Mapping Chromone-3-Phenylcarboxamide Pharmacophore: Quid Est Veritas?

Article abstract of DOI:10.1021/acs.jmedchem.1c00510

Chromone-3-phenylcarboxamides (Crom-1 and Crom-2) were identified as potent, selective, and reversible inhibitors of human monoamine oxidase B (hMAO-B). Since they exhibit some absorption, distribution, metabolism, and excretion (ADME)-toxicity liabilities, new derivatives were synthesized to map the chemical structural features that compose the pharmacophore, a process vital for lead optimization. Structure-activity relationship data, supported by molecular docking studies, provided a rationale for the contribution of the heterocycle's rigidity, the carbonyl group, and the benzopyran heteroatom for hMAO-B inhibitory activity. From the study, N-(3-chlorophenyl)-4H-thiochromone-3-carboxamide (31) (hMAO-B IC50 = 1.52 ± 0.15 nM) emerged as a reversible tight binding inhibitor with an improved pharmacological profile. In in vitro ADME-toxicity studies, compound 31 showed a safe cytotoxicity profile in Caco-2, SH-SY5Y, HUVEC, HEK-293, and MCF-7 cells, did not present cardiotoxic effects, and did not affect P-gp transport activity. Compound 31 also protected SH-SY5Y cells from iron(III)-induced damage. Collectively, these studies highlighted compound 31 as the first-in-class and a suitable candidate for in vivo preclinical investigation.

Full text of DOI:10.1021/acs.jmedchem.1c00510

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Article
Mapping Chromone-3-Phenylcarboxamide Pharmacophore: Quid
Est Veritas?
Francesco Mesiti, Alexandra Gaspar,* Daniel Chavarria, Annalisa Maruca, Roberta Rocca,
Eva Gil Martins, Sandra Barreiro, Renata Silva, Carlos Fernandes, Sheraz Gul, Oliver Keminer,
Stefano Alcaro, and Fernanda Borges*
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Supporting Information
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ABSTRACT: Chromone-3-phenylcarboxamides (Crom-1 and Crom-2) were identified as potent, selective, and reversible
inhibitors of human monoamine oxidase B (hMAO-B). Since they exhibit some absorption, distribution, metabolism, and excretion
(ADME)-toxicity liabilities, new derivatives were synthesized to map the chemical structural features that compose the
pharmacophore, a process vital for lead optimization. Structure−activity relationship data, supported by molecular docking studies,
provided a rationale for the contribution of the heterocycle’s rigidity, the carbonyl group, and the benzopyran heteroatom for
hMAO-B inhibitory activity. From the study, N-(3-chlorophenyl)-4H-thiochromone-3-carboxamide (31) (hMAO-B IC50 = 1.52
0.15 nM) emerged as a reversible tight binding inhibitor with an improved pharmacological profile. In in vitro ADME-toxicity
studies, compound 31 showed a safe cytotoxicity profile in Caco-2, SH-SY5Y, HUVEC, HEK-293, and MCF-7 cells, did not present
cardiotoxic effects, and did not affect P-gp transport activity. Compound 31 also protected SH-SY5Y cells from iron(III)-induced
damage. Collectively, these studies highlighted compound 31 as the first-in-class and a suitable candidate for in vivo preclinical
investigation.
DA catabolism, prolonging its action in the basal ganglia,3 but
INTRODUCTION
■
also prevents the formation of neurotoxic dopamine-derived
Human monoamine oxidases A and B (hMAO-A and hMAO-B,
oxidative products.6 The i-hMAO-B selegiline and rasagiline are
respectively) are mitochondrial flavoenzymes expressed in the
brain and peripheral tissues.1 They catalyze the oxidative
deamination of endogenous and dietary monoamines having a
currently used as monotherapy or as add-on therapy to L-DOPA
in PD.7 However, they present adverse effects and safety issues
often associated with their irreversible inhibition.5 Considerable
pivotal role in regulating the levels of monoamine neuro-
transmitters.2 Since hMAO-A and hMAO-B isoforms exhibit
efforts have been made to overcome these drawbacks, which
different affinities for specific neurotransmitters, their selective
culminated in safinamide approval, the only reversible i-hMAO-
inhibition is considered an attractive approach for the therapy of
B in the market.5 As a result, the discovery of novel reversible i-
several neurological disorders.3 hMAO-B is a pharmacological
hMAO-B is an active research area in drug discovery, and we
target for the treatment of Parkinson’s disease (PD)3 and has
also been described as a potential target for Alzheimer’s disease.4
report here novel outcomes based on a new scaffold.
During aging, the activity and expression of hMAO-B are
enhanced approximately 4-fold in most of the brain regions.5
Received: March 19, 2021
Published: July 16, 2021
This hMAO-B overexpression boosts dopamine (DA) catabo-
lism, with subsequent DA deficiency and generation of hydrogen
peroxide and toxic aldehydes, which contribute to increased
oxidative stress and neurodegeneration.5 As such, the use of
selective hMAO-B inhibitors (i-hMAO-B) not only decreases
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Figure 1. Rational design followed for the mapping of the chromone-3-phenylcarboxamide pharmacophore.
Benzopyrones, such as coumarins and chromones, are
considered valid scaffolds for the development of new i-
hMAO-B.3,8 Within this framework, benzopyrone-based
libraries were developed and screened toward hMAOs, and
relevant structure−activity relationships (SAR) were estab-
lished.9−11 From our preliminary studies, we observed that the
location and type of substituent at position C3 of the γ-pyrone
nucleus were crucial to obtain potent and selective i-hMAO-B.12
As a result, N-(3′,4′-dimethyl phenyl)-4-oxo-4H-chromene-3-
carboxamide (Crom-1, Figure 1) and N-(3′-chlorophenyl)-4-
oxo-4H-chromene-3-carboxamide (Crom-2, Figure 1) were
identified as the most potent, selective, and reversible i-hMAO-
B.10 Notably, the presence of the amide spacer and its direct
linkage to the pyrone ring were found to be crucial for hMAO-B
inhibitory activity. Moreover, the presence of 3,4-dimethyl and
3-chloro substituents at the exocyclic aromatic ring had
remarkable effects on the compounds’ inhibitory potency
against hMAO-B.10,11 Focusing on the lead optimization, we
evaluated the effect of substituents located on the aromatic ring
of benzopyrone (chromone and its bioisoster coumarin).11
From this study, we validated chromone-3-phenyl carboxamide
as a lead to develop new and reversible i-hMAO-B. Then,
crystallographic ligand−enzyme studies showed that both
Crom-1 and Crom-2 occupied the hydrophobic active site of
hMAO-B.13 However, despite the promising hMAO-B inhib-
ition activity, these compounds presented cytotoxic effects in
differentiated SH-SY5Y cells.
relevance of key structural features of the benzopyrone moiety
on bioactivity, namely, heterocycle rigidity, the presence/
absence of a carbonyl group, and the type of heteroatom.
Modifications of the substituents at the aromatic exocyclic ring
were also explored. Thus, following a rational design strategy
(Figure 1), here, we report the synthesis of a novel library and
new SAR studies guided by in silico and experimental studies.
The results were then rationalized using docking studies with
crystal structures of hMAO-A and hMAO-B. The most
promising i-hMAO-B followed through with preliminary
preclinical in vitro ADME-toxicity studies, in order to evaluate
its cytotoxicity against SH-SY5Y, Caco-2, HEK-293, and MCF-7
cells, cardiotoxicity (hERG inhibition), P-gp modulation, and
protection against iron(III)-induced oxidative stress in differ-
entiated neuroblastoma cells. Collectively, these studies allowed
the selection of i-hMAO-B with the best ADME-toxicity profile
suitable for progression to animal studies.
RESULTS AND DISCUSSION
■
Chemistry. Benzopyran-inspired libraries were obtained
following the synthetic strategies depicted in Scheme 1. In
accordance with our rational design strategy (Figure 1),
compounds lacking the carbonyl group and/or the double
bond at the benzopyran ring were synthesized. To assess the
relevance of the benzopyran oxygen on the compounds’
bioactivity, isosteric replacements by nitrogen and sulfur were
also performed. The new derivatives were designed with the
same types of substitution patterns of Crom-1 and Crom-2 at
the phenyl carboxamide ring (3,4-dimethyl and 3-chloro groups,
respectively).10 Additionally, 3,4-dimethoxy and 3,4-dichloro
substituents were incorporated at the phenyl exocyclic ring to
gather additional SAR data.
The data obtained gave us the rationale to develop selective
and potent i-hMAO-B based on the chromone-3-phenyl-
carboxamide pharmacophore with improved absorption, dis-
tribution, metabolism, and excretion (ADME)-toxicity proper-
ties. To achieve this goal, the first step includes the evaluation of
the chemical features that are part of the chromone-3-
phenylcarboxamide pharmacophore. We investigated the
Chromane and chromene derivatives (2−5 and 7−10,
respectively) were synthetized by a one-pot reaction using
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Scheme 1. Synthetic Strategy Pursued for Synthesis of (A) Chromane-3-phenylcarboxamides (2−5), (B) Chromene-3-
phenylcarboxamides (7−10), (C) Coumarin-3-phenylcarboxamides (14−17), (D) 2-Oxoquinoline-3-phenylcarboxamides (20−
a
22) and N-Methyl-2-oxoquinoline-3-phenylcarboxamides (23−25), and (E) Thiochromone-3-phenylcarboxamides (30 and 31)
a
Reagents and conditions: (A,B) (a) POCl3, DMF, appropriate arylamine, rt, 24 h; (C) (a) diethyl malonate, EtOH, pyrimidine, reflux, overnight;
(b) NaOH (ethanolic solution), reflux, 1 h; (c) POCl3, DMF, appropriate arylamine, rt, 24 h; (D) (a) CH3I, K2CO3, 60 °C, argon, 1 h; (b) DIPEA,
TBTU, appropriate arylamine, rt, 24 h; (E) (a) N,N-dimethylformamide dimethyl acetal, 115 °C, 1 h; (b) H2S, −30 °C, 4 h, argon; (c) HCl,
AcOH, reflux, 4 h; (d) POCl3, appropriate arylamine, rt, 24 h.
