Design and synthesis of a new series of 3,5-disubstituted isoxazoles active against Trypanosoma cruzi and Leishmania amazonensis

Article abstract of DOI:10.1016/j.ejmech.2017.01.029

Chagas disease and leishmaniasis are neglected tropical diseases (NTDs) endemic in developing countries. Although there are drugs available for their treatment, efforts on finding new efficacious therapies are continuous. The natural lignans grandisin (1)

Full text of DOI:10.1016/j.ejmech.2017.01.029

Accepted Manuscript
Design and synthesis of a new series of 3,5-disubstituted isoxazoles active against
Trypanosoma cruzi and Leishmania amazonensis
Rafael da Rosa, Milene Höehr de Moraes, Lara Almida Zimmermann, Eloir Paulo
Schenkel, Mario Steindel, Lílian Sibelle Campos Bernardes
PII:
S0223-5234(17)30039-9
10.1016/j.ejmech.2017.01.029
EJMECH 9180
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 November 2016
Revised Date: 18 December 2016
Accepted Date: 21 January 2017
Please cite this article as: R. da Rosa, M. Höehr de Moraes, L.A. Zimmermann, E.P. Schenkel, M.
Steindel, L.S.C. Bernardes, Design and synthesis of a new series of 3,5-disubstituted isoxazoles active
against Trypanosoma cruzi and Leishmania amazonensis, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.01.029.
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ACCEPTED MANUSCRIPT
ndisin and veraguensin isoxazole derivatives
Synthesized via microwave-assisted
1,3-dipolar cycloaddition
(20-77%)
-CH3
↓ Selectivity, trypanocidal
and leishmanicidal activity
-CH2OH
-CH2OAc
-H
Ring increases Trypanocidal activity
Group increases Trypanocidal activity
31
Most active compound
IC50= 1.13 µM (T. cruzi)
IC50= 5.08 µM (L. amazonensis)
SI= 36-161

ACCEPTED MANUSCRIPT
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Design and synthesis of a new series of 3,5-disubstituted isoxazoles active
against Trypanosoma cruzi and Leishmania amazonensis
3
4
Rafael da Rosaa, Milene Höehr de Moraesb, Lara Almida Zimmermanna, Eloir Paulo
Schenkela, Mario Steindelb, Lílian Sibelle Campos Bernardesa*
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aPharmaceutical and Medicinal Chemistry Laboratory, Department of Pharmaceutical
Sciences, Federal University of Santa Catarina, Florianópolis, 88040-900 SC, Brazil.
bProtozoology Laboratory, Department of Microbiology, Immunology and Parasitology,
Federal University of Santa Catarina, Florianópolis, 88040-900 SC, Brazil.
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*Corresponding author: l.bernardes@ufsc.br
Abstract: Chagas disease and leishmaniasis are neglected tropical diseases (NTDs)
endemic in developing countries. Although there are drugs available for their treatment,
efforts on finding new efficacious therapies are continuous. The natural lignans
grandisin (1) and veraguensin (2) show activity against trypomastigote T. cruzi and
their scaffold has been used as inspiration to design new derivatives with improved
potency and chemical properties. We describe here the planning and microwave-
irradiated synthesis of 26 isoxazole derivatives based on the structure of the lignans 1
and 2. In addition, the in vitro evaluation against culture trypomastigotes and
intracellular amastigotes of T. cruzi and intracellular amastigotes of L. amazonensis
and L. infantum is reported. Among the synthesized derivatives, compounds 17 (IC50=
5.26 µM for T. cruzi), 29 (IC50= 1.74 µM for T. cruzi) and 31 (IC50= 1.13 µM for T. cruzi
and IC50= 5.08 µM for L. amazonensis) were the most active and were also evaluated
against recombinant trypanothione reductase of T. cruzi in a preliminary study of their
mechanism of action.
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Keywords: natural product analogues; drug design; isoxazole; Trypanosoma cruzi;
Leishmania amazonensis; trypanothione reductase.
29
30
31
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Abbreviations: %GI, percentage of growth inhibition; AB, amphotericin B; BZN,
benznidazol; logP, partition coefficient octanol/water; MW, microwave; ND, not
determined due to absence of trypanocidal or leishmanicidal activity; NTD, neglected
tropical disease; SI, selectivity index; (r)TR, (recombinant) trypanothione reductase.
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1. Introduction
2
3
Chagas disease and leishmaniasis are neglected tropical diseases that affect
more than 15 million people worldwide, representing a major public health issue
especially in Latin American countries [1–3]. Benznidazole and nifurtimox are the only
drugs available for treatment of Chagas disease, while pentavalent antimonials,
amphotericin B, miltefosine, paromomycin and pentamidine are drugs for leishmaniasis
treatment. There are drawbacks associated with these drugs, such as high cost,
toxicity and limited efficacy resulting in high levels of treatment interruption. Resistant
strains of T. cruzi and Leishmania spp. are also emerging and might compromise even
more the limited therapeutic options [4–8].
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Through the centuries, biodiversity has been a useful source of medicinal
therapies. There are reports of traditional use of plants dating from more than two
thousand years ago and, currently, about 39% of the drugs on the market are natural
products or have a chemical structure directly based on natural products [9–12].
Several natural products as flavonoids, terpenes, quinones and lignans have been
tested against trypanosomatid parasites [13–15]. In spite of some identified hits, there
are still limitations associated to them, especially regarding the isolation of larger
quantities to conduct further biological investigations [16]. On the other hand,
secondary metabolites obtained from natural sources represent a library of diverse
chemical features that can give insights on the development of new chemical entities to
treat parasitic diseases [16,17].
The development of efficient drugs for treatment of Chagas disease and
leishmaniasis depends on the identification of new lead compounds [18–20]. Grandisin
(1) and veraguensin (2) are two tetrahydrofuran lignans isolated from Virola
surinamensis twigs and Piper solmsianum inflorescences that have been described as
having promising trypanocidal activity (IC50= 3.7 µM and 2.3 µM, respectively) against
the T. cruzi trypomastigote stage (Figure 1) [21,22].
Attempts on the total synthesis of lignans 1 and 2 were successful and exemplify
the importance of organic synthesis to the field of natural products [23–26]. Likewise,
the design of analogues applying distinct molecular modification strategies is a
valuable tool, and can be used to overcome problems in isolation, physicochemical
properties or to optimize the bioactivity of known natural product hits [27–31]. Our
research group has been working on different compounds based on the grandisin and
veraguensin scaffold. Between them, non-rigid 1,4-diaryl-1,4-diol analogues were
synthesized with the same methoxylation pattern of compounds 1 and 2, and displayed
IC50 values as low as 10 µM on T. cruzi trypomastigotes [32]. In another work, cyclic
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derivatives obtained through Michael addition-carbocyclization were planned aiming to
increase water solubility and also yielded compounds with good trypanocidal activity
[33]. These results confirm the potential of the tetrahydrofuran lignans scaffold and
encourage us to continue our research for bioactive compounds related to their
structure.
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4
5
6
In this work we describe the planning, synthesis and a preliminary evaluation of
the structure-activity relationship of a set of isoxazole derivatives structurally related to
the lignans 1 and 2 (Figure 1). The choice of the isoxazole ring as a bioisosteric
replacement is justified by its higher water solubility, what can improve biological and
chemical properties. The design of the compounds also relied on molecular
simplification, once the chiral tetrahydrofuran ring in compounds 1 and 2 gives place to
the aromatic isoxazole ring. Both strategies have been widely used in medicinal
chemistry on the study of molecules, affording new hits.
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12
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17
18
As the synthesis of compounds containing different substituents linked to the
isoxazole ring is feasible and straightforward, we prepared compounds substituted with
one and two aromatic rings bearing different R groups via a microwave-assisted
methodology.
OCH3
OCH3
H3CO
H3CO
O
OCH3
H3CO
Ring B
O
O
N
N
Ring A
Ring A
Grandisin 1
R''
R'
R
R
O
OCH3
OCH3
Veraguensin 2
H3CO
R= -H, -OMe, -F, -NO2, -OCH2O-
H3CO
R'= -CH2OH, -CH2N3, -COOH, -CH2OGlucose
R''= -CH2OH, -NHR, -CH3, -H, -CH2Cl, -CH2OAc
19
20
Fig 1. Structure of trypanocidal tetrahydrofuran lignans grandisin (1), veraguensin (2)
21
and proposed derivatives.
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23
24
25
26
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Thereafter, the derivatives were assessed against T. cruzi and L. amazonensis
intracellular amastigotes. The chemical variability present in the first synthesized series
allowed us to identify sites for punctual modifications that enhanced bioactivity and
selectivity, providing hints on the structure-activity relationship. The inhibitory potential
of the compounds on recombinant trypanothione reductase (rTR) was also evaluated,
due to its relevance as a target for anti-trypanosomatid drug design and its presence in
both genera Trypanosoma and Leishmania.
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2
2. Results and Discussion
2.1. Chemistry
3
4
Over the last years, microwave irradiation has been proved a powerful tool in
synthetic organic chemistry, allowing the preparation of compounds more efficiently in
comparison to traditional methods. Although there is a range of different applications
for its use, one of the main advantages of microwave irradiation is the possibility of
preparing potentially bioactive molecules within minutes [34–37].
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Based on the methodology described by Himo et al. (2005) [38], we focused on
synthesizing 3,5-disubstituted isoxazole derivatives via 1,3-dipolar cycloaddition, under
microwave irradiation in order to increase the speed of the synthesis (Scheme 1).
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Scheme 1. Reagents and conditions: i) NCS, DMF, MW: 30 ˚C, 150 W, 1 min; ii)
CuSO4, sodium ascorbate, NaHCO3, MW: 30 ˚C, 150 W, 10 min.
Our initial efforts synthesizing 5-hydroxymethyl-3-phenylisoxazole (6) have
shown that after 1 minute of irradiation (at 30 °C) it was possible to visualize by TLC
analysis the total consumption of the starting material, leading to a more apolar product
suggested to be the imidoyl chloride (Scheme 1, intermediate 4). Subsequently, when
the imidoyl chloride was irradiated with an alkyne and the final product was purified, we
could observe that the yield depends on the total time of irradiation (Table 1).
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20
21
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Table 1. Time-based yield optimization for 5-hydroxymethyl-3-phenylisoxazole (6)
synthesis.
Entry
R1
R2
Timea (min)
Yield (%)
i
ii
-H
-CH2OH
1
3
37
35
iii
iv
10
15
77
74
24
25
a. After addition of the alkyne. All the experiments were performed at 30 °C and the maximum potency was set to 150
W.
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27
The highest yield was obtained when the reaction was further irradiated for 10
minutes (Table 1, entry 6iii). As irradiation for 15 minutes did not enhance the yield of
4

