Synthesis of 3-aryl-4-(N-aryl)aminocoumarins via photoredox arylation and the evaluation of their biological activity

Article abstract of DOI:10.1016/j.bioorg.2021.105141

A new series of 3-aryl-4-(N-aryl)aminocoumarins was synthesized in two steps starting from the natural product 4-hydroxycoumarin using the photoredox catalysis for the key step. These conditions reactions allowed to make C[sbnd]C bonds is up to 95% yields

Full text of DOI:10.1016/j.bioorg.2021.105141

Bioorganic Chemistry 114 (2021) 105141
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of 3-aryl-4-(N-aryl)aminocoumarins via photoredox arylation and
the evaluation of their biological activity
a
b
,
c
a
a
Leonardo S.A. Carneiro , Fernando Almeida-Souza , Yanne S.C. Lopes , Rachel C.V. Novas ,
c
d
c
a,*
Kaique B.A. Santos , Carolina B.P. Ligiero , K a´ tia da S. Calabrese , Camilla D. Buarque
a
Departmento de Química, Pontifícia Universidade Cat o´ lica do Rio de Janeiro (PUC-Rio), Rua Marqu ˆe s de S a˜ o Vicente 225, sala 576L, G a´ vea, Rio de Janeiro, RJ
0551-031, Brazil
P ´o s-graduaç a˜ o em Ci ˆe ncia Animal, Universidade Estadual do Maranh a˜ o (UEMA), Cidade Universit a´ ria Paulo VI, S a˜ o Luís, MA 65055-310, Brazil
Laborat o´ rio de Imunomodulaç a˜ o e Protozoologia, Instituto Oswaldo Cruz (Fiocruz), Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ, 21040-900, Brazil
Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), Av. Athos da Silveira Ramos, 149, CT, Bl. A-622, Cid. Universit a´ ria, Ilha do Fund a˜ o, Rio de
2
b
c
d
Janeiro, RJ 21941-909, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of 3-aryl-4-(N-aryl)aminocoumarins was synthesized in two steps starting from the natural product
Aminocoumarin
Arylcoumarin
Arylation
4
-hydroxycoumarin using the photoredox catalysis for the key step. These conditions reactions allowed to make
C bonds is up to 95% yields in mild conditions, easy operation, in an environmentally benign way, and are
C
–
compatible with several patterns of substitution. The biological activity of the new compounds was tested in vitro
against MCF-7, MDA-MB-231, and CCD-1072Sk cancer cell lines, as soon as to promastigotes and intracellular
amastigotes of Leishmania amazonensis. Compounds 17d, 17s and 17x showed activity against promastigote
Photoredox catalysis
X-ray diffraction
Leishmaniasis
Cytotoxicity
In silico
forms (IC50 = 5.96 ± 3.210, 9.05 ± 2.855 and 5.65 ± 2.078
best activity against L. amazonensis amastigote intracellular form (IC50 = 9.6 ± 1.148
macrophage cytotoxicity at assayed concentrations (CC50 > 600 M), and high selectivity to parasites over the
μ
M respectively), and compound 17x presented the
μM), no BALB/c peritoneal
ADMET
μ
mammalian cells (Selectivity Index > 62.2). There was no expressive activity for the cancer cell lines. Single
crystal X-ray diffraction analysis was employed for structural elucidation of compounds 17a and 17s. In silico
analyses of physicochemical, pharmacokinetic, and toxicological properties suggest that compound 17x is a
potential candidate for anti-leishmaniasis drugs.
1
. Introduction
The 4-hydroxycoumarin (1) is a class of natural product obtained
hydroxycoumarin and anilines (3) can be considered as a hybrid of 4-
arylcoumarins (4) and 4-(N-aryl)aminoquinazolines (5) (Fig. 1), two
classes of compounds that exhibit activity as a modulator of P-gp and
BCRP ABC efflux pump[8] and anticancer [9], respectively. Compound
6 with no substitution at C3 position showed activity against lung car-
usually as a result of microbial fermentation processes by the action of
biphenyl synthase [1,2]. The tautomer bearing the heterocycle enhances
the possible chemical transformations at C3 and C4 [3]. Many of its
derivatives were studied to evaluate their biological activities. The most
known example is warfarin, which acts as an anticoagulant drug in the
treatment of venous thromboembolism [4]. Some reviews pointed out
the uses of coumarins and, also, 4-hydroxycoumarin derivatives as po-
tential candidates as neglected diseases drugs, such as malaria [5],
Chagas disease [6], and leishmaniasis [7].
cinoma with EC50 = 0.14
μ
M in A549 cells [10]. Later, Chen et al. (2016)
made a series of 4-(N-aryl)aminocoumarins as tubulin polymerization
inhibitors; compound 7 had the best results for this proposal with IC50
=
21 nM [11].
The free C3 position combined with the changing of the heteroatom
bonded to the C4 position opens to further functionalization (Fig. 2). The
compounds SS5020 (8), developed by AstraZeneca [12], and CHF4227
(9), from Chiesi Pharmaceuticals [13], act as down-regulator of the es-
trogen receptor (ER) and selective ER modulator, respectively. Scott et al
Recently, a class of 4-hydroxycoumarin derivatives gained attention
due to, mainly, their anticancer activities. The 4-(N-aryl)amino-
coumarins (2), which are obtained from the reaction of 4-
2
proposed the substitution of the CH group at 10a (the green highlighted
*
Corresponding author.
E-mail address: camilla-buarque@puc-rio.br (C.D. Buarque).
https://doi.org/10.1016/j.bioorg.2021.105141
Received 30 April 2021; Received in revised form 22 June 2021; Accepted 29 June 2021
Available online 2 July 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
one) by an O- or NH-linker. Besides the oxygen-linked 10b having given
the best improvement to ER MCF-7 down-regulation IC50 and lower
lipophilicity, the nitrogen-linked molecule 10c showed to be promising
for enhancing the biological activity. In 2017, Xiang’s group explored
these structures in three reports evaluating the anticancer activities.
Compounds 11 and 12 presented great results for four different types of
cancer (breast, liver, colon, and pancreas) [14]. Also, compound 12 has
also been evaluated for its cytotoxicity, showing good antiproliferative
activity against normal cell line (HUVEC) and selectivity. Further studies
reported that compound 13 had a better binding affinity to the MCF-7
and Ishikawa cell lines compared to tamoxifen [15]. Then, compound
15a to optimize the next step.
Coumarin’s arylation at position C3 has also been described by
photoredox catalysis. K o¨ nig et al (2012) showed the arylation of alkenes
and enones, but coumarin was just one example of their work [17].
Kojima et al (2015) obtained some new metal-free porphyrin-based
photocatalyst to arylate coumarins [18]. This methodology has a
disadvantage in the synthesis of the catalyst. The proposed porphyrin
was obtained in only a 7% yield and required microwave irradiation for
◦
12 h at 250 C. Gryko et al (2017) used the same kind of catalyst to
arylate heteroarenes and showed two examples with reasonable yields
[19]. Despite the disadvantages of the methods or the lack of more ex-
amples, these reports showed the feasibility of the photocatalytic
methodologies to obtain 3-arylcoumarins.