chromane-3-carboxylic (1) or chromene-3-carboxylic acids (6)
as starting materials. The amides were synthesized by an acyl
nucleophilic substitution reaction between the appropriate
arylamine and the corresponding acyl chloride intermediate,
which is generated in situ by the addition of phosphoryl chloride
(POCl3) (Scheme 1B,A).10
Coumarin derivatives (14−17) were obtained using a three-
step synthetic strategy (Scheme 1C). The reaction of
salicylaldehyde (11) with diethyl malonate afforded coumarin-
3-ethyl carboxylate (12, Scheme 1C, step a), which, after alkaline
hydrolysis, yielded coumarin-3-carboxylic acid (13, Scheme 1C,
step b).14 Finally, the POCl3-mediated coupling reaction of 13
with the appropriate arylamine led to the synthesis of coumarin
carboxamides (14−17, Scheme 1C, step c).10
The synthetic route used to obtain 2-oxoquinoline derivatives
(20−22) and their N-methyl counterparts (23−25) is outlined
in Scheme 1D. First, N-methyl-2-oxoquinoline-3-carboxylic acid
(19) was obtained via N-methylation of 18 with methyl iodide
under alkaline conditions (Scheme 1D, step a).15 Then,
carboxylic acids (18 or 19) reacted with the appropriate
arylamine, using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethy-
laminium tetrafluoroborate (TBTU) as a coupling agent
(Scheme 1D, step b).16 In this case, the use of POCl3 was not
suitable for the synthesis of these amides due to the formation of
several byproducts.17,18
Thiochromone derivatives (30 and 31) were obtained using
the synthetic strategy shown in Scheme 1E. The synthesis
started with the reaction of ethyl 3-(2-fluorophenyl)-3-
oxopropanoate (26) with N,N-dimethylformamide dimethyl
acetal affording the intermediate 27 (Scheme 1E, step a), which
in turn was reacted with hydrogen sulfide (H2S) to yield a
thiochromone ester (28, Scheme 1E, step b).19 Then, 4-
oxothiochromone-3-carboxylic acid (29) was synthesized from
the hydrolysis of 28 with HCl and acetic acid20 (Scheme 1E, step
c). A coupling reaction of 29 with the appropriate arylamine, in
the presence of POCl3, provided thiochromone carboxamides
(30 and 31, Scheme 1E, step d).10
Monoamine Oxidase Inhibition Studies. The evaluation
of i-hMAOs (2−5, 7−10, 14−17, 20−25, and 30 and 31) was
performed using a spectrophotometric assay using kynuramine
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Table 1. hMAO Inhibitory Activities of Chromane (2−5), Chromene (7−10), Coumarin (14−17), 2-Oxoquinoline (20−22), N-
Methyl-2-oxoquinoline (23−25), Thiochromone (30 and 31), and Chromone (Crom-1 and Crom-2) Derivatives and Reference
d
Inhibitors
a
b
c
SI: hMAO-B selectivity index [IC50 (hMAO-A)/IC50 (hMAO-B)]. Percentage of inhibition at 10 μM (highest concentration tested). Values
d
obtained under the assumption that the corresponding IC50 against hMAO-A is the highest concentration tested (10 μM). Asterisks (*) indicate
not soluble in phosphate buffer at concentrations higher than 10 μM.
as a substrate and recombinant hMAO-A and hMAO-B
isoforms.21,22 Since IC50 values of Crom-1 and Crom-2
reported by Reis et al. were determined using the Amplex Red
assay,10 their activity was reassessed under our experimental
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Figure 2. Synopsis of the SAR data obtained in the present work.
conditions. The hMAO inhibition potencies (IC50 values) and
the selectivity index (SI) of the compounds under study and
reference inhibitors (rasagiline and safinamide for hMAO-B and
clorgyline for hMAO-A) are presented in Table 1. The results
obtained indicate that the novel compounds that exhibited
hMAO inhibitory activity were more selective toward the
isoform B. The hMAO-B inhibitory activities of compounds
bearing 3-chloro (3, 8, 15, and 31) and 3,4-dichloro (5 and 10)
groups at the exocyclic aromatic ring were, in general, higher
than their 3,4-dimethyl counterparts (2, 7, 14, and 30) (Table
1). In contrast, the presence of 3,4-dimethoxy substituents
decreased (9) or even abolished (4) hMAO-B inhibition at the
highest concentration tested (Table 1).
2-oxoquinolines (20−22) and the N-methyl analogues (23−25)
were inactive toward hMAOs. The thiochromones (30 and 31)
displayed hMAO-B IC50 values within the same concentration
range as Crom-1 and Crom-2 (30, hMAO-B IC50 = 3.35 0.64
nM; 31, hMAO-B IC50 = 1.52 0.15 nM). Overall, the data
show that the bioisosteric replacement of O by NH or N−CH3
atoms at the benzopyrone core results in complete loss of
activity and that the amidic tautomerism, which can occur in the
2-oxoquinoline-based compounds, does not influence the
hMAOs inhibition properties. A recent work on 4-hydrox-
yquinoline and hMAOs reveals important findings that also
supported the data.17 In marked contrast, O to S isosteric
replacement was well-tolerated leading to potent and selective i-
hMAO-B. Of note, the thiochromone amide (31) was the most
potent i-hMAO-B, showing an IC50 value lower than safinamide
and the parent chromones (Crom-1 and Crom-2). Taking these
data together, the results obtained enabled the establishment of
robust SAR that is summarized in Figure 2.
Evaluation of the Enzyme−Ligand Inhibition Mecha-
nism. To further evaluate the type of binding between hMAO-B
and the most potent i-hMAO-B (31), time-dependent inhibition
studies were performed. The reference irreversible (rasagiline)
and reversible (safinamide) i-hMAO-Bs were also included in
these experiments (Figure 3). The data obtained with rasagiline
showed a decay in the residual enzyme activity after 30 min
incubation, which is consistent with irreversible hMAO-B
inhibition. In contrast, the results obtained with safinamide
showed an increase in hMAO-B activity over time indicating
With the exception of 4, chromane derivatives (2, 3, and 5)
acted as moderate i-hMAO-B (2, hMAO-B IC50 = 1779 130
nM; 3, hMAO-B IC50 = 957 40 nM; 5, hMAO-B IC50 = 78.8
6.3 nM). Since they are considerably less potent than Crom-1
(hMAO-B IC50 = 2.82 0.34 nM) and Crom-2 (hMAO-B IC50
= 2.50
0.30 nM), we concluded that the absence of the
carbonyl group and the double carbon bond led to a remarkable
decrease in MAO-B inhibitory activity. In marked contrast, the
chromene derivatives (7−10) were more active toward hMAO-
B than the chromane counterparts (7, hMAO-B IC50 = 45.7
3.0 nM; 8, hMAO-B IC50 = 26.4 2.8 nM; 9, hMAO-B IC50
=
2869 350 nM; 10, hMAO-B IC50 = 3.16 0.2 nM),
demonstrating that benzopyran rigidity is an essential chemical
feature for hMAO-B inhibition. The compound lacking the
carbonyl group but with two chloro substituents in the exocyclic
aromatic ring (10) was the most potent i-hMAO-B of the series.
Subsequently, the effect of the position of the carbonyl group
on the pyrone ring (14−17) on MAOs inhibitory activity was
investigated. Coumarins 14 (hMAO-B IC50 = 18.8 1.3 nM)
and 15 (hMAO-B IC50 = 10.2
1.1 nM) were potent and
selective i-hMAO-B. Although they were less active than the
chromone counterparts (Crom-1 and Crom-2, respectively),
they exhibited similar, or even lower, IC50 values than the
reference i-hMAO-Bs safinamide and rasagiline. When com-
pared with chromene derivatives (7 and 8), we can conclude
that the presence of a carbonyl group at positions C2
(coumarin) or C4 (chromone) improved potency and
selectivity. Since the presence of 3,4-dimethoxy substituents
was not required for hMAO-B inhibition and was associated
with poor solubility, this substitution pattern was not considered
when designing additional compounds for further studies.
In order to investigate the influence of different heteroatoms
on hMAO inhibition, 2-oxoquinoline derivatives (20−25) and
thiochromones (30 and 31) were synthesized and screened. The
Figure 3. Time-dependent enzyme inhibition studies for rasagiline,
safinamide, and compound 31. Enzymatic activity is expressed in the
amount of 4-hydroxyquinoline (4-HQ, nmol). Data are the mean SD
of three different experiments.
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Figure 4. (A) Double reciprocal plot of the initial velocity of hMAO-B at increasing substrate concentrations (10−100 μM) in the absence or the
presence of compound 31. (B) Effect of total enzyme concentration on hMAO-B inhibition by compound 31 (Ackermann−Potter plot).
reversible inhibition. Compound 31 exhibited an inhibition
profile similar to safinamide, suggesting that it acts as a reversible
i-hMAO-B. The reversible inhibition mechanism could
minimize the adverse effects and safety issues often associated
to irreversible i-hMAO-B.
Moreover, to clarify the inhibition mechanism of the most
potent i-hMAO-B (31), kinetic experiments were performed.
The assay was carried out by measuring the initial rates of the
hMAO-B activity at six different concentrations of kynuramine
(substrate) in the absence or presence of three different
concentrations of compound 31. The kinetic parameters of
the Michaelis−Menten reaction were analyzed using a double
reciprocal Lineweaver−Burk plot, which provides a better visual
assessment of enzyme inhibition data. As reported in Figure 4A,
the data were fitted to lines intersecting in the upper-left
quadrant of the Lineweaver−Burk plot indicating a mixed-type
inhibition mechanism.
Since this type of behavior can be indicative of “tight binding
inhibition”,23,24 the kinetics was additionally investigated by
using seven different hMAO-B concentrations in the absence or
presence of three different concentrations of compound 31 (SI).