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2
3
4
5
the desired product, 10 minutes was standardized for the synthesis of a series of
isoxazole derivatives 6-20 (Table 2). Furthermore, we compared the yield of the
products obtained via the microwave-assisted reaction (20-77%) with those obtained
via the reaction under conventional conditions [38] (18-72%) and it was not possible to
observe much difference besides a significant decrease in reaction time.
6
Table 2. Isoxazole derivatives initially synthetized and their yields (%)
Compound
R1
-H
R2
-H
R3
-CH2OH
Yield (%)a Yield MWb (%)
6
72
52
50
47
36
63
47
45
51
42
22
18
49
40
77
56
55
38
32
56
52
52
51
40
20
22
50
54
7
-OCH3
-OCH3
-F
-H
-CH2OH
8
-OCH3
-H
-CH2OH
9
-CH2OH
10
11
12
13
14
15
16
17
18
19
-NO2
-H
-CH2OH
-OCH2O-
-CH2OH
-H
-H
-H
-C6H4-4-CH2OH
-C6H4-4-CH2OH
-C6H4-4-CH2OH
-C6H4-4-CH2OH
-C6H4-4-CH2OH
-C6H4-4-CH2OH
-C6H4-4-NH2
-OCH3
-OCH3
-F
-OCH3
-H
-NO2
-H
-OCH2O-
-H
-H
-H
-H
(2,3,4,6-tetracetyl-α-D-
glucopyranose-1-yl)methyl
(2,3,4,6-tetracetyl-α-D-
glucopyranose-1-yl)methyl
20
-OCH2O-
46
48
7
8
a. Yield obtained in a non-irradiated reaction; i) NCS, DFM, rt.; ii) CuSO4, sodium ascorbate, NaHCO3, alkyne,
H2O/tBuOH, rt.
9
b. All microwave experiments were performed at 30 °C and the maximum potency was set to 150 W.
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11
12
13
14
15
Additionally, compound 6 was oxidized with Jones reagent, yielding compound
21 (Scheme 2), and the derivative 22 was synthesized from previously obtained
compound 7 through protection with tosyl chloride (compound 7a) followed by reaction
with sodium azide. Also, compounds 19 and 20 were synthesized from a glycosidic
alkyne, and further deprotected leading to 23 and 24 (Scheme 3) [39]. These further
5