Based on K o¨ nig’s methodology arylation [17], we decided to start
from these conditions to optimize the reaction (Table 1). It was noted
that 15a has poor solubility in DMSO, which made us decrease the re-
action concentration to solubilize all starting material. After the
moderated yield was obtained (53%, Entry 1), we changed the photo-
catalyst to organic dyes (Entries 2 to 4) trying to figure out a metal-free
method, but no improvement was achieved. Then, the solvent optimi-
zation (Entry 5 and 6) showed again the bad solubility of 15a at DMF
1
4 bound almost three times better MCF-7 cells than tamoxifen [16].
With these results, the authors suggested that the coumarin scaffold
mimics the estradiol one.
Despite these compounds showed important results for medicinal
chemistry, organic chemistry research has also contributed to improving
the proposed synthetic routes. The use of radicals to make natural
products and drug candidates was evaluated in many reviews over the
years [17–20]. Their unique reactivity and their use in making new C
–
C
bonds made them routine in organic synthesis. Photoredox catalysis was
a milestone for this chemistry with the introduction of a photocatalyst,
which can be organic dyes or Ru/Ir complexes when excited by visible
light can promote oxidations or reductions to produce organic radicals.
Advantages like mild reaction conditions, easy operation, environmen-
tally benign, selective excitation of the catalyst over reactants and sol-
vent, feasibility of scaling up, and combination with continuous flow
methods highlight this approach concerning traditional methodologies
like metal-catalyzed reactions, which usually need harsh conditions and
expensive and/or toxic catalysts and/or ligands.21
3
and CH CN and no product was obtained at these conditions. The re-
action with no light and no catalyst (Entries 7 and 8) did not work,
confirming that the reaction is photocatalyzed.
To evaluate the methodology, initially, we submitted reactions
varying the phenyldiazonium salts (Scheme 2). The reaction with phe-
nyldiazonium salts with groups at para-position is suitable. The presence
of stronger donating groups at 17b and 17c reduces the yield to 32 and
34%, respectively. A neutral group (17d) and halogens (17e and 17f)
have similar performance compared to the product with no substituents.
Electron withdrawing groups improved the reaction to achieve good
yields, as in 17g and 17h. The presence of a dioxazole group (17i) gave a
product in moderate yield. Based on this result, we evaluated sub-
stituents at meta- (17j and 17 k) and ortho-positions (17 l to 17o).
Surprisingly, the methodology was not tolerant to these groups.
Substituents at N-(aryl)amino group showed inversion of reactivity
In this present work, we focused on the synthesis of 3-aryl-4-(N-aryl)
aminocoumarins using photoredox arylation as the key step. These
compounds were evaluated for the biological activity against MCF-7,
MDA-MB-231, and CCD-1072Sk cancer cell lines, as well as promasti-
gote and intracellular amastigote forms of Leishmania amazonensis.
2
. Results and discussion
(
Scheme 3). The strong donating group OMe (17p) increased the yield to
2
.1. Chemistry
66%. Weaker donating groups like Me (17q) and halogens like F (17r)
and Br (17s) decreased the yield to 54, 53, and 50%, respectively. The
use of withdrawing groups decreased the yield to 42% (17t) and a
We began our work obtaining 4-(N-aryl)aminocoumarins (15a-i)
from the reaction of 1 and different anilines (3a-i) (Scheme 1) and the
products were obtained in up to 91% yield. The reactions were carried
out as a 1-gram scale in the absence of solvent and the obtained products
did not require any purification method. We decided to use coumarin
2
stronger group such as NO did not give products (17y). The presence of
substituents at ortho-position (17u) and meta-position (17v) and in both
rings (17w and 17x) was also tolerated giving the products in reason-
able yields (51 to 71%).
Fig. 1. The origin of 4-(N-aryl)aminocoumarins and some examples with biological activities.
2

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
The structural elucidation of the coumarins 17a and 17s was ach-
estradiol via cytoplasmic estrogen receptors, MDA-MB-231 is a cell
lineage hormone-independent [24–26]. The cell lineage CCD-1072Sk, a
human fibroblast cell obtained from skin taken from the normal foreskin
of a newborn, was used as a normal mammalian cell.
ieved by single-crystal X-ray diffraction. The collection of data was
realized with Cu K
smaller than 5%) are displayed in Fig. 3 with thermal ellipsoids. Both
structures were indexed in the P2 /n space group of the monoclinic
α
at 150 K. The refined structures (with residual factor
1
The CC50 values of the 3-aryl-4-(N-aryl)aminocoumarins compounds
against MCF-7, MBA-MB-231, and CCD-1072Sk are presented in Table 2.
Compounds 17d and 17g showed the lowest IC50 values against MCF-7
cells and were also the only compounds that presented values lower than
crystalline system. More information, such as crystal, instrumental, and
structural refinement parameters, in addition to selected bond distances
and angles, are presented in the Supplementary Information.
The analyzed compounds exhibit different orientations of the sub-
stituents relative to the coumarin core, as shown in Fig. 4a. For the group
200
μ
M. All the analyzed compounds showed cytotoxic effect at con-
M against MDA-MB-231 and CCD-1072Sk cells
centrations above 300
μ
◦
◦
in C3, the torsion on C4
–
C3
–
C9
–
C14 is ꢀ 57.28 for 17a and 57.05
line. Doxorubicin IC50 against MDA-MB-231 and CCD-1072Sk were 95.5
and 42.5-fold higher than against MCF-7 respectively, demonstrating its
antitumor effect against MCF-7 as expected. These results corroborate
the conclusion that these compounds did not present antitumor activity
for human breast cancer cell line or even to the not tumor cell line CCD-
1072 k, reassuring the low toxic effect in cell culture in vitro.
◦
for 17s. In the C4, the torsion on C15
–
N1
–
C4
–
C4a is ꢀ 53.30 for 17a
◦
and 50.68 for 17s.
Despite their remarkable electronegativities, halogens bonded to
organic moieties can act as Lewis acids giving origin to the halogen
bonds. This type of intermolecular interaction is a consequence of the
anisotropy in the distribution of the electrostatic potential over the
halogen atom, giving rise to an electron-poor region in the extension of
The two compounds, 17d and 17g, that showed antitumor activity
against MCF-7, displayed weaker activity than the reference drug
doxorubicin. Interestingly, Xiang’s group at their first work also studied
the same compound 17x described in this work [14]. The anti-
proliferative effect was evaluated to some cancer cell lines and 17x
the C
7s exhibits a halogen bond between the bromine and the C15 > C20
aromatic ring, with the site of interaction over the C18 C19 bond, as
–
X bond (X = halogen), known as sigma-hole [21]. The structure of
1
–
shown in Fig. 4b. The halogen bond connects the molecules in a linear
showed CC50 38.45
μ
M against MCF-7 lineage after 48 h of treatment, a
motif extended along with the plane 〈101〉 [22].
very different value to that observed by us (267.86
μ
M after 24 h of
Both 17a and 17s structures present hydrogen bonds between N1-
H1a and the carbonyl oxygen (O1) (Fig. 5). These interactions are
similar and originate infinite chain motives extended with the crystal-
lographic axis b. Such hydrogen bonds present distances smaller than the
treatment). Later, the authors showed structural modifications with the
insertion of basic groups at the C4 aminophenyl ring that contributed to
the desired biological activities [15,16]. The functionalization of such
ring could be considered important for the use of this class of compounds
for anticancer purposes.
