Discovery, biological evaluation, and structure-activity and -selectivity relationships of 6′-substituted (E)-2-(benzofuran-3(2H)-ylidene)-N- methylacetamides, a novel class of potent and selective monoamine oxidase inhibitors

Article abstract of DOI:10.1021/jm4000769

The use of selective inhibitors of monoamine oxidase A (MAO-A) and B (MAO-B) holds a therapeutic relevance in the treatment of depressive disorders and Parkinson's disease (PD), respectively. Here, the discovery of a new class of compounds acting as monoamine oxidase inhibitors (MAO-Is) and bearing a 6′-substituted (E)-2-(benzofuran-3(2H)-ylidene)-N-alkylacetamide skeleton is reported. 6′-Sulfonyloxy derivatives exhibited outstanding affinities to MAO-A (7.0 nM < IC₅₀ < 49 nM, much higher than moclobemide) and a pronounced MAO-A/B selectivity. The corresponding 6′-benzyloxy derivatives showed potent MAO-B inhibition and inverted selectivity profile. The rigid E-geometry of the exocyclic double bond allowed a more efficient binding conformation compared to more flexible and less active 2-(1-benzofuran-3-yl)-N- methylacetamide isomers and 4-N-methylcarboxamidomethylcoumarin analogues. Focused structural modifications and docking simulations enabled the identification of key molecular determinants for high affinity toward both MAO isoforms. These novel MAO-Is may represent promising hits for the development of safer therapeutic agents with a potential against depression, PD, and other age-related neurodegenerative pathologies.

Full text of DOI:10.1021/jm4000769

Article
pubs.acs.org/jmc
Discovery, Biological Evaluation, and Structure−Activity and
−Selectivity Relationships of 6′-Substituted (E)‑2-(Benzofuran-
3(2H)‑ylidene)‑N‑methylacetamides, a Novel Class of Potent and
Selective Monoamine Oxidase Inhibitors
Leonardo Pisani,† Maria Barletta,† Ramon Soto-Otero,‡ Orazio Nicolotti,† Estefania Mendez-Alvarez,‡
Marco Catto,† Antonellina Introcaso,† Angela Stefanachi,† Saverio Cellamare,† Cosimo Altomare,†
and Angelo Carotti*,†
†Dipartimento di Farmacia − Scienze del Farmaco, Universita
̀
degli Studi di Bari “Aldo Moro”, Via E. Orabona, 4, I-70125 Bari, Italy
‡Grupo de Neuroquimica, Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad de Santiago de
Compostela, San Francisco I, E-15782, Santiago de Compostela, Spain
S
* Supporting Information
ABSTRACT: The use of selective inhibitors of monoamine oxidase A
(MAO-A) and B (MAO-B) holds a therapeutic relevance in the
treatment of depressive disorders and Parkinson’s disease (PD),
respectively. Here, the discovery of a new class of compounds acting
as monoamine oxidase inhibitors (MAO-Is) and bearing a 6′-substituted
(E)-2-(benzofuran-3(2H)-ylidene)-N-alkylacetamide skeleton is re-
ported. 6′-Sulfonyloxy derivatives exhibited outstanding affinities to
MAO-A (7.0 nM < IC50 < 49 nM, much higher than moclobemide) and
a pronounced MAO-A/B selectivity. The corresponding 6′-benzyloxy
derivatives showed potent MAO-B inhibition and inverted selectivity
profile. The rigid E-geometry of the exocyclic double bond allowed a
more efficient binding conformation compared to more flexible and less
active 2-(1-benzofuran-3-yl)-N-methylacetamide isomers and 4-N-
methylcarboxamidomethylcoumarin analogues. Focused structural modifications and docking simulations enabled the
identification of key molecular determinants for high affinity toward both MAO isoforms. These novel MAO-Is may represent
promising hits for the development of safer therapeutic agents with a potential against depression, PD, and other age-related
neurodegenerative pathologies.
preferentially, and is blocked by low concentrations of
selegiline.3 Dopamine, adrenaline, and noradrenaline are
nonselectively degraded by both isoenzymes.4 The two
isoforms are unevenly present in most mammalian cell types
but erythrocytes,5 with the proportion and distribution varying
from species to species. The different density and distribution
in human brain regions have been mapped through positron
emission tomography (PET) studies.6 The highest enzymatic
activity has been found in hypothalamus and basal ganglia
(striatum), where the prevalent isoform is MAO-B (80% of
total human brain MAO). To a major extent, the mood-
elevating effect of MAOs inhibitors is mediated by the elevation
of serotonin level, as a consequence of the inhibition of MAO-A
in glial cells.3
INTRODUCTION
■
Monoamine oxidases (MAO, EC 1.4.3.4, amine−oxygen
oxidoreductase) are FAD-dependent enzymes bound to the
outer mitochondrial membrane, where they catalyze the
oxidative deamination of both exogenous and endogenous
amines.1 Since MAOs are involved in the catabolic trans-
formation of several neurotransmitters, including serotonin,
histamine, and catecholamines, they have been considered
attractive therapeutic targets in neuropharmacology.2 Several
decades after the seminal discovery of the antidepressant action
of MAOs inhibitors, recombinant DNA technology has allowed
the isolation, purification, and structural characterization of two
distinct enzymatic isoforms, named MAO-A and MAO-B.
Although exerting the same biological function and showing
nearly 70% homology, the two isoenzymes differ in their three-
dimensional structure, substrate specificity, and sensitivity to
inhibitors. MAO-A predominates in catecholaminergic neurons,
deaminates preferentially serotonin, and is selectively inhibited
by clorgyline, whereas the MAO-B isoform prevails in
serotonergic neurons, metabolizes 2-phenylethylamine (PEA)
The X-ray crystallographic resolution of the enzyme−
inhibitors complexes has elucidated the three-dimensional
structures of both MAO isoforms and delineated the binding
Received: January 16, 2013
Published: February 25, 2013
© 2013 American Chemical Society
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Chart 1. Chemical Structures of Nonselective (NS) and Selective Inhibitors of Monoamine Oxidases A (A) and B (B)
mode of reversible and irreversible inhibitors.7−10 The main
structural differences of MAOs have been found in their active
sites because of the diverse shape and volume of the substrates/
inhibitors binding pockets. In hMAO-A there is a single
hydrophobic cavity of nearly 550 Å3 volume in humans (450 Å3
in the rMAO-A). By contrast, the active site of hMAO-B is
characterized by a smaller entrance cavity (290 Å3 volume) and
a wider substrate cavity of 420 Å3 volume.10,11 In both
isoenzymes these cavities are narrow pockets lined with
hydrophobic residues, with the flavin ring of FAD at the distal
end. The replacement of Ile199 and Tyr326 residues in hMAO-
B (correspondingly, Phe208 and Ile335 residues in hMAO-A),
which can be observed within the region that separates the two
cavities, represents a key structural feature for isoform-selective
molecular recognition.10 In addition, Ile199 acts as a flexible
“gating switch”, changing conformation according to the bound
ligand and moving from an open to a closed arrangement with
the protein backbone.12 On this basis, selective inhibitors have
been designed by exploiting the structural differences of the
“gate-keeper” residues and of the shape of MAO-A substrate-
binding site that looks less elongated and not as flat as the
MAO-B substrate cavity.
Since the 1950s, MAOs have attracted the attention of
medicinal chemists. Iproniazid (Chart 1), initially conceived as
antituberculosis drug, was the first MAO-I developed as an
antidepressive agent. The first generation of MAO-Is (e.g.,
tranylcypromine, Chart 1) was represented by irreversible and
nonselective compounds, whose severe side effects (e.g.,
hypertensive crisis triggered by the so-called “cheese effect”13
and the hepatotoxicity of hydrazine-containing com-
pounds14,15) hampered their further development. Later, a
second generation of MAO-Is came into the market. They were
selective but still irreversible inhibitors, typically bearing a
propargylamine moiety (e.g., clorgyline and selegiline, Chart 1).
The last generation MAO-Is were selective and reversible, with
potentially lower adverse effects. Currently, reversible and
selective MAO-AIs (RIMA) represent the third/fourth line
treatment of depressive disorders16 (e.g., moclobemide, Chart
1), whereas some irreversible and selective MAO-BIs are
clinically used in the therapy of Parkinson’s disease (PD) as
monotherapy or as L-DOPA adjuvants (e.g., selegiline and
rasagiline).17
More recently, MAO-Is have been re-evaluated as potential
therapeutics against many age-related pathologies,18 for which
neurotoxicity, protein misfolding and/or aggregation, iron
accumulation, mitochondrial damage, and oxidative stress
have been described as major downstream causes, e.g., in
Alzheimer’s disease (AD), PD, amyotrophic lateral sclerosis
(ALS, Lou Gehrig’s disease), and Huntington’s disease (HD).
Several studies reported an increase of MAO-B activity in the
brain and blood platelets of patients suffering from PD and
AD.19,20 Inhibition of MAOs has the potential of modulating
the level of key neurotransmitters involved in the pathogenesis
or triggering the cognitive decline and motor disabilities of
some neurodegenerative disorders (NDs).21 Furthermore, toxic
radicals are produced by the MAO catalytic cycle itself, leading
to hydrogen peroxide that works as a harmful precursor of
reactive oxygen species (ROS).22 These mechanisms underlie
the considerable interest toward MAOs in the search of novel
therapeutic options against oxidative stress-related patholo-
gies.23,24 The inhibition of MAOs can reduce ROS production
directly or indirectly by limiting the Fenton reaction.25
Aiming at the discovery of novel and effective disease-
modifying agents to cope with NDs, the attention of many
researchers has been focused on MAO-Is by following two
main research lines. One consists of the study of already known
inhibitors (e.g., rasagiline, Chart 1) and of looking for
additional neuroprotective properties.26,27 Another elegant, as
well-promising, strategy is the design of multitarget directed
ligands (MTDLs) showing MAO inhibition as the core
activity28 (e.g., ladostigil, Chart 1).29,30 To date, the lack of a
disease-modifying therapy for NDs and other severe neuro-
logical illnesses linked to aging, as well as the safety issues
associated with currently available antidepressive and anti-PD
agents, stresses the urgency for novel, efficient, and less toxic
MAO-Is.3,16,31,32
Over the years, large numbers of heterocyclic scaffolds have
been exploited to design inhibitors targeting MAOs.33−39
Among them, nature-inspired oxygen heterocycles have
occupied a prominent role.40−45 In the past decade, our
research group has been successfully involved in the discovery
and optimization of novel MAO inhibitors (i.e., indeno-fused
azines46,47 and coumarins48), drawing helpful structure−activity
and structure−selectivity relationships (SARs and SSRs).49,50
The introduction of small basic moieties at position 4 of the
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Scheme 1. General Procedure for the Synthesis of 2H-1-Benzofuran-6-yl Derivatives 20−38, 43, and Benzofurans 39, 40, and
a
44
a
Reagents and conditions. (a) For Y = SO2: triethylamine, R2SO2Cl, dry acetonitrile, room temperature, 18 h. For Y = CH2: Cs2CO3, R2CH2Cl, dry
N,N-dimethylformamide, room temperature, 24 h. (b) For 20−36: suitable 4-chloromethylcoumarin 3−19, methylamine 2.0 N in THF, dry THF,
room temperature, 3 h. For 37: 4, ethylamine 2.0 N in THF, dry THF, room temperature, 18 h. For 38: 4, isopropylamine, dry THF, room
temperature, 24 h. For 43: 42, methylamine 2.0 N in THF, dry THF, room temperature, 3 h. (c) For 39−40: 21 (for 39) or 37 (for 40), DBU, dry
THF, room temperature, 24 h. For 44: 43, DBU, dry THF, reflux, 8 h.
