(E)-3-Heteroarylidenechroman-4-ones as potent and selective monoamine oxidase-B inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.03.081

A series of (E)-3-heteroarylidenechroman-4-ones (1a-r) was designed, synthesized and investigated in vitro for their ability to inhibit the enzymatic activity of both human monoamine oxidase (hMAO) isoforms, hMAO-A and hMAO-B. All the compounds were found to be selective hMAO-B inhibitors showing IC₅₀ values in the nanomolar or micromolar range. (E)-5,7-Dichloro-3-{[(2-(dimethylamino)pyrimidin-5-yl]methylene}chroman-4-one (1c) was the most interesting compound identified in this study, endowed with higher hMAO-B potency (IC₅₀ Combining double low line 10.58 nM) and selectivity (SI > 9452) with respect to the reference selective inhibitor selegiline (IC₅₀ Combining double low line 19.60 nM, IC₅₀ > 3431). Molecular modelling studies were performed for rationalizing at molecular level the target selective inhibition of our compounds, revealing a remarkable contribution of hydrogen bond network and water solvent.

Full text of DOI:10.1016/j.ejmech.2016.03.081

Accepted Manuscript
(E)-3-Heteroarylidenechroman-4-ones as potent and selective monoamine oxidase-B
inhibitors
Nicoletta Desideri, Luca Proietti Monaco, Rossella Fioravanti, Mariangela Biava,
Matilde Yáñez, Stefano Alcaro, Francesco Ortuso
PII:
S0223-5234(16)30264-1
10.1016/j.ejmech.2016.03.081
EJMECH 8506
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 August 2015
Revised Date: 23 March 2016
Accepted Date: 26 March 2016
Please cite this article as: N. Desideri, L. Proietti Monaco, R. Fioravanti, M. Biava, M. Yáñez, S. Alcaro,
F. Ortuso, (E)-3-Heteroarylidenechroman-4-ones as potent and selective monoamine oxidase-B
inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.081.
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ACCEPTED MANUSCRIPT
(E)-3-Heteroarylidenechroman-4-ones as potent and selective monoamine oxidase-B
inhibitors
Nicoletta Desideri, Luca Proietti Monaco, Rossella Fioravanti, Mariangela Biava, Matilde Yáñez,
Stefano Alcaro, Francesco Ortuso.

ACCEPTED MANUSCRIPT
(E)-3-Heteroarylidenechroman-4-ones as potent and
selective monoamine oxidase-B inhibitors
Nicoletta Desideri a,*, Luca Proietti Monaco a, Rossella Fioravanti a, Mariangela Biava a, Matilde Yáñez
b, Stefano Alcaro c, Francesco Ortuso c.
aDipartimento di Chimica e Tecnologie del Farmaco, Sapienza - Università di Roma, P.le Aldo Moro, 5,
00185 Rome, Italy
b Departamento de Farmacología, Facultad de Farmacia, Universidad de Santiago de Compostela,
Campus Universitario Sur, E-15782 Santiago de Compostela (La Coruña), Spain
cDipartimento di Scienze della Salute, Università “Magna Græcia” di Catanzaro, Campus Universitario
“S. Venuta”, Viale Europa, 88100, Catanzaro, Italy.
*
To whom correspondence should be addressed. Phone: +39-06-49913892. E-mail:
nicoletta.desideri@uniroma1.it
Abstract – A series of (E)-3-heteroarylidenechroman-4-ones (1a-r) was designed, synthesized and
investigated in vitro for their ability to inhibit the enzymatic activity of both human monoamine oxidase
(hMAO) isoforms, hMAO-A and hMAO-B. All the compounds were found to be selective hMAO-B
inhibitors showing IC50 values in the nanomolar or micromolar range. (E)-5,7-Dichloro-3-{[(2-
(dimethylamino)pyrimidin-5-yl]methylene}chroman-4-one (1c) was the most interesting compound
identified in this study, endowed with higher hMAO-B potency (IC50 = 10.58 nM) and selectivity (SI >
9452) with respect to the reference selective inhibitor selegiline (IC50 = 19.60 nM, IC50 > 3431).
Molecular modeling studies were performed for rationalizing at molecular level the target selective
inhibition of our compounds, revealing a remarkable contribution of hydrogen bond network and water
solvent.
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Keywords: Monoamine oxidases; Selective hMAO-B inhibitors; Chroman-4-ones; Homoisoflavonoids;
Docking studies
1. Introduction
Monoamine oxidases (MAOs) are flavin-containing enzymes that catalyze the oxidative
deamination of a variety of monoamines including neurotransmitters such as serotonin, norepinephrine,
epinephrine and dopamine, as well as dietary amines. Two different isoforms have been identified,
MAO-A and MAO-B, both associated with the outer membrane of mitochondria. The two isoenzymes
are encoded by separate genes and show different tissue distribution, and distinct substrate and inhibitor
specificities. MAO-A preferentially catalyzes the oxidative deamination of neurotransmitters, serotonin,
norepinephrine and epinephrine and it is selectively inhibited by clorgyline and moclobemide,
irreversible and reversible inhibitor, respectively. MAO-B preferentially metabolizes β-phenethylamine
and benzylamine and it is selectively and irreversibly inhibited by selegiline. Tyramine, tryptamine and
dopamine, are substrates for both isoforms [1]. MAO-A inhibitors are indicated for the treatment of
psychiatric disorders, while MAO-B inhibitors are used in the therapy of Parkinson’s disease and have
been proposed for the treatment of Alzheimer’s disease [2-4].
The MAO inhibitory activities of several classes of synthetic flavonoids were previously
investigated [5-10]. In particular, we reported the potent and selective hMAO-B inhibitory properties of
synthetic (E)-3-benzylidenechroman-4-ones, structurally related to natural homoisoflavonoids [7]. With
the aim to further explore the structure-activity relationships of this new class of MAO-B inhibitors, we
planned the replacement of the 3-benzylidene moiety with a variety of heteroarylidene substituents,
including either five-membered or six-membered heterocycles. Therefore, a series of (E)-3-
heteroarylidenechroman-4-ones 1a-r was synthesized and evaluated in vitro for their ability to inhibit
the enzymatic activity of both hMAO isoforms.
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2. Results and discussion
2.1 Chemistry
The most common synthetic procedure to obtain (E)-3-benzylidenechroman-4-ones involves acid
or base catalyzed condensation of chroman-4-ones with the appropriate benzaldehyde [11]. The
corresponding Z-isomers were usually obtained by photoisomerization of the synthesized E-isomers
1
[12-15]. The HNMR spectra allow the unambiguous assignment of E and Z configurations to these
stereoisomers. In particular, the signal of the olefinic proton in the E isomers appears at about 7.7 ppm,
due to the effect of the near carbonyl group, and the signal of C-2 protons was measured at about 5.3
ppm, owing to the proximity with the phenyl ring. Both signals were considerable upfield shifted for the
Z-isomers that show the signals at about 6.8 ppm and 4.9 ppm, respectively [12-15].
With similar synthetic procedures, several (E)-3-heteroarylidenechroman-4-ones were synthesized
starting from chroman-4-ones and different heteroaryl aldehydes [13,16-20]. As illustrated in Scheme 1,
we conveniently obtained the designed (E)-3-heteroarylidenechroman-4-ones 1a-r using pyrrolidine as
catalyst of the condensation. All the synthesized compounds were obtained as a single stereoisomer and
1
the HNMR spectra allow the assignment of the E configuration to the exocyclic double bond on the
basis of the chemical shifts of the olefinic proton (ranging from 7.59 ppm to 8.11 ppm) and of the C-2
methylene protons (ranging from 5.29 ppm to 5.85 ppm).
2.2 Biochemistry
The effects of the tested compounds on hMAO-A and hMAO-B enzymatic activities were
evaluated by measuring the inhibition of the production of hydrogen peroxide from p-tyramine, using
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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. The
hMAO activity was evaluated by measuring the fluorescence generated by resorufin following the
general procedure previously described by us [21]. In our experiments, hMAO-A displayed a Michaelis
constant (KM) of 514 ± 46.8 µM and a maximum reaction velocity (Vmax) of 301.4 ± 27.9 nM/min/mg
protein, whereas hMAO-B showed a KM of 104.7 ± 16.3 µM and a Vmax of 28.9 ± 6.3 nM/min/mg
protein (n = 5).
