Design, synthesis and antitrypanosomatid activities of 3,5-diaryl-isoxazole analogues based on neolignans veraguensin, grandisin and machilin G

Article abstract of DOI:10.1111/cbdd.13417

Using bioisosterism as a medicinal chemistry tool, 16 3,5-diaryl-isoxazole analogues of the tetrahydrofuran neolignans veraguensin, grandisin and machilin G were synthesized via 1,3-dipolar cycloaddition reactions, with yields from 43% to 90%. Antitrypanosomatid activities were evaluated against Trypanosoma cruzi, Leishmania (L.) amazonensis and Leishmania (V.) braziliensis. All compounds were selective for the Leishmania genus and inactive against T.?cruzi. Isoxazole analogues showed a standard activity on both promastigotes of L.?amazonensis and L.?braziliensis. The most active compounds were 15, 16 and 19 with IC₅₀ values of 2.0, 3.3 and 9.5?μM against L.?amazonensis and IC₅₀ values of 1.2, 2.1 and 6.4?μM on L.?braziliensis, respectively. All compounds were noncytotoxic, showing lower cytotoxicity (>250?μM) than pentamidine (78.9?μM). Regarding the structure–activity relationship (SAR), the methylenedioxy group was essential to antileishmanial activity against promastigotes. Replacement of the tetrahydrofuran nucleus by an isoxazole core improved the antileishmanial activity.

Full text of DOI:10.1111/cbdd.13417

PROFESSOR ADRIANO CESAR DE MORAIS BARONI (Orcid ID : 0000-0002-5371-3755)
Article type : Research Article
Design, synthesis and anti-trypanosomatid activities of 3,5-diaryl-isoxazole analogues based on
neolignans veraguensin, grandisin and machilin G
Ozildéia S. Trefzger,1a Amarith R. das Neves,1,2a Natália V. Barbosa,1,2 Diego B. Carvalho,1
Indiara C. Pereira,3 Renata T. Perdomo, 3 Maria F. C. Matos, 3 Nidia C. Yoshida,4 Massuo J.
Kato,5 Sérgio de Albuquerque,6 Carla C. P. Arruda2 and Adriano C. M. Baroni1*
aThese authors contributed equally to this work.
1
LASQUIM – Laboratório de Síntese e Química Medicinal, FACFAN - Faculdade de
Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal do Mato Grosso do
Sul, UFMS, Campo Grande/MS, 79070-900, Brazil
2 Laboratório de Biologia molecular e Cultura de Células, FACFAN - Faculdade de Ciências
Farmacêuticas, Alimentos e Nutrição, Universidade Federal do Mato Grosso do Sul, UFMS,
Campo Grande/MS, 79070-900, Brazil
3
Laboratório de Parasitologia Humana, INBIO - Instituto de Biologia, Universidade Federal do Mato
Grosso do Sul, 79070-900, Campo Grande, MS, Brazil.
4
Instituto de Química, Universidade Federal do Mato Grosso do Sul, UFMS, Campo Grande/MS,
79070-900, Brazil.
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.13417
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5 Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, 05508-000, São Paulo,
SP, Brazil.
6
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, USP, 14040-930, Ribeirão Preto, SP, Brazil.
Correspondence:
Adriano C. M. Baroni, LASQUIM – Laboratório de Síntese e Química Medicinal, FACFAN
- Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal do Mato
Grosso do Sul, UFMS, Campo Grande/MS, 79070-900, Brazil.
Tel.: 55673345-7365
Email: adriano.baroni@ufms.br
Abstract
Using bioisosterism as a medicinal chemistry tool, 16 3,5-diaryl-isoxazole analogues of the
tetrahydrofuran neolignans veraguensin, grandisin and machilin G, were synthesized via 1,3-
dipolar cycloaddition reactions, with yields from 43–90%. Anti-trypanosomatid activities
were evaluated against Trypanosoma cruzi, Leishmania (L.) amazonensis and Leishmania
(V.) braziliensis. All compounds were selective for the Leishmania genus and inactive against
T. cruzi. Isoxazole analogues showed a standard activity on both promastigotes of L.
amazonensis and L. braziliensis. The most active compounds were 15, 16 and 19 with IC50
values of 2.0, 3.3 and 9.5 µM against L. amazonensis and IC50 values of 1.2, 2.1 and 6.4 µM
on L. braziliensis, respectively. All compounds were non-cytotoxic, showing lower
cytotoxicity (>250 µM) than pentamidine (78.9 µM). Regarding the structure-activity
relationship (SAR), the methylenedioxy group was essential to antileishmanial activity
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against promastigotes. Replacement of the tetrahydrofuran nucleus by an isoxazole core
improved the antileishmanial activity.
Keywords: Neolignans, cycloaddition [3+2], isoxazole, bioisosterism.
1. Introduction
Leishmaniasis is among many neglected diseases prevailing in underdeveloped
tropical countries in Asia, Africa and Central America and South America.1 Ranking
just after malaria and with over one billion people worldwide at risk of infection,
leishmaniasis is a severe health problem. The main clinical forms of the disease are
cutaneous, mucocutaneous and visceral.1,2
The first-line treatment for leishmaniasis consists of administration of pentavalent
antimonials such as meglumine antimoniate (Glucantime®) and sodium
stibogluconate (Pentostam®). However, the use of antimonial therapy is limited due to
its hepatotoxicity, cardiotoxicity, and nephrotoxicity, as well as other diverse collateral
effects.3
When these drugs are ineffective or cannot be prescribed for other reasons, the second
line of treatment typically involves amphotericin B, pentamidine or paromomycin.
However, the adverse side effects and parasite resistance to all of these drugs
underscore the urgency of developing newer and safer therapeutic agents for the
treatment of leishmaniasis, as well as other neglected diseases.4
Chagas disease, caused by Trypanosoma cruzi, is another member of the neglected-
disease family. With a high mortality and morbidity rate, affecting over six million
people in Latin American countries, its treatment is restricted to two drugs,
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benznidazole, and nifurtimox, which are only useful during the acute phase of the
disease.5
Benznidazole is the first-choice drug in most cases, even though it presents problems
such as a narrow therapeutic window, side effects and different susceptibility of T.
cruzi strains.6
Currently available treatments for neglected diseases are far from ideal. It is therefore,
imperative to emphasize the need for continuous research on the development of new
and useful candidate drugs.
