Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds

Article abstract of DOI:10.1016/j.ejmech.2016.12.017

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2’-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, com

Full text of DOI:10.1016/j.ejmech.2016.12.017

Accepted Manuscript
Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds
Chiara Borsari, Nuno Santarem, Juan Torrado, Ana Isabel Olías, María Jesús Corral,
Catarina Baptista, Sheraz Gul, Markus Wolf, Maria Kuzikov, Bernhard Ellinger,
Jeanette Reinshagen, Pasquale Linciano, Annalisa Tait, Luca Costantino, Lucio
H. Freitas-Junior, Carolina B. Moraes, Pascoalino Bruno dos Santos, Laura Maria
Alcântara, Caio Haddad Franco, Claudia Danielli Bertolacini, Vanessa Fontana,
Paloma Tejera Nevado, Joachim Clos, José María Alunda, Anabela Cordeiro-da-
Silva, Stefania Ferrari, Maria Paola Costi
PII:
S0223-5234(16)31023-6
10.1016/j.ejmech.2016.12.017
EJMECH 9108
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 October 2016
Revised Date: 6 December 2016
Accepted Date: 8 December 2016
Please cite this article as: C. Borsari, N. Santarem, J. Torrado, A.I. Olías, M.J. Corral, C. Baptista, S.
Gul, M. Wolf, M. Kuzikov, B. Ellinger, J. Reinshagen, P. Linciano, A. Tait, L. Costantino, L.H. Freitas-
Junior, C.B. Moraes, P. Bruno dos Santos, L.M. Alcântara, C.H. Franco, C.D. Bertolacini, V. Fontana,
P. Tejera Nevado, J. Clos, J.M. Alunda, A. Cordeiro-da-Silva, S. Ferrari, M.P. Costi, Methoxylated 2'-
hydroxychalcones as antiparasitic hit compounds, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2016.12.017.
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ACCEPTED MANUSCRIPT
Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds
a
b
c
c,e
c,e
Borsari, Chiara ; Santarem, Nuno ; Torrado, Juan ; Olías, Ana Isabel ; Corral, María Jesús ; Baptista,
b
d
d
d
d
d
Catarina ; Gul, Sheraz ; Wolf, Markus ; Kuzikov, Maria ; Ellinger, Bernhard ; Reinshagen, Jeanette ;
a
a
a
f
f
Linciano, Pasquale ; Tait, Annalisa ; Luca Costantino ; Freitas-Junior, Lucio H. ; Moraes, Carolina B. ;
f
f
f
Pascoalino, Bruno dos Santos ; Alcântara, Laura Maria ; Franco, Caio Haddad ; Bertolacini, Claudia
f
f
g
g
c,e
Danielli ; Fontana, Vanessa ; Tejera Nevado, Paloma ; Clos, Joachim ; Alunda, José María ; Cordei-
b
a
a*
ro-da-Silva, Anabela ; Ferrari, Stefania ; Costi, Maria Paola .
a
University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy
b
Institute for Molecular and Cell Biology, 4150-180 Porto, Portugal and Instituto de Investigação e
Inovação em Saúde, Universidade do Porto and Institute for Molecular and Cell Biology, 4150-180
Porto, Portugal
c
Complutense University of Madrid, 28040 Madrid, Spain
d
Fraunhofer Institute for Molecular Biology and Applied Ecology Screening Port, Hamburg, Germany
e
Instituto de Investigación Hospital 12 de Octubre, Madrid
f
Brazilian Centre for Research in Energy and Materials, Campinas, Brazil.
g
Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany.
Abstract
Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2’-hydroxy
methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma
cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the
most potent and selective anti-T. brucei compounds (EC50 = 1.3 – 4.2 µM, selectivity index >10-fold).
Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of com-
1

ACCEPTED MANUSCRIPT
pound 4 in BALB/c mice using hydroxypropil-β-cyclodextrins formulation showed a 7.5 times increase
in oral bioavailability.
Keywords. Chalcones, Trypanosoma brucei, Trypanosoma cruzi, Leishmania infantum, Early Toxicity
studies, cyclodextrin formulations.
1
. Introduction
The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are respon-
sible for potentially fatal insect-borne diseases namely, respectively, Human African Trypanosomiasis
(
HAT), Chagas disease and Leishmaniasis [1-3]. Despite the serious health, economic and social con-
sequences of these parasitic infections, there are no available vaccines. Therefore, disease control relies
only on chemotherapy and prophylaxis. The available drugs suffer from many drawbacks including
toxicity, poor efficacy and drug resistance [4]. Hence, there is an urgent requirement for new, safe and
effective drugs. Phenotypic screening approaches have been successfully used in the field of neglected
diseases, particularly for the treatment of HAT [5]. Two compounds, discovered through a phenotypic
screening, have recently been progressed into clinical trials by DNDi: fexinidazole, a nitroimidazole,
and SCYX-7158, an oxaborole [6]. In the drug discovery process for new antileishmanial and antitryp-
anosomal drugs, phenotypic screening and follow up optimization were largely applied and provided a
wide range of chemical structures, including chalcones (1,3-diphenyl-2-propen-1-one derivatives) [7-
1
4]. Chalcones exist as E- or Z-isomers. The E-isomer is the thermodynamically most stable form;
thus, chalcones are usually isolated as E- stereoisomers after recrystallization of a Z-E mixture [15].
Chalcones display a broad spectrum of pharmacological activities including anti-oxidant, antifungal,
anti-infective, anti-inflammatory, anticancer and antinociceptive properties, and have been shown to
affect a great variety of parasitic targets [16-20]. The chalcone scaffold has been widely explored for
the antileishmanial activity [21,22], while a limited number of compounds bearing the 1,3-diphenyl-2-
2

