Elias Maccioni b, M. Cristina Cardia b, Matilde Yáñez c, Francisco Orallo c, Stefano Alcaro d,
Francesco Ortuso d, Roberto Cirilli e, Rosella Ferretti e, Simona Distinto d,g, Johannes Kirchmair f,
Thierry Langer g
*
a Dipartimento di Chimica e Tecnologie del Farmaco Università degli Studi di Roma ‘La Sapienza’, P.le A. Moro 5, 00185 Roma, Italy
b Dipartimento Farmaco Chimico Tecnologico, Università di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
c Departamento de Farmacología and Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur,
E-15782 Santiago de Compostela (La Coruña), Spain
d Dipartimento di Scienze Farmaco Biologiche ‘Complesso Ninì Barbieri’, Università degli Studi di Catanzaro ‘Magna Graecia’, 88021 Roccelletta di Borgia (CZ), Italy
e Istituto Superiore di Sanità, Dipartimento del Farmaco, V.le Regina Elena 299, 00161 Roma, Italy
f Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 52, A-6020 Innsbruck, Austria
g Inte:Ligand Software-Entwicklungs und Consulting GmbH, Clemens Maria Hofbauer-Gasse 6, A-2344 Maria Enzersdorf, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study reports on synthesis in high yields (70–99%), HPLC enantioseparation, inhibitory activ-
ity against human monoamino oxidases, and molecular modeling including 3D-QSAR studies, of a large
series of (4-aryl-thiazol-2-yl)hydrazones (1–45). Most of the synthesized compounds proved to be potent
and selective inhibitors of hMAO-B isoform in the micromolar or nanomolar range, thus demonstrating
that hydrazothiazole could be considered a good pharmacophore to design new hMAO-B inhibitors.
Due to the presence in some derivatives of a chiral center, we also performed a semipreparative chro-
matographic enantioseparation of these compounds obtained by a stereoconservative pattern. The sepa-
rated enantiomers were submitted to in vitro biological evaluation to point out the stereorecognition of
the active site of the enzyme towards these structures. Finally, a 3D-QSAR study was carried out using
Comparative Molecular Field Analysis (CoMFA), aiming to deduce rational guidelines for the further
structural modification of these lead compounds.
Received 17 March 2010
Revised 25 May 2010
Accepted 26 May 2010
Available online 1 June 2010
Keywords:
Hydrazothiazole derivatives
3D-QSAR
Human monoamine oxidase B inhibitors
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
with respect to substrate specificity, sensitivity to inhibitors, and
tissue distribution.
Mitochondrial monoamine oxidases (MAOs, EC 1.4.3.4) are
flavin-containing enzymes (FAD) that catalyze the oxidative deam-
ination of neurotransmitters and exogenous arylalkylamines. In
mammals, two different types of MAOs are present in most tissues,
namely, MAO-A and MAO-B. MAO-A preferentially deaminates
aromatic monoamines such as the neurotransmitters serotonin
(5-HT), noradrenaline (NA), and adrenaline (A), while MAO-B
Although MAOs are widely distributed in various organs, most
of the studies concerning their functional properties and involve-
ment in pathological processes have been mainly focused on the
central nervous system. In the periphery, MAO-A and MAO-B are
differently expressed in a variety of tissues: MAO-A is predominant
in heart, adipose tissue, and skin fibroblasts, MAO-B is the major
form found in platelets and lymphocytes, whereas both isoen-
zymes are expressed in kidney and liver.2 It has been recently iden-
tified an additional MAO mechanism which is involved in reactive
oxygen species (ROS) production and the consequent induction of
intracellular oxidative stress. Hydrogen peroxide (H2O2) is one of
the reaction products generated by MAOs during substrate
degradation. Except for the potential cytotoxic effect of H2O2 in ni-
gral cells in Parkinson’s disease, the cell events following MAO-
dependent H2O2 production in physiological conditions are still
mainly oxidizes
a-phenylethylamines and benzylamine. Both
isoforms act on dopamine (D) and tryptamine.1
These enzymes are tightly bound to the mitochondrial outer
membrane, sharing about 70% amino acid identity. They also differ
* Corresponding author. Tel.: +39 06 4991 3763; fax: +39 06 49913772.
