Discovery of a benzothiophene-flavonol halting miltefosine and antimonial drug resistance in Leishmania parasites through the application of medicinal chemistry, screening and genomics

Article abstract of DOI:10.1016/j.ejmech.2019.111676

Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous

Full text of DOI:10.1016/j.ejmech.2019.111676

European Journal of Medicinal Chemistry 183 (2019) 111676
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of a benzothiophene-flavonol halting miltefosine and
antimonial drug resistance in Leishmania parasites through the
application of medicinal chemistry, screening and genomics
Chiara Borsari a , María Dolores Jim eꢀ nez-Ant oꢀ n
,
1
,
2
b
,
c
,
1
d
d
, Julia Eick , Eugenia Bifeld ,
b
b,
c
b,
c
e
Juan Jos eꢀ Torrado , Ana Isabel Olías-Molero , María Jesús Corral , Nuno Santarem ,
Catarina Baptista , Leda Severi , Sheraz Gul , Markus Wolf , Maria Kuzikov ,
Bernhard Ellinger , Jeanette Reinshagen , Gesa Witt , Pasquale Linciano , Annalisa Tait ,
e
a
f
f
f
f
f
f
a
a
a
a
d
d
Luca Costantino , Rosaria Luciani , Paloma Tejera Nevado , Dorothea Zander-Dinse ,
Caio H. Franco g , Stefania Ferrari , Carolina B. Moraes , Anabela Cordeiro-da-Silva h,
,
2
a
g,
2
e,
a
d
,
**
b,
c
,
***
, Maria Paola Costi a
, *
Glauco Ponterini , Joachim Clos
University of Modena and Reggio Emilia, Via Campi 103, 41125, Modena, Italy
Complutense University of Madrid, 28040, Madrid, Spain
Instituto de Investigaci oꢀ n Hospital 12 de Octubre, Madrid, Spain
Bernhard Nocht Institute for Tropical Medicine, D-20359, Hamburg, Germany
, Jos eꢀ María Alunda
a
b
c
d
e
Instituto de Investigaç a~ o e Inovaç a~ o em Saúde, Universidade do Porto and Institute for Molecular and Cell Biology, University of Porto, 4150-180, Porto,
Portugal
f
Fraunhofer Institute for Molecular Biology and Applied Ecology Screening Port, Hamburg, Germany
Laborat oꢀ rio Nacional de Bioci ^e ncias (LNBio), Centro Nacional de Pesquisaem Energia e Materiais (CNPEM), 13083-100, Campinas, SP, Brazil
Departamento de Ci ^e ncias Biol oꢀ gicas, Faculdade de Farm aꢀ cia da Universidade do Porto (FFUP), Porto, Portugal
g
h
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 May 2019
Received in revised form
Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The
appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop
new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the
design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of
assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the
progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)-
1
August 2019
Accepted 2 September 2019
Available online 5 September 2019
3
(
7
-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp.
EC50 1.9 M for Leishmania infantum, 3.4 M for L. donovani, 6.7 M for L. major), Trypanosoma cruzi (EC50
.5 M) and T. brucei (EC50 0.8 M). Focusing on anti-Leishmania activity, compound 19 challenge in vitro
did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance
genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC50
Keywords:
Leishmaniasis
m
m
m
m
m
Flavonoid-like scaffold
Pharmacokinetic studies
Drug resistance
Genomic studies
Fluorescence
.
Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo
pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2 h; per os
(
PO): 46.9 h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no
toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectro-
scopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a
*
Corresponding author. University of Modena and Reggio Emilia, Via Campi 103, 41125, Modena, Italy.
*
*
* Corresponding author. Bernhard Nocht Institute for Tropical Medicine, D-20359, Hamburg, Germany.
** Corresponding author. Complutense University of Madrid, 28040, Madrid, Spain.
E-mail addresses: clos@bni-hamburg.de (J. Clos), jmalunda@ucm.es (J.M. Alunda), mariapaola.costi@unimore.it (M.P. Costi).
C.B. and M.D.J.A. contributed equally and are considered as co-first authors.
Present Address: C.B. Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland. C.H.F. University of Coimbra 3004-504, Coimbra,
1
2
Portugal. C.B.M Department of Microbiology, Institute of Biomedical Sciences, University of S a~ o Paulo, 05508-900 S a~ o Paulo e SP, Brazil.
https://doi.org/10.1016/j.ejmech.2019.111676
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
specific compound based on the screening against a protein set, following the intrinsic fluorescence
changes.
©
2019 Elsevier Masson SAS. All rights reserved.
1
. Introduction
flavonols and to secure intellectual property [17]. Despite their
well-known multitargeting behavior, flavonoids remain impor-
tant core structures in the medicinal chemistry field [18]. This
compound library was evaluated in vitro against L. infantum and
compound 19 displayed an interesting anti-leishmanial activity.
Sixteen additional derivatives (compounds 23-38) were subse-
quently designed and synthesized to explore the Structure Ac-
tivity Relationships (SAR) of this series. To assess its spectrum of
activity, compound 19 was also screened against L. donovani,
L. major, T. brucei and a panel of T. cruzi strains. Resistance studies
showed that no significantly resistant parasites could be selected
from a L. donovani population during a 78-day challenge with
compound 19 at the EC50/EC75, indicating a low propensity of the
parasites to develop resistance against 19. Pharmacokinetic
studies of compound 19 pointed out its long half-life and suitable
profile for in vivo evaluation in L. infantum infected hamsters. In
the mice model, we observed a decrease in parasite load in spleen.
Compound a low toxicity, while spectroscopic studies showed
high lipophilicity and high percentage of plasma binding, securing
inhibitor transport in the bloodstream. Compound 19 offers the
potential to expand the lead pipeline in anti-leishmanial therapy
by tackling the important challenge of drug resistance in the
discovery of new drugs for Leishmania infections.
Leishmania spp. are obligate protozoan parasites of cells of the
mononuclear phagocytic system and cause a wide spectrum of
diseases, from cutaneous lesions to potentially fatal visceral in-
fections [1]. Leishmaniasis represents a huge burden since an
estimated 12 million people are infected with different species of
Leishmania [2]. Despite the serious health, economic and social
consequences, effective vaccines are lacking and the limited arsenal
of anti-leishmanial drugs has severe drawbacks including toxicity,
poor efficacy and high treatment costs. Five main drugs are in
therapeutical use against leishmaniasis, as reported by the World
Health Organization. Miltefosine represents the first and only oral
drug approved for this disease. Among Neglected Tropical Diseases
(
NTDs), leishmaniasis is considered to be one of the most chal-
lenging infections and its control is hampered by its complex
epidemiology and by pathogen resistance [3]. The first line
antimony-containing drugs have been in use since the 1920s and
face wide-spread drug resistance (DR) in key endemic regions, and
even the newer drugs encounter therapy-resistant cases [4,5]. DR is
usually due to a decrease in the active drug concentration within
the parasite cell that results from increased efflux, reduced uptake
or amplified expression of the targeted enzyme. Resistance against
classic first-line antimonial therapy has been observed in patients
and produced in vitro to analyze the main mechanisms involved in
this process [6]. A decrease of Sb(III) inside the cell has been
observed in several Leishmania mutant strains developed in labo-
ratory [7]. Resistance to established second-line therapeutics, such
as pentamidine and amphotericin B (AmB) has also been induced
in vitro. Resistance to AmB has been associated with a variation in
cell membrane fluidity, while resistance to pentamidine has been
shown to be related to a change in arginine and polyamine con-
centrations in the cell [8]. The appearance of resistance to estab-
lished first- and second-line anti-leishmanial drugs stresses the
demand to develop novel, less toxic and more effective drugs that
would not likely develop spontaneous resistance [9]. To accelerate
the discovery of anti-leishmanial drugs, phenotypic approaches
offer the potential to identify compounds that are active against the
intracellular parasites because they take into account membrane
permeability, cell uptake and cell efflux mechanisms. Aiming to use
such approaches to select the most promising compounds, suitable
counter-screens, including screening against mammalian cells,
need to be performed to filter out cytotoxic compounds [10]. This
approach has been successfully implemented in NTD drug discov-
ery [11,12]. A wide range of chemical structures, including flavonols
2. Chemistry
The design, synthesis and in vitro evaluation of natural and
synthetic compounds against T. brucei and L. infantum have shown
that methoxylated and hydroxylated classical flavonols are potent
anti-T. brucei agents (EC50 1e20
against Leishmania spp. [16].
mM) and have limited activity
Herein, we report that replacement of the phenyl ring of the
classical flavonols with heteroaromatic rings and biphenyl moieties
leads to a novel anti-leishmanial scaffold. Specifically, we have
introduced furan, 3-/4-/5-methylthiophene, pyridine, quinoline,
indole, 1-methylindole, benzothiophene and 1,3-benzodioxole
rings (Fig. 1 and Table 1).
Compound 19 showed potent anti-leishmanial activity and a
SAR study on ring A and on the 3-OH of the chromen-4-one scaffold
has been carried out. The chemical structures of the synthesized
derivatives of compound 19 are shown in Table 2.
The synthetic procedure for the preparation of these com-
pounds is shown in Scheme 1.
The chalcone intermediates (39e58) were synthesized by
Claisen-Schmidt condensation using substituted acetophenones
and benzaldehydes in the presence of NaOH as the base. The re-
action was carried out in ethanol as previously reported [16]. For
the synthesis of compound 20, piperidine was used instead of a
strong base. In some cases, the chalcones were not isolated as pure
products and were used for the following step without further
purification (see the Experimental Part for details). The chalcones
were converted into the corresponding flavonol-like compounds (1,
(
3-hydroxy-2-phenylchromen-4-one), has been investigated in
drug discovery programs with the aim of identifying novel anti-
leishmanial and anti-trypanosomal agents [13e16]. Recently, we
proposed classic hydroxylated and methoxylated flavonols as
promising hits against trypanosomatidic infections [16]. However,
these compounds displayed only a low potency against Leishmania
species.
In the search for novel anti-leishmanial compounds, we report
herein the design and synthesis of 22 classical flavonols modified
with heteroaromatic rings and biphenyl moieties conferring
inherent chemical diversity. These structural modifications
allowed to explore a novel chemical space around classical
2, 5, 6, 8, 9, 11, 13, 17e20 and 22e32) using the Flynn-Algar-
Oyamada method for epoxidation and subsequent intramolecular
cyclization of the open-chain structure [19]. The reaction was per-
formed with hydrogen peroxide in aqueous base (1 M NaOH). The
hydroxylated flavonols (3, 4, 7, 10, 12 and 21) were synthesized by

C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
3
Fig. 1. Structure-Activity Relationship (SAR) studies.
Table 1
Structure of the synthesized flavonol-like compounds bearing heteroaromatic and biphenyl rings. Compounds 2, 3, 13, 17, 20 and 22 have been previously described in the
literature, while compounds 1, 4e12, 14-16, 18, 19 and 21 are novel structures and have not been previously reported.
Comp.
1
R
2
R
5
R
6
R
7
Comp.
12
R
2
R
5
R
6
R
7
H
OCH
3
H
H
H
OH
2
3
H
H
OCH
3
13
14
H
CH
3
H
H
H
H
OH
H
H
H
H
H
OCH
3
H
H
H
4
5
OH
H
15
16
OH
OCH
3
OCH
3
6
7
8
H
H
H
H
OCH
H
3
17
18
19
H
H
H
H
OCH
3
OH
OCH
3
3
H
CH
H
3
H
OCH
H
9
OCH
3
H
20
H
OCH
3
H
1
0
1
OH
H
H
H
H
21
22
H
H
OH
H
H
1
OCH
3
OCH
3
cleavage of the methoxy protecting groups using boron tribromide
of NaOH. The chalcones were not isolated as pure products but
were directly converted into the corresponding flavonols using the
procedure described above.
(Scheme 1).
The procedure for the synthesis of compounds 14e16 is shown
in Scheme S1 of the Supporting Information. The first step involved
a Suzuki cross-coupling reaction of alkyl boronic acid with 3-
bromobenzaldehyde. The synthesized aldehydes (59 and 60)
Compound 19 was synthesized as reported in Scheme 1 and
subsequently converted into the corresponding esters (33e35),
ethers (37 and 38) and carbamates (36). For the synthesis of esters
and carbamates, compound 19 was treated with an excess of acyl
0
0
reacted with 2 -hydroxy-5 -methoxyacetophenone in the presence

4
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
Table 2
Chemical structure of the novel derivatives of compound 19 (compounds 23e38).
Comp.
R
3
R
6
R
7
Comp.
R
3
R
6
R
7
23
24
25
OH
OH
OH
CH
Br
F
3
H
H
H
31
32
33
OH
OH
H
H
OCH
Cl
OCH
H
2 3
CH
3
26
OH
Cl
H
34
OCH
3
H
2
2
7
8
OH
OH
H
H
H
35
36
OCH
3
3
H
H
OCH
3
OCH
2
3
9
0
OH
OH
H
H
CH
F
3
37
38
OCH
3
3
H
H
OCH
ꢀ
2 2 3
O , NaOH (1 M), EtOH, r.t.; (iv) BBr (1 M in dry DCM), dry DMC, 0 C, r.t. With the
Scheme 1. Reagents and conditions: (i) NaOH (3 M), EtOH, r.t.; (ii) Piperidine, EtOH, reflux; (iii) H
exception of compound 46, all chalcones were synthesized following procedure (i).
chloride in dry dichloromethane (DCM) in the presence of trie-
thylamine. For the synthesis of ethers, an alkyl halide was added to
fact, compounds with a methyl-thiophene and a hydroxyl group
linked to ring A (10 and 12) did not inhibit T. brucei cell growth,
a solution of compound 19 in dry DMF and in the presence of K
Scheme S2 of the Supporting Information).
2
CO
3
with the exception of 7 (EC50 ¼ 4.9
mM). The SI of compounds with a
(
methyl thiophene were <10 with the exception of compound 8
which showed a SI of 36. Compounds 14e16, bearing a biphenyl
ring, had low micromolar potency against T. brucei with EC50 values
3
. Results
of 3.0, 1.7, 3.9 mM, respectively. Compound 15, which contains hy-
droxyl groups linked to the biphenyl ring, was the most potent and
selective biphenyl derivative (SI ¼ 15). Compound 19, possessing a
benzothiophene ring, was more active and selective than the cor-
3
.1. In vitro evaluation against T. brucei and L. infantum
With the exception of compound 22, the novel library of
responding methyl-thiophene derivative 5 (EC50 ¼ 0.8 and 5.7
SI ¼ 33 and 4, respectively). Replacement of the sulfur of compound
9 with a nitrogen (20) yielded a decrease in activity and a signif-
icant decrease in SI (EC50 ¼ 0.8 and 1.4 M, SI ¼ 33 and 8, respec-
tively). Compound 17, bearing a 1,3-benzodioxole, was the most
active and selective molecule towards T. brucei (EC50 ¼ 0.4 M,
mM,
flavonol-like compounds (1e21) was evaluated against L. infantum
intracellular amastigotes and T. brucei bloodstream forms. For
compounds causing a >70% reduction of parasite loads or a >70%
1
m
parasite growth inhibition at 10 mM, dose-response experiments
were performed to determine their potencies. In addition, the
compound series was assessed for cytotoxicity in THP1
macrophage-like cells to estimate the half maximal cytotoxicity
concentration (CC50) and the selectivity index (SI ¼ CC50/EC50
parasite). Most of the synthesized compounds displayed low
micromolar potency against T. brucei (Table S1 of the Supporting
Information). All compounds bearing a furan (1e4) and compound
m
SI ¼ 250). The most promising compounds are depicted in Fig. 2A.
Very recently, we have validated compound 17 as an anti-T. brucei
agent and have carried out SAR studies and discovered follow-up
hits [20]. Herein, we focus our attention on the identification of
flavonol-like compounds with a broad spectrum of activity, able to
inhibit Leishmania parasites. Therefore, the library of 22 com-
pounds was evaluated for the anti-parasitic activity against
L. infantum-infected macrophages. Compound 19 showed an EC50
for L. infantum comparable to that of miltefosine, the drug of choice
to treat Leishmania infections when resistance to antimonials is
18 bearing a quinoline were not active towards T. brucei in the
concentration range evaluated (from 78 nM to 10 M). Compounds
bearing a methylated thiophene showed activity only when a
methoxy group (5, 6, 9 and 11) or a methyl group (8 and 13) was
linked to ring A of the chromen-4-one scaffold (EC50 < 6 mM). In
m

C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
5
Fig. 2. (A) Selectivity index and EC50 of compounds showing activity towards T. brucei (upper panel) and L. infantum (bottom panel). The selectivity index is reported on the left
light color bars), while EC50 is reported on the right (dark color bars). (B) Inhibitory profile of compound 19 against T. brucei, Leishmania spp. and T cruzi strains. (C) Early toxi-
cological data of compounds 1-38. All the assays were performed at 10 M and the data ranges are reported using a traffic light system. An ideal compound (I.C.) possesses all the
(
m
parameters in green. The cells are colored in green when the percentage inhibition of CYP isoforms, hERG, Aurora B kinase and mitochondrial toxicity is 0e30%. Cells are colored in
red when data indicate toxicity (percentage of inhibition of CYP isoforms, hERG, Aurora B kinase and mitochondrial toxicity ꢁ60). Yellow color indicates inhibition values between
30 and 60% for slightly toxic compounds. Compounds are considered as non-cytotoxic (green) for A549 cell line when growth is 60e100%, cytostatic (yellow) when 0e59% and
cytotoxic (red) when <0. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
observed, and a higher SI (EC50 for 19 ¼ 1.88 ± 0.32
m
M, SI ¼ 17; EC50
3.3. Structure-Activity Relationship (SAR) studies around
compound 19
for miltefosine ¼ 2.6 ± 0.4
m
M, SI ¼ 5). Therefore, compound 19 was
screened against different Leishmania species and T. cruzi to
determine its spectrum of anti-parasitic activity.
Considering the anti-parasitic activity and selectivity of com-
pound 19, SAR studies were carried out for hit confirmation. Sixteen
derivatives of compound 19 (23e38) were designed, synthesized
and biologically evaluated towards T. brucei and L. infantum para-
sites (for chemical structures, see Table 2). With the exception of
five compounds (30, 32, 36e38), all molecules had significant ac-
3.2. Evaluation of compound 19 towards L. major, L. donovani and
T. cruzi
Compound 19 showed EC50 in the low micromolar range also
against L. donovani (EC50 ¼ 3.4 M) and L. major promastigotes
M). Thus, it represents a promising compound for both
tivity against T. brucei (EC50 < 6
3 and 35 showed activity towards L. infantum (EC50 < 6
Table S2 of Supporting Information). When the OCH in position 6
of compound 19 was replaced with CH , Br, F and Cl (compounds
3, 24, 25 and 26, respectively) anti-T. brucei activity was main-
mM), whilst only compounds 24-26,
m
3
mM)
(
EC50 ¼ 6.7
m
(
3
visceral (VL) and cutaneous leishmaniasis (CL). Moreover, com-
pound 19 was active against the T. cruzi infection of human glio-
3
2
blastoma cells (HG39), with an EC50 of 7.5
EC50 of 12.5 M for miltefosine (Fig. 2B). It was screened against the
intracellular amastigotes of a panel of T. cruzi strains: Silvio X10/1
mM, compared with an
tained (EC50 < 2
mM), and compounds 24-26, bearing halogens in
m
position 6, showed anti-Leishmania activity with EC50 < 6
mM.
Compounds bearing an unsubstituted ring A (27), a methoxy group
or a methyl group in position 7 (28 and 29) showed anti-
(
(
TcI), Y (TcII), ARMA13 cl1 (TcIII), 92.80 cl2 (TcV) and CL Brener
TcVI), and was found to be active against all five strains. The po-
trypanosomal activity (EC50 ¼ 2.6, 3.1 and 4.5
mM, respectively),
but were not active against L. infantum. The presence of an ester
tency of compound 19 was superior to the reference compound
benznidazole in all tested strains, except for strain 92.80 cl2 where
the EC50 values of both compounds were similar (Table 3).
instead of a hydroxyl group in position 3 led to activity towards
Table 3
Potency of compound 19 and the reference compound benznidazole against a panel of T. cruzi strains.
T. cruzi strains
Silvio X10/1
Y
ARMA13 cl1
92.80 cl2
CL Brener
EC50
EC50
(
(
m
M) comp. 19
0.3 ± 0.09
3.9 ± 0.98
0.9 ± 0.34
2.2 ± 1.81
1.2 ± 0.01
5.1 ± 3.11
0.7 ± 0.06
0.6 ± 0.05
0.6 ± 0.33
1.0 ± 0.31
mM) benznidazole
Data represent mean ± standard deviation of two independent experiments.