sum of van der Waals radii (74.6 and 79.0% for 17a and 17s, respec-
1
tively), with graph-set C (6). Non-classic C
–
H⋯O hydrogen bonds were
1
also observed and connect two molecules reciprocally, originating cyclic
2.2.2. In vitro antileishmanial activity
systems. In 17a the closer contact occurs with C8
–
H8⋯O2 and can be
The antileishmanial activity of the obtained compounds is presented
in Table 3. From all the compounds analyzed against L. amazonensis
promastigote forms, only the compounds 17d, 17s and 17x showed
antileishmanial activity below the highest assayed concentration (300
2
2
described as R (12). Otherwise, in 17s the involved molecules are slight
dislocated and the closer contact involves C8-H8⋯O1, with a smaller
2
2
ring with graph-set R (8) [23]. Other weak interactions such as car-
μ
M), with IC50 = 5.96 ± 3.210, 9.05 ± 2.855 and 5.65 ± 2.078
μ
M,
bonyl⋯
π
,
π
⋯π
–
and C H⋯
π
were also observed. A Hirshfeld Surface
respectively. The in vitro leishmanial activity against promastigotes is
widely used as a screening test in the search for new drugs due to its ease
of being performed in the laboratory. However, the assay against
intracellular amastigotes is required as the most accurate way to relate
them to in vitro activity [27].
analysis with the Fingerprint Plots is presented in the Supplementary
Information.
2
2
.2. Biological studies
These three compounds were selected to be evaluated against
intracellular amastigote forms. The compounds 17d and 17x showed
similarity and the best concentration-inhibition curve against promas-
tigote forms among the compounds analyzed (Fig. 6a), however only
.2.1. In vitro antitumor activity
The synthesized compounds were evaluated for the antitumor ac-
tivity against two human breast cancer cell lineage, the MCF7, and
MDA-MB-231. Both lineages were obtained from pleural effusion that
retains several characteristics of differentiated mammary epithelium,
however, while the MCF-7 line conserved the capacity to process
1
7x compound maintained this same inhibition pattern against intra-
cellular amastigote, presenting concentration-inhibition curve like mil-
tefosine (Fig. 6b) and the lower IC50 value among the four compounds
Fig. 2. Some functionalized 4-(N-aryl)aminocoumarins with anticancer activity.
3

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
Scheme 1. Synthesis of 4-(N-aryl)aminocoumarins 15a-i.
Table 1
Optimization of the photoredox arylation of the coumarin 15a.
a
Entry
Catalyst (mol%)
Solvent
Yield (%)
b
1
2
3
4
5
6
[Ru(bpy)
3
]Cl
2
(1.0)
DMSO
DMSO
DMSO
DMSO
DMF
53 (50 )
Eosin Y (2.0)
33
Rose Bengal (2.0)
PDI (2.0)
32
30
[Ru(bpy)
[Ru(bpy)
[Ru(bpy)
–
3
3
3
]Cl
2
]Cl
2
]Cl
2
(1.0)
(1.0)
(1.0)
NR
NR
NR
NR
3
CH CN
c
7
DMSO
DMSO
8
General reaction conditions: 0.2 mmol of benzenediazonium tetrafluoroborate, 1.0 mmol of 4-(aminophenyl)coumarin, 2.5
a
1
mL of solvent, 1.0 mol% of catalyst, and irradiation with Kessil Lamps 440 nm. Yields determined after workup by H NMR
b
using 1,3,5-trimethoxybenzene or 1,4-dimethoxybenzene as internal standard. Isolated yield after column chromatography.
c
No light. NR = no reaction.
analyzed. It is important to highlight the presence of fluorine in the (N-
aryl)amino group. Several reports showed that the presence of this high
electronegative atom can increase the anti-amastigote activity, like in
naphthoquinones [28], quinolones [29], and imidazo[1,2-a]pyrazines
0.0012, p = 0.0022, and p = 0.0106, respectively, Fig. 6m) than un-
treated and infected cells. On the other hand, only at 40 M the com-
μ
pounds 17d and 17s displayed amastigote number per 200 cells (p =
0.0220, Fig. 6c, and p = 0.0054, Fig. 6d, respectively), percentage of
infected cells (p = 0.0112, Fig. 6g, and p = 0.0156, Fig. 6h, respectively)
and amastigote mean per infected cells (p = 0.0134, Fig. 6k and p =
0.0047, Fig. 6l, respectively) lower than untreated and infected cells.
Miltefosine presented lower values of parameters of infection like that
[
30]. As 17s also showed activity and its crystal presented the formation
of halogen bonds, it suggests that this kind of interaction at that position
participates in some way of the activity. The “fluorine bond” is a special
kind of halogen bond in accordance with the literature [31].
All three compounds exhibited low cytotoxicity against BALB/c
peritoneal macrophage, being 17x the highest selectivity index,
demonstrating that this compound is more than 62.2-fold more selective
to intracellular amastigote than toxic to mammal cells. The selectivity
index is a well-established standard for the identification of potential
new compounds against infectious diseases such as leishmaniasis. Global
initiatives for the search of new treatments for neglected diseases, such
as the Drugs for Neglected Diseases initiative (DNDi) and the Japanese
Global Health Innovative Technology, defined that the SI of a hit or lead
compound should be greater than 10-fold [32,33]. Despite the com-
pounds 17d and 17s have demonstrated SI above 10, 17x is the com-
pound that fulfills these criteria by far, and thus it can be classified as a
hit compound.
observed to compound 17x, but not showed lower values at 10
μ
M, only
at 40 and 20
μ
M to amastigote number per 200 cells (p = 0.0003 and p
= 0.0173 respectively, Fig. 6f), percentage of infected cells (p = 0.0026,
and p = 0.070 respectively, Fig. 6j) and amastigote mean per infected
cells (p = 0.0105 and p = 0.0438 respectively, Fig. 6n) when compared
to untreated and infected cells. The treatment effect can be observed in
the images of infected cells treated with the analyzed compounds
(Fig. 6o).
2.2.3. In silico pharmacokinetic properties prediction
In silico predictions of physical-chemical (Supplementary material –
Table S8), drug-likeness (Supplementary material – Table S9), and me-
dicinal chemistry properties (Supplementary material – Table S10) were
carried out for 17d, 17s, and 17x. The physical-chemical characteristics
of the molar refractivity, polar surface area (TPSA), and lipophilicity
were quite similar. Compound 17x showed the best drug-likeness
properties among the three compounds, with no violation of any Lip-
inski, Veber, Egan, and Muegge rules. In the medicinal chemistry
properties prediction, two complementary pattern recognition methods
The parameters of infection analysis corroborated with intracellular
amastigote data. Compound 17x showed statistically significant lower
values of amastigote number per 200 cells at 40, 20 and 10
μM (p =
0
.0006, p = 0.0008, and p = 0.0106, respectively, Fig. 6e), percentage of
infected cells at 40 and 20
μ
M (p = 0.0030, and p = 0.0337, respectively,
Fig. 6i) and amastigote mean per infected cells at 40, 20 and 10
μ
M (p =
4

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
Scheme 2. Evaluation of the phenyldiazonium scope.
were used that allow identifying fragments that could be problematic:
pan-assay interference compounds (PAINS); and Brenk filters. None of
the compounds created an alert for PAINS, but all had an alert for Brenk,
the coumarin group.
oral toxicity in rats, but 17x compared to the other two compounds had
lower oral chronic toxicity in rats.