E-mail address: daniela.secci@uniroma1.it (D. Secci).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.070

5064
F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5063–5070
unknown. These findings, along with other results showing the
important role of ROS generated by MAOs in heart, adipose tissue,
macrophages, and skeletal muscle, pointed to the relevance also of
peripheral MAOs in various pathological processes and their poten-
tiality as pharmacological agents.3
The discovery of reversible and selective inhibitors has renewed
the interest towards MAO enzymes as drug targets. In fact, irre-
versible and/or nonselective inhibitors showed shortcomings
including cumulative effects, loss of selectivity after chronic
treatment, and interaction with tyramine-containing foods
(cheese-effect). Selective and reversible hMAO-A inhibitors
(hMAO-AIs) are currently used in the treatment of depression
and anxiety disorders, whereas hMAO-B inhibitors (hMAO-BIs)
are used as adjuncts in the treatment of Parkinson’s disease (PD).
MAO-B predominates over MAO-A in the human brain especially
after the 60th year of life, where hMAO-BIs have proven to be ben-
studies.11 The same structural features are found in dopamine, a
MAO-A and MAO-B substrate.
In the past decade, we have reported several series of reversible
and potent MAOIs,12,13 some of which allowed the 2-thiazolylhyd-
razone nucleus to be identified as scaffold for promising hMAO-B
selective inhibitors.14 The cyclo-thiosemicarbazone moiety repre-
sents the pharmacophore portion. Docking studies suggest that
the orientation of the two series, N1-thiocarbamoylpyrazolines
and 2-thiazolylhydrazones, can be compared within the enzymatic
active site as reported in Figure 2.
Furthermore, a large number of substituted hydrazines have
been studied as MAO inhibitors and their specific mechanism of
action was fully investigated.15
Substituents on the aromatic ring at C4 position of thiazole ring
also influenced the activity, as shown by the introduction of several
groups (NO2, CN, CH3, OCH3) or halogens (F, Cl) in the ortho and
para positions,14 while the introduction of more steric hindered
coumarin or naphthalene nuclei at C4 position of thiazole, led to
a decreased hMAO inhibitory activity.16 Different cycloaliphatic
moieties, probed onto the C@N group, allowed us to systematically
explore the steric influence of this substituent on the MAO inhibi-
tor activity. In the past other authors have investigated some hyd-
razothiazoles as regards their potency as rat MAO inhibitors, and
their thermotropic behavior in relation to the partition coefficient
in lipidic membrane models, correlating a different binding of
these inhibitors to the enzyme surface according to their electronic
or lipophilic characteristics.17
eficial in prolonging the anti-Parkinsonian action of
L-DOPA.
hMAO-BIs, which decrease the rate of MAO-B catalyzed oxidative
deamination and, consequently, the production of reactive oxygen
species (ROS), might contribute to the treatment of other neurode-
generative diseases, such as Alzheimer’s disease.4–6
The rational design of new agents targeted to MAOs could be
based on the recent description of the crystal structure of human
MAO-B and MAO-A with their corresponding inhibitors and aided
with theoretical calculations.7,8 The above studies elucidated some
factors responsible for selectivity against the A and B isoforms,
such as the lipophilicity of the inhibitor that is important for
achieving effective binding to MAO-B, the presence of electron-rich
aromatic moieties, typical of selective MAO-A inhibitors, and the
role played by some amino acid residues in the active sites, such
as Tyr326 for hMAO-B and Ile335 for hMAO-A.9
Since our major goal was to identify novel lead compounds with
hMAO inhibitory activity, in this paper we report on the synthesis,
the biological properties, and 3D-QSAR studies of 4-aryl-2-thia-
The aim of the present study was the identification of novel
potent and selective hMAO inhibitors that could serve as potential
lead molecules for drug discovery. An examination of the chemical
structures of MAOIs of clinical interest (Fig. 1) and the analysis of
patents dealing with new inhibitors10 led us to identify some struc-
tural features that characterize these compounds: (i) a basic nitro-
gen atom, sometimes incorporated in an alicyclic ring and (ii) an
electron-rich aromatic moiety that plays a pivotal role in the inter-
action with the biological target, as shown in recent QSAR
Cy
Cy
Cy
Cy
S
N
N
N
N
NH
N
NH
S
S
H2N
H2N
Figure 2. Conversion of N1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-(1H)-pyrazole in
cycloalkylidenhydrazothiazole derivative.