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1
2
3
4
5
6
7
8
modifications were planned to increase the diversity related to functional groups and
physicochemical properties. The ionized moieties in compounds 21 and 22 might be
subject to ionic interactions with molecular targets while the monosaccharide unit
present in derivatives 23 and 24 led to the most hydro soluble compounds in the series.
Carbohydrates are also known for acting on cellular signaling in both parasites and
humans [40–43], and glycosides for being responsible for different biological activities,
such as antiviral [44,45], antiparasitic [46,47], antibacterial [48–50] and cytotoxic
[51,52].
O
OH
O
OH
N
N
O
i)
21
6
O
O
O
N
OH
OTs
N3
N
N
ii)
iii)
H3CO
H3CO
H3CO
7
7a
22
9
10
11
12
Scheme 2. Reagents and conditions: i) Jones reagent 8N, acetone, 30 min, 0 °C; ii)
TsCl, Et3N, K2CO3, H2O/CH2Cl2, 2 h, rt.; iii) NaN3, DMF, MW, 7 min, 70 °C.
O
O
N
N
AcO
O
HO
O
OH
OH
OAc
OAc
O
O
i)
R1
R1
R2
AcO
R2
HO
19 and 20
23 and 24
19. R1 = -H, R2 = -H
20. R1 = -OCH2O - = R2
23. R1 = -H, R2 = -H
24. R1 = -OCH2O - = R2
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Scheme 3. Reagents and conditions: i) NaOMe, MeOH, pH= 9, 15 min, rt.
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16
17
18
All synthetized compounds were purified by column chromatography and
characterized by nuclear magnetic resonance (1H, 13C NMR) and mass spectrometry
(ESI-TOF). In addition, IR spectroscopy was employed in the characterization when
necessary. The spectral data is available as supplementary material.
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20
21
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2.2.
Biologic investigation and structural optimization.
2.2.1. Trypanocidal evaluation
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T. cruzi presents a complex life cycle and a wide range of mammalian hosts. Its
trypomastigote stage is the infective form both for triatomine vectors and mammals,
while its intracellular amastigote stage is the main form responsible for the disease
[53].
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6
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Considering the importance of the amastigote stage for the pathogenesis of
Chagas disease, compounds 6-24 were firstly screened (at 100 µM) against this life
stage of the parasite (Tulahuen strain). The compounds that led to a percentage of
growth inhibition (%GI) >50% were assayed in different concentrations to determine
the half maximal inhibitory concentration (IC50). The cytotoxicity to THP-1 cells (CC50)
was also assessed to calculate the selectivity index (SI).
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12
13
14
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17
18
19
20
21
22
23
24
25
26
27
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29
30
31
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The results showed that most of the compounds were active against intracellular
amastigotes to some extent, except for compounds 8 and 21 (Table 3). These results
allowed us to set the synthetic direction for obtaining new improved derivatives that will
be discussed in the session 2.2.2.
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Table 3. Trypanocidal activity of compounds 6-24 against intracellular amastigotes
Compound
SI
-
%GI (100 µM)
7.57 (±0.06)
9.32 (±0.49)
0.00 (±0.00)
8.27 (±0.54)
12.17 (±0.86)
1.95 (±0.06)
7.01 (±1.62)
5.77 (±0.06)
IC50 (µM)
>100
>100
ND
CC50 (µM)
ND
6
7
ND
-
8
ND
-
9
>100
>100
>100
>100
>100
ND
-
10
11
12
13
ND
-
ND
-
ND
-
ND
-
14
14.15 (±1,02)
10.57 (±1.41)
15.86 (±1.49)
65.04(±4.55)
72.97 (±2.58)
1.16 (±1.00)
3.15 (±1.13)
0.00 (±0.00)
60.35 (±4.64)
7.30 (±0.40)
2.93 (±0.32)
85.94 (±1.29)
>100
>100
ND
-
15
ND
-
16
>100
ND
70.18 (±14.94)
407.2 (±88.2)
ND
-
17
5.26 (±0.96)
90.39 (±14.49)
>100
13.3
18
4.5
19
-
20
>100
ND
-
21
ND
ND
-
22
23
ND
<15.6
-
>100
ND
-
-
24
>100
ND
10.18 (±0.3)
> 500
> 49.1
BZN 20 µM
2
3
The results are averages ± SD of triplicates. Compounds 17-18 were tested in triplicate in two independent
experiments.
4
5
In the series substituted with only one aromatic ring (ring A, compounds 6-11), a
para substitution on the aromatic ring led to a slight increase in activity when compared
with the unsubstituted compound 6. On the other hand, a 3,4-substitution on the A ring
led to a loss of activity (compound 8) or to a 3-fold decrease in activity (compound 11)
in this series.
6
7
8
9
In the series substituted with two aromatic rings (rings A and B, compounds 12-
18), except for compound 13, a substituent group on the A ring seems to contribute to
an increase in biological activity in comparison with compound 12. When the
methylenehydroxy group in the B ring is replaced by a primary amine (compound 18),
the activity also increases. These findings are summarized in Figure 2. Compounds 17
10
11
12
13
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and 18 were the most promising identified among compounds 6-24, with IC50 values of
5.26 µM and 90.39 µM, respectively.
1
2
3
4
5
Fig 2. Preliminary SAR for derivatives 6-24.
6
7
Regarding compound 17, the methylenedioxy group linked to the aromatic ring A
seems to be important for the activity. It was the A ring substituent that led to a higher
increase in activity in comparison with other substituents in compounds 12-16. Also,
the replacement of the methylenedioxy group in compound 17 by two methoxy groups
(compound 14), which maintains similar chemical properties, modifies the conformation
of the molecule due to a steric clash between the aromatic substituents, and this might
be the cause of the lower activity of compound 14 (Figure 3).
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9
10
11
12
13
14
15
16
Fig 3. 3D shape of compounds 17 (a) and 14 (b) generated using Marvin Suite (version
16.4.25.0, 2016, http://chemaxon.com/).
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2
Compounds 19-20 and 23-24 displayed only a small inhibition of growth of T.
cruzi. At the first experiment, compound 22 showed a percentage of growth inhibition
similar to compound 17, but when it was assayed again one week later, the activity was
lost. Mass spectrometry analysis (HRMS ESI+/TOF) of the stock solution (Supporting
Information, Figure S1) revealed the absence of a peak relative to the cationized
molecule or other adduct, suggesting that compound 22 degraded in the test solution.
However, two relevant peaks are observed at m/z 247.1470 and m/z 188.0713. The
3
4
5
6
7
+
8
first one might be relative to the oxidized compound 22 (calculated for C11H11N4O3 :
9
247.0826) and the second peak shows that oxidation is likely to have occurred at the
10
11
azide
moiety,
since
it
can
be
attributed
to
+
the
fragment
4-
methoxyphenylisoxazolylmethylene (calculated for C11H10NO2 : 188.0706).
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2.2.2. Synthesis of new derivatives
13
14
15
16
17
18
19
20
21
For a better understanding of the properties responsible for the activity of
compounds 17 and 18, derivatives 25-30 were synthetized using the methodology
already described in session 2.1. Otherwise, compound 31 was obtained directly from
17 through reaction with thionyl chloride, under N2 atmosphere, in excellent yield
(94%).
Compounds 25-27 were planned to verify the importance of the amine group for
compound 18 activity. Moreover, compounds 28-31 were synthesized to understand
the role of the para-substituted ring B for activity. The structure of derivatives 25-31
together with results of the trypanocidal evaluation are shown in Table 4.
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Table 4. Trypanocidal activity of compounds 25-31 against intracellular amastigotes.
O
N
R4
R1
R2
Compound
25
R1
R2
R4
SI
%GI (100 µM)
IC50 (µM)
CC50 (µM)
-H
-H
-H
29.40 (±2.21)
>100
ND
-
26
-H
-H
-H
-H
-NHAc
0.00 (±0.00)
0.00 (±0.00)
ND
ND
ND
-
27
-NHBoc
ND
>500
-
28
-OCH2O-
-CH2OAc 47.59 (±4.00)
23.21 (±3.19)
1.74 (±0.41)
23.02 (±6.94)
1.13 (±0.39)
10.18 (±0.3)
>21.5
46.7
>21.7
29
30
-OCH2O-
-OCH2O-
-OCH2O-
-CH3
-H
81.65 (±3.53)
64.77 (±3.54)
95.75 (±0.31)
85.94 (±1.29)
81.32 (±9.74)
>500
31
-CH2Cl
181.8 (±25.9) 160.9
> 500 > 49.1
BZN 20 µM
The results are averages ± SD of two independent experiments run in triplicate.
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2
Considering compound 18, acetylation and carbamatization of the primary amine
lead to a loss of trypanocidal activity in compounds 26 and 27, respectively. In both
derivatives, the nitrogen lone pair is delocalized into a carbonyl, resulting in a lack of
basicity. Besides, a reduction in activity is observed when the amine group is removed
(compound 25), suggesting therefore that a basic group might play an important role
for the bioactivity of compound 18 in T. cruzi (Figure 4).
3
4
5
6
7
When compound 17 was acetylated (compound 28) or had its methylenehydroxy
group removed (compound 30) the activity had a fourfold decrease. However, an
improvement of activity and selectivity was observed in derivatives where the hydroxyl
was removed (compound 29) or replaced by a chlorine atom (compound 31) (Figure 4).
The calculation of some chemical properties using Marvin (version 16.4.25.0, 2016,
http://chemaxon.com/, Supporting Information, Table S1) showed that compounds 17,
29 and 31 have a similar surface area, while compound 28 has a larger and compound
30 a smaller surface area. Previously, we observed that the presence of two benzene
rings linked to the isoxazole ring favored the inhibition of T. cruzi growth, and now our
results indicate that there is an ideal range of molecular size in order to optimize
bioactivity in this class of compounds.
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9
10
11
12
13
14
15
16
17
18
19
Fig 4. Compounds 25-30 contribution for SAR of 3,5-disubstituted isoxazoles.
20
21
22
23
24
25
26
An important aspect of compounds 17, 29 and 31 is their potency compared to
the reference drug benznidazole (Figure 5). The amastigote stage of T. cruzi is
predominately found during the chronic course of Chagas disease, which cannot be
effectively treated by benznidazole or other drugs. Therefore, these hits will be further
investigated aiming to design new efficacious and selective molecules to treat and
control the mortality and morbidity of Chagas disease.
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2
Fig 5. Inhibition curves (a) of compounds 17, 29, 31 and comparison of their IC50
3
4
values with BZN IC50 (b).
5
Finally, although 6 compounds had high activity against T. cruzi amastigotes (IC50<100
µM, compounds 17, 18, 28-31), none of them were active against the trypomastigote
stage of the parasite (Tulahuen) at the tested concentration. It is not uncommon for
compounds to have different potencies against different T. cruzi life stages [54–56],
implying that morphological changes experienced by the parasite during its life cycle
could affect its interactions with micromolecules.
6
7
8
9
10
11
2.2.3. Leishmanicidal evaluation
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Alongside Chagas disease, leishmaniasis is also a NTD that affects millions of
people and lacks options for efficient treatment. In the New World, the Leishmania
mexicana and Leishmania braziliensis complexes comprise 7 different species,
whereas L. braziliensis and L. guyanensis are the main species responsible for
mucocutaneous leishmaniasis; L. mexicana, L. amazonensis, L. venezuelensis, L.
panamensis and L. peruviana are causative agents for cutaneous leishmaniasis. Other
5 species are commonly found in the Old World, being L. donovani and L. infantum,
responsible for causing visceral leishmaniasis and L. major, L. tropica and L. aethiopica
etiological agents of cutaneous leishmaniasis [57]. As we obtained molecules with an
interesting bioactivity against T. cruzi, we found valuable to test compounds 6-31 also
against intracellular amastigotes of L. amazonensis (MHOM/BR/77/LTB0016). Similarly
to the trypanocidal screening, the IC50 and CC50 (THP-1 cells) values were only
determined for compounds that led to a percentage of growth inhibition >50% (%GI).
The results are summarized in Table 5.
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1
2
Table 5. Leishmanicidal activity of compounds 9, 10, 19, 20, 23-25, 28, 29 and 31
against L. amazonensis intracellular amastigotes.
Compound
SI
%GI (100 µM)
3.35 (±0.61)
4.98 (±0.09)
2.06 (±0.02)
2.05 (±1.09)
4.57 (±0.18)
3.82 (±0.51)
6.03 (±1.97)
15.65 (±8.29)
28.12 (±1.51)
57.67 (±1.85)
81.62 (±0.43)
IC50 (µM)
>100
CC50 (µM)
9
10
ND
-
>100
ND
-
19
>100
ND
-
20
>100
ND
-
23
>100
ND
-
24
>100
ND
ND
-
25
>100
-
-
28
>100
ND
29
>100
ND
-
31
5.08 (±1.31)
0.14 (±0.02)
181.8 (±25.9)
27.86 (± 0.98)
35.8
199
AB 2 µM
3
4
The results are averages ± SD of triplicates. Compound 31 was tested in triplicate in two independent
experiments.
5
6
While most of the compounds inhibited the growth of T. cruzi to some degree, in
this assay only derivatives 9, 10, 19, 20, 23-25, 28, 29 and 31 presented activity at 100
µM against L. amazonensis. Overall, it is not possible to observe a clear correlation
between their structure and leishmanicidal activity. However, regarding the derivatives
based on the structure of compound 17 (compounds 28, 29, 30 and 31), lipophilicity is
a factor that might be related with percentage of growth inhibition. While compound 17
does not show any activity against L. amazonensis, compounds 28, 29 and 31 are
increasingly more potent as their lipophilicity, indicated by a higher logP value, also
increases (Figure 6). Lipophilic compounds are more permeable to cellular
membranes, what could justify this high in vitro activity. Additionally, a para-substituted
B ring might also be important for activity, as compound 30 showed no growth inhibition
at 100 µM, even though its logP value is 3.56. From an electronic point of view, the
different substituents in compounds 17, 28, 29 and 31 modify the electron distribution
on the aromatic ring B and this characteristic, together with lipophilicity, could favor the
activity for these compounds.
7
8
9
10
11
12
13
14
15
16
17
18
19
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1
2
3
Fig 6. Positive correlation between logP and leishmanicidal activity for compounds 17,
28, 29 and 31.
4
5
6
7
8
In view of the promising leishmanicidal property of compound 31, we also tested
its activity against intracellular amastigotes of L. infantum (MHOM/BR/74/PP75) to gain
an insight into the spectrum of action of the compound. Despite the high activity
observed against L. amazonensis, compound 31 had only a weak activity against L.
infantum (%GI= 9.01 ± 0.75 at 50 µM).
9
In conclusion, compound 31 was found to be the most active one against
intracellular amastigotes of both T. cruzi and L. amazonensis.
10
11
2.2.4. Enzymatic assay
12
13
14
15
16
17
18
19
20
21
22
23
24
Since the essential role of trypanothione reductase (TR) to the redox metabolism
of trypanosomatids was established, it has become one of the main exploited targets in
T. cruzi and Leishmania spp [58–61]. Different inhibitors have been described in
literature, but up to date, none of them proceeded to the further step of drug
development [62–66].
In order to investigate potential molecular targets for the most promising
synthesized molecules (IC50< 10 µM), the rTR inhibitory activity of compounds 17, 29
and 31 was assessed. All compounds evaluated have IC50 values >100 µM suggesting
that TR is not the main target involved on their antiparasitic property. Consequently,
additional studies are necessary to elucidate the mechanism of action of the
compounds presented herein. Once a mechanism of cell death or molecular target is
identified, further chemical modifications might be planned to obtain isoxazole
derivatives with an optimized activity.
15