6
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
both T. brucei and L. infantum (compounds 33 and 35) (EC50 < 4
m
M),
were grown in vitro at the IC50 of compound 19 and miltefosine,
5
while compounds 36 and 37e38, bearing a carbamate or an ether,
respectively, did not possess significant anti-parasitic activity.
These results suggest that the free hydroxyl group in position 3 is
required for significant compound potency. As for the derivatives of
compound 19, the activity of esters can be related to enhanced
hydrolysis with respect to the ethers and carbamates and therefore
with DMSO as a negative control. Parasites were seeded (1 ꢂ 10 /
mL) into a medium containing the drugs and grown for 3 days
5
under selective pressure, then seeded again (1 ꢂ 10 cells/mL) into
a fresh medium containing the respective compounds. Three-day
in vitro passages were repeated 11 times (>50 generations).
Miltefosine-selected cultures showed increasing growth rates after
this selection, indicating the development/selection of resistant
parasites in the populations. By contrast, growth in the presence of
compound 19 remained at <50% throughout the 33 days of selec-
tion (Fig. 3B), excluding a spontaneous development of resistance.
With the caveat that the experiments were performed with
promastigotes in culture and not with the relevant, intracellular
amastigote stage, we conclude that exposure to compound 19 at
sub-effective concentrations do not lead to spontaneous resistance.
This indicates the absence of compound 19-tolerant L. donovani
clones within a population that harbors miltefosine-resistant in-
dividual promastigotes.
19 remains the most promising anti-leishmanial agent. The most
potent and selective compounds on T. brucei and L. infantum are
depicted in Fig. 2A, while a summary of the broad spectrum of
activity of compound 19 is shown in Fig. 2B. Miltefosine, pentam-
idine and benznidazole were selected as reference compounds for
comparison (Fig. 2B).
3.4. Early ADME-Toxicity profile
The ADME-Toxicity profile of the library of 38 compounds was
assessed at 10 mM in a panel of assays comprising hERG, Aurora B
kinase, cytochromes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and
CYP3A4), cytotoxicity (A549 cell line) and mitochondrial toxicity.
The data are reported in Fig. 2C using a traffic light system to
indicate value ranges and in Table S3 of the Supporting Information.
With the exception of compounds 10, 13, 20, 21 and 38, none
exhibited toxicity towards hERG, which is a pivotal parameter for
proceeding a compound further in the drug discovery process.
Overall, the compounds yielded significant inhibition of cyto-
chrome CYP1A2. Some compounds were cytostatic, but only 31 was
cytotoxic (cell growth A549 < 0%). With the exception of com-
pounds 31 and 38, no compound presented mitochondrial toxicity.
Reversible gene amplification occurs frequently in Leishmania
spp. and contributes to the appearance of drug resistance in vitro
and in the field [25,26]. This includes chromosome ploidy changes,
gene copy number variations, but also the extrachromosomal
amplification of genomic regions in the form of episomes. The latter
mechanism may be mimicked by a cosmid library screening or
functional cloning of dominant resistance genes [27]. This approach
was recently refined by a combination with next generation
sequencing (NGS), called Cos-Seq [28,29]. This strategy (Fig. 3C)
was used to identify possible gene markers of compound 19 re-
sistances in vitro. Fig. 3C shows the selection process and the
isolation of cosmids for NGS.
A L. donovani population, randomly transfected with a genomic
DNA cosmid library of L. donovani, was challenged with miltefosine
or compound 19 at EC50 using the same, periodic 3-day in vitro
passage intervals as above. Another population was passaged
identically using the solvent DMSO as control. Indeed, by day 37 of
the selection, parasites were showing >50% growth compared to
the DMSO control, both under miltefosine and under compound 19
challenge (Fig. 3D).
For compound 19, EC50 towards L. infantum was 1.9
the hERG IC50 (>100 M) was over 50-fold greater than the EC50
towards the parasite. With the exception of the CYP1A2 IC50
m
M (SI ¼ 33);
m
(
(
0.4
IC50: CYP2C9 >100
M), thus consistent with the parameters for com-
m
M), the IC50 against the cytochrome isoforms were >10
mM
mM; CYP2C19 > 100
mM; CYP2D6 ¼ 14.8
mM;
CYP3A4 >100
m
pound selection and prioritization as reported previously in a
similar compound series [16]. The compound slowed the growth of
W1-38 cells, but it was not cytotoxic (Fig. 2C).
All compounds were also screened using the FAFdrugs4 soft-
ware (http://fafdrugs4.mti.univ-paris-diderot.fr, Table S9 of the
Supporting Information), a software dedicated to evaluate the po-
tential liabilities as PAINS (pan assay interference compounds) [21].
Considering the above described comprehensive panel of assays
used for compounds screening, the flavonol-like scaffold over-
comes some of the liabilities typical of classical flavonols such as
CYP450 enzymes inhibition and hepatocytes inhibition effects [22].
This suggested that the chemical modifications introduced do not
induce the PAINS features in the scaffold. Therefore, compound 19
was selected for further characterization.
Cosmid DNA from populations selected at IC50 for miltefosine
and compound 19, and control cultures were recovered and
analyzed by Illumina NGS. Sequence reads (https://www.ncbi.nlm.
nih.gov/Traces/study/?acc¼PRJNA512372) were aligned to the
L. infantum genome database using Bowtie2 alignment. One major
and one minor alignment peak were recorded (Fig. S1 of the Sup-
porting Information), identifying gene regions on chromosome 36
(position 2,561,981 - 2,592,497) and chromosome 24 (position
357,326e392,031) as possible markers of compound 19 tolerance
(Table S4 and Fig. S1 of the Supporting Information).
The selected populations were subjected to further challenge
with miltefosine or compound 19 at their respective IC75 for
another 38 days (25 passages in total). At IC75, miltefosine-selected
parasites yielded >75% growth relative to DMSO controls. In
contrast, compound 19-preselected parasites showed no ability to
proliferate at the IC75 of the compound (Fig. 3D). This indicates that
none of the cosmid-based transgenes in the recombinant popula-
tion (Table S4 of the Supporting Information) is a dominant resis-
tance gene marker for compound 19, further supporting the notion
that compound 19 is comparatively impervious to spontaneous or
gene amplification-mediated resistance in Leishmania.
3.5. Drug resistance investigation in L. donovani
First, we tested whether compound 19 was effective against
antimony-resistant Leishmania parasites. Two field isolates of
L. donovani from Nepal, BPK091 and BPK190 [23] that were
respectively inherently sensitive and resistant to antimony treat-
ment, were used to infect mouse bone marrow-derived macro-
phages. Infected cells were subsequently treated with increasing
concentrations of compound 19 (Fig. 3A). Compound 19 is active
against both strains, showing 36% and 78% parasite load reduction
at 5
m
M, against BPK091 and BPK190, respectively, and 69% reduc-
M, indicating its capacity to eliminate
3.6. Half-life determination in vivo
tion for both strains at 10
m
antimony-resistant L. donovani from infected macrophages.
Afterwards, we investigated whether spontaneous resistance
may overcome compound 19 activities. L. donovani promastigotes
Compound 19 bioavailability and half-life were evaluated in
BALB/c mice treated IV with 1 mg/kg and PO with 20 mg/kg.
Compound 19 was characterized by a long half-life (IV):63.2 h;

C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
7
Fig. 3. (A) Activity of compound 19 against two strains of L. donovani, BPK091 (Sb sensitive, gray) and BPK190 (Sb resistant, black) [23]. Mouse bone marrow-derived macrophages
M. At 72 h p.i., the infected cells
were infected with stationary growth phase promastigotes at Multiplicity Of Infection (MOI) ¼ 10:1. At 24 h p.i., compound 19 was added at 0e10
m
were harvested, total DNA was isolated, and parasite load was determined by qPCR [24]. Activity [%] was defined as reduction of relative parasite load compared to the solvent
5
controls. Bars show the mean of 2 biological replicates. (B) Selection for resistance. L. donovani strain 1SR promastigotes were seeded at 1 ꢂ 10 cells/mL and grown for 3 days at EC50
5
of miltefosine or compound 19 with DMSO solvent controls and in biological duplicates. Cell densities were recorded, and cells were diluted to 1 ꢂ 10 cells/mL in drug-containing
medium. The selection cycles were repeated 10 times for a total of 33 days. Cell growth relative to DMSO controls is given in [%] at days 3, 18 and 33 of selection. Bars show the mean
with SD of 2 technical repeats, from 2 biological samples (#1 and #2) (C) Schematic representation of the functional cloning strategy, with pink and red circles symbolizing selected
cosmids. (D) Cosmid library selection. L. donovani strain BPK91 transfected with a genomic DNA cosmid library of strain BPK190 was selected essentially as described in (A). After 40
days, the selective pressures of miltefosine and compound 19 were increased to EC75. Cell growth relative to DMSO controls (% inhibition) at days 3, 18, 37 (EC50) and 43, 56, 78
(
EC75) of selection. Bars show the mean with SD of 2 technical repeats, from 2 biological samples (#1 and #2). (For interpretation of the references to color in this figure legend, the
reader is referred to the Web version of this article.)
(
1
PO): 46.9 h and a Cmax higher than the EC50 towards L. infantum (ca.
.9 M) (Table 4).
was tested on Syrian hamsters that had been infected with
L. infantum for 15 weeks. Infection with L. infantum did not cause
significant changes of food uptake and behavior, or clinical signs in
the test animals. Lesions were absent or mild and included
conjunctivitis, periorbital and abdominal alopecia. Animals showed
comparable live weight gain (P > 0.05) irrespective of their infec-
tion status (Fig. S2 of the Supporting information).
Treatment with compound 19, free (8% v/v DMSO) or formulated
with CDs (30% or 50%), did not elicit any noticeable adverse effect
along treatment days. In contrast, treatment-using miltefosine
m
3
.7. Clinical course of the infection and toxicity of compound 19 in
hamsters
Efficacy studies were performed in hamster as it is considered to
be the best rodent model for human VL [30] on the basis of pro-
gression of the disease including organs affected, lesions and final
outcome [31]. Cyclodextrins (CDs) have been shown to enhance
permeability and increase activity of several anti-leishmanial drugs
(20 mg/kg/day, 10 days) provoked some adverse effects in 6 out of 8
animals from the group, including distress, itching, dizziness and
nausea (Fig. 4A). These effects were transient and disappeared once
[
32,33]. Compound 19, formulated with or without CDs and
administered PO (40 mg/kg/day; 20 mg/kg/every 12 h) for 10 days,

8
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
Table 4
Pharmacokinetic parameters of compound 19.
Compound
Dose (mg/kg) and route
C
max (ng/mL)
C
max
(
m
M)
T
max (h)
AUCtot (ng/mL h)
AUCtot (nmol/mL h)
Half life (h)
1
1
9
9
1 (IV)
20 (PO)
1,540
630
4.76
1.96
0.08
1.00
5,280
12,500
16.28
38.54
63.2
46.9
the drug administration was completed. Plasma creatinine and urea
values were homogeneous within and among hamster groups. In
contrast, transaminases (ALT and AST), particularly AST, showed
intergroup and intragroup variations and some outliers. Although
most of the values were within the expected physiological range
significant differences (P < 0.01) were found in alkaline phospha-
tase levels, with the highest value being found in CDs 50% treated
hamsters. However, this difference lacks significance since animals
treated with CDs 50% plus compound 19 did not show any differ-
ence (Fig. S3 of the Supporting Information). Under necropsy, no
evident gross pathology lesions were observed in liver and spleen
of most animals. Only 3 out of 47 infected animals showed apparent
congestive hepatopathy, although no spleen or liver enlargement
was found (Table S5 of the Supporting Information).
antibody mean levels at the end-point of the experiment (14
weeks vs 18 weeks sampling, Fig. 4B). Wide intra-group varia-
tions among animals precluded the observation of significant
differences, with the exception for miltefosine (P < 0.05). Due to
the close correlation between leishmanial infection and specific
antibody response, these findings provided indirect evidence of
the anti-leishmanial activity of compound 19. Moreover, the
higher antibody levels in untreated animals suggested that the
reduction in treated hamsters was not due to the vehicle (DMSO
or CDs).
3.9. Anti-leishmanial efficacy of compound 19
Parasite burden (PB) was determined using Leishman-Donovan
units (LDU) by counting the amastigotes found in 500 cells in
spleen and liver (Table S6 of the Supporting information). None of
the uninfected control animals had L. infantum amastigotes at the
time point of euthanasia. Hamsters receiving the vehicle (8% v/v
The ability of 19 to evade resistance mechanisms upon drug
challenge in Leishmania parasites, its efficacy against Sb resistant
strains, its low toxicity and the preliminary PK data reported,
suggest the suitability for in vivo efficacy studies in hamsters.
6
DMSO) were infected in both liver and spleen (liver: 5.3 ± 3.0 ꢂ 10
6
3.8. Serum antibody response against L. infantum in hamsters
amastigotes/g; spleen: 9.1 ± 2.6 ꢂ 10 amastigotes/g). As expected
there was a general correlation between liver and spleen infection,
with higher PB in spleen. These findings confirm the spleen as the
main target for L. infantum in hamster and its value as surrogate
model for human infections [30]. Miltefosine elicited a strong
reduction of PB compared to the untreated infected controls in liver
(81.2 ± 28.3% reduction) and, particularly, in spleen (97.33 ± 3.9%
reduction, Fig. 4C) and four hamsters apparently cleared the
leishmanial infection after miltefosine treatment (#59, #14, #74,
#29). A wide intragroup variation was observed in animals infected
and treated with compound 19 þ CDX. The average PB in the spleen
Serum antibody response against Leishmania can give infor-
mation on the spread of infection and thus it is a convenient tool
to follow up infection and recovery in experimental infections in
hamsters [34,35]. The serum anti-Leishmania IgG response,
determined by ELISA, confirmed the efficacy of the inoculation
since all infected groups displayed a steady increase of specific
IgG. Untreated hamsters (only receiving DMSO or CDs) raised
their antibody levels, whereas animals treated with compound
1
9 or miltefosine showed, after medication, equal or lower
Fig. 4. (A) Score of immediate adverse effects observed after administration of miltefosine (20 mg/kg/day) to hamsters with Leishmania infantum infection undergoing ten days of
treatment. (þ): no adverse effects; (þþ): mild effects; (þþþ): significant effects. (B) Variation (mean ± standard error of the mean) of serum anti-Leishmania IgG levels (Optical
density values of pretreatment sampling e Optical density post treatment) in hamsters infected with L. infantum and treated with vehicles or vehicles þ compound 19, determined
by ELISA. (C) Reduction (%) of Leishmania burden in spleen of infected and treated hamsters. Values are mean ± standard error of the mean.

