In vitro and in vivo trypanocidal activities of 8-methoxy-3-(4-nitrobenzoyl)-6-propyl-2H-cromen-2-one, a new synthetic coumarin of low cytotoxicity against mammalian cells

Article abstract of DOI:10.1111/cbdd.13362

Natural and synthetic coumarins have been described as prototypes of new drug candidates against Chagas’ disease. During a typical screening with new compounds, we observed the potential of a new synthetic nitrobenzoylcoumarin (1) as trypanocidal against Trypanosoma cruzi epimastigotas. Then, we decided to prepare and evaluate a set of analogues from 1 to check the major structural requirements for trypanocidal activity. The structural variations were conducted in six different sites on the original compound and the best derivative (3) presented activity (IC₅₀ 28?±?3?μM) similar to that of benznidazole (IC₅₀ 25?±?10?μM). The enhancement of trypanocidal activity was conditioned to a change in the side chain at C6 (allyl to n-propyl group) and the preservation of coumarin nucleus and the nitrobenzoyl group at C3. Exposure of 3 to H9C2 cells showed low toxicity (CC₅₀?>?200?μM) and its activity on T.?cruzi amastigotes (IC₅₀ 13?±?0.3?μM) encouraged us to perform an evaluation of its potential when given orally to mice infected with trypomastigote forms. Derivative 3 was able to reduce parasitemia when compared to the group of untreated animals. Taken together, these results show the potential therapeutic application of the synthetic coumarins.

Full text of DOI:10.1111/cbdd.13362

PROFESSOR DIOGO TEIXEIRA CARVALHO (Orcid ID : 0000-0002-6362-2822)
Article type : Research Article
Title: In vitro and in vivo trypanocidal activities of 8-methoxy-3-
(4-nitrobenzoyl)-6-propyl-2H-cromen-2-one, a new synthetic
coumarin of low cytotoxicity against mammalian cells
Short running title: New synthetic coumarin with trypanocidal
activity
Key words: Coumarins, Nitro compounds, Trypanocidal agents, Trypanosoma cruzi, Chagas’
disease.
Guilherme Andrade Brancagliona, André Eidi Toyotaa, José Vaz Cardoso Machadoa, Antônio
Ávila Fernandes Júniora, Alberto Thalison Silveirab, Diego Fernandes Vilas Boasc, Elda
Gonçalves dos Santosc, Ivo Santana Caldasc, Diogo Teixeira Carvalhoa,*
aDepartamento de Alimentos e Medicamentos, Faculdade de Ciências Farmacêuticas, Universidade
Federal de Alfenas, Alfenas, MG, Brazil
bDepartamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas,
Universidade Federal de Alfenas, Alfenas, MG, Brazil
cDepartamento Patologia e Parasitologia, Instituto de Ciências Biomédicas, Universidade Federal de
Alfenas, Alfenas, MG, Brazil
*Corresponding author: Diogo Teixeira Carvalho.
Faculdade de Ciências Farmacêuticas,
Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva, 700, sala D-208e, Alfenas, MG,
37130-001, Brazil.
E-mail: diogo.carvalho@unifal-mg.edu.br. Phone
number: +55 35 3701-9213
Abstract
Natural and synthetic coumarins have been described as prototypes of new drug candidates
against Chagas’ disease. During a typical screening with new compounds, we observed the
potential of a new synthetic nitrobenzoylcoumarin (1) as trypanocidal against T. cruzi
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13362
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epimastigotas. Then we decided to prepare and evaluate a set of analogues from 1 to check
the major structural requirements for trypanocidal activity. The structural variations were
conducted in six different sites on the original compound and the best derivative (3)
presented activity (IC50 28 ± 3 M) similar to that of benznidazole (IC50 25 ± 10 M). The
enhancement of trypanocidal activity was conditioned to a change in the side chain at C6
(allyl to n-propyl group) and the preservation of coumarin nucleus and the nitrobenzoyl group
at C3. Exposure of 3 to H9C2 cells showed low toxicity (CC50>200 M) and its activity on T.
cruzi amastigotes (IC50 13 ± 0.3 M) encouraged us to perform an evaluation of its potential
when given orally to mice infected with trypomastigote forms. Derivative 3 was able to
reduce parasitemia when compared to the group of untreated animals. Taken together,
these results show the potential therapeutic application of the synthetic coumarins.
1. Introduction
Chagas disease is considered one of the most important parasitic diseases in South and
Central America and it is estimated that in the world there are approximately 7 million people
infected by this parasite, with around 7000 deaths per year [1]. Chagas disease is considered
a neglected disease and currently only two drugs are recommended for treatment: nifurtimox
and benznidazole [2]. In addition to the efficacy of these drugs being dependent on the stage
of infection in which treatment is introduced, they induce serious side effects in patients, so
considering the risk of therapeutic failure and death during the chronic phase, the
development of new drugs may be important not only to increase the cure rate of patients
but also to prevent the progression of the disease to severe clinical forms, even if
parasitological cure is not obtained [3].
Natural product research represents an important strategy in finding new drug candidates.
The pharmacological activity of these substances can be optimized by rationally oriented
structural modifications or they may assist as inspiration for new compounds with activities
other than the original. Coumarins are good examples of this, since besides being ubiquitous
in plant species, they present a wide range of pharmacological activities, as anti-
inflammatory, antioxidant, antiproliferative and antimicrobial. In addition, they can be easily
accessed by organic synthetic methodologies, which avoids issues inherent to obtaining
bioactive molecules by exploitation of natural resources [4, 5]
.
In the last years, some authors have revealed the trypanocidal potential of natural and
synthetic coumarins (Scheme 1) and this activity seems to be related mostly to the inhibition
of trypanothione reductase (TR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
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[5,
which are essential enzymes in T. cruzi metabolic pathways
6]. For instance, the
prenylated coumarin Mammea B/BA (A) extracted from Calophyllum brasiliense showed
good in vitro trypanocidal activity [7, 8], while chalepin (B), a coumarin present in the crude
extracts of Rutales species, is active as GAPDH inhibitor from T. cruzi [4, 9]. Umbelliferone
aminoalkyl derivative (C) inhibits the activity of oxidosqualene cyclase, an enzyme involved
in T. cruzi ergosterol biosynthesis [10]. Ethanolic extracts from Polygala sabulosa have
activity against epimastigote and trypomastigote forms of T. cruzi and this is associated to
the presence of 6-methoxy-7-prenyloxycoumarin (D) [11]. Ferula narthex exudate contains the
trypanocidal coumarins fesolol (E) and 10’-R-karatavacinol (F) [12]. Some synthetic
coumarins like 3-amino-4-hidroxi-6-metil-2H-cromen-2-ona (G) showed dual activity as
trypanocidal and antioxidant agents [13]. Other coumarins were synthetized as hybrids of
chalcones (H) or quinones (I) and interesting trypanocidal or antioxidant activity was
observed [14-17]
.