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1
3. Concluding remarks
2
3
By using bioisosterism and molecular simplification strategies, 26 isoxazole
derivatives analogues of grandisin and veraguensin were designed and synthesized.
The screening of their trypanocidal, leishmanicidal (intracellular amastigotes) and
cytotoxic activity (THP-1 cells) afforded 6 non-cytotoxic derivatives highly active against
T. cruzi (compounds 17, 18, 28, 29, 30 and 31) and one non-cytotoxic derivative highly
active against L. amazonensis (compound 31).
4
5
6
7
8
Different criteria for hit identification are described in the literature and include, for
example, IC50 values <1 µg/mL and SI>50 for Chagas disease [18,20,67]. Among our
compounds, three have IC50 values lower than the IC50 for benznidazole (compounds
17, 29 and 31), and two have IC50<1 µg/mL (compounds 29 and 31). Concerning
selectivity, compound 31 has a SI=160.9 for cells infected by T. cruzi and a SI=35.8 for
cells infected by L. amazonensis, making it the most promising compound described
herein for satisfying all the hit selection criteria. Compound 31, as well as other
compounds in this study, also fits drug-likeness requirements related to Lipinski’s rule
of five (Supporting Information, Table S1) suggesting potential for further development
and biological investigations, especially due to its action on both parasites [68].
9
10
11
12
13
14
15
16
17
18
19
4. Supporting Information
20
Representative copies of 1H and 13C spectra are available online.
21
22
5. Experimental section
5.1.
Chemistry
23
24
25
26
27
28
29
30
31
32
33
34
Melting points were measured in a melting point apparatus MQAPF- 301 and are
1
reported uncorrected. All H and 13C NMR spectra were obtained in Nuclear Bruker
Advance DPX 400 MHz, Bruker Fourier 300 MHz and Varian Oxford AS-400 using
TMS as internal standard, unless indicated otherwise. Mass spectra were performed in
ESI-TOF Bruker micrOTOF Q II and Waters Xevo G2-S QTOF. Reactions under
microwave irradiation were conducted in a Discovery - CEM Explorer microwave
reactor with cooling, pressure and gas addition systems. Solvents and reagents,
purchased from Sigma Aldrich, were treated and purified when necessary, according to
literature [69]. Finally, thin layer chromatography was performed on silica G60 gel
layers SILICYCLE® with fluorescence indicator F-254 and column chromatography
was performed using silica gel with particle size 40-63 and 63-200 µm (Sigma Aldrich)
and hexane:ethyl acetate (Tedia) as eluent.
16

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1
2
5.1.1. General procedure for preparation of isoxazoles 6-31 under microwave
irradiation.
3
4
To a solution of an aldoxime (0.1 mmol) in DMF (0.3 mL) in a microwave tube, N-
chloro succinimide (0.105 mmol) was slowly added to avoid overheating. The tube was
sealed and submitted to microwave irradiation (30 °C, 150 W) for 1 min, and then TLC
analysis showed the complete consumption of the starting material. An alkyne (0.105
mmol), copper (II) sulphate (2 mol%), sodium ascorbate (10 mol%), sodium
bicarbonate (0.4 mmol) and water (0.3 mL) were added and the tube was further
irradiated for 10 min (30 °C, 150 W). The solution was diluted with 15 mL of brine,
extracted with ethyl acetate (3 x 10 mL), dried (Na2SO4) and concentrated under
vacuum. The crude extract was purified by flash column chromatography on silica
(hexane:ethyl acetate 6:4) yielding the expected product.
5
6
7
8
9
10
11
12
13
5.1.2. General procedure for preparation of isoxazoles 6-20.
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
The compounds were synthesised based on procedure reported in the literature
[38]. For the preparation of the imidoyl chloride, N-chloro succinimide (0.1 mmol) was
slowly added to a solution of an aldoxime (0.105 mmol) in DMF (1 mL) and the reaction
was stirred until the starting material was not visible on the TLC analysis. After, the
reaction was diluted with brine (15 mL), extracted with ethyl ether (3x 10 mL), dried
over Na2SO4, concentrated under vacuum and utilized without any purification in the
next step. Following, propargylic alcohol (0.105 mmol), copper (II) sulphate (2 mol%),
sodium ascorbate (10 mol%), sodium bicarbonate (0.4 mmol) and 4 mL of H2O:t-BuOH
were added to the product obtained in the first part, and the reaction was further stirred
for 4 hours. Next, the reaction was diluted with brine (15 mL), extracted with ethyl
acetate (3x 10 mL), dried over Na2SO4, concentrated under vacuum and the crude
extract was purified by flash column chromatography on silica (hexane:ethyl acetate
6:4) yielding the expected product. After purification, they were compared via TLC
analysis to the respecting compounds synthesized under microwave irradiation, when
this comparison was desired, and characterized by NMR and mass spectrometry.
29
30
5.1.3. Structural characterization of compounds 6-31
5.1.3.1.
5-hydroxymethyl-3-phenylisoxazole (6)
1
31
32
33
34
Yield: 72%. Yield MW: 77%. Mp: 48.8-49.5 °C. H NMR (300 MHz, Methanol-d4): δ =
7.78-7.85 (m, 2H, Ar), 7.43-7.50 (m, 3H, Ar), 6.76 (s, 1H, Isoxazole), 4.71 (s, 2H, CH2).
13C NMR (75 MHz, Methanol-d4): δ = 171.7, 162.5, 130.1, 128.9, 128.8, 126.8, 100.0,
56.7. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C10H10NO2 176.0712; Found 176.0699.
17