The Ackermann−Potter plot (Figure 4B) clearly resulted in
asymptotic concave curves showing that compound 31 is a tight
binding inhibitor.
These results were in line with our previous findings and
strengthen the concept that mixed-type inhibition behavior may
occur when compounds bind to the enzyme active site in a “tight
binding inhibition” manner.10,13
Molecular Ligand−Enzyme Studies. Following the in
vitro studies, molecular modeling studies were carried out for the
best benzopyran-based i-hMAO-B to better explore the in silico
ligand−target interactions. Moreover, the binding mode of the
selected compounds on hMAO-A was studied to define their
strong hMAO-B selectivity. In the case of the chromane
derivatives (2, 3, and 5), both enantiomers R and S were
explored. The best thermodynamic complex, provided by the
application of the XP Glide protocol and by further
thermodynamic characterization through the MM-GBSA
(ΔGbind), was analyzed. Coulomb (coul) and van der Waals
(vdW) components were also examined to evaluate the
nonbonded interaction energy contribution (Table S1, SI).
In general, the data analysis showed good target−ligand
interactions. However, the theoretical binding energies,
considered as ΔGbind, did not perfectly match with the IC50
values obtained since in these calculations, translational,
rotational, and vibrational entropy changes were ignored.
However, it is interesting to observe that for each series, the
most active compound also showed the best ΔGbind. Overall, the
in silico prediction studies are in line with the selectivity of the
compounds toward the hMAO-B isoform than hMAO-A (Table
S1, SI). In particular, the vdW component strongly favored
hMAO-B binding when compared to hMAO-A (Table S1, SI).
Chromane (2, 3, and 5), chromene (7−10), coumarin (14 and
15), and thiochromone (30 and 31) derivatives shared a similar
docking profile into the hMAO-B binding pocket, with their
heterocycle facing FAD. The binding mode of the most potent
compounds for each series (Figure 5) is characterized by the
establishment of an H-bond with the side chain of Tyr435 and
several hydrophobic interactions with Tyr398, Tyr60, Gln206,
Leu171, Ile199, Leu164, Leu167, and Phe168, as observed in the
crystal structure of Crom-2 (Figure S1B). For the chromane
enantiomers R/S, an overlapping binding mode toward hMAO-
B was observed (Figure 5A,B and Figure S2, SI), except for the
enantiomer S of 2 (Figure S2C, SI), which showed the flipped
chromane ring by preventing the H-bond with Tyr435.
Interestingly, both enantiomers of the most active chromane
derivative (5) engage an additional halogen bond with Pro102
(Figure 5A,B).
In accordance with the biological results, chromene
derivatives showed several favorable interactions due to the
double bond present at C3-C4, endowing them with higher
planarity and leading to a better orientation in the hMAO-B
binding site (Figure 5C and Figure S3, SI). Moreover, 7 (Figure
S3A, SI) and 9 (Figure S3C, SI), the two low-potency molecules
of the series, lose the H-bond with Tyr435 due to the inverted
position of the chromene ring. Interestingly, the binding mode
of 10, the most active compound of this series, is favored by the
presence of the chlorine in meta, which establishes a halogen
bond with Pro102 (Figure 5C) as observed for 5.
Regarding the coumarin series, the best docking poses of 15
established a further π−π interaction with Phe343, by means of
the aromatic portion of the coumarin ring (Figure 5D).
Moreover, it was observed that the two methyl groups of
derivative 14 interact with the hydrophobic pocket created by
several residues, such as Phe168, Leu167, Ile316, Trp119,
Leu164, Pro104, Phe103, Pro102, Ile199, and Ile198 (Figure
S4A, SI), while 15 places the meta-chlorine similarly to Crom-2
in its crystal structure (Figure 5D and Figure S1B).
Finally, the thiochromone derivatives, 30 and 31, perfectly fit
the hydrophobic flat active site of the hMAO-B. Both complexes
exhibited an H-bond between the carbonyl group of the
thiochromone ring and the side chain of Tyr435, and the most
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Figure 5. Best docking pose of the most active compounds for each series (A) 5R, (B) 5S, (C) 10, (D) 15, and (E) 31 against hMAO-B. Ligands are
shown in green carbon ball-and-sticks, while the protein is represented as a gray surface and FAD in CPK. H-bonds, halogen, and π−π interactions are
displayed as dashed purple, violet, and cyan lines, respectively. Amino acid residues involved in the molecular interactions are shown as gray carbon
sticks.
active compound 31 engages a further π−π interaction with
Tyr326 and places the meta-chlorine similarly to Crom-2
(Figure 5E and Figure S1B, SI). As previously reported, docking
results on hMAO-A are reported in the Supporting Information
(Figures S5−S8, SI).
Furthermore, the pan-assay interference compound (PAINS)
in silico assay was run for the compounds studied, using two
different methods, namely, ZINC PAINS Pattern Identifier and
False Positive Remover tools. The analysis of the results
obtained indicated that the chromone (2, 3, and 5) chromene
(7−10), coumarin (14 and 15), and thiochromone (30 and 31)
derivatives were not potential PAINS.
Furthermore, we estimated the drug-like properties of Crom-
1, Crom-2, and the most potent derivatives (10, 15, 30, and 31)
to predict their capability to reach the central nervous system
(CNS). The drug-like properties were studied by estimating
some important physicochemical parameters including the
molecular weight (MW), topological polar surface area (tPSA
in Å2), number of hydrogen bond acceptors (HBA), number of
hydrogen bond donors (HBD), and number of rotatable bonds
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Figure 6. Cellular viability of differentiated SH-SY5Y (A,B) and Caco-2 cells (C,D) following treatment with Crom-1, Crom-2, and compounds 5, 7,
8, 10, 14, 15, 30, and 31 at 1 (black) and 10 μM (orange) for 24 h. Cytotoxicity was evaluated by measuring metabolic (A,C) and lysosomal activities
(B,D) using MTT reduction and NR uptake assays, respectively. Results are expressed as mean SEM from at least three independent experiments,
performed in triplicate. Statistical comparisons were performed by one-way ANOVA (A,B) and by Kruskal−Wallis test (C,D). In all cases, p values
lower than 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs untreated cells).
(RB). Overall, the values obtained were in line with the general
drug-likeness requirements of the CNS-active drugs (Table S2,
SI). An important physicochemical parameter used to estimate
the ability of a compound to cross the blood−brain barrier
(BBB) by passive diffusion is the logarithm of the ratio of the
compound concentration in the brain and in blood (log BB). It
was reported that compounds with log BB < −1 are unlikely to
act as CNS drugs due to their low distribution into the brain.25
The compounds under study showed log BB values suggesting >
−1, indicating that these derivatives may cross the BBB and
reach the CNS (Table S2, SI). It is important to note that based
on the predicted tPSA and log BB values, the thiochromone 31 is
more likely to cross the BBB than Crom-2.
Preliminary In Vitro Preclinical ADME-Toxicity Studies.
Following the discovery of novel, potent, and selective
benzopyran-based i-hMAO-B, preliminary in vitro preclinical
ADME-toxicity studies were performed. These included the
evaluation of the compounds’ cytotoxicity against SH-SY5Y,
Caco-2, HUVEC, HEK-293, and MCF-7 cells, cardiotoxicity
(hERG inhibition), P-gp modulation, and protection against
iron(III)-induced oxidative stress.
Cytotoxicity Studies in SH-SY5Y and Caco-2 Cells. The
cytotoxicity profile of the most promising i-hMAO-B (hMAO-B
IC50 < 80 nM) was assessed in differentiated neuroblastoma
(SH-SY5Y) and in colon adenocarcinoma (Caco-2) cells, two
cell lines extensively used to assess the safety of drug
candidates.26,27 Prior to conducting the cytotoxicity experiments
with the test compounds, SH-SY5Y cells were treated with
differentiation-inducing agents to obtain cells morphologically
similar to dopaminergic neurons.28 Since the clinical symptoms
of PD result mainly from the loss of dopaminergic neurons,
differentiated SH-SY5Y cells are a suitable cellular model to
evaluate the neurotoxicity profile of drug candidates for PD.29
Differentiated SH-SY5Y cells and Caco-2 cells were treated
with two different concentrations (1 and 10 μM) of compounds
5, 7, 8, 10, 14, 15, 30, and 31 for 24 h. Crom-1 and Crom-2 were
also included in these experiments for the establishment of
structure−toxicity relationships (STR). Cellular cytotoxicity
was determined using the methylthiazolyldiphenyltetrazolium
bromide (MTT) reduction assay and the neutral red (NR)
uptake assay. The MTT assay is based on the reduction of MTT
into the respective formazan by metabolically active cells,30
while the NR uptake assay is based on the lysosomal
accumulation of the NR dye in living cells.31 The results
obtained showed that treatment of differentiated SH-SY5Y cells
with Crom-1, Crom-2, and compound 14 at 10 μM led to a
marked decrease in MTT reduction (% MTT reduction was
<85%) (Figure 6A). Compound 15 also significantly decreased
MTT reduction in neuroblastoma cells at 1 (79.5%) and 10 μM
(71.8%) (Figure 6A). In contrast, incubation of differentiated
SH-SY5Y cells for 24 h with compounds at both concentrations
did not significantly affect NR uptake (Figure 6B). In Caco-2
cells, only compounds 8 (88.3%) and 15 (93.5%), at 10 μM,
slightly decreased the cellular metabolic activity (Figure 6C)
without any effect on lysosomal activity. The treatment with the
other compounds did not affect MTT reduction (Figure 6C)
and NR uptake (Figure 6D) when compared with control cells.
