Design, synthesis, antileishmanial, and antifungal biological evaluation of novel 3,5-disubstituted isoxazole compounds based on 5-nitrofuran scaffolds

Article abstract of DOI:10.1002/ardp.201900241

Nineteen 3,5-disubstituted-isoxazole analogs were synthesized based on nitrofuran scaffolds, by a [3 + 2] cycloaddition reaction between terminal acetylenes and 5-nitrofuran chloro-oxime. The compounds were obtained in moderate to very good yields (45–91%). The antileishmanial activity was assayed against the promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. Alkylchlorinated compounds 14p–r were active on both the promastigote and amastigote forms, with emphasis on compound 14p, which showed strong activity against the amastigote form (IC₅₀ = 0.6 μM and selectivity index [SI] = 5.2). In the alkyl series, compound 14o stands out with an IC₅₀ = 8.5 μM and SI = 8.0 on the amastigote form. In the aromatic series, the most active compounds were those containing electron-donor groups, such as trimethoxy isoxazole 14g (IC₅₀ = 1.2 μM and SI = 20.2); compound 14h, with IC₅₀ = 7.0 μM and SI = 6.1; and compound 14j containing the 4-SCH₃ group, with IC₅₀ = 5.7 μM and SI = 10.2. In addition, the antifungal activity of 19 nitrofuran isoxazoles was evaluated against five strains of Candida (C. albicans, C. parapsilosis, C. krusei, C. tropicalis, and C. glabrata). Eleven isoxazole derivatives were active against C. parapsilosis, and compound 14o was found to be the most active (minimal inhibitory concentration [MIC] = 3.4 μM) for this strain. Compound 14p was active against all the strains tested, with an MIC = 17.5 μM for C. glabrata, lower than that of the fluconazole used as the reference drug.

Full text of DOI:10.1002/ardp.201900241

Received: 18 August 2019
Revised: 27 October 2019
Accepted: 15 November 2019
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DOI: 10.1002/ardp.201900241
F U L L P A P E R
Design, synthesis, antileishmanial, and antifungal biological
evaluation of novel 3,5‐disubstituted isoxazole compounds
based on 5‐nitrofuran scaffolds
Ozildéia S. Trefzger1*
Amarith R. das Neves1,2
Antônio M. Honorato2
Renata T. Perdomo3
Adriano C. M. Baroni1
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Natália V. Barbosa1,2*
Maria L. F. S. Ortale1
Mariana R. Fragoso1
Maria F. C. Matos3 Marilene R. Chang4
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Renata L. Scapolatempo1*
Diego B. Carvalho1
Cristiane Y. K. Shuiguemoto1
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Carla C. P. Arruda2
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1LASQUIM—Laboratório de Síntese e Química
Medicinal, FACFAN—Faculdade de Ciências
Farmacêuticas, Alimentos e Nutrição,
Universidade Federal do Mato Grosso do Sul
(UFMS), Campo Grande, Brazil
Abstract
Nineteen 3,5‐disubstituted‐isoxazole analogs were synthesized based on nitrofuran
scaffolds, by a [3 + 2] cycloaddition reaction between terminal acetylenes and
5‐nitrofuran chloro‐oxime. The compounds were obtained in moderate to very good
yields (45–91%). The antileishmanial activity was assayed against the promastigote
and amastigote forms of Leishmania (Leishmania) amazonensis. Alkylchlorinated
compounds 14p–r were active on both the promastigote and amastigote forms, with
emphasis on compound 14p, which showed strong activity against the amastigote
form (IC50 = 0.6 μM and selectivity index [SI] = 5.2). In the alkyl series, compound 14o
stands out with an IC50 = 8.5 μM and SI = 8.0 on the amastigote form. In the aromatic
series, the most active compounds were those containing electron‐donor groups, such
as trimethoxy isoxazole 14g (IC50 = 1.2 μM and SI = 20.2); compound 14h, with
IC50 = 7.0 μM and SI = 6.1; and compound 14j containing the 4‐SCH3 group, with
IC50 = 5.7 μM and SI = 10.2. In addition, the antifungal activity of 19 nitrofuran
isoxazoles was evaluated against five strains of Candida (C. albicans, C. parapsilosis,
C. krusei, C. tropicalis, and C. glabrata). Eleven isoxazole derivatives were active against
C. parapsilosis, and compound 14o was found to be the most active (minimal inhibitory
concentration [MIC] = 3.4 μM) for this strain. Compound 14p was active against all
the strains tested, with an MIC = 17.5 μM for C. glabrata, lower than that of the
fluconazole used as the reference drug.
2Laboratório de Parasitologia Humana,
INBIO—Instituto de Biociências, Universidade
Federal do Mato Grosso do Sul (UFMS),
Campo Grande, Brazil
3Laboratório 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, Brazil
4Laboratório de Microbiologia Clínica,
FACFAN—Faculdade de Ciências
Farmacêuticas, Alimentos e Nutrição,
Universidade Federal do Mato Grosso do Sul
(UFMS), Campo Grande, Brazil
Correspondence
Dr. 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.
Email: adriano.baroni@ufms.br
Funding information
Fundação de Apoio ao Desenvolvimento do
Ensino, Ciência e Tecnologia do Estado de
Mato Grosso do Sul, Grant/Award Numbers:
23/200.462/2014, 23/200.708/2013,
59/300.109/2015; Conselho Nacional de
Desenvolvimento Científico e Tecnológico;
Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior
K E Y W O R D S
5‐nitrofuran scaffolds, antifungal activity, antileishmanial activity, drug design, isoxazole core
*Ozildéia S. Trefzger, Natália V. Barbosa, and Renata L. Scapolatempo contributed equally to this work.
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Arch Pharm Chem Life Sci. 2019;e1900241.
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900241

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replacing the tetrahydrofuran ring by an isoxazole core[11,12] and
others[13,14] (Figure 1).
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1
INTRODUCTION
Leishmaniasis is a neglected disease that affects mainly under-
developed tropical countries located in Africa, Asia, and Central and
South America. About one billion people around the world are at the
risk of contracting this disease, with a high mortality rate, if not
adequately treated. The main clinical manifestations of the disease
are cutaneous, mucocutaneous, and visceral.[1]
Isoxazoles are considered privileged scaffolds in drug discovery and
have a broad spectrum of activities—including antibacterial,[15] antivir-
al,[16] anti‐inflammatory,[17] antimycobacterial[18]—and more recently,
they have also demonstrated antitrypanosomal activity.[19]
This nucleus is present in antifungal compounds, such as
micafungin 7[20] and azoles derivatives 8 (Figure 2).[21] The presence
of the isoxazole nucleus increased the potency of micafungin 7 ten
times compared to the natural echinocandin.[20] The azole derivative
The current treatment is limited to pentavalent antimonials, such
as sodium stibogluconate, liposomal amphotericin B, miltefosine,
pentamidine, and paromomycin. The available treatment is not
optimal due to adverse effects, difficulty in use, parasite resistance,
safety, and price.[2–4] Considering the socioeconomic conditions of
the countries involved and increasing problems of resistance,
programs to develop new, accessible drugs for leishmaniasis are
imperative.
with isoxazole nucleus
concentration [MIC] = 2 µg/ml)
voriconazole‐resistant C. glabrata strains.[21]
8
showed activity (minimal inhibitory
against fluconazole and
On the other hand, the family of nitroheterocycles is relevant in the
treatment against neglected diseases and also as antibacterial agents. In
this family, it is important to highlight the compounds based on the 5‐
nitrofurans structures, such as nitrofurazone 9 and its hydroxylated
derivative 10, with potent antichagasic activity; nifurtimox 11, a drug
used to treat Chagas disease; and nifuroxazide 12, a nitrofuran with
potent antimicrobial and antiprotozoal activity (Figure 3).[22] Recently,
Kamal et al.[23] synthesized a 5‐nitrofuran–triazole congener 13 with
antifungal properties, extending the antimicrobial activities of this
scaffold.