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5.1.3.2.
5-hydroxymethyl-3-(4-methoxyphenyl)isoxazole (7)
2
3
4
5
6
Yield: 52%. Yield MW: 56%. Mp: 90.1-92.0 °C. 1H NMR (400 MHz, CDCl3): δ = 7.74 (d,
J= 9.1 Hz, 2H, Ar), 6.98 (d, J= 9.1 Hz, 2H, Ar), 6.52 (s, 1H, Isoxazole), 4.82 (s, 2H,
CH2), 3.86 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3): δ = 171.4, 162.1, 161.0, 128.2,
121.3, 114.3, 99.8, 56.7, 55.4. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C11H12NO3
206.0817; Found 206.0815.
7
5.1.3.3.
5-hydroxymethyl-3-(3,4-dimethoxyphenyl)isoxazole (8)
8
9
Yield: 50%. Yield MW: 55%. Mp: <25 °C. 1H NMR (400 MHz, Methanol-d4): δ = 7.42 (d,
J= 2.0 Hz, 1H, Ar), 7.39 (dd, J= 2.0, 8.3 Hz, 1H, Ar), 7.04 (d, J= 8.3 Hz, 1H, Ar), 6.73 (t,
J= 0.8 Hz, 1H, Isoxazole), 4.70 (d, J= 0.8 Hz, 2H, CH2), 3.89 (s, 3H, OCH3), 3.88 (s,
10
11
12
13
13
3H, OCH3). C NMR (100 MHz, Methanol-d4): δ = 174.4, 163.6, 152.3, 150.9, 122.9,
121.3, 112.9, 110.9, 100.8, 56.5 (x2), 56.4. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
C12H14NO4 236.0923; Found 236.0922.
14
5.1.3.4.
3-(4-fluorophenyl)-5-hydroxymethylisoxazole (9)
1
15
16
17
18
19
Yield: 47%. Yield MW: 38%. Mp: 78.8-81.8 °C. H NMR (300 MHz, CDCl3): δ = 7.79
(dd, J= 5.3, 8.6 Hz, 2H, Ar), 7.15 (t, J= 8.6 Hz, 2H, Ar), 6.54 (s, 1H, Isoxazole), 4.83 (s,
2H, CH2). 13C NMR (100 MHz, CDCl3): δ = 171.9, 163.8 (d, 1JCF= 249 Hz), 161.6, 128.7
(d, 3JCF= 8 Hz), 125.0, 116.1 (d, 2JCF= 22 Hz), 99.9, 56.6. HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C10H9FNO2 194.0617; Found 194.0600.
20
5.1.3.5.
5-hydroxymethyl-3-(4-nitrophenyl)isoxazole (10)
21
22
23
24
Yield: 36%. Yield MW: 32%. Mp: 156.0-157.5 °C. 1H NMR (400 MHz, CDCl3): δ = 8.00
(d, J= 9.0 Hz, 2H, Ar), 8.33 (d, J= 9.0 Hz, 2H, Ar), 6.67 (s, 1H, Isoxazole), 4.88 (s, 2H,
CH2). 13C NMR (100 MHz, CDCl3): δ = 172.9, 160.7, 148.7, 134.9, 127.7, 124.3, 100.1,
56.6. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C10H9N2O4 221.0562; Found 221.0555.
25
5.1.3.6.
3-(3,4-benzodioxole)-5-hydroxymethylisoxazole (11)
26
27
28
29
30
Yield: 63%. Yield MW: 56%. Mp: 76.0-77.7 °C. 1H NMR (300 MHz, CDCl3): δ = 7.28 (d,
J= 1.6 Hz, 1H, Ar), 7.24 (dd, J= 8.0, 1.6 Hz, 1H, Ar), 6.86 (d, J= 8.0 Hz, 1H, Ar), 6.47
(s, 1H, Isoxazole), 6.01 (s, 2H, OCH2O), 4.78 (s, 2H, CH2). 13C NMR (75 MHz, CDCl3):
δ = 171.8, 162.1, 149.2, 148.2, 122.7, 121.2, 108.6, 106.9, 101.5, 99.9, 56.5. HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C11H10NO4 220.0610; Found 220.0598.
31
18

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1
5.1.3.7.
5-(4-hydroxymethylphenyl)-3-phenylisoxazole (12)
2
3
4
5
6
Yield: 47%. Yield MW: 52%. Mp: 168.8-169.3 °C. 1H NMR (400 MHz, CDCl3): δ = 7.83-
13
7.89 (m, 4H, Ar), 7.45-7.52 (m, 5H, Ar), 6.84 (s, 1H, Isoxazole), 4.78 (s, 2H, CH2). C
NMR (100 MHz, CDCl3): δ = 170.1, 163.0, 143.1, 130.0, 129.1, 128.9, 127.4, 126.8,
126.7, 126.0, 97.5, 64.8. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C16H14NO2 252.1025;
Found 252.0987.
7
5.1.3.8.
5-(4-hydroxymethylphenyl)-3-(4-methoxyphenyl)isoxazole (13)
8
9
Yield: 45%. Yield MW: 52%. Mp: 157.0-157.9 °C. 1H NMR (300 MHz, CDCl3): δ = 7.82
(d, J= 6.6 Hz, 2H, Ar), 7.80 (d, J= 6.6 Hz, 2H, Ar), 7.49 (d, J= 8.4 Hz, 2H, Ar), 7.00 (d,
13
10
11
12
13
J= 8.4 Hz, 2H, Ar), 6.78 (s, 1H, Isoxazole), 4.78 (s, 2H, CH2), 3.87 (s, 3H, OCH3). C
NMR (75 MHz, CDCl3): δ = 169.9, 162.6, 161.0, 143.0, 128.2, 127.3, 126.8, 126.0,
121.6, 114.3, 97.2, 64.8, 55.4. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C17H16NO3
282.1130; Found 282.1139.
14
5.1.3.9.
5-(4-hydroxymethylphenyl)-3-(3,4-dimethoxyphenyl)isoxazole (14)
15
16
17
18
19
20
21
Yield: 51%. Yield MW: 51%. Mp: 142.6-145.5 °C. 1H NMR (400 MHz, CDCl3): δ = 7.83
(d, J= 8.4 Hz, 2H, Ar), 7.48 (d, J= 8.4 Hz, 2H, Ar), 7.47 (d, J= 2.0 Hz, 1H, Ar), 7.36 (dd,
J= 2.0, 8.4 Hz, 1H, Ar), 6.95 (d, J= 8.4 Hz, 1H, Ar), 6.79 (s, 1H, Isoxazole), 4.77 (s, 2H,
13
CH2), 3.98 (s, 3H, OCH3), 3.95 (s, 3H, OCH3). C NMR (100 MHz, CDCl3): δ = 170.0,
162.7, 150.6, 149.3, 143.1, 127.3, 126.7, 126.0, 121.8, 119.9, 111.0, 109.3, 97.3, 64.8,
56.0 (x2). HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C18H18NO4 312.1236; Found
312.1236.
22
5.1.3.10. 3-(4-fluorophenyl)-5-(4-hydroxymethylphenyl)isoxazole (15)
23
24
25
26
27
Yield: 42%. Yield MW: 40%. Mp: 179.0-179.6 °C. 1H NMR (400 MHz, CDCl3): δ = 7.82-
7.89 (m, 4H, Ar), 7.51 (d, J= 8.6 Hz, 2H, Ar), 7.18 (t, J= 8.6 Hz, 2H, Ar), 6.80 (s, 1H,
Isoxazole), 4.79 (s, 2H, CH2). 13C NMR (100 MHz, CDCl3): δ = 170.4, 162.1, 143.2,
3
2
128.7 (d, JCF= 8 Hz), 127.4, 126.1, 116.1 (d, JCF= 22 Hz), 97.3, 64.8. HRMS (ESI-
TOF) m/z: [M+H]+ Calcd for C16H13FNO2 270.0930; Found 270.0922.
28
5.1.3.11. 5-(4-hydroxymethylphenyl)-3-(4-nitrophenyl)isoxazole (16)
29
30
31
Yield: 22%. Yield MW: 20%. Mp: 189.3-191.2 °C. 1H NMR (400 MHz, CDCl3): δ = 8.28
(d, J= 9.0 Hz, 2H, Ar), 7.99 (d, J= 9.0 Hz, 2H, Ar), 7.77 (d, J= 8.4 Hz, 2H, Ar), 7.45 (d,
J= 8.4 Hz, 2H, Ar), 6.89 (s, 1H, Isoxazole), 4.65 (s, 2H, CH2). 13C NMR (100 MHz,
19