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propen- 1-one moiety and active towards T. brucei has been reported in literature. One of the most
studied antileishmanial chalcones is licochalcone A (Figure 1, compound A), a natural chalcone, that
inhibits the in-vitro growth of L. major promastigotes (IC50 = 21 µM) and L. donovani amastigotes
(
IC = 2.7 µM). Intraperitoneal administration of licochalcone A prevents the development of lesions
50
in BALB/c mice infected with L. major and reduces the parasite load in the spleen and liver of ham-
sters infected with L. donovani [23]. Licochalcone A was found to inhibit the activity of the parasite
mitochondrial dehydrogenase and to alter the ultrastructure of mithocondria [24]. Very recently, fla-
vokawin B, a 2’-hydroxy methoxylated natural chalcone (Figure 1, compound B), has been reported to
inhibit T. brucei parasite growth (T. brucei strain 427 IC50 = 6.2 µM) [25]. In our previous work, meth-
oxylated flavonols (Figure 1, scaffold C) proved to be active against T. brucei and T. cruzi showing
EC50 in the range of 1-8 µM [11]. The best compounds had shown a potency analogue or higher than
flavokavin B against T. brucei. Based on the mentioned results, we focused our attention on the open
analogues of the flavone heterocycle (Figure 1, scaffold D). Thirteen 2’- hydroxy methoxylated chal-
cones (1-13, Table 1) were synthesized and evaluated in-vitro towards cultured T. brucei, T. cruzi and
L. infantum aiming to develop novel antiparasitic compounds. We evaluated the cytotoxicity of the
compounds on THP-1 cells and we assessed a panel of in-vitro toxicological properties in order to iden-
tify safe hits. We selected compound 4 for pharmacokinetic studies in BALB/c mice and we investigat-
ed the use of cyclodextrins as drug delivery system.
2
. Results and discussion
2
.1. Chemistry
Herein, we synthesized a library of thirteen 2’-hydroxy chalcones bearing methoxy groups (1-13, Table
). Eight compounds (1-3, 5, 6, 9-11) are intermediates of the previously published flavonols11, while
1
five additional chalcones (4, 7, 8, 12 and 13) were synthesized to explore the Structure Activity Rela-
tionship (SAR) of chalcones showing methoxy groups in different positions of ring A and B. Com-
3

ACCEPTED MANUSCRIPT
pounds 1-3, 5-7, 9-12, 13 have been previously described in literature [11, 26-28], while compounds 4
and 8 are novel structures. The chalcones (1-13) were synthesized by aldol condensation of substituted
acetophenones and benzaldehydes in presence of NaOH (3M) as previously reported in literature [29].
The general synthetic procedure is reported in Scheme 1. LogP was calculated with ALOGPS 2.1 to
evaluate a possible effect of the lipophylicity on the biological activity. All compounds showed ana-
logue logP (~3.5, Table 1).
2
.2. Biological evaluation
All the synthesized compounds were assessed for their antiparasitic activity against the bloodstream
form of T. brucei (Table 1, Figure 2 and Table S1) and the intracellular T. cruzi at 10 µM (Figure 2 and
Table S3). The library was tested against L. infantum amastigotes (Figure 2 and Table S2) at a higher
concentration (50 µM) since it is usually more difficult to identify anti-leishmanial hits. Eight com-
pounds (1-4, 7, 8, 12 and 13) showed an antiparasitic activity towards T. brucei higher than 85%, thus
dose-response studies were performed (Table 1 and Table S1 Supporting Information). The eight com-
pounds showed EC50 values against T. brucei L427 WT bloodstream form in the low micromolar range
(
1.3-7.4 µM). In 3-methoxy substituted chalcones, the presence of methoxy groups on ring A did not
remarkably influence the activity towards T. brucei. In fact, compounds 1-4 showed a comparable ac-
tivity (EC = 1.3, 3.4, 2.1 and 4.4 µM, respectively), with compound 1 bearing no methoxy groups on
5
0
ring A being the most potent. It is worth noting that while the -OCH group at position 3 of ring B pro-
3
vides activity, the same group at position 4 renders the molecule inactive, as highlighted by comparing
the activity of compounds 2 and 3 with that of their corresponding 4-methoxy chalcones (compounds 5
and 6). In addition, the presence of -OCH group at both 3 and 4 positions on ring B makes compounds
3
9
and 10 almost inactive. A clear effect of the methoxy group on compounds antiparasitic activity
could not be observed for 3,4,5-trimethoxy-substituted compounds: compound 11 was inactive, while
compounds 12 and 13 showed low micromolar activity against T. brucei (EC = 4.4 and 7.4 µM, re-
50
4