<Scheme 1>
Following our interests in the synthesis of new bioactive coumarins [18] and taking advantage
of abundant natural products as eugenol, recently we synthesized a series of new
unpublished coumarins structurally related to eugenol in order to find new antibacterial and
antifungal compounds. They did not show relevant activity on these microorganisms, but one
of these coumarins (1, Scheme 2) presented good trypanocidal effect (IC50 66 M) in a
screening against epimastigote forms of T. cruzi, besides having low citotoxicity (>200 M).
This result encouraged us to accomplish structural changes in this coumarin in order to
obtaining derivatives that could be more effective and to understand the current structure-
activity relationships (SAR). For this purpose, it was decided to promote structural changes
in 1 in order to investigate the influence of hydrophobicity and of electronic and steric effects
on trypanocidal activity.
<scheme 2>
2. Methods and materials
2.1 Chemistry
All required chemicals were purchased from Sigma-Aldrich (São Paulo, Brazil) and used
without further purification. Thin-layer chromatography (TLC) on silica gel TLC plates
(ALUGRAM® Xtra Sil G/UV254, Macherey-Nagel) was used to check purity of the obtained
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compounds and to monitor the reactions progress. The spots were detected by exposure to
the UV light at 254 nm. For column chromatography, column grade (0.040-0.063 mm mesh
size) silica gel was employed (Sorbiline®). Melting points of the compounds were obtained
on PFM-II melting-point apparatus (MS Tecnopon, Piracicaba, Brazil) and are uncorrected.
IR spectra were recorded on FT-IR-Affinity-1 spectrometer with an ATR dispositive
(Shimadzu®, Kyoto, Japan). NMR spectra were recorded on a Bruker AC-300 spectrometer
operating at 300 MHz for 1H-NMR and 75 MHz for 13C-NMR spectra (Rheinstetten,
Germany). Chemical shifts are expressed as  (ppm) relative to TMS as internal standard.
The coupling constants (J) are given in Hertz. Multiplets are given as s (singlet), d (doublet),
dd (doublet of doublets), t (triplet), q (quartet), sex (sextet) and m (multiplet). Ultra-high
performance liquid chromatography system (UHPLC) model LC–MS 8030 equipped with a
triple-quadrupole mass analyzer was employed for compounds characterization (Shimadzu®,
Kyoto, Japan). The MS conditions were as follows: source block, 400°C; desolvation line,
250ºC; nebulizing gas, 2.0 L min−1; drying gas, 15.0 L min−1; electrospray ionization mode
(ESI). The acquisition of the precursors were in full scan (positive and negative ionization
mode, ranged from 200 to 500 m/z). Two microliters of solutions at 5 ppm of each analyte
dissolved in methanol were injected into the system. The mobile phase was MeOH:water
(95:5, v/v) at a flow rate of 0.2 mL min−1. ClogP values of synthesized compounds were
calculated using ChemDraw Ultra 11.0. Coumarin 1 was synthesized as reported elsewhere
[19]
.
2.1.1 General procedure for the synthesis of 5-allyl-2-hydroxy-3-methoxybenzaldehyde and
2-hydroxy-3-methoxy-5-propylbenzaldehyde [20]
Hexamine (5 eq) was solubilized in glacial acetic acid (40 mL) and this solution was stirred at
125 ºC for 10 minutes, then the specific phenol (1 eq) was added in one portion. Reaction
was maintained at 125 °C for five hours under stirring. Following, 2M HCl (10 mL) was
added and the reaction mixture was maintained as such for additional 30 minutes. Then, this
mixture was cooled down to room temperature, washed with aqueous NaHCO3 and
extracted with dichloromethane. The organic phase solvent was evaporated in a rotary
evaporator and the oil purified by column chromatography (hexanes:ethyl acetate, 9:1, v/v).
5-allyl-2-hydroxy-3-methoxybenzaldehyde
From 4-allyl-2-methoxyphenol. Light greenish oil; yield 57%. IR (ATR) 3200-2800 ( O-H
phenol), 3005 ( C-H ar), 2965 ( C-H sp3), 2850 ( C-H aldehyde), 1655 ( C=O aldehyde),
1593 ( C=C ar), 1265 ( C-O-C). 1H NMR (CDCl3) 3.37 (d, JH9,H10 6.6 Hz; 2H), 3.90 (s; 3H),
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5.07–5.13 (m; 1H), 5.87–6.01 (m; 2H), 6.94 (s; 1H), 6.97 (s; 1H), 9.87 (s; 1H), 10.96 (s; 1H).
13C NMR (CDCl3) 39.53, 56.41, 116.66, 118.82, 120.58, 123.83, 131.55, 136.85, 148.34,
150.19, 196.73.
2-hydroxy-3-methoxy-5-propylbenzaldehyde
From 2-methoxy-4-propylphenol. Yellowish oil; yield: 61%. IR (ATR) 3350-2750 ( O-H
phenol), 3003 ( C-H ar), 2957 ( C-H sp3), 2870 ( C-H aldehyde), 1647 ( C=O aldehyde),
1
1595 ( C=C ar), 1259 ( C-O-C). H NMR (CDCl3) 0.95 (t; JH11,H10 7.39 Hz; 3H), 1.64 (sex;
JH10,H9 7.53 Hz; JH10,H11 7.39 Hz; 2H), 2.57 (t; JH9,H10 7.53 Hz; 2H), 3.91 (s; 3H), 6.94 (s; 1H),
6.96 (s; 1H), 9.88 (s; 1H), 10.91 (s; 1H). 13C NMR (CDCl3) 13.80, 24.60, 37.47, 56.45,
118.89, 120.56, 123.72, 134.24, 148.19, 149.86, 196.83.
2.1.2 General procedure for the synthesis of derivatives 2 and 3
To a stirred solution of a specific salicylaldehyde (1 eq) in ethanol (20 mL) it was added ethyl
4-nitrobenzoylacetate (1 eq) and piperidine (3 drops). Then the mixture was heated under
reflux at 80 °C during three hours when a white solid precipitated as product. Then, the
mixture was cooled to room temperature and the solid product formed was collected by
vacuum filtration, washed throughout with cold ethanol and dried in a desiccator.