N
CH3
O
N
CH3
N
Cl
N
CH3
H
H
N
CH3
CH3
CH3
O
N
H
Cl
R-(-)-Deprenyl (I) (B)
Pargyline (I) (A/B)
Iproniazide (I) (A/B)
Clorgyline (I) (A)
NH
O
O
O
H
Cl
OH
H3CO
N
N
N
H3C
N
H
N
N
O
H
O
O
O
H3C
Br
Toloxatone (R) (A)
Isocarboxazide (I) (A/B)
Brofaromine (R) (A)
Moclobemide (R) (A)
O
N
NH2
H
N
NH
Cl
Lazabemide (R) (B)
R-Rasagiline (I) (B)
Figure 1. Irreversible (I), reversible (R), and/or selective hMAO-A and hMAO-B inhibitors (A and/or B).

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5063–5070
5065
zolylhydrazone derivatives (1–45) useful to assess a pharmaco-
phore motif common to the active compounds.
reaction was monitored by the same enantioselective HPLC system
used for the semipreparative enantioseparation. The cyclization of
the chiral synthon is stereoconservative as demonstrated by the
high ee values (95–98%) of the obtained thiazoles. The assignment
of the absolute configuration of the enantiomers of compounds
37–45 was determined by chemical correlation using as reference
compound the chiral synthon 2-MCET.
2. Chemistry
All (4-aryl-thiazol-2-yl)hydrazone derivatives 1–45 were syn-
thesized14,16 in high yields (70–99%) as reported in Scheme 1 and
listed in Table 1. Different carbonyl compounds reacted directly
with thiosemicarbazide in ethanol or in 2-propanol with catalytic
amounts of acetic acid, and the obtained thiosemicarbazones were
subsequently converted in (4-aryl-thiazol-2-yl)hydrazones by
4. Biological activity
The inhibitory activity of the test drugs (new compounds and
five reference inhibitors) against hMAOs was evaluated using a
general procedure previously described by us.20 The potential
inhibitory activity was investigated by measuring the effects of
new derivatives on the production of hydrogen peroxide (H2O2)
from p-tyramine (a common substrate for both hMAO-A and
hMAO-B), using the Amplex Red MAO assay kit and microsomal
MAO isoforms prepared from insect cells (BTI-TN-5B1-4) infected
with recombinant baculovirus containing cDNA inserts for
hMAO-A or hMAO-B (Table 1).
Hantzsch reaction with 2- or 2,4-substituted
a-bromo-acetophe-
nones in the same solvent. -Bromoacetophenones have been
a
synthesized by direct halogenation of the corresponding acetophe-
nones with bromine in chloroform.18
All the synthesized compounds were washed with petroleum
ether, hexane or diethyl ether and, if necessary, purified by chro-
matography before characterization by spectroscopic methods
(see Supplementary data).
3. Semipreparative HPLC enantioseparation
5. Reversibility and irreversibility experiments
Owing to the presence of a stereogenic carbon on the cyclohexyl
ring (37–45), some compounds exist as (R)- and (S)-enantiomers.
The first step towards the investigation of the impact of absolute
stereochemistry on the activity and selectivity was to isolate suffi-
cient quantities of the enantiomers of four of the most active com-
pounds to use in a parallel test in vitro. Our strategy was to
synthesize quantitatively the racemic forms and successively re-
solve them by HPLC on polysaccharide-based chiral stationary
phases (CPSs) on a semipreparative scale. A combined method
based on chemical correlation, single-crystal X-ray diffraction,
and enantioselective HPLC allowed us to assign the absolute con-
figuration of the isolated enantiomers. Knowledge of the absolute
configuration and in vitro biological activity of homochiral forms
were needed in the in silico study of the interactions involving
the receptor site. For the stereocontrolled synthesis of the pure
enantiomers of compounds 39, 41, 44, and 45, we used the
enantiopure forms of 2-methylcyclohexylidene thiosemicarbazone
(2-MCET) as chiral synthon, on the basis of a previous study by us
on the enantioseparation and configurational stability of this
compound.19 The racemic 2-MCET obtained in the first step of
the reaction was separated by enantioselective HPLC on a semipre-
parative scale, and the isolated pure enantiomers were used for the
second step. The absolute configuration of the chiral synthon was
established by a single-crystal X-ray diffraction analysis. From
the findings of the X-ray structure analysis, we could assign
unambiguously the (S)-configuration to the C2 stereogenic center
located on the cyclohexyl ring of the (+)-2-MCET enantiomer.