The hMAO inhibition data and the hMAO-B selectivity indexes (SI = IC50 MAO-A/IC50 MAO-B)
obtained for the (E)-3-arylidenechroman-4-ones 1a-r and for the reference inhibitors (clorgyline,
selegiline, iproniazid, moclobemide and isatin) are reported in Table 1. The new tested compounds
inhibited the hMAO-B enzymatic activity in the micromolar or submicromolar range, conversely, no
inhibition or poor efficacy was observed against hMAO-A, up to the highest concentration tested (100
µM).
In the series of (E)-3-heteroarylidenechroman-4-ones containing a five-membered heterocycle (1l-
r), the thiophene substituted analogues (1q and 1r) were the most potent and selective inhibitors of
hMAO-B activity (IC50 = 1.13 µM and 1.73 µM, SI = 88 and 58, respectively), whereas the furan-2-yl
derivative (1p) (IC50 = 3.77 µM, SI = 27) was about 3-fold less potent and selective than the
corresponding thiophen-2-yl analogue (1q) (IC50 = 1.13 µM, SI = 88). A further reduction in potency
and selectivity was observed for compounds containing a pyrrole (1l) or an imidazole ring (1m-o). A
comparison of the potency of the three imidazol-4-yl derivatives (1m-o) revealed that either the
introduction of a methoxy group at position 7 (1o) or two chlorine atoms at the positions 5 and 7 (1n) of
the chromanone ring, led to hMAO-B inhibitors about 2-fold more potent and selective than the
unsubstituted analogue 1m.
However, the replacement of the 3-benzylidene substituent with a five-membered heteroarylidene
moiety generally led to a reduction in potency toward hMAO-B with respect to previously tested (E)-3-
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benzylidenechroman-4-ones [7]. Conversely, highly potent and selective hMAO-B inhibitors were
obtained with the introduction of a six-membered heterocycle or a bulkier indole ring. (E)-5,7-Dichloro-
3-{[(2-(dimethylamino)pyrimidin-5-yl]methylene}chroman-4-one (1c) was the most potent and
selective compound (IC50 = 10.58 nM, SI > 9452) within the entire series of inhibitors, showing hMAO-
B potency and selectivity even better than the reference selective MAO-B inhibitor selegiline (IC50
19.60 nM, SI = 3431).
=
It is worth nothing that, either the presence of two chlorine atoms at the positions 5 and 7 of the
chroman-4-one ring or the 2-dimethylamino group on the pyrimidine ring are necessary for the potent
hMAO-B inhibitory activity. In fact, the substitution with a methoxy group at position 5 of the chroman-
4-one ring (1d) produced a dramatic loss of hMAO-B potency and selectivity (IC50 = 10.40 µM, SI >
9.6) whereas the removal of both the chlorine substituents resulted in compound 1b, about 22-fold less
potent and selective (IC50 = 241.11 nM, SI > 415) than 1c. Moreover, the replacement of the
dimethylamino group with the amino one provided a 75-fold less active and selective compound (1a)
(IC50 = 18.33 µM, SI > 5.5) with respect to 1b. A similar behaviour was also observed for the (E)-3-
benzylidenechroman-4-ones previously tested [7].
Among the pyridine substituted derivatives (1e-j), only the 3-[[6-(dimethylamino)pyridin-3-
yl]methylene] analogue (1i) showed activity in the submicromolar range (IC50 = 213.22 nM, SI > 469).
This compound exhibited hMAO-B potency and selectivity comparable to the corresponding
pyrimidinyl derivative (1b) (IC50 = 241.11 nM, SI > 415). Surprisingly, the introduction of chlorine
atoms at positions 5 and 7 of the chromone ring (1j) produced a marked reduction in potency and
selectivity when compared to 1i.
Finally, the substitution with a bulkier indole ring resulted in compound 1k, exhibiting
submicromolar potency and high selectivity toward hMAO-B (IC50 = 181.45 nM, SI = 551).
To investigate whether the most potent hMAO-B inhibitor (1c) is a reversible or irreversible
enzyme inhibitor, an effective dilution method was used [22]. Following this method, reversible
inhibitors show a linear progress with a slope equal to about 91% of the slope of the control sample,
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while irreversible inhibitors reach only about 9% of this slope. The results obtained for the tested
compound (1c), for the reversible inhibitor isatin and for the irreversible inhibitor selegiline are
presented in Figure 1. The data suggest that 1c is a partially reversible inhibitor. In fact, only a modest
recovery of MAO-B activity was observed after dilution of samples previously incubated with this
compound.
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Table 1. IC50 and SI values for the inhibitory effects of (E)-3-heteroarylidenechroman-4-ones 1a-r and reference inhibitors
on the enzymatic activity of human recombinant MAO isoforms expressed in baculovirus infected BTI insect cells.
hMAO-A (IC50)
hMAO-B (IC50)
Comp
R
H
R1
H
Ar
SIb
(µM)
(µM)
**
**
**
**
18.33 ± 1.76
> 5.5
1a
241.11 x 10-3 ±
14.18 x 10-3
H
Cl
H
H
Cl
> 415
> 9452
> 9.6
1b
1c
10.58 x 10-3
1.25 x 10-3
±
OCH3
10.40 ± 1.28
1d
H
H
H
H
H
H
**
**
**
2.94 ± 0.17
12.44 ± 0.67
8.23 ± 0.22
> 34
> 8
1e
1f
> 12
1g
N
NH2
H
H
H
H
**
**
2.39 ± 0.21
> 42
1h
1i
213.22 x 10-3 ±
24.43 x 10-3
> 469
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Cl
H
Cl
H
***
10.51 ± 1.87
9.5#
1j
181.45 x 10-3 ±
8.60 x 10-3
***
551#
1k
H
H
H
H
***
***
13.76 ± 0.57
20.20 ± 0.12
7.3#
1l
5#
1m
Cl
H
Cl
***
***
10.21 ± 1.36
10.62 ± 0.71
9.8#
9.4#
1n
1o
OCH3
H
H
H
H
***
***
3.77 ± 0.35
1.13 ± 0.24
27#
88#
1p
1q
H
H
***
1.73 ± 0.12
58#
1r
4.46 x 10-3 ± 0.32 x
10-3 a
61.35 ± 1.13
0.000073
3431
Clorgyline
Selegiline
Iproniazid
Moclobemide
Isatin
19.60 x 10-3 ±
67.25 ± 1.02 a
6.56± 0.76
361± 19.37
**
0.86 x 10-3
7.54 ± 0.36
*
0.87
< 0.36
34.39 ± 1.73
> 3
a
All IC50 values shown in this table are the mean ± S.E.M. from five experiments. Level of statistical significance: P < 0.01
b
versus the corresponding IC50 values obtained against MAO-B, as determined by ANOVA/Dunnett´s. SI: hMAO-B
selectivity index = IC50 (hMAO-A)/IC50 (hMAO-B). * Inactive at 1 mM (highest concentration tested). ** Inactive at 100
µM (highest concentration tested).*** 100 µM inhibits the corresponding MAO activity by approximately 40-50%. At
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higher concentration the compounds precipitate. # Values obtained under the assumption that the corresponding IC50 against
MAO-B is the highest concentration tested (100 µM).
15000
Control
10000
1c
Selegiline
5000
Isatin
0
0
5
10
15
t (min)
Figure 1. Recovery of hMAO-B enzymatic activity after dilution and following incubation (30 min at room temperature)
of the 100x-enzyme concentration with 10-fold IC50 concentration of compound 1c, isatin or selegiline. The control was
carried out by pre-incubating in the absence of inhibitor and diluting in the same way.