C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
9
of hamsters treated with compound 19 þ 50% CDs was lower
35.8 ± 28.7% reduction) than that observed in the hamsters treated
with free compound 19 (25.9 ± 24.4% reduction) or with 30% CDs
28.8 ± 30.1% reduction) although differences were not significant
due to the intragroup variations. These results indicate that a
limited leishmanicidal/leishmaniostatic activity of compound 19
can be observed, together with a modest effect of the CDs formu-
lations on compound efficacy.
3.11. Binding of compound 19 to proteins revealed by its
fluorescence
(
(
To set the basis for an understanding of the off-targets binding
profile of compound 19, in addition to the early-tox profile (section
3.4), we evaluated its ability to bind to a set of proteins taking
advantage from the enviromental dependence of its fluorescence
properties [36]. This, and the resulting possibility to discriminate
between the free and protein-bound states of 19 in cellular envi-
ronments, offer a valuable observable, alternative to the use of tags,
for fluorescence-based investigations of the binding of compound
19 with both catalytic and non-catalytic soluble proteins, to be
performed in subsequent stages of the drug discovery path (e.g.,
target and off-target engagement in cells). We described the
changes in fluorescence properties of this compound due to its
interaction with five proteins. When human or bovine serum al-
bumin (HSA and BSA) were added to a freshly prepared solution of
compound 19, changes in the steady-state emission spectrum and
anisotropy and in the lifetime profile were observed (Fig. 5AeD).
While the intensity of the free-compound emission at 450 nm
decreased, a new band with a maximum at 530e535 nm appeared
whose intensity correlated with albumin concentration (see
Figs. S6 and S7 of the Supporting Information for the case of HSA).
3.10. Plasma concentration in treated hamsters and lipophilicity of
compound 19
We investigated if the changes in blood levels of compound 19
could be related to the observed low in vivo efficacy. To this aim,
we determined the plasma levels of compound 19 and miltefosine
in infected and treated hamsters 72 h after the final treatment
(
Table S7 of the Supporting information). The results obtained in
the miltefosine-treated group (59.3 ± 13.7 M) were in agreement
m
with the long half-life of the molecule and the anti-leishmanial
efficacy achieved with the therapeutic regimen employed. On
the other hand, all the groups treated with compound 19 had
plasma concentrations of the drug lower than the estimated EC50
value for L. infantum (1.88 ± 0.32
used (DMSO 8%: 0.044 ± 0.024
0% CDs: 0.038 ± 0.026 M). For ethical reasons, no repeated
m
M) irrespective of the vehicle
At [19] ¼ 5
m
M, the emission intensity at 450 nm was halved at an
m
M; 30% CDs: 0.076 ± 0.125 M;
m
albumin concentration of 0.6 ± 0.1 m
M. The 530 nm emission band,
5
m
characteristic of a stable 19/protein complex, increased and was
blood samplings during treatment were performed. However, the
results suggest that the low efficacy of compound 19 in hamsters
infected with Leishmania is likely due to its low blood level,
possibly related to a too high lipophilicity and a poor intestinal
absorption.
We tackled the problem of the low plasma concentration of
compound 19 in hamsters by investigating whether it might be due
to high lipophilicity and/or chemical instability. The UVevis ab-
saturated at higher albumin concentrations, half of the saturating
intensity being reached at [HSA] ¼ 2
mM and [BSA] ¼ 12
mM. These
figures roughly corresponded to the dissociation constants of
compound 19 versus the two proteins and confirmed their ability to
keep it solubilized. The emission time decay of compound 19 in
aqueous buffer was a single exponential (90e95% abundance) with
a very short lifetime, 0.32 ± 0.05 ns (red in Fig. 5D). The decays of
the 19/protein complexes exhibited profiles with a new prominent
component with lifetimes of 5.1 ± 0.05 ns (60% abundance, HSA)
and 5.5 ± 0.07 ns (50% abundance, BSA) (Fig. 5D). Also, as a clear
result of a decrease in the rotational mobility of the fluorophore due
to its binding to BSA and HSA, the emission anisotropy increased
from 0.06 for the free-molecule to 0.21 ± 0.02 and 0.185 ± 0.02,
respectively. Addition of three other proteins, pepsin, trypsin and
casein, to compound 19 also increased emission in the 530 nm re-
gion (Fig. 5C) and the abundance of the 5.5 ± 0.07 ns lifetime
component (black and green in Fig. 5D). However, at fixed protein
sorption spectrum at 60 mM solution of compound 19 in DMSO,
kept at room temperature in the dark, was reproduced within 1%
one day after solution preparation. In contrast, a relatively rapid
decrease in the absorption was consistently observed in a solution
of compound 19 in PBS (pH 7 and pH 8), with no obvious effect of
the exposure to daylight. For a 60
m
M solution, a 80% decrease in the
ꢀ
maximum absorbance was observed at 25 C in 50 min (Fig. S4 of
the Supporting Information), and 20 min after solution preparation,
the absorbance was reduced by 50%. To test the hypothesis that a
water-assisted oxidation of compound 19 was responsible for its
disappearance, nitrogen was bubbled through the solution imme-
diately after preparation for 2 min to replace the dissolved oxygen
and prevent oxidation. During this process, an immediate fading of
the solution was observed resulting in >90% reduction in the
absorbance. The experiment was repeated by replacing nitrogen
with air, and the same result was obtained. These observations are
consistent with segregation of a large fraction of compound 19 from
the water bulk, where it is spectrophotometrically observable, to
the water/air(nitrogen) interface, where it is no more accessible to
the spectrophotometer beam, a migration mechanically accelerated
by gas bubbling. To support the view that the disappearance of
compound 19 from aqueous solutions is due to segregation of the
compound from bulk water, rather than to chemical instability, we
concentration (10 mM in Fig. 5C), these proteins showed weaker
effects than the two albumins, likely resulting from lower affinities
for compound 19.
In conclusion, among the five proteins assayed, only HSA
showed a K
nificance for specific versus non-specific binding (the latter corre-
sponding to K s higher than 12 M [36]).
d
below 10 mM and is therefore of high biological sig-
d
m
4. Discussion
Treatment and control of leishmaniasis relies on a small number
of available anti-parasitic drugs, most of which are compromised by
a limited activity spectrum, severe side effects and emerging drug
resistance [37e39]. This highlights the need for novel and more
effective drugs with a low risk of resistance. Herein, we report the
design, synthesis and biological evaluation of a novel library of 38
flavonol-like compounds. Compound 19, bearing a benzothio-
phene, was associated with an in vitro anti-parasitic activity against
observed a large recovery of absorption by adding 20 mM human
serum albumin to the nitrogen- or air-bubbled solutions of the
compound (Fig. S5 of the Supporting Information). Albumin offers a
suitable environment to this compound and, at least partially, re-
solubilizes it. Thus, the low levels of compound 19 found in
plasma may result from its lipophilicity and the related tendency to
escape bulk water. Albumin can bind 19 and transport it through
the bloodstream.
L.
infantum
comparable
to
that
of
miltefosine
mM) and an
(EC50 ¼ 1.88 ± 0.32
mM; EC50 miltefosine ¼ 2.65 ± 0.4
improved SI (19 SI ¼ 17, miltefosine SI ¼ 5). Compound 19 was
screened against L. major and L. donovani and was shown to be

10
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
cytochrome isoform (CYP1A2 EC50 ¼ 0.4
mM) and being slightly
toxic for A549. However, the A549 appear to be very sensitive to
antioxidants such as 19 and this might be the reason of its higher
toxicity with respect to miltefosine. The toxicity against THP1
mammalian cells is lower than that of miltefosine. The main ad-
vantages of compound 19 over miltefosine are the higher synthetic
tractability and its inability to induce spontaneous resistance. The
flavonoid scaffold allows a rapid optimization process and the
synthesis of a wide range of derivatives at a lower cost with respect
to miltefosine (drug cost is a pivotal parameter for NTDs that affect
resource-poor settings). Moreover, 19 has a broad spectrum of ac-
tivity on trypanosomatidic parasites, being more active than mil-
tefosine on T. brucei (EC50 ¼ 0.8 and 34
mM, respectively).
A Cos-Seq [28] functional cloning approach [41] yielded pop-
ulations with a tolerance against the IC50 of 19, but not against
higher concentrations. The resistance against 19 is not as easily
attained as against compounds such as miltefosine or antimony-
based drugs [26,42].
Pharmacokinetic studies indicated that compound 19 has a long
half-life (IV: 63.2 h; PO: 46.9 h) and allowed to proceed 19 to in vivo
studies. The efficacy trial in a hamster chronic model of infection
showed a reduction of PB in the spleen, the main target organ. This
occurred in spite of a low concentration of compound 19 in plasma,
likely related with low hydrosolubility. CDs formulation did not
significantly improve this feature and, given the absence of toxicity
of this compound, other formulations will have to be evaluated.
Finally, the environmental sensitivity of the fluorescence of com-
pound 19 results in large changes in both steady-state and time-
dependent properties following 19 binding to five test proteins.
This observation can be exploited in future investigations of 19
intracellular targets and off-targets, a strong asset in the rational
design of optimized derivatives, and in the understanding of its
mode of action in Leishmania parasites.
Fig. 5. Fluorescence properties of compound 19 in aqueous solution at pH 8: (A) ab-
sorption, emission, excitation (dashed,
em ¼ 450 nm) and emission anisotropy (red)
spectra; (B) emission spectra of 5 M 19 with 0, 0.5, 1, 2, 3, 4, 5, 7 and 10 M HSA and
emission anisotropy (red) of the final solution; (C) emission spectra of 5 M 19 alone
black curve with maximum at 455 nm) and with 10 M BSA (black curve with
l
m
m
m
(
m
In conclusion, compound 19 offers the potential to tackle drug
resistance in Leishmania, showing a broad activity spectrum against
Trypanosomatidic infections in vitro and not leading to sponta-
neous resistance as observed for miltefosine and antimonials drugs.
maximum at 533 nm), pepsin (blue), trypsin (green) and casein (red); (D) lifetime
distributions measured at the emission wavelengths of 450 nm for 19 alone (red), and
530 nm for 5
mM 19 with 10
mM HSA (blue) and BSA (magenta), with 130 mM pepsin
(
black) and with 80
m
M casein (green). The relative abundance of a component with a
given lifetime represents the fraction of all emitted photons corresponding to that
component; the 0.4 ns component in red ha a relative abundance of 0.93. For all
5. Material and methods
steady-state emission and anisotropy measurements,
resolved measurements,
in this figure legend, the reader is referred to the Web version of this article.)
l
exc ¼ 360 nm; for the time-
l
exc ¼ 340 nm. (For interpretation of the references to color
5.1. General experimental procedures
All commercial chemicals and solvents were reagent grade and
more potent than miltefosine towards both L. donovani (EC50
were used without further purification. TLC monitored reaction
1
9 ¼ 4.6
compound 19 ¼ 60%,
9 ¼ 5 M). Compound 19 was active also towards T. brucei
EC50 ¼ 0.8 M; EC50 pentamidine ¼ 1.5 nM) and, in a panel of
m
M, miltefosine ¼ 10.8
m
M) and L. major (% efficacy at 5
m
M:
progress on pre-coated silica gel 60 F254 plates (Merck), and
1
13
miltefosine ¼ 20%,
EC50
compound
visualization was accomplished with UV light (254 nm). H and
NMR spectra were recorded on a Bruker FT-NMR AVANCE 400.
Chemical shifts are reported as values (ppm) referenced to re-
sidual solvent (CHCl at 7.26 ppm, DMSO at 2.50 ppm, MeOD at
3.31 ppm); J values are given in Hz. When peak multiplicities are
C
1
(
m
m
d
T. cruzi strains (Silvio X10/1, Y, ARMA cl1, 92.80 cl 2, CL Brener), it
showed potency comparable to that of benznidazole, a drug
currently used for the treatment of Chagas’ disease. A panel of
in vitro early ADME-Toxicity properties was assessed for this series
of compounds and 19 yielded a satisfactory profile. The properties
of compound 19 and miltefosine, a marketed drug, were compared.
The specific assays included hERG, cytochrome P450 (1A2, 2C9,
3
d
d
d
given, the following abbreviations are used: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br, broadened signal. Silica gel
Merck (60e230 mesh) was used for column chromatography. Mass
spectra were obtained on a 6520 Accurate-Mass QTOF LC/MS and
6310A Ion Trap LC-MS(n).
2
C19, 2D6 and 3A4 isoforms), cell viability (A549 and WI38 cell-
lines) and mitochondrial toxicity. The early ADME-Toxicity evalu-
ation has a significant value in compound characterization since it
points out in vitro liabilities before moving to in vivo studies. The
different cut-off values were selected on the basis of accepted and
recognized profiles for the approved anti-leishmanial compounds.
According to the target product profile (TPP) description [40] (Fig. 6
and Table S8 of the Supporting Information), the profiles of the two
compounds, 19 and miltefosine, are comparable. Compound 19 has
a profile suitable for selection and prioritization, inhibiting only one
5.2. General procedures for synthesis of compounds
5.2.1. General procedure for the synthesis of biaryl aldehydes (59
and 60)
To a solution of 5.49 mmol of the aryl halide in 20 mL of water/
ethanol (1:1), 5.49 mmol of phenyl boronic acid, 13.73 mmol of
K CO and 293 mg of Palladium on carbon (5 mol%) were added.
2 3
ꢀ
The mixture was stirred for 5 h at 60 C. Then the reaction mixture
was filtered through Celite and concentrated in vacuum. The

C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
11
Fig. 6. Target product profile (TPP) of compound 19 (red line) and miltefosine (MIL) (blue line). For parameters requiring a yes/no answer, the cut-off was set at 50% of the y-axes.
For parameters requiring a numeric value, the marker line between proper and improper behavior was set to the corresponding cut-off value described in the main text. Pale green
color covers the area for which the compound parameters agree with the desired values, whereas pale red color covers the area with an improper profile. Red dots represent data
relative to compound 19, blue dots are related to MIL. Further details are reported in Table S8 of the Supporting Information. (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article.)
mixture was extracted with ethyl acetate, the organic layer was
dried over anhydrous sodium sulphate and evaporated under
reduced pressure. The crude product was purified by column
chromatography.
extracted with dichloromethane and washed with water and brine.
The organic layer was dried over Na SO and concentrated under
reduced pressure. Column chromatography was then utilized to
purify the desired product.
2
4
5
.2.2. General procedure for the synthesis of chalcones (39e58)
An aqueous solution of NaOH (3 M, 1.6 mL) was added to a so-
5.2.4. General procedure for the synthesis of demethylated flavonol-
like compounds (3, 4, 7, 10, 12, 15 and 21)
lution of aromatic ketone (1 mmol) and 3-methoxybenzaldehyde
1.2 eq), in EtOH (1e2 mL). The reaction was stirred at r.t for
8e24 h. The reaction mixture was cooled in an ice-water bath and
acidified to pH 2 with concentrated HCl (37%). The solid formed was
filtered, washed with ethanol and then further purified by recrys-
tallization from ethanol. When no precipitate occurred, the reac-
tion mixture was extracted with dichloromethane and washed
To a stirring solution of 14 (0.083 g, 0.205 mmol) in anhydrous
dichloromethane (5 mL) under nitrogen at 0 C, boron tribromide in
ꢀ
(
1
dichloromethane (1.0 M, 1.85 mL, 1.847 mmol, 9 eq) was added. The
mixture was allowed to warm at r.t. and was stirred for 2 days. The
ꢀ
reaction mixture was then cooled to 0 C and methanol (10 mL) was
added. The reaction mixture was concentrated in vacuo. Water
(10 mL) was added, the reaction sonicated and left to stand. The
solid was filtered to give the pure product 15.
2 4
with water and brine. The organic layer was dried over Na SO and
concentrated under reduced pressure. Column chromatography
was then utilized to purify the desired product.
(BBr
3
being widely used for the demethylation of methyl aryl
are used for each CeO bond cleavage).
ethers; 3 eq. of BBr
3
5
(
.2.5. General procedure for the synthesis of esters and carbamates
33e36)
To a suspension of 19 in dry DCM, triethylamine and an excess of
5
.2.3. General procedure for the synthesis of flavonol-like
compounds (1, 2, 5, 6, 8, 9, 11, 13, 14, 16e20 and 22e32)
An aqueous solution of H (30%, 250 L) was added to an ice-
2
O
2
m
acyl chloride was added. The mixture was stirred until the starting
material was totally consumed (1e4 h). After basic workup, the
crude was purified by column chromatography to obtain the
desired product.
cold suspension of the chalcone (1 mmol) in ethanol (5 mL) and 1 M
NaOH (2 mL). The mixture was allowed to warm at r.t. and was
stirred overnight. Then the reaction mixture was cooled in an ice
bath and distilled water (2e4 mL) was added. Concentrated HCl
(
37%) was added until pH 2. The solid formed was filtered, washed
with ethanol and then further purified by recrystallization from
5.2.6. General procedure for the synthesis of ethers (37 and 38)
ethanol. When no precipitate occurred, the reaction mixture was
2 3
To a solution of 19 in dry DMF placed in a MW tube, K CO (2.5