Overall, the data obtained indicate that chromones Crom-1
and Crom-2 and coumarins 14 and 15 negatively affected
cellular metabolic activity of differentiated SH-SY5Y cells. In
contrast, chromane derivative 5, chromene derivatives 7, 8, and
10, and thiochromones 30 and 31 did not induce significant
cytotoxic effects in both differentiated SH-SY5Y cells and Caco-
2 cells, exhibiting a broader safety window than Crom-1 and
Crom-2. Based on these preliminary results, we can conclude
that despite the slight improvement of hMAO-B inhibition
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Table 2. Preliminary Preclinical In Vitro ADME-Toxicity Studies: Cytotoxicity in a Panel of Cell Lines (HUVEC, HEK-293, and
MCF-7) for 24 h and Cardiotoxicity (hERG Inhibition) for Crom-1, Crom-2, 10, and 31
% cytotoxicity at 10 μM
cytotoxicity IC50 (μM)
% inhibition at 10 μM
hERG
compound
HUVEC
HEK-293
MCF-7
HUVEC
HEK-293
MCF-7
10
31
Crom-1
Crom-2
64.4 10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
8.4 2.5
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
<10
<10
<10
<10
<10
properties, the presence of a carbonyl group in the benzopyran
scaffold at positions C4 (chromones) or C2 (coumarins) of the
heterocyclic ring was associated with increased cytotoxicity.
However, thiochromones 30 and 31 with a carbonyl group at
position C4 did not show significant cytotoxicity effects in both
cellular models.
Cytotoxicity Studies in HUVEC, HEK-293, and MCF-7
Cells and Cardiotoxicity (hERG Inhibition). The two most
active compounds (10, hMAO-B IC50 = 3.16 0.2 nM and 31,
hMAO-B IC50 = 1.52 0.15 nM) and the parent chromones
(Crom-1 and Crom-2) were then selected for the evaluation of
their cytotoxicity in a panel of cell lines (HUVEC, HEK-293, and
MCF-7). Cells were treated with the test compounds at 10 μM
for 24 h exposure, and the cellular cytotoxicity was evaluated
using previously described methods.26,32 Briefly, cellular
cytotoxicity was determined in triplicate using a highly sensitive
luminescence-based commercial kit that measures intracellular
ATP levels (Promega). In general, negligible cytotoxic effects
were observed in all cell lines (<10% inhibition at 10 μM)
(Table 2). The only exception was compound 10, which was
associated with moderate toxicity in HUVEC cells (cellular
cytotoxicity at 10 μM, 64.4 10%; cytotoxicity IC50, 8.4 2.5
μM).
Figure 7. Evaluation of P-gp activity by fluorescence spectroscopy in
Caco-2 cells exposed to Crom-1, Crom-2, and compounds 10 and 31
(10 μM) during the incubation period with the fluorescent substrate
(RHO 123, 5 μM). Results are presented as mean SEM from at least
three independent experiments, performed in triplicate. Statistical
comparisons were performed by one-way ANOVA.
Compounds were also tested for potential cardiotoxicity using
the predictor hERG fluorescence polarization (FP) binding
assay (Thermo). The assay relies on the use of a homogenized
membrane solution containing hERG, which, in the presence of
a fluorescent tracer, yields a high FP signal. If compounds
displace the fluorescent tracer, then a reduction in the FP signal
indicating hERG inhibition is observed. In the case of Crom-1,
Crom-2, 10, and 31, negligible hERG inhibition at 10 μM was
observed (<10% inhibition) (Table 2).
Evaluation of P-glycoprotein (P-gp) Transport Activ-
ity. One of the main challenges for an effective drug transport to
the brain is the blood−brain barrier (BBB), the main interface
between the brain and the circulatory system.33 The BBB
restricts the permeability of CNS drugs,34 a problem that is
partially linked to the presence of efflux pumps such as the P-gp
at the luminal membrane of brain capillary endothelial cells.35
The P-gp uses adenosine triphosphate (ATP) to actively extrude
a wide variety of lipophilic drugs against their concentration
gradient,36 contributing to the high attrition observed for CNS
drugs and drug candidates.37 Caco-2 cells have been accepted as
a useful in vitro model to evaluate BBB drug permeability and P-
gp-mediated transport.36 Therefore, in this study, we evaluated
the P-gp modulatory activity of compounds 10 and 31 and the
reference chromones Crom-1 and Crom-2. The assay was
performed in Caco-2 cells using Rhodamine 123 (RHO 123), a
P-gp substrate, and zosuquidar, a specific third-generation P-gp
inhibitor.30 Overall, the pretreatment of Caco-2 cells with the
selected i-hMAO-B at 10 μM did not significantly affect P-gp
transport activity (Figure 7).
Protection against Iron(III)-Induced Damage in Neu-
roblastoma Cells. The association between iron and PD is
long-standing since daily exposure to elevated iron levels is a risk
factor for the development of this neurodegenerative disease.38
In fact, the development of new multitarget compounds that can
chelate iron and prevent ferroptosis is one of the strategies
currently used to develop new drug candidates for PD. With this
in mind, we evaluated whether Crom-1, Crom-2, and
compounds 10 and 31 can protect differentiated SH-SY5Y
cells against iron(III)-induced damage. After a pretreatment
with the test compounds at 1 and 10 μM for 30 min, cells were
treated with iron(III) at 1000 and 1500 μM for 24 h. Cellular
cytotoxicity was measured using the NR uptake assay. The
results obtained are shown in Figure 8.
Compared with nontreated cells, the 24 h treatments with
only iron(III) at 1000 and 1500 μM reduced 55.7 5.8% and
44.9 4.4% of cellular lysosomal activity, respectively. As shown
in Figure 8, when cells were coincubated with iron(III) at 1000
μM, no significant protection was observed for the compounds
Crom-1, Crom-2, and 10. Remarkably, a moderate but
significant increase in lysosomal activity was observed in cells
pretreated with compound 31 at 10 μM (63.0 7.0%, p < 0.01).
No significant protection was observed when cells were
coincubated with i-hMAO-B and iron(III) at 1500 μM.
Together with the data obtained from hMAOs inhibition
studies, these results indicate that compound 31 can act as a
multitarget agent able to prevent dopamine depletion and
iron(III)-induced damage.
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cell lines) and cardiotoxicity (hERG inhibition) indicated that
compound 31 has no obvious ADME-toxicity liability.
From the gathered data, N-(3-chlorophenyl)-4H-thiochro-
mone-3-carboxamide 31 stands out as the best potent and
selective “tight binding” i-hMAO-B, showing a safe cytotoxicity
profile in SH-SY5Y, Caco-2, HUVEC, HEK-293, and MCF-7
cells. Furthermore, compound 31 did not significantly affect P-
gp activity and protected differentiated neuroblastoma cells
against iron(III)-induced cell death. As such, compound 31 is a
valid candidate for in vivo pharmacological studies.
EXPERIMENTAL SECTION
Chemistry. Details on reagents, materials, and apparatuses are
provided in the SI.
■
Figure 8. Evaluation of the protective effect of i-hMAO-B in
differentiated SH-SY5Y cells against iron(III) (1000 and 1500 μM).
The dashed line is the control data obtained from nontreated cells,
while solid and squared bars are related with the data from cells exposed
to iron(III) at 1000 and 1500 μM, respectively. The data are expressed
as the means of three independent experiments together with the
standard deviation (mean SD). Statistical comparisons were made
using two-way ANOVA. In all cases, p values lower than 0.05 were
considered significant (####p < 0.0001 vs the control data; **p < 0.01 vs
iron(III) values).
Synthesis. Synthesis of 2-Oxo-2H-chromene-2-carboxylic Acid
(13). Salicylaldehyde (11) (16.37 mmol), diethyl malonate (16.37
mmol), and catalytic amounts of piperidine (0.5 mL) were refluxed in
ethanol (163.7 mL) overnight. After cooling to rt, the suspension was
filtered off, and ethyl-coumarin-3-caboxylate (12) was obtained
without further purification. 12 was refluxed in a 150 mL ethanolic
solution of 0.5% NaOH (m/v) for 1 h. Then, the reaction mixture was
acidified with 12 M HCl. The solid formed was isolated by filtration
under reduced pressure and washed with H2O to provide 2-oxo-2H-
1
chromene-2-carboxylic acid (13). Yield = 61%. H NMR (400 MHz,
DMSO) δ (ppm): 7.36 (1H, d, J = 7.7 Hz, H5), 7.10 (1H, s, H4), 6.98
(1H, dd, J = 7.3, 6.9 Hz, H7), 6.71 (1H, d, J = 8.1 Hz, H8), 6.64 (1H, dd,
J = 7.8, 6.8 Hz, H6).
CONCLUSIONS
■
A new library of benzopyran-based compounds was synthesized,
and relevant SAR and STR data were obtained. In general, the
derivatives bearing chloro substituents at the exocyclic ring
presented lower hMAO-B IC50 values than the 3,4-dimethyl
counterparts, while compounds bearing 3,4-dimethoxy groups
lacked or exhibited reduced inhibitory activity toward hMAO-B.
These observations reinforce the data obtained so far in our
previous works. Globally, our data showed for the first time the
importance of the ring rigidity for hMAO-B inhibition and that
the presence of a double bound and a carbonyl group at
positions C2 or C4 of the heterocyclic ring improves hMAO-B
inhibitory activity. We also demonstrated for the first time that
while the O to N isosteric replacement abolished hMAO-B
inhibitory activity, the O to S replacement in the chromone
moiety preserved the hMAO-B inhibition profile of compounds.
All the studies performed so far reinforce the superiority of the
chemical features of the previously reported i-hMAO-Bs N-
(3′,4′-dimethyl phenyl)-4-oxo-4H-chromene-3-carboxamide
(Crom-1, Figure 1) and N-(3′-chlorophenyl)-4-oxo-4H-chro-
mene-3-carboxamide (Crom-2, Figure 1). However, for the first
time, relevant data related with thiochromones emerged. They
were identified as potent and selective i-hMAO-Bs, with IC50
values within the low nM range. All the conclusions were
supported by in silico studies that provided a rationale for the
interactions of the active compounds toward hMAO-B and
hMAO-A, which are consistent with the observations from the
outputs of the in vitro biological assays.
