Synthesis of 3,5-diarylisoxazole derivatives and evaluation of in vitro trypanocidal activity

Article abstract of DOI:10.21577/0103-5053.20170137

Chagas disease is included in the neglected tropical diseases list and is endemic to 21 Latin American countries. The two drugs currently available for treating Chagas disease are nifurtimox and benznidazole and both result in many significant side effects. The study describes the synthesis and biological evaluation of 3,5-disubstituted isoxazoles. Isoxazoles were obtained by reaction of flavones and hydroxylamine and either alkylated at the free hydroxyl group and/or nitrated at the isoxazole ring. These compounds were evaluated for their in vitro anti-Trypanosoma cruzi activity against trypomastigote and amastigote forms of the parasite in T. cruzi-infected cell lineages. Benznidazole was used as a reference compound for the in vitro assay and mammalian L929 cells were employed to evaluate cytotoxicity. A majority of the compounds tested were very active and the most active isoxazole against amastigote and trypomastigotes of T. cruzi was slightly more potent than the current medicine benznidazole.

Full text of DOI:10.21577/0103-5053.20170137

http://dx.doi.org/10.21577/0103-5053.20170137
J. Braz. Chem. Soc., Vol. 29, No. 2, 269-277, 2018.
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Synthesis of 3,5-Diarylisoxazole Derivatives and Evaluation of in vitro Trypanocidal
Activity
Aline A. N. de Souza,a Viviane F. Xavier,a,b Gleicekelly S. Coelho,a
Policarpo A. Sales Junior,b Alvaro J. Romanha,b Silvane M. F. Murta,b
Claudia M. Carneiroc and Jason G. Taylor*,a
aDepartamento de Química, Instituto de Ciências Exatas e Biológicas (ICEB),
Universidade Federal de Ouro Preto, Campus Universitário Morro do Cruzeiro,
35400-000 Ouro Preto-MG, Brazil
bCentro de Pesquisas René Rachou, FIOCRUZ, 30190-002 Belo Horizonte-MG, Brazil
cLaboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas (NUPEB),
Universidade Federal de Ouro Preto, Campus Universitário Morro do Cruzeiro,
35400-000 Ouro Preto-MG, Brazil
Chagas disease is included in the neglected tropical diseases list and is endemic to 21 Latin
American countries. The two drugs currently available for treating Chagas disease are nifurtimox
and benznidazole and both result in many significant side effects. The study describes the synthesis
and biological evaluation of 3,5-disubstituted isoxazoles. Isoxazoles were obtained by reaction of
flavones and hydroxylamine and either alkylated at the free hydroxyl group and/or nitrated at the
isoxazole ring. These compounds were evaluated for their in vitro anti-Trypanosoma cruzi activity
against trypomastigote and amastigote forms of the parasite in T. cruzi-infected cell lineages.
Benznidazole was used as a reference compound for the in vitro assay and mammalian L929 cells
were employed to evaluate cytotoxicity. A majority of the compounds tested were very active and
the most active isoxazole against amastigote and trypomastigotes of T. cruzi was slightly more
potent than the current medicine benznidazole.
Keywords: isoxazole, azole, amastigote, trypomastigote, in vitro, Chagas disease
Introduction
demonstrated resistance to available drugs.5 Different
approaches have been taken towards the in vitro evaluation
of novel synthetic molecules that were tested for anti
T. cruzi activity. The epimastigote form that is present in
the midgut vector has been used for assessing anti-parasitic
activity.6-8 The fact that the intracellular amastigotes forms
of T. cruzi are present in the vertebrate host during the acute
and chronic phases of the disease makes the use of this form
of the parasite very attractive as screening method for the
evaluation of anti T. cruzi activity.9-11 The trypomastigote
form enters through the bite wound and is therefore initially
present in the blood and, for this reason, the trypomastigote
form has also been utilized for in vitro screening.12-14
Following the guidelines proposed by the Fiocruz
Program for Research and Technological Development on
Chagas Disease Initiative, the whole cell-based screening
methodology was utilized in the present study.15,16 This
allows us to study the parasite forms that are responsible for
Discovered by Brazilian scientist Carlos Chagas in
1909,1 Chagas disease has become endemic to 21 Latin
American countries.2 The transmission of this disease in
humans usually occurs by deposition of faeces contaminated
with the protozoan hemoflagellate Trypanosoma cruzi
after being bitten by the blood-sucking triatomine bugs.