2.3 Docking studies
To rationalize at molecular level the inhibition of hMAOs by the compounds, molecular
modelling studies were carried out. The binding capabilities of 1a-r were investigated, with respect to
hMAO-A and hMAO-B receptor models, by means of docking simulation (Experimental section). All
compounds recognized both isozymes active sites. The hMAO-B was the preferred target but none of
theoretical scoring functions, available in Glide software, was in accord to experimental IC50 data (Table
S1). Generally, even if head-tail configurations were observed, the binding modes of our molecules
reported the chroman-4-one moiety located towards the FAD cofactor establishing pi-pi stacking to
hMAO-A Tyr407 and/or Tyr444 and the corresponding hMAO-B Tyr398 and/or Tyr435. The Ar
substituent was positioned at the active site entrance area in well known hydrophobic regions suggesting
possible T-shape stacking contacts to the sidechain of hMAO-A Phe208 and hMAO-B Tyr407.
According to literature data [23], the hydrophobic area in hMAO-B is larger than in hMAO-A and could
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better accommodate Ar substituent of our compounds resulting in a superior stabilisation of the related
complexes. The graphical analysis of the docking results showed a hydrogen bond between the hMAO-
B Cys172 sidechain and the chroman-4-one common moiety of all ligands, except 1b that reported a
head-tail recognition with respect to 1c. In details, the previous interaction involved the sp3 oxygen
atom of 1c, 1d, 1f, 1j, 1m and 1n, whereas, due to different binding modes, 1e, 1g-i, 1k-l, 1o-r
established hydrogen bond to Cys172 sidechain by means of their sp2 chroman-4-one oxygen atom. The
equivalent residue in hMAO-A is Asn181 but its sidechain was involved in hydrogen bond to Phe177
backbone preventing interaction with the compounds. Taking into account the better accommodation
and hydrogen bond contribution in hMAO-B, the chroman-4-one and the Ar substituent could be
considered as the key moieties of the compounds to exert affinity and selectivity to MAOs. However,
the poor agreement among theoretical and experimental data prompted us to improve our study
including the water solvent effects to the ligand target recognition. As a consequence, the most stable
complex models of 1c into the hMAO-A and hMAO-B were explicitly solvated and submitted to
molecular dynamics (MD) simulation (Experimental section). Both MD starting structures revealed
water molecules into the active site engaging hydrogen bonds to target residues such as hMAO-A
Gly110, Ile207, Phe208, Ser209, Thr211 and Gly214 and hMAO-B Pro102, Thr201, Cys172, Ile198
and Tyr435. Notably, in both complexes, one solvent molecule was interacting with 1c reporting
hydrogen bond to chroman-4-one sp2 oxygen or to pyrimidine nitrogen in hMAO-A and hMAO-B
respectively. For a deeper investigation on both the role of solvent and the induced fit phenomena, we
submitted also the inhibitor free receptor models to the same MD protocol previously reported for the
complexes (Experimental section). Interestingly, the mere MD starting structures revealed a protein
desolvation penalty for the 1c hMAO-A recognition with respect to the hMAO-B. In fact, in order to be
accommodate into the active site, the inhibitor displaced 15 water molecules in the former case while in
the second one 9 only (Figure S1).
Our study pursued by investigating the enzymes structural modification induced by 1c. In fact, if a
ligand binding generates strong perturbation of the target conformational properties, the recognition
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could be energy penalized or prevented. In order to evaluate the 1c influence in target stability, all MD
trajectory frames were aligned to the corresponding enzyme starting structure and the root mean square
deviation (RMSd) of the target not hydrogen atoms was computed (Figure 2).
Figure 2. Root mean square deviation (in Å) of 1c bound and ligand free hMAO-A and hMAO-B MD trajectories.
The RMSd data remarked significant enzyme isoform dependent differences. Actually, the ligand
induced a perturbation of the hMAO-A stronger than hMAO-B as reported by RMSd values wider in the
former case with respect to the second one. Furthermore, the MD single trajectories RMSd analysis
revealed that 1c stabilized the hMAO-B conformers close to the starting structure while in hMAO-A the
ligand influence was completely opposite. (Figure S2)
After the above reported indication, MD trajectories of 1c complexes were examined for
highlighting other interactions useful to explain the experimentally observed hMAO selectivity. We
focused our attention on 1c targets stacking contacts, hydrogen and halogen bonds to both hMAOs and
solvent molecules (Figure S3-S5). Stacking contacts, already reported by docking, were stable during
MD. Our inhibitor spent 76% and 84% of the simulation time, in hMAO-A and hMAO-B respectively,
establishing pi-pi interaction between its chroman-4-one moiety and the FAD close tyrosine residues
while T-shape stacking contacts to hMAO-A Phe208 and hMAO-B Tyr407 sidechains, only suggested
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by docking, were simultaneously observed in 16% and 8% of the simulation time, respectively (Figure
S3). The stabilizing effect can be considered almost equivalent confirming our idea that stacking
contacts are related to 1c MAO affinity but have not a pivotal role in terms of enzyme isoform
selectivity. The same considerations can be done after the halogen bond (HalB) network analysis that
reported productive contribution of chlorine substituent and a solvent key role (Figure S4). In fact,
chlorine moiety showed a similar HalB frequency in both targets (52% in hMAO-A and 48% in hMAO-
B) but in the enzyme isoform B the productive interaction was often mediated by water molecules.
Conversely, hydrogen bond (HB), involving the target both directly or by means of water bridges,
should be considered the driving force of 1c MAO discrimination. Actually, our ligand established HB
to hMAO-B for a MD simulation time significantly longer in hMAO-B (80%) than in hMAO-A (52%)
and in the former case the 32% double HBs, one target directed and one water mediated, were observed
(Figure S5).
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Figure 3. Representative binding modes of 1c into the a) hMAO-A and b) hMAO-B. The inhibitor is reported in green
carbon polytube. Relevant interacting residues are in CPK polytube. Enzyme isoform specific amino acids are depicted in
CPK thin tube. FAD cofactor is showed in spacefill notation. Active site water molecules are represented in balls & sticks.
The rest of the enzyme residues are illustrated as yellow transparent surface. Yellow, purple and blue dotted lines indicate
hydrogen bond, halogen bond and stacking contacts respectively.
The reported interaction resulted in a similar orientation of 1c in both targets (Figure 3). The
graphical inspection of MD trajectories remarked the different shape of hMAO active sites. Actually,
the mere replacement of hMAO-A Phe208 and Ile335 with the corresponding hMAO-B Ile199 and
Tyr326 could be considered equivalent in terms of ligand interaction but such residue mutations opened
in hMAO-B a large hydrophobic area that is not available in hMAO-A.
Comparing all molecular modelling results to experimental IC50 data, it is possible to rationalize
structure activity relationship of our inhibitors. Actually, halogen atoms or hydrogen bond acceptors can
be suggested for the common scaffold R1 substituent. Such moieties can productively interact to hMAO-
A Tyr197 and hMAO-B Tyr188. In order to establish hydrogen bond to the solvent and pi-pi stacking
and in both isozymes, the Ar group should be an aromatic heterocycle. Its steric hindrance can
contribute to the selectivity, actually 5 member rings can easily fit in both isoform while amino or
dimethylamino p-substituted 6 member rings or indole can be accommodate in hMAO-B better than in
hMAO-A. Finally, as already reported [24], the replacement of hMAO-A Asn181 by the hMAO-B
Cys172 plays a pivotal role in isoform selectivity.
3. Conclusions
We have designed, synthesized and investigated the hMAO inhibitory activity of a series of (E)-3-
heteroarylidenechroman-4-ones, structurally related to homoisoflavonoids. All the compounds exhibited
selectivity for the hMAO-B isoform with potency in the nanomolar or micromolar range. Generally, the
introduction of a six-membered heterocycle or a bulkier indole ring led to highly potent and selective
hMAO-B inhibitors. In particular, compound 1c showed higher potency and selectivity than the
reference inhibitor selegiline. In addition, compound 1c was found to be a partially reversible inhibitor
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of hMAO-B enzymatic activity. Docking experiments coupled to explicit solvent MD, rationalized the
structure activity relationships of our inhibitors. Water solvent effect revealed a key role for
understanding the molecular recognition of both hMAO isoforms. The hydrogen bond established by
our ligand chroman-4-one moiety to Cys172 and the steric hindrance of Ar substituent can be
considered the main responsible of hMAO-B selectivity.