12
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
eq) and iodoethane/iodopropane (1.5 eq) was added. The mixture
was irradiated with microwaves at 80 C until the starting material
from compound 41. Compound 5 was isolated as a yellow solid in
ꢀ
ꢀ
1
73% yield. mp: 200 C. H NMR (CDCl , 400 MHz) d: 7.82 (d,
3
was consumed. The mixture was subsequently filtered and
concentrated and column chromatography was used to isolate the
pure compound.
J ¼ 1.1 Hz, 1H), 7.55 (d, J ¼ 3.1 Hz, 1H), 7.48 (d, J ¼ 9.2, 1H), 7.28 (dd,
J ¼ 9.2, 3.1 Hz, 1H), 7.20 (m, 1H), 3.92 (s, 3H), 2.36 (d, J ¼ 0.6 Hz, 3H).
13
3
C NMR (CDCl , 100 MHz) d: 172.06, 156.48, 149.98, 142.76, 138.85,
1
36.00, 132.58, 131.52, 125.58, 124.00, 121.51, 119.50, 104.00, 55.93,
þ
5
5
.3. Synthesis of compounds
15.70. ESI-HRMS calcd. for C15
89.0529. Anal. calcd. for C15
Found C, 62.43; H, 4.31; S, 11.09.
H
13
O
O
4
S [MþH] 289.0529, found
2
H
12
4
S: C, 62.49; H, 4.20; S, 11.12;
.3.1. 2-(furan-2-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one (1)
Compound 1 was prepared according to the general method for
the synthesis of flavonol-like compounds reported above starting
from compound 39. Compound 1 was isolated as a yellow solid in
5.3.6. 3-hydroxy-7-methoxy-2-(4-methylthiophen-2-yl)-4H-
chromen-4-one (6)
ꢀ
1
8
8
1
1% yield. mp: 197 C. H NMR (CDCl
3
, 400 MHz)
d
: 9.94 (br s, 1H),
Compound 6 was prepared according to the general method for
the synthesis of flavonol-like compounds reported above starting
from compound 42. Compound 6 was isolated as a brown solid in
.03 (dd, J ¼ 1.7, 0.7 Hz, 1H), 7.66 (d, J ¼ 9.2, 1H), 7,45 (d, J ¼ 3.1 Hz,
H), 7.38 (dd, J ¼ 9.1, 3.1 Hz, 1H), 7.28 (dd, J ¼ 0.7, 3.5 Hz, 1H), 6.79
13
ꢀ
1
(
dd, J ¼ 3.5, 1.7 Hz, 1H), 3.88 (s, 3H). C NMR (CDCl
3
, 100 MHz)
d:
29% yield. mp: 189 C. H NMR (DMSO, 400 MHz)
d: 10.02 (br s, 1H),
171.89, 156.49, 149.42, 145.62, 144.61, 139.66, 137.25, 123.71, 122.92,
7.97 (d, J ¼ 8.1 Hz, 1H), 7.75 (m, 1H), 7.46 (s, 1H), 7.19 (m, 1H), 7.02 (d,
13
1
20.28, 115.63, 113.26, 104.56, 56.19. ESI-HRMS calcd. for C14
H
11
O
: C,
5
J ¼ 8.1 Hz, 1H), 3.92 (s, 3H), 2.31 (s, 3H). C NMR (DMSO, 100 MHz)
d: 171.89, 164.03, 156.47, 143.07, 138.15, 136.86, 132.62, 130.28,
þ
[MþH] 258.0601, found 259.0611. Anal. calcd. for C14
H
10
O
5
6
5.12; H, 3.90; Found C, 65.34; H, 3.88.
126.59, 126.46, 116.05, 114.87, 100.59, 56.52. ESI-HRMS calcd. for
þ
C
C
15
H
H
13
O
O
4
S [MþH] 289.0529, found 289.0529. Anal. calcd. for
5.3.2. 2-(furan-2-yl)-3-hydroxy-7-methoxy-4H-chromen-4-one (2)
15
12
4
S: C, 62.49; H, 4.20; S, 11.12; Found C, 62.79; H, 4.18; S,
Compound 2 was prepared according to the general method for
10.92.
the synthesis of flavonol-like compounds reported above starting
from compound 40. Compound 2 was isolated as a yellow solid in
5.3.7. 3,6-dihydroxy-2-(4-methylthiophen-2-yl)-4H-chromen-4-
one (7)
ꢀ
1
2
8
3
3% yield. mp: 198 C. H NMR (CDCl , 400 MHz) d: 9.87 (br s, 1H),
3
.01 (dd, J ¼ 1.8, 0.7 Hz, 1H), 8.00 (d, J ¼ 8.9, 1H), 7,23 (dd, J ¼ 0.7,
.5 Hz, 1H), 7.19 (d, J ¼ 2.3 Hz, 1H), 7.05 (dd, J ¼ 8.9, 2.3 Hz, 1H), 6.79
Compound 7 was prepared according to the general method for
the synthesis of demethylated flavonols reported above starting
from compound 5. Compound 7 was isolated as a yellow solid in
13
(
dd, J ¼ 3.5, 1.8 Hz, 1H), 3.92 (s, 3H). C NMR (CDCl
3
, 100 MHz) d:
ꢀ
1
171.78, 164.08, 156.46, 145.34, 144.62, 139.27, 137.35, 126.66, 116.13,
91% yield. mp: 237 C. H NMR (DMSO, 400 MHz) d: 9.97 (br s, 1H),
1
15.05 (2C), 113.22, 100.70, 56.58. ESI-HRMS calcd. for C14
H
11
O
5
9.93 (br s, 1H), 7.76 (d, J ¼ 1.2 Hz, 1H), 7.56 (d, J ¼ 9.0 Hz, 1H), 7.47
(m, 1H), 7.35 (d, J ¼ 3.0 Hz, 1H), 7.23 (dd, J ¼ 9.0, 3.0 Hz, 1H), 2.30 (s,
þ
[MþH] 258.0601, found 259.0606. Anal. calcd. for C14
10 5
H O : C,
13
6
5.12; H, 3.90; Found C, 65.17; H, 3.94.
3H). C NMR (DMSO, 100 MHz) d: 172.10, 154.58, 148.49, 143.37,
1
38.23, 136.59, 132.74, 130.61, 126.67, 123.41, 123.02, 119.84, 107.48,
þ
5.3.3. 2-(furan-2-yl)-3,6-dihydroxy-4H-chromen-4-one (3)
15.75. ESI-HRMS calcd. for C14
275.0368. Anal. calcd. for C14
H
11
O
4
S [MþH] 275.0373, found
Compound 3 was prepared according to the general method for
10 4
H O S: C, 61.31; H, 3.67; S, 11.69;
the synthesis of demethylated flavonols reported above starting
from compound 1. Compound 3 was isolated as a yellow-brown
solid in quantitative yield. mp 305 C dec. H NMR (DMSO,
Found C, 61.30; H, 3.67; S, 11.49.
ꢀ
1
5.3.8. 3-hydroxy-6-methyl-2-(4-methylthiophen-2-yl)-4H-
chromen-4-one (8)
4
00 MHz)
d
: 9.89 (br s, 1H), 9.81 (br s, 1H), 8.02 (dd, J ¼ 1.8, 0.8 Hz,
1
H), 7.57 (d, J ¼ 9.1, 0.3 Hz,1H), 7.36 (dd, J ¼ 3.0, 0.3 Hz,1H), 7.26 (dd,
Compound 8 was prepared according to the general method for
the synthesis of flavonol-like compounds reported above starting
from compound 43. Compound 8 was isolated as a yellow solid in
J ¼ 3.5, 0.8 Hz, 1H), 7.23 (dd, J ¼ 9.1, 3.0 Hz, 1H), 6.78 (dd, J ¼ 3.5,
.8 Hz, 1H). 13C NMR (DMSO, 100 MHz)
: 171.97, 154.68, 148.46,
45.52, 144.71, 139.55, 137.01, 123.49, 123.13, 119.99, 115.48, 113.22,
1
1
d
ꢀ
1
56% yield. mp: 222 C. H NMR (400 MHz, DMSO)
d: 10.10 (s, 1H),
þ
1
2
6
07.45. ESI-HRMS calcd. for C13
45.0446. Anal. calcd. for C13
3.69; H, 3.30.
H
9
O
5
[MþH] 245.0444, found
7.88-7.86 (m, 1H), 7.76 (d, J ¼ 1.3 Hz, 1H), 7.58 (d, J ¼ 1.3 Hz, 2H),
13
H
8
O
5
: C, 63.94; H, 3.30; Found C, C,
7.48-7.46 (m, 1H), 2.43 (d, J ¼ 0.5 Hz, 3H), 2.31 (s, 3H). C NMR
(100 MHz, DMSO) : 172.30, 152.89, 143.45, 138.24, 137.12, 135.23,
d
1
34.46, 132.58, 130.69, 126.83, 124.37, 121.92, 118.31, 20.86, 15.74.
þ
5.3.4. 2-(furan-2-yl)-3,7-dihydroxy-4H-chromen-4-one (4)
ESI-HRMS calcd. for C15
Anal. calcd. for C15
H, 4.42; S, 11.70.
H
13
O
3
S [MþH] 273.0580, found 273.0583.
Compound 4 was prepared according to the general method for
the synthesis of demethylated flavonols reported above starting
from compound 2. Compound 4 was isolated as a gray solid in
12 3
H O S: C, 66.16; H, 4.44; S,11.77; Found C, 65.98;
ꢀ
1
quantitative yield. mp: 285 C. H NMR (DMSO, 400 MHz)
br s, 1H), 9.74 (br s, 1H), 8.00 (dd, J ¼ 1.8, 0.8 Hz, 1H), 7.95 (d,
J ¼ 8.7 Hz, 1H), 7.21 (dd, J ¼ 3.5, 0.8 Hz, 1H), 6.92 (dd, J ¼ 8.7, 2.2 Hz,
d
: 10.93
5.3.9. 3-hydroxy-5-methoxy-2-(4-methylthiophen-2-yl)-4H-
chromen-4-one (9)
(
Compound 9 was prepared according to the general method for
the synthesis of flavonol-like compounds reported above starting
from compound 44. Compound 9 was isolated as a yellow solid in
1
3
1
H), 6.89 (d, J ¼ 2.2 Hz, 1H), 6.77 (dd, J ¼ 3.5, 1.8 Hz, 1H). C NMR
(
DMSO, 100 MHz) : 171.79, 162.92, 156.47, 145.24, 144.70, 138.91,
d
ꢀ
1
1
37.09, 127.06, 115.28, 115.19, 114.85, 113.15, 102, 37. ESI-HRMS
25% yield. mp: 170e171 C. H NMR (DMSO, 400 MHz) d: 9.71 (br s,
þ
calcd. for C13
H
9
O
5
[MþH] 245.0444, found 245.0448. Anal. calcd.
1H), 7.71 (d, J ¼ 1.4 Hz, 1H), 7.66 (t, J ¼ 8.4 Hz, 1H), 7.45 (m, 1H), 7.19
(dd, J ¼ 8.4, 0.7 Hz, 1H), 7.95 (d, J ¼ 8.4 Hz, 1H), 3.94 (s, 3H), 2.30 (s,
8 5
for C13H O : C, 63.94; H, 3.30; Found C, C, 63.89; H, 3.33.
13
3
H). C NMR (DMSO, 100 MHz) d: 171.74, 159.67, 156.59, 140.71,
5
.3.5. 3-hydroxy-6-methoxy-2-(4-methylthiophen-2-yl)-4H-
138.23, 137.36, 134.37, 132.40, 130.16, 126.25, 112.59, 110.20, 106.44,
þ
chromen-4-one (5)
Compound 5 was prepared according to the general method for
the synthesis of flavonol-like compounds reported above starting
56.69, 15.76. ESI-HRMS calcd. for C15
found 289.0532. Anal. calcd. for C15
11.12; Found C, 62.71; H, 4.24; S, 11.17.
H
13
O
4
S [MþH] 289.0529,
12 4
H O S: C, 62.49; H, 4.20; S,

C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
13
5
.3.10. 3,5-dihydroxy-2-(4-methylthiophen-2-yl)-4H-chromen-4-
one (10)
Compound 10 was prepared according to the general method for
8.42 (s, 1H), 8.14 (d, J ¼ 8.0 Hz, 1H), 7.79 (d, J ¼ 9.1 Hz, 1H), 7.79 (m,
1H), 7.63 (t, J ¼ 8.0 Hz, 1H), 7.47 (d, J ¼ 3.0 Hz, 1H), 7.43 (dd, J ¼ 9.1,
3.0 Hz, 1H), 7.28 (s, 1H), 7.25 (dd, J ¼ 8.3, 1.9 Hz, 1H), 7.09 (d,
13
the synthesis of demethylated flavonols reported above starting
from compound 9. Compound 10 was isolated as a green solid in
J ¼ 8.3 Hz, 1H), 3.90 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H). C NMR
(DMSO, 100 MHz) d: 173.00, 156.47, 150.12, 149.63, 149.28, 145.69,
140.98,139.23,133.07,132.40,129.47,128.45,126. 60,126.12,124.06,
ꢀ
1
7
9% yield. mp: 180e181 C. H NMR (DMSO, 400 MHz)
s, 1H), 10.44 (br s, 1H), 7.81 (d, J ¼ 1.3 Hz, 1H), 7.62 (t, J ¼ 8.3 Hz, 1H),
.54 (m, 1H), 7.11 (dd, J ¼ 8.3, 0.6 Hz, 1H), 6.77 (dd, J ¼ 8.3, 0.6 Hz,
H), 2.31 (s, 3H). 13C NMR (DMSO, 100 MHz)
: 176.38, 159.40,
54.28, 144.85, 137.93, 135.24, 135.22, 131.40, 130.99, 127.47, 109.53,
d: 12.36 (br
122.39, 120.74, 119.57, 112.78, 111.17, 104.30, 56.21, 56.11, 56.12. ESI-
þ
7
1
1
HRMS calcd. for C24
H
21
O
6
[MþH] 405.1333, found 405.1339. Anal.
d
calcd. for C24H
20
O
6
: C, 71.28; H, 4.98; Found C, 71.35; H, 4.86.
þ
0
0
0
109.43, 107.25, 15.20. ESI-HRMS calcd. for C14
H
11
O
4
S [MþH]
5.3.15. 2-(3 ,4 -dihydroxy-[1,1 -biphenyl]-3-yl)-3,6-dihydroxy-4H-
chromen-4-one (15)
2
3
75.0373, found 289.0375. Anal. calcd. for C14
.67; S, 11.69; Found C, 61.01; H, 3.56; S, 11.41.
H O S: C, 61.31; H,
10 4
Compound 15 was prepared according to the general method for
the synthesis of demethylated flavonols reported above starting
from compound 14. Compound 15 was isolated as a brown solid in
5.3.11. 3-hydroxy-7-methoxy-2-(3-methylthiophen-2-yl)-4H-
ꢀ
1
chromen-4-one (11)
84% yield. mp: 302e303 C dec. H NMR (DMSO, 400 MHz) d: 9.97
Compound 11 was prepared according to the general method for
the synthesis of flavonol-like compounds reported above starting
from compound 45. Compound 11 was isolated as a brown solid in
(br s, 1H), 9.43 (br s, 1H), 9.11 (br s, 2H), 8.33 (s, 1H), 8.07 (d,
J ¼ 7.9 Hz, 1H), 7.67 (d, J ¼ 9.1 Hz, 1H), 7.64 (d, J ¼ 7.9 Hz, 1H), 7.58 (t,
J ¼ 7.7 Hz, 1H), 7.38 (d, J ¼ 3.0 Hz, 1H), 7.27 (dd, J ¼ 3.0, 9.1 Hz, 1H),
7.09 (d, J ¼ 2.2 Hz, 1H), 7.00 (dd, J ¼ 8.2, 2.2 Hz, 1H), 7.86 (d,
ꢀ
1
2
5% yield. mp: 177e178 C. H NMR (DMSO, 400 MHz)
d: 9.69 (br s,
13
1
H), 7.99 (d, J ¼ 8.8 Hz, 1H), 7.75 (d, J ¼ 4.9 Hz, 1H), 7.11 (m, 1H),
J ¼ 8.2 Hz, 1H). C NMR (DMSO, 100 MHz)
d: 173.00, 154.61, 149.52,
13
7.06-7.03 (m, 2H), 3.91 (s, 3H), 2.54 (s, 3H). C NMR (DMSO,
149.12, 146.16, 145.96, 145.55, 145.39, 144.35, 141.18, 138.92, 132.43,
1
00 MHz) : 171.99, 164.01, 156.66, 144.10, 139.70, 137.67, 131.66,
d
131.60, 129.44, 127.89, 125.99, 125.69, 123.77, 122.58, 120.41, 118.31,
þ
129.43, 126.62, 125.98, 116.05, 114.98, 100.49, 56.55, 17.03. ESI-
116.57, 114.54, 107.43. ESI-HRMS calcd. for C21
15
H O
6
[MþH]
þ
HRMS calcd. for C15
Anal. calcd. for C15
H, 4.11; S, 11.05.
H
13
O
4
S [MþH] 289.0529, found 289.0525.
363.0863, found 363.0865. Anal. calcd. for C21
3.89; Found C, 69.35; H, 4.01.
14 6
H O : C, 69.61; H,
12 4
H O S: C, 62.49; H, 4.20; S,11.12; Found C, 62.53;
5.3.16. 2-(3-(benzo[d] [1,3]dioxol-5-yl)phenyl)-3-hydroxy-6-
5.3.12. 3,7-dihydroxy-2-(3-methylthiophen-2-yl)-4H-chromen-4-
methoxy-4H-chromen-4-one (16)
one (12)
The intermediate chalcone was synthesized following the gen-
eral method for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-
phenylprop-2-en-1-ones and was used, without further purifica-
tion, for the synthesis of compound 16 according to the general
method for the synthesis of flavonol-like compounds reported
above. Compound 16 was isolated as a brown solid in 38% yield.
Compound 12 was prepared according to the general method
for the synthesis of demethylated flavonols reported above starting
from compound 11. Compound 12 was isolated as a yellow solid in
ꢀ
1
9
2% yield. mp: 278 C dec. H NMR (DMSO, 400 MHz)
d: 10.74 (br s,
1
H), 9.58 (br s, 1H), 7.94 (d, J ¼ 8.8 Hz, 1H), 7.74 (d, J ¼ 5.0 Hz, 1H),
ꢀ
1
7
.05 (d, J ¼ 5.0 Hz, 1H), 6.92 (dd, J ¼ 8.8, 2.2 Hz, 1H), 6.86 (d,
mp: 226 C. H NMR (400 MHz, DMSO) d: 9.60 (s, 1H), 8.39 (s, 1H),
1
3
J ¼ 2.2 Hz, 1H), 2.51 (s, 1H). C NMR (DMSO, 100 MHz)
d
: 172.00,
8.16 (d, J ¼ 7.9 Hz, 1H), 7.81 (d, J ¼ 9.1 Hz, 1H), 7.73 (d, J ¼ 7.8 Hz, 1H),
7.62 (t, J ¼ 7.8 Hz,1H), 7.47 (d, J ¼ 3.0 Hz, 1H), 7.43 (dd, J ¼ 9.1, 3.0 Hz,
1H), 7.31 (d, J ¼ 1.3 Hz,1H), 7.22 (d, J ¼ 8.0 Hz,1H), 7.06 (d, J ¼ 8.0 Hz,
162.83, 156.66, 143.69, 139.45, 137.39, 131.65, 129.30, 127.01, 126.12,
þ
1
2
3
15.27, 115.10, 102.21, 16.91. ESI-HRMS calcd. for C14
75.0373, found 289.0375. Anal. calcd. for C14
.67; S, 11.69; Found C, 60.99; H, 4.00; S, 11.23.
H
11
O
4
S [MþH]
13
H
10
O
4
S: C, 61.31; H,
1H), 6.10 (s, 2H), 3.90 (s, 3H). C NMR (100 MHz, DMSO) d: 173.05,
156.45, 150.10, 148.53, 147.60, 145.50, 140.70, 139.25, 134.54, 132.42,
29.53, 128.44, 126.74, 126.11, 124.04, 122.35,120.98, 120.75, 109.23,
1
þ
5.3.13. 3-hydroxy-6-methyl-2-(5-methylthiophen-2-yl)-4H-
107.75, 104.27, 101.71, 56.21. ESI-HRMS calcd. for C23
389.1020, found 389.1026. Anal. calcd. for C23
4.15; Found C, 71.06; H, 4.17.
H
17
O
6
[MþH]
chromen-4-one (13)
H O : C, C, 71.13; H,
16 6
Compound 13 was prepared according to the general method for
the synthesis of flavonol-like compounds reported above starting
from compound 46. Compound 13 was isolated as a yellow solid in
5.3.17. 2-(benzo[d] [1,3]dioxol-5-yl)-3-hydroxy-7-methoxy-4H-
chromen-4-one (17)
ꢀ
1
6
0% yield. mp: 185e186 C. H NMR (400 MHz, DMSO)
d: 9.79 (s,
1
H), 7.91-7.87 (m,1H), 7.77 (d, J ¼ 5.0 Hz,1H), 7.62-7.56 (m, 2H), 7.07
Compound 17 was prepared according to the general method for
the synthesis of flavonol-like compounds reported above starting
from compound 47. Compound 17 was isolated as a yellow solid in
13
(
d, J ¼ 5.0 Hz, 1H), 2.55 (s, 3H), 2.44 (s, 3H). C NMR (100 MHz,
DMSO) : 172.40, 153.06, 144.73, 139.98, 137.82, 135.20, 134.54,
d
ꢀ
1
131.75, 129.80, 125.92, 124.32, 121.89, 118.35, 20.89, 17.08. ESI-
52% yield. mp: 211 C. H NMR (400 MHz, DMSO) d: 9.39 (br s, 1H),
þ
HRMS calcd. for C15
Anal. calcd. for C15
H, 4.31; S, 11.80.
H
13
O
3
S [MþH] 273.0580, found 273.0585.
7.98 (d, J ¼ 8.9 Hz, 1H), 7.84 (dd, J ¼ 8.4, 1.7 Hz, 1H), 7.77 (d,
J ¼ 1.7 Hz, 1H), 7.32 (d, J ¼ 2.4 Hz, 1H), 7.12 (d, J ¼ 8.4 Hz, 1H), 7.04
H
12
O
3
S: C, 66.16; H, 4.44; S,11.77; Found C, 66.08;
13
(dd, J ¼ 8.9, 2.4 Hz, 1H), 6.14 (s, 2H), 3.92 (s, 3H). C NMR (100 MHz,
DMSO) d: 172.58, 164.04, 156.79, 148.80, 148.00, 144.87, 138.44,
0
0
0
5.3.14. 2-(3 ,4 -dimethoxy-[1,1 -biphenyl]-3-yl)-3-hydroxy-6-
126.51, 125.65, 122.90, 115.56, 115.03, 108.89, 107.75, 102.10, 100.82,
þ
methoxy-4H-chromen-4-one (14)
56.54. ESI-HRMS calcd. for C17
313.0704. Anal. calcd. for C17
65.51; H, 3.75.
H
13
O
6
[MþH] 313.0707, found
The intermediate chalcone was synthesized following the gen-
eral method for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-
phenylprop-2-en-1-ones and was used, without further purifica-
tion, for the synthesis of compound 14 according to the general
method for the synthesis of flavonol-like compounds reported
above. Compound 14 was isolated as a yellow solid in quantitative
12 6
H O : C, 65.39; H, 3.87; Found C,
5.3.18. 3-hydroxy-6-methoxy-2-(quinolin-6-yl)-4H-chromen-4-
one (18)
The intermediate chalcone was synthesized following the gen-
eral method for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-
ꢀ
1
yield. mp: 182e183 C. H NMR (DMSO, 400 MHz) d: 9.63 (br s, 1H),