Almost half of those infected either have or will develop
cardiomyopathy, digestive megasyndromes, or both.3 This
“neglected disease” is currently treated in Brazil with
benznidazole, but unfortunately, this drug causes many
significant side effects.4 One of the major problems related
to infectious diseases that humanity faces is the continuous
mutation that microorganisms suffer and the resistance
they develop to medicines. Certain strains of T. cruzi have
*e-mail: jason@iceb.ufop.br

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Synthesis of 3,5-Diarylisoxazole Derivatives and Evaluation of in vitro Trypanocidal Activity
J. Braz. Chem. Soc.
human infection, analyse novel compounds for anti T. cruzi
activity in infected cells whilst at the same time monitoring
their effects on amastigotes and trypomastigotes in the same
system. Different azole derivatives such as imidazoles,9,17
thiazoles,12,18 1,3,4-oxadiazoles,19 1,2,4-oxadiazoles20 and
1,2,3-triazoles21 have been evaluated for their anti T. cruzi
activity (Figure 1).
Isoxazoles and their derivatives are considered
important heterocyclic compounds due to their prolific
biological properties.22 This heterocycle can be encountered
in the chemical structure of approved medicines such as
Leflunomide, Cloxaciline, Sulfisoxazol, Isocarboxazide,
Broxaterole and Sitaxentane amongst others. To the best of
our knowledge, isoxazoles have not been explored for their
anti T. cruzi activity. Motivated by the need to develop more
efficient drug candidates for Chagas disease with a less
severe side effects profile, we have prepared 17 isoxazole
derivatives and tested them against both amastigote and
trypomastigote forms of T. cruzi in vitro.
involve the use of chalcones (α-β unsaturated ketones),
β-diketones, flavones and cyclocondensation of nitrile
oxides with alkynes.24 The key intermediate having
3,5-diarylisoxazole framework were synthesized from
flavones25 in four steps (Scheme 1). The synthesis of
the target isoxazoles began with the esterification of
2-hydroxyacetophenones with substituted benzoyl
chlorides to provide the corresponding esters 2 as illustrated
in Scheme 1. A Baker-Venkataraman rearrangement
ensued in the presence of KOH to afford 1,3-diketones,
which when isolated by precipitation and filtration, were
immediately subjected to a condensation reaction under
refluxing acetic acid to provide flavones 3. Many of the
ester, 1,3-diketone and flavone intermediates are known
compounds and were thus confirmed by comparison of
their melting point and nuclear magnetic resonance (NMR)
spectral data with literature values. All data for these
intermediates were in complete accordance with literature
values. Flavones 3 were reacted with hydroxylamine to
afford the required isoxazoles 4 in reasonable yields albeit,
on occasion, mixtures of isomers were formed. The two
isomers were either purified by recrystallization, separated
by column chromatography or were immediately subjected
to a Williamson type O-alkylation reaction to provide the
alkylated products 5-9 in good yield. The spectroscopic
data of known 3,5-diarylisoxazoles were in accordance with
those previously reported. Compounds 5, 6a-d, 7a-c, 8, 9,
10 are novel and were therefore characterized by Fourier
transform infrared spectroscopy (FTIR), NMR and mass
spectrometry (MS).
Results and Discussion
Chemistry
3,5-Diphenylisoxazole 1, was prepared from chalcone
according to literature procedures23 and used as a reference
in order to determine the importance of a free hydroxyl group
for anti T. cruzi activity (Scheme 1). A major challenge in
the synthesis of non-symmetrical 3,5-disubstitued
isoxazoles using hydroxylamine is to selectively form one
isomer from a substrate that bears two electrophilic centres.
The main reactions for obtaining 3,5-diarylisoxazoles
It has been well documented that benznidazole and
nifurtimox are prodrugs that require nitroreductase
Figure 1. Selected examples of azole compounds with anti T. cruzi activity.

Vol. 29, No. 2, 2018
de Souza et al.
271
Scheme 1. Synthetic route for the preparation of isoxazole derivatives: (i) NaOH, NH2OH.HCl, DMSO, 100 °C, 8 h; (ii) pyridine, rt, 1 h; (iii) pyridine,
KOH, 50 °C, 1 h; (iv) AcOH, H2SO4, reflux, 1 h; (v) NH2OH.HCl, pyridine, reflux, 14 h; (vi) R3X (X = Br or I), acetone, K2CO3, 60 °C, 18 h.
catalyzed activation within the parasite to have trypanocidal
effects. We synthesised a nitro bearing isoxazole for the
purpose of evaluating possible enhancement in activity
via this known mode of action involving nitroreductase.
Compound 9 was nitrated in the presence of acetic
anhydride and nitric acid to provide novel compound 10
in 32% yield (Scheme 2).
group increases potency significantly without rendering the
3,5-diarylisoxazole more cytotoxic. Introducing electron
donating groups on to the unsubstituted phenyl ring of
4a did not result in any remarkable improvements in anti
T. cruzi activity, but did, however, provide more cytotoxic
compounds (compare 4a to 4b-c). Completely substituting
the benzene ring for a furan moiety 4d gave similar results
to 4b and 4c. Next, we evaluated the effect of substituents on
the phenol ring and observed almost a doubling in potency
with the inclusion of two chlorine atoms (compare 4b to
4e). There may not necessarily be any important donor
hydrogen bonding interactions associated with the free
hydroxyl group of the phenol ring. Indeed, methylation
of 4c afforded compound 5 and a comparison of their
in vitro activities suggests that potency can be improved
when a bulkier substituent in the ortho position is present.