Candidiasis is a fungal infection caused by the Candida genus with
diverse clinical manifestations, which include invasive candidiasis, a
bloodstream infection that ranges from symptomatic candidaemia to
fulminant sepsis.[5] The risk factors are long‐term hospitalization of
patients in the intensive care unit and use of central venous catheters
and immunosuppressive agents.[6] Although Candida albicans is often
the main cause of candidiasis, a shift has been observed from this
species to nonalbicans Candida, mainly C. glabrata, C. parapsilosis, C.
tropicalis, and C. krusei.[7]
Despite the applicability of nitroheterocyclic derivatives as
antimicrobial and antiprotozoal agents, they have been the subject
of intense discussions about their mutagenicity and carcinogenicity
due to the bioreduction process of the nitro group.[24] However,
compound 10 (NFOH‐121) showed lower mutagenicity in the Ames
test than nitrofurazone 9,[22] and its nongenotoxicity was confirmed
by micronucleus assay.[24]
There are four classes of antifungal agents used in the treatment
of candidiasis: azoles, echinocandins, polyenes, and pyrimidine
analogues. However, they have several disadvantages in terms of
toxicity, pharmacokinetic profile, and resistant strains.[8] For in-
stance, C. glabrata shows the highest proportion of isolated strains
resistant to azoles, such as fluconazole.[9]
Other drug discovery programs based on molecular modifications
of nitroheterocyclic drugs resulted in the design of safe compounds
for the treatment of neglected diseases. In this scenario, fexinidazole,
an antitrypanosomal candidate drug, has been shown to be highly
safe in clinical trials in humans performed by DNDi.[25,26]
Our research group studied molecular modification of tetrahy-
drofuran neolignans[10] like veraguensin 1, grandisin 2, and machilin
G 3 with a potential antileishmanial activity using bioisosterism, as a
tool for molecular modification, to produce new derivatives by
FIGURE 1 Antileishmanial activities of
synthetic isoxazole 4–6 compounds based
on neolignans 1–3

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FIGURE 2 Antifungal isoxazole compounds micafungin 7 and azole derivative 8
FIGURE 3 Structures of the bioactive
nitrofuran compounds 9–13
Among the 5‐nitrofurans that are used as antiparasitic agents,
those with a structural profile based on semicarbazones, hydrazones,
acyl hydrazones, and modifications have been highlighted.[27]
To broaden the chemical space of the compounds obtained with
this scaffold, this study aims to prepare a series of compounds using
the isoxazole core as a linker between the 5‐nitrofuran scaffold and
various substituents R1, and perform assays to explore their
antileishmanial and antifungal activities (Figure 4).
base (Scheme 2, procedure a).[11–14] Subsequently, the compounds were
submitted to retro‐Favorski reaction with KOH and the terminal
acetylenes 22b–i were obtained in good yields (Scheme 2, procedure
b).[11–14] Phenylacetylene was obtained as a commercially available
reagent. Ethynyl‐1,2,3‐trimethoxybenzene 22i was synthesized by the
Corey–Fuchs procedure (Scheme 2, procedures c and d).[11–14] The
compound 25 was prepared through Sonogashira cross‐coupling reaction
between aryl iodide 23 and ethynyltrimethylsilane 24 (Scheme 2,
procedure e).[11–14]
Cycloaddition [3 + 2] reaction was performed between 5‐nitrofuran
chloro‐oxime 17 and terminal acetylenes 22a–i, and trimethylsilylace-
tylene 25, using a catalytic system of CuSO4·5H2O, sodium ascorbate,
and KHCO3, in a solvent mixture of THF/CH2Cl2 1:1 providing
nitrofuran isoxazoles 14a–j in good yields (Table 1).[29]
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Compounds were synthesized through a [3 + 2] cycloaddition reac-
tion between terminal acetylenes with 5‐nitrofuraldehyde chloro‐
oxime. The 5‐nitrofuraldehyde chloro‐oxime 17 was synthesized
from 5‐nitrofuraldehyde 15 in two steps (Scheme 1).[28]
The synthesis of alkylated and chloroalkylated substituted 3,5‐
isoxazoles 14k–r (Table 1), and 14t (Scheme 3) aimed to expand the
proposed series to obtain more information about the structure–activity
relationship (SAR). Alkyl‐ and chloroalkyl‐substituted acetylenes 22k–q
and 22s were obtained as commercially available reagents.
For the synthesis of aryl acetylenes, first, compounds with aromatic
substituents were prepared. Aryl acetylenes containing withdrawing and
donor electron groups were synthesized by the Sonogashira cross‐
coupling reaction between 2‐methyl‐3‐butyn‐2‐ol and aryl bromides or
iodides using Pd(PPh3)2Cl2, CuI in the presence of Et3N as the reaction
Compound 14t was synthesized in two reaction steps from the
[3 + 2] cycloaddition reaction of chloro‐oxime 17 with 3‐butyn‐1‐ol
22r, and subsequent reaction of 14s with cyanuric chloride
(Scheme 3).[30]
FIGURE 4 Design of novel
3,5‐isoxazole nitrofuran analogues

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SCHEME 1 Synthesis of 5‐nitrofuraldehyde chloro‐oxime 17. Reagents and conditions: (a) NH2OH.HCl, NaOH, H2O/EtOH (2:3), r.t., 3 hr
(yield = 80%); (b) NCS, DMF, 60°C, 2 hr (yield = 78%)
All synthesized compounds 14a–t were identified by 1H NMR,
13C NMR, and high‐resolution mass spectra (HRMS).
5‐methoxylated‐substituted derivatives 14f–g were considered
inactive. The activity profile changed when the series was evaluated
against the amastigote forms.
The 3,4,5‐methoxylated compound 14g was the most active (IC50
=
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2.2
Biology
1.2 μM and SI = 20.2) against amastigote forms and its 3,5‐methoxy
congener 14f showed an IC50 of 19.5 μM.
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2.2.1
Antileishmanial activity
Compound 14h, containing the methylenedioxy group, showed
activity against promastigotes and amastigotes with IC50 = 16.4 µM
and 7.0 μM, respectively (Table 2). Compound 14i (R1 = Ph–O–Ph,
Table 2) was more active against promastigote (IC50 = 1.7 μM) than
against amastigote forms (IC50 = 16.6 μM). Compound 14j, containing
the 4‐SCH3 group, a more apolar bioisoster of the compound 14d
(containing the 4‐OCH3 group), had an IC50 of 5.7 µM against the
amastigote forms of L. amazonensis.