4. Experimental section
4.1. Chemistry
Chemicals were generally purchased from Sigma-Aldrich and used without further purification. 2-
Aminopyrimidine-5-carboxaldehyde, and 6-(dimethylamino)nicotinaldehyde were purchased from
ChemBridge. Melting points were determined on a Stenford Research Systems OptiMelt (MPA-100)
1
13
apparatus and are uncorrected. H NMR and C NMR spectra were recorded in CDCl3 or DMSO-d6
on a Bruker AM-400 spectrometer, using TMS as internal standard. IR spectra were recorded in KBr
disks on an FT-IR PerkinElmer Spectrum 1000. The structures of the synthesized compounds were
characterized by elemental analysis, IR, 1HNMR and 13CNMR spectroscopy. Thin-layer
chromatography (TLC) with pre-coated Silica Gel F254 plates was routinely used for checking the
reactions. Components were visualised by UV light. Analyses of the elements (C,H,N,S,Cl) were within
± 0.4 % of the theoretical values.
4.1.1. Synthesis of 6-aminopyridine-3-carboxaldehyde.
A 1M solution of diisobutylaluminum hydride (DIBAL) in hexane (10 mL) was added to a
solution of 6-amino-5-cyanopyridine (10 mmol) in dry THF (50 mL) cooled at 0 °C. The ice-bath was
removed, and additional 7.1 mL of a 1M solution of DIBAL in hexane were added in two successive
portions. After stirring for additional 30 min, the reaction was quenched by the dropwise addition of dry
MeOH (15 mL). The mixture was partitioned between 100 mL of AcOEt and 70 mL of 2N HCl. The
aqueous layer was treated with 75 mL of 2N NaOH and extracted with AcOEt. The organic layers were
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washed with brine, dried over anhydrous Na2SO4, filtered and evaporated to dryness. The residue was
purified by column chromatography on silica gel eluting with CHCl3/AcOEt 1:4. Yield: 30%, mp = 174-
1
175 °C. IR (KBr): 3366, 3121, 1659 cm-1. H NMR (CD3OD, 400 MHz):
δ (ppm) 9.67 (s, 1H, CHO),
8.41 (d, 1H, H2, J2-4 = 1.6 Hz), 7.87 (dd, 1H, H4, J4-5 = 8.8 Hz, J2-4 = 1.6 Hz), 6.63 (d, 1H, H5, J4-5 = 8.8
Hz), 3.31 (s, 2H, NH2). 13C NMR (CDCl3, 100 MHz):
109.99.
δ (ppm) 191.15, 164.46, 155.78, 137.39, 123.92,
4.1.2. General procedure for the synthesis of (E)-3-heteroarylidenechroman-4-ones (1a-r)
METHOD A: Pyrrolidine (10 mmol) was added to a mixture of the appropriate chroman-4-one (6.7
mmol) and heteroaryl aldehyde (10 mmol) in dry MeOH (15 mL for 1a, 1b, 1e, 1h, 1k-r and 30 mL for
1c, 1d, 1i, 1j). The reaction mixture was stirred at room temperature for 1-24h (1h for 1f, 3h for 1l-q,
24h for 1a-c, 1h-j, 1n, and 1o). The mixture was diluted with ice-water, and the precipitate was filtered
off and washed with water. The crude solid was purified by column chromatography on silica gel,
eluting with a mixture of AcOEt and CHCl3 (3:1) (1a-g and 1i-r) or CHCl3 (1h) and crystallized from n-
hexane.
METHOD B: (synthesis of 1f and 1g) A mixture of chroman-4-one (6.7 mmol), the appropriate
pyridinecarbaldehyde (10 mmol) and pyrrolidine (10 mmol) was heated at 120 °C under stirring for 1h.
After cooling, the mixture was diluted with ice-water, and the precipitate was filtered off and washed
with water. The crude solid was purified by column chromatography on silica gel, eluting with a
mixture of AcOEt and CHCl3 (3:1), and crystallized from n-hexane.
4.1.2.1. (E)-3-[(2-Aminopyrimidin-5-yl)methylene]chroman-4-one (1a). Yield: 97%, mp = 260-261 °C.
IR (KBr): 3304, 3160, 1670 cm-1. 1H NMR (DMSO-d6, 400 MHz):
(d, 1H, H5, J5-6 = 7.6 Hz), 7.60-7.56 (m, 2H, =CH, H7), 7.34 (s, 2H, NH2), 7.12 (t, 1H, H6, J5-6 = J7.6
δ (ppm) 8.43 (s, 2H, H2’, H6’), 7.87
=
7.6 Hz), 7.05 (d, 1H, H8, J7-8 = 8.0 Hz), 5.43 (s, 2H, H2). 13C NMR (DMSO-d6, 100 MHz):
δ (ppm)
15

ACCEPTED MANUSCRIPT
180.43, 162.83, 160.43, 160.20, 135.88, 131.50, 127.67, 127.10, 121.78, 121.42, 117.18, 116.79, 67.57.
Anal. Calcd. for C14H11N3O2: C, 66.40; H, 4.38; N, 16.59. Found: C, 66.59; H, 4.29; N, 16.68.
4.1.2.2. (E)-3-{[2-(Dimethylamino)pyrimidin-5-yl]methylene}chroman-4-one (1b). Yield: 89%, mp =
194-195 °C. IR (KBr): 1662 cm-1. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 8.34 (s, 2H, H2’, H6’), 8.01
(ddd, 1H, H5, J5-6 = 8.0 Hz, J5-7 = 2.0 Hz, J5-8 = 0.4 Hz), 7.64 (t, 1H, =CH, Jall = 2.0 Hz), 7.47 (ddd, 1H,
H7, J7-8 = 8.4 Hz, J7-6 = 7.2 Hz, J5-7 = 2.0 Hz), 7.06 (ddd, 1H, H6, J5-6 = 8.0 Hz, J7-6 = 7.2 Hz, J6-8 = 1.2
Hz), 6.96 (ddd, 1H, H8, J7-8 = 8.4 Hz, J6-8 = 1.2 Hz, J5-8 = 0.4 Hz), 5.34 (d, 2H, H2, Jall = 2.0 Hz), 3.26
(s, 6H, 2CH3). 13C NMR (CDCl3, 100 MHz):
δ (ppm) 181.40, 161.19, 160.90, 159.27, 135.68, 131.70,
128.47, 127.91, 122.05, 121.94, 117.82, 116.34, 67.85, 37.22. Anal. Calcd. for C16H15N3O2: C, 68.31;
H, 5.37; N, 14.94; O, 11.37. Found: C, 68.16; H, 5.36; N, 14.80.
4.1.2.3. (E)-5,7-Dichloro-3-{[(2-(dimethylamino)pyrimidin-5-yl]methylene}chroman-4-one (1c). Yield:
69%, mp = 181-182 °C. IR (KBr): 1677 cm-1. 1H NMR (CDCl3, 400 MHz):
δ
(ppm): 8.30 (s, 2H, H2’,
13
H6’), 7.65 (s, 1H, =CH), 7.11 (s, 1H, H8), 6.95 (s, 1H, H6), 5.28 (s, 2H, H2), 3.26 (s, 6H, 2CH3), C
(CDCl3, 100 MHz):
δ (ppm) 178.63, 162.65, 161.29, 159.31, 139.91, 136.12, 132.82, 127.92, 125.70,
118.88, 117.31, 116.11, 68.09, 37.24. Anal. Calcd. for C16H13Cl2N3O2: C, 54.87; H, 3.74; Cl, 20.25;
N, 12.00. Found: C, 54.99; H, 3.80; Cl, 20.15; N, 11.87.