8-methoxy-3-(4-nitrobenzoyl)-2H-chromen-2-one (2)
From ortho-vanillin. Light greenish solid; yield: 89%; CLogP 2.80; M.p.: 266-267 ºC. IR
(ATR) 3105 ( C-H ar), 3075 ( C-H sp²), 1698 ( C=O ester), 1660 ( C=O ketone), 1574 (
C=C ar), 1514 cm-1 ( NO2). ¹H NMR (DMSO-d₆ ) 3.95 (s, 3H), 7.37 (t, JH6, H5 7.65 Hz; JH6,H7
7.92 Hz, 1H), 7.43-7.48 (m, 2H), 8.15 (d, JH3’,H2’ 9 Hz, 2H), 8.33 (d, JH2’,H3’ 9 Hz, 2H), 8.56 (s,
1H); ¹³C NMR (DMSO-d₆ ) 11.31, 56.28, 118.81, 121.20, 123.70, 124.97, 125.45, 130.64,
141.41, 143.86, 146.48, 147.65, 150.09, 157.81, 190.95. MS (ESI) calcd for C17H11NO6
[M+H]+ 326.27 found 326.40.
8-methoxy-3-(4-nitrobenzoyl)-6-propyl-2H-chromen-2-one (3)
From 2-hydroxy-3-methoxy-5-propylbenzaldehyde. White solid; yield 91%; CLogP 4.32;
M.p.: 143-144 ºC. IR (ATR) 3069 ( C-H ar), 3048 ( C-H sp2), 1705 ( C=O ester), 1682 (
C=O ketone), 1582 ( C=C ar), 1513 cm-¹ ( NO2). ¹H NMR (DMSO-d₆) 0.98 (t, JH15,H14 7.37
Hz, 3H), 1.69 (sex, JH14,H13 7.5 Hz; JH14,H15 7.37 Hz, 2H), 2.67 (t, JH13,H14 7.5 Hz, 2H), 3.99 (s,
3H), 7.01 (d, JH7,H5 1.71 Hz, 1H), 7.05 (d, JH5,H7 1.71 Hz, 1H), 7.95-7.99 (m , 2H), 8.23 (s, 1H),
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8.28-8.33 (m, 2H); ¹³C NMR (DMSO-d₆) 13.67, 24.43, 37.68, 56.40, 116.86, 118.40, 119.91,
123.68, 125.58, 130.18, 140.20, 141.52, 143.24, 147.05, 148.12, 150.39, 190.74. MS (ESI)
calcd for C20H17NO6 [M+H]+ 368.35 found 368.00.
2.1.3 Synthesis of 3-(4-aminobenzoyl)-8-methoxy-6-propyl-2H-chromen-2-one (4)
Derivative 3 (1 eq) and stannous chloride dihydrate (5 eq) were added to ethanol (20 mL)
and the mixture was stirred under heating to 85 ºC under reflux. After two hours, TLC
analysis (hexanes:ethyl acetate, 7:3, v/v) showed the reaction completion and the mixture
was cooled to room temperature. Aqueous NaHCO3 was then added to raise pH to 8 and the
mixture was extracted with ethyl acetate. The resulting organic phase was washed with
brine, dried with anhydrous sodium sulfate and the solvent evaporated in a rotary
evaporator.
Yellow solid; yield 91%; CLogP 3.6; M.p.: 215-216 ºC. IR (ATR) 3491 ( NH2), 3046 ( C-H
ar), 2952 ( C-H sp2), 1702 ( C=O ester), 1645 ( C=O ketone), 1580 cm-1 ( C=C ar). ¹H
NMR (DMSO-d6) 0.96 (t, JH15, H14 7.33 Hz, 3H), 1.67 (sex, JH14,H13 7.57 Hz; JH14, H15 7.33 Hz,
2H), 2.64 (t, JH13,H14 7.57 Hz, 2H), 3.97 (s, 3H), 4.23 (s, 2H), 6.61-6.64 (m, 2H), 6.92 (d,
JH7,H51.72 Hz, 1H), 6.97 (d, JH5,H7 1.72 Hz, 1H), 7.71-7.76 (m, 2H), 7.89 (s, 1H); ¹³C NMR
(DMSO-d6) 13.69, 24.48, 37.71, 56.34, 113.75, 115.52, 118.70, 119.28, 126.33, 128.25,
132.56, 139.55, 144.21, 146.92, 152.04, 158.41, 189.55. MS (ESI) calcd for C20H19NO4
[M+H]+ 338.37 found 338.05.
2.1.4 Synthesis of 4-((4-(8-methoxy-2-oxo-6-propyl-2H-chromene-3-carbonyl)phenyl)amino)-
4-oxobutanoic acid (5)
To a stirring solution of succinic anhydride (5 eq) in pyridine (5 mL) it was added the amino
derivative 4 (1 eq) and then the reaction mixture was heated at 90 ºC for 24 hours. When all
amine was consumed (TLC with hexanes: ethyl acetate: methanol, 6:3.75:0.25, v/v/v), the
reaction mixture was cooled to room temperature and the pH brought to 2 by addition of 1M
HCl. The formed solid was collected by vacuum filtration and was purified by trituration with
ethyl ether.
Brown solid; yield 42%; CLogP 3.47 ± 0,75; M.p 202-203 ºC. IR (ATR) 3278 ( N-H amide),
3058 ( C-H sp2), 3000-2800 ( O-H carboxylic acid), 2962 ( C-H ar), 1719 ( C=O
carboxylic acid), 1710 ( C=O ester), 1677 ( C=O amide), 1651 ( C=O ketone), 1584 com-1
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( C=C ar). ¹H NMR (DMSO-d₆) 0.86 (t, JH15,H14 7.33 Hz, 3H), 1.58 (sex, JH14,H13 7.53 Hz;
JH14,H15 7.33 Hz, 2H), 2.44-2.59 (m, 6H), 3.88 (s, 3H), 7.12 (d, JH7,H5 1.56 Hz, 1H), 7.20 (d,
JH5,H7 1.62 Hz, 1H), 7.67 (d, JH3’,H2’ 8.8 Hz, 2H), 7.83 (d, JH2’,H3’ 8.8 Hz, 2H), 8.20 (s, 1H), 10.33
(s, 1H). ¹³C NMR (DMSO-d₆) 13.61, 24.01, 28.65, 31.26, 36.88, 56.24, 116.07, 118.23,
118.49, 119.62, 126.93, 130.40, 131.14, 139.17, 141.73, 144.41, 144.74, 146.25, 158.00,
170.99, 173.79, 190.28. MS (ESI) calcd for C24H23NO7 [M-H]+ 436.44 found 436.25.