The aromatic compounds were first evaluated for antileishmanial
activity on the promastigote forms of Leishmania (Leishmania)
amazonensis. The initial objective was to screen 3,5‐diaryl‐
isoxazoles containing electron donors and withdrawing groups to
make a comparison with compound 14a, and thus to understand the
role of these groups in the possible antileishmanial activity (Table 2).
Compound 14a and its chlorinated derivative 14b were active
against promastigotes of L. amazonensis (IC50 = 7.9 µM and 7.0 µM,
respectively) and showed moderate activity against amastigote forms
with an IC50 = 34.0 µM and 23.0 μM, respectively. Compound 14c
was inactive against both forms (IC50 > 150 µM).
To expand the number of examples, alkylated homologous series
14k–o were tested, in which the carbon chain number ranged from
four to eight carbons.
SAR information reported in the literature supports that the
homologation approach can be adopted in the discovery of new drug
candidates.[31] The size of the carbon chain, in this case, affects
directly the lipophilicity parameter (Table 3). In compounds 14k–n,
The methoxylated‐substituted derivatives 14d–g had interesting
profiles of antileishmanial activity. Although the 4‐methoxy and 3,
4‐methoxyl substituted compounds 14d–e had relevant activity
against the promastigote forms (IC50 = 6.0 µM and 7.8 μM, respec-
tively), compared to pentamidine (IC50 = 8.9 μM). The 3,5‐ and 3,4,
there is
a tendency to increase antileishmanial activity over
promastigote forms when ClogP increases to the limit of 5 (Table 4).
However, the antileishmanial activity on amastigote forms showed an
SCHEME 2 Preparation of aryl acetylenes 22b–i and 25. Reagents and conditions: (a) 2‐methyl‐3‐butyn‐2‐ol, PdCl2(PPh3)2/CuI, Et3N, reflux,
24 hr (yield: 55–93%); (b) KOH, toluene, reflux, 24 hr (yield: 43–90%); (c) CBr4, PPh3, CH2Cl2, 0°C, 5 hr (yield: 74%); (d) THF, n‐BuLi, −25°C to
room temperature, 1 hr (22h, yield: 83%). (e) PdCl2(PPh3)2/CuI, Et3N, reflux, 24 hr (yield: 73%)

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TABLE 1 Synthesized 3,5‐nitrofuran isoxazoles 14a–r and 14t
OH
O
N
CuSO4.5H2O
N
X
O
sodium ascorbate
O2N
R1
O
+
Cl
R1
KHCO3
CH2Cl2/THF
48 hr, r.t.
O2N
14a–j
22a–q,25
17
X = H, -Si(CH3)3
Compounds 14a–r
R1
Yield (%)
89
14a
14b
14c
14d
14e
14f
14g
14h
14i
Ph
4‐Cl–Ph
55
4‐NO2–Ph
4‐OCH3–Ph
3,4‐di‐OCH3–Ph
3,5‐di‐OCH3–Ph
3,4,5‐tri‐OCH3–Ph
3,4‐methylenedioxy–Ph
Ph–O–Ph
4‐SCH3–Ph
–C4H9
45
83
85
72
81
74
64
14j
58
14k
14l
86
–C5H11
70
14m
14n
14o
14p
14q
14r
–C6H13
91
–C7H15
89
–C8H17
84
–CH2Cl
68
–(CH2)3Cl
–(CH2)4Cl
74
78
aChlorooxime 17 (2.0 mmol), terminal acetylenes 22a–r, 25 (2.0 mmol), CuSO4·5H2O (0.30 mmol), sodium ascorbate (0.50 mmol), KHCO3 (20 mmol), THF
(5 ml), CH2Cl2 (5 ml), r.t., 48 hr.
IC50 with a zig‐zag tendency, when the biological activity alternates
with the number of carbons present in the alkyl chain.[31] In this
series, compound 14o showed a relevant antileishmanial activity
against promastigote and amastigote forms (IC50 = 6.1 μM and
8.5 μM, respectively).
compounds 14o–r showed a tendency of higher potency asso-
ciated with increasing –CH2 groups in the homologous series.
Compound 14t was an exception with poor activity toward
promastigotes (Table 3). However, in the specific case of
antileishmanial activity against amastigote forms, compound 14p
Inspired by the positive results found in the alkylated series
(14k–o; Table 3), a homologous chlorinated alkyl series 14p–r and
14t was designed and tested to complement the SAR information.
The number of –CH2 groups ranged from one to four, and
antileishmanial activity assay showed compounds with a good
profile on both the promastigotes and amastigotes forms. Upon
analysis of the antipromastigote activity, it was found that
was the most active, with only one –CH2 group (IC50
0.6 μM, SI = 5.2).
=
Compounds 14q, 14r, and 14t had an IC50 between 3.2 μM and
4.1 μM, which is similar to the activity of the reference drug
pentamidine (IC50 = 3.3 μM) on the amastigote forms of L. amazo-
nensis, although the selectivity indexes (SI) were not satisfactory with
values close to 1.
SCHEME 3 Synthesis of nitrofuran isoxazole derivative 14t. Reagents and conditions: (a) CuSO4·H2O, sodium ascorbate, KHCO3, THF/
CH2Cl2 (1:1), r.t., 48 hr (yield: 50%); (b) cyanuric chloride, DMF, CH2Cl2, r.t., 4 hr (yield: 65%)

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TABLE 2 Antileishmanial activities of 3,5‐diaryl‐isoxazoles 14a–j
14a–j
NIH/3T3
Promastigote
Compound/R1
ClogPa
3.2
3.9
3.2
3.3
2.9
3.3
2.9
3.1
5.0
3.7
–
IC50 (µM) SDb
28.5 0.2
250 2.3
NC
IC50 (µM) SDc
SId
Amastigote IC50 (µM) SDe
SIf
14a: R1 = Ph
7.9 0.9
7.0 0.8
166 2.2
6.0 0.7
7.8 0.8
158 2.2
144 2.1
16.4 1.2
1.7 0.2
16.7 1.2
–
3.6
35.3
NC
25.4
4.7
2.6
0.2
2.6
142.0
3.5
–
34.0 1.5
23.0 1.3
155 1.2
31.8 1.5
14.6 1.1
19.5 1.3
1.2 0.0
7.0 0.8
16.6 1.2
5.7 0.5
–
0.8
14b: R1 = 4‐Cl–Ph
10.8
NC
0.8
14c: R1 = 4‐NO2–Ph
14d: R1 = 4‐OCH3–Ph
14e: R1 = 3,4‐di‐OCH3–Ph
14f: R1 = 3,5‐di‐OCH3–Ph
14g: R1 = 3,4,5‐tri‐OCH3–Ph
14h: R1 = 3,4‐methylenedioxy–Ph
14i: R1 = Ph–O–Ph
14j: R1 = 4‐SCH3–Ph
Doxorubicing
25.4 4.9
36.4 4.3
250 2.3
25.0 2.1
43.3 6.0
250 2.3
57.9 8.5
0.7 0.0
2.5
12.8
20.2
6.1
15.2
10.2
–
Pentamidineg
–
27.5 3.1
2.6 0.0
8.9 0.6
–
3.0
–
3.3 0.3
0.08 0.0
8.3
Amphotericin B
–
32.5
Abbreviations: IC50, half‐maximal inhibitory concentration; NC, not calculated; SD, standard deviation; SI, selectivity index.
aClogP, octanol/water partition coefficient.
bIC50 on fibroblast cells NIH/3T3.
cIC50 on promastigote of Leishmania amazonensis.
dSI, IC50 on mammal cells/IC50 on promastigotes.
eCI50 on intracellular amastigote of L. amazonensis.
fSI, IC50 on mammal cells/IC50 on intracellular amastigotes.
gPositive control, pentamidine, and amphotericin B for L. amazonensis and doxorubicin for fibroblasts.