Some outstanding drug-discovery programs are widely used to find leading
compounds, based on and inspired by the molecular modifications of natural products.
Recent studies have demonstrated the potential of this approach for the discovery of
new chemical entities in the treatment of neglected diseases.2,6–10
Tetrahydrofuran neolignans, derived from natural sources are considered privileged
structures since they show several biological activities including antibacterial,
antifungal, anti-tumour, and anti-inflammatory. The tetrahydrofuran neolignans
veraguensin (1), grandisin (2), and machilin G (3), for instance (Figure 1), have shown
anti-trypanosomal and anti-leishmanial activities (Figure 1).11–21
Regarding the biological activities of compounds 1-3, IC50 values were described of
2.3, 3.7 and 2.2 M, respectively, against T. cruzi.18-20 Veraguensin (1) and machilin G
(
3) showed an IC50 value of 18 g mL-1 (48.8 and 50.54 M respectively) against L.
donovani.13 These results indicate that neolignans 1-3 are more active against T. cruzi
than the leishmaniasis.
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Considering that our research group is interested in developing new ligands against
leishmaniasis and Chagas disease we developed compounds via molecular
modification of neolignans 1–3, using a bioisosterism approach to obtain analogues
with increased potency. Thus, the tetrahydrofuran core from the neolignans was
replaced by a 1,2,3-triazole moiety (Figure 2).
The position isomers of 1,2,3-triazole analogues showed diverse antileishmanial
activities. The triazole hybrid 4, from grandisin 2 and machilin G 3 was the most
active against the promastigotes and amastigotes of Leishmania (L.) amazonensis
(Figure 2).2,8
In order to increase the compound series based on the tetrahydrofuran neolignan
family, our research group is particularly interested in the isoxazole core. Several
compounds containing this nucleus have shown interesting biological activities
including antimicrobial, antiviral, anticancer, anti-inflammatory, immunomodulatory,
anticonvulsant, anti-diabetic and anti-trypanosomatid.22 The isoxazole core is a
bioisostere of some heterocyclic rings and was used in the design of valdecoxib.23 It
shows a structural resemblance to the 1,2,3-triazole nucleus, and is also a hydrogen-
bond acceptor.24,25
Therefore, we theorized that the isoxazole core must be a ring equivalent and
bioisostere of the tetrahydrofuran nucleus and the 1,2,3-triazole ring. In this way, the
aim of this study was to obtain 16 new, synthetic isoxazole hits inspired by neolignan
structures 1–3 and to evaluate their anti-trypanosomatid activities (Figure 2).
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2. Experimental
2.1. General remarks
Proton (1H) and carbon (13C) nuclear magnetic resonance (NMR) spectra were recorded on Brucker
75 MHz or 300 MHz spectrometer, with deuterated chloroform solution (CDCl3) or DMSOd6. For
NMR data, chemical shifts (δ) are expressed as parts per million (ppm) downfield with values related
to tetramethylsilane (TMS) used as an internal standard. High-resolution electrospray ionization (HR-
ESI-MS) measurements were carried out on a quadrupole time-of-flight instrument (UltrOTOF-Q,
Bruker Daltonics, Billerica, MA). Infrared spectrums (IR) were recorded on a Thermo Scientific
Nicolet iS5® system equipped with an iD3 attenuated total reflectance (ATR) attachment
(germanium crystal). All solvents were dried and distilled before use according to the standard
procedure. All reactions were performed under an atmosphere of dry nitrogen and monitored by
thin layer chromatography (TLC) using prepared plates (silica gel 60 F254 on aluminium). Flash
column chromatography was performed using silica gel 60 (particle size 200-400 mesh ASTM,
purchased from Sigma-Aldrich, (USA). Melting points (mp) were determined using a Fisatom 430D.
Terminal acetylenes, oximes, and chloro-oximes were synthesized using procedures from the
literature.2,8,26,33-39 The spectra data for all isoxazole analogs 5-20 as, 1H NMR, 13C NMR, IR and High-
Resolution Mass Spectrometry, is provided in the Supporting Information.
2.2. Synthesis
General procedure for the preparation of Isoxazole compounds 5-1626
To a solution of the respective chloro-oxime 29a-c (2 mmol) and the acetylenes 26a-d
(2.1mmol) in CH2Cl2/THF 1:1, 4 mL/mmol, CuSO4.5H2O (0.15 mmol), sodium ascorbate
(0.35 mmol) and KHCO3 (20 mmol) were added. The mixture was stirred at room
temperature for 48 hours. Extraction was performed with ethyl acetate and washed with a
saturated solution of NH4Cl (3 x 30 mL), the organic phase was dried over anhydrous
MgSO4, and solvent was removed under vacuum.
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3,5-bis-(4-methoxyphenyl) isoxazole 5.40 The product was purified by crystallization in
heated hexane/ethyl acetate solution (90:10). The product was obtained as a yellow solid, mp
= 160 °C. Yield: 90%.1H NMR (300 MHz, CDCl3) δ 3.84 (s, 3H, OCH3); 3.85 (s, 3H,
OCH3); 6.63 (s, 1H, CH); 6.97 (d, J = 8.7 Hz, 4H, Ar-H); 7.75 (d, J = 8.7 Hz, 2H, Ar-H);
7.78 (d, J = 8.7 Hz, 2H, Ar-H). 13C NMR (CDCl3, 75 MHz) δ 55.3; 55.4; 95.91; 114.3; 114.4;
120.4; 121.8; 127.4; 128.2; 160.9; 161.1; IR (ATR): 2842; 2922; 2851; 1575; 1502; 1436;
1260; 1123; 1123; 1021; 860; 841; 804 vmax(cm-1). HRMS (ESI+) m/z calcd for C17H15NO3
[M + H]: 282.1124; found: 282.1134.