4.1.2.4. (E)-3-{[2-(Dimethylamino)pyrimidin-5-yl]methylene}-7-methoxychroman-4-one (1d). Yield:
90%, mp = 186-187 °C. IR (KBr): 1669 cm-1. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 8.32 (s, 2H, H2’,
H6’), 7.94 (d, 1H, H5, J5-6 = 8.8 Hz), 7.61 (s, 1H, =CH), 7.94 (d, 1H, H6, J5-6 = 8.8 Hz), 6.41 (s, 1H,
13
H8), 5.33 (s, 2H, H2), 3.84 (s, 3H, OCH3), 3.26 (s, 6H, 2CH3). C NMR (CDCl3, 100 MHz):
δ (ppm)
180.18, 165.97, 162.85, 161.15, 159.12, 130.89, 129.67, 128.52, 116.46, 115.73, 110.41, 100.80, 68.43,
16

ACCEPTED MANUSCRIPT
55.87, 37.20. Anal. Calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50. Found: 65.41; H, 5.52; N,
13.47.
4.1.2.5. (E)-3-[(Pyridin-2-yl)methylene]chroman-4-one (1e) Yield: 77%, mp = 139-140 °C (lit. 142°C)
[17]. IR (KBr): 1673 cm-1. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 8.65 (d, 1H, H3’, J3’-4’ = 8.0 Hz), 8.02
(dd, 1H, H5, J5-6 = 8.0 Hz, J5-7 = 1.6 Hz), 7.74 (dt, 1H, H7, J7-6 = J7-8 = 8.0 Hz, J5-7 = 1.6 Hz), 7.71 (t,
1H, =CH, Jall = 2.0 Hz), 7.51-7.47 (m, 2H, H5’, H6’), 7.26-7.23 (m, 1H, H4’), 7.05 (t, 1H, H6, J5-6 = J7-6
= 8.0 Hz), 6.99 (d, 1H, H8, J7-8 = 8.0 Hz), 5.92 (d, 2H, H2, Jall = 2.4 Hz). 13C NMR (CDCl3, 100 MHz):
δ
(ppm) 182.26, 161.72, 154.18, 149.66, 136.54, 135.94, 134.33, 133.27, 127.95, 127.84, 123.15,
121.83, 121.63, 118.07, 68.41. Anal. Calcd. for C15H11NO2: C, 75.94; H, 4.67; N, 5.90. Found: C,
76.16; H, 4.69; N, 5.85.
4.1.2.6. (E)-3-[(Pyridin-4-yl)methylene]chroman-4-one (1f). Yield: 40%, mp = 132-133 °C. IR (KBr):
1676 cm-1. 1H NMR (DMSO-d6, 400 MHz): d (ppm) 8.72 (d, 2H, H3’, H5’, J2’-3’ = 6.0 Hz), 8.03 (dd,
1H, H5, J5-6 = 8.0 Hz, J5-7 = 1.6 Hz), 7.75 (s, 1H, =CH), 7.52 (dt, 1H, H7, J7-8 = J7-6 = 8.0 Hz, J5-7 = 1.6
Hz), 7.19 (d, 2H, H2’, H6’, J2’-3’ = 6.0 Hz), 7.10 (t, 1H, H6, J7-6 = J5-6 = 8.0 Hz), 6.99 (d, 1H, H8, J7-8
=
8.0 Hz), 5.29 (s, 2H, H2). 13C NMR (DMSO-d6, 100 MHz): d (ppm) 180.77, 160.74, 149.99, 140.92,
136.52, 133.99, 133.47, 127.26, 123.93, 122.07, 121.13, 117.96, 67.02. Anal. Calcd. for C15H11NO2:
C, 75.94; H, 4.67; N, 5.90. Found: C, 75.81; H, 4.75; N, 5.93.
4.1.2.7. (E)-3-[(Pyridin-3-yl)methylene]chroman-4-one (1g) Yield: 61%, mp = 120-122 °C. IR (KBr):
1671 cm-1. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 8.65 (dd, 1H, H4’, J4’-5’ = 4.8 Hz, J2’-4’ = 1.2 Hz), 8.59
(d, 1H, H2’, J2’-4’ = 1.2 Hz), 8.02 (dd, 1H, H5, J5-6 = 7.6 Hz, J5-7 = 1.6 Hz), 7.82 (s, 1H, =CH), 7.66 (d,
1H, H6’, J6’-5’ = 7.6 Hz), 7.51 (ddd, 1H, H7, J7-8 = 8.4 Hz, J7-6 = 7.6 Hz, J5-7 = 1.6 Hz), 7.41 (dd, 1H,
H5’, J6’-5’ = 7.6 Hz, J4’-5’ = 4.8 Hz), 7.09 (t, 1H, H6, J5-6 = J7-6 = 7.6 Hz), 6.98 (d, 1H, H8, J7-8 = 8.4 Hz),
17

ACCEPTED MANUSCRIPT
5.32 (d, 2H, H2, Jall = 2.0 Hz). 13C NMR (CDCl3, 100 MHz):
δ (ppm) 181.60, 161.15, 150.22, 149. 91,
137.04, 136.21, 133.23, 133.06, 130.43, 128.02, 123.62, 122.17, 121.79, 118.03, 67.28. Anal. Calcd. for
C15H11NO2: C, 75.94; H, 4.67; N, 5.90. Found: C, 75.78; H, 4.66; N, 5.89.
4.1.2.8. (E)-3-[(6-Aminopyrimidin-3-yl)methylene]chroman-4-one (1h). Yield: 84%, mp = 186-187 °C.
IR (KBr): 3387, 3117, 1657 cm-1. 1H NMR (DMSO-d6, 400 MHz):
δ (ppm) 8.11 (s, 1H, =CH), 7.86 (d,
1H, H5, J5-6 = 8.0 Hz), 7.59-7.53 (m, 3H, H2’, H6’, H7), 7.11 (t, 1H, H6, J5-6 = J7.6 = 7.2 Hz), 7.04 (d,
1H, H8, J7-8 = 8.4 Hz), 6.69 (s, 2H, NH2), 6.53 (d, 1H, H5’, J5’-6’ = 8.4 Hz), 5.43 (s, 2H, H2). 13C NMR
(DMSO-d6, 100 MHz):
δ (ppm) 180.52, 160.32, 160.27, 152.40, 138.60, 135.63, 134.72, 127.04,
126.11, 121.69, 121.63, 118.19, 117.63, 107.73, 67.64. Anal. Calcd. for C15H12N2O2: C, 71.42; H,
4.79; N, 11.10. Found: C, 71.52; H, 4.83; N, 11.07.
4.1.2.9. (E)-3-{[6-(Dimethylamino)pyridin-3-yl]methylene}chroman-4-one (1i). Yield: 96%, mp = 160-
162 °C. IR (KBr): 1655 cm-1. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 8.19 (d, 1H, H2’, J2’-6’ = 2.4 Hz),
8.00 (dd, 1H, H5, J5-6 = 7.6 Hz, J5-7 = 1.6 Hz), 7.74 (s, 1H, =CH), 7.47-7.42 (m, 2H, H6’, H7), 7.05 (t,
1H, H6, J5-6 = J7-6 = 7.6 Hz), 6.99 (d, 1H, H8, J7-8 = 8.0 Hz), 6.56 (d, 1H, H5’, J5’-6’ = 9.2 Hz), 5.38 (d,
2H, H2, Jall = 1.6 Hz), 3.15 (s, 6H, 2CH3). 13C NMR (CDCl3, 100 MHz):
δ (ppm) 181.73, 160.87,
158.93, 151.35, 138.73, 135.43, 135.14, 127.84, 127.23, 122.25, 121.77, 118.36, 117.73, 105.47, 68.04,
38.02. Anal. Calcd. for C17H16N2O2: C, 72.84; H, 5.75; N, 9.99. Found: C, 72.97; H, 5.77; N, 9.93.
4.1.2.10. (E)-5,7-Dichloro-3-[(6-(dimethylamino)pyridin-3-yl)methylene]chroman-4-one (1j). Yield:
11%, mp = 239-240 °C. IR (KBr): 1668 cm-1. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 8.01 (s, 1H, =CH),
7.30 (s, 1H, H2’), 7.24 (d, 1H, H6’, J5’-6’ = 7.6 Hz), 7.10 (d, 1H, H8, J6-8 = 1.6 Hz), 6.92 (d, 1H, H6, J6-8
= 1.6 Hz), 6.76 (d, 1H, H5’, J5’-6’ = 7.6 Hz), 5.38 (d, 2H, H2, Jall = 2.0 Hz), 3.07 (s, 6H, 2CH3). 13C
NMR (CDCl3, 100 MHz):
δ
(ppm) 179.24, 162.65, 151.19, 139.39, 139.17, 135.92, 132.59, 126.01,
18

ACCEPTED MANUSCRIPT
125.43, 123.38, 122.48, 119.29, 117.17, 112.13, 68.46, 40.29. Anal. Calcd. for C17H14Cl2N2O2: C,
58.47; H, 4.04; Cl, 20.30; N, 8.02. Found: C, 58.28; H, 3.95; Cl, 20.35; N, 8.09.