Starting from the enantiopure forms of 2-MCET of known stereo-
chemistry, we obtained enantioenriched forms of the target mole-
cules with correlated chirality. The stereochemical course of the
To evaluate whether these derivatives acted as reversible or
irreversible hMAO inhibitor, an effective centrifugation–ultrafiltra-
tion method (so-called repeated washing), previously described by
us, was used for the most active and selective derivative 44.14 The
data reported in Table 2 showed that this compound acted as an
irreversible inhibitor against both isoforms (using the same IC50
values reported in Table 1), because the percent enzymatic inhibi-
tion was constant after repeated washing as respect to R-(ꢀ)-
deprenyl (irreversible inhibitor), but different with respect to Moc-
lobemide (reversible inhibitor). These results displayed a strong
and long-lasting interaction with the enzyme which could not
restore its enzymatic activity in the presence of the inhibitor. In
most cases, the irreversible inhibitors establish a covalent interac-
tion with the active center of the enzyme.21 However, not all irre-
versible inhibitors form covalent adducts with their enzyme
targets but they may also act by other mechanisms. In fact, some
reversible inhibitors bind so tightly to their target enzyme that
they are essentially irreversible. These tight-binding inhibitors
may show kinetics similar to covalent irreversible inhibitors. In
these cases, some of these inhibitors rapidly bind to the enzyme
in a low-affinity enzyme–inhibitor (EI) complex and this then
undergoes a slower rearrangement to a very tightly bound EI* com-
plex. This kinetic behavior is called slow-binding. This slow rear-
rangement after binding often involves a conformational change
as the enzyme ‘clamps down’ around the inhibitor molecule.22
Also some MAO irreversible inhibitors (the so-called suicide
inhibitors) act as a substrate for the target enzyme, which finally
generates a new compound that irreversibly inhibits MAO activity.
Therefore, the initial interaction of these inhibitors with MAO may
O
H
R'
N
NH2
X
H2N
O
R'
N
NH2
S
n(H2C)
R
H
N
n(H2C)
R
H
N
n(H2C)
R
N
N
S
i
ii
S
R= H; 2-CH3; 4-CH3
n= 1, 2, 3
R'= H; 4-F; 4-Cl; 4-CN; 4-NO2;
4-CH3; 4-OCH3; 2,4-F; 2,4-Cl
X= Cl, Br
1-45
Scheme 1. Reagents and conditions: (i) CH3COOH (cat.), ethanol or 2-propanol, rt; (ii) ethanol, rt.

5066
F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5063–5070
Table 1
Structures, biological activity (IC50), and hMAO-B selectivity ratios for the inhibitory effects of tested compounds 1–45 and reference drugs on enzymatic activity of human
recombinant MAO isoforms expressed in baculovirus infected cells
Compd
R
R0
hMAOA IC50 (nM)
hMAOB IC50 (nM)
Ratio
1
2
3
4
5
6
7
8
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
H
4-Cl
4-F
2,4-Cl
2,4-F
4-CH3
4-OCH3
4-NO2
4-CN
H
7883 91*
296
262
40 0.9
7
8
27
27
111
192
773
15
6
4
3
417
14
438
>495b
70
6
0.6
281
91
132
6
1057
9
214
1.5
7160 640*
4436 212*
54,507 4123*
2318 161*
2019 74*
284 11
3
0.2
134
164 13
9
1055 42*
344 22*
94
3
9
644 21*
221
116
211
2
5
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
(S)-39
(R)-39
40
41
(S)-41
(R)-41
42
43
44
(S)-44
(R)-44
45
(S)-45
(R)-45
C
48,351 1433*
2911 171*
1752 21*
4-Cl
4-F
4
0.2
2,4-Cl
2,4-F
4-CH3
4-OCH3
4-NO2
4-CN
H
N.E.