3,5-bis-(3,4-dimethoxyphenyl) isoxazole 6. The product was purified by crystallization in a
heated hexane/ethyl acetate solution (90:10). The product was obtained as a white solid, mp =
1
131 °C. Yield: 55%. H NMR (300 MHz, CDCl3) δ 3.92 (s, 6H, 2xOCH3); 3.95 (s, 6H,
2xOCH3); 6.66 (s, 1H, CH); 6.93 (dd, J = 9.0 and 3.0 Hz, 2H, Ar-H); 7.53 (d, J = 1.5 Hz, 1H,
13
Ar-H); 7.66 (m, 2H, Ar-H); 8.03 (s, 1H, Ar-H). C NMR (CDCl3, 75 MHz) δ 55.9; 56.0;
56.0; 56.0; 96.2; 108.7; 109.4; 111.1; 111.3; 119.1; 119.9; 120.5; 122.0; 142.3; 150.6; 150.7;
162.7; 170.2. IR (ATR): 1608.; 1509; 1435; 1269.90; 1222; 1143; 1020; 862; 800; 765 vmax
(cm-1). HRMS (ESI+) m/z calcd for C19H19NO5 [M + H]: 342.1359; found: 342.1353.
3,5-bis-(1,3-benzodioxol-5-yl) isoxazole 7. The product was purified by crystallization in a
heated hexane/ethyl acetate (90:10) solution. The product was obtained as a yellow solid, mp
=193°C, Yield: 75%.1H NMR (300 MHz, CDCl3) δ 6.01 (s, 2H, CH2); 6.02 (s, 2H, CH2);
6.58 (s, 1H, CH); 6.86 (d, J = 8.1 Hz, 1H, Ar-H); 6.87 (d, J = 8.1 Hz, 1H, Ar-H); 7.37 (dd, J
= 8.3 and 1.9 Hz, 1H, Ar-H); 8.02 (d, J = 9.1 Hz, 2H, Ar-H); 8.42 (d, J = 8.9 Hz, 2H, Ar-
H).13C NMR (CDCl3, 75 MHz) δ 96.4; 101.4; 101.6; 106.1; 106.96; 108.6; 108.8; 120.4;
121.1; 121.6; 123.1; 148.2; 148.2; 149.1; 149.3; 162.5; 170.0 IR (ATR): 2921; 2851; 1592;
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1499; 1459.; 1253; 1038; 866; 817; 795 vmax(cm-1). HRMS (ESI+) m/z calcd for C17H11NO5
[M + H]: 310.0709; found: 310.0705.
5-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl) isoxazole 8. The product was purified by
crystallization in a heated hexane/ethyl acetate solution (90:10). The product was obtained as
an off white solid, mp= 111°C. Yield: 72%.1H NMR (300 MHz, CDCl3) δ 3.85 (s, 3H,
OCH3); 3.92 (s, 3H, OCH3); 3.95 (s, 3H, OCH3); 6.66 (s, 1H, CH); 6.93 (d, J = 8.4 Hz, 1H,
Ar-H); 6.97 (d, J = 8.7 Hz, 2H, Ar-H); 7.32 (d, J = 1.8 Hz, 1H, Ar-H); 7.39 (dd, J = 8.1 and
1.8 Hz, 1H, Ar-H); 7.78 (d, J = 8.7 Hz, 2H, Ar-H). 13C NMR (CDCl3, 75 MHz) δ 55.4; 56.0;
56.1; 96.2; 108.7; 111.3; 114.3; 119.1; 120.5; 121.7; 128.2; 149.3; 150.7; 162.0; 162.6;
170.1. IR (ATR): 2998; 2840; 1616; 1509; 1441; 1247; 1178; 1141; 861; 791 vmax(cm-1).
HRMS (ESI+) m/z calcd for C18H17NO4 [M + H]: 312,1236; found: 312.1239.
3-(3,4-dimethoxyphenyl)-5-(4-methoxyphenyl) isoxazole 9. The product was purified by
crystallization in a heated hexane/ethyl acetate solution (90:10). The product was an off white
solid, mp = 121 °C. Yield: 55%.1H NMR (300 MHz, CDCl3) δ 3.85 (s, 3H, OCH3); 3.92 (s,
3H, OCH3); 3.95 (s, 3H, OCH3); 6.66 (s, 1H); 6.92 (d, J = 9.0 Hz, 1H); 6.97 (d, J = 9.0 Hz,
2H); 7.33 (dd, J = 9.0 and 2.2 Hz, 1H); 7.45 (d, J = 2.1 Hz, 1H); 7.76 (d, J = 9.0 Hz, 2H). 13C
NMR (CDCl3, 75 MHz) δ 55.4; 56.0; 56.0; 96.0; 109.3; 111.0; 114.4; 119.9; 120.4; 122.0;
127.4; 149.3; 150.53; 161.1; 170.2. IR (ATR): 1616; 1432; 1251; 1172; 1144; 1024; 832; 809
vmax(cm-1). HRMS (ESI+) m/zcalcd for C18H17NO4 [M + H]: 312.1230; found: 312.1230.
5-(1,3-benzodioxol-5-yl)-3-(4-methoxyphenyl) isoxazole 10. The product was purified by
crystallization in a heated hexane/ethyl acetate solution (90:10). The product was obtained as
an off white solid, mp= 137°C. Yield: 68%.1H NMR (300 MHz, CDCl3) δ 3.84 (s, 3H,
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OCH3); 6.02 (s, 2H, CH2); 6.61 (s, 1H, CH); 6.88 (d, J = 8.1 Hz, 1H, Ar-H); 6.97 (d, J = 9.0
Hz, 2H, Ar-H); 7.26 (d, J = 1.5 Hz, 1H, Ar-H); 7.34 (dd, J = 8.1 and 1.5 Hz, 1H, Ar-H); 7.76
13
(d, J = 7.2 Hz, 2H, Ar-H). C NMR (CDCl3, 75 MHz) δ 55.3; 96.3; 101.6; 106.1; 108.8;
113.8; 114.3; 120.4; 121.7; 121.7; 128.2; 148.2; 149.2; 161.0.; 162.6; 169.9. IR (ATR): 1613;
1502; 1492; 1437; 1392.84; 1250.; 1179; 1109; 825; 804 vmax (cm-1). HRMS (ESI+) m/zcalcd
for C17H13NO4 [M + H]: 296.0917; found: 296.0923.