14
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
phenylprop-2-en-1-ones and was used, without further purifica-
tion, for the synthesis of compound 18 according to the general
method for the synthesis of flavonol-like compounds reported
above. Compound 18 was isolated as a yellow solid in 64% yield. mp
phenylprop-2-en-1-ones and was used, without further purifica-
tion, for the synthesis of compound 22 according to the general
method for the synthesis of flavonol-like compounds reported
above. Compound 22 was isolated as a yellow solid in 60% yield.
ꢀ
1
ꢀ
1
2
80e281 C. H NMR (400 MHz, DMSO)
d: 10.03 (s, 1H), 9.16 (dd,
mp: 290e293 C. H NMR (400 MHz, DMSO) d: 9.41 (br s, 1H), 8.43
J ¼ 4.7, 1.5 Hz, 1H), 9.00 (d, J ¼ 1.8 Hz, 1H), 8.91 (d, J ¼ 8.3 Hz, 1H),
(dd, J ¼ 7.2, 0.9 Hz, 1H), 8.35 (s, 1H), 7.87 (d, J ¼ 9.1 Hz, 1H), 7.59 (d,
J ¼ 7.7 Hz, 1H), 7.48 (d, J ¼ 3.1 Hz, 1H), 7.37 (dd, J ¼ 9.1, 3.1 Hz, 1H),
7.35 (m, 1H), 7.31 (m, 1H), 7.28 (dd, J ¼ 7.2, 1.1 Hz, 1H), 3.94 (s, 3H),
8
4
.73 (dd, J ¼ 9.1, 1.9 Hz, 1H), 8.38 (d, J ¼ 9.1 Hz, 1H), 7.87 (dd, J ¼ 8.3,
13
.7 Hz, 1H), 7.82-7.77 (m, 1H), 7.49-7.44 (m, 2H), 3.90 (s, 3H).
: 173.17, 156.52, 150.14, 148.75, 143.76,
43.22, 142.32, 140.20, 131.57, 131.09, 128.61, 128.47, 124.95, 124.45,
C
13
NMR (100 MHz, DMSO)
d
3.89 (s, 3H). C NMR (100 MHz, DMSO) d: 170.54, 156.36, 149.28,
1
148.13, 137.13, 136.15, 134.40, 125.44, 123.07, 123.01, 122.53, 122.35,
1
C
C
23.07, 122.36, 120.63, 104.29, 56.24. ESI-HRMS calcd. for
121.70, 120.08, 111.06, 105.87, 104.64, 56.17, 33.61. ESI-HRMS calcd.
þ
þ
19
H
14NO
13NO
4
[MþH] 320.0917, found 320.0910. Anal. calcd. for
for C19
H
16NO
4
[MþH] 322.1074, found 322.1072. Anal. calcd. for
19
H
4
: C, 71.47; H, 4.10; N, 4.39; Found C, 71.30; H, 4.16; N, 4.37.
C
4
19
H15NO
4
: C, 71.02; H, 4.71; N, 4.36; Found C, 71.13; H, 4.70; N,
.19.
5.3.19. 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methoxy-4H-
chromen-4-one (19)
The intermediate chalcone was synthesized following the gen-
eral method for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-
phenylprop-2-en-1-ones and was used, without further purifica-
tion, for the synthesis of compound 19 according to the general
method for the synthesis of flavonol-like compounds reported
above. Compound 19 was isolated as a yellow solid in 40% yield.
5.3.23. 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methyl-4H-
chromen-4-one (23)
Compound 23 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 49. Compound 23 was isolated as a pale
ꢀ
1
yellow solid in 41% yield. mp: 236e237 C. H NMR (400 MHz,
DMSO)
ꢀ
1
mp: 216e217 C. H NMR (400 MHz, DMSO)
d: 9.63 (s, 1H), 8.62 (s,
d
: 9.64 (s, 1H), 8.62 (s, 1H), 8.39 (dd, J ¼ 7.4, 0.9 Hz, 1H), 8.12
1
1
H), 8.39 (d, J ¼ 7.8 Hz,1H), 8.12 (d, J ¼ 7.8 Hz,1H), 7.78 (d, J ¼ 9.0 Hz,
(dd, J ¼ 7.2, 0.9 Hz, 1H), 7.95 (d, J ¼ 0.9 Hz, 1H), 7.71 (d, J ¼ 8.7 Hz,
1H), 7.64 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.57 - 7.51 (m, 1H), 7.51-7.45 (m,
13
H), 7.55 - 7.43 (m, 4H), 3.90 (s, 3H). C NMR (100 MHz, DMSO) d:
13
172.48,156.55, 149.70,144.68,139.58, 139.01,136.90, 132.60, 126.33,
1H), 2.46 (s, 3H). C NMR (100 MHz, DMSO) d: 172.28, 152.62,
1
25.61, 125.37, 124.85, 123.83, 123.51, 122.76, 120.44, 104.43, 56.22.
144.17, 139.10, 138.84, 136.41, 134.83, 134.18, 132.14, 125.84, 125.11,
þ
ESI-HRMS calcd. for C18
Anal. calcd. for C18
H, 3.75; S, 9.75.
H
13
O
4
S [MþH] 325.0529, found 325.0531.
124.88, 124.36, 123.88, 123.02, 121.39, 118.08, 20.41. ESI-HRMS
þ
12
H O
4
S: C, 66.66; H, 3.73; S, 9.88; Found C, 66.45;
calcd. for C18
calcd. for C18
.91; S, 10.31.
H
13
O
3
S [MþH] 309.0580, found 309.0582. Anal.
H
12
O
3
S: C, 70.11; H, 3.92; S, 10.40; Found C, 70.28; H,
3
5.3.20. 3-hydroxy-2-(1H-indol-3-yl)-6-methoxy-4H-chromen-4-
one (20)
Compound 20 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 48. Compound 20 was isolated as a yellow
5.3.24. 2-(benzo[b]thiophen-3-yl)-6-bromo-3-hydroxy-4H-
chromen-4-one (24)
Compound 24 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 50. Compound 24 was isolated as a yellow
ꢀ
1
solid in 54% yield. mp: 273 C dec. H NMR (400 MHz, DMSO) d:
1
1.96 (br s, 1H), 9.36 (br s, 1H), 8.44-8.40 (m, 1H), 8.36 (d, J ¼ 2.8 Hz,
ꢀ
1
1
H), 7.87 (d, J ¼ 9.1 Hz,1H), 7.57-7.53 (m,1H), 7.49 (d, J ¼ 3.0 Hz,1H),
solid in 66% yield. mp: 251 C. H NMR (400 MHz, DMSO) d: 9.90 (s,
13
7
.37 (dd, J ¼ 9.1, 3.0 Hz, 1H), 7.29-7.23 (m, 2H), 3.90 (s, 3H). C NMR
100 MHz, DMSO) : 170.55, 156.35, 149.30, 148.48, 136.57, 136.18,
30.79, 125.03, 123.00, 122.98, 122.54, 122.14, 121.43, 120.08, 112.72,
1H), 8.65 (s, 1H), 8.39 (dd, J ¼ 7.3, 1.2 Hz, 1H), 8.23 (d, J ¼ 2.5 Hz, 1H),
8.12 (dd, J ¼ 7.3, 1.2 Hz, 1H), 7.96 (dd, J ¼ 9.0, 2.5 Hz, 1H), 7.83 (d,
(
1
d
13
J ¼ 9.0 Hz, 1H), 7.56-7.46 (m, 2H). C NMR (100 MHz, DMSO)
d:
þ
106.82, 104.61, 56.15. ESI-HRMS calcd. for C18
H
13NO
14NO
4
[MþH]
171.69, 153.63, 145.36, 139.60, 139.57, 136.78, 136.53, 133.17, 127.23,
126.01, 125.62, 125.40, 124.89, 123.79, 123.49, 121.51, 117.46. ESI-
3
4
08.0917, found 308.0915. Anal. calcd. for C18
.26; N, 4.56; Found C, 70.45; H, 4.18; N, 4.51.
H
4
: C, 70.35; H,
7
9
þ
þ
HRMS calcd. for
72.9525. Calcd for C17
374.9498. Anal. calcd. for C17
Found C, 54.85; H, 2.41; S, 8.57.
C
17
H
11
O
H O
11 3
3
SBr
[MþH]
372.9529, found
3
SBr81 [MþH] 374.9509, found
5.3.21. 3,6-dihydroxy-2-(1H-indol-3-yl)-4H-chromen-4-one (21)
9 3
H BrO S: C, 54.71; H, 2.43; S, 8.59;
Compound 21 was prepared according to the general method
for the synthesis of demethylated flavonols reported above starting
from compound 20. Compound 21 was isolated as a yellow solid in
ꢀ
1
9
9
2% yield. Mp: 305 C. H NMR (400 MHz, DMSO)
d
11.94 (s, 1H),
5.3.25. 2-(benzo[b]thiophen-3-yl)-6-fluoro-3-hydroxy-4H-
chromen-4-one (25)
.87 (s, 1H), 9.22 (s, 1H), 8.40 (dd, J ¼ 6.6, 2.2 Hz, 1H), 8.34 (d,
J ¼ 2.9 Hz, 1H), 7.76 (d, J ¼ 9.0 Hz, 1H), 7.55 (dd, J ¼ 6.6, 2.2 Hz, 1H),
.40 (d, J ¼ 3.0 Hz, 1H), 7.27-7.23 (m, 1H), 7.24 (d, J ¼ 7.4 Hz, 1H), 7.22
Compound 25 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 51. Compound 25 was isolated as a pale
7
(
1
1
3
d, J ¼ 9.0, 3.0 Hz, 1H). C NMR (100 MHz, DMSO)
d: 170.18, 153.98,
ꢀ
1
47.89, 147.83, 136.08, 135.48, 130.17, 124.57, 122.71, 122.45, 121.88,
yellow solid in 70% yield. mp: 236 C. H NMR (400 MHz, DMSO) d:
1
21.61, 120.89, 119.24, 112.21, 107.00, 106.42. ESI-HRMS calcd. for
9.81 (br s, 1H), 8.64 (s, 1H), 8.38 (d, J ¼ 7.5 Hz, 1H), 8.12 (d, J ¼ 7.5 Hz,
1H), 7.91 (dd, J ¼ 9.2, 4.2 Hz, 1H), 7.81 (dd, J ¼ 8.5, 3.1 Hz, 1H), 7.74-
þ
C
C
4
17
H
H
12NO
11NO
4
[MþH] 294.0761, found 294.0763. Anal. calcd. for
13
17
4
: C, 69.62; H, 3.78; N, 4.78; Found C, 69.49; H, 3.96; N,
7.69 (m, 1H), 7.55 - 7.46 (m, 2H). C NMR (100 MHz, DMSO) d:
.68.
171.74 (d, J ¼ 2.4 Hz), 158.52 (d, J ¼ 243.5 Hz), 150.75, 144.80, 139.08,
138.62, 136.33, 132.59, 125.59, 125.14, 124.91, 124.39, 123.01, 122.72
5
.3.22. 3-hydroxy-6-methoxy-2-(1-methyl-1H-indol-3-yl)-4H-
(d, J ¼ 7.6 Hz), 121.86 (d, J ¼ 25.8 Hz), 121.12 (d, J ¼ 8.5 Hz), 109.01 (d,
þ
chromen-4-one (22)
The intermediate chalcone was synthesized following the gen-
eral method for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-
J ¼ 23.7 Hz). ESI-HRMS calcd. for C17
H10FO
3
S [MþH] 313.0329,
9 3
found 313.0329. Anal. calcd. for C17H FO S: C, 65.38; H, 2.90; S,
10.27; Found C, 65.19; H, 2.86; S, 10.17.