2.1.5 General procedure for the synthesis of derivatives 6, 7 and 8
A specific -ketoester or malonic acid (1 eq), 2-hydroxy-3-methoxy-5-propylbenzaldehyde (1
eq) and L-proline (0.1 eq) were mixture and heated to 90 °C in a glycerol bath when all the
solids melt. The heating was continued for an hour when TLC indicated the end of the
reaction (hexane:ethyl acetate; 8:2, v/v). The remaining slurry solid was purified by trituration
with diethyl ether until a fine powder was formed, which was isolated by simple filtration.
3-benzoyl-8-methoxy-6-propyl-2H-chromen-2-one (6)
From benzoyl acetoacetate. Light yellow solid; yield 65%; CLogP 4.36; M.p. 140-141 ºC. IR
(ATR) 3051 ( C-H ar), 2953 ( C-H sp2), 1716 ( C=O ester), 1676 ( C=O ketone), 1579
1
cm-1 ( C=C ar); H NMR (DMSO-d6) 0.97 (t, JH15, H14 7.35 Hz, 3H), 1.68 (sex, JH14, H13 7.58
Hz, JH14, H15 7.35 Hz, 2H), 2.65 (t, JH13, H14 7.58 Hz, 2H), 3.98 (s, 3H), 6.95 (s, 1H), 7.00 (s,
1H), 7.44-7.49 (m, 2H), 7.60 (tt, JH4’,H3’ 7.39 Hz, JH4’, H2’ 2.65 Hz, 1H), 7.85-7.89 (m, 2H), 8.02
(s, 1H). 13C NMR (DMSO-d6) 13.68, 24.46, 37.70, 56.36, 116.06, 118.50, 119.54, 127.16,
128.56, 129.62, 133.74, 136.31, 139.76, 142.85, 145.82, 146.98, 191.92. MS (ESI) calcd for
C20H18O4 [M+H]+ 323.35 found 323.10.
3-acetyl-8-methoxy-6-propyl-2H-chromen-2-one (7)
From ethyl acetoacetate. Dark yellow solid; yield 61%; CLogP 2.52; M.p. 109-110 ºC. IR
(ATR) 3069 ( C-H sp2), 2998 ( C-H ar), 1717 ( C=O ester), 1676 ( C=O ketone), 1570
1
cm-1 ( C=C ar). H NMR (CDCl3) 0.96 (t, JH15, H14 7.38 Hz, 3H), 1.68 (sex, JH14, H13 7.56 Hz,
JH14, H15 7.38 Hz, 2H), 2.64 (t, JH13, H14 7.56 Hz, 2H), 3.97 (s, 3H), 7.00 (s, 1H), 7.26 (s, 1H),
8.44 (s, 1H). 13C NMR (CDCl3) 13.79, 24.55, 30.76, 37.79, 56.49, 116.90, 118.70, 120.57,
124.67, 139.90, 143.54, 146.92, 148.02, 159.11, 195.95. MS (ESI) calcd for C15H16O4 [M+H]+
261.29 found 261.10.
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8-methoxy-2-oxo-6-propyl-2H-chromene-3-carboxylic acid (8)
From malonic acid. Yellow solid; yield 67%; CLogP 2.76; M.p. 158-159 ºC. IR (ATR) 3045 (
C-H sp2), 3003 ( C-H ar), 3000-2700 ( O-H carboxylic acid), 1742 ( C=O ester), 1671 (
C=O carboxylic acid), 1582 cm -1 ( C=C ar). 1H NMR (CDCl3) 0.95 (t, JH15, H14 7.35 Hz, 3H),
1.67 (sex, JH14, H13 7.57 Hz, JH14, H15 7.35 Hz, 2H), 2.66 (t, JH13, H14 7.57 Hz, 2H), 3.98 (s, 3H),
7.05-7.06 (m, 1H), 7.09-7.10 (m, 1H), 8.82 (s, 1H). 13C NMR (CDCl3) 13.73, 24.44, 37.73,
56.54, 114.97, 117.98, 118.90, 120.44, 141.41, 142.60, 147.16, 151.81, 162.86, 163.85. MS
(ESI) calcd for C14H14O5 [M-H]+ 261.26 found 260.95.
2.1.6 Synthesis of 8-methoxy-3-(4-nitrobenzoyl)-6-propylchroman-2-one (9)
The nitro derivative 3 (1 eq), sodium cianoborohydride (1.1 eq) and bromocresol green (1
mg) were added in methanol (10 mL). The mixture was kept at room temperature under
stirring while 1M HCl was added dropwise until the solution color changed to yellow. The
reaction mixture was maintained as such until TLC ((hexanes:ethyl acetate; 8:2, v/v) showed
the reaction ended after 3 hours. Then, the reaction mixture was diluted with water, cooled
and extracted with dichloromethane. The organic phase was dried with anhydrous sodium
sulfate and the solvent evaporated in a rotary evaporator.
White solid; yield 61%; CLogP 4,00; M.p.136-137 ºC. IR (ATR) 3106 ( C-H ar), 1738 (
C=O ester), 1692 ( C=O ketone), 1601 ( C=C ar), 1521 cm -1 ( NO2). ¹H (DMSO-d₆) 0.94
(t, JH15, H14 7.31 Hz, 3H), 1.63 (sex, JH14, H13 7.6 Hz; JH14, H15 7.31 Hz, 2H), 2.55 (t, JH13, H14 7.6
Hz, 2H), 3.07 (dd, JH4**.H4* 15,9 Hz, JH4**.H3 5,9 Hz; 1H), 3.56 (dd, JH4*.H4** 15,9 Hz, JH4*.H3 5,2
Hz; 1H), 3.89 (s, 3H), 4.61 (dd; JH3.H4* 5,2 Hz, JH3.H4** 5,9 Hz; 1H), 6.61 (s, 1H), 6.70 (s, 1H),
8.12 (d, JH2’,H3’ 8.56 Hz, 2H), 8.32 (d, JH3’,H2’ 8.56 Hz, 2H). ¹³C NMR (DMSO-d₆) 19.90, 24.71,
26.69, 37.97, 47.52, 56.21, 111.80, 119.38, 122.07, 124.10, 130.02, 140.25, 140.55, 147.41,
165.32, 174.18, 192.75. MS (ESI) calcd for C20H19NO6 [M-H]+ 368.37 found 368.10.
2.1.7 Synthesis of 8-hydroxy-3-(4-nitrobenzoyl)-6-propyl-2H-chromen-2-one (10)
Anhydrous aluminum chloride (1.1 eq) was added under stirring to a cold solution of the nitro
derivative 3 (1 eq) in anhydrous dichloromethane. The reaction mixture was allowed to
warm up to room temperature and was maintained as such for 1.5 hours, until TLC showed
the total consumption of the starting material (hexanes:ethyl acetate, 7:3, v/v). The reaction
was quenched by slow addition of aqueous sodium bicarbonate and the product was
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extracted with ethyl acetate, followed by drying with anhydrous sodium sulfate and
elimination of the solvent in a rotary evaporator.