To test this hypothesis, compounds 6-8 were prepared by
benzylation of their corresponding 2-(5-arylisoxazol-3-yl)
phenols. In general, the presence of electron withdrawing
groups such as fluorine or nitro groups on either aromatic
ring gave the best in vitro activities and selectivities. In
particular, compounds 6b, 7b and 8 were the stand out lead
compounds and demonstrated very similar anti T. cruzi
activity to reference compound benznidazole. Narsaiah and
Biology
Once the final products were purified and fully
characterized, we carried out in vitro bioassays using
trypomastigote and amastigote forms of Tulahuen-strain
T. cruzi, using the method described by Romanha et al.15
Benznidazole was used as positive control against T. cruzi
and cytotoxicity was determined in mammalian L929 cells
(Table 1).
Our reference compound provided an IC50 (50%
inhibitory concentration) of 3.8 μM and gave a selectivity
factor (SI) of 625 and was used as a benchmark value
for assessing the potency and selectivity of the isoxazole
derivatives. Many of the compounds described herein
were active at the tested concentrations. A comparison of
1 with 4a revealed that the presence of the free hydroxyl
Scheme 2. Nitration of isoxazole 9: (i) Ac2O, HNO3, 0.5 h, 0-5 °C, 32% yield.

272
Synthesis of 3,5-Diarylisoxazole Derivatives and Evaluation of in vitro Trypanocidal Activity
J. Braz. Chem. Soc.
Table 1. In vitro trypanocidal activity, cytotoxicity and selectivity index of bioactive isoxazoles
Trypanocide
IC50 / μM
Cytotoxicity
CC50 / μM
Compound No.
Compound
SI
log P
4.01
TPSA / Å2
26.03
1
53.3 0.9
80.0 1.5
1.5
4a
4b
4c
R = H
R = 4-OCH3
R = 3,4,5-(OCH3)3
33.5 1.0
22.8 1.6
30.2 1.6
80.0 1.5
50.0 4.1
55.0 6.1
2.4
2.2
1.8
3.75
3.80
3.38
46.26
55.49
73.96
4d
4e
26.2 1.5
13.7 1.5
40.0 1.9
10.0 1.0
1.5
1.4
2.89
5.06
59.40
55.49
5
11.7 1.7
40.0 4.5
3.4
3.65
62.97
6a
6b
6c
R = 4-OCH3
R = 4-F
R = 3,4,5-(OCH3)3
18.0 1.5
8.0 1.2
25.3 2.2
40.0 5.5
45.0 5.0
60.0 2.3
2.2
5.6
2.4
5.67
5.78
5.25
44.50
35.27
62.97
6d
inactive
ND
−
4.51
44.50
7a
7b
7c
R = H
R = 4-F
R = 4-OCH3
57 1.9
3.3 1.9
15 1.3
80.0 1.1
80.0 5.5
55.0 2.1
1.4
26.4
3.7
6.27
6.43
6.33
35.27
35.27
44.50
8
9
5.9 1.6
40.0 1.0
60.2 1.9
6.8
3.3
5.58
5.08
90.32
35.27
18.4 1.0
10
32.1 1.0
3.8
73.3 1.0
2381
2.3
4.96
81.09
Benznidazole
−
625
−
−
IC50: 50% inhibitory concentration; CC50: 50% cytotoxic concentration; ND: not determined; SI: selectivity index calculated from CC50/IC50; TPSA: total
polar surface area.

Vol. 29, No. 2, 2018
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273
co-workers26 have reported that 5-(3-alkylquinolin-2-yl)-
3-aryl isoxazole derivatives screened against four human
cancer cell lines (A549, COLO 205, MDA-MB 231 and
PC-3) display potent cytotoxicity against all the cell lines
at IC50 values of < 12 μM. Given the similarity in chemical
structure with isoxazoles 4-9, the cytotoxicity profile of the
compounds described in this report is supported by this
literature precedence. As a consequence, the selectivity of
the tested compounds was hampered by their cytotoxicity
towards mammalian cells. Activation of nitroheterocyclic
drugs, such as benznidazole, by T. cruzi has been associated
with the formation of reactive radical species responsible
for the death of the parasite. For this reason, we synthesised
a nitro bearing isoxazole 10 for the purpose of evaluating
possible enhancement in activity via this known mode
of action. Surprisingly, both anti T. cruzi activity and
cytotoxicity were lower than the parent compound 9 and
this result suggests that nitro-isoxazoles are not activated
by nitroreductase like their imidazole (benznidazole) and
furan (nifurtimox) counterparts. The physicochemical drug
descriptors of the molecular properties for the synthesized
compounds were calculated by Molinspiration software.27
The partition coefficient (log P: octanol/water partition
coefficient) describes the equilibrium distribution between
two liquid phases such as octanol and water and the total
polar surface area (TPSA) is a measure of the extent of
the molecules exposed polar area. No linear correlations
between hydrophobicity and bioactivity were observed.