202 16
652 22
3689 353
11,956 131
86
53
259
2411 30
43
9446 352
91 0.8
5477 461
31,197 1055
362 16
61 0.7
27 0.6
45,754 143*
23,731 324*
7509 213**
24,154 824*
4837 183*
34,223 1486*
15,217 355*
45,457 3119*
81,485 3766*
19,496 1127*
8109 442**
5763 197*
20,882 788*
6994 351*
14,851 739*
6182 173*
10,279 812*
59,280 2845*
7229 490*
9612 716*
3925 84**
17,853 1026*
4327 263*
41,236 3962*
35,220 1813*
43,556 3611*
4943 92*
Cyclohexyl
Cyclohexyl
3
2
4-Methylcyclohexyl
4-Methylcyclohexyl
4-Methylcyclohexyl
4-Methylcyclohexyl
4-Methylcyclohexyl
4-Methylcyclohexyl
4-Methylcyclohexyl
4-Methylcyclohexyl
4-Methylcyclohexyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
Cycloheptyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
2-Methylcyclohexyl
8
4-Cl
4-F
1
2,4-Cl
2,4-F
4-CH3
4-OCH3
4-NO2
4-CN
H
0.2
58
115
550
13
2570
63
452
10
0.8
4-Cl
4-F
477 15
4
0.2
2,4-Cl
2,4-F
4-CH3
4-OCH3
4-NO2
4-CN
H
4-Cl
4-F
4-F
4-F
2,4-Cl
2,4-F
2,4-F
2,4-F
4-CH3
4-OCH3
4-NO2
4-NO2
4-NO2
4-CN
4-CN
4-CN
940 13
16 0.4
920 31
5025 160
11 0.4
46
711 37
13,126 517
204
47
1623
94
58
2
3
8
214
105
161
2
2711
134
228
>699b
1.2
2
1
3536 125*
44,705 5234**
37,955 3413*
4813 35*
22
26,811 2745
14 0.3
36
30
143
2373 145
32
17 0.9
10 0.7
26
63
32
3
3
6834 148*
N.E.
2762 171
9
N.E.
2
>3125b
2489
4395
1194
115
172
0.00008
3363
0.9
42,316 2815*
43,953 1087*
31,034 2446*
7221 563*
5507 261*
1
4
2
5
0.3*
61,356 1137
20 0.9
7547 361
N.E.
De
I
M
67,250 1025*
6566 763
361,382 19,374
N.E.
<0.4a
>5b
Is
18,754 1242
C = clorgyline, De = R-(ꢀ)-deprenyl, I = iproniazid, M = moclobemide, Is = isatin. Ratio: hMAO-B selectivity index = IC50 (hMAO-A)/IC50 (hMAO-B). Each IC50 value is the mean
**
-
*
S.E.M. from five experiments. Level of statistical significance: P <0.01 or P <0.05 versus the corresponding IC50 values obtained against hMAO-B, as determined by ANOVA/
Dunnett’s. N.E. = Inactive at 100 M (highest concentration tested).
Values obtained under the assumption that the corresponding IC50 against hMAO-B is the highest concentration tested (1 mM).
l
a
b
Value obtained under the assumption that the corresponding IC50 against hMAO-A is the highest concentration tested (100 lM).
be different to the interaction obtained after several minutes of the
enzyme-inhibitor complex formation. R-(ꢀ)-deprenyl, for example,
first of all form a non-covalent complex with MAO as an initial,
reversible step. The subsequent interaction of R-(ꢀ)-deprenyl with
MAO leads to a reduction of the enzyme-bound flavin adenine
dinucleotide, and concomitant oxidation of the inhibitor. This oxi-
dized inhibitor then reacts with FAD at the N-5-position in a cova-
lent manner.23 The initial non-covalent binding to MAO has been
also described for other MAO inhibitors.24 Finally, it is possible
that, in some cases of irreversible inhibition, a steric hindrance
may drastically difficult the removal of the inhibitor from the enzy-
matic active center (although its interaction with this binding site
is either very weak or reversible). Bearing in mind all the above
considerations and taking into account that the docking studies

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5063–5070
5067
Table 2
Reversibility and irreversibility of hMAO inhibition of derivative 44 and reference inhibitorsa
Compd
% hMAO-A inhibition
After repeated washing
% hMAO-B inhibition
After repeated washing
Before washing
Before washing
Moclobemide (500
44 (100 lM)
lM)
86.75 4.34a
23.41 1.62
69.47 3.76
60.56 2.98
10.26 0.65
26.61 1.54
71.47 3.47
58.43 2.76
(S)-44 (100
lM)
(R)-44 (100
lM)
R-(ꢀ)-Deprenyl (20 nM)
48.67 2.34a
58.65 2.97
74.32 4.14
77.83 3.89
49.14 2.80
60.19 3.26
79.26 4.32
81.63 4.25
44 (50 nM)
(S)-44 (50 nM)
(R)-44 (50 nM)
Each value is the mean S.E.M. from five experiments (n = 5).
a
Level of statistical significance: P <0.01 versus the corresponding % hMAO-A or hMAO-B inhibition before washing, as determined by ANOVA/Dunnett’s.
adopted for deriving the linear equation from the resulting field
*
S
R
matrix using the cross-validate leave-one-out (LOO) method. Min-
imum sigma equal to 2 kcal/mol was applied in order to decrease
noise. Table 3 shows the calculated CoMFA statistical parameters.