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2.2.2
Antifungal activity
improvement in the biological activity due to the bioisosteric
replacement of the oxygen atom to a sulfur atom (Table 4).
The alkylated compounds with the odd number of methylene groups
14k, 14m, and 14o were active against C. parapsilosis. Compound 14o has
the highest ClogP of this series (Table 4), and it was found to be the most
active with an MIC of 3.4 µM, lower than fluconazole and close to the
amphotericin B, both used as positive controls for the test.
Initially, the disk diffusion method was performed for determining
the susceptibility of C. parapsilosis (ATCC 22019), C. albicans (ATCC
90028), C. krusei (ATCC 6258), C. glabrata (ATCC 2001), and C.
tropicalis (ATCC 750) to the nitrofuran isoxazole derivatives.
Nineteen nitrofuran isoxazole derivatives were tested, and
compounds 14a, 14d, 14g, 14h, 14k, 14m, 14o, 14p, 14q, and 14t
were active against one or more strains of Candida and were thus
selected for the broth microdilution test.
The chloroalkylated compounds 14p, 14q, and 14t showed
activity against two or more strains of Candida. In contrast to the
alkylated compound series, the compound 14p with the lowest ClogP
showed an MIC of 8.7 µM. This derivative was active against C. krusei
with MIC = 35 µM lower than the reference drug fluconazole (MIC =
52 µM) and also showed activity against all strains tested.
All 3,5‐nitrofuran isoxazole derivatives tested were active against C.
parapsilosis (Table 4). Although this strain is less virulent than other
Candida spp, this result is very interesting since C. parapsilosis is the most
common candidemia‐causing species in Latin America after C. albicans.[32]
These infections are related to the use of catheters and parenteral
nutrition probably because of the capacity of colonizing plastic surfaces
and forming of a biofilm. Moreover, an increase in the nonsusceptibility
of C. parapsilosis to azole and amphotericin B has been reported.[33]
For the aromatic series, compound 14g was found to be most
active against C. parapsilosis with a minimal inhibitory concentration
(MIC) of 5.7 µM, similar to fluconazole (MIC = 6.5 µM) used as the
standard drug. Compound 14d showed an MIC of 111 µM, whereas
compound 14j showed an MIC = 26.4 µM, which suggests an
For C. glabrata, compound 14p showed an MIC of 17.5 µM, lower
than fluconazole. This is a promising result since C. glabrata has its
mechanism of resistance related to azole compounds, such as
fluconazole, due to the efflux pump.[34]
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3
CONCLUSION
Isoxazole nitrofuran series with structural diversity was synthe-
sized with good yields. The antileishmanial activity showed that

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TABLE 3 Antileishmanial activity of alkyl and chloroalkyl 3,5‐disubstituted isoxazoles, 14k–r and 14t
14k–r and 14t
NIH/3T3
IC50 (µM) SDb
Promastigote
IC50 (µM) SDc
Intracellular amastigote
Compound
ClogPa
3.2
3.7
4.2
4.7
5.2
2.1
2.3
2.6
2.9
–
SId
0.1
NC
1.5
76.2
11.2
0.8
1.7
8.0
1.8
–
IC50 (µM) SDe
SIf
14k: R1 = C4H9
14l: R1 = C5H11
14m: R1 = C6H13
14n: R1 = C7H15
14o: R1 = C8H17
14p: R1 = CH2Cl
14t: R1 = (CH2)2Cl
14q: R1 = (CH2)3Cl
14r: R1 = (CH2)4Cl
Doxorubicing
24.2 1.6
NC
211 2.3
200 2.3
16.4 1.2
3.3 0.5
6.1 0.7
3.5 0.5
206 2.3
0.9 0.0
1.8 0.3
–
39.2 1.6
19.4 1.2
36.9 1.6
64.4 1.8
8.5 0.9
0.6 0.1
3.2 0.4
3.8 0.5
4.1 0.6
–
0.6
NC
0.6
3.9
8.0
5.2
1.6
1.8
0.8
–
24.0 1.5
250 2.3
68.5 5.8
3.1 0.1
5.0 2.3
7.0 3.7
3.3 0.4
0.7 0.0
27.5 3.1
2.6 0.0
Pentamidineg
–
8.9 0.6
–
3.0
–
3.3 0.3
0.08 0.0
8.3
32.5
Amphotericin Bg
–
Abbreviations: IC50, half‐maximal inhibitory concentration; NC, not calculated; SD, standard deviation; SI, selectivity index.
aClogP, octanol/water partition coefficient.
bIC50 on fibroblast cells NIH/3T3.
cIC50 on promastigote of Leishmania amazonensis.
dSI, IC50 on mammal cells/IC50 on promastigotes.
eCI50 on intracellular amastigote of L. amazonensis.
fSI, IC50 on mammal cells/IC50 on intracellular amastigotes.
gPositive control, pentamidine and amphotericin B for L. amazonensis and doxorubicin for fibroblasts.
TABLE 4 Anti‐Candida activity of 5‐nitrofuran isoxazole derivatives
Candida parapsilosis
Candida albicans
Candida krusei
Candida tropicalis
Candida glabrata
Compound
MIC (µM SD)
MIC (µM SD)
MIC (µM SD)
MIC (µM SD)
MIC (µM SD)
14a: R1 = Ph
31.2 1.5
111.0 2.0
5.7 0.7
–
–
–
–
14d: R1 = 4‐OCH3–Ph
14g: R1 = 3,4,5‐tri‐OCH3–Ph
14h: R1 = 3,4‐methylenedioxy–Ph
14j: R = SCH3–Ph
14k: R1 = C4H9
–
–
–
–
–
–
–
–
53.2 1.7
26.4 1.4
135.5 2.1
7.6 0.8
–
–
–
–
–
–
–
–
–
–
–
–
14m: R1 = C6H13
14o: R1 = C8H17
–
–
–
–
3.4 0.5
–
–
–
–
14p: R1 = CH2Cl
8.7 0.9
35.0 1.5
132.0 2.1
118.0 2.0
0.8 0.0
0.6 0.0
35.0 1.5
132.0 2.1
–
35.0 1.5
–
17.5 1.2
14t: R1 = (CH2)2Cl
14q: R1 = (CH2)3Cl
Fluconazolea
66.0 1.8
29.5 1.5
6.5 0.8
–
–
–
52.0 1.7
0.5 0.0
3.3 0.5
4.3 0.6
52.0 1.7
8.6 0.9
Amphotericin Ba
1.0 0.0
Abbreviations: MIC, minimal inhibitory concentration; SD, standard deviation.
aFluconazole and amphotericin B used as positive controls.
the chloroalkyl‐substituted compounds 14p–r were active for
both the promastigote and amastigote forms, emphasizing the
activity of compound 14p that showed IC50 = 0.6 μM and SI = 5.2
against the amastigote forms. In the alkyl series, compound 14o
stands out with an IC50 of 8.5 μM and SI of 8.0 for the amastigote
forms. In the aromatic series, the most active compounds were
those containing electron‐donor groups such as trimethoxy
isoxazole 14g (IC50 = 1.2 μM and SI = 20.2), 14h (IC50 = 7.0 μM

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TREFZGER ET AL.