ACCEPTED MANUSCRIPT
1
2
CDCl3): δ = 171.5, 161.4, 148.8, 144.3, 135.3, 127.8, 127.5, 126.1, 125.9, 124.4, 97.6,
64.0. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C16H13N2O4 297.0875; Found 297.0900.
3
5.1.3.12. 3-(3,4-benzodioxole)-5-(4-hydroxymethylphenyl)isoxazole (17)
4
5
6
7
8
9
Yield: 18%. Yield MW: 22%. Mp: 180.1-181.7 °C. 1H NMR (400 MHz, CDCl3): δ = 7.83
(d, J= 8.5 Hz, 2H, Ar), 7.50 (d, J= 8.5 Hz, 2H, Ar), 7.39 (d, J= 1.8 Hz, 1H, Ar), 7.34 (dd,
J= 1.8, 8.1 Hz, 1H, Ar), 6.91 (d, J= 8.1, 1H, Ar), 6.75 (s, 1H, Isoxazole), 6.04 (s, 2H,
OCH2O), 4.78 (s, 2H, CH2). 13C NMR (100 MHz, Methanol-d4): δ = 171.5, 164.1, 150.8,
149.7, 145.4, 128.4, 127.5, 126.8, 124.0, 122.4, 109.6, 107.6, 102.9, 98.6, 64.6. HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C17H14NO4 296.0923; Found 296.0891.
10
5.1.3.13. 5-(4-aminophenyl)-3-phenylisoxazole (18)
11
12
13
14
15
Yield: 49%. Yield MW: 50%. Mp: 130.6-132.4 °C. 1H NMR (400 MHz, CDCl3): δ = 7.80-
7.87 (m, 2H, Ar), 7.65 (d, J= 8.7 Hz, 2H, Ar), 7.74-7.50 (m, 3H, Ar), 6.75 (d, J= 8.7 Hz,
13
2H, Ar), 6.64 (s, 1H, Isoxazole), 3.95 (bs, 2H, NH2). C NMR (100 MHz, CDCl3): δ =
170.9, 162.8, 148.3, 129.8, 129.4, 128.9, 127.4, 126.8, 117.9, 114.9, 95.1. HRMS (ESI-
TOF) m/z: [M+H]+ Calcd for C15H13N2O 237.1028; Found 237.0993.
16
17
5.1.3.14. 5-[(2,3,4,6-tetraacetyl-α-D-glucopyranose-1-yl)methyl]-3-
phenylisoxazole (19)
18
19
20
21
22
23
24
25
26
Yield: 40%. Yield MW: 54%. Mp: 111.1-113.6 °C. 1H NMR (400 MHz, Methanol-d4): δ =
7.81-7.86 (m, 2H, Ar), 7.46-7.51 (m, 3H, Ar), 6.85 (s, 1H, Isoxazole), 5.28 (t, J= 9.6 Hz,
1H, H3’), 5.05 (t, J= 9.6 Hz, 1H, H4’), 4.95 (t, J= 8.4 Hz, 1H, H2’), 4.94 (d, J= 13.9 Hz,
1H, CH), 4.87 (d, J= 13.9 Hz, 1H, CH), 4.86 (t, J= 8.4 Hz, 1H, H1’), 4.29 (dd, J= 4.6,
12.4 Hz, 1H, H6’), 4.16 (dd, J= 2.5, 12.4 Hz, 1H, H6’), 3.92 (m, 1H, H5’), 2.04 (s, 3H,
CH3), 2.01 (s, 3H, CH3), 2.00 (s, 3H, CH3), 1.96 (s, 3H, CH3). 13C NMR (100 MHz,
Methanol-d4): δ = 170.6, 170.2, 169.4, 168.3, 130.2, 129.0, 126.8, 101.6, 99.9, 72.6,
72.1, 71.0, 68.2, 61.74, 61.72, 29.7, 20.73, 20.71, 20.6. HRMS (ESI-TOF) m/z: [M+H]+
Calcd for C24H28NO11 506.1618; Found 506.1655.
27
28
5.1.3.15. 5-[(2,3,4,6-tetraacetyl-α-D-glucopyranose-1-yl)methyl]-3-(3,4-
benzodioxole)isoxazole (20)
29
30
31
32
Yield: 46%. Yield MW: 48%. Mp: 110.4-112.0 °C. 1H NMR (400 MHz, Methanol-d4): δ =
7.37-7.31 (m, 2H, Ar), 6.93 (d, J= 8.3 Hz, 1H, Ar), 6.77 (s, 1H, Isoxazole), 6.03 (s, 2H,
OCH2O), 5.28 (t, J= 9.3 Hz, 1H, H3’), 5.05 (t, J= 9.3 Hz, 1H, H4’), 4.94 (t, J= 9.1 Hz,
1H, H2’), 4.93 (t, J= 13.4 Hz, 1H, CH), 4.84 (d, J= 9.1 Hz, 1H, H1’), 4.83 (d, J= 13.4 Hz,
20