The compound 17x showed medicinal chemistry properties adequate
to a hit compound, exhibiting no violation to Lipinski’s rules of five,
Veber (GSK), Egan (Pharmacia), and Muegge (Bayer) methods. This
indicates good prediction in the first step of absorption, such as good
solubility and intestinal permeability, that could result in good oral
bioavailability. Pharmacokinetics properties of compound 17x demon-
strated absorption, distribution, metabolism, and excretion prediction
similar or better than miltefosine. Toxicity prediction also exhibited
similar or even better results than the reference drug. These in silico
prediction results, together with the in vitro antileishmanial evaluation,
lead us to nominate the compound 17x as a promising candidate for
further studies of drug development against leishmaniasis.
Their pharmacokinetic properties and toxicity parameters were also
predicted (Supplementary material – Table S11). The three compounds
showed moderate water solubility, high permeability to Caco-2, high
intestinal absorption, low skin permeability, predicted as substrates for
P glycoprotein, and inhibition of P-glycoprotein I and II. The volume of
distribution at human steady-state (ssVD) was low in the three com-
pounds; none of the three compounds were predicted to be distributed
through the brain through the blood-brain barrier (BBB) but were pre-
dicted to penetrate the central nervous system (CNS). They showed
metabolism by the CYP3A4 isoform, but not by CYP2D6, demonstrated
inhibition for four cytochrome P450 isoforms, and only the compound
1
7d demonstrated inhibition for CYP2D6. No compound was predicted
3. Conclusion
as a substrate carrier for renal organic cation 2 (OCT2). In the prediction
of toxicity parameters, all three compounds showed mutagenic potential
and carcinogenic action potential by the Salmonella prediction/micro-
some mutagenicity assay (AMES), hepatotoxicity, high maximum
tolerated human dose (>0.477 log mg/kg/day), toxicity for minnow and
Tetrahymena piriformis, and inhibition for hERG II, but not inhibition for
hERG I or skin sensitization. All three compounds showed high acute
A new series of 3-aryl-4-(N-aryl)aminocoumarins were synthesized
in good yields, employing 4-hydroxycoumarin as the precursor, and
were evaluated for MCF-7, MDA-MB-231, and CCD-1072Sk cancer cell
lines, and to promastigotes and intracellular amastigotes forms of
L. amazonensis. The photoredox catalysis was consolidated as a good
strategy for obtaining these compounds since the reactions were
5

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
Scheme 3. Evaluation of the 4-(N-aryl)aminocoumarins scope.
Fig. 3. Thermal ellipsoids for 17a and 17s (50% of probability).
6

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
Fig. 4. (a) Superimposition of 17a (blue) and 17s (pink); (b) halogen bond in 17s with brown dashed lines; map of electrostatic potential plotted over electron
2
0.
density surface (B3LYP/6-31G(d,p); isovalue 0.008). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
Fig. 5. Hydrogen bonds (O⋯H) for 17a and 17s.
performed in few steps at room temperature and were compatible with
2
refluxing overnight over CaH followed by distillation (vacuum distil-
different patterns of substitution at the rings. Compounds 17d, 17s, and
lation for DMSO and DMF) and stored in molecular sieves. Reactions
were monitored by thin-layer chromatography using Merck TLC Silica
gel 60 F254. The chromatographic columns were performed over Merck
Silica gel 60 Å (particle size: 0.040–0.063 mm, 230–400 mesh ASTM)
treated with triethylamine before use. The columns were run at Bio-
tage®Isolera One instrument. 1H NMR and C NMR spectra were
recorded on a Bruker Avance III spectrometer, operating at 400 MHz for
1
7x showed activity against promastigote forms. Compound 17x was
highlighted for the best activity against L. amazonensis amastigote
intracellular form, no BALB/c peritoneal macrophage cytotoxicity at
assayed concentrations, and high selectivity to parasites over the
mammalian cells. Otherwise, in silico analyses of physicochemical,
pharmacokinetic, and toxicological properties suggest that compound
13
1
13
1
1
7s are potential candidates for anti-leishmaniasis drugs. Unfortunately,
there wasnt any expressive result for MCF-7, MDA-MB-231, and CCD-
072Sk cancer cell lines for any aminocoumarin.
H NMR and 100 MHz for C NMR ( H-decoupled) using CDCl
3
or
́
DMSO-d as solvents. The chemical shifts (δ) were given in parts per
6
1
million (ppm) and tetramethylsilane was used as an internal standard.
The multiplicities were reported as s =singlet, d = doublet, t = triplet, q
= quartet, m = multiplet, and br = broad signal. All coupling constants
4
. Experimental part
(
J values) were given in Hz. High-resolution mass spectra were obtained
4
.1. Materials and methods
by a BrukerMicrOTOF II instrument. Single crystal analysis was con-
ducted in a Bruker Venture II diffractometer employing Cu K radiation.
α
All reagents were bought from Sigma-Aldrich, Oakwood Chemicals,
or Isofar and used as received. DMSO, DMF, and acetonitrile were dried
7

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
Table 2
4.3. General procedure for the photoredox arylation of the coumarins
Antitumor activity of the compounds 17a-x and 18 after 24 h of treatment.
An oven-dried 2-dram vial was charged with 0.2 mmol of the dia-
Compounds
CC50 (µM)
zonium tetrafluoroborate salt, 1.0 mmol (5 eq) of the 4-(N-aryl)amino-
MCF-7
MDA-MB-231
CCD-1072Sk
3
coumarins, 0.002 mmol of [Ru(bpy) ]Cl2, and 2.5 mL of anhydrous
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
7a
7b
7c
7d
7e
7f
492.08 ± 4.368
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
>600
129.0 ± 2.325
DMSO. The vial was purged with argon and the reaction was carried out
at room temperature for 12 h under blue light (440 nm) irradiation with
a Kessil Lamp. After this time, the reaction was quenched with brine and
extracted with dichloromethane (3X30 mL). The organic phases were
>600
466.42 ± 4.506
178.05 ± 4.351
266.76 ± 3.996
437.47 ± 3.907
193.24 ± 3.671
416.25 ± 5.866
273.33 ± 3.822
>600
>600
>600
>600
>600
dried with Na
2
SO
4
and evaporated. The product was purified by column
N before use.
7g
7h
7i
>600
chromatography using silica treated with Et
3
336.6 ± 4.325
>600
7p
7q
7r
>600
4.3.1. 4-(N-phenyl)amino-3-phenylcoumarin (17a)
1
>600
>600
Yield 50%; yellow solid. H NMR (400 MHz, CDCl
): δ 7.51–7.30 (m,
3
>600
>600
8
H), 7.24 (t, 6.9 Hz, 2H), 7.09 (t, 7.4 Hz, 1H), 7.02 (t, 7.6 Hz, 1H), 6.92
7s
7t
538.17 ± 3.475
>600
>600
13
(
d, 7.8 Hz, 2H), 6.26 (br, 1H). C NMR (100 MHz): δ 162.0, 153.5,
48.8, 142.3, 132.3, 132.3, 131.6, 130.3, 129.5, 129.3, 128.6, 126.3,
>600
1
7u
7v
7w
7x
>600
>600
420.52 ± 3.959
229.15 ± 3.937
267.86 ± 3.588
3.03 ± 1.138
>600
124.3, 123.3, 121.9, 117.4, 115.6, 111.9. HRMS(ESI) m/z calcd for
[M+H]+ 314.1181; found 314.1173.