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and SI = 6.1), and 14j (IC50 = 5.7 μM and SI = 10.2). Thus, the
compounds 14o, 14p, 14g, 14h, and 14j are promising candidates
for future in vivo studies, due to their relevant activity mainly on
amastigotes, the target/aimed parasite form in this type of study.
For the antifungal activity, eleven compounds were active against
one or more strains of Candida in a broth microdilution test. All
the compounds tested were active against C. parapsilosis, and
compound 14o was found to be the most active (MIC = 3.4 μM) to
this strain. On the contrary, compound 14p was active against all
strains of Candida tested, with a minimal inhibitory concentration
(MIC) for C. krusei and C. glabrata of 35.5 μM and 17.5 μM,
respectively, lower than fluconazole.
(0.35 mmol) were added, and the mixture reaction was stirred for
10 min. Then, KHCO3 (20 mmol, 10 equiv.) was added, and the mixture
was stirred at room temperature for 48 hr. Extraction was performed
with ethyl acetate (3 × 30 ml) and washed with a saturated solution of
NH4Cl (3 × 30 ml). The organic phase was dried over anhydrous MgSO4,
and the solvent was removed under reduced pressure. The product was
purified by recrystallization in heated ethanol.
3‐(5‐Nitrofuran‐2‐yl)‐5‐phenylisoxazole (14a)[36]
The product was obtained as a yellow solid in 89% yield. Mp:
182–184°C. 1H NMR (300 MHz, CDCl3): δ 6.97 (s, 1H), 7.18 (d,
J = 3 Hz, 1H), 7.44 (d, J = 3 Hz, 1H), 7.49–7.51 (m, 3H), and 7.82–7.85
(m, 2H). 13C NMR (75 MHz, CDCl3): δ 99.15, 114.76, 114.93, 126.27,
126.47, 129.86, 131.53, 146.05, 152.64, 154.14, and 171.06. HRMS
(ESI+) m/z calcd for C13H18N2O4 [M+H]: 257.0562; found: 257.0578.
|
4
EXPERIMENTAL
Chemistry
4.1
5‐(4‐Chlorophenyl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14b)[37]
|
The product was obtained as a yellow solid in 55% yield. Mp: 211–213°C.
1H NMR (300 MHz, DMSO‐d6): δ 7.47 (d, J = 3.9 Hz, 1H), 7.62 (d,
J = 8.5 Hz, 2H), 7.68 (s, 1H), 7.86 (d, J = 3.9 Hz, 1H), and 7.94 (d, J = 8.5 Hz,
2H). 13C NMR (75 MHz, DMSO‐d6): 99.69, 114.76, 114.96, 125.31,
128.10, 129.99, 136.19, 145.87, 152.66, 154.22, and 169.96. HRMS
(ESI+) m/z calcd for C13H7ClN2O4 [M+H]: 291.0172; found: 291.0172.
|
4.1.1
General remarks
Anhydrous solvents were dried and distilled before use according
to the standard procedure.[35] All chemicals used for synthesis
were of reagent grade and used without purification unless noted
otherwise. Reactions were carried out under a nitrogen atmo-
sphere and monitored by thin‐layer chromatography using
prepared plates (silica gel 60 F254 on aluminum). The chromato-
grams were examined under both 254 and 360 nm ultraviolet
light or with the developing agent ethanolic vanillin and heat.
Flash‐column chromatography was performed on silica gel 60
(particle size 200–400 mesh ASTM, purchased from Aldrich).
Melting points were determined using Fisatom 430D equipment.
The 1H and 13C NMR spectra were recorded in CDCl3 solutions
using a Bruker 75‐ or 300‐MHz spectrometer, as noted. Chemical
shifts (δ) are expressed as parts per million (ppm) downfield from
3‐(5‐Nitrofuran‐2‐yl)‐5‐(4‐nitrophenyl)isoxazole (14c)
The product was obtained as a yellow solid in 45% yield. Mp: 230–232°C.
1H NMR (300 MHz, DMSO‐d6): δ 7.57 (d, J = 3 Hz, 1 H), 7.91 (d, J = 3 Hz,
1H), 7.98 (s, 1H), 8.24 (d, J = 9 Hz, 2H), 8.42 (d, J = 9 Hz, 2H). 13C NMR
(75 MHz, DMSO‐d6): δ 101.58, 114.34, 114.89, 124.70, 127.23, 131.47,
145.14, 148.55, 154.07, 168.48. HRMS (ESI+) m/z calcd for C13H7N3O6
[M+H]: 302.0413; found: 302.0469.
5‐(4‐Methoxyphenyl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14d)
The product was obtained as a brown solid in 83% yield. Mp: 164–166°C.
1H NMR (300 MHz, DMSO‐d6): δ 3.81 (s, 3H), 7.09 (d, J = 9 Hz, 2H),
7.45–7.47 (m, 2H), and 7.85–7.87 (m, 3H). 13C NMR (75 MHz, DMSO‐d6):
δ 55.90, 97.54, 114.69, 114.76, 115.25, 119.12, 128.04, 146.26, 152.58,
154.02, 161.75, and 171.13. HRMS (ESI+) m/z calcd for C14H10N2O5
[M+H]: 287.0668; found: 287.0664.
tetramethylsilane or deuterated solvent (CDCl3 1H δ 7.27 and 13
C
δ 77.0 ppm; dimethyl sulfoxide (DMSO)‐d6 1H δ 2.5 and 13C δ
39.51 ppm; acetone 1H δ 2.05 and 13C δ 29.8 ppm) as the internal
standard. HR‐ESI‐MS (high‐resolution electrospray ionization
mass spectrometry) measurements were carried out on
a
quadrupole time‐of‐flight instrument (UltrOTOF‐Q; Bruker Dal-
tonics, Billerica, MA).
5‐(3,4‐Dimethoxyphenyl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14e)
The product was obtained as a yellow solid in 85% yield. Mp: 240–242°C.