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
1H, CH), 4.29 (dd, J= 4.6, 12.4 Hz, 1H, H6’), 4.16 (dd, J= 2.3, 12.4 Hz, 1H, H6’), 3.92
(m, 1H, H5’), 2.05 (s, 3H, CH3), 2.01 (s, 3H, CH3), 2.00 (s, 3H, CH3), 1.96 (s, 3H, CH3).
13C NMR (100 MHz, Methanol-d4): δ = 172.4, 171.2, 171.6, 171.3, 170.3, 163.5, 150.9,
149.8, 123.9, 122.4, 109.7, 107.6, 103.0, 102.9, 101.3, 74.1, 73.0, 72.7, 69.7, 63.0,
62.7, 20.6, 20.59, 20.56, 20.54. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C25H28NO13
550.1561; Found 550.1522.
7
5.1.3.16. (3,5-diphenyl)isoxazole (25)
8
9
Yield: 42%. Mp: 141.2-143.8 °C. 1H NMR (300 MHz, CDCl3): δ = 7.83-7.89 (m, 4H, Ar),
13
7.47-7.51 (m, 6H, Ar), 6.84 (s, 1H, Isoxazole). C NMR (100 MHz, CDCl3): δ = 170.3,
10
11
162.9, 130.2, 130.0, 129.0, 128.9, 127.4, 126.8, 125.8, 97.4. HRMS (ESI-TOF) m/z:
[M+H]+ Calcd for C15H12NO 222.0913; Found 222.0912.
12
5.1.3.17. 5-(4-acetamidephenyl)-3-phenylisoxazole (26)
1
13
14
15
16
17
Yield: 19%. Mp: 179.0-182.0 °C. H NMR (300 MHz, Methanol-d4): δ = 7.85-7.91 (m,
2H, Ar), 7.84 (d, J= 8.8 Hz, 2H, Ar), 7.73 (d, J= 8.8 Hz, 2H, Ar), 7.46-7.52 (m, 3H, Ar),
7.10 (s, 1H, Isoxazole), 2.17 (s, 3H, CH3). 13C NMR (75 MHz, Methanol-d4): δ = 170.4,
170.1, 163.0, 140.5, 129.9, 128.7, 126.3, 126.1, 122.6, 119.6, 96.7, 23.3. HRMS (ESI-
TOF) m/z: [M+H]+ Calcd for C17H15N2O2 279.1134; Found 279.1139.
18
5.1.3.18. 5-(tert-butylcarbamatephenyl)-3-phenylisoxazole (27)
19
20
21
22
23
Yield: 48%. Mp: 188.3-192.9 °C. 1H NMR (300 MHz, CDCl3): δ = 7.84-7.90 (m, 2H, Ar),
7.77 (d, J= 8.9 Hz, 2H, Ar), 7.50 (d, J= 8.9 Hz, 2H, Ar), 7.45-7.50 (m, 3H, Ar), 6.76 (s,
1H, Isoxazole), 6.65 (bs, 1H, NH), 1.54 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3): δ =
170.0, 162.6, 140.2, 130.2, 130.0, 128.9, 126.8, 122.5, 118.4, 96.6, 53.5, 28.3. HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C20H21N2O3 337.1552; Found 337.1559.
24
5.1.3.19. 5-(4-acetoxymethylphenyl)-3-(3,4-benzodioxole)isoxazole (28)
1
25
26
27
28
29
30
Yield: 28%. Mp: 164.2-168.0 °C. H NMR (300 MHz, CDCl3): δ = 7.83 (d, J= 8.3 Hz,
2H, Ar), 7.48 (d, J= 8.3 Hz, 2H, Ar), 7.38 (d, J= 1.5 Hz, 1H, Ar), 7.35 (dd, J= 1.5, 8.1
Hz, 1H, Ar), 6.91 (d, J= 8.1 Hz, 1H, Ar), 6.76 (s, 1H, Isoxazole), 6.04 (s, 2H, CH2), 2.14
(s, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ = 170.8, 169.8, 162.6, 149.2, 148.3, 138.1,
128.7, 127.3, 126.0, 123.0, 121.2, 108.7, 107.0, 101.5, 97.6, 65.7, 21.0. HRMS (ESI-
TOF) m/z: [M+H]+ Calcd for C19H16NO5 338.1028; Found 338.1028.
31
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5.1.3.20. 3-(3,4-benzodioxole)-5-(4-methylphenyl)isoxazole (29)
1
2
3
4
5
6
7
Yield: 26 %. Mp: 141.9-144.9 °C. H NMR (300 MHz, CDCl3): δ = 7.71 (d, J= 8.2 Hz,
2H, Ar), 7.37 (d, J= 1.7 Hz, 1H, Ar), 7.33 (dd, J= 1.7, 8.0 Hz, 1H, Ar), 7.28 (d, J= 8.2
Hz, 2H, Ar), 6.91 (d, J= 8.0 Hz, 1H, Ar), 6.69 (s, 1H, Isoxazole), 6.03 (s, 2H, CH2), 2.41
(s, 3H, CH3). 13C NMR (75 MHz, CDCl3): δ = 170.4, 162.5, 149.1, 148.2, 140.5, 129.7,
125.7, 124.8, 123.2, 121.1, 106.6, 107.0, 101.5, 96.7, 21.5. HRMS (ESI-TOF) m/z:
[M+H]+ Calcd for C17H14NO3 280.0974; Found 280.0975.
8
5.1.3.21. 3-(3,4-benzodioxole)-5-phenylisoxazole (30)
1
9
10
11
12
13
14
Yield: 29%. Mp: 125.2-127.9 °C. H NMR (300 MHz, CDCl3): δ = 7.83 (dd, J= 1.9, 7.7
Hz, 2H, Ar), 7.52 – 7.44 (m, 2H, Ar), 7.39 (d, J= 1.70 Hz, 1H, Ar), 7.34 (dd, J= 1.7, 8.0
Hz, 1H, Ar), 6.90 (d, J= 8.0 Hz, 1H, Ar), 6.75 (s, 1H, Isoxazole), 6.04 (s, 2H, CH2). 13C
NMR (75 MHz, CDCl3): δ = 170.2, 162.6, 149.1, 148.2, 130.2, 129.0, 125.8, 123.1,
121.2, 108.6, 107.0, 101.5, 97.3. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C16H12NO3
266.0817; Found 266.0831.
15
5.1.4. General procedure for tosylation
16
17
18
The compounds were synthesized from primary alcohols following a methodology
described in literature [70]. Purification were done by column chromatography on silica
(hexane:ethyl acetate 8:2) yielding the desired products.
19
20
5.1.4.1.
3-(4-methoxyphenyl)-5-[(4-
methylbenzenesulfonate)methylphenyl)isoxazole (7a)
21
22
23
24
25
Yield: 76%. Mp: 81-82 °C. 1H NMR (300 MHz, CDCl3): δ = 7.81 (d, J= 8.3 Hz, 2H, Ar),
7.67 (d, J= 8,8 Hz, 2H, Ar), 6.98 (d, J= 8,3 Hz, 2H, Ar), 6.98 (d, J= 8,8 Hz, 2H, Ar), 6.50
13
(s, 1H, Isoxazole), 5.17 (s, 2H, CH2), 3.85 (s, 3H, OCH3), 2.41 (s, 3H, CH3). C NMR
(75 MHz, CDCl3): δ = 164.5, 162.1, 161.2, 145.5, 132.5, 130.0, 128.2, 128.0, 120.8,
114.4, 102.9, 61.2, 55.4, 21.7.
26
5.1.5. 3-phenylisoxazole-5-carboxylic acid (21)
27
28
29
30
31
32
Jones reagent 8 N (0.3 mmol) was added dropwise to a solution of compound 7
(0.1 mmol) in acetone (4 mL) in an ice bath. The solution was stirred for 30 minutes
when TLC analysis showed the total consumption of the starting material. The solution
was diluted in acetone (15 mL) filtered through celite and evapored under vacuum. The
crude extract was purified by flash column chromatography on silica (hexane:ethyl
acetate 3:7) affording compound 23 as a white solid. Yield: 69%. Mp: 155.0-57.4 °C.
22