>600
C
21
H15NO
2
>600
Doxorubicin
289.4 ± 2.485
4
.3.2. 4-(N-phenyl)amino-3-(4-methoxyphenyl)coumarin (17b)
1
Data represent means ±standard deviation of at least two independent experi-
ments carried out at least in triplicate. CC50: cytotoxic concentration for 50%
cells.
Yield 32%; yellow solid. H NMR (400 MHz, CDCl ): δ 7.48 (t, 7.7 Hz,
3
1
7
H), 7.41–7.32 (m, 4H), 7.30–7.24 (m, 2H), 7.11 (t, 7.4 Hz, 1H), 7.04 (t,
13
.6 Hz, 1H), 6.96 (m, 4H), 6.25 (br, 1H), 3.83 (s, 3H). C NMR (100
MHz): δ 162.2, 159.8, 153.4, 148.7,142.5, 131.6, 131.4, 129.5, 126.3,
1
24.2, 124.2, 123.2, 121.7, 117.4, 115.7, 115.7, 114.9, 112.0, 55.4.
Table 3
HRMS(ESI) m/z calcd for
C
22
H17NO
3
[M+H]+ 344.1286; found
Activity against promastigote and intracellular amastigote forms of Leishmania
amazonensis, cytotoxicity against BALB/c peritoneal macrophages, and selec-
tivity index of the compounds 17a-x and 18 after 24 h of treatment.
3
44.1279.
4
.3.3. 4-(N-phenyl)amino-3-(4-methylphenyl)coumarin (17c)
Compounds
L. amazonensis IC50 (µM)
Peritoneal
macrophage
CC50 (µM)
SI
1
Yield 34%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.48–7.43 (m,
Promastigote
Intracellular
1H), 7.38 (8.1 Hz, 1H), 7.34–7.22 (m, 7H), 7.10 (t, 7.4 Hz, 1H), 7.01 (t,
7
amastigote
1
3
.6 Hz, 1H), 6.93 (d, 7.9 Hz, 2H), 6.20 (br, 1H), 2.37 (s, 3H). C NMR
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
7a
7b
7c
7d
7e
7f
>300
–
>600
–
(
100 MHz): δ 162.1, 153.6, 148.8, 142.6, 138.7, 131.5, 130.2, 130.1,
>300
–
>600
–
1
29.6, 129.1, 26.5, 124.3, 123.2, 121.8, 117.4, 115.6, 112.4, 21.5.
>300
–
>600
–
HRMS(ESI) m/z calcd for
C
22
H17NO
2
[M+H]+ 328.1337; found
5.96 ± 3.210
>300
32.15 ± 1.216
>600
>18.6
–
>600
–
328.1329.
>300
–
>600
–
7g
7h
7i
>300
–
>600
–
4
.3.4. 4-(N-phenyl)amino-3-(4-thiomethylphenyl)coumarin (17d)
>300
–
8.80 ± 5.106
>600
–
1
Yield 61%; brown solid. H NMR (400 MHz, CDCl
3
): δ 7.49–7.43 (m,
>300
–
–
7p
7q
7r
>300
–
>600
–
1H), 7.38 (d, 8.2 Hz, 1H), 7.32 (t, 6.7 Hz, 4H), 7.29–7.22 (m, 4H), 7.10
(t, 7.4 Hz, 1H), 7.03 (t, 7.6 Hz, 1H), 6.92 (d, 7.9 Hz, 2H), 6.19 (br, 1H),
>300
–
>600
–
>300
–
>600
–
13
2
1
1
.48 (s, 3H). C NMR (100 MHz): δ 162.0, 153.6, 148.8, 142.4, 139.5,
7s
7t
9.05 ± 2.855
>300
24.55 ± 1.109
>600
>24.4
31.6, 130.7,129.6, 128.7, 127.1, 126.3, 124.4, 123.3, 123.3, 121.8,
–
>600
–
17.5, 115.6, 111.7, 15.6. HRMS(ESI) m/z calcd for C22
H
17NO
2
S
7u
7v
7w
7x
>300
–
349.2 ± 3.675
>600
–
+
>300
–
–
[M+H] 360.1058; found 360.1050.
>300
–
>600
–
5.65 ± 2.078
8.56 ± 0.695
9.64 ± 1.148
>600
>62.2
>23.6
4
.3.5. 4-(N-phenyl)amino-3-(4-fluorphenyl)coumarin (17e)
Miltefosine
12.71 ± 1.099
>300
1
Yield 50%; yellow solid. H NMR (400 MHz, CDCl ): δ 7.49 (m, 1H),
3
Data represent mean ±standard deviation of at least two independent experi-
ments carried out at least in triplicate. CC50: cytotoxic concentration for 50%
cells; IC50: inhibitory concentration for 50% parasites; SI: selectivity index ob-
tained for the ratio of peritoneal macrophage CC50 and intracellular amastigote
IC50; –: not determined.
7
1
1
1
3
.36 (m, 4H), 7.24 (m, 2H), 7.06 (m, 4H), 6.91 (d, 79 Hz, 2H), 6.20 (br,
13
H). C NMR (100 MHz): δ 164.0, 162.0, 161.5, 153.5, 148.8, 142.0,
32.4, 132.3, 131.7, 129.5, 123.4, 121.9, 117.5, 116.4, 116.2, 115.6,
[M+H]+ 332.1087; found
10.6. HRMS(ESI) m/z calcd for C21H14FNO
2
32.1078.
4
.2. General procedure for the synthesis of 4-(N-aryl)aminocoumarins
5a-j
4
.3.6. 4-(N-phenyl)amino-3-(4-bromophenyl)coumarin (17f)
1
1
Yield 55%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.54–7.45 (m,
3
H), 7.37 (d, 9.1 Hz, 2H), 7.28–7.21 (m, 4H), 7.13–7.02 (m, 2H), 6.90
A mixture of 4-hydroxycoumarin (1 g, 6.2 mmol) and the respective
13
(
d, 7.8 Hz, 2H), 6.25 (br, 1H). C NMR (100 MHz): δ 161.8, 153.5,
◦
aniline (2 eq) is heated in a 50 mL Becker at 180 C for 1 h under
vigorous stirring. Then, the solid is added to a solution containing 30 mL
1
1
48.8, 141.9, 132.4, 132.1, 131.8, 129.5, 126.0, 124.6, 123.5, 122.8,
21.9, 117.5, 115.5, 110.3. HRMS(ESI) m/z calcd for C21H14BrNO
2
ꢀ 1
of hot methanol and 30 mL of aqueous NaOH 1 mol L . The mixture is
stirred for 30 min and after this time filtered. The solid is washed with
water, oven-dried, and used without further purification. The com-
pounds 15a-j are known and were characterized according to the liter-
ature [34–37].
M+H]+ 391.0286; found 392.0278.