C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
15
5
.3.26. 2-(benzo[b]thiophen-3-yl)-6-chloro-3-hydroxy-4H-
s, 1H), 8.62 (s, 1H), 8.39 (d, J ¼ 7.5 Hz, 1H), 8.22 (dd, J ¼ 8.9, 6.4 Hz,
1H), 8.12 (d, J ¼ 7.5 Hz, 1H), 7.81 (dd, J ¼ 9.8, 2.3 Hz, 1H), 7.55-7.47
chromen-4-one (26)
13
Compound 26 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 52. Compound 26 was isolated as a yellow
(m, 2H), 7.40 (td, J ¼ 8.9, 2.3 Hz, 1H). C NMR (100 MHz, DMSO)
d:
171.83, 164.80 (d, J ¼ 251.3 Hz), 155.25 (d, J ¼ 14.2 Hz), 144.76,
139.07, 138.92, 136.32, 132.27, 127.63 (d, J ¼ 11.0 Hz), 125.56, 125.16,
ꢀ
1
solid in 63% yield. mp: 253e254 C. H NMR (400 MHz, DMSO)
d
:
124.91, 124.53, 122.98, 118.95, 113.62 (d, J ¼ 23.3 Hz), 104.95 (d,
þ
9
1
2
.89 (br s, 1H), 8.65 (s, 1H), 8.38 (d, J ¼ 7.5 Hz, 1H), 8.12 (d, J ¼ 7.5 Hz,
J ¼ 25.6 Hz). ESI-HRMS calcd. for C17
H
10FO
3
S [MþH] 313.0329,
H), 8.08 (d, J ¼ 2.5 Hz, 1H), 7.89 (d, J ¼ 9.0 Hz, 1H), 7.84 (dd, J ¼ 9.0,
found 313.0323. Anal. calcd. for C17
9 3
H FO S: C, 65.38; H, 2.90; S,
13
.5 Hz, 1H), 7.56-7.46 (m, 2H). C NMR (100 MHz, DMSO)
d: 171.34,
10.27; Found C, 65.35; H, 2.88; S, 10.04.
152.78, 144.87, 139.08, 139.06, 136.30, 133.39, 132.71, 129.12, 125.51,
1
25.15, 124.93, 124.41, 123.60, 123.02, 122.85, 120.85. ESI-HRMS
5.3.31. 2-(benzo[b]thiophen-3-yl)-7-chloro-3-hydroxy-4H-
chromen-4-one (31)
3
5
þ
calcd. For C17
calcd. for C17
calcd. for C17
H
10
O
3
SCl
[MþH] 329.0034, found 329.0036.
3
7
þ
H
10
O
3
SCl [MþH] 331.0005, found 331.0007. Anal.
Compound 31 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 57. Compound 31 was isolated as a yellow
9 3
H ClO S: C, 62.11; H, 2.76; S, 9.75; Found C, 62.33; H,
2
.73; S, 9.63.
ꢀ
1
solid in 69% yield. mp: 267e268 C. H NMR (400 MHz, DMSO)
d:
5
.3.27. 2-(benzo[b]thiophen-3-yl)-3-hydroxy-4H-chromen-4-one
9.85 (s, 1H), 8.64 (s, 1H), 8.43 (d, J ¼ 7.8 Hz, 1H), 8.18-8.10 (m, 2H),
13
(27)
8.05 (s, 1H), 7.58-7.46 (m, 3H). C NMR (100 MHz, DMSO) d: 171.82,
Compound 27 was prepared according to the general method
154.41, 144.66, 139.13, 139.05, 137.99, 136.31, 132.58, 126.67, 125.49,
125.28, 125.23, 124.94, 124.66, 122.97, 120.63, 118.26. ESI-HRMS
for the synthesis of flavonol-like compounds reported above
starting from compound 53. Compound 27 was isolated as a pale
3
5
þ
calcd. for C17
for C17
ClO
H
10
37
O
3
SCl [MþH] 329.0034, found 329.0036. Calcd
ꢀ
1
þ
brown solid in 37% yield. mp: 225 C. H NMR (400 MHz, DMSO)
d:
H
10
O
3
SCl [MþH] 331.0005, found 331.0007. Anal. calcd. for
9
(
.70 (s, 1H), 8.63 (s, 1H), 8.40 (m, 1H), 8.19-8.11 (m, 2H), 7.85-7.80
C
17
H
9
3
S: C, 62.11; H, 2.76; S, 9.75; Found C, 62.14; H, 2.79; S,
13
m, 2H), 7.57-7.47 (m, 3H). C NMR (100 MHz, DMSO)
d: 172.41,
9.65.
154.30, 144.33, 139.11, 138.92, 136.41, 133.61, 132.27, 125.79, 125.13,
1
24.90, 124.83, 124.69, 124.36, 123.04, 121.71, 118.31. ESI-HRMS
5.3.32. 2-(benzo[b]thiophen-3-yl)-7-ethoxy-3-hydroxy-4H-
chromen-4-one (32)
þ
calcd. for C17
calcd. for C17
3
H
11
O
3
S [MþH] 295.0423, found 295.0426. Anal.
H
10
O
3
S: C, 69.37; H, 3.42; S, 10.89; Found C, 69.18; H,
Compound 32 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 58. Compound 32 was isolated as a yellow
.43; S, 10.87.
ꢀ
1
5.3.28. 2-(benzo[b]thiophen-3-yl)-3-hydroxy-7-methoxy-4H-
solid in 25% yield. mp: 209e210 C. H NMR (400 MHz, DMSO) d:
chromen-4-one (28)
9.52 (s, 1H), 8.57 (s, 1H), 8.39 (d, J ¼ 7.5 Hz, 1H), 8.12 (d, J ¼ 7.5 Hz,
1H), 8.04 (d, J ¼ 8.9 Hz, 1H), 7.57 - 7.48 (m, 2H), 7.26 (d, J ¼ 2.3 Hz,
1H), 7.07 (dd, J ¼ 8.9, 2.3 Hz, 1H), 4.22 (q, J ¼ 7.0 Hz, 2H), 1.40 (t,
Compound 28 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 54. Compound 28 was isolated as a yellow
13
J ¼ 7.0 Hz, 3H). C NMR (100 MHz, DMSO)
d: 171.81, 162.88, 156.21,
ꢀ
1
solid in 12% yield. mp: 236 C. H NMR (400 MHz, DMSO)
H), 8.57 (s, 1H), 8.39 (d, J ¼ 7.8 Hz, 1H), 8.12 (d, J ¼ 7.8 Hz, 1H), 8.06
d, J ¼ 8.9 Hz,1H), 7.61-7.44 (m, 2H), 7.28 (d, J ¼ 2.2 Hz,1H), 7.09 (dd,
d
: 9.54 (s,
143.67, 139.08, 138.58, 136.40, 131.64, 126.17, 125.88, 125.11, 124.84,
1
(
124.47, 122.98, 115.45, 114.84, 100.66, 64.25, 14.36. ESI-HRMS calcd.
þ
for C19
H
15
O
4
S [MþH] 339.0686, found 339.0680. Anal. calcd. for
13
J ¼ 8.9, 2.2 Hz, 1H), 3.95 (s, 3H). C NMR (100 MHz, DMSO)
d:
19 14 4
C H O S: C, 67.44; H, 4.17; S, 9.47; Found C, 67.31; H, 4.13; S, 9.21.
171.84, 163.62, 156.24, 143.70, 139.09, 138.62, 136.40, 131.66, 126.19,
1
25.87, 125.10, 124.85, 124.44, 122.99, 115.56, 114.69, 100.22, 56.15.
5.3.33. 2-(benzo[b]thiophen-3-yl)-6-methoxy-4-oxo-4H-chromen-
3-yl acetate (33)
Compound 33 was prepared according to the general method
for the synthesis of ethers reported above starting from compound
þ
ESI-HRMS calcd. for C18
Anal. calcd. for C18
H, 3.64; S, 9.72.
H
13
O
4
S [MþH] 325.0529, found 325.0526.
H
12
O
4
S: C, 66.66; H, 3.73; S, 9.88; Found C, 66.90;
1
9. Compound 33 was isolated as a beige solid in 94% yield. mp:
ꢀ
1
5.3.29. 2-(benzo[b]thiophen-3-yl)-3-hydroxy-7-methyl-4H-
180 C dec. H NMR (400 MHz, CDCl ) d: 8.16-8.12 (m, 1H), 8.07 (s,
3
chromen-4-one (29)
1H), 7.97-7.93 (m,1H), 7.66 (d, J ¼ 3.1 Hz,1H), 7.52 (d, J ¼ 9.2 Hz,1H),
7.52-7.46 (m, 2H), 7.34 (dd, J ¼ 9.2, 3.1 Hz, 1H), 3.94 (s, 3H), 2.29 (s,
Compound 29 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 55. Compound 29 was isolated as a brown
13
3
3H). C NMR (100 MHz, CDCl ) d 170.70, 166.99, 156.12, 152.24,
149.40, 138.74, 135.49, 132.58, 130.42, 124.39, 124.25, 124.22,123.51,
ꢀ
1
solid in 21% yield. mp: 237 C. H NMR (400 MHz, DMSO)
H), 8.62 (s, 1H), 8.42-8.40 (m, 1H), 8.13 (d, J ¼ 7.7 Hz, 1H), 8.05 (d,
J ¼ 7.8 Hz, 1H), 7.67-7.47 (m, 1H, 3H), 7.33 (d, J ¼ 7.8 Hz, 1H), 2.51 (s,
d
: 9.62 (s,
123.27, 122.82, 121.78, 118.43, 104.09, 54.99, 19.53. ESI-HRMS calcd.
þ
1
for C20
H
15
O
5
S [MþH] 367.0635, found 367.0631. Anal. calcd. for
20 14 5
C H O S: C, 65.56; H, 3.85; S, 8.75; Found C, 65.40; H, 3.86; S, 8.61.
13
1
H). C NMR (100 MHz, DMSO)
d: 172.23, 154.41, 144.60, 143.94,
1
39.12, 138.77, 136.43, 132.06, 126.22, 125.81, 125.11, 124.89, 124.60,
5.3.34. 2-(benzo[b]thiophen-3-yl)-6-methoxy-4-oxo-4H-chromen-
3-yl pivalate (34)
Compound 34 was prepared according to the general method
for the synthesis of ethers reported above starting from compound
124.38, 123.03, 119.48, 117.73, 21.21. ESI-HRMS calcd. for C18
H
13
O
3
S
þ
[
7
MþH] 309.0580, found 309.0586. Anal. calcd. for C18
H
12
O
3
S: C,
0.11; H, 3.92; S, 10.40; Found C, 69.95; H, 4.11; S, 9.21.
1
9. Compound 34 was isolated as a white solid in 93% yield. mp:
ꢀ
1
5.3.30. 2-(benzo[b]thiophen-3-yl)-7-fluoro-3-hydroxy-4H-
169 C. H NMR (400 MHz, CDCl ) d: 8.15-8.10 (m, 1H), 8.01 (s, 1H),
3
chromen-4-one (30)
7.97-7.90 (m, 1H), 7.66 (d, J ¼ 3.1 Hz, 1H), 7.51 (d, J ¼ 9.1 Hz, 1H),
7.51-7.43 (m, 2H), 7.32 (dd, J ¼ 9.2, 3.1 Hz, 1H), 3.91 (s, 3H), 1.30 (s,
Compound 30 was prepared according to the general method
for the synthesis of flavonol-like compounds reported above
starting from compound 56. Compound 30 was isolated as a yellow
13
3
9H). C NMR (100 MHz, CDCl ) d 175.65, 171.72, 157.10, 153.13,
150.43, 139.69, 136.62, 133.89, 131.27, 125.37, 125.25, 125.21, 124.54,
124.25, 123.89, 122.78, 119.48, 104.96, 55.93, 39.07, 27.12. ESI-HRMS
ꢀ
1
solid in 70% yield. mp: 258 C. H NMR (400 MHz, DMSO) d: 9.78 (br

16
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
þ
calcd. for C23
21
H O
5
S [MþH] 409.1104, found 409.1102. Anal. calcd.
21 18 4
C H O S: C, 68.83; H, 4.95; S, 8.75; Found C, 69.18; H, 5.03; S, 8.53.
for C23 S: C, 67.63; H, 4.94; S, 7.85; Found C, 67.81; H, 5.05; S,
20 5
H O
7.67.
5.3.39. (E)-3-(furan-2-yl)-1-(2-hydroxy-5-methoxyphenyl)prop-2-
en-1-one (39)
5
3
.3.35. 2-(benzo[b]thiophen-3-yl)-6-methoxy-4-oxo-4H-chromen-
-yl pentanoate (35)
Compound 35 was prepared according to the general method
Compound 39 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
1-ones reported above. Compound 39 was isolated as a red powder
ꢀ
1
for the synthesis of ethers reported above starting from compound
9. Compound 35 was isolated as a pale yellow solid in 53% yield.
in 51% yield. mp: 73e74 C. H NMR (CDCl , 400 MHz) d: 12.51 (br s,
3
1
1H), 7.71 (d, J ¼ 15.1 Hz, 1H), 7.59 (d, J ¼ 1.8 Hz, 1H), 7.52 (d,
J ¼ 15.1 Hz, 1H), 7.38 (d, J ¼ 3.0 Hz, 1H), 7,17 (dd, J ¼ 9.1, 3.0 Hz, 1H),
6.99 (d, J ¼ 9.1 Hz, 1H), 6.81 (d, J ¼ 3.4 Hz, 1H), 6.58 (dd, J ¼ 1.8,
ꢀ
1
3
mp: 111e112 C. H NMR (400 MHz, CDCl ) d: 8.13-8.11 (m, 1H), 8.05
(
1
s, 1H), 7.96-7.93 (m, 1H), 7.66 (d, J ¼ 3.1 Hz, 1H), 7.51 (d, J ¼ 9.2 Hz,
13
H), 7.51-7.42 (m, 2H), 7.32 (dd, J ¼ 9.2, 3.1 Hz, 1H), 3.92 (s, 3H), 2.55
3
3.4 Hz,1H), 3.87 (s, 3H). C NMR (CDCl ,100 MHz) d: 192.94,157.99,
(
t, J ¼ 7.5 Hz, 2H), 1.69-1.61 (m, 2H), 1.38-1.28 (m, 2H), 0.88 (t,
151.76, 151.54, 145.47, 131.25, 124.12, 119.64, 119.32, 117.60, 117.24,
13
þ
-
J ¼ 7.4 Hz, 3H). C NMR (100 MHz, CDCl
3
)
d
: 171.79, 170.87, 157.12,
112.95, 112.56, 56.12. MS (ESI) m/z ¼ 245.1 [MþH] ; 243.0 [M ꢃ H] .
1
53.22, 150.44, 139.75, 136.56, 133.65, 131.39, 125.47, 125.25, 125.22,
1
2
24.55, 124.27, 123.88, 122.78, 119.46, 105.08, 55.99, 33.64, 26.88,
5.3.40. (E)-3-(furan-2-yl)-1-(2-hydroxy-4-methoxyphenyl)prop-2-
en-1-one (40)
Compound 40 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
þ
2.08, 13.67. ESI-HRMS calcd. for C23
H
21
O
5
S [MþH] 409.1104,
found 409.1100. Anal. calcd. for C23
Found C, 67.33; H, 4.83; S, 8.01.
H
20
O
5
S: C, 67.63; H, 4.94; S, 7.85;
1
-ones reported above. Compound 40 was isolated as an orange
ꢀ
1
5
3
.3.36. 2-(benzo[b]thiophen-3-yl)-6-methoxy-4-oxo-4H-chromen-
-yl dimethylcarbamate (36)
Compound 36 was prepared according to the general method
solid in 65% yield. mp: 118e120 C. H NMR (CDCl , 400 MHz) d:
3
13.51 (br s, 1H), 7.83 (d, J ¼ 8.7 Hz, 1H), 7.65 (d, J ¼ 15.1 Hz, 1H), 7.55
(d, J ¼ 1.7 Hz, 1H), 7.48 (d, J ¼ 15.1 Hz, 1H), 6.74 (d, J ¼ 3.4 Hz, 1H),
6.53 (dd, J ¼ 1.7, 3.4 Hz, 1H), 6.49 (dd, J ¼ 8.7, 2.5 Hz, 1H), 6.48 (d, 1H,
for the synthesis of carbamates reported above starting from
compound 19. Compound 36 was isolated as a yellow solid in 42%
13
J ¼ 2.5 Hz, 1H), 3.86 (s, 3H). C NMR (CDCl
3
, 100 MHz) d: 191.45,
ꢀ
1
yield. mp: 221e222 C. H NMR (400 MHz, CDCl
.5 Hz, 1H), 8.11 (s, 1H), 7.94 (dd, J ¼ 7.2, 1.5 Hz, 1H), 7.66 (d,
J ¼ 3.1 Hz, 1H), 7.51 (d, J ¼ 9.1 Hz, 1H), 7.51-7.43 (m, 2H), 7.32 (dd,
3
)
d: 8.18 (dd, J ¼ 7.2,
166.61, 166.18, 151.66, 145.14, 131.24, 130.22, 117.90, 116.51, 114.15,
þ
1
112.77, 107.68, 101.05, 55.57. MS (ESI) m/z ¼ 245.1 [MþH] ; 243.1
-
[M ꢃ H] .
13
J ¼ 9.1, 3.1 Hz, 1H), 3.92 (s, 3H), 3.09 (s, 3H), 3.01 (s, 3H). C NMR
100 MHz, CDCl : 172.70, 156.98, 153.67, 153.40, 150.40, 139.78,
36.77, 134.33, 131.58, 125.54, 125.13, 125.11, 124.63, 124.10, 123.96,
(
1
3
)
d
5.3.41. (E)-1-(2-hydroxy-5-methoxyphenyl)-3-(4-methylthiophen-
2-yl)prop-2-en-1-one (41)
1
22.76, 119.40, 105.08, 55.95, 37.07, 36.83. ESI-HRMS calcd. for
Compound 41 was prepared according to the general method for
the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-
ones reported above. Compound 41 was isolated as an orange
þ
C
C
4
21
H18NO
17NO
5
S [MþH] 396.0900, found 396.0908. Anal. calcd. for
21
H
5
S: C, 63.79; H, 4.33; N, 3.54; S, 8.11; Found C, 63.75; H,
ꢀ
1
.58; N, 3.50; S, 7.90.
solid in 73% yield. mp: 79 C. H NMR (CDCl , 400 MHz) d: 12.36 (br
3
s, 1H), 8.00 (d, J ¼ 15.1 Hz, 1H), 7.35 (d, J ¼ 15.1 Hz, 1H), 7.33 (d,
J ¼ 3.0 Hz, 1H), 7.23 (m, 1H), 7,15 (dd, J ¼ 9.0, 3.0 Hz, 1H), 7.08 (m,
5
.3.37. 2-(benzo[b]thiophen-3-yl)-3-ethoxy-6-methoxy-4H-
13
chromen-4-one (37)
Compound 37 was prepared according to the general method
for the synthesis of ethers reported above starting from compound
1H), 6.98 (d, J ¼ 9.0 Hz, 1H), 3.86 (s, 3H), 2.30 (d, J ¼ 0.7 Hz, 3H).
C
3
NMR (CDCl , 100 MHz) d: 192.79, 157.88, 151.69, 139.82, 139.29,
138.20, 134.81, 125.36, 123.66, 119.63, 119.28, 118.36, 112.93, 56.16,
þ
19. Compound 37 was isolated as a yellow solid in 77% yield. mp:
15.53. MS (ESI) m/z ¼ 275.1 [MþH] .
ꢀ
1
1
46e147 C. H NMR (400 MHz, CDCl
J ¼ 7.4, 0.9 Hz, 1H), 7.94 (dd, J ¼ 7.4, 1.1 Hz, 1H), 7.66 (d, J ¼ 3.1 Hz,
H), 7.52 (d, J ¼ 9.1 Hz, 1H), 7.51 (dd, J ¼ 7.0, 1.2 Hz, 1H), 7.44 (dd,
J ¼ 7.1, 0.9 Hz, 1H), 7.30 (dd, J ¼ 9.1, 3.1 Hz, 1H), 4.14 (q, J ¼ 7.0 Hz,
3
) d: 8.46 (s, 1H), 8.31 (dd,
5.3.42. (E)-1-(2-hydroxy-4-methoxyphenyl)-3-(4-methylthiophen-
2-yl)prop-2-en-1-one (42)
Compound 42 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
1-ones reported above. Compound 42 was isolated as a yellow solid
1
13
2
H), 3.94 (s, 3H),1.27 (t, J ¼ 7.0 Hz, 3H). C NMR (100 MHz, CDCl
3
) d:
174.56, 156.79, 153.58, 149.91, 140.06, 139.68, 136.91, 132.31, 125.97,
ꢀ
1
125.04, 124.93, 124.32, 123.79, 122.79, 119.28, 104.73, 68.54, 55.97,
in quantitative yield. mp 123e124 C. H NMR (CDCl
ppm: 13.68 (br s, 1H), 7.94 (d, J ¼ 15.1 Hz, 1H), 7.78 (d, J ¼ 8.7 Hz,
1H), 7.31 (d, J ¼ 15.1 Hz,1H), 7.17 (s,1H), 7.03 (s,1H), 6.49 (dd, J ¼ 8.7,
3
, 400 MHz)
þ
1
3
5.61. ESI-HRMS calcd. for C20
53.0845. Anal. calcd. for C20
Found C, 67.92; H, 4.81; S, 9.11.
H
17
O
4
S [MþH] 353.0842, found
d
H
16
O
4
S: C, 68.17; H, 4.58; S, 9.10;
13
2.5 Hz, 1H), 3.86 (s, 3H), 2.32 (s, J ¼ 0.7 Hz, 3H). C NMR (CDCl
00 MHz) : 191.29, 166.62, 166.15, 140.02, 139.15, 137.02, 134.35,
131.10, 124.85, 118.62, 114.04, 107.67, 101.06, 55.57, 15.53. MS (ESI)
3
,
1
d
5
.3.38. 2-(benzo[b]thiophen-3-yl)-6-methoxy-3-propoxy-4H-
þ
-
chromen-4-one (38)
Compound 38 was prepared according to the general method
for the synthesis of ethers reported above starting from compound
m/z ¼ 275.1 [MþH] ; 273.0 [M ꢃ H] .
5.3.43. (E)-1-(2-hydroxy-5-methylphenyl)-3-(4-methylthiophen-2-
yl)prop-2-en-1-one (43)
19. Compound 38 was isolated as an orange solid in 76% yield. mp:
ꢀ
1
137 C. H NMR (400 MHz, CDCl
3
)
d
: 8.33 (s, 1H), 8.20 (d, J ¼ 7.4 Hz,
Compound 43 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
1-ones reported above. Compound 43 was isolated as an orange
1
H), 7.85 (d, J ¼ 7.4, 1H), 7.57 (d, J ¼ 3.1 Hz, 1H), 7.44-7.33 (m, 2H),
7
.42 (d, J ¼ 9.2 Hz,1H), 7.21 (dd, J ¼ 9.1, 3.1 Hz,1H), 3.93 (t, J ¼ 6.7 Hz,
1
3
ꢀ
1
2
H), 3.85 (s, 3H), 1.66-1.50 (m, 2H), 0.77 (t, J ¼ 7.4 Hz, 3H). C NMR
: 174.51, 156.78, 153.41, 149.92, 140.31, 139.66,
36.95, 132.25, 125.95, 125.06, 125.02, 124.92, 124.27, 123.76,
solid in 60% yield. mp: 104e105 C. H NMR (400 MHz, CDCl ) d:
3
(
100 MHz, CDCl
3
)
d
12.71 (s, 1H), 7.99 (d, J ¼ 15.1 Hz, 1H), 7.65 (d, J ¼ 1.6 Hz, 1H), 7.41 (d,
J ¼ 15.1 Hz, 1H), 7.32 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.23 (s, 1H), 7.07 (s, 1H),
1
13
122.78, 119.28, 104.72, 74.56, 55.95, 23.35, 10.35. ESI-HRMS calcd.
6.94 (d, J ¼ 8.5 Hz, 1H), 2.38 (s, 3H), 2.31 (d, J ¼ 0.7 Hz, 3H). C NMR
þ
for C21
H
19
O
4
S [MþH] 367.0999, found 367.0998. Anal. calcd. for
3
(100 MHz, CDCl ) d: 193.07, 161.48, 139.92, 139.25, 137.85, 137.38,