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2
3
4
1H NMR (300 MHz, CDCl3): δ = 7.78-7.90 (m, 2H, Ar), 7.45-7.55 (m, 3H, Ar), 7.25 (s,
1H, Isoxazole). 13C NMR (75 MHz, CDCl3): δ = 166.9, 165.5, 162.3, 134.4, 133.0,
132.8, 131.9, 130.7, 111.3. HRMS (ESI-TOF) m/z: [M-H]- Calcd for C10H6NO3
188.0353; Found 188.0321.
5
5.1.6. 5-(azidomethyl)-3-(4-methoxyphenyl)isoxazole (22)
6
7
DMF (0.3 mL), sodium azide (0.15 mmol) and compound 8a (0.1 mmol) were
added to a microwave tube and submitted to irradiation for 7 minutes (150 W, 70 ºC).
At the end, the solution was diluted with 10 mL of brine, extracted with ethyl acetate (3
x 10 mL), dried (NaSO4) and concentrated under vacuum. The product (compound 22)
8
9
1
10
11
12
13
14
was obtained quantitatively (20 mg, 100%). Mp: 51.9-53.0 °C. H NMR (300 MHz,
CDCl3): δ = 7.75 (d, J= 9.0 Hz, 2H, Ar), 6.99 (d, J= 9.0 Hz, 2H, Ar), 6.55 (s, 1H,
Isoxazole), 4.50 (s, 2H, CH2), 3.87 (s, 3H, OCH3). 13C NMR (75 MHz, CDCl3): δ =
166.7, 161.2, 128.2, 121.0, 114.4, 101.1, 55.4, 45.5. IR (KBr): 2100 (N3) cm-1. HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C11H11N4O2 231.0882; Found 231.0872.
15
16
5.1.7. General
procedure
for
deacetylation
of
2,3,4,6-tetracetyl-α-D-
glucopyranosydes (19 and 20)
17
18
19
20
21
The protected glucoside (compound 19 or 20, 0.1 mmol) was solubilized in
methanol (1 mL) and NaOMe (1 M) was added until the pH of the solution reached 9.
The solution was stirred for 15 min, cooled to 0 ºC and then the resin Dowex 50WX-
200 was added until pH dropped to 7. The solution was filtered and the product (23 or
24) was obtained in good yields [39].
22
23
5.1.7.1.
5-[(2,3,4,6-tetrahydroxy-α-D-glucopyranose-1-yl)methyl]-3-
phenylisoxazole (23)
24
25
Yield: 95%. Mp: 149.0-152.0°C. HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for
C16H19NO7Na 360.1054; Found 360.1056.
26
27
5.1.7.2.
3-(3,4-benzodioxole)-5-[(2,3,4,6-tetrahydroxy-α-D-glucopyranose-1-
yl)methyl]isoxazole (24)
28
29
Yield: 80%. Mp: 186.3-188.5 °C. HRMS (ESI-TOF) m/z: [M+Na]+ Calcd for
C17H19NO9Na 404.0952; Found 404.0941.
30
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5.1.8. 3-(3,4-benzodioxole)-5-(4-chloromethylphenyl)isoxazole (31)
2
3
A solution of compound 17 (0.1 mmol) in thionyl chloride (100 µl) was stirred
under a nitrogen atmosphere for 1 hour when TLC analysis showed total consumption
of the starting material. After, the reaction was cooled down to 0 °C, quenched with
water and extracted with CH2Cl2 (3x 10 mL). The combined organic layers were then
washed with brine and dried under vacuum affording compound 31 as a pale yellow
powder (30 mg, 94%). Mp: 162.1-163.2 °C. 1H NMR (300 MHz, CDCl3): δ = 7.82 (d, J=
8.4 Hz, 2H, Ar), 7.51 (d, J= 8.4 Hz, 2H, Ar), 7.38 (d, J= 1.7 Hz, 1H, Ar), 7.34 (dd, J=
8.0, 1.7 Hz, 1H, Ar), 6.90 (d, J= 8.0 Hz, 1H, Ar), 6.76 (s, 1H, Isoxazole), 6.04 (s, 2H,
CH2), 4.63 (s, 2H, CH2). 13C NMR (75 MHz, CDCl3): δ = 169.6, 163.2, 149.2, 148.2,
139.4, 129.2, 127.4, 122.9, 121.2, 106.6, 101.5, 97.7, 46.6. HRMS (ESI-TOF) m/z:
[M+H]+ Calcd for C17H13ClNO3 314.0584; Found 314.0576.
4
5
6
7
8
9
10
11
12
13
14
5.2.
Biologic assays
5.2.1. Trypanocidal and Leishmanicidal screening in intracellular amastigotes.
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
THP-1 cells (ATCC TIB202) were grown in RPMI-1640 without phenol red
(Sigma-Aldrich, CO. St. Louis, MO, USA) supplemented with 10% FBS (Life
Technologies, USA), 12.5 mM HEPES, penicillin (100 U/ml), streptomycin (100 µg/ml)
and Glutamax (2 mM) (37 °C, 5% CO2).
L. amazonensis MHOM/BR/77/LTB0016 and L. infantum MHOM/BR/74/PP75
promastigotes, expressing β-galactosidase, were grown in Schneider's insect medium
(Sigma Chemical Co., St. Louis, MO, USA) supplemented with 5% of heat inactivated
FBS and 2% of human urine (26 °C, 5% CO2).
T. cruzi Tulahuen trypomastigotes (raised from infected L929 cell line),
expressing β-galactosidase, were provided by the Laboratory of Cellular and Molecular
Parasitology, Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte [71].
Afterwards, THP-1 cells were cultivated in 96 well plates (4.0 × 104 cells/well) in
supplemented RPMI-1640 and treated with 100 ng/ml of phorbol 12-myristate 13-
acetate (PMA) for 72 h (37 °C, 5% CO2) to allow cell differentiation into non-dividing
macrophages [72].
Four days culture promastigotes of L. amazonensis (4.0 × 106 parasites/ml) and
L. infantum (4.0 × 106 parasites/ml) were washed with phosphate buffered saline (PBS,
pH 7.4) and incubated in RPMI-1640 supplemented with 10% of heat-inactivated
human B+ serum for 1 h (34 °C) to parasite opsonization. THP-1 cells were then
incubated with a parasite/cell ratio of 10:1 for 3 h (34 °C, 5% CO2) for L. amazonensis
and L. infantum, and with a parasite/cell ratio of 2:1 overnight (37 °C, 5% CO2) for T.
24

ACCEPTED MANUSCRIPT
1
2
cruzi. After that, non-adherent parasites were removed by washing with PBS and
infected cells were incubated with 180 µl of full supplemented RPMI-1640 medium for
another 24 h (34 °C and 37 °C, 5% CO2) to allow the transformation of
promastigotes/trypomastigotes into intracellular amastigotes.
3
4
5
The infected cells were treated with 20 µl of each compound (2 µM of DMSO-
diluted stock solution in 18 µM of RPMI-1640) in triplicate, followed by incubation for 48
h (34 °C and 37 °C, 5% CO2). Subsequently, cells were carefully washed with PBS and
incubated for 16 h (34 °C, 5% CO2) with 250 µl of chlorophenolred-ß-d-
galactopyranoside (Sigma–Aldrich Co., St. Louis, MO, USA) (CPRG) at 100 µM and
Nonidet P-40 (Amresco Inc, Solon, Ohio, USA) (NP-40) 0.1%. Optical density was read
at 570/630 nm in an Infinite M200 TECAN, Austria. Amphotericin B (Bristol-Myers,
Squibb) and benznidazole (Sigma Aldrich) were used as positive control and DMSO
1% as negative control.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
5.2.2. Trypanocidal screening in trypomastigotes
Culture T. cruzi Tulahuen trypomastigotes (raised from infected L929 cell line),
were cultivated in 96 well plates (1.5 × 106 parasites/well) and treated with the
compounds (2 µM of DMSO-diluted stock solution in 18 µM of RPMI-1640) serially
diluted in concentrations ranging from 250 to 3.9 µM. After incubation for 72 h (37°C,
5% CO2), cell viability was assessed by the MTT assay, which consists in the
colorimetric measurement of the metabolization of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) to formazan by viable cells. DMSO 1% was used
as negative control, and benznidazole (Sigma Aldrich) as positive control. The optical
density was read at 540 nm in a Infinite M200 TECAN microplate reader immediately
after the dissolution of formazan crystals with DMSO [73,74].
27
5.2.3. Cytotoxicity assay (MTT)
28
29
30
31
32
THP-1 cells were grown and cultivated in 96 well plates (4.0 × 106 cells/well), as
described in 5.2.1., treated with the compounds (2 µM of DMSO-diluted stock solution
in 18 µM of RPMI-1640) serially diluted in concentrations ranging from 15.6 µM to 500
µM and incubated for 72 h (37 °C, 5% CO2). Cell viability was assessed as described in
5.2.2.
25

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5.2.4. Trypanothione reductase enzyme assay
2
3
4
Recombinant trypanothione reductase from T. cruzi (TcTR), was expressed in
Escherichia coli BL21DE3 and purified by affinity chromatography. TR assays were
performed as described by Hamilton et al. (2003) [75]. In 96 well micro plates (final
5
6
volume= 240 µL), TcTR (1 m-unit), HEPES (40 mM, pH 7.5), NADPH (0.15 mM),
DTNB (25 µM) and EDTA (1 mM) were incubated for 5 min (27 °C) before T(S)2 (1 µM)
and the tested compound (diluted in DMSO) were added. Compounds and controls
were pre-incubated at 27 oC for 30 min and 10 µL of DTNB was added to the reaction
mixture. Absorbance at 412 nm was measured for 30 min to determine the enzymatic
activity. DMSO 1% was used as negative control and clomipramine as positive control.
7
8
9
10
11
Acknowledgments
12
13
14
15
16
17
18
19
20
We gratefully acknowledge José Carlos Tomaz (Faculty of Pharmaceutical Sciences of
Ribeirão Preto, USP, Brazil), Luis Otávio Zamoner (Faculty of Pharmaceutical
Sciences of Ribeirão Preto, USP, Brazil), Vinícius Palaretti (Faculty of Philosophy,
Sciences and Letters of Ribeirão Preto, USP, Brazil) and Louis Pergaud Sandjo
(Graduate Program in Pharmacy, UFSC, Brazil) for the spectral analyses. This study is
part of the collaboration work within the Research Network Natural Products against
Neglected Diseases (ResNetNPND http://www.resnetnpnd.org/).
Funding
21
22
This work was supported by FAPESC/CNPq, Brazil (PRONEX, grant 2671/2012-9);
and CAPES, Brazil.
23
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