The cross-validated analysis, obtaining q2 greater than 0.3, was
considered for model generation, as with this q2 cut-off the proba-
bility of chance correlation is lower than 5%.25 PLS analysis was
processed without validation to derive the square of no-cross-val-
idated correlated coefficient (r2). The number of optimum compo-
nents (NOC) taken into account for the r2 calculation was assessed
by previous cross-validated analysis. This allows to de-noise data
and to avoid over-fitting. For all further analysis the same setting
described above were applied. The most important requirement
for CoMFA studies is that the 3D structures to be analyzed are
aligned according to a suitable conformational template, which is
assumed to be the ‘bioactive’ conformation. The pharmacophore-
based alignment represents another commonly used approach, in
particular when the substructure-based alignment method
fails.30–32 We generated the pharmacophore models by means of
the HipHop algorithm, as included in Catalyst package.33 HipHop
generates hypotheses which have common feature. Compounds
30 (principal2), 12, 5, 35, 21, 19 (principal1) were chosen for the
model generation. The pharmacophores differing from direction
of projection points were discarded, the remaining models were
used for the alignment procedure. Once the aligned compounds
were imported in Sybyl, their electrostatic charge distribution
was computed using the Gasteiger–Hückel method. Only the sta-
tistical values of the best pharmacophore-based alignment are
shown in Table 3.
*
N
*
*
*
N
H
*
R
N
*
Scheme 2. Substructure of the common core of the molecules used for rigid fitting
to the template.
make only a theoretical prediction of the initial possible interaction
inhibitor-enzyme, the results obtained in these docking studies
and in the reversibility experiments may be different.
6. Molecular modeling
The CoMFA25 study was performed using Sybyl v. 7.2.26 A well-
defined training set of 36 compounds was collected and used for
model generation. Compounds 37–45 show a chiral center as well
a E/Z double bond configuration. As the in vitro activity for all
respective configurations is not available and in order to assure
high quality data for the model generation, these configurations
were excluded for such compounds. Derivatives 39, 41, 44, and
45, whose activity data of separated enantiomeric forms are avail-
able, were employed as a test set for the model validation. Know-
ing that the alignment plays a fundamental role in the COMFA
studies, different alignments have been used to define the best
one for our set of compounds. First of all, we applied substruc-
ture-based alignment. All the molecules were submitted to full
geometry optimization by molecular mechanics using the standard
However the models based on this alignment exhibited only
minor improvements from the previous one. Therefore we decided
Tripos force field27 with a distance dependent dielectric (
e = 1) and
Powell conjugate gradient algorithm28 (convergence criterion of
to apply a receptor-based alignment using the docking program
0
34
GLIDE
,
as we found this approach to work reliably in our previous
0.001 kcal/mol ÅA). Atomic charges were calculated using the
Gasteiger–Hückel method.29 Compound 30 was used as template,
and the moiety considered for the alignment is shown with aster-
isked atoms in the Scheme 2.
study on the same target13b and in other studies reported in liter-
ature.35,36 The conformational properties of compounds have been
investigated by Monte Carlo (MC) search applying 5000 iterations
to all rotatable bonds. Each generated structure has been opti-
mized with at least 2000 interactions of the Polak Ribiere Conju-
gate Gradient algorithm using MMFFs force field and the implicit
solvation model GB/SA water, as implemented in MacroModel
The aligned molecules were included into a sufficiently large
0
box considering a grid spacing equal to 2.0 ÅA. The steric and elec-
trostatic fields were computed using an sp3 C-atom with explicit
electrostatic charge fixed to +1. Partial least square (PLS) was
Table 3
Statistical results of different models derived from the training set molecules using different alignments
Model
q2
NOC
SEP
r2
SEE
F
Steric%
Substructure based MAO-A
Substructure based MAO-B
Pharmacophore based MAO-A
Pharmacophore based MAO-B
Receptor based MAO-A
0.449
0.356
0.352
0.444
0.811
0.831
5
4
4
4
4
4
0.473
0.829
0.504
0.770
0.273
0.224
0.752
0.678
0.768
0.790
0.961
0.948
0.317
0.586
0.302
0.474
0.125
0.135
18227
16286
25603
29081
188759
141468
57
62
50
51
40
47
Receptor based MAO-B

5068
F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5063–5070
Figure 3. Correlation between experimental versus predicted activities of training and test set of MAO-A (a) and MAO-B (b). In addition, beside the line of ideal correlation,
dotted lines are given, which indicated deviation from the actual pIC50 by 1 logarithmic unit.