Scheme 2. Hypothetic Reaction Mechanism for the Reaction of 7-Benzyloxy- and 7-Sulfonyloxy-4-chloromethylcoumarins with
Primary Amines in THF
coumarin ring has led to the discovery of potent and selective
MAO-B inhibitors (e.g., NW-1772, Chart 1) with an out-
standing biopharmacological profile.51,52
cyclocondensation reaction with slight modifications.53 Com-
pounds 1 and 2 were reacted with the suitable (hetero)-
arylsulfonyl chloride or alkylsulfonyl chloride and subsequently
with appropriate amines. In place of the expected nucleophilic
substitution reaction, the coupling between 4-chloromethyl
derivatives 3−19 and primary amines yielded compounds 20−
38 (Scheme 1) as the unique products, deriving from the
opening reaction of the lactonic ring followed by a cyclization
reaction through the intramolecular nucleophilic attack of the
phenoxide anion on the chloromethyl group (Scheme 2). The
exocyclic double bond always exhibited an E-geometry. A
similar reaction was previously observed by others but under
more drastic conditions, and it led to benzofuran-3-acetic
acids.54
When the same reaction was performed on 7-(3′-
chlorobenzyloxy)-4-chloromethylcoumarin 42, the nucleophilic
substitution product, which is the already known MAO-BI NW-
1772,51 was isolated along with a side product (43) as described
for compounds 20−38 (Scheme 1). The different reaction
outcome can be attributed to the different electronic effect of
the substituent at position 7 of the coumarin ring. In fact, this
group modulates the electrophilic reactivity of the coumarin
lactonic carbonyl. To assess the stereochemistry of the
exocyclic double bond of 6′-substituted-(E)-2-(benzofuran-
3(2H)-ylidene)-N-alkylacetamides, NOESY NMR experiments
were performed (see Supporting Information). A strong NOE
effect between the exocyclic vinylic proton and the amide
Using the same design strategy, we tried to improve the
pharmacokinetic properties of diversely substituted 7-sulfony-
loxycoumarins, previously reported as potent, selective, but
highly lipophilic MAO-A inhibitors.50 While working on the
synthesis of the designed molecules, we faced an unexpected
side reaction and discovered serendipitously a novel and
effective scaffold for the inhibition of MAOs, that is (E)-2-
(benzofuran-3(2H)-ylidene)-N-methylacetamide. Depending
on the substituents placed at positions 6′ and 3′ of the
benzofuran backbone, potent and extremely selective MAO-A
or, to a lesser extent, MAO-B inhibitors were obtained, with
IC50 values in the nanomolar range. Herein, we report the
synthesis, structural assignment, and biological evaluation as
MAO-Is of a novel series of 6′-substituted-(E)-2-[benzofuran-
3(2H)-ylidene]-N-alkylacetamides, along with a modeling study
aimed at highlighting key molecular determinants for high
MAO affinity. In addition, more flexible 2-(1-benzofuran-3-yl)-
N-methylacetamide isomers and 4-N-methylcarboxamidome-
thylcoumarin analogues were prepared to extend our under-
standing of the SARs and SSRs.
CHEMISTRY
■
The preparation of starting 4-chloromethyl-7-hydroxycoumar-
ins 151 and 2 was carried out by the well-known von Pechmann
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a
Scheme 3. Synthesis of Amidocoumarin Derivatives 49 and 50
a
Reagents and conditions: (a) 2.0 N CH3NH2 in THF, 90 °C, sealed vessel, 96 h; (b) triethylamine, 3-chlorobenzenesulfonyl chloride, dry
acetonitrile, room temperature, 5−24 h.
a
Scheme 4. Synthesis of 4-Aminomethylcoumarin Derivatives 52 and 53
a
Reagents and conditions: (a) N-(diphenylmethyl)methylamine, anhydrous K2CO3, TBAI (cat.), dry THF, reflux, 7 h; (b) TES, TFA, reflux, 4 h; (c)
(i) hexamethylenetetramine, dry chloroform, reflux, 48 h; (ii) HCl 6% in ethanol, reflux, 4 h.
proton as well as with the endocyclic methylene along with the
absence of spatial coupling signal between the vinylic proton
and H4 of benzofuran ring fully confirmed the E-geometry. By
treatment of derivatives 21, 37, and 43 with a strong non-
nucleophilic base such as 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU), the aromatization to the corresponding less rigid
benzofuran-3-acetamides 39, 40, and 44 was accomplished in
high yields (Scheme 1). Compound 41 was prepared by
reacting the commercially available 6-hydroxy-1-benzofuran-
3(2H)-one with 3-chlorobenzenesulfonyl chloride as detailed in
Experimental Section.
In order to extend the SAR study of these newly discovered
scaffolds, a small number of amido- and aminocoumarin
congeners were also synthesized. The synthetic route to amido
derivatives 49 and 50 started from the condensation of
resorcinol with diethyl 1,3-acetonedicarboxylate or diethyl
oxalacetate sodium salt to obtain intermediates 45 and 46,
respectively. These esters underwent aminolysis with methyl-
amine in a sealed vessel before final sulfonylation to yield
compounds 49 and 50 (Scheme 3). To synthesize compound
52, a protected form of methylamine was required to
circumvent the lactone opening reaction. To this purpose, N-
(diphenylmethyl)methylamine was chosen as the starting
material, being a hindered secondary amine more amenable
to nucleophilic substitution rather than to lactonic ring-
opening. The reductive cleavage of benzydryl group with
trifluoroacetic acid (TFA) in the presence of triethylsilane
(TES) as radical scavenger furnished the desired amine
(Scheme 4). Primary amine 53 was prepared through a
Delepine reaction55 by hydrolyzing urotropine salt of chloride 4
under acidic conditions (Scheme 4).
ENZYME INHIBITION ASSAYS
■
MAO inhibition data of compounds 20−41, 43, 44, 49, 50, 52,
and 53 are reported in Tables 1 and 2. The inhibition of
monoamine oxidases A and B activity was measured in vitro
through a spectrophotometric method,56 by using crude rat
brain mitochondrial suspensions as previously reported.50
RESULTS AND DISCUSSION
■
Nineteen 6′-sulfonyloxy-(E)-2-(benzofuran-3(2H)-ylidene)-N-
alkylacetamides (20−38) were designed, synthesized, and
tested as MAO-Is. Most of them showed MAO-A inhibition
values in the nanomolar range and a clear-cut selectivity over
MAO-B (Table 1). To gain deeper insights into the SARs,
focused structural modifications were introduced on the
benzofuran ring at position 6 (sulfonate group) and at position
3 (amide N-substituent). In addition, the effect of a methyl
group at position 7 was studied in a few compounds.
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Table 1. Biological Data of 2H-1-Benzofuran-6-yl Derivatives 20−38 and 43, Benzofurans 39, 40, and 44, and Benzofuran-3-one
41
a
b
b
c
compd
Y
R1
R2
R3
double bond
MAO-A
MAO-B
0%
20
21
22
23
24
25
26
27
28
30
31
32
33
29
34
35
36
37
38
39
40
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
CH2
CH2
H
phenyl
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
exo
endo
endo
34 3.9
41 3.3
41 3.2
19 2.5
12 2.6
45 3.5
37 2.9
29 3.2
19 3.1
16 3.1
9.8 1.4
24 4.7
15 2.7
11 2.2
7.0 2.1
9.1 0.9
11 2.8
H
3′-chlorophenyl
3′-fluorophenyl
3′-cyanophenyl
4′-chlorophenyl
4′-cyanophenyl
4′-nitrophenyl
4′-acetylphenyl
4′-methylsulfonylphenyl
5′-chlorothien-2′-yl
phenyl
0%
6
H
0.5%
H
0%
6%
H
c
H
10 2.1%
13%
H
H
0%
H
0%
H
6
1.4%
0.5%
0.5%
0.5%
Me
Me
Me
H
5
3′-chlorophenyl
4′-cyanophenyl
4′-methoxyphenyl
4′-methoxyphenyl
3′,3′,3′-trifluoropropyl
3′,3′,3′-trifluoropropyl
3′-chlorophenyl
3′-chlorophenyl
3′-chlorophenyl
3′-chlorophenyl
3′-chlorophenyl
3′-chlorophenyl
3′-chlorophenyl
2
6
0%
0%
Me
H
17 2.4%
Me
H
5
0.8%
4200 1100
14 2.3%
409 8.2
0%
1
H
iPr
0.3%
0.7%
H
Me
Et
4
H
31 2.0%
17 2.6%
427 5.9
0%
0%
d
41
43
44
H
H
Me
Me
exo
36 2.0
284 7.4
0%
endo
3100 800
10000 2400
e
MOC
a
b
Exocylic (exo) or endocyclic (endo) double bond position. MAO-A and MAO-B inhibitory activities are expressed as IC50 SD (nM), unless
c
otherwise expressed as percentage of inhibition SD, measured at 10 μM. Values are the mean of two or three independent experiments. Values are
d
expressed as percentage of inhibition SD measured at 20 μM by mean of two or three independent experiments. Exact structure in table heading.
e
MOC = moclobemide.
substituents with varying lipophilic, electronic, and steric
properties on the phenyl ring of the sulfonate group at position
6 (derivatives 20−29, Table 1). The MAO-A inhibitory
potencies and MAO-A over MAO-B selectivities of compounds
21, 22, 25, 26, and 27 remained close to those observed for the
unsubstituted compound 20, which showed IC50 = 34 nM at
MAO-A and no MAO-B inhibition at 10 μM. Interestingly, 4′-
methoxy- (29), 4′-chloro- (24), and to a lesser extent 3′-cyano-
(23) and 4′-methylsulfonyl-benzenesulfonate (28) derivatives
exhibited a slightly higher MAO-A activity than 20. The
different stereoelectronic and lipophilic properties of these
substituents did not allow the derivation of significant insights
on the SARs and SSRs. As a further structural variation, the 3′-
chlorophenyl group of 21 was replaced with the isosteric 5′-
chlorothien-2′-yl group, affording compound 30 with a
significantly improved MAO-A affinity (IC50 = 16 nM vs 41
nM) and a high MAO-A selectivity.