[
4
.3.7. 4-(N-phenyl)amino-3-(4-ethoxycarbonylphenyl)coumarin (17g)
Yield 77%; yellow solid. 1H NMR (400 MHz, CDCl
3
): δ 8.03 (d, 8.2
Hz, 1H), 7.53–7.43 (m, 3H), 7.42–7.36 (m, 2H), 7.22 (t, 7.8 Hz, 2H),
8

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
Fig. 6. Antileishmanial activity of the obtained compounds. (A) Concentration-inhibition curves against promastigote; (B) Concentration-inhibition curves against
intracellular amastigote forms; (C to N) Parameters of infection; (O) Photomicrography of BALB/c peritoneal macrophages infected with L. amazonensis after 24 h of
treatment with 17d, 17s, 17x, and miltefosine. Data represent mean ±sD of at least two independent experiments carried out at least in triplicate. *p < 0.05, **p <
0
.01, ***p < 0.001, when compared with control infected and treated with 0.5% dimethyl sulfoxide by Kruskal–Wallis and Dunn’s test. The images are representative
of two independent experiments carried out at least in triplicate of control infected cells (treated with DMSO 0.5%) and infected cells treated with 17d, 17s, and 17x
compounds or miltefosine at 20 M. Amastigotes within macrophages (black arrows) and remains of amastigotes inside cells after treatment (red arrows). Giemsa, 40
objective. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
μ
×
9

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
7
2
1
1
.07 (t, 7.6 Hz, 2H), 6.90 (d, 7.8 Hz, 2H), 6.33 (br, 1H), 4.35 (q, 7.1 Hz,
H), 1.37 (t, 7.1 Hz, 3H). 13C NMR (100 MHz): δ 166.2, 161.7, 153.6,
48.9, 141.8,137.4, 131.9, 130.5, 130.2, 129.5,125.9,124.5, 123.5,
21.9, 117.5, 115.6, 110.3, 61.2, 14.4. HRMS(ESI) m/z calcd for
3H), 7.40–7.36 (m, 5H), 7.11–7.05 (m, 1H), 7.02–6.95 (m, 2H), 6.85 (d,
13
8.0 Hz, 1H), 6.75–6.72 (m, 1H), 6.27 (br, 1H), 3.79 (s, 3H). C NMR
(100 MHz): δ 165.8, 162.2, 153.3, 150.5, 148.5, 132.8, 132.7, 131.6,
131.2, 131.1, 130.4, 129.0, 128.4, 125.6, 124.2, 123.3, 121.1, 120.7,
C
24
H
19NO
4
[M+H]+ 385.1392; found 386.1383.
117.4, 116.2,111.0, 55.8. HRMS(ESI) m/z calcd for C22
44.1286; found 344.1171.
H
17NO
3
[M+H]+
3
4
.3.8. 4-(N-phenyl)amino-3-(4-nitrophenyl)coumarin (17h)
Yield 95%; yellow solid. 1H NMR (400 MHz, CDCl
): δ 8.14 (d, 8.6
3
4.3.16. 4-(N-(3-methoxyphenyl))amino-3-phenylcoumarin (17v)
1
Hz, 2H), 7.53 (m, 4H), 7,42 (d, 8.2 Hz, 1H), 7.17 (m, 3H), 7.04 (t, 7.2 Hz,
Yield 71%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.50–7.34 (m,
1
1
1
H), 6.88 (7.9 Hz, 2H), 6.35 (br, 1H). 13C NMR (100 MHz): δ 161.5,
8H), 7.14 (t, 8.1 Hz, 1H), 7.08–7.02 (m, 1H), 6.64 (dd, 8.3 and 2.1 Hz,
1H), 6.50 (d, 7.9 Hz, 1H), 6.45 (t, 2.0 Hz, 1H), 6.17 (br, 1H), 3.72 (s,
3H). 13C NMR (100 MHz): δ 162.0, 160.6, 153.5, 148.7, 143.7, 132.3,
131.6, 130.3, 129.3, 128.7, 126.4, 123.3, 117.4, 115.7, 114.2, 112.4,
53.6, 148.6, 147.3, 140.7, 140.2, 132.5, 131.7, 129.5, 125.1, 124.8,
14 2 4
23.9, 122.2, 117.8, 115.6, 107.5. HRMS(ESI) m/z calcd for C21H N O
M+H]+ 359.1032; found 359.1024.
[
1
09.7, 107.8, 107.8, 55.5. HRMS(ESI) m/z calcd for C22
H17NO
3
[M+H]+
4
.3.9. 4-(N-phenyl)amino-3-(3,4-dioxolephenyl)coumarin (17i)
344.1286; found 344.1277.
1
Yield 58%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.49–7.43 (m,
1
7
1
1
1
H), 7.37 (d, 7.5 Hz, 1H), 7.33–7.23 (m, 3H), 7.10 (t, 7.4 Hz, 1H),
.05–6.99 (m, 1H), 6.93 (d, 7.8 Hz, 2H), 6.88–6.83 (m, 3H), 6.27 (br,
H), 5.97 (s, 2H). 13C NMR (100 MHz): δ 162.1, 153.5, 149.0, 148.5,
48.0, 142.3, 131.6, 129.6, 126.3, 125.6, 124.5, 123.8, 123.3, 122.1,
17.5, 115.5, 111.5, 110.9, 109.3, 101.5. HRMS(ESI) m/z calcd for
4.3.17. 4-(N-(4-fluorphenyl))amino-3-(4-thiomethylphenyl)coumarin
(17w)
1
Yield 62%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.51–7.44 (m,
1H), 7.37 (dd, 8.3 and 1.0 Hz, 1H), 7.32–7.27 (m, 5H), 7.08–7.01 (m,
1H), 6.98–6.87 (m, 4H), 6.19 (br, 1H), 2.48 (s, 3H). C NMR (100
MHz): δ 161.9, 161.1, 158.6, 153.6, 148.8, 139.5, 138.2, 131.7, 130.7,
1
3
C
22
H
15NO
4
[M+H]+ 358.1079; found 358.1070.
1
28.7, 127.1, 125.9, 123.9, 123.8, 123.4, 117.6, 116.5, 116.2, 115.4,
4
.3.10. 4-(N-(4-methoxyphenyl))amino-3-phenylcoumarin (17p)
110.9, 15.6. HRMS(ESI) m/z calcd for C22
H16FNO
2
S [M+H]+ 378.0964;
1
Yield 66%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.47–7.41 (m,
found 378.0953.
5
H), 7.41–7.34 (m, 3H), 6.99 (t, 7.7 Hz, 1H), 6.92 (d, 8.9 Hz, 2H), 6.80
1
3
(
d, 8.8 Hz, 2H), 6.17 (br, 1H), 3.78 (s, 3H). C NMR (100 MHz): δ 162.0,
4.3.18. 4-(N-(4-fluorphenyl))amino-3-(4-methoxyphenyl)coumarin (17x)
1
1
1
57.2, 153.8, 149.6, 135.2, 132.6, 131.4, 131.0, 130.5, 129.5, 128.9,
Yield 62%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.50–7.44 (m,
28.6, 126.4, 124.6, 123.1, 117.5, 115.4, 114.8, 110.0, 29.8. HRMS
1H), 7.37 (d, 8.1 Hz, 1H), 7.35–7.25 (m, 3H), 7.04 (t, 7.6 Hz, 1H),
[M+H]+ 344.1286; found 344.1278.