On the other hand, all log P values for the most bioactive
compounds were found to be slightly greater than 5
(Lipinski’s rule of five). The largest TPSA values were all
below the limit of 140 Å2 (Lipinski’s rule of five); based
on the TPSA value alone, the most potent compounds, 6b
and 7b, would be expected to perform better in permeating
the parasites cell membranes. Thus, these results suggest
that penetration of cell membranes is not the critical factor
determining anti T. cruzi activity.
Experimental
All commercial reagents were used as received.
Anhydrous solvents were purchased from Sigma-Aldrich.
Flash column chromatography was performed using
silica gel 200-400 mesh. Thin layer chromatography
(TLC) analysis were performed using silica gel plates,
using ultraviolet (UV) light (254 nm), phosphomolybdic
acid or vanillin solution for visualization. Melting points
are uncorrected and were recorded on a Buchi B-540
apparatus. For NMR data, the chemical shifts are reported
in d (ppm) referenced to residual solvent protons and 13C
signals in deuterated chloroform. Coupling constants (J)
are expressed in hertz (Hz). Infrared spectra were obtained
on a Thermo Scientific Nicolet 380 FT-IR apparatus
(600-4000 cm−1, Nicolet Instrument Corp., Madison, WI,
USA) using attenuated total reflection (ATR). Mass spectra
were obtained by GC-MS, Shimadzu QP-2010 Plus model
(Shimadzu, Kyoto, Japan) and high resolution mass spectra
(HRMS) were obtained on a Shimadzu HPLC-ESI-IT-TOF.
SMILES notations of the isoxazole derivatives were
inputted into the online Molinspiration software (software
version v2015.01)27 and subjected to molecular properties
prediction by Molinspiration software. Flavones were
prepared according to literature methods.25
Typical experimental procedure for the synthesis of
isoxazoles 4-10
In a round bottom flask (50.0 mL) equipped with stir
bar and reflux condenser was added flavone (2.0 mmol),
pyridine (10.0 mL) and hydroxylamine hydrochloride
(6.0 mmol). The reaction was stirred at 100 °C for 14 h.
The reaction was then allowed to cool to room temperature
and poured in an ice-water mixture (20.0 mL). The
precipitate that formed was filtered and recrystallized from
ethanol. A selection of certain isoxazoles were alkylated
with either iodomethane, iodoethane or benzylbromide
as follows: in a round bottom flask (50.0 mL) equipped
with stir bar and reflux condenser were added isoxazole
(1.0 mmol), anhydrous K2CO3 (2.0 mmol) and acetone
(20.0 mL) at 0 °C. Next, the alkylhalide (1.5 mmol) was
added slowly at 0 °C with stirring and the reaction was then
warmed to 60 °C and allowed to stir at this temperature
overnight. Upon completion, the reaction mixture was
cooled to room temperature, concentrated under reduced
pressure and then taken up in to a separatory funnel
containing ethyl acetate (20.0 mL). The organic layer was
washed with distilled water (15.0 mL) and the organic
phases were worked up in the usual way. An oily residue
was obtained and purified by flash chromatography eluted
Conclusions
In conclusion, although the potency of many of the
isoxazole derivatives was less active than the positive
control, anti T. cruzi activity was significantly improved
by the inclusion of fluorine group on to either the phenol
of phenyl ring of the 3,5-diarylisoxazole. In one case,
the isoxazole derivative 7b was slightly more potent
than benznidazole. Further studies and investigations
by colorimetric methods and indirect analysis by light
microscopy are ongoing in order to discern the mechanism
of action of the most potent lead compounds uncovered in
the present study.

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Synthesis of 3,5-Diarylisoxazole Derivatives and Evaluation of in vitro Trypanocidal Activity
J. Braz. Chem. Soc.
in a gradient mixture of hexanes and ethyl acetate (1-10%
ethyl acetate in hexanes).
d 100.7, 111.8, 112.5, 114.0, 117.0, 119.9, 127.3, 132.1,
144.2, 145.3, 155.2, 155.3, 166.8; EI m/z: 227 (55%), 121
(100%), 105 (70%), 65 (90%); HRMS (ESI) m/z: [M + H]+
calcd. for C13H10NO3: 228.0661; found: 228.0656.