4.1.2.11. (E)-3-[(1H-Indol-3-yl)methylene]chroman-4-one (1k). Yield: 38%, mp = 221-222 °C. IR
(KBr): 3241, 1643 cm-1. 1H NMR (DMSO-d6, 400 MHz):
δ (ppm) 12.10 (bs, 1H, NH), 8.09 (s, 1H,
=CH), 7.91 (d, 1H, H5, J5-6 = 8.0 Hz), 7.82 (d, 1H, H7’, J6’-7’ = 7.6 Hz), 7.80 (s, 1H, H2’), 7.56 (t, 1H,
H7, J7-8 = J7-6 = 8.0 Hz), 7.51 (d, 1H, H4’, J4’-5’ = 7.6 Hz), 7.26 (t, 1H, H6’, J6’-7’ = J5’-6’ = 7.6 Hz), 7.20
(t, 1H, H5’, J5’-6’ = J4’-5’ = 7.6 Hz), 7.12 (t, 1H, H6, J5-6 = J7-6 = 8.0 Hz), 7.06 (d, 1H, H8, J7-8 = 8.0 Hz),
5.49 (s, 2H, H2). 13C NMR (DMSO-d6, 100 MHz):
δ (ppm) 180.03, 160.32, 136.11, 135.38, 129.53,
127.63, 127.50, 126.98, 124.23, 122.75, 121.76, 121.57, 120.80, 118.19, 117.63, 112.20, 110.36, 68.55.
Anal. Calcd. for C18H13NO2: C, 78.53; H, 4.76; N, 5.09. Found: C, 78.73; H, 4.71; N, 5.13.
4.1.2.12. (E)-3-[(1H-Pyrrol-2-yl)methylene]chroman-4-one (1l). Yield: 64%, mp = 203-204 °C. IR
(KBr): 3276, 1649 cm-1. 1H NMR (DMSO-d6, 400 MHz):
δ (ppm) 11.71 (bs, 1H, NH), 7.91 (d, 1H, H5,
J5-6 = 8.0 Hz), 7.69 (s, 1H, =CH), 7.56 (t, 1H, H7, J7-8 = J7-6 = 8.0 Hz), 7.21 (s, 1H, H3’), 7.10 (t, 1H,
H6, J5-6 = J7-6 = 8.0 Hz), 7.04 (d, 1H, H8, J7-8 = 8.0 Hz), 6.57 (s, 1H, H5’), 6.35 (s, 1H, H4’), 5.41 (s,
2H, H2). 13C NMR (DMSO-d6, 100 MHz):
δ (ppm) 179.86, 160.27, 135.35, 127.25, 126.88, 125.64,
124.09, 122.44, 121.63, 121.52, 117.59, 115.12, 111.58, 67.87. Anal. Calcd. for C14H11NO2: C, 74.65;
H, 4.92; N, 6.22. Found: C, 74.78; H, 4.97; N, 6.23.
4.1.2.13. (E)-3-[(1H-Imidazol-4-yl)methylene]chroman-4-one (1m). Yield: 78%, mp = 197-198 °C. IR
(KBr): 3385, 1667 cm-1. 1H NMR (DMSO-d6, 400 MHz):
δ (ppm) 12.49 (bs, 1H, NH), 7.93 (s, 1H,
=CH), 7.88 (dd, 1H, H5, J5-6 = 8.0 Hz, J5-7 = 1.6 Hz), 7.80 (s, 1H, H3’), 7.58-7.54 (m, 2H, H7, H5’),
7.11 (t, 1H, H6, J5-6 = J7-6 = 8.0 Hz), 7.06 (d, 1H, H8, J7-8 = 8.4 Hz), 5.85 (d, 2H, H2, Jall = 0.8 Hz). 13C
NMR (DMSO-d6, 100 MHz):
δ
(ppm) 181.18, 160.99, 137.86, 136.23, 135.48, 127.33, 127.10, 125.31,
19

ACCEPTED MANUSCRIPT
125.06, 121.87, 121.49, 117.76, 67.73. Anal. Calcd. for C13H10N2O2: C, 69.02; H, 4.46; N, 12.38.
Found: C, 69.21; H, 4.52; N, 12.33.
4.1.2.14. (E)-5,7-Dichloro-3-[(1H-imidazol-4-yl)methylene]chroman-4-one (1n) Yield: 23%, mp = 252-
253 °C from n-hexane. IR (KBr): 3248, 1667 cm-1. 1H NMR (DMSO-d6, 400 MHz):
δ (ppm) 12.76 (bs,
1H, NH), 7.91 (s, 1H, =CH), 7.81 (s, 1H, H3’), 7.55 (s, 1H, H8), 7.30 (s, 1H, H6), 7.24 (s, 1H, H5’),
5.81 (s, 2H, H2). 13C NMR (DMSO-d6, 100 MHz):
δ
(ppm) 192.25, 163.03, 138.33, 138.07, 136.29,
135.12, 135.73, 134.54, 125.11, 124.43, 118.84, 117.35, 68.07. Anal. Calcd. for C13H8Cl2N2O2: C,
52.91; H, 2.73; Cl, 24.03; N, 9.49. Found: C, 53.08; H, 2.77; Cl, 24.11; N, 9.53.
4.1.2.15. (E)-3-[(1H-imidazol-4-yl)methylene]-7-methoxychroman-4-one (1o). Yield: 63%, mp = 176-
178 °C from n-hexane. IR (KBr): 3112, 1679 cm-1. 1H NMR (DMSO-d6, 400 MHz):
δ (ppm) 12.59 (bs,
1H, NH), 7.90 (s, 1H, =CH), 7.79 (d, 1H, H5, J5-6 = 8.4 Hz), 7.74 (s, 1H, H3’), 7.51 (s, 1H, H5’), 6.67
(dd, 1H, H6, J5-6 = 8.4 Hz, J6-8 = 2.0 Hz), 6.57 (d, 1H, H8, J6-8 = 2.0 Hz), 5.82 (s, 2H, H2), 3.83 (s, 3H,
OCH3). 13C NMR (DMSO-d6, 100 MHz):
δ (ppm) 179.93, 165.18, 162.95, 137.65, 136.68, 128.82,
126.68, 125.32, 124.07, 115.47, 109.94, 100.83, 68.02, 55.68. Anal. Calcd. for C14H12N2O3: C, 65.62;
H, 4.72; N, 10.93. Found: C, 65.78; H, 4.78; N, 10.88.
4.1.2.16. (E)-3-[(Furan-2-yl)methylene]chroman-4-one (1p). Yield: 87%, mp = 118-119 °C (lit. 105 °C)
[19]. IR (KBr): 1668 cm-1. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 7.99 (d, 1H, H5, J5-6 = 8.0 Hz) 7.59 (s,
1H, =CH), 7.50 (s, 1H, H3’), 7.45 (t, 1H, H7, J7-8 = J7-6 = 8.0 Hz), 7.03 (t, 1H, H6, J5-6 = J7-6 = 8.0 Hz),
6.96 (d, 1H, H8, J7-8 = 8.0 Hz), 6.73 (s, 1H, H5’), 6.52 (s, 1H, H4’), 5.57 (s, 2H, H2). 13C NMR (CDCl3,
100 MHz):
δ (ppm) 181.53, 161.43, 151.37, 145.61, 135.59, 127.82, 126.80, 122.02, 121.75, 121.71,
118.50, 117.88, 112.66, 67.98. Anal. Calcd. for C14H10O3: C, 74.33; H, 4.46. Found: C, 74.54; H,
4.47.