C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
17
1
34.66, 129.19, 127.88, 125.19, 119.63, 118.50, 118.32, 20.64, 15.54.
12.48 (br s, 1H), 12.04 (br s, 1H), 8.24 (s, 1H), 8.19 (d, J ¼ 15.3 Hz, 1H),
8.11 - 8.07 (m, 1H), 7.76 (d, J ¼ 15.3 Hz, 1H), 7.61 (d, J ¼ 3.0 Hz, 1H),
þ
MS (ESI) m/z ¼ 259.1 [MþH] .
7
.54 - 7.50 (m, 1H), 7.29 - 7.23 (m, 2H), 7.19 (dd, J ¼ 9.0, 3.0 Hz, 1H),
13
5
2
.3.44. (E)-1-(2-hydroxy-6-methoxyphenyl)-3-(4-methylthiophen-
-yl)prop-2-en-1-one (44)
6.94 (d, J ¼ 9.0 Hz, 1H), 3.83 (s, 3H). C NMR (100 MHz, DMSO)
d:
193.03, 156.37, 152.03, 140.44, 137.96, 134.24, 125.78, 123.33, 123.16,
121.85, 121.27, 120.67, 118.98, 115.21, 113.75, 113.42, 113.00, 56.29.
Compound 44 was prepared according to the general method
-
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
-ones reported above. Compound 44 was isolated as a yellow solid
MS (ESI) m/z ¼ 292.0 [M ꢃ H] .
1
ꢀ
1
in quantitative yield. mp: 89 C. H NMR (CDCl
br s, 1H), 7.90 (d, J ¼ 15.3 Hz, 1H), 7.68 (d, J ¼ 15.3 Hz, 1H), 7.36 (t,
J ¼ 8.3 Hz, 1H), 7.15 (m, 1H), 7.00 (m, 1H), 6.62 (dd, J ¼ 8.3, 0.8 Hz,
3
, 400 MHz)
d: 13.11
5.3.49. (E)-3-(benzo[b]thiophen-3-yl)-1-(2-hydroxy-5-
methylphenyl)prop-2-en-1-one (49)
Compound 49 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
1-ones reported above. Compound 49 was isolated as an orange
(
13
1
(
1
H), 6.43 (d, J ¼ 0.8 Hz, 1H), 3.95 (s, 3H), 2.28 (s, 3H). C NMR
CDCl , 100 MHz) : 193.75, 164.90, 160.95, 140.71, 139.07, 135.91,
35.80, 133.90, 126.09, 124.38, 111.87, 110.90, 101.54, 55.89, 15.56.
3
d
ꢀ
1
solid in 55% yield. mp: 139 C. H NMR (400 MHz, CDCl
1H), 8.23 (d, J ¼ 15.5 Hz, 1H), 8.13 (d, J ¼ 8.1 Hz, 1H), 7.99 (s, 1H), 7.94
d, J ¼ 8.1 Hz, 1H), 7.77 (d, J ¼ 15.5 Hz, 1H), 7.72 (d, J ¼ 1.5 Hz, 1H),
7.57 - 7.53 (m, 1H), 7.49-7.45 (m, 1H), 7.36 (dd, J ¼ 8.5, 1.5 Hz, 1H),
3
) d: 12.71 (s,
þ
-
MS (ESI) m/z ¼ 275.1 [MþH] ; 273.0 [M ꢃ H] .
(
5
2
.3.45. (E)-1-(2-hydroxy-4-methoxyphenyl)-3-(3-methylthiophen-
-yl)prop-2-en-1-one (45)
13
6.98 (d, J ¼ 8.5 Hz, 1H), 2.39 (s, 3H). C NMR (100 MHz, CDCl
3
)
Compound 45 was prepared according to the general method
d:193.50, 161.58, 140.57, 137.52, 137.34, 136.65, 132.27, 129.26,
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
-ones reported above. Compound 45 was isolated as a yellow solid
129.15, 127.96, 125.28, 125.19, 123.13, 122.14, 120.72, 119.69, 118.45,
20.67. MS (ESI) m/z ¼ 295.1 [MþH] .
þ
1
ꢀ
1
3
in 49% yield. mp: 128e129 C. H NMR (CDCl , 400 MHz) d: 13.39
(
br s, 1H), 8.12 (d, J ¼ 15.0 Hz, 1H), 7.81 (d, J ¼ 8.8 Hz, 1H), 7.35 (d,
5.3.50. (E)-3-(benzo[b]thiophen-3-yl)-1-(5-bromo-2-
hydroxyphenyl)prop-2-en-1-one (50)
J ¼ 5.1 Hz, 1H), 7.32 (d, J ¼ 15.0 Hz, 1H), 6.94 (d, J ¼ 5.1 Hz, 1H), 6.51
(
3
dd, J ¼ 8.8, 2.5 Hz, 1H), 6.49 (d, J ¼ 2.5 Hz, 1H), 3.88 (s, 3H), 2.44 (s,
Compound 50 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
1-ones reported above. Compound 50 was isolated as an orange
13
3
H). C NMR (CDCl , 100 MHz) d: 191.39, 166.63, 166.12, 143.01,
1
5
35.28, 134.53, 131.53, 131.09, 127.68, 118.15, 114.07, 107.70, 101.06,
þ
ꢀ
1
5.59, 14.33. MS (ESI) m/z ¼ 275.1 [MþH] .
solid in 74% yield. mp: 159 C. H NMR (400 MHz, CDCl ) d: 12.83 (s,
3
1
H), 8.25 (d, J ¼ 15.4 Hz,1H), 8.11 (d, J ¼ 8.0 Hz,1H), 8.02 (s,1H), 8.02
5
.3.46. (E)-1-(2-hydroxy-5-methylphenyl)-3-(5-methylthiophen-2-
(d, J ¼ 2.4 Hz, 1H), 7.94 (dd, J ¼ 8.0, 0.8 Hz, 1H), 7.65 (d, J ¼ 15.4 Hz,
1H), 7.60 (dd, J ¼ 8.9, 2.4 Hz, 1H), 7.58-7.53 (m, 1H), 7.50-7.46 (m,
yl)prop-2-en-1-one (46)
Compound 46 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
13
1H), 6.97 (d, J ¼ 8.9 Hz, 1H). C NMR (100 MHz, CDCl
3
) d: 192.56,
162.54, 140.54, 138.95, 137.81, 137.21, 131.95, 131.75, 130.04, 125.40,
1
-ones reported above. Compound 46 was isolated as a light brown
125.34, 123.17, 122.08, 121.27, 120.70, 119.67, 110.49. MS (ESI) m/z
Br ¼ 359.1 [MþH] ; Br ¼ 361.1 [MþH] .
ꢀ
1
79
þ
81
þ
solid in 44% yield. mp: 113 C. H NMR (400 MHz, DMSO)
1
d
: 12.24 (s,
H), 8.01 (d, J ¼ 15.1, 1H), 7.89-7.88 (m, 1H), 7.74 (d, J ¼ 5.0 Hz, 1H),
7
.54 (d, J ¼ 15.1 Hz, 1H), 7.36 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.06 (d,
5.3.51. (E)-3-(benzo[b]thiophen-3-yl)-1-(5-fluoro-2-
hydroxyphenyl)prop-2-en-1-one (51)
13
J ¼ 5.0 Hz, 1H), 6.89 (d, J ¼ 8.4 Hz, 1H), 2.39 (s, 3H), 2.31 (s, 3H).
C
NMR (100 MHz, DMSO)
d: 192.38, 159.42, 143.64, 136.87, 135.24,
Compound 51 was prepared according to the general method for
the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-
ones reported above. Compound 51 was isolated as an orange
133.66, 131.74, 130.01, 129.58, 127.96, 120.54, 119.33, 117.48, 19.89,
þ
13.95. MS (ESI) m/z ¼ 259.1 [MþH] .
ꢀ
1
3
solid in 55% yield. mp: 168 C. H NMR (400 MHz, CDCl ) d: 12.62 (s,
5
.3.47. (E)-3-(benzo[d] [1,3]dioxol-5-yl)-1-(2-hydroxy-4-
1H), 8.26 (dd, J ¼ 15.4, 0.7 Hz, 1H), 8.12 (d, J ¼ 7.9 Hz, 1H), 8.01 (s,
1H), 7.96-7.93 (m, 1H), 7.65 (d, J ¼ 15.5 Hz, 1H), 7.61 (dd, J ¼ 9.1,
3.1 Hz, 1H), 7.56 (ddd, J ¼ 8.2, 7.1, 1.2 Hz, 1H), 7.51-7.46 (m, 1H), 7.31-
methoxyphenyl)prop-2-en-1-one (47)
Compound 47 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
13
7.26 (m, 1H), 7.04 (dd, J ¼ 9.1, 4.6 Hz, 1H). C NMR (100 MHz, CDCl
3
)
1
-ones reported above. Compound 47 was isolated as a yellow solid
d
:
192.72 (d, J ¼ 2.7 Hz), 159.80 (d, J ¼ 1.2 Hz), 154.90 (d,
ꢀ
1
in 44% yield. mp: 149 C. H NMR (400 MHz, CDCl
H), 7.73 (d, J ¼ 15.2 Hz, 1H), 7.45 (d, J ¼ 15.2 Hz, 1H), 7.21 (d,
J ¼ 1.6 Hz, 1H), 7.18 (dd, J ¼ 8.0, 1.6 Hz, 1H), 6.90 (d, J ¼ 8.0 Hz, 1H),
.52 (dd, J ¼ 9.0, 2.5 Hz, 1H), 6.51 (s, 1H), 6.08 (s, 2H), 3.90 (s, 3H).
3
)
d: 7.75-7.71 (m,
J ¼ 238.3 Hz), 140.60, 137.77, 137.15, 132.01, 130.17, 125.40, 125.34,
123.92 (d, J ¼ 23.6 Hz), 123.19, 122.13, 119.93 (d, J ¼ 7.3 Hz), 119.76,
119.52 (d, J ¼ 6.1 Hz), 114.45 (d, J ¼ 23.3 Hz). MS (ESI) m/z ¼ 299.2
1
þ
6
[MþH] .
1
3
3
C NMR (100 MHz, CDCl ) d: 191.75, 166.66, 166.12, 150.06, 148.48,
1
44.25, 131.11, 129.30, 125.39, 118.30, 114.14, 108.72, 107.66, 106.72,
5.3.52. (E)-3-(benzo[b]thiophen-3-yl)-1-(5-chloro-2-
hydroxyphenyl)prop-2-en-1-one (52)
þ
101.69, 101.10, 55.59. MS (ESI) m/z ¼ 297.0 [MþH] .
Compound 52 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
1-ones reported above. Compound 52 was isolated as an orange
5.3.48. (E)-1-(2-hydroxy-5-methoxyphenyl)-3-(1H-indol-3-yl)
prop-2-en-1-one (48)
0
0
ꢀ
1
To a suspension of 2 -hydroxy-4 -methoxyacetophenone (1.00 g,
.02 mmol) and 1H-indole-3-carbaldehyde (0.87 g, 6.02 mmol) in
solid in 76% yield. mp: 163e164 C. H NMR (400 MHz, CDCl ) d:
3
6
12.78 (s, 1H), 8.25 (d, J ¼ 15.4 Hz, 1H), 8.10 (d, J ¼ 8.0 Hz, 1H), 8.02 (s,
1H), 7.93 (d, J ¼ 8.0 Hz, 1H), 7.88 (d, J ¼ 2.5 Hz, 1H), 7.66 (d,
ethanol, piperidine (0.6 mL, 0.51 g, 6.02 mmol) was added. The re-
action mixture was refluxed for 24 h. After the completion of the
reaction, the mixture was cooled to 0 C and neutralized with acetic
acid. The resulting solid was collected by filtration, washed with
ethanol and recrystallized from ethanol to give compound 48 as an
J ¼ 15.4 Hz, 1H), 7.54 (ddd, J ¼ 8.1, 7.1, 1.1 Hz, 1H), 7.49-7.43 (m, 2H),
ꢀ
13
7.01 (d, J ¼ 8.9 Hz, 1H). C NMR (100 MHz, CDCl
3
) d: 192.67, 162.11,
140.57, 137.86, 137.20, 136.21, 131.98, 130.12, 128.74, 125.41, 125.35,
123.58, 123.19, 122.11, 120.62, 120.31, 119.70. MS (ESI) m/z
ꢀ
1
35
þ
37
þ
orange solid in 54% yield. mp 206 C. H NMR (400 MHz, DMSO)
d:
Cl ¼ 315.0 [MþH] ; Cl ¼ 317.0 [MþH] .