1H NMR (300 MHz, DMSO‐d6): δ 3.80 (s, 6H), 6.64 (t, J = 2.1 Hz, 1H), 7.07
(d, J = 2.1 Hz, 2H), 7.45 (d, J = 3.9 Hz, 1H), 7.69 (s, 1H), and 7.86 (d,
J = 3.9 Hz, 1H). 13C NMR (75 MHz, DMSO‐d6): δ 56.07, 99.71, 103.60,
104.13, 114.78, 128.10, 146.02, 152.65, 154.15, 161.51, 170.92. HRMS
(ESI+) m/z calcd for C15H12N2O6 [M+H]: 317.0773; found: 317.0765.
Additional data are provided as Supporting Information. The
InChI codes of the investigated compounds together with
some biological activity data are also provided as Supporting
Information.
|
4.1.2
General procedure for the preparation of
3,5‐nitrofuran isoxazole derivatives 14
5‐(3,5‐Dimethoxyphenyl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14f)
The product was obtained as a yellow solid in 72% yield. Mp:
216–218°C. 1H NMR (300 MHz, DMSO‐d6): δ 3.80 (s, 6H), 6.65
(s, 1H), 7.07 (d, J = 3.9 Hz, 2H), 7.45 (d, J = 3 Hz, 1H), 7.69 (s, 1H), and
7.85 (d, J = 3.9 Hz, 1H). 13C NMR (75 MHz, DMSO‐d6): δ 56.08, 99.71,
To a solution of the respective chloro‐oxime 17 (2 mmol, 1.0 equiv.) and
the acetylene of interest 22a–s, 25 (2.2 mmol, 1.1 equiv.) in CH2Cl2/THF
(50:50, 6 ml), CuSO4.5H2O (0.13 mmol) and sodium ascorbate

TREFZGER ET AL.
9 of 12
|
103.62, 104.17, 114.77, 128.12, 146.04, 152.15, 154.15, 161.53, and
170.94. HRMS (ESI+) m/z calcd for C15H12N2O6 [M+H]: 317.0773;
found: 317.0780.
(s, 1H), 7.42 (d, J = 3 Hz, 1H), and 7.81 (d, J = 3 Hz, 1H). 13C NMR
(75 MHz, DMSO‐d6): δ 14.21, 22.14, 26.19, 26.93, 30.99, 100.05,
114.66, 114.69, 146.45, 152.52, 153.28, and 175.76. HRMS (ESI+)
m/z calcd for C12H14N2O4 [M+H]: 251.1032; found: 251.1058.
3‐(5‐Nitrofuran‐2‐yl)‐5‐(3,4,5‐trimethoxyphenyl)isoxazole (14g)
The product was obtained as a yellow solid in 81% yield. Mp:
189–191°C. 1H NMR (300 MHz, CDCl3): δ 3.90 (s, 3H), 3.94 (s, 6H),
6.92 (s, 1H), 7.04 (s, 2H), 7.18 (d, J = 3 Hz, 1H), and 7.44 (d, J = 3 Hz,
1H). 13C NMR (75 MHz, CDCl3): δ 56.35, 61.04, 96.90, 103.31, 111.95,
113.09, 121.75, 140.49, 146.60, 153.80, 153.89, and 171.39. HRMS
(ESI+) m/z calcd for C16H14N2O7 [M+H]: 347.0879; found: 347.0884.
5‐Hexyl‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14m)
The product was obtained as a brown solid in 91% yield. Mp:
92–94°C. 1H NMR (300 MHz, CDCl3): δ 0.87–0.91 (t, J = 6 Hz, 3H),
1.35–1.36 (m, 6H), 1.69–1.77 (qt, J = 6 Hz, 2H), 2.81 (t, J = 6 Hz, 2H),
6.45 (s, 1H), 7.11 (d, J = 3 Hz, 1H), 7.42 (d, J = 3 Hz, 1H). 13C NMR
(75 MHz, DMSO‐d6): δ 14.32, 22.35, 26.21, 27.19, 28.44, 31.21,
100.14, 114.75, 114.85, 146.42, 152.46, 153.26, and 175.71. HRMS
(ESI+) m/z calcd for C13H16N2O4 [M+H]: 265.1188; found: 265.1192.
5‐(Benzo[d][1,3]dioxol‐5‐yl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14h)
The product was obtained as as brown solid in 74% yield. Mp:
162–164°C. 1H NMR (300 MHz, DMSO‐d6): δ 6.11 (s, 2H), 7.08 (d,
J = 6 Hz, 1H), 7.43–7.49 (m, 4H), and 7.85 (d, J = 3 Hz, 1H). 13C NMR
5‐Heptyl‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14n)
The product was obtained as a yellow solid in 89% yield. Mp:
95–97°C. 1H NMR (300 MHz, DMSO‐d6): δ 0.81 (m, 3H); 1.21–1.25
(m, 8H), 1.62 (t, J = 6 Hz, 2H), 2.78 (t, J = 6 Hz, 2H), 6.83 (s, 1H), 7.42
(d, J = 3.2 Hz, 1H), and 7.81 (d, J = 3.2 Hz, 1H). 13C NMR (75 MHz,
DMSO‐d6): δ 14.33, 22.47, 26.23, 27.26, 28.69, 28.77, 31.53, 100.03,
114.63, 114.68, 146.47, 152.50, 153.27, and 175.75. HRMS (ESI+)
m/z calcd for C14H18N2O4 [M+H]: 279.1345; found: 279.1347
(75 MHz, DMSO‐d6):
δ 98.06, 102.38, 106.42, 109.56, 114.66,
114.77, 120.47, 121.17, 146.16, 148.62, 150.04, 152.62, 154.06,
and 170.93. HRMS (ESI+) m/z calcd for C14H8N2O6 [M+H]: 301.0460;
found: 301.0456.
3‐(5‐Nitrofuran‐2‐yl)‐5‐(4‐phenoxyphenyl)isoxazole (14i)
The product was obtained as a yellow solid in 64% yield. Mp:
183–185°C. 1H NMR (300 MHz, acetone‐d6) δ 7.09–7.14 (m, 4H),
7.19–7.24 (t, J = 7.4 Hz, 1H), 7.32 (s, 1H), 7.37 (d, J = 3.8 Hz, 1H),
7.41–7.47 (m, 2H), 7.68 (d, J = 3.8 Hz, 1H), and 7.97 (d, J = 8.9 Hz, 2H).
13C NMR (75 MHz, acetone‐d6) δ 97.1, 113.1, 133.3, 118.4, 119.8,
121.4, 124.4, 127.9, 130.2, 146.3, 154.0, 155.9, 160.0, and 170.7.
HRMS (ESI+) m/z calcd for C19H12N2O5 [M+H]: 349.0824; found:
349.0814.
3‐(5‐Nitrofuran‐2‐yl)‐5‐octylisoxazole (14o)
The product was obtained as a yellow solid in 84% yield. Mp:
97–99°C. 1H NMR (300 MHz, DMSO‐d6):
δ 0.78–0.8 (m, 3H),
1.20–1.31 (m, 10H), 1.60‐1.65 (m, 2H), 2.78 (t, J = 7.4 Hz, 2H), 6.84
(s, 1H), 7.43 (d, J = 3.9 Hz, 1H), 7.82 (d, J = 3.9 Hz, 1H). 13C NMR
(75 MHz, DMSO‐d6): δ 14.37, 22.51, 26.23, 27.25, 28.82, 28.98,
29.00, 31.67, 100.04, 114.67, 114.71, 146.45, 152.50, 153.27,
175.75. HRMS (ESI+) m/z calcd for C15H20N2O4 [M+H]: 293.1501;
found: 293.1512.