20

ACCEPTED MANUSCRIPT
4.1.2.17. (E)-3-[(Thiophen-2-yl)methylene]chroman-4-one (1q). Yield: 85%, mp = 126-127 °C (lit. 125-
126 °C) [16]. IR (KBr): 1661 cm-1. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 8.01 (d, 1H, H5, J5-6 = 7.2
Hz), 7.98 (s, 1H, =CH), 7.59 (d, 1H, H3’, J3’-4’ = 4.4 Hz), 7.48 (t, 1H, H7, J7-8 = J7-6 = 7.2 Hz), 7.34 (d,
1H, H5’, J4’-5’ = 4.4 Hz), 7.16 (t, 1H, H4’, J3’-4’ = J4’-5’ = 4.4 Hz), 7.06 (t, 1H, H6, J5-6 = J7-6 7.2 Hz),
13
6.98 (d, 1H, H8, J7-8 = 7.2 Hz), 5.46 (d, 2H, H2, Jall = 1.2 Hz). C NMR (CDCl3, 100 MHz):
δ (ppm)
181.24, 161.02, 137.67, 135.69, 133.88, 130.94, 128.70, 128.20, 127.87, 127.28, 121.95, 121.89,
117.83, 67.81. Anal. Calcd. for C14H10O2S: C, 69.40; H, 4.16; S, 13.23. Found: C, 69.26; H, 4.16; S,
13.28.
4.1.2.18. (E)-3[-(Thiophen-3-yl)methylene]chroman-4-one (1r). Yield: 14%, mp = 105-106 °C. IR
(KBr): 1664 cm-1. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 7.98 (d, 1H, H5, J5-6 = 7.2 Hz), 7.78 (s, 1H,
=CH), 7.44 (t, 1H, H7, J7-8 = J7-6 = 7.2 Hz), 7.37-7.36 (m, 2H, H2’, H4’), 7.13 (d, 1H, H5’, J4’-5’ = 3.2
Hz), 7.02 (t, 1H, H6, J5-6 = J7-6 7.2 Hz), 6.93 (d, 1H, H8, J7-8 = 7.2 Hz), 5.35 (d, 2H, H2, Jall = 0.8 Hz).
13C NMR (CDCl3, 100 MHz):
δ (ppm) 181.67, 161.03, 136.02, 135.72, 130.26, 129.22, 129.04, 128.84,
127.86, 126.69, 121.99, 121.85, 117.87, 67.88. Anal. Calcd. for C14H10O2S: C, 69.40; H, 4.16; S,
13.23. Found: C, 69.31; H, 4.14; S, 13.20.
4.2. Biochemistry
4.2.1. Determination of hMAO Isoform Activity
The potential effects of the tested compounds on hMAO activity were investigated by measuring
their effects on the production of hydrogen peroxide (H2O2) from p-tyramine, using the Amplex Red
MAO assay kit (Molecular Probes, Inc., Eugene, Oregon, USA) and microsomal MAO isoforms
prepared from insect cells (BTI-TN-5B1-4) infected with recombinant baculovirus containing cDNA
inserts for hMAO-A or hMAO-B (Sigma-Aldrich Química S.A., Alcobendas, Spain). In this study
hMAO activity was evaluated using the above method following the general procedure previously
21

ACCEPTED MANUSCRIPT
described by us [21]. A brief description of the procedure is provided in Supplementary data. The tested
drugs (new compounds and reference inhibitors) themselves were unable to react directly with the
Amplex Red reagent, which indicates that these compounds do not interfere with the measurements.
4.2.2. Reversibility and Irreversibility Assays
To evaluate whether 1c is a reversible or irreversible hMAO-B inhibitor, a dilution method was
used [22]. A 100X concentration of the enzyme used in the above described experiments was incubated
with a concentration of inhibitor equivalent to 10-fold its IC50. After 30 min, the mixture was diluted
100-fold into reaction buffer containing Amplex® Red reagent, horseradish peroxidase and p-tyramine
and reaction was monitored for 15 minutes. Reversible inhibitors show a linear progress with a slope
equal to about 91% of the slope of the control sample while irreversible inhibition reaches only about
9% of this slope. Control tests were carried out by pre-incubating and diluting in the absence of
inhibitor.
4.3. Molecular Modeling.
Protein Data Bank (PDB) [25] crystallographic structures 2Z5X [26] and 2V5Z [27] were
considered for building our theoretical models of hMAO-A and hMAO-B respectively. In order to be
suitable in docking simulation, the original PDB entries required preliminary manipulations, following
the same approach of a previously reported manuscript [28]. The co-crystallised reversible ligands,
harmine for 2Z5X and safinamide for 2V5Z, and water molecules were removed. FAD cofactor bonds
order was fixed and hydrogen atoms were added to final models. In order to allow an optimal
positioning of the hydrogen atoms, modified structures were submitted to protein heavy atoms
constrained energy minimisation using 10,000 steps of the Polak Ribiere Conjugate Gradient algorithm
as implemented in MacroModel program [29]. The constant force applied to not hydrogen atoms was
equal to 50 kJ/mol·Ǻ.
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Optimised structures were considered as targets models of hMAO-A and hMAO-B and, in both
cases, the ligand binding site was considered as a regular box of 27,000 Å3 centred onto the N5 FAD
atom.
The theoretical structure of all our inhibitors was built by means of the Maestro GUI [30].
Glide software [31] was considered for docking simulation using the standard precision (SP)
search algorithms. The ligands possible conformations were taken into account by the Glide flexible
docking implementation. Default software scoring function (GScore) was applied for ranking docking
poses.
The hMAO-A and hMAO-B models, both including the docking top ranked 1c pose and ligand
free, were explicitly solvated with water molecules included by the “Soak” method as implemented in
Impact [32]. For all structures a regular box, of 64,000 Å3 centred onto the N5 FAD atom, was
considered for the preliminary water positioning. Finally, after default refinement, 244/326 and 289/304
solvent molecules were retrieved in hMAO-A and hMAO-B complexes/ligand free respectively.
The resulting models were considered as starting structures for explicit solvent Molecular
Dynamics (MD) simulation carried out using the Desmond software [33]. The SPC method was applied
for generating solvated systems suitable for MD. The overall electrostatic net charge of the starting
structures was neutralized including 3 Cl- ions into the hMAO-A models and 3 Na+ ions into the hMAO-
B ones. According to Desmond default protocol, solvated systems were equilibrated by means of 7 short
and low temperature runs followed by the production simulation carried out at 300 °K, up to 1.2 ns,
using an integration time step equal to 2 fs. The output MD trajectories consisted in 120 structures
sampled at regular time intervals equal to 10 ps.
Ligand target interactions were computed by means of Maestro GUI.
Appendix A. Supplementary data
Supplementary data related to this article can be found at …
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References
[1] A.S. Kalgutkar, D.K. Dalvie, N. Castagnoli, T.J. Taylor, Interactions of nitrogen-containing
xenobiotics with monoamine oxidase (MAO) isozymes A and B: SAR studies on MAO substrates
and inhibitors. Chem. Res. Toxicol. 14 (2001) 1139-1162.
[2] M. Yamada, H. Yasuhara, Clinical pharmacology of MAO inhibitors: safety and future,
Neurotoxicol. 25 (2004) 215-221.
[3] M.C. Carreiras, J.L. Marco, Recent approaches to novel anti-Alzheimer’s therapy, Curr. Pharm. Des.
10 (2004) 3167-3175.
[4] H.H. Fernandez, J.J. Chen, Monoamine oxidase-B inhibition in the Parkinson’s disease,
Pharmacother. 27 (2007) 174S-185S.