6.99–6.87 (m, 6H), 6.18 (br, 1H), 3.82 (s, 3H). C NMR (100 MHz): δ
13
(
ESI) m/z calcd for C22
H17NO
3
1
62.2, 161.0, 159.8, 158.6, 153.5, 148.8, 138.5, 138.4, 131.6, 131.5,
4
.3.11. 4-(N-(4-methylphenyl))amino-3-phenylcoumarin (17q)
126.0, 124.1, 123.8, 123.7, 123.3, 117.5, 116.4, 116.2, 115.4, 114.9,
1
[M+H]+ 362.1192;
Yield 54%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.43–7.31 (m,
111.4, 55.5. HRMS(ESI) m/z calcd for C22H16FNO
3
8
H), 7.14 (d, 8.4 Hz, 2H), 7.05 (t, 7.7 Hz, 1H), 6.85 (d, 8.4 Hz, 2H), 6.21
found 362.1184.
(
br, 1H), 2.45 (s, 3H). 13C NMR (100 MHz): δ 162.0, 153.5, 148.7, 139.8,
1
1
3
34.1, 132.2, 131.6, 130.3, 129.3, 128.7, 128.1, 126.2, 123.3, 122.5,
4.4. Biological evaluation
4.4.1. Ethics statement
17.4, 115.5, 111.8, 16.5. HRMS(ESI) m/z calcd for C22
H17NO
2
[M+H]+
28.1337; found 328.1329.
The protocols performed in this work that used animals were
4
.3.12. 4-(N-(4-fluorophenyl))amino-3-phenylcoumarin (17r)
approved by the Ethics Committee on the Use of Animals of the Oswaldo
Cruz Institute (Comiss a˜ o de E´ tica no Uso de Animais do Instituto
1
Yield 53%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.55–7.34 (m,
7
1
1
1
H), 7.34–7.27 (m, 1H), 7.12–7.04 (m, 1H), 7.01–6.89 (m, 4H), 6.21 (br,
Oswaldo Cruz, CEUA-IOC), license number CEUA/IOC–L053/2016.
1
3
H). C NMR (100 MHz): δ 162.0, 161.1, 158.6, 153.6, 148.8, 138.3,
38.2, 131.6, 130.4, 129.4, 126.0, 124.0, 123.9, 117.6, 116.4, 116.2,
4.4.2. Animals
15.4, 111.4. HRMS(ESI) m/z calcd for C21
H14FNO
2
[M+H]+ 332.1087;
BALB/c female mice 4–6 weeks old were provided by the Institute of
Science and Technology in Biomodels (Instituto de Ci ˆe ncia e Tecnologia
em Biomodelos), Instituto Oswaldo Cruz, Rio de Janeiro. The animals
were handled as recommended by the National Council for Control of
Animal Experimentation (Conselho Nacional de Controle de
Experimentaç a˜ o Animal – CONCEA), maintained in microisolators
under pathogen-free conditions, with food and water ad libitum and 12-h
light-dark cycle.
found 332.1077.
4
.3.13. 4-(N-(4-bromophenyl))amino-3-phenylcoumarin (17s)
1
Yield 53%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.52–7.45 (m,
1
H), 7.44–7.39 (m, 3H), 7.38–7.32 (m, 6H), 7.08 (ddd, 1.2, 7.3 and 8.3
1
3
Hz, 1H), 6.77 (d, 8.6 Hz, 2H), 6.15 (br, 1H). C NMR (100 MHz): δ
1
61.9, 153.5, 148.2, 141.6, 132.5, 132.1, 131.8, 130.2, 129.3, 128.8,
1
26.0, 123.5, 123.0, 117.5, 117.0, 115.5, 113.1. HRMS(ESI) m/z calcd
for C21
H
14BrNO
2
[M+H]+ 392.0286; found 392.0278.
4.4.3. Parasite
L. amazonensis (MHOM/BR/76/MA-76) was previously obtained
from a human case of diffuse leishmaniasis and characterized by
isoenzyme [38] and lectin techniques [39]. The parasite was maintained
in a murine experimental model in vivo by successive inoculations in
BALB/c footpad mice. The parasite was isolated from footpad lesions,
which were macerated to rupture the cells and release the amastigotes.
Macerated tissue lesions were then inoculated in biphasic Novy-
4
.3.14. 4-(N-(4-ethoxycarbonylphenyl))amino-3-phenylcoumarin (17t)
Yield 42%; yellow solid. 1H NMR (400 MHz, CDCl
): δ 7.91 (d, 8.5
Hz, 2H), 7.52 (t, 7.4 Hz, 1H), 7.45–7.31 (m, 7H), 7.10 (t, 7.5 Hz, 1H),
3
6
3
1
1
3
.84 (d, 8.5 Hz, 2H), 6.28 (br, 1H), 4.35 (q, 7.1 Hz, 2H), 1.37 (t, 7.1 Hz,
1
3
H). C NMR (100 MHz): δ 166.2, 161.8, 153.4, 147.4, 146.8, 132.0,
31.9, 131.2, 130.1, 129.2, 128.9, 126.0, 125.1, 123.7, 119.3, 117.5,
[M+H]+
◦
15.8, 115.2, 61.0, 14.5. HRMS(ESI) m/z calcd for C24
H
19NO
4
MacNeal-Nicolle (NNN) medium and maintained at 26 C in BOD
86.1392; found 386.1382.
incubator for amastigote forms to became promastigote forms. Pro-
mastigote forms were cultured in Schneider’s Insect medium (Sigma, St
Louis, MO, USA) supplemented with 10% of inactivated fetal bovine
serum (FBS; Gibco, Gaithersburg, MD, USA), 100 U/mL of penicillin
4
.3.15. 4-(N-(2-methoxyphenyl))amino-3-phenylcoumarin (17u)
1
Yield 51%; yellow solid. H NMR (400 MHz, CDCl
3
): δ 7.51–7.42 (m,
1
0

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
(
Sigma, St Louis, MO, USA) and 100
μ
g/mL of streptomycin (Sigma, St
4.4.6. Activity of the coumarins against L. amazonensis promastigote
◦
Louis, MO, USA), in BOD incubator at 26 C. The following experiments
were carry out with promastigote forms at maximum of ten in vitro
culture passages in Schneider’s Insect medium.