Synthesis of 1-Methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic
Acid (19). To a solution of 2-oxo-1,2-dihydroquinoline-3-carboxylic
acid (18) (4.56 mmol) in DMF (10 mL), K2CO3 (6.84 mmol) was
added, and the reaction mixture was stirred at rt in an argon atmosphere
for 20 min. Then, CH3−I (9.12 mmol) was added dropwise, and the
mixture was stirred at 60 °C for 1 h. Upon completion, water (15 mL)
was added, and the mixture was extracted with DCM (3 × 15 mL). The
organic layers were combined, dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The crude product was
1
recrystallized from DCM/ethyl ether. Yield = 70%. H NMR (400
MHz, DMSO) δ (ppm): 14.67 (1H, s, COOH), 8.97 (1H, s, H4), 8.11
(1H, dd, J = 7.9, 1.5 Hz, H8), 7.89 (1H, ddd, J = 8.6, 7.1, 1.5 Hz, H7)
7.78 (1H, dd, J = 8.6, 1.5 Hz, H5), 7.49 (1H, ddd, J = 7.9, 7.1, 1.2 Hz,
H6), 3.80 (3H, s, NCH3).
Synthesis of Ethyl-3-(dimethylamino)-2-(2-fluorobenzoyl)-
acrylate (27). N,N-Dimethylformamide dimethyl acetal (5.94 mmol)
was added to a solution of ethyl-(2-fluorobenzoyl)acetate (26) (2.73
mmol) in dry toluene (13 mL). The reaction mixture was stirred at 115
°C for 1 h. Then, the mixture was concentrated under reduced pressure.
Purification by flash column chromatography (DCM/ethyl acetate 1:1)
1
yielded compound 27. Yield = 90%. H NMR (400 MHz, CDCl3) δ
(ppm): 7.76 (1H, s, CH), 7.59 (1H, ddd, J = 7.5, 7.5, 1.8 Hz, H5), 7.38
(1H, dddd, J = 8.2, 7.1, 5.1, 1.9 Hz, H6), 7.16 (1H, ddd, J = 7.5, 7.5, 1.0
Hz, H4), 7.01 (1H, ddd, J = 10.4, 8.2, 1.1 Hz, H3), 3.96 (2H, q, J = 7.1
Hz, CH2), 3.39−2.80 (6H, m, CH3NCH3), 0.89 (3H, t, J = 7.1 Hz,
CH3).
Synthesis of Ethyl 4-Oxo-4H-thiochromene-3-carboxylate (28).
Hydrogen sulfide (H2S) was bubbled into a solution of ethyl-3-
(dimethylamino)-2-(2-fluorobenzoyl)acrylate (27) (3.20 mmol) in
THF (60 mL) at −30 °C for 4 h. The mixture was concentrated, and the
crude product was purified by chromatography to afford compound 28.
Yield = 80%. 1H NMR (400 MHz, DMSO) δ (ppm): 7.70 (1H, s, H2),
7.50−7.38 (2H, m, H5, H7), 7.23−7.19 (1H, m, H8), 7.16 (ddd, J =
10.7, 8.3, 1.1 Hz, H6), 3.84 (2H, q, J = 7.1 Hz, CH2), 0.84 (3H, t, J = 7.1
Hz, CH3).
Synthesis of 4-Oxo-4H-thiochromene-3-carboxylic Acid (29). A
mixture of ethyl 4-oxo-4H-thiochromene-3-carboxylate (28) (2.13
mmol), acetic acid (54 mL), H2O (15 mL), and HCl (1.5 mL) was
heated on a steam bath for 4 h. The reaction mixture was concentrated
under reduced pressure, and the product obtained was recrystallized
from DCM/petroleum ether to give the compound 29. Yield = 40%. 1H
NMR (400 MHz, CDCl3) δ (ppm): 15.12 (1H, s, COOH), 8.07 (1H, s,
Cytotoxicity studies showed that chromenes are safer than the
related carbonyl-containing benzopyran compounds (coumar-
ins and chromones) and that thiochromones 30 and 31 with a
carbonyl group at C4 did not display remarkable cytotoxicity
effects. Of particular note is N-(3-chlorophenyl)-4H-thiochro-
mone-3-carboxamide (31), which exhibited potent and selective
hMAO-B inhibition with an IC50 value within the low
nanomolar range (hMAO-B IC50 = 1.52
0.15 nM). The
selected i-hMAO-B did not significantly affect P-gp transport
activity at 10 μM. The preliminary preclinical in vitro ADME-
toxicity studies (cytotoxicity in HUVEC, HEK-293, and MCF-7
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H2), 8.25 (1H, ddd, J = 8.5, 1.3, 0.6 Hz, H5), 7.90 (1H, ddd, J = 8.5, 6.9,
1.3 Hz, H7), 7.70 (1H, ddd, J = 8.5, 1.3, 0.6 Hz, H8), 7.62 (1H, ddd, J =
8.5, 6.9, 1.3 Hz, H6).
6.92 (1H, ddd, J = 7.4, 7.4, 1.1 Hz, H6), 6.86 (1H, d, J = 8.1 Hz, H5′),
5.06 (2H, d, J = 1.3 Hz, H2), 2.24 (3H, s, 3′CH3), 2.22 (3H, s, 4′CH3).
13C NMR (100 MHz, CDCl3) δ (ppm): 163.2, 154.9, 137.4, 135.1,
133.1, 131.5, 130.1, 128.4, 127.4, 127.2, 121.8, 121.6, 120.8, 117.7,
116.2, 64.9, 19.9, 19.2. EI/MS m/z (%): 77 (23), 115 (28), 131 (22),
159 (100), 278 (15), 279 (M·+, 90), 280 (M·+ + 1, 18).
N-(3-Chlorophenyl)-2H-chromene-3-carboxamide (8). Com-
pound 8 was recrystallized from ethyl acetate/n-hexane. Yield = 28%.
1H NMR (400 MHz, CDCl3) δ (ppm): 7.69 (1H, dd, J = 2.1, 2.1 Hz,
H2′), 7.59 (1H, s, CONH), 7.42 (1H, ddd, J = 8.2, 2.1, 1.0 Hz, H6′),
7.28 (1H, ddd, J = 8.2, 1.5, 1.5 Hz, H4′), 7.24 (1H, dd, J = 6.05, 1.78,
H5), 7.14−7.10 (2H, m, H7, H5′), 7.08 (1H, s, H4), 6.94 (1H, ddd, J =
7.5, 7.5, 1.1 Hz, H6), 6.88 (1H, d, J = 8.1 Hz, H8), 5.06 (2H, d, J = 1.3
Hz, H2). 13C NMR (100 MHz, CDCl3) δ (ppm): 163.2, 155.0, 138.6,
134.8, 131.9, 130.1, 128.5, 127.9, 126.8, 124.7, 121.9, 120.6, 120.2,
118.0, 116.3, 64.7. EI/MS m/z (%): 51 (10), 77 (22), 115 (26), 131
(17), 159 (100), 160 (11), 285 (M·+, 45), 286 (M·+ + 1, 9), 287 (M·+ +
2, 15).
General Procedure A: Synthesis of Chromane (2−5),
Chromene (7−10), Coumarin (14−17), and Thiochromone (30
and 31) Derivatives. A solution of the appropriate heterocyclic
carboxylic acid (chromane-3-carboxylic acid (1), chromene-3-carbox-
ylic acid (6), 2-oxo-2H-chromene-3-carboxylic acid (13), or
thiochromone-3-carboxylic acid (29)) (2.8 mmol) and POCl3 (4.2
mmol) in DMF (4.2 mL) was stirred at rt for 15 min. Then, the
appropriate amine (5.61 mmol) was added, and the mixture was stirred
at rt overnight. Following the addition of DCM (50 mL), the mixture
was extracted with H2O (3 × 15 mL), 1 M HCl (3 × 15 mL) and
saturated aqueous solution of NaHCO3 (3 × 5 mL). The organic layers
were combined, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified
by recrystallization.
N-(3,4-Dimethylphenyl)chromane-3-carboxamide (2). Com-
1
pound 2 was recrystallized from DCM/ethyl ether. Yield = 56%. H
N-(3,4-Dimethoxyphenyl)-2H-chromene-3-carboxamide (9).
Compound 9 was recrystallized from ethyl acetate/n-hexane. Yield =
NMR (400 MHz, CDCl3) δ (ppm): 7.53 (1H, s, CONH), 7.28 (1H, d, J
= 2.3 Hz, H2′), 7.19 (1H, dd, J = 8.1, 2.4 Hz, H6′), 7.15−7.10 (1H, m,
H6), 7.08 (1H, dd, J = 7.7, 1.3 Hz, H5), 7.05 (1H, d, J = 8.1 Hz, H5′),
6.90 (1H, dd, J = 7.4, 1.3 Hz, H8), 6.87−6.84 (1H, m, H7), 4.48−4.17
(2H, m, H2), 3.22 (1H, dd, J = 16.4, 9.4 Hz, H3), 3.04−2.85 (2H, m,
H4), 2.22 (3H, s, 3′CH3), 2.21 (3H, s, 4′CH3). 13C NMR (100 MHz,
CDCl3) δ (ppm): 170.2, 153.8, 137.3, 135.1, 133.1, 130.0, 129.9, 127.7,
121.5, 121.0, 120.4, 117.6, 116.8, 67.2, 40.8, 28.4, 19.8, 19.1. EI/MS m/
z (%): 55 (12), 77 (24), 79 (12) 105 (22), 106 (16), 120 (16), 121
(92), 131 (13), 133 (13), 237 (12), 281 (M·+, 100), 282 (M·+ + 1, 19).