Greenish solid; yield 79%; CLogP 4.15; M.p. 213-214 ºC. IR (ATR) 3367 ( OH phenol),
3113 ( C-H sp2), 3083 ( C-H ar), 1725 ( C=O ester), 1654 ( C=O ketone), 1576 ( C=C
ar), 1521 cm-1 ( NO2). 1H NMR (CDCl3) 0.86 (t, JH15, H14 7.28 Hz, 3H), 1.56 (sex, JH14, H13 7.45
Hz, JH14, H15 7.28 Hz, 2H), 2.52 (t, JH13, H14 7.45 Hz, 2H), 7.04 (s, 1H), 7.09 (s, 1H), 8.10 (d,
JH2’, H3’ 8.71 Hz, 2H), 8.29 (d, JH3’, H2’ 8.71 Hz, 2H), 8.42 (s, 1H), 10.30 (s, 1H). 13C NMR
(CDCl3) 16.50, 23.89, 36.49, 118.83, 119.29, 120.72, 123.68, 125.06, 130.57, 139.13,
141.56, 144.35, 147.91, 150.01, 158.12, 191.12. MS (ESI) calcd for C20H19NO6 [M-H]+
368.37 found 368.10. MS (ESI) calcd for C19H15NO6 [M-H]+ 352.33 found 352.10.
2.1.8 Synthesis of 3-(hydroxyl-(4-nitrophenyl)methyl)-8-methoxy-6-propyl-2H-chromen-2-one
(11)
Sodium borohydride (1.1 eq) was solubilized in 0.01M NaOH (5 mL) and this was added
gradually to a stirred solution of the nitro derivative 3 (1 eq) in metanol (10 mL) at room
temperature. After 3 hours, when the reaction was over (TLC, hexanes:ethyl acetate, 7:3,
v/v), HCl 1M (10 mL) was added and the solution allowed to stir for another 30 minutes.
Then, the reaction mixture was neutralized with aqueous sodium bicarbonate and extracted
with ethyl acetate. After drying the organic phase with anhydrous sodium sulfate and
elimination the solvent in rotary evaporator, the final slurry was triturated with diethyl ether to
afford the product.
Light brown solid; yield 71%; CLogP 4.83; M.p.180-181 ºC. IR (ATR) 3378 ( OH alcohol),
3113 ( C-H sp2), 3067 ( C-H ar), 1683 ( C=O ester), 1588 ( C=C ar), 1522 cm-1 ( NO2).
1H NMR (CDCl3) 0.89 (t, JH15, H14 7.3 Hz, 3H), 1.62 (sex, JH14, H13 7.53 Hz, JH14, H15 7.3 Hz, 2H),
2.59 (t, JH13, H14 7.53 Hz, 2H), 3.87 (s, 3H), 5.77 (d, JH16, OH 4.24 Hz, 1H), 6.41 (d, JOH, H16 4.24
Hz, 1H), 7.13 (s, 1H), 7.16 (s, 1H), 7,68 (d, JH2’, H3’ 8.75 Hz, 2H), 8.13 (s, 1H), 8.18 (d, JH3’, H2’
8.75 Hz, 2H). 13C NMR (CDCl3) 13.58, 23.97, 36.90, 56.07, 68.79, 114.46, 118.90, 119.08,
123.36, 128.13, 130.71, 138.91, 140.34, 146.11, 146.76, 150.26, 159.25. MS (ESI) calcd for
C20H19NO6 [M-H]+ 368.37 found 368.00.
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2.2 Biological evaluation
2.2.1 Trypanosoma cruzi strain and culture procedure
The T. cruzi Y strain (DTU II), partially sensitive to benznidazol chemotherapy and
characterized by Filardi and Brener was used [21]. The epimastigote forms were maintained
in culture at 28 °C in LIT (Liver Infusion Triptose) medium, supplemented with 10% fetal
bovine serum (FBS). The trypomastigote forms of strain Y were maintained in liquid nitrogen
and periodically transferred to mice of the Swiss line, during successive passages. The
strain maintenance procedure was carried out at the Animal Facility of the Pathology and
Parasitology Department of the Federal University of Alfenas (UNIFAL-MG) to avoid
changes in the original characteristics of susceptibility and virulence.
2.2.2 In vitro anti-Trypanosoma cruzi assay
Stock solutions of coumarins and benznidazole (10 mg/mL) were prepared in dimethyl
sulfoxide (DMSO). For analysis of the effects against epimastigotes forms, the maximum
concentration was 200 µg/mL for each compound and benznidazole, and a seven-point
profile with 2-fold serial dilutions was used in triplicate. Then, epimastigotes were added to
the plate (1.5 x 106/mL) in a 72-h assay. The final DMSO concentration had no deleterious
effect on parasite growth. After incubation in a BOD oven at 28 °C for 72 hours, resazurin
(1mM) was added to the plates. After incubation for 12 hours, the results were read at 570
nm and 600 nm. The percentage of inhibition of proliferation induced by the compounds was
calculated using the formula: % inhibition = 100 – [A570-(A600xR0)
/ A570-(A600xR0)
Treated
Control+] x100, where A570= Absorbance a 570nm, A600= Absorbance a 600nm, where Control
+ represents the well containing epimastigotes, medium and resazurin, in the absence of the
compound. R0 is the correction factor, calculated from the absorbance values of the
negative control (C-), that is, only culture medium and resazurin in the absence of parasites
[Ro = (A570 /A600)C-]. The IC50 values were calculated from the percent inhibition of
compounds using Graph Pad Prism 5 software.
For analysis of the effects against intracellular parasites, the concentrations used were
120µM, 60µM, 30µM, 15µM, 7.5µM, 3.75µM and 1.87µM for 4-nitrobenzoyl coumarin or
benznidazole, in a 72 hours assay. Cells of H9c2 lineage (1x104 cells; American Type
Culture Collection, ATCC: CRL 1446) from neonatal rat cardiomyoblasts were cultured in
DMEM medium (supplemented with 10% FBS, 1% glutamine 2nM and 0.2% gentamicin
200μg / ml). After 24 h, the cells were infected with trypomastigotes (obtained from H9c2
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cells) at a ratio of 20 parasites to 1 H9c2 cell. After 24 h of incubation, the cultures were
exposed to the compounds for 3 days. All tissue culture plates were maintained at 37°C in a
5%CO2-air mixture. After 72 h, the cultures were fixed with methanol, stained with Giemsa
stain, and examined microscopically to determine the percentages of infected cells in treated
and untreated cultures. At least 200 cells were counted for determination of percentage of
infection and the percentage of growth inhibition were obtained by dividing the number of
infected cells in treated cultures by the mean value for infected control cultures.