Table 2. Biological Data of Coumarin Congeners 49, 50, 52,
and 53
a
a
compd
X
R
MAO-A
MAO-B
49
50
52
53
-CH2CO-
-CO-
Me
Me
Me
H
111 8.3
8.0 0.9%
5300 800
997 14
8300 2100
9.0 1.7%
1700 500
3300 500
-CH2-
-CH2-
a
MAO-A and MAO-B inhibitory activities are expressed as IC50 SD
(nM) unless otherwise expressed as percentage of inhibition SD,
measured at 10 μM. Values are the mean of two or three independent
experiments.
The introduction of a methyl group at position 7 was
conceived keeping in mind the remarkable change of affinity
against both MAO isoforms resulting from the introduction of a
methyl substituent at position 8 of 4,7-disubstituted coumarin
The modulation of MAO-A inhibitory activity of N-methyl
substituted amides was pursued by introducing a series of
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Figure 1. Key pharmacophoric elements for MAO-A inhibition.
derivatives.30 The working idea was rooted on the fact that
position 7 of benzofurans might be considered topologically
equivalent to position 8 of coumarins. Even though the change
of MAO-A affinity was not very pronounced, all of the 7-
methyl-substituted derivatives proved to be more active than
the corresponding 7-unsubstituted analogues (compare 32 vs
21, 33 vs 25, and 34 vs 29, Table 1). This structural
modification led to the most active compound of the series (34,
IC50 = 7.0 nM), bearing a 4′-methoxybenzenesulfonate moiety.
This MAO-AI was nearly 1000-fold more potent than
moclobemide (IC50 = 10 μM), which represents one of the
most popular RIMA used in the therapy of depression. The
positive effect of the methyl group at position 7 was not
confirmed by derivative 36 that was slightly less active than its
demethylated analogue 35 (IC50 = 11 nM and IC50 = 9.1 nM,
respectively), endowed with a flexible fluoroalkylsulfonate
group. Compounds 35 and 36, designed as safer and less
metabolically labile analogues of arylsulfonates, exhibited
outstanding potencies and an excellent MAO-A selectivity.
The homologation of the amide methyl group to ethyl causes a
dramatic drop (∼100-fold) of MAO-A affinity (37 vs 21). A
further increased steric hindrance in the isopropyl homologue
38 led to the complete loss of affinity to both MAO isoforms.
The biological evaluation of MAO inhibition was extended to
the benzofuran isomers 39 and 40 to address the importance of
a rigid geometry, allowed by the E-configuration of the
exocyclic double bond, for a regiospecific interaction at the
MAO-A binding site. Indeed, the increased flexibility of the
amide lateral chain at position 3 resulted in a consistent drop of
MAO-A affinity (39 vs 21, 40 vs 37), proving the importance of
the rigid “folded” conformation determined by the E-double
bond for a high inhibitory potency. Like their precursors 21 and
37, both benzofuran isomers 39 and 40 were not able to inhibit
MAO-B.
design strategy, compound 21 was chosen as the reference
compound because of its closer similarity with previously
reported potent coumarin MAO inhibitors50,51 that bear a m-
chlorophenyl ring as substituent at position 7. 4-N-Methyl-
carboxamidomethylcoumarin 49 showed lower MAO-A in-
hibitory potency (IC50 = 111 nM, Table 2) than its 2,3-
dihydrobenzofuran analogue 21 but greater than its benzofuran
analogue 39 (IC50 = 34 nM and IC50 = 409 nM, respectively),
and consequently, its selectivity ratio over MAO-B was much
lower. Conversely, the less flexible amido derivative 50 was
almost inactive on both isoenzymes when tested at 10 μM. The
insertion of a basic, protonatable group at position 4 of the
coumarin core led to moderate MAO inhibitors 52 and 53
exhibiting micromolar affinities toward MAO-A and MAO-B
and poor selectivity (Table 2). Interestingly, compound 52
showed a reversed, albeit low, selectivity, being more active on
MAO-B. Biological data found for the benzyloxy derivative 43
(IC50 = 427 nM and IC50 = 36 nM for MAO-A and MAO-B,
respectively, Table 1) confirm our previous findings concerning
the key role of the substituent at position 7 in modulating the
isoform selectivity for coumarin derivatives.50 A comparison
between inhibitor 43 and the corresponding benzofuran 44
points out that the exocyclic double bond is preferred for a high
MAO-B binding affinity in this series, too.
The E-geometry of the double bond freezes the compound in
a “folded” molecular shape, which is essential for an optimal
fitting into the MAOs substrate cavity. The unusual deshielding
of the benzofuran H4′ in 2,3-dihydrobenzofuran-3-yl com-
pounds ranging from 8.68 to 8.97 ppm can be attributed to the
strong anisotropic effect of the amide carbonyl that points
toward H4′ of the aromatic ring. As a matter of fact, the same
proton was found at 7.50 ppm in an open chain trans-cinnamic
acid derivative (see Supporting Information).
The SARs discussed above for these novel, potent, and
selective sulfonate MAO-A inhibitors allowed the identification
of the key structural requirements for an efficient MAO-A
binding, which are summarized in a pictorial representation in
Figure 1. The nature and size of the bridge linking the aromatic
or alkyl moiety to position 6 or 7 of the benzofuran and
coumarin rings, respectively, influenced mainly selectivity and
represented a crucial pharmacophoric feature along with the
rigid and folded E-geometry of the propenamide moiety, which
allowed an optimal spatial orientation of the molecule for
productive binding interactions within the MAO active site and
was essential for a high enzymatic affinity. The substituents on
the amide nitrogen impact a sterically sensitive enzymatic
A comparison between compound 21 and its benzofuran-3-
one analogue 41 (Table 1), lacking the exocyclic α,β-
unsaturated amide moiety and showing a very poor (as for
MAO-A) or totally absent (as for MAO-B) inhibitory activity,
highlighted the importance of the rigid 3-propenamide chain
for a strong binding interaction at the enzymatic active site.
To further validate this novel scaffold, a small series of
congeners of compound 21 was designed by replacing the
benzofuran bicyclic core of derivatives 20−38 with a coumarin
nucleus and by introducing small nitrogen-containing groups
(i.e., amine and amide groups) at position 4 while maintaining a
m-chlorobenzenesulfonate ester moiety at position 6. In our
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region. In fact, groups larger than methyl drastically reduced the
MAO-A inhibitory potency. Stringent lipophilic, electronic, and
steric requirements are absent in the arylsulfonyloxy group,
where electron-withdrawing and electron-donating substituents
in para as well as in meta position did not produce any relevant
shifts of affinity. On this basis, this region can be properly
modified in order to improve the pharmacokinetic properties of
this class of inhibitors.
Molecular Modeling Study. A molecular modeling study
was carried out to shed light on peculiar binding interactions
modulating MAO-A affinity, with the aim of explaining at least
the dramatic drop of affinity resulting from the introduction of
large-sized N-amide substituent at position 3 (i.e., changing the
N-substituent of the 3-propenamide group from methyl to
isopropyl, that is, from 21 to 38) and supporting the relevance
of maintaining an exocyclic double bond (i.e., a propenamide
moiety at position 3) for high MAO-A inhibitory potency
(compare 21 vs 39).
Since inhibition data were determined on mitochondria
MAOs from rat brain homogenates, docking simulations were
run on a rat model of the MAO-A isoform. The 3D structure
for rMAO-A was retrieved from the PDB (coded as 1O5W). As
reported in our previous papers,50,51 a number of structural
water molecules were explicitly considered in the docking
simulations carried out using GOLD. Among those available in
GOLD, the ChemScore scoring function proved to better rank
the binding affinity of ligands in the simulated enzyme−
inhibitor complexes.57 As a result, a good agreement was
observed between experimental inhibitory activities and
docking scoring values.
The first purpose of the docking study was that of explaining
the dramatic fall of MAO-A activity originated by increasing the
size of the N-substituent on the 3-propenamide group
(compare 21-NMe with 37-NEt and 38-NiPr derivatives). As
shown in Figure 2a,b, docking simulations showed that the
posing of 21 and 38 returned a similar binding mode with the
benzofuran moiety facing the region beneath the FAD and the
3′-chlorophenyl moiety placed in the cavity entrance, lined by
residues having a more pronounced hydrophobic character.
This binding topology was actually observed in all the poses
occurring during GOLD simulation. However, N-substituents
bulkier than methyl were less tolerated, presumably because of
a high chance of steric clashes with structural water molecules
in a protein region characterized by a number of tyrosine
residues. Very satisfactorily, docking simulations disclosed that
moving from methyl to isopropyl (21 vs 38) lowered the score
from 21.37 to 12.61 kJ/mol. In addition, the steric hindrance of
the bulkier isopropyl group could hinder the formation of the
hydrogen bond (HB) between the 3-propenamide carbonyl and
the Tyr444 hydroxyl along with other reinforcing interactions
mediated by two surrounding structural water molecules
(Figure 2b).
Figure 2. Docking pose of inhibitor 21 (a), 38 (b), and 39 (c) into
rMAO-A binding site. Relevant amino acid residues, FAD cofactor,
and inhibitors are represented as stick models colored according to the
following atom code: C atoms in white, yellow, and green for amino
acid residues, cofactor, and inhibitor, respectively. Red dashed lines
indicate the HB network established by the ligand with protein
residues and structural water molecules. The protein structure is
rendered as a white cartoon model.