2-(3-Phenylisoxazol-5-yl) phenol (4a)
Product obtained as a white solid in 47% yield; mp
221-222 °C (Lit.12 mp 232-236 °C); Rf (retention factor)
= 0.5 (ethyl acetate/hexane 3:7); IR (ATR) ν / cm-1 3402,
1614, 1456, 950, 756, 684; 1H NMR (300 MHz, DMSO)
d 6.91-7.65 (m, 7H), 7.82-8.01 (m, 3H), 10.06 (s, 1H);
13C NMR (75 MHz, DMSO) d 101.3, 114.6, 115.1, 117.2,
120.2, 121.9, 127.5, 128.8, 132.1, 155.4, 161.3, 162.6,
167.0; EI (electron ionization) m/z: 237 (25%), 207 (20%),
117 (100%), 73 (90%), 63 (77%), HRMS (ESI, electrospray
ionization) m/z: [M + H]+ calcd. for C15H12NO2: 238.0868;
found: 238.0782.
2,4-Dichloro-6-(3-(4-methoxyphenyl)isoxazol-5-yl)phenol
(4e)
Product obtained as a white solid in 47% yield; mp 206-
208 °C (Lit.31 mp 193 °C); Rf = 0.4 (ethyl acetate/hexane
3:7); IR (ATR) ν / cm-1 1701, 1359, 1224, 536; 1H NMR
(400 MHz, DMSO) d 3.80 (s, 3H), 7.10 (d, J 8.8 Hz, 2H),
7.40 (s, 1H); 7.70 (d, J 8.4 Hz, 1H), 7.80 (d, J 8.4 Hz,
1H), 7.90 (d, J 8.8 Hz, 2H); 13C NMR (100 MHz, DMSO)
d 55.8, 102.8, 115.0, 118.7, 121.2, 123.9, 124.4, 125.9,
128.6, 130.9, 149.8, 161.3, 162.6, 165.0; EI m/z: 335
(10%), 108 (100%); HRMS (ESI) m/z: [M + H]+ calcd. for
C16H12Cl2NO3: 336.0194; found: 336.0200.
2-(3-(4-Methoxyphenyl)isoxazol-5-yl)phenol (4b)
Product obtained as a yellow solid in 78% yield. mp
208-210 °C (Lit.28 mp 212-214 °C); Rf = 0.4 (ethyl acetate/
hexane 3:7); IR (ATR) ν / cm-1 3200, 1600, 1500, 1450,
1260, 1030; 1H NMR (400 MHz, DMSO) d 3.80 (s, 3H),
7.00 (t, J 8.4 Hz, 1H), 7.04-7.08 (m, 3H), 7.30 (s, 1H), 7.33
(d, J 8.4 Hz, 1H), 7.79 (d, J 7.8 Hz, 1H), 7.90 (d, J 9.0 Hz,
2H); 13C NMR (100 MHz, DMSO) d 55.9, 101.3, 114.6,
115.1, 117.2, 120.2, 121.9, 127.5, 128.8, 132.1, 155.4,
161.3, 162.6, 167.0; EI m/z: 267 (15%), 252 (10%), 236
(30%), 108 (100%); HRMS (ESI) m/z: [M + H]+ calcd. for
C16H14NO3: 268.0974; found: 268.0967.
5-(2-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)isoxazole
(5)
Product obtained as a white solid in 72% yield; mp
88-92 °C; Rf = 0.5 (ethyl acetate/hexane 3:7); IR (ATR)
ν / cm-1 1600, 1500, 1460, 1240, 1120; 1H NMR (500 MHz,
CDCl3) d 3.90 (s, 3H), 4.00 (s, 6H), 4.05 (s, 3H), 7.06-7.15
(m, 5H), 7.45-7.48 (m, 1H), 8.05 (d, J 7.5 Hz, 1H);
13C NMR (75 MHz, CDCl3) d 55.7, 56.4, 61.0, 101.4, 104.3,
111.3, 116.5, 121.0, 125.0, 127.8, 131.3, 139.6, 153.6,
156.2, 162.9, 166.5; EI m/z: 341 (100%), 326 (50%), 135
(90%); HRMS (ESI) m/z: [M + H]+ calcd. for C19H20NO5:
342.1341; found: 342.1333.
2-(3-(3,4,5-Trimethoxyphenyl)isoxazol-5-yl)phenol (4c)
Product obtained as a white solid in 82% yield; mp
218-219 °C (Lit.29 mp 235-238 °C); Rf = 0.3 (ethyl acetate/
hexane 3:7); IR (ATR) ν / cm-1 3160, 1600, 1500, 1420,
1250, 1010; 1H NMR (400 MHz, DMSO) d 3.70 (s, 3H),
3.90 (s, 6H), 6.95 (t, J 7.5 Hz, 1H), 7.10 (d, J 8.4 Hz, 1H),
7.20 (s, 2H), 7.31-7.42 (m, 2H), 7.81 (d, J 8.4 Hz, 1H),
10.65 (brs, 1H); 13C NMR (100 MHz, DMSO) d 56.8, 60.8,
101.8, 104.7, 114.6, 117.2, 120.2, 125.0, 127.6, 132.2,
139.6, 154.0, 155.4, 163.0, 167.3; EI m/z: 327 (5%), 312
(20%), 296 (10%), 170 (35%), 93 (100%); HRMS (ESI)
m/z: [M + H]+ calcd. for C18H18NO5: 328.1185; found:
328.1191.