5‐(4‐(Methylthio)phenyl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14j)
The product was obtained as a brown solid with 58% yield. Mp:
138–140°C. 1H NMR (300 MHz, DMSO‐d6): δ 2.51 (s, 3H), 7.38 (m,
4H), 7.50 (d, J = 9 Hz, 2H) and 7.88 (d, J = 3 Hz, 1H). 13C NMR
5‐(Chloromethyl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14p)
The product was obtained as a yellow solid in 68% yield. Mp:
87–89°C. 1H NMR (300 MHz, DMSO‐d6): δ 5.02 (s, 2H), 7.18 (s, 1H),
7.52 (d, J = 3.9 Hz, 1H), and 7.83 (d, J = 3.9 Hz, 1H). 13C NMR
(75 MHz, DMSO‐d6):
δ 14.50, 108.52, 114.69, 114.92, 124.31,
125.62, 126.64, 130.13, 142.95, 147.02, 152.59, 157.49, and
176.90. HRMS (ESI+) m/z calcd for C19H12N2O5 [M+H]: 303.0440;
found: 303.0433.
(75 MHz, DMSO‐d6):
δ 34.49, 103.07, 114.60, 115.46, 145.53,
152.68, 153.66, and 169.95. HRMS (ESI+) m/z calcd for C8H5ClN2O4
[M+H]: 229.0016; found: 229.0012.
5‐Butyl‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14k)
The product was obtained as a brown solid in 86% yield. Mp:
88–90°C. 1H NMR (300 MHz, DMSO‐d6): δ 0.91 (t, J = 6 Hz, 3H), 1.33
(q, J = 6 Hz, 2H), 1.68 (qt, J = 6 Hz, 2H), 2.84 (t, J = 6 Hz, 2H), 6.90 (s,
1H), 7.48 (d, J = 3 Hz, 1H), and 7.86 (d, J = 3 Hz, 1H). 13C NMR
(75 MHz, DMSO‐d6): δ 13.94, 21.93, 25.90, 29.30, 100.17, 114.77,
114.90, 146.42, 152.46, 153.28, and 175.72. HRMS (ESI+) m/z calcd
for C11H12N2O4 [M+H]: 237.0875; found: 237.0872.
5‐(3‐Chloropropyl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14q)
The product was obtained as yellow solid in 74% yield. Mp: 94–96°C.
1H NMR (300 MHz, DMSO‐d6): δ 2.11 (qt, J = 6 Hz, 2H), 2.96 (t,
J = 6 Hz, 2H), 3.68 (t, J = 6 Hz, 2H), 6.90 (s, 1H), 7.42 (d, J = 3.7 Hz,
1H), and 7.80 (d, J = 3.7 Hz, 1H). 13C NMR (75 MHz, DMSO‐d6): δ
23.81, 30.12, 44.67, 100.45, 114.66, 114.71, 146.32, 152.51, 153.36,
and 174.32. HRMS (ESI+) m/z calcd for C10H9ClN2O4 [M+H]:
257.0329; found: 257.0342.
3‐(5‐Nitrofuran‐2‐yl)‐5‐pentylisoxazole (14l)
The product was obtained as a yellow solid in 70% yield. Mp:
91–93°C. 1H NMR (300 MHz, DMSO‐d6): δ 0.83–0.85 (m, 3H),
1.27–1.30 (m, 4H), 1.64 (t, J = 6 Hz, 2H), 2.79 (t, J = 6 Hz, 2H), 6.85
5‐(4‐Chlorobutyl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14r)
The product was obtained as a yellow solid in 78% yield. Mp:
96–98°C. 1H NMR (300 MHz, DMSO‐d6): δ 1.74–1.79 (m, 4H),

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TREFZGER ET AL.
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2.84–2.88 (m, 2H), 3.63–3.68 (m, 2H), 6.88 (s, 1H), 7.44 (d, J = 3.6 Hz,
1H), and 7.82 (d, J = 3.6 Hz, 1H). 13C NMR (75 MHz, DMSO‐d6): δ
29.34, 30.21, 36.44, 50.05, 104.98, 119.47, 119.51, 151.11, 151.27,
158.09, and 180.08. HRMS (ESI+) m/z calcd for C11H11ClN2O4
[M+H]: 271.0485; found: 271.0482.
The percentage of activity was calculated by the following
formula:
% Activity = 100 – [(N – Y)/(N – P)] × 100,
where Y is the optical density reading of cells and wells with different
concentrations of the compounds, N is the optical density reading of
parasites in wells with 1.5% DMSO, and P is the optical density
reading of the wells with only culture medium.
2‐(3‐(5‐Nitrofuran‐2‐yl)isoxazol‐5‐yl)ethanol (14s)
The product was obtained as a yellow solid in 30%. 1H NMR
(300 MHz, DMSO‐d6): δ 2.95 (t, J = 6 Hz, 2H), 3.71 (q, J = 6 Hz, 2H),
4.91 (t, J = 6 Hz, 1H), 6.86 (s, 1H), 7.44 (d, J = 3 Hz, 1H), and 7.81 (d,
J = 3 Hz, 1H). 13C NMR (75 MHz, DMSO‐d6): δ 30.39, 58.78, 100.75,
114.65, 114.72, 146.46, 152.52; 153.28, and 174.00. HRMS (ESI+)
m/z calcd for C9H8N2O5 [M+H]: 225.0511; found: 225.0507.
Pentamidine (IC50) promastigotes = 8.9 μM.
Parasites and peritoneal macrophages—amastigotes anti‐Leishmania
evaluation[11–14,38–40]
In vitro antileishmanial activity was evaluated on peritoneal macrophages
infected with. L. (L.) amazonensis intracellular amastigotes. Parasites (IFLA/
BR/1967/PH8 strain) were routinely isolated from BALB/c mice and
maintained as promastigotes at 25°C in Schneider’s Insect Medium
(Sigma‐Aldrich®) supplemented with 20% fetal calf serum (FCS‐Cultilab®
Campinas, Brazil) and 140 µg/ml gentamicin (Sigma‐Aldrich®). Macro-
phages were obtained from the peritoneal wash of BALB/c mice after
euthanasia. 10 ml of RPMI‐1640 (Sigma‐Aldrich®) supplemented with 2%
L‐glutamine, 2.8% bicarbonate buffer, 100 U/ml penicillin and 100 mg/ml
streptomycin were injected into the peritoneal cavity. After the massage
area, the liquid was aspirated and transferred to tubes on ice. Peritoneal
cells were quantified in a Neubauer chamber after cellular exclusion with
Trypan Blue staining (Sigma‐Aldrich®).