Subsequently this result was multiplied by 100 [22]. IC50 values were calculated using
CalcuSyn software (Biosoft, United Kingdom) and Graph Pad Prism 5.
2.2.3 In vitro cell toxicity assay
Cells of H9c2 Lineage (1 X 103) were cultured in DMEM medium (supplemented with 10%
FBS, 1% glutamine 2nM and 0.2% gentamicin 200μg / ml). 1000 H9c2 cells were seeded
and incubated at 37°C, 5% CO2 for 24 hours. The culture medium was replaced and for
analysis of the effects toxic, the maximum concentration was 200 µg/mL for each compound
and benznidazole, and a seven-point profile with 2-fold serial dilutions was used in triplicate
for 72 hours. After incubation, resazurin (1mM) was added and the reading was carried out
at 570nm and 600nm after 12 hours hours [23]. The percentage of inhibition of proliferation
induced by the compounds was calculated using the same formula used in the assays
against epimastigote forms. From the percent inhibition of the compounds CC50 (50%
cytotoxic concentration) was calculated using the Graph Pad Prism 5.
2.2.4 In vivo activity
Female Swiss mice with about 30 days of age (20-25g), from the UNIFAL-MG, and
maintained in a temperature-controlled room with access to water and food ad libitum were
inoculated intraperitoneally with 5x103 blood trypomastigotes of the T. cruzi Y strain. This
strain is partially resistant to benznidazole and induces high levels of parasitemia
accompanied by 100% mortality at around 15 days of infection when in the absence of
treatment. After four days, tail blood was examined for the presence of parasites. Only when
T. cruzi was detected microscopically, were the mice submitted to a specific treatment. The
treatment was administered orally at the dosage of 50mg/kg. The compounds (derivative 3
and benznidazole) were each suspended in distilled water using 5% de Cremophor (Sigma)
and each animal received 0.2 mL of compound suspension daily by gavage for 6
consecutive days. Parasitaemia and mortality were checked every day until ten days after
infection.
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2.2.5 Ethics
All experimental protocols were conducted in accordance with the guidelines issued by the
Brazilian College of Animal Experimentation (COBEA) and approved by the Ethics
Committee in Animal Research at UNIFAL-MG (number 59/2017).
3. Results and discussion
3.1 Chemistry
The synthetic plan to obtain the intended coumarins is depicted in Scheme 3. Coumarins 1-3
and 6-8 were obtained by Knoevenagel reactions between specific aromatic aldehydes and
-ketoesters (1-3, 6 and 7) or malonic acid (8) employing the conditions described by Karade
[24,
et al (2007) or Vazquez-Rodriguez (2013)
24]. They were all obtained as pure and
crystalline solids with good yields. The analysis of their IR spectra showed bands between
1742-1645 cm-1 assigned to the carbonyl groups (lactone, ketone or carboxylic acid). In
1
addition, all their H NMR spectra showed the signal for H-4 of the coumarin nucleus in the
range 8.82-7.89 ppm. The confirmation of coumarins formation was also due to the
characteristic signals of two carbonyl carbons, found above 150 ppm, in their 13C NMR
spectra.
To obtain de amino derivative 4 it was employed the methodology proposed by Bellamy and
co-workers which uses stannous chloride in ethanol [26]. In this way, coumarin 4 was
smoothly obtained in high yields. The IR stretching bands at 3491 e 3373 cm-1 and an
expressive shielding effect on aromatic hydrogens noted in the NMR spectrum confirmed the
presence of amino group. The succinamic derivative 5 was synthesized by reacting the
amino coumarin 4 with succinic anhydride in refluxing pyridine. In its IR spectrum it was
possible to note the four expected carbonyl bands due to lactone, ketone, amide and
carboxylic acid groups. The insertion of the succinoyl chain was also confirmed by the
1
presence of a multiplete in the range 2.59-2.44 ppm in the H NMR spectra and also by the
two new methylene carbon signals in the 13C NMR spectrum.
<Scheme 3>
Next, sodium cianoborohydride was chosen to accomplish the reduction of the coumarin
alkene group to generate derivative 9, following what was described by Hutchins and
coworkers [27]. Isolated alkenes are inert to reduction by sodium cianoborohydride, though
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highly polarized double bonds, as in this case, may be reduced in acidic medium without
affecting the ketone group. One can note the diastereotopic hydrogens, originated from the
reduction, as double doublets in the 1H NMR spectrum. On the other hand, a new methylene
carbon signal was observed at 26.6 ppm in the 13C spectrum. Derivative 10 was obtained by
an O-demethylation reaction with coumarin 3 carried out by aluminum chloride in dry
dichloromethane, as an adaptation of a well-known methodology [28]. The loss of methyl
group could be perceived by the absence of typical methoxyl signals in NMR spectra.
Finally, coumarin 11 was achieved by a classical ketone reduction made by sodium
borohydride in alkaline media [29]. In its IR spectra one could see only a carbonyl stretching
band whereas the ketone signal was also absent in 13C NMR spectrum.
3.2 Biological evaluation
Structural analogues of coumarin 1 were devised to investigate structure-activity
relationships. Firstly, we investigated the trypanocidal effect of analogues 1-11 against T.
cruzi epimastigotes and the cytotoxicity of the most interesting derivatives on
cardiomyocytes (Table 1).
<table 1>
It is possible to suggest that the presence of an allylic (1) or propylic (3) side chain on the
coumarin nucleus positively affects the trypanocidal activity and that the saturated chain
leads to a more active derivative. This result allows inferring that hydrophobic interactions
between a flexible side chain of the ligand and the molecular target should be important for
the activity, due to the drastic drop in activity observed with the unsubstituted derivative 2.
Since derivative 3 showed a better selectivity index than its allylic analogue 1, it was chosen
for the other intended modifications. Therefore, the next changes aimed at evaluating the
pharmacophoric importance of the substituent group at the C-4’ position on the benzoyl
aromatic ring, besides the importance of this ring itself.
The results obtained with compounds 4 (amino derivative), 5 (succinamic derivative) and 6
(unsubstituted benzoyl derivative) showed that the nitro group, among the evaluated options,
is the better substituent in the ring. This may be related to the importance of the aromatic
nitro group in various antiparasitic groups (e.g., nitrofurazone, benznidazole, nifurtimox,
megazol, oxamniquine, praziquantel) wherein it receives the classification of parasitophoric
group [30]
.