3-(1,3-benzodioxol-5-yl)-5-(4-methoxyphenyl) isoxazole 11. The product was purified by
crystallization in a heated hexane/ethyl acetate (90:10) solution. The product obtained was a
1
yellow solid, mp = 162°C, Yield: 77%. H NMR (300 MHz, CDCl3) δ 3.85 (s, 3H, OCH3);
6.01 (s, 2H, CH2); 6.60 (s, 1H, CH); 6.88 (d, J = 9.0 Hz, 1H, Ar-H); 6.97 (d, J = 9.0 Hz, 2H,
Ar-H); 7.30 (dd, J = 9.0 and 1.5 Hz, 1H, Ar-H); 7.35 (d, J = 1.5 Hz, 1H, Ar-H); 7.74 (d, J =
13
6.0 Hz, 2H, Ar-H). C NMR (CDCl3, 75 MHz) δ 55.4; 96.0; 101.4; 107.0; 108.6; 114.4;
120.3; 121.1; 123.3; 127.4; 148.2; 149.05; 161.1; 162.6; 170.2. IR (ATR): 2921; 2851; 1612;
1504; 1457; 1250; 1180; 1114; 1030; 872; 825; 804 vmax (cm-1). HRMS (ESI+) m/z calcd for
C17H13NO4 [M + H]: 296,0917; found: 296,0932.
3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl) isoxazole 12. The product was purified
by crystallization in heated a hexane/ethyl acetate solution (90:10). The product was obtained
1
as an off white solid, mp= 156°C. Yield: 80%. H NMR (300 MHz, CDCl3) δ 3.84 (s, 3H,
OCH3); 3.88 (s, 3H, OCH3); 3.92 (s, 6H, 2xOCH3); 6.70 (s, 1H, CH); 6.96 (d, J 8.7 Hz, 2H,
Ar-H); 7.02 (s, 2H, Ar-H); 7.77 (d, J 8.7 Hz, 2H, Ar-H). 13C NMR (CDCl3, 75 MHz) δ 55.3;
56.3; 61.0; 97.0; 103.2; 114.3; 121.6; 122.9; 128.2; 139.8; 153.6; 161.0; 162.7; 169.9. IR
(ATR): 1615; 1571; 1498; 1418; 1185; 1125; 999; 839; 795 vmax (cm-1). HRMS (ESI+) m/z
calcd for C19H19NO5[M + H]: 342.1335; found: 342.1328.
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3-(3,4-dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl) isoxazole 13. The product was
purified by crystallization in a heated hexane/ethyl acetate solution (90:10). The product was
1
obtained as a yellow solid, mp = 107 °C, Yield: 52%. H NMR (300 MHz, CDCl3) δ 3.88 (s,
3H, OCH3); 3.91 (s, 3H, OCH3); 3.92 (s, 6H, 2xOCH3); 3.95 (s, 3H, OCH3); 6.70 (s, 1H,
CH); 6.92 (d, J = 8.4 Hz, 1H, Ar-H); 7.02 (s, 2H, Ar-H); 7.34 (dd, J = 8.4 and 1.8 Hz, 1H,
13
Ar-H); 7.44 (d, J = 1.8 Hz, 1H, Ar-H). C NMR (CDCl3, 75 MHz) δ 55.9; 56.0; 56.3; 61.0;
97.0; 103.2; 109.4; 110.0; 112.0; 119.9; 120.0; 120.9; 121.8; 122.9; 139.9; 149.3; 150.6;
153.6; 162.7, 170.0. IR (ATR): 2922; 2851; 1575; 1502; 1436; 1260; 1123; 1021; 860; 841;
804 vmax(cm-1). HRMS (ESI+) m/zcalcd for C20H21NO6 [M + H]: 372,1441; found: 372,1446.
5-(1,3-benzodioxol-5-yl)-3-(3,4-dimethoxyphenyl) isoxazole 14. The product was purified
by flash chromatography on silica gel using a hexane/ethyl acetate solution (90:10), as the
1
mobile phase which resulted a white solid, mp = 140 °C, Yield: 85%. H NMR (300 MHz,
CDCl3) δ 3.90 (s, 3H, OCH3); 3.93 (s, 3H, OCH3); 6.00 (s, 2H, CH2); 6.61 (s, 1H, CH); 6.86
(d, J = 8.1 Hz, 1H, Ar-H); 6.90 (d, J = 8.4 Hz, 1H, Ar-H); 7.23 (d, J = 1.2 Hz, 1H, Ar-H);
7.29 (dd, J = 8.7 and 1.2 Hz, 1H, Ar-H); 7.32 (dd, J = 8.1 and 1.5 Hz, 1H, Ar-H); 7.42 (d, J
13
= 1.8 Hz, 1H, Ar-H). C NMR (CDCl3, 75 MHz) δ 55.9; 56.0; 96.3; 101.6; 106.1; 108.8;
109.3; 111.0; 119.8; 120.4; 121.6; 121.9; 148.2; 149.2; 149.2; 150.57; 162.6; 169.9. IR
(ATR): 2922; 2851; 1607; 1495; 1434.; 1257; 1226; 1173; 1143; 1032; 852; 820; 804 vmax
(cm-1). HRMS (ESI+) m/z calcd for C18H15NO5 [M + H]: 326.1022; found: 326.1021.
3-(1,3-benzodioxol-5-yl)-5-(3,4-dimethoxyphenyl) isoxazole 15. The product was purified
by crystallization in heated hexane/ethyl acetate solution (90:10). The product was obtained
1
as a yellow solid, mp = 133°C, Yield: 80%. H NMR (300 MHz, CDCl3) δ 3.92 (s, 3H,
OCH3); 3.95 (s, 3H, OCH3); 6.01 (s, 2H, CH2); 6.62 (s, 1H, CH); 6.93 (d, J = 8.1 Hz, 1H, Ar-
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H); 6.88 (d, J = 8.4 Hz, 1H, Ar-H); 7.30 (dd, J = 9.0 and 1.5 Hz, 1H, Ar-H); 7.35 (d, J 1.5
Hz, 1H, Ar-H); 7.74 (d, J 6.0 Hz, 2H, Ar-H). 13C NMR (CDCl3, 75 MHz) δ 55.9; 56.0; 96.2;
101.4; 106.9; 108.5; 111.2; 119.0; 120.3; 121.0; 123.1; 148.1; 149.0; 149.2; 150.6; 162.5;
170.1. IR (ATR): 2922; 2851; 1607; 1508; 1465; 1252; 1139; 1035; 818; 809; 797 vmax (cm-
1). HRMS (ESI+) m/z calcd for C18H15NO5 [M+H]: 326,1022; found: 326,1032.