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spectively). Compounds 7 and 8 bearing methoxy groups at both positions 2 and 6 on ring B showed
EC50 in the low micromolar range (EC50 = 3.4 and 2.1 µM, respectively). With the exception of com-
pounds 11-13, the activity against T. brucei of methoxylated chalcones is guided by the position of
methoxy groups on ring B, while the presence of a methoxy group in position 4’ or 5’ of ring A does
not significantly influence the chalcones activity. We evaluated the cytotoxicity of methoxy-substituted
2
1
’-hydroxy chalcones on PMA-differentiated THP1 cells and CC (or NOAEL) was determined (Table
50
). Compounds 1, 3, 4, 7 and 8 showed a selectivity index (SI = CC /EC ) higher than 10, with com-
5
0
50
pound 7 being the most selective (29-fold). Most of the tested compounds were active against T.
brucei, while only compounds 11, 12 and 13, bearing a 3,4,5-trimethoxy substituted ring, had moderate
activity towards L. infantum (Figure 2). The EC50 values towards L. infantum of compounds 11, 12 and
1
3 were 30.3, 42.2 and 22.2 µM, respectively (Table S2, Supporting Information). Seven compounds
1, 2, 4, 5, 8, 9, 11) were able to reduce the parasite load of T. cruzi-infected HG39 cells by more than
0% at 10 µM, with compound 2 being the most potent (Figure 2). Compounds 2, 8, 9 and 11 showed
EC towards T. cruzi lower than 10 µM (Table S3, Supporting Information). These results indicate that
(
4
50
methoxylated 2’-hydroxy chalcones showed interesting antiparasitic activity in particular towards the
bloodstream form T. brucei.
2
.3.Early toxicology studies
The chalcones library was assessed at 10 µM in a panel of assays including hERG, Aurora B kinase,
cytochromes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity (A549 and W1-38
cell lines) and mitochondrial toxicity. Among the 13 compounds evaluated for hERG liability, only
compounds 6 and 11 decrease hERG activity by more than 25%. Most of the compounds inhibited at
least one cytochrome isoform. None of the compounds evaluated towards Aurora B kinase showed
inhibitory activity at the tested concentration. None of the compounds turned out to be either cytotoxic
or mitotoxic. These data have been summarised using a traffic light system in Figure 3 with absolute
5

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values being reported in Table S4 of the Supporting Information. After evaluating the data obtained
from the different assays, we selected compound 4 for further studies. The activity of compound 4 to-
wards hERG (IC50 >100 µM) was more than 20-fold greater than T. brucei EC50 (EC50 = 4.2 µM), thus
consistent with the parameters defined for progressing a compound from in-vitro studies to early phar-
macokinetic studies in mice (target-product profile) [11]. Compound 4 did not inhibit two cytochrome
isoforms (CYP2C9 IC = 42.1 µM and CYP3A4 IC = 27.4 µM), while it strongly inhibited CYP1A2
5
0
50
(
IC = 1.5 µM) and moderately inhibited CYP2C19 and CYP2D6 (IC = 7.7 and 6.8 µM, respective-
50 50
ly). The toxicological profile of pentamidine, the reference compound for T. brucei infections, is re-
ported in Table S4 of the Supporting Information.
2
.4. Solubilization of compound 4 with cyclodextrins and snapshot PK
The solubility of compound 4 was evaluated using UV-Vis spectroscopy. Pharmacokinetics of com-
pound 4 in BALB/c mice (1 mg/kg, IV) was characterized by low plasma concentrations (Figure 4A).
Thus, compound 4 was complexed with hydroxypropil-β-cyclodextrins (50%, w/v). The bioavailability
of compound 4 alone and formulated with cyclodextrins was determined in BALB/c mice using LC-
MS. The data are reported in Table S5 and S6 of the Supporting Information. Compound 4 adminis-
tered by IV injection showed a biphasic elimination behavior. The half life (t ½) of the free compound
after IV administration was 62.5 min (Figure 4A), while after oral administration was 12.6 min (Figure
4
B). The t ½ of compound 4 in cyclodextrins after oral administration was 36.7 min and the cyclodex-
trins increased by ca. 7.5 times the oral bioavailability of compound 4 (Figure 4B).
. Conclusion
3
Herein, we discovered methoxylated 2'-hydroxychalcones as potent anti-T. brucei agents. The best
compounds (1-4, 7, 8, 12, 13) showed similar or higher potency (EC = 1.3- 7.4 µM) with respect to
5
0
the recently isolated natural compound flavokawin B (T. brucei strain 427 IC = 6.2 µM) [25], with
5
0
compound 1 being over 4 times more potent than flavokawin B. To our knowledge none of our meth-
6

ACCEPTED MANUSCRIPT
oxylated 2'-hydroxychalcones has been previously reported in literature as anti-trypanosomatidic agent.
A panel of in-vitro early toxicological properties was evaluated. Chalcones showed a toxicological pro-
file similar to that of the previously identified flavonols [11] showing some effects on CYP isoforms.
The selectivity index of compound 1 is almost 5 times higher than that of the corresponding flavonol
(
Table S6 of the Supporting Information). Compound 4 (EC T. brucei = 4.2 µM, SI = 12) was select-
50
ed for pharmacokinetic studies. The formulation of compound 4 with cyclodextrins led to an increase
of the oral bioavailability of the molecule by 7.5 fold and to a half-life of 36.7 min. Hence, chemical
modifications together with further optimization of the drug delivery systems and the understanding of
the molecular targets could lead to fully exploit the anti-parasitic potential of methoxylated 2’-
hydroxychalcones.
4
. Material and methods
4
.1. Synthesis
The general procedure for the synthesis of (2E)-1-(2-hydroxyphenyl)- 3-phenylprop-2-en-1-ones is
reported in the Supporting Information together with the spectral data of all remaining compounds.
ALOGPS 2.1 program (http://www.vcclab.org/lab/alogps/) was used to calculate logP.
(
2E)-1-(2-hydroxy-4,5-dimethoxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one (4) was isolated as
1
an orange solid in a 40% yield. Mp. [119-120 °C]. H NMR (CDCl , 400 MHz) δ: 13.18 (s,1H, OH),
3
7
1
.88 (d, 1H, J = 15.4 Hz, H ), 7.51 (d, 1H, J = 15.4 Hz, H ), 7.38 (dd, 1H, J = 7.9 Hz, H-5), 7.28 (m,
B A
H, H-6), 7.27 (s, 1H, H-6') 7.19 (m, 1H, H-2), 7.00 (ddd, 1H, J 4,5 = 8.2 Hz, J = 2.6, 0.9 Hz, H-4), 6.53
1
3
(
s, 1H, H-3'), 3.96 (s, 1H, 4'-OCH ), 3.94 (s, 1H, 5'-OCH ), 3.89 (s, 3H, 3-OCH ). C NMR (CDCl ,
3 3 3 3
1
1
3
00 MHz) δ: 190.47, 161.87, 159.99, 157.19, 144.43, 142.00, 136.22, 130.03, 121.12, 120.74, 116.08,
+
14.00, 112.02, 111.10, 100.87, 57.01, 56.23, 55.43. ESI-HRMS calcd. for C18
15.1227, found 315.1228.
19 5
H O [M+H]
7