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The results of antiepimastigote activity obtained with derivative 6, 7 and 8, allow us to point
out the pharmacophoric importance of the benzoyl group at C-3 on the coumarin nucleus,
since lower values of trypanocidal activities were found when it was replaced by methyl (7)
or carboxyl (8) groups. This finding corroborates the high trypanocidal activities of coumarins
in the literature with bulky substituints in this region [15, 25, 30]
.
From these results derivative 3 was maintained as prototype for the last three planned
modifications. These aimed at an additional assessment of different functional groups in the
structure and even an attempt to improve the water solubility profile of the substance in
question. The drastic drop in activity observed for the derivative 9 (actually a 3,4-dihydro-
benzopiran-2-one derivative) suggests the importance of a coumarin nucleus intact for the
trypanocidal effect. At the same time, there was a significant increase in the cytotoxicity of
this coumarin against cardiomyocytes. This alkene reduction decreases the occurrence of
the flat conformation that the coumarin nucleus possessed and its increased flexibility may
have negatively affected the interaction with the binding site.
Coumarin 10 (O-demethylated derivative) is among the most active derivatives in this work.
In contrast, the exposure of the phenolic hydroxyl promoted an increase in cytotoxicity,
positioning it along with derivative 9 as derivatives in which the selectivity index is totally
inadequate. Thus, it was noted that the intact methoxyl group is important for the desired
profile of activity with this series.
Finally, the reduction of the ketone group (derivative 11) led to a slight decline in
trypanocidal activity and increase in cytotoxicity. However, it still gave a selectivity index
greater than five. The alcoholic group formed is interesting because it may be subject to new
modifications, especially those that aim to increase water solubility. It is worth mentioning
that the reduction products 9 and 11 were obtained as racemic mixtures, so the trypanocidal
potential of isolated enantiomers may be quite different and these studies are currently in
course in our group.
A comparison of the findings on anti-epimastigote and cytotoxicity activities permitted the
selection of coumarin 3 as the best derivative to conduct in vitro assays against amastigote
forms of T. cruzi. Although assays using epimastigotes are widely used for screening
[23,32,33,34]
trypanocidal substances
some studies show divergences between the observed
activity when using the three evolutionary stages of T. cruzi [35]. Despite the fact that
correlation has been reported between compound activity against epimastigote form and in
vivo activity using mice as experimental models [36], in this work, the evaluation of the cellular
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amastigote model was carried out before performing the in vivo evaluation. The results
obtained in this test are shown in Figure 1.
<figure 1>
There was a significant decrease in the number of cells infected by amastigote forms in both
treatments, with activity observed with benznidazole treatment (IC50 = 3μM) similar to that
observed with treatment with the derivative 3 (IC50 = 13μM). The decrease in the amount of
infected cells can be observed as the concentration of derivative 3 and benznidazole is
increased, in addition to the lower number of infected cells, it can be observed lower number
of amastigotes per cell. This shows a strong effect of decreasing infectivity and multiplication
of the parasite.
Thus, as derivative 3 was shown to be effective against amastigote forms and no cytotoxic
effects were observed in the host cells, a preliminary in vivo evaluation of the potential
efficacy of that compound in reducing parasitaemia was conducted in the sequence. This
efficacy was determined using the acute murine infection model with T. cruzi Y strain at oral
doses of 50 mg/kg for six consecutive days. Coumarin 3 was administered to the mice via
suspension, since even with the use of 5% Cremophor there was no complete dissolution of
the substance. The parasitaemia was reduced in 100% of the animals treated with derivative
3 or benznidazole. A reduction in parasitaemia peak and in the area under the curve in the
treated group with derivative 3 can be clearly observed (Figure 2). Although the in vitro
assay demonstrated efficacy of 3, animals receiving this compound had a peak of
parasitaemia with an order less than that of benznidazole. However, it is stressed that the
decrease of the area under the curve was of the same order for both, although greater for
benznidazole. It is worth mentioning that at the end of the tests all treated animals, both with
the derivative 3 and benznidazole, presented good appearance and good health, indicating
that the treatment with the substances possibly did not cause any type of apparent,
physiological or external damage. The animals had normal habits and there was no death.
It should be assumed that in vitro results against epimastigote forms do not assure activity of
a substance against amastigote and trypomastigote forms, due to the morphological and
physiological differences among the evolutionary forms of the parasite [21]. However, the
biological evaluations carried out investigated the activity of the substance against each of
these forms. The parasitaemia reduction assay is accomplished during the acute phase of
infection, which is characterized by the high concentration of trypomastigote forms in the
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bloodstream. Derivative 3 showed a similar trypanocidal profile against the three
evolutionary forms of T. cruzi.
<figure 2>
4. Conclusion
A significant increase in activity against T. cruzi epimastigote form was observed when the
allyl group on the C6 carbon of nitrobenzoylcoumarin (1) was changed to an n-propyl chain,
making it as active as benznidazole. This improvement in the activity was accompanied by
low toxicity, suitable activity against the amastigote forms and the ability in lowering
parasitaemia of mice infected by the Y strain of T. cruzi. Together, these results demonstrate
the trypanocidal potential of coumarin 3, which is now under studies in our group to enhance
its in vivo activity and to discover its mode of action.
Acknowledgements
This work was supported by grants from Fapemig, Brazil (APQ-01268-16).
Conflict of interest
The authors declare no conflict of interest.
References
[1] WHO, Media Centre. 2017, Chagas disease (American trypanosomiasis) fact sheet.
[2] J.C.P. Dias, Ann. Int. Med. 2006, 144, 772.
[3] J.A. Urbina, Acta Trop. 2010, 115, 55.
[4] J. Alvim Jr., R.L.A. Dias, M.S. Castilho, et al. J. Braz. Chem. Soc. 2005, 16, 763.
[5] J.C. Gomez-Verjan, E. Estrella-Parra, E.R. Vazquez-Martinez, et al. Food Chem. Toxicol.
2016, 91, 117.
[6] P.C. Vieira, J. Mafezoli, M.T. Pupo, et al. Pure Appl. Chem. 2001, 73, 617.
[7] E. Scio, A. Ribeiro, T.M.A. Alves, et al. J. Nat. Prod. 2003, 66, 634.
[8] R. Reyes-Chilpa, E. Estrada-Muñiz, E. Vega-Avila, et al. Mem. Inst. Oswaldo Cruz. 2008,
103, 431.
[9] F. Pavão, M.S. Castilho, M.T. Pupo, et al. FEBS Letters. 2002, 520, 13.
[10] S. Oliaro-Bosso, F. Viola, S. Taramino, et al. ChemMedChem. 2007, 2, 226.
[11] M.G. Pizzolatti, B.G. Mendes, A. Cunha Jr, et al. Braz J. Pharmacogn. 2008, 18, 177.
[12] A. Amin, E. Tuenter, P. Cos, et al. Molecules. 2016, 21, 1287.