3-(1,3-benzodioxol-5-yl)-5-(3,4,5-trimethoxyphenyl) isoxazole 16. The product was
purified by crystallization by heated in hexane/ethyl acetate solution (90:10). The product
1
was obtained as a yellow solid, mp = 138 °C, Yield: 71%. H NMR (300 MHz, CDCl3) δ
3.88 (s, 3H, OCH3); 3.91 (s, 6H, 2xOCH3); 6.01 (s, 2H, CH); 6.66 (s, 1H, CH2); 6.87 (d, J =
8.1 Hz, 1H, Ar-H); 7.01 (s, 2H, Ar-H); 7.30 (dd, J = 8.1 and 1.5 Hz, 1H, Ar-H); 7.35 (d, J =
13
1.5 Hz, 1H, Ar-H); C NMR (CDCl3, 75 MHz) δ 56.3; 61.0; 97.0; 101.5; 103.1; 107.0;
108.6; 121.1; 122.8; 123.0; 139.8; 148.2; 149.2; 153.6; 162.6; 170.0. IR (ATR): 2922; 2851;
1574; 1503; 1464; 1421; 1248; 1125; 1039; 875; 847.08; 823; 807 vmax (cm-1). HRMS (ESI+)
m/z calcd for C19H17NO6 [M + H]: 356.1128; found: 356.1128.
General procedure for the preparation of isoxazole compounds 17-20.32
A mixture of the N-chlorosuccinimide (2 mmol), pyridine (4 drops), and oxime 28d (2 mmol)
in CHCl3, was stirred by 12 h at room temperature. After terminal acetylenes 26a-d (2.1
mmol), followed by triethylamine (4mmol) were added. The mixture was stirred at room
temperature for 48 hours. Extraction was performed with ethyl acetate and washed with a
saturated solution of NH4Cl (3 x 50 mL). The organic phase was dried over anhydrous
MgSO4, and solvent was removed under vacuum.
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3-(3,4,5-trimethoxyphenyl)-5-(4-methoxyphenyl) isoxazole 17. The product was purified
by flash chromatography on silica gel using a hexane/ethyl acetate (90:10) solution as the
1
mobile phase. The product was obtained as an off-white solid, mp = 168°C, Yield: 49%. H
NMR (300 MHz, CDCl3) δ 3.88 (s, 3H, OCH3); 3.91 (s, 3H, OCH3); 3.96 (s, 6H, 2xOCH3);
6.69 (s, 1H, CH); 7.01 (d, J = 6.0 Hz, 2H, Ar-H); 7.09 (s, 2H, Ar-H); 7.79 (d, J = 6.0 Hz, 2H,
13
Ar-H). C NMR (CDCl3, 75 MHz) δ 55.4; 56.3; 60.9; 96.1; 104.1; 114.4; 120.2; 124.7;
127.4; 153.6; 161.2; 162.8; 170.5. IR (ATR): 2965; 2937; 1615; 1505; 1431; 1235; 1125;
1000; 841; 794 vmax (cm-1). HRMS (ESI+) m/z calcd for C19H19NO5 [M + H]: 342.1341;
found: 342.1359.
3-(3,4,5-trimethoxyphenyl)-5-(3,4-dimethoxyphenyl) isoxazole 18. The product was
purified by flash chromatography on silica gel using a hexane/ethyl acetate (90:10) solution
as the mobile phase. The product was obtained as a yellow solid, mp = 118°C. Yield: 51%.
1H NMR (300 MHz, CDCl3) δ 3.91 (s, 3H, OCH3); 3.96 (s, 9H, 3xOCH3); 3.99 (s, 3H,
OCH3); 6.70 (s, 1H, CH); 6.96 (d, J = 9.0 Hz, 1H, Ar-H); 7.09 (s, 2H, Ar-H); 7.35 (d, J = 1.0
Hz, 1H, Ar-H); 7.42 (dd, J = 9.0 and 1.0 Hz, 1H, Ar-H). 13C NMR (CDCl3, 75 MHz) δ 56.0;
56.1; 56.3; 61.0; 96.4; 104.1; 108.7; 111.3; 119.2; 120.37; 124.6; 149.3; 150.8; 153.6; 162.9;
170.4. IR (ATR): 2937; 2837; 1611; 1505; 1424; 1269.41; 1236; 1131; 1022; 858; 849; 786
vmax (cm-1). HRMS (ESI+) m/z calcd for C20H21NO6 [M + H]: 372.1447; found: 372.1441.
5-(1,3-benzodioxol-5-yl)-3-(3,4,5-trimethoxyphenyl) isoxazole 19. The product was
purified by flash chromatography on silica gel using a hexane/ethyl acetate (90:10) solution
as the mobile phase. The product was obtained as a white solid, mp= 142 °C. Yield: 45%. 1H
NMR (300 MHz, CDCl3) δ 3.87 (s, 3H, OCH3); 3.90 (s, 6H, 2xOCH3); 6.00 (s, 2H, CH2);
6.62 (s, 1H, CH); 6.86 (d, J = 9.0 Hz, 1H, Ar-H); 7.02 (s, 2H, Ar-H); 7.23 (d, J = 3.0 Hz, 1H,
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13
Ar-H); 7.31 (dd, J = 9.0 and 3.0 Hz, 1H, Ar-H); C NMR (CDCl3, 75 MHz) δ 56.2; 60.9;
96.5; 101.6; 104.0; 106.1; 108.8; 120.4; 121.5; 124.5; 139.6; 148.3; 149.3; 153.6; 162.8;
179.2. IR (ATR): 2905; 2853; 1581; 1505; 1493; 1469; 1451; 1423; 1265; 1228; 1112; 1037;
998; 840; 781 vmax (cm-1). HRMS (ESI+) m/z calcd for C19H17NO6 [M + H]: 356.1129; found:
356.1134.
3,5-bis-(3,4,5-trimethoxyphenyl) isoxazole 20. The product was purified by flash
chromatography on silica gel using a hexane/ethyl acetate (90:10) solution as the mobile
phase. The product was obtained as a yellow solid, mp = 143 °C. Yield: 43%. 1H NMR (300
MHz, CDCl3) δ 3.91 (s, 6H, 2xOCH3); 3.95 (s, 12H, 4xOCH3); 6.67 (s, 1H, CH); 7.05 (s, 2H,
Ar-H); 7.08 (s, 2H, Ar-H). 13C NMR (CDCl3, 75 MHz) δ 26.7; 56.3; 61.0; 97.2; 103.2; 104.1;
122.8; 153.6. IR (ATR): 2925; 2852; 1574; 1502; 1422; 1236; 1125; 999; 854; 833; 778 vmax
(cm-1). HRMS (ESI+) m/z calcd for C21H23NO7 [M + H]: 402.1553; found: 402.1549.