5‐(2‐Chloroethyl)‐3‐(5‐nitrofuran‐2‐yl)isoxazole (14t)
To a solution of the respective of 2,4,6‐trichloro‐(1,3,5)‐triazine (2 mmol,
1.05 eq.) in dimethylformamide (1 ml) under magnetic stirring until total
consumption of the starting material (approximately 10 min), then the
synthesized isoxazole 14s (2 mmol, 1.0 eq.) dissolved in CH2Cl2 (5 ml) was
added dropwise. This reaction mixture was stirred at room temperature
for 4 hr. Then, 30 ml of saturated NaCl solution was added and the
product was extracted with ethyl acetate (2 × 30 ml). The organic phase
was dried with anhydrous MgSO4 and the solvent evaporated under
reduced pressure. The product was purified by crystallization in heated
ethanol. The product was obtained as a yellow solid in 65% yield. Mp:
91–93°C. 1H NMR (300 MHz, DMSO‐d6): δ 3.33 (m, 2H), 3.96 (t, J = 6 Hz,
2H), 7.00 (s, 1H), 7.47 (d, J = 3.9 Hz, 1H), and 7.83 (d, J = 3.9 Hz, 1H). 13C
NMR (75 MHz, DMSO‐d6): δ 29.8, 42.02, 101.43, 114.71, 114.85, 146.11,
152.59, 153.43, and 172.30. HRMS (ESI+) m/z calcd for C9H7ClN2O4
[M+H]: 243.0172; found: 243.0138.
In vitro antileishmanial activity on intracellular amastigotes[11–14,38–40]
Peritoneal cells (1 × 105 cells/well) were added to 24‐well plates
containing circular coverslips. The plates were incubated for 1 hr
at 37°C/5% CO2 to allow cell adhesion and then 1 × 106 L. (L.)
amazonensis promastigotes were added to each well. Plates were
incubated at 35°C/5% CO2 for 4 hr and then cells were treated
for 24 hr with synthetic compounds 14a–t (6.25–50 µg/ml).
Amphotericin B (Sigma‐Aldrich®) was used as the reference
drug (0.25 to 2 µg/ml) and untreated cells were used as a negative
control. Coverslips were processed as described by Rizk et al.[39]
The overall number of amastigotes was determined by
counting 100 cells in six replicates. The half‐maximal inhibitory
concentration (IC50) was calculated using a nonlinear regression
curve. The infection index was obtained as described by
Paladi et al.[40]
|
4.2
Biological methods
|
4.2.1
Antileishmanial assays[11–14,38–40]
L. (L.) amazonensis promastigotes
In 96‐well microplates, the synthetic compounds 14a–t were tested in
quintuplicates, with concentrations from 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®, St. Louis, MO)
were added to the wells in the exponential phase with a concentration
of 2 × 105 parasites/ml. The plates were incubated at 26°C for 72 hr. To
evaluate the cell viability, 20 μl (5 mg/ml) of Thiazolyl Blue Tetrazolium
Bromide—MTT (Sigma‐Aldrich®) was added in each well and incubated
at 37°C, 5% CO2 for 2 hr. After that, 80 μl of DMSO was added to finish
the reaction.
Pentamidine (Sigma‐Aldrich®) was used as a positive control.
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
nonlinear dose–response regression curve was used to calculate the
half‐maximum inhibitory concentration (IC50).
|
4.2.2
Cytotoxicity assay[11–14,38–40]
Fibroblasts (NIH/3T3) obtained from Rio de Janeiro Cell Bank
(Brazil) were seeded in 96‐well plates (1 × 104 cells/ml)
and incubated with synthetic compounds at 37°C, 5% CO2 for
48 hr at the concentration of 0.25–250 μg/ml.[13] Doxorubicin
(Sigma‐Aldrich®) was used as the reference drug at concentrations
of 0.025–25 μg/ml. Cell growth was estimated by the sulforhoda-
mine B colorimetric method.[41] DMSO (Vetec) was used as a

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11 of 12
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negative control at a concentration necessary to solubilize the
highest concentration of the test sample and did not interfere with
cell viability. The percentage of growth was calculated as described
by Monks et al.[42] IC50 was determined by nonlinear regression
analysis (Microcal Origin Versão 6.0®) and Microsoft Office Excell
2007®. SI was calculated according to Medeiros et al.[38]
64 μg/ml in row 2 and 0.125 μg/ml in row 11 followed by the addition
of inoculum suspension (100 μl). RPMI‐1640 (200 μl) was added to
column 1 and used as the negative control. RPMI‐1640 (100 μl) and
the inoculum (100 μl) were added to the column 12 and used as the
positive control. Fluconazole was used at a concentration of 64 μg/ml
to 0.125 μg/ml. Amphotericin B was used at a concentration of
16–0.03 μg/ml. The plates were sealed and incubated at 35°C ( 2°C)
for 24 hr. The tests were performed in triplicate. The result was
|
4.2.3
Antifungal assays
observed in a spectrophotometer (Spectramax 190 microplate
reader) at 530 nm. The MIC was defined as the lowest concentration
that causes a reduction in the visible growth of a microorganism
when compared with the positive control.
Disk diffusion method
The strains of C. parapsilosis (ATCC 22019), C. albicans (ATCC
90028), C. krusei (ATCC 6258), C. glabrata (ATCC 2001), and C.
tropicalis (ATCC 750) were used for the disk diffusion test.
The procedure was performed according to the protocol M44‐A2
of the Clinical Laboratory Standards Institute (CLSI, 2009).[43]
The yeast was subcultured onto Sabouraud‐dextrose agar and
incubated at 35°C ( 2°C) for 24 hr. The inoculums of yeast strains
were prepared by selecting colonies from a 24‐hr‐old culture of
Candida spp and suspended in 5 ml of sterile 0.145 mol/l saline
(0.85% saline). Its turbidity was adjusted with a spectrometer
according to 0.5 McFarland standard resulting in a stock suspension
of 1 × 106 to 5 × 106 cells/ml. Each yeast strain was inoculated with a
sterile swab into Mueller–Hinton + GMB agar plates. A solution of
each nitrofuran isoxazole derivative at concentrations of 5 and
2.5 mg/ml was prepared and added to the paper discs placed onto the
inoculated plate. The plates were incubated at 35°C ( 2°C) for 24 hr,
and the fungal susceptibility was determined by measuring the
diameter of the inhibition zone. Assays were performed in duplicate,
and DMSO was used as the negative control, whereas fluconazole
and amphotericin B were used as positive controls.
ACKNOWLEDGMENTS
We thank Dr. 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 Uni-
versity of Mato Grosso do Sul for the HPLC‐DAD‐MS/MS analysis.
This study was supported by grants from FUNDECT‐MS (Process
numbers 59/300.109/2015, 23/200.462/2014, 23/200.708/2013),
CNPq, and CAPES.
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
ORCID
Adriano C. M. Baroni
http://orcid.org/0000-0002-5371-3755
Determination of minimal inhibitory concentration
The MIC was determined only for the active compounds that showed
a growth inhibition zone on the disk diffusion test. The procedure
was performed according to the protocol M27‐A3 of the Clinical
Laboratory Standards Institute.[44]
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Supporting Information section.
How to cite this article: Trefzger OS, Barbosa NV,
Scapolatempo RL, et al. Design, synthesis, antileishmanial, and
antifungal biological evaluation of novel 3,5‐disubstituted
isoxazole compounds based on 5‐nitrofuran scaffolds. Arch
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