N-(3-Chlorophenyl)chromane-3-carboxamide (3). Compound 3
was recrystallized from DCM/ethyl ether. Yield = 54%. 1H NMR (400
MHz, DMSO) δ (ppm): 10.37 (1H, s, CONH), 7.84 (1H, dd, J = 2.1,
2.1 Hz, H2′), 7.48 (1H, ddd, J = 8.2, 2.1, 1.0 Hz, H6′), 7.35 (1H, dd, J =
8.0, 8.0 Hz, H5′), 7.17−7.06 (3, m, H5, H6, H4′), 6.86 (1H, ddd, J =
7.4, 7.4, 1.2 Hz, H7), 6.79 (1H, dd, J = 8.2, 1.2 Hz, H8), 4.49−3.96 (2H,
m, H2), 3.11−2.91 (3H, m, H3, H4). 13C NMR (100 MHz, DMSO) δ
(ppm): 170.6, 153.6, 140.3, 133.0, 130.4, 129.7, 127.1, 123.0, 121.0,
120.3, 118.6, 117.5, 116.1, 66.8, 39.2, 27.3. EI/MS m/z (%): 51 (19), 55
(39), 77 (44), 79 (22), 103 (23), 129 (26), 131 (59), 132 (60), 133
(74), 134 (18), 243 (16), 287 (M·+, 100), 288 (M·+ + 1, 18), 289 (M·+ +
2, 33).
1
25%. H NMR (400 MHz, DMSO) δ (ppm): 9.91 (1H, s, CONH),
7.44 (1H, s, H4), 7.39 (1H, d, J = 2.4 Hz, H2′), 7.31−7.24 (2H, m, H5,
H7), 7.25 (1H, dd, J = 8.8, 2.1 Hz, H6′), 6.99 (1H, ddd, J = 7.4, 7.4, 1.1
Hz, H6), 6.92 (1H, d, J = 8.8 Hz, H5′), 6.88 (1H, dd, J = 7.8, 1.0 Hz,
H8), 4.98 (2H, d, J = 1.4 Hz, H2), 3.74 (3H, s, 3′OCH3), 3.73 (3H, s,
4′OCH3). 13C NMR (100 MHz, DMSO) δ (ppm): 163.4, 154.7, 148.9,
145.6, 132.8, 131.6, 129.0, 127.9, 127.5, 122.4, 121.7, 116.2, 112.6,
112.4, 105.7, 64.9, 56.2, 55.8. EI/MS m/z (%): 77 (17), 115 (33), 131
(20), 159 (100), 292 (13), 311 (M·+, 80), 312 (15).
N-(3,4-Dichlorophenyl)-2H-chromene-3-carboxamide (10).
Compound 10 was recrystallized from DCM/ethyl ether. Yield =
21%. 1H NMR (400 MHz, DMSO) δ (ppm): 10.29 (1H, s, CONH),
8.07 (1H, d, J = 2.4 Hz, H2′), 7.68 (1H, dd, J = 8.8, 2.4 Hz, H6′), 7.60
(1H, d, J = 8.8 Hz, H5′), 7.50 (1H, s, H4), 7.32−7.25 (2H, m, H7, H5),
7.00 (1H, ddd, J = 7.4, 7.4, 1.1 Hz, H6), 6.89 (1H, dd, J = 7.4, 1.1 Hz,
H8), 4.98 (2H, d, J = 1.3 Hz, H2). 13C NMR (100 MHz, DMSO) δ
(ppm): 164.1, 154.8, 139.5, 132.0, 131.3, 131.1, 129.2, 129.1, 126.8,
125.5, 122.4, 121.6, 121.4, 120.4, 116.3, 64.7. EI/MS m/z (%): 77 (24),
102 (9), 103 (10), 115 (34), 131 (17), 159 (100), 160 (11), 319 (16),
320 (M·+, 3), 321 (M·+ + 1, 10), 322 (M·+ + 2, 2).
N-(3,4-Dimethylphenyl)-2-oxo-2H-chromene-3-carboxamide
N-(3,4-Dimethoxyphenyl)chromane-3-carboxamide (4). Com-
pound 4 was recrystallized from ethyl acetate/n-hexane. Yield = 45%.
1H NMR (400 MHz, DMSO) δ (ppm): 10.04 (1H, s, CONH), 7.34
(1H, d, J = 2.4 Hz, H2′), 7.14 (1H, dd, J = 7.6, 1.6 Hz, H6′), 7.14−7.04
(2H, m, H5, H6), 6.89 (1H, d, J = 8.7 Hz, H5′), 6.86 (1H, ddd, J = 7.40,
7.39, 1.26 Hz, H7), 6.78 (1H, dd, J = 8.1, 1.2 Hz, H8), 4.50−3.93 (2H,
m, H2), 3.73 (3H, s, 3′OCH3), 3.72 (3H, s, 4′OCH3), 3.07−2.90 (3H,
m, H4, H3). 13C NMR (100 MHz, DMSO) δ (ppm): 170.3, 154.2,
149.0, 145.4, 133.1, 130.3, 127.6, 121.7, 120.8, 116.6, 112.5, 111.6,
104.9, 67.5, 56.2, 55.8, 39.6, 28.0. EI/MS m/z (%): 77 (11), 105 (17),
133 (14), 138 (52), 151 (11), 153 (57), 313 (M·+, 100), 314 (21).
N-(3,4-Dichlorophenyl)chromane-3-carboxamide (5). Com-
(14). Compound 14 was recrystallized from ethyl acetate/n-hexane.
1
Yield = 45%. H NMR (400 MHz, DMSO) δ (ppm): 10.55 (1H, s,
CONH), 8.91 (1H, s, H4), 8.01 (1H, dd, J = 7.7, 1.6 Hz, H5), 7.78 (1H,
ddd, J = 8.8, 7.4, 1.6 Hz, H7), 7.55 (1H, dd, J = 8.4, 2.2 Hz, H6′), 7.52−
7.47 (2H, m, H2′, H8), 7.46−7.44 (1H, m, H6), 7.13 (1H, d, J = 8.1 Hz,
H5′), 2.23 (3H, s, 3′CH3), 2.20 (3H, s, 4′CH3). 13C NMR (100 MHz,
DMSO) δ (ppm): 160.5, 159.4, 153.8, 147.2, 136.7, 135.6, 134.1, 132.1,
130.2, 129.8, 125.2, 120.9, 119.8, 118.4, 117.2, 116.1, 19.4, 18.7. EI/MS
m/z (%): 89 (13), 101 (16), 173 (100), 174 (11), 264 (10), 265 (14),
293 (M·+, 79), 294 (M·+ + 1, 16).
N-(3-Chlorophenyl)-2-oxo-2H-chromene-3-carboxamide (15).
Compound 15 was recrystallized from ethyl acetate/n-hexane. Yield
= 42%. 1H NMR (400 MHz, DMSO) δ (ppm): 10.76 (1H, s, CONH),
8.91 (1H, s, H4), 8.02 (1H, dd, J = 7.8, 1.6 Hz, H5), 7.98 (1H, dd, J =
2.0, 2.0 Hz, H2′), 7.79 (1H, ddd, J = 8.9, 7.4, 1.6 Hz, H7), 7.60−7.54
(2H, m, H4′, H6′), 7.48 (1H, ddd, J = 7.5, 7,52, 1.1 Hz, H6), 7.42 (1H,
dd, J = 8.1, 8.0 Hz, H5′), 7.22 (1H, ddd, J = 7.9, 2.1, 0.9 Hz, H8). 13C
NMR (100 MHz, DMSO) δ (ppm): 160.3, 160.0, 153.8, 147.4, 139.3,
134.3, 133.2, 130.6, 130.2, 125.2, 124.0, 119.8, 119.4, 118.4, 118.3,
116.2. EI/MS m/z (%): 63 (8), 89 (14), 101 (14), 173 (100), 174 (11),
299 (M·+, 26), 301 (M·+ + 2, 9).
1
pound 5 was recrystallized from DCM/ethyl ether. Yield = 36%. H
NMR (400 MHz, DMSO) δ (ppm): 10.47 (1H, s, CONH), 8.01 (1H,
d, J = 2.4 Hz, H2′), 7.58 (1H, d, J = 8.8 Hz, H5′), 7.51 (1H, dd, J = 8.8,
2.4 Hz, H6′), 7.14 (1H, dd, J = 7.6, 1.6 Hz, H5), 7.09 (1H, ddd, J = 8.1,
7.3, 1.7 Hz, H6), 6.86 (1H, ddd, J = 7.4, 7.4, 1.2 Hz, H7), 6.79 (1H, dd, J
= 8.1, 1.3 Hz, H8), 4.48−3.98 (2H, m, H2), 3.06−2.93 (3H, m, H3,
H4). 13C NMR (100 MHz, DMSO) δ (ppm): 170.7, 153.6, 138.9,
130.9, 130.6, 129.7, 127.1, 124.7, 121.0, 120.3 (2 × 13C), 119.2, 116.1,
66.7, 39.3, 27.2. EI/MS m/z (%): 77 (54), 78 (18), 79 (27), 103 (24),
105 (83), 131 (49), 132 (51), 133 (98), 134 (18), 160 (20), 161 (88),
163 (48), 277 (18), 321 (100), 322 (M·+, 18), 323 (M·+ + 1, 63), 324
(M·+ + 2, 12).
N-(3,4-Dimethoxyphenyl)-2-oxo-2H-chromene-3-carboxamide
(16). Compound 16 was recrystallized from ethyl acetate/n-hexane.