2.2. Biological Methods
2.2.1. Antileishmanial assays
Leishmania (Leishmania) amazonensis
In 96-well microplates, the synthetic compounds 5-20 were tested in quintuplicates, with final
concentrations of 0.78 μg/mL to 50 μg/mL. Promastigotes forms of L. amazonensis
(IFLA/BR/1967/PH8 strain) cultivated in Schneider’s Insect Medium(Sigma-Aldrich®) were
added to the wells in the exponential phase with a concentration of 2x105 parasites/mL. The
plates were incubated at 26°C for 72 h. To evaluate the cell viability, 20 μL (5mg/mL) of
Thiazolyl Blue Tetrazolium Bromide - MTT (Sigma-Aldrich®),41 were added in each well
and incubated 37°C, 5% CO2 for 2h. After that time, 80 μL of DMSO was added to finish the
reaction.
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Pentamidine (Sigma-Aldrich®) was used as positive control. Dimethyl sulfoxide (DMSO,
Sigma-Aldrich®), in Schneider’s Insect Medium, was used as negative control and did not
interfere with cell viability. The reaction results were obtained in a spectrophotometer
(Biotek) at 540 nm. A non-linear dose-response regression curve was used to calculate the
half maximum inhibitory concentration (IC50).
The percentage of activity was calculated by the following formula:
% Activity = 100 - [(N-Y) / (N-P)] * 100
where:
Y = optical density reading of cells and wells with different concentrations of the
compounds;
N = optical density reading of parasites in wells with 1.5% DMSO;
P = optical density reading of the wells with only culture medium.
Pentamidine (IC50) promastigotes = 8.9 μM
Leishmania (viannia) braziliensis
The compounds 5-20 were tested in triplicates (0.5; 2.0; 8.0, and 32.0 μM) against
promastigotes forms of L. braziliensis, previously adjusted to 1x106 cells/mL in 96 well plate.
The plate was incubated in a humidified atmosphere at 22°C for 72 hours. Subsequently, the
viability of the promastigotes was tested by Thiazolyl Blue Tetrazolium Bromide - MTT
(Sigma-Aldrich®),41which was added to 50mL of an XTT solution containing PMS
(phenazine methosulfate), in the proportion of 1 mg/mL of XTT to 0.001 mg/mL of PMS.
The plate was then incubated in a humid atmosphere at 37°C and 5% CO2 for 4 hours,
protected from light. At the conclusion of the test, the reaction results were obtained in a
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spectrophotometer (Biotek) at 450 nm. The positive control was Schneider’s medium and
parasites. DMSO 1.5% was used as negative control.
The percentage of activity was calculated by the following formula:
% Activity = 100 - [(N-Y) / (N-P)] * 100
where:
Y = optical density reading of cells and wells with different concentrations of the
compounds;
N = optical density reading of parasites in wells with 1.5% DMSO;
P = optical density reading of the wells with only culture medium.
Pentamidine (IC50) promastigotes = 10.1μM
2.2.2. Trypanocidal assay2
The assays against trypomastigotes forms of T. cruzi were carried out in 96 wells microplates.
The trypomastigotes were obtained from the supernatant of cellular culture. The
concentration of parasites was adjusted to 106 forms/mL in RPMI medium, supplemented by
antibiotics and fetal bovine serum. The compounds 5-20 were added at concentrations of 0.5,
2.0, 8.0 and 32.0μM. All the compounds were evaluated in triplicates.
The material was incubated for 24 hours at 4 °C. After this period, to each well was added a
10μL solution of FluoReporter lacZ/Galactosidase quantitative Kit (Life Technologies), and
the plates were incubated again for 30 minutes. The colorimetric reaction was quantified by
fluorescence microplates reader (BIOTEK), at 386 nm excitation and 448 nm emissions.
The percentages of parasite lysis were determined from the following formula.
% lysis = 100 - {[(X-CP) / (PC-NC)]} .100
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where:
X = optical density value of the samples;
CP = optical density value of the positive controls;
CN = optical density value of the negative controls.
Culture medium was used as positive control (CP) and medium with DMSO 0.6% as a
negative control.
Benznidazole (IC50): tripomastigotes = 7.32 μM
2.2.4. Cytotoxicity assay2
Fibroblasts (NIH/3T3) obtained from Rio de Janeiro Cell Bank (Brazil), were seeded in 96
well plates (1x104 cells mL-1) and incubated with synthetic compounds at 37°C, 5% CO2 for
48h at the concentrations of 0.25 to 250 µg/mL. Doxorubicin (Sigma-Aldrich®) was used as
the reference drug at concentrations of 0.025 to 25 µg/mL. Cell growth was estimated by the
sulforhodamine B colorimetric method (SRB).42DMSO (Vetec®) was used as a negative
control at the concentration necessary to solubilize the highest concentration of the test
samples and did not interfere with cell viability. The percentage of growth was calculated as
described by Monks et al.43 IC50 was determined by non-linear regression analysis (Microcal
Origin Versão 6.0® e Microsoft Office Excel 2007®). Selectivity index (SI) was calculated
according to Medeiros et al.44
3. Results and discussion
3.1. Chemistry
The isoxazole compounds were synthesized by a cycloaddition reaction [3+2] between
terminal acetylenes and chloro-oximes catalyzed by copper salts.26 Aryl bromides were
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obtained through the reaction of 21a-c with NBS, p-TsOH, CH2Cl2 and SiO2 (Scheme
1).2,27 More-reactive aryl iodides can also be used as starting materials.28
The cross-coupling reaction between aryl bromines 22a-c and 2-methyl-3-butyn-2-ol
in the presence of PdCl2(PPh3)2/CuI, Et3N, for a duration of 24 h, produced acetylene
alcohols 23a-c with a 75–85% yield (Scheme 1).2,29
The Retro-Favorski reaction between 23a-c with KOH under reflux in toluene
generated terminal acetylenes 26a-c with 65–85% yield.30 Ethynyl-1,2,3-
trimethoxybenzene 26d was synthesized by the Corey-Fuchs method (Scheme 1).31
Oximes were prepared by reaction of methoxy benzaldehydes 27a-c with NH2OH.HCl/KOH in
H2O/EtOH 1:1 within a yield range of 85–97%. Chloro-oximes 29a-c were synthesized by reaction
between oximes 28a-c and N-chlorosuccinimide (NCS) in DMF with significant yields (Scheme 2).26
The cycloaddition [3+2] reaction was performed between terminal acetylenes 26a-d
and chloro-oximes 29a-c using a catalytic system of CuSO4.5H2O, sodium ascorbate,
and KHCO3 in a solvent mixture of THF/CH2Cl2 1:1 (Table 1).26
Additional solvent mixtures were tested as well, and included: CH2Cl2/H2O, THF/H2O, t-BuOH/THF, t-
BuOH/H2O, CuI as copper salt and TEA or NaHCO3 as bases. However, the most promising results
were achieved using the method described above.