18
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
5
.3.53. (E)-3-(benzo[b]thiophen-3-yl)-1-(2-hydroxyphenyl)prop-2-
139.91, 139.90, 136.88, 135.95, 132.33, 131.99, 131.44, 125.32, 125.24,
123.37, 122.43, 122.15, 120.52, 119.49, 117.36. MS (ESI) m/z
Cl ¼ 315.0 [MþH] ; Cl ¼ 317.0 [MþH] .
en-1-one (53)
3
5
þ
37
þ
Compound 53 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
1
-ones reported above. Compound 53 was isolated as a yellow solid
5.3.58. (E)-3-(benzo[b]thiophen-3-yl)-1-(4-ethoxy-2-
hydroxyphenyl)prop-2-en-1-one (58)
ꢀ
1
in 58% yield. mp: 140 C. H NMR (400 MHz, CDCl
.22 (d, J ¼ 15.5 Hz, 1H), 8.11 (d, J ¼ 7.9 Hz, 1H), 7.96-7.91 (m, 3H),
.75 (d, J ¼ 15.5 Hz, 1H), 7.55-7.51 (m, 2H), 7.48-7.44 (m, 1H), 7.08
3
) d: 12.93 (s, 1H),
8
7
Compound 58 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
1-ones reported above. Compound 58 was isolated as a yellow solid
13
(
(
1
dd, J ¼ 8.4, 1.1 Hz, 1H), 6.99 (ddd, J ¼ 8.2, 7.2, 1.1 Hz, 1H). C NMR
100 MHz, CDCl : 193.57, 163.66, 140.59, 137.24, 136.91, 136.42,
32.19, 129.59 (2C), 125.31, 125.23, 123.16, 122.17, 120.37, 120.04,
ꢀ
1
3
)
d
in 52% yield. mp: 152 C. H NMR (400 MHz, CDCl ) d: 13.49 (s, 1H),
3
8.20 (d, J ¼ 15.5 Hz, 1H), 8.13 (d, J ¼ 7.9 Hz, 1H), 7.96-7.92 (m, 2H),
7.86 (d, J ¼ 8.9 Hz, 1H), 7.70 (d, J ¼ 15.5 Hz, 1H), 7.54 (ddd, J ¼ 8.2, 7.1,
þ
118.92, 118.70. MS (ESI) m/z ¼ 281.1 [MþH] .
1
.2 Hz, 1H), 7.49-7.45 (m, 1H), 6.52 (dd, J ¼ 8.9, 2.5 Hz, 1H), 6.50 (d,
13
5.3.54. (E)-3-(benzo[b]thiophen-3-yl)-1-(2-hydroxy-4-
J ¼ 2.5 Hz, 1H), 4.13 (q, J ¼ 7.0 Hz, 2H), 1.47 (t, J ¼ 7.0 Hz, 3H).
C
methoxyphenyl)prop-2-en-1-one (54)
3
NMR (100 MHz, CDCl ) d: 191.65, 166.75, 165.70, 140.59, 137.32,
Compound 54 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
135.87, 132.37, 131.15, 128.87, 125.22, 125.14, 123.12, 122.20, 120.80,
113.97, 108.20, 101.55, 63.99, 14.57. MS (ESI) m/z ¼ 325.2 [MþH] .
þ
1
-ones reported above. Compound 54 was isolated as a yellow solid
ꢀ
1
0
0
0
in 69% yield. mp 215 C. H NMR (400 MHz, CDCl
.20 (d, J ¼ 15.5 Hz, 1H), 8.12 (d, J ¼ 7.9 Hz, 1H), 7.97-7.91 (m, 2H),
.86 (d, J ¼ 8.7 Hz, 1H), 7.69 (d, J ¼ 15.5 Hz, 1H), 7.55-7.51 (m, 1H),
.48-7.44 (m, 1H), 6.55-6.51 (m, 2H), 3.89 (s, 3H). 13C NMR
100 MHz, CDCl : 191.70, 166.76, 166.27, 140.59, 137.31, 135.95,
32.34, 131.17, 128.94, 125.23, 125.14, 123.12, 122.19, 120.73, 114.10,
3
)
d
: 13.51 (s, 1H),
5.3.59. 3 ,4 -dimethoxy-[1,1 -biphenyl]-3-carbaldehyde (59)
The intermediate 59 was prepared according to the general
8
7
7
(
method for the synthesis of biaryl aldehydes reported above.
1
Compound 59 was isolated in 62% yield. H NMR (200 MHz, CDCl
3
)
3
)
d
d: 10.10 (s, 1H), 8.09-8.07 (m, 1H), 7.87-7.81 (m, 2H), 7.60 (t,
1
J ¼ 7.6 Hz, 1H), 7.21 (dd, J ¼ 8.2, 2.0 Hz, 1H), 7.15 (d, J ¼ 2.0 Hz, 1H),
þ
107.84, 101.13, 55.63. MS (ESI) m/z ¼ 311.1 [MþH] .
6.99 (d, J ¼ 8.2 Hz, 1H), 3.98 (s, 3H), 3.95 (s, 3H).
5.3.55. (E)-3-(benzo[b]thiophen-3-yl)-1-(2-hydroxy-4-
5.3.60. 3-(benzo[d] [1,3]dioxol-5-yl)benzaldehyde (60)
methylphenyl)prop-2-en-1-one (55)
The intermediate 60 was prepared according to the general
Compound 55 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
method for the synthesis of biaryl aldehydes reported above.
1
Compound 60 was isolated in 54% yield. H NMR (200 MHz, CDCl
3
)
1
-ones reported above. Compound 55 was isolated as an orange
d: 10.07 (s, 1H), 8.04-8.02 (m, 1H), 7.85-7.75 (m, 2H), 7.59 (d,
J ¼ 7.6 Hz, 1H), 7.13-7.07 (m, 2H), 6.91 (d, J ¼ 8.5 Hz, 1H), 6.02 (s, 2H).
ꢀ
1
solid in 58% yield. mp: 146 C. H NMR (400 MHz, CDCl
H), 8.19 (d, J ¼ 15.5 Hz, 1H), 8.10 (d, J ¼ 8.1 Hz, 1H), 7.93 (s, 1H), 7.91
d, J ¼ 8.1 Hz, 1H), 7.81 (d, J ¼ 8.2 Hz, 1H), 7.72 (d, J ¼ 15.5 Hz, 1H),
.53-7.49 (m, 1H), 7.47-7.41 (m, 1H), 6.85 (s, 1H), 6.77 (dd, J ¼ 8.2,
.2 Hz,1H), 2.38 (s, 3H). 13C NMR (100 MHz, CDCl
: 192.97,163.83,
48.15, 140.59, 137.29, 136.45, 132.28, 129.45, 129.20, 125.26, 125.18,
3
)
d
: 12.94 (s,
1
(
7
1
5.4. General bioassay procedures
3
)
d
5.4.1. In vitro evaluation of activity against L. infantum intra
macrophage amastigotes
1
1
23.13, 122.18, 120.64, 120.24, 118.74, 117.84, 22.04. MS (ESI) m/
The efficacy of compounds 1e13 against L. infantum intracellular
þ
z ¼ 295.1 [MþH] .
amastigotes at 10
16,43].
mM was determined according to the literature
[
5.3.56. (E)-3-(benzo[b]thiophen-3-yl)-1-(4-fluoro-2-
hydroxyphenyl)prop-2-en-1-one (56)
Compound 56 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
5.4.2. In vitro evaluation of activity against L. donovani and L.
major
Infections of bone marrow-derived macrophages and parasite
load quantification was performed as described in literature [16].
1
-ones reported above. Compound 56 was isolated as an orange
ꢀ
1
3
solid in 58% yield. mp: 172e173 C. H NMR (400 MHz, CDCl ) d:
1
3.18 (d, J ¼ 1.4 Hz, 1H), 8.14 (dd, J ¼ 15.4, 0.7 Hz, 1H), 8.03 - 8.00 (m,
5.4.3. In vitro evaluation of activity against T. brucei
1
H), 7.88-7.85 (m, 2H), 7.84-7.82 (m, 1H), 7.58 (d, J ¼ 15.5 Hz, 1H),
The efficacy of compounds against T. brucei bloodstream forms
was evaluated using a modified resazurin-based assay previously
described in literature [16]. The results are reported as the EC50
from the arithmetic average of at least two independent de-
terminations performed in triplicate.
7
.44 (ddd, J ¼ 8.2, 7.1,1.2 Hz,1H), 7.41-7.34 (m,1H), 6.65 (dd, J ¼ 10.4,
13
2
.5 Hz, 1H), 6.60 (ddd, J ¼ 8.9, 8.1, 2.5 Hz, 1H). C NMR (100 MHz,
: 192.42, 167.48 (d, J ¼ 257.0 Hz), 166.24 (d, J ¼ 14.3 Hz),
40.60, 137.20, 137.19, 132.10, 131.86 (d, J ¼ 11.9 Hz), 129.75, 125.35,
3
CDCl ) d
1
125.27, 123.18, 122.13, 120.16, 117.09 (d, J ¼ 2.2 Hz), 107.19 (d,
þ
J ¼ 22.8 Hz), 105.23 (d, J ¼ 23.5 Hz). MS (ESI) m/z ¼ 299.1 [MþH] .
5.4.4. In vitro evaluation of activity against T. cruzi
6
Infections were performed in 6-well plates (3 ꢂ 10 HG39 cells/
5.3.57. (E)-3-(benzo[b]thiophen-3-yl)-1-(4-chloro-2-
well). Confluent HG39 cells were infected with trypomastigotes of
T. cruzi Y strain at a 1:1 ratio. The method used to assess the growth
inhibition effect is based on the LGC Genomics, Berlin kit for the
gDNAs detection. TaqMan™ probe-based quantitative real-time
PCR was performed.
hydroxyphenyl)prop-2-en-1-one (57)
Compound 57 was prepared according to the general method
for the synthesis of (2E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-
1
-ones reported above. Compound 57 was isolated as a yellow solid
ꢀ
1
in 59% yield. mp: 194 C. H NMR (400 MHz, DMSO)
.68 (s, 1H), 8.24 (d, J ¼ 7.6 Hz, 1H), 8.19 (d, J ¼ 8.6 Hz, 1H), 8.13 (d,
J ¼ 15.6 Hz,1H), 8.11 (dd, J ¼ 7.2, 0.8 Hz,1H), 8.04 (d, J ¼ 15.6 Hz,1H),
.59 - 7.53 (m, 1H), 7.51 - 7.47 (m, 1H), 7.13 (d, J ¼ 2.1 Hz, 1H), 7.09
d
: 12.59 (s, 1H),
The assay against a T. cruzi panel of strains was performed as
previously described [44]. Activity values were processed with the
Graphpad Prism software (version 7), for generation of sigmoidal
dose-response (variable slope) non-linear curve fitting and deter-
mination of EC50 values by interpolation. The assay was performed
8
7
13
(
dd, J ¼ 8.6, 2.1 Hz, 1H). C NMR (100 MHz, DMSO)
d: 192.26, 161.91,

C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
19
in duplicate.
v/v CDs; G15: 50% v/v CDs). G16 (7 animals) was treated with free
compound 19 formulated with DMSO 8%; G17 (7 animals) received
the compound 19 with 30% v/v CDs; G18 (n ¼ 7) was treated with
compound 19 with 50% v/v CDs. Compound 19 was administered at
the dose of 40 mg/kg/day (20 mg/kg, every 12 h), for 10 days.
Infected groups G13-G15 received equal volume and followed the
same treatment schedule of vehicles solution than treated groups.
G20 (8 hamsters) was treated with miltefosine (Sigma) (20 mg/kg/
day, 10 days). All treatments were administered PO with 20G
feeding needle. In addition an uninfected and untreated control
group of six animals was included in the experiment (G12). During
the experiments, animals were observed daily and weighed
weekly; blood samples were taken every 15 days and clinical signs
and lesions recorded. Ten to sixteen days after the last treatment,
the animals were euthanatized by isoflurane overdose, bled by
intracardiac puncture and dissected.
5.4.5. Cytotoxicity assessment against THP-1 macrophages
The effect of compounds 1-13 on THP-1-derived macrophages
was assessed by the colorimetric MTT assay with (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The re-
sults are reported as the CC50 and NOAEL from the arithmetic
average of at least two independent determinations performed in
duplicate.
5.4.6. Early ADME-Toxicity assays
These made use of the hERG Predictor™ Fluorescence Polar-
isation Assay (Invitrogen), P450-Glo™ Cytochrome P450 1A2, 2C9,
C19, 2D6 and 3A4 Assays (Promega), CellTiter-Glo® Luminescent
2
Cell Viability Assay (Promega) in the A549 and WI38 cell-lines and
MitoTracker® Red chloromethyl-Xrosamine (CMXRos) uptake and
High Content Imaging for assessment of mitochondrial toxicity as
described in literature [45].
5.4.10. Toxicity of compound 19 and efficacy criteria in hamsters
Clinical course and lesions during the experimental infection
and treatment were recorded. In addition, immediate adverse ef-
fects after medication were annotated. Liver and spleen markers
(AST, ALT, AP, Creatinine and urea) were determined at the end of
the experiment. All animals, after euthanasia, were dissected and
the weight and gross pathology of major target visceral organs,
spleen and liver, were examined. Samples from both organs were
taken and the leishmanial burden determined (Leishman Donovan
Units, LDU) on spleen and liver smears stained with May-Grünwald
Giemsa. Amastigotes/500 host cell nuclei were determined and
efficacy was expressed as the reduction observed in the treated
animal groups compared to the infected and untreated control
hamsters.
5.4.7. Analytical procedure for miltefosine and compound 19
Compound 19 and miltefosine were quantified using Liquid
Chromatography-Mass Spectrometry (LC-MS) with a Gemini C18,
50 mm ꢂ 2.0 mm I.D., 5 m particle size column (Phenomenex
0G-4435-B0). Briefly, plasma samples were mixed 0.5 mL aceto-
nitrile to precipitate proteins. Samples were vortexed and then
centrifuged at 4,020g for 15 min, filtered (0.45 m mesh) (PTFE,
1
0
m
m
VWR) and assayed. Chromatographic separation was carried out
using a Shimadzu liquid cromatograph system consisting of two
pumps, column oven, degasser and autosampler. The HPLC system
was connected to a triple-quadrupole mass spectrometer equipped
with a turboionspray source operated with unit resolution in the
positive ion mode (ESI-QQQMS, Shimadzu LCMS-30) at the “CAI
Espectrometría de Masas”, Complutense University of Madrid
5.4.11. Serum antibody response in hamsters
(
Spain). Plasma samples from mice were used as blank samples for
Enzyme-Linked Immunosorbent Assay (ELISA) was performed
to monitor Leishmania-specific IgG antibodies in peripheral blood
snapshot studies. Calibration standards were freshly prepared
before each analytical run with acetonitrile and purified water (25,
of uninfected control and infected animals. Soluble antigen (40 mg/
5
0, 100, 250, 500 and 1000 ng/mL). The lowest limit of quantifica-
mL) of promastigotes cultures were used for coating Maxisorp® 96-
ꢀ
tion (LLOQ) was 17.6 ng/mL and the lowest limit of detection (LLOD)
was 5.15 ng/mL All procedures were carried out at r.t.
well plates (Nunc) 50
m
L/well at 4 C overnight. PBS-BSA 2% w/v (75
ꢀ
mL/well) was employed to block the plates for 1 h at 37 C. Sera
samples taken at different time points were added 50
50 dilution and incubated at for 2 h at 37 C. Goat anti-hamster IgG
m
L/well at a 1/
ꢀ
5.4.8. Snapshot of compound 19
The pharmacological characteristics of compound 19 were
evaluated by the snapshot method [46]. Compound 19 was
(H þ L)-HRP (Southern Biotech) was used as secondary antibody
(50 mL/well), 1/2000 diluted and incubated for 30 min at r.t. Two to
dispersed in saline isotonic solution plus DMSO (8% v/v) and
five washes with PBS-Tween 20 (0.05% v/v) were included between
steps [47]. O-phenylenediamine (1 mg/mL) (Sigma) plus H (1/
1000) solution was added 100 L/well as HRP-enzyme substrate
SO (3 N) 50 L/well.
ꢃ1
ꢃ1
administered at 1 mg Kg
(IV) and at 20 mg Kg
(PO). For IV
2 2
O
administration, 0.1 mL of the solution was administered to BALB/c
mice (Harlan Laboratories, Barcelona, Spain) by injection in the tail
vein. The volume for PO of compound 19 was 0.2 mL and given to
mice using a 22G feeding needle. Blood samples were taken from
facial vein at 0, 15, 30, 45 and 60 min and then at 3, 24, 48 and 72 h.
Additional sample at 5 min was included from IV administered
animals. Plasma was obtained and analyzed immediately or stored
m
and the reaction was stop with H
2
4
m
5.4.12. Resistance studies
L. donovani strains 1SR, BPK091 and BPK190 were cultivated in
supplemented Medium 199. Selection of strain 1SR was performed
5
by seeding promastigotes at 1 ꢂ 10 cells/mL into medium sup-
ꢀ
at ꢃ20 C until analysis.
plemented with solvent (DMSO) or empirically determined EC50 of
each compound. Cell density was determined daily using a Sch a€ rfe
5
.4.9. Evaluation of efficacy of compound 19 in hamsters
Male Syrian hamsters were obtained from Janvier Labs (France).
System CASY cell counter. After 3 days, cells were diluted into fresh
drug- or solvent-supplemented medium at 1 ꢂ 10 cells/mL. This
5
When the hamsters reached ca. 120 g they were IV infected with
procedure was repeated 11 times. A genomic DNA cosmid library
was prepared from the DNA of L. donovani strain BPK190 as
described before [27]. The cosmid DNA was prepared from that li-
brary and used to electrotransfect L. donovani BPK091 promasti-
gotes as described [27]. Using Cos-Seq DNA analysis [28,29]>90%
genome sequence coverage of the cosmid library in the recombi-
nant parasites was ascertained, and the recombinant population
was subjected to the drug/lead selection scheme outlined above.
Cosmids isolated from the selected populations were subjected to
8
1
0
promastigotes of L. infantum (BCN150) and infection was
allowed to progress for 15 weeks. CDs formulations were prepared
by dispersing compound 19 in hydroxypropyl- -cyclodextrin
Cavasol© W7 HP Pharma, Ashland, Switzerland) up to final con-
b
(
centrations of 30% v/v or 50% v/v CDs v/v in saline isotonic solution.
Infected animals were divided in a weight stratified way into 7
groups. Groups 13e15 (G13-G15), 6 animals each, were the infected
control groups receiving the vehicles (G13: DMSO 8% v/v; G14: 30%

20
C. Borsari et al. / European Journal of Medicinal Chemistry 183 (2019) 111676
NGS on an Illumina MiSeq system and the obtained reads were
aligned to the L. infantum genome (TriTrypDB-41_Linfan-
tumJPCM5_Genome.fasta)using the Bowtie2 algorithm within the
Assemble module of the MacVector™ software suite (versio 12.x).
T. cruzi
CDCl
Trypanosoma cruzi
deuterated trichloromethane
Ethanol
Methanol
sodium hydroxide
3
EtOH
MeOH
NaOH
TMS
IV
5.4.13. UVevisible and fluorescence investigation
Trimethylsilane
intravenous
The one-photon absorption spectra were measured with a Cary
1
00 UVevisible spectrophotometer using 1 cm quartz cuvettes. The
PBS
phosphate-buffered saline
acetonitrile
steady-state fluorescence spectra were obtained on a Spex Jobin-
Yvon FluoroMax3 spectrofluorometer and were corrected for the
spectral sensitivity of the excitation and the emission channels. The
time-resolved emission measurements were performed with a
Horiba FluoroMax4 time-correlated single-photon counting
equipment using a 340 nm emitting 1.3 ns laser diode. To obtain a
lifetime profile, each emission decay was described as the sum of 3
or 4 exponentials, each characterized by a pre-exponential factor
and a decay constant, the reciprocal of the lifetime. A least-squares
deconvolution procedure, based on the classical Marquardt algo-
rithm, was employed (DAS-6 Horiba software). The estimated time
resolution after deconvolution is 200 ps HSA (Sigma, ꢁ97% purity),
BSA (Sigma, ꢁ 99% purity), pepsin from porcine gastric mucosa
ACN
EC50
CC50
THP1
A549
HSA
BSA
half maximal effective concentration
half maximal cytotoxicity concentration
human monocytic cell line
human lung adenocarcinoma epithelial cell line;
human serum albumin
bovine serum albumin
Miltefosine.
MIL
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NTDs
HAT
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Human African trypanosomiasis
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DNDi
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