The second goal of the docking investigation was that of
elucidating the key role of the exocyclic double bond to
strengthen the binding interactions with MAO-A. Indeed a
significant drop of inhibitory activity was observed going from
an exocyclic to an endocyclic double bond in tautomeric
compounds belonging to both the 6′-sulfonyloxy and 6′-
benzyloxy classes (compare 21 vs 39 and 43 vs 44,
respectively). However, docking simulations were limited to
inhibitors 21 and 39 for which posing and scoring were
compared. As can be seen in Figure 2c, derivative 39, bearing
the endocyclic double bond, adopted a reversed binding mode
with the 3′-chlorophenyl moiety facing the FAD while the
benzofuran ring occupied the cavity entrance surrounded by a
number of phenylalanine residues. The reversed binding
topology shown by 39 was, to some extent, not completely
unexpected, as it was already observed in recent investigations
for some 7-substituted coumarin derivatives.50,51 As far as
inhibitor 39 is concerned, the observed binding topology was
likely assumed to ensure a larger room for the 3-
carboxamidomethyl group which is much more flexible than
the E-propenamide moiety of 21. The reversed posing of 39
was characterized by a lower score compared to 21 (i.e., 13.55
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use of dry solvents, the glassware was flame-dried and then cooled
under a stream of dry argon before use. Elemental analyses were
performed on the EuroEA 3000 analyzer only on the final compounds
tested as MAOs inhibitors and are reported in Supporting Information.
The measured values for C, H, and N agreed to within 0.40% of the
theoretical values. Nuclear magnetic resonance spectra were recorded
at 300 MHz on a Varian Mercury 300 instrument at ambient
temperature in the specified deuterated solvent. Chemical shifts (δ)
are quoted in parts per million (ppm) and are referenced to the
residual solvent peak. The coupling constants J are given in hertz (Hz).
The following abbreviations were used: s (singlet), d (doublet), dd
(doublet of doublet), dq (doublet of quadruplet), dt (doublet of
triplet), t (triplet), q (quadruplet), m (multiplet), br (broad signal).
Signals due to OH and NH protons were located by deuterium
exchange with D2O.
Synthesis of 4-(Chloromethyl)-7-hydroxy-8-methyl-2H-chro-
men-2-one (2). A slight modification of a reported procedure was
followed.51 2-Methylresorcinol (4.1 g, 33 mmol) was dissolved in
concentrated H2SO4 (40 mL) while cooling at 0 °C, and ethyl 4-
chloroacetoacetate (4.1 mL, 30 mmol) was slowly added in 15 min via
syringe. The mixture was kept at room temperature under magnetic
stirring for 24 h and then poured onto crushed ice (600 g) and filtered.
The resulting solid was crystallized from methanol/water. Yield: 62%.
1H NMR (300 MHz,DMSO-d6) δ: 2.14 (s, 3H), 4.93 (s, 2H), 6.40 (s,
kJ/mol vs 21.37 kJ/mol) in agreement with the observed
inhibitory potency. Unlike 21, the HB network involved the 6′-
sulfonyloxy group of the bridge joining the two aromatic
moieties with the effect of moving the 3′-chlorophenyl ring into
a less hydrophobic region mostly formed by structural water
molecules and tyrosine residues. On the other hand, the amide
NH group of 39 established an HB with the carbonyl backbone
of Phe208 in the entrance cavity of the enzyme.
CONCLUSION
■
During the course of our investigation aimed at enhancing the
pharmacokinetic features of already reported MAO-Is,50 an
unexpected lactone opening reaction of 7-substituted coumar-
ins with primary amines was observed. Serendipitously, this
reaction constituted a straightforward and very efficient
synthetic method for a scaffold hopping to 6′-substituted (E)-
2-(benzofuran-3(2H)-ylidene)-N-methylacetamides as a novel
class of MAO-Is. The nature and the size of the 6′-substituents
modulate MAO affinity and isoform selectivity. In particular, in
this report we described several (hetero)aryl sulfonate esters
showing outstanding affinity (IC50 in the nanomolar range) and
selectivity toward MAO-A (approximately, selectivity index of
≫500), comparable to that of esuprone (7-hydroxy-3,4-
dimethylcoumarin ethanesulfonate),58 taken as the reference
coumarin compound, and much greater than moclobemide,
which is a prototype reversible MAO-A inhibitor and one of the
most widely used antidepressive agents.
1H), 6.87 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 10.54 (s, 1H,
disappears with D2O).
General Procedure for the Synthesis of Intermediates 3−19.
The suitable 7-hydroxycoumarin derivative 151 (50 mmol) or 2 (10
mmol) was suspended in anhydrous acetonitrile (200 mL in the case
of 1; 40 mL in the case of 2) followed by the addition of triethylamine
(7.7 mL, 55 mmol in the case of 1; 1.5 mL, 11 mmol in the case of 2).
After complete dissolution of the starting coumarin derivatives, the
suitable sulfonyl chloride (50 mmol in the case of 1, 10 mmol in the
case of 2) was added. The mixture was stirred at room temperature for
18 h. After removal of the solvent under vacuum, the crude solid was
purified by flash chromatography (gradient eluent, different mixtures
of ethyl acetate in n-hexane).
Structural modifications mainly addressing the 6-arylsulfo-
nate group and the N-amide substituents at position 3 enabled
the detection of the crucial pharmacophoric elements
modulating MAO-A inhibitory potency, fully supported by a
docking study. The high MAO-A selectivity of the arylsulfo-
nates could be ascribed to the folded molecular shape, better
accommodated in MAO-A substrate-binding site, which is
wider and less flat than MAO-B. The obtainment of MAO-A-
targeting inhibitors devoid of dietary restrictions and toxic side
effects is still a desirable goal in the therapy of depressive
disorders. Remarkably, the substitution of the chemically and
metabolically sensitive, and even toxic, arylsulfonate moiety
with a fluoroalkylsulfonate group, afforded compounds 35 and
36 with a potentially safer pharmacological and toxicological
profile. The outstanding MAO-A inhibitory potency and MAO-
A over MAO-B selectivity of 35 and 36 should warrant further
preclinical investigation to exploit their potential as anti-
depressive agents.
Moreover, the new class of selective MAO-B inhibitors (such
as compound 43) reported herein would also deserve further
attention for their potential use in the treatment of behavior-
and age-related disorders associated with MAO activity and/or
with MAO byproducts toxicity.
A hit-to-lead development process to improve chemical and
metabolic stability of these novel scaffolds is ongoing and will
be reported in due time.
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl Benzenesulfonate
(3). 3 was purified by flash chromatography (gradient eluent, ethyl
1
acetate in n-hexane 0% → 40%). Yield: 90%. H NMR (300 MHz,
DMSO-d6) δ: 4.98 (s, 2H), 6.68 (s, 1H), 7.12 (dd, J1 = 2.2 Hz, J2 = 8.8
Hz, 1H), 7.17 (d, J = 2.2 Hz, 1H), 7.66−7.71 (m, 2H), 7.82−7.87 (m,
2H), 7.91−7.94 (m, 2H).
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 3-Chlorobenzene-
sulfonate (4). 4 was purified by flash chromatography (gradient
1
eluent, ethyl acetate in n-hexane 0% → 50%). Yield: 86%. H NMR
(300 MHz, DMSO-d6) δ: 4.99 (s, 2H), 6.69 (s, 1H), 7.17 (dd, J1 = 2.2
Hz, J2 = 8.8 Hz, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.68−7.73 (m, 1H),
7.88 (d, J = 8.8 Hz, 1H), 7.91−7.94 (m, 2H), 8.00−8.01 (m, 1H).
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 3-Fluorobenzene-
sulfonate (5). 5 was purified by flash chromatography (gradient
1
eluent, ethyl acetate in n-hexane 0% → 40%). Yield: 94%. H NMR
(300 MHz, DMSO-d6) δ: 4.99 (s, 2H), 6.69 (s, 1H), 7.16 (dd, J1 = 2.5
Hz, J2 = 8.8 Hz, 1H), 7.27 (d, J = 2.5 Hz, 1H), 7.71−7.78 (m, 3H),
7.83−7.87 (m, 1H), 7.87 (d, J = 8.8 Hz, 1H).
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 3-Cyanobenzene-
sulfonate (6). 6 was purified by flash chromatography (gradient
1
eluent, ethyl acetate in n-hexane 0% → 30%). Yield: 88%. H NMR
(300 MHz, DMSO-d6) δ: 5.00 (s, 2H), 6.69 (s, 1H), 7.15 (dd, J1 = 2.5
Hz, J2 = 8.8 Hz, 1H), 7.26 (d, J = 2.5 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H),
7.86 (d, J = 8.8 Hz, 1H), 8.16 (dq, J1 = 1.4 Hz, J2 = 8.3 Hz, 1H), 8.29
(dt, J1 = 1.4 Hz, J2 = 8.0 Hz, 1H), 8.46 (t, J = 1.4 Hz, 1H).
EXPERIMENTAL SECTION
Chemistry. Starting materials, reagents, and analytical grade
■
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 4-Chlorobenzene-
solvents were purchased from Sigma-Aldrich (Europe). The purity
sulfonate (7). 7 was purified by flash chromatography (gradient
1
1
of all the intermediates, checked by H NMR and HPLC, was always
eluent, ethyl acetate in n-hexane 0% → 30%). Yield: 91%. H NMR
better than 95%. Flash chromatographic separations were performed
on a Biotage SP1 purification system using flash cartridges prepacked
with KP-Sil 32−63 μm, 60 Å silica. All reactions were routinely
checked by TLC using Merck Kieselgel 60 F254 aluminum plates and
visualized by UV light or iodine. Regarding the reactions requiring the
(300 MHz, DMSO-d6) δ: 4.99 (s, 2H), 6.69 (s, 1H), 7.14 (dd, J1 = 2.5
Hz, J2 = 8.8 Hz, 1H), 7.25 (d, J = 2.5 Hz, 1H), 7.73−7.78 (m, 2H),
7.87 (d, J = 8.8 Hz, 1H), 7.90−7.95 (m, 2H).