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(
2E)-3-(2,6-dimethoxyphenyl)-1-(2-hydroxy-5-methoxyphenyl)prop-2-en-1-one (8) was isolated as
1
an orange solid in a quantitative yield. Mp. [151-152°C]. H NMR (CDCl
3
, 400 MHz) δ: 12.75 (br s,1H,
), 7.40 (d, 1H, J 4',6' = 2.9 Hz, H-
'), 7.34 (t , 1H, J = 8.4 Hz, H-4), 7.13 (dd, J 4',3' = 9.1, J 4',6' = 2.9 Hz, H-4'), 6.98 (d, 1H, J 3',4' = 9.1 Hz,
B A
OH), 8.41 (d, 1H, J B,A= 15.7 Hz, H ), 8.11 (d, 1H, J A,B = 15.7 Hz, H
6
1
3
H-3'), 6.61 (d, 1H, J = 8.4 Hz, H-2 + H-5), 3.96 (s, 3H, -OCH ), 3.85 (s, 3H, -OCH ). C NMR
3
3
(
CDCl , 100 MHz) δ: 195.11, 160.60, 157.84, 151.53, 136.57, 132.12, 123.31, 122.76, 120.13, 119.08,
3
+
1
3
13.12, 112.73, 103.80 (2C), 55.94 (3C). ESI-HRMS calcd. for C H O [M+H] 315.1227, found
1
8
19
5
15.1227.
4
4
.2. In vitro Antiparasitic evaluation
.2.1. In vitro evaluation of activity against L. infantum intramacrophage amastigotes
The efficacy of compounds 1-13 against L. infantum intracellular amastigotes at 10 µM was deter-
mined according to literature [30]. The Operetta high-content automated imaging system was used to
acquire images and the Harmony Software was optimized quantifying host cells number, infection ratio
and number of parasites per infected cell. The ratio between infected cells and total number of cells was
then calculated, and defined as the Infection Ratio (IR) [31].
4
.2.2. In vitro evaluation of activity against T. brucei
The efficacy of compounds against T. brucei bloodstream forms was evaluated using a modified resaz-
urin-based assay previously described in literature [32]. The reported EC50 are the arithmetic average of
at least two independent determinations done in triplicate.
4
.2.3. In-vitro evaluation of activity against T. cruzi
Infections were performed in 6-well plates (3 x 106 HG39 cells/well). Confluent HG39 cells were in-
fected with sanguineous 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 [33].
TaqMan™ probe-based quantitative real-time PCR was performed.
8

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4
4
.3. Early In vitro Toxicology study
.3.1. Cytotoxicity assessment against THP-1 macrophages
The effect of compounds 1-13 on THP-1-derived macrophages was assessed by the colorimetric MTT
assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The reported CC and NOAEL
5
0
are the arithmetic average of at least two independent determinations done in triplicate.
.3.2. hERG assay
4
This assay made use of Invitrogen’s Predictor™ hERG Fluorescence Polarisation Assay. The assay
uses a membrane fraction containing hERG channel (Predictor™ hERG Membrane) and a high-affinity
red fluorescent hERG channel ligand, or “tracer” (Predictor™ hERG Tracer Red), whose displacement
by test compounds can be determined in a homogenous, Fluorescence Polarisation (FP) based format
[
11].
4
.3.3. Cytochrome P450 1A2, 2C9, 2C19, 2D6 and 3A4 assays.
These assays made use of the PromegaP450-Glo™assayplatform. Each CYP450 assay made use of
microsomal preparations of cytochromes from baculovirus infected insect cells. Action of the CYP450
enzymes upon each substrate ultimately resulted in the generation of light and a decrease in this was
indicative of inhibition of the enzymes [11].
4
.3.4. Cytotoxicity assay against A549 and WI38 cells
The assays were performed using the Cell Titer-Glo assay from Promega. The assay detects cellular
ATP content with the amount of ATP being directly proportional to the number of the present cells.
The A549 cells were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany) and WI38cells were obtained from ATCC (ATCC® CCL- 75™) and were
grown in DMEMwith FCS (10% v/v), streptomycin (100 µg/ml) and penicillin G (100 U/ml) [11].
4
.3.5. Aurora B kinase assay
9