This article is protected by copyright. All rights reserved.

[13] R.F. Guíñez, M.J. Matos, S. Vazquez-Rodriguez, et al. Future Med. Chem. 2013, 5,
1911.
[14] F. Belluti, E. Uliassi, G. Veronesi, et al. ChemMedChem. 2014, 9, 371.
[15] S. Vazquez-Rodriguez, R.F. Guíñez, M.J. Matos, et al. Med. chem. 2015, 5, 173.
[16] E. Uliassi, G. Fiorani, R.L. Krauth-Siegel, et al. Eur. J. Med. Chem. 2017, 141, 138.
[17] F.Pérez-Cruz, S. Vazquez-Rodriguez, M. J.Matos, A. Herrera-Morales et al. J. Med.
Chem. 2013, 56 (15), 6136
[18] I. A. Rosa, L. de Almeida, K.F. Alves, et al. Med Chem Res. 2016, 26, 131.
[19] V. La Pietra, L. Marinelli, S. Cosconati. Eur. J. Med. Chem. 2012, 47, 143.
[20] N. Grimblat, A.M. Sarotti, T.S. Kaufman, et al. Org. Biomol. Chem. 2016, 14, 10496.
[21] L.S. Filardi, Z. Brener, Trans. R. Soc. Trop. Med. Hyg. 1987, 81, 755.
[22] M. T. Bahia, A. F. Nascimento, A. L. Mazzeti et al. Antimicrob Agents Chemother. 2014,
58 (8), 4362.
[23] M. Rolón, C. Veja, J. A. Escario et al. Parasitol. Res. 2006, 99 (2), 103.
[24] N.N. Karade, S.V. Gampawar, S.V. Shinde, et al. Chin. J. Chem. 2007, 25, 1686.
[25] S. Vazquez-Rodriguez, R.F. Guíñez, M.J. Matos, et al. Med. Chem. Commun. 2013, 4,
993.
[26] F.D. Bellamy, K. Ou, Tetrahedron Lett. 1984, 25, 839.
[27] R.O. Hutchins, D. Rotstein, N. Natale, et al. J. Org. Chem. 1976, 41, 3328.
[28] T.M. Kosak, H.A. Conrad, A.L. Korich, et al. Eur. J. Org. Chem. 2015, 34, 7460.
[29] L. Banfi, E. Narisano, R. Riva et al. Encyclopedia of Reagents for Organic Synthesis.
2004. J. Wiley & Sons, New York.
[30] M.S. Lopes, R.C.C.S. Pietra, T.F. Borgati, et al. Eur. J. Med. Chem. 2011, 46, 5443.
[31] F. Sanchez-Sancho, N.E. Campillo, J.A. Paez, Curr. Med. Chem. 2010, 17, 423.
[32] J. A. Urbina, G. Payares, C. Sanoja et al. Int. J. Antimicrob Agents. 2003, 21 (1), 27.
[33] M. A. Chena, S. Elizondo, L. Rodríguez-Páez et al. J. Enzyme Inhib. Med. Chem. 2005,
20 (2),189.
[34] M. Moreno, D. A. D'ávila, M. N. Silva et al. Mem. Inst. Oswaldo Cruz. 2010, 105 (7),
918.
[35] R. A. Neal, J. van Bueren. Trans. R. Soc. Trop. Med. Hyg. 1988, 82 (5), 709.
[36] S. L. Croft. Parasitol. Today.1986, 2, 64.
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Scheme 1: Natural and synthetic coumarins with trypanocidal activity. A) mammea B/BA type coumarin; B)
chalepin; C) umbelliferone aminoalkyl derivative; D) 6-methoxy-7-prenyloxycoumarin; E) fesolol; F) 10’-R-
karatavacinol; G) 3-amino-4-hydroxy-2H-chromen-2-one; H) (E)-3-(3-(2,5-dimethoxyphenyl)acryloyl)-2H-
chromen-2-one; I) 2-((3-(2-(dimethylamino)ethoxy)-2-oxo-2H-chromen-7-yl)oxy)anthracene-1,4-dione.
Scheme 2: Coumarin 1 and structural modification planned for structure-activity relationship studies.
Scheme 3: Synthetic route to coumarin 1 and its analogues 2-11. Reagents and conditions: (i) 1) hexamine,
glacial AcOH, 125 ºC, 5h, 2) HCl 2M, 0.5h; (ii) ethyl 4-nitrobenzoyl acetate, piperidine, EtOH, 80 ºC, 3h; (iii)
SnCl2. H2O, EtOH, 85 ºC, 2h; (iv) succinic anhydride, pyridine, 90 ºC, 24h; (v) benzoyl acetoacetate for derivative
6 or ethyl acetoacetate for derivative 7 or malonic acid for derivative 8, L-proline, 90ºC, 1h; (vi) NaBH3CN, HCl
1M, MeOH, r.t., 3h; (vii) AlCl3, anhydrous CH2Cl2, r.t., 1.5h; (viii) 1) NaBH4, NaOH 0.1M, MeOH, r.t., 3h, 2) HCl
1M, 0.5h.
Table 1 – Trypanocidal activity (IC50, µM) against T. cruzi epimastigote forms, citotoxicity on cardiomyocytes
H9C2 (CC50, µM) and selectivity index (SI, from the ratio CC50/IC50) for coumarins 1-11 and benznidazole.
Figure 1 - Slides and activity results of coumarin 3 and benznidazole against amastigote forms of T. cruzi. Cells
treated with coumarin 3: 60 μM (A), 15 μM (B) and 3.75 μM (C). Cells treated with benznidazole: 60 μM (D), 15
μM (E) and 3.75 μM (F). Test controls: uninfected cells (G) and untreated infected cells (H). The arrows point to
amastigotes. Bar indicates 50 μm.
Figure 2 - Influence of treatment with derivative 3 and benznidazole on parasitaemia. The lines represent the
means of parasitaemia values obtained in peripheral blood samples from mice infected with 5,000
trypomastigotes of the T. cruzi Y strain. Control: animals infected by T. cruzi and untreated. AUC: area under the
curve. Line different letters are significantly different (P<0.05).
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Compound
1
2
IC50 ± SD
66 ± 15
>500
CC50
>200
-
SI
>3.03
-
3
28 ± 3
>200
>7.14
4
>500
-
-
5
>500
-
-
6
7
8
9
10
11
176 ± 30
251 ± 43
318 ± 40
196 ± 58
71 ± 32
35 ± 12
25 ± 10
-
-
-
-
-
-
108 ± 37
58 ± 18
185 ± 70
>200
0.55
0.81
5.28
>8
Benznidazole
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`
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