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 4-Cyanobenzene-
sulfonate (8). 8 was purified by flash chromatography (gradient
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1
eluent, ethyl acetate in n-hexane 0% → 30%). Yield: 81%. H NMR
(300 MHz, DMSO-d6) δ: 4.99 (s, 2H), 6.70 (s, 1H), 7.15 (dd, J1 = 2.5
Hz, J2 = 8.8 Hz, 1H), 7.29 (d, J = 2.5 Hz, 1H), 7.87 (d, J = 8.8 Hz,
1H), 8.11 (d, J = 8.8 Hz, 2H), 8.17 (d, J = 8.8 Hz, 2H).
suspended in dry THF (8.0 mL), and a commercially available 2.0 N
solution of methylamine (for compounds 20−36) or ethylamine (for
compound 37) in THF (10 mL, 20 mmol) was added. For the
synthesis of compound 38 isopropylamine (1.7 mL, 20 mmol) was
previously dissolved in dry THF (2.0 mL) before addition to the
reaction mixture. The resulting mixture was stirred at room
temperature until completion of the reaction as checked by TLC
control (3−24 h). The inorganic precipitate was filtered off, and the
solution was concentrated to dryness and purified by flash
chromatography (gradient eluent, different mixtures of ethyl acetate
in n-hexane).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl Benzenesulfonate (20). 20 was purified by flash
chromatography (gradient eluent, ethyl acetate in n-hexane 20% →
80%). Yield: 86%. 1H NMR (300 MHz, DMSO-d6) δ: 2.63 (d, J = 4.7
Hz, 3H), 5.22 (d, J = 2.5 Hz, 2H), 5.84 (t, J = 2.5 Hz, 1H), 6.50−6.54
(dd, J1 = 2.2 Hz, J2 = 8.5 Hz, 1H), 6.62 (d, J = 2.2 Hz, 1H), 7.64−7.69
(m, 2H), 7.78−7.89 (m, 3H), 8.05 (br s, 1H, disappears with D2O),
8.82 (d, J = 8.5 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 3-Chlorobenzenesulfonate (21). 21 was purified
by flash chromatography (gradient eluent, ethyl acetate in n-hexane
10% → 80%). Yield: 91%. 1H NMR (300 MHz, DMSO-d6) δ: 2.64 (d,
J = 4.7 Hz, 3H), 5.23 (d, J = 2.5 Hz, 2H), 5.86 (t, J = 2.5 Hz, 1H), 6.58
(dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 6.70 (d, J = 2.2 Hz, 1H), 7.66−7.71
(m, 1H), 7.82−7.85 (m, 1H), 7.89−7.95 (m, 2H), 8.04 (br q, J = 4.7
Hz, 1H, disappears with D2O), 8.86 (d, J = 8.8 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 3-Fluorobenzenesulfonate (22). 22 was purified
by flash chromatography (gradient eluent, ethyl acetate in n-hexane
10% → 80%). Yield: 93%. 1H NMR (300 MHz, DMSO-d6) δ: 2.64 (d,
J = 4.4 Hz, 3H), 5.24 (d, J = 2.5 Hz, 2H), 5.86 (t, J = 2.5 Hz, 1H), 6.58
(dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 6.69 (d, J = 2.2 Hz, 1H), 7.69−7.73
(m, 3H), 7.78−7.80 (m, 1H), 8.04 (br q, J = 4.4 Hz, 1H, disappears
with D2O), 8.85 (d, J = 8.8 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 3-Cyanobenzenesulfonate (23). 23 was purified
by flash chromatography (gradient eluent, ethyl acetate in n-hexane
10% → 90%). Yield: 83%. 1H NMR (300 MHz, DMSO-d6) δ: 2.64 (d,
J = 4.6 Hz, 3H), 5.23 (d, J = 2.6 Hz, 2H), 5.86 (t, J = 2.6 Hz, 1H), 6.60
(dd, J1 = 2.2 Hz, J2 = 8.5 Hz, 1H), 6.74 (d, J = 2.2 Hz, 1H), 7.85 (t, J =
8.0 Hz, 1H), 8.05 (br q, J = 4.6 Hz, 1H, disappears with D2O), 8.16
(dq, J1 = 1.4 Hz, J2 = 8.3 Hz, 1H), 8.29 (dt, J1 = 1.4 Hz, J2 = 8.0 Hz,
1H), 8.46 (t, J = 1.4 Hz, 1H), 8.85 (d, J = 8.5 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 4-Chlorobenzenesulfonate (24). 24 was purified
by flash chromatography (gradient eluent, ethyl acetate in n-hexane
10% → 80%). Yield: 87%. 1H NMR (300 MHz, DMSO-d6) δ: 2.64 (d,
J = 4.7 Hz, 3H), 5.23 (d, J = 2.5 Hz, 2H), 5.85 (t, J = 2.5 Hz, 1H), 6.55
(dd, J1 = 2.2 Hz, J2 = 8.5 Hz, 1H), 6.67 (d, J = 2.2 Hz, 1H), 7.74 (d, J =
8.8 Hz, 2H), 7.88 (d, J = 8.8 Hz, 2H), 8.04 (br q, J = 4.7 Hz, 1H,
disappears with D2O), 8.85 (d, J = 8.5 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 4-Cyanobenzenesulfonate (25). 25 was purified
by flash chromatography (gradient eluent, ethyl acetate in n-hexane
20% → 90%). Yield: 98%. 1H NMR (300 MHz, DMSO-d6) δ: 2.64 (d,
J = 4.7 Hz, 3H), 5.22 (d, J = 2.5 Hz, 2H), 5.86 (t, J = 2.5 Hz, 1H), 6.56
(dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H), 8.03−8.08
(m, 3H, 1 proton disappears with D2O), 8.15 (d, J = 8.5 Hz, 2H), 8.85
(d, J = 8.8 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 4-Nitrobenzenesulfonate (26). 26 was purified
by flash chromatography (gradient eluent, ethyl acetate in n-hexane
20% → 80%). Yield: 90%. 1H NMR (300 MHz, DMSO-d6) δ: 2.64 (d,
J = 4.7 Hz, 3H), 5.24 (d, J = 2.5 Hz, 2H), 5.86 (t, J = 2.5 Hz, 1H), 6.56
(dd, J1 = 2.2 Hz, J2 = 8.5 Hz, 1H), 6.74 (d, J = 2.2 Hz, 1H), 8.04 (br q,
J = 4.7 Hz, 1H, disappears with D2O), 8.13−8.18 (m, 2H), 8.41−8.45
(m, 2H), 8.87 (d, J = 8.5 Hz, 1H).
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 4-Nitrobenzene-
sulfonate (9). 9 was purified by flash chromatography (gradient
1
eluent, ethyl acetate in n-hexane 0% → 50%). Yield: 77%. H NMR
(300 MHz, DMSO-d6) δ: 4.99 (s, 2H), 6.70 (s, 1H), 7.16−7.19 (m,
1H), 7.28−7.30 (m, 1H), 7.87 (d, J = 8.5 Hz, 1H), 8.19 (d, J = 8.3 Hz,
2H), 8.45 (d, J = 8.3 Hz, 2H).
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 4-Acetylbenzene-
sulfonate (10). 10 was purified by flash chromatography (gradient
1
eluent, ethyl acetate in n-hexane 0% → 40%). Yield: 95%. H NMR
(300 MHz, DMSO-d6) δ: 2.64 (s, 3H), 4.98 (s, 2H), 6.69 (s, 1H), 7.15
(dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 7.25 (d, J = 2.2 Hz, 1H), 7.87 (d, J =
8.8 Hz, 1H), 8.07 (d, J = 8.5 Hz, 2H), 8.18 (d, J = 8.5 Hz, 2H).
4 - ( C h l o r o m e t h y l ) - 2 - o x o - 2 H - c h r o m e n - 7 - y l 4 -
(Methylsulfonyl)benzenesulfonate (11). 11 was purified by flash
chromatography (gradient eluent, ethyl acetate in n-hexane 0% →
1
50%). Yield: 62%. H NMR (300 MHz, DMSO-d6) δ: 3.30 (s, 3H),
4.99 (s, 2H), 6.70 (s, 1H), 7.17 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.32
(d, J = 2.5 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 8.21 (s, 4H).
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 4-Methoxybenze-
nesulfonate (12). 12 was purified by flash chromatography (gradient
1
eluent, ethyl acetate in n-hexane 0% → 40%). Yield: 91%. H NMR
(300 MHz, DMSO-d6) δ: 3.85 (s, 3H), 4.98 (s, 2H), 6.68 (s, 1H), 7.10
(dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.16 (d, J = 2.5 Hz, 1H), 7.17 (d, J =
9.1 Hz, 2H), 7.83 (d, J = 9.1 Hz, 2H), 7.85 (d, J = 8.8 Hz, 1H).
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 5-Chlorothio-
phene-2-sulfonate (13). 13 was purified by flash chromatography
(gradient eluent, ethyl acetate in n-hexane 0% → 30%). Yield: 57%. 1H
NMR (300 MHz, DMSO-d6) δ: 5.01 (s, 2H), 6.72 (s, 1H), 7.22 (dd, J1
= 2.5 Hz, J2 = 8.8 Hz, 1H), 7.33 (d, J = 2.5 Hz, 1H), 7.41 (d, J = 4.1
Hz, 1H), 7.80 (d, J = 4.1 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H).
4-(Chloromethyl)-8-methyl-2-oxo-2H-chromen-7-yl Benze-
nesulfonate (14). 14 was purified by flash chromatography (gradient
1
eluent, ethyl acetate in n-hexane 0% → 40%). Yield: 85%. H NMR
(300 MHz, DMSO-d6) δ: 1.99 (s, 3H), 5.00 (s, 2H), 6.70 (s, 1H), 7.10
(d, J = 8.8 Hz, 1H), 7.68−7.76 (m, 3H), 7.85−7.93 (m, 3H).
4-(Chloromethyl)-8-methyl-2-oxo-2H-chromen-7-yl 3-Chlor-
obenzenesulfonate (15). 15 was purified by flash chromatography
(gradient eluent, ethyl acetate in n-hexane 0% → 50%). Yield: 84%. 1H
NMR (300 MHz, DMSO-d6) δ: 2.07 (s, 3H), 5.00 (s, 2H), 6.71 (s,
1H), 7.11 (d, J = 9.1 Hz, 1H), 7.71−7.76 (m, 2H), 7.88−7.92 (m,
1H), 7.95−7.98 (m, 1H), 8.00−8.01 (m, 1H).