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This assay made use of the ADP-Glo Kinase Enzyme System from Promega, which is a bioluminescent
assay that employs firefly luciferase in a coupled-enzyme assay format to enable detection of ADP lev-
els from ATPase assays [11].
4
.3.6. Assessment of mitochondrial toxicity
This assay made use of MitoTracker® Red chloromethyl-Xrosamine (CMXRos) uptake and High Con-
tent Imaging to monitor compound mediated mitochondrial toxicity in the 786-O (renal carcinoma) cell
line. Cells were maintained using RPMI-1640 medium containing 2 mM glutamine, FCS (10% v/v),
streptomycin (100 µg/ml) and penicillin G (100 U/ml) [11].
4
4
.4. Pharmacokinetic evaluation
.4.1. Solubility of compound 4 using UV-Vis spectroscopy
UV-visible spectroscopy was used to measure the solubility of compound 4. The experiments were
carried out with the instrument CARY 50. Six different solutions (3.125, 6.25, 12.5, 25, 50 and 100
µM) were prepared. The maximum of absorbance was found at 312 nm and epsilon was calculated.
Four saturated solutions were prepared: 100 µM in water, 100 µM in PBS, 200 µM in PBS + 10 %
DMSO and 10 mM in PBS + 50 % DMSO. The suspensions were left in incubation at 25°C for 18
hours, then filtered (0.2 µm). Maximum solubility in water, phosphate buffered saline (PBS) and PBS
with 10% or 50% of DMSO were, respectively, 0.91, 2.37 µM, 11.41 µM and 0.6 mM.
4
.4.2. Cyclodextrins solubilization of compound 4
Hydroxypropil-β-cyclodextrin (50%, w/v) was used to solubilize compound 4 as previously described
[
11].
4
.4.3. Pharmacokinetics of compound 4 using LC-MS
The snapshot PK studies were carried out according to literature [34]. BALB/c mice were treated with
compound 4 administered alone (8% DMSO) (1 mg/kg IV or 20 mg/kg per os), compound 4 solubil-
ized
1
0

ACCEPTED MANUSCRIPT
with hydroxypropil-β-cyclodextrin (50%) (20 mg/kg per os) or with a mixture of cyclodextrins (25%)
plus polyethylene glycol 400 (Quimidroga) (50%) (20 mg/kg per os). Plasma samples were analyzed
by LC-MS. Chromatographic separation was carried out using a Shimadzu LC system consisting of
two pumps, column oven, degasser and autosampler. Attached to this system was an analytical column
(
C18, Gemini 5 µm 110 A Phenomenex, 150 x 2 mm). The HPLC system was connected to a tri-
plequadrupole
mass spectrometer equipped with a turboionspray source operated with unit resolution in the positive
ion mode (ESI-QQQMS, Shimadzu LCMS-30). Under these conditions a retention time of approxi-
mately 4 minutes for the molecule was obtained. Mobile phase consisted of a mixture (80:20) of ace-
tonitrile: purified water flow at a 0.2 ml/min rate in isocratic mode. Run time was 10 minutes and the
injection volume was 5 µl. The mass transition of m/z was: Quantifier (m/z): 315.0 > 161.0 CE: - 20;
Qualifier (m/z): 315.0 > 132.9 CE: -34. Plasma samples of mice (NMRI and BALB/c) were obtained
by serial sampling from submandibular vein and stored at -20 ºC until analyzed.
Ethics Statement
The experimental design and housing conditions at UCM were approved by the Committee of Animal
Experimentation (Universidad Complutense de Madrid) and regional authorities (Community of Ma-
drid) (Ref. PROEX 169/15). Experiments were carried out at the animal house with official identifica-
tion code ES280790001164 following the 3Rs principles. Animal handling and sampling were per-
formed by trained and officially qualified personnel.
Supporting Information.
Antiparasitic activity against Trypanosoma brucei, Leishmania infantum and Trypanosoma cruzi (Ta-
ble S1, S2 and S3); ADME-Tox data (Table S4); Concentration of compound 4 in BALB/c mice plas-
ma at different sampling times after intravenous injection (IV) (1 mg/kg) (Table S5); Concentration of
compound 4 in BALB/c mice plasma at different sampling times after oral administration of 20 mg/kg
1
1

ACCEPTED MANUSCRIPT
compound 4 formulated with cyclodextrins, cyclodextrins + PEG 400 or administered free (Table S6);
Comparison of activity and selectivity between eight 2’- hydroxy methoxylated chalcones and their
corresponding methoxylated flavonols already published (Table S7); Experimental data of the synthe-
sized compounds (pp. S11- S16); 1H-NMR and 13C-NMR spectra of compounds 4 and 8 (pp. S17-
S20).
Molecular formula strings (CSV).
Corresponding Authors
*
M.P.C. phone, 0039-059-205-8579; E-mail, mariapaola.costi@unimore.it.
Acknowledgment
This project has received funding from the European Union’s Seventh Framework Programme for re-
search, technological development and demonstration under grant agreement n° 603240 (NMTrypI -
New Medicines for Trypanosomatidic Infections). http://www.nmtrypi.eu/. Ministry of Education and
Research (MIUR - project PRIN 2012, grant number 2012-74BNKN), COST ACTION 1307 for the
discussion on the data obtained. The Authors acknowledge CIGS (Centro Interdipartimentale Grandi
Strumenti) for assistance in NMR, MS analysis.
Abbreviations used
NTDs, Neglected Tropical diseases; HAT, Human African trypanosomiasis; DNDi, Drugs for Neglect-
ed Diseases initiative; L. infantum, Leishmania infantum; L. donovani, Leishmania donovani; T. brucei,
Trypanosoma brucei; T. cruzi, Trypanosoma cruzi; CDCl , deuterated trichloromethane, EtOH, Etha-
3
nol; NaOH, sodium hydroxide; TMS, Trimethylsilane; IV, intravenous; PBS, phosphate-buffered sa-
line; ACN, acetonitrile; FBS, fetal bovine serum; EC50, half maximal effective concentration; CC50
,
half maximal cytotoxicity concentration; THP1, human monocytic cell line; A549, human lung adeno-
carcinoma epithelial cell line; WI-38, fetal lung fibroblasts cell lines; NOAEL, no adverse effect level.
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References
1] K. Nussbaum, J. Honek, C.M. Cadmus, T. Efferth, Trypanosomatid parasites causing neglected
diseases, Curr. Med. Chem. 17 (2010) 1594-1617.
[
[
2] K.R. Matthews, The developmental cell biology of Trypanosoma brucei, J. Cell Sci. 118 (2005)
83-290.
2
[
3] E.K. Elmahallawy, A. Agil, Treatment of leishmaniasis: a review and assessment of recent research,
Curr. Pharm. Des. 21 (2015) 2259-2275.
[
4] E. Castillo, M.A. Dea-Ayuela, F. Bolás-Fernández, M. Rangel, M.E. González-Rosende, The kinet-
oplastid chemotherapy revisited: current drugs, recent advances and future perspectives. Curr. Med.
Chem. 17 (2010) 4027-4051.
[
5] M.L. Sykes, V.M. Avery, Approaches to protozoan drug discovery: phenotypic screening. J. Med.
Chem. 56 (2013) 7727-7740.
[
6] G. Eperon, M. Balasegaram, J. Potet, C. Mowbray, O. Valverde, F. Chappuis, Treatment options for
second-stage gambiense human African trypanosomiasis. Expert Rev. Anti. Infect. Ther. 12 (2014)
407-1417.
1
[
7] I.H. Gilbert, D. Leroy, J.A. Frearson, Finding new hits in neglected disease projects: target or phe-
notypic based screening? Curr Top Med Chem. 11 (2011) 1284-1291.
[
8] I.H. Gilbert, Drug Discovery for Neglected Diseases: Molecular Target-Based and Phenotypic Ap-
proaches. J Med Chem. 56 (2013) 7719–7726.
1
3