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The synthesis of positional isomers derived from trimethoxy oxime 28d was not successful because
this compound was chlorinated in the aromatic nucleus32a when NCS in DMF was used, providing 30
with 100% conversion (Scheme 3).
However, when the reaction was performed between trimethoxy oxime 28d and acetylenes 26a-d,
using NCS, CHCl3, pyridine, and Et3N at room temperature, in a one-pot procedure, isoxazoles 17-20
were obtained in moderated yields (Scheme 4).32b-c Chlorinated isoxazoles were also formed in a
minor proportion (15-20%), but the reaction mixture was readily purified by flash chromatography.
All isoxazole compounds 5-20, synthesized in our study were analysed using nuclear magnetic
resonance 1H NMR, 13C NMR, IR and High-Resolution Mass Spectrometry.
3.2. Biological evaluation
Antitrypanosomatid in vitro activity were targeted the stationary promastigote forms of Leishmania
(L.) amazonensis and the trypomastigote forms of Trypanosoma cruzi (Table 2).
All synthesized compounds showed limited or no biological activity against T. cruzi with IC50 values
over 150 µM. These results indicate a selectivity of isoxazole analogues for the Leishmania genus.
Regarding the antileishmanial activities against L. amazonensis strains, we observed that the
symmetrical compounds 5, 6, 7 and 20, exhibited different patterns of biological activities.
Compound 5 was inactive against L. amazonensis strains. Compound 6 showed an IC50 of 34 µM.
Compound 7, containing two methylenedioxy groups, had an IC50 of 13.9 µM. Compound 23, a
grandisin isoxazole derivative, showed an IC50 of 18.3 µM.
Hybrid asymmetrical compounds 8–19 showed a diverse set of biological activities against L.
amazonensis strains.
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Positional isomers such as hybrids 8 and 9, showed IC50 values higher than 160µM. Isomers 10 and
11 had IC50 values of 169.3 and 21.9 µM respectively. Isomers 12 and 17 had IC50 values of >146.4
and 32.6 µM, respectively.
Trimethoxy hybrid compounds 13 and 18 both showed biological activity at approximately 20 µM.
Machilin G isoxazole analogues 14 and 15 exhibited IC50 values of 63.1 and 2.0 µM, respectively.
Also, positional isomers 16 and 19 showed IC50 measurements of 3.3 and 9.5 µM respectively.
Among the compounds obtained, those with antileishmanial activity below 20 µM were tested
against the promastigotes of L. braziliensis, related to cases of mucocutaneous leishmaniasis, to
determine whether the isoxazole compounds could demonstrate biological activity against other
Leishmania species. Positional isomers 15, 16 and 19 were the most active compounds against L.
braziliensis, showing IC50 values of 1.2, 2.1 and 6.4 µM, respectively, and cytotoxicity >250 µM (Table
2).
Compounds 15, 16 and 19 were more active than the reference drug pentamidine for the two
species of Leishmania studied.
Grandisin showed an IC50 = 28 μM against the promastigote forms of L. amazonensis while its
derivative, the isoxazole analogue 20, showed higher activity (IC50 = 18.3 µM), indicating that the
presence of this nucleus improved the antileishmanial activity (Table 2).
The selectivity index, a crucial parameter in all drug-design studies, was established as the ratio of
IC50 for mammal cells (NIH/3T3) to the IC50 value of protozoans treated with synthetic compounds
obtained in an anti-trypanosomatid assay. Higher selectivity indexes correspond to increased
specificity and selectivity to parasites.34-35
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Synthetic compounds 15 and 16 were non-cytotoxic against mammal cells, with optimal selectivity
index (SI>75.8 and SI>125.0, respectively), when compared to first-line drugs such as pentamidine (SI
= 7.8), which has a higher cytotoxicity on murine fibroblast cells (NIH/ 3T3) (Table 2). Concerning the
structure-activity relationship (SAR), isoxazole analogues structured with methylenedioxy exhibited
impressive potency, highlighted by the machilin G isoxazole derivative 15 and positional isomers 16
and 19, hybrids between grandisin 2 and machilin G 3. The bioisosteric replacement of the
tetrahydrofuran nucleus by the isoxazole ring in 1-3, increased potency and selectivity of the
synthesized compounds over Leishmania species. These results showed similar behaviour, to the
replacement of this same core with the 1,2,3-triazole ring.2
3. Conclusions
In conclusion, 16 isoxazole analogues derived from neolignans 1–3 were synthesized. The most-
active compounds 15, 16 and 19, which contain the methylenedioxy group, appear to be essential
for anti-leishmanial activity. The anti-leishmanial activity of synthesized 3,5-diaryl-isoxazoles,
described herein, was similar to 1,4-diaryl-1,2,3-triazoles,2 indicating that these rings are equivalents
and interchangeable.
Acknowledgments
This study was supported by grants from CNPq (Process number 404201/2012-1),
FUNDECT-MS, PROPP-UFMS, and CAPES. We thank Dra Janet W. Reid (JWR
Associates) for her assistance with English corrections. Special thanks to the Laboratory of
Natural Products and Mass Spectrometry (LaPNEM) of the Federal University of Mato
Grosso do Sul for the HPLC-DAD-MS/MS analysis.
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Conflicts of interest
There are no conflicts to declare.