4-(Chloromethyl)-8-methyl-2-oxo-2H-chromen-7-yl 4-Cya-
nobenzenesulfonate (16). 16 was purified by flash chromatography
(gradient eluent, ethyl acetate in n-hexane 0% → 30%). Yield: 87%. 1H
NMR (300 MHz, DMSO-d6) δ: 2.05 (s, 3H), 5.00 (s, 2H), 6.72 (s,
1H), 7.10 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 8.13 (d, J = 8.3
Hz, 2H), 8.19 (d, J = 8.3 Hz, 2H).
4-(Chloromethyl)-8-methyl-2-oxo-2H-chromen-7-yl 4-Me-
thoxybenzenesulfonate (17). 17 was purified by flash chromatog-
raphy (gradient eluent, ethyl acetate in n-hexane 0% → 60%). Yield:
1
69%. H NMR (300 MHz, acetone-d6) δ: 2.10 (s, 3H), 3.95 (s, 3H),
4.98 (s, 2H), 6.65 (s, 1H), 7.14 (d, J = 8.8 Hz, 1H), 7.20 (d, J = 9.1
Hz, 2H), 7.76 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 9.1 Hz, 2H).
4-(Chloromethyl)-2-oxo-2H-chromen-7-yl 3,3,3-Trifluoro-
propane-1-sulfonate (18). 18 was purified by flash chromatography
(gradient eluent, ethyl acetate in n-hexane 0% → 40%). Yield: 71%. 1H
NMR (300 MHz, DMSO-d6) δ: 2.90−3.06 (m, 2H), 3.95−4.01 (m,
2H), 5.04 (s, 2H), 6.73 (s, 1H), 7.43 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz,
1H), 7.59 (d, J = 2.5 Hz, 1H), 7.96 (d, J = 8.8 Hz, 1H).
4-(Chloromethyl)-8-methyl-2-oxo-2H-chromen-7-yl 3,3,3-
Trifluoropropane-1-sulfonate (19). 19 was purified by flash
chromatography (gradient eluent, ethyl acetate in n-hexane 0% →
60%). Yield: 81%. H NMR (300 MHz, acetone-d6) δ: 2.43 (s, 3H),
2.99−3.15 (m, 2H), 3.98−4.04 (m, 2H), 5.01 (s, 2H), 6.69 (s, 1H),
1
7.45 (d, J = 8.8 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 4-Acetylbenzenesulfonate (27). 27 was purified
by flash chromatography (gradient eluent, ethyl acetate in n-hexane
General Procedure for the Synthesis of Final Compounds
20−38. A suitable sulfonate intermediate, 3−19 (2.0 mmol), was
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1
20% → 90%). Yield: 97%. 1H NMR (300 MHz, DMSO-d6) δ: 2.63 (d,
J = 4.7 Hz, 3H), 2.64 (s, 3H), 5.23 (d, J = 2.5 Hz, 2H), 5.85 (t, J = 2.5
Hz, 1H), 6.54 (dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 6.69 (d, J = 2.2 Hz,
1H), 8.01−8.05 (m, 3H, 1 proton disappears with D2O), 8.16 (d, J =
8.8 Hz, 2H), 8.83 (d, J = 8.8 Hz, 1H).
ethyl acetate in n-hexane 20% → 90%). Yield: 98%. H NMR (300
MHz, DMSO-d6) δ: 2.12 (s, 3H), 2.66 (d, J = 4.7 Hz, 3H), 2.89−3.01
(m, 2H), 3.95−4.01 (m, 2H), 5.28 (d, J = 2.5 Hz, 2H), 5.87 (t, J = 2.5
Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 8.03 (br q, J = 4.7 Hz, 1H,
disappears with D2O), 8.79 (d, J = 8.5 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 4-(Methylsulfonyl)benzenesulfonate (28). 28
was purified by flash chromatography (gradient eluent, ethyl acetate in
(3E)-3-[2-(Ethylamino)-2-oxoethylidene]-2,3-dihydro-1-ben-
zofuran-6-yl 3-Chlorobenzenesulfonate (37). 37 was purified by
flash chromatography (gradient eluent, ethyl acetate in n-hexane 10%
1
1
n-hexane 30% → 90%). Yield: 88%. H NMR (300 MHz, DMSO-d6)
→ 80%). Yield: 90%. H NMR (300 MHz, DMSO-d6) δ: 1.02 (t, J =
δ: 2.64 (d, J = 4.7 Hz, 3H), 3.32 (s, 3H), 5.24 (d, J = 2.5 Hz, 2H), 5.86
(t, J = 2.5 Hz, 1H), 6.59 (dd, J1 = 2.5 Hz, J2 = 8.5 Hz, 1H), 6.73 (d, J =
2.5 Hz, 1H), 8.05 (br q, J = 4.7 Hz, 1H, disappears with D2O), 8.16 (d,
J = 8.8 Hz, 2H), 8.20 (d, J = 8.8 Hz, 2H), 8.86 (d, J = 8.5 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 4-Methoxybenzenesulfonate (29). 29 was
purified by flash chromatography (gradient eluent, ethyl acetate in n-
hexane 20% → 90%). Yield: 94%. 1H NMR (300 MHz, DMSO-d6) δ:
2.63 (d, J = 4.7 Hz, 3H), 3.85 (s, 3H), 5.22 (d, J = 2.5 Hz, 2H), 5.84 (t,
J = 2.5 Hz, 1H), 6.50 (dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 6.62 (d, J = 2.2
Hz, 1H), 7.15 (d, J = 9.1 Hz, 2H), 7.78 (d, J = 9.1 Hz, 2H), 8.02 (br q,
J = 4.7 Hz, 1H, disappears with D2O), 8.82 (d, J = 8.8 Hz, 1H).
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 5-Chlorothiophene-2-sulfonate (30). 30 was
purified by flash chromatography (gradient eluent, ethyl acetate in n-
hexane 10% → 70%). Yield: 80%. 1H NMR (300 MHz, DMSO-d6) δ:
2.65 (d, J = 4.7 Hz, 3H), 5.28 (d, J = 2.5 Hz, 2H), 5.87 (t, J = 2.5 Hz,
1H), 6.64 (dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 6.75 (d, J = 2.2 Hz, 1H),
7.38 (d, J = 4.4 Hz, 1H), 7.75 (d, J = 4.4 Hz, 1H), 8.06 (br q, J = 4.7
Hz, 1H, disappears with D2O), 8.90 (d, J = 8.8 Hz, 1H).
7.2 Hz, 3H), 3.08−3.17 (m, 2H), 5.23 (d, J = 2.2 Hz, 2H), 5.80−5.88
(m, 1H), 6.58 (dd, J1 = 2.2 Hz, J2 = 8.5 Hz, 1H), 6.70 (d, J = 2.2 Hz,
1H), 7.67−7.70 (m, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.5 Hz,
1H), 7.94 (s, 1H), 8.10 (br t, J = 5.2 Hz, 1H, disappears with D2O),
8.86 (d, J = 8.5 Hz, 1H).
(3E)-3-[2-(Isopropylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 3-Chlorobenzenesulfonate (38). 38 was purified
by flash chromatography (gradient eluent, ethyl acetate in n-hexane
10% → 60%). Yield: 51%. 1H NMR (300 MHz, DMSO-d6) δ: 1.06 (d,
J = 6.6 Hz, 6H), 3.80−3.90 (m, 1H), 5.23 (d, J = 1.9 Hz, 2H), 5.80−
5.88 (m, 1H), 6.58 (dd, J1 = 2.2 Hz, J2= 8.8 Hz, 1H), 6.70 (d, J = 1.6
Hz, 1H), 7.66−7.70 (m, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.91 (d, J = 8.0
Hz, 1H), 7.94 (s, 1H), 7.98 (br d, J = 7.4 Hz, 1H, disappears with
D2O), 8.86 (d, J = 8.8 Hz, 1H).
General Procedure for the Synthesis of Benzofurans 39 and
40. The suitable derivative 21 or 37 (0.5 mmol) was dissolved in dry
THF (5.0 mL), and DBU (0.075 mL, 0.5 mmol) was added. The
mixture was stirred at room temperature for 24 h. The solvent was
removed under reduced pressure and the resulting oil was purified by
flash chromatography (gradient eluent, different mixtures of ethyl
acetate in n-hexane), yielding the desired final products.
3-[2-(Methylamino)-2-oxoethyl]-1-benzofuran-6-yl 3-Chlor-
obenzenesulfonate (39). 39 was purified by flash chromatography
(gradient eluent, ethyl acetate in n-hexane 20% → 80%). Yield: 88%.
1H NMR (300 MHz, DMSO-d6) δ: 2.56 (d, J = 4.4 Hz, 3H), 3.46 (s,
2H), 6.95 (dd, J1 = 1.9 Hz, J2 = 8.5 Hz, 1H), 7.37 (d, J = 1.9 Hz, 1H),
7.58 (d, J = 8.5 Hz, 1H), 7.64−7.70 (m, 1H), 7.79−7.82 (m, 1H), 7.89
(s, 1H), 7.92−7.93 (m, 2H), 8.00 (br q, J = 4.4 Hz, 1H, disappears
with D2O).
3-[2-(Ethylamino)-2-oxoethyl]-1-benzofuran-6-yl 3-Chloro-
benzenesulfonate (40). 40 was purified by flash chromatography
(gradient eluent, ethyl acetate in n-hexane 20% → 90%). Yield: 60%.
1H NMR (300 MHz, DMSO-d6) δ: 0.99 (t, J = 7.2 Hz, 3H), 3.00−3.10
(m, 2H), 3.45 (s, 2H), 6.95 (dd, J1 = 2.2 Hz, J2 = 8.5 Hz, 1H), 7.37 (d,
J = 2.2 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.64−7.69 (m, 1H), 7.79−
7.83 (m, 1H), 7.88−7.94 (m, 3H), 8.08 (br s, 1H, disappears with
D2O).