ACCEPTED MANUSCRIPT
[
9] A. Cavazzuti, G. Paglietti, W.H. Hunter, F. Gamarro, S. Piras, M. Loriga, S. Allecca, P. Corona, K.
McLuskey, L. Tulloch, F. Gibellini, S. Ferrari, M.P. Costi, Discovery of potent pteridine reductase in-
hibitors to guide antiparasite drug development. Proc Natl Acad Sci U S A. 105 (2008) 1448–1453.
[
10] P. Corona, F. Gibellini, A. Cavalli, P. Saxena, A. Carta, M. Loriga, R. Luciani, G. Paglietti, D.
Guerrieri, E. Nerini, S. Gupta, V. Hannaert, P.A. Michels, S. Ferrari, M.P. Costi, Structure-based selec-
tivity optimization of piper-idine-pteridine derivatives as potent Leishmania pteridine reductase inhibi-
tors. J Med Chem. 55 (2012) 8318-8329.
[
11] C. Borsari, R. Luciani, C. Pozzi, I. Pöhner, S. Henrich, M. Trande, A. Cordeiro-da-Silva, N. Santa-
rém, C. Baptista, A. Tait, F. Di Pisa, L. Dello Iacono, G. Landi, S. Gul, M. Wolf, M. Kuzikov, B. El-
linger, J. Reinshagen, G. Witt, P. Gribbon, M. Kohler, O. Keminer, B. Behrens, L. Costantino, P. Teje-
ra Nevado, E. Bifeld, J. Eick, J. Clos, J. Torrado, M.D. Jiménez- Antón, M.J. Corral, J.M. Alunda, F.
Pellati, R.C. Wade, S. Ferrari, S. Mangani, M.P. Costi, Profiling of flavonol derivatives for the devel-
opment of anti-trypanosomatidic drugs. J. Med. Chem. 59 (2016) 7598-7616.
[
12] I. Peña, M.P. Manzano, J. Cantizani, A. Kessler, J. Alonso- Padilla, A.I. Bardera, E. Alvarez, G.
Colmenarejo, I. Cotillo, I. Roquero, F. de Dios-Anton, V. Barroso, A. Rodriguez, D.G. Gray, M. Na-
varro, V. Kumar, A. Sherstnev, D.H. Drewry, J.M. Brown, M.J. Fiandor, J.J. Martin, New Compound
Sets Identified from High Throughput Phenotypic Screening Against Three Kinetoplastid Parasites: An
Open Resource. Scientific Reports 5 (2015) Article number: 8771
[
13] P. Boeck, C.A. Bandeira Falcão, P.C. Leal, R.A. Yunes, V.C. Filho, E.C. Torres-Santos, B. Rossi-
Bergmann, Synthesis of chalcone analogues with increased antileishmanial activity. Bioorg. Med.
Chem. 14 (2006) 1538-1545.
1
4

ACCEPTED MANUSCRIPT
[
14] L. Troeberg, X. Chen, T.M. Flaherty, R.E. Morty, M. Cheng, H. Hua, C. Springer, J.H. McKer-
row, G.L. Kenyon, J.D. Lonsdale-Eccles, T.H. Coetzer, F.E. Cohen, Chalcone, acyl hydrazide, and
related amides kill cultured Trypanosoma brucei brucei. Mol. Med. 6 (2000) 660-669.
[
15] M. Larsen, H. Kromann, A. Kharazmi, S.F. Nielsen, Conformationally restricted anti-plasmodial
chalcones. Bioorg. Med. Chem. Lett. 15 (2005) 4858-4861.
[
16] T. Hussain, H.L. Siddiqui, M. Zia-ur-Rehman, M. Masoom Yasinzai, M. Parvez, Anti-oxidant,
anti-fungal and antileishmanial ctivities of novel 3-[4-[1H-imidazol-1- yl]phenyl]prop-2-en-1-ones.
Eur. J. Med. Chem. 44 (2009) 4654-4660.
[
17] H.J. Shin, D.H. Shon, H.S. Youn, Isobavachalcone suppresses expression of inducible nitric oxide
synthase induced by Toll-like receptor agonists. Int. Immunopharmacol. 15 (2013) 38-41.
[
18] D.K. Mahapatra, S.K. Bharti, V. Asati, Anti-cancer chalcones: Structural and molecular target
perspectives. Eur. J. Med. Chem. 98 (2015) 69-114.
19] R. Corrêa, M.A. Pereira, D. Buffon, L. dos Santos, V. Cechinell Filho, A.R. Santos, R.J. Nunes,
Antinociceptive properties of chalcones. Structure-activity relationships. Arch. Pharm. [Weinheim].
34 (2001) 332-334.
[
3
[
20] D.K. Mahapatra, S.K. Bharti, V. Asati, Chalcone scaffold as anti-infective agents: structural and
molecular target perspectives. Eur. J. Med. Chem. 101 (2015) 496-524.
[
21] E.C. Torres-Santos, M.I. Sampaio-Santos, F.S. Buckner, K. Yokoyama, M. Gelb, J.A. Urbina, B.
Rossi-Bergmann, Altered sterol profile induced in Leishmania amazonensis by a natural dihydroxy
methoxylated chalcone. J. Antimicrob. Chemother. 63 (2009) 469-472.
1
5