[5] F. Chimenti, R. Fioravanti, A. Bolasco, P. Chimenti, D. Secci, F. Rossi, M. Yañez, F. Orallo, F.
Ortuso, S. Alcaro, Chalcones: A valid scaffold for Monoamine Oxidases inhibitors, J. Med. Chem.
52 (2009) 2818-2824.
[6] F. Chimenti, R. Fioravanti, A. Bolasco, P. Chimenti, D. Secci, F. Rossi, M. Yanez, F. Orallo, F.
Ortuso, S. Alcaro, A new series of flavones, thioflavones, and flavanones as selective monoamine
oxidase-B inhibitors, Bioorg. Med. Chem. 18 (2010) 1273-1279.
[7] N. Desideri, A. Bolasco, R. Fioravanti, L. Proietti Monaco, F. Orallo, M. Yañez, F. Ortuso, S.
Alcaro, Homoisoflavonoids: Natural scaffold with Potent and Selective Monoamine Oxidase-B
Inhibition Properties, J. Med. Chem. 54 (2011) 2155-2164.
[8]
N. Desideri, R. Fioravanti, L. Proietti Monaco, M. Biava, M. Yañez, F. Ortuso, S. Alcaro, 1,5-
Diphenylpenta-2,4-dien-1-ones as potent and selective monoamine oxidase-B inhibitors, Eur. J. Med.
Chem. 59 (2013) 91-100.
[9] S.J. Robinson, J.P. Petzer , A. Petzer , J.J. Bergh, A.C.U. Lourens, Selected furanochalcones as
inhibitors of monoamine oxidase, Bioorg. Med. Chem. Lett. 23 (2013) 4985–4989.
24

ACCEPTED MANUSCRIPT
[10] G. Jo, S. Ahn, B.G. Kim, H.R. Park, Y.H. Kim, H.A. Choo, D. Koh, Y. Chong, J.H. Ahn, Y.
Lim, Chromenylchalcones with inhibitory effects on monoamine oxidase-B, Bioorg. Med. Chem. 21
(2013) 7890-7897.
[11] A. Lévai, Synthesis of exocyclic α,β-unsaturated ketones, ARKIVOC (2004) (vii) 15-33.
[12] P. Bennett, J.A. Donnelly, D.C. Meaney, P. O’Boyle, Stereochemistry of cyclopropyl ketones
from the reaction of dimethylsulphoxonium methylide with 3-benzylidenechroman-4-ones, J. Chem.
Soc., Perkin Trans.1 (1972) 1554-1959.
[13] A. Lévai, C. Nemes, T. Patonay, Synthesis of new (Z)-3-arylidenechromanones by the
photoisomerization of (E)-3-arylidenechromanones, Heterocycl. Commun. 5 (1999) 441-444.
[14] V. Siddaiah, C.V. Rao, S. Venkateswarlu, A.V. Krishnaraju, G.V. Subbaraju, Synthesis,
stereochemical assignments, and biological activities of homoisoflavonoids, Bioorg. Med. Chem. 14
(2006) 2545-2551.
[15] B. Kupcewicz, G. Balcerowska-Czerniak, M. Małecka , P. Paneth , U. Krajewska , M. Rozalski,
Structure–cytotoxic activity relationship of 3-arylideneflavanone and chromanone (E,Z isomers) and
3-arylflavones, Bioorg. Med. Chem. Lett. 23 (2013) 4102–4106.
[16] A. Levai, Z. Szabo, Synthesis of exocyclic α,β-unsaturated ketones, Pharmazie, 47 (1992) 56-57.
[17] F.
Fournier,
J. Berthelot,
A.M.
Pavard,
Anticholinesterase compounds. Structure-
activity relationships of carbamates and phosphates, Eur. J. Med. Chem. 16 (1981) 48-58.
[18] U. Thapa, P. Thapa, R. Karki, M. Yun, J.H. Choi, Y. Jahng, E. Lee, K.H. Jeon, Y. Na, E.M. Ha,
W.J. Cho, Y. Kwon, E.S. Lee, Synthesis of 2,4-diaryl chromenopyridines and evaluation of their
topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship, Eur. J.
Med. Chem. 46 (2011) 3201-3209.
[19] K. M. Tapas, P. Rammohan, M. Rina, K.M. Asok, Facile condensation of aromatic aldehydes
with chroman-4-ones and 1-thiochroman-4-ones catalysed by Amberlyst-15 under microwave
irradiation condition, E-J. Chem. 8(2) (2011) 863-869.
25

ACCEPTED MANUSCRIPT
[20] B.L. Zhao, D.M. Du, Chiral Squaramide-Catalyzed Michael/Alkylation Cascade Reaction for
the Asymmetric Synthesis of Nitro-Spirocyclopropanes, Eur. J. Org. Chem. 24 (2015) 5350-5359.
[21] M. Yáñez, N. Fraiz, E. Cano, F. Orallo, Inhibitory effects of cis- and trans-resveratrol on
noradrenalina and 5-hydroxytryptamine uptake and on monoamine oxidase activity, Biochem.
Biophys. Res. Commun. 344 (2006) 688-695.
[22] R.A. Copeland, Evaluation of Enzyme Inhibitors in Drug Discovery, Wiley Interscience,
Hoboken, 2005.
[23] L. Basile, M. Pappalardo, S. Guccione, D. Milardi, R.R. Ramsay, Computational comparison of
imidazoline association with the I2 binding site in human monoamine oxidases. J. Chem. Inf.
Model. 54 (2014) 1200−1207.
[24] J. Juárez-Jiménez, E. Mendes, C. Galdeano, C. Martins, D.B. Silva, J. Marco-Contelles, M. do
Carmo Carreiras, F.J. Luque, RR. Ramsay, Exploring the structural basis of the selective inhibition
of monoamine oxidase A by dicarbonitrile aminoheterocycles: role of Asn181 and Ile335 validated
by spectroscopic and computational studies. Biochim. Biophys. Acta. 1844 (2014) 389-397.
[25] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E.
Bourne, The Protein Data Bank, Nucleic Acids Res. 28 (2000) 235-242.
[26] S.Y. Son, J. Ma, Y. Kondou, M. Yoshimura, E. Yamashita, T. Tsukihara, Structure of human
monoamine oxidase A at 2.2 Ǻ resolution: the control of opening the entry for substrates/inhibitors,
Proc. Natl. Acad. Sci. USA. 15 (2008) 5739-5744.
[27] C. Binda, J. Wang, L. Pisani, C. Caccia, A. Carotti, P. Salvati, D.E. Edmondson, A. Mattevi,
Structures of human monoamine oxidase B complexes with selective noncovalent inhibitors:
safinamide and coumarin analogs, J. Med. Chem. 23 (2007) 5848-5852.
[28] F. Chimenti, A. Bolasco, D. Secci, P. Chimenti, A. Granese, S. Carradori, M. Yanez, F. Orallo,
F. Ortuso, S. Alcaro, Investigations on the 2-thiazolylhydrazyne scaffold: synthesis and molecular
modeling of selective human monoamine oxidase inhibitors, Bioorg. Med. Chem. 18 (2010) 5715-
5723.
26

ACCEPTED MANUSCRIPT
[29] MacroModel, version 10.8, Schrödinger, LLC, New York, NY, 2015.
[30] Maestro, version 10.2, Schrödinger, LLC, New York, NY, 2015.
[31] Glide, version 6.7, Schrödinger, LLC, New York, NY, 2015.
[32] Impact version 6.7, Schrödinger, LLC, New York, NY, 2015.
[33] Desmond Molecular Dynamics System, version 4.2, D. E. Shaw Research, New York, NY,
2015.
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•
•
•
•
•
A series of (E)-3-heteroarylidenechroman-4-ones was synthesized.
The compounds were evaluated in vitro as inhibitors of both human MAO isoforms.
All the compounds were found to be selective hMAO-B inhibitors.
The most active compound showed MAO-B inhibitory activity in the nanomolar range.
Docking and molecular dynamics simulations proposed the binding mode of the best
compound.