v.7.2.37 All MC minimum energy conformers were submitted to
Table 4
rigid docking using GLIDE. PDB structures 2BXR38 and 1GOS39 were
Comparison between experimental (exp) and predicted (pred) pIC50 by CoMFA
models obtained with structure-based alignment
used as human MAO-A and MAO-B target structures, respectively.
A box of about 110 k Å3 centered onto the N5 FAD atom was
defined as binding site in GLIDE. For each compound, the ten best
docked poses were considered for the receptor-based alignment.
Starting from the highest ranked structure, the pose obtaining
the best q2 value was chosen for the alignment.40 The Figure 3 de-
picts the plot of the experimental activities versus the predicted
ones; the corresponding values are reported in Table 4,
respectively.
In order to investigate the robustness of our models, a group
cross-validation (10 groups, 20 times) was processed. The high
average q2 (Table 5) corroborates the stability of the models. Boot-
strapping procedure (100 runs) was considered to exclude the risk
of chance correlation.41 Also in this case, the high value of q2 and
the minimal standard deviation confirmed the robustness of these
models.
Furthermore, the predictive capability of our models was eval-
uated with the test set compounds: 10 conformers of each com-
pound, obtained from the docking studies, were considered; the
one with the highest predicted activity was defined the putative
bioactive conformation. All R/S and E/Z isomers of 39, 41, 44, and
45, have been taken into account. As reported in Table 4 and Figure
3 due to the possible E/Z interconversion at room temperature,
suggested by ab initio calculation,14b and considering that the
respective predicted activities of both E and Z configurations are
very similar, only one value was considered for these isomers.
The activity of the test set compounds was accurately predicted
by both models.
Compd
MAO-A
MAO-B
Config pIC50 exp pIC50 pred Config pIC50 exp pIC50 pred
Training set
1
2
3
4
5
6
7
8
5.10
5.14
5.35
4.26
5.64
5.70
5.98
6.46
6.19
4.31
5.54
5.76
4.00
4.34
4.62
5.12
4.62
5.32
4.47
4.82
4.34
4.09
4.71
5.09
5.24
4.68
5.15
4.83
5.21
4.99
4.23
5.14
5.02
5.41
4.75
5.36
5.31
5.16
5.57
4.15
5.59
5.67
6.08
6.40
6.14
4.28
5.69
5.39
3.97
4.39
4.71
5.02
4.66
5.32
4.44
4.86
4.60
4.06
4.69
4.92
5.19
4.73
5.05
4.89
5.30
5.26
4.28
5.16
5.00
5.21
4.73
5.28
6.53
6.58
7.39
6.60
8.49
6.87
6.79
7.02
6.65
6.93
6.67
8.42
6.69
6.18
5.96
4.92
7.07
7.28
6.59
5.62
7.36
5.02
7.04
5.26
4.50
6.44
7.21
7.56
6.32
8.45
6.03
7.79
6.04
5.30
7.94
7.34
6.67
6.71
7.63
6.45
8.58
6.81
6.72
7.17
6.55
6.99
7.03
8.18
6.69
5.84
6.12
4.77
7.28
7.21
6.39
5.76
7.23
5.12
7.22
5.28
4.42
6.45
7.29
7.06
6.21
7.90
5.94
7.60
6.55
5.55
8.21
7.28
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
7. Results and discussion
CoMFA results of the best models obtained were analysed in
order to highlight further modification onto the thiazole scaffold.