FIGURE LEGENDS
Figure 1. Tetrahydrofuran neolignans veraguensin 1, grandsin 2 and machilin G 3.
Figure 2. Design of 1,4-diaryl-1,2,3-triazole2,8 and 3,5-diaryl isoxazole analogues based on
tetrahydrofuran neolignans 1-3
Scheme 1. Synthesis of aryl acetylenes 26a-d. Reagents and reaction conditions: (a) NBS, p-TsOH,
CH2Cl2, SiO2, 22a = 81%, 22b = 87%, 22c = 79%; (b) 2-methyl-3-butyn-2-ol, PdCl2(PPh3)2/CuI, Et3N,
reflux, 24h, 23a = 85%, 23b =75%, 23c = 79%; (c) KOH, toluene, reflux, 24h, 26a = 65%, 26b = 85% ,
26c = 81%; (d) CBr4, PPh3/ CH2Cl2, 0°C, 5h., 25 = 74%; (e) THF, n-BuLi, -25 °C to room temperature, 1h,
26d = 83%.
Scheme 2. Synthesis of Chloro-oximes 29a-c. Reagents and reaction conditions: (a)
NH2OH.HCl / KOH, H2O / EtOH 1:1, 28a = 85%, 28b = 95%, 28c = 95%; (b) NCS,
DMF, 0 °C, 2 h, 29a = 72%, 29b = 75%, 29c = 83%.
Table 1. Synthesis of 3,5-diaryl-isoxazole 5-16.
Scheme 3. Synthesis of oxime 30.
Scheme 4. Synthesis of isoxazoles 17-20. Reagents and reaction conditions: (a) NCS, CHCl3, pyridine,
Et3N, rt, 48 h; yields, 17 = 49%; 18 = 51%; 19 = 45%; 20 = 43%.
Table 2. In vitro anti-trypanosomatid activity of 3,5-diaryl-isoxazole analogues 5–20.
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Figure 1. Tetrahydrofuran neolignans veraguensin 1, grandsin 2 and machilin G 3.
Figure 2. Design of 1,4-diaryl-1,2,3-triazole2,8 and 3,5-diaryl isoxazole analogues based on
tetrahydrofuran neolignans 1-3
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Scheme 1. Synthesis of aryl acetylenes 26a-d. Reagents and reaction conditions: (a) NBS, p-TsOH,
CH2Cl2, SiO2, 22a = 81%, 22b = 87%, 22c = 79%; (b) 2-methyl-3-butyn-2-ol, PdCl2(PPh3)2/CuI, Et3N,
reflux, 24h, 23a = 85%, 23b =75%, 23c = 79%; (c) KOH, toluene, reflux, 24h, 26a = 65%, 26b = 85% ,
26c = 81%; (d) CBr4, PPh3/ CH2Cl2, 0°C, 5h., 25 = 74%; (e) THF, n-BuLi, -25 °C to room temperature, 1h,
26d = 83%.
Scheme 2. Synthesis of Chloro-oximes 29a-c. Reagents and reaction conditions: (a)
NH2OH.HCl / KOH, H2O / EtOH 1:1, 28a = 85%, 28b = 95%, 28c = 95%; (b) NCS,
DMF, 0 °C, 2 h, 29a = 72%, 29b = 75%, 29c = 83%.
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Table 1. Synthesis of 3,5-diaryl-isoxazole 5-16.
aReactions conditions: 26a-d (2.0 mmol), 29a-c (2.0 mmol), CuSO4.5H2O (0,30 mmol), sodium ascorbate (0,50
mmol), KHCO3 (20 mmol), THF (3 mL), CH2Cl2 (3 mL), room temperature, 24h.
Scheme 3. Synthesis of oxime 30.
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Scheme 4. Synthesis of isoxazoles 17-20. Reagents and reaction conditions: (a) NCS, CHCl3, pyridine,
Et3N, rt, 48 h; yields, 17 = 49%; 18 = 51%; 19 = 45%; 20 = 43%.
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Table 2. In vitro anti-trypanosomatid activity of 3,5-diaryl-isoxazole analogues 5–20.
Compd
NIH/3T3
T. cruzi
SIc
L. amazonensis
IC50b / (µM) ±
SD
SIc
L. braziliensis
IC50b / (µM) ±
SD
SIc
IC50b / (µM) ± IC50b / (µM) ±
SD
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
SD
>200
>200
>200
>200
>200
NCd
>200
>200
NCd
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
13.2
NCd
177 ± 2.2
34.0 ± 1.5
13.9 ± 1.1
200 ± 2.3
160 ± 2.2
169 ± 2.2
21.9 ± 1.3
146 ± 2.1
20.2 ± 1.3
63.1 ± 1.8
2.0 ± 0.3
3.3 ± 0.5
32.6 ± 1.5
19.4 ± 1.2
9.5 ± 0.9
18.3 ± 1.2
8.9 ± 0.9
28.0 ± 1.4
NCd
>1.4
>7.4
>18
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
NCd
>208
>119
NCd
NCd
>39
>9.9
7.8
NCd
NCd
NC
NCd
>4.2
>9.4
>11.4
>11.2
>12.4
>4.0
>125
>75.8
>7.7
>12.9
>20
NCd
NCd
NCd
NCd
NCd
183.6 ± 2.2
NCd
NCd
1.2 ± 0.0
2.1 ± 0.3
NCd
>200
>200
>200
153.2 ± 2.1
>200
NCd
NCd
6.4 ± 0.8
25.2± 1.4
10.1 ± 1.0
NCd
>14.2
8.8
PEe
GRANf
BZNg
DOXh
78.7 ± 1.9
>250
96.1 ± 1.9
2.6± 0.4
NCd
8.9
NCd
NCd
NCd
7.3 ± 0.8
NCd
NCd
NCd
NCd
NCd
NCd
a IC50: half maximal inhibitory concentration on fibroblast cells; b IC50: half maximal inhibitory concentration on
c
T. Cruzi, L. amazonensis and L. braziliensis; SI: selectivity index: IC50 on mammal cells per IC50 on
trypanosomatids; d NC: values not calculated; e PE = pentamidine, positive control for L. Amazonensis and L.
braziliensis; f GRAN = grandisin, positive control of natural product for L. amazonensis ; g benznidazole, positive
control for T. Cruzi; h doxorubicin positive control for fibroblast cells.
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