ACCEPTED MANUSCRIPT
[
22] J. Piñero, R.M. Temporal, A.J. Silva-Gonçalves, I.A. Jiménez, I. Bazzocchi, A. Oliva, A. Perera,
L.L. Leon, B. Valladares, New administration model of trans-chalcone biodegradable polymers for the
treatment of experimental leishmaniasis. Acta Trop. 98 (2006) 59-65.
[
23] M. Chen, S.B. Christensen, T.G. Theander, A. Kharazmi, Antileishmanial activity of licochalcone
A in mice infected with Leishmania major and in hamsters infected with Leishmaniadonovani. ntimi-
crob. Agents Chemother. 38 (1994) 1339-1344.
[
24] L. Zhai, J. Blom, M. Chen, S.B. Christensen, A. Kharazmi, The antileishmanial agent licochal-
cone A interferes with the function of parasite mitochondria. Antimicrob. Agents Chemother. 39 (1995)
742-2748.
2
[
25] D.F. Rodrigues, D.A. Maniscalco, F.A. Silva, B.G. Chiari, M.V. Castelli, V.L. Isaac, R.M.
Cicarelli, S.N. López, Trypanocidal Activity of Flavokawin B, a Component of Polygonum ferrugine-
um Wedd. Planta Med.2016. [Epub ahead of print]
[
26] X.G. Bai, C.L. Xu, S.S. Zhao, H.W. He, Y.C. Wang, J.X. Wang, Synthesis and cytotoxic evalua-
tion of alkoxylated chalcones. Molecules 19 (2014) 17256-17278.
[
27] A.S. Albogami, U. Karama, A.A. Mousa, M. Khan, S.A. Al- Mazroa, H.Z. Alkhathlan, Simple and
efficient one step synthesis of functionalized flavanones and chalcones. Oriental Journal of Chemistry
8 (2012) 619-626.
2
[
28] G. Zheng, Z. Zhang, B. Kang, R. Yu, Y. Cao, S. Zhang, Synthesis and vasodilatation of homoiso-
flavones. Youji Huaxue 35 (2015) 1112-1122.
[
29] T.A. Dias, C.L. Duarte, C.F. Lima, M.F. Proença, C. Pereira-Wilson, Superior anticancer activity
of halogenated chalcones and flavonols over the natural flavonol quercetin. Eur. J. Med. Chem. 65
2013) 500-510.
(
1
6