5-(2-(Benzyloxy)phenyl)-3-(4-methoxyphenyl)isoxazole (6a)
Product obtained as a yellow solid in 62% yield; mp
95-98 °C; Rf = 0.6 (ethyl acetate/hexane 3:7); IR (ATR)
ν / cm-1 2925, 1608, 1512, 1458, 1249; 1H NMR (400 MHz,
CDCl3) d 3.90 (s, 3H), 5.30 (s, 2H), 7.00 (d, J 9.2 Hz,
2H), 7.06 (s, 1H), 7.10-7.16 (m, 2H), 7.40-7.50 (m,
4H), 7.55 (d, J 8.4 Hz, 2H), 7.70 (d, J 8.8 Hz, 2H),
8.10 (d, J 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3)
d 55.4, 70.7, 101.6, 112.6, 114.2, 117.0, 121.2, 122.1, 127.7,
127.8, 128.1, 128.4, 128.7, 131.0, 136.4, 155.3, 160.8, 162.5,
166.0; EI m/z: 357 (10%), 91 (100%); HRMS (ESI) m/z:
[M + H]+ calcd. for C23H20NO3: 358.1443; found: 358.1488.
2-(3-(Furan-2-yl)isoxazol-5-yl)phenol (4d)
Product obtained as a white solid in 55% yield; mp
198-199 °C (Lit.30 mp 237-239 °C); Rf = 0.5 (ethyl acetate/
hexane 3:7); IR (ATR) ν / cm-1 3120, 1600, 1500, 1370,
1240, 960; 1H NMR (500 MHz, DMSO) d 6.71-6.72 (m,
1H), 7.0 (t, J 8.0 Hz, 1H), 7.10 (d, J 8.0 Hz, 1H), 7.21-7.23
(m, 2H), 7.40 (t, J 8.5 Hz, 1H), 7.80 (d, J 8.0 Hz, 1H), 7.90
(brs, 1H), 10.70 (s, 1H, OH); 13C NMR (125 MHz, DMSO)
5-(2-(Benzyloxy)phenyl)-3-(4-fluorophenyl)isoxazole (6b)
Product obtained as a white solid in 27% yield; mp
145-148 °C; Rf = 0.66 (ethyl acetate/hexane 3:7); IR (ATR)
ν / cm-1 1500, 1442, 1236, 754, 698; 1H NMR (400 MHz,
CDCl3) d 5.30 (s, 2H), 7.05 (s, 1H), 7.11-7.17 (m, 4H),
7.40-7.55 (m, 6H), 7.75-7.80 (m, 2H), 8.10 (d, J 7.6 Hz,
1H); 13C NMR (100 MHz, CDCl3) d 70.8, 101.6, 112.6,

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116.0 (d, JC-F 11 Hz), 116.7, 121.3, 125.7 (d, JC-F 3 Hz),
127.7, 127.8, 128.4 , 128.5 (d, JC-F 9 Hz), 128.8, 131.2,
136.3, 155.4, 162.1, 163.7 (d, JC-F 248 Hz), 166.4; EI m/z:
345 (10%), 91 (100%); HRMS (ESI) m/z: [M + H]+ calcd.
for C22H17FNO2: 346.1243; found: 346.1231.
(300 MHz, CDCl3) d 5.20 (s, 2H), 7.00 (s, 2H), 7.10 (d,
J 8.7 Hz, 2H), 7.32-7.35 (dd, J 9.0, 2.4 Hz, 1H), 7.40-
7.47 (m, 5H), 7.68-7.72 (m, 2H), 8.0 (d, J 2.40 Hz, 1H);
13C NMR (75 MHz, CDCl3) d 71.1, 102.3, 113.9, 116.1 (d,
JC-F 21.75 Hz), 117.8, 126.4, 127.4, 127.6, 128.5 (d, JC-F
1.5 Hz), 128.6, 128.8, 130.8, 135.8, 153.8, 162.7, 162.1,
165.1, 165.4; EI m/z: 379 (15%), 120 (25%), 91 (100%),
HRMS (ESI) m/z: [M + H]+ calcd. for C22H16ClFO2:
380.0854; found: 380.0853.
5-(2-(Benzyloxy)phenyl)-3-(3,4,5-trimethoxyphenyl)
isoxazole (6c)
Product obtained as a white solid in 78% yield; mp
148-153 °C; Rf = 0.5 (ethyl acetate/hexane 3:7); IR (ATR)
ν / cm-1 1600, 1500, 1450, 1140, 1000; 1H NMR (300 MHz,
CDCl3) d 3.86 (s, 6H), 3.88 (s, 3H), 5.20 (s, 2H), 6.90-7.01
(m, 2H), 7.11-7.16 (m, 2H), 7.37-7.46 (m, 4H), 7.55 (d,
J 7.2 Hz, 2H), 8.03 (d, J 7.8 Hz, 1H); 13C NMR (75 MHz,
CDCl3) d 56.1, 60.9, 70.7, 101.7, 103.8, 112.3, 116.6,
121.2, 124.8, 127.7, 128.2, 128.3, 128.7, 131.2, 136.3,
139.4, 153.5, 155.4, 162.7, 166.2; EI m/z: 312 (100%), 297
(50%); HRMS (ESI) m/z: [M + H]+ calcd. for C25H24NO5:
418.1654; found: 418.1649.