1
Yield = 53%. H NMR (400 MHz, DMSO) δ (ppm): 10.53 (1H, s,
N-(3,4-Dimethylphenyl)-2H-chromene-3-carboxamide (7). Com-
pound 7 was recrystallized from DCM/ethyl ether. Yield = 43%. H
NMR (400 MHz, CDCl3) δ (ppm): 7.54 (1H, s, CONH), 7.35 (1H, d,
J = 2.3 Hz, H2′), 7.27 (1H, dd, J = 8.0, 2.3 Hz, H5), 7.22 (1H, ddd, J =
8.1, 7.4, 1.7 Hz, H7), 7.13−7.05 (2H, m, H6′, H8), 7.05 (1H, s, H4),
CONH), 8.91 (1H, s, H4), 8.01 (1H, dd, J = 7.8, 1.6 Hz, H5), 7.78 (1H,
ddd, J = 8.4, 7.3, 1.6 Hz, H7), 7.56 (1H, d, J = 8.4 Hz, H5′), 7.48 (1H,
ddd, J = 7.7, 7.7, 1.1 Hz, H6), 7.39 (1H, d, J = 2.4 Hz, H2′), 7.29 (1H,
dd, J = 8.4, 2.4 Hz, H6′), 6.96 (1H, dd, J = 7.8, 1.6, Hz, H8), 3.78 (3H, s,
3′OCH3), 3.75 (3H, s, 4′OCH3). 13C NMR (100 MHz, DMSO) δ
1
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(ppm): 160.4, 159.3, 153.8, 148.6, 147.2, 145.5, 134.1, 131.3, 130.2,
125.2, 119.8, 118.4, 116.1, 112.0, 111.9, 104.9, 55.6, 55.4. EI/MS m/z
(%): 89 (10), 101 (11), 173 (100), 174 (11), 310 (24), 325 (M·+, 82),
326 (17).
N-(3,4-Dichlorophenyl)-2-oxo-2H-chromene-3-carboxamide
(17). Compound 17 was recrystallized from ethyl acetate/n-hexane.
Yield = 25%. 1H NMR (400 MHz, MeOD) δ (ppm): 9.20 (1H, s, H4),
8.22 (1H, d, J = 2.4 Hz, H2′), 7.99 (1H, dd, J = 7.7, 1.6 Hz, H5), 7.95
(1H, ddd, J = 8.8, 7.3, 1.6 Hz, H7), 7.72 (1H, dd, J = 8.7, 2.5 Hz, H6′),
7.68−7.67 (1H, m, H5′), 7.66−7.64 (2H, m, H8, H6). 13C NMR and
DEPT 135 data (*). MS/EI m/z (%): 63 (12), 89 (22), 101 (21), 173
(100), 174 (11), 333 (15), 334 (M·+, 3), 335 (M·+ + 1, 10), 336 (M·+ +
2, 1).
162.6, 161.9, 145.3, 140.1, 140.0, 133.8, 133.7, 131.2, 130.5, 124.2,
123.6, 121.5, 119.8, 119.2, 118.8, 116.0. EI/MS m/z (%): 63 (8), 89
(23), 116 (46), 117 (8), 128 (11), 144 (3), 172 (100), 173 (11), 298
(M·+, 33), 299 (M·+ + 1, 7), 300 (M·+ + 2, 12).
N-(3,4-Dichlorophenyl)-2-oxo-1,2-dihydroquinoline-3-carboxa-
mide (22). Compound 22 was recrystallized from MeOH/ethyl ether.
1
Yield = 40%. H NMR (400, MHz, DMSO) δ (ppm): 12.60 (1H, s,
CONH), 12.05 (1H, s, NH), 8.45 (1H, s, H4), 7.90 (1H, d, J = 2.4 Hz,
H2′), 7.83 (1H, dd, J = 7.5, 1.7 Hz, H5), 7.75 (1H, dd, J = 7.1, 2.4 Hz,
H6′), 7.60 (1H, d, J = 7.1 Hz, H5′), 7.50 (1H, dd, J = 7.5, 1.5 Hz, H8),
7.30−7.26 (2H, m, H7, H6). 13C NMR and DEPT 135 data (*). EI/MS
m/z (%): 89 (23), 116 (40), 128 (10), 172 (100), 173 (11), 332 (29),
333 (M·+, 6), 334 (M·+ + 1, 19), 335 (M·+ + 2, 3), 337 (M·+ + 4, 6).
N-(3,4-Dimethylphenyl)-1-methyl-2-oxo-1,2-dihydroquinoline-3-
carboxamide (23). Compound 23 was recrystallized from ethyl
acetate/n-hexane. Yield = 35%. 1H NMR (400 MHz, DMSO) δ (ppm):
12.00 (1H, s, CONH) 8.95 (1H, s, H4), 8.07 (1H, dd, J = 7.8, 1.5 Hz,
H8), 7.82 (1H, ddd, J = 8.6, 7.1, 1.5 Hz, H7), 7.71 (1H, dd, J = 8.5 Hz,
1.5 Hz, H5), 7.53 (1H, dd, J = 8.1, 2.0 Hz, H6′), 7.46 (1H, d, J = 2.0 Hz,
H2′), 7.43 (1H, ddd, J = 7.9, 7.1, 1.2 Hz, H6), 7.13 (1H, d, J = 8.1 Hz,
H5′), 3.79 (3H, s, NCH3), 2.24 (3H, s, 3′CH3), 2.20 (3H, s, 4′CH3).
13C NMR (100 MHz, DMSO) δ (ppm): 161.4, 160.5, 143.4, 140.2,
N-(3,4-Dimethylphenyl)-4-oxo-4H-thiochromene-3-carboxa-
mide (30). Compound 30 was recrystallized from DCM/ethyl ether.
1
Yield = 25%. H NMR (400 MHz, CDCl3) δ (ppm): 11.28 (1H, s,
CONH), 9.07 (1H, s, H2), 8.34 (1H, dd, J = 8.0, 1.7 Hz, H5), 7.79 (1H,
ddd, J = 8.7, 7.1, 1.7 Hz, H7), 7.59 (1H, dd, J = 8.6, 1.1 Hz, H8), 7.56−
7.53 (1H, m, H6), 7.52 (1H, d, J = 1.2 Hz, H2′), 7.48 (1H, dd, J = 8.1,
2.4 Hz, H6′), 7.12 (1H, d, J = 8.1 Hz, H5′), 2.28 (3H, s, 3′CH3), 2.24
(3H, s, 4′CH3). 13C NMR (100 MHz, CDCl3) δ (ppm): 177.4, 162.7,
160.5, 156.2, 137.2, 135.7, 134.8, 132.8, 130.0, 126.4, 126.2, 124.1,
121.8, 118.5, 118.0, 116.1, 19.9, 19.2. EI/MS m/z (%): 53 (15), 77 (9),
121 (63), 122 (9), 145 (9), 173 (100), 174 (11), 293 (91), 294 (18).
N-(3-Chlorophenyl)-4-oxo-4H-thiochromene-3-carboxamide
(31). Compound 31 was recrystallized from DCM/ethyl ether. Yield =
20%. 1H NMR (400 MHz, CDCl3) δ (ppm): 11.49 (1H, s, CONH),
9.07 (1H, s, H2), 8.34 (1H, ddd, J = 6.4, 1.3, 0.4 Hz, H5), 7.90 (1H, dd,
J = 2.04, 2.04 Hz, H2′), 7.81 (1H, ddd, J = 8.7, 7.1, 1.7 Hz, H7), 7.61
(1H, dd, J = 7.8, 1.8 Hz, H8), 7.58−7.53 (2H, m, H6, H6′), 7.29 (1H,
dd, J = 8.13, 8.13 Hz, H5′), 7.12 (1H, ddd, J = 7.9, 2.0, 1.0 Hz, H4′). 13C
NMR (100 MHz, CDCl3) δ (ppm): 177.4, 163.0, 160.9 (CONH),
156.2 (C8a), 139.1, 135.0, 134.6, 129.9, 126.6, 126.3, 124.5, 124.0,
120.6, 118.6, 118.5, 115.7. EI/MS m/z (%): 53 (17), 89 (9), 120 (4),
121 (58), 123 (7), 145 (3), 172 (3), 173 (100), 174 (11), 299 (51), 300
(9), 301 (17), 315 (M·+, 2).
General Procedure B: Synthesis of 2-Oxo-1,2-dihydroquino-
line-3-carboxamide (20−22) and N-1-Methyl-2-oxo-1,2-dihy-
droquinoline-3-carboxamide (23−25) Derivatives. A solution of
the appropriate heterocyclic carboxylic acid (2-oxo-1,2-dihydroquino-
line-3-carboxylic acid (18) or N-1-methyl-2-oxo-1,2-dihydroquinoline-
3-carboxylic acid (19)) (1.05 mmol), TBTU (1.05 mmol), and DIPEA
(2.1 mmol) in DMF (10 mL) was stirred at rt for 15 min. Then, the
appropriate amine (1.26 mmol) was added, and the mixture was stirred
at room temperature overnight. Then, DCM (50 mL) was added, and
the mixture was extracted with H2O (3 × 5 mL), 1 M HCl (3 × 5 mL),
and saturated aqueous solution of NaHCO3 (3 × 5 mL). The organic
layers were combined, washed with aqueous solution of 10% NaOH
(m/v), dried over anhydrous Na2SO4, filtered, and concentrated. The
crude product was purified by recrystallization.
136.7, 135.9, 133.3, 131.8, 130.8, 129.8, 123.1, 120.9, 120.8, 119.2,
117.1, 115.3, 29.9, 19.4, 18.7. EI/MS m/z (%): 89 (19), 91 (5), 186
(100), 277 (11), 278 (10), 306 (M·+, 99), 307 (M·+ + 1, 19).
N-(3-Chlorophenyl)-1-methyl-2-oxo-1,2-dihydroquinoline-3-car-
boxamide (24). Compound 24 was recrystallized from MeOH/n-
1
hexane. Yield = 22%. H NMR (400 MHz, DMSO) δ (ppm): 12.22
(1H, s, CONH), 8.97 (1H, s, H4), 8.07 (1H, dd, J = 7.9, 1.3 Hz, H8),
8.03 (1H, dd, J = 2.0, 2.0 Hz, H2′), 7.83 (1H, ddd, J = 8.6, 7.1, 1.5 Hz,
H7), 7.72 (1H, d, J = 8.6 Hz, H5), 7.55 (1H, ddd, J = 8.1, 1.9, 1.5 Hz,
H6′), 7.43 (1H, ddd, J = 7.5, 7.5, 1.8 Hz, H6), 7.40 (1H, d, J = 8.1 Hz,
H5′), 7.20 (1H, ddd, J = 8.0, 2.0, 1.5 Hz, H4′), 3.80 (3H, s, NCH3). 13
C
NMR and DEPT 135 data (a). MS/EI m/z (%): 89 (13), 128 (3), 130
(4), 185 (3), 186 (100), 312 (M·+, 35), 313 (M·+ + 1, 6), 314 (M·+ + 2,
12).