(3E)-7-Methyl-3-[2-(methylamino)-2-oxoethylidene]-2,3-di-
hydro-1-benzofuran-6-yl Benzenesulfonate (31). 31 was purified
by flash chromatography (gradient eluent, ethyl acetate in n-hexane
20% → 80%). Yield: 94%. 1H NMR (300 MHz, DMSO-d6) δ: 1.82 (s,
3H), 2.64 (d, J = 4.7 Hz, 3H), 5.24 (d, J = 2.2 Hz, 2H), 5.84 (t, J = 2.2
Hz, 1H), 6.43 (d, J = 8.8 Hz, 1H), 7.66−7.71 (m, 2H), 7.81−7.90 (m,
3H), 8.02 (br s, 1H, disappears with D2O), 8.68 (d, J = 8.8 Hz, 1H).
(3E)-7-Methyl-3-[2-(methylamino)-2-oxoethylidene]-2,3-di-
hydro-1-benzofuran-6-yl 3-Chlorobenzenesulfonate (32). 32
was purified by flash chromatography (gradient eluent, ethyl acetate in
1
n-hexane 10% → 80%). Yield: 85%. H NMR (300 MHz, DMSO-d6)
δ: 1.89 (s, 3H), 2.65 (d, J = 4.7 Hz, 3H), 5.25 (d, J = 2.5 Hz, 2H), 5.85
(t, J = 2.5 Hz, 1H), 6.51 (d, J = 8.8 Hz, 1H), 7.69−7.75 (m, 1H),
7.85−7.87 (m, 1H), 7.93−7.96 (m, 2H), 8.03 (br q, J = 4.7 Hz, 1H,
disappears with D2O), 8.71 (d, J = 8.8 Hz, 1H).
(3E)-7-Methyl-3-[2-(methylamino)-2-oxoethylidene]-2,3-di-
hydro-1-benzofuran-6-yl 4-Cyanobenzenesulfonate (33). 33
was purified by flash chromatography (gradient eluent, ethyl acetate in
1
n-hexane 20% → 90%). Yield: 90%. H NMR (300 MHz, DMSO-d6)
Synthesis of 3-Oxo-2,3-dihydro-1-benzofuran-6-yl 3-Chlor-
obenzenesulfonate (41). Commercially available 6-hydroxy-1-
benzofuran-3(2H)-one (0.83 g, 5.5 mmol) was suspended in dry
THF (18 mL), and potassium carbonate (0.42 g, 3.0 mmol) was
added. 3-Chlorobenzenesulfonyl chloride (0.70 mL, 5.0 mmol) was
dissolved in dry THF (7.0 mL) and added dropwise while cooling at 0
°C with an external ice bath. The mixture was slowly kept at room
temperature and then the stirring was continued for an additional 18 h.
The solvent was evaporated under reduced pressure, and the resulting
mixture was purified by flash chromatography (gradient eluent, ethyl
δ: 1.88 (s, 3H), 2.64 (d, J = 4.7 Hz, 3H), 5.25 (d, J = 2.5 Hz, 2H), 5.85
(t, J = 2.5 Hz, 1H), 6.49 (d, J = 8.8 Hz, 1H), 8.02 (br q, J = 4.7 Hz, 1H,
disappears with D2O), 8.08 (d, J = 8.5 Hz, 2H), 8.17 (d, J = 8.5 Hz,
2H), 8.69 (d, J = 8.8 Hz, 1H).
(3E)-7-Methyl-3-[2-(methylamino)-2-oxoethylidene]-2,3-di-
hydro-1-benzofuran-6-yl 4-Methoxybenzenesulfonate (34). 34
was purified by flash chromatography (gradient eluent, ethyl acetate in
1
n-hexane 30% → 90%). Yield: 97%. H NMR (300 MHz, DMSO-d6)
δ: 1.85 (s, 3H), 2.64 (d, J = 4.7 Hz, 3H), 3.86 (s, 3H), 5.24 (d, J = 2.5
Hz, 2H), 5.83 (t, J = 2.5 Hz, 1H), 6.48 (d, J = 8.5 Hz, 1H), 7.17 (d, J =
8.8 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 8.01 (br q, J = 4.7 Hz, 1H,
disappears with D2O), 8.68 (d, J = 8.5 Hz, 1H).
1
acetate in n-hexane 20% → 40%). Yield: 39%. H NMR (300 MHz,
CDCl3) δ: 4.67 (s, 2H), 6.73 (dd, J1 = 1.9 Hz, J2 = 8.3 Hz, 1H), 6.89
(d, J = 1.9 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H),
7.66−7.70 (m, 1H), 7.75−7.78 (m, 1H), 7.88−7.90 (m, 1H).
Synthesis of 7-[(3-Chlorobenzyl)oxy]-4-(chloromethyl)-2H-
chromen-2-one (42). 7-Hydroxycoumarin derivative 151 (1.1 g, 5.0
mmol) was dissolved in dry DMF (25 mL), and then Cs2CO3 (1.9 g,
6.0 mmol) and 3-chlorobenzyl bromide (0.65 mL, 5.0 mmol) were
added. The mixture was stirred at room temperature for 24 h and then
poured onto crushed ice. The precipitate was washed several times
with water and used without further purification for the next steps.
Yield: 79%. Spectroscopic data are in agreement with those already
reported in the literature.8,51
(3E)-3-[2-(Methylamino)-2-oxoethylidene]-2,3-dihydro-1-
benzofuran-6-yl 3,3,3-Trifluoropropane-1-sulfonate (35). 35
was purified by flash chromatography (gradient eluent, ethyl acetate
1
in n-hexane 20% → 70%). Yield: 86%. H NMR (300 MHz, DMSO-
d6) δ: 2.66 (d, J = 4.7 Hz, 3H), 2.85−3.01 (m, 2H), 3.86−3.91 (m,
2H), 5.28 (d, J = 2.5 Hz, 2H), 5.88 (t, J = 2.5 Hz, 1H), 6.92 (dd, J1 =
2.2 Hz, J2 = 8.5 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 8.06 (br q, J = 4.7
Hz, 1H, disappears with D2O), 8.97 (d, J = 8.5 Hz, 1H).
(3E)-7-Methyl-3-[2-(methylamino)-2-oxoethylidene]-2,3-di-
hydro-1-benzofuran-6-yl 3,3,3-Trifluoropropane-1-sulfonate
(36). 36 was purified by flash chromatography (gradient eluent,
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Synthesis of (2E)-2-[6-[(3-Chlorobenzyl)oxy]-1-benzofuran-
3(2H)-ylidene]-N-methylacetamide (43). Intermediate 42 (0.67 g,
2.0 mmol) was dissolved in dry THF (4.0 mL), and the commercially
available 2.0 N solution of methylamine was added (10 mL, 20 mmol).
The resulting mixture was stirred at room temperature for 3 h. The
inorganic precipitate was filtered off and the solution was concentrated
to dryness and purified by flash chromatography (gradient eluent,
ethyl acetate in n-hexane 20% → 70%), isolating the nonfluorescent
spot at higher Rf (0.7, in ethyl acetate/n-hexane 8/2 v/v). Yield: 31%.
1H NMR (300 MHz, DMSO-d6) δ: 2.64 (d, J = 4.7 Hz, 3H), 5.14 (s,
4-[2-(Methylamino)-2-oxoethyl]-2-oxo-2H-chromen-7-yl 3-
Chlorobenzenesulfonate (49). 49 was purified by crystallization
from hot absolute ethanol. Yield: 84%. H NMR (300 MHz, DMSO-
1
d6) δ: 2.56 (d, J = 4.7 Hz, 3H), 3.68 (s, 2H), 6.45 (s, 1H), 7.13 (dd, J1
= 2.2 Hz, J2 = 8.8 Hz, 1H), 7.23 (d, J = 2.2 Hz, 1H), 7.67−7.73 (m,
1H), 7.77 (d, J = 8.8 Hz, 1H), 7.86−7.95 (m, 2H), 8.00−8.01 (m,
1H), 8.07 (br s, 1H, disappears with D2O).
4-[(Methylamino)carbonyl]-2-oxo-2H-chromen-7-yl 3-Chlor-
obenzenesulfonate (50). 50 was purified by flash chromatography
(gradient eluent, ethyl acetate in n-hexane 20% → 70%). Yield: 80%.
1H NMR (300 MHz, DMSO-d6) δ: 2.76 (d, J = 4.7 Hz, 3H), 6.59 (s,
1H), 7.11 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.31 (d, J = 2.5 Hz, 1H),
7.67−7.73 (m, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.81−7.88 (m, 1H),
7.92−7.95 (m, 1H), 8.00−8.01 (m, 1H), 8.83 (br q, J = 4.7 Hz, 1H,
disappears with D2O).
2H), 5.17 (d, J = 2.2 Hz, 2H), 5.67 (t, J = 2.2 Hz, 1H), 6.56−6.60 (m,
2H), 7.36−7.42 (m, 3H), 7.50 (s, 1H), 7.87 (br q, J = 4.7 Hz, 1H,
disappears with D2O), 8.83 (d, J = 8.8 Hz, 1H).
Synthesis of 2-{6-[(3-Chlorobenzyl)oxy]-1-benzofuran-3-yl}-
N-methylacetamide (44). Derivative 43 (0.099 g, 0.30 mmol) was
dissolved in dry THF (2.0 mL), and DBU (0.045 mL, 0.30 mmol) was
added. The mixture was heated at reflux for 8 h. The solvent was
removed under reduced pressure and the resulting oil was purified by
flash chromatography (gradient eluent, ethyl acetate in n-hexane 20%
Synthesis of 4-{[(Diphenylmethyl)(methyl)amino]methyl}-2-
oxo-2H-chromen-7-yl 3-Chlorobenzenesulfonate (51). Inter-
mediate 4 (0.39 g, 1.0 mmol) was dissolved in dry THF (10 mL)
before the addition of K2CO3 (0.14 g, 1.0 mmol), tetrabutylammo-
nium iodide (TBAI) (0.0070 g, 0.020 mmol), and N-
(diphenylmethyl)methylamine (0.22 g, 1.1 mmol). The mixture was
kept under reflux for 7 h, and then the solvent was evaporated under
reduced pressure. Purification by flash chromatography (gradient
eluent, ethyl acetate in n-hexane 0% → 30%) yielded the desired
1
→ 90%), yielding the desired final product 44. Yield: 62%. H NMR
(300 MHz, DMSO-d6) δ: 2.57 (d, J = 4.7 Hz, 3H), 3.42 (s, 2H), 5.15
(s, 2H), 6.95 (dd, J1 = 2.2 Hz, J2 = 8.5 Hz, 1H), 7.23 (d, J = 2.2 Hz,
1H), 7.35−7.42 (m, 3H), 7.46 (d, J = 8.5 Hz, 1H), 7.51 (s, 1H), 7.68
(s, 1H), 7.97 (br q, J = 4.7 Hz, 1H, disappears with D2O).