5-(2-(Benzyloxy)-5-chlorophenyl)-3-(4-methoxyphenyl)
isoxazole (7c)
Product obtained as a white solid in 32% yield; mp
174-174 °C; Rf = 0.4 (ethyl acetate/hexane 3:7); IR (ATR)
1
ν / cm-1 1600, 1500, 1360, 1250, 1020, 710; H NMR
(300 MHz, CDCl3) d 3.90 (s, 3H), 5.20 (s, 2H), 6.90-7.00
(m, 4H), 7.30-7.33 (dd, J 2.4, 9.0 Hz, 1H), 7.40-7.50
(m, 5H), 7.70 (d, J 8.1 Hz, 2H), 8.00 (d, 1H); 13C NMR
(75 MHz, CDCl3) d 55.3, 71.1, 102.3, 113.8, 114.2, 118.1,
121.7, 126.4, 127.4, 127.7, 128.1, 128.5, 128.8, 130.5,
135.0, 153.7, 160.9, 162.6, 164.6; EI m/z: 391 (20%), 108
(25%), 91 (100%); HRMS (ESI) m/z: [M + H]+ calcd. for
C23H19ClNO3: 392.1053; found: 392.1044.
5-(2-(Benzyloxy)phenyl)-3-(furan-2-yl)isoxazole (6d)
Product obtained as a white solid in 60% yield; mp
122-123 °C; Rf = 0.34 (ethyl acetate/hexane 3:7); IR (ATR)
ν / cm-1 1600, 1400, 1400, 1260, 1020; 1H NMR (400 MHz,
CDCl3) d 5.30 (s, 2H), 6.53-6.55 (m, 1H), 6.80 (d, J 3.2 Hz,
1H), 7.10 (s, 1H), 7.15-7.40 (m, 2H), 7.48-7.51 (m, 4H),
7.56 (d, J 8.8 Hz, 2H), 7.57 (brs, 1H), 8.10 (d, J 7.60 Hz,
1H); 13C NMR (100 MHz, CDCl3) d 70.7, 101.2, 110.0,
111.6, 112.6, 116.6, 121.2, 127.5, 128.0, 128.3, 128.7,
131.3, 136.3, 143.7, 144.8, 155.4, 155.4, 166.0; EI m/z:
317 (10%), 91 (100%); HRMS (ESI) m/z: [M + H]+ calcd.
for C20H16NO3: 318.1130; found: 318.1123.
5-(2-(Benzyloxy)-5-methoxyphenyl)-3-(3-nitrophenyl)
isoxazole (8)
Product obtained as a white solid in 63% yield; mp
150-152 °C; Rf = 0.30 (ethyl acetate/hexane 3:7); IR
1
(ATR) ν / cm-1 1600, 1500, 1360, 1230, 1070; H NMR
(300 MHz, CDCl3) d 3.90 (s, 3H), 5.30 (s, 2H), 7.0
(d, J 9.6 Hz, 1H), 7.10 (s, 1H), 7.30 (d, J 9.0 Hz, 1H),
7.33-7.43 (m, 4H), 7.50 (d, J 7.8 Hz, 2H), 7.60 (t, J 8.1 Hz,
1H), 8.10 (d, J 8.7 Hz, 1H), 8.30 (d, J 9.0 Hz, 1H), 8.70
(s, 1H); 13C NMR (75 MHz, CDCl3) d 55.8, 76.4, 94.8,
104.4, 118.6, 118.7, 119.3, 120.6, 124.5, 127.9, 128.3,
128.4, 129.7, 131.2, 134.7, 138.0, 143.4, 146.0, 148.5,
152.3, 156.7; EI m/z: 402 (5%), 91 (100%); HRMS (ESI)
m/z: [M + H]+ calcd. for C23H19N2O5: 403.1294; found:
403.1314.
5-(2-(Benzyloxy)-5-chlorophenyl)-3-phenylisoxazole (7a)
Product obtained as a white solid in 79% yield; mp
196-196 °C; Rf = 0.34 (ethyl acetate/hexane 3:7); IR (ATR)
ν / cm-1 1600, 1400, 1360, 1240, 1020; 1H NMR (300 MHz,
CDCl3) d 5.20 (s, 2H), 7.0 (d, J 8.7 Hz, 1H), 7.10 (s, 1H),
7.33-7.36 (d, J 2.4, 9.0 Hz, 1H), 7.43-7.47 (m, 8H), 7.74-
7.77 (m, 2H), 8.0 (d, J 2.40 Hz, 1H); 13C NMR (75 MHz,
CDCl3) d 70.1, 102.5, 113.8, 118.1, 126.4, 126.7, 127.4,
127.6, 128.5, 128.8, 128.8, 129.2, 129.9, 130.6, 135.8,
153.8, 163.0, 164.9; EI m/z: 361 (5%), 91 (100%), HRMS
(ESI) m/z: [M + H]+ calcd. for C22H17ClNO2: 362.0948;
found: 362.0965.