N-(3,4-Dichlorophenyl)-1-methyl-2-oxo-1,2-dihydroquinoline-3-
carboxamide (25). Compound 25 was recrystallized from MeOH/n-
1
hexane. Yield = 20%. H NMR (400 MHz, DMSO) δ (ppm): 12.14
(1H, s, CONH), 8.93 (1H, s, H4), 8.15 (1H, dd, J = 8.1, 2.0 Hz, H6′),
8.07 (1H, dd, J = 7.8, 1.5 Hz, H8), 7.82 (1H, dd, J = 7.9, 7.9 Hz, H7),
7.70 (1H, dd, J = 8.5 Hz, 1.5 Hz H5), 7.61 (1H, d, J = 6.08 Hz, H5′),
7.50−7.43 (2H, m, H6, H2′), 3.80 (3H, s, NCH3). 13C NMR and
DEPT 135 data (*). EI/MS m/z (%): 55 (19), 57 (20), 89 (22), 128
(9), 186 (100), 187 (14), 346 (36), 348 (M·+ + 1, 21).
(*) 13C NMR was not acquired due to solubility constraints.
The purity of the final products (>98%) was verified by high-
performance liquid chromatography (HPLC) equipped with a DAD
detector (Figures S9−S26).
Pharmacology. The details about monoamine oxidase inhibitory
assays, cytotoxicity studies, and computational simulations are provided
in the SI.
N-(3,4-Dimethylphenyl)-2-oxo-1,2-dihydroquinoline-3-carboxa-
mide (20). Compound 20 recrystallized from MeOH/n-hexane. Yield =
40%. 1H NMR (400 MHz, DMSO) δ (ppm): 12.63 (1H, s, CONH),
12.01 (1H, s, NH), 8.96 (1H, s, H4), 8.00 (1H, dd, J = 6.7, 1.1 Hz, H8),
7.70 (1H, ddd, J = 7.1, 6.0, 1.5 Hz, H7), 7.52 (1H, dd, J = 8.3, 2.1 Hz,
H5), 7.48 (1H, dd, J = 8.2, 2.2 Hz, H6′), 7.45 (1H, d, J = 2.2 Hz, H2′),
7.34 (1H, ddd, J = 7.0, 6.2, 1.5 Hz, H6), 7.13 (1H, d, J = 8.1 Hz, H5′),
2.24 (3H, s, 3′CH3), 2.20 (3H, s, 4′CH3). 13C NMR (100 MHz,
DMSO) δ (ppm): 162.1, 160.6, 144.2, 141.00, 139.4, 136.7, 135.9,
132.9 (2 × 13C), 131.7, 129.8, 127.5, 123.0, 120.8, 117.1, 115.4, 19.4,
18.7. EI/MS m/z (%): 57 (15), 89 (30), 116 (48), 121 (87), 128 (12),
172 (92), 173 (11), 292 (M·+, 100), 293 (22).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00510.
■
sı
Molecular formula strings (CSV)
PDB files of homology (ZIP)
Enzymatic assays, in vitro toxicology, computational
studies, supplementary tables, and figures (PDF)
N-(3-Chlorophenyl)-2-oxo-1,2-dihydroquinoline-3-carboxamide
(21). Compound 21 was recrystallized from ethyl acetate/n-hexane.
1
Yield = 50%. H NMR (400 MHz, DMSO) δ (ppm): 12.68 (1H, s,
AUTHOR INFORMATION
Corresponding Authors
■
CONH), 12.27 (1H, s, NH), 8.98 (1H, s, H4), 8.03−8.00 (2H, m, H8,
H2′), 7.72 (1H, ddd, J = 8.5, 7.1, 1.4 Hz, H7), 7.54 (1H, ddd, J = 8.2,
2.1, 1.0 Hz, H6′), 7.49 (1H, dd, J = 8.3, 1.3 Hz, H5), 7.41 (1H, dd, J =
8.0, 8.0 Hz, H5′), 7.35 (1H, ddd, J = 8.1, 7.1, 1.1 Hz, H6), 7.20 (1H,
ddd, J = 8.0, 2.2, 1.0 Hz, H4′). 13C NMR (100 MHz, DMSO) δ (ppm):
Alexandra Gaspar − CIQUP/Department of Chemistry and
Biochemistry, Faculty of Sciences, University of Porto, Porto
4169-007, Portugal; Email: alexandra.gaspar@fc.up.pt
11180
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Article
Notes
Fernanda Borges − CIQUP/Department of Chemistry and
Biochemistry, Faculty of Sciences, University of Porto, Porto
4169-007, Portugal; orcid.org/0000-0003-1050-2402;
Email: fborges@fc.up.pt
The authors declare no competing financial interest.
ABBREVIATIONS
■
(ATP), adenosine tri-phosphate; (BBB), blood−brain barrier;
(CNS), central nervous system; (Caco-2), colon adenocarcino-
ma cells; (TBTU), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetrame-
thylaminium tetrafluoroborate; (coul), Coulomb; (DCM),
dichloromethane; (Crom-2), N-(3′-chlorophenyl)-4-oxo-4H-
chromene-3-carboxamide; (Crom-1), N-(3′,4′-dimethyl phe-
nyl)-4-oxo-4H-chromene-3-carboxamide; (DIPEA), N,N-diiso-
propylethylamine; (DMF), dimethylformamide; (FP), fluores-
cence polarization; (PDB code 2Z5X), hMAO-A; (PDB code
6FW0), hMAO-B; (HBA), hydrogen bond acceptors; (MCF-7),
human breast adenocarcinoma cell-line Michigan Cancer
Foundation-7 cells; (HEK-293), human embryonic kidney
293 cells; (HUVEC), human umbilical vein endothelial cells;
(HBD), hydrogen bond donors; (MTT), methylthiazolyldiphe-
nyltetrazolium bromide; (MW), molecular weight; (MAO-A
and MAO-B), monoamine oxidases A and B; (i-hMAO-B),
MAO-B inhibitors; (SH-SY5Y), neuroblastoma cells; (NR),
neutral red; (PD), Parkinson’s disease; (RHO 123), Rhodamine
123; (RB), rotatable bonds; (SAR), structure−activity relation-
ship; (STR), structure−toxicity relationship; (tPSA in Å2),
topological polar surface area; (XP G-Score), XP Glide Score;
(vdW), van der Waals
Authors
Francesco Mesiti − Dipartimento di Scienze della Salute,
̀
Universita “Magna Græcia” di Catanzaro, Campus “Salvatore
Venuta”, Catanzaro 88100, Italy; Net4Science srl, Academic
̀
Spinoff, Universita “Magna Græcia” di Catanzaro, Campus
“Salvatore Venuta”, Catanzaro 88100, Italy; CIQUP/
Department of Chemistry and Biochemistry, Faculty of
Sciences, University of Porto, Porto 4169-007, Portugal
Daniel Chavarria − CIQUP/Department of Chemistry and
Biochemistry, Faculty of Sciences, University of Porto, Porto
4169-007, Portugal
Annalisa Maruca − Dipartimento di Scienze della Salute,
̀
Universita “Magna Græcia” di Catanzaro, Campus “Salvatore
Venuta”, Catanzaro 88100, Italy; Net4Science srl, Academic
̀
Spinoff, Universita “Magna Græcia” di Catanzaro, Campus
“Salvatore Venuta”, Catanzaro 88100, Italy
̀
Roberta Rocca − Net4Science srl, Academic Spinoff, Universita
“Magna Græcia” di Catanzaro, Campus “Salvatore Venuta”,
Catanzaro 88100, Italy; Department of Experimental and
Clinical Medicine, “Magna Græcia” University of Catanzaro,
Campus “S. Venuta”, Catanzaro 88100, Italy; orcid.org/
0000-0002-0680-7097
Eva Gil Martins − UCIBIO-REQUIMTE, Laboratory of
Toxicology, Department of Biological Sciences, Faculty of
Pharmacy, University of Porto, Porto 4050-313, Portugal
Sandra Barreiro − UCIBIO-REQUIMTE, Laboratory of
Toxicology, Department of Biological Sciences, Faculty of
Pharmacy, University of Porto, Porto 4050-313, Portugal
Renata Silva − UCIBIO-REQUIMTE, Laboratory of
Toxicology, Department of Biological Sciences, Faculty of
Pharmacy, University of Porto, Porto 4050-313, Portugal;
orcid.org/0000-0001-9962-7548
Carlos Fernandes − CIQUP/Department of Chemistry and
Biochemistry, Faculty of Sciences, University of Porto, Porto
4169-007, Portugal; orcid.org/0000-0002-0102-0703
Sheraz Gul − Fraunhofer Institute for Translational Medicine
and Pharmacology, Hamburg 22525, Germany; Fraunhofer
Cluster of Excellence for Immune-Mediated Diseases CIMD,
Hamburg 22525, Germany
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Oliver Keminer − Fraunhofer Institute for Translational
Medicine and Pharmacology, Hamburg 22525, Germany;
Fraunhofer Cluster of Excellence for Immune-Mediated
Diseases CIMD, Hamburg 22525, Germany
Stefano Alcaro − Dipartimento di Scienze della Salute,
̀
Universita “Magna Græcia” di Catanzaro, Campus “Salvatore
Venuta”, Catanzaro 88100, Italy
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00510
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This project was supported by the Foundation for Science and
Technology (FCT) and FEDER/COMPETE (Grants UID/
QUI/00081/2020 and PTDC/MED-QUI/29164/2017). This
article is based upon work from COST Action CA15135.
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J. Med. Chem. 2021, 64, 11169−11182

Journal of Medicinal Chemistry
pubs.acs.org/jmc
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