In 96-well flat plate, 50 L per well of the 3-aryl-4-(N-aryl)amino-
μ
coumarins at different concentrations (600–18.75
μ
M), obtained as
6
described in Section 4.4.5, were cultured with 50
μL of 2 × 10 /mL
L. amazonensis promastigote forms culture at exponential growth for 24
◦
4
.4.4. Cell culture
h at 26 C. Wells with culture medium only were used as a blank, wells
The human breast cancer cells lineage MCF-7 (ATCC® HTB-22™)
with parasites and DMSO 1% were used as control. Miltefosine was used
as a reference drug. After treatment time, an MTT viability assay with
and MDA-MB-231 (ATCC® HTB-26™) was kindly donated by the Lab-
orat o´ rio de Tecnologia de Anticorpos Monoclonais, Biomanguinhos,
Fiocruz-RJ. The mammary cells were cultured in sterile flasks containing
Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma, St Louis, MO,
USA) supplemented with 10% FBS, 2 mM glutamine (Sigma, St Louis,
MO, USA), antibiotic and antimycotic solution (100 × ) (Sigma, St Louis,
MO, USA) at a final concentration of 100 U/mL penicillin, 100 µg/mL
modifications was performed as described elsewhere [42]. Briefly, 10
of MTT solution at 5 mg/mL were added to each well and after 5 h it was
added 150 L of DMSO. The plate was shaken in a shaker plate for 15
μL
μ
min, absorbance was obtained and data used to calculate the inhibitory
concentration of 50% parasite growth (IC50) as described in Section
4.4.5.
streptomycin and 0.25 µg/mL amphotericin B, and incubated in humid
◦
atmosphere at 37 C and 5% CO
2
(Teles et al. 2020). The human
4.4.7. Activity of the coumarins against L. amazonensis intracellular
amastigote
fibroblast cell line CCD-1072Sk (ATCC® CRL-2088™) was purchased
from ATCC and maintained nitrogen-frozen at Laborat o´ rio de
Imunomodulaç a˜ o e Protozoologia of Instituto Oswaldo Cruz, Fiocruz-RJ.
CCD-1072Sk was cultured in sterile flasks containing Iscove’s modified
Dulbecco medium (Sigma, St Louis, MO, USA) supplemented similarly to
In a 24-well plate with coverslips, peritoneal macrophages at 106
cells/mL were infected with L. amazonensis promastigotes at 2:1 multi-
◦
plicity of infection (MOI) overnight at 35 C and 5% CO
2
. Non-
internalized promastigotes were removed by PBS wash and infected
cells were treated with different concentrations of 3-aryl-4-(N-aryl)
aminocoumarins compounds or miltefosine, both with concentrations
◦
DMEM used in the breast cancer cell, and incubated at 37 C and 5%
2
CO . The peritoneal macrophages were obtained from BALB/c mice
inoculated intraperitoneally with 3 mL sterile Brewer thioglycollate
medium (Sigma, St Louis, MO, USA). After 72 h, the animals were
obtained as described before in Section 4.4.6 range 40–2.5
μ
M. Wells
with infected cells treated with 0.5% DMSO were used as control. After
24 h, coverslips with cells were recovered from wells, stained with Bouin
solution, and stained with Giemsa’s azure-eosin-methylene blue (MERK,
Darmstadt, Germany). The infected cells were observed at light micro-
scopy and intracellular amastigotes, the percentage of infected cells and
mean of intracellular amastigotes in infected cells were counted in 200
cells. The IC50, parameters of infection, and selectivity index (SI) were
calculated as described elsewhere [43].
euthanized with 250
mg/mL ketamine (Syntec, S a˜ o Paulo, BRA) and 20 mg/mL xylazine
Syntec, S a˜ o Paulo, BRA). The peritoneal cavity was washed with 10 mL
μL intraperitoneal injection of a 1:1 mixture of 100
(
of chilled PBS pH 7.0, and the recovered PBS with peritoneal suspension
◦
cells was centrifuged at 1500 rpm for 5 min at 4 C. Peritoneal macro-
phages were suspended in RPMI 1640 medium (Sigma, St Louis, MO,
USA) supplemented with 10% inactivated FBS and 100 U/mL of peni-
◦
cillin and 100
μ
g/mL of streptomycin, and maintained at 37 C and 5%
CO (Teles et al. 2021).
2
4.4.8. In silico pharmacokinetics properties prediction of the coumarins
In silico prediction analysis of ADMET was carried out as described
elsewhere [32]. The pkCSM[44] and SwissADME[45] web tools were
used to in silico predictions using the simplified molecular-input line-
entry system (SMILES) file of the obtained structures.
4
.4.5. Cytotoxicity evaluation of the synthesized coumarins
For in vitro cytotoxicity evaluation, the monolayer of the cells lineage
MCF-7, MDA-MB-231, and CCD-1072Sk maintained in sterile culture
flasks were detached from bottom flasks by trypsinization with a 0.25%
(
w/v) trypsin solution plus 0.03% (w/v) EDTA (Sigma, St Louis, MO,
4.4.9. Statistical analysis
USA), while peritoneal macrophage was immediately used in experi-
Data were expressed as mean ±standard deviation and organized in
tables and/or graphs. The IC50 and CC50 were calculated from the non-
linear regression of concentration log versus normalized response, and
comparisons between groups were performed by Kruskal–Wallis and
multiple comparison Dunn’s test using GraphPad Prism 7.00 software
(GraphPad Software, San Diego, CA, USA).
5
ments right after its obtaining. The cells were set to 5x10 cells/mL and
1
00
μ
L of cells suspension in its respective culture medium were
◦
cultured in 96-well flat plate overnight at 37 C and 5% CO
2
. The non-
adherent cells were washed with PBS and it was added cell culture
medium with 3-aryl-4-(N-aryl)aminocoumarins at different concentra-
tions (600 to 18.75 M), obtained by serial dilution 1:2 from a stock
μ
solution in dimethyl sulfoxide (DMSO; Sigma, St Louis, MO, USA) 100x
higher than the highest analyzed concentration. Wells with culture
medium only were used as a blank, wells with cells and DMSO 1% were
used as control. Doxorubicin and miltefosine were used as reference
drugs to lineage cells and peritoneal macrophages, respectively. After
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
2
4 h of treatment, the cell viability was carried out with a colorimetric
assay based on cellular metabolic activity measurement with 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St
Acknowledgments
Louis, MO, USA) [40]. Briefly, 10
was added to each well. After 2 h, the supernatant was completely
removed from wells and 50 L of DMSO was added to solubilize for-
μ
L of MTT sterile solution at 5 mg/mL
This study was financed in part by the Coordenaç a˜ o de Aperfeiçoa-
mento de Pessoal de Nível Superior - Brasil (CAPES) – Finance Code 001
and by the Carlos Chagas Filho Foundation for Research Support of the
State of Rio de Janeiro (Fundaç a˜ o Carlos Chagas Filho de Amparo a`
Pesquisa do Estado do Rio de Janeiro; FAPERJ) grant numbers E-26/
010.001759/2019 and E_26/202.780/2019. We would like to
acknowledge LDRX (Laborat o´ rio Multiusu ´a rio de Difraç a˜ o de Raios- X
da UFF), and Centro Nacional de Biologia Estrutural e Bioimagem
(CENABIO) for the support with the X-ray diffraction facility (D8-Ven-
ture). The authors are also grateful to CAPLH/PUC-Rio for the use of the
NMR facilities. Dr. Fernando Almeida-Souza is a postdoctoral research
μ
mazan crystals with the aid of a shaker plate for 15 min. Absorbance was
obtained at 570 nm in a spectrophotometer EZ Read 400 (Biochrom,
Cambridge, UK) and the data were normalized with the formula: %
viable parasites = (sample absorbance-blank absorbance)/(control
absorbance-blank absorbance) × 100. The normalized data were used to
calculate the cytotoxic concentration of 50% cell growth (CC50) [41].
1
1

L.S.A. Carneiro et al.
Bioorganic Chemistry 114 (2021) 105141
fellow of CAPES grant number 88887.363006/2019-00.
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