5-(4-Chlorophenyl)-3-(2-ethoxyphenyl)isoxazole (9)
Product obtained as a white solid in 57% yield; mp
142-144 °C; Rf = 0.38 (ethyl acetate/hexane 3:7); IR (ATR)
ν / cm-1 1600, 1500, 1460, 1240, 1120; 1H NMR (300 MHz,
CDCl3) d 1.56 (t, J 6.0 Hz, 3H), 4.22 (q, J 6.0 Hz, 2H), 7.00-
7.11 (m, 3H), 7.38-7.47 (m, 3H), 7.80 (d, J 9.0 Hz, 2H),
8.02 (d, J 6.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 14.8,
64.1, 101.2, 111.9, 116.2, 120.8, 126.0, 127.7, 128.1, 129.1,
131.3, 135.7, 155.6, 161.9, 166.7; EI m/z: 299 (30%), 113
(10%), 77 (15%); HRMS (ESI) m/z: [M + H]+ calcd. for
C17H15ClNO2: 300.0791; found: 300.0831.
5-(2-(Benzyloxy)-5-chlorophenyl)-3-(4-fluorophenyl)
isoxazole (7b)
Product obtained as a white solid in 85% yield; mp
192-193 °C; Rf = 0.3 (ethyl acetate/hexane 3:7); IR (ATR)
ν / cm-1 1600, 1500, 1440, 1260, 1240, 1020, 740; 1H NMR

276
Synthesis of 3,5-Diarylisoxazole Derivatives and Evaluation of in vitro Trypanocidal Activity
J. Braz. Chem. Soc.
Nitration of isoxazole 9
interpolation. Quadruplicates were run in the same plate,
and the experiments were repeated at least once.
Synthesis of 3-(4-chlorophenyl)-5-(2-ethoxyphenyl)-
4-nitroisoxazole (10)
Supplementary Information
To a suspension of isoxazole 10 (230 mg, 0.69 mmol)
in Ac2O (7 mL) was added dropwise concentrated HNO3
(0.05 mL) at 0 °C with stirring. A temperature range of 0
and 5 °C was maintained until addition of nitric acid was
complete. Next, the reaction mixture was stirred for 70 h
at room temperature and then poured into ice (25 mL)
and extracted with dichloromethane (3 × 20 mL). The
organic extracts was dried with sodium sulfate, filtered and
concentrated under vacuum. The yellow solid obtained was
triturated with Et2O, filtered and then purified by column
chromatography (ethyl acetate/hexane 1:9) to yield 10 as a
pale yellow solid in 32% yield; mp 193-194 °C; Rf = 0.36
(ethyl acetate/hexane 1:9); IR (ATR) ν / cm-1 1504, 1429,
1346, 1286, 1089, 709; 1H NMR (300 MHz, CDCl3) d 1.67
(t, J 6.0 Hz, 3H), 4.39 (q, J 6.0 Hz, 2H), 7.10-7.07 (m,
2H), 7.47 (d, J 9.0 Hz, 2H), 7.80 (d, J 9.0 Hz, 2H), 8.30
(dd, J 9.0, 3.0 Hz, 1H), 8.89 (s, 1H); 13C NMR (75 MHz,
CDCl3) d 14.6, 65.6, 102.6, 111.9, 116.7, 123.6, 126.7,
127.4, 128.0, 129.2, 136.1, 141.1, 159.7, 162.2, 164.3;
EI m/z: 344 (5%), HRMS (ESI) m/z: [M + H]+ calcd. for
C17H14ClN2O4: 345.0642; found: 345.0654.
Supplementary information and NMR spectra are
available free of charge at http://jbcs.sbq.org.br as PDF file.
Acknowledgments
This work was supported by the Brazilian funding
agency Fundação de Amparo à Pesquisa do Estado de
Minas Gerais (FAPEMIG) under research grant project
code APQ-01629-16. Authors gratefully acknowledge the
generous financial support from the Universidade Federal
de Ouro Preto (UFOP), FAPEMIG and the Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq). The authors thank the Program for Technological
Development of Tools for Health-PDTIS-FIOCRUZ for use
of its facilities. The authors would also like to thank Prof
Dr Robson Jose de Cassia Afonso (UFOP) and Ananda
Lima Sanson (UFOP) for the excellent mass spectrometry
service.
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Submitted: April 1, 2017
Published online: July 13, 2017