ACCEPTED MANUSCRIPT
[
30] D. Sereno, M. Cavaleyra, K. Zemzoumi, S. Maquaire, A. Ouaissi, J.L. Lemesre, Axenically grown
amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony
mode of action. Antimicrob Agents Chemother. 42 (1998) 3097-3102.
[
31] J.L. Siqueira-Neto, S. Moon, J. Jang, G. Yang, C. Lee, H.K. Moon, E. Chatelain, A. Genovesio, J.
Cechetto, L.H. Freitas- Junior, An Image-Based High-Content Screening Assay for Compounds Tar-
geting Intracellular Leishmania donovani Amastigotes in Human Macrophages. PLoS Negl Trop Dis. 6
(
[
2012) e1671.
32] T. Bowling, L. Mercer, R. Don, R. Jacobs, B. Nare, Application of a resazurin-based high-
throughput screening assay for the identification and progression of new treatments for human African
trypanosomiasis. Int J Parasitol Drugs Resist. 2 (2012) 262-270.
[
33] E. Bifeld, P. Tejera Nevado, J. Bartsch, J. Eick, J. Clos, A versatile qPCR assay to quantify trypa-
nosomatidic infections of host cells and tissues. Med. Microbiol. Immunol. 205 (2016) 449-458.
[
34] B. Liu, J. Chang, W.P. Gordon, J. Isbell, Y. Zhou, T. Tuntland, Snapshot PK: a rapid rodent in
vivo preclinical screening approach. Drug Discov. Today. 13 (2008) 360-367.
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Scheme 1. Synthesis of the compounds 1-13.
Reaction conditions: (a) NaOH (3 M), EtOH, rt.
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Table 1. Chemical structure of the 13 synthesized 2’-hydroxy chalcones bearing methoxy groups, logP,
EC50 towards T. brucei, NOAEL (THP-1 cells) and selectivity index. * LogP licochalcone A = 4.74,
LogP flavokawin B = 3.90.
EC50
Selectivity
CC50
SD
±
Comp.
R
4’
R
5’
R
2
R
3
R
4
R
5
R
6
LogP*
T. brucei
index
(
µM)
(CC50/EC50)
1
2
3
4
5
6
7
8
9
H
H
H
H
OCH
OCH
OCH
OCH
H
3
3
3
3
H
H
H
H
H
H
3.94
3.86
3.87
3.59
3.88
3.87
3.51
3.52
3.53
3.52
3.49
3.47
3.50
1.3 ± 0.2
>25
>25
>25
>19
>7
>12
12
-
OCH
H
H
OCH
OCH
H
3.4 ± 0.8
3
H
H
H
H
2.1 ± 0
3
3
OCH
OCH
H
H
H
H
H
4.2 ± 0.2
51 ± 7
>100
>100
>100
>25
3
3
H
OCH
OCH
H
H
H
-
3
OCH
H
H
H
H
H
-
-
3
3
3
3
OCH
H
OCH
OCH
H
H
H
OCH
OCH
H
3.4 ± 0.1
>29
>12
-
3
3
3
3
3
3
OCH
H
3
H
H
H
3
2.1 ± 0.4
OCH
H
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
H
-
>100
>50
1
1
1
1
0
1
2
3
OCH
H
H
H
H
-
-
OCH
H
H
OCH
OCH
OCH
H
-
>100
>25
-
3
3
3
OCH
OCH
H
H
4.4 ± 0.4
7.4 ± 1.2
>6
>3
3
3
OCH
H
H
>25
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Figure 1. Chemical structure of licochalcone A (A) and flavokawin B (B), general chemical structure
of the compounds studied in the present paper (C) and in our previously published paper (D). The
common chemical features are shown in blue.
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1
00
8
6
4
2
0
0
0
0
0
Figure 2. Percentage of growth inhibition of the synthesized compounds against T. brucei at 10 µM
gray bar), T. cruzi at 10 µM (blue bar) and L. infantum at 50 µM (green bar). The reference com-
pounds were pentamidine (EC = 1.55 ± 0.24 nM) for T. brucei and miltefosine for L. infantum (EC
50
(
5
0
=
2.65 ± 0.4 µM), nifurtimox for T. cruzi (EC = 2.2 ± 0.3 µM). The data are reported in Table S1, S2
50
and S3 of the Supporting Information.
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Compound
11,30 13,60 17,64 8,52 2,66 40,00 18,83 -7,06 3,52 15,91 40,00 9,45 7,10
85,61 75,42 84,19 97,95 40,00 85,15 40,00 40,00 40,00 85,15 1,24 13,27 -3,10
17,11 40,00 -31,74 18,95 21,70 25,78 -25,55 40,00 10,69 -26,85 40,00 60,87 26,95
76,19 63,48 69,60 40,00 40,00 40,00 74,87 61,12 40,00 21,41 67,54 84,55 75,68
-8,37 40,00 40,00 58,00 40,00 79,61 17,11 11,68 92,65 93,71 65,10 88,32 -24,92
-1,38 -8,25 14,27 12,36 5,03 6,15 11,80 15,30 2,42 -2,68 -10,00 -3,06 18,72
21,54
%
Inh. hERG
0
,00
,00
,00
%
%
%
%
%
Inh. CYP1A2
Inh. CYP2C9
Inh. CYP2C19
Inh. CYP2D6
Inh. CYP3A4
0
0
60,95
0
,00
,00
-6,59 -10,58 -15,10 -6,83
NT
NT
NT
NT -10,37 -10,08 -23,55 -8,08 -4,18
0
%
Inh. Aurora B
Cell Growth A549
Cell Growth W1-38
-
1,00
%
105,04 109,59 115,45 95,51 107,16 106,68 40,00 103,82 98,78 115,46 127,26 107,51 114,88
118,11 63,91 87,49 96,14 40,00 40,00 48,18 40,00 127,83 113,69 78,10 73,93 88,46
111,72
%
%
Toxicity Mitochondria 6,16 -2,92 13,36 3,66 14,23 15,76 0,04 14,59 -5,76 -9,45 1,08 -6,35 9,97
5,91
%
Inh. T. brucei
100,00 99,00 97,00 100,00
7,00 10,00 100,00 99,00 100,0
1,6
1
8,2
8,9
99
29
90,5
12
Selectivity index
19
7
12,00 12,00
6,00 3,00
17
Figure 3. Early toxicological data of the thirteen synthesized compounds.The data are reported as a
traffic light system. An ideal compound (I. comp.) should have all the parameters green. The cells are
colored in green when the percentage of inhibition of T. brucei is between 60 and 100, while the per-
centage of inhibition of CYP isoforms, hERG, Aurora B kinase and mitochondrial toxicity is between 0
and 30. Cells are colored in red when data indicates toxicity (percentage of inhibition of CYP isoforms,
hERG, Aurora B kinase and mitochondrial toxicity ≥ 60) or inactivity (percentage of inhibition of T.
brucei is ≤ 30). Yellow stands for a borderline value (> 30 and < 60%): moderately active or slightly
toxic compound. Compounds are not cytotoxic (green) when the percentage of A549 and W1-38 cell
growth is between 60 and 100, cytostatic (yellow) when it is between 0 and 60 and cytotoxic (red)
when it is below 0. Cells are colored in green when the selectivity index (CC THP1/EC T. brucei) is
50
50
higher than 10. Gray: not evaluated.
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Figure 4. A) Plasma concentration in BALB/c mice of free compound 4 administered IV (1 mg/kg). B)
Plasma concentration in BALB/c mice of compound 4 after oral administration of 20 mg/kg formulated
with cyclodextrins (triangles), cyclodextrins + PEG 400 (circles) or free compound (squares).
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Graphical abstract
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4

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Highlights
•
•
Thirteen 2’-hydroxy methoxylated chalcones were synthesized.
Compounds 1, 3, 4, 7 and 8 showed higher potency than flavokawin B
towards T. brucei.
•
•
A panel of in-vitro early toxicological properties was evaluated.
The formulation of compound 4 with cyclodextrins increased the oral
bioavailability by 7.5 fold.