Test set
(S)-39
(R)-39
(S)-41
(R)-41
(S)-44
(R)-44
(S)-45
(R)-45
E
E
E
Z
E
E
E
Z
5.30
5.45
5.32
5.16
4.37
4.36
5.14
5.26
5.29
5.32
5.43
4.95
5.49
5.35
5.24
5.36
Z
E
E
E
Z
Z
E
E
7.32
7.66
7.18
7.32
7.78
8.00
7.20
7.49
7.30
7.25
7.14
7.10
7.93
8.15
7.42
7.34
Table 5
Validation of the stability of the receptor-based models
Model
Group cross-validation
Bootstrapping
Average q2
Average q2 Standard deviation q2
Model MAOA 0.732
Model MAOB 0.805
0.949
0.952
0.019
0.019

F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5063–5070
5069
Figure 4. Contour ‘stdev x coeff’ plot of (a) steric and (b) electrostatic properties for the CoMFA model for MAO-B in combination with exemplary compound 30 in capped
sticks and FAD in space fill style. Green contours indicate regions where an increase in steric bulk will enhance the activity; yellow contour indicates steric bulk unfavorable
area. Blue and red contours show regions of desirable positive and negative electrostatic interactions, respectively.
Figure 5. Contour ‘stdev x coeff’ plot of (a) steric and (b) electrostatic properties for the CoMFA model for MAO-A in combination with exemplary compound 8 in capped
sticks and FAD in space fill style. Green contours indicate regions where an increase in steric bulk will enhance the activity; yellow contour indicates steric bulk unfavorable
area. Blue and red contours show regions of desirable positive and negative electrostatic interactions, respectively.
The CoMFA contour map of MAO-B steric contribution, overlapping
compound 30, is depicted in Figure 4a.
which accommodates bigger compounds able to go through the
two cavities. This could explain the higher inhibitory activity on
MAO-B of this series of compounds. The analysis of the corre-
sponding maps for MAO-A (Fig. 5) confirms the relevance of the
hydrazo group for the MAO recognition.
The green contour map suggests that substitution with bulky
groups at the phenyl ring would lead towards MAO-A selective
inhibitors, while, the substitution with bulky groups in correspon-
dence of alicyclic portion does not seem favorable for MAO-A
inhibitory activity since small yellow areas are surrounding this
position. As above mentioned, this substitution would probably
enhance the activity towards MAO-B consequently increasing the
selectivity for B isoform. These considerations lead us to consider
appropriate modifications on this scaffold. Though some portions
seem to be essential, the maps clearly indicate some possibilities
for the improvement of activity and selectivity.
A large green contour around the alicyclic portion and ortho
position of phenyl ring, suggests that substitution with a more ste-
ric bulky groups at these positions may enhance activity. The small
yellow contour map surrounding the phenyl ring indicates that
occupancy of this sterically unfavorable area would have a detri-
mental effect on the inhibitory activity, for example, in 16 and
25 the substitution of fluorine with bulkier methoxy group leads
to lower activity compounds. The electrostatic CoMFA field is
shown in Figure 4b. Here, a small red contour map around the phe-
nyl ring para position remarks the relevance of an electronegative
group at this location for inhibitory activity.
Such a suggestion is consistent with higher activity of 3, 12, 21,
and 30, showing a fluorine, with respect to, for example, 15 and 33,
which have a methyl group. At the same time, the close yellow
map suggests that electronegative groups should not be too bulky.
Another red contour map is placed in correspondence of the hydra-
zo group, while a large blue contour map is wrapped around the
NH-position of this moiety. The first area indicates that an hydro-
gen bond acceptor is important at this position and the blue one
remarks the key role played by the hydrogen bond donor NH.
The docking experiment on MAO-A revealed that most of the
compounds showed orientation of the alicyclic part towards the
cofactor as it is shown in Figure 5. The difference of the putative
binding mode in the two isoforms could be due to shape and vol-
ume differences of the substrate cavities. The MAO-A isoform pre-
sents a large substrate cavity, while the MAO-B substrate cavity is
characterized by a narrow access cavity that precedes a larger one
The aim of this study was to rationalize the structure–activity
relationships of these new hMAO inhibitors by deriving a suit-
able selectivity model. The receptor-guided alignment allowed
us to obtain the most accurate model. The information deduced
will be taken into account for structural modification on this
scaffold.
Acknowledgments
This work was supported by MURST (Italy), Ministerio de Sani-
dad y Consumo (Spain; FISSPI061537) and Xunta de Galicia (Spain;
INCITE07PXI203039ES, INCITE08E1R203054ES, and 08CSA019
203PR).

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F. Chimenti et al. / Bioorg. Med. Chem. 18 (2010) 5063–5070
Chimenti, P.; Granese, A.; Carradori, S.; Alcaro, S.; Ortuso, F.; Yáñez, M.; Orallo,
Supplementary data
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