Synthesis of Ethyl (7-Hydroxy-2-oxo-2H-chromen-4-yl)-
acetate (45). Resorcinol (0.66 g, 6.0 mmol) was suspended in
diethyl 1,3-acetonedicarboxylate (1.2 mL, 6.6 mmol), and a few drops
of concentrated H2SO4 were added. The mixture was heated at 100 °C
for 45 min with vigorous stirring, then cooled at room temperature
and crystallized from hot acetic acid, furnishing the desired ester as an
off-white solid. Yield: 61%. 1H NMR (300 MHz, DMSO-d6) δ: 1.15 (t,
J = 7.2 Hz, 3H), 3.90 (s, 2H), 4.08 (q, J = 7.2 Hz, 2H), 6.21 (s, 1H),
6.71 (d, J = 2.2 Hz, 1H), 6.77 (dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 7.48
(d, J = 8.8 Hz, 1H), 10.57 (s, 1H, disappears with D2O).
Synthesis of Ethyl 7-Hydroxy-2-oxo-2H-chromene-4-carbox-
ylate (46). Resorcinol (0.66 g, 6.0 mmol) was suspended in absolute
ethanol (15 mL), and diethyl oxalacetate sodium salt (3.8 g, 18 mmol)
was added. The mixture was refluxed for 96 h, cooled at room
temperature, and concentrated to dryness. The resulting solid mixture
was treated with chloroform, and the insoluble residue was filtered-off.
The solution was concentrated under vacuum and purified by flash
chromatography (gradient eluent, ethyl acetate in n-hexane 10% →
70%), thus furnishing the desired ester as a yellow solid. Yield: 43%.
1H NMR (300 MHz, DMSO-d6) δ: 1.32 (t, J = 7.2 Hz, 3H), 4.36 (q, J
= 7.2 Hz, 2H), 6.58 (s, 1H), 6.76 (d, J = 2.2 Hz, 1H), 6.82 (dd, J1 = 2.2
Hz, J2 = 8.8 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 10.75 (s, 1H, disappears
with D2O).
General Procedure for the Synthesis of Amides 47 and 48.
Ester 45 or 46 (2.0 mmol) and commercially available 2.0 N solution
of methylamine in THF (30 mmol, 15 mL) were stirred at 90 °C in a
closed Pyrex vessel for 96 h. After cooling, the crude mixture was
concentrated to dryness and treated with CHCl3. The resulting
precipitate was filtered and washed several times with CHCl3, thus
obtaining the desired product with a satisfactory purity.
1
product as an amorphous solid. Yield: 56%. H NMR (300 MHz,
DMSO-d6) δ: 2.08 (s, 3H), 3.62 (s, 2H), 4.65 (s, 1H), 6.70 (s, 1H),
7.09 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.16−7.23 (m, 5H), 7.26−7.31
(m, 4H), 7.46−7.48 (m, 3H), 7.67−7.72 (m, 1H), 7.85−7.95 (m, 2H),
7.99−8.00 (m, 1H).
Synthesis of 4-[(Methylamino)methyl]-2-oxo-2H-chromen-
7-yl 3-Chlorobenzenesulfonate (52). Coumarin derivative 51
(0.22 g, 0.40 mmol) was preliminarily dissolved in trifluoroacetic
acid (5.0 mL) under irradiation with ultrasounds. Then triethylsilane
(0.13 mL, 0.80 mmol) was added and the mixture was refluxed for 4 h.
After dilution with saturated Na2CO3 (50 mL), the aqueous phase was
extracted with ethyl acetate (3 × 30 mL). The organic layers were
collected, dried over Na2SO4, concentrated to dryness, and the
obtained crude solid was purified by flash chromatography by using
1
ethyl acetate as the eluent. Yield: 72%. H NMR (300 MHz, DMSO-
d6) δ: 2.31 (s, 3H), 3.82 (s, 2H), 6.47 (s, 1H), 7.09 (dd, J1 = 2.5 Hz, J2
= 8.8 Hz, 1H), 7.23 (d, J = 2.5 Hz, 1H), 7.67−7.73 (m, 1H), 7.85−
7.95 (m, 2H), 7.86 (d, J = 8.8 Hz, 1H), 7.99−8.01 (m, 1H), NH not
detected.
Synthesis of 4-(Aminomethyl)-2-oxo-2H-chromen-7-yl 3-
chlorobenzenesulfonate Hydrochloride (53). Hexamethylene-
tetramine (0.28 g, 2.0 mmol) was added to a suspension of
intermediate 4 (0.77 g, 2.0 mmol) in dry chloroform (10 mL), and
the mixture was refluxed for 48 h. After the mixture was cooled to
room temperature, the obtained precipitate was separated from the
solution by filtration and used without further purification. Urotropine
salt of intermediate 4 (0.80 g, 1.5 mmol) was refluxed in 6% HCl in
ethanol (10 mL) until complete dissolution, and the mixture was
heated for an additional 1 h and then cooled to room temperature.
The resulting precipitate was filtered, dissolved in saturated Na2CO3
(40 mL), and extracted with ethyl acetate (3 × 60 mL). The organic
layers were collected, washed with brine (1 × 30 mL), dried over
NaSO4, and concentrated to dryness. The resulting oil was treated
with the commercially available solution of 4.0 N HCl in dioxane to
obtain the desired product as a hydrochloride salt. Overall yield: 69%.
1H NMR (300 MHz, DMSO-d6) δ: 4.35 (s, 2H), 6.54 (s, 1H), 7.17
(dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.34 (d, J = 2.5 Hz, 1H), 7.68−7.74
(m, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.87−7.96 (m, 2H), 8.00−8.01 (m,
1H), 8.50 (s, 3H, disappears with D2O).
Monoamine Oxidases Inhibition Assays. MAO inhibitory
activity of compounds in Tables 1 and 2 was assessed using a
continuous spectrophotometric assay,56 monitoring the rate of
oxidation of the nonselective nonfluorescent MAO substrate kynur-
amine to 4-hydroxyquinoline. Briefly, male Sprague−Dawley rats
(200−250 g) were sacrificed by decapitation. The brains were
immediately removed and washed in an ice-cold isotonic Na2HPO4/
2-(7-Hydroxy-2-oxo-2H-chromen-4-yl)-N-methylacetamide
1
(47). Yield: 89%. H NMR (300 MHz, DMSO-d6) δ: 2.57 (d, J = 4.7
Hz, 3H), 3.60 (s, 2H), 6.14 (s, 1H), 6.70 (d, J = 2.5 Hz, 1H), 6.77 (dd,
J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 8.06 (br s, 1H,
disappears with D2O), 10.53 (s, 1H, disappears with D2O).
7-Hydroxy-N-methyl-2-oxo-2H-chromene-4-carboxamide
1
(48). Yield: 73%. H NMR (300 MHz, DMSO-d6) δ: 2.77 (d, J = 4.7
Hz, 3H), 6.22 (s, 1H), 6.72 (d, J = 2.5 Hz, 1H), 6.77 (dd, J1 = 2.5 Hz,
J2 = 8.8 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 8.73 (br q, J = 4.7 Hz, 1H,
disappears with D2O), 10.72 (br s, 1H, disappears with D2O).
General Procedure for the Synthesis of Final Compounds 49
and 50. Amide 47 or 48 (1.0 mmol) was suspended in dry acetonitrile
(8.0 mL), and triethylamine (0.14 mL, 1.0 mmol) was added, followed
by 3-chlorobenzenesulfonyl chloride (0.17 mL, 1.2 mmol). The
mixture was stirred at room temperature for 5−24 h. After evaporation
of the solvent, the resulting crude oil was purified as indicated below.
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KH2PO4 buffer (pH 7.40) containing sucrose. A crude brain
mitochondrial fraction was then prepared by differential centrifuga-
tion59 and stored at −40 °C in an isotonic Na2HPO4/KH2PO4 buffer
(pH 7.4) containing KCl. MAO-A and MAO-B activities in
mitochondrial preparations (1 mg/mL) were assayed using as selective
and irreversible inhibitors clorgyline (250 nM) and (−)-L-deprenyl
(250 nM), respectively. After a preincubation for 5 min with the
assayed compound dissolved in DMSO at a final concentration of 5%
(v/v), kynuramine was added at a concentration equal to the
corresponding KM value (90 μM for MAO-A and 60 μM for MAO-B).
Then the rate of formation of 4-hydroxyquinoline was monitored at
314 nm for 5 min. Finally, IC50 values were determined by nonlinear
regression of MAO inhibition vs −log of the concentration plots, using
the program Origin, version 6.0 (Microcal Software Inc., North-
ampton, MA).
RIMA, reversible inhibitor of monoamine oxidase A; SSR,
structure−selectivity relationship; TBAI, tetrabutylammonium
iodide; TES, triethylsilane
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ASSOCIATED CONTENT
* Supporting Information
Elemental analysis data for compounds 20−41, 43, 44, 49, 50,
52, 53; synthesis and spectroscopic characterization of open
chain cinnamic acid derivatives; NOESY spectral data for
compounds 29 and 38. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
S
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Mattevi, A. Structure of Human Monoamine Oxidase B, a Drug Target
for the Treatment of Neurological Disorders. Nat. Struct. Mol. Biol.
2002, 9, 22−26.
AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed. Phone: +39-
080-5442782. Fax: +39-080-5442230. E-mail: angelo.carotti@
uniba.it.
■
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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The authors thank Prof. Renzo Luisi from the University of
Bari, Italy, for helpful discussions and execution of NOESY
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PRIN 20085HR5JK_005 and PRIN 2009ESXPT2_005), is
kindly acknowledged.
ABBREVIATIONS USED
■
HB, hydrogen bond; HD, Huntington’s disease; L-DOPA, L-3-
(3,4-dihydroxyphenyl)alanine; MAO-A, monoamine oxidase A;
MAO-B, monoamine oxidase B; MAO-I, monoamine oxidase
inhibitor; MTDL, multitarget directed ligand; ND, neuro-
degenerative disease/disorder; PEA, 2-phenylethylamine;
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